EA014647B1 - 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

Related Applications

The application for this patent claims priority for multi-owner provisional patent application US No. 11/201664, filed August 11, 2005, and υδδΝ 11/206903, filed August 17, 2005, both of which are entitled Hybrid Polypeptides with Selectable properties, and υδδΝ 11/301744, filed December 12, 2005, all of which are fully incorporated herein by reference. A paper copy of the sequence list and a machine-readable form of the sequence list on two CDs, entitled Sequence List, copy 1 and Sequence List, copy 2, each of which contains a 216 KB file, designated 0701 ^ O2_8st_151 10-26-06.1x1, created 26 October 2006, are also incorporated into this description by reference.

Technical field

This invention relates to peptide chemistry, and more particularly to hybrid polypeptides with selectable properties.

State of the art

The main in many metabolic diseases and disorders is the regulation of insulin levels and blood glucose levels. The secretion of insulin is partially modulated by hormones that enhance secretion, called incretins, which are produced by enteroendocrine cells. The hormone incretin, a glucagon-like peptide-1 (OR-1), is a peptide hormone secreted by intestinal cells, which, as shown by numerous studies, has an enhancing effect on insulin secretion. ORI-1 is produced from proglucagon in the intestine and enhances the secretion of insulin caused by nutrients (Kgsutapp V., e1 a1., Lapsse1, 2: 1300-1303 (1987)). It is known that various truncated forms of ORB-1 stimulate insulin secretion (insulinotropic effect) and cAMP formation [see, for example, Morow, δ., 1p1. 1. Rer. Rgo. Century., 40: 333-343 (1992)]. An interrelation was established between various laboratory experiments of ν ίΙΐΌ ин and insulinotropic reactions in mammals, especially humans, to the exogenous administration of ORB-1, ORB-1 (7-36) amide (δΕΟ ΙΌ ΝΟ: 61) and ORB-1 (7-37) acid (δΕΟ ΙΌ ΝΟ: 204) (see, for example, No.isk, M.A., e1 a1., 1% 1E1o1sc 36: 741-744 (1993); Oi1shak, M., e1 a1. 1. οί Meb., 326 (20): 1316-1322 (1992); Noisk, M.A., е1 а1., Ί. С1ш. 1пук1., 91: 301-307 (1993); apb Tyogepk, V ., e1 a1., I1aEeek, 42: 12191225 (1993)).

ORI-1 (7-36) amide (δΕΟ ΙΌ ΝΟ: 61) has a pronounced antidiabetogenic effect in patients with insulin-dependent diabetes by stimulating insulin sensitivity and by enhancing glucose-induced secretion of insulin in physiological concentrations (O1shak M., e1 a1., Νονν Епд 1. Meb., 326: 1316-1322 (1992)). When ORS-1 (7-36) is administered to patients with non-insulin-dependent diabetes, the amide (δΕΟ ΙΌ ΝΟ: 61) stimulates the release of insulin, reduces the secretion of glucagon, inhibits gastric emptying and improves glucose utilization (Noisk, 1993; Oi1shak, 1992; Noisk, 1993 ) However, the use of OPI-1 molecules for the long-term treatment of diabetes is complicated because the serum half-life for these peptides is too short.

More specifically, OR-1 is a 30 amino acid peptide derived from proglucagon, a pro-hormone of 160 amino acids. The action of various pro-hormone convertases in the pancreas and intestines leads to the formation of glucagon and other poorly defined peptides, while the cleavage of proglucagon causes the formation of OP1 and OP2, as well as two other peptides. The amino acid sequence of OR-1 is 100% homologous to all mammals currently studied, which means a critical physiological role. ORB-1 (7-37) acid is subjected to C-terminal truncation and amidated to give ORB-1 (7-36) ΝΗ ₂ (δΕΟ W ΝΟ: 61). The biological effects and metabolic renewal of the free acid ORB-1 (7-37) OH (δΕΟ ΙΌ ΝΟ: 204) and the amide, ORB-1 (7-36) ΝΗ ₂ (δΕΟ ΙΌ ΝΟ: 61), are indistinguishable. In the conversion, the numbering of amino acids is based on the obtained from proglucagon OR-1 (1-37) OH (δΕΟ ΙΌ ΝΟ: 59). The biologically active OR-1 is the result of further processing: OR-1 (7-36) ΝΗ ₂ (δ (ΕΟ ΝΟ: 61). Thus, the first amino acid ORF-1 (7-37) OH (δΕΟ W ΝΟ: 204) or ORB-1 (7-36) ΝΗ ₂ (δΕΟ ΙΌ ΝΟ: 61) is ⁷ Ηίκ.

In the gastrointestinal tract, OR-1 is produced by L cells of the intestinal, colon and rectal mucosa in response to stimulation by intraluminal glucose. The half-life of active ORB-1 in plasma is <5 min, the rate of its metabolic clearance is about 12-13 min (^ 151, Oak1noe1107 107 (6): 1848-55 (1994)). The main protease involved in ORB-1 metabolism is dipeptidyl peptidase (OPP) GU (СО26), which cleaves the Ν-terminal dipeptide Shk-A1a, thereby forming metabolites, ORB-1 (9-37) OH (δΕΟ ΙΌ ΝΟ: 205) or OR-1 (9-36) ΝΗ ₂ (δΕΟ ΙΌ ΝΟ: 206), which are described in various ways as inactive, weak agonists or antagonists of the OR-1 receptor. The OR1-receptor (OR1-1B) is an O protein-coupled receptor of 463 amino acids and is localized in the beta cells of the pancreas, in the lungs, and, to a lesser extent, in the brain, adipose tissue, and kidneys. Stimulation of ORB-1B with ORB-1 (7-37) OH (δΕΟ ΙΌ ΝΟ: 204) or ORB-1 (7-36) ΝΗ ₂ (δΕΟ ΙΌ ΝΟ: 61) leads to activation of adenylate cyclase, cAMP synthesis, membrane depolarization an increase in intracellular calcium and an increase in glucose-induced secretion

- 1 014647 insulin (Ηο1ζ е! А1., 1. Βΐοΐ. Сет. 270 (30): 17749-57 (1995)).

CPP-1 is an effective stimulator of insulin secretion, which is secreted by the intestinal mucosa in response to food intake. The complex incretin effect of CPB-1 is underestimated due to the fact that mice knockout for CRP-1K do not tolerate glucose. The incretin response to CPF-1 intravenous infusion persists in patients with diabetes, although the incretin response to oral glucose in such patients is impaired. The administration of CPP-1 by infusion or subcutaneous injection regulates fasting plasma glucose levels in patients with diabetes and maintains a threshold glucose level for insulin secretion (C! Shack e! A1., N. Epp. 1. 1. Mob. 326: 1316-22 (1992 ); No-shsk e! A1., E1ae !. Furniture 13: (9 8irr1 5): 839-843 (1996); Ushsk e! A1., 1. S1sh. Εηάοαίηοΐ. Me! Ab. 76: 912- 917 (1993)). CPP-1 has great potential as a therapeutic agent capable of increasing physiological insulin secretion while avoiding the hypoglycemia characteristic of sulfonylurea drugs.

Other important effects of CPB-1 on glucose homeostasis are inhibition of glucagon secretion and inhibition of gastric motor function. The inhibitory effects of CPB-1 on the secretion of glucagon by pancreatic alpha cells lead to a decrease in the production of glucose in the liver through a decrease in gluconeogenesis and glycogenolysis. This anti-glucagon effect of CPB-1 persists in patients with diabetes.

The so-called ileal inhibition effect, exerted by CP1, in which the gastric motor function and gastric secretion is inhibited, is achieved through vagal efferent receptors or through a direct effect on the smooth muscles of the intestine. Reducing gastric secretion using CPB-1 promotes a latent phase in the absorption of nutrients, which eliminates the need for a quick insulin response. In general, the action of CPI-1 on the gastrointestinal tract significantly slows down the absorption of glucose and fatty acid and modulates the secretion of insulin and glucose homeostasis.

It has also been shown that CPB-1 acts on beta-cell specific genes, such as the CBiT-1 transporter, insulin (via the interaction of PEX-1 with the promoter of the insulin gene) and hexokinase-1. Thus, CPB-1 can presumably abolish glucose intolerance, usually associated with aging, as shown by experiments in rodents. In addition, CPB-1 may promote beta cell neogenesis and increase beta cell mass, in addition to restoring beta cell function during beta cell failure states.

The central action of CPB-1 involves an increase in the feeling of fullness while reducing food intake, achieved through the action of CRP-1K in the hypothalamus. Continuous 48-hour subcutaneous infusion of CPP-1 in patients with type II diabetes reduces hunger and the amount of food consumed and enhances the feeling of satiety. Anorexic activity is also absent in SRB-1K knockout mice.

Exendins are another family of peptides involved in insulin secretion. Exendins are found in the saliva of puffer lizards, lizards found in Arizona, and Mexican pufferfish. Exendin-3 is present in the saliva of escarpone lizards, and exendin-4 is present in the saliva of Zhilatier’s lizards (Epd, I., e! A1., I. Βίο1. Сет., 265: 20259-62, 1990; Epd., I., e! a1., I. Βίο1. Sib., 267: 7402-05 (1992)). The exendins share some sequence similarity with some members of the glucagon-like peptide family, with the highest identity, 53%, found for CPB-1 (^ ke, e! A1., I. Βίο1. Sb., 268: 19650-55 (1993) )

Exendin-4 binds CP1 receptors on TC1 insulin-secreting cells, on dispersed guinea pig pancreas acinar cells, and on parietal cells of the stomach; the peptide also stimulates the release of somatostatin and inhibits the release of gastrin in isolated stomachs (Οοΐ ^. е! а1., I. Βίο1. Сет., 268: 19650-55 (1993); 8серр, е! а1., Еиг. I. Риток 69 : 183-91 (1994); E1 £ е e1e, e! A1., Bie 8c1., 55: 629-34 (1994)). It was found that exendin-3 and exendin4 bind the CPB-1 receptors, stimulate the formation of cAMP, and the release of amylase from acinar cells of the pancreas (MaPyuSh!! K., e! A1. 1992); KaiTtap, e! A1., I. 1ο1. Siet., 267: 21432-37 (1992); 8shdb, e! A1., Kedi1. Rer!., 53: 47-59 (1994)). The use of the insulinotropic activities of exendin-3 and exendin-4 has been proposed for the treatment of diabetes mellitus and the prevention of hyperglycemia (U.S. Patent No. 5,424,286).

It has been described that truncated exendin peptides such as exendin [9–39], a carboxyamidated molecule, and fragments 3–39–9–39 are effective and selective antagonists of CPb-1 (^ ke, e! A1., I. Βίο1. ., 268: 19650-55 (1993); KaiTtap, I.R., e! A1., I. Βίο1. Sib., 266: 2897-902 (1991); 8cerr, V., e! A1., Eig I. Pagt., 269: 183-91 (1994); Möηί ^ ο £ e-Kaί ^ ζάάάЬ, e! A1., Clae! E £, 45 (8pir1. 2): 152A (1996)). Exendin [9-39] (8EO GO N0: 207) blocks endogenous CPb-1 and νίνο, which leads to reduced insulin secretion (^ add, е! А1., I. С1ш. Ике ^., 95: 417-21 ( 1995); Ts'A1e £ £ Yu, e! A1., I. SBP. Ike ^., 97: 133-38 (1996)). The receptor, presumably responsible for the insulinotropic action of CPB-1, was cloned from islet cells of rat pancreas (Nyugo !! ^ V., Prgos. No. 111. Beab. 8sk I8A 89: 8641-8645 (1992)). Exendins and exendin [9-39] binds to the cloned CPB-1 receptor (CPB-1 receptor of rat pancreatic cells: Eeytapp N.S., e! A1., Rurbbek, 15 (3): 453-6 (1994);

- 014647 human receptor OR-1: Tyogepk V., e! A1., P1ae! e8, 42 (11): 1678-82 (1993)). In cells transfected with the cloned receptor OR-1, exendin-4 is an agonist, i.e. it increases cAMP, while exendin [9–39] (ZEO ΙΌ N0: 207) is an antagonist, i.e. it blocks the stimulating effect of exendin-4 and OR-1. 1b.

More specifically, exendin-4 is a 39-amino acid C-terminal amidated peptide found in saliva of Toadjack lizards (He1obegta 8I8res! It), with 53% amino acid sequence identity with the ORB-1 peptide sequence, see, e.g., Epd, 1 ., e! a1. LokShop aib S'yagas1ep / a1yup οί Yheepbsh-4, apb Yheepbsh-3 Apaodyod Got Ne1obegta 8I8res1it Uepot, 1. Βίο. Siet., 267: 11, p. 7402-7405 (1992), Uoyid, A.A., e! A1., CI1co5e-b0 \\ 'eppd aib 1n5iNp-Zep5Y1 / td Asyoik oG Exepb-4, b | aEe1e5. Yo1. 48, p. 1026-1034, Mau, 1999. From the point of view of its activity, exendin-4 is a highly specific agonist of the receptor OR-1 and, like OR-1, is able to stimulate insulin secretion. Therefore, like ORB-1, exendin-4 is considered an insulinotropic peptide.

However, unlike ORB-1, exendin-4 has a relatively long half-life in humans due to its resistance to dipeptidyl peptidase IV, which quickly destroys the sequence of ORB-1 and y1y0. Moreover, it was shown that, compared to OR1, exendin-4 has a stronger ability to stimulate insulin secretion and that lower concentrations of exendin-4 can be used to obtain such a stimulating effect, see, for example, US Pat. No. 5,424,286, incorporated in the present description by reference. Therefore, exendin-4 peptides or their derivatives (examples of such derivatives can be seen, for example, in US Pat. No. 6,528,486, incorporated herein by reference, and corresponding international application A0 01/04156) have greater potential use for treating conditions involving dysregulation. insulin levels (for example, conditions such as diabetes) than insulin or OR-1.

Another family of peptide hormones involved in diseases and metabolic disorders is the amylin family of peptide hormones, including amylin, calcitonin, a peptide encoded by the calcitonin gene, adrenomedullin, and interludes (also known as AEP-6). Amylin is a 37-amino acid peptide hormone. It was isolated, purified, and chemically characterized as the main component of amyloid deposits in the pancreatic islets of people with type 2 diabetes (Sooreg e! A1., Proc. No. 1. Asab. 8cE, I8A, 84: 8628-8632 (1987) ) The amylin molecule has two post-translational modifications: the C-terminal region is amidated, and the cysteines at positions 2 and 7 are cross-linked to form a Ν-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of the Lyuk-Agd proteolytic signal of the dibasic amino acid before the L-terminal codon for Ly3, and Ol before the Lyuk-Agd proteolytic signal at the SLA1M8-terminal position, a typical sequence for amidation using an amidating protein enzyme, RAM (Sooreg e! A1., Vusyet. Vyryuk. As! A, 1014: 247-258 (1989)).

Amylin is believed to regulate gastric emptying and inhibit glucagon secretion and food intake, thereby regulating the rate of glucose production in the bloodstream. This, apparently, complements the action of insulin, which regulates the rate of glucose disappearance from the bloodstream and its absorption by peripheral tissues. These actions are confirmed by experimental discoveries in rodents and humans, which show that amylin complements the effects of insulin in glucose control after eating using at least three independent mechanisms, all of which affect the rate of glucose production. Firstly, amylin inhibits the secretion of glucagon after meals. Compared to healthy adults, patients with type 1 diabetes do not have circulating amylin, and patients with type 2 diabetes have lower amylin concentrations after meals. Moreover, an infusion of amylin-specific monoclonal antibodies that bind circulating amylin again causes a significant increase in glucagon concentrations compared to the control. Both of these findings indicate the physiological role of endogenous amylin in the regulation of glucagon secretion after meals. Secondly, amylin slows down the gastrointestinal motility and gastric emptying. Finally, it has been shown that injections of rat amylin into the hypothalamus reduce the amount of food eaten by rats and alter the metabolism of the neurotransmitter in the hypothalamus. In some studies, food intake decreased significantly up to 8 hours after injection of rat amylin and rat COCR into the hypothalamus. In human studies, it has been shown that an amylin analog, pramlintide, reduces weight or weight gain. Amylin may be useful in treating metabolic conditions such as diabetes and obesity. Amylin can also be used to treat pain, bone disorders, gastritis, to modulate lipids, in particular triglycerides, or to influence physique, such as selective loss of fat and preservation of muscle tissue.

G hormone calcitonin (CT) is so named because of its secretion in response to hypercalcemia caused, and its rapid hypocalcemic effect. It is produced and secreted from neuroendocrine thyroid cells called C cells. The most studied effect of CT (1-32) (ZEO ΙΌ N0: 48) is its effect on osteoclast. The first steps of CT include the rapid loss of corrugated edges and reduced release of lysosomal enzymes. Ultimately, in

- 3 014647 bending osteoclast functions using CT gives a decrease in bone resorption. However, neither a chronic decrease in serum CT in the case of thyroidectomy, nor an elevated serum CT found in medullary thyroid cancer, are associated with a change in serum calcium or bone mass. Thus, it is most likely that the main function of CT (1-32) (8EO ΙΌ N0: 48) is to combat acute hypercalcemia in critical situations and / or to protect the skeleton during calcium stress, such as growth, pregnancy and breastfeeding (Overview at Weskeg, 1SEM, 89 (4): 1512-1525 (2004) and 8 ex! oi, United States! This is consistent with recent data from calcitonin gene knockout mice that have removed both calcitonin and COBP-пепти peptides, which show that mice have normal levels of basic calcium, but an increased calcemic response (Kstyata H, e! A1., Nureyek Century. 2003 Eb; 26 8irr1: 8105-8). CT has an effect on plasma calcium levels and inhibits osteoclast function and is widely used to treat osteoporosis. Therapeutically, it is likely that salmon CT (CTF) increases bone density and reduces the rate of cracking with minimal side effects. CT has also been used successfully over the past 25 years as a therapy for Paget's bone disease, which is a chronic bone disorder that can lead to an increase or deformation of bones in one or more parts of the skeleton. CT is also widely used due to the analgesic effect of bone pain that occurs with osteoporosis, although the mechanism of this action has not been studied in detail.

The peptide encoded by the calcitonin gene (COBP) is a neuropeptide whose receptors are widely distributed in the body, including the nervous system and the cardiovascular system. Apparently, this peptide modulates sensory neurotransmission and is one of the most effective endogenous vasodilating peptides found to date. The described biological actions of COBP include modulation of substance P during inflammation, nicotinic receptor activity in the neuromuscular junction, stimulation of pancreatic enzyme secretion, decreased gastric acid secretion, peripheral vasodilation, increased cardiac activity, neuromodulation, regulation of calcium metabolism, osteogenic stimulation, secretion insulin and an increase in body temperature and a decrease in food intake (^ 1sha1ateaika, Lshu1sh, sa1syop1p deie-te1a! ne reryye, sa1syoshp apy а: a reye iregGashyu Sty BEy №igoyu1 1997; 11 (2-3):.. 167-239). An important role of COCR is the control of blood flow to various organs due to its effective vasodilating effect, which is confirmed by a decrease in mean arterial pressure after intravenous administration of α-COBP. The vasodilating effect is also confirmed by a recent analysis of homozygous SOKR knockout mice, which demonstrates increased peripheral vascular stability and high blood pressure caused by increased peripheral sympathetic activity (KigSata N., e! A1. ! ws! bw1w1w1 w1k. Nureyepk Vek. 2003 Eb; 26 8irr1: 8105-8). Thus, it is likely that COX causes vasodilator effects, hypotensive effects, and increased cardiac activity, among other effects.

A prolonged infusion of COBP in patients with congestive heart failure showed a long-term beneficial effect on hemodynamic functions without side effects, which suggests its use in heart failure. Other indications for using COBP include renal failure, acute and chronic coronary artery ischemia, treatment of cardiac arrhythmias, other peripheral vascular diseases such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension. Presumably, pre-eclampic toxicosis of pregnant women and premature birth can also be treated (^ 1shaateaika, 1997). Recent therapeutic uses include the use of COBP antagonists for the treatment of migraine.

Adrenomedullin (ΑΌΜ) is almost universally expressed in most tissues containing the peptide than not. The published review ΑΌΜ (Nshkoi, EP. E! A1., Epiose Beu1e ^ k (2000) 21 (2): 138167) details its effects on the cardiovascular system, cell growth, central nervous system and endocrine system, where the biological Actions include vasodilation, cell growth, regulation of hormone secretion and natriuresis. Studies in rats, cereals, sheep and humans confirm that intravenous infusion ΑΌΜ gives effective and prolonged hypotension, and this action is comparable to the action of COBP. However, the hypotensive effect of ΑΌΜ on the mean arterial pressure in anesthetized rats is not inhibited by the COBP COBP _{8-37 antagonist} , which suggests that this effect is not mediated by COBP receptors. Acute or chronic administration of human ΑΌΜ rats, anesthetized, conscious or with high blood pressure, gives a significant decrease in total peripheral resistance, accompanied by a drop in blood pressure, while increasing heart rate, cardiac output and cardiac systolic volume.

ΑΌΜ has also been proposed as an important factor in embryonic development and differentiation, and as a survival factor in apoptosis of rat endothelial cells. These suggestions are supported by recent studies of ΑΌΜ knockout mice in which mice homozygous for gene loss

- 4 014647

ΑΌΜ, demonstrate impaired vascular formation during embryonic development and, thus, die in the middle stages of pregnancy. It has been described that ΆΌΜ +/- heterozygous mice have high blood pressure simultaneously with a predisposition to tissue damage (Kshtyata N., e! A1., Nureyepz Kek. 2003 Beb; 26 Birr1: § 105-8).

ΆΌΜ affects endocrine organs such as the pituitary gland, adrenal glands, reproductive organs, and pancreas. The peptide appears to play a role in inhibiting the release of ACTH from the pituitary gland. In the adrenal glands, it probably affects the secretory activity of the adrenal cortex in rats and humans, and it increases blood flow in the adrenal glands, acting as a vasodilator in the vascular bed of the adrenal glands in healthy rats. It was shown that ΑΌΜ is present in the reproductive system of females, and during normal pregnancy, its plasma levels are increased. Studies of a rat model of preeclampsia show that ΑΌΜ can reverse hypertension and reduce mortality in cubs given to rats in late pregnancy. Since it does not have a similar effect in animals in early pregnancy and in non-pregnant rats in the model of preeclampsia, it was suggested that ΑΌΜ may play an important regulatory role in the uteroplacental cardiovascular system. In the pancreas, ΑΌΜ most likely plays an inhibitory role, since it weakens and delays the insulin response to the oral sugar load, which gives initially elevated glucose levels. ΑΌΜ may also affect renal function. A peripherally administered bolus can significantly lower mean arterial pressure and increase blood flow in the kidneys, glomerular filtration rate, and urine flow. In some cases, an increase in Να + excretion is also noted.

ΑΌΜ also has other peripheral effects on the bones and lungs. For bones, studies have confirmed a role that goes beyond the cardiovascular system and fluid homeostasis and demonstrated that ΑΌΜ acts on the osteoblasts of embryos and adult rodents, increasing cell growth comparable to the effects of known growth factors of osteoblasts, such as transforming growth factor β . This is clinically important, since one of the main tasks in the study of osteoporosis is the development of a treatment that would increase bone mass through osteoblastic stimulation. In the lungs, ΑΌΜ not only causes vasodilation of the lungs, but also inhibits bronchostenosis caused by histamine or acetylcholine. Recent studies using aerosol ΑΌΜ to treat pulmonary hypertension in a rat model have shown that inhalation treatment of this condition is effective, as evidenced by the fact that arterial mean pulmonary pressure and total lung resistance are significantly lower in rats treated with ΑΌΜ compared to rats which was given saline. This result is achieved without changing the systemic arterial pressure or heart rate (Noschaua е. E! A1., Vol. 1. Ryu8y1 Neal Sie Ryu8yu. 2003; 285: H2125-31).

In healthy volunteers, intravenous infusion показано has been shown to lower blood pressure and stimulate heart contractions, cardiac output and plasma levels of cAMP, prolactin, norepinephrine and rennin. Such patients have a slight or no increase in urine volume or sodium excretion is observed. In patients with heart failure or chronic renal failure, intravenous administration of ΑΌΜ has the same effects as in healthy people, and also causes diuresis and natriuresis, depending on the dose administered (ΝίοΗο118, Μ6 e! A1. Rerybez. 2001; 22: 1745 -1752). It has also been shown that experimental treatment of ΑΌΜ is beneficial for arterial and pulmonary hypertension, septic shock and ischemic / reperfusion injury (Be11o \\ '8k | 1., Po1. 1. Ryatschaek 2004; 56: 5-27). Other indications for treatment ΑΌΜ include peripheral vascular disease, subarachnoid hemorrhage, hypertension, pre-eclampic toxicosis of pregnant women and premature birth, and osteoporosis.

Expression of LER-6 (for example, intermedin) mainly occurs in the pituitary gland and gastrointestinal tract. The specific receptor for EP-6 is not described; however, binding studies have shown that EP-6 binds to all known receptors in the amylin family. ΑEP-6 has been shown to increase cAMP production in ΞΚ-Ν-Μί cells. ' and b6 expressing endogenous SCCR receptors, and competes with labeled SCCR for binding to its receptors in these cells. In studies published by ίη νίνο, administration of SEP-6 causes a decrease in blood pressure in both normal rats and rats with spontaneous hypertension, most likely through interaction with CΚ ^ Κ / ΚΑΜР receptors. Ιη νίνο administration to mice suppresses gastric emptying and food intake (Cop e! A1. I. Bü1. Sesh. 2004 Eb 20; 279 (8): 7264-74.)

It has been described that the biological effects of the peptide hormones of the amylin family are mainly mediated through binding to two closely related C-protein type II receptors (SRSK), a calcitonin receptor (STK) receptor similar to calcitonin receptor (SBCK). Cloning and functional studies have shown that CCKP, ΑΌΜ and amylin interact with various combinations of CTK or SKKK and a protein that modifies receptor activity (KKP). Many cells express many K клетокP. It is believed that co-expression of KΑΜP and either STK or SBCK is required to create functional receptors for calcitonin, SCKR, ΑΌΜ, and amylin. The ΚΑΜ ^ family includes three members (KΑΜP1, -2, and -3), which have less than 30% identities

- 5 014647 sequence sequence, but have a common topological organization. Co-expression of CPPP and PAMP1 leads to the formation of a receptor for CCPP. Co-expression of CPPP and PAMP2 leads to the formation of a receptor for ΆΌΜ. Co-expression of CPPP and PAMP3 leads to the formation of a receptor for AEM and CCPP. Co-expression of 11CTP2 and PAMP1 leads to the formation of a receptor for amylin and CCPP. Co-expression of 11CTP2 and PAMP3 leads to the formation of a receptor for amylin.

Another family of peptide hormones involved in diseases and metabolic disorders is the leptin family. A mature form of circulating leptin is a protein of 146 amino acids, which is usually excluded from the central nervous system by the blood-brain barrier (BBB) and the blood-brain barrier, see, for example, ^ e <d1e eE a1., 1995. 1. С1ш ΙηνοδΙ 96: 2065-2070. Leptin is an afferent signal in the negative reverse cycle, regulating food intake and body weight. The leptin receptor is a member of the cytokine family. The anorexigenic effect of leptin depends on the binding to the homodimer of the OB-Pb isoform of this receptor, which encodes a long intracytoplasmic domain, which includes several motifs for protein-protein interaction. OB-Pb is largely expressed in the hypothalamus, suggesting that this area of the brain is an important site of leptin action. A mutation of the mouse O gene gene has been shown to cause a syndrome that exhibits pathophysiology, which includes: obesity, increased body fat deposition, hyperglycemia, hyperinsulinemia, hypothermia and impaired thyroid and reproductive functions in male and female homozygous ob / ob obese mice (see, for example, 1pdike, eE a1., 1950. 1. Negeb 41: 317-318). The therapeutic use of leptin or the leptin receptor includes (ί) diabetes (see, for example, PCT patent applications UO 98/55139, UO 98/12224 and XVO 97/02004); (i) hematopoiesis (see, for example, PCT patent application XVO 97/27286 and HUO 98/18486); (w) infertility (see, for example, PCT patent application ^ O 97/15322 and ^ O 98/36763) and (ίν) tumor suppression (see, for example, PCT patent application ^ O 98/48831), each of which are fully incorporated into this description by reference.

The leptin receptor gene (OB-P) was cloned (SepVapk Assexcup. AP098792) and mapped to the b locus (see, for example, TagEadNA, eE a1., 1995. Ce11 83: 1263-1271). Several transcripts of OB-P obtained by alternative splicing have also been identified. Disorders in the formation of OB-P cause a syndrome in mutated O / O mice with diabetes, which are phenotypically identical to O / O mice (see, for example, CI1atb1, eE a1., 1996. Proc. No. 111. Asab. 8c1. I8A 93: 6231-6235). In contrast to ob / mice mice, however, the administration of the recombinant leptin C57BK8 / 1-wb / mice to mice does not cause a decrease in food intake and body weight (see, for example, Pojeck apb Szheepdegd, 1996. SHEPEjup Rev. 54: 41-49 )

Most leptin-related studies describe the effect on weight loss with the introduction of recombinant leptin, fragments of leptin and / or variants of the leptin receptor with the introduction of these structures directly into the ventricles of the brain, see, for example, ^ e1d1e, eE a1., 1995.1. C11p 1pue81 96: 2065-2070; Wataky, eE a1., 1996. Epbostalod 137: 3144-3147.

Other studies have shown a significant effect on weight loss with the administration of leptin peptides when administered intraperitoneally (i.p.) to the test patients, see Sgakko eE a1., 1997. Eppospodod 138: 1413-1418. It has further been described that leptin fragments, more specifically, an 18 amino acid fragment containing residues derived from full-length human leptin, affect weight loss, but only when introduced directly through an implanted catheter into the lateral ventricle of the rat brain, see, for example, PCT Patent Application ^ O 97/46585, which is incorporated herein by reference in its entirety.

Another peptide hormone involved in diseases and metabolic disorders is cholecystokinin (CCK). According to reports, SSK was identified in 1928 from preparations of intestinal extracts due to its ability to stimulate gallbladder contraction. Since then others have been described CCK biological actions, including stimulation of pancreatic secretion, slow gastric emptying, stimulation of intestinal motility and stimulation of insulin secretion, see. Ne \ egke eE ^r a1., Ann. Ν.Υ. Asab. 8CT 713: 268-272 (1994). Reportedly, CCK actions also include actions on cardiovascular function, respiratory function, neurotoxicity and convulsions, cancer cell proliferation, analgesia, sleep, sexual and reproductive behavior, memory, anxiety, and dopamine-mediated behavior. SGA \\ '1eu apb SotGOp, Rurbbek 15: 731-755 (1994). Other described CCK actions include stimulating pancreatic growth, stimulating gallbladder contraction, inhibiting the secretion of gastric acid, releasing pancreatic polypeptide, and reducing the peristalsis component. Additional described CCK actions include vasodilation. ^ a1ky, Sa81gosh1e81sha1 Nogtopek, 1n Ryukyu1 about! Eeye SakEgo1n (ekNpa1 TgasE (3b eb. 1994; Paνеη Ргекк, №е \ т Υο ^ к).

Injections of combinations of glucagon, CCK, and bombesin have been described to enhance the inhibition of the consumption of concentrated milk test dishes in rats with free access to food, compared with the inhibition obtained using individual compounds. NshEop eE a1., Wtash Rec. Vi11. 17: 615-619 (1986). It has also been described that glucagon and CCK synergistically inhibit false nutrition in rats. Le8a1e1 apb Seagu, At. 1. Ryukyu 1. 253: P217-225 (1987); 8th APB C1bk, Appa1k Ν.Υ.

- 6 014647

Asab. 8s1. 713: 236-241 (1994). It has also been suggested that estradiol and SSC can have a synergistic effect on the feeling of satiety. Bik-pois e1 a1., RerPBev 15: 913-918 (1994); 8th ApB C | bb. higher. It was also suggested. what signals. coming from the small intestine in response to the ingestion of nutrients. can synergistically interact with CCK to reduce food intake. Sokh. Weiau. Vgash Kev. 38: 35-44 (1990). Besides. has been described. that SSC causes a feeling of fullness in some species. For example. has been described. that a decrease in the amount of nutrition is caused by the introduction of CCK intraperitoneally in rats. intraarterially in pigs. intravenously in cats and pigs. into the ventricles of the brain in monkeys. rats. dogs and sheep. intravenously in people with and without obesity. see Jeueve e1 a1 .. above. According to reports. research. carried out in several laboratories. confirmed the behavioral specificity of low doses of CCK during inhibition of nutrition. by comparing responses to food and responses to non-food stimulants in monkeys and rats. and that. what they showed. that CCK shows a sequence of behavior. usually observed after eating food (eg. saturation sequence. occurring after eating). Besides. comparing behavior after SSC with behavior after eating. in pure form or in combination with CCK. reportedly reveals a behavioral similarity between SSC eating. Sga \\ 1eu apB Sog \ ush. higher. It has also been described. that CCK in physiological plasma concentrations inhibits food intake and enhances the feeling of fullness in thin and fat people. see Jeueve e1 a1 .. above.

CCK was characterized in 1966 as a peptide of 33 amino acids. Sga \\ 1eu apB Sopush. higher. Species-specific molecular variants of the CCK amino acid sequence were identified. According to reports. a sequence of 33 amino acids and a truncated peptide. its C-terminal sequence (CCK-8) of 8 amino acids was identified in the pig. rats. Chicken. chinchillas. dogs and humans. The sequence of 39 amino acids was. according to available data. found in a pig. dogs and guinea pigs. A sequence of 58 amino acids was found in a cat. dogs and humans. The frog and the turtle. according to available data. have a sequence of 47 amino acids. homologous to CCK. and gastrin. More recently, it has been described. that the human intestines contain small amounts of even large molecules. named SSK-83. According to reports. the predominant intermediate form was identified in the rat and named SSK-22. Wa1vy. Sav1go1p1e8 (1pa1 Nailgun. 1n Ryuvuodu o £ 1e Sav1got1ev1ta1 Tgas1 (3B eB. 1994; Yaauep Rgevv. No. \ u Wogk). Unsulfated SSK-8 and tetrapeptide {named SSK-4 (SSK) (30-) in the rat brain. The C-terminal pentapeptide {named CCK-4 (CCK (29-33))} preserves the structural homology of CCK. as well as homology with the neuropeptide. gastrin. C-terminal sulfated octapeptide sequence. CCK-8. according to available data. relatively conservative between species. Cloning and sequence analysis of cDNA encoding preprocholecystokinin from shield cancer cells prominent prostatic rat. Brain pig and pig intestines. reportedly. show 345 nucleotides. precursor CCK encoding. which has 115 amino acids and contains all of the CCK sequences. previously described as dedicated. Cm \\ 1eu APB Sopush. above.

CCK was characterized in 1966 as a peptide of 33 amino acids. Sga \\ 1eu apB Sopush. higher. Species-specific molecular variants of the CCK amino acid sequence were identified. According to reports. a sequence of 33 amino acids and. truncated peptide. its 8-amino acid C-terminal sequence (CCK-8) was identified in a pig. rats. Chicken. chinchillas. dogs and humans. The sequence of 39 amino acids was. according to available data. found in a pig. dogs and guinea pigs. A sequence of 58 amino acids was found in a cat. dogs and humans. The frog and the turtle. according to available data. have a sequence of 47 amino acids. homologous and CCK. and gastrin. More recently, it has been described. that the human intestines contain small amounts of even large molecules. named SSK-83. According to reports. the predominant intermediate form was identified in the rat and named SSK-22. Wa1vy. Sav1go1p1e8 (1pa1 Nailgun. 1n Ryuvuodu o £ 1e Sav! Gosh1ev! Ta1 Tgas1 (3B eB. 1994; Eauep Rzhevv. Wogk). Unsulfated CCK-8 and tetrapeptide (named CCK-4 (CCK (30-33)) GO N0: 208) was found in the brain of the rat. The C-terminal pentapeptide (named CCK-4 (CCK (29-33)); 8E0 GO no: 209) retains the structural homology of CCK and also the homology with the neuropeptide gastrin. terminal sulfated octapeptide sequence. CCK-8. reportedly relatively conservative between species. Cloning and sequence analysis of cDNA encoding preprocholecystokinin and From rat thyroid cancer cells, pig brain and pig intestine, 345 nucleotides are known to be encoded by the precursor of CCK, which has 115 amino acids and contains all the CCK sequences previously described as isolated. SGA \ u1eu apB Sopush. above.

It is described. that CCK is distributed in the central nervous system and in the endocrine cells and intestinal nerves of the upper small intestine. SSK agonists include SSK itself (also called SSK33). SSK-8 (SSK (26-33); 8E0 GO N0: 55). not sulfated SSK-8. pentagastrin (CCK-5 or CCK (29-33); 8EO GO N0: 209) and tetrapeptide. SSK-4 (SSK (30-33); 8EO GO N0: 208). In the pancreatic CCK receptor CCK-8. according to available data. replaces binding with 1000-5000 times greater efficiency. than not sulfated SSK-8 or SSK-4. and has been described. that SSK-8 is about 1000 times more effective. than not sulfated SSK-8 or SSK-4 when stimulated

- 7 014647 secretion of pancreatic amylase. Sgh \\ 1eu apy ί'οπνίπ. higher. It has been described that in homogenates from the cerebral cortex, the binding of the CCK receptor, replaced by non-sulfated CCK-8 and CCK-4 at equimolar concentrations, is 10-100 times more than sulfated CCK-8. 1st.

Another family of peptide hormones involved in diseases and metabolic disorders is the family of pancreatic polypeptides (PPPs). Pancreatic polypeptide (PP) was discovered as a minor component of insulin extracts and was named according to the organ in which it was found, and not by its functional importance (K1shche1 e! A1., Episode 83: 1323-30 (1968)). PP is a 36-amino acid peptide containing characteristic structural motifs. Further, a related peptide was found in intestinal extracts and was named peptide ΥΥ (ΡΥΥ) due to Ν- and C-terminal tyrosines (Ta1esho1o, Prgos. Να! 1. Asai. 8c1. I8A 79: 2514-8 (1982)). The third related peptide was later found in brain extracts and was called the neuropeptide Υ (ΝΡΥ) (Ta1esho1o, Prgos. Νη11. Asai. 8c1. I8A 79: 5485-9 (1982); Ta1esho1o e! A1., Wing 296: 659-60 ( 1982)).

It has been described that these three related peptides have different biological effects. The actions of PP include inhibition of pancreatic secretion and relaxation of the gallbladder. Central administration of PP causes a moderate increase in food intake, which can be mediated by receptors localized in the hypothalamus and brain stem (review in CeJey, Proc. 8oc. Exp. Wu. May. 218: 7-22 (1998)).

ΡΥΥ Release occurs after food intake. An alternative molecular form of ΡΥΥ is ΡΥΥ (3-36) (8ETC ΙΌ ΝΟ: 58) (Eeyes e! A1., Reyyes 10: 797-803 (1989); Cgapy! E! A1., Kedi1.Rep !. 51: 151 -9 (1994)). This fragment represents approximately 40% of the total ΡΥΥ-like immunological reactivity of intestinal extracts of humans and dogs, and from about 36% of the total ΡΥΥ immunological reactivity in fasting plasma to a little more than 50% after eating. This is obviously a product of the cleavage of dipeptidyl peptidase-ΐν (ERP4) ΡΥΥ. ΡΥΥ (3-36) (8EC GO ΝΟ: 58) is reported to be a selective ligand at Υ2 and Υ5 receptors, which are pharmacologically unique when truncated in the Ν-terminal region (i.e., C-terminal fragments) are preferred Υ analogues. Peripheral administration of ΡΥΥ, according to reports, reduces the secretion of acid of the gastric juice, the motor function of the stomach, the secretion of exocrine of the pancreas (Uozypada e! A1., At. 1. Pb.uzyu.1.263: C695-701 (1992); Siap e! A1 ., Episode 1 128: 911-6 (1991); Rarraz e! A1., Saz! Goop! His1od 91: 1386-9 (1986)), contraction of the gall bladder and intestinal motility (8auade e! A1., Ci! 28: 166-70 (1987)). The effect of central administration ΡΥΥ on gastric emptying, gastric motor function, and gastric acid secretion obtained after direct administration in or near the rhomboid / brain stem (Syep ap Codejs, At. 1. Ryuzyu1. 269: K.787-92 (1995) ; Syep e! A1., Kedi 1.Rep !. 61: 95-98 (1996); Khapd apy Tasye, At. 1. Ryuzyu1. 268: S943-8 (1995); Syep e! A1., #Todaz! Toep ! eto1. Mo! 11. 9: 109-16 (1997)) may differ from the effect observed after peripheral administration. For example, centrally administered ΡΥΥ has some actions opposite to those described here for peripherally administered ввод (3-36) (8EC GO ΝΟ: 58), which consists in stimulating the secretion of gastric acid, and not inhibition. The gastric motor function suppresses only in combination with stimulation of TSC, and not when administered in its pure form, and in fact is stimulating at a higher dosage due to the alleged interaction with PP receptors. It has been shown that ΡΥΥ stimulates the consumption of food and water after central administration (Mog1eu e! A1., Vgat Kez. 341: 200-3 (1985); Cons. E! A1., At. 1. Ryuzyu.1. 259: K317-23 ( 1990)).

Diseases and metabolic disorders can take many forms, including obesity, diabetes, dyslipidemia, insulin resistance, cell apoptosis, etc. Obesity and related disorders are common and very serious public health problems in the United States and around the world. Upper body obesity is the strongest risk factor known for type 2 diabetes and is the strongest risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gall bladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, breast, prostate and colon cancer, and to increase the incidence of complications general anesthesia (see, for example, Kore1tap, No. 1Shge 404: 635-43 (2000)). It reduces life expectancy and carries a serious risk of the concomitant diseases listed above, as well as disorders such as infections, varicose veins, acantokeratoderma, eczema, exercise intolerance, insulin resistance, hypercholesterolemia with hypertension, gallstone disease, orthopedic injuries and thromboembolic disease ( K1zzapep e! A1., Bg. May. 1. 301: 835-7 (1990)). Obesity is also a risk factor for a group of conditions called insulin resistance syndrome, or Syndrome X. A recent estimate of the medical costs of treating obesity and related diseases totals $ 150 billion worldwide. It is believed that the pathogenesis of obesity is multifunctional, but the main problem is that in patients with obesity, the absorption of nutrients and energy consumption are not balanced as long as there is an excess of adipose tissue. Obesity is currently poorly treated, is chronic, in fact

- 8 014647 ski is an intractable metabolic disorder. Therapeutic drugs used to reduce the weight of obese patients can have a profound beneficial effect on their health.

Diabetes is a carbohydrate metabolism disorder characterized by hyperglycemia and glucosuria resulting from insufficient production or utilization of insulin. Diabetes greatly affects the quality of life of most of the population in developed countries. Insufficient insulin production is characterized as type 1 diabetes, and insufficient insulin utilization is characterized as type 2 diabetes. However, it is now generally accepted that there are many individual diabetes-related diseases that occur long before a patient is diagnosed with diabetes. Also, the effect of insufficient control of glucose metabolism in diabetes causes an increase in a wide range of related lipid and cardiovascular disorders.

Dyslipidemia, or abnormal plasma lipoprotein levels, often occurs in diabetics. Dyslipidemia is usually characterized by elevated plasma triglycerides, low levels of NE (cholesterol high density lipoproteins) cholesterol, normal or elevated (low density lipoproteins) cholesterol, and elevated levels of densely spaced particles of low cholesterol (low density lipoproteins) in the blood. Dyslipidemia is one of the main factors contributing to an increase in cases of coronary attacks and mortality in patients with diabetes. Epidemiological studies support this finding, demonstrating a multiple increase in mortality from coronary seizures among patients with diabetes compared with patients without diabetes. In patients with diabetes, several lipoprotein abnormalities have been described.

Insulin resistance is the weakened ability of insulin to exert a biological effect over a wide range of concentrations. With insulin resistance, the body secretes abnormally high amounts of insulin to compensate for this defect, and a state of impaired glucose tolerance develops. If it is impossible to compensate for the insufficient effect of insulin, the concentration of glucose in the plasma inevitably increases, which causes the clinical condition of diabetes. It is recognized that insulin resistance and related hyperinsulinemia play a contributing role in obesity, hypertension, atherosclerosis, and type 2 diabetes. The association of insulin resistance with obesity, hypertension, and angina pectoris is described as Syndrome X syndrome, which has insulin resistance as a common pathogenic link.

Apoptosis is an active process of self-destruction of cells, which is regulated by external and internal signals that occur during normal development. It is described in detail that apoptosis plays a key role in the regulation of pancreatic endocrine beta cells. There is increasing evidence that in adult mammals, the mass of beta cells is subject to dynamic changes to adapt insulin production to maintain normoglycemia in specific conditions, such as pregnancy and obesity. Beta cell mass control depends on a delicate balance between cell proliferation, growth, and programmed cell death (apoptosis). Violation of this balance can lead to poor glucose homeostasis. For example, it is noteworthy that glucose intolerance develops with age, when the rate of beta cell replication decreases, and a sectional pathological study of a person has repeatedly shown a 4060% decrease in beta-cell mass in patients with non-insulin-dependent diabetes, compared with patients without diabetes. It is generally recognized that insulin resistance invariably accompanies obesity, but that normoglycemia is supported by compensatory hyperinsulinemia until the beta cells are unable to meet the increasing need for insulin when type 2 diabetes begins.

Attempts to treat many of the diabetes-related abnormalities have been to administer several antidiabetic drugs to treat such abnormalities in various patients. Examples of antidiabetic drugs include proteins, such as insulin and insulin analogs, and small molecules, such as insulin sensitizing agents, insulin secretion enhancing agents, and appetite regulating compounds.

There remains a need for the development of polypeptides used in the above diseases, conditions and metabolic disorders. Therefore, the object of the present invention are hybrid polypeptides and methods for their preparation and use. The compounds of this invention find use in the diseases, conditions and metabolic disorders described above and below.

All documents to which this description refers are presented in this application as references in their entirety.

SUMMARY OF THE INVENTION

This invention generally relates to new, selectable hybrid polypeptides used as agents for the treatment and prevention of diseases and metabolic disorders, which can be facilitated by the regulation of plasma glucose levels, insulin levels and / or insulin secretion, such as diabetes and associated diabetes condition. Such conditions and disorders include,

- 9 014647, but not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity and diabetes mellitus of any type, including type 1 and type 2, and diabetes during pregnancy.

In one embodiment of the present invention, hybrid polypeptides exhibiting at least one hormonal activity are provided. The hybrid polypeptides of the invention comprise at least two bioactive peptide hormone modules covalently linked together, wherein at least one of the bioactive peptide hormone modules exhibits at least one hormonal activity of the complex peptide hormone. The bioactive modules of the peptide hormone are independently selected from complex peptide hormones, fragments of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones, analogues and derivatives of complex peptide hormones, which exhibit at least one hormonal activity of complex peptide hormones, fragments of analogues or derivatives of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones, and peptide enhancement moat.

In one embodiment of a hybrid polypeptide exhibiting at least one hormonal activity, the hybrid polypeptide comprises at least a first bioactive module of a peptide hormone covalently linked to at least one additional bioactive module of a peptide hormone; where the bioactive modules of the peptide hormone are independently selected from the group consisting of complex peptide hormones, fragments of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones; analogues and derivatives of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones; fragments of analogues and derivatives of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones; and peptide enhancers. Complex peptide hormones are usually independently selected from at least two of the group consisting of amylin, adrenomedullin (ΆΌΜ), calcitonin (CT), the peptide encoded by the calcitonin gene (CCCR), intermedin, cholecystokinin (CCK), leptin, peptide ΥΥ (ΡΥΥ ), glucagon-like peptide-1 (CPB-1), glucagon-like peptide 2 (CPB-2), oxintomodulin (OXM), natriuretic peptide and exendin-4. Typically, peptide enhancers are independently selected from the group consisting of structural motifs of complex peptide hormones that give the hybrid polypeptide the desired chemical stability, conformational stability, metabolic stability, bioavailability, orientation to the organ / tissue, interaction with the receptor, protease inhibition, plasma protein binding, or other pharmacokinetic characteristics, and structural motifs of analogues or derivatives of complex peptide hormones that confer hybrid polypeptides the desired chemical stability, conformational stability, metabolic stability, bioavailability, targeting to an organ / tissue, interaction with a receptor, protease inhibition, plasma protein binding, or other pharmacokinetic characterization. In yet another embodiment, at least one of the bioactive modules of the peptide hormone exhibits at least one hormonal activity of the complex peptide hormone. In further alternative embodiments, if at least one bioactive module of a peptide hormone that exhibits at least one hormonal activity of a complex peptide hormone is amylin, a fragment of amylin that exhibits at least one hormonal activity, an analog or derivative of amylin that exhibits at least one hormonal activity, or a fragment of an analog or derivative of amylin that exhibits at least one hormonal activity, and at least one other bioactive module of the peptide hormone is SSC, a SSC fragment that exhibits at least one hormonal activity, an SSC analog or derivative that exhibits at least one hormonal activity, a SSC analog or derivative fragment that exhibits at least one hormonal activity , CT, a fragment of CT that exhibits at least one hormonal activity, an analog or derivative of CT that exhibits at least one hormonal activity, or a fragment of an analog or derivative CT, which exhibits at least one hormonal activity, then the hybrid polypeptide may further comprise at least three bioactive peptide hormone modules selected from at least three different complex peptide hormones. In yet a further alternative embodiment, if at least one bioactive module of the peptide hormone that exhibits at least one hormonal activity of the complex peptide hormone is CPB-1, a fragment of CPB-1 that exhibits at least one hormonal activity, an analog or a derivative of CPB-1, which exhibits at least one hormonal activity, or a fragment of an analogue or derivative of CPB-1, which exhibits at least one hormonal activity, and at least about These other bioactive peptide hormone module is a peptidic enhancer comprising an exendin fragment, then the hybrid polypeptide may also comprise at least three bio-active peptide hormone module.

Complex peptide hormones in accordance with this invention include amylin, adrenoma

- 10 014647 dullin (ΑΌΜ), calcitonin (CT), peptide encoded by the calcitonin gene (SSBP), interludes, cholecystokinin (CCK), leptin, peptide ΥΥ (ΡΥΥ), glucagon-like peptide-1 (CPB-1), glucagon-like peptide 2 (CPB-2), oxyntomodulin (OXM), natriuretic peptides and exendin-4.

The peptide enhancers of the invention include structural motifs of complex peptide hormones that confer on the hybrid polypeptide the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue orientation, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic the characteristics and structural motifs of analogues or derivatives of complex peptide hormones that give the hybrid polypeptide the same aemuyu chemical stability, conformational stability, metabolic stability, bioavailability, directivity organ / tissue, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic characteristic.

In another aspect of the invention, methods of treating or preventing obesity are provided, wherein the methods comprise administering a therapeutically or prophylactically effective amount of a hybrid polypeptide of the invention to a patient. In a preferred embodiment, the patient is obese or overweight. Although obesity is usually defined as a body mass index in excess of 30, for the purposes of this description, any patients, including those who have a body mass index less than 30 but who need to reduce body weight or want to reduce body weight, are included in the definition of obesity. Patients who have insulin resistance, glucose intolerance or any form of diabetes mellitus (for example, type 1, type 2 diabetes or during pregnancy) can use this method.

In yet another aspect of the present invention, methods of reducing food intake, reducing nutrient absorption, causing weight loss, treating diabetes and diabetes-related conditions, and improving the lipid profile (including lowering LDL cholesterol and triglyceride levels and / or changing NOT levels of cholesterol) are provided where the methods include administering to the patient an effective amount of a hybrid polypeptide in accordance with this invention. In a preferred embodiment, the methods of the invention are used to treat or prevent conditions or disorders that can be alleviated by reducing the absorption of nutrients in a patient, comprising administering to the patient a therapeutically or prophylactically effective amount of a fusion polypeptide of the invention. In another embodiment, the methods of the invention are used to treat or prevent conditions or disorders that can be alleviated by controlling plasma glucose levels, insulin levels and / or insulin secretion. In yet another embodiment, the methods of the invention are used to treat diabetes and / or 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 type, including type I, type II and diabetes during pregnancy, diabetic complications, neuropathy ( based, for example, on the neurotrophic action of exendin-4), neuropathic pain (based on, for example, the action of amylin), retinopathy, nephropathy, pancreatic beta-cell mass deficiency states (based on, for example, effect on the neogenesis of exendin-4 and CPb-1 islets).

The present invention also relates to pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of at least one hybrid polypeptide of the invention or a pharmaceutically acceptable salt thereof, together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers used for the delivery of hybrid polypeptides.

These and other aspects of the present invention will be better understood with reference to the following preferred embodiments and a detailed description of the invention.

Brief Description of the Drawings

In FIG. 1 shows the effect of typical compounds in accordance with this invention in the analysis of EH mice.

In FIG. 2 shows the effect of typical compounds of the invention in the analysis of EH mice.

In FIG. 3A-3C show the effect of typical compounds of the invention in the analysis of EH mice.

In FIG. 4A, 4B show the effect of typical compounds in accordance with this invention in the analysis of food consumption compared with the original peptide compounds.

In FIG. 5A, 5B show the effect of typical compounds in accordance with this invention in the analysis of lowering blood glucose and analysis of food intake, respectively.

In FIG. 6 depicts structures of various typical linkers used.

In FIG. Figure 7 shows the structures of various typical linkers, as well as the sequence and νίίτο activity of typical hybrids of the ΡΥΥ family and amylins.

- 11 014647

In FIG. Figure 8 shows the structures of various typical linkers, as well as the sequence and the νί1th activity of other typical hybrids of the RUU / RUU family.

In FIG. Figure 9 shows weight loss using typical hybrids of the RUU family and amylins.

In FIG. 10 is a graph of calcium mobilization by hybrids of the CCK8 family in cell colonies having the CCK receptor.

In FIG. 11A, B and C show food inhibition by hybrids of the amylin family, for example, amylin-kST-amylin and RUU (for example, RUU№RU chimeras).

In FIG. 12A depicts a decrease in total food intake and FIG. 12B depicts weight loss using hybrids of the amylin-kST-amylin / RUU * family, meaning that P <0.05 compared to the carrier.

In FIG. 13 shows the inhibition of food intake by hybrids of the RUU / RUU family. Doses are 25 nmol / kg. The discrepancy of ~ 15-20% is statistically significant.

In FIG. 14A and 14B depict the inhibitory effect on food consumption of hybrids of the CCK family with a component of the family of either amylins or RUU. Doses are 25 nmol / kg. The discrepancy of ~ 15-20% is statistically significant.

In FIG. 15A and 15B depict the inhibitory effect on food consumption of hybrids of the Μ & 4 family with a component of the family of either amylines or RUU. Doses are 25 nmol / kg. The discrepancy of ~ 15-20% is statistically significant.

In FIG. 16A depicts a decrease in total food intake, and 16B depicts a decrease in body weight by using hybrids with a component of the семейства & 4 family and a component of the amylin family, for example, amylin-kST-amylin. * means that P <0.05 compared with the carrier.

In FIG. 17Α-17Ό shows the inhibition of food intake using hybrids containing a component of the ΕΝ-38 family and said component of the second family. FIG. 17A-ΕΝ38 with a component of the amylin family, amylin-kST-amylin chimera, compound 10. FIG. 7B - ΕΝ38 with a component of the RUU, RUU-3-36 family. FIG. 17С - ΕΝ38 with a component of the RUU family, RUU№RU chimera. FIG. 17 Ό 38 with a component of the CCK8, CCK8 family. Doses are 25 nmol / kg. The discrepancy of ~ 15-20% is statistically significant.

In FIG. 18A and 18B depict inhibition of food intake by hybrids containing components of the exendin family and the amylin family. Unless otherwise indicated, doses are 25 nmol / kg. The discrepancy of ~ 15-20% is statistically significant.

In FIG. 19A and 19B depict inhibition of food intake during meals in the dark and long-term inhibition of total food intake using hybrids of the exendin / amylin family.

In FIG. 20A and 20B depict chronic inhibition of food intake and the effects on fasting and weight loss of an empty stomach of a typical hybrid of the exendin / amylin-kST-amylin family compared to the parent molecules. * means that P <0.05 compared with the carrier.

In FIG. 21A-21C show a hybrid effect on metabolic parameters after 14 days of treatment in rats.

In FIG. 22-29 depict the ability of PPEs of polypeptide chimeras, for example RUU #RU of chimeric compounds 4883 and 5705, to reduce total food intake in the food consumption assays described herein.

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to new breeding hybrid polypeptides used as agents for the treatment and prevention of diseases and metabolic disorders, which can be facilitated by controlling plasma glucose levels, insulin levels and / or insulin secretion, such as diabetes and diabetes-related condition. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and diabetes mellitus of any type, including type 1, type 2, and diabetes during pregnancy.

In one aspect, the invention includes a modular construction of physiologically, metabolically and / or pharmacokinetically active peptide molecules that can be selected based on bioactivity, for example, therapeutic efficacy, spectrum of action, duration of action, physico-chemical properties and / or other pharmacokinetic properties.

Not limited to theory, this invention relates, at least in part, to an instrumental approach in which the bioactive modules of peptide hormones are linked in two-component, three-component or higher order combinations to produce new effective therapeutic agents with selectable properties. Bioactive modules of peptide hormones can include peptide hormones, peptide fragments with hormonal activity or structural motifs of peptide hormones that improve chemical, metabolic and / or other pharmacokinetic stability. Peptide hormones may include natural peptide hormones, as well as peptide hormone analogs and derivatives known in the art and described herein.

- 12 014647

In one aspect of the present invention, it was found that a combination of certain physicochemical characteristics of two or more peptide hormones in a single agent can facilitate the intervention at several points of a dysfunctional metabolic system. In one aspect of the invention, as such, rationally engineered hybrid polypeptides are provided that integrate selected biological actions into a single polypeptide agent. In one embodiment, breeding hybrid polypeptides in accordance with this invention may include the use of chemically stable linkers for covalent attachment of bioactive molecules. In another embodiment, breeding hybrid polypeptides in accordance with this invention may include the use of cleavable linkers, which alone can be or can form part of a bioactive molecule.

Again, not limited to theory, the creation of hybrid polypeptides in accordance with this invention typically may include: (1) identification, selection and coupling of bioactive modules of peptide hormones to obtain the desired effectiveness and therapeutic use, and (2) covalent binding of bioactive modules (e.g., natural peptide hormones, analogues and derivatives of peptide hormones with hormonal activity, fragments of peptide hormones with hormonal activity, stabilizing motifs, etc.) or directly either through the linker, without loss of bioactivity of the constituent modules. In certain embodiments, the selection criteria for the modules may include, but are not limited to: (a) the desired action for the desired therapeutic or prophylactic indication, such as an additive or synergistic effect; (B) optional synergies or dual effects of coupled modules for a variety of therapeutic and / or prophylactic indications / and / or (c) desired chemical stability, conformational stability, metabolic stability, bioavailability, targeting of the organ / tissue, interaction with the receptor, protease inhibition, plasma protein binding and / or other pharmacokinetic characteristics.

The section headings are used here solely for the purpose of streamlining and should not be construed as limiting the subject matter in any way.

Hybrid Polypeptides of the Invention

As indicated above, this invention partially relates to hybrid polypeptides containing at least two bioactive modules of the peptide hormone selected from the complex peptide hormones described herein. Hybrid polypeptides in accordance with this invention are usually used for the treatment and prevention of conditions and metabolic disorders. Hybrid polypeptides in accordance with this invention exhibit at least one hormonal activity of the complex peptide hormone and preferably can exhibit at least one additional biological activity of the second complex peptide hormone.

In one embodiment, the hybrid polypeptides of the invention may comprise at least two bioactive modules of the peptide hormone, wherein each of the at least two bioactive modules of the peptide hormone exhibits at least one hormonal activity of the complex peptide hormone. In another embodiment, the hybrid polypeptides in accordance with this invention may contain at least two bioactive modules of the peptide hormone, where at least one of these bioactive modules of the peptide hormone has at least one hormonal action of the complex peptide hormone, and at least one of these bioactive modules of the peptide hormone gives the desired chemical stability, confirmation stability, metabolic stability, bioavailability, focus on organ / tissue, vz receptor interaction, protease inhibition, plasma protein binding and / or other pharmacokinetic characteristics of the hybrid polypeptide.

In a preferred embodiment, the hybrid polypeptides in accordance with this invention may have comparable or greater efficacy in the treatment and / or prophylaxis of conditions and metabolic disorders compared with complex peptide hormones. In another embodiment, the hybrid polypeptides of the invention may have comparable or greater efficacy in the treatment and / or prophylaxis of diabetes and / or diabetes-related disorders compared to complex peptide hormones. Alternatively, preferred hybrid polypeptides of the invention may have improved processability, stability, and / or ease of formulation compared to complex peptide hormones.

More specifically, the hybrid polypeptides of the invention typically comprise a first bioactive peptide hormone module covalently linked to at least one additional bioactive peptide hormone module. The bioactive modules of the peptide hormone can be covalently linked to each other by any method known in the art, including, but not limited to, direct amide bonds or chemical linker groups, which are described in more detail below. In one embodiment, chemical linker groups may include peptide mimetics that induce or stabilize the polypeptide conformation.

The first bioactive module of the peptide hormone can be selected from the first complex peptide

- 13 014647 of a hormone and may be a peptide hormone (including natural peptide hormones, as well as their analogues and derivatives), a peptide fragment with hormonal activity (including fragments of natural peptide hormones, as well as their analogues and derivatives), or a structural motif of the peptide hormone ( including natural peptide hormones, as well as their analogues and derivatives), which give the desired chemical stability, conformational stability, metabolic stability, bioavailability, focus on organ / tissue, interactions tvie receptor, protease inhibition, binding to plasma proteins and / or other pharmacokinetic characteristic to the hybrid polypeptide. Thus, the additional bioactive peptide module (s) can be selected from complex peptide hormones, and can be a peptide hormone (including natural peptide hormones, as well as their analogues and derivatives), a peptide fragment with hormonal activity (including fragments of natural peptide hormones, and also their analogues and derivatives) or the structural motif of the peptide hormone (including natural peptide hormones, as well as their analogues and derivatives), which give the desired chemical stability, conformational stability, metabolic stability, bioavailability, targeting to organ / tissue, interaction with the receptor, protease inhibition, binding to plasma protein and / or other pharmacokinetic characteristics of the hybrid polypeptide. The first peptide hormone and the additional peptide hormone may be the same peptide hormones, may be from the same family of peptide hormones, or may be different peptide hormones depending on the desired characteristics of the bioactive modules of the peptide hormone.

As used herein, the term bioactive refers to (1) biological activity in at least one ίη νίνο hormonal pathway, or (2) modulation of therapeutic efficacy, spectrum of action, duration of action, physicochemical properties and / or other pharmacokinetic properties of such biological action. Biological effects can be evaluated using binding hormone receptor binding assays, or using metabolic studies that control physiological indications, as is known in the art and described herein. The modulation of therapeutic efficacy, spectrum of action, duration of action, physico-chemical properties and / or other pharmacokinetic properties of such a biological action can be carried out by changing, for example, chemical stability, conformational stability, metabolic stability, bioavailability, focus on organ / tissue, interactions with the receptor, protease inhibition, plasma protein binding and / or other pharmacokinetic characteristics.

In one embodiment, the hybrid polypeptides of the invention retain at least about 25%, preferably about 30, 40, 50, 60, 70, 80, 90, 95, 98, or 99% of the biological activity of the complex peptide hormone. Preferred hybrid polypeptides are those that demonstrate efficacy in one of the metabolism 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.), which is equal to or superior to the effectiveness of the complex peptide hormone in the same analysis. Alternatively, preferred hybrid polypeptides of the invention may have improved processability, stability, and / or ease of formulation compared to complex peptide hormones.

In another embodiment, the hybrid polypeptides in accordance with this invention retain at least about 25, preferably about 30, 40, 50, 60, 70, 80, 90, 95, 98, or 99% of the biological activity of the natural complex peptide hormone related to reducing digestibility nutrients, reducing food intake, the effect on weight gain and / or treatment and prevention of conditions and metabolic disorders. In yet another embodiment, the hybrid polypeptides in accordance with this invention have at least about 110, 125, 130, 140, 150, 200% or more of the biological effects of natural peptide hormone related to reducing the absorption of nutrients, reducing food intake, and the effect on weight gain body weight and / or treatment and prevention of conditions and metabolic disorders. In another embodiment, the hybrid polypeptides of this invention have an improved agonistic effect on the receptor for a complex peptide hormone.

Complex peptide hormones, analogues and derivatives

Complex peptide hormones typically include peptide hormones used to treat or prevent diseases and metabolic disorders, including: (a) a family of amylins, including amylin, adrenomedullin (ΑΌΜ), calcitonin (CT), a peptide encoded by the calcitonin gene (SSNR), interludes (also known as ΑΕΡ-6) and related peptides; (B) cholecystokinin (CCK); (c) a family of leptins, including leptins and leptin-like peptides; (ά) a family of pancreatic polypeptides, including pancreatic polypeptide (PP) and peptide ΥΥ (ΡΥΥ); (e) incretins and incretin mimetics, including: peptide hormones derived from the proglucagon gene, such as glucagon, glucagon-like peptide-1 (CPB-1), glucagon-like peptide 2 (CPB-2) and oxyntomodulin (OXM); and exendins, such as exendin-3 and exendin-4; and (D) natriuretic peptides

- 14 014647 dy, including А№, В \ Р, С№ and urodilatin, their precursors and peptides derived from (e) the urocortin family and (11) the neuromedin family, and their analogues, derivatives and fragments. As described herein, peptide hormones in accordance with this invention also include analogs and derivatives that retain the hormonal activity of such natural peptide hormones. In one embodiment, such analogs and derivatives are target hormone receptor agonists.

Amylin refers to the human peptide hormone designated as amylin and isolated from pancreatic beta cells and its species variations described in US Pat. No. 5,234,906 issued August 10, 1993, under the name Hyperglycemic Compositions, the contents of which are incorporated herein by reference. as a link. More specifically, amylin is a 37 amino acid polypeptide hormone that is usually secreted in conjunction with insulin by the pancreatic beta cells in response to nutrient intake (see, for example, Coia e! A1., Lapse! 339: 1179-1180, 1992). In this sense, the terms amylin, wild amylin and natural amylin, i.e. unmodified amylin, are used interchangeably.

Adrenomedullin or AOM refers to the human peptide hormone and its species variations. More specifically, AOM is obtained from a preprohormone of 185 amino acids through sequential enzymatic cleavage and amidation. This process ends with the release of a 52 amino acid bioactive peptide.

Calcitonin or CT refers to the human peptide hormone and its species variants, including salmon calcitonin (CST). More specifically, CT is a 32 amino acid peptide cleaved from the larger prohormone. It contains a single disulfide bond, which causes the ring shape of the amino-terminal region. Alternative splicing of calcitonin pre-mRNA can produce an mRNA encoding a peptide encoded by a calcitonin gene; presumably, this peptide acts in the nervous and vascular systems. The calcitonin receptor is cloned and demonstrated as a member of the seven-transmembrane family of C protein-coupled receptors.

A calcitonin-related peptide or SCCR gene refers to the human peptide hormone and its species variations in any physiological form.

Intermedia or AGR-6 refers to the human peptide hormone and its species variations in any physiological form.

By cholecystokinin or CCK is meant the human peptide hormone and its species variations. More specifically, CCK is a sequence of 33 amino acids, first identified in humans, and includes a w1uo C-terminal fragment of 8 amino acids (CCK8), which is reportedly found in pigs, rats, chicken, chinchillas, dogs and humans . Thus, the term CCK-33 generally refers to human CCK (1-33), and CCK-8 (CCK (2633); 8EC ΙΌ ΝΟ: 55) generally refers to a C-terminal octapeptide sulfated or non-sulfated if not specified otherwise. Further, pentagastrin or CCK-5 refers to the C-terminal peptide of cSK (29-33) (8EC ΙΌ ΝΟ: 209), and CCK-4 refers to the C-terminal tetrapeptide of CCK (30-33) (8EC ΙΌ ΝΟ: 208) . However, in this description, the term CCK refers to all naturally occurring hormone variations, including CCK-33, CCK-8, CCK-5 and CCK-4, in sulfated and non-sulfated forms, unless otherwise indicated.

By leptin is meant existing in nature leptins of any species, as well as biologically active Ό-isoforms, or fragments of naturally occurring leptin and its variations, and a combination of the above. Leptin is a polypeptide product of the b gene, as described in international patent publication No. XV 96/05309, which is incorporated herein by reference in its entirety. Prospective analogs and fragments of leptin are described in US Pat. No. 5,521,283, US Pat.

By PP is meant a human pancreatic peptide polypeptide or its species variations in any physiological form. Thus, the term PP includes the human complete peptide of 36 amino acids described in (8EC ΙΌ ΝΟ: 290) and species variations of PP, including, for example, PP of a mouse, hamster, chicken, bull, rat, and dog. In this sense, the terms PP, wild PP, and natural PP, i.e., not modified PP, are used interchangeably.

By ΦΥΥ is meant a human peptide ΥΥ a polypeptide or its species variations in any physiological form. Thus, the term 'ΈΥΥ includes the human full peptide of 36 amino acids and species variations of ΡΥΥ, including, for example, ΡΥΥ of the mouse, hamster, chicken, bull, rat, and dog. In this sense, the terms' ΈΥΥ and wild ΡΥΥ and natural ΡΥΥ, i.e. not modified ΡΥΥ, apply interchangeably. In the context of the present invention, all modifications described with reference to RUU similar polypeptides in accordance with this invention are based on the 36 amino acid sequence of natural human ΡUΥ.

By CPP-1 is meant the human glucagon-like peptide-1 or its species variations in any physiological form. The term CPP-1 includes human CPP-1 (1-37) (8EC ΙΌ ΝΟ: 59), CPP-1 (7-37) (8EO ΙΌ ΝΟ: 204), and CPP-1 (7-36) amide (8EO ΙΌ ΝΟ: 61), with reference to the full human CP-1 (1-37) (8EC ΙΌ ΝΟ: 59), and species variations of CP-1, including, for example, mouse PP,

- 15 014647 hamster, chicken, bull, rat and dog. In this sense, the terms CPB-1, wild CPB-1 and natural CPB-1, i.e. unmodified CPB-1, are used interchangeably.

By CPP-2 is meant the human glucagon-like peptide-2 or its species variations in any physiological form. More specifically, CPB-2 is a 33 amino acid peptide released together with CPB-1 from intestinal endocrine cells in the small and large intestines.

By OXM is meant human oxyntomodulin or its species variations in any physiological form. More specifically, OXM is a 37 amino acid peptide that contains a 29 amino acid glucagon sequence with the addition of a 8 amino acid carboxy-terminal extension segment.

By exendin is meant the peptide hormone found in the saliva of lizardfish, lizards that are found in Arizona, and Mexican toothfish, as well as its species variations. More specifically, exendin-3 is present in the saliva of escorpion lizards, and exendin-4 is present in the saliva of the Zhilatier lizards (Epd, I., e! A1., E ΒίοΙ. Сет., 265: 20259-62, 1990; Epd., I ., е! а1., Е ΒίοΙ. Сет., 267: 7402-05 (1992)). The exendins have some sequence similarity with some members of the glucagon-like peptide family, with the highest identity, 53%, to CPB-1 (Soke, e! A1., E ΙοΙ. Сет., 268: 19650-55 (1993)). In this sense, the terms exendin, wild exendin, and natural exendin, i.e. unmodified exendin, are used interchangeably.

By urocortin is meant the human urocortin peptide hormone or its species variants in any physiological form. More specifically, there are three human urocortins: isp-1, isp-2, and isp-3. For example, human urocortin 1 has the formula: Acre-Akp-Rgo-Zeg-Eeyzeg-11e-Akr-Eey-Tyr-Rye-N1k-Eey-Eey-Agd-Thg-Eey-Eey-S1i-Eey-A1a-Agd- Tyr-S1n-Zeg-S1n-Agd-S1i-Agd-A1aC1i-S1n-Akp-Agd-Pe-Pe-Pe-Akr-Zeg-Ya1-MN2 (WE WE GO N0: 294). The urocortin obtained from the rat is identical, but has 2 substitutions: Acr2 instead of Acp2 and Prgo4 instead of Zeg4. The human Isp-2 has the sequence N0: 399). Human Isp-3 has the sequence Pb, Th, Th, Zie, Ley, Acre, Va1, Rg, Th, Ac, 11e Me! Akp еи еи еи Р е Ак Ак Ак Akp 11e A1a ук А А ук а а а ук Ак Ак Ак Ак Ак Ак п п A1a С1п 11е (WEES GO N0: 299). Isp-3 is preferably in the form of an amide. Other urocortins and analogs are described in the literature, for example, US Pat. No. 6,214,797. Urocortins Isp-2 and Isp-3, which retain the properties of suppressing food intake and antihypertensive / cardioprotective / inotropic properties, are widely used in hybrids in accordance with this invention. Streskopin (Isp-3) and the peptide related to streskopin (Isp 2), so named because of its ability to suppress chronic activation of HPA in response to aspiration, such as diet / starvation, is specific for CHE receptor type 2 and does not activate SKE-K1 which mediates the release of ACTH. Hybrids containing urocortin, for example, Isp-2 or Isp-3, are especially useful due to the vasodilating effect and, thus, are used for the cardiovascular diseases described here, for example CHE. Hybrids containing urocortin in accordance with this invention find particular use in the treatment or prevention of conditions associated with stimulation of ACTH, hypertension due to vasodilating action, inflammation mediated by an increase other than ACTH, hyperthermia, impaired appetite, congestive heart failure, stress, anxiety and psoriasis. Such compounds are also used to obtain antiproliferative effects, such as the treatment or prevention of cancer or tumor growth. Of particular interest is the urocortin peptide hormone module, combined with the natriuretic peptide module, the amylin family, the exendin family, or the CPB1 family module, to obtain an improved beneficial effect on the cardiovascular system, for example, for treating CHE via a favorable vasodilator effect.

By neuromedin is meant a family of peptides of neuromedin, including the peptides neuromedin I and Z, more specifically their active hormone sequences. For example, the natural active human peptide hormone neuromedin I is neuromedin I25: PbE Agd Va1 Acre C1i C1i PbE C1p Zeg Prgo PbE A1a Zeg C1l Zeg Agd C1u Tug Pbie Bie Rye Agd Rgo Agd Akp (ZES GO N0: 30 WE GO N0: form. P1d I25 has the sequence E1 <U0EECCP1UZSIKKUEEKRKI (WEU GO N0: 314), especially its amide form. Other members of the family, and includes neuromedin listed under the symbol Z \ U1ZZ-RE0T and input numbers: mEii_SA№A (R34962), meii_saur0 (R34966), №ii_sshsk (R34963), NEυυ_nυmAN (R48645), IEii_B1TSE (R81872), MEIi_M0IZE (O9OHK8) . MEII_R1C (P34964), NЕυυ_ΚЛΒIТ (Р34965), MEISCHNAKGE (Р20056) and MEISCHNAT (Р12760). Of particular interest are their processed active peptide hormones and their analogues, derivatives and fragments. Various truncated or spliced variants are included in the family of neuromedins I, for example, Е ^ ЕНΥЗΚТ ^ Κ ^ СΚЗНУУЕЕ ^^ ЗРЕАЗ ^ ЗΚСΥЕ ^ ЕΚРΚN (ЗЕЕ) ГО N0: 300). A typical representative of the family of neuromedins Z is the human neuromedin Z, having the sequence 1ESKSSTAAUPETKKGONTATNOSKREEEKRS \ [(WEZ GO N0: 315), especially its amide form. Hybrids of the invention containing a neuromedin module possess anorexigen

- 16 014647 and thus are valuable for the treatment of obesity, diabetes, reduced food intake and other similar conditions and disorders described here. Of particular interest are the modules of neuromedin combined with the module of the peptide of the amylin family, the peptide of the exendin family or the peptide of the CP1 family.

As used herein, the term “analogue” refers to a peptide whose sequence is derived from the sequence of the main reference peptide (e.g., PP, ΡΥΥ, amylin, CPB-1, exendin, etc.), including insertions, substitutions, extensions and / or deletion of the reference sequence amino acids, preferably having 50 or 55% amino acid sequence identity with the main peptide, more preferably having at least 70, 80, 90 or 95% amino acid sequence identity with the main peptide. In one embodiment, such analogs may contain conservative or non-conservative amino acid substitutions (including non-natural amino acids and b and Ό forms).

A derivative is defined as a molecule having an amino acid sequence of a natural reference peptide or analog, but additionally having chemical modifications of one or more of the side groups of amino acids, α-carbon atoms, an amino terminal group or a carboxylic acid terminal group. Chemical modification includes, but is not limited to, the addition of chemical groups, the creation of new bonds and the removal of chemical groups. Modifications of the amino acid side groups include, but are not limited to, acylation of the ε-amino groups of lysine, Ν-alkylation of arginine, histidine, or lysine, alkylation of glutamine or aspartic carboxylic acid groups, and deamidation of glutamine or aspargin. Modifications to the terminal amino include, without limitation, desamino, Ν-lower alkyl, Ν-di-lower alkyl, restricted alkyls (e.g., branched, cyclic, condensed, adamantyl) and Ν-acyl modifications. Modifications to the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, restricted alkyls (e.g., branched, cyclic, fused, adamantyl), alkyl, dialkylamide, and lower alkyl ester modifications. Lower alkyl is _C1-C4 alkyl. Further, one or more of the side groups or end groups may be protected by protecting groups known to those skilled in the art of peptide chemistry. The α-carbon of an amino acid may be mono- or dimethylated.

By agonist is meant a compound that has the biological effect of a natural human reference peptide, preferably has a potency greater than that of the reference peptide, or has five-fold (plus / minus) potency compared to the reference peptide, more preferably 4-, 3-, 2- or 1-fold, evaluated using measurement methods known in the art, such as receptor binding / comparative studies. In one embodiment, the term refers to a compound that has a biological effect, the same as that of a natural human reference peptide, for example, compound (1), which acts in analyzes of food intake, gastric emptying, pancreatic secretion or weight loss, similar to a natural human reference peptide , or (2) which specifically binds in a reference receptor assay or in a comparative binding assay, to a labeled reference peptide. Preferably, agonists bind in such assays with an affinity of more than 1 μM, more preferably an affinity of more than 1-5 nM. In another embodiment, the term refers to a compound that has a biological effect in the treatment of diabetes or diabetes-related conditions or disorders. Such agonists may contain an active fragment of a reference peptide or a small chemical molecule.

By amino acid and amino acid residue are meant natural amino acids, non-natural amino acids and modified amino acids. Unless otherwise indicated, any reference to an amino acid, usually or specifically to a name, includes references to both the S and L stereoisomers, if its structure allows for such stereoisomeric forms. Natural amino acids include alanine (A1a), arginine (Agd), asparagine (Aki), aspartic acid (Acre), cysteine (Suk), glutamine (C1p), glutamic acid (C1i), glycine (C1u), histidine (Ηίκ), isoleucine (11e), leucine (Le), lysine (Luc), methionine (Me!), phenylalanine (Pb), proline (Rgo), serine (8eg), threonine (Tyg), tryptophan (Thr), tyrosine (Tug) and valine (Va1). Unnatural amino acids include, but are not limited to, homolysin, homoarginine, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminobutyric acid, 6 -aminoisobutyric acid, 3aminoisobutyric acid, 2-aminopimelic acid, tertiary butyl glycine, 2,4 diamino butobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, Ν-ethyl glycine, Ν-ethylsp omoproline, hydroxylisine, allohydroxylysine, 3hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, мет-methylalanine, метил-methylglycine, Νmethylisoleucine, Ν-methylpentylglycine, norin-tinlinecline, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, nornitholinicin, norinthynlein plyl-n-tinline-n-plinline-n-lignin-plynline-n-plinolen-yl-pentylene glycolin Additional unnatural amino acids include modified amino acid residues that are chemically blocked, reversibly or irreversibly, or chemically modified at the Ν-terminal amino group or side chain groups,

- 17 014647 for example, Ν-methylated Ό and b amino acids or residues where the functional groups of the side chain are chemically modified to other functional groups. For example, modified amino acids include methionine sulfoxide; methionine sulfone; (beta methyl ester) aspartic acid; a modified amino acid of aspartic acid; Ν-ethyl glycine, a modified glycine amino acid; or alanine carboxamide, a modified amino acid of alanine. Additional residues that may be introduced are described in 8app. EE a1., 1. Meb. Oh. 41: 248191, 1998.

In this specification, 5 Ara means 5 aminopentanoyl, 12 Abo means 12-aminododecanoyl, PEG (8) means 3,6, -dioioctanoyl and PEG (13) means succinimoyl 1-amino-4,7,10-trioxa-13tridecanamine.

As described herein, natural complex peptide hormones are known in the art, as are their analogues and derivatives. For reference, the sequence of some natural complex peptide hormones are presented in table. one.

Table 1

Typical Complex Peptide Hormones

44 amylin rat Κ0ΝΤΑΤ0ΑΤΩΚίΑΝΕίνΚ33ΝΝί6ΡνίΡΡΤΝνΘ3ΝΤΥ 45 ή-amylin Κ0ΝΤΑΤ0ΑΤ0βίΑΝΕίνΗ83ΝΝΕΘΑΙ138ΤΝνΘ3ΝΤΥ 46 B-ALM ΥΡΟδΜΝΝΡΟΘίΚ3ΡΟΟΕ! Ε6ΤΐσναΚΙΑΗαΐΥαΕΤΟΚΟΚΟΝνΑΡΕ! 3Κ Ι8ΡΟΘΥ 47 5-CT СЗЬ-ЗТСУЬЬ КЬЗОЬН ΚΙ αΤΥΡΕΙΤΝΤΟ3εΤΡ 48 B-CT ΟΟΝΙ 8ΤΟΜ1-ΟΤΥΤΟΟΡΝΚΕΗΤΕΡΟΤΑΙΘνΘΑΡ 49 b-sec a Α00ΤΑΤ0νΤΗΚίΑΘΙί8Κ30ΘννΚΝΝΓνΡΤΝνΘ3ΚΑΡ fifty ίι-SSKR β AC ΝΤΑΤΟνΤΗΚ ΙΑΘίί3Κ3ΘΘΜνΚ3 Ν ΕνΡΤΝνθδΚΑΕ 51 ή-ΑΕΡ-6 (1-47) Τ0ΑαίίΚνΘ0νΐ6Τ0αν0Νί3ΗΕ! Ϊ́νναΐΜΘΡΑ0Κα03ΑΡν0Ρ33ΡΗ 8Υ 52 ή-ΑΕΡ-6 (8-47) νΘ0νΐ © Τ00ναΝΙ ^ ΗΡΐνν0Ι.Μ (3ΡΑ (3Κα03ΑΡν0Ρ35ΡΗ8Υ 53 APP-6 mouse (1-47) ΡΗΑαΐίΗνΘ0νίΘΤ0αν0Νί8ΗΒΐνναΐ.νΗΡΑΘΗΗ05ΑΡν0Ρ88ΡΗ 5Υ 54 AEP-6 mice (8-47) νΘ0νΐθΤ00ν0ΝΙ 8ΗΚΙ.νναΐ.νΚΡΑ6ΚΚ08ΑΡν0Ρ88ΡΙ-Ι8Υ 55 sulfated SSK-8 ΟΥ (8Ο ₃ ) ΜθννΜΟΕ 56 ή-leptin ΜΗΜΟΤΙΟΟΕΙ ^ No. ΥΙΕ ¥ νθΑνΡΙΟΚνθΟΟΤΚΤίΙΚΤΐνΤΕ: ΐΝΟΙ3ΗΤ 03У83К0 КУТЬОР1Р6ЬН ΡΙ ЬЬЗКМООТГАУУОANТЗМРЕЕМЫУУ I 5Ν01.ΕΝ! _Ε! 01_Ι_ΗνΐΑΕ3Κ30ΗΙΡνΛΑ3ι31_ΕΤ! _05Ι1Ж1РΥ1.011Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж1Ж 57 ή-ΡΥΥ ΥΡΙΚΡΕΑΡΟΕΟΑ8ΡΕΕίΝβΥΥΑ3ίΕ? ΗΥίΝίνΤΚΟΚΥ 58 ή-ΡΥΥ (3-36) ΙΚΡΕΑΡΘΕΟΑ3ΡΕΕίΝΙ4ΥΥΑ3ίίΐΗΥίΝίνΤΚΟΚΥ With ΚΓΪΕΗ 4/4 ι ι- ι ί, ι-> l ι υΐΊΕΓΕΟυΛΓηΤΓΤ ^ ηΐ / ΟΟΤΙ ΕΤίΤϊι'ΛΛΛΙ ΕΕΊΛλΛΗ 1 1k / SL 1-.Ι \ Ι Ι / * ΛΙ — 1 1 1 «L-t V 4ί4Ρ 1 1 1 and Ча </ ~ и-М 1 1 ΙΓΤ.Υ ν 60 ΘΡί-1 frogs Η ΑΕ6ΤΥΤΝ ϋνΤΕΥίΕΕ ΚΑΑΚΕΠΕΜ / ίΙΚΟ ΚΡΚΚΙΚΥ3-0Η; 188 ΘΡί-1 frogs NABETTZOUZHOSHAKAAKEENEngi ΝΘΟ Ρ3ΚΕΙ13-ΟΗ 61 ή-ΘΡί-1 (7-36) ΗΑΕ6ΤΕΤ80ν38ΥίΕΘ0ΑΑίΕΕΙΑννίνΚ0Ρ 62 p-SR-2 ΗΑθε3Ε3ΟΕΜΝΤΙίϋΝίΑΑΚΟΕΙΝννίΙΕΤΚΙΤ0 63 ΘΡΙ.-2 frogs ΗΑΕΟΤΕΤΝϋΜΤΝΥΙΕΕΚΑΑΚΕΕνΘννίΙΚΟΚΡ-ΟΗ 64 OHM Η30ΰΤΕΤ80Υ5ΚΥ103ΚΚΑ00Εν0ννίΜΝΤΚΚΝΚΝΝΙΑ 65 Exendin-3 Η8ϋ (3ΤΕΤ30Ι.8Κ0ΜΕΕΕΑνΚΙΕΙΕννΐ-ΚΝΘ6Ρ386ΑΡΡΡ8 66 Exendin-4 ΗΘΕ (3ΤΕΤ30Ι 8Κ0ΜΕΕΕΑνΚΙ.ΡΙΕννΐ-ΚΝ <36Ρ88ΘΑΡΡΡ8 291 / rocortin II (mice) νΐ1810νΡΙΘίΙΗΙΙίΕ0ΑΚΥΚΑΑΗΝ0ΑΑΤΝΑΩ! ΙΑΗν-ΝΗ2 292 No. 24 (Urocortin) U / ZRZARMOBOSAEANSH-AEKER-ON 293 GU-18 (Urokortin) Τ080ΗΕΗΑΕ0ΝΒΙΙΕ08ν-ΝΗ2 294 Human urocortin 0ΝΡ813ΙΟ1_ΤΡΗ! _Ι_ΡΤΙΧΕΙΑΙΡΤΟ3ΟΕ! ΕΙΡΑΕΟΝΕΙΙΡ08ν-ΝΗ2 295 ZE-20 (Urokortin111 / Streskopin) 3ΕΗ ΥΙ_β3 ΚϋΑ83ΘΕΕΕΕΟΚΕ-ΟΗ 296 ΑΙ-13 (UrokortinN / Streskopin) ΑΟΑΑΑΝΑΗΙΜΑΩΙ-ΟΗ 297 ϋΑ-21 (Urocortin) 0ΝΡ8ί3Ι0ίΤΕΗΙΙΚΤΙΙΕΙΑ-0Η 298 TB-26 (UrokortinI / Streskopin) ΤΚΕΤ1.3Ι.0νΡΤΝΙΜΝ [-1-ΕΝΙΑΚΑΚΝΙ.-ΟΗ

- 18 014647

299 Urocortin III person ΕΤίδΙϋνΡΤΝ I ΜΝ1_Ι_ΡΝ I ΑΚΑΚΝίΗΑΟ ΑΑΑΝ ΑΗίΜΑΟΙ-Ν Η2 300 ΕΝ-38 (8Ι.Μ 14) ΡΙΕΗΥ8ΚΤαΚίΘΚ8ΝννΕΕίΟ8ΡΓΑ8αδΚβΥΡίΕΕίΡΚΝ-ΝΗ2 301 alpha-atrial natriuretic polypeptide (1-28) of the person, pig, Zyka 31ΚΚ830ΕΘΘΚΜϋΚΙΘΑα80Ι.30Ν8ΕΚΥ-ΟΗ 302 Brain rat natriuretic peptide; ΒΝΡ, <trot □ (Ν5ΚΜΑΗδ880ΡθαΚΙθΚΙΟΑνδΚΙΟΟΟΘΙΙ ^ Ρ) -ΟΗ 303 Brain Sodium Yuretic Peptide (ΒΝΡ) [Human] δΡΚΜναοδΘΟΕΟκκΜοκίδδδδΘίαεκνικκΗ-ΟΗ 304 S-TURA pig natriuretic peptide; ΟΝΡ pigs 3Ι_8Κ60Ε61ΚΙ-ΟΕΙΘδΜδΘΙ.ΘΟ-ΟΗ 305 neuromedin ΙΙ-8 (pigs) ΥΕίΕΚΡΕΝ-ΝΗ2 306 neuromedin and (rats) ΥΚνΝΕΥ0ΘΡνΑΡδΘΘΕΡΙΓΡΡΚΝ-ΝΗ2 307 neuromedin i-9 ΘΥΕΙΕΚΡΕΝ-ΝΗ2 308 neuromedin (025), of man ΓΕν0ΕΕΕ03ΡΕΑ303Ε (3ΥΕΙΡΚΡΕΝ-ΝΗ2

These peptides are usually amidated at the C-terminal region upon physiological expression, but this is not necessary for the purposes of this invention. In other words, the C-terminal region of such peptides, as well as the hybrid polypeptides of the invention, may have a free —OH or —ΝΗ ₂ group. These peptides may also have other post-translational modifications. One skilled in the art will recognize that the hybrid polypeptides of the invention can also be constructed with a methionine residue at the цевой -terminal region.

Typical peptide modules for use in accordance with this invention also include peptide modules extended at the кон-terminal region (and their analogs and fragments), including apelin, which exists in 2 forms, apelin 36 and 13, both of which are active at the A1P receptor (ECCRIBZHIBRBRA 'OBBRIKEYIIIOIRIEZ! 1KS1RMRE-0N (ZER) ΙΌ N0: 316) and REKRKEZNKORMRE0N (ZEO ΙΌ N0: 317)); a prolactin-secreting peptide that exists in 2 forms, RKP31 and RKP20, are equally active against ORK10 (ZKTKNZMEIKTR ^ INRA \ ΥAZKOIKRVOKE-NH2 (ZEO ΙΌ N0: 318) and TP ^ INRA \ ΥAZKOIKRVOKE-NH2 (ZEO 31 31O9); gastrin, which exists in the form of large gastrin and mini-gastrin, the region of activity of which is in the residues of pentagastrin (^^ OR ^ ORRN ^ VA ^ RZKK ^ OR \ ^ EEEEEAΥO \ M ^ E-NH2 (ZEO ΙΌ N0: 320);

pEOP \ ^ EEEEEAΥO \ M ^ E-NH2 (ZEO ΙΌ N0: 321); beta-AMOE-MH2 (ZEO ΙΌ N0: 322)); CCK, which exists as CCK33 or CCK8 (central versus peripheral; CARZOKMZIVKN ^^ N ^^ RZNKIZ ^ K ^ ΥMO \ M ^ E-NН2 (ЗЕО ΙΌ N0: 323); ^ Υ МО \ М ^ Е-NН2) (ЗЕО ΙΌ N0: 55); cortistatin, which exists in the form of cortistatin 17 or 29 (OEOARROOZAKKPKMRSK \ EEAKTEZZSK-0H (ZEO ΙΌ N0: 324) and 1) K \ 1RSI \ EE \\ 'KTEZZSK-011 (ZEO ΙΌ N0: 325)); somatostatin, which exists in the form of somastostatin 14 or 28 (ZA \ Z \ RLMLRIEIKL6SK \ EEKTETZS-0N (ZEO ΙΌ N0: 326); AS, SK \ EE \\ 'KTETZS-011 (ZEO ΙΌ N0: 327)); OCD, in which the main activity occurs in 10 amino acids of the C-terminal region (VR ^ RAOOOTV ^ TKMΥPKONH \ AVON ^ M-NH2 (ZEO ΙΌ N0: 328); ONH \ AVON ^ M-NH2 (ZEO ΙΌ N0: 329)); neuromedin B, in which 10 amino acids of the C-terminal region account for the main activity (ЕЗ ^ EXERCISE OF UNZKOME ^ ATONEM-MS (ZEO ΙΌ N0: 330); ON ^ \ ATONEM-NN2 (ZEO ΙΌ N0: 331)); neuromedin Z, in which 9 amino acids of the Skontsev region account for the main activity (Ш0В.686ТАЛ '^ ETKKONTAT \ 6KRE'EE'KKK№ ^ Н2 (ЗЕО ΙΌ N0: 315);

REEEEEKRK№ (No. 2 (ZEO ΙΌ N0: 332)); neuromedin, in which 9 amino acids of the C-terminal region account for the main activity (ECUOEeEnErAzEaEeEeRKN (Ж2 (ЗЕО ΙΌ N0: 308); САЕЕЕКРКН-НН2 (ЗЕО ΙΌ N0: 307)); neurotensin, which exists in long and short forms ( КИРΥI ^ КК ^^ Υ ЕНКРККРΥI ^ -ΟН (ЗЕО ΙΌ N0: 333); 0ЕУЕ \ КРРИ ¥ 1Е-0Н) (ЗЕО ΙΌ N0: 334); К188-1, whose activity mainly falls on the C-terminal region (OTZ ^ RRZRZOZR ^^ PO ^ ZARNZK ^ IРАР ^ ОАV ^ V ^ КЕК ^^ РNΥN \ NЗЕО ^ КЕ-NН2 (ЗЕО ΙΌ N0: 335); Е1 <ОЕРНАН \ УНЗЕОЕКЕ-НН2 (ЗЕО ΙΌ N0: 336 amd); КЕ -3, fragments of the C-terminal region of which have activity (ZAOATA ^ REKZOKMMEA L ¹ CQI ¹ URMERrKE-NH2 (ZEO ΙΌ N0: 337); VRN ^ P ^ KE-NH2 (ZEO ΙΌ N0: 338)); dynorphin, which exists in a plurality dynorphin (A), dynorphin B (rimorfin) (¥ 66EERI1IR <Е <\ П ^ КОТОЕЕККРЕК ¹ ¥ ^п ¥ Т-0Н (ЗЕО ΙΌ N0: 339) and Υ00Е ^ КК ^ ЕКVVТ-0Н (ЗЕО ΙΌ N0: 340)); ΓΥΥ, fragments of the C-terminal region of which are active in

- 19 014647 with respect to the receptor Υ2 (¥ Р1 КРЕАРЕСПА8РЕЕк \ К ¥¥ А8кКН ¥ 1. \ 1. УТК ('Ж ¥ - \ Н2 (8EP) ГО N0: 57); 8 ^ ΚΗΥ ^ N ^ УΓΚ ^ ΚΥ-NΗ2 ( 8E0 GO N0: 341)); AER-6, 7-47 regions of which remain active (TARKUSSUSSTSrurM'8KN№rMSRASKrP8ARUPR88RN8 ¥ -MN2 (8 EP) GO N0: 51);

USSU ^ STS ^ Y ^ N ^ 8ΗΚ ^ V ^^ MCPACΚ ^^ 8ARU ^ P88PΗ8Υ-NΗ2 (8EP) GO N0: 52)); the amylin family, including adrenomodullin, calcitonin, and SSCR; oxytocin, the C-terminal amide of which usually needs activity and may allow lengthening of the Zhkontsevy region.

Typical peptide modules for use in accordance with this invention also include peptide modules extendable in the C-terminal region, including endothelium I, II and III: ET1 (C8C88 ^ M ^ KΕCUΥRSΗ ^^ IIVУNТРΕΗУУРΥС ^ С8РΚ8-ΟΗ (8 EP) GO No: 342);

С8С88ЬМПКЕСУ ¥ РНЬППП-0Н (8 ЕР) ГО N0: 343)), ЕТП (С8С88V ^^ КΕСУΥРСΗ ^^ II \\ А '\ ТЕРР) Т А Р ¥ С.И. (.. 1 ^ 4 ^ -011 (8EP) GO N0: 344); С8С88№ППЕСУ ¥ РНЬППП№-0Н (8ЕР) ГО N0: 345)) and ЕТШ (СТСРТΥК ^ КΕСУΥ ¥ СΗ ^^ IIVINТРΕ ^ ТURΥС ^ 8NΥΚС8РΚ-NΗ2 (8EP) ГО N0: 346); STSRTΥK ^ KΕSUΥΥSΗ ^^ IIV-ΟΗ (8Е0 GO N0: 347)); ghrelin, in which the main activity occurs in the first 10 residues (P.88EE8P1E Р \ \ К)) WxE8KKRRAKRR) P-P) N (8EO GO N0: 348); C88E8RENR-0H (8E0 GO N0: 349)); glucagons, including oxyntomodulin, which is an elongated glucagon in the C-terminal region with glucagon-like activity (Н80СТРТ8О ¥ 8КΥ ^^ 8ΚΚА ^^ РУ ^ V ^ МNТКΚNΚNNIA-0Η (8 EP) GO N0: 350); 1180 (.00 (81 ^ 8KG ¥ 6N8KKARPEUR ^ LMET-0H (8E0 GO N0: 351)); CPB-1 / CPB-2, the activity of which persists with or without a C-terminal amide; SRH, which circulates in 2 forms, СГ1-42 and СГР1-30, both of which are fully active at the receptor (.. IR (ΥАΕСТРI8 ^ Υ8IАМ ^ КIΗ ^^^ РУНВ ^^ А ^ КСККN ^ VКΗNIТ ^ -ΟΗ (8ЕО ГО N0: 352); ΥАΕСТРI8 ^ Υ8IАМ ^ КIΗ ^^^ RUNV ^^ А ^ К-NΗ2 (8ЕО GO N0: 353)); neuropeptide V, which exists as ХР \ ¥ 23 and НР ^ 30, is equally active to СРК7 and 8 (№ ¥ КНУА8РК ¥ НТУКАКАСЬЬЬЬЬСЬКЬКК8Р ¥Ь No.-0H (8EO GO N0: 354);

No. ¥ КНУА8РК ¥ НТУКАКАСЬЬ-0Н (8ЕР) ГО N0: 355)); RASAR, which exists in 2 forms, RASAR27 and 38 (1181UIRT1) 8 ¥ 8P ¥ WxO \ PA \ LCD ¥ RAAURSKI ¥ K \ K- \ H2 (8EO GO N0: 356); Η8 ^ СIRТ ^ 8Υ8ΚΥΚК ^ МАУККΥ ^ ААУ ^ -NΗ2 (8 ЕР) ГО N0: 357)); RSh and RNU (ΗА ^ СVΕТ8 ^ Ρ8Κ ^^ С ^^ 8АΚΚΥ ^ Ε8 ^ МСΚКV88NI8Ε ^ РВРV-ΟΗ (8 ЕР) ГО N0: 358);

NAPSURT8PR8KbSr8AKK ¥ ЬЕ8ЬМ-МН2 (8EP) GO N0: 359)); SKR, which exists in 2 forms, SKR29 and SKR40 (ΥА ^ АИРТN8ΥπКУ ^ С ^^ 8АΚК ^^^^ IM8Κ ^^ СΕ8N ^ ΕΚCAΚАΚ ^ -NΗ2 (8ЕО GO N0: 360); ΥА ^ АИРТN8ΥΚКУ ^ С ^^ 8АΚ ^^^ IM8-ΟΗ (8ЕО GO N0: 361)); PTH 1-34 and 1-37 forms, which have the activity of full PTH 1-84 (8 \ U1Π (I \ 1ΗХΕ (..> К1 IΕХ8 \ 1ΕИУΕ \\ 'ΕIKKΕΟ ^ UI ^ \ RUАΕ (.. _I АРΕАРIЮА (. . _I 8ΟIRIKKΕ ^ \ UΕUΕ8 NEK8E (.. 1EA1ZHA1) \\\ P.TKAK8O (8EO GO N0: 362); 8U8ΕI MΗN ^ ^^ ^ SKΗ N8MΕKUΕV CCC ^ ^^^ UΗNRUA ^ -ΟΗ (EP 8) GO N0: 363);

8U8ΕI ^^ МΗN ^ SKΗ ^ N8МΕΚУΕV ^ ΚКК ^^^ УΗNР-ΟΗ (8EP) GO N0: 364)) РТН-КР, in which 1-36 has the activity of complete 1-86 (АУ8ΕΗ ^^^ Η ^ КСК8I ^^ ^ ΚΚΚРР ^ ΗΗ ^ IАΕIΗТАΕIΚАТ8ΕУ8РN8КР8РNТКNΗУРУКРС8 ^^ ΕСКΥ ^ Т ^ ΕТNКУΕТΥКΕ ^ Р ^ КТРСККККСКР-NΗ2 (8EP) ГО N0: 365); АУ8ΕΗ ^^^ Η ^ KSK8I ^^^ ΚΚΚРР ^ ΗΗ ^ IАΕIΗТАΕI-ΟΗ (8EP) GO N0: 366)) gamma-M8H, which has shorter gamma-M8H1 and longer gamma-M8H3 have the same activity (ΥУМСΗРКV ^ КРСКСС8888 ^ -0Η (8EO GO N0: 367); ΥΑΑ1 (.., Ι ПЖ \\ '1) IR- \ H2 (8EO GO N0: 368)); M8H, in which alpha-M8H is an active part of ACTH (8Υ8MΕΗRΚVKRUSKKΚΚRUKΥΥRNCAΕ ^ Ε8АΕАРР ^ ΕР-0Η (8ЕО ГО N0: 369); 8 ¥ 8MENRK№СКРУМН2 (8Е0 ГО N0: 370)); and endorphins, in which A, delta and γ endorphin are active peptide fragments of the larger β endorphin (ΥSSRMT8ΕK8 ^ TP ^ YT ^ PKNAIIKNAΥKKSΕ-0Η (8Е0 GO N0: 371); ΥSSMT8ΕK8 ^ TP ^ YT ^ PKNAIIKNΥ 0N 80NER0 : 372); ¥ SSRMT8EK8rTRUT-0H (8EO GO N0: 373); ¥ ’(.. 1 (.I'AIT8EK8OTRE \’ T-011 (8EO GO N0: 374)).

For example, melanocortins are peptides from the pro-opiomelanocortin gene, including alpha-melanocyte-stimulating hormone (alpha-M8H) and adrenocorticotrophic hormone (ACTH), and five melanocortin receptors, MC1-5K, are known. MS4K apparently plays a role in energy metabolism and obesity, see, for example, Apptegtop e! A1., Ehrei 0rsh. Thy. Ra! Ep! § 11: 15831592 (2001), 8rea e! A1., Exhr 0rt. Thy. Ra! Ep! § 12: 1631-1638 (2002), Bebpagek e! A1., Exhr 0rt. Thy. Ra! Ep! § 14: 327-336 (2004).

Analogues of the aforementioned complex peptide hormones are known in the art, but typically include modifications such as substitutions, deletions and additions of such complex peptide hormones to the amino acid sequence, and any combination thereof. Substitutions, inclusions and deletions may be in the Zhkontsevy or C-terminal regions, or may be in the inner part of the complex peptide hormone. In a preferred aspect, analogs of the complex peptide hormones of the invention comprise one or more modifications of a non-vital amino acid residue. In this context of the invention, a non-vital amino acid residue is a residue that can be modified, i.e. removed or substituted, in the natural human amino acid sequence, for example, a fragment of a complex peptide hormone, without canceling or significantly reducing the agonistic effect on the receptor of a complex peptide hormone in the obtained analogue.

- 20 014647

Preferred substitutions include stored amino acid substitutions. A stored amino acid substitution is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain or physicochemical characteristics (e.g., electrostatic, hydrogen bonding, isostereic, hydrophobic characteristics). Families of amino acid residues having similar side chains are known in the art. Such families include amino acids with major side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine , methionine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine , phenylalanine, tryptophan, hist idin).

This invention also relates to derivatives of complex peptide hormones. Such derivatives include complex peptide hormones and their analogues conjugated to one or more water-soluble polymer molecules, such as polyethylene glycol (PEG) or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, octanoyl, etc.), or to which are added polyamino acids such as poly-Yk, poly-agd, poly-1uk and poly-A1a. Modifications of complex peptide hormones or their analogues also include low molecular weight substituents, such as short alkyls and restricted alkyls (e.g., branched, cyclic, condensed, adamantyl) and aromatic groups. The water-soluble polymer molecules preferably have a molecular weight of from about 500 to about 20,000 Daltons.

Such modifications by conjugation with the polymer and low molecular weight substituents can be carried out singularly at the Ν or C-terminal region or on the side chains of amino acid residues in the sequence of hybrid polypeptides. Alternatively, there may be a plurality of derivative sites in the hybrid polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine can provide additional sites for derivatization, see, for example, US Pat. Nos. 5,824,784 and 5,824,778. Preferably, hybrid polypeptides can be coupled to one, two, or three polymer molecules.

The molecules of the water-soluble polymer are preferably linked to an amino, carboxyl or thiol group and can be linked through the Ν or C-terminal region or to the side chains of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, water-soluble polymer molecules may be coupled to diamine and dicarboxylic groups. In a preferred embodiment, the hybrid polypeptides of the invention are coupled to one, two, or three PEG molecules via the epsilon amino group of the lysine amino acid.

Derivatives in accordance with this invention also include complex peptide hormones or analogues with chemical changes in one or more amino acid residues. Such chemical changes include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization. Chemical changes can be carried out singularly at the Ν or C-terminal regions or in the side chains of amino acid residues in the PPE sequence of hybrid polypeptides. In one embodiment, the C-terminal region of such peptides may have free —OH or —IN ₂ groups. In another embodiment, the кон-terminal region may be covered by an isobutyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group (isocap), an octanyl group, an octylglycine group (O (0c!)), Or an 8-amino-octanoic acid group. In a preferred embodiment, the cyclization can be carried out through the formation of disulfide bridges. Alternatively, a plurality of chemical change sites may exist in a hybrid polypeptide.

Amylin Family

As described, the complex peptide hormones used in accordance with this invention include peptide hormones of the amylin family, including amylin, adrenomodullin (ΑΌΜ), calcitonin (CT), a peptide encoded by the calcitonin gene (COBP), interludes (also known as ΑEP-6 ) and related peptides. Natural peptide hormones of the amylin family are known in the art, as are their functional peptide analogues and derivatives. Certain preferred natural peptides, peptide analogs and derivatives are described herein, however, it should be understood that any known peptides of the amylin family that have hormonal effects known in the art can be used in accordance with this invention.

Any analog or derivative of amylin known in the art can be used in accordance with this invention. In one embodiment, the amylin analogs and derivatives exhibit at least one hormonal activity of the natural amylin. In certain embodiments, amylin analogs are receptor agonists with which natural amylin is able to specifically bind. Preferred amylin analogs and derivatives include those described in I8 2003/0026812 A1, which is incorporated herein by reference.

- 21 014647

Examples of amylin analogues include

Zeo Yu: 67 ^{23 '28.2E} Prgo ~ L-amylin (praylintide) 68 6es- ¹ bv5-b-amylin 69 ²⁵ Pro, ²⁶ Ua1, ^{2a '28} Pro-L-amylin 70 ^1a Agd, ^{2b, 28} Rgo-b-amylin ' 71 des- ¹ b3, ^1a Agd, ^{2b, 2a} Prgo-b — amylin 72 Arg ^{1a, 2b, 28 '2a} R _F O-b-amylin 73 bez- ¹ bz, ^1a agd, ²³ ' ²⁸ ' ^2a rgo-b-amylin 74 des- ¹ , bf ^ '^' ^ rgo-b-amylin 75 ²³ Rgo, ^2a Va1, ² “' ² ' Rgo-b-amylin 76 ²⁸ Prgo-L-amylin, 2, 7-СC1- [ ³ Azr, 'L3] -L-amylin 77 ² “ ³ 'b-amylin 78 ² A1a-b-amylin 79 ² A1a-b-amylin 80 ² ' ⁷ A1a-b-amylin 81 ¹ Zeg-b-amylin 82 ^2a Rgo-b-amylin 83 ^{23, ha} Rgo-N-amylin 84 des ¹ bj, ^{2 :! '28} Pro-L-amylin 85 “Lei, ^2b Rgo, ²⁶ Ba1, ²⁸ ' ^2E Rgo-b-amylin 86 ^2b Leu ²³ Pro ²⁸ Pro Ua1 ^2b-6-amylin 87 des Luz, ²³ Lei, ²⁵ Rgo, ^2e ”7a1, ^2a Rgo“ I-amylin 88 ^1a Agd, ²³ Lei, ^2b Prgo, ^{2 (,} \ 7a1, ^2B Prgo-b-amylin 89 ^1a Agd, ²³ Lei, ²⁵ ' ^2a ' ²⁸ Prgo-b-amylin 90 ¹⁸ Agd ²³ Ley, ^{2b, 2a} Rgo-H-amylin 91 ¹⁷ 11e, ²³ Lei, ^{2b, 2} ″ ^2a Rgc-11-amylin 92 ¹⁷ 11e, ^{28,28 '29} Pro-L-amylin 93 des Luz, ^r 'Phe, Leu ^{23, 25' 28 '29} Pro-I-ayilin 94 ¹ 'Not, ^1a Agd, ² Lei-b ~ amylin 95 ¹ 'Not, ^1a Agd, ²³ Lei, ²⁸ ¥ a1, ^2a Rgo-H-amylin 96 "No, ^1a Agde, ²³ Leu, Pro ^2b, Ua1 ^{26, 28 '29} Pro-L-amylin, 97 “Thr, ²¹ Sz, ² Lei, ^2a A1a, ^2a Lei, ^2a Rgo, ³¹ Azr-b-amylin 98 ^1a Tb, ²¹ H1z, ² Lei, ^ab A1a, ^2a Rgo, ³¹ Azr-b-amylin 99 without- ¹ b3, ¹³ bb, ²¹ h1z, ^and Lei, ²⁶ d1a, ^2a rg, ^a1 azr-b-amylin one hundred ¹³ Tb, ^1a Agd, ^az H1z, ^az Ley, ^al A1a, Prgo, ³¹ Azr-b-amylin 101 ¹³ Tb, ^1a Agd, ²¹ H1z, ^az b, ^{av, ae} rgo, ^a1 azr-b-amylin 102 ¹³ Tg, ^1a Arg ²¹ M1H, ² Lei, ^au Pro, A1a ^{2b, 2c, 29} Pro, Asp-anilin ^a1 |

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

Any analog or derivative ΆΌΜ known in the art may be used in accordance with this invention. In another embodiment, analogues and derivatives of ΆΌΜ exhibit at least one hormonal activity of natural ΆΌΜ. In certain embodiments, аналог analogues are receptor agonists with which natural ΆΌΜ is capable of binding specifically.

Any analogue or derivative of CT known in the art can be used in accordance with this invention. In one embodiment, the analogues and derivatives of CT show at least one hormonal activity of natural CT. In certain embodiments, CT analogues are receptor agonists with which natural CT is capable of binding specifically. Preferred analogs and derivatives of CT include those described in US patent No. 4652627; 4,606,856; 4,604,238; 4,597,900; 4,537,716; 4,497,731; 4,495,097; 4,444,981; 4,414,149; 4,401,593 and 4,397,780, which are incorporated herein by reference.

Typical CT analogues include

ZE <2 1B: 103 ⁱⁿ e1u-st 104 ² Lei-ST ' 105 ^a 51u, ^a 3d, ^a 61u, ^a2 des-Tug-ST 106 ¹⁴ C1i-zST, 107 ^1c Agde-FTA, 108 ^11.18 Agd-zST, 109 -% 1 and ^{1 =} _I .Agd- CT BUT ¹³ 61i, ^g1 ' ^1a Agd-zST

As is known in the art, such CT analogues are preferably amidated, but in the context of this invention may optionally be in the form of an acid, unless otherwise indicated.

Any analogue or derivative of SCCR known in the art can be used in accordance with this invention. In one embodiment, analogues and derivatives of SSCR exhibit at least one hormonal activity of natural SSCR. In certain embodiments, analogs of SCCR are receptor agonists with which natural SCCR is capable of binding specifically. Preferred analogues and derivatives of SCCR include those described in US patents

- 22 014647 No. 4697002 and 4687839. which are incorporated into this description by reference. Typical SSNR analogues include

ZEO Ιϋ: 111 ³⁶ P-Zeg-SSVR 112 ³⁶ 0-TNG-SSKR 113 0 ^{bond-Asp-S6KR} 114 ³⁶ Ts-Azp-S6KR 115 ^Z1, 3G-SSER 116 ³⁶ Nze-SSKR 117 ³⁶ Azr sec 119 D'ig-SSKR 119 ^3!> A5P-S6VR

Any analog or derivative of APP-6. known in the art. can be used in accordance with this invention. In one embodiment, analogs and derivatives of APP-6 exhibit at least one hormonal activity of natural APP-6. In certain embodiments, APP-6 analogues are receptor agonists. with which natural APP-6 is capable of binding specifically. Preferred APP-6 analogs and derivatives include those. which are described in XV0 2003/022304. which is incorporated herein by reference.

Typical APP-6 analogues include

Zeo Yu: 120 TOAOYKUSSEIZTSHOUONZKNOMSRASKOOZARUORZZRNZU 121 TOAOYUVUSSOTATSUOUZNNNEMERAECOZARUORZZRNZU 122 TOAOKUBMUSTMOUOMZNKNOEMERASVOOZARUORZZRNZU 123 TOAOYKUESUSTSUOOTSZNKOMOMSRASKOOZARUERZZRNZU 124 TOOYKUESUETSUOZZNNOMSRAEKOZARUERZRZRZRZU 125 TOOYKUESUSTSUOMUZNZYOJ ---- KOIZARUORZZRZZU 126 TOAOYKUSSUSSTSUOUZZNYIO ---- OZARUORZZRNZU 127 KUESUSTSUOMZNOIOMSRAEVOOZARUORZZRNZU 128 USSUSTUOUZZNOIOMSRASKOOZARUERZZRNZU 129 USUETSUONUZNZNYIO ---- Corzaruerszrzu 130 SECURITY COMMISSION 131 SUMMARY OF UNDERSTANDING ---- WOOZARUORZZRNZU 132 ectatooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo 133 SECURITY SECURITY ---- wobzaruerszrzu 134 SSSITSTSOUOUZZNVIOJO ---- KORZARUERZZRNZU 135 b) 136 SUSSTUOUZZNOIOMSRASKOOZARUORZZRNZU 137 OUOZZNYOMYRABKOOZARUORZZRNZU 138 UIOZNKYIOMSRASVOOZARUORZZRNZU 139 UOIZNK ---- ^^ ΜСΡΑСΚ ^^ ЗΑΡV ^ Ρ83ΡΗЗΥ 140 STMOUOUZZNYIO ---- KOZARUERZRNZU

As is known in the art. such APP-6 analogs are preferably amidated. but in the context of this invention may optionally be in the form of an acid. unless otherwise indicated.

SSK family

SSK. including ySSK and species variations. and various analogs are known in the art. Generally. CCKs have a sequence of 33 amino acids. first identified in humans. and include 1n νί \ Ό C-terminal fragment of 8 amino acids (CCK-8). which the. according to available data. was found in a pig. rats. Chicken. chinchillas. dogs and humans. Other species variations include a sequence of 39 amino acids. found in a pig. dogs and guinea pigs. and a sequence of 58 amino acids. found in a cat. dogs and humans. and sequences of 47 amino acids. homologous SSK and gastrin. The C-terminal tyrosine-sulfated octapeptide sequence (CCK-8) is relatively preserved in species and may be a minimal sequence. providing a biological effect in the peripheral system of rodents. Thus. the term CCK-33 usually refers to human CCK (1-33). and CCK-8 (CCK (26-33); 8EO ΙΌ N0: 55) refers to the C-terminal octapeptide. generally. in sulfated and non-sulfated forms. unless otherwise indicated. Further. pentagastrin or CCK-5 refers to the C-terminal peptide CCK (29-33) (8E0 ΙΌ N0: 209). and CCK-4 refers to the C-terminal tetrapeptide CCK (30-33) (8E0 ΙΌ N0: 208).

It has been described. that the receptor subtype of type A (CCCA) is selective for the sulfated octapeptide. A Type B receptor subtype (HSCW) has been identified in the brain and in the stomach. and. according to available data. does not require sulfation of all eight amino acids.

Various 1p νί \ Ό and 1p ν 11th screening methods for CCK analogues are known in the art. Examples include 1n νί \ Ό analyzes. comprising contracting the dog’s gallbladder or guinea pig after rapid intravenous administration of the compound. which is tested for CCK-like activity. and shuigo tests using scrapings of the rabbit gall bladder. see Va1vy. Сав (г1п (ев11п1 Ногтопев. 1п Рюувю1од о £ 1е Сав! Гот! Еюп1 Тгас! (3Б еБ. 1994; Яауеп Ргевв. № \ ν Уогк).

Certain typical SSCs and SSC analogues. possessing CCK action include

- 23 014647

5E <2 ΙΏ: 141 0Υ (OZN) ISIMOG 142 143 mimog 144 ΟΗΜϋΓ 145 YUYUG 146 ΚϋΥ (OZN) MY 147 K0UM6IM0G 148 kmsimog 149 ΚΕΗΜϋΕ 150 KIM№

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

Leptin family

The complex peptide hormones used in accordance with this invention also include peptide hormones of the leptin family. Natural peptide hormones of the leptin family are known in the art, as are their functional peptide analogues and derivatives. Certain preferred natural peptides, analogs and derivatives of the peptides are described herein, but it should be understood that any known peptide of the amylin family that has a hormonal effect known in the art can be used in accordance with this invention.

Any analog or derivative of leptin known in the art can be used in accordance with this invention. In one embodiment, leptin analogs and derivatives exhibit at least one hormonal activity of natural leptin. In certain embodiments, leptin analogues are receptor agonists with which natural leptin is capable of binding specifically. Preferred analogs and derivatives of leptin include those described, for example, in UO 2004/039832, UO 98/55139, UO 98/12224 and UO 97/02004, all of which are incorporated herein by reference. In one embodiment, leptin peptides include

Μ7ΡΙΟΚ (Zeo Yu

N0: 400), νΟϋβΤΚ (ЗОО ΙΏ N0: 400), ТЫК (ЗЕО Ю N0: 402), КνΤΚ (ЗЕО Ю N0: 403), ΙΝϋΙ5ΗΤ05753Κ (ЗЕО Ю N0: 404), 7Т0Ь0Г1Р6ЬНР1ЬЗЗК (ЗЕО Ν7ΙΟΙ3Ν01ΕΝ1Κ (ЗЕО Ю N0: 406), ОЫНУЬАГЗК (ЗЕО 10 N0: 407),

EXCLUSIVE EXCEPT7EA36UZTE77ABC (ZEO U N0: 408) and LOCZOOMOZNOZROS (ZEO U N0: 409), as described in HO97046585,

In one embodiment, the leptin peptide may have the amino acid sequence Haap-BegSu8-Haa1-Lei-Proc-Haa2-Haa3-Haap (BEP ΙΌ NO: 410), where Haap may be zero residues in length, or may be an adjacent fragment of the peptide residues obtained from complete human or mouse leptin sequences, a fragment between 1 and 7 in either the C-terminal or the либо-terminal regions, or where the leptin peptide comprises only 15 amino acids or less. In another embodiment, Xaa1, Xaa2 or Xaa3 may be any amino acid substituent. In yet another embodiment, Xaa1, Xaa2 or Xaa3 may be any conservative amino acid substituent of the corresponding residues in full mouse or human leptin. In another embodiment, Xaa1 may be selected from the group consisting of NL or Running, and Xaa2 or Xaa3 is any amino acid substituent. In another embodiment, Xaa2 may be selected from the group consisting of Tgr or C1l, and Xaa1 or Xaa3 is any amino acid substituent. In yet another embodiment, Xaa3 may be selected from the group consisting of D1a or Tyg, and Xaa1 or Xaa2 is any amino acid substituent. In another embodiment, Xaa1 may be selected from the group consisting of Hs or Beg, Xaa2 may be selected from the group consisting of Tgr or C1n, and Xaa3 may be selected from the group consisting of T1a or Tyg, see \ XO 04039832.

In one embodiment, the leptin peptide contains the C-terminal amino acid residues 116-122 of the natural full human or mouse leptin (corresponding to the provisions of 95-101 of their mature form) and its Ό-isoforms, fragments, derivatives, analogs and homologs that are capable of modulating mass homeostasis bodies in tested animals at. b. (intraperitoneal) administration. Certain murine замещ-substituted peptides with the sequence BSBRPT (BePr ΙΌ NO: 411) include [O-Beg-1] -, [O-Suz-2] -, [O-Beg-3] -, [P-Lei-4 ] -, [О-Рго-5] -, [О-61п-6] -, [О-Тиг-7] -БСБЬРрТ (БЕР ΙΌ NO: 411) and all [Ό] БСБРРрТ (БЕР ΙΌ NO: 411) . Certain human Ό-substituted BSNRRAD peptides (BEPr ΙΌ 412) include [Ό-BegL] -, [O-Suz-2] -, [O-H18-3] -, [O-Lei-4] -, [O- Рgo-5] -, [О-Тгр-6] -, [^ -Α1а-7] -БСН ^ Р№Α (БЕР ΙΌ 412) and all | О | -ССНЕРАТ (БЕР ΙΌ 412). In addition, BSNRCP (BEPr ΙΌ NO: 412) and BSBPrT (BEPr ΙΌ NO: 411) peptides may contain Ό-substituted amino acids at any two, three, four, five or six positions. Leptin-related peptides containing Ν-terminal amino acids 21-35, 31-45, 41-55 and 51-65 of natural leptin are also described.

- 24 014647 on and its fragments, derivatives, analogues and homologs. Additional leptin peptides in accordance with this invention include amino acid sequences 61-75, 71-85, 81-95, 91105, 106-120, 116-130, 126-140, 136-150, 146-160 and 156-167 of mouse and / or human total leptin, see \ UO 04039832.

In one embodiment, leptin has the sequence

Zeg Souz

Bb baaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

Vai bie si ab aa zeg siu tug zeg tg si

Еи а а ег у у З З З еи еи а а а Аз Аз Аз Аз а а а

Csu Suz (Zeo ΙΟ N0: 413), where Xaa in position 6 is Tgr or C1n; Xaa at position 36 is C1p or C1i; Xaa at position 40 is C1p or C1i; Xaa at position 42 is 11e, Le, Mc1 or methionine sulfoxide; Haa at position 44 is Tgr or C1p; and Haa at position 45 is C1p or C1i. In another embodiment, leptin is provided, such as described above, wherein Xaa at position 6 is Tgr; Haa at position 36 is C1p; Haa at position 40 is C1p; Xaa at position 42 is Me1; Haa at position 44 is Tgr; and Haa at position 45 is C1p, see US Pat. No. 5,521,283.

In one embodiment, leptins are natural sequences, including murine leptin:

Ya rgo Bb Bn Bz Yb Shp Azr Azr Tb Bz Tht Bie Not Bz Tht 1b Yb Tht Agd Bb Azp Azr Bb Zeg Hb Tht Haa Zeg Yb Zeg Zeg Bz Bn Bz Yb Tht Sue Bie Azr Pb Not Rgo Sue Bie Hb Rgo Bb Bie Tht Bie Zeg Bz Me Azr Bn Tht Bie Aba Yb Tug Bn Bn Not Bie Tht Zeg Me Rgo Zeg Agd Azp Yb Bb Bn Not Zeg Azp Azr Bie SI Azp Bie Agd Azr Bie Bie Hb Yb Bie Aba Pb Zeg Bz Zeg Suz Hb Bie Rgo Bn Aba Zeg Sue Bie SI Tht Bie SI Zeg Bie Sue Sue Yb Bie SI Aba Zeg Sue Tug Zeg Tht SI Yb Yb Aba Bie Zeg Agd Bie Bn C1u Zeg Bie Bn Azr Mei Bie Bn Bn Bie Azr Bie Zeg Rgo C1u Suz (3Εβ Yu N0: 414),

where Haa at position 28 is C1p or absent; pork leptin:

Yb . Rgo Lj Tgr Agd Ya Sb Azr Azr Tht Bz Tht Bie Not Bz Tht Bb Yb Tht Agd 1b Zeg Azr 1b Zeg Hb Me Bn Zeg Yb Zeg Zeg Bz Bn Agd Yb Tht Sue Bie Azr Pb Bb Rgo Sue Bie Hb Rgo Yb Bie Zeg Bie Zeg Bz Me Azr Bn Tht Bie Aba 1b Tug Bn Bn 1b Bie Tht Zeg Bie Rgo Zeg Agd Azp Yb 11th Bn Bb Zeg Azp Azr Bie SI Azp Bie Agd Azr Bie Bie Hb Bie Bie Aba Zeg Zeg Bz Zeg Suz Rgo Bie Rgo Bn A1a Agd Aba Bie SI Tht Bie SI Zeg Bie Sue Sue Yb Bie SI Aba Zeg Bie Tug Zeg Tht SI Yb Ya1 Aba Bie Zeg Agd Bie Bn Sue Aba Bie Bn Azr MeE Bie Agd Bn Bie Azr Bie Zeg Rgo Sue Suz (3ΕΏ 10 N0: 4 fifteen) ;

bovine leptin:

Va1 Rgo Not Suz Uy

Tht Bie 1b Bz Tht Bb Yb Tht Agd Bb Azp Azr Not Zeg Hb Tht Haa Zeg Yb Zeg Zeg Bz Bn Agd Yb Tht Sue Bie Azr Pb 1b Rgo Sue Bie Hb Rgo Bie Bie Zeg Bie Zeg Bz Me Azr Bn Tht Bie Aba Not Tug Bn Bn 11th Bie Tht Zeg Bie Rgo Zeg Agd Azp Ya1 Yb Bn Bb Zeg Azp Azr Bie SI Azp Bie Agd Azr Bie Bie H13 Bie Bie Aba Aba Zeg Bz Zeg Suz Rgo Bie Rgo Bn Yb Agd Aba Bie C1 and Zeg Bie SI Zeg Bie Sue Yb Yb Bie C1 and Aba Zeg Bie Tug Zeg Tht SI Yb Yb Aba Bie Zeg Agd Bie Bn Sue Zeg Bie Bn Azr MeE Bie Agd Bn Bie Azr Bie Zeg Rgo Sue Suz (WEU YU N0: 416)

where Haa at position 28 is C1p or absent;

- 25 014647 human leptin:

Yb Procrastin Bn Azr Azr Tht Bz Tht Bie S Bz Tht Bb Yb Tht Agd Bb Azp Azr Bb Zeg Hb Haa Haa Zeg Yb Zeg Zeg Bz Bn Bz Yb Tht Sue Bie Azr Pb Not Rgo Sue Bie Hb Rgo S Bie Tht Bie Zeg Bz Meb Azr Bn Tht Bie Aba Yb Tug Bn Bn 1b Bie Tht Zeg Meb Rgo Zeg Agd Azp Yb S Bn S Zeg Azp Azr Bie SI Azp Bie Agd Azr Bie Bie Hb Yb Bie Aba Pb Zeg Bz Zeg Suz Hb Bie Rgo Tgr A1a Zeg Sue Bie si Tht Bie Azr Zeg Bie Sue Sue Yb Bie SI Aba Zeg Sue Tug Zeg Tht C1 and Yb Yb Aba Bie Zeg Agd Bie Bn C1u Zeg Bie Bn Azr Meb Bie Tgr Bn Bie Azr Bie Zeg Rgo Sue Suz (Zeo Yu N0: 417),

where Haa at position 27 is Tyg or A1a; and Haa at position 28 is O1n or absent;

rhesus macaque leptin:

Bn Bz Yb Bn Zeg Azr Tht Bz Tht Bie Bb Bz Tht S Yb Tht Agd S Azp Azr Bb Zeg Hb Tht Bn Zeg Yb Zeg Zeg Bz Bn Agd Yb Tht Sue Bie Azr Pb S Rgo Sue Bie Hb Rgo Yb Bie Tht Bie Zeg Bn Meb Azr Bn Tht Bie Aa. Bb Tug Bn Bn S Bie Bb Azp Bie Rgo Zeg Agd Azp Yb Bb Bn Bb Zeg Azp Azr Bie SI Azp Bie Agd Azr Bie Bie Hb Bie Bie Aba Pb Zeg Bz Zeg Suz Hb Bie Rgo Bie Aba Zeg Sue Bie SI Tht Bie SI Zeg Bie Sue Azr Yb Bie SI Aba Zeg Bie Tug Zeg Tht SI Yb Yb Aba Bie Zeg Agd Bie Bn Sue Zeg Bie Bn Azr Meb Ley Tgr Ll Ley Azr Ley: 5eg Rgo Sue Suz (Zeo Yu N0: 418);

and rat leptin:

Yb Rgo Bb Hb Bz Yb Bn Azr Azr Tht Bz Tht Bie Bb Bz Tht Bb Yb Tht Agd Bb Azp Azr S Zeg Hb Tht Bn Zeg Yb Zeg Aba Agd Bn Agd Yb Tht Sue Bie Azr Pb S Rgo Sue Bie Hb Rgo Bb Bie Zeg Bie Zeg Bz Meb Azr Bn Tht Bie Aba Yb Tug Bn Bn Bb Bie Tht Zeg Bie Rgo Zeg Bn Azp Yb Bie Bn Bb Aba Hb Azr Bie SI Azp Bie Agd Azr Bie Bie Hb Bie Bie Aba Pb Zeg Bz Zeg Suz Zeg Bie Rgo Bn Tht Agd Sue Bie Bn Bz Rgo SI Zeg Bie Azr Sue Yb Bie SI Aba Zeg Bie Tug Zeg Tht SI Yb Yb Aba Bie Zeg Agd Bie Bn Sue Zeg Bie Bn Azr S Bie Bn Bn Bie Azr Bie Zeg Rgo SI Suz (EEO 10 N0: 419).

In another embodiment, leptin peptides have the sequence

Ba b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b Tht Bz Tht Bie S Bz Tht S Yb Tht Agd S Haa Azr S Zeg Hb Haa Haa Zeg Yb Zeg Zeg Bz Bn Bz Yb Tht Sue Bie Azr Pb S Rgo C1U Bie Hb Rgo S Bie Tht Bie Zeg Bz Haa Azr Bn Tht Bie Aba Yb Tug Bn Bn S Bie Tht Zeg Haa Rgo Zeg Agd Haa Yb S Bn S Haa Azp Azr Bie SI Azp Bie Agd Azr Bie Bie Hb Yb Bie Aba Pb Zeg Bz Zeg Suz Hb Bie Rgo Tgr Aba Zeg Sue Bie SI Tht Bie Azr Zeg Bie Sue Sue Yb Bie SI Aba Zeg Haa Tug Zeg Tht SI Yb Yb Aba Bie Zeg Agd Bie Bn Sue Zeg Bie Bn Azr Meb Bie Tgr Bn Bie Azr Bie Zeg Rgo Sue Suz (Zeo YU N0: 420),

where Haa at position 22 is Akp Akr or O1i; Haa at position 27 is Tyg or A1a; Xaa at position 28 is O1n, O1i or absent; Xaa at position 54 is Me1 or A1a; Haa at position 68 is Me1 or Lea; Haa in pos. 72 is Akp, Acre or O1i; Xaa at position 77 is δе ^ or A1a; Haa at position 118 is O1y or Ley; said protein has at least one substituent selected from the group consisting of: NK at position 97 is substituted by δе ^ or Рgo; Tgr in

- 26 014647 position 100 is replaced by C1n, A1a or Ley; A1a at position 101 is substituted with THg or Va1; 8eg at position 102 is replaced by Agd; C1y at position 103 is substituted by A1a; C1i at position 105 is substituted by C1p; TNG at position 106 is substituted by b3 or 8eg; Ley at position 107 is replaced by Rgo; Azr at position 108 is substituted by C1i; or C1y at position 111 is substituted with Azr.

In another embodiment, leptin has the sequence

Ya rgo

Not Bn Bz Yb Bn Azr Azr TNG Bz TNG Bie Bb Bz TNG Not Yb TNG Agd Not Azp Azr Bb Zeg Hb Haa Bn Zeg Uat Zeg Zeg Bz Bn Bz Yb TNG Sue Bie Azr Rke Not Rgo Sue Bie Hb Rgo Bb Bie TNG Bie Zeg Bz Mek Azr Bn TNG Bie ATa Yb Tug Bn Bn Bb Bie TNG 5eg Mek Rgo Zeg Agd Azp Yb 1b Bn Bb Haa Azp Azr Bie SI Azp Bie Agd Azr Bie Bie Hb Uat Bie Aba RNe Zeg Bz Zeg Suz Hb Bie Rgo Tgr Aba Zeg Sue Bie SI TNG Bie Azr Zeg Bie Sue Sue Yb Bie SI Aba Zeg Haa Tug Zeg TNG SI Yb Uat ATa Bie Zeg Agd Bie Bn Sue Zeg Bie Bn Azr Mek Bie Tgr Bn Bie Azr Bie Zeg Rgo Sue Suz (5Εζ> Yu ΝΟ: 421) g where:

Haa at position 27 is TNG or A1a; Xaa at position 77 is 8eg or A1a; Haa at position 118 is C1u or Lei; said protein has at least one substituent, preferably has from one to five substituents and, most preferably, from one to two substituents selected from the group consisting of: H1z at position 97 is replaced by 8eg; Tgr at position 100 is substituted by C1p; A1a at position 101 is substituted with TNg; C1i at position 105 is substituted by C1p; TNG at position 106 is replaced by b3; Ley at position 107 is replaced by Rgo; Azr at position 108 is substituted by C1i; or C1y at position 111 is substituted with Azr.

In other embodiments of the above sequence, Xaa at position 27 is TNG; Haa at position 77 is 8eg; Haa at position 118 is C1u; and amino acid residues at positions 97, 100, 101, 105, 106, 107, 108 and 111, such as in the table below.

97 one hundred 101 105 T06 107 108 111 natural human Hb Tgr Aba C1 and TNG Bie Azr Sue Zeg Tgr Aba SI TNG Bie Azr Sue Hb Bn ABA SI TNG Bie Azr Sue Hb Tgr TNG SI you Bie Azr Sue Hb Tgr Aba Bn TNG Bie Azr Sue Hb Tgr Aba SI Bz Bie Azr Sue Hb Tgr ATa SI TNG Rgo Azr Sue Hb Tgr ATa SI TNG Bie SI Sue Hb Tgr Aba SI TNG Bie Azr Azr Zeg Bn Aba SI TNG Bie Azr Sue Zeg Tgr TNG SI TNG Bie Azr Sue Zeg Tgr Aba Bn TNG Bie Azr Sue 5eg Tgr Aba SI Bz Bie Azr Sue Zeg Tgr ATa SI TNG Rgo Azr Sue Zeg Tgr Aba CU. TNG Bie SI Sue Zeg Tgr Aba SI TNG Bie Azr Azr Hb Bn TNG SI TNG Bie Azr Sue Hb Bn Aba Bn TNG Bie Azr Sue Hb Bn Aba SI Bz Bie Azr Sue Hb Bn Aba SI TNG Rgo Azr Sue Hb Bn ATa SI TNG Bie SI Sue H13 Bn Aba SI TNG Bie Azr | Azr H15 Tgr TNG Bn TNG Bie Azr Sue Hb Tgr TNG SI Bz Bie Azr Sue Hb Tgr TNG SI TNG Rgo Azr Sue Hb Tgr TNG SI TNG Bie SI Sue Hb Tgr TNG SI TNG Bie Azr Azr Hb Tgr ATa Bn Bz Bie Azr Sue Hb Tgr ATa Bn TNG Rgo Azr Sue Hb Tgr ATa Bn TNG Bie SI Sue

- 27 014647

ηϊ3 Tgr A1a C1p Tht Bie Azr Azr Ηί3 Tgr A1a C1 and Bz Rgo Azr C1u Η13 Tgr A1a C1 and Bz Bie C1 and C1u ΗΪ3 Tgr A1a E1i Bz Bie Azr Azr Η13 Tgr A1a E1i Tht Rgo C1 and E1u Ηί3 Tgr A1a C1 and Bj Rgo Azr Azr ΗΪ3 Tgr A1a C1 and Tyg Bie E1i Azr 5eg C1p Tht C1 and Tht Bie Azr eu 5eg C1p A1a E1p Tyg Bie Azr C1u Zeg E1p A1a E1i Bz Bie Azr C1u Zeg b1p A1a E1i Tht Rgo Azr E1u Zeg C1p A1a E1i TNG Bie C1 and C1u Zeg C1p A1a C1 and Tht Bie Azr Azr Zeg Tgr Tht C1p TNG Bie Azr C1u Zeg Tgr Tht E1i Bz Bie Azr C1u Zeg Tgr Bj E1i Tht Rgo Azr C1u Zeg Tgr Tht E1i Tht Bie C1 and 01y Zeg Tgr Bj E1i TIG Bie Azr Azr Zeg Tgr A1a E1p Bz Bie Azr E1u Zeg Tgr A1a E1p Tkg Rgo Azr 61y Zeg Tgr A1a C1p Tht Bie E1i C1u Zeg Tgr A1a E1p Tht Bie Azr Azr Zeg Tgr A1a C1 and Bz Rgo Azr E1u Zeg Tgr A1a E1i Bz Bie E1i C1u Zeg Tgr A1a E1i Bz Bie Azr Azr Zeg Tgr A1a E1i Tht Rgo E1i E1u Zeg Tgr A1a E1i Tht Rgo Azr Azr Zeg Tgr A1a E1i Tht Bie C1 and Azr ΗΪ5 C1p Tht SHP Tht Bie Azr 01y ΗΪ3 E1p Tht C1 and Bz Bie Azr C1u ΗΪ8 C1p Tht C1 and TPg Rgo Azr C1u H13 C1p TIG C1 and Tht Bie E1i C1u ΗΪ5 C1p TIG 01 and Bj Bie Azr Azr H1z E1p A1a E1p Bz Bie Azr E1u H13 C1p A1a E1p Tht Rgo Azr C1u H13 E1p A1a C1p TIG Bie E1i eu H1z C1p A1a E1p Tht Bie Azr Azr ΗΪ3 B1p A1a E1i Bz Rgo Azr C1u ΗΪ3 Θΐη A1a C1 and Bz Bie 61and E1u H13 C1p A1a C1 and Bz Bie Azr Azr H1z C1p A1a E1i TIG Rgo C1 and C1u H13 εΐη A1a E1i TIG Rgo Azr Azr H18 εΐη A1a 51and TIG Bie C1 and Azr ΗΪ3 Tgr Tht E1p Bz Bie Azr C1u Η13 Tgr Tht C1p Tht Rgo Azr C1u ΗΪ3 Tgr Tht C1p Tht Bie E1i C1u ΗΪ3 Tgr TIG E1p Tht Bie Azr Azr H13 Tgr TIG E1i Bz Rgo Azr C1u ΗΪ3 Tgr Tht E1i Bz Bie E1i E1u H1z Tgr Tkg Bz Bie Azr Azr H1z Tgr Tht E1i Tht Rgo E1i E1u

- 28 014647

ΗΪ5 Tgr Tht C1 and Tht Rgo Azr Azr H1z Tgr Tht C1 and Tht Bie C1 and Azr ΗΪΞ Tgr A1a BTp Bz Rgo Azr STU Ηί3 Tgr A1a C1p Bz Bie C1 and STU ΗίΞ Tgr A1a 51p Bz Bie Azr Azr Ηί3 Tgr ATa ετη Tht Rgo C1 and STU ΗΪ3 Tgr A1a STP Tht Rgo Azr Azr Ηίθ Tgr A1a C1p Tht Bie C1 and Azr Ηί3 Tgr A1a C1 and Bz Rgo C1 and STU Η1Ξ Tgr ATa C1 and Bz Rgo Azr Azr Ηί3 Tgr ATa ST Bz Bie C1 and Azr Nhz Tgr ATa ST Tht Rgo C1 and Azr 5eg C1p Tht STP Tht Bie Azr STU Zeg 51P Tht ST Bz Bie Azr STU Zeg C1p Tht ST Tht Rgo Azr STU Zeg C1p Tht ST Tht Bie ST STU 5eg C1p Tht ST Tht Bie Azr Azr Zeg C1p ATa STP Bz Bie Azr STU Zeg C1p ATa STP Tht Rgo Azr STU Zeg C1p ATa STP Tht Bie C1 and STU Zeg C1p ATa STP Bx Bie Azr Azr Zeg C1p ATa ST Bz Rgo Azr STU Zeg 61p ATa ST Bz Bie C1 and STU Zeg C1p ATa ST Bz Bie Azr Azr Zeg E1p ATa ST Tht Rgo C1 and STU Zeg C1p ATa ST Tht Rgo Azr Azr Zeg C1p A1a ST YOU Bie ST Azr Zeg Tgr Tht Bz Bie Azr STU Zeg Tgr Tht STP Tht Rgo Azr STU Zeg Tgr Tht STP Tht Bie C1 and STU Zeg Tgr Tht STP Tht Bie Azr Azr Zeg Tgr Tht ST Bz Rgo Azr STU Zeg Tgr Tht ST Bz Bie C1 and STU Zeg Tgr Tht ST Bz Bie Azr Azr Zeg Tgr Tht ST Tht Rgo C1 and STU Zeg Tgr Tht ST Tht Rgo Azr Azr Zeg Tgr Tht ST Tht Bie C1 and Azr Zeg Tgr ATa STP Bz Rgo Azr C1u Zeg Tgr ATa STP Bz Bie C1 and C1u Zeg Tgr ATa STP Bz Bie Azr Azr Zeg Tgr ATa STP Tht Rgo C1 and STU Zeg Tgr ATa STP Tht Rgo Azr Azr Zeg Tgr ATa STP Tht Bie C1 and Azr Zeg Tgr ATa ST Bz Rgo C1 and STU Zeg Tgr ATa ST Bz Rgo Azr Azr Zeg Tgr ATa ST Bz Bie C1 and Azr Zeg Tgr A1a C1 and Tht Rgo C1 and Azr N1E C1p Tht STP Bz Bie Azr STU H13 C1p Tht STP Tht Rgo Azr STU H1z C1p Tht STP Tht Bie C1 and STU H1z C1p Tht STP Tht Bie Azr Azr H ± 5 C1p Tht ST Bz Rgo Azr STU H1z Θΐη Tht ST Bz Bie C1 and STU H1z C1p Tht ST Bz Bie Azr Azr H13 C1p Tht ST Tht Rgo C1 and STU H1z C1p Tht ST Tht Rgo Azr Azr H1z C1p Tht ST Tht Bie C1 and Azr H13 C1p ATa STP Bz Rgo Azr STU H1z C1p ATa STP Bz Bie ST STU H1z C1p ATa STP Bz Bie Azr Azr ΗΪ3 C1p ATa STP Tht Rgo C1 and STU ΗΪ5 C1p ATa STP Tht Rgo Azr Azr H1z C1p ATa STP Tht Bie ST Azr

- 29 014647

In one embodiment, leptin has the sequence

Not Rgo C1u Bie H1z Rgo Not Bie Tht Bie Zeg Bz Haa Azr Haa Tkg Bie A1a Ya1 Tug Haa Haa Not Bie Bj Zeg Haa Rgo Zeg Agd Haa Ya1 Not Haa Not Zeg Haa Azr Bie C1 and Haa Bie Agd Azr Bie Bie H1z Ya1 Bie A1a RNe Zeg Bz Zeg Suz ΗΪ3 Bie Rgo Haa A1a Zeg C1u Bie C1 and Tkg Bie Azr Zeg Bie C1u C1u Ya1 Bie 61and A1a Zeg E1u Tug Zeg Tkg C1 and Ya1 Ya1 A1a Bie Zeg Agd Bie Haa C1u Zeg Bie Haa Avr Haa Bie Haa Haa Bie Azr Lie Zeg Rgo 61y Suz (ZE <3 U N0: 422),

where Haa in position 13 is Not, Lea, Me! or methionine sulfoxide; Xaa at position 15 is C1i or C1i; Xaa at position 21 is C1p or C1i; Xaa at position 22 is C1p or C1i; Haa in position 27 is 11th, Lei, Me! or methionine sulfoxide; Haa at position 31 is Aki, Acre or C1P; Xaa at position 34 is C1p or C1i; Haa at position 37 is Aki, Acre or C1i; Haa at position 41 is Aki, Acre or C1P; Haa at position 59 is Tgr or C1p; Xaa at position 89 is C1i or C1i; Xaa at position 93 is C1p or C1i; Haa in position 95 is 11th, Lei, Me! or methionine sulfoxide; Haa at position 97 is Tgr or C1p; and Haa at position 98 is C1p or C1i. In another embodiment, the leptin of the above Haa formula at position 13 is Me !; Xaa at position 15 is C1i; Haa at position 21 is C1p; Xaa at position 22 is C1i; Haa at position 27 is Me !; Haa at position 31 is Aki; Xaa at position 34 is C1i; Haa at position 37 is Aki; Haa at position 41 is Aki; Haa at position 59 is Tgr; Xaa at position 89 is O1i; Xaa at position 93 is C1i; Haa at position 95 is Me !; Haa at position 97 is Tgr; and Xaa at position 98 is O1, see US Pat. No. 5,532,336.

Typical leptin analogs include those in which the amino acid at position 43 is substituted by Acre or C1i; at position 48, substituted A1a; at position 49 is substituted C1 or absent; at position 75, A1a is substituted; at position 89 is replaced by lei; at position 93 is replaced by Acre or C1i; at position 98, substituted A1a; at position 117, 8eg is replaced, at position 139, Le is replaced, at position 167, 8eg is substituted, and any combinations thereof.

Certain exemplary leptins and leptin analogs having leptin action include

Zeo Yu: 151 ^ Azr-Leptin 152 ^ SNi-leptin 153 ^4U A1a-leptin 154 C1-leptin 155 ^4U 0es-AA-leptin 156 ^/:> A1a-leptin 157 ^ya lei leptin 158 ^AB Asp-leptin 159 ^av C1i-leptin 160 A1a ^{centuries-leptin} 161 ¹¹ 'Zeg Leptin' 162 ^1vv lei-leptin 163 ^{1b /} 5eg-leptin 164 ' ⁱⁿ Azr, ⁴ B1i-leptin 165 ^{, 3} Azr, ^{/! 1} A1a-leptin 166 ^ah lei, ^ 'zeg-leptin 167 ^al 61i, ¹⁶ '3g-leptin

PPP Family

The complex peptide hormones used in accordance with this invention also include the peptide hormones PPP, including PP and ΡΥΥ. Natural peptide hormones PPP are known in the art, as are their functional peptide analogues and derivatives. Certain natural peptides, analogs and derivatives of peptides are described herein, however, it should be understood that any known peptides of the amylin family that have hormonal effects can be used in accordance with this invention.

Any analogue or derivative of PPP known in the art may be used in accordance with this invention. In one embodiment, the analogs and derivatives of PPP exhibit at least one hormonal activity of the natural PPP polypeptide. In certain embodiments, the PPP analogs are receptor agonists to which the naturally occurring PPP method polypeptide specifically binds. Preferred analogues and derivatives of PPP include those described in XVO 03/026591 and νθ 03/057235, which are fully incorporated into this description by reference.

In one embodiment, preferred PPP analogs and derivatives that exhibit at least one hormonal PPP activity typically include at least two два motifs, including

- 30 014647 motif of polyproline and motive of the C-terminal tail. Such analogs are generally described in provisional application for US patent No. 60/543406, filed February 11, 2004, published as I8 2006 / 0135747A1 June 22, 2006, which is incorporated into this description by reference. Other preferred PPE analogues are described in PCT / I82005 / 004351, Motives of the Pancreatic Polypeptide Family and Containing Polypeptides thereof, published as AO 2005/077094 on August 25, 2005, the contents of which are incorporated herein by reference. Other preferred PPE analogs are described in PCT / I82005 / 045471, Motives of the Pancreatic Polypeptide Family, and Polypeptides and Methods Containing Them, published as AO 2006/066024 June 22, 2006, the contents of which are incorporated herein by reference. According to the prior art, studies have suggested that differences in the binding affinity of the receptor Υ are interconnected with structural differences of the second and third order, see, for example, Kaye e! A1., Vusyetschyu 2000, 39, 9935-9942. Natural pork ΡΥΥ was characterized as including two C-terminal helical segments from residues 17-22 and 25-33, separated by a fold at residues 23, 24 and 25, with a revolution around residues 12-14, and a Ν-terminal region bent around residues 30 and 31. Another complete pork ΡΥΥ was characterized as including a PP fold stabilized by hydrophobic interactions between residues in the Ν- and C-terminal regions, see ίά.

The '^ ΥΥ motif is usually a structural component, primary, secondary or tertiary, of a natural polypeptide of the PP family, which is critical for biological action, i.e. the biological effect is significantly reduced in the absence or damage of the motive. Preferred ΡΥΥ motifs include a Ν-terminal polyproline type II motif of a natural PP family polypeptide, a type II β-reverse motif of a natural PP family polypeptide, an α-helical motif at the C-terminal region of a natural PP family polypeptide, and a C-terminal tail motif of a natural polypeptide of the family PP More specifically, in the Ν-terminal region of the polyproline, the amino acids corresponding to residues 5 and 8 of the natural polypeptide of the PP family are usually stored as proline. Type II β-reverse motif typically includes amino acids corresponding to residues 12-14 of a natural polypeptide of the PP family. The α-helical motif can usually come from amino acids corresponding to approximately residue 14 of the natural polypeptide of the PP family at any point before and in the C-terminal region, and the α-helical motif includes a sufficient number of amino acid residues, such that allow the formation of an α-helical turn in In a solution, the α-helical motif may also include amino acid substituents, insertions and deletions in the sequence of natural PP, provided that they allow the formation of an α-helical coil in solution. The C-terminal tail motif typically includes amino acids corresponding to approximately the last 10 residues of the natural polypeptide of the PP family, more preferably the last 7, 6 or 5 residues of the natural polypeptide of the PP family, and even more preferably, amino acid residues 32-35.

Preferred ΡΥΥ analogues include analogues with internal deletions, insertions and substitutions in regions of the ΡΥΥ molecule that do not correspond to the polyproline motif and / or C-terminal tail motif. For example, internal deletions are provided for in positions 4, 6, 7, 9 or 10.

In another embodiment of interest, the complex hormone is a PPE polypeptide containing at least two PPE motifs, including at least the Ν-terminal motif of the polyproline PPE and the C-terminal tail PPE motif. In this description, the term motive refers to an amino acid sequence that is characteristic of a particular biochemical action, or defines an independently condensed domain. Additional PPE motifs may correspond to the motif of any polypeptide of the PP family, including PP, ΡΥΥ and No. Υ, for example, the motif of the β-reverse region of type II ΡΥΥ or the α-helical motif in the C-terminal region ΡΥΥ.

In yet another embodiment, a complex module of the PPE family is a chimeric PPE polypeptide containing a fragment of a PP, ΡΥΥ or No. полип polypeptide covalently linked to at least one additional fragment of a second PP, ΡΥΥ or No. полип polypeptide, where each fragment of a PP, ΡΥΥ or No. includes PPE motive. Alternatively, the PPE chimera may comprise a fragment of a PP family polypeptide linked to one, two, three, or four segments of a polypeptide, where at least one of the linked segments of the polypeptide is a fragment of a second polypeptide of the PP family. In certain embodiments, the PPE polypeptides do not include the Ν-terminal fragment of PP with the C-terminal fragment No. Υ. The complex module of the chimeric PPE polypeptide has at least 50% identical sequence with natural ΡUU (3-36) along the entire length of ΡΥΥ (3-36). In some embodiments, such a chimeric PPE polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, or at least 97% identical sequence with natural ΡUU (3-36) along the entire length of ΡUU (3-36). Such chimeric PPE polypeptides may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, or at least at least 97% identical sequence with natural PP. In yet another embodiment, such a chimeric PPE polypeptide may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94 % or at least

- 31 014647

97% identical sequence with natural ΝΡΥ. In some embodiments, the PPP chimeric polypeptides may include at least the Ν-terminal PPP polyproline motif and the PPP C-terminal tail motif. Such PPP chimeras, as well as other analogues of PP and PP, are described in I8 2006 / 0135747A1, published June 22, 2006. In one embodiment, any of the peptides of the PPP family, if it is not a hybrid component, can be represented as a second agent, i.e. . as a second anti-obesity agent, where the hybrid is as described above.

Again, the chimeric PPP polypeptide typically retains, at least in part, the biological effect of natural human PP, PΥΥ or ΝΈΥ. In some embodiments, the PPP chimeric polypeptide has a biological effect in the treatment and prevention of conditions and metabolic disorders.

The polypeptide fragments of the PPP chimeras can be covalently linked together by any method known in the art, including, but not limited to, direct amide bonds or chemical linker groups. Chemical linker groups may include peptide mimetics that induce or stabilize the conformation of the polypeptide. PPP chimeric polypeptides include the chimeras PP-PP, PP-NP, PP-PP, PP-ΝΤΥ, Τ-1Τ or ΝΤΥΦΤ.

The PPP chimera may have at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids. In some embodiments, analogs of the P полип polypeptides include only natural L amino acid residues and / or modified natural L amino acid residues. In some embodiments, analogues of the PΥΥ polypeptides do not include non-natural amino acid residues.

In some embodiments, the PPP chimeric polypeptides include

LRP (1-7) -ρΝΡΥ, LRP (1-17) -ρΝΡΥ, LRP (19-23) -ρΝΡΥ, LRP (19-23) Рgo ³⁴ рЫУ, LРР (19-23) -Ηί3 ³⁴ ρΝΡΥ, ГРР ( 19-23) -ρΝΡΥ, ГРР (19-23) Рро ³⁴ рЫРУ, ГРР (19-23) -Ηί5 ³⁴ ρΝΡΥ, LРР (1-17) -Ηί5 ³⁴ ρΝΡΥ, ρΝΡΥ (1-7) LРР, ρΝΡΥ (1 -7, 19-23) -PPP, cPP (1-7) -ρΝΡΥ (19-23) -PPP, cPP (1-7) ΝΡΥ (19-23) -H1z ³⁴ LPP, LRP (1-17) - H ± s ³⁴ pYRU, LRP (19-23) -ρΝΡΥ,

LRP (19-23) -RGO ³⁴ rRRU, ρΝΡΥ (1-7) -LRR, ρΝΡΥ (19-23) -LRR, ρΝΡΥ (1923) -O1n ³⁴ LRR, ρΝΡΥ (19-23) -Ηίδ ³⁴ Η₽Ρ , ρΝΡΥ (19-23) -ΡΗβ ⁶ Ο1η ^{3, |} ΗΡΡ, ρΝΡΥ (19-23) -Pе ⁶ Н1з ³⁴ LРР, ρΝΡΥ (1-7,19-23) -РРР, ρΝΡΥ (1-7,19-23} С1п ^{3! |} LРР, срР (20-23) -ΡΓθ ³⁴ -ρΝΡΥ, сРР (21-23) -Ρτο ³⁴ -ρΝΡΥ, сРР (2223) -ΡΓθ ³ '-ρΝΡΥ, сРР (1-7) -Рго ³⁴ -РРУ, срР (20-23) -Рго ³⁴ -RSRU, SRR (1-7,20-23) -ΡΓθ ³ '-ρΝΡΥ, SRR (1-7) -ρΝΡΥ (19-23) -RRR, SRR (1-7) ρΝΡΥ (19-23) -Ndz ³⁴ LRP, cPP (1-7) -PPP (19-23) -PPP, cPP (1-7) -ρΝΡΥ (1923) -A1a ³¹ A1b ³² S1n ³⁴ -PP, cPP (1-7) -ρΝΡΥ (19 -23) -A1a ³¹ A1b ³² H1z ³⁴ -PPPPP (1-7) -A1a ³¹ A1b ³² -pcru, PPP (1-17) -A1a ³¹ A1b ³² -pPYU, ρΝΡΥ (1-7) A1a ³¹ A1b ³² C1n ³⁴ -PP or ρΝΡΥ (1-7, 19-23) -A1a ³¹ A1b ³² C1p ^{3, |} -PPP.

In some embodiments, the PPP chimeric polypeptides may comprise fragments of analogues of the PP family of polypeptides. For example, PPP chimeric polypeptides may contain the PPP polypeptide analogs described herein, as well as PP polypeptide analogues and аналоги polypeptide analogs.

Analogues of the PΥΥ polypeptide that can be used in the hybrids of this invention or as a second agent are those that are active in the assays described herein (including assays for food intake, gastric emptying, pancreatic secretion, body composition, or weight loss), which is equal to or exceeds the activity of NPΥ, PΥΥ, or PΥΥ (3–36) in the same assays. In some embodiments, analogs of PP полип polypeptides can be used in the treatment of metabolic diseases, such as, for example, obesity, insulin resistance syndrome (Syndrome X), or diabetes mellitus. In some embodiments, analogues of PΥΥ polypeptides may have improved manufacturability, stability, and / or ease of formulation compared to PP, ΝΓΝ, PΥΥ, or PΥΥ (3-36).

In some embodiments, the PPP chimeric polypeptides retain at least about 25, or about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 95, about 98, or about 99% of the biological effects of natural human PΥΥ in in relation to reducing the absorption of nutrients, reducing food intake, effects on weight gain and / or treatment and prevention of conditions and metabolic disorders. In another embodiment, the PPP chimeric polypeptides exhibit an improved agonistic effect on PP. In some embodiments, the PPP chimeric polypeptides have at least about 110, about 125, about 130, about 140, about 150, about 200% or more of the biological effects of natural human PΥΥ in reducing nutrient absorption, reducing food intake, and weight gain body and / or treatment and prevention of conditions and metabolic disorders.

More specifically, in one aspect, the PPP chimeric polypeptides comprise a PP fragment linked to

- 32 014647 fragment of RUU. In one embodiment, the PPE chimeric polypeptides comprise an I-terminal fragment of PP or an analogue of a PP polypeptide linked in the C-terminal region to a C-terminal fragment of an RUU or analogue of an RUU polypeptide. In another embodiment, the PPE chimeric polypeptides comprise a terminal fragment of RUU, RUU (3-36) or an analogue of the RUU polypeptide linked in the C-terminal region to the C-terminal fragment of PP or an analogue of the PP polypeptide.

In some embodiments, the PPE chimeric polypeptides comprise an RUU fragment linked to No. U fragment. In one embodiment, the PPE chimeric polypeptides contain a terminal fragment of RUU, RUU (3-36) or an analogue of the RUU polypeptide linked in the C-terminal region to the C-terminal fragment of NPΥ or an analogue of No. U polypeptide. In another embodiment, the PPE chimeric polypeptides contain a ^ terminal fragment # Y or an analogue of a polypeptide # U linked in the C-terminal region to the C-terminal fragment of the RUU or analog of the RUU polypeptide.

In some embodiments, the PPE chimeric polypeptides comprise a PP fragment linked to a No. Y fragment. In one embodiment, the PPE chimeric polypeptides comprise a ^ terminal fragment of PP or an analog of a PP polypeptide linked in the C-terminal region to a C-terminal fragment of # Y or an analog of an NPΥ polypeptide. In another embodiment, the PPE chimeric polypeptides comprise a terminal fragment # Y or an analogue of a No. U polypeptide linked in the C-terminal region to the C-terminal fragment of PP or an analog of the PP polypeptide.

In some embodiments, a fragment of PP, an analog of a polypeptide of PP, RUU, RUU (3-36), an analog of a polypeptide of RUU, No. Y, or an analog of an NPP polypeptide is a fragment containing anywhere from 4 to 20 amino acid residues of PP, an analog of a polypeptide of PP, RUU , RUU (3-36), an analogue of the RUU polypeptide, No. U, or an analog of the No.U polypeptide. In some embodiments, the length of the fragment is chosen so as to obtain the final chimeric PPE polypeptide containing at least 34 amino acids.

Chimeric PPE polypeptides may also contain other modifications, including, but not limited to, substitutions, deletions and insertions into the amino acid sequence of such chimeric PPE polypeptides and any combinations thereof. In some embodiments, the PPE chimeric polypeptides comprise one or more modifications of non-essential amino acid residues. A non-vital amino acid residue is a residue that can be changed, i.e. deleted or substituted, in the natural human amino acid sequence without canceling or significantly reducing the target effect.

Derivatives of chimeric PPE polypeptides are also used. Such derivatives include chimeric PPE polypeptides conjugated to one or more molecules of a water-soluble polymer, such as polyethylene glycol (PEG) or chains of fatty acids of various lengths (e.g., stearyl, palmitoyl, octanoyl, oleoyl, etc.), or with the addition of polyamino acids, such as poly-b1k, poly-agd, poly-1uk and poly-a1a. Modifications of the PPE chimeric polypeptides may also include low molecular weight substituents, such as short alkyls and limited alkyls (e.g., branched, cyclic, fused, adamantyl) and aromatic groups. In some embodiments, the water soluble polymer molecules have a molecular weight of from about 500 to about 20,000 Daltons.

Such modifications by conjugation with a polymer or low molecular weight substituents can be performed singularly on the N or C-terminal region or on the side chains of amino acid residues that are not involved in the formation of the hybrid, in the PPE sequence of chimeric polypeptides. Alternatively, in a PPE chimeric polypeptide, there may be multiple derivative sites. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine may provide additional sites for derivatization, see, for example, US Pat. Nos. 5,824,784 and 5,824,778. In some embodiments of the PPE, chimeric polypeptides may be coupled to one, two, or three molecules. polymer.

In some embodiments, the molecules of the water-soluble polymer are preferably linked to an amino, carboxyl, or thiol group and may be linked through the N or C-terminal region, or to the side chains of lysine, aspartic acid, glutamic acid, or cysteine. Alternatively, water-soluble polymer molecules may be coupled to diamine and dicarboxylic groups. In some embodiments of the PPE, the chimeric polypeptides of the invention are coupled to one, two, or three PEG molecules via the epsilon amino group of the lysine amino acid.

PPE chimeric polypeptides also include complex PPE chimeric polypeptides with chemical changes in one or more amino acid residues. Such chemical changes include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization. Chemical changes can be carried out singularly in the N or C-terminal regions or in the side chains of amino acid residues in the PPE sequence of chimeric polypeptides. In one embodiment, the C-terminal region of such peptides may have free —OH or —MH ₂ groups. In another embodiment, the N-terminal region may be covered by an isobutyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group (isocap), an octanyl group, an octylglycine group (C (0c!)) Or an amino octanoic acid group. In a preferred embodiment, the cyclization can be carried out through

- 33 014647 formation of disulfide bridges. Alternatively, in the analogue of the ΡUP polypeptide, there can be many places of chemical change.

In some embodiments, the PPE chimeric polypeptides include those that have the amino acid sequence 8E0 GO ΝΟ: 238-347 from I82006 / 0135747A1.

Typical chimeric PPE polypeptides include polypeptides of formula (VI):

Haah Haag Haaz Xs1s * 4 Rgo 31i Haa ₇ Rgo Haad

Azr Haagg Haahz Haaz C1i Haahe Haap Haahd Haa _1e Tug

Haagh Haa ₂ 2 Haa ₂ s Lye Haa ₂₅ Haage Tug Haage Azp Haa ₃₀

Haa ₃₁ Thr Agd 61p Haa ₃₅ Haa ₃₆ (5E0 Yu N0: 423) where Haa1 is Tug or absent;

Haa ₂ is He, Rgo or absent;

Haa ₃ is He, BH (Bo1! Op-Nip! Eg) -modified Luke, Luke, Va1 or Prgo;

Xaa ₄ is bk, the BH-modified bk, A1a, 8eg or Arg;

Haa ₇ is A1a, C1u or H1k;

Haa9 is C1u or A1a;

Haa1 ₂ is A1a or Prgo;

Haa1 ₃ is 8eg or Rgo;

Haatz is Rgo, A1a or 8eg;

Haa1 ₆ is C1i or Acre;

Haa1 ₇ is Ley or He;

Haa18 is Akp or A1a;

Xaa19 is Arg, bk, BH-modified bk, C1n or N (Si) A1a;

Haa ₂ 1 is Ty, A1a, Pb, Lb or BH-modified Lj;

Haa ₂₂ is A1a or 8eg;

Haa ₂₃ is 8eg, A1a or Acre;

Haa ₂₅ is Arg, bk or BH-modified bk;

Haa ₂₆ is H1k, A1a or Agd;

Haa ₂₈ is Ley or Not;

Haa ₃₀ is Lei or Me !;

Haa ₃ 1 is Ba1, He or Ley;

Haa ₃₅ is bk, BH-modified bk or agd; and

Haa ₃₆ is Tug, Thr or Pb.

In one embodiment, the PPE polypeptide of formula VI may be a natural polypeptide

PPE, ΡΥΥ (2-36), Уа13ЬРУ ¥ (3-36),

Luz25YRUU (3-36), Luz2511e28YRUU (3-36), Luz25Ι1ε31! ΙΡΥΥ (3-36), Luz25yeu31URUU (3-36), Luz25REu36NRUU (3-36), 361ε28ήΡΥΥ (3-36), 11-36YU , BRAZIROU (3-36), BRAZBIROU (3-36),

Leyzkhrёz6ΝΡΥΥ (3-36) or PrgoKhZAhAh4H1RUU.

As will be appreciated by one of skill in the art, the polypeptides of formula VI may be in the form of a free acid or may be amidated at the C-terminal region.

In some embodiments, the PPE polypeptide may contain a Ν-terminal fragment, consisting mainly of the first 17 amino acid residues of natural human

ΡΥΥ (Tug Rgo

Not Lus Rgo 31. and AXa Rgo SXu SHi Azr AHa Zeg Rgo 51i 61i Lye Azp

Agd Tug Tug AXa Zeg Ley Agd ΗΪ3 Tug Ley Azp Ley 7aX Tg Agd Cxn Agd Tug) (ZE <2 10 N0: 58), associated with the C-terminal fragment, consisting mainly of amino acid residues 18-36 of the natural human

ΝΡΎ) Tug Rgo Zeg Luz Rgo Azr Azp Rgo

ЕХУ СХи Azr A1a Рго АХа СХи Azr Meb A1a Agd Tug Tug Zeg A1a Ле

Agd (From Tug 11e Azp Ley Not Tg Agd Cxn Agd Tug) (5ES U N0: 457), where one or more of the amino acid residues in the Ν-terminal region of ΡΥΥ can be deleted or absent and where in each of ΡΥΥ and fragments be one, two, three, four, five, six, seven, eight, nine or ten amino acid substituents. In some embodiments, the Ν-terminal fragment, consisting mainly of the first 17 amino acids of the PPE polypeptide, can have 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 first 17 amino acids of natural ΡUU. In some embodiments, C-terminal

- 34 014647 a fragment of the PPE polypeptide, consisting mainly of amino acid residues 18-36, can have 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 with amino acids 18-36 of natural NPΥ. In some embodiments, amino acids at the кон-terminal fragment of the RUU (for example, proline at positions 5 and 8, glutamates at positions 6, 10 and 15, or aspartate at position 11) and / or amino acids at the C-terminal fragment of ΝΈΥ (for example, tyrosines 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, PPE polypeptides include those that have an amino acid sequence of BEP ΙΌ NO: 266, 267, 274, 282, 320, and 436-480 from IB2006 / 0135747D1. In some embodiments, the PPE polypeptides also include a Ν-terminal cap. Examples of such PPE polypeptides include BEP ΙΌ NO: 282, 320, 437, 441, 444, 445-447, 452, 454459, 461-464, 466, 468-470, and 472-480 from IB2006 / 0135747D1.

Other PPE polypeptides include polypeptides of the formula (CN)

Haa1 Haa ₂ Rgo Haa ₄ Rgo Haa ₆ N1z Rgo Haae Haayu

Haat Haahg Haai Haa ₁₄ Haa _{] 5} Haa1 ₆ Haat A1a Haa _4E Tug

Haa21 Haagg Haa Haa Leu _{23 2} e Haa Haa ₂ b _{2 July} Haag Haa ₂₉ Haase

Haa ₃ 1 Thb Agd S1n Agd Tug (5E <2 U N0: 424) where Haa1 is Tug or absent;

Haa ₂ is He, Rgo or absent;

Xaa ₄ is bk, the BH-modified bk, D1a, Beg or Dgd;

Haa ₆ is C1i, C1p, D1a, Dkp, Αφ or Ya1;

Haa ₉ is C1u or D1a;

Haa1 ₀ is C1i, D1a, Αφ. Dkp, C1p, C1y, Prgo or D1b;

Haatz is C1i, D1a, Dkr, Dkp, C1p, C1u, Prgo or D1b;

Haa1 ₂ is D1a or Prgo;

Haatz is Running or Rgo;

Haa1 ₄ is Rgo, D1a, og Run;

Haa15 is C1i, D1a, Dkr, Dkp, C1p, C1u, Prgo or D1b;

Haa1 ₆ is C1i or Αφ;

Haatz is Lei or Not;

Xaa19 is Dgd, bk, BH-modified bk, C1n or N (Μе) Α1a;

Haa ₂ 1 is Tug, ΑΠ, PHe, LUK or BH-modified LUK;

Haa ₂₂ is Α1η or Running;

Haa ₂₃ is Running, Α1η or Αφ;

Xaa ₂₅ is Αγ§, bk or BH-modified bk;

Haa ₂₆ is H18, Α1η or Αγ§;

Haa ₂₇ is Tug or RNe;

Haa ₂₈ is Ley or Not;

Haa ₂₉ is Αδπ or C1p;

Haa ₃₀ is Ley or Μеΐ; and

Haa ₃ 1 is Wa1, He or Lei.

As will be understood by a person skilled in the art, the polypeptides of the formula CN can be in the form of a free acid or can be amidated in the C-terminal region.

In some embodiments, the PPE polypeptide may comprise a Ν-terminal fragment consisting primarily of the first 17 amino acid residues of natural human P человеческого (BEr р NO: 2 of из ^ 2006/013574771) linked to a C-terminal fragment consisting mainly of amino acid residues 18 -36 natural human ΝΓΥ (BEr ΙΌ NO: 4 from IS 2006 / 0135747A1), where one or more of the amino acid residues in the области-terminal region of the PΥΥ fragment can be deleted or absent and where in each of the P один and ΝΓΥ fragments there can be one, two, three, four, five, six, seven, sun NIL, nine or ten amino acid substitutions. In some embodiments, the Ν-terminal fragment, consisting mainly of the first 17 amino acids of the PPE polypeptide, can have 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 first 17 amino acids of natural PΥΥ. In some embodiments, the C-terminal fragment of the PPE polypeptide, consisting mainly of amino acid residues 18-36, may have 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 with amino acids 18-36 of natural ΝΓΥ. In some embodiments, amino acids at the Ν-terminal fragment of PΥΥ (for example, proline at positions 3, 5 and 8 or histidine at position 7) and / or amino acids at the C-terminal fragment of ΝΓΥ (for example, alanine at position 18, tyrosines at positions 20 and 36, leucine in position

- 35 014647

24, threonine at position 32, arginine at position 33, glutamine at position 34, or arginine at position 35) are not substituted. In some embodiments, the PPE polypeptides include those that have the amino acid sequence of the ΡUΥ-NΡΥ chimera, such as 8EC ΙΌ ΙΌ: 266, 437, 438, 439, 442, 462, 469, 470, 471 and 472 of I8 2006 / 0135747A1, or a compound with amino acid sequence

11th

Rgo CU Hb Yeah Aba Agde Alp, Yeah Not

Bz

In some embodiments, the PPE polypeptides also include a Ν-terminal cap. Examples of such PPE polypeptides include 8EC ΙΌ ΝΟ: 437, 462, 469, 470 and 472 from I82006 / 0135747A1. For example, sequence 438 of I82006 / 0135747A1 is

Rgo

Bue

one component after In one embodiment, the hybrid of this invention includes the sequences 438 of I82006 / 0135747A1, especially in hybrids used to treat obesity, weight loss, reduce or redistribute fat, and reduce calorie intake. Such a hybrid may also contain an amylinomimetic component, or a leptin component, or both.

PRE polypeptides and PPE chimeras, used alone or as a second agent, or as a component of a hybrid according to the invention, find use in methods involving changing the patient’s body composition, which include administering to a patient a compound (PPE polypeptide or PPE chimera , in its pure form, in the form of a second agent or as a component of a hybrid in accordance with this invention), where the compound changes the ratio of fat and muscle tissue, thereby changing the composition of the body. The PPE polypeptide may contain an amino acid sequence selected from the group comprising the ΡУΥ-NΡΥ chimera designated 5705 and the following sequences from I82006 / 0135747A1: 8ЕО ΙΌ ΝΟ: 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 muscle mass is maintained or increased in the body. In one embodiment, body fat and muscle mass are measured as the percentage of fat mass and the percentage of muscle mass in the body, respectively. In another embodiment, reduced body weight. In another embodiment, body weight is maintained or increased. Compounds may be administered peripherally. The PPE polypeptide or the PPE chimera or PPE-containing hybrid can be used in a method that also includes administering to the patient at least one other agent selected from the group consisting of amylin, amylin agonist or analog of an amylin agonist, salmon calcitonin, cholecystokinin (CCK) or agonist CCK, leptin (OB protein) or a leptin agonist, exendin, or an analog of an exendin agonist, CPB-1, a CPB-1 agonist or analogue of a CPB-1 agonist, CCK or a CCK agonist, calcitonin, calcitonin agonist, low molecular weight cannabinoid receptor antagonist B1, rimonabant, an inhibitor 1 beta hydroxysteroid dehydrogenase, sibutramine and phentermine. In one embodiment, the object is overweight or obesity. In yet another embodiment, the PPE polypeptides and the PPE chimeras, used alone or as a second agent or as a component of a hybrid in accordance with this invention, are used in methods, including a method for preferentially reducing plasma triglycerides in a patient, comprising administering the patient an amount of a compound effective to lower plasma triglycerides, where cholesterol levels are reduced to a lesser extent. In another embodiment, lower triglycerides and do not lower cholesterol. In another embodiment, lower levels of triglycerides and do not lower levels of bb cholesterol. In another embodiment, triglyceride levels are reduced, and bb cholesterol levels are reduced to a lesser extent. In another embodiment, amylase levels are also reduced. In yet another embodiment, the PPE polypeptides and the PPE chimeras, used alone or as a second agent or as a component of a hybrid in accordance with this invention, are used in methods comprising a method of reducing body fat mass or increasing body fat mass while maintaining or increasing lean body mass, including administering to a patient an amount of a compound effective to reduce body fat or increase body fat, while maintaining or increasing lean body mass. In another embodiment, the PPE polypeptides and the PPE chimeras used in pure form, either as a second agent or as a component of the hybrid in accordance with this invention, are used in methods including a method of reducing fat mass in internal organs, including administering to a patient an amount of a compound, effective to reduce body fat in the internal organs, while maintaining or increasing lean body mass. In another embodiment, the PPE polypeptides and the PPE chimeras used in pure form, either as a second agent or as a component of a hybrid in accordance with this invention, are found

- 36 014647 application in the methods. comprising a method for changing the distribution of fat in a patient. In one aspect, the change occurs as a result of improved metabolism of fat in the internal organs or ectopic fat. or both. in a patient. In another embodiment, the PPP polypeptides and the PPP chimeras. applied in pure form. or as a second agent. or as a component of a hybrid in accordance with this invention. find application in methods. comprising a method for increasing β-oxidation of a fatty acid. maintaining or increasing muscle mass. comprising administering to the patient an amount of the compound. effective for increasing β-oxidation of fatty acids. maintaining or increasing muscle mass. In another embodiment, the PPP polypeptides and the PPP chimeras. applied in pure form. or as a second agent. or as a component of a hybrid in accordance with this invention. find application in methods. comprising a method of treating non-alcoholic steatohepatate or lipodystrophy in a patient. comprising administering to the patient an amount of the compound. effective for non-alcoholic steatohepatitis or lipodystrophy. Hybrids of particular interest in the applications described above may contain the PPP chimera described herein in combination with a component from the leptin family or the amylin family. such as the amylin-bST-amylin hybrid. or both. A PPP chimera / leptin hybrid or a PPP chimera / amylin-bST-amylin hybrid has an effect. superior to the action of each of them individually. In yet another embodiment, the PPP chimer / leptin hybrid is administered with an amylinomimetic. such as the chimera amylin-bST-amylin. or a PPP chimera / amylin-bST-amylin hybrid is administered with leptin.

If they are not a component of the hybrid module. the PPP polypeptide chimeras indicated herein may be administered alone or as a second agent. preferably in combination with a hybrid in accordance with this invention. They may or may not contain a pharmaceutically acceptable carrier or excipient. in one or more doses. Such pharmaceutical compounds may be combined with pharmaceutically acceptable carriers or diluents. as well as with any other known adjuvants and excipients. using conventional techniques. such as those described in Ref. see also Vapd. Υ. G apB Napvop. M. A. Ragep! Eg1 Rogt1a! Yupv o £ Prgo! Etw apB ReyBev: 81aY1yu apB 81aYb / egv. IOIT1 O GAREPE! EEG1 8AEPSE APPLICATION. Keynote 1. 10.8irr. 42:28 (1988). included in the present description by reference. PPP polypeptides may be in unit dosage form. For example. therapeutically effective amounts of the PPP polypeptide for influencing body composition vary with a variety of factors. including age and body weight of the patient. physical condition of the patient. use in combination with other treatments. ultimate goal. to be achieved. such as a general decrease in body weight and / or maintenance or increase of muscle mass. as well as other factors. However, typical doses (if not a component of the hybrid) may include from a lower limit to about 0.05 mcg. about 0.1 mcg. about 1 mcg. about 5 mcg. about 10 mcg. about 50 mcg. about 75 mcg or about 100 mcg to the upper limit of about 50 mcg. about 100 mcg. about 500 mcg. about 1 mg. about 5 mg. about 10 mg. about 15 mg. about 50 mg. about 100 mg or about 150 mg of the pharmaceutical compound per day. Other dosage ranges are also included within the scope of the invention. such as from 0.1 μg to 1 mg of compound per dose or from about 0.001 μg / kg to about 500 μg / kg per dose. In some embodiments, the PPP polypeptide chimera is administered peripherally at a dose of about 0.5 μg to about 5 mg per day in one or more doses, or by controlled continuous release. or from about 0.01 to about 500 mcg / kg per dose. or from about 0.05 to about 250 mcg / kg. In some embodiments, the PPP polypeptide chimera is administered at a dose below about 50 μg / kg. Doses at indicated intervals. of course. can vary depending on the effectiveness of each analog or derivative and can be easily determined by a person skilled in the art. Daily doses can be divided into separate unit doses. administered continuously for 24 hours or during any period of the specified 24 hours. The number of doses per day can be from 1 to about 4 per day. although there may be more. Continuous administration may be in the form of a continuous infusion. Other contemplated typical dosages and rates of infusion include from 0.005 to about 20 nmol / kg per single dose. or from about 0.01 to about 10 pmol / kg / min with continuous infusion. The indicated doses and infusions may be administered by any known or future peripheral route. e.g. intravenous (intravenous). intradermally. intramuscularly. intramammary. intraperitoneal. intrathecal. retrobulbar. intrapulmonary (secreted over time); subcutaneous administration (SC). oral administration. sublingual. nasal. anal. vaginal or percutaneous administration. or by surgical implantation in a specific place. An example of a total intravenous dose / administration of a pharmaceutical composition is from about 1 μg to about 8 mg per day. and the total dose / administration of the pharmaceutical composition is from about 6 mcg to about 16 mg per day.

Incretins and incretin mimetics

Complex peptide hormones. used in accordance with this invention. also include peptide hormones CPb-1. Natural peptide hormones CP-1. including CPP-1 (1-37) (8EO ΙΌ N0: 59). CPB-1 (7-37) (81b) ΙΌ N0: 204) and CPB-1 (7-36) amide (81 G) ΙΌ N0: 61). known in the art. as well as their functional peptide analogues and derivatives. In this description

- 37 014647

CPB-1 refers to all natural forms of the peptide hormones CPB-1. Certain preferred natural peptides, peptide analogs and derivatives are described herein, although it should be understood that any known CPB-1 peptides that have a hormonal effect known in the art can be used in accordance with this invention.

Any analogue or derivative of the CPB-1 peptide known in the art can be used in accordance with this invention. In one embodiment, analogs and derivatives of the CPB-1 peptide exhibit at least one hormonal activity of the naturally occurring CPB-1 peptide. In certain embodiments, CPB-1 peptide analogs and derivatives are receptor agonists to which the naturally-occurring CPB-1 peptide can specifically bind. Preferred analogs and derivatives of the CPB-1 peptide include those described, for example, in νΟ 91/11457, which is incorporated herein by reference.

Analogs of CPB-1 known in the art include

Zeo Yu: 168 ⁹ C1P-SR-1 (7-37) 169 0- ^а С1п -СР-1 (7-37) 170 ¹⁶ Thr- ¹⁸ bf “CP-1 (7-37) '' 171 ¹⁸ LUZ-SR-1 (7-37) 172 “C1u-SR-1 (7-36) 173 ^and С1п-СРЬ-1 (7-37) 174 O- ^e C1p-CPb-1 (7-37) 175 ace! y1- ^y bue-cp-1 (7-37) 176 ⁵ Tg-CP-1 (7-37) 177 Tg 0- ^9-SR-1 (7-37): 178 ⁹ AZP-SR-1 (7-37) 179 L- ⁸ Azp-SR-1 (7-37) 180 ²² 5eg ²⁸ Agd ²⁴ Agd ²⁸ C1p-CPb-1 (7-37) 181 ¹⁶ Thr ¹⁸ bz-sr-1 (7-37) 182 ¹⁸ bz-sr-1 (7-37) '''' 183 ²⁸ Agd-CP-1 (7-37) 184 ²⁴ Agd-CP-1 (7-37)

As is known in the art, such CPB-1 analogs may preferably be amidated, but in the context of this invention may optionally be in the form of an acid, unless otherwise indicated.

Other analogs and derivatives of CP-1 are described in US Pat. No. 5,545,618, which is incorporated herein by reference. A preferred group of analogs and derivatives of CPB-1 include those described in US patent No. 6747006, which is fully incorporated into this description by reference. The use in this invention of the molecule described in US patent No. 5188666, which is incorporated into this description by reference, is also considered. Another group of molecules used in this invention includes compounds described in US patent No. 5512549, which is incorporated into this description by reference. Another preferred group of CPB-1 compounds used in this invention is described in νΟ 91/11457, which is incorporated herein by reference.

Complex peptide hormones in accordance with this invention also include peptide hormones CPB-2. Natural peptide hormones CPB-2, for example, rat CPB-2 and its homologues, including oxides CPB-2, pork CPB-2, CPB-2 degu, bovine CPB-2, CPB-2 guinea pig, CPB-2 hamster, human CPB -2, CPB-2 rainbow trout and chicken CPB-2, are known in the art, as are their analogues and derivatives. Certain natural peptides, analogues and derivatives of the peptides are described herein, however, it should be understood that any known CPB-2 peptides that have a hormonal effect known in the art can be used in accordance with this invention.

Any analogue or derivative of the CPB-2 peptide known in the art can be used in accordance with this invention. In one embodiment, analogs and derivatives of the CPB-2 peptide exhibit at least one hormonal activity of the natural CPB-2 peptide. In certain embodiments, analogs and derivatives of the CPB-2 peptide are receptor agonists to which the naturally occurring CPB-2 peptide can specifically bind. Preferred analogs and derivatives of the CPB-2 peptide include those described, for example, in 8-8eg. No. 08/669791 and PCT / CA97 / 00252, both of which are incorporated herein by reference. Certain CPB-2 analogues known in the art include rat or human CPB-2, altered at position 2 to fix resistance to LBP-Ιν by substituting C1y for A1a.

The complex peptide hormones used in accordance with this invention also include peptide hormones oxyntomodullin (OXM). Natural peptide hormones OXM are known in the art, as are their functional peptide analogues and derivatives. Certain preferred natural peptides, analogs and derivatives of peptides are described herein, however, it should be understood that any known OXM peptides that have a hormonal effect known in the art can be used in accordance with this invention.

- 38 014647

Any analog or derivative of an OXM peptide known in the art can be used in accordance with this invention. In one embodiment, analogs and derivatives of the OXM peptide exhibit at least one hormonal activity of the natural OXM peptide. In certain embodiments, the OXM peptide analogs and derivatives are agonists of a receptor to which the naturally occurring OXM peptide can specifically bind.

The complex peptide hormones used in accordance with this invention also include exendin peptide hormones. Natural peptide hormones of exendin are known in the art, as are peptide analogs and derivatives. Certain preferred natural peptides, analogues and derivatives of the peptide are described herein, however, it should be understood that any known exendin peptides that have hormonal effects known in the art can be used in accordance with this invention.

Any analog or derivative of an exendin peptide known in the art can be used in accordance with this invention. In one embodiment, exendin peptide analogs and derivatives exhibit at least one hormonal activity of the naturally occurring exendin peptide. In certain embodiments, exendin peptide analogs and derivatives are receptor agonists to which a naturally occurring exendin peptide can specifically bind.

Exemplary exendin analogues include

ΞΕΟ Yu: 185 ^1, 1ey, ^2b Pb-exendin-4 186 ^s A1a, ^1a Leu, "Rie-exendin-W 187 ^1, 1ey, ²² A1a, ^2!> Pb-exendin-4

As is known in the art, such exendin analogs may preferably be amidated, but in the context of this invention may not necessarily be in the form of an acid, unless otherwise indicated.

Additional typical exendin analogs and derivatives are described in PCT Application No. PCT / IZ98 / 16387, filed August 6, 1998, entitled New Exendin Agonist Compounds, which claims priority to US Patent Application No. 60/055404, filed August 8, 1997. , both included in the present description by reference. Other analogs and derivatives of exendin are described in PCT application No. PCT / IZ98 / 24210, filed November 13, 1998, entitled New compounds of exendin agonists, which claims priority to provisional application for US patent No. 60/065442, filed November 14, 1997 , both included in the present description by reference. Other analogs and derivatives of exendin are described in PCT application No. PCT / IZ98 / 24273, filed November 13, 1998, entitled New compounds of exendin agonists, which claims priority to provisional patent application US No. 60/066029, filed November 14, 1997 , both included in the present description by reference. Other exendin analogs and derivatives are described in PCT Application No. PCT / IZ97 / 14199, filed August 8, 1997, entitled Methods of Regulating Gastrointestinal Activity, which is a partial continuation of US Patent Application No. 08/694954, filed August 8, 1996. , both included in the present description by reference. Other exendin analogs and derivatives are described in PCT application No. PCT / IZ98 / 00449, filed January 7, 1998, entitled Use of exendins and their agonists to reduce food intake, which claims priority to provisional patent application US No. 60/034905, filed January 7, 1997, both are incorporated into this description by reference. Other exendin analogs and derivatives are described in IZ 2004/0209803 A1, filed December 19, 2003, entitled Composition for the Treatment and Prevention of Neuropathy, which is incorporated herein by reference.

Natriuretic peptides

Natriuretic peptides are a family of hormones that consists of the atrial natriuretic peptide (A #) of the cerebral natriuretic peptide (BCP), C-type natriuretic peptides (C \ P |. They are synthesized and stored as 3 separate prohormone precursors, which are A # of 126 amino acids, BCH of 108 amino acids and C'XR of 104 amino acids, each encoded by separate genes and has separate synthesis sites and regulatory mechanisms. ayut

ZEO Ιϋ N0: 375 human ΑΝΡ 151 amino acids lreprogsrmon MZZGZTTUZEZ'EOZOTOKRUMUMAUZKAPEKOM EEONEEKMRIEVETUPOUZORZEEASAEREZERURRIT OEUZRASKIBSAKSKR WEU ΙΏ ΝΟ: 376 HUMAN ΒΝΡ PREPROHORMONE OF 134 AMINO ACIDS MOROTARZAEJЬЬГААГГГРРРРРРРЗЗЗРСРСРСАЗЗЗЗЗЗЗЗ BOEOKI ΗΕςϋΚΣ3ΕΣ0νΕ0Τ ZEEROEZ RKRTS \ 7IKZEEEUAT ES1NSNKKM7UTKARN5RKM70SZSSESSEKM0K15835S SKUKNNN ZE <2 10 N0: 377 HUMAN CHEESE BROADCAST FROM 126 AMINO ACIDS ΜΗΙЛС'1., 1АСАГΙ, Τ, ΤΙ.Τ, 5 ЬЕРЗ ЕАКРСАРРЮТКТР ΡΆΕΕ1ΑΕ РОААСССОКОКАРССОСАМКСОЗКЗКЬКУКУОКЗЗАА ИАКЬОЕНЫАКАКУКСАЙККС1, ЗКССESSЬКУСЬЬ

The main site of synthesis of prohormone A # is the atrial myocyte, where it is synthesized as a preprohormone of 151 amino acids. Removal of a signal peptide of 25 amino acids from its N

- 39 014647 of the terminal region occurs in the endoplasmic reticulum, leaving the prohormone ANP (RgoANP) of 126 amino acids, the main stored form of ΛΝΓ in the heart. Prohormone consists of 4 biologically active peptide segments: amino acids 1-30 (Progoame 1-30, also known as a long-acting stimulant Να), 31-67 (Progoame 31-67, also known as a vasodilator), 79-98 (Progoame 7998 , also known as excreting potassium) and 99-126 (ΛΝΕ, also known as atrial natriuretic factor).

B \ P was originally isolated from the pig brain, but in humans it is synthesized and isolated from the left ventricle. Sequence analysis shows that pre-RtoVIR consists of 134 residues and is cleaved to PrgoBN of 108 amino acids. Cleavage of the 32-amino acid sequence from the end region of PrgoBN gives human BNP (77-108), which is a physiologically active form in plasma.

CPR is the third member of the natriuretic peptide system, and it is primarily found in human vascular endothelial cells, kidneys and the pig brain. High concentrations of CPR are also found in the human hypothalamus and midbrain. In humans, prepro-С№ is a precursor of 126 amino acids, converted into pro-С№ by cleavage of 23 residues from the цевой-terminal region. This 23 amino acid sequence serves as a signal peptide. The terminal 22 (105-126) amino acids are cleaved from the pro-CGOR to give a biologically active form of CPR.

Urodilatin is a member of the family of natriuretic peptides obtained from the kidneys, and it is formed from the same prohormone A # and consists of amino acids 95-126. With the exception of lengthening 4 amino acids of the Ν-terminal region, it is identical to AME (99-126). Urodilatin is considered an important regulator of sodium and water retention in the kidneys, as well as a mediator of sodium excretion in patients with congestive heart failure (CHE).

Natriuretic peptides exert their biological effect through binding to receptors with high affinity on the surface of target cells. Three subtypes of NRPk-NRP-A, NRP-B and No.PC were distinguished. Therefore, in one embodiment of the present invention, a method for screening hybrids for binding and / or activating natriuretic receptors is provided. Natriuretic peptides, including prohormones, can confer many functions of the natriuretic peptide hormone to the hybrids of this invention. In other interesting embodiments, hybrids of natriuretic antagonists are provided. Natriuresis is the removal of excess large amounts of sodium in the urine. Natriuresis is similar to diuresis (the release of an unusually large amount of urine), except that with natriuresis, urine is exceptionally salty. Natriuresis occurs with certain diuretics and diseases (such as the adrenal glands) and can lead to the appearance of an adrenogenital syndrome with loss of salts, characterized by dehydration, vomiting, low blood pressure and the risk of sudden death. The exogenous administration of 4 independent circulating peptides of the prohormone AYR (1-30, 31-67, 79-98 and 99-126) w νί \ Ό causes vasodilation, diuresis, suppression of the renin-angiotensin-aldosterone system and increased natriuresis and / or potassium urine. Each of RGOANE 1-30, RGOANE 31-67 and ΛΝΕ 99-126 has a natriuretic, pressure-lowering and diuretic effect, and RGOAKE 31-67 and AOT 99-126 have a strong effect on blood pressure. There is a changing effect of AOT peptides on potassium homeostasis: ProgoAE 79-98 stimulates potassium excretion, while ProCoE 31-67 restores potassium loss by inhibiting Να / K ATPase in the cells of the medullary collecting ducts. Specific to AOT 99-126 is the dosage-dependent inhibition of angiotensin II-mediated secretion of aldosterone, while Progoane 31-67 has the property of inducing natriuresis through the formation of prostaglandin.

B \ P has a biological effect, the same as AOT in normal people. B \ P infusion in a normal person causes a 2-fold increase in sodium excretion, a 50% decrease in plasma renin, secretion of angiotensin II and aldosterone, as well as a decrease in plasma volume.

С№ has a cardiovascular effect, the same as other natriuretic peptides, but, apparently, does not mediate any renal effects. When С№ is infused into anesthetized dogs at doses equivalent to ANE, plasma cMSP increases with a concomitant decrease in mean arterial pressure, pressure in the right atrium and cardiac output, and glomerular filtration rate, renal blood flow, and sodium excretion decrease.

Natriuretic peptides can have a therapeutic effect on heart failure. Congestive heart failure (CHE) is associated with an increase in vasopressin, endothelin and activation of the renin-angiotensin-aldosterone system and the sympathetic nervous system, mediating vasodilation, retention of sodium and water, and negative correction of the heart and blood vessels. This effect occurs due to elevated levels of natriuretic peptides in patients with heart failure. In one embodiment of the present invention, hybrids are provided that provide elevated or therapeutic serum levels of the activity of a natriuretic peptide for the treatment or prevention of heart disease and conditions, including CHE. Although AOT infusion in normal people can give a slower increase in sodium excretion and urine flow rate, a noticeable favorable response reduction can be obtained in patients with heart failure.

- 40 014647 kidneys. Infusion of Bn significantly increases the excretion of sodium in patients with heart failure and has a significant favorable hemodynamic effect. Compared with A #, the diuretic and natriuretic action of B # is much stronger. BH is cleaved more slowly than AH and has a different effect, including suppression of aldosterone secretion and an increase in AH levels in plasma. ΒNP peptides can also provide a favorable decrease in pressure in terminal pulmonary capillaries, systemic vascular resistance, pressure in the right atrium and arterial systolic pressure, with an increase in cardiac index in patients hospitalized with symptoms of CHE. In patients with decompensated heart failure, natriuretic peptide hybrids can provide a beneficial reduction in pressure in the terminal pulmonary capillaries and an improvement in dyspnea (shortness of breath is an unpleasant feeling of difficulty breathing, usually associated with the early stages of heart failure). Hybrids having one, two or three functions of a natriuretic hormone represent methods of administering pharmaceutically active compositions that are useful for the prophylactic and therapeutic treatment of patients with CHE, preferably decompensated CHE patients, patients with chronic CHE, and patients with hypertension. The natriuretic part (s) of the hybrid is sufficient to provide a therapeutically effective amount of a natriuretic peptide in such patients when administered in a therapeutically effective dose over a therapeutically effective period.

As described herein, any member of the family of therapeutically effective natriuretic peptides or their analogues can be used. Useful natriuretic peptides include, for example, atrial natriuretic peptide (A #), brain natriuretic peptide (B # or B-type natriuretic peptide) and C-type natriuretic peptide (C #).

Sequences of useful forms of natriuretic peptides are described in US Patent Publication 20010027181, which is incorporated herein by reference. Examples of A # include human A # (Kapda ^ a e! A1., VVKS 118: 131 (1984)) or various types of this peptide, including pork and rat A # (Kapda ^ a e! A1., VVKS 121: 585 ( 1984)). Such Ao include 28 amino acids. Such Ao can be introduced in the form of a peptide having an A№ ring structure (formation of a disulfide bond based on Suk) and a C-terminal region after the ring structure. An example of such a peptide is a peptide having amino acid residues at positions 7 to 28 A #, described in US Patent Application Publication No. 20010027181. Another example is a frog A #. Specific examples of BHI that can be used in the methods of this invention include human BH (yBH). Human No. contains 32 amino acids and includes the formation of a disulfide bond (Ziboy e! A1., VVKS 159: 1420 (1989)) and US patents No. 5114923, 5674710, 5674710 and 5948761, each of which is incorporated into this description by reference. Various non-human ΒNPs, including porcine B и and rat B№, are also known and can be used. Another example is chicken no. Examples of C # that can be used in the methods of this invention include pork C #. Pork С№ contains 22 amino acids and includes the formation of a disulfide bond, as well as ANP and В№ described above (Ziboy e! A1., VVKS 168: 863 (1990)) (human and rat have the same amino acid sequences), chicken С№ (Aptiga e! A1., VVKS 174: 142 (1991)). The frog number # okYyaga e can also be used! A1., VVKS 173: 591 (1990)). As described herein, one skilled in the art will be able to make modifications, such as deletion, substitution, addition or insertion, and / or chemical modifications with the amino acid residues of the amino acid sequence of known natriuretic peptides, if desired, by known methods. The resulting compound is a compound that has an effect on the receptor of the original A #, B # or C #. Therefore, analogues having such activity are included in hybrids using in accordance with the methods of this invention.

In another embodiment, hybrids having one or more natriuretic functions can be used to treat hypertension. In one embodiment, the natriuretic hybrid will not adversely affect heart rate and is not associated with arrhythmia. In one embodiment, the hybrid has at least one, two, or three functions of a natriuretic peptide, for example, A # and B # activity. One or more functions of the natriuretic hormone may be combined with any other hormonal functions or peptide enhancers, as described herein. In another embodiment, the natriuretic moiety is a more stable analogue having a longer half-life of 1n yuo compared to the natural natriuretic peptide. Analogs are also provided that prevent undesired degradation by enzymes such as NEP. Hybrids containing natriuretic are also associated with a decrease in hypertension, inducing diuresis, inducing natriuresis, vasodilation or relaxation, binding to natriuretic peptide receptors (such as No.K-A), inhibition of renin secretion from the kidneys, inhibition of adrenal secretion from the adrenal gland, and treatment of the heart -vascular diseases and disorders, decreased, stopped or reversed heart correction in case of congestive heart failure, treatment of kidney diseases and disorders; treating or preventing ischemic attack and treating asthma. Hybrids may be administered to patients for whom inducing natriuresis, di

- 41 014647 cutting and vasodilation. Hybrids may be administered alone or in combination with one or more of the following types of compounds: ACE inhibitors. beta blockers. diuretics. spironolactone. digoxin. anticoagulant and antiplatelet agents. and angiotensin receptor blockers. Other diseases or conditions include renal disorders and diseases. asthma hypertension and pulmonary hypertension. erectile dysfunction and hypercholesterolemia.

Bioactive modules of the peptide hormone

As described here. the hybrid polypeptides of this invention typically include at least two bioactive peptide hormone modules. covalently bonded together. The bioactive modules of the peptide hormone can be: (a) natural complex peptide hormones. (B) analogues or derivatives of natural complex peptide hormones. which retain a hormonal effect. (c) fragments of natural complex peptide hormones. which retain hormonal activity. (B) fragments of analogues or derivatives of natural complex peptide hormones. which retain a hormonal effect. (e) structural motifs of natural complex peptide hormones. which give the desired chemical stability. conformational stability. metabolic stability. bioavailability. focus on organ / tissue. interaction with the receptor. protease inhibition. binding to plasma protein d / or other pharmacokinetic characteristics of the hybrid polypeptide; or (ί) structural motifs of analogues or derivatives of natural complex peptide hormones. which give the desired chemical stability. conformational stability. metabolic stability. bioavailability. focus on organ / tissue. interaction with the receptor. protease inhibition. plasma protein binding and / or other pharmacokinetic characteristics of the hybrid polypeptide. Structural motifs (e) and (ί) are collectively referred to herein as peptide enhancers.

Preferred bioactive modules of the peptide hormone include natural hormones. selected from amylin. ABM ST SSR. intermedina. SSK (1-33). SSK-8. leptin. PΥΥ (1-36) (8EC ΙΌ N0: 57). PΥΥ (3-36) (8ES) ΙΌ N0: 58). CPP-1 (1-37) (8ES) ΙΌ N0: 59). CPP-1 (7-37) (8ES) ΙΌ N0: 204). CPP-1 (7-36) (8E0 ΙΌ N0: 61). CP2. 0XM. exendin-3. exendin-4. natriuretic peptide hormones. peptides of the urocortin family. e.g. Isp-2 and Isp-3. peptides of the neuromedin family. for example neuromedin I25 or spliced variants. and AHR. VNR. ATS or urodilatin.

Other preferred bioactive modules of the peptide hormone include peptide hormone analogs and derivatives. selected from: amylin. ABM ST SSR. intermedina. SSK. leptin. PΥΥ (1-36) (8ES) ΙΌ N0: 57). PΥΥ (3-36) (8ES) ΙΌ N0: 58). CPP-1 (1-37) (8ES) ΙΌ N0: 59). CPP-1 (7-37) (8ES) ΙΌ N0: 204). CPP-1 (7-36) (81+) ΙΌ N0: 61). CP2. OXM. exendin-3 and exendin-4. natriuretic peptide hormones. peptides of the urocortin family. e.g. Isp-2 and Isp-3. peptides of the neuromedin family. for example neuromedin I25 or spliced variants. and AIR. BNP. ATS or urodilatin. where the analogue or derivative has at least one hormonal action of the complex peptide hormone. An analogue may contain one or more inserts. deletions or substitutions of the amino acid sequence of a complex peptide hormone. and the derivative may contain one or more chemical modifications of the amino acid residue of an analog or a complex peptide hormone. as described in more detail here and in the art.

More specific. analogues and derivatives can be selected from any. described above and known in the art. Particularly preferred analogs and derivatives. which exhibit at least one hormonal activity. used as a bioactive module of the peptide hormone in accordance with this invention. are

Amylin: ² A1a-th-amylin (ZEO U N0: 79), ² '' AXa-b-amylin (ZEO U N0: 80), ²⁰ Рgo “11-amylin (ZEO U N0: 189), ^25,2е Рgo-Iamilin (ZEO Yu N0: 83), ²⁵ '28 ^{, 29} Rgo-L-amylin (ZEO Yu N0: 67), ²⁵ ₽go, ²⁶ Ya1, ^2E ' ²⁹ Rgo-L-amylin (ZEO 10 N0: 69), ^1е arg, Pro-11, ^25,26-amylin (ZEO Yu N0: 70), ¹⁸ arg ^{25 '23' 2E} Pro "L-amylin (ZEO Yu N0: 72), ²⁵ Pro, ² Ua1, ^{28 '29} Rgo- B-amylin (ZEO 10 N0: 75), ¹⁰ Agd, ^2E Ley, ²⁵ ' ^2a ' ²⁹ Prgo-L-amylin (ZEO KB N0: 89), ¹⁸ Agd ²³ Lye, ^25.28 Rgo-L-amylin (ZEO Yu N0: 90), and 2.7Cus1- [ ² Azr, ⁷ Luz] ~ b-amylin (ZEO Yu N0: 76)

- 42 014647

ST: ¹⁴ C1I “ZST (ZEY U N0: 106), ^{1 in} Arg-zST (ZEY 10 N0: 107), ^11.18 DGD-zST (ZEY 10 N0: 108), ¹⁴ C1i, ¹⁸ DGD ~ zST (ZEY U N0: 109), ¹⁴ C1i, ^11.1v Agd-zST (ZEY Yu N0: 110) SSKR: ^si 0-Zeg-SSKR (ZEY Yu N0: 111), ^ E-TIG-SSKR (ZEY Yu N0: 112), ³⁶ 0-Azr-SSKR (ZEY Yu N0: 113), ^e6 0-Azp-SSER (ZEY Yu N0: 114), ^Z6 3eg-C6EP (ZEY Yu N0: 115), ³⁶ NZeSSER (ZEY Yu N0: 116), ³⁶ Azr-S <ZER (ZEY Yu N0: 117), ³⁶ Tg-SOKR (ZEY Yu N0 : 118), ^zb Azp-S0ER (ZEY Yu N0: 119) AGR-6: TYAYEUSSSNZTSY7NYZNYYMBRASEYOZARUORZZRNZU (ZEY! □ N0: 120}, TYAYUUUZSOTATUZNYMBRASEYOZARUORZZRNZU (ZEY YU N0: 121) TAYAJEUSMUETMYUZNYZMERASAYOZARUORZZRNZT (ZEY YU N0: 122) TYAYEEUBSUOTSYNZNYMORASKYOZARUERZRZRNZU (ZEY Ιϋ N0: 123) TIAYKUSSUSSYUYZNZNYMSRESEYESARUERZRZRNZU (ZEY YU N0: 124) SSK: 0Υ (OZOZN) MOCKET (ZEY U N0: 141), EUMZHMEG (ZEY U N0: 142), MBIMOG (ZEY U N0: 143), SMMOG (ZEY U N0: 144), KIPE (ZEY U N0: 145), ΚΏΥ (О5О ₃ Н) ISIMOG (ZEY U N0: 146), ΚΟΥΜΞΗΜϋΓ (ZEY U N0: 147), KMBIMO (ZEY 10 N0: 148), KSIMOE (ZEY U N0: 149), KIMBG (ZEY U N0: 150) leptin: ⁴³ Azr-leptin (ZEY U N0: 151), ⁴³ 61i-leptin (ZEY U N0: 152), ⁴⁸ A1a-leptin (ZEY U N0: 153), ⁴⁹ 61-leptin (ZEY U N0: 154), ⁴⁹ 0 -AA-leptin (ZEY U N0: 155), ⁷⁵ A1a-leptin (ZEY U N0: 156), ^8E Ley-leptin (ZEY U N0: 157), ^ez Azr-leptin (ZEY U N0: 158), ⁹³ < 31i-leptin (ZEY U N0: 159), ⁹⁸ A1a-leptin (ZEY U N0: 160), ¹³⁹ Leu-leptin (ZEY U N0: 162),

- 43 014647

ΡΥΥ: ³ Ley-RUU (Zeo Yu N0: 192), ³ Wa1-RUU (Zeo Yu N0: 193), Agd-ΡΥΥ (Zeo Yu N0: 194), ⁴ 01η-ΡΥΥ (Zeo Yu N0: 195), ⁴ Azp- RUU (Zeo Yu N0: 196), ²⁵ Lue-RUU (Zeo Yu N0: 197), ³⁴ Rgo-RUU (Zeo Yu N0: 198), ³⁴ Η1β-ΡΥΥ (Zeo Yu N0: 199), ^1.36 Tug- RUU (ЗЕО Ю N0: 57), ¹³ Рgo ¹⁴ А1а-РУУ (ЗЕО Ю N0: 200), ³¹ Рё ³⁴ Рго-РУУ (ЗЕО 10 N0: 201), с! Ез-АА-4-РУУ (ЗЕО Ю N0 : 202) sr-1 ^and S1p-SR-1 (7-37) (ZEO Yu N0: 168), B- ^and S1p -ER-1 (737) (ZEO Ιϋ N0: 169) ^{16 18} TAg- uz SR-1 (7-37 ) (Zeo Yu N0: 170), ^1e Lue-CPb-1 (7-37) (Zeo Yu N0: 171), ⁸ 61y6Pb-1 (7-36) (Zeo 10 N0: 172), ⁹ 61n-6Pb- 1 (7-37) (ZEO Yu N0: 173), 0- ^e С1п-СРЬ-1 (7-37) (ЗЕО Yu N0: 174), acEyu1- ⁹ LU-CPI-1 (7-37) (ЗЕО 10 N0: 175), ⁹ Tg-CP1 (7-37) (ZEO 10 N0: 176), 0- ⁹ Tg-CPB-1 (7-37) (ZEO 10N0: 177), ⁵ Azp-6Pb-1 (7-37) (Zeo Yu N0: 178), O-AzpSr-1 (7-37) (Zeo Yu N0: 179), ²² 5eg ²³ Agd ²⁴ Agd ²⁶ ClpSr-1 (7-37) (Zeo Yu N0: 180), ¹⁶ TNG ¹⁸ LZ-CPB-1 (7-37) (ZEO Yu N0: 181), ^1a LZ-6PB-1 (7-37) (ZEO Yu N0: 182), ²³ Arg-6PB- 1 (7-37) (Zeo Yu N0: 183), ²⁴ Agd-6Pb-1 (7-37) (Zeo Yu N0: 184) exendin ¹⁴ Ley, ²³ Lye-exendin-4 (Zeo Yu N0: 185), ¹⁴ Ley, Gyeckendzn-4 (Zeo Yu N0: 185), ⁵ L1a, ¹⁴ Ley, ²⁵ Ρ7ιθexendin-4 (Zeo 10 N0: 186) and ¹⁴ Ley, ²² A1a, ²⁵ Pyeeksendin-4 (Zeo Yu N0: 187).

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

Other preferably bioactive modules of the peptide hormone include fragments of a complex peptide hormone selected from amylin, ΑΌΜ, CT, CCCR, intermedin, CCK, leptin, PΥΥ (1-36) (BEP ΙΌ \ O: 57), PΥΥ (3-36) ( BEP ΙΌ \ O: 58), CPP-1 (1-37) (BEP ΙΌ \ O: 59), CPP-1 (7-37) (BEP ΙΌ \ O: 204), CPP-1 (7-36) (BEr ΙΌ MO: 61), CPB-2, OXM, a natriuretic peptide, exendin-3 and exendin-4, peptides of the urocortin family, for example Isp-2 and Isp-3, peptides of the neuromedin family, for example I25 neuromedin or spliced variants, and ΑΝΈ, ΒΝΓ, SDR or urodilatin, where the fragment has ayney least one hormonal action of a component peptide hormone.

Other preferably bioactive modules of the peptide hormone include fragments of analogues or derivatives of a complex peptide hormone selected from amylin, ΑΌΜ, CT, CCCR, intermedin, CCK, leptin, PΥΥ (1-36) (BEr ΙΌ HO: 57), PΥΥ (3-36 ) (BEr ΙΌ HO: 58), CPP-1 (1-37) (BEr ΙΌ HO: 59), CPP-1 (7-37) (BEr ΙΌ KO: 204), CPP-1 (7-36) ( BEPR ΙΌ KO: 61), CPB-2, OXY, ΑΝΓ, ΒΝΒ, CB1P, urodilatin, exendin-3, exendin-4, the hormone of the natriuretic peptide, and peptides of the urocortin family, for example Isp-2 and Isp-3, peptides of the neuromedin family for example, neuromedin I25 or spliced vari ntov and ΑNR, VIR urodilatina or NRI, wherein the fragment exhibits at least one hormonal action of a component peptide hormone. Again, the analogue may contain one or more insertions, deletions or substitutions of the amino acid sequence of the complex peptide hormone, and the derivative may contain one or more chemical modifications of the amino acid residue of the analogue or complex peptide hormone, as described in more detail here and in the art.

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

- 44 014647

Amylin: aniline (1-36) (ZEO Ιϋ N0: 210), amylin (1-35) (ZEO Yu N0: 211), amylin (1-20) (ZEO Yu N0: 212), amylin (1-18) (ZEO 10 N0: 213), amylin (1-17) (ZEO 1B N0: 214), aniline (1-16) (ZEO U N0: 215), amylin (1-15) (ZEO U N0: 216), amylin (1-7) (ZEO 10 N0: 217) ST: ST (8-32) (ZEO U N0: 218), ST (8-27) (ZEO U N0: 219), ST (8-26) (ZEO U N0: 220), ST (8-10) (ZEO Yu N0: 221), ST (18-26) (ZEO Yu N0: 222), ST (18-27) (ZEO Yu N0: 223) AGR-6: APP-6 (18-27) (Zeo Yu N0: 224) SSK: SSK-8, SSK-5, SSK-4 leptin: leptin (22-167) (Zeo Yu N0: 225), leptin (56-73) (Zeo Yu N0: 226) ΡΥΥ: ΡΥΥ (1-35) (ZEO 10 N0: 227), ΡΥΥ {1-30) (ZEO Yu N0: 228), ΡΥΥ (1-25) (ZEO Yu N0: 229), ΡΥΥ (1-15) (ZEO Yu N0: 230), ΡΥΥ (Ι-ΙΟ) (Zeo Yu N0: 231), ΡΥΥ (2-36) (ZEO Yu N0: 232), ΡΥΥ (3-36) (ZEO 10 N0: 58), ΡΥΥ (4- 36) (Zeo Yu N0: 233), ΡΥΥ (5-36) (Zeo Yu N0: 234) SR-1 СРЬ-1 (7-37) (ЗЕО ю N0: 204), СРЬ-1 (7-36) (ЗЕО ю N0: 61), CP-1 (7-35) (Zeo Yu N0: 235) exendin exendin-4 (1-27) (Zeo Yu N0: 236), exendin-4 (1-28) (Zeo Yu N0: 237), exendin-4 (1-29) (Zeo Yu N0: 238), exendin- 4 (1-30) (Zeo Yu N0: 239) or longer

Again, as is known in the art, such peptide compounds may preferably be amidated, but in the context of this invention may optionally be in the form of an acid, unless otherwise indicated. Further, the above preferred fragments can be combined with any analogs or derivatives described herein or known in the art. For example, preferred fragment analogs may include ⁵ A1a, ¹⁴ Ley, ²⁵ Pb-exendin-4 (1-28) (8 EU) II) ΝΟ: 240), ¹⁴ Ley, ²⁵ Pb-exendin-4 (1-27) ( 8 EU) GO ΝΟ: 241), ⁵ A1a, ¹⁴ Ley, ²⁵ Pb-exendin-4 (1-28) (8EO GO ΝΟ: 240), ¹⁴ Ley, ²⁵ Pb-exendin-4 (1-27) (8EO GO ΝΟ: 241), or any other combination of the described fragments, analogs and derivatives.

Other preferred bioactive peptide modules include a peptide enhancer, i.e. structural motifs of complex peptide hormones (including analogs and derivatives) that confer the desired chemical stability, conformational stability, metabolic stability, bioavailability, orientation to the organ / tissue, interaction with the receptor, protease inhibition, binding to plasma protein and / or other pharmacokinetic characteristics of the hybrid a polypeptide. Exemplary peptide enhancers include the following. Again, it must be understood that combinations of the above analogs and derivatives with the bioactive peptide modules described below are also contemplated. For example, the last six amino acid residues of analogues and derivatives of the peptide hormone of the amylin family, known in the art and / or described above, are also considered as preferred bioactive peptide modules.

- 45 014647

amylin family AMILIN (32-37) (Zeo Yu N0: 242), Amilin (33-37) (Zeo Yu N0: 243), Amilin (34-37) (Zeo Yu N0: 244), AMILIN (35-37), AMILIN (36-37), AMILIN (37), ΑϋΜ (4752) (ЗОО N N0: 245), ΑϋΜ (48-52) (ЗЕО Ιϋ N0: 246), ΑΏΜ (49-52 ) (ZEO Ιϋ N0: 247), ΑϋΜ (50-52), ΑϋΜ (5152), ΑϋΜ (52), ST {27-32) (ZEO U N0: 248), ST (2732} (ZEO U N0: 249 ), ST {28-32) (Zeo Yu N0: 250), ST (29-32), ST (30-32), ST (31-32), ST (32), SSKR (3237) (ZEO Yu N0: 251), S6KR (33-37) (ZEO Yu N0: 252 ), SSKR (34-37) (ZEO Ιϋ N0: 253}, SSKR (35-37), SSKR (36-37), SSVR (37), INTERMEDIN (42-47) (Zeo Yu N0: 254), INTERMEDIN (43-47) (Zeo Yu N0: 255), INTERMEDIN (44-47) (Zeo Yu N0 : 256), INTERMEDIN (45-47), INTERMEDIN (46-47), INTERMEDIN (47) ΡΥΥ ΡΥΥ (25-36) (Zeo Yu N0: 257), ΡΥΥ (26-36) (Zeo Yu N0: 258), ΡΥΥ (27-36) (Zeo Yu N0: 259), ΡΥΥ (28-36) (Zeo Yu N0: 260), ΡΥΥ (29-36) (Zeo Yu N0: 261), ΡΥΥ (30-36) (Zeo Yu N0: 262), ΡΥΥ (31-36) (Zeo Yu N0: 263), ΡΥΥ (32-36) (Zeo Yu N0: 264), ΡΥΥ (25-35) (ZEO 10 N0: 265), ΡΥΥ (26-35) (ZEO Yu N0: 266), ΡΥΥ (27-35) (Zeo Yu N0: 267), ΡΥΥ (28-35) (Zeo Yu N0: 268), ΡΥΥΙ29-35) (Zeo Yu N0: 269), ΡΥΪ (30-35) (Zeo Yu N0: 270), ΡΥΥ (31-35) (Zeo Yu N0: 271), ΡΥΥ (32-35) (Zeo Yu N0: 272) - CPF-1 and 2 frog CPY-1 (29-37) (Zeo Yu N0: 273); frog С ₽ Ь-1 (30-37) (Zeo Yu N0: 274); frog CPY-2 (24-31) (ZEO Yu N0: 275), frog CPY-2 (2531) (ZEO 10 N0: 276) Exendin-4 exendin-4 (31-39) (ZEO · 10 N0: 277), exendin-4 (3239) (ZEO 10 N0: 278), exendin-4 (33-39) (ΞΕΟ Yu N0: 279), exendin-4 ¢ 34-39) (ZEO 10 N0: 280), exendin-4 (35-39) (ZEO 10 N0: 281), exendin-4 (3639) (ZEO Yu N0: 282), exendin-4 (37-39 ), exendin4 (38-39), exendin-4 (39)

The principles of the choice of the peptide module, spacers and linking groups.

Hybrid polypeptides in accordance with this invention typically contain at least two bioactive modules of the peptide hormone in accordance with this invention, where at least one of the bioactive modules of the peptide hormone has at least one hormonal effect. A bioactive module of a peptide hormone that exhibits at least one hormonal activity can be located in the Ν-terminal region of the hybrid polypeptide, the terminal region of the hybrid polypeptide, or, if the hybrid polypeptide contains more than two bioactive modules of the peptide hormone, can be located in the inner parts of a hybrid polypeptide.

In certain embodiments, it may be preferable to position the bioactive module of the peptide hormone having at least one hormonal action, so that the end region of the bioactive module of the peptide hormone is amidated. Amidation of the C-terminal region of the bioactive module of the peptide hormone can be achieved by placing the module in the C-terminal region of the hybrid peptide, or by positioning the module in the C-terminal to the Ν-terminal direction in the кон-terminal region of the hybrid polypeptide. In both configurations, the end region of the bioactive module of the peptide hormone is available for amidation. Certain complex peptide hormones, which may preferably be C-terminal amidation, include peptide hormones of the amylin family, CCK, ΓΥΥ, LBP-1 (7-36) (8E0 GO NO: 61) and LBP-2. Specific complex peptide hormones in which C-terminal amidation is not preferred (in other words, where extension in the C-terminal region of the module is acceptable) include exendin-4, exendin-4 (1-28) (8E0 GO NO: 237) , CPY-1 (7-37) (8E0 GO NO: 204), the frog CPY-1 (7-36) (8EO GO NO: 283) and the frog CPY-2. However, if these complex peptide hormones are located in the C-terminal region of the hybrid polypeptide, they can optionally be amidated, and, in fact, preferably, can be optionally amidated.

The bioactive modules of the peptide hormone can be covalently linked by any method known in the art. Stable bonds or cleavable bonds may be used. In one embodiment, the carboxy in the first module may be directly linked to the amino of the second module. In another embodiment, linking groups may be used to attach the modules. Further, if desired, spacers or revolution inducers known in the art can be used to stabilize communication. As an example, if amidation of the C-terminal region of the Ν-terminal region of the bioactive module of the peptide hormone is undesirable, the module can be attached to the second module directly or using any suitable linking group known in the art, such as alkyl; PEG; amino acid, for example, bk, C1i, b-A1a; polyamino acid, for example, poly-Yk, poly-agd, poly-1uk, poly-a1a, C1u-Luk-Atd (SKR), etc .; a bifunctional linker (see, for example, Yegse CaEa1od, RoscTogb, II); aminocaproyl (Aca), β-alanyl, 8-amino-3,6-dioxaoctanoyl or other cleavable and non-cleavable linkers known in the art. Separately described

- 46 014647, as if each was explicitly described, variants of specific hybrids in which the linker, in each presented hybrid containing the linker, is replaced by the C1u linker. especially options in which the C1u linker is C1u-C1u-C1u. As an example, for the presented species, ²⁹ 5 AraEksendin (1-28) - ¹ back-Luk-y-Amilin (1-7) - ^11.18 Agd-kS! (8-27) -y-Amilin (33-37 ) (8ЕО ГО N0: 32) (see tables), their species analogue with the linker С1у is also separately included and described. Such species include ²⁹ 61u61u61u-Exendin (1-28) - ¹ beck-yuk-amilin (1-7) - ^11.18 Agd-kS! (8-27) th-Amylin (33-37) (8Е0 ГО N0: 313), where three glycines are located after the exendin sequence (1-28). In one embodiment, the linker or spacer consists of 1-30 residues, in another embodiment, from 2-30 residues, and in another embodiment, from 330, including any integer length of 2-30; where each whole unit is considered, for example 2, 3, 4, 5, 6, 7, etc. In one embodiment, a C1u linker is used, and in a specific embodiment, a linker of three residues C1u61u-61u is used.

If amidation of the C-terminal region of the Ν-terminal region of the bioactive module of the peptide hormone is desired, the module can be attached to the second module using any suitable linking group known in the art. More specifically, in the case where the bioactive module of the peptide hormone having at least one hormonal action is located in the C-terminal - Ν-terminal direction giving the amino amino bond, preferred linking groups include dicarboxylic acids, alkyls, PEG and amino acids such as Luc , Bitch and C1i.

As indicated above, hybrid polypeptides can also preferably include a spacer to further stabilize the bond of the bioactive modules of the peptide hormone. Any spacers or revolution inducers may be used. As an example, said β-turn mimetics include facial expression A and facial expression B, shown below, as well as dipeptides A1a-A1b and A1a-Pro. Their names are GORAS Mimic A: N- (38.68.98) -2-oxo-3-amino-1-azabicyclo [4.3.0] nonane-9-carboxylic acid. Mimic B: Ν (38.68.9B) -2-oxo-3-amino-7-thia-1-azabicyclo [4.3.0] nonane-9-carboxylic acid.

MIMICA facial expression V

Other typical combinations and specific options.

Typical combinations of bioactive modules of the peptide hormone for producing hybrid polypeptides in accordance with this invention include combinations of two or more bioactive modules of the peptide hormone selected from natural peptide hormones, analogues and derivatives of peptide hormones that exhibit at least one hormonal activity, fragments of natural peptide hormones that exhibit at least one hormonal activity, fragments of analogues or derivatives of peptide hormones that exhibit of at least one hormonal activity, and peptidic enhancers, with the proviso that at least one module has at least one hormonal activity.

Hybrid polypeptides in accordance with this invention include at least two bioactive modules of the peptide hormone, where each module consists of complex peptide hormones. In the context of this invention, the complex peptide hormones of the hybrid polypeptide may be the same or different, provided that at least two of the complex peptide hormones are different. In a preferred embodiment, at least two of the complex peptide hormones belong to different families of peptide hormones, for example, the amylin family, CCK, the leptin family, PPP, the proglucagon family, the natriuretic peptide family, the urocortin family peptides, for example, Isi-2 and Isi-3, peptides neuromedin families, for example, I25 neuromedin or spliced variants, and A№, B№, CPR or urodilatin, and CRP-1 exendin family.

In certain embodiments, the hybrid polypeptides of the invention may comprise two or more modules that exhibit at least one hormonal activity. For example, a hybrid polypeptide may comprise a fragment of a first peptide hormone or an analog that exhibits at least one hormonal activity covalently linked to a fragment of at least one other peptide hormone analogue. The additional fragment (s) may optionally have at least one hormonal effect. The first peptide hormone may be the same or different from the additional peptide hormone (s), provided that at least one of the additional peptide hormones is different from the first peptide hormone, and the first hormonal action may be the same or different from the optional additional hormonal action.

In other embodiments, the hybrid polypeptides of the invention may comprise one or more modules that exhibit at least one hormonal activity, in combination with one or more peptide enhancer modules. For example, a fragment of a first peptide hormone that exhibits at least one hormonal activity may be covalently

- 47 014647 is associated with a peptide enhancer, or a fragment of a first peptide hormone that exhibits at least one hormonal activity can be covalently linked to a second peptide hormone that exhibits at least one hormonal activity, which, in turn, is associated with a peptide enhancer.

Alternatively, the peptide enhancer may be located between the two modules of the peptide hormone as a stabilizing spacer. Again, the first peptide hormone may be the same or different from the second peptide hormone, and the first hormonal action may be the same or different from the second hormonal action.

In another embodiment, the hybrid polypeptides of the invention may contain two, three, four or more bioactive modules of the peptide hormone. Typical combinations include a hormone-acting module in combination with one, two, or three peptide enhancers; two hormone-acting modules in combination with one or two peptide enhancers; three modules with hormonal action in combination with one peptide enhancer, etc.

Compound peptide hormones are preferably selected from amylin, adrenomedullin, calcitonin, a peptide related to the calcitonin gene, intermedin, cholecystokinin, ΥΥ leptin peptide, glucagon-like peptide-1, glucagon-like peptide 2, oxintomodulin, ΑΝ₽, ΒΝ род ат д д д д urocortin family, for example, Isi2 and isp-3, peptides of the neuromedin family, for example, I25 neuromedin or spliced variants, and ΑΝ₽, ΒΝΓ, ί'ίΓ or urodilatin, or exendin-4.

More specifically, preferred combinations of modules include those containing combinations of exendin, amylin (and / or cST), ΒΝΈ and PΥΥ as complex peptide hormones. Particular combinations include combinations of exendin-4 / ΓΥΥ and PΥΥ / exendin-4, with or without spacers and linking groups. Other combinations include exendin / amylin and amylin / exendin combinations with or without spacers and linking groups. Other combinations include combinations of amylin / ΓΥΥ and PΥΥ / amylin with or without spacers and linking groups.

In one aspect, preferred combinations of modules include those that comprise a first module comprising exendin-4, an exendin-4 fragment that exhibits at least one hormonal activity, an analog or derivative of exendin-4 that exhibits at least one hormonal activity, or a fragment of an exendin-4 analog that exhibits at least one hormonal activity, in combination with at least one additional bioactive module of the peptide hormone. In one embodiment, the first module is associated with one, two, or three additional bioactive modules of the peptide hormone.

In preferred embodiments, a first module containing an exendin-4 peptide is coupled to a second bioactive peptide hormone module containing an amylin peptide (and / or cST) that exhibits at least one hormonal activity. In another embodiment, the second module is also associated with a third bioactive module of the peptide hormone containing a calcitonin peptide that exhibits at least one hormonal activity. In yet another embodiment, the third module may also be associated with a fourth bioactive module of the peptide hormone containing a peptide enhancer selected from amylin peptides. In one embodiment, the first module may be located in the Cnth region of the hybrid polypeptide. Alternatively, the first module may be located in the “terminal region of the hybrid polypeptide. In certain embodiments, spacers and linkers, such as βΑ1η. can be inserted, if desired, for binding modules.

Preferred exendin-4 peptides include exendin-4, exendin-4 (1-27) (8E0 ΙΌ ΙΌ0: 236), exendin-4 (1-28) (8 EC) ΙΌ Ν0: 237), ¹⁴ Ley, ²⁵ Rye exendin-4 (1-28) (8 EU) ΙΌ Ν0: 284) and 41 ^ / ^ 70 ^, 470-exendin-4 (1-28) (8Е0 ΙΌ Ν0: 240). Exendin (7-15) and its analogue 8et2, H8EOTET8E (8E0 ΙΌ Ν0: 378) are also used. Preferred amylin peptides that exhibit at least one hormonal activity include amylin, amylin fragments such as amylin (1-17) (8E0 ΙΌ Ν0: 214), amylin (1-16) (8Ep ΙΌ Ν0: 215), amylin (1-15) (870 ΙΌ Ν0: 216) and amylin (1-7) (870 ΙΌ Ν0: 217), and amylin analogues such as pramlintide, 41a-11-amylin (8E0 ΙΌ Ν0: 79), ² L1a -11-amylin (8Е0 ΙΌ Ν0: 80), and its fragments. Preferred calcitonin peptides that exhibit at least one hormonal activity include cct, fragments of cct, such as cct (8-10), cct (8-27) (8E0 ΙΌ Ν0: 288), and calcitonin analogs, such as ^{11, 18} Α ^ d-cST (8E0 ΙΌ Ν0: 108), '^ dg-cST (8E0 ΙΌ Ν0: 107), ¹⁴ O1i, ¹⁸ Α ^ d-cST (8E0 ΙΌ Ν0: 109), ¹⁴ O1i, ^11.18 Α ^ d-cST (8E0 ΙΌ Ν0: 110), and their fragments. Preferred amylin-based peptide enhancers include amylin (32-37) (8E0 ΙΌ Ν0: 242), amylin (3337) (8E0 ΙΌ Ν0: 243) and amylin (34-37) (8E0 ΙΌ Ν0: 244), and their analogues . Amylin-KST combinations used in accordance with this invention include those described in PCT / I82005 / 004 631, an amylin agonist, attorney dossier No. 18528.835, which is incorporated herein by reference. The chimera amylin-kST, especially useful for creating hybrids in accordance with this invention, includes compound 10 (described here and in PCT / I82005 / 004631) and its analogues and derivatives.

In one aspect, preferred combinations of modules include those containing exendin-4, an exendin-4 fragment that exhibits at least one hormonal activity, ana

- 48 014647 log or derivative of exendin-4, which exhibits at least one hormonal activity, in combination with a peptide enhancer. Preferred exendin-4 compounds include exendin-4, exendin-4 (1-27) (8EO GO ΝΟ: 236), exendin-4 (1-28) (8ES) GO ΝΟ: 237), ¹⁴ Leu, ²⁵ Rye exendin -4 (1-28) (8ETs GO ΝΟ: 284) and ⁵ A1a, ¹⁴ Lei, ²⁵ Rye-exendin-4 (1-28) (8ETs GO ΝΟ: 240). Preferred peptide enhancers include PΥΥ (25-36) (8EO GO ΝΟ: 257), PΥΥ (30-36) (8EO GO ΝΟ: 262) and PΥΥ (31-36) (8EO GO ΝΟ: 263). In one embodiment, the first module is located at the C-terminal region of the hybrid polypeptide and the peptide enhancer is located at the Ν-terminal region of the hybrid polypeptide. Alternatively, the first module may be located at the Ν-terminal region of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal region of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as βA1a. can be inserted, if desired, for connecting modules.

In another embodiment, preferred combinations of modules include those that have a first module containing exendin-4, an exendin-4 fragment that exhibits at least one hormonal activity, an analog or derivative of exendin-4 that exhibits at least one hormonal activity, or a fragment of an exendin-4 analogue that exhibits at least one hormonal activity, in combination with a second module containing SSC, a SSC fragment that exhibits at least one hormonal activity, an analog or pro a CCK derivative that exhibits at least one hormonal activity, or a fragment of a CCK analog that exhibits at least one hormonal activity. Again, preferred exendin-4 compounds include exendin-4, exendin-4 (1-27) (8EC GO ΝΟ: 236), exendin-4 (1-28) (8EC GO ΝΟ: 237), ¹⁴ Eyey, ²⁵ Rye exendin-4 (1-28) (8EO GO ΝΟ: 284), ⁵ A1a, ¹⁴ Eey, ²⁵ Rye-exendin-4 (1-28) (8EO GO ΝΟ: 240) and ¹⁴ Lye-exendin-4 (1- 28) (8ETS GO ΝΟ: 190). Preferred CCK compounds include CCK-8 and CCK8 (Τ1κ (ί.Ή28Ο ₃ ,)). In one embodiment, the first module is located in the C-terminal region of the hybrid polypeptide and the second module is located in the Ν-terminal region of the hybrid polypeptide. Alternatively, the first module may be located at the Ν-terminal region of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal region of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as βΛ1; · ι. can be inserted, if desired, for connecting modules.

In another embodiment, preferred combinations of modules include those that comprise a first module comprising amylin, an amylin fragment that exhibits at least one hormonal activity, an amylin analog or derivative that exhibits at least one hormonal activity, or an amylin analog fragment that exhibits at least one hormonal activity, in combination with a second module comprising a peptide enhancer, such as PΥΥ (25-36) (8EC GO ΝΟ: 257) or PΥΥ (30-36) (8EC GO ΝΟ: 262). In one embodiment, the first module is located at the C-terminal region of the hybrid polypeptide and the peptide enhancer is located at the Ν-terminal region of the hybrid polypeptide. Alternatively, the first module may be located at the Ν-terminal region of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal region of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as bA1a, can be inserted, if desired, to connect the modules. In another embodiment, a preferred combination of modules includes those that contain a first module, amylin, an amylin fragment that exhibits at least one hormonal activity, an amylin analog or derivative that exhibits at least one hormonal activity, or an amylin analog fragment that exhibits at least one hormonal activity, in combination with a second module comprising a peptide enhancer, such as PΥΥ (25-36) or PΥΥ (30-36). In one embodiment, the first module is located in the C-terminal region of the hybrid polypeptide and the peptide enhancer is located in the кон-terminal region of the hybrid polypeptide. Alternatively, the first module may be located at the Ν-terminal region of the hybrid polypeptide and the peptide enhancer may be located at the C-terminal region of the hybrid polypeptide. In certain embodiments, spacers or linkers, such as bA1a, can be inserted, if desired, to connect the modules.

Other preferred combinations of modules include combinations of exendin and CCK or amylin, calcitonin and CCK as a tertiary composition. Particular combinations include exendin / CCK and CCK / exendin, with or without the use of spacers and linkers and linking groups. Other combinations include CCK / amylin / calcitonin and CCK / amylin / calcitonin / amylin, with or without the use of spacers and linking groups. Each module may independently be a peptide enhancer, or may have a hormonal effect, depending on the desired properties of the hybrid polypeptide. In one embodiment, amylin / calcitonin / amylin is presented as the chimera amylin / calcitonin / amylin, such as compound 10.

Other preferred combinations of modules include combinations of exendin, amylin and calcitonin in the form of tertiary and tetrahybrid molecules. Typical combinations include exendin / amylin / calcitonin combinations; exendin / amylin / calcitonin / amylin; amylin / calcitonin / exendin; and amylin / calcitonin / amylin / exendin, with or without the use of spacers or linking groups. Every mo

- 49 014647 barrel independently can be a peptide enhancer, or may have a hormonal effect, depending on the desired properties of the hybrid polypeptide. In one embodiment, amylin / calcitonin / amylin is presented as the chimera amylin / calcitonin / amylin, such as compound 10.

In one embodiment, if one of the bioactive modules of the peptide hormone that exhibits at least one hormonal activity is amylin or its analogue or fragment, and the second bioactive module of the peptide hormone includes CCK, then the hybrid polypeptide should preferably contain a third bioactive module of the peptide hormone selected from various complex peptide hormones. An exemplary third bioactive module of the peptide hormone includes calcitonins, more preferably salmon calcitonin, analogs and fragments thereof.

In another embodiment, if one of the bioactive modules of the peptide hormone that exhibits at least one hormonal activity is amylin, or its analogue, or fragment, and the second bioactive module of the peptide hormone includes CT, then the hybrid polypeptide should preferably contain a third bioactive module of the peptide hormone selected from various complex peptide hormones. A typical third bioactive module of the peptide hormone includes exendin-4, its analogues or fragments.

In yet another embodiment, if one of the bioactive modules of the peptide hormone that exhibits at least one hormonal activity is CPB-1 or an analogue or fragment thereof, and the second bioactive module of the peptide hormone is a peptide enhancer containing an exendin fragment, then the hybrid polypeptide should preferably contain third bioactive module of the peptide hormone. A typical third bioactive module of the peptide hormone includes ΡΥΥ, including its analogues, derivatives and fragments, and CCK including its analogues, derivatives and fragments.

It is understood that in each of the combinations described here, a reference to a complex peptide hormone includes links to its analogues, derivatives, fragments, as well as peptide enhancers related to it.

In a preferred aspect, the hybrid polypeptides include

ZEY Yu N0: one Exendin-4-ΡΥΥ (22-36) 2 Exendin-4-ΡΥΥ (25-36) 3 Exendin-4-ΡΥΥ (18-36) 4 Exendin-4-pA1a-ZA1a-RUU (22-36) 5 Exendin-4-pA1a-pA1a-RUU (25-36) 6 Exendin-4-pA1a-₽A1a-RUU (31-36) 7 Exyondin-4 (1-28) -ΡΥΥ (22-36) 8 Exendin-4 (1-28) -ΡΥΥ (25-36) nine Exendin-4 (1-28) -ΡΥΥ (18-36) 10 Exendin-4 (1-28) -pA1a-pA1a-RUU (22-36) eleven Exendin-4 (1-28) -pA1a-pA1a-RUU (25-36) 12 EXENDIN-4 (1-28) -βΆΙΑ-βΑΙΑ-ΡΥΥ (31-36) thirteen ⁵ A1a, ¹⁴ Lei, “P11e-Exendin-4 (1-28) -ΡΥΥ (18-36) 14 ^s A1a, ei ^{14, Re-2b} Exendin-4 (1-28) -ΡΥΥ (22-36) fifteen ⁵ A1a, ¹⁴ yoy7 ²⁵ RIyo-Exendin-4 (G-28) -ΡΥΥ (25-36) sixteen ^b A1a, ¹⁴ bj, ²⁵ bb-exendin-4 (1-17) -ΡΥΥ (18-36) 17 ⁵ A1a, ¹⁴ Ley, ²⁵ Pye-Exendin-4 (1-28) -PA1a-pA1a-RUU (22-36) eighteen ⁵ D1a, ¹⁴ Ley, ²⁵ Rke-Exendin-4 (1-28) -βΑΙθ-βΑΙθ-ΡΥΥ (25-36) nineteen ⁵ A1a, ¹⁴ Ley, ²⁵ Rye-Exendin-4 (1-28) -pA1a-pA1a-RUU (31-36) twenty Exendin-4-SSK-8 21 Exendin-4 (1-28) -SSK-8 22 Exendin-4 (1-28) -SSK-8 (Pb (CH ₂ ZO ₃ )) 23 Exendin-4 (1-28) - (8-amino-3,6-dioxoctanoyl) -SSK-8 24 Exendin-4 (1-28) - (8-amino-3,6-dioxaoctanoyl) -SCC-8 (Pb (CH ₂ ZO ₃ )) 25 Exendin-4 (1-27) -NAmilin (1-7) - ^{11 '1a} Arg-ECT (8-27), amylin (33-37). 26 Ekoendin-4 (1-27) - ² ' ⁷ A1a-L Amylin (1-7) -StStB-10)

- 50 014647

27 ^ Y 12 Άάο-Exendin (1-28) -L Amylin (1-7) - 1.18 Agd-5C £ (8-27) L Amilin (33-37) 28 29 12 Ayo-Exendin (1-28) -Mez-Euz-L Amilin (1-7) - 11.18 ArgdCS (8-27) -L Amilin (33-37) 29th 2a dioksaoktanoil-3,6-Exendin (1-28) -Amilin (1-7) - n 'is Arg-18 zS1: (8-27) -Amilin (33-37) thirty gu 3, 6-dioksaoktanoil-Exendin (1-2 8) -'Mez-uz-IAmilin (1- 7), etc. '18 Arg-zSO (8-27) -Amilin (33-37) 31 29 5 Ara-Exendin (1-28) -L Amylin (1-7) - 11 '“Agd-sSD (8-27) L Amylin (33-37) 32 2nd 5 Ara 'ExendNein 71-28) -Nes-bzChlmilin (1-7) - 1118 ArgdSs (8-27) -L Amilin (33-37) 33 23 ₽A1a-₽A1a-Ekaendin (1-28), L Amilin (1-7) - 11.18 Agd-sCb (8-27) -L Amilin (33-37) 34 2Y £ A1a-pA1-Exendin (1-23) -Mez-Lua-L Amilin (1-7) and ' 1e Ard-sC £ (8-27) -Amilin (33-37) 35 2a 4,7,10-trioxa-13-tridecanamine succinimidyl Exendin (1-28) -L Amylin (1-7) - 11, 18 Arg-3Cb (8-27) L Amylin (33-37) the ^ 4, Ί, 10-trioxa-13-tridecanamine succinimidyl- Exendin (1-28) -Mez-b3-Amylin (1-7) - 11.18 Agd-cCb (8-27} b-Amylin (33-37) 37 SSK-8-6KV ~ 1! 5 61i ~ L Amylin (1-17) - 18 ygd-sST (18-26) amylin (32-37) 38 amylin (1-18) -ΡΥΥ (19-36) 39 isocaproyl-ZTAU- (A1b) -K (formyl) -ZOE- (A1b) - K (formyl) -BOT-ΡΥΥ (18-36) 40 isocaproyl-3TAU- (A1b) -K (formyl) -B5 <2E- (A1b) - K (formyl) -B-ΡΥΥ (16-36) 41 SSK-8- [Succinoyl-Suz] -ΡΥΥ (3-36) 42 SSK-8- [Βΐε-Souz (Ν-Acetyl)] - ΡΥΥ (3-36) 43 SSK-8- [C1u-Aminoxymethylcarbonyl] -ΡΥΥ (3-36) 379 Exendin-4 (1-27) -A amylin (1-7) - 14 01i , 11.1a Agd-sST (8-27) amylin (33-37) 380 aa 12 Αάο-Exendin (1-28) -L Amylin (1-7) - 14 C1i, 1a ArgcSL (8-27) -L Amylin (33-37) 381 ^ y 12 Αάο-Exendin (1-28) -Mez-b3-Amylin (1-7) - 14 C1i, 11.13 Agd-sST (8-27) -L Amilin (33-37) 382 2a 3,6-dioxaoctanoyl-Exendin (1-28) -L Amylin (1-7) - 14 <31i, 11.18 Agd-sCl: (8-27) -L Amylin (33-37) 383 2a dioksaoktanoil-3,6-Exendin (1-28) -'Zez-uz-Amilin (1- 7), 14, 61i, 11 '18 Arg-zS1 :( 8-27) -Amilin (33-37) 384 29 5 Ara-Exendin (1-28) -L Amylin (1-7) - 14 C1i, 1111a Agd-cSb (8-27) -L Amylin (33-37) 385 ^ at 5 Ara-Exendin (1-28) -Mez-b3-Amylin (1-7) - 14 C1i, 11 (1b Arg-sCb (8-27) -B amylin (33-37) 386 29 8A1a-pA1a-Exendin (1-28), L Amylin (1-7) - 14 01i , 11.1a ArgcSb (8-27) -L Amylin (33-37) 387 23 ZA1a-₽A1a-Exendin (1-28) - 1 des Luz-Amilin (1-7) - 14 Cli, 11.18 Agd-5Sb (8-27) -L Amilin (33-37) |

388 ^2Y gΊ 4 _{g of} 10-trioxa-13-succinimidyl tridekanamin Exendin (1-28) -Amilin (1-7) - ¹ '01, and ^Arg-11,18 8SKH (8-27) Amilin (33-37) 389 ^{2 !,} 4,7,10-trioxa-13-tridecanamine succinimidyl Exendin (1-28) -Mez-Euz-L Amilin (1-7) -ZZ.i, ^11.18 ArgdSb (8-27) -L Amilin (33 -37) 390 ^CCK-8-S1i-1b yKY- Amilin (1-17) - _^"g 6Tn- ^1a Arg-FTA (18-26), amylin (32-37)

Typical exendin and neuromedin hybrids include

Exendin- (1-28) -BeLa-A1a-LeBa-A1a-EY-38:

NSE6ETET30b5K0MEEEAUKG1EIEKI-beta-A1a-beta-A1aΓΒΓΗΥ5ΚΤ0ΚΣΘΚ5ΝννΕΕΒ05Ρϊ · Α305Κ6ΥΕΧΕ’Κ.ΡΚΝ-ΝΗ2 (ЗЕ <2 Ю N0: 391);

Exendin- (1-28) -beta-A1a-beta-A1a-neuromedin (Ts25 :)

NESESTTZOZZMEEEKUKG1ENKN-beta-A1a-beta-A1aΓΒνϋΕΕΓ05ΡΓΑ503Κ6ΥίΊιΓΚΡίίΝ-ΝΗ2 (ЗЕО Ю N0: 392); and

Exendin- (1-28) -beta-A1a-beta-A1a-Neuromedin (P-9): H6ESTRT50ZKOMEEEA ¥ ВГ1НЬКЫ-beta-А1а-beta-А1а-0 ¥ ГГКРКНΝΗ2 (ЗЕО 10 N0: 393).

The beta-A1a-beta-A1a spacer is optional and can be replaced by a C1u-C1u-C1u spacer, a mini-PEG group, or another linker known in the art, especially the one described herein.

- 51 014647

Exemplary exendin and natriuretic peptide hybrids include exendin-HBIR peptide hybrids, including

Exyondin - (1-28) -beta-A1a-beta-A1a-bbp:

NESESTGGZOZK0MEEEUKG1EIIKI-beta-A1a-beta-A1aЗРКМУ0СЗССГСККМОК133533Ь5СКУЬККН (ЗЕО Ю N0: 394); and

Exendin-beta-A1a-beta-A1a-11BMR: NESESTGT50ZZK0MEEEUNUNGTEIKYS6RZZSAR₽RZbeta-A1a-beta-A1aZRKM70eZSSRSCKM0K153336SSKUKKN (ZE3) 39E5.

As with all hybrids of the invention, the beta-A1a-beta-A1a spacer is optional and may be substituted by O1y-O1y-O1y, a mini-PEG group or other linker known in the art, especially as described herein .

Hybrid polypeptides in accordance with this invention may also contain further modifications, including, but not limited to, substitution, deletion and insertion into the amino acid sequence of such hybrid polypeptides, and any combination thereof. In a preferred embodiment, the hybrid polypeptides of the invention comprise one or more modifications of a non-essential amino acid residue. In the context of this invention, a non-vital amino acid residue is a residue that can be modified, i.e. deleted or substituted, in the natural human amino acid sequence of a fragment, for example, a fragment of a complex peptide hormone, without canceling or not significantly reducing the agonistic effect of the hybrid polypeptide on the receptor of the complex peptide hormone.

Preferred substitutions include conservative amino acid substitutions. A conservative amino acid substitution is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain or physicochemical characteristics (e.g., electrostatic parameters, hydrogen bonding, isosteric, hydrophobic characteristics). Families of amino acid residues having similar side chains are known in the art. Such families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side sections (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, methionine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenyl alanine, tryptophan, histidine).

This invention also relates to derivatives of hybrid polypeptides. Such derivatives include hybrid polypeptides conjugated to one or more molecules of a water-soluble polymer, such as polyethylene glycol (PEG) or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, octanoyl, etc.), or obtained by the addition of polyamino acids, such as poly , poly-agd, poly-1uk and poly-A1a. Modifications of hybrid polypeptides may also include low molecular weight substituents, such as short alkyls and restricted alkyls (e.g., branched, cyclic, fused, adamantyl) and aromatic groups. The water-soluble polymer molecules preferably have a molecular weight of from about 500 to about 20,000 Daltons.

Such modifications by conjugation with the polymer and low molecular weight substituents can be carried out singularly at the Ν or C-terminal region or on the side chains of amino acid residues in the sequence of hybrid polypeptides. Alternatively, there may be a plurality of derivative sites in the hybrid polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine may provide additional sites for derivatization, see, for example, US Pat. Nos. 5,824,784 and 5,824,778. Preferably, hybrid polypeptides can be coupled to one, two, or three polymer molecules.

The molecules of the water-soluble polymer are preferably linked to an amino, carboxyl or thiol group and may be linked through the Ν or C-terminal region, or to the side chains of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, water-soluble polymer molecules may be coupled to diamine and dicarboxylic groups. In a preferred embodiment, the hybrid polypeptides of the invention are coupled to one, two, or three PEG molecules via the epsilon amino group of the lysine amino acid.

Derivatives of the hybrid polypeptide in accordance with this invention also include hybrid polypeptides with chemical changes of one or more amino acid residues. Such chemical changes include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization. Chemical changes can be carried out singularly at the Ν or C-terminal regions or in the side chains of amino acid residues in the PPP sequence of hybrid polypeptides. In one embodiment, the C-terminal region of such peptides may have free-ΟΗ or -ΝΗ ₂ groups. In another embodiment, the Ν-terminal region may be covered by an isobutyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group,

- 52 014647 with an ethoxycarbonyl group, an isocaproyl group (isocap), an octanyl group, an octyl glycine group (O (0c!)) Or an 8-amino-octanoic acid group. In a preferred embodiment, the cyclization can be carried out through the formation of disulfide bridges. Alternatively, a plurality of chemical change sites may exist in a hybrid polypeptide.

In another embodiment, hybrids do not include any hybrids described in \ 0 2005/077072. Therefore, in one embodiment, new hybrids are claimed. In another embodiment, a new use is claimed, as described herein, of any of the hybrids described herein in \ 0 2005/077072 or elsewhere. Examples of hybrid polypeptides in accordance with this invention are presented in the list of sequences, and are also discussed in the experimental part of the description.

The use of hybrid polypeptides for the treatment or prevention of conditions and metabolic disorders

Hybrids in accordance with this invention can be used to reduce food intake, reduce appetite, reduce calorie intake, induce a feeling of fullness, reduce the absorption of nutrients, cause a decrease in body weight, affect the composition of body tissues, change the energy supply or energy consumption of the body, improve lipid profile (including lowering levels of bb cholesterol and triglycerides and / or changes in levels of bb cholesterol), slowing gastrointestinal motility, slowing gastric emptying, reducing changes in blood glucose after eating, preventing or inhibiting the secretion of glucagon and lowering blood pressure. In one embodiment, such hybrids contain exendin, OR1, amylin and / or cct part.

Thus, in certain embodiments, the hybrids of the invention are used to treat or prevent conditions or disorders that can be alleviated by reducing the absorption of nutrients, which involves administering to a patient a therapeutically or prophylactically effective amount of a compound of the invention. Such conditions and disorders include, but are not limited to, eating disorders, insulin resistance, obesity, abnormal postprandial hyperglycemia, any type of diabetes, including Ι type, ΙΙ type and diabetes during pregnancy, metabolic syndrome, dropping cider, hypertension, dyslipidemia , cardiovascular disease, hyperlipidemia, attacks of sleep apnea, pulmonary hypertension, cholecystitis and osteoarthritis. In one embodiment, such hybrids contain exendin, OR1, amylin and / or cST parts.

Typical conjugations of the peptide module include cardioactive / protective peptides, for example urocortin with OR1-1 or exendin, A #, B # or CAP with OR-1 or exendin, and urocortin with A #, ΒNP or C #. Such hybrids are cardioprotective and are especially useful for the related diseases and conditions described here, including acute or chronic CHE, ischemic reperfusion, myocardial infarction, and also have a vasodilating effect and are useful for the treatment or prevention of hypotensive indications and angina pectoris. Use 2 and 3 are especially useful in hybrids in accordance with this invention.

Non-limiting examples of a cardiovascular condition or disease include hypertension, myocardial ischemia, and myocardial reperfusion. The compounds of this invention can also be used in the treatment or prevention of other conditions related to obesity, including stroke, cancer (e.g., cancer of the endometrium, breast, prostate and colon), gall bladder disease, sleep apnea, decreased fertility and osteoarthritis (see Luchshsk! e! a1., At. Rat. Ryuk. 63: 2185, 2001). In other embodiments, the compounds of this invention can be used to alter the composition of body tissues for aesthetic purposes, to improve the patient’s physical abilities, or to provide a leaner diet. Hybrids are used to change the composition of body tissues through a decrease in fat mass without a significant decrease in muscle mass, which gives the desired loss of fat mass while maintaining muscle mass. In one embodiment, such hybrids contain exendin, OR1, amylin and / or cct part.

In another general aspect, hybrids of the invention can be used to inhibit ghrelin secretion. Therefore, the compounds of this invention can use this mechanism to treat or prevent ghrelin-related disorders such as Prader-Willy cider, all types of diabetes and their complications, obesity, hyperphagia, hyperlipidemia, or other overeating disorders. In one embodiment, such hybrids contain exendin, OR1, amylin and / or cct part.

In another general aspect, it has been found that hybrids containing amylin and / or cST parts can be used to treat or prevent Barrett's esophagus, gastroesophageal reflux disease (GERD) and related conditions. Such conditions may include, but are not limited to, heartburn, heartburn, accompanied by a discharge of stomach / intestines into the mouth or lungs, difficulty swallowing, coughing, intermittent wheezing and inflammation of the vocal folds (conditions associated with GERD), esophageal erosion, and esophageal ulcer , esophageal stricture, Barrett metaplasia (replacing the normal epithelium of the esophagus with abnormal epithelium), Barrett adenocarcinoma, and lung aspiration. Such hybrids have antisecretory properties, such as inhibition of

- 53 014647 dic acids, inhibition of bile acids and inhibition of pancreatic enzymes. Moreover, such hybrids may have a gastroprotective effect, which makes them especially useful in the treatment or prophylaxis of Barrett's esophagus and / or GERD and related or related conditions described herein.

In another general aspect, hybrids can also be used to treat or prevent pancreatitis, pancreatic carcinoma and gastritis, especially for the treatment and prevention of pancreatitis in patients who have undergone endoscopic retrograde cholangeopancreatography (ERCP). Hybrid agonists containing amylin and / or §ST may have an unexpectedly excellent therapeutic effect when combined with somatostatin. Therefore, in certain embodiments, methods for treating or preventing pancreatitis include administering such hybrids and administering somatostatin and somatostatin agonists to the patient.

In another general aspect, hybrids are used to reduce bone resorption, reduce plasma calcium and cause analgesic effects, especially for the treatment of bone disorders such as osteogenesis disturbance and osteoporosis. In other embodiments, hybrids are used to treat pain and painful neuropathy. In one embodiment, such hybrids contain exendin, CRP1, amylin and / or §ST part.

In another aspect of the present invention, methods of treating or preventing obesity are provided, wherein the method comprises administering a therapeutically or prophylactically effective amount of a hybrid polypeptide to a patient in need thereof. In a preferred embodiment, the patient is obese or overweight. Although obesity is usually defined as a body mass index in excess of 30, for the purposes of this description, any patients, including those who have a body mass index less than 30 but who need or want to reduce body weight, are included in the definition of obesity. Patients who have insulin resistance, glucose intolerance or any form of diabetes mellitus (for example, type 1, type 2 diabetes or during pregnancy) can use this method. In one embodiment, such hybrids contain exendin, CRP1, amylin and / or §ST part.

In yet another embodiment, a method for reducing weight in patients with morbidly obese is presented, which includes first reducing the patient's weight to a level lower than morbidly obese, then administering to the patient a combination of anti-obesity agents in amounts effective to further reduce the patient's weight. Ways to reduce patient weight below pathological obesity include reducing calorie intake, increasing physical activity, drug therapy, bariatric surgery, such as bypass gastric anastomosis, or any combination of the above methods. In one aspect, administering a combination of anti-obesity agents further reduces the weight of the patient. In another embodiment, methods for reducing a body mass index in a patient having a body mass index of 40 or less are provided by administering a combination of anti-obesity agents in effective amounts to further reduce the weight of the patient.

By weight loss is meant that the patient loses part of his / her total body weight during the treatment process, where the course of treatment can last days, weeks, months or years. Alternatively, weight loss can be defined as a decrease in the proportion of fat to muscle mass (in other words, the patient loses fat mass, but maintains or increases muscle mass, optionally with a corresponding loss of total body weight). An effective amount of anti-obesity agents administered in conjunction with this invention is an amount effective to reduce the patient’s body weight during treatment, or, alternatively, an amount effective to reduce the percentage of fat mass of the patient during treatment. In certain embodiments, the patient’s body weight during treatment is reduced by at least about 1%, at least about 5%, at least about 10%, at least about 15%, or at least about 20%. Alternatively, the percentage of fat mass in the patient is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%.

In yet another aspect of the present invention, methods of reducing food intake, reducing nutrient absorption, causing weight loss, affecting body tissue composition and changing energy reserves or increasing energy expenditure, treating diabetes mellitus and improving lipid profile (including lowering EBL cholesterol and triglycerides) are provided. and / or a change in the NLO levels of cholesterol), where the methods include administering to the patient an effective amount of a hybrid polypeptide in accordance with this invention. In a preferred embodiment, the methods of the invention are used to treat or prevent conditions or disorders that can be alleviated by reducing the absorption of nutrients in a patient in need thereof, comprising administering to said patient a therapeutically or prophylactically effective amount of a hybrid polypeptide of the invention. 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, CRP1, amylin and / or §ST part.

Not wishing to be limited by theory, it is believed that the effect of introduced peripherally hybrid polypeptides in accordance with this invention on the reduction of food intake, on the slowdown

- 54 014647 gastric emptying. to reduce the absorption of nutrients and weight loss is determined by interactions with one or more separate classes of receptors in or similar classes of receptors of the PP family. More specific. believe. that the receptor or receptors are involved. similar to PΥΥ-preferred (or Υ7) receptors.

Of particular interest are hybrids. used against obesity. for weight loss. to reduce food intake. to increase the rate of measurement of substances and to reduce fat mass and / or to redistribute fat mass. which include at least one. preferably two components. which act on the central nervous system. Separate areas of the forebrain (derivatives of the cerebrum and diencephalon constituting the brain) and the rhomboid brain or brain stem (including the midbrain. Varolian bridge and medulla) were identified. as involved in controlling energy balance. Structures of the forebrain or nucleus. located in the hypothalamus. involved in food intake and / or modulation of body weight. include. eg. arc nuclei (ANN). paraventricular nuclei (RUH). dorsomedial hypothalamus (BMN). ventromedial nucleus (UMN) and the nucleus of the lateral hypothalamus (LNA). Structures or nuclei of the rhomboid. located in the brain stem. those involved in food intake and / or modulation of body weight include. eg. single path cores (X8T). the posterior field of the rhomboid fossa (AR). lateral parabrachial nuclei (1PBX). The nuclei of the rhomboid. which control the elements of the final control system. also controlled by projection of the first or second order from such areas of the head stem. both N8 ^ AR and 1RVU deserves attention then. what was shown. that ar. N81 and 1PBN (together or separately) have their own integrative abilities.

Many CNS-related anti-obesity agents act on such forebrain structures. located in the hypothalamus. involved in food intake and / or modulation of body weight. Besides. CNS-related anti-obesity agents act on the structures of the rhomboid. located in the brain stem. involved in food intake and / or modulation of body weight. Examples of such anti-obesity agents are described herein. see the table below. which presents examples of modules of peptide families. which may be combined to produce a hybrid anti-obesity agent and may be combined to produce a hybrid anti-obesity agent. acting on the forebrain and rhomboid. Such components include. eg. antagonists of the neuropeptide receptor Υ1 (NPΥ1). NPΥ5 receptor antagonists. leptin and leptin agonists. ciliary neurotrophic factor (ATS) and agonists of an ATS. peptide ΥΥ (₽ΥΥ) and PΥΥ agonists. exendin and exendin agonists. CPP-1 and CPP-1 agonist. ghrelin and ghrelin antagonists. cholecystokinin (CCK) and CCK agonists and amylin amylin agonists. including those described here. Other components of the peptide family and clinical methodology are described in PCT / I806 / 17529, co-pending patent application filed by this applicant. which is incorporated herein by reference.

Separate obesity goals and location

Signal system CNS area Role in food absorption Anti-obesity agents Neuropeptide Υ (ΝΡΥ) Forebrain (AKC / ΡνΝ) Increases absorption receptor antagonists ΝΡΥ1 and ΝΡΥ5 Leptin Forebrain (AKC) Reduces absorption Leptin or agonists Ciliary Neurotrophic Factor (SAT) Forebrain (ARS) Reduces absorption SKTE Peptide ΥΥ (ΡΥΥ) Forebrain (AKC) Reduces absorption agonists ΡΥΥ (3-36) Glucagon-like peptide-1 (CPB-1) Forebrain (ΡνΝ) Reduces absorption Exenatide and other CPI-1 ligands, ΌΡΡ-ΐν inhibitors Ghrelin Forebrain (AKC) Increases absorption Ghrelin antagonists Cholecystokinin (SSK) Rhomboid Brain (AR) Reduces absorption Agonists SSK Amilin Rhomboid Brain (AR) Reduces absorption Amylin agonists, pramlintide, amylin analogues Melanocortins (MS) Forebrain (ΡνΝ / AKC) Agonists reduce absorption Agonists MS4

In certain embodiments, the hybridoma is an anti-obesity agent. which may include one or more components of the peptide family. predominantly acting in the forebrain. In other embodiments. a hybrid is an anti-obesity agent. which may include one or more anti-obesity agents. acting. predominantly. to the forebrain. Examples of peptide families

- 55 014647 probes and components include an ΝΓΥ1 receptor antagonist, a ΝΓΥ5 receptor antagonist, a leptin or agonist or leptin analog, CNTE, a аг2 receptor agonist (e.g., PΥΥ (3-36) or a PΥΥ (336) agonist, an exendin or agonist, or an exendin analog , OR-1 or an agonist or analog of OR-1, a ghrelin antagonist, CCK or an agonist or an analog of CCK and an amylin or agonist or an analog of amylin.

In certain embodiments, the hybrid and the method of its use include the first component, which is mainly aimed at the centers of the hypothalamus, which are responsible for the energy balance, such as Α ^ ΒΗ, ₽ν, νΜ and НН. In one embodiment, the hybrid contains one or more other components of the peptide family, which is also aimed at the hypothalamus, but at a different location, or has a different mechanism of action, unlike the first component. If the hybrid contains more than one other component of the peptide family, and it also targets the hypothalamus, more than one other component of the peptide family can be directed to the same place and have the same mechanism of action as the first, or they can be directed to different places and / or have different mechanisms of action. In another embodiment, the hybrid, if desired, contains one or more other components of the peptide family, which gives one or more additional therapeutic effects, including effects on obesity through a site or mechanism of action different from the first component and each other, control of blood glucose, cardioprotection and / or control of hypertension. In certain embodiments, an additional component of the peptide family is one that is predominantly directed towards the centers of the forebrain responsible for the energy balance, such as Ν8Ϊ, AP, and 1ΓΒΝ.

In certain embodiments, the hybrid and the method of its use include the first component, which is mainly aimed at the centers of the forebrain responsible for the energy balance, such as ΝδΧ AR and 1ΓΒΝ. In one embodiment, the hybrid contains one or more other components of the peptide family, which is also directed to the hypothalamus, but to a different place and through a different mechanism of action than the first component and each other. In another embodiment, the hybrid contains one or more other components of the peptide family, which gives one or more additional therapeutic effects, including acting on obesity through a site or mechanism of action different from the first component and each other, monitoring blood glucose levels, cardioprotection and / or hypertension control. In certain embodiments, an additional component of the peptide family is one that is primarily aimed at the hypothalamus, which are responsible for the energy balance, such as LΒC, RJ νΜ and LN.

As used herein, an anti-obesity agent that acts on the structure of the forebrain involved in food intake and / or modulation of body weight, stimulates or inhibits the activity of certain areas, such as a specific nucleus and / or neural circuit, in the forebrain. Such stimulation or suppression of the forebrain leads to a decrease in the absorption of nutrients by the body. An anti-obesity agent that acts on the structure of the rhomboid brain, involved in food intake and / or modulation of body weight, stimulates or inhibits the activity of certain regions, for example, a specific nucleus and / or neural chain, in the rhomboid. Such stimulation or suppression of the diamond-shaped brain leads to a decrease in the absorption of nutrients by the body.

In another aspect, methods of reducing fat mass by increasing a patient's metabolic rate are provided, wherein the methods include administering a hybrid anti-obesity in amounts effective to reduce fat mass by increasing a patient's metabolic rate. Fat mass can be expressed as a percentage of total body weight. In some aspects, fat mass is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% in the course of treatment. In one aspect, the patient’s muscle mass is not reduced during treatment. In another aspect, the patient’s muscle mass is maintained or increased during treatment. In another aspect, the patient is on a low-calorie diet or a restricted diet. By a low-calorie diet is meant that the patient takes fewer calories per day than with the patient’s usual diet. In one embodiment, the patient takes at least 50 calories less per day. In other embodiments, the patient takes 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 calories less per day.

In one embodiment, methods are presented that are used to alter the distribution of fat, reduce fat mass, or both, in a patient. Therefore, patients for whom a change in the composition of body tissues is favorable can also use these methods. Changing the composition of body tissues in this description includes the loss or maintenance of body fat while minimizing loss, maintenance or muscle building. In such cases, the weight may increase or decrease. Therefore, patients may be thin, overweight or obese in the sense in which these terms are used in the art. Presents methods also include reducing fat in non-adipose tissue, while maintaining muscle mass. Applications for this method include diseases such as non-alcoholic steatohepatitis (ΝΑ & Π) or lipodystrophy.

In one embodiment, a method for changing a fat distribution in a patient is provided, wherein the method comprises administering an anti-obesity hybrid in amounts effective to alter the distribution of fat

- 56 014647 in the patient. In one aspect, the change is due to an increased metabolism of visceral or ectopic fat, or both, in the patient. Under the distribution of fat understand the location of body fat in the body. Such fat deposits include, for example, subcutaneous, visceral or ectopic fat deposits. Under the subcutaneous fat is understood, the deposition of fat immediately below the surface of the skin. The amount of subcutaneous fat in a patient can be measured using any method available to measure subcutaneous fat. Methods for measuring subcutaneous fat are known in the art, for example, described in US Pat. No. 6,530,886, which is incorporated herein by reference in its entirety. Ectopic body fat refers to body fat in and around the tissues and organs that make up the lean body mass (e.g., skeletal muscle, heart, liver, pancreas, kidneys, blood vessels). Usually ectopic body fat is called the accumulation of fat outside the classic body fat deposits. Visceral fat is understood to mean fat deposition as an intra-abdominal adipose tissue. Visceral fat surrounds vital organs and can be metabolized in the liver to produce blood cholesterol. Visceral fat is associated with an increased risk of conditions such as polycystic ovary syndrome, metabolic syndrome and cardiovascular disease. In some embodiments, the method comprises metabolizing visceral or ectopic fat, or both, at a rate of at least about 5, 10, 15, 20, 25, 30, 40, or 50% more than subcutaneous fat. In one aspect, the methods provide a favorable distribution of fat. In one embodiment, a favorable distribution of fat is an increased ratio of subcutaneous fat to visceral fat, ectopic fat, or both. In one aspect, the method includes increasing muscle mass, for example, as a result of an increase in muscle cell mass.

In another embodiment, methods for reducing the amount of subcutaneous fat in a patient are provided, wherein the method comprises administering to a patient in need thereof an anti-obesity hybrid in amounts effective to reduce subcutaneous fat in a patient. In one case, the amount of subcutaneous fat in a patient is reduced by at least about 5%. In other cases, the amount of subcutaneous fat in a patient is reduced by at least about 10, 15, 20, 25, 30, 40, or 50% compared with the condition of the patient before the introduction of the anti-obesity hybrid.

The methods described herein can be used to reduce visceral fat in a patient. In one case, the amount of visceral fat in a patient is reduced by at least about 5%. In other cases, the amount of visceral fat in a patient is reduced by at least about 10, 15, 20, 25, 30, 40, or 50% compared with the condition of the patient before the introduction of the anti-obesity hybrid. Visceral fat can be measured by any methods available to determine the amount of visceral fat in a patient. Such methods include, for example, abdominal tomography using CT scintigraphy and NMR. Other methods for determining visceral fat are described, for example, in US patent No. 6864415, 6850797 and 6487445.

In one embodiment, a method is presented for preventing the accumulation of ectopic fat or reducing the amount of ectopic fat in a patient, the method comprising administering to a patient in need thereof an anti-obesity hybrid in amounts effective to prevent the accumulation of ectopic fat or to reduce the amount of ectopic fat in a patient. In one case, the amount of ectopic fat in a patient is reduced by at least about 5% compared with the condition of the patient before the introduction of the anti-obesity hybrid. In other cases, the amount of ectopic fat in a patient is reduced by at least about 10% or at least about 15, 20, 25, 30 40, or 50%. Alternatively, the amount of ectopic fat is proportionally reduced by 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100% compared with the subcutaneous fat in the patient. Ectopic fat in a patient can be measured using any method available to measure ectopic fat.

In another embodiment, methods for achieving a more patient-friendly distribution of fat are provided, wherein the method comprises administering to the patient a hybrid that is effective as an anti-obesity agent in an amount effective to achieve a favorable distribution of fat. In one embodiment, administering an anti-obesity hybrid reduces the amount of visceral fat or ectopic fat, or both, in a patient. In one embodiment, an anti-obesity hybrid is introduced that contains at least one family module that acts on the forebrain structures involved in food intake or modulation of body weight, or both with at least one family module that acts on the rhomboid structures involved in food intake or modulation of body weight, or both. In one embodiment, the methods advantageously reduce the amount of visceral or ectopic fat, or a combination thereof, to a greater extent than they reduce subcutaneous fat. Such methods give a greater ratio of subcutaneous fat to visceral fat or ectopic fat. Such improved ratios can help 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 rate of 5% more than subcutaneous fat. In other embodiments, ectopic or visceral fat is metabolized at a rate of at least 10, 15, 20, 25, 30, 50, 60, 70, 80, 90, or 100% more than subcutaneous fat.

- 57 014647

Of particular interest for the treatment methods described here associated with obesity, body weight and fat composition are hybrids containing modules of the amylin family (for example, the amylin-kST-amylin chimera), leptin and / or PPE (for example, ΓΥΥ analogues or the RRUGORU chimera) . For example, a module of the amylin family can be attached to a module of the leptin family, and administered either separately, or, in another embodiment, administered in combination (eg, alone or as a mixture) with a compound of the PPE family. In another embodiment, the hybrid contains a combination of leptin-PPE, which is administered alone or in combination with a compound of the amylin family. In another embodiment, the hybrid comprises an amylin-PPE combination that is administered alone or in combination with a compound of the leptin family. In yet another embodiment, the hybrid contains all three modules of the peptide families. For example, the amylin-PPE hybrid can be presented in the form of a sterile, pharmaceutically acceptable solution, which is then used to dissolve the lyophilized or powdered compound of the leptin family, as in a two-chamber delivery system.

In yet another aspect, a method is provided for administering a therapeutically effective amount of a hybrid effective as an anti-obesity agent administered in combination with glucocorticosteroids. Glucocorticosteroids have the side effect of increasing fat mass and decreasing muscle mass. Therefore, it is contemplated that a combination of an anti-obesity agent can be used in conjunction with glucocorticosteroids under conditions in which the use of a glucocorticosteroid is useful to counteract the side effect of glucocorticosteroid.

Further, with regard to lipid reduction, the hybrids of the invention are used to lower blood lipids, such as triglycerides, total cholesterol, cholesterol cholesterol and VLDL cholesterol, and to provide a more favorable lipid profile in patients when such treatment is necessary. Therefore, in one embodiment, a method is presented for lowering blood triglycerides, total cholesterol, Lb cholesterol, VLDL cholesterol, or any combination thereof, comprising administering to a patient in need thereof a lipid-lowering hybrid. In another embodiment, the lipid-lowering hybrid may comprise a component of the exendin (incretin) family, a component of the amylin family, a component of the PPE or ΓΥΥ (ΝΈΥ) family, or any combination thereof. In one embodiment of the method, the reduced lipid is plasma triglyceride, in another embodiment, plasma cholesterol, in another embodiment, LbO cholesterol, in another embodiment, VLDL cholesterol. A hybrid may be dramatically and / or continuously effective to maintain or reduce fasting lipid levels and / or reduce lipid movement (especially triglyceride) after a meal. A patient in need of such treatment may have elevated cholesterol, elevated VLDL cholesterol, or any combination thereof. Such patients may include those who, without external signs, have diabetes or are in a prediabetic state, are obese, have a lipid disease or condition such as dyslipidemia, hypercholesterolemia or hypertriglyceridemia, and / or have cardiovascular disease. This effect of hybrids is beneficial in reducing cardiac and atherosclerotic risk in patients at high risk, for example, who are genetically predisposed, are obese, have diabetes, etc. Thus, the patient may be a patient suffering from atherosclerosis with elevated levels of lipid or cholesterol. This method is a method of reducing triglyceride movements after eating, reducing circulating lipid levels, treating dyslipidemia, improving circulating lipid profile, treating hypertriglyceridemia, treating hypercholesterolemia and / or lowering triglyceride concentrations after a patient’s food intake, which comprises administering an effective amount of the hybrid in accordance with this invention to a patient in need of such treatment. As used herein, a lipid profile means the balance, proportion or actual concentration of circulating lipids, including triglyceride, HOB, Lb, cholesterol, etc. In a preferred aspect, the methods of the invention are used to reduce triglyceride levels in a patient, and include administering an effective amount of an amylin or amylin agonist. Total lipid or triglyceride levels can be reduced using this method, for example, fasting levels, peak levels after eating and movement of the total lipid / triglyceride level after eating (for example, measured by the area under the curve (AIS), the increase in triglyceride after eating by compared with an increase in a condition not treated with an amylin agonist). Certain clinically important indicators, such as fasting lipid levels (including triglyceride, cholesterol, NEB and LEO, etc.) and lipid after ingestion (e.g., triglyceride) are also reduced when using the methods in accordance with this invention. Patients with dyslipidemia or altered lipid levels compared to normal can be treated with amylin or amylin agonists. Patients with diabetes or obesity, as well as those who are genetically predisposed to dyslipidemia or cardiovascular disease, are particularly suitable for treatment by the methods in accordance with this invention. As used herein, treating elevated triglyceride levels in a patient means preventing the increase in these levels or lowering such levels relative to the levels before treatment. In this description, a decrease in triglyceride movements after a meal means a decrease in peak concentration and the total area under the triglyceride concentration curve that patients receive after a meal. This usually means a decrease in total area.

- 58 014647 di under a curve in a graph such as that shown in FIG. 2-4 within an hour after eating. As used herein, a decrease in circulating lipid levels in a patient means a decrease in a measurable amount of blood lipids compared to the level before treatment. As used herein, the treatment of dyslipidemia means improving or recovering to a level, ratio, profile or balance close to that defined in medicine as normal and / or healthy, of any or all of the clinically measurable parameters of a lipid or lipoprotein. They include, but are not limited to, triglyceride levels, Lb, Hb, LBF, VLDL, total cholesterol, apolipoproteins, etc. As used herein, improving a patient’s circulating lipid profile means a change in the concentration of one or more lipids found in the blood to change the patient’s total lipid content to a preferred state. This definition also includes a shift in the distribution of lipids in relation to different fractions of lipoprotein without changing the total content / concentration of circulating lipids. As used herein, treating a patient’s hypertriglyceridemia means lowering the concentration of triglycerides found in the patient’s blood at one or more appropriate times, for example, on an empty stomach or after eating. In this description, a decrease in circulating triglycerides after a patient’s food intake means a decrease in a measurable amount of triglycerides in the circulatory system after a meal (for example, about 4-6 hours after a meal) relative to the level of such lipids after a meal in patients with or without treatment when taking the same kind of food.

Hybrids and chimeras of RUU in accordance with this invention can be used with other drugs that reduce lipids. Lipid lowering drugs include any compound capable of lowering plasma lipid levels. Examples of lipid lowering drugs include, but are not limited to, statins, such as atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin or rosuvastatin; bile acid binding agents such as cholestyramine or colestipol; peroxisome proliferator activated receptor agonists (PPH), such as tezaglitazar, vitamin E; cholesterol ester transfer protein inhibitors (CETP) such as ezetemibe, LT-705 and torcetrapib.

Additional tests used in accordance with this invention include those that determine the effect of hybrid compounds on the composition of body tissues. Examples of analyzes include those that use a model of metabolic disease in mice with alimentary obesity (ΌΙ0). Prior to treating male mice, C57BB / 61 mice are fed a high-fat diet (# 12331, 58% calories from fats; Kekeags O1e! K, 1ps.) For 6 weeks starting at 4 weeks of age. During the study, the mice continue to receive a high fat diet. During the study, water is provided ab 11Y! Um. One group of non-obese mice of the same age receives a low-fat diet (# Ό12329, 11% calories from fat) in order to compare metabolic parameters with ΌΙ0 groups.

ΌΙ0 mice may be implanted with subcutaneous (PC) intra-osmotic osmotic pumps to deliver either a vehicle (50% dimethyl sulfoxide (DMSO) in water) or a compound of the invention. The pumps of the latter group can be configured to deliver any quantity, for example, 1000 μg / kg / s of the compound in accordance with this invention within 7-28 days.

Body weight and food intake are measured at regular intervals throughout the study period. Respiratory rate (DC runningaway defined as C0 ₂ -r O ₂ consumption) and metabolic rate can be determined using indirect calorimetry intact animals (0hutah, So1itik 1pk1gntep1k. So1itik, OH). Mice are killed by an overdose of isoflurane and the obesity ratio (weight of the bilateral epididymal fat body) is measured. Moreover, before determining the epididymal weight, the composition of the body tissues (muscle mass, fat mass) of each mouse can be analyzed using the En1 Epegdu X-hau Alkogrutute! Gu (OEXA) apparatus according to the manufacturer's instructions (Lipag P1x1tik, CE 1tadtd Zuk! em). In the methods of the invention, preferred hybrid polypeptides of the invention are those that are effective in one of the assays described herein (preferably assays for food intake, gastric emptying, pancreatic secretion, weight loss or body tissue composition), which exceeds the effectiveness of the complex peptide hormone in the same analysis.

In addition to alleviating hypertension in patients in need thereof, by reducing food intake, weight loss or treating obesity, the compounds of this invention can be used to treat hypotension.

The compounds of this invention can also be used to potentiate, induce, improve, or restore glucose susceptibility in pancreatic islets and cells. Such actions may be useful for the treatment or prophylaxis of conditions associated with metabolic disorders, such as those described above and in US patent application No. IZ20040228846. Assays to determine such activity are known in the art. For example, US Published Patent Application Publication No. IZ20040228846 (fully incorporated by reference herein) describes assays for isolating islets and culture, as well as for

- 59 014647 determining the maturity of germinal islets. In the examples of US patent application No. 820040228846, intestinal hormonal peptides, including pancreatic polypeptide (1111), neuropeptide Υ (ΠΈΥ), neuropeptide K (NPK), 6ΥΥ, secretin, glucagon-like peptide-1 (CP1-1) and bombesin are bought at 81dta. Type XI collagenase is obtained from 81 dt. СредуPMI 1640 culture medium and fetal bovine serum are obtained from Cbt. A radioimmunoassay kit containing an anti-insulin antibody ([ ¹²⁵ T] -RIA kit) is purchased from Mpto, 8! 6οι.ιί5.

The islet of rat islets in the postpartum period is carried out in one-year-old R-02 rats. The islands of adult rats take rats aged 6-8 weeks. The islands of rat embryos are taken as follows. Pregnant female rats are killed at gestational age E21. A fetus is removed from the uterus. 10-14 segments of the pancreas are excised from each litter and washed twice in Hanks buffer. Pancreas segments are combined, suspended in 6 ml of 1 mg / ml collagenase (type XI, 81dta) and incubated at 37 ° C for 8-10 minutes with constant shaking. Cleavage is stopped by adding 10 volumes of Hanks ice buffer followed by three washes with Hanks buffer. The islets are then purified with a Ficoll gradient and cultured in 10% fetal bovine serum (PBS) / PMI medium with or without the addition of 1 μM HMX. At the end of the fifth day, 20 islets are manually collected in each tube and analyzed for static insulin secretion. In general, the islets are first washed with KKR buffer and then incubated with 1 ml KKR buffer containing 3 mM (low) glucose for 30 min at 37 ° C with constant shaking. After collecting the supernatant, the islands are incubated with 17 mM (high) glucose for 1 h at 37 ° C. Insulin secretion with low or high glucose stimulation is analyzed by radioimmunoassay (RIA) using the [ ¹²⁵ T] -RIL kit. E21 germinal islets are cultured for 5 days in the presence of 200 ng / ml PΥΥ, PP, CCK, cattle, секрет, secretin, CPB-1 or bombesin.

A typical w ν шνο analysis is also carried out using male rats 2iskeg O1aBe! 1s Rabu (ΖΌΡ), an inbred (> P30 generation) rat model that spontaneously causes diabetes for all Ga / Ga males that receive standard food for rodents Rsgpa 5008. In males ΖΌΡ Ga-Ga hyperglycemia begins to develop around the age of seven weeks with glucose (feed) levels typically reaching 500 mg / DL from 10 to 11 weeks old. Insulin levels (feed) increase during the development of diabetes. However, at the age of 19 weeks, insulin drops to about the level of thin control rat pups. Triglyceride and cholesterol levels in obese rats are usually higher than in lean rats. In the analysis, three groups of 7-week-old rats ΖΌΡ, each of which includes 6 individuals, were infused via a Λ6 · ΖΛ pump for 14 days: 1) control vehicle, 2) and 3) РΥΥ in two different doses, 100 and 500 pmol / kg / h, respectively. Four measurements are taken before and after infusion on days 7 and 14: 1) plasma glucose level, 2) plasma insulin level and 3) plasma triglycerides (TG) level, and oral glucose tolerance testing (0CTT) is also carried out. Accordingly, these assays can be used to test the desired effect of the compounds of this invention.

Other uses contemplated for hybrid polypeptides include methods for reducing the concentration of aluminum (A1) in the central nervous system (see US Pat. No. 6,734,166, incorporated herein by reference in its entirety) for the treatment, prevention, or delay of Alzheimer's disease. Assays for determining the effect on A1 are known in the art and can be found in US Pat. No. 6,734,166 using diploid and T§ mice. These mice are separately placed in exchange or polypropylene chambers of brand No. v ^ 1'ι® and given three days to get used to the chambers before starting the experiment. The mice are given free access to food (LaBl1e! ® MN Ka! And My £ £ / Λи! Ο 6Р5К52, 8 !. Γοιι® Мо.) And water during the experiment, except for 16 hours before sacrifice, when food is not given. Mice are given daily subcutaneous injections of either the active compound or saline. Mice are euthanized at the end of 13 days in one experiment and 3 days in another and samples are taken. Mouse brain samples were weighed in pure Teflon pads and prepared for analysis by microwave digestion in nitric acid with a low trace content. Then, the samples are analyzed for A1 content using inductively coupled plasma mass spectrometry (Pibab e! A1., Atak οΓ C11n1sa1 apb ΓηόοηΙοΓν 8с1epse 25, 3, 264-271 (1995)). Work with all tissues during analysis is carried out in a clean room using HEPA air filtration systems to minimize background pollution. Hybrids in accordance with this invention are used for the prevention and treatment of nephropathy, including hypertensive and diabetic nephropathy and nephropathy associated with insulin resistance and metabolic syndrome. Hybrids achieve these goals by, inter alia, improving or preventing the deterioration of hypertension, endothelial function, renal function, and glomerulosclerosis. In one embodiment, the invention provides a method for preventing or treating nephropathy or nephropathy associated with insulin resistance, comprising administering a compound of the invention. Hybrids can also be used to improve endothelial function in patients with reduced vasodilating functional activity, or having glomerulosclerosis or any other decrease in glomerular current. Such an improvement in endothelial function serves both to reduce hypertension and to improve the function of glomerular capillaries. In additional

- 60 014647 variants of the molecule in accordance with this invention are used to prevent the development of nephropathy to ΕδΚΌ, to prevent, slow the development, treatment or alleviation of proteinuria and / or glomerulosclerosis. Hybrids are used to reduce the risk of occurrence, prevention or treatment of cardiac arrhythmias. Hybrids can have an antiarrhythmic effect in patients with cardiac ischemia, cardiac ischemia-reperfusion, and congestive heart failure. For example, ORI-1 has been found to reduce heart damage and improve recovery in patients with these disorders. The incretins, including ORB-1, are glucose-dependent insulinotropic hormones. OR-1 and exendin effectively improve peripheral glucose uptake without causing dangerous hypoglycemia. They also strongly suppress the secretion of glucagon regardless of their insulinotropic effect, and thereby powerfully reduce plasma free fatty acid (FFA) levels to a greater extent than insulin does. High FFA levels are the main toxic mechanism during myocardial ischemia. In another embodiment, hybrids are used for the prevention and treatment of cardiac arrhythmias, which significantly reduces the damage associated with reperfusion and ischemia, and improves patient recovery. In yet another embodiment, treating with hybrids after an acute stroke and hemorrhage, preferably by intravenous administration, is a means to optimize insulin secretion by increasing brain anabolism, improving insulin efficiency by suppressing glucagon, and maintaining normoglycemia or mild hypoglycemia without the risk of acute hypoglycemia or other negative side effects. . In one embodiment, such hybrids contain a portion of OR1 or exendin. In another embodiment, the module of the OP1 family or exendins is combined with the module of the natriuretic peptide family, the amylin family peptide, the urocortin family peptide, to provide improved treatment or prevention of cardiovascular conditions or diseases, including SIR, as described herein.

Congestive heart failure is one of the most significant causes of morbidity and mortality in developed countries. It occurs as a late manifestation in various cardiovascular diseases characterized by loss of contractile tissue and / or volume overload or excessive pressure (Roglipo, Yureyepkup 38: 1406-1412 (2001)). Numerous studies have suggested that cardiac correction is the main determinant during CRF, regardless of its etiology (Rebak, Satbuyuaxiat Ra1bod1od 14: 1-11 (2005)). Therefore, cardiac correction is an attractive target in the treatment of congestive heart failure. As such, agents that prevent or reduce cardiac correction are desirable. In fact, the need for molecules that can weaken cardiac correction has been identified in the literature (Roglipo, Yaurepekkup 38: 1406-1412 (2001)). Literature indicates that weakening of ventricular correction also increases survival after myocardial stroke, although treatments that worsen correction have been associated with poorer outcomes, even if they have improved systolic function (see δοtakipbagat, Meb. S1sh. At. 88 : 1193-1207 (2004)).

Therefore, methods of treating cardiovascular disease are presented, in one embodiment, heart failure, acute or chronic, in another embodiment, myocardial infarction, in another embodiment of ischemic heart failure, and in another embodiment, congestive heart failure. In one embodiment, such treatment is achieved by preventing or alleviating damage to the cardiovascular system caused by hyperglycemia. In one embodiment, such treatment is achieved by a cardioprotective effect. In another embodiment, such treatment is achieved by prevention, delayed onset, weakening or facilitating cardiac correction. In general, cardiac correction refers to a change in structure and a change in the shape of any of the chambers of the heart. In one embodiment, cardiac correction refers to a change in structure and a change in the shape of the ventricles. As described above, and not wanting to be limited by theory, cardiac correction can be described as genomic changes in the myocardium after a stroke, followed by cellular and interstitial changes, leading to a change in the structure and shape of the heart chambers. Such a change in structure and shape change can manifest itself clinically as changes in the size, shape and function of the heart. Cardiac correction may occur in response to any stimulation or combination of myocardial stimuli. In one embodiment, cardiac correction is the result of a myocardial stroke. By way of non-limiting example, cardiac correction may occur in response to a myocardial stroke due to myocardial infarction, hypertension, volume overload (e.g., with aortic regurgitation), infection, inflammation, diabetes, viral cardiomyopathy, and idiopathic cardiomyopathy.

In one aspect, cardiac correction is prevented, slowed, weakened or facilitated by the introduction of a hybrid in accordance with this invention. A hybrid may have the ability to alleviate (improve) at least one of the following heart parameters: diastolic function of the left ventricle, E-wave to A-wave ratio, final diastolic pressure in the left ventricle, cardiac output, cardiac contractility, left ventricular mass, weight ratio the left ventricle to body weight, the volume of the left ventricle, the volume of the left atrium, the size of the left ventricle in a state of diastole or systole, the size of a heart attack, the ability to tolerate physical activity and whether any indicator of systolic and / or diastolic function of the heart; or weaken, slow down or prevent the enlargement of the heart chambers or the negative impact on

- 61 014647 any of the above parameters of the heart. In one embodiment, the hybrid comprises a member of the incretin family, for example, exendin-4, which binds to the CPB-1 or exendin receptor. In the context of the presented methods, the prevention or amelioration of cardiac correction may include reducing cardiac correction to any extent. In one embodiment, prophylaxis or relief of cardiac correction is associated with a reduced risk of congestive heart failure.

In one embodiment, cardiac correction is facilitated or decreased to less than about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the degree of cardiac correction in the absence of a hybrid. In another embodiment, the cardiac correction can be slightly reduced, moderately reduced, significantly reduced or significantly eliminated compared with cardiac correction that occurs in the absence of the introduction of the hybrid. In this description, a slight decrease in cardiac correction refers to a decrease in cardiac correction of about 25% or less compared with cardiac correction that occurs when there is no introduction of a hybrid. A moderate decrease in cardiac correction refers to a decrease in cardiac correction of about 50% or less compared to cardiac correction that occurs when there is no introduction of a hybrid. A significant reduction in cardiac correction refers to a decrease in cardiac correction of about 80% or less compared to cardiac correction that occurs when there is no introduction of a hybrid. Significant elimination of cardiac correction refers to a reduction in cardiac correction of 80% or more compared to cardiac correction that occurs in the absence of a hybrid.

Any means available to a person skilled in the art can be used to assess the degree of prophylaxis, relief, weakening, or slowdown of cardiac correction. For example, cardiac correction can be assessed using tests that include, but are not limited to, histological examination of the heart, LV mass, or, during a patient’s life, by measuring chamber size and wall thickness and movement, such as echocardiography, or by measuring the diastolic function of the left ventricle (LV) using the ratio of the peak velocity of the E wave and A wave (E / A ratio).

In one embodiment, the patient, who can benefit from the introduction of the hybrid for the prevention, relief, weakening or slowing of heart correction, can be determined by a specialist taking into account the conditions and risk factors specific to the patient. In one embodiment, the patient may need to be prevented, relieved, weakened or slowed down by heart correction. In another embodiment, prophylaxis, relief, amelioration, or slowdown of cardiac correction may be desirable for the patient. One of the risk factors may be a genetic predisposition of the heart to cardiac correction. Examples of samples and patients for the methods provided herein include those that have suffered, suffer, or have a risk of a condition associated with cardiac correction. A condition associated with cardiac correction can be any condition or disorder in which it is known that cardiac correction occurs or there is a risk of cardiac correction. Conditions associated with cardiac correction include, for example, myocardial infarction, inflammation, ischemia / reperfusion, oxidative stress, pulmonary heart, deep glycation end products, abnormal heart wall tension, sympathetic stimulation, myocarditis, hypertension, viral cardiomyopathy, idiopathic cardiomyopathy, transplant and heart surgery.

As indicated above, the hybrid can be introduced in acute or chronic condition. Regardless of whether the condition is acute or chronic, the methods provided herein include continuous treatment of hybridomas. Thus, the duration of ongoing treatment may include the time when the attack has already passed and the patient is considered to be in a state of recovery from an acute attack or recovery from a chronic condition.

Continuous administration or treatment of hybridomas to prevent, attenuate, slow down, or alleviate cardiac correction may be warranted unless a specific transient attack or transient condition associated with cardiac correction has been identified. Continuous administration involves administering the hybrid for a continuous but indefinite period of time based on a general predisposition to cardiac correction that is not transient (for example, a condition that is not transient, may not be identified or cannot be transformed, such as diabetes). The hybrid may be administered continuously in the methods provided herein for the prevention of cardiac correction in a patient who has congestive heart failure, regardless of etiology. The continuous introduction of a hybrid for the prevention or relief of heart failure can also be used in diabetics who are at risk of congestive heart failure. The hybrid can also be administered on an ongoing basis to preserve the transplanted organ in patients undergoing heart transplantation. If the hybrid is administered continuously, administration can continue for any period of time. However, continuous administration often takes place over an extended period of time. For example, in a typical embodiment, continuous administration lasts for 6 months, 1 year, 2 years or longer.

In another embodiment, methods are presented that improve the contractility of the heart. Improving cardiac contractility may include Socra cardiomyocyte ability

- 62 014647 To evaluate the improvement in cardiac contractility, any method of assessment can be used. For example, clinical observation, such as an increase in cardiac output or a decrease in heart rate, may be the basis for determining increased cardiac contractility. Alternatively, w νί \ Ό, increased contractility of the heart can be assessed by determining the increased partial contraction of the left ventricle. The partial contraction of the left ventricle can be determined by any available means, such as an echocardiograph. When assessing the increased contractility of the heart, an increase in the partial contraction of the left ventricle can be any increase compared with partial seduction before the introduction of the hybrid. For example, the increase in reduction may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more than about 200%.

In yet another aspect, a method for reducing or preventing atrial correction by administering a hybrid is provided. The reduction or prophylaxis of atrial correction can be evaluated in comparison with atrial correction before the introduction of the hybrid. The therapeutic effect of such a reduction or prevention of atrial correction includes reducing atrial fibrillation. In yet another aspect, a method for reducing or preventing ventricular correction by administering a hybrid is provided. Reduction or prevention of ventricular correction can be evaluated in comparison with the correction of the ventricle before the introduction of the hybrid.

In another aspect, therapeutic and prophylactic methods are provided. Treatment is considered on a short-term and ongoing basis. In addition, treatment on a short-term basis may be extended to treatment on an ongoing basis, if indicated. Continuous treatment is considered as treatment for more than 2 weeks. In certain embodiments, ongoing treatment may be longer than 1 month, 3 months, 6 months, 1 year, 2 years, 5 years, or throughout life. In one aspect, a method for treating or preventing a condition associated with cardiac correction in a patient in need thereof is provided. The method generally includes administering to the patient an amount of a hybrid effective to prevent or ameliorate cardiac correction, where the condition associated with the correction is thereby improved, prevented, or slowed down. As described here, the introduction of the hybrid can be carried out by any method, including the introduction of other agents that have a favorable cardiovascular effect.

In yet another embodiment, the methods provided herein also include identifying a patient in need of treatment. Any effective criteria can be used to determine whether the patient will benefit from the introduction of the hybrid. Diagnostic methods for heart disease and / or diabetes, for example, as well as methods for identifying patients at risk for these conditions, are well known to those skilled in the art. Such techniques may include a clinical examination, a physical examination, a personal interview, and a family history assessment.

In yet another embodiment, hybrids that are capable of decreasing insulin resistance or increasing insulin sensitivity are used to treat polycystic ovary syndrome (PC08). The introduction of hybrids in accordance with this invention can reduce or prevent insulin resistance in a patient suffering from PC08. In yet another embodiment, hybrids prevent the onset of type 2 diabetes in a patient suffering from PC08. Other hybrids can restore regular menstruation, ovulation, or fertility in patients with PC08. In one embodiment, such hybrids contain a portion of OP1 or exendin to bind and activate the OP1 receptor.

Compounds in accordance with this invention have a wide range of biological effects, some relate to their antisecretory and antioxidant properties. The compounds can inhibit secretion in the gastrointestinal tract through direct interaction with epithelial cells or, possibly, by inhibiting the secretion of hormones or neurotransmitters that stimulate secretion in the intestine. Antisecretory properties include inhibition of the secretion of the stomach and / or pancreas and can be used to treat or prevent diseases and disorders including gastritis, pancreatitis, Barrett's esophagus and gastroesophageal reflux disease.

The compounds in accordance with this invention are used to treat any number of gastrointestinal disorders (see, for example, Natkop'k Rgshs1p1ek o £! P1et1 Μο ^ ο ^, ΜсО ^ аν-НШ МСО, Ναν Wogk, 12111 Her.), Which associated with excessive secretion of electrolyte and water in the intestine, as well as with reduced absorption, for example, infectious diarrhea, inflammatory diarrhea, shortened colon syndrome or diarrhea, which usually occurs after surgery, such as ileostomy. Examples of infectious diarrhea include, but are not limited to, acute viral diarrhea, acute bacterial diarrhea (e.g., with salmonella, campylobacter infection and clostridia, or due to protozoal infections), or traveler's diarrhea (e.g., with Norvalk virus or rotavirus). Examples of inflammatory diarrhea include, but are not limited to, malabsorption syndrome, tropical diarrhea, chronic pancreatitis, Crohn's disease, diarrhea, and cider of an irritated colon. It was also found that the peptides in accordance with this invention can be used to treat acute or life-threatening situations associated with a gastrointestinal disturbance, for example, after surgery or in cholera.

- 63 014647

The compounds of this invention can also be used to treat or prevent bowel damage, as opposed to only treating symptoms associated with bowel damage (e.g., diarrhea). Such intestinal damage may result from ulcerative colitis, inflammatory bowel disease, colon atrophy, loss of colon mucosa and / or loss of function of the colon mucosa (see \ 0 03/105763, incorporated herein by reference in its entirety). In analyzes of such an action, as described in \ 0 03/105763, male Nb3 rats are used at the age of 11 weeks, weighing 250-300 g, which are contained in a 12:12 cycle of alternating light and dark, and which give access to 11b1! Um to standard food for rodents (Tek1ab LM 485, Mab1kop, \ Ι) and water. Animals are not fed for 24 hours before the experiment. A simple and reproducible rat model of chronic inflammation of the colon and colon was previously described by Motk O.R., e! A1., Nar! eptbiseb tobe1 oG sygoshs tPattayop apb i1segayop t! ye ha! coop. Oak! Goyep! His1odu. 1989; 96: 795-803. It demonstrates a relatively long-lasting inflammation and ulceration, which makes it possible to study the pathophysiology of inflammation of the colon and colon in a certain controlled manner and to evaluate new methods of treatment that are supposedly used for inflammation of the colon in humans.

Rats are anesthetized with 3% isofluorane and placed on a temperature-controlled pillow at 37 ° C. The probe needle is inserted rectally into the colon by 7 cm. Trinitrobenzenesulfonic acid (TNBS) hapten dissolved in 50% ethanol (vol./about.), Is fed into the colon cavity through the probe needle at a dose of 30 mg / kg, in a total volume of 0 0.4-0.6 ml, as described by Maheip, e! A1., 1i! op No.gu Zuk !. 1998; 73:38 45. The control groups receive physiological saline (No. C1 0.9%) inside the colon.

Four days after the induction of colitis, the colon is excised in anesthetized rats, which are then killed by decapitation. The weight of the excised colon and spleen is measured and samples of the colon are photographed to assess the total morphological damage. Inflammation is defined as areas of redness and thickening of the colon wall.

The hybrid polypeptides of the invention can also be used to treat or prevent pancreatic tumors (e.g., inhibit the proliferation of pancreatic tumors). Methods in accordance with this invention include reducing the proliferation of tumor cells. Types of benign pancreatic tumor cells that can be treated in accordance with this invention include serous cyst adenomas, microcystic tumor, and solid cystic tumor. The method is also effective in reducing the proliferation of pancreatic cancer malignant cells, such as carcinomas arising from ducts, acini, or pancreatic islets. US Pat. No. 5,574,010 (incorporated herein by reference in its entirety) provides representative assays for testing antiproliferative properties. For example, the '010 patent states that PANC-1 and M1aPaCa-2 are two colonies of human pancreatic adenocarcinoma cells, which are commercially available from suppliers such as Atepsap Ture Cuyige Co11ec! Two colonies of tumor cells are grown in the culture medium KPM640 with the addition of 10% fetal calf serum, 29.2 mg / l glutamine, 25 μg gentamicin, 5 ml penicillin, streptomycin and fungicone solution (1KN Vyukaepsek, Lepekha, Kapk.) At 37 degrees Celsius in a NARC0 incubator with a water jacket of 5% CO ₂ . All cell colonies are separated by the addition of 0.25% trypsin (C1ope! 1sk, Zap P1edo, Saig.) Once or twice a week, when a confluent monolayer of tumor cells is obtained. Cells are pelleted by centrifugation for 7 min at 500 d in a cooled centrifuge at 4 degrees Celsius, and resuspended in trypsin-free enriched culture medium KPMI 1640. Viable cells are counted on a hemocytometer from trypan blue microscopic specimens.

10,000, 20,000, 40,000 and 80,000 cells of each type are added to 96-well microculture plates (Sok! Ag, Satbpbde, Mack.) In a total volume of 200 μl of culture medium per well. The cells are allowed to adhere for 24 hours before the addition of PΥΥ or the test peptide. Fresh culture medium is changed before adding peptides. The incubation of pancreatic tumor cells with PΥΥ or the test compound is continued for 6 hours and 36 hours. PΥΥ is added to the cells at doses of 250 pmol, 25 pmol and 2.5 pmol per well (N = 14). The test compound is added to cell cultures at doses of 400 pmol, 40 pmol and 4 pmol per well. 2 μl of 0.9% saline solution was added to control wells to simulate the volume and physical excitation of glued tumor cells. Each 96-well plate contains 18 control wells for comparison in each plate during the experiment. 96-well plates were repeated 6 times with different concentrations of PΥΥ and test compound for both PANC-1 and M1aPaCa-2 cells.

At the end of the incubation period, 3- (4,5-dimethylthiazolyl-2-yl) -2,5-diphenyltetrazolium bromide, MTg tetrazolium bromide (Z1dta, Z !. Loshk, Mo.) are added to the fresh culture medium at a concentration of 0.5 mg / ml The culture medium is changed and tumor cells are incubated for 4 hours with MTT tetrazolium bromide at a temperature of 37 ° C. At the end of the incubation, the culture medium is aspirated. The precipitate of formazone crystals was dissolved in 200 μl of dimethyl sulfoxide (Z1dta, Z !. Loshk, Mo.). The calculation of solubilized formazone is carried out by obtaining absorption indicators at a wavelength

- 64 014647 500 nm on an EIB8A geabeg reader (Mo1ci1ag Eeu1sec, Mep1o Ragk, Ca11 £.). In the MTT assay, dehydrogenase activity is measured depending on mitochondrial NADH, and this assay is considered one of the most sensitive and reliable methods for quantifying the w ν response of tumor cells to chemotherapy (A11еу, М.С., е! А1., Sapseg Kek., 48 : 589-601, 1988; Sagtbae1, I., e! A1., Sapseg Kek., 47: 936-942, 1987; MsNa1e, A.R., e! A1., Sapseg Keb., 41: 315-321 , 1988; apb 8ax! Op, K.E., e! A1., I. Cl. B.Lakeg Meb. apb 8igd., 10 (5): 331-336, 1992). The analysis of absorbance at 550 nm is carried out by grouping cells with the same test conditions and comparing the differences that arise between the control and treatment with different concentrations of the peptide by one-way analysis of variance (ANVA).

A typical w νί \ Ό analysis is also presented. Human pancreatic duct adenocarcinoma M1a Rasa-2 was tested for inhibition of growth by using peptide ΥΥ and the test compound. From 70,000 to 100,000 human M1a RaCa-2 cells are orthotopically transplanted into 48 male athymic mice. After one week, the animals are treated with either ΡUU or the test compound at a dose of 200 pmol / kg / h using mini-osmotic pumps for four weeks. Paired cultures receive physiological saline. After killing, the size and mass of the tumor are measured. Control mice have significant growth of human cancer in the pancreas, as evidenced by histological sections. After 9 weeks, 90% of the control mice had a significant metastatic tumor. Tumor mass was reduced by 60.5% in mice treated with the test compound and 27% in mice treated with ΡUU.

Hybrids are also used for the therapeutic and prophylactic treatment of neurological and nervous system disorders associated with the loss or dysfunction of neurons, including but not limited to Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, stroke, RDD and neuropsychiatric syndromes, and to improve or promoting learning, memory, and cognitive ability in mammals. In this regard, hybrids containing the active part of exendin or CPb1, more specifically containing at least the Ν-terminal 7-15 amino acids or their analogue, for example, H8ESTET8O (8EO GO ΝΟ: 378), are especially useful.

For all indications, in preferred embodiments, the hybrid polypeptide of the invention is administered peripherally at a dose of from about 0.5 μg to about 5 mg per day in one or more doses or by controlled continuous release, or from about 0.01 to about 500 μg / kg per dose, preferably from about 0.05 to about 250 μg / kg, most preferably below 50 μg / kg. Doses within these ranges, of course, can vary depending on the effectiveness of each analogue or derivative and can be determined by a person skilled in the art.

In the methods of the invention, the hybrid polypeptides of the invention can be administered alone or together with one or more other compounds and compositions that have a long-term or short-term effect on reducing the absorption of nutrients, including, but not limited to, other compounds or compositions that contain an amylin or an amylin analog agonist, salmon calcitonin, cholecystokinin (CCK) or a CCK agonist, leptin (OB protein) or a leptin agonist, exendin agonis or exendin analog agonist SR-1 SR-1 analogue. Suitable amylin agonists include, for example, ^[,, Tgo-] human amylin (8EO GO ΝΟ: 67) (also known as pramlintide and described in U.S. Patents 5,686,511 and 5,998,367 №). The CCK used is preferably the CCK octapeptide (CCK-8), more preferably in sulfated form. Leptin is described, for example, in Re11utoip! Ee e! A1., 8c1epse 269: 540-3 (1995); Na1aak e! A1., 8aepse 269: 543-6 (1995); Satreye1b e! A1., 8aepse 269: 546-9 (1995). Suitable exendins include exendin-3 and exendin-4, and exendin agonist compounds include, for example, those described in PCT Publication Nos. 99/07404, 99/25727 and 99/25728.

As described here, the hybrid in accordance with this invention can be introduced separately or together with one or more other agents to obtain additional benefits or to improve the action of either a hybrid or another agent. For example, an anti-obesity hybrid may be administered with an anti-obesity agent or a cardioprotective or antihypertensive agent, depending on the risk factors inherent in the patient in need of treatment and the desired treatment outcome. Examples of anti-obesity agents for administration (either alone, or in a mixture; either before, or simultaneously, or after) with the hybrid include serotonin (5HT) transport inhibitors, including, but not limited to, paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline and imipramine. Anti-obesity agents also include selective serotonin reuptake inhibitors, including, but not limited to, dexfenfluramine, fluoxetine, sibutramine (e.g., MENGOM®) and those described in US Pat. No. 6,365,633 and PCT Patent Application Publication No. 01/2706060. and ΑΟ 01/162341, which are fully incorporated into this description by reference. Such 5HT transport inhibitors and serotonin reuptake inhibitors, their analogues, derivatives, preparations, compositions, pharmaceutical compositions, doses and methods of administration have been described previously.

Anti-obesity agents also include selective serotonin agonists and selective 5-HT2C receptor agonists, including but not limited to those described in US Pat. No. 3,914,250; and

- 65 014647 publications of PCT patent applications No. XX'O 02/36596, XX'O 02/48124, XX'O 02/10169, XX'O 01/66548, XVO 02/44152; XVO 02/51844, XVO 02/40456 and XVO 02/40457, which are incorporated herein by reference in their entireties. Such selective serotonin agonists and 5-HT2C receptor agonists, compositions containing such agonists, and administration methods suitable for use in the present methods are known in the art, see, for example, Na1Bog eί a1. (2005) Sigg. Aegis Tagdei 6: 201-213 (1984) AgsI. 1p1egp. May. 144: 1143-1148.

Anti-obesity agents also include antagonists / inverse agonists of central cannabinoid receptors (CB-1 receptors), including, but not limited to, rimonabant (8apoy 8uyye1ao) and 8Ha-147778 (8apoy 8uyye1abo). Antagonists / inverse agonists of CB-1, their derivatives, preparations, compositions, pharmaceutical compositions, doses and methods of administration are described previously, for example, in US patent No. 6344474, 6028084, 5747524, 5596106, 5532237, 4973587, 5013837, 5081122, 5112820, 5292736 5624941; European patent applications EP-656354 and EP-658546; and publications of PCT applications No. ХХ'О 96/33159, ХХ'О 98/33765, ХХ'О 98/43636, ХХ'О 98/43635, ХХ'О 01/09120, ХХ'О 98/31227, ХХ'О 98/41519, ХХ'О 98/37061, ХХ'О 00/10967, ХХ'О 00/10968, ХХ'O 97/29079, ХХ'О 99/02499, ХХ'О 01/58869 and ХХ'О 02 / 076949, which are fully incorporated into this description by reference.

Anti-obesity agents also include melanocortins and melanocortin agonists. It is believed that the MC4K receptor plays a role in energy balance and obesity, see, for example, Lpiegkop e1, Exhr Orsh. Tieg., Ra1ep18 11: 1583-1592, (2001), 8reak ea a1., Exp. Orsh. Tieg. Ra1ep18 12: 1631-1638 (2002), Weipagek eί a1., ExPr Orsh. Tieg. Ra1ep18 14: 327-336 (2004). Melanocortin agonists, including but not limited to MC4B agonists and compositions containing such agonists suitable for use in the present methods, are known in the art. MCB agonists, MC4B agonists, their derivatives, preparations, compositions, pharmaceutical compositions, doses and methods of administration are described previously, for example, in the following PCT patent applications, which are incorporated herein by reference in their entirety: XO 03/007949, XO 02/068388, XO 02/068387, ХХО 02/067869, ХХ'О 03/040117, ХХ'О 03/066587, ХХ'O 03/068738, ХХ'O 03/094918 and ХХ'О 03/031410.

Anti-obesity agents also include antagonists of the metabotropic glutamate receptor subtype 5 (tC1iJ8), including, but not limited to, compounds such as 2-methyl-6 (phenylethinyl) pyridine (MPEP) and 3 - [(2-methyl-1, 3-thiazol-4-yl) ethynyl] pyridine (MTER) and the compounds described in Lpiegeopen et al. 1. Eig. 1. Ryagtaso 1. 473: 35-40 (2003); Create it a1. Vuogd. May. C1. BeP. 13 (3): 351-4 (2003); apy apyegedp e1 a1. 1. Ryagtaso 1. Exp Tieg. 303: 1044-1051 (2002).

Anti-obesity agents also include topiramate (TOR1MAX® (Og11u Msuya RyagtaseeysaN)), which is prescribed as an anticonvulsant and anticonvulsant, but also enhancing weight loss. The agent may also include lipase inhibitors such as orlistat.

Anti-obesity agents also include antagonists of neuropeptide Υ1 (ΝΓΥ1) and antagonists of ΝΓΥ5. The antagonists ΝΓΥ1 and ΝΓΥ5 are known in the art, see, for example, OnIain and its a1. (2000) Sap. 1. Rb.y8y1. Ryagt. 78: 173-185, and US patent No. 6124331, 6214853 and 6340683. Antagonists No. 1 and No. 5, their derivatives, preparations, compositions, pharmaceutical compositions, doses and methods of administration are described previously. Antagonists No. 1 used in the present compositions and methods include US patent No. 6001836; and PCT applications publication No. ХХО 96/14307, ХХО 01/23387, ХХО 99/51600, ХХ'О 01/85690, ХХ'О 01/85098, ХХ'О 01/85173 and ХХ'О 01/89528, which is fully incorporated herein by reference. Antagonists No. 5 used in the present compositions and methods of use include, but are not limited to, the compounds described in: US patent No. 6140354, 6191160, 6258837, 6313298, 6337332, 6329395, 6340683, 6326375 and 6335345; European patents No. EP-01010691 and EP-01044970; and PCT Patent Publications No. ХХО 97/19682, ХХО 97/20820, ХХ'О 97/20821, ХХ'О 97/20822, ХХ'О 97/20823, ХХ'О 98/27063, ХХ'О 00/64880, ХХ'О 00/68197, ХХ'О 00/69849, ХХ'О 01/09120, ХХ'О 01/85714, ХХ'О 01/85730, ХХ'О 01/07409, ХХ'О 01/02379, ХХ 'О 01/23388, ХХ'О 01/23389, ХХ'О 01/44201, ХХ'О 01/62737, ХХ'О 01/62738, ХХ'О 01/09120, ХХ'О 02/22592, ХХ' O 0248152, XX'O 02/49648 and XX'O 01/14376.

Anti-obesity agents also include melanin-concentrating hormone (MCH) antagonists, including melanin-concentrating hormone 1 receptor antagonists (MCH1B), such as T226296 (Takeya) and melanin-concentrating hormone 2 receptor antagonists (MCH2B). MCH receptor antagonists, their derivatives, preparations, compositions, pharmaceutical compositions, doses and methods of administration are described previously, for example, in US Patent Application Publications No. 2005/0009815, 2005/0026915, 2004/0152742, 2004/0209865; PCT Patent Application Publications No. XX'O 01/82925, XX'O 01/87834, XX'O 02/06245, XX'O 02/04433 and XX'O 02/51809; and Japanese Patent Application No. DR 13226269, which are incorporated herein by reference in their entirety.

Anti-obesity agents also include opioid antagonists, including, but not limited to those described in PCT application No. XXO 00/21509. Specific opioid antagonists used in the present compositions and methods of use include, but are not limited to, nalmefene (KEUEX®), 3-methoxynaltrexone, naloxone, naltrexone, naloxonazine, beta-funntrexamine, delta1 [B-A1a2, Bei5, Sui5 ,86, 86, 86] enkephalin (WABCE), natrindole isothiocyanate and norbinaltrofamine.

- 66 014647

Anti-obesity agents also include orexin antagonists, including but not limited to those described in PCT patent applications No. XV XV 01/96302, XVO 01/68609, XVO 02/51232 and Χνϋ 02/51838. Specific orexin antagonists used in the present compositions and methods of use include, but are not limited to, SV-334867-A.

Anti-obesity agents also include neuropeptide agonists Υ2 (ΝΡΥ2), including, but not limited to, compounds such as ΡΥΥ3-36 (for example, Vayegyat e! A1. (2003) No. 1ige 418: 650-654), ΝΡΥ3-36 and others Υ2 agonists, such as N acetyl [Ley (28.31)] ΝΡΥ 24-36 ^ yye-Ztyy e! a1. (1999) No. igorer! 1Bek 33: 526-533, TAZR-U (Mike e! A1. (1999) Vg. 1. Ryagtaso. 126: 989-996), cyclo- (28/32) -Ac | Sy.132 | - (25-36) -RZHU (CaLe1e e! A1. (2000) 1. Pep !. Zc1. 6: 97-122), which can either be a discussed component of the hybrid, or can be introduced separately. Presented anti-obesity agents also include neuropeptide agonists Υ4 (No. Υ4), including, but not limited to, compounds such as pancreatic peptide (PP) (for example, Vayegyat e! A1. (2003) 1. SBP. EpBosgto1. Me! Ab . 88: 3989-3992) and other agonists Υ4, such as 1229I91 (Yaaroktyo e! A1. (2000) No. igyosBosgto1ogu 71:27). Agonists No. 2 and agonists No. 4, their derivatives, preparations, compositions, pharmaceutical compositions, doses and methods of administration are described previously, for example, in US Patent Publication No. 2002/0141985 and PCT Application Publication No. νϋ 2005/077094.

Anti-obesity agents also include histamine 3 (H3) antagonists / inverse agonists, including but not limited to those described in PCT application No. νϋ 02/15905, O- [3- (1 Nimidazol-4-yl) propanol] carbamates (1 <forest-1 <opopo \\ '1s / e! A1. (2000) Ryagta / 1e 55: 349-355) containing piperidine histamine H3 receptor antagonists (Baxhe \\ kka e! A1. (2001) Ryagta / 1e 56: 927-932), benzophenone derivatives and related compounds (Zacke e! A1. (2001) Agsy. Reag. (^ Etyyp) 334: 45-52), substituted β-phenylcarbamates (Ree10e1k1eg e! A1. (2000 ) Ryagta / 1е 55: 83-86), and proxyphane derivatives (Zakke e! A1. (2000) 1. MeB. Set. 43: 3 335-3343). Specific H3 antagonists / inverse agonists used in the present compositions and methods of use include, but are not limited to, thioperamide, 3- (1H-imidazol-4-yl) propyl No. (4-pentenyl) carbamate, clobenpropite, iodfenpropit, imoproxiphan and ST2394 (SBa! Yes).

Anti-obesity agents also include cholecystokinin (CCK) and CCK agonists. Useful cholecystokinin-A agonists (CCK-A) include, but are not limited to, those described in US patent No. 5739106. Specific CCK-A agonists include, but are not limited to, AJ-Z 15849, C1 181771, LMU-180 , A-71378, A-71623, and ZA146131.

Anti-obesity agents also include ghrelin antagonists, such as those described in PCT application publications No. νϋ 01/87335 and νϋ 02/08250. Ghrelin antagonists are also known as CVD antagonists (a receptor that enhances the secretion of growth hormone). The compositions and methods presented herein encompass the use of CHD antagonists instead of ghrelin antagonists.

Anti-obesity agents include obestatin and obestatin analogs and agonists. Obestatin is a peptide derived from the same precursor from which ghrelin, preprogreline is obtained, see, for example, 2apd e! a1. (2005). Zaepse 310: 996-999; Shdiyak e! a1. (2005) Zaepse 310: 985986; Rap e! a1. (2006) RerBBeck 27: 911-916. In contrast to the action of ghrelin, it is believed that obestatin acts as an anorectic hormone by lowering food intake, emptying the stomach, moving the jejunum and increasing body weight. Applicable obestatin peptides include, but are not limited to, those described in 2nd e! a1. (2005) Zaepse 310: 996-999.

Amylinomimetics, for example, pramlintide, amylin-kST-amylin (compound 10), incretins, for example, exendin-4, and analogues аналоги, are anti-obesity agents that can also be administered as anti-obesity agents together with the hybrid. For example, a leptin-compound 10 hybrid may be administered with exendin-4, analog ΡΥΥ, or both. In another embodiment, the hybrid leptin-analog ΡUU can be introduced with exendin-4, amylinomimetic, or both.

Of particular interest in the treatment of diabetes and related diseases described herein are hybrids containing the exendin family and the alpha-MH module, and exendin and a member of the amylin family. Of particular interest are hybrids in which exendin is exendin4 or an analog or derivative thereof, and the amylin component is pramlintide or the chimera amylin-kST-amylin.

In particularly interesting treatment options for obesity and related diseases and conditions (reduction in body fat mass) described herein, hybrids containing the exendin family in combination with the alpha-MH module, exendin with a member of the amylin family, a member of the amylin family with a member of the family представлены , a member of the amylin family with a member of the CCK family, a member of the amylin family with a member of the alpha-MZN family, a member of the 38-38 family, a member of the amylin family with a member of the ΡUU family, a member of the ΡΥΥ family with a different, identical or different, member of the family -keeping ΡΥΥ, ΡΡΥ family member with a member of the family of CCK, a member of the family of a member of the family ΡUU ΕΝ-38, a member of the SSC family member with ΕΝ-38 family. Of particular interest are hybrids in which exendin-4 is exendin-4 or its analogue or derivative, the amylin component is pramlintide or chimera amylin-kST-amylin, членом38 is a member of its family or its analogue or derivative, ΡУУ chimera is ΡУУ-NΡΥ chimera described here, such as ZEO W Νϋ: 266, 437, 438, 439, 442, 462, 469,

- 67 014647

470, 471 and 472 from I8 2006 / 0135747A1 and the chimera ΤΥΥ-ΝΈΥ 5705, for example.

Obtaining and purification of the polypeptide

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

Hybrid polypeptides in accordance with this invention can be synthesized in solution or on a solid substrate in accordance with conventional methods. Various automatic synthesizers are commercially available and can be used with known protocols. For example, see 8Ee \\ ^r agE apb Υοиηд, 8оНб РНаке РеРе1бе 8упЕНек1к, 2б. eb., Negse SNetua1 Co. (1984); Tat eE a1., I. At. Oh. 8os. 105: 6442 (1983); Metieb, 8aepse 232: 341-7 (1986); and Vagapu apb Metieb, THe Rerbeck, Sgokk apb Me1bOe, ebk., Asabetu Rgekk, Yoet Υο ^ k, 1-284 (1979). Solid-phase synthesis of peptides can be carried out using an automatic peptide synthesizer (for example, Mobe1 430A, Arrieb VyukukEtk Sh., EokEyg Syu, SaShogsha) using the UMR / NOVE system (option 1) and the chemistry of Evos or Etos (see, Arrieb Vyukuket instructions 'to Mapia1 for ΛВI 430А РерЕ1бе 8упШек1 / ег, Уегкюп 1.3В Л.11у 1, 1988, section 6, pp. 49-70, ArrIEb VyukukEetk, Sh., EokEeg Syu, SayEogsha) with capping. Peptides can also be assembled using Abνaηηеб СН СН Т есНет Т Т Т Т Т 8 8 8упупупупуп ((((((((М М М М М М М М М М М М М Мобобобобобобобобоб11е1111РР8888888888888888,, К К К К К К К К К))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))). Peptides can be purified by RP-HPLC (preparative and analytical) using, for example, ^ aEegk Ee1Ea Rger 3000 system and preparative columns C4, C8 or C18 (10 microns, 2.2x25 cm; Uubas, Nekrep, SaShogsh). The active peptide can be easily synthesized and then filtered using screening assays designed to identify reactive peptides.

Hybrid polypeptides in accordance with this invention can alternatively be obtained by recombinant methods well known in the art, see, for example, 8bbc eE a1., Mo1ci1a1C1cpd: Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa, (1989). Such hybrid polypeptides obtained by recombinant methods can be expressed from a polynucleotide. One skilled in the art will understand that polynucleotides, including DNA and RNA, which encode various fragments of hybrid polypeptides, can be obtained from wild cDNAs, taking into account the degradation of the use of the codon, or can, if desired, be obtained by genetic engineering. Such polynucleotide sequences can introduce codons that facilitate transcription and translation of mRNA into microbial carriers. Such producing sequences can be easily constructed using methods well known in the art, see, for example, ^ O 83/04053. The polynucleotides described above may also optionally encode the Ν-terminal methionyl residue. Non-peptide compounds used in accordance with this invention can be obtained by methods known in the art. For example, phosphate-containing amino acids and peptides containing such amino acids can be prepared by methods known in the art, see, for example, Vagiei apb Lapbep, Wuogd. Oh. 14: 356-77 (1986).

A variety of expression vector / carrier systems can be used to contain and express a sequence encoding a hybrid polypeptide. They include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMU; tobacco mosaic virus, TMU) or transfected with bacterial expression vectors (e.g. T1 or pBP322 plasmids); or animal cell systems. Mammalian cells that are used in recombinant protein production methods include, but are not limited to, UEPO cells, CE11K cells, Chinese hamster ovary cell colonies (CHO), CO8 cells (such as CO8-7), VI 38 cells, BHK, HepC2 , 3T3, RSh, MOSK, A549, PC12, K562, and 293. Typical protein recombinant expression protocols are described herein.

As such, the polynucleotide sequences of this invention are used to create new and useful viral and plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including culture, bacterial, yeast and mammalian cells), and new and useful methods for culture growing such host cells capable of expressing the hybrid polypeptides of the invention. Polynucleotide sequences encoding hybrid polypeptides can be used for gene therapy in cases where insufficient production of the complex peptide hormone (s) of the chimera is facilitated, or, if necessary, at elevated levels thereof.

The present invention also provides recombinant DNA methods for producing the hybrid polypeptides of the invention. A method for producing hybrid polypeptides from a host cell containing nucleic acids encoding such hybrid polypeptides is provided, comprising: (a) culturing said host cells containing polynucleotides encoding such hybrid polypeptides under conditions conducive to the expression of such a DNA molecule ; and (b) the receipt of such hybrid polypeptides.

Host cells can be prokaryotic or eukaryotic and include bacterial, mammalian (such as Chinese hamster ovary (CHO) cells, monkey cells, newborn hamster kidney cells, cancer cells or other cells), yeast cells and insect cells.

Mammalian host systems for expressing a recombinant protein are also well known to those skilled in the art. The strains of the host cells can be selected based on the specific ability to process the expressed protein or give certain post-translational modifications that will be useful to ensure the action of the protein. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves the prepro form of the protein, can also be important for proper insertion, looping and / or function. Various host cells, such as CHO, HeLa, ΜΌΟΕ, 293, Ι Ι38, and the like, have a specific cellular complex and a characteristic mechanism of such post-translational action, and can be selected to ensure the correct modification and processing of the introduced foreign protein.

Alternatively, the yeast system can be used to create hybrid polypeptides in accordance with this invention. The coding region of the cDNA of the hybrid polypeptide is expanded by PCR. DNA encoding the pre-pro-alpha leader sequence of the yeast is expanded from the genomic DNA of the yeast in a PCR reaction using one primer containing nucleotides 1-20 of the alpha crossed factor gene, and another primer complementary to nucleotides 255-235 of this gene (Kig) Apb Negkko ^ yh, Ce11, 30: 933-43 (1982)). The pre-pro-alpha leader coding sequence and fragments of the hybrid polypeptide coding sequence are ligated into a plasmid containing the yeast alcohol dehydrogenase (DON2) promoter in such a way that the promoter directs the expression of a fusion protein consisting of a pre-pro-alpha factor fused to mature a hybrid polypeptide. As described by Koke apb Vgoasy, Μеΐй. Eph. 185: 234-79, Soebbe1 eb., Dsabetu Rgekk, Shs., Bap P1edo, SayGogsha (1990), the vector further includes the transcription terminator ΑΌΜ descending to the cloning site, the origin of replication of yeast 2-micron, yeast 1ei-2b gene, yeast KEP1 and KEP2 genes, E. soy β-lactamase gene, and E. co11 replication origin. The β-lactamase and 1ei-2b genes provide selection in bacteria and yeast, respectively. The 1ei-2b gene also contributes to an increase in the number of copies of the plasmid in yeast, causing increased levels of expression. The KEP1 and KEP2 genes encode proteins involved in the regulation of plasmid copy number.

The DNA construct described in the previous paragraph is converted into yeast cells using a known method, for example, treatment with lithium acetate (Batek e! A1., He. Ye. 185: 280-97 (1990)). The promoter ΑΌΜ is stimulated by the expenditure of glucose in the growth medium (Pse e! A1., Cepé 55: 287 (1987)). The pre-pro-alpha sequence affects the secretion of fusion protein from cells. At the same time, KEX2 yeast protein cleaves the pre-pro sequence from mature PΥΥ analog polypeptides (Wy! Ee! A1., Prg. No. 11. Dsab. Bsk 81: 5330-4 (1984)).

Hybrid polypeptides in accordance with this invention can also be recombinantly expressed in yeast using commercially available expression systems, such as Pyrrhia Exrgeccup Boulesh (Jodogep, Bap P1edo, Saigogsha), according to the manufacturer's instructions. This system is also based on a pre-pro-alpha sequence for directing secretion, but the transcription of the insert is driven by the alcohol oxidase promoter (AOX1) when stimulated with methanol. The isolated hybrid polypeptide is purified from yeast growth medium using, for example, methods used to purify the hybrid polypeptide from bacterial and mammalian cell supernatants.

Alternatively, cDNA encoded hybrid polypeptides can be cloned into the expression vector of the baculovirus pUb1393 Bap P1edo, CaNogogsha). Such a vector containing a hybrid polypeptide is then used according to the manufacturer’s instructions to infect Brobor! Ega Ggischregba cells in a protein-free medium in kE9 medium and to produce a recombinant protein. The protein is purified and concentrated from the medium using a heparin-sepharose column (Ryattaaa, P1xa! A ^ ay, Νο \ ν 1gkeu) and the next column to determine the size of the molecules ^ m ^ op, Veueg1u, Μπκсакикикик Нк) and resuspended in RFPB. The analysis of B ^ B-RDSE shows one lane and confirms the size of the protein, and Edman's dance method on Prospect 2090 Rurbbe Beschischepseg confirms its terminal sequence.

For example, a DNA sequence encoding a hybrid polypeptide can be cloned into a plasmid containing the desired promoter and, optionally, a leader sequence (see, Vejeg e! A1., Baepse 240: 1041-3 (1988)). The sequence of this design can be confirmed by automatic sequencing. Then the plasmids are transformed into E. coli strain 11, 061, using standard techniques that use incubation with CaCl2 and heat treatment of bacteria (Batok e! A1., Above). Transformed bacteria are grown in LB medium supplemented with carbenicillin, and the production of the expressed protein is caused by growing in a suitable

- 69 014647 Wednesday. If present, the leader sequence will affect the secretion of the hybrid polypeptide and will be cleaved during secretion. The isolated recombinant protein is purified from the bacterial culture medium as described herein.

Alternatively, the hybrid polypeptides of the invention may be expressed in an insect system. Insect systems for protein expression are well known to those skilled in the art. In one of these systems, the nuclear polyhedrosis virus Liΐod ^ aryk saigogshsa (LsNPV) is used as a vector for the expression of foreign genes in 8royor ega 1gid1regya cells or Tpsjorya larvae. The hybrid polypeptide coding sequence is cloned into a minor region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Subsequent insertion of the fusion polypeptide renders the gene inactive and causes the recombinant virus to lack a coat protein. Then, recombinant viruses are used to infect cells 8. Hydridregia or the larvae of Tpsyor1ik1a, where a hybrid polypeptide is expressed (8th e! A1., 1. νπΌ1. 46: 584 (1983); Epdechag e! A1., Proc. No.! 1. Lsai. 8α. Υ8Α 91: 3224-7 (1994)).

In another example, the DNA sequence encoding the hybrid polypeptide can be increased by PCR and cloned into a suitable vector, for example, pOEX-3X (Ptyagast, P1xa! A ^ ay, No. \ ν 1egkeu). The pOEX vector is designed to produce a fusion protein containing glutathione-8-transferase (O8T) encoded by the vector, and the protein encoded by the DNA fragment is inserted into the cloning point of the vector. Primers designed for PCR may include, for example, suitable cleavage points. The recombinant fusion protein may be cleaved from a portion of the O8T fusion protein. The pOEX-3X / PΥΥ analog polypeptide construct is transformed into E. coli XB-1 B1ie cells (8! ha! adepe, ba 1o11a, SayGogsha), and individual transformants are isolated and grown at 37 ° C in the middle of bb (with the addition of carbenicillin) to an optical density at a wavelength of 600 nm equal to 0.4, followed by incubation for 4 h in the presence of 0.5 mM isopropyl β-β-thiogalactopyranoside (81dta Syetua1 Co., 8ΐ. Loshk, Μ ^ kkoi ^^). The plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the insert of the desired gene encoding the PPE hybrid polypeptide in the right direction.

A fusion protein, which is to be formed as an insoluble intracellular body in a bacterium, can be purified as follows. Cells are harvested by centrifugation; washed in 0.15 M No. C1, 10 mm Tick, pH 8, 1 mm EDTA; and treated with 0.1 mg / ml lysozyme (81dta Syetua1 Co.) for 15 minutes at room temperature. The lysate is purified by ultrasound and cell debris is precipitated by centrifugation for 10 min at 12000hd. The precipitate containing the fusion protein was resuspended in 50 mM Teak, pH 8, and 10 mM EDTA, layered in 50% glycerol and centrifuged for 30 min at 6000 xd. The precipitate was resuspended in standard saline with phosphate buffer (PBS) that did not contain Μд ⁺⁺ and Ca ⁺⁺ . The fusion protein is then purified by separation of the resuspended residue in a denaturing 8-8 polyacrylamide gel. (8tyagok e! A1., Above). The gel is impregnated in 0.4 M KC1 to visualize the protein, which is removed and electroeluted in gel buffer containing 8-8. If the O8T / PΥΥ fusion protein of an analog polypeptide is obtained in bacteria as a soluble protein, it can be purified using O8T RipDsayop иοйі1е (Ryagtasta Vu! Es).

The fusion protein can be digested to cleave O8T from the PPE of the hybrid polypeptide. The cleavage reaction mixture (20-40 μg of fusion protein, 20-30 units of human thrombin (4000 U / mg (81 dt) in 0.5 ml of PBS) is incubated for 16-48 hours at room temperature and loaded into a denaturing 8 ^ 8-RLOE gel for separation of reaction products into fractions. The gel is impregnated in 0.4 M KCl to visualize protein bands. Identity of protein bands with the expected molecular weight of the hybrid polypeptide can be confirmed by partial analysis of the amino acid sequence using an automated sequencer ^ Ryu VukuMetk Μοйе1 473Α, Еок! егSyu, SaiGogsha).

In a particularly preferred recombinant expression method of the hybrid polypeptides of the invention, 293 cells can be co-transfected with plasmids containing the cDNA of the hybrid polypeptide in the pCV vector (5 'CΜV promoter, 3' HOH poly A sequence) and p8V2ie (containing the resistance neo gene ) by calcium phosphate. Preferably, the vectors should be linearized with 8caI before transfection. An alternative construct using a similar pCV vector with an incorporated neogen can also be used. Stable cell colonies are selected from single cell clones by limited dilution in a growth medium containing 0.5 mg / ml O418 (neomycin-like antibiotic) for 10-14 days. Cell colonies are sorted according to the degree of expression of the hybrid polypeptide using either Western blotting and colonies of cells with a high degree of expression are increased for growth on a larger scale.

It is preferable to use transformed cells for long-term formation of a protein in high yield, and, as such, stable expression is desired. Once such trans cells

- 70 014647 formed with vectors that contain selectable markers together with the desired polygenic expression cluster, cells can be grown for 1-2 days in an enriched medium before transferring to a selective medium. The selectable marker is designed to confer resistance to selection, and its presence allows the growth and isolation of cells, which then express the presented sequences. Resistant clusters of stably transformed cells can be proliferated using tissue culture techniques applicable to the cell.

Many selection systems can be used to isolate cells that have been transformed to isolate a recombinant protein. Such selection systems include, but are not limited to, the HSV thymidine kinase genes, hypoxanthine guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes in! K, idry or ary cells, respectively. Also, anti-metabolic resistance can be used as a basis for breeding which give resistance to methotrexate; others !, which confer resistance to mycophenolic acid; IEOs that confer resistance to aminoglycoside, 0418; A1co, which confer resistance to chlorosulfuron; and hydro, which give resistance to hydromycin. Additional breeding genes that can be used include! GrV, which allows cells to use indole instead of tryptophan, or Lyco, which allows cells to use histinol instead of histidine. Markers that provide a visual indication for identifying transformants include anthocyanins, β-glucuronidase and its substrate, 0I8, and luciferase and its substrate, luciferin.

Most of the hybrid polypeptides in accordance with this invention can be obtained using a combination of automated peptide synthesis and recombinant techniques. For example, a hybrid polypeptide may comprise a combination of modifications, including deletion, substitution, and PEGylation insertion. Such a hybrid polypeptide can be obtained in several stages. In a first step, an intermediate polypeptide containing modifications consisting of deletion, substitution, insertion and any combination thereof can be obtained by recombinant techniques, if desired. Then, after the optional purification step described here, the intermediate polypeptide is PEGylated by chemical modification with a suitable PEGylating reagent (for example, NoK! Ag Tyegareiysk, 8Ai Cag1ok, Ca11 £ ogsh) to obtain the desired hybrid polypeptide. One skilled in the art will understand that the above technique can be generalized to apply to a hybrid polypeptide comprising a combination of modifications selected from deletion, substitution, insertion, and other modification tools well known in the art and contemplated by this invention.

Purification of hybrid polypeptides prepared according to the invention may be desirable. Peptide purification techniques are well known to those skilled in the art. Such techniques include, at one level, pre-fractionating the surrounding cell environment into and without polypeptide fractions. After separating the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatography and electrophoresis techniques to partially or completely purify (or purify to uniformity). Analytical methods that are particularly suitable for preparing a pure peptide include ion exchange chromatography, gel chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly effective method for peptide purification is reverse phase HPLC, followed by identification of the purified product by liquid chromatography / mass spectrometry (LC / MS) and mass spectrometry Ma! Pkh-Akk1k! Ebakeg Oekogryoi [os / aboy (MAbO1). Additional confirmation of purity is carried out using a determining analysis of amino acids.

Some aspects of the present invention relate to the purification and, in particular embodiments, the thorough purification of the encoded protein or peptide. The term purified peptide, as used here, refers to a composition isolated from other components, where the peptide is purified to any degree with respect to the state in which it is obtained in nature. Therefore, the term purified peptide also refers to a peptide that does not contain the environment in which it exists in nature.

In general, the term purified refers to a peptide composition that is fractionated to remove other components, and wherein the composition substantially retains its expressed biological effect. If the term substantially purified is used, this definition refers to a composition in which the peptide is the main component of the composition, for example, the peptide is 50, about 60, about 70, about 80, about 90, about 95 or more of the composition.

Various techniques suitable for use in peptide purification are known in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like; thermal denaturation followed by centrifugation; chromatography steps such as ion exchange, gel chromatography, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and a combination of these and other techniques. As is well known in the art, it is believed that the order of the various purification steps can be changed, or that certain steps can be skipped and still constitute a suitable method for preparing a substantially purified protein or peptide.

- 71 014647

There is no general requirement that peptides are always obtained in their most purified state. In fact, it is believed that less refined products may be used in certain embodiments. Partial purification can be carried out using fewer purification steps in combination, or using different forms of the same general purification scheme. For example, it is understood that chromatography on a cation exchange column using an HPLC apparatus usually gives a more refined purification than the same technique as the low pressure chromatography system. Methods giving a lower degree of relative purification may have advantages in the overall isolation of the protein product, or in maintaining the activity of the expressed protein.

You can optionally purify and isolate such hybrid polypeptides from other components obtained in the method. Methods for purifying a polypeptide can be found in US patent No. 5849883. This document describes certain typical methods for the isolation and purification of C-C8E compositions that can be used for the isolation and purification of hybrid polypeptides in accordance with this invention. Based on the description of these patents, it is obvious that a person skilled in the art will be able to take many 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 prepare purified hybrid polypeptide compositions in accordance with this invention.

Pharmaceutical Compositions

The invention also relates to pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of at least one hybrid polypeptide of the invention or a pharmaceutically acceptable salt thereof, together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers used for the delivery of hybrid polypeptides. Such compositions may include diluents containing various buffers (eg, TPZ-HC1, acetate, phosphate), pH, and ionic strength; additives, such as detergents and solubilizing agents (for example, Те уеп 80, Ролзогъа! e 80), antioxidants (for example, ascorbic acid, sodium metabisulfite), preservatives (for example, thimersol, benzyl alcohol) and substances that add volume (for example, lactose, beckons); introducing the product into partial compositions of polymeric compounds, such as polylactic acid, polyglycolic acid, etc., or together with liposomes. Such compositions will affect the physical condition, stability, rate of recovery and the speed of clearance of hybrid polypeptides in accordance with this invention, see, for example, Ketshd! Op'z Rägtaseijsa1 8c1epsez 1435-712, 18! Riyzypd Co., Eaz! Op, Reppzukasha (1990).

In general, the hybrid polypeptides of the invention are used in the same way that individual complex polypeptides are used based on their pharmacological properties. One preferred use is the peripheral administration of such hybrid polypeptides for the treatment or prophylaxis of conditions and metabolic disorders. In particular, the compounds of this invention have effects as agents for reducing the absorption of nutrients, reducing food intake, suppressing appetite, and inducing weight loss. In another embodiment, the preferred use is the introduction of such hybrid polypeptides for the treatment of diabetes or diabetes-related conditions and disorders.

Hybrid polypeptides in accordance with this invention can be formulated for peripheral administration, including compositions for injection, oral administration, nasal administration, pulmonary administration, local administration or other types of administration known to a person skilled in the art. More specifically, the administration of pharmaceutical compositions in accordance with this invention can be carried out through any well-known path, if the target tissue is accessible through this path. In a preferred embodiment, the pharmaceutical compositions may be administered to a patient by any conventional peripheral method, for example, intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g., delayed release); by oral, sublingual, nasal, anal, vaginal or transdermal delivery methods, or through surgical implantation at a specific location. Treatment may consist of a single dose or multiple doses over a period of time. Controlled sustained release of the compositions of this invention is also contemplated.

The composition may be liquid or may be solid, for example, lyophilized for reconstitution. Aqueous compositions in accordance with this invention contain 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 forms and compositions that do not cause harmful, allergic or other adverse reactions when administered to an animal or human. As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion medium, coatings, antibacterials

72 014647 and antifungal agents, isotonic and absorption delaying agents and the like. The use of such a medium and agents for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active ingredient, their use in therapeutic compositions is also contemplated. Additional active ingredients may also be included in the composition. In some cases, it will be convenient to obtain a hybrid polypeptide according to this invention and another food reducing agent, a diabetes treatment agent, a plasma glucose lowering agent, or a plasma lipid modifying agent such as amylin, an analogue of an amylin agonist, CCK, or an agonist CCK or leptin or a leptin agonist or exendin or an analog of an exendin agonist, as a single composition or solution for administration together. In other cases, it may be advantageous to administer an additional agent separately from said hybrid polypeptide.

The hybrid polypeptide of the invention can be prepared for administration as free base solutions or pharmacologically acceptable salts in water, suitably mixed with a surfactant such as hydroxypropyl cellulose. Pharmaceutically acceptable salts include acid addition salts (obtained with the free amino groups of the protein), and salts which are obtained with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, and almond and the like. Salts obtained with free carboxyl groups can also be obtained from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and organic bases such as, for example, isopropylamine, trimethylamine, histidine, procaine and the like. . Such products are readily prepared by methods well known to those skilled in the art. Dispersions can also be obtained in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, such formulations contain a preservative to prevent the growth of microorganisms.

In one embodiment, the pharmaceutical compositions of this invention are formulated to be suitable for parenteral administration, for example, by injection or infusion. Preferably, the hybrid polypeptide is suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of from about 3.0 to about 8.0, preferably at a pH of from about 3.5 to about 7.4, from 3.5 to 6.0, or from 3.5 to about 5.0. Used buffers include buffers based on sodium citrate — citric acid and sodium phosphate — phosphoric acid, and sodium acetate / acetic acid. A delayed release composition, such as a depot form or depot, may be used so that therapeutic amounts of the composition enter the bloodstream within a few hours or days after a percutaneous injection or installation.

Suitable pharmaceutical compositions for injection include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions immediately prior to administration. In all cases, the form must be sterile and must be fluid to the extent that it can be easily inserted through a syringe. It is also desirable that the hybrid polypeptide of the invention is stable under the conditions of manufacture and storage and should be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (e.g. glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by applying a coating such as lecithin, maintaining the desired particle size in the dispersion, and using surfactants. Prevention of exposure to microorganisms can be achieved by the use of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to add isotonic agents (for example, sugars or sodium chloride). Prolonged absorption of the administered compositions can be achieved by the use of absorption inhibiting agents (for example, aluminum monostearate and gelatin) in the compositions.

Sterile injectable solutions can be prepared by administering the active compounds in the required amount in a suitable solvent with the various other ingredients mentioned above, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the desired other ingredients selected from the above. In preparing sterile powders for the preparation of sterile injectable solutions, preferred manufacturing methods include vacuum drying and freeze drying, which provide the powder of the active ingredient, plus any additional desired ingredients from the sterile filtered solutions described above.

In general, the therapeutically or prophylactically effective amount of hybrid polypeptides in accordance with this invention is determined based on the age, weight and condition or severity of the patient’s diseases, conditions or disorders, see, for example, Vet1pd1op'k Ryagtasei11ca1 δc ^ epsex 697-773, see also \ Wapd apb Jaapkop, Ragep1eg1 Rogt1a1yupk o £ Рг11шк apb Rerybek: δΙη ^ 1ίΙν apb δΙηΝΗζ

- 73 014647 EGK, 1OIGPA1O аг RAGEP1EG1 8c1epse apb Tébnoduod, Técnoca1 Keroy Νο. 10, 8irr. 42:28 (1988). Typically, a dose of from about 0.001 μg / kg body weight / day to about 1000 μg / kg body weight / day is used, but a smaller or larger dose as determined by a person skilled in the art can be used. Doses may be administered one or more times per day and may be combined with other compositions as described herein. It should be noted that the invention is not limited to the doses indicated here.

Suitable dosages can be determined using recognized assays to determine the level of conditions or metabolic disorders in conjunction with appropriate dosage data. The final dosing regimen can be determined by the attending physician taking into account factors that change the effect of drugs, for example, the specific effect of the drug, the severity of the damage and reaction of the patient, age, condition, body weight, gender and diet of the patient, severity of infection, time of administration and other clinical factors. After research, additional information appears regarding the appropriate dosage levels and duration of treatment for specific diseases and conditions.

An effective dose is usually from about 1 to 30 μg to about 5 mg / day, preferably from about 10 to 30 μg to about 2 mg / day, and more preferably from about 5 to 100 μg to about 1 mg / day, most preferably from about 5 mcg to about 500 mcg / day, for a patient weighing 50 kg, when administered once or repeatedly. Preferably, the dose is from about 0.01 to about 100 μg / kg / dose. The exact dose administered can be determined by a person skilled in the art and depends on the effectiveness of the particular compound, as well as on the age, weight and condition of the patient. The introduction should be started at the moment when, for example, suppression of the absorption of nutrients, food intake, mass, modulation of blood glucose or plasma lipids, becomes desirable, for example, at the first sign of symptoms or immediately after the diagnosis of obesity, diabetes mellitus or insulin resistance syndrome . The introduction can be carried out in any way, for example, by injection, preferably subcutaneously or intramuscularly, orally, nasally, percutaneously, etc. Doses for certain modes of administration, for example, oral administration, can be increased due to reduced bioavailability by 5-100 times.

Parenteral administration can be carried out using a bolus followed by continuous infusion to maintain therapeutic circulating levels of the drug. A person skilled in the art will easily optimize effective dosages and regimens based on good medical practice and the clinical condition of an individual patient.

The frequency of dosing depends on the pharmacokinetic parameters of the agents and methods of administration. The optimal pharmaceutical composition is determined by a person skilled in the art, depending on the route of administration and the desired dose, see, for example, KettUp'k Ryagtaseijs1 8c1epse5, above, p. 1435-1712. Such compositions may affect the physical condition, stability, release rate w νί \ Ό, and clearance speed w y | o of the administered agents. Depending on the route of administration, a suitable dose may be calculated based on body weight, body surface areas and organ size. Further refinement of the calculations necessary to determine the appropriate dose for treatment is usually carried out by a person skilled in the art without undue experimentation, especially in light of the dosage information and analyzes described here, as well as the pharmacokinetic data obtained in clinical studies in animals and humans.

It is understood that pharmaceutical compositions and methods of treatment in accordance with this invention can be used in medicine and veterinary medicine. Thus, the patient to be treated may be a mammal, preferably a human or other animal. For veterinary purposes, the patient may be, for example, livestock, including cows, sheep, pigs, horses and goats, domestic animals such as dogs and cats, exotic and / or zoo animals, laboratory animals, including mice, rats, rabbits guinea pigs and hamsters; and poultry such as chickens, turkeys, ducks and geese.

In addition, the invention includes a kit comprising a hybrid polypeptide of the invention, components suitable for preparing said hybrid polypeptide of the invention for pharmaceutical use, and instructions for using said hybrid polypeptide and components for pharmaceutical use.

To facilitate understanding of the present invention, the following examples are provided. The experiments related to this invention, of course, should not be construed as limiting the invention, and such variations of the invention, known or future, that are within the competence of a person skilled in the art, are considered to be included in the scope of the invention described herein and in the claims .

Examples

The invention is described in more detail in the following non-limiting examples, which serve to more fully illustrate the invention, but should not be construed as limiting its scope. The examples show the preparation of hybrid polypeptides in accordance with this invention and the testing of such hybrid polypeptides in accordance with this invention w yyugo and / or w yyy. A person skilled in the art will understand that the techniques described

74 014647 in these examples are the methods described by the inventors of the present invention that work well in the practice of the present invention and, as such, are preferred methods for practicing it. However, it should be clear that a person skilled in the art in the light of this description will understand that many changes can be made to the specific methods described, while obtaining the same or comparable result without departing from the essence and scope of the present invention.

Example 1. Obtaining hybrid polypeptides.

The peptides in accordance with this invention can be assembled in a Zutpopu peptide synthesizer (Procodibus Tepinopod, 1 ps.) Using Rinca Schowabe amide amide resin) at a load of 0.430.49 mmol / g at 0.050-0.100 mmol or pre-loading of a Wang resin (Etoc- Tug (1Bi) - Wang resins) 0.63 mmol / g Schowal). Ethos amino acid (5.0 eq., 0.250-0.500 mmol) residues are dissolved at a concentration of 0.10 M in 1-methyl-2-pyrrolidinone. All other reagents (NVTI, 1-hydroxybenzotriazole hydrate and N, N-diisopropylethylamine) are prepared as 0.55 M solutions in dimethylformamide. The protected Ethos amino acids are then combined with the resin bound amino acid using NVTI (2.0 eq., 0.100-0.200 mmol), 1-hydroxybenzotriazole hydrate (1.8 eq., 0.090-0.18 mmol),

N, N-diisopropylethylamine (2.4 equiv., 0.120-0.240 mmol) for 2 hours. After combining the last amino acid, the peptide is deprotected with 20% (v / v) piperidine in dimethylformamide for 1 hour. How only the peptide sequence becomes complete, the peptide synthesizer is programmed to cleave the resin. Cleavage of the peptide from the resin using trifluoroacetic acid (TFA) is carried out using 93% TFA, 3% phenol, 3% water and 1% triisopropylsilane for 1 hour. The cleaved peptide is precipitated using tert-butyl methyl ether, precipitated by centrifugation and lyophilized. The precipitate was redissolved in water (10-15 ml), filtered and purified by reverse phase HPLC using a C18 column and an acetonitrile / water gradient containing

O, 1% TFA.

The general procedure for ping peptides in accordance with this invention with fatty acids (for example, octanoic and stearic acids) is as follows: a peptide on a rink amide resin (0.1 mmol) is suspended in NMR (5 ml). In a separate flask NVTI (0.3 mmol), Н0В! (0.3 mmol) was dissolved in DMF (5 ml) followed by the addition of E1EA (0.6 mmol). This solution is added to the resin and the resulting suspension is shaken for 2 hours. The solvent is filtered and thoroughly washed with MMP (5 ml x4) and CH ₂ Cl ₂ (20 ml), dried and subjected to TFA cleavage for 1 hour. The yield of the desired peptide is about 40 mg after cleavage and purification.

PEG modification can be carried out in solution on the free epsilon-amino group of lysine or the terminal amino group of the purified peptide using commercially available activated PEG esters. The resulting PEGylated derivatives are purified to homogeneity by reverse phase HPLC and the purity is confirmed by LC / MS and MABOY ^

Certain typical hybrid polypeptides in accordance with this invention are shown in table. 1-1. Various modifications of the obtained compounds are obvious, such as chemical modifications, such as glycosylation, PEG modifications, etc .; amino acid modifications such as substitutions, insertions and deletions, etc. Further, although the present compounds are amidated at the C-terminal region, it is understood that the hybrid polypeptides of the invention can alternatively be in the form of a free acid.

- 75 014647

Table 1-1

Certain Exemplary Hybrid Compounds of the Invention

5ΕΩ 10: one NESESTGTZO8KOMEEEEUKG1EEEKMSSR38SARRRZ-AZKNU1L1OTKZKU-YN2 2 NSESTETZO5KSMEEEEUKYE1ENKSSR55SARRZ-KNUMUTNOKU-UN ₂ 3 Η (3ΕΟΤΓΤ3ΟΓ3ΚΰΜΕΕΕΑνΗΒΓΙΕΗΕΚΝσ5Ρ585ΑΡΡΡ3-ΝΚΥΥΑ3ΕΡΗΥΕΝΐνΤΚ (2ΚΥ-ΝΗ2 4 N <ZEBTGTZOZK0MEEEAUIG1EI1K№СР55САРР₽В-рА1а-рА1а-А.5ЬКН ¥ ЛЫ1УТР.0НУ-МН ₂ 5 Nooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo 6 Η0Ε0ΤΓΤ30Ε3Κ3ΜΕΕΕΑνΚΕΓΙΕΗΕΚΝσεΡ33ΞΑΡΡΡ3-βΑ1Η-βΑ13-νΤΚ0ΗΥ-ΝΗ ₂ Ί ΗΟΕ6ΤΓΤ8ΟΕ5ΚαΜΕΕΕΆνΚΕΕΙΕΝΕΚΝ “ΑΞΕΚΕΥ1ΝΣνΤΚςΚΥ-ΝΗ2 8 ΗεΕΘΤΕΤ3ϋΕ5Κ0ΜΕΕΕΑνΗΒΓΙΞΗ1ΚΝ-ΚΗΥ5ΝΕνΤΚΏΗΥ-ΝΗ ₂ nine N6ESTET5 OZKOMEEEAUVG1EKM-MKUUAZNENNYEUTK2KU-MN ₂ 10 NBBETTZRZEZMEEEUKUKHTEIKI ^ AZA-RAZA-AZEKNUYUUTKOKU-YNG eleven NEE6TETZEZK0MEEEA7KE1EIEKI-RA1a-pA1a-KNUIN7TVKK ¥ -НН ₂ 12 ΗδΕΟΤΓΤΞϋΣΒΚΟΜΕΕΕΑνΚΕΓΙΕΜΕΚΝ-βΑΙθ-βΑΙθ-νΤΚςΕΥ-ΝΗζ thirteen NOESAΓΤ8ΟΣ8Κ £ ΕΕΕΕΑνΕΕΕΙΕΡΓΚΝΝΡΥΥΑ8ΕΗΗΥΕΝΕνΤΒ2ΗΥ-ΝΗ ₂ 14 NESESAGTZPZZ'KOEEAURPEEKNAZARNU ^ UTROKU-YN ₂ fifteen NESESETZPZZEZEEEEUU ^ EPEEKKNUEMOUVOZVOKU-YNZ sixteen NESESET805K0EEENUAZUKNUKNUTSRU-MN ₂ 17 NESESAGTZSZYEEEUAUKG1EEKY-PA1a-pA1a-AZKNUEKUTK <2KU-EN _g eighteen Η0Ε0ΑΓΤ5ϋΕ5Κ0ΕΕΕΕΑνΚΕΕ · ΙΕΕΊ.ΚΝ-βΑ13-βΑ13-ΚΗΥ1ΝΙ · νΤΚ0ΚΪ-ΝΗ ₂ nineteen ΗαΕεΑΓΤδΟΕΞΚίΙΙ, ΕΕΕΑνΒΕΕΊΕΕΈΚΝ-βΑΙβ-βΑΙδ-νΤΗΩΚΥ-Ν ΝΗ ₂ twenty ΗΟΕ6ΤΕΤ30Ε5Κ0ΜΞΕΕΑνΡΕΕΙΕ & Π, ΚΝΟ6ΡΞ56ΑΡ₽ΡΞ-ΏΥ (ЗО ₃ ) MSIMOG-IN ₂ 21 NSESTGTZOZEKOMEEUKUKPEYKY-OU (5Ο ₃ ) Μ6ΝΜΌΓ-ΝΗ ₂ 22 NESESTGTZO8K2MEEEAUKG1ENKN-OE (CH ₂ 8Oz) MSIMOG-ON2 23 NSESTGTZOZE MEEAUKUZPEKI ”[8-amino-3, 6-dioxo-octanoyl]-З (ЗО ₃ ) ΜΟΗΜΟΕ-ΝΗ ₂ 24 Η0Ε6ΤΕΤ50Ε5Κζ) ΜΕΕΕΑνΕΣΓΙΕΜΕΚΝ- [8-amino-3,6-dioxoctanoyl] -OG (CH ₂ 8О ₃ ) Μ0ΚΜ0Γ-ΝΗ ₂ 25 NESESTGTZOZKOMEEEUKUKPEIKKSYTATSUKSZNESNRSTURRTMTSZKTU-KNg 26 NSESTET508SKMEEEAUNEE1EEKKAYTATAUYS-IN ₂ 27 NSE2TRTZOZKSMEEEAUKE1ENKa-12-Ac1o-KSMTATSUYONZOENKYOTURKTYTSZKTU-K1N ₂ 28 H6E6TET50L5KMEEEEEUEH1EIJKM-12-Mo-S »1TATS <Жъ50Е1НКЬ0ТУРКТМТ63ЫТУ-МН2 29th Nooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo thirty NSEETZOZZMEEEKUKG1EIBK1YA-3, 6-dioxoctanoyl-SNTATSTYKYZ () ЕНВЬ <2 ТURКТАТЦЗЫТ ¥ -ЫН ₂ 31 NSCEST50E5K0MEEEA7KE1ET, KY-5-Ara-KSOTATSU6KEKE50ENNO0T ¥ РКТ11ТСЗМТУ-ЕН ₂ 32 NSESTET80'5KSMEEEEUKYE1EIKK-5-Ara-SMTATS7SKS8SEENK2TURKTNTS8NTU-MN ₂ 33 NESTETSOZK (2MEEEEAUKE1EIEKI-рА1а-рА1а-ΚΕΝΤΑΤσνΕ5ΚΕ30ΕΑΗΒ.10ΤΥΡΕΤΝΤ63ΝΤΎ-ΝΗ ₂ 34 NSEETGTZOZKOMEEEUKUKI-pA1a-pA1a-SNTATSUYKZOEZNOKTURKTYTSZLTU-NN ₂ 35 H6ESTGT5OLL5K £ MEEEAUKG1EIBK-4.7, U-triox-13-tridecanamine succinimidyl- ΚΟΝΤΑΤ € νΒΟΡΕ30ΕΕΗΗΕΟΤΥΡΚΤΝΤΟ5ΝΤΥ-ΝΗ ₂ 36 NON-TETZOZK0MEEEAUK1E1EIBKM-4,7,10-trioxa-13-tridecanamine succinimidyl- ΟΝΤΑΤθνΕ <3ΚΕ30ΕΑΗΚΣΟΤΎΡΚΤΝΤε3ΝΤΥ-ΝΗ ₂ |

- 76 014647

203 OG (SN25OZ) MeiMPG-bKV-KSMTATSATOKYAYUYUOTURVUTYUSSKTU-Yng 38 ΚΟΝΤΑΤΟΑΤΟΚΟΆ№ΐνΚ-ΚΥΥΑ5Ι, ΚΗΥΙ, No.νΤί <ς [<ϊ-ΝΗ2 39 isocaproyl-ETAU- (A1b) -K (formyl) -B3CEL- (A1b) -K (formyl) -BCT-MK ¥ УА5ЬВН ¥ ЬЬУТИОКУ-Un ₂ 40 IEOkaproil-ZTAU- (A1b) -K (formyl) -YOE- (A1b) -K (formyl) -Ο-ΕΟΝΗΥΥΑ3ΕΗΗΥΣΝΣντΚζ) ΚΥ-ΝΗ ₂ 41 CH ⁰ H > I _{κρΕΑΡ0Ε1} - _{] Α5} ρ _ΕΕΚΝΚΥ γ _{Α & ΕΚΗΥΕΝΕντκ} ρ _κ _ _Ν ⁷ 'Α ^ _ ₀ ° ° Ύ ° ° ⁰ О 42 a 3 О О НМ- ^ ⁴ РНЗ Μ V b М-- ° ХМ А 0 = 5 _h (Г ^{1 КРЕАР0Е0А5 РЕ} Е ^Н N К ϊ Υ А η К Η Υ 1 Ν ί V Т К 0 К -ЫЧ- О θ СН о О

- 77 014647

311 □ o about _{0 60} ..,. _e1mv ,,, _{vi ν 1n} . ν-ΓΙΡ .. 1 * and X? ° ' ⁰ X ^x OH 27 ^2E 12 Αάο-Exendin (1-28) -I-Amylin (1-7) - ^11/18 Agd-sCb (8-27) -B Amylin (33-37) 28 ²⁹ 12 Αάο-Exendin (1-28) -Mea-Lub-L Amilin (1-7) - ^11,1I Agd-bs (8-27) -L Amilin (33-37) 29th ²⁹ 3, 6-dioxaoctanoyl-Exendin (1-28) -g.Amilin (1-7) - ^ '^ Agd-sCb (8-27) -Namilin (33-37) AP ²⁹ 3, 6-dioxaoctanoyl-Exendin (1-28) - ^x "1ez-b3-11Amilin (1-7), ^11/15 Agd-sC ^ (8-27) -Namylin (33-37) 31 ²⁹ 5 Ara-Ekaendin (1-28) -Namylin (1-7) - ^11.18 Agd-sCl (8-27) -L Amylin (33-37) 32 ²⁹ 5 Ara-Exendin (1-28) -Mez-b3-Amylin (1-7) - ^11.1a Agd-sCb (8-27) -Namilin (33-37) 33 ^2E betaA1a-betaA1a-Exendin (1-28), L Amylin (1-7) - ¹¹ ' ^1B Agd-sC1 (8-27) -A Aglk ^ in (33-37) 34 ^2U betaA1a-betaA1a-Exendin (1-28) -Mee-uz-Amilin (1-7) - ^11.1V Agd-sCb (27) -L Amilin (33-37) 35 ^2U 4.7, Yu-trioxa-13-tridecanamine succinimidyl-Exendin (1-28) -L Amylin (1-7) ^11.18 Arg-sCb (8-27) - L Amilin (33-37) 36 _g 7,10-trioxa-13-tridekanamin succinimidyl-Exendin (1-28) - + es-uz-Amilin (1-7) - ¹¹ 'Agd- zS ¹ (8-27) -Amilin (33-37) 312 ²⁹ (61u-01u-S1u) -Ekzendin (1-28) NAmilin (1-7) - ^{11 '1c} Arg-SPR (8-27) -Amilin (33-37) 313 ²⁹ (S1u-S1u-S1u) -Ekzendin (1-28) uz-mesyl-Amilin (1-7) - ^{11 '18} Arg-zS (27) -Amilin (33-37)

Other typical hybrids containing various combinations of families of complex peptides are presented in the following table. Their analogues and derivative hybrids described herein are also contemplated. The sign '' # '' indicates the location of the link for each component. These linkers are as described herein. CCK-8 is ^ Υ (§O3H) MCVM ^ Ρ-NH2, where `` CCK-8 * '' is the sulfated phenylalanine form ^ Ρ (CH2§O3H) MCVM ^ Ρ-NH2.

Connection No. Family Name / Sequence 4240 Exendin amylin Exendin4 (1-27) LATU (1-7), IAGD) CST (8-27), LATU (33-77) NSESTETZOZK0MEEEAUK1G »KKSYTATSUSKY50EKNK 0ΤΥΡΚΤΝΤ03ΝΤΥ-ΝΗ2 (Zeo Yu N0: 426) 4487 Exendin SSK8 EX (1-28) SSK8 (Υ2Υ303Η) NOESTETZOZZKOMEOBAUKPEEKNOU (ZOZN) M0IM0E-MN2 (ZEO 10 N0: 427} 4489 Exendin SSK8 EX (1-28} SSK8 (Υ2Γ0Η2303Η NSESTGTZOZEKMEEEUKUKPEYEKMOU (СН2303В) MYOEMΝΗ2 (ЗЕО Ю N0: 428) 5133 Exendin Amylin Exendin-4- (1-28) 01u-b1u-C1u-NAtu- (1-7) - [Agd11, Agd18] zST- (8- 27) -BAtu- (33-37), amide NON-TETOZEZOEMEAUKGGTEI ΕΗΚΟ0ΤΥΡΚΤΝΤΟ3ΝΤΪ-ΝΗ2 (Zeo Yu N0: 429) 5453 Exendin4 amylin Exendin-4- (1-28) -BA1a-bA1a-Ba1; amylin, amide ΗΟΕΟΤΓΤ30Ε3Κ0ΜΕΕΕΆνΚ1ΕΙΕΝ1ΚΝ (beta-A) (beta- Α) ΚΟΝΤΑΤΟΑΤ0Κ1ΑΝΓΕνΚ33ΝΝ1ΟΡνΐΡΡΤΝν03ΝΤΥ-ΝΗ2 (ZEO AND N0: 430) 5138 Exendin4 amylinsST-amylin Exendin-4- (1-28) -6etA1a-betaA1a-compound 10- amide NSESTETZOZEZEMEAUKUCHEECHEI (BETA-A) (BETA- Α) Κ0ΝΤΑΤ0νΐ. (3ΚΧι80ΕΙ.ΗΚΙι0ΤΥΡΚΤΝΤΟ5ΝΤΥ-ΝΗ2 (ZEO 10 N0: 431} 5401 ΡΥΥ · (3-36 amidinsST-amylin [# 5is- (LA1a) -ΡΥΥ- (3-36) _g amide] [# MH-be £ -Ly1-compound 10-amide] [# ΝΗ-ΟΝΤΑΤ0νθ5ΚΕ30ΕΕΗΚΕ0ΤΥΡΚΤΝΤ0ΞΝΤΥ-ΝΗ2] (ЗОО Yu N0: 432} [# 3is- (beta Α) ΙΚΡΕΑΡ0Ε0Α3ΡΕΕΙ, ΝΗΥΥΑ3ΕΚΗΥΕΝ0νΤΚ0ΚΥ-ΝΗ2] (ЗЕО U N0: 433)

- 78 014647

5403 ΡΥΥ (3-36) AMILINSST-amylin [# Ma1-B- (LA1a) -ΡΥΥ- (3-36), amide] [# 5-Prg- (LA1a) -connection 10, amide] [# 3-Rgr- (beta A) ΚΟΝΤΑΤθνΒεΗ130ΕΒΗΕ10ΤΥΡΚΤΝΤ6ΞΒΤΥ-ΝΗ2] (5Εζ> Yu N0: 434) [# Ma1-V- (beta- A) ΙΚΡΕΑΡΘΕΟΑ3ΡΕΕΟΝΕΥΥΑ31ΚΗΥΕΝθνΤΚ0ΚΥ-ΝΗ2] (ЗЕО U N0: 435) 5426 ΡΥΥ 13-36) amylin [# 3is- (LA1a) -ΡΥΎ- (3-36), amide] [# KH-0ez-L3l-Ba1: amylin, amide] [# ΝΗ-0ΝΤΑΤ0ΑΤ0Ε1ΑΝΡ0νΡ3ΕΝΝ00ΡνΐΡΡΤΝν6ΕΝΤΥ-ΝΗ2] (Zeo Yu N0: 436) [# 3is- (beta A) GKREARSEPAZREEDUUAZKNYUUTKOKU-ΝΗ2] (ZEO 10 N0: 433) 5457 amylinsST-amylin SSK8 [# 3-Prg- (bAl) compound 10, amide) [# Ma1-B- (bA1a) -CCC-8, amide] [# Ma1-B- (beta-A) SOUTH (СН2303Н) Μ6ΗΜ0Γ-ΝΗ2] (ЗЕО 10 N0: 437) [# E-Prg- (beta A) KSYTATS7SKS80EKN0TURKTIT68NTU-11N2] (ZEO U N0: 434) 4277 CCK8 Amylin 27 (p-CH2303) -PeSSKZ (26- 33), С, К, К, 15С1и, ААу (1-17), 18АГД, 8σϊ (18- 26), bAtu (32-37 , BAtu (1-17), 18Agd, 3cb (18-26), bAtu (32-37 ϋΡ (ρ- 0Η23Ο3) Μ <3 ^ ΟΓ6ΚΚΚΟΝΤΑΤ € ΑΤ0ΚΙ, ΑΝΕΙ.νΚΙ, ΟΤΥΡΒΤΝνδ5Ν ΤΥ-ΝΗ2 (Zeo Yu N0: 438) 5498 amylin 5ST-amylin [# HH-yez-b1-compound 10-amide] SSK8 [# 3is- (bA1a) -CCC8, amide] [# ΝΗ-0ΝΤΑΤθνθ0ΚΕ30ΕΕΗΕΕ0ΤΥΡΚΤΝΤ65ΝΤΥ-ΝΗ2] (ЗОО 10 N0: 432) [# 3is- (beta-A) OG (СН2303Н) Μ2ΝΜ0Γ- ΝΗ2] (Zeo Yu N0: 439) 5666 amylinvST-amylin [# IN-s1e2-b1l-compound 10, amide] SSK8 [# 3is- (LA1a) -Rye (CH25OZN) 2-0ez-MSYUM0E-SSK-8, amide] [# ΝΗ-0ΝΤΆΤ0νΟ0ΗΟ30ΕΑΗΚΟ0ΤΥΡΚΤΝΤΕ3ΝΤΥ-ΝΗ2] (ЗЕО Yu N0: 432) [# 3is- (beta-A) OG (CH2503H) -ΝΗ2 5493 AMZN Exendin4 [Ac-a1pAa-MZN- (bA1a) - (bA1a) -K = bcK] [Exendin-4- (1-28) - (bA1a) - (bA1a) -b3 (# b3b), amide [Αο-3Υ3ΜΕΗΓΚΗ5ΚΡν (beta-А) (beta-А) К #] (ЗЕО Ю N0: 440) [NESTROSZOKMEEEAUV.Р1ЕИ1КМ (betaА) (beta-А) К (# С) -ΝΗ2] (ЗЕО Ю N0: 441 )

- 79 014647

5499 AM5N amylinsST-amylin Ac-alpha-MZN-bA1a-bA1a-compound 10-amide Ac-3UVMENRKISKRU (beta-A) (beta- A) ΚΟΝΤΑΤθνΙ. (3ΚΙ> 30ΕΙιΗΒΙι0ΤΥΡΚΤΝΤ63ΝΤΥ-ΝΗ2 (Zeo Yu N0: 442) 5527 AMZN ΡΥΥ (3-36) [Ac-alpha-M8H-bA1a-bA1a-RUU- (3-36) _g amide] As-5UZMENRKN6KRU (beta-A) (beta- A) 1KREARSE0AZREYINUUAZINNYUTK0KU-K1N2 (Zeo HZ N0: 443) 5522 ΓΝ38 SSK8 [# 3-₽gr- (bA1a) -ΡΝ38, amide] [# Ma1-B- (bA1a) -CCC8, amide] [# 3-₽gr- (bata- A) RGNUZKTOKZZNUEEZZRGAZOZE (ZUEGGKRKI-НН2] (ЗЕО 10 N0: 444) [# Ma1-В- (Lе £ - A) EE '(CH2303H) MSIM0R-KH2] (ZEO Yu N0: 437) 5528 ΕΝ38 amylinsST-amylin [# Zis-bA1a-GY38, amide] [# MH-s1ez-b31-compound 10-amide] [# 3is- (beta-A) ΡΕΕΗΥ3ΚΤΟΚί, ΟΚ8ΝννΕΕΙ _Ι (23Ρί ^, Α503ΗΟΥΕ ^, Ι.ΕΗΡΚΝ-ΝΗ2] (Zeo Yu N0: 445) [# IN-Tsez-Zuz1- SSTATSYUZYOZNYOUNTURKTTS5UZU-4N2-MN2) 5497 ΓΝ38 [# $ - pgr- (bA1a) -ΓΝ38, amide] ΡΥΥ (336) [# Ma1-B- (bA1a) -ΡΥΥ- (3-36), amide] [# 3-Rgr- (beta A) GGNUZKT0KBKZYAUUEE 03RRA325K6 UGRKRVYA-UN2] (Zeo Yu N0: 444) [# Ma1-B- (beta- A) ΙΚΡΕΆΡ6ΕΟΑ3ΡΕΕΕΝΕΥΥΑ51ΚΗΥΕΝΕνΤΚ0ΚΥ-ΝΗ2] (Zeo Yu N0: 435) 5561 ΡΥΥ-ΝΡΥ amylins ST amylin [# 3is-compound 4883 / amide] [# LN- <5ec-b3l-compound 10, amide] [# Zis-? KRLENRS-EVAZREYAEUUA.5RL ”1MYTE0R.1-MN2 '(ZEO Yu N0: 446) [# ΝΗ- ΟΝΤΑΤθνΐ _Ι ΟΕ.Ε30ΕΕί1ΚΙ, 0ΤΥΡΜ'ΝΤΰ3ΝΤΥ-ΝΗ2] (ZEO Yu N0: 432) 5562 ΡΥΥ-ΝΡΥ amylinsST-amylin [# 5is-bA1a-compound 4883, amide] [# HH-s1ez-1 | y51-s €> unity 10, amide] [# 3is (beta A) ΡΚΡΕΗΡ0ΕΌΑ3ΡΕΕΕΑΚΥΥΑ3ΕΚΆΥΙΝΕΙΤΚ0ΚΥ-ΝΗ2] (Zeo Yu N0: 447) [# ΝΗ- ΟΝΤΆΤθνΕΟΕΕ30ΕΕΗΚΕΟΤΥΡΡΤΝΤΕ3ΝΤΥ-ΝΗ2] (Zeo Yu N0: 432)

- 80 014647

5582 amylinsST-amylin ΡΥΥ (3-36) [# Res-lib1 compound 10, amide] [Azr (#) - Rgo-Rgo-RgO-Rgo-Rgo-RGO-ΡΥΥ (3-36), amide] [# 0ΝΤΑΤ0νΕεΚΕ30ΕΕΗΒΕ0ΤΥΡΡΤΝΤ05ΝΤΥ-ΝΗ2] (ЗОО 10 N0: 432} [ϋ (#) РРРРРР1КРЕАРСО0АЗЕРЕЫНУУЗВУНУЩУТУКОЕУ- ΝΗ2] (ZEO 10 N0: 448) 5598 amylinsST-amylin ΡΥΥ (3-36) [# 0e5-lib3 ~ compound 10, amide] [Azr (#) - miniPEGZ-C1u-RUU (3-36), amide] [# 0ΝΤΑΤθνθ2ΕΙ, 30ΕΙ, ΗΗΙ, 0ΤΥΡΚΤΝΤ53ΝΤΥ ~ ΝΗ2] (ЗЕО ГО N0: 432) [ϋ (#) - miniPEGZ- εΐΚΡΕΑΡΟΕΟΆ5ΡΕΕΟΝΚΥΥΑ3ΟΚΗΥΟΝθνΤΕ0ΚΥ-ΝΗ2] (ЗЕ <3 Ю N0: 449) 5454 ΡΥΥ (3-36) ssk [# Ma1-B- (LA1a) -ΡΥΥ- (3-36), amide] [# 3-Prg- (LA1a) -CCC-8, amide] [# 3-Pgr- (beta-Ά) ϋΕ (0Η2503Η) Μ0ΜΜϋΓ-ΝΗ2] (ЗЕО ГО N0: 450) [# МА1-Вi- (beta- A) ΙΚΡΕΆΡ3ΕΟΑ3ΡΕΕΙ, ΝΚΥΥΑ3Ι, ΚΗΥΙ, ΝΕντΚ0ΕΥ-ΝΗ2] (ZEO GO N0: 437) 5603 SSK8 * ΡΥΥ (3-36) [# 2-Prg- (LA1a) -Pe (CH22OZN) 2-CCK8- (1-2), amide] [# Ma1-Bc- (LA1a) -ΡΥΥ (3-36) _{ amide] [# 5-Pgr- (beta-A) BG (CH25OZN) -ΝΗ2] [# Ma1-Vi (beta-Ά) 1KREARSE0AZREENKUAZKNUYUUTU0KU- ΝΗ2] (ZEO GO N0: 437} 5604 SSK8 * ΡΥΥΙ3-36) [# 3-Prg- (LA1a) -Tug2-SSC-8, amide] [# Ma1-B- (LA1a) -ΡΥΥ (3-36), amide] [# 3-Pgr- (beta-A) 0ΥΜ6ΉΜΒΕ-ΝΗ2] (Zeo Yu N0: 451) [# Ma1-Vi- (betλ- α) ΙΚΡΕΑΡ6ΕΟΑ3ΡΕΕ1ΝΚΥΥΑ3ΣΒΗΥ1ΝΐνΤΚ0ΚΥ-ΝΗ2] (ЗОО ТО N0: 437) 5550 SSK-8 [# 3-Prg- (bA1a) -CCC-8, amide] ΡΥΥ-ΝΡΥ [# Ma1-B- (bA1a) compound 4883, amide] [# 3-Pgr- (beta-A) PE (CH2303H) MSTSMVG-YN2] (Zeo Yu N0: 450} [# Ma1-B- (beta- A} ΡΚΡΕΗΡΟΕΟΑ3ΡΕΕ1ΆΡΥΥΑΞΕΚΑΥΙΝΕΙΤΕΟΚΥ-ΝΗ2] (ZEO 10 N0: 452) 5626 ΓΝ38 ΡΥΥ-ΝΡΥ [# 5is- (bA1a) - ΓΝ38, amide] [ΪΝΗ-Οΐγ- (bA1a) compound 4883, amide] [ΪΝΗ-C (beta A} RKRENR6EOAZRVEAYUAZAZU1YYTV0EU-UN2] (ZEO GO N0: 456) [# 3is- (BA1a) ΓΟΕΗΥ3ΚΤ0Κ0εκ3ΝννΕΕΟ03ΡΕΑ50εκ3ΥΡΟΕΕΡΕΝ- ΝΗ2] (ZEO GO N0: 445) 5567 ΡΥΥ (3-36) ΡΥΥ (3-36) [# Suz-C1- (31u-RUU (3-36), amide] [Azr (#) - Rgo-Rgo-Rgo-Rgo-Rgo-Rgo-ΡΥΥ (3-36), amide] [# 0ε0ΙΚΡΕΑΡεΕ0Α5ΡΕΕ0ΝΚΥΥΑ51ΚΗΥ0Ν1νΤΚ0ΚΥ] (ЗЕО GO N0: 453) Щ (#) ΡΡΡΡΡΡΙΚΡΕΑΡ0Ε0Ά3₽ΕΕΣΝΒΥΥΑ3ΣΒ.ΗΥ1ΝΣνΤΚ0ΕΥ] (ЗОО 10 N0: 448) 5568 ΡΥΥ {3-36) ΡΥΥ (3-36) [Azr (#) - miniPEG-01u-RUU (3-36), amide] [# Suz-C1u-01u-RUU (3-36), amide] [# ССС1КРЕА₽СООЗРЕЕЕЫНУУУУ31, КНУШЛТК0РУ] (ЗЕО ГО N0: 453} [ϋ (#) - miniPEG- 6ΙΚΡΕΑΡΟΕΟΑ3ΡΕΕΕΝΕΥΥΆ3ΙΕΗΥΕΝΕνΤΕΟΚΥ] (5Е0 Tb N0: 454) 5581 ΡΥΥ (3-36) ΡΥΥ (3-36) [# Suz-C1u-C1u-RUU (3-36}, amide] [Azr (#) - miniPEGZ-C1u-RUU (3-36), amide] [# С5С1КРЕРАОЕОЗРЕЕЫУУУЗЬУУУУ7ТК0КУ-ЕН2] (ЗЕО ГО N0: 453) [ϋ (#) miniPEGZ- εΐΚΡΕΑΡεΕϋΑ3ΡΕΕΣΝΚΥΥΑ3ΕΚΗΥΕΝΕνΤΚ0ΚΥ-ΝΗ2] (ЗЕО 10 N0: 449) 5425 ΡΥΥ (3-36} ΡΥΥ (3-36) [# YN-Suz- (bA1a) -ΡΥΥ- (3-36), amide] [# 3is- (bA1a) -ΡΥΥ- (3-36), amide] [# ΝΗ-0 (beta Α) 1KREARSBAZREYNUUAZIENY7TE0EU-UN2] (Zeo GO N0: 455) [# 3is- (beta A) ΙΚΡΕΑΡ0Ε6Α3ΡΕΕΝΕΥΥΑ3ΕΕΗΥΕΝΣνΤΚ0ΚΥ-ΝΗ2] (ZEO GO N0: 433)

Example 2. Binding Assays.

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

- 81 014647

Amylin binding assay.

Evaluation of the binding of certain typical compounds in accordance with this invention with amylin receptors can be carried out as follows in the membranes of adjacent nuclei obtained from rat brain. Male 8gadie-Oaayeteu® rats (200-250) grams are killed by decapitation. They take out the brain and place it in cold saline with phosphate buffer (PBS). Sections from the ventral surface are taken rostrally to the hypothalamus, connected laterally by the olfactory paths and located at an angle of 45 ° medially to these paths. The obtained basal sections of the forebrain, containing the adjacent nuclei and surrounding areas, are weighed and homogenized in 20 mM HEPE8 ice buffer (20 mM HEPE8 acid, pH adjusted to 7.4 by adding No. at 23 ° C). Membranes are washed three times in fresh buffer by centrifugation for 15 min at 48000 xd. The final membrane pellet was resuspended in 20 mM HEPE8 buffer containing 0.2 mM phenylmethylsulfonyl fluoride (PFMS).

To measure the binding of ¹²⁵ Lamylin (see Veitop! K. e! A1. Sap 1 RNuzyu 1 RNagtaso1. 1995 E.11; 73 (7): 1025-9), 4 mg tissue raw membranes were incubated with ¹²⁵ Vasilin at 12 -16 pM in 20 mM HEPE8 buffer containing 0.5 mg / ml bacitracin, 0.5 mg / ml bovine serum albumin and 0.2 mM FFMS. The solutions are incubated for 60 min at a temperature of 2 ° C. The incubation is interrupted by filtration through CE / B glass fiber filters (Na! Tap Shs., C11yop, Ν.Ι), which are pre-soaked for 4 hours in 0.3% polyethyleneimine to reduce the non-specific binding of radiolabeled peptides. Filters are washed immediately before filtration with 5 ml of cold FFB and immediately after filtration with 15 ml of cold FFB. Filters are removed and radioactivity is evaluated on a gamma radiometer with a count efficiency of 77%. Competition success curves are built on the basis of measuring binding in the presence of 10 ^-12 to 10 ^-6 M unlabeled test compound, and analyzed using curvilinear regression using a logistic equation with 4 parameters Cpr1o! rogdat; SgarNRAI 8oigage, 8ap I1edo).

SSCR receptor binding assay.

Evaluation of the binding of the compounds of this invention to the SCKR receptors is carried out in much the same way as described for amylin, except for the use of membranes derived from δΚ-Ν-MS cells, known as expressing SCKR receptors (Mi £$, K. e! A1 ., App ΝΥ Asai. 8sk 1992: 657, 106-16). Binding assays were performed as described for amylin, with the exception of using 13,500 cpm / hr-SCCR / well or 21.7 pM / well (AtegNat).

Adrenomodullin binding assay.

Adrenomodullin receptor binding can be investigated using EHECs that contain an adrenomedullin receptor (Ka! O e! A1., Eig I Rnagtaso.1. 1995, 289: 383-5) using the Regct E1tag A1p8aGepe ™ analysis for cyclic AMP using optimum 25-30000 cells per cell. The increase in cAMP levels in EHF does not exceed the same indicator for CHO cells. If desired, as such, CHO cells can be selected as a negative control, since they do not express an adrenomedullin receptor.

Calcitonin binding assay.

Calcitonin receptor binding can be tested using CHO cells or T47I cells that also express calcitonin receptor (Mi £ K. e! A1., App Ν Asai 8sk 1992, 657: 106-16 and Kiezsczeg K.E. ! a1. Mo1. RNagtaso. 1994, 46: 246-55), as is known in the art.

Leptin binding assay.

For W νίΙΐΌ bioassay, it is usually used to assess leptin binding and receptor activation (see, for example, νΜ ^, е! А1., 1997. Proceedings of IAI. Asai. 8sk I8A 94: 10657-10662). The alkaline phosphatase (AP) -leptin ('' ΟΒ) (AP-ΟΒ) fusion protein can be used to measure inhibition of leptin binding in the absence or presence of recombinant mouse leptin (positive control) or peptide in CΟδ-7 cells transfected with long ( signal) form of the mouse receptor ΟΒ (ΟΒ-КЕ). Signal transduction assays can be performed in CT1-7 cells transfected with an AR reporter and ΟΒ-KE constructs. The activity of secreted alkaline phosphatase (8EAP) in response to stimulation with murine leptin or a peptide can be measured by chemiluminescence.

Analysis of receptor binding Υ1.

Membranes are obtained from fused cell cultures of δΚ-Ν-MS cells, which endogenously express neuropeptide receptors Υ1. Membranes are incubated with 60 pM [^^ - human peptide ΥΥ (2200 Ci / mmol, Regk1pE1teg1 £ e 8aepsez) and with unlabeled active compound for 60 min at ambient temperature in a 96-well polystyrene plate. Then, the contents of the wells are collected in a 96-well glass fiber plate using a Regkt E1teg cell collection plate. The dried fiberglass plates are combined with a scintillator and counted on a Regkt E1tag scintillation counter.

- 82 014647

Тора2 Receptor Binding Assay.

Membranes are obtained from fused cultures of ZK-S-BE cells, which endogenously express neuropeptide receptors Υ2. The membranes are incubated with 30 pM of the [ ¹²⁵ 1] -human peptide ΥΥ (2200 Ci / mmol, Regk1iE1teg1Ge Zaepsek) and with an unlabeled active compound for 60 min at ambient temperature in a 96-well polystyrene plate. Then, the contents of the wells are collected in a 96-well glass fiber plate using a Regkt E1teg cell collection plate. The dried fiberglass plates are combined with a scintillator and counted on a Regkt E1tag scintillation counter.

Тора4 Receptor Binding Assay.

CHO-K1 cells are temporarily transfected with cDNA encoding the neuropeptide Υ4 gene and then after 48 hours the membranes are obtained from fusion cell cultures. Membranes are incubated with 18 pM of Inhuman Pancreatic Polypeptide (2200 Ci / mmol, Regk1eE1teg1Ge Zaepsek), and with the active compound for 60 min at ambient temperature in a 96-well polystyrene plate. Then, the contents of the wells are collected in a 96-well glass fiber plate using a Regkt E1teg cell collection plate. The dried fiberglass plates are combined with a scintillator and counted on a Regkt E1tag scintillation counter.

Тора5 Receptor Binding Assay.

CHO-K1 cells are temporarily transfected with cDNA encoding the neuropeptide да5 gene and then after 48 hours the membranes are obtained from fusion cell cultures. The membranes are incubated with 44 pM of the Inhuman peptide ΥΥ (2200 Ci / mmol, Regk1eE1teHGE Zaepsek) and with the active compound for 60 min at ambient temperature in a 96-well polystyrene plate. Then, the contents of the wells are collected in a 96-well glass fiber plate using a Regkt E1teg cell collection plate. The dried fiberglass plates are combined with a scintillator and counted on a Regkt E1tag scintillation counter.

Assay of binding to the CPP-1 receptor.

The activity of binding to the CPB-1 receptor and affinity can be measured using a binding rearrangement assay in which the source of the receptor is K1Bt5E cell membranes and the ligand is [ ¹²⁵ 1] CPB-1. Homogenized K1Bt5E cell membranes are incubated in 20 mM NEREZ buffer with a 40,000 cpm label [ ¹²⁵ 1] CPb-1, and changing the concentration of the test compound for 2 hours at 23 ° C with constant stirring. The reaction mixtures are filtered through a glass filter, previously impregnated with a 0.3% PE1 solution and washed with ice-cold saline solution with phosphate buffer. Bound pulses are determined using a scintillation counter. Binding affinities are calculated using the SgARRab Rpkt software (SgarRab Zoyuage, 1ps., Zap O1edo, CA).

Ίπ νίΙΐΌ receptor binding and receptor activation, including specific receptor activation by complex modules, for typical hybrids are shown in the table below.

Connection No. / name Receptor Binding Receptor activation SR-1 amylin SSR.R ct Υ2 CP1 cyclase CPI- 1 / C1P / ST cyclase ST cyclase Υ1 SSK --- source --- Rat amylin > 1000 0.05 21 3 > 1000 5 326 Human ΝΡΥ 0, 2 Compound 10 > 1000 0,03 2,3 0,03 > 1000 1.4 0.7 1000 Exendan-4 amide 0.5 > 1000 > 1000 0.3 10,000 1000 Exendin- (1-28) amide 0, 6 > 1000 > 1000 > 1000 0.8 Οοά ΡΥΥ (3-36) 0.08 874 1000 rat TsMi 24 SSK8 (sulfated tyrosine) 7.8 ΕΉ-38 SSK8 1000 - - - hybrids - - 4240 1.7 0.6 178 0, 5 > 1000 8 28 4277 4.4 0.04 1000 ΝΗ2- NbESTGTEZZKOME Cd > 1000 > 1000 > 1000 2.9

- 83 014647

EEAUKPENKKAM ΤΑΤΑνΤ, ΟΝΗ2 4487 49 1000 1000 1000 42, 1 4489 31 yoo 229, 5 5133 0.4 0, 2 63 0.03 6.4 8 5138 0, 3 0.2 113 oh 1 7.4 7.8 5401 0.09 0, 48 8.3 5403 0,052 0, 2 7.5 5425 643 12 2271 5426 46 0, 39 64 5 5453 6, 6 5.8 108.3 5454 93.9 83.3 5455 5457 0.21 sixteen 58 5493 ' thirteen 5497 0.163 54 98 0, 5 0.8 57 5499 0.02 3 5522 61.7 5527 0.2 5528. 2, 9 5550 151.6 175 5561 0 .11 2,3 27 5562 0,084 1.3 3,7 5567 0.8 5568 0.8 5581 0.8 5582 0,064 0.5 10 5598 0, 3 7, 6 5603 12,2 1000 5604 44.8 1000 5626 0.2 5666 0.16 0.43 yoo

Example 3. Analysis of food intake by mice.

Hybrid polypeptides in accordance with this invention can be tested for suppressing appetite in the analysis of food intake by mice, and for the effect on weight gain in mice with alimentary obesity (ΌΙ0). The experimental protocols are described below.

Female MH / 8 \\) kk mice (age 8-24 weeks) are grouped into cells with a 12-hour cycle of alternating light and darkness with the start of the light period at 06.00. Water and standard granular food for mice give ay Nyyit, except as indicated. Animals stop feeding at approximately 15.00 1 day before the start of the experiment. On the morning of the start of the experiment, the mice are divided into experimental groups. In a typical analysis, n = 4 cells with 3 mice / cell are used.

At time = 0 min, all animals were injected intraperitoneally with a carrier or compound, usually in an amount of about 10 to 75 nmol / kg, and immediately pre-weighed amounts (10-15 g) of standard feed were given. Food is removed and weighed at various points in time, usually after 30, 60 and 120 minutes, to determine the amount of food absorbed (Μο ^ 1еу, Е1оой е! А1., Lt. 1. Ryukyu.1.267: B178-B184, 1994). Food consumption is calculated by subtracting the weight of the food remaining at the point in time, for example, 30, 60, 120, 180 and / or 240 min, from the weight of the food that was given initially, at time = 0. Significant treatment effects identify ΑΝ0νΑ (p <0.05). Where there is a significant difference, the average values of the tested groups are compared with the average values of the control groups using the Dunnett test (Rpkt ν. 2.01, OgaryRay 8th ^ ake Shs., 8ap P1edo, Ca11Gogsha).

The activity in the analysis of food consumption and the sequence of source molecules used for the synthesis of hybrids are as follows:

- 84 014647

Food intake mice,% of the original Description Ya connect. 60 min EO50 (nmol / kg) Sequence 30 min 60 min 120 min 180 min Dose ΡΥΥ (3-36) one 3 ΙΚΡΕΑΡ6Ε0Α8ΡΕΕΙ.ΝΚΥΥ Α8ίΡΗΥίΝίνΤΚΟΚΥ-ΝΗ2 (ЗЕО IО N0: 58) -31 -38 • 40 -26 10 nmol / kg Exendin-4 5 NOESTRZOZZKOMEEEA νΚΙ_Ρ1Ε \ Νυ <Ν <3ΟΡ58 (3ΑΡ ΡΡ8-ΝΗ2 (5ЕО Ю N0: 66) -41 -60 -61 60 4.8 nmol / kg Exendin-4 (1-28) eleven 0.3 NESESTRZOEKOMEEEEA νΡίΡΙΕννΐΚΝ-ΝΗ2 (5ЕО Ю N0: 237) -fifty -62 -49 -49 16.3 nmol / kg Exendin-4 (1-28) [A1a5,1_ei14, Pbе25] 12 thirteen ΗΟΕ <3ΑΕΤ8ΰ15ΚΟΙ_ΕΕΕΑ νΚΙ_ΡΙΕΡΙΚΝ-ΝΗ2 (8Е0 Ю N0: 240) -53 -61 -fifty -53 16.7 nmol / kg Rat amylin nine ΚΟΝΤΑΤΟΑΤΟΡΕΑΝΡίνΚ 88ΝΝΙΘΡνίΡΡΤΝνθ8ΝΤ Υ-ΝΗ2 (Zeo Yu N0: 44) -58 -40 -36.5 -35.5 25 nmol / kg 1t Amylin (1-7) - ¹¹ ' ^1c Agd-zST (8-27) Amylin (33-37) 10 26 ΚΟΝΤΑΤΟνίΟΡίδΟΕΙΗΚ Ι.0ΤΥΡΚΤΝΤ <38ΝΤΥ-ΝΗ2 (8ЕО Yu N0: 396) -60 -47 -42.5 -32 25 nmol / kg SSK-8 26 ϋΥ (8Ο3) Μή / νΜϋΡ-ΝΗ2 (Zeo Yu N0: 55) -92 -56 -27 10 nmol / kg

Example 4. Analysis of body weight, redistribution of adipose tissue and muscle tissue.

Analyzes of body weight and related actions can be carried out as follows.

Alimentary obesity (ΌΙ0) in 8gadie-Oa ^ 1еу rats is a useful model for studying obesity and regulating energy homeostasis. These rats are obtained from a colony of rats (Cr1: CO® (8P) VK), which are prone to obesity in food with a relatively high fat content and energy, see, for example, Bett (1994) At. 1. Ruku 1. 267: K527-K535, But e! a1. (1997) At. 1. R11ukyu1. 273: K725-K730. Male rats ΌΙ0 receive from Cialis erythrocytes, guardianship, 1 fig. ^ 11ttd! Oi, MA). Rats were placed separately in small cages at a temperature of 22 ° C and a 12/12 hour cycle of alternating light and dark. Rats are given free access to food with a relatively high fat content (32% kcal from fats; Kekeags E | e1k к1226Β). Animals usually reach a body mass of about 500 g. Rats Leut 0 are taught to cells for 7 days. During 3 nights of addiction, animals receive one intraperitoneal (WB) injection of the vehicle. On the day of testing, rats receive one WB injection of the compound or vehicle (10% DMSO) when the dark cycle occurs for 6-14 subsequent nights. Food consumption is measured using an automated system for measuring food consumption (VOELO, Kekeags E | e1k) at intervals of 5 s throughout the course of the study. Body mass is recorded every night.

The composition of body tissues is measured before and after treatment with the drug using NMR (Ec1u Meb1cu8uetk, Noikoi, TX). To measure the body tissue composition of rats for a short time (~ 1 min), they are placed in well-ventilated plexiglass tubes, which are then inserted into a specialized rodent NMR apparatus. This allows you to calculate changes in actual grams of adipose and muscle tissue (for example, grams of fat after treatment - grams of initial fat mass = change in grams of fat mass) and changes in% of body tissue composition for fat and muscle tissue (for example,% of fat after treatment -% of initial fat mass = change in% of fat mass). All data are presented as mean values ± 8EM. Deviation analysis (ANOVA) and secondary tests are used to test for group differences. A value of P <0.05 is considered significant. Statistical analysis and plotting of the curves is carried out using PK18M® 4 ог og V V ^ ηbo \ νк (СарЬРаб 8о £ Щаге, 1p., 8a and E / edo, CA). In some Bole early studies used for analysis 8Υ8TAT® £ og \ V ^ ηbo \ νk (8uk! And! 8o £ Schage, 1is., Roth! Shstoib CA). For hybrids containing amylinomimetic, exendin, an analogue of ΡΥΥ aia1od and / or leptin, changes in body fat mass are not accompanied by a significant decrease in muscle mass. Graphs and results are usually presented as media-adjusted changes in percent body weight, fat mass, and change in protein mass.

In other experiments, male C57BB / 6 mice (at the start of the study, age 4 weeks) were fed foods high in fat (HP, 58% kcal as fat). After 4 weeks at this feed, an osmotic pump (ANe! # 2002) is implanted in each mouse, which subcutaneously delivers a predetermined dose of the hybrid polypeptide continuously for two weeks. Body weight and food intake are measured weekly (8ιΐΓ \ νίΙ е! А1., Ме! Аобо11кт-С11шса1 аиб Ехрптеи! А1, 44: 645-51, 1995). The actions of the tested compounds are expressed as mean values of +/- CO% change in body weight (for example,% change in initial weight) in at least 14 mice per treatment group (p <0.05 ANOVA, Dunnett test, Rpct ν. 2.01, CarbPab 8oP \ uage 1is., 8ai E / edo, SaShogsha).

- 85 014647

Hybrid Exendin / RΥΥ.

Typical hybrid polypeptides in accordance with this invention are synthesized using limited in the C-terminal region of exendins (for example, exendin-4 (1-28) or AC ^ LeyurNeexendin-4 (1-28)) and limited in the Ν-terminal region of P bending from 18-36 to 31-36 areas. As such, typical hybrid polypeptides typically contain two modules, where the first module is a fragment of an exendin-4 analog and the second module is a peptide enhancer selected from the restrictions of PΥΥ. For comparison, β-alanine dipeptide spacers are also introduced between the blocks constituting the peptide in several variants (see table 4-1).

Table 4-1 Exendin / PΥΥ Hybrids, Receptor Binding Data, and Their Effect in Food Consumption Analysis

Receptor Binding (1C50) nM Mouse food intake% of baseline Description No. conn. Υ2 HIDDEN thirty min 60 min 120 min Dose RUU (Z-Zb) (ZEO 10 N0: 58) 0.04 - -31 -38 -40 -26 'A1a, ¹⁴ Ley, ²⁵ Pye - exendin-4 (1-28) (Zeo Yu N0: 240) - 1.9 -fifty -62 -49 -49 ^b A1a, ¹⁴ 1st, ²⁵ Rieksendin-4 (1-17) ΡΥΥ (18-36) (Zeo Yu N0: 16} - 1.7 1000 -4 -eleven -10 10 nmol / kg ^b A1a, ¹⁴ bj, ²⁵ Ryeeksendin-4 (1-28) RUU (22-36) (Zeo Yu N0: 14) - 17 2.1 21 nine "4 10 nmol / kg ⁵ A1a, ¹⁴ Ley, ²⁵ Pyeeksendin-4 (1-28) -RYA1arA1a-RUU (22-36) (Zeo Yu N0: 17) - nine 0, 81 -3 -5 -22 10 nmol / kg ⁵ A1a, ¹⁴ Ley, ^{2 !,} Pyeeksendin-4 (1-28) ΡΥΥ (25-36) (8Е0 10 N0: 15) 2 BUT BUT 8 -thirteen -thirty 10 nmol / kg ^s A1a, Leu ^{14, r5} Reekzendin-4 (1-28) -0A1a₽A1a-Ruu (25-36) (ZEO 10 N0: 18) 3 thirteen 0, 22 -nine -25 -42 10 nmol / kg ⁵ A1a, ¹⁴ Ley, ^r5 RPeeksendin-4 (1-28) -pA1arA1a-RUU (31-36) (Zeo Yu N0: 19) 4 1000 0.25 -14 -36 -52 10 nmol / kg exendin-4 (1-28) - ΡΥΥ {25-36) (5E £ 10 N0: 8) - sixteen 0.29 -thirty -37 -45 10 nmol / kg exendin-4 (1-28) -pA1aβΆ13-ΡΥΥ (25-36} (8Е0 U N0: 5) - 7.8 0, 16 -24 -40 -52 10 nmol / kg exendin-4 (1-28) - pA1arA1a-RUU (31-36) (Zeo Yu N0: 6) - 1000 0.19 -49 -56 -61 10 nmol / kg

As shown in the table. 4-1, certain exemplary compounds of the invention show efficacy in food intake analysis. Certain peptides were also tested at a dose of 75 nmol / kg in an EH assay, and they were more effective than PΥΥ (Fig. 1). According to data obtained for other hybrids, hybrids can retain the ability to bind to one, two or more receptors that recognize the original molecules. Hybrids are designed to recognize at least one receptor from each parent molecule, or from only one parent molecule, if desired. According to data obtained for the other hybrids presented here, the use of a linker (which can act as a spacer between each adjacent part of the hormones) can provide an improved effect, including binding to the receptor (s) and 1p νίΙΐΌ and 1p νί \ Ό action, such as weight loss . The results presented here show that the H-region PΥΥ can modulate the action.

Exendin / Amylin Hybrids.

Other typical hybrid polypeptides in accordance with this invention are obtained from a C-terminal region of exendin (1-27) (BEP ΙΌ NO: 236) bounded in the C-terminal region of amylin peptides (e.g., amylin (1-7) (BEP ΙΌ NO: 217), ^ Ma-amylin (1-7) (BEP ΙΌ NO: 285) and amylin (33-27) (BEP ΙΌ NO: 243), and optional fragments of ccT (for example, ccT (8-10) , ^11.18 Αγ§kST (8-27) (BEr ΙΌ NO: 289) and ^ NW / ^ Agd-kST (8-27) (BEr ΙΌ NO: 286) If both hybrid polypeptides were very active in suppressing appetite (see tab. 4-2), superior to the rat

- 86 014647 amylin in the same dose, the onset of action differs from the activity profiles of the starting molecules (data not shown). At a dose of 1 nmol / kg, compound 5 is as effective as rat amylin.

Table 4-2

Exendin / Amylin Hybrids and Their Effect in P1 Analysis

Receptor Binding Assay Mouse food intake% of baseline Description LR conn. SR-1 amylin CORR CT thirty min 60 min 120 min Dose Exendin-4 (1271-amylmn (1'7) - ^11.18 AgdzST (8-27) amylin (33-37) 5 (ΞΕΟ 10 Ysj 25) -24 -40 -48 25. nmol / kg Exendin-4 (1- 27) - ^{2 &} apos A1aanilin ⁷ (1-7) FTZ (8-10) 6 (5Е0 Yu ΝΟ. 26) -40 -59 -66 25 nmol / kg Exendin-4 (128) -Beta1AbetaA1aamylin (1-7) 11.18AhdeST (8-27) Amylin (33-37) 14 (5Е0 Yu N0: 33) 0, 3 0.2 113 0.1 -5 -thirty -51 3 nmol / kg Exendin-4 (1- 28) “C1u-C1u- C1u-amylin (1- 7) -11.18 Agg- 5CT (8-27) ~ amylin (33-37) 15 (£ E <2 U N0: 312} 0.4 0.2 63 0.03 -8 -36 -51 3 nmol / kg Exendin-4 (127) -amylin (17) -11.18 AgdvST (827) “amylin (33-37) 5 (ZEO W N0: 25) 1/7 0.6 178 0.5 -twenty -10 -26 3 nmol / kg

Both compounds also showed excellent screening efficacy in assay ΌΙΟ (FIG. 2).

Other typical compounds are analyzed for effects on blood glucose levels and in the analysis of food intake. These tests include compounds 14 and 15. Compound 14, which includes the A1A linker, is ²⁹ BA1a-bA1a-exendin (1-28), 1-amylin (1-7) - ^11.18 Agd-5C1 (8-27) - y-amylin (33-37) (8EC ΙΌ ΝΟ: 33), (alternatively called exendin (1-28) -BA1a-bA1a-1-amylin (1-7) - ^11.18 Agd-5C1 (8-27) β-amylin (33-37), and compound 15 contains a C1u linker: ²⁹ C1uC1uC1u-exendin (1-28), β-amylin (1-7) - ^11.18 Agd-5C1 (827) -1-amylin ( 33-37) (8EC ΙΌ ΝΟ: 312) A longer exendin (1-28) provides improved action compared to exendin (1-27).

A blood glucose test is performed to test the effect on lowering blood glucose levels. Female mice ΝΙΗ / 8 \ νί55 (age 8-20) are placed in cages with a 12: 12-hour cycle of light and darkness, including light at 06.00. Water and standard granular feed for mice give ab Nb, except as indicated. On the morning of the experiment, the animals are divided into experimental groups and stop feeding at approximately 06.30 hours. In a typical study, cells are used in an amount of n = 2 with 3 mice / cell. At time = 0 min, a blood glucose sample is taken immediately after that, an intraperitoneal injection of the carrier or compound in an amount of about 1 to 25 nmol / kg is made. Blood glucose is measured at 30, 60, 120, 180 and 240 minutes. The percentage of the level before treatment is calculated by dividing blood glucose at a point in time, for example, 30, 60, 120, 180 and / or 240 min by blood glucose at time = 0 t. A significant treatment effect is identified with ANVA (p <0.05). If there are significant differences, the average values of the tests are compared with the average values of the control using the Dunnett test (Рпзт ν. 4.01, SgaryRab 8оП \ гage 1пс., 8ап П1едо, СайГогша). The results for typical compounds are shown in FIG. 5A. The points represent the mean ± SD. The peptide is administered intraperitoneally (WB) at time ί = 0 immediately after collection of the initial sample from 2 h fasting mice ΝΙΗ / 8 \ νίδ5. Samples are taken at 1 = 30, 60, 120, 180 and 240 minutes. Blood glucose is measured on Οικ ToisN® and Iga® (L1 £ e8sap, 1 ps., Goipkop &. Gobkop Sotrapu, Mbryak, CA). * p <0.05 to the carrier (control); ANVA, Dunnett test.

Analysis of food intake is carried out as described above. The results are presented in FIG. 5B. Dots mean mean ± SD for n = 4 cells (3 mice / cell). The peptide is administered intraperitoneally (WB) in 1 = 0 starving mice ΝΙΗ / 8 \ νίδ5 starving overnight. Food is given immediately after injection and the amount of food eaten is measured at 1 = 30, 60 and 120 minutes. * p <0.05 to the carrier (control); ANVA, Dunnett test.

Starting compound 10 and exendin compounds have the opposite effect in glucose analysis. Their combination is expected to cause a neutralizing effect or, at best, a decrease in the effect of a more powerful source compound. However, in the analysis of glucose typical hybrid

- 87 014647 compounds were as effective as exendin (1-28), and showed a longer effect.

Based on data on food intake, typical compounds are anorexigenic. Their activity is usually higher than that of the starting compounds, which give separately. The activity is comparable to the activity of the starting compounds given together, but at a dose of half the dose of the drug (3 nmol / kg of hybrid versus 6 nmol / kg of total for the jointly introduced starting compounds); which also demonstrates the superiority of hybrids. Adding a linker increases the activity of hybrids. The linker C1u-C1u-C1u is more efficient than the linker ZAa-ZAa in this case.

Hybrids of exendin / SSK-8.

Other typical hybrid polypeptides in accordance with this invention are obtained from the complete or restricted in the C-terminal region of exendin-4 by attaching to the кон-terminal region of CCK8 either directly or through a linker, while retaining the Ν-terminal amide CCK-8. (tab. 4-3). Further, certain hybrids are prepared by incorporating the natural Tyr (LP ₃₎ and the other hybrids obtained by introducing a more stable group Rye _(CH2 LP _3). All obtained hybrid polypeptides are active in inhibiting food intake (Table 4-3).

Table 4-3

Exendin / SSK-8 Hybrids and Their Effects in the Analysis of Food Consumption

Receptor Binding Assay (1C50) iM Mouse food intake § FROM THE INITIAL Description yes conn Th 30 min 60 min 120 min Dose Ekeendin-4 [1-28) amide 11 (ZEO 10 N0: 237} 0, 63 Exendin-4-SSK-Z - (Zeo Yu N0. twenty) -12 -28 -28 10 nmol / kg Exendin ~ 4 (1-28) - SSK-9 7) Zeo Yu N0. 21) 75 -twenty -36 -45 10 nmol / kg Exendin-4 (1-28) SSK-8 [Pb (CH ₂ ZO ₃ ) 1 8 (ZEO 10 N0. 22). 31 -24 -47 -66 10 nmol / kg Exendin-4 (1-28) [8-amino-3,6-dioxaoctanoyl] CCK-8 9) Zeo Yu N0. 23) -12 -28 -40 10 nmol / kg

Typical exendin / CCK-8 hybrid polypeptides are tested in the analysis ΌΙϋ at a dose of 25 nmol / kg (Fig. 3A and 3B). The data show initial weight loss, followed by the opposite effect for all compounds. Interestingly, the opposite effect can be reduced in hybrids containing a more hydrolytically stable Pye residue (CH ₂ ZO ₃ ) (compare Figs. 3A and 3C), as well as in hybrids containing a linker, for example, an 8-amino-3,6 linker -dioxoctanoyl between the residues of exendin and CCK (compare Fig. 3A and 3B). A tenfold amount of SSK-8 (250 nmol / kg / day) is necessary to obtain a change of 2.8% on day 2, when you return to the control level of NOT a diet on day 7.

Amylin Hybrid / ΡUU.

Amylin / ΡUU hybrid polypeptide is synthesized in such a way that it contains limited segments of each peptide. 1n ^ sho action in the analysis of food consumption is shown in table. 4-4.

Table 4-4

Amylin Hybrid / ΡUU

Mouse food intake% of baseline Description thirty MIN 60 min 120 min Dose Amilin (1-18) -ΡΥΥ (19-36) N0. 38) (Zeo Yu -thirteen -14 -thirteen 25 nmol / kg

To make sure that the typical hybrid polypeptides of the invention are more effective than their parent compound peptide hormones, the type compounds are tested in a food intake analysis at the minimum effective dose of a more active parent molecule. The results are shown in FIG. 4A and 4B, where the action of the combined parent peptides is also compared (compounds 1, 11 and 12 are complex peptide hormones or fragments thereof). The data show that several peptides are at least as effective as the combined parent peptides. In parallel with studies of w νί \ Ό. 1p ν 11th receptor binding and functional analysis (cyclase activity) is performed for all compounds (data not shown).

Amylin-KST / Leptin Hybrids.

Get other typical hybrids that contain a fragment of the leptin peptide connected to compound 10, described here by the chimera amylin-kST-amylin. Compound 16 is [Zeg117, Beyi119] leptin (116-122) -amylin (1-7) - [ ^11.18 Agd] kST (8-27) -amylin (33-37) (ZEO ΙΌ Νϋ: 397). The compound binds (ACP = receptor binding assay) amylin and CT receptors and, to some extent, binds to the SCCR receptor. The compound is also capable of activating the CT receptor (C1A assay).

Compound Connection No Analysis 1C50 [Zeg117, ybey119] leptin (116-122) amylin (1-7) - ( ^11.10 Agd] cST (8-27) amylin (33-37) (ZE £) N0: 397) sixteen Amp Amylin 0.04 nM sixteen ASR SSKR 81 nM sixteen ST 0Υ0ΣΑ5Ε (Ο1Α) 2.2 nM sixteen ASR ST (C1A) 0.063 nM sixteen ASR SR (ΚΙΝ) 1000 nM

The action of this type of molecule is tested in an analysis of food intake, as described here. Although leptin is not active in this test, compound 16 is anorexigenic at 1 mg / kg. Compound 16 also surpasses rat amylin (at a dose of 25 nmol / kg) in anorexigenic action. Although compound 10 reduces food intake by 91-95% compared to control, it is much more effective / compound 16 reduces total absorption by 34-38% of control. Other typical examples include the direct connection of the кон-terminal region of the leptin peptide with the same amylin (1-7) [ ^11.18 Agd] kST (8-27) -amylin (3 3-37) compounds.

Hybrids SSK / amylin-kST.

A typical CCK hybrid with the amylin-KST chimera demonstrates the specificity and activation of the corresponding receptor. A typical compound having the IR sequence (PC11; 5O _; ) \ 1С \ ХЛМ) 1 <11ОШС \ ТЛТСЛТО1и.А \ Е1Л1иПТ ¥ РНТ \\ 'С5 \ Т ¥ (8Еф Ш ΝΘ: 398) has 1С50 0.044 nM in the binding analysis with CT receptor, 4.4 nM in the analysis of binding to the SSCP receptor, 0.083 nM in the analysis of binding to the amylin receptor and 1000 nM in the analysis of the CPb receptor cyclase (ΚΙΝ).

Example 5. Reducing food intake, body weight and weight gain.

Typical hybrid polypeptides in accordance with this invention are further tested for suppression of appetite, weight loss and weight gain in mice and rats, as described here. The starting compounds are also analyzed, either in pure form or in combination. In FIG. Figures 9-21 show the νί \ Ό action of typical hybrids in the analyzes described here.

1p νί \ Ό inhibition of food intake in mice by typical hybrids and their starting compounds is shown in the following table. Values are the percent inhibition of food intake after infusion at indicated time periods, nz does not mean significantly compared to the vehicle.

Compound / Dose Time (min) Rat amylin thirty 60 120 180 240 25 nmol / kg 46 35 21 17 7 250 nmol / kg 70 53 38 thirty 300 nmol / kg 88 90 51 21 Human ΝΡΥ 37.5 nmol / kg 21 29th 0 0

- 89 014647

Compound 10 thirty 60 120 180 240 10 nmol / kg 24 26 27 21 25 nmol / kg 57 46 31 nineteen nine 30 nmol / kg 64 55 33 100 nmol / kg 81 89 55 300 nmol / kg 93 96 88 Exendin-4 Amide thirty 60 120 180 240 3 nmol / kg 40 48 61 10 nmol / kg 46 61 67 67 30 nmol / kg 48 61 68 Exendin-4 (1-28) amide thirty 60 120 180 240 3 nmol / kg 7 24 16, 3 10 nmol / kg 23 41 41 30 nmol / kg 34 51 60 100 nmol / kg 45 60 72 300 nmol / kg 41 57 65 RUU (3-36) thirty 60 120 180 240 3 nmol / kg NA 7.3 10 6 0 7.5 nmol / kg 32 39 34 31 8 nmol / kg 48 40 twenty thirteen 10 nmol / kg 28 36 38 thirty thirty 25 nmol / kg 40 51 54 43 30 nmol / kg 45 33 58 54 46 100 nmol / kg 41 35 56 56 52 300 nmol / kg 37 34 52 51 51 SSK8 sulfated Tug thirty 60 120

- 90 014647

) 3 nmol / kg 49 26 0 10 nmol / kg 92 56 27 30 nmol / kg 92 73 43 100 nmol / kg one hundred 78 59 sska thirty 60 120 1000 mcg / kg ns ns ns —HYBRIDS— 4240 thirty 60 120 180 240 10 nmol / kg 37 fifty 60 25 nmol / kg 24 40 48 4277 thirty 60 120 180 240 172 nmol / kg (1000 mcg / kg) 76 70 fifty ΝΗ2- ΗεΕ6ΤΕΤΞΏΕ3Κζ) ΜΕΕΕΑνΗΕ GIEEKKAITATΑνΣΟ-ΝН2 thirty 60 120 180 240 10 nmol / kg 33 49 57 25 nmol / kg 40 59 66 4437 thirty 60 120 10 nmol / kg 0 36 45 4489 thirty 60 120 3 nmol / kg NA 25 ns 10 nmol / kg NA 47 66 5133 thirty 60 120 180 240 3 nmol / kg 7.5 36 51 5138 thirty 60 120 180 240 3 nmol / kg 4.7 thirty 38 5401 thirty 60 120 180 240 25 nmol / kg 32 52 59 57 5403 thirty 60 120 180 240 25 nmol / kg 52 68 65 55 5425 thirty 60 120 180 240 25 nmol / kg 8 27 31 eighteen

- 91 014647

5426 thirty 60 120 180 240 2 5 nmol / kg 49 66 55 45 5453 thirty 60 120 180 240 25 nmol / kg 55 66 71 61 5454 thirty 60 120 180 240 25 nmol / kg 39 33 28 twenty 5455 thirty 60 120 180 240 25 nmol / kg 28 33 39 5457 thirty 60 120 180 240 25 nmol / kg ns ns ns 5493 thirty 60 120 180 240 25 nmol / kg 54 59 68 64 5497 thirty 60 120 180 240 25 nmol / kg 54 63 41 ns 5498 thirty 60 120 180 240 25 nmol / kg ns ns ns ns 5499 thirty 60 120 180 240 25 nmol / kg 40 32 25 5522 thirty 60 120 180 240 25 nmol / kg 5527 thirty 60 120 180 240 25 nmol / kg 40 32 25 5528 thirty 60 120 180 240 25 nmol / kg 56 55 fifty 5550 thirty 60 120 180 240 25 nmol / kg 0 34 0 5561 thirty 60 120 180 240 25 nmol / kg 0 42 47 5562 thirty 60 120 180 240 25 nmol / kg 5567 thirty 60 120 180 240 25 nmol / kg 0 0 0 5568 thirty 60 120 180 240 25 nmol / kg 0 39 48 5581 thirty 60 120 180 240 25 nmol / kg 0 45 32 5582 thirty 60 120 180 240 25 nmol / kg 0 58 57 5598 thirty 60 120 180 240 25 nmol / kg 0 fifty 58 5603 thirty 60 120 180 240 25 nmol / kg 0 0 0 5604 thirty 60 120 180 240 25 nmol / kg 0 0 0

W νί \ Ό body weight inhibition in the модели0 mouse model for typical hybrids and their parent compounds is shown in the table below. Values are given as percent inhibition of body weight after infusion at indicated time periods.

- 92 014647

Day Compound Units 2 7 14 21 28 35 42 49 56 63 70 77 84 RAT AMILINE Zol / kg / s thirty 1.9 1,5 2.1 2,5 2.2 75 1,5 1.4 2.1 one hundred 1,6 1.8 3, 1 3.8 4.4 6.9 8.6 9, 6 10, 4 10.3 11,4 12 13,4 300 1,1 -0.7 -0, 9 -1.5 -1.5 2 7 SOED 10 nmol / kg / s 150 2.2 -2.4 2 7 14 EXENDIN-4 nmol / kg / s 2,5 7 6.3 7.6 ΡΥΥ (3-36) nmol / kg / s 2 7 14 25 2.6 3,1 75 4.6 4.7 5.9 one hundred 4.4 3.4 3.9 250 nine 10.1 4240 nmol / kg / s 2 7 75 9.6 10.1 ΝΗ2 “ NBEETHTZOZE MORE EAUKPEYEKKAYTA ΓΆνΣ (1-ΝΗ2 nmol / kg / s 2 7 75 11.6 12, 1

The inhibition of body weight was demonstrated in the rat model No. 0 for typical hybrids and their parent compounds in the following table. Values are presented as percent inhibition of body weight after infusion at indicated time periods.

Day Compound / Dose (nmol / kg / s) 3 7 14 21 28 35 42 49 56 63 70 77 84 RAT Adilin 0, 7 sixteen thirteen sixteen 2, 1 1 s 2.2 2, 2 2.2 2.2 2, 3 4 5, 4 5,4 6.2 5, 6 2, 9 3.9 4.2 4.8 7 5.7 7.3 7.9 8.3 18,4 5.9 7.5 6, 7 6.5 23 7.8 9,4 8, 8 8.8 9.5 10 9.8 9.1 69 7.6 9.3 11.1 12,2 14,4 14, 9 15.4 15, 7 16,2 15, 8 70 7, 6 8, 9 10,2 11.8 3 7 14 21 28 35 42 49 56 63 70 77 84 SOED 10 0, 29 2, 3 3.8 0, 9 5.1 7.1 2, 9 7.7 10.0 12.0 11, 5 11, 6 12.1 11.7 12, 3 7.5 11, 6 14, 9 17.9 19, 5 20, 8 22.1 23.3 24.2 26.2 26 eleven 8, 5 9, 9 69 11, 5 13.7 15.6 17 69 11.5 13, 7 15.6 17 3 7 14 21 28 EXENDIN4 (1-28) 0, 8 nz 2, 6 14 nz nz nz 3 7.5 8, 3 8.5 9.2 eleven 6.5 6.8 26 10, 4 thirteen 14.6 15.7 3 7 14 21 28 35 42 49 56 63 70 77 84 EXENDIN-4 0.7 9, 6 8, 4 6, 3 4.3 2, 4 12.1 10, 8 10 8.4 7 13.9 12.8 11, 4 10, 6 6.3 12, 2 13.7 14,4 14,4 19.1 18.6 18, 9 18, 5 nineteen 18, 1 21 11.9 13,2 13.3 12, 6 RAT ΝΜ0 24 2 7 300 2, 3 1,6

- 93 014647

3 7 14 21 28 ΡΥΥΟ-36) 67 6.7 7, 3 6.2 5, 8 67 5.5 7, 3 7.8 nine 67 4.9 5.2 5 4, 6 01-38 2 7 300 3,7 2.7 5138 ¹¹ . 7.5 9.3

The effect of typical hybrids and their parent compounds on inhibition of body weight in the rat model (daily injections) in the following table is demonstrated. Values are given as percent inhibition of body weight. Bold I indicates a significant deviation from the media.

Dose DAY one ! Day ! 5 Day 6 Day 7 Day 10 DAY 14 RAT AMILIN 25 nmol / kg nz 1 nz NZ NZ NZ NZ COMPOUND 10 15 nmol / kg 1.7 3.2 4,5 6.2 EXENDIN-4 15 nmol / kg 2,8 ¹ 6/1 7.8 9.3 12.0 5138 15 nmol / kg 1.3 1 5.2 5.8 7.9 10.7 5401 15 nmol / kg 5403 15 nmol / kg 5426 15 nmol / kg 5454 15 nmol / kg 5455 fifteen nmol / kg 0, 7 > 2.6 3.2 5493 15 nmol / kg one 3.3 5.1 5499 15 nmol / kg 0, 5 0.9 one 1.3 5568 15 nmol / kg 0.5 '0.5 0, 5 5581 fifteen nmol / kg 0.6 0.8 1,2 5582 15 nmol / kg 0.3 1 0.7 one one one 5598 15 nmol / kg 0.7 1.7 2,5

W νί \ Ό decrease in body weight and weight gain was demonstrated for typical hybrids and their starting compounds for inhibition of body weight in rat models for both infusion during the indicated period and for injections once a day during the indicated period, presented in the table below . Values are given as percent inhibition of body weight. The percent inhibition of food intake during the 8-hour nighttime feeding period on day 6 is also measured. Hybrids were given at a dose of 15 nmol / kg / day, unless otherwise indicated. Body weight: ~ 2% difference is statistically significant. Food Consumption: ~ 20% difference is statistically significant.

- 94 014647

Continuous infusion 1 week, MT loss Daily single injection 1 n & d. MT loss Inthib and PP & 8 h Source Exenatide eleven% 8% 42-5-52% amylin 6% 0% 0% Compound 10 eleven% 3% 32% -57% ΡΥΥ3-36 0-15 0% 0% Compound 4883 BUT BUT ΡϊΎ-amylin 5401 ΡΥΥ-compound 10 8% 5403 ΡΥΥ- connection 10 7% 5582 ΡΥΥ- connection 10 one% fifteen% 5598 ΡΥΥ- connection 10 3% 54% 5561 ΡΥΥ / ΝΡΥ-connection 10 7¾ 5562 ΡΥΥ / ΝΡΥ-connection 10 nine% 5426 RUU-amylin one% Exendin amylin 5138 EX-connection 10 10¾ (11 nmol) 6% 54% 5133 EX-connection 10 5454 Ex amylin thirteen% 12%

Example 7. Decrease in blood glucose.

The effect of typical hybrids and their starting compounds on the decrease in blood glucose in the CTTT analysis during the indicated periods in Leush rats was shown in the table below. Values are given as a percentage of a decrease in blood glucose. Highlighting in bold means significantly different from media.

% reduction Compound Dose Time (day) Source fifteen thirty 60 120 RAT AMILIN 23 nmol / kg / s day 26 4 BUT 2,3 19, 2 2.1 69 nmol / kg / s day 14 6 -0.2 eleven 5.9 1,2 COMPOUND 10 11 nmol / kg / s day 7 5, 1 thirteen 14 thirty 26 69 nmol / kg / s day 14 5 7 5, 5 9.8 5.9 EXENDIN4 (1-28) 7.8 nmol / kg day 2 6 11, 6 BUT -21.4 2 -fifteen 11 nmol / kg / s day 7 fifteen -one -8 5 -one 5138 11 nmol / kg / s day 7 thirteen -one 2.6 14 sixteen

Example 8. The effect on the emptying of the stomach.

The effect of typical hybrids and their parent compounds on gastric emptying in Lut rats during the periods indicated in the table below is shown. Values are given as percent inhibition of gastric emptying and percentage of missed acetaminophen.

- 95 014647

Compound Dose Time research % skipped % inhibition RAT AMIPIN 69 nmol / kg / s 21 day 55 45 100 mcg / kg / s 26 days 76 24 COMPOUND 10 69 nmol / kg / s 21 day 87 thirteen EXENDIN-4 (1-28) 30 mcg / kg / s 26 days 94 6

Example 9. PPP polypeptides. strongly and chronically suppressing food intake.

Female mice MH ^ hzvv (age 8-24) are placed in cells with a 12: 12-hour cycle of light and darkness. including lighting at 06.00. Water and standard granular feed for mice give ab 11b1! Um. except as noted. Animals stop feeding at approximately 15.00 h 1 day before the start of the experiment. On the morning of the experiment, animals are divided into experimental groups. In a typical study, cells are used in an amount of n = 4 in 3 mice / cell.

At time = 0 min, all animals receive an intraperitoneal injection of a carrier or compound in an amount of about 10 to 100 nmol / kg. and immediately after that they are given a pre-weighed amount (10-15 g) of standard food. The food is removed and weighed at 30. 60 and 120 minutes to determine the amount of food eaten (Mogueu. P1ooB e! A1 .. At. E Pbu1. 267: Ya 178Ya184. 1994). To study the strong effect of PPP polypeptides on food intake, the amount of ingested food is calculated by subtracting the weight of the food. remaining at 30. 60. 120 and 180 min of the weight of the food. provided initially at time = 0. Same. water absorption is calculated by subtracting grams of water. remaining at specified times. of the weight of water in grams. provided initially. To study the effect of polypeptides on long-term (chronic) food intake and / or body tissue composition. measure the amount of food. absorbed in two weeks. To conduct a PPP study of polypeptides for long-term food intake and / or body tissue composition. mice are placed individually for 1 week before treatment. During the experiment, food intake and body weight are monitored daily. During the treatment period, the carrier (50% dimethyl sulfoxide in water) and PΥΥ (3-36) (1 mg / kg / day) are administered by continuous subcutaneous (SC) infusion using osmotic pumps A1he1® (Biges! Cons .. Siregipo SA; models 1003B. 2001 and 2004 for research during

3. 7 and 28 days respectively). placed under the scapula under isoflurane anesthesia. At the end of each study, animals are killed after 2-4 hours of fasting with an overdose of isoflurane. Blood is collected in syringes washed with β-heparin through a puncture of the heart and the plasma is immediately frozen. In some studies. the composition of body tissues is determined using x-ray absorptiometry with two energy sources (BEXA; P1x1Miv. CE Lipag). In some studies, the composition of body tissues (for example, the amount of fat and protein) is measured before and after treatment using a rodent NMR apparatus (EcNoMB [-700 ™). where the animals are placed in a restriction tube and then placed in an NMR apparatus for 2 minutes and the amount of fat and muscle mass (in grams) is calculated. The bilateral epididymal fat layer and subscapular drilling adipose tissue (BAT) can be excised from animals and weighed. Excised liver samples can be placed in K.XAe1eg (Amyup. AivBp. TX) and stored at -20 ° C.

In FIG. 22-29 show the ability of chimeras PPP polypeptides. especially the chimeras PPΥ-NPΥ. eg. compounds 4883 and 5705. to reduce the total food intake in the analysis of food consumption. described above.

In FIG. 22 shows a decrease in total food intake within 3 hours after administration of compound 5705 compared to the vehicle. In FIG. 23A and 24A show the change in food intake (in grams). and Figures 23B and 24B show a change in body weight (change in percent of body weight adjusted on a carrier). in mice. which continuously introduced the carrier or compounds 4883 or 5705 (at doses of 500 μg / kg / s) for 13 days. On the first day after the start of the administration of PPP polypeptide, there was a tendency to decrease food intake and body weight in animals. treated with compound 4883 or compound 5705.

In FIG. 25 and 26 show changes in daily water absorption and urination with the administration of compound 4883 or compound 5705 in mice. continuous administration of the carrier or compounds 4883 or 5705 (at doses of 500 μg / kg / s) for 13 days.

In FIG. 27 shows the effect of continuous administration of a carrier or compounds 4883 or 5705 (at doses of 500 μg / kg / s). within 13 days. on the composition of body tissues. specific NMR.

In FIG. 28A and 28B show water changes in grams. as well as percentage. in mice. which continuously introduced the carrier or compounds 4883 or 5705 (at doses of 500 μg / kg / s) for 13 days.

In FIG. 29 shows the effect of 14 days of continuous infusion of compounds 4883 or 5705 (in doses

- 96 014647

500 mcg / kg / s) for urine electrolytes.

Although the invention has been described using preferred examples and variations, it should be understood that variations and modifications can be made by one skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that are included within the scope of the claimed invention.

Claims (30)

CLAIM 1. A hybrid polypeptide exhibiting at least one hormonal activity, wherein said hybrid polypeptide comprises a first bioactive module of a peptide hormone covalently linked to at least one additional bioactive module of a peptide hormone; where the bioactive modules of the peptide hormone are independently selected from the group consisting of complex peptide hormones, fragments of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones, analogues and derivatives of complex peptide hormones, which exhibit at least one hormonal activity of complex peptide hormones hormones, fragments of analogues and derivatives of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones c, and peptide enhancers; complex peptide hormones are independently selected from at least two of the group consisting of amylin, adrenomedullin (ΑΌΜ), calcitonin (CT), the peptide encoded by the calcitonin gene (SCCR), intermedin, cholecystokinin (CCK), leptin, peptide ΥΥ (RUU) , glucagon-like peptide-1 (CPB-1), glucagon-like peptide 2 (CPB-2), oxyntomodulin (OXM), natriuretic peptide, peptide of the urocortins family, for example isp-2 and isp-3, peptide of the neuromedin family, for example I25 neuromedin or spliced options, and AIR, VIR, SIR or urodilatin and exendin-4; peptide enhancers are independently selected from the group consisting of structural motifs of complex peptide hormones that give the hybrid polypeptide the desired chemical stability, conformational stability, metabolic stability, bioavailability, orientation to the organ / tissue, interaction with the receptor, protease inhibition, binding to plasma protein or other pharmacokinetic characterization and structural motifs of analogues and derivatives of complex peptide hormones that confer the hybrid polypeptide available chemical stability, conformational stability, metabolic stability, bioavailability, targeting to an organ / tissue, interaction with a receptor, protease inhibition, binding to a plasma protein or other pharmacokinetic characteristic; and at least one of the bioactive modules of the peptide hormone exhibits at least one hormonal activity of the complex peptide hormone. 2. The hybrid polypeptide according to claim 1, where the peptide enhancers are independently selected from the group consisting of amylin (32-37) (8E0 ΙΌ N0: 242), amylin (33-37) (8E0 ΙΌ N0: 243), amylin (34 -37) (8Е0 GO N0: 244), amylin (35-37), amylin (36-37), amylin (37), ΑΌΜ (47-52) (8ЕО GO N0: 245), ΑΌΜ (4852) (8 BUT GO N0: 246), ΑΌΜ (49-52) (8EO GO N0: 247), ΑΌΜ (50-52), ΑΌΜ (51-52), ΑΌΜ (52), ST (27-32) (8EO GO N0 : 248), ST (27-32) (8EO GO N0: 248), ST (28-32) (8EO GO N0: 249), ST (29-32) (8EO GO N0: 250), ST (30- 32), ST (31-32), ST (32), SSKR (32-37) (8EO GO N0: 251), SSKR (33-37) (8EO GO N0: 252), SSKR (34-37) ( 8EO GO N0: 253), SSKR (35-37), SSKR (36-37), SSKR (37), intermediate (42-47) (8EO GO N0: 254), intermediate (43-47) (8E0 GO N0 : 255), inter dyne (44-47) (8E0 GO N0: 256), intermediate (45-47), intermediate (46-47), intermediate (47), RUU (25-36) (8EO GO N0: 257), RUU (26 -36) (8EO GO N0: 258), RUU (27-36) (8EO GO N0: 259), RUU (28-36) (8EO GO N0: 260), RUU (29-36) (8EO GO N0: 261), RUU (30-36) (8EO GO N0: 262), RUU (31-36) (8EO GO N0: 263), RUU (32-36) (8EO GO N0: 264), RUU (25-35 ) (8EO GO N0: 265), RUU (26-35) (8EO GO N0: 266), RUU (27-35) (8EO GO N0: 267), RUU (28-35) (8EO GO N0: 268) , RUU (29-35) (8EO GO N0: 269), RUU (30-35) (8EO GO N0: 270), RUU (31-35) (8EO GO N0: 271), RUU (32-35) ( 8EO GO N0: 272), CPY-1 (29-37) frogs (8EO GO N0: 273), CPY-1 (30-37) frogs (8EO GO N0: 274), CPY-2 (24-31) frogs (8EO GO N0: 275), exendin-4 (31-39) (8EO GO N0: 277), exendin4 (32-39) (8EO GO N0: 278), exend na-4 (33-39) (8EO GO N0: 279), exendin-4 (34-39) (8EO GO N0: 280), exendin-4 (35-39) (8E0 GO N0: 281), exendin- 4 (36-39) (8Е0 ГО N0: 282), exendin-4 (37-39), exendin-4 (38-39), exendin-4 (39) and their analogues. 3. The hybrid polypeptide according to claim 1, where at least one of the first bioactive modules of the peptide hormone or at least one of the additional bioactive modules of the peptide hormone is a complex peptide hormone or a fragment of a complex peptide hormone that exhibits at least one hormonal activity of a complex peptide hormone. 4. The hybrid polypeptide according to claim 1, where at least one of the first bioactive modules of the peptide hormone or at least one of the additional bioactive modules of the peptide hormone is an analog or derivative of a complex peptide hormone that exhibits at least one hormonal activity, or a fragment peptide analogue or derivative - 97 014647 hormones that exhibit at least one hormonal activity of a complex peptide hormone. 5. The hybrid polypeptide according to claim 1, where at least one of the first bioactive modules of the peptide hormone or at least one additional bioactive module of the peptide hormone is a peptide enhancer. 6. The hybrid polypeptide according to claim 1, where the complex peptide hormones are independently selected from the group consisting of amylin, calcitonin, CCK, ΡUU, a peptide of the urocortin family, a peptide of the neuromedin family and A№, ΒΝΓ, CNΡ or urodilatin and exendin-4. 7. The hybrid polypeptide according to claim 1, where at least one bioactive module of a peptide hormone that exhibits at least one hormonal activity is located at the Ν-terminal portion of the hybrid polypeptide. 8. The hybrid polypeptide according to claim 7, where at least one bioactive module of a peptide hormone that exhibits at least one hormonal activity located in the Ν-terminal part of the hybrid polypeptide is oriented in the direction from the C-terminal region to the Ν-terminal region . 9. The hybrid polypeptide of claim 8, where the Ν-terminal portion of the hybrid polypeptide is amidated. 10. The hybrid polypeptide according to claim 1, where at least one bioactive module of a peptide hormone that exhibits at least one hormonal activity is located at the C-terminal portion of the hybrid polypeptide. 11. The hybrid polypeptide of claim 10, where the C-terminal region of the hybrid polypeptide is amidated. 12. The hybrid polypeptide according to claim 1, where the C-terminal region of one bioactive module of the peptide hormone is attached directly to the Ν-terminal region of another bioactive module of the peptide hormone with the formation of a covalent bond. 13. The hybrid polypeptide according to claim 1, where the bioactive modules of the peptide hormone are covalently linked using one or more linker groups independently selected from the group consisting of alkyls; PEG dicarboxylic acids; amino acids; polyamino acids; bifunctional linkers; aminocaproyl (Aca), C1u, β-alanyl, 8-amino-3,6-dioxaoctanoyl and C1u-Luc-Agd (CQA). 14. The hybrid polypeptide according to claim 1, where the first bioactive module of the peptide hormone is selected from the group consisting of exendin-4, a fragment of exendin-4, which exhibits at least one hormonal activity, an analog or derivative of exendin-4, which exhibits at least at least one hormonal activity, and a fragment of an exendin-4 analog that exhibits at least one hormonal activity; and at least one additional bioactive module of the peptide hormone is independently selected from the group consisting of amylin, an amylin fragment that exhibits at least one hormonal activity, an amylin analog or derivative that exhibits at least one hormonal activity, or an amylin analog fragment, which exhibits at least one hormonal activity, CCK, a fragment of CCK, which exhibits at least one hormonal activity, an analog or derivative of CCK, which exhibits at least one hormonal activity, a fragment of an analogue of CCK that exhibits at least one hormonal activity, CT, a fragment of CT, which exhibits at least one hormonal activity, an analogue or derivative of CT, which exhibits at least one hormonal activity, a fragment of an analogue of CT which exhibits at least one hormonal activity, and a peptide enhancer. 15. The hybrid polypeptide according to 14, where the first bioactive module of the peptide hormone is selected from the group consisting of exendin-4, exendin-4 (1-27) (ZEO ΙΌ Νϋ: 236), exendin-4 (1-28) ( ZEO ΙΌ Νϋ: 237), ¹⁴ ZEI, ²⁵ Rye-exendina-4 (1-28) (ZEO ΙΌ Νϋ: 284); ⁵ A1a, ¹⁴ Ley, ²⁵ Rye-exendin-4 (1-28) (ZEO ΙΌ Νϋ: 240) and ¹⁴ Lye-exendin-4 (1-28) (ZEO ΙΌ Νϋ: 190); and at least one additional bioac- 25,28,29 tive peptide hormone module is independently selected from the group consisting ^of,, MGO-d-amylin (ZEO ΙΌ Νϋ: 67), amylin (1-7) (ZEO ΙΌ Νϋ: 217), ^2.7 A1a- Amylin (1-7) (ZEO ΙΌ Νϋ: 285), KST (8-10), KST (8-27) (ZEO ΙΌ Νϋ: 288), ^11D8 Arg-kST (8-27) (ZEO ΙΌ Νϋ: 289), ¹⁴ C1i, ^11D8 Arg-kST (8-27) (ZEO ΙΌ Νϋ: 286), SSK-8, Pye ² SSK-8 (ZEO ΙΌ Νϋ: 287), amylin (33-37) (ZEO ΙΌ Νϋ: 243), ΡUU (25-36) (ZEO ΙΌ Νϋ: 257), ΡUU (30-36) (ZEO ΙΌ Νϋ: 262) and ΡUU (31-36) (ZEO ΙΌ Νϋ: 263). 16. The hybrid polypeptide according to 14, where the hybrid polypeptide contains at least three bioactive modules of the peptide hormone. 17. The hybrid polypeptide according to 14, where the hybrid polypeptide contains at least four bioactive modules of the peptide hormone. 18. The hybrid polypeptide according to 14, where the first bioactive module of the peptide hormone is located in the C-terminal region of the hybrid polypeptide and at least one additional bioactive module of the peptide hormone is located in the Ν-terminal region of the hybrid polypeptide. 19. The hybrid polypeptide according to 14, where the first bioactive module of the peptide hormone is located in the Ν-terminal region of the hybrid polypeptide and at least one additional bioactive module of the peptide hormone is located in the C-terminal region of the hybrid polypeptide. 20. The hybrid polypeptide according to claim 1, where the first bioactive module of the peptide hormone is selected from - 98 014647 of a group consisting of amylin, an amylin fragment that exhibits at least one hormonal activity, an amylin analog or derivative that exhibits at least one hormonal activity, and an amylin analog fragment that exhibits at least one hormonal activity; and at least one additional bioactive module of the peptide hormone is a peptide enhancer independently selected from the group consisting of ΡUU (25-36) (8EO GO ΝΟ: 257), ΡUU (26-36) (8EO GO ΝΟ: 258), ΡUU (27-36) (8EE) GO ΝΟ: 259), ΡUU (28-36) (8EE) GO ΝΟ: 260), ΡUU (29-36) (8EE) GO ΝΟ: 261), ΡUU (30-36 ) (8EE) GO ΝΟ: 262), ΡUU (31-36) (8EE) GO ΝΟ: 263), ΡUU (32-36) (8EE) GO ΝΟ: 264), ΡUU (25-35) (8EE) GO ΝΟ: 265), ΡUU (26-35) (8EE) GO ΝΟ: 266), ΡUU (2735) (8 IU) GO ΝΟ: 267), ΡUU (28-35) (8EE) GO ΝΟ: 268), ΡUU (29-35) (8EE) GO ΝΟ: 269), ΡUU (30-35) (8EE) GO ΝΟ: 270), ΡUU (31-35) (8EE) GO ΝΟ: 271), ΡUU (32-35) (8EE) GO ΝΟ: 272) and their analogues. 21. A hybrid polypeptide exhibiting at least one hormonal activity, wherein said hybrid polypeptide comprises a first bioactive module of a peptide hormone covalently linked to a second bioactive module of a peptide hormone; where the bioactive modules of the peptide hormone are independently selected from the group consisting of complex peptide hormones, fragments of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones, analogues and derivatives of complex peptide hormones, which exhibit at least one hormonal activity of complex peptide hormones hormones, fragments of analogues and derivatives of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones c, and peptide enhancers; complex peptide hormones are independently selected from at least two of the group consisting of amylin, UY and exendin-4; peptide enhancers are independently selected from the group consisting of structural motifs of complex peptide hormones that give the hybrid polypeptide the desired chemical stability, conformational stability, metabolic stability, bioavailability, orientation to the organ / tissue, interaction with the receptor, protease inhibition, binding to plasma protein or other pharmacokinetic characteristics, and structural motifs of analogues or derivatives of complex peptide hormones that give the hybrid polypeptide the same bark chemical stability, conformational stability, metabolic stability, bioavailability, targeting to an organ / tissue, interaction with a receptor, protease inhibition, binding to a plasma protein or other pharmacokinetic characteristic; and at least one of the bioactive modules of the peptide hormone exhibits at least one hormonal activity of the complex peptide hormone. 22. The hybrid polypeptide according to item 21, where the hybrid polypeptide contains a combination of bioactive modules of the peptide hormone selected from the group consisting of exendin-4 / ΡUU, ΡΥΥ / exendin-4, exendin / amylin, amylin / exendin, amylin / ΡUU and ΡΥΥ / amylin bioactive modules of the peptide hormone. 23. A hybrid polypeptide exhibiting at least one hormonal activity, wherein said hybrid polypeptide comprises a first bioactive module of a peptide hormone covalently linked to at least a second bioactive module of a peptide hormone; where the bioactive modules of the peptide hormone are independently selected from the group consisting of complex peptide hormones, fragments of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones, analogues and derivatives of complex peptide hormones, which exhibit at least one hormonal activity of complex peptide hormones hormones, fragments of analogues and derivatives of complex peptide hormones that exhibit at least one hormonal activity of complex peptide hormones c, and peptide enhancers; the first complex peptide hormone contains leptin; at least one second bioactive module of the peptide hormone contains a polypeptide independently selected from exendin or CP1; and at least one of the bioactive modules of the peptide hormone exhibits at least one hormonal activity of its complex peptide hormone. 24. The hybrid polypeptide according to item 23, where the module of the peptide hormone leptin contains a polypeptide selected from the group consisting of leptin, fragments of leptin that exhibit at least one hormonal activity, analogues and derivatives of leptin that exhibit at least one hormonal activity , and fragments of analogues and leptin derivatives that exhibit at least one hormonal activity. 25. The hybrid polypeptide according to item 23, where the module of the peptide hormone leptin contains a polypeptide selected from the group consisting of leptin, fragments of leptin that exhibit at least one hormonal activity, analogues and derivatives of leptin that exhibit at least one hormonal activity , fragments of analogues and derivatives of leptin that exhibit at least one hormonal activity, and where at least one module of the peptide hormone - 99 014647 exendin contains a polypeptide selected from the group consisting of exendin-4, a fragment of exendin-4, which exhibits at least one hormonal activity, an analog or derivative of exendin-4, which exhibits at least one hormonal activity, and a fragment of the analog exendin-4, which exhibits at least one hormonal activity. 26. The hybrid polypeptide according to item 23, where the exendin is selected from the group consisting of exendin-4, Le, Pe-exendin-4, A1a, Le, Pe-exendin-4, Le, A1a, Pe-exendin-4 and them active fragments. 27. A method of treating a patient suffering from a disease or metabolic disorder, comprising administering to the patient a therapeutically effective amount of the hybrid polypeptide according to item 23. 28. The method of claim 27, wherein the disease or metabolic disorder is diabetes, type 1 diabetes, type 2 diabetes, obesity, hypertension, atherosclerosis, dyslipidemia, congestive heart failure, stroke, hypercholesterolemia, cardiovascular disease, myocardial ischemia, reperfusion myocardium, eating disorders, diabetes during pregnancy, diabetic neuropathy, pulmonary hypertension, or diseases associated with an insufficient mass of pancreatic beta cells. 29. The hybrid according to any one of claims 1 to 26, where the hybrid is not a hybrid described in ν0 2005/077072. 30. The method according to claims 27, 28, wherein the hybrid is not a hybrid described in ν0 2005/077072. The effect of treatment on body weight in fatty C57B1 mice. / 6 ha £ £