JP2009504681A - Hybrid polypeptides with selectable properties - Google Patents

Abstract

  The present invention generally relates to agents for the treatment and prevention of metabolic diseases and disorders that can be alleviated by controlling plasma glucose levels, insulin levels and / or insulin secretion, such as diabetes and diabetes related conditions. It relates to useful new, selectable hybrid polypeptides. Such conditions and disorders include hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and all types of diabetes, including type 1, type 2 and gestational diabetes, It is not limited to these.

Description

Related Applications This application is filed in common with US Provisional Patent Application No. 11 / 201,664, filed August 11, 2005, and US Patent Application No. 11 / 206,903, filed August 17, 2005 (these Both of which are entitled “Hybrid Polypeptides with Selectable Properies”), as well as US patent application Ser. No. 11 / 301,744, filed Dec. 12, 2005. All of the aforementioned applications are incorporated herein by reference in their entirety.

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

  Central to many metabolic diseases and disorders is the regulation of insulin levels and blood glucose levels. Insulin secretion is regulated in part by a secretagogue hormone called incretin produced by enteroendocrine cells. The incretin hormone, glucagon-like peptide-1 (“GLP-1”), is a peptide hormone secreted by intestinal cells that has been shown in many studies to produce an enhancing effect on insulin secretion. . GLP-1 is processed from proglucagon in the intestine and enhances nutrient-induced insulin release (Krcymann B., et al., Lancet, 2: 1300-1303 (1987)). Various truncated forms of GLP-1 are known to stimulate insulin secretion (insulin secretion stimulatory action) and to stimulate cAMP formation [eg Mojsov, S., Int. J. Pep. Pro. Res. ., 40: 333-343 (1992)]. To various ex vivo laboratory experiments and exogenous administration of GLP-1, GLP-1 (7-36) amide (SEQ ID NO: 61), and GLP-1 (7-37) acid (SEQ ID NO: 204) A relationship with the insulinotropic response of mammals, particularly humans, has been established (eg, Nauck, MA, et al., Diabetologia, 36: 741-744 (1993); Gutniak, M., et al., New Eng. J. of Med., 326 (20): 1316-1322 (1992); Nauck, MA, et al., J. Clin. Invest., 91: 301-307 (1993); and Thorens, B. , et al., Diabetes, 42: 1219-1225 (1993)).

  GLP-1 (7-36) amide (SEQ ID NO: 61) is a prominent anti-diabetic in insulin-dependent diabetes by stimulating insulin sensitivity and by enhancing glucose-induced insulin release at physiological concentrations It exerts an inducing action (Gutniak M., et al., New Eng. J. Med., 326: 1316-1322 (1992)). When administered to non-insulin dependent diabetics, GLP-1 (7-36) amide (SEQ ID NO: 61) stimulates insulin release, decreases glucagon secretion, suppresses gastric emptying, and uses glucose Strengthen the ability (Nauck, 1993; Gutniak, 1992; Nauck, 1993). However, the use of GLP-1 type molecules for long-term treatment of diabetes was complicated by the short half-life of these peptides.

More particularly, GLP-1 is a 30-amino acid peptide derived from proglucagon, a 160-amino acid prohormone. The action of various prohormone convertases in the pancreas and intestine results in the production of glucagon and other ambiguous peptides, but the cleavage of proglucagon produces GLP-1 and GLP-2 and two other peptides Give rise to The amino acid sequence of GLP-1 is 100% homologous in all mammals studied so far, which represents an important physiological role. GLP-1 (7-37) acid is truncated at the C-terminus and amidated to form CLP-1 (7-36) NH ₂ (SEQ ID NO: 61). The biological action and turnover of this free acid GLP-1 (7-37) OH (SEQ ID NO: 204), amide, GLP-1 (7-36) NH ₂ (SEQ ID NO: 61) are distinct. Absent. By convention, amino acid numbering is based on GLP-1 (1-37) OH (SEQ ID NO: 59) processed from proglucagon. Biologically active GLP-1 is the result of further processing: GLP-1 (7-36) NH ₂ (SEQ ID NO: 61). Thus, the first amino acid of GLP-1 (7-37) OH (SEQ ID NO: 204) or GLP-1 (7-36) NH ₂ (SEQ ID NO: 61) is ⁷ His.

In the gastrointestinal tract, L-cells of the intestine, colon and rectal mucosa produce GLP-1 in response to stimulation with intraluminal glucose. The plasma half-life of active GLP-1 is <5 minutes and its metabolic clearance rate is about 12-13 minutes (Holst, Gastroenterology 107 (6): 1848-55 (1994)). The major protease involved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV (CD26), which cleaves the N-terminal His-Ala dipeptide, eg, metabolites GLP-1 (9-37 ) OH (SEQ ID NO: 205) or GLP-1 (9-36) NH ₂ (SEQ ID NO: 206) is produced. These metabolites are variously described as GLP-1 receptor inactivity, weak agonists or antagonists. The GLP-1 receptor (GLP-1R) is a 463 amino acid G protein-coupled receptor and is located in the pancreatic beta cells, in the lung, and to a lesser extent in the brain, adipose tissue and kidney. Stimulation of GLP-1R by GLP-1 (7-37) OH (SEQ ID NO: 204) or GLP-1 (7-36) NH ₂ (SEQ ID NO: 61) activates adenylate cyclase and synthesizes cAMP. Cause membrane depolarization, elevated intracellular calcium and increased glucose-induced insulin secretion (Holz et al., J. Biol. Chem. 270 (30): 17749-57 (1995)).

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

  Another important effect of GLP-1 on glucose homeostasis is suppression of glucagon secretion and suppression of gastric motility. GLP-1 inhibitory action of glucagon on pancreatic alpha cell secretion results in decreased hepatic glucose production by reducing gluconeogenesis and glycogenolysis. In diabetic patients, this anti-glucagon effect of GLP-1 is maintained.

  The so-called ileal braking effect of GLP-1, in which gastric motility and gastric fluid secretion are suppressed, is brought about by vagal nerve efferent receptors or by direct action on intestinal smooth muscle. The decrease in gastric acid secretion by GLP-1 contributes to a late phase of nutrient utilization, thus eliminating the need for a rapid insulin response. In summary, the gastrointestinal action of GLP-1 contributes significantly to delayed glucose and fatty acid absorption and modulates insulin secretion and glucose homeostasis.

  It has also been demonstrated that GLP-1 induces beta cell specific genes such as GLUT-1 transporter, insulin (due to the interaction of PDX-1 and insulin gene promoter), and hexokinase-1. Thus, GLP-1 has the potential to reverse the glucose intolerance normally associated with aging, as demonstrated by rodent experiments. In addition, GLP-1 can contribute to beta cell neogenesis and increase beta cell mass in addition to restoring beta cell function during beta cell deficiency.

  The major effects of GLP-1 include reduced food intake and paired satiety caused by the action of the hypothalamic GLP-1R. 48-hour continuous SC infusion of GLP-1 in type II diabetic subjects decreased hunger and food intake and increased satiety. These anorectic effects were not present in GLP-1R knockout mice.

  Exendins are another family of peptides involved in insulin secretion. Exendins can be found in the saliva of the American lizard (Gila-monster), the lizards native to Arizona, and the Mexican beaded lizard (Mexican Beaded Lizard). Exendin-3 is present in the saliva of the Mexican lizard (scientific name: Heloderma horridum), and exendin-4 is present in the saliva of the American lizard (scientific name: Heloderma suspectum) (Eng, J., et al., J Biol. Chem., 265: 20259-62, 1990; Eng., J., et al., J. Biol. Chem., 267: 7402-05 (1992)). Exendin has some sequence similarity with several members of the glucagon-like peptide family, with the highest identity, 53%, with GLP-1 (Goke, et al., J. Biol. Chem., 268: 19650-55 (1993)).

  Exendin-4 binds to GLP-1 receptors on insulin-secreting TC1 cells, diffuse acinar cells from the guinea pig pancreas, and mural cells from the stomach; this peptide also stimulates somatostatin release Suppresses gastrin release in the isolated stomach (Goke, et al., J. Biol. Chem., 268: 19650-55 (1993); Schepp, et al., Eur. J. Pharmacol., 69: 183 -91 (1994); Eissele, et al., Life Sci., 55: 629-34 (1994)). Exendin-3 and exendin-4 were found to bind to the GLP-1 receptor on pancreatic acinar cells, stimulate cAMP production in pancreatic acinar cells, and amylase release from pancreatic acinar cells (Malhotra, R., et al., Relulatory Peptides, 41: 149-56 (1992); Raufman, et al., J. Biol. Chem., 267: 21432-37 (1992); Singh, et al., Regul. Pept., 53: 47-59 (1994)). The use of exendin-3 and exendin-4 insulin secretion stimulating activity for the treatment of diabetes and prevention of hyperglycemia has been reported (Eng, US Pat. No. 5,424,286).

  Truncated exendin peptides, such as exendin [9-39], carboxyamidated molecules, and fragments 3-39 to 9-39, have been reported to be potent and selective antagonists of GLP-1. (Goke, et al., J. Biol. Chem., 268: 19650-55 (1993); Raufman, JP, et al., J. Biol. Chem., 266: 2897-902 (1991); Schepp, W., et al., Eur. J. Pharm., 269: 183-91 (1994); Montrose-Rafizadeh, et al., Diabetes, 45 (Suppl. 2): 152A (1996)). Exendin [9-39] (SEQ ID NO: 207) blocks endogenous GLP-1 in vivo, resulting in decreased insulin secretion (Wang, et al., J. Clin. Invest., 95: 417-21 (1995); D'Alessio, et al., J. Clin. Invest., 97: 133-38 (1996)). A receptor that appears to be responsible for the insulinotropic effect of GLP-1 has been cloned from rat islet cells (Thorens, B., Proc. Natl. Acad. Sci. USA 89: 8641-8645 (1992)). . Exendin and exendin [9-39] bind to this cloned GLP-1 receptor (rat pancreatic cell GLP-1 receptor: Fehmann HC, et al., Peptides, 15 (3): 453-6 ( 1994); human GLP-1 receptor: Thorens B, et al., Diabetes, 42 (11): 1678-82 (1993)). In cells transfected with its cloned GLP-1 receptor, exendin-4 is an agonist, ie, increases cAMP, but exendin [9-39] (SEQ ID NO: 207) is antagonistic It is a drug, ie blocks the stimulatory action of exendin-4 and GLP-1. Id.

  More specifically, exendin-4 is a 39 amino acid C-terminal amidated peptide found in saliva of the American lizard (Heloderma suspectum) and has 53% amino acid sequence identity with the GLP-1 peptide sequence. For example, Eng, J., et al. “Isolation and Characterization of Exendin-4, and Exendin-3 Analogue from Heloderma suspectum Venom” J. Bio. Chem., 267: 11, p. 7402-7405 (1992), Young , AA, et al., “Glucose-Lowering and Insulin-Sensitizing Actions of Exendin-4,” Diabetes, Vol. 48, p. 1026-1034, May, 1999. In view of its activity, exendin-4 is a very specific agonist for the GLP-1 receptor and, like GLP-1, can stimulate insulin secretion. Thus, like GLP-1, exendin-4 is considered an insulin secretion stimulating peptide.

  However, unlike GLP-1, exendin-4 has a relatively long half-life in humans due to its resistance to dipeptidyl peptidase IV, which rapidly degrades the GLP-1 sequence in vivo. Furthermore, compared to GLP-1, it was demonstrated that exendin-4 has a strong ability to stimulate insulin secretion and that such stimulatory activity can be obtained using low exendin-4 concentrations. See, for example, US Pat. No. 5,424,286, which is incorporated herein by reference. Thus, exendin-4 peptides or derivatives thereof (see, eg, US Pat. No. 6,528,486 (incorporated herein by reference) and its corresponding international application WO01 / 04156 for examples of such derivatives) ) Has greater potential utility than insulin or GLP-1 for the treatment of conditions involving dysregulation of insulin levels (eg, conditions such as diabetes).

  Another family of peptide hormones involved in metabolic diseases and disorders includes amylin, calcitonin, calcitonin gene-related peptides, adrenomedullin and intermedin (also known as “AFP-6”) The amylin family of peptide hormones. Amylin is a 37-amino acid peptide hormone. It was isolated, purified and chemically characterized as the main component of amyloid deposits in the islets of human type 2 diabetics (Cooper et al., Proc. Natl. Acad. Sci., USA, 84: 8628 -8632 (1987)). The amylin molecule has two posttranslational modifications: the C-terminus is amidated and the cysteines at positions 2 and 7 are bridged to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene is the presence of the Lys-Arg dibasic amino acid proteolytic cleavage signal before the Lys N-terminal codon and the Gly before the Lys-Arg proteolytic signal at the CLAIMS terminal position. The typical sequence for the presence of C, amidation of protein, amidation with PAM (Cooper et al., Biochem. Biophys. Acta, 1014: 247-258 (1989)) is shown.

  Amylin is thought to regulate gastric emptying, suppress glucagon secretion and food intake, and thus regulate the rate of glucose appearance in the circulation. It appears to complement the action of insulin that regulates the rate of disappearance of glucose from the circulation and its uptake by peripheral tissues. These actions indicate that amylin complements the effects of insulin on postprandial glucose control by at least three independent mechanisms, indicating that all of these mechanisms affect the rate of glucose appearance. Supported by findings from experiments. First, amylin suppresses postprandial glucagon secretion. Compared to healthy adults, patients with type 1 diabetes do not have circulating amylin, and patients with type 2 diabetes have reduced postprandial amylin levels. Furthermore, injection of amylin-specific monoclonal antibodies that bind to circulating amylin also results in greatly increased glucagon concentrations relative to controls. Both these results point to the physiological role of endogenous amylin in the regulation of postprandial glucagon secretion. Second, amylin slows gastrointestinal motility and gastric emptying. Finally, intrahypothalamic injection of rat amylin has been shown to reduce rat feeding and alter neurotransmitter metabolism in the hypothalamus. In one study, food intake was significantly reduced up to eight hours after intrahypothalamic injection of rat amylin and rat CGRP. Human studies have shown that the amylin analog, pramlintide, decreases body weight or weight gain. Amylin can be beneficial in the treatment of metabolic conditions such as diabetes and obesity. Amylin should be used to treat pain, bone disease, gastritis, to adjust lipids, especially triglycerides, or to affect body components such as preferential reduction of fat and saving lean tissue You can also.

  The hormone calcitonin (CT) was named for its secretion in response to induced hypercalcemia and its rapid hypocalcemic effect. CT is produced in and secreted from neuroendocrine cells within the thyroid. Therefore, those cells were named C cells. The most well-studied action of CT (1-32) (SEQ ID NO: 48) is its effect on osteoclasts. In vitro effects of CT include rapid loss of wavy edges and reduced release of lysosomal enzymes. Ultimately, inhibition of osteoclast function by CT results in decreased bone resorption. However, neither the chronic decrease in serum CT in the case of thyroidectomy nor the increase in serum CT seen in myeloid thyroid cancer appears to be related to changes in serum calcium and body weight. Therefore, the primary function of CT (1-32) (SEQ ID NO: 48) is to strive to control acute hypercalcemia during emergencies and / or “calcium stress” such as growth, pregnancy and breastfeeding It is likely that the skeleton is protected during this period (Reviewed in Becker, JCEM, 89 (4): 1512-1525 (2004) and Sexton, Current Medicinal Chemistry 6: 1067-1093 (1999)). Recent data from a calcitonin gene knockout mouse that removes both calcitonin and the CGRP-I peptide, showing that mice have normal levels of basal calcium-related values but an increased calcemia response ( Kurihara H, et al., Hypertens Res. 2003 Feb; 26 Suppl: S105-8) agrees with this. CT affects plasma calcium levels, inhibits osteoclast function, and is widely used to treat osteoporosis. Therapeutically, salmon CT (sCT) appears to increase bone density and decrease fracture rate with minimal side effects. CT has also been used successfully over the past 25 years as a treatment for Paget's disease of bone, a chronic skeletal disease that can cause expanded or deformed bone in one or more regions of the skeleton. CT is also widely used for its analgesic effect on bone pain experienced during osteoporosis, but the mechanism of this effect is not clearly understood.

  Calcitonin gene-related peptide (CGRP) is a neuropeptide and its receptors are widely distributed in the body including the nervous system and cardiovascular system. This peptide appears to modulate sensory neurotransmission and is one of the most potent endogenous vasodilator peptides discovered to date. Reported biological effects of CGRP include modulation of substance P during inflammation, nicotinic receptor activity at the neuromuscular junction, stimulation of pancreatic enzyme secretion, decreased gastric acid secretion, peripheral vasodilation, Heart rate acceleration, neuromodulation, regulation of calcium metabolism, osteogenic stimulation, insulin secretion, increased body temperature and decreased food intake. (Wimalawansa, Amylin, calcitonin gene-related peptide, calcitonin and ADM: a peptide superfamily. Crit Rev Neurobiol. 1997; 11 (2-3): 167-239). An important role of CGRP is the control of blood flow to various organs through its potent vasodilatory action as evidenced by a decrease in mean arterial pressure following intravenous administration of α-CGRP. Vasodilatory activity was recently analyzed in homozygous knockout CGRP mice that demonstrated high peripheral vascular resistance and hypertension due to increased peripheral sympathetic nerve activity (Kurihara H, et al., Targeted disruption of ADM and αCGRP genes reveals their distinct biological Roles. Hypertens Res. 2003 Feb; 26 Suppl: S105-8). Thus, CGRP appears to cause vasodilatory effects, blood pressure lowering effects and heart rate increases among other actions.

  Continuous infusion of CGRP in patients with congestive heart failure showed sustained efficacy on hemodynamic function without side effects, indicating use in heart failure. Other indications for using CGRP include renal failure, acute and chronic coronary ischemia, treatment of cardiac arrhythmias, other peripheral vascular diseases such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension. It has the potential to treat preeclamptic preeclampsia and premature labor (Wimalawansa, 1997). Recent therapeutic uses include the use of CGRP antagonists for the treatment of migraine.

Adrenomedullin (ADM) is expressed almost everywhere, and much more in tissues that contain this peptide than in those that do not. A review of published ADMs (Hinson, JP et al., Endocrine Reviews (2000) 21 (2): 138-167) covers a range of areas including vasodilation, cell proliferation, regulation of hormone secretion and natriuresis. Along with the biological effects, its effects on the cardiovascular system, cell proliferation, central nervous system and endocrine system are detailed. Studies in rats, cats, sheep and humans have confirmed that intravenous infusion of ADM produces strong sustained hypotension and is comparable to that of CGRP. However, the blood lowering effect of ADM on mean arterial pressure in anesthetized rats is not inhibited by the CGRP antagonist CGRP _8-37 , suggesting that this effect is not mediated by the CGRP receptor. Acute or chronic administration of human ADM in anesthetized, conscious, or hypertensive rats is associated with a significant reduction in total peripheral resistance with hypotension and concomitant heart rate, cardiac output and stroke volume. Increase.

  ADM has also been proposed as an important factor in embryogenesis and differentiation and as an apoptosis survival factor for rat endothelial cells. This is supported by a recent mouse ADM knockout study in which homozygous mice, due to loss of the ADM gene, showed poor angiogenesis during embryogenesis and therefore died in midgestation. ADM +/− heterozygous mice have been reported to have high blood pressure and are susceptible to tissue damage (Kurihara H, et al., Hypertens Res. 2003 Feb; 26 Suppl: S105-8).

  ADM affects endocrine organs such as the pituitary gland, adrenal gland, reproductive organs and pancreas. This peptide appears to play a role in ACTH release from the pituitary gland. In the adrenal gland, it appears to affect the secretory activity of the adrenal cortex in both rats and humans, and in intact rats it acts as a vasodilator in the adrenal vascular bed, increasing adrenal blood flow. ADM has been demonstrated to exist throughout the female reproductive tract and plasma levels are elevated during normal pregnancy. Studies in a rat model of pre-eclampsia have demonstrated that ADM can reverse hypertension and reduce pup mortality when given to rats during late pregnancy. This suggests that ADM may play an important regulatory role in the uteroplacental cardiovascular system, since this preeclampsia model did not have a similar effect in early and non-pregnant animals. ing. In the pancreas, ADM is most likely to play an inhibitory role because it attenuates and delays the response of insulin to an oral glucose load that produces an initial high glucose level. ADM can also affect renal function. A peripherally administered bolus can significantly reduce mean arterial pressure and increase renal blood flow, glomerular filtration rate and urine flow. In some cases, Na + excretion is also increased.

  ADM also has other peripheral effects on bone and lung. For bone, research supports a role beyond cardiovascular and fluid homeostasis, where ADM acts on fetal and adult rodent osteoblasts to increase cell proliferation, which It proved to be comparable to that of known osteoblast growth factors such as transforming growth factor-β. This is clinically important because one of the major challenges in osteoporosis research is the development of treatments that increase bone mass by osteoblast stimulation. In the lung, ADM not only causes pulmonary vasodilation, but also inhibits bronchoconstriction induced by histamine or acetylcholine. A recent study using aerosolized ADM to treat pulmonary hypertension in a rat model showed that mean pulmonary artery pressure and total lung resistance were significantly lower in rats treated with ADM than in rats given saline Evidence shows that inhalation therapy under this condition is effective. This result was achieved without changes in systemic arterial pressure or heart rate (Nagaya N et al., Am J Physiol Heart Circ Physiol. 2003; 285: H2125-31).

  In healthy volunteers, intravenous infusion of ADM has been shown to reduce arterial pressure and stimulate heart rate, cardiac output, cAMP, prolactin, norepinephrine and renin plasma levels. In these patients, little or no increase in urine output and sodium excretion was observed. In patients with heart failure or chronic renal failure, intravenous ADM has effects similar to those seen in normal subjects, and also induced diuresis and natriuresis, depending on the dose administered (Nicholls, MG et al. Peptides. 2001; 22: 1745-1752). Experimental ADM treatment has also proved beneficial in arterial and pulmonary hypertension, septic shock and ischemia / reperfusion injury (Beltowski J., Pol J Pharmacol. 2004; 56: 5-27). Other indications for ADM treatment include peripheral vascular disease, subarachnoid hemorrhage, hypertension, preeclamptic preeclampsia and preterm delivery, and osteoporosis.

  AFP-6 (ie, intermedin) expression is primarily in the pituitary and gastrointestinal tract. No specific receptor for AFP-6 has been reported. However, binding studies indicate that AFP-6 binds to all known receptors of the amylin family. AFP-6 has been shown to increase cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptor and to compete with labeled CGRP for binding to that receptor in these cells. In an in vivo study published in the publication, AFP-6 administration resulted in hypotension, most likely by interaction with CRLR / RAMP receptors, in both normal and spontaneously hypertensive rats . In vivo administration in mice resulted in suppression of gastric emptying and food intake (Roh et al. J Biol Chem. 2004 Feb 20; 279 (8): 7264-74).

  The biological effects of amylin family peptide hormones are generally determined by binding to two closely related type II G protein-coupled receptors (GPCR), calcitonin receptor (CTR), and calcitonin receptor-like receptor (CRLR). It has been reported to be mediated. Cloning and functional studies have demonstrated that CGRP, ADM and amylin interact with various combinations of CTR or CRLR and receptor activity modifying protein (RAMP). Many cells express a large number of RAMPs. It is believed that co-expression of RAMP and either CRT or CRLR is required to generate functional receptors for calcitonin, CGRP, ADM and amylin. The RAMP family includes three members (RAMP 1, -2 and -3), which share less than 30% sequence identity but have a common topological configuration. Co-expression of CRLR and RAMP1 results in the formation of CGRP receptors. Co-expression of CRLR and RAMP2 results in the formation of ADM receptors. Co-expression of CRLR and RAMP3 results in the formation of ADM and CGRP receptors. Co-expression of hCTR2 and RAMP1 results in the formation of amylin and CGRP receptors. Co-expression of hCTR2 and RAMP3 results in the formation of amylin receptors.

  Yet another peptide hormone family involved in metabolic diseases and abnormalities is the leptin family. The mature form of circulating leptin is a 146-amino acid protein that is normally excluded from the CNS by the blood brain barrier (BBB) and the blood-CSF barrier. See, for example, Weigle et al., 1995. J Clin Invest 96: 2065-2070. Leptin is an afferent signal in negative feedback that regulates dietary intake and weight. The leptin receptor is a member of the cytokine receptor family. The anorexic effect of leptin depends on the binding of the receptor's Ob-Rb isoform to a homodimer, encoding a long cytoplasmic domain containing several motifs for protein-protein interactions . Ob-Rb is highly expressed in the hypothalamus, suggesting that this brain region is an important site of leptin action. Mutations in the mouse ob gene include pathologies including obesity, increased body fat accumulation, hyperglycemia, hyperinsulinemia, hypothermia and thyroid and reproductive dysfunction in both male and female homozygous ob / ob obese mice It has been demonstrated to produce physiological syndromes (see, for example, Ingalis, et al., 1950. J Hered 41: 317-318). Therapeutic uses for leptin or leptin receptor include (i) diabetes (see, eg, PCT patent applications W0 98/55139, W0 98/12224, and W0 97/02004); (ii) hematopoiesis (eg, PCT patents) (Iii) infertility (eg, PCT patent applications W0 97/15322 and W0 98/36763); and (iv) tumor suppression (eg, PCT patent application W0 98/48831). ). Each of the aforementioned patent applications is hereby incorporated by reference in its entirety.

  The leptin receptor (OB-R) gene has been cloned (GenBank accession number AF098792) and mapped to the db locus (see, eg, Tartaglia, et al., 1995. Cell 83: 1263-1271) . Several transcripts of OB-R obtained from variable splicing have also been identified. Deletion of OB-R results in certain syndromes in mutant diabetic ob / ob mice that are phenotypically identical to ob / ob mice (eg, Ghilardi, et al., 1996. Proc. Natl. Acad Sci. USA 93: 6231-6235). However, in contrast to ob / ob mice, administration of recombinant leptin to C57BLKS / Jm ob / ob mice does not result in dietary intake and weight loss (eg, Roberts and Greengerg, 1996. Nutrition Rev. 54 : 41-49).

  Most leptin-related studies that can report weight loss activity from administration of recombinant leptin, leptin fragments and / or leptin receptor variants are those in which the construct is administered directly into the ventricle. See, for example, Weigle, et al., 1995. J Clin Invest 96: 2065-2070; Barash, et al., 1996. Endocrinology 137: 3144-3147.

  Other studies have demonstrated significant weight loss activity due to administration of leptin peptide by intraperitoneal (i.p.) administration to subjects. See Grasso et al., 1997. Endocrinology 138: 1413-1418. Furthermore, leptin fragments, most particularly 18 amino acid fragments containing residues taken from full-length human leptin, can function with respect to weight loss only by direct administration via an implanted cannula into the rat posterior ventricle. It has been reported. See, for example, PCT Patent Application W0 97/46585, which is incorporated herein by reference in its entirety.

  Another peptide hormone associated with metabolic diseases and abnormalities is cholecystokinin (CCK). According to reports, CCK was identified in 1928 from a prepared sample of intestinal extract due to its ability to stimulate gallbladder contraction. Since then, other biological effects of CCK have been reported, including stimulation of pancreatic juice secretion, delayed gastric emptying, stimulation of intestinal motility and stimulation of insulin secretion. See Lieverse et al., Ann. N.Y. Acad. Sci. 713: 268-272 (1994). Reportedly, CCK actions also include effects on cardiovascular function, respiratory function, neurotoxins and seizures, cancer cell proliferation, analgesia, sleep, sexual and reproductive behavior, memory, anxiety and dopamine-mediated behavior. Crawley and Corwin, Peptides 15: 731-755 (1994). Other reported effects of CCK include stimulation of pancreatic growth, stimulation of gallbladder contraction, gastric acid secretion, pancreatic polypeptide release and suppression of contractile components of peristalsis. Additional reported effects of CCK include vasodilation. Walsh, “Gastrointestinal Hormones,” In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York).

  Injections of the combination of glucagon, CCK and bombesin have been reported to enhance the suppression of condensed milk test food intake in non-deprived rats over that observed with individual compounds. Hinton et al., Brain Res. Bull. 17: 615-619 (1986). Glucagon and CCK have also been reported to synergistically suppress pseudonutrient burden in rats. LeSauter and Geary, Am. J. Physiol. 253: R217-225 (1987); Smith and Gibbs, Annals N.Y. Acad. Sci. 713: 236-241 (1994). It has also been suggested that estradiol and CCK may have a synergistic effect on satiety. Dulawa et al., Peptides 15: 913-918 (1994); Smith and Gibbs, (supra). It has also been reported that signals generated from the small intestine in response to nutrients therein interact synergistically with CCK to reduce contact. Cox, Behav. Brain Res. 38: 35-44 (1990). In addition, CCK has been reported to induce satiety in several species. For example, intraperitoneally injected in rats, injected intraarterially in pigs, injected intravenously in cats and pigs, injected into the ventricles in monkeys, rats, dogs and sheep, and in obese and non-obese humans Intravenous CCK has been reported to cause loss of appetite. See Lieverse et al., (Above). Reports from several laboratories have reported that by comparing responses to food supplements and non-food supplements in both monkeys and rats, as well as the sequence of behaviors normally observed after food intake ( That is, by confirming that CCK elicits the postprandial satiety order), we confirmed the behavioral specificity of low doses of CCK for suppression of eating. In addition, according to reports, comparison of behavior after CCK with behavior after food intake alone or in combination with CCK showed behavioral similarity between CCK and food intake. Crawley and Corwin, (above). Physiological plasma concentrations of CCK have also been reported to suppress food intake and increase satiety in both lean and obese people. See Lieverse et al., (Above).

  CCK was characterized in 1966 as a 33-amino acid peptide. Crawley and Corwin, (above). A species-specific molecular variant of the amino acid sequence of CCK has been identified. Reports have identified a 33-amino acid sequence and a truncated peptide, its 8-amino acid C-terminal sequence (CCK-8), in pigs, rats, chickens, chinchillas, dogs and humans. According to reports, 39-amino acid sequences were found in pigs, dogs and guinea pigs. It was reported that 58-amino acid sequences were found in cats, dogs and humans. According to reports, frogs and turtles showed a 47-amino acid sequence homologous to both CCK and gastrin. The very fresh human intestine was reported to contain a small amount of an even longer molecule called CCK-83. According to reports, an important intermediate form has been identified in rats, called CCK-22. Walsh, “Gastrointestinal Hormones,” In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York). Non-sulfated CCK-8 and tetrapeptide (CCK-4 (referred to as CCK (30-33)) have been reported in rat brain.C-terminal pentapeptide (CCK-4 (CCK (29- 33)) preserves the structural homology of CCK and preserves the homology with neuropeptide and gastrin, according to reports, its C-terminal sulfated octapeptide sequence, CCK-8, According to reports, cloning and sequence analysis of cDNA encoding preprocholecystokinin from rat thyroid cancer, porcine brain and porcine intestine is 115 amino acids, previously 345 nucleotides encoding CCK precursor containing all CCK sequences reported to have been isolated, Crawley and Corwin, supra.

  CCK was characterized in 1966 as a 33-amino acid peptide. Crawley and Corwin, (above). A species-specific molecular variant of the amino acid sequence of CCK has been identified. Reports have identified a 33-amino acid sequence and a truncated peptide, its 8-amino acid C-terminal sequence (CCK-8), in pigs, rats, chickens, chinchillas, dogs and humans. According to reports, 39-amino acid sequences were found in pigs, dogs and guinea pigs. It was reported that 58-amino acid sequences were found in cats, dogs and humans. According to reports, frogs and turtles showed a 47-amino acid sequence homologous to both CCK and gastrin. The very fresh human intestine has been reported to contain a small amount of an even longer molecule called CCK-83. According to reports, an important intermediate form has been identified in rats, called CCK-22. Walsh, “Gastrointestinal Hormones,” In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York). Non-sulfated CCK-8 and tetrapeptide (CCK-4 (referred to as CCK (30-33); SEQ ID NO: 208) have been reported in rat brain. C-terminal pentapeptide (CCK-4 (Referred to as CCK (29-33); SEQ ID NO: 209) preserves the structural homology of CCK and the homology with the neuropeptide, gastrin, according to reports. The sulfated octapeptide sequence, CCK-8, is relatively conserved across species, according to reports, cloning and sequencing of a cDNA encoding preprocholecystokinin from rat thyroid cancer, pig brain and pig intestine. Analysis revealed 115 amino acids and 345 nucleotides encoding a precursor of CCK containing all previously reported CCK sequences that were previously isolated Crawley and Corwin, supra.

  CCK is said to be distributed throughout the central nervous system and in endocrine cells and in the enteric nerves of the upper small intestine. CCK agonists include CCK itself (also called CCK-33), CCK-8 (CCK (26-33); SEQ ID NO: 55), non-sulfated CCK-8, pentagastrin (CCK-5 or CCK ( 29-33); SEQ ID NO: 209), and the tetrapeptide CCK-4 (CCK (30-33); SEQ ID NO: 208). According to reports, at the pancreatic CCK receptor, CCK-8 displaces the bond with 1000-5000 greater force than non-sulfated CCK-8 or CCK-4, and CCK-8 is related to stimulation of pancreatic amylase secretion. It is reported to be about 1000 times more potent than non-sulfated CCK-8 or CCK-4. Crawley and Corwin, (above). In homogenates from cerebral cortex, CCK receptor binding is said to be displaced by non-sulfated CCK-8 and by equimolar concentrations of CCK-4 at concentrations 10 or 100 times greater than non-sulfated CCK-8. It has been broken.

  Yet another family of peptide hormones involved in metabolic diseases and disorders is the pancreatic polypeptide family (“PPF”). Pancreatic polypeptide ("PP") was discovered as an impurity in insulin extract and was named by its source organ rather than functional significance (Kimmel et al., Endocrinology 83: 1323-30 (1968)). PP is a 36-amino acid peptide containing individual structural motifs. Later, a related peptide was found in intestinal extracts and named for peptide YY (“PYY”) because of its N- and C-terminal tyrosines (Tatemoto, Proc. Natl. Acad. Sci. USA 79: 2514-8 (1982)). A third related peptide was later found in brain extracts and named neuropeptide Y (“NPY”) (Tatemoto, Proc. Natl. Acad. Sci. USA 79: 5485-9 (1982); Tatemoto et al., Nature 296: 659-60 (1982)).

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

  The release of PYY occurs after a meal. An alternative molecular form of PYY is PYY (3-36) (SEQ ID NO: 58) (Eberlein et al., Peptides 10: 797-803 (1989); Grandt et al., Regul. Pept. 51: 151- 9 (1994)). This fragment constitutes about 40% of PYY-like immunoreactivity in human and dog intestinal extracts, and about 36% of fasted total plasma PYY immunoreactivity to slightly more than 50% after meals Configure. It is apparently a cleavage product of PYY dipeptidyl peptidase-IV (DPP4). Reportedly, PYY (3-36) (SEQ ID NO: 58) is a selective ligand at the Y2 and Y5 receptors, which is an NPY analog truncated at the N-terminus (ie, of NPY). It seems to be pharmacologically unique in that it prefers the C-terminal fragment. Peripheral administration of PYY reportedly reduced gastric acid secretion, gastric motility, and exocrine pancreatic secretion (Yoshinaga et al., Am. J. Physiol. 263: G695-701 (1992); Guan et al. , Endocrinology 128: 911-6 (1991); Pappas et al., Gastroenterology 91: 1386-9 (1986)), reducing gallbladder contraction and intestinal motility (Savage et al., Gut 28: 166-70 ( 1987)). Effects of central injection of PYY on gastric emptying, gastric motility and gastric acid secretion as seen after direct injection into or around the hindbrain / brain stem (Chen and Rogers, Am. J. Physiol. 269: R787- 92 (1995); Chen et al., Regul. Pept. 61: 95-98 (1996); Yang and Tache, Am. J. Physiol. 268: G943-8 (1995); Chen et al., Neurogastroenterol. Motil 9: 109-16 (1997)) may differ from the effects observed after peripheral injection. For example, centrally administered PYY stimulates and does not inhibit gastric acid secretion in a number that conflicts with that described herein for peripherally injected PYY (3-36) (SEQ ID NO: 58). Had an effect. Gastric motility was only suppressed in conjunction with TRH stimulation, not when administered alone, and was actually irritating due to putative interactions with PP receptors at higher doses. PYY has been shown to stimulate food and water intake after central administration (Morley et al., Brain Res. 341: 200-3 (1985); Corp et al., Am. J. Physiol. 259: R317-23 (1990)).

  Metabolic diseases and disorders take many forms, including obesity, diabetes, dyslipidemia, insulin resistance, cell apoptosis and the like. Obesity and its related diseases are a common and very serious public health problem in the United States and throughout the world. Upper body obesity is the strongest risk factor known for type 2 diabetes and a strong risk factor for cardiovascular disease. Obesity is hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, genital diseases such as polycystic ovary syndrome, breast cancer, prostate cancer, colon cancer, and general anesthesia Is a recognized risk factor for an increased incidence of complications (see, for example, Kopelman, Nature 404: 635-43 (2000)). This shortens lifespan, upper complications, as well as infections, varicose veins, melanoma, eczema, exercise intolerance, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, orthopedic injury, and thrombus Has a serious risk of diseases such as embolic disease (Rissanen et al., Br. Med. J. 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 for health care costs for obesity and related diseases is $ 1,500,000,000 worldwide. Although the etiology of obesity is believed to have many factors, the fundamental problem is that nutrient utilization and energy expenditure in obese patients are not balanced until adipose tissue is in excess. Obesity is a chronic, essentially refractory metabolic disease that cannot be adequately treated recently. A therapeutic agent useful for weight loss in obese people should have a significant and beneficial effect on their health.

  Diabetes is a disease of carbohydrate metabolism characterized by hyperglycemia and diabetes due to insufficient production or utilization of insulin. Diabetes severely affects the quality of life for the majority of the population in developed countries. Insufficient production of insulin is characterized as type 1 diabetes, and insufficient use of insulin is type 2 diabetes. However, it is now widely recognized that there are many distinct diabetes-related diseases that develop well before patients are diagnosed with overt diabetes. Also, the effects of suboptimal glucose metabolism control in diabetes give rise to a wide range of related lipids and cardiovascular diseases.

  Dyslipidemia, ie abnormal lipoprotein levels in plasma, is a frequent occurrence in diabetic patients. Dyslipidemia generally consists of high plasma triglycerides, low HDL (high density lipoprotein) cholesterose, normal to high levels of LDL (low density lipoprotein) cholesterol, and low specific gravity in blood, LDL (low density lipoprotein) Characterized by increased particle levels. Dyslipidemia is one of the main causes of increased incidence of coronary events and death among diabetic subjects. Epidemiological studies confirmed this by demonstrating a several-fold increase in coronary artery death among diabetic subjects when compared to non-diabetic subjects. Several lipoprotein abnormalities have been described among diabetic subjects.

  Insulin resistance is the reduced ability of insulin to exert its biological action over a wide range of concentrations. In insulin resistance, the body secretes an abnormally high amount of insulin to make up for this deficiency and impaired glucose resistance. Failure to compensate for the lack of insulin action inevitably increases plasma glucose levels, resulting in a clinical state of diabetes. Insulin resistance and relative hyperinsulinemia have an incentive role in obesity, hypertension, atherosclerosis and type 2 diabetes. Obesity, hypertension and the association of angina and insulin resistance have been described as syndrome X, a syndrome with insulin resistance as a common pathogen-related factor.

  Apoptosis is an active process of cell self-destruction that is regulated by extrinsic and intrinsic signals that occur during normal development. It is well documented that apoptosis plays an important role in the regulation of pancreatic endocrine beta cells. There is also increasing evidence that adult mammals dynamically alter beta cell mass to produce insulin to maintain normoglycemia during certain conditions such as pregnancy and obesity. The control of beta cell mass depends on a delicate balance between cell proliferation, growth and cell self-death (apoptosis). This disruption of balance can lead to impaired glucose homeostasis. For example, glucose resistance develops with aging when beta cell replication rate is reduced, and human autopsy studies show that beta cell mass in patients with non-insulin-dependent diabetes compared to non-diabetic subjects It is worth noting that the -60% reduction was repeated. Insulin resistance is a permanent component of obesity, but normoglycemia is maintained by compensatory hyperinsulinemia until the start of type 2 diabetes, where beta cells cannot meet the increased demand for insulin That is generally accepted.

  Attempts to treat a number of abnormalities associated with diabetes have led to the administration of several anti-diabetic drugs to address these abnormalities in various patients. Examples of anti-diabetic drugs are proteins such as insulin and insulin analogs, and small molecules such as insulin sensitizers, insulin secretagogues and appetite regulating compounds.

  There remains a need for the development of polypeptides useful in the metabolic diseases, conditions and metabolic disorders described above. Accordingly, it is an object of the present invention to provide hybrid polypeptides and methods for their production and use. The compounds of the present invention can be used in the metabolic diseases, metabolic conditions and metabolic disorders described above and described herein.

  All documents mentioned in this specification are hereby incorporated by reference as if fully set forth herein.

  Summary of the Invention

  In general, the present invention is useful as an agent for the treatment and prevention of metabolic diseases and disorders, such as diabetes and diabetes related conditions, which can be alleviated by controlling plasma glucose levels, insulin levels and / or insulin secretion It relates to novel, selectable hybrid polypeptides. These conditions and abnormalities include hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and all types of diabetes, including type 1, type 2 and gestational diabetes, It is not limited to these.

  In one embodiment of the invention, a hybrid polypeptide exhibiting at least one hormonal activity is provided. The hybrid polypeptide of the present invention comprises at least two bioactive peptide hormone modules covalently linked to each other, wherein at least one of the bioactive peptide hormone modules exhibits at least one hormonal activity of a component peptide hormone. . Biologically active peptide hormone module includes component peptide hormone, component peptide hormone fragment exhibiting at least one hormone activity of component peptide hormone, analog and derivative of component peptide hormone exhibiting at least one hormone activity of component peptide hormone, component peptide Ingredients that exhibit at least one hormonal activity of the hormone are selected independently from peptide hormone analogues and derivatives fragments, and peptide enhancers.

  In one embodiment, the hybrid polypeptide exhibits at least one hormonal activity, and the hybrid polypeptide is a first bioactive peptide hormone module covalently linked to at least one additional bioactive peptide hormone module. These biologically active peptide hormone modules comprise: component peptide hormones; component peptide hormone fragments that exhibit at least one hormone activity of component peptide hormones; analogs of component peptide hormones that exhibit at least one hormone activity of component peptide hormones Independently from the group consisting of analogs and derivatives of component peptide hormones that exhibit at least one hormonal activity of the component peptide hormones; and peptide enhancers It is selected. These component peptide hormones are typically amylin, adrenomedullin (ADM), calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin, cholecystokinin (“CCK”), leptin, peptide Independent of at least two of the group consisting of YY (PYY), glucagon-like peptide-1 (GLP-1), glucagon-like peptide 2 (GPL-2), oxyntomodulin (OXM), natriuretic peptide and exendin-4 Selected. Typically, a peptide enhancer has a desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding or others in a hybrid polypeptide. The structural motifs of peptide hormones that confer the pharmacokinetic properties of and the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition to hybrid polypeptides Independently selected from the group consisting of structural motifs of analogs or derivatives of component peptide hormones that confer plasma protein binding or other pharmacokinetic properties. In still further embodiments, at least one of the bioactive peptide hormone modules exhibits at least one hormone activity of the component peptide hormone. In yet a further alternative embodiment, at least one bioactive peptide hormone module exhibiting at least one hormonal activity of the component peptide hormone is amylin, a fragment of amylin exhibiting at least one hormone activity, amylin exhibiting at least one hormone activity. Or a fragment of an analog or derivative of amylin that exhibits at least one hormone activity, and at least one other bioactive peptide hormone module is CCK, a fragment of CCK that exhibits at least one hormone activity CCK analogs or derivatives exhibiting at least one hormonal activity, fragments of CCK analogs or derivatives exhibiting at least one hormonal activity, CT, CT When a fragment, an analog or derivative of CT that exhibits at least one hormonal activity, or a fragment of an analog or derivative of CT that exhibits at least one hormonal activity, the hybrid polypeptide is selected from at least three different component peptide hormones May further contain at least three bioactive peptide hormone modules. In yet a further alternative embodiment, at least one bioactive peptide hormone module exhibiting at least one hormone activity of the component peptide hormone is GLP-1, a fragment of GLP-1 exhibiting at least one hormone activity, at least one hormone. An analog or derivative of GLP-1 that exhibits activity, or a fragment of an analog or derivative of GLP-1 that exhibits at least one hormonal activity, and at least one other biologically active peptide hormone module may comprise an exendin fragment When comprising a peptide enhancer, the hybrid polypeptide may further contain at least three bioactive peptide hormone modules.

  Component peptide hormones of the present invention include amylin, adrenomedullin (ADM), calcitonin (CT), calcitonin gene related peptide (CGRP), intermedin, cholecystokinin (`` CCK ''), leptin, peptide YY (PYY ), Glucagon-like peptide-1 (GLP-1), glucagon-like peptide 2 (GPL-2), oxyntomodulin (OXM), natriuretic peptide and exendin-4.

  Peptide enhancers of the present invention include hybrid polypeptides with the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding or other Structural motifs of the component peptide hormones that confer pharmacokinetic properties, and desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, Structural motifs of analogs or derivatives of the component peptide hormones that confer plasma protein binding or other pharmacokinetic properties.

  In another aspect of the invention, a method for treating or preventing obesity is provided, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a hybrid polypeptide of the invention. In preferred embodiments, the subject is an obese or overweight subject. “Obesity” is generally defined as a body mass index greater than 30, but for the purposes of this disclosure, any subject, including a person with a body mass index less than 30, that requires or desires weight loss. In the “obesity” scope. Subjects who are insulin resistant, glucose intolerant, or have any form of diabetes (eg, type 1, type 2 or gestational diabetes) can benefit from this method.

  In yet another aspect of the invention, methods for reducing dietary intake, methods for reducing nutrient utilization, methods for causing weight loss, methods for treating diabetes or conditions associated with diabetes, and improving lipid profiles Methods (including methods for lowering LDL cholesterol and triglyceride levels and / or methods for altering HDL cholesterol levels) comprising administering an effective amount of a hybrid polypeptide of the invention to a subject. Including. In a preferred embodiment, the methods of the invention are used to treat or prevent conditions or abnormalities that can be alleviated by reducing nutrient availability in a subject, and these methods are used to hybridize polypeptides of the invention. Administering to the subject a therapeutically or prophylactically effective amount of In another embodiment, the methods of the invention are used to treat or prevent conditions or abnormalities 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 conditions associated with diabetes. These conditions and abnormalities include hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and all types of diabetes, including type I, type II and gestational diabetes, diabetic complications (neuropathy) (Eg, based on the neurotropic effects of exendin-4), neuropathic pain (eg, based on amylin action), retinopathy, nephropathy, pancreatic beta cell deficiency (eg, exendin-4 and GLP) -1) based on -1 isogenic action)), but is not limited thereto.

  The present invention provides a therapeutically or prophylactically effective amount of at least one hybrid polypeptide of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable diluent, preservative useful for delivery of the hybrid polypeptide. It also relates to pharmaceutical compositions comprising solubilizers, emulsifiers, adjuvants and / or carriers.

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

  In general, the present invention is useful as an agent for the treatment and prevention of metabolic diseases and disorders, such as diabetes and diabetes related conditions, which can be alleviated by controlling plasma glucose levels, insulin levels and / or insulin secretion. Relates to novel, selectable hybrid polypeptides. Such conditions and disorders include hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and all types of diabetes, including type 1, type 2 and gestational diabetes, It is not limited to these.

  In one embodiment, the present invention may be selected based on “biological activity”, eg, therapeutic efficacy, range of function, duration of action, physiochemical properties, and / or other pharmacokinetic properties, A modular assembly of peptide modules that are active, metabolically and / or pharmacokinetically active.

  While not intending to be limited by theory, the present invention provides a novel, efficacious therapeutic agent with selectable properties in which bioactive peptide hormone modules are linked in a secondary, tertiary or higher order combination. Make, at least in part, a “toolbox” approach. A “bioactive peptide hormone module” can be a peptide hormone, a peptide fragment having hormonal activity, or a structural motif of a peptide hormone that confers chemical stability, metabolic stability and / or other pharmacokinetic stability. Peptide hormones can include natural peptide hormones and peptide hormone analogs and derivatives as are known in the art and described herein.

  In one embodiment of the invention, it has been found that combining certain physiochemical properties of two or more peptide hormones into a single modality can facilitate intervention at several points in the dysfunctional metabolic circuit. did. Accordingly, in one embodiment of the invention, a rationally designed hybrid polypeptide is provided that integrates a selectable biological activity into a single polypeptide agent. In one embodiment, a selectable hybrid polypeptide of the invention can include the use of a chemically stable linker to attach a bioactive module covalently. In another embodiment, the selectable hybrid polypeptides of the present invention are of cleavable linkers that may themselves be part of a bioactive module or may form part of a bioactive module. Use can be included.

  Again, without intending to be limited by theory, the design of the hybrid polypeptides of the invention generally includes (1) identification, selection and pairing of bioactive peptide hormone modules for the desired efficacy and therapeutic use. And (2) loss of biological activity of component modules of biological activity modules (eg, natural peptide hormones, peptide hormone analogs or derivatives with hormonal activity, peptide hormone fragments with hormonal activity, stabilization motifs, etc.) No, direct or linker covalent linkage may be included. In certain embodiments, the module selection criteria include: (a) in vivo efficacy desired for the desired therapeutic or prophylactic application, eg, additive or synergistic effects; (b) for multiple therapeutic or prophylactic applications Optimal synergism or dual action of the linking module; and / or (c) desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, Plasma protein binding and / or other pharmacokinetic properties include but are not limited to.

This section uses section titles for organizational purposes only and should not be construed as limiting the subject matter described in any way.
Hybrid polypeptide of the present invention

  As noted above, the present invention relates in part to hybrid polypeptides comprising at least two bioactive peptide hormone modules that can be selected from the component peptide hormones described herein. The hybrid polypeptides of the invention will generally be useful in the treatment and prevention of metabolic conditions and metabolic disorders. The hybrid polypeptides of the invention will exhibit at least one hormonal activity of the component peptide hormone, and preferably may include at least one additional biological activity of the second component peptide hormone.

  In one embodiment, a hybrid polypeptide of the invention can comprise at least two bioactive peptide hormone modules, each of which has at least one hormone activity of a component peptide hormone. Show. In another embodiment, the hybrid polypeptide of the invention can comprise at least two bioactive peptide hormone modules, wherein at least one of the bioactive peptide hormone modules is at least one hormonal activity of a component peptide hormone. Wherein at least one of the biologically active peptide hormone modules has a desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, Providing protease inhibition, plasma protein binding, and / or other pharmacokinetic properties.

  In preferred embodiments, the hybrid polypeptides of the invention have comparable or higher efficacy in the treatment and / or prevention of metabolic conditions and metabolic disorders when compared to component peptide hormones. In another embodiment, the hybrid polypeptides of the invention have comparable or higher efficacy in the treatment and / or prevention of diabetes and / or diabetes related diseases when compared to the component peptide hormones. Alternatively, preferred hybrid polypeptides of the present invention can exhibit improved manufacturability, stability, and / or ease of formulation compared to component peptide hormones.

  More particularly, the hybrid polypeptides of the invention will generally comprise a first bioactive peptide hormone module covalently linked to at least one additional bioactive peptide hormone module. Bioactive peptide hormone modules include each other in any manner known in the art, including (but not limited to) direct amide bonds or chemical linker groups, as described in further detail herein. It can be linked by a covalent bond. In one embodiment, the chemical linker group can include a peptidomimetic that induces or stabilizes the conformation of the polypeptide.

  The first bioactive peptide hormone module can be selected from the first component peptide hormones, can be peptide hormones (including natural peptide hormones and analogs and derivatives thereof), and peptide fragments ( Natural peptide hormones and fragments of analogs and derivatives thereof) or hybrid polypeptides of which the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting It may also be a structural motif of peptide hormones (including natural peptide hormones and their analogs and derivatives) that confer receptor interactions, protease inhibition, plasma protein binding or other pharmacokinetic properties. Similarly, the additional bioactive peptide module (s) can be selected from the component peptide hormones and can be peptide hormones (including natural peptide hormones and their analogs and derivatives), and hormone activity Peptide fragments (including fragments of natural peptide hormones and their analogs and derivatives), or the desired chemical stability, conformational stability, metabolic stability, bioavailability of the hybrid polypeptide May be a structural motif of peptide hormones (including natural peptide hormones and their analogs and derivatives) that confer organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding or other pharmacokinetic properties . The first peptide hormone and the additional peptide hormone may be the same peptide hormone, may be from the same family of peptide hormones, or different depending on the desired properties of the bioactive peptide hormone module It may be a peptide hormone.

  As used herein, the term “biological activity” refers to (1) biological activity in at least one in vivo hormonal pathway, or (2) therapeutic efficacy, functional range, duration of action, physiology of such biological activity. Refers to modification of chemical properties and / or other pharmacokinetic properties. Biological activity can be assessed by target hormone receptor binding assays, as known in the art and described herein, or by metabolic studies that monitor physiological indicators. Modification of the therapeutic efficacy, functional range, duration of action, physiochemical properties and / or other pharmacokinetic properties of such biological activity is, for example, chemical stability, conformational stability, metabolic stability, bioavailability Can be modified by altering organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding and / or pharmacokinetic properties.

  In one embodiment, the hybrid polypeptide of the invention has at least about 25%, preferably about 30%, 40%, 50%, 60%, 70%, 80%, of the biological activity of the component peptide hormones, Hold 90%, 95%, 98%, or 99% percent. Preferred hybrid polypeptides have a potency that is equal to or greater than that of the component peptide hormones in the same assay in one of the metabolic-related assays known in the art or described herein (eg, receptor Binding, food intake, gastric emptying, pancreatic secretion, insulin secretion, hypoglycemia, weight loss, etc.). Alternatively, preferred hybrid polypeptides of the present invention can exhibit improved manufacturability, stability, and / or ease of formulation compared to component peptide hormones.

In another embodiment, the hybrid polypeptides of the present invention may be used for natural component peptide hormones with respect to reduced nutrient utilization, reduced food intake, effects of weight gain, and / or treatment and prevention of metabolic conditions and metabolic disorders. Retain at least about 25%, preferably about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% percent of the biological activity. In yet another embodiment, the hybrid polypeptide of the invention is a natural peptide hormone with respect to reduced nutrient utilization, reduced food intake, effects of weight gain, and / or treatment and prevention of metabolic conditions and metabolic disorders. Exhibit at least about 110%, 125%, 130%, 140%, 150%, 200% or more of the biological activity of In another embodiment, the hybrid polypeptides of the invention exhibit improved component peptide hormone receptor agonist activity.
Component peptide hormones, analogs and derivatives

  In general, component peptide hormones include (a) amylin, adrenomedullin (“ADM”), calcitonin (“CT”), calcitonin gene-related peptide (“CGRP”), and intermedin (“AFP-6”). The amylin family, including known peptides); and (b) cholecystokinin ("CCK"); (c) the leptin family, including leptin and leptin-like peptides; (d) pancreatic polypeptides ("PP") ) And peptide YY (“PYY”); (e) peptide hormones derived from the proglucagon gene, such as glucagon, glucagon-like peptide-1 (“GLP-1”), glucagon-like peptide 2 ("GPL-2") and oxyntomodulin ("OXM"), and exendins such as exendin-3 and exendin-4 Cretin and incretin mimetics; (f) natriuretic peptides including ANP, BNP, CNP and urodilatin, their precursor forms and peptides derived therefrom, (g) the urocortin family and (h) the neuromedin family, and Peptide hormones useful for the treatment or prevention of metabolic diseases and disorders, including their analogs, derivatives and fragments. The component peptide hormones of the present invention discussed herein also include analogs and derivatives that retain the hormonal activity of these natural peptide hormones. In one embodiment, such analogs and derivatives are agonists of the target hormone receptor.

  “Amylin” is as described in US Pat. No. 5,234,906 for “Hypoglycemic hormone” issued August 10, 1993, the contents of which are incorporated herein by reference. , Called amylin, means human peptide hormone secreted from pancreatic beta cells and its species variants. More particularly, amylin is a 37-amino acid polypeptide hormone that is normally co-secreted with insulin by pancreatic beta cells in response to nutrient intake (eg, Koda et al., Lancet 339: 1179-1180, 1992 reference). In this sense, “amylin”, “wild-type amylin”, and “natural amylin” or unmodified amylin are used interchangeably.

  “Adrenomedullin” or “ADM” means human peptide hormones and species variants thereof. More specifically, ADM is produced from a 185 amino acid preprohormone by sequential enzymatic cleavage and amidation. This process reaches its highest point with the release of 52 amino acid bioactive peptides.

  “Calcitonin” or “CT” refers to human peptide hormones and species variants thereof including salmon calcitonin (“sCT”). More specifically, CT is a 32 amino acid peptide cleaved from a larger prohormone. It contains a single disulfide bond, and due to this bond, its amino terminus takes the form of a ring. Variable splicing of calcitonin pre-mRNA can give rise to mRNA encoding a calcitonin gene-related peptide, which appears to function in the nervous and vasculature. The calcitonin receptor has been cloned and shown to be a member of the seven-transmembrane G protein coupled receptor family.

  “Calcitonin gene-related peptide” or “CGRP” means human peptide hormone and species variants thereof in any physiological form.

  “Intermedin” or “AFP-6” refers to human peptide hormones and species variants thereof in any physiological form.

  “Cholecystokinin” or “CCK” refers to human peptide hormones and species variants thereof. More specifically, CCK is the first identified 33-amino acid sequence in humans, an 8-amino acid in vivo C-terminal fragment (reported in pigs, rats, chickens, chinchillas, dogs and humans) "CCK-8"). Thus, the term CCK-33 generally refers to human CCK (1-33), while CCK-8 (CCK (26-33); SEQ ID NO: 55) is genetically sulfated unless otherwise indicated. Refers to both the sulfated and non-sulfated forms of the C-terminal octapeptide. Furthermore, pentagastrin or CCK-5 refers to the C-terminal peptide CCK (29-33) (SEQ ID NO: 209), and CCK-4 refers to the C-terminal tetrapeptide CCK (30-33) (SEQ ID NO: 208). Point to. However, as used herein, CCK generally refers to hormonal, including sulfated and non-sulfated forms, CCK-33, CCK-8, CCK-5 and CCK-4, unless otherwise indicated. Refers to all naturally occurring variants.

  “Leptin” means naturally occurring leptin and biologically active D-isoforms from any species, or fragments of naturally occurring leptin and variants thereof, and combinations of the foregoing. Leptin is a peptide product of the ob gene as described in International Patent Publication No. WO 96/05309, which is hereby incorporated by reference in its entirety. Putative analogs and fragments of leptin are described in US Pat. No. 5,521,283, US Pat. No. 5,532,336, PCT / US96 / 22308 and PCT / US96 / 01471, each of which is incorporated herein by reference in its entirety. Are reported).

  “PP” means a human pancreatic peptide polypeptide or species variant thereof in any physiological form. Thus, the term “PP” refers to the full-length, 36 amino acid peptide as shown in (SEQ ID NO: 290) and species variants of PP, including, for example, mouse, hamster, chicken, bovine, rat and dog PP. Includes both. In this sense, “PP”, “wild-type PP” and “natural PP”, ie unmodified PP, are used interchangeably.

  “PYY” means a human YY polypeptide or species variant thereof in any physiological form. Thus, the term “PYY” encompasses both human full-length, 36 amino acid peptides and species variants of PYY including, for example, mouse, hamster, chicken, cow, rat and dog PYY. In this sense, “PYY”, “wild-type PYY” and “natural PYY”, ie, unmodified PYY, are used interchangeably. In the context of the present invention, all modifications discussed with respect to the PYY analog polypeptides of the invention are based on the 36 amino acid sequence of native human PYY.

  “GLP-1” means human glucagon-like peptide-1 or species variant thereof in any physiological form. The term “GLP-1” refers to human GLP-1 (1-37) (SEQ ID NO: 59), its full length human GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204) and GLP-1 (7-36) amide (SEQ ID NO: 61), and GLP-1 including, for example, mouse, hamster, chicken, cow, rat and dog PP Includes species variants. In this sense, “GLP-1”, “wild-type GLP-1” and “natural GLP-1”, ie, unmodified GLP-1, are used interchangeably.

  “GLP-2” means human glucagon-like peptide-2 or species variant thereof in any physiological form. More particularly, GLP-2 is a 33 amino acid peptide that is co-secreted with GLP-1 from enteroendocrine cells in the small and large intestines.

  “OXM” means human oxyntomodulin or species variant thereof in any physiological form. More particularly, OXM is a 37 amino acid peptide containing the 29 amino acid sequence of glucagon followed by an 8 amino acid carboxy terminal extension.

  “Exendin” refers to a peptide hormone found in the saliva of the American lizard (Gila-monster), the lizards native to Arizona, and the Mexican Beaded Lizard. More specifically, exendin-3 is present in the saliva of the Mexican lizard (scientific name: Heloderma horridum) and exendin-4 is present in the saliva of the American lizard (scientific name: Heloderma suspectum) (Eng, J., et al., J. Biol. Chem., 265: 20259-62, 1990; Eng., J., et al., J. Biol. Chem., 267: 7402-05 (1992)). Exendin has some sequence similarity with several members of the glucagon-like peptide family, with the highest 53% identity with GLP-1 (Goke, et al., J. Biol Chem., 268: 19650-55 (1993)). In this sense, “exendin”, “wild type exendin” and “natural exendin”, ie unmodified exendin, are used interchangeably.

  “Urocortin” means human urocortin peptide hormone or species variant thereof in any physiological form. More specifically, there are three human urocortins: Ucn-1, Ucn-2 and Ucn-3. For example, human urocortin 1 has the formula Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu- It has Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 (SEQ ID NO: 294). Urocortin from rat is identical except for two substitutions: Asn2 is replaced with Asp2 and Aer4 is replaced with Pro4. Human Ucn-2 has the sequence Ile Val Leu Ser Leu Asp Val Pro Ile Gly Leu Leu Gln Ile Leu Leu Glu Gln Ala Arg Ala Arg Ala Ala Arg Glu Gln Ala Thr Thr Asn Ala Arg Ile Leu Ala Arg Val Gly His Cys Number: 399). Human Ucn-3 has the sequence Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe Asn Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala His Leu Met Ala Gln Ile (SEQ ID NO: 299 ). Ucn-3 is preferably in the amide form. Additional urocortin and analogs are described in the literature, eg US Pat. No. 6214797. Urocortin Ucn-2 and Ucn-3, which retain dietary intake suppression and antihypertensive / cardioprotective / muscular inotropic properties, can be used in particular in the hybrids of the present invention. Named for their ability to suppress chronic HPA activation following stressful stimuli such as feeding / fasting, stress copine (Ucn-3) and stress copine-related peptide (Ucn 2) are CRF type 2 receptor It is specific to the body and does not activate CRF-R1, which mediates ACTH release. Hybrids comprising urocortin, such as Ucn-2 or Ucn-3, are particularly useful for vasodilation and thus for cardiovascular applications such as those described herein, such as CHF. The urocortin-containing hybrids of the present invention are particularly associated with conditions associated with stimulation of ACTH release, hypertension due to vasodilatory effects, inflammation mediated by other than ACTH elevation, hyperthermia, appetite abnormalities, congestive heart failure, stress, anxiety and Can be used for the treatment or prevention of psoriasis. Such compounds are also useful for anti-proliferative effects, for example to treat or prevent cancer or tumor growth. Urocortin in combination with natriuretic peptide module, amylin family, exendin family or GLP1 family module to provide enhanced cardiovascular benefits, for example to treat CHF by providing beneficial vasodilatory effects, etc. Of particular interest is the peptide hormone module.

  “Neuromedin” means neuromedin U and S peptides, and more particularly the neuromedin family of peptides comprising their active hormone sequences. For example, the naturally active human neuromedin U peptide hormone is Neuromedin-U25: Phe Arg Val Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe Leu Phe Arg Pro Arg Asn (SEQ ID NO: 308), in particular Its amide form. Pig U25 has the sequence: FKVDEEFQGPIVSQNRRYFLFRPRN (SEQ ID NO: 314), in particular its amide form. Other neuromedin U family members include their SWISS-PROT symbols and registration numbers listed below: NEUU_CANFA (P34962), NEUU_CAVPO (P34966), NEUU_CHICK (P34963), NEUU_HUMAN (P48645), NEUU_LITCE ( P81872), NEUU_MOUSE (Q9QXK8), NEUU_PIG (P34964), NEUU_RABIT (P34965), NEUU_RANTE (P20056), and NEUU_RAT (P12760). Of particular interest are these processed peptide hormones and their analogs, derivatives and fragments. Various truncated or splice variants, for example FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN (SEQ ID NO: 300) are included in the neuromedin U family. An example of the neuromedin S family is human neuromedin S, in particular its amide form, having the sequence ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN (SEQ ID NO: 315). The hybrids of the invention with a neuromedin module are beneficial in anorexia-inducing effects and thus in obesity, treatment of diabetes, reduced food intake and other related conditions and disorders as described herein Will have value. Of particular interest are neuromedin modules in combination with amylin family peptides, exendin peptide families or GLP1 peptide family modules.

  As used herein, an “analog” is an insertion of a reference amino acid sequence whose sequence is derived from the sequence of a basic reference peptide (eg, PP, PYY, amylin, GLP-1, exendin, etc.), Peptides containing substitutions, extensions and / or deletions, preferably have at least 50 or 55% amino acid sequence identity with the base peptide, more preferably at least 70%, 80%, 90 with the base peptide Refers to peptides with% or 95% amino acid sequence identity. In one embodiment, such analogs can include conservative or non-conservative amino acid substitutions (including unnatural amino acids and L and D forms).

  A “derivative” has the amino acid sequence of a natural reference peptide or analog, but additionally has one or more chemical modifications of its amino acid side groups, α-carbon atoms, terminal amino groups or terminal carboxylic acid groups. It is defined as a molecule having. Chemical modifications include, but are not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at the amino acid side groups include acylation of lysine ε-amino groups, N-alkylation of arginine, histidine or lysine, alkylation of glutamic acid or aspartic acid carboxylic acid groups, and deamidation of glutamine or asparagine. However, it is not limited to these. Terminal amino modifications include deamination, N-lower alkyl, N-di-lower alkyl, constrained alkyl (eg, branched alkyl, cyclic alkyl, fused alkyl, adamantyl alkyl) and N-acyl modifications, It is not limited to these. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, constrained alkyl (eg, branched, cyclic, fused, adamantyl) alkyl, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. In addition, one or more side groups or terminal groups can be protected by protecting groups known to the skilled peptide chemist. The α-carbon of the amino acid may be mono- or dimethylated.

  An “agonist” is a compound that elicits the biological activity of a natural human reference peptide, preferably better potency than the reference peptide when evaluated by means known in the art such as receptor binding / competition testing. Or a compound having a potency within 5 orders of magnitude (plus or minus) compared to its reference peptide, more preferably 4, 3, 2 or 1 order. In one embodiment, these terms are compounds that elicit a biological effect similar to that of a natural human reference peptide, e.g., (1) in a dietary intake, gastric emptying, pancreatic juice secretion or weight loss assay. A compound that has activity similar to a human reference peptide or (2) specifically binds in a reference receptor assay or in a competitive binding assay with a labeled reference peptide. Preferably, the agonist will bind in such an assay with an affinity of greater than 1 μM, more preferably with an affinity of greater than 1-5 nM. In another embodiment, these terms refer to compounds that elicit a biological effect in the treatment of diabetes or a condition or disease associated with diabetes. Such agonists can include a polypeptide comprising a reference peptide or an active fragment of a small chemical molecule.

  “Amino acid” and “amino acid residue” mean natural amino acids, unnatural amino acids, and modified amino acids. Unless stated to the contrary, any reference to an amino acid refers to a reference to both the D and L stereoisomers, generally or specifically, if their structure allows such stereoisomeric forms. Included. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His) , Isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) And valine (Val). Non-natural amino acids include homo-lysine, homo-arginine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6 -Aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, t-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3 -Diaminopropionic acid, N-ethylglycine, N-ethylalparagine, homoproline, hydroxysilin, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methyl Glycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphtha Min, norvaline, norleucine, ornithine, pentylglycine, including but pipecolic acid and thioproline, without limitation. Additional non-natural amino acids include, for example, N-terminal amino acids such as N-methylated D and L amino acids or residues whose side chain functional groups are chemically modified to another functional group Examples include modified amino acid residues that are reversibly, irreversibly or chemically modified wherein the groups or their side chain groups are chemically blocked. For example, modified amino acids include methionine sulfoxide; methionine sulfone; aspartic acid- (beta-methyl ester), modified amino acid of aspartic acid; modified amino acid of N-ethylglycine, glycine; or modified amino acid of alanine carboxamide, alanine . Additional residues that can be incorporated are described in Sandberg et al., J. Med. Chem. 41: 2481-91, 1998.

  As used herein, “5 Apa” means 5 amino-pentanoyl, “12 Ado” means 12-aminododecanoyl, and “PEG (8)” means 3,6, -di- Means oxyoctanoyl, and “PEG (13)” means 1-amino-4,7,10-trioxa-13-tridecanamine succinimoyl.

The component peptide hormones discussed herein are known in the art and their analogs and derivatives are also known. For reference, sequences of some natural component peptide hormones are provided in Table 1.
Table 1: Exemplary component peptide hormones

These peptides are generally C-terminal amidated when expressed physiologically, but need not be for the purposes of the present invention. In other words, the C-terminus of these peptides and the hybrid polypeptides of the invention may have a free —OH or —NH ₂ group. These peptides may have other post-modification modifications. One skilled in the art will appreciate that the hybrid polypeptides of the present invention may be composed of an N-terminal methionine residue.

  Exemplary peptide modules for use in the present invention include apelin (LVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF-OH (SEQ ID NO: 316) and pERPRLSHKGPMPF-OH, which exist in two forms, apelin 36 and 13, both active at the AJP receptor. SEQ ID NO: 317)); prolactin-releasing peptides (SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH2 (SEQ ID NO: 318) and TPDINPAWYASRGIRPVGRF-NH2 (SEQ ID NO: 319)) present in two forms, PRP31 and PRP20 and equally active at GPR10; Gastrin (QLGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDF-NH2 (SEQ ID NO: 320); pEGPWLEEEEEAYGWMDF-NH2 (SEQ ID NO: 321); (SEQ ID NO: 322)); CCK (central vs. peripheral; KAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF-NH2 (CCK33 or CCK8) SEQ ID NO: 323); DYMGWMDF-NH2) (SEQ ID NO: 55); Corstatin (QEGAPPQQSARRDRMPCRNFFWKTFSSCK-OH (SEQ ID NO: 324) and DRMPCRNFFWKTFSSCK-OH (SEQ ID NO: 325)) present as cortisatin 17 or 29; somatostatin 14 Or somastatin (SANSNPAMAPRERKAGCKNFFWKTFTSC-OH (SEQ ID NO: 326); AGCKNFFWKTFTSC-OH (SEQ ID NO: 327)); GRP (VPLPAGGGTVLTKMYPRGNHWAVGHLM-NH2 (SEQ ID NO: 328); GNHWAVGHLM-NH2 (SEQ ID NO: 329)); Neuromedin B in which the C-terminal 10 amino acid region has most of its activity (LSWDLPEPRSRASKIRVHSRGNLWATGHFM-NH2 (SEQ ID NO: 330); GNLWATGHFM-NH2 (SEQ ID NO: 331)) Neuromedin S (ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN-NH2 (SEQ ID NO: 315); PFFLFRPRN-NH2 (SEQ ID NO: 332)) whose C-terminal 9 amino acid region has most of its activity Neuromedin U (FRVDEEFQSPFASQSRGYFLFRPRN-NH2 (SEQ ID NO: 308); GYFLFRPRN-NH2 (SEQ ID NO: 307)); neurotensin existing as a long and short form (KIPYILKRQLYENKPRRPYIL- OH (SEQ ID NO: 333); QLYENKPRRPYIL-OH (SEQ ID NO: 334)); Kiss-1 (GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF-NH2 (SEQ ID NO: 335); EKDLPNYNWNSFGLRF-NH2 (SEQ ID NO: GLRF-NH2) 336)); RF-amide-3 whose C-terminal fragment is active (SAGATANLPLRSGRNMEVSLVRRVPNLPQRF-NH2 (SEQ ID NO: 337); VPNLPQRF-NH2 (SEQ ID NO: 338)); large dynorphin (A) of dynorphin B (rimorphin) Dinorphine (YGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVT-OH (SEQ ID NO: 339) and YGGFLRRQFKVVT-OH (SEQ ID NO: 340)); its C-terminus is active at the Y2 receptor A certain PYY (YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH2 (SEQ ID NO: 57); SLRHYLNLVTRQRY-NH2 (SEQ ID NO: 341)); AFP-6 (TQAQLLRVGCVLGTCQVQSYL-VQSQ) NH2 (SEQ ID NO: 52)); amylin family including adrenomodulin, calcitonin and CGRP; N-terminal extension is possible, including oxytocin, whose C-terminal amide is generally required for activity and can tolerate N-terminal extension Further included are modules (and their analogs and derivatives).

  Exemplary peptide modules for use in the present invention include Endothelin I, II and III: ETI (CSCSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSPRS-OH (SEQ ID NO: 342); SEQ ID NO: 344); CSCSSWLDKECVYFCHLDIIW-OH (SEQ ID NO: 345)) and ETIII (CTCFTYKDKECVYYCHLDIIWINTPEQTVPYGLSNYRGSFR-NH2 (SEQ ID NO: 346); CTCFTYKDKECVYYCHLDIIW-OH (SEQ ID NO: 347)); Ghrelin (GSSFLSPEHQRVQQRKESKKPPAKLQP-OH (SEQ ID NO: 348); GSSFLSPEHQ-OH (SEQ ID NO: 349); Glucagon (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-OH (SEQ ID NO: 349); HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 351)); GLP-1 / GLP-2 retained with or without; GIP (YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH (SEQ ID NO: 352), which circulates in two forms, GIP1-42 and GIP1-30, both fully active at the GIP receptor ); YAEGTFISDYSIAMDKIHQQDFVNWLLAQK-NH2 (SEQ ID NO: 353)); Neuropeptide W present as NPW23 and NPW30 and is equally active in GPR7 and 8 (WYKHVASPRYHTVGRAAGLLMGLRRSPYLW-OH (SEQ ID NO: 354); WYKHVASPRYHTVGOH AGL (SEQ ID NO: 354); PACAP (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK-NH2 (SEQ ID NO: 356); HSDGIFTDSYSRYRKQMAVKKYLAAVL-NH2 (SEQ ID NO: 357)); PHI and PHV (HADGVFTSDFSKLLGQVSAKLES HADGVFTSDFSKLLGQLSAKKYLESLM-NH2 (SEQ ID NO: 359)); GRF present in two forms GRF29 and GRF40 (YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL-NH2 (SEQ ID NO: 360); YADAIFTNSYRKVLGQLSAR KLLQDIMS-OH (SEQ ID NO: 361)); PTH 1-34 and 1-37 forms with full-length PTH 1-84 activity (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO: 362); SVSEIQLMHN -OH (SEQ ID NO: 364)); PTH-RP (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKP-NH2 (SEQ ID NO: 365); AVSEHQRRHDFLHSIH Gamma-MSH1 and longer gamma-MSH3 have similar activities Gamma-MSH (YVMGHFRWDRFGRRNSSSSGSSGAGQ-OH (SEQ ID NO: 367); YVMGHFRWDRF-NH2 (SEQ ID NO: 368)); Alpha-MSH is the active part of ACTH MSH (SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF-OH (SEQ ID NO: 369); SYSMEHFRWGKPV-NH2 (SEQ ID NO: 370); and A, Delta and Endorphins (YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE-OH (SEQ ID NO: 371); YGGFMTSEKSQTPLVTLFKNAIIKNAY-OH (SEQ ID NO: 372); YGGFMTSEKSQTPLVTL-OH (GF: MTSE; Y)) (SEQ ID NO: 374)) and other C-terminal extendible peptides are further included.

  For example, melanocortin is a peptide from the pro-opiomelanocortin gene, including alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH), and five melanocortin receptors are known, MC1 -5R. MC4R appears to play a role in energy balance and obesity. For example, Anderson et al., Expert Opin. Ther. Patents 11: 1583-1592 (2001), Speake et al., Expert Opin. Ther. Patents 12: 1631-1638 (2002), Bednarek et al., Expert Opin. See Ther. Patents 14: 327-336 (2004).

  Analogs of the above component peptide hormones are known in the art, but generally include modifications such as substitutions, deletions and insertions into the amino acid sequence of such component peptide hormones and any combination thereof. Substitutions, insertions and deletions can be at the N-terminal tip, at the C-terminal tip, or can be an internal part of its component peptide hormones. In a preferred embodiment, the analogs of the component peptide hormones of the invention comprise one or more modifications of “non-essential” amino acid residues. In the context of the present invention, “non-essential” amino acid residues are altered, ie, deleted or substituted, without completely destroying or substantially reducing the component peptide hormone receptor agonist activity of the resulting analog. A residue within the natural human amino acid sequence of the fragment, eg, a component peptide hormone fragment, that can be.

  Preferred substitutions include conservative amino acid substitutions. A “conservative amino acid substitution” is a substitution of an amino acid residue with an amino acid residue having similar side chain or physiochemical properties (eg, electrostatic, hydrogen-bonded, isosteric, hydrophobic characteristics) It is to be done. Families of amino acid residues with similar side chains are known in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg, glycine) , Asparagine, glutamine, serine, threonine, tyrosine, methionine, cysteine), amino acids with non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), amino acids with β-branched side chains (For example, threonine, valine, isoleucine) and amino acids having an aromatic side chain (for example, tyrosine, phenylalanine, tryptophan, histidine).

  The invention also relates to derivatives of the component peptide hormones. Such derivatives include one or more water-soluble polymer molecules such as polyethylene glycol (“PEG”) or fatty acid chains of various lengths (eg, stearyl, palmitoyl, octanoyl, etc.) or polyamino acids, For example, the component peptide hormones and their analogs conjugated by addition of poly-his, poly-arg, poly-lys, and poly-ala. Modifications to component peptide hormones or analogs thereof may include small molecule substituents such as short alkyls and constrained alkyls (eg, branched alkyl, cyclic alkyl, fused alkyl, adamantyl alkyl), and aromatic groups . The water soluble polymer molecule will preferably have a molecular weight ranging from about 500 to about 20,000 daltons.

  Such polymer conjugation and small molecule substituent modifications can be performed once at the N- or C-terminus within the sequence of the hybrid polypeptide or at the side chain of an amino acid residue. Alternatively, there may be multiple derivatization sites along the hybrid polypeptide. Additional derivatization sites can be generated by substituting one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine. See, for example, US Pat. Nos. 5,824,784 and 5,824,778. Preferably, the hybrid polypeptide can be conjugated to one, two or three polymer molecules.

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

The derivatives of the present invention also include component peptide hormones or analogs that have a chemical modification to one or more amino acid residues. Such chemical modifications include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization. These chemical modifications can be made at the N- or C-terminus within the sequence of the PPF hybrid polypeptide or at the side chain of amino acid residues. In one embodiment, the C-terminus of these peptides may have a free —OH or —NH ₂ group. In another embodiment, the N-terminal tip has an isobutyloxycarbonyl group, isopropyloxycarbonyl group, n-butyloxycarbonyl group, ethoxycarbonyl group, isocaproyl group (isocap), octanyl group, octylglycine group (G (Oct )), Or may be capped with an 8-aminooctanoic acid group. In preferred embodiments, the cyclization may be by formation of a disulfide bridge. Alternatively, there may be multiple chemical modification sites along the hybrid polypeptide.
Amylin family

  The component peptide hormones useful in the present invention discussed herein include amylin, adrenomedullin (“ADM”), calcitonin (“CT”), calcitonin gene related peptide (“CGRP”), intermedin (“ And amylin family peptide hormones, including AFP-6 ”) and related peptides. Natural amylin family peptide hormones are known in the art, and functional peptide analogs and derivatives are also known. Although certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any amylin family peptide exhibiting hormonal activity known in the art can be used in conjunction with the present invention.

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

Exemplary amylin analogs include the following:

  As is known in the art, such amylin analogs are preferably amidated, but may be in their acid form in some cases unless otherwise indicated in connection with the present invention.

  Any ADM analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, these ADM analogs and derivatives have at least one hormonal activity of natural ADM. In certain embodiments, these ADM analogs are receptor agonists to which native ADM can specifically bind.

  Any CT analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, these CT analogs and derivatives have at least one hormonal activity of native CT. In certain embodiments, these CT analogs are agonists of receptors to which native CT can specifically bind. Preferred CT analogs and derivatives include U.S. Patent Nos. 4,652,627, 4,606,856, 4,604,238, 4,597,900, 4,537,716, 4,497,731, 4,495,097, No. 4,414,149, No. 4,401,593, and No. 4,397,780, which are incorporated herein by reference.

Exemplary CT analogs include the following:

  As is known in the art, such CT analogs are preferably amidated but may optionally be in their acid form unless otherwise indicated in connection with the present invention.

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

Exemplary CGRP analogs include the following:

  Any AFP-6 analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, these AFP-6 analogs and derivatives have at least one hormonal activity of native AFP-6. In certain embodiments, these AFP-6 analogs are receptor agonists to which native AFP-6 can specifically bind. Preferred AFP-6 analogs and derivatives include those described in WO 2003/022304, which is incorporated herein by reference.

Exemplary AFP-6 analogs include the following:

As is known in the art, such AFP-6 analogs are preferably amidated, but in the context of the present invention, even in their acid form, unless otherwise indicated. Good.
CCK family

  CCK (including hCCK and species variants and species variants) and their various analogs are known in the art. In general, CCK has a 33-amino acid sequence originally identified in humans, and reported 8-amino acid in vivo C-terminal fragment ("CCK- 8 ”). Other species variants include the 39-amino acid sequence found in pigs, dogs and guinea pigs, and the 58-amino acid sequence found in cats, dogs and humans, and the 47-amino acid sequence homologous to both CCK and gastrin. The C-terminal tyrosine sulfated octapeptide sequence (CCK-8) is relatively conserved across species and may be the minimal sequence for biological activity in the rodent periphery. Thus, the term CCK-33 generally refers to human CCK (1-33), while CCK-8 (CCK (26-33); SEQ ID NO: 55) is genetically sulfated unless otherwise indicated. Refers to both the sulfated and non-sulfated forms of the C-terminal octapeptide. Furthermore, pentagastrin or CCK-5 refers to the C-terminal peptide CCK (29-33) (SEQ ID NO: 209), and CCK-4 refers to the C-terminal tetrapeptide CCK (30-33) (SEQ ID NO: 208). Point to.

The type A receptor subtype (CCK _A ) has been reported to be selective for sulfated octapeptides. The type B receptor subtype (CCK _B ) has been identified throughout the brain and stomach and reportedly does not require sulfation and all eight amino acids.

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

Certain exemplary CCK and CCK analogs having CCK activity include the following:

As is known in the art, such CCK analogs are preferably amidated but may optionally be in their acid form unless otherwise indicated in connection with the present invention.
Leptin family

  Component peptide hormones useful in the present invention also include leptin family peptide hormones. Natural leptin family peptide hormones are known in the art, and functional peptide analogs and derivatives are also known. Although certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any known amylin family peptide exhibiting hormonal activity known in the art can be used in conjunction with the present invention. .

  Any leptin analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, these leptin analogs and derivatives have at least one hormonal activity of natural leptin. In certain embodiments, these leptin analogs are agonists of receptors to which natural leptin can specifically bind. Preferred leptin analogs and derivatives are described, for example, in WO 2004/039832, WO 98/55139, WO 98/12224, and WO 97/02004, all of which are incorporated herein by reference. What is being done is mentioned.

  In one embodiment, the leptin peptides include MVPIQK, VQDDTK, TLIK, TIVTR, INDISHTQSVSSK, VTGLDFIPGLHPILTLSK, NVIQISNDLENLR, DLLHVLAFSK, SCHLPWASGLETLDSLGGVLEASGYSTEVPGR and LQGSLDLSMLW as described in WO 97046585.

  In one embodiment, the leptin peptide can have the amino acid sequence Xaan-Ser-Cys-Xaa1-Leu-Pro-Xaa2-Xaa3-Xaan, where Xaan is a residue of zero length Or a continuous extension of peptide residues derived from a full-length human or mouse leptin sequence, an extension between 1 and 7 at either the C-terminal or N-terminal, or This leptin peptide has a total length of 15 or less amino acids. In another embodiment, Xaa1, Xaa2 or Xaa3 can be any amino acid substitution. In yet another embodiment, Xaa1, Xaa2 or Xaa3 can be any conservative amino acid substitution of the respective residue in full-length mouse or human leptin. In further embodiments, Xaa1 can be selected from the group consisting of His or Ser, and Xaa2 or Xaa3 is any amino acid substitution. In another embodiment, Xaa2 can be selected from the group consisting of Trp or Gln, and Xaa1 or Xaa3 is any amino acid substitution. In yet another embodiment, Xaa3 can be selected from the group consisting of Ala or Thr, and Xaa1 or Xaa2 is any amino acid substitution. In another embodiment, Xaa1 is selected from the group consisting of His or Ser, Xaa2 is selected from the group consisting of Trp or Gln, and Xaa3 is selected from the group consisting of Ala or Thr. See WO 04039832.

  In one embodiment, the leptin peptide is a natural full-length human or mouse leptin C-terminal amino acid residue 116-122 having the ability to modulate body weight homeostasis in a test animal when administered ip (intraperitoneally) As well as their D-isoforms, fragments, derivatives, analogs and homologs). Specific mouse D-substituted peptides of the sequence SCSLPQT include [D-Ser-l]-, [D-Cys-2]-, [D-Ser-3]-, [D-Leu-4]-, [D-Pro-5]-, [D-Gln-6]-, [D-Thr-7] -SCSLPQT and all [D] SCSLPQT. Specific human D-substituted peptides of SCHLPWA include [D-Ser-l]-, [D-Cys-2]-, [D-His-3]-, [D-Leu-4]-, [ D-Pro-5]-, [D-Trp-6]-, [D-Ala-7] -SCHLPWA and all [D] -SCHLPWA. In addition, the SCHLPWA and SCSLPQT peptides may contain D-substituted amino acids at any two, three, four, five or six positions. Also disclosed are leptin-related peptides comprising the N-terminal amino acids 21-35, 31-45, 41-55 and 51-65 of natural leptin, and fragments, derivatives, analogs and homologues thereof. Further leptin peptides of the invention are amino acid sequences 61-75, 71-85, 81-95, 91-105, 106-120, 116-130, 126-140, 136-150 of mouse and / or human full length leptin. , 146-160, and 156-167. See WO 04039832.

  In one embodiment, the leptin has the sequence Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys, in which Xaa at position 6 is Trp or Gln; Xaa at position 36 is Gln or Glu; Xaa at position 40 is Xaa at position 42 is Ile, Leu, Met or methionine sulfoxide; Xaa at position 44 is Trp or Gln; and Xaa at position 45 is Gln or Glu. In another embodiment, the above leptin is: Xaa at position 6 is Trp; Xaa at position 36 is Gln; Xaa at position 40 is Gln; Xaa at position 42 is Met; Xaa is Trp; and Xaa at position 45 is Gln. See US Pat. No. 5,521,283.

In one embodiment, the leptin is a native sequence including:
Mouse Leptin: Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Gln Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Gln Gln Leu Asp Leu Ser Pro Gly Cys ( In the sequence, Xaa at position 28 is Gln or absent);
Butapeptin: Val Pro Ile Trp Arg Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Ser Asp Ile Ser His Met Gln Ser Val Ser Ser Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys Pro Leu Pro Gln Ala Arg Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ala Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro Gly Cys;
Bovine Leptin: Val Pro Ile Cys Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Leu Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser Cys Pro Leu Pro Gln Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro Gly Cys Wherein Xaa at position 28 is Gln or absent);
Human leptin: Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Xaa Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys Xaa at position 27 is Thr or Ala; and Xaa at position 28 is Gln or absent); Rhesus monkey leptin: Val Pro Ile Gln Lys Val Gln Ser Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val Leu Thr Leu Ser Gln Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile Leu Ile Asn Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys His Leu Pro Leu Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Asp Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys; and Rat Leptin: Val Pro Ile His Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ala Arg Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln Ile Ala His Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys Ser Leu Pro Gln Thr Arg Gly Leu Gln Lys Pro Glu Ser Leu Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Leu Ser Pro Glu Cys.

In another embodiment, the leptin peptide has the sequence:
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val Ser Ser Ly Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Xaa Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Gln Ile Xaa Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys In the sequence, Xaa at position 22 is Asn, Asp or Glu; Xaa at position 27 is Thr or Ala; Xaa at position 28 is Gln or Glu or absent; Xaa at position 68 is Met or Leu; Xaa at position 72 is Asn, Asp or Glu; Xaa at position 77 is Ser or Ala; Xaa, Gly Or the Leu; the protein has at least one substitution selected from the group consisting of: His at position 97 is replaced with Ser or Pro; Trp at position 100 is Gln, Ala or Leu Ala at position 101 is replaced with Thr or Val; Ser at position 102 is replaced with Arg; Gly at position 103 is replaced with Ala; Glu is replaced with Gln; Thr at position 106 is replaced with Lys or Ser; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu Or Gly at position 111 is replaced with Asp.

In another embodiment, the leptin has the sequence: Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Xaa Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys, in the sequence, Xaa at position 27 is Thr or Ala; Xaa at position 77 is Ser or Ala; Xaa at position 118 is Gly or Leu Said protein has at least one substitution selected from the group consisting of: preferably 1 to 5 substitutions, most preferably Has two substitutions: His at position 97 is replaced with Ser; Trp at position 100 is replaced with Gln; Ala at position 101 is replaced with Thr; Glu is replaced with Gln; Thr at position 106 is replaced with Lys; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu; or Gly at position 111 is replaced with Asp. In further embodiments of the above sequence, Xaa at position 27 is Thr; Xaa at position 77 is Ser; Xaa at position 118 is Gly; and positions 97, 100, 101, 105, 106 , 107, 108 and 111 amino acid residues are as shown in the following table:

  In one embodiment, the leptin is Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa Ile Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Xaa Ile Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys, in this sequence, position 13 is Ile, Leu, Met or methionine sulfoxide; position 15 Xaa is Gln or Glu Xaa at position 21 is Gln or Glu; Xaa at position 22 is Gln or Glu; Xaa at position 27 is Ile, Leu, Met or methionine sulfoxide; Xaa at position 31 is Asn, Asp or Gln; Xaa at position 34 is Gln or Glu; Xaa at position 37 is Asn, Asp or Gln; Xaa at position 41 is Asn, Asp or Gln; Xaa at position 59 is Trp or Gln; Xaa at position 89 is Gln or Glu; Xaa at position 93 is Gln or Glu; Xaa at position 95 is Ile, Leu, Met, or methionine sulfoxide; Xaa at position 97 is Trp or Gln; and Xaa at position 98 is Gln or Glu. In another embodiment, the leptin of the above formula has Xaa at position 13 as Met; Xaa at position 15 is Gln; Xaa at position 21 is Gln; Xaa at position 22 is Gln; Xaa at position 27 is Met; Xaa at position 31 is Asn; Xaa at position 34 is Gln; Xaa at position 37 is Asn; Xaa at position 41 is Asn; Xaa at position 59 is Trp Xaa at position 89 is Gln; Xaa at position 93 is Gln; Xaa at position 95 is Met; Xaa at position 97 is rp; and Xaa at position 98 is Gln. See US Pat. No. 5,532,336.

  Exemplary leptin analogs include those in which the amino acid at position 43 is replaced with Asp or Glu; position 48 is replaced with Ala; position 49 is replaced with Glu or absent; Position 75 is substituted with Ala; Position 89 is replaced with Leu; Position 93 is replaced with Asp or Glu; Position 98 is replaced with Ala; Position 117 is Ser Substituted at position 139 with Leu; position 167 substituted with Ser.

PPFファミリー Certain exemplary leptin and leptin analogs having leptin activity include the following:

PPF family

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

  Any PPF analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, these PPF analogs and derivatives have at least one hormonal activity of a native PPF polypeptide. In certain embodiments, these PPF analogs are agonists of receptors to which the native PPF polypeptide can specifically bind. Preferred PPF analogs and derivatives include those described in WO 03/026591 and WO 03/057235, which are incorporated herein by reference.

  In one embodiment, preferred PFF analogs and derivatives that exhibit at least one PPF hormone activity generally comprise at least two PYY motifs, including a polyproline motif and a C-terminal tail motif. Such analogs are generally described in US Provisional Application No. 60 / 543,406, filed Feb. 11, 2004, published as US 2006 / 013547A1 on Jun. 22, 2006 (see references Is incorporated herein by reference). Another preferred PPF analog is disclosed in PCT / US2005 / 004351 entitled “Pancreatic Polypeptide Family Motifs and Polypeptides Comprising the Same”, published as WO 2005/077094 on August 25, 2005. Is incorporated herein by reference. Other preferred PPF analogs are disclosed in PCT / US2005 / 045471 entitled "Pancreatic Polypeptide Family Motifs, Polypeptides and Methods Comprising the Same", published as WO 2006/066024 on June 22, 2006, This content is incorporated herein by reference. As background, studies suggest that differences in Y receptor binding affinity correlate with differences in secondary and tertiary structure. See, for example, Keire et al., Biochemistry 2000, 39, 9935-9942. Natural porcine PYY is a two C-terminal helix segment from residues 17-22 and 25-33 separated by a twist at residues 23, 24 and 25, a turn centered around residues 12-14, and Characterized to include an N-terminus that folds around residues 30 and 31. Furthermore, full-length porcine PYY has been characterized as containing a PP fold stabilized by hydrophobic interactions between residues at the N and C termini. See the same book.

  In general, a “PYY motif” is essential for biological activity, ie, primary, secondary, natural PP family polypeptides whose biological activity is substantially reduced upon the absence or interference of the motif, Or it is a tertiary structure component. Preferred PYY motifs include the N-terminal polyproline type II motif of the natural PP family polypeptide, the type II β-turn motif of the natural PP family polypeptide, the α-helix motif at the C-terminal tip of the natural PP family polypeptide, and C-terminal tail motif of natural PP family polypeptide. More particularly, within the N-terminal polyproline region, amino acids corresponding to residues 5 and 8 of a natural PP family polypeptide are generally conserved as proline. Type II β-turn motifs generally include amino acids corresponding to residues 12-14 of a native PP family polypeptide. In general, the α-helix motif can span any point from the amino acid corresponding approximately to residue 14 of the natural PP family polypeptide to the C-terminal tip, including the C-terminal tip, provided that the α-helix motif is It is provided that it contains a sufficient number of amino acid residues to form an α-helix turn in the dissolved state. An α-helix motif may include amino acid substitutions, insertions and deletions into its native PP family sequence, provided that an α-helix turn is still formed in the dissolved state. The C-terminal tail motif is generally an amino acid corresponding to the last about 10 residues of a natural PP family polypeptide, more preferably the last 7, 6 or 5 residues of a natural PP family polypeptide, and more preferably, Contains amino acid residues 32-35.

  Preferred PYY analogs include those with internal deletions, insertions and substitutions in regions of the PYY molecule that do not correspond to a polyproline motif and / or a C-terminal tail motif. For example, an internal deletion at position 4, 6, 7, 9 or 10 is contemplated.

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

  In yet another embodiment, the PPF family component module comprises a fragment of PP, PYY or NPY polypeptide covalently linked to at least one additional fragment of a second PP, PYY or NPY polypeptide, each A PPF chimeric polypeptide in which a PP, PYY or NPY fragment contains a PPF motif. Alternatively, a PPF chimera may comprise a fragment of a PP family polypeptide linked to one, two, three or four polypeptide segments, in which case one of the linked polypeptide segments Is a fragment of a second PP polypeptide. In certain embodiments, the PPF polypeptide does not include an N-terminal PP fragment and a C-terminal NPY fragment. A PPF chimeric polypeptide component module will exhibit at least 50% sequence identity with native PYY (3-36) over its entire length of PYY (3-36). In some embodiments, such a PPF chimeric polypeptide is at least 60%, at least 70%, at least 80%, at least 90%, at least 90% with native PYY (3-36) over its entire length of PYY (3-36). 92%, at least 94% or at least 97% sequence identity can be shown. Such PPF chimeric polypeptides may exhibit at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% sequence identity with native PP. it can. In yet another embodiment, such a PPF chimeric polypeptide has 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 with native NPY. Identity can be shown. In some embodiments, the PPF chimeric polypeptide can comprise at least an N-terminal polyproline PPF motif and a C-terminal tail PPF motif. These PPF chimeras and other PYY and PP analogs are described in US 2006 / 013547A1, published June 22, 2006. In one embodiment, any PPF family peptide can be supplied with a hybrid described herein as a second agent, eg, as an anti-obesity agent, when not included as a hybrid component.

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

  PPF chimeric polypeptide fragments can be covalently linked to each other in any manner known in the art, including but not limited to direct amide bonds or chemical linker groups. Chemical linker groups can include peptidomimetics that induce or stabilize the polypeptide conformation. PPF chimeric polypeptides can include PYY-PP, PYY-NPY, PP-PYY, PP-NPY, NPY-PP or NPY-PYY chimeras.

  The PPF chimera has a length of at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids. In some embodiments, the PYY analog polypeptide comprises only natural L amino acid residues and / or modified natural L amino acid residues. In some embodiments, the PYY analog polypeptide does not contain unnatural amino acid residues.

In some embodiments, PPF chimeric polypeptides include hPP (1-7) -pNPY, hPP (1-17) -pNPY, hPP (19-23) -pNPY, hPP (19-23) -Pro ³⁴ pNPY, hPP (19-23) -His ³⁴ pNPY, rPP (19-23) -pNPY, rPP (19-23) -Pro ³⁴ pNPY, rPP (19-23) -His ³⁴ pNPY, hPP (1-17) -His ³⁴ pNPY, pNPY (1-7) -hPP, pNPY (1-7,19-23) -hPP, cPP (1-7) -pNPY (19-23) -hPP, cPP (1-7)- NPY (19-23) -His ³⁴ hPP, hPP (1-17) -His ³⁴ pNPY, hPP (19-23) -pNPY, hPP (19-23) -Pro ³⁴ pNPY, pNPY (1-7) -hPP , PNPY (19-23) -hPP, pNPY (19-23) -Gln ³⁴ hPP, pNPY (19-23) -His ³⁴ hPP, pNPY (19-23) -Phe ⁶ Gln ³⁴ hPP, pNPY (19-23 ) -Phe ⁶ His ³⁴ hPP, pNPY (1-7,19-23) -hPP, pNPY (1-7,19-23) -Gln ³⁴ hPP, cPP (20-23) -Pro ³⁴ -pNPY, cPP ( 21-23) -Pro ³⁴ -pNPY, cPP (22-23) -Pro ³⁴ -pNPY, cPP (1-7) -Pro ³⁴ -pNPY, cPP (20-23) -Pro ³⁴ -pNPY, cPP (1- 7,20-23) -Pro ³⁴ -pNPY, cPP (1-7) -pNPY (19-23) -hPP, cPP (1-7) -pNPY (19-23) -His ³⁴ hPP, cPP (1- 7) -gPP (19-23) -hPP, cPP (1-7) -pNPY (19-23) -Ala ³¹ Aib ³² Gln ³⁴ -hPP, cPP (1-7) -pNPY (19-23) -Ala ³¹ Aib ³² His ³⁴ -hPP hPP (1-7) -Ala ³¹ Aib ³² -pNPY, hPP (1-17) -Ala ³¹ Aib ³² -pNPY, pNPY (1-7) -Ala ³¹ Aib ³² Gln ³⁴ -hPP, or pNPY (1-7,19-23) -Ala ³¹ Aib ³² Gln ³⁴ -hPP.

  In some embodiments, the PPF chimeric polypeptide may comprise a fragment of a PP family analog polypeptide. For example, a PPF chimeric polypeptide can include the PPF analog polypeptides described herein, as well as PP analog polypeptides and NYP analog polypeptides.

  PYY analog polypeptides for use in the hybrids of the invention or as second agents include assays described herein (including dietary intake, gastric emptying, pancreatic juice secretion, body composition or weight loss assays). One of which has a potency equal to or greater than that of NPY, PYY or PYY (3-36) in the same assay. In some embodiments, PPY analog polypeptides may be useful in the treatment of metabolic diseases such as obesity, insulin resistance syndrome (syndrome X) or diabetes. In some embodiments, the PYY analog polypeptide exhibits improved manufacturability, stability, and / or ease of formulation compared to PP, NYP, PYY or PYY (3-36). Can do.

  In some embodiments, the PPF chimeric polypeptide has a biology of natural human PYY with respect to reduced nutrient utilization, reduced food intake, effects of weight gain, and / or treatment and prevention of metabolic conditions and metabolic disorders. At least about 25%, or about 30%, to about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 99% Hold the percentage. In another embodiment, the PPF chimeric polypeptide exhibits improved PYY agonist activity. In some embodiments, the PPF chimeric polypeptide has a biology of natural human PYY with respect to reduced nutrient utilization, reduced food intake, effects of weight gain, and / or treatment and prevention of metabolic conditions and metabolic disorders. Exhibit at least about 110%, about 125%, about 130%, about 140%, about 150%, about 200% or more of the active activity.

  More particularly, in one embodiment, the PPF chimeric polypeptide comprises a fragment of PP linked to a fragment of PYY. In one embodiment, the PPF chimeric polypeptide comprises an N-terminal fragment of PP or PP analog polypeptide linked at its C-terminal tip to a C-terminal fragment of PYY or PYY analog polypeptide. In another embodiment, the PPF chimeric polypeptide comprises a PYY, PYY (3-36) or N-terminal fragment of a PYY analog linked at its C-terminal tip to a C-terminal fragment of a PP or PP analog polypeptide. Including.

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

  In some embodiments, the PPF chimeric polypeptide comprises a fragment of PP linked to a fragment of NPY. In one embodiment, the PPF chimeric polypeptide comprises an N-terminal fragment of PP or PP analog polypeptide linked at its C-terminal tip to a C-terminal fragment of NPY or NPY analog polypeptide. In another embodiment, the PPF chimeric polypeptide comprises an N-terminal fragment of NPY or NPY analog polypeptide linked at its C-terminal tip to a C-terminal fragment of PP or PP analog polypeptide.

  In some embodiments, PP, PP analog polypeptide, PYY, PYY (3-36), PYY analog polypeptide, NPY or a fragment of NPY analog polypeptide is its PP, PP analog polypeptide, A fragment comprising approximately 4 to 20 amino acid residues of a PYY, PYY (3-36), PYY analog polypeptide, NPY or NPY analog polypeptide. In some embodiments, this fragment length is selected to obtain a final PPF chimeric polypeptide of at least 34 amino acids in length.

  PPF chimeric polypeptides may include further modifications including, but not limited to, amino acid sequence substitutions, deletions and insertions of these PPF chimeric polypeptides and any combination thereof. In some embodiments, the PPF chimeric polypeptide comprises one or more modifications of “non-essential” amino acid residues. “Non-essential” amino acid residues are those residues within the native human amino acid sequence that can be modified, ie, deleted or substituted, without completely destroying or substantially reducing the activity of interest.

  Derivatives of PPF chimeric polypeptides are also useful. Such derivatives include conjugated to one or more water-soluble polymer molecules such as polyethylene glycol (“PEG”) or fatty acid chains of various lengths (eg, stearyl, palmitoyl, octanoyl, oleoyl, etc.), or PPF chimeric peptides conjugated by addition of polyamino acids such as poly-his, poly-arg, poly-lys, and poly-ala are included. Modifications to PPF chimeric polypeptides may also include small molecule substituents such as short alkyls and constrained alkyls (eg, branched alkyls, cyclic alkyls, fused alkyls, adamantyl alkyls), and aromatic groups. In some embodiments, the water soluble polymer molecule will have a molecular weight ranging from about 500 to about 20,000 daltons.

  Such polymer conjugation and small molecule substituent modifications can be made once in the sequence of the PPF chimeric polypeptide, at the N or C terminus, or at the side chain of amino acid residues that are not involved in the formation of the hybrid. Alternatively, there may be multiple derivatization sites along the PPF chimeric polypeptide. Additional derivatization sites can be generated by substituting one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine. See, for example, US Pat. Nos. 5,824,784 and 5,824,778. In some embodiments, the PPF chimeric polypeptide can be conjugated to one, two or three polymer molecules.

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

PPF chimeric polypeptides also include PPF chimeric polypeptides that have a chemical modification to one or more amino acid residues. Such chemical modifications include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization. These chemical modifications can be made once at the N- or C-terminus within the sequence of the PPF chimeric polypeptide or at the side chains of amino acid residues. In one embodiment, the C-terminus of these peptides may have a free —OH or —NH ₂ group. In another embodiment, the N-terminal tip has an isobutyloxycarbonyl group, isopropyloxycarbonyl group, n-butyloxycarbonyl group, ethoxycarbonyl group, isocaproyl group (isocap), octanyl group, octylglycine group (G (Oct )), Or may be capped with an 8-aminooctanoic acid group. In some embodiments, cyclization can be by formation of a disulfide bridge. Alternatively, there may be multiple chemical modification sites along the PYY analog polypeptide.

  In some embodiments, the PPF chimeric polypeptides include those having the amino acid sequence of SEQ ID NOs: 238-347 of US 2006 / 013547A1.

Exemplary PPF chimeric polypeptides include formula (IV):
Xaa ₁ Xaa ₂ Xaa ₃ Xaa ₄ Pro Glu Xaa ₇ Pro Xaa ₉ Glu
Asp Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Glu Xaa ₁₆ Xaa ₁₇ Xaa ₁₈ Xaa ₁₉ Tyr
Xaa ₂₁ Xaa ₂₂ Xaa ₂₃ Leu Xaa ₂₅ Xaa ₂₆ Tyr Xaa ₂₈ Asn Xaa ₃₀
Xaa ₃₁ Thr Arg Gln Xaa ₃₅ Xaa ₃₆
And the polypeptide of the formula:
Xaa ₁ is Tyr or absent;
Xaa ₂ is Ile, Pro or absent;
Xaa ₃ is Ile, BH-modified Lys, Lys, Val or Pro;
Xaa ₄ is, Lys, BH- modified Lys, Ala, Ser, or Arg;
Xaa _{7 is,} Ala, be Gly or His;
Xaa ₉ is Gly or Ala;
Xaa ₁₂ is Ala or Pro;
Xaa ₁₃ is Ser or Pro;
Xaa ₁₄ is Pro, Ala or Ser;
Xaa ₁₆ is Glu or Asp;
Xaa ₁₇ is Leu or Ile;
Xaa ₁₈ is Asn or Ala;
Xaa ₁₉ is Arg, Lys, BH-modified Lys, Gln or N (Me) Ala;
Xaa ₂₁ is Tyr, Ala, Phe, Lys or BH-modified Lys;
Xaa ₂₂ is Ala or Ser;
Xaa ₂₃ is Ser, Ala or Asp;
Xaa ₂₅ is Arg, Lys or BH-modified Lys;
Xaa ₂₆ is His, Ala or Arg;
Xaa ₂₈ is Leu or Ile;
Xaa ₃₀ is Leu or Met;
Xaa ₃₁ is Val, Ile or Leu;
Xaa ₃₅ is, Lys, be BH- modified Lys or Arg; and
Xaa ₃₆ is Tyr, Trp or Phe.

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

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

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

Other PPF polypeptides include those of formula (VII):
Xaa ₁ Xaa ₂ Pro Xaa ₄ Pro Xaa ₆ His Pro Xaa ₉ Xaa ₁₀
Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Tyr
Xaa ₂₁ Xaa ₂₂ Xaa ₂₃ Leu Xaa ₂₅ Xaa ₂₆ Xaa ₂₇ Xaa ₂₈ Xaa ₂₉ Xaa ₃₀
Xaa ₃₁ Thr Arg Gln Arg Tyr
And the polypeptide of the formula:
Xaa ₁ is Tyr or absent;
Xaa ₂ is Ile, Pro or absent;
Xaa ₄ is, Lys, BH- modified Lys, Ala, Ser, or Arg;
Xaa ₆ is Glu, Gln, Ala, Asn, Asp or Val;
Xaa ₉ is Gly or Ala;
Xaa _{10 is, Glu, Ala, Asp, Asn} , Gln, Gly, be Pro or Aib;
Xaa _{11 is, Glu, Ala, Asp, Asn} , Gln, Gly, be Pro or Aib;
Xaa ₁₂ is Ala or Pro;
Xaa ₁₃ is Ser or Pro;
Xaa ₁₄ is Pro, Ala or Ser;
Xaa ₁₅ is Glu, Ala, Asp, Asn, Gln, Gly, Pro or Aib;
Xaa ₁₆ is Glu or Asp;
Xaa ₁₇ is Leu or Ile;
Xaa ₁₉ is Arg, Lys, BH-modified Lys, Gln or N (Me) Ala;
Xaa ₂₁ is Tyr, Ala, Phe, Lys or BH-modified Lys;
Xaa ₂₂ is Ala or Ser;
Xaa ₂₃ is Ser, Ala or Asp;
Xaa ₂₅ is Arg, Lys or BH-modified Lys;
Xaa ₂₆ is His, Ala or Arg;
Xaa ₂₇ is Tyr or Phe;
Xaa ₂₈ is Leu or Ile;
Xaa ₂₉ is Asn or Gln;
Xaa ₃₀ is Leu or Met; and
Xaa ₃₁ is Val, Ile or Leu.

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

  In some embodiments, the PPF polypeptide is of natural human PYY linked to a C-terminal fragment consisting essentially of amino acid residues 18-36 of natural human NPY (SEQ ID NO: 4 of US 2006 / 013547A1). May include an N-terminal fragment consisting essentially of the first 17 amino acid residues (SEQ ID NO: 2 of US 2006 / 013547A1), in which case one or more residues at the N-terminus of the PYY fragment are missing. May be missing or absent, and one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions may be made in each of the PYY and NPY fragments is there. In some embodiments, the N-terminal fragment consisting essentially of the first 17 amino acids of the PPF polypeptide is at least 50%, at least 60%, at least 70%, at least 80 with the first 17 amino acids of native PYY. %, At least 90%, at least 92%, at least 94% or at least 97% sequence identity. In some embodiments, the C-terminal fragment of the PPF polypeptide consisting essentially of amino acid residues 18-36 is at least 50%, at least 60%, at least 70%, at least 80 with amino acids 18-36 of native NPY. %, At least 90%, at least 92%, at least 94% or at least 97% sequence identity. In some embodiments, amino acids within the N-terminal fragment of PYY (eg, proline at positions 3, 5 and 8 or histidine at position 7) and / or amino acids within the C-terminal fragment of NPY (eg, position 18 Alanine, tyrosine at positions 20 and 36, leucine at position 24, threonine at position 32, arginine at position 33, glutamine at position 34, or arginine at position 35) is not substituted. In some embodiments, the PPF polypeptide comprises an amino acid sequence of a PYY-NPY chimera, such as SEQ ID NOs: 266, 437, 438, 439, 442, 462, 469, 470, 471 and 472 of US 2006 / 013547A1. Or amino acid sequence Ile, Lys, Pro, Glu, His, Pro, Gly, Glu, Asp, Ala, Ser, Pro, Glu, Glu, Leu, Ala, Arg, Tyr, Tyr, Ala, Ser, Leu, Examples include compounds having Arg, Ala, Tyr, Ile, Asn, Leu, Ile, Thr, Arg, Gln, Arg, and Tyr-NH2. In some embodiments, the PPF polypeptide further comprises an N-terminal cap. Examples of these PPF polypeptides include SEQ ID NOs: 437, 462, 469, 470 and 472 of US 2006 / 013547A1. For example, sequence 438 of US 2006 / 013547A1 is Pro Lys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Ala Arg Tyr Tyr Ala Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr In one embodiment, the hybrid of the present invention comprises one component, sequence 438 of US2006 / 013547A1, particularly for hybrids useful for the treatment of obesity, weight loss, fat loss or redistribution, and caloric intake reduction. Such hybrids may contain an amylin mimetic component or a leptin component or both.

  When PPF polypeptides and PPF chimeras are used alone or as a second agent or as a component of a hybrid of the invention, they can be used in a method that involves modification of a subject's body composition, Administering the subject compound (PPF polypeptide or PPF chimera alone, as a second agent, or as a component of a hybrid of the invention) to the subject, wherein the compound modifies the fat to lean ratio As a result, the body composition is altered. The PPF polypeptide is a PYY-NPY chimera called 5705 and the following sequences from US2006 / 013547A1: 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 may comprise an amino acid selected from the group consisting of: In one embodiment, fat is reduced and lean body mass is maintained or increased. In one embodiment, body fat and lean body mass are measured as body fat percentage and lean body mass percentage, respectively. In one further embodiment, weight is lost. In another embodiment, weight is maintained or increased. The compound can be administered peripherally. PPF polypeptides or PPF chimeras or PPF-containing hybrids are amylin, amylin agonist or amylin analog agonist, salmon calcitonin, cholecystokinin (CCK) or CCK agonist, leptin (OB protein) or leptin agonist, exendin or Exendin analog agonist, GLP-1, GLP-1 agonist or GLP-1 analog agonist, CCK or CCK agonist, calcitonin, calcitonin agonist, small molecule cannabinoid CB1 receptor antagonist, rimonabant, 11 beta- It can be used in a method further comprising administering to the subject at least one other agent selected from the group consisting of a hydroxysteroid dehydrogenase-1 inhibitor, sibutramine and phentermine. In one embodiment, the subject is overweight or obese. In yet another embodiment, a method of preferentially lowering a subject's plasma triglyceride levels when a PPF polypeptide and a PPF chimera are used alone or as a second agent or as a component of a hybrid of the invention. Which can be used in a method comprising administering to a subject an amount of a compound effective to lower plasma triglyceride levels, wherein the method lowers cholesterol levels to a lesser extent. In further embodiments, triglyceride levels are reduced and cholesterol levels are not reduced. In a further embodiment, the triglyceride level is decreased and the LDL cholesterol level is not decreased. In further embodiments, triglyceride levels are reduced and LDL cholesterol levels are reduced to a lesser extent. In a further embodiment, amylase levels are also reduced. In yet another embodiment, while PPF polypeptides and PPF chimeras are used alone or as a second agent or as a component of a hybrid of the invention, while reducing body fat or body fat increase in a subject Can be used in a method that includes a method for maintaining or increasing lean body mass, wherein the method comprises an amount effective to maintain or increase lean body weight while decreasing body fat or body fat gain. Administering the compound to the subject. In another embodiment, a PPF polypeptide and a PPF chimera are used in a method comprising a method of reducing visceral body fat in a subject when used alone or as a second agent or as a component of a hybrid of the invention. The method includes administering to the subject an amount of a compound effective to reduce visceral body fat and maintain or increase lean body mass. In another embodiment, PPF polypeptides and PPF chimeras are used in methods, including methods of altering a subject's fat distribution when used alone or as a second agent or as a component of a hybrid of the invention. be able to. In one embodiment, the modification occurs as a result of increased metabolism of visceral or ectopic fat or both in the subject. In another embodiment, the PPF polypeptide and PPF chimera, when used alone or as a second agent or as a component of a hybrid of the invention, increases lean fatty acid weight while increasing fatty acid β-oxidation in a subject. Can be used in a method, including a method of maintaining or increasing fat, which comprises administering to a subject an amount of a compound effective to maintain or increase lean body mass while increasing fatty acid β-oxidation including. In another embodiment, treating non-alcoholic steatohepatitis or lipodystrophy in a subject when the PPF polypeptide and PPF chimera are used alone or as a second agent or as a component of a hybrid of the invention The method comprises administering to the subject an amount of a compound effective to treat nonalcoholic steatohepatitis or lipodystrophy. Hybrids of particular interest in the above use can contain the PPF chimeras described herein in combination with components from the leptin family or amylin family, eg, amylin-sCT-amylin hybrids, or both. PPF-chimera / leptin hybrids or PPF-chimera / amylin-sCT-amylin hybrids will provide superior effects than either compound alone. In still further embodiments, the PPF-chimera / leptin hybrid is administered with an amylin mimic, such as an amylin-sCT-amylin chimera, or the PPF-chimera / amylin-sCT-amylin hybrid is administered with leptin.

When not a component module of a hybrid, the PPF polypeptide chimeras described herein can be administered alone or as a second agent, preferably in combination with a hybrid of the invention. They can be provided in single or multiple doses with or without a pharmaceutically acceptable carrier or excipient. These pharmaceutical compounds are formulated with pharmaceutically acceptable carriers or diluents and other known adjuvants and excipients related to the prior art, such as those disclosed by EW Martin in Remington's Pharmaceutical Sciences. be able to. See also Wang, YJ and Hanson, MA "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S (1988). The PPF polypeptide can be provided in dosage unit form. For example, a therapeutically effective amount of a PPF polypeptide to affect body composition is the age and weight of the patient, the patient's physical condition, their combination with other therapeutic agents, the final goal to be achieved, such as overall weight loss And / or will depend on a number of factors, including lean body weight maintenance or gain, and other factors. However, typical doses (when not a component of a hybrid) are about 0.05 μg, about 0.1 μg, about 1 μg, about 5 μg, about 10 μg, about 50 μg, about 75 μg or about 100 of the pharmaceutical compound per day. From the lower limit of μg to the upper limit of about 50 μg, about 100 μg, about 500 μg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 50 mg, about 100 mg or about 150 mg Can do. Dose ranges such as 0.1 μg to 1 mg of the compound per dose, or about 0.001 μg / kg to about 500 μg / kg per dose are also contemplated. In some embodiments, the PPF polypeptide chimera is administered at a dose of about 0.5 μg to about 5 mg per day in a single or divided dose or controlled sustained release, or from about 0.01 μg / kg to about 500 μg / kg per dose. Peripherally administered in kg, or from about 0.05 μg / kg to about 250 μg / kg. In some embodiments, the PPF polypeptide chimera is administered at a dose of less than about 50 μg / kg. The dosage in these ranges will vary depending on the potency of each analog or derivative and, of course, can be readily determined by one skilled in the art. The daily dose can be delivered in individual unit doses and can be continuously supplied for 24 hours or any portion thereof. The number of doses per day is from 1 to about 4 per day, but may be higher. Sustained delivery can be in the form of continuous infusion. Other possible exemplary doses and infusion rates include 0.005 nmol / kg to about 20 nmol / kg per individual dose, or about 0.01 / pmol / kg / min to about 10 pmol / kg / min for continuous infusion. These doses and infusions can be any known conventional or future developed peripheral method such as intravenous (iv), intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary (eg , Limited time release), subcutaneous administration (sc), oral, sublingual, nasal, anal, vaginal or transdermal delivery, or by surgical implantation at a specific site. An exemplary total dose / delivery of a pharmaceutical composition administered intravenously may be from about 1 μg to about 8 mg per day, whereas a total dose / delivery of a pharmaceutical composition administered subcutaneously is one It can be from about 6 μg to about 16 mg per day.
Incretin and incretin mimetics

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

  Any GLP-1 peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, GLP-1 peptide analogs and derivatives have at least one hormonal activity of a native GLP-1 peptide. In certain embodiments, these GLP-1 peptide analogs are agonists of receptors to which the native GLP-1 peptide can specifically bind. Preferred GLP-1 peptide analogs and derivatives include those described in WO 91/11457, which is incorporated herein by reference.

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

  As is known in the art, such GLP-1 analogs are preferably amidated, but in the context of the present invention, even in their acid form, unless otherwise indicated. Good.

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

  Component peptide hormones useful in the present invention also include GLP-2 peptide hormone. Natural GLP-2 peptide hormones such as rat GLP-2 and its homologues (bovine GLP-2 (ox GLP-2), porcine GLP-2, degu GLP-2, bovine GLP-2), Guinea pig GLP-2, hamster GLP-2, human GLP-2, rainbow trout GLP-2, and chicken GLP-2 are known in the art, and functional peptide analogs and derivatives are also known. Although certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any known GLP-2 peptide exhibiting hormonal activity known in the art can be used in conjunction with the present invention. .

  Any GLP-2 peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, GLP-2 peptide analogs and derivatives have at least one hormonal activity of a native GLP-2 peptide. In certain embodiments, these GLP-2 peptide analogs are agonists of receptors to which the native GLP-2 peptide can specifically bind. Preferred GLP-2 peptide analogs and derivatives are described, for example, in US patent application Ser. No. 08 / 669,791 and PCT application PCT / CA97 / 00252, both of which are incorporated herein by reference. The one that is. Specific BLP-2 analogs known in the art include rat or human GLP-2 modified at position 2 to confer DPP-IV resistance by replacing Ala with Gly.

  Component peptide hormones useful in the present invention also include oxyntomodulin (OXM) peptide hormones. Natural OXM peptide hormones are known in the art, and functional peptide analogs and derivatives are also known. Although certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any known OXM peptide exhibiting hormonal activity known in the art can be used in conjunction with the present invention.

  Any OXM peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, the OXM peptide analogs and derivatives have at least one hormonal activity of a native OXM peptide. In certain embodiments, these OXM peptide analogs are agonists of receptors to which the native OXM peptide can specifically bind.

  Component peptide hormones useful in the present invention also include exendin peptide hormones. Natural exendin peptide hormones are known in the art, and functional peptide analogs and derivatives are also known. Although certain preferred natural peptides, peptide analogs and derivatives are described herein, it should be understood that any known exendin peptide exhibiting hormonal activity known in the art can be used in conjunction with the present invention.

  Any exendin peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, exendin peptide analogs and derivatives have at least one hormonal activity of a natural exendin peptide. In certain embodiments, an exendin peptide analog is an agonist of a receptor to which a natural exendin peptide can specifically bind.

Exemplary exendin analogs include the following:

  As is known in the art, such exendin analogs are preferably amidated, but may optionally be in their acid form unless otherwise indicated in connection with the present invention.

  Additional exemplary exendin analogs and derivatives include a PCT entitled “Novel Exendin Agonist Compounds” filed Aug. 6, 1998, claiming the benefit of US Provisional Application No. 60 / 055,404, filed Aug. 8, 1997. Application No. PCT / US98 / 16387, both of which are incorporated herein by reference. Another exendin analog and derivative is a PCT application entitled "Novel Exendin Agonist Compounds" filed November 13, 1998, claiming the benefit of US Provisional Application No. 60 / 065,442, filed November 14, 1997. No. PCT / US98 / 24210, both of which are hereby incorporated by reference. Yet another exendin analog and derivative is a PCT application entitled "Novel Exendin Agonist Compounds" filed November 13, 1998, claiming the benefit of US Provisional Application No. 60 / 066,029 filed November 14, 1997. No. PCT / US98 / 24273, both of which are hereby incorporated by reference. Still other exendin analogs and derivatives include `` Methods for Regulating Gastrointestinal Activity '' filed Aug. 8, 1997, which is a partially pending application of U.S. Provisional Application No. 08 / 694,954 filed Aug. 8, 1996. PCT application number PCT / US97 / 14199, both of which are hereby incorporated by reference. Still other exendin analogs and derivatives are listed in `` Use of Exendins and Agonists Thereof for filing Jan. 7, 1998, claiming priority to U.S. Provisional Application No. 60 / 034,905, filed Jan. 7, 1997. PCT application number PCT / US98 / 00449, entitled “The Reduction of Food Intake”, both of which are incorporated herein by reference. Still other exendin analogs and derivatives are described in US 2004/0209803 A1 entitled “Compositions for the Treatment and Prevention of Neuropathy” filed on December 19, 2003, which is hereby incorporated by reference. It is incorporated in the book.

Natriuretic peptides Natriuretic peptides are a family of hormones consisting of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). These are synthesized and stored as three separate precursor prohormones, 126 amino acid ANP, 108 amino acid BNP and 104 amino acid CNP. These are each encoded by a separate gene and have distinct synthesis sites and regulatory mechanisms. Parental natriuretic peptide sequences include the following:

  The main synthetic site of ANP prohormone is atrial myocyte, which is synthesized as a 151-amino acid preprohormone. Removal of the 25-amino acid signal peptide from its N-terminal tip occurs in the endoplasmic reticulum, resulting in a 126-amino acid ANP prohormone (ProANP), the main storage form in the heart. This prohormone has four biologically active peptide segments: amino acids 1-30 (ProANF 1-30, also known as long-acting Na-stimulating factor), 31-67 (ProANF 31-67, as vasodilator) 79-98 (ProANF 79-98, also known as potassium excretion factor) and 99-126 (ANF, also known as atrial natriuretic factor).

  BNP was originally isolated from porcine brain, but is also synthesized in humans and secreted from the left ventricle. Sequence analysis shows that preproBNP consists of 134 residues and is cleaved to 108-amino acid ProBNP. Cleavage of the 32-amino acid sequence from the C-terminal tip of the ProBNP residue yields human BNP (77-108), which is a bioactive form in plasma.

  CNP is the third member of the natriuretic peptide system and is found primarily in human vascular endothelial cells, kidney and pig brain. High concentrations of CNP are also found in the human hypothalamus and midbrain. In humans, prepro-CNP is a 126-amino acid precursor that is processed into pro-CNP by cleavage of 23 residues from its N-terminal tip. This 23-amino acid sequence serves as a signal peptide. The terminal 22 (105-126) amino acids are cleaved from pro-CNP to produce a biologically active form of CNP.

  Urodilatin is a kidney-derived member of the natriuretic peptide family, formed from the same ANP prohormone and consisting of amino acids 95-126. Same as ANF (99-126) except for 4 amino acid N-terminal extension. Urodilatin appears to be an important regulator of sodium and water treatment in the kidney and a mediator of sodium excretion in patients with congestive heart failure (CHF).

  Natriuretic peptides exert their biological effects by binding to high affinity receptors on the surface of target cells. Three subtypes of NPR-NPR-A, NPR-B and NPRC- have been isolated. Accordingly, in one embodiment, a method for screening hybrids for natriuretic receptor binding and / or activation is provided. Natriuretic peptides containing prohormone variants can confer a great deal of natriuretic peptide hormone activity to the hybrids of the present invention. In other embodiments, natriuretic antagonist hybrids are of interest. Natriuresis is the excretion of excessive amounts of sodium into the urine. Natriuresis is similar to diuresis (excessive excretion of very large amounts of urine), but natriuresis has a very high urinary salt content. Natriuresis occurs with some diuretics and illnesses (such as adrenal illness) and can result in a salt loss syndrome characterized by the risk of dehydration, vomiting, hypotension, and sudden death. Exogenous administration of four independent circulating peptides of the ANP prohormone (1-30, 31-67, 79-98 and 99-126) can help in vivo vasodilation, diuresis, inhibition of the renin-angiotensin-aldosterone system, and sodium Causes diuresis and / or potassium urine enhancement. Pro ANF 1-30, Pro ANF 31-67 and ANF 99-126 have natriuretic properties, blood pressure lowering properties and diuretic properties, respectively, and Pro ANF 31-67 and ANF 99-126 against blood pressure The greatest influence. The effects of ANP peptides on potassium homeostasis vary: pro-ANF 79-98 stimulates potassium excretion, whereas pro-ANF 31-67 inhibits Na / K ATPase in medullary collecting duct cells Refrain from potassium loss. Dose-dependent inhibition of angiotensin II-mediated aldosterone secretion is specific for NAF 99-126, whereas pro-ANF 31-67 has the property of inducing natriuresis by producing prostaglandins.

  BNP produces biological effects similar to ANF in normal humans. Infusion of BNP in normal humans results in a 2-fold increase in sodium excretion, a 50% decrease in plasma renin, angiotensin II and aldosterone secretion, and a decrease in plasma volume.

  CNP induces cardiovascular effects similar to other natriuretic peptides but does not appear to mediate any renal effects. Infusion of CNF at a dose equivalent to ANF in anesthetized dogs increases plasma cGMP with a concomitant decrease in mean arterial pressure, right atrial pressure and cardiac output, but glomerular filtration rate, renal blood flow and Sodium excretion decreases.

  Natriuretic peptides can provide a therapeutic benefit during heart failure. Congestive heart failure (CHF) increases vasopressin, endothelin, and activation of the renin-angiotensin-aldosterone and parasympathetic nervous systems that mediate vasoconstriction, sodium and water retention, and vascular and cardiac negative remodeling. Accompanying. These effects occur despite high natriuretic peptide levels in patients with heart failure. The hybrid in one embodiment of the invention is a hybrid that provides increased or therapeutic serum levels of natriuretic peptide activity for the treatment or prevention of heart related diseases and conditions including CHF. ANF infusion can produce a sustained increase in sodium excretion and urine flow in normal individuals, but can achieve a significant and beneficial reduction in renal response in heart failure patients. BNP infusion significantly increases sodium excretion in patients with heart failure and exerts a significant and beneficial hemodynamic effect. Compared to ANP, the diuretic and natriuretic effects of BNP are significantly greater. BNP is removed more slowly than ANP and exerts other effects including suppression of aldosterone secretion and increased serum ANP levels. BNP peptides can also result in increased cardiac index in patients hospitalized for symptomatic CHF, along with beneficial reductions in pulmonary capillary wedge pressure, systemic vascular resistance, and right atrial pressure. In patients with decompensated heart failure, natriuretic peptide hybrids can result in a beneficial reduction in lung capillary wedge pressure and an improved dyspnea score. (Dyspnea is a discomfort that makes breathing difficult and is generally associated with early heart failure). Hybrids with one, two or three natriuretic hormone functions are useful for both prophylactic and therapeutic treatment of CHF patients, preferably decompensated CHF patients, chronic CHF patients, and hypertensive patients Resulting in a method of administering a pharmaceutically active composition. The natriuretic part (s) of the hybrid are sufficient to provide such patients with a therapeutically effective amount of natriuretic peptide when administered at a therapeutically effective dose over a therapeutically effective period. is there.

  Any family of therapeutically effective natriuretic peptides or their analogs discussed herein can be used. Useful natriuretic peptides include, for example, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP or B-type natriuretic peptide) and C-type natriuretic peptide (CNP). Useful forms of natriuretic peptide sequences are disclosed in US Patent Publication No. 20010027181, which is incorporated herein by reference. Examples of ANPs include human ANP (Kangawa et al., BBRC 118: 131 (1984)) or from various species including porcine and rat ANP (Kangawa et al., BBRC 121: 585 (1984). )). Such ANPs contain 28 amino acids. Such ANPs can be administered as peptides having the ANP ring structure (Cys-based disulfide bond formation) and the C-terminal portion following the ring structure. An example of such a peptide is a peptide having amino acid residues at positions 7 to 28 of ANP and is provided in US Patent Application Publication No. 20010027181. Another example is the frog ANP. Specific examples of BNP that can be used in the method of the present invention include human BNP (hBNP). Human BNP contains 32 amino acids and is associated with the formation of disulfide bonds (Sudoh et al., BBRC 159: 1420 (1989) and US Pat. Nos. 5,114,923, 5,674,710, 5,674,710 and 5,948,761) Each of which is incorporated herein by reference). Various BNPs of non-human origin are also known and can be used, including porcine BNP and rat BNP. A further example is chicken BNP. An example of a CNP that can be used in the methods of the present invention includes porcine CNP. Porcine CNP contains 22 amino acids, ANP and BNP as described above (Sudoh et al., BBRC 168: 863 (1990)) (human and rat have the same amino acid sequence), chicken CNP (Arimura et al ., BBRC 174: 142 (1991)) with disulfide bond formation. Frog CNP (Yoshihara et al., BBRC 173: 591 (1990)) can also be used. As discussed herein, one of skill in the art will modify, eg, delete, substitute, add or insert, and / or amino acid residues within the amino acid sequence of known natriuretic peptides as described, by known methods. Or chemical modifications can be applied. The resulting compound is a compound having activity that acts on the receptor for the starting ANP, BNP or CNP. Accordingly, analogs having this activity are included in hybrids for use in accordance with the methods of the invention.

In another embodiment, a hybrid having one or more natriuretic functions can be used to treat hypertension. In one embodiment, the natriuretic hybrid is not associated with arrhythmia, which has a deleterious effect on heart rate. In one embodiment, the hybrid will contain two or three natriuretic peptide functions, eg, both ANP activity and BNP activity. One or more natriuretic hormone functions can be used in combination with any other hormone function or peptide enhancer as described herein. In another embodiment, the natriuretic moiety (s) is a more stable analog that has an extended in vivo half-life when compared to that of a natural natriuretic peptide. Analogs that prevent undesired cleavage by endogenous enzymes such as NEP are also contemplated. Natriuretic factor-containing hybrids can further lower hypotension, induce diuresis, induce natriuresis, dilate or relax vascular conduction, bind natriuretic peptide receptors (eg, NPR-A), inhibit renin secretion from the kidney, and from the adrenal gland For suppressing aldosterone secretion, treating cardiovascular disease and cardiovascular disorders, reducing cardiac remodeling in congestive heart failure, stopping or reversing, treating kidney disease and disorders, treating or preventing ischemic stroke, and treating asthma Also related. Hybrids can be administered to patients who would benefit from induction of natriuresis, diuresis and vasodilation. The hybrid can be administered alone or in combination with one or more of the following types of compounds: ACE inhibitors, beta-blockers, diuretics, spironolactone, digoxin, anticoagulant Drugs, antiplatelet substances, and angiotensin receptor blockers. Additional diseases or conditions include kidney damage, kidney disease, asthma, hypertension and pulmonary hypertension. The hybrid is also useful for the treatment of diseases related to inflammation, erectile dysfunction and anticholesterolemia.
Bioactive peptide hormone module

  The hybrid polypeptides of the invention as discussed herein generally comprise at least two bioactive peptide hormone modules that are covalently linked to each other. These bioactive peptide hormone modules are: (a) a natural component peptide hormone, (b) an analog or derivative of a natural component peptide hormone that retains hormone activity, (c) a fragment of a natural component peptide hormone that retains hormone activity, (d) Fragments of analogs or derivatives of natural component peptide hormones that retain hormonal activity, (e) desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting for hybrid polypeptides Natural component peptide hormone structural motifs that confer, receptor interaction, protease inhibition, plasma protein binding and / or other pharmacokinetic properties, or (f) desired chemical stability, conformational stability to the hybrid polypeptide , Metabolic stability, bioavailability, organ / tissue Getingu, receptor interaction, protease inhibition, can be a structural motif analog or derivative of native component peptide hormones that impart a plasma protein binding and / or other pharmacokinetic properties. The structural motifs (e) and (f) are collectively referred to herein as “peptide enhancers”.

  Preferred bioactive peptide hormone modules include amylin, ADM, CT, CGRP, Intelmedin, CCK (1-33), CCK-8, leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3 -36) (SEQ ID NO: 58), GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (sequence) No. 61), GLP-2, OXM, Exendin-3, Exendin-4, Natriuretic peptide hormone, Urocortin family peptides such as Ucn-2 and Ucn-3, Neuromedin family peptides such as Neuromedin U25 or Splice variants and natural peptide hormones selected from ANP, BNP, CNP or urodilatin.

  Other preferred bioactive peptide hormone modules include amylin, ADM, CT, CGRP, Intelmedin, CCK, leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3-36) (SEQ ID NO: 58), GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (SEQ ID NO: 61), GLP- 2, OXM, exendin-3, and exendin-4, natriuretic peptide hormones, urocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splice variants, and ANP Analogs and derivatives of component peptide hormones selected from BNP, CNP or urodilatin, wherein the analogs or derivatives exhibit at least one hormonal activity of the component peptide hormones. Analogs may include one or more insertions, deletions or substitutions of the amino acid sequence of the component peptide hormones, and derivatives as described more fully herein and as known in the art. May include one or more chemical modifications of the amino acid residues of the analog or component peptide hormone.

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

  As is known in the art, such peptidic compounds are preferably amidated, but may be in their acid form in some cases unless otherwise indicated in connection with the present invention.

  Still other preferred bioactive peptide hormone modules include amylin, ADM, CT, CGRP, Intelmedin, CCK, leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3-36) (SEQ ID NO: : 58), GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (SEQ ID NO: 61), GLP -2, OXM, natriuretic peptide, exendin-3, and exendin-4, urocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splice variants, and ANP Or a fragment of a component peptide hormone selected from BNP, CNP or urodilatin, wherein the fragment exhibits at least one hormonal activity of the component peptide hormone.

  Still other preferred bioactive peptide hormone modules include amylin, ADM, CT, CGRP, Intelmedin, CCK, leptin, PYY (1-36) (SEQ ID NO: 57), PYY (3-36) (SEQ ID NO: : 58), GLP-1 (1-37) (SEQ ID NO: 59), GLP-1 (7-37) (SEQ ID NO: 204), GLP-1 (7-36) (SEQ ID NO: 61), GLP -2, OXM, ANP, BNP, CNP, urodilatin, exendin-3, exendin-4, natriuretic peptide hormone, urocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin Examples include U25 or splice variants, as well as fragments of analogs or derivatives of component peptide hormones selected from ANP, BNP, CNP, wherein the fragment exhibits at least one hormonal activity of the component peptide hormones. Again, analogs may include one or more insertions, deletions or substitutions in the amino acid sequence of the component peptide hormones, and derivatives are described more fully herein and are known in the art. Some may include one or more chemical modifications of the amino acid residues of the analog or component peptide hormone.

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

Again, as is known in the art, such peptide compounds are preferably amidated, but in the context of the present invention, even in their acid form, unless otherwise indicated. Good. Furthermore, the above preferred fragments can be combined with any of the analogs or derivatives discussed herein or known in the art. For example, preferred analog fragments include ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27) (SEQ ID NO: : 241), ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27) (SEQ ID NO: 241), book Mention may be made of any other combinations of the disclosed fragments, analogs and derivatives.

ペプチドモジュール選択の考察、スペーサーおよび連結基 Still other preferred bioactive peptide modules include “peptide enhancers”, ie, the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction for the hybrid polypeptide. , Structural motifs of component peptide hormones (including analogs and derivatives thereof) that confer protease inhibition, plasma protein binding and / or other pharmacokinetic properties. Exemplary peptide enhancers include the following: Again, it should be understood that combinations of the above analogs and derivatives with the following bioactive peptide modules are possible. For example, the last six amino acid residues of the amylin family peptide hormone analogs and derivatives known in the art and / or described above are also considered preferred bioactive peptide modules.

Peptide module selection considerations, spacers and linking groups

  The hybrid polypeptides of the invention generally comprise at least two bioactive peptide hormone modules of the invention, at least one of the peptide hormone modules exhibit at least one hormone activity. The bioactive peptide hormone module exhibiting at least one hormonal activity may be located at the N-terminal tip of the hybrid polypeptide, or at the C-terminal tip of the hybrid polypeptide, or the hybrid polypeptide has more than two organisms. When an active peptide hormone module is included, it may be arranged inside the hybrid polypeptide.

  In certain embodiments, it may be preferable to position a bioactive peptide hormone module that exhibits at least one hormonal activity such that the C-terminal tip of the bioactive peptide hormone module is amidated. The amidation of the C-terminal tip of a bioactive peptide hormone module can be achieved by placing the module at the C-terminal tip of the hybrid peptide or at the N-terminus of the hybrid polypeptide in the C-terminal to N-terminal orientation Can be accomplished by arranging In both arrangements, the C-terminal tip of the bioactive peptide hormone module is available for amidation. Specific component peptide hormones where C-terminal amidation may be suitable include amylin family peptide hormones, CCK, PYY, hGLP-1 (7-36) (SEQ ID NO: 61) and hGLP-2 . C-terminal amidation is not necessarily preferred (in other words, when extension at the C-terminal of the module is not easily allowed), specific component peptide hormones include exendin-4, exendin-4 (1-28) (SEQ ID NO: 237), GLP-1 (7-37) (SEQ ID NO: 204), frog GLP-1 (7-36) (SEQ ID NO: 283) and frog GLP-2. However, when these component peptide hormones are located at the C-terminus of the hybrid polypeptide, in some cases they can still be amidated, and in fact preferably, in some cases, can be amidated.

The bioactive peptide hormone module can be covalently linked in any manner known in the art. Any suitable linkage can be used, or a cleavable linkage can be used. In one embodiment, the carboxy of the first module can be directly linked to the amino of the second module. In another embodiment, a linking group can be used to attach to the module. Further, if desired, the linkage can be stabilized using spacers or turn inducers known in the art. By way of example, if amidation of the C-terminal tip of a bioactive peptide hormone module located at the N-terminus is not desired, the module can be directly attached to the second module or any suitable linking group known in the art, For example, alkyl; PEG; amino acids such as Lys, Glu, β-Ala; polyamino acids such as poly-his, poly-arg, poly-lys, poly-ala, Gly-Lys-Arg (GKR), etc .; Functional linkers (see, eg, Pierce catalog, Rockford, Il); aminocaproyl (“Aca”), β-alanyl, 8-amino-3,6-dioxaoctanoyl, or other known in the art It can be attached using cleavable and non-cleavable linkers. Specifically described herein as if each was specifically written is a specific hybrid embodiment in which the linker in each exemplified linker-containing hybrid is replaced by a Gly linker, in particular In another embodiment, the Gly linker is Gly-Gly-Gly. As an example, illustrative species ²⁹ 5 Apa- exendin ^{(1-28) - 1 des-Lys} -h -amylin ^{(1-7) - 11,18 Arg-sCt} (8-27) -h -amylin (33-37) (SEQ No. 32) (see table herein) also specifically designates and discloses species analogs of the Gly linker. This type, ²⁹ GlyGlyGly- exendin ^{(1-28) - 1 des-Lys} -h -amylin ^{(1-7) - 11,18 Arg-sCt} (8-27) -h -amylin (33-37) (SEQ ID NO: 313), in which case the three glycines are placed after the exendin (1-28) sequence. In one embodiment, the linker or spacer is 1 to 30 residues long, in another embodiment 2 to 30 residues, and in another embodiment 3-30 residues long. And an arbitrary integer from 2 to 30 (including 2 and 30, each integer unit, for example, 2, 3, 4, 5, 6, 7, etc. is conceivable). In one embodiment, a Gly linker is used, and in certain embodiments, a three residue linker Gly-Gly-Gly is used.

  If amidation of the C-terminal tip of a bioactive peptide hormone module located at the N-terminus is desired, the module is also linked to the second module using any suitable linking group known in the art. Can be made. More specifically, when bioactive peptide hormone modules exhibiting at least one hormone activity are arranged in the C-terminal to N-terminal orientation to form an amino-amino linkage, preferred linking groups include dicarboxylic acid, alkyl , PEG, and amino acids such as Lys, Cys and Glu.

さらなる例示的組み合わせおよび具体的な実施形態 As noted above, the hybrid polypeptide can preferably include a spacer to further stabilize the linkage of the bioactive peptide hormone module. Any spacer or turn inducer known in the art can be used. By way of example, preferred β-turn mimetics include Mimic A and Mimic B, illustrated below, and also include Ala-Aib and Ala-Pro dipeptides. Their IUPAC name is Mimic A: N- (3S, 6S, 9S) -2-oxo-3-amino-1-azabicyclo [4.3.0] -nonane-9-carboxylic acid, Mimic B: N- (3S, 6S, 9R) -2-oxo-3-amino-7-thia-1-azabicyclo [4.3.0] -nonane-9-carboxylic acid.

Further exemplary combinations and specific embodiments

  Exemplary combinations of biologically active peptide hormone modules to form the hybrid polypeptides of the invention include natural peptide hormones, analogs and derivatives of peptide hormones that exhibit at least one hormone activity, natural that exhibit at least one hormone activity. Peptide hormone fragments, peptide hormone analog and derivative fragments exhibiting at least one hormone activity, and combinations of two or more bioactive peptide hormone modules selected from peptide enhancers, provided that at least one The module is subject to at least hormonal activity.

  The hybrid polypeptides of the invention will comprise at least two bioactive peptide hormone modules, each module being composed of component peptide hormones. In the context of the present invention, the component peptide hormones of the hybrid polypeptide may be the same or different, provided that at least two of the component peptide hormones are different. . In a preferred embodiment, at least two of the component peptide hormones are different peptide hormone families such as the amylin family, CCK, leptin family, PPF, proglucagon family, natriuretic peptide family, urocortin family peptides such as Ucn-2 and From Ucn-3, neuromedin family peptides such as neuromedin U25 or splice variants, and ANP, BNP, CNP or urodilatin, and GLP-1 and exendin families.

  In certain embodiments, the hybrid polypeptides of the invention may comprise two or more modules that exhibit at least one hormonal activity. For example, the hybrid polypeptide may comprise a first peptide hormone or analog fragment that exhibits at least hormonal activity covalently linked to at least one additional peptide hormone analog fragment. The additional fragment (s) may in some cases exhibit at least one hormonal activity. The first peptide hormone may be the same as or different from the additional peptide hormone (s), provided that at least one of the additional peptide hormones is Subject to being different, as well as the first hormonal activity may be the same or different from the optional additional hormonal activity.

  In other embodiments, the hybrid polypeptides of the invention may comprise one or more modules exhibiting 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 can be covalently linked to a peptide enhancer, or a fragment of the first peptide hormone that exhibits at least one hormonal activity has at least one hormonal activity. It can be covalently linked to the second peptide hormone shown, and it is also linked to a peptide enhancer. Alternatively, a peptide enhancer can be placed as a stabilizing spacer between two peptide hormones. Again, the first peptide hormone may be the same as or different from the second peptide hormone, and the first hormone activity may be the same as or different from the second hormone activity. is there.

  In another embodiment, the hybrid polypeptides of the invention may comprise two, three, four or more bioactive peptide hormone modules. Exemplary combinations include one module with hormonal activity in combination with one, two or three peptide enhancers; two modules with hormonal activity in combination with one or two peptide enhancers Three modules with hormonal activity in combination with one peptide enhancer.

  The component peptide hormone is preferably amylin, adrenomedullin, calcitonin, calcitonin gene related peptide, intermedin, cholecystokinin, leptin peptide YY, glucagon-like peptide-1, glucagon-like peptide-2, oxyntomodulin, ANP , BNP, CNP, urodilatin, natriuretic peptide hormone, urocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splice variants, and ANP, BNP, CNP or urodilatin or exendin -4 is selected.

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

  In one embodiment, preferred module combinations include exendin-4, a fragment of exendin-4 that exhibits at least one hormonal activity, an exendin-4 analog or derivative that exhibits at least hormonal activity, or at least one hormonal activity. A first module containing a fragment of exendin-4 analog is included in combination with at least one additional bioactive peptide hormone module. In one embodiment, the first module is linked to one, two or three additional bioactive peptide hormone modules.

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

Preferred exendin-4 peptides include exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe-exendin- 4 (1-28) (SEQ ID NO: 284), and ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240). Exendin (7-15) and its Ser2 analog, HSEGTFTSD (SEQ ID NO: 378) are also useful. Preferred amylin peptides exhibiting at least one hormonal activity include amylin, amylin fragments such as amylin (1-17) (SEQ ID NO: 214), amylin (1-16) (SEQ ID NO: 215), amylin (1- 15) (SEQ ID NO: 216), and amylin (1-7) (SEQ ID NO: 217), and amylin analogs such as pramlintide, ² Ala-h-amylin (SEQ ID NO: 79), ^2,7 Ala- h-Amylin (SEQ ID NO: 80), and fragments thereof. Preferred calcitonin peptides exhibiting at least one hormonal activity include sCT, sCT fragments such as sCT (8-10), sCT (8-27) (SEQ ID NO: 288), and calcitonin analogs such as ^11,18 Arg-sCT (SEQ ID ^{NO: 108), 18 Arg-sCT} ( SEQ ID ^{NO: 107), 14 Glu, 18} Arg-sCT ( SEQ ID ^{NO: 109), 14 Glu, 11,18} Arg-sCT ( SEQ ID NO: 110) And fragments thereof. Preferred amylin peptide enhancers include amylin (32-37) (SEQ ID NO: 242), amylin (33-37) (SEQ ID NO: 243) and amylin (34-37) (SEQ ID NO: 244) and analogs thereof. Is mentioned. Useful amylin-sCT combinations in the context of the present invention are disclosed in PCT / US 2005/004631, Amylin Family Agonist, Attorney Docket No. 18528.835, which is incorporated herein by reference. Are listed. An amylin-sCT chimera that is particularly useful for the generation of the hybrids of the invention is compound 10 (described herein and in PCT / US 2005/004631) and analogs and derivatives thereof.

In one embodiment, preferred module combinations include exendin-4, a fragment of exendin-4 that exhibits at least one hormone activity, an exendin-4 analog or derivative that exhibits at least one hormone activity, or at least one hormone activity And a first module comprising a fragment of an exendin-4 analog exhibiting a combination with a peptide enhancer. Preferred exendin-4 compounds include exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe-exendin -4 (1-28) (SEQ ID NO: 284), and ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240). Preferred peptide enhancers include PYY (25-36) (SEQ ID NO: 257), PYY (30-36) (SEQ ID NO: 262) and PYY (31-36) (SEQ ID NO: 263). In one embodiment, the first module is placed at the C-terminal tip of the hybrid polypeptide and the peptide enhancer is placed at the N-terminal tip of the hybrid polypeptide. Alternatively, the first module can be placed at the N-terminal tip of the subject hybrid polypeptide and the peptide enhancement can be placed at the C-terminal tip of the hybrid polypeptide. In certain embodiments, if desired, spacers or linkers such as βAla can be inserted to link the modules.

In another embodiment, preferred module combinations include exendin-4, a fragment of exendin-4 that exhibits at least one hormonal activity, an exendin-4 analog or derivative that exhibits at least one hormonal activity, or at least one hormonal activity. A first module comprising a fragment of an exendin-4 analog exhibiting CCK, a fragment of CCK exhibiting at least one hormonal activity, a CCK analog or derivative exhibiting at least one hormonal activity, or exhibiting at least one hormonal activity Including in combination with a second module containing a fragment of the CCK analog. Again, preferred exendin-4 compounds include exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe. -Exendin-4 (1-28) (SEQ ID NO: 284), ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240), and ¹⁴ Leu-Exendin-4 (1- 28) (SEQ ID NO: 190). Preferred CCK compounds include CCK-8 and CCK-8 (Phe (CH ₂ SO ₃ )). In one embodiment, the first module is placed at the C-terminal tip of the hybrid polypeptide and the second module is placed at the N-terminal tip of the hybrid polypeptide. Alternatively, the first module can be placed at the N-terminal tip of the hybrid polypeptide and the peptide enhancement can be placed at the C-terminal tip of the hybrid polypeptide. In certain embodiments, if desired, spacers or linkers such as βAla can be inserted to link the modules.

  In another embodiment, preferred module combinations include amylin, a fragment of amylin that exhibits at least one hormonal activity, an amylin analog or derivative that exhibits at least one hormonal activity, or an amylin analog that exhibits at least one hormonal activity. In combination with a second module composed of a peptide enhancer, eg, PYY (25-36) (SEQ ID NO: 257) or PYY (30-36) (SEQ ID NO: 262) Including. In one embodiment, the first module is placed at the C-terminal tip of the hybrid polypeptide and the peptide enhancer is placed at the N-terminal tip of the hybrid polypeptide. Alternatively, the first module can be placed at the N-terminal tip of the hybrid polypeptide and the peptide enhancement can be placed at the C-terminal tip of the hybrid polypeptide. In certain embodiments, if desired, spacers or linkers such as βAla can be inserted to link the modules. In another embodiment, preferred module combinations include amylin, a fragment of amylin that exhibits at least one hormonal activity, an amylin analog or derivative that exhibits at least one hormonal activity, or an amylin analog that exhibits at least one hormonal activity. Examples include those comprising a first module comprising a fragment in combination with a peptide enhancer, eg, a second module composed of PYY (25-36) (SEQ ID NO: 257) or PYY (30-36). In one embodiment, the first module is placed at the C-terminal tip of the hybrid polypeptide and the peptide enhancer is placed at the N-terminal tip of the hybrid polypeptide. Alternatively, the first module can be placed at the N-terminal tip of the hybrid polypeptide and the peptide enhancement can be placed at the C-terminal tip of the hybrid polypeptide. In certain embodiments, if desired, spacers or linkers such as βAla can be inserted to link the modules.

  Other preferred module combinations include those that include a combination of exendin and CCK or amylin, calcitonin, and CCK as a ternary combination. Specific combinations include exendin / CCK and CCK / exendin with and without a spacer or linker and linking group. Other combinations include CCK / amylin / calcitonin and CCK / amylin / calcitonin / amylin with and without spacers or linkers and linking groups. Depending on the desired properties of the hybrid polypeptide, each module can independently be a peptide enhancer or exhibit hormonal activity. In one embodiment, amylin / calcitonin / amylin is provided as an amylin / calcitonin / amylin chimera, such as in the case of Compound 10.

  Still other preferred module combinations include those containing combinations of exendin, amylin and calcitonin as ternary and tetra-hybrid molecules. Exemplary combinations include exendin / amylin / calcitonin; exendin / amylin / calcitonin / amylin; / amylin / calcitonin / exendin; Can be mentioned. Depending on the desired properties of the hybrid polypeptide, each module can independently be a peptide enhancer or exhibit hormonal activity. In one embodiment, amylin / calcitonin / amylin is provided as an amylin / calcitonin / amylin chimera, such as in the case of Compound 10.

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

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

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

  Within each of the combinations described herein, reference to the component peptide hormone is understood to include analogs, derivatives, fragments and peptide enhancers associated therewith.

In preferred embodiments, the hybrid polypeptides include the following:

Exemplary exendin and neuromedin hybrids include:
Exendin- (1-28) -beta-Ala-beta-Ala-FN-38:
HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN-NH2 (SEQ ID NO: 391);
Exendin- (1-28) -beta-Ala-beta-Ala-neuromedin (U25):
HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-FRVDEEFQSPFASQSRGYFLFRPRN-NH2 (SEQ ID NO: 392); and exendin- (1-28) -beta-Ala-beta-Ala-neuromedin (U-9):
HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-GYFLFRPRN-NH2 (SEQ ID NO: 393)
Is mentioned. The beta-Ala-beta-Ala spacer is optional and may be substituted with Gly-Gly-Gly, mini-PEG groups, or other linkers known in the art, particularly those described herein. it can.

Exemplary exendin and natriuretic peptide hybrids include:
Exendin- (1-28) -beta-Ala-beta-Ala-hBNP:
HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 394); and exendin-beta-Ala-beta-Ala-hBNP:
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-beta-Ala-beta-Ala-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 395)
And exendin-hBNP peptide hybrids.

  The beta-Ala-beta-Ala spacer, as in all hybrids of the present invention, is optional and includes Gly-Gly-Gly, mini-PEG groups, or other linkers known in the art, particularly those described herein. Can be substituted.

  The hybrid polypeptides of the present invention may include further modifications including, but not limited to, substitutions, deletions and insertions into the amino acid sequence of such hybrid polypeptides, and combinations thereof. In preferred embodiments, the hybrid polypeptides of the invention comprise one or more modifications of “non-essential” amino acid residues. In the context of the present invention, “non-essential” amino acid residues may be altered, ie deleted or substituted, without completely destroying or substantially reducing the component peptide hormone receptor agonist activity of the hybrid polypeptide. A residue in the natural human amino acid sequence of a possible fragment, eg, a component peptide hormone fragment.

  Preferred substitutions include conservative amino acid substitutions. A “conservative amino acid substitution” is a substitution of an amino acid residue with an amino acid residue having similar side chain or physiochemical properties (eg, electrostatic, hydrogen-bonded, isosteric, hydrophobic characteristics) It is to be done. Families of amino acid residues with similar side chains are known in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg, glycine) , Asparagine, glutamine, serine, threonine, tyrosine, methionine, cysteine), amino acids with non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), amino acids with β-branched side chains (For example, threonine, valine, isoleucine) and amino acids having an aromatic side chain (for example, tyrosine, phenylalanine, tryptophan, histidine).

  The invention also relates to derivatives of hybrid polypeptides. Such derivatives include one or more water-soluble polymer molecules such as polyethylene glycol (“PEG”) or fatty acid chains of various lengths (eg, stearyl, palmitoyl, octanoyl, etc.) or polyamino acids, For example, hybrid polypeptides conjugated by addition of poly-his, poly-arg, poly-lys, and poly-ala. Modifications to hybrid polypeptides may also include small molecule substituents such as short alkyls and constrained alkyls (eg, branched alkyls, cyclic alkyls, fused alkyls, adamantyl alkyls), and aromatic groups. The water soluble polymer molecule will preferably have a molecular weight ranging from about 500 to about 20,000 daltons.

  Such polymer conjugation and small molecule substituent modification can be performed once at the N- or C-terminus, or at the side chain of amino acid residues, within the sequence of the hybrid polypeptide. Alternatively, there may be multiple derivatization sites along the hybrid polypeptide. Additional derivatization sites can be generated by substituting one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine. See, for example, US Pat. Nos. 5,824,784 and 5,824,778. Preferably, the hybrid polypeptide can be conjugated to one, two or three polymer molecules.

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

The hybrid polypeptide derivatives of the present invention also include hybrid polypeptides having chemical modifications to one or more amino acid residues. Such chemical modifications include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization. These chemical modifications can be made at the N- or C-terminus, or at the side chain of amino acid residues, within the sequence of the PPF hybrid polypeptide. In one embodiment, the C-terminus of these peptides may have a free —OH or —NH ₂ group. In another embodiment, the N-terminal tip has an isobutyloxycarbonyl group, isopropyloxycarbonyl group, n-butyloxycarbonyl group, ethoxycarbonyl group, isocaproyl group (isocap), octanyl group, octylglycine group (G (Oct )), Or may be capped with an 8-aminooctanoic acid group. In preferred embodiments, the cyclization may be by formation of a disulfide bridge. Alternatively, there may be multiple chemical modification sites along the hybrid polypeptide.

In a further embodiment, the hybrid does not include any of the hybrids disclosed in WO 2005/077072. Accordingly, in one embodiment, a novel hybrid is claimed. In another embodiment, a novel use as disclosed herein of any hybrid disclosed in WO 2005/077072 or elsewhere is also claimed herein. Examples of hybrid polypeptides of the invention are provided in the sequence listing and are discussed further in the Examples section herein.
Use of hybrid polypeptides in the treatment or prevention of metabolic conditions or metabolic disorders

  The hybrids of the present invention have reduced dietary intake, reduced appetite, reduced caloric intake, induction of satiety, reduced nutrient utilization, reduced body weight, effects on body composition, body energy content or Altered energy consumption, improved lipid profile (including reduced LDL and triglyceride levels, and / or altered HDL cholesterol levels), slow gastrointestinal motility, delayed gastric emptying, slowed postprandial blood glucose excursion, It may be useful in preventing or suppressing glucagon secretion and lowering blood pressure. In one embodiment, such hybrids contain exendin, CLP1, amylin, and / or cST moieties.

  Accordingly, in certain embodiments, the hybrids of the invention are of a condition or disease that can be alleviated by reducing nutrient utilization, including administering to a subject a therapeutically or prophylactically effective amount of a compound of the invention. Useful for treatment or prevention. These conditions and diseases include eating disorders, insulin resistance, obesity, abnormal postprandial hyperglycemia, all types of diabetes (including type I, type II and gestational glucouric disease), metabolic syndrome, dumping syndrome, hypertension, Examples include, but are not limited to, dyslipidemia, cardiovascular disease, hyperlipidemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and osteoarthritis. In one embodiment, such hybrids contain exendin, CLP1, amylin and / or sCT moieties.

  Exemplary peptide module pairings include cardioactive / protective peptides such as urocortin and GLP-1 or exendin, ANP, BNP or CNP and CLP-1 or exendin, and urocortin and ANP, BNP or CNP. Can be mentioned. Such hybrids will be cardioprotective, in particular for related diseases and conditions described herein including acute or chronic CHF, ischemic reperfusion, myocardial infarction, and antihypertensive indications and angina. It would be useful for vasodilation useful for treatment or prevention. Ucn2 and 3 are particularly useful in the hybrids of the present invention.

  Non-limiting examples of cardiovascular conditions or cardiovascular diseases are hypertension, myocardial ischemia, and myocardial reperfusion. The compounds of the present invention are useful for obesity including stroke, cancer (eg, endometrial cancer, breast cancer, prostate cancer and colon cancer), gallbladder disease, sleep apnea, decreased fertility, and osteoarthritis. It is useful for the treatment or prevention of other concomitant conditions (see, for example, Lyznicki et al, Am. Fam. Phys. 63: 2185, 2001). In other embodiments, the compounds of the invention can be used to alter body composition for cosmetic reasons, enhance physical performance, or produce a less fat meat source. The hybrid is useful for altering body composition by reducing fat without significantly reducing muscle mass and thus producing desirable body fat loss while maintaining lean body mass. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

  In another general aspect, the hybrids of the invention can be used to inhibit ghrelin secretion. Thus, the compounds of the present invention take advantage of this mechanism for ghrelin-related diseases such as Prader-Willi syndrome, all types of diabetes and its complications, obesity, bulimia, hyperlipidemia, or overnutrition. Other related diseases can be treated or prevented. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

  In another general embodiment, it is now recognized that hybrids containing amylin and / or sCT moieties may be useful for the treatment or prevention of Barrett's esophagus, gastroesophageal reflux disease (GERD) and related conditions. It has been. Such conditions include heartburn, heartburn with reflux of stomach / intestinal contents to mouth or lungs, dysphagia, cough, intermittent wheezing and vocal cord inflammation (related to GERD), esophageal erosion, esophageal ulcer, esophageal stenosis , Barretized positive (replacement of normal esophageal epithelium with abnormal epithelium), Barrett's adenocarcinoma, and lung aspiration, but are not limited to these. Such hybrids have antisecretory properties such as gastric acid inhibition, bile acid inhibition and pancreatic enzyme inhibition. Furthermore, such hybrids may have a gastroprotective effect, which makes them particularly useful in the treatment or prevention of Barrett's esophagus and / or GERD and related or concomitant conditions as described herein.

  In another general embodiment, the hybrid is further used in the treatment or prevention of pancreatitis, pancreatic cancer and gastritis, particularly in the treatment and prevention of pancreatitis in patients who have undergone endoscopic retrograde cholangiopancreatic imaging (ERCP). Useful in some cases. Amylin and / or sCT containing hybrid agonists may have a surprisingly superior therapeutic effect when combined with somastatin. Accordingly, in certain embodiments, a method of treating or preventing pancreatitis comprises administration of such a hybrid and somastatin and somastatin agonists to a subject.

  In another general embodiment, the hybrid is useful for reducing bone resorption, reducing plasma calcium, and inducing analgesic effects, and particularly useful for treating bone diseases such as osteopenia and osteoporosis. In yet other embodiments, the hybrid is useful for the treatment of pain and painful neuropathy. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

  In another aspect of the invention, a method of treating or preventing obesity is provided, which method comprises administering to a subject in need thereof a therapeutic or prophylactically effective amount of a hybrid polypeptide. In preferred embodiments, the subject is an obese or overweight subject. “Obesity” is generally defined as a body mass index greater than 30, but for purposes of the present invention, includes subjects having a body mass index less than 30 that require or desire to lose weight. Any subject is included in the “obesity” range. Subjects who are insulin resistant, glucose intolerant, or have any form of diabetes (eg, type 1, type 2 or gestational diabetes) can benefit from these hybrids. In one embodiment, such hybrids contain exendin, PYY, GLP1, amylin and / or sCT moieties.

  In yet another embodiment, the subject's weight is first reduced to a level below that of morbid obesity, and then the anti-obesity drug combination is added in an amount effective to further reduce the subject's weight. Provided is a method for reducing the weight of a morbidly obese patient by administering to a subject. Methods for reducing the subject's weight below that of morbidly obese include methods of reducing caloric intake, methods of increasing physical activity, medication, bariatric surgery such as gastric bypass surgery, or the aforementioned Any combination of methods may be mentioned. In one embodiment, administration of the anti-obesity drug combination further reduces the subject's weight. In another embodiment, a method for reducing body mass index in a subject having a body mass index of 40 or less by administering an amount of an anti-obesity drug combination effective to further reduce the subject's weight I will provide a.

  Weight loss refers to whether the subject is part of his / her overall weight throughout the course of treatment, whether it is days, weeks, months, or years. Means to lose. Alternatively, weight loss can be defined as a decrease in the ratio of fat mass to slow fat mass (in other words, a subject does not necessarily have a corresponding overall weight loss and has lost fat mass, Maintained or increased the amount of degreasing). The effective amount of an anti-obesity agent administered in combination in this embodiment is an amount effective to reduce the subject's body weight throughout the course of treatment, or reduces the percentage of fat in the subject throughout the course of treatment. It is an effective amount. In certain embodiments, the weight of the subject is reduced by at least about 1%, at least about 5%, at least about 10%, at least about 15%, or at least about 20% throughout the course of treatment. Alternatively, the percentage of fat mass of the subject is reduced by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% throughout the course of treatment.

  In other aspects of the invention, methods of reducing food intake, methods of reducing nutrient utilization, methods of causing weight loss, methods of affecting body composition, altering body energy content or increasing energy expenditure , Methods for treating diabetes, and methods for improving lipid profiles (including reduction of LDL cholesterol and triglyceride levels and / or alteration of HDL cholesterol levels), which methods comprise the hybrid poly Administering an effective amount of the peptide to the subject. In preferred embodiments, the methods of the invention can be used to treat or prevent a condition or disease in a subject in need thereof that can be alleviated by reducing nutrient utilization. Administering to the subject a therapeutically or prophylactically effective amount of a hybrid polypeptide of the invention. Such conditions and diseases include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity and all types of diabetes. In one embodiment, such hybrids contain exendin, GLP1, amylin and / or sCT moieties.

  While not intending to be limited by theory, of the prophylactically administered hybrid polypeptides of the present invention relating to causing reduced food intake, delayed gastric emptying, reduced nutrient utilization, and weight loss The effect is thought to be determined by interaction with one or more unique receptor classes within or similar to those within the PP family. More particularly, it appears to involve receptors similar to receptors or PYY palatability (or Y7) receptors.

  Of particular interest as anti-obesity, weight loss, diet loss, increased metabolic rate and body fat reduction and / or fat redistribution hybrids are those having at least one, preferably two, components that act on the CNS. Specific regions of the forebrain (components derived from the telencephalon and diencephalon) and the hindbrain or brainstem (including the midbrain, pons and marrow) are identified as regions involved in controlling energy balance. Forebrain structures or nuclei present in the hypothalamus that are involved in regulating food intake and / or body weight include, for example, the arcuate nucleus (ARC), paraventricular nucleus (PVN), hypothalamic dorsolateral (DMH), These include the compound inner nucleus (VMH) and the hypothalamic outer nucleus (LHA). Rhizobic structures or nuclei present in the brainstem that are involved in regulating dietary intake and / or body weight include, for example, the solitary nucleus (NST), the last cortex (AP), and the lateral paracerebellar limb nucleus (IPBN) It is done. The brainstem nucleus that controls the elements of the complete motor control system is likely to be controlled by primary or secondary projections from brainstem regions such as NST, AP and IPBN. It is noteworthy that AP, NST and IPBN have all been proven to have their own integration capabilities (collectively or independently).

Various CNS-specific anti-obesity agents act on these forebrain structures present in the hypothalamus that are involved in regulating dietary intake and / or weight. In addition, CNS-specific anti-obesity drugs act on the hindbrain structures present in the brain stem that are involved in regulating dietary intake and / or body weight. Examples of such anti-obesity agents are described herein. For further examples of peptide family modules that can contribute to the formation of anti-obesity drug hybrids and that can be used in combination to form anti-obesity drugs that have activity against both the forebrain and hindbrain, see below. Please refer to the table. Such components include, for example, those described herein, neuropeptide Y1 (NPY1) receptor antagonists, NPY5 receptor antagonists, leptin and leptin agonists, ciliary neurotrophic factor (CNTF) ) And CNTF agonists, peptide YY (PYY) and PYY agonists, exendin and exendin agonists, GLP-1 and GLP-1 agonists, ghrelin and ghrelin antagonists, cholecystokinin (CCK) and CCK agonists, and Amylin and amylin agonists. Additional peptide family components and clinical guidance can be found in Applicant's co-pending patent application PCT / US06 / 17529, which is hereby incorporated by reference.

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

  In certain embodiments, the hybrid and method for using the same include a first component that primarily targets the hypothalamic energy balance center, eg, ARC, PVN, VM, and LH. In one embodiment, the hybrid contains one or more other peptide family components that are at different positions than the first component or by different mechanisms of action but also target the hypothalamus. When hybrids contain more than one peptide family component, and these also target the hypothalamus, those more than one other peptide family component target the same location by the same mechanism of action with each other In some cases, different locations and / or different mechanisms of action may be targeted. In another embodiment, the hybrid is different from the first component and one or more additional, including anti-obesity action, blood glucose control, cardioprotection, and / or hypertension control by different positions or mechanisms of action. Containing one or more other peptide family components that optionally provide the beneficial therapeutic effects of In certain embodiments, the additional peptide family components are primarily targeted to the hindbrain energy balance center, eg, NST, AP and IPBN.

  In certain embodiments, the hybrid and methods for using it include a first component that primarily targets the brain balance energy center, eg, NST, AP, and IPBN. In one embodiment, the hybrid contains one or more other peptide family components that are different from the first component and that also target the hypothalamus at different positions or by different mechanisms of action. In another embodiment, the hybrid is different from the first component and one or more additional, including anti-obesity action, blood glucose control, cardioprotection, and / or hypertension control by different positions or mechanisms of action. Containing one or more other peptide family components that optionally provide the beneficial therapeutic effects of In certain embodiments, the additional peptide family components are primarily targeted to the hypothalamic energy balance center, eg, ARC, PVN, VM and LH.

  As used herein, an anti-obesity agent that “acts on forebrain structures involved in regulating dietary intake and / or weight” refers to a specific region within the forebrain, such as, in particular, the nucleus and / or nerves. Stimulate or inhibit circuit activity. This forebrain stimulation or suppression results in decreased nutrient utilization to the body. Anti-obesity drugs “acting on the lobe brain structure involved in the regulation of dietary intake and / or body weight” stimulate or inhibit the activity of specific regions within the lobe brain, in particular the nuclear and / or neural circuits To do. This hindbrain stimulation or suppression results in reduced nutrient utilization to the body.

  In another aspect, methods are provided for reducing fat mass by increasing metabolic rate in a subject, and these methods are effective in reducing fat mass by increasing subject metabolic rate. And administering an anti-obesity hybrid. Fat mass can be expressed as a percentage of total body weight. In some embodiments, the amount of fat is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% throughout the course of treatment. In one embodiment, the subject's lean mass is not decreased throughout the course of treatment. In another embodiment, the subject's lean mass is maintained or increased throughout the course of treatment. In another embodiment, the subject continues on a low calorie diet or restricted diet. “Low calorie diet” means that a subject consumes a lower amount of calories per day compared to the subject's normal diet. In one example, the subject is consuming at least 50 calories lower per day. In other examples, the subject is consuming 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 calories lower per day.

  In one embodiment, a method of use is provided that modifies fat distribution, reduces fat mass, or both in a subject. Thus, subjects who benefit from modification of body composition can also benefit from the method. As intended herein, modified body composition includes the reduction or maintenance of body fat with the minimization, maintenance or increase of lean body mass loss. In these situations, weight may increase or decrease. Thus, when these terms are used in the art, the subject may be lean, overweight, or obese. The provided method can also include a method of reducing fat while leaving lean mass in non-fat tissue. Applications of such methods include the treatment of diseases such as non-alcoholic steatohepatitis (NASH) or lipodystrophy.

  In one embodiment, a method for altering fat distribution in a subject is provided, the method comprising administering an anti-obesity drug hybrid in an amount effective to alter fat distribution in the subject. In one embodiment, the modification occurs as a result of increased metabolism of visceral fat or ectopic fat or both. “Fat distribution” means the position of fat accumulation in the body. Examples of such fat accumulation positions include subcutaneous, visceral and ectopic fat reservoirs. “Subcutaneous fat” means a lipid deposit just below the skin surface. The amount of subcutaneous fat in a subject can be measured using any method available for measuring subcutaneous fat. Methods for measuring subcutaneous fat are known in the art, for example, as described in US Pat. No. 6,530,886, which is hereby incorporated by reference in its entirety. “Ectopic fat storage” means lipid deposits in and around the tissues and organs that make up lean body mass (eg, skeletal muscle, heart, liver, pancreas, kidneys, blood vessels). In general, ectopic fat storage is the accumulation of fat outside the typical adipose tissue repository in the body. “Visceral fat” means fat accumulation as abdominal adipose tissue. Visceral fat surrounds living organs and can be metabolized by the liver to produce blood cholesterol. Visceral fat has been associated with increased risk of conditions such as polycystic ovary syndrome, metabolic syndrome and cardiovascular disease. In some embodiments, the method comprises visceral or ectopic fat at a rate of at least about 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50% greater than subcutaneous fat or Includes both metabolisms. In one embodiment, these methods result in a suitable fat distribution. In some embodiments, a suitable fat distribution is an increased ratio of subcutaneous fat to visceral fat, ectopic fat, or both. In one embodiment, the method includes increasing lean body mass, for example, as a result of increasing muscle cell mass.

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

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

  In one embodiment, a method is provided for preventing visceral fat accumulation or reducing the amount of ectopic fat in a subject, the method providing to a subject in need thereof ectopic fat accumulation in the subject. Administration of an anti-obesity hybrid in an amount effective to prevent or reduce the amount of ectopic fat. In one example, the amount of ectopic fat is reduced by at least about 5% compared to the subject prior to administration of the anti-obesity hybrid. In other examples, the amount of ectopic fat is reduced by at least about 10%, or at least about 15%, 20%, 25%, 30%, 40% or 50% in the subject. Alternatively, the amount of ectopic fat is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% compared to the subcutaneous fat in the subject Decrease proportionally by 90% or 100%. Ectopic fat can be measured in a subject using any method available for measuring ectopic fat.

  In another embodiment, a method for producing a more suitable fat distribution in a subject is provided, wherein the method comprises a hybrid that is effective as an anti-obesity agent in an amount effective to produce a suitable fat distribution. Administration to a subject. In one embodiment, administration of the anti-obesity hybrid reduces the amount of visceral fat or ectopic fat or both in the subject. In one embodiment, at least one family module that acts on forebrain structures involved in dietary intake or body weight adjustment or both, An anti-obesity hybrid is administered, comprising in combination with at least one family module acting. In one embodiment, these methods preferentially reduce the amount of visceral fat or ectopic fat or a combination of both over subcutaneous fat reduction. Such a method results in a higher subcutaneous fat to visceral fat or ectopic fat ratio. This improved ratio can consequently 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 5% higher than subcutaneous fat. In other embodiments, ectopic or visceral fat is 10%, 15%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90% or 100% higher than subcutaneous fat. It is metabolized in.

  Of particular interest for anti-obesity, weight and fat composition related treatments as discussed herein are amylin (eg, amylin-sCT-amylin chimera), leptin and / or PPF (eg, PYY analogs or PPY / NPY chimera) is a hybrid containing a family module. For example, an amylin family module can be administered alone attached to a leptin family module, or, in further embodiments, can be administered in combination (eg, separately or mixed together) with a PPF family compound. In another embodiment, the hybrid contains a leptin-PPF combination that is administered alone or in combination with an amylin family compound. In another embodiment, the hybrid contains an amylin-PPF combination that is administered alone or in combination with a leptin family compound. In still further embodiments, the hybrid contains all three family modules. For example, as in the case of a multi-chamber delivery system, the amylin-PPF hybrid can be supplied in a pharmaceutically acceptable sterile solution that is used to dissolve lyophilized or powdered leptin family compounds. The

  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 a glucocorticosteroid. Glucocorticosteroids have the side effect of increasing fat mass and decreasing lean mass. Therefore, it is considered that this anti-obesity drug combination agent can be used in combination with glucocorticosteroids under conditions where the use of glucocorticosteroids is beneficial in order to prevent the side effects of glucocorticosteroids.

  Furthermore, with respect to lipid lowering, the hybrids of the present invention are in reducing blood lipid levels such as triglycerides, total cholesterol, LDL cholesterol and VLDL cholesterol, and in generating more beneficial lipid profiles in subjects in need of such treatment. Can be used for Accordingly, in one embodiment, a method is provided for lowering blood triglycerides, total cholesterol, LDL cholesterol, VLDL cholesterol, or any combination thereof, comprising administering a lipid-lowering hybrid to a subject in need thereof. To do. In further embodiments, the lipid-lowering hybrid may contain an exendin (incretin) family component, an amylin family component, a PPF or PPF (NPY) family component, or any combination thereof. In one embodiment of this method, the lipid to be reduced is plasma triglycerides. In another embodiment, it is total plasma cholesterol. In another embodiment, it is LDL cholesterol. In another embodiment, it is VLDL cholesterol. The hybrid can be effective acutely and / or long-term to maintain or reduce fasting lipid levels and / or reduce postprandial lipid (particularly triglyceride) excursions. Patients in need of such treatment can have high triglycerides, high LDL cholesterol, high VLDL cholesterol, or any combination thereof. Such patients may otherwise appear normal, have diabetes or pre-diabetes, have obesity, have a lipid disease or condition, such as dyslipidemia, hypercholesterolemia or hypertriglyceridemia, and / or Mention of patients with cardiovascular disease. This effect of the hybrid is beneficial in reducing the risk of heart disease and atherosclerosis in patients at higher risk, such as patients with genetic predisposition, obese patients, diabetic patients, etc. . For example, the patient can be a patient suffering from atherosclerosis with high lipid or cholesterol levels. The method reduces postprandial triglyceride excursions in subjects, reduces circulating lipid levels, treats dyslipidemia, improves circulating lipid profile, treats hypertriglyceridemia, treats hypercholesterolemia And / or a method of reducing postprandial triglyceride levels comprising administering to a subject in need of such treatment an effective amount of a hybrid of the invention. As used herein, “lipid profile” means the balance, ratio or actual concentration of circulating lipids including triglyceride levels, HDL, LDL, cholesterol, and the like. In a preferred embodiment, the methods of the invention comprising administering an effective amount of amylin or an amylin agonist are useful for reducing triglyceride levels in a patient. Total lipid or triglyceride levels, such as fasting levels, postprandial peak levels, and total postprandial lipid / triglyceride level excursions (eg, area under the curve for postprandial triglyceride increases compared to increases in untreated amylin agonists) (As measured by (AUC)) can be reduced by this method. Individual clinically relevant measurements, such as fasting lipid levels (including triglycerides, cholesterol, HDL and LDL, etc.) and postprandial lipid (eg, triglycerides) levels are also reduced by the methods of the invention. Dyslipidemias, ie patients with lipid levels that are modified compared to normal levels can be treated by administration of amylin or amylin agonists. Diabetic and obese patients, and patients with a genetic predisposition to dyslipidemia or cardiovascular disease are particularly suitable for treatment with the methods of the invention. As used herein, “treatment of high triglyceride levels in a patient” refers to either preventing an increase in those levels or causing a decrease in those levels relative to the level prior to treatment. means. As used herein, “reduction in postprandial triglyceride excursion” means a reduction in both the peak concentration seen in a patient after meal intake and the area under the total curve of the triglyceride concentration curve. Typically this will mean a reduction in the area under the entire curve for graphs such as those provided in FIGS. 2-4 within that time after meal. As used herein, “reduced circulating lipid levels” in a patient means a decrease in blood lipid levels that can be measured relative to the level prior to treatment. As used herein, “treatment of dyslipidemia” is a level that is closer to what is medically defined as any or all lipid or lipoprotein parameters that are clinically measurable normal and / or healthy Means improvement or recovery to ratio, profile or balance. This includes, but is not limited to, levels of triglycerides, LDL, HDL, IDL, VLDL, total cholesterol, apolipoprotein, and the like. As used herein, “improving circulating lipid profile in a patient” causes a change in the concentration of one or more lipids found in the blood to alter the patient's total blood lipid content to a favorable state. Means that. This may include shifting the lipid distribution across different lipoprotein fractions without changing the total content / concentration of circulating lipids. As used herein, “treatment of hypertriglyceridemia” in a patient causes a decrease in the concentration of triglycerides found in the patient's blood at one or more relevant time points, for example, on an empty stomach or after a meal. Means. As used herein, “decrease in postprandial circulating triglycerides” refers to a measurable amount of triglycerides in the blood circulation after a meal (eg, within a period of about 4-6 hours after a meal) before treatment for a similar meal or Meaning to reduce relative to the level of such lipids found after meals in untreated patients.

  The hybrids and PYY chimeras of the present invention can be used with other lipid-lowering drugs. Lipid lowering agents include any compound that can lower plasma lipid levels. Exemplary lipid-lowering agents include statins such as atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin or rosuvastatin; bile acid binders such as cholestyramine or colestipol; peroxisome proliferator-activated receptor (PPAR) agonists Examples include, but are not limited to, tesaglitazar, vitamin E; cholesterol ester transfer protein (CETP) inhibitors such as ezetimibe, JTT-705 and torcetrapib.

  Additional assays useful in the present invention include those that can determine the effect of a hybrid compound on body composition. An exemplary assay may include the use of a diet-induced obesity (DIO) mouse model for metabolic diseases. Prior to the treatment period, male C57BL / 6J mice can be fed a high fat diet (# D12331, 58% of fat to calories; Research Diets, Inc.) for the first 6 weeks at the age of 4 weeks. During this test, mice can continue to eat their high fat diet. Water is freely available throughout this test. A group of non-obese mice of similar age can be given a low fat diet (# D12329, 11% of calories from fat) for comparison of metabolic parameters with the DIO group.

  DIO mice can be implanted with a subcutaneous (SC) intrascapular osmotic pump to deliver either vehicle (in water, 50% dimethyl sulfoxide (DMSO)) or a compound of the invention. The group of pumps described below can be set to deliver any amount of a compound of the invention, eg, 1000 μg / kg / day, for 7-28 days.

Body weight and food intake can be measured at regular intervals throughout the study period. Respiratory quotient (defined as RQ, CO ₂ output ÷ O ₂ consumption) and metabolic rate can be determined using total animal indirect calorimetry (Oxymax, Columbus Instruments, Columbus, OH). Mice can be euthanized by isoflurane overdose and the body mass index (bilateral epididymal fat pad weight) can be measured. In addition, prior to determination of epididymal weight, a double X-ray energy absorption measurement (DEXA) device was used according to the manufacturer's instructions (Lunar Piximus, GE Imaging System) for body composition (exclusion). Fat mass, fat mass) can be analyzed. In the methods of the invention, preferred hybrid polypeptides of the invention are used in one of the assays described herein (preferably dietary intake, gastric emptying, pancreatic juice secretion, weight loss or body composition assay). Which has a potency greater than that of the component peptide hormones in the same assay.

  In addition to improving hypertension as a result of reduced food intake, weight loss or obesity treatment in a subject in need thereof, the compounds of the present invention can be used to treat hypotension.

The compounds of the present invention are also useful for enhancing, inducing, enhancing or restoring glucose responsiveness in islets or pancreatic cells. These effects may be useful in the treatment or prevention of conditions associated with metabolic diseases such as those described above and those described in US Patent Application No. US 20040228846. Assays for determining such activity are known in the art. For example, US Patent Application Publication No. US 20040228846, which is incorporated by reference in its entirety, describes assays for islet isolation and culture and determination of fetal islet maturity. Examples of patent application US 20040228846 include pancreatic polypeptide (PP), neuropeptide Y (NPY), neuropeptide K (NPK), PYY, secretin, glucagon-like peptide-1 (GLP-1) and bombesin Intestinal derived hormone peptides were purchased from Sigma. Type XI collagenase was obtained from Sigma. RPMI 1640 medium and fetal bovine serum were obtained from Gibco. A radioimmunoassay kit ([ ¹²⁵ I] -RIA kit) containing an anti-insulin antibody was purchased from Linco, St Louis.

Postpartum rat islets were obtained from age P-02 rats. Adult rat islets were obtained from rats 6-8 weeks old. Fetal rat islets were obtained as follows. Pregnant female rats were sacrificed on gestational day E21. The fetus was removed from the uterus. 10-14 pancreas were excised from each litter and washed twice with Hanks buffer. The pancreas were pooled, suspended in 6 mL 1 mg / mL collagenase (type XI, Sigma) and incubated for 8-10 minutes at 37 ° C. with constant shaking. Digestion was stopped by addition of 10 volumes of ice-cold Hanks buffer and then washed 3 times with Hanks buffer. The islets were then purified by Ficoll gradient and cultured in 10% fetal bovine serum (FBS) / RPMI medium with or without 1 μM IBMX. At the end of the fifth day, 20 islets were hand-picked and transferred to each tube and analyzed for static insulin release. In general, islets were first washed with KRP buffer and then incubated with 1 mL KRP buffer containing 3 mM (low) glue for 30 minutes at 37 ° C. with constant shaking. After collecting the supernatant, the islets were incubated with 17 mM (high) glucose for 1 hour at 37 ° C. Insulin released by low and high glucose stimulation was assayed by radioimmunoassay (RIA) using [ ¹²⁵ I] -RIA kit. E21 fetal islets were cultured for 5 days in the presence of 200 ng / mL PYY, PP, CCK, NPK, NPY, secretin, CLP-1 or bombesin.

  Zucker diabetic obese (ZDF) male rats, also provided in vivo assays using inbred (> F30 generation) rat models that spontaneously develop diabetes in all fa / fa males fed the rodent standard diet Purina 5008 To do. In male ZDF fa / fa, hyperglycemia begins to develop at about 7 weeks of age, and typically glucose levels (feeding) reach 500 mg / DL by 10 to 11 weeks of age. Insulin levels (feeding) are high during the development of diabetes. However, by 19 weeks of age, insulin falls near the level of lean control litter brothers. Triglyceride and cholesterol levels in obese rats are usually higher than those in lean rats. In this assay, three groups of 7 week-old ZDF rats (6 rats per group) were infused with ALZA pump for 14 days: 1) vehicle control, 2) and 3), two different doses, respectively, PYY at 100 pmol / kg / hour and 500 pmol / kg / hour was applied. Four measurements were made before infusion and on days 7 and 14 after infusion: 1) plasma glucose level, 2) plasma insulin level, 3) plasma triglyceride (TG) level, and oral glucose tolerance (OGTT) test. Thus, these assays can be used with the compounds of the invention to test for the desired activity.

  Other possible uses for hybrid polypeptides include methods of reducing the concentration of aluminum (Al) in the central nervous system to treat, prevent or delay the onset of Alzheimer's disease (see, eg, US Pat. No. 6,734,166). (Which is incorporated by reference in its entirety)). Assays for determining effects on Al using diploid and Ts mice are known in the art and can be found in US Pat. No. 6,734,166. These mice were individually housed in Nalgene® brand metabolism or polypropylene cages and given 3 days prior to the experiment to acclimate to the cage. Mice are free to feed (LabDiet® NIH Rat and Moust / Auto 6F5K52, St. Louis, Mo.) and water during the experiment, except for 16 hours before euthanasia without food. It was. Mice were given daily subcutaneous injections of either active compound or saline. Mice were sacrificed at day 13 for some experiments and at the end of day 3 for other experiments and samples were collected. Mouse brain samples were weighed in a clean Teflon liner and prepared for analysis by microwave digestion in low trace element grade nitric acid. The samples were then analyzed for Al content using Inductively Coupled Plasma Mass Spectrometry (Nuttall et al., Annals of Clinical and Laboratory Science 25, 3, 264-271 (1995)). All tissues under analysis were handled in a clean room environment using a HEPA air filtration system to minimize background contamination. The hybrids of the present invention are useful for the prevention and treatment of hypertensive and diabetic nephropathy and renal disorders, including renal disorders associated with insulin resistance and metabolic syndrome. Hybrids achieve these objectives by, among other things, improving and preventing exacerbation of hypertension, endothelial function, renal function and glomerulosclerosis. In one embodiment, the invention provides for preventing or treating renal disorders, including hypertensive and diabetic renal disorders or renal disorders associated with insulin resistance, comprising administering a compound of the invention. Provide a method. Furthermore, the hybrid can be used to improve endothelial function in patients with reduced vasodilatory capacity, or patients with glomerulosclerosis or any other decrease in glomerular flow. These improvements in endothelial function are useful both for reducing hypertension and for improving the function of glomerular capillaries. In additional embodiments, the molecules of the invention are used to prevent, slow, treat or ameliorate progression of proteinuria and / or glomerulosclerosis to prevent progression of renal injury to ESRD. Useful. The hybrid is useful for reducing the risk of suffering from cardiac arrhythmia, preventing or treating cardiac arrhythmia. The hybrid can produce antiarrhythmic effects in patients with cardiac ischemia, cardiac ischemia-reperfusion, and congestive heart failure. For example, GLP-1 has been found to reduce cardiac damage and enhance recovery in patients with these diseases. Incretins such as GLP-1 are glucose-dependent insulinotropic hormones. GLP-1 and exendin effectively enhance peripheral glucose uptake without inducing dangerous hypoglycemia. They also strongly suppress glucagon secretion, which is independent of its insulinotropic effect, so that plasma free fatty acid (FFA) levels can be reduced substantially more than can be achieved with insulin. High FFA levels have been implicated as a major toxic mechanism during myocardial ischemia. In another embodiment, the hybrid is useful for the prevention and treatment of cardiac arrhythmias that reliably reduce injuries associated with reperfusion and ischemia and enhance patient recovery. In yet a further embodiment, hybrid therapy, preferably intravenous administration after acute stroke or bleeding, optimizes insulin secretion, increases brain anabolism, improves insulin efficacy by suppressing glucagon, and severely low Provides a means to maintain normoglycemia or mild hypoglycemia without the risk of blood sugar or other adverse side effects. In one embodiment, such a hybrid contains a GLP1 or exendin moiety. In further embodiments, GLP1 or exendin family modules are combined with natriuretic family peptides, amylin family peptides, urocortin family peptide modules, or cardiovascular conditions, including CHF, as described herein, or Achieve improved treatment or prevention of cardiovascular disease.

  Congestive heart failure is one of the most significant causes of morbidity and mortality in developed countries. It occurs as a late onset in various cardiovascular diseases and is characterized by decreased contractility and / or volume or pressure overload (Fortuno, Hypertension 38: 1406-1412 (2001)). Numerous studies suggest that cardiac remodeling is a major determinant of the clinical course of CHF, regardless of its etiology (Fedak, Cardiovascular Pathology 14: 1-11 (2005)). Cardiac remodeling is therefore an attractive target for the treatment of congestive heart failure. Accordingly, agents that act to prevent or reduce cardiac remodeling are desired. In fact, the literature identifies the need for molecules that can attenuate cardiac remodeling (Fortuno, Hypertension 38: 1406-1412 (2001)). Literature reports show that attenuation of ventricular remodeling also improves survival after myocardial injury, but treatments that exacerbate remodeling are associated with poor results even when systolic function is improved. (See Somasundaram, Med. Clin. N. Am., 88: 1193-1207 (2004)).

  Thus, a method for treating cardiovascular disease, in one embodiment acute or chronic heart failure, in another embodiment myocardial infarction, in another embodiment ischemic heart failure, and in yet another embodiment is a congestive heart failure. Provide here. In one embodiment, the treatment is administered by preventing or ameliorating cardiovascular injury induced by hyperglycemia. In one embodiment, the treatment is administered by providing a cardioprotective effect. In another embodiment, the treatment is achieved by preventing cardiac remodeling, delaying the onset of cardiac remodeling, diminishing or improving cardiac remodeling. In general, cardiac remodeling refers to the reconstruction and remodeling of any ventricle of the heart. In one embodiment, cardiac remodeling refers to ventricular reconstruction and remodeling. As explained above and without intending to be limited by theory, cardiac remodeling is a post-injury genome to the myocardium that later involves molecular, cellular and intestinal changes leading to ventricular remodeling and remodeling. Can be explained as Such reconstruction and remodeling can be clinically proven as a change in heart size, shape and function. Cardiac remodeling can occur in response to any stimulus or combination of stimuli to the myocardium. In one embodiment, cardiac remodeling is the result of myocardial injury. As a non-limiting example, cardiac remodeling occurs as a result of myocardial infarction, hypertension, volume overload (eg, from aortic regurgitation), infection, inflammation, diabetes, viral cardiomyopathy, and idiopathic cardiomyopathy It can occur in response to myocardial injury.

  In one embodiment, cardiac remodeling is prevented, delayed, attenuated or ameliorated by administration of the hybrids of the invention. Hybrid has the following cardiac parameters: left ventricular contraction diastolic function, E wave to A wave ratio, left ventricular end diastolic pressure, cardiac output, cardiac contractility, left ventricular volume, left ventricular volume to body weight ratio, left ventricle Improve at least one of volume, left atrial volume, left ventricular end-diastolic or end-diastolic diameter, infarct size, exercise capacity, exercise efficiency, or any measure of systolic and / or diastolic function Ability to (enhance); or may include the ability to attenuate, delay or prevent adverse effects on ventricular dilation or one of the above cardiac parameters. In one embodiment, the hybrid contains a family member such as exendin-4 that binds to GLP-1 or exendin receptors. In connection with this method, prevention or improvement of cardiac remodeling can include any reduction in cardiac remodeling. In one embodiment, prevention or amelioration of cardiac remodeling is achieved by reducing the risk of congestive heart failure.

  In one embodiment, cardiac remodeling is about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50% of the amount of cardiac remodeling in the absence of hybrid administration. Improve or reduce to an amount that is less than 60%, 70%, 80% or 90%. In another embodiment, cardiac remodeling is slightly reduced, moderately reduced, substantially reduced, or substantially compared to the occurrence of cardiac remodeling in the absence of hybrid administration. Can be eliminated. As used herein, a slight reduction in cardiac remodeling refers to cardiac remodeling that is reduced by about 25% or less compared to cardiac remodeling in the absence of hybrid administration. Moderate reduction in cardiac remodeling refers to cardiac remodeling that is reduced by about 50% or less compared to cardiac remodeling in the absence of hybrid administration. Substantial reduction in cardiac remodeling refers to cardiac remodeling that is reduced by about 80% or less compared to cardiac remodeling in the absence of hybrid delivery. Substantial elimination of cardiac remodeling refers to cardiac remodeling that is reduced by about 80% or more compared to cardiac remodeling in the absence of hybrid administration.

  Any means available to those skilled in the art can be utilized to assess the extent to which cardiac remodeling is prevented, ameliorated, attenuated or delayed. For example, cardiac remodeling measures chamber dimensions and wall thickness and movement by cardiac histology, analysis including (but not limited to) LV mass, or throughout the life of a subject, for example, by echocardiography. Or by quantifying left ventricular (LV) diastolic function using the peak velocity ratio (E / A ratio) of E and A waves.

  In one embodiment, one of skill in the art will identify subjects that can benefit from administration of the hybrid that prevent, ameliorate, attenuate or delay cardiac remodeling in view of the conditions and risk factors associated with the subject. be able to. In one embodiment, the subject is in need of prevention, improvement, attenuation or delay of cardiac remodeling. In another embodiment, the subject may be one who wishes to prevent, improve, attenuate or delay cardiac remodeling. One risk factor may be a genetic predisposition for the heart undergoing cardiac remodeling. Exemplary samples and subjects of the methods provided herein include those that have experienced, experienced, or at risk of experiencing a condition associated with cardiac remodeling. The condition associated with cardiac remodeling can be any condition or disease where cardiac remodeling is known to occur or is considered a risk. Conditions associated with cardiac remodeling include, for example, myocardial infarction, inflammation, ischemia / reperfusion, oxidative stress, pulmonary heart, late glycation reaction products, cardiac wall tension abnormalities, sympathetic nerve stimulation, myocarditis, hypertension, Viral cardiomyopathy, idiopathic cardiomyopathy, heart transplantation, and cardiac surgical procedures.

  As noted above, the hybrid can be administered as a result of an acute event or a chronic condition. Whether it is an acute event or a chronic condition, the methods provided herein include chronic treatment with a hybrid. For example, the length of the chronic treatment can include the time that the event occurred and the time that the subject thinks he has recovered from an acute event or has recovered from a chronic condition.

  Chronic administration of hybrids or chronic treatment with hybrids to prevent, attenuate, delay or improve cardiac remodeling is permitted if no specific transient events or transient conditions associated with cardiac remodeling are confirmed Is done. Chronic administration is based on a genetic predisposition to cardiac remodeling or to a condition that forms a non-transient predisposition (eg, a non-transient condition that cannot be confirmed and easily eliminated, eg, diabetes) On the basis of administering the hybrid over an unclear but continuous period. The hybrid can be administered chronically in the methods provided herein to prevent cardiac remodeling in subjects exhibiting congestive heart failure, regardless of etiology. Chronic administration of hybrids to prevent or ameliorate cardiac remodeling can also be associated with diabetic patients at risk for congestive heart failure. The hybrid can also be administered on a chronic basis to protect the transplanted organ in an individual who has undergone a heart transplant. When the hybrid is administered chronically, administration can continue for any length of time. However, chronic administration is often performed for a long time. For example, in exemplary embodiments, chronic administration continues for 6 months, 1 year, 2 years or longer.

  In another embodiment, the methods disclosed herein provide improved cardiac contractility. An improvement in cardiac contractility can include the ability of cardiomyocytes to contract. Any evaluation mode can be used to evaluate the improvement in cardiac contractility. For example, clinical observations such as increased cardiac output or decreased heart rate or both lead to a determination of increased cardiac contractility. Alternatively, an increase in contractility of the heart in vivo can be assessed by determining an increase in left ventricular fractional shortening. The left ventricular segmentation shortening can be observed by any available means such as echocardiography. When assessing increased cardiac contractility, the increase in left ventricular fractional shortening can be any amount of increase when compared to fractional shortening prior to administration of the hybrid. For example, this increase in shortening is greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or about 200% Sometimes.

  In yet another aspect, a method for reducing or preventing atrial remodeling by administration of a hybrid is provided. Reduction or prevention of atrial remodeling can be assessed relative to atrial remodeling prior to administration of the hybrid. The therapeutic effect of such reduction or prevention of atrial remodeling includes reduction of atrial fibrillation. In yet another aspect, a method for reducing or preventing ventricular remodeling by administration of a hybrid is provided. Reduction or prevention of ventricular remodeling can be assessed relative to ventricular remodeling prior to administration of the hybrid.

  In further embodiments, prophylactic and therapeutic methods are provided. Treatment on an acute or chronic basis is contemplated. In addition, when directed to do so, treatment on an acute basis can be extended to chronic treatment. Chronic treatment is considered longer than 2 weeks. In certain embodiments, chronic treatment may be longer than 1 month, 3 months, 6 months, 1 year, 2 years, 5 years, or may be lifelong. In one embodiment, provided herein is a method for treating or preventing a condition associated with cardiac remodeling in a subject in need thereof. The method generally includes administering to the subject an amount of the hybrid effective to prevent or ameliorate cardiac remodeling and ameliorate, prevent or delay the condition associated with the remodeling. As described herein, administration of the hybrid can occur in any manner, including in combination with other agents that provide cardiovascular benefits.

  In yet another embodiment, the methods provided herein include identifying a subject in need of treatment. Any effective criteria can be used to determine that the subject will benefit from administration of the hybrid. For example, methods for diagnosing heart disease and / or diabetes, as well as procedures for identifying individuals at risk of developing these conditions are known to those skilled in the art. Such procedures can include clinical trials, physical examinations, personal interviews and family history assessments.

  In still further embodiments, hybrids that can decrease insulin resistance or increase insulin sensitivity are useful for the treatment of polycystic ovary syndrome (PCOS). Administration of the hybrids of the present invention can reduce or prevent insulin resistance in subjects suffering from PCOS. In yet another embodiment, the hybrid prevents the development of type 2 diabetes in a subject suffering from PCOS. Furthermore, the hybrid can restore regular menstruation, ovulation or fertility in subjects suffering from PCOS. In one embodiment, such hybrids contain a GLP1 or exendin moiety for binding or activating the GLP1 receptor.

  The compounds of the present invention exhibit a broad range of biological activities, some related to their antisecretory and anti-motility properties. The compounds can inhibit gastrointestinal secretion by direct interaction with epithelial cells or possibly by inhibiting the secretion of hormones or neurotransmitters that stimulate intestinal secretion. Anti-secretory properties include inhibition of gastric and / or pancreatic juice secretion and may be useful and useful for the treatment or prevention of diseases and disorders including gastritis, pancreatitis, Barrett's esophageal and gastroesophageal reflux disease.

  The compounds of the present invention may be used in any number of gastrointestinal diseases associated with intestinal electrolytes and hypersecretion and decreased absorption, such as infectious diarrhea, inflammatory diarrhea, short bowel syndrome, or generally surgical procedures such as ileal fistula formation. It is useful for the treatment of diarrhea that occurs after surgery (see, for example, Harrison's Principles of Internal Medicine, McGraw-Hill Inco, New York, 12th Ed.). Examples of infectious diarrhea include acute viral diarrhea, acute bacterial diarrhea (eg, due to Salmonella, Campylobacter and Clostridium, or protozoal infections) or traveler's diarrhea (eg, Norwalk virus or retrovirus) However, it is not limited to these. Examples of inflammatory diarrhea include, but are not limited to, dyspepsia syndrome, tropical diarrhea, chronic pancreatitis, Crohn's disease, diarrhea and irritable bowel syndrome. It has also been found that the peptides of the present invention can be used to treat emergency or life-threatening conditions involving gastrointestinal disorders, for example, post-surgery or cholera-induced conditions.

  The compounds of the present invention may also be useful in the treatment or prevention of intestinal damage as opposed to merely treating symptoms associated with intestinal damage (eg, diarrhea). Such damage to the intestine can be or can be the result of ulcerative colitis, inflammatory bowel disease, intestinal atrophy, intestinal mucosal loss, and / or loss of intestinal mucosal function (see WO 03/105763 (in its entirety) Is incorporated herein by reference)). An assay for such activity, as described in WO 03/105763, is maintained in the 12:12 light: dark cycle and allows free feeding in rodent standard diet (Teklad LM 485, Madison, WI) and water. Including 11 week old male HSD rats in the range of 250-300 grams. The animals were fasted for 24 hours before the experiment. A simple and reproducible rat model of chronic colitis has been previously described by Morris GP, et al., "Hapten- induced model of chronic inflammation and ulceration in the rat colon." Gastroenterology. 1989; 96: 795-803 Explained. It shows a relatively long period of inflammation and ulceration, so it has a new potential to study the pathophysiology of colon inflammatory diseases in a specifically controlled manner and to be applicable to inflammatory bowel diseases in humans Opportunity to evaluate treatment.

  Rats were anesthetized with 3% isofluorane and placed on a conditioned heating pad set at 37 ° C. A gavage needle was inserted into the rectum into the colon 7 cm. As described in Mazelin, et al., Juton Nerv Syst. 1998; 73: 38 45, hapten trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol (v / v) And delivered to the colon lumen by its gavage needle at a dose of 30 mg / kg. In the control group, a saline solution (NaCl 0.9%) was administered intracolonically.

  Four days after the induction of colitis, the colon was excised from anesthetized rats and then the rats were euthanized by decapitation. The excised colon and spleen were weighed and pictures of the colon were taken to score for gross morphological damage. Inflammation was defined as the area of hyperemia and intestinal wall thickening.

The hybrid polypeptides of the invention can also be used to treat or prevent pancreatic tumors (eg, suppress the growth of pancreatic tumors). The methods of the invention include reducing the growth of tumor cells. The types of benign pancreatic tumor cells that can be treated according to the present invention include serous cystadenoma, microcystic tumor, and solid-cystic tumor. The method is also effective in reducing the growth of carcinomas arising from malignant pancreatic tumor cells, such as pancreatic ducts, acini or islets. US Pat. No. 5,574,010, which is incorporated by reference in its entirety, provides an exemplary assay for testing anti-proliferative properties. For example, the '010 patent provides two human pancreatic adenocarcinoma cancer cell lines, PANC-1 and MiaPaCa-2, which are supplied by the US Microbiology Preservation Agency, ATCC (Rockville, Md.), Etc. Commercially available. These two tumor cells were placed in a NAPCO water jacketed 5% CO ₂ incubator at 37 degrees Celsius, 10% fetal calf serum, 29.2 mg / L glutamine, 25 μg gentamicin, 5 ml penicillin, streptomycin and fungison solution (JRH Biosciences, Lenexa, Kans.) Was grown in RPMI-1640 medium supplemented. All cell lines were detached with 0.25% trypsin (Clonetics, San Diego, Calif.) Once or twice a week once a confluent monolayer of tumor cells was achieved. Cells were pelleted at 500 g for 7 minutes in a 4 ° C. refrigerated centrifuge and resuspended in enriched RPMI1640 medium with trypsin negative. Viable cells were counted with a hemocytometer using trypan blue.

  10,000, 20,000, 40,000 and 80,000 cells of each type were added to a 96 well microtiter plate (Costar, Cambridge, Mass.) In a total volume of 200 μL of medium per well. Cells were allowed to adhere for 24 hours prior to addition of PYY or test peptide. Before adding the peptide, it was replaced with fresh medium. In vitro incubation of pancreatic tumor cells with either PYY or test compound was continued for 6 and 36 hours in length. PYY was added to the cells at doses of 250, 25 and 2.5 pmol per well (N = 14). Test compounds were added to the cell culture at doses of 400 pmol, 40 pmol and 4 pmol per well. Control wells received 2 μL of 0.9% saline to mimic volume and physical damage to attached tumor cells. Each 96 well plate contained 18 control wells to allow comparison within each plate during the experiment. For both PANC-1 and MiaPaCa-2 cells, ninety-six (96) well plates were repeated six times with various concentrations of PYY and test compound.

  At the end of the incubation period, 3- (4,5-dimethylthiazolyl-2-yl) -2,5-diphenyltetrazolium bromide and MTr tetrazolium bromide (Sigma, St. Louis, Mo.) were renewed at 0.5 mg / mL. Added to fresh medium. The medium was changed and the tumor cells were incubated with MTT tetrazolium bromide for 4 hours at 37 ° C. At the end of the incubation, the medium was aspirated. The formazone crystal precipitate was dissolved in 200 μL of dimethyl sulfoxide (Sigma, St. Louis, Mo.). Quantification of solubilized formazone was performed by obtaining an absorbance reading at a wavelength of 500 nm using an ELISA reader (Molecular Devices, Menlo Park, Calif.). Mitochondrial NADH-dependent dehydrogenase activity was measured by MTT assay. This assay was one of the most sensitive and reliable methods for quantifying the in vitro chemotherapy response of tumor cells. (Alley, MC, et al., Cancer Res., 48: 589-601, 1988; Carmichael, J., et al., Cancer Res., 47: 936-942, 1987; McHale, AP, et al., Cancer Lett., 41: 315-321, 1988; and Saxton, RE, et al., J. Clin. Laser Med. And Surg., 10 (5): 331-336, 1992). Analysis of the absorbance reading at 550 nm was analyzed by grouping wells with the same test conditions and verifying the difference that occurred between the control and various peptide concentration treatments by one-sided ANOVA.

  An exemplary in vivo assay is also provided. Human pancreatic ductal adenocarcinoma Mia Paca-2 was tested for in vivo growth inhibition by YY and test compounds. 70,000 to 100,000 human Mia PaCa-2 cells were orthotopically transplanted into 48 male athymic mice. One week later, the animals were treated with PYY or test compound for 4 weeks at 200 pmol / kg / hr with an osmotic minipump. Paired cultures were given saline. At sacrifice, both tumor size and mass were measured. Control mice had significant human cancer growth in the pancreas as evidenced by tissue sections. At nine weeks, ninety percent (90%) of control mice had substantial metastatic disease. Tumor mass was reduced by 60.5% in test treated mice and 27% in PYY treated mice.

  Hybrids include therapeutics for neurological disorders and nervous system abnormalities associated with neuronal loss or dysfunction, including (but not limited to) Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, stroke, ADD and neuropsychiatric syndrome And useful for prophylactic treatment, and for strengthening or facilitating learning, memory and cognition in mammals. Particularly useful in this regard are hybrids containing exendin or CLP1 active moieties, more specifically hybrids comprising at least the N-terminal 7-15 amino acids or their analogs, eg HSEGTFTSD (SEQ ID NO: 378).

  For all indications, in preferred embodiments, the hybrid polypeptides of the invention are administered at a dose of about 0.5 μg to about 5 mg per day in single or divided doses or continuous controlled release, or about 0.01 μg per dose. Peripherally administered at from about / kg to about 500 μg / kg, more preferably from about 0.05 μg / kg to about 250 μg / kg, most preferably less than about 50 μg / kg. Dosages within these ranges will, of course, vary depending on the potency of each analog or derivative, and such dosages can be determined by one skilled in the art.

In the method of the present invention, the hybrid polypeptide of the present invention is an amylin or amylin analog agonist, salmon calcitonin, cholecystokinin (CCK) or CCK agonist, leptin (OB protein) or leptin agonist, exendin or exendin analog One that exhibits long-term or short-term effects that reduce nutrient utilization, including but not limited to body agonists, or other compounds and compositions including GLP-1 or GLP-1 analog agonists It can be administered separately or together with one or more other compounds and compositions. Suitable amylin agonists include, for example, [ ^25,28,29 Pro-] human amylin (SEQ ID NO: 67) (also known as “pramlintide” and described in US Pat. Nos. 5,686,511 and 5,998,367). Is). The CCK used is preferably CCK octapeptide (CCK-8), more preferably its sulfated form. Leptin is, for example, (Pelleymounter et al., Science 269: 540-3 (1995); Halaas et al., Science 269: 543-6 (1995); Campfield et al., Science 269: 546-9 (1995) ). Suitable exendins include exendin-3 and exendin-4, and exendin agonist compounds include, for example, those described in PCT publications WO 99/07404, WO 99/25727 and WO 99/25728. It is done.

  As discussed herein, the hybrids of the invention can be administered separately or together with one or more other agents to gain additional benefits or enhance the effectiveness of the hybrid or other agent. be able to. For example, an anti-obesity hybrid can be administered with an anti-obesity agent or cardioprotective or anti-hypertensive agent, depending on the risk factors associated with the subject in need of treatment and desiring treatment outcome. Exemplary anti-obesity agents for administration with the hybrid (administered separately or mixed; administered prior to, simultaneously with, or following the hybrid) include paroxetine, fluoxetine, fenfluramine, fluvoxamine , Serotonin (5HT) transport inhibitors, including but not limited to sertraline and imipramine. Anti-obesity agents include dexfenfluramine, fluoxetine, sibutramine (eg, MERIDIA®), and US Pat. No. 6,365,633 and PCT patent application publication numbers WO 01/27060 and WO 01/162341 (these are those Also included are selective serotonin uptake inhibitors, including (but not limited to) those described in (incorporated herein by reference in their entirety). Such 5HT transport inhibitors and serotonin uptake inhibitors, analogs, derivatives, formulations, formulations, pharmaceutical compositions, dosages and routes of administration have been previously described.

  Anti-obesity agents include U.S. Pat.No. 3,914,250; and PCT Application Publication Nos. Selective serotonin agonists and selective 5-HT2C receptor agonists, including but not limited to 40456 and WO 02/40457, which are hereby incorporated by reference in their entirety. Also mentioned. Such selective serotonin agonists and 5-HT2C receptor agonists, compositions containing such agonists and routes of administration suitable for use in the provided methods are known in the art. See, for example, Halford et al. (2005) Curr. Drug Targets 6: 201-213 and Weintraub et al. (1984) Arch. Intern. Med. 144: 1143-1148.

  Anti-obesity drugs include but are not limited to rimonabant (Sanofi Synthelabo) and SR-147778 (Sanofi Synthelabo) antagonists / inverse agonists of central cannabinoid receptors (CB-1 receptors) Also mentioned. CB-1 antagonists / inverse agonists, derivatives, formulations, formulations, pharmaceutical compositions, doses and routes of administration have previously been described, for example, in US Pat. Nos. 6,344,474, 6,028,084, 5,747,524, 5,596,106, 5,532,237, 4,973,587, 5,013,837, 5,081,122, 5,112,820, 5,292,736, 5,624,941; European patent application numbers EP-656354 and EP-658546; and PCT application publication numbers WO 96/33159, WO 98/33765, WO 98/43636, WO 98/43635, WO 01/09120, WO 98/31227, WO98 / 41519, WO 98/37061, WO 00/10967, WO 00 / 10968, WO 97/29079, WO 99/02499, WO 01/58869 and WO 02/076949, which are hereby incorporated by reference in their entirety.

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

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

  Anti-obesity agents also include topiramate (TOPIMAX® (Ortho McNeil Pharmaceuticals)), which is indicated as an anticonvulsant and anticonvulsant, but has also been shown to increase weight loss. The drug also includes a lipase inhibitor such as orlistat.

  Anti-obesity agents also include neuropeptide Y1 (NPY1) antagonists and NPY5 antagonists. NPY1 and NPY5 antagonists are known in the art. See, for example, Duhault et al. (2000) Can. J Physiol. Pharm. 78: 173-185, and U.S. Patent Nos. 6,124,331, 6,214,853, and 6,340,683. PY1 and NPY5 antagonists, derivatives, formulations, formulations, pharmaceutical compositions, doses and routes of administration have been previously described. NPY1 antagonists useful in the provided compositions and methods include US Pat.No. 6,001,836; and PCT application publication numbers WO 96/14307, WO 01/23387, WO 99/51600, WO 01/85690, WO 01/85098. WO 01/85173 and WO 01/89528. NPY5 antagonists useful in the compositions and methods of use provided herein include U.S. Patent Nos. 6,140,354, 6,191,160, 6,258,837, 6,313,298, 6,337,332, 6,329,395. No. 6,340,683, No. 6,326,375 and No. 6,335,345; European Patent Nos. EP-01010691 and EP-01044970; and PCT Patent Publication Nos. WO 97/19682, WO 97/20820, WO 97/20821, WO 97 / 20822, WO 97/20823, WO 98/27063, WO 00/64880, WO 00/68197, WO 00/69849, WO 01/09120, WO 01/85714, WO 01/85730, WO 01/07409, WO 01 / 02379, WO 01/02379, WO 01/23388, WO, 01/23389, WO 01/44201, WO 01/62737, WO 01/62738, WO 01/09120, WO 02/22592, WO 0248152, WO 02/49648 And compounds described in WO 01/14376, but are not limited thereto.

  Anti-obesity drugs, including melanin-concentrating hormone 1 receptor (MCH1R) antagonists, such as T-226296 (Takeda) (Takeda), and melanin-concentrating hormone 2 receptor (MCH1R) antagonists, Also included are melanin-concentrating hormone (MCH) antagonists. MCH receptor antagonists, derivatives, formulations, formulations, pharmaceutical compositions, doses and routes of administration have been previously described in, for example, U.S. Patent Application Publication Nos. 2005/0009815, 2005/0026915, 2004/0152742. PCT Patent Application Publication Numbers WO 01/82925, WO 01/87834, WO 02/06245, WO 02/04433 and WO 02/51809; and Japanese Patent Application JP-A-13-226269 (these 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 number WO 00/21509. Specific opioid antagonists useful in the compositions and methods of use provided herein include nalmefene (REVEX®), 3-methoxynaltrexone naloxone, naltrexone, naloxonazine, beta-funaltrexamine, delta 1 ([D-Ala2, Leu5, Cys6] -enkephalin (DALCE)), naltrindole isothiocyanate, and nor-binaltorfamine.

  Anti-obesity agents include, but are not limited to, those described in PCT patent application numbers WO 01/96302, WO 01/68609, WO 02/51232 and WO 02/51838. Drugs are also mentioned. Specific orexin antagonists useful in the provided compositions and methods of use include, but are not limited to, SB-334867-A.

  Anti-obesity agents include PYY3-36 (eg Batterham et al. (2003) Nature 418: 650-654), NPY3-36 and other Y2 agonists such as N-acetyl [Leu (28,31)] NPY24- 36 (White-Smith et al. (1999) Neuropeptides 33: 526-533), TASP-V (Malis et al. (1999) Br. J. Pharmacol. 126: 989-996), cyclo- (28/32) Including but not limited to compounds such as -Ac- [Lys28-Glu32]-(25-36) -pNPY (Cabrele et al. (2000) J. Pept. Sci. 6: 97-122) ), Neuropeptide Y2 (NPY2) agonists, which may be hybrid components as discussed or may be administered separately. Anti-obesity drugs provided include pancreatic peptides (PP) (eg Batterham et al. (2003) J. Clin. Endocrinol. Metab. 88: 3989-3992) and other Y4 antagonists such as 1229U91 (Raposinho et al. (2000) Neuroendocrinology 71: 2-7) and the like, including but not limited to neuropeptide Y4 (NPY4) agonists. NPY2 agonists and NPY4 agonists, derivatives, formulations, formulations, pharmaceutical compositions, doses and routes of administration have been previously described, for example, in US Patent Application Publication No. 2002/0141985 and PCT Application Publication Number WO 2005/077094. Has been.

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

  Anti-obesity agents also include cholecystokinin (CCK) and CCK agonists. Cholecystokinin-A (CCK-A) agonists used include, but are not limited to, those described in US Pat. No. 5,739,106. Specific CCK-A agonists include, but are not limited to, AR-R15849, GI181771, JMV-180, A-71378, A-71623 and SR146131.

  Anti-obesity agents also include ghrelin antagonists such as those described in PCT Application Publication No. 01/87335 and WO 02/08250. Ghrelin antagonists are also known as GHS (growth hormone secretagogue receptor) antagonists. Accordingly, the provided compositions and methods may also use GHS antagonists instead of ghrelin antagonists.

  Anti-obesity agents include obestatin and obestatin analogs and agonists. Obestatin is a peptide derived from prepregrelin, the same precursor from which ghrelin is derived. See, for example, Zhang et al. (2005) Science 310: 996-999; Nogueiras et al. (2005) Science 310: 985-986; Pan et al. (2006) Peptides 27: 911-916. In contrast to ghrelin activity, obestatin appears to act as an appetite suppressive hormone by reducing food intake, gastric emptying activity, jejunal motility, and weight gain. Obestatin peptides used include, but are not limited to, those described in Zhang et al. (2005) Science 310: 996-999.

  And amylin mimetics such as pramlintide, amylin-sCT-amylin (compound 10), increstin such as exendin-4, and PYY analogs are anti-obesity drugs that can also be administered with the hybrid as an anti-obesity drug. For example, a leptin-compound 10 hybrid can be administered with exendin-4, a PYY analog, or both. In another embodiment, the leptin-PYY analog hybrid can be administered with exendin-4, an amylin mimetic, or both.

  Particularly interesting embodiments for treating diabetes and related disorders as discussed herein include hybrids comprising exendin family and alpha-MSH modules, as well as exendin and amylin family members. Of particular interest are hybrids in which exendin is exendin-4 or an analogue or derivative thereof and the amylin component is pramlintide or amylin-sCT-amylin chimera.

Particularly interesting embodiments for treating obesity and related diseases and conditions as discussed herein (loss of body fat) include exendin family, exendin and amylin family members, amylin family in combination with alpha-MSH module Members and PYY Family Members, Amylin Family Members and CCK Family Members, Amylin Family Members and Alpha-MSH Family Members, FN-38 Family Members, Amylin Family Members and PYY Family Members, Same or Different PYY Family Members Different from PYY Family Members , Hybrids including PPY family members and CCK family members, PYY family members and FN-38 family members, CCK family members and FN-38 family members That. Exendin is exendin-4 or an analogue or derivative thereof, the amylin component is pramlintide or amylin-sCT-amylin chimera, the FN38 family member is FN38 or an analogue or derivative thereof, and a PYY chimera is described herein PYY-NPY chimeras as described, for example, hybrids that are SEQ ID NOs 266, 437, 438, 439, 442, 462, 469, 470, 471 and 472 of US 2006 / 013547A1, and PYY-NPY chimera 5705 are particularly Interesting.
Peptide production and purification

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

  The hybrid polypeptides of the invention can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. For example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc. 105: 6442 (1983); Merrifield, Science 232: 341 -7 (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, 1-284 (1979). Solid phase peptide synthesis is performed using the NMP / HOBt (Option 1) system and tBoc or Fmoc chemistry (Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B July 1, 1988, section 6, pp. 49-70, Applied Biosystems , Inc., Foster City, California) and capping using an automated peptide synthesizer (eg, Model 430A, Applied Biosystems Inc., Foster City, California). Peptides can also be assembled using the Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Kentucky). Peptides can be purified by RP-HPLC (preparative and analytical) using, for example, a Waters Delta Prep 3000 system and a C4, C8 or C18 preparative column (10μ, 2.2x25cm; Vydac, Hesperia, California) Can do. Active peptides can be readily synthesized and then screened in screening assays designed to identify reactive peptides.

  Alternatively, the hybrid polypeptides of the present invention can be produced by recombinant techniques well known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor (1989). These hybrid polypeptides produced by recombinant techniques can be expressed from polynucleotides. Polynucleotides, including DNA and RNA, encoding such various fragments of hybrid polypeptides can be obtained from wild-type cDNAs, taking into account the use of codon degeneracy, or designed and made as desired. Those skilled in the art will understand that this is possible. These polynucleotide sequences can be incorporated into codons that facilitate transcription and translation of mRNA in the microbial host. Such production sequences can be easily constructed according to methods well known in the art. See, for example, WO 83/04053. The above polynucleotide may optionally encode an N-terminal methionyl residue. Non-peptide compounds useful in the present invention can be made by methods known in the art. For example, phosphoric acid-containing amino acids and peptides containing such amino acids can be produced using methods known in the art. See, for example, Bartlett and Landen, Bioorg. Chem. 14: 356-77 (1986).

  A variety of expression vector / host systems may be utilized to accommodate and express the hybrid polypeptide coding sequence. These include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insects infected with viral expression vectors (eg, baculovirus) Cell lines; plant cell lines transfected with viral expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg Ti or pBR322 plasmid); or animal cells Examples include, but are not limited to, systems. Mammalian cells useful in recombinant protein production include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (eg, COS-7), WI38, BHK, HepG2, 3T3, RIN, MDCK , A549, PC12, K562 and 293 cells. Exemplary protocols for recombinant expression of proteins are described herein.

  Thus, the polynucleotide sequences provided by the present invention provide for the production of new and useful viral and plasmid DNA vectors, new and useful transformations and transfected prokaryotic and eukaryotic host cells (bacteria grown in culture, Useful in novel and useful methods for the production of yeast and mammalian cells (including yeast and mammalian cells) and in culture growth of such host cells capable of expressing the hybrid polypeptides of the invention. The polynucleotide sequences encoding the hybrid polypeptides herein are useful for gene therapy to alleviate the shortage of production of the chimeric component peptide hormone (s) or to meet the need for increased levels of such It can be.

  The invention also provides a process for recombinant DNA production of hybrid polypeptides. Provided is a process for producing a hybrid polypeptide from a host cell containing a nucleic acid encoding such a hybrid polypeptide, the process comprising: (a) said host cell containing a polynucleotide encoding such a hybrid polypeptide Culturing under conditions that facilitate expression of such DNA molecules; and (b) obtaining such hybrid polypeptides.

  The host cell may be prokaryotic or eukaryotic, and may be a bacterium, mammalian cell (eg, Chinese hamster ovary (CHO) cell, monkey cell, baby hamster kidney cell, cancer cell or others). Cells), yeast cells and insect cells.

  Mammalian host systems for the expression of recombinant proteins are also well known to those skilled in the art. Host strains can be selected for their particular ability to process the expressed protein or produce certain post-translational modifications that would be useful in generating protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glucosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” form of the protein may also be important for correct insertion, folding and / or function. Various host cells, such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activity, and ensure that the introduced heterologous protein is correctly modified and You can choose to process.

  Alternatively, the hybrid polypeptide of the present invention can be produced using a yeast system. The coding region of the hybrid polypeptide cDNA is amplified by PCR. DNA encoding the yeast pre-pro-alpha leader sequence is obtained from yeast genomic DNA with one primer containing nucleotides 1-20 of the alpha matching factor gene and another complementary to nucleotides 255-235 of the gene. Amplification is performed in a PCR reaction using primers (Kurjan and Herskowitz, Cell, 30: 933-43 (1982)). The pre-pro-alpha leader coding sequence and hybrid polypeptide coding sequence fragment are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter so that the promoter is fused to the mature hybrid polypeptide. Directs the expression of a fusion protein consisting of a leader factor. As taught by Rose and Broach, Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, California (1990), this vector is an ADH2 transcript downstream of its cloning site. It further includes a terminator, a yeast “2-micron” replication origin, a yeast leu-2d gene, a yeast REP1 and REP2 gene, an E. coli β-lactamase gene, and an E. coli replication origin. The β-lactamase and leu-2d genes address selection in bacteria and yeast, respectively. The leu-2d gene also helps increase plasmid copy number in yeast and induces higher expression levels. The REP1 and REP2 genes encode proteins that are involved in the regulation of plasmid copy number.

  The DNA construct described in the previous paragraph is transformed into yeast cells using known methods, for example, lithium acetate treatment (Steams et al., Meth. Enz. 185: 280-97 (1990)). The ADH2 promoter is triggered upon depletion of glucose in the growth medium (Price et al., Gene 55: 287 (1987)). The pre-pro-alpha sequence affects the secretion of the fusion protein from the cell. Concomitantly, the yeast KEX2 protein cleaves its pre-pro sequence from the mature PYY analog polypeptide (Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330-4 (1984)).

  The hybrid polypeptides of the invention can also be recombinantly expressed in yeast using a commercially available expression system, such as the Pichia Expression System (Invitrogen, San Diego, California) according to the manufacturer's instructions. . This system also directs secretion depending on the pre-pro-alpha sequence, but transcription of the insertion is performed by the alcohol oxidase (AOX1) promoter based on induction by methanol. The secreted hybrid polypeptide is purified from the yeast growth medium, for example, by the methods used to purify the hybrid polypeptide from bacterial and mammalian cell supernatants.

  Alternatively, cDNA encoding the hybrid polypeptide can be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego, California). This hybrid polypeptide-containing vector is then used according to the manufacturer's instructions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free medium to produce recombinant protein . The protein is purified and concentrated from the medium using a heparin-sepharose column (Pharmacia, Piscataway, New Jersey) and sequential molecular weight sizing columns (Amicon, Beverly, Massachusetts) and resuspended in PBS. By SDS-PAGE analysis, single bond is proved, the size of the protein is confirmed, and N-terminal sequence is confirmed by Edman sequencing with Proton 2090 Peptide Sequencer.

For example, a DNA sequence encoding a hybrid polypeptide can be cloned into a plasmid containing the desired promoter and optionally a leader sequence (see, eg, Better et al., Science 240: 1041-3 (1988)). ). The sequence of this construct can be confirmed by automated base sequencing. The plasmid is then transformed into E. coli, strain MC1061, using standard procedures (Sambrook et al., Supra) utilizing CaCl ₂ incubation and bacterial heat shock treatment. Transformed bacteria are grown in LB medium supplemented with carbenicillin and induced production of expressed protein by growth in an appropriate medium. When present, the leader sequence facilitates secretion of the hybrid polypeptide and will be cleaved during secretion. The secreted recombinant protein is purified from the bacterial medium by the methods described herein.

  Alternatively, the hybrid polypeptides of the invention can be expressed in insect systems. Insect systems for protein expression are well known to those skilled in the art. In one such system, a heterologous gene is expressed in larvae or Trichoplusia larvae using Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. The hybrid polypeptide coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the hybrid polypeptide inactivates its polyhedrin gene, producing a recombinant virus that lacks the coat protein coat. These recombinant viruses are then used to infect S. frugiperda cells or nettle larvae and express hybrid polypeptides in them (Smith et al., J. Virol. 46 : 584 (1983); Engelhard et al., Proc. Natl. Acad. Sci. USA 91: 3224-7 (1994)).

  In another embodiment, the DNA sequence encoding the hybrid polypeptide can be amplified by PCR and cloned into an appropriate vector, such as pGEX-3X (Pharmacia, Piscataway, New Jersey). This pGEX vector is designed to produce the fusion protein containing the glutathione-S-transferase (GST) encoded by the vector and the protein encoded by the DNA fragment inserted into the cloning site of the vector. ing. Primers for PCR can be made, for example, to include appropriate cleavage sites. The recombinant fusion protein can then be cleaved from the GST portion of the fusion protein. The pGEX-3X / PYY analog polypeptide construct was transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla, California), individual transformants were isolated, and in LB medium (supplemented with carbenicillin) Grown at 37 ° C. to an optical density of 0.4 at a wavelength of 600 nm, followed by 4 hours in the presence of 0.5 mM isopropyl β-D-thiogalactopyranoside (Sigma Chemical Co., St. Louis, Missouri) Incubate further. Purification of plasmid DNA from individual transformants and partial sequencing using an automated sequencing instrument to ensure the correct orientation of the gene insert encoding the desired PPF hybrid polypeptide Confirm.

Fusion proteins that are expected to be produced as insoluble inclusion bodies in bacteria can be purified as follows. Cells are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; treated with 0.1 mg / mL lysozyme (Sigma Chemical Co.) for 15 minutes at room temperature. The lysate is clarified by sonication and the cell debris is pelleted by centrifugation at 12,000 xg for 10 minutes. The fusion protein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered with 50% glycerol and centrifuged for 30 minutes at 6000 × g. The pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg ⁺⁺ and Ca ⁺⁺ . The fusion protein is further purified by fractionation of the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., Supra). The gel is soaked in 0.4 M KCl to visualize the protein, which is excised and electroeluted with SDS without gel-running buffer. If the GST / PYY analog polypeptide fusion protein is produced as a soluble protein in bacteria, it can be purified using the GST purification module (Pharmacia Biotech).

  The fusion protein is subjected to digestion to cleave GST from the PPF hybrid polypeptide. The digestion reaction (20-40 μg fusion protein, 20-30 units human thrombin (4000 U / mg (Sigma)) in 0.5 mL PBS) was incubated for 16-48 hours at room temperature and loaded onto a denaturing SDS-PAGE gel. The protein product is visualized by soaking the gel in 0.4 M KCl, and the identity of the protein band corresponding to the expected molecular weight of the hybrid polypeptide is determined by an automated sequencing device (Applied Biosystems Model 473A, Foster City, California) can be confirmed by partial amino acid sequence analysis.

  In a particularly preferred recombinant expression method of the hybrid polypeptide of the invention, 293 cells are obtained by the calcium phosphate method in a pCMV vector (5′CMV promoter, 3′HGH poly A sequence) and pSV2neo (containing a neo resistance gene), Co-transfect with a plasmid containing the hybrid polypeptide cDNA. Preferably, these vectors should be linearized with ScaI prior to transfection. Similarly, alternative constructions using similar pCMVs that incorporate the neo gene can be used. Stable cell lines are selected from single cell clones by limiting dilution in growth medium containing 0.5 mg / mL G418 (neomycin-like antibiotic) for 10-14 days. Cell lines are screened for expression of the hybrid polypeptide by ELISA or Western blot, and highly expressing cell lines are amplified for large scale expansion.

  For long-term, high-yield protein production, it is preferable to use transformed cells, and as such, stable expression is desirable. Once such cells are transformed with a vector containing a selectable marker along with the desired expression cassette, the cells are left to grow for 1-2 days in nutrient enriched media and then switched to selective media. Selectable markers are designed to confer resistance to selection, and their presence allows for the growth and recovery of cells that successfully express the introduced sequence. Resistant clumps of stably transformed cells can be propagated using tissue culture techniques suitable for the cells.

  A number of selection systems can be used to recover the cells that have been transformed for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphororibosyltransferase gene and adenine phosphororibosyltransferase gene in tk-, hgprt- or aprt-cells, respectively. Antimetabolite resistance also dhfr confers resistance to methotrexate; gpt confers resistance to mycophenolic acid; confers resistance to aminoglycosides, G418, and also confers resistance to chlorsulfuron; and resistance to hygromycin Can be used as a selection criterion for hygro. Additional selectable genes that may be useful include trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine. Markers that provide visual indicators for identification of transformants include anthocyanins, β-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

  Many of the hybrid polypeptides of the invention can be produced using a combination of both automated peptide synthesis and recombinant techniques. For example, the hybrid polypeptides of the invention contain a combination of modifications including deletions, substitutions and insertions due to PEGylation. Such hybrid polypeptides can be produced in stages. In the first step, an intermediate polypeptide comprising modifications of deletions, substitutions, insertions and any combination thereof can be produced by recombinant techniques as described. The intermediate polypeptide is then PEGylated by chemical modification with an appropriate PEGylation reagent (eg, from Nektar Therapeutics, San Carlos, California) after an optional purification step as described herein. To yield the desired hybrid polypeptide. To apply to hybrid polypeptides comprising combinations of modifications selected from deletions, substitutions, insertions, inductions and other modification means well known in the art and contemplated by the present invention, the procedures described above are used. Those skilled in the art will understand that generalization is possible.

  It may be desirable to purify the hybrid polypeptide produced by the present invention. Peptide purification techniques are well known to those skilled in the art. These techniques include, at a certain level, a crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. When the polypeptide is separated from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve mild or complete purification (ie, purification to homogeneity). Analytical methods that are particularly suitable for the production of pure peptides are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis and isoelectric focusing. Particularly effective peptide purification methods are the characterization of purified products by reverse phase HPLC followed by liquid chromatography / mass spectrometry (LC / MS) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Further confirmation of purification is achieved by determination of amino acid analysis.

  Certain aspects of the present invention relate to the purification of encoded proteins or peptides, and in certain embodiments, substantial purification. As used herein, the term “purified peptide” is intended to refer to a composition that can be isolated from other components, in which case the peptide is arbitrary based on its naturally obtainable state. It is refined to the extent. Thus, a purified peptide also refers to a peptide in which there is no environment in which it can occur naturally.

  In general, "purified" refers to a peptide composition that has been fractionated to remove various other components that substantially retains its expressed biological activity. Will. When the term “substantially purified” is used, this manifestation indicates that the peptide forms the major component of the composition, eg, the peptide is about about the peptide in the composition. It will refer to a composition comprising 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more.

  Various techniques suitable for use in peptide purification will be well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc .; heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite and affinity chromatography; isoelectric convergence Methods; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, the order in which the various purification steps are performed can be changed, or certain steps can be eliminated, and nevertheless substantially purified protein or It is believed that a suitable method for producing a peptide is obtained.

  It is generally not necessary to always provide the peptides in their most purified state. Indeed, it is believed that in certain embodiments, a product that is not substantially purified will be utilized. Mild purification can be achieved by using fewer purification steps in combination or by utilizing different forms of the same general purification scheme. For example, cation exchange column chromatography performed using an HPLC instrument will generally result in a “-times” better purification than the same technique using a low pressure chromatography system. A method that exhibits a low degree of relative purification may be advantageous in terms of total recovery of the protein product or maintaining the activity of the expressed protein.

  In some cases, such hybrid polypeptides may be purified and isolated from other components obtained in the process. Polypeptide purification methods can be found in US Pat. No. 5,849,883. These references describe specific exemplary methods for the isolation and purification of G-CSF compositions that may be useful for the isolation and purification of the hybrid polypeptides of the invention. From the disclosure of these patents, it is clear that those skilled in the art are well aware of the vast number of purification techniques that can be used to purify hybrid polypeptides from a given source.

It is also considered that the purified hybrid polypeptide composition of the present invention can be produced using a combination of anion exchange chromatography and immunoaffinity chromatography.
Pharmaceutical composition

  The invention includes a pharmaceutically acceptable diluent useful for delivery of the hybrid polypeptides, together with a therapeutically or prophylactically effective amount of at least one hybrid polypeptide of the invention, or a pharmaceutically acceptable salt thereof, It also relates to pharmaceutical compositions comprising preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions have various buffer content (eg Tris-HCl, acetate, phosphate), pH and ionic strength diluents; additives such as surfactants and solubilizers (eg Tween 80 Polysorbate 80), antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, thymelsol, benzyl alcohol), and fillers (eg, lactose, mannitol); polylactic acid, polyglycolic acid, etc. May include the incorporation of materials into particulate formulations of polymeric compounds or in conjunction with liposomes. Such compositions will affect the physical state, stability, in vivo release rate and in vivo clearance rate of the hybrid polypeptide. See, for example, Remington's Pharmaceutical Sciences 1435-712, 18th ed., Mack Publishing Co., Easton, Pennsylvania (1990).

  In general, hybrid polypeptides are as useful as individual component polypeptides are useful in view of their pharmacological properties. One preferred use is the peripheral administration of such hybrid polypeptides for the treatment or prevention of metabolic conditions and metabolic disorders. In particular, the compounds of the present invention have pharmacological activity that reduces nutrient utilization, reduces food intake, suppresses appetite, and causes weight loss. In another embodiment, a preferred use is the administration of such hybrid polypeptides for the treatment of diabetes or diabetes related conditions and abnormalities.

  Hybrid polypeptides may be formulated for peripheral administration, including formulations for injection, oral administration, nasal administration, pulmonary administration, topical administration, or other types of administration that would be known to those skilled in the art. it can. More specifically, administration of the pharmaceutical composition according to the present invention may be via any common route so long as the target tissue is available via that route. In preferred embodiments, the pharmaceutical composition is administered by any conventional peripheral method, for example, intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary (eg, limited time release). By delivery; by oral, sublingual, nasal, anal, vaginal or transdermal delivery; or by surgical implantation at a specific site can be introduced into the subject. This treatment may consist of a single dose or may consist of multiple doses over a period of time. Continuous controlled release of the composition of the present invention is also contemplated.

  The formulation may be a liquid or a solid for reconstitution, such as lyophilized. The aqueous composition of the present invention comprises an effective amount of the hybrid polypeptide dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or cautiously acceptable” refers to molecular units and compositions that do not cause side effects, allergic reactions or other adverse reactions when administered to animals or humans. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, bactericides and fungicides, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional vehicle or drug is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions. In some cases, a hybrid polypeptide of the invention and another dietary intake reduction, diabetes treatment, plasma glucose lowering or plasma lipid modifying agent such as amylin, amylin agonist analogs, CCK or CCK agonists, or leptin Alternatively, it may be convenient to provide a leptin agonist, or exendin or exendin agonist analog, in a single composition or solution for administration together. In other cases, it may be advantageous to administer an additional agent separately from the hybrid polypeptide.

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

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

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form should be sterile and should be fluid to the extent that easy syringability exists. It is also desirable that the hybrid polypeptides of the present invention be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions can be prepared by formulating the required amount of the active compound in a suitable solvent, optionally with various other ingredients listed above, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization resulting in a powder of the active ingredients and any additional desired ingredients from a solution that has been previously sterile filtered It is.

  In general, a therapeutically or prophylactically effective amount of a hybrid polypeptide will be determined by the age, weight and condition of the recipient or the severity of the disease, condition or disorder. See, for example, Remington ’s Pharmaceutical Sciences 697-773. See also Wang and Hanson, Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S (1988). Typically, dosages of between about 0.001 μg / kg body weight / day to about 1000 μg / kg body weight / day can be used, but as will be appreciated by those skilled in the art, higher or lower dosages may be used. It can also be used. Dosing may be more than once a day, or less frequently, as well as with other compositions as described herein. It should be noted that the present invention is not limited to the dosages listed herein.

  Appropriate dosages can be ascertained by using established assays for determining the level of metabolic conditions or metabolic abnormalities with associated dose-response data. The final drug regimen is a factor that modifies the action of the drug, such as specific activity of the drug, severity of injury and patient responsiveness, patient age, condition, weight, gender and diet, any infection The attending physician will take into account the severity, number of doses, and other clinical factors. As studies are conducted, more information will emerge regarding appropriate dosage levels and duration of treatment for specific diseases and conditions.

  Effective doses administered in a single or divided dose for a 50 kg patient are generally about 1 to 30 μg to about 5 mg / day, preferably about 10 to 30 μg to about 2 mg / day, more preferably about 5 From about 100 μg to about 1 mg / day, most preferably from about 5 μg to about 500 μg / day. Preferably, the dosage is between about 0.01 and about 100 μg / kg / dose. The exact dose to be administered can be determined by one skilled in the art and will depend on the potency of the individual compounds and the age, weight and condition of the individual. Administration is, for example, whenever suppression of nutrient utilization, adjustment of dietary intake, body weight, blood glucose or plasma lipids is desired, eg at the first sign of symptoms or immediately after diagnosis, eg obesity, diabetes or insulin resistance syndrome Should start. Administration can be by any route, eg, injection, preferably subcutaneous or intramuscular injection, oral route, nasal route, transdermal route, and the like. Dosages for certain routes, such as oral administration, may be increased by, for example, about 5-100 fold, taking into account reduced bioavailability.

  Parenteral administration can be done initially with a bolus and then with continuous infusion to maintain therapeutic circulating levels of the drug. Those of ordinary skill in the art will readily optimize effective dosages and dosing schedules as judged by sufficient medical experience and clinical status of the individual patient.

  The frequency of dosing will depend on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation will be determined by one skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, (supra), pages 1435-1712. Such formulation can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the administered drug. Depending on the route of administration, a suitable dose can be calculated according to body weight, body surface area or organ size. Further improvements in the calculations necessary to determine an appropriate therapeutic dose are particularly excessive in light of the dosage information and assays disclosed herein and the pharmacokinetic data observed in animal or human clinical trials. It is carried out routinely by a person skilled in the art without experimentation.

  It will be appreciated that the pharmaceutical compositions and methods of treatment of the present invention may be useful in the fields of human medicine and veterinary medicine. Thus, a subject that can be treated can be a mammal, preferably a human or other animal. Subjects for veterinary use include, for example, domestic animals including cattle, sheep, pigs, horses and goats, pets such as dogs and cats, foreign and / or zoo animals, mice, rats, rabbits, Laboratory animals including guinea pigs and hamsters and poultry such as chickens, turkeys, ducks and geese.

  In addition, the present invention provides a hybrid polypeptide of the present invention, a component suitable for the production of the hybrid polypeptide of the present invention for pharmaceutical use, and the use of the hybrid polypeptide and component for pharmaceutical use. Intended for kits containing instructions for use.

  To assist in understanding the present invention, the following examples are included. Experiments relating to the present invention should, of course, not be construed as specifically limiting the present invention, and variations of the present invention as currently known or later developed will be within the scope of those skilled in the art. It is considered that the invention falls within the scope of the invention as described herein and as set forth in the claims below.

  The invention will now be described in further detail with reference to the following non-limiting examples. These examples are provided to more fully illustrate the present invention and should not be construed as limiting the scope of the invention. These examples illustrate the production of the present hybrid polypeptides and the in vitro and / or in vivo testing of these hybrid polypeptides of the present invention. The techniques described in these examples are representative of the techniques described by the inventors to function well in the practice of the invention and are therefore preferred mode components for the practice of the invention. It will be understood by those skilled in the art. However, one of ordinary skill in the art, in view of the present disclosure, may make many modifications to the specific methods disclosed without departing from the spirit and scope of the present invention and still obtain similar or similar results. It will be understood that it should be understood.

Example 1
Preparation of Hybrid Polypeptides The peptides of the present invention are Rink amide resin (Novabiochem), or preloaded Wang Resin (Fmoc-Tyr (tBu) -Wang resin) at a loading of 0.43-0.49 mmol / g at 0.050-0.100 mmol. It can be assembled on a Symphony peptide synthesizer (Protein Technologies, Inc.) using 0.63 mmol / g (Novabiochem). Fmoc amino acid (5.0 equivalents, 0.250-.500 mmol) residues are dissolved in 1-methyl-2-pyrrolidinone at a concentration of 0.10M. All other reagents (HBTU, 1-hydroxybenzotriazole hydrate and N, N-diisopropylethylamine) are prepared as 0.55M dimethylformamide solutions. Then use HBTU (2.0 eq, 0.100-0.200 mmol), 1-hydroxybenzotriazole hydrate (1.8 eq, 0.090-0.18 mmol), N, N-diisopropylethylamine (2.4 eq, 0.120-0.240 mmol). And couple the Fmoc protected amino acid to the resin bound amino acid for 2 hours. After coupling of the last amino acid, the peptide is deprotected using 20% (v / v) piperidine in dimethylformamide for 1 hour. Once the peptide sequence is complete, the Symphony peptide synthesizer is programmed to cleave the resin. Trifluoroacetic acid (TFA) cleavage of the peptide from the resin is performed using 93% TFA, 3% phenol, 3% water and 1% triisopropylsilane for 1 hour. The cleaved peptide is precipitated using t-butyl methyl ether, pelleted by centrifugation and lyophilized. The pellet is redissolved in water (10-15 mL), filtered, and purified by reverse phase HPLC using a C18 column and an acetonitrile / water gradient containing 0.1% TFA.

The general procedure for N-capping peptides of the invention with fatty acids (eg, octanoic acid and stearic acid) is as follows: Suspend peptide (0.1 mmol) on rink amide resin in NMP (5 mL) Let In a separate vial, HBTU (0.3 mmol), HOBt (0.3 mmol) are dissolved in DMF (5 mL) and then DIEA (0.6 mmol) is added. This solution is added to the resin and the suspension is shaken for 2 hours. The solvent is filtered, washed thoroughly with NMP (5 mL × 4) 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.

  Commercially available activated PEG esters can be used in solution to effect PEG modifications on the free epsilon-amino group of lysine or the terminal amino group of the purified peptide. The resulting PEGylated derivative is purified to homogeneity by reverse phase HPLC and its purity is confirmed by LC / MS and MALDI-MS.

Certain exemplary hybrid polypeptides of the invention are shown in Table 1-1. Various modifications to the working compound are contemplated, such as chemical modifications such as glycosylation, PEG modifications, etc .; amino acid modifications such as substitutions, insertions and deletions. Further, it is interpreted that the hybrid polypeptides of the invention can alternatively be in the free acid form, even though the C-terminus is expressed as amidated.
Table 1-1: Certain exemplary hybrid compounds of the invention

Additional exemplary hybrids containing various combinations of component peptide families can be found in the following table. Those analogs and derivative hybrids as discussed herein are also specifically contemplated. The symbol “#” indicates a connection position for each component. The linkers shown are as described herein. CCK-8 is DY (SO3H) MGWMDF-NH2, whereas "CCK-8 ^* " is sulfated phenylalanine form DF (CH2SO3H) MGWMDF-NH2.

(Example 2)
Binding Assays The hybrid polypeptides of the invention can be tested in various receptor binding assays using binding assay methodologies generally known to those skilled in the art. Such assays include those described herein.

Amylin binding assay : Assessment of the binding of some exemplary compounds of the invention to the amylin receptor can be performed on the nucleus accumbens membrane prepared from rat brain as follows. Male Sprague-Dawley® rats (200-250) grams are sacrificed by decapitation. The brain is removed and placed in cold phosphate buffered saline (PBS). An incision is made from the abdominal surface to the rostral side of the hypothalamus, with the olfactory tracts as lateral borders, extending from these olfactory tracts at an internal 45 ° angle. This basal forebrain tissue, including the nucleus accumbens and surrounding area, is weighed and homogenized in ice-cold 20 mM HEPES buffer (20 mM HEPES acid, pH adjusted to 7.4 with NaOH at 23 ° C.). Wash the membrane three times in fresh buffer by centrifugation at 48,000 xg for 15 minutes. The final membrane pellet is resuspended in 20 mM HEPES buffer containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF).

To measure ¹²⁵ I-amylin binding (see Beaumont K et al. Can J Physiol Pharmacol. 1995 Jul; 73 (7): 1025-9), a membrane from 4 mg raw wet weight tissue was 0.5 mg / mL. Incubate with 12-16 pM of ¹²⁵ I-amylin in 20 mM HEPES buffer containing 10 mg of bacitracin, 0.5 mg / mL bovine serum albumin and 0.2 m PMSF. The solution is incubated for 60 minutes at 2 ° C. To reduce non-specific binding of radiolabeled peptide, the incubation was performed by filtration through a GF / B glass fiber filter (Whatman Inc., Clifton, NJ) pre-soaked in 0.3% polyethyleneimine for 4 hours. End. The filter is washed with 5 mL cold PBS immediately before filtration and with 15 mL cold PBS immediately after filtration. Remove the filter and assay for radioactivity in a gamma-counter with 77% counting efficiency. Competitive curves are generated by identifying binding in the presence of 10 ^-12 to 10 ^-6 M unlabeled test compound and analyzed by non-linear regression using a 4-parameter theory (Inplot program; GraphPAD Software, San Diego) .

CGRP Receptor Binding Assay : Assessment of binding of compounds of the present invention to the CGRP receptor is based on SK-N-MC cells known to express the CGRP receptor (Muff, R. et.al., Ann NY Acadine Sci. 1992: 657, 106-16) Except for the use of membranes, essentially as described for amylin. The binding assay is performed as described for amylin, except using 13,500 cpm 125 I-hCGRP / well or 21.7 pM / well (Amersham).

Adrenomedullin binding assay : Binding to the adrenomedullin receptor was performed using HUVEC containing the adrenomedullin receptor (Kato J et.al., Eur J Pharmacol. 1995, 289: 383-5). The Perkin Elmer AlphaScreen ™ assay for click AMP can be used to investigate using optimal conditions of 25-30,000 cells per well. The assessment of cAMP levels for HUVEC is not great compared to CHO cells. Therefore, CHO cells can be chosen as a negative control. This is because CHO cells do not express the adrenomedullin receptor as desired.

Calcitonin receptor binding assay : Binding to calcitonin receptor can be investigated using CHO cells or T47D cells, as known in the art, and these cells also express calcitonin receptor ( Muff R. et.al, Ann NY Acad Sci. 1992, 657: 106-16 and Kuestner RE et. Al. Mol Pharmacol. 1994, 46: 246-55).

Leptin binding assays : Two in vitro bioassays are routinely used to assess leptin binding and receptor activation (eg, White, et al., 1997. Proc. Natl. Acad. Sci. USA 94: 10657- 10662). Long (signaling) form in the absence or presence of recombinant mouse leptin (positive control) or peptide using alkaline phosphatase ("AP")-leptin ("OB") fusion protein ("AP-OB") Inhibition of leptin binding by COS-7 cells transfected with the mouse OB receptor ("OB-RL") can be measured. Signaling assays can be performed in GT1-7 cells co-transfected with AP reporter and OB-RL construct. Alkaline phosphatase (“SEAP”) activity secreted in response to stimulation with mouse leptin or peptide can be measured by chemiluminescence.

Y1 receptor binding assay : Membranes are made from confluent cultures of SK-N-MC cells that endogenously express the neuropeptide Y1 receptor. Membranes are incubated in a 96-well polystyrene plate with 60 pM [ ¹²⁵ I] -human peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences) and with unlabeled active compound for 60 minutes at ambient temperature. The well contents are then collected into 96-well glass fiber plates using a Perkin Elmer plate harvester. The dried glass fiber plate is combined with scintillant and counted in a Perkin Elmer scintillation counter.

Y2 receptor binding assay : Membranes are made from confluent cultures of SK-N-BE cells that endogenously express the neuropeptide Y2 receptor. Membranes are incubated in a 96-well polystyrene plate with 30 pM [ ¹²⁵ I] -human peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences) and with unlabeled active compound for 60 minutes at ambient temperature. The well contents are then collected on a 96-well glass fiber plate using a Perkin Elmer plate harvester. The dried glass fiber plate is combined with scintillant and counted in a Perkin Elmer scintillation counter.

Y4 receptor binding assay : CHO-K1 cells are transiently transfected with cDNA encoding the neuropeptide Y4 gene and, 48 hours later, membranes are made from confluent cell cultures. Membranes are incubated in 96-well polystyrene plates with 18 pM [ ¹²⁵ I] -human pancreatic polypeptide (2200 Ci / mmol, PerkinElmer Life Sciences) and with unlabeled active compound for 60 minutes at ambient temperature. The well contents are then collected on a 96-well glass fiber plate using a Perkin Elmer plate harvester. The dried glass fiber plate is combined with scintillant and counted in a Perkin Elmer scintillation counter.

Y5 receptor binding assay : CHO-K1 cells are transiently transfected with cDNA encoding the neuropeptide Y5 gene, and after 48 hours, membranes are made from confluent cell cultures. Membranes are incubated in a 96 well polystyrene plate with 44 pM [ ¹²⁵ I] -human peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences) and with active compound for 60 minutes at ambient temperature. The well contents are then collected on a 96-well glass fiber plate using a Perkin Elmer plate harvester. The dried glass fiber plate is combined with scintillant and counted in a Perkin Elmer scintillation counter.

GLP-1 receptor binding assay : GLP-1 receptor binding activity and affinity may be measured using a binding displacement assay where the source of the receptor is RINm5F cell membrane and the ligand is [ ¹²⁵ I] GLP-1. it can. Homogenized RINm5F cell membranes are incubated for 2 hours at 23 ° C. with constant mixing with 40,000 cpm [ ¹²⁵ I] GLP-1 tracer and various concentrations of test compound in 20 mM HEPES buffer. Filter the reaction mixture through a glass filter pad pre-wetted with 0.3% PEI solution and rinse with ice-cold phosphate buffered saline. Determine the binding count using a scintillation counter. Binding affinity is calculated using GraphPad Prism software (GraphPad Software, Inc., San Diego, Calif.).

In vitro receptor binding and receptor activation, including specific receptor activation of component modules, for exemplary hybrids is shown in the following table.

(Example 3)
Mouse Dietary Intake Assay The hybrid polypeptides of the invention can be tested for appetite suppression in a mouse dietary intake assay and for their effect on weight gain in diet-induced obese (DIO) mice. The experimental protocol for screening is described below.

  Female NIH / Swiss mice (8-24 weeks old) are housed in a 12:12 hour light: dark cycle that lights at 06:00. Water and standard pelleted chow chow diet can be freely fed unless otherwise noted. One day before the experiment, start fasting the animals at approximately 15:00. On the morning of the experiment, animals are divided into experimental groups. In a typical study, n = 4 cages with 3 mice / cage.

At time = 0, all animals are given an intraperitoneal injection of vehicle or compound, typically in an amount ranging from about 10 nmol / kg to 75 nmol / kg, and a pre-weighed amount (10-15 g) standard. Give Chau immediately. Food is removed and weighed at various time points, typically 30, 60 and 120 minutes to determine the amount of food consumed (Morley, Flood et al., Am. J. Physiol. 267: R178-R184, 1994). Food intake is calculated by subtracting the weight of food remaining at the time of, for example, 30, 60, 120, 180 and / or 240 minutes from the weight of food initially fed at time = 0. Significant therapeutic effects are identified by ANOVA (p <0.05). If there is a significant difference, the test mean is compared to the control mean using Dunnett's test (Prism v. 2.01, GraphPad Software Inc., San Diego, California).
The sequences of the parent molecules used for activity and hybrid synthesis in the food intake assay here are as follows:

(Example 4)
Body weight, fat redistribution, and lean body mass assay Assays for body weight and related effects can be performed as follows.

  Diet-induced obesity (DIO) in Sprague-Dawley rats is a model available for the study of obesity and energy homeostasis regulation. These rats were grown from a strain of rats prone to obesity (Crl: CD® (SD) BR) using diets with relatively high fat and energy. See, for example, Levin (1994) Am. J. Physiol. 267: R527-R535, Levin et al. (1997) Am. J. Physiol. 273: R725-R730. DIO male rats are obtained from Charles River Laboratories, Inc. (Wilmington, MA). Rats are individually housed in a shoebox cage with a light / dark cycle of 12/12 hours at 22 ° C. Rats are kept on a free diet using a moderately high fat diet (32% kcal from fat; Research Diets D1226B). Animals typically reach an average weight of about 500 g. Acclimate LevinDIO rats to cage rearing for 7 days. During three nights of acclimatization, animals receive a single intraperitoneal (IP) injection of vehicle. On the day of the test, rats receive a single IP injection of compound or vehicle (10% DMSO) at the start of the dark cycle over 6-14 consecutive nights. Food intake is measured at 5-second intervals throughout the course of the study by an automated food intake measurement system (BioDAQ, Research Diets). Body weight is recorded every night.

  Body composition was measured before and after treatment using NMR (Echo Medical Systems, Houston, TX). For body composition measurements, rats were briefly placed (about 1 minute) into a well-ventilated plexiglas tube, after which it was inserted into a dedicated rodent NMR machine. This is a calculation of the exact gram change in fat and dry lean tissue (eg, gram of body fat after treatment-grams of body fat at baseline = change in grams of body fat) and fat And calculation of% change in body composition for dry lean tissue (eg% body fat after treatment-% body fat at baseline =% change in body fat). All data are expressed as mean ± SEM. We tested for differences between groups using analysis of variance (ANOVA) and multiple comparison tests. P-value <0.05 was considered significant. Statistical analysis and graphing were performed using PRISM® 4 for Windows (GraphPad Software, Inc., San Diego, Calif.). In some early studies, SYSTAT® for Windows (Systat Software, Inc., Point Richmond CA) was used for analysis. For hybrids containing amylin mimics, exendins, PYY analogs and / or leptin, changes in body fat were not accompanied by a significant reduction in lean tissue. Graphs and results are typically presented as vehicle corrected changes in weight percent, changes in body fat and body protein.

  In other experiments, male C57BL / 6 mice (4 weeks of age at the start of the study) were fed high-fat chow (HF, 58% of dietary kcal as fat) or low-fat chow (LF, 11% of dietary kcal as fat). Feed. After 4 weeks using chow, each mouse is implanted with an osmotic pump (Alzet # 2002) that delivers subcutaneously a predetermined dose of hybrid polypeptide continuously for 2 weeks. Body weight and food intake are measured weekly (Surwit et al., Metabolism-Clinical and Experimental, 44: 645-51, 1995). The effect of the test compound is expressed as the mean +/− sd of% body weight change (ie% change from starting body weight) of at least 14 mice per treatment group (p <0.05 ANOVA, Dunnett's test, Prism v. 2.01, GraphPad Software Inc., San Diego, California).

Exendin / PYY hybrid . C-terminal truncated exendins (eg, exendin-4 (1-28) or ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-exendin-4 (1-28)) and N-terminal truncated forms spanning the 18-36 region to the 31-36 region PYY was used to synthesize exemplary hybrid polypeptides of the invention. Thus, these exemplary hybrid polypeptides generally comprise two modules, where the first module is an exendin-4 analog and the second module is a peptide selected from a PYY truncation Enhancer. For comparison, β-alanine dipeptide spacer was also incorporated between the peptide building blocks in some variants (see Table 4-1).
Table 4-1: Exendin / PYY hybrids, receptor binding data, and their effects in food intake assays

  As shown in Table 4-1, certain exemplary compounds of the present invention have shown efficacy in food intake assays. Certain peptides were tested even at 75 nmol / kg in the DIO assay and proved to be more potent than PYY (Figure 1). As observed for other hybrids here, the hybrid can retain binding to one, two or more receptors that recognize the parent molecule. If desired, hybrids were designed that recognize at least one receptor from each parent or from only one parent. As observed for other hybrids here, the use of a linker (which can serve as a spacer between each adjacent hormone moiety) allows for receptor (s) binding as well as in vitro and in vivo activity, such as body weight. An increase in activity, including a decrease, can be brought about. The results here show that the C-terminal part of PYY can modify the activity.

Exendin / amylin hybrid . Exendin / amylin hybrid. Further exemplary hybrid polypeptides of the invention include C-terminal truncated exendin (1-27) (SEQ ID NO: 236), C-terminal truncated amylin peptide (eg, amylin (1-7) (SEQ ID NO: 217) ^2,7 Ala-amylin (1-7) (SEQ ID NO: 285) and amylin (33-27) (SEQ ID NO: 243)), and optional sCT fragments (eg, sCT (8-10), ^{11 , 18 Arg-sCT (8-27)} ( SEQ ID NO: 289) and ^{14 Glu, 11,18 Arg-sCT (} 8-27) ( SEQ ID NO: 286). was prepared from both hybrid polypeptides, anorectic It was very active in terms of (see Table 4-2) and was superior to the same dose of rat amylin, but the onset of action was different from the activity profile of the parent molecule (data not shown). At a dose of / kg, Compound 5 was as effective as rat amylin.
Table 4-2: Exendin / amylin hybrids and their effects in FI assays

  Both compounds showed excellent efficacy when screened in the DIO assay (Figure 2).

Additional exemplary compounds were assayed in a food intake assay for effects on blood glucose levels. These tests included compounds 14 and 15. Compound 14 comprising beta Ala linker, ²⁹ βAla-βAla- exendin (1-28), h amylin ^{(1-7) - 11,18 Arg-sCt} (8-27) -h -amylin (33-37) (SEQ ID NO: 33), (or exendin (1-28) - is described as ^11,18 Arg-sCt (8-27) -h-amylin (33-37) - βAla-βAla- h -amylin (1-7) ), and on the other hand, compound 15 contains a Gly linker: ²⁹ GlyGlyGly- exendin (1-28), h amylin ^{(1-7) - 11,18 Arg-sCt} (8-27) -h -amylin (33 -37) (SEQ ID NO: 312). Longer exendin (1-28) resulted in increased activity compared to exendin (1-27).

  A blood glucose assay was performed to test the effect on blood glucose reduction. Female NIM / Swiss mice (8-20 weeks of age) were housed in a 12:12 hour light: dark cycle that lights at 06:00. Water and standard pelleted chow chow diets were available for free except where noted. On the morning of the experiment, the animals were divided into experimental groups and started fasting at approximately 06:30. In a typical study, n = 2 cages with 3 mice / cage. At time = 0, blood glucose samples were taken and immediately followed by intraperitoneal injections of vehicle or compounds in amounts ranging from about 1 nmol / kg to 25 nmol / kg. Blood glucose was measured at 30, 60, 120, 180 and 240 minutes. For example, the percent pre-treatment was calculated by dividing blood glucose at 30, 60, 120, 180 and / or 240 minutes and blood glucose at time = 0 minutes. Significant therapeutic effects were identified by ANOVA (p <0.05). If there was a significant difference, the test mean was compared to the control mean using Dunnett's test (Prism v. 4.01, GraphPad Software Inc., San Diego, California). Results for exemplary compounds are presented in FIG. 5A. Points represent mean ± sd. NIH / Swiss mice fasted for 2 hours were injected intraperitoneally (IP) with the peptide at time t = 0 immediately after baseline sampling. Samples were taken at t = 30, 60, 120, 180 and 240 minutes. Blood glucose was measured with OneTouch® Ultra® (LifeScan, Inc., a Johnson & Johnson Company, Milpitas, Calif.). * p <0.05 vs vehicle control; ANOVA, Dunnett test.

  The food intake assay was performed as previously described herein. Results are presented in FIG. 5B. Points represent the mean ± sd of n = 4 cages (3 mice / cage). NIH / Swiss mice fasted overnight were injected intraperitoneally (IP) with the peptide at time t = 0. Immediately after injection, food was introduced and consumption was measured at t = 30, 60 and 120 minutes. * p <0.05 vs vehicle control; ANOVA, Dunnett test.

  Parent compound 10 and exendin compounds have opposite results in this blood glucose assay. Together they are expected to result in neutralization or, at best, dilution of the effects seen with the more potent parent compound. However, in this blood glucose assay, the exemplary hybrid compound was just as potent as exendin (1-28) and had a longer duration of action.

  From dietary intake data, these exemplary compounds were anorexic. The activity was generally better than the parent compound administered individually. The activity was comparable to the parent compound administered together but at half drug concentration (3 nmol / kg hybrid vs. 6 nmol / kg co-administered parent compound), thus further demonstrating that the hybrid is superior. . The addition of a linker increased the activity of these hybrids. In this case, the Gly-Gly-Gly linker was more effective than the beta Ala-beta Ala linker.

Exendin / CCK-8 hybrid . Still further exemplary hybrid polypeptides of the invention were made from full-length or C-terminal truncated exendin-4 attached directly or via a linker to the N-terminus of CCK-8, retaining the N-terminal amide of CCK-8. In addition, certain hybrids incorporating naturally occurring Tyr (SO ₃ ) were made, while other hybrids incorporating more stable Phe (CH ₂ SO ₃ ) groups were also made. All of the prepared hybrid polypeptides were active in suppressing food intake (Table 4-3).
Table 4-3: Exendin / CCK-8 hybrids and their effects in food intake assays

Exemplary exendin / CCK-8 hybrid polypeptides were tested at 25 nmol / kg in the DIO assay (FIGS. 3A and 3B). The data showed initial weight loss followed by a rebound effect for all compounds. Interestingly, this rebound effect is due to a more hydrolytically stable hybrid incorporating Phe (CH ₂ SO ₃ ) residues (compare FIGS. 3A and 3C), as well as a linker between exendin and CCK residues, For example, it appears to be reduced for hybrids incorporating the linker 8-amino-3,6-dioxaoctanoyl (compare FIGS. 3A and 3B). Ten times higher amounts of CCK-8 (250 nmol / kg / day) were required to produce a change of about -2.8% on day 2 and rebounded to the HF diet control level on day 7.

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

  In order to ascertain whether the exemplary hybrid polypeptides of the invention are more potent than their parent component peptide hormones, the exemplary compounds were tested at the least effective dose of the more active parent molecule in a food intake assay. The results are shown in FIGS. 4A and 4B. This also compares the effects of the pooled parent peptide (compounds 1, 11 and 12 are component peptide hormones, analogs or fragments thereof). The data shows that some peptides are at least as potent as the pooled parent peptide. In parallel with in vivo studies, in vitro receptor binding and functional assays (cyclase activity) were performed for all compounds (data not shown).

Amylin-sCT / leptin hybrid . A further exemplary hybrid was made containing compound 10, a leptin peptide fragment linked to an amylin-sCT-amylin chimera as described herein. Compound 16, [Ser117, dLeu119] Leptin (116-122) - amylin (33-37) (SEQ ID NO: 397) amylin ^{(1-7) - - [11,18 Arg} ] sCT (8-27) is . The compound bound to amylin and CT receptor (RBA = receptor binding assay) and some bound to CGRP receptor. This compound was also able to activate the CT receptor (C1A assay).

This representative molecule was tested for activity in a food intake assay as described herein. Leptin was not active in this assay, but compound 16 was anorexic at 1 mg / kg. Compound 16 was also superior to rat amylin (at 25 mg / kg) in its anorexia-inducing effect. Compound 10 reduced food intake by 91-95% much more effectively than control, while compound 16 reduced cumulative intake to 34-38% of control. Additional exemplary embodiment, N-terminal and amylin leptin peptide ^{(1-7) - [11,18 Arg]} sCT (8-27) - as head coupling - head and amylin (33-37) Compound .

CCK / amylin-sCT hybrid . An exemplary hybrid of CCK and amylin-sCT chimera demonstrated reasonable receptor specificity and activation. An exemplary compound having the sequence DF (P-CH ₂ SO ₃ ) MGWMDFGKR KCNTATCATQRLANELVRLQTYPRTNVGSNTY (SEQ ID NO: 398) is 0.044 nM in CT receptor binding assay, 4.4 nM in CGRP receptor binding assay, 0.083 in amylin receptor binding assay. An IC50 of 1000 nM was demonstrated in the nM and GLP receptor cyclase (RIN) assays.

(Example 5)
Reduced Dietary Intake, Body Weight and Weight Gain Exemplary hybrid polypeptides of the invention were assayed for appetite suppression, weight loss and weight gain reduction in mice and rats as described herein. The parent compound was also assayed alone or in combination. Figures 9-21 demonstrate the in vivo effect of an exemplary hybrid in an assay as described herein.

The in vivo activity of food intake suppression in mice for exemplary hybrids and their parent compounds is shown in the following table. Values, if provided, are provided as percent inhibition of food intake after infusion for the indicated period. “Ns” means “not significant” compared to vehicle.

The in vivo activity of weight suppression in the mouse DIO model for exemplary hybrids and their parent compounds is shown in the following table. Values, if provided, are provided as percent inhibition of body weight after infusion for the indicated period.

The in vivo activity for weight suppression in the rat DIO model for exemplary hybrids and their parent compounds is shown in the following table. Values, if provided, are provided as percent inhibition of body weight after infusion for the indicated period.

The in vivo activity for exemplary hybrids and their parent compounds for suppression of body weight in a rat model (daily injection) is shown in the following table. Values are given as percent inhibition of body weight. Bold values indicate significance from vehicle.

Of reduced body weight and weight gain for the exemplary hybrids and their parent compounds with respect to weight suppression in a rat model for both infusion over the indicated period and once-daily injection over the indicated period In vivo activity is shown in the following table. Values are given as percent inhibition of body weight. The percent inhibition of food intake for the 8 hour night intake period on day 6 was also measured. Hybrids were supplied at 15 nmol / kg / day unless otherwise noted. Body weight: The difference of about 2% is statistically significant. Dietary intake: The difference of about 20% is statistically significant.

(Example 7)
Hypoglycemia
The in vivo activities of exemplary hybrids and their parent compounds for hypoglycemia in the OGTT assay for the indicated duration in Levin rats are shown in the following table. The value is the blood sugar level

Give as a percentage drop. Bold values indicate significance from vehicle.

(Example 8)
Gastric emptying activity The in vivo activities of exemplary hybrids and their parent compounds for gastric emptying in Levin rats for the indicated periods are shown in the following table. Values are given as percent inhibition of gastric emptying and percent acetaminophen passed.

Example 9
PPF Polypeptide Suppresses Acute and Chronic Food Intake Female NIH / Swiss mice (8-24 weeks of age) were reared in a 12:12 hour light: dark cycle at 06:00. Water and standard pelleted chow chow diet were free to eat except where noted. One day before the experiment, the animals started fasting at approximately 15:00. On the morning of the experiment, the animals were divided into experimental groups. In a typical study, n = 4 cages with 3 mice / cage.

  At time = 0, all animals are given an intraperitoneal injection of vehicle or compound in an amount ranging from about 10 nmol / kg to 100 nmol / kg and immediately receive a pre-weighed amount (10-15 g) of standard chow. Gave. Food was removed and weighed at 30, 60 and 120 minutes to determine the amount of food consumed (Morley, Flood et al., Am. J. Physiol. 267: R178-R184, 1994). To test the acute effect of a PPF polypeptide on food intake, subtract the weight of the remaining food at 30, 60, 120 and 180 minutes from the weight of the food initially fed at time = 0 The food intake was calculated by Similarly, water intake was calculated by subtracting the weight in grams of water remaining at these time points from the weight of water in grams initially supplied. To test the effect of PPF polypeptides on long-term (chronic) food intake and / or body composition, the amount of food consumed over a two-week period was measured. For these studies on the effect of PPF polypeptides on long-term food intake and / or body composition, mice were individually housed for 1 week prior to the start of treatment. Throughout this experiment, food intake and body weight were monitored daily. During the treatment period, vehicle (50% dimethyl sulfoxide in water) and PYY (3-36) (1 mg / kg / day) were placed in the intrascapular region under isoflurane anesthesia with an Alzet® osmotic pump (Durect Corp., Cupertino, CA; for the 3, 7 and 28 day studies, administration was by subcutaneous (sc) continuous infusion using Models 1003D, 2001 and 2004, respectively. At the end of each study, animals were sacrificed by isoflurane overdose after 2-4 hours of fasting. Blood was collected by cardiac puncture into a syringe flushed with Na-heparin, and the plasma was immediately frozen. In some studies, body composition was determined using a double X-ray energy absorption assay (DEXA; PixiMus, GE Lunar). In some studies, body composition (eg, fat and protein levels) was measured before and after treatment using a rodent NMR machine (EchoMRI-700 ™). In this case, the animal is placed in a restraint tube and placed in its NMR for 2 minutes to quantify the amount of fat and lean body mass in grams. Bilateral epididymal fat pads and intrascapular brown adipose tissue (BAT) can be excised from the animals to determine the weight of these organs. The extracted liver sample can be placed in RNALater (Ambion, Austin, TX) and stored at -20 ° C.

  FIGS. 22-29 show the ability of PPF polypeptide chimeras, particularly PPY-NPY chimeras such as compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described above.

  FIG. 22 shows the reduction in cumulative food intake over 3 hours based on administration of Compound 5705 compared to vehicle alone. Figures 23A and 24A show changes in food intake (grams), and Figures 23B and 24B show continuous vehicle or compound 4883 or 5705 (at a dose of 500 μg / kg / day) for 13 days. 2 shows changes in body weight (changes in vehicle-corrected body weight percentage) in mice administered to 1). Animals treated with Compound 4883 or Compound 5705 appear to have a tendency for food intake and weight loss as early as a day after the start of PPF polypeptide administration.

  Figures 25 and 26 show daily water intake based on administration of Compound 4883 or Compound 5705 in mice continuously administered vehicle or Compound 4883 or 5705 (at a dose of 500 μg / kg / day) for 13 days. And changes in urinary excretion.

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

  FIG. 29 shows the effect of 14 days of continuous administration of compound 4883 or 5705 (at a dose of 500 μg / kg / day) on urinary electrolytes.

  While the invention has been described in terms of preferred examples and embodiments, those skilled in the art will appreciate that variations and modifications will occur. Accordingly, the appended claims are intended to be construed as including all such equivalent variations that fall within the scope of the invention as defined by the claims.

FIG. 2 demonstrates the effect of exemplary compounds of the invention in the DIO mouse assay. FIG. 2 demonstrates the effect of exemplary compounds of the invention in the DIO mouse assay. FIG. 2 demonstrates the effect of exemplary compounds of the invention in the DIO mouse assay. FIG. 2 demonstrates the effect of exemplary compounds of the invention in the DIO mouse assay. FIG. 2 demonstrates the effect of exemplary compounds of the invention in the DIO mouse assay. FIG. 3 demonstrates the effect of exemplary compounds of the invention in a food intake assay compared to a parent peptide compound. FIG. 3 demonstrates the effect of exemplary compounds of the invention in a food intake assay compared to a parent peptide compound. FIG. 3 demonstrates the effect of exemplary compounds of the invention in a hypoglycemic assay and a food intake assay, respectively. FIG. 3 demonstrates the effect of exemplary compounds of the invention in a hypoglycemic assay and a food intake assay, respectively. FIG. 3 shows the structure of various exemplary linkers used herein. FIG. 6 shows the structure of various exemplary linkers, as well as the sequence and in vitro activity of exemplary PYY and amylin family hybrids. FIG. 3 shows the structure of various exemplary linkers, as well as the sequence and in vitro activity of additional exemplary PYY / PYY family hybrids. FIG. 6 shows weight loss by exemplary PYY and amylin family hybrids. Figure 2 is a graph of calcium mobilization by CCK8 family hybrids in cell lines with CCK receptors. FIG. 5 shows diet suppression by amylin, such as amylin-sCT-amylin, and PYY (eg, PYY-NPY chimera) family hybrids. FIG. 5 shows diet suppression by amylin, such as amylin-sCT-amylin, and PYY (eg, PYY-NPY chimera) family hybrids. FIG. 5 shows diet suppression by amylin, such as amylin-sCT-amylin, and PYY (eg, PYY-NPY chimera) family hybrids. FIG. 12A shows a decrease in total food intake by amylin-sCT-amylin / PYY family hybrid, and FIG. 12B shows a decrease in body weight. “ ^* ” Indicates P <0.05 compared to vehicle. FIG. 12A shows a decrease in total food intake by amylin-sCT-amylin / PYY family hybrid, and FIG. 12B shows a decrease in body weight. “ ^* ” Indicates P <0.05 compared to vehicle. It is a figure which shows suppression of the food intake by PYY / PYY family hybrid. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. It is a figure which shows suppression of the food intake activity of the CKK family hybrid which has either amylin or a PYY family component. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. It is a figure which shows suppression of the food intake activity of the CKK family hybrid which has either amylin or a PYY family component. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. It is a figure which shows suppression of the food intake activity of MSH family hybrid which has either amylin or a PYY family component. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. It is a figure which shows suppression of the food intake activity of MSH family hybrid which has either amylin or a PYY family component. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. FIG. 16A shows a decrease in total food intake by a hybrid with an MSH family component and amylin, eg, amylin-sCT-amylin, family component, and FIG. 16B shows a decrease in body weight. “ ^* ” Indicates P <0.05 compared to vehicle. FIG. 16A shows a decrease in total food intake by a hybrid with an MSH family component and amylin, eg, amylin-sCT-amylin, family component, and FIG. 16B shows a decrease in body weight. “ ^* ” Indicates P <0.05 compared to vehicle. FIG. 3 shows suppression of food intake by a hybrid containing the FN-38 family component and the indicated second family component. FIG. 17A-FN38 and amylin family components, amylin-sCT-amylin chimeric compound 10. FIG. 17B--FN38 and PYY family components, PYY-3-36. FIG. 17C--FN38 and PYY family components, PYY-NPY chimera. Figure 17D--FN38 and CCK8 family components, CCK8. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. FIG. 3 shows suppression of food intake by a hybrid containing the FN-38 family component and the indicated second family component. FIG. 17A-FN38 and amylin family components, amylin-sCT-amylin chimeric compound 10. FIG. 17B--FN38 and PYY family components, PYY-3-36. FIG. 17C--FN38 and PYY family components, PYY-NPY chimera. Figure 17D--FN38 and CCK8 family components, CCK8. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. FIG. 3 shows suppression of food intake by a hybrid containing the FN-38 family component and the indicated second family component. FIG. 17A-FN38 and amylin family components, amylin-sCT-amylin chimeric compound 10. FIG. 17B--FN38 and PYY family components, PYY-3-36. FIG. 17C--FN38 and PYY family components, PYY-NPY chimera. Figure 17D--FN38 and CCK8 family components, CCK8. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. FIG. 3 shows suppression of food intake by a hybrid containing the FN-38 family component and the indicated second family component. FIG. 17A-FN38 and amylin family components, amylin-sCT-amylin chimeric compound 10. FIG. 17B--FN38 and PYY family components, PYY-3-36. FIG. 17C--FN38 and PYY family components, PYY-NPY chimera. Figure 17D--FN38 and CCK8 family components, CCK8. The dose was 25 nmol / kg. A difference of -15-20% is statistically significant. It is a figure which shows suppression of food intake by the hybrid containing an exendin family and an amylin family component. If not indicated otherwise, the dose was 25 nmol / kg. A difference of -15-20% is statistically significant. It is a figure which shows suppression of food intake by the hybrid containing an exendin family and an amylin family component. If not indicated otherwise, the dose was 25 nmol / kg. A difference of -15-20% is statistically significant. It is a figure which shows the long-term suppression of the food intake suppression by the exendin / amylin family hybrid during a dark cycle breeding, and the whole food intake. It is a figure which shows the long-term suppression of the food intake suppression by the exendin / amylin family hybrid during a dark cycle breeding, and the whole food intake. FIG. 7 shows long-term suppression of dietary intake and lean-sparing weight loss and fat loss activity of an exemplary exendin / amylin-sCT-amylin family hybrid compared to the parent molecule. “ ^* ” Indicates P <0.05 compared to vehicle. FIG. 7 shows long-term suppression of dietary intake and lean-sparing weight loss and fat loss activity of an exemplary exendin / amylin-sCT-amylin family hybrid compared to the parent molecule. “ ^* ” Indicates P <0.05 compared to vehicle. FIG. 5 demonstrates the effect of hybrids on metabolic parameters after 14 days treatment in rats. FIG. 5 demonstrates the effect of hybrids on metabolic parameters after 14 days treatment in rats. FIG. 5 demonstrates the effect of hybrids on metabolic parameters after 14 days treatment in rats. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein. FIG. 3 shows the ability of PPF polypeptide chimeras, eg, PYY-NPY chimeric compounds 4883 and 5705, to reduce cumulative food intake in the food intake assay described herein.

Claims (103)

A hybrid polypeptide exhibiting at least one hormonal activity comprising a first bioactive peptide hormone module covalently linked to at least one additional bioactive peptide hormone module comprising:
Biologically active peptide hormone module includes component peptide hormone, component peptide hormone fragment exhibiting at least one hormone activity of component peptide hormone, analog and derivative of component peptide hormone exhibiting at least one hormone activity of component peptide hormone, component peptide A component that exhibits at least one hormonal activity of the hormone, selected independently from the group consisting of peptide hormone analogs and derivatives fragments, and peptide enhancers;
Component peptide hormones are amylin, adrenomedullin (ADM), calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin, cholecystokinin (“CCK”), leptin, peptide YY (PYY), glucagon-like Peptide-1 (GLP-1), glucagon-like peptide 2 (GPL-2), oxyntomodulin (OXM), natriuretic peptide, urocortin family peptide such as Ucn-2 and Ucn-3, neuromedin family peptide Independently selected from at least two of the group consisting of, for example, neuromedin U25 or splice variants, and ANP, BNP, CNP or urodilatin, and exendin-4;
Peptide enhancers can produce the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding or other pharmacokinetic properties to the hybrid polypeptide. Peptide hormone structural motifs that confer and the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding to hybrid polypeptides Or independently selected from the group consisting of structural motifs of analogs or derivatives of component peptide hormones that confer other pharmacokinetic properties; and at least one of the bioactive peptide hormone modules is at least one of the component peptide hormones A hybrid polypeptide that exhibits the hormonal activity of.   Peptide enhancers are amylin (32-37) (SEQ ID NO: 242), amylin (33-37) (SEQ ID NO: 243), amylin (34-37) (SEQ ID NO: 244), amylin (35-37), Amylin (36-37), Amylin (37), ADM (47-52) (SEQ ID NO: 245), ADM (48-52) (SEQ ID NO: 246), ADM (49-52) (SEQ ID NO: 247) , ADM (50-52), ADM (51-52), ADM (52), CT (27-32) (SEQ ID NO: 248), CT (27-32) (SEQ ID NO: 248), CT (28- 32) (SEQ ID NO: 249), CT (29-32) (SEQ ID NO: 250), CT (30-32), CT (31-32), CT (32), CGRP (32-37) (SEQ ID NO: : 251), CGRP (33-37) (SEQ ID NO: 252), CGRP (34-37) (SEQ ID NO: 253), CGRP (35-37), CGRP (36-37), CGRP (37), Intel Medin (42-47) (SEQ ID NO: 254), Intel Medin (43-47) (SEQ ID NO: 255), Intel Medin (44-47) (SEQ ID NO: 256), Intel Medin (45- 47), Intel Medin (46-47), Intel Medin (47), PYY (25-36) (SEQ ID NO: 257), PYY (26-36) (SEQ ID NO: 258), PYY (27-36 ) (SEQ ID NO: 259 ), PYY (28-36) (SEQ ID NO: 260), PYY (29-36) (SEQ ID NO: 261), PYY (30-36) (SEQ ID NO: 262), PYY (31-36) (SEQ ID NO: : 263), PYY (32-36) (SEQ ID NO: 264), PYY (25-35) (SEQ ID NO: 265), PYY (26-35) (SEQ ID NO: 266), PYY (27-35) ( SEQ ID NO: 267), PYY (28-35) (SEQ ID NO: 268), PYY (29-35) (SEQ ID NO: 269), PYY (30-35) (SEQ ID NO: 270), PYY (31-35) ) (SEQ ID NO: 271), PYY (32-35) (SEQ ID NO: 272), Frog GLP-1 (29-37) (SEQ ID NO: 273), Frog GLP-1 (30-37) (SEQ ID NO: 274), frog GLP-2 (24-31) (SEQ ID NO: 275), exendin-4 (31-39) (SEQ ID NO: 277), exendin-4 (32-39) (SEQ ID NO: 278), exendin -4 (33-39) (SEQ ID NO: 279), exendin-4 (34-39) (SEQ ID NO: 280), exendin-4 (35-39) (SEQ ID NO: 281), exendin-4 (36- 39) (SEQ ID NO: 282), exendin-4 (37-39), exendin-4 (38-39), exendin-4 (39), and analogs thereof. 2. The hybrid polypeptide of claim 1, wherein the hybrid polypeptides are independently selected.   At least one of the first bioactive peptide hormone module or at least one additional bioactive peptide hormone module is a component peptide hormone or a fragment of a component peptide hormone that exhibits at least one hormone activity of the component peptide hormone. Item 2. The hybrid polypeptide according to Item 1.   At least one of the first bioactive peptide hormone module or at least one additional bioactive peptide hormone module is an analogue or derivative of a component peptide hormone or at least one hormone of a component peptide hormone exhibiting at least one hormone activity 2. The hybrid polypeptide according to claim 1, which is a fragment of an analog or derivative of a component peptide hormone exhibiting activity.   2. The hybrid polypeptide according to claim 1, wherein at least one of the first bioactive peptide hormone module or at least one additional bioactive peptide hormone module is a peptide enhancer.   The component peptide hormone is independently selected from the group consisting of amylin, calcitonin, CCK, PYY, urocortin family peptide, neuromedin family peptide, and ANP, BNP, CNP or urodilatin and exendin-4. A hybrid polypeptide according to 1.   2. The hybrid polypeptide according to claim 1, wherein at least one bioactive peptide hormone module exhibiting at least one hormonal activity is located at the N-terminal portion of the hybrid polypeptide.   8. The hybrid polypeptide according to claim 7, wherein at least one bioactive peptide module exhibiting at least one hormonal activity located in the N-terminal portion of the hybrid polypeptide is configured in a C-terminal to N-terminal orientation.   9. The hybrid polypeptide according to claim 8, wherein the N-terminal portion of the hybrid polypeptide is amidated.   2. The hybrid polypeptide of claim 1, wherein at least one bioactive peptide hormone module exhibiting at least one hormone activity is located at the C-terminal portion of the hybrid polypeptide.   11. The hybrid polypeptide according to claim 10, wherein the C-terminus of the hybrid polypeptide is amidated.   2. The hybrid polypeptide of claim 1, wherein the C-terminus of one bioactive peptide hormone module is attached directly to the N-terminus of another bioactive peptide hormone module to form a covalent attachment.   The bioactive peptide hormone module is alkyl; dicarboxylic acid PEG; amino acid; polyamino acid; bifunctional linker; aminocaproyl (Aca), Gly, β-alanyl, 8-amino-3,6-dioxaoctanoyl, and 2. The hybrid polypeptide of claim 1, wherein the hybrid polypeptide is attached covalently using one or more linking groups independently selected from the group consisting of Gly-Lys-Arg (GKR). The first bioactive peptide hormone module is exendin-4, a fragment of exendin-4 that exhibits at least one hormone activity, an exendin-4 analog or derivative that exhibits at least one hormone activity, and an exendin that exhibits at least one hormone activity Selected from the group consisting of -4 analog fragments;
At least one additional biologically active peptide hormone module of amylin, a fragment of amylin exhibiting at least one hormone activity, an amylin analog or derivative exhibiting at least one hormone activity, or an amylin analog exhibiting at least one hormone activity Fragment, CCK, fragment of CCK exhibiting at least one hormonal activity, CCK analog or derivative exhibiting at least one hormonal activity, fragment of CCK analog exhibiting at least one hormonal activity, CT, exhibiting at least one hormonal activity 2. An independent selection from the group consisting of a fragment of CT, a CT analog or derivative exhibiting at least one hormonal activity, a fragment of a CT analog exhibiting at least one hormonal activity, and a peptide enhancer. Hybrid polypeptides described. The first bioactive peptide hormone module is exendin-4, exendin-4 (1-27) (SEQ ID NO: 236), exendin-4 (1-28) (SEQ ID NO: 237), ¹⁴ Leu, ²⁵ Phe-exendin -4 (1-28) (SEQ ID NO: 284); ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28) (SEQ ID NO: 240) and ¹⁴ Leu-Exendin-4 (1-28) ( Selected from the group consisting of SEQ ID NO: 190); and at least one additional bioactive peptide hormone module is ^25,28,29 Pro-h-amylin (SEQ ID NO: 67), amylin (1-7) (sequence number: 217), ^2,7 Ala- amylin (1-7) (SEQ ID NO: 285), sCT (8-10) , sCT (8-27) ( SEQ ID ^{NO: 288), 11,18 Arg-sCT} ( 8-27) (SEQ ID ^{NO: 289), 14 Glu, 11,18} Arg-sCT (8-27) ( SEQ ID NO: 286), CCK-8, Phe 2 CCK-8 ( SEQ ID NO: 287), amylin ( 33-37) (SEQ ID NO: 243), PYY (25-36) (SEQ ID NO: 257), PYY (30-36) (SEQ ID NO: 262) and PYY (31-36) (SEQ ID NO: 263) Completion It is independently selected from the group, the hybrid polypeptide of claim 14.   15. A hybrid polypeptide according to claim 14, comprising at least three bioactive hormone modules.   15. A hybrid polypeptide according to claim 14, comprising at least four bioactive hormone modules.   15. The hybrid polypetide of claim 14, wherein the first bioactive peptide hormone module is located at the C-terminus of the hybrid polypeptide and at least one additional bioactive peptide hormone module is located at the N-terminus of the hybrid polypeptide. peptide.   15. The hybrid polypeptide of claim 14, wherein the first bioactive peptide hormone module is located at the N-terminus of the hybrid polypeptide and at least one bioactive peptide hormone module is located at C of the hybrid polypeptide. The first bioactive peptide hormone module comprises amylin, a fragment of amylin exhibiting at least one hormone activity, an amylin analog or derivative exhibiting at least one hormone activity, and a fragment of amylin analog exhibiting at least one hormone activity And at least one additional bioactive peptide hormone module is selected from PYY (25-36) (SEQ ID NO: 257), PYY (26-36) (SEQ ID NO: 258), PYY (27-36) (SEQ ID NO: 259), PYY (28-36) (SEQ ID NO: 260), PYY (29-36) (SEQ ID NO: 261), PYY (30-36) (SEQ ID NO: 262), PYY (31- 36) (SEQ ID NO: 263), PYY (32-36) (SEQ ID NO: 264), PYY (25-35) (SEQ ID NO: 265), PYY (26-35) (SEQ ID NO: 266), PYY ( 27-35) (SEQ ID NO: 267), PYY (28-35) (SEQ ID NO: 268), PYY (29-35) (SEQ ID NO: 269), PYY (30-35) (SEQ ID NO: 270), PYY (31-35) (SEQ ID NO: 271), PYY (32 -35) (SEQ ID NO: 272), and a hybrid polypeptide according to claim 1, which is a peptide enhancer independently selected from the group consisting of analogs thereof. A hybrid polypeptide exhibiting at least one hormonal activity comprising a first bioactive peptide hormone module covalently linked to a second bioactive peptide hormone module comprising:
Biologically active peptide hormone module includes component peptide hormone, component peptide hormone fragment exhibiting at least one hormone activity of component peptide hormone, analog and derivative of component peptide hormone exhibiting at least one hormone activity of component peptide hormone, component peptide A component that exhibits at least one hormonal activity of the hormone, selected independently from the group consisting of peptide hormone analogs and derivatives fragments, and peptide enhancers;
The component peptide hormone is independently selected from at least two of the group consisting of amylin, PYY, and exendin-4;
Peptide enhancers provide the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interactions, protease inhibition, plasma protein binding or other pharmacokinetic properties for hybrid polypeptides The structural motif of the component peptide hormone to be imparted, and the desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding, or hybrid polypeptide Independently selected from the group consisting of structural motifs of analogs or derivatives of component peptide hormones that confer other pharmacokinetic properties; and at least one of the bioactive peptide hormone modules is at least one of the component peptide hormones A hybrid polypeptide exhibiting hormonal activity.   Peptide enhancers are amylin (32-37) (SEQ ID NO: 242), amylin (33-37) (SEQ ID NO: 243), amylin (34-37) (SEQ ID NO: 244), amylin (35-37), Amylin (36-37), Amylin (37), PYY (25-36) (SEQ ID NO: 257), PYY (26-36) (SEQ ID NO: 258), PYY (27-36) (SEQ ID NO: 259) , PYY (28-36) (SEQ ID NO: 260), PYY (29-36) (SEQ ID NO: 261), PYY (30-36) (SEQ ID NO: 262), PYY (31-36) (SEQ ID NO: 263), PYY (32-36) (SEQ ID NO: 264), PYY (25-35) (SEQ ID NO: 265), PYY (26-35) (SEQ ID NO: 266), PYY (27-35) (sequence) Number: 267), PYY (28-35) (SEQ ID NO: 268), PYY (29-35) (SEQ ID NO: 269), PYY (30-35) (SEQ ID NO: 270), PYY (31-35) (SEQ ID NO: 271), PYY (32-35) (SEQ ID NO: 272), Exendin-4 (31-39) (SEQ ID NO: 277), Exendin-4 (32-39) (SEQ ID NO: 278), Exendin-4 (33-39) (SEQ ID NO: 279), Exendin-4 (34-39) (SEQ ID NO: 280), Exendin-4 (35-39) (SEQ ID NO: 281) , Exendin-4 (36-39) (SEQ ID NO: 282), exendin-4 (37-39), exendin-4 (38-39), exendin-4 (39), and analogs thereof 24. The hybrid polypeptide of claim 21, which is independently selected.   23. The hybrid polypeptide of claim 21, wherein the first bioactive peptide hormone module is located at the C-terminus of the hybrid polypeptide.   24. The hybrid polypeptide of claim 21, wherein the first bioactive peptide hormone module is located at the N-terminus of the hybrid polypeptide.   A bioactive peptide hormone module selected from the group consisting of exendin-4 / PYY, PYY / exendin-4, exendin / amylin, amylin / exendin, amylin / PYY, and PYY / amylin bioactive peptide hormone module. The hybrid polypeptide according to 21. A hybrid polypeptide exhibiting at least one hormonal activity comprising a first bioactive peptide hormone module covalently linked to at least one second bioactive peptide hormone module;
Biologically active peptide hormone module includes component peptide hormone, component peptide hormone fragment exhibiting at least one hormone activity of component peptide hormone, analog and derivative of component peptide hormone exhibiting at least one hormone activity of component peptide hormone, component peptide A component that exhibits at least one hormonal activity of the hormone, selected independently from the group consisting of peptide hormone analogs and derivatives fragments, and peptide enhancers;
The first component peptide hormone comprises leptin;
At least one second bioactive peptide hormone module is independently selected from exendin or GLP1;
A hybrid polypeptide wherein at least one bioactive peptide hormone module exhibits at least one hormonal activity of its component peptide hormones.   The group of pre-leptin peptide hormone modules consisting of leptin, leptin fragments exhibiting at least one hormone activity, leptin analogs and derivatives exhibiting at least one hormone activity, and fragments of leptin analogs and derivatives exhibiting at least one hormone activity 27. The hybrid polypeptide of claim 26, comprising a more selected polypeptide.   27. The hybrid polypeptide of claim 26, wherein the first and second bioactive peptide hormone modules exhibit at least one hormonal activity of a component peptide hormone.   Leptin contains the sequence MHWGTLCGFLWLWPYLFYVQAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLPGPGMLQ   The leptin contains the sequence VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC, or a peptide whose claim is a poly.   A leptin analog has an amino acid substitution at position 43 for ASP or Glu, an amino acid substitution at position 48 for Ala, an amino acid substitution at position 49 or no amino acid substitution, an amino acid substitution at position 75 for Ala , Amino acid substitution to Leu at position 89, amino acid substitution to Asp or Glu at position 93, amino acid substitution to Ala at position 98, amino acid substitution to Ser at position 117, to Leu at position 139 Selected from the group consisting of an amino acid substitution and an amino acid substitution to Ser at position 167, at position 43, 48, 49, 75, 89, 93, 98, 117, 139 or 167 or the corresponding position in the analog 27. The hybrid polypeptide of claim 26, comprising one or more amino acid substitutions.   Leptin is 43Asp-leptin, 43Glu-leptin, 48Ala-leptin, 49Glu-leptin, 49Des-AA-leptin, 75Ala-leptin, 89Leu-leptin, 93Asp-leptin, 93Glu-leptin, 98Ala-leptin, 117Ser-leptin, 139Leu 27. selected from the group consisting of -leptin, 167Ser-leptin, 43Asp, 49Glu-leptin, 43Asp, 75Ala-leptin, 89Leu, 117Ser-leptin, 93Glu, 167Ser-leptin, and 117Ser, D-119Leu-leptin A hybrid polypeptide according to 1. The leptin is a fragment selected from the group consisting of leptin (22-167), leptin (116-122), ¹¹⁷ Ser, D- ¹¹⁹ Leu-leptin (116-12) and leptin (56-73) Item 27. The hybrid polypeptide according to Item 26. The leptin peptide hormone module comprises leptin, a leptin fragment that exhibits at least one hormonal activity, a leptin analog and derivative that exhibits at least one hormonal activity, a fragment of leptin analogs and derivatives that exhibit at least one hormonal activity,
At least one GLP1 peptide hormone module comprising glucagon-like peptide-1 (GLP-1), a fragment of GLP1 exhibiting at least one hormone activity, an analog or derivative of GLP1 exhibiting at least one hormone activity, and at least one hormone Selected from the group consisting of fragments of analogs of GLP1 that exhibit activity,
27. A hybrid polypeptide according to claim 26.   GLP1 is selected from the group consisting of GLP1 (1-37), GLP1 (1-36), GLP1 (7-37), GLP1 (7-36), GLP1 (7-35) and analogs or derivatives thereof. 27. The hybrid polypeptide of claim 26.   27. The hybrid polypeptide of claim 26, wherein GLP1 is from a mouse, hamster, chicken, cow, rat, frog or dog.   27. The hybrid polypeptide of claim 26, wherein GLP1 is from a human or frog. GLP1
HDEFERHAEGTFTSDVSSTLEGQAALEFIAWLVKGRG,
HAEGTYTNDVTEYLEEKAAKEFIEWLIKGKPKKIRYS;
HAEGTFTSDVTQQLDEKAAKEFIDWLINGGPSKEIIS, and
HAEGTFTSDVSSYLEGQAALEFIAWLVKGR
27. The hybrid polypeptide of claim 26, selected from the group consisting of: Exendin is ⁹ Gln-GLP-1 (7-37), D- ⁹ Gln-GLP-1 (7-37), ¹⁶ Thr- ¹⁸ Lys ^- GLP-1 (7-37), ¹⁸ Lys-GLP-1 (7-37), ⁸ Gly-GLP-1 (7-36), ⁹ Gln-GLP-1 (7-37), D- ⁹ Gln-GLP-1 (7-37), Acetyl- ⁹ Lys-GLP -1 (7-37), ⁹ Thr-GLP-1 (7-37), D- ⁹ Thr-GLP-1 (7-37), ⁹ Asn-GLP-1 (7-37), D- ⁹ Asn -GLP-1 (7-37), ²² Ser ²³ Arg ²⁴ Arg ²⁶ Gln-GLP-1 (7-37), ¹⁶ Thr ¹⁸ Lys-GLP-1 (7-37), ¹⁸ Lys-GLP-1 (7 27. The hybrid polypeptide of claim 26, selected from the group consisting of: -37), ²³ Arg-GLP-1 (7-37), and ²⁴ Arg-GLP-1 (7-37).   The leptin peptide hormone module is selected from the group consisting of leptin, leptin fragments exhibiting at least one hormonal activity, leptin analogs and derivatives exhibiting at least one hormonal activity, fragments of leptin analogs and derivatives exhibiting at least one hormonal activity As well as at least one exendin peptide hormone module, exendin-4, a fragment of exendin-4 exhibiting at least one hormone activity, an exendin-4 analog or derivative exhibiting at least one hormone activity, 27. The hybrid polypeptide of claim 26, comprising a polypeptide selected from the group consisting of and a fragment of an exendin-4 analog exhibiting at least one hormonal activity. Exendin consists of Exendin-4, ¹⁴ Leu, ²⁵ Phe-Exendin-4, ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4, ¹⁴ Leu, ²² Ala, ²⁵ Phe-Exendin-4, and active fragments thereof 27. The hybrid polypeptide of claim 26, selected from the group.   27. The hybrid polypeptide of claim 26, wherein the exendin comprises the sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS or an active fragment thereof. Exendin is exendin (7-15), ² Ser-exendin (7-15), exendin-4 (1-27), exendin (1-28), exendin-4 (1-29), exendin-4 (1 -30), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27), ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-27), ¹⁴ Leu, ²² Ala, ²⁵ Phe-Exendin-4 ( 1-27), ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28); ⁵ Ala, ¹⁴ Leu, ²⁵ Phe-Exendin-4 (1-28), and ¹⁴ Leu-Exendin-4 (1-28) 27. The hybrid polypeptide of claim 26, selected from the group consisting of:   27. The hybrid polypeptide according to claim 26, wherein the C-terminus is amidated.   27. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 70% sequence identity with its base peptide.   27. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 80% sequence identity with its base peptide.   27. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 90% sequence identity with its base peptide.   27. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules has at least 95% sequence identity with its base peptide.   27. The hybrid polypeptide of claim 26, wherein any one of the component peptide hormone modules comprises a D-amino acid.   27. The hybrid polypeptide of claim 26, wherein the leptin peptide hormone module is linked at its N-terminus to the C-terminus of at least one exendin and / or GLP1 peptide hormone module.   27. The hybrid polypeptide of claim 26, wherein the leptin peptide hormone module is covalently linked to at least one exendin and / or GLP1 peptide hormone module by a linker.   52. The hybrid polypeptide of claim 51, wherein the linker is chemically stable.   52. The leptin peptide hormone module or at least one exendin and / or GLP1 peptide hormone module comprises substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine to create a linker site. The hybrid polypeptide.   The linker comprises a moiety selected from the group consisting of alkyl, PEG, amino acid, polyamino acid, bifunctional linker; aminocaproyl, β-alanyl, and 8-amino-3,6-dioxaoctanoyl. Item 52. The hybrid polypeptide according to Item 51.   The linker is amino acid Lys, Glu, Gly, Cys, or β-Ala and polyamino acid poly-his, poly-arg, poly-lys, poly-ala, beta Ala-beta Ala, Gly-Gly-Gly, or Gly- 52. The hybrid polypeptide of claim 51, comprising a portion selected from the group consisting of Lys-Arg.   52. The hybrid polypeptide of claim 51, wherein the linker is 1 to 30 residues long, 2 to 30 residues long, or 3 to 30 residues long.   52. The hybrid polypeptide of claim 51, wherein the linker is 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues long.   27. The hybrid polypeptide of claim 26, wherein at least one component peptide hormone is produced recombinantly.   27. The hybrid polypeptide of claim 26, produced recombinantly.   27. The hybrid polypeptide of claim 26, wherein at least one component peptide hormone is chemically synthesized.   27. The hybrid polypeptide of claim 26, which is chemically synthesized.   27. A hybrid polypeptide according to claim 26 comprising two, three or four exendins and / or GLP1 peptide hormone modules.   27. A method for treating a patient having a metabolic disease or disorder comprising administering to a patient in need thereof a therapeutically effective amount of the hybrid polypeptide of claim 26.   64. The method of claim 63, wherein the patient is in need of dietary intake adjustment.   64. The method of claim 63, wherein the patient is in need of weight control.   64. The method of claim 63, wherein the patient is in need of hematopoiesis.   64. The method of claim 63, wherein the hybrid provides a therapeutically effective anorexia-inducing effect.   64. The method of claim 63, wherein the hybrid provides a therapeutically effective glucose lowering effect.   64. The method of claim 63, wherein the hybrid provides a therapeutically effective enhancement of insulin secretion.   64. The method of claim 63, wherein the patient is in need of one or more of dietary intake regulation, weight regulation, hematopoiesis, anorexia-inducing action, glucose reduction, insulin secretion enhancement or pancreatic beta cell mass increase.   Metabolic disease or abnormality is type 2 diabetes, type 1 diabetes, obesity, hypertension, atherosclerosis, dyslipidemia, congestive heart failure, stroke, hypercholesterolemia, cardiovascular disease, myocardial ischemia, myocardial relapse 64. The method of claim 63, wherein the method is perfusion, eating disorder, gestational diabetes, diabetic neuropathy, pulmonary hypertension, or is accompanied by insufficient pancreatic beta cell mass.   A method of treating obesity comprising administering an anti-obesity hybrid to a subject in need of such treatment.   75. The method of claim 72, wherein the subject loses weight at least 10%.   75. The method of claim 72, wherein the subject reduces body fat mass.   75. The method of claim 72, wherein at least one component of the hybrid acts on structures in the forebrain that are involved in dietary intake or weight adjustment.   75. The method of claim 72, wherein at least one component of the hybrid acts on structures in the hindbrain that are involved in dietary intake or weight adjustment.   The hybrid comprises at least one component that acts on structures in the forebrain that are involved in dietary intake or weight adjustment, and at least one component that acts on structures in the hindbrain that are involved in dietary intake or weight adjustment, 73. The method of claim 72.   NPY1 receptor antagonist, NPY5 receptor antagonist, NPY2 receptor agonist, NPY4 receptor agonist, leptin, leptin derivative, leptin agonist, CNTF, CNTF agonist / modulator, CNTF derivative, MCH1R antagonist, MCH2R antagonist Drug, melanocortin 4 agonist, MC4 receptor agonist, cannabinoid receptor (CB-1) antagonist / reverse agonist, ghrelin antagonist, 5HT2c agonist, serotonin reuptake inhibitor, serotonin transport inhibitor, exendin, exendin Derivatives, exendin agonists, GLP-1, GLP-1 analogs, GLP-1 agonists, DPP-IV inhibitors, opioid antagonists, orexin antagonists, metabolite-producing glutamate subtype 5 receptor antagonists, histamine Further administration of an anti-obesity agent selected from the group consisting of 3 antagonists / inverse agonists, topiramate, CCK, CCK analogs, CCK agonists, amylin, amylin analogs, and amylin agonists. 73. The method of claim 72, comprising.   80. The method of claim 78, wherein the antiobesity agent administered is phentermine, rimonabant, sibutramine or pramlintide.   79. The method of claim 78, wherein the anti-obesity agent administered is ADM, an ADM analog or ADM agonist, leptin, a leptin derivative or leptin agonist, or a PPF chimera or derivative thereof.   The hybrid acts on forebrain structures involved in dietary intake or weight adjustment, and the anti-obesity agent acts on hindbrain structures involved in dietary intake or body weight regulation, or the hybrid acts on dietary intake 79. The method of claim 78, wherein the method acts on a hindbrain structure involved in weight adjustment, and the anti-obesity agent acts on a forebrain structure involved in food intake or weight adjustment.   A method of treating cardiovascular disease by attenuating, delaying or preventing cardiac remodeling in a subject in need thereof, effective cardioprotection to prevent adverse effects on the subject or to improve at least one cardiac parameter A method comprising administering a therapeutically effective amount of a sex hybrid, wherein the subject has experienced, is experiencing, or has a risk of experiencing myocardial injury, thereby attenuating, delaying or preventing cardiac remodeling.   The cardiac parameters are left ventricular diastolic function, E / A ratio, left ventricular end diastolic pressure, cardiac output, cardiac contractility, left ventricular mass, left ventricular mass to body weight ratio, left ventricular volume, left atrial volume, 84. The method of claim 82, wherein the method is selected from the group consisting of left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), infarct size, exercise capacity, exercise efficiency, and heart chamber size.   84. The method of claim 83, wherein the heart chamber size is not increased with respect to dimensions and wall thickness.   84. The method of claim 83, wherein the E / A ratio is increased after myocardial infarction.   84. The method of claim 83, wherein the infarct size is reduced.   84. The method of claim 83, wherein athletic performance is increased.   84. The method of claim 83, wherein the exercise efficiency is increased.   84. The method of claim 83, wherein the cardiac blood flow is normalized after myocardial infarction.   The myocardial injury is the result of a condition selected from the group consisting of heart valve disease, myocardial infarction, cardiomyopathy, hypertension, infection, inflammation, surgery, genetic predisposition, volume overload, pulmonary heart and pulmonary hypertension 84. The method of claim 82.   94. The method of claim 90, wherein the cardiomyopathy is dilated cardiomyopathy, viral cardiomyopathy, or idiopathic cardiomyopathy.   92. The method of claim 91, wherein the subject is also afflicted with diabetes.   92. The method of claim 91, wherein the cardioprotective hybrid is acutely administered to the subject.   92. The method of claim 91, wherein the cardioprotective hybrid is administered chronically to the subject.   Administering a therapeutically effective amount of a cardioprotective hybrid effective to prevent or reduce atrial or ventricular remodeling to a subject in need thereof, said subject having experienced myocardial injury A method for preventing or reducing atrial or ventricular remodeling that is at or at risk of experiencing.   For treating or preventing a condition associated with or resulting from cardiac remodeling in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a cardioprotective hybrid effective to prevent cardiac remodeling A method wherein the subject has experienced, is experiencing, or has a risk of experiencing myocardial injury, and the condition associated with cardiac remodeling is thereby improved.   Said condition is myocardial infarction, inflammation, ischemia / reperfusion, oxidative stress, pulmonary heart, late glycation reaction product, cardiac wall tension abnormality, sympathetic nerve stimulation, myocarditis, hypertension, heart transplantation, cardiac surgical procedure, Left ventricular hypertrophy, coronary artery disease, essential hypertension, acute hypertension, cardiomyopathy, heart failure, exercise tolerance, chronic heart failure, arrhythmia, cardiac rhythm abnormalities, sudden death, dizziness, atherosclerosis, mild chronic heart failure, angina, 99. The method of claim 96, wherein the method is cardiac bypass re-occlusion, intermittent claudication, diastolic dysfunction and / or systolic dysfunction.   A method of preventing cardiac remodeling in a subject exhibiting congestive heart failure comprising administering to the subject an amount of a cardioprotective hybrid effective to prevent cardiac remodeling.   99. The method of any one of claims 82 to 98, wherein the cardioprotective hybrid comprises an incretin family module capable of binding to GLP-1 or exendin receptors.   100. The method of claim 99, wherein the hybrid further comprises a peptide family module capable of producing beneficial cardiovascular effects, reduced glucose-induced tissue damage, reduced hypertension, or weight loss.   For reducing or treating or preventing high lipid levels, high triglyceride levels or high cholesterol in a subject in need thereof, comprising administering a therapeutically effective amount of a lipid-lowering agent alone or in combination with a second lipid-lowering agent Method.   The hybrid according to any one of the above claims, which is not the hybrid disclosed in WO2005 / 077072.   The method according to any of the preceding claims, which is not the hybrid disclosed in WO2005 / 077072.

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