JP2006501820A - Modified GLP-1 receptor agonists and their pharmacological uses - Google Patents

Abstract

The present invention relates to modified GLP-1 receptor agonists comprising GLP-1 receptor agonists linked to a polyethylene glycol polymer having a molecular weight greater than 30 kD, and related formulations and formulations and their administration for therapeutic purposes Law is provided. More specifically, these modified GLP-1 receptor agonists, compositions and methods induce metabolic disorders such as diabetes and glucose tolerance by inducing glucose-dependent insulin secretion without reducing gastrointestinal motility. Useful for providing therapeutic options to individuals suffering from pre-diabetic conditions such as impaired performance and fasting glycemic disorders.

Description

This application claims the benefit of Patent Document 1 filed on September 6, 2002 and Patent Document 2 filed on January 9, 2003, all of which are incorporated herein by reference. To do.
FIELD OF THE INVENTION The present invention relates to modified GLP-1 receptor agonists comprising GLP-1 receptor agonists linked to polyethylene glycol polymers having a molecular weight greater than 30 kD, and related formulations for therapeutic purposes, It relates to formulation and administration methods. More specifically, these modified GLP-1 receptor agonists, compositions and methods can be used to induce diabetes, glucose tolerance by inducing glucose-dependent insulin secretion without the side effects of reducing or inhibiting gastrointestinal motility limiting treatment. Useful for providing treatment options to individuals suffering from metabolic disorders such as disorders, or metabolic syndrome, pre-diabetic conditions.

Background of Related Art Diabetes is characterized by inadequate insulin secretion manifested by elevated glucose levels, particularly in diabetic patients. Diabetes is divided into two main groups by distinct deficits: type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM) (this occurs when there is no insulin produced by β-cells in the patient's pancreas), and 2 Type diabetes, or non-insulin-dependent diabetes mellitus (NIDDM), which occurs in patients with insufficient beta-cell insulin secretion and changes in insulin action.

  Patients with type 1 diabetes are currently being treated with insulin, while patients with type 2 diabetes are treated with agents that stimulate β-cell function or with agents that enhance the patient's tissue sensitivity towards insulin. Can do. Over time, nearly half of type 2 diabetics lose their response to these drugs and must then be treated with insulin. The drugs currently used to treat type 2 diabetes are described below.

  Alpha-glucosidase inhibitors (eg, PRECOSE ™, VOGLIBOSE ™ and MIGLITOL ™) reduce postprandial glucose excretion by delaying glucose absorption from the gut. These drugs are safe and provide treatment for mild to moderately afflicted diabetic patients. However, the literature reports side effects in the gastrointestinal tract and limits their efficacy.

  Insulin sensitizers are drugs that enhance the body's response to insulin. Thiazolidinedione, such as Avandia ™ (rosiglitazone) and Actos ™, activates peroxisome proliferator-activated receptor (PPAR) and activates peroxisome proliferator-activated receptor (PPAR). Modulates the activity of a group of genes that have not been described in Rezulin ™ (troglitazone) (the first drug in this class) was withdrawn due to elevated liver enzyme levels and drug-induced liver toxicity. These liver effects do not appear to be a significant problem for patients using Avandia ™ and Actos ™. Even so, liver enzyme testing is recommended every 2 months in the first year of treatment and periodically thereafter. Avandia ™ and Actos ™ treatment is associated with fluid retention, edema and weight gain. Avandia ™ has not been shown to be used with insulin due to concerns of congestive heart failure.

  Insulin secretagogues such as sulfonylureas (SFU) and non-sulfonylureas (eg, Nateglinide and Pepaglinide) act through ATP-dependent K + channels and cause glucose-dependent insulin secretion. These drugs are standard treatments for type 2 diabetes with mild to moderate fasting blood glucose. SFU has limitations including the potential to induce hypoglycemia, weight gain and high primary and secondary failure rates. 10-20% of initially treated patients failed to show a significant therapeutic effect (primary failure). Secondary failure is indicated by an additional 20-30% loss of therapeutic effect after 6 months with SFU. Insulin treatment is required in 50% of SFU responders after 5-7 years of treatment (1). Dateglinide and Pepaglinide are short-acting drugs that require ingestion three times a day. They are used only for postprandial glucose control and not for fasting glucose control.

  GLUCOPHAGE ™ (metformin HCl) is a biguanide that lowers blood glucose by decreasing hepatic glucose output and increasing peripheral glucose uptake and utilization. This drug is effective in lowering blood glucose in mildly and moderately affected individuals and has no potential to induce weight gain side effects or hypoglycemia. However, GLUCOPHAGE ™ has a number of side effects including gastrointestinal disorders and lactic acidosis. GLUCOPHAGE ™ is contraindicated in diabetic patients over 70 years of age and in individuals with insufficient kidney or liver function. Finally, GLUCOPHAGE ™ has the same primary and two-letter failure rates as SFU.

  Insulin treatment is begun after diet, exercise and oral medication have not been able to adequately control blood glucose. This treatment, which is injectable, can produce hypoglycemia and causes weight gain, has drawbacks. The possibility of inducing hypoglycemia with insulin limits the extent to which hyperglycemia can be controlled.

  Due to the problems associated with current treatments, new therapies are needed to treat type 2 diabetes. In particular, new treatments are needed to maintain normal (glucose-dependent) insulin secretion. Such new drugs should have the following characteristics: glucose dependence to promote insulin secretion (ie, insulin secretion occurs only when elevated blood glucose is present); low primary and Secondary failure rate; and preservation of islet cell function. The method of developing a novel therapy disclosed herein is based on the cyclic adenosine monophosphate (cAMP) signaling mechanism and its effect on insulin secretion.

  Glucose is a major regulator of the insulin secretion process. This increase in sugar promotes the closure of K + channels after an increase in ATP. Closure of the K ++ channel results in cell depolarization followed by Ca ++ channel opening, which in turn leads to exocytosis of insulin particles. The slight, if any, effect on insulin secretion occurs in the absence of low glucose concentrations (Non-Patent Document 2). The secretagogue-like GLP-1 utilizes the cAMP system to regulate insulin secretion via this glucose-dependent mechanism (Non-patent Document 3; Non-patent Document 4; Non-patent Document 5). Insulin secretagogues via cAMP elevation can also enhance insulin synthesis in addition to insulin release (Non-Patent Document 6; Non-Patent Document 7).

  GLP-1 (glucagon-like peptide 1) is released from intestinal L-cells after a meal and functions as an incretin hormone (ie, it enhances glucose-induced insulin release from pancreatic β-cells). This is a 37 amino acid peptide that is differentially expressed by the glucagon gene depending on the tissue type. Clinical data was collected with GLP-1 supporting a beneficial effect of increasing cAMP levels in β-cells. Infusion of GLP-1 into uncontrolled type 2 diabetes normalizes their fasting blood glucose levels (8), and longer infusions are associated with β-cell function against these normal individuals. (Non-Patent Document 9). Recent reports have shown that GLP-1 improves the ability of β-cells to respond to glucose in individuals with impaired glucose tolerance (10). However, all these effects are short-lived due to the short half-life of the peptide.

  In addition to the short half-life of GLP-1 peptide, GLP-1 reduces intestinal motility (see, for example, Non-Patent Document 11 and Non-Patent Document 12), which are then significant such as nausea and nausea. Causes gastrointestinal side effects. Such gastrointestinal side effects were also shown with GLP-1 agonists such as NN-2211 (Non-patent document 13) and Exendin-4 (Non-patent document 14). Intestinal motility causing gastrointestinal side effects has also been studied in rodent models (Non-Patent Document 15). The inhibitory effect of GLP-1 on gastrointestinal motility and secretion has been shown to be mediated, at least in part, by the central nervous system (Non-Patent Document 16). Systemically injected GLP-1 acquires access from the periphery to the brain by simple diffusion (Non-patent Document 17).

Thus, there is a need for improved peptides that have the glucose-dependent insulinotropic activity of GLP-1 but without the side effects that limit GLP-1 based treatment.
US Provisional Patent Application No. 60 / 408,696 US Provisional Patent Application No. 60 / 439,369 Schen, et al. Diabetes Res. Clin. Pract. 6: 533-543, 1989 Weinhaus, et al. Diabetes 47: 146-1435, 1998. Komatsu, et al. Diabetes 46: 1928-1938, 1997. Filissson et al. , Diabetes 50: 1959-1969, 2001. Drucker, Endocrinology 142: 521-527, 2001 Skogland, et al. , Diabetes 49: 1156-1164,2000 Borbon, et al. , Endocrinology 140: 5530-5537, 1999. Gutriak, et al. , New Eng. J. et al. Med. 326: 1316-1322, 1992 Rachman, et al. Diabetes 45: 1524-1530, 1996. Byrne, et al. Diabetes 47: 1259-1265, 1998. Kieffer and Habener, Endocrine Reviews 20: 876-913, 1999 Drucker, Gastroenterology 122: 531-544, 2002 Agerso, et al. Diabetologia 45: 195-202, 2002. Amylin abstract, American Diabetes Association meeting, 2001 Lotti, et al. , Life Sci. 39: 1631-1638, 1986 Imeryuz, et al. , Am. J. et al. Physiol 273: G920-G927, 1997 Kastin, et al. , J .; Mol. Neurosci. 18: 7-14, 2002

SUMMARY OF THE INVENTION The present invention is a modified GLP-1 receptor comprising a GLP-1 receptor agonist linked to a polyethylene glycol polymer having a molecular weight greater than 30 kD and retaining the ability to agonize the GLP-1 receptor. It relates to agonists. These modified GLP-1 agonists are effective in treating metabolic disorders such as diabetes or impaired glucose tolerance, and prediabetes. Furthermore, the modified GLP-1 agonists of the present invention can treat metabolic disorders without inhibiting gastrointestinal motility, thereby reducing gastrointestinal side effects such as nausea and nausea and greater efficacy. Be able to administer appropriate doses.

  In particular, one aspect of the present invention is a polypeptide that functions as a GLP-1 receptor agonist. Examples of GLP-1 receptor agonists include, but are not limited to, the polypeptides shown in FIG. 1 and those polypeptides selected from the group consisting of SEQ ID NOs: 1-10, and SEQ ID NOs: 1-10 Polypeptide fragments and variants that function as agonists of the GLP-1 receptor at substantially the same level as the listed polypeptides (collectively polypeptides of the invention).

  Another aspect of the invention is a polynucleotide encoding a GLP-1 receptor agonist polypeptide, and associated vectors and host cells necessary for recombinant expression of the polypeptide of the invention.

  Another aspect of the present invention is a book that functions as a GLP-1 receptor agonist at substantially the same level as a polypeptide of the present invention, modified by linking to a polyethylene glycol polymer having a molecular weight greater than 30 kD. A modified GLP-1 receptor agonist comprising one of the polypeptides of the invention, or a fragment or variant thereof (collectively “modified polypeptides of the invention”). Examples of modified GLP-1 receptor agonists include, but are not limited to, the modified polypeptides shown in FIG. 2 and those polypeptides selected from the group consisting of SEQ ID NOs: 13-14 and 16-31 .

  The present invention is also directed to methods for making the GLP-1 agonist polypeptides of the invention (both recombinant and synthetic) and methods for making the modified GLP-1 agonist polypeptides of the invention.

Also, treating diabetes and / or other diseases or conditions in a mammal without causing gastrointestinal side effects, comprising administering to the mammal a therapeutically effective amount of a modified polypeptide of the invention. A method is disclosed.
DESCRIPTION OF THE INVENTION The present invention relates to a modified GLP-1 receptor agonist comprising a GLP-1 receptor agonist linked to a polyethylene glycol (PEG) polymer having a molecular weight greater than 30 kD, and its administration for therapeutic purposes. Law is provided. More specifically, these modified GLP-1 receptor agonists and compositions induce diabetes, hyperglycemia, impaired glucose tolerance, impaired fasting glycemia by inducing glucose-dependent insulin secretion without reducing gastrointestinal motility. And function as a GLP-1 receptor agonist in vivo to prevent and / or treat diseases or conditions such as obesity.

  Peptides having GLP-1 receptor agonist activity have been identified and are, for example, GLP-1 (7-36), GLP-1 (7-37), Exendin-4 and other GLP-1 agonists (eg, WO 98 No. / 43658, 00/15224, 00/16797, 01/98331, US Pat. No. 5,545,618, US Pat. No. 5,118,666, and US See patent application 60 / 395,738; all of which are incorporated herein by reference).

  The sequence superimposed below shows the primary structural relationship between several known GLP-1 agonist peptides:

  The one-letter abbreviations, their corresponding amino acids and three-letter abbreviations for specific amino acids used herein 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 (ser); T, threonine (thr); V, valine (val); W, tryptophan (trp); and Y, tyrosine (tyr).

  These polypeptides play a role in glucose homeostasis, and in particular, these polypeptides function as GLP-1 receptor agonists by lowering plasma glucose concentrations. Given the role of GLP-1 in the promotion of pancreatic glucose-regulated insulin secretion, GLP-1 receptor agonists are potentially valuable for the treatment of metabolic disorders and other diseases. To date, however, GLP-1 receptor agonists have had serious side effects: reduced gastrointestinal motility, which in turn can lead to nausea and nausea.

  In the art, PEGylation of drugs such as small molecules, peptides or proteins improves plasma half-life, physical solubility and stability, and resistance to protease degradation, and reduces immunogenicity It is well known that Furthermore, it is known in the art that PEGylation can reduce the degree of adverse side effects by lowering the drug's trough versus peak levels resulting from sustained plasma concentrations. However, it was not known in the art that pegylation can limit the access of drugs to specific tissues.

  Surprisingly, the tissue distribution of the administered modified peptide or protein can be selectively influenced by modifying the peptide or protein with a polymer of a specific size or structure, such as PEG. For example, modification of a GLP-1 agonist with linear 22 kD PEG does not increase the therapeutic index (glucose lowering vs intestinal motility) when compared to unmodified GLP-1 agonists and C16-fatty acid modified agonists. Modification with linear 30 kD PEG slightly improved the therapeutic index, but modification with branched 43 kD PEG greatly reduced intestinal motility mediated by CNS. Interestingly, a 43 kD-pegylated GLP-1 agonist was able to induce intestinal motility when injected directly into the brain. That is, the size and structure of PEG are the main determinants of such selective processes.

  Despite the many advantages resulting from pegylation, one important drawback is the bulky PEG hindrance in the interaction between the peptide and its receptor that results in reduced functional activity. Using GLP-1 and glucagon as models, a “twisted helix” model was developed, and based on this model, the position located within the C-terminus of the peptide was selected for PEGylation. These positions were expected to be on the opposite side of the peptide-receptor interaction.

Here, the inventors typically modify GLP-1 receptor agonists by linking a polyethylene glycol polymer having a molecular weight greater than 30 kD to the GLP-1 receptor agonist. And found to suppress the accompanying decrease in gastrointestinal motility. Without being bound by theory, here we use pegylation techniques to increase the size of a GLP-1 receptor agonist, passing the GLP-1 receptor agonist to the blood brain barrier, and In other words, it is considered to prevent access to the central nervous system. As a result, the ability of GLP-1 agonists to cause gastrointestinal side effects, which appear to be mediated by the central nervous system, is reduced. However, pegylated GLP-1 receptor agonists have the desirable activity to treat type 2 diabetes which approaches the pancreas, ie lowers blood glucose.
GLP-1 receptor agonist:
The polypeptides of the present invention are GLP-1 receptor agonists and are determined as their ability to activate the GLP-1 receptor. A GLP-1 receptor agonist activates the GLP-1 receptor in one or more in vitro or in vivo assays for GLP-1 receptor activation. Examples of such assays include, but are not limited to, in vitro assays for induction of cAMP in RINm5F cells, in vitro assays for induction of insulin secretion from pancreatic β-cells, as described in the specific examples below. In vivo assays for lowering plasma glucose levels and in vivo assays for increases in plasma insulin levels.

  Examples of GLP-1 receptor agonists include, but are not limited to, polypeptides selected from the group consisting of SEQ ID NOs: 1-10 and substantially the same levels as the polypeptides listed in SEQ ID NOs: 1-10. And its fragments, derivatives, variants and analogs that function as agonists of the GLP-1 receptor.

A polypeptide of the present invention that is a GLP-1 receptor agonist may be a naturally occurring polypeptide, a recombinant polypeptide or a synthetic peptide.
Fragments, derivatives, variants and analog fragments, derivatives, variants and analog polypeptides of GLP-1 receptor agonists retain substantially the same biological function or activity as, for example, the polypeptides shown in SEQ ID NOs: 1-10 To do. “Substantially the same biological function or activity” is each about 30% to about 100% (ie, 30%) exhibited by the polypeptides being compared when the biological activity of each polypeptide is measured by the same procedure. , 40, 50, 60, 70, 80, 90 or 100%) biological activity within or greater than.

  Fragments are shorter than full-length polypeptides such as those shown in SEQ ID NOs: 1-10 that retain substantially similar functional activity, as shown in the in vitro and in vivo models disclosed herein.

  Derivatives include polypeptides that have been chemically modified to provide additional structure and / or function. For example, fatty acids can be added to the polypeptide to improve half-life. Fusion polypeptides that confer targeted specificity or additional activity can also be constructed as described in more detail below.

  Derivatives can be modified either by natural processes such as post-translational processing or by chemical modification techniques, both of which are well known in the art. Modifications can occur anywhere in the polypeptide including the peptide backbone, amino acid side chains, and amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Variants can also contain one or more different types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.

  Other chemical modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol Covalent bond, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cysteine formation, pyroglutamate formation, formulation, gamma-carboxylation, glycosylation, GPI anchor Amino acids mediated by transfer RNA such as formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfuration, alginate of Including protein addition and ubiquitination (eg, TE Creighton, protein, structure and molecular properties (PROTEINS, STRUCTURES AND MOLECULAR PROPERIES), 2nd edition, WH Freeman and Company, New York (1993) Posttranslational covalent modification of proteins (POST TRANSLATIONAL COVALENTATION OF PROTEINS), edited by BC Johnson, Academic Publishing, New York, pages 1-14 (1983); Seifter, et al., Meth. Enzymol 182: 626. Rattan, et al., Ann.N.Y.Acad.Sci.663: 48-62,19. It should be 2 in the reference).

  Derivatives also include mature polypeptides fused to another polypeptide, such as human serum albumin, to improve their pharmacokinetic profile. The fusion of the two polypeptides can be performed by means known to those skilled in the art. For example, DNA encoding human serum albumin and DNA sequences encoding a polypeptide of the invention can be cloned into mammalian expression vectors known to those of skill in the art. Since free N-terminal histidine appears to be necessary for GLP-1 receptor activity, the polypeptide of the present invention is preferably located N-terminal to another polypeptide (Kawa, Endocrinology 124 (49) : 1768-73, 1989). The resulting recombinant fusion protein can then be expressed by transforming an appropriate cell line such as HKB or CHO with the vector and expressing the fusion protein.

  Variants of polypeptides of the present invention include polypeptides having changes in one or more amino acid sequences with respect to the amino acid sequences shown in SEQ ID NOs: 1-10. Variants can have amino acids linked to each other by modified peptide bonds (ie, peptide isostere) and may include amino acids other than the 20 naturally occurring amino acids.

  Preferably, the variant comprises one or more conservative amino acid substitutions (ie 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions), preferably in non-essential amino acid residues. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering its biological activity, whereas an “essential” amino acid is required for biological activity. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar amino acid side chain. Families of amino acid residues with similar side chains have been defined in the art. These families 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). Non-polar 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, phenylalanine, tryptophan) , Histidine) containing amino acids. Non-conservative substitutions are not made at conserved amino acid residues or amino acid residues that are within conserved protein domains.

  Variants also include polypeptides that differ in amino acid sequence due to mutagenesis. Variants that function as GLP-1 receptor agonists can be identified by screening a combinatorial library of variants, eg, one or more positions (ie 1, 2, 3, 4, 5, 6, 7, Polypeptide variants with conservative substitutions at positions 8, 9, or 10) can be generated using the methods well known in the art and described in Examples 3, 4, 5 and 6. Can be screened for body agonist activity.

Analogs include propolypeptides comprising the amino acid sequence of a polypeptide of the invention. The active polypeptides of the present invention can be cleaved from additional amino acids in the propolypeptide molecule by natural in vivo processes or by procedures well known in the art such as by enzymatic or chemical cleavage.
Methods of Producing Polynucleotides and Polypeptides of the Invention Any polynucleotide sequence encoding a polypeptide of the invention can be used to express a polypeptide. A polynucleotide can consist solely of a coding sequence for a polypeptide or can include additional coding and / or non-coding sequences.

  Polynucleotide sequences encoding the 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. 215-23, 1980; see Horn, et al., Nucl. Acids Res. Symp. Ser. 225-32, 1980). The polynucleotide encoding the polypeptide can then be cloned into an expression vector to express the polypeptide, or cloned into cloning beta- to propagate the polynucleotide.

  As will be appreciated by those skilled in the art, it may be advantageous to produce nucleotide sequences that encode polypeptides having non-naturally occurring codons. For example, selecting a suitable codon for a particular prokaryotic or eukaryotic host to increase the rate of polypeptide expression or such as a half-life longer than that of a transcript produced from a naturally occurring sequence RNA transcripts with the desired properties can be produced.

  The nucleotide sequences disclosed herein can be manipulated using methods commonly known in the art to include, but are not limited to, cloning, processing and / or expression of a polypeptide or mRNA product. The polypeptide-encoding sequence can be altered for a variety of reasons, including modifications to modify. Nucleotide sequences can be manipulated 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, alter glycosylation patterns, change codon preferences, generate splice variants, introduce mutations, etc. .

  The present invention also includes cloning and expression vectors comprising one or more nucleotide sequences encoding a polypeptide of the present invention. Nucleotide sequences can be inserted in the forward or reverse direction. The DNA sequence can be inserted into the vector by various procedures. In general, DNA sequences are known in the art at appropriate restriction endonuclease sites and are described in Sambrook et al. Molecular Cloning: A Laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), 2nd Edition, (Cold Spring Harbor, New York, 1989). Such procedures and others are deemed to be within the skill of the artisan.

  Examples of cloning vectors include, but are not limited to, pBR322, pUC18, pUC19, pSport and pCRII.

The expression vector can further comprise a regulatory sequence including, for example, a promoter operably linked to the coding sequence. A number of suitable expression beta- and promoters are known to those of skill in the art and are commercially available. The following expression vectors are provided as examples. Although not limited to bacterial expression vectors, pQE70, pQE60, pQE-9 (Qiagen); pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (stratage) And pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia). Eukaryotic expression vectors include, but are not limited to, pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene); and pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other cloning or expression vector can be used as long as it is replicable and viable in the desired host. The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) expression vector or other vector containing a selectable marker. Two suitable vectors are pKK232-8 and pCM7. Particularly mentioned are bacterial promoters include laci, lacZ, T3, T7, gpt, lambda _P R, the _{P L} and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art.

The expression vector may also contain a ribosome binding site for translation initiation, translation termination and a suitable sequence for amplifying expression. Expression vectors are phenotypes for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance for culture in E. coli. Genes for providing traits can be included.

  In one embodiment, the library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by an altered gene library. A library of variants is expressed, for example, as a degenerate set of potential variant amino acid sequences as individual polypeptides or as a set of longer fusion proteins (eg, in phage display) containing the set of sequences therein. Alternatively, it can be generated by enzymatically linking a mixture of synthetic oligonucleotides to a gene sequence.

  There are a variety of methods that can be used to generate a library of potential variants from a degenerate oligonucleotide sequence. Chemical synthesis of degenerate gene sequences can be performed automatically on a DNA synthesizer, and then the synthetic gene can be ligated into an appropriate expression vector. By using a degenerate set of genes, all sequences encoding the desired set of potential similar sequences can be prepared in one mixture. Methods for synthesizing degenerate oligonucleotides are known in the art (eg, Narang, Tetrahedron 39: 3, 1983; Itura, et al., Annu. Rev. Biochem. 53: 323, 1984; Itakura, et al. Science 198: 1056, 1984; Ike, et al., Nucleic Acids Res. 11: 477, 1983).

  Several techniques are known in the art for screening gene products of combinatorial libraries created by point mutations or truncations, and for screening cDNA libraries of gene products with selected properties. ing. Such techniques are applicable to rapid screening of gene libraries generated by the combinatorial mutagenesis of the polypeptides of the present invention. The most widely used techniques available for high-throughput analysis to screen large gene libraries have typically cloned gene libraries into replicable expression vectors and generated appropriate cells Transforming with a library of vectors and expressing the combined gene under conditions that facilitate the isolation of the vector in which the loss of the desired activity facilitates the isolation of the vector encoding the gene of the product to be detected. Reversible ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutations in a library, can be used in combination with screening assays to identify desired variants.

  The present invention also provides a host cell comprising the above vector. The host cell can be a higher eukaryotic cell such as a mammalian cell, or a lower eukaryotic cell such as a yeast cell. Alternatively, the host cell can be a prokaryotic cell such as a bacterial cell.

  Host cells can be genetically engineered with the cloning or expression vectors of the invention (transduction, transformation or transfection). The vector can be, for example, in the form of a plasmid, viral particle or phage. Engineered host cells can be cultured in customary nutrient media appropriately modified to activate the promoter or select transformants. Selection of appropriate culture conditions such as temperature and pH is within the skill of one of ordinary skill in the art.

Representative examples of appropriate hosts include, but are not limited to E. (E. coli), Salmonella typhimurium (Salmonella typhimurium) or bacterial cells, such as Streptomyces (Streptomyces); fungal cells, such as yeast; Drosophila ( insect cells such as Drosophila) S2 and Spodoptera (Spodoptera) Sf9; or CHO, including mammalian cells such as COS or Bowes (Bowes) melanoma. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the techniques herein.

  Introduction of the construct into the host cell can be accomplished, for example, by calcium phosphate transfection, DEAE-dextran mediated transfection or electroporation (Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY, 1986). The construct in the host cell can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.

  The mature protein can be expressed in mammalian cells, yeast, bacteria or other cells under the control of a suitable promoter. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use in eukaryotic and prokaryotic hosts are described above and in Sambrook et al. Molecular Cloning: A Laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), Second Edition, (Cold Spring Harbor, New York, 1989).

Transcription of the DNA encoding the polypeptide of the present invention by higher eukaryotes can be increased by inserting an enhancer sequence into the expression vector. Enhancers are elements that act on the cis of DNA, usually about 10-300 bp, and act to increase transcription at the promoter. Examples include the late SV40 enhancer of origin of replication (100-270 bp), the cytomegalovirus early promoter enhancer, the polyoma enhancer late of the origin of replication, and the adenovirus enhancer. In general, recombinant expression vectors contain an origin of replication and a selectable marker that allow transformation of the host cell (eg, the E. coli ampicillin resistance gene and yeast ( S. cerevisiae ) TRP1 gene), and downstream structural sequences. Contains a promoter derived from a highly expressed gene to control the transcription of. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phases with translation, initiation and termination sequences, and preferably leader sequences that can govern the secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein comprising an N-terminal identification peptide that confers desired properties (eg, simplified purification of the recombinant product expressed or stabilized).

  After transformation of the appropriate host strain and growth of the host strain to the appropriate cell density, the selected promoter is derepressed by appropriate means (eg temperature shift or chemical induction) and the cells are cultured for an additional period of time. To do. Cells are typically harvested by centrifugation and disrupted by physical or chemical means. The resulting crude extract is retained for further purification. The microbial cells used for protein expression can be disrupted by any conventional method, including freeze-thaw cycles, sonication, mechanical disruption or the use of cytolytic enzymes.

  Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 strain of monkey kidney fibroblasts (Gluzman, Cell 23: 175, 1981) and other cell lines capable of expressing compatible vectors include, for example: Includes C127, 3T3, CHO, HeLa and HBK cell lines.

  Polypeptides of the present 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. It can be recovered and purified from recombinant cell culture by methods well known in the art. High performance liquid chromatography (HPLC) can be used as the final purification step.

  The polypeptides of the present invention can be conveniently isolated. A purified polypeptide is at least 70% pure, i.e., an isolated polypeptide is substantially free of cellular material and has less than about 30% (by dry weight) of non-polypeptide material. Preferably the preparation is 85% to 99% (ie 85, 87, 89, 91, 93, 95, 96, 97, 98 and 99%) pure. The purity of the preparation can be assessed by means known in the art such as SDS-polyacrylamide gel electrophoresis, mass spectrometry and liquid chromatography.

  Depending on the host used in the recombinant production method, the polypeptides of the invention can be glycosylated with mammalian or other eukaryotic carbohydrates, or may be non-glycosylated. The polypeptides of the invention can also include an initiating methionine amino acid residue.

  Alternatively, a polypeptide of the invention can be produced using chemical synthesis methods such as direct peptide synthesis using solid phase techniques to synthesize its amino acid sequence (see, eg, Merrifield, J. Am. Chem. Soc. 85: 2149-2154, 1963; see Robert, et al., Science 269: 202-204, 1995). Polypeptide synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished using, for example, an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Optionally, polypeptide fragments can be synthesized separately and combined using chemical methods to produce a full-length molecule.

Newly synthesized polypeptides can be substantially purified by preparative high performance liquid chromatography (eg, Creighton, Proteins: Structures and Molecular Principles), WH Freeman and Company, See New York, NY, 1983). The composition of the synthetic polypeptide of the present invention can be confirmed by amino acid analysis or by sequencing, for example by Edman degradation (see Creighton, ibid.). In addition, any portion of the amino acid sequence of the polypeptide can be modified directly during synthesis and / or in combination with sequences derived from other proteins using chemical methods to produce variant or fusion polypeptides. be able to.
Modified GLP-1 receptor agonists and production methods The modified GLP-1 receptor agonists of the present invention comprise a GLP-1 receptor agonist, or a fragment, derivative, variant or analog thereof, which has a molecular weight greater than 30 kD. Is linked to a polyethylene glycol (PEG) polymer having

  Many suitable PEG polymers are commercially available or may be made by techniques well known to those skilled in the art. The PEG polymer has a molecular weight greater than 30 kD, preferably a molecular weight greater than 30 kD, more preferably greater than 40 kD, and even more preferably a branched structure such as a branched PEG-peptide of 43 kD (share water ( Shearwater) 2001 catalog # 2D3X0T01, mPEG2-MAL).

  Attachment of PEG onto the complete peptide can be accomplished by attaching PEG to the opposite side of the peptide surface that interacts with the receptor. Preferably, PEG attachment occurs on the GLP-1 agonist at positions 22-28 and 30-31, numbered according to GLP-1 (7-37), and beyond the C-terminus; ie positions 32-37 . More preferably, the attachment of PEG is on the GLP-1 agonist, positions 24, 28, 30 and 31 numbered according to GLP-1 (7-37), and positions beyond the C-terminus; ie 32, 34, 36 And takes place in 37th place. Even more preferably, the attachment of PEG occurs at the C-terminus numbered according to GLP-1 (7-37) on the GLP-1 agonist;

  There are several methods for coupling PEG to peptides (see, eg, Veronese, Biomaterials 22: 405-417, 2001), all of which are incorporated herein by reference. Accordingly, those skilled in the art can utilize such well-known techniques for linking the PEG polymers described herein to GLP-1 receptor agonists.

  Briefly, cysteine pegylation is one method of site-specific pegylation and is often utilized when peptides have few or no cysteine residues. For example, in the case of natural GLP-1 (7-37), there is no cysteine residue. Thus, PEGylation of native GLP-1 (7-37) or GLP-1 agonists that do not have a cysteine residue results in specific sites on the native GLP-1 (7-37) or GLP-1 agonist identified above. One can introduce a single cysteine mutation and then react the resulting derivative with a PEGylation reagent specific for cysteine, such as PEG-maleimide.

  However, it may be necessary to mutate the GLP-1 agonist of the present invention to allow site-specific pegylation. For example, if GLP-1 agonists contain cysteine residues, these need to be replaced with conservative amino acids to ensure site-specific pegylation. In addition, rigid linkers including but not limited to “GGS” (SEQ ID NO: 32), “GGSGGS” (SEQ ID NO: 33), and “PPPS” (SEQ ID NO: 34) are attached to the C-terminus of the GLP-1 agonist, but PEG It can be added before the addition site (ie only one cysteine residue).

  Examples of modified GLP-1 receptor agonists of the present invention include, but are not limited to, polypeptides selected from the group consisting of SEQ ID NOs: 13-14 and 16-31. The most preferred modified GLP-1 receptor agonist of the present invention is SEQ ID NO: 26.

  The polypeptides of the invention can be used to treat diabetes, including both type 1 and type 2 diabetes (non-insulin dependent diabetes). Such treatment can also delay the onset of diabetes and diabetic complications. The polypeptide can be used to prevent individuals with impaired glucose tolerance from progressing to type 2 diabetes. In the methods of the invention, other diseases and conditions that can be treated or prevented using the compounds of the invention are: adult-onset diabetes of the young (MODY) (Herman, et al., Diabetes 43:40, 1994); adult potential autoimmune diabetes (LADA) (Zimmet et al., Diabetes Med, 11: 299, 1994); impaired glucose tolerance (IGT) (Expert Committee on Classification of Diabetes) Melitus), Diabetes Care 22 (Supp. 1): S5, 1999); Fasting Glucose Disorder (IFG) (Charles, et al., Diabetes 40: 796, 1991); Diabetes Mellitus (Me) Including and metabolic syndrome X; 197,1991): zger, Diabetes 40.

  The polypeptides of the present invention may be used to treat disorders such as obesity and atherosclerosis, hyperlipidemia, hypercholesterolemia, low HDL levels, hypertension, cardiovascular disease (atherosclerosis, coronary heart disease, coronary artery disease, and (Including hypertension), cerebrovascular disease and peripheral vascular disease.

  The compounds of the present invention can also be useful for treating physiological disorders associated with, for example, cell differentiation to produce cells that accumulate lipids, modulation of insulin sensitivity and blood glucose levels. For example, abnormal pancreatic β-cell function, insulin-secreting tumors and / or autoantibodies to insulin, autoantibodies to insulin receptors, or autoimmune hypoglycemia by autoantibodies that are stimulators to pancreatic β-cells, atherosclerotic plaques Macrophage differentiation leading to formation, inflammatory response, carcinogenesis, hyperplasia, adipocyte gene expression, adipocyte differentiation, pancreatic β-cell mass reduction, insulin secretion, tissue sensitivity to insulin, liposarcoma cell proliferation, polycystic ovarian disease Chronic anovulation, hyperandrogen hyperplasia, progesterone production, steroidogenesis, cellular acidity Reduction potential and oxidative stress, nitric oxide synthase (NOS) production, increased gamma Guru Tamil trans peptidase, catalase, plasma triglycerides, HDL, is involved in LDL cholesterol levels and the like.

  The polypeptides of the invention can be used in the methods of the invention to treat secondary causes of diabetes (Special Committee on Diabetes Classification, Diabetes Care 22 (Supp. 1): S5, 1999). . Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma and drug-induced diabetes. Agents used to treat pyriminyl, nicotinic acid, glucocorticoid, phenytoin, thyroid hormone, β-adrenergic agents, α-interferon and HIV infections, including but not limited to agents capable of inducing diabetes including.

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

  The polypeptide of the present invention treats diabetes, including PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α-glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose output lowering compounds, insulin and anti-obesity drugs Can be administered in combination with other known therapeutic agents. Such therapeutic agents can be administered before, simultaneously with, or after administration of the polypeptides of the present invention. Insulin includes both long and short acting forms and formulations of insulin. PPAR agonists can include agonists of any PPAR subunit or combinations thereof. For example, a PPAR agonist can include PPAR-α, PPAR-γ, PPAR-δ agonists, or a combination of two or three PPAR subunits. PPAR agonists include, for example, rosiglitazone and pioglitazone. Sulfonylurea drugs include, for example, glyburide, glimepiride, chlorpropamide and glipizide. Alpha-glucosidase inhibitors that may be useful for treating diabetes when administered with the polypeptides of the invention include acarbose, miglitol and voglibose. Insulin sensitizers that can be useful in the treatment of diabetes include thiazolidinediones and non-thiazolidinediones. Hepatic glucose output lowering compounds that may be useful for treating diabetes when administered with the polypeptides of the invention include metformin such as Glucophage and Glucophage XR. Insulin secretagogues that may be useful for treating diabetes when administered with the polypeptides of the invention include sulfonylureas and non-sulfonylureas; GIP, secretin, nateglinide, meglitinide, repaglinide, glibenclamide (Glibenclamide), glimepiride, chlorpropamide, glipizide. In one aspect of the invention, the polypeptide of the invention is used in combination with an insulin secretagogue to increase pancreatic β-cell sensitivity to an insulin secretagogue.

  The polypeptides of the present invention can also be used in the methods of the present invention in combination with antiobesity agents. Anti-obesity drugs include beta-3 agonists, CB-1 antagonists, appetite suppressants such as sibutramine (Meridia), and lipase inhibitors such as orlistat (Xenical).

  The polypeptides of the present invention can also be used in the methods of the present invention in combination with drugs commonly used to treat lipid disorders in diabetic patients. Such agents include, but are not limited to, HMG-CoA reductase inhibitors, nicotinic acid, bile acid secretion inhibitors, and fibric acid derivatives. The polypeptides of the present invention can also be used in combination with antihypertensive agents such as β-blockers and ACE inhibitors.

  Such co-therapy can be administered in any combination of two or more agents (eg, a compound of the invention in combination with an insulin sensitizer and an anti-obesity agent). Such co-therapy may be administered in the form of a pharmaceutical composition as described above.

  Various terms as used herein are defined below.

  When introducing elements of the present invention or preferred embodiments (s) thereof, the articles “a”, “an”, “the” and “said” indicate that there are one or more elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

  As used herein, the term “individual” includes mammals (eg, humans and animals).

  As used herein, the term “treatment” includes any process, action, application, therapy, etc., where individuals including humans are directly or indirectly, for the purpose of improving the condition of the individual. Or medical assistance to slow the progression of the individual's condition or disorder.

  The term “combination therapy” or “simultaneous therapy” means administering two or more therapeutic agents to treat a diabetic condition and / or disorder. Such administration includes co-administering two or more therapeutic agents, such as a single capsule having a fixed ratio of active ingredients or multiple individual capsules for each inhibitor drug, in a substantially simultaneous manner. . Such administration further includes the use of various therapeutic agents in a sequential manner.

  The phrase “therapeutically effective” means the amount of each drug administered that achieves the goal of improving the severity of the diabetic condition or disorder, but at the same time avoids or minimizes the adverse side effects associated with the therapeutic treatment. To do.

  The term “pharmaceutically acceptable” means that an individual item is suitable for use in medicine.

  Based on well-known assays used to determine efficacy for treatment of the conditions identified above in mammals and compare these results with the results of known drug therapies used to treat these conditions Thus, an effective dosage of a polypeptide of the present invention can be readily determined for the treatment of each desired indication. The amount of active ingredient (eg, polypeptide) administered in one treatment of these conditions depends on the particular compound and unit dosage used, the mode of administration, the duration of treatment, the age and sex of the patient being treated, and the condition being treated. It can vary widely according to considerations such as nature and degree.

  The total amount of active ingredient to be administered generally ranges from about 0.0001 mg / kg to about 200 mg / kg, and preferably from about 0.01 mg / kg to about 200 mg / kg body weight per day. A unit dose can contain from about 0.05 mg to about 1500 mg of active ingredient, and can be administered one or more times per day. Daily dosages for administration by intravenous, intramuscular, subcutaneous, and parenteral injection, and use of infusion techniques can be from about 0.01 to about 200 mg / kg. Daily rectal dosing can range from 0.01 to 200 mg / kg total body weight. The transdermal concentration may be that required to maintain a daily dose of 0.01 to 200 mg / kg.

  Of course, the specific initial and continuous dosing regimen for each patient will be the nature and severity of the condition as determined by the attending physician, the activity of the specific polypeptide used, the patient's age, the patient's diet, the timing of administration, It will vary according to the route of administration, drug excretion rate, drug combination, etc. The desired mode of treatment and frequency of polypeptide administration of the invention can be ascertained by one skilled in the art using routine treatment tests.

  Desired pharmacological effects can be achieved using the polypeptides of the invention by administering a suitably formulated pharmaceutical composition to a patient in need thereof. For purposes of the present invention, a patient is a mammal, including a human in need of treatment for a particular condition or disease. Accordingly, the present invention includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the polypeptide. Since pharmaceutically acceptable carriers are relatively non-toxic and harmless to the patient at a concentration consistent with the effective activity of the active ingredient, side effects due to the carrier are beneficial effects of the active ingredient. Will not be damaged. A therapeutically effective amount of a polypeptide is an amount that results in or exerts an effect on the particular condition being treated. The polypeptides described herein are pharmaceutically acceptable carriers using effective conventional unit dosage forms including, for example, immediate and timed release preparations, oral, parenteral, topical, etc. Can be administered.

  For oral administration, the polypeptides can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions or emulsions, and pharmaceutical compositions It can be prepared according to methods known in the art for the manufacture of products. Solid unit dosage forms are capsules that can be conventional hard or soft shell gelatin types containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate and corn starch. Good.

  In another embodiment, the polypeptides of the present invention may be formulated into conventional tablet bases such as lactose, sucrose, and corn starch, binders such as gum arabic, corn starch or gelatin; such as potato starch, alginic acid, corn starch and guar gum. Disintegrants intended to assist tablet breakage and dissolution after successful administration; improve tablet granulation flow and prevent tablet material from adhering to the surface of tablet dies and punches, eg talc, stearic acid or Lubricants such as magnesium stearate, calcium or zinc; dyes; colorants; and can be formulated in combination with flavors intended to enhance the aesthetic quality of the tablets and make them more acceptable to the patient. Excipients suitable for use in oral liquid dosage forms include water and diluents such as alcohol, e.g. ethanol, benzyl alcohol and polyethylene alcohol, pharmaceutically acceptable surfactants, suspending agents or emulsifiers. Contains with or without addition. A variety of other materials may be present as coatings or to modify the physical form of the unit dose. For instance tablets, pills or capsules may be coated with shellac, sugar or both.

  Dispersible powders and particles are suitable for the preparation of aqueous suspensions. They provide the active ingredient in a mixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients may also be present, such as the sweetening, flavoring and coloring agents described above.

  The pharmaceutical composition of the present invention may 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 (1) naturally occurring gums such as gum arabic and tragacanth, (2) naturally occurring phosphatides such as soybean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides. For example, sorbitan monooleate, and (4) a condensation product of the partial ester with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may contain sweetening and flavoring agents.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil; or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. The suspension may contain one or more preservatives, such as ethyl or n -propyl p-hydroxybenzoate; one or more colorants; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin. .

  Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent and a preservative, flavoring and coloring agents.

  The polypeptides of the invention can also be administered parenterally, i.e., subcutaneously, intravenously, intramuscularly, or intraperitoneally, a sterile liquid or solution of water, saline, aqueous dextrose and related sugars. Liquid mixtures such as: 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; oils; fatty acids; fatty acid esters or glycerides; or further pharmaceutically acceptable surfactants such as soaps or surfactants, pectin, carbomers, methylcellulose, hydroxypropyl Methyl cell In a physiologically acceptable diluent comprising a pharmaceutical carrier which may be an acetylated fatty acid glyceride, with or without suspending agents such as glucose or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants As an injectable dose of the compound.

  Examples of oils that can be used in the parenteral formulations of the present invention are oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petroleum and mineral oils . Suitable fatty acids include oleic acid, stearic acid and isostearic acid. Suitable fatty acid esters include, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty alkali metals, ammonium and triethanolamine salts, and suitable surfactants include cationic surfactants such as dimethyldialkylammonium halides, alkylpyridinium halides and alkylamine acetates; anionic surfactants Agents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates and sulfosuccinates; nonionic surfactants such as fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; and both Includes ionic surfactants such as alkyl-beta-aminopropionate and 2-alkylimidazoline quaternary ammonium salts and mixtures

  The parenteral compositions of the invention can typically contain from about 0.5% to about 25% by weight of active ingredient in solution. Preservatives and buffers can also be used advantageously. To minimize or eliminate inflammation at the site of injection, such compositions may include a nonionic surfactant having a hydrophilic lipophilic balance (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 can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB.

  Examples of surfactants for use in parenteral formulations are high molecular weight adducts of ethylene oxide containing a class of polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and a hydrophobic substrate formed by condensation of propylene oxide and propylene glycol. is there.

  The pharmaceutical composition may be in the form of a sterile injectable aqueous suspension. Such suspensions are known using suitable dispersing or wetting agents and suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic. Can be formulated according to the method; the dispersing or wetting agent can be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide and a fatty acid such as polyoxyethylene stearate, ethylene oxide and a long chain aliphatic alcohol. Condensation products such as condensation products of ethylene oxide such as heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate with partial esters derived from fatty acids and hexitol, Condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.

  The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Diluents and solvents that can be used are, for example, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils can usually be employed as a solvent or suspending medium. For this, any blend, fixed oil containing synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid can 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 consist of a drug (eg, a polypeptide) and a suitable non-inflammatory excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Can be prepared by mixing. Such materials are cocoa butter and polyethylene glycols, for example.

  Another formulation employed in the methods of the present invention uses transdermal delivery devices (“patches”). Such transdermal patches can be used to provide continuous or intermittent infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for drug delivery is well known in the art (see, eg, US Pat. No. 5,023,252, incorporated herein by reference). Such patches can be constructed to deliver the drug continuously, pulsatile, or on demand.

  It may be desirable or necessary to introduce the pharmaceutical composition to the patient via a mechanical delivery device. The construction and use of mechanical delivery devices for delivering formulations is well known in the art. For example, direct techniques for administering drugs directly to the brain typically involve placing a drug delivery catheter in the patient's vasculature to bypass the blood brain barrier. One such implantable delivery system used to deliver drugs to specific anatomical regions of the body is described in US Pat. No. 5,011,472, which is incorporated herein by reference. Are listed.

  The compositions of the present invention may also include other customary pharmaceutically acceptable mixed materials, commonly referred to as carriers or diluents, as necessary or desired. Any composition of the present invention can be preserved by the addition of an antioxidant such as ascorbic acid or by other suitable preservatives. Conventional procedures for preparing such compositions into suitable dosage forms can be used.

  Commonly used pharmaceutical materials that can be used as appropriate for formulating the composition for the intended route of administration include: acidifying agents such as, but not limited to, acetic acid, citric acid Fumaric acid, hydrochloric acid, nitric acid; and but not limited to ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine, etc. An alkalinizing agent.

Other pharmaceutical materials include, but are not limited to, adsorbents (eg, powdered cellulose and activated carbon); aerosol propellants (eg, carbon dioxide, CCl ₂ F ₂ , F ₂ ClC—CClF ₂ and CClF ₃ ). Air displacement agents (eg, nitrogen and argon); antibacterial and mold preservatives (eg, benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate); antimicrobial preservatives (eg, benzal chloride) Luconium, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal); antioxidants (eg ascorbic acid, ascorbyl palmitate, Tillated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, isomeric sodium bisulfite); 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, anhydrous sodium citrate and sodium citrate dihydrate) Carrying agents (eg acacia syrup, aromatic syrup, aromatic elixir, cherry syrup, cacao syrup, orange syrup, syrup, corn) Shii oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection and bacteriostatic water for injection); complexing agents (eg disodium edetate and edetic acid); colorants (eg FD & C Red No. 3) , FD & C Red No. 20, FD & C Yellow No. 6, FD & C Blue No. 2, D & C Green No. 5, D & C Orange No. 5, D & C Red No. 8, Caramel and Iron Oxide Red); Purification agent (clarifying agent) (E.g. bentonite); emulsifiers (but not limited to acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyethylene 50 stearate); encapsulating agents (e.g. gelatin and phthalic acetate phthalate) Cellulose); flavoring agent (for example, Anise oil, cinnamon oil, cacao, menthol, orange oil, peppermint oil and vanillin); humectants (eg, glycerin, propylene glycol and sorbitol); levitation 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) (eg monohydroxy or polyhydroxy alcohols, saturated or unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, Farin, terpenes, amides, ethers, ketones and ureas); plasticizers (eg, diethyl phthalate and glycerin); solvents (eg, alcohol, corn oil, cottonseed oil, glycerin, isopropyl alcohol, mineral oil, oleic acid, peanut oil, refined) Water, water for injection, sterile water for injection and sterile water for irrigation); stiffeners (eg cetyl alcohol, cetyl ester wax, microcrystalline wax, paraffin, stearyl alcohol, white wax, yellow wax); suppository group Materials (eg cocoa butter and polyethylene glycol (mixtures)); surfactants (eg benzalkonium chloride, nonoxynol 10, oxoxynol 9, polysorbate 80, sodium lauryl sulfate and sorbitan monopa Suspending agents (eg, agar, bentonite, carbomer, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, kaolin, methylcellulose, tragacanth and beegum); sweeteners such as aspartame, dextrose, glycerin, mannitol , Propylene glycol, sodium saccharin, sorbitol and sucrose); tablet anti-adhesives (eg, magnesium stearate and talc); tablet binders (eg, gum arabic, alginic acid, sodium carboxymethylcellulose, compressible sugar, ethyl cellulose, gelatin) Liquid glucose, methylcellulose, povidone and gelatinized starch); tablets and tablets Cell 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, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetate phthalate and shellac); tablet direct compression excipients (eg, dibasic calcium phosphate); tablet disintegrants (eg, alginic acid, Carboxymethylcellulose calcium, microcrystalline cellulose, polacrilin potassium, sodium alginate, sodium glycolate starch Tablet glidants (eg colloidal silica, corn starch and talc); tablet lubricants (eg potassium stearate, magnesium stearate, mineral oil, stearic acid and zinc stearate); tablet / capsule opacifiers (E.g. titanium dioxide); tablet abrasives (e.g. carnauba wax and white wax); thickeners (e.g. beeswax, cetyl alcohol and paraffin); tonicity modifiers (e.g. dextrose and sodium chloride); viscosity increasing agents (Eg, alginic acid, bentonite, carbomer, sodium carboxymethylcellulose, methylcellulose, povidone, sodium alginate and tragacanth gum); and wetting agents (eg, heptadecaethyleneoxysetanol, lecithin, polyethylenesol Bitol monooleate, polyoxyethylene sorbitol monooleate and polyoxyethylene stearate).

  The polypeptides described herein can be administered as a single formulation or in combination with one or more other formulations where the combination does not cause undesirable side effects. For example, the polypeptides of the present invention can be combined with known anti-obesity drugs, or known anti-diabetic drugs, or other symptomatic drugs, and mixtures and combinations thereof.

  The polypeptides described herein can be utilized in free base form or in compositions, for research and diagnosis, or as a reference standard for analysis. Accordingly, the present invention includes a composition comprising an inert carrier and an effective amount of a compound identified by the methods described herein, or a salt or ester thereof. An inert carrier is any material that does not interact with the compound being transported and attaches to the transported compound a support, means of transport, bulk, traceable material, and the like. An effective amount of a compound is that amount that produces a result or exerts an influence on the particular procedure being performed.

  Polypeptides are known to undergo hydrolysis, deamidation, oxidation, racemization and isomerization in aqueous and non-aqueous environments. Degradation such as hydrolysis, deamidation or oxidation can be easily detected by capillary electrophoresis. Despite enzymatic degradation, polypeptides with long plasma half-life or biological residence time must be at least stable in aqueous solution. It is essential that the polypeptide exhibits less than 10% degradation per day at body temperature. Furthermore, the polypeptide preferably exhibits less than 5% degradation per day at body temperature. Since treatment is lifelong in chronic diabetic patients, it is more preferred that these therapeutic agents be conveniently administered less frequently when by the parenteral route. Stability over several weeks at body temperature (ie, less than a few percent degradation) allows for lower dosing frequencies. Stability on a scale of several years at refrigerated temperatures allows the manufacturer to present a liquid formulation, i.e. avoids the inconvenience of reconstitution. In addition, stability in organic solvents will provide the formulation of polypeptides into new dosage forms such as implants.

  Formulations suitable for subcutaneous, intravenous, intramuscular, etc .; suitable pharmaceutical carriers; and formulation and administration techniques can be prepared by any method well known in the art (eg, Remington's Pharmaceutical Sciences). 's Pharmaceutical Science), Mac Publishing Company, Easton, PA, 20th edition, 2000).

The following examples are presented to illustrate the invention described herein, but should not be construed as limiting the scope of the invention in any way.
Capsule preparation Capsule formulation:
10 mg of the polypeptide of the present invention
Starch 109mg
Magnesium stearate 1mg
Prepare from

The ingredients are blended, sieved through a suitable mesh and filled into hard gelatin capsules.
Pill preparation pills
25 mg of the polypeptide of the present invention
Cellulose, microcrystals 200mg
Colloidal silicon dioxide 10mg
Stearic acid 5.0mg
Prepare from

The ingredients are mixed and compressed into tablets. Appropriate aqueous and non-aqueous coatings can be applied to increase palatability, improve quality and stability, or delay absorption.
Sterile IV solution
A mg / mL solution of the desired compound of the invention is made using sterile injectable water and the pH adjusted as necessary. Solutions are diluted with sterile 5% dextrose for administration and administered as an IV infusion.
Intramuscular suspension The following intramuscular suspension is prepared:
Polypeptide of the present invention 50 μg / mL
Sodium carboxymethyl cellulose 5mg / mL
TWEEN 80 4mg / mL
Sodium chloride 9mg / mL
Benzyl alcohol 9mg / mL
The suspension is administered intramuscularly.
Hard Shell Capsules A number of unit capsules are prepared by filling standard two-piece hard gelatin capsules, each capsule containing powdered active ingredient, 150 mg lactose, 50 mg cellulose and 6 mg magnesium stearate. .
Soft gelatin capsules A mixture of active ingredients is prepared in a digestible oil such as soybean oil, cottonseed oil or olive oil and injected into molten gelatin by a displacement pump to form a soft gelatin capsule containing the active ingredients. The 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 drug mix.
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 drug. The active ingredient is mixed with a liquid containing ingredients such as sugar, gelatin, pectin and sweeteners. These liquids are solidified into solid tablets or capsules by freeze drying and solid state extraction techniques. The drug compound can be compressed with viscoelastic and thermoelastic sugars and polymers or effervescent ingredients to produce a porous matrix intended for immediate release without the need for water.

It will be apparent to those skilled in the art that changes and modifications can be made to the present invention without departing from the spirit or scope of the invention as described herein.
[Example]
In order that this invention be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. All publications mentioned in this specification are incorporated by reference.

Peptide Synthesis Method The following general procedure was followed to synthesize some of the polypeptides of the present invention. Peptide synthesis was carried out according to the FMOC / t-butyl method (Peptide Synthesis Protocols, Vol. 35, Michael W. Pennington & Ben M. Dunn, 1994), and Rapp-Polymer PEG-polystyrene resin (wrap-polymer (Rapp-polymer)). Polymere), Tuebingen, Germany) under continuous flow conditions. At the completion of synthesis, the peptide was cleaved from the resin and deprotected using TFA / DTT / H ₂ O / triisopropylsilane (88/5/5/2). The peptide was precipitated from the cleavage cocktail using cold diethyl ether. The precipitate was washed three times with cold ether and then dissolved in 5% acetic acid and lyophilized. Peptide identity was determined by reverse phase chromatography on a YMC-Pack ODS-AQ column (YMC, Wilmington, NC) Waters ALLIANCE ™ system (Waters, Milford, MA). A VOYAGER DE ™ MALDI mass spectrometer (Model 5-2386-00, Perceptive Bio) using water / acetonitrile with 3% TFA in a gradient from 0% to 100% acetonitrile and by MALDI mass spectrometry. Confirmed by Systems (PerSeptive BioSystems, Framingham, Mass.) Matrix buffer (50/50 dH ₂ O / acetonitrile with 3% TFA) peptide samples Those peptides that did not meet the> 95% purity criteria were purified by reverse phase chromatography on a Waters Delta Prep 4000 HPLC system (Waters, Milford, Mass.).

  Table 1 contains some selected polypeptides made according to the peptide synthesis protocol discussed above.

Pegylated pegylation of peptides can be performed by any method known to those skilled in the art. In this case, however, pegylation uses two different methods to introduce a unique cysteine mutation at the peptide or at the C-terminus of the peptide, followed by peptide sulfhydryl and methoxy-PEG-maleimide reagents (inhale). The cysteine was PEGylated via a stable thioether linkage with the maleimide group of (/ Sharewater). It is preferred to introduce a unique cysteine at the C-terminus of the peptide.

  In the first method, a 2-fold molar excess of mPEG-mal (MW 22 kD, 30 kD and 43 kD) reagent is added to 1 mg of peptide and the reaction buffer (0.1 M Na phosphate / 0.1 M NaCl / 0.1 M EDTA at pH 6). ). After 0.5 hours at room temperature, the reaction was stopped with a 2-fold molar excess of DTT over mPEG-mal. The peptide-PEG-mal reaction mixture was added to the cation exchange column to remove residual PEG reagent, followed by removal of remaining free peptide by gel filtration column. Purity, mass and number of PEGylation sites were determined by SDS-PAGE and MALDI-TOF mass spectrometry.

  In the second method, a 10-fold molar excess of mPEG-mal (MW to 22 kD or 43 kD) reagent was dissolved in 50 μM of a pH 6 reagent buffer (0.1 M Na phosphate / 0.1 M NaCl / 0.1 M EDTA). Added to peptide. After 0.5 hours at room temperature, the reaction was stopped with a 2-fold molar excess of cysteine over mPEG-mal. The crude peptide-PEG-mal reaction mixture was assayed in vitro without further purification.

  Table 2 contains a number of selected polypeptides made according to the pegylation techniques discussed herein. Note that the underlined amino acids represent the position where the PEG polymer is attached to the peptide.

Measurement of Peptide Signaling via GLP-1 Receptor Using Cyclic AMP Scintillation Proximal (SPA) Assay For the modified GLP-1 receptor agonists of the present invention, the “activity of GLP-1 receptor in cAMP scintillation proximal assay” Is "at least 50%, more preferably at least 70%, even more preferably at least 80%, and even more preferably at least 90% of the maximum activity induced by native GLP-1 (7-36) -amide Is the induction of maximal activity. The EC50 values of the modified GLP-1 receptor agonists of the present invention are between 0.1 and 1000 nM. “EC50” is defined herein as the polypeptide concentration of the invention at which 50% of maximum activity is achieved.

RINm5F cells were seeded at 1.5 × 10 ⁵ cells / well in 96-well plates (Costar) and at 37 ° C. for 24 hours, RPMI 1640, 5% FBS, antibiotic / antibacterial fungi (Gibco ) BRL). The medium was removed and the cells were washed twice with PBS. Cells were incubated for 15 minutes at 37 ° C. in HEPES-PBS containing 1% BSA and 100 μM IBMX with peptides in a concentration range of 1 × 10 ⁻¹² to 1 × 10 ⁻⁵ M. Incubation buffer was removed and cells were lysed in the lysis reagent provided by the cAMP scintillation proximal assay (SPA) direct screening assay system (Amersham Pharmacia Biotech, Piscataway, NJ). The amount of cAMP (in picomoles) present in the lysate was determined according to the instructions provided in the kit. The amount of cAMP produced (in picomoles) at each peptide concentration was plotted and analyzed by non-linear regression using Prizm software to determine the EC50 for each peptide.

  The results of this assay, including a control (indicated by an asterisk) and a representative polypeptide of the invention are shown in the table below.

Insulin secretion from dispersed rat islet cells In this assay, the increase in insulin secretion from dispersed rat islet cells is at least a 1.5-fold increase. The modified GLP-1 agonists of the invention at least about 1.5-fold to about 10-fold insulin secretion from dispersed islet cells (ie 1.5, 2.0, 3.0, 4.0, 5. 0, 6.0, 7.0, 8.0, 9.0 or 10 times).

  Islets of Langerhans were isolated from SD rats (200-250 g) via a digestion procedure using collagenase. Dispersed islet cells were prepared via treatment with trypsin, seeded into 96V bottom plates and pelleted. Cells were cultured overnight in media with or without the peptides of the invention. The medium was aspirated and the cells were preincubated with Krebs-Ringer-HEPES buffer containing 3 mM glucose for 30 minutes at 37 ° C. The preincubation buffer was removed and cells were stimulated with Krebs-Ringer-HEPES buffer containing the appropriate concentration of glucose (eg, 8 mM) with or without peptide for an appropriate time at 37 ° C. In some experiments, appropriate concentrations of GLP-1 were also included. A portion of the supernatant was removed and its insulin content was measured by SPA. Results were expressed as fold over control (FOC). At a concentration of 50 nM, a modified GLP-1 agonist having the amino acid sequence set forth in SEQ ID NO: 26 containing 43 kD PEG linked to the C-terminus increased insulin secretion approximately 3 fold from dispersed islet cells.

Measurement of elevated plasma insulin levels during IVGTT in fasted Wistar rats The increase in plasma insulin levels in this assay is at least about 2-fold. Preferably, the modified GLP-1 receptor agonists of the present invention reduce rat insulin secretion as measured by elevated plasma insulin levels during an in vivo glucose tolerance test in fasted Wistar rats as a control. Times to about 5 times, more preferably about 2 times to about 10 times, and even more preferably about 2 times to about 20 times (ie 2.0, 3.0, 4.0, 5.0). 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times).

  Male Wistar rats are fasted overnight and then anesthetized with isoflurane gas. Rats are supplemented with 0.4 g / kg glucose plus vehicle (0.9% saline + 1% albumin) or 1 nmol / kg GLP-1 (positive control) or 1 nmol / kg polypeptide of the invention. Give a tail vein injection. Rats are then bled 1 min later and plasma is assayed for insulin using an ELISA kit (Alpco Diagnostics, Windham, NH).

Effect of PEGylated peptides of the invention on intraperitoneal glucose tolerance in mice The reduction in blood glucose levels measured by this assay is a reduction of at least about 10%. Preferably, the modified GLP-1 receptor agonist of the present invention has a mouse blood glucose level of from about 10% to about 60%, as measured by an intraperitoneal glucose tolerance test in rats or mice as controls. Preferably it is reduced from about 10% to about 80%, and even more preferably from about 10% to about 100% (ie 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%).

  The in vivo activity of the modified peptide of the present invention (pegylated) when administered subcutaneously was investigated in mice. Mice fasted overnight were injected subcutaneously with control or modified peptide (100 μg / kg). Three hours later, basal blood glucose was measured and rats were given 2 g / kg glucose intraperitoneally. Blood glucose was measured again after 30 and 60 minutes.

  Representative modified peptides of the present invention significantly reduced blood glucose levels after IPGTT compared to vehicle (36% -54% reduction in glucose AUC “area under the curve”). This indicates that the modified peptide has prolonged glucose lowering activity in vivo. In addition to glucose-lowering activity, the modified peptides of the present invention also exhibit a prolonged peptide half-life in vivo. Unmodified GLP-1 has a very short half-life in vivo (<10 min UTES). The ability of the modified peptide of the present invention to lower blood glucose 3 hours after administration of the peptide reveals that the peptide is in circulation at this point and thus has a longer half-life compared to unmodified GLP-1. Shown in

Measurement of gastrointestinal motility in mice by subcutaneous injection The modified GLP-1 receptor agonists of the present invention have a therapeutic index of at least 5 times. Preferably, the modified GLP-1 receptor agonist of the present invention is about 5 to 10 times, more preferably about 5 to about 20 times, even more preferably about 5 to about 50 times, even more preferably 5 to about It has a therapeutic index of 100 times, and most preferably about 5 to 200 times. The therapeutic index is the minimum concentration of peptide (ie agonist) required to reduce bowel motility by at least 20% divided by the minimum glucose concentration required to reduce blood glucose AUC by at least 20%. Yes (see FIG. 6).

  Gastrointestinal motility in mice was tested using representative modified GLP-1 agonists of the present invention (GLP-1 agonist linked to 43 kD PEG, GLP-1 agonist linked to 30 kD PEG, 22 kD) GLP-1 agonist peptide linked to PEG and GLP-1 agonist linked to fatty acid). Gastrointestinal motility was measured as follows: Male Balb / c mice were first fasted overnight and peptide (3-100 μg / kg) depending on the time interval between dosing and motility measurements. Or the vehicle was either given by subcutaneous injection or they were first given the peptide or vehicle and then fasted overnight. At the appropriate time after dosing, mice were fed a charcoal diet by oral gavage and 5 minutes later were sacrificed by cervical dislocation. The small intestine was cut out and the length of the intestine was measured, as well as the distance the activated charcoal moved across the pyloric sphincter. The% migration was calculated by dividing the distance of activated charcoal moved by the total length of the small intestine and multiplying by 100.

In one example, GLP1 modified with a fatty acid (R ³⁴ -GLP-1 (7-37) containing K26 modified with γ-L-glutamoyl (Nα-hexadecanoyl)) was added 3 hours before charcoal diet. Gives to fasted mice. Fatty acid-GLP1 decreased gastrointestinal motility in a dose-dependent manner (FIG. 3A).

  In a second example, GLP-1 conjugated to 22 kD PEG was given to mice fasted overnight 3 hours prior to a charcoal diet. GLP1 linked to 22 kD PEG also reduced gastrointestinal motility in a dose-dependent manner (FIG. 3B).

  In a third example, GLP-1 conjugated to 43 kD PEG was given to mice fasted overnight 3 hours prior to a charcoal diet. GLP1 linked to 43 kD PEG showed no significant effect on gastrointestinal motility (FIG. 3B).

  In the fourth example, GLP-1 agonist linked to 22 kD PEG was given to mice fasted overnight 3 hours before charcoal diet. A GLP1 agonist linked to 22 kD PEG also reduced gastrointestinal motility in a dose-dependent manner (FIG. 4).

  In the fifth example, GLP-1 agonist linked to 30 kD PEG was given to mice fasted overnight 3 hours before charcoal diet. A GLP1 agonist linked to 30 kD PEG also reduced gastrointestinal motility in a dose-dependent manner (FIG. 4).

  In a sixth example, a representative modified GLP-1 agonist of the present invention (GLP-1 agonist linked to 43 kD PEG) was given to mice fasted overnight 3 hours prior to a charcoal diet. Representative modified GLP-1 agonists of the present invention did not show significant effects on gastrointestinal motility (FIG. 4).

Measurement of gastrointestinal motility in mice by ICV injection A stainless steel guide cannula aimed at the third ventricle of the brain was implanted into male Wistar rats (275-350 g) anesthetized with isoflurane. One 21G cannula was aimed at the third ventricle of the brain using a stereotaxic device and the following coordinates: -2.2 mm posterior from Bregma and -7.5 mm ventral from the dura mater. The cannula was secured to the skull with jewel screws and dental cement. One week after surgery, cannula placement was tested by infusion of 1 μl of 10 ng / μl concentration of Angiotensin II. Animals that drink more than 5 ml of water during the hour were left in the experiment. On the day of gastric exercise experiment, rats fasted overnight were placed C-terminal with vehicle (PBS), 0.5 μg / rat GLP-1 (7-36) amide peptide or 43 kDa PEG in the third ventricle. 20 μg / rat SEQ ID NO: 26 modified with the above were injected in a volume of 10 μl using an infusion pump (Harvard Apparatus). The peptide or vehicle was infused for 2 minutes and the needle was kept in place for an additional minute after the infusion. Five minutes after injection, rats received oral administration of 10% activated carbon, 5% gum arabic and 1% carboxymethylcellulose in a 0.8 ml volume. Five minutes after activated carbon, rats were sacrificed by CO ₂ inhalation and decapitation. The intestine was excised and the distance of activated charcoal that moved beyond the pyloric sphincter was measured. After the experiment, the correct placement of the cannula was confirmed by Evans Blue injection and brain incision.

  SEQ ID NO: 26 modified with 43 kDa PEG at 20 μg / rat produced a significant reduction in gastric motility. This effect was comparable to that produced by 0.5 μg / rat GLP-1 (7-36) amide peptide (FIG. 5). These data show that GLP-1 receptor agonists linked to PEG polymers with molecular weights greater than 30 kDa (ie, modified GLP-1 receptor agonists of the present invention) are different from many GLP-1 agonists in cerebral blood vessels. Indicates that it cannot pass through the barrier. As a result, the modified GLP-1 receptor agonists of the present invention can prevent the reduction in gastrointestinal motility typically associated with GLP-1 receptor agonists.

Pharmaceutical composition-IV and SC formulation Sterile IV injectable formulation is derived from a derivatized peptide equivalent to 4 mg peptide content (eg SEQ ID NO: 28 linked to 43 kDa PEG) and 1 liter sterile saline. Created using manufacturing methods well known in the art. SC formulations may require a higher concentration of derivatized polypeptide.

  All publications and patents mentioned in the above specification are herein incorporated 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 invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. Those skilled in the art will recognize, and be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

It represents the amino acid sequence of the polypeptide identified in SEQ ID NOs: 1 to 10, which is an example of the polypeptide of the present invention, GLP-1 receptor agonist. 2 represents the amino acid sequences of the polypeptides of SEQ ID NOs: 13 to 14 and 16 to 31, which are examples of the modified polypeptide of the present invention and the modified GLP-1 agonist. FIG. 3A is a line graph showing that GLP-1 modified with fatty acids reduced gastrointestinal motility. 3B is a line graph showing that GLP-1 modified with 22 kD PEG reduced gastrointestinal motility, whereas GLP-1 modified with 43 kD PEG did not reduce gastrointestinal motility. A GLP-1 agonist of the present invention (SEQ ID NO: 26) modified with 22 kD or 30 kD PEG reduced gastrointestinal motility, whereas the same GLP-1 agonist peptide modified with 43 kD PEG reduced gastrointestinal motility It is a line graph which shows not having been made. 4 is a bar graph showing that a GLP-1 agonist of the present invention (SEQ ID NO: 26) modified with 43 kD PEG reduces gastrointestinal motility when injected with ICV.

[Sequence Listing]

Claims (26)

  A polypeptide selected from the group consisting of SEQ ID NOs: 1, 2, 4, 10, 13, 14, and 16-31 and functionally equivalent fragments, derivatives and variants thereof.   The polypeptide of claim 1, wherein the polypeptide is linked to a polyethylene glycol polymer.   The polypeptide of claim 2, wherein the polyethylene glycol has a molecular weight of at least 30 kD.   The polypeptide according to claim 3, wherein the polyethylene glycol is branched.   A polynucleotide encoding the polypeptide sequence of claim 1, or a degenerate variant thereof.   A vector comprising the polynucleotide according to claim 5.   A host cell comprising the vector of claim 6. a) culturing the host cell of claim 7 under conditions suitable for expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
A method for producing a polypeptide comprising:   A method for reducing or inhibiting gastrointestinal side effects of a GLP-1 receptor agonist, comprising a step of linking a polyethylene glycol polymer to a GLP-1 receptor agonist.   The method of claim 9, wherein the polyethylene glycol has a molecular weight of at least 30 kD.   The method according to claim 10, wherein the polyethylene glycol is branched.   10. The GLP-1 receptor agonist according to claim 9, selected from the group consisting of SEQ ID NOs: 1, 2, 4, 10, 13, 14, and 16-31 and functionally equivalent fragments, derivatives and variants thereof. The method described.   A pharmaceutical comprising a therapeutically effective amount of a polypeptide of any one of claims 1 to 4 or functionally equivalent fragments, derivatives and variants thereof in combination with a pharmaceutically acceptable carrier. Composition.   A therapeutically effective amount of a polypeptide of any one of claims 1 to 4 or functionally equivalent fragments, derivatives and variants thereof in combination with a pharmaceutically acceptable carrier and one or more agents. A pharmaceutical composition comprising:   The above drugs are PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α-glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose output lowering compounds, insulin, anti-obesity drugs, HMG CoA reductase inhibitors, nicotinic acid 15. The pharmaceutical composition according to claim 14, selected from the group consisting of a bile acid inhibitor, a fibric acid derivative and an antihypertensive agent.   A composition comprising an effective amount of a polypeptide of any one of claims 1 to 4 or functionally equivalent fragments, derivatives and variants thereof in combination with an inert carrier.   A method of treating diabetes in an individual comprising administering a therapeutically effective amount of a polypeptide of any one of claims 1 to 4 to the individual in need of treatment.   18. The method of claim 17, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, juvenile adult-onset diabetes, adult potential autoimmune diabetes, gestational diabetes, and syndrome X.   18. The method of claim 17, wherein the polypeptide is administered in combination with one or more agents.   The drug is selected from the group consisting of PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α-glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose output lowering compounds, insulin and anti-obesity drugs, The method of claim 19.   18. The method of claim 17, wherein the polypeptide is administered in combination with one or more agents selected from the group consisting of HMG CoA reductase inhibitors, nicotinic acid, bile acid inhibitors, fibric acid derivatives and antihypertensive agents. .   24. The method of any one of claims 19 to 21, wherein the polypeptide and one or more agents are administered as a single pharmaceutical formulation.   The polypeptide according to any one of claims 1 to 4, for the treatment and / or prevention of diabetes.   A medicament comprising at least one polypeptide according to any one of claims 1 to 4 in combination with at least one pharmaceutically acceptable pharmaceutically safe carrier or excipient.   Use of the polypeptide according to claim 1 for the manufacture of a medicament for the treatment and / or prevention of diabetes.   25. A medicament according to claim 24 for the treatment and / or prevention of diabetes.

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