Compositions Containing Multi-Agonist Peptides and Methods of Manufacture and Use

Abstract

The present invention generally relates to novel multi - agonist peptides useful as agents for the treatment and prevention of metabolic diseases and disorders, such as those that can be alleviated by control of weight loss, plasma glucose and lipid levels, insulin levels, and / or insulin secretion, positive inotropic action, reduction of catabolism, deceleration of gastric emptying, and prevention of neurodegeneration. Such conditions and disorders include, but are not limited to, food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive inotropic action, reduction of catabolism, deceleration of gastric emptying, obesity, diabetes and diabetes - related conditions, control of 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 disorders of obesity, such as Prader - Willi syndrome, critical care, insulin resistance and its disorders, such as polycystic ovary syndrome, obesity, all types of diabetes including type 1, type 2 and gestational diabetes, and CNS disorders, such as prevention of neurodegeneration, depression, alcoholism, Alzheimer's disease and Parkinson's disease, and non - alcoholic steatohepatitis (NASH).

Description

Technical Field

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

[0002] Field of the Invention The present invention generally relates to novel multi - agonist peptides useful as agents for the treatment and prevention of metabolic diseases and disorders, such as those that can be alleviated by weight loss, control of plasma glucose and lipid levels, insulin levels, and / or insulin secretion, positive inotropic action, reduction of catabolism, deceleration of gastric emptying, and prevention of neurodegeneration. Such conditions and disorders include, but are not limited to, food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or control of insulin secretion, positive inotropic action, reduction of catabolism, deceleration 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 disease, eating disorders, and rare genetic disorders of obesity, such as Prader - Willi syndrome, critical care, insulin resistance and its disorders, such as polycystic ovary syndrome, obesity, all types of diabetes, including type 1, type 2 and gestational diabetes, and CNS disorders, such as prevention of neurodegeneration, depression, alcoholism, Alzheimer's disease and Parkinson's disease, and non - alcoholic steatohepatitis (NASH).

Background Art

[0003] Incretin peptides are hormones, and peptidomimetics are glucose regulators that cause an increase in the amount of insulin released when glucose levels are normal or, particularly, when they are elevated. These incretin peptides have other actions beyond the first incretin effect defined by insulin secretion. For example, incretin peptides may also have actions that reduce glucagon production, increase satiety, slow gastric emptying, regulate white and brown adipose tissue, and cause weight loss. In addition, incretin peptides may have an action to improve insulin sensitivity, and they can increase the regeneration of pancreatic islet cells, i.e., the formation of new pancreatic islets.

[0004] The concept of the incretin effect originated from the observation that the insulin response to oral glucose exceeded the insulin response measured after intravenous administration of an equal amount of glucose. It was concluded that a factor derived from the gastrointestinal tract, i.e., incretin, affected postprandial insulin release. When nutrients enter the stomach and proximal gastrointestinal tract, the release of incretin hormones is induced, which then stimulates insulin secretion. This insulinotropic property, i.e., the ability to stimulate insulin secretion, can be quantified by comparing the insulin or C-peptide responses to oral versus intravenous glucose loading. By this method, it has been shown that the incretin effect is involved in approximately 50% - 70% of the insulin response to oral glucose in healthy individuals.

[0005] Many postprandial hormones have incretin-like activity, but the predominant incretin peptides include glucose-dependent insulinotropic polypeptide, which is also known as gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and exendin peptides (non-endogenous incretin mimetics). Both GIP and GLP-1 belong to the glucagon peptide superfamily and thus have amino acid sequence homology. GIP and GLP-1 are secreted by specialized cells in the gastrointestinal tract and have receptors located on other tissues in addition to pancreatic islet cells. Similar to incretin, both are secreted from the intestine in response to nutrient ingestion, thereby enhancing insulin secretion. The insulinotropic actions of GIP and GLP-1 are dependent on an increase in ambient glucose. Both are rapidly inactivated by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV).

[0006] Native human GIP is a single 42-amino acid peptide synthesized and secreted by specialized enteroendocrine K cells. These cells are concentrated mainly in the duodenum and proximal jejunum, but they can also be found throughout the intestine. The major stimulatory factor for GIP secretion is the ingestion of a carbohydrate- and lipid-rich meal. After ingestion, circulating plasma GIP levels increase 10- to 20-fold. The half-life of intact GIP is estimated to be approximately 7.3 minutes in healthy subjects and 5.2 minutes in diabetic subjects.

[0007] The physiological actions of GIP are being elucidated using GIP receptor antagonists, GIP peptide antagonists, and GIP receptor knockout mice in addition to GIP infusion protocols. Blocking GIP binding to its receptor results in attenuation of glucose-dependent insulin secretion after oral glucose loading in rats and mice. Similarly, administration of GIP antagonists or GIP antiserum significantly reduces postprandial insulin release in rats. GIP receptor knockout mice exhibit normal fasting glucose levels but mild glucose intolerance after oral glucose loading. Interestingly, GIP receptor knockout mice also show resistance to diet-induced obesity after several months of high-fat feeding. In addition, in leptin-deficient ob / ob mice, the GIP receptor knockout genotype appears to reduce the degree of developing obesity.

[0008] GIP also has many non-incretin actions. Activation of the GIP receptor by GIP has an anti-emetic effect. Unlike other insulin secretagogues, GIP stimulates beta-cell proliferation and cell survival in INS-1 pancreatic cell line studies. Furthermore, animal experiments have suggested a role for GIP in lipid metabolism by stimulating lipoprotein lipase activity, inducing fatty acid uptake into adipose tissue, and stimulating fatty acid synthesis. GIP also appears to stimulate glucagon secretion from isolated perfused rat pancreas, but no significant effect on glucagon secretion has been demonstrated in human studies. Additionally, GIP appears to act by promoting gastric emptying rather than inhibiting gastrointestinal motility, unlike GLP-1.

[0009] Despite its potent glucose regulatory action via glucose-dependent stimulation of insulin secretion, the insulin secretagogue action of GIP in diabetic subjects is significantly reduced compared to normal individuals (16-18). As a result, the clinical use of GIP has not progressed much. Further, there is still a need to develop additional diabetes treatment modalities, as well as treatments for metabolic diseases, conditions, and disorders. Accordingly, the present invention encompasses the peptides of the invention and methods for their use for treating or preventing metabolic diseases, conditions, and disorders.

[0010] GLP-1 is a potent insulin secretagogue secreted from the intestinal mucosa in response to food intake. The significant incretin effect of GLP-1 is emphasized by the fact that GLP-1 receptor knockout mice are glucose intolerant. The incretin response to intravenous GLP-1 is preserved in diabetic subjects, but the incretin in response to oral glucose in these patients is impaired. Administration of GLP-1 by infusion or subcutaneous injection controls fasting glucose levels in diabetic patients and maintains the glucose threshold for insulin secretion. GLP-1 has shown great potential as a therapeutic agent capable of increasing insulin secretion in a physiological manner while avoiding hypoglycemia associated with sulfonylurea drugs. Other important actions of GLP-1 on glucose homeostasis are the suppression of glucagon secretion and the inhibition of gastric motility. The inhibitory effect of GLP-1 on pancreatic alpha cell secretion of glucagon causes a decrease in hepatic glucose production via a reduction in gluconeogenesis and glycogenolysis. This anti-glucagon effect of GLP-1 is preserved in diabetic patients. The so-called ileal brake effect of GLP-1 inhibits gastric motility and gastric juice secretion, which is affected via receptors of the vagus nerve efferent pathway or by a direct action on intestinal smooth muscle. The reduction of gastric acid secretion by GLP-1 contributes to the lag phase in nutrient availability, thus eliminating the need for a rapid insulin response. In summary, the gastrointestinal actions of GLP-1 significantly contribute to the delay in glucose and fatty acid absorption and modulate insulin secretion and glucose homeostasis. GLP-1 has also been shown to induce beta cell-specific genes such as the GLUT-1 transporter, insulin (via the interaction of PDX-1 and the insulin gene promoter), and hexokinase-1. Therefore, GLP-1 may reverse the glucose intolerance associated with normal aging, as demonstrated in rodent experiments. In addition, GLP-1 may contribute to beta cell regeneration and increase the beta cell mass in addition to restoring beta cell function during a state of beta cell dysfunction.The central actions of GLP-1 include an increase in satiety combined with a decrease in food intake mediated by the action of GLP-1 receptors in the hypothalamus. These appetite-suppressing effects were not present in GLP-1R knockout mice. In addition, GLP-1 receptor signaling has been shown to be involved in the regulation of energy consumption and thermogenesis in brown adipose tissue. (Beiroa D et al., Diabetes 63:3346 - 3358, 2014).

[0011] Glucagon is a 29 - amino acid peptide hormone that is produced by pancreatic alpha cells and released into the bloodstream when circulating glucose is low. The important physiological role of glucagon is to stimulate glucose output in the liver, which is a process that provides a major counter - regulatory mechanism to insulin in maintaining glucose homeostasis in vivo.

[0012] However, since glucagon receptors are also expressed in extra - hepatic tissues such as the kidney, heart, adipocytes, lymphocytes, brain, retina, adrenal glands, and gastrointestinal tract, a broader physiological role beyond glucose homeostasis is suggested. Therefore, recent studies have reported that glucagon has therapeutically positive effects on energy management, including stimulation of energy consumption and thermogenesis associated with reduced food intake and weight loss. In summary, stimulation of the glucagon receptor may be useful in the treatment of obesity and other disorders.

[0013] Another family of peptide hormones involved in 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 islets of human type 2 diabetes (Cooper et al., Proc. Natl. Acad. Sci., USA, 84:8628-8632 (1987)). The amylin molecule has two post-translational modifications: the C-terminus is amidated, and the cysteines at positions 2 and 7 are cross-linked to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of a proteolytic cleavage signal for Lys-Arg dibasic amino acids before the N-terminal codon for Lys, and a typical sequence for PAM, for amidation by protein amidating enzyme, before the Lys-Arg proteolytic signal at the C-terminal position (Cooper et al., Biochem. Biophys. Acta, 1014:247-258 (1989)).

[0014] Amylin is thought to regulate gastric emptying, suppress glucagon secretion and food intake, and thus regulate the rate of appearance of glucose in the circulation. Amylin is thought to complement the action of insulin and thereby regulate the rate of disappearance of glucose from the circulation and its uptake by peripheral tissues. These actions are supported by experimental findings in rodents and humans indicating that amylin complements the action of insulin by at least three independent mechanisms that all affect the rate of appearance of glucose. First, amylin suppresses postprandial glucagon secretion. Patients with type 1 diabetes have no circulating amylin, and patients with type 2 diabetes have reduced postprandial amylin concentrations, compared to healthy adults. Furthermore, infusion of an amylin-specific monoclonal antibody that binds circulating amylin resulted in glucagon concentrations that were significantly elevated compared to controls. Both of these results represent the physiological role of endogenous amylin in the regulation of postprandial glucagon secretion. Second, amylin slows gastrointestinal motility and gastric emptying. Finally, intracerebroventricular injection of rat amylin has been shown to reduce food intake in rats and alter neurotransmitter metabolism in the hypothalamus. In one particular study, food intake was significantly reduced for up to 8 hours after intracerebroventricular injection of rat amylin and rat CGRP. In human trials, pramlintide, an amylin analog, has been shown to either reduce or increase body weight. Amylin may be beneficial in treating metabolic conditions such as diabetes and obesity. Amylin may also be used to affect body composition, such as treating pain, bone disorders, alcohol addiction, gastritis, modulating lipids, particularly triglycerides, or preferentially losing fat while leaving lean tissue intact.

[0015] Calcitonin was named after its secretion in response to induced hypercalcemia and its action in rapidly lowering hypocalcemia. Since calcitonin is produced and then secreted by neuroendocrine cells in the thyroid gland, these neuroendocrine cells are called C cells. The most well-studied action of calcitonin is its action on osteoclasts. The in vitro actions of calcitonin include the rapid loss of the ruffled border and a decrease in lysosomal enzyme release. Ultimately, the inhibition of osteoclast function by calcitonin causes a decrease in bone resorption. However, neither the chronic reduction of serum calcitonin in cases of thyroidectomy nor the increase in serum calcitonin found in medullary thyroid cancer seems to be related to changes in serum calcium or bone mass. Therefore, the main function of calcitonin is most likely to fight acute hypercalcemia in emergencies and / or to protect the skeleton during periods of "calcium stress" such as growth, pregnancy, and lactation. Calcitonin has an effect on plasma calcium levels, inhibits osteoclast function, and is widely used in the treatment of osteoporosis. Therapeutically, salmon calcitonin seems to increase bone density and decrease the fracture rate while minimizing adverse effects. Calcitonin has also been successfully used as a therapeutic agent for Paget's disease of bone, a chronic bone disorder that can cause bone enlargement or deformation in one or more regions of the skeleton, for over 25 years. Calcitonin is also widely used for its analgesic effect on the bone pain experienced during osteoporosis, but the mechanism of this action is not clearly understood. Salmon calcitonin has actions beyond those related to bone metabolism. In human studies, salmon calcitonin inhibits postprandial gastric emptying and gastrin release while inducing dose-dependent relaxation of the gallbladder in both the postprandial and fasting states. In mice and monkeys, salmon calcitonin acts to cause anorexia and weight loss after a single dose. In long-term studies, oral preparations of salmon calcitonin also reduce food intake and body weight in rat models of obesity and diabetes.

[0016] Metabolic diseases and disorders present in many forms such as obesity, diabetes, dyslipidemia, insulin resistance, fatty liver, steatohepatitis, apoptosis of cells, etc. 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 most well-known strong risk factor for type 2 diabetes and a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for diabetes, hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, cancers of the breast, prostate, and colon, and an increased incidence of complications of general anesthesia (see, e.g., Kopelman, Nature 404:635-43 (2000)). Obesity shortens lifespan and poses a serious risk of disorders such as the above-mentioned co-morbidities, in addition to infections, varicose veins, seborrheic keratosis, 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 group of conditions called the insulin resistance syndrome, or "syndrome X". Estimated recent medical costs for obesity and related disorders are two trillion dollars worldwide. The etiology of obesity is thought to be multifactorial, but the underlying problem in obese subjects is that the availability of nutrients and energy consumption do not balance until adipose tissue becomes excessive. Obesity is currently a chronic, essentially refractory metabolic disorder that cannot be adequately treated. Therapeutic agents useful in weight reduction in obese persons may have a significant beneficial effect on their health status.

[0017] Diabetes is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria resulting from insufficient production or utilization of insulin. Diabetes significantly affects the quality of life of a large portion of the population in developed countries. Insufficient production of insulin is characterized as type 1 diabetes, and insufficient utilization of insulin is type 2 diabetes. However, it is widely recognized that there are many distinct diabetes-related disorders that develop well before a patient is diagnosed with overt diabetes. Also, the effects of suboptimal control of glucose metabolism in diabetes cause a wide range of associated lipid disorders and cardiovascular disorders.

[0018] Dyslipidemia, or abnormal levels of lipoproteins in the plasma, frequently occurs among diabetics. Dyslipidemia is typically characterized by elevated plasma triglycerides, low HDL (high-density lipoprotein) cholesterol, levels of LDL (low-density lipoprotein) cholesterol elevated from normal levels, and increased levels of low-density LDL (low-density lipoprotein) particles in the blood. Dyslipidemia is one of the major contributing factors to the increased incidence of coronary events and death among diabetic subjects. Epidemiological studies have confirmed this by showing that the incidence of coronary death is several times higher in diabetic subjects compared to non-diabetic subjects. Among diabetic subjects, numerous lipoprotein abnormalities have been described.

[0019] Insulin resistance is a decrease in the ability of insulin to exert its biological action 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 occurs. If the defective insulin action cannot be compensated for, an increase in plasma glucose concentration is inevitable, resulting in a clinical state of diabetes. Insulin resistance and relative hyperinsulinemia are recognized to have a contributory role in obesity, hypertension, atherosclerosis, and type 2 diabetes. The association between insulin resistance and obesity, hypertension, and angina has been explained as a syndrome with insulin resistance as a common etiological association, i.e., syndrome X.

[0020] In order to address these abnormalities in various patients, attempts to treat multiple abnormalities associated with diabetes have been promoted by the administration of numerous antidiabetic agents. Examples of antidiabetic agents are proteins such as insulin and insulin analogs, GLP-1 analogs, and small molecules such as insulin sensitizers, insulin secretagogues, sodium glucose cotransporter-2 (SGLT-2) inhibitors, DPPIV inhibitors, and compounds that regulate appetite.

[0021] Non-alcoholic fatty liver disease (NAFLD) is an umbrella term that encompasses everything from mere accumulation of fat in the liver to more progressive steatosis associated with related hepatitis, fibrosis, cirrhosis, and in some cases hepatocellular carcinoma. NAFLD consists of non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NAFL is characterized by fatty liver degeneration involving more than 5% of the parenchyma and no evidence of hepatocyte injury [2]. In contrast, NASH is a necroinflammatory process and is defined from a histological point of view in which liver cells are damaged against the background of steatosis. The natural progression of NAFLD has not yet been fully characterized. Studies indicate that the incidence of NAFLD is increasing in concert with the rising incidence of metabolic syndrome. Patients with type 2 diabetes show a 2- to 4-fold increased risk of fatty liver-related complications in addition to a very high risk of developing NASH. Currently, there is no approved treatment for NASH.

[0022] There is still a need to develop therapeutic agents useful in the metabolic diseases, conditions, and disorders described above. Accordingly, the present invention provides novel multiagonist peptides and methods for producing and using them. The peptides of the present invention are utilized in the metabolic diseases, conditions, and disorders described above and herein.

Prior Art Documents

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Summary of the Invention

[0025] The present invention provides, inter alia, novel multi - agonist peptides and methods for treating and preventing related disorders such as obesity, metabolic disorders, and liver disorders.

[0026] The present invention generally relates to novel peptides useful 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 action, reduction of catabolism, deceleration 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, eating disorders, and rare genetic disorders of obesity such as Prader - Willi syndrome, critical care medicine, insulin resistance and its disorders such as polycystic ovary syndrome, obesity, all types 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 includes "peptides of the present invention" comprising two or more component peptides independently selected from at least two peptides including bioactive components such as, for example, but not limited to, amylin, glucose - dependent insulinotropic polypeptide (GIP), glucagon - like peptide - 1 (GLP - 1), and calcitonin, as defined herein.

[0028] The present invention encompasses a peptide exhibiting at least one hormonal activity. The peptide of the present invention contains at least two bioactive peptides covalently linked to each other via a linker group, and at least one of the bioactive peptides exhibits at least one hormonal activity of the component peptides. The bioactive peptides are independently selected from component peptides (e.g., GIP, amylin, GLP-1), fragments of component peptides that exhibit at least one hormonal activity of the component peptides, analogs and derivatives of component peptides that exhibit at least one hormonal activity of the component peptides, and fragments of analogs and derivatives of component peptides that exhibit at least one hormonal activity of the component peptides.

[0029] In one embodiment, the peptide exhibits at least one hormonal activity, and the peptide contains at least a first peptide covalently linked to at least one additional peptide; the peptide is independently selected from the group consisting of component peptides (e.g., GIP, GLP-1, amylin); fragments of component peptides that exhibit at least one hormonal activity of the component peptides; analogs and derivatives of component peptides that exhibit at least one hormonal activity of the component peptides; and fragments of analogs and derivatives of component peptides that exhibit at least one hormonal activity of the component peptides.

[0030] The present invention encompasses the peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, and the peptide of the present invention comprises or consists of the amino acid sequence of formula (I):

[0031]

Chemical formula

[0032] wherein, X 1 is Tyr, (d)Tyr, His, or ImPrA; X 2is 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 is Ile or Leu; X 7 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 is Pro or Gly; X 9 is absent 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 is absent or Gly or Cys; X 11 is absent or Ala, Cys, or Ser; X 12 is absent or Pro or Asn; X 13 is absent or Pro, Thr, or Leu; X 14 is absent or Pro, Ala, or Ser; X 15 is absent or Ser, Thr, or Cys; X 16 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 Val, Leu, 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 is absent or is His, Leu, Asn, or Arg; X 32is absent or is Arg, His, Phe, or Ser; X 33 is absent or is Leu, Arg, Gly, or Ser; X 34 is absent or is Gln, Leu, Pro, Ser, or Asn; X 35 is absent or is 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, 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 is absent or is Tyr, Gly, or Ser; X 48 is absent or is Ser or His; X 49 is absent or is His or Thr; X 50 is absent or is Thr or Pro; X 51 is absent or is Pro; wherein R 1 and R 2 are each independently 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.

[0033] In certain embodiments, R 3 is -CO2H.

[0034] In certain embodiments, R 3 is -CO2CH3.

[0035] In certain embodiments, R 3 is -CO2NH2.

[0036] In certain embodiments, R 3 is -CO2NHCH3.

[0037] In certain embodiments, R 3 is -CO2N(CH3)2.

[0038] In certain embodiments, R 3 is -CH3.

[0039] In certain embodiments, R 3 is -NH2.

[0040] In certain embodiments, n is 12.

[0041] In certain embodiments, n is 13.

[0042] In certain embodiments, n is 14.

[0043] In certain embodiments, n is 15.

[0044] In certain embodiments, n is 16.

[0045] In certain embodiments, n is 17.

[0046] In certain embodiments, n is 18.

[0047] In certain embodiments, n is 19.

[0048] In certain embodiments, n is 20.

[0049] In other embodiments, peptide components of the peptides of the present invention can include calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin, oxyntomodulin (OXM), and exendin-4.

[0050] In certain embodiments, the peptides of the present invention include structural motifs of component peptides that confer to the peptides desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic characteristics, and structural motifs of analogs or derivatives of component peptides that confer to the peptides of the present invention desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic characteristics. In further additional embodiments, at least one of the peptides exhibits at least one hormonal activity of the component peptides.

[0051] In a further embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is GIP, a fragment of GIP exhibiting at least one hormonal activity, an analog or derivative of GIP exhibiting at least one hormonal activity, or a fragment of an analog or derivative of GIP exhibiting at least one hormonal activity, and at least one other peptide.

[0052] In a further embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is GLP-1, a fragment of GLP-1 exhibiting at least one hormonal activity, an analog or derivative of GLP-1 exhibiting at least one hormonal activity, or a fragment of an analog or derivative of GLP-1 exhibiting at least one hormonal activity, and at least one other peptide.

[0053] In a further embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is amylin, a fragment of amylin exhibiting at least one hormonal activity, an analog or derivative of amylin exhibiting at least one hormonal activity, or a fragment of an analog or derivative of amylin exhibiting at least one hormonal activity, and at least one other peptide.

[0054] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is calcitonin, a fragment of calcitonin exhibiting at least one hormonal activity, an analog or derivative of calcitonin exhibiting at least one hormonal activity, or a fragment of an analog or derivative of calcitonin exhibiting at least one hormonal activity, and at least one other peptide.

[0055] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is calcitonin gene-related peptide, a fragment of calcitonin gene-related peptide exhibiting at least one hormonal activity, an analog or derivative of calcitonin gene-related peptide exhibiting at least one hormonal activity, or a fragment of an analog or derivative of calcitonin gene-related peptide exhibiting at least one hormonal activity, and at least one other peptide.

[0056] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is intermedin, a fragment of intermedin exhibiting at least one hormonal activity, an analog or derivative of intermedin exhibiting at least one hormonal activity, or a fragment of an analog or derivative of intermedin exhibiting at least one hormonal activity, and at least one other peptide.

[0057] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is cholecystokinin, a fragment of cholecystokinin exhibiting at least one hormonal activity, an analog or derivative of cholecystokinin exhibiting at least one hormonal activity, or a fragment of an analog or derivative of cholecystokinin exhibiting at least one hormonal activity, and at least one other peptide.

[0058] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is glucagon-like peptide 2, a fragment of glucagon-like peptide 2 exhibiting at least one hormonal activity, an analog or derivative of glucagon-like peptide 2 exhibiting at least one hormonal activity, or a fragment of an analog or derivative of glucagon-like peptide 2 exhibiting at least one hormonal activity, and at least one other peptide.

[0059] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is oxyntomodulin, a fragment of oxyntomodulin exhibiting at least one hormonal activity, an analog or derivative of oxyntomodulin exhibiting at least one hormonal activity, or a fragment of an analog or derivative of oxyntomodulin exhibiting at least one hormonal activity, and at least one other peptide.

[0060] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is a natriuretic peptide, a fragment of a natriuretic peptide exhibiting at least one hormonal activity, an analog or derivative of a natriuretic peptide exhibiting at least one hormonal activity, or a fragment of an analog or derivative of a natriuretic peptide exhibiting at least one hormonal activity, and at least one other peptide.

[0061] In a further alternative embodiment, at least one component peptide exhibiting at least one hormonal activity of the component peptides is exendin-4, a fragment of exendin-4 exhibiting at least one hormonal activity, an analog or derivative of exendin-4 exhibiting at least one hormonal activity, or a fragment of an analog or derivative of exendin-4 exhibiting at least one hormonal activity, and at least one other peptide.

[0062] 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; a gastric inhibitory polypeptide (GIP) receptor ligand.

[0063] In other embodiments, the GIP portion of the peptides of the invention is combined with an EGF receptor ligand; a calcitonin receptor ligand; a CGRP receptor ligand, a gastrin / CCK receptor ligand; a keratinocyte growth factor (KGF) receptor 1 ligand; a dipeptidyl peptidase IV inhibitor; a REG protein receptor ligand; a growth hormone receptor ligand; a prolactin (PRL) receptor ligand; an insulin-like growth factor (IGF) receptor ligand; a PTH-related protein (PTHrP) receptor ligand; a hepatocyte growth factor (HGF) receptor ligand; a fibroblast growth factor 19 (FGF19) receptor ligand; a fibroblast growth factor 21 (FGF21) receptor ligand; a bone morphogenetic protein (BMP) receptor ligand, a transforming growth factor (TGF) receptor ligand; a laminin receptor ligand; a vasoactive intestinal peptide (VIP) receptor ligand; a fibroblast growth factor (FGF) receptor ligand; a nerve growth factor (NGF) receptor ligand; an islet neogenesis associated protein (INGAP) receptor ligand; an activin A receptor ligand; a vascular endothelial growth factor (VEGF) receptor ligand; an erythropoietin (EPO) receptor ligand; a pituitary adenylate cyclase activating polypeptide (PACAP) receptor ligand; a granulocyte colony stimulating factor (G-CSF) receptor ligand; a granulocyte macrophage colony stimulating factor (GM-CSF); a platelet-derived growth factor (PDGF) receptor ligand, a cannabinoid CB1 receptor antagonist, and a secretin receptor ligand.

[0064] In another embodiment, the GIP peptide component included in the peptide of the present invention comprises an N-terminal GIP or a novel GIP analog fragment in combination with a C-terminal peptide or a fragment thereof having the ability to inhibit or reduce hypoglycemic activity (e.g., antidiabetic agent, amylin) or gastric emptying. Such GIP peptides of the present invention comprise an N-terminal GIP fragment or a novel GIP analog or derivative fragment in combination with amylin, gastrin, PYY, secretin, CCK, GRP, neuromedin, urocortin, C-terminus, calcitonin or salmon calcitonin, natriuretic peptide (e.g., ANP, BNP, CNP, urodilatin), or an analog (e.g., amylin-sCT-amylin chimera), derivative or fragment thereof.

[0065] In other embodiments, the GIP peptide component included in the peptide of the present invention comprises a C-terminal GIP or a novel GIP analog fragment in combination with an N-terminal polypeptide or a fragment thereof having the ability to inhibit or reduce hypoglycemic activity (e.g., antidiabetic agent, amylin) or gastric emptying. In such embodiments, the chimeric peptide may comprise a C-terminal GIP, a novel GIP analog, or a fragment thereof in combination with an N-terminal exendin, GLP-1, amylin, CCK, gastrin, PYY, secretin, GRP, neuromedin, urocortin, calcitonin or salmon calcitonin, natriuretic peptide, or an analog, derivative or fragment thereof.

[0066] In one embodiment, the peptide of the present invention includes a C-terminal tail or terminal extension portion that is heterologous to the GIP portion. Similar to the other GIP peptides described herein, in one embodiment of the peptide of the present invention, the GIP portion may be native GIP, an active fragment thereof, or an analog or derivative thereof. In another aspect, the GIP component of the peptide of the present invention includes at least one modification, substitution, deletion or addition that provides one or more enhanced properties, such as an increase in resistance to proteolytic digestion (thus lengthening the half-life), derivatization of a fatty acyl that reduces renal clearance. In one embodiment, the tail includes a Trp-cage motif sequence. In another embodiment, examples of the GIP analog peptide portion include unnatural amino acids such as D-amino acids that inhibit or reduce the rate of proteolysis by DPP-IV.

[0067] The present invention also encompasses using the peptides of the present invention for the treatment and prevention of metabolic diseases and disorders and liver diseases and disorders, in particular, for the treatment and prevention of such 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 action, reduction of catabolism, deceleration 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, eating disorders, and rare genetic disorders of obesity, such as Prader-Willi syndrome, critical care medicine, insulin resistance and its disorders, such as polycystic ovary syndrome, obesity, all types 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). In addition, complications due to diabetes (e.g., neuropathy (e.g., treatment with the peptides of the present invention including GIP peptides containing exendin family components), neuropathic pain (e.g., treatment with the peptides of the present invention including GIP peptides containing amylin family hormone modules), retinopathy, nephropathy, insufficient pancreatic beta cell mass (e.g., based on the islet regeneration action of exendin-4 and GLP-1)) are included.

[0068] Accordingly, there is provided a method for treating or preventing such conditions, the method comprising administering a therapeutically or prophylactically effective amount of a peptide of the present invention. In certain embodiments, the peptides of the present invention include GIP or an analog or derivative thereof, such as a novel GIP analog of the present invention, etc., to a subject in need thereof, or a GIP peptide of the present invention, such as one or more additional peptides or peptide fragments, etc.

[0069] In one embodiment, the peptide of the invention can be provided as a monotherapy. In another embodiment for treating obesity, diabetes or a condition associated with increased body weight or elevated glucose levels, the peptide of the invention can be administered in an adjuvant therapy using a method of reducing food intake or a hypoglycemic agent (e.g., an anti-diabetic agent) or an agent or method that inhibits or reduces gastric emptying. Examples of such agents are presented herein. For example, in one embodiment, a method of adjuvant therapy for reducing the body weight or blood glucose level of a subject, e.g., a subject having obesity, type 1, type 2 or gestational diabetes, comprising administering to the subject a therapeutically effective amount of the peptide of the invention, wherein the agonist is a peptide, and in addition, an adjuvant therapy using GIP or a novel GIP analog of the invention, or an effective amount of GIP-GLP-1- amylin is provided.

[0070] The peptide of the invention may also be useful alone or in combination with a hypoglycemic agent (e.g., an anti-diabetic agent) or an agent or method that inhibits or reduces gastric emptying to enhance, induce, potentiate or restore glucose responsiveness in pancreatic islets or cells. These actions can also be used to treat or prevent conditions associated with metabolic disorders such as those described above.

[0071] In another embodiment, a method for treating or preventing obesity is provided, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the peptide of the invention. In certain embodiments, the peptide of the invention includes GIP or an analog or derivative thereof, such as a novel GIP analog of the invention, or a GIP hybrid of the invention, such as a GIP-GLP-1- amylin hybrid.

[0072] In another embodiment, the subject is an obese or overweight subject. "Obesity" is generally defined, for the purposes of this disclosure, as any subject having a body mass index exceeding 30, although this includes subjects with a body mass index of less than 30 who need or desire to reduce their weight and are included within the scope of "obesity". Subjects who are insulin resistant, glucose intolerant, or have 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 useful in treating or preventing other conditions associated with obesity such as stroke, cancer (e.g., endometrial, breast, prostate, and colon cancer), gallbladder disease, sleep apnea, reduced fertility, and osteoarthritis (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001). Where the condition is associated with elevated glucose or hyperglycemia, the method includes administering a therapeutically or prophylactically effective amount of the peptide of the present invention, alone or in combination with a hypoglycemic agent (e.g., an anti-diabetic agent) or an agent or method that inhibits or reduces gastric emptying.

[0073] In yet another aspect, the peptides of the present invention, particularly the GIP hybrids of the present invention, can be used in a method of reducing food intake, a method of reducing appetite, a method of inducing satiety, a method of reducing nutrient availability, a method of reducing calorie efficiency, a method of causing weight loss, a method of affecting body composition, a method of altering body energy content or energy consumption, and a method of improving lipid profile (such as reducing LDL cholesterol and triglyceride levels and / or altering HDL cholesterol levels, etc.), where the method includes administering an effective amount of the peptide of the present invention, e.g., a GIP hybrid of the peptide of the present invention, to a subject. In one embodiment, the method of the present 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 and includes administering a therapeutically or prophylactically effective amount of the peptide of the present invention to the subject.

[0074] The states and disorders include, but are not limited to, food intake, weight loss, energy metabolism, plasma glucose level, insulin level and / or insulin secretion, positive inotropic effect, reduction of catabolism, deceleration of gastric emptying, obesity, diabetes and diabetes-related states, control of liver fat-related inflammation and injury. Such states and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, and rare genetic disorders of obesity, such as Prader-Willi syndrome, life-saving emergency medical treatment, insulin resistance and its disorders, such as polycystic ovary syndrome, obesity, all types 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), diabetes complications (neuropathy (e.g., based on the neurodifferentiation-inducing action of exendin-4), neuropathic pain (e.g., based on the action of amylin), retinopathy, nephropathy, insufficient pancreatic beta cell mass (e.g., based on the islet regeneration action of exendin-4 and GLP-1). If the state is related to an increase in glucose or hyperglycemia, the method includes administering a therapeutically or prophylactically effective amount of the peptide of the invention alone or in combination with an antihyperglycemic agent (e.g., an antidiabetic agent) or an agent or method that inhibits or reduces gastric emptying.

[0075] In addition to alleviating hypertension in a subject in need thereof as a result of reducing food intake, weight loss, and / or treating obesity, the peptides of the invention can be used to treat or prevent hypotension and conditions associated therewith.

[0076] The peptides of the invention, such as the GIP moiety, include GIP hybrids with an increased half-life (e.g., DPP-IV cleavage resistant, e.g., D-Tyr1, D-Ala2, N-acetyl or N-pyroglutamyl analogs), and optionally further include peptides such as heterologous C-terminal tails. The peptides of the invention include other hormone modules known to provide beneficial cardiovascular effects and are useful for treating cardiovascular diseases and related conditions. As disclosed herein, the peptides of the invention can increase cardiac contractility (dp / dt), decrease blood pressure (e.g., by acute vasodilation), decrease systolic pressure, decrease diastolic pressure, and provide a direct beneficial effect on cardiomyocytes. The peptides of the invention also improve cardiac function through metabolic effects, such as weight loss, glucose lowering, insulin secretion, and beta cell proliferation. However, the peptides of the invention are surprisingly even more beneficial by providing a direct effect on the cardiovascular system.

[0077] The peptides of the present invention are also useful in the treatment or prevention of various gastrointestinal disorders associated with decreased absorption in addition to excessive gastric secretion, excessive intestinal electrolyte and water secretion, such as infectious (e.g., viral or bacterial) diarrhea, inflammatory diarrhea, short bowel syndrome, or diarrhea typically occurring after surgery, e.g., ileostomy (see, e.g., Harrison's principles of Internal Medicine, McGraw Hill Inc., New York, 12th edition). Examples of infectious diarrhea include, but are not limited to, acute viral diarrhea, acute bacterial diarrhea (e.g., Salmonella, Campylobacter, and Clostridium), or diarrhea resulting from protozoal infection, or traveler's diarrhea (e.g., Norwalk virus or rotavirus). Examples of inflammatory diarrhea include, but are not limited to, malabsorption syndrome, tropical sprue, chronic pancreatitis, Crohn's disease, diarrhea, and irritable bowel syndrome. The GIP and GIP compounds of the present invention can be used, for example, to treat or prevent emergency or life-threatening situations including gastrointestinal disorders after surgery or resulting from cholera. Further, the compounds can be used to treat intestinal dysfunction in patients with acquired immunodeficiency syndrome (AIDS), particularly during periods of cachexia.

[0078] The peptides of the present invention are also useful in mammals for inhibiting the secretion of intestinal fluid and electrolytes, increasing nutrient transport, in addition to increasing cell proliferation in the gastrointestinal tract, for example, regulating lipolysis in adipose tissue, and regulating blood flow. The peptides of the present invention are useful for treating or preventing the above conditions by their gastrointestinal protective activity (e.g., inhibition of gastric juice secretion). Thus, the peptides of the present invention can be used to treat gastrointestinal or mucosal damage. Exemplary types of damage include, but are not limited to, inflammatory bowel disease, intestinal atrophy, conditions characterized by loss of intestinal mucosa or intestinal mucosal function, and other conditions of the gastrointestinal tract such as those that may be caused by exposure to cytotoxic substances, radiation, toxins, infection and / or injury. Further, the peptides of the present invention may be combined with analgesics, anti-inflammatory agents, growth hormones, heparin, or any other therapy that can be used to treat inflammatory bowel disease or other conditions enumerated above.

[0079] In another embodiment, the peptides of the present invention are useful for treating or preventing gastritis, pancreatitis, Barrett's esophagus, gastroesophageal reflux disease (GERD) and related conditions. Such conditions include, but are not limited to, heartburn, heartburn associated with reflux of gastric / intestinal contents into the chest, mouth or lungs, dysphagia, cough, intermittent stridor and inflammation of the vocal cords (conditions related to GERD), esophageal erosion, esophageal ulcer, esophageal stricture, Barrett's metaplasia (replacement of normal esophageal epithelium with abnormal epithelium), and aspiration. In certain embodiments, the peptides of the present invention may have anti-secretory properties such as inhibition of gastric acid, inhibition of bile acids, and inhibition of pancreatic enzymes. Further, the peptides of the present invention may also have a gastric protective effect. Thus, the peptides of the present invention may be particularly useful in the treatment or prevention of gastritis, pancreatitis, Barrett's esophagus, and / or GERD and related or associated conditions.

[0080] 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, together with a pharmaceutically acceptable diluent, preservative, solubilizing agent, emulsifying agent, adjuvant and / or carrier useful in the delivery of the peptide of the present invention.

[0081] These and other aspects of the invention will be more clearly understood with reference to the following embodiments and detailed description.

[0082] The present invention encompasses the treatment methods disclosed herein, comprising one or more peptides of the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the present invention comprises or consists of the amino acid sequence of formula (I):

[0083]

Chemical formula

[0084] Wherein, 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 is Ile or Leu; X 7 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 is; X 8 is Pro or Gly; X 9 is absent or 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 is; X 10 is absent or is Gly or Cys; X 11 is absent or is Ala, Cys, or Ser; X 12 is absent or is Pro or Asn; X 13 is absent or is Pro, Thr, or Leu; X 14 is absent or is Pro, Ala, or Ser; X 15 is absent or is Ser, Thr, or Cys; X 16 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 is; 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 Thr, Ala, Arg, or Cys; X 21 is Cys, Leu, Thr, or Ala; X 22is 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 Val, Leu, 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 is absent or His, Leu, Asn, or Arg; X 32 is absent or Arg, His, Phe, or Ser; X 33 is absent or Leu, Arg, Gly, or Ser; X 34 is absent or Gln, Leu, Pro, Ser, or Asn; X 35 is absent or 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 40is 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 is absent or is Tyr, Gly, or Ser; X 48 is absent or is Ser or His; X 49 is absent or is His or Thr; X 50 is absent or is Thr or Pro; X 51 is absent or is Pro; wherein R 1 and R 2 are each independently 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.

[0085] In certain embodiments, R 3 is -CO2H.

[0086] In certain embodiments, R 3 is -CO2CH3.

[0087] In certain embodiments, R 3 is -CO2NH2.

[0088] In certain embodiments, R 3 is -CO2NHCH3.

[0089] In certain embodiments, R 3 is -CO2N(CH3)2.

[0090] In certain embodiments, R 3 is -CH3.

[0091] In certain embodiments, R 3 is -NH2.

[0092] In certain embodiments, n is 12.

[0093] In certain embodiments, n is 13.

[0094] In certain embodiments, n is 14.

[0095] In certain embodiments, n is 15.

[0096] In certain embodiments, n is 16.

[0097] In certain embodiments, n is 17.

[0098] In certain embodiments, n is 18.

[0099] In certain embodiments, n is 19.

[0100] In certain embodiments, n is 20.

[0101] In certain embodiments, any of the peptides of the invention described herein includes 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.

[0102] In certain embodiments, any of the peptides of the invention described herein further includes a conjugated chemical substituent. In certain embodiments, the conjugated chemical substituent is a lipophilic substituent or a polymer moiety, such as, for example, Ac, Palm, gamma-Glu-Palm, isoGlu-Palm, PEG2-Ac, PEG4-isoGlu-Palm, (PEG)5-Palm, succinic acid, glutaric acid, pyroglutamic acid, benzoic acid, IVA, octanoic acid, 1,4-diaminobutane, isobutyl, Alexa488, Alexa647, or biotin. In certain embodiments, the conjugated chemical substituent is polyethylene glycol having a molecular weight of from 400 Da to 40,000 Da.

[0103] In related aspects, the present invention is a peptide of the present invention comprising at least two peptide components connected via one or more linker moieties, wherein each peptide subunit comprises a sequence of formula (I)-(V) or any other sequence or structure described herein. In certain embodiments, the linker is any of those 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 polyethylene glycol having a molecular weight of 400 Da to 40,000 Da. In certain embodiments, the linker is an Fc protein molecule. In certain embodiments, the N-terminus of each peptide monomer subunit is connected by a linker moiety. In certain embodiments, the C-terminus of each peptide monomer subunit is connected by a linker moiety. In other embodiments, the linker connects at least one internal amino acid residue of a peptide component to the N-terminus, C-terminus, or internal amino acid residue of another peptide component.

[0104] In further related embodiments, the present invention comprises a sequence encoding a peptide of the present invention or one or more peptide subunits of a peptide of the present invention. The present invention also includes a vector comprising a polynucleotide.

[0105] 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 certain embodiments, the pharmaceutical composition comprises an enteric coating. In certain embodiments, the enteric coating protects and releases the pharmaceutical composition in the digestive system of the subject.

[0106] In certain embodiments, the pharmaceutical composition is provided to a subject by an oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, intraocular, inhalation, intravaginal, or topical route of administration. In certain embodiments, the pharmaceutical composition is provided to a subject topically, parenterally, intravenously, subcutaneously, peritoneally, or intravenously to treat obesity.

Brief Description of the Drawings

[0107]

Fig. 1A

Fig. 1B

Fig. 2

Fig. 3

Fig. 4A

Fig. 4B

Fig. 5A

Fig. 5B

Modes for Carrying Out the Invention

[0108] Definitions Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those skilled in the art. Generally, the academic names and related techniques described herein in connection with chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry are well-known and commonly used in the art.

[0109] The following terms, when used in this specification, shall have the meanings given to them unless otherwise specified.

[0110] Throughout this specification, variations such as the words "comprise", "comprises", or "comprising" are understood to necessarily imply the inclusion of the stated element or group of elements, but do not exclude the inclusion of any other elements or groups of elements.

[0111] The singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise.

[0112] The term "including" is used to mean "including, but not limited to". "Including" and "including, but not limited to" are used synonymously.

[0113] The term "hybrid" is used to mean a peptide that includes a combination or portion of two or more peptides, for example, GLP-1-GIP-amylin; or GIP-amylin.

[0114] The terms "patient", "subject", and "individual" can be used synonymously and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, domestic animals (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats).

[0115] As used herein, the term "peptide" generally refers to a sequence of two or more amino acids joined together by peptide bonds. This term is not intended to include polymers of a specific length of amino acids, nor to imply or distinguish whether the polypeptide is produced using recombinant technology, chemical synthesis, or enzymatic synthesis, or whether it is naturally occurring. The term "peptide" also includes cyclic peptides.

[0116] Recitation of "sequence identity", "percent identity", "percent homology", or, for example, "a sequence that is 50% identical to ~", as used herein, refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a window of comparison. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which 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) are present in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0117] Calculation of array similarity or array identity (these terms are used synonymously herein) can be performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). 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%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecule is identical at that position.

[0118] The percent identity between two sequences is a function of the number of identical positions the sequences share, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0119] The comparison of arrays and determination of percent identity between two arrays can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453) incorporated into the GAP program in the GCG software package, using either a Blossum62 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 yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, 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 exemplary set of parameters includes a Blossum62 score 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 algorithm of E. Meyers and W. Miller (1989, Cabios, 4:11-17) 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.

[0120] The peptide sequences described herein can be used as "query sequences" for performing searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol, 215: 403-10). The BLAST nucleotide search can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. The BLAST protein search can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0121] The term "conservative substitution" as used herein means that one or more amino acids have been replaced with another biologically similar residue. Examples include substitutions of amino acid residues having similar characteristics, such as substitutions of small 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 some embodiments of the invention, one or more Met residues have been replaced with norleucine (Nle), which is a bioisostere of Met but is not readily oxidized as opposed to Met. Another example of a conservative substitution with a residue not normally found in endogenous mammalian peptides and proteins is the conservative substitution of Arg or Lys with, for example, ornithine, canavanine, aminoethylcysteine, or another basic amino acid. In some embodiments, one or more cysteines of the peptide analogs of the invention may be replaced with another residue, such as serine. For further information regarding phenotypically 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: Acid and amide, III: Basic, IV: Hydrophobic, V: Aromatic, bulky amino acids.

[0122]

Table 1

[0123] In the following scheme, conservative substitutions of amino acids are grouped by physicochemical properties. VI: Neutral or hydrophobic, VII: Acidic, VIII: Basic, IX: Polar, X: Aromatic.

[0124]

Table 2

[0125] The term "amino acid" or "any amino acid", as used herein, refers to any and all amino acids, including naturally occurring amino acids (e.g., α-amino acids), non-naturally occurring amino acids, modified amino acids, and unnatural amino acids. This term includes both D- and L-amino acids. Naturally occurring amino acids include those found in nature, such as the 23 amino acids that combine to form the building blocks of countless proteins by forming peptide chains, etc. These are mainly L-stereoisomers, although small amounts of D-amino acids occur in bacterial envelopes and some antibiotics. The above table lists the 20 "standard" naturally occurring amino acids. "Non-standard" naturally occurring amino acids are pyrrolysine (found in methanogens and other eukaryotes), selenocysteine (present in many prokaryotes, as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Non-naturally occurring" or "unnatural" amino acids are amino acids that do not constitute proteins (i.e., are not naturally encoded or found in the genetic code), whether they occur naturally or are chemically synthesized. Over 140 non-naturally occurring amino acids are known, and thousands of combinations are possible. Examples of "non-naturally occurring" amino acids include β-amino acids (β 3 and β 2) Homologous amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N-methyl amino acids are included. 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 contain one or more groups or chemical moieties that do not naturally occur on the amino acid. According to certain embodiments, the peptide contains an intramolecular bond between two amino acid residues present in the peptide. It is understood that the amino acid residues forming the bond are expected to change somewhat when bonded to each other compared to when not bonded to each other. Reference to a particular amino acid means including that amino acid in both its unbonded and bonded states. For example, the amino acid residue homoserine (hSer) or homoserine (Cl), when participating in an intramolecular bond according to the present invention in its unbonded form, can take the form of 2-aminobutyric acid (Abu).

[0126] Typically, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the nomenclature rules suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as described in "Nomenclature of α-Amino Acids (Recommendations, 1974)", Biochemistry, 14(2), (1975). When the names and abbreviations of amino acids and aminoacyl residues employed in this specification and the appended claims differ from such suggestions, they will be apparent to the reader. Some of the abbreviations useful in the description of the present invention are defined below in Table 1 (Table 3).

[0127]

Table 3A

[0128]

Table 3B

[0129]

Table 3C

[0130]

Table 3D

[0131]

Table 3E

[0132]

Table 3F

[0133] The one-letter and three-letter abbreviations of the naturally occurring amino acids described in the present invention are defined below in Table 2 (Table 4).

[0134]

Table 4

[0135] Throughout this specification, when naturally occurring amino acids are not referred to by their full names (e.g., alanine, arginine, etc.), they are represented by their conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). Unless otherwise specified, the three-letter and one-letter abbreviations of amino acids refer to the L-isomer form of the amino acid in question. The term "L-amino acid," as used herein, refers to the "L" isomer form of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomer form of a peptide (e.g., Dasp, (d)Asp or D-Asp; Dphe, (d)Phe or D-Phe). Amino acid residues in the D-isomer form may be substituted with any L-amino acid residue as long as the desired function is retained by the peptide. D-amino acids, when referred to using one-letter abbreviations, may be denoted in lower case by convention.

[0136] In the case of amino acids that are less common or not naturally occurring, when they are not referred to by their full names (e.g., sarcosine, ornithine, etc.), the three- or four-letter codes that are often adopted are used for their residues. Such codes include Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Dab (2,4-diaminobutyric acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutyric acid), β-Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-aminobutyric acid), βhPro (β-homoproline), βhPhe (β-homophenylalanine) and Bip (β,β-diphenylalanine), as well as Ida (iminodiacetic acid), etc.

[0137] As will be apparent to those skilled in the art, the peptide sequences disclosed herein are shown as proceeding 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 sequences in which either a "Hy-" moiety is incorporated at the amino terminus (N-terminus) of the sequence and either an "-OH" moiety or an "-NH2" moiety is incorporated at the carboxy terminus (C-terminus) of the sequence. In such cases, unless otherwise specified, the "Hy-" moiety at the N-terminus of the sequence in question represents a hydrogen atom corresponding to the presence of a free primary or secondary amino group at the N-terminus, while the "-OH" or "-NH2" moiety at the C-terminus of the sequence represents a hydroxy group or an amino group, respectively, corresponding to the presence of an amide (CONH2) group at the C-terminus. In each sequence of the present invention, the C-terminal "-OH" moiety may be replaced by the C-terminal "-NH2" moiety, and vice versa.

[0138] Those skilled in the art will understand that certain amino acids and other chemical moieties are modified when they are attached to another molecule. For example, an amino acid side chain may be modified when it forms an intramolecular bridge with another amino acid side chain, e.g., when one or more hydrogens are removed or replaced by a bond. Thus, as used herein, references to amino acids or modified amino acids present in the peptides of the present invention are meant to include such amino acids or modified amino acids in the form present in the peptide both before and after the formation of intramolecular bonds.

[0139] The term "NH2", as used herein, may refer to a free amino group present at the amino terminus of a polypeptide. The term "OH", as used herein, may refer to a free carboxy group present at the carboxy terminus of a peptide. Further, the term "Ac", as used herein, refers to acetyl protection via acylation of the C or N terminus of a polypeptide. In a particular peptide shown herein, NH2 located at the C-terminus of the peptide represents an amino group.

[0140] The term "carboxy", as used herein, refers to -CO2H.

[0141] The term "isostearic replacement", as used herein, refers to any amino acid or other analog moiety having chemical and / or structural properties similar to the specified amino acid. In certain embodiments, the isostearic replacement is a conservative substitution or an analog of the specified amino acid.

[0142] The term "cyclized", as used herein, refers to a portion of a peptide molecule being linked to another portion of the peptide molecule to form a closed ring, for example, by forming a disulfide bridge or a thioether bond.

[0143] The terms "constituent" or "subunit" are used synonymously and refer to one of a pair of peptide monomers that are joined together to form the peptides of the present invention.

[0144] The term "linker moiety", as used herein, generally refers to a chemical structure capable of linking or joining two peptide subunits to each other or of linking a peptide to another group, such as a fatty acid.

[0145] The term "pharmaceutically acceptable salt", as used herein, refers to salts or zwitterionic forms of the peptides or the peptides of the invention, which are water-soluble or oil-soluble or water-dispersible or oil-dispersible, suitable for the treatment of diseases without causing adverse toxicity, irritation, and allergic responses; those with an appropriate benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compound or separately 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, paratoluenesulfonate, and undecanoate. The amino groups in the compounds of the invention may also be quaternized with chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl; dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; chlorides, bromides, and iodides of decyl, lauryl, myristyl, and steryl; and bromides of benzyl and phenethyl. Examples of acids that can be employed 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. The pharmaceutically acceptable salts may preferably be salts selected, for example, from among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts.Examples of basic salts include salts in which the cation is an alkali metal cation such as sodium or potassium ion, an alkaline earth metal cation such as calcium or magnesium ion, and in addition, a substituted ammonium ion such as an N(R1)(R2)(R3)(R4)+ type ion (wherein each of R1, R2, R3 and R4 independently typically represents hydrogen), optionally substituted C. 1~6 -alkyl or optionally substituted C 2~6 -alkenyl, including salts selected therefrom. Related C 1~6 Examples of -alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups. C with possible relevance 2~6 Examples of -alkenyl groups include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its more recent editions), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66:2 (1977). Also, for a general review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Other suitable base salts are formed from bases that form non-toxic salts. Representative examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Acid and base hemisalts may also be formed, for example, hemisulfate and hemicalcium salts.

[0146] The term "N(alpha)methylation", as used herein, describes the methylation of the alpha amine of an amino acid and is also commonly referred to as N-methylation.

[0147] The term "sym methylation" or "Arg-Me-sym", as used herein, describes the symmetric methylation of the two nitrogens of the guanidine group of arginine. Further, the term "asym methylation" or "Arg-Me-asym" describes the methylation of a single nitrogen of the guanidine group of arginine.

[0148] The term "acylated organic compound", as used herein, refers to various compounds having a carboxylic acid functional group used to acylate the N-terminus of an amino acid or peptide component, such as a monomeric subunit, prior to formation of the 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(Tetrahedro)-2H-pyran-4-carboxylic acid.

[0149] The term "alkyl" includes straight-chain or branched, acyclic or cyclic saturated aliphatic hydrocarbons containing from 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 representative saturated branched 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 representative unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl, cyclohexenyl, etc.

[0150] "Halo" or "halogen" refers to a bromo (Br), chloro (Cl), fluoro (F), or iodo (I) substituent.

[0151] The term "haloalkyl" includes an alkyl structure in which at least one hydrogen is replaced by a halogen atom. In certain embodiments where two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms are all the same as each other. In other embodiments where two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms are not all the same as each other.

[0152] The "alkoxy" group refers to an (alkyl)O- group, where alkyl is as defined herein.

[0153] The "aryloxy" group refers to an (aryl)O- group, where aryl is as defined herein.

[0154] "Aminocarbonyl" or "carboxamide" refers to a -CONH2 group.

[0155] "2-Aminoethoxy" refers to a -OCH2CH2-NH2 group.

[0156] "2-Acetylaminoethoxy" refers to a -OCH2CH2-N(H)C(O)Me group.

[0157] The term "mammal" refers to any mammalian species such as, for example, humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, etc.

[0158] The "therapeutically effective amount" of the peptides of the present invention, as used herein, means an amount sufficient to treat or prevent any of the diseases and disorders described herein (e.g., to treat obesity). In certain embodiments, the therapeutically effective amount is expected to achieve the desired benefit / risk ratio applicable to any drug therapy.

[0159] An "analog" of an amino acid, e.g., a "Phe analog" or a "Tyr analog", means an analog of the referenced amino acid. Various amino acid analogs such as Phe and Tyr analogs are known and available in the art. In certain embodiments, an amino acid analog, e.g., a Phe analog or a Tyr analog, contains 1, 2, 3, 4, or 5 substitutions compared to Phe or Tyr, respectively. In certain embodiments, the substitutions 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 is Hy, OH, CH3, CO2H, CONH2, CONH2OCH2CH2NH2, t-Bu, OCH2CH2NH2, phenoxy, OCH3, O allyl, Br, Cl, F, NH2, N3, or guanadino. In certain embodiments, R 2 is CONH2OCH2CH2NH2, OCH3, CONH2, OCH3, or CO2H. Examples of Phe analogs include, but are not limited to, hPhe, Phe(4-OMe), α-Me-Phe, hPhe(3,4-dimethoxy), Phe(4-CONH2), Phe(4-phenoxy), Phe(4-guanidino), Phe(4-tBu), Phe(4-CN), Phe(4-Br), Phe(4-OBzl), Phe(4-NH2), BhPhe(4-F), Phe(4-F), Phe(3,5DiF), Phe(CH2CO2H), Phe(penta-F), Phe(3,4-Cl2), Phe(3,4-F2), Phe(4-CF3), ββ-diPheAla, Phe(4-N3), Phe[4-(2-aminoethoxy)], 4-phenylbenzylalanine, Phe(4-CONH2), Phe(3,4-dimethoxy), Phe(4-CF3), Phe(2,3-Cl2), and Phe(2,3-F2). Examples of Tyr analogs include, but are not limited to, hTyr, N-Me-Tyr, Tyr(3-tBu), Tyr(4-N3), and βhTyr.

[0160] The present invention generally relates to the peptides of the present invention for treating or preventing various metabolic diseases and disorders and liver diseases and disorders. In certain embodiments, this invention demonstrates a new paradigm for treating obesity, metabolic disorders, and liver disorders, as well as other diseases and disorders by administration of the peptides of the present invention. Administration of the peptides of the present invention is expected to maximize drug levels in diseased tissues while limiting circulating drug concentrations, thereby providing effective, safe, and long-lasting delivery for the lifelong treatment of obesity and metabolic and liver diseases and disorders.

[0161] In certain embodiments, the peptides of the present invention include various peptides, or peptide hetero- or homo-monomeric subunits that optionally form a cyclic structure via disulfide bonds or other bonds. In certain embodiments, the disulfide bond or other bond is an intramolecular bond. The cyclic structure of the peptide has been shown to increase the potency 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 that link two peptide subunits within the peptide.

[0162] In certain embodiments, the peptides of the present 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% compared to a negative control peptide.

[0163] In some embodiments, the half-life is measured in vitro using any suitable method known in the art. For example, in some embodiments, the stability of the peptides of the invention is determined by incubating the peptides with pre-warmed human serum or plasma at 37°C (one of ordinary skill in the art would presumably understand that serum or plasma from other species (e.g., rat, mouse, etc.) may also be used). Samples are taken at various time points, typically up to 24 hours, and the stability of the samples is analyzed by separating the peptides from the serum or plasma proteins and then analyzing for the presence of the peptide of interest using LC-MS.

[0164] In some embodiments, the peptides of the invention exhibit improved solubility or reduced aggregation characteristics compared to a control peptide. Solubility can be determined via any suitable method known in the art. In some embodiments, suitable methods known in the art for determining solubility include incubating the peptide 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, acetate pH 5.0, and others known in the art) and testing for aggregation or solubility using standard techniques. Examples of these include, but are not limited to, visible precipitation, dynamic light scattering, circular dichroism, and fluorescent dyes for measuring surface hydrophobicity and detecting aggregation or fibrillation. In some embodiments, improved solubility means that the peptide is more soluble in a given liquid than the control peptide. In some embodiments, reduced aggregation means that the peptide aggregates less than the control peptide under a given set of conditions in a given liquid.

[0165] In some embodiments, the peptides of the present invention have, for example, over a predetermined period of time, less degradation (i.e., higher degradation stability) than the control peptide, for example, at least about 10% less, at least about 20% less, at least about 30% less, at least about 40% less, or at least about 50% less degradation. In some embodiments, the degradation stability is determined via any suitable method known in the art. In some embodiments, the degradation is enzymatic degradation. For example, in certain embodiments, the peptides of the present invention have a reduced sensitivity to degradation by trypsin, chymotrypsin, or elastase. In some embodiments, suitable methods known in the art for determining degradation stability include those described in Hawe et al., J Pharm Sci, Vol. 101, No. 3, 2012, pp. 895 - 913, which is hereby incorporated by reference in its entirety. Such methods are used in some embodiments to select potent peptide sequences with enhanced shelf life.

[0166] The various peptides of the present invention may be constructed of only natural amino acids. Alternatively, the peptides of the present invention may include non - natural amino acids such as, but not limited to, modified amino acids. In certain embodiments, modified amino acids include natural amino acids that are chemically modified to include one group, multiple groups, or chemical moieties not naturally present in the amino acid. The peptides of the present invention may additionally include one or more D - amino acids. Still further, the peptides of the present invention may include amino acid analogs.

[0167] In certain embodiments, the peptides of the present invention include one or more modified or non - native amino acids. In some embodiments of the present invention, the peptides of the present invention include one or more non - natural amino acids shown in Table 1 (Table 3). In certain embodiments, the peptides of the present invention include any of those described herein, including, but not limited to, any of those containing the amino acid sequences or peptide structures shown herein.

[0168] The present invention also includes any of the peptides of the present invention described herein, either in free form or in the form of a salt. Accordingly, any embodiment of the peptides of the present invention (and related methods of use thereof) described herein includes a pharmaceutically acceptable salt of the peptide of the present invention.

[0169] The present invention also includes any variant of any of the peptides of the present invention described herein, examples of which include, but are not limited to, any of those containing the sequences shown in any one of the tables described herein, wherein one or more L-amino acid residues are replaced with the D-isomer form of the amino acid residue, for example, L-Ala is replaced with D-Ala.

[0170] Examples of peptides of the present invention described herein include peptides labeled with isotopes. In certain embodiments, the disclosure of the present invention provides peptides of the present invention having various formulas and structures as shown herein or identical to any of those listed herein, except that one or more atoms are replaced with atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into the peptides of the present 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 Cl. Certain isotopically labeled compounds described herein, such as 3 H and 14 C, etc., incorporated with radioactive isotopes are useful in tissue distribution assays of drugs and / or substrates. Furthermore, deuterium, i.e., 2Substitution with isotopes such as H can result in certain therapeutic advantages that lead to greater metabolic stability, such as increased in vivo half-life or the requirement for reduced dosing.

[0171] The present invention also includes any of the peptide components described herein linked to a linker portion that includes any of the specific linker portions described herein. In certain embodiments, the linker is attached to the N-terminal or C-terminal amino acid, while in other embodiments, the linker is attached to an internal amino acid. In certain embodiments, the linker is attached to two internal amino acids, for example, internal amino acids in each of two monomeric subunits. In some embodiments, the peptides of the invention include one or more linker portions.

[0172] The present invention also includes peptides that include a peptide 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 include a core peptide sequence, as well as one or more N-terminal and / or C-terminal modifications (e.g., Ac and NH2), and / or one or more conjugated linker portions and / or portions that lengthen the half-life. The core peptide sequence, as used herein, is the amino acid sequence of the peptide component without such modifications and conjugates.

[0173] In certain embodiments, the peptides of the present invention include, 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 portions.

[0174] In certain embodiments, the peptides of the present invention (or components thereof), including but not limited to those of any of the embodiments of Formulas I-V, etc., 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, for example, 35 to 80 amino acids. In certain embodiments, the peptides (or component monomer subunits) of the present invention 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 present 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 certain embodiments, the monomer subunits of the peptides of the present invention comprise or consist of 10 to 75 amino acid residues, and optionally, one or more additional non-amino acid moieties, such as conjugated chemical moieties, for example, PEG or lipidated amino acid residues.

[0175] In certain embodiments of the present invention, the amino acid sequence of the peptides of the present invention is not present in an antibody or is not present within the V H or V L region of the antibody.

[0176] The peptides of the present invention The peptides of the present invention include peptides comprising or consisting of any of the amino acid sequences described herein, and peptides having any of the structures described herein.

[0177] The present invention encompasses the peptides of the present invention or pharmaceutically acceptable salts or solvates thereof, and the peptides of the present invention comprise or consist of the amino acid sequence of formula (I):

[0178]

Chemical formula

[0179] wherein, 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 is Ile or Leu; X 7 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 is Pro or Gly; X 9 is absent or is Ser, Lys, Lys-g-Glu-g-Glu-C=O(CH2) n R 3 , or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ; X 10 is absent or is Gly or Cys; X 11 is absent or is Ala, Cys, or Ser; X 12 is absent or is Pro or Asn; X 13 is absent or is Pro, Thr, or Leu; X 14 is absent or is Pro, Ala, or Ser; X 15 is absent or is Ser, Thr, or Cys; X 16 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 Thr, Ala, 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 Val, Leu, 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 either does not exist or is His, Leu, Asn, or Arg; X 32 either does not exist or is Arg, His, Phe, or Ser; X 33 either does not exist or is Leu, Arg, Gly, or Ser; X 34 either does not exist or is Gln, Leu, Pro, Ser, or Asn; X 35 either does not exist or is 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; X47 is absent or is Tyr, Gly, or Ser; X 48 is absent or is Ser or His; X 49 is absent or is His or Thr; X 50 is absent or is Thr or Pro; X 51 is absent or is Pro; wherein R 1 and R 2 are each independently 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.

[0180] In certain embodiments, R 3 is -CO2H.

[0181] In certain embodiments, R 3 is -CO2CH3.

[0182] In certain embodiments, R 3 is -CO2NH2.

[0183] In certain embodiments, R 3 is -CO2NHCH3.

[0184] In certain embodiments, R 3 is -CO2N(CH3)2.

[0185] In certain embodiments, R 3 is -CH3.

[0186] In certain embodiments, R 3is -NH2.

[0187] In certain embodiments, n is 12.

[0188] In certain embodiments, n is 13.

[0189] In certain embodiments, n is 14.

[0190] In certain embodiments, n is 15.

[0191] In certain embodiments, n is 16.

[0192] In certain embodiments, n is 17.

[0193] In certain embodiments, n is 18.

[0194] In certain embodiments, n is 19.

[0195] In certain embodiments, n is 20.

[0196] In certain embodiments, the peptide of the present invention is a peptide of Formula I or a pharmaceutically acceptable salt thereof, wherein: X 1 is Tyr; X 2 is Ala; X 3 is Glu; X 4 is Ala; X 5 is Gln; X 6 is Ile; X 7 is Gly; X 8 is Pro; X 9 is absent or Ser; X 10 either does not exist or is Gly; X 11 either does not exist or is Ala; X 12 either does not exist or is Pro; X 13 either does not exist or is Pro; X 14 either does not exist or is Pro; X 15 either does not exist or is Ser; X 16 is Cys; X 17 is Asn; X 18 is Thr; X 19 is Ala; X 20 is Thr; X 21 is Cys; X 22 is Val; X 23 is Leu; X 24 is Gly; X 25 is Arg; X 26 is Leu; X 27 is Ser; X 28 is Gln; X 29 is Glu; X 30 is Leu; X 31 either does not exist or is His; X 32 either does not exist or is Arg; X 33 either does not exist or is Leu; X 34 either does not exist or is Gln; X 35 is absent or is Thr; X 36 is Tyr; X 37 is Pro; X 38 is Arg; X 39 is Thr; X 40 is Asn; X 41 is Thr; X 42 is Gly; X 43 is Ser; X 44 is Asn; X 45 is Thr; X 46 is Tyr; X 47 is absent or is Tyr; X 48 is absent or is Ser; X 49 is absent or is His; X 50 is absent or is Thr; and / or X 51 is absent or is Pro.

[0197] In another embodiment, the present invention includes a peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, and the peptide of the present invention has the formula (II):

[0198]

Chemical formula

[0199] comprises or consists of the amino acid sequence of, wherein, X 1 is Gln, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R3 or Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3 wherein; X 2 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 wherein; In the formula, R 1 and R 2 are each independently 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.

[0200] In certain embodiments, the peptide of the invention is a peptide of formula II or a pharmaceutically acceptable salt thereof, wherein: X 1 is Gln; and / or X 2 is Ser.

[0201] In certain embodiments, the peptide of the invention is a peptide of formula II or a pharmaceutically acceptable salt thereof, wherein: X 1 is Lys - γ - Glu - γ - Glu - C=O(CH2) n R 3 wherein; and / or X 2 is Ser.

[0202] In certain embodiments, the peptide of the invention is a peptide of formula II or a pharmaceutically acceptable salt thereof, wherein: X 1 is Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3and / or X 2 is Ser.

[0203] In certain embodiments, the peptide of the invention is a peptide of formula II or a pharmaceutically acceptable salt thereof, wherein: X 1 is Gln; and / or X 2 is Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 thereof.

[0204] In certain embodiments, the peptide of the invention is a peptide of formula II or a pharmaceutically acceptable salt thereof, wherein: X 1 is Gln; and / or X 2 is Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 thereof.

[0205] In another embodiment, the invention encompasses a peptide of the invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the invention comprises or consists of the amino acid sequence of formula (III):

[0206]

Chemical formula

[0207] wherein, X 1 is Tyr or (d)Tyr; X 2 is Aib or Ala; X 3 is Aib or Ala; X 4 is Ile or Leu; X 5 is Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3, or Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3 wherein; X 6 is Pro or Gly; X 7 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 wherein; In the formula, R 1 and R 2 are each independently 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.

[0208] In certain embodiments, the peptide of the invention is a peptide of formula III or a pharmaceutically acceptable salt thereof, wherein: X 1 is Tyr; X 2 is Aib; X 3 is Aib; X 4 is Ile; X 5 is Gly; X 6 is Pro; and / or 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 wherein.

[0209] In certain embodiments, the peptide of the invention is a peptide of Formula III or a pharmaceutically acceptable salt thereof, wherein: X 1 is (d)Tyr; X 2 is Ala; X 3 is Aib; X 4 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 5 is Gly; X 6 is Gly; and / or X 7 is Ser.

[0210] The present invention encompasses a peptide of the invention or a pharmaceutically acceptable salt or solvate thereof, and the peptide of the invention comprises or consists of the amino acid sequence of Formula (IV):

[0211]

Chemical formula

[0212] wherein, X 1 is His or ImPrA; X 2 is Gly, 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 3 is Gly or Pro; X 4 is absent or Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R3 or Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3 ; X 5 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 ; In the formula, R 1 and R 2 are each independently 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.

[0213] In certain embodiments, the peptide of the present invention is a peptide of Formula IV or a pharmaceutically acceptable salt thereof, wherein: X 1 is His; X 2 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 3 is Gly; X 4 is absent; and / or X 5 is Ser.

[0214] In certain embodiments, the peptide of the present invention is a peptide of Formula IV or a pharmaceutically acceptable salt thereof, wherein: X 1 is ImPrA; X 2 is Lys; X 3 is Pro; X 4 does not exist; and / or X 5 is Ser.

[0215] In certain embodiments, the peptide of the invention is a peptide of formula IV or a pharmaceutically acceptable salt thereof, wherein: X 1 is ImPrA; X 2 is Gly; X 3 is Pro; X 4 is Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 and / or X 5 is Ser.

[0216] In certain embodiments, the peptide of the invention is a peptide of formula IV or a pharmaceutically acceptable salt thereof, wherein: X 1 is His; X 2 is Gly; X 3 is Pro; X 4 is Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 and X 5 is Ser.

[0217] In another embodiment, the present invention includes the peptide of the present invention or a pharmaceutically acceptable salt or solvate thereof, and the peptide of the present invention comprises or consists of the amino acid sequence of formula (V):

[0218]

Chemical formula

[0219] wherein X 1 is Tyr or (d)Tyr; X 2 is Aib or Ala; X 3 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 4 is absent or Gly; X 5 is Cys or Ala; X 6 is Asn or Pro; X 7 is Thr or Pro; X 8 is Ala or Pro; X 9 is Thr or Ser; X 10 is Cys, 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 11 is Val or Cys; X 12 is Leu or Asn; X 13 is Gly or Thr; X14 is Arg or Ala; X 15 is Leu or Thr; X 16 is Ser or Cys; X 17 is Gln or Val; X 18 is Glu or Leu; X 19 is Leu or Gly; X 20 is His or Arg; X 21 is Arg or Leu; X 22 is Leu or Ser; X 23 is Thr or Glu; X 24 is absent or Leu; X 25 is absent or His; X 26 is absent or Arg; X 27 is absent or Leu; X 28 is absent or Gln; X 29 is Tyr or Thr; X 30 is Pro or Tyr; X 31 is Arg or Pro; X 32 is Thr or Arg; X 33 is Asn or Thr; X 34 is Thr or Asn; X 35 is Gly or Thr; X 36 is Ser, Gly, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3, or Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3 is; X 37 is Asn or Ser; X 38 is Thr or Asn; X 39 is Tyr or Thr; X 40 is absent or Tyr; wherein, R 1 and R 2 are each independently 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.

[0220] In certain embodiments, the peptide of the present invention X 1 is Tyr; X 2 is Aib; X 3 is Ser; X 4 is absent; X 5 is Cys; X 6 is Asn; X 7 is Thr; X 8 is Ala; X 9 is Thr; X 10 is Cys; X 11 is Val; X 12 is Leu; X 13 is Gly; X 14is Arg; X 15 is Leu; X 16 is Ser; X 17 is Gln; X 18 is Glu; X 19 is Leu; X 20 is His; X 21 is Arg; X 22 is Leu; X 23 is Thr; X 24 does not exist; X 25 does not exist; X 26 does not exist; X 27 does not exist; X 28 does not exist; X 29 is Tyr; X 30 is Pro; X 31 is Arg; X 32 is Thr; X 33 is Asn; X 34 is Thr; X 35 is Gly; X 36 is Ser; X 37 is Asn; X 38 is Thr; X 39 is Tyr; and / or X 40 is a peptide of formula V or a pharmaceutically acceptable salt thereof that does not exist.

[0221] In certain embodiments, the peptide of the invention is X 1 is (d)Tyr; X 2 is Ala; X 3 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 4 is Gly; X 5 is Ala; X 6 is Pro; X 7 is Pro; X 8 is Pro; X 9 is Ser; X 10 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 11 is Cys; X 12 is Asn; X 13 is Thr; X 14 is Ala; X 15 is Thr; X 16 is Cys; X 17 is Val; X 18 is Leu; X 19 is Gly; X 20 is Arg; X 21is Leu; X 22 is Ser; X 23 is Glu; X 24 is Leu; X 25 is His; X 26 is Arg; X 27 is Leu; X 28 is Gln; X 29 is Thr; X 30 is Tyr; X 31 is Pro; X 32 is Arg; X 33 is Thr; X 34 is Asn; X 35 is Thr; X 36 is Gly; X 37 is Ser; X 38 is Asn; X 39 is Thr; and / or X 40 is Tyr, a peptide of formula V or a pharmaceutically acceptable salt thereof.

[0222] 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 the amino acid sequence of formula (VI):

[0223]

Chemical formula

[0224] wherein, X1 is His or Tyr; X 2 is Aib or Ser; X 3 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 4 is Gly, 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 5 is Pro or Gly; X 6 is absent or Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 , or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ; wherein, R 1 and R 2 are each independently 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, z is 0 or 1.

[0225] In certain embodiments, the peptide of the invention X 1 is Tyr; X 2 is Aib; X 3 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 4 is Gly; X 5 is Pro; and / or X 6 is absent, or Lys, Lys - γ - Glu - γ - Glu - C=O(CH2) n R 3 or Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3 ; a peptide of formula VI or a pharmaceutically acceptable salt thereof.

[0226] In certain embodiments, the peptide of the invention is X 1 is Tyr; X 2 is Aib; X 3 is Gln; X 4 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 5 is Gly; and / or X 6 is absent, or Lys, Lys - γ - Glu - γ - Glu - C=O(CH2) n R 3 or Lys(AEEAc - AEEAc - γ - Glu - C=O(CH2) n R 3 ; a peptide of formula VI or a pharmaceutically acceptable salt thereof.

[0227] In certain embodiments, the peptide of the invention is X 1 is His; X 2 is Ser; X 3 is Gln; X 4 is Gly; X 5 is Pro; and / or X 6 is absent or is Lys or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or a pharmaceutically acceptable salt thereof, of the peptide of formula VI.

[0228] In other embodiments, the invention encompasses a peptide of the invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the invention comprises or consists of the amino acid sequence of formula (VII):

[0229]

Chemical formula

[0230] wherein X 1 is Gly, 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 2 is Gly or Pro; X 3 is absent or 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 4 is absent or is Cys; X 5 is Ser or Cys; X 6 is Leu or Thr; X 7 is Ser or Ala; X 8 is Met, Val, or Ala; X 9 is Leu or Thr; X 10 is Gly or Gln; X 11 is Ser or Ala; X 12 is Gln or Glu; X 13 is Asp or Phe; X 14 is His or Arg; X 15 is Arg or His; X 16 is Leu or Ser; X 17 is Gln or Ser; X 18 is Thr or Asn; X 19 is absent or Asn; X 20 is absent or Phe; X 21 is absent or Gly; X 22 is absent or Pro; X 23 is absent or Ile; X 24 is Tyr or Leu; X 25 is Lys, Pro, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 、or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ; X 26 is Asp or Asn; X 27 is Ala or Ser; X 28 is Asn or His; X 29 is Ala or Thr; wherein, R 1 and R 2 are each independently 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.

[0231] In certain embodiments, the peptide of the invention X 1 is Gly; X 2 is Gly; X 3 is absent or 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 4 is absent or Cys; X 5 is Ser; X 6 is Leu; X 7 is Ser; X 8 is Met; X 9 is Leu; X 10 is Gly; X 11 is Ser; X 12 is Gln; X 13 is Asp; X 14 is His; X 15 is Arg; X 16 is Leu; X 17 is Gln; X 18 is Thr; X 19 is absent or is Asn; X 20 is absent or is Phe; X 21 is absent or is Gly; X 22 is absent or is Pro; X 23 is absent or is Ile; X 24 is Tyr; X 25 is Lys; X 26 is Asp; X 27 is Ala; X 28 is Asn; and / or X 29 is a peptide of formula VII or a pharmaceutically acceptable salt thereof, wherein X is Ala.

[0232] In certain embodiments, the peptide of the invention is X 1 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 wherein; X 2 is Pro; X 3 is Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3which is the peptide of formula VII or a pharmaceutically acceptable salt thereof.

[0233] X 4 is Cys; X 5 is Cys; X 6 is Thr; X 7 is Ala; X 8 is Val or Ala; X 9 is Thr; X 10 is Gln; X 11 is Ala; X 12 is Glu; X 13 is Phe; X 14 is Arg; X 15 is His; X 16 is Ser; X 17 is Ser; X 18 is Asn; X 19 is Asn; X 20 is Phe; X 21 is Gly; X 22 is Pro; X 23 is Ile; X 24 is Leu; X 25 is Pro, Lys, Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 and; X 26is Asn; X 27 is Ser; X 28 is His; and / or X 29 is Thr.

[0234] In another embodiment, the invention encompasses a peptide of the invention or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide of the invention comprises or consists of the amino acid sequence of formula (VIII):

[0235]

Chemical formula

[0236] wherein X 1 is Tyr, His, or ImPra; X 2 is Aib or Gly; X 3 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 4 is Gly, 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 5 is Pro or Gly; X 6 is absent or 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 ; X7 is either absent or Gly; X 8 is either absent or Ala or Cys; X 9 is either absent or Pro or Asn; X 10 is either absent or Pro or Thr; X 11 is either absent or Pro or Ala; X 12 is either absent or Ser, Cys, or Thr; X 13 is Cys or Asn; X 14 is Asn, Thr, or Val; X 15 is Thr, Ala, or Leu; X 16 is Ala, Thr, or Gly; X 17 is Thr, Cys, or Arg; X 18 is Cys, Ala, or Leu; X 19 is Ala, Thr, or Ser; X 20 is Thr or Gln; X 21 is Gln, Arg, or Glu; X 22 is Arg or Leu; X 23 is Leu, Ala, or His; X 24 is Ala, Glu, or Arg; X 25 is Glu, Phe, or Leu; X 26 is Phe, Leu, or Gln; X 27 is Leu, Arg, or Thr; X 28is absent, or is Arg or His; X 29 is absent, or is His or Ser; X 30 is absent, or is Ser; X 31 is absent, or is Ser or Asn; X 32 is absent, or is Asn; X 33 is Asn, Phe, or Tyr; X 34 is Phe, Gly, or Pro; X 35 is Gly, Pro, or Arg; X 36 is Pro, Ile, or Thr; X 37 is Ile, Leu, or Asn; X 38 is Leu, Pro, or Thr; X 39 is Pro or Gly; X 40 is 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 41 is Thr or Asn; X 42 is Asn, Val, or Thr; X 43 is Val, Gly, or Tyr; X 44 is absent, or is Gly or Ser; X 45 is absent, or is Ser or His; X 46 is absent, or is His or Thr; X47 is absent, or is Thr or Pro; X 48 is absent, or is Pro; wherein, R 1 and R 2 are each independently 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.

[0237] In certain embodiments, the peptide of the invention X 1 is Tyr; X 2 is Aib; X 3 is Gln; X 4 is Gly; X 5 is Pro; X 6 is absent, or is Ser; X 7 is absent, or is Gly; X 8 is absent, or is Ala; X 9 is absent, or is Pro; X 10 is absent, or is Pro; X 11 is absent, or is Pro; X 12 is absent, or is Ser; X 13 is Cys; X 14 is Asn; X 15 is Thr; X 16 is Ala; X 17 is Thr; X 18 is Ala; X 19 is Thr; X 20 is Gln; X 21 is Arg; X 22 is Leu; X 23 is Ala; X 24 is Glu; X 25 is Phe; X 26 is Leu; X 27 is Arg; X 28 is absent or Arg; X 29 is absent or His; X 30 is absent or Ser; X 31 is absent Ser; X 32 is absent or Asn; X 33 is Phe; X 34 is Gly; X 35 is Pro; X 36 is Ile; X 37 is Leu; X 38 is Pro; X 39 is Pro; X 40 is Thr; X 41 is Asn; X 42 is Val; X 43is Val; X 44 is absent or is Gly; X 45 is absent or is Ser; X 46 is absent or is His; X 47 is absent or is Thr; and / or X 48 is a peptide of formula VIII or a pharmaceutically acceptable salt thereof, where X is absent or is Pro.

[0238] In certain embodiments, the peptide component of an exemplary peptide of the invention is a fragment of a component peptide hormone selected from 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, urocortin family peptides such as Ucn-2 and Ucn-3, neuromedin family peptides such as neuromedin U25 or splice variants, exendin-3, and exendin-4, where the fragment exhibits at least one hormonal activity of the component peptide.

[0239] As peptide components of still other exemplary peptides of the present invention, fragments of analogs or derivatives of component peptide hormones selected from amylin, ADM, CT, CGRP, intermedin, CCK, leptin, GLP-1(1-37), GLP-1(7-37), GLP-1(7-36), GLP-2, human catestatin, OXM, ANP, BNP, CNP, urodilatin, FGF-19, FGF-21, Ucn-2 and Ucn-3, neuromedin U25 or splice variants, neuromedin S, exendin-3 and exendin-4 are included, and this fragment exhibits at least one hormonal activity of the component peptide hormone. Here too, as more fully described herein and as further known in the art, an analog may include one or more insertions, deletions, or substitutions of the amino acid sequence of the component peptide hormone, and a derivative may include one or more chemical modifications of the amino acid residues of the analog or component peptide hormone.

[0240] Certain exemplary fragments that exhibit at least one hormonal activity include the following.

[0241] 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) GLP-1: GLP-1(7-37), GLP-1(7-36), GLP-1(7-35) GIP: GIP(1-14), GIP(1-28), GIP(1-30) or longer, GIP(1-39) or longer Exendin: exendin-4(1-27), exendin-4(1-28), exendin-4(1-29), exendin-4(1-30) or longer.

[0242] The peptide of the present invention may be amidated, but for the present invention, unless otherwise specified, it may optionally be in the acid form. Further, the above exemplary peptides may be combined with any analogs or derivatives discussed herein or known in the art. For example, exemplary analog fragments 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 the disclosed fragments, analogs, and derivatives.

[0243] Still other exemplary peptide components include structural motifs of peptide hormones (including their analogs and derivatives) that impart 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 peptide. Exemplary peptide components of the peptides of the present invention include the following.

[0244] 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), intermedin(42-47), intermedin(43-47), intermedin(44-47), intermedin(45-47), intermedin(46-47), intermedin(47).

[0245] GLP-1 and 2: GLP-1(29-37); GLP-1(30-37); GLP-2(24-31), GLP-2(25-31).

[0246] 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).

[0247] 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).

[0248] In certain embodiments, combinations of GIP analogs and derivatives with the peptide components described herein are contemplated. For example, the last six amino acid residues of amylin family peptide hormones analogs and derivatives known in the art and / or described above are also contemplated as exemplary peptide components. For example, as further discussed herein, the short tail of the peptide enhancer Ex-4, an exemplary Trp-cage sequence, or an analog thereof, is added to the C-terminus of any GIP analog, and in further embodiments, the peptide enhancer is attached using a linker.

[0249] In one aspect, the peptide of the present invention comprises a 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), and a GIP moiety having 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 over the entire length of the GIP moiety.

[0250] Thus, in certain embodiments, the peptide of the present invention may comprise a trp-cage motif. In certain embodiments, the peptide of the present invention comprises an N-terminal GIP or a novel GIP analog fragment in combination with a C-terminal polypeptide or a fragment thereof having the ability to reduce body weight or hypoglycemic activity (e.g., antidiabetic agents, exendin) or inhibit or reduce gastric emptying. In certain embodiments, the peptide of the present invention comprises an N-terminal GIP fragment or a novel GIP analog fragment in combination with a C-terminal exendin, GLP1, amylin, CCK, gastrin, secretin, GRP, neuromedin, urocortin, calcitonin or salmon calcitonin, or a fragment thereof. In other embodiments, the peptide of the present invention comprises a C-terminal GIP or a novel GIP analog fragment in combination with an N-terminal polypeptide or a fragment thereof having the ability to reduce body weight or hypoglycemic activity (e.g., antidiabetic agents, exendin) or inhibit or reduce gastric emptying. In certain embodiments, the peptide of the present invention comprises a C-terminal GIP, a novel GIP analog (in this case, a sequence forming a Trp-cage is present), or a fragment thereof, in combination with an N-terminal exendin, GLP1, amylin, CCK, gastrin, secretin, GRP, neuromedin, urocortin, calcitonin or salmon calcitonin, or a fragment thereof.

[0251] In other embodiments, the peptide component of the present invention is combined with a gastrin / CCK receptor ligand; an amylin receptor ligand; a calcitonin receptor ligand; a CGRP receptor ligand, an EGF receptor ligand; a glucagon-like peptide 1 receptor ligand; a glucagon-like peptide 2 receptor ligand; a gastric inhibitory polypeptide (GIP) receptor ligand; a keratinocyte growth factor (KGF) receptor 1 ligand; a dipeptidyl peptidase IV inhibitor; a REG protein receptor ligand; a growth hormone receptor ligand; a prolactin (PRL) receptor ligand; an insulin-like growth factor (IGF) receptor ligand; a PTH-related protein (PTHrP) receptor ligand; a hepatocyte growth factor (HGF) receptor ligand; an osteogenic protein (BMP) receptor ligand, a transforming growth factor (TGF) receptor ligand; a laminin receptor ligand; a vasoactive intestinal peptide (VIP) receptor ligand; a fibroblast growth factor (FGF) receptor ligand; a nerve growth factor (NGF) receptor ligand; a pancreatic islet neogenesis-associated protein (INGAP) receptor ligand; an activin A receptor ligand; a vascular endothelial growth factor (VEGF) receptor ligand; an erythropoietin (EPO) receptor ligand; a pituitary adenylate cyclase-activating polypeptide (PACAP) receptor ligand; a granulocyte colony-stimulating factor (G-CSF) receptor ligand; a granulocyte macrophage colony-stimulating factor (GM-CSF); a platelet-derived growth factor (PDGF) receptor ligand, and a secretin receptor ligand.

[0252] The peptides of the present invention are expected to preferably retain at least partially the biological activity of native human GIP. For example, the peptides of the present invention are generally expected to be GIP agonists or antagonists. In one embodiment, the peptides of the present invention are expected to exhibit biological activity in the treatment and prevention of metabolic states and disorders. Further, the novel GIP analogs of the peptides of the present invention may contain an internal linker compound, may contain chemical modifications to internal amino acid residues, or may be chemically modified at the N-terminal or C-terminal residues. In yet another embodiment, the peptides of the present invention contain 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 contain non-natural amino acid residues.

[0253] In an exemplary embodiment, the GIP portion of the peptides of the present invention comprises an N-terminal region of GIP that has been modified or substituted to provide greater DPP-IV resistance than that of native GIP.

[0254] In an exemplary embodiment, the peptides of the present invention comprise GIP or a novel GIP analog combined with an amylin family peptide, including amylin, adrenomedullin ("ADM"), calcitonin ("CT"), calcitonin gene-related peptide ("CGRP"), intermedin (also known as "AFP-6") and related peptides. Native amylin family peptide hormones are known in the art as functional peptide analogs and derivatives. Certain specific exemplary native peptides, peptide analogs and derivatives are described herein, but it should be recognized that any known amylin family peptide exhibiting hormonal activity known in the art can be used in combination with the present invention. Any amylin analog or derivative known in the art can be used in combination with the present invention.

[0255] The amylin family of peptide hormones is involved in metabolic diseases and disorders and includes, for example, amylin, calcitonin, calcitonin gene-related peptide, adrenomedullin, and intermedin (also known as "AFP-6"). In certain embodiments, the peptides of the invention include, as component peptides, one or more of the amylin family 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 islets of human type 2 diabetes (Cooper et al., Proc. Natl. Acad. Sci., USA, 84:8628-8632 (1987)). The amylin molecule has two post-translational modifications: the C-terminus is amidated and the cysteines at positions 2 and 7 are cross-linked to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of a proteolytic cleavage signal of the Lys-Arg dibasic amino acid before the N-terminal codon for Lys, and before the Lys-Arg proteolytic signal at the N-terminal position, the presence of Gly, a typical sequence for amidation by protein amidating enzyme PAM (Cooper et al., Biochem. Biophys. Acta, 1014:247-258 (1989)). "Adrenomedullin" or "ADM" means the human peptide hormone and variants of its species. More specifically, ADM is produced from a 185-amino acid preprohormone via successive enzymatic cleavage and amidation. This process ultimately leads to the release of a 52-amino acid bioactive peptide. "Calcitonin" or "CT" means the human peptide hormone and variants of its species, including salmon calcitonin ("sCT"). More specifically, CT is a 32-amino acid peptide cleaved from a larger prohormone. CT contains a single disulfide bond, and this disulfide bond is thought to shape the amino terminus into a loop. Alternative splicing of the pre-mRNA of calcitonin can result in an mRNA encoding calcitonin gene-related peptide, which is thought to function in the nervous and vascular systems.Calcitonin receptors have been cloned and shown to be members of the seven transmembrane G-protein coupled receptor family. "Calcitonin gene-related peptide" or "CGRP" means the human peptide hormone and variants of that species in any physiological form. "Intermedin" or "AFP-6" means the human peptide hormone and variants of that species in any physiological form.

[0256] In certain embodiments, the peptides of the invention include amylin peptides as component peptides. Amylin is thought to regulate gastric emptying, suppress glucagon secretion and food intake, and thus regulate the rate of appearance of glucose in the circulation. Amylin is thought to complement the action of insulin and thereby regulate the rate of disappearance of glucose from the circulation and its uptake by peripheral tissues. These actions are supported by experimental findings in rodents and humans indicating that amylin complements the action of insulin by at least three independent mechanisms that all affect the rate of appearance of glucose in postprandial glucose control. First, amylin suppresses postprandial glucagon secretion. Patients with type 1 diabetes do not have circulating amylin, and patients with type 2 diabetes have decreased postprandial amylin concentrations compared to healthy adults. Furthermore, infusion of an amylin-specific monoclonal antibody that binds circulating amylin again resulted in glucagon concentrations that were significantly elevated compared to controls. Both of these results represent the physiological role of endogenous amylin in the regulation of postprandial glucagon secretion. Second, amylin delays gastrointestinal motility and gastric emptying. Finally, intracerebroventricular injection of rat amylin has been shown to reduce food intake in rats and alter neurotransmitter metabolism in the hypothalamus. In certain studies, food intake was significantly reduced for up to 8 hours after intracerebroventricular injection of rat amylin and rat CGRP. In human trials, the amylin analog pramlintide has been shown to either reduce or increase body weight. Amylin may be beneficial in treating metabolic conditions such as diabetes and obesity. Amylin may also be used to treat pain, bone disorders, gastritis, modulate lipids, particularly triglycerides, or affect body composition such as preferentially losing fat or leaving lean tissue behind.

[0257] In certain embodiments, the peptide of the invention comprises calcitonin as a component peptide. The hormone calcitonin (CT) was named after its secretion in response to induced hypercalcemia and its rapid hypocalcemic action. Calcitonin is produced and then secreted by neuroendocrine cells in the thyroid gland, and thus these neuroendocrine cells are called C cells. The most well-studied action of CT(1-32) is its action on osteoclasts. Actions of CT in vitro include rapid loss of the ruffled border and decreased release of lysosomal enzymes. Ultimately, inhibition of osteoclast function by CT causes a decrease in bone resorption. However, neither chronic reduction of serum CT in cases of thyroidectomy nor the increase in serum CT found in medullary thyroid cancer seems to be associated with changes in serum calcium or bone mass. Thus, the main function of CT(1-32) is most likely to fight acute hypercalcemia in emergencies and / or to protect the skeleton during periods of "calcium stress" such as growth, pregnancy, and lactation. (Reviewed in Becker, JCEM, 89(4):1512-1525(2004) and Sexton, Current Medicinal Chemistry 6:1067-1093(1999)). Consistent with this, recent data from calcitonin gene knockout mice that remove both calcitonin and CGRP-I peptides have revealed that these mice had normal levels of basal calcium-related values but an increased hypercalcemic response. (Kurihara H et al., Hypertens Res. February 2003;26, Suppl:S105-8).

[0258] CT has an effect on plasma calcium levels, inhibits osteoclast function, and is widely used in the treatment of osteoporosis. Therapeutically, salmon CT (sCT) appears to increase bone density and decrease fracture rates while minimizing adverse effects. CT has also been successfully used as a therapeutic agent for Paget's disease of bone, a chronic bone disorder that can cause bone enlargement or deformation in one or more regions of the skeleton over the past 25 years. CT is also widely used for its analgesic effect on bone pain experienced during osteoporosis, but the mechanism of this effect is not clearly understood.

[0259] In human studies, salmon calcitonin inhibits postprandial gastric emptying and gastrin release while inducing dose-dependent relaxation of the gallbladder in both postprandial and fasting states. In mice and monkeys, salmon calcitonin acts to cause anorexia and weight loss after single administration. In long-term studies, oral preparations of salmon calcitonin also reduce food intake and body weight in rat models of obesity and diabetes. Therefore, having a calcitonin component in a therapeutic agent can be beneficial for weight loss.

[0260] 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, such as in the nervous system and cardiovascular system. This peptide appears to modulate sensory neurotransmission and is one of the most potent endogenous vasodilatory peptides discovered to date. Reported biological actions of CGRP include modulation of substance P in inflammation, nicotinic receptor activity at the neuromuscular junction, stimulation of pancreatic enzyme secretion, reduction of gastric acid secretion, peripheral vasodilation, cardiac acceleration, neuromodulation, regulation of calcium metabolism, stimulation of bone formation, insulin secretion, elevation of body temperature, and reduction of food intake. (Wimalawansa, Amylin, calcitonin gene-related peptide, calcitonin and 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 by its potent vasodilatory action, as demonstrated by the decrease in mean arterial pressure after intravenous administration of α-CGRP. The vasodilatory action is also supported by recent analysis of homozygous knockout CGRP mice that showed an increase in peripheral vascular resistance and hypertension caused by increased peripheral sympathetic nerve activity. (Kurihara H et al., Targeted disruption of ADM and αCGRP genes reveals their distinct biological roles. Hypertens Res. February 2003;26, supplement:S105-8). Thus, CGRP appears to elicit, among other actions, particularly vasodilation, hypotensive effects, and an increase in heart rate.

[0261] Infusion of CGRP for a long time to patients with congestive heart failure has been suggested for use in heart failure because it has shown a continuous beneficial effect on hemodynamic function without causing harmful effects. Other indications for the use of CGRP include the treatment of renal failure, acute and chronic coronary ischemia, cardiac arrhythmias, other peripheral vascular diseases such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension. Preeclamptic toxemia and preterm labor during pregnancy may also be treatable. (Wimalawansa, 1997). Recent therapeutic uses include the use of CGRP antagonists for the treatment of migraine.

[0262] In certain embodiments, the peptide of the present invention includes adrenomedullin as a component peptide. Adrenomedullin (ADM) is expressed almost ubiquitously in far more tissues containing peptides than those that do not. The published review of ADM (Hinson, J.P. et al., Endocrine Reviews (2000) 21(2):138 - 167) details its actions on the cardiovascular system, cell growth, central nervous system, and endocrine system, as well as various biological actions such as vasodilation, regulation of cell growth, hormone secretion, and natriuresis. Studies in rats, cats, sheep, and humans confirm that intravenous infusion of ADM produces a potent and sustained hypotension comparable to that of intravenous infusion of CGRP. However, since the blood pressure - lowering effect of ADM on mean arterial pressure in anesthetized rats is not inhibited by CGRP8 - 37, a CGRP antagonist, it is suggested that this effect is not mediated by the CGRP receptor. Acute or chronic administration of human ADM in anesthetized, conscious, or hypertensive rats results in a marked decrease in total peripheral resistance accompanied by hypotension and an increase in co - existing heart rate, cardiac output, and stroke volume.

[0263] ADM has also been proposed as an important factor in embryogenesis and differentiation, and further as an apoptosis survival factor for rat endothelial cells. This is supported by recent mouse ADM knockout studies showing that homozygous mice for ADM gene loss exhibited defective angiogenesis during embryogenesis and thus died in mid-pregnancy. It has been reported that ADM+ / - heterozygous mice had hypertension along with increased sensitivity to tissue injury (Kurihara H, et al., Hypertens Res. February 2003;26, Suppl:S105-8).

[0264] ADM affects endocrine organs such as the pituitary, adrenal, genital, and pancreas. The peptide is thought to have a role in inhibiting ACTH release from the pituitary. In the adrenal, the peptide is thought to affect the secretory activity of the adrenal cortex in both rats and humans, and acts as a vasodilator of the adrenal vascular bed in intact rats since it increases adrenal blood flow. ADM has been shown to be present throughout the female reproductive organs and plasma levels increase in normal pregnancy. Studies in a rat model of preeclampsia indicate that ADM can reverse hypertension and reduce fetal mortality when given to rats during mid-pregnancy. Since ADM did not have a similar effect in early pregnant animals or non-pregnant rats in the preeclampsia model, this suggests that ADM may play an important regulatory role in the uteroplacental cardiovascular system. In the pancreas, ADM most likely plays an inhibitory role in causing an initial rise in glucose levels by attenuating and delaying the insulin response to an oral glucose load. ADM may also affect renal function. A peripherally administered bolus can significantly lower the mean arterial pressure and increase renal blood flow, glomerular filtration rate, and urine flow. In some cases, an increase in Na+ excretion may also occur.

[0265] ADM also has other peripheral actions in bone and lung. Regarding bone, studies have supported a role beyond cardiovascular and fluid homeostasis, demonstrating that ADM has the effect of increasing cell growth in fetal and adult rodent osteoblasts comparable to the action of known osteoblast growth factors such as transforming growth factor-alpha. This is clinically important as one of the major challenges in osteoporosis research is to develop therapies that increase bone mass through osteoblast stimulation. In the lung, ADM not only causes pulmonary vasodilation but also inhibits bronchoconstriction induced by histamine or acetylcholine. Recent studies using aerosolized ADM to treat rat model pulmonary hypertension have shown that inhaled treatment of this condition is effective, as demonstrated by the fact that mean pulmonary artery pressure and total pulmonary vascular resistance are significantly lower in rats treated with ADM than in rats given saline. This result was achieved without changing systemic arterial pressure or heart rate (Nagaya N et al., Am J Physiol Heart Circ Physiol. 2003;285:H2125-31).

[0266] In healthy volunteers, i.v. infusion of ADM has been shown to reduce arterial pressure and stimulate plasma levels of heart rate, cardiac output, cAMP, prolactin, norepinephrine, and renin. Little increase in urine volume or sodium excretion was observed in these patients. In patients with heart failure or chronic renal insufficiency, i.v. ADM had similar effects to those seen in normal subjects and also induced diuresis and natriuresis depending on the dose administered (Nicholls, M G et al., Peptides. 2001;22:1745~1752). Experimental ADM treatment has also been shown to be beneficial in arterial and pulmonary hypertension, septic shock, and ischemia / reperfusion injury (Beltowski J., Pol J. Pharmacol. 2004;56:5~27). Other indications for ADM treatment include peripheral vascular disease, subarachnoid hemorrhage, hypertension, preeclamptic toxemia and premature labor in pregnancy, and osteoporosis.

[0267] AFP-6 (i.e., Intermedin) is mainly expressed in the pituitary gland and the gastrointestinal tract. Although no specific receptor for AFP-6 has been reported, 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 that express the endogenous CGRP receptor, and competes with labeled CGRP for binding to its receptor in these cells. In published in vivo studies, administration of AFP-6 resulted in a reduction in blood pressure in both normal and spontaneously hypertensive rats, which is most likely mediated through interaction with the CRLR / RAMP receptor. In vivo administration in mice resulted in inhibition of gastric emptying and food intake. (Roh et al., J Biol. Chem. February 20, 2004; 279(8):7264-74).

[0268] The biological actions of amylin family peptide hormones have generally been reported to be mediated by binding to two closely related type II G protein-coupled receptors (GPCRs), the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CRLR). Cloning and functional studies have shown that CGRP, ADM, and amylin interact with different combinations of CTR or CRLR and receptor activity-modifying proteins (RAMPs). Many cells express multiple RAMPs. Co-expression of a RAMP with either CTR or CRLR is thought to be necessary to generate functional receptors for calcitonin, CGRP, ADM, and amylin. The RAMP family includes three members (RAMP1, -2, and -3), which have less than 30% sequence identity but share a common topological structure. Co-expression of CRLR and RAMP1 results in the formation of the CGRP receptor. Co-expression of CRLR and RAMP2 results in the formation of the ADM receptor. Co-expression of CRLR and RAMP3 results in the formation of receptors for ADM and CGRP. Co-expression of hCTR2 and RAMP1 results in the formation of receptors for amylin and CGRP. Co-expression of hCTR2 and RAMP3 results in the formation of the amylin receptor.

[0269] In certain embodiments, the peptides of the invention comprising an amylin family hormone module can, in addition to GIP function, provide functions and uses associated with amylin family modules, such as amylin, amylin / sCT / amylin, ADM, CGRP, as discussed.

[0270] In one embodiment, amylin analogs and derivatives have at least one hormonal activity of native amylin. In certain embodiments, the amylin analog is an agonist of a receptor to which native amylin can specifically bind. Exemplary amylin analogs and derivatives include those described in US2003 / 0026812 A1, which is incorporated herein by reference.

[0271] In certain embodiments, the peptides of the invention include CCK as a component peptide. CCK includes hCCK (cholecystokinin) as well as variants and various analogs of that species, and is known in the art. Generally, CCK has a 33 amino acid sequence first identified in humans and is reported to include an 8 amino acid C-terminal fragment in vivo ("CCK-8") 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, as well as a 58 amino acid sequence found in cats, dogs, and humans, and a 47 amino acid sequence homologous to both CCK and gastrin. The C-terminal tyrosine-sulfated octapeptide sequence (CCK-8) is relatively conserved across species and may be the minimal sequence for biological activity in the rodent periphery. Thus, the term CCK-33 generally refers to human CCK (1-33), while CCK-8 (CCK(26-33)) is generally expected to refer to the C-terminal octapeptide in both sulfated and non-sulfated forms, unless otherwise specified. Further, pentagastrin or CCK-5 is expected to refer to the C-terminal peptide CCK(29-33), and CCK-4 is expected to refer to the C-terminal tetrapeptide CCK(30-33).

[0272] CCK was identified from preparations of intestinal extracts by its ability to stimulate gallbladder contraction. Subsequently, other biological actions of CCK, such as stimulation of pancreatic secretion, delay of gastric emptying, stimulation of intestinal motility, and stimulation of insulin secretion, have been reported. See Lieverse et al., Ann. N.Y. Acad. Sci. 713:268-272 (1994). Also reported as actions of CCK are effects on cardiovascular function, respiratory function, neurotoxicity and seizures, cancer cell proliferation, analgesia, sleep, sexual and reproductive behavior, memory, anxiety, and behavior mediated by dopamine. Crawley and Corwin, Peptides 15:731-755 (1994). Other reported actions of CCK include stimulation of pancreatic growth, stimulation of gallbladder contraction, inhibition of gastric acid secretion, pancreatic polypeptide release, and contractile elements of peristalsis. An additional reported action of CCK is vasodilation. Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal Tract (3rd ed., 1994; Raven Press, New York).

[0273] The injection of a combination of glucagon, CCK, and bombesin has been reported to enhance the inhibition of food intake in the milk-intake test in rats that are not in a starved state more than the inhibition observed with the individual compounds. Hinton et al., Brain Res. Bull. 17:615 - 619 (1986). Glucagon and CCK have also been 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). Estradiol and CCK have also been suggested to have a synergistic effect in satiety. Dulawa et al., Peptides 15:913 - 918 (1994); Smith and Gibbs, supra. Signals originating from the small intestine in response to nutrients in the small intestine have also been proposed to interact synergistically with CCK to reduce food intake. Cox, Behav. Brain Res. 38:35 - 44 (1990). In addition, CCK has been reported to induce satiety in numerous species. For example, feeding suppression has been reported to be caused by CCK injected intraperitoneally in rats, intraarterially in pigs, intravenously in cats and pigs, intracerebroventricularly in monkeys, rats, dogs, and sheep, and intravenously in obese and non-obese humans. See Lieverse et al., supra. Studies from numerous laboratories have further confirmed the behavioral specificity of low doses of CCK for inhibition of feeding by comparing responses to food with responses to non-food reinforcers in both monkeys and rats and by showing that CCK elicits the chain of behaviors normally observed after meal ingestion (i.e., the postprandial satiety chain). In addition, comparison of the behavior after CCK with the behavior after food intake has been reported to elucidate the behavioral similarities between CCK and food intake, either alone or in combination with CCK. CCK at physiological plasma concentrations has also been reported to inhibit food intake and increase satiety in both lean and obese humans.

[0274] CCK is characterized as a 33 - amino acid peptide. Species - specific molecular variants of the CCK amino acid sequence have been identified. The 33 - amino acid sequence and a shortened peptide, its 8 - amino acid C - terminal sequence (CCK - 8), have been reported to be identified in pig, rat, chicken, chinchilla, dog, and human. The 39 - amino acid sequence has been reported to be found in pig, dog, and guinea pig. The 58 - amino acid sequence has been reported to be found in cat, dog, and human. Frogs and turtles have been reported to exhibit a 47 - amino acid sequence that is homologous to both CCK and gastrin. Very fresh human intestine has been reported to contain a small amount of a larger molecule called CCK - 83. In rats, a major intermediate form has been reported to be identified, which is called CCK - 22. Walsh, “Gastrointestinal Hormones,” In Physiology of the Gastrointestinal Tract (3rd ed., 1994; Raven Press, New York). Nonsulfated CCK - 8 and a tetrapeptide (called CCK - 4 (CCK(30 - 33))) have been reported in rat brain. The C - terminal pentapeptide (called CCK - 4 (CCK(29 - 33))) preserves the structural homology of CCK and also preserves the homology with the neuropeptide gastrin. The C - terminal sulfated octapeptide sequence, CCK - 8, has been reported to be relatively conserved across species. Cloning and sequence analysis of cDNA encoding preprocholecystokinin from rat thyroid carcinoma, pig brain, and pig intestine have been reported to have elucidated 345 nucleotides encoding the precursor of CCK, which is 115 amino acids and contains all of the previously reported CCK sequences. Crawley and Corwin, supra.

[0275] CCK is said to be distributed throughout the central nervous system and also in endocrine cells and the enteric nerves of the upper small intestine. CCK agonists include CCK itself (also referred to as CCK-33), CCK-8 (CCK(26-33)), desulfated CCK-8, pentagastrin (CCK-5 or CCK(29-33)), and the tetrapeptide, CCK-4 (CCK(30-33)). At pancreatic CCK receptors, CCK-8 has been reported to displace binding with 1000- to 5000-fold greater potency than desulfated CCK-8 or CCK-4, and CCK-8 has been reported to be approximately 1000-fold more effective than desulfated CCK-8 or CCK-4 in stimulating pancreatic amylase secretion. Crawley and Corwin, supra. In homogenates from cerebral cortex, CCK receptor binding was said to be displaced by desulfated CCK-8 and CCK-4 at equimolar, 10-fold, or 100-fold greater concentrations than sulfated CCK-8. Id. CCK receptors have been reported to be identified in various tissues, and two primary subtypes have been described as the A-type receptor and the B-type receptor. The A-type receptor has been reported in peripheral tissues such as the pancreas, gallbladder, pyloric sphincter, and afferent vagal fibers, and also in other regions of the brain. The A-type receptor subtype (CCKA) has been reported to be selective for sulfated octapeptides. The B-type receptor subtype (CCKB) has been identified throughout the brain and stomach and has been reported to not require sulfation or all eight amino acids. Reidelberger, J. Nutr. 124(8, suppl)1327S-1333S(1994); see Crawley and Corwin, supra.

[0276] A variety of in vivo and in vitro screening methods for CCK analogs are known in the art. Examples include in vivo assays, including the contraction of the canine or guinea pig gallbladder after rapid intravenous injection of a compound to be tested for CCK-like activity, and in vitro assays using strips of rabbit gallbladder. See Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal Tract (3rd ed., 1994; Raven Press, New York).

[0277] Peptide hormones useful as components of the peptides of the present invention include the GLP-1 peptide hormone. Native GLP-1 peptide hormones, such as GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36) amide, etc., are known in the art to be functional peptide analogs and derivatives. GLP-1, as used herein, refers to all native forms of the GLP-1 peptide hormone. Certain exemplary native peptides, peptide analogs, and derivatives are described herein, but it should be recognized that any known GLP-1 peptide exhibiting hormonal activity known in the art can be used in conjunction with the present invention.

[0278] At the center of metabolic diseases and disorders is the regulation of insulin levels and blood glucose levels. Insulin secretion is partially modulated by incretin, a secretagogue hormone produced by enteroendocrine cells. Glucagon-like peptide-1 (「GLP-1」), an incretin hormone, is a peptide hormone secreted by intestinal cells and has been shown in multiple studies to have a potentiating effect on insulin secretion. GLP-1 is processed from proglucagon in the gastrointestinal tract and enhances nutrient-induced insulin release (Krcymann B., et al., Lancet, 2:1300-1303 (1987)). Various truncated forms of GLP-1 are known to stimulate insulin secretion (insulin secretagogue effect) and cAMP formation (see, for example, Mojsov, S., Int. J. Pep. Pro. Res., 40:333-343 (1992)). The relationship between various in vitro laboratory experiments on the exogenous administration of GLP-1, GLP-1(7-36) amide, and GLP-1(7-37) acid and the insulin secretagogue response in mammals, particularly humans, has been established (see, for example, Nauck, M. A., et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., New Eng. J. of Med., 326(20):1316-1322 (1992); Nauck, M. A. et al., J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes, 42:1219-1225 (1993)).

[0279] GLP-1(7-36) amide exerts a remarkable preventive effect on diabetes 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 diabetes, GLP-1(7-36) amide stimulates insulin release, reduces glucagon secretion, inhibits gastric emptying, and enhances glucose utilization (Nauck, 1993; Gutniak, 1992; Nauck, 1993). However, the use of GLP-1 type molecules for the long-term treatment of diabetes has been difficult because the serum half-life of such peptides is extremely short.

[0280] More specifically, GLP-1 is a 30-amino acid peptide derived from proglucagon, a 160-amino acid prohormone. The action of various prohormone convertases in the pancreas and intestine results in the production of glucagon and other poorly defined peptides, while the cleavage of proglucagon results in the production of GLP-1 and GLP-2 in addition to two other peptides. The amino acid sequence of GLP-1 is 100% homologous in all mammals, suggesting that it has an important physiological role. GLP-1(7-37) acid is truncated at the C-terminus and amidated to form GLP-1(7-36)NH2. The biological actions 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 amino acid numbering 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 His at position 7.

[0281] In the digestive tract, GLP-1 is produced by L cells in the intestine, colon, and rectal mucosa in response to luminal glucose stimulation. The plasma half-life of active GLP-1 is <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, thus producing GLP-1(9-37)OH or GLP-1(9-36)NH2 as metabolites, 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 that is localized in pancreatic beta cells and the lung, and to a lesser extent in the brain, adipose tissue, and kidney. Stimulation of GLP-1R by GLP-1(7-37)OH or GLP-1(7-36)NH2 results in activation of adenylate cyclase, cAMP synthesis, membrane depolarization, an increase in intracellular calcium, and an increase in glucose-induced insulin secretion (Holz et al., J. Biol. Chem. 270(30):17749-57(1995)).

[0282] GLP-1 is a potent insulin secretagogue secreted from the intestinal mucosa in response to food intake. The critical incretin effect of GLP-1 is highlighted by the fact that GLP-1 receptor knockout mice are glucose intolerant. The incretin response to intravenous GLP-1 is preserved in diabetic subjects, but the incretin response to oral glucose is impaired in these patients. Administration of GLP-1 by infusion or subcutaneous injection controls fasting 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 has shown great potential as a therapeutic agent capable of increasing insulin secretion in a physiological manner while avoiding sulfonylurea drug-related hypoglycemia.

[0283] Other important actions of GLP-1 on glucose homeostasis are the suppression of glucagon secretion and the inhibition of gastric motility. The inhibitory effect of GLP-1 on pancreatic alpha cell secretion of glucagon causes a decrease in hepatic glucose production via a reduction in gluconeogenesis and glycogenolysis. This anti-glucagon action of GLP-1 is preserved in diabetic patients.

[0284] The so-called ileal brake effect of GLP-1 inhibits gastric motility and gastric juice secretion, which is affected via receptors of the vagus nerve efferent pathway or by a direct action on intestinal smooth muscle. The reduction of gastric acid secretion by GLP-1 contributes to the lag phase in nutrient availability, thereby eliminating the need for a rapid insulin response. In summary, the gastrointestinal actions of GLP-1 significantly contribute to the delay in glucose and fatty acid absorption and modulate insulin secretion and glucose homeostasis.

[0285] GLP-1 has also been shown to induce GLUT-1 transporters, insulin (through the interaction of PDX-1 with the insulin gene promoter), and beta cell-specific genes such as hexokinase-1. Therefore, GLP-1 may reverse glucose intolerance associated with normal aging, as demonstrated in rodent experiments. In addition, GLP-1 may contribute to beta cell regeneration and increase the beta cell mass, in addition to restoring beta cell function during states of beta cell dysfunction.

[0286] A central action of GLP-1 is an increase in satiety combined with a decrease in food intake mediated through the action of GLP-1R in the hypothalamus. Continuous SC infusion of GLP-1 for 48 hours in type II diabetic subjects decreased hunger and food intake and increased satiety. These anorectic effects were not present in GLP-1R knockout mice. Therefore, GIP hybrids containing an incretin family hormone module can provide functions and uses related to the incretin family module, such as exendin-4, GLP1, GLP2, in addition to having GIP function, as discussed.

[0287] Any GLP-1 peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, the GLP-1 peptide analogs and derivatives have at least one hormonal activity of 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 WO91 / 11457, which is incorporated herein by reference.

[0288] In certain embodiments, the peptides of the invention are any one or contain any one of the amino acid sequences listed below.

[0289]

Table 5A

[0290]

Table 5B

[0291]

Table 5C

[0292]

Table 5D

[0293]

Table 5E

[0294] * - attached AA is a linking group (e.g., Lys-γGlu-γGlu-C(=O)(CH2) n R 3 (where R 3 and n are as defined above); or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 ) indicates its presence.

[0295] In certain embodiments, the peptide of the invention is any one or includes any one of the amino acid sequences having the exemplified lipidated substituents listed below:

[0296]

Table 6A

[0297]

Table 6B

[0298] Any of the peptides of the present invention can be further defined, for example, as described hereinafter. It is understood that each of the further defining features described herein can be applied to any of the peptides of the present invention when the amino acid at a particular position chemically permits the presence of the further defining feature. In certain embodiments, these features can be present in any of the peptides of the present invention of formulas (I) to (V).

[0299] In various embodiments, any nitrogen group of an amino acid of a peptide of the present invention may optionally be substituted with C1-C6 alkyl, C6-C12 aryl, C6-C12 aryl C1-C6 alkyl, or C1-C20 alkanoyl, and the pegylated version may be included alone or as a spacer of any of the foregoing, such as acetyl. It is understood that N-substitution may not be present. In certain embodiments, a peptide of the present invention includes an N-terminus selected from hydrogen, C1-C6 alkyl, C6-C12 aryl, C6-C12 aryl C1-C6 alkyl, or C1-C20 alkanoyl, and the pegylated version may be included alone or as a spacer of any of the foregoing, such as acetyl. In certain embodiments of any of the peptides of the present invention described herein, the N-terminal portion is hydrogen.

[0300] In certain embodiments of any of the peptides of the present invention having any of the various formulas described herein, K* is Lys or the following group: Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 , or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH2) n R 3 and is one of them, where R 3 is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2, and n is an integer from 12 to 20. In certain embodiments, K* is Lys-γ-Glu-γ-Glu-C=O(CH2) 18 CO2H, Lys-γ-Glu-γ-Glu-C=O(CH2)18 It is CH3, or Lys(AEEAc - AEEAc - γ - Glu - 19 - carboxynonadecanoyl).

[0301] In certain embodiments of any of the peptides of the invention having any of the various formulas described herein, the N - substituted moiety is selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanyl, and the conjugated amides of lauric acid, hexadecanoic acid, and γ - Glu - hexadecanoic acid. In certain embodiments, the N - substituted moiety is pGlu. In certain embodiments, the N - substitution is acetyl, whereby the peptide of the invention is acylated at its N (e.g., to cap or protect the N - terminal amino acid residue, e.g., the N - terminal Pen residue).

[0302] In certain embodiments of any of the peptides of the invention described herein, the N - substituted moiety is an acid. In certain embodiments, the N - substituted moiety is an acid selected from 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, glucoheptonic acid, 3 - phenylpropionic acid, trimethylacetic acid, tertiary - butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, alkylsulfonic acids and arylsulfonic acids.

[0303] 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.

[0304] 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.

[0305] In certain embodiments, the peptide of the present invention includes one or more linker groups that covalently link one peptide component to a second subunit. The subunits are optionally linked via their C-terminus.

[0306] The peptides of the present invention generally include at least two peptide components, and at least one of the peptide components, such as the GIP component, exhibits at least one hormonal activity. In the context of the present invention, at least one of the peptide components is expected to be composed of a GIP peptide, an analog, a derivative, a fragment, or a peptide enhancer. The peptide component exhibiting at least one hormonal activity may be located at the N-terminus of the peptide, at the C-terminus of the peptide, or, in the event that the peptide contains more than two peptide components, for example, at an internal portion of the peptide.

[0307] In certain embodiments, it may be preferable to arrange a peptide component exhibiting at least one hormonal activity such that the C-terminus of the peptide component is amidated. Amidation of the C-terminus of the peptide component can be achieved by arranging a module at the C-terminus of the hybrid peptide or by designing the peptide in the C-terminus to N-terminus direction at the N-terminus of the peptide. In both configurations, the C-terminus of the peptide component is available for amidation. Specific component peptides for which C-terminal amidation may be preferred include amylin family peptides, CCK, PYY, hGLP-1(7-36), and hGLP-2. Specific component peptides where C-terminal amidation is not necessarily typical (in other words, where extension at the C-terminus of the module is readily tolerated) include exendin-4, exendin-4(1-28), GIP, GLP-1(7-37), frog GLP-1(7-36), and frog GLP-2. However, when these component peptides are arranged at the C-terminus of the peptide, they may still be optionally amidated and, in practice, may preferably be optionally amidated.

[0308] The peptides of the component peptides of the present invention may be linked covalently by any method known in the art. Stable linkages may be used, or cleavable linkages may be used. In one embodiment, the carboxy of the first peptide may be directly linked to the amino of the second peptide. In another embodiment, the linking group can be used to attach the module. Further, if necessary, spacers or turn-inducers known in the art may be employed to stabilize the linkage. As an example, when amidation of the C-terminus of a peptide component located at the N-terminus is not desired, the module may be attached directly to the second module, or any suitable linking group known in the art, such as alkyl; PEG; amino acids, such as Lys, Glu, beta-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, Ill.); aminocaproyl ("Aca"), beta-alanyl, 8-amino-3,6-dioxaoctanoyl, or other cleavable and non-cleavable linkers known in the art may be used to attach to the second module. Specific hybrid embodiments in which the linker in a hybrid containing each of the exemplified linkers is replaced with a Gly linker, particularly embodiments in which the Gly linker is Gly-Gly-Gly, are specifically described herein as if each were explicitly recited. In one embodiment, the linker or spacer is 1 to 30 residues in length, in another embodiment 2 to 30 residues, in yet another embodiment 3 to 30 residues in length, and any integer length from 2 to 30 including the upper and lower limits; each integer unit is expected to be, for example, 2, 3, 4, 5, 6, 7, etc. In one embodiment, a Gly linker is used, and in a particular embodiment, Gly-Gly-Gly, a three-residue linker, is used.

[0309] In certain embodiments, the peptide components of the invention may be linked by suitable linking moieties, such as disulfide bridges between two cysteine residues, one for each peptide subunit, or by other suitable linker moieties such as those defined herein but not limited thereto. In certain embodiments, the subunits may be modified to exclude either the free amine at the C or N terminus, thereby allowing dimerization at the remaining free amine. Further, in some cases, the termini of one or more monomeric subunits are acylated with an acylating organic compound selected from the group consisting of trifluoropentyl, acetyl, octonyl, butyl, pentyl, hexyl, palmitoyl, trifluoromethylbutyric acid, cyclopentanecarboxylic acid, cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, tetrahydro-2H-pyran-4-carboxylic acid, succinic acid, and glutaric acid. In some cases, the subunits contain both a free carboxy terminus and a free amino terminus, whereby the user can selectively modify the subunits such that dimerization at the desired terminus is achieved. Those skilled in the art will understand that the subunits of the invention can be selectively modified such that a single specific amine for the desired linkage is achieved.

[0310] Furthermore, it is understood that the C-terminal residue of the subunits disclosed herein is optionally an amide. Further, in certain embodiments, it is understood that dimerization at the C terminus is facilitated by using a suitable amino acid having a side chain with amine functionality, as generally understood in the art. With respect to the N-terminal residue, coupling may be achieved via the free amine of the terminal residue or, as generally understood in the art, by using a suitable amino acid side chain having a free amine.

[0311] The linker portion for connecting the subunits may have any structure, length, and / or size that conforms to the teachings described herein. In at least one embodiment, the linker portion 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-phenylenediacetic acid, 1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, triazine, Boc-triazine, IDA-biotin, PEG4-biotin, AADA, suitable aliphatic, aromatic compounds, heteroaromatic compounds, and polyethylene glycol-based linkers having a molecular weight of approximately 400 Da to approximately 40,000 Da. In certain embodiments, PEG2 is HO2CCH2CH2OCH2CH2OCH2CH2CO2H.

[0312] Table 2 (Table 7) provides non-limiting examples of suitable linker portions.

[0313] [Table 7A]

[0314] [Table 7B]

[0315] [Table 7C]

[0316] [Table 7D]

[0317] [Table 7E]

[0318] In some embodiments, the peptides of the invention include a linker portion. In some embodiments, the peptide subunits of the invention are joined by intermolecular disulfide bonds formed between two cysteine residues, one for each subunit. In some embodiments, the peptides of the invention include both a linker portion and intermolecular disulfide bonds formed between two cysteine residues. In some embodiments, the intramolecular bond is a thioether, lactam, triazole, selenoether, diselenide, or olefin instead of a disulfide bond.

[0319] One of ordinary skill in the art will understand that the linker (e.g., C- and N-terminal linkers) portions disclosed herein are non-limiting examples of suitable linkers and that the invention may include any suitable linker portion. Thus, some embodiments of the invention are composed of two monomer subunits selected from the peptides shown in any of the tables described herein, or include or consist of homo- or heterodimeric peptides comprising the sequences presented in any of the tables described herein, where the C- or N-terminus of each subunit (or internal amino acid residue) is linked by any suitable linker portion such that the peptides of the invention are provided. In certain embodiments, the linker binds to the N- or C-terminus of one subunit that makes up the peptide of the invention and an internal amino acid residue of the other subunit. In certain embodiments, the linker binds to an internal amino acid residue of one monomer subunit that makes up the peptide of the invention and an internal amino acid residue of the other monomer subunit. In further embodiments, the linker binds to the N- or C-terminus of both subunits.

[0320] In certain embodiments, one or more of the subunits comprise any one of the sequences or structures of formulas (I)-(V) or any of the peptides described herein. In certain embodiments of the peptides of the present invention, the linker portion is any of the linkers 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-Ala-IDA) linker, or a PEG linker.

[0321] In various embodiments of any of the peptides of the present invention, each of the peptide subunits is attached to the linker portion via its N-terminus, C-terminus, or internal amino acid residue. In certain embodiments of any of the peptides of the present invention, the N-terminus of each peptide subunit is connected by the linker portion. In certain embodiments of any of the peptides of the present invention, the C-terminus of each peptide subunit is connected by the linker portion. In certain embodiments of any of the peptides of the present invention, each peptide subunit is connected by a linker portion attached to an internal amino acid.

[0322] 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 also be referred to herein as half-life enhancing moieties. Without wishing to be bound by any particular theory, it is believed that lipophilic substituents bind to albumin in the bloodstream, thereby protecting the peptides of the present invention from enzymatic degradation and thus enhancing their half-life. In addition, polymer moieties are thought to enhance the half-life and reduce clearance in the bloodstream.

[0323] In additional embodiments, any of the peptides of the present invention, such as the peptides of formulas (I)-(V), further comprises a linker moiety attached to an amino acid residue present in the peptide. For example, the linker 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.

[0324] In additional embodiments, any of the peptides of the present invention, such as the peptides of formulas (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.

[0325] In additional embodiments, any of the peptides of the present invention, such as the peptides of formulas (I)-(V), further comprises a half-life extending moiety attached to a linker moiety attached to an amino acid residue present in the peptide. For example, the half-life extending moiety can be attached to the 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.

[0326] In certain embodiments, the peptide comprises a half-life extending moiety having the structure shown below, where n = 0-24 or n = 14-24:

[0327]

Chemical formula

[0328] In certain embodiments, the peptide of the present invention comprises a half-life extending moiety shown in Table 3 (Table 8).

[0329]

Table 8A

[0330]

Table 8B

[0331] In certain embodiments, the moiety that lengthens the half-life is directly attached to the peptide, while in other embodiments, the moiety that lengthens the half-life is attached to the peptide via a linker moiety, such as via any of those depicted herein.

[0332]

Table 9A

[0333]

Table 9B

[0334]

Table 9C

[0335] In certain embodiments, the peptides of the invention include any of the linker moieties shown herein and any of the moieties that lengthen the half-life shown herein, examples of which include any of the following combinations shown in Table 5 (Table 10).

[0336]

Table 10A

[0337]

Table 10B

[0338]

Table 10C

[0339]

Table 10D

[0340] In some embodiments, there may be a plurality of linkers between the peptide and the conjugated moiety, such as a moiety that prolongs the half-life, as depicted, for example, in Table 6 (Table 11).

[0341] [Table 11]

[0342] In certain embodiments, the half-life of the peptides of the invention comprising a conjugated chemical substituent, i.e., a moiety that prolongs the half-life, 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 except having no conjugated chemical substituent. In certain embodiments, the lipophilic substituent and / or polymer moiety enhance the permeability of the peptide across the epithelium and / or its retention in the lamina propria of the mucosa. In certain embodiments, the permeability of the peptides of the invention comprising a conjugated chemical substituent across the epithelium and / or its retention in the lamina propria of the mucosa 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 except having no conjugated chemical substituent.

[0343] In certain embodiments, the side chains of one or more amino acid residues (e.g., Lys residues) in the peptides of the invention are conjugated (e.g., attached by a covalent bond) to a lipophilic substituent. The lipophilic substituent may be covalently bonded to an atom in the amino acid side chain or, alternatively, may be conjugated to the amino acid side chain via one or more spacers. The spacer, if present, can provide a spacing between the peptide subunit and the lipophilic substituent. In certain embodiments, the peptides of the invention include any of the conjugated moieties disclosed herein.

[0344] In certain embodiments, the lipophilic substituent may include a hydrocarbon chain having from 4 to 30 carbon atoms, such as at least 8 or 12 carbon atoms, preferably 24 carbon atoms or fewer, or 20 carbon atoms or fewer. The hydrocarbon chain may be straight or branched and may be saturated or unsaturated. In certain embodiments, the hydrocarbon chain is substituted with a moiety that forms part of the attachment to the amino acid side chain or spacer, such as an acyl group, sulfonyl group, N atom, O atom, or S atom. In some embodiments, the hydrocarbon chain is substituted with an acyl group and thus the hydrocarbon chain may form part of an alkanoyl group, such as palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

[0345] The lipophilic substituent may be conjugated to any amino acid side chain in the peptide of the present invention. In certain embodiments, the amino acid side chain includes carboxy, hydroxyl, thiol, amide or amine groups to form an ester, sulfonyl ester, thioester, amide or sulfonamide with a spacer or a lipophilic substituent. For example, the lipophilic substituent may be conjugated to 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 to Lys. The amino acid shown as Lys in the formulas provided herein may be replaced, for example, with Dbu, Dpr or Orn when a lipophilic substituent is added.

[0346] In certain embodiments, the peptides of the invention may be modified via conjugation of chemical moieties to one or more amino acid side chains within the peptide, for example, to enhance stability, increase permeability, or enhance drug-like characteristics. For example, the N(epsilon) of lysine N(epsilon), the beta-carboxyl of aspartic acid, or the gamma-carboxyl of glutamic acid may be appropriately functionalized. Thus, the amino acids within the peptide may be appropriately modified to produce the modified peptides. Further, in some cases, the side chains are acylated with an acylated organic compound selected from the group consisting of trifluoropentyl, acetyl, octonyl, butyl, pentyl, hexyl, 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 bile acids. One of ordinary skill in the art will understand that a series of conjugates may be linked, for example, PEG4, isoglu, and combinations thereof. One of ordinary skill in the art will understand that the amino acids in the peptide may be isoelectronically replaced, for example, Lys may be replaced with Dap, Dab, alpha-MeLys, or Orn. Table 7 (Table 12) shows examples of modified residues within the peptide.

[0347]

Table 12A

[0348]

Table 12B

[0349] In a further embodiment of the present invention, alternatively or in addition, the side chains of one or more amino acid residues in the peptides of the present invention are conjugated to a polymer moiety, for example, to increase solubility and / or half-life and / or bioavailability in vivo (e.g., in plasma). Such modifications are also known to reduce the clearance (e.g., renal clearance) of therapeutic proteins and peptides.

[0350] "Polyethylene glycol" or "PEG", as used herein, is a polyether compound of the general formula H-(O-CH2-CH2)n-OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on their molecular weights. PEO, PEE, or POG, as used herein, refers to oligomers or polymers of ethylene oxide. Although the three names are chemically synonymous, PEG tends to refer to oligomers and polymers having a molecular weight less than 20,000 Da, PEO tends to refer to polymers having a molecular weight greater than 20,000 Da, and POE tends to refer to polymers of any molecular weight. PEG and PEO are liquids or low melting point solids depending on their molecular weights. Throughout this disclosure, the three names are used interchangeably. PEG is prepared by the polymerization of ethylene oxide and is commercially available over a wide range of molecular weights from 300 Da to 10,000,000 Da. PEGs and PEOs with different molecular weights are utilized in different applications and have different physical properties (e.g., viscosity) due to the influence of the chain length, but their chemical properties are substantially the same. The polymer moiety is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Suitable polymer moieties include polyethylene glycol (PEG), homo- or co-polymers of PEG, monomethyl-substituted polymers of PEG (mPEG), or polyoxyethylene glycerol (POG). See, for example, Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992). Also included are PEGs prepared for the purpose of increasing half-life, for example, mono-activated alkoxy-terminated polyalkylene oxides (POA), such as monomethoxy-terminated polyethylene glycol (mPEG); bis-activated polyethylene oxide (glycol) or other PEG derivatives are also contemplated.Suitable polymers are expected to vary substantially by mass in the range of about 200 Da to about 40,000 Da or about 200 Da to about 60,000 Da and are typically selected according to the purpose of the present invention. In certain embodiments, PEG having a molecular weight of 200 to 2,000 or 200 to 500 is used. Different forms of PEG can also be used depending on the initiator used in the polymerization process, and common initiators are monofunctional methyl ether PEG, or methoxypoly(ethylene glycol) abbreviated as mPEG.

[0351] Lower molecular weight PEGs are also available as pure oligomers, which are referred to as monodisperse, uniform, or discrete. These are used in certain embodiments of the present invention.

[0352] PEGs with different geometric arrangements are also available: branched PEGs have 3 to 10 PEG chains extending from a central core group; star PEGs have 10 to 100 PEG chains extending from a central core group; comb PEGs usually have multiple PEG chains grafted onto a polymer backbone. PEGs can also be linear. Often the numerical values included in the name of PEGs indicate their average molecular weight, and (for example, PEG with n = 9) will have an average molecular weight of approximately 400 Daltons and will be labeled PEG400.

[0353] "PEGylation", as used herein, is the act of covalently coupling a PEG structure to a peptide of the present invention, and this peptide is referred to as a "PEGylated peptide". In certain embodiments, the PEG of the PEGylated side chain is PEG having a molecular weight of about 200 to about 40,000. In certain embodiments, the PEG of the PEGylation spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, the PEG of the PEGylation spacer is PEG3 or PEG8.

[0354] 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 may be linear or branched. In some embodiments, the polymer moiety has a molecular weight of 500-40,000 Da, such as 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, or 20,000-40,000 Da.

[0355] In some embodiments, the peptide of the invention may contain two or more such polymer moieties, in which case the total molecular weight of all such moieties is generally expected to be within the ranges provided above.

[0356] In some embodiments, the polymer moiety is coupled (by covalent linkage) 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 included.

[0357] Those skilled in the art will be well aware of suitable techniques that can be used to perform the coupling reaction. For example, a PEG moiety having a methoxy group can be coupled to a Cys thiol group by maleimide linkage using reagents commercially available from Nektar Therapeutics AL. See also WO2008 / 101017, and the references cited above, for details of suitable chemistries. Also, maleimide-functionalized PEG can be conjugated to the sulfhydryl group of the side chain of a Cys residue.

[0358] Oxidation of disulfide bonds, as used herein, may be performed within a single step or may be a two-step process. In the case of a single oxidation step, as used herein, a trityl protecting group is often employed during assembly, deprotected during cleavage, and subsequent solution oxidation is possible. If a second disulfide bond is required, there is an option of native oxidation or selective oxidation. In the case of selective oxidation requiring orthogonal protecting groups, Acm and trityl are used as protecting groups for cysteine. Oxidation of one protecting pair of cysteine is made possible by removing one of the protecting pairs of cysteine by cleavage. Subsequently, a second oxidative deprotection step of cysteine protected with the Acm group is performed. In the case of native oxidation, use of a trityl protecting group for all cysteines allows for the natural folding of the peptide. Those skilled in the art will be well aware of suitable techniques that can be used to perform the oxidation step.

[0359] Numerous chemical moieties, such as poly(ethylene) glycol, react with functional groups present in the 20 naturally occurring amino acids, such as the epsilon amino group in a lysine amino acid residue, the thiol present in a cysteine amino acid residue, or other nucleophilic amino acid side chains, etc. When multiple naturally occurring amino acids in a peptide react, these non-specific chemical reactions result in the final peptide of the present invention containing many isomers of the peptide conjugated to one or more poly(ethylene) glycol strands at different positions within the peptide of the present invention.

[0360] One advantage of certain embodiments of the present invention is the ability to add one or more chemical moieties (e.g., PEG) by incorporating one or more unnatural amino acids having a unique functional group that reacts with activated PEG by a chemical method that does not react with the naturally occurring amino acids present in the peptides of the present invention. For example, azide and alkyne groups do not react with all naturally occurring functional groups in proteins. Thus, unnatural amino acids can be incorporated into one or more specific sites in the peptides of the present invention where PEG or another modification is desired without causing unwanted non-specific reactions. In certain embodiments, the specific chemistry involved in this reaction results in a stable covalent linkage between the PEG strand and the peptide of the present invention. In addition, such reactions can be carried out under mild aqueous conditions that do not damage most peptides. In certain embodiments, the unnatural amino acid residue is AHA.

[0361] The chemical moieties attached to natural amino acids are limited in number and scope. In contrast, the chemical moieties attached to unnatural amino acids can utilize a much broader range of useful chemistries for attaching chemical moieties to target molecules. Essentially all target molecules, including any protein (or portions thereof) containing unnatural amino acids, such as unnatural amino acids containing reactive sites or side chains to which chemical moieties, such as aldehyde or keto-derivatized amino acids, can be attached, may serve as substrates for attaching chemical moieties.

[0362] A very large number of chemical moieties can be ligated or conjugated to a particular molecule via various known methods in the art. A variety of such methods are described in U.S. Patent No. 8,568,706. As an illustrative example, an azide moiety may be useful in conjugating chemical moieties such as PEG or others described herein. The azide moiety serves as a reactive functional group and is not present in most naturally occurring compounds (and thus it does not react with the native amino acids of naturally occurring compounds). Azides also undergo selective ligation with a limited number of reaction partners, and azides are small and can be introduced into biological samples without significantly altering the molecular size. One reaction that enables the incorporation or introduction of azides into molecules is the copper-mediated Huisgen [3+2] cycloaddition of azides. 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., J. Am. Chem. Soc., 2003, 125, 4686).

[0363] In another embodiment, the peptide of the present invention may have one or more amino acid residues 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 analog or peptide of the present invention may have one or more amino acid residues deleted from the N-terminus or C-terminus of native GIP. In another embodiment, the peptide of the present invention may have one or more amino acid residues deleted at amino acid positions 1 to 42 of 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. Such deletions can include more than one consecutive or non-consecutive deletion. In an exemplary embodiment, one or fewer, two or fewer, three or fewer, four or fewer, or five or fewer 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, as in the case where the region is, for example, exendin(31-39) or exendin(27-39). In one embodiment, the native GIP is human, rat, mouse, pig or bovine.

[0364] In one embodiment of the peptide of the present invention, when any of the GIP peptides, analogs, derivatives or hybrids is intended to be used as an agonist, it does not contain any deletion at any one of positions 1 to 15 corresponding to positions YAEGTFISDYSIAMD of the N-terminal sequence of GIP. In other words, each of the corresponding positions 1 to 15 of GIP is expected to be present, but they may be substituted or derivatized. In a further embodiment, the agonist GIP compound does not contain any deletion at any one of positions 4 to 15 corresponding to positions GTFISDYSIAMD of the C-terminal sequence of GIP. In other words, each of the corresponding positions 4 to 15 of GIP is expected to be present, but they may be substituted or derivatized. Accordingly, in embodiments of the agonist GIP compound, each of positions 1 to 15 or positions 4 to 15 is present and is expected to be occupied by the amino acid present at that position in the naturally occurring GIP species, or by a substitute or derivative thereof. In yet another embodiment of the agonist GIP compound, GIP compounds that do not exhibit sufficient receptor binding activity or receptor activation activity as shown are excluded from the various embodiments described herein.

[0365] In one embodiment of the peptide of the present invention, when any of the GIP peptides, analogs, derivatives or hybrids is intended to be used as an agonist, it does not contain any deletion at any one of positions 1 to 15 corresponding to positions YAEGTFISDYSIAMD of the N-terminal sequence of GIP. In other words, each of the corresponding positions 1 to 15 of GIP is expected to be present, but they may be substituted or derivatized. In a further embodiment, the agonist GIP compound does not contain any deletion at any one of positions 4 to 15 corresponding to positions of the C-terminal sequence of GIP. In other words, each of the corresponding positions 4 to 15 of GIP is expected to be present, but they may be substituted or derivatized.

[0366] In another embodiment of the peptides of the invention, the GIP analog or hybrid may have one or more amino acid residues inserted 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, alone or in combination with one or more deletions and / or substitutions. In one aspect, the invention relates to a GIP analog or hybrid peptide having a single insertion of more than one amino acid residue, or contiguous or non-contiguous insertions, 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, rat, mouse, porcine or bovine.

[0367] In another embodiment of the peptides of the invention, the GIP analog or hybrid may contain one or more non-natural amino acids and / or non-amino acid insertions 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 a further such embodiment, the native GIP may be human, rat, mouse, porcine or bovine.

[0368] Accordingly, the compounds are shown using optional linking groups, and in one embodiment of the sequences described herein, the linker is a Gly linker, such as Gly-Gly-Gly, or a beta Ala linker, such as beta Ala-beta Ala; all of these are specifically contemplated. Particularly interesting linker molecules include aminocaproyl ("Aca"), beta-alanyl, and 8-amino-3,6-dioxaoctanoyl. Further, in other embodiments, beta turn mimics are used, examples of which include mimic A: N-(3S,6S,9S)-2-oxo-3-amino-1-azabicyclo[4.3.0]-nonane-9-carboxylic acid, mimic B: N-(3S,6S,9R)-2-oxo-3-amino-7-thia-1-azabicyclo[4.3.0]-nonane-9-carboxylic acid, and further Ala-Aib and Ala-Pro dipeptides are also included.

[0369] In another embodiment of the peptides of the invention, the GIP analog or hybrid may include the insertion of a polyamino acid sequence (e.g., poly-his, poly-arg, poly-lys, poly-ala, etc.) at either end of the polypeptide, known as an "extension" or "tail".

[0370] In some embodiments, the novel peptides of the invention include amino acid sequence insertions, examples of which include 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 alanine substitutions at each amino acid position along the length of region S, such as exendin(27-39) and exendin(31-39).

[0371] 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 (e.g., stearyl, palmitoyl, octanoyl, etc.), or GIP analogs and hybrid polypeptides by the addition of polyamino acids such as poly-his, poly-arg, poly-lys, and poly-ala. Modifications to the polypeptide can include small molecule substituents such as short alkyl and constrained alkyl (e.g., branched, cyclic, fused adamantyl), and aromatic groups. The water-soluble polymer molecule is expected to preferably have a molecular weight in the range of about 500 to about 20,000 daltons.

[0372] Such polymer conjugation and small molecule substituent modification can be done within the sequence of the GIP analog and hybrid polypeptide, either alone at the N or C terminus, or at the side chain of an amino acid residue. Alternatively, there may be multiple sites of derivatization along the GIP analog and hybrid peptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine can provide additional sites for derivatization. See, for example, U.S. Pat. Nos. 5,824,784 and 5,824,778. In one embodiment, the peptide of the present invention may be conjugated to 1, 2, or 3 polymer molecules.

[0373] The water-soluble polymer molecule is preferably linked to an amino, carboxyl, or thiol group, and may be linked by the N or C terminus, or by the side chain of lysine, aspartic acid, glutamic acid, or cysteine. Alternatively, the water-soluble polymer molecule may be linked by diamine and dicarboxylic acid groups. In an exemplary embodiment, the peptide of the present invention is conjugated to 1, 2, or 3 PEG molecules via the epsilon amino group on a lysine amino acid.

[0374] The peptides of the present invention also include chemical modifications to one or more amino acid residues. Such chemical modifications include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. Chemical modifications can be made, for example, within the sequences of GIP analogs and hybrid peptides, either at the N or C terminus alone, or on the side chains of amino acid residues. In one embodiment, the C terminus of these peptides may have a free -OH or -NH2 group. In another embodiment, the N terminus may be capped with an isobutyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group (iso cap), an octanyl group, an octylglycine group (G(Oct)), or an 8-aminooctanoic acid group or an Fmoc group. In an exemplary embodiment, cyclization may be via the formation of a disulfide bridge. Alternatively, there may be multiple sites of chemical modification along the peptides of the present invention.

[0375] The number of mimetic peptide bonds is generally described as having no effect on the structure and biological activity of peptides. An example of this approach is for replacing retro-inverso type mimetic peptide bonds (Rivier, J. E. and Marshall, G. R. (eds.) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), pp. 722-773, "Biologically active retroinverso analogues of thymopentin", Sisto A. et al.) and Dalpozzo et al. (1993), Int. J. Peptide Protein Res., 41:561-566, which are incorporated herein by reference). According to this modification, the component peptides of the peptides of the present invention can be identical to, for example, the sequence of GIP described herein, except that one or more of the peptide bonds are replaced with retro-inverso type mimetic peptide bonds. Such substitutions are preferably made for most of the N-terminal peptide bonds, since they are expected to confer resistance to proteolysis by exopeptidases acting on the N-terminus. Further modifications can also be made by replacing the chemical groups of the amino acids with other chemical groups having similar structures. Another suitable mimetic peptide bond known to enhance stability to enzymatic cleavage without or with little loss of biological activity is the reduced isostere mimetic peptide bond (Couder et al. (1993), Int. J. Peptide Protein Res., 41:181-184, which is incorporated herein by reference in its entirety).

[0376] Accordingly, the amino acid sequences of these peptides may be identical to, for example, the sequences of novel GIP analogs and hybrid peptides, except that one or more of the peptide bonds are replaced with isostere pseudopeptide bonds. Such substitutions are expected to confer resistance to proteolysis by exopeptidases acting on the N-terminus, and preferably most of the N-terminal peptide bonds are replaced. The synthesis of peptides having one or more reduced isostere pseudopeptide 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.

[0377] In another embodiment, the bond between the second and third residues that is a target for cleavage by DPP-IV is replaced with a protease-resistant bond as disclosed herein.

[0378] The peptoid derivatives of the peptides of the present invention are representative examples of another class of peptidomimetics that retain important structural determinants for biological activity but confer resistance to proteolysis by eliminating peptide bonds (Simon et al., Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992), which is hereby incorporated by reference in its entirety). Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups corresponding to the side chains of natural amino acids have been described (Simon et al. (1992), cited above). Some or all of the amino acids of the GIP peptide may be replaced with N-substituted glycines corresponding to the replaced amino acids.

[0379] In one embodiment, the peptides of the present invention include the modifications described above, i.e., combinations of deletions, insertions, and substitutions.

[0380] Component peptides in which the indicated amino acid residues are chemically modified or derivatized (e.g., via fatty acid derivatization, pegylation, amidation, glycolation, etc.) are also included within the scope of the present invention. Exemplary embodiments include the derivatization of lysine residues, particularly lysine residues at positions 16 or 30. D - amino acid residues of the indicated amino acids are also expected to be within the scope of the present invention. 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 sites described herein.

[0381] Exemplary peptides of the present invention may include substitutions of non - natural amino acids. For example, exemplary derivatives of the GIP analogs or hybrid peptides of the present invention include GIP analogs or hybrid peptides conjugated with polymers, wherein the GIP analog or hybrid peptide includes any of the insertions, deletions, substitutions, or combinations thereof described above, and the polymer molecule is conjugated via 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 compared to human GIP or analogs. For example, octyl - glycine at methionine increases the duration of action of the compound in vivo. By this modification, the duration of action is increased by at least 4 hours. Thus, in one embodiment, the peptides of the present invention are conjugated to one or more water - soluble polymer molecules such as polyethylene glycol ( "PEG") or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, octanoyl, etc.), or are conjugated by the addition of polyamino acids such as poly - his, poly - arg, poly - lys, poly - glu, and poly - ala. Modifications to the polypeptide may include small molecule substituents such as short alkyl and constrained alkyl (e.g., branched, cyclic, fused adamantyl), and aromatic groups.

[0382] More specifically, D-Tyr1 and D-Ala2 variants of each peptide component described herein are contemplated. In still other embodiments, variants of each of the above-described sequences in which the peptides of the invention are modified by 1, 2, or 3 modifications described herein are contemplated. Exemplary modifications are modifications at the 1st, 2nd, or 3rd N-terminal amino acids of GIP that confer DPP-IV resistance superior to that of native GIP. In further additional embodiments, the novel peptides of the invention contain a C-terminal amide.

[0383] In a further embodiment, the peptides of the invention have a half-life of at least 2-fold that 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 certain embodiments, the half-life may be at least 24 hours.

[0384] Another embodiment is a pharmaceutically acceptable salt of the peptide of the invention. The peptides of the invention can be formulated in the form of a composition containing a pharmaceutically acceptable carrier.

[0385] Synthesis of the Peptides of the Invention The peptides of the present invention can be synthesized by many 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 present invention provides a method for producing a peptide of the present invention (or a subunit thereof), including, but not limited to, chemically synthesizing a peptide having an amino acid sequence described herein, such as any of the amino acid sequences of Formulas I-V or any of the amino acid sequences described in the tables set forth herein, consisting of, or consisting essentially of, such a peptide. In other embodiments, the peptide is synthesized recombinantly instead of being chemically synthesized. In certain embodiments, the peptides and methods of the present invention include synthesizing both monomeric subunits of the peptide of the present invention and then linking the two subunits to produce the peptide of the present invention. In various embodiments, the coupling or linking is achieved via any of the various methods described herein.

[0386] In certain embodiments, a method for producing a peptide of the present invention (or a monomeric subunit thereof) further includes cyclizing the peptide of the present invention (or a monomeric subunit thereof) after its synthesis. In certain embodiments, the cyclization is achieved via any of the various methods described herein. In certain embodiments, the present invention provides a method for producing a peptide (or a monomeric subunit thereof), including, but not limited to, introducing an intramolecular bond, such as a disulfide, amide, or thioether bond, between two amino acid residues in a peptide having an amino acid sequence described herein, such as any of the amino acid sequences of Formulas (I)-(V), the attached examples, or the tables, consisting of, or consisting essentially of, such a peptide.

[0387] The peptides of the present invention can be prepared using standard recombinant techniques or chemical peptide synthesis techniques known in the art, for example, using an automated or semi-automated peptide synthesizer, or both.

[0388] The peptides of the present invention may be synthesized in solution or on a solid support according to the prior art. Such methods are described, for example, herein, further in U.S. Patent No. 6,610,824 and U.S. Patent No. 5,686,411, and further in Patent Application No. 454,533 (filed on December 6, 1999), the entireties of which are incorporated herein by reference. Various automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd Edition, 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, edited by Gross and Meienhofer, Academic Press, New York, 1-284 (1979). Solid-phase peptide synthesis may be performed using an automated peptide synthesizer that uses an NMP / HOBt (Option 1) system (e.g., Model 430A, Applied Biosystems Inc., Foster City, Calif.), or tBoc or Fmoc chemistry in capping (see the Users Manual of Applied Biosystems for ABI430A Peptide Synthesizer, Version 1.3B, July 1, 1988, Section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.). The peptides may also be assembled using an Advanced Chem Tech synthesizer (Model MPS350, Louisville, Ky.). The peptides can be purified, for example, by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system and a C4, C8, or C18 preparative column (10μ, 2.2×25 cm; Vydac, Hesperia, Calif.).The polypeptide can be synthesized by convergent methods such as "native chemical ligation" and modified methods thereof, in which two or more peptide fragments having termini with appropriate orthogonal reactivity are ligated by formation of native amide bonds. The newly formed peptide can be further ligated to produce an even longer polypeptide. The individual starting peptides may be derivatized, if required, or derivatized after the ligation step.

[0389] Peptide analogs can be synthesized on 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 equivalents, 1.0 mmol) residues are activated using 4.0 equivalents of HBTU, 4.0 equivalents of HOBT, and 8.0 equivalents of DIEA and coupled to the resin over 1 hour. The Fmoc group is removed by treatment with 20% (v / v) piperidine in dimethylformamide. Final deprotection and cleavage of the peptide from the solid support is effected by treating the resin with reagent B (93% TFA, 3% phenol, 3% water, and 1% triisopropylsilane) for 2 - 3 hours. The cleaved peptide is precipitated using tert-butyl methyl ether, pelleted by centrifugation, and lyophilized. The pellet is redissolved in water (10 - 15 mL), filtered, and purified via reverse phase HPLC using an acetonitrile / water gradient containing a C-18 column and 0.1% TFA. The purified product is lyophilized and analyzed by ESI-LC / MS and analytical HPLC, and shown to be pure (>98%). All mass results were in agreement with the calculated values.

[0390] Alternatively, peptides are assembled on a Symphonyl peptide synthesizer (Protein Technologies, Inc., Woburn, Mass.) using Rink amide resin (Novabiochem, San Diego, Calif.) with a loading of 0.43 - 0.49 mmol / g at 0.050 - 0.100 mmol. Fmoc amino acids (Applied Biosystems, Inc., 5.0 equivalents, 0.250 - 0.500 mmol) residues are dissolved at a concentration of 0.10 M in 1-methyl-2-pyrrolidinone. All other reagents (HBTU, HOBT and N,N-diisopropylethylamine) are prepared as 0.55 M dimethylformamide solutions. The Fmoc-protected amino acids are then coupled to the amino acids bound to the resin over 2 hours using HBTU (2.0 equivalents, 0.100 - 0.200 mmol), HOBT (1.8 equivalents, 0.090 - 0.18 mmol), N,N-diisopropylethylamine (2.4 equivalents, 0.120 - 0.240 mmol). After the last amino acid coupling, the peptide is deprotected over 1 hour using 20% (v / v) piperidine in dimethylformamide. Once the peptide sequence is complete, the Symphonyl peptide synthesizer is programmed to cleave the resin. Trifluoroacetic acid (TFA) cleavage of the peptide from the resin is performed using a reagent mixture consisting of 93% TFA, 3% phenol, 3% water and 1% triisopropylsilane. The cleaved peptide is precipitated using tert-butyl methyl ether, pelleted by centrifugation and lyophilized. The pellet is dissolved in acetic acid, lyophilized, then dissolved in water, filtered and purified via reverse phase HPLC using an acetonitrile / water gradient containing a C18 column and 0.1% TFA. Analytical HPLC is used to assess the purity of the peptide and identity is confirmed by LC / MS and MALDI-MS.

[0391] Active proteins can be readily synthesized and then screened in a screening assay designed to identify reactive peptides.

[0392] Alternatively, the peptides of the present invention may be produced by recombinant techniques well known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor (1989). The peptides of the present invention produced by recombinant techniques can be expressed from polynucleotides. Those skilled in the art will understand that polynucleotides containing DNA and RNA encoding such GIP analogs or hybrid peptides may be obtained from wild-type cDNAs, such as GIP, GLP1, amylin, taking into account the degeneracy of codon occurrence, or may be engineered as required. These polynucleotide sequences may incorporate codons that facilitate transcription and translation of mRNA in a microbial host. Such produced sequences can be readily constructed according to methods well known in the art. See, for example, WO83 / 04053. The above polynucleotides may also optionally encode an N-terminal methionyl residue. Non-peptide compounds useful in the present invention can be prepared by methods known in the art. For example, amino acids containing phosphoric acid and peptides containing such amino acids can be prepared using methods known in the art. See, for example, Bartlett and Landen, Bioorg. Chem. 14:356-77 (1986).

[0393] A variety of expression vector / host systems can be utilized to contain and express the coding sequence of the peptides of the present invention. Examples of these include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell lines infected with virus expression vectors (e.g., baculovirus); plant cell lines transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell lines. Mammalian cells useful in recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g., COS-7), WI38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for recombinant expression of proteins are described herein.

[0394] Accordingly, the polynucleotide sequences provided by the present invention are useful in generating novel and useful viral and plasmid DNA vectors, novel and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacteria, yeast, and mammalian cells grown in culture), and novel and useful methods for culturing and growing such host cells capable of expressing the GIP polypeptides of the present invention. Polynucleotide sequences encoding the GIP analogs or hybrids described herein may be useful in gene therapy when a deficiency in the production of hybrid GIP or other component peptide hormones is expected to be alleviated, or when a need for an increase in the levels of such is expected to be met.

[0395] The host cell may be a prokaryotic cell or a eukaryotic cell, examples of which 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.

[0396] Mammalian host systems for the expression of recombinant proteins are also well known to those skilled in the art. Host cell lines can be selected with respect to their ability to process the expressed protein or to provide certain post-translational modifications that are expected to be useful in the provision of protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing cleaves the "prepro" form of the protein, which 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 such post-translational activities and can be selected to ensure correct modification and processing of the introduced foreign protein.

[0397] Alternatively, a yeast system may be employed 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 prepro alpha leader sequence is amplified from yeast genomic DNA in a PCR reaction using one primer containing nucleotides 1 to 20 of the alpha mating factor gene and another primer complementary to nucleotides 255 to 235 of this gene (Kurjan and Herskowitz, Cell, 30:933-43 (1982)). The prepro alpha leader coding sequence and the GIP polypeptide coding sequence fragment are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter such that the promoter directs the expression of a fusion protein consisting of the prepro alpha factor fused to the mature GIP polypeptide. As taught by Rose and Broach, Meth. Enz. 185:234-79, Goeddel, ed., Academic Press, Inc., San Diego, Calif. (1990), the vector further contains an ADH2 transcription terminator, a yeast "2-micron" origin of replication, a yeast leu-2d gene, yeast REP1 and REP2 genes, an Escherichia coli (E. coli) beta-lactamase gene, and an E. coli origin of replication downstream of the cloning site. The beta-lactamase and leu-2d genes provide selection in bacteria and yeast, respectively. The leu-2d gene also facilitates an increase in the copy number of the plasmid in yeast to induce higher levels of expression. The REP1 and REP2 genes encode proteins involved in the regulation of the plasmid copy number.

[0398] The DNA construct described in the foregoing paragraph is transformed into yeast cells using known methods, such as lithium acetate treatment (Steams et al., Meth. Enz. 185:280-97 (1990)). The ADH2 promoter is induced when the glucose in the growth medium is depleted (Price et al., Gene 55:287 (1987)). The preproalpha sequence results in the secretion of the fusion protein from the cell. Concurrently, the yeast KEX2 protein cleaves the prepro sequence from the mature GIP-polypeptide (Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330-4 (1984)).

[0399] The peptides of the present invention may also be recombinantly expressed in yeast using a commercially available expression system, such as the Pichia expression system (Invitrogen, San Diego, Calif.), according to the manufacturer's instructions. This system also relies on the preproalpha sequence to support secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter when induced by methanol. The secreted peptides are purified from the yeast growth medium by methods such as those used to purify peptides from bacterial and mammalian cell supernatants.

[0400] Alternatively, the cDNA encoding the peptide of the present invention can be cloned into an expression vector, such as the baculovirus expression vector pVL1393. Then the vector encoding this GIP compound is used according to the manufacturer's instructions (Sigma Chemical Co., St. Louis, MO) to infect Spodoptera frugiperda cells in a sF9 protein-free medium to produce the recombinant protein. The protein is purified from the medium using a heparin-sepharose column (Pharmacia, Piscataway, N.J.) and sequential molecular sizing columns (Amicon, Beverly, Mass), concentrated, and resuspended in PBS. SDS-PAGE analysis shows a single band to confirm the size of the protein, and Edman sequencing with a Proton 2090 peptide sequencer confirms its N-terminal sequence.

[0401] 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, N.J.). The pGEX vector is designed to produce a fusion protein containing glutathione-5-transferase (GST) encoded by the vector and a protein encoded by a DNA fragment inserted into the cloning site of the vector. Primers for PCR can be generated, for example, to include appropriate cleavage sites. The recombinant fusion protein can then be cleaved from the GST portion of the fusion protein. For example, the pGEX-3X / GIP analog peptide construct is transformed into E. coli XL-1 blue cells (Agilent, Santa Clara, CA), individual transformants are isolated, grown in LB medium (supplemented with carbenicillin) at 37 °C until the optical density at a wavelength of 600 nm reaches 0.4, and then further incubated for 4 hours in the presence of 0.5 mM isopropyl beta-D-thiogalactopyranoside (Sigma Chemical Co., St. Louis, MO.). Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the gene insert encoding the desired peptide in the appropriate orientation.

[0402] Fusion proteins expected to be produced as insoluble inclusion bodies in bacteria can be purified as follows. Cells are harvested by centrifugation, washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA, and treated with 0.1 mg / mL lysozyme (Sigma Chemical Co.) for 15 minutes at room temperature. The lysate is clarified by sonication, and dead cell debris is pelleted by centrifugation at 12,000 × g for 10 minutes. The pellet containing the fusion protein is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered on 50% glycerol, and centrifuged at 6,000 × g for 30 minutes. The pellet is resuspended in a standard phosphate-buffered saline (PBS) without Mg++ and Ca++. The fusion protein is further purified by fractionating the pellet resuspended in denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is immersed in 0.4 M KCl to visualize the protein, which is excised and electroeluted in SDS-free gel electrophoresis buffer. When, for example, a GST / GIP polypeptide fusion protein is produced as a soluble protein in bacteria, this can be purified using a GST purification module (Pharmacia Biotech).

[0403] The fusion protein may be subjected to digestion to cleave GST from the mature GIP analog or hybrid peptide. The digestion reaction (in 0.5 mL PBS, 20 - 40 μg of fusion protein, 20 - 30 units of human thrombin (4000 U / mg (Sigma))) is incubated at room temperature for 16 - 48 hours and loaded onto a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is immersed in 0.4 M KCl to visualize the protein bands. The identity of the protein band corresponding to the expected molecular weight of the peptide can be confirmed by partial amino acid sequence analysis using an Orbitrap Exploris 490 mass spectrometer (ThermoFisher Scientific, Waltham, MA).

[0404] The transformed cells are preferably used for long-term high-yield protein production, and thus stable expression is desirable. When such cells are transformed with a vector containing a selectable marker along with the desired expression cassette, the cells can grow in the enrichment medium for 1-2 days before the enrichment medium is switched to the selection medium. The selectable marker is designed to confer resistance to selection, and its presence enables the growth and recovery of cells that successfully express the introduced sequence. Resistant aggregates of stably transformed cells can be grown using appropriate tissue culture techniques for the cells.

[0405] Various selection systems can be used to recover cells transformed for recombinant protein production. Such selection systems include, but are not limited to, the HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes in tk, hgprt, or aprt cells, respectively. Antimetabolite resistance can also be used as the basis for selection for dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G418 and also confers resistance to chlorosulfuron; and hygro, which confers resistance to hygromycin. Additional selectable genes that may be useful include trpB, which enables cells to utilize indole instead of tryptophan, or hisD, which enables cells to utilize histinol instead of histidine. Markers that visually represent the identification of transformants include anthocyanin, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin.

[0406] The peptides of the present invention can be produced using a combination of both automated peptide synthesis and recombinant techniques. For example, the GIP peptide may contain a combination of modifications such as deletions, substitutions, and insertions by pegylation. Such GIP peptides can be produced stepwise. In the first step, an intermediate GIP peptide containing modifications of deletions, substitutions, insertions, and any combination thereof can be produced by recombinant techniques as described. Then, after any optional purification steps as described herein, the intermediate GIP peptide can be pegylated via chemical modification with a suitable pegylation reagent (e.g., from NeKtar Transforming Therapeutics, San Carlos, Calif.) to obtain the desired GIP peptide. One of ordinary skill in the art will understand that the procedures described above can be generalized to apply to the peptides of the present invention containing a combination of modifications selected from deletions, substitutions, insertions, derivatizations, and other means of modification well known in the art and contemplated by the present invention.

[0407] It may be desirable to purify the GIP polypeptide produced by the present invention. Peptide purification techniques are well known to those of ordinary skill in the art. These techniques include, at one level, the fractionation of the crude product of the cellular environment into peptide and non-peptide fractions. Once the peptide has been separated from other proteins, chromatographic and electrophoretic techniques may be used to further purify the peptide of interest to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly efficient method for purifying peptides 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 purity confirmation is achieved by determining amino acid analysis.

[0408] Certain aspects of the invention relate to purification, and in certain embodiments, to substantial purification of the encoded protein or peptide. The term "purified peptide," as used herein, is intended to refer to a composition separable from other components, where the peptide is purified to any degree compared to its naturally occurring state. Thus, a purified peptide also refers to a peptide that has been removed from its natural environment. Generally, "purified" is expected to refer to a peptide composition that has been fractionated to remove various other components and that substantially retains its expressed biological activity. When the term "substantially purified" is used, this expression is expected to refer to a composition in which the peptide is the major component of the composition, e.g., a composition consisting of about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the peptide in the composition.

[0409] A variety of techniques suitable for use in peptide purification are expected to be well known to those of skill in the art. Examples of these 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, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, the order of execution of the various purification steps can be changed or a particular step can be omitted and still a suitable method for preparing a substantially purified protein or peptide will result.

[0410] There is no general requirement that the peptide be always provided in its most purified state. In fact, even products that are not so well purified are expected to be useful in certain embodiments. Partial purification may be achieved by using a combination of fewer purification steps, or by utilizing different forms of the same general purification scheme. For example, it is understood that cation exchange column chromatography performed using an HPLC apparatus generally provides a greater "magnitude" of purification than the same technique using a low-pressure chromatography system. Methods that exhibit a lower degree of relative purification may have advantages in total protein product recovery or in maintaining the activity of the expressed protein.

[0411] Optionally, such peptides of the invention may be purified and isolated from other components obtained in the process. Methods for purifying polypeptides can be found in U.S. Patent No. 5,849,883. These documents describe specific exemplary methods for the isolation and purification of G-CSF compositions that may be useful in isolating and purifying the GIP polypeptides of the invention. In view of the disclosures of these patents, it is clear that one of ordinary skill in the art is well aware of a great many purification techniques that can be used to purify the peptides of the invention from a given source.

[0412] It is expected that a combination of anion exchange and immunoaffinity chromatography can be employed to produce the purified peptides of the invention.

[0413] Methods of treatment The peptides of the invention are useful in treating and preventing metabolic diseases and disorders and liver diseases and disorders.

[0414] Metabolic diseases and disorders present in many forms such as, for example, obesity, diabetes, dyslipidemia, insulin resistance, apoptosis of cells, etc. Obesity and its related disorders are common and very serious public health problems in the United States and throughout the world. Upper body obesity is the most well-known strong risk factor for type 2 diabetes and a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for increased incidence of hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, cancers of the breast, prostate, and colon, and complications of general anesthesia (see, for example, Kopelman, Nature 404:635-43 (2000)). Obesity shortens lifespan and poses a serious risk of disorders such as the above co-morbidities, in addition to infections, aneurysms, seborrheic keratosis, 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 group of conditions called the insulin resistance syndrome, or "syndrome X". Estimated recent medical costs for obesity and related disorders are two trillion dollars worldwide. The etiology of obesity is thought to be multifactorial, but the underlying problem is that in obese subjects, the availability of nutrients and energy consumption do not balance until adipose tissue becomes excessive. Obesity is currently a chronic, essentially intractable metabolic disorder that cannot be adequately treated. Therapeutic agents useful in weight reduction in obese persons may have a significant beneficial effect on their health status.

[0415] Diabetes is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria resulting from inadequate production or utilization of insulin. Diabetes significantly affects the quality of life of a large portion of the population in developed countries. Inadequate production of insulin is characterized as type 1 diabetes, and inadequate utilization of insulin is type 2 diabetes. However, it is widely recognized that there are many distinct diabetes-related disorders that develop well before a patient is diagnosed with overt diabetes. Also, the effects of suboptimal control of glucose metabolism in diabetes cause a wide range of related lipid disorders and cardiovascular disorders.

[0416] Dyslipidemia, or abnormal levels of lipoproteins in the plasma, occurs frequently among diabetics. Dyslipidemia is typically characterized by elevated plasma triglycerides, low HDL (high-density lipoprotein) cholesterol, elevated levels of LDL (low-density lipoprotein) cholesterol from normal levels, and increased levels of low-density LDL (low-density lipoprotein) particles in the blood. Dyslipidemia is one of the major contributing factors to the increased incidence of coronary events and death among diabetic subjects. Epidemiological studies have confirmed this by showing that the incidence of coronary death is several times higher in diabetic subjects compared to non-diabetic subjects. Among diabetic subjects, numerous lipoprotein abnormalities have been described.

[0417] Insulin resistance is a decrease in the ability of insulin to exert its biological action 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 occurs. If the defective insulin action cannot be compensated for, an increase in plasma glucose concentration is inevitable, resulting in a clinical state of diabetes. Insulin resistance and relative hyperinsulinemia are recognized to have a contributory role in obesity, hypertension, atherosclerosis, and type 2 diabetes. The association between insulin resistance and obesity, hypertension, and angina has been explained as a syndrome with insulin resistance as a common etiological association, i.e., syndrome X.

[0418] Non-alcoholic fatty liver disease (NAFLD) is an umbrella term that encompasses everything from mere fat accumulation in the liver to more progressive steatosis associated with related hepatitis, fibrosis, cirrhosis, and in some cases hepatocellular carcinoma. NAFLD is becoming increasingly prevalent worldwide, particularly in Western European countries. In the United States, NAFLD is the most common form of chronic liver disease, affecting approximately one-quarter of the population. Some individuals with NAFLD are at risk of developing non-alcoholic steatohepatitis (NASH), a highly malignant form of fatty liver disease, which is characterized by hepatitis and can progress to progressive scarring (cirrhosis) and liver failure. This damage is similar to that caused by heavy alcohol consumption. NAFLD consists of non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NAFL is characterized by hepatic steatosis involving more than 5% of the parenchyma, with no evidence of hepatocyte injury. In contrast, NASH is defined from a histological perspective as a necroinflammatory process by which liver cells become injured against the background of steatosis. The natural progression of NAFLD has not yet been fully characterized. Studies indicate that the incidence of NAFLD is increasing in concert with the rising incidence of metabolic syndrome. Patients with type 2 diabetes show a more than two-fold to four-fold increased risk of fatty liver-related complications in addition to a very high risk of developing NASH.

[0419] Apoptosis is an active process of self-destruction of cells that is regulated by extrinsic and intrinsic signals that occur during normal development. It is well established that apoptosis plays a major role in the regulation of pancreatic endocrine beta cells. In adult mammals, there is increasing evidence that the beta cell mass is subject to dynamic changes to adapt insulin production to maintain euglycemia in specific states such as pregnancy and obesity. The control of the beta cell mass depends on a delicate balance between cell proliferation, growth, and programmed cell death (apoptosis). Disruption of this balance can potentially compromise glucose homeostasis. For example, notably, glucose intolerance occurs as the replication rate of beta cells decreases with aging, and human autopsy studies have repeatedly shown a 40 - 60% reduction in the beta cell mass in patients with insulin-independent diabetes compared to non-diabetic subjects. Generally, insulin resistance is always associated with obesity, but it has been recognized that euglycemia is maintained by compensatory hyperinsulinemia until beta cells can no longer meet the increased demand for insulin, at which point type 2 diabetes begins.

[0420] To address these abnormalities in various patients, attempts to treat multiple diabetes-related abnormalities have been facilitated by the administration of numerous anti-diabetic drugs. However, the peptides of the invention discussed herein are useful when administered in a therapeutically effective amount, either as monotherapy or as adjuvant therapy, in treating or preventing these and other diseases and conditions discussed overall.

[0421] In an embodiment of the invention, when hyperglycemia reduction occurs in a treated diabetic patient (e.g., by a GLP-1 mimetic such as exenatide), an intervention is prepared. The chronic hyperglycemic state in type 2 diabetic patients weakens the insulin secretory response of GIP, whereas the improvement in blood glucose control resulting from exenatide treatment is expected to restore the responsiveness of pancreatic beta cells to GIP stimulation. Administration of the peptides of the invention is expected to result in desired normoglycemia in diabetic patients or patients suffering from conditions associated with elevated glucose.

[0422] Currently prescribed anti-diabetic agents (metformin, sulfonylurea, TZD, SGLT2 inhibitors, etc.) can achieve varying degrees of blood glucose control. Therefore, a combination of the peptides of the invention with any of these therapies should also elicit an improved response that results in normalization of glucose levels.

[0423] Accordingly, in one embodiment, the method of the invention is based on the concept that prior glucose lowering by other anti-diabetic agents, such as GLP-1, GLP-1 analogs or exendin-4, or other agents, such as metformin, sulfonylureas, thiazolidinediones (TZD), pramlintide, insulin, acarbose, dipeptidyl peptidase (DPP-IV) inhibitors, and SGLT-2 inhibitors, prepares the patient for therapy. DPP-IV inhibitors are well known and are described, for example, in published application US20050004117, U.S. Patent No. 6,710,040, and U.S. Patent No. 6,645,995, which are incorporated herein by reference with respect to those compounds. An example of a sulfonylurea (SFU) that acts on pancreatic tissue to produce insulin is glimepiride.

[0424] The peptides of the present invention can be useful for reducing food intake, reducing appetite, reducing calorie intake, inducing satiety, reducing nutrient availability, causing weight loss, affecting body composition, altering body energy content or energy consumption, improving lipid profile (such as reducing LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels, etc.), delaying gastrointestinal motility, slowing gastric emptying, blunting postprandial blood glucose spikes, preventing or inhibiting glucagon secretion, and lowering blood pressure. In one embodiment, such GIP peptides contain an exendin, GLP1, amylin and / or sCT moiety.

[0425] Particularly interesting as peptides for anti-obesity, weight reduction, reduced food intake, increased metabolic rate, and reduction and / or redistribution of body fat are peptides of the invention that effectively reduce food intake, alter body composition, redistribute fat, and / or reduce weight, such as GLP-1-GIP receptor co-agonists (i.e., dual amylin calcitonin receptor agonists) like GIP, GLP-1, exendin, amylin, etc., and peptides of the invention containing a leptin family module. Particularly interesting embodiments for treating obesity and related diseases and conditions (reduction of body fat) discussed herein are peptides of the invention comprising GIP, exendin-4 or an analog or derivative thereof, an amylin component, such as pramlintide or a dual amylin calcitonin receptor agonist, an FN38 family member, such as 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. Specific regions of the forebrain (components derived from the telencephalon and diencephalon of the brain) and the hindbrain or brainstem (including the midbrain, pons, and medulla oblongata) have been identified as being involved in the control of energy balance. Structures or nuclei of the forebrain present in the hypothalamus involved in the regulation of food intake and / or body weight include, for example, the arcuate nucleus (ARC), the paraventricular nucleus (PVN), the dorsomedial hypothalamic nucleus (DMH), the ventromedial nucleus (VMH), and the lateral hypothalamus nucleus (LHA). Structures or nuclei of the hindbrain present in the brainstem involved in the regulation of food intake and / or body weight include, for example, the nucleus of the solitary tract (NST), the area postrema (AP), and the lateral parabrachial nucleus (IPBN). Nuclei of the brainstem that control elements of the motor control system of consummatory behavior are likely to be controlled by primary or secondary projections from brainstem regions such as NST, AP, and IPBN. Notably, AP, NST, and IPBN have all been shown to have their own integrative capabilities (collectively and independently).

[0426] Various CNS-directed anti-obesity agents act on these forebrain structures that are present in the hypothalamus and are involved in the regulation of food intake and / or body weight. In addition, CNS-directed anti-obesity agents act on the hindbrain structures that are present in the brainstem and are involved in the regulation of food intake and / or body weight. Such component peptides include, for example, leptin and leptin agonists, ciliary neurotrophic factor (CNTF) and CNTF agonists, peptide YY (PYY) and PYY agonists, exendin and exendin agonists, GLP-1 and GLP-1 agonists, ghrelin and ghrelin antagonists, cholecystokinin (CCK) and CCK agonists, and amylin and amylin agonists, such as those described herein.

[0427] In certain embodiments, the peptides of the invention and methods for their use include a first component that preferentially targets energy balance centers in the hypothalamus, such as the ARC, PVN, VM, and LH. In one embodiment, the peptide of the invention contains GIP or an analog or derivative thereof, and one or more other peptide family components that similarly target the hypothalamus but at a different location than the first component or via a different mechanism of action. If the GIP peptide contains more than one other peptide family component, and these also target the hypothalamus, the more than one other peptide family components may target the same location via the same mechanism of action as each other, or they may target different locations and / or different mechanisms of action. In another embodiment, the GIP peptide then contains one or more other peptide family components that provide one or more additional beneficial therapeutic effects, such as anti-obesity effects, control of blood glucose, cardioprotection, and / or control of hypertension, via a location or mechanism of action different from that of the first component. In certain embodiments, the additional peptide family components preferentially target energy balance centers in the hindbrain, such as the NST, AP, and IPBN.

[0428] In certain embodiments, the peptides of the invention and methods for their use include a first component that preferentially targets energy balance centers of the hindbrain, such as NST, AP, and IPBN. In one embodiment, the GIP peptide further contains one or more other peptide family components that similarly target the hypothalamus, but at a different location than the first component or via a different mechanism of action. In another embodiment, the peptides of the invention then contain one or more additional beneficial therapeutic components that provide one or more additional beneficial therapeutic effects, such as anti-obesity effects, control of blood glucose, cardioprotection, and / or control of hypertension, via a location or mechanism of action different from that of the first component. In certain embodiments, the additional peptide family components preferentially target energy balance centers of the hypothalamus, such as ARC, PVN, VM, and LH.

[0429] As used herein, an anti-obesity agent that "acts on forebrain structures involved in the regulation of food intake and / or body weight" stimulates or inhibits the activity of specific regions in the forebrain, such as specific nuclei and / or neural circuits. This stimulation or inhibition of the forebrain results in a reduction in the availability of nutrients in the body. An anti-obesity agent that "acts on hindbrain structures involved in the regulation of food intake and / or body weight" stimulates or inhibits the activity of specific regions in the hindbrain, such as specific nuclei and / or neural circuits. This stimulation or inhibition of the hindbrain results in a reduction in the availability of nutrients in the body.

[0430] In another aspect, there is provided a method for reducing fat mass by increasing the metabolic rate in a subject, the method comprising administering a peptide of the 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 weight. In some embodiments, the fat mass is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% over the course of treatment. In one aspect, the fat-free mass of the subject does not decrease over the course of treatment. In another aspect, the fat-free mass of the subject is maintained or increased over the course of treatment. In another aspect, the subject is on a low-calorie diet or a restricted diet. A "low-calorie diet" means that the subject consumes fewer calories per day compared to the normal diet of the same subject. In one example, the subject is consuming at least 50 fewer calories per day. In other examples, the subject is consuming at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 fewer calories per day.

[0431] In another embodiment, methods of use for altering fat distribution, reducing fat mass, or both in a subject are provided. Thus, subjects for whom it is beneficial to alter body composition may also benefit from the methods of the invention. Altering body composition includes loss or maintenance of body fat, with minimization, maintenance, or gain of fat-free body mass, as is the purpose herein. In such situations, body weight may increase or, similarly, decrease. Thus, the subject may be a lean, overweight, or obese subject, as these terms are commonly used in the art. The methods provided may also include reducing fat in non-fat tissue while maintaining fat-free mass. Use of this method includes treating diseases such as non-alcoholic steatohepatitis (NASH) or lipodystrophy.

[0432] In another embodiment, a method for altering fat distribution in a subject is provided, the method comprising administering an anti-obesity peptide of the invention in an amount effective to alter fat distribution in the subject. In one aspect, the alteration results from an increase in the metabolism of visceral or ectopic fat or both in the subject. "Fat distribution" means the location of fat deposits in the body. Such locations of fat deposits include, for example, subcutaneous, visceral, and ectopic fat storage. "Subcutaneous fat" means the deposition of lipids just below the skin surface. The amount of subcutaneous fat in a subject can be measured using any method available for measuring subcutaneous fat. Methods for measuring subcutaneous fat are known in the art and include, for example, those described in U.S. Patent No. 6,530,886, which is incorporated herein by reference in its entirety. "Ectopic fat storage" means the deposition of lipids within and around the tissues and organs that make up fat-free mass (e.g., skeletal muscle, heart, liver, pancreas, kidney, blood vessels). Generally, ectopic fat storage is the accumulation of lipids outside of the storage portion of classical adipose tissue in the body. "Visceral fat" means the deposition of fat as adipose tissue within the abdominal cavity. Visceral fat surrounds the organs necessary for life and can be metabolized by the liver to produce blood cholesterol. Visceral fat has been shown to be associated with an increased risk of conditions such as polycystic ovary syndrome, metabolic syndrome, and cardiovascular disease. In some embodiments, the method includes the metabolism of visceral or ectopic fat or both at a rate that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% greater than in the case of subcutaneous fat. In one aspect, the method results in a favorable fat distribution. In one embodiment, a favorable fat distribution is an increased proportion of subcutaneous fat relative to visceral fat, ectopic fat, or both. In one aspect, the method includes an increase in fat-free mass, such as an increase in fat-free mass as a result of an increase in muscle mass.

[0433] In another embodiment, a method for reducing the amount of subcutaneous fat in a subject, the method comprising administering an anti-obesity peptide of the present invention in an amount effective to reduce the amount of subcutaneous fat in the subject to a subject in need thereof. In one example, the amount of subcutaneous fat is reduced by at least about 5% in the subject. In other examples, the amount of subcutaneous fat is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50% compared to the subject prior to administration of the anti-obesity peptide of the present invention.

[0434] The methods described herein can be used to reduce the amount of visceral fat in a subject. In one example, visceral fat is reduced by at least about 5% in the subject. In other examples, visceral fat is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50% in the subject compared to the subject prior to administration of the anti-obesity peptide of the present invention. Visceral fat can be measured via any means available for determining the amount of visceral fat in a subject. Such methods include, for example, abdominal tomography by CT scanning or MRI. Other methods for determining visceral fat are described, for example, in U.S. Pat. Nos. 6,864,415, 6,850,797, and 6,487,445.

[0435] In another embodiment, a method for preventing the accumulation of ectopic fat in a subject or reducing the amount of ectopic fat, the method comprising administering to a subject in need thereof an anti-obesity peptide of the present invention in an amount effective to prevent the accumulation of ectopic fat in the subject or reduce the amount of ectopic fat. In one example, the amount of ectopic fat is reduced by about 5% in the subject as compared to the subject prior to administration of the anti-obesity peptide of the present invention. In other examples, the amount of ectopic fat is reduced by at least about 10%, or at least about 15%, 20%, 25%, 30%, 40%, or 50% in the subject. Alternatively, the amount of ectopic fat is reduced proportionally by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to subcutaneous fat in the subject. Ectopic fat can be measured in the subject using any available method for measuring ectopic fat.

[0436] In another embodiment, a method for providing a more favorable fat distribution in a subject, the method comprising administering to the subject a peptide of the invention that is effective as an anti-obesity agent in an amount effective to provide a favorable fat distribution is provided. In one embodiment, administration of the anti-obesity peptide of the invention reduces the amount of visceral fat, ectopic fat, or both, in the subject. In one embodiment, an anti-obesity peptide of the invention is administered in combination with at least one family module that acts on a forebrain structure involved in food intake, body weight regulation, or both, and at least one family module that acts on a hindbrain structure involved in food intake, body weight regulation, or both. In one embodiment, the method preferentially reduces the amount of visceral or ectopic fat, or a combination thereof, rather than subcutaneous fat. Such a method results in a higher ratio of subcutaneous fat to visceral or ectopic fat. Such an improved ratio can result in a reduced risk of developing cardiovascular disease, polycystic ovary syndrome, metabolic syndrome, or any combination thereof. In one embodiment, ectopic or visceral fat is metabolized at a rate 5% higher than subcutaneous fat. In other embodiments, ectopic or visceral fat is metabolized at a rate at least 10%, 15%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than subcutaneous fat.

[0437] Particularly interesting are the peptides of the invention for the treatment of obesity, body weight and fat composition related discussed herein, for example, containing GIP, exendin, amylin (i.e., dual amylin calcitonin receptor agonist), leptin, or GLP-1-GIP receptor co-agonist. In a further additional embodiment, the peptide of the invention contains at least two of these peptide family modules. The peptides of the invention can be administered either alone or in combination with a second anti-obesity agent such as an amylin, leptin, or exendin family peptide.

[0438] In yet another aspect, a method for administration of a therapeutically effective amount of a peptide of the invention effective as an anti-obesity agent in combination with a glucocorticosteroid is provided. Glucocorticosteroids have deleterious effects of increasing fat mass and decreasing fat-free mass. Thus, it is expected that the combination of anti-obesity agents can be used with glucocorticosteroids under conditions where the use of glucocorticosteroids is beneficial in order to counteract the deleterious effects of glucocorticosteroids.

[0439] As discussed herein, the peptides of the invention can be administered separately or in combination with one or more other agents to obtain additional benefits or to enhance the action of either the peptides of the invention or any of the other agents. For example, the anti-obesity peptides of the invention can be administered in combination with anti-obesity agents or cardioprotective agents or antihypertensive agents depending on the risk factors for the subject in need of treatment and the desired treatment outcome. Exemplary anti-obesity agents (either separately or in any mixed form; either before, concomitant with, or after) for administration with the peptides of the invention include, but are not limited to, serotonin (5HT) transport inhibitors such as paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline, and imipramine. Also included as anti-obesity agents are selective serotonin reuptake inhibitors, examples of which include, but are not limited to, dexfenfluramine, fluoxetine, sibutramine and combinations thereof, and those described in U.S. Patent No. 6,365,633 and PCT Published Patent Publications WO01 / 27060 and WO01 / 162341, which are hereby incorporated by reference in their entirety. Such 5HT transport inhibitors and serotonin reuptake inhibitors, analogs, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration have been described heretofore.

[0440] Also included as anti-obesity drugs are selective serotonin agonists and selective 5-HT2C receptor agonists, examples of which include, but are not limited to, U.S. Patent No. 3,914,250, which is hereby incorporated by reference in its entirety; and PCT application publications WO02 / 36596, WO02 / 48124, WO02 / 10169, WO01 / 66548, WO02 / 44152, WO02 / 51844, WO02 / 40456, and WO02 / 40457, etc. Suitable routes of administration for such selective serotonin agonists and 5-HT2C receptor agonists, compositions containing such agonists, and use in the provided methods are known in the art. See, for example, Halford et al. (2005) Curr. Drug Targets 6:201-213 and Weintraub et al. (1984) Arch. Intern. Med. 144:1143-1148.

[0441] Also included as anti-obesity agents are antagonists / inverse agonists of the central cannabinoid receptor (CB-1 receptor), examples of which include, but are not limited to, rimonabant (Sanofi Synthelabo), and SR-147778 (Sanofi Synthelabo), etc. CB-1 antagonists / inverse agonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration are described, for example, in U.S. Patent 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, which are hereby incorporated by reference in their entirety; European Patent Applications EP656354 and EP658546; and PCT Application Publications WO96 / 33159, WO98 / 33765, WO98 / 43636, WO98 / 43635, WO01 / 09120, WO98 / 31227, WO98 / 41519, WO98 / 37061, WO00 / 10967, WO00 / 10968, WO97 / 29079, WO99 / 02499, WO01 / 58869, and WO02 / 076949, which have been described heretofore.

[0442] Anti-obesity drugs also include melanocortin and melanocortin agonists. The receptor MC4R is thought 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 suitable for use in the provided methods, such as but not limited to MC4R agonists, etc., and compositions containing such agonists are known in the art. MCR agonists, MC4R agonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration are, for example, described in the following PCT patent applications WO03 / 007949, WO02 / 068388, WO02 / 068387, WO02 / 067869, WO03 / 040117, WO03 / 066587, WO03 / 068738, WO03 / 094918, and WO03 / 031410, which are hereby incorporated by reference in their entirety.

[0443] Anti-obesity drugs also include metabotropic glutamate subtype 5 receptor (mGluR5) antagonists, such as 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), etc., and the compounds 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).

[0444] Also included as anti-obesity agents are topiramate, phentermine, or a combination thereof, which are also designated as anti-convulsants and have also been shown to increase weight loss.

[0445] Also included as anti-obesity agents are 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. Patents Nos. 6,124,331, 6,214,853, and 6,340,683. NPY1 and NPY5 antagonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration have been described previously. NPY1 antagonists useful in the provided compositions and methods include U.S. Patent No. 6,001,836, which is hereby incorporated by reference in its entirety; and PCT Application Publications WO96 / 14307, WO01 / 23387, WO99 / 51600, WO01 / 85690, WO01 / 85098, WO01 / 85173, and WO01 / 89528. NPY5 antagonists useful in the compositions and methods of use provided herein include, but are not limited to, U.S. Patent Nos. 6,140,354, 6,191,160, 6,258,837, 6,313,298, 6,337,332, 6,329,395, 6,340,683, 6,326,375, and 6,335,345; European Patents EP01010691 and EP01044970; and compounds described in PCT Patent Publications WO97 / 19682, WO97 / 20820, WO97 / 20821, WO97 / 20822, WO97 / 20823, WO98 / 27063, WO00 / 64880, WO00 / 68197, WO00 / 69849, WO01 / 09120, WO01 / 85714, WO01 / 85730, WO01 / 07409, WO01 / 02379, WO01 / 02379, WO01 / 23388, WO01 / 23389, WO01 / 44201, WO01 / 62737, WO01 / 62738, WO01 / 09120, WO02 / 22592, WO0248152, WO02 / 49648, and WO01 / 14376.

[0446] Anti-obesity agents also include melanin-concentrating hormone (MCH) antagonists, examples of which include melanin-concentrating hormone 1 receptor (MCH1R) antagonists such as T-226296 (Takeda Pharmaceutical Company) and melanin-concentrating hormone 2 receptor (MCH2R) antagonists. MCH receptor antagonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0009815, 2005 / 0026915, 2004 / 0152742, 2004 / 0209865, which are hereby incorporated by reference in their entirety; PCT Publication Nos. WO01 / 82925, WO01 / 87834, WO02 / 06245, WO02 / 04433, and WO02 / 51809; and Japanese Patent Application JP13226269, which have been described heretofore.

[0447] Anti-obesity agents also include opioid antagonists, examples of which include, but are not limited to, those described in PCT Application WO00 / 21509. Specific opioid antagonists useful in the compositions and methods of use provided herein include, but are not limited to, nalmefene (REVEX®), 3-methoxynaltrexone, naloxone, naltrexone, bupropion, naloxonazine, beta-funaltrexamine, delta1 ([D-Ala2,Leu5,Cys6]-enkephalin (DALCE), naltrindole isothiocyanate, and nor-binaltorphamine or combinations thereof.

[0448] Anti-obesity agents also include orexin antagonists, examples of which include, but are not limited to, those described in PCT Patent Applications WO01 / 96302, WO01 / 68609, WO02 / 51232, and WO02 / 51838. Specific orexin antagonists useful in the compositions and methods of use provided herein include, but are not limited to, SB-334867-A.

[0449] Anti-obesity agents also include neuropeptide Y2 (NPY2) agonists. Examples include, but are not limited to, 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)], NPY24-36 (White-Smith et al. (1999) Neuropeptides 33:526-533), TASP-V (Malis et al. (1999) Br. J. Pharmacol. 126:989-996), cyclo-(28 / 32)-Ac-[Lys28-Glu32]-(25-36)-pNPY (Cabrele et al. (2000) J. Pept. Sci. 6:97-122), and other compounds. These may be either the peptide components of the present invention as discussed or administered separately. Anti-obesity agents provided also include neuropeptide Y4 (NPY4) agonists, such as, 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, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration have been previously described, for example, in U.S. Patent Publication No. 2002 / 0141985 and PCT Application Publication No. WO2005 / 077094.

[0450] Anti-obesity agents also include histamine 3 (H3) antagonists / inverse agonists, examples of which include, but are not limited to, those described in PCT application WO02 / 15905, O-[3-(1H-imidazol-4-yl)propanol]carbamate (Kiec-Kononowicz et al. (2000) Pharmazie 55:349-355), histamine H3-receptor antagonists containing piperidine (Lazewska et al. (2001) Pharmazie 56:927-932), benzophenone derivatives and related compounds (Sasse et al. (2001) Arch. Pharm. (Weinheim) 334:45-52), substituted N-phenylcarbamates (Reidemeister et al. (2000) Pharmazie 55:83-86), and proxifan derivatives (Sasse et al. (2000) J. Med. Chem. 43:3335-3343), etc. Specific H3 antagonists / inverse agonists useful 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, clobenpropit, iodofenpropit, imoproxifan, and GT2394 (Gliatech).

[0451] Anti-obesity agents also include cholecystokinin (CCK) and CCK agonists. Useful cholecystokinin-A (CCK-A) agonists 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, GI181771, JMV-180, A-71378, A-71623, and SR146131.

[0452] Also included as anti-obesity agents are ghrelin antagonists, such as those described in PCT Application Publications WO01 / 87335 and WO02 / 08250. Ghrelin antagonists are also known as GHS (growth hormone secretagogue receptor) antagonists. Therefore, the provided compositions and methods contemplate the use of GHS antagonists instead of ghrelin antagonists.

[0453] Anti-obesity agents include obestatin, as well as obestatin analogs and agonists. Obestatin is a peptide derived from preproghrelin, the same precursor as ghrelin. 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 ghrelin, obestatin is thought to act as an anorectic hormone by decreasing food intake, gastric emptying activity, jejunal motility, and weight gain. Useful obestatin peptides include, but are not limited to, those described in Zhang et al. (2005) Science 310:996-999.

[0454] Furthermore, amylinomimetics (e.g., amylin-calcitonin receptor coagonists, such as davalintide), GLP-1-GIP receptor coagonists, such as incretins (e.g., exendin-4, leptin, and PYY analogs) are anti-obesity agents that can also be administered as anti-obesity agents together with GIP containing the peptides of the present invention.

[0455] Accordingly, in certain embodiments, the peptides of the invention are useful for treating or preventing conditions or disorders that can be alleviated by reducing nutrient availability, and include administering to the subject a therapeutically or prophylactically effective amount of a compound of the invention. Such conditions and disorders include, but are not limited to, food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels and / or insulin secretion, positive inotropic action, reduction of catabolism, deceleration of gastric emptying, obesity, diabetes and diabetes-related conditions, control of liver fat-related inflammation and injury. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, and rare genetic disorders of obesity, such as Prader-Willi syndrome, critical care, insulin resistance and its disorders, such as polycystic ovary syndrome, obesity, all types 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). In addition, complications due to diabetes (e.g., neuropathy (e.g., treatment with the peptides of the invention including GIP peptides containing exendin family components), neuropathic pain (e.g., treatment with the peptides of the invention including GIP peptides of the invention containing amylin family hormone modules), retinopathy, nephropathy, insufficient pancreatic beta cell mass (e.g., based on the islet regeneration action of exendin-4 and GLP-1), metabolic syndrome, dumping syndrome, polycystic ovary syndrome, hypertension, dyslipidemia, cardiovascular disease, hyperlipidemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis, and osteoarthritis are included).

[0456] Non-limiting examples of cardiovascular conditions or diseases are hypertension, myocardial ischemia, and myocardial reperfusion. The compounds of the present invention may also be useful in treating or preventing stroke, cancer (e.g., endometrial, breast, prostate, and colon cancer), gallbladder disease, sleep apnea, reduced fertility, and other conditions associated with obesity such as osteoarthritis (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001). In other embodiments, the peptides of the present invention can be used to alter body composition for cosmetic reasons, enhance physical performance, or produce a leaner meat source. The peptides of the present invention are useful for changing body composition by reducing fat without causing a significant loss of muscle mass, thus resulting in a desirable loss of body fat while maintaining lean body mass. In one embodiment, such peptides of the present invention contain exendin, GLP1, amylin, and / or an sCT moiety.

[0457] In another aspect of the present invention, there is provided a method for treating or preventing obesity, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a peptide of the present invention. In an exemplary embodiment, the subject is an obese or overweight subject. "Obesity" is generally defined, for the purposes of this disclosure, as any subject having a body mass index greater than 30, but also includes subjects having a body mass index less than 30 who need or desire to lose weight and are included within the scope of "obesity". Subjects having 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 peptides of the present invention contain GIP, an amylin calcitonin receptor agonist such as davalintide, a GLP-1-GIP receptor co-agonist such as exendin, GLP1, amylin, and / or an sCT moiety.

[0458] In another aspect of the present invention, provided are methods for reducing food intake, reducing nutrient availability, causing weight loss, affecting body composition, and altering body energy content or increasing energy consumption, treating diabetes, and improving lipid profiles (such as reducing LDL cholesterol and triglyceride levels and / or altering HDL cholesterol levels), which methods comprise administering to a subject an effective amount of a peptide of the present invention. In an exemplary embodiment, the methods of the present invention are used to treat or prevent a condition or disorder that can be alleviated by reducing nutrient availability in a subject in need thereof, and comprise administering to the subject a therapeutically or prophylactically effective amount of a peptide of the present invention. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and all types of diabetes. In one embodiment, such peptides of the present invention contain a GIP, exendin, GLP1, amylin, and / or sCT moiety.

[0459] Additional assays useful in the present invention include assays capable of determining the effect of the peptides of the present invention, particularly those containing a GIP, exendin, GLP1, amylin, and / or sCT moiety, on body composition. An exemplary assay can be an assay that involves the use of a diet-induced obesity (DIO) mouse model for metabolic diseases. Prior to the treatment period, male C57BL / 6J mice can be fed a high-fat diet (#D12331, 58% of calories from fat; Research Diets, Inc.) starting at 4 weeks of age for 6 weeks. During the study, the mice can continue to eat their high-fat diet. Water can be provided as appropriate during the study. For the purpose of comparing the DIO group with metabolic parameters, one group of similarly aged lean mice can be fed a low-fat diet (#D12329, 11% of calories from fat).

[0460] To deliver either vehicle (50% dimethyl sulfoxide (DMSO) in water) or the peptides of the invention, a subcutaneous (SC) osmotic pump may be implanted into the scapular region of DIO mice. The pumps of the latter group can be set to deliver any amount of, for example, 1000 μg / kg / d of the peptides of the invention for 7 - 28 days. Body weight and food intake can be measured at regular intervals over the course of the study. Respiratory quotient (RQ, defined as CO2 production ÷ O2 consumption) and metabolic rate can be determined using indirect calorimetry of the whole animal (Oxymax, Columbus Instruments, Columbus, Ohio). Mice can be euthanized by overdose of isoflurane and the index of adiposity (adipose tissue mass of bilateral epididymal fat) can be measured. Additionally, before determining the mass of the epididymis, the body composition (lean mass, fat mass) of each mouse may be analyzed using a dual energy X-ray absorptiometry (DEXA) instrument according to the manufacturer's instructions (Lunar Piximus, GE Imaging System). In the method of the invention, a peptide of the invention having a potency greater than that of the component peptide hormone in the same assay in one of the assays described herein (preferably a food intake, gastric emptying, pancreatic secretion, weight loss or body composition assay), particularly one containing GIP, exendin, PPF, PYY, GLP1, amylin and / or an sCT moiety can be identified.

[0461] In addition to alleviating hypertension in a subject in need thereof as a result of reduced food intake, weight loss, or treatment of obesity, the peptides of the invention can be used to treat hypotension.

[0462] In another general aspect, the peptides of the invention can be used to inhibit the secretion of ghrelin. Accordingly, the peptides of the invention may utilize this mechanism to treat or prevent ghrelin-related disorders such as Prader-Willi syndrome, all types of diabetes and its complications, obesity, polyphagia, hyperlipidemia, or other disorders associated with overnutrition.

[0463] The peptides of the present invention may also be useful for enhancing, inducing, strengthening, or restoring glucose responsiveness in pancreatic islets or cells. These actions may be useful for treating or preventing conditions associated with metabolic disorders, such as those described above and those described in U.S. Patent Application US2004 / 0228846. Assays for determining such activities are known in the art. For example, in the published U.S. Patent Application US2004 / 0228846 (which is incorporated herein by reference in its entirety), assays are described regarding the isolation and culture of pancreatic islets, as well as the determination of the maturation of fetal pancreatic islets. In the examples of Patent Application US2004 / 0228846, enteric hormone peptides such as 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. A radioimmunoassay kit ([125I]-RIA kit) containing anti-insulin antibody was purchased from Linco, St. Louis.

[0464] The peptides of the present invention are useful for the prevention and treatment of kidney diseases such as hypertensive and diabetic nephropathy, as well as kidney diseases associated with insulin resistance and metabolic syndrome. The peptides of the present invention achieve these objectives, among other things, by improving or preventing the worsening of hypertension, endothelial function, renal function, and glomerulosclerosis. In one embodiment, the present invention provides a method for preventing or treating kidney diseases such as hypertensive and diabetic nephropathy, or kidney diseases associated with insulin resistance, the method comprising administering a compound of the present invention. The peptides of the present invention have a further use for improving endothelial function in patients having a reduced vasodilatory capacity, or a reduced glomerulosclerosis or any other glomerular flow rate. Such improvement in endothelial function serves both to reduce hypertension and to improve the function of the glomerular capillaries. In additional embodiments, the molecules of the present invention are useful for preventing the progression of kidney disease to ESRD, preventing the progression of proteinuria and / or glomerulosclerosis, slowing their progression, treating or alleviating them.

[0465] The peptides of the present invention are useful for reducing the risk of suffering from cardiac arrhythmia, preventing cardiac arrhythmia, or treating cardiac arrhythmia. The peptides of the present invention can provide an anti-arrhythmic effect in patients with cardiac ischemia, cardiac ischemia-reperfusion, and congestive heart failure. For example, the incretin GLP-1 has been found to reduce heart injury and enhance recovery in patients with these disorders. Incretins including GLP-1 are glucose-dependent insulinotropic hormones. GLP-1 and exendin effectively enhance peripheral glucose uptake without inducing dangerous hypoglycemia. It also strongly inhibits glucagon secretion independent of its insulinotropic action, thereby strongly reducing plasma free fatty acid (FFA) levels much more than can be achieved with insulin. High FFA levels have been suggested to be a major toxic mechanism during myocardial ischemia. In another embodiment, the peptides of the present invention that reliably reduce reperfusion- and ischemia-related injury and enhance patient recovery are useful for preventing and treating cardiac arrhythmia. In a further additional embodiment, treatment after acute cerebral infarction or hemorrhage, preferably intravenous administration, optimizes insulin secretion by suppressing glucagon, increases cerebral assimilation, enhances the effectiveness of insulin, and provides a means for maintaining normoglycemia or mild hypoglycemia without incurring the risk of severe hypoglycemia or other adverse side effects. In one embodiment, such peptides of the present invention contain a GIP, GLP1, or exendin moiety.

[0466] In a further additional embodiment, the peptides of the invention capable of reducing insulin resistance or increasing insulin sensitivity are useful for treating polycystic ovary syndrome (PCOS). Administering the peptides of the invention can reduce or prevent insulin resistance in a subject suffering from PCOS. In yet another embodiment, the peptides of the invention prevent the onset of type 2 diabetes in a subject suffering from PCOS. Further peptides of the invention can restore regular menstruation, ovulation, or fertility in a subject suffering from PCOS. In one embodiment, GIP containing such peptides of the invention also contains a GLP1 or exendin moiety for binding to and activating the GLP1 receptor.

[0467] By selecting the peptides of the present invention, a wide range of biological activities can be exhibited, some of which relate to their antisecretory and gut motility inhibitory properties. The peptides of the present invention can suppress gastrointestinal endocrinology by direct interaction with epithelial cells or perhaps by inhibiting the secretion of hormones or neurotransmitters that stimulate enteroendocrine. The antisecretory properties include inhibition of gastric and / or pancreatic secretion, gastritis, pancreatitis, Barrett's esophagus, and gastroesophageal reflux disease, in addition to heartburn, heartburn accompanied by reflux of gastric / intestinal contents into the chest, mouth or lungs, dysphagia, cough, intermittent stridor and inflammation of the vocal cords (conditions associated with GERD), esophageal erosion, esophageal ulcer, esophageal stricture, Barrett's metaplasia (replacement of normal esophageal epithelium with abnormal epithelium), Barrett's esophageal adenocarcinoma, and conditions associated therewith such as aspiration, and may be useful in the treatment or prevention of diseases and disorders including these. In another embodiment, the GIP peptides of the present invention containing amylin and / or sCT moieties may be useful in treating or preventing these diseases and conditions, such as Barrett's esophagus, gastroesophageal reflux disease (GERD) and conditions associated therewith disclosed herein. Such peptides of the present invention have antisecretory properties such as particularly effective inhibition of gastric acid, inhibition of bile acids, and inhibition of pancreatic enzymes. Furthermore, such peptides of the present invention may have a gastric protective effect, whereby the peptides of the present invention are particularly useful in the treatment or prevention of intestinal diseases and conditions, and the treatment or prevention of Barrett's esophagus and / or GERD and related or attendant conditions described herein.

[0468] In another general aspect, the peptides of the present invention are useful for reducing bone resorption, reducing plasma calcium, and / or inducing an analgesic effect, particularly for treating bone disorders such as osteopenia and osteoporosis. In yet other embodiments, the peptides of the present invention are useful for treating pain and painful neuropathies. In one embodiment, such peptides of the present invention contain an exendin, GLP1, amylin, and / or sCT moiety. For example, the GIP-sCT or GIP-amylin / sCT peptides of the present invention may have selectable properties of salmon calcitonin or amylin / sCT / amylin chimeras, such as properties that reduce bone loss and bone resorption, or pr...

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide has the amino acid sequence of formula (I): 【Chemical 1】 (wherein, 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(CH 2 ) n R 3 , or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) n R 3 ; X 6 is Ile or Leu; X 7 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 ; X 8 is Pro or Gly; X 9 is absent or 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 ; X 10 is absent or is Gly or Cys; X 11 is absent or is Ala, Cys, or Ser; X 12 is absent or is Pro or Asn; X 13 is absent or is Pro, Thr, or Leu; X 14 is absent or is Pro, Ala, or Ser; X 15 is absent or is Ser, Thr, or Cys; X 16 is Cys, Asn, Lys, 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 ; 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 Thr, Ala, 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 is absent or is His, Leu, Asn, or Arg; X 32 is absent or is Arg, His, Phe, or Ser; X 33 is absent or is Leu, Arg, Gly, or Ser; X 34 is absent or is Gln, Leu, Pro, Ser, or Asn; X 35 is absent or is 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, Pro, Gly, or Lys; 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(CH 2 ) n R 3 , or Lys(AEEAc-AEEAc-γ-Glu-C=O(CH 2 ) 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 is absent or is Tyr, Gly, or Ser; X 48 is absent or is Ser or His; X 49 is absent or is His or Thr; X 50 is absent or is Thr or Pro; X 51 does not exist or is Pro; wherein, R 1 and R 2 each independently is H or C 1～5 alkyl, R 3 is -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) A peptide or a pharmaceutically acceptable salt thereof comprising the same.

2. X 1 is Tyr; X 2 is Ala; X 3 is Glu; X 4 is Ala; X 5 is Gln; X 6 is Ile; X 7 is Gln; X 8 is Pro; X 9 does not exist; X 10 does not exist; X 11 does not exist; X 12 does not exist and is Asn; X 13 does not exist; X 14 does not exist; X 15 does not exist; X 16 is Cys; X 17 is Asn; X 18 is Thr; X 19 is Ala; X 20 is Thr; X 21 is Cys; X 22 is Val; X 23 is Leu; X 24 is Gly; X 25 is Arg; X 26 is Leu; X 27 is Ser; X 28 is Gln; X 29 is Glu; X 30 is Leu; X 31 does not exist; X 32 does not exist; X 33 does not exist; X 34 does not exist; X 35 does not exist; X 36 is Tyr; X 37 is Pro; X 38 is Arg; X 39 is Thr; X 40 is Asn; X 41 is Thr; X 42 is Gly; X 43 is Ser; X 44 is Asn; X 45 is Thr; X 46 is Tyr; X 47 does not exist; X 48 does not exist; X 49 does not exist; X 50 does not exist; X 51 The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, which does not exist.

3. X 1 is (d)Tyr, His, or ImPrA, X 2 is (d)Ala, Aib, Ser, or Gly; X 3 is Gln; X 4 is Aib; X 5 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 ; X 6 is Leu; 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 ; and X 8 is Gly; X 9 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 ; 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 is 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 ; 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 is Thr or Pro; X 43 is 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 ; and 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; X 51 The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, which is Pro.

4. X 5 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 , Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl, or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl)), the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

5. X 7 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 、 Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl, or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl)), the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

6. X 9 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 、 Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl, or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl)), the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

7. X 16 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 、Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl, or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl), the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

8. X 43 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 、 Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl, or Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl)), the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

9. The following sequence: 【Table 1A】 【Table 1B】 【Table 1C】 【Table 1D】 (wherein, K* 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 )(is) A peptide or a pharmaceutically acceptable salt thereof according to Claim 1 having the same.

10. The following sequence: 【Table 2A】 【Table 2B】 【Table 2C】 A peptide or a pharmaceutically acceptable salt thereof according to Claim 1 having the same.

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

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

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

14. The method according to Claim 12 for treating obesity.

15. The method according to Claim 12, wherein the metabolic disorder is diabetes.

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

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