RU2247575C2 - Method for inhibiting glucagon - Google Patents

Abstract

FIELD: medicine, endocrinology, pharmacy.

SUBSTANCE: invention relates to a pharmaceutical compound used for preparing a medicinal agent for reducing plasma blood glucagon level in a patient. Compound is taken among a group consisting of eczendin, its analog, modified eczendin and modified eczendin analog wherein indicated compound involves at least 31 modified amino acids occurring in the nature. Invention provides reducing level of glucagons and/or inhibition of glucagons in both insulin-dependent (type 1) and non-insulin-dependent (type 2) of diabetes mellitus.

EFFECT: improved inhibiting method, valuable medicinal properties of agent.

15 cl, 27 dwg

Description

This application claims the priority of provisional patent application US 60/116380, entitled "New preparations of exendin agonists and methods for their administration", filed January 14, 1999 (and the corresponding PCT application filed January 14, 2000, serial number [not yet assigned]), US Provisional Patent Application 60/132017, entitled “Methods for Suppressing Glucagon,” filed April 30, 1999, and US Provisional Patent Application 60 / [number not yet assigned], entitled “Use of Exendins and Its Agonists to Modulate Trigly Levels” of itscerides and treatment of dyslipidemia ”, filed January 10, 2000: their contents are fully included here for information in the form of bibliographic references.

FIELD OF THE INVENTION

The present invention relates to methods for suppressing and / or reducing glucagon levels in a subject, comprising administering an ekendin, an exendin agonist, or a modified ekendin or an exendin agonist, in which an exendin peptide or an exendin agonist is coupled to one or more polyethylene glycol polymers or another compound used to reduce renal clearance source peptide. Such methods are applicable, for example, for the treatment of hyperglucagonemia and other conditions in which a decrease in glucagon levels or suppression of glucagon secretion is beneficial.

Background

The following description contains information that can be used to understand the present invention. This is not a recognition that any of the information presented here is a prototype for the invention claimed here, as well as any of the publications that are directly or allegedly mentioned in this text, is a prototype for the invention.

Exendins are peptides that are found in the salivary secretions of the gillier (Gilean tooth tooth) and escortion (Mexican tooth tooth) - lizards that live in Arizona and northern Mexico. Exendin-3 [SEQ ID NO 1: His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Ser-NH ₂ ] is present in the salivary secretions of Heloderma horridum (escortion, Mexican puffer), and exendin-4 [SEQ ID NO 2: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe lle Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Ser-NH ₂ ] is present in the salivary secretions of the Heloderma suspectum (vest, Gil's tooth tooth) (J. Eng et al., 1990, J. Biol. Chem., 265, 20259 -20262; J. Eng et al., 1992, J. Biol. Chem., 267, 7402-7405). The amino acid sequence of exendin-3 is shown in figure 1. The amino acid sequence of exendin-4 is shown in figure 2. It was initially believed that exendin-4 is a (potentially toxic) component of the poison. Now it seems that exendin-4 is not associated with toxicity, but in fact is formed in the salivary glands of the Ghillian tooth tooth.

Exendins exhibit a certain degree of similarity with some members of the glucagon-like peptide family with the highest homology level — 53% — with the GLP-1 peptide [7-36] NH ₂ [SEQ ID NO 3] (Goke et al., 1993, J. Biol Chem., 268, 19650-19655). GLP-1 [7-36] HN ₂ , also sometimes referred to as proglucagon [78-107] or simply as “GLP-1”, which is most often used in this text, has an insulinotropic effect that stimulates the secretion of insulin by pancreatic beta cells ; GLP-1 has also been reported to suppress the secretion of glucagon by pancreatic alpha cells (Orsov et al., 1993, Diabetes, 42, 658-661; D'Alessio et al., 1996, J. Clin. Invest., 97, 133-138). GLP-1 has been reported to suppress gastric emptying (B.Willms et al., 1996, K. Clin. Endocrinol. Metab. 81 (1), 327-332; A.Wettergren et al., 1993, Dig. Dis. Sci., 38 (4), 665-673) and gastric acid secretion (BTSchjoldager et al., 1989, Dig. Dis. Sci., 34 (5), 703-708; DJO'Halloran et al., 1990, J Endocrinol., 126 (1), 169-173; A.Wettergren et al., 1993, Dig. Dis. Sci., 38 (4), 665-673). GLP-1 [7-37], which includes an additional glycine residue at its C-terminus, has been reported to stimulate insulin secretion in humans (Orskov et al., 1993, Diabetes, 42, 658-661). The transmembrane adenylate cyclase-linked G-protein receptor, considered to be responsible for at least part of the insulinotropic action of GLP-1, was cloned from the beta cell line (Thorens, 1992, Proc. Natl. Acad. Sci. USA, 89, 8641-8645) . GLP-1 has been the focus of intensive research in recent years due to a report on its effect on the stimulation of stimulated insulin production (MMByrne, B. Goke, "Lessons from human studies with glucagon-like peptide-1: Potential of the gut hormone for clinical use" , In: HCFehmann, B. Goke, "Insulinotropic Gut Hormone Glukagon-Like Peptide 1", Basel, Switzerland, Karger, 1997, pp. 219-233).

Other reports relate to suppression of gastric emptying. Shortened GLP-1 (proglucagon 78-107 amide) inhibits the functions of the stomach and pancreas in humans (A.Wettergren et al. 1993, Dig. Dis. Sci., 38 (4), 665-673), inhibits the secretion of glucagon ( WOC Creutzfeldt et al., 1996, "Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide 1 (7-36) amide in type I diabetic patients". Diabetes Care, 19 (6), 580-586) and presumably participates in appetite control (MDTurton et al., 1996, "A role for glucagon-like peptide-1 in the central regulation of feeding". Nature, 379 (6560), 69-72).

It has also been reported that GLP-1 restores the sensitivity of islets of Langerhans to glucose in aging rats by increasing their glucose tolerance to levels characteristic of young rats (JMEgan et al., 1997, "Glucagon-like peptide-1 restores acute-phase insulin release to aged rats ", Diabetologia, 40 (suppl. 1), A130). However, the short duration of the biological effect of GLP-1 in vivo is a property of the peptide that makes it difficult to use as a medicine. Various methods have been tried to increase the half-life of GLP-1 or GLP-1 (7-37), including attempts to change their amino acid sequence and deliver them using different forms of drugs (see, for example, the European patent application entitled “Prolonged delivery of peptides) ”, Filed by Darley et al., Publication Number 0-619322-A2, regarding the incorporation of polyethylene glycol in formulations containing GLP-1 (7-37)).

Pharmacological studies on the material of dispersed acinous cells of the guinea pig pancreas and parietal cells of the stomach have led to reports that exendin-4 may act on GLP-1 receptors on some insulin-secreting cells; it has also been reported that the peptide stimulates somatostatin secretion and inhibits gastrin secretion in isolated stomachs (Goke et al., 1993, J. Biol. Chem., 268, 19650-19655; Schepp et al., 1994, Eur. J. Pharmacol ., 69, 183-191; Eissele et al., 1994, Life Sci., 55, 629-634). It was found that exendin-3 and exendin-4 stimulate the production of cAMP in the acinous cells of the pancreas and their secretion of amylase (R. Malhotra et al., 1992, Regulatory Peptides, 41, 149-156; Raufman et al., 1992, J Biol. Chem., 267, 21432-21437; Singh et al., 1994, Regul. Pept., 53, 47-59). In addition, exendin-4 has a significantly longer duration compared to GLP-1. For example, in one experiment, the decrease in glucose levels by the action of exendin-4 in diabetes mice was reported to continue for several hours, and up to 24 hours depending on the dose (J. Eng, 1996, "Prolonged effect of exendin-4 on hyperglycemia of db / db mice. "Diabetes, 45 (suppl. 2), 152A (abstract 554)). Given their insulinotropic activity, the use of exendin-3 and exendin-4 was suggested for the treatment of diabetes mellitus and the prevention of hyperglycemia (Eng, US patent 5424286).

The results of a study of whether exendins are mammalian GLP-1 homologs are published by Chen & Drucker, who cloned the Gilesian toothooth exendin gene (J. Biol. Chem., 272 (7), 4108-4115 (1997)). The discovery of the fact that the Ghillian toothfish also has independent proglucagon genes (from which GLP-1 is formed), which are more similar to mammalian proglucagon than exendin, indicates that exendins are not the only GLP-1 homologs in these species.

To date, funds that serve to delay gastric emptying, in general, find their place in medicine as diagnostic tools for x-ray studies of the gastrointestinal tract. For example, glucagon is a polypeptide hormone that is produced by the alpha cells of the pancreatic islets of Langerhans. It is a hyperglycemic agent that mobilizes glucose by activating glycogenolysis in the liver. It is less able to stimulate the secretion of pancreatic insulin. Glucagon is used in the treatment of insulin-induced hypoglycemia, for example, when intravenous glucose is not possible. However, since glucagon weakens the motility of the gastrointestinal tract, it is also used as a diagnostic tool for X-ray studies of the gastrointestinal tract. Glucagon has also been used in several studies on the treatment of various diseases of the gastrointestinal tract, characterized by severe pain caused by cramping. It has been reported (Daniel et al., 1974, Br. Med. J., 3, 720) that the symptoms of acute diverticulitis are more likely to lessen in patients treated with glucagon compared with patients treated with analgesics or antispasmodics. A review (Glauser et al., 1979, J. Am. Coll. Emergency Physicians, 8, 228) described the weakening of acute food blockage of the esophagus after treatment with glucagon. In another study, glucagon significantly reduced pain and soreness in 21 patients with bile duct disease compared with 22 patients given a placebo (M.J. Power et al., 1982, Br. J. Surg., 69, 591-592).

Methods for regulating gastrointestinal motility using amylin agonists are described in International Patent Application No. PCT / US 94/10225, published March 16, 1995.

Methods for regulating gastrointestinal motility using exendin agonists are described in US patent application Ser. No. 08/908867, filed August 8, 1997, entitled “Methods for regulating gastrointestinal motility,” the application being partly extending the patent US Application Serial No. 08/694954, filed August 8, 1996.

Methods for reducing food intake using exendin agonists are described in US Patent Application Serial No. 09/003869, filed January 7, 1998, entitled “Use of Exendin and Its Agonists to Reduce Food Consumption,” which claims the priority of U.S. provisional patent applications. No. 60/034905, filed January 7, 1997, 60/055404, filed August 7, 1997, 60/065442, filed November 14, 1997, and 60/066029, filed November 14, 1997

New exendin agonist compounds are described in PCT Application Serial No. PCT / US 98/16387, filed August 6, 1998, entitled “New Exendin Agonist Compounds,” which claims the priority of US patent application Serial No. 60/055404, filed August 8, 1997

Other new exendin agonists are described in PCT Application Serial No. PCT / US 98/24210, filed November 13, 1998, entitled “New Exendin Agonist Compounds,” which claims the priority of provisional patent application US No. 60/065442, filed November 14, 1997

The following new eekendin agonists are described in PCT Application Serial No. PCT / US 98/24273, filed November 13, 1998, entitled “New Exendin Agonist Compounds,” which claims the priority of provisional patent application US No. 60/066029, filed November 14, 1997

Other recent advances in exendin-based technology are described in US Provisional Patent Application Serial No. 60/075122, filed February 13, 1998, entitled “Inotropic and Diuretic Effects of Exendin and GLP-1,” and in US Provisional Patent Application Serial No. 60/116380, filed January 14, 1998, entitled “New Exendin Agonist Drugs and Methods of Their Administration”.

Modification of therapeutic peptides and proteins with polyethylene glycol (PEG) can lead to both advantages and disadvantages. While the PEG modification can lead to improved circulation time, decreased antigenicity and immunogenicity, improved solubility, resistance to proteolytic cleavage, increased bioavailability, reduced toxicity, improved stability and easier preparation of such peptides (see Francis et al. , 1998, International Journal of Hematology, 68, 1-18), in most cases, the problem accompanying “PEG” is a significant decrease in biological activity (cit. Ibid.). In addition, in most methods, linkers are used that are characterized by various types of adverse side effects, including immunogenicity, instability, toxicity and reactivity (cit. Ibid.).

Glucagonoma (a tumor of cells secreting glucagon) in addition to glucose intolerance causes a specific skin condition - necrolytic migratory erythema. This is manifested in the appearance of a peeling red rash, sometimes the formation of blisters and, possibly, scabs located on the face, abdomen, limbs and perineum. It can also be associated with inflammation of the tongue and mouth, damage to the nails and thinning of the hair. It has been reported that this condition responds to octreotide, an analogue of glucagonostatic hormone. The compounds described herein are also useful as glucagonostatic agents and, accordingly, in the treatment of this disease, which was first described in 1966 (LKKaplan, "Endocrine Tumors of the Gastrointestinal Tract and Pancreas", Ch. 262, p. 1392, In "Harrison's Principles of Internal Medicine", 12th edition, McGraw-Hill Inc., New York, 1991). Compounds described herein as useful for lowering glucagon levels and / or suppressing glucagon secretion include exendin, exendin agonists and modified exendins and exendin agonists, as well as related drugs and dosage forms.

The contents of the above-mentioned articles, patents and patent applications and all other documents referred to or cited in this text, in their entirety are included here for information in the form of bibliographic references. Applicants retain the right to physically include in this application any and all material and information from any such articles, patents, patent applications or other documents referred to or cited in this text.

Summary of invention

The present invention relates to methods for reducing glucagon levels and / or suppressing glucagon secretion in a subject. It also relates to the treatment of hyperglucagonemia and conditions in which administration of glucagonostatic agents is beneficial, including, but not limited to, necrolytic migratory erythema.

Thus, in one aspect, the invention relates to the use of an exendin, an exendin agonist or a modified exendin or an exendin agonist, in which an exendin or an exendin agonist is coupled to one or more polyethylene glycol polymers or other molecules that increase molecular weight in order to reduce glucagon levels in a subject.

In another aspect, the invention relates to the use of an exendin, an exendin agonist or a modified exendin or an exendin agonist in which an exendin or an exendin agonist is coupled to one or more polyethylene glycol polymers or other compounds used to reduce the renal clearance of the parent peptide in order to suppress the secretion of glucagon in a subject.

In yet another aspect, the invention relates to the use of an exendin, an exendin agonist, or a modified exendin or an exendin agonist in which an exendin or an exendin agonist is coupled to one or more polyethylene glycol polymers or other molecular weight enhancing molecules for treating conditions associated with hyperglucagonemia.

In a further aspect, the invention relates to the use of an exendin, an exendin agonist or a modified exendin or an exendin agonist in which an exendin or an exendin agonist is coupled to one or more polyethylene glycol polymers or other molecular weight enhancing molecules to treat a subject with glucagon or necrolytic migratory erythema.

In preferred embodiments, the exendin is exendin-4. In other preferred embodiments, the modified exendin or exendin agonist has a molecular weight that is greater than the molecular weight of the exendin or exendin agonist (preferably 10%, 50% or 90% more), the modified exendin or exendin agonist has a negative charge that is greater than the negative charge of exendin or an exendin agonist (preferably 10%, 50% or 90% more), a modified exendin or an exendin agonist is characterized by lower renal clearance compared to an exendi renal clearance on either an exendin agonist (preferably 10%, 50% or 90% less), the half-life of the modified exendin or exendin agonist is longer than the half-life of an exendin or exendin agonist (preferably 10%, 50% or 90% more), immunogenicity / antigenicity of the modified exendin or exendin agonist is less than the immunogenicity / antigenicity of exendin or exendin agonist, the solubility of the modified exendin or exendin agonist is greater than the solubility of the exendin or exendin agonist (preferably 10%, 50 more % or 90%), the rate of proteolytic cleavage of the modified exendin or exendin agonist is less than the proteolysis rate of the exendin or exendin agonist (preferably less than 10%, 50% or 90%), the toxicity of the modified exendin or exendin agonist is less than the toxicity of the exendin or exendin agonist, the stability of the modified an exendin or exendin agonist is superior to the stability of an exendin or exendin agonist and the permeability / biological functionality of the modified exendin or exendin agonist bo less or less than the permeability / biological functionality of the exendin or exendin agonist (preferably 10%, 50% or 90% more or less).

An exendin or exendin agonist may be combined with one, two, or three polyethylene glycol polymers. Polyethylene glycol polymers can preferably have a molecular weight in the range of 500-20000. In a preferred embodiment, the modified exendin or exendin agonist is one of compounds 201-217, more preferably one of compounds 209, 210 and 213 or one of compounds 201 and 202, or one of compounds 216 and 217 (see example 4 below).

Polyethylene glycol polymers are preferably attached to an amino group, carboxyl group or thio group and can be attached at the N- or C-terminus to the side chains of lysine, aspartic acid, glutamic acid or cysteine, or, alternatively, polyethylene glycol polymers can be attached using diamine and dicarboxyl groups. Preferably, the exendin or exendin agonist is coupled to polyethylene glycol polymers via the ε-amino group of the lysine amino acid in the exendin or exendin agonist.

By “exendin agonist” is meant a compound that mimics the action of exendins, for example, on intestinal motility and gastric emptying (namely, a compound that effectively binds to a receptor with respect to which exendins themselves are active, affecting intestinal motility and gastric emptying, preferably derivative or analog of exendin) or a compound that, for example, mimics the effect of exendin on reducing food intake by binding to a receptor or receptors, through which exendin mediates its action. Preferred exendin agonist compounds are those described in US Serial Application Serial No. 90/003869, entitled “Use of Exendin and Its Agonists to Reduce Food Consumption,” filed January 7, 1998 (and in priority applications), rights property, which belongs to the applicants of this application and which is included in this application in full as a bibliographic reference. The effects of exendins and exendin agonists can be detected, evaluated, or screened using the methods described herein or other methods known in the art in connection with establishing the effects of exendin.

In another aspect, a therapeutically effective amount of an amylin agonist is also administered to a subject. In a preferred aspect, the amylin agonist is an amylin analog or an analog of an amylin agonist, such as ^25,28,29 human proamylin (also known as “pramlintide”, previously referred to as “AC-137” and described in US patent No. 5686511 “Peptide agonists amylin and their use ”, issued November 11, 1997) or salmon calcitonin.

Preferably, the subject is a vertebrate, more preferably a mammal, and most preferably a human. In preferred aspects, an exendin, an exendin agonist, or a modified exendin or an exendin agonist of the present invention is administered parenterally, more preferably by injection. In a most preferred aspect, the injection is peripheral injection. Preferably, from about 1-30 μg to about 5 mg of the modified exendin or exendin agonist of the present invention is administered per day. More preferably, from about 1-30 μg to about 2 mg or from about 1-30 μg to about 1 mg of the modified exendin or exendin agonist of the present invention is administered per day. Most preferably, from about 3 μg to about 500 μg of the modified exendin or exendin agonist of the present invention is administered per day.

Preferred exendins or exendin agonists for modification and use are:

exendin-4 (1-30) [SEQ ID NO 4: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly];

exendin-4 (1-30) -amide [SEQ ID NO 5: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe He Glu Trp Leu Lys Asn Gly Gly-NH ₂ ] ;

exendin-4 (1-28) -amide [SEQ ID NO 6: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH ₂ ];

¹⁴ Leu, ²⁵ Phe-exendin-4-amide [SEQ ID NO 7: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe lle Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH ₂ ];

¹⁴ Leu, ²⁵ Phe-exendin-4- (l-28) -amide, [SEQ ID NO 8: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe lle Glu Phe Leu Lys Asn-NH ₂ ]; and

¹⁴ Leu, ²² Ala, ²⁵ Phe-exendin-4- (1-28) -amide [SEQ ID NO 9: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Ala Ile Glu Phe Leu Lys Asn-NH ₂ ].

Definitions

In accordance with the present invention and for use in this text, the following terms are defined as having the following meanings, unless specifically stated otherwise.

The term “amino acid” means natural amino acids, non-native amino acids and analogs of amino acids and all their D and L stereoisomers, if their structure allows such stereoisomeric forms to exist. Natural amino acids are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trr), tyrosine (Tur) and Valine Native amino acids include, but are not limited to, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2 -aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butyl glycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagin, homoproline, hydroxylysis in, allohydroxylisine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvalin, naphthylalanine, norvaline, norleucine, tyrnilpinin, pinnacillin, tyrnilpinin, tyrnilpinin, tyrnilpinin . Analogues of amino acids are natural and non-native amino acids that are chemically, reversibly or irreversibly blocked or modified by their N-terminal amino group or by groups of their side chains, such as methionine sulfoxide, methionine sulfone, S- (carboxymethyl) -cysteine, S- ( carboxymethyl) cysteinesulfoxide; and S- (carboxymethyl) cysteinesulfone.

The term “amino acid analogue” means an amino acid in which a C-terminal carboxyl group, an N-terminal amino group or a side chain functional group has been chemically modified to another functional group. For example, aspartic acid β-methyl ester is an amino acid analogue of aspartic acid; N-ethyl glycine is an analog of the amino acid glycine or alanine carboxamide is an analog of the amino acid alanine.

The term “amino acid residue” means radicals having the structure: (1) —C (O) —R — NH—, where R is usually —CH (R ^′ ) -, where R ′ is an amino acid side chain, typically H or carbon Deputy; or 2)

where p is 1, 2 or 3, which represents residues of azetidine carboxylic acid, proline or pipecolic acid, respectively.

The term “lower” as used herein with respect to organic radicals, such as alkyl groups, defines those groups that include up to and including 6 carbon atoms, preferably up to and including 4 and preferably 1 or two carbon atoms. Such groups may be a straight chain or branched chain.

A “pharmaceutically acceptable salt” encompasses salts of the compounds of the present invention derived from a combination of such compounds with an organic or inorganic acid. In practice, using a salt form is equivalent to using a base form. The compounds of the present invention are applicable both in the form of a base and in the form of a salt, while both such forms are considered to be within the scope of the present invention.

In addition, these abbreviations denote the following:

“ACN” or “CH ₃ CN” refers to acetonitrile.

“Boc”, “tBoc” or “Tboc” means tert-butoxycarbonyl.

“DCC” means N, N'-dicyclohexylcarbodiimide.

“Fmoc” means fluorenylmethoxycarbonyl.

“HBTU” means 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate.

“HOBt” means 1-hydroxybenzotriazole monohydrate.

“HomoP” or “hPro” means homoproline.

“MeAla” or “Nme” means N-methylalanine.

“Naph” means naphthylalanine.

“PG” or “pGly” refers to pentyl glycine.

“TBuG” means tert-butyl glycine.

“ThioP” or “tPro” means thioproline.

“3Nur” means 3-hydroxyproline.

“4Nur” means 4-hydroxyproline.

“NAG” means N-alkyl glycine.

“NAPG” means N-alkylpentylglycine.

“Norval” means norvaline.

“Norleu” means norleucine.

Other features and advantages of the invention will be apparent from the following description of its preferred embodiments, as well as from the claims.

Brief Description of the Drawings

The figure 1 shows the amino acid sequence of exendin-3 [SEQ ID NO 1].

The figure 2 shows the amino acid sequence of exendin-4 [SEQ ID NO 2].

The figure 3 shows the amino acid sequences of some exendin agonist compounds useful in the present invention [SEQ ID NO 10-40].

The figure 4 shows the amino acid sequences of some compounds of the present invention - compounds 1-174.

Figure 5 is a graph showing the effect of functional nephrectomy on clearance of exendin-4.

Figure 6 is a graph depicting the final breakdown of the plasma levels of exendin-4 in nephrectomized and simulated subjects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for suppressing and / or reducing glucagon levels in a subject, comprising administering an exendin, an exendin agonist or a modified exendin or an exendin agonist, in which an exendin peptide or an exendin agonist is coupled to one or more polyethylene glycol polymers or other compounds used to increase molecular weight . Such methods are applicable, for example, for the treatment of hyperglucagonemia and other conditions in which a decrease in glucagon levels or suppression of glucagon secretion will benefit. Such conditions include, but are not limited to, glucagonoma and necrolytic migratory erythema.

Modified Exendins and Exendin Agonists

The modified exendins and exendin agonists of the present invention include, for example, one or more PEG polymers attached to an exendin or an exendin agonist, such as a naturally occurring exendin, a synthetic exendin, or an exendin agonist.

Exendin-4

Exendin-4 is a naturally-occurring peptide isolated from the salivary secretions of the Gil's Toothpaste. An analysis of the effect of exendin-4 on animals showed that its ability to lower blood glucose levels persists for several hours. Exendin-4, which is a polypeptide consisting of 39 amino acids, is synthesized using solid-phase synthesis as described here and, as shown, such a synthetic material is identical to native exendin-4.

As described in this text, the non-clinical pharmacology of exendin-4 was studied. In the brain, exendin-4 binds specifically to the areas of the area postrema and nucleus tractus solitarius of the hindbrain and the subcutaneous region of the forebrain. Exendin-4 binding has been detected in the brain and kidneys of rats and mice. The structures with which exendin-4 binds in the kidneys have not been established.

In various experiments, the biological activity of exendin-4 and GLP-1 was compared, and they show the existence of a more preferred spectrum of properties for exendin-4. A single subcutaneous administration of exendin-4 reduced plasma glucose in mice db / db (with diabetes) and ob / ob (with diabetes and obesity) by 40%. In diabetic and obese rats, Diabetic Fatty Zucker (ZDF) administration of exendin-4 for 5 weeks resulted in a reduction of HbA _1c (a measure of glycosylated hemoglobin used to estimate plasma glucose levels) by 41%. Insulin susceptibility also improved by 76% after a 5-week treatment of obese ZDF rats. In primates with glucose intolerance, a dose-dependent decrease in plasma glucose was also detected.

The insulinotropic effect of exendin-4 was also found in rodents with an improvement in the insulin response to glucose by more than 100% in non-starving Harlan Sprague Dawley (HSD) rats and up to about 10 times in non-starving db / db mice. Higher glucose levels before treatment were associated with more pronounced effects on lowering glucose levels. Thus, the revealed effect on the decrease in glucose level in exendin-4, apparently, is dependent on glucose and is minimally expressed if the animal is already characterized by euglycemia.

Exendin-4 in a dose-dependent manner slowed the gastric emptying in rats of HSD and was approximately 90 times stronger in this action compared to GLP-1. It was also shown that exendin-4 reduces food intake in NIH / Sw (Swiss) mice after peripheral administration and is at least 1000 times stronger in this regard than GLP-1. Exendin-4 reduced plasma glucagon concentrations by approximately 40% in ZDF rats under anesthesia in the state of hyperinsulin / hyperglycemic fixation, but did not affect the plasma glucagon concentration in the euglycemic state in normal rats. As shown, exendin-4 in a dose-dependent manner reduces body weight in obese ZDF rats, while in ZDF rats without obesity, the detected decrease in body weight appears to be temporary.

Considering the effects of decreasing glucagon levels and suppressing the secretion of glucagon, exendins, exendin agonists and modified exendins or exendin-4 exendin agonists will be used, for example, for people who may benefit from lowering glucagon levels, such as patients with glucagon and necrolytic erythema migrans, as well as patients with diabetes, regardless of whether or not they retained the ability to secrete insulin: see example 5.

The toxicology of exendin-4 was studied with a single administration to mice, rats and monkeys, with repeated injections (up to 28 consecutive daily doses) to rats and monkeys and in vitro tests for mutagenicity and chromosome aberration. To date, there have been no cases of death and no changes related to the introduction of hematology, clinical chemistry and large or microscopic changes in tissues have been found. It was shown that exendin-4 is not a mutagen and does not induce chromosomal aberrations at the studied concentrations (up to 5000 μg / ml).

A series of immunological tests have been developed to support studies of non-clinical pharmacokinetics and metabolism of exendin-4. A radioimmunoassay test with limited sensitivity (approximately 100 pM) was used in the initial pharmacokinetic studies. After that, the two-site IRMA test for exendin-4 was tested with a lower limit of quantitative detection - 15 pcM. According to radioimmunological analysis, the bioavailability of subcutaneous exendin-4 was determined at a level of approximately 50-80%. This is similar to what was established with intraperitoneal administration (48-60%). Peak plasma concentrations (C _max ) were observed between the 30th and 43rd minutes (T _max ). The values of both C _max and AUC were directly dose dependent. The observed final half-life for subcutaneous exendin-4 was approximately 90-110 minutes. This is significantly more than those 14-41 minutes that are detected after intravenous administration. Similar results were obtained using the IRMA test. An analysis of the destruction of exendin-4 compared to GLP-1 shows that exendin-4 is relatively resistant to destruction.

Exendin agonists

The relationship between the structure and activity (SAR analysis) of exendin was studied in connection with structures that may be associated with the antidiabetic activity of exendin, with its resistance to metabolic processes and with the improvement of its physical parameters, especially in connection with peptide stability and availability for alternative delivery systems As a result, various compounds of exendin agonist peptides have been invented. Exendin agonists include peptide analogs of exendin, in which one or more native amino acids are deleted or replaced with another amino acid (s). Preferred exendin agonists are exendin-4 agonist analogues. Particularly preferred exendin agonists are those described in PCT application Serial PCT / US98 / 16387, filed August 6, 1998, entitled "New Exendin Agonist Compounds," which claims the priority of US Patent Application Serial No. 60 / 055404, filed August 8, 1997; PCT Application Serial No. PCT / US 98/24210, filed November 13, 1998, entitled "New Exendin Agonist Compounds," which claims to prioritize US provisional patent application No. 60/065442, filed November 14, 1997; and PCT Application Serial No. PCT / US 98/24273, filed November 13, 1998, entitled “New Exendin Agonist Compounds,” which claims priority to provisional US patent application No. 60/066029, filed November 14, 1997. , all of them in their entirety, including any drawings, are included here for information in the form of bibliographic references.

The activity of exendin agonists can be determined, for example, by activity in the tests described hereinafter. The effect of exendins or exendin agonists on gastric motility and gastric emptying can be detected, evaluated or screened using the methods described in this application or other methods known in the art or equivalent methods for determining gastric motility. Negative receptor tests or screening systems for exendin agonist compounds or putative exendin agonist compounds, such as an amylin receptor screening test using an amylin receptor preparation as described in US Pat. No. 5,264,372 issued November 23, 1993, contents which is included here for information in the form of a bibliographic reference, screening tests with one or more calcitonin receptors using, for example, T47D and MCF7 breast carcinoma cells that carry calcium etseptory dependent stimulation of adenylate cyclase activity, and / or a screening test with CGRP receptor using, for example, SK-N-MC cells.

One such method for use in identifying or evaluating the ability of a compound to attenuate gastric motility includes: (a) contacting the test sample and the test system, where the test sample includes one or more test compounds, and the test system includes a system for evaluating gastric motility, moreover, the system is characterized in that it exhibits, for example, increased plasma glucose in response to the introduction of glucose or food into the system; and (b) determining the presence or degree of increase in plasma glucose in the system. Positive and / or negative controls may also be included.

Also included within the scope of the present invention are pharmaceutically acceptable salts of the compounds of formula (I-VIII) and pharmaceutical compositions containing said compounds and their salts.

Formula I

Exendin agonist compounds also include those described in provisional patent application US No. 60/065442, including compounds of formula (I) (SEQ ID NO 41):

Xaa ₁ Xaa ₂ Haa ₃ Gly Xaa ₅ Haa ₆ Haa ₇ Xaa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Haa ₂₃ Haa ₂₄ Xaa ₂₅ Xaa ₂₆ Xaa ₂₇ Xaa ₂₈ -Z ₁ ; Where

Xaa ₁ is His, Arg or Tur;

Xaa ₂ represents Ser, Gly, Ala or Thr;

Xaa ₃ represents Asp or Glu;

Xaa ₅ is Ala or Thr;

Xaa ₆ is Ala, Phe, Tur or naphthylalanine;

Xaa ₇ represents Thr or Ser;

Xaa ₈ represents Ala, Ser or Thr;

Xaa ₉ represents Asp or Glu;

Haa ₁₀ represents Ala, Leu, Ile, Val, pentyl glycine or Met;

Xaa ₁₁ represents Ala or Ser;

Xaa ₁₂ is Ala or Lys;

Xaa ₁₃ is Ala or Gln;

Xaa ₁₄ represents Ala, Leu, Ile, pentyl glycine, Val or Met;

Xaa ₁₅ is Ala or Glu;

Xaa ₁₆ represents Ala or Glu;

Xaa ₁₇ represents Ala or Glu;

Xaa ₁₉ represents Ala or Val;

Haa ₂₀ represents Ala or Arg;

Xaa ₂₁ represents Ala or Leu;

Xaa ₂₂ represents Ala, Phe, Tur or naphthylalanine;

Haa ₂₃ represents Ile, Val, Leu, pentyl glycine, tert-butyl glycine or Met;

Haa ₂₄ represents Ala, Glu or Asp;

Haa ₂₅ represents Ala, Trp, Phe, Tur or naphthylalanine;

Haa ₂₆ represents Ala or Leu;

Haa ₂₇ represents Ala or Lys;

Haa ₂₈ represents Ala or Asn;

Z ₁ represents —OH,

-NH ₂ ,

Gly-Z ₂ ,

Gly Gly-Z ₂ ,

Gly Gly Xaa ₃₁ -Z ₂ ,

Gly Gly Haa ₃₁ Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ , or

Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ;

Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ independently represent Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and

Z ₂ represents —OH or —NH ₂ ;

providing that no more than three of Xaa _3, Xaa _5, Xaa _6, Xaa _8, Xaa _10, Xaa _11, Xaa _12, Xaa _13, Xaa _14, Xaa _15, Xaa _16, Xaa _17, Xaa _19, Xaa _20, Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa ₂₈ are Ala. Preferred N-alkyl groups for N-alkyl glycine, N-alkyl pentyl glycine and N-alkylalanine are lower alkyl groups, preferably from 1 to about 6 carbon atoms, more preferably from 1 to 4 carbon atoms.

Preferred exendin agonist compounds are those in which Xaa ₁ is His or Tyr. More preferably, Xaa ₁ is His.

Preferred are those compounds in which Xaa ₂ represents Gly.

Preferred are those compounds in which Xaa ₁₄ is Leu, pentyl glycine or Met.

Preferred are those compounds in which Xaa ₂₅ are Trp or Phe.

Preferred are those compounds in which Xaa ₆ is Phe or naphthylalanine; Xaa ₂₂ represents Phe or naphthylalanine and Haa ₂₃ represents Ilé or Val.

Preferred are those compounds in which Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from Pro, homoproline, thioproline and N-alkylalanine.

Preferably Z ₁ is —NH ₂ .

Preferably Z ₂ is —NH ₂ .

In accordance with one aspect, preferred are compounds of formula (I) wherein Xaa ₁ is His or Tyr, more preferably His; Xaa ₂ represents Gly; Haa ₆ represents Phe or naphthylalanine; Xaa ₁₄ represents Leu, pentyl glycine or Met; Haa ₂₂ represents Phe or naphthylalanine; Haa ₂₃ represents Ilé or Val; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from Pro, homoproline, thioproline or N-alkylalanine. More preferably, Z ₁ is —NH ₂ .

In a particularly preferred aspect, particularly preferred compounds are those compounds of formula (I) in which Xaa ₁ is His or Arg; Haa ₂ represents Gly or Ala; Xaa ₃ represents Asp or Glu; Xaa ₅ is Ala or Thr; Xaa ₆ is Ala, Phe or naphthylalanine; Xaa ₇ represents Thr or Ser; Xaa ₈ is Ala, Ser or Thr; Xaa ₉ represents Asp or Glu; Haa ₁₀ represents Ala, Leu or pentyl glycine; Xaa ₁₁ is Ala or Ser; Xaa ₁₂ is Ala or Lys; Xaa ₁₃ is Ala or Gln; Xaa ₁₄ represents Ala, Leu or pentyl glycine; Xaa ₁₅ is Ala or Glu; Xaa ₁₆ represents Ala or Glu; Xaa ₁₇ represents Ala or Glu; Xaa ₁₉ represents Ala or Val; Xaa ₂₀ represents Ala or Arg; Xaa ₂₁ represents Ala or Leu; Haa ₂₂ represents Phe or naphthylalanine; Haa ₂₃ represents Ilé, Val or tert-butyl glycine; Haa ₂₄ represents Ala, Glu or Asp; Xaa ₂₅ represents Ala, Trp or Phe; Haa ₂₆ represents Ala or Leu; Xaa ₂₇ represents Ala or Lys; Xaa ₂₈ represents Ala or Asn; Z ₁ is -OH, -NH _2, Gly-Z _2, Gly Gly-Z _2, Gly Gly Xaa ₃₁ -Z _2, Gly Gly Xaa ₃₁ Ser-Z _2, Gly Gly Xaa ₃₁ Ser Ser-Z _2, Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Xaa ₃₈ -Z ₂ ; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ independently represent Pro, homoproline, thioproline or N-methylalanine; and Z ₂ represents —OH or —NH ₂ ; providing that no more than three of Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa ₂₈ are Ala. Particularly preferred compounds are those compounds that are listed in PCT Patent Application Serial No. PCT / US 98/24210, filed November 13, 1998, entitled “New Exendin Agonist Compounds”, identified therein as compounds 2-23.

According to a particularly preferred aspect, compounds are provided in which Xaa ₁₄ is Leu, Ile, Val or pentyl glycine, more preferably Leu or pentyl glycine, and Xaa ₂₅ is Phe, Tur or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will be less sensitive to oxidative degradation both in vitro and in vivo, as well as during the synthesis of the compound.

Formula II

Exendin agonist compounds also include those described in provisional patent application US No. 60/066029, including compounds of formula (II) [SEQ ID NO 42]:

Xaa ₁ Xaa ₂ Haa ₃ Haa ₄ Xaa ₅ Xaa ₆ Haa ₇ Haa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Haa ₂₃ Xaa ₂₄ Xaa ₂₅ Haa ₂₆ Haa ₂₇ Xaa ₂₈ -Z ₁ ; Where

Xaa ₁ represents His, Arg, Tur, Ala, Norval, Val or Norleu;

Xaa ₂ represents Ser, Gly, Ala or Thr;

Xaa ₃ represents Ala, Asp or Glu;

Xaa ₄ represents Ala, Norval, Val, Norleu or Gly;

Xaa ₅ is Ala or Thr;

Xaa ₆ is Phe, Tur or naphthylalanine;

Haa ₇ represents Thr or Ser;

Xaa ₈ represents Ala, Ser or Thr;

Xaa ₉ represents Ala, Norval, Val, Norleu, Asp or Glu;

Haa ₁₀ represents Ala, Leu, Ile, Val, pentyl glycine or Met;

Xaa ₁₁ is Ala or Ser;

Xaa ₁₂ is Ala or Lys;

Xaa ₁₃ is Ala or Gln;

Haa ₁₄ represents Ala, Leu, lle, pentyl glycine, Val or Met;

Xaa ₁₅ is Ala or Glu;

Xaa ₁₆ represents Ala or Glu;

Xaa ₁₇ represents Ala or Glu;

Xaa ₁₉ represents Ala or Val;

Xaa ₂₀ represents Ala or Arg;

Xaa ₂₁ represents Ala or Leu;

Xaa ₂₂ represents Phe, Tur or naphthylalanine;

Haa ₂₃ represents Ile, Val, Leu, pentyl glycine, tert-butyl glycine or Met;

Haa ₂₄ represents Ala, Glu or Asp;

Xaa ₂₅ represents Ala, Trp, Phe, Tug or naphthylalanine;

Haa ₂₆ represents Ala or Leu;

Haa ₂₇ represents Ala or Lys;

Haa ₂₈ represents Ala or Asn;

Z ₁ represents —OH,

-NH ₂ ,

Gly-Z ₂ ,

Gly Gly-Z ₂ ,

Gly Gly Xaa ₃₁ -Z ₂ ,

Gly Gly Xaa ₃₁ Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Xaa ₃₈ -Z ₂ ; or

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ Haa ₃₉ -Z ₂ ;

Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ independently represent Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and

Z ₂ represents —OH or —NH ₂ ;

providing that no more than three of Haa ₃ , Haa ₄ , Xaa ₅ , Xaa ₆ , Haa ₈ , Haa ₉ , Haa ₁₀ , Haa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Haa ₂₆ , Haa ₂₇ and Haa ₂₈ represent Ala; and providing also that, if Xaa ₁ is His, Arg or Tyr, then at least one of Xaa _3, Xaa ₄ and Xaa ₉ is Ala.

Preferred N-alkyl groups for N-alkyl glycine, N-alkyl pentyl glycine and N-alkylalanine are lower alkyl groups, preferably from 1 to about 6 carbon atoms, more preferably from 1 to 4 carbon atoms. Suitable compounds of formula (II) are those described in Serial Application No. PCT / US98 / 24273, filed November 13, 1998, entitled “New Exendin Agonist Compounds” identified therein in Examples 1-89 (“compounds 1-89 ”, respectively), as well as those compounds that correspond to the compounds identified in it in examples 104 and 105.

Preferred exendin agonist compounds are those in which Xaa ₁ is His, Ala or Norval. More preferably, Xaa ₁ is His or Ala. Most preferably, Xaa ₁ is His.

Preferred are those compounds of formula (II) in which Xaa ₂ is Gly.

Preferred are those compounds of formula (II) in which Xaa ₃ is Ala.

Preferred are those compounds of formula (II) in which Xaa ₄ is Ala.

Preferred are those compounds of formula (II) in which Xaa ₉ is Ala.

Preferred are those compounds of formula (II) in which Xaa ₁₄ is Leu, pentyl glycine or Met.

Preferred compounds of formula (II) are those in which Xaa ₂₅ is Trp or Phe.

Preferred compounds of formula (II) are those in which Xaa ₆ is Ala, Phe or naphthylalanine; Xaa ₂₂ is Phe or naphthylalanine; and Haa ₂₃ represents Ilé or Val.

Preferred are compounds of formula (II) in which Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from Pro, homoproline, thioproline and N-alkylalanine.

Preferably Z ₁ is —NH ₂ .

Preferably Zz is —NH ₂ .

In one aspect, compounds of formula (II) are preferred in which Xaa ₁ is Ala, His or Tur, more preferably Ala or His; Haa ₂ represents Ala or Gly; Haa ₆ represents Phe or naphthylalanine; Xaa ₁₄ represents Ala, Leu, pentyl glycine or Met; Xaa ₂₂ is Phe or naphthylalanine; Haa ₂₃ represents Ilé or Val; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from Pro, homoproline, thioproline and N-alkylalanine; and Haa ₃₉ is Ser or Tur, more preferably Ser. More preferably, Z ₁ is –NH ₂ .

In accordance with a particularly preferred aspect, particularly preferred compounds are those compounds of formula (II) in which: Xaa ₁ is His or Ala; Haa ₂ represents Gly or Ala; Xaa ₃ represents Ala, Asp or Glu; Haa ₄ represents Ala or Gly; Xaa ₅ is Ala or Thr; Haa ₆ represents Phe or naphthylalanine; Haa ₇ represents Thr or Ser; Xaa ₈ represents Ala, Ser or Thr; Xaa ₉ represents Ala, Asp or Glu; Haa ₁₀ represents Ala, Leu or pentyl glycine; Xaa ₁₁ represents Ala or Ser; Xaa ₁₂ is Ala or Lys; Xaa ₁₃ is Ala or Gln; Xaa ₁₄ represents Ala, Leu, Met or pentyl glycine; Xaa ₁₅ is Ala or Glu; Haa ₁₆ represents Ala or Glu; Xaa ₁₇ represents Ala or Glu; Xaa ₁₉ represents Ala or Val; Xaa ₂₀ represents Ala or Arg; Xaa ₂₁ represents Ala or Leu; Haa ₂₂ represents Phe or naphthylalanine; Haa ₂₃ represents Ile, Val or tert-butyl glycine; Haa ₂₄ represents Ala, Glu or Asp; Xaa ₂₅ represents Ala, Trp or Phe; Haa ₂₆ represents Ala or Leu; Xaa ₂₇ represents Ala or Lys; Xaa ₂₈ represents Ala or Asn; Z ₁ is -OH, -NH _2, Gly-Z _2, Gly Gly-Z _2, Gly Gly Xaa ₃₁ -Z _2, Gly Gly Xaa ₃₁ Ser-Z _2, Gly Gly Xaa ₃₁ Ser Ser-Z _2, Gly Gly Haa ₃₁ Ser Ser Gly-Z ₂ , Gly Gly Haa ₃₁ Ser Ser Gly Ala-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z _2, or Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ Xaa ₃₉ -Z _2; Haa ₃₁ , Haa ₃₆ , Haa ₃₇ and Haa ₃₈ independently represent Pro, homoproline, thioproline or N-methylalanine; and Z ₂ represents —OH or —NH ₂ ; providing that no more than three of Xaa _3, Xaa _5, Xaa _6, Xaa _8, Xaa _10, Xaa _11, Xaa _12, Xaa _13, Xaa _14, Xaa _15, Xaa _16, Xaa _17, Xaa _19, Xaa _20, Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa _{28 are} represented by Ala; and providing also that, if Xaa ₁ is His, Arg or Tyr, then at least one of Xaa _3, Xaa ₄ and Xaa ₉ is Ala. Particularly preferred compounds of formula (II) are those described in serial application No. PCT / US 98/24273, filed November 13, 1998, entitled “New exendin agonist compounds as characterized by the amino acid sequences SEQ ID NO 5 shown therein. -93.

According to a particularly preferred aspect, there are provided compounds of formula (II) in which Xaa ₁₄ represents Ala, Leu, Ile, Val or pentyl glycine, more preferably Leu or pentyl glycine, and Xaa ₂₅ represents Ala, Phe, Tur or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will be less sensitive to oxidative degradation both in vitro and in vivo, as well as during the synthesis of the compound.

Formula III

Also within the scope of the present invention are narrower classes of compounds including peptides of various lengths, for example classes of compounds that do not include peptides having 28, 29 or 30 amino acid residues in length, respectively. In addition, the present invention encompasses narrower classes of compounds described in PCT Application Serial No. PCT / US 98/24210, filed November 13, 1998, entitled “New Exendin Agonist Compounds,” and characterized by specific amino acid sequences, for example, compounds of the formula ( III) [SEQ ID NO 43]:

Xaa ₁ Xaa ₂ Haa ₃ Gly Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₈ Xaa ₁₉ Xaa ₂₀ Xaa ₂₁ Haa ₂₂ Haa ₂₃ Xaa ₂₄ Xaa ₂₅ Xaa ₂₆ Xaa ₂₇ Xaa ₂₈ -Z ₁ ;

Where

Xaa ₁ is His or Arg;

Xaa ₂ represents Gly or Ala;

Xaa ₃ represents Asp or Glu;

Xaa ₅ is Ala or Thr;

Xaa ₆ represents Ala, Phe or naphthylalanine;

Haa ₇ represents Thr or Ser;

Xaa ₈ is Ala, Ser or Thr;

Xaa ₉ represents Asp or Glu;

Haa ₁₀ represents Ala, Leu or pentyl glycine;

Xaa ₁₁ represents Ala or Ser;

Xaa ₁₂ is Ala or Lys;

Xaa ₁₃ is Ala or Gln;

Xaa ₁₄ represents Ala, Leu or pentyl glycine;

Xaa ₁₅ is Ala or Glu;

Haa ₁₆ represents Ala or Glu;

Xaa ₁₇ represents Ala or Glu;

Xaa ₁₉ represents Ala or Val;

Haa ₂₀ represents Ala or Arg;

Xaa ₂₁ represents Ala or Leu;

Haa ₂₂ represents Phe or naphthylalanine;

Haa ₂₃ represents Ilé, Val or tert-butyl glycine;

Haa ₂₄ represents Ala, Glu or Asp;

Haa ₂₅ represents Ala, Trg or Phe;

Haa ₂₆ represents Ala or Leu;

Haa ₂₇ represents Ala or Lys;

Haa ₂₈ represents Ala or Asn;

Z ₁ represents —OH,

-NH ₂ ,

Gly-Z ₂ ,

Gly Gly-Z ₂ ,

Gly Gly Xaa ₃₁ -Z ₂ ,

Gly Gly Haa ₃₁ Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ , or

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ;

Haa ₃₁ , Haa ₃₆ , Haa ₃₇ and Haa _{38 are} independently selected from the group consisting of Pro, homoproline, thioproline or N-methylalanine; and

Z ₂ represents —OH or —NH ₂ ;

providing that no more than three of Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa ₂₈ are Ala; and their pharmaceutically acceptable salts.

Formula IV

In addition, the present invention encompasses narrower classes of peptide compounds described in PCT Application Serial No. PCT / US 98/24273, filed November 13, 1998, entitled “New Exendin Agonist Compounds” as having specific amino acid sequences, for example, compounds of the formula (IV) [SEQ ID NO 44]:

Xaa ₁ Xaa ₂ Haa ₃ Haa ₄ Xaa ₅ Xaa ₆ Xaa ₇ Haa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₈ Xaa ₁₉ Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Haa ₂₃ Haa ₂₄ Haa ₂₅ Haa ₂₆ Haa ₂₇ Xaa ₂₈ -Z ₁ ; Where

Xaa ₁ is His or Ala;

Xaa ₂ represents Gly or Ala;

Xaa _h represents Ala, Asp or Glu;

Xaa ₄ represents Ala or Gly;

Haa ₅ represents Ala or Thr;

Haa ₆ represents Phe or naphthylalanine;

Haa ₇ represents Thr or Ser;

Xaa ₈ is Ala, Ser or Thr;

Xaa ₉ represents Ala, Asp or Glu;

Haa ₁₀ represents Ala, Leu or pentyl glycine;

Xaa ₁₁ represents Ala or Ser;

Xaa ₁₂ is Ala or Lys;

Xaa ₁₃ is Ala or Gln;

Xaa ₁₄ represents Ala, Leu, Met or pentyl glycine;

Xaa ₁₅ is Ala or Glu;

Xaa ₁₆ represents Ala or Glu;

Xaa ₁₇ represents Ala or Glu;

Xaa ₁₉ represents Ala or Val;

Haa ₂₀ represents Ala or Arg;

Xaa ₂₁ represents Ala or Leu;

Haa ₂₂ represents Phe or naphthylalanine;

Haa ₂₃ represents Ilé, Val or tert-butyl glycine;

Haa ₂₄ represents Ala, Glu or Asp;

Haa ₂₅ represents Ala, Trg or Phe;

Haa ₂₆ represents Ala or Leu;

Haa ₂₇ represents Ala or Lys;

Haa ₂₈ represents Ala or Asn;

Z ₁ represents —OH,

-NH ₂ ,

Gly-Z ₂ ,

Gly Gly-Z ₂ ,

Gly Gly Xaa ₃₁ -Z ₂ ,

Gly Gly Haa ₃₁ Ser-Z ₂ ,

Gly Gly Xaa ₃₁ Ser Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ;

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ Ser-Z ₂ ;

Haa ₃₁ , Haa ₃₆ , Haa ₃₇ and Haa ₃₈ independently represent Pro, homoproline, thioproline or N-methylalanine; and

Z ₂ represents —OH or —NH ₂ ;

providing that no more than three of Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Haa ₁₅ , Xav ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Haa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Haa ₂₆ , Haa ₂₇ and Haa ₂₈ represent Ala and providing that if Xaa ₁ represents His, Arg or Tur, then at least one of Haa ₃ , Haa ₄ and Haa ₉ represents Ala and their pharmaceutically acceptable salts.

Preferred compounds of formula (IV) are those in which Xaa ₁ is His, Ala, Norval or 4-imidazopropionyl. Preferably, Xaa ₁ is His or 4-imidazopropionyl, or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (IV) are those in which Xaa ₂ represents Gly.

Preferred compounds of formula (IV) are those compounds in which Xaa ₄ is Ala.

Preferred compounds of formula (IV) are those in which Xaa ₉ is Ala.

Preferred compounds of formula (IV) are those in which Xaa ₁₄ is Leu, pentyl glycine or Met.

Preferred compounds of formula (IV) are those in which Xaa ₂₅ is Trg or Phe.

Preferred compounds of formula (IV) are those in which Xaa ₆ is Ala, Phe or naphthylalanine; Haa ₂₂ represents Phe or naphthylalanine and Haa ₂₃ represents Ilé or Val.

Preferred compounds of formula (IV) are those in which Z ₁ is —NH ₂ .

Preferred compounds of formula (IV) are those in which Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (IV) are those in which Xaa ₃₉ is Ser or Tyr, preferably Ser.

Preferred compounds of formula (IV) are those in which Z ₂ is —NH ₂ .

Preferred compounds of formula (IV) are those 42 compounds in which Z ₁ is —NH ₂ .

Preferred compounds of formula (IV) are those in which Xaa ₂₁ is Lys-NH ⁶ R, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl.

Preferred compounds of formula (IV) are those in which X ₁ is Lys Asn, Lys-NH ⁶ R Asn or Lys-NH ⁶ -R Ala, where R is Lys, Arg, C _1-10 straight or branched alkanoyl chain. Preferred compounds of formula (IV) are those characterized by the amino acid sequence described in PCT Application Serial No. PCT / US 98/24273, filed November 13, 1998, entitled “New Exendin Agonist Compounds” as selected from SEQ ID NO 95-110 of this application.

Formula V

Also provided are compounds described in PCT application PCT / US 98/24210, filed November 13, 1998, entitled “New exendin agonist compounds”, including compounds of formula (V) [SEQ ID NO 45]:

5 10

Xaa ₁ Xaa ₂ Haa ₃ Gly Xaa ₅ Haa ₆ Haa ₇ Xaa ₈ Xaa ₉ Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Haa ₂₃ Haa ₂₄ Xaa ₂₅ Xaa ₂₆ X ₁ -Z ₁ ; Where

Xaa ₁ is His, Arg or Tur, or 4-imidazopropionyl;

Haa ₂ represents Ser, Gly, Ala or Thr;

Xaa ₃ represents Asp or Glu;

Haa ₅ represents Ala or Thr;

Haa ₆ represents Ala, Phe, Tug or naphthylalanine;

Haa ₇ represents Thr or Ser;

Xaa ₈ represents Ala / Ser or Thr;

Xaa ₉ represents Asp or Glu;

Haa ₁₀ represents Ala, Leu, Ile, Val, pentyl glycine or Met;

Xaa ₁₁ represents Ala or Ser;

Xaa ₁₂ is Ala or Lys;

Xaa ₁₃ is Ala or Gln;

Xaa ₁₄ represents Ala, Leu, Ile, pentyl glycine, Val or Met;

Xaa ₁₅ is Ala or Glu;

Haa ₁₆ represents Ala or Glu;

Xaa ₁₇ represents Ala or Glu;

Xaa ₁₉ represents Ala or Val;

Xaa ₂₀ represents Ala or Arg;

Xaa ₂₁ is Ala, Leu or Lys-NH ⁶ -R, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl or cycloalkylalkanoyl;

Haa ₂₂ represents Phe, Tur or naphthylalanine;

Haa ₂₃ represents Ilé, Val, Leu, pentyl glycine, tert-butyl glycine or Met;

Xaa ₂₄ represents Ala, Glu or Asp;

Xaa ₂₅ represents Ala, Trp, Phe, Tur or naphthylalanine;

Haa ₂₆ represents Ala or Leu;

X ₁ represents Lys Asn, Asn Lys, Lys-NH ⁶ -R Asn, Asn Lys-NH ⁶ -R, Lys-NH ⁶ -R Ala, Ala Lys-NH ⁶ -R, where R represents Lys, Arg, C _{1 -10} straight or branched alkanoyl or cycloalkylalkanoyl;

Z ₁ represents —OH,

-NH ₂ ,

Gly-Z ₂ ,

Gly Gly-Z ₂ ,

Gly Gly Xaa ₃₁ -Z ₂ ,

Gly Gly Haa ₃₁ Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ , or

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ;

Where

Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and

Z ₂ represents —OH or —NH ₂ ;

providing that no more than three of Xaa _3, Xaa _5, Xaa _6, Xaa _8, Xaa _10, Xaa _11, Xaa _12, Xaa _13, Xaa _14, Xaa _15, Xaa _16, Xaa _17, Xaa _19, Xaa _20, Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ and Xaa ₂₆ represent Ala. Also within the scope of the present invention are pharmaceutically acceptable salts of the compounds of formula (V) and pharmaceutical compositions containing said compounds and their salts.

Preferred exendin agonist compounds of formula (V) are those in which Xaa ₁ is His, Tyr or 4-imidazopropionyl. More preferably, Xaa ₁ is His.

Preferred are those compounds of formula (V) in which Xaa ₁ is 4-imidazopropionyl.

Preferred are those compounds of formula (V) in which Xaa ₂ is Gly.

Preferred compounds of formula (V) are those in which Xaa ₁₄ is Leu, pentyl glycine or Met.

Preferred compounds of formula (V) are those in which Xaa ₂₅ is Trg or Phe.

In accordance with one aspect, compounds of formula (V) are preferred in which Xaa ₆ is Phe or naphthylalanine; and Haa ₂₂ represents Phe or naphthylalanine; and Haa ₂₃ represents Ilé or Val. More preferably, Z ₁ is —NH ₂ . In accordance with one aspect, those compounds of formula (V) are particularly preferred wherein Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, thioproline and N-alkylalanine. More preferably, Z ₂ is —NH ₂ .

Preferred compounds of formula (V) are those in which X ₁ is Lys Asn, Lys-NHε-R Asn or Lys-NIε-R Ala, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl . Preferred compounds of formula (V) are those described in PCT Application Serial No. PCT / US98 / 24210, filed November 13, 1998, entitled “New Exendin Agonist Compounds”, and identified therein as compounds No. 62-69.

Such preferred exendin agonist compounds are those in which Xaa ₁ is His, Ala or Norval. More preferably, Xaa ₁ is His or Ala. Most preferably, Xaa ₁ is His.

Preferred are those compounds of formula (V) in which Xaa ₂ is Gly.

Preferred are those compounds of formula (V) in which Xaa ₃ is Ala.

Preferred are those compounds of formula (V) in which Xaa ₄ is Ala.

Preferred are those compounds of formula (V) in which Xaa ₉ is Ala.

Preferred are those compounds of formula (V) in which Xaa ₁₄ is Leu, pentyl glycine or Met.

Preferred compounds of formula (V) are those in which Xaa ₂₅ is Trg or Phe.

Preferred compounds of formula (V) are those in which Xaa ₆ is Ala, Phe or naphthylalanine; Haa ₂₂ represents Phe or naphthylalanine and Haa ₂₃ represents Ilé or Val.

Preferred are compounds of formula (V) in which Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from Pro, homoproline, thioproline and N-alkylalanine.

Preferably Z ₁ is —NH ₂ .

Preferably Z ₂ is —NH ₂ .

In accordance with one aspect, preferred are compounds of formula (V) wherein Xaa ₁ is Ala, His or Tur, more preferably Ala or His; Xaa ₂ represents Ala or Gly; Xaa ₆ is Phe or naphthylalanine; Xaa ₁₄ represents Ala, Leu, pentyl glycine or Met; Haa ₂₂ represents Phe or naphthylalanine; Haa ₂₃ represents Ilé or Val; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from Pro, homoproline, thioproline or N-alkylalanine; and Haa ₃₉ is Ser or Tur, more preferably Ser. More preferably, Z ₁ is —NH ₂ .

In a particularly preferred aspect, particularly preferred compounds are those compounds of formula (V) in which Xaa ₁ is His or Ala; Haa ₂ represents Gly or Ala; Xaa ₃ represents Ala, Asp or Glu; Xaa ₄ represents Ala or Gly; Xaa ₅ is Ala or Thr; Haa ₆ represents Phe or naphthylalanine; Haa ₇ represents Thr or Ser; Xaa ₈ is Ala, Ser or Thr; Xaa ₉ represents Ala, Asp or Glu; Haa ₁₀ represents Ala, Leu or pentyl glycine; Xaa ₁₁ represents Ala or Ser; Xaa ₁₂ is Ala or Lys; Xaa ₁₃ is Ala or Gln; Xaa ₁₄ represents Ala, Leu, Met or pentyl glycine; Xaa ₁₅ is Ala or Glu; Xaa ₁₆ represents Ala or Glu; Xaa ₁₇ represents Ala or Glu; Xaa ₁₉ represents Ala or Val; Xaa ₂₀ represents Ala or Arg; Xaa ₂₁ represents Ala or Leu; Xaa ₂₂ is Phe or naphthylalanine; Haa ₂₃ represents Ilé, Val or tert-butyl glycine; Haa ₂₄ represents Ala, Glu or Asp; Haa ₂₅ represents Ala, Trp or Phe; Haa ₂₆ represents Ala or Leu; Haa ₂₇ represents Ala or Lys; Haa ₂₈ represents Ala or Asn; Z ₁ is -OH, -NH _2, Gly-Z _2, Gly Gly-Z _2, Gly Gly Xaa ₃₁ -Z _2, Gly Gly Xaa ₃₁ Ser-Z _2, Gly Gly Xaa ₃₁ Ser Ser-Z _2, Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ -Z ₂ , Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Xaa ₃₇ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ or Gly Gly Xaa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ Xaa ₃₉ -Z ₂ ; Haa ₃₁ , Haa ₃₆ , Haa ₃₇ and Haa ₃₈ independently represent Pro, homoproline, thioproline or N-methylalanine; and Z ₂ represents —OH or —NH ₂ ; providing that no more than three of Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ , Xaa ₂₈ are Ala; and providing also that, if Xaa ₁ is His, Arg or Tyr, then at least one of Xaa _3, Xaa ₄ and Xaa ₉ is Ala. Particularly preferred compounds of formula (V) are those compounds that were described in PCT application serial No. PCT / US 98/24210, filed November 13, 1998, entitled "New exendin agonist compounds", and characterized by the amino acid sequences identified therein as SEQ ID NO 5-93.

According to a particularly preferred aspect, there are provided compounds of formula (V) in which Xaa ₁₄ is Ala, Leu, Ile, Val or pentyl glycine, more preferably Leu or pentyl glycine, and Xaa ₂₅ is Ala, Phe, Tur or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will be less sensitive to oxidative degradation both in vitro and in vivo, as well as during the synthesis of the compound.

Formula VI

Also provided are peptide compounds described in PCT Application Serial No. PCT / US 98/24273, filed November 13, 1998, entitled “New Exendin Agonist Compounds”, including compounds of formula (VI) [SEQ ID NO 46]:

5 10

Xaa ₁ Haa ₂ Haa ₃ Haa ₄ Haa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Haa ₂₃ Xaa ₂₄ Xaa ₂₅ Haa ₂₆ X ₁ -Z ₁ ; Where

Xaa ₁ represents His, Arg, Tur, Ala, Norval, Val, Norleu or 4-imidazopropionyl;

Haa ₂ represents Ser, Gly, Ala or Thr;

Xaa ₃ represents Ala, Asp or Glu;

Xaa ₄ represents Ala, Norval, Val, Norleu or Gly;

Xaa ₅ is Ala or Thr;

Haa ₆ represents Phe, Tur or naphthylalanine;

Haa ₇ represents Thr or Ser;

Xaa ₈ is Ala, Ser or Thr;

Xaa ₉ represents Ala, Norval, Val, Norleu, Asp or Glu;

Haa ₁₀ represents Ala, Leu, He, Val, pentyl glycine or Met;

Xaa ₁₁ is Ala or Ser;

Xaa ₁₂ is Ala or Lys;

Xaa ₁₃ is Ala or Gln;

Xaa ₁₄ represents Ala, Leu, Ile, pentyl glycine, Val or Met;

Xaa ₁₅ is Ala or Glu;

Xaa ₁₆ represents Ala or Glu;

Xaa ₁₇ represents Ala or Glu;

Xaa ₁₉ represents Ala or Val;

Haa ₂₀ represents Ala or Arg;

Xaa ₂₁ is Ala, Leu or Lys-NHε-R, where R is Lys, Agd, straight or branched chain C _1-10 alkanoyl or cycloallylalkanoyl;

Haa ₂₂ represents Phe, Tur or naphthylalanine;

Haa ₂₃ represents Ilé, Val, Leu, pentyl glycine, tert-butyl glycine or Met;

Haa ₂₄ represents Ala, Glu or Asp;

Haa ₂₅ represents Ala, Trg, Phe, Tur or naphthylalanine;

Haa ₂₆ represents Ala or Leu;

X ₁ represents Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, Lys-NHε-R Ala, Ala Lys-NHε-R, where R represents Lys, Arg, C _1-10 alkanoyl straight or branched chain or cycloalkylalkanoyl;

Z ₁ represents —OH,

-NH ₂ ,

Gly-Z ₂ ,

Gly Gly-Z ₂ ,

Gly Gly Xaa ₃₁ -Z ₂ ,

Gly Gly Haa ₃₁ Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala-Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ -Z ₂ ,

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ,

or

Gly Gly Haa ₃₁ Ser Ser Gly Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ Haa ₃₉ -Z ₂ ;

Where

Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and

Z ₂ represents —OH or —NH ₂ ;

providing that no more than three of Xaa _3, Xaa _4, Xaa _5, Xaa _6, Xaa _8, Xaa _9, Xaa _10, Xaa _11, Xaa _12, Xaa _13, Xaa _14, Xaa _15, Xaa _16, Xaa _17, Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ represent Ala; and providing also that, if Xaa ₁ is His, Arg, Tyr or 4-imidazopropionil, then at least one _of Xaa, Xaa ₄ and Haa ₉ is Ala.

Preferred compounds of formula (VI) are those in which Xaa ₁ is His, Ala, Norval or 4-imidazopropionyl. Preferably, Xaa ₁ is His or 4-imidazopropionyl, or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (VI) are those in which Xaa ₂ is Gly.

Preferred compounds of formula (VI) are those in which Xaa ₄ is Ala.

Preferred compounds of formula (VI) are those in which Xaa ₉ is Ala.

Preferred compounds of formula (VI) are those in which Xaa ₁₄ is Leu, pentyl glycine or Met.

Preferred compounds of formula (VI) are those in which Xaa ₂₅ is Trg or Phe.

Preferred compounds of formula (VI) are those in which Xaa ₆ is Ala, Phe or naphthylalanine; Haa ₂₂ represents Phe or naphthylalanine and Haa ₂₃ represents Ilé or Val.

Preferred compounds of formula (VI) are those in which Z ₁ represents —NH ₂ .

Preferred compounds of formula (VI) are those in which Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (VI) are those in which Xaa ₃₉ is Ser or Tyr, preferably Ser.

Preferred compounds of formula (VI) are those in which Z ₂ is —NH ₂ .

Preferred compounds of formula (VI) are those 42 compounds in which Z ₁ is —NH ₂ .

Preferred compounds of formula (VI) are those in which Xaa ₂₁ is Lys-NHε-R, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl.

Preferred compounds of formula (VI) are those in which X ₁ is Lys Asn, Lys-NHε-R Asn or Lys-NHε-R Ala, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl .

Preferred compounds of formula (VI) are those compounds that are described in PCT application serial No. PCT / US 98/24273, filed November 13, 1998, entitled "New exendin agonist compounds", as characterized by an amino acid sequence selected from those which identified in this application as SEQ ID NO 95-110.

Formula VII

Compounds specifically applicable in accordance with the present invention are exendin agonist compounds described in US Patent Application Serial No. 09/003869, filed January 7, 1998, entitled “Use of Exendins and Their Agonists to Reduce Food Consumption”, including compounds of the formula (VII) [SEQ ID NO 47]:

5 10

Xaa ₁ Haa ₂ Haa ₃ Gly Thr Xaa ₄ Xaa ₅ Haa ₆ Xaa ₇ Xaa ₈

15 20

Ser Lys Gin Haa ₉ Glu Glu Glu Ala Val Arg

25 30

Leu Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Leu Lys Asn Gly Gly

35

Xaa ₁₄ Ser Ser Gly Ala Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Xaa ₁₈ -Z,

where Xaa ₁ represents His, Arg or Tur; Xaa ₂ represents Ser, Gly, Ala or Thr; _Of Xaa represents Asp or Glu; Xaa ₄ represents Phe, Tug or naphthylalanine; Xaa ₅ is Thr or Ser; Xaa ₆ is Ser or Thr; Haa ₇ represents Asp or Glu; Xaa ₈ represents Leu, Ile, Val, pentyl glycine or Met; Haa ₉ represents Leu, Ile, pentyl glycine, Val or Met; Haa ₁₀ represents Phe, Tur or naphthylalanine; Xaa ₁₁ represents Ilé, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Xaa ₁₂ represents Glu or Asp; Xaa ₁₃ is Trg, Phe, Tur or naphthylalanine; Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ independently represent Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; Xaa ₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH ₂ , provided that the compound does not have the formula SEQ ID NO 1 or 2. Preferred N-alkyl groups for N-alkyl glycine, N-alkyl pentyl glycine and N-alkylalanine are lower alkyl groups, preferably from 1 to about 6 carbon atoms, more preferably 1-4 carbon atoms. Suitable compounds are those compounds that are characterized by the amino acid sequences of SEQ ID NO 10-40. Also pharmaceutically acceptable salts of the compounds of formula (VII) are useful in the present invention.

Preferred exendin agonist compounds are those in which Xaa ₁ is His or Tyr. More preferably, Xaa ₁ is His.

Preferred are those compounds in which Xaa ₂ represents Gly.

Preferred are those compounds in which Xaa ₉ is Leu, pentyl glycine or Met.

Preferred are those compounds in which Xaa ₁₃ is Trp or Phe.

Also preferred are compounds in which Xaa ₄ is Phe or naphthylalanine; Xaa ₁₁ is Ilé or Val, and Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa _{17 are} independently selected from Pro, homoproline, thioproline or N-alkylalanine. Preferably, N-akylalanine has an N-alkyl group comprising from 1 to about 6 carbon atoms.

In a particularly preferred aspect, Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ are the same amino acid residues.

Preferred are compounds in which Xaa ₁₈ is Ser or Tur, more preferably Ser.

Preferably Z is —NH ₂ .

In accordance with one aspect, compounds of formula (VII) are preferred in which Xaa ₁ is His or Tyr, more preferably His; Xaa ₂ represents Gly; Haa ₄ represents Phe or naphthylalanine; Haa ₉ represents Leu, pentylglycine or Met; Haa ₁₀ represents Phe or naphthylalanine; Haa ₁₁ represents Ilé or Val; Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa _{17 are} independently selected from Pro, homoproline, thioproline or N-alkylalanine; and Xaa ₁₈ is Ser or Tur, more preferably Ser. More preferably Z is —NH ₂ .

In a particularly preferred aspect, particularly preferred compounds are those compounds of formula (VII) in which Xaa ₁ is His or Arg; Haa ₂ represents Gly; Xaa ₃ represents Asp or Glu; Haa represents Phe or naphthylalanine; Xaa ₅ is Thr or Ser; Haa ₆ represents Ser or Thr; Haa ₇ represents Asp or Glu; Xaa ₈ represents Leu or pentyl glycine; Haa ₉ represents Leu or pentyl glycine; Haa ₁₀ represents Phe or naphthylalanine; Xaa ₁₁ is Ilé, Val or tert-butyl glycine; Xaa ₁₂ represents Glu or Asp; Xaa ₁₃ represents Trp or Phe; Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ independently represent Pro, homoproline, thioproline or N-methylalanine; Xaa ₁₈ is Ser or Tur and Z is —OH or —NH ₂ , provided that the compound does not have the formula SEQ ID NO 1 or 2. More preferably Z is —NH ₂ . Particularly preferred compounds are those compounds that have the amino acid sequence of SEQ ID NO 10, 11, 22, 23, 24, 27, 29, 36, 37, and 40.

According to a particularly preferred aspect, compounds are provided in which Xaa ₉ is Leu, Ile, Val or pentyl glycine, more preferably Leu or pentyl glycine, and Xaa ₁₃ is Phe, Tur or naphthylalanine, more preferably Phe or naphthylalanine. These compounds are believed to exhibit a preferred duration of action and will be less susceptible to oxidative degradation both in vitro and in vivo, as well as during synthesis of the compound.

Formula VIII

Also provided are compounds described in PCT Application Serial PCT / US 98/16387, filed August 6, 1998, entitled “New Exendin Agonist Compounds”, including compounds of formula (VIII) [SEQ ID NO 48]:

5 10

Xaa ₁ Xaa ₂ Haa ₃ Gly Thr Xaa ₄ Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈

15 20

Ser Lys Gln Xaa ₉ Glu Glu Glu Ala Val Arg

25 30

Leu Haa ₁₀ Haa ₁₁ Xaa ₁₂ Xaa ₁₃ Leu X ₁ Gly Gly Xaa ₁₄

35

Ser Ser Gly Ala Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Xaa ₁₈ -Z,

where Xaa ₁ represents His, Arg, Tyr or 4-imidazopropionyl; Haa ₂ represents Ser, Gly, Ala or Tur; Xaa ₃ represents Asp or Glu; Haa ₄ represents Phe, Tur or naphthylalanine; Xaa ₅ is Thr or Ser; Haa ₆ represents Ser or Thr; Haa ₇ represents Asp or Glu; Xaa ₈ represents Leu, Ile, Val, pentyl glycine or Met; Haa ₉ represents Leu, He, pentylglycine, Val or Met; Haa ₁₀ represents Phe, Tur or naphthylalanine; Xaa ₁₁ represents Ilé, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Xaa ₁₂ represents Glu or Asp; Xaa ₁₃ represents Trp, Phe, Tur or naphthylalanine; X ₁ is Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl or cycloalkylalkanoyl; Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ independently represent Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; Xaa ₁₈ is Ser, Thr or Tyr and Z is —OH or —NH ₂ ; provided that the compound does not have the formula SEQ ID NO 1 or 2. Suitable compounds of formula (VIII) are those described in PCT Application Serial No. PCT / US98 / 16387, filed August 6, 1998, entitled “New Agonist Compounds” exendin ”characterized by the amino acid sequences contained therein of SEQ ID NO 37-40.

Preferred exendin agonist compounds of formula (VIII) are those compounds in which Xaa ₁ is His, Tyr or 4-imidazopropionyl. More preferably, Xaa ₁ is His or 4-imidazopropionyl.

Preferred are those compounds of formula (VIII) in which Xaa ₂ is Gly.

Preferred are those compounds of formula (VIII) in which Xaa ₉ is Leu, pentyl glycine or Met.

Preferred are those compounds of formula (VIII) in which Xaa ₁₃ is Trp or Phe.

Preferred are those compounds of formula (VIII) in which X ₁ is Lys Asn or Lys-NHε-R Asn, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl.

Also preferred are compounds of formula (VIII) in which Xaa ₄ is Phe or naphthylalanine; Haa ₁₀ represents Phe or naphthylalanine; Xaa ₁₁ is Ilé or Val, and Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa _{17 are} independently selected from Pro, homoproline, thioproline or N-alkylalanine. In a particularly preferred aspect, Xaa ₁₈ is Ser or Tur. Preferred are those compounds in which Xaa ₁₈ is Ser. Preferably Z is —NH ₂ .

In accordance with one preferred aspect, preferred are compounds of formula (VIII) in which Xaa ₄ is Phe or naphthylalanine; Haa ₁₀ represents Phe or naphthylalanine; Haa ₁₁ represents Ilé or Val; X ₁ is Lys Asn or Lys-NHε-R Asn, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl, Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa _{17 are} independently selected from Pro, homoproline, thioproline or N-alkylalanine.

Obtaining modified exendins and exendin agonists

The modified exendins and exendin agonists of the present invention can be obtained by attaching one or more polyethylene glycol polymers to an exendin or an exendin agonist. The synthesis of exendins and exendin agonists is thus described first, followed by a methodology for the addition of polyethylene glycol polymer (s) to an exendin or exendin agonist.

Obtaining exendins and exendin agonists

Exendins and exendin agonist compounds, such as exendin analogs and exendin derivatives described herein, can be prepared by protein purification as described, for example, in Eng et al., 1990, J. Biol. Chem., 265, 20259-20262; and Eng et al., 1992, J. Biol. Chem., 267, 7402-7405, which is incorporated herein by reference in the form of bibliographic references. Alternatively, exendins and exendin agonist peptides can be obtained using approaches known to those skilled in the art, for example, as described by Raufman et al., 1992, J. Biol. Chem., 267, 21432-21437, which is incorporated herein by reference as a bibliographic reference based on standard solid-phase peptide synthesis methods and preferably using an automatic or semi-automatic peptide synthesizer. Compounds that are active components of the preparations and dosage forms of the present invention can be obtained using standard methods of solid-phase peptide synthesis and preferably using an automatic or semi-automatic peptide synthesizer. Typically, using such methods, an α-N-carbamoyl protected amino acid and an amino acid attached to a growing peptide chain on a polymer are combined at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride, in the presence of binding agents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, in the presence of a base such as diisopropylethylamine. The α-N-carbamoyl protecting group is removed from the resulting peptide-polymer bond using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction is repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, among which tert-butoxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) are preferred.

Solvents, amino acid derivatives, and the 4-methylbenzhydrylamine polymer used in the peptide synthesizer can be purchased from Applied Biosystems Inc. (Foster City, CA). The following side chain protected amino acids can be obtained from Applied Biosystems Inc .: BSD-112344.1 - Arg (Pmc), Boc-Thr (Bzl), Fmoc-Thr (t-Bu), Boc-Ser (Bzl), Fmoc- Ser (t-Bu), Boc-Tyr (BrZ), Fmoc-Tyr (t-Bu), Boc-Lys (Cl-Z), Fmoc-Lys (Boc), Boc-Glu (Bzl), Fmoc-Glu ( t-Bu), Fmoc-His (Trt), Fmoc-Asn (Trt) and Fmoc-GIn (Trt). Boc-His (BOM) is available from Applied Biosystems Inc. or from Bachem Inc. (Torrance, CA). Anisole, dimethyl sulfide, phenol, ethanedithiol and thioanisole can be obtained from Aldrich Chemical Co. (Milwaukee, WI). Air Products & Chemicals (Allentown, PA) supplies HF. Ethyl ether, acetic acid and methanol can be purchased from Fisher Scientific (Pittsburgh, PA).

Solid-phase peptide synthesis can be performed on an automatic peptide synthesizer (Model 430A: Applied Biosystems Inc., Foster City, CA) using the NMP / HOBt system (Option I) and tBOC and Fmoc chemistry (see the Applied Biosystems User Guide for ABI Peptide Synthesizer 430A, vers. 1.3B of July 1, 1988, section 6, pages 49-70: Applied Biosystems Inc., Foster City, CA) with blocking of active groups. The Boc peptide complexes with the polymer can be cleaved with HF (from -50 to 0 ° C for 1 hour). The peptide can be extracted from the polymer by changing water and acetic acid, followed by lyophilization of the filtrates. Complexes of the Fmoc peptide and polymer can be cleaved using standard methods (Introduction to Cleavage Techniques, Applied Biosystems Inc., 1990, pp. 6-12). Peptides can also be assembled using an Advanced Chem Tech synthesizer (Model MPS-350; Louisville, KE).

Peptides can be purified using RP-HPLC (preparative and analytical) using the Waters Delta Prep 3000 system. A preparative column with a C4, C8 or C18 sewn on (10 μm, 2.2 × 25 cm; Vydac, Hesperia, CA) can be used to isolate peptides, and purity can be determined using an analytical column C4, C8 or C18 (5 μm, 0.46 × 25 cm; Vydac). Solvents (A = 0.1% TFA / water and B = 0.1% TFA / CH ₃ CN) can be added to the analytical column at a flow rate of 1.0 ml / min and to the preparative column 15 ml / min. Amino acid analysis can be carried out in the Waters Pico Tag system with data processing in the Maxima program. Peptides can be hydrolyzed by hydrolysis in acid vapors (115 ° C, 20-24 hours). Hydrolysates can be derivatized and analyzed by standard methods (Cohen et al., 1998, "The Pico Tag Method: A Manual of Advanced Techniques for Amino Acid Analysis", pp. 11-52, Millipore Corporation, Milford, MA). Fast atom bombardment analysis can be performed using M-Scan Incorporated (West Chester, PA). Mass calibration can be performed using cesium iodide or cesium iodide / glycerol. Plasma desorption ionization analysis using flight time determination can be performed on an Applied Biosystems Bio-Ion 20 mass spectrometer. Electrospray mass spectroscopy can be performed on a VG-Trio device.

Peptide compounds useful in the preparations and dosage forms of the present invention as active components can also be obtained using recombinant DNA technology using methods currently known in the art: see, for example, Sambrook et al., 1989 , "Molecular Cloning: A Laboratory Manual," 2d ed., Cold Spring Harbor. Alternatively, such compounds can be prepared by peptide synthesis in a homogeneous phase. Non-peptide compounds useful in the present invention can be prepared using methods known in the art. For example, phosphate-containing amino acids and peptides including such amino acids can be prepared using methods known in the art: see, for example, Bartlett & Landen, 1986, Bioorg. Chem., 14, 356-377.

Addition of polyethylene glycol polymers

There are several strategies for attaching PEG to peptides / proteins: see Int. J. Hematology, 68, 1 (1998); Bioconjugate Chem., 6, 150 (1995) and Crit. Rev. Therap. Drug Carrier Sys., 9m 249 (1992): all of them are fully included here for information in the form of bibliographic references.

Therefore, a person skilled in the art will be able to apply such well-known methods to attach one or more polyethylene glycol polymers to the exendins and exendin agonists described in this text. Suitable polyethylene glycol polymers are usually commercially available or can be made using methods well known to those skilled in the art. Preferably, the polyethylene glycol polymers have molecular weights from 500 to 20,000 and may be straight or branched chain polymers.

The attachment of PEG to an intact peptide or protein can be carried out by combination with amino groups, carboxyl groups or thiol groups. Typically, such groups must be N- and C-terminal or located on the side chains of natural amino acids such as lysine, aspartic acid, glutamic acid and cysteine. Since exendin-4 and other exendins and exendin agonists can be obtained using solid-phase peptide chemistry methods, various components containing diamino groups and dicarboxyl groups with orthogonal protective groups can be introduced for PEG addition.

The present invention also provides the attachment of an exendin or an exendin agonist to one or more polymers other than polyethylene glycol, which are able to regulate renal clearance by a mechanism similar to that of polyethylene glycol. Examples of such polymers are albumin and gelatin: see Gombotz & Pettit, 1995, Bioconjugate Chem., 6, 332-351, which is fully incorporated herein by reference.

Application

The drugs and dosage forms described herein are used based on their pharmacological properties. In particular, the compounds described herein are active as agents for lowering glucagon levels and suppressing glucagon secretion, as evidenced by the ability to lower glucagon levels in animals and humans. They can be used to treat conditions or diseases in which relief can be achieved by lowering glucagon levels and suppressing glucagon secretion.

The compounds listed above can form salts by reacting with various inorganic and organic acids and bases. Such salts are salts obtained with organic and inorganic acids, for example, HCl, HBr, H ₂ SO ₄ , H ₃ PO ₄ , trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, toluene sulfonic acid, maleic acid, fumaric acid and camphorsulfonic acid. Salts obtained with the help of bases are ammonium salts, alkali metal salts, for example sodium and potassium salts, and alkaline earth metal salts, for example calcium and magnesium salts. Acetate, hydrochloride and trifluoroacetate salts are preferred. Salts can be prepared by standard methods by reacting a free acid or base in which the product is reacted with 1 or more equivalents of a suitable base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is then removed in vacuo. or by freeze-drying, or by replacing the ions of an existing salt with another ion on a suitable ion-exchange resin.

Preparation of compositions and administration

The preparations (compositions) and dosage forms of the modified exendin or exendin agonist of the present invention are applicable in terms of their exendin-like effects and can be prepared in a standard manner in the form of preparations suitable for parenteral (including intravenous, intramuscular and subcutaneous) administration. Also provided in this application are formulations and dosage forms suitable for alternative delivery routes, including oral, nasal, buccal, sublingual and pulmonary administration.

The feasibility of alternative exendin-4 delivery routes has been tested by measuring circulating blood levels of exendin-4 in parallel with monitoring a biological response, such as a decrease in plasma glucose levels after administration in animals with diabetes. The penetration of exendin-4 through several surfaces was studied - in the respiratory tract (the path through the nasal cavity, trachea and lungs) and the digestive system (sublingual, gastric and intraduodenal). The biological effect and appearance of exendin-4 in the blood was observed with each method of administration through the respiratory tract and with the introduction of the peptide into the digestive tract sublingually and through the stomach. All routes of administration have been described intratracheal administration, nasal administration, administration through the intestines and administration under the tongue.

In some cases, a suitable approach is to present the modified exendin or exendin agonist and other anti-glucagon agent, such as amylin or amylin agonist, in a common composition or solution for co-administration. In other cases, administration of another antiglucagon agent separately from an exendin, an exendin agonist, and a modified exendin or an exendin agonist may be of greater advantage. In some other cases, it may be beneficial to present an exendin, an exendin agonist, or a modified exendin or an exendin agonist either in the general composition or separately from other glucagon-lowering agents such as amylin. A suitable administration schedule can best be determined by the practitioner for each patient individually. Suitable pharmaceutically acceptable carriers and their use in the preparation of preparations are described in standard pharmaceutical courses, for example, "Remington's Pharmaceuticals Sciences", E.W. Martin; also see Y. J. Wang & M. A. Hanson, "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers", Journal of Parenteral Science and Technology, Technical Report No. 10, suppl. 42: 2S (1988).

Compounds useful in the present invention may be presented as parenteral compositions for injection or infusion. They can, for example, be suspended in an inert oil, for example a vegetable oil such as sesame, peanut, olive oil, or another suitable vehicle. Preferably, they can be suspended in an aqueous carrier, for example in an isotonic buffer solution, at a pH of from about 5.6 to 7.4. These compositions may be sterilized using standard sterilization methods or may be sterilized by filtration. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH-controlling buffering agents. Suitable buffers are, for example, sodium acetate / acetic acid buffers. A “depot” form with slow secretion of the drug can be used so that therapeutically effective amounts of the drug enter the bloodstream for many hours or days after transdermal injection or delivery.

The desired isotonicity can be achieved using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, polyalcohols (such as mannitol and sorbitol) or other inorganic or organic dissolved substances. Sodium chloride is preferred, in particular for buffers containing sodium ions.

The inventive compounds can also be included in the preparation in the form of pharmaceutically acceptable salts (for example, acid addition salts) and / or their complexes. Pharmaceutically acceptable salts are salts that are non-toxic at the concentrations at which they are administered. The preparation of such salts may facilitate pharmacological use by changing the physicochemical properties of the composition without losing the expected physiological effect of the composition from the composition. Examples of changes in physical properties used are lowering the melting point to facilitate administration through the mucous membranes and increasing solubility to facilitate the administration of higher concentrations of the drug.

Pharmaceutically acceptable salts are acid addition salts, such as those that include sulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, ρ-toluenesulfonate, cyclohexyl sulfamate and hinate. Pharmaceutically acceptable salts can be prepared with acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, ρ- toluenesulfonic acid, cyclohexylsulfamic acid and quinic acid. Such salts can be obtained, for example, by reacting a free acid or a base in which the product is reacted with 1 or more equivalents of a suitable base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is then removed in a vacuum, or by freeze-drying, or by replacing the ions of an existing salt with another ion on a suitable ion-exchange resin.

Carriers or excipients may also be used to facilitate administration of the compound. Examples of carriers and excipients are calcium carbonate, calcium phosphate, various sugars such as lactose, glucose or sucrose, or varieties of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. The compositions or pharmaceutical composition can be administered in various ways, including intravenous, intraperitoneal, subcutaneous and intramuscular, oral, local route or through the mucosa.

If desired, solutions of the above compositions may be densified with a densifying agent such as methyl cellulose. They can be prepared in the form of an emulsion, both oil-water and oil-in-water emulsions. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be used, including, for example, acacia powder, a nonionic surfactant (such as Tween), or an ionic surfactant (such as alkaline sulfates or polyester alcohol sulfonates, for example, Triton).

Compositions useful in the present invention are prepared by mixing the components in accordance with generally accepted procedures. For example, the selected compounds can be conventionally mixed in a mixer or other standard device to obtain a concentrated mixture, which can then be adjusted to a final concentration and viscosity by adding water or a densifying agent and, possibly, a pH control buffer or additional solute to control tonicity .

For use by a physician, the compounds should be presented in unit dosage form containing an amount of exendin, an exendin agonist, or a modified exendin or an exendin agonist with or without other antiglucagon agent. Therapeutically effective amounts of an exendin, an exendin agonist, or a modified exendin or an exendin agonist for use in the control of glucagon and conditions in which a decrease or regulation of glucagon levels are advantageous are those amounts that desirably lower blood glucagon levels after ingestion. In patients with diabetes or glucose intolerance, plasma glucagon levels may be higher than in healthy people. In such patients, an advantageous reduction or “smoothing” of blood glucagon levels after a meal can be achieved. As will be clear to those skilled in the art, the effective amount of the drug will vary depending on many factors, including the age and body weight of the patient, the physical condition of the patient, the level of glucagon or the level of suppression of glucagon to be achieved, and other factors.

Such pharmaceutical compositions are applicable in cases where the patient needs to lower the level of glucagon, and can also be used for other diseases in which lowering the level or suppression of glucagon can benefit.

An effective daily anti-glucagon dose of the compounds will usually be in the range of from 0.01 or 0.03 to about 5 mg / day, preferably from about 0.01 or 0.5 to 2 mg / day, or more preferably from about 0.01 or 0 , 1 to 1 mg / day for a patient weighing 70 kg, with a single dose or in divided doses. The exact dose that you need to enter is determined by the attending physician, and it depends on whether a particular compound falls into the above ranges, as well as on the age, body weight and condition of the patient. The introduction should begin when the first symptoms appear or shortly after the diagnosis, for example, diabetes mellitus, which manifests itself in an increased level of glucagon. Administration may be by injection, preferably subcutaneous or intramuscular. Orally active compounds can be administered orally, however, dosages should be increased 5-10 times.

In general, in the treatment and prophylaxis of elevated, inappropriate, or undesirable blood glucagon levels after a meal, the compounds of the present invention can be administered to patients in need of such treatment in dose ranges similar to those described above, however, the compounds are administered more frequently for example once, twice or three times a day. Particularly preferred are exendin and exendin agonist preparations, as well as dosages and routes for their administration, as described in US Provisional Patent Application 60/116380, entitled “New Exendin Agonist Formulations and Methods of Administration”, filed January 14, 1999 ( and in the corresponding PCT application stating its priority, filed January 14, 2000, serial number has not yet been assigned)) and in provisional patent application US 60 / (not yet assigned), entitled “Use of exendins and their agonists for modulation and triglyceride levels, and treatment of dyslipidemia, "filed January 14, 1999, which claims priority of the present application and the contents of this application is incorporated herein by reference in the form of a bibliographic reference in its entirety if it is reproduced in full here.

The optimal preparation and route of administration to the patient of the compounds of this application depends on factors known in the art, such as the particular disease or disorder, the desired effect, and the type of patient. Although the compounds will usually be used to treat humans, they can also be used to treat similar or identical diseases in other vertebrates, such as other primates, farm animals such as pigs, cattle and poultry, as well as sports animals and domestic animals. animals such as horses, cats and dogs.

To aid in understanding the present invention, the following examples are included in order to describe the results of a series of experiments. The experiments relating to the present invention, of course, should not be construed as limiting the invention in any way, and such variants of the present invention, already known or developed later, which will be covered by the outlook of specialists in the art, are considered to fall within the scope of the present invention in accordance with as described above and as claimed in the claims below.

Example 1

Getting exendin-3

His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro-NHz [SEQ ID NO I].

The above amidated peptide was assembled on 4- (2'-4'-dimethoxyphenyl) -Fmoc-aminomethylphenoxyacetamidnorleucine-MBHA polymer (Novabiochem; 0.55 mmol / g) using Fmoc-protected amino acids (Applied Biosystems Inc.). In general, single coupling reaction cycles were used during the synthesis using Fast MoC chemistry (activation using HBTU). Deprotection (removal of the Fmoc group) from the growing peptide chain was carried out using piperidine. The final deprotection of the completed peptide on the polymer was carried out using a mixture of triethylsilane (0.2 ml), ethanedithiol (0.2 ml), anisole (0.2 ml), water (0.2 ml) and trifluoroacetic acid (15 ml) in accordance with standard methods (Introduction to Cleavage Techniques, Applied Biosystems Inc.). The peptide was precipitated in ether / water (50 ml) and centrifuged. The precipitate was diluted in glacial acetic acid and lyophilized. The lyophilized peptide was dissolved in water. The initial purity was approximately 75%.

Solvent A (0.1% TFA in water) and solvent B (0.1% TFA in ACN) were used in the purification and analysis steps.

The solution containing the peptide was loaded onto a C-18 preparative column and purified (10% -40% of solvent B in solvent A for 40 minutes). The purity of the fractions was determined under the same conditions using a C-18 analytical column. Pure fractions were combined to give the peptide at the end of the above. Analytical RP-HPLC (gradient of 30-60% solvent B in solvent A for 30 minutes) of the lyophilized peptide gave a peptide product whose detected retention time was 19.2 minutes.

Example 2

Getting exendin-4

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NHz [SEQ ID NO 2].

The above amidated peptide was assembled on 4- (2'-4'-dimethoxyphenyl) -Fmoc-aminomethylphenoxyacetamidnorleucine-MBHA-polymer (Novabiochem; 0.55 mmol / g) using Fmoc-protected amino acids (Applied Biosystems Inc.), separated from polymer, deprotected and purified in the same way as exendin-3, as described in example 1. In the analysis, solvent A (0.1% TFA in water) and solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient of 36-46% solvent B in solvent A for 30 minutes) of the lyophilized peptide gave a peptide product whose detected retention time was 14.9 minutes. Mass spectrometry with electrospray (M): calculated 4186.6; found - 4186.0-4186.8 (four samples).

Example 3

Renal clearance

, легко отфильтровываются. В случае молекул с большей молекулярной массой их фильтрация происходит на более ограничивающей основе, где учитываются молекулярная масса, структура и общий заряд. Точка отсечки для клубочковой фильтрации проходит между альбумином (67 кД), который сохраняется, и гемоглобином (68 кД), который отфильтровывается. Альбумин, эффективный радиус которого составляет примерно 36

, отфильтровывается клубочком менее чем на 1%.The kidneys can play a key role in the excretion of certain molecules (drugs, peptides, proteins). For some molecules, this process begins when the kidneys filter the blood in the glomeruli to produce the ultrafiltrate described below. The glomerular filter not only works on the basis of molecular weight, but also acts as a negatively charged selective barrier, contributing to the preservation of anionic compounds. A free fraction of plasma molecules (not bound to proteins) having a molecular weight of less than 5 kD and an effective radius of less than 15

are easily filtered out. In the case of molecules with a higher molecular weight, their filtration takes place on a more restrictive basis, which takes into account the molecular weight, structure and total charge. The glomerular filtration cut-off point passes between albumin (67 kD), which is retained, and hemoglobin (68 kD), which is filtered out. Albumin with an effective radius of approximately 36

, is filtered by a glomerulus less than 1%.

Once in the glomerulus, the molecule advances into the proximal tubule, where it either readsorbed or passes through the Henle loop to the distal tubule, from where the direct renal duct directs the filtrate into the bladder. Filtered proteins and peptides are usually cleaved by enzymes of the brush border in the proximal tubule, from where they are efficiently extracted with the participation of sodium / amine carriers (“cleaning pumps”). On the other hand, polar, ionized and high molecular weight molecules are not re-absorbed. Using this process, the metabolizing enzymes of the cortical substance of the kidney (proximal tubules) can also destroy the molecule into more polar molecules, thereby increasing the likelihood of its excretion in the urine. Many peptide hormones (for example, amylin, calcitonins) are destroyed by passing through the renal circulation, presumably due to vascular ectoenzymes delivered to the plasma, regardless of the glomerular filtration process. In these examples, the rate of peptide clearance from plasma is similar to the rate of plasma passage through the kidneys, which is three times higher than the rate of glomerular filtration.

Studies conducted to identify exendin-4 metabolites circulating in plasma have shown very little evidence of proteolytic cleavage; after large-scale intravenous administration to animals, an HPLC analysis of the plasma showed the presence of only intact exendin and the appearance of minor “daughter” peaks indicating the formation of degradation products. This contrasts with other peptides studied (for example, amylin and GLP-1), in which the disappearance of the “parent” HPLC peak was associated with the appearance of “daughter” HPLC peaks, subsequently identified as subpeptide destruction products. The absence of exendin destruction products in the plasma indicates a slight proteolysis at any site, including renal circulation, or its complete absence. This means that any clearance with the participation of the kidneys occurs in a non-proteolytic way, namely due to filtration or active excretion (as happens with paraaminogippurat).

Initial measurements of exendin clearance in humans (120-130 ml / min), monkeys (approximately 9 ml / min) and rats (3.2-4.4 ml / min) correspond to published glomerular filtration rates in these species. To analyze whether renal filtration could be a key route for exendin excretion, studies were conducted in male rats with removed kidneys starving overnight with a constant level of exendin infusion. A constant plasma level of exendin-4 increased sharply in nephrectomized rats compared with rats that retained normal kidneys. The data obtained showed that the kidneys are responsible for at least 80% of the total clearance level of exendin-4 (see FIGS. 5 and 6). Exendin clearance rates in intact rats were again similar to the glomerular filtration rate expected in such rats (4.2 ml / min). After summarizing, these results show that in the body as a whole, the metabolism of exendin-4 is extremely limited and that the bulk of its clearance occurs through renal filtration (but not due to proteolysis inside the vessels of the kidneys). A low amount of immunoreactive full-sized exendin in the urine is consistent with the fact that it is cleaved by the enzymes of the brush border in the proximal tubule after filtering.

Example 4

Exendin-4 decreases glucagon secretion during hyperglycemic fixation in Zucker rats with diabetes and obesity.

Absolute or relative hyperglucagonemia is often a sign, for example, of type 1 and type 2 diabetes mellitus and the suppression of the excessive secretion of glucagon in these and other conditions described or mentioned here can potentially be useful in the treatment with glucagonostatic agents. In this example, the effect of exendin-4 on glucagon secretion was analyzed in male Zucher diabetic and obese (ZDF) rats under anesthesia. Using the fixation protocol of hyperinsulinemia and hyperglycemia, factors that tended to affect glucagon secretion were kept constant. Plasma glucagon was fixed at about 34 mM 60 minutes before the start of intravenous saline (n = 7) or exendin-4 (0.21 μg + 2.1 μg / ml / hour: n = 7). Plasma glucagon concentrations measured before these injections were similar in both groups (306 ± 30 pM vs. 252 ± 32 pM, respectively: the difference was not significant).

The average plasma glucagon concentration in rats that were injected with glucagon was about half lower than in rats that were injected with saline in the last 60 minutes of fixation (165 ± 18 pcM versus 298 ± 26 pcM, respectively: P <0.002). The hyperglycemic fixation protocol also allowed insulin sensitivity to be determined. The rate of glucose administration during fixation increased by 111 ± 7% in rats treated with exendin-4, compared with control individuals (P <0.001). In other words, exendin-4 exerted a glucagonostatic effect in ZDF rats in a hyperglycemic fixation assay, and this effect will be beneficial in the treatment of diabetes in humans.

Example 5

The metabolic effects of exendin-4 on glucagon secretion in people with type 2 diabetes

In this example, the safety, tolerability, and efficacy of synthetic exendin-4 were evaluated in a group of 8 male patients with type 2 diabetes (without insulin therapy) who were interrupted by another diabetes treatment for at least 7 days. Each patient received a subcutaneous (s / c) injection of placebo (RVO) and 0.1, 0.2 and 0.3 μg / kg of exendin-4 with a 48-hour interval in the cross-section of the placebo-controlled, simple, blind experiment with increasing doses. Five patients also received a dose of 0.4 mcg / kg. Plasma glucose, insulin, and glucagon concentrations were evaluated during fasting and in response to Sustacal® (7 kcal / kg) administered concurrently with exendin-4 / PBO injection. Gastric emptying was evaluated by measuring serum acetaminophen concentrations after oral administration of 20 mg / kg of liquid acetaminophen administered with Sustacal®. He did not find any safety problems, as indicated by the data recorded negative manifestations, ECG and laboratory safety monitoring. Doses of 0.3 and 0.4 μg / kg caused a dose-dependent increase in nausea, and vomiting was observed at the highest dose.

Plasma glucose concentrations decreased at all doses of exendin-4 compared with PBO, although insulin concentrations did not differ at a statistically significant level. Mean ± s.e. the change in AIS plasma glucose compared to the baseline after 8 hours was + 391 ± 187, -263 ± 108, -247 ± 64, -336 ± 139 and -328 ± 70 mg · h / dl, respectively, for doses of PBO 0.1 , 0.2, 0.3 and 0.4 μg / kg. Changes in the amount of plasma glucagon after 3 hours were + 128.0 ± 19.2, -5.6 ± 10.5, -29.4 ± 18.6, -40.5 ± 24.5 and + 6.9 ± 38 6 pkg · h / ml, respectively. The rate of gastric emptying decreased at all doses, and the average level of acetaminophen absorbed over 6 hours decreased by 51%, 50%, 57 and 79% compared with PBO for doses of 0.1, 0.2, 0.3 and 0.4 μg / kg respectively. In general, an injection of SC exendin-4 in patients showed no safety problems, was tolerated at doses ≤ 0.3 μg / kg, reduced plasma glucose and glucagon and decreased gastric emptying rate. These observations support the use of exendin to treat conditions in which a decrease in glucagon levels and / or suppression of glucagon is useful, including but not limited to type 1 and type 2 diabetes.

Example 6

PEG-modified exendin-4

In the case of exendin-4, a 39-amino acid peptide having a molecular weight of 4187, modifications that increase its size and / or increase its anionic nature should reduce its ability to be filtered by the kidneys. Since clearance of exendin-4 mainly occurs in the kidneys, this will effectively increase its half-life. Other “PEG” properties (increased plasma half-lives due to the avoidance of renal and / or cellular clearance mechanisms that may exist; reduced immunogenicity and antigenicity; increased solubility; resistance to proteolytic cleavage; reduced toxicity (avoiding peak dose potential); improved thermal and mechanical stability; improved permeability through mucous or epithelial cell layers and selective control over specific biological function) akzhe will give potential advantage of exendin-4 and exendin agonists.

In particular, since the authors have revealed numerous pharmacological manifestations (apparently caused by the existence of multiple receptor subtypes), various spectra of biological activities of exendin-4 can be selected by placing PEG in the corresponding positions. The loss or change in biological activity has been described for “pegylated” proteins, which may be due to the presence of the PEG chain itself, a specific site occupied by the PEG chain, or combination conditions that have a negative effect on the protein.

The primary factors in the case of PEG modification in terms of renal filtration of exendin and exendin agonists are size and charge. Being unmodified, exendin-4 has a molecular weight of about 4.2 kD and is anionic in nature with a total charge of approximately –2 at physiological pH. One, two or three PEG components can be covalently attached to exendin-4 or an analog of exendin-4, for example, while it is preferable to have one PEG component. The size of the PEG can vary in molecular weight from 500 to 20,000, preferably from 5,000 to 12,000.

Many PEG covalent attachment processes employ the ε-amino group of lysine. Exendin-4 contains two lysine residues that can be modified by addition of PEG. Alanine scanning AC3177 (Leu ¹⁴ , Phe ²⁵ (1-28) -exendin-4) - a shortened analog of exendin-4 - indicated positions that are sensitive to alanine substitution. Two lysine residues at positions 12 and 27 are slightly suppressed by such a substitution: this confirms that the loss of the lysine-specific R-group of the side chain (methylene chain plus ε-amino group) is permissible. As for the full-size peptide, exendin-4, two positions of lysine are suitable for PEG attachment (see compounds 1 and 2). In addition, depending on the chemistry used to attach the PEG, the ε-amino groups at these positions can be masked, which will lead to an increase in the anionic nature of the peptide.

(201) HGEGTFTSDLSK (PEG) QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(202) HGEGTFTSDLSKQMEEEAVRLFIEWLK (PEG) NGGPSSGAPPPS-NH ₂

Based on the results of an alanine scan, other possible positions that can be modified by inserting a Lys-PEG or equivalent, for example, are as follows:

(203) HK (PEG) EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(204) HGEGK (PEG) FTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(205) HGEGTFTKtPEODLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(206) HGEGTFTSDK (PEG) SKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(207) HGEGTFTSDLK (PEG) KQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(208) HGEGTFTSDLSKK (PEG) MEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(209) * HGEGTFTSDLSKQMEK (PEG) EAVRLFIEWLKNGGPSSGAPPPS-NH ₂

(210) * HGEGTFTSDLSKQMEEK (PEG) AVRLFIEWLKNGGPSSGAPPPS-NH ₂

(211) HGEGTFTSDLSKQMEEEAK (PEG) RLFIEWLKNGGPSSGAPPPS-NH ₂

(212) HGEGTFTSDLSKQMEEEAVRK (PEG) FIEWLKNGGPSSGAPPPS-NH ₂

(213) * HGEGTFTSDLSKQMEEEAVRLFIK (PEG) WLRNGGPSSGAPPPS-NH ₂

(214) HGEGTFTSDLSKQMEEEAVRLFIEK (PEG) LKNGGPSSGAPPPS-NH ₂

(215) HGEGTFTSDLSKQMEEEAVRLFIEWLKK (PEG) GGPSSGAPPPS-NH ₂

The three positions noted above *, normally including glutamic acid, which are indicated for modification K (PEG), can also be modified by attaching to the carboxyl group of the side chain of glutamic acid E (PEG).

Another analogue to which Lys-PEG can be added is in the proposed GlyGly rotation:

(216) HGEGTFTSDLSKQMEEEAVRLFIEWLKNK (PEG) GPSSGAPPPS-NH ₂

(217) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGK (PEG) PSSGAPPPS-NH ₂

Positions 29-39 of exendin-4 may not be key for glucose-lowering activity, as indicated by the parameters of AC 3177, which has almost equipotent activity compared to exendin-4, and any of them, individually or in combination, may be substituted on K (PEG) or equivalent.

For a person skilled in the art it should be obvious that the present invention is fully adapted to achieve its objects and obtain the advantages described above, as well as what is inherent in them. Molecular complexes and methods, procedures, treatments, molecules, specific compounds described herein, in this case represent preferred options, serve as examples and are not intended to serve as limitations on the scope of the present invention. Changes in it and other ways of using that will come to the person skilled in the art that correspond to the essence of the invention are defined as being included in the scope of the present invention.

One skilled in the art will appreciate that various replacements and modifications can be made to the invention claimed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the application are determined at the level that is understood by specialists in this field of technology to which the present invention is directed. All patents and publications are hereby incorporated by reference to the same extent that each individual publication is individually and specifically defined as being included for information in the form of a bibliographic reference.

The invention, illustratively described here, can accordingly be put into practice in the absence of any element or elements, limitations or restrictions that are not specifically specified here. So, for example, in each case, here, any of the terms “including”, “essentially including” and “consisting of” can be replaced by any of the other two terms. The terms and expressions that were used here were used in terms of description and not limitation, and there was no intention to use such terms and expressions to exclude any equivalents of the properties shown and described here, or parts thereof, but it is clear that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that, although the present invention has been specifically described in the form of preferred options and discretionary properties, one skilled in the art may resort to modifications and changes to the concepts described herein and such modifications and changes are considered to be within the scope of the present inventions as defined in the attached claims.

Furthermore, when the properties or aspects of the present invention are described in terms of Markush groups, one skilled in the art will appreciate that the invention is also described in terms of any individual member or subgroup of members from a Markush group. For example, if “X” is described as being selected from the group consisting of bromine, chlorine, and iodine, then the statement that X represents bromine and the statement that X represents bromine and chlorine are fully taken into account.

In this application, the invention has been described in a broad and general sense. Each of the narrower objects and subclasses that make up the general description also forms part of the present invention. This includes a general description of the invention, under a condition or a negative attribute, that removes any material from the claimed class of objects, regardless of whether a particular material to be deleted is mentioned in this text or not.

Other options fall within the scope of the following claims.

Claims (15)

1. A pharmaceutical compound for use in the manufacture of a medicament for lowering plasma glucagon level in a subject selected from the group consisting of exendin, an exendin analog, a modified exendin, and a modified exendin analog, wherein said compound includes at least 31 naturally occurring or modified amino acid residues. 2. A pharmaceutical compound for use in the manufacture of a medicament according to claim 1, wherein said compound is selected from the group consisting of exendin and an exendin analog. 3. A pharmaceutical compound for use in the manufacture of a medicament according to claim 1, wherein said compound is exendin-4. 4. A pharmaceutical compound for use in the manufacture of a medicament for reducing plasma glucagon levels in a subject, said compound having the formula (I) (SEQ ID NO 41): Haa ₁ Haa ₂ Haa ₃ Cla Haa ₅ Haa ₆ Haa ₇ Haa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Haa ₁₂ Haa ₁₃ Haa ₁₄ Haa ₁₅ Haa ₁₆ Haa ₁₇ Ala Haa ₁₉ Haa ₂₀ Haa ₂₁ Haa ₂₂ Haa ₂₃ Haa ₂₄ Haa ₂₅ Haa ₂₆ Haa ₂₇ Haa ₂₈ -Z ₁ ; Where Xaa ₁ is His, Arg or Tur; Haa ₂ represents Ser, Gl, Ala or Thr; Xaa ₃ represents Asp or Cl; Haa ₅ represents Ala or Thr; Xaa ₆ represents Ala, Phe, Tur or naphthylalanine; Haa ₇ represents Thr or Ser; Haa ₈ represents Ala, Ser or Thr; Haa ₉ represents Asp or Glu; Haa ₁₀ represents Ala, Leu, Ilé, Val, pentyl glycine or Met; Xaa ₁₁ is Ala or Ser; Haa ₁₂ represents Ala or Ls; Xaa ₁₃ is Ala or Gln; Haa ₁₄ represents Ala, Leu, Ile, pentylglycine, Val or Met; Haa ₁₅ represents Ala or Glu; Haa ₁₆ represents Ala or Glu; Haa ₁₇ represents Ala or Glu; Haa ₁₉ represents Ala or Val; Haa ₂₀ represents Ala or Arg; Haa ₂₁ represents Ala or Leu; Haa ₂₂ represents Ala, Phe, Tur or naphthylalanine; Haa ₂₃ represents Ile, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Haa ₂₄ represents Ala, Glu or Asp; Haa ₂₅ represents Ala, Tr, Phe, Tur or naphthylalanine; Haa ₂₆ represents Ala or Leu; Haa ₂₇ represents Ala or Lus; Haa ₂₈ represents Ala or Asn; Z ₁ represents —OH, -NH ₂ , Gl-Z ₂ , Glü Glü-Z ₂ , Glü Glü Haa ₃₁ -Z ₂ , Glü Glü Haa ₃₁ Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl Gl Ala-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Gla Al Haa ₃₆ -Z ₂ , Glü Glü Haa ₃₁ Ser Ser Hl Ala Haa ₃₆ Haa ₃₇ -Z ₂ , or Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ Haa _{338- Z 2} ; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ independently represent Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and Z ₂ represents —OH or —NH ₂ ; provided that no more than three of Haa ₃ , Haa ₅ , Haa ₆ , Haa ₈ , Haa ₁₀ , Haa ₁₁ , Haa ₁₂ , Haa ₁₃ , Haa ₁₄ , Haa ₁₅ , Haa ₁₆ , Haa ₁₇ , Haa ₁₉ , Haa ₂₀ , Haa ₂₁ , Haa ₂₄ , Haa ₂₅ , Haa ₂₆ , Haa ₂₇ and Haa ₂₈ represent Ala. 5. A pharmaceutical compound for use in the manufacture of a medicament for lowering plasma glucagon levels in a subject, said compound having the formula (II) [SEQ ID NO 42]: Haa ₁ Haa ₂ Haa ₃ Haa ₄ Haa ₅ Haa ₆ Haa ₇ Haa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Haa ₁₂ Haa ₁₃ Haa ₁₄ Haa ₁₅ Haa ₁₆ Haa ₁₇ Ala Haa ₁₉ Haa ₂₀ Haa ₂₁ Haa ₂₂ Haa ₂₃ Haa ₂₄ Haa ₂₅ Haa ₂₆ Haa ₂₇ Haa ₂₈ -Z ₁ ; Where Haa ₁ represents His, Arg, Tur, Ala, Norval, Val or Norulu; Haa ₂ represents Ser, Gl, Ala or Thr; Haa ₃ represents Ala, Asp or Glu; Haa ₄ represents Ala, Norval, Val, Norluu or Gl; Haa ₅ represents Ala or Thr; Xaa ₆ represents Phe, Tur or naphthylalanine; Haa ₇ represents Thr or Ser; Xaa ₈ represents Ala, Ser or Thr; Haa ₉ represents Ala, Norval, Val, Norleu, Asp or Glu; Haa ₁₀ represents Ala, Leu, Ilé, Val, pentyl glycine or Met; Xaa ₁₁ is Ala or Ser; Haa ₁₂ represents Ala or Ls; Xaa ₁₃ is Ala or Gln; Haa ₁₄ represents Ala, Leu, Ile, pentylglycine, Val or Met; Haa ₁₅ represents Ala or Glu; Haa ₁₆ represents Ala or Glu; Haa ₁₇ represents Ala or Glu; Haa ₁₉ represents Ala or Val; Haa ₂₀ represents Ala or Arg; Haa ₂₁ represents Ala or Leu; Haa ₂₂ represents Phe, Tur or naphthylalanine; Haa ₂₃ represents Ilé, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Haa ₂₄ represents Ala, Glu or Asp; Haa ₂₅ represents Ala, Tr, Phe, Tur or naphthylalanine; Haa ₂₆ represents Ala or Leu; Haa ₂₇ represents Ala or Lus; Haa ₂₈ represents Ala or Asn; Z ₁ represents —OH, -NH ₂ , Gl-Z ₂ , Glü Glü-Z ₂ , Glü Glü Haa ₃₁ -Z ₂ , Glü Glü Haa ₃₁ Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl Gl Ala-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Gla Al Haa ₃₆ -Z ₂ , Glou Gly Haa ₃₁ Ser Ser Glou Ala Haa ₃₆ Haa ₃₇ -Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ; or Glü Glü Haa ₃₁ Ser Ser Gl Gl Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ Ser-Z ₂ ; Haa ₃₁ , Haa ₃₆ , Haa ₃₇ and Haa ₃₈ independently of each other are Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and Z ₂ represents —OH or —NH ₂ ; provided that no more than three of Haa ₃ , Haa ₄ , Haa ₅ , Haa ₆ , Haa ₈ , Haa ₉ , Haa ₁₀ , Haa ₁₁ , Haa ₁₂ , Haa ₁₃ , Haa ₁₄ , Haa ₁₅ , Haa ₁₆ , Haa ₁₇ , Haa ₁₉ , Haa ₂₀ , Haa ₂₁ , Haa ₂₄ , Haa ₂₅ , Haa ₂₆ , Haa ₂₇ and Haa ₂₈ represent Ala; and also provided that if Xaa ₁ represents His, Arg or Tur, then at least one of Xaa ₃ , Xaa ₄ and Haa ₉ represents Ala. 6. A pharmaceutical compound for use in the manufacture of a medicament for lowering plasma glucagon levels in a subject, said compound having the formula (III) [SEQ ID NO 43]: Haa ₁ Haa ₂ Haa ₃ Gla Haa ₅ Haa ₆ Haa ₇ Haa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Haa ₁₂ Haa ₁₃ Haa ₁₄ Haa ₁₅ Haa ₁₆ Haa ₁₇ Ala Haa ₁₉ Haa ₂₀ Haa ₂₁ Haa ₂₂ Haa ₂₃ Haa ₂₄ Haa ₂₅ Haa ₂₆ Haa ₂₇ Haa ₂₈ -Z ₁ ; Where Xaa ₁ is His or Arg; Haa ₂ represents Gl or Ala; Haa ₃ represents Asp or Glu; Haa ₅ represents Ala or Thr; Haa ₆ represents Ala, Phe or naphthylalanine; Haa ₇ represents Thr or Ser; Xaa ₈ represents Ala, Ser or Thr; Haa ₉ represents Asp or Glu; Haa ₁₀ represents Ala, Leu or pentyl glycine; Xaa ₁₁ is Ala or Ser; Haa ₁₂ represents Ala or Ls; Xaa ₁₃ is Ala or Gln; Haa ₁₄ represents Ala, Leu or pentyl glycine; Haa ₁₅ represents Ala or Glu; Haa ₁₆ represents Ala or Glu; Haa ₁₇ represents Ala or Glu; Haa ₁₉ represents Ala or Val; Haa ₂₀ represents Ala or Arg; Haa ₂₁ represents Ala or Leu; Haa ₂₂ represents Phe or naphthylalanine; Haa ₂₃ represents Ilé, Val or tert-butyl glycine; Haa ₂₄ represents Ala, Glu or Asp; Haa ₂₅ represents Ala, Trp or Phe; Haa ₂₆ represents Ala or Leu; Haa ₂₇ represents Ala or Lus; Haa ₂₈ represents Ala or Asn; Z ₁ represents —OH, -NH ₂ , Gl-Z ₂ , Glü Glü-Z ₂ , Glü Glü Haa ₃₁ -Z ₂ , Glü Glü Haa ₃₁ Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl Gl Ala-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Gla Al Haa ₃₆ -Z ₂ , Glü Glü Haa ₃₁ Ser Ser Hl Ala Haa ₃₆ Haa ₃₇ -Z ₂ , or Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, thioproline or N-methylalanine; and Z ₂ represents —OH or —NH ₂ ; provided that no more than three of Haa ₃ , Haa ₅ , Haa ₆ , Haa ₈ , Haa ₁₀ , Haa ₁₁ , Haa ₁₂ , Haa ₁₃ , Haa ₁₄ , Haa ₁₅ , Haa ₁₆ , Haa ₁₇ , Haa ₁₉ , Haa ₂₀ Haa ₂₁ Haa ₂₄ Haa ₂₅ Haa ₂₆ Haa ₂₇ and Haa ₂₈ represent Ala; and their pharmaceutically acceptable salts; and characterized in that the indicated use is use for the manufacture of a medicament for reducing plasma glucagon levels in a subject. 7. A pharmaceutical compound for use in the manufacture of a medicament for reducing plasma glucagon in a subject, said compound having the formula (IV) [SEQ ID NO 44]: Xaa ₁ Xaa ₂ Xaa _h Xaa ₄ Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Xaa ₉ Xaa ₁₀ Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Xaa ₂₃ Xaa ₂₄ Xaa ₂₅ Xaa ₂₆ Haa ₂₇ Haa ₂₈ -Z ₁ ; Where Xaa ₁ is His or Ala; Haa ₂ represents Gl or Ala; _Of Xaa is Ala, Asp or Glu; Xaa ₄ represents Ala or Gl; Haa ₅ represents Ala or Thr; Xaa ₆ represents Phe or naphthylalanine; Haa ₇ represents Thr or Ser; Xaa ₈ represents Ala, Ser or Thr; Haa ₉ represents Ala, Asp or Glu; Haa ₁₀ represents Ala, Leu or pentyl glycine; Xaa ₁₁ is Ala or Ser; Haa ₁₂ represents Ala or Ls; Xaa ₁₃ is Ala or Gln; Haa ₁₄ represents Ala, Leu, Met or pentyl glycine; Haa ₁₅ represents Ala or Glu; Haa ₁₆ represents Ala or Glu; Haa ₁₇ represents Ala or Glu; Haa ₁₉ represents Ala or Val; Haa ₂₀ represents Ala or Arg; Haa ₂₁ represents Ala or Leu; Haa ₂₂ represents Phe or naphthylalanine; Haa ₂₃ represents Ilé, Val or tert-butyl glycine; Haa ₂₄ represents Ala, Glu or Asp; Haa ₂₅ represents Ala, Trp or Phe; Haa ₂₆ represents Ala or Leu; Haa ₂₇ represents Ala or Lus; Haa ₂₈ represents Ala or Asn; Z ₁ represents —OH, -NH ₂ , Gl-Z ₂ , Glü Glü-Z ₂ , Glü Glü Haa ₃₁ -Z ₂ , Glü Glü Haa ₃₁ Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl Gl Ala-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Gla Al Haa ₃₆ -Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ -Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ ; Glü Glü Haa ₃₁ Ser Ser Gl Gl Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ Ser-Z ₂ ; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ independently represent Pro, homoproline, thioproline or N-methylalanine; and Z ₂ represents —OH or —NH ₂ ; provided that no more than three of Haa ₃ , Haa ₅ , Haa ₆ , Haa ₈ , Haa ₁₀ , Haa ₁₁ , Haa ₁₂ , Haa ₁₃ , Haa ₁₄ , Haa ₁₅ , Haa ₁₆ , Haa ₁₇ , Haa ₁₉ , Haa ₂₀ Haa ₂₁ Haa ₂₄ Haa ₂₅ Haa ₂₆ Haa ₂₇ and Haa ₂₈ represent Ala; and provided that if Xaa ₁ is His, Arg, or Tur, then at least one of Xaa ₃ , Xaa _4, and Xaa ₉ is Ala; and their pharmaceutically acceptable salts. 8. A pharmaceutical compound for use in the manufacture of a medicament for reducing plasma glucagon levels in a subject, said compound having the formula (V) [SEQ ID NO 45]: Haa ₁ Haa ₂ Haa ₃ Gla Haa ₅ Haa ₅ Haa ₇ Haa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Haa ₁₂ Haa ₁₃ Haa ₁₄ Haa ₁₅ Haa ₁₆ Haa ₁₇ Ala Haa ₁₉ Haa ₂₀ Haa ₂₁ Haa ₂₂ Haa ₂₃ Haa ₂₄ Haa ₂₅ Haa ₂₆ X ₁ -Z ₁ ; Where Xaa ₁ is His, Arg or Tyr, or 4-imidazopropionyl; Haa ₂ represents Ser, Gl, Ala or Thr; Haa ₃ represents Asp or Glu; Haa ₅ represents Ala or Thr; Haa ₆ is Ala, Phe, Tur or naphthylalanine; Haa ₇ represents Thr or Ser; Xaa ₈ represents Ala, Ser or Thr; Haa ₉ represents Asp or Glu; Haa ₁₀ represents Ala, Leu, Ilé, Val, pentyl glycine or Met; Xaa ₁₁ is Ala or Ser; Haa ₁₂ represents Ala or Ls; Xaa ₁₃ is Ala or Gln; Haa ₁₄ represents Ala, Leu, Ile, pentylglycine, Val or Met; Haa ₁₅ represents Ala or Glu; Haa ₁₆ represents Ala or Glu; Haa ₁₇ represents Ala or Glu; Haa ₁₉ represents Ala or VaL; Haa ₂₀ represents Ala or Arg; Xaa ₂₁ is Ala, Leu or Lys-NHε-R, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl or cycloalkylalkanoyl; Haa ₂₂ represents Ile, Val or naphthylalanine; Haa ₂₃ represents Ile, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Haa ₂₄ represents Ala, Glu or Asp; Xaa ₂₆ represents Ala or Leu; X ₁ represents Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, Lys-NHε-R Ala, Ala Lys-NHε-R, where R represents Lys, Arg, C _1-10 alkanoyl straight or branched chain or cycloalkylalkanoyl; Z ₁ represents —OH; -NH ₂ , Gly-Z ₂ , Gly Gly-Z ₂ , Gly Gly Xaa ₃₁ -Z ₂ , Gly Gly Xaa ₃₁ Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ ; or Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ ; Where Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and Z ₂ represents —OH or —NH ₂ ; provided that no more than three of Haa ₃ , Haa ₅ , Haa ₆ , Haa ₈ , Haa ₁₀ , Haa ₁₁ , Haa ₁₂ , Haa ₁₃ , Haa ₁₄ , Haa ₁₅ , Haa ₁₆ , Haa ₁₇ , Haa ₁₉ , Haa ₂₀ Haa ₂₁ , Haa ₂₄ , Haa ₂₅ and Haa ₂₆ represent Ala; and characterized in that the indicated use is use for the manufacture of a medicament for reducing plasma glucagon levels in a subject. 9. A pharmaceutical compound for use in the manufacture of a medicament for reducing plasma glucagon levels in a subject, said compound having the formula (VI) [SEQ ID NO 46]: Haa ₁ Haa ₂ Haa ₃ Haa ₄ Haa ₅ Haa ₆ Haa ₇ Haa ₈ Haa ₉ Haa ₁₀ Haa ₁₁ Haa ₁₂ Haa ₁₃ Haa ₁₄ Haa ₁₅ Haa ₁₆ Haa ₁₇ Ala Haa ₁₉ Haa ₂₀ Haa ₂₁ Haa ₂₂ Haa ₂₃ Haa ₂₄ Haa ₂₅ Haa ₂₆ X ₁ -Z ₁ ; Where Haa ₁ represents His, Arg, Tur, Ala, Norval, Val, Norleu or 4-imidazopropionyl; Haa ₂ represents Ser, Gl, Ala or Thr; Haa ₃ represents Ala, Asp or Glu; Xaa ₄ represents Ala, Norval, Val, Norleu or Glu; Haa ₅ represents Ala or Thr; Xaa ₆ represents Phe, Tur or naphthylalanine; Xaa ₇ represents Thr or Ser; Xaa ₈ is Ala, Ser or Thr; Xaa ₉ represents Ala, Norval, Val, Norleu, Asp or Glu; Xaa ₁₀ represents Ala, Leu, Ile, Val, pentyl glycine or Met; Xaa ₁₁ is Ala or Ser; Xaa ₁₂ is Ala or Lys; Xaa ₁₃ is Ala or Gln; Xaa ₁₄ represents Ala, Leu, Ile, pentyl glycine, Val or Met; Xaa ₁₅ is Ala or Glu; Xaa ₁₆ represents Ala or Glu; Xaa ₁₇ represents Ala or Glu; Xaa ₁₉ represents Ala or Val; Xaa ₂₀ represents Ala or Arg; Xaa ₂₁ is Ala, Leu or Lys-NHε-R, where R is Lys, Arg, C _1-10 straight or branched chain alkanoyl or cycloalleylalkanoyl; Xaa ₂₂ is Phe, Tyr or naphthylalanine; Xaa ₂₃ represents Ile, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Xaa ₂₄ represents Ala, Glu or Asp; Xaa ₂₅ represents Ala, Trp, Phe, Tyr or naphthylalanine; Xaa ₂₆ represents Ala or Leu; X ₁ represents Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, Ls-NНε-R Ala, Ala Ls-NНε-R, where R represents Ls, Arg, C _1-10 alkanoyl straight or branched chain or cycloalkylalkanoyl; Z ₁ represents —OH, -NH ₂ , Gl-Z ₂ , Glü Glü-Z ₂ , Glü Glü Haa ₃₁ -Z ₂ , Glü Glü Haa ₃₁ Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Gl Gl Ala-Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Gla Al Haa ₃₆ -Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ -Z ₂ , Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ -Z ₂ , or Glü Glü Haa ₃₁ Ser Ser Ser Glü Ala Haa ₃₆ Haa ₃₇ Haa ₃₈ Haa ₃₉ -Z ₂ ; Where Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa _{38 are} independently selected from the group consisting of Pro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; and Z ₂ represents —OH or —NH ₂ ; provided that no more than three of Haa ₃ , Haa ₄ , Haa ₅ , Haa ₆ , Haa ₈ , Haa ₉ , Haa ₁₀ , Haa ₁₁ , Haa ₁₂ , Haa ₁₃ , Haa ₁₄ , Haa ₁₅ , Haa ₁₆ , Haa ₁₇ , Haa ₁₉ , Haa ₂₀ , Haa ₂₁ , Haa ₂₄ , Haa ₂₅ , Haa ₂₆ represent Ala; and also provided that if Xaa ₁ is His, Arg, Tur or 4-imidazopropionyl, then at least one of Xaa ₃ , Xaa _4, and Xaa ₉ is Ala; and characterized in that the indicated use is use for the manufacture of a medicament for reducing plasma glucagon levels in a subject. 10. A pharmaceutical compound for use in the manufacture of a medicament for lowering plasma glucagon levels in a subject, said compound having the formula (VII) [SEQ ID NO 47]: Haa ₁ Haa ₂ Haa ₃ Gla Thr Haa ₄ Haa ₅ Haa ₆ Haa ₇ Haa _in Ser Lus Gln Haa ₉ Glu Glu Glu Ala Val Arg Leu Haa ₁₀ Haa ₁₁ Haa ₁₂ Haa ₁₃ Leu As As Glu Gla Haa ₁₄ Ser Sеr Glu Ala ₁₅ Haa ₁₆ Haa ₁₇ Haa ₁₈ -Z, where Haa ₁ represents His, Arg or Tur; Haa ₂ represents Ser, Gl, Ala or Thr; Haa ₃ represents Asp or Glu; Xaa ₄ represents Phe, Tur or naphthylalanine; Xaa ₅ represents Thr or Ser; Haa ₆ represents Ser or Thr; Haa ₇ represents Asp or Glu; Haa ₈ represents Leu, Ilé, Val, pentyl glycine or Met; Haa ₉ represents Leu, Ilé, pentyl glycine, Val or Met; Haa ₁₀ represents Phe, Tur or naphthylalanine; Haa ₁₁ represents Ilé, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Xaa ₁₂ represents Glu or Asp; Xaa ₁₃ represents Trp, Phe, Tur or naphthylalanine; Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ independently represent Rro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; Haa ₁₈ represents Ser, Thr or Tur; and Z is —OH or —NH ₂ ; provided that the compound does not have the formula SEQ ID NO 1 or 2. 11. A pharmaceutical compound for use in the manufacture of a medicament for lowering plasma glucagon level in a subject, said compound having the formula (VIII) [SEQ ID NO 48]: Haa ₁ Haa ₂ Haa ₃ Gla Thr Haa ₄ Haa ₅ Haa ₆ Haa ₇ Haa ₈ Ser Lus Gln Haa ₉ Glu Glu Glu Ala Val Arg Leu Haa ₁₀ Haa ₁₁ Haa ₁₂ Haa ₁₃ Leu X ₁ Glu Gla Haa ₁₄ Ser Ser Ser Al Haaa ₁₅ Haa ₁₆ Haa ₁₇ Haa ₁₈ -Z, where Xaa ₁ is His, Arg, Tyr or 4-imidazopropionyl; Xaa ₃ represents Ser, Gl, Ala or Tur; Haa ₃ represents Asp or Glu; Xaa ₄ represents Phe, Tur or naphthylalanine; Xaa ₅ represents Thr or Ser; Xaa ₆ is Ser or Thr; Haa ₇ represents Asp or Glu; Haa ₈ represents Leu, Ilé, Val, pentyl glycine or Met; Haa ₉ represents Leu, Ilé, pentyl glycine, Val or Met; Haa ₁₀ represents Phe, Tur or naphthylalanine; Haa ₁₁ represents Ilé, Val, Leu, pentyl glycine, tert-butyl glycine or Met; Xaa ₁₂ represents Glu or Asp; Haa ₁₃ represents Trp, Phe, Tur or naphthylalanine; X ₁ is Ls Asn, Asn Ls, Ls-NНε-R Asn, Asn Ls-Nsε-R, where R is Ls, Arg, C _1-10 straight or branched chain alkanoyl or cycloalkylalkanoyl; Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ independently represent Rro, homoproline, 3Nur, 4Nur, thioproline, N-alkyl glycine, N-alkyl pentyl glycine or N-alkylalanine; Haa ₁₈ represents Ser, Thr or Tur; and Z is —OH or —NH ₂ ; provided that the compound does not have the formula SEQ ID NO 1 or 2. 12. A pharmaceutical compound for use in the manufacture of a medicament for lowering plasma glucagon level in a subject, said compound having a formula selected from the group of compounds consisting of SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216 and SEQ ID NO: 217. 13. A pharmaceutical compound for use in the manufacture of a medicament according to any one of claims 1-12, wherein said subject suffers from necrolytic migratory erythema. 14. A pharmaceutical compound for use in the manufacture of a medicament according to any one of claims 1-12, wherein said subject has glucagonoma. 15. A pharmaceutical compound for use in the manufacture of a medicament according to any one of claims 1-14, wherein said subject is a human.

Images