Compositions Comprising Multiagonist Peptides and Methods of Making and Using Them - Patent application

Abstract

The present invention relates generally 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, plasma glucose and lipid levels, insulin levels, and / or insulin secretion, control of positive inotropy, reduction of catabolism, slowing of gastric emptying, and prevention of neurodegeneration, including, but not limited to, food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels, and / or insulin secretion, control of positive inotropy, reduction of catabolism, slowing of gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 222,747, filed July 16, 2021, and incorporated herein by reference in its entirety.

[0002] Description of sequence listing The sequence listing accompanying this application is provided in ST.26XML format and is incorporated herein by reference. The name of the text file containing the sequence listing is Pep2-001_01US_ST26.xml. The text file is approximately 46KB, was created on July 13, 2022, and has been submitted electronically.

[0003] FIELD OF THEINVENTION The present invention relates generally 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, plasma glucose and lipid levels, insulin levels, and / or insulin secretion, control of positive inotropy, reduction of catabolism, slowing of gastric emptying, and prevention of neurodegeneration, including, but not limited to, food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels, and / or insulin secretion, control of positive inotropy, reduction of catabolism, slowing of gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, rare genetic disorders of eating disorders and obesity, such as Prader-Willi syndrome, critical care, insulin resistance and disorders thereof, such as polycystic ovarian syndrome, obesity, diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes, and CNS disorders, such as prevention of neurodegeneration, depression, alcohol addition, Alzheimer's and Parkinson's disease, and non-alcoholic steatohepatitis (NASH). [Background technology]

[0004] 2. Background of the Invention Incretin peptides are hormones and peptidomimetics are glucoregulatory agents that cause an increase in the amount of insulin released when glucose levels are normal or especially when they are elevated. These incretin peptides have other effects beyond the initial incretin effect defined by insulin secretion. For example, they can also act to reduce glucagon production, increase satiety, delay gastric emptying, regulate white and brown adipose tissue, and induce weight loss. In addition, they can act to improve insulin sensitivity, and they can increase islet cell neogenesis, the formation of new islets.

[0005] The concept of the incretin effect developed from the observation that the insulin response to oral glucose exceeds that measured after intravenous administration of an equivalent amount of glucose. It was concluded that gut-derived factors, or incretins, affect postprandial insulin release. Nutrient influx into the stomach and proximal gastrointestinal tract causes the release of incretin hormones, which then stimulate insulin secretion. This insulinotropic effect, or ability to stimulate insulin secretion, can be quantified by comparing insulin or C-peptide responses to oral versus intravenous glucose loads. Thus, the incretin effect has been shown to be responsible for approximately 50%-70% of the insulin response to oral glucose in healthy individuals.

[0006] While many postprandial hormones have incretin-like activity, the major incretin peptides include glucose-dependent insulinotropic polypeptide, also known as gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and exendin peptides, which are non-endogenous incretin mimetics. Both GIP and GLP-1 belong to the glucagon peptide superfamily and therefore share amino acid sequence homology. GIP and GLP-1 are secreted by specialized cells in the gastrointestinal tract and have receptors localized on pancreatic islet cells and other tissues. Like the incretins, both are secreted from the intestine in response to nutrient ingestion, resulting in enhanced insulin secretion. The insulinotropic effects of GIP and GLP-1 are dependent on elevated ambient glucose. Both are rapidly inactivated by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV).

[0007] Native human GIP is a single 42 amino acid peptide synthesized and secreted by specialized enteroendocrine K cells. These cells are primarily concentrated in the duodenum and proximal jejunum, but they can also be found throughout the intestine. The primary stimulus for GIP secretion is ingestion of a meal high in carbohydrates and lipids. After ingestion, circulating plasma GIP levels increase 10-20 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.

[0008] The physiological effects of GIP have been elucidated using GIP infusion protocols as well as GIP receptor antagonists, GIP peptide antagonists, and GIP receptor knockout mice. Blockade of 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 demonstrate normal fasting glucose levels but mild glucose intolerance after oral glucose loading. Interestingly, they also show resistance to food-induced obesity after several months of high-fat feeding. Furthermore, in leptin-deficient ob / ob mice, the GIP receptor knockout genotype appears to reduce the degree of obesity that develops.

[0009] GIP also has many non-incretin effects. GIP activation of the GIP receptor has an antiemetic effect. Unlike other insulin secretagogues, GIP stimulates beta cell proliferation and cell survival in studies of the INS-1 pancreatic islet cell line. Furthermore, animal studies suggest 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, although human studies have not demonstrated any significant effect on glucagon secretion. Furthermore, unlike GLP-1, GIP appears to act by accelerating gastric emptying rather than inhibiting gastrointestinal motility.

[0010] Despite its potent glucoregulatory effect via glucose-dependent stimulation of insulin secretion, the insulinotropic effect of GIP is significantly reduced in diabetic subjects compared to normal individuals (16-18). As a result, the clinical use of GIP has not progressed significantly. Furthermore, there remains a need to develop additional diabetes treatment modalities and treatments for metabolic diseases, conditions, and disorders. Thus, the present invention encompasses the peptides of the present invention and methods of use thereof for treating or preventing metabolic diseases, conditions, and disorders.

[0011] GLP-1 is a potent insulin secretagogue secreted from the intestinal mucosa in response to food intake. The profound incretin effect of GLP-1 is underestimated by the fact that GLP-1R knockout mice are glucose intolerant. The incretin response to intravenously infused GLP-1 is preserved in diabetic subjects, whereas the incretin response to oral glucose in these subjects is impaired. GLP-1 administration by infusion or sc injection controls fasting glucose levels and maintains the glucose threshold for insulin secretion in diabetic patients. GLP-1 shows great potential as a therapeutic agent that can enhance insulin secretion in a physiological manner while avoiding the hypoglycemia associated with sulfonylurea drugs. Other important effects of GLP-1 on glucose homeostasis are the suppression of glucagon secretion and inhibition of gastric motility. GLP-1 inhibitory action on pancreatic alpha cell secretion of glucagon leads to a decrease in hepatic glucose production via reduced gluconeogenesis and glycogenolysis. This antiglucagon effect of GLP-1 is preserved in diabetic patients. The so-called ideal brake effect of GLP-1, where gastric motility and gastric secretion are inhibited, is influenced via efferent vagus receptors or by direct action on intestinal smooth muscle. Reduction of gastric acid secretion by GLP-1 contributes to a lag phase in nutrient availability, thus eliminating the need for a rapid insulin response. Taken together, the gastrointestinal effects of GLP-1 contribute significantly to the delay of glucose and fatty acid absorption, modulating insulin secretion and glucose homeostasis. GLP-1 has also been shown to induce beta cell specific genes, such as the GLUT-1 transporter, insulin (through the interaction of PDX-1 with the insulin gene promoter), and hexokinase-1. Thus, GLP-1 could potentially reverse the glucose intolerance normally associated with aging, as demonstrated by rodent experiments. Furthermore, GLP-1 could contribute to beta cell neogenesis and increase beta cell mass, in addition to restoring beta cell function during conditions of beta cell failure. The central effects of GLP-1 include increased satiety coupled with reduced food intake, which occurs via the action of hypothalamic GLP-1 receptors. These appetite suppressing effects were absent in GLP-1R knockout mice.Furthermore, GLP-1 receptor signaling has been implicated in the regulation of energy expenditure and thermogenesis in brown adipose tissue (Beiroa D, et al. Diabetes 63:3346-3358, 2014).

[0012] Another family of peptide hormones involved in metabolic diseases and disorders is the amylin family of peptide hormones, which includes amylin, calcitonin, calcitonin gene-related peptide, adrenomedullin, and intermedin (also known as "AFP-6"). Amylin is a 37 amino acid peptide hormone. It was isolated, purified, and chemically characterized as the major component of amyloid deposits in the pancreatic islets of human type 2 diabetic patients (Cooper et al., Proc. Natl. Acad. Sci., USA, 84:8628-8632 (1987)). The amylin molecule has two post-translational modifications: 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 Lys-Arg dibasic amino acid proteolytic cleavage signal preceding the N-terminal codon for Lys, and a Gly preceding the Lys-Arg proteolytic signal in the CLAIMS terminal position, a typical sequence for amidation by the protein amidating enzyme PAM (Cooper et al., Biochem. Biophys. Acta, 1014:247-258 (1989)).

[0013] Amylin is believed to regulate gastric emptying, inhibit glucagon secretion and food intake, and thus regulate the rate of glucose appearance in the circulation. It is believed to complement the action of insulin, regulating the rate of glucose disappearance from the circulation and its uptake by peripheral tissues. These actions are supported by experimental findings in rodents and humans, which indicate that amylin complements the effects of insulin in postprandial glucose control by at least three independent mechanisms that all affect the rate of glucose appearance. First, amylin inhibits postprandial glucagon secretion. Compared to healthy adults, patients with type 1 diabetes have no circulating amylin and patients with type 2 diabetes have reduced postprandial amylin concentrations. Furthermore, infusion of an amylin-specific monoclonal antibody that binds to circulating amylin also resulted in significantly elevated glucagon concentrations relative to controls. Both of these results point to a physiological role for endogenous amylin in regulating postprandial glucagon secretion. Second, amylin slows gastrointestinal motility and gastric emptying. Finally, it has been shown that intrahypothalamic injection of rat amylin reduces feeding in rats and alters neurotransmitter metabolism in the hypothalamus. In one study, food intake was significantly reduced up to 8 hours after intrahypothalamic injection of rat amylin and rat CGRP. In human studies, the amylin analog pramlintide has been shown to reduce body weight or weight gain. Amylin may be beneficial in the treatment of metabolic conditions, such as diabetes and obesity. Amylin may also be used to treat pain, bone disorders, gastritis, modulate lipids, especially triglycerides, or affect body composition, such as preferential loss of fat and sparing of lean tissue, and attenuate alcohol-related behavior.

[0014] Calcitonin was named for its secretion in response to induced hypercalcemia and its rapid calcium lowering effect. It is produced in and secreted from neuroendocrine cells in the thyroid gland, hereafter referred to as C-cells. Calcitonin's most studied action is its effect on osteoclasts. In vitro effects of calcitonin include rapid loss of ruffled borders and reduced release of lysosomal enzymes. Finally, inhibition of osteoclast function by calcitonin results in reduced bone resorption. However, neither the chronic reduction of serum calcitonin in cases of thyroidectomy nor the increased serum calcitonin found in medullary thyroid carcinoma appears to be associated with changes in serum calcium or bone mass. Thus, the primary function of calcitonin is most likely to be effective in acute hypercalcemia in emergency situations and / or to protect the skeleton during periods of "calcium stress", e.g., growth, pregnancy, and lactation. Calcitonin has effects on plasma calcium levels, inhibits osteoclast function, and is widely used to treat osteoporosis. Therapeutically, salmon calcitonin is believed to increase bone density and reduce fracture rates with minimal adverse effects. Over the past 25 years, calcitonin has also been used successfully as a treatment for Paget's disease of bone, a chronic skeletal disorder that can result in bone hypertrophy or deformation in one or more regions of the skeleton. Calcitonin is also widely used for its analgesic effect on bone pain observed in osteoporosis, although the mechanism for this effect is not clearly understood. Salmon calcitonin has effects beyond those related to bone metabolism. In human studies, salmon calcitonin inhibits gastric emptying and gastrin release after a meal, while inducing dose-dependent relaxation of the gallbladder in both the postprandial and fasting states. In mice and monkeys, salmon calcitonin acts in an anorectic manner, causing weight loss after a single dose. In chronic studies, oral preparations of salmon calcitonin also reduce food intake and body weight in rat models of obesity and diabetes.

[0015] Metabolic diseases and disorders take many forms, including obesity, diabetes, dyslipidemia, insulin resistance, fatty liver, steatohepatitis, cell apoptosis, and the like. Obesity and its associated disorders are common and extremely serious public health problems in the United States and around the world. Upper body obesity is the strongest known risk factor for type 2 diabetes mellitus, 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 ovarian syndrome, breast cancer, prostate cancer, and colon cancer, as well as an increased incidence of complications of general anesthesia (see, e.g., Kopelman, Nature 404:635-43 (2000)). Obesity reduces life expectancy and carries a serious risk of the above comorbidities as well as disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholelithiasis, orthopedic injuries, and thromboembolic disease (Rissanen et al., Br. Med. J. 301:835-7 (1990)). Obesity is also a risk factor for certain types of cancer and for a group of conditions called insulin resistance syndrome, or "syndrome X". Recent estimates for the medical costs of obesity and related disorders are $2 trillion worldwide. Although the etiology of obesity is thought to be multifactorial, the fundamental problem is that nutrient availability and energy expenditure do not reach an equilibrium state until there is excess adipose tissue in the obese subject. Obesity is currently a difficult to treat, chronic, and essentially intractable metabolic disorder. Therapeutic agents useful in reducing the weight of obese individuals could have significant beneficial effects on their health.

[0016] Diabetes mellitus is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria resulting from insufficient production or utilization of insulin. Diabetes severely impacts 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 characterized as type 2 diabetes. However, it is now widely recognized that there are many distinct diabetes-related diseases that develop long before a patient is diagnosed with overt diabetes. Effects from suboptimal control of glucose metabolism in diabetes also result in a wide range of associated lipid and cardiovascular disorders.

[0017] Dyslipidemia, or abnormal levels of lipoproteins in plasma, occurs frequently among diabetic patients. Dyslipidemia is typically characterized by elevated plasma triglycerides, low HDL (high density lipoprotein) cholesterol, normal to elevated levels of LDL (low density lipoprotein) cholesterol, and increased levels of small dense LDL (low density lipoprotein) particles in the blood. Dyslipidemia is one of the main contributing factors to the increased incidence of coronary events and deaths among diabetic subjects. Epidemiological studies support this by showing a several-fold increase in coronary deaths among diabetic subjects compared to nondiabetic subjects. Several lipoprotein abnormalities have been described among diabetic subjects.

[0018] Insulin resistance is the reduced ability of insulin to exert its biological action over a wide range of concentrations. In insulin resistance, the body secretes abnormally large amounts of insulin to compensate for this deficiency, and a state of impaired glucose tolerance develops. When the deficiency in insulin action cannot be compensated for, plasma glucose concentrations inevitably rise, resulting in the clinical state of diabetes. It is recognized that insulin resistance and relative hyperinsulinemia have a contributing role in obesity, hypertension, atherosclerosis, and type 2 diabetes. The association of insulin resistance with obesity, hypertension, and angina pectoris has been described as Syndrome X, a syndrome with insulin resistance as the common etiological link.

[0019] Attempts to treat the multiple abnormalities associated with diabetes have prompted the administration of several antidiabetic drugs to address those abnormalities in different patients. Examples of antidiabetic drugs are proteins such as insulin and insulin analogues, GLP-1 analogues, and small molecules such as insulin sensitizers, insulin secretagogues, alpha-glucosidase inhibitors, sodium glucose cotransporter-2 (SGLT-2) inhibitors, DPP-IV inhibitors, and appetite regulating compounds.

[0020] Nonalcoholic fatty liver disease (NAFLD) is a collective term that encompasses a range of conditions from simple deposition of fat in the liver to more progressive steatosis associated with hepatitis, fibrosis, cirrhosis, and in some cases hepatocellular carcinoma. NAFLD consists of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). NAFL is characterized by hepatic steatosis involving more than 5% of the parenchyma and no evidence of hepatocellular injury [2]. NASH, on the other hand, is defined by histological terms, which is a necroinflammatory process that leads to hepatocyte damage on a background of steatosis. The natural history of NAFLD remains incompletely characterized. Studies show that the incidence of NAFLD increases in concert with the rising rates of metabolic syndrome. Patients with type 2 diabetes present an extremely high risk of developing NASH and a 2- to 4-fold increased risk of fatty liver-related complications. Currently, there is no approved treatment for NASH.

[0021] There remains a need to develop therapeutic agents useful in the above metabolic diseases, conditions, and disorders. Thus, the present invention provides novel multi-agonist peptides and methods for their production and use. The peptides of the present invention are used in the metabolic diseases, conditions, and disorders described above and herein. Summary of the Invention [Means for solving the problem]

[0022] SUMMARY OF THE PRESENT APPLICATION The present invention provides, inter alia, novel multi-agonist peptides and methods for treating and preventing obesity and related disorders, including metabolic and liver disorders.

[0023] The present invention generally relates to novel peptides useful as drugs for the treatment and prevention of metabolic diseases and disorders that can be alleviated by, for example, controlling food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels, and / or insulin secretion, positive inotropic effects, reducing catabolism, slowing gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, rare genetic disorders of eating disorders and obesity, such as Prader-Willi syndrome, critical care, insulin resistance and its disorders, such as polycystic ovarian syndrome, obesity, any type of diabetes mellitus, including type 1, type 2, and gestational diabetes, alcoholism, and CNS disorders, such as prevention of neurodegeneration, depression, alcohol addiction, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH).

[0024] The present invention includes "peptides of the invention" as defined herein, which comprise two or more component peptides independently selected from at least two peptides comprising biologically active components including, for example, but not limited to, amylin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and calcitonin.

[0025] The present invention encompasses peptides that exhibit at least one hormonal activity. The peptides of the present invention include at least two bioactive peptides covalently linked together, optionally via a linker group, where at least one of the bioactive peptides exhibits at least one hormonal activity of the constituent peptides. The bioactive peptides are independently selected from the constituent peptides (e.g., GIP, amylin, GLP-1), fragments of the constituent peptides that exhibit at least one hormonal activity of the constituent peptides, analogs and derivatives of the constituent peptides that exhibit at least one hormonal activity of the constituent peptides, and fragments of the analogs and derivatives of the constituent peptides that exhibit at least one hormonal activity of the constituent peptides.

[0026] In one embodiment, the peptide exhibits at least one hormonal activity, the peptide containing at least a first peptide covalently linked to at least one additional peptide; the peptides are independently selected from the group consisting of component peptides (e.g., GIP, amylin, GLP-1); fragments of the component peptides that exhibit at least one hormonal activity of the component peptides; analogs and derivatives of the component peptides that exhibit at least one hormonal activity of the component peptides; and fragments of the component peptide analogs and derivatives that exhibit at least one hormonal activity of the component peptide hormone.

[0027] The present invention relates to a peptide of the present invention or a pharma- ceutically acceptable salt or solvate thereof, comprising the peptide of formula (I): X 1 -X 2 -EGTFX 3 -SDYSIX 4 -X 5 -DKIX 6 -QX 7 -X 8 -FVX 9 -WLX 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16-CNTATCX 17 -X 18 -X 19 -X 20 -LX 21 -X 22 -X 23 -LX 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 X 34 -PX 35 -TNX 36 -GX 37 -NTY-(NR 1 R 2 ) (I) (In the formula, X 1 is Tyr or (d) Tyr; X 2 is Ala, (d) Ala, or Aib; X 3 is Ile or Thr; X 4 is Ala, Aib, or Gln; X 5 is Met, Leu, or Val; X 6 is Ala or His; X 7 is Gln, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 8 is Ala or Asp; X 9 is Asn or Gln; X 10 is Leu, Val, or Ile; X 11 is Ala or Val; X 12 is Gly or Gln; X13 is Gly, Lys, Arg, Ser, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 14 is Pro, Gly, Lys, or Lys-γ-Glu-γ-Glu-C═O(CH2) n R 3 and; X 15 is Ser, Gly, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 16 is absent or is Gly or Ser; X 17 is Ala, Met or Val; X 18 is Thr or Leu; X 19 is Gln or Gly; X 20 is Arg, Lys, Gln, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 21 is Ala or Ser; X 22 is Asn or Gln; X 23 is Phe or Glu; X 24 is His or Val; X 25 is His or Arg; X 26 is Ser or Leu; X 27 is Ser or Gln; X 28 is Asn or Thr; X 29 is absent or is Asn or Gln; X 30 is absent or is Phe; X31 is absent or is Gly; X 32 is absent or is Pro; X 33 is absent or is Ile, Lys, or Lys-γ-Glu-γ-Glu-C═O(CH2) n R 3 and; X 34 is Leu or Tyr; X 35 is Pro, Lys, Arg, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 36 is Val or Thr; X 37 is Ser, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; R 1 and R 2 are each independently H or C 1~5 is alkyl, R 3 is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2; n is an integer from 12 to 20. The peptide of the present invention includes a peptide comprising or consisting of the amino acid sequence of

[0028] In other embodiments, the peptide component of the peptides of the invention may also include calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin, oxyntomodulin (OXM), and exendin-4.

[0029] In certain embodiments, the peptides of the invention comprise structural motifs of the constituent peptides that confer desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic characteristics to the peptide, and structural motifs of analogs or derivatives of the constituent peptides that confer desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic characteristics to the peptide of the invention. In still further embodiments, at least one of the peptides exhibits at least one hormonal activity of the constituent peptides.

[0030] In further embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptides is GIP, a fragment of GIP that exhibits at least one hormonal activity, an analog or derivative of GIP that exhibits at least one hormonal activity, or a fragment of an analog or derivative of GIP that exhibits at least one hormonal activity, and at least one other peptide.

[0031] In a further embodiment, the 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 analogue or derivative of GLP-1 exhibiting at least one hormonal activity, or a fragment of an analogue or derivative of GLP-1 exhibiting at least one hormonal activity, and at least one other peptide.

[0032] In a further embodiment, at least one component peptide that exhibits at least one hormonal activity of the component peptides is amylin, a fragment of amylin that exhibits at least one hormonal activity, an analog or derivative of amylin that exhibits at least one hormonal activity, or a fragment of an analog or derivative of amylin that exhibits at least one hormonal activity, and at least one other peptide.

[0033] In further alternative embodiments, the 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.

[0034] In further alternative embodiments, the 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.

[0035] In further alternative embodiments, 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.

[0036] In further alternative embodiments, the 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 analogue or derivative of cholecystokinin exhibiting at least one hormonal activity, or a fragment of an analogue or derivative of cholecystokinin exhibiting at least one hormonal activity, and at least one other peptide.

[0037] In further alternative embodiments, the 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.

[0038] In further alternative embodiments, at least one component peptide that exhibits at least one hormonal activity of the component peptides is oxyntomodulin, a fragment of oxyntomodulin that exhibits at least one hormonal activity, an analog or derivative of oxyntomodulin that exhibits at least one hormonal activity, or a fragment of an analog or derivative of oxyntomodulin that exhibits at least one hormonal activity, and at least one other peptide.

[0039] In further alternative embodiments, the at least one component peptide that exhibits at least one hormonal activity of the component peptides is a natriuretic peptide, a fragment of a natriuretic peptide that exhibits at least one hormonal activity, an analog or derivative of a natriuretic peptide that exhibits at least one hormonal activity, or a fragment of an analog or derivative of a natriuretic peptide that exhibits at least one hormonal activity, and at least one other peptide.

[0040] In further alternative embodiments, the 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.

[0041] In certain embodiments, the GIP component portion of the peptide of the invention is combined with an amylin receptor ligand; or a glucagon-like peptide 1 receptor ligand.

[0042] In other embodiments, the GIP portion of the peptide of the invention is 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; an oxytocin 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 trabecular bone marrow growth factor (TBD) receptor ligand; a cytoplasmic endothelial cell line (CTL ... Transforming growth factor (TGF) receptor ligands; laminin receptor ligands; vasoactive intestinal peptide (VIP) receptor ligands; fibroblast growth factor (FGF) receptor ligands; nerve growth factor (NGF) receptor ligands; islet neogenesis associated protein (INGAP) receptor ligands; activin-A receptor ligands; vascular endothelial growth factor (VEGF) receptor ligands; erythropoietin (EPO) receptor ligands; pituitary adenylate cyclase-activating polypeptide (PACAP) receptor ligands; granulocyte colony-stimulating factor (G-CSF) receptor ligands; granulocyte-macrophage colony-stimulating factor (GM-CSF); platelet-derived growth factor (PDGF) receptor ligands, cannabinoid CB1 receptor antagonists, and secretin receptor ligands.

[0043] In another embodiment, the GIP peptide component included in the peptide of the present invention includes N-terminal GIP or novel GIP analog fragments in combination with C-terminal peptides or fragments thereof having glucose lowering activity (e.g., antidiabetic drugs, amylin) or the ability to inhibit or reduce gastric emptying. Such GIP peptides of the present invention include N-terminal GIP fragments or novel GIP analog or derivative fragments in combination with amylin, gastrin, PYY, secretin, CCK, GRP, neuromedin, urocortin, C-terminus, calcitonin or salmon calcitonin, natriuretic peptides (e.g., ANP, BNP, CNP, urodilatin) or analogs thereof (e.g., amylin-sCT-amylin chimera), derivatives or fragments thereof.

[0044] In other embodiments, the GIP peptide component contained within the peptide of the present invention includes a C-terminal GIP or novel GIP analog fragment in combination with an N-terminal polypeptide or fragment thereof having glucose lowering activity (e.g., antidiabetic drugs, amylin) or the ability to inhibit or reduce gastric emptying. In such embodiments, the chimeric peptide may include a C-terminal GIP, novel GIP analog, or 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.

[0045] In one embodiment, the peptide of the present invention comprises a heterologous C-terminal tail or terminal extension to the GIP portion. In one embodiment of the peptide of the present invention, with other GIP peptides described herein, the GIP portion can be native GIP, an active fragment thereof, or an analog or derivative thereof. In another aspect, the GIP component of the peptide of the present invention comprises at least one modification, substitution, deletion, or addition that provides one or more improved properties, such as increased resistance to proteolytic digestion (and thus extended half-life), fatty acyl derivatization to reduce renal clearance. In one embodiment, the tail comprises a Trp-cage motif sequence. In another embodiment, the GIP analog peptide portion comprises unnatural amino acids, such as D amino acids, such as those that inhibit or reduce the rate of proteolysis by DPP-IV.

[0046] The present invention also encompasses the use of the peptides of the present invention for the treatment and prevention of metabolic and liver diseases and disorders, particularly those that can be alleviated by controlling food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels, and / or insulin secretion, positive inotropic effect, reduced catabolism, slowing gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, rare genetic disorders of eating disorders and obesity, e.g., Prader-Willi syndrome, critical care, insulin resistance and disorders thereof, e.g., polycystic ovarian syndrome, obesity, diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes, and CNS disorders, e.g., prevention of neurodegeneration, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH), as well as complications from diabetes, e.g., neuropathy (e.g., treated with a peptide of the invention comprising a GIP peptide containing an exendin family component), neuropathic pain (e.g., treated with a peptide of the invention comprising a GIP peptide containing an amylin family hormone module), retinopathy, nephropathy, conditions of insufficient pancreatic beta cell mass (e.g., based on the islet neogenesis action of exendin-4 and GLP-1).

[0047] Thus, there is provided a method of treating or preventing such conditions, comprising administering a therapeutically or prophylactically effective amount of a peptide of the invention. In one embodiment, the peptide of the invention comprises GIP or an analog or derivative thereof, including the novel GIP analogs of the invention, or a GIP peptide of the invention, including one or more additional peptides or peptide fragments, to a subject in need thereof.

[0048] In one embodiment, the peptide of the present invention can be provided as a monotherapy. In another embodiment for treating obesity, diabetes, or conditions associated with weight gain or elevated glucose levels, the peptide of the present invention can be administered in adjunctive therapy with food intake reducing or glucose lowering agents (e.g., antidiabetic drugs) or agents or methods that inhibit or reduce gastric emptying. Examples of such agents are provided herein. For example, in one embodiment, a method of adjunctive treatment for reducing the weight or blood glucose level of a subject, for example, a subject with obesity, type 1, type 2, or gestational diabetes mellitus, is provided, comprising administering to the subject a therapeutically effective amount of the peptide of the present invention, for example, the agonist is a peptide of the present invention, comprising adjunctive therapy with GIP or a novel GIP analog of the present invention, or an effective amount of GIP-GLP-1-amylin.

[0049] The peptides of the invention may also be useful to enhance, induce, improve or restore glucose responsiveness in pancreatic islets or cells, either alone or in combination with glucose lowering agents (e.g., antidiabetic agents) or agents or methods that inhibit or reduce gastric emptying. These actions may also be used to treat or prevent conditions associated with metabolic disorders, such as those mentioned above.

[0050] In another embodiment, a method for treating or preventing obesity is provided, comprising administering a therapeutically or prophylactically effective amount of a peptide of the present invention to a subject in need thereof. In one embodiment, the peptide of the present invention comprises GIP or an analog or derivative thereof, including a novel GIP analog of the present invention, or a GIP-hybrid of the present invention, e.g., a GIP-GLP-1-amylin hybrid.

[0051] In another embodiment, the subject is an obese or overweight subject. While "obese" is generally defined as a body mass index greater than 30, for the purposes of this disclosure, any subject with a body mass index less than 30 and who needs or desires to lose weight is included in the scope of "obese". Subjects who are insulin resistant, glucose intolerant, or have any form of diabetes mellitus (e.g., type 1, 2, or gestational diabetes) may benefit from this method. The peptides of the present invention may also be useful in the treatment or prevention of other conditions associated with obesity, including stroke, cancer (e.g., endometrial, breast, prostate, and colon cancer), gallbladder disease, sleep apnea, subfertility, and osteoarthritis (see Lyznicki et al, Am.Fam.Phys.63:2185,2001). Where the condition is associated with elevated glucose or hyperglycemia, the methods include administering a therapeutically or prophylactically effective amount of a peptide of the invention alone or in combination with a glucose-lowering agent (e.g., an antidiabetic agent) or an agent or method that inhibits or reduces gastric emptying.

[0052] In yet another aspect, the peptide of the present invention, particularly the GIP hybrid of the present invention, can also be used in a method for reducing food intake, reducing appetite, inducing satiety, reducing nutrient availability, reducing caloric efficiency, causing weight loss, affecting body composition, modifying total body energy content or energy expenditure, improving lipid profile (including reducing LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels), comprising administering to a subject an effective amount of the peptide of the present invention, for example the GIP hybrid of the peptide of the present invention.In one embodiment, the method of the present invention comprises administering to a subject a therapeutically or prophylactically effective amount of the peptide of the present invention to treat or prevent a condition or disorder that can be alleviated by reducing nutrient availability in a subject in need of such treatment or prevention.

[0053] Conditions and disorders include, but are not limited to, metabolic diseases and disorders that may be alleviated by food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels, and / or insulin secretion, positive inotropic control, reduced catabolism, slowing gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, rare genetic disorders of eating disorders and obesity, e.g., Prader-Willi syndrome, critical care, insulin resistance and disorders thereof, e.g., polycystic ovarian syndrome, obesity, diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes, and CNS disorders, e.g., prevention of neurodegeneration, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH), diabetic complications (neuropathy (e.g., based on the neurotrophic action of exendin-4), neuropathic pain (e.g., based on the amylin action), retinopathy, nephropathy, conditions of insufficient pancreatic beta cell mass (e.g., based on the islet neogenesis action of exendin-4 and GLP-1). When the condition is associated with elevated glucose or hyperglycemia, the method includes administering a therapeutically or prophylactically effective amount of the peptide of the invention alone or in combination with a glucose lowering agent (e.g., an antidiabetic agent) or an agent or method that inhibits or reduces gastric emptying.

[0054] In addition to improving hypertension in subjects in need of such improvement as a result of reducing food intake, weight loss, and / or treating obesity, the peptides of the present invention may be used to treat or prevent hypertension and its associated conditions.

[0055] The peptides of the present invention, e.g., GIP components, include peptides, e.g., half-life extended GIP hybrids, optionally further comprising a heterologous C-terminal tail (e.g., DPP-IV cleavage resistant, e.g., D-Tyr1, D-Ala2, N-acetyl or N-pyroglutamyl analogs). The peptides of the present invention include other hormone modules known to provide beneficial cardiovascular effects, and are useful for treating cardiovascular disease and related conditions. As disclosed herein, the peptides of the present invention can increase myocardial contractility (dp / dt), reduce blood pressure (e.g., by acute vasodilation), reduce systolic blood pressure, reduce diastolic blood pressure, and provide direct beneficial effects on cardiac cells. The peptides of the present invention also improve cardiac function through metabolic effects, e.g., weight reduction, glucose lowering, insulin secretion, beta cell proliferation. However, by also providing direct effects on the cardiovascular system, the peptides of the present invention are surprisingly even more beneficial.

[0056] The peptides of the present invention are also useful in the treatment or prevention of any gastrointestinal disorder associated with excess gastric secretion, excess intestinal electrolyte and water secretion, and reduced absorption, such as infectious (e.g., viral or bacterial) diarrhea, inflammatory diarrhea, short bowel syndrome, or diarrhea that typically occurs after surgery, such as ileostomy (see, e.g., Harrison's principles of Internal Medicine, McGraw Hill Inc., New York, 12th ed.). Examples of infectious diarrhea include, but are not limited to, acute viral diarrhea, acute bacterial diarrhea (e.g., Salmonella, Campylobacter, and Clostridium) or diarrhea caused by 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 spue, chronic pancreatitis, Crohn's disease, diarrhea, and irritable bowel syndrome. GIP and the GIP compounds of the present invention can be used to treat or prevent emergency or life-threatening situations involving, for example, postoperative gastrointestinal disorders or gastrointestinal disorders due to cholera. Additionally, the compounds can be used to treat intestinal dysfunction in patients with acquired immune deficiency syndrome (AIDS), especially during cachexia.

[0057] The peptides of the present invention are also useful for inhibiting intestinal fluid and electrolyte secretion and enhancing nutrient transport, as well as increasing cell proliferation in the gastrointestinal tract, for example, regulating lipolysis in adipose tissue and regulating blood flow in mammals. The peptides of the present invention are useful for treating or preventing the above conditions due to their gastrointestinal protective activity (e.g., inhibiting gastric 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 mucosa function, and other conditions of the gastrointestinal tract, including conditions that may result from exposure to cytotoxic agents, radiation, toxicity, infection, and / or injury. Furthermore, the peptides of the present invention can be combined with anesthetics, anti-inflammatory agents, growth hormones, heparin, or any other therapy that may be used to treat inflammatory bowel disease or other conditions listed above.

[0058] 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 conditions associated therewith. Such conditions may include, but are not limited to, heartburn, heartburn with reflux of stomach / intestinal contents into the mouth or lungs, dysphagia, coughing, intermittent wheezing and vocal cord inflammation (conditions associated with GERD), esophageal erosion, esophageal ulcer, esophageal stenosis, Barrett's metaplasia (replacement of normal esophageal epithelium with abnormal epithelium), and aspiration. In some embodiments, the peptides of the present invention may have antisecretory properties, such as inhibition of gastric acid, inhibition of bile acid, and inhibition of pancreatic enzymes. In addition, the peptides of the present invention may also have gastroprotective effects. Thus, the peptides of the present invention may be particularly useful in treating or preventing gastritis, pancreatitis, Barrett's esophagus, and / or GERD and related or associated conditions.

[0059] 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 pharma- ceutically acceptable salt thereof, together with pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers useful in the delivery of the peptide of the present invention.

[0060] These and other aspects of the present invention will be more clearly understood with reference to the following embodiments and detailed description.

[0061] The present invention encompasses the peptides of the present invention or pharma- ceutically acceptable salts or solvates thereof, and in one embodiment, the peptides of the present invention have the formula (I): X 1 -X 2 -EGTFX 3 -SDYSIX 4 -X 5 -DKIX 6 -QX 7 -X 8 -FVX 9 -WLX 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -CNTATCX 17 -X 18 -X 19 -X 20 -LX 21 -X 22 -X 23 -LX 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 X 34 -PX 35 -TNX 36 -GX 37 -NTY-(NR 1 R 2 ) (I) (In the formula, X 1 is Tyr or (d) Tyr; X 2 is Ala, (d) Ala, or Aib; X 3 is Ile or Thr; X4 is Ala, Aib, or Gln; X 5 is Met, Leu, or Val; X 6 is Ala or His; X 7 is Gln, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 8 is Ala or Asp; X 9 is Asn or Gln; X 10 is Leu or Ile; X 11 is Ala or Val; X 12 is Gly or Gln; X 13 is Gly, Lys, Arg, Ser, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 14 is Pro, Gly, Lys, or Lys-γ-Glu-γ-Glu-C═O(CH2) n R 3 and; X 15 is Ser, Gly, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 16 is absent or is Gly or Ser; X 17 is Ala, Met or Val; X 18 is Thr or Leu; X 19 is Gln or Gly; X 20 is Arg, Lys, or Gln; X 21 is Ala or Ser; X22 is Asn or Gln; X 23 is Phe or Glu; X 24 is His or Val; X 25 is His or Arg; X 26 is Ser or Leu; X 27 is Ser or Gln; X 28 is Asn or Thr; X 29 is absent or is Asn or Gln; X 30 is absent or is Phe; X 31 is absent or is Gly; X 32 is absent or is Pro; X 33 is absent or is Ile, Lys, or Lys-γ-Glu-γ-Glu-C═O(CH2) n R 3 and; X 34 is Leu or Tyr; X 35 is Pro, Lys Arg, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 36 is Val or Thr; X 37 is Ser, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and R 1 and R 2 are each independently H or C 1~5 is alkyl, R 3is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2; n is an integer from 12 to 20. The amino acid sequence of the present invention may comprise, consist essentially of, or consist of the amino acid sequence of

[0062] In certain embodiments, any of the peptides of the invention described herein comprises one or more half-life extending moieties and / or one or more linker moieties conjugated to the peptide. In certain embodiments, the half-life extending moieties are conjugated to the peptide component of the peptide of the invention via one or more linker moieties.

[0063] In certain embodiments, any of the peptides of the invention described herein further comprises a conjugate chemical substituent. In certain embodiments, the conjugate chemical substituent is a lipophilic substituent or polymer moiety, such as Ac, Palm, gamma-Glu-Palm, isoGlu-Palm, PEG2-Ac, PEG4-isoGlu-Palm, (PEG)5-Palm, succinic acid, glutaric acid, pyroglutaric acid, benzoic acid, IVA, octanoic acid, 1,4 diaminobutane, isobutyl, Alexa488, Alexa647, or biotin. In certain embodiments, the conjugate chemical substituent is a polyethylene glycol having a molecular weight of 400 Da to 40,000 Da.

[0064] In a related aspect, the invention provides a peptide of the invention comprising at least two peptide components linked via one or more linker moieties, each peptide subunit comprising 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 a 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 linked by a linker moiety. In certain embodiments, the C-terminus of each peptide monomer subunit is linked by a linker moiety. In other embodiments, the linker links 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.

[0065] In further related embodiments, the invention includes sequences encoding the peptides of the invention or one or more peptide subunits of the peptides of the invention.The invention also includes vectors comprising the polynucleotides.

[0066] In another aspect, the invention includes a pharmaceutical composition comprising a peptide of the invention and a pharma- ceutically 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 gastrointestinal system of a subject.

[0067] In some embodiments, the pharmaceutical composition is provided to a subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, nebulization, sublingual, buccal, parenteral, rectal, ocular, inhalation, vaginal or topical routes of administration. In some embodiments, the pharmaceutical composition is provided to a subject topically, parenterally, intravenously, subcutaneously, intraperitoneally or intravenously to treat obesity. [Brief description of the drawings]

[0068] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] Figure 1A illustrates the reduction in blood glucose after a single administration of an exemplary peptide of the invention. Blood glucose was measured after 30 minutes (Figure 1A). [Figure 1B] Figure 1B illustrates the reduction in blood glucose after a single administration of an exemplary peptide of the invention. Blood glucose was measured after 120 minutes (Figure 1B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description of the Invention definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0070] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0071] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of the stated component or group of components, but not the exclusion of any other component or group of components.

[0072] The singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0073] The term "including" is used to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0074] 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.

[0075] The terms "patient," "subject," and "individual" are used interchangeably and may refer to either human or non-human animals. These terms include mammals, such as humans, primates, livestock animals (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats).

[0076] The term "peptide" as used herein broadly refers to a sequence of two or more amino acids that are connected together by peptide bonds. It should be understood that the term does not refer to a polymer of amino acids of a specific length, and is not intended to imply or distinguish whether the polypeptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The term peptide also includes cyclic peptides.

[0077] As used herein, the terms "sequence identity", "percent identity", "percent homology", or including, for example, "a sequence that is 50% identical to", refer to the degree to which sequences are identical on a nucleotide basis or on an amino acid basis over a comparison window. Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0078] Calculation of sequence similarity or sequence identity (these terms are used interchangeably herein) between sequences 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., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In one embodiment, 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 as the corresponding position in the second sequence, the molecules are identical at that position.

[0079] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap.

[0080] The comparison of sequences and the determination of percent identity between two sequences 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 in 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 a 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 with 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) as incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4.

[0081] The peptide sequences described herein can be used, for example, as a "query sequence" to perform searches against public databases 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). BLAST nucleotide searches 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. BLAST protein searches 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 used as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0082] The term "conservative substitution" as used herein indicates that one or more amino acids are replaced by another biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. See, for example, the table below. In some embodiments of the invention, one or more Met residues are replaced with norleucine (Nle), which is a biological equivalent for Met but, in contrast to Met, is not easily oxidized. Another example of a conservative substitution with a residue not normally found in endogenous mammalian peptides and proteins is, for example, the conservative substitution of Arg or Lys with ornithine, canavanine, aminoethylcysteine, or another basic amino acid. In some embodiments, one or more cysteines of the peptide analogs of the invention can be replaced with another residue, such as serine. For more information on 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.

[0083] [Table 1]

[0084] 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.

[0085] [Table 2]

[0086] 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., a-amino acids), non-naturally occurring amino acids, modified amino acids, and unnatural amino acids. This includes both D- and L-amino acids. Natural amino acids include those found in nature, such as the 23 amino acids that combine into peptide chains to form the building blocks of countless proteins. These are primarily L stereoisomers, although some D-amino acids occur in bacterial envelopes and some antibiotics. The 20 "standard" natural amino acids are listed in the table above. The "nonstandard" natural amino acids are pyrrolysine (found in methanogens and other eukaryotes), selenocysteine ​​(present in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Unnatural" or "non-natural" amino acids are non-proteinogenic amino acids (i.e., not naturally encoded or found in the genetic code), either naturally occurring or chemically synthesized. Over 140 unnatural amino acids are known, with thousands of additional combinations possible. Examples of "unnatural" amino acids include the β-amino acids (β 3 and β 2), homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N-methyl amino acids. Non-natural or unnatural amino acids also include modified amino acids. "Modified" amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups or chemical moiety that does not naturally occur on the amino acid. According to certain embodiments, the peptide comprises an intramolecular bond between two amino acid residues present in the peptide. It is understood that the amino acid residues that form the bond are somewhat altered when they are bound to each other compared to when they are not bound to each other. Reference to a particular amino acid is meant to encompass both the amino acid in its unbound and bound state. For example, the amino acid residue homoserine (hSer) or homoserine (Cl) in its unbound form may take the form of 2-aminobutyric acid (Abu) when involved in an intramolecular bond according to the present invention.

[0087] Typically, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions proposed by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set forth in "Nomenclature of α-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975). The names and abbreviations of amino acids and aminoacyl residues used in this specification and the appended claims are made clear to the reader only to the extent that they differ from such indications. Some abbreviations useful in describing the present invention are defined below in Table 1 below.

[0088] [Table 3]

[0089] [Table 4]

[0090] [Table 5]

[0091] [Table 6]

[0092] [Table 7]

[0093] [Table 8]

[0094] The one-letter and three-letter abbreviations for the naturally occurring amino acids described in this invention are defined below in Table 2 below.

[0095] [Table 9]

[0096] Throughout this specification, naturally occurring amino acids, when not referred to by their full names (e.g., alanine, arginine, etc.), 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 indicated, the three-letter and one-letter abbreviations of amino acids refer to the L-isomer form of the amino acid. As used herein, the term "L-amino acid" refers to the "L" isomeric form of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomeric form of a peptide (e.g., Dasp, (d)Asp or D-Asp; Dphe, (d)Phe or D-Phe). The D-isomer form of an amino acid residue can be substituted for any L-amino acid residue, so long as the desired function is retained by the peptide. D-amino acids, when referred to using one-letter abbreviations, can be conventionally indicated by lowercase letters.

[0097] In the case of rare or non-naturally occurring amino acids, unless referred to by their full name (e.g., sarcosine, ornithine, etc.), the frequently used three-letter or four-letter code for the residue is used, including Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutanoic 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), and Ida (iminodiacetic acid).

[0098] As will be appreciated by those of skill in the art, peptide sequences disclosed herein are presented 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. Some sequences disclosed herein incorporate a "Hy-" moiety at the amino terminus (N-terminus) of the sequence and either an "-OH" or "-NH2" moiety at the carboxy terminus (C-terminus) of the sequence. In such cases, unless otherwise indicated, the "Hy-" moiety at the N-terminus of the sequence indicates 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 indicates a hydroxyl or amino group, respectively, corresponding to the presence of an amide (CONH2) group at the C-terminus. In each of the sequences of the invention, the C-terminal "-OH" moiety can be substituted for the C-terminal "-NH2" moiety, and vice versa.

[0099] Those skilled in the art recognize 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, for example, one or more hydrogens may be removed or replaced by the bond. Thus, as used herein, reference to an amino acid or modified amino acid present in a peptide of the present invention is meant to include the form of such amino acid or modified amino acid present in the peptide both before and after forming an intramolecular bond.

[0100] 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. Additionally, the term "Ac" as used herein refers to acetyl protection via acylation of the C- or N-terminus of a polypeptide. In certain peptides presented herein, NH2 located at the C-terminus of the peptide represents an amino group.

[0101] The term "carboxy" as used herein refers to -CO2H.

[0102] As used herein, the term "equivalent substitution" refers to any amino acid or other analog moiety that has similar chemical and / or structural properties to a specified amino acid. In certain embodiments, an equivalent substitution is a conservative substitution or analog of the specified amino acid.

[0103] The term "cyclization" as used herein refers to the attachment of one portion of a polypeptide molecule to another portion of the peptide molecule to form a closed ring, for example, by forming a disulfide bridge or a thioether bond.

[0104] The terms "component" or "subunit" are used interchangeably and refer to one of a pair of polypeptide monomers that are linked to form a peptide of the invention.

[0105] The term "linker moiety" as used herein refers broadly to a chemical structure that can bond or connect two peptide subunits together.

[0106] The term "pharmaceutically acceptable salt" as used herein refers to a salt or zwitterionic form of a peptide or peptide of the invention that is water or oil soluble or dispersible, suitable for the treatment of disease without undue toxicity, irritation, and allergic response; commensurate with a reasonable benefit / risk ratio and effective for the intended use. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting the 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, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Also, the amino group in the compound of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids, such as oxalic acid, maleic acid, succinic acid, and citric acid. The pharmaceutically acceptable salts can be suitably selected, for example, from among acid addition salts and base salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts.Examples of basic salts include those 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 a substituted ammonium ion, such as N(R1)(R2)(R3)(R4)+, where each of R1, R2, R3, and R4 is independently typically hydrogen, optionally substituted C. 1~6 Alkyl or optionally substituted C 2~6 The salts are selected from the group consisting of ions of the type C 1~6 Examples of alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups. 2~6 Examples of alkenyl groups include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharma-ceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and 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 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 which form non-toxic salts. Representative examples include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts.

[0107] The term "N (alpha) methylation" as used herein describes the methylation of the alpha amine of an amino acid, also commonly referred to as N-methylation.

[0108] As used herein, the term "sym methylation" or "Arg-Me-sym" describes the symmetric methylation of the two nitrogens of the guanidine group of arginine. Additionally, the term "asym methylation" or "Arg-Me-asym" describes the methylation of a single nitrogen of the guanidine group of arginine.

[0109] The term "acylating organic compound" as used herein refers to various compounds having a carboxylic acid functional group that are used to acylate the N-terminus of an amino acid or peptide component, e.g., a monomeric subunit, prior to forming a C-terminal dimer. Non-limiting examples of acylated organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, 3,3,3-trifluoropropeonic acid, 3-fluoromethylbutyric acid, and tetrahydro-2H-pyran-4-carboxylic acid.

[0110] The term "alkyl" includes straight or branched chain, acyclic or cyclic saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

[0111] "Halo" or "halogen" refers to a bromo (Br), chloro (Cl), fluoro (F), or iodo (I) substituent.

[0112] The term "haloalkyl" includes alkyl structures in which at least one hydrogen has been replaced with a halogen atom. In some embodiments in which two or more hydrogen atoms have been replaced with halogen atoms, the halogen atoms are all the same as each other. In other embodiments in which two or more hydrogen atoms have been replaced with halogen atoms, the halogen atoms are not all the same as each other.

[0113] An "alkoxy" group refers to a (alkyl)O- group, where alkyl is as defined herein.

[0114] An "aryloxy" group refers to an (aryl)O- group, where aryl is as defined herein.

[0115] "Aminocarbonyl" or "carboxamide" refers to the group -CONH2.

[0116] "2-aminoethoxy" refers to the group -OCH2CH2-NH2.

[0117] "2-Acetylaminoethoxy" refers to the group -OCH2CH2-N(H)C(O)Me.

[0118] The term "mammal" refers to any mammalian species, e.g., humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals.

[0119] As used herein, a "therapeutically effective amount" of a peptide of the invention is meant to describe an amount sufficient to treat or prevent any of the diseases and disorders described herein (e.g., to treat obesity). In certain embodiments, a therapeutically effective amount achieves a desired benefit / risk ratio applicable to any medical treatment.

[0120] An "analog" of an amino acid, e.g., a "Phe analog" or a "Tyr analog," means an analog of the referenced amino acid. A variety of amino acid analogs, including Phe and Tyr analogs, are known in the art and are available. In some 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 some embodiments, the substitutions are in the side chain of the amino acid. In some embodiments, a Phe analog has the structure Phe(R 2 ) and R 2 is Hy, OH, CH, COH, CONH, CONHOCHCHNH, t-Bu, OCHCHNH, phenoxy, OCH, Oallyl, Br, Cl, F, NH, N, or guanadino. 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-guanadino), Phe(4-tBu), Phe(4-CN), Phe(4-Br), Phe(4-OBzl), Phe(4-NH2), BhPhe(4-F), Phe(4-F), Phe(3,5Di Examples of Tyr analogs include, but are not limited to, hTyr, N-Me-Tyr, Tyr(3-tBu), Tyr(4-N3), and βhTyr.

[0121] The present invention generally relates to the peptides of the present invention for treating or preventing various metabolic and hepatic diseases and disorders.In some embodiments, the present invention demonstrates a new paradigm for the treatment of obesity, metabolic and hepatic disorders, and other diseases and disorders by administering the peptides of the present invention.It is predicted that the administration of the peptides of the present invention maximizes drug levels in diseased tissues while limiting drug concentrations in circulation, thereby providing effective, safe and sustained delivery for the lifelong treatment of obesity and metabolic and hepatic diseases and disorders.

[0122] In some embodiments, the peptides of the present invention include various peptides, or peptide hetero- or homomonomer subunits, which optionally form a cyclized structure via disulfide or other bonds. In some embodiments, the disulfide or other bonds are intramolecular bonds. Cyclization of the peptides has been shown to increase the potency and selectivity of the peptides of the present invention. In some embodiments, the peptides of the present invention may include one or more intermolecular bonds that connect two peptide subunits within the peptide.

[0123] In certain embodiments, the peptides of the invention reduce the presence of a disease or disorder by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a negative control peptide.

[0124] In some embodiments, 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. (The skilled artisan will recognize that serum or plasma from other species (e.g., rat, mouse, etc.) can be used.) Samples are typically taken at various time points 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.

[0125] In some embodiments, the peptides of the invention exhibit improved solubility or reduced aggregation characteristics compared to a control peptide. Solubility may 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, acetic acid (pH 5.0 and other pHs known in the art) and testing for aggregation or solubility using standard techniques. These include, but are not limited to, visual precipitation, dynamic light scattering, circular dichroism and fluorescent dyes to measure surface hydrophobicity and detect, for example, aggregation or fibrillation. In some embodiments, improved solubility means that the peptide is more soluble in a given liquid than a control peptide. In some embodiments, reduced aggregation means that the peptide aggregates less in a given liquid than a control peptide under given conditions.

[0126] In some embodiments, the peptides of the present invention are less degraded (i.e., more degradatively stable) than a control peptide over a period of time, e.g., about 10% or more less, about 20% or more less, about 30% or more less, about 40% or more less, or about 50% or more less degraded. In some embodiments, the degradative stability is determined via any suitable method known in the art. In some embodiments, the degradative is enzymatic degradative. For example, in some embodiments, the peptides of the present invention have reduced susceptibility to degradative by trypsin, chymotrypsin, or elastase. In some embodiments, suitable methods known in the art for determining degradative stability include those described in Hawe et al., J Pharm Sci, VOL.101, No.3,2012, p895-913, which is incorporated herein in its entirety. Such methods are used in some embodiments to select potent peptide sequences with improved shelf life.

[0127] Various peptides of the present invention may be constructed from only natural amino acids. Alternatively, peptides of the present invention may include unnatural amino acids, including but not limited to modified amino acids. In some embodiments, modified amino acids include natural amino acids that have been chemically modified to include a group, groups, or chemical moieties that do not naturally occur on the amino acid. Peptides of the present invention may further include one or more D-amino acids. Furthermore, peptides of the present invention may include amino acid analogs.

[0128] In some embodiments, the peptides of the invention comprise one or more modified or non-naturally occurring amino acids. In some embodiments of the invention, the peptides of the invention comprise one or more non-naturally occurring amino acids as shown in Table 1. In some embodiments, the peptides of the invention comprise any of those described herein, including, but not limited to, any of those comprising the amino acid sequences or peptide structures shown herein.

[0129] The present invention also includes any of the peptides of the invention described herein, either in free or salt form. Thus, any embodiment of the peptides of the invention (and related methods of use thereof) described herein includes pharma- ceutically acceptable salts of the peptides of the invention.

[0130] The present invention also includes variants of any of the peptides of the invention described herein, including but not limited to any of those comprising a sequence shown in any one of the tables herein, in which one or more L-amino acid residues are replaced with the D-isomer form of that amino acid residue, e.g., L-Ala is replaced with D-Ala.

[0131] The peptides of the present invention described herein include isotopically labeled peptides. In certain embodiments, the present disclosure provides peptides of the present invention that are identical to any of the peptides with various formulas and structures presented herein or cited therein, except for the fact that one or more atoms are replaced by atoms having atomic masses or mass numbers different from those usually 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, respectively, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, for example, are labeled with radioactive isotopes, e.g., 3 H and 14 C is useful in drug and / or substrate tissue distribution assays. Additionally, isotopes such as deuterium, i.e. 2 Substitution with H may result in certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.

[0132] The present invention also includes any of the peptide components described herein linked to a linker moiety, including any of the specific linker moieties described herein. In certain embodiments, the linker is linked to an N-terminal or C-terminal amino acid, while in other embodiments, the linker is linked to an internal amino acid. In certain embodiments, the linker is linked to two internal amino acids, for example, to an internal amino acid in each of two monomer subunits. In some embodiments, the peptides of the present invention include one or more linker moieties.

[0133] The present invention also includes peptides, including peptides having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the peptide sequences of the peptides of the present invention described herein. In certain embodiments, the peptides of the present invention comprise a core peptide sequence and one or more N-terminal and / or C-terminal modifications (e.g., Ac and NH2) and / or one or more conjugate linker moieties and / or half-life extending moieties. As used herein, a core peptide sequence is the amino acid sequence of the peptide component absent such modifications and conjugates.

[0134] In certain embodiments, the peptides of the invention comprise, consist essentially of, or consist of 10-90 amino acid residues, 15-80 amino acid residues, 20-75 amino acid residues, 25-70 amino acid residues, 30-65 amino acid residues, 35-60 amino acid residues, 40-55 amino acid residues, 45-50 amino acid residues, and optionally one or more additional non-amino acid moieties, such as conjugate chemical moieties, such as PEG or linker moieties.

[0135] In certain embodiments, peptides of the invention (or constituent monomeric subunits thereof), including but not limited to those of any embodiment of Formulas I-V, 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, e.g., 35-80 amino acids. In certain embodiments, peptides of the invention (or constituent monomeric subunits thereof) 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, constituent monomeric subunits of peptides of the invention comprise or consist of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues. In certain embodiments, a monomeric subunit of a peptide of the invention comprises or consists of 10-75 amino acid residues and, optionally, one or more additional non-amino acid moieties, e.g., conjugated chemical moieties, e.g., PEG or lapidated amino acid residues.

[0136] In certain embodiments of the invention, the amino acid sequence of the peptide of the invention is not present in an antibody or is not present in the V H There is also V in the area. L It doesn't exist in the area either.

[0137] Peptides of the Invention 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.

[0138] In one embodiment, the present invention provides a peptide of the present invention or a pharma- ceutically acceptable salt or solvate thereof, comprising the formula (I): X 1 -X 2 -EGTFX 3 -SDYSIX 4 -X 5 -DKIX 6 -QX 7 -X8 -FVX 9 -WLX 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -CNTATCX 17 -X 18 -X 19 -X 20 -LX 21 -X 22 -X 23 -LX 24 -X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 X 34 -PX 35 -TNX 36 -GX 37 -NTY-(NR 1 R 2 ) (I) (In the formula, X 1 is Tyr or (d) Tyr; X 2 is Ala, (d) Ala, or Aib; X 3 is Ile or Thr; X 4 is Ala, Aib, or Gln; X 5 is Met, Leu, or Val; X 6 is Ala or His; X 7 is Gln, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 8 is Ala or Asp; X 9 is Asn or Gln; X 10 is Leu, Val, or Ile; X 11 is Ala or Val; X 12 is Gly or Gln; X 13 is Gly, Lys, Arg, Ser, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 14 is Pro, Gly, Lys, or Lys-γ-Glu-γ-Glu-C═O(CH2) n R 3 and; X 15 is Ser, Gly, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 16 is absent or is Gly or Ser; X 17 is Ala, Met or Val; X 18 is Thr or Leu; X 19 is Gln or Gly; X 20 is Arg, Lys, Gln, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 21 is Ala or Ser; X 22 is Asn or Gln; X 23 is Phe or Glu; X 24 is His or Val; X 25 is His or Arg; X 26 is Ser or Leu; X 27 is Ser or Gln; X28 is Asn or Thr; X 29 is absent or is Asn or Gln; X 30 is absent or is Phe; X 31 is absent or is Gly; X 32 is absent or is Pro; X 33 is absent or is Ile, Lys, or Lys-γ-Glu-γ-Glu-C═O(CH2) n R 3 and; X 34 is Leu or Tyr; X 35 is Pro, Lys Arg, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 36 is Val or Thr; X 37 is Ser, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and R 1 and R 2 are each independently H or C 1~5 is alkyl, R 3 is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2; n is an integer from 12 to 20. The present invention also includes peptides of the present invention that comprise, consist essentially of, or consist of the amino acid sequence of

[0139] In one embodiment, R 3 is -COH.

[0140] In one embodiment, R 3is -CO2CH3.

[0141] In one embodiment, R 3 is -CO2NH2.

[0142] In one embodiment, R 3 is -CO2NHCH3.

[0143] In one embodiment, R 3 is -CO2N(CH3)2.

[0144] In one embodiment, R 3 is -CH3.

[0145] In one embodiment, R 3 is -NH2.

[0146] In one embodiment, n is 12.

[0147] In one embodiment, n is 13.

[0148] In one embodiment, n is 14.

[0149] In one embodiment, n is 15.

[0150] In one embodiment, n is 16.

[0151] In one embodiment, n is 17.

[0152] In one embodiment, n is 18.

[0153] In one embodiment, n is 19.

[0154] In one embodiment, n is 20.

[0155] In one embodiment, the peptide of the present invention comprises X 1 is Tyr; X 2 is Aib; X 3 is Ile; X 4 is Ala; X 5 is Leu; X 6 is His; X 7 is Lys or Lys-γ-Glu-γ-Glu-C=O(CH2) n CH3; X 8 is Asp; X 9 is Asn; X 10 is Leu; X 11 is Ala; X 12 is Gln; X 13 is Lys or Lys-γ-Glu-γ-Glu-C=O(CH2) n CH3; X 14 is absent or is Pro; X 15 is absent or is Ser; X 16 is absent or Ser or X 14 , X 15 , and X 16 are both linking groups; X 17 is Ala; X 18 is Thr; X 19 is Gln; X 20 is Arg; X 21 is Ala; X 22 is Asn; X 23 is Phe; X 24 is Val; X 25 is His; X 26 is Ser; X 27 is Ser; X 28 is Asn; X 29 is absent or is Asn; X 30 is absent or is Phe; X 31 is absent or is Gly; X 32 is absent or is Pro; X 33 is absent or is Ile; X 34 is Leu; X 35 is a Pro; X 36 is Val; X 37 is Ser A peptide of Formula I or a pharma- ceutically acceptable salt thereof.

[0156] In another embodiment, the present invention provides a peptide of the present invention or a pharma- ceutically acceptable salt or solvate thereof, comprising the formula (II): X 1 -X 2 -EGTFX 3 -SDYSIAX 4 -DKIX 5 -QX 6 -X 7 -FVX 8 -WLLAQX 9 -X 10 -X 11 -X 12 -CNTATCATQRLANFLVHSSNN-FGPX 13 -LPPTNVGX14 -NTY(NR 1 R 2 ) (II) (In the formula, X 1 is Tyr or (d)-Tyr; X 2 is Ala, (d) Ala, or Aib; X 3 is Thr or Ile; X 4 is Met, Leu, or Val; X 5 is His or Ala; X 6 is Gln, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 7 is Asp or Ala; X 8 is Asn or Gln; X 9 is Gln, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 10 is any naturally occurring amino acid; X 11 is any naturally occurring amino acid; X 12 is any naturally occurring amino acid; X 13 is Ile, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 14 is Ser, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and R 1 and R 2 are each independently H or C 1~5 is alkyl, R 3is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2; n is an integer from 12 to 20. The peptide of the present invention includes a peptide comprising or consisting of the amino acid sequence of

[0157] In one embodiment, the peptide of the present invention comprises X 1 is Tyr; X 2 is Ala; X 3 is Leu; X 4 is Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 5 is Asn; X 6 is Gln; X 7 is any amino acid; X 8 is any amino acid; X 9 is any amino acid; X 10 is Ile; X 11 is Ser A peptide of Formula II or a pharma- ceutically acceptable salt thereof.

[0158] In certain embodiments, naturally occurring amino acids can be substituted with non-naturally occurring amino acids and the peptides of the invention retain biological activity.

[0159] In another embodiment, the present invention provides a peptide of the present invention or a pharma- ceutically acceptable salt or solvate thereof, comprising the formula (III): X 1 -X 2 -EGTFX 3 -SDYSIX 4 -LDKIAQX 5-AFVQWLX 6 -AGGPSCNTATCVLGRLSQELH-RLQTYPRTNTGX 7 -NTY(NR 1 R 2 ) (III) (In the formula, X 1 is (d) Tyr or Tyr; X 2 is Ala, (d) Ala or Aib; X 3 is Ile or Thr; X 4 is Ala or Aib; X 5 is Gln, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and; X 6 is Leu or Ile; X 7 is Ser, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 and R 1 and R 2 are each independently H or C 1~5 is alkyl, R 3 is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2; n is an integer from 12 to 20. The peptide of the present invention includes a peptide comprising or consisting of the amino acid sequence of

[0160] In one embodiment, the peptide of the present invention comprises X 1 is (d) Tyr; X 2 is Aib; X 3is Thr; X 4 is Aib; X 5 is Gln; X 6 is Ile; X 7 is Lys-γ-Glu-γ-Glu-C=O(CH2) n R 3 is A peptide of formula IV or a pharma- ceutically acceptable salt thereof.

[0161] In certain embodiments, exemplary peptide components of the peptides of the invention include fragments 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, wherein the fragment exhibits at least one hormonal activity of the component peptide.

[0162] Still other exemplary peptide components of the peptides of the invention include analog or derivative fragments of a component peptide hormone 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, wherein the fragment exhibits at least one hormonal activity of the component peptide hormone. Again, as described in more detail herein and known in the art, analogs may include one or more insertions, deletions, or substitutions in the amino acid sequence of the component peptide hormone, and derivatives may include one or more chemical modifications of the amino acid residues of the analog or component peptide hormone.

[0163] Certain exemplary fragments that exhibit at least one hormonal activity include the following:

[0164] 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)

[0165] GLP-1: GLP-1(7-37), GLP-1(7-36), GLP-1(7-35)

[0166] GIP: GIP(1-14), GIP(1-28), GIP(1-30) or longer, GIP(1-39) or longer

[0167] Exendin: Exendin-4(1-27), Exendin-4(1-28), Exendin-4(1-29), Exendin-4(1-30) or longer.

[0168] The peptides of the present invention may be amidated, but may optionally be in acid form, unless otherwise specified within the context of the present invention.Furthermore, the above exemplary peptides may be combined with any of the analogs or derivatives discussed herein or known in the art.For example, exemplary analog fragments may include 5Ala, 14Leu, 25Phe-Exendin-4(1-28), 14Leu, 25Phe-Exendin-4(1-27), 5Ala, 14Leu, 25Phe-Exendin-4(1-28), 14Leu, 25Phe-Exendin-4(1-27), or any other combination of the disclosed fragments, analogs, and derivatives.

[0169] Still other exemplary peptide moieties include structural motifs of constituent peptide hormones (including analogs and derivatives thereof) that confer desirable 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 moieties of the peptides of the invention include:

[0170] 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).

[0171] GLP-1 and 2: GLP-1(29-37); GLP-1(30-37); GLP-2(24-31), GLP-2(25-31).

[0172] 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).

[0173] 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)

[0174] In some embodiments, the combination of GIP analogs and derivatives used together with the peptide moieties described herein is contemplated.For example, the last six amino acid residues of the amylin family peptide hormone analogs and derivatives known in the art and / or described above are also contemplated as exemplary peptide moieties.For example, as further discussed herein, the exemplary Trp-cage sequence, peptidic enhancer Ex-4 short tail, or its analog, is added to the C-terminus of any GIP analog, and in further embodiments, the peptidic enhancer is linked using a linker.

[0175] In one embodiment, a peptide of the invention comprises a GIP portion that exhibits at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity over the entire length of the GIP portion to native GIP(1-30), native GIP(1-26), native GIP(1-14), native GIP(1-39), native GIP(19-30), native GIP(19-26), native GIP(19-39), native GIP(19-42) or native GIP(1-42).

[0176] Thus, in some embodiments, the peptides of the present invention may include a trp-cage motif. In some embodiments, the peptides of the present invention include an N-terminal GIP or novel GIP analog fragment in combination with a C-terminal polypeptide or fragment thereof having weight-lowering or glucose-lowering activity (e.g., antidiabetic drugs, exendins) or the ability to inhibit or reduce gastric emptying. In some embodiments, the peptides of the present invention include an N-terminal GIP or novel GIP analog fragment in combination with a C-terminal exendin, GLP1, amylin, CCK, gastrin, secretin, GRP, neuromedin, urocortin, calcitonin, or salmon calcitonin, or fragments thereof. In other embodiments, the peptides of the present invention include a C-terminal GIP or novel GIP analog fragment in combination with an N-terminal polypeptide or fragment thereof having weight-lowering or glucose-lowering activity (e.g., antidiabetic drugs, exendins) or the ability to inhibit or reduce gastric emptying. In certain embodiments, the peptides of the invention comprise C-terminal GIP, a novel GIP analog (when the Trp-cage forming sequence is present), or a fragment thereof, in combination with N-terminal exendin, GLP1, amylin, CCK, gastrin, secretin, GRP, neuromedin, urocortin, calcitonin, or salmon calcitonin, or a fragment thereof.

[0177] In other embodiments, the peptide component of the peptide of the present invention is a gastrin / CCK receptor ligand; amylin receptor ligand; calcitonin receptor ligand; CGRP receptor ligand, EGF receptor ligand; glucagon-like peptide 1 receptor ligand; glucagon-like peptide 2 receptor ligand; gastric inhibitory polypeptide (GIP) receptor ligand; keratinocyte growth factor (KGF) receptor 1 ligand; dipeptidyl peptidase IV inhibitor; REG protein receptor ligand; growth hormone receptor ligand; prolactin (PRL) receptor ligand; insulin-like growth factor (IGF) receptor ligand; PTH-related protein (PTHrP) receptor ligand; hepatocyte growth factor (HGF) receptor ligand; bone morphogenetic protein (BMP) receptor ligand. and secretin receptor ligands.

[0178] The peptides of the present invention preferably at least partially retain the biological activity of natural human GIP, for example, the peptides of the present invention are generally GIP agonists or antagonists. In one embodiment, the peptides of the present invention exhibit biological activity in the treatment and prevention of metabolic conditions and disorders. Furthermore, the novel GIP analogs of the peptides of the present invention may contain internal linker compounds, may contain chemical modifications at internal amino acid residues, or may be chemically modified at the N- 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 unnatural amino acid residues.

[0179] In an exemplary embodiment, the GIP portion of the peptide of the invention comprises a GIP N-terminal region that has been modified or substituted to provide greater DPP-IV resistance than native GIP.

[0180] In an exemplary embodiment, the peptides of the present invention comprise GIP or novel GIP analogs in combination with amylin family peptides, including amylin, adrenomedullin ("ADM"), calcitonin ("CT"), calcitonin gene-related peptide ("CGRP"), intermedin (also known as "AFP-6") and related peptides. Natural amylin family peptide hormones are known in the art, as are functional peptide analogs and derivatives. Although certain exemplary natural peptides, peptide analogs and derivatives are described herein, it should be recognized that any known amylin family peptide that exhibits hormonal activity known in the art may be used with the present invention. Any amylin analog or derivative known in the art may be used with the present invention.

[0181] The amylin family of peptide hormones, including amylin, calcitonin, calcitonin gene-related peptide, adrenomedullin, and intermedin (also known as "AFP-6"), are involved in metabolic diseases and disorders. In certain embodiments, the peptides of the present invention include one or more of the amylin family peptides as constituent peptides. Amylin is a 37 amino acid peptide hormone. It was isolated, purified, and chemically characterized as the major component of amyloid deposits in the pancreatic islets of human type 2 diabetic patients (Cooper et al., Proc. Natl. Acad. Sci., USA, 84:8628-8632 (1987)). The amylin molecule has two post-translational modifications: 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 Lys-Arg dibasic amino acid proteolytic cleavage signal before the N-terminal codon for Lys, and a Gly before the Lys-Arg proteolytic signal at the N-terminal position, a typical sequence for amidation by the protein amidating enzyme PAM (Cooper et al., Biochem. Biophys. Acta, 1014:247-258 (1989)). "Adrenomedullin" or "ADM" refers to the human peptide hormone and species variants thereof. More specifically, ADM is generated from a 185 amino acid preprohormone through successive enzymatic cleavage and amidation. This process terminates with the liberation of a 52 amino acid bioactive peptide. "Calcitonin" or "CT" refers to the human peptide hormone and species variants thereof, including salmon calcitonin ("sCT"). More specifically, CT is a 32 amino acid peptide that is cleaved from the larger prohormone. It contains a single disulfide bond that gives it a ring shape at the amino terminus. Alternative splicing of the calcitonin pre-mRNA can give rise to an mRNA that codes for calcitonin gene-related peptide; the peptide is thought to function in the nervous and vascular systems. The calcitonin receptor has been cloned and shown to be a member of the seven-transmembrane G protein-coupled receptor family."Calcitonin gene-related peptide" or "CGRP" refers to the human peptide hormone in any physiological form and species variants thereof. "Intermedin" or "AFP-6" refers to the human peptide hormone in any physiological form and species variants thereof.

[0182] In one embodiment, the peptide of the present invention comprises an amylin peptide as a constituent peptide. Amylin is believed to regulate gastric emptying, inhibit glucagon secretion and food intake, and thus regulate the rate of glucose appearance in the circulation. It is believed to complement the action of insulin, regulating the rate of glucose disappearance from the circulation and its uptake by peripheral tissues. These actions are supported by experimental findings in rodents and humans, which show that amylin complements the effect of insulin in postprandial glucose control by at least three independent mechanisms, all of which affect the rate of glucose appearance. First, amylin suppresses postprandial glucagon secretion. Compared to healthy adults, patients with type 1 diabetes have no circulating amylin, and patients with type 2 diabetes have reduced postprandial amylin concentrations. Furthermore, infusion of an amylin-specific monoclonal antibody that binds to circulating amylin also resulted in significantly elevated glucagon concentrations relative to controls. Both of these results point to a physiological role for endogenous amylin in regulating postprandial glucagon secretion. Secondly, amylin slows gastrointestinal motility and gastric emptying. Finally, it has been shown that intrahypothalamic injection of rat amylin reduces feeding in rats and alters neurotransmitter metabolism in the hypothalamus. In one study, food intake was significantly reduced up to 8 hours after intrahypothalamic injection of rat amylin and rat CGRP. In human studies, the amylin analog pramlintide has been shown to reduce body weight or weight gain. Amylin may be beneficial in the treatment of metabolic conditions, such as diabetes and obesity. Amylin may also be used to treat pain, bone disorders, gastritis, modulate lipids, especially triglycerides, or affect body composition, such as preferential fat loss and sparing of lean tissue.

[0183] In one embodiment, the peptide of the present invention comprises calcitonin as a constituent peptide. The hormone calcitonin (CT) was named for its secretion in response to induced hypercalcemia and its rapid calcium lowering effect. It is produced in and secreted from neuroendocrine cells in the thyroid gland, hereafter referred to as C-cells. The most studied action of CT(1-32) is its effect on osteoclasts. In vitro effects of CT include rapid loss of ruffled borders and reduced release of lysosomal enzymes. Finally, inhibition of osteoclast function by CT results in reduced bone resorption. However, neither the chronic reduction of serum CT in cases of thyroidectomy nor the increased serum CT found in medullary thyroid carcinoma appears to be associated with changes in serum calcium or bone mass. Thus, the primary function of CT(1-32) is most likely to be effective in acute hypercalcemia in emergency situations and / or to protect the skeleton during periods of "calcium stress", e.g., growth, pregnancy, and lactation. (Reviewed in Becker, JCEM, 89(4):1512-1525(2004) and Sexton, Current Medicinal Chemistry 6:1067-1093(1999)). This is consistent with recent data from calcitonin gene knockout mice, which remove both calcitonin and CGRP-I peptides, revealing that the mice had normal levels of basal calcium-related values ​​but increased calcemic responses (Kurihara H, et al., Hypertens Res. 2003 February;26 Suppl:S105-8).

[0184] CT has effects on plasma calcium levels, inhibits osteoclast function, and is widely used to treat osteoporosis. Therapeutically, salmon CT (sCT) is believed to increase bone density and reduce fracture rates with minimal adverse effects. Over the past 25 years, CT has also been used successfully as a treatment for Paget's disease of bone, a chronic bone disorder that can result in enlargement or deformation of bone in one or more areas of the skeleton. CT is also widely used for its analgesic effect on bone pain observed in osteoporosis, although the mechanism for this effect is not clearly understood.

[0185] In human studies, salmon calcitonin inhibits gastric emptying and gastrin release after a meal, while inducing dose-dependent relaxation of the gallbladder in both postprandial and fasting states.In mice and monkeys, salmon calcitonin acts as anorectic and causes weight loss after a single dose.In chronic studies, oral preparations of salmon calcitonin also reduce food intake and body weight in obese and diabetic rat models.Therefore, having a calcitonin component in a therapeutic agent may be beneficial for weight loss.

[0186] In one embodiment, the peptide of the present invention comprises calcitonin gene-related peptide as a constituent peptide. Calcitonin gene-related peptide (CGRP) is a neuropeptide whose receptors are widely distributed throughout the body, including the nervous and cardiovascular systems. This peptide is believed to modulate sensory neurotransmission and is one of the most potent endogenous vasodilatory peptides ever discovered. Reported biological effects 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, increase in 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). An important role of CGRP is to control blood flow to various organs through its potent vasodilatory action, as evidenced by the reduction in mean arterial pressure after intravenous administration of α-CGRP. The vasodilatory action is also supported by the recent analysis of homozygous knockout CGRP mice, which demonstrated increased peripheral vascular resistance and higher blood pressure caused by increased peripheral sympathetic nerve activity (Kurihara H, et al., Targeted disruption of ADM and αCGRP genes reveals their distinct biological roles. Hypertens Res. 2003 February;26 Suppl:S105-8). Thus, CGRP is thought to induce vasodilatory, hypotensive, and increased heart rate effects, among other actions.

[0187] Long-term infusion of CGRP into patients with congestive heart failure shows sustained beneficial effects on hemodynamic function without adverse effects, suggesting its use in heart failure.Other indications for CGRP use include renal failure, acute and chronic coronary ischemia, treatment of cardiac arrhythmias, other peripheral vascular diseases such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension.Preeclampsia and preterm labor in pregnancy are also potentially treatable.(Wimalawansa,1997).Recent therapeutic uses include the use of CGRP antagonists for the treatment of migraine headaches.

[0188] In one embodiment, the peptide of the present invention comprises adrenomedullin as a component peptide. Adrenomedullin (ADM) is nearly ubiquitously expressed in many more tissues that contain the peptide than in tissues that do not. A published review of ADM, (Hinson, JP et al., Endocrine Reviews (2000) 21(2):138-167), details its effects on the cardiovascular system, cell growth, central nervous system, and endocrine system, with a range of biological actions including vasodilation, cell growth, regulation of hormone secretion, and natriuresis. Studies in rats, cats, sheep, and humans confirm that intravenous infusion of ADM produces potent and sustained hypotension comparable to that of CGRP. However, the hypotensive effect of ADM on mean arterial pressure in anesthetized rats is not inhibited by the CGRP antagonist CGRP8-37, suggesting that this effect is not mediated through the CGRP receptor. Acute or chronic administration of human ADM in anesthetized, conscious or hypertensive rats produces a significant decrease in total peripheral vascular resistance accompanied by a fall in blood pressure, with concomitant increases in heart rate, cardiac output and stroke volume.

[0189] ADM has been proposed as a key factor in embryonic development and differentiation, as well as an apoptotic survival factor for rat endothelial cells. This is supported by recent mouse ADM knockout studies, in which mice homozygous for loss of the ADM gene demonstrated defects in angiogenesis during embryonic development and subsequently died during pregnancy. ADM+ / -heterozygous mice were reported to have high blood pressure along with susceptibility to tissue damage (Kurihara H, et al., Hypertens Res. 2003 February; 26 Suppl: S105-8).

[0190] ADM affects endocrine organs such as the pituitary, adrenal, reproductive and pancreatic glands. This peptide is believed to have a role in inhibiting ACTH release from the pituitary. In the adrenal gland, it is believed to affect the secretory activity of the adrenal cortex in both rats and humans, where it increases adrenal blood flow in intact rats and acts as a vasodilator in the adrenal vascular bed. ADM has been shown to be present throughout the female reproductive tract, and plasma levels are elevated in normal pregnancy. Studies in a rat model of preeclampsia show that ADM can reverse hypertension and reduce offspring lethality when given to rats during late pregnancy. This did not have a similar effect in early pregnant animals or non-pregnant rats in the preeclampsia model, suggesting that ADM may play an important regulatory role in the uteroplacental cardiovascular system. In the pancreas, ADM most likely plays an inhibitory role, as it attenuated and delayed the insulin response to an oral glucose challenge, resulting in an early rise in glucose levels. ADM may also affect renal function. A peripherally administered bolus can significantly lower mean arterial pressure and increase renal blood flow, glomerular filtration rate and urine flow. In some cases, Na+ excretion is also increased.

[0191] ADM also has other peripheral effects on bone and lung. For bone, studies support a role beyond cardiovascular and fluid homeostasis, demonstrating that ADM acts on fetal and adult rodent osteoclasts to increase cell growth comparable to known osteoclast growth factors, such as transforming growth factor-alpha. This is clinically important, since one of the main challenges in osteoporosis research is to develop a treatment that increases bone mass through osteoblast stimulation. In the lung, ADM not only causes pulmonary vasodilation, but also inhibits bronchoconstriction induced by histamine or acetylcholine. A recent study using aerosolized ADM to treat pulmonary hypertension in a rat model shows that inhalation treatment of this condition is effective, as evidenced by the fact that mean pulmonary artery pressure and total pulmonary vascular resistance are significantly lower in rats treated with ADM than those given saline. This result was achieved without any changes in systemic arterial pressure or heart rate (Nagaya N et al., Am J Physiol Heart Circ Physiol. 2003;285:H2125-31).

[0192] In healthy subjects, iv infusion of ADM has been shown to reduce arterial pressure and stimulate heart rate, cardiac output, cAMP, prolactin, norepinephrine and rennin plasma levels. Little or no increase in urine volume or sodium excretion was observed in these patients. In patients with heart failure or chronic renal failure, ivADM showed effects similar to those seen in normal subjects, also inducing diuresis and natriuresis depending on the dose administered (Nicholls, MG 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, preeclampsia and preterm labor of pregnancy, and osteoporosis.

[0193] AFP-6 (i.e., intermedin) is expressed primarily in the pituitary gland and gastrointestinal tract. No specific receptor has been reported for AFP-6; however, binding studies show 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 endogenous CGRP receptors, in which it competes with labeled CGRP for binding to its receptor. In published in vivo studies, AFP-6 administration resulted in blood pressure reduction in both normal and spontaneously hypertensive rats, most likely via 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. 2004 Feb. 20; 279(8): 7264-74).

[0194] The biological actions of amylin family peptide hormones are generally reported to be mediated through 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. It is believed that co-expression of RAMP and either CTR or CRLR is required to generate functional receptors for calcitonin, CGRP, ADM, and amylin. The RAMP family includes three members (RAMP1, -2, and -3) that share less than 30% sequence identity but have a common topological organization. Co-expression of CRLR and RAMP1 results in the formation of a receptor for CGRP. Co-expression of CRLR and RAMP2 results in the formation of a receptor for ADM. 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 a receptor for amylin and CGRP Co-expression of hCTR2 and RAMP3 results in the formation of a receptor for amylin.

[0195] In certain embodiments, peptides of the invention that include an Amylin family hormone module may provide GIP functions as well as functions and uses associated with Amylin family modules, e.g., amylin, amylin / sCT / amylin, ADM, CGRP, as discussed.

[0196] In one embodiment, amylin analogs and derivatives have at least one hormonal activity of natural amylin. In some embodiments, amylin analogs are agonists of receptors to which natural amylin can specifically bind. Exemplary amylin analogs and derivatives include those described in U.S. Patent Application Publication No. 2003 / 0026812A1, which is incorporated herein by reference.

[0197] In some embodiments, the peptides of the present invention include CCK as a component peptide. CCK, including hCCK (cholecystokinin) and species variants, and various analogs thereof, are known in the art. In general, CCK has a 33 amino acid sequence that was first identified in humans, and includes an 8 amino acid in vivo C-terminal fragment ("CCK-8") that has been reportedly 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, and a 58 amino acid sequence found in cats, dogs, and humans, and a 47 amino acid sequence that is 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)) refers generically to both sulfated and non-sulfated C-terminal octapeptides unless otherwise specified. Additionally, pentagastrin or CCK-5 refers to the C-terminal peptide CCK(29-33), and CCK-4 refers to the C-terminal tetrapeptide CCK(30-33).

[0198] CCK was identified from preparations of intestinal extracts by its ability to stimulate gallbladder contraction. Other biological actions of CCK have since been reported, including stimulation of pancreatic secretion, slowing of gastric emptying, stimulation of intestinal motility, and stimulation of insulin secretion. See Lieverse et al., Ann. NY Acad. Sci. 713:268-272 (1994). Actions of CCK also reportedly include effects on cardiovascular function, respiratory function, neurotoxicity and seizures, cancer cell growth, analgesia, sleep, sexual and reproductive behavior, memory, anxiety, and dopamine-mediated behaviors. Crawley and Corwin, Peptides 15:731-755 (1994). Other reported effects of CCK include stimulation of pancreatic growth, stimulation of gallbladder contraction, inhibition of gastric acid secretion, pancreatic polypeptide release, and the contractile component of peristalsis. Additional reported effects of CCK include vasodilation. Walsh, “Gastrointestinal Hormones,” In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York).

[0199] It has been reported that injection of a combination of glucagon, CCK, and bombesin enhanced the inhibition of intake of a condensed milk test meal in nonfasted rats compared to the inhibition observed with the individual compounds. Hinton et al., Brain Res. Bull. 17:615-619 (1986). It has also been reported that glucagon and CCK synergistically inhibit sham feeding in rats. LeSauter and Geary, Am. J. Physiol. 253:R217-225 (1987); Smith and Gibbs, Annals NYAcad. Sci. 713:236-241 (1994). It has also been suggested that estradiol and CCK may have synergistic effects on satiety. Dulawa et al., Peptides 15:913-918 (1994); Smith and Gibbs, supra. It has also been proposed that signals emanating from the small intestine in response to nutrients therein may synergistically interact with CCK to reduce food intake. Cox, Behav. Brain Res. 38:35-44 (1990). Additionally, CCK has been reported to induce satiety in several species. For example, feeding suppression has been reported to be caused by CCK injected intraperitoneally in rats, intra-arterially 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 several laboratories have reportedly supported the behavioral specificity of low doses of CCK for inhibition in feeding by comparing responding for food with responding for non-food reinforcers in both monkeys and rats, and by showing that CCK induces the behavioral sequence normally observed after a meal (i.e., the postprandial satiety sequence) Furthermore, comparison of behavior after CCK with behavior after food ingestion reportedly reveals behavioral similarities between CCK alone or in combination with CCK and food intake. It has also been reported that CCK at physiological plasma concentrations inhibits food intake and increases satiety in both non-obese and obese humans.

[0200] CCK is characterized as a 33 amino acid peptide. Species-specific molecular variants of the amino acid sequence of CCK have been identified. The 33 amino acid sequence and a truncated peptide, its 8 amino acid C-terminal sequence (CCK-8), have reportedly been identified in pig, rat, chicken, chinchilla, dog, and human. A 39 amino acid sequence has reportedly been found in pig, dog, and guinea pig. A 58 amino acid sequence has reportedly been found in cat, dog, and human. Frogs and turtles reportedly exhibit a 47 amino acid sequence that is homologous to both CCK and gastrin. Extremely fresh human intestine is reported to contain small amounts of a larger molecule designated CCK-83. In rats, a major intermediate form has reportedly been identified and is designated CCK-22. Walsh, “Gastrointestinal Hormones,” In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York). Nonsulfated CCK-8 and a tetrapeptide, designated CCK-4 (CCK(30-33)), have been reported in rat brain. The C-terminal pentapeptide, designated CCK-4 (CCK(29-33)), conserves structural homology to CCK and also homology to the neuropeptide, gastrin. The C-terminal sulfated octapeptide sequence, CCK-8, is reportedly relatively conserved across species. Cloning and sequence analysis of cDNAs encoding preprocholecystokinin from rat thyroid carcinoma, pig brain, and pig intestine reportedly revealed a 345 nucleotide sequence encoding a precursor of CCK, which is 115 amino acids and contains all of the CCK sequence previously reported to have been isolated. Crawley and Corwin, supra.

[0201] CCK is said to be distributed throughout the central nervous system, as well as in the endocrine cells and enteric nerves of the upper small intestine. CCK agonists include CCK itself (also called CCK-33), CCK-8 (CCK(26-33)), non-sulfated CCK-8, pentagastrin (CCK-5 or CCK(29-33)), and the tetrapeptide, CCK-4 (CCK(30-33)). At the pancreatic CCK receptor, CCK-8 reportedly displaces binding with 1000-5000 times greater potency than non-sulfated CCK-8 or CCK-4, and CCK-8 is reported to be approximately 1000 times more potent than non-sulfated 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 10- or 100-fold more by nonsulfated CCK-8 and CCK-4 than by sulfated CCK-8 at equimolar concentrations. Id. Receptors for CCK have reportedly been identified in a variety of tissues, and two major subtypes have been described, type A and type B receptors. Type A receptors have been reported in peripheral tissues including the pancreas, gallbladder, pyloric sphincter and afferent vagus nerve fibers, as well as in discrete areas of the brain. The type A receptor subtype (CCKA) has been reported to be selective for sulfated octapeptides. The type B receptor subtype (CCKB) has been identified throughout the brain and in the stomach, and reportedly does not require either sulfation or all eight amino acids. See Reidelberger, J. Nutr. 124(8 Suppl.) 1327S-1333S (1994); Crawley and Corwin, supra.

[0202] Various in vivo and in vitro screening methods for CCK analogs are known in the art. Examples include in vivo assays of dog or guinea pig gallbladder contraction after rapid intravenous injection of a compound to be tested for CCK-like activity, and in vitro assays using rabbit gallbladder strips. See Walsh, "Gastrointestinal Hormones", In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York).

[0203] Constituent peptide hormones useful as components of the peptides of the present invention include GLP-1 peptide hormones. Natural GLP-1 peptide hormones, including GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36)amide, are known in the art, as are functional peptide analogs and derivatives. As used herein, GLP-1 refers to all natural forms of GLP-1 peptide hormones. Although certain exemplary natural peptides, peptide analogs and derivatives are described herein, it should be recognized that any known GLP-1 peptide that exhibits hormonal activity known in the art can be used with the present invention.

[0204] At the heart of many metabolic diseases and disorders is the regulation of insulin levels and blood glucose levels. Insulin secretion is modulated in part by secretagogue hormones called incretins, which are produced by enteroendocrine cells. The incretin hormone, glucagon-like peptide-1 ("GLP-1"), is a peptide hormone secreted by enterocytes that has been shown in several studies to produce an enhancing effect on insulin secretion. GLP-1 is processed from proglucagon in the intestine and enhances nutrient-induced insulin release (Krcymann B., et al., Lancet, 2:1300-1303 (1987)). Various truncated forms of GLP-1 are known to stimulate insulin secretion (insulinotropic action) and cAMP formation (see, e.g., Mojsov, S., Int. J. Pep. Pro. Res., 40:333-343 (1992)). A variety of in vitro laboratory experiments have been established to correlate the insulin secretion stimulatory response of mammals, particularly humans, to exogenous administration of GLP-1, GLP-1(7-36)amide, and GLP-1(7-37)acid (see, e.g., Nauck, MA, et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., New Eng. J. of Med., 326(20):1316-1322 (1992); Nauck, MA, et al., J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes, 42:1219-1225 (1993)).

[0205] GLP-1(7-36)amide exerts excellent antidiabetic effects in insulin-dependent diabetic patients by stimulating insulin sensitivity and by improving glucose-induced insulin release at physiological concentrations (Gutniak M., et al., New Eng. J. Med., 326:1316-1322(1992)). When administered to non-insulin-dependent diabetic patients, GLP-1(7-36)amide stimulates insulin release, reduces glucagon secretion, inhibits gastric emptying, and improves glucose utilization (Nauck, 1993; Gutniak, 1992; Nauck, 1993). However, the serum half-life of such peptides is rather short, making it difficult to use GLP-1 type molecules for long-term therapy of diabetes.

[0206] More specifically, GLP-1 is a 30 amino acid peptide derived from proglucagon, a 160 amino acid prohormone. The action of different prohormone convertases in the pancreas and intestine results in the production of glucagon and other unknown peptides, while cleavage of proglucagon results in the production of GLP-1 and GLP-2 as well as two other peptides. The amino acid sequence of GLP-1 is 100% homologous in all mammals, implying an important physiological role. GLP-1(7-37) acid is truncated and amidated at the C-terminus to form GLP-1(7-36)NH2. The biological effects and metabolic turnover of the free acid GLP-1(7-37)OH and the amide, GLP-1(7-36)NH2, are indistinguishable. By convention, the numbering of the amino acids is based on the processed GLP-1(1-37)OH from proglucagon. Biologically active GLP-1 is the result of further processing: GLP-1(7-36)NH2. Thus, the first amino acid of GLP-1(7-37)OH or GLP-1(7-36)NH2 is 7His.

[0207] In the gastrointestinal tract, GLP-1 is produced by L-cells of the intestinal, colonic and rectal mucosa in response to stimulation by intraluminal glucose. The plasma half-life of active GLP-1 is <5 min, and its metabolic clearance rate is approximately 12-13 min (Holst, Gastroenterology 107(6):1848-55(1994)). The main protease involved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP-IV or CD26), which cleaves the N-terminal His-Ala dipeptide and thus produces the metabolites GLP-1(9-37)OH or GLP-1(9-36)NH2, 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 located in pancreatic beta cells, lung, and to a lesser extent in the brain, adipose tissue, and kidney. Stimulation of the GLP-1R by GLP-1(7-37)OH or GLP-1(7-36)NH2 results in adenylate cyclase activation, cAMP synthesis, membrane depolarization, elevation of intracellular calcium, and an increase in glucose-induced insulin secretion (Holz et al., J. Biol. Chem. 270(30):17749-57(1995)).

[0208] GLP-1 is a potent insulin secretagogue secreted from the intestinal mucosa in response to food intake. The profound incretin effect of GLP-1 is underestimated by the fact that GLP-1R knockout mice are glucose intolerant. The incretin response to iv-infused GLP-1 is preserved in diabetic subjects, whereas the incretin response to oral glucose in these subjects is impaired. GLP-1 administration by infusion or sc injection controls fasting glucose levels and maintains the glucose threshold for insulin secretion in diabetic patients (Gutniak et al., N. Engl. J. Med. 326:1316-22 (1992); Nauck et al., Diabet. Med. 13:(9 Suppl 5):S39-S43 (1996); Nauck et al., J. Clin. Endocrinol. Metab. 76:912-917 (1993)). GLP-1 has shown great potential as a therapeutic agent that can enhance insulin secretion in a physiological manner while avoiding the hypoglycemia associated with sulfonylurea drugs.

[0209] Other important effects of GLP-1 on glucose homeostasis are suppression of glucagon secretion and inhibition of gastric motility. GLP-1 inhibitory action on pancreatic alpha cell secretion of glucagon leads to a decrease in hepatic glucose production through a reduction in gluconeogenesis and glycogenolysis. This antiglucagon effect of GLP-1 is preserved in diabetic patients.

[0210] The so-called ideal brake effect of GLP-1, in which gastric motility and gastric secretion are inhibited, is exerted via efferent vagus nerve receptors or by direct action on intestinal smooth muscle. The reduction of gastric acid secretion by GLP-1 contributes to the lag phase of nutrient availability, thus eliminating the need for a rapid insulin response. In summary, the gastrointestinal effects of GLP-1 contribute significantly to the delay of glucose and fatty acid absorption, modulating insulin secretion and glucose homeostasis.

[0211] GLP-1 has also been shown to induce beta cell specific genes, such as the GLUT-1 transporter, insulin (through the interaction of PDX-1 with the insulin gene promoter), and hexokinase-1. Thus, GLP-1 could potentially reverse the glucose intolerance normally associated with aging, as demonstrated by rodent experiments. Furthermore, GLP-1 may contribute to beta cell neogenesis and increase beta cell mass, in addition to restoring beta cell function during conditions of beta cell failure.

[0212] The central effect of GLP-1 is the increase in satiety coupled with the decrease in food intake that occurs via the action of hypothalamic GLP-1R. Continuous SC infusion of GLP-1 for 48 hours in type II diabetic subjects decreased hunger and food intake and increased satiety. These appetite suppressing effects were absent in GLP-1R knockout mice. Thus, in addition to having GIP functions, GIP hybrids containing incretin family hormone modules can provide the functions and uses associated with incretin family modules, such as exendin-4, GLP1, GLP2, as discussed.

[0213] Any GLP-1 peptide analog or derivative known in the art can be used with the present invention. In one embodiment, the GLP-1 peptide analog and derivative has at least one hormonal activity of a native GLP-1 peptide. In some 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 those described in, for example, WO 91 / 11457, which is incorporated herein by reference.

[0214] In certain embodiments, a peptide of the invention comprises or is any one of the amino acid sequences listed below.

[0215] [Table 10]

[0216] [Table 11]

[0217] [Table 12]

[0218] Any of the peptides of the invention may be further defined, for example, as follows. It is understood that each of the additional defining features described herein may also be applied to any of the peptides of the invention where the amino acids at the particular positions chemically allow for the presence of the additional important features. In certain embodiments, these features may be present in any of the peptides of the invention of formulas (I)-(V).

[0219] In various embodiments, the nitrogen group of any of the amino acids of the peptides of the invention may be optionally substituted with C1-C6 alkyl, C6-C12 aryl, C6-C12 aryl C1-C6 alkyl, or C1-C20 alkanoyl, including PEGylated versions alone or as any of the above, e.g., acetyl, as a spacer. It is understood that N-substitutions may be absent. In certain embodiments, the peptides of the invention include an N-terminus selected from hydrogen, C1-C6 alkyl, C6-C12 aryl, C6-C12 aryl C1-C6 alkyl, or C1-C20 alkanoyl, including PEGylated versions alone or as any of the above, e.g., acetyl, as a spacer. In certain embodiments of any of the peptides of the invention described herein, the N-terminal moiety is hydrogen.

[0220] In some embodiments of any of the peptides of the present 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 conjugate amides of lauric acid, hexadecanoic acid, and γ-Glu-hexadecanoic acid. In some embodiments, the N-substituted moiety is pGlu. In certain embodiments, the N-substituted moiety is acetyl, whereby the peptides of the present invention are acylated at their N (e.g., to cap or protect the N-terminal amino acid residue, e.g., the N-terminal Pen residue).

[0221] In some embodiments of any of the peptides of the invention described herein, the N-substituted moiety is an acid.In some embodiments, the N-substituted moiety is 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 sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, alkylsulfonic acids and arylsulfonic acids.

[0222] 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.

[0223] 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.

[0224] In certain embodiments, the peptides of the invention include one or more linker groups that covalently link one peptide moiety to a second subunit. The subunits are optionally linked via their C-terminus.

[0225] The peptide of the present invention generally comprises at least two peptide components, and at least one of the peptide components, e.g., the GIP component, exhibits at least one hormonal activity. Within the context of the present invention, at least one of the peptide components is comprised of a GIP peptide, analog, derivative, fragment, or peptidic enhancer. The peptide component exhibiting at least one hormonal activity can be located at the N-terminus of the peptide, the C-terminus of the peptide, or in the internal portion of the peptide, e.g., in the event that the peptide comprises three or more peptide components.

[0226] In some embodiments, it may be preferred to position at least one hormonally active peptide component such that the C-terminus of the peptide component is amidated. Amidation of the C-terminus of the peptide component can be achieved by positioning the module at the C-terminus of the hybrid peptide, or by constructing the peptide in a C-terminus-N-terminus orientation at the N-terminus of the peptide. In both configurations, the C-terminus of the peptide component is available for amidation. Specific component peptides with C-terminal amidation may preferably include amylin family peptides, CCK, PYY, hGLP-1(7-36) and hGLP-2. Specific component peptides with C-terminal amides, not necessarily exemplary (extensions at the C-terminus of the module are readily accommodated when otherwise stated), 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 located at the C-terminus of the peptide, they may still be optionally amidated, and indeed may preferably be optionally amidated.

[0227] The constituent peptides of the peptide of the present invention may be covalently linked in any manner known in the art. Stable bonds may be used or cleavable bonds 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, a linking group may be used for the linked module. Additionally, spacers or turn inducers known in the art may be used to stabilize the linkage, if desired. By way of example, if amidation of the C-terminus of an N-terminally located peptide component is not desired, the module may be attached to a second module directly or using any suitable linking group known in the art, such as alkyl; PEG; amino acids, e.g., Lys, Glu, beta-Ala; polyamino acids, e.g., poly-his, poly-arg, poly-lys, poly-ala, Gly-Ser-Gly, Gly-Gly-Pro-Ser, Ala-Lys-Ala, Gly-Lys-Arg (GKR), and the like; 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. As each is explicitly indicated herein, specific hybrid embodiments are described in which the linker in each exemplified linker-containing hybrid is replaced by a Gly linker, particularly those in which the Gly linker is Gly-Gly-Gly. In one embodiment, the linker or spacer is 1-30 residues long, in another embodiment 2-30 residues long, and in yet another embodiment 3-30 residues long, any integer length between 2-30 (inclusive); each integer increment is contemplated, e.g., 2, 3, 4, 5, 6, 7, etc. In one embodiment, a Gly linker is used, and in a particular embodiment, the three residue linker Gly-Gly-Gly is used.

[0228] In some embodiments, the peptide components of the invention may be linked by a suitable linking moiety, such as a disulfide bridge between two cysteine ​​residues, one in each peptide subunit, or by another suitable linker moiety, including but not limited to those defined herein. In some embodiments, the subunits may be modified to eliminate either the C- or N-terminal free amine, thereby allowing dimerization at the remaining free amine. Furthermore, in some examples, the termini of one or more of the monomeric subunits are acylated with an acylation organic compound selected from the group consisting of trifluoropentyl, acetyl, octanyl, butyl, pentyl, hexyl, palmityl, 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 examples, the subunits contain both a free carboxy terminus and a free amino terminus, allowing the user to selectively modify the subunits to achieve dimerization at the desired terminus. One of skill in the art will recognize that the subunits of the present invention can be selectively modified to yield a single defined amine for desired attachment.

[0229] It is further understood that the C-terminal residue of the subunits disclosed herein is optionally an amide. It is further understood that in some embodiments, dimerization at the C-terminus is facilitated by using a suitable amino acid having a side chain with an amine functionality, as is generally understood in the art. With respect to the N-terminal residue, it is generally understood that coupling can be achieved via the free amine of the terminal residue, or by using a suitable amino acid side chain with a free amine, as is generally understood in the art.

[0230] The linker moiety linking the subunits may comprise any structure, length, and / or size that is compatible with the teachings herein. In at least one embodiment, the linker moiety is selected from the non-limiting group consisting of cysteine, lysine, DIG, PEG4, PEG4-biotin, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, ADA, Boc-IDA, glutaric acid, isophthalic acid, 1,3-phenylene diacetic acid, 1,4-phenylene diacetic acid, 1,2-phenylene diacetic acid, triazine, Boc-triazine, IDA-biotin, PEG4-biotin, AADA, suitable aliphatic compounds, aromatic compounds, heteroaromatic compounds, and polyethylene glycol-based linkers having a molecular weight of approximately 400 Da to approximately 40,000 Da. In one embodiment, PEG2 is HO2CCH2CH2OCH2CH2OCH2CH2CO2H.

[0231] Non-limiting examples of suitable linker moieties are provided in Table 3.

[0232] [Table 13]

[0233] [Table 14]

[0234] [Table 15]

[0235] [Table 16]

[0236] [Table 17]

[0237] In some embodiments, the peptide of the present invention comprises a linker moiety. In some embodiments, the subunits of the peptide of the present invention are connected via an intermolecular disulfide bond formed between two cysteine ​​residues, one in each subunit. In some embodiments, the peptide of the present invention comprises both a linker moiety and an intermolecular disulfide bond 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.

[0238] Those skilled in the art will recognize that the linker (e.g., C- and N-terminal linkers) moieties disclosed herein are non-limiting examples of suitable linkers, and that the present invention may include any suitable linker moiety. Thus, some embodiments of the present invention include homo- or heterodimeric peptides composed of two monomeric subunits selected from the peptides shown in any of the tables herein or comprising or consisting of a sequence presented in any of the tables herein, with the C- or N-terminus (or internal amino acid residue) of each subunit being linked by any suitable linker moiety to provide the peptide of the present invention. In some embodiments, the linker is attached to the N- or C-terminus of one subunit and to an internal amino acid residue of the other subunit that constitutes the peptide of the present invention. In some embodiments, the linker is attached to an internal amino acid residue of one monomeric subunit and to an internal amino acid residue of the other monomeric subunit that constitutes the peptide of the present invention. In further embodiments, the linker is attached to the N- or C-terminus of both subunits.

[0239] In certain embodiments, one or more of the subunits comprises a sequence or structure of any one of formulas (I)-(V) or any of the peptides described herein. In certain embodiments of the peptides of the invention, the linker moiety is any of the linkers described herein. In some 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.

[0240] In various embodiments of any of the peptides of the invention, each of the peptide subunits is linked to a linker moiety through its N-terminus, C-terminus, or an internal amino acid residue. In some embodiments of any of the peptides of the invention, the N-terminus of each peptide subunit is linked by a linker moiety. In some embodiments of any of the peptides of the invention, the C-terminus of each peptide subunit is linked by a linker moiety. In some embodiments of any of the peptides of the invention, each peptide subunit is linked by a linker moiety that is attached to an internal amino acid.

[0241] In some embodiments, the peptide of the present invention comprises one or more conjugate chemical substituents, such as lipophilic substituents and polymer moieties, which may be referred to herein as half-life extending moieties.Without being bound to any particular theory, it is believed that the lipophilic substituents bind to albumin in the bloodstream, thereby protecting the peptide of the present invention from enzymatic degradation, and thus improving its half-life.Furthermore, it is believed that the polymer moiety improves half-life and reduces clearance in the bloodstream.

[0242] In additional embodiments, the peptides of the invention, e.g., any of the peptides of Formulae (I)-(V), further comprise a linker moiety attached to an amino acid residue present in the peptide, e.g., the linker moiety may 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.

[0243] In additional embodiments, the peptides of the invention, e.g., any of the peptides of Formulae (I)-(V), further comprise a half-life extending moiety attached to an amino acid residue present in the peptide, e.g., the half-life extending moiety may 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.

[0244] In additional embodiments, the peptides of the invention, e.g., any of the peptides of Formulae (I)-(V), further comprise a half-life extending moiety attached to a linker moiety that is attached to an amino acid residue present in the peptide, e.g., the half-life extending moiety may be attached to the side chain of any amino acid of the peptide, to the N-terminal amino acid of the peptide, or to a linker moiety that is attached to the C-terminal amino acid of the peptide.

[0245] In certain embodiments, the peptide comprises a half-life extending moiety having the structure shown below, where n=0-24 or n=14-24: [ka]

[0246] In certain embodiments, the peptides of the invention comprise a half-life extending moiety as shown in Table 4.

[0247] [Table 18]

[0248] [Table 19]

[0249] In some embodiments, the half-life extending moiety is directly attached to the peptide, while in other embodiments, the half-life extending moiety is attached to the peptide via a linker moiety, such as any of those described herein.

[0250] [Table 20]

[0251] [Table 21]

[0252] [Table 22]

[0253] In certain embodiments, the peptides of the invention comprise any of the linker moieties presented herein and any of the half-life extending moieties presented herein, including any of the following combinations shown in Table 6:

[0254] [Table 23]

[0255] [Table 24]

[0256] [Table 25]

[0257] [Table 26]

[0258] In some embodiments, there may be multiple linkers present between peptide conjugate moieties, e.g., half-life extending moieties (e.g., as shown in Table 7).

[0259] [Table 27]

[0260] In certain embodiments, the half-life of a peptide of the invention comprising a conjugated chemical substituent, i.e., a half-life extending moiety, is at least 100%, at least 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% of the half-life of the same peptide but without the conjugated chemical substituent. In certain embodiments, the lipophilic substituent and / or polymer moiety enhances the permeability of the peptide through epithelia and / or its retention in the lamina propria. In certain embodiments, the permeability of a peptide of the invention comprising a conjugated chemical substituent through epithelia and / or its retention in the lamina propria is 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 but without the conjugated chemical substituent.

[0261] In certain embodiments, the side chain of one or more amino acid residues (e.g., Lys residues) in the peptides of the invention is conjugated (e.g., covalently bonded) 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, may provide spacing between the peptide subunit and the lipophilic substituent. In certain embodiments, the peptides of the invention comprise any of the conjugate moieties disclosed herein.

[0262] In certain embodiments, the lipophilic substituent may comprise a hydrocarbon chain having 4 to 30 C atoms, for example at least 8 or 12 C atoms, preferably 24 or less C atoms, or 20 or less C atoms. The hydrocarbon chain may be linear or branched, saturated or unsaturated. In certain embodiments, the hydrocarbon chain is substituted with a moiety that forms part of the bond to the amino acid side chain or spacer, such as an acyl group, a sulfonyl group, an N atom, an O atom, or an S atom. In some embodiments, the hydrocarbon chain is substituted with an acyl group, such that the hydrocarbon chain may form part of an alkanoyl group, such as palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

[0263] The lipophilic substituent may be conjugated to any amino acid side chain in the peptide of the present invention. In some embodiments, the amino acid side chain comprises a carboxy, hydroxyl, thiol, amide or amine group to form an ester, sulfonyl ester, thioester, amide or sulfonamide with a spacer or 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 some embodiments, the lipophilic substituent is conjugated to Lys. The amino acid shown as Lys in any of the formulas provided herein may be replaced by, for example, Dbu, Dpr or Orn to which the lipophilic substituent is added.

[0264] In certain embodiments, the peptides of the present invention can be modified, for example, to improve stability, increase permeability, or improve drug-like characteristics, via the conjugation of chemical moieties to one or more amino acid side chains in the peptide. For example, the N(epsilon) of lysine N(epsilon), the β-carboxyl of aspartic acid, or the γ-carboxyl of glutamic acid can be appropriately functionalized. Thus, to produce a modified peptide, the amino acids in the peptide can be appropriately modified. Furthermore, in some instances, the side chains are acylated with an acylation organic compound selected from the group consisting of trifluoropentyl, acetyl, octanyl, butyl, pentyl, hexyl, palmityl, trifluoromethylbutyric acid, cyclopentane carboxylic acid, cyclopropyl acetic acid, 4-fluorobenzoic acid, 4-fluorophenyl acetic acid, 3-phenylpropionic acid, tetrahydro-2H-pyran-4 carboxylic acid, succinic acid, glutaric acid, or bile acid. Those skilled in the art will recognize that a range of conjugates can be attached, such as PEG4, isoGlu, and combinations thereof. Those skilled in the art will recognize that amino acids having a peptide may be substituted with equivalents, for example, Lys may be substituted with Dap, Dab, α-MeLys, or Orn. ​​Examples of modified residues within the peptides are shown in Table 8.

[0265] [Table 28]

[0266] [Table 29]

[0267] [Table 30]

[0268] In a further embodiment of the invention, or in addition, the side chains of one or more amino acid residues in the peptides of the invention are conjugated to polymer moieties, e.g., to increase solubility and / or in vivo (e.g., plasma) half-life and / or bioavailability. Such modifications are also known to reduce the clearance (e.g., renal clearance) of therapeutic proteins and peptides.

[0269] As used herein, "polyethylene glycol" or "PEG" 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), and as used herein, PEO, PEE, or POG refer to oligomers or polymers of ethylene oxide, depending on their molecular weight. Although the three names are chemically synonymous, PEG tends to refer to oligomers and polymers with molecular weights less than 20,000 Da, PEO to polymers with molecular weights greater than 20,000 Da, and POE to polymers of any molecular weight. Depending on their molecular weight, PEG and PEO are liquids or low melting solids. Throughout this disclosure, the three names are used interchangeably. PEG is prepared by polymerization of ethylene oxide and is commercially available over a wide range of molecular weights, from 300 Da to 10,000,000 Da. PEG and PEO with different molecular weights are used in different applications and have different physical properties (e.g., viscosity) due to the effect of chain length, but their chemical properties are nearly identical. The polymer moiety is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharma- ceutically inert. Suitable polymer moieties include polyethylene glycol (PEG), homo- or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG), or polyoxyethylene glycerol (POG). See, e.g., Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992). PEG prepared for the purpose of half-life extension, e.g., mono-activated alkoxy-terminated polyalkylene oxide (POA), e.g., mono-methoxy-terminated polyethylene glycol (mPEG), are also contemplated; bis-activated polyethylene oxide (glycol) or other PEG derivatives are also contemplated. Suitable polymers vary considerably in weight, ranging from about 200 Da to about 40,000 Da or from about 200 Da to about 60,000 Da, and are typically selected for purposes of the present invention. In certain embodiments, PEG having a molecular weight of 200-2,000 or 200-500 is used.Different forms of PEG can also be used depending on the initiator used in the polymerization process; a common initiator is methoxypoly(ethylene glycol), abbreviated as monofunctional methyl ether PEG, or mPEG.

[0270] Low molecular weight PEG is also available as pure oligomers, referred to as monodisperse, uniform, or discrete, which find use in certain embodiments of the invention.

[0271] PEGs with different geometries are also available: branched PEGs have 3-10 PEG chains emanating from a central core group; star PEGs have 10-100 PEG chains emanating from a central core group; and comb PEGs usually have multiple PEG chains grafted onto the polymer backbone. PEGs can be linear. The number often included in the name of PEG indicates its average molecular weight, (e.g., PEG with n=9) has an average molecular weight of approximately 400 daltons and is labeled PEG400.

[0272] As used herein, "PEGylation" refers to the act of covalently coupling a PEG structure to a peptide of the invention, which is then referred to as a "PEGylated peptide." In some embodiments, the PEG of the PEGylated side chain is a PEG having a molecular weight of about 200 to about 40,000. In some embodiments, the PEG of the PEGylated spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In some embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8.

[0273] Other suitable polymer moieties include polyamino acids, such as polylysine, polyaspartic acid, and polyglutamic acid (see, e.g., Gombotz, et al. (1995), Bioconjugate Chem., vol. 6:332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57, and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73,:721-729. The polymer moiety can be linear or branched. In some embodiments, it has a molecular weight of 500 to 40,000 Da, e.g., 500 to 10,000 Da, 1000 to 5000 Da, 10,000 to 20,000 Da, or 20,000 to 40,000 Da.

[0274] In some embodiments, the peptides of the invention may include two or more such polymeric moieties, in which case the total molecular weight of all such moieties will generally be within the ranges provided above.

[0275] In some embodiments, the polymer moiety is coupled (covalently) to an amino, carboxyl or thiol group of an amino acid side chain. Some examples are the thiol group of Cys residues and the epsilon amino group of Lys residues, although the carboxyl groups of Asp and Glu residues may also be involved.

[0276] Those skilled in the art will be familiar with suitable techniques that can be used to carry out coupling reactions.For example, PEG moieties carrying methoxy groups can be coupled to Cys thiol groups via maleimide bonds using commercially available reagents from Nektar Therapeutics AL.For details of suitable chemical reactions, see also WO 2008 / 101017 and the references cited above.Maleimide-functionalized PEG can also be conjugated to the side chain sulfhydryl groups of Cys residues.

[0277] As used herein, disulfide bond oxidation can occur in a single step or is a two-step process. As used herein, for a single oxidation step, a trityl protecting group is often used during assembly, which allows for deprotection during cleavage and subsequent solution oxidation. If a second disulfide bond is required, there are options for native or selective oxidation. For selective oxidation, which requires orthogonal protecting groups, Acm and trityl are used as protecting groups for cysteines. Cleavage results in the removal of one cysteine ​​of the protecting pair, allowing oxidation of this pair. A second oxidative deprotection step of the cysteine ​​protecting Acm group is then performed. For native oxidation, a trityl protecting group is used for all cysteines, allowing native folding of the peptide. Those skilled in the art will appreciate the suitable techniques that can be used to perform the oxidation step.

[0278] Some chemical moieties, including poly(ethylene) glycol, react with functional groups present in the 20 naturally occurring amino acids, such as the epsilon amino group in lysine amino acid residues, the thiols present in cysteine ​​amino acid residues, or other nucleophilic amino acid side chains. When multiple naturally occurring amino acids react in a peptide, their non-specific chemical reactions result in a final peptide of the invention that contains many isomers of the peptide conjugated to one or more poly(ethylene) glycol chains at different positions within the peptide of the invention.

[0279] One advantage of certain embodiments of the present invention includes the ability to add one or more chemical moieties (e.g., PEG) by incorporating one or more unnatural amino acids that have inherent functional groups that react with activated PEG through chemicals that are unreactive with the naturally occurring amino acids present in the peptides of the present invention. For example, azide and alkyne groups are unreactive with all naturally occurring functional groups in proteins. Thus, unnatural amino acids can be incorporated at one or more defined sites in the peptides of the present invention where PEG or another modification is desired without undesired non-specific reactions. In certain embodiments, the specific chemicals involved in the reaction result in a stable covalent bond between the PEG chain and the peptides of the present invention. Moreover, such reactions can be carried out in mild aqueous conditions that do not damage most peptides. In certain embodiments, the unnatural amino acid residue is AHA.

[0280] Chemical moieties that are attached to natural amino acids are limited in number and scope. In contrast, chemical moieties that are attached to unnatural amino acids can utilize a significantly broader range of useful chemistries that attach chemical moieties to target molecules. Essentially any target molecule can serve as a substrate for attaching a chemical moiety, including any protein (or portion thereof) that contains an unnatural amino acid, e.g., an unnatural amino acid that contains a reactive site or side chain to which a chemical moiety can be attached, e.g., an aldehyde or keto derivatized amino acid.

[0281] A large number of chemical moieties can be connected or attached to a particular molecule through various methods known in the art. Various such methods are described in U.S. Pat. No. 8,568,706. As an illustrative example, an azide moiety can be useful for conjugating chemical moieties, such as PEG or others described herein. The azide moiety serves as a reactive functional group and is absent in most naturally occurring compounds (hence it is non-reactive with the natural 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 allows the incorporation or introduction of azides into molecules is the copper-mediated Huisgen [3+2] cycloaddition of azides. This reaction can be used for selective PEGylation of peptides. (Tornoe et al., J.Org.Chem.67:3057,2002; Rostovtsev et al.,Angew.Chem.,Int.Ed.41:596,2002; and Wang et al.,J.Am.Chem.Soc.125:3192,2003,Speers et al. al., J. Am. Chem. Soc., 2003, 125, 4686).

[0282] In another embodiment, the peptide according to the present invention may have one or more amino acid residues deleted from the native peptide or the amino acid sequence of 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 from 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 at amino acid positions 1 to 42 of region S. Such deletions may include two or more consecutive or non-consecutive deletions. In exemplary embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five 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, where the region is, for example, exendin(31-39) or exendin(27-39). In one embodiment, the native GIP is human, rat, mouse, porcine, or bovine.

[0283] In one embodiment of the peptide of the invention, any of the GIP peptides, analogs, derivatives or hybrids, when intended for use as agonists, do not contain a deletion in any one of positions 1-15 corresponding to positions YAEGTFISDYSIAMD in the N-terminal sequence of GIP. In other words, each of the corresponding positions 1-15 in GIP are present, but they may be substituted or derivatized. In a further embodiment, the agonist GIP compound does not contain a deletion in any one of positions 4-15 corresponding to positions GTFISDYSIAMD in the C-terminal sequence of GIP. In other words, each of the corresponding positions 4-15 in GIP are present, but they may be substituted or derivatized. Thus, in an embodiment of the agonist GIP compound, each of positions 1-15 or 4-15 are present and occupied by an amino acid present at that position in the naturally occurring GIP species, or by a substitution or derivative thereof. In yet another embodiment of the agonist GIP compound, the various embodiments described herein exclude GIP compounds that did not demonstrate adequate receptor binding activity or receptor activation activity as indicated.

[0284] In one embodiment of the peptide of the invention, any of the GIP peptides, analogs, derivatives or hybrids, when intended for use as agonists, do not contain a deletion at positions 1-15 corresponding to positions YAEGTFISDYSIAMD of the N-terminal sequence of GIP. In other words, each of the corresponding positions 1-15 of GIP are present, but they may be substituted or derivatized. In a further embodiment, the agonist GIP compound does not contain a deletion at any one of positions 4-15 corresponding to positions of the C-terminal sequence of GIP. In other words, each of the corresponding positions 4-15 of GIP are present, but they may be substituted or derivatized.

[0285] In another embodiment of the peptide 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 or region S 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), either alone or in combination with one or more deletions and / or substitutions. In one aspect, the invention relates to 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 GIP analogs or hybrid peptides with single insertions, or consecutive or non-consecutive insertions of two or more amino acid residues into the amino acid sequence of region S, e.g., exendin(27-39) and exendin(31-39). In one embodiment, the native GIP is human, rat, mouse, porcine or bovine.

[0286] In another embodiment of the peptide of the invention, the GIP analog or hybrid may comprise one or more unnatural 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, e.g., exendin(27-39) and exendin(31-39). In exemplary embodiments, the unnatural 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, e.g., exendin(27-39) and exendin(31-39), can be a beta-turn mimetic or a linker molecule. In further such embodiments, the native GIP can be human, rat, mouse, porcine or bovine.

[0287] Thus, while compounds with optional linking groups are shown, in one embodiment of the sequences herein, the linker is a Gly linker, e.g., Gly-Gly-Gly, or a beta Ala linker, e.g., beta Ala-beta Ala, all of which are specifically contemplated. Particularly interesting linker molecules include aminocaproyl ("Aca"), beta-alanyl, and 8-amino-3,6-dioxaoctanoyl. In yet other embodiments, beta-turn mimetics are used, including 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 even Ala-Aib and Ala-Pro dipeptides.

[0288] In another embodiment of the peptide of the invention, the GIP analog or hybrid may include the insertion of polyamino acid sequences (e.g., poly-his, poly-arg, poly-lys, poly-ala, etc.) at either end of the polypeptide, known as the "extension" or "tail."

[0289] In some embodiments, the novel peptides of the invention comprise amino acid sequence insertions, including alanine substitutions at each amino acid position along the length of native GIP, GIP(1-30), GIP(1-14), GIP(1-26), GIP(1-39), GIP(19-30), GIP(19-26), GIP(19-39) or GIP(19-42), or region S, e.g., exendin(27-39) and exendin(31-39).

[0290] 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 by addition of polyamino acids, such as poly-his, poly-arg, poly-lys, and poly-ala. Modifications of the polypeptides may also include small molecule substituents, such as short chain alkyls and constrained alkyls (e.g., branched, cyclic, fused, adamantyl), and aromatic groups. The water-soluble polymer molecules preferably have a molecular weight in the range of about 500 to about 20,000 daltons.

[0291] Such polymer conjugation and small molecule substituent modification may occur singly at the N- or C-terminus of the GIP analog and hybrid polypeptides or at the side chains of amino acid residues within the sequence. Alternatively, there may be multiple sites of derivatization along the GIP analog and hybrid peptides. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine ​​may provide additional sites for derivatization. See, for example, U.S. Patent Nos. 5,824,784 and 5,824,778. In one embodiment, the peptide of the present invention may be conjugated to one, two, or three polymer molecules.

[0292] The water-soluble polymer molecules are preferably attached to amino, carboxyl, or thiol groups and may be attached by the N- or C-terminus or at the side chains of lysine, aspartic acid, glutamic acid, or cysteine. Alternatively, the water-soluble polymer molecules may be attached to diamine and dicarboxylic acid groups. In an exemplary embodiment, the peptides of the invention are conjugated to one, two, or three PEG molecules via the epsilon amino group on a lysine amino acid.

[0293] The peptides of the invention also include chemical modifications of one or more amino acid residues. Such chemical modifications include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. Chemical modifications may occur singly, for example, at the N- or C-terminus of the GIP analogs and hybrid peptides or at the side chains of amino acid residues within the sequence. In one embodiment, the C-terminus of these peptides may have a free -OH or -NH2 group. In another embodiment, the N-terminus may be capped with an isobutyloxycarbonyl group, an isopropylbutyloxycarbonyl group, an n-butoxyoxycarbonyl group, an ethoxyoxycarbonyl group, an isocaproyl group (isocap), 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 modifications along the peptides of the invention.

[0294] In general, numerous pseudopeptide bonds have been described that do not affect peptide structure and biological activity. One example of this approach is to replace it with a retroinverso pseudopeptide bond (see "Biologically active retroinverso analogues of thymopentin", Sisto A. et al in Rivier, JE and Marshall, GR (eds) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), pp. 722-773) and Dalpozzo, et al. (1993), Int. J. Peptide Protein Res., 41: 561-566, incorporated herein by reference). According to this modification, the constituent peptides of the peptides of the present invention can be identical to the sequence of, for example, GIP described herein, except that one or more of the peptide bonds are replaced by retroinverso pseudopeptide bonds. Preferably, most of the N-terminal peptide bonds are replaced. This is because such substitutions 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 of similar structure. Another suitable pseudopeptide bond that is known to improve stability against enzymatic cleavage with little or no loss of biological activity is the reducing equivalent pseudopeptide bond (Couder, et al. (1993), Int. J. Peptide Protein Res., 41:181-184, incorporated herein by reference in its entirety).

[0295] Thus, the amino acid sequences of these peptides may be identical to those of, for example, novel GIP analogs and hybrid peptides, except that one or more of the peptide bonds are replaced by equivalent pseudopeptide bonds. Preferably, most of the N-terminal peptide bonds are replaced, since such replacement confers resistance to proteolysis by exopeptidases acting on the N-terminus. The synthesis of peptides with one or more reducing equivalent 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.

[0296] In another embodiment, the bond between the second and third residues that is targeted for cleavage by DPP-IV is replaced with a peptidase-resistant bond disclosed herein.

[0297] Peptoid derivatives of the peptides of the present invention represent another class of peptidomimetics that retain important structural determinants of biological activity but eliminate peptide bonds, thereby conferring resistance to proteolysis (Simon, et al., Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992), incorporated herein by reference in its entirety). Peptoids are oligomers of N-substituted glycines. A large number of N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid (Simon, et al. (1992), cited above). Some or all of the amino acids of the GIP peptide may be replaced with N-substituted glycines corresponding to the amino acid being replaced.

[0298] In one embodiment, the peptide of the invention contains a combination of the above modifications, ie, deletions, insertions, and substitutions.

[0299] Also included within the scope of the invention are constituent peptides in which the indicated amino acid residues have been chemically modified or derivatized (e.g., via fatty acid derivatization, PEGylation, amidation, glycolation, etc.). Exemplary embodiments include derivatization of lysine residues, particularly at positions 16 or 30. D-amino acid residues of the indicated amino acids are also contemplated within the scope of the invention. In another embodiment, exemplary GIP analog or hybrid polypeptides include polypeptides of the formulas having internal deletions, particularly in regions not corresponding to the active site as described herein.

[0300] Exemplary peptides of the invention may include substitutions of unnatural amino acids. For example, exemplary derivatives of the GIP analog or hybrid polypeptide of the invention include polymer-conjugated GIP analog or hybrid polypeptides that include any of the above insertions, deletions, substitutions, or combinations thereof, and the polymer molecule is conjugated at the lysine residue. In one embodiment, the peptide of the invention includes a derivative or substitution of methionine and has a longer duration of action compared to human GIP or analog. For example, octyl-glycine at methionine increases the in vivo duration of action of the compound. The duration of action was increased to at least 4 hours by this modification. Thus, in one embodiment, there are peptides of the invention that 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 by the addition of polyamino acids, such as poly-his, poly-arg, poly-lys, poly-glu, and poly-ala. Polypeptide modifications can also include small molecule substitutions, such as short chain alkyls and constrained alkyls (eg, branched, cyclic, fused, adamantyl), and aromatic groups.

[0301] D-Tyr1 and D-Ala2 variants of each peptide component herein are further specifically contemplated. In yet another embodiment, variants of each of the above sequences are contemplated, in which the peptide of the invention is modified with one, two or three modifications as described herein. Exemplary modifications are at the first, second or third N-terminal amino acid of GIP, which confer better DPP-IV resistance than native GIP. In yet a further embodiment, the novel peptide of the invention comprises a C-terminal amide.

[0302] In further embodiments, the peptides of the invention comprise a half-life that is at least twice 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.

[0303] In another embodiment, there is a pharma- ceutically acceptable salt of the peptide of the present invention.The peptide of the present invention may be formulated in a composition comprising a pharma- ceutically acceptable carrier.

[0304] Synthesis of the Peptides of the Invention The peptides of the invention can be synthesized by a number of techniques known to those of skill in the art. In certain embodiments, peptide subunits are synthesized, purified, and dimerized using techniques known in the art. In certain embodiments, the invention provides methods of producing the peptides of the invention (or subunits thereof), comprising chemically synthesizing a peptide that comprises, consists of, or consists essentially of a peptide having an amino acid sequence described herein, including, but not limited to, any of Formulas I-V or any of the amino acid sequences described in the tables herein. In other embodiments, the peptides are recombinantly synthesized instead of chemically synthesized. In certain embodiments, the peptides and methods of the invention comprise synthesizing both monomeric subunits of the peptide of the invention and then coupling the two subunits to produce the peptide of the invention. In various embodiments, the coupling or conjugation is accomplished via any of the various methods described herein.

[0305] In certain embodiments, the method of producing a peptide of the invention (or a monomeric subunit thereof) further comprises cyclizing the peptide of the invention (or a monomeric subunit thereof) after its synthesis. In certain embodiments, cyclization is accomplished via any of the various methods described herein. In certain embodiments, the invention provides a method of producing a peptide (or a monomeric subunit thereof) comprising introducing an intramolecular bond, e.g., a disulfide, amide, or thioether bond, between two amino acid residues in a peptide that comprises, consists of, or consists essentially of a peptide having an amino acid sequence described herein, including but not limited to any of Formulas (I)-(V), any of the amino acid sequences described in the accompanying Examples or Tables.

[0306] The peptides of the invention can be prepared using standard recombinant or chemical peptide synthesis techniques known in the art, for example, using automated or semi-automated peptide synthesizers, or both.

[0307] The peptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. Such methods are described, for example, in U.S. Pat. Nos. 6,610,824 and 5,686,411 and U.S. Patent Application Serial No. 454,533 (filed December 6, 1999), which are incorporated herein by reference in their entirety. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d.ed., Pierce Chemical Co. (1984); Tam et al, J. Am. Chem. Soc. 105:6442 (1983); Merrifield, Science 232:341-7 (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, 1-284 (1979). Solid phase peptide synthesis can be performed on an automated peptide synthesizer (e.g., Model 430A, Applied Biosystems Inc., Foster City, Calif.) using the NMP / HOBt (Option 1) system and tBoc or Fmoc chemistry with capping (see Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.). Peptides can also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.). Peptides can be purified by RP-HPLC (preparative and analytical) using, for example, a Waters Delta Prep 3000 system and a C4, C8, or C18 preparative column (10μ, 2.2×25 cm; Vydac, Hesperia, Calif.).Polypeptides can be synthesized by convergent methods, such as "native chemical ligation," and variations thereof, in which two or more peptide fragments with suitable orthogonal reactive termini are ligated via native amide bond formation. The newly formed peptides can be further ligated to create even longer polypeptides. The individual starting peptides can be derivatized as desired or can be derivatized after the ligation step.

[0308] Peptide analogs can be synthesized using PAL-PEG-PS resin (Applied Biosystems) with a loading of 0.2 mmol / g (0.25 mmol scale) on a Pioneer continuous flow peptide synthesizer (Applied Biosystems). Fmoc amino acid (4.0 equiv., 1.0 mmol) residues are activated using 4.0 equiv. HBTU, 4.0 equiv. HOBT, 8.0 equiv. DIEA and coupled to the resin for 1 h. 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 performed by treatment of the resin with Reagent B (93% TFA, 3% phenol, 3% water and 1% triisopropylsilane) for 2-3 h. 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 a C-18 column and an acetonitrile / water gradient containing 0.1% TFA. The purified product was lyophilized and analyzed by ESI-LC / MS and analytical HPLC and demonstrated to be pure (>98%). Mass results were all in agreement with calculated values.

[0309] Alternatively, peptides are assembled on a Symphonyl peptide synthesizer (Protein Technologies, Inc., Woburn, Mass.) using Rink amide resin (Novabiochem, San Diego, Calif.) at 0.050-0.100 mmol with a loading of 0.43-0.49 mmol / g. Fmoc amino acid (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 solutions in dimethylformamide. The Fmoc-protected amino acid is then coupled to the resin-bound amino acid using HBTU (2.0 equiv., 0.100-0.200 mmol), HOBT (1.8 equiv., 0.090-0.18 mmol), and N,N-diisopropylethylamine (2.4 equiv., 0.120-0.240 mmol) for 2 h. After the last amino acid coupling, the peptide is deprotected using 20% ​​(v / v) piperidine in dimethylformamide for 1 h. 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 composed 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 a C18 column and an acetonitrile / water gradient containing 0.1% TFA. Analytical HPLC is used to assess the purity of the peptides, and identity is confirmed by LC / MS and MALDI-MS.

[0310] Active proteins can be readily synthesized and then screened in screening assays designed to identify reactive peptides.

[0311] 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, 2d ed., Cold Spring Harbor (1989). The peptides of the present invention produced by recombinant techniques may be expressed from polynucleotides. Those skilled in the art will recognize that polynucleotides including DNA and RNA encoding such GIP analogs or hybrid peptides can be obtained from wild-type cDNAs, e.g., GIP, GLP1, amylin, taking into account the degeneracy of codon usage, or can be artificially engineered as desired. These polynucleotide sequences may incorporate codons that facilitate the transcription and translation of mRNA in a microbial host. Such manufacturing sequences can be readily constructed according to methods well known in the art. See, for example, WO 83 / 04053. The polynucleotides may also optionally code for an N-terminal methionyl residue. Non-peptide compounds useful in the present invention may be prepared by methods known in the art. For example, phosphate-containing amino acids and peptides containing such amino acids may be prepared using methods well known in the art. See, e.g., Bartlett and Landen, Bioorg. Chem. 14:356-77 (1986).

[0312] A variety of expression vector / host systems can be utilized to contain and express the coding sequence of the peptide of the present invention. 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 systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Mammalian cells useful in 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.

[0313] Thus, the polynucleotide sequences provided by the present invention are useful in the generation of new and useful viral and plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacteria, yeast, and mammalian cells grown in culture), and new and useful methods of culturing and growing such host cells capable of expressing the present GIP polypeptides.Polynucleotide sequences encoding the GIP analogs or hybrids herein may be useful in gene therapy where underproduction of GIP or other component peptide hormones of the hybrid is alleviated or a need for increased levels of such is met.

[0314] Host cells can be prokaryotic or eukaryotic, and include bacteria, mammalian cells (e.g., Chinese hamster ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells), yeast cells, and insect cells.

[0315] 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 for their particular ability to process expressed proteins or produce certain post-translational modifications useful in providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing cleaves the "prepro" form of a protein and can also be important for correct insertion, folding and / or function. Various host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular machinery and characteristic mechanisms of such post-translational activities and can be selected to ensure correct modification and processing of the introduced foreign protein.

[0316] Alternatively, a yeast system can be used to produce the peptides of the invention. For example, the coding region of the GIP polypeptide cDNA is amplified by PCR. DNA encoding the yeast pre-pro-alpha leader sequence is amplified from yeast genomic DNA using one primer that includes nucleotides 1-20 of the alpha mating factor gene and another primer that is complementary to nucleotides 255-235 of this gene in a PCR reaction (Kurjan and Herskowitz, Cell, 30:933-43 (1982)). The pre-pro-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 expression of a fusion protein consisting of the pre-pro-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 comprises the ADH2 transcription terminator downstream of the cloning site, the yeast "2-micron" origin of replication, the yeast leu-2d gene, the yeast REP1 and REP2 genes, the E. coli beta-lactamase gene, and the E. coli origin of replication. The beta-lactamase and leu-2d genes provide selection in bacteria and yeast, respectively. The leu-2d gene also facilitates increased copy number of the plasmid in yeast to induce higher levels of expression. The REP1 and REP2 genes encode proteins involved in regulating plasmid copy number.

[0317] The DNA construct described in the preceding paragraph is transformed into yeast cells using known methods, for example, lithium acetate treatment (Steams et al., Meth. Enz. 185:280-97 (1990)). The ADH2 promoter is induced upon depletion of glucose in the growth medium (Price et al., Gene 55:287 (1987)). The pre-pro-alpha sequence results in secretion of the fusion protein from the cell. Concomitantly, the yeast KEX2 protein cleaves the pre-pro sequence from the mature GIP polypeptide (Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330-4 (1984)).

[0318] The peptides of the invention can also be recombinantly expressed in yeast using commercially available expression systems, such as the Pichia Expression System (Invitrogen, San Diego, Calif.), following the manufacturer's instructions. This system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction with methanol. Secreted peptides are purified from the yeast growth medium, for example, by methods used to purify peptides from bacterial and mammalian cell supernatants.

[0319] Alternatively, a cDNA encoding the peptide of the invention can be cloned into an expression vector, such as the baculovirus expression vector pVL1393. This GIP compound-encoding vector is then used to infect Spodoptera frugiperda cells in sF9 protein-free medium according to the manufacturer's instructions (Sigma Chemical Co., St. Louis, MO) to produce recombinant protein. The protein is purified and concentrated from the medium using a heparin-Sepharose column (Pharmacia, Piscataway, NJ) and sequential molecular sizing columns (Amicon, Beverly, Mass.) and resuspended in PBS. SDS-PAGE analysis shows a single band, confirming the size of the protein, and Edman sequencing on a Proton 2090 Peptide Sequencer confirms its N-terminal sequence.

[0320] In another example, a DNA sequence encoding a peptide of the invention can be amplified by PCR and cloned into a suitable vector, e.g., pGEX-3X (Pharmacia, Piscataway, NJ). The pGEX vector is designed to produce a fusion protein that contains 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 contain 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, Calif.), and individual transformants are isolated and grown at 37° C. in LB medium (supplemented with carbenicillin) to an optical density of 0.4 at 600 nm, followed by an additional incubation of 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.

[0321] The fusion protein is expected to be produced as an insoluble inclusion body in bacteria and 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.) at room temperature for 15 minutes. The lysate is cleared by sonication and 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 6000×g for 30 minutes. The pellet is resuspended in standard phosphate-buffered saline solution (PBS) without Mg++ and Ca++. The fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 M KCl to visualize the protein, which is excised and electroeluted in gel running buffer lacking SDS. For example, if the GST / GIP polypeptide fusion protein is produced in bacteria as a soluble protein, it can be purified using the GST Purification Module (Pharmacia Biotech).

[0322] The fusion protein can be subjected to digestion to cleave the GST from the mature GIP analog or hybrid peptide. The digestion reaction (20-40 μg of fusion protein, 20-30 units of human thrombin (4000 U / mg (Sigma) in 0.5 mL of PBS) is incubated at room temperature for 16-48 h 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 bands corresponding to the predicted 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).

[0323] Transformed cells are preferably used for long-term, high-yield protein production, and therefore stable expression is desirable. Once such cells have been transformed with a vector containing a selectable marker along with the desired expression cassette, the cells can be grown in rich medium for 1-2 days before switching to selective medium. The selectable marker is designed to confer resistance to the selection, and its presence allows for the growth and recovery of cells that express the introduced sequence well. Resistant, stably transformed cell masses can be propagated using tissue culture techniques appropriate to the cells.

[0324] Many selection systems can be used to recover transformed cells for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine 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 aminoglycosides; G418, which confers resistance to chlorsulfuron; and hygro, which confers resistance to hygromycin. Additional selection genes that may be useful include trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine. Markers which provide a visual indication for identification of transformants include anthocyanins, beta-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

[0325] The peptides of the present invention can be produced using a combination of both automated peptide synthesis and recombinant techniques. For example, GIP peptides can contain a combination of modifications including deletion, substitution, and insertion by PEGylation. Such GIP polypeptides can be produced in stages. In a first stage, intermediate GIP polypeptides containing deletion, substitution, insertion, and any combination of modifications thereof can be produced by recombinant techniques described. Then, after an optional purification step described herein, the intermediate GIP peptide is PEGylated via chemical modification with a suitable PEGylation reagent (e.g., from NeKtar Transforming Therapeutics, San Carlos, Calif.) to produce the desired GIP peptide. Those skilled in the art will recognize that the above procedure can be generalized to apply to peptides of the present invention containing a combination of modifications selected from deletion, substitution, insertion, derivation, and other means of modification well known in the art, and are contemplated by the present invention.

[0326] It may be desirable to purify the GIP polypeptide produced according to the invention. Peptide purification techniques are well known to those skilled in the art. These techniques include, at one level, crude fractionation of the cellular milieu to peptide and non-peptide fractions. Once the peptide has been separated from other proteins, the peptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly efficient method of purifying peptides is reversed-phase HPLC followed by characterization of the purified product by liquid chromatography / mass spectrometry (LC / MS) and matrix-assisted laser desorption / ionization (MALDI) mass spectrometry. Additional confirmation of purity can be obtained by determining amino acid analysis.

[0327] Certain aspects of the invention relate to the purification, and in certain embodiments, the substantial purification, of an encoded protein or peptide. The term "purified peptide" as used herein is intended to refer to a composition that is isolable from other components, and the peptide is purified to any degree relative to its naturally occurring state. Thus, a purified peptide also refers to a peptide that is free from the environment in which it may naturally occur. In general, "purified" refers to a peptide composition that has been subjected to fractionation to remove various other components and that substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a composition in which the peptide forms the major component of the composition, for example, constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the peptide in the composition.

[0328] Various techniques suitable for use in peptide purification are well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc.; heat denaturation followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is contemplated that the order in which the various purification steps are performed may be altered or certain steps may be omitted and still result in a method suitable for the preparation of a substantially purified protein or peptide.

[0329] It is generally not required that peptides be provided in their most purified state at all times. In fact, it is contemplated that products purified to a substantially lesser extent may have utility in certain embodiments. Partial purification may be achieved by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is recognized that cation exchange column chromatography performed using HPLC equipment generally results in a "-fold" greater purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in overall recovery of protein product, or in maintaining activity of the expressed protein.

[0330] Optionally, such peptides of the present invention can 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 isolating and purifying G-CSF compositions that can be useful in isolating and purifying the GIP polypeptides of the present invention. In view of the disclosures of these patents, it is clear that those skilled in the art will fully appreciate the numerous purification techniques that can be used to purify the peptides of the present invention from a given source.

[0331] It is contemplated that a combination of anion exchange and immunoaffinity chromatography may be used to produce purified peptides of the invention.

[0332] Treatment Method The peptides of the present invention are useful in the treatment and prevention of metabolic and liver diseases and disorders.

[0333] Metabolic diseases and disorders take many forms, including obesity, diabetes, dyslipidemia, insulin resistance, cell apoptosis, and the like. Obesity and its associated disorders are common and extremely serious public health problems in the United States and around the world. Upper body obesity is the strongest known risk factor for type 2 diabetes mellitus, and a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, breast cancer, prostate cancer, and colon cancer, as well as an increased incidence of complications of general anesthesia (see, e.g., Kopelman, Nature 404:635-43 (2000)). Obesity reduces life span and carries a serious risk of the above comorbidities as well as disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholelithiasis, orthopedic injuries, and thromboembolic disease (Rissanen et al., Br. Med. J. 301:835-7 (1990)). Obesity is also a risk factor for a group of conditions called insulin resistance syndrome, or "syndrome X". Recent estimates of medical costs for obesity and related disorders are $2 trillion worldwide. Although the etiology of obesity is thought to be multifactorial, the fundamental problem is that nutrient availability and energy expenditure do not reach an equilibrium state until there is excess adipose tissue in the obese subject. Obesity is currently an undertreated, chronic, and essentially intractable metabolic disorder. Therapeutic agents useful in reducing the weight of obese individuals could have significant beneficial effects on their health.

[0334] Diabetes mellitus is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria resulting from insufficient production and utilization of insulin. Diabetes severely impacts 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 characterized as type 2 diabetes. However, it is now widely recognized that there are many distinct diabetes-related diseases that develop long before a patient is diagnosed with overt diabetes. Effects from suboptimal control of glucose metabolism in diabetes also result in a wide range of associated lipid and cardiovascular disorders.

[0335] Dyslipidemia, or abnormal levels of lipoproteins in plasma, occurs frequently among diabetic patients. Dyslipidemia is typically characterized by elevated plasma triglycerides, low HDL (high density lipoprotein) cholesterol, normal to elevated levels of LDL (low density lipoprotein) cholesterol, and increased levels of small dense LDL (low density lipoprotein) particles in the blood. Dyslipidemia is one of the main contributing factors to the increased incidence of coronary events and deaths among diabetic patients. Epidemiological studies support this by showing a several-fold increase in coronary deaths among diabetic patients compared to nondiabetic subjects. Several lipoprotein abnormalities have been described among diabetic patients.

[0336] Insulin resistance is the reduced ability of insulin to exert its biological action over a wide range of concentrations. In insulin resistance, the body secretes abnormally large amounts of insulin to compensate for this deficiency, and a state of impaired glucose tolerance develops. When the deficiency in insulin action cannot be compensated for, plasma glucose concentrations inevitably rise, resulting in the clinical state of diabetes. It is recognized that insulin resistance and relative hyperinsulinemia have a contributing role in obesity, hypertension, atherosclerosis, and type 2 diabetes. The association of insulin resistance with obesity, hypertension, and angina pectoris has been described as Syndrome X, a syndrome with insulin resistance as the common etiological link.

[0337] Nonalcoholic fatty liver disease (NAFLD) is a general term that encompasses everything from simple deposition of fat in the liver to more progressive steatosis associated with hepatitis, fibrosis, cirrhosis, and in some cases hepatocellular carcinoma. NAFLD is becoming more common worldwide, especially in Western countries. In the United States, it is the most common form of chronic liver disease, affecting about one-quarter of the population. Some individuals with NAFLD may develop an aggressive form of fatty liver disease, nonalcoholic steatohepatitis (NASH), which is characterized by inflammation of the liver and can progress to advanced scarring (cirrhosis) and liver failure. This damage is similar to that caused by heavy alcohol consumption. NAFLD includes nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). NAFL is characterized by hepatic steatosis involving more than 5% of the parenchyma, without evidence of hepatocellular injury. NASH, on the other hand, is defined by histological terms, which is a necroinflammatory process that leads to hepatocyte damage on a background of steatosis. The natural history of NAFLD remains incompletely characterized. Studies indicate that the incidence of NAFLD increases in concert with rising rates of metabolic syndrome. Patients with type 2 diabetes present an extremely high risk of developing NASH and a two- to four-fold increased risk of fatty liver-related complications.

[0338] Apoptosis is an active cellular self-destruction process regulated by extrinsic and intrinsic signals occurring during normal development. It is well documented that apoptosis plays a key role in the regulation of pancreatic endocrine beta cells. In adult mammals, there is growing evidence that beta cell mass is subject to dynamic changes to adapt insulin production to the maintenance of euglycemia in certain conditions, e.g., pregnancy and obesity. The control of beta cell mass depends on a delicate balance between cell proliferation, growth and programmed cell death (apoptosis). Disturbance of this balance can result in the impairment of glucose homeostasis. For example, impaired glucose tolerance develops with age, when the rate of beta cell replication decreases, and it should be noted that human autopsy studies have repeatedly shown a 40-60% reduction in beta cell mass in patients with non-insulin-dependent diabetes mellitus compared to non-diabetic subjects. Although insulin resistance is indispensable with obesity, it is generally agreed that euglycemia is maintained by compensatory hyperinsulinemia until the point at which beta cells are no longer able to meet the increased demand for insulin and type 2 diabetes begins.

[0339] Attempts to treat the multiple disorders associated with diabetes have prompted the administration of several antidiabetic pharmaceuticals to address these disorders in different patients. However, the peptides of the invention discussed herein, when administered in therapeutically effective amounts, find use as either monotherapy or adjunctive therapy in the treatment or prevention of these and other diseases and conditions discussed throughout.

[0340] In an embodiment of the invention, reduction of hyperglycemia in treated diabetic patients (e.g., by a GLP-1 mimetic, e.g., exenatide) is the starting point for intervention. Chronic hyperglycemic conditions in type 2 diabetic patients attenuate the insulinotropic stimulation response of GIP, while improved glycemic control resulting from exenatide treatment restores the responsiveness of pancreatic beta cells to GIP stimulation. Administration of the peptides of the invention results in the desired normoglycemia in diabetic patients or patients suffering from conditions associated with elevated glucose.

[0341] Currently prescribed antidiabetic agents (metformin, sulfonylureas, TZDs, SGLT2 inhibitors, etc.) can achieve varying degrees of glycemic control, so the combination of the peptides of the present invention with any of these therapies should also elicit an improved response leading to normalization of glucose levels.

[0342] Thus, in one embodiment, the method of the present invention is based on the concept that patients can be primed for therapy through prior glucose lowering with other antidiabetic agents, such as GLP-I, GLP-I analogs or exendin-4, or other agents, such as metformin, sulfonylureas, thiazolidinediones (TZDs), pramlintide, insulin, acarbose, dipeptidyl peptidase (DPP-IV) inhibitors, and SGLT-2 inhibitors.DPP-IV inhibitors are well known, and are described, for example, in US Patent Publication No. 20050004117, US Patent No. 6,710,040, and US Patent No. 6,645,995, which are incorporated herein by reference, for their compounds.An example of a sulfonylurea (SFU) that acts on pancreatic tissue to produce insulin is glimepiride.

[0343] The peptides of the present invention may be useful for reducing food intake, reducing appetite, reducing caloric intake, inducing satiety, reducing nutrient utilization, causing weight loss, affecting body composition, altering whole body energy content or energy expenditure, improving lipid profile (including reducing LDL cholesterol and triglyceride levels and / or altering HDL cholesterol levels), slowing gastrointestinal motility, delaying gastric emptying, attenuating postprandial blood glucose excursions, preventing or inhibiting glucagon secretion, and reducing blood pressure. In one embodiment, such GIP peptides contain exendin, GLP1, amylin and / or sCT moieties.

[0344] Those peptides of the present invention that contain GIP, GLP-1, exendin, GLP-1-GIP receptor coagonist, e.g., amylin (i.e., dual amylin calcitonin receptor agonist), leptin family module, which can effectively reduce food intake, change body composition, redistribute fat, and / or reduce body weight, are particularly interesting as anti-obesity, weight reduction, food reduction, metabolic rate increase, and body fat reduction and / or fat redistribution peptides. In an embodiment that is particularly interesting for the treatment of obesity and related diseases and conditions discussed herein (body fat reduction), there are peptides of the present invention that contain GIP, exendin-4 or analogs or derivatives thereof, amylin components, e.g., pramlintide or dual amylin calcitonin receptor agonists, FN38 family members, e.g., FN38 or analogs or derivatives thereof. In another embodiment, the peptides of the present invention may have at least one, preferably two components that act on the CNS. Specific regions of the forebrain (telencephalon- and diencephalon-derived components of the brain) and hindbrain or brainstem (including the midbrain, pons, and medulla) have been identified as involved in the control of energy balance. Forebrain structures or nuclei present in the hypothalamus involved in food intake and / or body weight modulation include, for example, the arcuate nucleus (ARC), paraventricular hypothalamic nucleus (PVN), dorsomedial hypothalamus (DMH), ventromedial nucleus (VMH), and lateral hypothalamic nucleus (LHA). Hindbrain structures or nuclei present in the brainstem involved in food intake and / or body weight modulation include, for example, the nucleus of the solitary tract (NST), area postrema (AP), and lateral parabrachial nucleus (IPBN). Brainstem nuclei that control elements of the consummatory motor control system are likely controlled by primary or secondary projections from hindbrain regions such as the NST, AP, and lPBN. It should be noted that the AP, NST, and lPBN have all been shown to have their own integrative capabilities (collectively and independently).

[0345] Various CNS-directed anti-obesity agents act on those forebrain structures present in the hypothalamus that are involved in food intake and / or body weight modulation.Furthermore, CNS-directed anti-obesity agents act on hindbrain structures present in the brainstem that are involved in food intake and / or body weight modulation.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, including those described herein.

[0346] In one embodiment, the peptides of the present invention and methods of use thereof comprise a first component that primarily targets the hypothalamic energy balance centers, such as ARC, PVN, VM, and LH. In one embodiment, the peptides of the present invention contain GIP or an analog or derivative thereof and one or more other peptide family components that also target the hypothalamus, but at a different location or via a different mechanism of action than the first component. When a GIP peptide contains two or more other peptide family components that also target the hypothalamus, the two or more 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 optionally contains one or more other peptide family components that provide one or more additional beneficial therapeutic effects, including anti-obesity effects, blood glucose control, cardioprotection, and / or hypertension control, via different locations or mechanisms of action from the first component and from each other. In certain embodiments, the additional peptide family members primarily target the energy balance centers of the hindbrain, such as the NST, AP, and lPBN.

[0347] In one embodiment, the peptide of the present invention and its method of use comprises a first component that primarily targets the energy balance centers of the hindbrain, such as NST, AP and lPBN. In one embodiment, the GIP peptide further comprises one or more other peptide family components that also target the hypothalamus, but at a different location or through a different mechanism of action from the first component and each other. In another embodiment, the peptide of the present invention then optionally comprises one or more other peptide family components that provide one or more additional beneficial therapeutic effects, including anti-obesity effects, blood glucose control, cardioprotection, and / or hypertension control, at a different location or through a different mechanism of action from the first component and each other. In one embodiment, the additional peptide family components primarily target the energy balance centers of the hypothalamus, such as ARC, PVN, VM, and LH.

[0348] As used herein, an anti-obesity agent that "acts on forebrain structures involved in food intake and / or body weight modulation" 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 reduced nutrient availability to the body. An anti-obesity agent that "acts on hindbrain structures involved in food intake and / or body weight modulation" 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 reduced nutrient availability to the body.

[0349] In another aspect, a method is provided for reducing fat mass by increasing metabolic rate in a subject, comprising administering an effective amount of a peptide of the present invention to reduce fat mass by increasing metabolic rate of the subject. Fat mass can be expressed as a percentage of total body weight. In some embodiments, fat mass is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% over the course of treatment. In one aspect, the subject's lean mass is not reduced over the course of treatment. In another aspect, the subject's lean mass is maintained or increased over the course of treatment. In another aspect, the subject is on a reduced or restricted calorie diet. "Reduced calorie diet" means that the subject is consuming 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 instances, the subject is consuming at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 fewer calories per day.

[0350] In another embodiment, a method of use is provided for modifying fat distribution, reducing fat mass, or both in a subject. Thus, subjects who would benefit from modifying body composition can also benefit from this method. The modification of body composition contemplated herein includes losing or maintaining body fat and minimizing the loss, maintenance, or gain of lean body mass. In this situation, weight can be increased as well as decreased. Thus, a subject can be non-obese, overweight, or obese, as those terms are commonly used in the art. The method provided can also include reducing fat in non-adipose tissue while sparing lean mass. Use of this method includes treating diseases, such as non-alcoholic steatohepatitis (NASH) or lipodystrophy.

[0351] In another embodiment, a method of altering fat distribution in a subject is provided, comprising administering an effective amount of the anti-obesity peptide of the present invention 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" refers to the location of fat deposits in the body. Such locations of fat deposits include, for example, subcutaneous, visceral, and ectopic fat depots. "Subcutaneous fat" refers to deposits of lipids just below the surface of the skin. The amount of subcutaneous fat in a subject can be measured using any method available for measuring subcutaneous fat. Methods of measuring subcutaneous fat are known in the art, such as those described in U.S. Patent No. 6,530,886, which is incorporated herein by reference in its entirety. "Ectopic fat storage" refers to lipid deposits in or around tissues and organs that constitute lean body mass (e.g., skeletal muscle, heart, liver, pancreas, kidneys, blood vessels). In general, ectopic fat storage is the accumulation of lipids outside of classical adipose tissue depots in the body. "Visceral fat" refers to the deposition of fat as intraperitoneal adipose tissue. Visceral fat surrounds vital organs and can be metabolized by the liver to produce blood cholesterol. Visceral fat is associated with increased risk of pathologies, such as polycystic ovarian syndrome, metabolic syndrome, and cardiovascular disease. In some embodiments, the method includes metabolism of visceral or ectopic fat, or both, at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% greater than metabolism for subcutaneous fat. In one aspect, the method results in favorable fat distribution. In one embodiment, favorable fat distribution is an increased ratio of subcutaneous fat to visceral fat, ectopic fat, or both. In one aspect, the method includes an increase in lean body mass, for example, as a result of an increase in muscle cell mass.

[0352] In another embodiment, a method for reducing the amount of subcutaneous fat in a subject is provided, comprising administering to a subject in need thereof an amount of an anti-obesity peptide of the present invention effective to reduce the amount of subcutaneous fat in the subject. In one example, the amount of subcutaneous fat is reduced by at least about 5% in the subject. In another example, the amount of subcutaneous fat is reduced by at least about 10%, 15%, 20%, 25%, 30% 40%, or 50% compared to the subject before administration of the anti-obesity peptide of the present invention.

[0353] 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 a subject. In another example, visceral fat is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50% in a subject compared to the subject before administration of the anti-obesity peptide of the present invention. Visceral fat can be measured through any means available for determining the amount of visceral fat in a subject. Such methods include, for example, CT scan and MRI abdominal tomography. Other methods of determining visceral fat are described, for example, in U.S. Patent Nos. 6,864,415, 6,850,797, and 6,487,445.

[0354] In another embodiment, a method for preventing ectopic fat accumulation or reducing the amount of ectopic fat in a subject is provided, comprising administering to a subject in need thereof an amount of the anti-obesity peptide of the present invention effective to prevent ectopic fat accumulation or reduce the amount of ectopic fat in the subject. In one example, the amount of ectopic fat is reduced in the subject by at least about 5% compared to the subject before administration of the anti-obesity peptide of the present invention. In another example, the amount of ectopic fat is reduced in the subject by at least about 10%, or at least about 15%, 20%, 25%, 30%, 40%, or 50%. Alternatively, the amount of ectopic fat is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to the subcutaneous fat in the subject. Ectopic fat can be measured in the subject using any method available for measuring ectopic fat.

[0355] In another embodiment, a method for producing a more favorable fat distribution in a subject is provided, comprising administering to the subject an effective amount of a peptide of the present invention effective as an anti-obesity agent to produce a favorable fat distribution. In one embodiment, administration of the anti-obesity peptide of the present invention reduces the amount of visceral fat or ectopic fat, or both, in the subject. In one embodiment, the anti-obesity peptide of the present invention is administered, which comprises a combination of at least one family module acting on a forebrain structure involved in food intake or body weight modulation, or both, and at least one family module acting on a hindbrain structure involved in food intake or body weight modulation, or both. In one embodiment, the method preferentially reduces the amount of visceral or ectopic fat, or a combination of both, compared to reducing 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, cystic ovarian 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 least 10%, 15%, 20%, 25%, 30% 50%, 60%, 70%, 80%, 90%, or 100% more rapidly than subcutaneous fat.

[0356] For example, peptides of the invention containing GIP, exendin, amylin, (i.e., dual amylin calcitonin receptor agonists), leptin, or GLP-1-GIP receptor coagonists are of particular interest for anti-obesity, weight and fat composition related treatments as discussed herein. In yet further embodiments, peptides of the invention contain at least two of these peptide family modules. The peptides of the invention can be administered alone or in combination with a second anti-obesity agent, such as an amylin, leptin, or exendin family peptide.

[0357] In yet another aspect, a method is provided for administering a therapeutically effective amount of the peptide of the present invention effective as an anti-obesity agent administered in combination with glucocorticosteroids. Glucocorticosteroids have the adverse effect of increasing fat mass and decreasing lean mass. Thus, it is contemplated that this combination of anti-obesity agents can be used with glucocorticosteroids under conditions where glucocorticosteroid use is beneficial to counteract the adverse effects of glucocorticosteroids.

[0358] As discussed herein, the peptide of the present invention can be administered separately or together with one or more other drugs to obtain additional benefits or to improve the effect of either the peptide of the present invention or other drugs.For example, the anti-obesity peptide of the present invention can be administered with anti-obesity drugs or cardioprotective or antihypertensive drugs depending on the risk factors of the subject who needs treatment and the desired treatment outcome.Exemplary anti-obesity drugs for administration (either separately or mixed; either before, simultaneously or after) with the peptide of the present invention include serotonin (5HT) transport inhibitors, including but not limited to paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline, and imipramine. Antiobesity agents also include selective serotonin reuptake inhibitors, including, but not limited to, dexfenfluramine, fluoxetine, sibutramine, and combinations thereof, as well as those described in U.S. Patent No. 6,365,633 and PCT Patent Application Publications WO 01 / 27060 and WO 01 / 162341, which are incorporated herein 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 previously described.

[0359] Anti-obesity agents also include selective serotonin agonists and selective 5-HT2C receptor agonists, including, but not limited to, those described in U.S. Patent No. 3,914,250, which is incorporated herein by reference in its entirety; and PCT published applications WO 02 / 36596, WO 02 / 48124, WO 02 / 10169, WO 01 / 66548, WO 02 / 44152; WO 02 / 51844, WO 02 / 40456, and WO 02 / 40457.Such selective serotonin agonists and selective 5-HT2C receptor agonists, compositions containing such agonists, and suitable routes of administration for use in the provided methods are known in the art. See, e.g., Halford et al. (2005) Curr. Drug Targets 6:201-213 and Weintraub et al. (1984) Arch. Intern. Med. 144:1143-1148.

[0360] Anti-obesity agents also include antagonists / inverse agonists of the central cannabinoid receptor (CB-1 receptor), including, but not limited to, rimonabant (Sanofi Synthelabo), and SR-147778 (Sanofi Synthelabo). CB-1 antagonists / inverse agonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration are described in, e.g., U.S. Pat. Nos. 6,344,474, 6,028,084, 5,747,524, 5,596,106, 5,532,237, 4,973,587, 5,013,837, 5,081,122, 5,112,820, 5,292,736, 5,624,941; European Patent Application No. 656354 and European Patent Application No. 656354, which are incorporated herein by reference in their entirety. No. 658546; and published PCT applications WO 96 / 33159, WO 98 / 33765, WO 98 / 43636, WO 98 / 43635, WO 01 / 09120, WO 98 / 31227, WO 98 / 41519, WO 98 / 37061, WO 00 / 10967, WO 00 / 10968, WO 97 / 29079, WO 99 / 02499, WO 01 / 58869, and WO 02 / 076949.

[0361] Anti-obesity agents also include melanocortins and melanocortin agonists. The receptor MC4R is believed to play a role in energy balance and obesity. See, for example, Anderson et al., Expert Opin.Ther.Patents 11:1583-1592 (2001); Speake et al., Expert Opin.Ther.Patents 12:1631-1638 (2002); Bednarek et al., Expert Opin.Ther.Patents 14:327-336 (2004). Melanocortin agonists, including but not limited to MC4R agonists, and compositions containing such agonists suitable for use in the provided methods are known in the art. MCR agonists, MC4R agonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages and routes of administration have been previously described, for example, in the following PCT patent applications WO 03 / 007949, WO 02 / 068388, WO 02 / 068387, WO 02 / 067869, WO 03 / 040117, WO 03 / 066587, WO 03 / 068738, WO 03 / 094918, and WO 03 / 031410, which are incorporated herein by reference in their entireties.

[0362] Anti-obesity agents also include metabotropic glutamate subtype 5 receptor (mGluR5) antagonists, including compounds such as 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine) (MTEP) and 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).

[0363] Anti-obesity agents also include topiramate, phentermine or combinations thereof, which have been indicated as anticonvulsants and anticonvulsants, but have also been shown to increase weight loss.

[0364] Anti-obesity agents also include neuropeptide Y1 (NPY1) antagonists and NPY5 antagonists.NPY1 and NPY5 antagonists are known in the art.See, for example, Duhault et al. (2000) Can.J.Physiol.Pharm.78:173-185, and U.S. Patent Nos. 6,124,331, 6,214,853, and 6,340,683.NPY1 and NPY5 antagonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and administration routes have been described. NPY1 antagonists useful in the provided compositions and methods include those described in U.S. Pat. No. 6,001,836, which is incorporated herein by reference in its entirety; and PCT publications WO 96 / 14307, WO 01 / 23387, WO 99 / 51600, WO 01 / 85690, WO 01 / 85098, WO 01 / 85173, and WO 01 / 89528.NPY5 antagonists useful in the compositions and methods of use provided herein include, but are not limited to, those disclosed in 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 Patent Nos. 01010691 and 01044970; and PCT published patent applications WO 97 / 19682, WO 97 / 20820, WO 97 / 20821, WO 97 / 20822, WO 97 / 20823, WO 98 / 2 7063, WO 00 / 64880, WO 00 / 68197, WO 00 / 69849, WO 01 / 09120, WO 01 / 85714, WO 01 / 85730, WO 01 / 07409, WO 01 / 02379, WO 01 / 02379, WO 01 / 2338 8, WO 01 / 23389, WO 01 / 44201, WO 01 / 62737, WO 01 / 62738, WO 01 / 09120, WO 02 / 22592, WO 0248152, WO 02 / 49648, and WO 01 / 14376.

[0365] Anti-obesity agents also include melanin-concentrating hormone (MCH) antagonists, including melanin-concentrating hormone 1 receptor (MCH1R) antagonists, such as T-226296 (Takeda), and melanin-concentrating hormone 2 receptor (MCH2R) antagonists. MCH receptor antagonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration have been previously described, for example, in U.S. Patent Application Publication Nos. 2005 / 0009815, 2005 / 0026915, 2004 / 0152742, 2004 / 0209865; PCT Patent Application Publication Nos. WO 01 / 82925, WO 01 / 87834, WO 02 / 06245, WO 02 / 04433, and WO 02 / 51809; and Japanese Patent Application JP 13226269, which are incorporated herein by reference in their entireties.

[0366] Anti-obesity agents also include opioid antagonists, including, but not limited to, those described in PCT Application WO 00 / 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, delta 1 ([D-Ala2,Leu5,Cys6]-enkephalin (DALCE), naltrindole isothiacinate, and nor-binaltorfamine, or combinations thereof.

[0367] Anti-obesity agents also include orexin antagonists, including, but not limited to, those described in PCT Patent Applications WO 01 / 96302, WO 01 / 68609, WO 02 / 51232, and WO 02 / 51838. Particular orexin antagonists useful in the provided compositions and methods of use include, but are not limited to, SB-334867-A.

[0368] Anti-obesity agents 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 ... Also included are neuropeptide Y2 (NPY2) agonists, including compounds such as those described in, for example, Batterham et al. (2003) J. Clin. Endocrinol. Metab. 88:3989-3992, and other Y4 agonists, such as 1229U91 (Raposinho et al. (2000) Neuroendocrinology 10:111-112, which may be co-administered with the peptides of the invention or administered separately. Anti-obesity agents provided include, but are not limited to, 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 10:111-112, ... Also included are neuropeptide Y4 (NPY4) agonists, including compounds such as NPY2 agonists, NPY4 agonists, derivatives, preparations, formulations, pharmaceutical compositions, dosages, and routes of administration, which have been previously described, for example, in U.S. Patent Application Publication No. 2002 / 0141985 and PCT Publication No. WO 2005 / 077094.

[0369] Anti-obesity agents include, but are not limited to, those described in PCT Application WO 02 / 15905, O-[3-(1H-imidazol-4-yl)propanol]carbamates (Kiec-Kononowicz et al. (2000) Pharmazie 55:349-355), piperidine-containing histamine H3 receptor antagonists (Lazewska et al. (2001) Pharmazie 56:927-932), benzophenone derivatives and related compounds (Sasse et al. (2001) Arch. Pharm. (Weinheim) 334:45-52), substituted N-phenylcarbamates (Reidemeister et al. (2000) Pharmazie 55:83-86), and prosixiphan derivatives (Sasse et al. (2001) Pharmazie 55:83-86). Also included are histamine 3 (H3) antagonists / inverse agonists, including those described herein (Bach et al. (2000) J. Med. Chem. 43:3335-3343). 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, iodophenpropit, imoproxyfan, and GT2394 (Gliatech).

[0370] Anti-obesity agents also include cholecystokinin (CCK) and CCK agonists. The cholecystokinin-A (CCK-A) agonists used include, but are not limited to, those described in U.S. Patent No. 5,739,106. Specific CCK-A agonists include, but are not limited to, AR-R15849, GI181771, JMV-180, A-71378, A-71623 and SR146131.

[0371] Anti-obesity agents also include ghrelin antagonists, such as those described in PCT Publications WO 01 / 87335 and WO 02 / 08250. Ghrelin antagonists are known as GHS (growth hormone secretagogue receptor) antagonists. Thus, the provided compositions and methods contemplate the use of GHS antagonists in place of ghrelin antagonists.

[0372] Anti-obesity agents include obestatin and obestatin analogs and agonists. Obestatin is a peptide derived from the same precursor, preproghrelin, from which ghrelin is derived. See, for example, Zhang et al. (2005) Science 310:996-999; Nogueiras et al. (2005) Science 310:985-986; Pan et al. (2006) Peptides 27:911-916. In contrast to the activity of ghrelin, obestatin is believed to act as an appetite suppressant hormone by reducing food intake, gastric emptying activity, jejunal motility, and weight gain. Obestatin peptides that can be used include, but are not limited to, those described in Zhang et al. (2005) Science 310:996-999.

[0373] Amylinomimetics (e.g., amylin-calcitonin receptor coagonists, e.g., davalintide), GLP-1-GIP receptor coagonists, e.g., incretins (e.g., exendin-4, leptin, and PYY analogs), are also anti-obesity agents that may be administered as anti-obesity agents in the same manner as the GIP-containing peptides of the present invention.

[0374] Thus, in certain embodiments, the peptides of the present invention are useful for the treatment or prevention of conditions or disorders that can be alleviated by reducing nutrient availability, comprising administering to said subject a therapeutically or prophylactically effective amount of a compound of the present 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, control of positive inotropy, reduction of catabolism, slowing of gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, rare genetic disorders of eating disorders and obesity, e.g., Prader-Willi syndrome, critical care, insulin resistance and disorders thereof, e.g., polycystic ovarian syndrome, obesity, diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes, and CNS disorders, e.g., prevention of neurodegeneration, Alzheimer's disease and Parkinson's disease, and non-alcoholic steatohepatitis (NASH), and complications from diabetes, e.g., neuropathy (e.g., epilepsy, rheumatoid arthritis ... and conditions such as inflammatory bowel disease, osteoarthritis, and osteoarthritis. These conditions include, for example, conditions such as osteoarthritis, osteoporosis, and osteoarthritis. These conditions include conditions such as osteoarthritis, osteoporosis, and osteoarthritis. These conditions include conditions such as osteoarthritis, osteoporosis, and osteoarthritis. These conditions include conditions such as osteoarthritis, osteoporosis, and osteoarthritis.

[0375] Non-limiting examples of cardiovascular conditions or diseases are hypertension, myocardial ischemia, and myocardial reperfusion. Compounds of the invention may also be useful in the treatment or prevention of stroke, cancer (e.g., endometrial, breast, prostate, and colon cancer), gallbladder disease, sleep apnea, low fertility, and other conditions associated with obesity, including osteoarthritis (see Lyznicki et al, Am. Fam. Phys. 63:2185, 2001). In other embodiments, peptides of the invention may be used to alter body composition for aesthetic reasons, to improve the physical performance of a person, or to produce a lean meat source. Peptides of the invention are useful for altering body composition by reducing fat without a significant loss of muscle mass, thus resulting in desirable loss of body fat while preserving lean body mass. In one embodiment, such peptides of the invention contain exendin, GLP1, amylin, and / or sCT moieties.

[0376] In another aspect of the present invention, a method for treating or preventing obesity is provided, comprising administering a therapeutically or prophylactically effective amount of a peptide of the present invention to a subject in need thereof. In an exemplary embodiment, the subject is an obese or overweight subject. While "obesity" is generally defined as a body mass index greater than 30, for the purposes of this disclosure, any subject with a body mass index less than 30, including those who need or desire to lose weight, is included in the scope of "obesity". Subjects who are insulin resistant, glucose intolerant, or have any form of diabetes mellitus (e.g., type 1, 2, or gestational diabetes) may benefit from these peptides of the present invention. In one embodiment, such peptides of the present invention contain GIP, amylin calcitonin receptor agonists, such as davalintide, GLP-1-GIP receptor coagonists, such as exendin, GLP1, amylin, and / or sCT moieties.

[0377] In another aspect of the present invention, there is provided a method of reducing food intake, reducing nutrient availability, inducing weight loss, affecting body composition, and altering total body energy content or increasing energy expenditure, treating diabetes mellitus, and improving lipid profile (including reducing LDL cholesterol and triglyceride levels and / or changing HDL cholesterol levels), comprising administering to a subject an effective amount of a peptide of the present invention. In an exemplary embodiment, a 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 of such treatment or prevention, comprising administering to said 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 any type of diabetes mellitus. In one embodiment, such a peptide of the present invention contains a GIP, exendin, GLP1, amylin and / or sCT moiety.

[0378] Additional assays useful in the present invention include those that can determine the effect of the peptides of the present invention, particularly those containing GIP, exendin, GLP1, amylin and / or sCT moieties, on body composition. An exemplary assay can involve the use of a diet-induced obese (DIO) mouse model for metabolic disease. Prior to the treatment period, male C57BL / 6J mice can be fed a high fat diet (#D12331, 58% calories from fat; Research Diets, Inc.,) for 6 weeks, starting at 4 weeks of age. During the study, the mice can continue to eat their high fat diet. Water can be provided ad libitum throughout the study. A group of age-matched non-obese mice can be fed a low fat diet (#D12329, 11% calories from fat) for the purpose of comparing metabolic parameters to the DIO group.

[0379] DIO mice can be implanted with subcutaneous (SC) intrascapular osmotic pumps to deliver either vehicle (50% dimethylsulfoxide (DMSO) in water) or a peptide of the invention. Pumps for the peptide of the invention group can be set to deliver any amount, for example, 1000 μg / kg / day of a peptide of the invention for 7-28 days. Body weight and food intake can be measured at regular intervals throughout the study period. Respiratory quotient (RQ, defined as CO2 production ÷ O2 consumption) and metabolic rate can be determined using indirect calorimetry (Oxymax, Columbus Instruments, Columbus, Ohio) in individual animals. Mice can be euthanized by isoflurane overdose and indices of adiposity (bilateral epididymal fat pad weights) can be measured. Furthermore, prior to determining epididymal weight, body composition (lean mass, fat mass) for each mouse can be analyzed using a dual energy X-ray absorptiometry (DEXA) machine according to the manufacturer's instructions (Lunar Piximus, GE Imaging System). In the methods of the invention, peptides of the invention, particularly those containing GIP, exendin, PPF, PYY, GLP1, amylin and / or sCT moieties, can be identified that have a higher potency in one of the assays described herein (preferably food intake, gastric emptying, pancreatic secretion, weight loss or body composition assays) than the potency of the constituent peptide hormones in that same assay.

[0380] In addition to improving hypertension in subjects in need of such improvement as a result of reduced food intake, weight loss, or treatment of obesity, the peptides of the present invention may be used to treat hypotension.

[0381] In another general aspect, the peptides of the invention may be used to inhibit the secretion of ghrelin, and thus may exploit this mechanism to treat or prevent ghrelin-related disorders, such as Prader-Willi syndrome, all types of diabetes and its complications, obesity, hyperphagia, hyperlipidemia, or other disorders associated with overnutrition.

[0382] The peptides of the present invention may also be useful for enhancing, inducing, improving 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 in U.S. Patent Application Publication No. 2004 / 0228846. Assays for determining such activities are known in the art. For example, in published U.S. Patent Application Publication No. 2004 / 0228846 (incorporated by reference in its entirety), islet isolation and culture and assays for determining fetal islet maturation are described. In the examples of patent application U.S. Patent Application Publication No. 2004 / 0228846, gut-derived hormone peptides including secretin, glucagon-like peptide-1 (GLP-I) and bombesin were purchased from Sigma. Collagenase type XI was obtained from Sigma. RPMI1640 culture medium and fetal bovine serum were obtained from Gibco. A radioimmunoassay kit containing anti-insulin antibody ([125I]-RIA kit) was purchased from Linco, St Louis.

[0383] The peptides of the invention are useful for the prevention and treatment of nephropathy, including hypertensive and diabetic nephropathy, and nephropathy associated with insulin resistance and metabolic syndrome. The peptides of the invention achieve these objectives by improving or preventing the deterioration of hypertension, endothelial function, renal function, and glomerulosclerosis, among others. In one embodiment, the invention provides a method for preventing or treating nephropathy, including hypertensive and diabetic nephropathy, or nephropathy associated with insulin resistance, comprising administering a compound of the invention. The peptides of the invention are further used for improving endothelial function in patients with reduced vasodilatory capacity or with glomerulosclerosis or any other reduction in glomerular flow. Such improvement of endothelial function serves both to reduce hypertension and to improve the function of the glomerular capillaries. In additional embodiments, the molecules of the invention are useful for preventing the progression of nephropathy to ESRD or for preventing, slowing the progression of, treating or ameliorating proteinuria and / or glomerulosclerosis.

[0384] The peptides of the present invention are useful for reducing the risk of suffering from, preventing, or treating cardiac arrhythmias. The peptides of the present invention may provide antiarrhythmic effects in patients with cardiac ischemia, cardiac ischemia-reperfusion, and congestive heart failure. For example, the incretin GLP-I has been found to reduce cardiac damage and improve recovery in patients with these disorders. Incretins, including GLP-I, are glucose-dependent insulinotropic hormones. GLP-I and exendins effectively improve peripheral glucose uptake without inducing dangerous hypoglycemia. They also strongly suppress glucagon secretion independent of their insulinotropic effect, thereby reducing plasma free fatty acid (FFA) levels significantly more potently than can be achieved with insulin. High FFA levels have been implicated as the main toxicity mechanism during myocardial ischemia. In another embodiment, the peptides of the present invention are useful for preventing and treating cardiac arrhythmias, as they reliably reduce the damage associated with reperfusion and ischemia and improve patient recovery. In yet further embodiments, treatment following acute stroke or hemorrhage, preferably by intravenous administration, provides a means to optimize insulin secretion, increase brain anabolism, improve insulin efficacy through suppression of glucagon, and maintain normoglycemia or mild hypoglycemia without the risk of severe hypoglycemia or other adverse side effects. In one embodiment, such peptides of the invention contain a GIP, GLP1 or exendin moiety.

[0385] In yet a further embodiment, the peptide of the present invention, which can lower insulin resistance or increase insulin sensitivity, is useful for treating polycystic ovarian syndrome (PCOS). The administration of the peptide of the present invention can reduce or prevent insulin resistance in a subject suffering from PCOS. In yet another embodiment, the peptide of the present invention prevents the onset of type 2 diabetes in a subject suffering from PCOS. The further peptide of the present invention can restore regular menstruation, ovulation, or fertility in a subject suffering from PCOS. In one embodiment, such a peptide of the present invention containing GIP also contains a GLP1 or exendin moiety for binding and activating GLP1 receptor.

[0386] Depending on the selection of the peptide of the present invention, it may exhibit a wide range of biological activities, some of which are related to its antisecretory and antimotility properties.The peptide of the present invention may suppress gastrointestinal secretion by direct interaction with epithelial cells, or perhaps by inhibiting the secretion of hormones or neurotransmitters that stimulate intestinal secretion.Antisecretory properties include inhibition of gastric juice and / or pancreatic secretion, which may be useful in treating or preventing diseases and disorders including gastritis, pancreatitis, Barrett's esophagus, and gastroesophageal reflux disease, and the associated conditions thereof, including heartburn, heartburn with reflux of stomach / intestinal contents into the mouth or lungs, dysphagia, cough, intermittent wheezing, and vocal cord inflammation (conditions associated with GERD), esophageal erosion, esophageal ulcer, esophageal stenosis, Barrett's metaplasia (replacement of normal esophageal epithelium with abnormal epithelium), Barrett's esophageal adenocarcinoma, and aspiration. In another embodiment, the GIP peptides of the invention containing amylin and / or sCT moieties may be useful in the treatment or prevention of these diseases and conditions, such as Barrett's esophagus, gastroesophageal reflux disease (GERD) and associated conditions disclosed herein. Such peptides of the invention have particularly effective antisecretory properties, such as inhibition of gastric acid, inhibition of bile acids, and inhibition of pancreatic enzymes. Furthermore, such peptides of the invention may have a gastroprotective effect, making them particularly useful in the treatment or prevention of intestinal diseases and conditions, and Barrett's esophagus, and / or GERD and associated or associated conditions described herein.

[0387] In another general aspect, the peptides of the 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 another embodiment, the peptides of the invention are useful for treating pain and painful neuropathies. In one embodiment, such peptides of the invention contain an exendin, GLP1, amylin and / or sCT moiety. For example, the GIP-sCT or GIP-amylin / sCT peptides of the invention may have the selectable properties of salmon calcitonin or amylin / sCT / amylin chimeras, such as reducing bone loss and bone resorption or reducing cartilage turnover (chondroprotection), and the properties of GIP, such as lowering plasma glucose (associated with an anti-catabolic state as described herein) and / or inhibiting bone resorption and maintaining or increasing bone density. The peptides of the invention with such selectable properties may improve the treatment of osteoporosis or conditions of high cartilage turnover, particularly in those who would also benefit from glycemic control, such as subjects with diabetes or in critical care.

[0388] Pharmaceutical Compositions In certain embodiments, the peptide of the present invention or pharmaceutical composition comprising the peptide of the present invention is suspended in a sustained release matrix. A sustained release matrix as used herein is a matrix made of a material, usually a polymer, that is degradable by enzymatic or acid-base hydrolysis or by dissolution. When inserted into the body, the matrix is ​​acted upon by enzymes and body fluids. The sustained release matrix is ​​preferably selected from biocompatible materials, such as liposomes, polylactides (polylactic acids), polyglycolides (polymers of glycolic acid), polylactide-co-glycolides (copolymers of lactic and glycolic acids), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. One embodiment of a biodegradable matrix is ​​a matrix of either polylactide, polyglycolide, or polylactide-co-glycolide (copolymers of lactic and glycolic acid).

[0389] In one embodiment, the present invention includes pharmaceutical compositions comprising one or more peptides of the present invention and a pharma- ceutically acceptable carrier, diluent, or excipient. A pharma- ceutically acceptable carrier, diluent, or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation auxiliary. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like.

[0390] In some embodiments, the composition is administered orally, parenterally, intravesically, intravaginally, intraperitoneally, intrarectally, topically (by powder, ointment, eye drops, suppository, or transdermal patch), by inhalation (e.g., intranasal spray), ocularly (e.g., intraocular), or bucally. As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intraarticular injection and infusion. Thus, in some embodiments, the composition is formulated for delivery by any of these routes of administration.

[0391] In some embodiments, pharmaceutical compositions for parenteral injection include sterilized pharma- ceutical acceptable aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, β-cyclodextrin, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. For example, proper fluidity can be maintained by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0392] Injectable depot forms include those made by forming microencapsule matrices of the peptides of the invention in one or more biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters), poly(anhydrides), and (poly)glycols, such as PEG. Depending on the ratio of peptide to polymer and the nature of the particular polymer used, the release rate of the peptide inhibitor can be controlled. Depot injectable formulations are also prepared by entrapping the peptide in liposomes or microemulsions that are compatible with body tissues.

[0393] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water, or other sterile injectable medium immediately before use.

[0394] Topical administration includes administration to the skin or mucous membranes, including the lungs and the surface of the eye. Compositions for topical pulmonary administration, including those for inhalation and intranasal use, can include solutions and suspensions in aqueous and non-aqueous formulations, and can be prepared as dry powders, which can be pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredient can be in finely divided form, and can be used in admixture with larger sized pharma- ceutically acceptable inert carriers, including, for example, particles with a diameter of up to 100 micrometers. Suitable inert carriers include sugars, such as lactose.

[0395] Alternatively, the composition may be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant. The liquefied propellant medium, and indeed the total composition, may be such that the active ingredient does not dissolve therein to any substantial extent. The pressurized composition may also contain a surfactant, such as a liquid or solid non-ionic surfactant, or may be a solid anionic surfactant. It is preferred to use a solid anionic surfactant in the form of a sodium salt.

[0396] A further form of local administration is administration to the eye. The peptide of the present invention can be delivered in a pharma- ceutically acceptable ophthalmic vehicle so that the peptide is maintained in contact with the ocular surface for a period of time sufficient for the peptide to penetrate the cornea and inner regions of the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid / retina and sclera. The pharma-ceutically acceptable ophthalmic vehicle can be, for example, an ointment, vegetable oil or an encapsulating material. Alternatively, the peptide of the present invention can be directly injected into the vitreous and aqueous humor.

[0397] Compositions for rectal or vaginal administration preferably include suppositories, which may be prepared by mixing the peptides of the present invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which is solid at room temperature but liquid at body temperature and therefore will melt in the rectal or vaginal cavity and release the active compound.

[0398] The peptides of the present invention may also be administered in liposomes or other lipid-based carriers. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes may be used. The present composition in liposomal form may contain stabilizers, preservatives, excipients, etc., in addition to the peptides of the present invention. In some embodiments, lipids include phospholipids, including phosphatidylcholines (lecithins) and serine, both natural and synthetic. Methods of forming liposomes are known in the art.

[0399] The peptides of the present invention may also be administered using the FluidCrystal® technology described by Camurus. These formulations are based on a unique combination of endogenous polar lipids that spontaneously form liquid crystal nanostructures in aqueous environments; on tissue surfaces, or within the body. Such FluidCrystal® injectable depots are designed to provide therapeutic efficacy over an extended period (days to months) with a single injection, potentially reducing the burden of daily medication while increasing compliance with therapy.

[0400] FluidCrystal® injectable depots contain a lipid-based liquid with the peptide of the invention dissolved therein that can be easily injected subcutaneously using a conventional syringe with a fine needle. Upon contact with body fluids in tissue, the lipid solution transforms into a liquid crystalline gel, which effectively encapsulates the peptide. The drug compound is slowly released as the liquid crystalline matrix gradually degrades in the tissue, and release can be controlled over a period of days to weeks or months, depending on the composition.

[0401] Pharmaceutical compositions to be used in the present invention suitable for parenteral administration may comprise sterile aqueous solutions and / or suspensions of the peptide inhibitors, generally made isotonic with the blood of the recipient using sodium chloride, glycerin, glucose, mannitol, sorbitol, or the like.

[0402] In some embodiments, the present invention provides pharmaceutical compositions for oral delivery. The compositions and peptides of the present invention can be prepared for oral administration according to any of the methods, techniques, and / or delivery vehicles described herein. Furthermore, those skilled in the art will recognize that the peptides of the present invention can be modified or integrated into systems or delivery vehicles that are not disclosed herein but are well known in the art and are compatible for use in oral delivery of peptides.

[0403] In some embodiments, the formulation for oral administration may contain an auxiliary agent for artificially increasing the permeability of the intestinal wall (e.g., resorcinol and / or a nonionic surfactant, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether), and / or an enzyme inhibitor for inhibiting enzymatic degradation (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) or trasylol). In some embodiments, the peptide of the present invention in a solid dosage form for oral administration may be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer, or glyceride. These dosage forms may also contain other types of additives, such as inert diluents, lubricants, e.g., magnesium stearate, parabens, preservatives, e.g., sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants, e.g., cysteine, disintegrants, binders, thickening agents, buffers, pH adjusting agents, sweetening agents, flavoring agents, or perfuming agents.

[0404] In certain embodiments, oral dosage forms or unit doses suitable for use with the peptides of the present invention may include a mixture of peptides and non-drug components or excipients, as well as other disposable materials that may be considered as either ingredients or packaging. Oral compositions may include at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, oral dosage forms are provided that include an effective amount of the peptides of the present invention, including at least one of pills, tablets, capsules, gels, pastes, drinks, syrups, ointments, and suppositories. In some examples, oral dosage forms are provided that are designed and configured to achieve delayed release of the peptide in the small intestine and / or colon of a subject.

[0405] In some embodiments, oral pharmaceutical compositions comprising the peptides of the present invention comprise an enteric coating designed to delay the release of the peptides of the present invention in the small intestine. In at least some embodiments, pharmaceutical compositions are provided that comprise the peptides of the present invention and a protease inhibitor, e.g., aprotinin, in a delayed release pharmaceutical formulation. In some instances, pharmaceutical compositions of the present invention comprise an enteric coat that is soluble in gastric juice at a pH of about 5.0 or higher. In at least one embodiment, pharmaceutical compositions are provided that comprise an enteric coating that comprises a polymer with a dissociable carboxylic acid group, e.g., derivatives of cellulose, including hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate, and similar derivatives of cellulose, as well as other carbohydrate polymers.

[0406] In some embodiments, pharmaceutical compositions comprising the peptides of the present invention are provided in an enteric coating, which is designed to protect and release the pharmaceutical composition in a controlled manner in the lower gastrointestinal system of a subject and to avoid systemic side effects. In addition to enteric coatings, the peptides of the present invention can be encapsulated, coated, engaged or otherwise associated in any compatible oral drug delivery system or component. For example, in some embodiments, the peptides of the present invention are provided in lipid carrier systems, including at least one of polymer hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

[0407] To overcome peptide degradation in the small intestine, some embodiments of the present invention include a hydrogel polymer carrier system in which the peptide of the present invention is contained, where the hydrogel polymer protects the peptide from proteolytic degradation in the small intestine and / or colon. The peptide of the present invention can further be formulated for use in compatibility with carrier systems designed to increase dissolution kinetics and improve intestinal absorption of the peptide. These methods include the use of liposomes, micelles, and nanoparticles to increase GI tract penetration of the peptide.

[0408] Various bioresponsive systems may also be combined with one or more of the peptides of the present invention to provide a pharmaceutical agent for oral delivery. In some embodiments, the peptides of the present invention are used in combination with a bioresponsive system, such as hydrogels and mucoadhesive polymers with hydrogen-bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, Eudragit®, chitosan, and alginate) to provide a therapeutic agent for oral administration. Other embodiments include methods for optimizing or extending drug residence time for the peptides of the present invention disclosed herein, which involve modifying the surface of the peptide to include mucoadhesive properties via hydrogen bonds, polymers with bound mucin, or / and hydrophobic interactions. These modified peptide molecules may demonstrate increased drug residence time within a subject in accordance with the desired features of the present invention. Additionally, targeted mucoadhesive systems may specifically bind receptors on the enterocyte and M-cell surfaces, thereby further increasing uptake of particles containing the peptide.

[0409] Another embodiment includes a method for oral delivery of the peptides of the invention, where the peptide is provided to a subject in combination with a permeation enhancer that promotes transport of the peptide across the intestinal mucosa by increasing paracellular or transcellular permeation. Various permeation enhancers and methods for oral delivery of therapeutic agents are described in Brayden, DJ, Mrsny, RJ, 2011. Oral peptide delivery: prioritizing the leading technologies. Ther. Delivery 2(12), 1567-1573.

[0410] In some embodiments, the pharmaceutical compositions and formulations of the present invention comprise the peptides of the present invention and one or more permeation enhancers. Examples of absorption enhancers may include, for example, bile salts, fatty acids, surfactants (anionic, cationic, and non-anionic) chelators, zonular OT, esters, cyclodextrins, dextran sulfate, azones, crown ethers, EDTA, sucrose esters, and phosphotidyl choline. Absorption enhancers are typically not carriers themselves, but are also widely associated with other carriers to transport peptides and proteins across the intestinal mucosa, thereby improving oral bioavailability. Such substances may be added or incorporated into the formulation as excipients to form non-specific interactions with the intended peptides of the present invention.

[0411] Dietary constituents and / or other naturally occurring substances that enhance tight junction permeability and have been identified as generally recognized as safe (GRAS) include, for example, acylglycerides, acylcarnitines, bile salts, and medium-chain fatty acids. Sodium salts of medium-chain fatty acids (MCFAS) have also been suggested to be permeability enhancers. The most extensively studied MCFAS is sodium caprate, a salt of capric acid, which contains 2-3% fatty acids in the milk fat fraction. To date, sodium caprate has been used primarily as an excipient in suppository formulations (Doktacillin™) to improve rectal ampicillin absorption. The permeability properties of another dietary MCFAS, sodium caprylate (8 carbons), have been shown to be poorer in vitro compared to sodium caprate. Sodium caprylate and peptide drugs were formulated as a mixture with other excipients in oil to produce an oil suspension (OS) that improves permeability (Tuvia, S. et al., Pharmaceutical Research, Vol. 31, No. 8, pp. 2010-2021 (2014)).

[0412] For example, in some embodiments, a permeation enhancer is combined with the peptide of the present invention, the permeation enhancer comprising at least one of a medium chain fatty acid, a long chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelating agent. In some embodiments, the medium chain fatty acid salt enhances absorption by increasing the paracellular permeability of the intestinal epithelium. In some embodiments, a permeation enhancer comprising sodium N-[hydroxybenzoyl)amino]caprylate is used to form a weak non-covalent association with the peptide of the present invention, which favors membrane transport and further dissociation upon reaching the blood circulation. In some embodiments, the peptide of the present invention is conjugated to oligoarginine, thereby increasing the cellular penetration of the peptide into various cell types. Furthermore, in at least one embodiment, a non-covalent bond is provided between the peptide of the present invention and a permeation enhancer selected from the group consisting of cyclodextrins (CDs) and dendrimers, which reduces peptide aggregation and increases stability and solubility for the peptide of the present invention.

[0413] In one embodiment, a pharmaceutical composition or formulation comprises a peptide of the invention and a transient permeability enhancer (TPE). The permeability enhancer and the TPE can be used to increase the oral bioavailability of the peptide. One example of a TPE that can be used is an oil suspension formulation that disperses a powder containing sodium caprylate and a therapeutic agent (Tuvia, S. et al., Pharmaceutical Research, Vol. 31, No. 8, pp. 2010-2021 (2014)).

[0414] In certain embodiments, pharmaceutical compositions and formulations may include a peptide of the invention and one or more absorption enhancers, enzyme inhibitors, or mucoadhesive polymers.

[0415] In certain embodiments, the peptides of the invention are formulated in formulation vehicles such as, for example, emulsions, liposomes, microspheres, or nanoparticles.

[0416] Another embodiment of the invention provides a method of treating a subject with a peptide of the invention having an increased half-life. In one aspect, the invention provides peptides having a half-life of at least several hours to a day in vitro or in vivo (e.g., when administered to a human subject) sufficient for once-daily (qd) or twice-daily (bid) administration in a therapeutically effective amount. In some embodiments, the peptides of the invention have a half-life of 3 days or more sufficient for once-weekly (qw) administration in a therapeutically effective amount. In some embodiments, the peptides of the invention have a half-life of 8 days or more sufficient for biweekly (biw) or monthly administration in a therapeutically effective amount. In some embodiments, the peptides of the invention are derivatized or modified to have a longer half-life compared to the underivatized or unmodified peptide. In some embodiments, the peptides contain one or more chemical modifications to increase serum half-life.

[0417] When used in at least one of the treatment or delivery systems described herein, the peptides of the present invention may be used in pure form or in a pharma- ceutically acceptable salt form, if such form exists.

[0418] The total daily usage of the peptides and compositions of the present invention can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject depends on a variety of factors, including: a) the disorder being treated and the severity of the disorder; b) the activity of the specific compound used; c) the specific composition used, the age, weight, general health, sex and diet of the patient; d) the administration time, route of administration and excretion rate of the specific peptide of the present invention used; e) duration of treatment; f) drugs used in combination or concomitantly with the specific peptide of the present invention used, and similar factors well known in the medical field.

[0419] In certain embodiments, the total daily dose of the peptides of the invention to be administered to a human or other mammalian host in single or divided doses may be, for example, in an amount of 0.0001-300 mg / kg body weight per day, or 1-300 mg / kg body weight per day. EXAMPLES

[0420] Examples of the present invention Example 1: Peptide synthesis Peptides were synthesized on a Gyros Protein Technologies Symphony X synthesizer using polystyrene or PEG-based resins via solid-phase peptide chemistry (SPPS) / Fmoc chemistry. Fmoc chemistry uses an Fmoc (fluorenylmethyloxycarbonyl) protecting group at the N-terminus of each amino acid, which is removed by base (usually 20% piperidine in DMF) before the next coupling cycle. As an example, (peptide no. 12) was made using TentaGel RAM resin (with a loading of 0.18 mmol / g) and successive couplings and deprotections of Fmoc-protected amino acid derivatives. A lipidated extension lysine residue was introduced at position 20 of the sequence as a single preformed Fmoc derivative.

[0421] Upon completion of SPPS, TFA-mediated cleavage from the resin was performed with simultaneous removal of protecting groups, followed by isolation via precipitation with diethyl ether and filtration to give 285 mg of crude material. Disulfide bond formation was effected using hydrogen peroxide in aqueous solution at pH 8.6.

[0422] Preparative reversed-phase HPLC was performed on a Teledyne Isco ACCQ Prep HP150 System with a variable wavelength UV absorbance detector, and the material was lyophilized to provide 91 mg of purified peptide. Analytical reversed-phase HPLC performed on a Dionex UltiMate 3000system determined a purity of 80.2%, and mass identity was confirmed on a Kratos Axima CFR Plus MALDI-MS. The measured mass of 7537.39 matched the calculated mass of 7537.46, and the product was further characterized by its amino acid content of 93.1%. Table 9 describes the analytical characteristics of exemplary peptides of the present invention.

[0423] [Table 31]

[0424] Example 2: Biological activity of peptides in a cell-based cyclic AMP activity assay The peptides were tested in cAMP cell-based assays to determine potency against the calcitonin, AMY3, GLP-1 and GIP human receptors. Peptide activation of these various receptors results in the downstream production of the cAMP second messenger, which can be measured in functional activity assays.

[0425] The potency (EC) of peptides for different human receptors was determined using stably transfected CHO-K1 cells overexpressing untagged human calcitonin, AMY3, GLP-1 and GIP receptors. 50 ) were evaluated and determined in GPCR cell-based cAMP assays including CT Human Calcitonin GPCR Cell-Based Agonist cAMP Assay (Cat. 86-0007P-2245AG), AMY3 (CT / RAMP3) Human Calcitonin GPCR Cell-Based Agonist cAMP Assay (Cat. 86-0007P-2276AG), GIP Human Glucagon GPCR Cell-Based Agonist cAMP Assay, (Cat. 86-0007P-2308AG), and GLP-1 Human Glucagon GPCR Cell-Based Agonist cAMP Assay, (Cat. 86-0007P-2309AG), all from Eurofins (Fremont, Calif.).

[0426] Experimental protocol: Cells were seeded in white-walled 384-well microplates in a total volume of 20 μL and incubated overnight at 37° C. before testing. Cell plating medium was replaced with 10 μL of assay buffer (HBSS+10 mM HEPES) before testing. All compounds were run in assay buffer containing 0.1% casein. Briefly, intermediate dilutions of sample stocks were performed to generate 4× samples in assay buffer. 5 μL of 4× samples were added to cells and incubated at 37° C. for 30 minutes. Final assay vehicle concentration was 1%. After appropriate compound incubation, assay signal was developed via 1 hour incubation with 5 ul of antibody and 20 ul of cAMP XS+ED / CL lysis cocktail followed by 2 hours incubation at room temperature with 20 ul of cAMP XS+EA reagent. After signal development, microplates were read on a PerkinElmer Envision instrument for chemiluminescent signal detection. Data were normalized to the maximum and minimum responses observed in the presence of control ligand and vehicle.

[0427] Results are expressed as percent efficacy relative to the maximal response of the control ligand.

[0428] The compounds showed activity against the different human receptors tested, as demonstrated in the cyclic AMP assay. In vitro results for control peptides and exemplary peptides of the invention, EC 50 A summary is provided in Table 10.

[0429] [Table 32]

[0430] Example 3: Acute food intake study in non-obese mice Acute feeding effects of peptides were evaluated using non-obese C57BL / 6j male mice. Six-week-old C57BL / 6j male mice were purchased from Charles River (Spain) and housed individually. Mice were kept in a temperature (22°C) and humidity (45–55%) controlled room with a 12-h light / dark cycle (lights on at 8 a.m.). Mice were fed a standard rodent chow diet (CHD) (A04, U8220G10R, SAFE) ad libitum. At 8 weeks of age, mice were acclimated to a reversed light / dark cycle for at least 7 days prior to peptide administration.

[0431] Peptides No. 12, No. 14, No. 15, and No. 17 were reconstituted in the vehicle 20 mM Tris-HCl, 7.5 mg / mL mannitol, pH 7.0, and Peptide No. 16 was reconstituted at 1 mg / ml in the vehicle 5 mM NaAc, 2.5 mg / mL mannitol, pH 5.0. All peptide solutions were aliquoted and stored at -20°C until use. Dilutions for injection were made in saline buffer.

[0432] Mice were randomized into experimental groups according to body weight. For peptides #12, #14, #15 and #17, 12-hour fasted mice received a single subcutaneous (sc) injection 2 hours before lights off at the doses indicated in each experiment (n=6 mice per group). As controls, mice received a single injection of the corresponding vehicle at an equal volume. For peptide #16, 12-hour fasted mice received a single sc injection 10 minutes before lights off. As controls, mice received a single injection of the corresponding vehicle at an equal volume (n=5 mice per group). Food was replaced immediately after lights off. Food intake was measured using a precision balance at different time intervals up to 72 hours after injection. Cumulative food intake was calculated for the indicated time periods.

[0433] All data shown are mean ± standard error of the mean (SEM). Statistical evaluation of the data was performed using one-way ANOVA followed by Bonferroni post-hoc test to determine any statistically significant differences between vehicle and peptide-treated groups. Differences were considered statistically significant at p<0.05. Data analysis was performed with GraphPad software (GraphPad Prism).

[0434] The results are shown in Table 11. The exemplary peptides induced reduced food intake in non-obese mice after a single dose.

[0435] [Table 33]

[0436] Example 4: Effect on blood glucose after a single dose of peptide The glucose lowering effect of the peptides was used to demonstrate that the incretin portion of the molecule has an in vivo effect. Non-fasted mice (n=5 per group) received a single sc injection of peptides, all at 50 nmol / kg or an equivalent volume of the respective vehicle. Blood samples were collected from the tail clip at 0, 30 and 120 min after injection for glucose measurement. Glucose using a blood glucose meter (Accu-chek Aviva, Roche Diagnostics). Calculation of glucose lowering at 30 and 120 min was made relative to 0 h.

[0437] All data shown are mean ± standard error of the mean (SEM). Statistical evaluation of the data was performed using one-way ANOVA followed by Bonferroni post-hoc test to determine any statistically significant differences between vehicle and peptide-treated groups. Differences were considered statistically significant at p<0.05. Data analysis was performed with GraphPad software (GraphPad Prism).

[0438] The peptides of the present invention significantly reduced blood glucose at 30 and 120 minutes compared to 0 hours as shown in Table 12 and Figures 1A and 1B, whereas Cagrilitide (a dual calcitonin / amylin agonist) did not.

[0439] [Table 34]

[0440] Example 5: Acute food intake and weight reduction in non-obese mice Male Sprague Dawley rats (5 weeks old) were purchased from Charles River (Spain) and housed in isolation for individual registration of food consumption. Rats were acclimated to a reversed light-dark cycle (12 h light and 12 h dark) and housed in isolation in a controlled room with controlled temperature conditions (22°C) and humidity (45-55%) at least 5 days prior to testing. Rats were fed a standard rodent diet (CHD) (A04, U8220G10R, SAFE) ad libitum.

[0441] Sprague Dawley rats (200-250 g), randomized into different experimental groups by body weight, were fasted for 8 h before lights out (8 pm). Two hours before the onset of the dark cycle, rats were injected (sc) with peptides (peptide no. 12, peptide no. 14, and peptide no. 17) or the corresponding vehicle at the doses indicated for each experiment (n=5 per group). For peptide no. 16 and its corresponding vehicle control (n=5 per group), 8-h fasted mice received a single sc injection 10 min before the onset of the dark cycle. For all experimental groups, food was replaced immediately after lights out.

[0442] Food intake was measured using a precision balance at different time intervals up to 72 hours after injection. Cumulative food intake was calculated for the indicated time periods. Body weight was measured at the indicated time periods. The mean cumulative food intake in each treatment group was compared to vehicle and reported as a percentage of the mean food intake in the vehicle group, defined as 100%.

[0443] All data shown are mean ± standard error of the mean (SEM). The results are shown in Tables 13, 14, and 15. The peptide induces a reduction in food intake and body weight in non-obese mice after a single administration.

[0444] [Table 35]

[0445] [Table 36]

[0446] [Table 37]

[0447] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referenced herein are hereby incorporated by reference in their entirety.

[0448] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims.

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide has the formula (I): X １ -X ２ -E-G-T-F-X ３ -S-D-Y-S-I-X ４ -X ５ -D-K-I-X ６ -Q-X ７ -X ８ -F-V-X ９ -W-L-X １０ -X １１ -X １２ -X １３ -X １４ -X １５ -X １６ -C-N-T-A-T-C-X １７ -X １８ -X １９ -X ２０ -L-X ２１ -X ２２ -X ２３ -L-X ２４ -X ２５ -X ２６ -X ２７ -X ２８ -X ２９ -X ３０ -X ３１ -X ３２ -X ３３ X ３４ -P-X ３５ -T-N-X ３６ -G-X ３７ -N-T-Y-(NR １ R ２ ) (I) (In the formula,[[]] X １ is Tyr or (d)Tyr; X ２ is Ala, (d)Ala, or Aib; X ３ is Ile or Thr; X ４ is Ala, Aib, or Gln; X ５ is Met, Leu, or Val; X ６ is Ala or His; X ７ is Gln, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ R ３ and; X ８ is Ala or Asp; X ９ is Asn or Gln; X １０ is Leu, Val, or Ile; X １１ is Ala or Val; X １２ is Gly or Gln; X １３ is Gly, Lys, Arg, Ser, or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ R ３ and; X １４ is Pro, Gly, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ R ３ and; X １５ is Ser, Gly, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH ２ ), ｎ R ３ wherein; X １６ is absent or is Gly or Ser; X １７ is Ala, Met or Val; X １８ is Thr or Leu; X １９ is Gln or Gly; X ２０ is Arg, Lys, Gln, or Lys-γ-Glu-γ-Glu-C=O (C H ２ ) ｎ R ３ and; X ２１ is Ala or Ser; X ２２ is Asn or Gln; X ２３ is Phe or Glu; X ２４ is His or Val; X ２５ is His or Arg; X ２６ is Ser or Leu; X ２７ is Ser or Gln; X ２８ is Asn or Thr; X ２９ is absent or is Asn or Gln; X ３０ is either absent or is Phe; X ３１ is absent or is Gly; X ３２ is non-existent or is Pro; X ３３ is absent or is Ile, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ R ３ ; X ３４ is Leu or Tyr; X ３５ is Pro, Lys, Arg, or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ R ３ and; X ３６ is Val or Thr; X ３７ is Ser, Lys, or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ R ３ and 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 amino acid sequence of.

2. X １ is Tyr; X ２ is Ala in (d); X ３ is Ille; X ４ is Ala; X ５ is Leu; X ６ is His; X ７ is Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ CO ２ H, and n is 18; X ８ is Asp; X ９ is Asn; X １０ is Leu; X １１ is Ala; X １２ is Gln; X １３ is Lys-γ-Glu-γ-Glu-C=O(CH ２ ) ｎ CO ２ H, and n is 18; X １４ is Pro; X １５ is Ser; X １６ is Ser; X １７ is Ala; X １８ is Thr; X １９ is Gln; X ２０ is Arg; X ２１ is Ala; X ２２ is Asn; X ２３ is Phe; X ２４ is Val; X ２５ is His; X ２６ is Ser; X ２７ is Ser; X ２８ is Asn; X ２９ is Asn; X ３０ is Phe; X ３１ is Gly; X ３２ is Pro; X ３３ is Ille; X ３４ is Leu; X ３５ is Pro; X ３６ is Val; X ３７ is Ser The peptide or a pharmaceutically acceptable salt thereof according to Claim 1.

3. X １３ is Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CO ２ H or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CH ３ The peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

4. X １４ is Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CO ２ H or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CH ３ The peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

5. X １５ is Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CO ２ H or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CH ３ The peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

6. X ３３ is Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CO ２ H or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CH ３ and the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

7. X １３ The peptide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is Gly.

8. X １４ is the peptide according to claim 1 which is Pro or a pharmaceutically acceptable salt thereof.

9. X １５ The peptide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is Ser.

10. The peptide or a pharmaceutically acceptable salt thereof according to Claim 1, having the following sequence: 【Table 1】 【Table 2】 【Table 3】

11. K ＊ is Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CO ２ H or Lys-γ-Glu-γ-Glu-C=O(CH ２ ) １８ CH ３ The peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising the peptide or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 11, and a pharmaceutically acceptable carrier, excipient, or diluent.

13. For use in a method of treating obesity, metabolic disorder, or liver disorder in a subject in need thereof, a pharmaceutical composition comprising an effective amount of the peptide or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 11.

14. The pharmaceutical composition according to Claim 13, wherein the peptide or a pharmaceutically acceptable salt thereof is provided to the subject by an oral, parenteral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, nebulization, sublingual, buccal, parenteral, rectal, intraocular, inhalation, topical, vaginal, or local route of administration.

15. The pharmaceutical composition according to Claim 13, wherein the method is for treating obesity.

16. For use in a method of treating diabetes in a subject in need thereof, a pharmaceutical composition comprising an effective amount of the peptide or a pharmaceutically acceptable salt thereof according to Claim 1.

17. The pharmaceutical composition according to Claim 13, 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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