Compositions comprising multi-agonist peptides, methods of manufacture and uses thereof

Abstract

The present invention relates generally to novel multi-agonist peptides useful as agents for the treatment and prevention of metabolic diseases and disorders, for example, diseases and disorders that can be ameliorated by controlling weight loss, plasma glucose and lipid levels, insulin levels and / or insulin secretion, positive myodynamia, reducing catabolism, slowing gastric emptying, and preventing neurodegeneration. Such conditions and disorders include, but are not limited to, control of food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive myodynamia, reduction of catabolism, slowing of gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and injury. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular diseases, dietetic disorders, and rare obesity genetic diseases such as Prallard-Willi Syndrome, intensive care, insulin resistance and related disorders such as polycystic ovarian Syndrome, obesity, any type of diabetes, including type 1, type 2 and gestational diabetes, as well as diabetes mellitus, such as diabetes mellitus, diabetes mellitus, diabetes mellitus, diabetes mellitus, and diabetes mellitus. And CNS disorders such as prevention of neurodegeneration, depression, alcohol addiction, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH).

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 389,769, filed on July 15, 2022, and U.S. Provisional Application No. 63 / 435,723, filed on December 28, 2022, each of which is incorporated herein by reference in its entirety. Field of the Invention

[0002] The present invention generally relates to novel multi-agonist peptides, which can be used as agents for the treatment and prevention of metabolic diseases and disorders, such as those that can be alleviated by controlling weight loss, plasma glucose and lipid levels, insulin levels and / or insulin secretion, positive inotropic effects, reducing catabolism, slowing gastric emptying and preventing neurodegeneration. Such conditions and disorders include, but are not limited to, controlling food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive inotropic effects, reducing catabolism, slowing gastric emptying, obesity, diabetes and diabetes-related conditions, inflammation and damage associated with liver fat. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders and rare genetic diseases of obesity such as Prader-Willi syndrome, intensive care, insulin resistance and related disorders such as polycystic ovary syndrome, obesity, any type of diabetes, including type 1, type 2 and gestational diabetes, and CNS disorders such as preventing neurodegeneration, depression, alcohol addiction, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH). Background of the Invention

[0004] Incretin peptides are hormones and peptide mimetics that are glucose regulators that can increase the amount of insulin released when glucose levels are normal or particularly elevated. These incretin peptides have other effects in addition to the initial incretin effect limited by insulin secretion. For example, they may also have the effect of reducing glucagon production, increasing satiety, delaying gastric emptying, regulating white and brown adipose tissue and causing weight loss. In addition, they may have the effect of improving insulin sensitivity, and they may increase islet cell neogenesis - the formation of new islets.

[0005] The concept of the incretin effect comes from the observation that oral glucose exceeds the insulin response following an equal amount of glucose injected intravenously. The conclusion drawn is that gut-derived factors, or incretins, influence postprandial insulin release. Entry of nutrients into the stomach and proximal gastrointestinal tract causes the release of incretin hormones, which stimulate insulin secretion. This insulinotropic effect, or the ability to stimulate insulin secretion, can be quantified by comparing the insulin or C-peptide response to oral versus intravenous glucose loads. In this way, the incretin effect has been shown to account for approximately 50% to 70% of the insulin response to oral glucose in healthy individuals.

[0006] Although many postprandial hormones have incretin-like activity, the main incretin peptides include glucose-dependent insulin secretagogue polypeptide, also known as gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and exenatide (which is a non-endogenous incretin mimetic). Both GIP and GLP-1 belong to the glucagon peptide superfamily and therefore have amino acid sequence homology. GIP and GLP-1 are secreted by specialized cells in the gastrointestinal tract and have receptors in pancreatic islet cells and other tissues. As incretins, both are secreted by the intestine in response to the intake of nutrients, which leads to enhanced insulin secretion. The insulin secretagogue effect of GIP and GLP-1 depends on the increase of environmental glucose. Both are rapidly inactivated by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV).

[0007] Native human GIP is a single-chain 42 amino acid peptide synthesized and secreted by specialized enteroendocrine K cells. These cells are primarily concentrated in the duodenum and proximal jejunum, although they can also be found throughout the intestine. The major stimulus for GIP secretion is the ingestion of a meal rich in carbohydrates and lipids. Following ingestion, circulating plasma GIP levels increase 10- to 20-fold. The half-life of intact GIP in healthy subjects is estimated to be approximately 7.3 minutes and in diabetic subjects to be approximately 5.2 minutes.

[0008] The physiological role of GIP has been elucidated using GIP receptor antagonists, GIP peptide antagonists, and GIP receptor knockout mice, as well as GIP infusion protocols. Blocking the binding of GIP to its receptor results in attenuated glucose-dependent insulin secretion in rats and mice after an oral glucose load. Similarly, administration of a GIP antagonist or GIP antiserum significantly reduced postprandial insulin release in rats. GIP receptor knockout mice exhibit normal fasting glucose levels but develop mild glucose intolerance after an oral glucose load. Interestingly, they also exhibit resistance to diet-induced obesity after months of a high-fat diet. Furthermore, in leptin-deficient ob / ob mice, the GIP receptor knockout genotype appears to reduce the extent of obesity development.

[0009] GIP also has many non-incretin effects. GIP has anti-emetic effects by activating the GIP receptor. Unlike other insulin secretagogues, GIP stimulated beta cell proliferation and cell survival in studies of the INS-1 islet cell line. In addition, animal studies have shown that GIP plays a role in lipid metabolism by stimulating lipoprotein lipase activity, inducing fatty acid uptake in adipose tissue, and stimulating fatty acid synthesis. GIP also appears to stimulate glucagon secretion in isolated perfused rat pancreas, but human studies have not shown any significant effects on glucagon secretion. In addition, unlike GLP-1, GIP appears to work by accelerating gastric emptying rather than inhibiting gastrointestinal motility.

[0010] Despite its potent glucose-regulating effects through stimulation of glucose-dependent insulin secretion, the insulin secretagogue effect of GIP is significantly reduced in diabetic subjects compared to normal individuals (16-18). Therefore, the clinical application of GIP has not been significantly advanced. In addition, there is still a need to develop other diabetes treatment modalities and treatments for metabolic diseases, conditions and disorders. Therefore, the present invention encompasses the peptides of the present invention and methods for treating or preventing metabolic diseases, conditions and disorders.

[0011] GLP-1 is a potent insulin secretagogue that is secreted by the intestinal mucosa in response to food intake. The significant incretin effect of GLP-1 is reflected by the fact that GLP-1R knockout mice are glucose intolerant. Although these patients have impaired incretin responses to oral glucose, the incretin response to intravenous infusion of GLP-1 is preserved in diabetic subjects. GLP-1 administered by infusion or subcutaneous injection controls fasting blood glucose levels in diabetic patients and maintains the glucose threshold for insulin secretion. GLP-1 shows great potential as such a therapeutic agent that can enhance insulin secretion in a physiological manner while avoiding hypoglycemia associated with sulfonylureas. Other important effects of GLP-1 on glucose homeostasis are the inhibition of glucagon secretion and the inhibition of gastric motility. GLP-1's inhibitory effect on pancreatic alpha cell secretion of glucagon leads to reduced glucose production in the liver by reducing gluconeogenesis and glycogenolysis. This anti-glucagon effect of GLP-1 is preserved in diabetic patients. The so-called ileal brake effect of GLP-1 is achieved by inhibiting gastric motility and gastric secretion through vagal efferent receptors or directly acting on intestinal smooth muscle. The reduction of gastric acid secretion by GLP-1 contributes to the lag phase of nutrient availability, thereby eliminating the need for a rapid insulin response. In summary, the gastrointestinal effects of GLP-1 significantly delay the absorption of glucose and fatty acids and modulate insulin secretion and glucose homeostasis. GLP-1 has also been shown to induce β-cell-specific genes such as GLUT-1 transporter, insulin (through the interaction of PDX-1 with the insulin gene promoter), and hexokinase-1. Therefore, GLP-1 can potentially reverse the glucose intolerance commonly associated with aging, which has been demonstrated by rodent experiments. In addition, GLP-1 may contribute to β-cell neogenesis and increase β-cell mass, and can also restore β-cell function in states of β-cell insufficiency. The central effects of GLP-1 include increased satiety and an associated reduction in food intake, which is achieved through the action of hypothalamic GLP-1 receptors. These anorectic effects are abolished in GLP-1R knockout mice. In addition, GLP-1 receptor signaling is thought to play a role in the regulation of energy expenditure and brown adipose tissue thermogenesis (Beiroa D, et al. Diabetes 63:3346-3358, 2014).

[0012] Glucagon is a 29 amino acid peptide hormone produced by pancreatic alpha cells and released into the blood when circulating glucose levels are low. An important physiological role of glucagon is to stimulate glucose output from the liver, a process that provides the major antagonistic regulatory mechanism to insulin that maintains glucose homeostasis in the body.

[0013] However, glucagon receptors are also expressed in extrahepatic tissues such as the kidney, heart, adipocytes, lymphoblasts, brain, retina, adrenal glands, and gastrointestinal tract, suggesting a broad range of physiological roles that are not limited to glucose homeostasis. Thus, recent studies have reported that glucagon has therapeutically active effects in energy management, including stimulation of energy expenditure and thermogenesis, with concomitant reductions in food intake and weight loss. Overall, stimulation of the glucagon receptor may be useful in the treatment of obesity and other diseases.

[0014] Another family of peptide hormones associated with metabolic diseases and disorders is the amylin family of peptide hormones, including amylin, calcitonin, calcitonin gene-related peptide, adrenomedullin, and intermedin (also known as "AFP-6"). Amylin is a 37 amino acid peptide hormone. It was isolated, purified, and chemically characterized as the major component of amyloid deposits in the pancreatic islets of human type 2 diabetic patients (Cooper et al., Proc. Natl. Acad. Sci., USA, 84: 8628-8632 (1987)). The amylin molecule has two post-translational modifications: C-terminal amidation, and cross-linking of cysteines at positions 2 and 7 to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of a Lys-Arg dibasic amino acid proteolytic cleavage signal before the N-terminal codon of Lys, and a Gly before the Lys-Arg proteolytic signal at the N(CLAIMS)-terminal position, which is a typical sequence for amidation by protein amidase PAM (Cooper et al., Biochem. Biophys. Acta, 1014: 247-258 (1989)).

[0015] Amylin is thought to regulate gastric emptying, inhibit glucagon secretion and food intake, and thus regulate the rate of glucose appearance in the circulation. It appears to complement the actions of insulin, which regulates the rate at which glucose disappears from the circulation and is taken up by peripheral tissues. These actions are supported by experimental findings in rodents and humans, which show that amylin complements the role of insulin in postprandial glucose control by at least three independent mechanisms, all of which affect the rate of glucose appearance. First, amylin inhibits postprandial glucagon secretion. Compared with healthy adults, patients with type 1 diabetes have no circulating amylin, and patients with type 2 diabetes have decreased postprandial amylin concentrations. Furthermore, infusion of an amylin-specific monoclonal antibody that binds to circulating amylin again results in a substantial increase in glucagon concentrations relative to controls. These results all suggest a physiological role for endogenous amylin in regulating postprandial glucagon secretion. Second, amylin slows gastrointestinal motility and gastric emptying. Finally, intrahypothalamic injection of rat amylin has been shown to reduce food intake and alter neurotransmitter metabolism in the hypothalamus. In some studies, food intake was significantly reduced for up to eight hours after intrahypothalamic injection of rat amylin and rat CGRP. In human trials, the amylin analog pramlintide has been shown to reduce body weight or weight gain. Amylin may be beneficial in treating metabolic conditions such as diabetes and obesity. Amylin may also be used to treat pain, bone disease, alcohol addiction, gastritis, for regulating lipids, especially triglycerides, or for influencing body composition, such as preferentially reducing fat and preserving lean tissue.

[0016] Calcitonin is named for its secretion in response to induced hypercalcemia and its rapid calcium-lowering effects. It is produced and secreted by neuroendocrine cells in the thyroid gland, which have come to be known as C cells. The best-studied effect of calcitonin is its effect on osteoclasts. In vitro effects of calcitonin include rapid loss of the ruffled border and decreased release of lysosomal enzymes. Ultimately, inhibition of osteoclast function by calcitonin results in decreased bone resorption. However, neither the chronic decrease in serum calcitonin induced in the setting of thyroidectomy nor the increase in serum calcitonin found in medullary thyroid carcinoma appear to be associated with changes in serum calcium or bone mass. Therefore, it is most likely that the primary function of calcitonin is to counteract acute hypercalcemia in emergency situations and / or to protect bone during periods of “calcium stress” such as growth, pregnancy, and lactation. Calcitonin has an effect on plasma calcium levels and inhibits osteoclast function and is widely used to treat osteoporosis. Therapeutically, salmon calcitonin appears to increase bone density and reduce fracture rates with minimal adverse effects. Over the past 25 years, calcitonin has also been used successfully to treat Paget's disease of the bone, a chronic bone disorder that can cause bones to enlarge or deform in one or more areas. Calcitonin is also widely used to relieve bone pain experienced during osteoporosis, but the mechanism of this action is unclear. The effects of salmon calcitonin extend beyond those related to bone metabolism. In human studies, salmon calcitonin inhibited postprandial gastric emptying and gastrin release while causing dose-dependent relaxation of the gallbladder in both the postprandial and fasting states. In mice and monkeys, salmon calcitonin exhibited anorectic effects and caused weight loss after a single administration. In chronic studies, oral formulations of salmon calcitonin also reduced food intake and body weight in rat models of obesity and diabetes.

[0017] Metabolic diseases and disorders have many forms, including obesity, diabetes, dyslipidemia, insulin resistance, fatty liver, steatohepatitis, apoptosis, etc. Obesity and its related disorders are common and very serious public health problems in the United States and the world. Upper body obesity is the strongest known risk factor for type 2 diabetes and a strong risk factor for cardiovascular disease. Obesity is a known risk factor for diabetes, hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, breast cancer, prostate cancer and colon cancer, and the increased incidence of general anesthesia complications (see, for example, Kopelman, Nature 404:635-43 (2000)). It reduces life expectancy and carries with it the above-mentioned serious comorbidity risks, as well as disorders such as infection, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension, hypercholesterolemia, gallstones, orthopedic injuries, and thromboembolic disease (Rissanen et al., Br. Med. J. 301: 835-7 (1990)). Obesity is also a risk factor for a group of conditions known as insulin resistance syndrome or "Syndrome X". Recent estimates of medical costs for obesity and related disorders are $2 trillion worldwide. The pathogenesis of obesity is believed to be multifactorial, but the basic problem is that the nutritional supply and energy expenditure of obese subjects do not reach a balance until there is an excess of adipose tissue. At present, obesity is a chronic, essentially incurable metabolic disorder that is difficult to treat. Therapeutic drugs useful for weight loss in obese people may have a profound beneficial effect on their health.

[0018] Diabetes mellitus is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria, resulting from insufficient production or utilization of insulin. Diabetes mellitus severely affects the quality of life of a large portion of the population in developed countries. Insufficient insulin production is characterized as type 1 diabetes, whereas insufficient insulin utilization is characterized as type 2 diabetes. However, it is now generally accepted that there are many different diabetes-related disorders that begin long before a patient is diagnosed with overt diabetes. Furthermore, the effects of suboptimal control of glucose metabolism in diabetes give rise to a range of associated lipid and cardiovascular disorders.

[0019] Dyslipidemia, or abnormal plasma lipoprotein levels, frequently occurs in diabetic patients. Dyslipidemia is usually characterized by elevated plasma triglycerides, low HDL (high-density lipoprotein) cholesterol, normal to elevated LDL (low-density lipoprotein) cholesterol levels, and increased levels of small, dense LDL (low-density lipoprotein) particles in the blood. Dyslipidemia is one of the main causes of increased coronary events and mortality in diabetic subjects. Epidemiological studies have demonstrated this by showing that coronary deaths in diabetic subjects are several times higher than in non-diabetic subjects. Several lipoprotein abnormalities have been described in diabetic subjects.

[0020] Insulin resistance refers to the reduced ability of insulin to exert its biological effects over a wide range of concentrations. In insulin resistance, the body secretes abnormally high amounts of insulin to compensate for this defect, leading to the development of a state of impaired glucose tolerance. Failure to compensate for the defect in insulin action inevitably results in elevated plasma glucose concentrations, leading to the clinical state of diabetes. Insulin resistance and relative hyperinsulinemia are known to play a contributing role in obesity, hypertension, atherosclerosis, and type 2 diabetes. The association of insulin resistance with obesity, hypertension, and angina has been described as a syndrome, Syndrome X, with insulin resistance as a common etiology.

[0021] Attempts to treat the multiple abnormalities associated with diabetes have led to the administration of several antidiabetic drugs to address these abnormalities in different patients. Examples of antidiabetic drugs are proteins such as insulin and insulin analogs, GLP-1 analogs, and small molecules such as insulin sensitizers, insulin secretagogues, sodium-glucose co-transporter 2 (SGLT-2) inhibitors, DPPIV inhibitors, and appetite regulating compounds.

[0022] Nonalcoholic fatty liver disease (NAFLD) is an umbrella term and covers conditions ranging from simple fat deposition in the liver to more advanced steatosis associated with hepatitis, fibrosis, cirrhosis, and even hepatocellular carcinoma in some cases. NAFLD includes nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). NAFL is characterized by steatosis of the liver involving more than 5% of the parenchyma without evidence of hepatocellular damage [2]. NASH, on the other hand, is defined in histological terms as a necroinflammatory process in which hepatocytes become damaged in the context of steatosis. The natural history of NAFLD has not been fully characterized. Studies have shown that the incidence of NAFLD increases with the prevalence of metabolic syndrome. Patients with type 2 diabetes show a particularly high risk of developing NASH and a two- to four-fold increased risk of complications associated with fatty liver. Currently, there are no approved treatments for NASH.

[0023] There remains a need to develop therapeutic drugs useful for the above-mentioned metabolic diseases, conditions and disorders. Therefore, the present invention provides novel multi-agonist peptides and methods of producing and using the same. The peptides of the present invention are useful for the metabolic diseases, conditions and disorders described above and herein. SUMMARY OF THE INVENTION

[0025] The present invention provides, among other things, novel multi-agonist peptides and methods for treating and preventing obesity and related disorders, including metabolic and hepatic disorders.

[0026] The present invention generally relates to novel peptides that can be used as agents for the treatment and prevention of metabolic diseases and disorders that can be alleviated by controlling food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive inotropic effects, reducing catabolic effects, slowing gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, and rare genetic diseases of obesity such as Prader-Willi syndrome, intensive care, insulin resistance and its related disorders such as polycystic ovary syndrome, obesity, any type of diabetes, including type 1, type 2 and gestational diabetes, alcohol addiction, and CNS disorders such as prevention of neurodegeneration, depression, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH).

[0027] The present invention encompasses "peptides of the present invention" as defined herein, which include two or more component peptides independently selected from at least two peptides, including, for example, but not limited to, biologically active components including amylin, glucose-dependent insulin secretagogue polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and calcitonin.

[0028] The present invention encompasses peptides having at least one hormonal activity. The peptides of the present invention include at least two bioactive peptides covalently linked together, optionally covalently linked by a linker group, wherein at least one bioactive peptide exhibits at least one hormonal activity of a component peptide. The bioactive peptides are independently selected from the following: component peptides (such as GIP, amylin, GLP-1), fragments of component peptides that exhibit at least one hormonal activity of the component peptides, component peptide analogs and derivatives that exhibit at least one hormonal activity of the component peptides, and fragments of component peptide analogs and derivatives that exhibit at least one hormonal activity of the component peptides.

[0029] In one embodiment, the peptide exhibits at least one hormonal activity, the peptide comprising at least one first peptide covalently linked to at least one additional peptide; wherein the peptides are independently selected from: a component peptide (such as GIP, GLP-1, amylin); a fragment of a component peptide that exhibits at least one hormonal activity of a component peptide; a component peptide analog and derivative that exhibits at least one hormonal activity of a component peptide; and a fragment of a component peptide analog and derivative that exhibits at least one hormonal activity of a component peptide.

[0030] The present invention encompasses the peptides of the present invention or pharmaceutically acceptable salts or solvates thereof, wherein the peptides of the present invention comprise or consist of an amino acid sequence of formula (I):

[0031] X 1 -X 2 -X 3 -GTFTSDYSIX4 -L-D-K-I-A-Q-X 5 -A-F-V-Q-W-L-X 6 -A-G-X 7 -X 8 -S-X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -X 39 -X 40 -X 41 -X 42 -X 43 -X 44 -X 45 -X 46 -X 47 -X 48 -X 49 -X 50 -X 51 -(NR 1 R 2 )

[0032] (I)

[0033] wherein

[0034] X 1 is Tyr, (d)Tyr, His or ImPrA;

[0035] X 2 is Ala, (d)Ala, Aib, Ser or Gly;

[0036] X 3is Glu or Gln;

[0037] X 4 is Ala or Aib;

[0038] X 5 Is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0039] X 6 It is Ile or Leu;

[0040] X 7 It is Gly, Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0041] X 8 It is Pro or Gly;

[0042] X 9 Missing or Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0043] X 10 Missing either Gly or Cys;

[0044] X 11 The deletion is either Ala, Cys or Ser;

[0045] X 12 The deletion is either Pro or Asn;

[0046] X 13 Missing or Pro, Thr or Leu;

[0047] X 14 Missing or Pro, Ala or Ser;

[0048] X 15 Missing or Ser, Thr or Cys;

[0049] X 16 It is Cys, Asn, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0050] X 17 is Asn, Val, Met, Ala, or Thr;

[0051] X 18 is Thr, Leu, Asn, or Ala;

[0052] X 19 is Ala, Gly, Thr or Gln;

[0053] X 20 is Ala, Thr, Arg or Cys;

[0054] X 21 is Cys, Leu, Thr or Ala;

[0055] X 22 is Val, Ser, Cys, Ala, or Thr;

[0056] X 23 is Leu, Gln, Val, Glu or Thr;

[0057] X 24 is Gly, Glu, Leu, Asp, Phe, Gln or Arg;

[0058] X 25 is Arg, Leu or Gly;

[0059] X 26 is Val, Leu, His, Arg or Ala;

[0060] X 27 is Ser, Arg, Leu, His, Ala or Glu;

[0061] X 28 is Gln, Leu, Ser, Glu or Phe;

[0062] X 29is Glu, Gln, Ser, Phe or Leu;

[0063] X 30 is Leu, Thr, Glu, Asn or Arg;

[0064] X 31 Deletion or His, Leu, Asn or Arg;

[0065] X 32 Deletion or Arg, His, Phe or Ser;

[0066] X 33 Deletion or Leu, Arg, Gly or Ser;

[0067] X 34 Gln, Leu, Pro, Ser or Asn is missing or present;

[0068] X 35 The deletion is either Thr, Gln, Ile or Asn;

[0069] X 36 is Tyr, Thr, Leu, Asn or Phe;

[0070] X 37 is Pro, Tyr, Phe or Gly;

[0071] X 38 is Arg, Lys, Pro or Gly;

[0072] X 39 is Thr, Arg, Pro or Ile;

[0073] X 40 is Asn, Thr, Asp, Ile, or Leu;

[0074] X 41 is Thr, Asn, Val, Leu, or Pro;

[0075] X 42 is Gly, Thr or Pro;

[0076] X 43 It is Ser, Gly, Ala, Pro, Thr, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0077] X 44 is Asn, Ser, His, or Thr;

[0078] X 45 is Thr, Asn, Ala, or Val;

[0079] X 46 is Tyr, Thr, Pro, Val, or Gly;

[0080] X 47 The deletion is either Tyr, Gly or Ser;

[0081] X 48 Deletion is either Ser or His;

[0082] X 49 The deletion is either His or Thr;

[0083] X 50 Absent or Thr or Pro;

[0084] X 51 Missing or Pro;

[0085] in

[0086] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0087] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0088] n is an integer from 12 to 20.

[0089] In certain embodiments, R 3 Yes-CO 2 H.

[0090] In certain embodiments, R 3 Yes-CO 2 CH 3 .

[0091] In certain embodiments, R 3 Yes-CO 2 NH 2 .

[0092] In certain embodiments, R 3 Yes-CO 2 NHCH 3 .

[0093] In certain embodiments, R 3 Yes-CO 2 N(CH 3 ) 2 .

[0094] In certain embodiments, R 3 Yes-CH 3 .

[0095] In certain embodiments, R 3 Yes-NH 2 .

[0096] In certain embodiments, n is 12.

[0097] In certain embodiments, n is 13.

[0098] In certain embodiments, n is 14.

[0099] In certain embodiments, n is 15.

[0100] In certain embodiments, n is 16.

[0101] In certain embodiments, n is 17.

[0102] In certain embodiments, n is 18.

[0103] In certain embodiments, n is 19.

[0104] In certain embodiments, n is 20.

[0105] In other embodiments, the peptide components of the peptides of the present invention may also include calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin, oxyntomodulin (OXM) and exendin-4.

[0106] In certain embodiments, the peptides of the invention include structural motifs of the component peptides that confer 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 peptides, as well as structural motifs of analogs or derivatives of the component peptides that confer 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 peptides of the invention. In further embodiments, at least one peptide exhibits at least one hormonal activity of the component peptide.

[0107] In a further embodiment, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is GIP, a fragment of GIP that exhibits at least one hormonal activity, a GIP analog or derivative that exhibits at least one hormonal activity, or a fragment of a GIP analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0108] In a further embodiment, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is GLP-1, a fragment of GLP-1 that exhibits at least one hormonal activity, a GLP-1 analog or derivative that exhibits at least one hormonal activity, or a fragment of a GLP-1 analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0109] In further embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is 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 a fragment of an amylin analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0110] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is calcitonin, a fragment of calcitonin that exhibits at least one hormonal activity, a calcitonin analog or derivative that exhibits at least one hormonal activity, or a fragment of a calcitonin analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0111] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is a calcitonin gene-related peptide, a fragment of a calcitonin gene-related peptide that exhibits at least one hormonal activity, a calcitonin gene-related peptide analog or derivative that exhibits at least one hormonal activity, or a fragment of a calcitonin gene-related peptide analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0112] In further alternative embodiments, at least one component peptide that exhibits at least one hormonal activity of the component peptide is an interferon, a fragment of an interferon that exhibits at least one hormonal activity, an interferon analog or derivative that exhibits at least one hormonal activity, or a fragment of an interferon analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0113] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is cholecystokinin, a fragment of cholecystokinin that exhibits at least one hormonal activity, a cholecystokinin analog or derivative that exhibits at least one hormonal activity, or a fragment of a cholecystokinin analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0114] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is glucagon-like peptide 2, a fragment of glucagon-like peptide 2 that exhibits at least one hormonal activity, a glucagon-like peptide 2 analog or derivative that exhibits at least one hormonal activity, or a fragment of a glucagon-like peptide 2 analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0115] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is oxyntomodulin, a fragment of oxyntomodulin that exhibits at least one hormonal activity, an oxyntomodulin analog or derivative that exhibits at least one hormonal activity, or a fragment of an oxyntomodulin analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0116] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is a natriuretic peptide, a fragment of a natriuretic peptide that exhibits at least one hormonal activity, a natriuretic peptide analog or derivative that exhibits at least one hormonal activity, or a fragment of a natriuretic peptide analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0117] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptide is 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 a fragment of an exendin-4 analog or derivative that exhibits at least one hormonal activity, and at least one other peptide.

[0118] In certain embodiments, the GIP component portion of the peptides of the invention is combined with an amylin receptor ligand, a glucagon-like peptide 1 receptor ligand, or a gastric inhibitory polypeptide (GIP) receptor ligand.

[0119] In other embodiments, the GIP portion of the peptides of the invention binds to EGF receptor ligands, calcitonin receptor ligands, CGRP receptor ligands, gastrin / CCK receptor ligands, keratinocyte growth factor (KGF) receptor 1 ligands, dipeptidyl peptidase IV inhibitors, REG protein receptor ligands, growth hormone receptor ligands, prolactin (PRL) receptor ligands, insulin-like growth factor (IGF) receptor ligands, PTH-related protein (PTHrP) receptor ligands, hepatocyte growth factor (HGF) receptor ligands, fibroblast growth factor 19 (FGF19) receptor ligands, fibroblast growth factor 21 (FGF21) receptor ligands, bone morphogenetic protein (BMP) receptor ligands, transforming growth factor (TGF) receptor ligands. It binds to leukocytes, laminin receptor ligands, vasoactive intestinal peptide (VIP) receptor ligands, fibroblast growth factor (FGF) receptor ligands, nerve growth factor (NGF) receptor ligands, islet neogenesis-associated protein (INGAP) receptor ligands, activin A receptor ligands, vascular endothelial growth factor (VEGF) receptor ligands, erythropoietin (EPO) receptor ligands, pituitary adenylate cyclase-activating polypeptide (PACAP) receptor ligands, granulocyte colony-stimulating factor (G-CSF) receptor ligands, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor ligands, platelet-derived growth factor (PDGF) receptor ligands, cannabinoid CB1 receptor antagonists and secretin receptor ligands.

[0120] In another embodiment, the GIP peptide component contained in the peptide of the present invention includes N-terminal GIP or novel GIP analog fragments combined with C-terminal peptides or fragments thereof having glucose-lowering activity (e.g., antidiabetic agents, amylin) or the ability to inhibit or reduce gastric emptying. Such GIP peptides of the present invention include N-terminal GIP fragments or novel GIP analogs or derivative fragments combined with amylin, gastrin, PYY, secretin, CCK, GRP, neurotonin, urocortin, C-terminal, calcitonin or salmon calcitonin, natriuretic peptides (e.g., ANP, BNP, CNP, urodilatin) or analogs (e.g., amylin-sCT-amylin chimeras), derivatives or fragments thereof.

[0121] In other embodiments, the GIP peptide component included in the peptide of the present invention includes a C-terminal GIP or a novel GIP analog fragment combined with an N-terminal polypeptide or fragment thereof having hypoglycemic activity (such as an antidiabetic agent, amylin) or the ability to inhibit or reduce gastric emptying. In such embodiments, the chimeric peptide may include a C-terminal GIP, a novel GIP analog or fragment thereof combined with an N-terminal exenatide, GLP-1, amylin, CCK, gastrin, PYY, secretin, GRP, neurotonin, corticotropin, calcitonin or salmon calcitonin, natriuretic peptide or its analog, derivative or fragment thereof.

[0122] In one embodiment, the peptides of the invention include a heterologous C-terminal tail or terminal extension after the GIP portion. As with other GIP peptides described herein, in one embodiment of the peptides of the invention, the GIP portion can be native GIP, an active fragment thereof, or an analog or derivative thereof. In another aspect, the GIP component of the peptides of the invention comprises at least one modification, substitution, deletion, or addition that provides one or more enhanced properties, such as increased resistance to proteolytic digestion (thereby extending half-life), fatty acyl derivatization to reduce renal clearance. In one embodiment, the tail comprises a Trp-cage motif sequence. In another embodiment, the GIP analog peptide portion comprises a non-natural amino acid, such as a D amino acid, such as a D amino acid that inhibits or reduces the rate of DPP-IV proteolysis.

[0123] The present invention also encompasses the use of the peptides of the present invention to treat and prevent metabolic and liver diseases and disorders, particularly those that can be alleviated by controlling food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive inotropic effects, reduced catabolic effects, slowed gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders and rare genetic diseases of obesity such as Prader-Willi syndrome, intensive care, insulin resistance and its related disorders such as polycystic ovary syndrome, obesity, any type of diabetes, including type 1, type 2 and gestational diabetes, and CNS disorders such as prevention of neurodegeneration, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH), as well as complications of diabetes (e.g., neuropathy (treated with peptides of the invention including GIP peptides containing members of the exendin family), neuropathic pain (treated with peptides of the invention including GIP peptides containing amylin family hormone module), retinopathy, nephropathy, conditions of insufficient pancreatic beta cell mass (based on the islet neogenesis effects of, for example, exendin-4 and GLP-1).

[0124] Therefore, methods for treating or preventing such conditions are provided, wherein the method comprises administering a therapeutically or prophylactically effective amount of a peptide of the invention to a subject. In certain embodiments, the peptide of the invention comprises GIP or an analog or derivative thereof, including a novel GIP analog of the invention, or a GIP peptide of the invention comprising one or more additional peptides or peptide fragments, to a subject in need thereof.

[0125] In one embodiment, the peptides of the present invention can be provided as monotherapy. In another embodiment for the treatment of obesity, diabetes, or conditions associated with weight gain or elevated glucose levels, the peptides of the present invention can be administered in combination with treatments or hypoglycemic agents (such as antidiabetic drugs) that reduce food intake or agents or methods that inhibit or reduce gastric emptying. Examples of such agents are provided herein. For example, in one embodiment, a method of combining treatments to reduce the weight or blood glucose level of a subject (e.g., a subject with obesity, type 1, type 2, or gestational diabetes) is provided, comprising administering to the subject a therapeutically effective amount of the peptides of the present invention, for example, wherein the agonist is a peptide comprising a combination therapy with the GIP of the present invention or a novel GIP analog, or an effective amount of GIP-GLP-1-amylin.

[0126] The peptides of the present invention, alone or in combination with hypoglycemic agents (such as antidiabetic drugs) or agents or methods that inhibit or reduce gastric emptying, can also be used to strengthen, induce, enhance or restore glucose responsiveness in pancreatic islets or cells. These effects can also be used to treat or prevent conditions associated with metabolic disorders, such as those described above.

[0127] In another embodiment, a method for treating or preventing obesity is provided, wherein the method comprises administering a therapeutically or prophylactically effective amount of a peptide of the present invention to a subject in need thereof. In certain embodiments, the peptide of the present invention comprises GIP or an analog or derivative thereof, including a novel GIP analog of the present invention, or a GIP hybrid of the present invention, for example, a GIP-GLP-1-amylin hybrid.

[0128] In another embodiment, the subject is an obese or overweight subject. Although "obesity" is generally defined as a body mass index of more than 30, for the purpose of the present disclosure, any subject who needs or wishes to lose weight, including a subject with a body mass index of less than 30, is included in the scope of "obesity". Subjects with insulin resistance, glucose intolerance or any form of diabetes (e.g., type 1, type 2 or gestational diabetes) can benefit from this method. The peptides of the present invention can also be used to treat or prevent other conditions associated with obesity, including stroke, cancer (e.g., endometrial cancer, breast cancer, prostate cancer and colon cancer), gallbladder disease, sleep apnea, reduced fertility and osteoarthritis (see Lyznicki et al, Am. Fam. Phys. 63: 2185, 2001). For conditions associated with elevated glucose or hyperglycemia, the method includes administering a therapeutic or preventive effective amount of the peptides of the present invention alone or in combination with a hypoglycemic agent (e.g., an antidiabetic drug) or an agent or method that inhibits or reduces gastric emptying.

[0129] In another aspect, the peptides of the invention, in particular the GIP hybrids of the invention, can be used in a method of reducing food intake, reducing appetite, inducing satiety, reducing nutrient availability, reducing caloric efficiency, causing weight loss, affecting body composition, altering body energy content or energy expenditure, and improving lipid profiles (including lowering LDL cholesterol and triglyceride levels and / or altering HDL cholesterol levels), wherein the method comprises administering to a subject an effective amount of a peptide of the invention, e.g., a GIP hybrid of a peptide of the invention. In one embodiment, the method of the invention is used to treat or prevent a condition or disorder that can be alleviated by reducing nutrient availability in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of a peptide of the invention.

[0130] These conditions and disorders include, but are not limited to, control of food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive inotropic effects, reduced catabolic effects, slowed gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders and rare genetic diseases of obesity such as Prader-Willi syndrome, intensive care, insulin resistance and its related disorders such as polycystic ovary syndrome, obesity, any type of diabetes, including type 1, type 2 and gestational diabetes, and CNS disorders such as prevention of neurodegeneration, Alzheimer's disease and Parkinson's disease, alcohol addiction, and non-alcoholic steatohepatitis (NASH), diabetic complications (neuropathy (based on the neurotrophic effects of, for example, exendin-4), neuropathic pain (based on, for example, amylin effects), retinopathy, nephropathy, conditions of insufficient pancreatic β-cell mass (based on, for example, the islet neogenesis effects of exendin-4 and GLP-1). For conditions associated with elevated glucose or hyperglycemia, the method comprises administering a therapeutically or prophylactically effective amount of the peptides of the present invention alone or in combination with a glucose-lowering agent (such as an antidiabetic drug) or an agent or method that inhibits or reduces gastric emptying.

[0131] In addition to improving hypertension in a subject in need of such treatment by reducing food intake, reducing body weight and / or treating obesity, the peptides of the invention are also useful for treating or preventing hypotension and its related conditions.

[0132] The peptides of the present invention, for example, GIP components, include GIP hybrids that extend half-life (e.g., DPP-IV-resistant cleavage such as D-Tyr1, D-Ala2, N-acetyl or N-pyroglutamyl analogs), optionally further including peptides such as heterologous C-terminal tails. The peptides of the present invention include other hormone modules known to provide beneficial cardiovascular effects, and can be used to treat cardiovascular diseases and related conditions. As disclosed herein, the peptides of the present invention increase cardiac contractility (dp / dt), lower blood pressure (e.g., by rapid vasodilation), lower systolic pressure, lower diastolic pressure, and can provide direct beneficial effects on cardiac cells. The peptides of the present invention also improve cardiac function through metabolic effects such as weight loss, lowering blood sugar, insulin secretion, and beta cell proliferation. However, the peptides of the present invention are unexpectedly more beneficial by also having a direct effect on the cardiovascular system.

[0133] The peptides of the invention can also be used to treat or prevent a variety of gastrointestinal disorders associated with gastric hypersecretion, intestinal electrolyte and water hypersecretion, and reduced absorption, such as infectious (e.g., viral or bacterial) diarrhea, inflammatory diarrhea, short bowel syndrome, or diarrhea that typically occurs after surgery such as ileostomy (see, e.g., Harrison's principles of Internal Medicine, McGraw Hill Inc., New York, 12th ed.). Examples of infectious diarrhea include, but are not limited to, acute viral diarrhea, acute bacterial diarrhea (e.g., Salmonella, Campylobacter, and Clostridium), or diarrhea due to protozoan infection, or traveler's diarrhea (e.g., Norwalk virus or rotavirus). Examples of inflammatory diarrhea include, but are not limited to, malabsorption syndrome, tropical stomatitis, chronic pancreatitis, Crohn's disease, diarrhea, and irritable bowel syndrome. The GIP and GIP compounds of the invention can be used to treat or prevent emergency or life-threatening situations involving gastrointestinal disorders, such as after surgery or due to cholera. In addition, these compounds can be used to treat intestinal dysfunction in patients with acquired immune deficiency syndrome (AIDS), especially during cachexia.

[0134] The peptide of the present invention can also be used to inhibit small intestinal fluid and electrolyte secretion, and increase the transport of nutrients, and increase gastrointestinal cell proliferation, regulate lipolysis in adipose tissue, and regulate blood flow in mammals. The peptide of the present invention is used to treat or prevent the above-mentioned patient's condition by its gastrointestinal protective activity (such as inhibiting gastric secretion). Therefore, the peptide of the present invention can be used to treat gastrointestinal tract or mucosal damage. Exemplary injury types include but are not limited to inflammatory bowel disease, intestinal atrophy, the patient's condition characterized by intestinal mucosa or intestinal mucosa function loss, and other patient's conditions of the gastrointestinal tract, including those that may be due to exposure to cytotoxic agents, radiation, toxicity, infection and / or injury caused by the patient's condition. In addition, the peptide of the present invention can be combined with analgesics, anti-inflammatory agents, growth hormone, heparin or any other therapy that can be used to treat inflammatory bowel disease or the above-mentioned other patient's conditions.

[0135] In another embodiment, the peptides of the present invention can be used to treat or prevent gastritis, pancreatitis, Barrett's esophagus, gastroesophageal reflux disease (GERD) and related conditions thereof. Such conditions may include, but are not limited to, heartburn, heartburn with gastric / intestinal contents refluxed to the oral cavity or lungs, dysphagia, cough, intermittent wheezing and vocal cord inflammation (conditions associated with GERD), esophageal erosion, esophageal ulcer, esophageal stenosis, Barrett's metaplasia (normal esophageal epithelium is replaced by abnormal epithelium), and pulmonary aspiration. In certain embodiments, the peptides of the present invention may have anti-secretory properties, such as inhibiting gastric acid, inhibiting bile acid and inhibiting pancreatic enzymes. In addition, the peptides of the present invention may also have a gastric protective effect. Therefore, the peptides of the present invention are particularly useful in treating or preventing gastritis, pancreatitis, Barrett's esophagus and / or GERD and related or related conditions.

[0136] The present invention also relates to a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of at least one peptide of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or a carrier useful in delivering the peptide of the present invention.

[0137] These and other aspects of the invention will be more clearly understood from the following embodiments and detailed description.

[0138] The present invention encompasses the therapeutic methods disclosed herein, comprising one or more peptides of the present invention or pharmaceutically acceptable salts or solvates thereof, wherein the peptides of the present invention comprise or consist of an amino acid sequence represented by formula (I):

[0139] X 1 -X 2 -X 3 -GTFTSDYSIX 4 -LDKIAQX 5 -AFVQWLX 6 -AGX 7 -X 8 -SX 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26-X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -X 39 -X 40 -X 41 -X 42 -X 43 -X 44 -X 45 -X 46 -X 47 -X 48 -X 49 -X 50 -X 51 -(NR 1 R 2 )

[0140] (I)

[0141] in

[0142] X 1 is Tyr, (d)Tyr, His or ImPrA;

[0143] X 2 is Ala, (d)Ala, Aib, Ser or Gly;

[0144] X 3 is Glu or Gln;

[0145] X 4 is Ala or Aib;

[0146] X 5 Is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0147] X 6 It is Ile or Leu;

[0148] X 7 It is Gly, Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0149] X 8 It is Pro or Gly;

[0150] X 9 Missing or Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0151] X 10 Missing either Gly or Cys;

[0152] X 11 The deletion is either Ala, Cys or Ser;

[0153] X 12 The deletion is either Pro or Asn;

[0154] X 13 Missing or Pro, Thr or Leu;

[0155] X 14 Missing or Pro, Ala or Ser;

[0156] X 15 Missing or Ser, Thr or Cys;

[0157] X 16 It is Cys, Asn, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0158] X 17 is Asn, Val, Met, Ala, or Thr;

[0159] X 18 is Thr, Leu, Asn, or Ala;

[0160] X 19 is Ala, Gly, Thr or Gln;

[0161] X 20 is Ala, Thr, Arg or Cys;

[0162] X 21 is Cys, Leu, Thr or Ala;

[0163] X 22 is Val, Ser, Cys, Ala, or Thr;

[0164] X 23 is Leu, Gln, Val, Glu or Thr;

[0165] X 24 is Gly, Glu, Leu, Asp, Phe, Gln or Arg;

[0166] X 25 is Arg, Leu or Gly;

[0167] X 26 is Val, Leu, His, Arg or Ala;

[0168] X 27 is Ser, Arg, Leu, His, Ala or Glu;

[0169] X 28 is Gln, Leu, Ser, Glu or Phe;

[0170] X 29 is Glu, Gln, Ser, Phe or Leu;

[0171] X 30 is Leu, Thr, Glu, Asn or Arg;

[0172] X 31 Deletion or His, Leu, Asn or Arg;

[0173] X 32 Deletion or Arg, His, Phe or Ser;

[0174] X 33 Deletion or Leu, Arg, Gly or Ser;

[0175] X 34 Gln, Leu, Pro, Ser or Asn is missing or present;

[0176] X 35The deletion is either Thr, Gln, Ile or Asn;

[0177] X 36 is Tyr, Thr, Leu, Asn or Phe;

[0178] X 37 is Pro, Tyr, Phe or Gly;

[0179] X 38 is Arg, Lys, Pro or Gly;

[0180] X 39 is Thr, Arg, Pro or Ile;

[0181] X 40 is Asn, Thr, Asp, Ile, or Leu;

[0182] X 41 is Thr, Asn, Val, Leu, or Pro;

[0183] X 42 is Gly, Thr or Pro;

[0184] X 43 It is Ser, Gly, Ala, Pro, Thr, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0185] X 44 is Asn, Ser, His, or Thr;

[0186] X 45 is Thr, Asn, Ala, or Val;

[0187] X 46 is Tyr, Thr, Pro, Val, or Gly;

[0188] X 47 The deletion is either Tyr, Gly or Ser;

[0189] X 48 Deletion is either Ser or His;

[0190] X 49 The deletion is either His or Thr;

[0191] X 50Absent or Thr or Pro;

[0192] X 51 Missing or Pro;

[0193] in

[0194] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0195] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0196] n is an integer from 12 to 20.

[0197] In certain embodiments, R 3 Yes-CO 2 H.

[0198] In certain embodiments, R 3 Yes-CO 2 CH 3 .

[0199] In certain embodiments, R 3 Yes-CO 2 NH 2 .

[0200] In certain embodiments, R 3 Yes-CO 2 NHCH 3 .

[0201] In certain embodiments, R 3 Yes-CO 2 N(CH 3 ) 2 .

[0202] In certain embodiments, R 3 Yes-CH 3 .

[0203] In certain embodiments, R 3 Yes-NH 2 .

[0204] In certain embodiments, n is 12.

[0205] In certain embodiments, n is 13.

[0206] In certain embodiments, n is 14.

[0207] In certain embodiments, n is 15.

[0208] In certain embodiments, n is 16.

[0209] In certain embodiments, n is 17.

[0210] In certain embodiments, n is 18.

[0211] In certain embodiments, n is 19.

[0212] In certain embodiments, n is 20.

[0213] In certain embodiments, any of the peptides of the invention described herein comprises one or more half-life extending moieties and / or one or more linker moieties conjugated to the peptide. In certain embodiments, the half-life extending moiety is conjugated to the peptide component of the peptide of the invention via one or more linker moieties.

[0214] In certain embodiments, any of the peptides of the invention described herein further comprises a conjugated chemical substituent. In a specific embodiment, the conjugated chemical substituent is a lipophilic substituent or a polymer moiety, for example, Ac, Palm, gamma-Glu-Palm, isoGlu-Palm, PEG2-Ac, PEG4-isoGlu-Palm, (PEG) 5 -Palm, succinic acid, glutaric acid, pyroglutaric acid, benzoic acid, IVA, octanoic acid, 1,4-diaminobutane, isobutyl, Alexa 488, Alexa 647 or biotin. In certain embodiments, the conjugated chemical substituent is polyethylene glycol with a molecular weight of 400 Da to 40,000 Da.

[0215] In related aspects, the present invention includes peptides of the present invention, which include at least two peptide components connected by one or more linker moieties, wherein each peptide subunit comprises formula (I)-(V) or any other sequence or structure listed herein. In certain embodiments, the linker is any linker described herein. In certain embodiments, the linker moiety is a diethylene glycol linker, an iminodiacetic acid (IDA) linker, a β-Ala-iminodiacetic acid (β-Ala-IDA) linker, or a PEG linker. In certain embodiments, the PEG linker is a polyethylene glycol with a molecular weight of 400Da to 40,000Da. In certain embodiments, the linker is an Fc protein molecule. In specific embodiments, the N-terminus of each peptide monomer subunit is connected by a linker moiety. In specific embodiments, the C-terminus of each peptide monomer subunit is connected by a linker moiety. In other embodiments, the linker connects the internal amino acid residues of at least one peptide component to the N-terminus, C-terminus, or internal amino acid residues of another peptide component.

[0216] In further related embodiments, the present invention includes sequences encoding a peptide of the present invention or one or more peptide subunits of the present invention. The present invention also includes a vector comprising the polynucleotide.

[0217] In another aspect, the present invention includes a pharmaceutical composition comprising a peptide of the present invention and a pharmaceutically acceptable carrier, excipient or diluent. In a specific embodiment, the pharmaceutical composition comprises an enteric coating. In certain embodiments, the enteric coating protects and releases the pharmaceutical composition in the gastrointestinal system of a subject.

[0218] In certain embodiments, the pharmaceutical composition is provided to a subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, atomization, sublingual, buccal, parenteral, rectal, intraocular, inhalation, vaginal, or topical administration. In certain embodiments, the pharmaceutical composition is provided to a subject by topical, parenteral, intravenous, subcutaneous, intraperitoneal, or intravenous routes to treat obesity.

[0219] BRIEF DESCRIPTION OF THE DRAWINGS

[0220] Figure 1A and Figure 1B Figure 2 shows the reduction of blood glucose after a single administration of an exemplary peptide of the present invention. Blood glucose was Figure 1A ) and 240 minutes ( Figure 1B ) and then measure.

[0221] Figure 2 Shown is a reduction in food intake following chronic administration of exemplary peptides of the invention in DIO mice.

[0222] Figure 3 Shown is the reduction in body weight following chronic administration of exemplary peptides of the invention.

[0223] Figure 4A and Figure 4B shows the total fat mass ( Figure 4A ) but lean body mass ( Figure 4B ) was not reduced. MRI analysis.

[0224] Figure 5A and Figure 5B shows that blood glucose ( Figure 5A ) and insulin ( Figure 5B ) reduction. Detailed description of the invention

[0226] definition

[0227] Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature and techniques associated with chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0228] Unless otherwise stated, the following terms used herein have the meanings ascribed to them.

[0229] Throughout the specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated ingredient or group of ingredients but not the exclusion of any other ingredient or group of ingredients.

[0230] The singular forms "a", "an" and "the" include plural forms unless the context clearly requires otherwise.

[0231] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" can be used interchangeably.

[0232] The term "hybrid" is used to refer to a peptide comprising a combination or portions of two or more peptides, for example, GLP-1-GIP-amylin; or GIP-amylin.

[0233] The terms "patient," "subject," and "individual" are used interchangeably to refer to humans or non-human animals. These terms include mammals such as humans, primates, livestock (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats).

[0234] The term "peptide" as used herein refers broadly to a sequence of two or more amino acids linked by peptide bonds. It should be understood that the term does not imply a specific length of an amino acid polymer, nor is it intended to imply or distinguish whether a polypeptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally present. The term peptide also includes cyclic peptides.

[0235] As used herein, the terms "sequence identity", "percent identity", "percent homology" or, for example, "a sequence that is 50% identical to ... " refer to the degree of identity of a sequence on a nucleotide-to-nucleotide or amino acid-to-amino acid basis over a comparison window. Thus, the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appears in the two sequences can be determined by comparing two optimally aligned sequences over the comparison window to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and then multiplying the result by 100 to obtain the percentage sequence identity.

[0236] The calculation between sequence similarity or sequence identity can be performed as follows. In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison (e.g., a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides of the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0237] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences, and the length of each gap.

[0238] Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In certain embodiments, the Needleman and Wunsch (1970, J. Mol. Biol. 48: 444-453) algorithm (which has been incorporated into the GAP program in the GCG software package) is used to determine the percent identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the GAP program in the GCG software package is used to determine the percent identity between two nucleotide sequences using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. Another example parameter set includes a Blossum 62 scoring matrix, a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller (1989, Cabios, 4: 11-17) algorithm, which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0239] The peptide sequences described herein can be searched as "query sequences" to identify other family members or related sequences in public databases. For example, these searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can use the NBLAST program with score = 100 and wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can use the XBLAST program with score = 50 and wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0240] The term "conservative substitution" as used herein means that one or more amino acids are replaced by another biologically similar residue. Examples include substitutions of amino acid residues with similar characteristics, for example, small molecule amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. See, for example, the following table. In certain embodiments of the present invention, one or more Met residues are substituted with norleucine (Nle), which is a biological isostere of Met, but unlike Met, it is not easily oxidized. Another example of conservative substitution with residues that are not usually found in endogenous mammalian peptides and proteins is, for example, conservative substitution of Arg or Lys with ornithine, canavanine, aminoethylcysteine ​​or other basic amino acids. In certain embodiments, one or more cysteines of the peptide analogs of the present invention can be substituted by another residue, such as serine. For further information on phenotypic silent substitutions in peptides and proteins, see, for example, Bowie et.al.Science 247, 1306-1310, 1990. In the following scheme, conservative substitutions of amino acids are grouped by physicochemical properties. I: neutral, hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.

[0241] I II III IV V A N H M F S D R L Y T E K I W P Q V G C

[0242] In the scheme below, conservative substitutions of amino acids are grouped by physicochemical properties: VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic.

[0243] VI VII VIII IX X A E H M F L D R S Y I K T W P C G N V Q

[0244] As used herein, the term "amino acid" or "any amino acid" refers to any and all amino acids, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and unnatural amino acids. It includes D- and L-amino acids. Natural amino acids include those found in nature, for example, 23 amino acids that are combined into peptide chains to form a large number of protein building blocks. These are mainly L conformers, but small amounts of D-amino acids are present in bacterial envelopes and certain antibiotics. The above table lists 20 "standard" natural amino acids. "Non-standard" natural amino acids are pyrrolysine (found in methanogens and other eukaryotic organisms), selenocysteine ​​(present in many non-eukaryotic organisms as well as most eukaryotic organisms) and N-formylmethionine (encoded by the start codon AUG of bacteria, mitochondria, and chloroplasts). "Unnatural" or "unnatural" amino acids are non-proteinogenic amino acids (i.e., those that are not naturally encoded or found in the genetic code), which either occur naturally or are chemically synthesized. More than 140 unnatural amino acids are known, and there are thousands of possible combinations. Examples of "unnatural" amino acids include β-amino acids (β 3 and β 2 ), homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, α-methyl amino acids and N-methyl amino acids. Non-natural or unnatural amino acids also include modified amino acids. "Modified" amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, multiple groups or chemical moieties that are not naturally present on the amino acid. According to certain embodiments, the peptide includes an intramolecular bond between two amino acid residues present in the peptide. It should be understood that when the amino acid residues are bonded to each other to form a bond, these residues will change compared to when they are not bonded to each other. Reference to a specific amino acid is intended to cover the amino acid in its unbonded and bonded state. For example, the amino acid residue homoserine (hSer) or homoserine (Cl) in its unbonded form can take the form of 2-aminobutyric acid (Abu) in an intramolecular bond according to the present invention.

[0245] In general, the names of natural and non-natural aminoacyl residues used herein follow the naming conventions recommended by the IUPAC Organic Nomenclature Commission and the IUPAC-IUB Biochemical Nomenclature Commission, as described in "Nomenclature of α-AminoAcids (Recommendations, 1974)" Biochemistry, 14(2), (1975). In the event that the names and abbreviations of amino acids and aminoacyl residues used in this specification and the appended claims differ from these recommendations, the reader will be clear about them. Some abbreviations useful in describing the present invention are defined in Table 1 below.

[0246] Table 1. Abbreviations of exemplary non-natural amino acids and chemical moieties (for amino acid derivatives)

[0247]

[0248]

[0249]

[0250] The one-letter and three-letter abbreviations for the naturally occurring amino acids described in the present invention are defined in Table 2 below.

[0251] Table 2

[0252]

[0253] Throughout the specification, unless naturally occurring amino acids (e.g., alanine, arginine, etc.) are referred to by their full names, they are represented by conventional three-letter or single-letter abbreviations (e.g., Ala or A represents alanine, Arg or R represents arginine, etc.). Unless otherwise indicated, the three-letter and single-letter abbreviations of amino acids refer to the L-isomerism of the amino acid in question. As used herein, the term "L-amino acid" refers to the "L" isomerism of a peptide, whereas the term "D-amino acid" refers to the "D" isomerism of a peptide (e.g., Dasp, (d)Asp or D-Asp; Dphe, (d)Phe or D-Phe). Amino acid residues in the D isomerism can replace any L-amino acid residues as long as the peptide retains the desired function. When single-letter abbreviations are used, D-amino acids can be habitually represented by lowercase letters.

[0254] In the case of less common or non-naturally occurring amino acids, unless referred to by full name (e.g., sarcosine, ornithine, etc.), three-letter or four-letter codes are generally used to represent their residues, including Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Dab (2,4-diaminobutyric acid), Dapa (2,3-diaminopropionic acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutyric acid), β-Pro (pyrrolidine-3-carboxylic acid), 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-aminobutyric acid), βhPro (β-homoproline), βhPhe (β-homophenylalanine) and Bip (β,β-diphenylalanine), and Ida (iminodiacetic acid).

[0255] As is clearly known to those skilled in the art, the peptide sequences disclosed herein are shown from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end of the sequence being the C-terminus of the peptide. The sequences disclosed herein include a "Hy-" moiety at the amino terminus (N-terminus) of the sequence and a "-OH" moiety or "-NH" at the carboxyl terminus (C-terminus) of the sequence. 2 " portion of the sequence. In such cases and unless otherwise indicated, the "Hy-" portion at the N-terminus of the sequence in question represents a hydrogen atom, corresponding to a free primary or secondary amino group present at the N-terminus, while the "-OH" or "-NH" portion at the C-terminus of the sequence represents a hydrogen atom. 2 " respectively represent hydroxyl or amino groups, corresponding to the amide (CONH 2 In each sequence of the present invention, the C-terminal "-OH" part can replace the C-terminal "-NH 2 ” part and vice versa.

[0256] Those skilled in the art will appreciate that certain amino acids and other chemical moieties are modified when they are bound to another molecule. For example, an amino acid side chain may be modified when an amino acid side chain forms an intramolecular bridge with another amino acid side chain, such as when one or more hydrogens may be removed or replaced to form a bond. Therefore, as used herein, reference to an amino acid or modified amino acid present in a peptide of the present invention is meant to include such amino acid or modified amino acid in a peptide both before and after forming an intramolecular bond.

[0257] The term "NH 2 " can refer to the free amino group present at the amino terminus of the peptide. The term "OH" as used herein can refer to the free carboxyl group present at the carboxyl terminus of the peptide. In addition, the term "Ac" as used herein refers to acetyl protection by acylation of the C-terminus or N-terminus of the polypeptide. In some of the peptides shown herein, NH 2Located at the C-terminus of the peptide, representing the amino group.

[0258] The term "carboxyl" as used herein refers to -CO 2 H.

[0259] The term "isostere replacement" as used herein refers to any amino acid or other analog moiety having similar chemical and / or structural properties to a specified amino acid. In certain embodiments, an isostere replacement is a conservative substitution of a specified amino acid or analog.

[0260] As used herein, the term "cyclization" refers to the joining of one part of a peptide molecule to another part of a peptide molecule to form a closed ring, for example, by forming a disulfide bridge or a thioether bond.

[0261] The terms "component" or "subunit", which are used interchangeably, refer to one of a pair of peptide monomers that are linked to form a peptide of the present invention.

[0262] As used herein, the term "linker moiety" refers broadly to a chemical structure capable of linking or bonding two peptide subunits together or linking a peptide to another group (eg, a fatty acid).

[0263] The term "pharmaceutically acceptable salt" as used herein means a salt or zwitterionic form of a peptide or peptide of the present invention, which is water or oil soluble or dispersible, suitable for treating a disease without producing excessive toxicity, irritation and allergic response; commensurate with a reasonable benefit / risk ratio, and effective for its intended use. These salts can be prepared during the final isolation and purification of the compound, or separately prepared by reacting an amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. In addition, the amino groups in the compounds of the present invention can be quaternized with methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dimethyl, diethyl, dibutyl and diamyl sulfates; decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and benzyl and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. Pharmaceutically acceptable salts can be appropriately selected, such as acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrates and acetates. Examples of basic salts include salts in which the cation is selected from alkali metal cations such as sodium or potassium ions, alkaline earth metal cations such as calcium or magnesium ions, and substituted ammonium ions such as N(R 1 )(R 2 )(R 3 )(R 4 )+ type ions, where R 1 , R 2 , R 3 and R 4 Each of which independently represents hydrogen, optionally substituted C 1-6 Alkyl or optionally substituted C 2-6 Alkenyl. Related C 1-6 Examples of alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups. 2-6Examples of alkenyl groups include vinyl, 1-propenyl and 2-propenyl. Examples of other pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and updated versions thereof), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007 and J. Pharm. Sci. 66:2 (1977). In addition, for a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Other suitable alkaline salts are formed by bases that form non-toxic salts. Representative examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tris and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulphate and hemicalcium salts.

[0264] The term "N(α) methylation" as used herein describes the methylation of the α-amine of an amino acid, also commonly referred to as N-methylation.

[0265] As used herein, the term "symmetrical methylation" or "Arg-Me-sym" describes the symmetrical methylation of the two nitrogens of the guanidine group of arginine. In addition, the term "asymmetric methylation" or "Arg-Me-asym" describes the methylation of a single nitrogen of the guanidine group of arginine.

[0266] The term "acylated organic compound" as used herein refers to various compounds having a carboxylic acid function, which are used to acylate the N-terminus of an amino acid or peptide component, for example, a monomer subunit before forming a C-terminal dimer. Non-limiting examples of acylated organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, 3,3,3-trifluoropropionic acid, 3-fluoromethylbutyric acid, tetrahydro-2H-pyran-4-carboxylic acid.

[0267] The term "alkyl" includes straight or branched, non-cyclic or cyclic, saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc., while saturated branched chain alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, etc. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., while unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl, cyclohexenyl, etc.

[0268] "Halo" or "halogen" refers to a bromine (Br), chlorine (Cl), fluorine (F), or iodine (I) substituent.

[0269] The term "haloalkyl" includes alkyl structures in which at least one hydrogen is replaced by a halogen atom. In certain embodiments, two or more hydrogen atoms are replaced by halogen atoms, which are identical to one another. In other embodiments, two or more hydrogen atoms are replaced by halogen atoms, which are different from one another.

[0270] An "alkoxy" group refers to an (alkyl)O- group wherein alkyl is as defined herein.

[0271] An "aryloxy" group refers to an (aryl)O- group in which aryl is as defined herein.

[0272] "Carbamoyl" or "amide" refers to CONH 2 Free radicals.

[0273] "2-aminoethoxy" refers to -OCH 2 CH 2 -NH 2 Free radicals.

[0274] "2-Acetamidoethoxy" refers to -OCH 2 CH 2 -N(H)C(O)Me free radical.

[0275] The term "mammal" refers to any mammalian species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.

[0276] As used herein, a "therapeutically effective amount" of a peptide of the invention refers to an amount sufficient to treat or prevent any of the diseases and disorders described herein (e.g., to treat obesity). In certain embodiments, a therapeutically effective amount will achieve the desired benefit / risk ratio applicable to any medical treatment.

[0277] An amino acid "analog," such as a "Phe analog" or a "Tyr analog," refers to an analog of the referenced amino acid. Various amino acid analogs are known and available in the art, including Phe and Tyr analogs. In certain embodiments, an amino acid analog, such as a Phe analog or a Tyr analog, comprises one, two, three, four, or five substituents compared to Phe or Tyr, respectively. In certain embodiments, the substituents are present in the side chain of the amino acid. In certain embodiments, a Phe analog has the structure Phe(R 2 ), where R 2 Hy, OH, CH 3 , CO 2 H.CONH 2 ,CONH 2 OCH 2 CH 2 NH 2 , t-Bu, OCH 2 CH 2 NH 2 , phenoxy, OCH 3 , O-allyl, Br, Cl, F, NH 2 、N 3 In certain embodiments, R 2 It is CONH 2 OCH 2 CH 2 NH 2 、OCH 3 ,CONH 2 、OCH 3 or CO 2 Examples of Phe analogs include, but are not limited to, hPhe, Phe(4-OMe), α-Me-Phe, hPhe(3,4-dimethoxy), Phe(4-CONH 2 )、Phe(4-phenoxy), Phe(4-guanidino), Phe(4-tBu), Phe(4-CN), Phe(4-Br), Phe(4-Obenzyl), Phe(4-NH 2 ), BhPhe(4-F), Phe(4-F), Phe(3,5 diF), Phe(CH 2 CO 2 H), Phe(pentaF), Phe(3,4-Cl 2 )、Phe(3,4-F 2 )、Phe(4-CF 3 )、ββ-diPheAla、Phe(4-N 3 ), Phe[4-(2-aminoethoxy)], 4-phenylbenzylalanine, Phe(4-CONH2 )、Phe(3,4-dimethoxy), Phe(4-CF 3 )、Phe(2,3-Cl 2 ) and Phe(2,3-F 2 Examples of Tyr analogs include, but are not limited to, hTyr, N-Me-Tyr, Tyr(3-tBu), Tyr(4-N 3 ) and βhTyr.

[0278] The present invention generally relates to peptides of the present invention for use in treating or preventing various metabolic and hepatic diseases and disorders. In certain embodiments, the present invention presents a new paradigm for treating obesity, metabolic disorders, and hepatic disorders, and other diseases and disorders, by administering the peptides of the present invention. It is expected that administration of the peptides of the present invention will maximize drug levels in diseased tissues while limiting drug concentrations in the circulation, thereby providing effective, safe, and durable delivery for lifelong treatment of obesity and metabolic and hepatic diseases and disorders.

[0279] In certain embodiments, the peptides of the present invention encompass various peptides or heterologous or homologous monomeric subunits of peptides, which optionally form cyclized structures through disulfide bonds or other bonds. In certain embodiments, the disulfide bonds or other bonds are intramolecular bonds. The cyclized structures of peptides have been shown to increase the effectiveness and selectivity of the peptides of the present invention. In certain embodiments, the peptides of the present invention may include one or more intermolecular bonds to connect two peptide subunits within the peptide.

[0280] In certain embodiments, the peptides of the invention reduce the presence of a disease or disorder by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to a negative control peptide.

[0281] In certain embodiments, half-life is measured in vitro using any suitable method known in the art, for example, in certain embodiments, the stability of the peptides of the invention is determined by incubating the peptides with pre-warmed 37°C human serum or plasma (one skilled in the art will recognize that serum or plasma from other species (e.g., rat, mouse, etc.) can be used). Samples are taken at various time points, typically up to 24 hours, and the samples are analyzed for stability by separating the peptides from serum or plasma proteins and then analyzing the presence of the peptide of interest using LC-MS.

[0282] In certain embodiments, the peptides of the present invention show improved solubility or reduced aggregation characteristics relative to a control peptide. Solubility can be determined by any appropriate method known in the art. In certain embodiments, methods known in the art for determining solubility are included in various buffers (acetate pH 4.0, acetate pH 5.0, phosphate / citrate pH 5.0, phosphate citrate pH 6.0, phosphate pH 6.0, phosphate pH 7.0, phosphate pH 7.5, strong PBS pH 7.5, Tris pH 7.5, Tris pH 8.0, glycine pH 9.0, water, acetic acid (pH 5.0 and other known) incubated peptides, and standard techniques are used to test aggregation or solubility. For example, these methods include but are not limited to visual precipitation, dynamic light scattering, circular dichroism and fluorescent dyes to measure surface hydrophobicity, and detect aggregation or fibrillation. In certain embodiments, improved solubility means that the solubility of the peptide in a given liquid is higher than that of the control peptide. In certain embodiments, reduced aggregation means that the aggregation of the peptide in a given liquid under given conditions is less than that of the control peptide.

[0283] In certain embodiments, the peptides of the present invention have less degradation than the control peptide, for example, over a period of time (i.e., higher degradation stability), for example, less degradation than the control peptide by greater than or about 10%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50%. In certain embodiments, degradation stability is determined by any suitable method known in the art. In certain embodiments, degradation is enzymatic degradation. For example, in certain embodiments, the peptides of the present invention have reduced degradation sensitivity to trypsin, chymotrypsin or elastase. In certain embodiments, suitable methods known in the art for determining degradation stability include the methods described in Hawe et al., J Pharm Sci, VOL. 101, No. 3, 2012, p 895-913, the entire contents of which are incorporated herein. These methods are used in certain embodiments to select effective peptide sequences with enhanced shelf life.

[0284] The various peptides of the present invention can be constructed entirely from natural amino acids. Alternatively, the peptides of the present invention can include non-natural amino acids, including but not limited to, modified amino acids. In certain embodiments, modified amino acids include natural amino acids that have been chemically modified to include a non-naturally occurring group, multiple groups or chemical moieties on the amino acid. The peptides of the present invention can also include one or more D-amino acids. In addition, the peptides of the present invention can include amino acid analogs.

[0285] In certain embodiments, the peptides of the invention include one or more modified or unnatural amino acids. In certain embodiments of the invention, the peptides of the invention include one or more unnatural amino acids shown in Table 1. In certain embodiments, the peptides of the invention include any peptide described herein, including but not limited to, including any peptide comprising an amino acid sequence or peptide structure shown herein.

[0286] The present invention also includes any of the peptides of the present invention described herein, in free form or in salt form. Therefore, embodiments of any of the peptides of the present invention (and their related methods of use) described herein include pharmaceutically acceptable salts of the peptides of the present invention.

[0287] The present invention also includes variants of any of the peptides of the present invention described herein, including but not limited to any peptide comprising a sequence shown in any of the tables herein, wherein one or more L-amino acid residues are substituted with the D-enantiomeric form of the amino acid residue, for example, L-Ala is substituted with D-Ala.

[0288] The peptides of the invention described herein include isotopically labeled peptides. In certain embodiments, the disclosure provides peptides of the invention that are identical to any of the various formulas and structures described herein, but one or more atoms are replaced by atoms having an atomic mass or mass number different from that usually found in nature. Examples of isotopes that can be incorporated into the peptides of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F. 36 Certain isotopically labeled compounds described herein, for example, by incorporation of radioactive isotopes such as 3 H and 14 C compounds are useful in drug and / or substrate tissue distribution assays. In addition, isotopes such as deuterium (i.e. 2 H) Substitutions may offer certain therapeutic advantages, such as increased in vivo half-life due to greater metabolic stability or reduced dosage requirements.

[0289] The present invention also includes any peptide component described herein connected to a linker moiety, including any specific linker moiety described herein. In specific embodiments, the linker is connected to the N-terminal or C-terminal amino acid, while in other embodiments, the linker is connected to an internal amino acid. In specific embodiments, the linker is connected to two internal amino acids, such as one internal amino acid in each monomer subunit. In some embodiments, the peptides of the present invention include one or more linker moieties.

[0290] The present invention also includes peptides comprising a peptide sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the peptide sequence of the peptides of the present invention described herein. In certain embodiments, the peptides of the present invention comprise a core peptide sequence and one or more N-terminal and / or C-terminal modifications (e.g., Ac and NH 2 ) and / or one or more conjugated linker moieties and / or half-life extension moieties. The core peptide sequence used in this article refers to the amino acid sequence of the peptide component without these modifications and conjugates.

[0291] In certain embodiments, the peptides of the invention comprise, consist essentially of, or consist of: 10 to 90 amino acid residues, 15 to 80 amino acid residues, 20 to 75 amino acid residues, 25 to 70 amino acid residues, 30 to 65 amino acid residues, 35 to 60 amino acid residues, 40 to 55 amino acid residues, 45 to 50 amino acid residues, and optionally one or more additional non-amino acid moieties, such as conjugated chemical moieties, such as PEG or linker moieties.

[0292] In certain embodiments, the peptides of the invention (or their components), including but not limited to the peptides of any embodiment of formula IV, are more than 10, more than 12, more than 15, more than 20, more than 25, more than 30 or more than 35 amino acids, such as 35 to 80 amino acids. In certain embodiments, the peptides of the invention (or their component monomer subunits) are less than 90, less than 75, less than 60, less than 45, less than 30, less than 25, less than 20 or less than 10 amino acids. In certain embodiments, the component monomer subunits of the peptides of the invention comprise or consist of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acid residues. In a specific embodiment, a monomeric subunit of a peptide of the invention comprises or consists of 10 to 75 amino acid residues, and optionally one or more additional non-amino acid moieties, such as conjugated chemical moieties, such as PEG or lipidated amino acid residues.

[0293] In a specific embodiment of the invention, the amino acid sequence of the peptide of the invention is not present in an antibody, or is not present in the V or V region of an antibody. H or V L District.

[0294] Peptides of the present invention

[0295] The peptides of the present invention include peptides comprising or consisting of any amino acid sequence described herein, and peptides having any structure described herein.

[0296] The present invention encompasses the peptides of the present invention or pharmaceutically acceptable salts or solvates thereof, wherein the peptides of the present invention comprise or consist of an amino acid sequence of formula (I):

[0297] X 1 -X 2 -X 3 -GTFTSDYSIX 4 -LDKIAQX 5 -AFVQWLX 6 -AGX 7 -X 8 -SX 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -

[0298] X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -X 39 -X 40 -X 41 -X 42 -X 43 -X 44 -X 45 -X 46 -X 47 -X 48 -X 49 -X 50 -X 51 -(NR 1 R 2 )

[0299] (I)

[0300] in

[0301] X 1 is Tyr, (d)Tyr, His or ImPrA;

[0302] X 2 is Ala, (d)Ala, Aib, Ser or Gly;

[0303] X 3 is Glu or Gln;

[0304] X 4 is Ala or Aib;

[0305] X 5 Is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0306] X 6 It is Ile or Leu;

[0307] X 7 It is Gly, Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0308] X 8 It is Pro or Gly;

[0309] X 9 Missing or Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0310] X 10 Missing either Gly or Cys;

[0311] X 11The deletion is either Ala, Cys or Ser;

[0312] X 12 The deletion is either Pro or Asn;

[0313] X 13 Missing or Pro, Thr or Leu;

[0314] X 14 Missing or Pro, Ala or Ser;

[0315] X 15 Missing or Ser, Thr or Cys;

[0316] X 16 It is Cys, Asn, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0317] X 17 is Asn, Val, Met, Ala, or Thr;

[0318] X 18 is Thr, Leu, Asn, or Ala;

[0319] X 19 is Ala, Gly, Thr or Gln;

[0320] X 20 is Ala, Thr, Arg or Cys;

[0321] X 21 is Cys, Leu, Thr or Ala;

[0322] X 22 is Val, Ser, Cys, Ala, or Thr;

[0323] X 23 is Leu, Gln, Val, Glu or Thr;

[0324] X 24 is Gly, Glu, Leu, Asp, Phe, Gln or Arg;

[0325] X 25 is Arg, Leu or Gly;

[0326] X 26is Val, Leu, His, Arg or Ala;

[0327] X 27 is Ser, Arg, Leu, His, Ala or Glu;

[0328] X 28 is Gln, Leu, Ser, Glu or Phe;

[0329] X 29 is Glu, Gln, Ser, Phe or Leu;

[0330] X 30 is Leu, Thr, Glu, Asn or Arg;

[0331] X 31 Deletion or His, Leu, Asn or Arg;

[0332] X 32 Deletion or Arg, His, Phe or Ser;

[0333] X 33 Deletion or Leu, Arg, Gly or Ser;

[0334] X 34 Gln, Leu, Pro, Ser or Asn is missing or present;

[0335] X 35 The deletion is either Thr, Gln, Ile or Asn;

[0336] X 36 is Tyr, Thr, Leu, Asn or Phe;

[0337] X 37 is Pro, Tyr, Phe or Gly;

[0338] X 38 is Arg, Lys, Pro or Gly;

[0339] X 39 is Thr, Arg, Pro or Ile;

[0340] X 40 is Asn, Thr, Asp, Ile, or Leu;

[0341] X 41 is Thr, Asn, Val, Leu, or Pro;

[0342] X 42 is Gly, Thr or Pro;

[0343] X 43 It is Ser, Gly, Ala, Pro, Thr, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0344] X 44 is Asn, Ser, His, or Thr;

[0345] X 45 is Thr, Asn, Ala, or Val;

[0346] X 46 is Tyr, Thr, Pro, Val, or Gly;

[0347] X 47 The deletion is either Tyr, Gly or Ser;

[0348] X 48 Deletion is either Ser or His;

[0349] X 49 The deletion is either His or Thr;

[0350] X 50 Absent or Thr or Pro;

[0351] X 51 Missing or Pro;

[0352] in

[0353] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0354] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0355] n is an integer from 12 to 20.

[0356] In certain embodiments, R 3 Yes-CO 2 H.

[0357] In certain embodiments, R 3 Yes-CO 2 CH 3 .

[0358] In certain embodiments, R 3 Yes-CO 2 NH 2 .

[0359] In certain embodiments, R 3 Yes-CO 2 NHCH 3 .

[0360] In certain embodiments, R 3 Yes-CO 2 N(CH 3 ) 2 .

[0361] In certain embodiments, R 3 Yes-CH 3 .

[0362] In certain embodiments, R 3 Yes-NH 2 .

[0363] In certain embodiments, n is 12.

[0364] In certain embodiments, n is 13.

[0365] In certain embodiments, n is 14.

[0366] In certain embodiments, n is 15.

[0367] In certain embodiments, n is 16.

[0368] In certain embodiments, n is 17.

[0369] In certain embodiments, n is 18.

[0370] In certain embodiments, n is 19.

[0371] In certain embodiments, n is 20.

[0372] In certain embodiments, the peptide of the present invention is a peptide of Formula I or a pharmaceutically acceptable salt thereof, wherein:

[0373] X 1 It's Tyr;

[0374] X 2 It's Ala;

[0375] X 3 It is Glu;

[0376] X 4 It's Ala;

[0377] X 5 It is Gln;

[0378] X 6 It is Ile;

[0379] X 7 It's Gly;

[0380] X 8 It is Pro;

[0381] X 9 missing or Ser;

[0382] X 10 missing or Gly;

[0383] X 11 missing or Ala;

[0384] X 12 Missing or Pro;

[0385] X 13 Missing or Pro;

[0386] X 14 Missing or Pro;

[0387] X 15 missing or Ser;

[0388] X 16 It is Cys;

[0389] X 17 It is Asn;

[0390] X 18 It is Thr;

[0391] X 19 It's Ala;

[0392] X 20 It is Thr;

[0393] X 21 It is Cys;

[0394] X 22 It's Val;

[0395] X 23 It's Leu;

[0396] X 24 It's Gly;

[0397] X 25 is Arg;

[0398] X 26 It's Leu;

[0399] X 27 It is Ser;

[0400] X 28 It is Gln;

[0401] X 29 It is Glu;

[0402] X 30 It's Leu;

[0403] X 31 missing or His;

[0404] X 32 missing or Arg;

[0405] X 33 Missing or Leu;

[0406] X 34 missing or Gln;

[0407] X 35 missing or Thr;

[0408] X 36 It's Tyr;

[0409] X 37 It is Pro;

[0410] X 38 is Arg;

[0411] X 39 It is Thr;

[0412] X 40 It is Asn;

[0413] X 41 It is Thr;

[0414] X 42 It's Gly;

[0415] X 43 It is Ser;

[0416] X 44It is Asn;

[0417] X 45 It is Thr;

[0418] X 46 It's Tyr;

[0419] X 47 missing or Tyr;

[0420] X 48 missing or Ser;

[0421] X 49 missing or His;

[0422] X 50 Absent or Thr; and / or

[0423] X 51 Missing or Pro.

[0424] In another embodiment, the present invention encompasses a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of an amino acid sequence of formula (II):

[0425] Y-Aib-EGTFTSDYSI-Aib-LDKIAQX 1 -AFVQWLIAGGPSSGAPPPSCN-TATCVLGRLSQELHRLQTYPR-TNTGX 2 -NTY-(NR 1 R 2 )

[0426] (II)

[0427] in

[0428] X 1 Is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0429] X 2 It is Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0430] in

[0431] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0432] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0433] n is an integer from 12 to 20.

[0434] In certain embodiments, the peptide of the present invention is a peptide of Formula II or a pharmaceutically acceptable salt thereof, wherein:

[0435] X 1 is Gln; and / or

[0436] X 2 It's Ser.

[0437] In certain embodiments, the peptide of the present invention is a peptide of Formula II or a pharmaceutically acceptable salt thereof, wherein:

[0438] X 1 Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 ; and / or

[0439] X 2 It's Ser.

[0440] In certain embodiments, the peptide of the present invention is a peptide of Formula II or a pharmaceutically acceptable salt thereof, wherein:

[0441] X 1 is Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3) and / or

[0442] X 2 It's Ser.

[0443] In certain embodiments, the peptide of the present invention is a peptide of Formula II or a pharmaceutically acceptable salt thereof, wherein:

[0444] X 1 is Gln; and / or

[0445] X 2 Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 .

[0446] In certain embodiments, the peptide of the present invention is a peptide of Formula II or a pharmaceutically acceptable salt thereof, wherein:

[0447] X 1 is Gln; and / or

[0448] X 2 is Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 ).

[0449] In another embodiment, the present invention encompasses a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of an amino acid sequence of formula (III):

[0450] X 1 -X 2 -EGTFTSDYSIX 3 -LDKIAQQAFVQWLX 4 -AGX 5 -X 6 -SCNTATCVLGRLSQELHRLQT-YPRTNTGX 7 -NTY-(NR 1 R 2 )

[0451] (III)

[0452] in

[0453] X 1 is Tyr or (d)Tyr;

[0454] X 2It is Aib or Ala;

[0455] X 3 It is Aib or Ala;

[0456] X 4 It is Ile or Leu;

[0457] X 5 Is Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0458] X 6 It is Pro or Gly;

[0459] X 7 It is Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0460] in

[0461] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0462] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0463] n is an integer from 12 to 20.

[0464] In certain embodiments, the peptide of the present invention is a peptide of Formula III or a pharmaceutically acceptable salt thereof, wherein:

[0465] X1 It's Tyr;

[0466] X 2 It is Aib;

[0467] X 3 It is Aib;

[0468] X 4 It is Ile;

[0469] X 5 It's Gly;

[0470] X 6 is a Pro; and / or

[0471] X 7 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 ).

[0472] In certain embodiments, the peptide of the present invention is a peptide of Formula III or a pharmaceutically acceptable salt thereof, wherein:

[0473] X 1 Yes (d) Tyr;

[0474] X 2 It's Ala;

[0475] X 3 It is Aib;

[0476] X 4 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0477] X 5 It's Gly;

[0478] X 6 is Gly; and / or

[0479] X 7 It's Ser.

[0480] The present invention encompasses a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of an amino acid sequence of formula (IV):

[0481] X 1 -Aib-EGTFTSDYSI-Aib-LDKIAQQAFVQWLIAGX 2 -X 3 -SX 4 -CNTATCVLGRLSQELHRLQTY-PRTNTGX 5 -NTY-(NR 1 R 2 )

[0482] (IV)

[0483] in

[0484] X 1 is His or ImPrA;

[0485] X 2 Is Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0486] X 3 It is Gly or Pro;

[0487] X 4 Missing or Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0488] X 5 It is Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R3 );

[0489] in

[0490] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0491] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0492] n is an integer between 12 and 20.

[0493] In certain embodiments, the peptide of the present invention is a peptide of Formula IV or a pharmaceutically acceptable salt thereof, wherein:

[0494] X 1 It is His;

[0495] X 2 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0496] X 3 It's Gly;

[0497] X 4 missing; and / or

[0498] X 5 It's Ser.

[0499] In certain embodiments, the peptide of the present invention is a peptide of Formula IV or a pharmaceutically acceptable salt thereof, wherein:

[0500] X 1 It is ImPrA;

[0501] X 2 It's Lys;

[0502] X3 It is Pro;

[0503] X 4 missing; and / or

[0504] X 5 It's Ser.

[0505] In certain embodiments, the peptide of the present invention is a peptide of Formula IV or a pharmaceutically acceptable salt thereof, wherein:

[0506] X 1 It is ImPrA;

[0507] X 2 It's Gly;

[0508] X 3 It is Pro;

[0509] X 4 Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 ); and / or

[0510] X 5 It's Ser.

[0511] In certain embodiments, the peptide of the present invention is a peptide of Formula IV or a pharmaceutically acceptable salt thereof, wherein:

[0512] X 1 It is His;

[0513] X 2 It's Gly;

[0514] X 3 It is Pro;

[0515] X 4 Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );and

[0516] X 5 It's Ser.

[0517] In another embodiment, the present invention encompasses a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of an amino acid sequence of formula (V):

[0518] X 1 -X 2 -EGTFTSDYSI-Aib-LDKIAQQAFVQWLIAGGPSX 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -QX 23 -X 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -X 39 -X 40 (NR 1 R 2 )

[0519] Formula (V)

[0520] in

[0521] X 1 is Tyr or (d)Tyr;

[0522] X 2 It is Aib or Ala;

[0523] X 3It is Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0524] X 4 missing or Gly;

[0525] X 5 is Cys or Ala;

[0526] X 6 is Asn or Pro;

[0527] X 7 It is Thr or Pro;

[0528] X 8 It is Ala or Pro;

[0529] X 9 is Thr or Ser;

[0530] X 10 is Cys, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0531] X 11 is Val or Cys;

[0532] X 12 is Leu or Asn;

[0533] X 13 is Gly or Thr;

[0534] X 14 is Arg or Ala;

[0535] X 15 is Leu or Thr;

[0536] X 16 is Ser or Cys;

[0537] X 17 is Gln or Val;

[0538] X18 is Glu or Leu;

[0539] X 19 is Leu or Gly;

[0540] X 20 is His or Arg;

[0541] X 21 is Arg or Leu;

[0542] X 22 is Leu or Ser;

[0543] X 23 is Thr or Glu;

[0544] X 24 Missing or Leu;

[0545] X 25 missing or His;

[0546] X 26 missing or Arg;

[0547] X 27 Missing or Leu;

[0548] X 28 Missing or Gln.

[0549] X 29 is Tyr or Thr;

[0550] X 30 Is Pro or Tyr;

[0551] X 31 is Arg or Pro;

[0552] X 32 is Thr or Arg;

[0553] X 33 is Asn or Thr;

[0554] X 34 is Thr or Asn;

[0555] X 35 is Gly or Thr;

[0556] X 36 It is Ser, Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2 ) n R 3 );

[0557] X 37 is Asn or Ser;

[0558] X 38 is Thr or Asn;

[0559] X 39 is Tyr or Thr;

[0560] X 40 missing or Tyr;

[0561] in

[0562] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0563] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0564] n is an integer from 12 to 20.

[0565] In certain embodiments, the peptide of the present invention is a peptide of Formula V or a pharmaceutically acceptable salt thereof, wherein:

[0566] X 1 It's Tyr;

[0567] X 2 It is Aib;

[0568] X 3 It is Ser;

[0569] X 4 missing;

[0570] X 5 It is Cys;

[0571] X 6 It is Asn;

[0572] X 7 It is Thr;

[0573] X 8 It's Ala;

[0574] X 9 It is Thr;

[0575] X 10 It is Cys;

[0576] X 11 It's Val;

[0577] X 12 It's Leu;

[0578] X 13 It's Gly;

[0579] X 14 is Arg;

[0580] X 15 It's Leu;

[0581] X 16 It is Ser;

[0582] X 17 It is Gln;

[0583] X 18 It is Glu;

[0584] X 19 It's Leu;

[0585] X 20 It is His;

[0586] X 21 is Arg;

[0587] X 22 It's Leu;

[0588] X 23 It is Thr;

[0589] X 24 missing;

[0590] X 25 missing;

[0591] X 26 missing;

[0592] X 27 missing;

[0593] X 28 missing;

[0594] X 29 It's Tyr;

[0595] X 30 It is Pro;

[0596] X 31 is Arg;

[0597] X 32 It is Thr;

[0598] X 33 It is Asn;

[0599] X 34 It is Thr;

[0600] X 35 It's Gly;

[0601] X 36 It is Ser;

[0602] X 37 It is Asn;

[0603] X 38 It is Thr;

[0604] X 39 is Tyr; and / or

[0605] X 40 Missing.

[0606] In certain embodiments, the peptide of the present invention is a peptide of Formula V or a pharmaceutically acceptable salt thereof, wherein:

[0607] X 1 Yes (d) Tyr;

[0608] X 2 It's Ala;

[0609] X 3 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0610] X 4 It's Gly;

[0611] X 5 It's Ala;

[0612] X 6 It is Pro;

[0613] X 7 It is Pro;

[0614] X 8 It is Pro;

[0615] X 9 It is Ser;

[0616] X 10 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0617] X 11 It is Cys;

[0618] X 12 It is Asn;

[0619] X 13 It is Thr;

[0620] X 14 It's Ala;

[0621] X 15 It is Thr;

[0622] X 16 It is Cys;

[0623] X 17 It's Val;

[0624] X 18 It's Leu;

[0625] X 19 It's Gly;

[0626] X 20 is Arg;

[0627] X 21 It's Leu;

[0628] X 22 It is Ser;

[0629] X 23 It is Glu;

[0630] X 24 It's Leu;

[0631] X 25 It is His;

[0632] X 26 is Arg;

[0633] X 27 It's Leu;

[0634] X 28 It is Gln;

[0635] X 29 It is Thr;

[0636] X 30 It's Tyr;

[0637] X 31 It is Pro;

[0638] X 32 is Arg;

[0639] X 33 It is Thr;

[0640] X 34 It is Asn;

[0641] X 35 It is Thr;

[0642] X 36 It's Gly;

[0643] X 37 It is Ser;

[0644] X 38 It is Asn;

[0645] X 39 is Thr; and / or

[0646] X 40 It's Tyr.

[0647] In another embodiment, the present invention encompasses a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of an amino acid sequence of formula (VI):

[0648] X 1 -X 2 -QGTFTSDYSI-Aib-LDKIAQX 3 -AFVQWLIAGX 4 -X 5 -SX 6 -CNTATCVLGRLSQELHRLQTY-PRTNTGSNTY-(NR 1 R 2 )

[0649] (VI)

[0650] in

[0651] X 1 It is His or Tyr;

[0652] X 2 It is Aib or Ser;

[0653] X 3 Is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0654] X 4 Is Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0655] X 5 It is Pro or Gly;

[0656] X 6 Missing or Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0657] in

[0658] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0659] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0660] n is an integer from 12 to 20, and

[0661] z is 0 or 1.

[0662] In certain embodiments, the peptide of the present invention is a peptide of Formula VI or a pharmaceutically acceptable salt thereof, wherein:

[0663] X 1 It's Tyr;

[0664] X 2 It is Aib;

[0665] X 3 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0666] X 4 It's Gly;

[0667] X 5 is a Pro; and / or

[0668] X 6 Missing or Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 ).

[0669] In certain embodiments, the peptide of the present invention is a peptide of Formula VI or a pharmaceutically acceptable salt thereof, wherein:

[0670] X 1 It's Tyr;

[0671] X 2 It is Aib;

[0672] X 3 It is Gln;

[0673] X 4is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0674] X 5 is Gly; and / or

[0675] X 6 Missing or Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 ).

[0676] In certain embodiments, the peptide of the present invention is a peptide of Formula VI or a pharmaceutically acceptable salt thereof, wherein:

[0677] X 1 It is His;

[0678] X 2 It is Ser;

[0679] X 3 It is Gln;

[0680] X 4 It's Gly;

[0681] X 5 is a Pro; and / or

[0682] X 6 Missing or Lys or Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 .

[0683] In other embodiments, the present invention encompasses a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of an amino acid sequence of formula (VII):

[0684] Y-Aib-EGTFTSDYSI-Aib-LDKIAQQAFVQWLIAGX 1 -X 2 -SX 3 -X4 -X 5 -NX 6 -X 7 -TCX 8 -X 9 -X 10 -RLX 11 -X 12 -X 13 -LX 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -PX 25 -TX 26 -VGX 27 -X 28 -X 29 -P-(NR 1 R 2 )

[0685] (VII)

[0686] in

[0687] X 1 Is Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0688] X 2 It is Gly or Pro;

[0689] X 3 Missing or Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0690] X 4 Missing or Cys;

[0691] X 5is Ser or Cys;

[0692] X 6 is Leu or Thr;

[0693] X 7 is Ser or Ala;

[0694] X 8 is Met, Val or Ala;

[0695] X 9 is Leu or Thr;

[0696] X 10 is Gly or Gln;

[0697] X 11 is Ser or Ala;

[0698] X 12 is Gln or Glu;

[0699] X 13 is Asp or Phe;

[0700] X 14 is His or Arg;

[0701] X 15 is Arg or His;

[0702] X 16 is Leu or Ser;

[0703] X 17 is Gln or Ser;

[0704] X 18 is Thr or Asn;

[0705] X 19 missing or Asn;

[0706] X 20 missing or Phe;

[0707] X 21 missing or Gly;

[0708] X 22 Missing or Pro;

[0709] X 23 Missing or Ile;

[0710] X 24 It is Tyr or Leu;

[0711] X 25It is Lys, Pro, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0712] X 26 is Asp or Asn;

[0713] X 27 is Ala or Ser;

[0714] X 28 is Asn or His;

[0715] X 29 is Ala or Thr;

[0716] in

[0717] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0718] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0719] n is an integer from 12 to 20.

[0720] In certain embodiments, the peptide of the present invention is a peptide of Formula VII or a pharmaceutically acceptable salt thereof, wherein:

[0721] X 1 It's Gly;

[0722] X 2 It's Gly;

[0723] X 3 Missing or Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0724] X 4 Missing or Cys;

[0725] X 5 It is Ser;

[0726] X 6 It's Leu;

[0727] X 7 It is Ser;

[0728] X 8 It's Met;

[0729] X 9 It's Leu;

[0730] X 10 It's Gly;

[0731] X 11 It is Ser;

[0732] X 12 It is Gln;

[0733] X 13 It is Asp;

[0734] X 14 It is His;

[0735] X 15 is Arg;

[0736] X 16 It's Leu;

[0737] X 17 It is Gln;

[0738] X 18 It is Thr;

[0739] X 19 missing or Asn;

[0740] X 20 missing or Phe;

[0741] X 21 missing or Gly;

[0742] X 22 Missing or Pro;

[0743] X 23 Missing or Ile;

[0744] X 24 It's Tyr;

[0745] X 25 It's Lys;

[0746] X 26 It is Asp;

[0747] X 27 It's Ala;

[0748] X 28 is Asn; and / or

[0749] X 29 It's Ala.

[0750] In certain embodiments, the peptide of the present invention is a peptide of Formula VII or a pharmaceutically acceptable salt thereof, wherein:

[0751] X 1 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0752] X 2 It is Pro;

[0753] X 3 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 ).

[0754] X 4 It is Cys;

[0755] X 5 It is Cys;

[0756] X 6 It is Thr;

[0757] X 7 It's Ala;

[0758] X 8 It is Val or Ala;

[0759] X 9 It is Thr;

[0760] X 10 It is Gln;

[0761] X 11 It's Ala;

[0762] X 12 It's Glu.

[0763] X 13 It is Phe;

[0764] X 14 is Arg;

[0765] X 15 It is His;

[0766] X 16 It is Ser;

[0767] X 17 It is Ser;

[0768] X 18 It is Asn;

[0769] X 19 It is Asn;

[0770] X 20 It is Phe;

[0771] X 21 It's Gly;

[0772] X 22 It is Pro;

[0773] X 23 It is Ile;

[0774] X 24 It's Leu;

[0775] X 25 It is Pro, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0776] X 26 It is Asn;

[0777] X 27 It is Ser;

[0778] X 28 is His; and / or

[0779] X 29 It's Thr.

[0780] In another embodiment, the present invention encompasses a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of an amino acid sequence having formula (VIII):

[0781] X 1 -X 2 -EGTFTSDYSI-Aib-LDKIAQX 3 -AFVQWLIAGX 4 -X 5 -SX 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -X 39 -X 40 -X 41 -X 42 -X 43 -X 44 -X 45 -X 46 -X 47 -X 48 (NR 1 R 2 )

[0782] (VIII)

[0783] in

[0784] X 1 is Tyr, His, or ImPra;

[0785] X 2 is Aib or Gly;

[0786] X 3 Is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0787] X 4 Is Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0788] X 5 It is Pro or Gly;

[0789] X 6 Missing or Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0790] X 7 missing or Gly;

[0791] X 8 The deletion is either Ala or Cys;

[0792] X 9 The deletion is either Pro or Asn;

[0793] X 10 Missing either Pro or Thr;

[0794] X 11 Missing either Pro or Ala;

[0795] X 12 Missing or Ser, Cys or Thr;

[0796] X 13 is Cys or Asn;

[0797] X 14 is Asn, Thr, or Val;

[0798] X 15 is Thr, Ala, or Leu;

[0799] X 16 is Ala, Thr or Gly;

[0800] X 17 is Thr, Cys or Arg;

[0801] X 18 is Cys, Ala or Leu;

[0802] X 19 is Ala, Thr or Ser;

[0803] X 20 is Thr or Gln;

[0804] X 21 is Gln, Arg or Glu;

[0805] X 22 is Arg or Leu;

[0806] X 23 is Leu, Ala or His;

[0807] X 24 is Ala, Glu or Arg;

[0808] X 25 is Glu, Phe or Leu;

[0809] X 26 is Phe, Leu or Gln;

[0810] X 27 is Leu, Arg, or Thr;

[0811] X 28 The deletion is either Arg or His;

[0812] X 29 The deletion is either His or Ser;

[0813] X 30 missing or Ser;

[0814] X 31 Missing either Ser or Asn;

[0815] X 32 missing or Asn;

[0816] X 33 is Asn, Phe or Tyr;

[0817] X 34 is Phe, Gly or Pro;

[0818] X 35 is Gly, Pro or Arg.

[0819] X 36 is Pro, Ile or Thr;

[0820] X 37 is Ile, Leu or Asn;

[0821] X 38 is Leu, Pro or Thr;

[0822] X 39 It is Pro or Gly;

[0823] X 40 It is Pro, Thr, Lys, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 );

[0824] X 41 is Thr or Asn;

[0825] X 42 is Asn, Val, or Thr;

[0826] X 43 is Val, Gly, or Tyr;

[0827] X 44 The deletion is either Gly or Ser;

[0828] X 45 Deletion is either Ser or His;

[0829] X 46 The deletion is either His or Thr;

[0830] X 47Absent or Thr or Pro;

[0831] X 48 Missing or Pro;

[0832] in

[0833] R 1 and R 2 Each independently is H or C 1-5 alkyl,

[0834] R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 、-CH 3 or -NH 2 ,

[0835] n is an integer from 12 to 20.

[0836] In certain embodiments, the peptide of the present invention is a peptide of Formula VIII or a pharmaceutically acceptable salt thereof, wherein:

[0837] X 1 It's Tyr;

[0838] X 2 It is Aib;

[0839] X 3 It is Gln;

[0840] X 4 It's Gly;

[0841] X 5 It is Pro;

[0842] X 6 missing or Ser;

[0843] X 7 missing or Gly;

[0844] X 8 missing or Ala;

[0845] X 9 Missing or Pro;

[0846] X 10 Missing or Pro;

[0847] X11 Missing or Pro;

[0848] X 12 missing or Ser;

[0849] X 13 It is Cys;

[0850] X 14 It is Asn;

[0851] X 15 It is Thr;

[0852] X 16 It's Ala;

[0853] X 17 It is Thr;

[0854] X 18 It's Ala;

[0855] X 19 It is Thr;

[0856] X 20 It is Gln;

[0857] X 21 is Arg;

[0858] X 22 It's Leu;

[0859] X 23 It's Ala;

[0860] X 24 It is Glu;

[0861] X 25 It is Phe;

[0862] X 26 It's Leu;

[0863] X 27 is Arg;

[0864] X 28 missing or Arg;

[0865] X 29 missing or His;

[0866] X 30 missing or Ser;

[0867] X 31 missing or Ser;

[0868] X 32 missing or Asn;

[0869] X 33 It is Phe;

[0870] X 34 It's Gly;

[0871] X 35 It is Pro;

[0872] X 36 It is Ile;

[0873] X 37 It's Leu;

[0874] X 38 It is Pro;

[0875] X 39 It is Pro;

[0876] X 40 It is Thr;

[0877] X 41 It is Asn;

[0878] X 42 It's Val;

[0879] X 43 It's Val;

[0880] X 44 missing or Gly;

[0881] X 45 missing or Ser;

[0882] X 46 missing or His;

[0883] X 47 Absent or Thr; and / or

[0884] X 48 Missing or Pro.

[0885] In certain embodiments, exemplary peptide components of the peptides of the present invention include fragments of component peptide hormones selected from the following: amylin, ADM, CT, CGRP, intermedin, CCK, leptin, PYY (1-36), PYY (3-36), GLP-1 (1-37), GLP-1 (7-37), GLP-1 (7-36), GLP-2, OXM, natriuretic peptides, corticotropin family peptides (e.g., Ucn-2 and Ucn-3), neuromedin family peptides (e.g., neuromedin U25 or splice variants), exendin-3 and exendin-4, wherein the fragment exhibits at least one hormonal activity of the component peptide.

[0886] Other exemplary peptide components of the peptides of the present invention include fragments of analogs or derivatives of the component peptide hormones selected from the group consisting of amylin, ADM, CT, CGRP, intermedin, CCK, leptin, GLP-1 (1-37), GLP-1 (7-37), GLP-1 (7-36), GLP-2, human catecholamine inhibitor, OXM, ANP, BNP, CNP, urodilatin, FGF-19, FGF-21, Ucn-2 and Ucn-3, neuregulin U25 or splice variants, neuregulin S, exendin-3 and exendin-4, wherein the fragment exhibits at least one hormonal activity of the component peptide hormone. Similarly, analogs may include one or more insertions, deletions or substitutions of the amino acid sequence of the component peptide hormone, and derivatives may include one or more chemical modifications of the amino acid residues of the analog or component peptide hormone, as more fully described herein and known in the art.

[0887] Certain exemplary fragments that exhibit at least one hormonal activity include the following.

[0888] Amylin: Amylin (2-37), Amylin (1-35), Amylin (1-20), Amylin (1-18), Amylin (1-17), Amylin (1-16), Amylin (1-15), Amylin (1-7)

[0889] GLP-1:GLP-1(7-37),GLP-1(7-36),GLP-1(7-35)

[0890] GIP: GIP(1-14), GIP(1-28), GIP(1-30) or longer, GIP(1-39) or longer

[0891] Exendin: Exendin-4(1-27), Exendin-4(1-28), Exendin-4(1-29), Exendin-4(1-30) or longer.

[0892] The peptides of the present invention may be amidated, but in the context of the present invention, unless otherwise indicated, may optionally be in acid form. In addition, the above exemplary peptides may be combined with any analogs or derivatives discussed herein or known in the art. For example, exemplary analog fragments may include 5Ala, 14Leu, 25Phe-Exendin-4 (1-28), 14Leu, 25Phe-Exendin-4 (1-27), 5Ala, 14Leu, 25Phe-Exendin-4 (1-28), 14Leu, 25Phe-Exendin-4 (1-27), or any other combination of disclosed fragments, analogs, and derivatives.

[0893] Other exemplary peptide components include structural motifs of component peptide hormones (including analogs and derivatives thereof) that confer desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding and / or other pharmacokinetic characteristics to the peptides. Exemplary peptide components of the peptides of the present invention include the following.

[0894] Amylin family: Amylin (32-37), Amylin (33-37), Amylin (34-37), Amylin (35-37), Amylin (36-37), Amylin (37), ADM (47-52), ADM (48-52), ADM (49-52), ADM (50-52), ADM (51-52), ADM (52), CT (27-32), CT (27-32), CT (28-32), CT ( 29-32),CT(30-32),CT(31-32),CT(32),CGRP(32-37),CGRP(33-37),CGRP(34-37),CGRP(35-37),CGRP(36-37),CGRP(37),interleukin(42-47),interleukin(43-47),interleukin(44-47),interleukin(45-47),interleukin(46-47),interleukin(47).

[0895] GLP-1 and 2: GLP-1(29-37); GLP-1(30-37); GLP-2(24-31), GLP-2(25-31).

[0896] GIP:GIP(31-42),GIP(32-42),GIP(33-42),GIP(34-42),GIP(35-42),GIP(36- 42),GIP(37-42),GIP(38-42),GIP(39-42),GIP(40-42),GIP(41-42),GIP(42).

[0897] Exendin-4: Exendin-4(31-39), Exendin-4(32-39), Exendin-4(33-39), Exendin-4(34-39), Exendin-4(35-39), Exendin-4(36-39), Exendin-4(37-39), Exendin-4(38-39), Exendin-4(39)

[0898] In certain embodiments, combinations of GIP analogs and derivatives described herein with peptide components are contemplated. For example, the last six amino acid residues of the amylin family peptide hormone analogs and derivatives known in the art and / or described above are also considered exemplary polypeptide components. For example, as further discussed herein, the peptide enhancer Ex-4 short tail, which is an exemplary Trp-Cage sequence, or an analog thereof, is added to the C-terminus of any GIP analog, and in further embodiments, a linker is used to connect the peptide enhancer.

[0899] In one aspect, the peptides of the invention include a GIP portion that exhibits at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to native GIP (1-30), native GIP (1-26), native GIP (1-14), native GIP (1-39), native GIP (19-30), native GIP (19-26), native GIP (19-39), native GIP (19-42) or native GIP (1-42) over the entire length of the GIP portion.

[0900] Therefore, in certain embodiments, the peptides of the present invention may comprise a Trp-Cage motif. In certain embodiments, the peptides of the present invention include N-terminal GIP or novel GIP analog fragments in combination with a C-terminal polypeptide or fragment thereof, having weight loss or blood sugar lowering activity (e.g., antidiabetic drugs, exenatide) or the ability to inhibit or reduce gastric emptying. In certain embodiments, the peptides of the present invention include N-terminal GIP fragments or novel GIP analog fragments in combination with: C-terminal exenatide, GLP1, amylin, CCK, gastrin, secretin, GRP, neuromodulin, corticotropin, calcitonin or salmon calcitonin or fragments thereof. In other embodiments, the peptides of the present invention include C-terminal GIP or novel GIP analog fragments in combination with an N-terminal polypeptide or fragment thereof, having weight loss or blood sugar lowering activity (e.g., antidiabetic drugs, exenatide) or the ability to inhibit or reduce gastric emptying. In certain embodiments, the peptides of the invention include a C-terminal GIP, a novel GIP analog (in which case a Trp-Cage forming sequence is present), or a fragment thereof in combination with an N-terminal exenatide, GLP1, amylin, CCK, gastrin, secretin, GRP, neuregulin, corticotropin, calcitonin, or salmon calcitonin, or a fragment thereof.

[0901] In other embodiments, the peptide components of the peptides of the present invention are combined with: gastrin / CCK receptor ligands; amylin receptor ligands; calcitonin receptor ligands; CGRP receptor ligands, EGF receptor ligands; glucagon-like peptide 1 receptor ligands; glucagon-like peptide 2 receptor ligands; gastric inhibitory polypeptide (GIP) receptor ligands; keratinocyte growth factor (KGF) receptor 1 ligands; dipeptidyl peptidase IV inhibitors; REG protein receptor ligands; growth hormone receptor ligands; prolactin (PRL) receptor ligands; insulin-like growth factor (IGF) receptor ligands; PTH-related protein (PTHrP) receptor ligands; hepatocyte growth factor (HGF) receptor ligands; bone morphogenetic protein (BMP) receptors ligands; transforming growth factor (TGF) receptor ligands; laminin receptor ligands; vasoactive intestinal peptide (VIP) receptor ligands; fibroblast growth factor (FGF) receptor ligands; nerve growth factor (NGF) receptor ligands; islet neogenesis-associated protein (INGAP) receptor ligands; activin A receptor ligands; vascular endothelial growth factor (VEGF) receptor ligands; erythropoietin (EPO) receptor ligands; pituitary adenylate cyclase activating polypeptide (PACAP) receptor ligands; granulocyte colony stimulating factor (G-CSF) receptor ligands; granulocyte-macrophage colony stimulating factor (GM-CSF); platelet-derived growth factor (PDGF) receptor ligands; and secretin receptor ligands.

[0902] The peptides of the present invention preferably retain at least in part the biological activity of natural human GIP, for example, the peptides of the present invention are generally GIP agonists or antagonists. In one embodiment, the peptides of the present invention exhibit biological activity in the treatment and prevention of metabolic diseases and disorders. In addition, the novel GIP analogs of the peptides of the present invention may include internal linker compounds, may include chemical modifications at internal amino acid residues, or may be chemically modified at the N-terminal or C-terminal residues. In another embodiment, the peptides of the present invention include only natural L amino acid residues and / or modified natural L amino acid residues. Alternatively, in another embodiment, the peptides of the present invention do not include non-natural amino acid residues.

[0903] In an exemplary embodiment, the GIP portion of the peptides of the invention comprises a modified or substituted N-terminal region of GIP to provide improved DPP-IV resistance over native GIP.

[0904] In an exemplary embodiment, the peptides of the invention include GIP or novel GIP analogs in combination with amylin family peptides, including amylin, adrenomedullin ("ADM"), calcitonin ("CT"), calcitonin gene-related peptide ("CGRP"), interferon (also known as "AFP-6"), and related peptides. Natural amylin family peptide hormones are known in the art, as well as functional peptide analogs and derivatives. Certain exemplary natural peptides, peptide analogs, and derivatives are described herein, but it should be recognized that any amylin family peptide known in the art that has hormonal activity may be used in conjunction with the present invention. Any amylin analog or derivative known in the art may be used in conjunction with the present invention.

[0905] Amylin family peptide hormones are involved in metabolic diseases and disorders, including amylin, calcitonin, calcitonin gene-related peptide, adrenomedullin and intermedin (also known as "AFP-6"). In certain embodiments, the peptides of the present invention comprise one or more amylin family peptides as component peptides. Amylin is a 37 amino acid peptide hormone. It was isolated, purified and chemically characterized as the major component of amyloid deposits in the pancreatic islets of human type 2 diabetic patients (Cooper et al., Proc. Natl. Acad. Sci., USA, 84: 8628-8632 (1987)). The amylin molecule has two post-translational modifications: C-terminal amidation, and cross-linking of cysteines at positions 2 and 7 to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of a Lys-Arg dibasic amino acid proteolytic cleavage signal before the N-terminal codon of Lys, and a Gly before the Lys-Arg proteolytic signal at the N-terminal position, which is a typical sequence for amidation by the protein amidating enzyme PAM (Cooper et al., Biochem. Biophys. Acta, 1014: 247-258 (1989)). "Adrenomedullin" or "ADM" refers to a human peptide hormone and its species variants. More specifically, ADM is generated from a 185 amino acid preprohormone by sequential enzymatic cleavage and amidation. This process ultimately releases a 52 amino acid bioactive peptide. "Calcitonin" or "CT" refers to a human peptide hormone and its species variants, including salmon calcitonin ("sCT"). More specifically, CT is a 32 amino acid peptide cleaved from a larger prohormone. It contains a single disulfide bond, which makes the amino terminus cyclic. Alternative splicing of the calcitonin pre-mRNA can generate an mRNA encoding the calcitonin gene-related peptide; the peptide appears to play a role in the nervous and vascular systems. 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" refers to the human peptide hormone and its species variants, in any physiological form. "Intercalcin" or "AFP-6" refers to the human peptide hormone and its species variants, in any physiological form.

[0906] In certain embodiments, the peptides of the present invention include amylin peptides as component peptides. Amylin is believed to regulate gastric emptying, inhibit glucagon secretion and food intake, thereby regulating the rate of glucose appearance in the circulation. It appears to complement the action of insulin, which regulates the rate of disappearance of glucose from the circulation and its uptake in peripheral tissues. These actions are supported by experimental findings in rodents and humans, which show that amylin complements the role of insulin in postprandial blood glucose control through at least three independent mechanisms, all of which affect the rate of glucose appearance. First, amylin inhibits postprandial glucagon secretion. Compared with healthy adults, patients with type 1 diabetes have no circulating amylin, and patients with type 2 diabetes have reduced postprandial amylin concentrations. In addition, infusion of amylin-specific monoclonal antibodies that bind to circulating amylin again resulted in glucagon concentrations that were significantly higher than those in the control group. Both results indicate the physiological role of endogenous amylin in regulating postprandial glucagon secretion. Secondly, amylin slows gastrointestinal motility and gastric emptying. Finally, intrahypothalamic injections of rat amylin have been shown to reduce the amount of food eaten by rats and to alter neurotransmitter metabolism in the hypothalamus. In certain studies, food intake was significantly reduced for up to eight hours following intrahypothalamic injections of rat amylin and rat CGRP. In human trials, the amylin analog pramlintide has been shown to reduce body weight or prevent weight gain. Amylin may be useful in treating metabolic conditions such as diabetes and obesity. Amylin may also be used to treat pain, bone disorders, gastritis, for regulating lipids, especially triglycerides, or for influencing body composition, such as preferentially reducing fat and preserving lean tissue.

[0907] In certain embodiments, the peptides of the present invention include calcitonin as a component peptide. The hormone calcitonin (CT) is named for its secretion in response to induced hypercalcemia and its rapid calcium-lowering effect. It is produced and secreted by neuroendocrine cells in the thyroid gland, which are later called C cells. The most fully studied effect of CT (1-32) is its effect on osteoclasts. The in vitro effects of CT include rapid loss of ruffled borders and reduced release of lysosomal enzymes. Ultimately, the inhibition of osteoclast function by CT leads to reduced bone resorption. However, neither the chronic reduction of serum CT caused by thyroidectomy nor the increase in serum CT found in medullary thyroid cancer seems to be related to changes in serum calcium or bone mass. Therefore, it is most likely that the main function of CT (1-32) is to fight acute hypercalcemia in emergency situations and / or to protect bones during "calcium stress" such as growth, pregnancy and lactation. (For review, see Becker, JCEM, 89(4):1512-1525 (2004) and Sexton, Current Medicinal Chemistry 6:1067-1093 (1999)). Consistent with this, recent data from calcitonin knockout mice (which have both calcitonin and CGRP-I peptide deleted) show that mice have normal basal calcium-related values ​​but increased calcium responses (Kurihara H, et al., Hypertens Res. 2003 February; 26 Suppl:S105-8).

[0908] CT has an effect on plasma calcium levels and inhibits osteoclast function and is widely used to treat osteoporosis. Therapeutically, salmon CT (sCT) appears to increase bone density and reduce fracture rates with minimal adverse effects. Over the past 25 years, CT has also been used successfully to treat Paget's disease of bone, a chronic bone disorder that can cause bones to enlarge or deform in one or more skeletal areas. CT is also widely used to relieve bone pain experienced during osteoporosis, but the mechanism of this action is unclear.

[0909] In human studies, salmon calcitonin inhibited postprandial gastric emptying and gastrin release while causing dose-dependent relaxation of the gallbladder in both the fed and fasted states. In mice and monkeys, salmon calcitonin exhibited anorectic effects and resulted in weight loss after a single administration. In chronic studies, oral formulations of salmon calcitonin also reduced food intake and body weight in rat models of obesity and diabetes. Therefore, including a calcitonin component in treatment may aid in weight loss.

[0910] In certain embodiments, the peptides of the present invention include calcitonin gene-related peptide as a component peptide. Calcitonin gene-related peptide (CGRP) is a neuropeptide whose receptors are widely distributed in the body, including the nervous system and cardiovascular system. This peptide appears to regulate sensory neurotransmission and is one of the strongest endogenous vasodilator peptides discovered to date. The reported biological effects of CGRP include: regulation of substance P in inflammation, nicotinic receptor activity at neuromuscular junctions, stimulation of pancreatic enzyme secretion, reduction of gastric acid secretion, peripheral vasodilation, cardiac acceleration, neuromodulation, calcium metabolism regulation, osteogenic stimulation, insulin secretion, increased body temperature and reduced food intake. (Wimalawansa, Amylin, calcitonin gene-related peptide, calcitoninand ADM: a peptide superfamily. Crit. Rev Neurobiol. 1997; 11 (2-3): 167-239). The important role of CGRP is to control blood flow to various organs through its potent vasodilator effect, as evidenced by the decrease in mean arterial pressure after intravenous administration of α-CGRP. Recent analysis of homozygous knockout CGRP mice also supports this vasodilator effect, showing increased 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 February; 26 Suppl: S105-8). Therefore, among other effects, CGRP appears to cause vasodilator effects, hypotensive effects, and increased heart rate.

[0911] Prolonged CGRP infusions into patients with congestive heart failure showed sustained beneficial effects on hemodynamic function without adverse effects, suggesting its use in heart failure. Other indications for the use of CGRP include renal failure, acute and chronic coronary ischemia, treatment of arrhythmias, other peripheral vascular diseases such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension. Preeclampsia and preterm labor during pregnancy also have potential therapeutic possibilities (Wimalawansa, 1997). Recent therapeutic uses include the use of CGRP antagonists to treat migraine.

[0912] In certain embodiments, the peptides of the present invention include adrenomedullin as a component peptide. Adrenomedullin (ADM) is expressed in almost all tissues, and its content is much higher than that of tissues that do not contain the peptide. The published review article of ADM (Hinson, JP et al., Endocrine Reviews (2000) 21 (2): 138-167) details its effects on the cardiovascular system, cell growth, central nervous system and endocrine system, and has a series of biological effects, including vasodilation, cell growth, hormone secretion regulation and sodium diuresis. Studies in rats, cats, sheep and humans have confirmed that intravenous infusion of ADM leads to a potent and lasting hypotensive effect, which is comparable to CGRP. However, the hypotensive effect of ADM on the mean arterial pressure of anesthetized rats is not inhibited by the CGRP antagonist CGRP8-37, indicating that this effect is not mediated by the CGRP receptor. Acute or chronic administration of human ADM in anesthetized, awake or hypertensive rats leads to a significant decrease in total peripheral resistance, accompanied by a decrease in blood pressure, and an increase in heart rate, cardiac output and stroke volume.

[0913] ADM has also been proposed as an important factor in embryogenesis and differentiation and a cell apoptosis survival factor for rat endothelial cells. This has been supported by recent mouse ADM knockout studies, in which mice with homozygous deletion of the ADM gene showed defects in angiogenesis during embryogenesis and therefore died during mid-pregnancy. It is reported that ADM+ / -heterozygous mice have a tendency to hypertension and susceptibility to tissue damage (Kurihara H, et al., Hypertens Res. 2003 February; 26 Suppl: S105-8).

[0914] ADM affects endocrine organs such as the pituitary, adrenal glands, reproductive organs, and pancreas. The peptide appears to play a role in inhibiting the release of ACTH from the pituitary. In the adrenal glands, it appears to affect the secretory activity of the rat and human adrenal cortex and increase adrenal blood flow, acting as a vasodilator in the intact rat adrenal vascular bed. ADM has been shown to be present throughout the female reproductive tract, with elevated plasma levels during normal pregnancy. In studies of a rat model of preeclampsia, ADM reversed hypertension and reduced pup mortality when given to rats in late pregnancy. The absence of similar effects in early pregnant or nonpregnant rat animals in the preeclampsia model suggests that ADM may play an important regulatory role in the utero-placental cardiovascular system. In the pancreas, most likely, ADM plays an inhibitory role because it attenuates and delays the insulin response to an oral glucose load, resulting in an initial increase in blood glucose levels. ADM can also affect renal function. Peripherally administered bolus doses 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.

[0915] ADM also has other peripheral effects on bones and lungs. For bones, studies support its role outside the cardiovascular system and fluid balance, and prove that ADM has an effect comparable to known osteoblast growth factors (such as transforming growth factor-α) on the increase in cell growth of fetal and adult rodent osteoblasts. This is clinically important because one of the main challenges of osteoporosis research is to develop a therapy that increases bone mass by osteogenic stimulation. In the lungs, ADM not only causes pulmonary vasodilation, but also inhibits bronchoconstriction caused by histamine or acetylcholine. Recent studies using aerosol ADM to treat pulmonary hypertension in rat models have shown that inhalation treatment of this condition is effective, as demonstrated by the following facts: compared with rats given saline, the mean pulmonary artery pressure and total pulmonary resistance of rats treated with ADM are significantly lower. This result was achieved without changing systemic arterial pressure or heart rate (Nagaya N et al., Am J Physiol Heart CircPhysiol.2003; 285: H2125-31).

[0916] In healthy volunteers, intravenous infusion of ADM has been shown to reduce arterial pressure and stimulate heart rate, cardiac output, plasma cAMP, prolactin, norepinephrine and rennin levels. In these patients, little or no increase in urine volume 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 induces diuresis and natriuresis, depending on the dose administered (Nicholls, MG et al. Peptides. 2001; 22: 1745-1752). Experimental ADM treatment has also shown beneficial effects 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, preeclampsia and premature birth during pregnancy, and osteoporosis.

[0917] AFP-6 (i.e., intercalin) is expressed 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 receptors and compete with labeled CGRP for receptor binding in these cells. In published in vivo studies, administration of AFP-6 resulted in a decrease in blood pressure in normal and spontaneously hypertensive rats, most likely through interaction with CRLR / RAMP receptors. In vivo administration to mice resulted in inhibition of gastric emptying and food intake (Roh et al. J Biol. Chem. 2004 Feb. 20; 279(8): 7264-74).

[0918] It is reported that the biological actions of amylin family peptide hormones are generally mediated by binding to two closely related type II G protein-coupled receptors (GPCRs): calcitonin receptor (CTR) and calcitonin receptor-like receptor (CRLR). Cloning and functional studies have shown that CGRP, ADM and amylin interact with different combinations of CTR or CRLR and receptor activity modifying proteins (RAMPs). Many cells express multiple RAMPs. It is believed that co-expression of RAMP and CTR or CRLR is required for the generation of functional receptors for calcitonin, CGRP, ADM and amylin. The RAMP family includes three members (RAMP1, -2 and -3) with less than 30% sequence identity but a common topological organization. Co-expression of CRLR and RAMP1 leads to the formation of CGRP receptors. Co-expression of CRLR and RAMP2 leads to the formation of ADM receptors. Co-expression of CRLR and RAMP3 leads to the formation of receptors for ADM and CGRP. Co-expression of hCTR2 and RAMP1 leads to the formation of receptors for amylin and CGRP. Co-expression of hCTR2 and RAMP3 leads to the formation of amylin receptors.

[0919] In certain embodiments, peptides of the invention comprising an amylin family hormone module can provide functions and uses associated with an amylin family module, such as amylin, amylin / sCT / amylin, ADM, CGRP, as discussed, in addition to GIP functions.

[0920] In one embodiment, amylin analogs and derivatives have at least one hormonal activity of native amylin. In certain embodiments, amylin analogs are agonists that are capable of specifically binding to receptors of native amylin. Exemplary amylin analogs and derivatives include those described in US2003 / 0026812 A1, which is incorporated herein by reference.

[0921] In certain embodiments, the peptides of the present invention include CCK as a component peptide. CCK (including hCCK (cholecystokinin) and species variants, and various analogs thereof) is known in the art. In general, CCK has a 33 amino acid sequence first identified in humans, and includes an 8 amino acid C-terminal fragment ("CCK-8") in vivo, which has been reported to be demonstrated in pigs, rats, chickens, chinchillas, dogs and humans. Other species variants include a 39 amino acid sequence found in pigs, dogs and guinea pigs, a 58 amino acid sequence found in cats, dogs and humans, and a 47 amino acid sequence homologous to CCK and gastrin. The C-terminal tyrosine sulfated octapeptide sequence (CCK-8) is relatively conservative between species and may be the smallest sequence for peripheral biological activity in rodents. Therefore, unless otherwise specified, the term CCK-33 will generally refer to human CCK (1-33), while CCK-8 (CCK (26-33)) will generally refer to the C-terminal octapeptide regardless of whether it is sulfated. Furthermore, pentagastrin or CCK-5 will refer to the C-terminal peptide CCK(29-33), while CCK-4 will refer to the C-terminal tetrapeptide CCK(30-33).

[0922] CCK was identified from an intestinal extract preparation by its ability to stimulate gallbladder contraction. Since then, it has been reported that CCK also has other biological effects, including stimulating pancreatic secretion, delaying gastric emptying, stimulating intestinal motility and stimulating insulin secretion. See Lieverse et al., Ann.NY Acad. Sci. 713: 268-272 (1994). It is reported that the effects of CCK also include the effects on cardiovascular function, respiratory function, neurotoxicity and epilepsy, cancer cell proliferation, analgesia, sleep, sexual and reproductive behavior, memory, anxiety and dopamine-mediated behavior. Crawley and Corwin, Peptides 15: 731-755 (1994). It is reported that other effects of CCK include stimulating pancreatic growth, stimulating gallbladder contraction, inhibiting gastric acid secretion, pancreatic polypeptide release and peristalsis contraction components. It is reported that the additional effects of CCK include vasodilation. Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal Tract (3ded. 1994; Raven Press, New York).

[0923] It is reported that the combination of injection of glucagon, CCK and bombesin enhances the inhibitory effect of non-deprived rats on the intake of a concentrated milk test meal compared to the inhibitory effect observed with the individual compounds. Hinton et al., Brain Res. Bull. 17: 615-619 (1986). Glucagon and CCK are also reported to synergistically inhibit sham feeding 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 is suggested that estradiol and CCK also have a synergistic effect on satiety. Dulawa et al., Peptides 15: 913-918 (1994); Smith and Gibbs, supra. It is also suggested that signals generated by the small intestine in response to nutrients therein may work synergistically with CCK to reduce food intake. Cox, Behav. Brain Res. 38: 35-44 (1990). In addition, it is reported that CCK causes satiety in several species. For example, it is reported that intraperitoneal injection in rats, intraarterial injection in pigs, intravenous injection in cats and pigs, injection into the ventricle in monkeys, rats, dogs and sheep, and intravenous injection of CCK in obese and non-obese people leads to feeding inhibition. See Lieverse et al., the same. It is reported that studies from multiple laboratories, by comparing the response of monkeys and rats to food with the response to non-food reinforcers, and showing that CCK causes the sequence behavior (i.e., postprandial satiety sequence) commonly observed after eating, confirm the behavioral specificity of low-dose CCK to feeding inhibition. In addition, it is reported that by comparing the behavior after CCK with the behavior after food intake alone or CCK combination, the behavioral similarity between CCK and food intake is revealed. It is also reported that CCK inhibits food intake and increases the satiety of thin and obese people at physiological plasma concentrations.

[0924] CCK is characterized as a 33 amino acid peptide. Species-specific molecular variants of the CCK amino acid sequence have been identified. It is reported that a 33 amino acid sequence and a truncated peptide, i.e., its 8 amino acid C-terminal sequence (CCK-8), have been identified in pigs, rats, chickens, chinchillas, dogs, and humans. It is reported that a 39 amino acid sequence has been found in pigs, dogs, and guinea pigs. It is reported that a 58 amino acid sequence has been found in cats, dogs, and humans. It is reported that frogs and turtles show a 47 amino acid sequence homologous to CCK and gastrin. It is reported that very fresh human intestines contain a small amount of even larger molecules, called CCK-83. It is reported that a major 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). It is reported that unsulfated CCK-8 and a tetrapeptide (called CCK-4 (CCK (30-33))) have been found in rat brain. The C-terminal pentapeptide, termed CCK-5 (CCK(29-33)), retains structural homology to CCK and also retains homology to the neuropeptide gastrin. The C-terminal sulfated octapeptide sequence (CCK-8) is reported to be relatively conserved between species. Cloning and sequence analysis of cDNA encoding preprocholecystokinin from rat thyroid carcinoma, pig brain and pig intestine reportedly revealed that 345 nucleotides encoded a CCK precursor of 115 amino acids and contained all previously reported isolated CCK sequences. Crawley and Corwin, supra.

[0925] CCK is said to be distributed throughout the central nervous system as well as in endocrine cells and enteric nerves of the upper small intestine. CCK agonists include CCK itself (also known as CCK-33), CCK-8 (CCK (26-33)), unsulfated CCK-8, pentagastrin (CCK-5 or CCK (29-33)) and tetrapeptide CCK-4 (CCK (30-33)). CCK-8 is reported to bind 1000-5000 times more potently than unsulfated CCK-8 or CCK-4 at the pancreatic CCK receptor, while CCK-8 is reported to be approximately 1000 times more potent than unsulfated CCK-8 or CCK-4 in stimulating pancreatic amylase secretion. Crawley and Corwin, supra. It is said that in homogenates of the cerebral cortex, CCK receptor binding substituted by unsulfated CCK-8 at equimolar concentrations with sulfated CCK-8 and by CCK-4 at equimolar concentrations with sulfated CCK-8 is 10 or 100 times higher than that of sulfated CCK-8 at equimolar concentrations. See above. It is reported that receptors for CCK have been identified in various tissues, and two major subtypes have been described: type A receptors and type B receptors. Type A receptors are reported to be present in peripheral tissues including the pancreas, gallbladder, pyloric sphincter, and afferent vagal nerve fibers, as well as discrete regions of the brain. Type A receptor subtypes (CCKA) are reported to be selective for sulfated octapeptides. Type B receptor subtypes (CCKB) have been identified throughout the brain and stomach, and are reported to not require sulfation or all eight amino acids. See Reidelberger, J. Nutr. 124 (8 Suppl.) 1327S-1333S (1994); Crawley and Corwin, supra.

[0926] Various in vivo and in vitro methods for screening CCK analogs are known in the art. Examples include in vivo assays involving dog or guinea pig gallbladder contraction following rapid intravenous injection of a compound 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).

[0927] Useful component peptide hormones as components of the peptides of the present invention include GLP-1 peptide hormone. Natural GLP-1 peptide hormones are known in the art, including GLP-1 (1-37), GLP-1 (7-37) and GLP-1 (7-36) amide, as well as functional peptide analogs and derivatives. GLP-1 used herein refers to all natural forms of GLP-1 peptide hormones. Certain exemplary natural peptides, peptide analogs and derivatives are described herein, but it should be recognized that any known GLP-1 peptide known in the art to exhibit hormonal activity can be used in conjunction with the present invention.

[0928] At the heart of many metabolic diseases and disorders is the regulation of insulin levels and blood glucose levels. Insulin secretion is regulated in part by secretagogue hormones called incretins, which are produced by enteroendocrine cells. The incretin hormone, glucagon-like peptide-1 ("GLP-1"), is a peptide hormone secreted by intestinal cells, and multiple studies have shown that it has an enhancing effect on insulin secretion. GLP-1 is processed from proglucagon in the intestine and enhances nutrition-induced insulin release (Krcymann B., et al., Lancet, 2: 1300-1303 (1987)). Various truncated forms of GLP-1 are known to stimulate insulin secretion (insulinogenic effect) and cAMP formation (see, e.g., Mojsov, S., Int. J. Pep. Pro. Res., 40: 333-343 (1992)). A relationship has been established between various in vitro laboratory experiments and the insulinotropic response of mammals, particularly humans, to exogenously administered GLP-1, GLP-1(7-36)amide, and GLP-1(7-37)acid (see, e.g., 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)).

[0929] GLP-1 (7-36) amide exhibits significant antidiabetic effects in insulin-dependent diabetic patients by stimulating insulin sensitivity and enhancing glucose-induced insulin release at physiological concentrations (Gutniak M., et al., New Eng. J. Med., 326: 1316-1322 (1992)). When administered to non-insulin-dependent diabetic patients, GLP-1 (7-36) amide stimulates insulin release, decreases glucagon secretion, inhibits gastric emptying and enhances glucose utilization (Nauck, 1993; Gutniak, 1992; Nauck, 1993). However, the use of GLP-1 type molecules for long-term treatment of diabetes has been complicated by the relatively short serum half-life of this peptide.

[0930] More specifically, GLP-1 is a 30 amino acid peptide derived from proglucagon, a 160 amino acid prohormone. The action of different prohormone convertases in the pancreas and intestine results in the production of glucagon and other undefined peptides, while cleavage of proglucagon results in the production of GLP-1 and GLP-2, as well as two additional peptides. The amino acid sequence of GLP-1 is 100% homologous in all mammals, suggesting that it has a critical physiological role. GLP-1(7-37) acid is truncated at the C-terminus and amidated to form GLP-1(7-36)NH2. The biological effects and metabolic turnover of the free acid GLP-1(7-37)OH and the amide GLP-1(7-36)NH2 are indistinguishable. By convention, the numbering of the amino acids is based on GLP-1(1-37)OH processed from proglucagon. Biologically active GLP-1 is the result of further processing: GLP-1(7-36)NH2. Therefore, the first amino acid of GLP-1(7-37)OH or GLP-1(7-36)NH2 is 7His.

[0931] In the gastrointestinal tract, GLP-1 is produced by L cells of the intestinal, colonic, and rectal mucosa in response to intraluminal glucose stimulation. The plasma half-life of active GLP-1 is less than 5 minutes, and its metabolic clearance rate is approximately 12-13 minutes (Holst, Gastroenterology 107(6):1848-55 (1994)). The major protease involved in GLP-1 metabolism is dipeptidyl peptidase (DPP-IV or CD26), which cleaves the N-terminal His-Ala dipeptide to produce the metabolites GLP-1(9-37)OH or GLP-1(9-36)NH2, which are variously described as inactive, weak agonists, or antagonists of the GLP-1 receptor. The GLP-1 receptor (GLP-1R) is a 463 amino acid G protein-coupled receptor localized in pancreatic β cells, lungs, and to a lesser extent in the brain, adipose tissue, and kidneys. Stimulation of GLP-1R by GLP-1(7-37)OH or GLP-1(7-36)NH2 leads to activation of adenylate cyclase, synthesis of cAMP, membrane depolarization, increase in intracellular calcium and increase in glucose-induced insulin secretion (Holz et al., J. Biol. Chem. 270(30):17749-57 (1995)).

[0932] GLP-1 is a potent insulin secretagogue that is secreted by the intestinal mucosa in response to food intake. The significant incretin effect of GLP-1 is reflected by the fact that GLP-1R knockout mice are glucose intolerant. Although these patients have impaired incretin responses to oral glucose, the incretin response to intravenous infusion of GLP-1 is preserved in diabetic subjects. GLP-1 administered by infusion or subcutaneous injection controls fasting blood glucose levels in diabetic patients and maintains the 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 shows great potential as a therapeutic agent that can enhance insulin secretion in a physiological manner while avoiding the hypoglycemia associated with sulfonylureas.

[0933] Other important effects of GLP-1 on glucose homeostasis are inhibition of glucagon secretion and inhibition of gastric motility. The inhibitory effect of GLP-1 on glucagon secretion from pancreatic α cells leads to decreased hepatic glucose production by reducing gluconeogenesis and glycogenolysis. This anti-glucagon effect of GLP-1 is retained in patients with diabetes.

[0934] The so-called ileal brake effect of GLP-1 is achieved by inhibiting gastric motility and gastric secretion through vagal efferent receptors or directly acting on intestinal smooth muscle. The reduction of gastric acid secretion by GLP-1 contributes to the lag phase of nutrient availability, thereby eliminating the need for a rapid insulin response. In summary, the gastrointestinal effects of GLP-1 significantly delay the absorption of glucose and fatty acids and modulate insulin secretion and glucose homeostasis.

[0935] GLP-1 has also been shown to induce β-cell-specific genes such as the GLUT-1 transporter, insulin (via interaction of PDX-1 with the insulin gene promoter), and hexokinase-1. Thus, GLP-1 could potentially reverse glucose intolerance commonly associated with aging, as demonstrated in rodents. Furthermore, GLP-1 may contribute to β-cell neogenesis and increase β-cell mass, in addition to restoring β-cell function in states of β-cell insufficiency.

[0936] The central effects of GLP-1 include increased satiety and an associated reduction in food intake, which is achieved through the action of hypothalamic GLP-1 receptors. In subjects with type II diabetes, continuous SC infusion of GLP-1 for 48 hours reduced hunger and food intake and increased satiety. These anorectic effects were abolished in GLP-1R knockout mice. Therefore, as discussed, GIP hybrids containing incretin family hormone modules can provide functions and uses related to incretin family modules, such as exendin-4, GLP1, GLP2, in addition to GIP functions.

[0937] Any GLP-1 peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, the GLP-1 peptide analog and derivative have at least one hormonal activity identical to that of the native GLP-1 peptide. In certain embodiments, the GLP-1 peptide analog is an agonist of a receptor to which the native GLP-1 peptide can specifically bind. Exemplary GLP-1 peptide analogs and derivatives include, for example, those described in WO 91 / 11457, which is incorporated herein by reference.

[0938] In certain embodiments, the peptide of the present invention comprises or is any of the amino acid sequences listed below:

[0939]

[0940]

[0941]

[0942]

[0943] AA with an * indicates the presence of a linker (e.g., Lys-γGlu-γGlu-C(═O)(CH 2 ) n R 3 , where R 3 and n are defined above; or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 )).

[0944] In certain embodiments, the peptides of the invention comprise or are any of the amino acid sequences listed below with exemplary lipidation substituents:

[0945]

[0946]

[0947]

[0948] Any peptide of the invention may be further defined, for example, as described below. It should be understood that each further defining feature described herein may be applied to any peptide of the invention, where the amino acid at a particular position chemically permits the presence of the further defined feature. In a particular embodiment, these features may be present in any peptide of the invention of formula (I)-(V).

[0949] In various embodiments, the nitrogen group of any amino acid of the peptides of the invention may be optionally substituted with C1-C6 alkyl, C6-C12 aryl, C6-C12 arylC1-C6 alkyl or C1-C20 alkanoyl, including PEGylated forms alone or as a spacer for any of the foregoing, e.g., acetyl. It should be understood that N-substitution may not be present. In certain embodiments, the peptides of the invention comprise an N-terminus selected from hydrogen, C1-C6 alkyl, C6-C12 aryl, C6-C12 arylC1-C6 alkyl or C1-C20 alkanoyl, including PEGylated forms alone or as a spacer for any of the foregoing, e.g., acetyl. In specific embodiments of any of the peptides of the invention described herein, the N-terminal moiety is hydrogen.

[0950] In certain embodiments of any of the peptides of the invention described herein having any of the various formulae, K* is Lys or any of the following groups: Lys-γ-Glu-γ-Glu-C=O(CH 2 ) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) nR 3 ), where R 3 Yes-CO 2 H, -CO 2 CH 3 , -CO 2 NH 2 , -CO 2 NHCH 3 , -CO 2 N(CH 3 ) 2 , -CH 3 or -NH 2 , and n is an integer from 12 to 20. In certain embodiments, K* is Lys-γ-Glu-γ-Glu-C=O(CH 2 ) 18 CO 2 H, Lys-γ-Glu-γ-Glu-C=O(CH 2 ) 18 CH 3 or Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl).

[0951] In certain embodiments of any peptide of the invention with any various formulas described herein, the N-substituted part is selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanoyl, and lauric acid, hexadecanoic acid, and the conjugated amide of γ-Glu-hexadecanoic acid. In certain embodiments, the N-substituted part is pGlu. In specific embodiments, the N substitution is an acetyl group, whereby the peptide of the invention is acetylated at its N-terminus (e.g., capping or protecting the N-terminal amino acid residue, e.g., the N-terminal Pen residue).

[0952] In certain embodiments of any of the inventive peptides described herein, the N-substituted moiety is an acid. In certain embodiments, the N-substituted moiety is an acid selected from the group consisting of acetic acid, formic acid, benzoic acid, trifluoroacetic acid, isovaleric acid, isobutyric acid, octanoic acid, lauric acid, hexadecanoic acid, 4-biphenylacetic acid, 4-fluorophenylacetic acid, gallic acid, pyroglutamic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, 4-methylbicyclo(2.2.2)-oct-2-ene-1-carboxylic acid, glucoheptanoic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, alkylsulfonic acids, and arylsulfonic acids.

[0953] In certain embodiments, the N-substituted moiety is an alkylsulfonic acid selected from methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, and 2-hydroxyethanesulfonic acid.

[0954] In certain embodiments, the N-substituted moiety is an arylsulfonic acid selected from benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, and camphorsulfonic acid.

[0955] In certain embodiments, the peptides of the invention include one or more linker groups that covalently link one peptide component to a second subunit. The subunits are optionally linked via their C-termini.

[0956] The peptides of the present invention generally include at least two peptide components, wherein at least one peptide component, such as a GIP component, exhibits at least one hormonal activity. In the context of the present invention, at least one peptide component will include a GIP peptide, analog, derivative, fragment or peptide enhancer. The peptide component exhibiting at least one hormonal activity can be located at the N-terminus of the peptide, the C-terminus of the peptide, or, in the case where the peptide comprises more than two peptide components, can be located, for example, in the internal part of the peptide.

[0957] In certain embodiments, it may be preferred to position the peptide component that exhibits at least one hormonal activity so that the C-terminus of the peptide component is amidated. C-terminal amidation of the peptide component can be achieved by positioning the module at the C-terminus of the hybrid peptide, or by configuring the peptide to be at the N-terminus of the peptide in a C-terminal to N-terminal direction. In both configurations, the C-terminus of the peptide component is available for amidation. Specific component peptides in which the C-terminus is amidated may preferably include amylin family peptides, CCK, PYY, hGLP-1 (7-36) and hGLP-2. Specific component peptides in which C-terminal amidation is not necessarily exemplified (in other words, in the case where the C-terminus of the module easily tolerates extension) include exendin-4, exendin-4 (1-28), GIP, GLP-1 (7-37), frog GLP-1 (7-36) and frog GLP-2. However, if these component peptides are located at the C-terminus of the peptide, they can still be optionally amidated, and in fact can preferably be optionally amidated.

[0958] The peptide component peptides of the present invention can be covalently linked in any manner known in the art. A stable link can be used, or a cleavable link can be used. In one embodiment, the carboxyl group of the first peptide can be directly linked to the amino group of the second peptide. In another embodiment, a linking group can be used to connect the modules. In addition, if desired, a spacer or a turn inducing agent known in the art can be used to stabilize the link. As an example, where C-terminal amidation of the N-terminally positioned peptide component is not necessary, the module can be directly linked to the second module, or any suitable linker known in the art can be used, such as alkyl; PEG; amino acids, such as Lys, Glu, β-Ala; polyamino acids, such as poly-his, poly-arg, poly-lys, poly-ala, Gly-Ser-Gly, Gly-Gly-Pro-Ser, Ala-Lys-Ala, Gly-Lys-Arg (GKR), etc.; bifunctional linkers (see, e.g., Pierce catalog, Rockford, 111.); aminocaproyl ("Aca"), β-alanyl, 8-amino-3,6-dioxaoctanoyl, or other cleavable and non-cleavable linkers known in the art. Specifically described herein (as if each is explicitly set forth) are embodiments of specific hybrids in which the linker in each hybrid containing the exemplary linker is replaced by a Gly linker, particularly embodiments in which the Gly linker is Gly-Gly-Gly. In one embodiment, the linker or spacer is 1 to 30 residues long, in another embodiment 2 to 30 residues long, in another embodiment 3-30 residues long, and any integer length from 2 to 30 inclusive; each integer unit is contemplated, e.g., 2, 3, 4, 5, 6, 7, etc. In one embodiment, a Gly linker is used, and in a specific embodiment, a three-residue linker Gly-Gly-Gly is used.

[0959] In certain embodiments, the peptide components of the present invention can be connected by a suitable linking moiety, such as a disulfide bridge between two cysteine ​​residues (one cysteine ​​residue in each peptide subunit), or by another suitable linker moiety, including but not limited to those defined herein. In certain embodiments, the subunit can be modified to eliminate the C- or N-terminal free amino group, thereby allowing dimerization at the remaining free amino group. In addition, in certain cases, the end of one or more monomer subunits is selected from the following acylated organic compound acylation: trifluoropentyl, acetyl, octanoyl, butyryl (butyl), pentyl (pentyl), hexanoyl (hexyl), palmitoyl, trifluoromethylbutyric acid, cyclopentanecarboxyl, cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, tetrahydro-2H-pyran-4 carboxylic acid, succinic acid and glutaric acid. In certain cases, the subunit comprises a free carboxyl terminal and a free amino terminal, so that the user can selectively modify the subunit to achieve dimerization at the desired terminal. Those skilled in the art will appreciate that the subunits of the invention can be selectively modified to allow for a single, specific amino group to be available for desired linkage.

[0960] It is also understood that the C-terminal residue of the subunits disclosed herein is optionally an amide. In addition, it is understood that in certain embodiments, C-terminal dimerization is promoted by using appropriate amino acids with amino-functional side chains, which is generally understood in the art. With respect to the N-terminal residue, it is generally understood that coupling can be achieved through the free amino group of the terminal residue, or by using appropriate amino acid side chains with free amino groups, which is generally understood in the art.

[0961] The linker moiety connecting the subunits may include any structure, length and / or size compatible with the teachings herein. In at least one embodiment, the linker moiety is selected from the non-limiting group consisting of cysteine, lysine, DIG, PEG4, PEG4-biotin, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, ADA, Boc-IDA, glutaric acid, isophthalic acid, 1,3-benzenedioic acid, 1,4-benzenedioic acid, 1,2-benzenedioic acid, triazine, Boc-triazine, IDA-biotin, PEG4-biotin, AADA, suitable aliphatic, aromatic, heteroaromatic and polyethylene glycol-based linkers with a molecular weight of about 400 Da to about 40,000 Da. In certain embodiments, PEG2 is HO 2 CCH 2 CH 2 OCH 2 CH 2 OCH 2 CH2 CO 2 H.

[0962] Non-limiting examples of suitable linker moieties are provided in Table 2.

[0963] Table 2. Illustrative Linker Parts

[0964]

[0965]

[0966]

[0967]

[0968] In some embodiments, the peptides of the present invention include a linker moiety. In some embodiments, the peptide subunits of the present invention are connected by an intermolecular disulfide bond formed between two cysteine ​​residues (one cysteine ​​residue in each subunit). In some embodiments, the peptides of the present invention include a linker moiety and an intermolecular disulfide bond formed between two cysteine ​​residues. In some embodiments, the intramolecular bond is a thioether, a lactam, a triazole, a selenoether, a diselenide or an olefin, rather than a disulfide bond.

[0969] Those skilled in the art will appreciate that the joints disclosed herein (e.g., C-terminal and N-terminal joints) are partly non-limiting examples of suitable joints, and the present invention may include any suitable joint moiety. Therefore, some embodiments of the present invention include homologous or heterologous dimeric peptides composed of two monomer subunits, the monomer subunits being selected from the peptides shown in any table herein or comprising or consisting of the sequence shown in any table herein, wherein the C-terminal or N-terminal (or internal amino acid residues) of each subunit are connected by any suitable joint moiety to provide a peptide of the present invention. In certain embodiments, the joint combines the N-terminal or C-terminal of a subunit and the internal amino acid residues of another subunit to form a peptide of the present invention. In certain embodiments, the joint combines the internal amino acid residues of a monomer subunit and the internal amino acid residues of another monomer subunit to form a peptide of the present invention. In a further embodiment, the joint combines the N-terminal or C-terminal of two subunits.

[0970] In certain embodiments, one or more subunits comprise a sequence or structure of any one of formulas (I)-(V), or any peptide described herein. In certain embodiments of the peptides of the invention, the linker moiety is any linker described herein. In certain embodiments, the linker is a lysine linker, a diethylene glycol linker, an iminodiacetic acid (IDA) linker, a β-Ala-iminodiacetic acid (β-alanine-IDA) linker, or a PEG linker.

[0971] In various embodiments of any peptide of the present invention, each peptide subunit is connected to a linker moiety via its N-terminus, C-terminus or internal amino acid residue. In certain embodiments of any peptide of the present invention, the N-terminus of each peptide subunit is connected via a linker moiety. In certain embodiments of any peptide of the present invention, the C-terminus of each peptide subunit is connected via a linker moiety. In certain embodiments of any peptide of the present invention, each peptide subunit is connected by a linker moiety connected to an internal amino acid.

[0972] In certain embodiments, the peptides of the present invention include one or more conjugated chemical substituents, such as lipophilic substituents and polymer moieties, which may be referred to herein as half-life extension moieties. Without wishing to be bound by any particular theory, it is believed that the lipophilic substituents bind to albumin in the bloodstream, thereby protecting the peptides of the present invention from enzymatic degradation, thereby extending their half-life. In addition, it is believed that the polymer moiety extends the half-life and reduces clearance in the bloodstream.

[0973] In additional embodiments, any peptide of the invention, such as a peptide of formula (I)-(V), further comprises a linker portion attached to an amino acid residue present in the peptide, for example, the linker portion can be bound to the side chain of any amino acid of the peptide, to the N-terminal amino acid of the peptide, or to the C-terminal amino acid of the peptide.

[0974] In additional embodiments, any peptide of the invention, such as a peptide of Formula (I)-(V), further comprises a half-life extending moiety attached to an amino acid residue present in the peptide, for example, the half-life extending moiety can be attached to the side chain of any amino acid of the peptide, to the N-terminal amino acid of the peptide, or to the C-terminal amino acid of the peptide.

[0975] In additional embodiments, any of the peptides of the invention, such as the peptides of Formula (I)-(V), further comprises a half-life extending moiety attached to a linker moiety that is attached to an amino acid residue present in the peptide, for example, the half-life extending moiety can be attached to a linker moiety that is attached to the side chain of any amino acid of the peptide, to the N-terminal amino acid of the peptide, or to the C-terminal amino acid of the peptide.

[0976] In certain embodiments, the peptide comprises a half-life extending moiety having the structure shown below, wherein n=0 to 24 or n=14 to 24:

[0977]

[0978] In certain embodiments, the peptides of the invention include a half-life extending moiety as shown in Table 3.

[0979] Table 3. Illustrative Half-Life Extension Moieties

[0980]

[0981]

[0982] In certain embodiments, the half-life extending moiety is directly conjugated to the peptide, while in other embodiments, the half-life extending moiety is conjugated to the peptide via a linker moiety, such as any of the linker moieties set forth herein.

[0983] Table 4. Illustrative Linker Parts

[0984]

[0985]

[0986]

[0987] In specific embodiments, the peptides of the invention include any linker moiety set forth herein and any half-life extending moiety set forth herein, including any combination shown in Table 5.

[0988] Table 5. Illustrative combinations of linkers and half-life extending moieties in peptides of the invention

[0989]

[0990]

[0991] In some embodiments, multiple linkers can be present between the peptide and the conjugated moiety (e.g., half-life extending moiety), for example, as shown in Table 6.

[0992] Table 6. Illustrative combinations of linkers and half-life extending moieties in peptides of the invention

[0993]

[0994] In certain embodiments, the half-life of the peptide of the present invention comprising a conjugated chemical substituent (i.e., a half-life extending moiety) is at least 100%, at least 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% of the half-life of the same peptide not comprising a conjugated chemical substituent. In certain embodiments, the lipophilic substituent and / or polymer moiety enhances the permeability of the peptide through the epithelium and / or its retention in the lamina propria. In certain embodiments, the permeability of the peptide of the present invention comprising a conjugated chemical substituent through the epithelium and / or the retention in the lamina propria is at least 100%, at least 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% of the half-life of the same peptide not comprising a conjugated chemical substituent.

[0995] In certain embodiments, the side chains of one or more amino acid residues (e.g., Lys residues) of the peptides of the present invention are conjugated (e.g., covalently linked) to lipophilic substituents. The lipophilic substituents can be covalently bonded to atoms in the amino acid side chains, or conjugated to the amino acid side chains through one or more spacers. When there is a spacer, the spacer can provide a gap between the peptide subunit and the lipophilic substituent. In a specific embodiment, the peptides of the present invention include any conjugated moieties disclosed herein.

[0996] In certain embodiments, the lipophilic substituent can comprise a hydrocarbon chain with 4 to 30 C atoms, for example at least 8 or 12 C atoms, and preferably 24 C atoms or less, or 20 C atoms or less. The hydrocarbon chain can be linear or branched, and can be saturated or unsaturated. In certain embodiments, the hydrocarbon chain is substituted by a part to form a part or spacer connected to the amino acid side chain, for example acyl, sulfonyl, N atom, O atom or S atom. In some embodiments, the hydrocarbon chain is substituted by an acyl group, so the hydrocarbon chain can form a part for an alkanoyl, for example palmitoyl, caproyl, lauroyl, myristoyl or stearyl.

[0997] Lipophilic substituents can be conjugated with any amino acid side chain in the peptide of the present invention. In certain embodiments, the amino acid side chain includes a carboxyl group, a hydroxyl group, a thiol group, an amide group or an amino group, for forming an ester, a sulfonyl ester, a thioester, an amide or a sulfonamide with a spacer or a lipophilic substituent. For example, the lipophilic substituent can be conjugated with Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys or Dbu, Dpr or Orn. ​​In certain embodiments, the lipophilic substituent is conjugated with Lys. The amino acid shown as Lys in any formula provided herein can be replaced by, for example, Dbu, Dpr or Orn, wherein a lipophilic substituent is added.

[0998] In certain embodiments, the peptides of the present invention can be modified by conjugating a chemical moiety to one or more amino acid side chains in the peptide, for example, to enhance stability, increase permeability or enhance drug-like properties. For example, the N (ε) of lysine N (ε), the β-carboxyl of aspartic acid or the γ-carboxyl of glutamic acid can be appropriately functionalized. Therefore, in order to produce a modified peptide, the amino acid in the peptide can be appropriately modified. In addition, in some cases, the side chain is acylated by an acylated organic compound selected from the following groups: trifluoropentanoyl, acetyl, octanoyl, butyryl, valeryl, hexanoyl, palmitoyl, trifluoromethylbutyric acid, cyclopentanecarboxylic acid, cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, tetrahydro-2H-pyran-4-carboxylic acid, succinic acid, glutaric acid or cholic acid. It will be appreciated by those skilled in the art that a series of conjugates can be connected, for example, such as PEG4, isoglu and combinations thereof. Those skilled in the art will appreciate that amino acids within a peptide may be replaced isoelectronically, for example, Lys may be replaced by Dap, Dab, α-MeLys or Orn. ​​Examples of modified residues within a peptide are shown in Table 7.

[0999] Table 7. Examples of modified lysine, aspartic acid, and asparagine within peptides

[1000]

[1001]

[1002]

[1003] In a further embodiment of the invention, alternatively or additionally, the side chains of one or more amino acid residues in the peptides of the invention are conjugated to a polymer moiety, e.g., to increase solubility and / or in vivo (e.g., in plasma) half-life and / or bioavailability. Such modifications are known to also reduce the clearance (e.g., renal clearance) of therapeutic proteins and peptides.

[1004] As used herein, "polyethylene glycol" or "PEG" is a polyol of the general formula H-(O-CH 2 -CH 2)n-OH polyether compound. Depending on its molecular weight, PEG is also called polyethylene oxide (PEO) or polyoxyethylene (POE). PEO, PEE or POG used in this article refer to oligomers or polymers of ethylene oxide. These three names are chemically synonymous, but PEG tends to refer to oligomers and polymers with molecular weights lower than 20,000Da, PEO refers to polymers with molecular weights higher than 20,000Da, and POE refers to polymers of any molecular weight. PEG and PEO can be liquids or low melting point solids, depending on their molecular weight. Throughout this disclosure, these three names are used interchangeably. PEG is prepared by the polymerization of ethylene oxide and is commercially available in a wide range of molecular weights from 300Da to 10,000,000Da. Although PEG and PEO with different molecular weights have different uses in different applications and have different physical properties (such as viscosity) due to chain length effects, their chemical properties are almost the same. The polymer moiety is preferably water soluble (amphiphilic or hydrophilic), non-toxic and pharmaceutically inert. Suitable polymer moieties include polyethylene glycol (PEG), homopolymers or copolymers of PEG, monomethyl substituted polymers of PEG (mPEG) or polyoxyethylene glycerol (POG). See, e.g., Int. J. Hematology 68: 1 (1998); Bioconjugate Chem. 6: 150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9: 249 (1992). Also contemplated are PEGs prepared for the purpose of extending half-life, e.g., monoactivated, alkoxy-terminated polyolefin oxides (POA) such as monomethoxy-terminated polyethylene glycol (mPEG); diactivated polyethylene oxide (ethylene glycol) or other PEG derivatives are also contemplated. Suitable polymers generally have molecular weights ranging from about 200 Da to about 40,000 Da or from about 200 Da to about 60,000 Da and are generally selected for the purposes of the present invention. In certain embodiments, PEGs having molecular weights of 200 to 2,000 or 200 to 500 are used. Different forms of PEG may also be used depending on the initiator used in the polymerization process - a commonly used initiator is a monofunctional methyl ether PEG, or methoxy poly(ethylene glycol), abbreviated as mPEG.

[1005] Low molecular weight PEGs can also be provided as pure oligomers, referred to as monodisperse, homogeneous or discrete. These are used in certain embodiments of the invention.

[1006] PEG can also have different geometric shapes: branched PEG has three to ten PEG chains emanating from a central core group; star PEG has ten to one hundred PEG chains emanating from a central core group; comb PEG has multiple PEG chains usually grafted onto a polymer backbone. PEG can also be linear. The number usually included in the PEG name indicates its average molecular weight (e.g., a PEG with n=9 would have an average molecular weight of about 400 Daltons and would be labeled PEG 400).

[1007] As used herein, "PEGylation" refers to the act of covalently coupling a PEG structure to a peptide of the invention, which is then referred to as a "PEGylated peptide". In certain embodiments, the PEG of the PEGylated side chain is a PEG having a molecular weight of about 200 to about 40,000. In certain embodiments, the PEG of the PEGylated spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8.

[1008] Other suitable polymer moieties include polyamino acids such as polylysine, polyaspartic acid and polyglutamic acid (see, for example, Gombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57 and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73,: 721-729). The polymer moiety can be linear or branched. In certain embodiments, its molecular weight is 500-40,000 Da, for example 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da or 20,000-40,000 Da.

[1009] In certain embodiments, the peptides of the invention may comprise two or more such polymer moieties, in which case the total molecular weight of all of these moieties will generally fall within the ranges provided above.

[1010] In certain embodiments, the polymer moiety is coupled (by covalent attachment) to an amino, carboxyl or thiol group of an amino acid side chain. Certain examples are the thiol group of a Cys residue and the epsilon amino group of a Lys residue, and the carboxyl groups of Asp and Glu residues may also be involved.

[1011] Those of ordinary skill in the art will be well aware of suitable techniques for carrying out coupling reactions. For example, PEG moieties bearing methoxy groups can be coupled to Cys thiol groups via maleimide linkages using commercially available reagents from Nektar Therapeutics AL. See also WO 2008 / 101017 and the references cited above for details of suitable chemistry. Maleimide-functionalized PEGs can also be conjugated to side chain thiols of Cys residues.

[1012] The disulfide bond oxidation used herein can occur in a single step or be a two-step process. As used herein, for a single oxidation step, a trityl protecting group is usually used in the assembly process, deprotection is allowed during the cleavage process, and then solution oxidation is performed. When a second disulfide bond is needed, natural oxidation or selective oxidation can be selected. For selective oxidation requiring an orthogonal protecting group, Acm and trityl are used as protecting groups for cysteine. Cutting results in the removal of a cysteine ​​protection pair, allowing oxidation of this pair. Then the second oxidation deprotection step of the cysteine ​​protection Acm group is performed. For natural oxidation, all cysteines use trityl protecting groups to allow the natural folding of peptides. Those skilled in the art will be well aware of the appropriate techniques that can be used to perform the oxidation step.

[1013] Several chemical moieties, including poly(ethylene glycol), react with functional groups present in the twenty naturally occurring amino acids, such as the epsilon amino group in lysine amino acid residues, the thiol in cysteine ​​amino acid residues, or other nucleophilic amino acid side chains. When multiple naturally occurring amino acids are reacted in a peptide, these non-specific chemical reactions result in the final peptide of the invention comprising multiple peptide isomers conjugated to one or more poly(ethylene glycol) chains at different positions within the peptide of the invention.

[1014] An advantage of certain embodiments of the present invention includes the ability to add one or more chemical moieties (such as PEG) by adding one or more non-natural amino acids with unique functional groups that react chemically with activated PEG but do not react with natural amino acids present in the peptides of the present invention. For example, azide and alkynyl groups do not react with all naturally occurring functional groups in proteins. Therefore, non-natural amino acids can be added to one or more specific sites of the peptides of the present invention, where PEG or other modifications are desired, without undesirable non-specific reactions. In certain embodiments, the specific chemical results involved in the reaction are the formation of a stable covalent linkage between the PEG chain and the peptide of the present invention. In addition, these reactions can be carried out under mild aqueous conditions without damaging most peptides. In certain embodiments, the non-natural amino acid residue is AHA.

[1015] Chemical moieties connected to natural amino acids are limited in number and scope. In contrast, chemical moieties connected to non-natural amino acids can utilize significantly more extensive useful chemical methods to connect chemical moieties to target molecules. Essentially any target molecule, including any protein (or portion thereof) comprising non-natural amino acids, for example, non-natural amino acids comprising reactive sites or side chains to which chemical moieties can be connected, such as aldehyde or ketone derived amino acids, can be used as substrates for connecting chemical groups.

[1016] By various methods known in the art, many chemical moieties can be combined or connected to specific molecules. Some such methods are described in U.S. Patent number 8,568,706. As an illustrative example, the azide moiety may be useful when conjugating chemical moieties such as PEG or other parts described herein. The azide moiety is a reactive functional group and is not present in most naturally occurring compounds (so it does not react with the natural amino acids of naturally occurring compounds). Azide is also selectively connected with a limited number of reaction partners, and azide is small and can be introduced into biological samples without significantly changing molecular size. A method for allowing azide to be incorporated into or introduced into molecules is the copper-mediated Huisgen [3+2] cycloaddition reaction of azide. This reaction can be used for the selective PEGylation of peptides. (Tornoe et al., J. Org. Chem. 67:3057, 2002; Rostovtsev et al., Angew. Chem., Int. Ed. 41:596, 2002; and Wang et al., J. Am. Chem. Soc. 125:3192, 2003, Speers et al. al., J. Am. Chem. Soc., 2003, 125, 4686).

[1017] In another embodiment, the peptides of the invention may be deleted from the amino acid sequence of the native peptide or region S, either alone or in combination with one or more insertions or substitutions. In one aspect, the GIP analogs or peptides of the invention may be deleted from the N-terminus or C-terminus of the native GIP. In another embodiment, the peptides of the invention may be deleted from amino acid positions 1 to 42 of the native GIP, GIP (1-14), GIP (1-26), GIP (1-30), GIP (1-39), GIP (19-26), GIP (19-30), GIP (19-39) or GIP (19-42) or region S. These deletions may include more than one consecutive or non-consecutive deletion. In an exemplary embodiment, no more than 1, no more than 2, no more than 3, no more than 4 or no more than 5 amino acids are deleted from native GIP, from GIP (1-30), GIP (1-14), GIP (1-26), GIP (1-39), GIP (19-30), GIP (19-26), GIP (19-39) or GIP (19-42) or from region S (e.g., when the region is exendin (31-39) or exendin (27-39)). In one embodiment, the native GIP is human, rat, mouse, porcine or bovine.

[1018] In one embodiment of the peptides of the invention, when the GIP peptide (whether an analog, derivative or hybrid) is intended for use as an agonist, it does not include a deletion at any of positions 1-15 (corresponding to positions YAEGTFISDYSIAMD of the N-terminal sequence of GIP). In other words, each of the corresponding positions 1-15 of GIP will be present, but they may be substituted or derivatized. In a further embodiment, the agonist GIP compound does not include a deletion at any of positions 4-15 (corresponding to positions GTFISDYSIAMD of the C-terminal sequence of GIP). In other words, each of the corresponding positions 4-15 of GIP will be present, but they may be substituted or derivatized. Therefore, in an embodiment of the agonist GIP compound, each of positions 1-15 or 4-15 will be present and occupied by an amino acid or a substitution or derivative thereof at that position of a naturally occurring GIP species. In another embodiment of the agonist GIP compound, GIP compounds excluded from the various embodiments described herein are those GIP compounds that do not exhibit sufficient receptor binding activity or receptor activation activity as shown.

[1019] In one embodiment of the peptides of the invention, when the GIP peptide (whether an analog, derivative or hybrid) is intended for use as an agonist, it does not include a deletion at any of positions 1-15 (corresponding to positions YAEGTFISDYSIAMD of the N-terminal sequence of GIP). In other words, each of the corresponding positions 1-15 of GIP will be present, but they may be substituted or derivatized. In a further embodiment, the agonist GIP compound does not include a deletion at any of positions 4-15 (corresponding to positions of the C-terminal sequence of GIP). In other words, each of the corresponding positions 4-15 of GIP will be present, but they may be substituted or derivatized.

[1020] In another embodiment of the peptide of the present invention, the GIP analog or hybrid may have one or more amino acid residues inserted into the amino acid sequence of GIP(1-30), GIP(1-14), GIP(1-26), GIP(1-39), GIP(19-30), GIP(19-26), GIP(19-39) or GIP(19-42) or region S of native GIP, either alone or in combination with one or more deletions and / or substitutions. In one aspect, the present invention relates to a GIP analog or hybrid having a single insertion or more than one amino acid residue inserted continuously or non-continuously into the amino acid sequence of native GIP, GIP(1-30), GIP(1-14), GIP(1-26), GIP(1-39), GIP(19-30), GIP(19-26), GIP(19-39) or GIP(19-42) or region S, such as exendin(27-39) and exendin(31-39). In one embodiment, the native GIP is human, mouse, murine, porcine or bovine.

[1021] In another embodiment of the peptide of the present invention, the GIP analog or hybrid may have one or more unnatural amino acids and / or non-amino acids inserted into the sequence of GIP, GIP (1-30), GIP (1-14), GIP (1-26), GIP (1-39), GIP (19-30), GIP (19-26), GIP (19-39) or GIP (19-42) or region S, such as Exendin (27-39) and Exendin (31-39). In an exemplary embodiment, the non-natural amino acid inserted into the sequence of GIP, GIP(1-30), GIP(1-14), GIP(1-26), GIP(1-39), GIP(19-30), GIP(19-26), GIP(19-39) or GIP(19-42) or region S, such as Exendin(27-39) and Exendin(31-39) can be a beta turn mimetic or a linker molecule. In further such embodiments, the native GIP can be human, mouse, rat, porcine or bovine.

[1022] Thus, while the compounds are shown with optional linking groups, in one embodiment of the sequences herein, the linker is a Gly linker, such as Gly-Gly-Gly, or a βAla linker, such as βAla-βAla; all of which are specifically contemplated. Linker molecules of particular interest include aminohexanoyl ("Aca"), β-alanyl, and 8-amino-3,6-dioxaoctanoyl. β-Turn mimetics are also used in other embodiments, including mimetic A: N-(3S,6S,9S)-2-oxo-3-amino-1-azabicyclo[4.3.0]-nonane-9-carboxylic acid, mimetic B: N-(3S,6S,9R)-2-oxo-3-amino-7-thio-1-azabicyclo[4.3.0]-nonane-9-carboxylic acid, and Ala-Aib and Ala-Pro dipeptides.

[1023] In another embodiment of the peptide of the invention, the GIP analog or hybrid may include a polyamino acid sequence (eg, poly-his, poly-arg, poly-lys, poly-ala, etc.) inserted at either end of the polypeptide, referred to as an "extension" or "tail".

[1024] In certain embodiments, the novel peptides of the invention comprise amino acid sequence insertions including alanine substitutions at every amino acid position along the length of native GIP, GIP(1-30), GIP(1-14), GIP(1-26), GIP(1-39), GIP(19-30), GIP(19-26), GIP(19-39) or GIP(19-42) or region S, e.g., Exendin(27-39) and Exendin(31-39).

[1025] The peptides of the present invention also include derivatives of GIP analogs and hybrid peptides. Such derivatives include GIP analogs and hybrid polypeptides conjugated to one or more water-soluble polymer molecules (such as polyethylene glycol ("PEG") or fatty acid chains of various lengths (such as stearoyl, palmitoyl, octanoyl, etc.) or by adding polyamino acids (such as poly-his, poly-arg, poly-lys and poly-ala). Modifications to the polypeptide can also include small molecule substituents, such as short alkyl and restricted alkyl (such as branched, cyclic, fused, adamantyl) and aromatic groups. The molecular weight of the water-soluble polymer molecule is preferably between about 500 and about 20,000 Daltons.

[1026] Such polymer conjugation and small molecule substituent modification can occur alone at the N-terminus or C-terminus of the GIP analogs and hybrid polypeptides or at the side chains of amino acid residues within the sequence. Alternatively, there may be multiple derivatization sites along the GIP analogs and hybrid peptides. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine ​​can provide additional derivatization sites. See, for example, U.S. Patent Nos. 5,824,784 and 5,824,778. In one embodiment, the peptides of the present invention can be conjugated to one, two or three polymer molecules.

[1027] The water-soluble polymer molecules are preferably linked to amino, carboxyl or sulfhydryl groups and can be linked via the N-terminus or C-terminus or at the side chains of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, diamine and dicarboxyl groups can be used to link water-soluble polymer molecules. In an exemplary embodiment, the peptide of the invention is conjugated to one, two or three PEG molecules via the epsilon amino group on the lysine amino acid.

[1028] The peptides of the present invention also include chemical alterations to one or more amino acid residues. Such chemical alterations include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. Chemical alterations can occur alone, for example, at the N-terminus or C-terminus of GIP analogs and hybrid peptides, or at the side chains of amino acid residues within the sequence. In one embodiment, the C-terminus of these peptides may have a free -OH or -NH 2 In another embodiment, the N-terminus can be capped with an isobutyloxycarbonyl, isopropyloxycarbonyl, n-butyloxycarbonyl, ethoxycarbonyl, isohexanoyl (isocap), octanoyl, octylglycine group (G(Oct)), 8-aminooctanoic acid group or Fmoc group. In an exemplary embodiment, cyclization can be achieved by forming a disulfide bridge. Alternatively, chemical changes can be made at multiple positions along the peptide of the present invention.

[1029] Some pseudo-peptide bonds have been described, which generally do not affect the structure and biological activity of the peptide. An example of this method is to replace with a retro-isomer pseudo-peptide bond ("Biologically active retroinversoanalogues of thymopentin", Sisto A. et al in Rivier, JE and Marshall, GR (eds) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), pp.722-773 and Dalpozzo, et al. (1993), Int. J. Peptide Protein Res., 41: 561-566, incorporated herein by reference). According to this modification, the component peptides of the peptides of the present invention can be identical to the sequence of, for example, GIP described herein, but one or more peptide bonds are replaced by a retro-isomer pseudo-peptide bond. It is preferred to replace the most N-terminal peptide bond because such a replacement will confer resistance to proteolysis to exopeptidases acting on the N-terminus. Further modification can also be carried out by replacing the chemical group of amino acid with other structurally similar chemical groups. Another known pseudo-peptide bond suitable for enhancing stability to enzymatic cleavage without loss of biological activity or with only a small amount of biological activity loss is a reduced isosteric pseudo-peptide bond (Couder, et al. (1993), Int. J. Peptide Protein Res., 41: 181-184, the entire contents of which are incorporated herein by reference).

[1030] Thus, the amino acid sequence of these peptides may be identical to that of, for example, a novel GIP analog and hybrid peptide, but one or more peptide bonds are replaced by an isosteric pseudo-peptide bond. It is preferred to replace the most N-terminal peptide bond, since such a replacement will confer resistance to proteolysis by exopeptidases acting at the N-terminus. The synthesis of peptides containing one or more reduced isosteric pseudo-peptide bonds is known in the art (Couder, et al. (1993), cited above). Other examples include the introduction of ketomethylene or methylsulfide bonds to replace peptide bonds.

[1031] In another embodiment, the bond between the second and third residues that is a target for DPP-IV cleavage is replaced with a peptidase-resistant bond as disclosed herein.

[1032] Peptoid derivatives of the peptides of the present invention represent another class of peptide mimetics that retain important structural determinants of biological activity but eliminate peptide bonds, thereby conferring resistance to proteolysis (Simon, et al., Proc. Natl. Acad. Sci. USA, 89: 9367-9371 (1992), the entire contents of which are incorporated herein by reference). Peptides are oligomers of N-substituted glycines. Many N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid (Simon, et al. (1992), cited above). Some or all of the amino acids of the GIP peptide can be replaced by the N-substituted glycines corresponding to the replaced amino acids.

[1033] In one embodiment, the peptides of the invention include a combination of the above modifications, ie, deletions, insertions and substitutions.

[1034] The scope of the invention also includes component peptides in which the indicated amino acid residues are chemically modified or derivatized (e.g., by fatty acid derivatization, PEGylation, amidation, glycosylation, etc.). Exemplary embodiments include derivatization of lysine residues, particularly at positions 16 or 30. Also contemplated within the scope of the invention are D-amino acid residues of the indicated amino acids. In another embodiment, exemplary GIP analogs or hybrid polypeptides include polypeptides of the formula having internal deletions, particularly in regions that do not correspond to the active site, as described herein.

[1035] Exemplary peptides of the present invention may include substitutions of non-natural amino acids. Exemplary derivatives such as GIP analogs or hybrid peptides of the present invention include polymer-conjugated GIP analogs or hybrid peptides, wherein the GIP analogs or hybrid peptides include any of the above insertions, deletions, substitutions or combinations thereof, and the polymer molecule is conjugated to a lysine residue. In one embodiment, the peptides of the present invention include derivatives or substitutions of methionine and have a longer duration of action than human GIP or analogs. For example, octylglycine at methionine increases the duration of action of the compound in vivo. With this modification, the duration of action is increased to at least 4 hours. Therefore, in one embodiment, it is a peptide of the present invention conjugated to one or more water-soluble polymer molecules (such as polyethylene glycol ("PEG") or fatty acid chains of various lengths (such as stearoyl, palmitoyl, octanoyl, etc.) or by adding polyamino acids (such as poly-his, poly-arg, poly-lys, poly-glu and poly-ala). Modifications to polypeptides may also include small molecule substituents, such as short alkyl and restricted alkyl (e.g., branched, cyclic, fused, adamantyl) and aromatic groups.

[1036] Further specifically contemplated are D-Tyr1 and D-Ala2 variants of each peptide component herein. In other contemplated embodiments are variants of each of the above sequences, wherein the peptides of the invention are modified by one, two or three modifications described herein. Exemplary modifications are those located at the first, second or third N-terminal amino acid of GIP, conferring superior DPP-IV resistance over native GIP. In further embodiments, the novel peptides of the invention include a C-terminal amide.

[1037] In a further embodiment, the half-life of the peptides of the invention is at least twice the half-life of human GIP (1-30) amide. Further, the half-life may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 72 or 96 hours. In a specific embodiment, the half-life may be at least 24 hours.

[1038] In another embodiment, it is a pharmaceutically acceptable salt of the peptide of the present invention.The peptide of the present invention can be formulated in a composition comprising a pharmaceutically acceptable carrier.

[1039] Synthesis of the peptides of the present invention

[1040] The peptide of the present invention can be synthesized by a variety of techniques known to those skilled in the art. In certain embodiments, the peptide subunits are synthesized, purified and dimerized using techniques known in the art. In certain embodiments, the invention provides methods for producing the peptide of the present invention (or its subunit), including chemical synthesis comprising, consisting of or consisting mainly of a peptide of an amino acid sequence described herein, the amino acid sequence including but not limited to any amino acid sequence listed in any formula IV or this paper table. In other embodiments, the peptide is synthesized by recombinant synthesis, rather than chemical synthesis. In certain embodiments, the peptide and method of the present invention include two monomer subunits of the peptide of the present invention, which are then connected to produce the peptide of the present invention. In various embodiments, coupling or connection is achieved by any of the various methods described herein.

[1041] In certain embodiments, the method for producing a peptide (or its monomeric subunit) of the present invention further comprises cyclizing the peptide (or its monomeric subunit) of the present invention after its synthesis. In certain embodiments, the cyclization is achieved by any of the various methods described herein. In certain embodiments, the present invention provides a method for producing a peptide (or its monomeric subunit) comprising introducing an intramolecular bond, such as a disulfide bond, an amide bond, or a thioether bond, between two amino acids in the peptide, the peptide comprising, consisting of, or consisting essentially of a peptide having an amino acid sequence as described herein, the amino acid sequence including, but not limited to, any of the amino acid sequences listed in Formula (I)-(V), the accompanying Examples, or the Tables.

[1042] The peptides of the invention can be prepared using standard recombinant techniques known in the art or chemical peptide synthesis techniques, for example using an automated or semi-automated peptide synthesizer, or both.

[1043] The peptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. Such methods are described, for example, herein and in U.S. Patent No. 6,610,824 and U.S. Patent No. 5,686,411 and in patent application serial number 454,533 (submitted on December 6, 1999), the entire contents of which are incorporated herein by reference. Various automatic synthesizers are commercially available and can be used according to known operating protocols. See, 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 can be performed using an automatic peptide synthesizer (e.g., Model 430A, Applied Biosystems Inc., Foster City, Calif.), using an NMP / HOBt (option 1) system and tBoc or Fmoc chemistry (see Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) and end-capping. Peptides can also be assembled using an Advanced Chem Tech synthesizer (Model MPS 350, Louisville, Ky.). Peptides can be purified by RP-HPLC (preparation and analysis), for example, using a Waters Delta Prep 3000 system and a C4, C8 or C18 preparative column (10 μ, 2.2 × 25 cm; Vydac, Hesperia, Calif.). Polypeptides can be synthesized by convergent methods such as "native chemical ligation" and variations thereof, in which two or more peptide fragments with appropriately orthogonal reactive termini are linked by formation of native amide bonds. The newly formed peptides can be further linked to produce longer polypeptides. Individual starting peptides can be derivatized as desired, or can be derivatized after the ligation step.

[1044] Peptide analogs can be synthesized using a Pioneer continuous flow peptide synthesizer (Applied Biosystems) using PAL-PEG-PS resin (Applied Biosystems) with a loading of 0.2 mmol / g (0.25 mmol scale). Fmoc amino acid (4.0 eq, 1.0 mmol) residues were activated using 4.0 eq HBTU, 4.0 eq HOBT, 8.0 eq DIEA and coupled to the resin for 1 hour. The Fmoc group was removed by treatment with 20% (v / v) piperidine in dimethylformamide. The resin was treated with reagent B (93% TFA, 3% phenol, 3% water and 1% triisopropylsilane) for 2-3 hours for final deprotection and peptide cleavage from the solid support. The cleaved peptide was precipitated using tert-butyl methyl ether, precipitated by centrifugation and freeze-dried. The precipitate was redissolved in water (10-15 mL), filtered and purified by reverse phase HPLC using a C-18 column and an acetonitrile / water gradient containing 0.1% TFA. The purified product was freeze dried and analyzed by ESI-LC / MS and analytical HPLC and shown to be pure (>98%). The mass results were consistent with the calculated values.

[1045] Alternatively, peptides were assembled on a Symphonyl peptide synthesizer (Protein Technologies, Inc., Woburn, Mass.) using Rink amide resin (Novabiochem, San Diego, Calif.) at loadings of 0.43-0.49 mmol / g and scales of 0.050-0.100 mmol. Fmoc amino acids (Applied Biosystems, Inc., 5.0 eq, 0.250-0.500 mmol) residues were dissolved in 1-methyl-2-pyrrolidone at a concentration of 0.10 M. All other reagents (HBTU, HOBT and N,N-diisopropylethylamine) were prepared as 0.55 M solutions in dimethylformamide. The Fmoc-protected amino acids were then coupled to the resin-bound amino acids using HBTU (2.0 eq, 0.100-0.200 mmol), HOBT (1.8 eq, 0.090-0.18 mmol), N,N-diisopropylethylamine (2.4 eq, 0.120-0.240 mmol) for 2 hours. After the last amino acid was coupled, the peptide was deprotected using 20% ​​(v / v) piperidine in dimethylformamide for 1 hour. Once the peptide sequence was completed, the Symphonyl peptide synthesizer was programmed to cleave the resin. The peptide was synthesized using a 93% Trifluoroacetic acid (TFA) cleavage of the peptide from the resin was performed with a reagent mixture consisting of TFA, 3% phenol, 3% water, and 1% triisopropylsilane. The cleaved peptide was precipitated using tert-butyl methyl ether, precipitated by centrifugation and freeze-dried. The precipitate was dissolved in acetic acid, freeze-dried and then dissolved in water, filtered and purified by reverse phase HPLC using a C18 column and an acetonitrile / water gradient containing 0.1% TFA. The purity of the peptide was assessed using analytical HPLC and the identity was confirmed by LC / MS and MALDI-MS.

[1046] Active proteins can be readily synthesized and then screened in screening assays designed to identify reactive peptides.

[1047] The peptides of the present invention may be produced by recombinant techniques known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor (1989). The peptides of the present invention produced by recombinant techniques may be expressed from polynucleotides. Those skilled in the art will appreciate that polynucleotides encoding such GIP analogs or hybrid peptides, including DNA and RNA, may be obtained from wild-type cDNAs, such as GIP, GLP1, amylin, taking into account the degeneracy of codon usage, or may be engineered as needed. These polynucleotide sequences may incorporate codons that promote transcription and translation of mRNA in a microbial host. These manufacturing sequences may be easily constructed according to methods well known in the art. See, for example, WO 83 / 04053. The above polynucleotides may also optionally encode an N-terminal methionyl residue. Non-peptide compounds useful in the present invention may be prepared by methods known in the art. For example, phosphate-containing amino acids and peptides containing these amino acids may be prepared using methods known in the art. See, for example, Bartlett and Landen, Bioorg. Chem. 14: 356-77 (1986).

[1048] A variety of expression vectors / host systems can be used to contain and express the peptide coding sequences of the present invention. These systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Mammalian cells useful in the production of recombinant proteins include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g., COS-7), WI 38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Exemplary schemes for recombinantly expressing the protein are described herein.

[1049] Thus, the polynucleotide sequences provided herein are useful in generating new and useful viral and plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacteria, yeast and mammalian cells grown in culture), and new and useful methods for growing such host cells capable of expressing the GIP polypeptides of the present invention. The polynucleotide sequences encoding GIP analogs or hybrids herein may be useful in gene therapy to alleviate the problem of insufficient production of GIP or other component peptide hormones of the hybrids, or to meet the need for increased levels of these hormones.

[1050] Host cells can be prokaryotic or eukaryotic, and include bacteria, mammalian cells (such as Chinese hamster ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells), yeast cells, and insect cells.

[1051] Mammalian host systems for expressing recombinant proteins are also well known to those skilled in the art. Host cell strains can be selected based on a certain specific ability to process the expressed protein or produce certain useful post-translational modifications in providing protein activity. Modifications of these polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, such as cutting the "pre-pro" form of the protein, may also be important for correct insertion, folding, and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular machinery and characteristic mechanisms for these post-translational activities, and can be selected to ensure the correct modification and processing of the introduced foreign protein.

[1052] Alternatively, a yeast system can be used to generate the peptides of the present invention. For example, the coding region of the GIP polypeptide cDNA is amplified by PCR. DNA encoding the yeast pre-pro-alpha leader sequence is amplified by PCR from yeast genomic DNA, using a primer comprising nucleotides 1-20 of the alpha mating factor gene and another primer complementary to nucleotides 255-235 of the gene (Kurjan and Herskowitz, Cell, 30:933-43 (1982)). The pre-pro-alpha leader coding sequence and the GIP polypeptide coding sequence fragment are connected to a plasmid containing a yeast alcohol dehydrogenase (ADH2) promoter, so that the promoter directs the expression of a fusion protein fused to a mature GIP polypeptide by the pre-pro-alpha factor. As taught by Rose and Broach, Meth.Enz.185:234-79, Goeddeled., Academic Press, Inc., San Diego, Calif. (1990), the vector further includes an ADH2 transcription terminator downstream of the cloning site, a yeast "2-micron" replication origin, a yeast leu-2d gene, yeast REP1 and REP2 genes, an E. coli β-lactamase gene, and an E. coli replication origin. The β-lactamase and leu-2d genes provide selection in bacteria and yeast, respectively. The leu-2d gene also promotes an increase in the copy number of the plasmid in yeast to induce a higher level of expression. The REP1 and REP2 genes encode proteins that play a role in plasmid copy number regulation.

[1053] The DNA construct described in the previous paragraph is transformed into yeast cells using known methods (e.g., lithium acetate treatment (Steams et al., Meth. Enz. 185:280-97 (1990)). The ADH2 promoter is induced when glucose is depleted in the culture medium (Price et al., Gene 55:287 (1987)). The pre-pro-α sequence causes the fusion protein to be secreted from the cells. At the same time, the yeast KEX2 protein cleaves the pre-pro sequence from the mature GIP polypeptide (Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330-4 (1984)).

[1054] The peptides of the present invention can also be recombinantly expressed in yeast using commercially available expression systems such as the Pichia expression system (Invitrogen, San Diego, Calif.) according to the manufacturer's instructions. This system also relies on pre-pro-alpha sequences to direct secretion, but transcription of the insert is driven by a methanol-inducible alcohol oxidase (AOX1) promoter. The secreted peptide can be purified from yeast culture medium by methods such as those used to purify peptides from bacterial and mammalian cell supernatants.

[1055] Alternatively, the cDNA encoding the peptide of the present invention can be cloned into an expression vector, for example, the baculovirus expression vector pVL1393. The vector encoding the GIP compound is then used to infect Spodoptera frugiperda cells in sF9 protein-free medium according to the manufacturer's instructions (Sigma Chemical Co., St. Louis, MO), and produce recombinant protein. The protein is purified and concentrated from the culture medium using a heparin-Sepharose column (Pharmacia, Piscataway, NJ) and a continuous molecular sieve column (Amicon, Beverly, Mass.), and resuspended in PBS. SDS-PAGE analysis shows a single band and confirms the size of the protein, and Edman sequencing on a Proton 2090 peptide sequencer confirms its N-terminal sequence.

[1056] In another example, the DNA sequence encoding the peptide of the present invention can be amplified by PCR and cloned into a suitable vector, such as pGEX-3X (Pharmacia, Piscataway, NJ). The pGEX vector is designed to produce a fusion protein comprising a glutathione-S-transferase (GST) encoded by the vector and a protein encoded by a DNA fragment inserted into the vector cloning site. PCR primers can be generated to include, for example, an appropriate cleavage site. The recombinant fusion protein can then be cut from the GST portion of the fusion protein. For example, the pGEX-3X / GIP analog peptide construct is transformed into Escherichia coli XL-1Blue cells (Agilent, Santa Clara, CA), and a single transformant is isolated and grown in LB medium (supplemented with carbenicillin) at 37°C to an optical density of 0.4 at a wavelength of 600nm, and then further incubated for 4 hours in the presence of 0.5mM isopropyl β-D-thiogalactopyranoside (Sigma Chemical Co., St.Louis, Mo.). Plasmid DNA from individual transformants was purified and partially sequenced using an automated sequencer to confirm the presence of the desired peptide encoding gene inserted in the correct orientation.

[1057] Fusion proteins that are expected to be produced in bacteria as insoluble inclusion bodies can be purified as follows. Cells were collected by centrifugation; washed in 0.15M NaCl, 10mM Tris (pH 8), 1mM EDTA; and treated with 0.1mg / mL lysozyme (Sigma Chemical Co.) at room temperature for 15 minutes. The lysate was clarified by ultrasonic treatment, and cell debris was precipitated by centrifugation at 12,000×g for 10 minutes. The pellet containing the fusion protein was resuspended in 50mM Tris (pH 8) and 10mM EDTA, layered on 50% glycerol, and centrifuged at 6000×g for 30 minutes. The pellet was resuspended in standard phosphate buffered saline solution (PBS) without Mg++ and Ca++. The fusion protein was further purified by resuspending the pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel was soaked in 0.4M KCl to visualize the protein, cut out and electroeluted in gel running buffer without SDS. If, for example, a GST / GIP polypeptide fusion protein produced in bacteria is a soluble protein, it can be purified using the GST purification module (Pharmacia Biotech).

[1058] The fusion protein can be digested to cut GST from mature GIP analogs or hybrid peptides. The digestion reaction (20-40 μg fusion protein, 20-30 units of human thrombin (4000U / mg (Sigma) in 0.5mL PBS) was incubated at room temperature for 16-48 hours, and the reaction products were loaded on a denaturing SDS-PAGE gel. The gel was soaked in 0.4M KCl to visualize the protein bands. The identity of the protein bands corresponding to the peptides of the expected molecular weight can be confirmed by partial amino acid sequence analysis using an Orbitrap Exploris 490 mass spectrometer (ThermoFisher Scientific, Waltham, MA).

[1059] Transformed cells are preferably used for long-term, high-yield protein production, so stable expression is desirable. Once a vector containing a selectable marker and a desired expression cassette is transformed into these cells, the cells can be grown in a rich medium for 1-2 days and then switched to a selection medium. Selectable markers are designed to confer resistance to selection, and their presence allows 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.

[1060] Many selection systems can be used to recover cells transformed for recombinant protein production. These selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes in tk-, hgprt-, or aprt- cells, respectively. Similarly, antimetabolite resistance can be used as the basis for the following selections: dhfr, which confers methotrexate resistance; gpt, which confers mycophenolic acid resistance; neo, which confers aminoglycoside G418 resistance; als (also), which confers chlorsulfuron resistance; and hygro, which confers hygromycin resistance. Other potentially useful selectable genes include trpB, which enables cells to utilize indole instead of tryptophan, or hisD, which enables cells to utilize histinol instead of histidine. Markers that provide visual indications for transformant identification include anthocyanins, β-glucosidase and its substrate GUS, and luciferase and its substrate luciferin.

[1061] The peptides of the present invention can be produced using a combination of automated peptide synthesis and recombinant technology. For example, the GIP peptide can include a combination of modifications including deletions, substitutions, and insertions by PEGylation. Such GIP peptides can be produced in stages. In the first stage, an intermediate GIP peptide modified by deletion, substitution, insertion, and any combination thereof is produced by recombinant technology as described. Then, after the optional purification step described herein, the intermediate GIP peptide is chemically modified with a suitable PEGylation reagent (e.g., from NeKtar Transforming Therapeutics, San Carlos, Calif.) to generate the desired GIP peptide. Those skilled in the art will appreciate that the above-mentioned procedure can be extended to peptides of the present invention comprising a combination of modifications selected from deletions, substitutions, insertions, derivatizations, and other well-known and presently contemplated modification means.

[1062] It may be necessary to purify the GIP polypeptides produced by the present invention. Peptide purification techniques are well known to those skilled in the art. These techniques involve, at one level, the crude fractionation of the cellular environment into peptide and non-peptide fractions. After the peptide is separated from the other proteins, the peptide of interest can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly efficient peptide purification method is reverse phase HPLC followed by characterization of the purified product by liquid chromatography / mass spectrometry (LC / MS) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Additional confirmation of purity is obtained by determining amino acid analysis.

[1063] Certain aspects of the present invention relate to the purification of encoded proteins or peptides, and in specific embodiments, to the substantial purification of encoded proteins or peptides. The term "purified peptide" refers herein to a composition that can be separated from other components, wherein the peptide is purified to any extent relative to its naturally obtainable state. Therefore, a purified peptide also refers to a peptide that is free from the environment in which it can naturally exist. Typically, "purification" will refer to a peptide composition that has been graded to remove various other components, and such a composition substantially retains the biological activity of its expression. When the term "substantially purified" is used, this designation will refer to a composition in which the peptide constitutes the major component in the composition, such as a composition in which the peptide accounts for about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more.

[1064] Various techniques suitable for peptide purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc.; heat denaturation followed by centrifugation; chromatographic steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite, and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that the order in which the various purification steps are performed can be changed, or that certain steps can be omitted, and still result in a method suitable for preparing a substantially purified protein or peptide.

[1065] Peptides are not generally required to be provided in the purest state at all times. In fact, it is considered that in certain embodiments, products with lower substantial purification will have practicality. Partial purification can be achieved by using fewer purification step combinations or by using different forms of the same general purification scheme. For example, it is understood that the cation exchange column chromatography performed using HPLC equipment generally produces a higher "fold" of purification than the same technology using a low pressure chromatography system. The method showing a lower degree of relative purity may have advantages in the total recovery of the protein product or in maintaining the activity of the expressed protein.

[1066] Such peptides of the invention may optionally be purified and isolated from other components obtained in the process. Methods for polypeptide purification may be found in U.S. Pat. No. 5,849,883. These documents describe specific exemplary methods for the isolation and purification of G-CSF compositions, which may be useful in isolating and purifying the GIP polypeptides of the invention. In view of the disclosure of these patents, it will be apparent to those skilled in the art that many purification techniques may be used to purify the peptides of the invention from a given source.

[1067] It is contemplated that a combination of anion exchange and immunoaffinity chromatography may be employed to produce purified peptides of the invention.

[1068] Treatment

[1069] The peptides of the present invention are useful in the treatment and prevention of metabolic and hepatic diseases and disorders.

[1070] Metabolic diseases and disorders come in many forms, including obesity, diabetes, dyslipidemia, insulin resistance, apoptosis, and the like. Obesity and its related disorders are common and very serious public health problems in the United States and around the world. Upper body obesity is the strongest known risk factor for type 2 diabetes and an important risk factor for cardiovascular disease. Obesity is a recognized risk factor for reproductive diseases such as hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, polycystic ovary syndrome, breast cancer, prostate cancer, and colon cancer, as well as an increased incidence of complications of general anesthesia (see, e.g., Kopelman, Nature 404: 635-43 (2000)). Obesity reduces life expectancy and brings the above-mentioned serious comorbidity risks, as well as disorders such as infection, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, orthopedic injuries, and thromboembolic diseases (Rissanen et al., Br. Med. J. 301: 835-7 (1990)). Obesity is also a risk factor for a class of conditions known as insulin resistance syndrome or "Syndrome X". The latest estimate of the medical costs of obesity and its related disorders is $2 trillion worldwide. The pathogenesis of obesity is believed to be multifactorial, but the basic problem is that nutrient supply and energy expenditure in obese subjects are not balanced until there is excess adipose tissue. Obesity is currently a difficult to treat, chronic, and essentially incurable metabolic disorder. A weight loss treatment drug useful for obese people could have a profound beneficial effect on their health.

[1071] Diabetes mellitus is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria, caused by insufficient production or utilization of insulin. Diabetes mellitus severely affects the quality of life of a large portion of the population in developed countries. Insufficient insulin production is characterized as type 1 diabetes, while inadequate insulin utilization is characterized as type 2 diabetes. However, it is now generally accepted that there are many different diabetes-related diseases that begin long before overt diabetes is diagnosed. Furthermore, suboptimal control of glucose metabolism in diabetes mellitus leads to a range of associated lipid and cardiovascular disorders.

[1072] Dyslipidemia, or abnormal levels of lipoproteins in the plasma, is a common condition in diabetes. Dyslipidemia is usually characterized by elevated plasma triglycerides, low HDL (high-density lipoprotein) cholesterol, normal to elevated LDL (low-density lipoprotein) cholesterol levels, and increased levels of small, dense LDL (low-density lipoprotein) particles in the blood. Dyslipidemia is one of the main causes of increased coronary events and mortality in diabetic subjects. Epidemiological studies have confirmed this by showing that the coronary mortality rate in diabetic subjects is several times higher than that in non-diabetic subjects. Several lipoprotein abnormalities have been described in diabetic subjects.

[1073] Insulin resistance refers to the diminished ability of insulin to exert its biological effects over a wide range of concentrations. In insulin resistance, the body secretes abnormally high amounts of insulin to compensate for this defect, and a state of impaired glucose tolerance develops. If the defective insulin action cannot be compensated, plasma glucose concentrations inevitably rise, leading to the clinical state of diabetes. The contribution of insulin resistance and relative hyperinsulinemia to obesity, hypertension, atherosclerosis, and type 2 diabetes is being recognized. The association of insulin resistance with obesity, hypertension, and angina has been described as a syndrome, Syndrome X, with insulin resistance as the common pathogenic link.

[1074] Nonalcoholic fatty liver disease (NAFLD) is an umbrella term and covers everything from simple fat deposits in the liver to more advanced steatosis associated with hepatitis, fibrosis, cirrhosis, and even hepatocellular carcinoma in some cases. NAFLD is becoming increasingly common worldwide, especially in Western countries. In the United States, it is the most common form of chronic liver disease, affecting about a quarter of the population. Some individuals with NAFLD may develop nonalcoholic steatohepatitis (NASH), an aggressive fatty liver disease marked by hepatitis that may progress to severe scarring (cirrhosis) and liver failure. This damage is similar to that caused by heavy alcohol consumption. NAFLD includes nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). NAFL is characterized by fatty degeneration of the liver involving more than 5% of the parenchyma without evidence of hepatocellular damage. NASH, on the other hand, is defined in histological terms as a necroinflammatory process in which hepatocytes become damaged against a background of steatosis. The natural history of NAFLD has not been fully characterized. Studies have shown that the incidence of NAFLD increases with the incidence of metabolic syndrome. Patients with type 2 diabetes show a particularly high risk of developing NASH and a two- to four-fold increased risk of fatty liver-related complications.

[1075] Apoptosis is an active process of cellular self-destruction regulated by exogenous and endogenous signals that occurs during normal development. It is well documented that apoptosis plays a key role in regulating pancreatic endocrine β cells. Increasing evidence suggests that in adult mammals, β-cell mass changes dynamically to adjust insulin production for the maintenance of normoglycemia under specific conditions, such as pregnancy and obesity. Control of β-cell mass depends on a delicate balance between cell proliferation, growth, and programmed cell death (apoptosis). Disturbance of this balance may result in impaired glucose homeostasis. Notably, for example, glucose intolerance occurs with aging when the rate of β-cell replication decreases, and human autopsy studies have repeatedly shown that patients with non-insulin-dependent diabetes have 40-60% less β-cell mass than non-diabetic subjects. It is generally accepted that insulin resistance is an inevitable concomitant of obesity, but normoglycemia is maintained by compensatory hyperinsulinemia until the β-cells are unable to meet the increased insulin demand, at which point type 2 diabetes begins.

[1076] Attempts to treat the various abnormalities associated with diabetes have led to the administration of several antidiabetic drugs in order to address these abnormalities in different patients. However, the peptides of the present invention as discussed herein, when administered in a therapeutically effective amount, whether as a monotherapy or as an adjunct therapy, can be used to treat or prevent these and other diseases and conditions discussed throughout this article.

[1077] In one embodiment of the invention, reducing hyperglycemia in treated diabetic patients (e.g., by GLP-1 mimetics such as exenatide) provides a basis for intervention. Since chronic hyperglycemia in patients with type 2 diabetes impairs the insulin secretion response of GIP, improved glycemic control by exenatide treatment will restore the response of pancreatic β cells to GIP stimulation. Administration of the peptides of the invention will enable diabetic patients or patients with conditions associated with elevated blood sugar to achieve desired normal blood sugar levels.

[1078] Since currently prescribed antidiabetic agents (such as metformin, sulfonylureas, TZDs, SGLT2 inhibitors, etc.) are able to achieve varying degrees of glycemic control, the combination of the peptides of the present invention with these therapies should also elicit an improved response, leading to normalization of blood glucose levels.

[1079] Thus, in one embodiment, the methods of the invention are based on the concept that patients can be primed for treatment by prior glucose lowering with other antidiabetic agents such as GLP-1, GLP-1 analogs or exenatide or other agents such as metformin, sulfonylureas, thiazolidinediones (TZDs), pramlintide, insulin, acarbose, dipeptidyl peptidase (DPP-IV) inhibitors and SGLT-2 inhibitors. DPP-IV inhibitors are well known and described, for example, in published applications US20050004117, U.S. Pat. No. 6,710,040 and U.S. Pat. No. 6,645,995, which are incorporated herein by reference for their compounds. An example of a sulfonylurea (SFU) is glimepiride, which acts on pancreatic tissue to produce insulin.

[1080] The peptides of the present invention can be used to reduce food intake, reduce appetite, reduce caloric intake, induce satiety, reduce nutrient supply, cause weight loss, affect body composition, change body energy content or energy expenditure, improve lipid profile (including reducing LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels), slow gastrointestinal motility, delay gastric emptying, regulate postprandial blood sugar fluctuations, prevent or inhibit glucagon secretion and lower blood pressure. In one embodiment, these GIP polypeptides include exenatide, GLP1, amylin and / or sCT parts.

[1081] Of particular interest in anti-obesity, weight loss, reduced food intake, increased metabolic rate, and reduced body fat and / or fat redistribution are those peptides of the invention that contain GIP, GLP-1, exenatide, GLP-1-GIP receptor co-agonists (e.g., amylin, i.e., dual amylin calcitonin receptor agonists), leptin family modules that are effective in reducing food intake, altering body composition, redistributing fat, and / or reducing body weight. Of particular interest in treating obesity and related diseases and conditions discussed herein (body fat loss) are peptides of the invention that contain GIP, exenatide-4 or an analog or derivative thereof, an amylin component (e.g., pramlintide or dual amylin calcitonin receptor agonists), a FN38 family member (e.g., FN38 or an analog or derivative thereof). In another embodiment, the peptides of the invention may have at least one, preferably two, components that act on the CNS. It has been confirmed that the forebrain (brain components derived from the telencephalon and diencephalon) and the specific areas of the hindbrain or brainstem (including the midbrain, pons and medulla oblongata) are involved in controlling energy balance. The forebrain structures or nuclei located in the hypothalamus are relevant to food intake and / or weight regulation, including, for example, the arcuate nucleus (ARC), the paraventricular nucleus (PVN), the dorsomedial hypothalamus (DMH), the ventromedial nucleus (VMH) and the lateral hypothalamic nucleus (LHA). The hindbrain structures or nuclei located in the brainstem are relevant to food intake and / or weight regulation, including, for example, the solitary tract nucleus (NST), the postrema (AP) and the lateral accessory pontine nucleus (IPBN). The brainstem nuclei that control the elements of the completion motor control system may be controlled by the first or second order projections from brainstem areas such as NST, AP and IPBN. It is noteworthy that AP, NST and IPBN have all been shown to have their own integration capabilities (collectively and independently).

[1082] A variety of anti-obesity agents for the CNS act on these forebrain structures located in the hypothalamus that are related to food intake and / or weight regulation. In addition, anti-obesity agents for the CNS act on hindbrain structures located in the brainstem that are related to food intake and / or weight regulation. These peptide components include, for example, leptin and its agonists, ciliary neurotrophic factor (CNTF) and its agonists, peptide YY (PYY) and its agonists, exenatide and its agonists, GLP-1 and its agonists, growth hormone releasing peptide and its antagonists, cholecystokinin (CCK) and its agonists, and amylin and its agonists, including those described herein.

[1083] In certain embodiments, the peptides of the present invention and methods of use thereof include a first component that primarily targets hypothalamic energy balance centers such as ARC, PVN, VM, and LH. In one embodiment, the peptides of the present invention contain GIP or an analog or derivative thereof and one or more other peptide family components that also target the hypothalamus but are located in different locations or through different mechanisms of action that are different from the first component. When the GIP peptide contains more than one other peptide family component and these components also target the hypothalamus, these more than one other peptide family components can target the same location through the same mechanism of action as each other, or they can target different locations and / or different mechanisms of action. In another embodiment, the GIP peptide also contains one or more other peptide family components, which provide one or more additional beneficial therapeutic effects as needed, including anti-obesity effects, blood sugar control, cardioprotection, and / or hypertension control through locations or mechanisms of action that are different from the first component and each other. In certain embodiments, the additional peptide family component is a peptide family component that primarily targets energy balance centers such as NST, AP, and IPBN in the hindbrain.

[1084] In certain embodiments, the peptides of the present invention and methods of use thereof include a first component that primarily targets hindbrain energy balance centers such as NST, AP, and IPBN. In one embodiment, the GIP peptide further comprises one or more other peptide family components that also target the hypothalamus but are located in different locations or through different mechanisms of action from the first component and each other. In another embodiment, the peptides of the present invention then comprise one or more other peptide family components that provide one or more additional beneficial therapeutic effects as needed, including anti-obesity effects, blood sugar control, cardioprotection, and / or hypertension control through locations or mechanisms of action that are different from the first component and each other. In certain embodiments, the additional peptide family components are peptide family components that primarily target energy balance centers of the hypothalamus such as ARC, PVN, VM, and LH.

[1085] As used herein, an anti-obesity agent that "acts on the forebrain structures involved in food intake and / or weight regulation" stimulates or inhibits the activity of specific areas (e.g., specific nuclei and / or neural circuits) in the forebrain. Such stimulation or inhibition of the forebrain results in a reduction in nutrient availability to the body. An anti-obesity agent that "acts on the hindbrain structures involved in food intake and / or weight regulation" stimulates or inhibits the activity of specific areas (e.g., specific nuclei and / or neural circuits) in the hindbrain. Such stimulation or inhibition of the hindbrain results in a reduction in nutrient availability to the body.

[1086] On the other hand, methods for reducing fat mass by increasing the metabolic rate of the subject are provided, wherein these methods include administering the peptides of the present invention in an amount effective to reduce fat mass by increasing the metabolic rate of the subject. Fat mass can be expressed as a percentage of total body mass. In some embodiments, fat mass is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20% or at least 25% during treatment. In one aspect, the lean body mass of the subject does not decrease during treatment. On the other hand, the lean body mass of the subject is maintained or increased during treatment. On the other hand, the subject is in a diet or restrictive diet that reduces calories. The so-called "diet that reduces calories" refers to a subject's daily intake of calories less than the normal diet of the same subject. In one case, the subject's daily intake of calories is at least reduced by 50 calories. In other cases, the subject's daily intake of calories is at least reduced by 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 calories.

[1087] In another embodiment, a method for changing the fat distribution, reducing fat mass, or both in a subject is provided. Therefore, subjects for whom changing body composition is beneficial may also benefit from this method. Changing body composition as referred to herein includes reducing or maintaining body fat, and minimizing the reduction, maintenance, or increase in lean body mass. In this case, body weight may increase or decrease. Therefore, the subject may be thin, overweight, or obese, as these terms are commonly used in the art. The method provided may also include reducing fat in non-fat tissue while retaining lean body mass. The use of this method includes treating diseases such as non-alcoholic steatohepatitis (NASH) or lipodystrophy.

[1088] In another embodiment, a method for changing the distribution of fat in a subject is provided, wherein the method includes administering an anti-obesity peptide of the present invention in an amount that effectively changes the distribution of fat in a subject. In one aspect, the change is due to an increase in the metabolism of the subject's visceral or ectopic fat, or both. "Fat distribution" refers to the location of fat deposition in the body. Such fat deposition locations include, for example, subcutaneous, visceral and ectopic fat depots. "Subcutaneous fat" refers to lipid deposition just below the surface of the skin. Any method that can be used to measure subcutaneous fat can be used to measure the amount of subcutaneous fat in a subject. Methods for measuring subcutaneous fat are known in the art, for example, the method described in U.S. Patent No. 6,530,886, the entire contents of which are incorporated herein by reference. "Ectopic fat storage" refers to lipid deposition in and around tissues and organs (e.g., skeletal muscle, heart, liver, pancreas, kidney, blood vessels) that make up lean body mass. Typically, ectopic fat storage is the accumulation of lipids outside the classic adipose tissue depot in the body. "Visceral fat" refers to fat deposition as intra-abdominal adipose tissue. Visceral fat surrounds important organs and can be metabolized by the liver to produce blood cholesterol. Visceral fat is associated with an increased risk of conditions such as polycystic ovary syndrome, metabolic syndrome, and cardiovascular disease. In certain embodiments, the method relates to visceral or ectopic fat or both at a rate greater than subcutaneous fat at least about 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50% of metabolism. In one aspect, these methods result in a favorable fat distribution. In one embodiment, a favorable fat distribution is an increase in the ratio of subcutaneous fat to visceral fat, ectopic fat, or both. In one aspect, the method relates to an increase in lean body mass, for example, due to an increase in muscle cell mass.

[1089] In another embodiment, a method for reducing the amount of subcutaneous fat in a subject is provided, wherein the method comprises administering an anti-obesity peptide of the present invention to a subject in need thereof in an amount effective to reduce the amount of subcutaneous fat in the subject. In one case, the amount of subcutaneous fat in the subject is at least reduced by about 5%. In other cases, the amount of subcutaneous fat is at least reduced by about 10%, 15%, 20%, 25%, 30%, 40% or 50% compared to the subject before administration of the anti-obesity peptide of the present invention.

[1090] The methods described herein can be used to reduce the amount of visceral fat in a subject. In one case, the amount of visceral fat in a subject is at least reduced by about 5%. In other cases, the amount of visceral fat in a subject is at least reduced by about 10%, 15%, 20%, 25%, 30%, 40% or 50% compared to the subject before the administration of the peptide of the present invention for anti-obesity. Visceral fat can be measured by any means that can be used to determine the amount of visceral fat in a subject. These methods include, for example, abdominal tomography performed by CT scanning and MRI. Other methods for determining visceral fat are described, for example, in U.S. Patent Nos. 6,864,415, 6,850,797 and 6,487,445.

[1091] In another embodiment, a method for preventing ectopic fat accumulation or reducing ectopic fat amount in a subject is provided, wherein the method includes administering an anti-obesity peptide of the present invention to a subject in need in an amount effective to prevent ectopic fat accumulation or reduce ectopic fat amount in the subject. In one case, the ectopic fat amount of the subject is at least reduced by about 5% compared to the subject before the administration of the anti-obesity peptide of the present invention. In other cases, the ectopic fat amount of the subject is at least reduced by about 10%, or at least reduced by about 15%, 20%, 25%, 30%, 40% or 50%. Alternatively, the ectopic fat amount is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the subcutaneous fat of the subject. Any method that can be used to measure ectopic fat can be used to measure the ectopic fat of the subject.

[1092] In another embodiment, a method for producing a more favorable fat distribution in a subject is provided, wherein the method includes administering a peptide of the present invention that is effective as an anti-obesity agent to the subject in an amount that effectively produces a favorable fat distribution. In one embodiment, the anti-obesity peptide of the present invention is administered to reduce the visceral fat or ectopic fat of the subject, or both. In one embodiment, the anti-obesity peptide of the present invention is administered, which includes a combination of at least one family module that acts on the forebrain structure involved in food intake or weight regulation or both and at least one family module that acts on the hindbrain structure involved in food intake or weight regulation or both. In one embodiment, these methods preferentially reduce the amount of visceral or ectopic fat or a combination of the two, rather than the reduction of subcutaneous fat. Such a method produces a higher ratio of subcutaneous fat to visceral or ectopic fat. Such an improved ratio can lead to a reduced risk of development of cardiovascular disease, polycystic ovary syndrome, metabolic syndrome, or any combination thereof. In one embodiment, the metabolic rate of ectopic or visceral fat is 5% higher than that of subcutaneous fat. In other embodiments, the metabolic rate of ectopic or visceral fat is at least 10%, 15%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that of subcutaneous fat.

[1093] Of particular interest for anti-obesity, weight and fat composition related treatments discussed herein are peptides of the invention containing, for example, GIP, exenatide, amylin (i.e., a dual amylin calcitonin receptor agonist), leptin or a GLP-1-GIP receptor co-agonist. In a further embodiment, the peptides of the invention contain at least two of these peptide family modules. The peptides of the invention can be administered alone or in combination with a second anti-obesity agent (e.g., amylin, leptin or an exenatide family peptide).

[1094] On the other hand, a method is provided, which uses a peptide of the present invention that is effective as an anti-obesity agent with a therapeutically effective amount, which is used in combination with a glucocorticoid. The untoward reaction of a glucocorticoid is to increase fat mass and reduce lean body mass. Therefore, it is considered that the anti-obesity agent combination can be combined with a glucocorticoid to counteract the untoward reaction of a glucocorticoid when the use of a glucocorticoid is useful.

[1095] As described herein, the peptides of the present invention can be administered separately or together with one or more other agents to obtain additional benefits or enhance the effects of the peptides of the present invention or other agents. For example, the anti-obesity peptides of the present invention can be administered together with anti-obesity agents or cardioprotective or antihypertensive agents, depending on the risk factors of the subject to be treated and the desired treatment results. Exemplary anti-obesity agents for administration (separate or mixed administration; administration before, simultaneously or after the peptides of the present invention) include 5-hydroxytryptamine (5HT) transport inhibitors, including but not limited to paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline and imipramine. Anti-obesity agents also include selective 5-hydroxytryptamine reuptake inhibitors, including but not limited to dexfenfluramine, fluoxetine, sibutramine and combinations thereof, as well as those described in U.S. Patent No. 6,365,633 and PCT Patent Application Publication No. WO 01 / 27060 and WO 01 / 162341, the entire contents of which are incorporated herein by reference. The 5HT transport inhibitors and serotonin reuptake inhibitors, analogs, derivatives, formulations, formulations, pharmaceutical compositions, dosages and routes of administration of the present invention have been previously described.

[1096] Anti-obesity agents also include selective 5-hydroxytryptamine receptor agonists and selective 5-HT2C receptor agonists, including but not limited to U.S. Pat. No. 3,914,250; and PCT Application Publication Nos. WO 02 / 36596, WO 02 / 48124, WO 02 / 10169, WO 01 / 66548, WO 02 / 44152; WO 02 / 51844, WO 02 / 40456 and WO 02 / 40457, the entire contents of which are incorporated herein by reference. Such selective 5-hydroxytryptamine receptor agonists and 5-HT2C receptor agonists, compositions comprising such agonists, and routes of administration suitable for the provided methods are known in the art. See, e.g., Halford et al. (2005) Curr. Drug Targets 6:201-213 and Weintraub et al. (1984) Arch. Intern. Med. 144:1143-1148.

[1097] Anti-obesity agents also include antagonists / inverse agonists of central cannabinoid receptors (CB-1 receptors), including but not limited to rimonabant (Sanofi Synthelabo) and SR-147778 (Sanofi Synthelabo). CB-1 antagonists / inverse agonists, derivatives, formulations, formulations, pharmaceutical compositions, dosages and routes of administration have been previously described, for example, in U.S. 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 Nos. EP-656 354 and EP-658 546; and PCT Application Publication Nos. WO 96 / 33159, WO 98 / 33765, WO 98 / 43636, WO 98 / 43635, WO 01 / 09120, WO98 / 31227, WO98 / 41519, WO98 / 37061, WO00 / 10967, WO00 / 10968, WO97 / 29079, WO99 / 02499, WO 01 / 58869 and WO 02 / 076949, the entire contents of which are incorporated herein by reference.

[1098] Anti-obesity agents also include melanocortin and melanocortin agonists. MC4R receptors appear to play a role in energy balance and obesity. See, 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. Ther. Patents 14: 327-336 (2004). Melanocortin agonists including but not limited to MC4R agonists suitable for the provided methods and compositions comprising such agonists are known in the art. MCR agonists, MC4R agonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages and routes of administration have been previously described, for example in the following PCT patent applications, the entire contents of which are incorporated herein by reference: 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.

[1099] Anti-obesity agents also include metabotropic glutamate subtype 5 receptor (mGluR5) antagonists, including but not limited to compounds such as 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine) (MTEP) and those described in Anderson et al. J. Eur. J. Pharmacol. 473:35-40 (2003); Cosford et al. Bioorg. Med. Chem. Lett. 13(3):351-4 (2003); and Anderson et al. J. Pharmacol. Exp. Ther. 303:1044-1051 (2002).

[1100] Anti-obesity agents also include topiramate, phentermine, or a combination thereof, which are indicated as anticonvulsants but have also been shown to enhance weight loss.

[1101] 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. Pat. Nos. 6,124,331, 6,214,853 and 6,340,683. NPY1 and NPY5 antagonists, derivatives, formulations, formulas, pharmaceutical compositions, dosages and routes of administration have been previously described. NPY1 antagonists for use in the provided compositions and methods include U.S. Pat. No. 6,001,836; and PCT Application Publication Nos. WO 96 / 14307, WO 01 / 23387, WO 99 / 51600, WO 01 / 85690, WO 01 / 85098, WO 01 / 85173 and WO 01 / 89528, the entire contents of which are incorporated herein by reference. NPY5 antagonists useful in the compositions and methods of use provided herein include, but are not limited to, compounds described in U.S. Pat. Nos. 6,140,354, 6,191,160, 6,258,837, 6,313,298, 6,337,332, 6,329,395, 6,340,683, 6,326,375, and 6,335,345; European Patent Nos. EP-01010691 and EP-01044970; 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 WO 01 / 14376.

[1102] Anti-obesity agents also include melanin concentrating hormone (MCH) antagonists, including melanin concentrating hormone 1 receptor (MCH1R) antagonists, such as T-226296 (Takeda) and melanin concentrating hormone 2 receptor (MCH2R) antagonists. MCH receptor antagonists, derivatives, formulations, formulations, pharmaceutical compositions, dosages and routes of administration have been previously described, for example, in U.S. Patent Application Publication Nos. 2005 / 0009815, 2005 / 0026915, 2004 / 0152742, 2004 / 0209865; PCT Patent Application Publication Nos. WO 01 / 82925, WO 01 / 87834, WO 02 / 06245, WO 02 / 04433 and WO 02 / 51809 and Japanese Patent Application No. JP 13226269, the entire contents of which are incorporated herein by reference.

[1103] Anti-obesity agents also include opioid antagonists, including but not limited to those described in PCT Application No. WO 00 / 21509. Specific opioid antagonists for use in the compositions and methods of use provided herein include but are not limited to nalmefene 3-methoxynaltrexone, naloxone, naltrexone, bupropion, naloxazine, β-fulnaltrexamine, delta 1 ([D-Ala2, Leu5, Cys6]-enkephalin (DALCE)), naltrindole isothiocyanate and nor-binaltorphamine or a combination thereof.

[1104] Anti-obesity agents also include orexin antagonists, including but not limited to those described in PCT Patent Application Nos. WO 01 / 96302, WO 01 / 68609, WO 02 / 51232 and WO 02 / 51838. Specific orexin antagonists for use in the provided compositions and methods of use include but are not limited to SB-334867-A.

[1105] Anti-obesity agents also include neuropeptide Y2 (NPY2) agonists, including but not limited to compounds such as PYY3-36 (e.g., Batterham et al. (2003) Nature 418:650-654), NPY3-36 and other Y2 agonists, such as N-acetyl [Leu (28,31)] NPY 24-36 (White-Smith et al. (1999) Neuropeptides 33:526-533), TASP-V (Malis et al. (1999) Br. J. Pharmacol. 126:989-996), cyclo-(28 / 32)-Ac-[Lys28-Glu32]-(25-36)-pNPY (Cabrele et al. (2000) J. Pept. Sci. 6:97-122), which can be the peptide components of the invention as discussed or administered separately. Anti-obesity agents provided also include neuropeptide Y4 (NPY4) agonists, including but not limited to compounds such as pancreatic peptide (PP) (e.g., Batterham et al. (2003) J. Clin. Endocrinol. Metab. 88: 3989-3992) and other Y4 agonists such as 1229U91 (Raposinho et al. (2000) Neuroendocrinology 71: 2-7). NPY2 agonists and NPY4 agonists, derivatives, formulations, formulations, pharmaceutical compositions, dosages and routes of administration have been described, for example, in U.S. Patent Publication No. 2002 / 0141985 and PCT Application Publication No. WO2005 / 077094.

[1106] Anti-obesity agents also include histamine 3 (H3) antagonists / inverse agonists, including but not limited to those described in PCT Application No. WO 02 / 15905, O-[3-(1H-imidazol-4-yl)propanol]carbamates (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-phenyl carbamates (Reidemeister et al. (2000) Pharmazie 55:83-86), and proxifen derivatives (Sasse et al. (2001) Arch. Pharm. (Weinheim) 334:45-52). al. (2000) J. Med. Chem. 43:3335-3343). Specific H3 antagonists / inverse agonists for use in the provided compositions and methods of use include, but are not limited to, thioperamide, 3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate, clofopil, iofopil, imoproxifen, and GT2394 (Gliatech).

[1107] 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 U.S. Patent No. 5,739,106. Specific CCK-A agonists include but are not limited to AR-R15849, GI 181771, JMV-180, A-71378, A-71623 and SR146131.

[1108] Anti-obesity agents also include growth hormone releasing peptide antagonists, such as those described in PCT application publication numbers WO 01 / 87335 and WO02 / 08250. Growth hormone releasing peptide antagonists are also known as GHS (growth hormone secretagogue receptor) antagonists. Therefore, the compositions and methods provided contemplate the use of GHS antagonists instead of growth hormone releasing peptide antagonists.

[1109] Anti-obesity agents include obestatin and its analogs and agonists. Obestatin is a peptide derived from the same precursor, preproghrelin, from which growth hormone releasing peptide 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 the activity of growth hormone releasing peptide, obestatin appears to act as an anorectic 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.

[1110] In addition, amylin mimetics (e.g., amylin-calcitonin receptor co-agonists such as davalinide), GLP-1-GIP receptor co-agonists such as incretins (e.g., exendin-4, leptin and PYY analogs) are also anti-obesity agents that can be administered as anti-obesity agents with GIP comprising the peptides of the invention.

[1111] Therefore, in certain embodiments, the peptides of the present invention are used to treat or prevent conditions or disorders that are alleviated by reducing nutrient availability, comprising administering to the subject a therapeutically or prophylactically effective amount of a compound of the present invention. These conditions and disorders include, but are not limited to, controlling food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive inotropic effects, reducing catabolic effects, slowing gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. These conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders and rare genetic diseases of obesity such as Prader-Willi syndrome, intensive care, insulin resistance and its related disorders such as polycystic ovary syndrome, obesity, any type of diabetes, including type 1, type 2 and gestational diabetes, and CNS disorders such as prevention of neurodegeneration, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH) and complications of diabetes (e.g., neuropathy (e.g., treated with peptides of the invention including GIP peptides comprising members of the exenatide family), neuropathic pain (e.g., treated with GIP peptides of the invention including modules of the amylin family of hormones), retinopathy, nephropathy, conditions of insufficient pancreatic beta cell mass (e.g., islet neogenesis based on exenatide-4 and GLP-1), metabolic syndrome, dumping syndrome, polycystic ovary syndrome, hypertension, dyslipidemia, cardiovascular disease, hyperlipidemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and osteoarthritis.

[1112] Non-limiting examples of cardiovascular conditions or diseases are hypertension, myocardial ischemia and myocardial reperfusion. The compounds of the present invention may also be used to treat or prevent other conditions associated with obesity, including stroke, cancer (e.g., endometrial cancer, breast cancer, prostate cancer and colon cancer), gallbladder disease, sleep apnea, reduced fertility and osteoarthritis (see Lyznicki et al, Am. Fam. Phys. 63: 2185, 2001). In other embodiments, the peptides of the present invention may be used to change body shape for aesthetic reasons, enhance physical ability, or produce leaner meat sources. The peptides of the present invention are used to change body composition by reducing fat without significantly reducing muscle mass, thereby producing an ideal reduction in body fat while maintaining lean body mass. In one embodiment, such a peptide of the present invention comprises exenatide, GLP1, amylin and / or sCT moieties.

[1113] In another aspect of the present invention, a method for treating or preventing obesity is provided, wherein the method comprises administering a therapeutically or prophylactically effective amount of a peptide of the present invention to a subject in need thereof. In an exemplary embodiment, the subject is an obese or overweight subject. Although "obesity" is generally defined as a body mass index of more than 30, for the purposes of the present disclosure, any subject who needs or wishes to lose weight, including those with a body mass index of less than 30, is included within the scope of "obesity". Subjects with insulin resistance, glucose intolerance, or any form of diabetes (e.g., type 1, type 2, or gestational diabetes) can benefit from these peptides of the present invention. In one embodiment, such a peptide of the present invention comprises a GIP, amylin calcitonin receptor agonist such as davalintide, a GLP-1-GIP receptor co-agonist such as exenatide, GLP1, amylin, and / or sCT moiety.

[1114] In other aspects of the invention, methods of reducing food intake, reducing the availability of nutrients, causing weight loss, affecting body composition, and changing body energy content or increasing energy expenditure, treating diabetes and improving lipid profiles (including reducing LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels) are provided, wherein these methods include administering an effective amount of the peptides of the invention to a subject. In an exemplary embodiment, the methods of the invention are used to treat or prevent a condition or disorder that can be alleviated by reducing the availability of nutrients in a subject in need thereof, comprising administering a therapeutically or prophylactically effective amount of the peptides of the invention to the subject. These conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and any type of diabetes. In one embodiment, these peptides of the invention comprise GIP, exenatide, GLP1, amylin, and / or sCT moieties.

[1115] Additional assays useful in the present invention include those that can determine the effects of the peptides of the present invention, particularly those comprising a GIP, exenatide, GLP1, amylin and / or sCT portion, on body composition. An exemplary assay can be an assay involving the use of a diet-induced obesity (DIO) mouse model for metabolic disease. Male C57BL / 6J mice can be fed a high-fat diet (#D12331, 58% of calories from fat; Research Diets, Inc.) for 6 weeks beginning at 4 weeks of age prior to the treatment period. The mice can continue to consume their high-fat diet during the study. Water can be provided freely throughout the study. A group of non-obese mice of similar age can be fed a low-fat diet (#D12329, 11% of calories from fat) for comparison of metabolic parameters with the DIO group.

[1116] DIO mice can be delivered vehicle (50% dimethyl sulfoxide (DMSO) dissolved in water) or peptides of the present invention by subcutaneous (SC) intrascapular implantation of osmotic pumps. The pumps of the latter group can be set to deliver any amount, such as 1000 μg / kg / d of the peptides of the present invention, for 7-28 days. Body weight and food intake can be measured regularly throughout the study. Respiratory quotient (RQ, defined as CO2 production ÷ O2 consumption) and metabolic rate can be determined using whole animal indirect calorimetry (Oxymax, Columbus Instruments, Columbus, Ohio). Mice can be killed by isoflurane overdose and fat gain index (bilateral epididymal fat pad weight) can be measured. In addition, before determining the epididymal weight, the body composition (lean body mass, fat mass) of each mouse can be analyzed using a dual-energy X-ray absorptiometry (DEXA) instrument according to the manufacturer's instructions (Lunar Piximus, GE Imaging System). In the methods of the invention, peptides of the invention, particularly those comprising a GIP, exenatide, PPF, PYY, GLP1, amylin and / or sCT portion, can be identified whose potency in one of the assays described herein (preferably a food intake, gastric emptying, pancreatic secretion, weight loss or body composition assay) is greater than the potency of a component peptide hormone in the same assay.

[1117] In addition to improving hypertension in a subject in need thereof due to reduced food intake, weight loss or treatment of obesity, the peptides of the invention may also be used to treat hypotension.

[1118] In another general aspect, the peptides of the present invention can be used to inhibit the secretion of growth hormone releasing peptide. Therefore, the peptides of the present invention can use this mechanism to treat or prevent disorders associated with growth hormone releasing peptide, such as Prader-Willi syndrome, various types of diabetes and its complications, obesity, hyperphagia, dyslipidemia or other disorders associated with overnutrition.

[1119] The peptides of the present invention can also be used to strengthen, induce, enhance or restore the responsiveness of pancreatic islets or cells to glucose. These effects can be used to treat or prevent conditions associated with metabolic disorders, such as the above-mentioned conditions and the conditions described in U.S. Patent Application No. US2004 / 0228846. Determining the determination of this activity is known in the art. For example, in the published U.S. Patent Application No. US2004 / 0228846 (which is incorporated herein by reference in its entirety), islet separation and culture and determination of fetal pancreatic islet maturation are described. In the embodiment of patent application US2004 / 0228846, enteric hormone peptides including secretin, glucagon-like peptide-1 (GLP-1) and bombesin were purchased from Sigma. Type XI collagenase was obtained from Sigma. RPMI 1640 culture medium and fetal bovine serum were obtained from Gibco. Radioimmunoassay kits ([125I]-RIA kits) containing anti-insulin antibodies were purchased from Linco, St Louis.

[1120] The peptides of the present invention can be used to prevent and treat kidney diseases, including hypertension and diabetic nephropathy, and kidney diseases associated with insulin resistance and metabolic syndrome. The peptides of the present invention achieve these purposes by improving or preventing the deterioration of hypertension, endothelial function, renal function and glomerulosclerosis. In one embodiment, the present invention provides a method for preventing or treating kidney diseases including hypertension and diabetic nephropathy or kidney diseases associated with insulin resistance, comprising administering a compound of the present invention. The peptides of the present invention are also used to improve endothelial function in patients with reduced vasodilatory capacity or with glomerulosclerosis or any other reduced glomerular blood flow. This improvement in endothelial function can both reduce hypertension and improve the function of glomerular capillaries. In other embodiments, the molecules of the present invention can be used to prevent the progression of kidney disease to ESRD, prevent, slow progression, treat or improve proteinuria and / or glomerulosclerosis.

[1121] The peptides of the present invention can be used to reduce the risk of suffering from arrhythmias, prevent or treat arrhythmias. The peptides of the present invention can provide antiarrhythmic effects in patients suffering from cardiac ischemia, cardiac ischemia-reperfusion and congestive heart failure. For example, it has been found that the incretin GLP-1 can reduce cardiac damage and enhance the recovery of patients with these disorders. Incretins (including GLP-1) are glucose-dependent insulinotropic hormones. GLP-1 and exenatide effectively enhance the uptake of peripheral glucose without causing dangerous hypoglycemia. They also strongly inhibit the secretion of glucagon, regardless of its insulinotropic effect, thereby significantly reducing plasma free fatty acid (FFA) levels more effectively than what insulin can achieve. High FFA levels are considered to be the main toxic mechanism during myocardial ischemia. In another embodiment, the peptides of the present invention can be used to prevent and treat arrhythmias, which reliably reduce the damage associated with reperfusion and ischemia, and enhance the recovery of patients. In another embodiment, treatment after acute stroke or hemorrhage, preferably administered intravenously, provides a means to optimize insulin secretion, increase cerebral assimilation, enhance insulin effectiveness by inhibiting glucagon, and maintain normoglycemia or mild hypoglycemia without the risk of severe hypoglycemia or other adverse side effects. In one embodiment, such a peptide of the invention comprises a GIP, GLP1 or exenatide portion.

[1122] In another embodiment, the peptides of the present invention can reduce insulin resistance or increase insulin sensitivity and can be used to treat polycystic ovary syndrome (PCOS). Administration of the peptides of the present invention can reduce or prevent insulin resistance in PCOS patients. In another embodiment, the peptides of the present invention can prevent the occurrence of type 2 diabetes in PCOS patients. In addition, the peptides of the present invention can also restore normal menstruation, ovulation or fertility in PCOS patients. In one embodiment, the peptides of the present invention comprising GIP also comprise a GLP1 or exenatide portion for binding to and activating the GLP1 receptor.

[1123] By selecting the peptides of the present invention, a wide range of biological activities can be exhibited, some of which are related to their anti-secretory and anti-motility properties. The peptides of the present invention can inhibit gastrointestinal secretions by directly interacting 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 secretion and can be used to treat or prevent diseases and disorders including gastritis, pancreatitis, Barrett's esophagus and gastroesophageal reflux disease, and conditions related thereto include heartburn, heartburn with reflux of gastric / intestinal contents into the mouth or lungs, dysphagia, cough, intermittent wheezing and vocal cord inflammation (conditions associated with GERD), esophageal erosions, esophageal ulcers, esophageal stenosis, Barrett's metaplasia (normal esophageal epithelium is replaced by abnormal epithelium), Barrett's esophageal adenocarcinoma, and pulmonary aspiration. In another embodiment, the GIP peptides of the present invention comprising amylin and / or sCT moieties can be used to treat or prevent these diseases and conditions, such as Barrett's esophagus, gastroesophageal reflux disease (GERD) and related conditions disclosed herein. These peptides of the invention have particularly effective anti-secretory properties, such as gastric acid inhibition, bile acid inhibition, and pancreatic enzyme inhibition. In addition, these peptides of the invention also have a gastroprotective effect, making them particularly useful in treating or preventing the intestinal diseases and conditions described herein, as well as Barrett's esophagus and / or GERD and related or associated conditions.

[1124] In another general aspect, the peptides of the invention can be used to reduce bone resorption, reduce plasma calcium and / or induce analgesia, particularly for the treatment of bone disorders such as osteopenia and osteoporosis. In other embodiments, the peptides of the invention can be used to treat pain and painful neuropathy. In one embodiment, these peptides of the invention comprise exenatide, GLP1, amylin and / or sCT moieties. For example, the GIP-sCT or GIP-amylin / sCT peptides of the invention can have the selectable properties of salmon calcitonin or amylin / sCT / amylin chimeras, such as reducing bone loss and bone resorption or reducing cartilage turnover (chondroprotection), as well as the properties of GIP, such as reducing plasma glucose (with the anti-catabolic aspects described herein) and / or inhibiting bone resorption and maintaining or increasing bone density. The peptides of the invention with these selectable properties can enhance the treatment of osteoporosis or high cartilage turnover conditions, particularly in subjects who can also benefit from glycemic control, such as subjects with diabetes or receiving intensive care.

[1125] Pharmaceutical composition

[1126] In a specific embodiment, peptide of the present invention or the pharmaceutical composition comprising peptide of the present invention is suspended in a sustained-release matrix. The sustained-release matrix used in this article refers to a matrix made of a degradable material (usually a polymer) by enzymolysis or acid-base hydrolysis or dissolution. Once inserted into the body, the matrix can be subject to the effect of enzymes and body fluids. The sustained-release matrix preferably selects biocompatible materials, such as liposomes, polylactide (polylactic acid), polyglycolide (glycolic acid polymer), polylactide-glycolide copolymer (lactic acid and glycolic acid copolymer), polyanhydrides, poly (neighboring) esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyethylene propylene, polyvinyl pyrrolidone and silicone. An embodiment of biodegradable matrix is ​​a matrix of a kind of in polylactide, polyglycolide or polylactide-glycolide copolymer (lactic acid and glycolic acid copolymer).

[1127] In certain embodiments, the present invention includes pharmaceutical compositions comprising one or more peptides of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. Pharmaceutically acceptable carriers, diluents or excipients refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. Various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. can be included to ensure that the action of microorganisms is prevented. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc.

[1128] In certain embodiments, the composition is administered orally, parenterally, intracisternal, intravaginal, intraperitoneal, rectal, topically (e.g., by powders, ointments, drops, suppositories, or transdermal patches), by inhalation (e.g., nasal sprays), ocularly (e.g., intraocularly), or buccally. The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intraarticular injections and infusions. Thus, in certain embodiments, the composition is formulated for delivery by any of these routes of administration.

[1129] In certain embodiments, the pharmaceutical composition for parenteral injection includes pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, for reconstitution into sterile injection solutions or dispersions before use. Examples of applicable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and their appropriate mixtures, beta-cyclodextrin, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions can also include adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. By including agents such as aluminum stearate and gelatin that delay absorption, extended absorption of injectable drug forms can be achieved.

[1130] Injectable reservoir forms include reservoir forms made by forming the peptides of the present invention into microcapsule matrices of one or more biodegradable polymers such as polylactide-polyglycolide, poly(o-)esters, poly(anhydrides) and (poly)ethylene glycol (such as PEG). Depending on the ratio of peptide to polymer and the properties of the specific polymer used, the release rate of the peptide can be controlled. Reservoir injectable formulations are also prepared by embedding the peptide in liposomes or microemulsions compatible with body tissues.

[1131] For example, the injectable formulations can be sterilized by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use.

[1132] Topical administration includes administration to the skin or mucous membranes, including the lungs and ocular surfaces. Compositions for topical pulmonary administration, including compositions for inhalation and intranasal administration, may involve solutions and suspensions in aqueous and non-aqueous formulations, and may be prepared as dry powders, which may be pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredient may be in a subdivided form that may be mixed with a larger size of a pharmaceutically acceptable inert carrier containing particles, the size of which may be, for example, up to 100 microns in diameter. Suitable inert carriers include sugars such as lactose.

[1133] Alternatively, the composition may be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant. The liquefied propellant medium and indeed the entire composition may be such that the active ingredient is not dissolved therein to any significant extent. The pressurized composition may also contain a surfactant, such as a liquid or solid nonionic surfactant, or may be a solid anionic surfactant. Preferably, a solid anionic surfactant is used in the form of a sodium salt.

[1134] Another topical administration form is for the eye. The peptides of the present invention can be delivered in a pharmaceutically acceptable ophthalmic vehicle so that the peptides maintain a sufficiently long contact time with the ocular surface to allow the peptides to penetrate the cornea and intraocular regions, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humour, cornea, iris / ciliary body, lens, choroid / retina and sclera. Pharmaceutically acceptable ophthalmic vehicles can be, for example, ointments, vegetable oils or encapsulating materials. Alternatively, the peptides of the present invention can be injected directly into the vitreous body and aqueous humor.

[1135] Compositions for rectal or vaginal administration include suppositories, which can be prepared by mixing the peptides of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[1136] The peptides of the present invention can also be administered in liposomes or other lipid-based carriers. As is known in the art, liposomes are usually derived from phospholipids or other lipid substances. Liposomes are formed by monolayer or multilayer hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. In addition to comprising the peptides of the present invention, the compositions of the present invention in liposome form can also include stabilizers, preservatives, excipients, etc. In certain embodiments, lipids include phospholipids, which include phosphatidylcholine (lecithin) and serine, whether natural or synthetic. Methods for forming liposomes are known in the art.

[1137] The peptides of the present invention can also be used as described by Camurus These formulations are based on a special combination of endogenous polar lipids that spontaneously form liquid crystal nanostructures in aqueous environments on tissue surfaces or in vivo. Injectable depots are designed to deliver therapeutic effects over extended periods of time (from days to months) via a single injection and have the potential to reduce the burden of daily medication while increasing treatment compliance.

[1138] The injection reservoir contains a lipid-based liquid in which the peptide of the present invention is dissolved, and can be easily injected subcutaneously using a conventional syringe with a fine needle. Upon contact with the fluid in the tissue, the lipid solution is transformed into a liquid crystal gel that effectively encapsulates the peptide. As the liquid crystal matrix gradually degrades in the tissue, the drug compound is slowly released, and the release can be controlled from a few days to a few weeks or months depending on the composition.

[1139] Pharmaceutical compositions for use in the present invention suitable for parenteral administration may comprise sterile aqueous solutions and / or suspensions of the peptide inhibitors isotonic with the blood of the recipient, typically using sodium chloride, glycerol, glucose, mannitol, sorbitol, or the like.

[1140] In some embodiments, the present invention provides a pharmaceutical composition for oral delivery. Compositions and peptides of the present invention can be prepared for oral administration according to any method, technique and / or delivery vehicle described herein. In addition, it will be appreciated by those skilled in the art that the peptides of the present invention can be modified or incorporated into systems or delivery vehicles that are not disclosed herein but are well known in the art and are compatible with oral delivery of peptides.

[1141] In certain embodiments, the formulation for oral administration may include adjuvants (e.g., resorcinol and / or nonionic surfactants such as polyoxyethylene oleyl alcohol ether and n-hexadecyl polyoxyethylene ether) to artificially increase the permeability of the intestinal wall, and / or enzyme inhibitors (e.g., trypsin inhibitors, diisopropyl fluorophosphate (DFF) or trasylol) to inhibit enzymatic degradation. In certain embodiments, the solid dosage form of the peptide of the present invention for oral administration may be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides. These dosage forms may also contain other types of additives, for example, chemically inactive diluents, lubricants such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid, α-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffers, pH adjusters, sweeteners, flavoring agents or fragrances.

[1142] In certain embodiments, oral dosage forms or unit doses compatible with the peptides of the present invention may include a mixture of the peptides with non-drug ingredients or excipients, and other non-reusable materials that may be considered as ingredients or packaging. Oral compositions may include at least one liquid,...

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid sequence of formula (I): X 1 -X 2 -X 3 -G-T-F-T-S-D-Y-S-I-X 4 -L-D-K-I-A-Q-X 5 -A-F-V-Q-W-L-X 6 -A-G-X 7 -X 8 -S-X 9 - X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 - X 31 -X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -X 39 -X 40 -X 41 -X 42 -X 43 -X 44 -X 45 -X 46 -X 47 -X 48 -X 49 -X 50 -X 51 -(NR 1 R 2 ) (I) in X 1 is Tyr, (d)Tyr, His or ImPrA; X 2 is Ala, (d)Ala, Aib, Ser or Gly; X 3 is Glu or Gln; X 4 is Ala or Aib; X 5 Is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc- AEEAc-γ-Glu-C=O(CH2) n R 3 ); X 6 It is Ile or Leu; X 7 It is Gly, Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ); X 8 It is Pro or Gly; X 9 Missing or Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ); X 10 Missing either Gly or Cys; X 11 The deletion is either Ala, Cys or Ser; X 12 The deletion is either Pro or Asn; X 13 Missing or Pro, Thr or Leu; X 14 Missing or Pro, Ala or Ser; X 15 Missing or Ser, Thr or Cys; X 16 It is Cys, Asn, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ); X 17 is Asn, Val, Met, Ala, or Thr; X 18 is Thr, Leu, Asn, or Ala; X 19 is Ala, Gly, Thr or Gln; X 20 is Ala, Thr, Arg or Cys; X 21 is Cys, Leu, Thr or Ala; X 22 is Val, Ser, Cys, Ala, or Thr; X 23 is Leu, Gln, Val, Glu or Thr; X 24 is Gly, Glu, Leu, Asp, Phe, Gln or Arg; X 25 is Arg, Leu or Gly; X 26 is Leu, Val, His, Arg or Ala; X 27 is Ser, Arg, Leu, His, Ala or Glu; X 28 is Gln, Leu, Ser, Glu or Phe; X 29 is Glu, Gln, Ser, Phe or Leu; X 30 is Leu, Thr, Glu, Asn or Arg; X 31 Deletion or His, Leu, Asn or Arg; X 32 Deletion or Arg, His, Phe or Ser; X 33 Deletion or Leu, Arg, Gly or Ser; X 34 Gln, Leu, Pro, Ser or Asn is missing or present; X 35 The deletion is either Thr, Gln, Ile or Asn; X 36 is Tyr, Thr, Leu, Asn or Phe; X 37 is Pro, Tyr, Phe or Gly; X 38 is Arg, Lys, Pro or Gly; X 39 is Thr, Arg, Pro or Ile; X 40 is Asn, Thr, Asp, Ile, or Leu; X 41 is Thr, Asn, Val, Leu, or Pro; X 42 is Gly, Thr or Pro; X 43 is Ser, Gly, Ala, Pro, Thr, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ); X 44 is Asn, Ser, His, or Thr; X 45 is Thr, Asn, Ala, or Val; X 46 is Tyr, Thr, Pro, Val, or Gly; X 47 The deletion is either Tyr, Gly or Ser; X 48 Deletion is either Ser or His; X 49 The deletion is either His or Thr; X 50 Absent or Thr or Pro; and X 51 Missing or Pro; in R 1 and R 2 Each independently is H or C 1-5 alkyl, R 3 is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3 or -NH2, n is an integer from 12 to 20.

2. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein: X 1 It's Tyr; X 2 It's Ala; X 3 It is Glu; X 4 It's Ala; X 5 It is Gln; X 6 It is Ile; X 7 It is Gln; X 8 It is Pro; X 9 missing; X 10 missing; X 11 missing; X 12 missing or Asn; X 13 missing; X 14 missing; X 15 Missing; X 16 It is Cys;X 17 is Asn;X 18 It is Thr;X 19 It's Ala;X 20 It is Thr;X 21 Cys;X 22 It's Val;X 23 It's Leu;X 24 It is Gly;X 25 is Arg;X 26 It's Leu;X 27 It is Ser;X 28 It is Gln;X 29 It is Glu;X 30 It's Leu;X 31 Missing; X 32 Missing; X 33 Missing; X 34 Missing; X 35 Missing; X 36 It's Tyr;X 37 It's Pro;X 38 is Arg;X 39 It is Thr;X 40 is Asn;X 41 It is Thr;X 42 It is Gly;X 43 It is Ser; X 44 It is Asn; X 45 It is Thr; X 46 It's Tyr; X 47 missing; X 48 missing; X 49 missing; X 50 missing; and X 51 Missing.

3. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein X 1 is (d) Tyr, His or ImPrA, X 2 (d) Ala, Aib, Ser or Gly; X 3 It is Gln; X 4 It is Aib; X 5 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc- γ-Glu-C=O(CH2) n R 3 ); X 6 It's Leu; X 7 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc- γ-Glu-C=O(CH2) n R 3 ); X 8 It's Gly; X 9 It is Ser, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc- AEEAc-γ-Glu-C=O(CH2) n R 3 ); X 10 is Gly or Cys; X 11 is Ala, Cys or Ser; X 12 is Pro or Asn; X 13 is Pro, Thr or Leu; X 14 is Pro, Ala or Ser; X 15 is Ser, Thr or Cys; X 16 It is Asn, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc- AEEAc-γ-Glu-C=O(CH2) n R 3 ); X 17 is Val, Met, Ala, or Thr; X 18 is Leu, Asn or Ala; X 19 is Gly, Thr or Gln; X 20 is Arg or Cys; X 21 is Leu, Thr, or Ala; X 22 is Ser, Cys, Ala or Thr; X 23 is Gln, Val, Glu or Thr; X 24 is Glu, Leu, Asp, Phe, Gln or Arg; X 25 is Leu or Gly; X 26 is His, Arg or Ala; X 27 is Arg, Leu, His, Ala or Glu; X 28 is Leu, Ser, Glu or Phe; X 29 is Gln, Ser, Phe or Leu; X 30 is Thr, Glu, Asn or Arg; X 31 is His, Leu, Asn or Arg; X 32 is Arg, His, Phe or Ser; X 33 is Leu, Arg, Gly or Ser; X 34 is Gln, Leu, Pro, Ser or Asn; X 35 is Thr, Gln, Ile or Asn; X 36 is Thr, Leu, Asn or Phe; X 37 is Tyr, Phe or Gly; X 38 is Pro, Lys, Pro or Gly; X 39 is Arg, Pro or Ile; X 40 is Thr, Asp, Ile or Leu; X 41 is Asn, Val, Leu or Pro; X 42 It is Thr or Pro; X 43 It is Gly, Ala, Pro, Thr, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3 ); X 44 is Ser, His, or Thr; X 45 is Asn, Ala or Val; X 46 is Thr, Pro, Val or Gly; X 47 is Tyr, Gly or Ser; X 48 is Ser or His; X 49 is His or Thr; X 50 is Thr or Pro; and X 51 It's Pro.

4. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein X 5 is Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CO2H, Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CH3, Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl) or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl).

5. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein X 7 is Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CO2H, Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CH3, Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl) or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl).

6. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein X 9 is Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CO2H, Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CH3, Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl) or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl).

7. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein X 16 is Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CO2H, Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CH3, Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl) or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl).

8. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein X 43 is Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CO2H, Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CH3, Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl) or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl).

9. The peptide or pharmaceutically acceptable salt thereof according to claim 1, which has the following sequence: Where K* is Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 Or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ).

10. The peptide or pharmaceutically acceptable salt thereof according to claim 1, which has the following sequence:

11. A pharmaceutical composition comprising the peptide according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient or diluent.

12. A method for treating obesity, metabolic disorders or liver disorders in a subject in need thereof, the method comprising providing an effective amount of the peptide or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 10 to the subject.

13. The method of claim 12, wherein the peptide or a pharmaceutically acceptable salt thereof is provided to the subject by an oral, parenteral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, aerosolization, sublingual, buccal, parenteral, rectal, intraocular, inhalation, topical, vaginal or local administration route.

14. The method of claim 12, for treating obesity.

15. The method of claim 12, wherein the metabolic disorder is diabetes.

16. The method of claim 12, wherein the effective amount of the peptide or a pharmaceutically acceptable salt thereof or the pharmaceutical composition is from about 0.0001 to about 300 mg / kg body weight per day.

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