KR20050018988A - Pituitary adenylate cyclase activating peptide (pacap) receptor (vpac2) agonists and their pharmacological methods of use - Google Patents

Abstract

The present invention provides novel peptides that act as agonists of the VPAC2 receptor in vivo. These insulin secretagogue polypeptides appear to lower blood glucose more than the control in vivo upon glucose challenge. Polypeptides of the invention are also stable in formulations and have long half-lives. The peptides of the present invention provide novel therapies for patients with reduced endogenous insulin secretion, particularly type 2 diabetes. In particular, the invention is a polypeptide selected from a specific group of VPAC2-related polypeptides, or functional equivalents thereof. The present invention also relates to a method of treating metabolic disease in a mammal comprising administering to said mammal a therapeutically effective amount of an insulin secretagogue peptide. Also described herein are methods for preparing peptides by both recombinant and synthetic.

Description

Pituitary ADENYLATE CYCLASE ACTIVATING PEPTIDE (PACAP) RECEPTOR (VPAC2) AGONISTS AND THEIR PHARMACOLOGICAL METHODS OF USE}

This application claims the priority of U.S. Provisional Patent Application No. 60 / 395,738, filed Jul. 12, 2002, the content of which is incorporated herein by reference in its entirety.

The present invention relates to newly identified polypeptides and their use for therapeutic purposes. More particularly, the polypeptides of the invention are useful for promoting the release of insulin from pancreatic β-cells in a glucose-dependent manner, thereby suffering from metabolic diseases such as diabetes or impaired glucose tolerance or prediabetic conditions. Provide treatment options for the individual.

Diabetes is characterized by impaired glucose metabolism itself, especially elevated blood glucose levels, in diabetic patients. Due to acupuncture, diabetes mellitus is associated with type 1 diabetes or insulin dependent diabetes (IDDM), which occurs when the pancreatic gland lacks insulin-producing β-cells, and impaired changes in β-cell function and insulin action. There are two main groups, type 2 diabetes or non-insulin dependent diabetes mellitus (NIDDM), which occur in patients.

Patients with type 1 diabetes are generally treated with insulin, but the majority of type 2 diabetes is treated with drugs that promote β-cell function or with drugs that enhance the patient's tissue sensitivity to insulin. Over time, nearly half of Type 2 diabetic individuals lose their response to these agents, which requires insulin therapy. Drugs currently used to treat type 2 diabetes are described below.

Alpha-glucosidase inhibitors (eg, Precose®, Voglibose ™ and Miglitol®) reduce the excursion of postprandial glucose by delaying the absorption of glucose from the intestine. These drugs are safe and provide treatment for diabetic individuals with mild to moderate illness. However, gastrointestinal side effects have been reported in the literature.

Insulin sensitizers are drugs that enhance the body's response to insulin. Thiozolidinediones, such as Avandia ™ (Rosiglitazone) and Actos ™, activate peroxysome proliferator-activated receptor (PPAR) gamma subtypes and inhibit the activity of a set of less well-known genes. Modulate. This class of first drug, Rezulin ™ (troglitazone), was recovered because it elevates liver enzyme levels and drug induced hepatotoxicity. These liver effects do not appear to be a significant problem in patients using Avandia ™ and Actos ™. Even so, hepatic enzyme testing is recommended every two months in the first year of treatment and periodically thereafter. Avandia ™ and Actos ™ appear to be associated with fluid retention and edema. Avandia ™ is not directed for use with insulin due to concerns about congestive heart failure. Another potential side effect is weight gain.

Insulin secretagogues (eg, sulfonylureas (SFU) and other drugs acting by ATP-dependent K + channels) are another drug type currently used to treat type 2 diabetes. SFU is the standard treatment for type 2 diabetes with mild to moderate fasting glycemia. SFU has the limitation of including hypoglycemia, weight gain and the potential to induce high primary and secondary failure rates. 10 to 20% of patients treated primarily fail to show a significant therapeutic effect (primary failure). Secondary failure is evidenced by an additional 20-30% loss of treatment after 6 months of treatment with SFU. Insulin treatment is required in 50% of SFU responders after 5-7 years of treatment (Scheen, et al., Diabetes Res. Clin. Pract. 6: 533-543, 1989).

Glucophage ™ (metformin HCl) is a biguanide that lowers blood glucose by reducing hepatic glucose output and increasing peripheral glucose uptake and utilization. This drug is effective in lowering blood sugar in mild to moderately ill individuals and has no potential to induce side effects of weight gain or hypoglycemia. Glucophages, however, have a number of side effects, including gastrointestinal disorders and lactic acidosis. Glucophage is contraindicated in individuals over 70 years of age and individuals with impaired kidney or liver function. Finally, glucophages have the same primary and secondary failure rates as SFUs.

Insulin treatment is performed after diet, exercise and oral medications fail to properly control blood glucose. This treatment has the disadvantage that it is for injection, can cause hypoglycemia and cause weight gain.

Due to problems with commonly used therapies, new therapies for treating type 2 diabetes are needed. In particular, new therapies are needed to maintain normal (glucose-dependent) insulin secretion. Such new drugs should have the following characteristics: dependence on glucose (ie, developing insulin secretion only in the presence of increased blood sugar) to promote insulin secretion; Low primary and secondary failure rates; And preservation of islet cell function. The strategy for developing new therapies described in the present invention is based on cyclic adenosine monophosphate (cAMP) signaling mechanisms and their effects on insulin secretion.

Cyclic AMP is the main regulator of insulin secretion. The increase in signaling molecules promotes the closure of K + channels following activation of the protein kinase A pathway. Closure of the K + channel leads to cell depolarization and then opening of the Ca ⁺⁺ channel, which in turn leads to extracellular outflow of insulin granules. Any effect on insulin secretion does not occur in the absence of low glucose concentrations (Weinhaus, et al., Diabetes 47: 1426-1435, 1998). Secretion promoters such as the pituitary adenylate cyclase activating peptide ("PACAP") and GLP-1 (glucose-like peptide 1) use the cAMP system to regulate insulin secretion in a glucose-dependent fashion (Komatsu, et al. , Diabetes 46: 1928-1938, 1997; Filipsson, et al., Diabetes 50: 1959-1969, 2001; Drucker, Endocrinology 142: 521-527, 2001). Insulin secretagogues that act through increased cAMP, such as GLP-1 and PACAP, can also increase insulin synthesis in addition to insulin release (Skoglund, et al., Diabetes 49: 1156-1164, 2000; Borboni, et al. , Endocrinology 140: 5530-5537, 1999).

PACAP is a potent promoter of glucose-dependent insulin secretion from pancreatic β-cells. Three different PACAP receptor types (PAC1, VPAC1 and VPAC2) are known (Harmer, et al., Pharmacol. Reviews 50: 265-270, 1998; Vaudry, et al., Pharmacol. Reviews 52: 269-324, 2000). PACAP has equivalent activity and potency at all three receptors and does not exhibit receptor selectivity. PAC1 is mainly located in the CNS, while VPAC1 and VPAC2 are more widely distributed. VPAC1 is located in the liver, lungs and intestines as well as the CNS. VPAC2 is located in the CNS, pancreas, skeletal muscle, heart, kidney, adipose tissue, testes and stomach. Recent studies have claimed that VPAC2 is responsible for insulin secretion from β-cells (Inagaki, et al., Proc. Natl. Acad. Sci. USA 91: 2679-2683, 1994; Tsutsumi, et al., Diabetes 51 : 1453-1460, 2002). This insulin-directed action of PACAP is mediated by GPT binding protein Gs. Accumulation of intracellular cAMP in turn activates non-selective cation channels in β-cells that increase [Ca ⁺⁺ ] and promote extracellular leakage of insulin-containing secretory granules.

PACAP is the latest member of the superfamily of metabolic, neuroendocrine and neurotransmitter peptide hormones that exert their actions through the cAMP-mediated signaling pathway (Arimura, Regul. Peptides 37: 287-303, 1992). . Biologically active peptides are liberated from biosynthetic precursors in two molecular forms as 38-amino acid peptides (PACAP-38) and / or as 27-amino acid peptides (PACAP-27) having amidated carboxyl termini (Arimura, supra).

The highest concentrations of both forms of peptides are found in the brain and testes (Arimura, supra). PACAP-27, a shorter form of peptide, exhibits 68% structural homology to vasoactive intestinal polypeptide (VIP). However, the distribution of PACAP and VIP in the central nervous system suggests that these structurally related peptides have distinct neurotransmitter functions (Koves, et al., Neuroendocrinology 54: 159-169, 1991).

Recent studies have shown that PACAP-38's role in reproduction (McArdle, Endocrinology 135: 815-817, 1994) has the ability to promote insulin secretion (Yada, et al., J. Biol. Chem. 269: 1290-1293, Various biological effects have been demonstrated up to 1994). PACAP also provides hormonal regulation of lipid and carbohydrate metabolism (Gray, et al., Mol. Endocrinol. 15: 1739-47, 2001); Daily cycle function (Harmar, et al., Cell 109: 497-508, 2002); And immune system, growth, energy homeostasis, and male reproductive function (Asnicar, et al., Endocrinol. 143: 3994-4006, 2002); Regulation of appetite (Tachibana, et al., Neurosci. Lett. 339: 203-206, 2003); And acute and chronic inflammatory diseases, septic shock and autoimmune diseases (eg systemic lupus erythematosus) (Pozo, Trends Mol. Med. 9: 211-217, 2003).

Vascular Intestinal Peptides (VIPs) are 28 amino acid peptides first isolated from the upper small intestine of pigs (Said and Mutt, Science 169: 1217-1218, 1970; US Pat. No. 3,879,371). This peptide belongs to the class of structurally related small polypeptides including hellodermin, sacritin, somatostatin and glucagon. The biological effect of VIP is mediated by the activation of membrane-bound receptor proteins that couple to intracellular cAMP signaling systems. These receptors were originally known as VIP-R1 and VIP-R2, but they were later found to be the same receptors as VPAC1 and VPAC2. VIP shows equivalent activity and potency in VPAC1 and VPAC2.

To improve the stability of VIP in human lung fluids, Bolin et al. (Bolin, et al., Biopolymers 37: 57-66, 1995) enhance the helical tendency of this peptide and reduce proteolytic degradation. A series of VIP variants designed to make Substitutions were concentrated at positions 8, 12, 17 and 25-28 which appear to be insignificant for receptor binding. In addition, the "GGT" sequence was labeled on the C-terminus of the VIP mutein with the expectation to cap the helix more effectively. Finally, several cyclic variants were synthesized to further stabilize the helix (US Pat. No. 5,677,419). Although these efforts were not for receptor selectivity, they obtained two homologues with at least 100-fold VPAC2 selectivity (Gourlet, et al., Peptides 18: 403-408, 1997; Xia, et al., J Pharmacol.Exp. Ther., 281: 629-633, 1997).

GLP-1 is released from intestinal L-cells after a meal and acts as an incretin hormone (ie, it enhances glucose-derived insulin release from pancreatic β-cells). It is a 37-amino acid peptide that is expressed differently by the glucagon gene depending on the tissue type. Clinical data were collected by GLP-1 supporting the beneficial effects of raising cAMP levels in β-cells. Infusion of GLP-1 in poorly regulated type 2 diabetes normalized their fasting blood glucose levels (Gutniak, et al., New Eng. J. Med. 326: 1316-1992), and by longer periods of infusion β-cell function was improved to that of normal individuals (Rachman, et al., Diabetes 45: 1524-1530, 1996). Recent reports have shown that GLP-1 improves the ability of β-cells to respond to glucose in individuals with impaired glucose tolerance (Byrne, et al., Diabetes 47: 1259-1265, 1998). However, all these effects are short-lived due to the short half-life of the peptide.

Amylin Pharmaceuticals is conducting Phase III experiments using Exendin 4 ™ (AC2993), a 39 amino acid peptide that was first identified in Gila Monster. Amylin reported that clinical trials showed improved glycemic control in type 2 diabetes treated with Exendin 4 ™. However, the incidence of nausea and vomiting was significant.

Applicants have described novel polypeptides that act in vivo, such as agonists of VPAC2 receptors in WO 01/23420, the disclosure of which is incorporated herein by reference, and in particular, applicants have identified VPAC2 agonists identified as R3P66. Is described. However, the polypeptides described herein, including R3P66, are not suitable commercial candidates if there are problems with stability associated with the polypeptide in the formulation and problems with the short half-life of the polypeptide.

Improved peptides with glucose-dependent insulin secretagogue activity of PACAP, GLP-1 or Exendin 4 ™ but with fewer side effects, preferably improved peptides with stable and long plasma half-life in the formulation There is a need for this. In addition, further control of plasma glucose levels can prevent long-term diabetes complications. Thus, new diabetic drugs should provide an improved quality of life for the patient.

Summary of the Invention

The present invention provides novel polypeptides that act as agonists of the VPAC2 receptor (hereinafter VPAC2) in vivo and are effective in the treatment of diseases and conditions that may be ameliorated by agents having VPAC2 agonist activity. Preferably, the polypeptides of the invention are selective VPAC2 agonists with greater potency in VPAC2 than in VPAC1 and PAC1. For example, but not by way of limitation, these polypeptides promote release from pancreatic β-cells in insulin synthesis and glucose-dependent modalities, followed by plasma glucose reduction. These secretagogue polypeptides appear to lower blood glucose in vivo during glucose challenge than the vehicle control. Even more preferably, the polypeptides of the present invention are stable in the formulation and have long plasma half-life and long duration of action in vivo when derivatized.

Polypeptides of the present invention are for example metabolic diseases, such as diseases caused by reduced endogenous insulin secretion, especially patients with type 2 diabetes, or patients with impaired glucose tolerance, prediabetic conditions with minor changes in insulin secretion. Provide a new treatment for In addition, the polypeptides of the present invention can be used for type 1 diabetes, gestational diabetes mellitus, growth stage expression diabetes (MODY), latent autoimmune adult diabetes (LADA), and related diabetic dyslipidemia and other diabetic complications. , And hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, syndrome X, and insulin resistance.

Polypeptides of the invention can also be used for obesity (eg, control of appetite and food intake), atherosclerotic diseases, hyperlipidemia, hypercholesterolemia, low HDL levels, hypertension, cardiovascular diseases (atherosclerosis, coronary heart disease, coronary) Prevention and / or treatment of arterial disease, and hypertension), cerebrovascular and peripheral vascular disease, and lupus, polycystic ovary syndrome, carcinogenesis, and hyperplasia, asthma, male reproductive disorders, ulcers , Immune disorders including sleep disorders, disorders of lipid and carbohydrate metabolism, daily cycle dysfunction, growth disorders, disorders of energy homeostasis, autoimmune diseases (eg systemic lupus erythematosus), and acute and This can be used to prevent and / or treat chronic inflammatory diseases, septic shock, and other conditions identified herein, or to function in other ways as described below in the present invention. Can be used.

In particular, one aspect of the invention relates to fragments, derivatives and variants thereof, including SEQ ID NOs: 1 to 152, and functional equivalents thereof, which exhibit at least one biological function substantially identical to the polypeptides of SEQ ID NO. A polypeptide selected from the group consisting of (collectively, "polypeptide of the present invention"). Preferred embodiments of the invention include fragments, derivatives and variants thereof which exhibit at least one biological function substantially identical to SEQ ID NOs: 1 to 38, SEQ ID NOs: 115 to 152, and the polypeptides of SEQ ID NO mentioned. Polypeptides selected from the group consisting of: More preferred embodiments of the invention include SEQ ID NOs: 1 to 5 and SEQ ID NOs: 115 to 119, and functional equivalents thereof which exhibit at least one biological function substantially identical to the polypeptides of SEQ ID NO mentioned. Polypeptides selected from the group consisting of fragments, derivatives and variants thereof. Most preferred embodiments of the invention are fragments, derivatives thereof, including SEQ ID NOs: 1, 2, 115 and 116, and functional equivalents thereof, which exhibit at least one biological function substantially identical to the polypeptides of SEQ ID NO mentioned. And a polypeptide selected from the group consisting of variants.

Another embodiment of the invention is a polypeptide encoding a polypeptide of the invention, and a dedicated vector and host cell required to recombinantly express the polypeptide of the invention. These polynucleotide sequences include SEQ ID NOs: 154-264.

Also provided are antibodies and antibody fragments that selectively bind polypeptides of the invention. Such antibodies are useful for detecting polypeptides of the invention and can be identified and made by procedures well known in the art. Polyclonal N-terminal IgG antibodies and monoclonal C-terminal Fab antibodies that recognize the polypeptides of the invention were generated.

The invention also provides for administering to a mammal a diabetes, diabetes mellitus-in mammal, comprising administering to the mammal a therapeutically effective amount of any of the polypeptides of the invention such as SEQ ID NOs: 1 to 152 or any polypeptide active in VPAC2. Related disorders and / or methods for treating a disease or condition affected by a polypeptide of the invention, preferably affected by a VPAC2 agonist of a polypeptide of the invention.

Also described are methods for making polypeptides of the invention, both recombinant and synthetic.

1A-1D show amino acid sequences of polypeptides of SEQ ID NOs: 1-152. SEQ ID NOs: 115-152 represent peptides PEGylated at C-terminal cysteines via maleimide linkages. PEG can be 22 kD linear PEG or 43 kD branched PEG.

FIG. 2 depicts a DNA sequence cloned into pGEX-6P-1 (SEQ ID NO: 153) to produce the amino acid sequence of SEQ ID NO: 1. FIG. The underlined restriction enzyme sites BamHI and XhoI allow for in-frame cloning into the pGEX-6P-1 expression vector. The 12mer DNA sequences encoding factor Xa recognition sites and the two stop codons are highlighted in bold. The middle unhighlighted sequence encodes SEQ ID NO: 1 (amino acid sequence). Codons mutated from VIP are shown by small cap nucleotides.

3A-3H depict nucleic acid sequence SEQ ID NOs: 154-264. These nucleic acid sequences encode polypeptides of the invention.

FIG. 4 is a bar chart illustrating insulin secretion of dispersed islet cells after exposure to the fezylated peptide of the present invention.

FIG. 5 is a bar chart showing enhanced glucose placement in rats by the subcutaneous (SC) route of administration of the fezylated peptide of the present invention.

FIG. 6 shows the stability trends of three VPAC2 homologues (P5, P7, and control, R3P66) at 1 mg / ml in an aqueous solution containing 150 mM NaCl and 20 mM phosphate at pH 8.0 during incubation at 40 ° C. will be. Samples were analyzed by capillary electrophoresis to determine the purity of the peptides. Purity at the 2- and 4-week time points was normalized by the initial purity percentage.

FIG. 7 shows the stability trends of three VPAC2 homologues (P5, P7, and control, R3P66) at 2 mg / ml in dimethylsulfoxide (DMSO) during incubation at 40 ° C. Samples were analyzed by capillary electrophoresis to determine the purity of the peptides. Purity at the 2- and 4-week time points was normalized by the initial purity percentage.

Figure 8 illustrates the selective recognition of full length phenylated peptides as compared to similar phenylated peptides with single amino acid detection at the N-terminus.

Detailed description of the invention

The present invention provides novel polypeptides and fragments, derivatives and variants thereof that exhibit at least one biological function that is substantially identical to the polypeptides of FIGS. 1A-1D (collectively, polypeptides of the present invention). Polypeptides of the invention can be used in vivo to include both type 1 and type 2 diabetes in diabetes, gestational diabetes, young adult growth-type diabetes mellitus (MODY) (Herman, et al., Diabetes 43:40, 1994), Latent autoimmune adult diabetes (LADA) (Zimmet, et al., Diabetes Med. 11: 299, 1994), and related diabetic dyslipidemia and other diabetic complications, and hyperglycemia, hyperinsulinemia, impaired glucose It can act as a VPAC2 agonist or otherwise to prevent and / or treat diseases or conditions such as resistance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, X syndrome, and insulin resistance.

In addition, the polypeptides of the present invention may also be used for obesity (eg, control of appetite and food intake), atherosclerotic diseases, hyperlipidemia, hypercholesterolemia, low HDL levels, hypertension, cardiovascular diseases (atherosclerosis, coronary heart disease) , Prophylactic and / or treatment of cerebrovascular and peripheral vascular diseases, and coronary artery disease, and hypertension, and male reproductive disorders including lupus, polycystic ovary syndrome, carcinogenesis, and hyperplasia, asthma, human sperm motility Can be used to prevent and / or treat ulcers, sleep disorders, and other conditions identified herein, or to function in other ways as described below in the present invention.

Preferably, the polypeptides of the invention promote insulin release from pancreatic β-cells in a glucose-dependent fashion. Even more preferably, the polypeptides of the present invention are stable in both aqueous and non-aqueous formulations and exhibit a plasma half-life of at least 1 hour.

The polypeptide of the invention is a VPAC2 agonist. Preferably, they are selective VPAC2 agonists with at least 10-fold selectivity to VPAC2 relative to VPAC1 and / or PAC1. More preferably, they are selective VPAC2 agonists with at least 100-fold selectivity to VPAC2 relative to VPAC1 and / or PAC1. Most preferably, they promote insulin release into the plasma in a glucose-dependent fashion without inducing, for example, stopping or increasing plasma glucose levels that are adverse effects on the treatment of type 2 diabetes. In addition, the polypeptides of the present invention are plasma as selective agonists of the VPAC2 receptor, being selective against other receptors that cause undesirable or dangerous side effects such as gastrointestinal water retention, and / or unwanted cardiovascular effects such as increased heart rate. It is desirable to cause an increase in insulin release into.

Polypeptides of the invention are also stable in aqueous and non-aqueous formulations. Preferably, the polypeptides of the invention exhibit less than 10% degradation at 37-40 ° C. over a period of one week when dissolved in water (pH 7-8) or non-aqueous organic solvent. Even more preferably, the polypeptides of the invention exhibit less than 5% degradation at 37-40 ° C. over a period of one week when dissolved in water (pH 7-8) or non-aqueous organic solvent. In addition, the compositions and formulations of the present invention may contain a polypeptide of the present invention and about 2% to about 30% DMSO. In another embodiment of the invention, the compositions and formulations may optionally comprise about 0.2% to about 3% (w / v) of additional solvents such as propylene glycol, dimethyl formamide, propylene carbonate, polyethylene glycol, and triglycerides have.

Finally, the derivatized polypeptides of the invention exhibit at least one hour plasma half-life in rats after IV injection, more preferably plasma half-life is at least 2 hours, and even more preferably plasma half-life is at least 3 hours. .

The polypeptides of the present invention provide new therapies for patients with reduced endogenous insulin secretion or impaired glucose tolerance, especially type 2 diabetes. That is, the polypeptides of the present invention are long-acting VPAC2 agonists that can be used to maintain, improve and restore glucose-stimulated insulin secretion. In addition, selective peptide agonists of the VPAC2 receptor enhance glucose-dependent insulin secretion in the pancreas without causing side effects associated with non-selective activation of other PACAP receptors.

Certain terms used throughout this specification are defined below, and others are defined as introduced. Single letter abbreviations, corresponding amino acids, and three letter abbreviations for specific amino acids are as follows: A, alanine (ala); C, cysteine (cys); D, aspartic acid (asp); E, glutamic acid (glu); F, phenylalanine (phe); G, glycine (gly); H, histidine (his); I, isoleucine (ile); K, lysine (lys); L, leucine (leu); M, methionine (met); N, asparagine (asn); P, proline (pro); Q, glutamine (gln); R, arginine (arg); S, serine; T, threonine (thr); V, valine; W, tryptophan (trp); Y, tyrosine (tyr).

The term “polynucleotide encoding a polypeptide” includes polynucleotides comprising only the coding sequence for the polypeptide, and polynucleotides comprising additional coding and / or non-coding sequences. The invention further relates to a polynucleotide which hybridizes to the above-described sequences when there is at least about 70%, preferably at least about 90%, more preferably at least about 95% identity between the sequences. will be. The present invention relates in particular to polynucleotides encoding polypeptides which hybridize to the above-described polynucleotides under stringent conditions. As used herein, the term "stringent condition" means "strict hybridization condition". Preferably, hybridization occurs only if there is at least about 90%, preferably about 95% to 97% identity between the sequences. In a preferred embodiment, the polynucleotides hybridizing to the above-described polynucleotides encode polypeptides having substantially the same biological function or activity as the mature polypeptides encoded by cDNAs.

A "functional equivalent" and "substantially the same biological function or activity" means a biological activity of about 30% to about 100% or more of that biological activity, when the biological activity of each polypeptide is measured by the same procedure. Activity means that this is represented by the polypeptide being compared. For example, a polypeptide that is functionally equivalent to the polypeptide of FIG. 1 is a CHO cell expressing human VPAC2 receptor when tested by the cyclic AMP (cAMP) scintillation proximity assay of Example 7. CAMP accumulation in the line.

Polypeptides of the invention that are VPAC2 agonists exhibit about 30% to about 100% or more of the maximum PACAP-27 VPAC2 agonist activity when tested by the protocol of Example 7. Preferred polypeptides of the invention which are selective agonists for VPAC2 over PACAP, VPAC1 and PAC1 receptors have a ratio of VPAC2 agonist activity to VPAC1 activity of about 10: 1 or more, more preferably about 100: 1 or more. PAC1 receptor activity of about 10: 1 or more, more preferably about 100: 1 or more when the polypeptide is tested according to the protocol of Example 7 using cells expressing and / or expressing an appropriate receptor A polypeptide exhibiting the ratio of VPAC2 agonist activity to.

“Strict hybridization conditions” include 50% formamide, 5 × SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × denhardt solution, 10% dextran sulfate and 20 μg. Two polynucleotides (or fragments) to hybridize at 42 ° C. overnight in a solution containing / ml denatured sheared salmon sperm DNA, followed by washing the filter with 0.1 × SSC at 65 ° C.

When referring to the polypeptide of FIG. 1, the terms “fragment”, “derivative” and “variant” refer to fragments, derivatives of polypeptides having substantially the same biological function or activity as such polypeptides, as further described below. And variants.

Homologs include pro-polypeptides containing therein the amino acid sequence of the polypeptide of the invention. The active polypeptides of the present invention can be cleaved from additional amino acids that complete the pro-polypeptide molecule by natural in vivo processes or by procedures well known in the art such as enzymatic or chemical cleavage. For example, the 28-amino acid natural peptide VIP is naturally expressed as a much larger polypeptide, which is then processed in vivo to release the 28-amino acid active mature peptide.

A fragment is a portion of a polypeptide that possesses a functional activity substantially similar to that described in the in vivo model described herein.

Derivatives substantially preserve the functions described in the present invention, and all modifications to the polypeptides including additional structural and minor functions (eg, phenylated polypeptides with greater half-life), and further described below. Fusion polypeptides that confer additional activity, such as targeting specificity or toxicity, to a target of interest.

Polypeptides of the invention may be recombinant polypeptides, naturally purified polypeptides, or synthetic polypeptides.

Fragments, derivatives or variants of the polypeptides of the invention are (i) substituted by amino acid residues (preferably conserved amino acid residues) in which one or more amino acid residues are conserved or non-conserved, and such substituted amino acid residues are genetic code Compounds that may or may not be encoded by (ii) one or more amino acid residues comprise substituent groups, or (iii) increase the half-life of the polypeptide to the mature polypeptide (eg, polyethylene glycol Fusion with another compound such as), or (iv) an additional amino acid fused to a mature polypeptide such as a leader or secretory sequence or a sequence used for purification of a mature or propolypeptide sequence. One, or (v) a polypeptide sequence with a larger polypeptide (eg, an increased effect of It may be fused to human albumin, an antibody or Fc) for a deceleration time. Such fragments, derivatives and variants and homologs are considered to be within the scope of those skilled in the art from the guidance of the invention.

Preferably, the derivatives of the present invention may contain conservative amino acid substitutions (defined further below) which are made from one or more predicted, preferably non-essential amino acid residues. "Non-essential" amino acid residues are those residues that can be changed from the wild-type sequence of a protein without changing biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are those in which amino acid residues are substituted with amino acid residues having similar side chains. Classes of amino acid residues with similar side chains are defined in the art. These classes include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine ), Nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine) Amino acids having phenylalanine, tryptophan, histidine). Non-conservative substitutions are made either to conservative amino acid residues or to amino acid residues residing within conserved protein regions such as residues 19 and 27, which are essential residues for protein activity such as VPAC2 activity and / or VPAC2 selectivity. Do not. Fragments, or biologically active proteins, include polypeptide fragments suitable for use as medicaments, for producing antibodies, as research agents, and the like. A fragment is an amino acid sequence sufficiently similar to or derived from an amino acid sequence of a polypeptide of the invention and exhibits at least one activity of the polypeptide, but which is smaller than the full length polypeptide described herein. It includes a peptide comprising a. In general, a biologically active moiety comprises a moiety or region having at least one activity of a polypeptide. The biologically active portion of the polypeptide can be, for example, a peptide that is 5 or more amino acids in length. Such biologically active moieties may be prepared synthetically or by recombinant techniques and may be used for one or more functional activities of the polypeptides of the invention by methods described herein and / or as are well known in the art. Can be evaluated.

In addition, preferred derivatives of the invention include mature polys fused with another compound, such as a compound that increases the half-life of the polypeptide and / or reduces the potential immunogenicity of the polypeptide (eg, polyethylene glycol, “PEG”). Peptides are included. In the case of pegylation, fusion of the polypeptide to PEG can be performed by any of the methods known to those skilled in the art. For example, pegylation can be performed by first inducing cysteine mutations in the polypeptide to provide a linker for attaching PEG, followed by site-specific derivatization with PEG-maleimide. Cysteine can be added to the C-terminus of the peptide and is a preferred site in the present invention (see, eg, Tsutsumi, et al., Proc. Natl. Acad. Sci. USA 97 (15): 8548-53, 2000; Veronese , Biomaterials 22: 405-417, 2001; Goodsoon & Katre, Bio / Technology 8: 343-346, 1990). Variants of the polypeptides of the invention include polypeptides having an amino acid sequence or region thereof sufficiently similar to the amino acid sequence of SEQ ID NO of FIG. 1. The term “sufficiently similar” means that the first and second amino acid sequences have a sufficient number of amino acid residues that are the same or equivalent to the second amino acid sequence so that the first and second amino acid sequences have a common structural region and / or common functional activity. Or to contain the minimum number. For example, amino acid sequences containing at least 45%, preferably about 75% to 98%, of the same common structural region are defined as sufficiently similar in the present invention. Preferably, the variant will be sufficiently similar to the amino acid sequence of the preferred polypeptide of the present invention. Variants include variants of polypeptides encoded by polynucleotides that hybridize to the polynucleotides of the present invention or their complements under stringent conditions. Such variants generally retain the functional activity of the polypeptides of the invention. Libraries of fragments of polynucleotides can be used to generate diversified populations of fragments for screening and subsequent selection. For example, a library of fragments may be used to treat a double-helix PCR fragment of polynucleotides with nucleases under conditions where nicking occurs approximately once per molecule, denature double-stranded DNA, denature DNA, and Form double-helix DNA, which may include sense / antisense pairs, from the nicked product, remove single-helix portions from the reshaped duplexes by treatment with S1 nucleases, and generate the resulting fragment library Can be generated by linking into an expression vector. This method allows anyone to derive an expression library encoding the N-terminal and internal fragments of various sizes of the polypeptides of the present invention.

Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variants that act as VPAC2 agonists can be identified by screening the combinatorial libraies, eg truncation mutants, of mutants of the polypeptides of the invention for VPAC2 agonist activity.

In one embodiment, the diversified library of homologues is generated by molecular diversity mutagenesis at the nucleic acid level and encoded by various genetic libraries. A diversified library of variants may include, for example, a larger set of fusion proteins (eg, phage representations) in which a modified set of potential variant amino acid sequences contains the individual polypeptides, or alternatively, the set of sequences therein. Synthetic oligonucleotides can be produced by enzymatic linkage into the gene sequence for expression. There are a variety of methods that can be used to produce a library of potential variants from denatured oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, which then links the synthetic genes into appropriate expression vectors. The use of a degenerate set of genes makes it possible to provide in one mixture all the sequences encoding the desired set of potential variant sequences. Methods of synthesizing modified oligonucleotides are known in the art (see, for example, Narang, Tetrahedron 39: 3, 1983; Itakura, et al., Annu. Rev. Biochem. 53: 323, 1984; Itakura, et al. , Science 198: 1056, 1984; Ike, et al., Nucleic Acid Res. 11: 477, 1983).

Several techniques are known in the art for screening gene products of molecular diversity libraries made by point mutations or cleavage, and for screening cDNA libraries for gene products with selected properties. This technique can be applied for the rapid screening of gene libraries generated by molecular diversity mutagenesis of R-agonist polypeptides. The most widely used techniques that can be applied to high throughput assays for screening large gene libraries generally include cloning the gene library into replicable expression vectors, transforming the appropriate cells with the library of generated vectors, and Expression of a molecular diversity gene is included under the condition that detection of activity facilitates the isolation of the vector encoding the gene whose product is detected. Recursive ensemble mutagenesis (REM), a technique for enhancing the frequency of functional mutants within the library, can be used in conjunction with screening tests to identify desired variants.

The invention also provides chimeric or fusion polypeptides. The targeting sequence is designed to localize delivery of the polypeptide to the pancreas in order to minimize potential side effects. Polypeptides of the invention may consist of amino acids bound to each other by peptide bonds or modified peptide bonds (ie, peptide isotopes) and may contain amino acids other than 20 gene-encoded amino acids. Polypeptides can be modified by natural methods such as post-translational treatment, or by chemical modification techniques well known in the art. Such modifications are also described in the basic text, and in more detailed monographs, and in various literatures. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid-chains and amino or carboxy termini. It will be appreciated that the same type of modification may exist at the same or varying degrees at several positions within a given polypeptide. In addition, certain polypeptides may contain many types of modifications. Polypeptides can be branched, for example, as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may be produced by natural methods after translation, or may be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme components, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives. Covalent attachment of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinking, cysteine formation, pyroglutamate formation, formulation, gamma-carboxylation, glyco Misfire, GPI anchor formation, hydroxylation, iodide, methylation, myristoylation, oxidation, pegylation, proteolytic treatment, phosphorylation, prenylation, racemization, selenoylation, sulfated, argi Transition-RNA mediated addition of amino acids to proteins, such as nilation, and ubiquitination (see, eg , Proteins, Structure and Molecular Properties , 2nd ed, TE Creighton, WH Freeman and Company, Ne). w York (1993); Posttranslational Covalent Modification of Proteins , BC Johnson, ed., Academic Press, New York, pgs. 1-12 (1983); Seifter, et al., Meth.Enzymol 182: 626-646, 1990; Rattan, et al., Ann. NY Acad. Sci. 663: 48-62, 1992).

Polypeptides of the invention include the polypeptides of FIG. 1 (SEQ ID NOs: 1 to 152), and those sequences having meaningless variations in sequence from them. "Insubstantial variation" means substantially at least one biological function of the polypeptide of the invention, preferably VPAC2 agonist activity, more preferably selective VPAC2 agonist activity, most preferably described herein. Addition, substitution or deletion variants of any sequence that maintain insulin secretion activity. These functional equivalents are preferably at least about 90% identity to the polypeptide of FIG. 1, more preferably at least 95% identity to the polypeptide of FIG. 1, even more preferably to the polypeptide of FIG. 1. Polypeptides having at least 97% identity to, and include portions of such polypeptides having substantially the same biological activity. However, any polypeptide having an insignificant variation in the amino acid sequence from the polypeptide of FIG. 1 exhibiting functional equivalents as described further in the present invention is included within the description of the present invention.

As is known in the art, “similarity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substituents against the sequence of the second polypeptide. Such conservative substitutions are those described above and those described by Dayhoff ( The Atlas of Protein Sequence and Structure 5, 1978) and by Argos (EMBO J. 8: 779-785, 1989). Included. For example, amino acids belonging to one of the following groups show conservative changes:

ala, pro, gly, gln, asn, ser, thr;

cys, ser, tyr, thr;

-val, ile, leu, met, ala, phe;

lys, arg, his;

phe, tyr, trp, his; And

-asp, glu.

The invention also provides for the production of polynucleotides encoding polypeptides of the invention, and vectors comprising these polynucleotides, host cells genetically engineered by the vectors of the invention, and polypeptides of the invention by recombinant techniques. It is about. The host cell can be genetically engineered (transduced, transformed, or transfected) by a vector of the invention, which can be, for example, a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, viral particles, phage or the like. The engineered host cell can be cultured in a conventional nutrient medium modified as needed to activate the promoter or to select a transformant. Culture conditions such as temperature, pH, etc. are already used by the host cell selected for expression, and will typically be apparent to the skilled practitioner. Polypeptides of the invention can be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide sequence can be included in a vector or plasmid for expressing any one of a variety of expression vehicles, especially polypeptides. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences (eg, derivatives of SV40); Bacterial plasmids; Phage DNA; Yeast plasmids; Vectors derived from the combination of plasmid and phage DNA; Viral DNAs such as vaccinia, adenovirus, poultry pox virus, and pseudorabies. However, any other vector or plasmid can be used as long as they are replicable in the host and viable.

Appropriate DNA sequences can be inserted into the vector by various procedures. Generally, DNA sequences are inserted into appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are considered to be within the scope of those skilled in the art. DNA sequences in the expression vectors are operably linked to appropriate expression control sequence (s) (promoter) to direct mRNA synthesis. Representative examples of such promoters include the LTR or SV40 promoter, E. E. coli lac or trp, phage lambda P _L promoters, and other promoters known to modulate expression of genes in prokaryotic or eukaryotic cells or their viruses are included, but are not limited to these. Expression vectors may also contain ribosomal binding sites for initiation of translation and termination of transcription. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vector is preferably dihydrofolate reductase or neomycin resistance, or E. coli for eukaryotic cell culture. It contains genes that provide phenotypic traits for the selection of transformed host cells such as tetracycline or ampicillin resistance in E. coli. Vectors containing appropriate DNA sequences and appropriate promoters or regulatory sequences as described above can be used to transform the appropriate host to allow the host to express the protein. Representative examples of suitable hosts are E. coli. Bacterial cells such as E. coli, Salmonella typhimurium, Streptomyces; Fungal cells such as yeast; Insect cells such as Drosophila S2 and Spodoptera Sf9; Animal cells such as CHO, COS or Bowes melanoma; Adenovirus; Plant cells and the like, but are not limited to these. Selection of the appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

The invention also includes recombinant constructs containing one or more sequences as broadly described herein. This construct comprises a vector, such as a plasmid or viral vector, in which the sequence of the invention is inserted in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further contains regulatory sequences, including, for example, a promoter operably linked to the sequence. Many suitable vectors and promoters are known to those skilled in the art and are available commercially. The following vectors are provided as examples. Bacterial: pQE70, pQE60, pQE-9, pBS, phagescript, psiX174, pBluescript SK, pBsKs, pNH8a, pNH16a, pNH18a, pNH46a, pTRC99A, pKK223-3, pKK233-3, pDR540 and PRIT5. Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG and PSVL. However, any other plasmid or vector can be used as long as they are replicable and viable in the host. Promoter moieties can be selected from genes of interest using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two suitable vectors are pKK232-8 and pCM7. Specially designated bacterial promoters include laci, lacZ, T3, T7, gpt, lambda P _R , P _L and trp. Eukaryotic promoters include CMV immediate early (CMV immediate early), HSV thymidine kinase, early and late SV40, LTRs from retroviruses and mouse metallothionein-1. Selection of appropriate vectors and promoters is well within the level of techniques common in the art.

The present invention also relates to a host cell containing the construct described above. The host cell may be a higher eukaryotic cell such as a mammalian cell or a lower eukaryotic cell such as a yeast cell, or the host cell may be a prokaryotic cell such as a bacterial cell. Introduction of the construct into host cells can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (Davis, et al., Basic Methods in Molecular Biology, 1986). ). Constructs in host cells are used in a conventional manner to produce gene products encoded by recombinant sequences. Alternatively, the polypeptides of the present invention can be produced synthetically by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of an appropriate promoter. Cell-free translation systems may also be used to generate such proteins using RNAs derived from the DNA constructs of the invention. Cloning and expression vectors suitable for use by prokaryotic and eukaryotic hosts are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, NY, 1989), and their descriptions. The contents are incorporated by reference in the present invention.

Transcription of the DNA encoding the polypeptide of the invention by higher eukaryotic cells is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA from about 10 to about 300 bp that act on the promoter to increase transcription. Examples include SV40 enhancer (100-270 bp) late in the replicator, cytomegalovirus early promoter enhancer, polyoma enhancer in the latter half of the replicator, and adenovirus enhancer. In general, recombinant expression vectors are replicators and selectable markers that allow transformation of host cells (eg, the ampicillin resistance gene of E. coli or S. cerevisiae). TRP1 gene), and a promoter derived from a highly-expressed gene for directing transcription of downstream structural sequences. Such promoters may be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α factor, acid phosphatase or heat shock protein, among others. The heterologous structural sequences are combined into the cytoplasmic space or extracellular medium with the translational, initiation and termination sequences in the appropriate phase, and preferably a leader sequence capable of directing the secretion of the translated protein. Optionally, the heterologous sequence may encode a fusion protein comprising an N-terminal sequence that confers the desired characteristics (eg, stabilization or simplified purification of the expressed recombinant product).

Useful expression vectors for bacterial use can be constructed by inserting a structural DNA sequence encoding a protein of interest on an operable readout with a functional promoter with suitable translation, initiation and termination signals. The vector may contain one or more phenotypic selectable markers, and a replication source to ensure maintenance of the vector and, if necessary, to provide amplification in the host. Suitable prokaryotic hosts for transformation include, for example, E. coli. Various species of the genus E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas, Streptomyces and Staphylococcus are included. It can be used according to your choice. Useful expression vectors for bacterial use may include bacterial replication sources derived from commercially available plasmids comprising selectable markers and genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega, Madison, Wis., USA). These pBR322 "backbone" compartments can be combined with appropriate promoters and the structural sequences to be expressed.

After transforming the appropriate host strain and growing the host strain to the appropriate cell density, the selected promoters are deinhibited by appropriate means (eg, temperature shift or chemical induction) and the cells are cultured for an additional period of time. Cells are generally harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used for expression of proteins can be disrupted by conventional methods, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysates.

Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts described by Gluzman (Cell 23: 175, 1981), and other cell lines capable of expressing conformation vectors, such as For example C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors are clones, suitable promoters and enhancers, and also necessary ribosomal binding sites, polyadenylation sites, splice donor and receptor sites, transcription termination sequences, and 5 'flanking sequences. It may contain. DNA sequences derived from the SV40 viral genome, such as early promoters, enhancers, splices and polyadenylation sites of SV40 origin, can be used to provide the necessary non-transcribed genetic elements.

Polypeptides of the invention include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography Can be recovered and purified from recombinant cell culture by the methods used so far. If necessary, a protein refolding step can be used to complete the arrangement of mature proteins. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

Polypeptides of the invention may be products of chemical synthesis procedures or may be produced by recombinant techniques from prokaryotic or eukaryotic hosts (eg bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production procedure, the polypeptides of the invention may be glycosylated by mammalian or other eukaryotic carbohydrates, or may be aglycosylated. Polypeptides of the invention may also comprise initial methionine amino acid residues. Isolated or purified polypeptides of the present invention, or biologically active portions thereof, contain substantially no other cellular material, or culture medium when produced by recombinant technology, or chemical precursors or other when chemically synthesized. It is substantially free of chemicals. Preferably, the isolated polypeptides of the present invention are substantially free of cellular material and have less than about 30% (dry weight basis) of non-polypeptides or contaminants. If the polypeptide of the invention or a biologically active portion thereof is produced recombinantly, the culture medium preferably represents less than about 30% of the volume of the polypeptide preparation. If the present invention is produced by chemical synthesis, preferably the formulation contains less than about 30% of chemical precursors, or chemicals not of the present invention, based on dry weight.

Polypeptides of the present invention may be conveniently isolated as described in the specific examples below. The formulation of the purified polypeptide is at least about 70% pure, preferably the formulation is about 85% to about 99% pure. Purity of the formulation can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis and mass spectrometry / liquid chromatography.

Polynucleotide sequences encoding polypeptides of the invention can be synthesized in whole or in part using chemical methods well known in the art (see, eg, Caruthers, et al., Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn, et al., Nucl.Acids Res. Symp. Ser. 225-232, 1980). The polynucleotides encoding the polypeptides can then be cloned into expression vectors to express the polypeptides.

As will be appreciated by those skilled in the art, this may be beneficial for producing polypeptide-encoding nucleotide sequences having codons that do not exist naturally. For example, codons preferred by certain prokaryotic or eukaryotic hosts increase the rate of polypeptide expression or have RNA transcripts with desired properties such as half-lives longer than half-lives of transcripts generated from naturally occurring sequences. May be selected to generate.

The nucleotide sequences described herein are intended for altering polypeptide-coding sequences for a variety of reasons, including but not limited to changes that alter the closing, processing, and / or expression of the polypeptide or mRNA product. It can be manipulated using methods generally known in the art. Nucleotide sequences can be engineered using DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides. For example, site-directed mutagenesis can be used to insert new restriction sites, change glycosylation patterns, change codon preferences, generate splice variants, introduce mutations, and the like.

Also provided are related compounds, such as chemical analogs, organic analogs or peptido analogs, to the extent understood by those skilled in the art. As used herein, the terms "minetic", "peptide mimetic", "peptidomimetic", "organomimetic" and "chemical similar agent ( chemical mimetic) "includes peptide derivatives, peptide homologs, and chemical compounds having an atomic arrangement in three-dimensional orientation equivalent to the peptides of the present invention. As used herein, the phrase “equivalent to” has the same substitution or substitution of a particular atom or chemical moiety in a peptide and the same or similar to that of the atom and moiety in a similar agent compound to have the biological function of the peptide of the invention. It is understood to include compounds having bond lengths, bond angles, and arrangements that produce sufficiently similar arrangements or orientations. The three-dimensional arrangement of chemical components in the peptide analog of the invention is such that of the peptide of the invention having significant biological activity, structurally and / or functionally equivalent to the three-dimensional arrangement of the peptide backbone and the component amino acid side chains in the peptide. It produces peptido-, organic- and chemical analogs. These terms are described, for example, in Fachere, Adv. Drug Res. 15:29, 1986; Vever & Freidinger, TINS p. 392, 1985; and Evans, et al., J. Med. Chem. 30: 1229, 1987), as is understood in the art.

It is understood that pharmacophores exist for the biological activity of each peptide of the present invention. Drug action groups are understood in the art to include an ideal three-dimensional definition of the structural requirements for biological activity. Peptido-, organic- and chemical analogs can be designed such that each drug acting group is compatible with current computer aided drug design. The analog can be produced by structure-function analysis based on positional information from substituent atoms in the peptides of the invention.

Peptides provided by the present invention can advantageously be synthesized by chemical synthesis techniques known in the art, in particular solid phase synthesis techniques using, for example, automated peptide synthesizers available commercially. The analogous agents of the present invention can be synthesized by solid phase or solution phase methods commonly used for the synthesis of peptides (see, eg, Merrifield, J. Amer. Chem. Soc. 85: 2149-54, 1963; Carpino, Acc. Chem. Res. 6: 191-98, 1973; Birr, Aspects of the Merrifield Peptide Synthesis, Springer-Verlag: Heidelberg, 1978; The Peptides: Analysis, Synthesis, Biology, Vols. 1, 2, 3, and 5 ( Gross & Meinhofer, eds.), Academic Press: New York, 1979; Stewart, et al., Solid Phase Peptide Synthesis, 2nd.ed., Pierce Chem. Co .: Rockford, III., 1984; Kent, Ann. Rev. Biochem. 57: 957-89, 1988; and Gregg, et al., Int. J. Peptide Protein Res. 55: 161-214, 1990, which are incorporated herein by reference in their entirety).

Preference is given to using a solid phase method. Briefly, N-protected C-terminal amino acid residues are selected from divinylbenzene crosslinked polystyrene, polyacrylamide resin, Kieselguhr / polyamide (pepsyn K), controlled pore glass, cellulose, To insoluble supports such as polypropylene membranes, acrylic acid-coated polyethylene rods, and the like. A cycle of deprotection, neutralization and coupling of successive protected amino acid derivatives is used to bind amino acids from the C-terminus according to the amino acid sequence. For some synthetic peptides, the FMOC method using acid-sensitive resins may be used. Preferred solid supports in this regard are chloromethyl resins, hydroxymethyl resins, paraacetamidomethyl resins, benzhydrylamine (BHA) resins, 4-methylbenzhydrylamine (MBHA) resins, oxime resins, 4-alkoxy Benzyl alcohol resin (Wang resin), 4- (2 ', 4'-dimethoxyphenylaminomethyl) -phenoxymethyl resin, 2,4-dimethoxybenzhydryl-amine resin, and 4- (2 Di, which is available commercially in various functionalized forms, including ', 4'-dimethoxyphenyl-FMOC-amino-methyl) -phenoxyacetamidonorloyl-MBHA resin (Rink amide MBHA resin) Vinylbenzene crosslinked polystyrene resins. A particularly preferred protecting group for alpha amino acids is base-labile 9-fluorenylmethoxy-carbonyl (FMOC).

Suitable protecting groups for the side chain functionality of BOC (t-butyloxycarbonyl) and amino acids chemically compatible with FMOC groups are well known in the art. The following protected amino acid derivatives are preferred when using FMOC chemistry: FMOC-Cys (Trit), FMOC-Ser (But), FMOC-Asn (Trit), FMOC-Leu, FMOC-Thr (Trit), FMOC -Val, FMOC-Gly, FMOC-Lys (Boc), FMOC-Gln (Trit), FMOC-Glu (OBut), FMOC-His (Trit), FMOC-Tyr (But), FMOC-Arg (PMC (2, 2,5,7,8-pentamethylchroman-6-sulfonyl)), FMOC-Arg (BOC) ₂ , FMOC-Pro and FMOC-Trp (BOC). Amino acid residues are DIC (diisopropyl-carbodiimide), DCC (dicyclohexylcarbodiimide), BOP (benzotriazolyl-N-oxytrisdimethylaminophosphonium hexafluorophosphate), PyBOP (benzotriazole- By direct coupling with 1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate), PyBrOP (bromo-tris-pyrrolidinophosphonium hexafluorophosphate); Via the formation of symmetric anhydrides; Via an active ester such as pentafluorophenyl ester; Or via the formation of a BOBt (1-hydroxybenzotriazole) active ester; Or by using FMOC-amino acid fluoride and chloride, or by using various coupling agents and chemistry known in the art, such as using FMOC-amino acid-N-carboxy anhydride. HBTU (2- (1H-benzotriazol-1-yl), 1,1,3,3-tetramethyluronium hexafluorophosphate) in the presence of HOBt or HOAt (7-azahydroxybenztriazole) or Activation by HATU (2- (1H-7-aza-benzotriazol-1-yl), 1,1,3,3-tetramethyluronium lexafluoro-phosphate) is preferred.

Solid phase methods can be performed manually, but automated synthesis on a commercially available peptide synthesizer (eg, Applied Biosystems 431A, etc .; Applied Biosystems, Foster City, CA) is preferred. In a typical synthesis, the first (C-terminal) amino acid is loaded onto the chlorotrityl resin. Peptide sequences can be generated using continuous deprotection (using 20% piperidine / NMP (N-methylpyrrolidone)) and coupling cycles according to the ABI FastMoc protocol (Applied Biosystems). Double and triple couplings with capping with acetic anhydride may be used.

Synthetic similar agent peptides can be cleaved from the resin and deprotected by treatment with TFA (trifluoroacetic acid) containing the appropriate scavenger. Many such cleavage agents can be used, such as Reagent K (0.75 g crystalline phenol, 0.25 ml ethanedithiol, 0.5 ml thioanisole, 0.5 myri deionized water, 10 ml TFA) and the like. Peptides are separated from the resin by filtration and isolated by ether precipitation. Further purification can be carried out by conventional methods such as gel filtration and reversed phase HPLC (high performance liquid chromatography). Synthetic analogs according to the invention may be in the form of base-addition salts, including pharmaceutically acceptable salts, in particular salts of organic and inorganic bases. Base-addition salts of acidic amino acid residues are prepared by treating the peptides with the appropriate base or inorganic base according to procedures well known to those skilled in the art, or the desired salt is obtained directly by lyophilization of the appropriate base. May be

In general, those skilled in the art will appreciate that peptides as described herein are modified by a variety of chemical techniques that produce peptides that have essentially the same activity as unmodified peptides and optionally have other desirable properties. Can be. For example, the carboxylic acid group of the peptide may be provided in the form of a pharmaceutically-acceptable cation. The amino groups in the peptides may be in the form of pharmaceutically-acceptable acid addition salts such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid and other organic acid salts, or may be converted to amides. Thiols may be protected by any one of a number of well-known protecting groups such as acetamide groups. Those skilled in the art will also recognize how to introduce cyclic structures into the peptides of the present invention so that they can be closer to the natural binding orientation. For example, carboxyl terminus or amino terminus cysteine residues can be added to the peptide such that when oxidized the peptide contains a disulfide bond to produce a cyclic peptide. Other methods of ring closure include the formation of thioether and carboxyl- and amino-terminal amides and esters.

Specifically, various techniques for constructing peptide derivatives and homologues that have the same or similar desired biological activity as the corresponding peptide compound, but which have more desirable activity than the peptide in view of solubility, stability and susceptibility to hydrolysis and proteolysis This can be used. Such derivatives and homologues include peptides that are modified in the N-terminal amino group, C-terminal carboxyl group, and / or have changed one or more amido bonds in the peptide to non-amido bonds. Two or more such modifications can be coupled to one peptide mimetic structure (eg, including a modification in a C-terminal carboxyl group and a -CH ₂ -carbamate bond between two amino acids in the peptide). .

Amino terminal modifications include alkylation, acetylation, addition of carbobenzoyl groups, and formation of succinimide groups. Specifically, the N-terminal amino group is reacted to react an amide group of formula RC (O) NH-, wherein R is alkyl, preferably lower alkyl, and reacts with an acid halide, RC (O) Cl or an acid anhydride. Added by). Generally, the reaction is inert, containing approximately equimolar or excess (eg, about 5 equivalents) of acid halide, preferably containing an excess (eg, about 10 equivalents) of tertiary amine, such as diisopropylethylamine. This may be accomplished by contacting the peptide in a diluent (eg dichloromethane) to clean up the acid generated during the reaction. Reaction conditions are conventional in other respects (eg room temperature for 30 minutes). The terminal amino group may be alkylated to provide lower alkyl N-substitution, followed by reaction with an acid halide as described above to provide an N-alkyl amide group of formula RC (O) NR—. Alternatively, the amino terminus can be covalently bound to the succinimide group by reacting with succinic anhydride. Approximately equimolar amounts or excess succinic anhydride (eg, about 5 equivalents) are used, and the terminal amino groups are selected from suitable inert solvents (eg, as described in Wolfenberg, et al., US Pat. No. 4,612,132). For example, dichloromethane) is converted to succinimide by methods well known in the art, including the use of excess (eg, 10 equivalents) of tertiary amines, such as diisopropylethylamine. It is incorporated by reference into the invention. In addition, succinic acid groups can be substituted, for example, with C ₂ -to C ₆ -alkyl or -SR substituents prepared in a conventional manner to provide substituted succinimides at the N-terminus of the peptide. Such alkyl substituents can be prepared by reacting lower olefins (C ₂ -to C ₆ -alkyl) with maleic anhydride in the manner described by Wolenberg et al., Wherein the -SR substituent is RSH (where R is As defined above) can be prepared by reacting with maleic anhydride. In another advantageous embodiment, amino termini can be derivatized to form benzyloxycarbonyl-NH- or substituted benzyloxycarbonyl-NH- groups. This derivative is preferably reacted with an appropriate equivalent or excess of benzyloxycarbonyl chloride (CBZ-Cl) or substituted CBZ-Cl in a suitable inert diluent (e.g. dichloromethane) containing tertiary amines during the reaction. It can be produced by cleaning the generated acid. In another derivative, the N-terminus reacts the sulfonamide group by converting the terminal amine into sulfonamide by reacting with an equivalent or excess (eg 5 equivalents) of RS (O) ₂ Cl in a suitable inert diluent (dichloromethane). (Where R is alkyl, preferably lower alkyl). Preferably, the inert diluent contains an excess of tertiary amine (eg 10 equivalents) such as diisopropylethylamine to clean up the acid produced during the reaction. Reaction conditions are conventional in other respects (eg room temperature for 30 minutes). Carbamate groups are equivalent or excess (eg 5 equivalents) of R-OC (O) Cl or R-OC (O) OC ₆ H ₄ -in a suitable inert diluent (eg dichloromethane) at the amino terminus. It can be produced by reacting with p-NO ₂ to convert terminal amines to carbamate (where R is alkyl, preferably lower alkyl). Preferably, the inert diluent contains an excess (eg 10 equivalents) of tertiary amine, such as diisopropylethylamine, to clean any acid produced during the reaction. Reaction conditions are conventional in other respects (eg room temperature for 30 minutes). The urea group reacts the terminal amine with urea (ie, RNHC (O) NH) at an amino terminus with an equivalent or excess (eg 5 equivalents) of RN = C═O in a suitable inert diluent (eg dichloromethane). -) Can be formed by conversion to a group (where R is as defined above). Preferably, the inert diluent contains an excess (eg, about 10 equivalents) of tertiary amine, such as diisopropylethylamine. Reaction conditions are conventional in other respects (eg, room temperature for about 30 minutes).

When a C-terminal carboxyl group produces a peptide mimetic that can be substituted by an ester (e.g., -C (O) OR, where R is alkyl, preferably lower alkyl), The resin used for the preparation can be used and the side chain protected peptide can be cleaved with a base and an appropriate alcohol (eg methanol). The side chain protecting group can be removed in a conventional manner by treatment with hydrogen fluoride to give the desired ester. Benzhydrylamine resins are used as a solid support for peptide synthesis when the C-terminal carboxyl group produces a peptide like agent substituted with amide -C (O) NR ₃ R ₄ . Once the synthesis is complete, the hydrogen fluoride treatment to liberate the peptide from the support produces a free peptide amide (ie, C-terminus -C (O) NH ₂ ) directly. In another way, the use of chloromethylated resins in peptide synthesis coupled with the reaction with ammonia to cleave the side chain protected peptide from the support results in side chain protected alkylamides or dialkylamides (ie, C-terminal -C (O) NRR ₁ , wherein R and R ₁ are alkyl, preferably lower alkyl. The side chain protection is then removed in a conventional manner by treatment with hydrogen fluoride to give free amides, alkylamides or dialkylamides.

In another alternative embodiment, the ring closure is achieved by introducing a C-terminal carboxyl group or C-terminal ester to replace each of the -OH or ester (-OR) of the carboxyl group or ester with an N-terminal amino group to form a cyclic peptide. You can. For example, after synthesis and cleavage to provide a peptide acid, the free acid is combined with dicyclohexylcarbodiimide in solution, for example in methylene chloride (CH ₂ Cl ₂ ), dimethylformamide (DMF) or mixtures thereof. It is converted to the activated ester by the same appropriate carboxyl group activator. The cyclic peptide is then formed by replacing the activated peptide with an N-terminal amine. Ring closure reactions other than polymerization can be enhanced by using very dilute solutions according to methods well known in the art.

Peptide mimetics as understood in the art and provided by the present invention are structurally similar to the peptides of the invention, but are known in the art and described in the following documents, each of which is incorporated by reference herein. -CH ₂ NH-, -CH ₂ S-, -CH ₂ CH _2- , -CH = CH- (both cis and trans isomers), -COCH _2- , -CH (OH) CH _2- And one or more peptide bonds optionally substituted by a bond selected from the group consisting of -CH ₂ SO-: Spatola, Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, (Weinstein, ed.), Marcel Dekker: New York , p. 267, 1983; Spatola, Peptide Backbone Modifications 1: 3, 1983; Morley, Trends Pharm. Sci. pp. 463-468, 1980; Hudson, et al., Int. J. Pept. Prot. Res. 14: 177-185, 1979; Spatola, et al., Life Sci. 38: 1243-1249, 1986; Hann, J. Chem. Soc. Perkin Trans. 1307-314, 1982; Almquist, et al., J. Med. Chem. 23: 1392-1398, 1980; Jennings-White, et al., Tetrahedron Lett. 23: 2533, 1982; Szelke, et al., EP 045665A; Holladay, et al., Tetrahedron Lett. 24: 4401-4404, 1983; And Hruby, Life Sci. 31: 189-199, 1982. Such peptide analogs are more economical than polypeptide embodiments, for example, are more economical to production, and have greater chemical stability or enhanced pharmacological properties (eg, half-life, absorption, potency, Efficacy, etc.), and reduced antigenicity and other properties.

Analogous homologues of the peptides of the invention can also be obtained using the principles of conventional or ideal drug design (see, eg, Andrews, et al., Proc. Alfred Benzon Symp. 28: 145-165, 1990; McPherson, Eur. J. Biochem. 189: 1-24, 1990; Hol, et al., In Molecular Recognition: Chemical and Biochemical Problems, (Roberts, ed.); Royal Society of Chemistry; pp. 84-93, 1989a; Hol , Arzneim-Forsch. 39: 1016-1018, 1989b; Hol, Agnew Chem. Int. Ed. Engl. 25: 767-778, 1986, the disclosures of which are incorporated herein by reference).

According to conventional drug design, the desired analog agent molecule can be obtained by randomly testing a molecule whose structure has properties in common with the structure of the "natural" peptide. Quantitative contributions due to changes in certain groups of binding molecules can be determined by measuring the biological activity of putative analogs compared to the activity of the peptide. In a preferred embodiment of the ideal drug design, the agonist is designed to bear the properties of the most stable three-dimensional structure of the peptide. That is, for example, the agonist may be designed to have chemical groups oriented in a manner sufficient to cause ionic, hydrophobic or van der Waals interactions similar to those represented by the peptides of the invention as described herein. Can be.

Preferred methods for carrying out the ideal analog design are described in Hol, 1989a; Hol, 1989b; And computer systems capable of forming representations of three-dimensional structures of peptides such as those exemplified by Hol, 1986. The molecular structure of the peptido-, organic- and chemical analogs of the peptides of the invention can be produced using computer-assisted design programs available commercially in the art. Examples of such programs include SYBYL 6.5 ™, HQSAR ™, and ALCHEMY 2000 ™ (Tripos); GALAXY ™ and AM2000 ™ (AM Technologies, Inc., San Antonio, TX); CATALYST ™ and CERIUS ™ (Molecular Simulations, Inc., San Diego, CA); CACHE PRODUCTS ™, TSAR ™, AMBER ™, and CHEM-X ™ (Oxford Molecular Products, Oxford, CA) and CHEMBUILDER3D ™ (Interactive Simulations, San Diego, CA).

For example, peptido-, organic- and chemical analogs produced using the peptides described herein using molecular modeling programs recognized in the art can be produced using conventional chemical synthesis techniques. And most preferably can be designed to control high throughput screening, including molecular diversity chemistry. Molecular diversity methods useful for the production of peptido-, organic- and chemical analogs of the invention include, for example, Cidco (SIDDCO, Tuscon, Arizona); Tripos, Inc .; Calbiochem / Novabiochem, San Diego, CA; Symyx Technologies (Symyx Technologies, Inc., Santa Clara, Calif.); Medichem Research, Inc., Lemont, IL; Pharm-Eco Laboratories, Inc., Bethlehem, PA; Or phage display arrays, solid phase synthesis, and molecular diversity chemical arrays, such as those provided by organon (N.V. Organon, Oss, Netherlands). Molecular diversity chemical production of peptido-, organic- and chemical analogs of the present invention is described by Terrett, (Combinatorial Chemistry, Oxford University Press, London, 1998); Gallop, et al., J. Med. Chem. 37: 1233-51, 1994; Gordon, et al., J. Med. Chem. 37: 1385-1401, 1994; Look, et al., Bioorg.Med. Chem. Lett. 6: 707-12, 1996; Ruhland, et al., J. Amer. Chem. Soc. 118: 253-4, 1996; Gordon, et al., Acc. Chem. Res. 29: 144-54, 1996; Thompson & Ellman, Chem. Rev. 96: 555-600, 1996; Fruchtel & Jung, Angew. Chem. Int. Ed. Engl. 35: 17-42, 1996; Pavia, "The Chemical Generation of Molecular Diversity", Network Science Center, www.netsci.org, 1995 Adnan, et al., "Solid Support Combinatorial Chemistry in Lead Discovery and SAR Optimization," Id., 1995; Davies and Briant, "Combinatorial Chemistry Library Design using Pharmacophore Diversity, 'Id., 1995; Pavia," Chemically Generated Screening Libraries: Present and Future, "Id., 1996; and US Patent Nos. 5,880,972; 5,463,564; 5,331,5 73 and 5,573,905), including, but not limited to, those produced according to methods known in the art.

The newly synthesized polypeptide can be substantially purified by preparative high performance liquid chromatography (see, eg, Creighton, Proteins: Structures And Molecular Principles, WH Freeman and Co., New York, N.Y., 1983). The composition of the synthetic polypeptides of the present invention can be identified, for example, by amino acid analysis or sequencing by Edman degradation procedure (Creighton, supra). In addition, any portion of the amino acid sequence of a polypeptide can be changed during direct synthesis and / or combined with sequences derived from other proteins using chemical synthesis to produce variant polypeptides or fusion polypeptides.

Polypeptides of the invention can also be used according to the invention by expressing such polypeptides in vivo, which is often referred to as "gene therapy." Thus, for example, cells can be manipulated ex vivo with polynucleotides encoded for a polypeptide (DNA or RNA), after which the engineered cells can be provided to a patient to be treated with the polypeptide. Such methods are well known in the art. For example, cells can be engineered by procedures known in the art by using retroviral particles containing RNA encoded for the polypeptides of the invention.

Local delivery of insulin secretagogues using gene therapy can provide therapeutic agents to the target site (eg, the pancreas). For example, pancreas-specific promoters were used to generate β-cell pancreatic tumor mouse models (Hanahan, Nature 315 (6015): 115-22, 1985).

Both in vitro and in vivo gene therapy methodologies are envisioned. Several methods of transferring potentially therapeutic genes to defined cell populations are known (see, eg, Mulligan, Science 260: 926-31, 1993). These methods include, for example, 1) direct gene transfer (see, eg, Wolff, et al., Science 247: 1465-68, 1990); 2) liposome-mediated DNA transfer (see, eg, Caplen, et al., Nature Med. 3: 39-46, 1995; Crystal, Nature Med. 1: 15-17, 1995; Gao and Huang, Biochem. Res. Comm. 179: 280-85, 1991); 3) retrovirus-mediated DNA transfer (see, eg, Kay, et al., Science 262: 117-19, 1993; Anderson, Science 256: 808-13, 1992); 4) DNA virus-mediated DNA transfer. Such DNA viruses include, for example, adenoviruses (preferably Ad-2 or Ad-5 base vectors), herpes viruses (preferably herpes simplex virus base vectors), and parvoviruses (preferably "deletions ( defective ”or non-autonomous parvovirus base vector, more preferably adeno-associated viral base vector, most preferably AAV-2 base vector) (see, eg, Ali, et al). Gene Therapy 1: 367-84, 1994; US Pat. No. 4,797,368; US Pat. No. 5,139,941; these are incorporated herein by reference).

The choice of a particular vector system for transferring the gene of interest can depend on a variety of factors. One important factor is the nature of the target cell population. Retroviral vectors have been extensively studied and used in many gene therapy applications, but these vectors are generally unsuitable for infecting non-differentiated cells. Retroviruses also have the potential for oncogenicity. However, recent developments in the field of lentiviral vectors may circumvent some of these limitations (see, for example, Naldini, et al., Science 272: 263-67, 1996).

Retroviruses from which retroviral plasmid vectors can be derived include Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Retroviruses such as Rus Sarcoma Virus, Harvey Sarcoma Virus , But not limited to, chicken avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary gland virus Do not.

Adenoviruses have the advantage that they have a broad host range, can infect inactive or terminally differentiated cells, such as neurons or hepatocytes, and are essentially non-tumorigenic (see, eg, Ali, et al. , 1994). Adenovirus does not appear to integrate into the host genome. Since they are extrachromosomal, the risk of insertional mutagenesis is greatly reduced (Ali, et al., 1994).

Adeno-associated viruses show advantages similar to adenovirus-based vectors. However, AAVs exhibit site-specific integration on human chromosome 19 (see Ali, et al., 1994).

In a preferred embodiment, the DNA encoding the polypeptide insulin secretagogue of the invention is used in gene therapy for diseases such as diabetes and diabetes-related diseases.

According to the present invention, gene therapy with DNA encoding a polypeptide insulin secretagogue or mutein of the present invention is provided to a patient in need of such treatment either concurrently with or shortly after diagnosis.

The skilled practitioner will appreciate that any suitable gene therapy containing polypeptide insulin secretagogues, DNA or DNA fragments, derivatives, or variants of polypeptide insulin secretagogues may be used in accordance with these embodiments. Techniques for constructing such vectors are well known in the art (see, eg, Anderson, Nature 392: 25-30, 1998; Verma, et al., Nature 389: 239-242, 1998). The introduction of the polypeptide insulin secretory herbicidal DNA-containing vector into the target site can be performed using known techniques.

The vector may contain one or more promoters. Suitable promoters that can be used include retroviral LTR, SV40 promoter; Human cytomegalovirus (CMV) promoters (including Miller, et al., Biotechniques 7 (9): 980-990, 1989), or other promoters (e.g., histone, pol III, and β-actin promoters) But not limited to, cellular promoters such as eukaryotic promoters), but are not limited to these. Other viral promoters that can be used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of suitable promoters will be apparent to those skilled in the art from the guidance contained herein.

Nucleic acid sequences encoding polypeptides of the invention are present under the control of a suitable promoter. Suitable promoters that can be used include adenovirus promoters such as adenoviral major late promoters; Or heterologous promoters such as cytomegalovirus (CMV) promoters; Respiratory syncytial virus (RSV) promoter; Inducible promoters such as the MMT (metallothionein) promoter; Heat shock promoters; Albumin promoter; ApoAI promoter; Human globin promoter; Viral thymidine kinase promoters such as the herpes simplex thymidine kinase promoter; Retroviral LTRs (including the modified retroviral LTRs described above); β-actin promoter; And human growth hormone promoters. The promoter may also be a natural promoter that regulates the gene encoding the polypeptide.

Retroviral plasmid vectors can be used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected are PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP + E-86, GP + envAm12 And DNA cell lines as described in Miller, Hum. Gene Ther. 1: 5-14, 1990, which is incorporated herein by reference in its entirety. Vectors can transduce packaging cells by any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or coupled to lipids and then administered to a host. Producer vertical lines produce infectious retroviral vector particles comprising nucleic acid sequence (s) encoding a polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells in vitro or in vivo. Transduced eukaryotic cells may express nucleic acid sequence (s) encoding a polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells and bronchial epithelial cells.

A different approach to gene therapy is "transkaryotic therapy" in which a patient's cells are treated ex vivo to induce dormant chromosomal genes and then reintroduced to the patient to produce the subject protein. Transcarotitic therapy assumes that the individual has the normal complement of the genes required for activation. Transcarotitic therapy involves the introduction of promoters or other exogenous regulatory sequences capable of activating genes of the neonatal phase in vitro into the chromosomal DNA of the patient's cells, incubating, selecting active protein-producing cells, Reintroducing the activated cells to the patient for the purpose of ensuring that they are then fully established. Subsequently, the "gene activated" cells produce the protein of interest for some significant amount of time, perhaps for as long as the patient's lifetime (see, eg, US Pat. Nos. 5,641,670 and 5,733,761, incorporated herein by reference in their entirety). .

The invention also includes antibodies and antibody fragments that selectively bind the polypeptides of the invention. Any type of antibody known in the art can be produced using methods well known in the art. For example, antibodies can be generated that specifically bind to the epitopes of the polypeptides of the invention. As used herein, "antibody" includes fully immunoglobulin molecules, and fragments thereof such as Fab, F (ab ') ₂ and Fv capable of binding epitopes of polypeptides of the invention. In general, at least 6, 8, 10 or 12 contiguous amino acids may be required to form an epitope. However, epitopes comprising non-adjacent amino acids may require more amino acids, for example at least 15, 25 or 50 amino acids.

Antibodies that specifically bind to the epitopes of the polypeptides of the invention are not only therapeutically effective, but also Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or else known in the art. It can be used in immunochemical test methods such as immunochemical test methods. Many protocols for competitive binding or immunoradiometric testing are well known in the art. Such immunoassays generally include the determination of complex formation between an immunogen and an antibody that specifically binds to the immunogen.

In general, an antibody that specifically binds to a polypeptide of the invention provides a detection signal that is at least 5-, 10- or 20-fold greater than the detection signal provided by other proteins when used in immunochemical assays. do. Preferably, the antibody that specifically binds to the polypeptide of the present invention does not detect other proteins in the immunochemical test method, and can immunoprecipitate the polypeptide of the present invention from solution.

The polypeptides of the invention can be used to immunize mammals such as mice, rats, rabbits, guinea pigs, monkeys or humans to produce polyclonal antibodies. If desired, the polypeptides of the invention can be conjugated to carrier proteins such as bovine serum albumin, tyroglobulin and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include Freund's adjuvant, inorganic gels (e.g., aluminum hydroxide), and surface active materials (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions). , Keyhole limpet hemocyanides and dinitrophenols), but is not limited thereto. Among the adjuvant used in humans are BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are particularly useful.

Monoclonal antibodies that specifically bind to the polypeptides of the invention may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV hybridoma technology (Kohler, et al., Nature 256: 495-97, 1985; Kozbor, et al., J. Immunol.Methods 81: 3142, 1985; Cote, et al., Proc. Natl. Acad. Sci. 80: 2026-30, 1983; Cole, et al., Mol. Cell Biol. 62: 109-20, 1984).

In addition, techniques developed for the production of “chimeric antibodies”, ie, the splicing of mouse antibody genes against human antibody genes, will be used to obtain molecules with appropriate antigen specificity and biological activity. (Morrison, et al., Proc. Natl. Acad. Sci. 81: 6851-55, 1984; Neuberger, et al., Nature 312: 604-08, 1984; Takeda, et al., Nature 314: 452 -54, 1985). Monoclonal antibodies and other antibodies can also be “humanized” to prevent the patient from initiating an immune response to the antibody when the antibody is used therapeutically. Such antibodies may be sufficiently similar to human antibodies whose sequences are used directly in therapy, or may require changes in several key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues different from them in the human sequence by site directed mutagenesis of individual residues, or by grating the entire complementarity crystallization moiety. Alternatively, humanized antibodies can be produced using recombinant methods (see, for example, GB2188638B). Antibodies that specifically bind to the polypeptides of the invention may contain partially or fully humanized antigen binding sites as described in US Pat. No. 5,565,332.

Alternatively, the techniques described for the production of single chain antibodies can be adjusted to produce single chain antibodies that specifically bind to the polypeptides of the invention using methods known in the art. Antibodies with related specificities, but with distinct genetic factor compositions, can be generated by chain shuffling from a random molecular diversity immunoglobulin library (Burton, Proc. Natl. Acad. Sci. 88: 11120-23, 1991).

Single-chain antibodies can also be constructed using DNA amplification methods such as PCR using hybridoma cDNA as a template (Thirion, et al., Eur. J. Cancer Prev. 5: 507-11, 1996). Single chain antibodies may be mono- or bispecific and may be bivalent or tetravalent. The construction of tetravalent bispecific single chain antibodies is taught, for example, in Coloma & Morrison, Nat. Biotechnol. 15: 159-63, 1997. The construction of bivalent bispecific single chain antibodies is taught in Mallender & Voss, J. Biol. Chem. 269: 199-206, 1994.

Nucleotide sequences encoding single-chain antibodies can be constructed using manual or automated nucleotide synthesis, cloned into expression constructs using standard recombinant DNA methods, and introduced into cells to express coding sequences, as described below. have. Alternatively, single-chain antibodies are described, for example, in filamentous phage technology; Verhaar, et al., Int. J. Cancer 61: 497-501, 1995; Nicholis, et al., J. Immunol. Meth. 165: 81-91, 1993).

Antibodies that specifically bind to the polypeptides of the invention can also be induced by in vitro production in lymphocyte populations or by Orlandi, et al., Proc. Natl. Acad. Sci. 86: 38333-37, 1989; It may also be produced by screening immunoglobulin libraries or panels of high specific binding reagents as described in Winter, et al., Nature 349: 293-99, 1991).

Other types of antibodies can also be constructed and used therapeutically in the methods of the invention. For example, chimeric antibodies can be constructed as described in WO 93/03151. Binding proteins derived from immunoglobulins and that are multivalent and multispecific, such as "diabodies" can also be prepared (see, eg, WO 94/13804).

Human antibodies with the ability to bind the polypeptides of the invention may also be identified from the MorphoSys HuCAL ™ library as follows. Polypeptides of the invention may be coated onto microtiter plates and incubated with MorphoSys HuCAL ™ Fab phage library. These phage-bound Fabs that do not bind to the polypeptide of the invention can be removed by washing from the plate leaving only those phages that are tightly bound to the polypeptide of the invention. Bound phage may, for example, change the pH, or. Elution by eluting with E. coli, E. coli. E. coli hosts can be infected and amplified. This panning method can be repeated once or twice to enrich the population of antibodies that bind tightly to the polypeptide of the invention. The Fabs from the enriched pools are then expressed, purified and screened for ELISA testing.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passing over a column to which a polypeptide of the invention is bound. The bound antibody can then be eluted from the column using a buffer with high salt concentration.

Polypeptides of the invention include both type 1 and type 2 diabetes (non-insulin dependent diabetes mellitus) as a result of their ability to promote insulin secretion from pancreatic islet cells in vitro and by causing a decrease in blood glucose in vivo. It can be used to treat diabetes. Such treatment may also delay the onset of diabetes and diabetic complications. The polypeptide can be used to prevent the progression of developing type 2 diabetes in subjects with impaired glucose tolerance. Other diseases and conditions that can be treated or prevented using the compounds of the present invention in the methods of the present invention include the following: Growth stage phenotypic diabetes mellitus (MODY) in adolescents (Herman, et al., Diabetes 43: 40, 1994); Latent autoimmune adult diabetes (LADA) (Zimmet, et al., Diabetes Med. 11: 299, 1994); Impaired glucose tolerance (IGT) (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1): S5, 1999); Impaired fasting glucose (IFG) (Charles, et al., Diabetes 40: 796, 1991); Gestational diabetes (Metzger, Diabetes, 40: 197, 1991); And metabolic syndrome X.

Polypeptides of the invention also include diseases such as obesity, and atherosclerotic diseases, hyperlipidemia, hypercholesterolemia, low HDL levels, hypertension, cardiovascular diseases (including atherosclerosis, coronary heart disease, coronary artery disease, and hypertension) For the treatment of cerebrovascular diseases and peripheral vascular diseases; And lupus, polycystic ovary syndrome, carcinogenesis, and hyperplasia, asthma, male reproductive disorders, ulcers, sleep disorders, disorders of lipid and carbohydrate metabolism, daily cycle dysfunction, growth disorders, disorders of energy homeostasis, autoimmune diseases (eg For example, immune diseases including systemic lupus erythematosus), and acute and chronic inflammatory diseases, and septic shock.

The compounds of the present invention may also be used for example for cell differentiation to produce lipid accumulating cells, for example abnormal pancreatic β-cell function, insulin secreting tumors and / or autoantibodies to insulin, autoantibodies to insulin receptors, or Insulin sensitivity and blood glucose levels implicated in autoimmune hypoglycemia due to autoantibodies stimulating to pancreatic β-cells, macrophage differentiation, inflammatory response, carcinogenesis, hyperplasia, adipocyte genes that induce atherosclerotic plaque formation Expression, adipocyte differentiation, reduction of pancreatic β-cell mass, insulin secretion, tissue sensitivity to insulin, liposarcoma cell growth, polycystic ovarian disease, chronic ovulation, hyperandrogenosis, progesterone production, steroid production, potential redox in cells Sexual and oxidative stress, nitric oxide synthase (NOS) production, increased gamma glutamyl transpeptida , Catalase, plasma triglycerides, may be useful in the treatment of physiological disorders associated with such HDL and LDL cholesterol levels.

Compounds of the invention can also be used in the methods of the invention to treat secondary causes of diabetes (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1): S5, 1999). These secondary causes include excess glucocorticoids, excess growth hormones, chromaffin cell tumors, and drug-induced diabetes. Drugs that can induce diabetes include, but are not limited to, pyriminyl, nicotinic acid, glucocorticoids, phenytoin, thyroid hormones, β-adrenergic agonists, α-interferon, and drugs used to treat HIV infection.

In addition, the polypeptides of the present invention may be used for asthma (Bolin, et al., Biopolymer 37: 57-66, 1995; US Pat. No. 5,677,419; polypeptide R3P0 is active in relaxing guinea pig bronchial smooth muscle), induction of hypotension ( VIP induces hypotension, tachycardia and facial scarring in asthma patients; Morice, et al., Peptides 7: 279-280, 1986; Morice, et al., Lancet 2: 1225-1227, 1983), male reproductive problems ( Siow, et al., Arch. Androl. 43 (1): 67-71, 1999); As anti-cell death / neuroprotective agent (Brenneman, et al., Ann. N. Y. Acad. Sci. 865: 207-12, 1998); Cardioprotection during ischemic development (Kalfin, et al., J. Pharmacol. Exp. Ther. 1268 (2): 952-8, 1994; Das, et al., Ann. NY Acad. Sci. 865: 297-308, 1998), manipulation of the daily periodic clock and associated diseases (Hamar, et al., Cell 109: 497-508, 2002; Shen, et al., Proc. Natl. Acad. Sci. 97: 11575-80, 2000 ) And finally as an antiulcer agent (Tuncel, et al., Ann. NY Acad. Sci. 865: 309-22, 1998).

The polypeptides of the invention can be used alone or in combination with additional therapies and / or compounds known to those skilled in the art in the treatment of diabetes and related diseases. Alternatively, the methods and compounds described herein can be used partially or completely in combination therapy.

Polypeptides of the invention may also contain PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α-glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose excretory compounds, insulin And anti-obesity drugs, and may be administered in combination with other therapies known to treat diabetes. Such therapy may be administered prior to, concurrent with, or after administration of the polypeptide of the invention. Insulin includes both long and short acting forms and preparations of insulin. PPAR agonists include agonists or combinations of PPAR subunits. For example, a PPAR agonist may include a combination of two or three of agonists of PPAR-α, PPAR-γ, PPAR-δ, or subunits of PPAR. PPAR agonists can include, for example, rosiglitazone and pioglitazone. Sulfonylurea drugs include, for example, glyburide, glymepyride, chlorpropamide, and glyphide. Α-glucosidase inhibitors that may be useful in treating diabetes when administered with the polypeptides of the present invention include acarbose, miglitol and boglibose. Insulin sensitizers that may be useful for treating diabetes include thiazolidinedione and non-thiazolidinedione. Hepatic glucose excretory compounds that may be useful in treating diabetes when administered with the polypeptides of the invention include metformin, such as Glucophage and Glucophage XR. Insulin secretagogues that may be useful in the treatment of diabetes when administered with the polypeptides of the present invention include sulfonylurea and non-sulfonylurea drugs GLP-1, GIP, secretin, nateglinide, meglitin Amides, repaglinides, glybenclamides, glymepyrides, chlorpropamides, glyphamides. GLP-1 includes derivatives of GLP-1 having a longer half-life than native GLP-1, such as fatty acid derivatized GLP-1 and exendin, for example. In one embodiment of the present invention, the polypeptide of the present invention is used in combination with an insulin secretagogue to increase the sensitivity of pancreatic β-cells to the insulin secretagogue.

Polypeptides of the invention can also be used in the methods of the invention in combination with anti-obesity drugs. Anti-obesity drugs include β-3 agonists, CB-1 antagonists, such as appetite suppressants such as silbutramine (Meridia), and lipase inhibitors such as, for example, orlistat (Xenical). Included.

The polypeptides of the invention may also be used in the methods of the invention in combination with drugs commonly used to treat lipid disorders in diabetic patients. Such drugs include, but are not limited to, HMG-CoA reductase inhibitors, nicotinic acid, bile acid sequestrants, and fibric acid derivatives. Polypeptides of the invention may also be used in combination with anti-hypertensive drugs such as, for example, β-blockers and ACE inhibitors.

Such combination-therapy may be administered in any combination with two or more drugs (eg, compounds of the invention in combination with insulin sensitizers and anti-obesity drugs). Such combination-therapy may be administered in the form of a pharmaceutical composition as described above.

As used herein, various terms are defined as follows.

When referring to elements of the invention or preferred embodiment (s) thereof, the articles "a", "an", "the" and "said" are used for the purpose of indicating that one or more elements are present. The terms "containing", "comprising" and "having" are meant to be inclusive and mean that there may be additional elements other than the listed elements.

As used herein, the term "subject" includes mammals (eg, humans and animals).

The term "treatment" includes any method, action, application, treatment, etc., provided with medical help for the purpose of directly or indirectly improving the condition of an individual, or slowing the progress of a condition or disease in an individual, including a human. do.

The term "combination therapy" or "combination therapy" means the administration of two or more therapeutic agents to treat a diabetic condition and / or disease. Such administration involves co-administering two or more therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed proportion of active ingredient or multiple separate capsules for each inhibitor. Such administration also includes the use of each type of therapeutic agent in a continuous manner.

The phrase “therapeutically effective” means the amount of each agent administered to achieve the goal of improvement in diabetes status or disease severity while avoiding or minimizing the deleterious side effects associated with the desired therapeutic treatment.

The term "pharmaceutically acceptable" means that the item of interest is suitable for use in a pharmaceutical product.

Based on the well-known test methods used to determine the efficacy for the treatment of the identified conditions in mammals, and comparing these results with the results of known medications used to treat these conditions, Effective doses of the polypeptides of the invention for treatment can be readily determined. The amount of active ingredient (eg, polypeptide) to be administered in the treatment of one of these conditions depends on the particular compound and dosage unit used, mode of administration, duration of treatment, age and sex of the treated patient, and Considerations such as the nature and extent of the state can vary widely.

The total amount of active ingredient to be administered may generally range from about 0.0001 mg / kg to about 200 mg / kg, preferably from about 0.01 mg / kg to about 200 mg / kg, per kg body weight per day. Unit dosages may contain from about 0.05 mg to about 1500 mg of active ingredient and may be administered once or more per day. Daily dosages for administration by injection, including intravenous, intramuscular, subcutaneous and extra-intestinal injections, and the use of infusion techniques can be from about 0.01 to about 200 mg / kg. Daily rectal dosing can be from 0.01 to 200 mg / kg of body weight. Percutaneous concentrations may be necessary to maintain a daily dose of 0.01 to 200 mg / kg.

Specific initial and continuous dosing regimens for each patient include the nature and severity of the condition as determined by the attending physician, the activity of the particular polypeptide used, the age of the patient, the meal of the patient, the timing of administration, the route of administration, the It may vary depending on the rate of excretion, drug combination, and the like. The mode of administration desired and the number of doses of the polypeptides of the invention can be ascertained by one of ordinary skill in the art using routine therapeutic trials.

The desired pharmacological effect can be obtained by administering to a patient in need thereof in a pharmaceutical composition suitably formulated using the polypeptide of the present invention. Patients for the purposes of the present invention are mammals including humans in need of treatment for a particular condition or disease. Accordingly, the present invention includes pharmaceutical compositions containing a pharmaceutically acceptable carrier and a therapeutically effective amount of a polypeptide. Pharmaceutically acceptable carriers may be any carrier that is relatively nontoxic and harmless to a patient at a concentration consistent with the effective activity of the active ingredient such that any side effects due to the carrier do not impair the beneficial effect of the active ingredient. A therapeutically effective amount of a polypeptide is an amount that produces a result or effects on a particular condition to be treated. The polypeptides described herein can be administered with pharmaceutically-acceptable carriers orally, parenterally, topically, etc., using conventional, effective dosage unit forms including, for example, immediate or sustained preparations. .

For oral administration, the polypeptide may be formulated in a solid or liquid formulation, such as, for example, capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions or emulsions, for the preparation of pharmaceutical compositions Can be prepared according to methods known in the art. The solid unit dosage form can be, for example, a capsule that can be a conventional hard- or soft-shell gelatin type containing surfactants, lubricants and inert fillers such as lactose, sucrose, calcium phosphate and corn starch.

In another embodiment, a polypeptide of the invention is a binder such as acacia, corn starch or gelatin; Disintegrants aimed at helping to break down and dissolve the tablets after administration such as potato starch, alginic acid, corn starch and guar gum; Lubricants, for example talc, stearic acid, or magnesium, calcium or zinc stearate, aimed at improving the flow of tablet granules and preventing the attachment of tablet material to the surface of tablet dies and punches ; dyes; coloring agent; Tablets can be made using conventional tableting bases such as lactose, sucrose and corn starch with fragrances aimed at enhancing the aesthetic quality of the tablets and making them more acceptable to patients. Excipients suitable for use in oral liquid dosage forms include water and alcohols with or without pharmaceutically acceptable surfactants, suspending agents or emulsifiers, for example diluents such as ethanol, benzyl alcohol and polyethylene alcohol. . Various other materials may be present to modify the physical form of the dosage form into a coating or otherwise. For example, tablets, pills, or capsules may be coated with shellac, sugar or both.

Dispersible powders and granules are suitable for the preparation of aqueous suspensions. They provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents have been exemplified by those already mentioned above. Additional excipients may also be present, for example sweeteners, fragrances and colorants as described above.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifiers are derived from (1) naturally occurring rubbers such as acacia rubber and tragacanth rubber, (2) naturally occurring phosphatides such as soybean and lecithin, and (3) fatty acids and hexitol anhydrides. Esters or partial esters such as sorbitan monooleate, and (4) condensation products of said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and fragrances.

Oily suspensions may contain the active ingredient in, for example, vegetable oils such as peanut oil, olive oil, horsetail oil or palm oil; Or by suspending in mineral oil such as liquid paraffin. The suspension may also contain one or more preservatives, for example ethyl or n-propylbenzoate; One or more colorants; One or more fragrances; And one or more sweetening agents such as sucrose or saccharin.

Syrups and elixirs can be formulated using sweetening agents such as, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain demulcents and preservatives, fragrances and colorants.

Polypeptides of the invention may also contain pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or other emulsifiers, and other Water, aqueous dextrose and related sugar solutions with or without pharmaceutical auxiliaries; Alcohols such as ethanol, isopropanol or hexadecyl alcohol; Glycols such as propylene glycol or polyethylene glycol; Glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly (ethylene glycol) 400; oil; fatty acid; Fatty acid esters or glycerides; Or parenterally, ie, subcutaneously, intravenously, intramuscularly, or intraperitoneally as an injectable dosage form of a compound in a physiologically acceptable diluent with a pharmaceutical carrier, which may be a sterile liquid or mixture of liquids, such as acetylated fatty acid glycerides. It can be administered into.

Examples of oils that can be used in the parenteral preparations of the invention are oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. Suitable fatty acids include oleic acid, stearic acid, and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty alkali metal, ammonium and triethanolamine salts, and suitable detergents include cationic detergents such as dimethyl dialkyl ammonium halides, alkyl pyridinium halides and alkylamine acetates; Anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefins, ethers and monoglyceride sulfates and sulfosuccinates; Nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylenepolypropylene copolymers; And amphoteric detergents such as alkyl-beta-aminopropionate, and 2-alkylimidazoline quaternary ammonium salts, and mixtures thereof.

Parenteral compositions of the present invention may generally contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers can also be used advantageously. In order to minimize or eliminate irritation at the site of injection, such compositions may contain nonionic surfactants having a hydrophilic-lipophilic equilibrium (HLB) of about 12 to about 17. The amount of surfactant in such formulations ranges from about 5% to about 15% by weight. The surfactant may be a single component having the above HLB, or may be a mixture of two or more components having the desired HLB.

Examples of surfactants used in parenteral preparations include a class of polyethylene sorbitan fatty acid esters, such as sorbitan monooleate, and high ethylene oxide and hydrophobic bases formed by condensation of propylene oxide with propylene glycol. Molecular weight adduct.

The pharmaceutical composition may be in the form of a sterile injectable aqueous suspension. Such suspensions include, for example, dispersing or wetting agents and suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth rubber and acacia rubber; Naturally occurring phosphatides, such as lecithin, condensation products of alkali oxides and fatty acids, for example polyoxyethylene stearate, condensation products of ethylene oxide and long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, Condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, for example polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydride, for example polyoxy It may be formulated according to known methods using a dispersing or wetting agent which may be ethylene sorbitan monooleate.

Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Diluents and solvents that can be used are, for example, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be used including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.

The compositions of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug (eg, polypeptide) with a solid, at room temperature but liquid at rectal temperature, and with a suitable non-irritating excipient that can liberate the drug in the rectum. Such materials are, for example, cocoa butter and polyethylene glycols.

Another formulation used in the method of the invention uses a percutaneous delivery device ("patch"). Such transdermal patches can be used to provide continuous or discontinuous infusion of the compounds of the invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical ingredients are well known in the art (see, eg, US Pat. No. 5,023,252, incorporated herein by reference). Such patches may be configured for continuous, pulsatile, or as needed delivery of the pharmaceutical component.

It may be desirable or necessary for the pharmaceutical composition to be introduced into the patient via a mechanical delivery device. The construction and use of mechanical delivery devices for the delivery of pharmaceutical formulations are well known in the art. For example, direct techniques for administering drugs directly to the brain typically involve placing a drug delivery catheter within the patient's ventricular system to bypass the blood-brain barrier. One such implantable delivery system, used for the delivery of a medicament to a particular anatomical part of the body, is described in US Pat. No. 5,011,472, incorporated herein by reference.

The compositions of the present invention may also contain other conventional pharmaceutically acceptable formulations, generally referred to as carriers or diluents, where necessary or desirable. Any composition of the present invention may be preserved by the addition of an antioxidant such as ascorbic acid or by other suitable preservatives. Conventional procedures for preparing such compositions in suitable dosage forms can be used.

Commonly used pharmaceutical ingredients that can be suitably used in formulating a composition for the desired route of administration include the following: Acidifying agents, such as, but not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid Does not); And alkalizing agents such as, but not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine.

Other pharmaceutical ingredients include, for example, adsorbents (eg, powdered cellulose and activated charcoal); Aerosol propellants (eg, carbon dioxide, CCl ₂ F ₂ , F ₂ ClC-CClF ₂ and CClF ₃ ); Air substituents (eg, nitrogen and argon); Antifungal preservatives (eg, benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate); Antimicrobial preservatives (eg benzalkonium chloride, benzetonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal); Antioxidants (e.g. ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphoric acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium Formaldehyde sulfoxylate, sodium metabisulfite); Binding materials (eg block polymers, natural and synthetic rubbers, polyacrylates, polyurethanes, silicones and styrene-butadiene copolymers); Buffers (eg potassium metaphosphate, monobasic potassium phosphate, sodium acetate, sodium citrate anhydride and sodium citrate dihydrate); Carrying agents (e.g., acacia syrup, aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orange syrup, syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection and bacteriostatic strains) Used water); Chelating agents (eg, edetate disodium and edetic acid); Pigments (eg, FD & C Red 3, FD & C Red 20, FD & C Yellow 6, FD & C Blue 2, D & C Green 5, D & C Orange 5, D & C Red 8, Caramel and Red Iron Oxide); Clarifying agents (eg bentonite); Emulsifiers (acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyethylene 50 stearate, but not limited to these); Encapsulating agents (eg, gelatin and cellulose acetate phthalate); Fragrances (eg, anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin); Humectants (eg glycerin, propylene glycol and sorbitol); Levigating agents (eg, mineral oil and glycerin); Oils (eg, peanut oil, mineral oil, olive oil, peanut oil, sesame oil and vegetable oil); Ointment bases (eg lanolin, hydrophilic ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white ointment, yellow ointment and rose water ointment); Penetration enhancers (transdermal delivery) (e.g. mono or polyhydric alcohols, saturated or unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalins, terpenes, amides, ethers) , Ketones and urea); Plasticizers (eg, diethyl phthalate and glycerin); Solvents (eg, alcohol, corn oil, cottonseed oil, glycerin, isopropyl alcohol, mineral oil, oleic acid, peanut oil, purified water, water for injection, sterile water for injection and sterile water for irrigation); Stiffening agents (eg, cetyl alcohol, cetyl ester waxes, microcrystalline waxes, paraffins, stearyl alcohols, white lead and lead); Suppository bases (eg, cocoa butter and polyethylene glycols (mixtures)); Surfactants (eg benzalkonium chloride, nonoxynol 10, octoxynol 9, polysorbate 80, sodium lauryl sulfate and sorbitan monopalmitate); Suspending agents (eg, agar, bentonite, carbomer, carboxymethylcellulose sodium, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, kaolin, methylcellulose, tragacanth and veegum); Sweetening agents (eg, aspartame, dextrose, glycerin, mannitol, propylene glycol, saccharin sodium, sorbitol and sucrose); Tablet anti-adherents (eg, magnesium stearate and talc); Tablet binders (eg, acacia, alginic acid, carboxymethylcellulose sodium, compressible sugars, ethylcellulose, gelatin, liquid glucose, methylcellulose, povidone and pregelatinized starch); Tablet and capsule diluents (eg, dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sodium carbonate, sodium phosphate, sorbitol and starch); Tablet coatings (eg, liquid glucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, cellulose acetate phthalate and shellac); Tablet direct compression excipients (eg, dibasic calcium phosphate); Tablet disintegrants (eg, alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose, polyacrylic potassium, sodium alginate, sodium starch glycolate and starch); Tablet glidants (eg, colloidal silica, corn starch and talc); Tablet lubricants (eg, calcium stearate, magnesium stearate, mineral oil, stearic acid and zinc stearate); Tablet / capsule opaquants (eg titanium dioxide); Tablet polishes (eg, carnuba wax and white lead); Thickening agents (eg beeswax, cetyl alcohol and paraffin); Tonicity agents (eg dextrose and sodium chloride); Viscosity increasing agents (eg, alginic acid, bentonite, carbomer, carboxymethylcellulose sodium, methylcellulose, povidone, sodium alginate and tragacanth); And wetting agents (eg, heptadecaethylene oxycetanol, lecithin, polyethylene sorbitol monooleate, polyoxyethylene sorbitol monooleate and polyoxyethylene stearate).

The polypeptides described herein may be administered as the only pharmaceutical component or in combination with one or more other pharmaceutical components, wherein the combination does not cause unacceptable adverse effects. For example, the polypeptides of the present invention may be combined with known anti-obesity agents, known antidiabetic agents or other indications, and the like and mixtures and combinations thereof.

The polypeptides described in the present invention may also be used in freebase form or composition, during research and diagnosis, or as analytical reference material or the like. Accordingly, the present invention encompasses compositions containing an inert carrier and an effective amount of a compound identified by the method described herein, or a salt or ester thereof. An inert carrier may be any material that does not interact with the compound to be conveyed and provides support for the compound to be conveyed, means of transport, bulk, traceable material, and the like. An effective amount of a compound is the amount of compound that yields or affects the procedure in the particular procedure being performed.

Polypeptides are known to cause hydrolysis, deamidation, oxidation, racemization and isomerization in aqueous and non-aqueous environments. Degradation such as hydrolysis, deamidation or oxidation can be readily detected by capillary electrophoresis. Despite enzymatic degradation, polypeptides with long plasma half-life or biological residence time should be stable in at least an aqueous solution. It is essential that the polypeptides exhibit less than 10% degradation over a one day period at body temperature. More preferably, the polypeptide exhibits less than 5% degradation over a period of one day at body temperature. Due to lifelong treatment in chronic diabetics, it is much more convenient to administer these therapeutic agents, also rarely by parenteral route. Stability over a period of weeks at body temperature (ie less than a few percent degradation) may allow for less frequent dosing. The stability over the years at the freezing temperature allows the manufacturer to prepare liquid formulations, thereby avoiding the inconvenience of re-formulation. In addition, stability in organic solvents can provide novel dosage forms such as implants.

Suitable for subcutaneous, intravenous, intramuscular, etc .; Suitable pharmaceutical carriers; And formulations suitable for the art of formulation and administration can be prepared by any method well known in the art (see, eg, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20 ^th edition, 2000).

The following examples are presented to illustrate the invention described herein, but are not to be considered as limiting the scope of the invention in any way.

Capsule formulation

Capsule formulations are prepared from:

10 mg of polypeptide of the present invention

Starch 109 mg

Magnesium Stearate 1mg

The ingredients are blended, passed through an appropriate mesh sieve and filled into hard gelatin capsules.

Tablet formulation

Tablets are made from:

25 mg of polypeptide of the present invention

Microcrystalline cellulose 200 mg

10 mg colloidal silicon dioxide

Stearic acid 5.0 mg

The ingredients are mixed and compressed to form tablets. Appropriate aqueous and non-aqueous coatings can be applied to increase flavor, improve appearance and stability, or delay absorption.

Sterile IV Solution

A 1 mg / ml solution of the preferred compounds of the invention is prepared using sterile water for injection and the pH is adjusted as necessary. This solution is diluted by sterile 5% dextrose for administration by IV infusion.

Intramuscular suspension

The following intramuscular suspensions are prepared:

50 μg / ml polypeptide of the invention

Sodium Carboxymethylcellulose 5 mg / ml

Tween 80 4mg / ml

Sodium chloride 9mg / ml

Benzyl alcohol 9mg / ml

This suspension is administered intramuscularly.

Hard outer capsule

Many unit capsules are prepared by filling each of the standard two-piece hard gelatin capsules with powdered active ingredient, 150 mg lactose, 50 mg cellulose and 6 mg magnesium stearate.

Soft gelatin capsule

Mixtures of the active ingredients in digestible oils such as soybean oil, cottonseed oil or olive oil are prepared and infused into molten gelatin using a positive displacement pump to form soft gelatin capsules containing the active ingredient. This capsule is washed and dried. The active ingredient can be dissolved in a mixture of polyethylene glycol, glycerin and sorbitol to prepare a water-miscible pharmaceutical mixture.

Immediate release tablets / capsules

These are solid oral dosage forms made by conventional and novel methods. These units are taken orally without water for immediate dissolution and delivery of the medication. The active ingredient is mixed in a liquid containing ingredients such as sugar, gelatin, pectin and sweeteners. These liquids are solidified into solid tablets or caplets by lyophilization and solid phase extraction techniques. The drug combination can be compressed with viscoelastic and thermoelastic sugars and polymers or effervescent components to produce a porous matrix for immediate release without the use of water.

It will be apparent to those skilled in the art that changes and modifications to the present invention can be made without departing from the spirit or scope of the invention as described herein.

In order to better understand the present invention, the following examples are described. These examples are for illustrative purposes only and are not to be construed as contributing to the scope of the invention in any way. All documents mentioned herein are incorporated by reference in their entirety.

Example 1 Peptide Synthesis Method

Polypeptides of the present invention are designed to improve the stability and prolong half-life of these peptides. Specifically, these peptides in non-mutated form (see, for example, WO 01/23420, incorporated intact in the present invention) are characterized by N-terminal hydrolysis, deamidation and dimerization and in aqueous and non-aqueous environments. Trimerization was shown. Asparagine residues (positions 9 and 28) were mutated to glutamine residues to improve stability and minimize hydrolysis, deamidation and dimerization / trimerization. In addition, valine at position 17, alanine at position 19, lysine at position 29, arginine at position 30, and tyrosine at position 31 were also mutated. In addition, as described below, the polypeptides of the present invention were also pegylated to prolong half-life.

Several polypeptides of the invention were synthesized by the following general procedure:

Peptide synthesis was carried out using the FMOC / t-butyl method (Pennington & Dunn, Peptide Synthesis Protocols, Volume 35) under continuous flow conditions using Rapp-Polymere PEG-polystyrene resin (Rapp-Polymere, Tubingen, Germany). , 1994). Once synthesis is complete, the peptide is cleaved from the resin and deprotected using TFA / DTT / H ₂ O / triisopropyl silane (88/5/5/2). Peptides were precipitated from cleavage cocktails using cold diethyl ether. The precipitate was washed three times with cold ether and then dissolved in 5% acetic acid before lyophilization. YMC-Pack ODS on Waters ALLIANCE ™ System (Waters Corporation, Milford, Mass.) Using water / acetonitrile with 3% TFA with acetonitrile gradient from 0% to 100% By reversed phase chromatography on an AQ column (YMC, Inc., Wilmington, NC), or on a VOYAGER DE ™ MALDI mass spectrometer (Model 5-2386-00, PerSeptive BioSystems, Framingham, Mass.) Examination by Maldi (MALDI) mass spectrometry. Peptide samples were added to matrix buffer (acetonitrile containing 50/50 dH ₂ O / 3% TFA) at a ratio of 1/1. These peptides that do not meet the purity criteria of> 95% are purified by reverse phase chromatography on a Waters Delta Prep 4000 HPLC system (Waters Corporation, Milford, Mass.).

Example 2. Peptide Pegylation

The half-life of a peptide in vivo can be increased by attaching a polyethylene glycol (PEG) moiety to the peptide, thereby reducing the clearance rate of the peptide by the kidney and reducing the protease degradation of the peptide. The use of VPAC2 receptor agonist peptides is severely limited due to their short half-life in vivo, but the attachment of PEG moieties to peptides (pegylation) is sufficient to allow one / day to one / week treatment Prolonged its half-life.

Pegylation can be performed by any method known to those skilled in the art. In this example, however, pegylation introduces a unique cysteine mutation into the peptide, followed by the cysteine to the peptide's sulfhydryl and methoxy-PEG-maleimide reagent (Nektar (Inhale / Shearwater), San Carlos, CA). This was done by pegylation through stable thioether bonds between the mid groups. It is desirable to introduce only cysteine at the C-terminus of the peptide to minimize the potential reduction in activity by pegylation.

Specifically, a 2-fold molar excess of mPEG-mal (MW 22 kD and 43 kD) reagent was added to 1 mg of peptide (eg SEQ ID NO: 1 with cysteine mutations at the C-terminus of the peptide) , pH 6.0 in reaction buffer (0.1 M Na phosphate / 0.1 M NaCl / 0.1 M EDTA). After 0.5 h at room temperature, the reaction was terminated by a 2-fold molar excess of DTT against mPEG-mal. The peptide-PEG-mal reaction mixture was applied to a cation exchange column to remove residual PEG reagent, followed by gel free column to remove residual free peptide. Purity, mass, and number of pegylation sites were measured by SDS-PAGE and Malti-TOF mass spectrometers. When 22 kD PEG was attached to the peptide of the present invention, strong VPAC2 receptor activation was maintained. In addition, VPAC2 versus VPAC1 and PAC1 selectivity of receptor activation was also maintained. Pegylation with smaller PEGs (eg linear 22 kD PEG) may reduce the activity of the peptide less, while larger PEGs (eg branched 43 kD PEG) will further reduce the activity. Can be. However, larger PEGs can further increase plasma half-life to allow weekly injections (Harris, et al., Clin. Pharmacokinet. 40: 539-551, 2001).

Example 3. Peptide Cloning

To recombinantly express these peptides, the DNA sequence encoding the peptide was C-terminal cloned against glutathione S-transferase (GST) using a single factor Xa recognition site that separates the monomeric peptide from GST. The gene encoding the factor Xa recognition site fused to the DNA sequence of the peptide to be produced is composed of two overlapping single-stranded DNA fragments containing Bam HI or Xho I restriction sites near 5 'to the DNA sequence of the gene to be cloned. -90mers) and then synthesized by DNA synthesis of the opposite helix through a large fragment of DNA polymerase (Life Technologies, Inc., Gaithersburg, MD). The DNA sequence selected for each gene was based on the reverse reading of the designed amino acid sequence of each peptide. In some cases, the gene encoding the peptide is a PCR mutagenesis of a gene already made by the method described above (Picard, et al., Nucleic Acids Res 22: 2587-91, 1994; Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, 1989). The double stranded product was then digested by Bam HI and Xho I and linked to pGEX-6P-1 (Amersham Pharmacia Biotech, Piscataway, NJ), which was also degraded by Bam HI and Xho I. For example, the designed DNA sequence (FIG. 2) was cloned into pGEX-6P-1 to express SEQ ID NO: 1 as a fusion with GST.

Example 4. Peptide Recombinant Expression and Purification

BL21 cells (Stratagene, La Jolla, Calif.) Transformed with GST-peptide fusion-containing plasmids were grown at 37 ° C. until the OD ₆₀₀ reached 0.6-1.0, and then the cells were grown at 37 ° C. for 2 hours. Incubated with 1 mM IPTG (Life Technologies, Carlsbad, Calif.). Cells (2 L) were centrifuged at 7,700 g for 15 minutes, weighed and stored at −20 ° C. for at least 3 hours. Frozen cell pellets are resuspended in 100 ml ice-cold PBS containing 250 μl of protease inhibitor cocktail (Sigma Chemical, St. Louis, Mo.) per cell gram, 3 times sonication with 15 seconds rest for 1 minute. Treated. Cells were then centrifuged at 10,000 g for 20 minutes. The supernatant was mixed with 2 ml of 50% glutathione Sepharose 4B resin (Pharmacia) on a shaker overnight at 4 ° C. The supernatant / resin is centrifuged at 1500 g for 15 minutes, filled into an empty Poly-Prep chromatography column (Bio-Rad, Hercules, Calif.), 30 ml of PBS followed by 10 ml of factor Wash with Xa buffer (1 mM CaCl ₂ , 100 mM NaCl, and 50 mM Tris-HCl, pH 8.0). Peptides were cleaved from the column by addition of 60 units of Factor Xa (Pharmacia) in 1 ml of Factor Xa buffer, incubated overnight at 4 ° C. and 2 ml / min according to the following program using a 2 ml loop. Was separated by C18 HPLC (Beckman System Gold) at a flow rate of: 10 min with Buffer A (0.1% TFA / H ₂ O), 30 min with gradient to Buffer B (0.1% TFA / ACN), 10 with Buffer A Minutes, gradient 10 minutes, and buffer A 10 minutes. Peak fractions (1 ml each) were collected and screened by 10-20% Tricine-SDS gel electrophoresis. Fractions containing the peptide of FIG. 1 were combined and dried. Typical yield is this. Hundreds of micrograms of free peptide per liter of E. coli culture. Recombinant peptides have been shown to have the same activity as their synthetic versions.

Example 5 Insulin Secretion from Distributed Rat Islet Cells

Insulin secretion of dispersed rat islet cells mediated by multiple peptides of the invention was measured as follows. Langelhans isolated from SD rats (200-250 g) was digested using collagenase. Scattered islet cells were treated with trypsin, seeded in 96 V-bottomed plates and pelleted. The cells were then cultured overnight in medium with or without the peptides of the invention. The medium was aspirated and cells were pre-incubated for 30 minutes at 37 ° C. with Krebs-Ringer-HEPES buffer containing 3 mM glucose. The pre-culture buffer was removed and cells were incubated at 37 ° C. for an appropriate time with Krebs-Ringer-HEPES buffer containing the appropriate glucose concentration (eg 8 mM) in the presence or absence of the peptide. Some studies also included appropriate concentrations of GLP-1. A portion of the supernatant was removed and its insulin content was measured by SPA. Results were expressed as "fold over control (FOC)". Polypeptides with amino acid sequence SEQ ID NO: 1 + cys + PEG (43 kD) at a concentration of 300 nM increased insulin secretion from dispersed islet cells approximately 1.7-fold (FIG. 3).

The increase in insulin secretion from dispersed rat islet cells in this test was defined as an increase of 1.4-fold or more. The VPAC2 receptor agonist element of the polypeptides of the invention increased insulin secretion from dispersed islet cells at least 1.4- to about 1.7-fold.

Example 6 Effect of Pezylated Peptides on Intraperitoneal Glucose Tolerance in Rats

In vivo activity of the phenylated peptides of the invention administered subcutaneously was tested in rats. Rats starved overnight were injected subcutaneously with control or phenylated peptide (1-100 μg / kg). After 3 hours, the baseline blood glucose was measured and rats received 2 g / kg glucose intraperitoneally. Blood glucose was measured after 15, 30 and 60 minutes. Representative phenylated peptides of the present invention significantly reduced blood glucose levels relative to vehicle according to IPGTT (intraperitoneal glucose tolerance test) and showed a 17% -28% decrease in glucose AUC (FIG. 4). This demonstrates that the phenylated peptide has prolonged glucose lowering activity in vivo. In addition to the glucose lowering activity of the phenylated peptides of the present invention, this also suggests extended peptide half-life in vivo. PACAP-27 has a very short in vivo half-life (<10 minutes). The ability of the fezylated peptides of the present invention to lower blood glucose three hours after peptide administration clearly indicates that the peptide is in circulation at this point and therefore has an extended half-life compared to PACAP-27.

Example 7 Cyclic AMP SPA

CHO cells expressing VPAC2 peptides were plated in 96-well plates at a concentration of 8 × 10 ⁴ cells / well and αMEM, nucleosides, glutamine (Gibco / BRL, Rockville, MD), 5% FBS, 100 μg / Grows in 37 Pen / Strep, 0.4 mg / ml Hygromycin, and 1.5 mg / ml Geneticin (Gibco / BRL) at 37 ° C. for 24 hours. The medium was removed and the plates washed with PBS. Cells with peptide (10 mM Hepes, 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 1.2 mM MgSO ₄ , 25 mM NaHCO ₃ (pH with 1% BSA and 100 μM IBMX) 7.4)) at 37 ° C. for 15 minutes. Cyclic AMP in the cell extracts was quantified using the cAMP SPA direct screening test system (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The amount of cAMP present in the lysate was determined according to the instructions provided with this kit. The amount of cAMP present in lysate (in pmol) at each peptide concentration was plotted and analyzed by nonlinear regression using Prism software to determine the EC ₅₀ for each peptide.

Polypeptides of the invention are designed based on VIPs that have been shown to lack activity in PAC1 (Vaudry, et al., Pharmacol. Rev. 52: 269-324, 2000). Thus, it is believed that the polypeptides of the present invention do not have a detectable degree of activity in PAC1.

The results of this test using representative polypeptides of the invention are shown in Table 1 below. Peptides identified as P5, P7, P8, P12 and P12 + PEG are all potent agonists of VPAC2 receptors that activate receptors up to 100% of the maximum level of receptor activation achieved by the endogenous peptide PACAP-27. In addition, peptides identified as P5, P7, P8, P12 and P12 + PEG are selective VPAC2 receptor agonists with very weak agonist activity against VPAC1. PACAP-27 is a potent agonist of VPAC1.

Peptide SEQ ID NO. VPAC2EC ₅₀ (nM) VPAC1EC ₅₀ (nM) PACAP-27 SEQ ID NO: 116 0.09 0.35 P5 SEQ ID NO: 1 0.33 232.5 P7 SEQ ID NO: 5 7.81 > 1000 P8 SEQ ID NO: 2 0.19 130.5 P12 SEQ ID NO: 1 + C-terminal Cys 0.38 > 1000 P12 + 22 kD PEG 1.32 > 1000 P12 + 43 kD PEG 4.19 > 1000

Example 8.Pharmaceutical Compositions-IV and SC Formulations

Sterile IV injectable preparations are well known in the art using 4 mg of SEQ ID NO: 1, or derivatized polypeptides having an equivalent amount of 4 mg polypeptide, and 1 L sterile saline. It was prepared using the method. Higher concentrations of polypeptide can be used for SC formulations. In the case of the polypeptide or derivatized polypeptide represented by SEQ ID NO: 1, 4 mg was dissolved in 100 ml saline or DMSO and sterile vial after aseptic filtration was filled with the composition.

Example 9. Stability of Formulations Containing Peptides of the Invention

The formulation described in Example 8 was placed in a constant stability chamber. Samples were periodically removed for analysis by capillary electrophoresis, the most sensitive method of detecting degradation of polypeptides in these formulations. The areas of the various peaks are summed and the area for the peaks of the parent polypeptide divided by the total peak area (FIGS. 5 and 6). The share is% purity. Since impurities are present in the fresh polypeptide, the purity change is normalized by dividing the purity at various times by the initial purity.

Example 10 Generation of Peptide Specific Antibodies and Measurement of Peptides by ELISA

Polyclonal antibodies specific for the polypeptide of the invention were generated by synthesizing specific fragments of the polypeptide of the invention using an ABI 433A peptide synthesizer. Peptides were then cleaved from the resin and purified on a Beckman System Gold Analytical anc Preparative HPLC system. The correct product was identified using a perspective MALDI mass spectrometer system. Peptides were dried using a lyophilizer. Peptides (2 mg) were then conjugated to keyhole limpet hemocyanin (KLH) via free sulfhydryl groups on Cys.

Female New Zealand white rabbits were immunized on days 0, 14, 35, 56 and 77. On day 0 each rabbit was injected subcutaneously with 250 μg peptide and complete Freund's adjuvant. Subsequent immunization used 125 μg of peptide per rabbit. Blood collection began at 21 days and then continued at 21-day intervals. Purification of the anti-peptide antibody was performed by passing the hemoserum through a specific peptide affinity purification column. Antibody titers were determined by ELISA.

96-well Imulon 4HBX plates were coated with C-terminal Morphosys F (ab) antibodies specific for the peptides of the invention and incubated overnight at 4 ° C. The plate was then blocked to prevent non-specific binding. Peptide standards (2500 ng / ml-160 pg / ml) were then diluted with 33% plasma, samples were diluted 1: 3 in buffer and then incubated for 1.5 hours at room temperature. After washing, polyclonal N-terminal antibodies specific for the peptides of the invention were incubated for 1 hour on plates. Horseradish peroxidase (HRP) -donkey-anti-rabbit antibody was then added and the samples and standards were incubated for an additional hour. Incubation is assessed by incubating with 3,3 ', 5,5'-tetramethylbenzidine (TMB) solution and plates are read at OD ₄₅₀ (FIG. 7).

Demonstration of the activity of the polypeptides of the invention can be carried out through in vitro, ex vivo and in vivo test methods well known in the art. For example diabetes and related diseases such as syndrome X, impaired glucose tolerance, impaired fasting glucose, and hyperinsulinemia; Related diseases such as atherosclerosis and hypertriglyceridemia and hypercholesterolemia; And the following test methods may be used to demonstrate the efficacy of the pharmaceutical component for the treatment of obesity.

How to measure blood sugar levels

db / db mice (obtained from Jackson Laboratories, Bar Harbor, ME) were bled (by eye or tail vein) and grouped according to equivalent mean blood glucose levels. They were orally administered the test polypeptide once daily for 14 days (by gavage in a pharmaceutically acceptable vehicle). At this point the animal was bleeding again by the eye or tail vein and blood glucose levels were measured. In each case glucose levels were measured with Glucometer Elite XL (Bayer Corporation, Elkhart, In.).

How to measure triglyceride levels

hApoA1 mice (obtained from Jackson Laboratories, Bar Harbor, ME) were bled (by eye or tail vein) and grouped according to equivalent mean serum triglyceride levels. They were orally administered the test polypeptide once daily for 8 days (by Gabbaz in a pharmaceutically acceptable vehicle). The animals were then bleeding again by eye or tail vein and serum triglyceride levels were measured. In each case triglyceride levels were measured with a Technicon Axon Autoanalyzer (Bayer Corporation, Elkhart, IN).

How to measure HDL-cholesterol level

To determine plasma HDL-cholesterol levels, hApoA1 mice were bleeded and grouped according to equivalent mean plasma HDL-cholesterol levels. Mice were orally administered vehicle or test polypeptide once daily for 7 days and bleeding again on day 8. Plasma was analyzed for HDL-cholesterol using the Synchron Clinical System (CX4) (Beckman Coulter, Fullerton, Calif.).

How to measure total cholesterol, HDL-cholesterol, triglycerides and glucose levels

In another in vivo test method, obese monkeys were bleeded, followed by oral administration of vehicle or test polypeptide once daily for four weeks, followed by bleeding again. Serum was analyzed for total cholesterol, HDL-cholesterol, triglycerides, and glucose using the Synchronous Clinical System (Beckman Coulter, Fullerton, Calif.). Lipoprotein subclass analysis was performed by NMR spectroscopy as described in Oliver, et al., Proc. Natl. Acad. Sci. USA 98: 5306-5311, 2001.

How to measure the effect on cardiovascular parameters

Cardiovascular parameters (eg heart rate and blood pressure) were also evaluated. SHR rats were administered orally once daily with vehicle or test polypeptides for two weeks. Blood pressure and heart rate were measured using the tail-cuff method as described in Grinsell, et al., Am. J. Hypertens. 13: 370-375, 2000. Blood pressure and heart rate in monkeys were monitored as described in Shen, et al., J. Pharmacol. Exp. Therap. 278: 1435-1443, 1996.

Evaluation of the Efficacy of Compounds on Reduced Feed Intake (Inhibition of Appetite) in Dried Rats Fasting Overnight

Fasting-Refeed Acute Feeding Test

The purpose of this protocol is to determine the effect of a single dose of an unknown compound on feed consumption of dry rats fasted overnight. Fasting-feeding rat models are frequently used in the field of obesity to identify compounds with potential for anorexiagenic effects. This animal model has been successfully used to identify and characterize the efficacy profile of a compound that has been or has been used in the management of body weight in obese humans (see Balvet, et al., Gen. Pharmacol. 13: 293-297, 21982). Grignaschi, et al., Br. J. Pharmacol. 127: 1190-1194, 1999; McTavish and Heel, Drug 43: 713-733, 1992; Rowland, et al., Life Sci. 36: 2295-2300, 1985; ).

Representative trials included 60-80 male rats (n = 10 / treatment group) with an average body weight of about 280 g. Rats were placed in a standard animal room under controlled temperature and humidity, and a 12/12 light cycle. Rats are placed alone in suspended cages with a mesh bottom. Unless the animals were fasted for testing, water and feed were made available continuously.

Vehicle test : Rats were classified based on their performance on vehicle test. Vehicle tests were performed between 2 and 7 days prior to efficacy testing. Rats were fasted overnight during the dark phase (about 16-18 hours total). One hour after dosing the pre-weighed feed jar was placed back into the animal cage. Rats were allowed 1 hour of feeding time. After 1 hour, the spillage is placed back into the feed jar and the amount of feed consumed is measured. Groups of rats were assigned such that the mean and standard error of the mean of 1-hour feed consumption were similar between groups.

Efficacy test : Rats were fasted overnight during cancer (approximately 16-18 hours). Animals were treated with the indicated doses of polypeptide. One hour after dosing, the pre-balanced feed jar was placed in the cage again. Feed intake was recorded 30, 60, 90, 180 and 240 minutes after feed was reloaded. The spilled at each time point was put back into the feed jar and weighed. The amount of feed consumed is measured at each time point. Differences between treatment groups were determined using appropriate statistical analysis.

Evaluation of the Efficacy of Compounds on Weight and Reduction of Feed and Water Consumption in Obese Zucker fa / fa Rats

Chronic Meal Test

The purpose of this protocol is to measure the effect of chronic administration of unknown compounds on body weight and feed and water consumption in obese Zucker fa / fa rats. Obesity Zucker fa / fa rats are frequently used to measure compound efficacy in weight loss. This animal model has been successfully used to identify and characterize the efficacy profile of compounds that have been or have been used in the management of body weight in obese humans (see, eg, Al-Barazanji, et al., Obes Res. 8: 317-323, 2000; Assimacopoulos-Jeannet, et al., Am. J. Physiol. 260 (2 Pt 2): R278-283, 1991; Dryden, et al., Horm. Metab.Res. 31: 363-366, 1999; Edwards and Stevens, Pharmacol. Biochem.Behav. 47: 865-872, 1994; Grinker, et al., Pharmacol. Biochem.Behav. 12: 265-275, 1980).

Representative trials included 60-80 male Zucker fa / fa (n = 10 / treatment group) with an average body weight of about 550 g. Rats were placed in a standard animal room under controlled temperature and humidity, and a 12/12 light cycle. Water and feed were made available continuously. Rats were placed alone in a large rat shoebox with a lattice bottom. Animals were adapted to the grid bottom and gastro-dosed with test vehicle for at least 4 days prior to recording 2-day baseline measurements of body weight and 24-hour feed and water consumption. Rats are assigned to one of 6-8 treatment groups based on their weight at baseline. The groups were set up so that the mean of body weight and the standard error of the mean were similar.

Animals orally gabbed their designated dose of polypeptide for a pre-determined number of days (typically 6-14 days) before the dark period of the LD / cycle. At this point body weight, feed and water consumption were measured. On the last day animals were euthanized by CO ₂ inhalation and weighed.

The efficacy of the polypeptides of the invention on weight loss or control could be measured by using this chronic feeding test.

All documents and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with certain preferred embodiments, it is understood that the claimed invention is not overly limited to this particular embodiment. Indeed, the foregoing modes of carrying out the invention which are obvious to those skilled in the field of molecular biology and related fields are to be construed as being within the scope of the following claims. Those skilled in the art will recognize or identify many equivalents to the specific embodiments of the invention described herein without using more than routine experimentation. Such equivalents are also construed as being included in the following claims.

SEQUENCE LISTING <110> Bayer Pharmaceuticals Corporation FROLAND, Wayne KELNER, Drew DUMAS, Michael PAN, Clark WHELAN, James WANG, John WANG, Wei <120> PITUITARY ADENYLATE CYCLASE ACTIVATING PEPTIDE (PACAP) RECEPTOR 3 (VPAC2) AGONISTS AND THEIR PHARMACOLOGICAL METHODS OF USE <130> MSB-7295 <150> US 60 / 395,738 <151> 2002-07-12 <160> 264 <170> PatentIn version 3.2 <210> 1 <211> 31 <212> PRT <213> Homo sapiens <400> 1 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 2 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) <223> Ac is acetyl <400> 2 Ac-His Thr Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 3 <211> 31 <212> PRT <213> Homo sapiens <400> 3 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 4 <211> 29 <212> PRT <213> Homo sapiens <400> 4 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys 20 25 <210> 5 <211> 31 <212> PRT <213> Homo sapiens <400> 5 His Thr Glu Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 6 <211> 31 <212> PRT <213> Homo sapiens <400> 6 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Gln Asp Ile Lys Gln Gly Gly Thr 20 25 30 <210> 7 <211> 30 <212> PRT <213> Homo sapiens <400> 7 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 8 <211> 31 <212> PRT <213> Homo sapiens <400> 8 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 9 <211> 31 <212> PRT <213> Homo sapiens <400> 9 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 10 <211> 31 <212> PRT <213> Homo sapiens <400> 10 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala His Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 11 <211> 31 <212> PRT <213> Homo sapiens <400> 11 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys His Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 12 <211> 30 <212> PRT <213> Homo sapiens <400> 12 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 13 <211> 30 <212> PRT <213> Homo sapiens <400> 13 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 14 <211> 30 <212> PRT <213> Homo sapiens <400> 14 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Arg Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 15 <211> 30 <212> PRT <213> Homo sapiens <400> 15 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 16 <211> 30 <212> PRT <213> Homo sapiens <400> 16 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Gln Lys Arg 20 25 30 <210> 17 <211> 30 <212> PRT <213> Homo sapiens <400> 17 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Gln Gln Lys Arg 20 25 30 <210> 18 <211> 30 <212> PRT <213> Homo sapiens <400> 18 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Arg Gln Lys Arg 20 25 30 <210> 19 <211> 30 <212> PRT <213> Homo sapiens <400> 19 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Arg 20 25 30 <210> 20 <211> 30 <212> PRT <213> Homo sapiens <400> 20 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ala 20 25 30 <210> 21 <211> 30 <212> PRT <213> Homo sapiens <400> 21 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Phe 20 25 30 <210> 22 <211> 30 <212> PRT <213> Homo sapiens <400> 22 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys His 20 25 30 <210> 23 <211> 30 <212> PRT <213> Homo sapiens <400> 23 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ile 20 25 30 <210> 24 <211> 30 <212> PRT <213> Homo sapiens <400> 24 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Lys 20 25 30 <210> 25 <211> 30 <212> PRT <213> Homo sapiens <400> 25 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Leu 20 25 30 <210> 26 <211> 30 <212> PRT <213> Homo sapiens <400> 26 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Met 20 25 30 <210> 27 <211> 30 <212> PRT <213> Homo sapiens <400> 27 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Pro 20 25 30 <210> 28 <211> 30 <212> PRT <213> Homo sapiens <400> 28 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Gln 20 25 30 <210> 29 <211> 30 <212> PRT <213> Homo sapiens <400> 29 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ser 20 25 30 <210> 30 <211> 30 <212> PRT <213> Homo sapiens <400> 30 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Thr 20 25 30 <210> 31 <211> 30 <212> PRT <213> Homo sapiens <400> 31 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Val 20 25 30 <210> 32 <211> 30 <212> PRT <213> Homo sapiens <400> 32 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Trp 20 25 30 <210> 33 <211> 30 <212> PRT <213> Homo sapiens <400> 33 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Tyr 20 25 30 <210> 34 <211> 30 <212> PRT <213> Homo sapiens <400> 34 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile 20 25 30 <210> 35 <211> 30 <212> PRT <213> Homo sapiens <400> 35 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile 20 25 30 <210> 36 <211> 30 <212> PRT <213> Homo sapiens <400> 36 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile 20 25 30 <210> 37 <211> 30 <212> PRT <213> Homo sapiens <400> 37 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Gln Arg Ile 20 25 30 <210> 38 <211> 30 <212> PRT <213> Homo sapiens <400> 38 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Arg Gln Arg Ile 20 25 30 <210> 39 <211> 31 <212> PRT <213> Homo sapiens <400> 39 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 40 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) <223> Ac is acetyl <400> 40 Ac-His Thr Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 41 <211> 31 <212> PRT <213> Homo sapiens <400> 41 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 42 <211> 29 <212> PRT <213> Homo sapiens <400> 42 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys 20 25 <210> 43 <211> 31 <212> PRT <213> Homo sapiens <400> 43 His Thr Glu Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 44 <211> 31 <212> PRT <213> Homo sapiens <400> 44 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Gln Asp Ile Lys Gln Gly Gly Thr 20 25 30 <210> 45 <211> 30 <212> PRT <213> Homo sapiens <400> 45 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 46 <211> 31 <212> PRT <213> Homo sapiens <400> 46 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 47 <211> 31 <212> PRT <213> Homo sapiens <400> 47 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 48 <211> 31 <212> PRT <213> Homo sapiens <400> 48 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala His Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 49 <211> 31 <212> PRT <213> Homo sapiens <400> 49 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys His Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr 20 25 30 <210> 50 <211> 30 <212> PRT <213> Homo sapiens <400> 50 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 51 <211> 30 <212> PRT <213> Homo sapiens <400> 51 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 52 <211> 30 <212> PRT <213> Homo sapiens <400> 52 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Arg Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 53 <211> 30 <212> PRT <213> Homo sapiens <400> 53 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg 20 25 30 <210> 54 <211> 30 <212> PRT <213> Homo sapiens <400> 54 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Gln Lys Arg 20 25 30 <210> 55 <211> 30 <212> PRT <213> Homo sapiens <400> 55 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Gln Gln Lys Arg 20 25 30 <210> 56 <211> 30 <212> PRT <213> Homo sapiens <400> 56 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Arg Gln Lys Arg 20 25 30 <210> 57 <211> 30 <212> PRT <213> Homo sapiens <400> 57 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Arg 20 25 30 <210> 58 <211> 30 <212> PRT <213> Homo sapiens <400> 58 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ala 20 25 30 <210> 59 <211> 30 <212> PRT <213> Homo sapiens <400> 59 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Phe 20 25 30 <210> 60 <211> 30 <212> PRT <213> Homo sapiens <400> 60 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys His 20 25 30 <210> 61 <211> 30 <212> PRT <213> Homo sapiens <400> 61 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ile 20 25 30 <210> 62 <211> 30 <212> PRT <213> Homo sapiens <400> 62 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Lys 20 25 30 <210> 63 <211> 30 <212> PRT <213> Homo sapiens <400> 63 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Leu 20 25 30 <210> 64 <211> 30 <212> PRT <213> Homo sapiens <400> 64 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Met 20 25 30 <210> 65 <211> 30 <212> PRT <213> Homo sapiens <400> 65 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Pro 20 25 30 <210> 66 <211> 30 <212> PRT <213> Homo sapiens <400> 66 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Gln 20 25 30 <210> 67 <211> 30 <212> PRT <213> Homo sapiens <400> 67 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ser 20 25 30 <210> 68 <211> 30 <212> PRT <213> Homo sapiens <400> 68 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Thr 20 25 30 <210> 69 <211> 30 <212> PRT <213> Homo sapiens <400> 69 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Val 20 25 30 <210> 70 <211> 30 <212> PRT <213> Homo sapiens <400> 70 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Trp 20 25 30 <210> 71 <211> 30 <212> PRT <213> Homo sapiens <400> 71 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Tyr 20 25 30 <210> 72 <211> 30 <212> PRT <213> Homo sapiens <400> 72 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile 20 25 30 <210> 73 <211> 30 <212> PRT <213> Homo sapiens <400> 73 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile 20 25 30 <210> 74 <211> 30 <212> PRT <213> Homo sapiens <400> 74 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile 20 25 30 <210> 75 <211> 30 <212> PRT <213> Homo sapiens <400> 75 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Gln Arg Ile 20 25 30 <210> 76 <211> 30 <212> PRT <213> Homo sapiens <400> 76 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Arg Gln Arg Ile 20 25 30 <210> 77 <211> 31 <212> PRT <213> Homo sapiens <400> 77 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 78 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) <223> Ac is acetyl <400> 78 Ac-His Thr Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 79 <211> 31 <212> PRT <213> Homo sapiens <400> 79 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 80 <211> 29 <212> PRT <213> Homo sapiens <400> 80 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys 20 25 <210> 81 <211> 31 <212> PRT <213> Homo sapiens <400> 81 His Thr Glu Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 82 <211> 31 <212> PRT <213> Homo sapiens <400> 82 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Gln Asp Ile Lys Asn Gly Gly Thr 20 25 30 <210> 83 <211> 30 <212> PRT <213> Homo sapiens <400> 83 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg 20 25 30 <210> 84 <211> 31 <212> PRT <213> Homo sapiens <400> 84 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 85 <211> 31 <212> PRT <213> Homo sapiens <400> 85 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 86 <211> 31 <212> PRT <213> Homo sapiens <400> 86 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala His Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 87 <211> 31 <212> PRT <213> Homo sapiens <400> 87 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys His Tyr Leu Gln Ser Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 88 <211> 30 <212> PRT <213> Homo sapiens <400> 88 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg 20 25 30 <210> 89 <211> 30 <212> PRT <213> Homo sapiens <400> 89 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg 20 25 30 <210> 90 <211> 30 <212> PRT <213> Homo sapiens <400> 90 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Arg Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg 20 25 30 <210> 91 <211> 30 <212> PRT <213> Homo sapiens <400> 91 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg 20 25 30 <210> 92 <211> 30 <212> PRT <213> Homo sapiens <400> 92 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Asn Lys Arg 20 25 30 <210> 93 <211> 30 <212> PRT <213> Homo sapiens <400> 93 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Gln Asn Lys Arg 20 25 30 <210> 94 <211> 30 <212> PRT <213> Homo sapiens <400> 94 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Arg Asn Lys Arg 20 25 30 <210> 95 <211> 30 <212> PRT <213> Homo sapiens <400> 95 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Arg Arg 20 25 30 <210> 96 <211> 30 <212> PRT <213> Homo sapiens <400> 96 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Ala 20 25 30 <210> 97 <211> 30 <212> PRT <213> Homo sapiens <400> 97 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Phe 20 25 30 <210> 98 <211> 30 <212> PRT <213> Homo sapiens <400> 98 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys His 20 25 30 <210> 99 <211> 30 <212> PRT <213> Homo sapiens <400> 99 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Ile 20 25 30 <210> 100 <211> 30 <212> PRT <213> Homo sapiens <400> 100 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Lys 20 25 30 <210> 101 <211> 30 <212> PRT <213> Homo sapiens <400> 101 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Leu 20 25 30 <210> 102 <211> 30 <212> PRT <213> Homo sapiens <400> 102 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Met 20 25 30 <210> 103 <211> 30 <212> PRT <213> Homo sapiens <400> 103 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Pro 20 25 30 <210> 104 <211> 30 <212> PRT <213> Homo sapiens <400> 104 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Gln 20 25 30 <210> 105 <211> 30 <212> PRT <213> Homo sapiens <400> 105 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Ser 20 25 30 <210> 106 <211> 30 <212> PRT <213> Homo sapiens <400> 106 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Thr 20 25 30 <210> 107 <211> 30 <212> PRT <213> Homo sapiens <400> 107 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Val 20 25 30 <210> 108 <211> 30 <212> PRT <213> Homo sapiens <400> 108 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Trp 20 25 30 <210> 109 <211> 30 <212> PRT <213> Homo sapiens <400> 109 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Tyr 20 25 30 <210> 110 <211> 30 <212> PRT <213> Homo sapiens <400> 110 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Asn Arg Ile 20 25 30 <210> 111 <211> 30 <212> PRT <213> Homo sapiens <400> 111 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Tyr Leu Gln Ser Ile Lys Asn Arg Ile 20 25 30 <210> 112 <211> 30 <212> PRT <213> Homo sapiens <400> 112 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Asn Arg Ile 20 25 30 <210> 113 <211> 30 <212> PRT <213> Homo sapiens <400> 113 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Asn Arg Ile 20 25 30 <210> 114 <211> 30 <212> PRT <213> Homo sapiens <400> 114 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Arg Asn Arg Ile 20 25 30 <210> 115 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 115 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 116 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) Ac is acetyl; PEG is polyethylene glycol <400> 116 Ac-His Thr Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 117 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 117 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 118 <211> 30 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (30) PEG is polyethylene glycol <400> 118 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Cys-PEG 20 25 30 <210> 119 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 119 His Thr Glu Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 120 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 120 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Gln Asp Ile Lys Gln Gly Gly Thr Cys-PEG 20 25 30 <210> 121 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 121 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Cys-PEG 20 25 30 <210> 122 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 122 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Leu Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 123 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 123 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Thr Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 124 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 124 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala His Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 125 <211> 32 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (32) PEG is polyethylene glycol <400> 125 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys His Tyr Leu Gln Ser Ile Lys Gln Lys Arg Tyr Cys-PEG 20 25 30 <210> 126 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 126 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Cys-PEG 20 25 30 <210> 127 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 127 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Cys-PEG 20 25 30 <210> 128 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 128 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Arg Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Cys-PEG 20 25 30 <210> 129 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 129 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Arg Cys-PEG 20 25 30 <210> 130 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 130 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Gln Lys Arg Cys-PEG 20 25 30 <210> 131 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 131 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Gln Gln Lys Arg Cys-PEG 20 25 30 <210> 132 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 132 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Arg Gln Lys Arg Cys-PEG 20 25 30 <210> 133 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <133> 133 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Arg Cys-PEG 20 25 30 <210> 134 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 134 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ala Cys-PEG 20 25 30 <210> 135 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 135 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Phe Cys-PEG 20 25 30 <210> 136 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 136 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys His Cys-PEG 20 25 30 <210> 137 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 137 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ile Cys-PEG 20 25 30 <210> 138 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 138 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Lys Cys-PEG 20 25 30 <139> <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 139 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Leu Cys-PEG 20 25 30 <210> 140 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 140 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Met Cys-PEG 20 25 30 <210> 141 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 141 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Pro Cys-PEG 20 25 30 <210> 142 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 142 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Gln Cys-PEG 20 25 30 <210> 143 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 143 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Ser Cys-PEG 20 25 30 <210> 144 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 144 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Thr Cys-PEG 20 25 30 <210> 145 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 145 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Val Cys-PEG 20 25 30 <210> 146 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 146 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Trp Cys-PEG 20 25 30 <210> 147 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 147 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Gln Lys Tyr Cys-PEG 20 25 30 <210> 148 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 148 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Gly Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile Cys-PEG 20 25 30 <210> 149 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 149 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Lys Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile Cys-PEG 20 25 30 <210> 150 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 150 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Lys Gln Arg Ile Cys-PEG 20 25 30 <210> 151 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 151 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ala Lys Lys Tyr Leu Gln Ser Ile Pro Gln Arg Ile Cys-PEG 20 25 30 <210> 152 <211> 31 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (31) PEG is polyethylene glycol <400> 152 His Ser Asp Ala Val Phe Thr Asp Gln Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Ser Lys Lys Tyr Leu Gln Ser Ile Arg Gln Arg Ile Cys-PEG 20 25 30 <210> 153 <211> 123 <212> DNA <213> Homo sapiens <400> 153 ggatccatcg aaggtcgtca ctccgacgct gttttcaccg accagtacac gcgtctgcgt 60 aaacaggttg ctgcaaagaa atacctgcag tccatcaagc agaagcgtta ctaatgactc 120 gag 123 <210> 154 <211> 93 <212> DNA <213> Homo sapiens <400> 154 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 155 <211> 93 <212> DNA <213> Homo sapiens <400> 155 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 156 <211> 87 <212> DNA <213> Homo sapiens <400> 156 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gcagaag 87 <210> 157 <211> 93 <212> DNA <213> Homo sapiens <400> 157 cacaccgaag ctgttttcac cgaccagtac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 158 <211> 93 <212> DNA <213> Homo sapiens <400> 158 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagct ggctgttaag 60 aaatacctgc aggacatcaa gcagggcggt acc 93 <210> 159 <211> 90 <212> DNA <213> Homo sapiens <400> 159 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 160 <211> 93 <212> DNA <213> Homo sapiens <400> 160 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagct ggctgcaaag 60 aaatacctgc agaccatcaa gcagaagcgt tac 93 <210> 161 <211> 93 <212> DNA <213> Homo sapiens <400> 161 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agaccatcaa gcagaagcgt tac 93 <210> 162 <211> 93 <212> DNA <213> Homo sapiens <400> 162 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcacac 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 163 <211> 93 <212> DNA <213> Homo sapiens <400> 163 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 cactacctgc agtccatcaa gcagaagcgt tac 93 <210> 164 <211> 90 <212> DNA <213> Homo sapiens <400> 164 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctggcaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 165 <211> 90 <212> DNA <213> Homo sapiens <400> 165 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctaaaaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 166 <211> 90 <212> DNA <213> Homo sapiens <400> 166 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctcgtaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 167 <211> 90 <212> DNA <213> Homo sapiens <400> 167 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 168 <211> 90 <212> DNA <213> Homo sapiens <400> 168 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccc ccagaagcgt 90 <210> 169 <211> 90 <212> DNA <213> Homo sapiens <400> 169 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcca gcagaagcgt 90 <210> 170 <211> 90 <212> DNA <213> Homo sapiens <400> 170 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccg tcagaagcgt 90 <210> 171 <211> 90 <212> DNA <213> Homo sapiens <400> 171 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagcgtcgt 90 <210> 172 <211> 90 <212> DNA <213> Homo sapiens <400> 172 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaaggca 90 <210> 173 <211> 90 <212> DNA <213> Homo sapiens <400> 173 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagttc 90 <210> 174 <211> 90 <212> DNA <213> Homo sapiens <400> 174 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcac 90 <175> 175 <211> 90 <212> DNA <213> Homo sapiens <400> 175 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagatc 90 <210> 176 <211> 90 <212> DNA <213> Homo sapiens <400> 176 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagaag 90 <210> 177 <211> 90 <212> DNA <213> Homo sapiens <400> 177 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagctg 90 <210> 178 <211> 90 <212> DNA <213> Homo sapiens <400> 178 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagatg 90 <210> 179 <211> 90 <212> DNA <213> Homo sapiens <400> 179 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagccc 90 <210> 180 <211> 90 <212> DNA <213> Homo sapiens <400> 180 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcag 90 <210> 181 <211> 90 <212> DNA <213> Homo sapiens <400> 181 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagtcc 90 <210> 182 <211> 90 <212> DNA <213> Homo sapiens <400> 182 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagacc 90 <210> 183 <211> 90 <212> DNA <213> Homo sapiens <400> 183 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaaggtt 90 <210> 184 <211> 90 <212> DNA <213> Homo sapiens <400> 184 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagtgg 90 <210> 185 <211> 90 <212> DNA <213> Homo sapiens <400> 185 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagtac 90 <210> 186 <211> 90 <212> DNA <213> Homo sapiens <400> 186 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctggtaag 60 aaatacctgc agtccatcaa gcagcgtatc 90 <210> 187 <211> 90 <212> DNA <213> Homo sapiens <400> 187 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctaaaaag 60 aaatacctgc agtccatcaa gcagcgtatc 90 <210> 188 <211> 90 <212> DNA <213> Homo sapiens <400> 188 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatcaa gcagcgtatc 90 <210> 189 <211> 90 <212> DNA <213> Homo sapiens <400> 189 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccc ccagcgtatc 90 <210> 190 <211> 90 <212> DNA <213> Homo sapiens <400> 190 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatccg tcagcgtatc 90 <210> 191 <211> 93 <212> DNA <213> Homo sapiens <400> 191 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 192 <211> 93 <212> DNA <213> Homo sapiens <400> 192 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 193 <211> 87 <212> DNA <213> Homo sapiens <400> 193 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gcagaag 87 <210> 194 <211> 93 <212> DNA <213> Homo sapiens <400> 194 cacaccgaag ctgttttcac cgacaactac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 195 <211> 93 <212> DNA <213> Homo sapiens <400> 195 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagct ggctgttaag 60 aaatacctgc aggacatcaa gcagggcggt acc 93 <210> 196 <211> 90 <212> DNA <213> Homo sapiens <400> 196 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 197 <211> 93 <212> DNA <213> Homo sapiens <400> 197 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagct ggctgcaaag 60 aaatacctgc agaccatcaa gcagaagcgt tac 93 <210> 198 <211> 93 <212> DNA <213> Homo sapiens <400> 198 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agaccatcaa gcagaagcgt tac 93 <210> 199 <211> 93 <212> DNA <213> Homo sapiens <400> 199 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcacac 60 aaatacctgc agtccatcaa gcagaagcgt tac 93 <210> 200 <211> 93 <212> DNA <213> Homo sapiens <400> 200 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 cactacctgc agtccatcaa gcagaagcgt tac 93 <210> 201 <211> 93 <212> DNA <213> Homo sapiens <400> 201 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 cactacctgc agtccatcaa gcagaagcgt tac 93 <210> 202 <211> 90 <212> DNA <213> Homo sapiens <400> 202 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctaaaaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 203 <211> 90 <212> DNA <213> Homo sapiens <400> 203 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctcgtaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 204 <211> 90 <212> DNA <213> Homo sapiens <400> 204 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatcaa gcagaagcgt 90 <210> 205 <211> 90 <212> DNA <213> Homo sapiens <400> 205 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccc ccagaagcgt 90 <206> 206 <211> 90 <212> DNA <213> Homo sapiens <400> 206 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcca gcagaagcgt 90 <210> 207 <211> 90 <212> DNA <213> Homo sapiens <400> 207 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccg tcagaagcgt 90 <210> 208 <211> 90 <212> DNA <213> Homo sapiens <400> 208 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagcgtcgt 90 <210> 209 <211> 90 <212> DNA <213> Homo sapiens <400> 209 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaaggca 90 <210> 210 <211> 90 <212> DNA <213> Homo sapiens <400> 210 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagttc 90 <210> 211 <211> 90 <212> DNA <213> Homo sapiens <400> 211 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcac 90 <210> 212 <211> 90 <212> DNA <213> Homo sapiens <400> 212 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagatc 90 <210> 213 <211> 90 <212> DNA <213> Homo sapiens <400> 213 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagaag 90 <210> 214 <211> 90 <212> DNA <213> Homo sapiens <400> 214 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagctg 90 <210> 215 <211> 90 <212> DNA <213> Homo sapiens <400> 215 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagatg 90 <210> 216 <211> 90 <212> DNA <213> Homo sapiens <400> 216 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagccc 90 <210> 217 <211> 90 <212> DNA <213> Homo sapiens <400> 217 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagcag 90 <210> 218 <211> 90 <212> DNA <213> Homo sapiens <400> 218 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagtcc 90 <210> 219 <211> 90 <212> DNA <213> Homo sapiens <400> 219 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagacc 90 <210> 220 <211> 90 <212> DNA <213> Homo sapiens <400> 220 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaaggtt 90 <210> 221 <211> 90 <212> DNA <213> Homo sapiens <400> 221 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagtgg 90 <210> 222 <211> 90 <212> DNA <213> Homo sapiens <400> 222 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gcagaagtac 90 <210> 223 <211> 90 <212> DNA <213> Homo sapiens <400> 223 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctggtaag 60 aaatacctgc agtccatcaa gcagcgtatc 90 <210> 224 <211> 90 <212> DNA <213> Homo sapiens <400> 224 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctaaaaag 60 aaatacctgc agtccatcaa gcagcgtatc 90 <210> 225 <211> 90 <212> DNA <213> Homo sapiens <400> 225 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatcaa gcagcgtatc 90 <210> 226 <211> 90 <212> DNA <213> Homo sapiens <400> 226 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccc ccagcgtatc 90 <210> 227 <211> 90 <212> DNA <213> Homo sapiens <400> 227 cactccgacg ctgttttcac cgacaactac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatccg tcagcgtatc 90 <210> 228 <211> 93 <212> DNA <213> Homo sapiens <400> 228 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gaacaagcgt tac 93 <210> 229 <211> 93 <212> DNA <213> Homo sapiens <400> 229 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagcgt tac 93 <210> 230 <211> 87 <212> DNA <213> Homo sapiens <400> 230 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gaacaag 87 <210> 231 <211> 93 <212> DNA <213> Homo sapiens <400> 231 cacaccgaag ctgttttcac cgaccagtac acgcgtctgc gtaaacaggt tgctgcaaag 60 aaatacctgc agtccatcaa gaacaagcgt tac 93 <210> 232 <211> 93 <212> DNA <213> Homo sapiens <400> 232 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagct ggctgttaag 60 aaatacctgc aggacatcaa gaacggcggt acc 93 <210> 233 <211> 90 <212> DNA <213> Homo sapiens <400> 233 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagcgt 90 <210> 234 <211> 93 <212> DNA <213> Homo sapiens <400> 234 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagct ggctgcaaag 60 aaatacctgc agaccatcaa gaacaagcgt tac 93 <210> 235 <211> 93 <212> DNA <213> Homo sapiens <400> 235 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agaccatcaa gaacaagcgt tac 93 <210> 236 <211> 93 <212> DNA <213> Homo sapiens <400> 236 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcacac 60 aaatacctgc agtccatcaa gaacaagcgt tac 93 <210> 237 <211> 93 <212> DNA <213> Homo sapiens <400> 237 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 cactacctgc agtccatcaa gaacaagcgt tac 93 <210> 238 <211> 90 <212> DNA <213> Homo sapiens <400> 238 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctggcaag 60 aaatacctgc agtccatcaa gaacaagcgt 90 <210> 239 <211> 90 <212> DNA <213> Homo sapiens <400> 239 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctaaaaag 60 aaatacctgc agtccatcaa gaacaagcgt 90 <210> 240 <211> 90 <212> DNA <213> Homo sapiens <400> 240 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctcgtaag 60 aaatacctgc agtccatcaa gaacaagcgt 90 <210> 241 <211> 90 <212> DNA <213> Homo sapiens <400> 241 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatcaa gaacaagcgt 90 <210> 242 <211> 90 <212> DNA <213> Homo sapiens <400> 242 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccc caacaagcgt 90 <210> 243 <211> 90 <212> DNA <213> Homo sapiens <400> 243 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcca gaacaagcgt 90 <210> 244 <211> 90 <212> DNA <213> Homo sapiens <400> 244 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccg taacaagcgt 90 <210> 245 <211> 90 <212> DNA <213> Homo sapiens <400> 245 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaaccgtcgt 90 <210> 246 <211> 90 <212> DNA <213> Homo sapiens <400> 246 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaaggca 90 <210> 247 <211> 90 <212> DNA <213> Homo sapiens <400> 247 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagttc 90 <210> 248 <211> 90 <212> DNA <213> Homo sapiens <400> 248 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagcac 90 <210> 249 <211> 90 <212> DNA <213> Homo sapiens <400> 249 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagatc 90 <210> 250 <211> 90 <212> DNA <213> Homo sapiens <400> 250 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagaag 90 <210> 251 <211> 90 <212> DNA <213> Homo sapiens <400> 251 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagctg 90 <210> 252 <211> 90 <212> DNA <213> Homo sapiens <400> 252 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagatg 90 <210> 253 <211> 90 <212> DNA <213> Homo sapiens <400> 253 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagccc 90 <210> 254 <211> 90 <212> DNA <213> Homo sapiens <400> 254 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagcag 90 <210> 255 <211> 90 <212> DNA <213> Homo sapiens <400> 255 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagtcc 90 <210> 256 <211> 90 <212> DNA <213> Homo sapiens <400> 256 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagacc 90 <210> 257 <211> 90 <212> DNA <213> Homo sapiens <400> 257 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaaggtt 90 <210> 258 <211> 90 <212> DNA <213> Homo sapiens <400> 258 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagtgg 90 <210> 259 <211> 90 <212> DNA <213> Homo sapiens <400> 259 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatcaa gaacaagtac 90 <210> 260 <211> 90 <212> DNA <213> Homo sapiens <400> 260 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctggtaag 60 aaatacctgc agtccatcaa gaaccgtatc 90 <210> 261 <211> 90 <212> DNA <213> Homo sapiens <400> 261 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctaaaaag 60 aaatacctgc agtccatcaa gaaccgtatc 90 <210> 262 <211> 90 <212> DNA <213> Homo sapiens <400> 262 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatcaa gaaccgtatc 90 <210> 263 <211> 90 <212> DNA <213> Homo sapiens <400> 263 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggctgcaaag 60 aaatacctgc agtccatccc caaccgtatc 90 <210> 264 <211> 90 <212> DNA <213> Homo sapiens <400> 264 cactccgacg ctgttttcac cgaccagtac acgcgtctgc gtaaacagat ggcttccaag 60 aaatacctgc agtccatccg taaccgtatc 90

Claims (53)

A polypeptide selected from the group consisting of SEQ ID NOs: 1 to 152 and functionally equivalent fragments, derivatives and variants thereof. A polynucleotide encoding the polypeptide sequence of claim 1, or a variant thereof. A vector containing the polynucleotide of claim 2. A host cell containing the vector of claim 3. a) culturing the host cell of claim 4 under conditions suitable for expression of the polypeptide; b) recovering the polypeptide from the host cell culture. Purified antibody that specifically binds to the polypeptide of claim 1. 2. The polypeptide of claim 1 selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4 and 5. The polypeptide of claim 1 selected from the group consisting of SEQ ID NOs: 115, 116, 117, 118 and 119. A polynucleotide selected from the group consisting of SEQ ID NOs: 154 to 264. A pharmaceutical composition containing a therapeutically effective amount of the polypeptide of claim 1, or functionally equivalent fragments, derivatives and variants thereof, with a pharmaceutically acceptable carrier. A pharmaceutical composition according to claim 10 containing from about 2% to about 30% DMSO and optionally a solvent selected from the group consisting of propylene glycol, dimethyl formamide, propylene carbonate, polyethylene glycol and triglycerides. The pharmaceutical composition of claim 10, wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 115, 116, 117, 118 and 119. 12. A pharmaceutical composition containing a therapeutically effective amount of the polypeptide of claim 1, or functionally equivalent fragments, derivatives and variants thereof, with a pharmaceutically acceptable carrier and one or more pharmaceutical ingredients. 14. The compound according to claim 13, wherein the pharmaceutical component is a PPAR agonist, sulfonylurea drug, non-sulfonylurea secretagogue, α-glucosidase inhibitor, insulin sensitizer, insulin secretagogue, hepatic glucose excretory compound , A pharmaceutical composition selected from the group consisting of insulin, anti-obesity agents, HMG CoA reductase inhibitors, nicotinic acid, bile acid sequestrants, fibric acid derivatives, and anti-hypertensive agents. A composition comprising an effective amount of the polypeptide of claim 1, or functionally equivalent fragments, derivatives and variants thereof, with an inert carrier. A method of treating diabetes comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. 17. The method of claim 16, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, growth stage expression diabetes of adolescents, latent autoimmune adult diabetes and gestational diabetes. A method of treating syndrome X, comprising administering a therapeutically effective amount of the polypeptide of claim 1 to an individual in need thereof. A method of treating a diabetes-related disorder, comprising administering a therapeutically effective amount of the polypeptide of claim 1 to an individual in need thereof. The method of claim 19, wherein the diabetes-related disease is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia and insulin resistance. A method of treating diabetes comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1 together with one or more pharmaceutical ingredients. The compound according to claim 21, wherein the pharmaceutical component is a PPAR agonist, sulfonylurea drug, non-sulfonylurea secretagogue, α-glucosidase inhibitor, insulin sensitizer, insulin secretagogue, hepatic glucose excretion reducing compound , Insulin and anti-obesity agent. 23. The method of claim 22, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, growth stage expression diabetes of adolescents, latent autoimmune adult diabetes and gestational diabetes. A method of treating syndrome X, comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1 together with one or more pharmaceutical ingredients. The compound according to claim 24, wherein the pharmaceutical component is a PPAR agonist, sulfonylurea drug, non-sulfonylurea secretagogue, α-glucosidase inhibitor, insulin sensitizer, insulin secretagogue, hepatic glucose excretion inhibitory compound , Insulin and anti-obesity agent. A method of treating a diabetes-related disorder, comprising administering a therapeutically effective amount of the polypeptide of claim 1 together with one or more pharmaceutical ingredients to an individual in need thereof. 27. The method of claim 26, wherein the diabetes-related disorder is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia and insulin resistance. 28. The compound according to claim 27, wherein the pharmaceutical component is a PPAR agonist, a sulfonylurea drug, a non-sulfonylurea secretagogue, an α-glucosidase inhibitor, an insulin sensitizer, an insulin secretagogue, a liver glucose lowering compound , Insulin and anti-obesity agent. A therapeutically effective amount of the polypeptide of claim 1 is administered to an individual in need thereof with one or more agents selected from the group consisting of HMG CoA reductase inhibitors, nicotinic acid, bile acid sequestrants, fibric acid derivatives, and anti-hypertensive agents. How to treat diabetes, syndrome X or diabetes-related diseases. The method of claim 29, wherein the diabetes-related disease is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia and insulin resistance. 31. The method of any one of claims 21-30, wherein the polypeptide of claim 1 and one or more pharmaceutical ingredients are administered in a single pharmaceutical dosage form. A method of treating or preventing a secondary cause of diabetes comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. 33. The method of claim 32, wherein the secondary cause is selected from the group consisting of glucocorticoid excess, growth hormone excess, chromaffin cell tumor, and drug-induced diabetes. A method of treating or preventing a secondary cause of diabetes comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1 together with one or more pharmaceutical ingredients. 35. The compound according to claim 34, wherein the pharmaceutical ingredient is a PPAR agonist, a sulfonylurea drug, a non-sulfonylurea secretagogue, an α-glucosidase inhibitor, an insulin sensitizer, an insulin secretagogue, a hepatic glucose excretory compound , Insulin and anti-obesity agent. A method of treating a respiratory disease comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating obesity, comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of modulating appetite comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating cardiovascular disease, comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating a disorder of lipid and carbohydrate metabolism comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating a sleep disorder comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating male reproductive disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating a growth disorder or disorder of energy homeostasis comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating an immune disorder comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating acute and chronic inflammatory diseases comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of treating septic shock, comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1. A method of promoting insulin release in a glucose-dependent manner in a subject by administering to the subject in need thereof. A gene therapy composition comprising the polynucleotide of claim 2 together with a therapeutically effective gene therapy vector. The polypeptide of claim 1 for the treatment and / or prevention of diabetes and diabetes-related diseases. A pharmaceutical comprising one or more polypeptides according to claim 1 together with one or more pharmaceutically acceptable and pharmaceutically safe carriers or excipients. Use of the polypeptide of claim 1 for the manufacture of a medicament for the treatment and / or prevention of diabetes and diabetes-related diseases. 52. A medicament according to claim 51 for the treatment and / or prevention of diabetes.