KR20080045185A - 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 polypeptide with selectable properties {HYBRID POLYPEPTIDES WITH SELECTABLE PROPERTIES}

Related Applications

This specification discloses U.S. Provisional Application No. 11 / 201,664, filed Aug. 11, 2005, and co-owned with U.S. Appl. No. 11 / 206,903, filed Aug. 17, 2005. Both are "Hybrid Polypeptides with Selectable Properties" and US 11 / 301,744, filed Dec. 12, 2005, all of which are incorporated herein by reference in their entirety.

The present invention relates to peptide chemicals, more particularly to hybrid polypeptides having selectable properties.

For many metabolic diseases and disorders the regulation of insulin levels and blood glucose levels is very important. Insulin secretion is regulated in part by secretory stimulating hormone called incretin, which is produced by enteroendocrine cells. The incretin hormone glucagon-like peptide-1 (“GLP-1”) is a peptide hormone secreted by intestinal cells, and many studies have shown that it has the effect of improving insulin secretion. GLP-1 is processed from proglucagon in the intestines and enhances nutrient-induced insulin release (Krcymann B., et al., Lancet, 2: 1300-1303 (1987)). Various truncated forms of GLP-1 are known to stimulate insulin secretion (insulin stimulation) and cAMP formation (eg, Mojsov, S., Int. J. Pep. Pro. Res., 40 : 333-343 (1992). Insulin stimulation in mammals, particularly humans, for a variety of in vitro laboratory experiments and exogenous administration of GLP-1, GLP-1 (7-36) amide (SEQ ID NO: 61) and GLP-1 (7-37) acid (SEQ ID NO: 204) Relationships between responses have been established (see, eg, Nauck, MA, et al., Diabetologia, 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)).

GLP-1 (7-36) amide (SEQ ID NO: 61) exerts a significant anti-diabetic effect in insulin dependent diabetes by stimulating insulin sensitivity and enhancing glucose-induced insulin release at physiological concentrations [Gutniak M., et al., New Eng. J. Med., 326: 1316-1322 (1992). When administered to insulin non-dependent diabetes mellitus, GLP-1 (7-36) amide (SEQ ID NO: 61) stimulates insulin release, lowers glucagon secretion, inhibits gastric fasting and improves glucose utilization (Nauck, 1993). , Gutniak, 1992, Nauck, 1993). However, GLP-1 type molecules have very short serum half-lives, making it difficult to use these peptides for long term therapy of diabetes.

More particularly, GLP-1 is a 30 amino acid peptide derived from proglucagon, which is a 160 amino acid prohormone. In the pancreas and intestinal tract, the action of various prohormone converting enzymes produces glucagon and other unknown peptides, whereas cleavage of proglucagon produces GLP-1 and GLP-2 and also two other peptides. The amino acid sequence of GLP-1 is 100% homologous in all mammals studied so far, suggesting a significant physiological role. GLP-1 (7-37) acid is truncated at the C-terminus and amidated to form GLP-1 (7-36) NH ₂ (SEQ ID NO: 61). The biological effects and metabolic turnover of the free acid GLP-1 (7-37) OH (SEQ ID NO: 204) and the amide GLP-1 (7-36) NH ₂ (SEQ ID NO: 61) are indistinguishable. Typically, the numbering of amino acids is based on GLP-1 (1-37) OH (SEQ ID NO: 59) processed from proglucagon. GLP-1 (GLP-1 (7-36) NH ₂ ) (SEQ ID NO: 61) with biological activity is the result of further processing. Thus, the first amino acid of GLP-1 (7-37) OH (SEQ ID NO: 204) or GLP-1 (7-36) NH ₂ (SEQ ID NO: 61) is ⁷ His.

In the gastrointestinal tract, GLP-1 is produced by L-cells of the intestinal, colon and rectal mucosa in response to stimulation of intraluminal glucose. The plasma half-life of active GLP-1 is <5 minutes and its metabolic clearance is about 12 to 13 minutes [Holst, Gastroenterology 107 (6): 1848-55 (1994)]. The main protease involved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV (CD26), which cleaves the N-terminal His-Ala dipeptides to the metabolite GLP-1 (9-37) OH ( SEQ ID NO: 205) or GLP-1 (9-36) NH ₂ (SEQ ID NO: 206), which is variously described as a weakly inactive agonist or antagonist of the GLP-1 receptor. GLP-1 receptor (GLP-1R) is a 463 amino acid G protein coupling receptor, localized to pancreatic beta cells and lungs and to a lesser extent localized to brain, adipose tissue and kidneys. Stimulation of GLP-1R by GLP-1 (7-37) OH (SEQ ID NO: 204) or GLP-1 (7-36) NH ₂ (SEQ ID NO: 61) may result in adenylate cyclase activation, cAMP synthesis, membrane depolarization, cell Raises calcium in the blood and increases glucose-induced insulin secretion [Holz et al., J. Biol. Chem. 270 (30): 17749-57 (1995).

GLP-1 is a potent insulin secretagogue that is secreted from the intestinal mucosa in response to food intake. The sufficient incretin effect of GLP-1 is understood from the fact that GLP-1R knockout mice are glucose intolerant. I.v. in diabetic patients. The incretin response of the injected GLP-1 is preserved, but the incretin response to oral glucose is impaired in these patients. GLP-1 administration by infusion or sc injection controls fasting glucose levels in diabetic patients and maintains glucose thresholds for insulin secretion (Gutniak et al., N. Engl. J. Med. 326: 1316- 22 (1992), Nauck et al., Diabet. Med. 13: (9 Suppl 5): S39-S43 (1996), Nauck et al., J. Clin. Endocrinol. Metab. 76: 912- 917 (1993)]. GLP-1 has shown great potential as a therapeutic agent that can increase insulin secretion in a physiological manner while avoiding hypoglycemia associated with sulfonylurea drugs.

Another important effect of GLP-1 on glucose homeostasis is inhibition of glucagon secretion and inhibition of gastric motility. GLP-1's inhibitory action on pancreatic alpha cell secretion of glucagon reduces hepatic glucose production through the reduction of glucose synthesis and glycogen breakdown. The anti-glucagon effect of GLP-1 is preserved in diabetic patients.

The so-called ileal brake effect of GLP-1, which inhibits gastric motility and gastric secretion, is achieved either through the vagus nerve centrifugal receptor or by direct action on the intestinal smooth muscle. Reduction of gastric acid secretion by GLP-1 contributes to a lag phase in nutrient utilization, thus eliminating the need for a rapid insulin response. In summary, the gastrointestinal effects of GLP-1 significantly contribute to delayed glucose and fatty acid uptake and modulate insulin secretion and glucose homeostasis.

GLP-1 has also been shown to induce beta cell specific genes such as GLUT-1 transporter, insulin (via interaction of PDX-1 with insulin gene promoter) and hexokinase-1. Thus, GLP-1 can potentially reverse glucose intolerance normally associated with aging as demonstrated by rodent experiments. GLP-1 may also contribute to beta cell angiogenesis and increase beta cell volume, in addition to restoring beta cell function during beta cell dysfunction.

Central effects of GLP-1 include increased satiety associated with decreased food intake exerted through the action of hypothalamic GLP-1R. For 48 hours of continuous SC infusion of GLP-1 in type II diabetic patients, they reduced hunger and food intake and increased satiety. This loss of appetite was not seen in GLP-1R knockout mice.

Exendin is another family of peptides involved in insulin secretion. Exendin is found in the saliva of the Gila-monster and Mexican Beaded Lizards, which are native to Arizona. Exendin-3 is present in saliva of Heloderma horridum and exendin-4 is present in 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). Exendin has some sequence similarity to some members of the glucagon-like peptide family and 53% highest identity to GLP-1 [Goke, et al., J. Biol. Chem., 268: 19650-55 (1993).

Exendin-4 binds to GLP-1 receptors present in insulin-secreting TC1 cells, interspersed acinar cells of guinea pig pancreas and gastric wall cells, and the peptides also stimulate somatostatin release and inhibit gastric release in isolated stomach Inhibit (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)). Exendin-3 and exendin-4 have been shown to bind to the GLP-1 receptor on pancreatic acinar cells to stimulate cAMP production and amylase release from pancreatic acinar cells (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)]. It has been proposed to use the insulin stimulatory activity of exendin-3 and exendin-4 for the treatment of diabetes mellitus and for prevention of hyperglycemia (Eng, US Pat. No. 5,424,286).

Truncated exendin peptides such as exendin [9-39], carboxyamidation molecules, and fragments 3-39 to 9-39 have been reported to be potent and selective antagonists of GLP-1 ([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] (SEQ ID NO: 207) blocks endogenous GLP-1 in vivo to reduce insulin secretion (Wang, et al., J. Clin. Invest., 95: 417-21 (1995) , D'Alessio, et al., J. Clin. Invest., 97: 133-38 (1996)). Receptors explicitly responsible for the insulin stimulatory effect of GLP-1 were cloned from rat pancreatic islet cells [Thorens, B., Proc. Natl. Acad. Sci. USA 89: 8641-8645 (1992). Exendin and exendin [9-39] bind to cloned GLP-1 receptors (rat pancreatic cell GLP-1 receptor [Fehmann HC, et al., Peptides, 15 (3): 453-6 (1994)] , Human GLP-1 receptor [Thorens B, et al., Diabetes, 42 (11): 1678-82 (1993)]. In cells transfected with cloned GLP-1 receptors, exendin-4 increases cAMP as an agonist, whereas exendin [9-39] (SEQ ID NO: 207) stimulates exendin-4 and GLP-1 as antagonists Block (see above).

More particularly, Exendin-4 is a 39 amino acid C-terminal amidated peptide found in the saliva of American lizard (Helderma suspectum) and has 53% amino acid sequence identity to the GLP-1 peptide sequence. See, eg, 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, AA, et al., "Glucose-Lowering and Insulin-Sensitizing Actions of Exendin-4," Diabetes, Vol. 48, p. 1026-1034, May, 1999). In terms of activity, exendin-4 is a highly specific agonist for the GLP-1 receptor and can stimulate insulin secretion like GLP-1. Thus, exendin-4 is considered to be an insulin stimulating peptide like GLP-1.

However, exendin-4, unlike GLP-1, has a relatively long half-life in humans because it is resistant to dipeptidyl peptidase IV, which rapidly degrades GLP-1 sequences in vivo. In addition, exendin-4 has greater insulin secretion stimulation than GLP-1, and it has been shown that exendin-4 can be used at lower concentrations to achieve this stimulating activity. See, eg, US Pat. No. 5,424,286, which is incorporated herein by reference. Thus, exendin-4 peptides or derivatives thereof (see, for example, US Pat. No. 6,528,486 and its corresponding international patent application WO 01/04156, which are incorporated herein by reference for example), are associated with difficulty in regulating insulin levels. It has greater potential utility than insulin or GLP-1 in the treatment of comprising conditions (such as diabetes, for example).

Another family of peptide hormones associated with metabolic diseases and disorders are those of the amylin family, including amylin, calcitonin, calcitonin gene related peptides, adrenomedulin and intermedin (also known as "AFP-6"). It is a peptide hormone. Amylin is a 37 amino acid peptide hormone. It has been isolated, purified and chemically characterized as a major component of amyloid deposits in pancreatic islets of human type 2 diabetes [Cooper et al., Proc. Natl. Acad. Sci., USA, 84: 8628-8632 (1987). The amylin molecule has two post-translational modifications, such that the C-terminus is amidated and the cysteines at positions 2 and 7 crosslink to form an N-terminal loop. The open reading frame sequence of the human amylin gene is Lys-Arg dibasic amino acid proteolytic cleavage signal in front of the N-terminal codon of Lys, and Lys- at the CLAIMS-terminal position (a sequence typical for amidation by protein amidation enzyme PAM). The presence of Gly before Arg proteolysis signals [Cooper et al., Biochem. Biophys. Acta, 1014: 247-258 (1989).

Amylin is believed to regulate gastric emptying, inhibit glucagon secretion and food intake, thereby controlling the rate of circulating glucose. It is believed that this complements the action of insulin to regulate glucose extinction rate from circulating blood and its uptake by peripheral tissues. This action is supported by experimental results in rodents and humans, indicating that amylin supplements the effect of insulin on postprandial glucose control by at least three independent mechanisms affecting glucose abundance. First, amylin inhibits glucagon secretion after meals. Compared to healthy adults, there was no circulating amylin in patients with type 1 diabetes and decreased postprandial amylin concentrations in patients with type 2 diabetes. In addition, injection of amylin-specific monoclonal antibodies that bind to circulating amylin again resulted in significantly increased glucagon concentration compared to the control. These two results all point to the physiological role of endogenous amylin in the regulation of post-prandial glucagon secretion. Second, amylin slows down gastrointestinal motility and gastric emptying. Finally, hypothalamic injections of rat amylin have been shown to reduce food intake in rats and alter neurotransmitter metabolism in the hypothalamus. In certain studies, food intake was significantly reduced by 8 hours after intrahypothalamic injections of rat amylin and rat CGRP. In human testing, amylin analog pramlintide has been shown to reduce body weight or weight gain. Amylin may be used to treat metabolic diseases such as diabetes and obesity. Amylin may also be used for changes in body composition, such as treatment of pain, bone disorders and gastritis, modification of lipids, especially triglycerides, or preferential loss of fat and reduced use of lean tissue.

The hormone calcitonin (CT) has been named based on its secretion in response to induced hypercalcemia and its rapid low calcium effect. It is produced and secreted by neuroendocrine cells in the thyroid gland called C cells. The best studied action of CT (1-32) (SEQ ID NO: 48) is the effect on osteoclasts. In vitro effects of CT include rapid loss of ruffled border and reduced release of lysosomal enzymes. Ultimately, inhibition of osteoclast function by CT reduces bone uptake. However, in the case of thyroidectomy, neither chronic reduction of serum CT nor increased serum CT found in medulla thyroid cancer are considered to be associated with changes in serum calcium or bone mass. Thus, the primary function of CT (1-32) (SEQ ID NO: 48) is most likely to protect against acute hypercalcemia in emergency situations and / or to protect the skeleton during "calcium stress" periods, such as growth, pregnancy and lactation. (Reviewed in Becker, JCEM, 89 (4): 1512-1525 (2004) and Sexton, Current Medicinal Chemistry 6: 1067-1093 (1999)). The recent data found that the calcitonin gene knockout mice with both calcitonin and CGRP-I peptides removed showed normal levels of calcium-related values but increased calcium blood responses in mice [Kurihara H, et al., Hypertens]. Res. 2003 Feb; 26 Suppl: S105-8]. CT has an effect on plasma calcium levels, inhibits osteoclast function, and is widely used for the treatment of osteoporosis. Therapeuticly, salmon CT (sCT) increases bone density and reduces fracture rate and side effects are believed to be minimal. CT has also been used successfully over the last 25 years as a therapy for Paget's disease of chronic skeletal impaired bone, which can result in bones that have become larger or deformed in one or more areas of the skeleton. CT is also widely used because of the analgesic effect on bone pain experienced during osteoporosis, but the mechanism for this effect is not clearly understood.

Calcitonin gene related peptides (CGRP) are neuropeptides whose receptors are widely distributed in the body, including the nervous and cardiovascular systems. The peptide is believed to modulate sensory neurotransmission and is one of the most potent endogenous vasodilator peptides found to date. The reported biological effects on CGRP include: modulation of substance P upon inflammation, nicotine receptor activity at neuromuscular junctions, stimulation of pancreatic enzyme secretion, reduction of gastric acid secretion, peripheral vasodilation, cardiac acceleration, neuro-modulation, regulation of calcium metabolism , Stimulating bone formation, secreting insulin, increasing body temperature and reducing 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 CGRP is to control blood flow to various organs by its potent vasodilation, as evidenced by a decrease in mean arterial pressure after intravenous administration of α-CGRP. Vasodilation is also supported by recent analyzes of homozygous knockout CGRP mice showing elevated peripheral vascular resistance and high blood pressure caused by increased peripheral sympathetic nerve activity [Kurihara H, et al., Targeted disruption of ADM and αCGRP genes reveals their distinct biological roles. Hypertens Res. 2003 Feb; 26 Suppl: S105-8]. Therefore, CGRP is believed to cause vasodilation effect, blood pressure lowering effect and increased heart rate, among other actions.

Long-term infusion of CGRP in patients with congestive heart failure has a sustained beneficial effect on hemodynamic function without side effects, suggesting its use in heart failure. Other examples of CGRP use include renal failure, acute and chronic coronary ischemia, treatment of cardiac arrhythmias, other peripheral vascular diseases such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension and pulmonary hypertension. Preeclampsia in pregnancy and early delivery are also potentially treatable [Wimalawansa, 1997]. Recent therapeutic uses include the use of CGRP antagonists for the treatment of migraine headaches.

Adrenomedulin (ADM) is expressed almost everywhere in more tissues containing peptides than does not contain peptides. In a review reviewing ADM [Hinson, JP et al., Endocrine Reviews (2000) 21 (2): 138-167], a range of biological functions including vasodilation, cell growth, regulation of hormone secretion, and increased sodium excretion Its effects on cardiovascular, cell growth, central nervous and endocrine systems have been described in detail. Studies in rats, cats, sheep and humans have confirmed that intravenous infusion of ADM results in a strong and persistent blood pressure drop, similar to that of CGRP. However, the hypotensive effect of ADM on mean arterial pressure in anesthetized rats was not inhibited by the CGRP antagonist CGRP _{8 -37} , suggesting that the effect is not mediated through the CGRP receptor. Short-term or long-term administration of human ADM in anesthetized, conscious or hypertensive rats results in a significant decrease in total peripheral tolerance accompanied by a drop in blood pressure, accompanied by a rise in heart rate, cardiac output and single stroke.

ADM has also been proposed as an important factor in embryonic formation and differentiation and as apoptosis survival factor for rat endothelial cells. This is supported by recent mouse ADM knockout studies, in which mice homozygous for ADM gene loss exhibited incomplete angiogenesis during embryonic death and died mid-pregnancy. ADM +/- heterozygous mice have been reported to have high blood pressure with susceptibility to tissue damage [Kurihara H, et al., Hypertens Res. 2003 Feb; 26 Suppl: S105-8].

ADM affects endocrine organs such as the pituitary gland, adrenal glands, reproductive organs and pancreas. The peptide is believed to play a role in inhibiting ACTH release from the pituitary gland. In the adrenal glands, the peptides are believed to affect the secretory activity of the adrenal cortex in both rats and humans, and act as vasodilators in the adrenal vascular layer in intact rats to increase adrenal blood flow. ADM has been found to be present throughout the female reproductive tract, with elevated plasma levels in normal pregnancy. Studies in a rat model of preeclampsia show that ADM can reverse hypertension and reduce pup mortality when administered to rats during late pregnancy. This suggests that ADM may play an important regulatory role in the uterine-placental cardiovascular system because it does not have a similar effect on non-pregnant rats in early pregnancy or in preeclamptic models. In the pancreas, ADM is considered to be the most potent because it attenuates and delays insulin response to oral glucose attempts, thereby initially raising glucose levels. ADM can also affect renal function. Peripherally administered bolus can significantly lower mean arterial pressure and increase renal blood flow, glomerular filtration rate, and urine flow. In some cases, Na ⁺ emissions are also increased.

ADM also has other peripheral effects in bone and lung. Studies on bone have supported a role beyond cardiovascular and fluid homeostasis, and ADM acts on fetal and adult rodent osteoblasts to promote cell growth similar to known osteoblast growth factors such as transforming growth factor-β. Prove to increase. This is clinically important because one of the major challenges in osteoporosis research is to develop therapies that increase bone mass through osteoblast stimulation. In the lung, ADM not only causes pulmonary vasodilation but also inhibits bronchial contraction induced by histamine or acetylcholine. Recent studies using aerosolized ADM to treat pulmonary hypertension in a rat model demonstrate that the average pulmonary arterial pressure and total lung resistance are significantly lower in rats treated with ADM than in rats receiving saline. As shown, inhalation treatment of the disease is effective. This result was achieved without fluctuations in systemic arterial pressure or heart rate [Nagaya N et al., Am J Physiol Heart Circ Physiol. 2003; 285: H 2125-31.

In healthy volunteers, i.v. of ADM. Infusion has been shown to reduce arterial pressure and stimulate heart rate, cardiac output, and plasma levels of cAMP, prolactin, norepinephrine and renin. Little or no increase in urine volume or sodium excretion was observed in these patients. In patients with heart failure or chronic renal failure, i.v. ADM had an effect similar to that seen in normal subjects, and also induced diuresis and sodium excretion, depending on the dose administered [Nicholls, MG et al. Peptides. 2001; 22: 1745-1752. Experimental ADM treatment has also been shown to be beneficial for arterial and pulmonary hypertension, septic shock and ischemia / reperfusion injury [Beltowski J., Pol J Pharmacol. 2004; 56: 5-27. Other examples of ADM treatment include peripheral vascular disease, subarachnoid hemorrhage, high blood pressure, preeclampsia and osteoporosis in pregnancy and early delivery.

Expression of AFP-6 (ie intermedin) occurs mainly in the pituitary and gastrointestinal tract. No specific receptors for AFP-6 have been reported, but binding studies indicate that AFP-6 binds to all known receptors of the amylin family. AFP-6 has been shown to increase cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and to compete with labeled CGRP for binding to their receptors in these cells. In published in vivo studies, AFP-6 administration caused blood pressure reduction in both normal and spontaneous hypertensive rats, most likely due to interaction with CRLR / RAMP receptors. In vivo administration in mice results in inhibition of gastric emptying and food intake [Roh et al. J Biol Chem. 2004 Feb 20; 279 (8): 7264-74.

The biological action of amylin family peptide hormones is generally mediated through binding to two closely related type II G protein-coupling receptors (GPCRs), the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CRLR). It has been reported. Cloning and functional studies have shown that CGRP, ADM and amylin interact with several combinations of CTR or CRLR and receptor active modified protein (RAMP). Many cells express many RAMPs. Coexpression of RAMP and CTR or CRLR is believed to be necessary for the production of functional receptors for calcitonin, CGRP, ADM and amylin. The RAMP family includes three members (RAMP1, -2 and -3) with less than 30% sequence identity but with a common topological organ. Co-expression of CRLR and RAMP1 forms a receptor for CGRP. Coexpression of CRLR and RAMP2 forms a receptor for ADM. Coexpression of CRLR and RAMP3 forms receptors for ADM and CGRP. Coexpression of hCTR2 and RAMP1 forms receptors for amylin and CGRP. Co-expression of hCTR2 and RAMP3 forms a receptor for amylin.

Another peptide hormone family associated with metabolic diseases and disorders is the leptin family. The mature form of circulating leptin is a 146 amino acid protein that is normally blocked from the CNS by the vascular-brain barrier (BBB) and the blood-CSF barrier. See, eg, Weigle et al., 1995. J Clin Invest 96: 2065-2070. Leptin is a centripetal signal in the negative feedback loop that regulates food intake and weight. Leptin receptors are members of the cytokine receptor family. The appetite suppressing effect of leptin depends on the binding of the receptor to the homodimer of the Ob-Rb isoform, which encodes a long intracellular domain containing a plurality of motifs for protein-protein interactions. Ob-Rb is highly expressed in the hypothalamus, suggesting that the brain region is an important site of leptin action. Mutations in the mouse ob gene have been demonstrated to cause syndromes showing obesity, increased body fat deposition, hyperglycemia, hyperinsulinemia, hypothermia, and impaired thyroid and reproductive function in male and female homozygous ob / ob obese mice (See, eg, Ingalis, et al., 1950. J Hered 41: 317-318). Therapeutic uses of leptin or leptin receptors include (i) diabetes (eg PCT Application Publication WO 98/55139, WO 98/12224 and WO 97/02004), (ii) Hematopoiesis (eg PCT Application Publication WO 97 / 27286 and WO 98/18486), (iii) infertility (eg PCT application publication WO 97/15322 and WO 98/36763), and (iv) tumor inhibition (eg PCT application publication WO 98/48831 (Each of which is incorporated herein by reference in its entirety).

The leptin receptor (OB-R) gene was cloned (GenBank Accession No. AF098792) and mapped to the db locus (see, eg, Tartaglia, et al., 1995. Cell 83: 1263-1271). . In addition, multiple transcripts of OB-R have been identified due to alternative splicing. Defects in OB-R produce syndromes in mutant diabetic ob / ob mice whose phenotype is the same as ob / ob mice (see, eg, Ghilardi, et al., 1996. Proc. Natl. Acad. Sci. USA 93). : 6231-6235). However, unlike ob / ob mice, administration of recombinant leptin to C57BLKS / Jm ob / ob mice does not cause food intake and weight loss (eg, Roberts and Greengerg, 1996. Nutrition Rev. 54: 41- 49].

Most leptin-related studies that can report weight loss activity due to administration of recombinant leptin, leptin fragments and / or leptin receptor variants have been administered directly into the ventricles. See, eg, Weigle, et al., 1995. J Clin Invest 96: 2065-2070, Barash, et al., 1996. Endocrinology 137: 3144-3147.

Another study showed significant weight loss activity due to leptin peptide administered to the test subjects intraperitoneally (i.p.). See Grasso et al., 1997. Endocrinology 138: 1413-1418. In addition, fragments of 18 amino acids, including leptin fragments, and most particularly residues taken from full length human leptin, have been reported to function in weight loss only upon direct administration via cannula implanted in the rat ventricle. See, eg, PCT patent application WO 97/46585, which is incorporated by reference in its entirety.

Another peptide hormone associated with metabolic diseases and disorders is cholecystokinin (CCK). CCK was reported to have been identified in the intestinal extract formulation in 1928 due to gallbladder contractile stimulation. Later, other biological actions of CCK have been reported, including stimulation of pancreatic secretion, delayed gastric emptying, stimulation of intestinal motility and stimulation of insulin secretion. Lieverse et al., Ann. N. Y. Acad. Sci. 713: 268-272 (1994). CCK action has also been reported to include effects on cardiovascular function, respiratory function, neurotoxicity and seizures, cancer cell proliferation, analgesia, sleep, sexual and reproductive behavior, memory, anxiety and dopamine mediated behavior [Crawley and Corwin, Peptides 15: 731-755 (1994). Other reported effects of CCK include stimulation of pancreatic growth, stimulation of gallbladder contraction, inhibition of gastric acid secretion, pancreatic polypeptide release, and contractile components of peristalsis. Additional reported effects of CCK include vasodilation [Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal Tract 3d ed. 1994; Raven Press, New York.

Injection of a combination of glucagon, CCK and bombesin has been reported to enhance the inhibition of intake of condensed milk test meals in non-deprived rats compared to the inhibition 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 sham feeding in rats (LeSauter and Geary, Am. J. Physio. 253: R217-225 (1987)), Smith and Gibbs, Annals NY 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 suggested that signals generated in the small intestine in response to nutrients in the small intestine can synergistically interact with CCK to reduce food intake [Cox, Behav. Brain Res. 38: 35-44 (1990). In addition, CCK has been reported to induce satiety in several species. Inhibition of food intake, for example, by CCK injected intravenously in rats, intraperitoneally in pigs, intravenously in cats and pigs, intraventricularly in monkeys, rats, dogs and sheep, and intravenously in obese and non-obese humans. Has been reported. See Lieverse et al., Supra. Studies reported in several laboratories have shown that in both monkeys and rats, the response to food is compared to the response to non-food fortifications and that CCK induces a sequence of behaviors commonly observed after food intake (ie post-prandial satiety order). As a result, it was reported to confirm the behavioral specificity of the low dose of CCK for suppressing food intake. In addition, it has also been reported that behavioral similarity between CCK and food intake has been found in comparison to the behavior after CCK in combination with food intake itself or with CCK [Crawley and Corwin, supra]. It has been reported that physiological plasma concentrations of CCK inhibit food intake and increase satiety in both lean and obese humans. See Lieverse et al., Supra.

CCK was characterized as a 33 amino acid peptide in 1966 [Crawley and Corwin, supra]. Species-specific molecular variants of the amino acid sequence of CCK have been identified. The 33 amino acid sequence and the truncated peptide, its 8 amino acid C-terminal sequence (CCK-8), have been reported to have been identified in pigs, rats, chickens, chinchillas, dogs and humans. 39 amino acid sequences were reported to be found in pigs, dogs, and guinea pigs. 58 amino acid sequences have been reported to be found in cats, dogs, and humans. Frogs and turtles have been reported to exhibit 47 amino acid sequences that are homologous to both CCK and gastrin. Very fresh human intestines have been reported to contain small amounts of even larger molecules called CCK-83. In rats, major intermediate forms have been reported to be identified and called CCK-22 [Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York) .Non-sulphurized CCK -8 and tetrapeptides (CCK-4 (called CCK (30-33)) have been reported in the rat brain. C-terminal pentapeptides (CCK-4 (called CCK (29-33))) are structural phases of CCK Homology, and also homology with neuropeptides, gastrins The C-terminal sulfated octapeptide sequence CCK-8 has been reported to be relatively conserved in the entire species Prep from rat thyroid carcinoma, pig brain, and swine intestine Cloning and sequencing of cDNA encoding prepro cholecystokinin has been reported to reveal 345 nucleotides encoding the CCK precursor of 115 amino acids containing all CCK sequences previously reported as isolated [Crawley and Corwin, These doors It is.

CCK was characterized as a 33 amino acid peptide in 1966 [Crawley and Corwin, supra]. Species-specific molecular variants of the amino acid sequence of CCK have been identified. The 33 amino acid sequence and the truncated peptide, its 8 amino acid C-terminal sequence (CCK-8), have been reported to have been identified in pigs, rats, chickens, chinchillas, dogs and humans. 39 amino acid sequences were reported to be found in pigs, dogs, and guinea pigs. 58 amino acid sequences have been reported to be found in cats, dogs, and humans. Frogs and turtles have been reported to exhibit 47 amino acid sequences that are homologous to both CCK and gastrin. Very fresh human intestines have been reported to contain small amounts of even larger molecules called CCK-83. In rats, major intermediate forms have been reported to be identified and called CCK-22 [Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York) .Non-sulphurized CCK -8 and tetrapeptide (CCK-4 (called CCK (30-33), SEQ ID NO: 208) have been reported in rat brain. C-terminal pentapeptide (CCK-4 (called CCK (29-33), SEQ ID NO: 209) ) Preserves the structural homology of CCK and also its homopeptides with neuropeptides and gastrins: The C-terminal sulfated octapeptide sequence, CCK-8, has been reported to be relatively conserved in all species. Cloning and sequencing of cDNA encoding preprocholesystokinin from porcine intestine, and 345 nucleotides encoding CCK precursors of 115 amino acids containing all CCK sequences previously reported as isolated have been reported [Crawley and Cor win, supra.

CCK is distributed throughout the central nervous system and in the endocrine cells and the visceral nerve of the upper small intestine. CCK agonists include CCK itself (also referred to as CCK-33), CCK-8 (CCK (26-33), SEQ ID NO: 55), non-sulfated CCK-8, pentagastrin (CCK-5 or CCK (29-) 33), SEQ ID NO: 209) and tetrapeptide, CCK-4 (CCK (30-33), SEQ ID NO: 208). In pancreatic CCK receptors, CCK-8 has been reported to replace binding with a potency of 1000-5000 times greater than non-sulfurized CCK-8 or CCK-4, and CCK-8 is non-stimulating in stimulating pancreatic amylase secretion. It has been reported to be approximately 1000 times more potent than sulfated CCK-8 or CCK-4 (Crawley and Corwin, supra). In cerebral cortical homogenates, CCK receptor binding was replaced by non-sulphurized CCK-8 and CCK-4 at equimolar or 10- or 100-fold higher concentrations with sulfated CCK-8 (supra).

Another family of peptide hormones associated with metabolic diseases and disorders is the pancreatic polypeptide family ("PPF"). Pancreatic polypeptide ("PP") was found as a contaminant of insulin extracts and was named after the organ of origin, not its functional significance (Kimmel et al., Endocrinology 83: 1323-30 (1968)). PP is a 36 amino acid peptide containing a unique structural motif. Subsequently, a related peptide was found in the intestinal extract and was named peptide YY (“PYY”) because of the N-terminal and C-terminal tyrosine [Tatemoto, Proc. Natl. Acad. Sci. USA 79: 2514-8 (1982). Subsequently a third related peptide was found in brain extracts and was named neuropeptide Y ("NPY") (Tatemoto, Proc. Natl. Acad. Sci. USA 79: 5485-9 (1982)), Tatemoto et al., Nature 296: 659-60 (1982)].

These three related peptides have been reported to exert a variety of biological effects. The effects of PP include inhibition of pancreatic secretion and relaxation of the gallbladder. Centrally administered PP causes a moderate increase in food intake, which can be mediated by receptors localized in the hypothalamus and brainstem (Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22 (1998)].

Release of PYY occurs after meals. An alternative molecular type of PYY is PYY (3-36) (SEQ ID NO: 58) (Eberlein et al., Peptides 10: 797-803 (1989)), Grandt et al., Regul. Pept. 51: 151- 9 (1994)]. The fragment constitutes about 40% of the total PYY-like immunoreactivity in human and dog intestinal extracts, and the total plasma PYY immunoreactivity constitutes about 36% in the fasted state to more than 50% after meals. This is clearly the dipeptidyl peptidase-IV (DPP4) cleavage product of PYY. PYY (3-36) (SEQ ID NO: 58) has been reported to be a selective ligand at the Y2 and Y5 receptors, which is pharmacologically unique in providing N-terminal truncated NPY analogs (ie, C-terminal fragments thereof). Is considered. Peripheral administration of PYY may include gastric acid secretion, gastric motility, and 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)], and gall bladder contraction and intestinal motility [Savage et al., Gut 28: 166-70 (1987)]. Effect of central injection of PYY on gastric emptying, gastric motility and gastric acid secretion as observed after direct injection into the hindbrain / brain stem (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 Mot. 9: 109-16 (1997)] may differ from the effects observed after peripheral injection. For example, centrally administered PYY has some effects contrary to those described herein for peripherally injected PYY (3-36) (SEQ ID NO: 58) in that gastric acid secretion is stimulated without inhibition. Gastric exercise was inhibited only with TRH stimulation and not when administered alone, but was actually stimulating at higher doses, presumably through interaction with PP receptors. PYY has been shown to stimulate food and water intake after central administration (Morley et al., Brain Res. 341: 200-3 (1985), Corpet al., Am. J. Physiol. 259: R317-23) (1990)].

Metabolic diseases and disorders take many forms, including obesity, diabetes, dyslipidemia, insulin resistance, apoptosis of cells, and the like. Obesity and its related disorders are common and very serious public health problems in the United States and around the world. Upper body obesity is the largest known risk factor for type 2 diabetes mellitus and a strong risk factor for cardiovascular disease. Obesity is recognized in association with increased complications of hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, breast cancer, prostate cancer and colon cancer, and general anesthesia Risk factors (see, eg, Kopelman, Nature 404: 635-43 (2000)). It shortens lifespan, and also accompanies such disorders as infections, varicose veins, melanoma, eczema, exercise intolerance, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, orthopedic damage, and thromboembolic diseases Carries a serious risk to Rissanen et al., Br. Med. J. 301: 835-7 (1990). Obesity is also a risk factor for a group of conditions, also called insulin resistance syndrome or "syndrome X". Recent estimates of the medical costs of obesity and related disorders are $ 150 billion worldwide. The pathogenesis of obesity is considered to be multifactorial, but the basic problem is that in obese subjects, nutrient utilization and energy consumption are not balanced until excess adipose tissue is present. Obesity is a metabolic disorder that is currently difficult to treat, chronic and inherently refractory. Therapeutic drugs useful for weight loss in obese people may have a beneficial effect on their health.

Diabetes is a carbohydrate metabolic disorder characterized by hyperglycemia and diabetes due to insufficient production or use of insulin. Diabetes severely affects the quality of life of a large part of the population of developed countries. Insufficient insulin production is a hallmark of type 1 diabetes, and insufficient insulin utilization is a hallmark of type 2 diabetes. However, it is now widely recognized that there are many other diabetes-related diseases that develop long before certainty that diabetes is diagnosed. In addition, the effects of suboptimal control of glucose metabolism in diabetes leads to a broad spectrum of related lipid and cardiovascular disorders.

Dyslipidemia, or abnormal levels of lipoproteins in plasma, occur frequently in diabetes. Dyslipidemia typically features elevated plasma triglycerides in the blood, low levels of HDL (high density lipoprotein) cholesterol, normal to elevated levels of LDL (low density lipoprotein) cholesterol, and increased levels of small, dense LDL (low density lipoprotein) particles. It is done. Dyslipidemia is one of the major causes of increased incidence of coronary artery events and death in diabetics. Epidemiological studies have confirmed this by showing a multi-fold increase in coronary death in diabetic patients compared to non-diabetic subjects. Some lipoprotein abnormalities have been known in diabetic subjects.

Insulin resistance is a reduction in insulin's ability to exert biological action over a wide range of concentrations. In insulin resistance, the body secretes abnormally large amounts of insulin to compensate for this deficiency, and a damaged glucose tolerance state develops. Failure to compensate for defective insulin action, plasma glucose concentrations inevitably rise, leading to the clinical condition of diabetes. It is recognized that insulin resistance and related hyperinsulinemia contribute to obesity, hypertension, atherosclerosis and type 2 diabetes. The association of insulin resistance with obesity, hypertension and angina has been described as syndrome X, a syndrome with insulin resistance as a common pathogenesis.

Apoptosis is an active process of cell self-destruction, regulated by external and intrinsic signals that occur during normal development. Apoptosis is well demonstrated to play an important role in the regulation of pancreatic endocrine beta cells. There is increasing evidence that in adult mammals beta cell mass varies significantly to produce insulin so that normal blood glucose is maintained at certain conditions, such as pregnancy and obesity. Control of beta cell volume depends on the delicate balance between cell proliferation, growth and programmed cell death (apoptosis). This disturbance of balance can cause impairment of glucose homeostasis. For example, it is noteworthy that glucose intolerance develops with aging when the rate of beta cell replication decreases, and human necropsy studies show 40% to 60% beta cell volume in insulin-dependent diabetes mellitus patients compared to non-diabetic subjects. It has been shown to decrease repeatedly. Insulin resistance is invariably accompanied by obesity, but normal blood glucose is maintained by complementary hyperinsulinemia until beta cells, at the beginning of type 2 diabetes, cannot meet the increased demand for insulin. It is generally a common opinion.

Attempts to treat many abnormalities associated with diabetes have prompted several patients to administer several anti-diabetic medications to address these abnormalities. Examples of anti-diabetic medications are proteins such as insulin and insulin analogues, and small molecules such as insulin sensitizers, insulin secretagogues and appetite control compounds and the like.

There is still a need for the development of polypeptides useful for the metabolic diseases, conditions and disorders described above. It is therefore an object of the present invention to provide hybrid polypeptides and methods of making and using them. The compounds of the present invention are useful for the metabolic diseases, conditions and disorders described above herein.

All documents mentioned herein are incorporated herein by reference as if fully set forth herein.

Summary of the Invention

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

In one aspect of the invention, hybrid polypeptides are provided that exhibit one or more hormonal activities. Hybrid polypeptides of the invention comprise two or more bioactive peptide hormone modules covalently linked together, wherein the one or more bioactive peptide hormone modules exhibit one or more hormonal activities of the component peptide hormones. The bioactive peptide hormone module includes component peptide hormones, fragments of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, analogs and derivatives that exhibit one or more hormone activities of component peptide hormones, and one or more hormones of component peptide hormones. Fragments of analogs and derivatives of component peptide hormones that exhibit activity, and peptide enhancers are independently selected.

In one embodiment, the hybrid polypeptide exhibits at least one hormonal activity and contains at least one first bioactive peptide hormone module covalently linked to at least one additional bioactive peptide hormone module, wherein the bioactive peptide hormone module is Component peptide hormones, fragments of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, analogs and derivatives of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, and one or more hormone activities of component peptide hormones Fragments of analogs and derivatives of the component peptide hormones shown, and peptide enhancers are independently selected. Component peptide hormones are typically amylin, adrenomedulin (ADM), calcitonin (CT), calcitonin gene related peptide (CGRP), intermedin, cholecystokinin ("CCK"), leptin, peptide YY (PYY), glucagon- Independently selected from two or more of the group consisting of analogous peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), auxintomodulin (OXM), sodium excretion peptide and exendin-4 do. Typically, a peptide enhancer is a structural component of a component peptide hormone that imparts the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties to the hybrid polypeptide. Motifs and analogs or derivatives of component peptide hormones that impart to the hybrid polypeptide the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties. Independently from the group consisting of structural motifs. In further embodiments, the one or more bioactive peptide hormone modules exhibit one or more hormonal activities of the component peptide hormones. In a further alternative embodiment, the at least one bioactive peptide hormone module exhibiting at least one hormonal activity of the component peptide hormone is amylin, a fragment of amylin showing at least one hormonal activity, at least one hormonal activity. Analogs or derivatives of amylin, or fragments of analogs or derivatives of amylin exhibiting at least one hormonal activity, and at least one other bioactive peptide hormone module is CCK, fragment of CCK exhibiting at least one hormone activity, 1 Analogs or derivatives of CCK that exhibit at least one hormone activity, fragments of analogs or derivatives of CCK that exhibit one or more hormone activities, CTs, fragments of CT that exhibit one or more hormone activities, analogs of CT that exhibit one or more hormone activities Or derivatives or analogs or derivatives of CT that exhibit one or more hormonal activities In the case of a fragment of a conductor, the hybrid polypeptide may further contain three or more bioactive peptide hormone modules selected from three or more different component peptide hormones. In a further alternative embodiment, the at least one bioactive peptide hormone module exhibiting at least one hormone activity of the component peptide hormone is GLP-1, a fragment of GLP-1 exhibiting at least one hormone activity, at least one hormone activity An analogue or derivative of GLP-1 that represents a protein fragment or a fragment of an analogue or derivative of GLP-1 that exhibits one or more hormonal activities, and the one or more other bioactive peptide hormone modules is a peptide enhancer comprising an exendin fragment, The hybrid polypeptide may further contain three or more bioactive peptide hormone modules.

The component peptide hormones of the present invention are amylin, adrenomodulin (ADM), calcitonin (CT), calcitonin gene related peptide (CGRP), intermedin, cholecystokinin ("CCK"), leptin, peptide YY (PYY), glucagon -Like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), auxintomodulin (OXM) and exendin-4.

Peptide enhancers of the present invention are directed to component peptide hormones that impart to the hybrid polypeptide the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties. Structural motifs and analogs or derivatives of component peptide hormones that confer hybrid chemicals to desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties It includes the structural motif of.

In another aspect of the invention, there is provided a method of treating or preventing obesity comprising administering to a subject in need thereof a therapeutic or prophylactically effective amount of a hybrid polypeptide of the invention. In a preferred embodiment, the subject is obese or overweight. “Obesity” is generally defined as a body mass index of greater than 30 for purposes herein, but any subject, including subjects with a body mass index of less than 30, who needs or wants to lose weight, It is included in a range. Subjects with insulin resistance, glucose intolerance or any form of diabetes mellitus (eg, type 1, type 2 or gestational diabetes) may benefit from the methods of the present invention.

In another aspect of the invention, reducing food intake, reducing nutrient utilization, inducing weight loss, treating diabetes mellitus or a diabetes-related condition, comprising administering to a subject an effective amount of a hybrid polypeptide of the invention Methods for improving the profile (including decreasing LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels) are provided. In a preferred embodiment, the method of the present invention comprises administering to a subject in need thereof a therapeutic or prophylactically effective amount of a hybrid polypeptide of the invention to a condition or disorder that can be alleviated by reducing nutrient utilization. In the treatment or prevention of conditions or disorders that can be alleviated by reducing nutrient utilization. In other embodiments, the methods of the present invention are used to treat or prevent a condition or disorder that can be alleviated by control of plasma glucose levels, insulin levels, and / or insulin secretion. In another embodiment, the methods of the present invention are used to treat diabetes and / or diabetes-related conditions. These conditions and disorders include hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and any type of diabetes mellitus, diabetes complications, neuropathy (eg, type I, type II, and pregnancy diabetes, etc.) For example, based on the neurotrophic action of exendin-4), neuropathic pain (eg based on amylin action), retinopathy, nephropathy, conditions of insufficient pancreatic beta cell volume (eg exendin- 4 and GLP-1 based on islet angiogenesis), and the like.

The invention also provides a therapeutically or prophylactically effective amount of one or more hybrid polypeptides of the invention or a pharmaceutically acceptable salt thereof with a carrier useful for the delivery of a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or hybrid polypeptide. It relates to a pharmaceutical composition comprising together.

These and other aspects of the invention will be more clearly understood by reference to the following detailed description of the preferred embodiments and inventions.

1 demonstrates the effect of exemplary compounds of the invention in a DIO mouse assay.

2 demonstrates the effect of exemplary compounds of the invention in a DIO mouse assay.

3A-3C demonstrate the effect of exemplary compounds of the invention in DIO mouse assays.

4A and 4B demonstrate the effect of exemplary compounds of the invention in food intake assays compared to parent peptide compounds.

5A and 5B demonstrate the effect of exemplary compounds of the invention in the blood glucose lowering assay and the food intake assay, respectively.

6 illustrates the structure of various exemplary linkers used herein.

7 shows the structure of various exemplary linkers as well as the sequence and in vitro activity of exemplary PYY and amylin family hybrids.

8 shows the structure of various exemplary linkers as well as the sequence and in vitro activity of additional exemplary PYY / PYY family hybrids.

9 shows weight loss by exemplary PYY and amylin family hybrids.

10 shows a graph of calcium recruitment by CCK8 family hybrids in cell lines with CCK receptors.

11A, 11B and 11C show food inhibition by amylin (eg amylin-sCT-amylin) and PYY (eg PYY-NPY chimeric) family hybrids.

FIG. 12A shows total food intake reduction by amylin-sCT-amylin / PYY family hybrids, and FIG. 12B shows weight loss by amylin-sCT-amylin / PYY family hybrids. "*" Indicates p <0.05 compared to vehicle.

Figure 13 shows inhibition of food intake by PYY / PYY family hybrids. Dose was 25 nmol / kg. A difference of about 15-20% is statistically significant.

14A and 14B show inhibition of food intake activity by CCK family hybrids with amylin or PYY family components. Dose was 25 nmol / kg. A difference of about 15-20% is statistically significant.

15A and 15B show inhibition of food intake activity by MSH family hybrids with amylin or PYY family components. Dose was 25 nmol / kg. A difference of about 15-20% is statistically significant.

FIG. 16A shows the reduction in total food intake by hybridization with MSH family components and amylin, eg, amylin-sCT-amylin family components, and FIG. 16B shows MSH family components and amylin, eg amylin Weight loss by hybridization with the -sCT-amylin family component is shown. "*" Indicates p <0.05 compared to vehicle.

17A-17D show inhibition of food intake by a hybrid containing the FN-38 family component and the indicated second family component. Figure 17A-FN38 and Amylin family component, Amylin-sCT-Amiline chimeric compound 10. Figure 17B-FN38 and PYY family component, PYY-3-36. Figure 17C-FN38 and PYY family components, PYY-NPY chimeras. 17D-FN38 and CCK8 family components, CCK8. Dose was 25 nmol / kg. A difference of about 15-20% is statistically significant.

18A and 18B show inhibition of food intake by hybrids containing the exendin family and amylin family components. Unless otherwise indicated, the dose was 25 nmol / kg. A difference of about 15-20% is statistically significant.

19A and 19B show inhibition of food intake during cancer-cycle food intake and prolonged total food intake by exendin / amylin family hybrids.

20A and 20B show chronic suppression of food intake and lean-sparing weight loss and fat loss activity by exemplary exendin / amylin-sCT-amylin family hybrids as compared to the parent molecule. To show. "*" Indicates p <0.05 compared to vehicle.

21A, 21B and 21C demonstrate the effect on metabolic parameters after 14 days of treatment of hybrids in rats.

22-29 show the ability to reduce cumulative food intake by PPF polypeptide chimeras such as PYY-NPY chimeric compounds 4883 and 5705 in the food intake assays described herein.

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

In one aspect, the present invention provides physiological, metabolic and / or bioavailability, which may be selected based on therapeutic efficacy, range of function, duration of action, physicochemical properties and / or other pharmacodynamic properties. Module assembly of pharmacodynamically active peptide modules.

Without wishing to be bound by theory, the present invention provides a tool for linking a bioactive peptide hormone module into a two-component, three-component or more combination of components to create an effective new therapeutic agent that has at least partially selectable properties. Toolbox "method. A "bioactive peptide hormone module" can be a peptide hormone, a peptide fragment having hormonal activity, or a structural motif of a peptide hormone that confers chemical, metabolic and / or other pharmacodynamic stability. Peptide hormones may include natural peptide hormones as well as peptide hormone analogs and derivatives as known in the art and described herein.

In one aspect of the invention, it has been found that combining certain physicochemical characteristics of two or more peptide hormones in a single mode may facilitate intervention at various points in the dysfunctional metabolic circuit. Thus, in one aspect of the invention there is provided a hybrid polypeptide of rational design that incorporates selectable bioactivity into a single polypeptide agent. In one embodiment, the selectable hybrid polypeptides of the present invention may involve the use of chemically stable linkers to covalently attach the bioactive module. In another embodiment, the selectable hybrid polypeptides of the invention may involve the use of a cleavable linker that may itself be part of or form part of a bioactive module.

Again, without wishing to be bound by theory, the design of a hybrid polypeptide of the present invention generally involves (1) identifying, selecting, and pairing a bioactive peptide hormone module for a desired efficacy and therapeutic use, and (2) Share bioactive modules (eg, natural peptide hormones, peptide hormone analogs or derivatives with hormonal activity, peptide hormone fragments with hormonal activity, and stabilizing motifs, etc.) directly or via linkers without losing the bioactivity of the component modules May involve connecting. In certain embodiments, the module selection criteria include (a) any desired in vivo efficacy, eg, additive or synergistic effect, for the desired therapeutic or prophylactic indication, (b) any of the linked modules for multiple therapeutic or prophylactic indications. Synergistic or dual action of and / or (c) desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding and / or other pharmacodynamic properties, etc. There is, but is not limited to this.

It is to be understood that the headings of the paragraphs are used herein for purposes of distinction only and do not in any way limit the subject matter described herein.

Of the present invention hybrid  Polypeptide

As noted above, the present invention relates in part to a hybrid polypeptide comprising two or more bioactive peptide hormone modules selectable from the component peptide hormones described herein. Hybrid polypeptides of the invention will generally be useful for the treatment and prevention of metabolic conditions and disorders. The hybrid polypeptides of the present invention will exhibit one or more hormonal activities of the component peptide hormones, and may preferably comprise additional one or more biological activities of the second component peptide hormones.

In an embodiment, a hybrid polypeptide of the invention can comprise two or more bioactive peptide hormone modules, wherein each of the two or more bioactive peptide hormone modules exhibits one or more hormonal activities of the component peptide hormones. In another embodiment, a hybrid polypeptide of the present invention may comprise two or more bioactive peptide hormone modules, wherein one or more of the bioactive peptide hormone modules exhibit one or more hormonal activities of the component peptide hormones, One or more of the bioactive peptide hormone modules incorporate the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding and / or other pharmacodynamic properties to the hybrid polypeptide. Grant.

In a preferred embodiment, the hybrid polypeptides of the present invention may have a potency similar or higher than the component peptide hormones in the treatment and / or prevention of metabolic conditions and disorders. In another embodiment, the hybrid polypeptides of the present invention may have a potency similar or higher than the component peptide hormones in the treatment and / or prevention of diabetes and / or diabetes-related disorders. Alternatively, preferred hybrid polypeptides of the invention may appear to have improved ease of manufacture, stability, and / or formulation compared to component peptide hormones.

More particularly, the hybrid polypeptides of the present invention will generally comprise a first bioactive peptide hormone module covalently linked to one or more additional bioactive peptide hormone modules. The bioactive peptide hormone modules may be covalently linked together in any manner known in the art as described in detail below, including but not limited to direct amide linkages or chemical linker groups. In an embodiment, the chemical linker group can comprise peptide mimetics that induce or stabilize the polypeptide form.

The first bioactive peptide hormone module may be selected from the first component peptide hormones and may include desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding and / or other Peptide hormones (including natural peptide hormones and also analogs and derivatives thereof) that confer pharmacodynamic characteristics to the hybrid polypeptide, peptide fragments (including fragments of natural peptide hormones and also analogs and derivatives thereof) or structural motifs of peptide hormones that have hormonal activity (Including natural peptide hormones and also analogs and derivatives thereof). Likewise, additional bioactive peptide module (s) can be selected from the component peptide hormones and can be selected from desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding and / or Or peptide hormones (including natural peptide hormones and also analogs and derivatives thereof) that impart other pharmacodynamic characteristics to the hybrid polypeptide, peptide fragments (including fragments of natural peptide hormones and also analogs and derivatives thereof) or hormone peptides having hormonal activity Structural motifs, including natural peptide hormones and also analogs and derivatives thereof. The first peptide hormone and the additional peptide hormone may be the same peptide hormone, or may be the same family of peptide hormones, or different peptide hormones, depending on the desired characteristics of the bioactive peptide hormone module.

As used herein, the term “biological activity” refers to (1) biological activity in one or more hormonal pathways in vivo, or (2) therapeutic efficacy, range of action, duration of action, physicochemical properties, and / or such biological activity. Or adjustment of other pharmacodynamic properties. Biological activity can be assessed through metabolic studies that monitor target hormone receptor binding assays or physiological signs as known in the art and described herein. Modifications of therapeutic efficacy, range of action, duration of action, physicochemical properties and / or other pharmacodynamic properties of such biological activity are, for example, chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, Modification through protease inhibition, plasma protein binding and / or other pharmacodynamic characteristics.

In one embodiment, a hybrid polypeptide of the present invention comprises at least about 25%, preferably about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the biological activity of the component peptide hormones. , 98% or 99%. Preferred hybrid polypeptides are used in the same assay in one of the metabolic related assays known in the art or described herein (e.g., receptor binding, food intake, gastric emptying, pancreatic secretion, insulin secretion, lowering blood glucose, weight loss, etc.). The component of the same as or greater than the effect of the peptide hormone. Alternatively, preferred hybrid polypeptides of the invention may appear to have improved ease of manufacture, stability, and / or formulation compared to component peptide hormones.

In another embodiment, a hybrid polypeptide of the present invention comprises about 25% of the biological activity of the natural component peptide hormone in terms of reducing nutrient utilization, reducing food intake, weight gain effects and / or treating and preventing metabolic conditions and disorders. Or more preferably about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%. In another embodiment, the hybrid polypeptides of the present invention comprise at least about 110% of the biological activity of natural peptide hormones in terms of reducing nutrient utilization, reducing food intake, weight gain effects and / or treating and preventing metabolic conditions and disorders. , 125% or more, 130% or more, 140% or more, 150% or more, 200% or more, or higher activity. In another embodiment, the hybrid polypeptides of the present invention exhibit improved component peptide hormone receptor agonist activity.

Component Peptide Hormones, Analogs and Derivatives

Component peptide hormones generally include (a) the amylin family, such as amylin, adrenomedulin ("ADM"), calcitonin ("CT"), calcitonin gene related peptide ("CGRP"), intermedin (" And related peptides, (b) cholecystokinin ("CCK"), (c) leptin family, such as leptin and leptin-like peptide, (d) pancreatic polypeptide family, such as pancreatic polypeptide ("PP") and peptide YY ("PYY"), (e) incretin and incretin mimetics such as peptide hormones derived from proglucagon genes, such as glucagon, glucagon-like peptide-1 (" GLP-1 "), glucagon-like peptide-2 (" GLP-2 ") and oxintomodulin (" OXM "), and exendines such as exendin-3 and exendin-4, (f) Sodium excretion peptides, including ANP, BNP, CNP, and urodilatin, their precursor forms and peptides derived therefrom, (g) eurocortin family And (h) includes the neuromedin family, and their analogues, peptide hormones useful in the treatment or prevention of, including derivatives and fragments, etc., metabolic diseases and disorders. As discussed herein, the component peptide hormones of the invention also include analogs and derivatives that retain the hormonal activity of these natural peptide hormones. In one embodiment, such analogs and derivatives are agonists of target hormone receptors.

"Amiline" refers to amylin, as described in U.S. Patent No. 5,234,906 issued August 10, 1993 (in English title: "Hyperglycemic Compositions", the contents of which are incorporated herein by reference). Human peptide hormone and its species variants secreted from the beta cells of the pancreas. More particularly, amylin is a 37-amino acid polypeptide hormone normally co-secreted with insulin by pancreatic beta cells in response to nutrient uptake (see, eg, Koda et al., Lancet 339: 1179-1180, 1992). Reference). In this sense, "amylin", "wild type amylin" and "natural amylin", ie unmodified amylin, may be used interchangeably.

"Adrenomedulin" or "ADM" refers to human peptide hormones and species variants thereof. More particularly, ADM is produced by the cleavage and amidation of a continuous enzyme from 185 amino acids preprohormones. This process results in a bioactive peptide of 52 amino acids.

"Calcitonin" or "CT" refers to human peptide hormones and species variants thereof such as salmon calcitonin ("sCT"). More particularly, CT is a peptide of 32 amino acids cleaved from larger prohormones. It contains one disulfide bond that allows the amino termini to be assembled in the form of a ring. Selective splicing of calcitonin pre-mRNA can produce mRNA encoding calcitonin gene related peptides, which peptides are believed to function in the nervous and vascular systems. Calcitonin receptors have been cloned and found to be members of the 7-membrane, G protein-coupled receptor family.

"Calcitonin gene related peptide" or "CGRP" means any physiological form of human peptide hormone and species variants thereof.

"Intermedin" or "AFP-6" refers to any physiological form of human peptide hormone and species variants thereof.

"Cholecystokinin" or "CCK" refers to human peptide hormones and species variants thereof. More particularly, CCK is the first 33 amino acid sequence identified in humans, and the 8 amino acid in vivo C-terminal fragment ("CCK-8") reported to have been demonstrated in pigs, rats, chickens, chinchillas, dogs and humans. Include. Thus, the term CCK-33 generally refers to human CCK (1-33), while CCK-8 (CCK (26-33), SEQ ID NO: 55) is both sulfated and non-sulphated unless otherwise specified. Means the C-terminal octapeptide of. Additionally, pentagastrin or CCK-5 refers to the C-terminal peptide CCK (29-33) (SEQ ID NO: 209), and CCK-4 refers to the C-terminal tetrapeptide CCK (30-33) (SEQ ID NO: 208). . However, as used herein, CCK generally refers to all natural variants of the hormone, including CCK-33, CCK-8, CCK-5, and CCK-4 in sulfated and non-sulfurized forms, unless otherwise specified. do.

"Leptin" means any species of natural leptin, and biologically active D-isotypes, or fragments of native leptin and variants thereof, and combinations thereof. Leptin is the polypeptide product of the ob gene described in WO 96/05309, which is incorporated herein by reference in its entirety. Putative analogs and fragments of leptin are reported in US Pat. Nos. 5,521,283, 5,532,336, PCT / US96 / 22308 and PCT / US96 / 01471, each of which is incorporated herein by reference in its entirety.

"PP" refers to any physiological form of human pancreatic peptide polypeptide or species variant thereof. Thus, the term “PP” includes both the full-length human 36 amino acid peptides described in SEQ ID NO: 290 and species variants of PP, such as murine, hamster, chicken, bovine, rat and dog PP. In this sense, "PP", "wild type PP" and "natural PP", ie unmodified PP, can be used interchangeably.

"PYY" means any physiological form of human peptide YY polypeptide or species variant thereof. Thus, the term “PYY” includes full length human 36 amino acid peptides, and species variants of PYY, such as murine, hamsters, chickens, cattle, rats, and dogs PYY. In this sense, "PYY" and "wild type PYY" and "natural PYY", ie unmodified PYY, may be used interchangeably. In the context of the present invention, all modifications discussed for the PYY analogue polypeptides of the invention are based on the 36 amino acid sequence of native human PYY.

"GLP-1" refers to any physiological form of human glucagon-like peptide-1 or species variants thereof. The term “GLP-1” refers to human GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204) in connection with full length human GLP-1 (1-37) (SEQ ID NO: 59). And GLP-1 (7-36) amides (SEQ ID NO: 61), and species variants of GLP-1, such as murine, hamsters, chickens, cattle, rats, and dogs GLP-1. In this sense, "GLP-1", "wild type GLP-1" and "natural GLP-1", that is, unmodified GLP-1 may be used interchangeably.

"GLP-2" refers to any physiological form of human glucagon-like peptide-2 or species variant thereof. More particularly, GLP-2 is a 33 amino acid peptide co-secreted with GLP-1 from enteral endocrine cells in the small and large intestine.

"OXM" refers to any physiological form of human auxintomodulin or species variant thereof. More particularly, OXM is a 37 amino acid peptide comprising the 29 amino acid sequence of glucagon and the carboxy terminal extension of the 8 amino acids behind it.

"Exendin" refers to peptide hormones and species variants thereof found in the saliva of American lizards (lizards in Arizona, USA) and Mexican bead lizards. More particularly, exendin-3 is present in saliva of Helloderma horridum and exendin-4 is present in saliva of Helloderma suspectum (Eng, J., et al., J. Biol. Chem. 265: 20259-62, 1990, Eng., J., et al., J. Biol. Chem., 267: 7402-05 (1992). Exendin has some sequence similarity to some members of the glucagon-like peptide family and 53% highest identity to GLP-1 [Goke, et al., J. Biol. Chem., 268: 19650-55 (1993). In this sense, "exendin", "wild type exendin" and "natural exendin", ie unmodified exendin, may be used interchangeably.

"Eurocortin" means any physiological form of human eurocortin peptide hormone or species variant thereof. More particularly, there are three human eurocortins, Ucn-1, Ucn-2 and Ucn-3. For example, human eurocortin 1 is Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu- Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH ₂ (SEQ ID NO: 294) . Eurocortin derived from the rat is the same except for two substitutions, Asp2 instead of Asn2 and Pro4 instead of Ser4. Human Ucn-2 is Ile Val Leu Ser Leu Asp Val Pro Ile Gly Leu Leu Gln Ile Leu Leu Glu Gln Ala Arg Ala Arg Ala Ala Arg Glu Gln Ala Thr Thr Asn Ala Arg Ile Leu Ala Arg Val Gly His Cys (SEQ ID NO: 399) Has a sequence of. Human Ucn-3 sequenced Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe Asn Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala His Leu Met Ala Gln Ile (SEQ ID NO: 299) Have Ucn-3 is preferably in the amide form. Additional eurocortins and analogs are described in the literature, for example in US Pat. No. 6,214,797. Eurocortin Ucn-2 and Ucn-3 possess food intake inhibition and anti-hypertension / myocardial protection / muscular contraction properties and are particularly useful in the hybrid of the present invention. Stress Coffin (Ucn-3) and Stress Coffin-Related Peptides (Ucn-2) are named after their inhibition of chronic HPA activation after stress stimulation such as diet / fasting and are specific for CRF type 2 receptors and are responsible for ACTH release. It does not activate the mediating CRF-R1. Hybrids comprising urocortin, for example Ucn-2 or Ucn-3, are particularly useful for vasodilation and are therefore useful for cardiovascular applications such as CHF as described herein. Eurocortin containing the hybrid of the present invention treats conditions associated with stimulating ACTH release, hypertension due to vasodilatation effect, inflammation mediated by other than elevated ACTH, high fever, appetite disorders, congestive heart failure, stress, anxiety and psoriasis And particularly useful for prevention. Such compounds are also useful for anti-proliferative effects, such as the treatment or prevention of cancer or tumor growth. Of particular interest are eurocortin peptide hormone modules that provide a beneficial vasodilator effect in combination with the sodium excretion peptide module, amylin family, exendin family, or GLP1 family module to provide enhanced cardiovascular benefits, such as CHF treatment. to be.

"Nuromedin" refers to peptides of the neuromedin family comprising neuromedin U and S peptides, more particularly their active hormone sequences. For example, naturally active human neuromedin U peptide hormones are neuromedin-U25, in particular amides of Phe Arg Val Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe Leu Phe Arg Pro Arg Asn (SEQ ID NO: 308). Form. Porcine U25 has a sequence of FKVDEEFQGPIVSQNRRYFLFRPRN (SEQ ID NO: 314), in particular its amide form. Other neuromedin U family members include those listed by SWISS-PROT name and entry number below: NEUU_CANFA (P34962), NEUU_CAVPO (P34966), NEUU_CHICK (P34963), NEUU_HUMAN (P48645), NEUU_LITCE (P81872) ), NEUU_MOUSE (Q9QXK8), NEUU_PIG (P34964), NEUU_RABIT (P34965), NEUU_RANTE (P20056), and NEUU_RAT (P12760). Of particular interest are their processed active peptide hormones and their analogs, derivatives and fragments. The neuromedin U family includes various truncation variants or splice variants, for example FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN (SEQ ID NO: 300). An example of the neuromedin S family is ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN (SEQ ID NO: 315), especially human neuromedin S having a sequence in its amide form. Since the hybrids of the present invention having neuromedin modules have an appetite suppressing effect, they will be of valuable value for the treatment of obesity, diabetes, reduced food intake and other related conditions and disorders described herein. Of particular interest are neuromedin modules in combination with amylin family peptides, exendin peptide family or GLP1 peptide family modules.

As used herein, an “analog” has a sequence derived from the sequence of a base reference peptide (eg, PP, PYY, amylin, GLP-1, exendin, etc.) and a reference amino acid sequence. Preferably at least 50 or 55% amino acid sequence identity with said reference peptide, more preferably at least 70%, 80%, 90% or 95% amino acid with said reference peptide, including insertion, substitution, extension and / or deletion of Refers to peptides having sequence identity. In one embodiment, such 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 having the amino acid sequence of a natural reference peptide or analog, but further having a chemical modification on at least one of its amino acid side chain, α-carbon atom, terminal amino group or terminal carboxylic acid group. Chemical modifications include, but are not limited to, addition of chemical moieties, creation of new bonds, and removal of chemical moieties. Modifications at the amino acid side chains include, but are not limited to, acylation of lysine ε-amino groups, N-alkylation of arginine, histidine or lysine, alkylation of glutamic or aspartic acid carboxylic acid groups and deamidation of glutamine or asparagine . Modifications of terminal amino include, but are not limited to, desamino, N-lower alkyl, N-di-lower alkyl, constrained alkyls (e.g., branched chain, cyclic, fused, adamantyl) and N-acyl modifications . Modifications of the terminal carboxyl groups include, but are not limited to, amides, lower alkyl amides, constrained alkyls (eg branched chains, cyclic, fused, adamantyl), dialkyl amides and lower alkyl ester modifications. Lower alkyl is C ₁ -C ₄ alkyl. In addition, one or more side chain groups or end groups may be protected with protecting groups known to those skilled in the art of peptide chemistry. The α-carbons of amino acids may be monomethylated or dimethylated.

"Agonist" means a compound that elicits the biological activity of a natural human reference peptide, and preferably has superior potency than the reference peptide as assessed by means known in the art, eg, receptor binding / competitive studies. Or on a 5-fold scale (increase or decrease) relative to the reference peptide, more preferably on a 4, 3, 2 or 1-fold scale. In one embodiment, the term refers to a compound that causes a biological effect similar to a natural human reference peptide, eg, (1) has similar activity to a natural human reference peptide in food intake, gastric emptying, pancreatic secretion or weight loss assays, or Or (2) a compound that specifically binds in a reference receptor assay or a competitive binding assay using a labeled reference peptide. Preferably, an agonist will bind with an affinity greater than 1 μM, more preferably greater than 1-5 nM in this assay. In another embodiment, the term refers to a compound that elicits a biological effect in the treatment of diabetes or a diabetes-related condition or disorder. Such agonists may include polypeptides comprising an active fragment or small chemical molecule of a reference peptide.

"Amino acid" and "amino acid residue" refer to natural amino acids, non-natural amino acids, and modified amino acids. Unless stated otherwise, any particular reference to amino acids, either generically or by name, includes references to both of these stereoisomers when they have structures that allow D- and L-stereoisomers. 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 (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) and Valine. Non-natural amino acids include homo-lysine, homo-arginine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmocin, 2, 2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxy Proline, isodesmocin, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentyl Including but not limited to glycine, pipecolic acid and thioproline. Additional non-natural amino acids are chemically blocked reversibly or irreversibly, or N-terminal amino groups or side chains, such as, for example, N-methylated D- and L-amino acids or residues in which the side chain functional group is chemically modified with another functional group. Groups include modified amino acid residues that are chemically modified. For example, modified amino acids include methionine sulfoxide; Methionine sulfone; Aspartic acid- (beta-methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, modified amino acid of glycine; Or alanine carboxamides, modified amino acids of alanine. Additional residues that may be included 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, and “PEG (8)” means 3,6-dioxyocta By noil, "PEG (13)" means 1-amino-4,7,10-thioxa-13-tridecanamine succinimmoyl.

As discussed herein, natural component peptide hormones and analogs and derivatives thereof are known in the art. For reference, the sequences of the various natural component peptide hormones are provided in Table 1 below.

TABLE 1

These peptides are generally amidated at the C-terminus when expressed physiologically, but need not be for the purposes of the present invention. That is, not only these peptides but also the C-terminus of the hybrid polypeptide of the present invention may have a free -OH group or -NH ₂ group. These peptides may also undergo other post-translational modifications. Those skilled in the art will appreciate that the hybrid polypeptides of the invention may also consist of N-terminal methionine residues.

Exemplary peptide modules for use in the present invention exist in two forms, Apelin 36 and Apelin 13, both of which are active at the AJP receptor (LVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF-OH (SEQ ID NO: 316) and pERPRLSHKGPMPF-OH) SEQ ID NO: 317)), a prolactin releasing peptide (SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH ₂ (SEQ ID NO: 318) and TPDINPAWYASRGIRPVGRF-NH ₂ (SEQ ID NO: 319)), which is present in two forms, PRP31 and PRP20, is equally active in GPR10. Gastrins present (QLGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDF-NH ₂ (SEQ ID NO: 320), pEGPWLEEEEEAYGWMDF-NH ₂ (SEQ ID NO: 321), beta-AWMDF-NH ₂ (SEQ ID NO: 322)), CCK33 or CCK33 vs. peripheral, CCK, cortistatin 17 or cortistatin 29 (QEGAPPQQSARRDRMPCRNFFWKTFSSCK-OH) present as KAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF-NH ₂ (SEQ ID NO: 323), DYMGWMDF-NH ₂ (SEQ ID NO: 55) 324) and cortistatin, somatostatin 14 or somatostatin 28 (SANSNPAMAPRERKAGCKNFFWKTFTSC-OH (SEQ ID NO: 326), AGCKNFFWKTFTSC-OH (SEQ ID NO: 327)) present as DRMPCRNFFWKTFSSCK-OH (SEQ ID NO: 325), somatodan 10 amino acids, GRP where the sequence retains most of the activity (VPLPAGGGTVLTKMYPRGNHWAVGHLM-NH ₂ (SEQ ID NO: 328), GNHWAVGHLM-NH ₂ (SEQ ID NO: 329)), neuromedin B (LSWDLPEPRSRASKIRVHSRGNLHATN-NH), where the C-terminal 10 amino acid region retains most of the activity ₂ (SEQ ID NO: 330), GNLWATGHFM-NH ₂ (SEQ ID NO: 331), Neuromedin S (ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN-NH ₂ (SEQ ID NO: 315), PFFLFRPRN-NH ₂ (SEQ ID NO: 332), wherein the C-terminal 9 amino acid region retains most of the activity. )), Neuromedin U (FRVDEEFQSPFASQSRGYFLFRPRN-NH ₂ (SEQ ID NO: 308), GYFLFRPRN-NH ₂ (SEQ ID NO: 307)), in which the C-terminal 9 amino acid region retains most of the activity, neurotensin present in long and short form (KIPYILKRQLYENKPRRPYIL-OH (West 333), QLYENKPRRPYIL-OH) (SEQ ID NO: 334), Kiss-1 (GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF -NH , which is active predominantly in the C- terminal ₂ (SEQ ID NO: 335), EKDLPNYNWNSFGLRF-NH ₂ (SEQ ID NO: 336)), C- terminal fragment is active RF-amide-3 (SAGATANLPLRSGRNMEVSLVRRVPNLPQRF-NH ₂ (SEQ ID NO: 337), VPNLPQRF-NH ₂ (SEQ ID NO: 338)), dinorphine (YGGFLRRIRPKLKWQQKKVVFL-NH2), VPNLPQRF-NH ₂ (SEQ ID NO: 338) OH (SEQ ID NO: 339) and YGGFLRRQFKVVT-OH (SEQ ID NO: 340), PYY where the C-terminal fragment is active at the Y2 receptor (YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH ₂ (SEQ ID NO: 57), SLRHYLNLVTRQRY-NH ₂ (SEQ ID NO: 341) region) AFP-6 with this activity (TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY-NH ₂ (SEQ ID NO: 51), VGCVLGTCQVQNLS HRLWQLMGPAGRQDSAPVDPSSPHSY-NH ₂ (SEQ ID NO: 52)), adrenomerine, including adrenaldinin and G-DRP Is generally required and will allow N-terminal extension N- terminal extension peptide modules including such as oxytocin, which further comprises a (and their analogues and fragments).

Exemplary peptide modules for use in the present invention include endothelin I, II, and III: ETI (CSCSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSPRS-OH (SEQ ID NO: 342), CSCSSLMDKECVYFCHLDIIW-OH (SEQ ID NO: 343)), ETII (CSCSSWLDKECVYPECHLD-CHFCHLDW-CSF-CSF-LD) -OH (SEQ ID NO: 345)) and ETIII (CTCFTYKDKE CVYYCHLDIIWINTPEQTVPYGLSNYRGSFR-NH ₂ (SEQ ID NO: 346), CTCFTYKDKECVYYCHLDIIW-OH (SEQ ID NO: 347)), ghrelin whose activity is mainly present in the first 10 residues (GSSFLSPEHQKV SEQ ID NO: 348) GSSFLSPEHQ-OH (SEQ ID NO: 349)), Glucagon, e.g. C-terminal extended glucagon with glucagon-like activity, oxintomodulin (HSQGTFTSDYSKYLDSRRAQDFV QWLMNTKRNRNNIA-OH (SEQ ID NO: 350), HSQGTFTSDYSKYLDSRRAQDFV, WH) GIP (YAEGTFISDYSIAMDKIHQQDFV, which circulates in two forms, GLP-1 / GLP-2, GIP1-42 and GIP1-30, whose activity is retained with or without C-terminal amide, and is both fully active at the GIP receptor NWLLAQKGKKNDWKHNITQ-OH (SEQ ID NO: 352), YAEGTFISDYSIAM DKIHQQDFVNWLLAQK-NH ₂ (SEQ ID NO: 353), the neuropeptide W (WYKHVASPRYHTVGRHSPYLHSPYLHSPGRWHVAGLGL) 355)), PACAP (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK-NH ₂ (SEQ ID NO: 356), HSDGIFTDS YSRYRKQMAVKKYLAAVL-NH ₂ (SEQ ID NO: 357)), PHI and PHSDSDKFSVLSHHVVHHAG -NH ₂ (SEQ ID NO: 359)), GRF (YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERG ARARL -NH 2 ( SEQ ID NO: 360) present in the two types of GRF29 and GRF40, YADAIFTNSYRKVLGQLSARKLLQDIMS-OH (SEQ ID NO: 361)), to retain the activity of full-length PTH 1-84 PTH 1-34 and 1-37 forms (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN FVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO: 362), SVSEIQLMHNLGK HLNSMERVEWLRKKLQDVHNHLVHLNHLVEHLHHLVHLNHLVHLHHHL) 364)), the 1-36 PTH-RP (AVSEHQLLHDKGKSIQDLRRR FFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKP-NH 2 for holding the activity of the full-length 1-86 (SEQ ID NO: 365), AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEI-OH (SEQ ID NO: 366)), shorter and longer gamma -MSH1 gamma- Gamma-MSH with similar activity MSH3 (YVMGHFRWDRFGRRNSSSSGS SGAGQ-OH (SEQ ID NO: 367), YVMGHFRWDRF-NH ₂ (SEQ ID NO: 368)), MSH, where alpha-MSH is the active part of ACTH (SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESGSEPSEFPLE-SEH-SEHH) NH ₂ (SEQ ID NO: 370)), and endorphins (YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE-OH (SEQ ID NO: 371), YGGFMTSEKSQTPLVTLFKNAIIKNASE-OH (SEQ ID NO: GFLVTL372), and Α, delta, and γ endorphins, which are active subpeptides with larger β endorphins ), YGGFMTSEKSQTPLVT-OH (SEQ ID NO: 374)), and the like.

Melanocortin, for example, is a peptide derived from the pro-opiomelanocortin gene and includes, for example, alpha-melanin cell-stimulating hormone (alpha-MSH) and adrenal cortical stimulating hormone (ACTH), and MC1R to MC5R. Five melanocortin receptors are known. MC4R is believed to play a role in energy balance and obesity. See, eg, Anderson et al., Expert Opin. Ther. Patents 11: 1583-1592 (2001), Speake et al., Expert Opin. Ther. Patents 12: 1631-1638 (2002), [ Bednarek et al., Expert Opin. Ther. Patents 14: 327-336 (2004).

Analogs of the component peptide hormones are known in the art, but generally include modifications to the amino acid sequences of such component peptide hormones, such as substitutions, deletions and insertions, and any combination thereof. Substitutions, insertions, and deletions may occur at the N-terminal or C-terminal end, or may occur within the component peptide hormones. In a preferred aspect, analogs of the component peptide hormones of the invention comprise one or more modifications of "non-essential" amino acid residues. In the context of the present invention, "non-essential" amino acid residues are altered in the natural human amino acid sequence of a fragment, eg, component peptide hormone fragment, without destroying or substantially reducing the component peptide hormone receptor agonist activity of the resulting analog. Residues which may be ie deleted or substituted.

Preferred substitutions include conservative amino acid substitutions. A "conservative amino acid substitution" is one in which amino acid residues are replaced with amino acid residues having similar side chain or physicochemical properties (eg, electrostatic, hydrogen bonding, isosteric, hydrophobic features). Family of amino acid residues with similar side chains are known in the art. These families include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, methionine , Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine) Amino acids having phenylalanine, tryptophan, histidine).

The present invention also relates to derivatives of the component peptide hormone. Such derivatives are conjugated to one or more water soluble polymer molecules, for example polyethylene glycol ("PEG") or fatty acid chains of various lengths (e.g. stearyl, palmitoyl, octanoyl, etc.) or polyamino acids such as poly component peptide hormones and analogues added with -his, poly-arg, poly-lys, and poly-ala. Modifications of component peptide hormones or analogs thereof may also include small molecule substituents such as short chain alkyl and constrained alkyl (eg branched chain, cyclic, fused, adamantyl) and aromatic groups. The molecular weight of the water soluble polymer molecule will preferably range from about 500 to about 20,000 Daltons.

Such polymer-conjugation and small molecule substituent modifications may occur once at the N-terminus or C-terminus or side chain of an amino acid residue in the sequence of a hybrid polypeptide. Alternatively, there may be multiple sites of derivatization along the hybrid polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine may provide additional derivatization sites. See, for example, US Pat. No. 5,824,784 and US Pat. No. 5,824,778. Preferably, the hybrid polypeptide may be conjugated to one, two or three polymer molecules.

The water soluble polymer molecule is preferably linked to an amino, carboxyl or thiol group and may be linked by the N-terminus or C-terminus or in the side chain of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, the water soluble polymer molecule may be linked to diamine and dicarboxyl groups. In a preferred embodiment, the hybrid polypeptides of the invention are conjugated to one, two or three PEG molecules via epsilon amino groups on lysine amino acids.

In addition, derivatives of the invention include component peptide hormones or analogs having chemical alterations to one or more amino acid residues. Such chemical alterations include amidation, glycosylation, acylation, sulfated, phosphorylation, acetylation and cyclization. Chemical alterations may occur once at the N-terminus or C-terminus or side chain of an amino acid residue in the sequence of a PPF hybrid polypeptide. In one embodiment, the C-terminus of these peptides may have free -OH groups or -NH ₂ groups. In another embodiment, the N-terminal end isobutyloxycarbonyl group, isopropyloxycarbonyl group, n-butyloxycarbonyl group, ethoxycarbonyl group, isocaproyl group (isocap), octanyl group, octyl glycine group (G (Oct)) Or capped with an 8-aminooctanoic acid group. In a preferred embodiment, cyclization can be carried out through the formation of disulfide bridges. Alternatively, there may be multiple sites chemically altered along the hybrid polypeptide.

Amylin family

As discussed herein, the component peptide hormones useful in the present invention are amylin, adrenomedulin ("ADM"), calcitonin ("CT"), calcitonin gene related peptide ("CGRP"), intermedin ("AFP- Amyloid family peptide hormones, also known as 6 ") and related peptides. Natural amylin family peptide hormones and functional peptide analogs and derivatives are known in the art. Preferred specific natural peptides, peptide analogs and derivatives are described herein. However, it should be understood that any known amylin family peptide exhibiting hormonal activity known in the art may be used in connection with the present invention.

Any amylin analog or derivative known in the art can be used in connection with the present invention. In one embodiment, amylin analogs and derivatives retain one or more hormonal activities of natural amylin. In certain embodiments, the amylin analog is an agonist of a receptor to which natural amylin can specifically bind. Preferred amylin analogs and derivatives include those described in US 2003/0026812 A1, which is incorporated herein by reference.

Exemplary amylin analogs include the following:

As known in the art, such amylin analogs are preferably amidated, but may optionally be in acid form in the context of the present invention unless otherwise specified.

Any ADM analog or derivative known in the art can be used in connection with the present invention. In one embodiment, ADM analogs and derivatives retain one or more hormonal activities of native ADM. In certain embodiments, an ADM analog is an agonist of a receptor to which native ADM can specifically bind.

Any CT analog or derivative known in the art can be used in connection with the present invention. In one embodiment, the CT analogs and derivatives retain one or more hormonal activities of native CT. In certain embodiments, the CT analog is an agonist of a receptor to which native CT can specifically bind. Preferred CT analogs and derivatives are US Pat. 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 US Pat. Nos. 4,401,593 and 4,397,780, which are incorporated herein by reference.

Exemplary CT analogs include the following:

As is known in the art, such CT analogs are preferably amidated, but may be optionally in acid form within the context of the present invention unless otherwise specified.

Any CGRP analog or derivative known in the art can be used in connection with the present invention. In one embodiment, CGRP analogs and derivatives retain one or more hormonal activities of native CGRP. In certain embodiments, CGRP analogs are agonists of receptors to which native CGRP can specifically bind. Preferred CGRP analogs and derivatives include those described in US Pat. Nos. 4,697,002 and 4,687,839, which are incorporated herein by reference.

Exemplary CGRP analogs include the following:

Any AFP-6 analog or derivative known in the art can be used in connection with the present invention. In one embodiment, AFP-6 analogs and derivatives retain one or more hormonal activities of native AFP-6. In certain embodiments, the AFP-6 analog is an agonist of a receptor to which native AFP-6 can specifically bind. Preferred AFP-6 analogs and derivatives include those described in WO 2003/022304, which is incorporated herein by reference.

Exemplary AFP-6 analogs include the following:

As known in the art, such AFP-6 analogs are preferably amidated, but may be optionally in acid form in the context of the present invention unless otherwise specified.

CCK family

CCK and various analogs thereof, including hCCK and species variants, are known in the art. In general, CCK has the first 33 amino acid sequences identified in humans and is an in vivo C-terminal fragment (“CCK-8”) of 8 amino acids reported to have 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, 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 tyrosine-sulfated octapeptide sequence (CCK-8) is relatively conserved across species and may be the minimum sequence for biological activity in a few rodents. Thus, the term CCK-33 generally means human CCK (1-33), while CCK-8 (CCK (26-33), SEQ ID NO: 55) is both sulfated and non-sulphated unless otherwise specified. The C-terminal octapeptide of is referred to generically. Additionally, pentagastrin or CCK-5 refers to the C-terminal peptide CCK (29-33) (SEQ ID NO: 209), and CCK-4 refers to the C-terminal tetrapeptide CCK (30-33) (SEQ ID NO: 208). .

Type A receptor subtypes (CCK _A ) have been reported to be selective for sulfated octapeptide. Type B receptor subtypes (CCK _B ) have been identified throughout the brain and in the stomach and have been reported not to require sulfated or all eight amino acids.

Various in vivo and in vitro screening methods for CCK analogs are known in the art. Examples include in vitro assays using strips of rabbit gallbladder and in vivo assays involving contraction of dog or guinea pig gallbladder following rapid intravenous injection of the compound to be tested for CCK-like activity. See Walsh, "Gastrointestinal Hormones", In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York).

Exemplary specific CCKs, and CCK analogs with CCK activity, include the following:

As is known in the art, such CCK peptides are preferably amidated, but may optionally be in acid form in the context of the present invention unless otherwise specified.

Leptin family

Component peptide hormones useful in the present invention also include leptin family peptide hormones. Natural leptin family peptide hormones and functional peptide analogs and derivatives are known in the art. While certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any known amylin family peptide that exhibits hormone activity known in the art may be used in connection with the present invention.

Any leptin analog or derivative known in the art can be used in connection with the present invention. In one embodiment, leptin analogs and derivatives retain one or more hormonal activities of natural leptin. In certain embodiments, the leptin analog is an agonist of a receptor to which natural leptin can specifically bind. Preferred leptin analogs and derivatives include, for example, those described in WO 2004/039832, WO 98/55139, WO 98/12224 and WO 97/02004, all of which are incorporated herein by reference.

In one embodiment, the leptin peptide comprises MVPIQK, VQDDTK, TLIK, TIVTR, INDISHTQSVSSK, VTGLDFIPGLHPILTLSK, NVIQISNDLENLR, DLLHVLAFSK, SCHLPWASGLETLDSLGGVLEASGYSTEVVALSRD and LQQLDSLQ as disclosed in WO 97046585.

In one embodiment, the leptin peptide has an amino acid sequence Xaan-Ser-Cys-Xaa1-Leu-Pro-Xaa2-Xaa3-Xaan, wherein Xaan may be zero residues in length or derived from a full length human or mouse leptin sequence The peptide residues may have contiguous stretches (which may be 1 to 7 stretches at the C-terminus or N-terminus), or the leptin peptide may be up to 15 amino acids in total. In another embodiment, Xaa1, Xaa2 or Xaa3 can be any amino acid substitution. In another embodiment, Xaa1, Xaa2 or Xaa3 may be any conservative amino acid substitution of each residue in full length mouse or human leptin. In further embodiments, Xaa1 can be selected from the group consisting of His or Ser, and Xaa2 or Xaa3 is any amino acid substitution. In another embodiment, Xaa 2 can be selected from the group consisting of Trp or Gln, and Xaa 1 or Xaa 3 is any amino acid substitution. In another embodiment, Xaa 3 can be selected from the group consisting of Ala or Thr, and Xaa 1 or Xaa 2 is any amino acid substitution. In another embodiment, Xaa 1 is selected from the group consisting of His or Ser, Xaa 2 is selected from the group consisting of Trp or Gln, and Xaa 3 is selected from the group consisting of Ala or Thr. See WO 04039832.

In one embodiment, the leptin peptide comprises the C-terminal amino acid residues 116-122 (corresponding to positions 95-101 of their mature form) and D-isotypes, fragments, derivatives, analogs of native full-length human or mouse leptin and Homolog, which is ip Retains the ability to adjust body mass homeostasis in test animals upon (intraperitoneal) administration Specific mouse D-substituted peptides of the sequence SCSLPQT include [D-Ser-1]-, [D-Cys-2]-, [D-Ser-3]-, [D-Leu-4]-, [D- Pro-5]-, [D-Gln-6]-, [D-Thr-7] -SCSLPQT and all [D] SCSLPQT. Specific human D-substituted peptides of SCHLPWA include [D-Ser-1]-, [D-Cys-2]-, [D-His-3]-, [D-Leu-4]-, [D-Pro -5]-, [D-Trp-6]-, [D-Ala-7] -SCHLPWA, and all [D] -SCHLPWA. In addition, the SCHLPWA and SCSLPQT peptides may contain D-substituted amino acids in any of two, three, four, five or six positions. Also disclosed are leptin-related peptides comprising the N-terminal amino acids 21-35, 31-45, 41-55 and 51-65 and fragments, derivatives, analogs and homologs thereof of natural leptin. Further leptin peptides of the invention include amino acid sequences 61-75, 71-85, 81-95, 91-105, 106-120, 116-130, 126-140, 136-150, of mouse and / or human full-length leptin. 146-160, and 156-167. See WO 04039832.

In one embodiment, the sequence of leptin is as follows:

Wherein Xaa in position 6 is Trp or Gln, Xaa in position 36 is Gln or Glu, Xaa in position 40 is Gln or Glu, Xaa in position 42 is Ile, Leu, Met or methionine sulfoxide, and Xaa at 44 is Trp or Gln and Xaa at position 45 is Gln or Glu). In another embodiment, Xaa in position 6 is Trp, Xaa in position 36 is Gln, Xaa in position 40 is Gln, Xaa in position 42 is Met, Xaa in position 44 is Trp, and Xaa in position 45 Is said leptin. See US Pat. No. 5,521,283.

In one embodiment, the natural sequence of leptin comprises:

In another embodiment, the sequence of the leptin peptide is as follows:

Wherein Xaa at position 22 is Asn, Asp or Glu, Xaa at position 27 is Thr or Ala, Xaa at position 28 is Gln or Glu or is absent, Xaa at position 54 is Met or Ala, Xaa at position 68 is Met or Leu, Xaa at position 72 is Asn, Asp or Glu, Xaa at position 77 is Ser or Ala and Xaa at position 118 is Gly or Leu). The protein has one or more substitutions selected from the group consisting of: His at position 97 is replaced by Ser or Pro, Trp at position 100 is replaced by Gln, Ala or Leu, Ala at position 101 Thr or Val Replaced with Ser in position 102 replaced by Arg, Gly in position 103 replaced by Ala, Glu in position 105 replaced by Gln, Thr in position 106 replaced by Lys or Ser, Leu in position 107 Replaced by this Pro, Asp at position 108 replaced by Glu, or Gly at position 111 replaced by Asp.

In another embodiment, the sequence of leptin is as follows:

Wherein Xaa at position 27 is Thr or Ala, Xaa at position 77 is Ser or Ala, and Xaa at position 118 is Gly or Leu. The protein has one or more substitutions selected from the group consisting of: preferably 1 to 5 substitutions, most preferably 1 to 2 substitutions: His at position 97 is replaced by Ser, at position 100 Trp replaced by Gln, Ala in position 101 replaced by Thr, Glu in position 105 replaced by Gln, Thr in position 106 replaced by Lys, Leu in position 107 replaced by Pro, in position 108 Asp is replaced by Glu, or Gly in position 111 is replaced by Asp. In further embodiments of the sequence, Xaa at position 27 is Thr, Xaa at position 77 is Ser, Xaa at position 118 is Gly and at positions 97, 100, 101, 105, 106, 107, 108 and 111 Amino acid residues are shown in the table below:

In one embodiment, the sequence of leptin is as follows:

Wherein Xaa at position 13 is Ile, Leu, Met or methionine sulfoxide, Xaa at position 15 is Gln or Glu, Xaa at position 21 is Gln or Glu, Xaa at position 22 is Gln or Glu, position Xaa at 27 is Ile, Leu, Met or methionine sulfoxide, Xaa at position 31 is Asn, Asp or Gln, Xaa at position 34 is Gln or Glu, Xaa at position 37 is Asn, Asp or Gln, and Xaa at 41 is Asn, Asp or Gln, Xaa at position 59 is Trp or Gln, Xaa at position 89 is Gln or Glu, Xaa at position 93 is Gln or Glu, and Xaa at position 95 is Ile, Leu, Met or methionine sulfoxide, Xaa at position 97 is Trp or Gln, and Xaa at position 98 is Gln or Glu). In another embodiment, the leptin of the formula is Xaa at position 13 is Met, Xaa at position 15 is Gln, Xaa at position 21 is Gln, Xaa at position 22 is Gln, and Xaa at position 27 is Met , Xaa at position 31 is Asn, Xaa at position 34 is Gln, Xaa at position 37 is Asn, Xaa at position 41 is Asn, Xaa at position 59 is Trp, Xaa at position 89 is Gln, position Xaa at 93 is Gln, Xaa at position 95 is Met, Xaa at position 97 is Trp, and Xaa at position 98 is Gln. See US Pat. No. 5,532,336.

Exemplary leptin analogs include amino acids at position 43 substituted with Asp or Glu, Ala substituted at amino acid at position 48, amino acids substituted or absent with Glu at position 49, amino acids at Ala at position 75 Substituted, amino acid at position 89 substituted with Leu, amino acid at position 93 substituted with Asp or Glu, amino acid at position 98 substituted with Ala, amino acid at position 117 substituted with Ser, Amino acid at position 139 is substituted with Leu, amino acid at position 167 is substituted with Ser, and any combination thereof.

Exemplary specific leptin, and leptin analogs having leptin activity include the following:

PPF family

Component peptide hormones useful in the present invention also include PPF peptide hormones, including PP and PYY. Natural PPF peptide hormones and functional peptide analogs and derivatives are known in the art. While certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any known amylin family peptide that exhibits hormone activity known in the art may be used in connection with the present invention.

Any PPF analog or derivative known in the art can be used in connection with the present invention. In one embodiment, the PPF analogs and derivatives retain one or more hormonal activities of the native PPF polypeptide. In certain embodiments, the PPF analog is an agonist of a receptor to which a native PPF polypeptide can specifically bind. Preferred PPF analogs and derivatives include those described in WO 03/026591 and WO 03/057235, which are incorporated by reference in their entirety.

In one embodiment, preferred PPF analogs and derivatives exhibiting at least one PPF hormonal activity generally comprise two or more PYY motifs, including polyproline motifs and C-terminal tail motifs. Such analogs are generally described in US Provisional Application No. 60 / 543,406, filed February 11, 2004, published as US 2006/013547 A1 on June 22, 2006, which is incorporated herein by reference. . Other preferred PPF analogs are disclosed in PCT / US2005 / 004351, entitled “Pancreatic Polypeptide Family Motifs and Polypeptides Comprising the Same”, published on WO 25/2005 as WO 2005/077094. The contents are incorporated herein by reference. Other preferred PPF analogs are disclosed in PCT / US2005 / 045471 (Pancreatic Polypeptide Family Motifs, Polypeptides and Methods Comprising the Same, published WO 2006/066024 on June 22, 2006). The contents of the literature are incorporated herein by reference. As a background, the study suggested that differences in Y receptor binding affinity correlated with secondary and tertiary structural differences. See, eg, Keire et al., Biochemistry 2000, 39, 9935-9942. Natural pig PYY is divided into two C-terminal helix segments of residues 17-22 and 25-33 separated by kinks at residues 23, 24 and 25, a turn around residues 12-14, And an N-terminus folded near residues 30 and 31. In addition, full-length pig PYY was characterized by comprising PP folds stabilized by hydrophobic interactions in residues at the N- and C-terminus. See above document.

A "PYY motif" is generally a primary, secondary or tertiary structural component of a natural PP family polypeptide that is very important for biological activity. That is, in the absence or obstruction of the motif, biological activity is substantially reduced. Preferred PYY motifs include the N-terminal polyproline type II motif of the natural PP family polypeptide, the type II β-turn motif of the natural PP family polypeptide, the α-helix motif at the C-terminal end of the natural PP family polypeptide, and C-terminal tail motifs of native PP family polypeptides. More particularly, in the N-terminal polyproline region, amino acids corresponding to residues 5 and 8 of the native PP family polypeptide are generally conserved with proline. Type II β-turn motifs will generally comprise amino acids corresponding to residues 12-14 of the native PP family polypeptide. As long as the α-helix motif contains a sufficient number of amino acid residues to form an α-helix turn in solution, the α-helix motif is generally from the amino acid to the C-terminal end corresponding to about residue 14 of the native PP family polypeptide. May extend to any point of and to any point including the termination. α-helix motifs may also include amino acid substitutions, insertions, and deletions for native PP family sequences, as long as α-helix turns are still in solution. C-terminal tail motifs generally comprise amino acids corresponding to the last about 10 residues, more preferably the last 7, 6 or 5 residues, and even more preferably amino acid residues 32 to 35 of the native PP family polypeptide. do.

Preferred PYY analogues include those having internal deletions, insertions and substitutions in regions of the PYY molecule that do not correspond to polyproline motifs and / or C-terminal tail motifs. For example, internal deletions at positions 4, 6, 7, 9 or 10 are contemplated.

In another embodiment of particular interest, the component hormone is a PPF polypeptide containing at least two PPF motifs, including at least an N-terminal polyproline PPF motif and a C-terminal tail PPF motif. As used herein, a "motif" refers to an amino acid sequence characterized by a particular biochemical function, or defines an independently folded domain. Additional PPF motifs may correspond to the motifs of any PP family polypeptide, including PP, PYY, NPY, and the like, for example, the type II α-turn region motif of PYY or the α-helix motif of the PYY C-terminal termination. .

In another embodiment, the PPF family component module is a PPF chimeric polypeptide comprising a fragment of a PP, PYY or NPY polypeptide covalently linked to one or more additional fragments of a second PP, PYY or NPY polypeptide, wherein each PP , PYY or NPY fragments include PPF motifs. Alternatively, the PPF chimera may comprise a fragment of a PP family polypeptide linked to one, two, three or four polypeptide segments, wherein at least one of the linked polypeptide segments is a fragment of a second PP family polypeptide. . In certain embodiments, the PPF polypeptide does not comprise an N-terminal PP fragment and a C-terminal NPY fragment. The PPF chimeric polypeptide component module exhibits at least 50% sequence identity to native PYY (3-36) over the entire length of native PYY (3-36). In some embodiments, such PPF chimeric polypeptides comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, in natural PYY (3-36) over the entire length of the native PYY (3-36), At least 94% or at least 97% sequence identity can be exhibited. Such PPF chimeric polypeptides may exhibit at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% sequence identity to the natural PP. In another embodiment, such PPF chimeric polypeptides may exhibit at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, or at least 97% sequence identity to native NPY. have. In some embodiments, the PPF chimeric polypeptide may comprise at least an N-terminal polyproline PPF motif and a C-terminal tail PPF motif. These PPF chimeras and also other PYY and PP analogs are described in US 2006/013547 A1 published June 22, 2006. In one embodiment, any PPF family peptide may be provided as a second agent, eg, a second anti-obesity agent, in conjunction with a hybrid, as described herein, unless it is contained as a hybrid component.

Again, the PPF chimeric polypeptide will generally retain at least some of the biological activity of native human PP, PYY, or NPY. In some embodiments, the PPF chimeric polypeptide exhibits biological activity in the treatment and prevention of metabolic conditions and disorders.

Polypeptide fragments of the PPF chimera may be covalently linked together in any manner known in the art, including but not limited to direct amide linkages or chemical linker groups and the like. Chemical linker groups may include peptide mimetics that induce or stabilize polypeptide forms. PPF chimeric polypeptides include PYY-PP, PYY-NPY, PP-PYY, PP-NPY, NPY-PP or NPY-PYY chimeras.

PPF chimeras may be at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids long. . In some embodiments, the PYY analogue polypeptide comprises only native L-amino acid residues and / or modified native L-amino acid residues. In some embodiments, the PYY analogue polypeptide does not comprise non-natural amino acid residues.

In some embodiments, the PPF chimeric polypeptide comprises:

In some embodiments, the PPF chimeric polypeptide may comprise a fragment of a PP family analog polypeptide. For example, a PPF chimeric polypeptide may include not only the PPF analog polypeptides described herein, but also PP analog polypeptides, and NPY analog polypeptides.

PYY analogue polypeptides for use in a hybrid of the invention or for use as a second agent may be used in the same assay in one of the assays described herein (eg, food intake, gastric emptying, pancreatic secretion, body composition or weight loss assays). Have the same or greater potency than NPY, PYY, or PYY (3-36). In some embodiments, the PPY analog polypeptide may be useful for the treatment of metabolic diseases such as obesity, insulin resistance syndrome (syndrome X) or diabetes mellitus. In some embodiments, PYY analogue polypeptides may appear to have improved ease of manufacture, stability, and / or formulation compared to PP, NPY, PYY, or PYY (3-36).

In some embodiments, the PPF chimeric polypeptide reduces the nutrient utilization, decreases food intake, weight gain effects, and / or reduces the biological activity of native human PYY by about 25% or about 30 with respect to the treatment and prevention of metabolic conditions and disorders. %, About 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 99% or more. In another embodiment, the PPF chimeric polypeptide exhibits improved PYY agonist activity. In some embodiments, the PPF chimeric polypeptide comprises at least about 110% of the biological activity of native human PYY in connection with a decrease in nutrient utilization, a decrease in food intake, a weight gain effect, and / or the treatment and prevention of metabolic conditions and disorders. It exhibits at least 125%, at least about 130%, at least about 140%, at least about 150%, at least about 200% or higher activity.

More particularly, in one aspect, the PPF chimeric polypeptide comprises a fragment of PP linked to a fragment of PYY. In one embodiment, the PPF chimeric polypeptide is an N-terminal fragment of a PP or PP analog polypeptide, wherein the N-terminal fragment of the PP or PP analog polypeptide is linked to the C-terminal fragment of the PYY or PYY analog polypeptide at its C-terminal end. It includes. In another embodiment, the PPF chimeric polypeptide is PYY, wherein the N-terminal fragment of the PYY, PYY (3-36) or PYY analog polypeptide is linked to the C-terminal fragment of the PP or PP analog polypeptide at its C-terminal end, N-terminal fragments of PYY (3-36) or PYY analogue polypeptides.

In some embodiments, the PPF chimeric polypeptide comprises a fragment of PYY linked to a fragment of NPY. In one embodiment, the PPF chimeric polypeptide is PYY, PYY, wherein the N-terminal fragment of the PYY, PYY (3-36) or PYY analog polypeptide is linked to the C-terminal fragment of the NPY or NPY analog polypeptide at its C-terminal end. (3-36) or an N-terminal fragment of a PYY analogue polypeptide. In another embodiment, the PPF chimeric polypeptide is an N-terminus of an NPY or NPY analogue polypeptide, wherein the N-terminal fragment of the NPY or NPY analogue polypeptide is linked to the C-terminal fragment of the PYY or PYY analogue polypeptide at its C-terminal end. Contains fragments.

In some embodiments, the PPF chimeric polypeptide comprises a fragment of PP linked to a fragment of NPY. In one embodiment, the PPF chimeric polypeptide is an N-terminal fragment of a PP or PP analog polypeptide, wherein the N-terminal fragment of the PP or PP analog polypeptide is linked to the C-terminal fragment of the NPY or NPY analog polypeptide at its C-terminal end. It includes. In another embodiment, the PPF chimeric polypeptide is an N-terminus of an NPY or NPY analogue polypeptide, wherein the N-terminal fragment of the NPY or NPY analogue polypeptide is linked to the C-terminal fragment of the PP or PP analogue polypeptide at its C-terminal end. Contains fragments.

In some embodiments, a fragment of a PP, PP analog polypeptide, PYY, PYY (3-36), PYY analog polypeptide, NPY or NPY analog polypeptide is a PP, PP analog polypeptide, PYY, PYY (3-36), PYY analog polypeptide , A fragment comprising any 4-20 amino acid residues of an NPY or NPY analog polypeptide. In some embodiments, the length of the fragment is selected such that a final PPF chimeric polypeptide having a length of at least 34 amino acids is obtained.

The PPF chimeric polypeptide may also further comprise modifications to the amino acid sequence of such PPF chimeric polypeptide, including but not limited to, for example, substitutions, deletions and insertions, and any combination thereof. In some embodiments, a PPF chimeric polypeptide comprises one or more modifications of “non-essential” amino acid residues. A "non-essential" amino acid residue is a residue that can be altered, ie deleted or substituted, within the natural human amino acid sequence without destroying or substantially reducing the activity of interest.

Derivatives of PPF chimeric polypeptides are also useful. Such derivatives may be conjugated to one or more water soluble polymer molecules, such as polyethylene glycol ("PEG") or fatty acid chains of various lengths (eg stearyl, palmitoyl, octanoyl, oleoyl, etc.) or polyamino acids, eg PPF chimeric polypeptides with added poly-his, poly-arg, poly-lys and poly-ala, for example. Modifications to the PPF chimeric polypeptide may also include small molecule substituents such as short chain alkyl and constrained alkyl (eg branched chain, cyclic, fused, adamantyl) and aromatic groups. In some embodiments, the molecular weight of the water soluble polymer molecule will preferably range from about 500 to about 20,000 Daltons.

Such polymer-conjugation and small molecule substituent modifications may occur once at the N-terminus or C-terminus or side chain of amino acid residues not involved in hybrid formation in the sequence of the PPF chimeric polypeptide. Alternatively, there may be multiple sites of derivatization along the PPF chimeric polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine may provide additional derivatization sites. See, for example, US Pat. No. 5,824,784 and US Pat. No. 5,824,778. In some embodiments, the PPF chimeric polypeptide may be conjugated to one, two or three polymer molecules.

In some embodiments, the water soluble polymer molecule may be linked to an amino, carboxyl or thiol group and may be linked by the N-terminus or C-terminus or in the side chain of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, the water soluble polymer molecule may be linked to diamine and dicarboxyl groups. In some embodiments, the PPF chimeric polypeptide is conjugated to one, two or three PEG molecules via epsilon amino groups on lysine amino acids.

PPF chimeric polypeptides also include PPF chimeric polypeptides with chemical alterations to one or more amino acid residues. Such chemical alterations include amidation, glycosylation, acylation, sulfated, phosphorylation, acetylation and cyclization. Chemical alterations may occur once at the N-terminus or C-terminus or side chain of an amino acid residue in the sequence of a PPF chimeric polypeptide. In one embodiment, the C-terminus of these peptides may have free -OH groups or -NH ₂ groups. In another embodiment, the N-terminal end isobutyloxycarbonyl group, isopropyloxycarbonyl group, n-butyloxycarbonyl group, ethoxycarbonyl group, isocaproyl group (isocap), octanyl group, octyl glycine group (G (Oct)) Or an 8-aminooctanoic acid group. In some embodiments, cyclization can be performed through the formation of disulfide bridges. Alternatively, there may be multiple sites chemically altered along the PYY analogue polypeptide.

In some embodiments, the PPF chimeric polypeptide comprises one having the amino acid sequence of SEQ ID NOs: 238 to SEQ ID NO: 347 of US 2006/013547 A1.

Exemplary PPF chimeric polypeptides include polypeptides of Formula VI:

<Expression VI>

here,

Xaa ₁ is Tyr or absent,

Xaa ₂ is Ile or Pro or absent,

Xaa ₃ is Ile, BH-modified Lys, Lys, Val or Pro,

Xaa ₄ is Lys, BH-modified Lys, Ala, Ser or Arg,

Xaa ₇ is Ala, Gly, or His,

Xaa ₉ is Gly or Ala,

Xaa ₁₂ is Ala or Pro,

Xaa ₁₃ is Ser or Pro,

Xaa ₁₄ is Pro, Ala or Ser,

Xaa ₁₆ is Glu or Asp,

Xaa ₁₇ is Leu or Ile,

Xaa ₁₈ is Asn or Ala,

Xaa ₁₉ is Arg, Lys, BH-modified Lys, Gln or N (Me) Ala,

Xaa ₂₁ is Tyr, Ala, Phe, Lys or BH-modified Lys,

Xaa ₂₂ is Ala or Ser,

Xaa ₂₃ is Ser, Ala, or Asp,

Xaa ₂₅ is Arg, Lys or BH-modified Lys,

Xaa ₂₆ is His, Ala or Arg,

Xaa ₂₈ is Leu or Ile,

Xaa ₃₀ is Leu or Met,

Xaa ₃₁ is Val, Ile or Leu,

Xaa ₃₅ is Lys, BH-modified Lys or Arg,

Xaa ₃₆ is Tyr, Trp, or Phe.

In one embodiment, the PPF polypeptide of Formula VI is a native PPF polypeptide, PYY (2-36), Val3hPYY (3-36), Lys25hPYY (3-36), Lys25Ile28hPYY (3-36), Lys25Ile31hPYY (3-36), Lys25Leu31hPYY (3-36), Lys25Phe36hPYY (3-36), Ile28hPYY (3-36), Ile31hPYY (3-36), Leu31hPYY (3-36), Phe36hPYY (3-36), Leu31Phe36hPYY (3-36) or Pro13Ala14hPYY Can be.

As will be appreciated by those skilled in the art, the polypeptide of Formula VI may be in free acid form or may be C-terminal amidated.

In some embodiments, the PPF polypeptide is a native human NPY (Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr) Natural human PYY (Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu) linked to a C-terminal fragment consisting essentially of amino acid residues 18-36 Val Thr Arg Gln Arg Tyr) may comprise an N-terminal fragment consisting essentially of the first 17 amino acid residues, wherein one or more amino acid residues of the N-terminal of the PYY fragment may be deleted or absent, and PYY And one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in each of the NPY fragments. In some embodiments, the N-terminal fragment consisting essentially of the first 17 amino acids of the PPF polypeptide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 92 to the first 17 amino acids of native PYY. % Or more, 94% or more, or 97% or more of sequence identity can be shown. In some embodiments, the C-terminal fragment of the PPF polypeptide consisting essentially of amino acid residues 18-36 comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at amino acids 18-36 of the native NPY, 92% or more, 94% or more, or 97% or more of sequence identity can be exhibited. In some embodiments, amino acids present in the N-terminal fragment of PYY (eg, proline at position 5 and position 8, glutamate at position 6, position 10 and position 15 or aspartate at position 11) and / or of PYY The amino acids present in the C-terminal fragment (eg tyrosine at positions 20 and 27, leucine at position 24, asparagine at position 29, threonine at position 32, arginine at position 33 or glutamine at position 34) are not substituted . In some embodiments, the PPF polypeptide comprises one having the amino acid sequences of SEQ ID NOs: 266, 267, 274, 282, 320, and 436 to 480 of US 2006/013547 A1. In some embodiments, the PPF polypeptide further comprises an N-terminal cap. Examples of these PPF polypeptides include SEQ ID NOs: 282, 320, 437, 441, 444, 445 to 447, 452, 454 to 459, 461 to 464, 466, 468 to 470, and 472 to 480 of US 2006/013547 A1.

Other PPF polypeptides include polypeptides of Formula VII:

(VII)

here,

Xaa ₁ is Tyr or absent,

Xaa ₂ is Ile or Pro or absent,

Xaa ₄ is Lys, BH-modified Lys, Ala, Ser or Arg,

Xaa ₆ is Glu, Gln, Ala, Asn, Asp or Val,

Xaa ₉ is Gly or Ala,

Xaa ₁₀ is Glu, Ala, Asp, Asn, Gln, Gly, Pro or Aib,

Xaa ₁₁ is Glu, Ala, Asp, Asn, Gln, Gly, Pro or Aib,

Xaa ₁₂ is Ala or Pro,

Xaa ₁₃ is Ser or Pro,

Xaa ₁₄ is Pro, Ala or Ser,

Xaa ¹⁵ is Glu, Ala, Asp, Asn, Gln, Gly, Pro or Aib,

Xaa ₁₆ is Glu or Asp,

Xaa ₁₇ is Leu or Ile,

Xaa ₁₉ is Arg, Lys, BH-modified Lys, Gln or N (Me) Ala,

Xaa ₂₁ is Tyr, Ala, Phe, Lys or BH-modified Lys,

Xaa ₂₂ is Ala or Ser,

Xaa ₂₃ is Ser, Ala, or Asp,

Xaa ₂₅ is Arg, Lys or BH-modified Lys,

Xaa ₂₆ is His, Ala or Arg,

Xaa ₂₇ is Tyr or Phe,

Xaa ₂₈ is Leu or Ile,

Xaa ₂₉ is Asn or Gln,

Xaa ₃₀ is Leu or Met,

Xaa ₃₁ is Val, Ile or Leu.

As will be appreciated by those skilled in the art, the polypeptide of Formula VII may be in free acid form or may be C-terminal amidated.

In some embodiments, the PPF polypeptide is a native human PYY (SEQ ID NO: 2 of US 2006/013547 A1) linked to a C-terminal fragment consisting essentially of amino acid residues 18-36 of native human NPY (SEQ ID NO: 4 of US 2006/013547 A1). ) May comprise an N-terminal fragment consisting essentially of the first 17 amino acid residues of), wherein one or more amino acid residues of the N-terminal of the PYY fragment may be deleted or absent and may be present in each of the PYY and NPY fragments. There may be one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions. In some embodiments, the N-terminal fragment consisting essentially of the first 17 amino acids of the PPF polypeptide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 92 to the first 17 amino acids of native PYY. % Or more, 94% or more, or 97% or more of sequence identity can be shown. In some embodiments, the C-terminal fragment of the PPF polypeptide consisting essentially of amino acid residues 18-36 comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at amino acids 18-36 of the native NPY, 92% or more, 94% or more, or 97% or more of sequence identity can be exhibited. In some embodiments, amino acids present in the N-terminal fragment of PYY (eg, proline at positions 3, 5 and 8, or histidine at position 7) and / or amino acids present in the C-terminal fragment of NPY ( For example alanine at position 18, tyrosine at position 20 and 36, leucine at position 24, threonine at position 32, arginine at position 33, glutamine at position 34 or arginine at position 35). In some embodiments, the PPF polypeptides are those having the amino acid sequence of PYY-NPY chimera such as SEQ ID NOs: 266, 437, 438, 439, 442, 462, 469, 470, 471 and 472 of US 2006/013547 A1 or Ile, Lys , Pro, Glu, His, Pro, Gly, Glu, Asp, Ala, Ser, Pro, Glu, Glu, Leu, Ala, Arg, Tyr, Tyr, Ala, Ser, Leu, Arg, Ala, Tyr, Ile, Asn And compounds having the amino acid sequence of Leu, Ile, Thr, Arg, Gln, Arg, Tyr-NH ₂ . In some embodiments, the PPF polypeptide further comprises an N-terminal cap. Examples of these PPF polypeptides include SEQ ID NOs: 437, 462, 469, 470 and 472 of US 2006/013547 A1. For example, SEQ ID NO: 438 of US 2006/013547 A1 is Pro Lys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Ala Arg Tyr Tyr Ala Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr. In one embodiment, hybrids of the invention, particularly hybrids useful for treating obesity, losing weight, reducing or redistributing fat, and reducing calorie intake, comprise SEQ ID NO: 438, which is a component of US 2006/013547 A1. Such hybrids may contain an amylinomimetic component or leptin component or both.

When a PPF polypeptide and a PPF chimera are used alone or as a component of a second agent or a hybrid of the present invention, it is a compound (PPF polypeptide or PPF chimera alone) that alters the body composition by altering the fat: antifat ratio in the subject. Or used as a component of a second agent or a hybrid of the present invention), in a method comprising altering the body composition of a subject. The PPF polypeptide may comprise an amino acid sequence selected from the group consisting of PYY-NPY chimera called 5705 and the following sequence of US 2006/013547 A1: SEQ ID NOs: 266, 267, 274, 282, 320, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479 and 480. In one embodiment, body fat is reduced and lean body mass ) Is maintained or increased. In one embodiment, body fat and lean body weight are measured in percent body fat percentage and percent lean body weight percentage, respectively. In one further embodiment, the weight is reduced. In another embodiment, body weight is maintained or increased. The compound may be administered peripherally. PPF polypeptides or PPF chimeras or PPF-containing hybrids may be used in subjects with amylin, amylin agonist or amyloid analog agonists, salmon calcitonin, cholecystokinin (CCK) or CCK agonists, leptin (OB protein) or leptin agonists Dean or Exendin analog agonists, GLP-1, GLP-1 agonists or GLP-1 analog agonists, CCK or CCK agonists, calcitonin, calcitonin agonists, small molecule cannabinoid CB1 receptor antagonists, limonabant, 11 beta- It can be used in a method further comprising administering one or more other agents selected from the group consisting of hydroxysteroid dehydrogenase-1 inhibitors, sibutramine, and phentermin. In one embodiment, the subject is overweight or obese. In another embodiment, when the PPF polypeptide and PPF chimera are used alone or as components of a second agent or a hybrid of the present invention, this comprises administering to the subject an effective amount of a compound that lowers plasma triglyceride levels, It is used in methods that include a method of preferentially lowering plasma triglyceride levels in a subject, where cholesterol levels are lowered to a lower extent. In further embodiments, triglyceride levels are lowered but cholesterol levels are not lowered. In further embodiments, triglyceride levels are lowered but LDL cholesterol levels are not lowered. In further embodiments, triglyceride levels are lowered and LDL cholesterol levels are lowered to a lower extent. In further embodiments, amylase levels are also lowered. In another embodiment, when the PPF polypeptide and the PPF chimera are used alone or as a component of a second agent or a hybrid of the present invention, it is effective for the subject to reduce body fat or body fat gain while maintaining or increasing lean body weight. A method comprising reducing a body fat or an increase in body fat while maintaining or increasing lean body weight in a subject, comprising administering an amount of said compound. In another embodiment, when the PPF polypeptide and PPF chimera are used alone or as a component of a second agent or a hybrid of the invention, it is effective for the subject to reduce visceral body fat and preserve or increase lean body weight. It is used in a method comprising a method of reducing visceral body fat in a subject, comprising administering the compound. In another embodiment, when the PPF polypeptide and PPF chimera are used alone or as a component of a second agent or a hybrid of the present invention, it is used in a method comprising a method of altering fat distribution in a subject. In one aspect, the alteration is due to an increase in metabolism of visceral or ectopic fat, or both in the subject. In another embodiment, when the PPF polypeptide and PPF chimera are used alone or as a component of a second agent or a hybrid of the invention, it is effective for the subject to preserve or increase lean body weight while increasing fatty acid β-oxidation. It is used in a method comprising administering an amount of said compound, including increasing fatty acid β-oxidation while preserving or increasing lean body weight. In another embodiment, when the PPF polypeptide and PPF chimera are used alone or as a component of a second agent or a hybrid of the present invention, it is effective in treating the subject with non-alcoholic steatohepatitis or lipodystrophy. It is used in a method comprising the method of treating non-alcoholic steatohepatitis or lipodystrophy in a subject comprising administering a compound. Hybrids of particular interest in this use are PPF chimeras as described herein, in combination with components of the leptin family or amylin family, such as the amylin-sCT-amylin hybrid, or both. PPF-chimeric / leptin hybrids or PPF-chimeric / amylin-sCT-amylin hybrids provide better effects than either compound when used alone. In a further embodiment, the PPF-chimera / leptin hybrid is administered in combination with an amyloid mimetic, for example, the amylin-sCT-amylin chimera or the PPF-chimera / amylin-sCT-amylin hybrid is combined with leptin. Administered.

If the PPF polypeptide chimera referred to herein is not a component module of the hybrid, it may be administered alone or as a second agent, preferably in combination with the hybrid of the invention. These may be provided with pharmaceutically acceptable carriers or excipients in single or multiple administrations or without pharmaceutically acceptable carriers or excipients. These pharmaceutical compounds may be formulated according to conventional techniques as disclosed in Remington's Pharmaceutical Sciences by E. W. Martin with pharmaceutically acceptable carriers or diluents as well as any other known auxiliaries and excipients. See also Wang, Y. J. and Hanson, M. A. "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S (1988), which is incorporated herein by reference. PPF polypeptides may be provided in dosage unit form. For example, a therapeutically effective amount of PPF polypeptide that affects body composition may include the age and weight of the patient, the physical condition of the patient, whether it is in combination with other therapeutic agents, the ultimate goal to be achieved, such as overall weight loss and / or It will depend on many factors including the maintenance or increase of lean body weight, and also other factors. However, typical dosages (if not a hybrid component) are about 0.05 μg, about 0.1 μg, about 1 μg, about 5 μg, about 10 μg, about 50 μg, about 75 μg or about 100 μg of the pharmaceutical compound per day. It can contain an upper limit of about 50 μg, about 100 μg, about 500 μg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 50 mg, about 100 mg, or about 150 mg. Other dosage ranges are also contemplated, for example 0.1 μg to 1 mg of compound per dose, or about 0.001 μg / kg to about 500 μg / kg per dose. In some embodiments, the PPF polypeptide chimera is administered in single or multiple doses or controlled sustained release at a dosage of about 0.5 μg to about 5 mg per day, or about 0.01 μg / kg to about 500 μg / kg per dose. Or, from about 0.05 μg / kg to about 250 μg / kg. In some embodiments, the PPF polypeptide chimera is administered at a dosage of less than about 50 μg / kg. Administration of these ranges will of course depend on the potency of each analog or derivative and can be readily determined by one skilled in the art. The daily dose may be delivered in separate unit doses provided continuously for a period of 24 hours or any portion of 24 hours. The number of daily doses may range from 1 to about 4 times per day, but may be greater. Continuous delivery may be in the form of continuous infusion. Other exemplary dosages and infusion rates contemplated include continuous infusion from 0.005 nmol / kg to about 20 nmol / kg or from about 0.01 / pmol / kg / min to about 10 pmol / kg / min per separate dosage. Such dosages and infusions may be in any known conventional or future way to be developed, such as intravenous (iv), intradermal, intramuscular, intramammary, intraperitoneal, intradural, post-ocular, intrapulmonary (eg For example, by term release, subcutaneous administration (sc), oral, sublingual, nasal, anal, vaginal or transdermal delivery, or surgical implantation at specific sites. . i.v. An example of the total dosage per delivery of the pharmaceutical composition administered can be from about 1 μg to about 8 mg per day, and s.c. The total dosage per single delivery of the pharmaceutical composition administered can be from about 6 μg to about 16 mg per day.

Incretin  And Incretin Mimic

Component peptide hormones useful in the present invention also include GLP-1 peptide hormones. Natural GLP-1 peptide hormones and functional including GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), and GLP-1 (7-36) amide (SEQ ID NO: 61) Peptide analogs and derivatives are known in the art. As used herein, GLP-1 refers to all natural forms of the GLP-1 peptide hormone. While certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any known GLP-1 peptide that exhibits hormone activity known in the art may be used in connection with the present invention.

Any GLP-1 peptide analog or derivative known in the art can be used in connection with the present invention. In one embodiment, the GLP-1 peptide analogs and derivatives retain one or more hormonal activities of the native GLP-1 peptide. In certain embodiments, the GLP-1 peptide analog is an agonist of a receptor to which a native GLP-1 peptide can specifically bind. Preferred GLP-1 peptide analogs and derivatives include, for example, those described in WO 91/11457, which is incorporated herein by reference.

GLP-1 analogs known in the art include the following:

As is known in the art, such GLP-1 analogs are preferably amidated, but may optionally be in acid form in the context of the present invention unless otherwise specified.

Other GLP-1 analogs and derivatives are disclosed in US Pat. No. 5,545,618, which is incorporated herein by reference. Preferred groups of GLP-1 analogs and derivatives include those described in US Pat. No. 6,747,006, which is incorporated by reference in its entirety. The use of the molecules described in US Pat. No. 5,188,666 in the present invention is also contemplated, which is expressly incorporated herein by reference. Another group of molecules for use in the present invention includes the compounds described in US Pat. No. 5,512,549, which is expressly incorporated herein by reference. Another preferred group of GLP-1 compounds for use in the present invention are disclosed in WO 91/11457, which is incorporated herein by reference.

Component peptide hormones useful in the present invention also include GLP-2 peptide hormones. Natural GLP-2 peptide hormones such as rat GLP-2 and homologues thereof such as bull 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 and functional peptide analogs and derivatives are known in the art. While certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be appreciated that any known GLP-2 peptide that exhibits hormone activity known in the art may be used in connection with the present invention.

Any GLP-2 peptide analog or derivative known in the art can be used in connection with the present invention. In one embodiment, the GLP-2 peptide analogs and derivatives retain one or more hormonal activities of the native GLP-2 peptide. In certain embodiments, the GLP-2 peptide analog is an agonist of a receptor to which a native GLP-2 peptide can specifically bind. Preferred GLP-2 peptide analogs and derivatives include, for example, those described in US patent application Ser. No. 08 / 669,791 and PCT application PCT / CA97 / 00252, both of which are incorporated herein by reference. Certain GLP-2 analogs known in the art include rat or human GLP-2 modified at position 2 by replacing Ala with Gly to confer DPP-IV resistance.

In addition, component peptide hormones useful in the present invention also include auxintomodulin (OXM) peptide hormones. Natural OXM peptide hormones and functional peptide analogs and derivatives are known in the art. While certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be appreciated that any known OXM peptide that exhibits hormone activity known in the art may be used in connection with the present invention.

Any OXM peptide analog or derivative known in the art can be used in connection with the present invention. In one embodiment, the OXM peptide analogs and derivatives retain one or more hormonal activities of the native OXM peptide. In certain embodiments, the OXM peptide analog is an agonist of a receptor to which a native OXM peptide can specifically bind.

Component peptide hormones useful in the present invention also include exendin peptide hormones. Natural exendin peptide hormones and functional peptide analogs and derivatives are known in the art. While certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be appreciated that any known exendin peptide that exhibits hormonal activity known in the art may be used in connection with the present invention.

Any exendin peptide analog or derivative known in the art can be used in connection with the present invention. In one embodiment, the exendin peptide analogs and derivatives retain one or more hormonal activities of the native exendin peptide. In certain embodiments, the exendin peptide analog is an agonist of a receptor to which a natural exendin peptide can specifically bind.

Exemplary exendin analogs include the following:

As known in the art, such exendin analogs are preferably amidated, but may be optionally in acid form in the context of the present invention unless otherwise specified.

Examples of additional exendin analogs and derivatives are described in PCT Application No. PCT / US98 / 16387, filed Aug. 6, 1998, claiming the benefit of U.S. Patent Application No. 60 / 055,404, filed Aug. 8, 1997 ( The English name of the invention: “Novel Exendin Agonist Compounds”, both of which are incorporated herein by reference. Other exendin analogs and derivatives are described in PCT Application No. PCT / US98 / 24210, filed November 13, 1998, claiming the benefit of US Provisional Application No. 60 / 065,442, filed November 14, 1997. : "Novel Exendin Agonist Compounds", both of which are incorporated herein by reference. Another exendin analog and derivative is PCT Application No. PCT / US98 / 24273, filed November 13, 1998, claiming the benefit of US Provisional Application No. 60 / 066,029, filed November 14, 1997. Name: "Novel Exendin Agonist Compounds", both of which are incorporated herein by reference. Another exendin analog and derivative is PCT Application No. PCT / US97 / 14199, filed Aug. 8, 1997, as part of US Patent Application No. 08 / 694,954, filed Aug. 8, 1996. The English name: "Methods for Regulating Gastrointestinal Activity", both of which are incorporated herein by reference. Another exendin analog and derivative is PCT Application No. PCT / US98 / 00449, filed January 7, 1998 claiming the benefit of US Provisional Application No. 60 / 034,905, filed January 7, 1997. Name: "Use of Exendins and Agonists Thereof for the Reduction of Food Intake", both of which are incorporated herein by reference. Another exendin analogs and 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. Included as.

Sodium excretion peptide

Sodium excretion peptides are a family of hormones consisting of atrial sodium excretion peptides (ANP), brain sodium excretion peptides (BNP) and type C sodium excretion peptides (CNP). These are synthesized and stored as three separate precursor prohormones of 126 amino acids ANP, 108 amino acids BNP and 104 amino acids CNP. Each of these is encoded by a separate gene, with different synthetic and regulatory mechanism sites. Parental sodium excretion peptide sequences include:

The main synthetic site of ANP prohormones is atrial myocytes, where they are synthesized with 151 amino acids preprohormones. Removal of the 25 amino acid signal peptide from the N-terminal end occurs in the endoplasmic reticulum, resulting in 126 amino acid ANP prohormones (proANPs), the main storage form of ANP in the heart. The prohormones consist of four biologically active peptide segments: amino acids 1-30 (proANF 1-30, also known as persistent Na stimulators), 31-67 (proANF 31-67, also vasodilation) Also known as a factor), 79-98 (proANF 79-98, also known as potassium excretory factor), and 99 to 126 (ANF, also known as atrial sodium excretion factor).

BNP was first isolated in the pig brain, but is synthesized and secreted from the left ventricle in humans. Sequence analysis revealed that preproBNP consists of 134 residues and is cleaved into 108 amino acid proBNPs. 32 amino acid sequence cleavage from the proBNP C-terminal end results in human BNP (77-108), which is a physiologically active form in plasma.

CNP is the third member of the sodium excretion peptide system and is found primarily in human vascular endothelial cells, kidneys, and porcine brain. High concentrations of CNP are also found in the human hypothalamus and midbrain. In humans, preproCNPs are 126 amino acid precursors that are cleaved at 23 N residues and processed into proCNPs. The 23 amino acid sequence acts as a signal peptide. The terminal 22 (105-126) amino acids are cleaved from the proCNP to produce a biologically active form of CNP.

Eurodilatin is a kidney-derived member of the sodium excretion peptide family and is formed from the same ANP prohormones and consists of amino acids 95-126. This is identical to ANF (99-126) except for the N-terminal extension of four amino acids. Eurodilatin is not only an important regulator of sodium and water treatment in the kidneys, but also a mediator of sodium excretion in patients with congestive heart failure (CHF).

Sodium excretion peptides bind to high affinity receptors on the target cell surface and exert their biological effects. Three subtypes of NPR-NPR-A, NPR-B and NPR-C-have been isolated. As a result, one embodiment provides a method for screening hybrids for sodium excretion receptor binding and / or activation. Sodium excretion peptides, including prohormone variants, can impart numerous sodium excretion peptide hormone activities to the hybrids of the present invention. Of interest in other embodiments are sodium excretion antagonist hybrids. An increase in sodium excretion is the release of excessive amounts of sodium into the urine. Increased sodium excretion is similar to diuresis (significantly high urine output), except that urine is very salty. Increased sodium excretion occurs with the use of some diuretics and with diseases (such as diseases of the adrenal glands) and can lead to loss of salt syndrome characterized by dehydration, vomiting, hypotension, and sudden risk of death. Exogenous administration of four independent circulating peptides (1-30, 31-67, 79-98 and 99-126) of ANP prohormones has been shown to be effective in vasodilation, diuresis, inhibition of the renin-angiotensin-aldosterone system and enhanced sodium in vivo. Increases excretion and / or potassium excretion. ProANF 1-30, ProANF 31-67 and ANF 99-126 each have increased sodium excretion, lowered blood pressure and diuretic properties, and proANF 31-67 and ANF 99-126 have the greatest effect on blood pressure. The effect of ANP peptides on potassium homeostasis varies: proANF 79-98 stimulates potassium excretion, while proANF 31-67 inhibits Na / K ATPase in medulla collection cells to reduce potassium loss. Specific to ANF 99-126 is dose-dependent inhibition of angiotensin II-mediated aldosterone secretion, but proANF 31-67 is inducible to increase sodium excretion through the production of prostaglandins.

BNP exerts a biological effect similar to ANF in normal humans. When BNP was injected into normal, sodium excretion was doubled, plasma renin, angiotensin II and aldosterone secretion were reduced by 50% and plasma volume was also reduced.

CNP induces a cardiovascular effect similar to other sodium excretion peptides, but does not appear to mediate any renal effects. When CNP is infused in anesthesia at an equivalent dose to ANF, mean arterial pressure, right atrium pressure, and cardiac output decrease at the same time plasma cGMP rises, but glomerular filtration rate, renal blood flow, and sodium excretion are reduced.

Sodium excretion peptides can provide therapeutic benefits for heart failure. Congestive heart failure (CHF) is associated with increased vasopressin and endothelin, activation of the Lenin-Angiotensin-Aldosterone system and sympathetic nervous system and mediates vasoconstriction, sodium and water retention, and harmful vascular and heart remodeling. This effect occurs even with increased levels of sodium excretion peptides in heart failure patients. One embodiment of the invention relates to a hybrid that provides an increase in sodium excretion peptide activity or provides a therapeutic serum level thereof for the treatment or prevention of heart related diseases and conditions, including CHF. ANF infusion in normal individuals can lead to a sustained increase in sodium excretion and urine flow, but in patients with heart failure a markedly beneficial reduction in renal response can be achieved. BNP infusion significantly increases sodium excretion in heart failure patients and has a significant beneficial hemodynamic effect. Compared with ANP, the diuretic and sodium excretion effects of BNP are significantly higher. BNPs are removed more slowly than ANPs and have other effects, including inhibition of aldosterone secretion and increased serum levels of ANPs. BNP peptides may also provide a beneficial decrease in pulmonary capillary wedge pressure, systemic vascular resistance, right atrium pressure and systolic blood pressure and increase cardiac index in patients undergoing hospital treatment with symptomatic CHF. In non-targeted heart failure patients, increased sodium excretion peptide hybrids can provide a beneficial reduction in pulmonary capillary wedge pressure and improved dyspnea scores. (Difficulty breathing is an unpleasant sensation with difficulty breathing and is typically associated with early stages of heart failure). Hybrids with one, two, or three sodium excretory hormone functions are useful for both prophylactic and curative treatment of CHF patients, preferably CHF patients, chronic CHF patients, and hypertensive patients. Provided are methods of administration of the composition. The sodium excretion portion (s) of the hybrid is sufficient to provide a therapeutically effective amount of sodium excretion peptides to such patients when administered at a therapeutically effective dose over a therapeutic expiration date.

As discussed herein, any family of therapeutically effective sodium excretion peptides or analogs thereof can be used. Examples of useful sodium excretion peptides include atrial sodium excretion peptides (ANP), brain sodium excretion peptides (BNP or type B sodium excretion peptides) and type C sodium excretion peptides (CNPs). Sequences of useful forms of sodium excretion peptides are disclosed in US Patent Publication 20010027181, which is incorporated herein by reference. Examples of ANPs include human ANPs [Kangawa et al., BBRC 118: 131 (1984)] or those derived from various species, such as pig and rat ANPs [Kangawa et al., BBRC 121: 585 (1984)], etc. There is this. This ANP contains 28 amino acids. Such ANPs can be administered as peptides having the ring structure of the ANP (disulfide bond formation based on Cys) and the C-terminal portion following the ring structure. Examples of such peptides are peptides having amino acid residues at the 7- to 28-positions of the ANP, which are provided in US Patent Application Publication No. 20010027181. Another example is the frog ANP. Specific examples of BNPs that can be used in the methods of the invention include human BNPs (hBNPs). Human BNP comprises 32 amino acids and includes the formation of disulfide bonds (Sudoh et al., BBRC 159: 1420 (1989)) and US Pat. Nos. 5,114,923, 5,674,710, 5,674,710 and 5,948,761, each of which is incorporated herein by reference). Various BNPs originating outside of human beings are also known, including porcine BNP and rat BNP, and these may also be used. A further example is chicken BNP. Examples of CNPs that can be used in the methods of the invention include porcine CNPs. Porcine CNP comprises 22 amino acids, the ANP and BNP described above (Sudoh et al., BBRC 168: 863 (1990)) (human and rat have the same amino acid sequence) and chicken CNP [Arimura et al., BBRC 174: 142 (1991), including the formation of disulfide bonds. Frog CNP (Yoshihara et al., BBRC 173: 591 (1990)) may also be used. As discussed herein, those skilled in the art can, if desired, make modifications such as deletions, substitutions, additions or insertions, and / or chemical modifications to the amino acid residues of the amino acid sequence of known sodium excretion peptides using known methods. The resulting compound is a compound having agonistic activity at the receptor of the starting ANP, BNP or CNP. Thus, analogs with such activity are included in the hybrids to be used in accordance with the methods of the present invention.

In another embodiment, hybrids having at least one sodium excretory function can be used to treat hypertension. In one embodiment, the sodium excretory hybrid has no deleterious effect on heart rate and is not associated with arrhythmia. In one embodiment, the hybrid comprises at least one, at least two, or at least three of the sodium excretion peptide functions, such as both ANP and BNP activity. One or more sodium excretion hormone functions may be combined with any other hormone function or peptide enhancer as described herein. In another embodiment, the sodium excretion moiety (s) is a more stable analogue with a longer in vivo half-life compared to the native sodium excretion peptides. Analogues are also contemplated to prevent undesirable cleavage by endogenous enzymes such as NEP and the like. Hybrids of increased sodium excretion also reduce hypertension, induce diuresis, induce sodium excretion, dilate or relax vasculature, increase sodium excretion peptide receptors (eg NPR-A), inhibit renin secretion from the kidney, from adrenal glands It is also associated with inhibition of aldosterone secretion, treatment of cardiovascular diseases and disorders, reduction, arrest or repair of cardiac remodeling in congestive heart failure, treatment of renal and renal disorders, treatment or prevention of ischemic stroke, and treatment of asthma. The hybrid can be administered to patients benefiting from increased sodium excretion, diuresis and vasodilation. The hybrid may be administered alone or in combination with one or more of the following types of compounds: ACE inhibitors, beta-blockers, diuretics, spironolactone, digoxin, anti-coagulant agents, anti-platelet agents, And angiotensin receptor blockers. Additional diseases or conditions include renal disorders and kidney disease, asthma, hypertension and pulmonary hypertension. Hybrids are also useful for the treatment of inflammation-related diseases, erectile dysfunction and hypercholesterolemia.

Bioactive Peptide Hormone Module

As discussed herein, hybrid polypeptides of the invention generally comprise two or more bioactive peptide hormone modules covalently linked together. The bioactive peptide hormone module comprises (a) a natural component peptide hormone, (b) an analog or derivative of a natural component peptide hormone retaining hormonal activity, (c) a fragment of a natural component peptide hormone retaining hormonal activity, and (d) a hormone Fragments of analogs or derivatives of naturally occurring peptide hormones that retain activity, (e) desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding and / or other Structural motifs of naturally occurring peptide hormones that confer pharmacodynamic properties to the hybrid polypeptide, or (f) desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding, and Natural to confer hybrid pharmacodynamics to the hybrid polypeptide Minutes peptide may be a structural motifs of analogs or derivatives of the hormone. The structural motifs of (e) and (f) will be referred to collectively herein as "peptide enhancers".

Preferred bioactive peptide hormone modules include amylin, ADM, CT, CGRP, intermedin, CCK (1-33), CCK-8, leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3-36) ( SEQ ID NO: 58), GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (SEQ ID NO: 61), GLP-2, OXM, Exen Dean-3, Exendin-4, Sodium Excretion Peptide Hormones, Eurocortin Family Peptides such as Ucn-2 and Ucn-3, Neuromedin Family Peptides such as Neuromedin U25 or Splicing Variants and ANP Natural peptide hormones selected from BNP, CNP or urodilatin.

Other preferred bioactive peptide hormone modules include amylin, ADM, CT, CGRP, intermedin, CCK, leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3-36) (SEQ ID NO: 58), GLP-1 ( 1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (SEQ ID NO: 61), GLP-2, OXM, Exendin-3, and Exendin-4 , Sodium excretion peptide hormones, eurocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splicing variants and ANP, BNP, CNP or eurodilatin Analogs or derivatives of the component peptide hormones selected from, wherein the analogs or derivatives exhibit one or more hormonal activities of the component peptide hormones. The analog may comprise one or more insertions, deletions or substitutions of the amino acid sequence of the component peptide hormone, and the derivatives may be one or more of the amino acid residues of the analog or component peptide hormone as fully described herein and known in the art. And chemical modifications.

More specifically, analogs and derivatives may be selected from any of those described above and / or known in the art. Particularly preferred analogs and derivatives exhibiting at least one hormonal activity useful as the bioactive peptide hormone module of the present invention include:

As is known in the art, such peptide compounds are preferably amidated, but may optionally be in acid form in the context of the present invention unless otherwise specified.

Another preferred bioactive peptide hormone module is Amylin, ADM, CT, CGRP, Intermedin, CCK, Leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3-36) (SEQ ID NO: 58), GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (SEQ ID NO: 61), GLP-2, OXM, Sodium Excretion Peptide, Exendin- 3 and exendin-4, urocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splicing variants and ANP, BNP, CNP or eurodilatin A fragment of the component peptide hormone selected from wherein said fragment exhibits at least one hormonal activity of the component peptide hormone.

Another preferred bioactive peptide hormone module is Amylin, ADM, CT, CGRP, Intermedin, CCK, Leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3-36) (SEQ ID NO: 58), GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (SEQ ID NO: 61), GLP-2, OXM, ANP, BNP, CNP, Eurodil Latin, exendin-3, exendin-4, sodium excretion peptide hormones, eurocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splicing Variants and fragments of analogs or derivatives of component peptide hormones selected from ANP, BNP, CNP or urodilatin, wherein said fragments exhibit one or more hormonal activities of the component peptide hormones. Again, the analog may comprise one or more insertions, deletions or substitutions of the amino acid sequence of the component peptide hormone, and the derivatives may be one of the amino acid residues of the analog or component peptide hormone as fully described herein and known in the art. Or more than one chemical modification.

Specific preferred fragments exhibiting at least one hormonal activity include the following. However, it should be understood that combinations of fragments known in the art are also contemplated, including the analogs and derivatives described above and the preferred fragments described below.

Again, as is known in the art, such peptide compounds may preferably be amidated, but may optionally be in acid form in the context of the present invention unless otherwise specified. In addition, such preferred fragments may be combined with any analogues or derivatives discussed herein or known in the art. For example, preferred analog fragments are ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27) (SEQ ID NO: 241) , ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27) (SEQ ID NO: 241) or disclosed fragments, analogs and derivatives And any other combination of.

Another preferred bioactive peptide module provides a hybrid polypeptide with the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding and / or other pharmacodynamic properties. Peptide enhancers ", ie structural motifs of the component peptide hormones, including analogs and derivatives thereof. Exemplary peptide enhancers include the following. Again, it should be understood that combinations of the above analogs and derivatives with the following bioactive peptide modules are also contemplated. For example, the last six amino acid residues of the amylin family peptide hormone analogs and derivatives known in the art and / or described above are also contemplated as preferred bioactive peptide modules.

Peptide module selection considerations, Spacer  And coupler

Hybrid polypeptides of the invention generally comprise at least two bioactive peptide hormone modules, wherein at least one of the bioactive peptide hormone modules exhibits at least one hormone activity. A bioactive peptide hormone module exhibiting one or more hormonal activities may be located at the N-terminal end of the hybrid polypeptide, the C-terminal end of the hybrid polypeptide, or the hybrid polypeptide may comprise more than two bioactive peptide hormone modules. If included, it may be located in an internal portion of the hybrid polypeptide.

In certain embodiments, it may be desirable to position a bioactive peptide hormone module that exhibits at least one hormonal activity so that the C-terminal end of the bioactive peptide hormone module is amidated. Amidation of the C-terminal end of the bioactive peptide hormone module can be accomplished by placing the module at the C-terminal end of the hybrid polypeptide or by arranging the module in the C-terminal N-terminal orientation at the N-terminal end of the hybrid polypeptide. Can be achieved. In both of these arrangements, the C-terminal end of the bioactive peptide hormone module can be used for amidation. Specific component peptide hormones for which C-terminal amidation may be preferred include amylin family peptide hormones, CCK, PYY, hGLP-1 (7-36) (SEQ ID NO: 61), and hGLP-2. Specific component peptide hormones for which C-terminal amidation is not necessarily preferred (ie, extension at the C-terminal end of the module are readily acceptable) include exendin-4, exendin-4 (1-28) (SEQ ID NO: 237). ), GLP-1 (7-37) (SEQ ID NO: 204), frog GLP-1 (7-36) (SEQ ID NO: 283), frog GLP-2, and the like. However, when these component peptide hormones are located at the C-terminal end of the hybrid polypeptide, they may still be optionally amidated, and in fact it may be desirable to be optionally amidated.

The bioactive peptide hormone module can be covalently linked in any manner known in the art. Stable connections may be used, or cuttable connections may be used. In an embodiment, the carboxy of the first module can be directly linked to the amino of the second module. In another embodiment, the linker can be used in an attached module. In addition, if desired, spacers or turn inducers known in the art may be used to stabilize the connection. For example, if you do not want to amidate the C-terminal end of the N-terminal bioactive peptide hormone module, the module may be attached directly to the second module, or any suitable known in the art. Linking groups such as alkyl; PEG; Amino acids such as Lys, Glu, β-Ala; Polyamino acids such as poly-his, poly-arg, poly-lys, poly-ala, Gly-Lys-Arg (GKR) and the like; Bifunctional linkers (see, eg, Pierce catalog, Rockford, Ill.); Aminocaproyl (“Aca”), β-alanyl, 8-amino-3,6-dioxaoctanoyl, or other cleavable linkers and non-cleavable linkers known in the art may be attached. . Described specifically herein, and specifically expressed herein, are embodiments of certain hybrids in which the linker in each of the illustrated linker-containing hybrids is replaced with a Gly linker, in particular the Gly linker is Gly-Gly-Gly. For example, the exemplified species ²⁹ 5 Apa-exendin (1-28) ^-1 des-Lys-h amylin (1-7) ^-11,18 Arg- ^sCt (8-27) -h amylin (33 -37) (SEQ ID NO: 32) (see table herein), its Gly linker species analogues are also specifically intended and described. These species include ²⁹ GlyGlyGly-Exendin (1-28) ^-1 des-Lys-h ^Amylin (1-7) ^-11,18 Arg- ^sCt (8-27) -h ^Amylin (33-37) 313), wherein the three glycines are located after the exendin (1-28) sequence. In one embodiment the linker or spacer is from 1 to 30 residues in length, in another embodiment from 2 to 30 residues, in another embodiment from 3 to 30 residues in length and from 2 to 30 2, 3, 4, 5, 6, 7, and the like, for example, are considered to be arbitrary integer lengths belonging to each integer unit. In one embodiment the Gly linker is used, and in a particular embodiment the three residue linker Gly-Gly-Gly is used.

If amidation of the C-terminal end of a bioactive peptide hormone module located at the N-terminus is desired, the module can again be attached to the second module using any suitable linker known in the art. More specifically, preferred linking groups are those in which the bioactive peptide hormone module exhibiting at least one hormonal activity is arranged in the C-terminus to the N-terminus orientation to create an amino-amino linkage, such as dicarboxylic acid, alkyl, PEG and amino acid. , For example Lys, Cys and Glu.

As mentioned above, the hybrid polypeptide may preferably comprise a spacer to further stabilize the ligation of the bioactive peptide hormone module. Any spacer or turn inducer known in the art can be used. For example, β-turn mimetics include mimetic A and mimic B illustrated below, and also Ala-Aib and Ala-Pro dipeptides. In these IUPAC names, mimic A is N- (3S, 6S, 9S) -2-oxo-3-amino-1-azabicyclo [4.3.0] -nonane-9-carboxylic acid, mimic B Is N- (3S, 6S, 9R) -2-oxo-3-amino-7-thia-1-azabicyclo [4.3.0] -nonane-9-carboxylic acid.

Additional exemplary Combination  And certain embodiments

Exemplary combinations of bioactive peptide hormone modules for forming hybrid polypeptides of the invention include natural peptide hormones, analogs and derivatives of peptide hormones that exhibit one or more hormonal activities, fragments of natural peptide hormones that exhibit one or more hormonal activities. , A fragment of analogs and derivatives of peptide hormones exhibiting one or more hormonal activities, and a combination of two or more bioactive peptide hormone modules selected from peptide enhancers, wherein the one or more modules exhibit one or more hormonal activities. Indicates.

Hybrid polypeptides of the invention comprise two or more bioactive peptide hormone modules, wherein each module is selected from component peptide hormones. In the context of the present invention, component peptide hormones of a hybrid polypeptide may be the same or different, but two or more component peptide hormones are different. In a preferred embodiment, the two or more component peptide hormones are of different peptide hormone families, eg, amylin family, CCK, leptin family, PPF, proglucagon family, sodium excretion peptide family, eurocortin family peptide, for example Ucn-2 and Ucn-3, neuromedin family peptides, such as neuromedin U25 or splicing variants and ANP, BNP, CNP or urodilatin and GLP-1 and exendin families.

In certain embodiments, hybrid polypeptides of the invention may comprise two or more modules that exhibit one or more hormonal activities. For example, a hybrid polypeptide may comprise a fragment of a first peptide hormone or analog that exhibits at least one hormonal activity covalently linked to a fragment of one or more additional peptide hormone analogs. The additional fragment (s) may optionally exhibit one or more hormonal activities. The first peptide hormone may be the same as or different from the additional peptide hormone (s), but the at least one additional peptide hormone is different from the first peptide hormone and the first hormone activity is equal to any additional hormone activity. Can be different.

In other embodiments, a hybrid polypeptide of the present invention may comprise one or more modules that exhibit one or more hormonal activities in combination with one or more peptide enhancer modules. For example, a fragment of a first peptide hormone that exhibits one or more hormonal activities may be covalently linked to a peptide enhancer, or a fragment of the first peptide hormone that exhibits one or more hormonal activities may exhibit a second or more hormone activity. Covalently linked to a peptide hormone, which may be linked to a peptide enhancer. Alternatively, the peptide enhancer may be located between two peptide hormone modules as stabilizing spacers. Again, the first peptide hormone may be the same or different than the second peptide hormone, and the first hormonal activity may be the same or different from the second hormonal activity.

In another embodiment, the hybrid polypeptides of the present invention may comprise two, three, four or more bioactive peptide hormone modules. Exemplary combinations include one, two or three peptide enhancers and one module with hormonal activity, one or two peptide enhancers and two modules with hormonal activity, one peptide enhancer and three with hormonal activity. Modules and the like.

The component peptide hormone is preferably amylin, adrenomodulin, calcitonin, calcitonin gene related peptide, intermedin, cholecystokinin, leptin peptide YY, glucagon-like peptide-1, glucagon-like peptide-2, auxintomodulin, ANP, BNP, CNP, eurodilatin, sodium excretion peptide hormone, eurocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splicing variants and ANP, BNP, CNP or urodilatin or exendin-4.

More particularly, preferred modular combinations include those including component combinations of exendin, amylin (and / or sCT), BNP and PYY as component peptide hormones. Specific combinations include exendin-4 / PYY and PYY / exendin-4 combinations with or without spacers or linking groups. Other combinations include exendin / amylin and amylin / exendin combinations with or without spacers or linking groups. Still other combinations include amylin / PYY and PYY / amylin combinations with or without spacers or linking groups.

In one aspect, preferred module combinations comprise exendin-4, a fragment of exendin-4 that exhibits one or more hormonal activities, an exendin-4 analog or derivative that exhibits one or more hormonal activities, or one or more hormonal activities. And including a first module comprising a fragment of the exendin-4 analog that is shown, with one or more additional bioactive peptide hormone modules. In one embodiment, the first module is linked to one, two or three additional bioactive peptide hormone modules.

In a preferred embodiment, the first module comprising exendin-4 peptide is linked to a second bioactive peptide hormone module comprising an amylin (and / or sCT) peptide exhibiting at least one hormonal activity. In another embodiment, the second module is further linked to a third bioactive peptide hormone module comprising a calcitonin peptide exhibiting at least one hormone activity. In another embodiment, the third module can be further linked to a fourth bioactive peptide hormone module comprising a peptide enhancer selected from amylin peptides. In an embodiment, the first module can be located at the C-terminal end of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminal end of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as βAla, may be inserted if desired to be linked to the module.

Preferred exendin-4 peptides include exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 284) and ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240). Also useful are exendin (7-15) and its Ser2 analogue, HSEGTFTSD (SEQ ID NO: 378). Preferred amylin peptides exhibiting at least one hormonal activity include amylin, amylin fragments such as amylin (1-17) (SEQ ID NO: 214), amylin (1-16) (SEQ ID NO: 215), amylin (1- 15) (SEQ ID NO: 216) and Amylin (1-7) (SEQ ID NO: 217) and amylin analogues, such as frraminide, ² Ala-h-amylin (SEQ ID NO: 79), ^2,7 Ala-h-amy Milin (SEQ ID NO: 80), and fragments thereof. Preferred calcitonin peptides exhibiting at least one hormonal activity include sCT, sCT fragments such as sCT (8-10), sCT (8-27) (SEQ ID NO: 288) and calcitonin analogs such as ^11,18 Arg-sCT ( SEQ ID NO: 108), ¹⁸ Arg-sCT (SEQ ID NO: 107), ¹⁴ Glu, ¹⁸ Arg-sCT (SEQ ID NO: 109), ¹⁴ Glu, ^11,18 Arg-sCT (SEQ ID NO: 110) and fragments thereof. Preferred amylin peptide enhancers include amylin (32-37) (SEQ ID NO: 242), amylin (33-37) (SEQ ID NO: 243) and amylin (34-37) (SEQ ID NO: 244) and analogs thereof. Amylin-sCT combinations useful in connection with the present invention include those disclosed in PCT / US2005 / 004631 (Amylin Family Agonist, Agent Control No. 18528.835), which is incorporated herein by reference. Particularly useful amylin-sCT chimeras for the hybrid production of the invention are Compound 10 (described herein and in PCT / US2005 / 004631) and analogs and derivatives thereof.

In one aspect, preferred module combinations comprise exendin-4, a fragment of exendin-4 that exhibits one or more hormonal activities, an exendin-4 analog or derivative that exhibits one or more hormonal activities, or one or more hormonal activities. And including with a peptide enhancer a first module comprising a fragment of the exendin-4 analog that represents. Preferred exendin-4 compounds are exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 284) and ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240). Preferred peptide enhancers include PYY (25-36) (SEQ ID NO: 257), PYY (30-36) (SEQ ID NO: 262) and PYY (31-36) (SEQ ID NO: 263). 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-terminal end of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminal end of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal end of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as βAla, may be inserted if desired to be linked to the module.

In another aspect, preferred module combinations include exendin-4, fragments of exendin-4 that exhibit one or more hormonal activities, exendin-4 analogs or derivatives that exhibit one or more hormonal activities, or one or more hormonal activities. A first module comprising a fragment of an exendin-4 analog that represents a CCK, a fragment of CCK that exhibits one or more hormonal activities, a CCK analog or derivative that exhibits one or more hormonal activities, or a CCK that exhibits one or more hormonal activities Including with a second module comprising a fragment of the analog. Again, preferred exendin-4 compounds are exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe-Exendin -4 (1-28) (SEQ ID NO: 284), ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240) and ¹⁴ Leu-Exendin-4 (1-28) (SEQ ID NO: 190 ). Preferred CCK compounds include CCK-8 and CCK-8 (Phe (CH ₂ SO ₃ )). 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-terminal end of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal end of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as βAla, may be inserted if desired to be linked to the module.

In another aspect, preferred module combinations are amylin, a fragment of amylin that exhibits one or more hormonal activity, an amyloid analog or derivative that exhibits one or more hormonal activity, or a fragment of amylin analog that exhibits one or more hormonal activity. And including a first module comprising a peptide enhancer, eg, with a second module comprising PYY (25-36) (SEQ ID NO: 257) or PYY (30-36) (SEQ ID NO: 262). 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-terminal end of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminal end of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal end of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as βAla, may be inserted if desired to be linked to the module. In another aspect, preferred module combinations include amylin, fragments of amylin that exhibit one or more hormonal activities, amylin analogs or derivatives that exhibit one or more hormonal activities, or amylin analogs that exhibit one or more hormonal activities. Including a first module comprising a fragment with a peptide enhancer, eg, a second module comprising 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-terminal end of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminal end of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal end of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as βAla, may be inserted if desired to be linked to the module.

Other preferred modular combinations include those including exemplin and CCK or combinations of amylin, calcitonin and CCK as ternary combinations. Specific combinations include exendin / CCK and CCK / exendin with or without spacers or linkers and linkers. Other combinations include CCK / amylin / calcitonin and CCK / amylin / calcitonin / amylin with or without spacers or linking groups. Each module may independently be a peptide enhancer or exhibit hormonal activity, depending on the desired properties of the hybrid polypeptide. In one embodiment, for example as in compound 10 Amylin / calcitonin / amylin is provided as chimera of amylin / calcitonin / amylin.

Still other preferred module combinations include those including combinations of exendin, amylin and calcitonin as ternary and quaternary hybrid molecules. Exemplary combinations include exendin / amylin / calcitonin, exendin / amylin / calcitonin / amylin, amylin / calcitonin / exendin, and amylin / calcitonin / amylin / with or without spacers or linking groups. Exendin combinations. Each module may independently be a peptide enhancer or exhibit hormonal activity, depending on the desired properties of the hybrid polypeptide. In one embodiment, for example as in compound 10 Amylin / calcitonin / amylin is provided as chimera of amylin / calcitonin / amylin.

In one embodiment, a hybrid polypeptide is preferred if one of the bioactive peptide hormone module (s) exhibiting at least one hormonal activity is amylin or an analog or fragment thereof and the second bioactive peptide hormone module comprises CCK. Preferably a third bioactive peptide hormone module selected from different component peptide hormones. Exemplary third bioactive peptide hormone modules include calcitonin, more preferably salmon calcitonin, analogs or fragments thereof.

In another embodiment, if one of the bioactive peptide hormone module (s) exhibiting at least one hormonal activity is amylin or an analog or fragment thereof and the second bioactive peptide hormone module comprises CT, the hybrid polypeptide is It should preferably comprise a third bioactive peptide hormone module selected from different component peptide hormones. Exemplary third bioactive peptide hormone modules include exendin-4, analogs or fragments thereof.

In another embodiment, a peptide enhancer wherein one of the bioactive peptide hormone module (s) exhibiting at least one hormonal activity is GLP-1 or an analog or fragment thereof and the second bioactive peptide hormone module comprises an exendin fragment. In one case, the hybrid polypeptide should preferably comprise a third bioactive peptide hormone module. Exemplary third bioactive peptide hormone modules include PYY (including analogs, derivatives and fragments thereof) and CCK (including analogs, derivatives and fragments thereof).

With respect to each combination described herein, reference to component peptide hormones also refers to analogs, derivatives, fragments, and also peptide enhancers associated therewith. It should be understood to include.

In a preferred aspect, the hybrid polypeptide includes the following:

Exemplary exendin and neuromedin hybrids include the following:

Exendin- (1-28) -beta-Ala-beta-Ala-FN-38:

Exendin- (1-28) -beta-Ala-beta-Ala-neuromedine (U25 :)

Exendin- (1-28) -beta-Ala-beta-Ala-neuromedine (U-9):

The beta-Ala-beta-Ala spacers are optional and may be replaced with Gly-Gly-Gly, mini-PEG groups or other linkers known in the art, in particular those described herein.

Exemplary exendin and sodium excretion peptide hybrids include exendin-hBNP peptide hybrids, including:

Exendin- (1-28) -beta-Ala-beta-Ala-hBNP:

Exendin-beta-Ala-beta-Ala-hBNP:

As with all hybrids of the invention, the beta-Ala-beta-Ala spacers are optional and may be replaced with Gly-Gly-Gly, mini-PEG groups or other linkers known in the art, in particular those described herein. have.

Hybrid polypeptides of the present invention may also further comprise modifications to the amino acid sequences of such hybrid polypeptides, including but not limited to, for example, substitutions, deletions and insertions, and any combination thereof. In a preferred aspect, the hybrid polypeptides of the present invention comprise one or more modifications of "non-essential" amino acid residues. In the context of the present invention, a "non-essential" amino acid residue is altered within the natural human amino acid sequence of a fragment, eg, component peptide hormone fragment, without destroying or substantially reducing the component peptide hormone receptor agonist activity of the hybrid polypeptide. Residues which may be ie deleted or substituted.

Preferred substitutions include conservative amino acid substitutions. A "conservative amino acid substitution" is one in which amino acid residues are replaced with amino acid residues having similar side chains or physicochemical properties (eg, electrostatic, hydrogen bonding, isomeric, hydrophobic features). Family of amino acid residues with similar side chains are known in the art. These families include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, methionine , Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine) Amino acids having phenylalanine, tryptophan, histidine).

The present invention also relates to derivatives of the component peptide hormone. Such derivatives are conjugated to one or more water soluble polymer molecules, for example polyethylene glycol ("PEG") or fatty acid chains of various lengths (e.g. stearyl, palmitoyl, octanoyl, etc.) or polyamino acids such as poly hybrid polypeptides to which -his, poly-arg, poly-lys and poly-ala have been added. Modifications to hybrid polypeptides may also include small molecule substituents such as short chain alkyl and constrained alkyl (eg branched chain, cyclic, fused, adamantyl) and aromatic groups. The molecular weight of the water soluble polymer molecule will preferably range from about 500 to about 20,000 Daltons.

Such polymer-conjugation and small molecule substituent modifications may occur once at the N-terminus or C-terminus or side chain of an amino acid residue in the sequence of a hybrid polypeptide. Alternatively, there may be multiple sites of derivatization along the hybrid polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine may provide additional derivatization sites. See, for example, US Pat. No. 5,824,784 and US Pat. No. 5,824,778. Preferably, the hybrid polypeptide may be conjugated to one, two or three polymer molecules.

The water soluble polymer molecule may preferably be linked to amino, carboxyl or thiol groups and may be linked by the N-terminus or C-terminus or in the side chain of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, the water soluble polymer molecule may be linked to diamine and dicarboxyl groups. In a preferred embodiment, the hybrid polypeptides of the invention are conjugated to one, two or three PEG molecules via epsilon amino groups on lysine amino acids.

In addition, hybrid polypeptide derivatives of the invention include hybrid polypeptides having chemical alterations to one or more amino acid residues. Such chemical alterations include amidation, glycosylation, acylation, sulfated, phosphorylation, acetylation and cyclization. Chemical alterations may occur once at the N-terminus or C-terminus or side chain of an amino acid residue in the sequence of a PPF hybrid polypeptide. In one embodiment, the C-terminus of these peptides may have free -OH groups or -NH ₂ groups. In another embodiment, the N-terminal end isobutyloxycarbonyl group, isopropyloxycarbonyl group, n-butyloxycarbonyl group, ethoxycarbonyl group, isocaproyl group (isocap), octanyl group, octyl glycine group (G (Oct)) Or an 8-aminooctanoic acid group. In a preferred embodiment, cyclization can be carried out through the formation of disulfide bridges. Alternatively, there may be multiple sites chemically altered along the hybrid polypeptide.

In a further embodiment, the hybrid does not comprise any hybrid disclosed in WO 2005/077072. Thus, in one embodiment a novel hybrid is claimed. In another embodiment, as disclosed herein, claims novel uses of any hybrid disclosed herein or in WO 2005/077072 and the like. Examples of hybrid polypeptides of the invention are presented in the Sequence Listing and are further discussed in the Examples section below.

In the treatment or prevention of metabolic conditions or disorders hybrid  Use of Polypeptides

Hybrids of the present invention can reduce food intake, loss of appetite, reduction of calorie intake, induction of satiety, reduction of nutrient utilization, induction of weight loss, change of body composition, change of body energy content or energy consumption, improvement of lipid profile (eg For example, lowering LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels), slowing gastrointestinal motility, delaying gastric emptying, adjusting blood glucose excursion after meals, inhibiting or inhibiting glucagon secretion and reducing blood pressure. . In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

Thus, in certain embodiments, the hybrids of the present invention are useful for the treatment or prevention of a condition or disorder that can be alleviated by a decrease in nutrient utilization, comprising administering to a subject a therapeutic or prophylactically effective amount of a compound of the present invention. These conditions and disorders include any kind of diabetes, metabolic syndrome, dumping syndrome, hypertension, dyslipidemia, including eating disorders, insulin resistance, obesity, abnormal postprandial hyperglycemia, such as type I, type II and gestational diabetes, etc. , Cardiovascular disease, hyperlipidemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis, and osteoarthritis. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

Exemplary peptide module pairings include cardiac action / protective peptides such as eurocortin and GLP-1 or exendin, ANP, BNP or CNP and GLP-1 or exendin, and eurocortin and ANP, BNP or CNP It includes. Such hybrids are myocardial and are particularly useful for the related diseases and conditions described herein, including acute or chronic CHF, ischemic reperfusion, myocardial infarction, etc. useful. Ucn2 and Ucn3 are particularly useful for the hybrid of the present invention.

Non-limiting examples of cardiovascular conditions or diseases are hypertension, myocardial ischemia and myocardial reperfusion. The compounds of the present invention may also be useful in the treatment or prevention of other conditions associated with obesity, such as stroke, cancer (eg endometrial cancer, breast cancer, prostate cancer and colon cancer), gallbladder disease, sleep apnea, decreased fertility and osteoarthritis. (See Lyznicki et al, Am. Fam. Phys. 63: 2185, 2001). In other embodiments, the compounds of the present invention can be used to alter body composition for aesthetic reasons, to improve one's physical ability, or to make a leaner meat source. Hybrids are useful for changing body composition by reducing fat without significant loss of muscle mass, so that desirable body fat loss occurs while maintaining lean body weight. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

In another general aspect, the hybrid of the present invention can be used to inhibit secretion of ghrelin. Thus, the compounds of the present invention are in a mechanism for the treatment or prevention of ghrelin-related disorders, such as Prader-Willi syndrome, all types of diabetes and its complications, obesity, bulimia, hyperlipidemia, or other disorders associated with hypertrophy. Can be used. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

In another general aspect, it is now recognized that hybrids containing amylin and / or a portion of sCT may be useful for treating or preventing Barrett's esophagus, gastroesophageal reflux disease (GERD) and conditions associated therewith. These conditions include chest pain, chest pain associated with backflow into the mouth or lungs of gastric / intestinal contents, difficulty swallowing, coughing, intermittent wheezing and vocal cord inflammation (a condition associated with GERD), esophageal erosion, Esophageal ulcers, esophageal narrowing, Barrett metaplasia (normal esophageal epithelium has been replaced with abnormal epithelium), Barrett's adenocarcinoma, and lung aspiration. Such hybrids possess anti-secretory properties such as gastric acid inhibition, bile acid inhibition, and pancreatic enzyme inhibition. In addition, such hybrids may have a gastric protective effect, which makes them particularly useful for the treatment or prevention of Barrett's esophagus, and / or GERD and related or related conditions as described herein.

In another general aspect, the hybrid may be further useful for the treatment or prevention of pancreatitis, pancreatic carcinoma and gastritis, in particular for the treatment and prevention of pancreatitis in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP). Hybrid agonists containing amylin and / or sCT can have surprisingly good therapeutic effects when combined with somatostatin. Thus, in certain embodiments, a method of treating or preventing pancreatitis comprises administering such a hybrid to a subject and administering a somatostatin and a somatostatin agonist.

In another general aspect, hybrids are useful for the treatment of bone disorders such as decreased bone resorption, reduced plasma calcium, and induction of analgesic effects, especially osteopenia and osteoporosis. In other embodiments, the hybrids are useful for the treatment of pain and painful neuropathy. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

In another aspect of the invention, there is provided a method of treating or preventing obesity, comprising administering a therapeutically or prophylactically effective amount of a hybrid polypeptide to a subject in need thereof. In a preferred embodiment, the subject to be administered is obese or overweight. “Obesity” is generally defined as a body mass index of greater than 30 for purposes herein, but any subject, including those with a body mass index of less than 30, that needs or wants to lose weight is included in the range of “obesity”. Such hybrids may be beneficial to subjects with insulin resistance, glucose intolerance or any form of diabetes mellitus (eg, type 1, type 2 or gestational diabetes). In one embodiment, such hybrids contain exendin, PYY, GLP1, amylin and / or sCT moieties.

In another embodiment, the body weight of a morbidly obese subject is first reduced by reducing the body weight of the morbidly obese subject to a level below the weight of the morbidly obese, then administering to the subject an effective amount of an anti-obesity combination that further reduces the weight of the subject. It provides a method to reduce. Methods for reducing the subject's weight below pathological obesity include reducing calorie intake, increasing physical exertion, drug therapy, obesity surgery, such as gastric bypass surgery, or any combination of these methods. In one aspect, administration of the anti-obesity agent combination further reduces the subject's weight. In another embodiment, a method of reducing body mass index in a subject having a body mass index of 40 or less is administered by administering an effective amount of an anti-obesity combination that further reduces body weight of a subject having a body mass index of 40 or less.

Reducing weight means that the subject loses some of its total body weight over the course of the treatment, whether it is days, weeks, months or years. Alternatively, weight loss can be defined as a reduction in the ratio of fat mass to lean mass (ie, in the subject, fat mass is lost but lean mass is maintained or increased so that a corresponding loss in total body weight is not necessarily achieved). Not entailed). The effective amount of anti-obesity agent administered in combination in this embodiment is an amount effective to reduce the subject's weight over the course of the treatment, or alternatively an amount effective to reduce the percentage of fat mass of the subject over the course of the treatment. In certain embodiments, the subject's weight is reduced by at least about 1%, at least about 5%, at least about 10%, at least about 15% or at least about 20% over the course of the treatment. Alternatively, the percent fat mass of the subject is reduced by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% over the course of the treatment.

In another aspect of the invention, reducing food intake, reducing nutrient utilization, inducing weight loss, changing body composition and altering body energy content or energy, comprising administering to a subject an effective amount of a hybrid polypeptide of the invention Methods are provided for increasing consumption, treating diabetes mellitus, and improving lipid profiles (including decreasing LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels). In a preferred embodiment, the method of the present invention comprises administering to a subject in need thereof a therapeutic or prophylactically effective amount of a hybrid polypeptide of the invention in a subject in need of treatment or prevention of a condition or disorder that can be alleviated by reducing nutrient utilization. It is used to treat or prevent a condition or disorder that can be alleviated by reducing nutrient utilization. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and any type of diabetes mellitus. In one embodiment, such hybrids contain exendin, PYY, GLP1, amylin and / or sCT moieties.

Without wishing to be bound by theory, the effects of the peripherally administered hybrid polypeptides of the present invention on reducing food intake, delaying gastric emptying, decreasing nutrient utilization and weight loss are those belonging to or similar to the PP family. It is believed to be determined by the interaction with the class. More particularly, it is believed that similar receptor (s) are related to the PYY-preferred (or Y7) receptor.

Of particular interest as anti-obesity, weight loss, food loss, increased metabolic rate, and body fat reduction and / or fat redistribution hybrid are those having one or more, preferably two or more, components that act on the CNS. It has been shown that certain areas of the brain (from the brain and hepatic brain) and the brain or brain stem (including the midbrain, the brain and the medulla) are involved in energy balance control. Examples of hypothalamus nucleus or forebrain structure involved in food intake and / or weight control include arch nucleus (ARC), chamber nucleus (PVN), hypothalamus ventral medialosis (DMH), peritoneal nucleus (VMH), and hypothalamus Lateral nucleus (LHA) and the like. Examples of the nucleus or posterior brain structures of the brainstem involved in food intake and / or weight control include the fast nucleus (NST), the lowermost zone (AP), and the lateral brachial nucleus (lPBN). It is believed that the brainstem nucleus that controls the elements of the consummatory movement control system is controlled by primary or secondary protrusions from the brainstem region, such as NST, AP, and lPBN. It is noteworthy that AP, NST and lPBN have all been found to possess inherent integrated capabilities (both together and independently).

Various anti-obesity agents targeting the CNS act on these forebrain structures present in the hypothalamus involved in food intake and / or weight control. In addition, anti-obesity agents targeting the CNS act on the posterior brain structures present in the brain stem involved in food intake and / or weight control. Examples of such anti-obesity agents are described herein. See the following table for further examples of peptide family modules that can be combined to form an anti-obesity hybrid and can be combined to form an anti-obesity hybrid having activity in both the front and back brain. Examples of such components include neuropeptide Y1 (NPY1) receptor antagonists, NPY5 receptor antagonists, leptin and leptin agonists, ciliary neurotrophic factor (CNTF) and CNTF agonists, peptide YY (PYY) and PYY agonists, exendin and exen Dean agonists, GLP-1 and GLP-1 agonists, ghrelin and ghrelin antagonists, cholecystokinin (CCK) and CCK agonists, and amylin and amylin agonists such as those described herein and the like. Additional peptide family components and clinical guidance can be found in our co-pending patent application PCT / US06 / 17529, which is incorporated herein by reference.

Individual Anti-Obesity Targets and Locations Signaling system CNS  domain On food intake Role term- Obesity Neuropeptide Y (NPY) Whole brain (ARC / PVN) Increased intake NPY1 and NPY5 receptor antagonists Leptin Whole brain (ARC) Reduced intake Leptin, or agonist Ciliary Neurotrophic Factor (CNTF) Whole brain (ARC) Reduced intake CNTF Peptide YY (PYY) Whole brain (ARC) Reduced intake PYY (3-36) agonists Glucagon-like peptide-1 (GLP-1) Whole brain (PVN) Reduced intake Exenatide and other GLP-1 ligands, DPP-IV inhibitors Ghrelin Whole brain (ARC) Increased intake Ghrelin antagonist Cholecystokinin (CCK) Hindbrain (AP) Reduced intake CCK agonists Amylin Hindbrain (AP) Reduced intake Amylin agonists, frramintides, amylin analogs Melanocortin (MC) Whole brain (PVN / ARC) Agonists Reduce Intake MC4 agonists

In certain embodiments, the hybrid is an anti-obesity agent that may include one or more peptide family components that primarily act on the whole brain. In other embodiments, the hybrid is an anti-obesity agent that may include one or more peptide family components that primarily act on the back brain. Exemplary peptide families and components include NPY1 receptor antagonists, NPY5 receptor antagonists, leptin or leptin agonists or analogs, CNTF, NPY2 receptor agonists (eg, PYY (3-36) or PYY (3-36) agonists), Exendin or exendin agonists or analogs, GLP-1 or GLP-1 agonists or analogs, ghrelin antagonists, CCK or CCK agonists or analogs, and amylin or amylin agonists or analogs.

In certain embodiments, hybrids and methods of use thereof primarily comprise a first component that targets the hypothalamic energy balance center, such as ARC, PVN, VM, LH, and the like. In one embodiment, the hybrid contains one or more other peptide family components that also target the hypothalamus but function at different locations or through different mechanisms of action than the first component. If the hybrid contains more than one other peptide family component and they also target the hypothalamus, the more than one other peptide family component may target the same position through the same mechanism of action, or different positions And / or target different mechanisms of action. In another embodiment, the hybrid in this case is one or more of the desired components, including anti-obesity effects, control of blood glucose, control of myocardial protection, and / or control of hypertension through different and different positions or mechanisms of action from the first component. It contains one or more other peptide family components that provide additional beneficial therapeutic effects. In certain embodiments, additional peptide family components are primarily targeted to the center of energy balance of the posterior brain, such as NST, AP, lPBN, and the like.

In certain embodiments, the hybrid and methods of use thereof primarily comprise a first component that targets the center of energy balance of the posterior brain, such as NST, AP, lPBN, and the like. In one embodiment, the hybrid also contains one or more other peptide family components that target the hypothalamus but which are different from the first component and that function at different positions or through different mechanisms of action. In another embodiment, the hybrid in this case is one or more of the desired components, including anti-obesity effects, control of blood glucose, control of myocardial protection, and / or control of hypertension through different and different positions or mechanisms of action from the first component. It contains one or more other peptide family components that provide additional beneficial therapeutic effects. In certain embodiments, additional peptide family components are primarily targeted to the hypothalamic energy balance center, such as ARC, PVN, VM, and LH.

As used herein, an anti-obesity agent "acting on the forebrain structure involved in food intake and / or weight control" stimulates or inhibits the activity of certain regions of the brain, such as certain nuclei and / or neural circuits. do. This whole brain stimulation or inhibition causes a decrease in the utilization of nutrients into the body. Anti-obesity agents "acting on the posterior brain structures involved in food intake and / or weight control" stimulate or inhibit the activity of certain regions of the posterior brain, such as specific nuclei and / or neural circuits. Such posterior brain stimulation or inhibition results in a decrease in the utilization of nutrients into the body.

In another aspect, a method of reducing fat mass by increasing metabolic rate in a subject is provided, the method comprising administering an anti-obesity hybrid in an amount effective to increase the metabolic rate of the subject to reduce fat mass. Fat mass can be expressed as a percentage of total body mass. In some aspects, the fat mass is reduced by at least 1%, at least 5%, at least 10%, at least 15%, 20% or 25% over the course of the treatment. In one aspect, the lean mass of the subject does not decrease over the course of the treatment. In another aspect, the lean mass of the subject is maintained or increased over the course of the treatment. In another aspect, the subject has a low calorie or limited diet. "Low calorie" means that the subject's daily calorie intake is lower than that of the same subject's normal meal. In one example, the subject consumes at least 50 calories less calories per day. In another example, the subject consumes at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 calories less calories per day.

In one embodiment, methods are provided for altering fat distribution, reducing fat mass, or both in a subject. Thus, the method of the present invention may be beneficial to a subject who would benefit from altering body composition. As intended herein, altered body composition includes losing or maintaining body fat along with minimizing, maintaining or increasing loss of lean body weight. In this situation, body weight may increase or decrease. Thus, the subject may be lean, overweight or obese (as these terms are commonly used in the art). In addition, the methods provided herein can also include reducing fat and reducing utilization of lean body mass in non-fat tissue. Use of the method includes the treatment of diseases such as non-alcoholic steatohepatitis (NASH) or lipodystrophy and the like.

In one embodiment, a method of altering fat distribution in a subject is provided, the method comprising administering an effective amount of an altered fat distribution in the subject. In one aspect, the alteration is due to increased metabolism of visceral fat or ectopic fat, or both in the subject. "Fat distribution" means the location of fat deposits in the body. Examples of such fat deposit locations include subcutaneous, visceral and ectopic fat stores. "Subcutaneous fat" means a lipid deposit just below the skin surface. The subcutaneous fat amount of a subject can be measured using any method available for subcutaneous fat measurement. Subcutaneous fat determination methods are known in the art and are described, for example, in US Pat. No. 6,530,886, which is incorporated herein by reference in its entirety. "Ectopic fat store" means a lipid deposit that is present in and around tissues and organs and constitutes lean body weight (eg skeletal muscle, heart, liver, pancreas, kidneys, blood vessels). In general, ectopic fat storage is the accumulation of lipids outside the normal adipose tissue stores in the body. "Visceral fat" means fatty deposits as abdominal adipose tissue. Visceral fat surrounds life support organs and can be metabolized by the liver to produce blood cholesterol. Visceral fat has been associated with increased risk of conditions such as polycystic ovary syndrome, metabolic syndrome and cardiovascular disease. In some embodiments, the method comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% higher percentage of visceral or ectopic fat or both compared to subcutaneous fat. It involves metabolizing. In one aspect, an advantageous fat distribution is obtained with this method. In one embodiment, the advantageous fat distribution is an increase in the ratio of subcutaneous fat to visceral fat, ectopic fat, or both. In one aspect, the method involves lean body weight gain, such as, for example, increased muscle cell mass.

In another embodiment, a method for reducing subcutaneous fat in a subject is provided, the method comprising administering to the subject in need thereof a subcutaneous fat reduction effective amount of an anti-obesity hybrid. In one example, the subcutaneous fat amount of the subject is reduced by at least about 5%. In another example, the subcutaneous fat mass is reduced by at least about 10%, 15%, 20%, 25%, 30% 40% or 50% compared to prior to administering the anti-obesity hybrid to the subject.

The methods described herein can be used to reduce the amount of visceral fat in a subject. In one example, the subject's visceral fat is reduced by at least about 5%. In another example, the subject's visceral fat is reduced by at least about 10%, 15%, 20%, 25%, 30% 40% or 50% compared to prior to administering the anti-obesity hybrid to the subject. Visceral fat can be measured by any means available for measuring the amount of visceral fat in a subject. Examples of such methods include CT scanning and abdominal tomography using MRI. Other methods of measuring visceral fat are described, for example, in US Pat. Nos. 6,864,415, 6,850,797 and 6,487,445.

In one embodiment, a method of preventing the accumulation of ectopic fat or reducing the amount of ectopic fat in a subject is provided, said method comprising the step of ectopic fat in a subject in need thereof to prevent accumulation of ectopic fat or reduce the amount of ectopic fat in the subject. Administering an anti-accumulative effective amount or an ectopic fat reduction effective amount. In one example, the ectopic fat amount of the subject is reduced by at least about 5% compared to prior to administering the anti-obesity hybrid to the subject. In another example, the ectopic fat amount of the subject is reduced by at least about 10% or at least about 15%, 20%, 25%, 30% 40% or 50%. Alternatively, the ectopic fat mass is proportionally 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% when compared to the subcutaneous fat of the subject. Or 100% reduction. Ectopic fat in a subject can be measured using any method available for ectopic fat determination.

In another embodiment, a method of achieving a more favorable fat distribution in a subject is provided, the method comprising administering to the subject an effective amount of a hybrid that achieves an effective fat distribution. In one embodiment, administration of the anti-obesity hybrid reduces the amount of visceral fat or ectopic fat or both in a subject. In one embodiment, an anti-obesity hybrid comprising at least one family module that acts on the forebrain structure involved in food intake or weight control or both, is a hindbrain involved in food intake or weight control or both. It is administered in combination with one or more family modules that act on the structure. In one embodiment, the method preferentially reduces visceral fat amount or ectopic fat amount or a combination of both compared to the reduction of subcutaneous fat. In this way, the ratio of subcutaneous fat to visceral or ectopic fat is higher. Such improved proportions may reduce the risk of developing cardiovascular disease, polycystic ovary syndrome, metabolic syndrome, or any combination thereof. In one embodiment, ectopic or visceral fat is metabolized at a 5% higher rate than subcutaneous fat. In other embodiments, the ectopic or visceral fat is at least 10% 15%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90% or 100% higher than subcutaneous fat. Is metabolized.

Of particular interest in the treatment of anti-obesity, weight and fat composition as discussed herein include amylin (eg amylin-sCT-amylin chimera), leptin and / or PPF (eg PYY analogues or PPY / NPY chimeric) family module. For example, the amylin family module may be attached to the leptin family module, the amylin family module may be administered alone, or in further embodiments in combination administration with the PPF family compound (eg, separately or mixed together). May be administered). In another embodiment, the hybrid contains a leptin-PPF combination administered alone or in combination with an amylin family compound. In another embodiment, the hybrid contains an amylin-PPF combination administered alone or in combination with a leptin family compound. In further embodiments, the hybrid contains all three peptide family modules. For example, the amylin-PPF hybrid may be provided in a pharmaceutically acceptable sterile solution which is then used to dissolve the lyophilized or powdered leptin family compound in a dual chamber delivery system or the like.

In another aspect, provided is a method of administering a therapeutically effective amount of a hybrid effective as an anti-obesity agent, administered in combination with a glucocorticosteroid. Glucocorticosteroids have the side effects of increasing fat mass and decreasing lean body mass. Thus, it is contemplated that anti-obesity combinations may be used with glucocorticosteroids under conditions where glucocorticosteroid use is beneficial so that the side effects of glucocorticosteroids are offset.

In addition, with respect to lipid lowering, the hybrids of the present invention provide lower lipid levels and provide more beneficial lipid profiles in subjects in need of treatment of lowering blood lipid levels such as triglycerides, total cholesterol, LDL cholesterol and VLDL cholesterol. Used. Thus, in one embodiment, triglycerides in blood, total cholesterol, LDL, comprising administering a lipid-lowering hybrid to a subject in need of lowering blood triglycerides, total cholesterol, LDL cholesterol, VLDL cholesterol, or any combination thereof. Methods of lowering cholesterol, VLDL cholesterol, or any combination thereof are provided. In further embodiments, the lipid-lowering hybrid may contain an exendin (incretin) family component, an amylin family component, a PPF or PYY (NPY) family component, or any combination thereof. In one embodiment of the method, the lipid to be degraded is plasma triglycerides. In another embodiment, this is total plasma cholesterol. In another embodiment, this is LDL cholesterol. In another embodiment, this is VLDL cholesterol. Hybrids may be effective in the short and / or long term in maintaining or reducing fasting lipid levels and / or reducing postprandial lipid (particularly triglyceride) fluctuations. Patients in need of such treatment may have elevated triglycerides, elevated LDL cholesterol, elevated VLDL cholesterol, or any combination thereof. Such patients may include subjects who have a lipid disease or condition, such as diabetes or prediabetes, obesity, dyslipidemia, hypercholesterolemia or hypertriglyceridemia, and / or cardiovascular disease and may otherwise be considered normal. This effect of the hybrid is beneficial in lowering the risk of heart and atherosclerosis in patients with high risk of heart and atherosclerotic, for example, patients with genetic predisposition, obesity, or diabetes. Thus, the patient may be a person suffering from atherosclerosis with elevated lipid or cholesterol levels. The methods of the present invention provide for reducing postprandial triglyceride fluctuations, reducing circulating lipid levels, treating dyslipidemia, improving circulating lipid profile, treating hypertriglyceridemia, treating hypercholesterolemia, and / or reducing postprandial triglyceride concentrations in a subject. A method is provided, comprising administering an effective amount of a hybrid of the invention to a subject in need of such treatment. As used herein, “lipid profile” means the balance, proportion or actual concentration of circulating lipids, including triglyceride levels, HDL, LDL, cholesterol, and the like. In a preferred aspect, the methods of the present invention comprising administering an effective amount of amylin or amylin agonist are useful for lowering triglyceride levels in a patient. In non-amylinic agonist treatment conditions as measured by overall lipid or triglyceride levels, such as fasting levels, postprandial peak levels, and overall postprandial lipid / triglyceride level fluctuations (eg, area under the curve (AUC) of postprandial triglyceride increase) Can be reduced in this way. Individual clinically relevant measures such as fasting lipid levels (including triglycerides, cholesterol, HDL and LDL, etc.) and postprandial lipids (eg triglycerides) are also reduced by the methods of the present invention. Patients with dyslipidemia or altered lipid levels relative to normal can be treated with amylin or amylin agonist administration. Patients with genetic predisposition to dyslipidemia or cardiovascular disease, as well as patients with diabetes and obesity, are particularly suitable for treatment by the methods of the present invention. As used herein, “treatment of elevated triglyceride levels in a patient” means preventing or causing a decrease at this level relative to the level prior to treatment. As used herein, "reduced postprandial triglyceride fluctuation" means that both the highest concentration in the curve and the area under the total curve in the curve for triglyceride concentrations seen in postprandial patients are reduced. This typically means a decrease in the area under the overall curve for the graph, such as the one provided for postprandial times in FIGS. As used herein, “reduction of circulating lipid levels” in a patient means that the measurable amount of lipids in the blood is lower than the pretreatment level. As used herein, “treatment of dyslipidemia” improves to a level, ratio, profile, or balance that is closer to what is medically defined as any or all normal and / or healthy lipid or lipoprotein parameters that are clinically measurable. It means to recover or to recover. This includes, but is not limited to, triglycerides, LDL, HDL, IDL, VLDL, total cholesterol, levels of apolipoproteins, and the like. As used herein, "improvement of circulating lipid profile" in a patient means causing a change in the concentration of one or more lipids in the blood such that the patient's overall blood lipid content changes to a desired state. It may also include shifting the lipid distribution across the various lipoprotein fractions without changing the overall content / concentration of circulating lipids. As used herein, "treatment of hypertriglyceridemia" in a patient means to cause a decrease in the concentration of triglycerides found in the blood of the patient at one or more related times, for example, on an empty stomach or after a meal. As used herein, “reduction of postprandial circulating triglycerides” in a patient is determined after meals (eg for a period of about 4 to 6 hours postprandial) prior to treatment for meals with similar measurable amounts of triglycerides in the circulation. It is impaired relative to the level of these lipids observed postprandial in untreated patients.

The hybrid and PYY chimeras of the invention can be used with other lipid-lowering drugs. Lipid-lowering drugs include any compound capable of reducing plasma lipid levels. Exemplary lipid-lowering drugs include statins such as atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin or rosuvastatin; Bile acid binders such as cholestyramine or cholestipol; Peroxide proliferative activating receptor (PPAR) agonists such as tesaglitazar, vitamin E; Cholesterol ester transport protein (CETP) inhibitors such as but not limited to ezetimibe, JTT-705 and torcetrapib.

Additional assays useful in the present invention include those capable of determining the effects of hybrid compounds on body composition. Exemplary assays may involve the use of a diet-induced obesity (DIO) mouse model for metabolic disease. Prior to the treatment period, male C57BL / 6J mice can be fed a high fat diet (# D12331, 58% calories due to fat; Research Diets, Inc.) for 6 weeks starting at 4 weeks of age. . During the study, mice can continue to eat high fat foods. Water can be provided arbitrarily throughout the study. To compare metabolic parameters with the DIO group, a group of similar weekly non-obese mice can be fed a low fat diet (# D12329, 11% calories from fat).

Vehicles (50% dimethylsulfoxide (DMSO) in water) or subcutaneous (SC) intrascapular osmotic pumps delivering the compounds of the invention can be implanted in DIO mice. The pump for delivering a compound of the present invention may be set to deliver any amount of compound of the present invention, for example 1000 μg / kg / day, for 7 to 28 days.

Body weight and food intake can be measured at regular intervals throughout the study. Respiratory rate (defined as RQ, CO ₂ production ÷ O ₂ consumption) and metabolic rate are measured using indirect calorimetry (Oxymax, Columbus Instruments, Columbus, Ohio) throughout the animal Can be. Mice can be euthanized by isofluran overdose and the obesity index (both epididymal fat pad weight) can be measured. In addition, prior to measurement of the epididymal weight, the body composition (fat mass, fat mass) for each mouse was determined according to the manufacturer's instructions (Lunar Piximus, GE Imaging System). (Dual Energy X-ray Absorptiometry, DEXA). In the methods of the invention, the preferred hybrid polypeptides of the invention are the effects of the component peptide hormones in the same assay in one of the assays described herein (preferably food intake, gastric emptying, pancreatic secretion, weight loss or body composition assays). It is more effective than that.

In addition to reducing food intake, weight loss, or reducing hypertension due to obesity treatment in subjects in need of alleviation of hypertension, the compounds of the present invention may be used to treat hypotension.

The compounds of the present invention may also be useful for enhancing, inducing, enhancing or restoring glucose reactivity in pancreatic islets or cells. This action may be useful for the treatment or prevention of metabolic disorders, such as those associated with those described above and in US Patent Application US20040228846. Assays to determine this activity are known in the art. For example, published US patent application US20040228846, which is incorporated by reference in its entirety, describes assays for islet isolation and culture and determination of fetal islet maturation. Examples of patent application US20040228846 include intestinal-derived hormones including pancreatic polypeptide (PP), neuropeptide Y (NPY), neuropeptide K (NPK), PYY, secretin, glucagon-like peptide-1 (GLP-1) and bombesin Peptides were purchased from Sigma. Collagenase type XI was obtained from Sigma. RPMI-1640 culture medium and fetal bovine serum were obtained from Gibco. Radioimmunoassay kits containing anti-insulin antibodies ([ ¹²⁵ I] -RIA kits) were purchased from Linco (St. Louis).

Postpartum rat islands were obtained from P-02 age rats. Adult rat islands were obtained from 6 to 8 week old rats. Fetal rat islets were obtained as follows. Pregnant female rats were sacrificed on day 21 of pregnancy. The fetus was removed from the uterus. Ten to fourteen pancreas from each fetus were dissected and washed twice in Hanks buffer. The pancreas was collected and suspended in 6 mL of 1 mg / mL collagenase (type XI, Sigma) and incubated at 37 ° C. for 8-10 minutes with constant shaking. Digestion was stopped by the addition of a 10-fold volume of ice cold Hank buffer and washed three times with Hank buffer. The islets were then purified with a Ficoll gradient and cultured in 10% fetal bovine serum (FBS) / RPMI medium with or without 1 μM IBMX. At the end of 5 days, 20 islets were picked up by hand and placed in each tube and assayed for static insulin release. In general, the islands were first washed with KRP buffer and then incubated at 37 ° C. for 30 minutes with 1 mL KRP buffer containing 3 mM (low concentration) glucose with constant shaking. After harvesting the supernatant, the islands were incubated with 17 mM (high concentration) glucose for 1 hour at 37 ° C. Insulin released due to high or low glucose stimulation was assayed by radioimmunoassay (RIA) using the [ ¹²⁵ I] -RIA kit. E21 fetal islets were cultured for 5 days in the presence of 200 ng / mL PYY, PP, CCK, NPK, NPY, Secretin, GLP-1 or Bombesin.

In addition, an example was used using ZDF (Zucker Diabetic Fatty) male rats, a model of co-breeding (> F30 generation) rats that spontaneously develop diabetes in all fa / fa males fed the standard rodent feed Purina 5008. In vivo assays are provided. In ZDF fa-fa males, hyperglycemia began to develop at about 7 weeks of age, and glucose levels (fed) typically reached 500 mg / DL by 10-11 weeks of age. Insulin levels (fed) were high during diabetes development. However, at 19 weeks of age, insulin dropped to levels of nearly lean control male pups. Triglyceride and cholesterol levels in obese rats are typically higher than in lean rats. In the assay, 7 weeks old ZDF rats of 3 groups (6 rats per group) were treated with ALZA pump for 14 days each 1) vehicle control, 2) PYY at 100 pmol / kg / hour and 3) 500 PYY (ie two different doses) at pmol / kg / hour was infused. Four measurements of pre-infusion and post-infusion 7 and 14 were obtained: 1) plasma glucose levels, 2) plasma insulin levels, and 3) plasma triglyceride (TG) levels, and oral glucose tolerance (OGTT) test values. Thus, compounds of the invention can be used in these assays to test for desired activity.

Other uses contemplated for hybrid polypeptides include methods for reducing the aluminum (Al) concentration in the central nervous system for the treatment, prevention or delaying onset of Alzheimer's disease (see US Pat. No. 6,734,166, which is incorporated by reference in its entirety). Incorporated herein by reference). Assays to determine the effect on Al are known in the art and can be found in US Pat. No. 6,734,166 using diploid and Ts mice. Put the mouse individually naljin (Nalgene) ^® brand metabolism or polypropylene us, it gave three days of time to adjust to us before the experiment. Mice had free access to food (LabDiet ^® NIH rats and Mouse / Auto 6F5K52, St. Louis, Missouri, USA) and water during the experiment, provided they were fed for 16 hours before euthanasia. Did not give. Mice were injected subcutaneously with active compound or saline daily. Mice were sacrificed and samples collected at the end of Day 13 in one experiment and at the end of Day 3 in another experiment. Mouse brain samples were weighed with clean Teflon liners and prepared for analysis by microwave digestion in low trace element grade nitric acid. 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)). During the analysis all tissues were handled in a clean room environment using a HEPA air filtration system to minimize background contamination. The hybrid of the present invention is useful for the prevention and treatment of nephropathy, including hypertensive and diabetic nephropathy, and nephropathy associated with insulin resistance and metabolic syndrome. Hybrids achieve this goal, among others, by improving or preventing exacerbations of hypertension, endothelial function, renal function and glomerulosclerosis. In one embodiment, the present invention provides a method of preventing or treating nephropathy, including administering a compound of the present invention, including nephropathy associated with hypertensive and diabetic nephropathy or insulin resistance, and the like. Hybrids are also used to improve endothelial function in patients with reduced vasodilation performance, glomerulosclerosis, or any other decrease in glomerular flow rate. This improvement in endothelial function serves to reduce hypertension and also to improve the capillary function of glomeruli. In a further embodiment, the molecules of the present invention are useful for preventing the progression of nephropathy to ESRD, for preventing or delaying, treating or alleviating proteinuria and / or glomerulosclerosis. Hybrids are useful for reducing the risk of developing cardiac arrhythmias, or for preventing or treating cardiac arrhythmias. Hybrids can provide anti-arrhythmic effects in patients with cardiac ischemia, cardiac ischemia-reperfusion, and congestive heart failure. For example, GLP-1 has been shown to reduce heart damage and improve recovery of patients with these disorders. Incretin, including GLP-1, is a glucose-dependent insulin stimulating hormone. GLP-1 and exendin effectively enhance peripheral glucose uptake without inducing dangerous hypoglycemia. They also strongly inhibit glucagon secretion, independent of their insulin stimulatory action, substantially reducing plasma free fatty acid (FFA) levels significantly more than insulin can perform. High FFA levels are associated as the major toxic mechanism during myocardial ischemia. In another embodiment, hybrids are useful for preventing and treating cardiac arrhythmias by reliably reducing damage associated with reperfusion and ischemia and improving patient recovery. In a further embodiment, hybrid treatment, preferably intravenous administration after acute stroke or bleeding, optimizes insulin secretion, increases brain assimilation, inhibits glucagon and risks of normal blood sugar or severe hypoglycemia or other harmful side effects. It provides a means to improve insulin effect by maintaining minor hypoglycemia. In one embodiment, such hybrids contain a portion of GLP1 or exendin. In a further embodiment, the GLP1 or exendin family module, as described herein, can be used to increase sodium excretion family peptide, amylin family peptide, eurocortin family peptide to achieve improved treatment or prevention of cardiovascular conditions or diseases, including CHF. Combined with the module.

Congestive heart failure is one of the most significant causes of morbidity and mortality in developed countries. This occurs as a late symptom in various cardiovascular diseases, characterized by loss of contractility and / or overload of volume or pressure [Fortuno, Hypertension 38: 1406-1412 (2001)]. Numerous studies have suggested that cardiac remodeling is a major determinant of the clinical progression of CHF and has nothing to do with its etiology [Fedak, Cardiovascular Pathology 14: 1-11 (2005)]. Thus, cardiac remodeling is an attractive target in the treatment of congestive heart failure. As such, there is a need for agents that function to prevent or reduce cardiac remodeling. In fact, the literature describes the need for molecules that can attenuate cardiac remodeling (Fortuno, Hypertension 38: 1406-1412 (2001)). Reports in the literature indicate that attenuating ventricular remodeling improves survival after myocardial seizures, but treatments that worsen remodeling are associated with worse outcomes even if they improve systolic function (Somasundaram, Med. Clin. N. Am., 88: 1193-1207 (2004).

Thus, herein, the treatment of cardiovascular disease, in one embodiment the treatment of acute or chronic heart failure, in another embodiment the treatment of myocardial infarction, in another embodiment the treatment of ischemic heart failure, and in another embodiment the treatment of congestive heart failure. Provide a method of treatment. In one embodiment, such treatment is provided by the prevention or alleviation of hyperglycemia-induced damage to the cardiovascular system. In one embodiment, such treatment is provided by providing a myocardial protective effect. In another embodiment, such treatment is achieved by the prevention, delayed onset, attenuation or alleviation of cardiac remodeling. In general, cardiac remodeling refers to the restructuring and reshaping of any cardiac chamber of the heart. In one embodiment, cardiac remodeling refers to restructuring and reshaping of the ventricles. As described above, but also without wishing to be bound by theory, cardiac remodeling may be described as a genome change after a myocardial attack, followed by molecular, cellular and interstitial changes, resulting in restructuring and reshaping of the ventricles. have. Such restructuring and reshaping can be expressed clinically as changes in the size, shape, and function of the heart. Cardiac remodeling can occur in response to any stimulus or combination of stimuli to the myocardium. In one embodiment, cardiac remodeling is the result of myocardial attack. As a non-limiting example, cardiac remodeling may occur in response to myocardial infarction due to myocardial infarction, hypertension, volume overload (eg due to aortic reflux), infection, inflammation, diabetes, viral cardiomyopathy, and idiopathic cardiomyopathy. Can be.

In one aspect, cardiac remodeling may be prevented, delayed, attenuated or alleviated by the hybrid administration of the present invention. The hybrid has a diastolic function of the left ventricle, the ratio of E wave to A wave, the diastolic pressure of the left ventricle, cardiac output, cardiac contractility, the mass of the left ventricle, the mass of the left ventricle, the ratio of the mass of the left ventricle, the left ventricular volume, the left atrium volume, Ability to relieve (improve) one or more of the cardiac parameters of any measurement of pulp or systolic dimensions, magnitude of infarction, athletic performance, athletic efficiency or cardiac systolic and / or diastolic function or hypertrophy of the ventricles or cardiac parameters described above It may include the ability to attenuate, delay, or prevent a deleterious effect on either. In one embodiment, the hybrid contains a member of the incretin family that binds to GLP-1 or exendin receptor, for example exendin-4. In the method of the present invention, preventing or alleviating cardiac remodeling may comprise reducing cardiac remodeling by any amount. In one embodiment, the prevention or alleviation of cardiac remodeling is performed with a reduced risk of congestive heart failure.

In one embodiment, the cardiac remodeling comprises about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, of the amount of cardiac remodeling when no hybrid is administered. Reduced or reduced by 80% or 90% less. In another embodiment, cardiac remodeling may be slightly reduced, moderately reduced, substantially reduced, or substantially eliminated as compared to occurrence of cardiac remodeling when no hybrid is administered. As used herein, a slight reduction in cardiac remodeling refers to a cardiac remodeling that is reduced by about 25% or less as compared to cardiac remodeling when no hybrid is administered. Moderate reductions in cardiac remodeling refer to cardiac remodeling that has been reduced by about 50% or less as compared to cardiac remodeling when no hybrids are administered. Substantial reduction in cardiac remodeling refers to cardiac remodeling that is reduced by about 80% or less as compared to cardiac remodeling when no hybrids are administered. Substantial elimination of cardiac remodeling refers to cardiac remodeling that has been reduced by at least about 80% as compared to cardiac remodeling when no hybrids are administered.

In order to assess the extent to which cardiac remodeling is prevented, alleviated, attenuated or delayed, any means available to those skilled in the art can be used. For example, cardiac remodeling may be performed to include, but is not limited to, histological examination of the heart, amount of LV, or analysis while the subject is alive, or for example ventricular dimensions and wall thickness and Mobility can be measured or the left ventricular (LV) dilator function can be quantified and assessed using the peak velocity ratio (E / A ratio) of the E and A waves.

In an embodiment, a subject that would benefit from hybridization, preventing, alleviating, attenuating, or delaying cardiac remodeling can be identified by one of skill in the art in terms of conditions and risk factors associated with the subject. In one embodiment, the subject may need to be prevented or alleviated or attenuated or delayed cardiac remodeling. In another embodiment, the subject may wish to have cardiac remodeling prevented, alleviated, attenuated or delayed. One of the risk factors may be a genetic predisposition to heart remodeling occurring in the heart. Exemplary samples and subjects of the methods of the invention provided herein include samples and subjects who have experienced, have experienced, or are at risk of experiencing a condition associated with cardiac remodeling. The condition associated with cardiac remodeling may be any condition or disorder in which cardiac remodeling is known to occur or is believed to be at risk. Examples of conditions associated with cardiac remodeling include myocardial infarction, inflammation, ischemia / reperfusion, oxidative stress, pulmonary heart, advanced glycation endproduct, abnormal wall tension, sympathetic stimulation, myocarditis, hypertension, Viral cardiomyopathy, idiopathic cardiomyopathy, heart transplantation, and cardiac surgery.

As mentioned above, the hybrid may be administered as a result of an acute event or chronic condition. Whether acute or chronic, the methods provided herein include long term treatment of hybrids. Thus, the length of the long term treatment period may include the time that the event has passed and the subject is considered to have recovered from an acute event or recovered from a chronic condition.

Prolonged administration or treatment of the hybrid for the prevention, attenuation, delay or alleviation of cardiac remodeling can be done when it is confirmed that there are no particular transient events or transient conditions associated with cardiac remodeling. Long-term administration may be based on a general predisposition to cardiac remodeling or on a predisposition of a non-temporal state (eg, a non-temporary state may be unidentified or not eliminated, eg diabetes). Hybridization over continuous but vague time. The hybrid may be administered for a long time to prevent cardiac remodeling in a subject exhibiting congestive heart failure regardless of the etiology in the methods provided herein. Long-term administration of hybrids for the prevention or alleviation of cardiac remodeling may also be associated with diabetes at risk of congestive heart failure. Hybrids may be administered for a long time to preserve the transplanted organs in a subject who has had a heart transplant. If the hybrid is administered for a long time, such administration can last for any time. However, long term administration often occurs for a longer time. For example, in exemplary embodiments, long term administration lasts 6 months, 1 year, 2 years or more.

In another embodiment, the methods disclosed herein result in an improvement in cardiac contractility. Improvements in cardiac contractility may include contractility of cardiac myocytes. In order to evaluate the improvement of cardiac contractility, any evaluation scheme can be used. For example, an increase in cardiac contractility may be determined from clinical observation, such as an increase in cardiac output or a decrease in heart rate, or both. Alternatively, in vivo, the increased contractile force of the heart can be assessed by determining the increased fractional shortening of the left ventricle. The segmental shortening rate of the left ventricle can be observed by any available means, such as echocardiography. In assessing increased cardiac contractility, the increase in compartment shortening of the left ventricle may be any amount increase as compared to compartment shortening before hybrid administration. For example, the increase in shortening rate can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or about 200 May be greater than%.

In another aspect, a method of reducing or preventing atrial remodeling by administering a hybrid is provided. Reduction or prevention of atrial remodeling can be assessed compared to atrial remodeling prior to hybrid administration. The therapeutic effect of this reduction or prevention in atrial remodeling includes a reduction in atrial fibrillation. In another aspect, a method of reducing or preventing ventricular remodeling by administration of a hybrid is provided. Reduction or prevention of ventricular remodeling can be assessed in comparison to ventricular remodeling prior to hybrid administration.

In a further aspect, methods of prevention and treatment are provided. Short or long term treatment is considered. In addition, acute treatment can be extended to long-term treatment as directed. Long term treatment is considered to be greater than two weeks. In certain embodiments, long term treatment may be one month, three months, six months, one year, two years, five years, or more, or throughout life. In one aspect, provided herein is a method of treating or preventing a condition associated with cardiac remodeling in a subject in need of treatment or prevention of a condition associated with cardiac remodeling. Generally, the method comprises administering to the subject a prophylactic or alleviating effective amount of a hybrid of cardiac remodeling, thereby improving, preventing or delaying a condition associated with the remodeling. As described herein, hybrid administration can be performed in any manner, including in combination with other agents that provide cardiovascular benefits.

In another embodiment, the methods provided herein further comprise the identification of the subject in need of treatment. Any effective criterion may be used to determine whether it may be beneficial to administer the hybrid to a subject. For example, methods for diagnosing heart disease and / or diabetes and also procedures for identifying individuals at risk of developing this condition are known to those skilled in the art. Such procedures may include clinical trials, physical examinations, personal interviews, and assessment of family history.

In further embodiments, hybrids capable of lowering insulin resistance or increasing insulin sensitivity are useful for the treatment of polycystic ovary syndrome (PCOS). Hybrid administration of the present invention can reduce or prevent insulin resistance in a subject suffering from PCOS. In another embodiment, the hybrid prevents the development of type 2 diabetes in a subject suffering from PCOS. In addition, hybrids can restore periodic menstruation, ovulation, or fertility in subjects with PCOS. In one embodiment, such hybrids contain a portion of GLP1 or exendin for binding and activation of the GLP1 receptor.

The compounds of the present invention exhibit a wide range of biological activities, some of which are associated with anti-secretory and anti-migrating properties. The compound may inhibit gastrointestinal secretion by direct interaction with epithelial cells or perhaps by inhibiting the secretion of hormones or neurotransmitters that stimulate intestinal secretion. Anti-secretory properties include inhibition of gastric and / or pancreatic secretion and may be useful for the treatment or prevention of diseases and disorders, including gastritis, pancreatitis, Barrett's esophagus, and gastroesophageal reflux disease and the like.

Compounds of the present invention are described in any number of gastrointestinal disorders associated with excess enteric electrolytes and reduced water secretion and absorption (see, for example, Harrison's Principles of Internal Medicine, McGraw-Hill Inco, New York, 12th Ed.). ), For example, infectious diarrhea, inflammatory diarrhea, short bowel syndrome, or diarrhea that typically occurs after surgical operations such as ileostomy. Examples of infectious diarrhea include acute viral diarrhea, acute bacterial diarrhea (eg, Salmonella, campylobacter , and diarrhea due to clostridium or protozoan infection), or traveler's diarrhea (eg, Norwalk). (Norwalk) virus or rotavirus), but is not limited thereto. Examples of inflammatory diarrhea include, but are not limited to, malabsorption syndrome, tropical sprue, chronic pancreatitis, Crohn's disease, diarrhea, and irritable bowel syndrome. It has also been found that the peptides of the present invention can be used, for example, in the treatment of emergency or fatal conditions involving post-operative or gastrointestinal disorders by cholera.

In addition, the compounds of the present invention may be useful not only for the treatment of symptoms (eg diarrhea) associated with intestinal damage but also for the treatment or prevention of intestinal damage. Such intestinal damage may be, or may be a result of ulcerative colitis, inflammatory bowel disorders, intestinal atrophy, intestinal mucosal function, and / or loss of intestinal mucosa function (see WO 03/105763, which is incorporated herein in its entirety). Included for reference). Assays for this activity as described in WO 03/105763 were applied to the 12:12 light: cancer cycle and were free to eat standard rodent food (Teklad LM 485, Madison, Wisconsin) and water. And 250 to 300 g of 11 week old male HSD rats. The animals were starved for 24 hours before the experiment. A simple and reproducible rat model of chronic colitis is described in Morris GP, et al., "Hapten-induced model of chronic inflammation and ulceration in the rat colon." Gastroenterology. 1989; 96: 795-803. This model represents a relatively long duration of inflammation and ulcer formation and offers the opportunity to study the pathophysiology of colon inflammatory diseases in a specially controlled manner and to evaluate new therapies potentially applicable to inflammatory bowel disease in humans.

Rats were anesthetized with 3% isofluorane and placed in an adjustable heating pad set at 37 ° C. A Gavaz needle was inserted 7 cm through the rectum into the colon. See Mazelin, et al., Juton Nerv Syst. 1998; 73: 38-45, with a total volume of 0.4-0.6 mL of hapten trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol (v / v) at a dose of 30 mg / kg via Gabbaz's needle As delivered into the lumen of the colon. Saline solution (NaCl 0.9%) was administered intracolon to the control group.

Four days after inducing colitis, the rats were euthanized after cutting the colon from anesthetized rats. The weight of the resected colon and spleen was measured and photographed of the colon to score overall morphological damage. Inflammation has been defined as the region of hyperemia and barrier thickening.

Hybrid polypeptides of the invention may also be used to treat or prevent pancreatic tumors (eg, to inhibit the proliferation of pancreatic tumors). The methods of the invention include reducing the proliferation of tumor cells. Types of benign pancreatic tumor cells that can be treated according to the present invention include serous cysts, microcystic tumors, and solid cystic tumors. The methods of the present invention are also effective in reducing the proliferation of malignant pancreatic tumor cells, such as carcinomas arising from the ducts, glands or islets of the pancreas. US Pat. No. 5,574,010, which is incorporated by reference in its entirety, provides an exemplary assay for testing antiproliferative properties. For example, the '010 patent discloses two human pancreas available from PANC-1 and MiaPaCa-2 from a supplier, for example, the American Type Culture Collection (ATCC, Rockville, MD). Adenocarcinoma cancer cell line. The two tumor cells described above were 10% fetal bovine serum, 29.2 mg / L glutamine, 25 μg gentamicin, 5 mL penicillin, streptomycin, in a 37% 5% C0 ₂ incubator jacketed with NAPCO water. And RPMI-1640 culture medium supplemented with fungizone solution (JRH Biosciences, Renex, Kansas, USA). All cell lines were detached with 0.25% trypsin (Clonetics, San Diego, Calif.) Once or twice weekly when tumor growth monolayers of tumor cells were obtained. Cells were pelleted at 500 g for 7 minutes in a 4 ° C. refrigerated centrifuge and resuspended in trypsin-free enriched RPMI-1640 culture medium. Surviving cells were counted on hemocytometer slides using trypan blue.

Each type of 10,000, 20,000, 40,000 and 80,000 cells was added to a 96 well microculture plate (Costar, Cambridge, Mass.) In a total volume of 200 μl of culture medium per well. Cells were allowed to attach for 24 hours prior to addition of PYY or test peptide. Replace with fresh culture medium before peptide addition. In vitro incubation of pancreatic tumor cells with PYY or test compound was continued for 6 and 36 hours. PYY was added to the cells at doses of 250 pmol, 25 pmol and 2.5 pmol per well (N = 14). Test compounds were added to the cell culture at doses of 400 pmol, 40 pmol and 4 pmol per well. To the control wells, 2 μl of 0.9% saline was added to mimic the volume and physical invasion of the attached tumor cells. Each 96 well plate contained 18 comparative control wells in each plate during the experiment. 96 well plates were repeated six times with varying concentrations of PYY and test compounds in both PANC-1 and MiaPaCa-2 cells.

At the end of the incubation period, 3- (4,5-dimethylthiazolyl-2-yl) -2,5-diphenyltetrazolium bromide, MTr tetrazolium bromide (Sigma, St. Louis, MO) was added to fresh culture medium. Add at 0.5 mg / mL. The culture medium was replaced and tumor cells were incubated at 37 ° C. with MTT tetrazolium bromide for 4 hours. At the end of incubation, the culture medium was aspirated. Formazone crystalline precipitate was dissolved in 200 μl of dimethyl sulfoxide (Sigma, St. Louis, MO). Quantification of solubilized formazone was performed by obtaining absorption readings at 500 nm wavelength on an ELISA reader (Molecular Devices, Menlo Park, Calif.). The MTT assay measures mitochondrial NADH dependent dehydrogenase activity, which is one of the most sensitive and reliable methods for quantitative in vitro chemotherapy response of tumor cells (Alley, MC, 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). Analysis of absorption readings at 550 nm was analyzed by dividing wells of the same test conditions into groups and demonstrating the differences occurring between control and various peptide concentration treatments by one-way ANOVA.

Exemplary in vivo assays are also provided. Human pancreatic ductal adenocarcinoma Mia Paca-2 was investigated for in vivo growth inhibition by peptide YY and test compounds. 70,000 to 100,000 human Mia PaCa-2 cells were orthotopically transplanted into 48 athymic male mice. After 1 week, animals were treated with PYY or test compound for 4 weeks through a mini-osmotic pump at 200 pmol / kg / hour. Paired cultures were administered saline. Mice were sacrificed to measure both tumor size and mass. Control mice had significant human cancer growth in the pancreas, as evidenced by tissue sections. At 9 weeks, 90% of the control mice had substantial metastatic disease. Tumor mass was reduced by 60.5% in test compound treated mice and 27% in PYY treated mice.

Hybrids also provide therapeutic and prophylactic treatment for neurological and neurological disorders associated with nerve loss or dysfunction, including but not limited to Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, stroke, ADD and neuropsychiatric syndromes. It is also useful for treating and improving or facilitating learning, memory and cognition in mammals. Particularly useful in this regard are hybrids containing an exendin or GLP1 active moiety, more specifically a moiety comprising at least N-terminal 7-15 amino acids or an analog thereof, for example HSEGTFTSD (SEQ ID NO: 378).

For all indications, in a preferred embodiment, the hybrid polypeptides of the invention are in a single or divided dose or controlled continuous release at a dosage of about 0.5 μg to about 5 mg / day, from about 0.01 μg / kg per dose Peripheral administration of about 500 μg / kg, more preferably about 0.05 μg / kg to about 250 μg / kg, most preferably less than about 50 μg / kg. Doses in this range will of course depend on the potency of each analog or derivative and can be determined by one skilled in the art.

In the method of the present invention, the hybrid polypeptide of the present invention is an amylin or amylin analog agonist, salmon calcitonin, cholecystokinin (CCK) or CCK agonist, leptin (OB protein), which exhibits long-term or short-term action of reducing nutrient utilization. Or separately or in combination with one or more other compounds and compositions, including but not limited to, leptin agonists, exendin or exendin analog agonists, or other compounds and compositions comprising GLP-1 or GLP-1 analog agonists May be administered. Suitable amylin agonists are, for example, [ ^25,28,29 Pro-] human amylin (SEQ ID NO: 67) (also known as "pramlinetide" and described in US Pat. Nos. 5,686,511 and 5,998,367). It includes. The CCK used is preferably CCK octapeptide (CCK-8), more preferably its sulfated form. 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.

As discussed herein, a hybrid of the present invention may be administered separately or in combination with one or more other agents to obtain additional benefits or enhance the effectiveness of the hybrid or other agents. For example, an anti-obesity hybrid may be administered in combination with an anti-obesity agent or myocardial protectant or anti-hypertensive agent depending on the risk factors involved in the subject in need of treatment and the desired therapeutic outcome. Exemplary anti-obesity agents for combination administration with the hybrid (separately or in combination, before administration of the hybrid, concurrent with or after administration of the hybrid) include paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline, and Serotonin (5HT) transport inhibitors, including but not limited to imipramine and the like. Anti-obesity agents are also Dex penpeulruramin, fluoxetine, sibutramine (e.g. Meridia (MERIDIA) ^®), and United States Patent No. 6,365,633 and PCT Patent Application Publication No. WO 01/27060 described the call and copper. WO 01/162341 No. And selective serotonin reuptake inhibitors, including but not limited to, which is incorporated herein by reference in its entirety. Such 5HT transport inhibitors and serotonin reuptake inhibitors, analogs, derivatives, drugs, formulations, pharmaceutical compositions, dosages and routes of administration have already been described.

Anti-obesity agents are also selective serotonin agonists, and US Patent Nos. 3,914,250 and PCT Application Publications WO 02/36596, WO 02/48124, WO 02/10169, WO 01 / 66548, WO 02/44152, WO 02/51844, WO 02/40456 and WO 02/40457, all of which are incorporated herein by reference in their entirety. But also includes optional 5-HT2C receptor agonists as described in the literature, but not limited to these. Such selective serotonin agonists and 5-HT2C receptor agonists, compositions containing such agonists, and routes of administration suitable for use in the methods provided herein are known in the art. See, eg, Halford et al. (2005) Curr. Drug Targets 6: 201-213 and Weintraub et al. (1984) Arch. Intern. Med. 144: 1143-1148.

Anti-obesity agents also include antagonists / reverse agonists of central cannabinoid receptors (CB-1 receptors), including, but not limited to, limonabant (Sanofi Synthelabo) and SR-147778 (Sanofi Synthelabo). Include. CB-1 antagonists / reverse agonists, derivatives, drugs, formulations, pharmaceutical compositions, dosages and routes of administration are described, for example, in US Pat. Nos. 6,344,474, 6,028,084, 5,747,524, 5,596,106, and 5,532,237, 4,973,587, 5,013,837, 5,081,122, 5,112,820, 5,292,736, 5,624,941, European Patent Application EP-656 354 and EP-658546, And PCT application publications WO 96/33159, WO 98/33765, WO 98/43636, WO 98/43635, WO 01/09120, WO 98/31227 WO 98/41519, WO 98/37061, WO 00/10967, WO 00/10968, WO 97/29079, WO 99/02499, WO 01/58869 and WO 02/076949, the disclosures of which are incorporated herein by reference in their entirety.

Anti-obesity agents also include melanocortin and melanocortin agonists. Receptor MC4R is believed to play a role in energy balance and obesity. See, eg, Anderson et al., Expert Opin. Ther. Patents 11: 1583-1592 (2001), Speake et al., Expert Opin. Ther. Patents 12: 1631-1638 (2002), [ Bednarek et al., Expert Opin. Ther. Patents 14: 327-336 (2004). Melanocortin agonists, including but not limited to MC4R agonists and the like, and compositions containing such agonists suitable for use in the methods provided herein are known in the art. MCR agonists, MC4R agonists, derivatives, medicaments, formulations, pharmaceutical compositions, dosages, and routes of administration have already been described, for example, in the following PCT patent applications, which are incorporated by reference in their entirety: WO 03/007949, WO 02/068388, WO 02/068387, WO 02/067869, WO 03/040117, WO 03/066587, WO 03/068738, WO 03/094918 and WO 03/031410.

Anti-obesity agents are also 2-methyl-6- (phenylethynyl) -pyridine (MPEP) and (3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine) (MTEP And others (Anderson et al. J. Eur. J. Pharmacol. 473: 35-40 (2003)), Cosford et al. Bioorg. Med. Chem. Lett. 13 (3): 351- 4 (2003) and Anderson et al. J. Pharmacol. Exp. Ther. 303: 1044-1051 (2002)), including, but not limited to, metabotropic glutamate subtype 5 receptors (mGluR5) antagonists also include.

Anti-obesity agents also include topiramate (TOPIMAX ^® (Ortho McNeil Pharmaceuticals), known as anti-convulsant but shown to increase weight loss. Lipase inhibitors such as these may also be included.

Anti-obesity agents also include neuropeptide Y1 (NPY1) antagonists and NPY5 antagonists. NPY1 and NPY5 antagonists are known in the art. See, eg, Duhault et al. (2000) Can. J Physiol. Pharm. 78: 173-185 and US Pat. No. 6,124,331, 6,214,853 and 6,340,683. NPY1 and NPY5 antagonists, derivatives, drugs, formulations, pharmaceutical compositions, dosages and routes of administration have already been described. NPY1 antagonists useful in the compositions and methods provided herein include US Pat. No. 6,001,836, and PCT Application Publication Nos. WO 96/14307, WO 01/23387, WO 99/51600, WO 01 / 85690, WO 01/85098, WO 01/85173 and WO 01/89528, which are incorporated by reference in their entirety. NPY5 antagonists useful in the compositions and methods of use provided herein include U.S. Patent Nos. 6,140,354, 6,191,160, 6,258,837, 6,313,298, 6,337,332, 6,329,395, 6,340,683, 6,326,375 and 6,335,345, EP-01010691 and EP-01044970, and PCT Patent Publication WO 97/19682, WO 97/20820, WO 97/20821 WO 97/20822, WO 97/20823, WO 98/27063, WO 00/64880, WO 00/68197, WO 00/69849, WO 01/09120, WO 01/85714, WO 01/85730, WO 01/07409, WO 01/02379, WO 01/02379, WO WO 01/23388, WO 01/23389, WO 01/44201, WO 01/62737, WO 01/62738, WO 01/09120, WO 02 / 22592, WO 02/48152, WO 02/49648, and WO 01/14376, and the like, but are not limited thereto. Don't.

Anti-obesity agents are also melanin-concentrating hormone (MCH) including melanin-concentrating hormone 1 receptor (MCH1R) antagonists such as T-226296 (Takeda) and melanin-concentrating hormone 2 receptor (MCH2R) antagonists and the like. Include antagonists. MCH receptor antagonists, derivatives, drugs, formulations, pharmaceutical compositions, dosages and routes of administration are described, for example, in US Patent Application Publication Nos. 2005/0009815, 2005/0026915, 2004/0152742, 2004/01/2004. 0209865, PCT Patent Application Publication WO 01/82925, WO 01/87834, WO 02/06245, WO 02/04433 and WO 02/51809 and Japanese Patent Application JP 13226269, et al., Which are incorporated by reference in their entirety.

Anti-obesity agents also include opioid antagonists, including but not limited to those described in PCT Application WO 00/21509. Useful specific opioid antagonist in the compositions and methods of use provided herein, nalmefene (Le Bex (REVEX) ^®), 3- methoxy-naltrexone naloxone, naltrexone, nalrok hydrogen or binary, beta-punal agent Rex amine, delta 1 ([D-Ala2, Leu5, Cys6] -enkephalin (DALCE), naltrindol isothiocyanate, and nor-vinaltorphamine, and the like.

Anti-obesity agents also include orexin antagonists, including but not limited to those described in PCT Patent Application WO 01/96302, WO 01/68609, WO 02/51232, and WO 02/51838. Also includes. Specific orexin antagonists useful in the compositions and methods of use provided herein include, but are not limited to, SB-334867-A and the like.

Anti-obesity agents are also PYY3-36 (see, eg, Batterham et al. (2003) Nature 418: 650-654), NPY3-36 and other Y2 agonists such as N-acetyl [Leu (28, 31)] NPY 24-36 [White-Smith et al. (1999) Neuropeptides 33: 526-533, TASP-V [Malis et al. (1999) Br. J. Pharmacol. 126: 989-996], cyclo- (28/32) -Ac- [Lys28-Glu32]-(25-36) -pNPY [Cabrele et al. (2000) J. Pept. Sci. 6: 97-122, including but not limited to neuropeptide Y2 (NPY2) agonists, which may be hybrid components as discussed, or may be administered separately. Anti-obesity agents provided herein also provide for pancreatic peptide (PP) (eg, Batterham et al. (2003) J. Clin. Endocrinol. Metab. 88: 3989-3992) and other Y4 agonists, eg For example 1229U91 [Raposinho et al. (2000) neuroendocrinology 71: 2-7, including but not limited to neuropeptide Y4 (NPY4) agonists. NPY2 agonists and NPY4 agonists, derivatives, medicaments, formulations, pharmaceutical compositions, dosages and routes of administration have already been described, for example, in US Patent Publication No. 2002/0141985 and PCT Application Publication No. WO 2005/077094.

Anti-obesity agents are also described in PCT Application WO 02/15905, O- [3- (1H-imidazol-4-yl) propanol] carbamate [Kiec-Kononowicz et al. (2000) Pharmazie 55: 349-355], piperidine-containing histamine H3-receptor antagonists [Lazewska et al. (2001) Pharmazie 56: 927-932], benzophenone derivatives and related compounds [Sasse et al. (2001) Arch. Pharm. (Weinheim) 334: 45-52, substituted N-phenylcarbamate [Reidemeister et al. (2000) Pharmazie 55: 83-86] and proxy plate derivatives [Sasse et al. (2000) J. Med. Chem. 43: 3335-3343, including, but not limited to, histamine 3 (H3) antagonists / reverse agonists. Specific H3 antagonists / reverse agonists useful in the compositions and methods of use provided herein include thioperamide, 3- (1H-imidazol-4-yl) propyl N- (4-pentenyl) carbamate, clobenpro Pits, iodophenprop, imoproxyfan, and GT2394 (Gliatech) and the like.

Anti-obesity agents also include cholecystokinin (CCK) and CCK agonists. Cholecystokinin-A (CCK-A) agonists used include, but are not limited to, those described in US Pat. No. 5,739,106. Specific CCK-A agonists include, but are not limited to, AR-R 15849, GI 181771, JMV-180, A-71378, A-71623, and SR146131.

Anti-obesity agents also include ghrelin antagonists such as those described in PCT Application Publication Nos. WO 01/87335 and WO 02/08250. Ghrelin antagonists are also known as GHS (growth hormone secretagogue receptor) antagonists. Thus, the compositions and methods provided herein contemplate the use of GHS antagonists instead of ghrelin antagonists.

Anti-obesity agents include ovestatin and ovestatin analogs and agonists. Obesstatin is a peptide derived from preprogrelin, the same precursor as ghrelin. See, eg, Zhang et al. (2005) Science 310: 996-999, Nogueiras et al. (2005) Science 310: 985-986, Pan et al. (2006) Peptides 27: 911- 916). In contrast to the activity of ghrelin, ovestatin is believed to act as an appetite depleting hormone through reduced food intake, gastric fasting activity, factory exercise, and weight gain. Obesstatin peptides used are described in Zhang et al. (2005) Science 310: 996-999, and the like.

In addition, amiminomimetics such as frraminide, amylin-sCT-amylin (compound 10), incretins such as exendin-4, and PYY analogs are anti-obesity agents administered in combination with hybrids It is an anti-obesity agent that may be administered. For example, leptin-compound 10 hybrid can be administered in combination with exendin-4, a PYY analog or both. In another embodiment, the leptin-PYY analog hybrid can be administered in combination with exendin-4, amilinomimetic, or both.

Of particular interest in embodiments of treating diabetes and related diseases as discussed herein are hybrids comprising exendin family and alpha-MSH modules, and exendin and amylin family members. Of particular interest is the exendin is an exendin-4 or an analog or derivative thereof and the amylin component is frraminide or amylin-sCT-amylin chimeric hybrid.

Of particular interest in embodiments that treat obesity and related diseases and conditions (reduce body fat) as discussed herein are the exendin family and alpha-MSH modules, exendin and amylin family members, amylin family members and PYY. Family members, amylin family members and CCK family members, amylin family members and alpha-MSH family members, FN-38 family members, amylin family members and PYY family members, another identical or different PYY family member than the PYY family member It is a hybrid that includes PPY family members, CCK family members, PYY family members, FN-38 family members, CCK family members, and FN-38 family members. As described herein, of particular interest is the exendin is exendin-4 or an analog or derivative thereof, the amylin component is frraminide or amylin-sCT-amylin chimera, and the FN38 family member is FN38 or Analogs or derivatives thereof, wherein the PYY chimera is a PYY-NPY chimera such as SEQ ID NO: 266, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 442, SEQ ID NO: 462, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, and US Pat. Hybrid of SEQ ID NO: 472 and for example PYY-NPY chimeric 5705.

Polypeptide Generation and Purification

Hybrid polypeptides described herein can be prepared using, for example, chemical peptide synthesis techniques or standard recombinant techniques known in the art using automated or semi-automated peptide synthesizers or both.

Hybrid polypeptides of the invention can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols. See, eg, Stewart and Young, Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co. (1984), Tam et 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 is described by NMP / HOBt (Option 1) system with capping and tBoc or Fmoc chemistry (Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B July 1, 1988, section 6, pp. 49-70). (See Applied Biosystems Inc., Foster City, CA) using an automated peptide synthesizer (e.g., Model 430A, Applied Biosystems Inc., Foster City, CA, USA). Can be done. Peptides can also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, KY). Peptides are for example RP- using Waters Delta Prep 3000 system and C4, C8 or C18 preparative columns (10 μ, 2.2 × 25 cm; Vydac, Hesperia, CA, USA). Purification by HPLC (preparative and analytical). Active peptides can be easily synthesized and then screened in screening assays designed to identify reactive peptides.

Hybrid polypeptides of the invention may alternatively be produced by recombinant techniques known in the art. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor (1989). Hybrid polypeptides produced by such recombinant techniques can be expressed from polynucleotides. Those skilled in the art will appreciate that polynucleotides, including DNA and RNA, which encode various fragments of the hybrid polypeptide, may be obtained from wild-type cDNA in consideration of the degeneracy of codon usage, or may be genetically engineered if desired. Such polynucleotide sequences may incorporate codons that facilitate transcription and translation of mRNA in microbial hosts. Such preparation sequences can be readily constructed according to methods known in the art. See for example WO 83/04053. The polynucleotide may also optionally encode an N-terminal methionyl residue. Non-peptide compounds useful in the present invention can be prepared by methods known in the art. For example, phosphate-containing amino acids and peptides containing such amino acids can be prepared using methods known in the art. See, eg, Bartlett and Landen, Bioorg Chem. 14: 356-77 (1986).

Various expression vectors / host systems can be used to contain and express hybrid polypeptide coding sequences. These include 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 (eg baculovirus); Plant cell systems transfected with virus expression vectors (eg Cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg Ti or pBR322 plasmid); Or animal cell systems. Mammalian cells useful for producing recombinant proteins include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g. COS-7), WI 38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells, including but not limited to. Exemplary protocols for recombinant expression of proteins are described herein.

As such, the polynucleotide sequences provided by the present invention include novel and useful viral and plasmid DNA vectors, novel and useful transformed and transfected prokaryotic and eukaryotic host cells (including cultured and grown bacterial, yeast and mammalian cells), And novel and useful methods for the growth of cultures of such host cells capable of expressing a hybrid polypeptide of the invention. Polynucleotide sequences encoding hybrid polypeptides herein may be useful for gene therapy in instances where the low production of the component peptide hormone (s) of the chimera is alleviated or the need for increased levels thereof is met.

The invention also provides a method for producing recombinant DNA of a hybrid polypeptide of the invention. A method of producing a hybrid polypeptide from a host cell containing a nucleic acid encoding such a hybrid polypeptide is provided, the method comprising (a) containing a polynucleotide encoding such a hybrid polypeptide under conditions that facilitate expression of such DNA molecules. Culturing the host cell, and (b) obtaining such a hybrid polypeptide.

Host cells can be prokaryotic or eukaryotic, and include bacteria, mammalian cells (eg, Chinese hamster ovary (CHO) cells, monkey cells, young hamster kidney cells, cancer cells or other cells), yeast cells and insect cells. Include.

Mammalian host systems for the expression of recombinant proteins are also known to those skilled in the art. Host cell strains may be selected for their particular ability to process expressed proteins or to produce specific post-translational modifications useful for providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” form of a protein may also be important for correct insertion, folding and / or function. Various host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular mechanisms and characteristic mechanisms for this post-translational activity and can be chosen to ensure that the correct modification and processing of the foreign protein introduced takes place. .

Alternatively, yeast systems can be used to produce the hybrid polypeptides of the present invention. The coding region of the hybrid polypeptide cDNA is amplified by PCR. The DNA encoding the yeast pre-pro-alpha leader sequence is the yeast genomic DNA in a PCR reaction using a primer comprising nucleotides 1 to 20 of the alpha conjugation factor gene and another primer complementary to nucleotides 255 to 235 of the gene. Amplified from Kurjan and Herskowitz, Cell, 30: 933-43 (1982). The pre-pro-alpha leader coding sequence and hybrid polypeptide coding sequence fragments may be characterized by the yeast alcohol dehydrogenase (ADH2) promoter such that the promoter directs expression of the fusion protein consisting of the pre-pro-alpha factor fused to the mature hybrid polypeptide. It is ligated to the containing plasmid. Rose and Broach, Meth. Enz. 185: 234-79, Goeddeled., Academic Press, Inc., San Diego, California (1990), the vector is a downstream of the cloning site, the ADH2 transcription terminator, yeast "2-micron" origin of replication, Yeast leu-2d gene, yeast REP1 and REP2 genes, Lee. E. coli β-lactamase gene and E. coli Further comprises an E. coli origin of replication. β-lactamase and leu-2d genes are for selection in bacteria and yeast, respectively. In addition, the leu-2d gene facilitates increased copy number of plasmids in yeast, leading to higher levels of expression. The REP1 and REP2 genes encode proteins involved in plasmid copy number regulation.

The DNA constructs described in the previous paragraph are transformed into yeast cells using known methods, such as lithium acetate treatment [Steams et al., Meth. Enz. 185: 280-97 (1990). ADH2 promoter is induced upon glucose depletion in growth medium (Price et al., Gene 55: 287 (1987)). The pre-pro-alpha sequence results in secretion of the fusion protein from the cell. At the same time, the yeast KEX2 protein cleaves the pre-pro sequence from the mature PYY analogue polypeptide [Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330-4 (1984).

Hybrid polypeptides of the invention may also be recombinantly expressed in yeast using commercially available expression systems, such as the Pichia expression system (Invitrogen, San Diego, Calif.) According to the manufacturer's instructions. It may be. The system also depends on the pre-pro-alpha sequence that directs secretion, but transcription of the insert is driven by an alcohol oxidase (AOX1) promoter upon induction by methanol. Secreted hybrid polypeptides are purified from yeast growth media, for example by methods used to purify hybrid polypeptides from bacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding the hybrid polypeptide can be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego, CA, USA). The hybrid polypeptide-containing vector is then used according to the manufacturer's instructions (Pharmingen) to infect Spodoptera frugiperda cells in sF9 protein-free medium and generate recombinant protein. Proteins were removed from the medium using a Heparin-Sepharose column (Pharmacia, Piscataway, NJ) and a sequential molecular sizing column (Amicon, Beverly, Mass.) Purify and concentrate and resuspend in PBS. SDS-PAGE analysis shows a single band and confirms the size of the protein and confirms its N-terminal sequence through Edman sequencing on the Proton 2090 Peptide Sequencer.

For example, a DNA sequence encoding a hybrid polypeptide can be cloned into a plasmid containing the desired promoter and optionally the leader sequence (see, eg, Better et al., Science 240: 1041-3 (1988)). . The sequence of the construct can be confirmed by automated sequencing. The plasmid was then subjected to E. coli according to standard procedures using bacterial CaCl ₂ incubation and heat shock treatment. Transformation with E. coli strain MC1061 (Sambrook et al., Supra). Transformed bacteria are grown in LB medium supplemented with carbenicillin, and the production of expressed proteins is induced by growth in a suitable medium. If a leader sequence is present, it will affect the secretion of the hybrid polypeptide and will be cleaved during secretion. Secreted recombinant protein is purified from bacterial culture medium according to the methods described herein below.

Alternatively, the hybrid polypeptides of the invention can be expressed in insect systems. Insect systems for protein expression are known to those skilled in the art. In one such system, Autographa californica nuclear polyhedron virus (AcNPV) is a vector that expresses a foreign gene in S. dopplera prugiferda cells or in Trichoplusia larvae . Used. The hybrid polypeptide coding sequence is cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the hybrid polypeptide will inactivate the polyhedrin gene and produce a recombinant virus lacking the envelope protein envelope. Subsequently, recombinant viruses are used to infect either Spodoptera pruperfera cells or Tricoflucia lar embryo, wherein the hybrid polypeptide is expressed (Smith et al., J. Virol. 46: 584 (1983)), Engelhard et al., Proc. Natl. Acad. Sci. USA 91: 3224-7 (1994).

In another example, DNA sequences encoding hybrid polypeptides can be amplified by PCR and cloned into an appropriate vector, such as pGEX-3X (Pmascia, Piscataway, NJ). The pGEX vector is designed to produce a fusion protein comprising a glutathione-S-transferase (GST) encoded by the vector and a protein encoded by a DNA fragment inserted at the cloning site of the vector. Primers for PCR can be generated, for example, to include appropriate cleavage sites. The recombinant fusion protein can then be cleaved from the GST portion of the fusion protein. pGEX-3X / PYY analogue polypeptide constructs; Transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla, Calif.), And isolated individual transformants at 37 ° C. in wavelength 600 nm in LB medium (supplemented with carbenicillin) After growing to an optical density of 0.4, incubation for an additional 4 hours in the presence of 0.5 mM isopropyl β-D-thiogalactopyranoside (Sigma Chemical Co., St. Louis, MO) Let's do it. Plasmid DNA from individual transformants is purified and partially sequenced with an automated sequence analyzer to confirm that the desired PPF hybrid polypeptide-coding gene is present in the proper orientation.

Fusion proteins that are expected to be produced as insoluble inclusion bodies in bacteria can be purified as follows. Cells are harvested by centrifugation, washed with 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA and treated with 0.1 mg / mL lysozyme (Sigma Chemical Company) for 15 minutes at room temperature. Lysates are sonicated to become clear and cell debris is pelleted by centrifugation at 12,000 × g for 10 minutes. Fusion protein-containing pellets are resuspended in 50 mM Tris, pH 8 and 10 mM EDTA and stacked on 50% glycerol and centrifuged at 6000 × g for 30 minutes. The pellet is resuspended in standard phosphate buffered saline solution (PBS) without Mg ⁺⁺ and Ca ⁺⁺ . The fusion protein is further purified by fractionation of the resuspended pellet into denaturing SDS polyacrylamide gels [Sambrook et al., Supra]. The gel is immersed in 0.4 M KCl to visualize the protein, cut and electroeluted in gel-run buffer without SDS. If GST / PYY analog polypeptide fusion proteins are produced as soluble proteins in bacteria, they can be purified using the GST purification module (Pharmacia Biotech).

Fusion proteins can be digested to cleave GST from PPF hybrid polypeptides. Digestion reaction (20-40 μg fusion protein, 20-30 units of human thrombin (4000 U / mg (Sigma) in 0.5 mL PBS)) was incubated for 16-48 hours at room temperature and loaded on a denatured SDS-PAGE gel The reaction product is fractionated The gel is immersed in 0.4 M KCl to visualize the protein band The identity of the protein band corresponding to the expected molecular weight of the hybrid polypeptide is determined using an automated sequence analyzer (Applied Biosystems Model 473A). Partial amino acid sequencing can be confirmed.

In a particularly preferred method of recombinant expression of the hybrid polypeptide of the present invention, 293 cells are plasmid containing pLDV vector (5 'CMV promoter, 3' HGH poly A sequence) in the pCMV vector (5 'CMV promoter, 3' HGH poly A sequence) and pSV2neo (neo resistance gene) May be cotransfected. Preferably, the vector should be linearized with ScaI before transfection. Similarly, another construct can be used that uses similar pCMV vectors with neo genes inserted. Stable cell lines are selected from single cell clones via limiting dilution in growth medium containing 0.5 mg / mL G418 (neomycin-like antibiotic) for 10-14 days. Cell lines are screened for hybrid polypeptide expression using ELISA or Western blot and high expressing cell lines are propagated for large scale growth.

The transformed cells are preferably used for long term high yield protein production, and therefore stable expression is desirable. Once these cells have been transformed with a vector containing a selectable marker with the desired expression cassette, the cells can be grown for one to two days in nutrient enhancing media before switching to selection medium. Selectable markers are designed to confer resistance for selection, the presence of which allows the growth and recovery of cells that successfully express the introduced sequence. Resistant aggregates of stably transformed cells can be propagated using tissue culture techniques appropriate to the cells.

Numerous selection systems can be used to recover transformed cells for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyl transferase and adenine phosphoribosyl transferase genes in tk-, hgprt- or aprt- cells, respectively. In addition, dhfr confers resistance to methotrexate, gpt confers resistance to mycophenolic acid, neo confers resistance to aminoglycoside G418 and also confers resistance to chlorsulfuron, and to hygromycin. Anti-metabolite resistance can be used as the basis for screening for hygro conferring resistance. Additional selectable genes that may be useful include trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine. Markers that provide a visual indication for the identification of transformants include anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin.

Many hybrid polypeptides of the invention can be produced using a combination of automated peptide synthesis and recombinant techniques. For example, the hybrid polypeptides of the present invention may contain a combination of modifications and PEGylation reactions, including deletions, substitutions, and insertions. Such hybrid polypeptides can be generated step by step. In the first step, an intermediate polypeptide containing modifications of deletions, substitutions, insertions, and any combination thereof can be produced by recombinant techniques as described. Subsequently, after any purification step as described herein, the intermediate polypeptide is subjected to chemical modification using an appropriate PEGylation reagent (eg, from Nektar Therapeutics, Carlos, CA, USA). PEGylation yields the desired hybrid polypeptide. Those skilled in the art appreciate that the above procedures can be generalized for application to hybrid polypeptides containing combinations of modifications selected from deletions, substitutions, insertions, derivatizations, and other modification means known in the art and contemplated by the present invention. something to do.

It may be desirable to purify the hybrid polypeptides produced by the present invention. Peptide purification techniques are known to those skilled in the art. These techniques, at one level, include crude fractionation into polypeptide and non-polypeptide fractions of the cellular environment. Separation of the polypeptide from other proteins allows the polypeptide of interest to be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Particularly suitable assay methods for the preparation of pure peptides are ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Particularly efficient methods of peptide purification include purification of products purified by liquid chromatography / mass spectroscopy (LC / MS) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy after performing reversed phase HPLC. To characterize. Further purity confirmation is obtained with amino acid analysis crystals.

Certain aspects of the present invention relate to the purification of encoded proteins or peptides, and in particular embodiments to substantial purification. As used herein, the term "purified peptide" is intended to refer to a composition that is isolateable from other components, wherein the peptide is purified to any degree relative to the conditions available in nature. Thus, purified peptides also refer to peptides that are released from the environment that can occur in nature.

In general, "purified" refers to a peptide composition that is fractionated such that various other components are removed and substantially retains its expressed biological activity. When the term “substantially purified” is used, this expression indicates that the peptide forms the main component of the composition, such as, for example, about 50%, about 60%, about 70%, about 80%, about 90%, It refers to a composition that constitutes at least about 95%.

Various techniques suitable for use in peptide purification will be known to those skilled in the art. These techniques include, for example, precipitation using ammonium sulfate, PEG, antibodies, and the like; Thermal denaturation and subsequent centrifugation; Chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; Isoelectric point electrophoresis; Gel electrophoresis; Combinations of these and other techniques. As is generally known in the art, the order of performing the various purification steps may be varied or certain steps may be omitted, and it is still believed to produce methods suitable for the preparation of substantially purified proteins or peptides.

There is no general requirement that peptides always be provided in the best purified state. In practice, it is contemplated that substantially less purified products will be useful in certain embodiments. Partial purification can be achieved by using fewer purification steps in combination, or by using the same general purification regime in different forms. For example, cation-exchange column chromatography performed using HPLC apparatus is generally understood to produce larger “-drain” purifications than the same technique using low pressure chromatography systems. Lower relative purification may be advantageous in terms of overall recovery of the protein product or maintenance of expressed protein activity.

Optionally, such hybrid polypeptides can be purified and isolated from other components obtained in the process. Methods for purifying polypeptides can be found in US Pat. No. 5,849,883. This document describes specific exemplary methods for the isolation and purification of G-CSF compositions that may be useful for isolating and purifying hybrid polypeptides of the present invention. It is clear from the disclosure of this patent that one skilled in the art will be familiar with the numerous purification techniques that can be used to purify hybrid polypeptides from a given source.

It is also contemplated that a combination of anion exchange and immunoaffinity chromatography can be used to produce the purified hybrid polypeptide composition of the present invention.

Pharmaceutical composition

The invention also provides a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier useful for delivering a therapeutic or prophylactically effective amount of one or more hybrid polypeptides of the invention, or a pharmaceutically acceptable salt thereof, to a hybrid polypeptide. It relates to a pharmaceutical composition comprising together. Such compositions include diluents of various buffer contents (eg Tris-HCl, acetate, phosphate), pH and ionic strength; Additives such as detergents and solubilizers (e.g. Tween 80, Polysorbate 80), anti-oxidants (e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g. Thimersol, benzyl alcohol), and extenders (e.g. lactose, mannitol), and the material may be incorporated into or combined with liposomes in particulate preparations such as polymerizable compounds, e.g. polylactic acid, polyglycolic acid. Can be incorporated. Such compositions will affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the hybrid polypeptides of the present invention (see, eg, Remington's Pharmaceutical Sciences 1435-712, 18th ed., Mack Publishing Co. , Easton, Pennsylvania (1990)).

In general, the hybrid polypeptides of the present invention will be useful in the same way that individual component polypeptides are useful in terms of their pharmacological properties. One of the preferred uses is the peripheral administration of such hybrid polypeptides for the treatment or prevention of metabolic diseases and disorders. In particular, the compounds of the present invention have activity as agents for reducing nutrient utilization, reducing food intake, inhibiting appetite, and causing weight loss. In another embodiment, a preferred use is to administer such hybrid polypeptides for the treatment of diabetes or diabetes-related conditions and disorders.

Hybrid polypeptides of the invention may be formulated for peripheral administration, including formulations for injection, oral administration, nasal administration, pulmonary administration, topical administration, or other types of administration as would be appreciated by those skilled in the art. More particularly, administration of the pharmaceutical composition according to the invention can be by any general route as long as the target tissue is available. In a preferred embodiment, the pharmaceutical composition is administered to the subject by any conventional peripheral method, e. It may be introduced by oral, sublingual, nasal, anal, vaginal or transdermal delivery, or by surgical implantation at specific sites. Treatment can consist of a single administration or a plurality of administrations over time. Controlled sustained release of the compositions of the present invention is also contemplated.

The formulation may be liquid or solid, for example lyophilized for reconstitution. The aqueous composition of the present invention comprises an effective amount of a 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, when administered to an animal or human, do not cause deleterious, allergic or other inappropriate reactions. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. In some cases, the hybrid polypeptides of the present invention and other food intake reducing agents, antidiabetic agents, plasma glucose lowering agents or plasma lipid modifying agents such as amylin, amylin agonist analogs, CCK or CCK agonists, or leptin Or it will be convenient to provide the leptin agonist or exendin or exendin agonist analog in a single composition or solution for combination administration. In other cases, it may be more advantageous to administer additional agents separately from the hybrid polypeptide.

Hybrid polypeptides of the invention can be prepared for administration as a solution of a free base or pharmacologically acceptable salt in water suitably mixed with a surfactant, for example hydroxypropylcellulose. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. Such products are readily prepared by procedures known to those skilled in the art. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent microbial growth.

In one embodiment, the pharmaceutical compositions of the invention are formulated to be suitable for parenteral administration, eg, by injection or infusion. Preferably, the hybrid polypeptide is suspended in an aqueous carrier, for example in an isotonic buffer solution of pH about 3.0 to about 8.0, preferably pH about 3.5 to about 7.4, 3.5 to 6.0 or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphate, and sodium acetate / acetic acid buffer. Depot or “reservoir” sustained release formulation forms may be used to deliver a therapeutically effective amount of the formulation into the bloodstream over several hours or days after transdermal injection or delivery.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the instant preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It is preferred that the hybrid polypeptides of the present invention be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved 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 isotonic agents (eg sugars or sodium chloride). Prolonged absorption of the injectable compositions can be achieved by the use of agents in the composition, agents which delay absorption (eg aluminum monostearate and gelatin).

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent, if desired, with the various other ingredients enumerated above, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients in a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization techniques which yield a powder of the active ingredient + any further desired ingredient from a sterile filtered solution.

In general, the therapeutic or prophylactically effective amount of a hybrid polypeptide of the invention will depend on the age, weight, and condition or severity of the disease, condition or disorder of the recipient. See, eg, 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, dosages of from about 0.001 μg / day to about 1000 μg / day per kg of body weight may be used, although more or less amounts may be used, as will be appreciated by those skilled in the art. Administration may be one or more times daily, or may be less frequent, and may be administered in combination with other compositions, as described herein. It is to be understood that the invention is not limited to the dosages described herein.

Appropriate dosages can be established using established assays to determine the level of metabolic state or disorder along with relevant dose-response data. Final dosing includes factors that modify the action of the drug, such as the specific activity of the drug, the severity of the injury and the patient's response, the age, condition, weight, sex and diet of the patient, the severity of any infection, the time of administration and other Your doctor will determine the clinical factors. As the study is conducted, additional information regarding the appropriate dosage levels and duration of treatment for specific diseases and conditions will be found.

Effective dosages are typically from about 1 to 30 μg to about 5 mg / day, preferably from about 10 to 30 μg to about 2 mg / day, more preferably for 50 kg patients administered in single or divided doses. Will be from about 5 to 100 μg to about 1 mg / day, most preferably from about 5 μg to about 500 μg / day. Preferably, the dosage is about 0.01 to about 100 μg / kg per dose. The exact dosage to be administered can be determined by one skilled in the art and depends on the potency of the particular compound and the age, weight and condition of the individual. Administration is for example whenever nutrient utilization is inhibited, food intake is inhibited, weight gain is inhibited, blood glucose or plasma lipid modulation is desired, for example at the first sign of symptoms or after diagnosis of obesity, diabetes mellitus, or insulin resistance syndrome You should start soon. Administration can be by any route, for example by injection, preferably subcutaneously or intramuscularly, oral, nasal, transdermal and the like. The dosage for a particular route, for example oral administration, may be increased, for example, from about 5 to 100 times in view of the reduced bioavailability.

Parenteral administration can be performed with an initial bolus followed by continuous infusion to maintain the therapeutic circulation level of the drug. Those skilled in the art will readily optimize effective dosages and dosing regimens as determined by good medical practice and the clinical condition of the individual patient.

The frequency of administration will depend on the pharmacodynamic parameters of the agent and the route of administration. The optimal pharmaceutical formulation will be determined by one skilled in the art according to the route of administration and desired dosage. See, eg, Remington's Pharmaceutical Sciences, supra, pages 1435-1712. Such formulations may affect the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agent. Depending on the route of administration, suitable dosages can be calculated according to body weight, body surface area or organ size. Further detail of the calculations necessary to determine the appropriate therapeutic dose can be routinely performed by one of ordinary skill in the art without unnecessary experiments, particularly in view of the dosage information and assays disclosed herein, and the pharmacodynamic data observed in animal or human clinical trials. .

It will be appreciated that the pharmaceutical compositions and methods of treatment of the present invention may be useful in the fields of human medicine and veterinary medicine. Thus, the subject to be treated may be a mammal, preferably a human or other animal. For veterinary purposes, the subject is a livestock, for example cattle, sheep, pigs, horses and goats, pets, for example dogs and rabbits, foreign and / or zoo animals, laboratory animals, for example mice, rats, rabbits , Guinea pigs and hamsters, and poultry such as chickens, turkeys, ducks, and geese.

The invention also contemplates a kit comprising the hybrid polypeptide of the invention, a component suitable for preparing the hybrid polypeptide of the invention for pharmaceutical use, and instructions for using the hybrid polypeptide and the component for pharmaceutical use.

To help the understanding of the present invention, the following examples are included. Experiments concerning the present invention, of course, should not be construed as specifically limiting the present invention, and modifications of the present invention which are now known or later developed within the scope of those skilled in the art are within the scope of the invention described herein and claimed later. It is considered to belong.

The invention is described in more detail by referring to the following non-limiting examples which are provided to more fully illustrate the invention but should not be construed as limiting the scope of the invention. The Examples illustrate the preparation of hybrid polypeptides of the invention, and in vitro and / or in vivo testing of these hybrid polypeptides of the invention. Those skilled in the art will understand that the techniques described in these examples are representative of the techniques described by the inventors, which function well in the practice of the present invention and thus constitute the preferred manner for the practice of the present invention. However, one of ordinary skill in the art appreciates that many changes can be made in the specific methods disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example  One: hybrid  Polypeptide manufacturing

Peptides of the present invention are Rink amide resins (Novabiochem) or pre-loaded Wang resins (Fmoc-Tyr (tBu)) loaded from 0.050 to 0.100 mmol at 0.43 to 0.49 mmol / g. -Wang resin) can be assembled on a Symphony peptide synthesizer (Protein Technologies, Inc.) using 0.63 mmol / g (Novabiochem). Fmoc amino acid (5.0 equiv, 0.250 to 0.500 mmol) residues were dissolved in 1-methyl-2-pyrrolidinone at a concentration of 0.10 M. All other reagents (HBTU, 1-hydroxybenzotriazole hydrate and N, N-diisopropylethylamine) were prepared as 0.55 M dimethylformamide solution. The Fmoc protected amino acid was then purified by HBTU (2.0 equiv, 0.100 to 0.200 mmol), 1-hydroxybenzotriazole hydrate (1.8 equiv, 0.090 to 0.18 mmol), N, N-diisopropylethylamine (2.4 equiv, 0.120 To 0.240 mmol) was coupled to the resin bound amino acid for 2 hours. After the last amino acid coupling, the peptide was deprotected for 1 hour with 20% (v / v) piperidine in dimethylformamide. Once the peptide sequence was completed, the symphony peptide synthesizer was programmed to cleave the resin. Trifluoroacetic acid (TFA) cleavage of the peptide from the resin was performed for 1 hour using 93% TFA, 3% phenol, 3% water and 1% triisopropylsilane. Cleaved peptides were precipitated using tert-butyl methyl ether, pelleted by centrifugation and lyophilized. The pellet was redissolved in water (10 mL to 15 mL), filtered and purified via reverse phase HPLC using an acetonitrile / water gradient containing C18 column and 0.1% TFA.

The general procedure for N-capping the peptides of the invention with fatty acids (eg octanoic acid and stearic acid) was as follows: Peptides (0.1 mmol) on link amide resin were suspended in NMP (5 mL). In a separate vial, HBTU (0.3 mmol), HOBt (0.3 mmol) was dissolved in DMF (5 mL) before DIEA (0.6 mmol) was added. The solution was added to the resin and the suspension was shaken for 2 hours. The solvent was filtered, washed thoroughly with NMP (5 mL × 4) and CH ₂ Cl ₂ (20 mL), dried and TFA cleaved for 1 hour. The yield of the desired peptide was about 40 mg after cleavage and purification.

PEG modifications can be carried out in solution using commercially available activating PEG esters against the free epsilon-amino group of lysine or the terminal amino group of the purified peptide. The resulting PEGylated derivative was purified homogeneously by reversed phase HPLC and purity was confirmed by LC / MS and MALDI-MS.

Certain exemplary hybrid polypeptides of the invention are shown in Table 1-1 below. Various modifications to the embodied compounds, such as chemical modifications such as glycosylation, PEG modifications, and the like; Amino acid modifications such as substitutions, insertions, and deletions have been made. In addition, although the C-terminus has been shown to be amidated, it is understood that the hybrid polypeptides of the present invention may alternatively also exist in free acid form.

TABLE 1-1

Additional exemplary hybrids comprising various combinations of component peptide families are set forth in the table below. As discussed herein, analogs and derivative hybrids thereof are also specifically contemplated. "#" Indicates the position of linkage for each component. The linkers shown are as described herein. CCK-8 is DY (SO ₃ H) MGWMDF- NH _2, but “CCK-8 ^* ” is the sulfated phenylalanine form DF (CH ₂ SO ₃ H) MGWMDF-NH ₂ .

Example  2: binding test

Hybrid polypeptides of the invention can be tested in a variety of receptor binding assays using binding assay methods generally known to those skilled in the art. Such assays include those described herein.

Amylin Binding Assay : Binding assessment of some exemplary compounds of the invention to amylin receptors can be performed as follows on nuclueus accumbens membranes prepared from rat brain. Male Sprague-Dawley ^® rats (200-250 g) were barely sacrificed. The brain was taken out and placed in cold phosphate buffered saline (PBS). From the ventral surface, a lateral incision was made in the hypothalamus, which the abutment abuts and extends 45 degrees from the latter to the center. The basal forebrain tissue containing the nucleus and surrounding area of the septum was weighed and homogenized in ice cold 20 mM HEPES buffer (20 mM HEPES acid, adjusted to pH 7.4 with NaOH at 23 ° C.). Membranes were washed three times by centrifugation at 48,000 × g in fresh buffer for 15 minutes. The final membrane pellet was resuspended in 20 mM HEPES buffer containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF).

To measure ¹²⁵ I-amylin binding (see Beaumont K et al. Can J Physiol Pharmacol. 1995 Jul; 73 (7): 1025-9), 0.5 mg of membrane obtained from 4 mg of original wet weight tissue Incubated with 12-16 pM of ¹²⁵ I-amylin in 20 mM HEPES buffer containing / mL bacitracin, 0.5 mg / mL bovine serum albumin and 0.2 mM PMSF. The solution was incubated at 2 ° C. for 60 minutes. Incubation by filtration with a GF / B glass fiber filter (Whatman Inc., Clifton, NJ) pre-soaked in 0.3% polyethyleneimine for 4 hours to reduce nonspecific binding of radiolabeled peptides Terminated. The filter was washed with 5 mL of cold PBS immediately before filtration and 15 mL of cold PBS immediately after filtration. The filter was removed and the radioactivity was evaluated with a counting efficiency of 77% in a gamma-counter. Competition curves were generated by measuring binding in the presence of unlabeled test compounds of 10 ⁻¹² to 10 ⁻⁶ M and analyzed by nonlinear regression using 4-parameter logistic equations (Inplot program, GraphPad software). (GraphPAD Software), San Diego, CA, USA.

CGRP Receptor Binding Assay : Evaluation of the binding of compounds of the invention to CGRP receptors has been demonstrated in SK-N-MC cells known to express CGRP receptors [Muff, R. et. al., Ann NY Acad. Sci. 1992: 657, 106-16 is essentially the same as described for amylin except using membranes prepared from. Binding assays were performed as described for amylin except using 13,500 cpm ¹²⁵ I-hCGRP / well or 21.7 pM / well (Amersham).

Adrenemodulin Binding Assay : Binding to the adrenomedulin receptor is performed using a Perkin Elmer AlphaScreen ™ assay for cyclic AMP using optimal 25 to 30,000 cells per well. Can be investigated using HUVECs containing adrenomedulin receptor [Kato J et. al., Eur J Pharmacol. 1995, 289: 383-5. In the case of HUVECs, the elevation of cAMP levels is not significant compared to CHO cells. Thus, CHO cells do not express an adrenomedulin receptor and thus can be selected as a negative control if desired.

Calcitonin Receptor Binding Assay : Binding to calcitonin receptors can be examined using CHO cells or T47D cells that also express calcitonin receptors (Muff R. et. Al, Ann NY Acad Sci. 1992). , 657: 106-16 and Kuestner RE et. Al. Mol Pharmacol. 1994, 46: 246-55).

Leptin Binding Assay : Two in vitro biological assays were used in a conventional manner to assess leptin binding and receptor activation (see, eg, White, et al., 1997. Proc. Natl. Acad. Sci. USA 94: 10657-10662). An alkaline phosphatase ("AP")-leptin ("OB") fusion protein ("AP-OB") is used in the long (signaling) form of the mouse OB receptor in the absence or presence of recombinant mouse leptin (positive control) or peptide ( "OB-RL") can be used to measure the inhibition of leptin binding by COS-7 cells transfected with. Signal transduction assays can be performed 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 mouse leptin or peptide can be measured by chemiluminescence.

Y1 Receptor Binding Assay : Membranes were prepared from confluent cultures of SK-N-MC cells endogenously expressing the neuropeptide Y1 receptor. The membranes were incubated in 96-well polystyrene plates for 60 minutes at ambient temperature with 60 pM [ ¹²⁵ I] -human peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences) and unlabeled active compounds. Well contents were then harvested onto 96 well glass fiber plates using a Perkin Elmer plate collector. The dried glass fiber plates were mixed with scintillant and counted on a Perkin Elmer Scintillation Counter.

Y2 Receptor Binding Assay : Membranes were prepared from confluent cultures of SK-N-BE cells endogenously expressing the neuropeptide Y2 receptor. Membranes were incubated in 96-well polystyrene plates for 60 minutes at ambient temperature with 30 pM [ ¹²⁵ I] -human peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences) and unlabeled active compounds. Well contents were then harvested onto 96 well glass fiber plates using a Perkin Elmer plate collector. The dried glass fiber plates were mixed with scintillant and counted on a Perkin Elmer Scintillation Counter.

Y4 Receptor Binding Assay : CHO-K1 cells were transiently transfected with cDNA encoding the neuropeptide Y4 gene, and then 48 hours later membranes were prepared from confluent cell cultures. Membranes were incubated in 96-well polystyrene plates for 60 minutes at ambient temperature with 18 pM [ ¹²⁵ I] -human pancreatic polypeptide (2200 Ci / mmol, PerkinElmer Life Sciences) and unlabeled active compounds. Well contents were then harvested onto 96 well glass fiber plates using a Perkin Elmer plate collector. The dried glass fiber plates were mixed with scintillant and counted on a Perkin Elmer Scintillation Counter.

Y5 Receptor Binding Assay : CHO-K1 cells were transiently transfected with cDNA encoding the neuropeptide Y5 gene, and then 48 hours later membranes were prepared from confluent cell cultures. Membranes were incubated in 96-well polystyrene plates at ambient temperature for 60 minutes with 44 pM [ ¹²⁵ I] -human peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences) and active compound. Well contents were then harvested onto 96 well glass fiber plates using a Perkin Elmer plate collector. The dried glass fiber plates were mixed with scintillant and counted on a Perkin Elmer Scintillation Counter.

GLP- 1 Receptor Binding Assay : GLP-1 receptor binding activity and affinity can be measured using a binding replacement assay wherein the receptor source is RINm5F cell membrane and the ligand is [ ¹²⁵ I] GLP-1. Homogenized RINm5F cell membranes were incubated with continuous mixing at 23 ° C. for 2 hours with varying concentrations of test compounds in 20 mM HEPES buffer containing 40,000 cpm [ ¹²⁵ I] GLP-1 tracer. The reaction mixture was filtered through a glass filter pad pre-soaked with 0.3% PEI solution and washed with ice cold phosphate buffered saline. Coupling coefficients were determined using a scintillation counter. Binding affinity was calculated using GraphPad Prism software (GraphPad Software, Inc., San Diego, Calif.).

Receptor activation, including in vitro receptor binding and component receptor specific receptor activation for exemplary hybrids, is shown in the table below.

Example  3: mouse food intake assay

Hybrid polypeptides of the invention can be tested for appetite inhibition in a mouse food intake assay and for their effect on weight gain in diet-induced obesity (DIO) mice. Experimental protocols for screening are described below.

Female NIH / Swiss mice (8-24 weeks old) were grouped in a 12 hour: 12 hour light: dark cycle with light at 0600. Water and standard pelleted mouse food could be eaten at will, except as noted. One day before the experiment, the animals were starved from approximately 1500 o'clock. The animals were divided into experimental groups on the morning of the experiment. In a typical study, n = 4 cages (3 mice / cage).

At time = 0 min all animals are injected intraperitoneally with vehicle or compound typically in an amount in the range of about 10 nmol / kg to 75 nmol / kg, and immediately after that in a pre-weighed amount (10-15 g) of standard The food was fed. The food was cleared and weighed at various times, typically at 30, 60, and 120 minutes to determine food consumption [Morley, Flood et al., Am. J. Physio. 267: R178-R184, 1994]. Food intake was calculated by subtracting the weight of food left at the 30th, 60th, 120th, 180th and / or 240th time points, for example, from the weight of the food first given at 0th minute. Significant treatment effect was confirmed with ANOVA (p <0.05). If there were significant differences, Dunnett's test (Prism v. 2.01, GraphPad Software Inc., San Diego, Calif.) Was used to compare the test mean to the control mean.

The activity and sequence in the food intake assay of the parent molecule used in the hybrid synthesis herein are as follows:

Example  4: weight, fat redistribution, and Fat loss  Weight loss black

Assays for weight and related effects can be performed as follows:

Diet-induced obesity (DIO) in Sprague-Dawley rats is a useful model for studying the regulation of obesity and energy homeostasis. These rats were developed from a family of rats (Crl: CD ^® (SD) BR) susceptible to obesity when providing relatively high fat and energy foods. See, eg, Levin (1994) Am. J. Physiol. 267: R527-R535, Levin et al. (1997) Am. J. Physiol. 273: R725-R730. DIO male rats were obtained from Charles River Laboratories, Inc. (Wilmington, Mass.). The rats were individually confined in a 22 ° C. box cage with a 12 hour / 12 hour light: dark cycle. Rats maintained free access to moderate, high fat food (32% calories from fat, research diet D1226B). The animal typically had an average body weight of about 500 g. Levin DIO rats were adapted to our environment for 7 days. During a three-day nightly adaptation, animals were injected with vehicle alone intraperitoneally (IP). On test days, rats were administered a compound or vehicle (10% DMSO) as a single IP injection at the start of the cancer cycle for 6 to 14 nights consecutive nights. Food intake was measured every 5 seconds with an automated food intake measurement system (BioDAQ, Research Diet) throughout the study period. Body weights were recorded nightly.

Body composition was measured using NMR (Echo Medical Systems, Houston, TX) before and after drug treatment. To measure body composition, rats were placed briefly (approximately 1 minute) in a well-ventilated plexiglass tube and placed in a special NMR instrument for rodents. This results in a change in actual grams (g) of fat and fat-free (dry) tissue (e.g., grams of body fat after treatment (g)-grams of body fat at baseline (g) = grams of body fat (g) Amount of change), and percentage of change in body composition (%) of body composition relative to fat and fat-free (dry) tissue (e.g., percentage of body fat after treatment (%)-percentage of body fat at baseline (%) = change in percentage of body fat (%)) ) Could be calculated. All data are expressed as mean ± SEM. Group differences were tested using analysis of variance (ANOVA) and post-hoc tests. P-value <0.05 was considered significant. Statistical analysis and graph construction were performed using Prism ^® 4 for Windows (GraphPad Software, Inc., San Diego, CA, USA). Some early studies were analyzed using the Windows System tart (SYSTAT) ^® (cis tart Software, Inc.. (SYSTAT Software, Inc.), Point Richmond, Calif material) for. In the case of hybrids containing aminomimetics, exendin, PYY analogues and / or leptin, body fat changes did not involve significant reductions in lean fat tissue. Graphs and results are typically presented in body weight, vehicle-corrected percent change in body fat, and percent percent change in body protein.

In another experiment, male C57BL / 6 mice (4 weeks of age at the start of the study) were fed high fat (HF, 58% of kcal fat) or low fat (LF, 11% of kcal fat). Was supplied. At 4 weeks later, each mouse was implanted with an osmotic pump (Alzet # 2002) that delivered a subcutaneous delivery of a predetermined dose of hybrid polypeptide for 2 weeks. Body weight and food intake were measured weekly (Surwit et al., Metabolism-Clinical and Experimental, 44: 645-51, 1995). The effect of the test compound is expressed as the mean ± standard deviation of the percent change in body weight (ie, percent change from starting body weight) of 14 or more mice per treatment group (p <0.05 ANOVA, Dunnett's Test, Prism v. 2.01) , GraphPad Software Inc., San Diego, CA, USA.

Exendin / PYY Hybrid : Exemplary hybrid polypeptides of the invention are C-terminal truncated exendin (eg exendin-4 (1-28) or ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-exendin-4 (1-28) ) And N-terminal truncated PYY spanning 18 to 36 to 31 to 36 regions. Thus, exemplary hybrid polypeptides generally comprise two modules, where 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, β-alanine dipeptide spacers were also incorporated between peptide building blocks in a plurality of variants (see Table 4-1).

Table 4-1

As shown in Table 4-1, certain exemplary compounds of the invention have shown efficacy in food intake assays. Certain peptides were also tested at 75 nmol / kg in the DIO assay and proved to be more potent than PYY (FIG. 1). As observed for other hybrids herein, the hybrid may retain binding to one, two or three receptors that recognize the parent molecule. Hybrids were designed to recognize one or more receptors from each parent molecule or, if desired, from only one parent molecule. As observed for other hybrids herein, the use of linkers (which can act as spacers between each adjacent hormone moiety) include receptor (s) binding and in vitro and in vivo activity, such as weight loss. It can provide increased activity. The results herein show that the C-terminal portion of PYY can modulate activity.

Exendin / Amylin Hybrid : Additional exemplary hybrid polypeptides of the invention include C-terminal truncated exendin (1-27) (SEQ ID NO: 236), C-terminal truncated amylin peptide (e.g. amylin (1-7) (SEQ ID NO: 217), ^2,7 Ala-Amyline (1-7) (SEQ ID NO: 285) and Amylin (33-27) (SEQ ID NO: 243), and any sCT fragment (e.g., sCT (8-10), ^{Prepared from 11,18} Arg-sCT (8-27) (SEQ ID NO: 289) and ¹⁴ Glu, ^11,18 Arg-sCT (8-27) (SEQ ID NO: 286) Both hybrid polypeptides are very active against appetite inhibition. (See Table 4-2), but superior to rat amylin at the same dose and initiation of action was different from the activity profile of the parent molecule (data not shown) Compound 5 was administered at a dose of 1 nmol / kg It was as effective as Amylin.

Table 4-2

In addition, both compounds showed excellent efficacy in screening in the DIO assay (FIG. 2).

Additional exemplary compounds were assayed in the effect on blood glucose levels and in food intake assays. These tests included Compound 14 and Compound 15. Compound 14 comprising a beta Ala linker is composed of ²⁹ βAla-βAla-exendin (1-28), h amylin (1-7) ^-11,18 Arg- ^sCt (8-27) -h amylin (33-37). (SEQ ID NO: 33) (Alternatively, exendin (1-28) -β Ala-β Ala-h amylin (1-7) ^-11,18 Arg- ^sCt (8-27) -h amylin (33-). 37), and compound 15 contained a Gly linker: ²⁹ GlyGlyGly-exendin (1-28), h amylin (1-7) ^-11,18 Arg- ^sCt (8-27)- h amylin (33-37) (SEQ ID NO: 312). Longer exendin (1-28) provided increased activity than exendin (1-27).

Blood glucose assays were performed to test for the effect of lowering blood glucose levels. Female NIH / Swiss mice (8-20 weeks old) were grouped in a 12 hour: 12 hour light: dark cycle with light at 0600. Water and standard pelleted mouse food could be eaten at will, except as noted. On the morning of the experiment the animals were divided into experimental groups and starved starting at approximately 0630. In a typical study, n = 2 cages (3 mice / cage). Blood glucose samples were taken at time = 0 min, followed immediately by intraperitoneal injection of vehicle or compound in an amount ranging from about 1 nmol / kg to 25 nmol / kg. Blood glucose was measured at 30, 60, 120, 180 and 240 minutes. The percent pretreatment was calculated by dividing, for example, blood glucose at the 30th, 60th, 120th, 180th and / or 240th minute points by blood glucose at time = 0 min. . Significant treatment effect was confirmed with ANOVA (p <0.05). If there were significant differences, Dunnett's test (Prism v. 4.01, GraphPad Software Inc., San Diego, Calif.) Was used to compare the test mean to the control mean. Results for exemplary compounds are shown in FIG. 5A. Dots represent mean ± standard deviation. Reference samples were injected into NIH / Swiss mice starved for 2 hours immediately after intraperitoneal (IP) injection of the peptide at t = 0. Samples were taken at t = 30 min, 60 min, 120 min, 180 min and 240 min. Blood glucose was measured by OneTouch ^® Ultra ^® (LifeScan, Inc., Johnson & Johnson Company, Milpitas, Calif.). * p <0.05 vs. Vehicle control; ANOVA, Dunnett Exam.

Food intake assays were performed as previously described. The results are shown in Figure 5B. The points represent the mean ± standard deviation of n = 4 cages (3 mice / cage). Intraperitoneal (IP) injections of NIH / Swiss starved overnight at t = 0. Food was introduced immediately after injection and the consumption was measured at t = 30 min, 60 min and 120 min. * p <0.05 vs. Vehicle control; ANOVA, Dunnett Exam.

Parent compound 10 and exendin compound showed opposite effects in glucose assay. It was predicted that linking these would result in opposite effects or, at best, attenuate the effects observed in the more potent parent compound. However, in the glucose assay the exemplary hybrid compound was as efficacious as exendin (1-28) and the duration of action was longer.

In the food intake data, the exemplary compound described above showed appetite suppressing action. Activity was generally better than the parent compound administered individually. The activity was similar to the parent compound with combined dose but half drug concentration (Hybrid 3 nmol / kg vs. co-administered parent compound 6 nmol / kg total), thus further demonstrating hybrid excellence. The addition of linker increased the activity of the hybrid. In this case, the Gly-Gly-Gly linker was more effective than the beta Ala-betaAla linker.

Exendin / CCK- 8 Hybrids : Additional exemplary hybrid polypeptides of the invention can be prepared from full length exendin-4, or directly at the N-terminus of CCK-8 while preserving the N-terminal amide of CCK-8. Prepared from C-terminal truncated exendin-4, attached via a linker (Table 4-3). In addition, specific hybrids incorporating native Tyr (S0 ₃ ) were made, and another hybrid incorporating more stable Phe (CH ₂ SO ₃ ) groups. All hybrid polypeptides produced were active in inhibiting food intake (Table 4-3).

Table 4-3

Exemplary exendin / CCK-8 hybrid polypeptides were tested in the DIO assay at 25 nmol / kg (FIGS. 3A and 3B). The data showed an initial weight loss followed by a rebound effect on all compounds. Interestingly, the recoil effect is not only a hybrid incorporating Phe (CH ₂ SO ₃ ) residues that are more stable to hydrolysis (compare FIGS. 3A and 3C), but also linkers between exendin and CCK residues, for example linker 8-amino- It was also shown to decrease in hybrids incorporating 3,6-dioxaoctanoyl (compare FIG. 3A and FIG. 3B). To produce about -2.8% change on day 2, 10 times more CCK-8 (250 nmol / kg / day) was required, which rebounded to HF feeding control levels on day 7.

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

Table 4-4

To ascertain whether exemplary hybrid polypeptides of the invention are more potent than their parent component peptide hormones, exemplary compounds were tested in a food intake assay at the minimum potent dose of the parent molecule with greater activity. The results are shown in FIGS. 4A and 4B and also compare the effects of the parent peptides collected (compounds 1, 11 and 12 are component peptide hormones, analogs or fragments thereof). The data indicate that the various peptides have at least the same potency as the parent peptide collected. In parallel with in vivo studies, in vitro receptor binding and function assays (cyclase activity) were performed for all compounds (data not shown).

Amylin - sCT / Leptin Hybrid : Additional exemplary hybrids containing leptin peptide fragments linked to amylin-sCT-amylin chimeric compound 10 described herein were prepared. Compound 16 is [Ser117, d Leu 119] leptin (116-122) -amylin (1-7)-[ ^11,18 Arg] sCT (8-27) ^-amylin (33-37) (SEQ ID NO: 397). The compound bound to amylin and CT receptor (RBA = receptor binding assay) and slightly bound to CGRP receptor. The compound could also activate the CT receptor (C1A assay).

The representative molecule was tested for activity in a food intake assay as described herein. Leptin was not active in this assay, but compound 16 showed appetite suppressing activity at 1 mg / kg. Compound 16 was superior to rat amylin (25 nmol / kg) in appetite suppression effect. While compound 10 was much more effective by reducing food intake by 91-95% compared to control, compound 16 reduced cumulative intake by 34-38% of the control. A further exemplary embodiment is a head-to-leptin peptide N-terminus and a head-to- ^length of the amylin (1-7)-[ ^11,18 Arg] sCT (8-27) ^-amylin (33-37) compound. -head) contains a connection.

CCK / Amylin - sCT Hybrids : Exemplary hybrids of CCK and amylin-sCT chimeras demonstrated relevant receptor specificity and activation. Exemplary compounds having the sequence DF (P-CH ₂ SO ₃ ) MGWMDFGKR KCNTATCATQRLANELVRLQTYPRTNVGSNTY (SEQ ID NO: 398) are 0.044 nM in CT receptor binding assay, 4.4 nM in CGRP receptor binding assay, 0.083 nM in amylin receptor binding assay, and GLP receptor Cyclase (RIN) assay showed an IC ₅₀ of 1000 nM.

Example  5: reduction of food intake, weight and weight gain

As described herein, exemplary hybrid polypeptides of the invention were further tested in connection with decreased appetite inhibition, weight loss and weight gain in mice and rats. Parent compounds were also assayed alone or in combination. 9-21 demonstrate the in vivo working of exemplary hybrids in the assay as described herein.

In vivo activity of inhibition of food intake by exemplary hybrids and their parent compounds in mice is shown in the table below. Values are expressed as percent inhibition of food intake after infusion for the indicated time. "ns" means "not significant" compared to vehicle.

In vivo activity of weight inhibition by exemplary hybrids and their parent compounds in a mouse DIO model is shown in the table below. Values are expressed as percent body weight inhibition after infusion for the indicated time.

In vivo activity of weight inhibition by exemplary hybrids and their parent compounds in a mouse DIO model is demonstrated in the table below. Values are expressed as percent body weight inhibition after infusion for the indicated time.

In vivo activity of weight inhibition by exemplary hybrids and their parent compounds in rat model (daily injection) is demonstrated in the table below. Values are expressed as percent body weight inhibition. The value in bold indicates that it is significant compared to the vehicle.

In vivo activity of body weight and weight gain reduction by exemplary hybrids and their parent compounds for weight inhibition in a rat model is shown in the table below for both infusion over the indicated period and once daily injection over the indicated period. Is proven. Values are expressed as percent body weight inhibition. Percent inhibition of food intake on day 6 was also measured in night-time food intake over an 8 hour period. Unless indicated otherwise, hybrids were provided at 15 nmol / kg / day. The difference in body weight is about 2% statistically significant. There is a statistically significant difference in food intake between about 20%.

Example  7: blood Glucose  Lowering

The in vivo activity of blood glucose lowering by exemplary hybrids and their parent compounds in OGTT assays performed in Levin rats over the indicated periods is demonstrated in the table below. Values are expressed as percent reduction in blood glucose levels. The value in bold indicates that it is significant compared to the vehicle.

Example  8: stomach fasting active

In vivo activity against gastric fasting by exemplary hybrids and their parent compounds, performed in Levin rats over the indicated periods, is demonstrated in the table below. Values are expressed as gastric emptying inhibition rate (%) and acetaminophen pass rate (%).

Example  9: PPF  Inhibition of short and long term food intake by polypeptides

Female NIH / Swiss mice (8 to 24 weeks old) were housed in a 12 hour: 12 hour light: dark cycle with light at 0600. Water and standard pelleted mouse food could be eaten at will, except as noted. One day before the experiment, the animals were starved from approximately 1500 o'clock. The animals were divided into experimental groups on the morning of the experiment. In a typical study, n = 4 cages (3 mice / cage).

At time = 0 min all animals are injected intraperitoneally with vehicle or compound in an amount ranging from about 10 nmol / kg to 100 nmol / kg, and immediately afterwards a pre-weighed amount (10-15 g) of standard food Supplied. The food was cleared and weighed at 30, 60 and 120 minutes to determine food consumption [Morley, Flood et al., Am. J. Physiol. 267: R178-R184, 1994]. When studying the short-term effects of PPF polypeptide on food intake, food intake subtracts the weight of food left at the 30th, 60th, 120th and 180th minutes from the weight of the food first given at 0th minute. Calculated by Similarly, water intake was calculated by subtracting the number of grams (g) of water remaining at that time from the weight (g) of water initially provided. When studying the effects of PPF polypeptide on longer term (long-term) food intake and / or body composition, food consumption was measured over a two week period. To study this effect of PPF polypeptides on longer food intake and / or body composition, mice were individually confined for one week before the start of treatment. Daily food intake and body weight were monitored throughout the experiment. During the treatment period, vehicle (50% in water-dimethyl sulfoxide) and PYY (3- 36) a jet Al ^® osmotic pumps arranged to (1 mg / kg / day) to shoulder the region under isoflurane anesthesia (dew collected Corporation (Durect Corp.), Cupertino, Calif., Model 1003D, 2001, and 2004; for 3, 7, and 28-day studies, respectively), administered as a continuous subcutaneous (sc) infusion. At the end of each study, animals were sacrificed rapidly after 2-4 hours with isoflurane overdose. Blood was collected in a Na-heparin-washed syringe with a cardiac puncture and plasma was frozen immediately. In some studies, body composition was measured using dual energy X-ray absorptiometry (DEXA; PixiMus, GE Lunar). In some studies, body composition (eg, fat and post-treatment) was used using a rodent NMR instrument (EchoMRI-700) ™, where animals were placed in restraint tubes and subjected to NMR for 2 minutes. Amount of protein) was measured to quantify the amount of fat and lean body mass (g). Both epididymal fat pads and scapula brown adipose tissue (BAT) can be excised from the animals and the weight of these organs is measured. The excised liver sample can be stored in RNALater (Ambion, Austin, TX) at -20 ° C.

22-29 show the ability to reduce cumulative food intake by PPF polypeptide chimeras, particularly PPY-NPY chimeras such as compounds 4883 and 5705, in the food intake assays described herein.

FIG. 22 shows that cumulative food intake was reduced over 3 hours when Compound 5705 was administered compared to vehicle alone. 23A and 24A show changes in food intake (g), and FIGS. 23B and 24B show mice continuously administered vehicle or compound 4883 or compound 5705 (dose at 500 μg / kg / day) for 13 days Weight change in vehicle (% vehicle-corrected weight change). Early in the day after the start of the PPF polypeptide, food intake and body weight tended to decrease in animals treated with Compound 4883 or Compound 5705.

25 and 26 show daily water uptake and urine excretion upon administration of Compound 4883 or Compound 5705 in mice continuously administered vehicle or Compound 4883 or Compound 5705 (dose of 500 μg / kg / day) for 13 days. Shows the change.

27 shows NMR evaluation of the effect of continuous administration of vehicle or compound 4883 or compound 5705 (dose at 500 μg / kg / day) on body composition for 13 days. 28A and 28B show water change and percent change as measured in grams (g) in mice continuously administered vehicle or compound 4883 or compound 5705 (dose of 500 μg / kg / day) for 13 days. .

FIG. 29 shows the effect of continuous administration of Compound 4883 or Compound 5705 (dose of 500 μg / kg / day) on urine electrolyte for 14 days.

Although the invention has been described in terms of preferred examples and embodiments, it should be understood by those skilled in the art that modifications and variations of the invention will be apparent. Accordingly, the appended claims are intended to cover all such equivalent variations that fall within the scope of the claims.

                               SEQUENCE LISTING (26Oct06) .txt <110> LEVY, ODILE ESTHER       HANLEY, MICHAEL R.       JODKA, CAROLYN M.       LEWIS, DIANA Y.       SOARES, CHRISTOPHER J.       GHOSH, SOUMITRA S.       D'SOUZA, LAWRENCE J.       PARKES, DAVID       MACK, CHRISTINE M.       FOROOD, BRUCE B. <120> HYBRID POLYPEPTIDES WITH SELECTABLE PROPERTIES <130> 0701WO2 <140> 11 / 201,664 <141> 2005-08-11 <140> 11 / 206,903 <141> 2005-08-17 <140> 11 / 301,744 <141> 2005-12-12 <160> 457 <170> Patent In Ver. 3.3 <210> 1 <211> 54 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 1 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser Ala Ser Leu Arg His Tyr Leu Asn Leu          35 40 45 Val Thr Arg Gln Arg Tyr      50 <210> 2 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 2 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser Arg His Tyr Leu Asn Leu Val Thr Arg          35 40 45 Gln Arg Tyr      50 <210> 3 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 3 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser Asn Arg Tyr Tyr Ala Ser Leu Arg His          35 40 45 Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr      50 55 <210> 4 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (40) .. (41) <223> Beta-alanine <220> <223> C-term amidated <400> 4 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser Xaa Xaa Ala Ser Leu Arg His Tyr Leu          35 40 45 Asn Leu Val Thr Arg Gln Arg Tyr      50 55 <210> 5 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (40) .. (41) <223> Beta-alanine <220> <223> C-term amidated <400> 5 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser Xaa Xaa Arg His Tyr Leu Asn Leu Val          35 40 45 Thr Arg Gln Arg Tyr      50 <210> 6 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (40) .. (41) <223> Beta-alanine <220> <223> C-term amidated <400> 6 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser Xaa Xaa Val Thr Arg Gln Arg Tyr          35 40 45 <210> 7 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 7 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Ala Ser Leu Arg              20 25 30 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 8 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 8 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Arg His Tyr Leu              20 25 30 Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 9 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 9 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Asn Arg Tyr Tyr              20 25 30 Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 45 <210> 10 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 10 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Ala Ser              20 25 30 Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 45 <210> 11 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 11 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Arg His              20 25 30 Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 12 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 12 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 13 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 13 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Asn Arg Tyr Tyr              20 25 30 Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 45 <210> 14 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 14 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Ala Ser Leu Arg              20 25 30 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 15 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 15 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Arg His Tyr Leu              20 25 30 Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 16 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <223> C-term amidated <400> 16 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 17 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 17 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Xaa Xaa Ala Ser              20 25 30 Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 45 <210> 18 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 18 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Xaa Xaa Arg His              20 25 30 Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 19 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 19 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Xaa Xaa Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 20 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES <222> (41) <223> Tyr (SO 3) <220> <223> C-term amidated <400> 20 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser Asp Tyr Met Gly Trp Met Asp Phe          35 40 45 <210> 21 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES <222> (30) <223> Tyr (SO 3) <220> <223> C-term amidated <400> 21 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Asp Tyr Met Gly              20 25 30 Trp Met Asp Phe          35 <210> 22 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES <222> (30) Phe (CH 2 SO 3) <220> <223> C-term amidated <400> 22 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Asp Phe Met Gly              20 25 30 Trp Met Asp Phe          35 <210> 23 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES <222> (29) 8-amino-3,6-dioxactoanoyl <220> <221> MOD_RES <222> (31) <223> Tyr (SO 3) <220> <223> C-term amidated <400> 23 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Asp Tyr Met              20 25 30 Gly Trp Met Asp Phe          35 <210> 24 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard and human construct <220> <221> MOD_RES <222> (29) 8-amino-3,6-dioxactoanoyl <220> <221> MOD_RES <222> (31) Phe (CH 2 SO 3) <220> <223> C-term amidated <400> 24 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Asp Phe Met              20 25 30 Gly Trp Met Asp Phe          35 <210> 25 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <223> C-term amidated <400> 25 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Lys Cys Asn Thr Ala              20 25 30 Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln Thr          35 40 45 Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 <210> 26 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <223> C-term amidated <400> 26 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Lys Ala Asn Thr Ala              20 25 30 Thr Ala Val Leu Gly          35 <210> 27 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) <223> 12-Ado <220> <223> C-term amidated <400> 27 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn              20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu          35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60 <210> 28 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) <223> 12-Ado <220> <223> C-term amidated <400> 28 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr              20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln          35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60                                                                   <210> 29 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) 3223-dioxactoanoyl <220> <223> C-term amidated <400> 29 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn              20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu          35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60 <210> 30 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) 3223-dioxactoanoyl <220> <223> C-term amidated <400> 30 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr              20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln          35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60                                                             <210> 31 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) <223> 5-Apa <220> <223> C-term amidated <400> 31 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn              20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu          35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60 <210> 32 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) <223> 5-Apa <220> <223> C-term amidated <400> 32 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr              20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln          35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60                                                                   <210> 33 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 33 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Lys Cys              20 25 30 Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg          35 40 45 Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60 <210> 34 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (30) <223> Beta-alanine <220> <223> C-term amidated <400> 34 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Cys Asn              20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu          35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60 <210> 35 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) <223> 4,7,10-trioxa-13-tridecanamine succinimidyl <220> <223> C-term amidated <400> 35 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn              20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu          35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60 <210> 36 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric       lizard, human and salmon construct <220> <221> MOD_RES <222> (29) <223> 4,7,10-trioxa-13-tridecanamine succinimidyl <220> <223> C-term amidated <400> 36 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr              20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln          35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr      50 55 60                                                                   <210> 37 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric human       and salmon construct <220> <221> MOD_RES <222> (2) <223> Tyr (SO 3) <220> <223> C-term amidated <400> 37 Asp Tyr Met Gly Trp Met Asp Phe Gly Lys Arg Lys Cys Asn Thr Ala   1 5 10 15 Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu Val Arg Leu Gln Thr              20 25 30 Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr          35 40 <210> 38 <211> 36 <212> PRT <213> Homo sapiens <220> <223> C-term amidated <400> 38 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val Arg Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 39 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       polypeptide <220> <221> MOD_RES <222> (1) <223> Isocaproyl-Ser <220> <221> MOD_RES <222> (6) <223> Aib <220> <221> MOD_RES <222> (7) <223> Lys (formyl) <220> <221> MOD_RES <222> (13) <223> Aib <220> <221> MOD_RES <222> (14) <223> Lys (formyl) <220> <223> C-term amidated <400> 39 Ser Thr Ala Val Leu Xaa Lys Leu Ser Gln Glu Leu Xaa Lys Leu Gln   1 5 10 15 Thr Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 40 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       polypeptide <220> <221> MOD_RES <222> (1) <223> Isocaproyl-Ser <220> <221> MOD_RES <222> (6) <223> Aib <220> <221> MOD_RES <222> (7) <223> Lys (formyl) <220> <221> MOD_RES <222> (13) <223> Aib <220> <221> MOD_RES <222> (14) <223> Lys (formyl) <220> <223> C-term amidated <400> 40 Ser Thr Ala Val Leu Xaa Lys Leu Ser Gln Glu Leu Xaa Lys Leu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 41 <211> 43 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (1) <223> Amidated terminus <220> <221> MOD_RES <222> (7) <223> Sulfonated Tyr <220> <221> MOD_RES <222> (9) <223> Succinoyl-Cys <400> 41 Phe Asp Met Trp Gly Met Tyr Asp Cys Ile Lys Pro Glu Ala Pro Gly   1 5 10 15 Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg              20 25 30 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 42 <211> 43 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (1) <223> Amidated terminus <220> <221> MOD_RES <222> (7) <223> Sulfonated Tyr <220> <221> MOD_RES <222> (9) <223> Bis-Cys (N-Acetyl) <400> 42 Phe Asp Met Trp Gly Met Tyr Asp Cys Ile Lys Pro Glu Ala Pro Gly   1 5 10 15 Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg              20 25 30 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 43 <211> 43 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (1) <223> Amidated terminus <220> <221> MOD_RES <222> (7) <223> Sulfonated Tyr <220> <221> MOD_RES <222> (9) Gly-Aminoxymethylcarbonyl <220> <223> C-term amidated <400> 43 Phe Asp Met Trp Gly Met Tyr Asp Gly Ile Lys Pro Glu Ala Pro Gly   1 5 10 15 Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg              20 25 30 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr          35 40 <210> 44 <211> 37 <212> PRT <213> Rattus sp. <400> 44 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 45 <211> 37 <212> PRT <213> Homo sapiens <400> 45 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 46 <211> 52 <212> PRT <213> Homo sapiens <400> 46 Tyr Arg Gln Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys   1 5 10 15 Arg Phe Gly Thr Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln              20 25 30 Phe Thr Asp Lys Asp Lys Asp Asn Val Ala Pro Arg Ser Lys Ile Ser          35 40 45 Pro Gln Gly Tyr      50 <210> 47 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 47 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu   1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro              20 25 30                                                                  <210> 48 <211> 32 <212> PRT <213> Homo sapiens <400> 48 Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe   1 5 10 15 Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro              20 25 30                                                                   <210> 49 <211> 37 <212> PRT <213> Homo sapiens <400> 49 Ala Cys Asp Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Val Val Lys Asn Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Ala Phe          35 <210> 50 <211> 37 <212> PRT <213> Homo sapiens <400> 50 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Ala Phe          35 <210> 51 <211> 47 <212> PRT <213> Homo sapiens <400> 51 Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly              20 25 30 Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr          35 40 45 <210> 52 <211> 40 <212> PRT <213> Homo sapiens <400> 52 Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg   1 5 10 15 Leu Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala Pro Val              20 25 30 Asp Pro Ser Ser Pro His Ser Tyr          35 40 <210> 53 <211> 47 <212> PRT <213> Mus musculus <400> 53 Pro His Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Val Arg Pro Ala Gly              20 25 30 Arg Arg Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr          35 40 45 <210> 54 <211> 40 <212> PRT <213> Mus musculus <400> 54 Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg   1 5 10 15 Leu Trp Gln Leu Val Arg Pro Ala Gly Arg Arg Asp Ser Ala Pro Val              20 25 30 Asp Pro Ser Ser Pro His Ser Tyr          35 40 <210> 55 <211> 8 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (2) <223> Tyr (SO 3) <400> 55 Asp Tyr Met Gly Trp Met Asp Phe   1 5 <210> 56 <211> 167 <212> PRT <213> Homo sapiens <400> 56 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 57 <211> 36 <212> PRT <213> Homo sapiens <400> 57 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 58 <211> 34 <212> PRT <213> Homo sapiens <400> 58 Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn   1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln              20 25 30 Arg Tyr         <210> 59 <211> 37 <212> PRT <213> Homo sapiens <400> 59 His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val   1 5 10 15 Ser Ser Thr Leu Glu Gly Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu              20 25 30 Val Lys Gly Arg Gly          35 <210> 60 <211> 37 <212> PRT <213> Xenopus laevis <220> <221> MOD_RES <222> (37) <223> Ser-OH <400> 60 His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu   1 5 10 15 Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Pro Lys              20 25 30 Lys Ile Arg Tyr Ser          35 <210> 61 <211> 30 <212> PRT <213> Homo sapiens <400> 61 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg              20 25 30 <210> 62 <211> 33 <212> PRT <213> Homo sapiens <400> 62 His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn   1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Glu Thr Lys Ile Thr              20 25 30 Asp     <210> 63 <211> 31 <212> PRT <213> Xenopus sp. <220> <221> MOD_RES <222> (31) <223> Pro-OH <400> 63 His Ala Glu Gly Thr Phe Thr Asn Asp Met Thr Asn Tyr Leu Glu Glu   1 5 10 15 Lys Ala Ala Lys Glu Phe Val Gly Trp Leu Ile Lys Gly Arg Pro              20 25 30 <210> 64 <211> 37 <212> PRT <213> Homo sapiens <400> 64 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser   1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn              20 25 30 Arg Asn Asn Ile Ala          35 <210> 65 <211> 39 <212> PRT <213> Heloderma suspectum <400> 65 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser          35 <210> 66 <211> 39 <212> PRT <213> Heloderma suspectum <400> 66 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser          35 <210> 67 <211> 37 <212> PRT <213> Homo sapiens <400> 67 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 68 <211> 36 <212> PRT <213> Homo sapiens <400> 68 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val   1 5 10 15 His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 69 <211> 37 <212> PRT <213> Homo sapiens <400> 69 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro Val Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 70 <211> 37 <212> PRT <213> Homo sapiens <400> 70 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val Arg Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 71 <211> 36 <212> PRT <213> Homo sapiens <400> 71 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val   1 5 10 15 Arg Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 72 <211> 37 <212> PRT <213> Homo sapiens <400> 72 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val Arg Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 73 <211> 36 <212> PRT <213> Homo sapiens <400> 73 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val   1 5 10 15 Arg Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 74 <211> 36 <212> PRT <213> Homo sapiens <400> 74 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val   1 5 10 15 His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 75 <211> 37 <212> PRT <213> Homo sapiens <400> 75 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro Val Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 76 <211> 37 <212> PRT <213> Homo sapiens <220> 2,7-Cyclo bridge <400> 76 Lys Asp Asn Thr Ala Thr Lys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Pro Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 77 <211> 36 <212> PRT <213> Homo sapiens <400> 77 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val   1 5 10 15 His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 78 <211> 37 <212> PRT <213> Homo sapiens <400> 78 Ala Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 79 <211> 37 <212> PRT <213> Homo sapiens <400> 79 Lys Ala Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 80 <211> 37 <212> PRT <213> Homo sapiens <400> 80 Lys Ala Asn Thr Ala Thr Ala Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 81 <211> 37 <212> PRT <213> Homo sapiens <400> 81 Ser Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 82 <211> 37 <212> PRT <213> Homo sapiens <400> 82 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 83 <211> 37 <212> PRT <213> Homo sapiens <400> 83 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 84 <211> 36 <212> PRT <213> Homo sapiens <400> 84 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val   1 5 10 15 His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 85 <211> 37 <212> PRT <213> Homo sapiens <400> 85 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 86 <211> 37 <212> PRT <213> Homo sapiens <400> 86 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 87 <211> 36 <212> PRT <213> Homo sapiens <400> 87 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val   1 5 10 15 His Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Ser Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 88 <211> 37 <212> PRT <213> Homo sapiens <400> 88 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 89 <211> 37 <212> PRT <213> Homo sapiens <400> 89 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro Ile Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 90 <211> 37 <212> PRT <213> Homo sapiens <400> 90 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro Ile Leu Pro Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 91 <211> 37 <212> PRT <213> Homo sapiens <400> 91 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Ile His Ser Ser Asn Asn Leu Gly Pro Ile Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 92 <211> 37 <212> PRT <213> Homo sapiens <400> 92 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Ile His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 93 <211> 36 <212> PRT <213> Homo sapiens <400> 93 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Ile   1 5 10 15 His Ser Ser Asn Asn Leu Gly Pro Ile Leu Pro Pro Thr Asn Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 94 <211> 37 <212> PRT <213> Homo sapiens <400> 94 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Ile Arg Ser Ser Asn Asn Leu Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 95 <211> 37 <212> PRT <213> Homo sapiens <400> 95 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Ile Arg Ser Ser Asn Asn Leu Gly Ala Val Leu Ser Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 96 <211> 37 <212> PRT <213> Homo sapiens <400> 96 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Ile Arg Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Pro Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 97 <211> 37 <212> PRT <213> Homo sapiens <400> 97 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu   1 5 10 15 Val His Ser Ser His Asn Leu Gly Ala Ala Leu Leu Pro Thr Asp Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 98 <211> 37 <212> PRT <213> Homo sapiens <400> 98 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu   1 5 10 15 Val His Ser Ser His Asn Leu Gly Ala Ala Ser Leu Pro Thr Asp Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 99 <211> 36 <212> PRT <213> Homo sapiens <400> 99 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu Val   1 5 10 15 His Ser Ser His Asn Leu Gly Ala Ala Leu Pro Ser Thr Asp Val Gly              20 25 30 Ser Asn Thr Tyr          35 <210> 100 <211> 37 <212> PRT <213> Homo sapiens <400> 100 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu   1 5 10 15 Val Arg Ser Ser His Asn Leu Gly Ala Ala Leu Ser Pro Thr Asp Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 101 <211> 37 <212> PRT <213> Homo sapiens <400> 101 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu   1 5 10 15 Val Arg Ser Ser His Asn Leu Gly Ala Ile Leu Pro Pro Thr Asp Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 102 <211> 37 <212> PRT <213> Homo sapiens <400> 102 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu   1 5 10 15 Val Arg Ser Ser His Asn Leu Gly Pro Ala Leu Pro Pro Thr Asp Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 103 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 103 Cys Ser Asn Leu Ser Thr Cys Gly Leu Gly Lys Leu Ser Glu Glu Leu   1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Arg              20 25 30                                                                  <210> 104 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 104 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Glu Glu Leu   1 5 10 15 His Lys Leu Gln Thr Leu Pro Arg Thr Asn Thr Gly Ser Gly Thr Arg              20 25 30                                                                   <210> 105 <211> 31 <212> PRT <213> Oncorhynchus sp. <400> 105 Cys Gly Ser Leu Ser Thr Cys Gly Leu Gly Lys Leu Ser Glu Glu Leu   1 5 10 15 His Lys Leu Gln Thr Pro Arg Thr Asn Thr Gly Ser Gly Thr Arg              20 25 30 <210> 106 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 106 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Glu Glu Leu   1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro              20 25 30                                                                   <210> 107 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 107 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu   1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro              20 25 30                                                                  <210> 108 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 108 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu   1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro              20 25 30                                                                   <210> 109 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 109 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Glu Glu Leu   1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro              20 25 30                                                                   <210> 110 <211> 32 <212> PRT <213> Oncorhynchus sp. <400> 110 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu   1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro              20 25 30                                                                  <210> 111 <211> 37 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (36) <223> D-Ser <400> 111 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Ser Phe          35 <210> 112 <211> 37 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (36) <223> D-Thr <400> 112 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Thr Phe          35 <210> 113 <211> 37 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (36) <223> D-Asp <400> 113 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Asp Phe          35 <210> 114 <211> 37 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (36) <223> D-Asn <400> 114 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Asn Phe          35 <210> 115 <211> 37 <212> PRT <213> Homo sapiens <400> 115 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Ser Phe          35 <210> 116 <211> 37 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (36) <223> Hse <400> 116 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Xaa Phe          35 <210> 117 <211> 37 <212> PRT <213> Homo sapiens <400> 117 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Asp Phe          35 <210> 118 <211> 37 <212> PRT <213> Homo sapiens <400> 118 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Thr Phe          35 <210> 119 <211> 37 <212> PRT <213> Homo sapiens <400> 119 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu   1 5 10 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val              20 25 30 Gly Ser Lys Asn Phe          35 <210> 120 <211> 48 <212> PRT <213> Mus musculus <400> 120 Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Gly Asn Leu Ser Thr Cys   1 5 10 15 Gln Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala              20 25 30 Gly Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr          35 40 45                                                                   <210> 121 <211> 47 <212> PRT <213> Mus musculus <400> 121 Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Asp Thr Ala Thr Cys Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly              20 25 30 Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr          35 40 45 <210> 122 <211> 47 <212> PRT <213> Mus musculus <400> 122 Thr Gln Ala Gln Leu Leu Arg Val Gly Met Val Leu Gly Thr Met Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly              20 25 30 Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr          35 40 45 <210> 123 <211> 47 <212> PRT <213> Mus musculus <400> 123 Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly              20 25 30 Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro His Ser Tyr          35 40 45 <210> 124 <211> 47 <212> PRT <213> Mus musculus <400> 124 Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly              20 25 30 Arg Gln Glu Ser Ala Pro Val Glu Pro Ser Ser Pro His Ser Tyr          35 40 45 <210> 125 <211> 42 <212> PRT <213> Mus musculus <400> 125 Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Arg Gln Asp Ser Ala              20 25 30 Pro Val Asp Pro Ser Ser Pro His Ser Tyr          35 40 <210> 126 <211> 40 <212> PRT <213> Mus musculus <400> 126 Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln   1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Asp Ser Ala Pro Val              20 25 30 Asp Pro Ser Ser Pro His Ser Tyr          35 40 <210> 127 <211> 41 <212> PRT <213> Mus musculus <400> 127 Arg Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His   1 5 10 15 Arg Leu Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala Pro              20 25 30 Val Asp Pro Ser Ser Pro His Ser Tyr          35 40 <210> 128 <211> 40 <212> PRT <213> Mus musculus <400> 128 Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg   1 5 10 15 Leu Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala Pro Val              20 25 30 Glu Pro Ser Ser Pro His Ser Tyr          35 40 <210> 129 <211> 35 <212> PRT <213> Mus musculus <400> 129 Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg   1 5 10 15 Leu Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro              20 25 30 His Ser Tyr          35 <210> 130 <211> 39 <212> PRT <213> Mus musculus <400> 130 Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg Leu   1 5 10 15 Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala Pro Val Asp              20 25 30 Pro Ser Ser Pro His Ser Tyr          35 <210> 131 <211> 34 <212> PRT <213> Mus musculus <400> 131 Gly Cys Asn Thr Ala Thr Cys Gln Val Gln Asn Leu Ser His Arg Leu   1 5 10 15 Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His              20 25 30 Ser tyr         <210> 132 <211> 34 <212> PRT <213> Mus musculus <400> 132 Gly Cys Asn Thr Ala Thr Cys Gln Val Gln Asn Leu Ser His Arg Leu   1 5 10 15 Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro His              20 25 30 Ser tyr         <210> 133 <211> 35 <212> PRT <213> Mus musculus <133> 133 Gly Cys Ser Asn Leu Ser Thr Cys Gln Val Gln Asn Leu Ser His Arg   1 5 10 15 Leu Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro              20 25 30 His Ser Tyr          35 <210> 134 <211> 35 <212> PRT <213> Mus musculus <400> 134 Gly Cys Gly Asn Leu Ser Thr Cys Gln Val Gln Asn Leu Ser His Arg   1 5 10 15 Leu Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro              20 25 30 His Ser Tyr          35 <210> 135 <211> 34 <212> PRT <213> Mus musculus <400> 135 Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg Leu   1 5 10 15 Trp Gln Leu Arg Gln Glu Ser Ala Pro Val Glu Pro Ser Ser Pro His              20 25 30 Ser tyr         <210> 136 <211> 38 <212> PRT <213> Mus musculus <400> 136 Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg Leu Trp   1 5 10 15 Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala Pro Val Asp Pro              20 25 30 Ser Ser Pro His Ser Tyr          35 <210> 137 <211> 32 <212> PRT <213> Mus musculus <400> 137 Gln Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala   1 5 10 15 Gly Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr              20 25 30 <210> 138 <211> 31 <212> PRT <213> Mus musculus <400> 138 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly   1 5 10 15 Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr              20 25 30 <139> <211> 30 <212> PRT <213> Mus musculus <400> 139 Val Gln Asn Leu Ser His Arg Leu Gln Leu Met Gly Pro Ala Gly Arg   1 5 10 15 Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr              20 25 30 <210> 140 <211> 30 <212> PRT <213> Mus musculus <400> 140 Gly Thr Met Gln Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Arg   1 5 10 15 Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro His Ser Tyr              20 25 30 <210> 141 <211> 8 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (2) <223> Tyr (SO 3 H) <400> 141 Asp Tyr Met Gly Trp Met Asp Phe   1 5 <210> 142 <211> 8 <212> PRT <213> Homo sapiens <400> 142 Asp Tyr Met Gly Trp Met Asp Phe   1 5 <210> 143 <211> 6 <212> PRT <213> Homo sapiens <400> 143 Met Gly Trp Met Asp Phe   1 5 <210> 144 <211> 5 <212> PRT <213> Homo sapiens <400> 144 Gly Trp Met Asp Phe   1 5 <210> 145 <211> 4 <212> PRT <213> Homo sapiens <400> 145 Trp Met Asp Phe   One <210> 146 <211> 9 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (3) <223> Tyr (SO 3 H) <400> 146 Lys Asp Tyr Met Gly Trp Met Asp Phe   1 5 <210> 147 <211> 9 <212> PRT <213> Homo sapiens <400> 147 Lys Asp Tyr Met Gly Trp Met Asp Phe   1 5 <210> 148 <211> 7 <212> PRT <213> Homo sapiens <400> 148 Lys Met Gly Trp Met Asp Phe   1 5 <210> 149 <211> 6 <212> PRT <213> Homo sapiens <400> 149 Lys Gly Trp Met Asp Phe   1 5 <210> 150 <211> 5 <212> PRT <213> Homo sapiens <400> 150 Lys Trp Met Asp Phe   1 5 <210> 151 <211> 167 <212> PRT <213> Homo sapiens <400> 151 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asp Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 152 <211> 167 <212> PRT <213> Homo sapiens <400> 152 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Glu Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 153 <211> 167 <212> PRT <213> Homo sapiens <400> 153 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Ala          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 154 <211> 167 <212> PRT <213> Homo sapiens <400> 154 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Glu Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 155 <211> 166 <212> PRT <213> Homo sapiens <400> 155 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly      50 55 60 Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val  65 70 75 80 Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile                  85 90 95 Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe             100 105 110 Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp         115 120 125 Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val     130 135 140 Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu 145 150 155 160 Asp Leu Ser Pro Gly Cys                 165 <210> 156 <211> 167 <212> PRT <213> Homo sapiens <400> 156 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Ala Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 157 <211> 167 <212> PRT <213> Homo sapiens <400> 157 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 158 <211> 167 <212> PRT <213> Homo sapiens <400> 158 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asp Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 159 <211> 167 <212> PRT <213> Homo sapiens <400> 159 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Glu Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 160 <211> 167 <212> PRT <213> Homo sapiens <400> 160 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ala Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 161 <211> 167 <212> PRT <213> Homo sapiens <400> 161 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Ser His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 162 <211> 167 <212> PRT <213> Homo sapiens <400> 162 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 163 <211> 167 <212> PRT <213> Homo sapiens <400> 163 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Ser                 165 <210> 164 <211> 167 <212> PRT <213> Homo sapiens <400> 164 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asp Asp Ile Ser His Thr          35 40 45 Glu Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 165 <211> 167 <212> PRT <213> Homo sapiens <400> 165 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asp Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Ala Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 166 <211> 167 <212> PRT <213> Homo sapiens <400> 166 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Ser His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys                 165 <210> 167 <211> 167 <212> PRT <213> Homo sapiens <400> 167 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu   1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys              20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr          35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro      50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala  65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Glu Val Ile Gln                  85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala             100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu         115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val     130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Ser                 165 <210> 168 <211> 31 <212> PRT <213> Homo sapiens <400> 168 His Ala Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 169 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (3) <223> D-Gln <400> 169 His Ala Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 170 <211> 31 <212> PRT <213> Homo sapiens <400> 170 His Ala Glu Gly Thr Phe Thr Ser Asp Thr Ser Lys Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 171 <211> 31 <212> PRT <213> Homo sapiens <400> 171 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 172 <211> 30 <212> PRT <213> Homo sapiens <400> 172 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg              20 25 30 <210> 173 <211> 31 <212> PRT <213> Homo sapiens <400> 173 His Ala Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 174 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (3) <223> D-Gln <400> 174 His Ala Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <175> 175 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (3) 223 acetyl-Lys <400> 175 His Ala Lys Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30                                                         <210> 176 <211> 31 <212> PRT <213> Homo sapiens <400> 176 His Ala Thr Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 177 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (3) <223> D-Thr <400> 177 His Ala Thr Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 178 <211> 31 <212> PRT <213> Homo sapiens <400> 178 His Ala Asn Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 179 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (3) <223> D-Asn <400> 179 His Ala Asn Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 180 <211> 31 <212> PRT <213> Homo sapiens <400> 180 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Ser   1 5 10 15 Arg Arg Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 181 <211> 31 <212> PRT <213> Homo sapiens <400> 181 His Ala Glu Gly Thr Phe Thr Ser Asp Thr Ser Lys Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 182 <211> 31 <212> PRT <213> Homo sapiens <400> 182 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 183 <211> 31 <212> PRT <213> Homo sapiens <400> 183 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Arg Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 184 <211> 31 <212> PRT <213> Homo sapiens <400> 184 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly   1 5 10 15 Gln Arg Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 185 <211> 39 <212> PRT <213> Heloderma suspectum <400> 185 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser          35 <210> 186 <211> 39 <212> PRT <213> Heloderma suspectum <400> 186 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser          35 <210> 187 <211> 39 <212> PRT <213> Heloderma suspectum <400> 187 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Ala Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Pro Ser          35 <210> 188 <211> 37 <212> PRT <213> Xenopus laevis <220> <221> MOD_RES <222> (37) <223> Ser-OH <400> 188 His Ala Glu Gly Thr Phe Thr Ser Asp Val Thr Gln Gln Leu Asp Glu   1 5 10 15 Lys Ala Ala Lys Glu Phe Ile Asp Trp Leu Ile Asn Gly Gly Pro Ser              20 25 30 Lys Glu Ile Ile Ser          35 <210> 189 <211> 37 <212> PRT <213> Homo sapiens <400> 189 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Pro Ser Thr Asn Val              20 25 30 Gly Ser Asn Thr Tyr          35 <210> 190 <211> 28 <212> PRT <213> Heloderma suspectum <400> 190 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn              20 25 <210> 191 <211> 36 <212> PRT <213> Homo sapiens <400> 191 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 192 <211> 36 <212> PRT <213> Homo sapiens <400> 192 Tyr Pro Leu Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 193 <211> 36 <212> PRT <213> Homo sapiens <400> 193 Tyr Pro Val Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 194 <211> 36 <212> PRT <213> Homo sapiens <400> 194 Tyr Pro Ile Arg Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 195 <211> 36 <212> PRT <213> Homo sapiens <400> 195 Tyr Pro Ile Gln Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 196 <211> 36 <212> PRT <213> Homo sapiens <400> 196 Tyr Pro Ile Asn Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 197 <211> 36 <212> PRT <213> Homo sapiens <400> 197 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Lys His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 198 <211> 36 <212> PRT <213> Homo sapiens <400> 198 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 199 <211> 36 <212> PRT <213> Homo sapiens <400> 199 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg His Arg Tyr          35 <210> 200 <211> 36 <212> PRT <213> Homo sapiens <400> 200 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Pro Ala Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 201 <211> 36 <212> PRT <213> Homo sapiens <400> 201 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 202 <211> 35 <212> PRT <213> Homo sapiens <400> 202 Tyr Pro Ile Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu   1 5 10 15 Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg              20 25 30 Gln Arg Tyr          35 <210> 203 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chimeric human       and salmon construct <220> <221> MOD_RES <222> (2) Phe (CH 2 SO 3) <400> 203 Asp Phe Met Gly Trp Met Asp Phe Gly Lys Arg Lys Cys Asn Thr Ala   1 5 10 15 Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu Val Arg Leu Gln Thr              20 25 30 Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr          35 40 <210> 204 <211> 31 <212> PRT <213> Homo sapiens <400> 204 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Thr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 205 <211> 29 <212> PRT <213> Homo sapiens <400> 205 Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Thr Leu Glu Gly Gln Ala   1 5 10 15 Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 <206> 206 <211> 28 <212> PRT <213> Homo sapiens <400> 206 Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Thr Leu Glu Gly Gln Ala   1 5 10 15 Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg              20 25 <210> 207 <211> 31 <212> PRT <213> Heloderma suspectum <400> 207 Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu   1 5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser              20 25 30 <210> 208 <211> 4 <212> PRT <213> Homo sapiens <400> 208 Trp Met Asp Phe   One <210> 209 <211> 5 <212> PRT <213> Homo sapiens <400> 209 Gly Trp Met Asp Phe   1 5 <210> 210 <211> 36 <212> PRT <213> Homo sapiens <400> 210 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly ser asn thr          35 <210> 211 <211> 35 <212> PRT <213> Homo sapiens <400> 211 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val              20 25 30 Gly ser asn          35 <210> 212 <211> 20 <212> PRT <213> Homo sapiens <400> 212 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val His Ser Ser              20 <210> 213 <211> 18 <212> PRT <213> Homo sapiens <400> 213 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val his         <210> 214 <211> 17 <212> PRT <213> Homo sapiens <400> 214 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 Val     <210> 215 <211> 16 <212> PRT <213> Homo sapiens <400> 215 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu   1 5 10 15 <210> 216 <211> 15 <212> PRT <213> Homo sapiens <400> 216 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe   1 5 10 15 <210> 217 <211> 7 <212> PRT <213> Homo sapiens <400> 217 Lys Cys Asn Thr Ala Thr Cys   1 5 <210> 218 <211> 25 <212> PRT <213> Homo sapiens <400> 218 Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro   1 5 10 15 Gln Thr Ala Ile Gly Val Gly Ala Pro              20 25 <210> 219 <211> 20 <212> PRT <213> Homo sapiens <400> 219 Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro   1 5 10 15 Gln Thr Ala Ile              20 <210> 220 <211> 19 <212> PRT <213> Homo sapiens <400> 220 Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro   1 5 10 15 Gln thr ala             <210> 221 <211> 3 <212> PRT <213> Homo sapiens <400> 221 Met leu gly   One <210> 222 <211> 9 <212> PRT <213> Homo sapiens <400> 222 Lys Phe His Thr Phe Pro Gln Thr Ala   1 5 <210> 223 <211> 10 <212> PRT <213> Homo sapiens <400> 223 Lys Phe His Thr Phe Pro Gln Thr Ala Ile   1 5 10 <210> 224 <211> 10 <212> PRT <213> Homo sapiens <400> 224 Gln Asn Leu Ser His Arg Leu Trp Gln Leu   1 5 10 <210> 225 <211> 146 <212> PRT <213> Homo sapiens <400> 225 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr   1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser              20 25 30 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile          35 40 45 Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile      50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu  65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys                  85 90 95 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly             100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 226 <211> 18 <212> PRT <213> Homo sapiens <400> 226 Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr   1 5 10 15 Leu ser         <210> 227 <211> 35 <212> PRT <213> Homo sapiens <400> 227 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg          35 <210> 228 <211> 30 <212> PRT <213> Homo sapiens <400> 228 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu              20 25 30 <210> 229 <211> 25 <212> PRT <213> Homo sapiens <400> 229 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg              20 25 <210> 230 <211> 15 <212> PRT <213> Homo sapiens <400> 230 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu   1 5 10 15 <210> 231 <211> 10 <212> PRT <213> Homo sapiens <400> 231 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu   1 5 10 <210> 232 <211> 35 <212> PRT <213> Homo sapiens <400> 232 Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu   1 5 10 15 Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg              20 25 30 Gln Arg Tyr          35 <210> 233 <211> 33 <212> PRT <213> Homo sapiens <400> 233 Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg   1 5 10 15 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg              20 25 30 Tyr     <210> 234 <211> 32 <212> PRT <213> Homo sapiens <400> 234 Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr   1 5 10 15 Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 30                                                                   <210> 235 <211> 29 <212> PRT <213> Homo sapiens <400> 235 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Thr Leu Glu Gly   1 5 10 15 Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly              20 25 <210> 236 <211> 27 <212> PRT <213> Heloderma suspectum <400> 236 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys              20 25 <210> 237 <211> 28 <212> PRT <213> Heloderma suspectum <400> 237 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn              20 25 <210> 238 <211> 29 <212> PRT <213> Heloderma suspectum <400> 238 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly              20 25 <210> 239 <211> 30 <212> PRT <213> Heloderma suspectum <400> 239 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly              20 25 30 <210> 240 <211> 28 <212> PRT <213> Heloderma suspectum <400> 240 His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn              20 25 <210> 241 <211> 27 <212> PRT <213> Heloderma suspectum <400> 241 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys              20 25 <210> 242 <211> 6 <212> PRT <213> Homo sapiens <400> 242 Val Gly Ser Asn Thr Tyr   1 5 <210> 243 <211> 5 <212> PRT <213> Homo sapiens <400> 243 Gly Ser Asn Thr Tyr   1 5 <210> 244 <211> 4 <212> PRT <213> Homo sapiens <400> 244 Ser Asn Thr Tyr   One <210> 245 <211> 6 <212> PRT <213> Homo sapiens <400> 245 Ile Ser Pro Gln Gly Tyr   1 5 <210> 246 <211> 5 <212> PRT <213> Homo sapiens <400> 246 Ser Pro Gln Gly Tyr   1 5 <210> 247 <211> 4 <212> PRT <213> Homo sapiens <400> 247 Pro Gln Gly Tyr   One <210> 248 <211> 6 <212> PRT <213> Homo sapiens <400> 248 Ile Gly Val Gly Ala Pro   1 5 <210> 249 <211> 5 <212> PRT <213> Homo sapiens <400> 249 Gly Val Gly Ala Pro   1 5 <210> 250 <211> 4 <212> PRT <213> Homo sapiens <400> 250 Val Gly Ala Pro   One <210> 251 <211> 6 <212> PRT <213> Homo sapiens <400> 251 Val Gly Ser Lys Ala Phe   1 5 <210> 252 <211> 5 <212> PRT <213> Homo sapiens <400> 252 Gly Ser Lys Ala Phe   1 5 <210> 253 <211> 4 <212> PRT <213> Homo sapiens <400> 253 Ser Lys Ala Phe   One <210> 254 <211> 6 <212> PRT <213> Homo sapiens <400> 254 Ser Ser Pro His Ser Tyr   1 5 <210> 255 <211> 5 <212> PRT <213> Homo sapiens <400> 255 Ser Pro His Ser Tyr   1 5 <210> 256 <211> 4 <212> PRT <213> Homo sapiens <400> 256 Pro His Ser Tyr   One <210> 257 <211> 12 <212> PRT <213> Homo sapiens <400> 257 Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr   1 5 10 <210> 258 <211> 11 <212> PRT <213> Homo sapiens <400> 258 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr   1 5 10 <210> 259 <211> 10 <212> PRT <213> Homo sapiens <400> 259 Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr   1 5 10 <210> 260 <211> 9 <212> PRT <213> Homo sapiens <400> 260 Leu Asn Leu Val Thr Arg Gln Arg Tyr   1 5 <210> 261 <211> 8 <212> PRT <213> Homo sapiens <400> 261 Asn Leu Val Thr Arg Gln Arg Tyr   1 5 <210> 262 <211> 7 <212> PRT <213> Homo sapiens <400> 262 Leu Val Thr Arg Gln Arg Tyr   1 5 <210> 263 <211> 6 <212> PRT <213> Homo sapiens <400> 263 Val Thr Arg Gln Arg Tyr   1 5 <210> 264 <211> 5 <212> PRT <213> Homo sapiens <400> 264 Thr Arg Gln Arg Tyr   1 5 <210> 265 <211> 11 <212> PRT <213> Homo sapiens <400> 265 Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg   1 5 10 <210> 266 <211> 10 <212> PRT <213> Homo sapiens <400> 266 His Tyr Leu Asn Leu Val Thr Arg Gln Arg   1 5 10 <210> 267 <211> 9 <212> PRT <213> Homo sapiens <400> 267 Tyr Leu Asn Leu Val Thr Arg Gln Arg   1 5 <210> 268 <211> 8 <212> PRT <213> Homo sapiens <400> 268 Leu Asn Leu Val Thr Arg Gln Arg   1 5 <210> 269 <211> 7 <212> PRT <213> Homo sapiens <400> 269 Asn Leu Val Thr Arg Gln Arg   1 5 <210> 270 <211> 6 <212> PRT <213> Homo sapiens <400> 270 Leu Val Thr Arg Gln Arg   1 5 <210> 271 <211> 5 <212> PRT <213> Homo sapiens <400> 271 Val Thr Arg Gln Arg   1 5 <210> 272 <211> 4 <212> PRT <213> Homo sapiens <400> 272 Thr Arg Gln Arg   One <210> 273 <211> 9 <212> PRT <213> Xenopus sp. <400> 273 Gly Lys Pro Lys Lys Ile Arg Tyr Ser   1 5 <210> 274 <211> 8 <212> PRT <213> Xenopus sp. <400> 274 Lys Pro Lys Lys Ile Arg Tyr Ser   1 5 <210> 275 <211> 8 <212> PRT <213> Xenopus sp. <400> 275 Gly Trp Leu Ile Lys Gly Arg Pro   1 5 <210> 276 <211> 7 <212> PRT <213> Xenopus sp. <400> 276 Trp Leu Ile Lys Gly Arg Pro   1 5 <210> 277 <211> 9 <212> PRT <213> Heloderma suspectum <400> 277 Pro Ser Ser Gly Ala Pro Pro Pro Ser   1 5 <210> 278 <211> 8 <212> PRT <213> Heloderma suspectum <400> 278 Ser Ser Gly Ala Pro Pro Pro Ser   1 5 <210> 279 <211> 7 <212> PRT <213> Heloderma suspectum <400> 279 Ser Gly Ala Pro Pro Pro Ser   1 5 <210> 280 <211> 6 <212> PRT <213> Heloderma suspectum <400> 280 Gly Ala Pro Pro Pro Ser   1 5 <210> 281 <211> 5 <212> PRT <213> Heloderma suspectum <400> 281 Ala Pro Pro Pro Ser   1 5 <210> 282 <211> 4 <212> PRT <213> Heloderma suspectum <400> 282 Pro Pro Pro Ser   One <210> 283 <211> 30 <212> PRT <213> Xenopus sp. <400> 283 Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu Lys Ala Ala Lys Glu Phe   1 5 10 15 Ile Glu Trp Leu Ile Lys Gly Lys Pro Lys Lys Ile Arg Tyr              20 25 30 <210> 284 <211> 28 <212> PRT <213> Heloderma suspectum <400> 284 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn              20 25 <210> 285 <211> 7 <212> PRT <213> Homo sapiens <400> 285 Lys Ala Asn Thr Ala Thr Ala   1 5 <210> 286 <211> 20 <212> PRT <213> Oncorhynchus sp. <400> 286 Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu Gln Thr Tyr Pro   1 5 10 15 Arg Thr Asn Thr              20 <210> 287 <211> 8 <212> PRT <213> Homo sapiens <400> 287 Asp Phe Met Gly Trp Met Asp Phe   1 5 <210> 288 <211> 20 <212> PRT <213> Oncorhynchus sp. <400> 288 Val Leu Gly Lys Leu Ser Glu Glu Leu His Lys Leu Gln Thr Tyr Pro   1 5 10 15 Arg Thr Asn Thr              20 <210> 289 <211> 20 <212> PRT <213> artificial <220> <223> Chimeric human and salmon construct <400> 289 Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln Thr Tyr Pro 1 5 10 15 Arg Thr Asn Thr             20 <210> 290 <211> 36 <212> PRT <213> homo sapiens <400> 290 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln 1 5 10 15 Met Ala Gln Tyr Ala Ala Asp Leu Arg Arg Tyr Ile Asn Met Leu Thr             20 25 30 Arg Pro Arg Tyr         35 <210> 291 <211> 38 <212> PRT <213> mus musculus <220> <221> misc_feature <223> C-term amidated <400> 291 Val Ile Leu Ser Leu Asp Val Pro Ile Gly Leu Leu Arg Ile Leu Leu 1 5 10 15 Glu Gln Ala Arg Tyr Lys Ala Ala Arg Asn Gln Ala Ala Thr Asn Ala             20 25 30 Gln Ile Leu Ala His Val         35 <210> 292 <211> 24 <212> PRT <213> Homo sapiens <400> 292 Trp Ser Pro Gly Ala Arg Asn Gln Gly Gly Gly Ala Arg Ala Leu Leu 1 5 10 15 Leu Leu Leu Ala Glu Arg Phe Pro             20 <210> 293 <211> 18 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 293 Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln Asn Arg Ile Ile Phe Asp 1 5 10 15 Ser val          <210> 294 <211> 40 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> C-term amidated <400> 294 Asp Asn Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg Thr 1 5 10 15 Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln             20 25 30 Asn Arg Ile Ile Phe Asp Ser Val         35 40 <210> 295 <211> 20 <212> PRT <213> Homo sapiens <400> 295 Ser Phe His Tyr Leu Arg Ser Arg Asp Ala Ser Ser Gly Glu Glu Glu 1 5 10 15 Glu Gly Lys Glu             20 <210> 296 <211> 13 <212> PRT <213> Homo sapiens <400> 296 Ala Gln Ala Ala Ala Asn Ala His Leu Met Ala Gln Ile 1 5 10 <210> 297 <211> 21 <212> PRT <213> Homo sapiens <400> 297 Asp Asn Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg Thr 1 5 10 15 Leu Leu Glu Leu Ala             20 <210> 298 <211> 26 <212> PRT <213> Homo sapiens <400> 298 Thr Lys Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu 1 5 10 15 Leu Phe Asn Ile Ala Lys Ala Lys Asn Leu             20 25 <210> 299 <211> 38 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 299 Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe 1 5 10 15 Asn Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala             20 25 30 His Leu Met Ala Gln Ile         35 <210> 300 <211> 38 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 300 Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe             20 25 30 Leu Phe Arg Pro Arg Asn         35 <210> 301 <211> 28 <212> PRT <213> Homo sapiens <400> 301 Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly 1 5 10 15 Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr             20 25 <210> 302 <211> 32 <212> PRT <213> rat <400> 302 Asn Ser Lys Met Ala His Ser Ser Ser Cys Phe Gly Gln Lys Ile Asp 1 5 10 15 Arg Ile Gly Ala Val Ser Arg Leu Gly Cys Asp Gly Leu Arg Leu Phe             20 25 30 <210> 303 <211> 32 <212> PRT <213> Homo sapiens <400> 303 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His             20 25 30 <210> 304 <211> 22 <212> PRT <213> porcine <400> 304 Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser 1 5 10 15 Met Ser Gly Leu Gly Cys             20 <210> 305 <211> 8 <212> PRT <213> porcine <220> <221> MISC_FEATURE <223> C-term amidated <400> 305 Tyr Phe Leu Phe Arg Pro Arg Asn 1 5 <210> 306 <211> 23 <212> PRT <213> rat <220> <221> MISC_FEATURE <223> C-term amidated <400> 306 Tyr Lys Val Asn Glu Tyr Gln Gly Pro Val Ala Pro Ser Gly Gly Phe 1 5 10 15 Phe Leu Phe Arg Pro Arg Asn             20 <210> 307 <211> 9 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 307 Gly Tyr Phe Leu Phe Arg Pro Arg Asn 1 5 <210> 308 <211> 25 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 308 Phe Arg Val Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg 1 5 10 15 Gly Tyr Phe Leu Phe Arg Pro Arg Asn             20 25 <210> 309 <211> 36 <212> PRT <213> Artificial <220> <223> Hybrid of human CCK and human PYY <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MISC_FEATURE <223> N-terminal (head) to N-terminal (head) linked <220> <221> MOD_RES (222) (1) .. (1) <223> C-term amidated <220> <221> MOD_RES (222) (7) .. (7) <223> Sulfated Tyrosine <220> <221> MOD_RES (222) (9) .. (9) <223> Succinoyl-Cys <400> 309 Phe Asp Met Trp Gly Met Tyr Asp Cys Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr             20 25 30 Arg Gln Arg Tyr         35 <210> 310 <211> 36 <212> PRT <213> Artificial <220> <223> Hybrid of human CCK and human PYY <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MISC_FEATURE <223> N-terminal (head) to N-terminal (head) linked <220> <221> MOD_RES (222) (1) .. (1) <223> C-term amidated <220> <221> MOD_RES (222) (7) .. (7) <223> Sulfated Tyrosine <220> <221> MOD_RES (222) (9) .. (9) <223> Bis-Cys (N-Acetyl) <400> 310 Phe Asp Met Trp Gly Met Tyr Asp Cys Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr             20 25 30 Arg Gln Arg Tyr         35 <210> 311 <211> 36 <212> PRT <213> Artificial <220> <223> Hybrid of human CCK and human PYY <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MISC_FEATURE <223> N-terminal (head) to N-terminal (head) linked <220> <221> MOD_RES (222) (1) .. (1) <223> C-term amidated <220> <221> MOD_RES (222) (7) .. (7) Para (CH2SO3) Phe <220> <221> MOD_RES (222) (9) .. (9) Gly-Aminoxymethylcarbonyl <400> 311 Phe Asp Met Trp Gly Met Phe Asp Gly Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr             20 25 30 Arg Gln Arg Tyr         35 <210> 312 <211> 63 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <400> 312 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Gly Lys             20 25 30 Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His         35 40 45 Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 313 <211> 62 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <400> 313 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Gly Cys             20 25 30 Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg         35 40 45 Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 314 <211> 25 <212> PRT <213> porcine <400> 314 Phe Lys Val Asp Glu Glu Phe Gln Gly Pro Ile Val Ser Gln Asn Arg 1 5 10 15 Arg Tyr Phe Leu Phe Arg Pro Arg Asn             20 25 <210> 315 <211> 33 <212> PRT <213> Homo sapiens <400> 315 Ile Leu Gln Arg Gly Ser Gly Thr Ala Ala Val Asp Phe Thr Lys Lys 1 5 10 15 Asp His Thr Ala Thr Trp Gly Arg Pro Phe Phe Leu Phe Arg Pro Arg             20 25 30 Asn      <210> 316 <211> 36 <212> PRT <213> Homo sapiens <400> 316 Leu Val Gln Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gln Gly 1 5 10 15 Gly Arg Arg Lys Phe Arg Arg Gln Arg Pro Arg Leu Ser His Lys Gly             20 25 30 Pro Met Pro Phe         35 <210> 317 <211> 14 <212> PRT <213> Homo sapiens <400> 317 Pro Glu Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe 1 5 10 <210> 318 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 318 Ser Arg Thr His Arg His Ser Met Glu Ile Arg Thr Pro Asp Ile Asn 1 5 10 15 Pro Ala Trp Tyr Ala Ser Arg Gly Ile Arg Pro Val Gly Arg Phe             20 25 30 <210> 319 <211> 20 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 319 Thr Pro Asp Ile Asn Pro Ala Trp Tyr Ala Ser Arg Gly Ile Arg Pro 1 5 10 15 Val Gly Arg Phe             20 <210> 320 <211> 34 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 320 Gln Leu Gly Pro Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys 1 5 10 15 Lys Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met             20 25 30 Asp phe          <210> 321 <211> 18 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MOD_RES (222) (1) .. (1) <223> pyroGlutamine <400> 321 Pro Glu Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met 1 5 10 15 Asp phe          <210> 322 <211> 5 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MOD_RES (222) (1) .. (1) <223> bAla <400> 322 Ala Trp Met Asp Phe 1 5 <210> 323 <211> 33 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 323 Lys Ala Pro Ser Gly Arg Met Ser Ile Val Lys Asn Leu Gln Asn Leu 1 5 10 15 Asp Pro Ser His Arg Ile Ser Asp Arg Asp Tyr Met Gly Trp Met Asp             20 25 30 Phe      <210> 324 <211> 29 <212> PRT <213> Homo sapiens <400> 324 Gln Glu Gly Ala Pro Pro Gln Gln Ser Ala Arg Arg Asp Arg Met Pro 1 5 10 15 Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys             20 25 <210> 325 <211> 17 <212> PRT <213> Homo sapiens <400> 325 Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 1 5 10 15 Lys      <210> 326 <211> 28 <212> PRT <213> Homo sapiens <400> 326 Ser Ala Asn Ser Asn Pro Ala Met Ala Pro Arg Glu Arg Lys Ala Gly 1 5 10 15 Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys             20 25 <210> 327 <211> 14 <212> PRT <213> Homo sapiens <400> 327 Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 1 5 10 <210> 328 <211> 27 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 328 Val Pro Leu Pro Ala Gly Gly Gly Thr Val Leu Thr Lys Met Tyr Pro 1 5 10 15 Arg Gly Asn His Trp Ala Val Gly His Leu Met             20 25 <210> 329 <211> 10 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 329 Gly Asn His Trp Ala Val Gly His Leu Met 1 5 10 <210> 330 <211> 30 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 330 Leu Ser Trp Asp Leu Pro Glu Pro Arg Ser Arg Ala Ser Lys Ile Arg 1 5 10 15 Val His Ser Arg Gly Asn Leu Trp Ala Thr Gly His Phe Met             20 25 30 <210> 331 <211> 10 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 331 Gly Asn Leu Trp Ala Thr Gly His Phe Met 1 5 10 <210> 332 <211> 9 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 332 Pro Phe Phe Leu Phe Arg Pro Arg Asn 1 5 <210> 333 <211> 21 <212> PRT <213> Homo sapiens <400> 333 Lys Ile Pro Tyr Ile Leu Lys Arg Gln Leu Tyr Glu Asn Lys Pro Arg 1 5 10 15 Arg Pro Tyr Ile Leu             20 <210> 334 <211> 13 <212> PRT <213> Homo sapiens <400> 334 Gln Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 5 10 <210> 335 <211> 54 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 335 Gly Thr Ser Leu Ser Pro Pro Pro Glu Ser Ser Gly Ser Pro Gln Gln 1 5 10 15 Pro Gly Leu Ser Ala Pro His Ser Arg Gln Ile Pro Ala Pro Gln Gly             20 25 30 Ala Val Leu Val Gln Arg Glu Lys Asp Leu Pro Asn Tyr Asn Trp Asn         35 40 45 Ser Phe Gly Leu Arg Phe     50 <210> 336 <211> 16 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 336 Glu Lys Asp Leu Pro Asn Tyr Asn Trp Asn Ser Phe Gly Leu Arg Phe 1 5 10 15 <210> 337 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 337 Ser Ala Gly Ala Thr Ala Asn Leu Pro Leu Arg Ser Gly Arg Asn Met 1 5 10 15 Glu Val Ser Leu Val Arg Arg Val Pro Asn Leu Pro Gln Arg Phe             20 25 30 <210> 338 <211> 8 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 338 Val Pro Asn Leu Pro Gln Arg Phe 1 5 <210> 339 <211> 32 <212> PRT <213> Homo sapiens <400> 339 Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys Trp Asp Asn 1 5 10 15 Gln Lys Arg Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Val Thr             20 25 30 <210> 340 <211> 13 <212> PRT <213> Homo sapiens <400> 340 Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Val Thr 1 5 10 <210> 341 <211> 14 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amide <400> 341 Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 1 5 10 <210> 342 <211> 38 <212> PRT <213> Homo sapiens <400> 342 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp Val Asn Thr Pro Glu His Val Val Pro Tyr Gly             20 25 30 Leu Gly Ser Pro Arg Ser         35 <210> 343 <211> 21 <212> PRT <213> Homo sapiens <400> 343 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp             20 <210> 344 <211> 37 <212> PRT <213> Homo sapiens <400> 344 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp Val Asn Thr Pro Glu Gln Thr Ala Pro Tyr Gly             20 25 30 Leu Gly Asn Pro Pro         35 <210> 345 <211> 21 <212> PRT <213> Homo sapiens <400> 345 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp             20 <210> 346 <211> 41 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 346 Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His 1 5 10 15 Leu Asp Ile Ile Trp Ile Asn Thr Pro Glu Gln Thr Val Pro Tyr Gly             20 25 30 Leu Ser Asn Tyr Arg Gly Ser Phe Arg         35 40 <210> 347 <211> 21 <212> PRT <213> Homo sapiens <400> 347 Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His 1 5 10 15 Leu Asp Ile Ile Trp             20 <210> 348 <211> 27 <212> PRT <213> Homo sapiens <400> 348 Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro             20 25 <210> 349 <211> 10 <212> PRT <213> Homo sapiens <400> 349 Gly Ser Ser Phe Leu Ser Pro Glu His Gln 1 5 10 <210> 350 <211> 37 <212> PRT <213> Homo sapiens <400> 350 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn             20 25 30 Arg Asn Asn Ile Ala         35 <210> 351 <211> 29 <212> PRT <213> Homo sapiens <400> 351 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr             20 25 <210> 352 <211> 42 <212> PRT <213> Homo sapiens <352> 352 Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10 15 Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys             20 25 30 Lys Asn Asp Trp Lys His Asn Ile Thr Gln         35 40 <210> 353 <211> 30 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 353 Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10 15 Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys             20 25 30 <210> 354 <211> 30 <212> PRT <213> Homo sapiens <400> 354 Trp Tyr Lys His Val Ala Ser Pro Arg Tyr His Thr Val Gly Arg Ala 1 5 10 15 Ala Gly Leu Leu Met Gly Leu Arg Arg Ser Pro Tyr Leu Trp             20 25 30 <210> 355 <211> 23 <212> PRT <213> Homo sapiens <400> 355 Trp Tyr Lys His Val Ala Ser Pro Arg Tyr His Thr Val Gly Arg Ala 1 5 10 15 Ala Gly Leu Leu Met Gly Leu             20 <210> 356 <211> 38 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 356 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys             20 25 30 Gln Arg Val Lys Asn Lys         35 <210> 357 <211> 27 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 357 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu             20 25 <210> 358 <211> 42 <212> PRT <213> Homo sapiens <400> 358 His Ala Asp Gly Val Phe Thr Ser Asp Phe Ser Lys Leu Leu Gly Gln 1 5 10 15 Leu Ser Ala Lys Lys Tyr Leu Glu Ser Leu Met Gly Lys Arg Val Ser             20 25 30 Ser Asn Ile Ser Glu Asp Pro Val Pro Val         35 40 <210> 359 <211> 27 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 359 His Ala Asp Gly Val Phe Thr Ser Asp Phe Ser Lys Leu Leu Gly Gln 1 5 10 15 Leu Ser Ala Lys Lys Tyr Leu Glu Ser Leu Met             20 25 <210> 360 <211> 44 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 360 Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln 1 5 10 15 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly             20 25 30 Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu         35 40 <210> 361 <211> 28 <212> PRT <213> Homo sapiens <400> 361 Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln 1 5 10 15 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser             20 25 <210> 362 <211> 84 <212> PRT <213> Homo sapiens <400> 362 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His             20 25 30 Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser         35 40 45 Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu     50 55 60 Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys 65 70 75 80 Ala Lys Ser Gln                  <210> 363 <211> 37 <212> PRT <213> Homo sapiens <400> 363 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His             20 25 30 Asn Phe Val Ala Leu         35 <210> 364 <211> 34 <212> PRT <213> Homo sapiens <400> 364 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His             20 25 30 Asn phe          <210> 365 <211> 93 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 365 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His             20 25 30 Thr Ala Glu Ile Arg Ala Thr Ser Glu Val Ser Pro Asn Ser Lys Pro         35 40 45 Ser Pro Asn Thr Lys Asn His Pro Val Arg Gly Ser Asp Asp Glu Gly     50 55 60 Arg Tyr Leu Thr Gln Glu Thr Asn Lys Val Glu Thr Tyr Lys Glu Gln 65 70 75 80 Pro Leu Lys Thr Pro Gly Lys Lys Lys Lys Gly Lys Pro                 85 90 <210> 366 <211> 36 <212> PRT <213> Homo sapiens <400> 366 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His             20 25 30 Thr Ala Glu Ile         35 <210> 367 <211> 26 <212> PRT <213> Homo sapiens <400> 367 Tyr Val Met Gly His Phe Arg Trp Asp Arg Phe Gly Arg Arg Asn Ser 1 5 10 15 Ser Ser Ser Gly Ser Ser Gly Ala Gly Gln             20 25 <210> 368 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 368 Tyr Val Met Gly His Phe Arg Trp Asp Arg Phe 1 5 10 <210> 369 <211> 39 <212> PRT <213> Homo sapiens <400> 369 Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val Gly Lys Lys 1 5 10 15 Arg Arg Pro Val Lys Val Tyr Pro Asn Gly Ala Glu Asp Glu Ser Ala             20 25 30 Glu Ala Phe Pro Leu Glu Phe         35 <210> 370 <211> 13 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-term amidated <400> 370 Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val 1 5 10 <210> 371 <211> 31 <212> PRT <213> Homo sapiens <400> 371 Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr 1 5 10 15 Leu Phe Lys Asn Ala Ile Ile Lys Asn Ala Tyr Lys Lys Gly Glu             20 25 30 <210> 372 <211> 27 <212> PRT <213> Homo sapiens <400> 372 Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr 1 5 10 15 Leu Phe Lys Asn Ala Ile Ile Lys Asn Ala Tyr             20 25 <210> 373 <211> 17 <212> PRT <213> Homo sapiens <400> 373 Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr 1 5 10 15 Leu      <210> 374 <211> 16 <212> PRT <213> Homo sapiens <400> 374 Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr 1 5 10 15 <210> 375 <211> 151 <212> PRT <213> Homo sapiens <400> 375 Met Ser Ser Phe Ser Thr Thr Thr Val Ser Phe Leu Leu Leu Leu Ala 1 5 10 15 Phe Gln Leu Leu Gly Gln Thr Arg Ala Asn Pro Met Tyr Asn Ala Val             20 25 30 Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu         35 40 45 Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser     50 55 60 Asp Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val 65 70 75 80 Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala                 85 90 95 Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys             100 105 110 Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser         115 120 125 Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu     130 135 140 Gly Cys Asn Ser Phe Arg Tyr 145 150 <210> 376 <211> 134 <212> PRT <213> Homo sapiens <400> 376 Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu Leu Leu Leu Phe 1 5 10 15 Leu His Leu Ala Phe Leu Gly Gly Arg Ser His Pro Leu Gly Ser Pro             20 25 30 Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly Leu Gln Glu Gln Arg Asn         35 40 45 His Leu Gln Gly Lys Leu Ser Glu Leu Gln Val Glu Gln Thr Ser Leu     50 55 60 Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr Gly Val Trp Lys Ser Arg 65 70 75 80 Glu Val Ala Thr Glu Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr                 85 90 95 Thr Leu Arg Ala Pro Arg Ser Pro Lys Met Val Gln Gly Ser Gly Cys             100 105 110 Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys         115 120 125 Lys Val Leu Arg Arg His     130 <210> 377 <211> 126 <212> PRT <213> Homo sapiens <400> 377 Met His Leu Ser Gln Leu Leu Ala Cys Ala Leu Leu Leu Thr Leu Leu 1 5 10 15 Ser Leu Arg Pro Ser Glu Ala Lys Pro Gly Ala Pro Pro Lys Val Pro             20 25 30 Arg Thr Pro Pro Ala Glu Glu Leu Ala Glu Pro Gln Ala Ala Gly Gly         35 40 45 Gly Gln Lys Lys Gly Asp Lys Ala Pro Gly Gly Gly Gly Ala Asn Leu     50 55 60 Lys Gly Asp Arg Ser Arg Leu Leu Arg Asp Leu Arg Val Asp Thr Lys 65 70 75 80 Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg                 85 90 95 Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly             100 105 110 Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys         115 120 125 <210> 378 <211> 9 <212> PRT <213> Lizard <400> 378 His Ser Glu Gly Thr Phe Thr Ser Asp 1 5 <210> 379 <211> 59 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <400> 379 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Lys Cys Asn Thr Ala             20 25 30 Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu Gln Thr         35 40 45 Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 <210> 380 <211> 61 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) <223> 12-Ado <400> 380 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn             20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu         35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 381 <211> 60 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) <223> 12-Ado <400> 381 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr             20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu Gln         35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 382 <211> 61 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) 3223-dioxactoanoyl <400> 382 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn             20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu         35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 383 <211> 60 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) 3223-dioxactoanoyl <400> 383 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr             20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu Gln         35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 384 <211> 61 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) <223> 5-Apa <400> 384 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn             20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu         35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 385 <211> 60 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) <223> 5-Apa <400> 385 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr             20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu Gln         35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 386 <211> 62 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (30) <223> bAla <400> 386 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Lys Cys             20 25 30 Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg         35 40 45 Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 387 <211> 61 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (30) <223> bAla <400> 387 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Cys Asn             20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu         35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 388 <211> 61 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) <223> 4,7,10-trioxa-13-tridecanamine succinimidyl <400> 388 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Lys Cys Asn             20 25 30 Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu         35 40 45 Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 389 <211> 60 <212> PRT <213> Artificial <220> <223> Hybrid lizard, human and salmon construct <220> <221> MOD_RES (222) (29) .. (29) <223> 4,7,10-trioxa-13-tridecanamine succinimidyl <400> 389 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Cys Asn Thr             20 25 30 Ala Thr Cys Val Leu Gly Arg Leu Ser Glu Glu Leu His Arg Leu Gln         35 40 45 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 390 <211> 43 <212> PRT <213> Artificial <220> <223> Hybrid human and salmon construct <220> <221> MOD_RES (222) (2) .. (2) <223> Tyr (SO 3) <400> 390 Asp Tyr Met Gly Trp Met Asp Phe Gly Lys Arg Lys Cys Asn Thr Ala 1 5 10 15 Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu Val Arg Leu Gln Thr             20 25 30 Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr         35 40 <210> 391 <211> 68 <212> PRT <213> Artificial <220> <223> Hybird lizard human construct <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MOD_RES (222) (29) .. (30) <223> bAla <400> 391 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Phe Leu             20 25 30 Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser Asn Val Val Glu         35 40 45 Glu Leu Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe Leu Phe     50 55 60 Arg Pro Arg Asn 65 <210> 392 <211> 55 <212> PRT <213> Artificial <220> <223> Hybrid lizard and human construct <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MOD_RES (222) (29) .. (30) <223> bAla <400> 392 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Phe Arg             20 25 30 Val Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr         35 40 45 Phe Leu Phe Arg Pro Arg Asn     50 55 <210> 393 <211> 39 <212> PRT <213> Artificial <220> <223> Hybrid lizard and human construct <220> <221> MISC_FEATURE <223> C-term amidated <220> <221> MOD_RES (222) (29) .. (30) <223> bAla <400> 393 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Gly Tyr             20 25 30 Phe Leu Phe Arg Pro Arg Asn         35 <210> 394 <211> 62 <212> PRT <213> Artificial <220> <223> Hybrid lizard and human construct <220> <221> MOD_RES (222) (29) .. (30) <223> bAla <400> 394 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Ser Pro             20 25 30 Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile         35 40 45 Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His     50 55 60 <210> 395 <211> 73 <212> PRT <213> Artificial <220> <223> Hybrid lizard and human construct <220> <221> MOD_RES (222) (40) .. (41) <223> bAla <400> 395 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser             20 25 30 Ser Gly Ala Pro Pro Pro Ser Xaa Xaa Ser Pro Lys Met Val Gln Gly         35 40 45 Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly     50 55 60 Leu Gly Cys Lys Val Leu Arg Arg His 65 70 <210> 396 <211> 32 <212> PRT <213> Artificial <220> <223> Hybrid salmon and human construct <220> <221> MISC_FEATURE <223> C-term amidated <400> 396 Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr             20 25 30 <210> 397 <211> 39 <212> PRT <213> Artificial <220> <223> Hybrid human and salmon construct <220> <221> MOD_RES (222) (4) .. (4) <223> d-Leucine <400> 397 Ser Ser Ser Leu Pro Gln Thr Lys Cys Asn Thr Ala Thr Cys Val Leu 1 5 10 15 Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln Thr Tyr Pro Arg Thr             20 25 30 Asn Thr Gly Ser Asn Thr Tyr         35 <210> 398 <211> 43 <212> PRT <213> Artificial <220> <223> Hybrid human and salmon construct <220> <221> MOD_RES (222) (2) .. (2) Para (CH2SO3) Phe <400> 398 Asp Phe Met Gly Trp Met Asp Phe Gly Lys Arg Lys Cys Asn Thr Ala 1 5 10 15 Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu Val Arg Leu Gln Thr             20 25 30 Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr         35 40 <210> 399 <211> 41 <212> PRT <213> Homo sapiens <400> 399 Ile Val Leu Ser Leu Asp Val Pro Ile Gly Leu Leu Gln Ile Leu Leu 1 5 10 15 Glu Gln Ala Arg Ala Arg Ala Ala Arg Glu Gln Ala Thr Thr Asn Ala             20 25 30 Arg Ile Leu Ala Arg Val Gly His Cys         35 40 <210> 400 <211> 6 <212> PRT <213> Homo sapiens <400> 400 Met Val Pro Ile Gln Lys 1 5 <210> 401 <211> 6 <212> PRT <213> Homo sapiens <400> 401 Val Gln Asp Asp Thr Lys 1 5 <210> 402 <211> 4 <212> PRT <213> Homo sapiens <400> 402 Thr Leu Ile Lys One <210> 403 <211> 5 <212> PRT <213> Homo sapiens <400> 403 Thr Ile Val Thr Arg 1 5 <210> 404 <211> 13 <212> PRT <213> Homo sapiens <400> 404 Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys 1 5 10 <210> 405 <211> 18 <212> PRT <213> Homo sapiens <400> 405 Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu 1 5 10 15 Ser lys          <210> 406 <211> 13 <212> PRT <213> Homo sapiens <400> 406 Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg 1 5 10 <210> 407 <211> 10 <212> PRT <213> Homo sapiens <400> 407 Asp Leu Leu His Val Leu Ala Phe Ser Lys 1 5 10 <210> 408 <211> 34 <212> PRT <213> Homo sapiens <400> 408 Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 1 5 10 15 Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu             20 25 30 Ser arg          <210> 409 <211> 18 <212> PRT <213> Homo sapiens <400> 409 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro 1 5 10 15 Gly cys          <210> 410 <211> 9 <212> PRT <213> Artificial <220> <223> genus <220> <221> MISC_FEATURE <222> (1) .. () <223> Xaa can be contiguous mouse or human leptin sequence <220> <221> MISC_FEATURE <222> (4) .. () <223> Xaa can be any amino acid <220> <221> MISC_FEATURE (222) (7) .. () <223> Xaa can be any amino acid <220> <221> MISC_FEATURE (222) (8) .. () <223> Xaa is any amino acid <220> <221> MISC_FEATURE <222> (9) .. () <223> Xaa can be contigous mouse or human leptin sequence <400> 410 Xaa Ser Cys Xaa Leu Pro Xaa Xaa Xaa 1 5 <210> 411 <211> 7 <212> PRT <213> Mus musculus <400> 411 Ser Cys Ser Leu Pro Gln Thr 1 5 <210> 412 <211> 7 <212> PRT <213> Homo sapiens <400> 412 Ser Cys His Leu Pro Trp Ala 1 5 <210> 413 <211> 52 <212> PRT <213> Artificial <220> <223> synthetic <220> <221> MISC_FEATURE (222) (6) .. () <223> Xaa is Trp or Gln <220> <221> MISC_FEATURE <222> (36) .. () <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE (222) (40) .. () <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE <222> (42) .. () <223> Xaa is Ile, Leu, Met or methionine sulfoxide <220> <221> MISC_FEATURE <222> (44) .. () <223> Xaa is Trp or Gln <220> <221> MISC_FEATURE (222) (45) .. () <223> Xaa is Gln or Glu <400> 413 Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 1 5 10 15 Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu             20 25 30 Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu         35 40 45 Ser Pro Gly Cys     50 <210> 414 <211> 146 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (28) .. () <223> Xaa is Gln or absent <400> 414 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser             20 25 30 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile         35 40 45 Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile     50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys                 85 90 95 His Leu Pro Gln Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly             100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Gln Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 415 <211> 146 <212> PRT <213> porcine <400> 415 Val Pro Ile Trp Arg Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Ser Asp Ile Ser His Met Gln Ser Val Ser Ser             20 25 30 Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val         35 40 45 Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile     50 55 60 Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys                 85 90 95 Pro Leu Pro Gln Ala Arg Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly             100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ala Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 416 <211> 146 <212> PRT <213> bovine <220> <221> MISC_FEATURE <222> (28) .. () <223> Xaa is Gln or absent <400> 416 Val Pro Ile Cys Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser             20 25 30 Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Leu         35 40 45 Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile     50 55 60 Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gln Ile Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser Cys                 85 90 95 Pro Leu Pro Gln Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val             100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 417 <211> 146 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (27) .. () <223> Xaa is Thr or Ala <220> <221> MISC_FEATURE <222> (28) .. () <223> Xaa is Gln or absent <400> 417 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Xaa Ser Val Ser Ser             20 25 30 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile         35 40 45 Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile     50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys                 85 90 95 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly             100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 418 <211> 146 <212> PRT <213> rhesus <400> 418 Val Pro Ile Gln Lys Val Gln Ser Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser             20 25 30 Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val         35 40 45 Leu Thr Leu Ser Gln Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile     50 55 60 Leu Ile Asn Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys                 85 90 95 His Leu Pro Leu Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Asp             100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 419 <211> 146 <212> PRT <213> Rattus norvegicus <400> 419 Val Pro Ile His Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ala             20 25 30 Arg Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile         35 40 45 Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile     50 55 60 Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln Ile Ala His Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys                 85 90 95 Ser Leu Pro Gln Thr Arg Gly Leu Gln Lys Pro Glu Ser Leu Asp Gly             100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Leu Ser Pro     130 135 140 Glu cys 145 <210> 420 <211> 146 <212> PRT <213> Artificial <220> <223> leptin genus <220> <221> MISC_FEATURE <222> (22) .. () <223> Xaa is Asn, Asp or Glu <220> <221> MISC_FEATURE <222> (27) .. () <223> Xaa is Thr or Ala <220> <221> MISC_FEATURE <222> (28) .. () <223> Xaa is Gln, Glu, or absent <220> <221> MISC_FEATURE <222> (54) .. () <223> Xaa is Met or Ala <220> <221> MISC_FEATURE <222> (68) .. () <223> Xaa is Met or Leu <220> <221> MISC_FEATURE <222> (72) .. () <223> Xaa is Asn, Asp or Glu <220> <221> MISC_FEATURE <222> (77) .. () <223> Xaa is Ser or Ala <220> <221> MISC_FEATURE <222> (118) .. () <223> Xaa is Gly or Leu <400> 420 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val Ser Ser             20 25 30 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile         35 40 45 Leu Thr Leu Ser Lys Xaa Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile     50 55 60 Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Gln Ile Xaa Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys                 85 90 95 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly             100 105 110 Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 421 <211> 146 <212> PRT <213> Artificial <220> <223> leptin genus <220> <221> MISC_FEATURE <222> (27) .. () <223> Xaa is Thr or Ala <220> <221> MISC_FEATURE <222> (77) .. () <223> Xaa is Ser or Ala <220> <221> MISC_FEATURE <222> (118) .. () <223> Xaa is Gly or Leu <400> 421 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Gln Ser Val Ser Ser             20 25 30 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile         35 40 45 Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile     50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Xaa Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys                 85 90 95 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly             100 105 110 Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg         115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro     130 135 140 Gly cys 145 <210> 422 <211> 105 <212> PRT <213> Artificial <220> <223> leptin genus <220> <221> MISC_FEATURE (222) (13) .. () <223> Xaa is Ile, Leu, Met or methionine sulfoxide <220> <221> MISC_FEATURE <222> (15) .. () <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE (222) (21) .. () <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE <222> (22) .. () <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE <222> (27) .. () <223> Xaa is Ile, Leu, Met or methionine sulfoxide <220> <221> MISC_FEATURE <222> (31) .. () <223> Xaa is Asn, Asp or Gln <220> <221> MISC_FEATURE <222> (34) .. () <223> Xaa is Gln or Glun <220> <221> MISC_FEATURE (222) (37) .. () <223> Xaa is Asn, Asp or Gln <220> <221> MISC_FEATURE (222) (41) .. () <223> Xaa is Asn, Asp or Gln <220> <221> MISC_FEATURE <222> (59) .. () <223> Xaa is Trp or Gln <220> <221> MISC_FEATURE <222> (89) .. () <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE (222) (93) .. () <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE <222> (95) .. () <223> Xaa is Ile, Leu, Met or methionine sulfoxide <220> <221> MISC_FEATURE <222> (97) .. () <223> Xaa is Trp or Gln <220> <221> MISC_FEATURE <222> (98) .. () <223> Xaa is Gln or Glu <400> 422 Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Xaa Asp Xaa Thr 1 5 10 15 Leu Ala Val Tyr Xaa Xaa Ile Leu Thr Ser Xaa Pro Ser Arg Xaa Val             20 25 30 Ile Xaa Ile Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val         35 40 45 Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu     50 55 60 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr 65 70 75 80 Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu                 85 90 95 Xaa Xaa Leu Asp Leu Ser Pro Gly Cys             100 105 <210> 423 <211> 36 <212> PRT <213> Artificial <220> <223> PPF genus <220> <221> MISC_FEATURE <222> (1) .. () <223> Xaa is Tyr or absent <220> <221> MISC_FEATURE (222) (2) .. () <223> Xaa is Ile, Pro, or absent <220> <221> MISC_FEATURE (222) (3) .. () X223 is Ile, Bolton-Hunter-modified Lys, Lys, Val, or Pro <220> <221> MISC_FEATURE <222> (4) .. () <223> Xaa is Lys, Bolton-Hunter-modified Lys, Ala, Ser, or Arg <220> <221> MISC_FEATURE (222) (7) .. () <223> Xaa is Ala, Gly, or His <220> <221> MISC_FEATURE <222> (9) .. () <223> Xaa is Gly or Ala <220> <221> MISC_FEATURE (222) (12) .. () <223> Xaa is Ala or Pro <220> <221> MISC_FEATURE (222) (13) .. () <223> Xaa is Ser or Pro <220> <221> MISC_FEATURE <222> (14) .. () <223> Xaa is Pro, Ala, or Ser <220> <221> MISC_FEATURE <222> (16) .. () <223> Xaa is Glu or Asp <220> <221> MISC_FEATURE <222> (17) .. () <223> Xaa is Leu or Ile <220> <221> MISC_FEATURE <222> (18) .. () <223> Xaa is Asn or Ala <220> <221> MISC_FEATURE <222> (19) .. () <223> Xaa is Arg, Lys, Bolton-Hunter-modified Lys, Gln, or N (Me) Ala <220> <221> MISC_FEATURE (222) (21) .. () <223> Xaa is Tyr, Ala, Phe, Lys or Bolton-Hunter-modified Lys <220> <221> MISC_FEATURE <222> (22) .. () <223> Xaa is Ala or Ser <220> <221> MISC_FEATURE <222> (23) .. () <223> Xaa is Ser, Ala, or Asp <220> <221> MISC_FEATURE (222) (25) .. () X223 is Arg, Lys or Bolton-Hunter-modified Lys <220> <221> MISC_FEATURE <222> (26) .. () <223> Xaa is His, Ala, or Arg <220> <221> MISC_FEATURE <222> (28) .. () <223> Xaa is Leu or Ile <220> <221> MISC_FEATURE (222) (30) .. () <223> Xaa is Leu or Met <220> <221> MISC_FEATURE <222> (31) .. () <223> Xaa is Val, Ile, or Leu <220> <221> MISC_FEATURE <222> (35) .. () <223> Xaa is Lys, Bolton-Hunter-modified Lys, or Arg <220> <221> MISC_FEATURE <222> (36) .. () <223> Xaa is Tyr, Trp, or Phe <400> 423 Xaa Xaa Xaa Xaa Pro Glu Xaa Pro Xaa Glu Asp Xaa Xaa Xaa Glu Xaa 1 5 10 15 Xaa Xaa Xaa Tyr Xaa Xaa Xaa Leu Xaa Xaa Tyr Xaa Asn Xaa Xaa Thr             20 25 30 Arg Gln Xaa Xaa         35 <210> 424 <211> 36 <212> PRT <213> Artificial <220> <223> PPF genus <220> <221> MISC_FEATURE <222> (1) .. () <223> Xaa is Tyr or absent <220> <221> MISC_FEATURE (222) (2) .. () <223> Xaa is Ile, Pro, or absent <220> <221> MISC_FEATURE <222> (4) .. () <223> Xaa is Lys, Bolton-Hunter-modified Lys, Ala, Ser, or Arg <220> <221> MISC_FEATURE (222) (6) .. () <223> Xaa is Glu, Gln, Ala, Asn, Asp, or Val <220> <221> MISC_FEATURE <222> (9) .. () <223> Xaa is Gly or Ala <220> <221> MISC_FEATURE <222> (10) .. () <223> Xaa is Glu, Ala, Asp, Asn, Gln, Gly, Pro, or Aib <220> <221> MISC_FEATURE <222> (11) .. () <223> Xaa is Glu, Ala, Asp, Asn, Gln, Gly, Pro, or Aib <220> <221> MISC_FEATURE (222) (12) .. () <223> Xaa is Ala or Pro <220> <221> MISC_FEATURE (222) (13) .. () <223> Xaa is Ser or Pro <220> <221> MISC_FEATURE <222> (14) .. () <223> Xaa is Pro, Ala, or Ser <220> <221> MISC_FEATURE <222> (15) .. () <223> Xaa is Glu, Ala, Asp, Asn, Gln, Gly, Pro, or Aib <220> <221> MISC_FEATURE <222> (16) .. () <223> Xaa is Glu or Asp <220> <221> MISC_FEATURE <222> (17) .. () <223> Xaa is Leu or Ile <220> <221> MISC_FEATURE <222> (19) .. () <223> Xaa is Arg, Lys, BH-modified Lys, Gln, or N (Me) Ala <220> <221> MISC_FEATURE (222) (21) .. () <223> Xaa is Tyr, Ala, Phe, Lys, or BH-modified Lys <220> <221> MISC_FEATURE <222> (22) .. () <223> Xaa is Ala or Ser <220> <221> MISC_FEATURE <222> (23) .. () <223> Xaa is Ser, Ala, or Asp <220> <221> MISC_FEATURE (222) (25) .. () X223 is Arg, Lys or Bolton-Hunter-modified Lys <220> <221> MISC_FEATURE <222> (26) .. () <223> Xaa is His, Ala, or Arg <220> <221> MISC_FEATURE <222> (27) .. () <223> Xaa is Tyr or Phe <220> <221> MISC_FEATURE <222> (28) .. () <223> Xaa is Leu or Ile <220> <221> MISC_FEATURE (222) (29) .. () <223> Xaa is Asn or Gln <220> <221> MISC_FEATURE (222) (30) .. () <223> Xaa is Leu or Met <220> <221> MISC_FEATURE <222> (31) .. () <223> Xaa is Val, Ile, or Leu <400> 424 Xaa Xaa Pro Xaa Pro Xaa His Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa Tyr Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr             20 25 30 Arg Gln Arg Tyr         35 <210> 425 <211> 34 <212> PRT <213> Artificial <220> <223> PPY-NPY chimera <400> 425 Pro Lys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Ala 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gln             20 25 30 Arg Tyr          <210> 426 <211> 59 <212> PRT <213> Artificial <220> <223> hybrid <400> 426 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Lys Cys Asn Thr Ala             20 25 30 Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln Thr         35 40 45 Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 <210> 427 <211> 36 <212> PRT <213> Artificial <220> <223> hybrid <220> <221> MISC_FEATURE (222) (30) .. (30) <223> sulfated (SO3H) tyrosine <400> 427 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Asp Tyr Met Gly             20 25 30 Trp Met Asp Phe         35 <210> 428 <211> 36 <212> PRT <213> Artificial <220> <223> hybrid <220> <221> MISC_FEATURE (222) (30) .. (30) <223> sulfated (CH2SO3H) tyrosine <400> 428 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Asp Tyr Met Gly             20 25 30 Trp Met Asp Phe         35 <210> 429 <211> 63 <212> PRT <213> Artificial <220> <223> hybrid <400> 429 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Gly Lys             20 25 30 Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His         35 40 45 Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 430 <211> 67 <212> PRT <213> Artificial <220> <223> hybrid <220> <221> MISC_FEATURE (222) (29) .. (30) <223> beta-Alanine beta-Alanine linker <400> 430 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Lys Cys             20 25 30 Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val Arg         35 40 45 Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Pro Thr Asn Val Gly Ser     50 55 60 Asn Thr Tyr 65 <210> 431 <211> 62 <212> PRT <213> Artificial <220> <223> hybrid <220> <221> MISC_FEATURE (222) (29) .. (30) <223> beta-Alanine beta-Alanine linker <400> 431 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Lys Cys             20 25 30 Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg         35 40 45 Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr     50 55 60 <210> 432 <211> 31 <212> PRT <213> Artificial <220> <223> hybrid component <400> 432 Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His 1 5 10 15 Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr             20 25 30 <210> 433 <211> 35 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (1) .. (1) <223> Xaa is succinimide beta-Alanine <400> 433 Xaa Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu 1 5 10 15 Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg             20 25 30 Gln Arg Tyr         35 <210> 434 <211> 33 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (1) .. (1) <223> Xaa is thiopropyl beta-Alanine <400> 434 Xaa Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu 1 5 10 15 Leu His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr             20 25 30 Tyr      <210> 435 <211> 35 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (1) .. (1) <223> Xaa is maleimide butyl beta-Alanine <400> 435 Xaa Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu 1 5 10 15 Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg             20 25 30 Gln Arg Tyr         35 <210> 436 <211> 36 <212> PRT <213> Artificial <220> <223> hybrid component <400> 436 Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val 1 5 10 15 Arg Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Pro Thr Asn Val Gly             20 25 30 Ser Asn Thr Tyr         35 <210> 437 <211> 9 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (1) .. (1) <223> Xaa is maleimide butyl beta-Alanine <220> <221> MISC_FEATURE (222) (3) .. (3) <223> sulfated (CH2SO3H) phenylalanine <400> 437 Xaa Asp Phe Met Gly Trp Met Asp Phe 1 5 <210> 438 <211> 43 <212> PRT <213> Artificial <220> <223> hybrid <220> <221> MISC_FEATURE (222) (2) .. (2) <223> sulfated (para-CH2SO3) phenylalanine <400> 438 Asp Phe Met Gly Trp Met Asp Phe Gly Lys Arg Lys Cys Asn Thr Ala 1 5 10 15 Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu Val Arg Leu Gln Thr             20 25 30 Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr         35 40 <210> 439 <211> 9 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (1) .. (1) <223> Xaa is succinimide beta-ALanine <220> <221> MISC_FEATURE (222) (3) .. (3) <223> sulfated (CH2SO3H) phenylalanine <400> 439 Xaa Asp Phe Met Gly Trp Met Asp Phe 1 5 <210> 440 <211> 16 <212> PRT <213> Artificial <220> <223> hybird component <220> <221> MISC_FEATURE (222) (1) .. (1) N-terminal acetyl <220> <221> MISC_FEATURE (222) (14) .. (15) <223> beta-Alanine beta-Alanine <400> 440 Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val Xaa Xaa Lys 1 5 10 15 <210> 441 <211> 32 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (29) .. (30) <223> beta-Alanine beta-Alanine linker <400> 441 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Xaa Xaa Lys Cys             20 25 30 <210> 442 <211> 47 <212> PRT <213> Artificial <220> <223> hybrid <220> <221> MISC_FEATURE <222> (1) .. () N-terminal acetyl <220> <221> MISC_FEATURE (222) (14) .. (15) <223> beta-Alanine beta-Alanine <400> 442 Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val Xaa Xaa Lys 1 5 10 15 Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His             20 25 30 Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr         35 40 45 <210> 443 <211> 49 <212> PRT <213> Artificial <220> <223> hybrid <220> <221> MISC_FEATURE <222> (1) .. () N-terminal acetyl <220> <221> MISC_FEATURE (222) (14) .. (15) <223> beta-Alanine beta-Alanine <400> 443 Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val Xaa Xaa Ile 1 5 10 15 Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg             20 25 30 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg         35 40 45 Tyr      <210> 444 <211> 39 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE <222> (1) .. () <223> thiopropyl beta-Alanine <400> 444 Xaa Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser Asn 1 5 10 15 Val Val Glu Glu Leu Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr             20 25 30 Phe Leu Phe Arg Pro Arg Asn         35 <210> 445 <211> 39 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE <222> (1) .. () <223> succinimide beta-Alanine <400> 445 Xaa Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser Asn 1 5 10 15 Val Val Glu Glu Leu Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr             20 25 30 Phe Leu Phe Arg Pro Arg Asn         35 <210> 446 <211> 34 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE <222> (1) .. () <223> N-terminal succinimide <400> 446 Pro Lys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Ala 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gln             20 25 30 Arg Tyr          <210> 447 <211> 35 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE <222> (1) .. () <223> succinimide beta-Alanine <400> 447 Xaa Pro Lys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu 1 5 10 15 Ala Arg Tyr Tyr Ala Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg             20 25 30 Gln Arg Tyr         35 <210> 448 <211> 41 <212> PRT <213> Artificial <220> <223> hybrid component <400> 448 Asp Pro Pro Pro Pro Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp 1 5 10 15 Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr             20 25 30 Leu Asn Leu Val Thr Arg Gln Arg Tyr         35 40 <210> 449 <211> 37 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (2) .. () <223> Xaa is mini-PEG3 linker <400> 449 Asp Xaa Gly Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu 1 5 10 15 Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val             20 25 30 Thr Arg Gln Arg Tyr         35 <210> 450 <211> 9 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE <222> (1) .. () <223> Xaa is thiopropyl beta-Alanine <220> <221> MISC_FEATURE (222) (3) .. () <223> sulfated (CH2SO3H) phenylalanine <400> 450 Xaa Asp Phe Met Gly Trp Met Asp Phe 1 5 <210> 451 <211> 9 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE <222> (1) .. () <223> Xaa is thiopropyl beta-Alanine <400> 451 Xaa Asp Tyr Met Gly Trp Met Asp Phe 1 5 <210> 452 <211> 35 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE <222> (1) .. () <223> maleimide butyl beta-Alanine <220> <221> MISC_FEATURE <222> (1) .. () <223> Xaa is maleimide butyl beta-Alanine <400> 452 Xaa Pro Lys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu 1 5 10 15 Ala Arg Tyr Tyr Ala Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg             20 25 30 Gln Arg Tyr         35 <210> 453 <211> 37 <212> PRT <213> Artificial <220> <223> hybrid component <400> 453 Cys Gly Gly Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu 1 5 10 15 Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val             20 25 30 Thr Arg Gln Arg Tyr         35 <210> 454 <211> 37 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (2) .. () <223> Xaa is mini-PEG linker <400> 454 Asp Xaa Gly Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu 1 5 10 15 Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val             20 25 30 Thr Arg Gln Arg Tyr         35 <210> 455 <211> 35 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (2) .. () <223> Xaa is beta-Alanine <400> 455 Cys Xaa Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg             20 25 30 Gln Arg Tyr         35 <210> 456 <211> 36 <212> PRT <213> Artificial <220> <223> hybrid component <220> <221> MISC_FEATURE (222) (2) .. () <223> Xaa is beta-Alanine <400> 456 Gly Xaa Pro Lys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 Leu Ala Arg Tyr Tyr Ala Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr             20 25 30 Arg Gln Arg Tyr         35 <210> 457 <211> 36 <212> PRT <213> Homo sapiens <400> 457 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr             20 25 30 Arg Gln Arg Tyr         35 One  

Claims (103)

A hybrid polypeptide exhibiting at least one hormonal activity and comprising a first bioactive peptide hormone module covalently linked to at least one additional bioactive peptide hormone module, wherein The bioactive peptide hormone module comprises component peptide hormones, fragments of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, analogs and derivatives of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, component peptide hormones Independently selected from the group consisting of analogues and derivatives of component peptide hormones exhibiting at least one hormone activity, and peptide enhancers, Component peptide hormones are amylin, adrenomedulin (ADM), calcitonin (CT), calcitonin gene related peptide (CGRP), intermedin, cholecystokinin ("CCK"), leptin, peptide YY (PYY), glucagon-like peptide -1 (GLP-1), glucagon-like peptide-2 (GLP-2), auxintomodulin (OXM), sodium excretion peptides, eurocortin family peptides such as Ucn-2 and Ucn-3, Neuromedin family peptides, for example neuromedin U25 or splicing variants, and independently selected from two or more of the group consisting of ANP, BNP, CNP or urodilatin and exendin-4, Peptide enhancers are structural motifs of the component peptide hormones that impart to the hybrid polypeptide the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties, And structural motifs of analogues or derivatives of the component peptide hormones that impart to the hybrid polypeptide the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties. Independently selected from the group consisting of At least one bioactive peptide hormone module exhibits at least one hormonal activity of the component peptide hormone. The peptide enhancer of claim 1, wherein the peptide enhancer is amylin (32-37) (SEQ ID NO: 242), amylin (33-37) (SEQ ID NO: 243), amylin (34-37) (SEQ ID NO: 244), amylin (35-). 37), amylin (36-37), amylin (37), ADM (47-52) (SEQ ID NO: 245), ADM (48-52) (SEQ ID NO: 246), ADM (49-52) (SEQ ID NO: 247), ADM (50-52), ADM (51-52), ADM (52), CT (27-32) (SEQ ID NO: 248), CT (27-32) (SEQ ID NO: 248), CT (28-32) (SEQ ID NO: 249 ), CT (29-32) (SEQ ID NO: 250), CT (30-32), CT (31-32), CT (32), CGRP (32-37) (SEQ ID NO: 251), CGRP (33-37) ( SEQ ID NO: 252), CGRP (34-37) (SEQ ID NO: 253), CGRP (35-37), CGRP (36-37), CGRP (37), intermedin (42-47) (SEQ ID NO: 254), intermedin (43 -47) (SEQ ID NO: 255), Intermedin (44-47) (SEQ ID NO: 256), Intermedin (45-47), Intermedin (46-47), Intermedin (47), PYY (25-36) (SEQ ID NO: 257), PYY (26-36) (SEQ ID NO: 258), PYY (27-36) (SEQ ID NO: 259), PYY (28-36) (SEQ ID NO: 260), PYY (29-36) (SEQ ID NO: 261), PYY (30 -36) (SEQ ID NO: 262), PYY (31-36) (SEQ ID NO: 263), PYY (32-36) (SEQ ID NO: 264), PYY (25-35) (SEQ ID NO: 265), PYY (26-35) (SEQ ID NO: 266) ), PYY (27-35) (SEQ ID NO: 267), PYY (28-35) (SEQ ID NO: 268), P YY (29-35) (SEQ ID NO: 269), PYY (30-35) (SEQ ID NO: 270), PYY (31-35) (SEQ ID NO: 271), PYY (32-35) (SEQ ID NO: 272), Frog GLP-1 ( 29-37) (SEQ ID NO: 273), Frog GLP-1 (30-37) (SEQ ID NO: 274), Frog GLP-2 (24-31) (SEQ ID NO: 275), Exendin-4 (31-39) (SEQ ID NO: 277) , Exendin-4 (32-39) (SEQ ID NO: 278), exendin-4 (33-39) (SEQ ID NO: 279), exendin-4 (34-39) (SEQ ID NO: 280), exendin-4 (35- 39) (SEQ ID NO: 281), Exendin-4 (36-39) (SEQ ID NO: 282), Exendin-4 (37-39), Exendin-4 (38-39), Exendin-4 (39) and these A hybrid polypeptide independently selected from the group consisting of analogs of. The method of claim 1, wherein the at least one first bioactive peptide hormone module or at least one additional bioactive peptide hormone module is a component peptide hormone or a fragment of a component peptide hormone exhibiting at least one hormone activity of the component peptide hormone. Hybrid polypeptides. The component peptide of claim 1, wherein the at least one first bioactive peptide hormone module or at least one additional bioactive peptide hormone module is an analog or derivative of a component peptide hormone that exhibits at least one hormonal activity of the component peptide hormone. A hybrid polypeptide that is a fragment of an analog or derivative of a component peptide hormone that exhibits one or more hormonal activities of the hormone. The hybrid polypeptide of claim 1, wherein at least one first bioactive peptide hormone module or at least one additional bioactive peptide hormone module is a peptide enhancer. The method of claim 1 wherein the component peptide hormones are independent from the group consisting of amylin, calcitonin, CCK, PYY, eurocortin family peptide, neuromedin family peptide, and ANP, BNP, CNP or eurodilin and exendin-4 The hybrid polypeptide selected. The hybrid polypeptide of claim 1, wherein at least one bioactive peptide hormone module exhibiting at least one hormonal activity is located at the N-terminal portion. The hybrid polypeptide of claim 7, wherein at least one bioactive peptide hormone module located in the N-terminal portion and exhibiting at least one hormonal activity is arranged in the C-terminus in the N-terminal orientation. The hybrid polypeptide of claim 8, wherein the N-terminal portion is amidated. The hybrid polypeptide of claim 1, wherein at least one bioactive peptide hormone module exhibiting at least one hormonal activity is located at the C-terminal portion. The hybrid polypeptide of claim 10, wherein the C-terminal end is amidated. The hybrid polypeptide of claim 1, wherein the C-terminal end of one bioactive peptide hormone module is directly attached to the N-terminal end of another bioactive peptide hormone module to form a covalent attachment. The bioactive peptide hormone module of claim 1, wherein the bioactive peptide hormone module is an alkyl, dicarboxylic acid, PEG, amino acid, polyamino acid, bifunctional linker, aminocaproyl (Aca), Gly, β-alanyl, 8-amino-3 , 6-dioxaoctanoyl and Gly-Lys-Arg (GKR), wherein the hybrid polypeptide is covalently attached using at least one linker independently selected from the group consisting of. The method of claim 1, The first bioactive peptide hormone module exhibits exendin-4, a fragment of exendin-4 that exhibits at least one hormone activity, an exendin-4 analog or derivative that exhibits one or more hormone activities, and at least one hormone activity. Is selected from the group consisting of fragments of exendin-4 analogs, One or more additional bioactive peptide hormone modules comprise amylin, a fragment of amylin that exhibits one or more hormonal activities, amylin analogue or derivative that exhibits one or more hormonal activities, or amylin analog that exhibits one or more hormonal activities Fragment, CCK, fragment of CCK that exhibits one or more hormonal activity, CCK analog or derivative that exhibits one or more hormonal activity, fragment of CCK analog that exhibits one or more hormonal activity, CT, CT that exhibits one or more hormonal activities A hybrid polypeptide independently selected from the group consisting of: fragments of, CT analogs or derivatives exhibiting at least one hormone activity, fragments of CT analogs exhibiting at least one hormone activity, and peptide enhancers. The method of claim 14, The first bioactive peptide hormone module is exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe-Exendin- 4 (1-28) (SEQ ID NO: 284), ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240) and ¹⁴ Leu-Exendin-4 (1-28) (SEQ ID NO: 190) Is selected from the group consisting of One or more additional bioactive peptide hormone modules include ^25,28,29 Pro-h- ^Amyline (SEQ ID NO: 67), ^Amylin (1-7) (SEQ ID NO: 217), ^2,7 Ala- ^Amyline (1-7) ) (SEQ ID NO: 285), sCT (8-10), sCT (8-27) (SEQ ID NO: 288), ^11,18 Arg-sCT (8-27) (SEQ ID NO: 289), ¹⁴ Glu, ^11,18 Arg-sCT ( 8-27) (SEQ ID NO: 286), CCK-8, Phe ² CCK-8 (SEQ ID NO: 287), Amylin (33-37) (SEQ ID NO: 243), PYY (25-36) (SEQ ID NO: 257), PYY (30- 36) A hybrid polypeptide independently selected from the group consisting of (SEQ ID NO: 262) and PYY (31-36) (SEQ ID NO: 263). The hybrid polypeptide of claim 14, comprising three or more bioactive peptide hormone modules. The hybrid polypeptide of claim 14 comprising four or more bioactive peptide hormone modules. The hybrid polypeptide of claim 14, wherein the first bioactive peptide hormone module is located at the C-terminal end and the at least one additional bioactive peptide hormone module is located at the N-terminal end. The hybrid polypeptide of claim 14, wherein the first bioactive peptide hormone module is located at the N-terminal end and the at least one additional bioactive peptide hormone module is located at the C-terminal end. The method of claim 1, The first bioactive peptide hormone module comprises amylin, a fragment of amylin that exhibits one or more hormonal activities, an amyloid analog or derivative that exhibits one or more hormonal activities, and a fragment of amylin analog that exhibits one or more hormonal activities. Selected from the group consisting of, One or more additional bioactive peptide hormone modules include PYY (25-36) (SEQ ID NO: 257), PYY (26-36) (SEQ ID NO: 258), PYY (27-36) (SEQ ID NO: 259), PYY (28-36) (SEQ ID NO: 260), PYY (29-36) (SEQ ID NO: 261), PYY (30-36) (SEQ ID NO: 262), PYY (31-36) (SEQ ID NO: 263), PYY (32-36) (SEQ ID NO: 264), PYY (25-35) (SEQ ID NO: 265), PYY (26-35) (SEQ ID NO: 266), PYY (27-35) (SEQ ID NO: 267), PYY (28-35) (SEQ ID NO: 268), PYY (29-35) ( A peptide enhancer independently selected from the group consisting of SEQ ID NO: 269), PYY (30-35) (SEQ ID NO: 270), PYY (31-35) (SEQ ID NO: 271), PYY (32-35) (SEQ ID NO: 272), and analogs thereof Hybrid polypeptides. A hybrid polypeptide exhibiting at least one hormone activity and comprising a first bioactive peptide hormone module covalently linked to a second bioactive peptide hormone module, wherein The bioactive peptide hormone module comprises component peptide hormones, fragments of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, analogs and derivatives of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, component peptide hormones Independently selected from the group consisting of analogues and derivatives of component peptide hormones exhibiting at least one hormone activity, and peptide enhancers, Component peptide hormones are independently selected from two or more of the group consisting of amylin, PYY and exendin-4, Peptide enhancers are structural motifs of component peptide hormones that confer hybrid chemicals to desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties, and Structural motifs of analogues or derivatives of the component peptide hormones that impart to the hybrid polypeptide the desired chemical stability, morphological stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacodynamic properties. Independently selected from the group consisting of, At least one bioactive peptide hormone module exhibits at least one hormonal activity of the component peptide hormone. The peptide enhancer of claim 21, wherein the peptide enhancer is amylin (32-37) (SEQ ID NO: 242), amylin (33-37) (SEQ ID NO: 243), amylin (34-37) (SEQ ID NO: 244), amylin (35-). 37), amylin (36-37), amylin (37), PYY (25-36) (SEQ ID NO: 257), PYY (26-36) (SEQ ID NO: 258), PYY (27-36) (SEQ ID NO: 259), PYY (28-36) (SEQ ID NO: 260), PYY (29-36) (SEQ ID NO: 261), PYY (30-36) (SEQ ID NO: 262), PYY (31-36) (SEQ ID NO: 263), PYY (32-36) (SEQ ID NO: 264), PYY (25-35) (SEQ ID NO: 265), PYY (26-35) (SEQ ID NO: 266), PYY (27-35) (SEQ ID NO: 267), PYY (28-35) (SEQ ID NO: 268), PYY (29-35) (SEQ ID NO: 269), PYY (30-35) (SEQ ID NO: 270), PYY (31-35) (SEQ ID NO: 271), PYY (32-35) (SEQ ID NO: 272), Exendin-4 (31-) 39) (SEQ ID NO: 277), Exendin-4 (32-39) (SEQ ID NO: 278), Exendin-4 (33-39) (SEQ ID NO: 279), Exendin-4 (34-39) (SEQ ID NO: 280), Exendin Dean-4 (35-39) (SEQ ID NO: 281), Exendin-4 (36-39) (SEQ ID NO: 282), Exendin-4 (37-39), Exendin-4 (38-39), Exendin- 4 (39) and a hybrid polypeptide independently selected from the group consisting of analogs thereof. The hybrid polypeptide of claim 21, wherein the first bioactive peptide hormone module is located at the C-terminal end. The hybrid polypeptide of claim 21, wherein the first bioactive peptide hormone module is located at the N-terminal end. 22. The method of claim 21, wherein from the group consisting of exendin-4 / PYY, PYY / exendin-4, exendin / amylin, amylin / exendin, amylin / PYY and PYY / amylin bioactive peptide hormone modules A hybrid polypeptide comprising a selected bioactive peptide hormone module combination. A hybrid polypeptide exhibiting at least one hormone activity and comprising a first bioactive peptide hormone module covalently linked to at least one second bioactive peptide hormone module, wherein The bioactive peptide hormone module comprises component peptide hormones, fragments of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, analogs and derivatives of component peptide hormones that exhibit one or more hormonal activities of component peptide hormones, component peptide hormones Independently selected from the group consisting of analogues and derivatives of component peptide hormones exhibiting at least one hormone activity, and peptide enhancers, The first component peptide hormone comprises leptin, At least one second bioactive peptide hormone module comprises a polypeptide independently selected from exendin or GLP1, At least one bioactive peptide hormone module exhibits at least one hormonal activity of its component peptide hormones. The leptin peptide hormone module of claim 26, wherein the leptin peptide hormone module comprises leptin, a leptin fragment that exhibits at least one hormonal activity, a leptin analog and derivative that exhibits at least one hormonal activity, and a fragment of a leptin analog and derivative that exhibits at least one hormonal activity. A hybrid polypeptide comprising a polypeptide selected from the group consisting of. The hybrid polypeptide of claim 26, wherein the first and second bioactive peptide hormone modules exhibit one or more hormonal activities of the component peptide hormones. The method of claim 26, wherein the leptin is

A hybrid polypeptide comprising a sequence of or an active fragment thereof. The method of claim 26, wherein the leptin is

A hybrid polypeptide comprising a sequence of or an active fragment thereof. The leptin analog of claim 26, wherein the leptin analog comprises one or more amino acid substitutions at positions 43, 48, 49, 75, 89, 93, 98, 117, 139 or 167, or the analog to position Asp or Glu at position 43. , Amino acid substitution from position 48 to Ala, amino acid substitution from position 49 to Glu or amino acid substitution from position 49, amino acid substitution from position 75 to Ala, amino acid substitution from position 89 to Leu, Asp at position 93 Or a corresponding amino acid substitution from Glu, an amino acid substitution from position 98 to Ala, an amino acid substitution from position 117 to Ser, an amino acid substitution from position 139 to Leu, and an amino acid substitution from position 167 to Ser Hybrid polypeptide comprising at least one amino acid substitution at a position. The method of claim 26, wherein the leptin is ⁴³ Asp-leptin, ⁴³ Glu-leptin, ⁴⁸ Ala-leptin, ⁴⁹ Glu-leptin, ⁴⁹ Des-AA-leptin, ⁷⁵ Ala-leptin, ⁸⁹ Leu-leptin, ⁹³ Asp-leptin, ⁹³ Glu-Leptin, ⁹⁸ Ala-Leptin, ¹¹⁷ Ser-Leptin, ¹³⁹ Leu-Leptin, ¹⁶⁷ Ser-Leptin, ⁴³ Asp, ⁴⁹ Glu-Leptin, ⁴³ Asp, ⁷⁵ Ala-Leptin, ⁸⁹ Leu, ¹¹⁷ Ser-Leptin, ^A hybrid polypeptide selected from the group consisting of ⁹³ Glu, ¹⁶⁷ Ser-leptin, and ¹¹⁷ Ser, D- ¹¹⁹ Leu-leptin. The leptin of claim 26, wherein the leptin is a fragment selected from the group consisting of leptin (22-167), leptin (116-122), ¹¹⁷ Ser, D- ¹¹⁹ Leu-leptin (116-12) and leptin (56-73). Hybrid polypeptides. The method of claim 26, The leptin peptide hormone module comprises a polypeptide selected from the group consisting of leptin, leptin fragments exhibiting at least one hormone activity, leptin analogs and derivatives exhibiting at least one hormone activity, leptin analogs and fragments exhibited at least one hormone activity and, One or more GLP1 peptide hormone modules are used for glucagon-like peptide-1 (GLP-1), fragments of GLP1 that exhibit one or more hormonal activities, GLP1 analogs or derivatives that exhibit one or more hormonal activities, and one or more hormone activities. A hybrid polypeptide comprising a polypeptide selected from the group consisting of fragments of GLP1 analogs that are indicated. 27. The composition of claim 26, wherein GLP1 consists of GLP1 (1-37), GLP1 (1-36), GLP1 (7-37), GLP1 (7-36), GLP1 (7-35) and analogs or derivatives thereof. A hybrid polypeptide selected from the group. The hybrid polypeptide of claim 26, wherein GLP1 is derived from murine, hamster, chicken, bovine, rat, frog or dog. The hybrid polypeptide of claim 26, wherein GLP1 is derived from a human or a frog. The method of claim 26, wherein GLP1 is

Hybrid polypeptide selected from the group consisting of. The method of claim 26, wherein the exendin is

Hybrid polypeptide selected from the group consisting of. The method of claim 26, The leptin peptide hormone module comprises a polypeptide selected from the group consisting of leptin, leptin fragments exhibiting at least one hormone activity, leptin analogs and derivatives exhibiting at least one hormone activity, leptin analogs and fragments exhibited at least one hormone activity and, One or more exendin peptide hormone modules are used for exendin-4, a fragment of exendin-4 that exhibits one or more hormonal activities, an exendin-4 analog or derivative that exhibits one or more hormonal activities, and one or more hormone activities. A hybrid polypeptide comprising a polypeptide selected from the group consisting of fragments of exendin-4 analogs represented. The method of claim 26, wherein the exendin is exendin-4, ¹⁴ Leu, ²⁵ Phe-exendin-4, ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-exendin-4, ¹⁴ Leu, ²² Ala, ²⁵ Phe-exendin -4, and a hybrid polypeptide selected from the group consisting of active fragments thereof. The method of claim 26, wherein the exendin is

A hybrid polypeptide comprising a sequence of or an active fragment thereof. The method of claim 26, wherein the exendin is exendin (7-15), ² Ser-exendin (7-15), exendin-4 (1-27), exendin (1-28), exendin-4 (1-29), Exendin-4 (1-30), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27), ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27) , ¹⁴ Leu, ²² Ala, ²⁵ Phe-Exendin-4 (1-27), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28), ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 ( 1-28) and ¹⁴ Leu-exendin-4 (1-28). The hybrid polypeptide of claim 26, wherein the C-terminus is amidated. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 70% or more sequence identity with a base peptide thereof. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 80% or more sequence identity with a reference peptide thereof. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 90% or more sequence identity with the reference peptide thereof. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 95% or more sequence identity with the reference peptide thereof. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules comprises D-amino acid. The hybrid polypeptide of claim 26, wherein the leptin peptide hormone module is linked to the C-terminus of at least one exendin and / or GLP1 peptide hormone module at its N-terminus. The hybrid polypeptide of claim 26, wherein the leptin peptide hormone module is covalently linked via a linker to one or more exendin and / or GLP1 peptide hormone modules. The hybrid polypeptide of claim 51, wherein the linker is chemically stable. 52. The method of claim 51, wherein the leptin peptide hormone module or one or more exendin and / or GLP1 peptide hormone modules generated a linker site, wherein the one or more amino acids were substituted with lysine, aspartic acid, glutamic acid or cysteine. Hybrid polypeptide. The moiety of claim 51, wherein the linker is selected from the group consisting of alkyl, PEG, amino acids, polyamino acids, bifunctional linkers, aminocaproyl, β-alanyl, and 8-amino-3,6-dioxaoctanoyl Hybrid polypeptide comprising a. The poly-his, poly-arg, poly-lys, poly-ala, beta-Ala-betaAla, Gly-Gly of claim 51, wherein the linker is an amino acid Lys, Glu, Gly, Cys or β-Ala and a polyamino acid. A hybrid polypeptide comprising a moiety selected from the group consisting of -Gly or Gly-Lys-Arg. The hybrid polypeptide of claim 51, wherein the linker is 1 to 30 residues in length, 2 to 30 residues in length, or 3 to 30 residues in length. The hybrid polypeptide of claim 51, wherein the linker is 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues in length. The hybrid polypeptide of claim 26, wherein the one or more component peptide hormones are recombinantly produced. The hybrid polypeptide of claim 26, wherein the recombinant polypeptide is produced. The hybrid polypeptide of claim 26, wherein the one or more component peptide hormones are chemically synthesized. The hybrid polypeptide of claim 26, which is chemically synthesized. 27. The hybrid polypeptide of claim 26 comprising two, three or four exendin and / or GLP1 peptide hormone modules. A method of treating a patient with metabolic disease or metabolic disorder comprising administering to the patient in need thereof a therapeutically effective amount of the hybrid polypeptide of claim 26. The method of claim 63, wherein the patient requires food intake control. The method of claim 63, wherein the patient requires weight control. The method of claim 63, wherein the patient requires hematopoietic control. The method of claim 63, wherein the hybrid provides a therapeutically effective appetite suppressing effect. The method of claim 63, wherein the hybrid provides a therapeutically effective glucose lowering effect. The method of claim 63, wherein the hybrid provides therapeutically effective insulin secretion enhancement. 64. The method of claim 63, wherein the patient requires one or more of food intake control, weight control, hematopoiesis, appetite suppressing effect, decreased glucose, improved insulin secretion, or an increase in pancreatic beta cell mass. 66. The method of claim 63, wherein the metabolic disease or metabolic disorder is diabetes, type 2 diabetes, type 1 diabetes, obesity, hypertension, atherosclerosis, dyslipidemia, congestive heart failure, stroke, hypercholesterolemia, cardiovascular disease, myocardial ischemia, myocardium Reperfusion, eating disorders, gestational diabetes, diabetic neuropathy or pulmonary hypertension, or in connection with insufficient pancreatic beta cell mass. A method of treating obesity, comprising administering an anti-obesity hybrid to a subject in need thereof. The method of claim 72, wherein the weight of the subject is reduced by at least 10%. The method of claim 72, wherein the body fat mass of the subject is reduced. 73. The method of claim 72, wherein at least one component of the hybrid acts on the structure of the whole brain involved in food intake or weight control. 73. The method of claim 72, wherein at least one component of the hybrid acts on the structure of the posterior brain involved in food intake or weight control. 73. The method of claim 72, wherein the hybrid comprises at least one component that acts on the structure of the forebrain involved in food intake or weight control and at least one component acts on the structure of the posterior brain involved in food intake or weight control. Way. 73. The method of claim 72, wherein the NPY1 receptor antagonist, NPY5 receptor antagonist, NPY2 receptor agonist, NPY4 receptor agonist, leptin, leptin derivative, leptin agonist, CNTF, CNTF agonist / modulator, CNTF derivative, MCH1R antagonist, MCH2R antagonist, Melanocortin 4 agonists, MC4 receptor agonists, cannabinoid receptor (CB-1) antagonists / reverse agonists, ghrelin antagonists, 5HT2c agonists, serotonin reuptake inhibitors, serotonin transport inhibitors, exendin, exendin derivatives, Exendin agonists, GLP-1, GLP-1 analogs, GLP-1 agonists, DPP-IV inhibitors, opioid antagonists, orexin antagonists, metabotropic glutamate subtype 5 receptor antagonists, histamine 3 antagonists / reverses The method further comprises the administration of an anti-obesity agent selected from the group consisting of agonists, topiramate, CCK, CCK analogs, CCK agonists, amylin, amylin analogs and amylin agonists. 79. The method of claim 78, wherein the anti-obesity agent administered is phentermin, limonabant, sibutramine or pramlinetide. 79. The method of claim 78, wherein the anti-obesity agent administered is an ADM, ADM analog or ADM agonist, leptin, leptin derivative or leptin agonist, or PPF chimera or derivatives thereof. 79. The method of claim 78, wherein the hybrid acts on the structure of the forebrain involved in food intake or weight control and the anti-obesity agent acts on the structure of the posterior brain involved in food intake or weight control, or the hybrid acts on food intake or weight control. Acting on the structure of the involved posterior brain and the anti-obesity agent acting on the structure of the entire brain involved in food intake or weight control. Administering a therapeutically effective amount of a myocardial protective hybrid effective to prevent or improve the deleterious effects on one or more heart parameters in a subject in need of treatment for cardiovascular disease, thereby causing cardiac remodeling to attenuate, delay Or prevented, wherein the subject has experienced a myocardial attack, or is at risk of experiencing a myocardial attack, or attenuates, delays or prevents cardiac remodeling in a subject in need of treatment of a cardiovascular disease. Method of treating cardiovascular disease by. 83. The cardiac parameters of claim 82, wherein the cardiac parameters include diastolic function of the left ventricle, E / A ratio, diastolic pressure at the left ventricle, cardiac output, cardiac contractility, mass of the left ventricle, ratio of mass to weight of the left ventricle, left ventricular volume, left atrial volume, left ventricle Distal diastolic dimension (LVEDD) or left ventricular distal dimension (LVESD), infarct size, athletic performance, athletic efficiency or ventricular size. 84. The method of claim 83, wherein the ventricular size is not increased in dimension or wall thickness. 84. The method of claim 83, wherein the E / A ratio is increased after myocardial infarction. 84. The method of claim 83, wherein the size of the infarction is reduced. 84. The method of claim 83, wherein athletic performance is increased. 84. The method of claim 83, wherein exercise efficiency is increased. 84. The method of claim 83, wherein the cardiac output is normalized after myocardial infarction. 83. The method of claim 82, wherein the myocardial attack is selected from the group consisting of heart valve disease, myocardial infarction, cardiomyopathy, hypertension, infection, inflammation, surgery, genetic predisposition, volume overload, pulmonary heart and pulmonary hypertension. Due to. 91. The method of claim 90, wherein the cardiomyopathy is dilated cardiomyopathy, viral cardiomyopathy or idiopathic cardiomyopathy. 92. The method of claim 91, wherein the subject is also suffering from diabetes. 92. The method of claim 91, wherein the myocardial hybrid is short-term administered to the subject. 92. The method of claim 91, wherein the myocardial hybrid is long term administered to the subject. Administering to a subject in need or prevention of atrial or ventricular remodeling a therapeutically effective amount of a myocardial hybrid effective to prevent or reduce atrial or ventricular remodeling, wherein the subject has experienced a myocardial attack A method of preventing or reducing atrial or ventricular remodeling, wherein the patient has been, is experiencing a myocardial attack, or is at risk of experiencing a myocardial attack. Administering a therapeutically effective amount of a myocardial protective hybrid effective to prevent cardiac remodeling to a subject in need of treatment or prevention of a condition associated with cardiac remodeling or a condition resulting from cardiac remodeling, thereby causing a condition associated with cardiac remodeling. Where the subject has experienced a myocardial attack, or is at risk of experiencing a myocardial attack, or a condition associated with a cardiac remodeling or a condition due to a cardiac remodeling in the subject. Method of treatment or prevention. 98. The method of claim 96, wherein the condition is myocardial infarction, inflammation, ischemia / reperfusion, oxidative stress, pulmonary heart, advanced glycation endproduct, abnormal wall tension, sympathetic stimulation, myocarditis, hypertension, heart transplantation, cardiac surgery, Left ventricular hypertrophy, coronary artery disease, essential hypertension, acute hypertensive emergency, cardiomyopathy, heart failure, exercise tolerance, chronic heart failure, arrhythmia, cardiac dysrhythmia, sudden death, fainting, atherosclerosis, Mild chronic heart failure, angina pectoris, cardiac bypass reocclusion, intermittent claudication, diastolic dysfunction and / or systolic dysfunction. A method of preventing heart remodeling in a subject exhibiting congestive heart failure, comprising administering to the subject exhibiting congestive heart failure a prophylactic heart remodeling hybrid. 99. The method of any one of claims 82-98, wherein the myocardial protection hybrid comprises an incretin family module capable of binding to GLP-1 or exendin receptors. 107. The method of claim 99, wherein the hybrid further comprises a peptide family module capable of providing beneficial cardiovascular effects, reduction of glucose-induced tissue damage, reduction of hypertension or weight loss. There is a need to reduce, prevent or treat elevated levels of lipids, elevated triglycerides, or elevated cholesterol, including administering a therapeutically effective amount of a lipid-lowering hybrid alone or in combination with a second lipid-lowering agent. Elevated lipid levels, elevated triglyceride levels, or reduced elevated cholesterol levels in a subject. Hybrid according to any of the preceding claims, not as disclosed in WO 2005/077072. The method of claim 1, wherein the hybrid is not a hybrid disclosed in WO 2005/077072.

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