WO2014049610A2 - Peptides as gip, glp-1 and glucagon receptors triple-agonist - Google Patents

Description

PEPTIDES AS GIP, GLP-1 AND GLUCAGON RECEPTORS TRIPLE-AGONIST FIELD OF INVENTION

The present invention relates to novel peptides of general formula (I), their tautomeric forms, their pharmaceutically acceptable salts and pharmaceutical compositions containing them.

A-Z₁-Z₂-Z₃-Z₄-Z5-Z₆-Z₇-Zg-Z9-Z₁0-Z₁₁-Z₁2-Z₁₃-Z₁₄-Z₁5-Z₁₆-Z₁₇-Z₁g-Z₁₉-Z2₀-Z2₁-Z2₂-Z₂₃-

Z₂₄-Z2₅-Z₂₆-Z₂₇-Z28-Z₂₉-B

(I)

The present invention also relates to a process for preparing peptides of general formula (I), their tautomeric forms, their pharmaceutically acceptable salts and pharmaceutical compositions containing them.

BACKGROUND OF THE INVENTION

Worldwide, incidence of type 2 diabetes mellitus (T2DM) is increasing rapidly due to rise in the prevalence of obesity, combined with ageing populations and a trend towards urbanization. The simultaneous rise in these two diseases has resulted in a new term, 'diabesity,' to describe individuals who have obesity and T2DM. These patients are at increased risk of multiple co-morbidities (particularly cardiovascular disease) arid as such represent a huge economic burden on .health services.

Diabetes is characterized by impaired insulin secretion from pancreatic β-cells, insulin resistance or both (Cavaghan, M.K., et al., J. Clin. Invest. 2000, 106, 329). Majority of T2DM patients can be treated with agents that reduces hepatic glucose production, stimulate β-cell function (insulin secretagogues) or with agents that e,nhance the tissue sensitivity of diabetic patients towards insulin (insulin sensitizer). The drugs presently used to treat T2DM include a-glucosidase inhibitors, insulin sensitizers, insulin secretagogues and K_AT_P channel blocker (Chehade, J. M., et al., Drugs, 2000, 60, 95). However, almost one-half of type 2 diabetic subjects lose their response to these agents, over a period of time and thereby require insulin therapy (Burge, M.R., Diabetes Obes. Metab., 1999, 1, 199). One of the major problems with currently available therapies for diabetes is that most conventional treatments (e.g. sulfonylureas, thiazolidinediones, and insulin) promote weight gain. Treatment-induced weight gain might promote further insulin resistance and aggravate other co-morbidities associated with obesity. Obesity is characterized by increased body mass index (BMI), which occurs due to imbalance involved in appetite regulation and energy expenditure. A weight loss of 10% of the initial body weight in both overweight and obese adults significantly decreases risk of hypertension, hyperlipidemia and hyperglycemia. Diet and exercise promote weight loss; however, overweight and obese individuals often cannot lose weight effectively, just by diet control and exercise. Currently several anti-obesity agents have been approved that can be used as part of a comprehensive weight loss program. However, many of these drugs have serious adverse side-effects. Thus problems with the current diabesity treatment necessitate new therapies to treat obesity and diabetes.

In this regard, oxyntomodulin (OXM) peptide, which act as a dual agonist of Glucagon like peptide (GLP1) and glucagon (GCGR) receptors were found to be therapeutically potential. OXM is a 37 amino acid peptide; it's a member of the glucagon superfamily, comprising the entire 29 amino acid sequence of glucagon, with an eight amino acid carboxy terminal extension, resulting from the tissue-specific processing of the pre-pro-glucagon precursor in the brain and gut (Hoist, Ann. Rev. Physiol., 1997, 59, 257). It has been suggested that the dual agonistic activity of OXM is essential for the antidiabesity effect.

OXM activates both the glucagon and the GLP-lr with a two-fold higher potency for the glucagon receptor over the GLP-1 receptor, but is less potent than native glucagon and GLP-1 on their respective receptors. Repeated intracerebro ventricular (ICV) administration of OXM in rat's results in elevated core temperature and reduced weight gain compared to pair-fed animals, suggesting effects on both caloric intake and energy expenditure (Dakin et al., Am. J. Physiol. Endocrinol. Metab., 283, El 173, 2002). Central or peripheral administration of OXM in rats decreases feed intake with minimal effects on gastric emptying (Dakin et al., Endocrinology, 142, 2001, 4244; Dakin et al., Endocrinology, 145, 2004, 2687). Treatment of obese rodents with OXM also improves their glucose tolerance (Parlevliet e₍t al., Am. J. Physiol Endocrinol. Metab, 294, 2008, El 42) and suppresses body weight gain (WO 2003/ 022304). Studies in humans have shown that intravenously infused OXM is an effective appetite suppressant (Cogen et al., J. Clin. Endocrinol. Metab, 2003, 88(10), 4696). In a study of the effects of OXM on weight loss in humans it was found that subcutaneous injections of OXM (1.8 mg) to human volunteers (three times daily, for 28 days), resulted in a significant reduction of body weight (Wynne et al., Diabetes, 2005, 54, 2390).

OXM represent potential treatment for metabolic disorders such as diabesity. However, OXM has a very short half-life and is rapidly inactivated by the cell surface dipeptidyl peptidase IV (DPP-IV). Thus, because of poor in vivo stability of OXM, there exists a need to develop OXM derivatives that can be safely and efficaciously administered for the treatment of metabolic diseases such as diabetes and obesity. Several attempts have been made to develop a useful medicament based on the native human OXM sequence or modifications thereof (WO2003/057235, WO2003/022304, WO2004/062685, WO2007/100535, US2009/0298757, US2010/0144617, WO2010/096142, WO2010/096052, US2011/0034374, US2011/0152182 and WO2011/087671, WO2012/173422, WO2012/169798, WO2012/166951, US2012/0165503). The anorectic effects of OXM can be mimicked by analogues of glucagon, which have dual (GLP-1 and glucagon) agonistic activity (WO2008/101017, WO2009/155258, US2010/0190701, US2010/0204105 and WO2011/075393). Thus, in the recent years, there has been considerable interest in identifying a single ligand, which act as GCGR and GLP-1 receptor co-agonists (Claus, T. H., J. Endocrinology, 2007, 192, 371; Pan C.Q., JBC, 2006, 281, 12506).

Glucagon and GLP-1 are members of structurally related peptide hormone family (secretin family). Glucagon and GLP-1 constitute a highly homologous set of peptides because these two hormones originate from a common precursor, preproglucagon, which upon tissue-specific processing leads to the production of GLP- 1, predominantly in the intestine and glucagon in the pancreas (Jiang, G., et al., Am. J. Physiol. Endocrinol. Metab., 2003, 284, E671-678). The receptors for these two peptides are homologous (58 % identity) and belong to the class B family of G-protein coupled receptors (GPCRs).

Glucagon is a 29-amino acid peptide hormone processed from proglucagon in pancreatic a-cells by PC2. Glucagon acts via a seven transmembrane GPCRs, consisting of 485 amino acids. Glucagon is released into the bloodstream when circulating glucose is low. The main physiological role of glucagon is to stimulate hepatic glucose output, thereby leading to increase in glycemia (Tan, K., et al., Diabetologia, 1985, 28, 435). Glucagon provides the major counterregulatory mechanism for insulin in maintaining glucose homeostasis in vivo.

The GLP-1 (7-36) amide is a product of the preproglucagon gene, which is secreted from intestinal L-cells, in response to the ingestion of food. GLP-1 exerts multiple actions by stimulating insulin secretion from pancreatic β-cells, in a glucose dependent manner (insulinotropic action). GLP-1 lowers circulating plasma glucagon concentration, by inhibiting its secretion (production) from a-cells (Drucker D. J., Endocrinology, 2001, 142, 521-527). GLP-1 also exhibits properties like stimulation of β-cell growth, appetite suppression, delayed gastric emptying and stimulation of insulin sensitivity ( auck, M.A., Horm. Metab. Res., 2004, 36, 852).

The effector system of glucagon and GLP-1 receptors is the Adenylyl Cyclase (AC) enzyme. Interaction of glucagon or GLP-1 agonist with glucagon or GLP-1 receptors (GLP-1 R) respectively causes activation of AC, which converts ATP to cAMP. Increase in the intracellular cAMP level raises the ratio of ADP/ATP, thereby initiating the cell depolarization (due to closure of KATP channel). Increase in the intracellular cAMP level also activates Protein Kinase (PK-A & PK-C), which raises the cystolic Ca²⁺ concentration, by opening of L-type of Ca²⁺ channel. An increase in the intracellular Ca leads to exocytosis (Fehmann, H.C., Endocr. Rev., 1995, 16, 390).

Structure-activity relationship (S AR) studies have been reported in the literature to determine the role of individual amino acids in both the glucagon and GLP-1 sequences (Runge, S., JBC, 2003, 278, 28005; Mann, R., Biochem. Soc. Trans., 2007, 35, 713). Glucagon and GLP-1 have no defined structure in aqueous solution, but in the presence of micelles or in the membrane mimetic environment, they adopt an a-helical structure in the mid-section, with flexible N- and C-terminal regions (Thornton, K., Biochemistry, 1994, 33, 3532; Neidigh, J. W., Biochemistry, 2001, 40, 13188). This suggests that the helical structure is required for binding of peptide ligands to their respective receptors.

Glucagon and GLP-1 acutely regulate glucose control in opposite directions. Glucagon acts directly at the liver to raise blood glucose by stimulating gluconeogenesis and glycogenolysis, whereas GLP-1 acts by multiple mechanisms to lbwer glucose, most notably by enhancing glucose stimulated insulin synthesis and secretion at the pancreas (Hare et al., J. Clinical Endocrinology & Metabolism 94 (2009), 4679-4687; Zander et al., Lancet. 2002; 359, 824-830).

Human glucagon is capable of activating both receptors, though with a strong preference for the glucagon receptor over the GLP-1 receptor, GLP-1 on the other hand is not capable of activating glucagon receptors. Thus the molecular basis for selectivity between these two hormones and their receptors is of physiological and medicinal importance (Day et al., Biopolymers. 2012; 98(5):443-50). The application of co- agonists to enhance body weight reduction and correct multiple abnormalities associated with the metabolic syndrome has recently been reported (Alessandro et al., Diabetes, 14, 2009; Alessandro et al., J Endocrinol, 1, 2012, 335-346).

Glucagon acts primarily at hepatic GCGR to increase plasma glucose, while GLP-1 functions during nutrient ingestion at pancreatic β-cell GLP-1 receptors to enhance insulin synthesis and secretion. Acute glucagon administration reduces food intake in animals and humans and some reports indicate that sustained GCGR activation not only decreases food intake but also promotes lipolysis and weight loss. More recently, the unique pharmacology of GCGR/GLP-1R Gastric inhibitory polypeptide (GIP) triple-agonists/ GIP-oxyntomodulin hybrid peptide was reported to promote enhanced weight loss and effective blood glucose control when compared to selective GLP-1 R and / GCGR co-agonists (Bhat et al., Diabetologia. 2013, 56(6), 1417-24; Biochem Pharmacol. 2013, 1, 85(11), 1655-62). Thus the GLP- 1/glucagon/GIP receptors mixed/ triple agonists represents upcoming and promising therapeutic approach for the effective treatment of diabesity (Green et al., Curr Pharm Des., 2004,10(29), 3651-62).

Gastric inhibitory polypeptide (GIP) also designated as glucose-dependent insulinotropic polypeptide, is a peptide hormone of 42 amino acid residues, posttranslationally processed from a precursor prepro-GIP of 153 amino acid residues (Brown et al., Can. J. Biochem., 49, 867-872, 1971). GIP is a member of a family of structurally related hormones that includes secretin, glucagon and vasoactive intestinal peptide. The GIP moiety is flanked by a signal peptide of 21 residues and a peptide of 30 amino acids, and a peptide of 60 amino acids at its NH₂- and COOH-termini, respectively (Takeda et al., Proc. Natl. Acad. Sci., 84, 7005-7008, 1987). Prohormone convertase 1/3 is essential and sufficient for endoproteolytic processing to produce mature GIP. GIP is secreted from specific endocrine cells (K-cells), which are scattered in the epithelium of the upper part of small intestine after ingestion of a meal. Once released, GIP is subjected to NH₂-terminal degradation by dipeptidyl peptidase-IV (DPP-IV), yielding GIP metabolite, which acts as a GIP receptor antagonist (Yamada et al., Diabetes 55 (Suppl. 2), S86-S91, 2006; Ugleholdt et ah, J. Biol. Chem., 281, 11050- 11057,2006).

GIP exerts its effects by binding to its specific receptors, GIP receptors, activating adenylyl cyclase and increasing intracellular cAMP concentrations. The GIP receptor belongs to Class-B GPCR. In vitro studies using perfused pancreas or isolated islets have clearly demonstrated that GIP stimulates insulin secretion. Furthermore, administration of GIP in vivo has been revealed to increase insulin secretion in the presence of hyperglycemia (Dupre et al., J. Clin. Endocrinol. Metab, 37,826-828, 1973).

The present invention provides novel peptide of formula (I), which primarily acts as a GIP, GLP-1 and GCGR (triple-agonist), and thereby, such peptides can be useful for the treatment of metabolic disorders such as diabetes and obesity. Various peptides reported in this invention showed different level of affinity/selectivity towards glucagon, GIP and GLP-1 receptors. Furthermore, these peptides showed increased stability to proteolytic cleavage, especially against DPP-IV enzyme with improved half-life, making them suitable candidate for the treatment / mitigation / prophylaxis of diabesity and related metabolic disorders.

GIP, GLP-1 and glucagon sequences alignment shown below represent the primary structural relationships:

Glucagon: NH₂-1HSQGTFTSD⁹YSKYLDSRRAQDFVQW-L²⁶MNT²⁹-CONH₂ GLP-l(7-36): NH₂-¹HAEGTFTSD⁹VSSYLEGQAAKEFIAWLVKGR-CONH₂

GIP(1-42):NH₂- ^lYAEGTFISD⁹YSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-CONH₂ Single-letter abbreviations for amino acids can be found in Zubay, G., Biochemistry 2^nd ed., 1988, MacMillan Publishing, New York, p. 33.

Prior art

A series of human GLP-1 and glucagon co-agonist have been reported with varying degree of selectivity. Published patents: WO2009/155258, US2010/0190699, WO2010/011439, WO2010/148089, US2011/0098217, US20110166062 and WO2011/075393, discloses glucagon/GLP-1 receptor co-agonist for the treatment of metabolic diseases such obesity and diabetes. WO2003/057235, WO2004/062685, WO2007/100535, WO2003/022304, US2009/0298757, US2007666835, WO2006/134340, WO2008/071972, US2011/0152182, WO2011/087671, US 2010/0144617, EP0795562, US2000/588975, WO2010/096052 and WO2010/096142, discloses oxyntomodulin derivatives as anti-obesity agents. Zealand Pharma (WO2008/152403, WO2010/070251, WO2010/070252, WO2010/070253, WO 2010/070255, US2010/0204105, WO2011/006497, US 2013/0090286), discloses glucagon derivatives as anti-obesity agents. The unique pharmacology of GCGR/GLP- 1R/ Gastric inhibitory polypeptide (GIP) triple-agonists/ GIP-oxyntomodulin hybrid peptide has been reported to promote enhanced weight loss and effective blood glucose control when compared to selective GLP-1 R and / GCGR co-agonists (Bhat et al., Diabetologia. 2013, 56(6), 1417-24; Bhat et al., Biochem Pharmacol. 2013, 1, 85(l l),1655-62).

SUMMARY OF THE INVENTION

The present invention describes novel peptides that function as an agonist of the GIP, GLP-1 and glucagon receptors (triple-agonists/ GIP-oxyntomodulin hybrid peptide), having different degree of affinity/selectivity towards these receptors and are useful for reducing circulating glucose levels and for the treatment of diabetes, obesity and metabolic disorders. These peptides are defined by the general formula (I) as given below. The peptides of the present invention are useful in the treatment of the human or animal body, by regulation of GIP, GLP-1 and glucagon receptors. The peptides of this invention are therefore suitable for the treatment/mitigation/regulation or prophylaxis of diabetes, obesity and associated metabolic disorders.

A preferred embodiment of the present invention is to provide novel peptides of general formula (I), their tautomeric forms, novel intermediates involved in their synthesis, their pharmaceutically acceptable salts, their pharmaceutically acceptable solvates and pharmaceutical compositions containing them or their mixtures, suitable for the treatment/mitigation/regulation of diabetes and obesity (diabesity).

In another preferred embodiment is provided a process for the preparation of novel peptides of general formula (I), their tautomeric forms, their pharmaceutically acceptable salts, pharmaceutically acceptable solvates and pharmaceutical compositions containing them.

In a further preferred embodiment, is provided pharmaceutical compositions containing peptides of general formula (I), their tautomeric forms, their pharmaceutically acceptable salts, solvates and their mixtures having pharmaceutically acceptable carriers, solvents, diluents, excipients and other media normally employed in their manufacture.

In a still further preferred embodiment is provided the use of the novel peptides of the present invention as antidiabetic and antiobesity agents, by administering a therapeutically effective & non-toxic amount of the peptides of formula (I), or their pharmaceutically acceptable compositions to the mammals those are in need of such treatment.

Abbreviations used

The following abbreviations are employed in the examples and elsewhere herein:

Aib = a-Aminoisobutyric acid,

APP A = 2-Amino-5phenyl-pentanoic acid,

ACN = Acetonitrile,

AC₃C = 1 -amino cyclopropane carboxylic acid,

AC₅C = 1-amino-cyclopentanecarboxylic acid,

aMe-Gln = alpha-methyl-Glutamine,

ccMe-Glu = alpha-methyl-Glutamic acid,

aMe-Asp = alpha-methyl-Aspartic acid}

oiMe-Phe = alpha-methyl-phenylalanine,

aMe-2F-Phe = alpha-methyl-2-flubrophenylalanine,

aMe-2,6-F-Phe = alpha-methyl-2,6-diflurophenylalanine,

Bn = Benzyl,

Boc = tert-Butoxycarbonyl,

Bu^l= O-tert-butyl group,

cAMP= Adenosine 3 \5 '-cyclic monophosphate,

DCM = Dichloromethane,

DMF = N,N-Dimethylformamide,

DIPCDI= Di-isopropylcarbodiimide, DIPEA= Diisopropylethylamine,

Et = Ethyl,

Et₂0 = Diethyl ether,

2F-Phe = 2-fluorophenylalanine,

Fmoc = Fluorenylmethoxycarbonyl,

g = Gram (s),

GLP-1R = Glucagon Like Peptide-1 Receptor,

Glucagon R= Glucagon receptor,

h = Hour (s),

HOBt = Hydroxybenzotriazole,

HOAt= 7-Aza-hydroxybenzotriazole,

HBTU = 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyl aminium hexafluorophosphate,

HPLC = High Performance Liquid Chromatography,

i.p.= intraperitonial,

L = Liter,

LC /MS = Liquid Chromatography / Mass Spectrometry,

Me = Methyl,

Min = minute (s),

mL = milliliter,

μΐ = microliter,

mg = milligram (s),

mmol = millimole (s),

MS= Mass Spectrometry,

□Me-Gln = N-methyl-Glutamine,

DMe-Glu = N-methyl-Glutamic acid,

□Me- Asp = N-methyl-Aspartic acid,

PyBOP = Benzotriazole- 1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, SPPS = Solid Phase Peptide Synthesis,

sc = subcutaneous,

TMS = Trimethylsilyl,

TIPS = Triisopropylsilane,

TFA = Trifluoroacetic acid, TBTU= 2-( 1 H-benzotriazole- 1 -yl)- 1 , 1 ,3 ,3 -tetramethylaminium tetrafiuoroborate, Trt= Trityl group.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, synthetic peptides having the structural formula (I) showed GIP, GLP-1 and glucagon receptors triple-agonistic activity. These peptides exhibit increased stability to proteolytic cleavage, especially against DPP-IV (Dipeptidyl peptidase-IV) enzyme. These peptides can be delivered by parenteral routes of administration, for the treatment or prevention of diabetes, obesity and related metabolic disorders.

The present invention thus discloses novel peptides as GIP, GLP-1 and glucagon receptors triple-agonist having the following structure (I)

A-Z₁-Z2-Z3-Z₄-Z -Z₆-Z₇-Zg-Z9-Z₁₀-Z₁₁-Z₁₂-Z₁₃-Z₁₄-Z₁₅-Z₁₆-Z₁₇-Z₁g-Z₁9-Z₂₀-Z₂₁-Z2₂-Z23-

Z24-Z25-Z26-Z27-Z28-Z29-B

< (I)

Wherein,

'A' represents the groups -NH-Rt or R3-CO-NEL wherein Ri represents hydrogen or optionally substituted linear or branched (Cns) alkyl chain; R₃ is selected from optionally substituted linear or branched (Cns) alkyl chain, (C₁-₆)alkoxy, (C₃-C₆) cycloalkyl, aryl, heteroaryl or arylalkyl groups;

In a preferred embodiment, the aryl group is selected from phenyl, napthyl, indanyl, fluorenyl or biphenyl, groups; the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, indolyl, benzofuranyl groups;

'B' represents -COOR₂, -CONHR2 or CH₂OR₂, wherein R₂ represents H;

( Z_\ represents Histidine (H);

Each of Z2, Z₃, Z₄, Z₁₆ & Z₁₉ independently represents a naturally or unnaturally occurring amino acid selected from the group comprising of Glycine, L-Serine, D- Serine, L-alanine, D-alanine, a-amino-isobutyric acid (Aib), 1 -amino cyclopropane carboxylic acid (ACP), 1-amino-cyclopentanecarboxylic acid (AC₅C), 1-amino- cyclohexanecarboxylic acid (AC₆C), Glutamic acid (Glu; E), Glutamine (Glu; Q) having the following structures;

1 -amino-cyclopropane carboxyl ic acid (ACP) l-aminocyclopentanecaiboxylic acid (AC₅C) 1 -aminocyclohexanecarboxylic acid (AC₆C)

Each of Z₅, Z_7j Z₈ & Zn independently represents a naturally or non-naturally occurring amino acid comprising a hydroxyl side chain; a preferred Z₅, Z_7j Z & Zn is threonine or serine;

Each of Z₆ & Z₂₂ independently represents a naturally or unnaturally occurring amino acid having a disubstituted alpha carbon having two side chains, wherein each of them may independently be an optionally substituted alkyl or aryl or an aralkyl group wherein the substituents on each of them may be independently selected from one or more alkyl groups or one or more halo groups. Preferred Z₆ & Z₂₂ are represented by Phe (F), alpha-methyl-phenylalanine (-a-Me-Phe-), alpha-methyl-2- fluorophenylalanine (-a-Me-2F-Phe-) or alpha-methyl-2,6-diflurophenylalanine (-a- Me- -F-Phe-) or 2-fluorophenylalanine (-2F-Phe-) having the following structures.

alpha-methyl- alpha-methyl- alpha-methyl- phenylalanine 2-fluorophenylalanine 2,6-difluorophenylalanine 2-fluorophenylalanine

Each of Z9, Z₁₅, Z₂o & Z₂i independently represent a naturally or non-naturally occurring amino acid having an\ amino acid side chain comprising an acidic or amide group. Preferred Z₉, Z₁₅, Z₂₀ & Z₂i are selected from Aspartic acid, Glutamic acid, Asparagine, Glutamine;

Each of Z10 & Z₁₃ & Z₂ independently represents a naturally or unnaturally occurring amino acid selected from the group comprising of Tryptophan (W), D-Tryptophan (iAV), alpha-methyl -tryptophan (a-Me-Trp), N-methyl tryptophan (N-Me-Trp), Tyrosine (Y), D-Tyrosine (dY), alpha-methy-tyrosine (a-Me-Tyr), N-methyl tyrosine (N-Me-Tyr), alpha-methyl-phenylalanine (a-Me-Phe), alpha-methyl-2- fluofophenylalanine (a-Me-2F-Phe), alpha-methyl-2,6-diflurophenylalanine (a-Me-2,6- F-Phe), 2-fluorophenylalanine (2F-Phe) having the following structures:

N-Me-Tyr a- e-Tyr N- e-Trp

Each of Z₁₂ & Z₁₇ & Zi independently represents a naturally or non-naturally occurring amino acid selected from the group comprising of Lysine (K), D-Lysine (dK), Lysine(Octyl), Lysine(Decyl), Lysine(Dodecyl), Lysine(Myristyl), Lysine(Palmityl), D-Lysine(Octyl), D-Lysine(Decyl), D-Lysine(Dodecyl), D- Lysine(Myristyl), D-Lysine(Palmityl), N-Lysine (N-Lys), N-Arginine (N-Arg), Arginine (R), Arginine(Nitro); (Arg(N0₂)), N-Homoarginine (N-Har), Homoarginine (Har), Homoarginine(Nitro); (Har(N0₂), D-Homoarginine (c/Har), beta-homoarginine (β-Har), Ornithine (Orn), Citrulline (Cit), Homocitrulline (HoCit), L-alanine, D- alanine, a-amino-isobutyric acid (Aib), 1 -amino cyclopropane carboxylic acid (ACP), 1-amino-cyclopentanecarboxylic acid (AC₅C), 1-amino-cyclohexanecarboxylic acid (AC₆C);

Each of Z₁₄ , Z₂3 & Z₂₆ represents a naturally or unnaturally occurring amino acid selected from the group comprising of Leu (L), He (I), alpha-methyl-isoleucine (ar Me-Ile), Val (V), Nle (Norleucine), HoLeu (Homoleucine);

Z₂₄ represents a naturally or unnaturally occurring amino acid selected from the group comprising of Cystine (C), D-Cystine (dC), alpha-methyl-cystine (a-Me-Cys), N-methyl Cystine (N-Me-Cys), Glutamic acid (Glu; E), Glutamine (Glu; Q);

Z₂₇ represents a naturally or unnaturally occurring amino acid selected from the group comprising of Methonine (M), D- Methonine (dM), alpha-methyl-Methonine (a- Me-Met), N-methyl Methonine (N-Me-Met), Leu (L), He (I), alpha-methyl-isoleucine (μ-Me-Ile), Val (V), Nle (Norleucine), alpha-methyl-norleucine (a-Me-Nle), HoLeu (Homoleucine);

Z₂₈ may be present or absent, if present, it represents a naturally or unnaturally occurring amino acid selected from the group comprising of Glutamine (Glu; Q), Aspargine (Asn; N); Z₂9 may be present or absent, if present, it represents a naturally or nonnaturally occurring amino acid comprising a hydroxyl side chain; a preferred Z₂₉ is threonine; Definitions:

The term 'natural amino acids' indicates all those twenty amino acids, which are present in nature.

The term 'unnatural amino acids' or 'non-natural amino acids' represents either replacement of L-amino acids with corresponding D-amino acids such as replacement of L-Ala with D-Ala and the like or suitable modifications of the L or D amino acids, amino alkyl acids, either by

- a-alkylation such as substitution of Ala with a-methyl Ala (Aib), replacement of Phe with a-methyl-Phe;

D-alkylation such as substitution of APPA with D-methyl-APPA (NMe-APPA), replacement of Bip(OMe) with D-methyl-Bip(OMe);

- substitution on the side chain of amino acid such as substitution of aromatic amino acid side chain with halogen, (C₁-C₃)alkyl, aryl groups, more specifically the replacement of Phe with 2 & 6-halo Phe;

The various groups, radicals and substituents used anywhere in the specification are described in the following paragraphs.

Unless otherwise indicated, the term 'amino acid' as employed herein alone or as part of another group includes, without limitation, an amino group and a carboxyl group linked to the same carbon, referred to as 'a' carbon.

The absolute 'S' configuration at the 'a' carbon is commonly referred to as the 'L' or natural configuration. The 'R' configuration at the 'a' carbon is commonly referred to as the 'D' amino acid. In the case where both the 'a-substituents' is equal, such as hydrogen or methyl, the amino acids are Gly or Aib and are not chiral.

The term 'GLP-1 receptor modulator or agonist' refers to a compound that acts at the GLP-1 receptor to alter its ability to regulate downstream signaling events, such as cAMP production and insulin release. Example of receptor modulators includes agonist, partial agonist, inverse agonist and allosteric potentiators.

The term 'GIP receptor modulator or agonist' refers to a compound that acts at the GIP receptor to alter its ability to regulate downstream signaling events, such as cAMP production and insulin release. Example of receptor modulators includes agonist, partial agonist, inverse agonist and allosteric potentiators.

The term 'glucagon (GCGR) receptor modulator or agonist' refers to a compound that acts at the GCGR receptor to alter its ability to regulate downstream signaling events, such as cAMP production and insulin release. Example of receptor modulators includes agonist, partial agonist, inverse agonist and allosteric potentiators.

In accordance with the present invention, the synthetic isolated peptides described herein primarily act as GLP-1 /glucagon receptors (GCGR)/GIP triple- agonists. These synthetic peptides exhibit desirable in vitro glucagon receptor as well as GLP-1 & GIP receptors agonistic activities in CHO cells transfected with human glucagon or GLP-1 or GIP receptors (H Glucagon R or HGLP-1R or HGIPR), in the range of 1- 100 nM concentrations. The H GLP-1 R/GCGR and GIPR agonistic activity is assessed by estimation of amount of cAMP released. These new classes of peptides can be administered by parenteral routes of administration.

The present invention provides peptides of formula (I), pharmaceutical compositions employing such peptides either alone or in combination and methods of using such peptides. In particular, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of peptides of formula (I), alone or in combination(s), with a pharmaceutically acceptable carrier.

Further provided is a method for treating or delaying the progression or onset of diabetes and obesity, especially type 2 diabetes and obesity, including complications of diabetes, including retinopathy, neuropathy, nephropathy and delayed wound healing and related diseases such as insulin resistance (impaired glucose homeostasis), hyperglycemia, hyperinsulinemia, elevated blood levels of fatty acids or glycerol, hyperlipidemia including hypertriglyceridemia, syndrome X, atherosclerosis and hypertension, wherein a therapeutically effective amount of a peptides of formula (I) or their combination(s) are administered to a mammal, example, human, a patient in need of treatment.

Preparation of the peptides:

Several synthetic routes can be employed to prepare the peptides of the present invention well known to one skilled in the art of peptide synthesis. The peptides of formula (I), where all symbols are as defined earlier can be synthesized using the methods described below, together with conventional techniques known to those skilled in the art of peptide synthesis, or variations thereon as appreciated by those skilled in the art. Referred methods include, but not limited to those described below.

The peptides thereof described herein may be produced by chemical synthesis using suitable variations of various solid-phase techniques generally known such as those described in G. Barany & R. B. Merrifield, "The peptides: Analysis, synthesis, Biology"; Volume 2- "Special methods in peptide synthesis, Part A", pp. 3-284, E. Gross & J. Meienhofer, Eds., Academic Press, New York, 1980; and in J. M. Stewart and J. D. Young, "Solid-phase peptide synthesis" 2nd Ed., Pierce chemical Co., Rockford, II, 1984.

The preferred strategy for preparing the peptides of this invention is based on the use of Fmoc-based SPPS approach, wherein Fmoc (9-Fluorenyl-methyl-methyloxycarbonyl) group is used for temporary protection of the a-amino group, in combination with the a'cid labile protecting groups, such as t-butyloxy carbonyl (Boc), tert-butyl (Bu^l), Trityl (Trt) groups (Figure 1), for temporary protection of the amino acid side chains (see for example E. Atherton & R.C. Sheppard, "The Fluorenylmethoxycarbonyl amino protecting group", in "The peptides: Analysis, synthesis, Biology"; Volume 9 - "Special methods in peptide synthesis, Part C", pp. 1-38, S. Undenfriend & J. Meienhofer, Eds., Academic Press, San Diego, 1987).

The peptides can be synthesize in a stepwise manner on an insoluble polymer support (resin), starting from the C-terminus of the peptide. In an embodiment, the synthesis is initiated by appending the C-terminal amino acid of the peptide to the resin through formation of an amide, ester or ether linkage. This allows the eventual release of the resulting peptide as a C-terminal amide, carboxylic acid or alcohol, respectively.

In the Fmoc-based SPPS, the C-terminal amino acid and all other amino acids used in the synthesis are required to have their a-amino groups and side chain functionalities (if present) differentially protected (orthogonal protection), such that the a-amino protecting group may be selectively removed during Jhe synthesis, using suitable base such as 20% piperidine solution, without any premature cleavage of peptide from resin or deprotection of side chain protecting groups, usually protected with the acid labile protecting groups. The coupling of an amino acid is performed by activation of its carboxyl group as an active ester and reaction thereof with unblocked a-amino group of the N-terminal amino acid appended to the resin. After every coupling and deprotection, peptidyl-resin was washed with the excess of solvents, such as DMF, DCM and diethyl ether. The sequence of a-amino group deprotection and coupling is repeated until the desired peptide sequence is assembled (Scheme 1). The peptide is then cleaved from the resin with concomitant deprotection of the side chain functionalities, using an appropriate cleavage mixture, usually in the presence of appropriate scavengers to limit side reactions. The resulting peptide is finally purified by reverse phase HPLC.

The synthesis of the peptidyl-resins required as precursors to the final peptides utilizes commercially available cross-linked polystyrene polymer resirts (Novabiochem, San Diego, CA). Preferred for use in this invention is Fmoc-PAL-PEG-PS resin, 4-(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methyl benzhydrylamine resin (Fmoc-Rink amide MBHA resin), 2-chloro-Trityl-chloride resin or p- benzyloxybenzyl alcohol resin (HMP resin) to which the C-terminal amino acid may or may not be already attached. If the C-terminal amino acid is not attached, its attachment may be achieved by HOBt active ester of the Fmoc-protected amino acid formed by its reaction with DIPCDI. In case of 2-Chloro-trityl resin, coupling of first Fmoc-protected amino acid was achieved, using DIPEA. For the assembly of next amino acid, N- terminal protection of peptidyl resin was selectively deprotected using a solution of 10- 20 % piperidine solution. After every coupling and deprotection, excess of amino acids and coupling reagents were removed by washing with DMF, DCM and ether. Coupling of the subsequent amino acids can be accomplished using HOBt or HO AT active esters produced from DIPCDI/ HOBt or DIPCDI/HOAT, respectively. In case of some difficult coupling, especially coupling of those amino acids, which are hydrophobic or amino acids with bulky side chain protection, complete coupling can be achieved using a combination of highly efficient coupling agents such as HBTU, PyBOP or TBTU, with additives such as DIPEA.

Fmoc =

Fmoc-His(Trt)-OH Fmoc-Gln(Trt)-OH Fmoc-Thr(Bu')-OH

Fmoc-Ser(Bu')-OH Fmoc-Asp(Bu')-OH Fmoc-Phe-OH

Fmoc-Ala-OH Fmoc-Aib-OH Fmoc-GIy-OH

Figure 1: Examples of orthogonally protected amino acids used in Fmoc based-solid phase peptide synthesis (SPPS) of peptides.

The synthesis of the peptides described herein can be carried out by using batchwise or continuous flow peptide synthesis apparatus, such as CS-Bio or AAPPTEC peptide synthesizer, utilizing the Fmoc/t-butyl protection strategy. The non- natural non-commercial amino acids present at different position were incorporated into the peptide chain, using one or more methods known in the art. In one approach, an Fmoc-protected non-natural amino acid was prepared in solution, using appropriate literature procedures. For example, the Fmoc-protected a-methylated amino acids were prepared using asymmetric Strecker synthesis (Boesten, W.H.J., et al., Org. Lett., 2001, 3(8), 1 121). The resulting derivative was then used in the step-wise synthesis of the peptide. Alternatively, the required non-natural amino acid was built on the resin directly using synthetic organic chemistry procedures and a linear peptide chain were build.

20% Piperidine

Deprotection

'

Peptide + Resin

Scheme 1: General Scheme for Fmoc-Based SPPS

The peptide-resin precursors for their respective peptides may be cleaved and deprotected using suitable variations of any of the standard cleavage procedures _v described in the literature (King, D. S., et al., Int. J. Peptide Protein Res., 1990, 36, 255). A preferred method for use in this invention is the use of TFA cleavage mixture, in the presence of water and TIPS as scavengers. Typically, the peptidyl-resin was incubated in TFA / Water /TIPS (94:3:3; V: V: V; 10 ml / 100 mg of peptidyl resin) for 1.5-2 hrs at room temperature. The cleaved resin is then filtered off; the TFA solution is concentrated or dried under reduced pressure. The resulting crude peptide is either precipitated or washed with Et₂0 or is re-dissolved directly into DMF or 50 % aqueous acetic acid for purification by preparative HPLC.

Peptides with the desired purity can be obtained by purification using preparative HPLC. The solution of crude peptide is injected into a semi-Prep column (Luna 10μ; d₈; 100 A ), dimension 250 X 50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1 % TFA, using a flow rate of 15 -50 ml /min with effluent monitoring by PDA detector at 220 nm. The structures of the purified peptides can be confirmed by Electrospray Mass Spectroscopy (ES-MS) analysis.

All the peptide prepared were isolated as trifluoro-acetate salt, with TFA as a counter ion, after the Prep-HPLC purification. However, some peptides were subjected for desalting, by passing through a suitable ion exchange resin bed, preferably through anion-exchange resin Dowex SBR P(C1) or an equivalent basic anion-exchange resin. In some cases, TFA counter ions were replaced with acetate ions, by passing through suitable ion-exchange resin, eluted with dilute acetic acid solution. For the preparation of the hydrochloride salt of peptides, in the last stage of the manufacturing, selected peptides, with the acetate salt was treated with 4 M HC1. The resulting solution was filtered through a membrane filter (0.2 μηι) and subsequently lyophilized to yield the white to off-white HC1 salt. Following similar techniques and/or such suitable modifications, which are well within the scope of persons skilled in the art, other suitable pharmaceutically acceptable salts of the peptides of the present invention were prepared.

General method of preparation of peptides, using SPPS approach:

Assembly of peptides on resin:

Sufficient quantity (50-100 mg) of Fmoc-PAL-PEG-PS resin or Fmoc-Rink amide MBHA resin, loading: 0.5-0.6 mmol / g were swelled in DMF (1-10 ml /100 mg of resin) for 2-10 minutes. The Fmoc-group on resin was then removed by incubation of resin with 10-30 % piperidine in DMF (10-30 ml / 100 mg of resin), for 10-30 minutes. Deprotected resin was filtered and washed excess of DMF, DCM and ether (50 ml X 4). Washed resin was incubated in freshly distilled DMF (1 ml / 100 mg of resin), under nitrogen atmosphere for 5 minutes. A 0.5 M solution of first Fmoc- piOtected amino acid (1-3 eq.), pre-activated with HOBt (1-3 eq.) and DIPCDI (1-2 eq.) in DMF was added to the resin, and the resin was then shaken for 1-3 hrs, under nitrogen atmosphere. Coupling completion was monitored using a qualitative ninhydrin test. After the coupling of first amino acid, the resin was washed with DMF, DCM and Diethyl ether (50 ml X 4). For the coupling of next amino acid, firstly, the Fmoc- protection on first amino acid, coupled with resin was deprotected, using a 10-20% piperidine solution, followed by the coupling the Fmoc-protected second amino acid, using a suitable coupling agents, and as described above. The repeated cycles of deprotection, washing, coupling and washing were performed until the desired peptide chain was assembled on resin, as per general Scheme 1 above.

Finally, the Fmoc-protected peptidyl-resin prepared above was deprotected by 20% piperidine treatment as described above and the peptidyl-resins were washed with DMF, DCM and Diethyl ether (50 ml X 4). Resin containing desired peptide was dried under nitrogen pressure for 10-15 minutes and subjected for cleavage/ deprotection. Using above protocol and suitable variations thereof which are within the scope of a person skilled in the art, the peptides designed in the present invention were prepared, using Fmoc-SPPS approach. Furthermore, resin bound peptides were cleaved and deprotected, purified and characterized using following protocol.

Representative example of automated solid phase synthesis of peptide sequence ID No. 80:

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKEFICWLM The novel peptide, H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKEFICWLM was assembled on an automated CS-Bio 536 PepSynthesiser™ using Fmoc solid phase peptide synthesis (SPPS) approach (Scheme 2). The Fmoc amino acids and the 2-(lH- Benzotriazol-l-yl)-l,l,3,3-tetramethyluroniumtetrafluoroborate (TBTU) were packed together in vials and positioned in the amino acid module of the synthesizer.

A stock solution of diisopropylethylamine (DIPEA; 0.9 M) and DMF were stored in reagent bottles, under dry nitrogen atmosphere. The resin, Fmoc-PAL-PEG- PS (0.38 mmol/g; lg) was dried over P₂0_5j in vacuo (1 hr) and swollen in freshly distilled DMF (5 mL). The swollen resin was slurry packed into a glass column and positioned in the synthesizer. All the synthetic cycles were carried out at a flow rate of 5 mL min^'1, Table 1. The resin was washed with freshly distilled DMF for 10 minutes. Deprotection of Fmoc group was performed with 20% pipeiidine in DMF for 10 minutes and the deprotection was monitored by UV detection of the column effluent at 304 nm.

Table 1. Automated cycles for solid phase peptide synthesis

Excess piperidine was removed by three auxiliary wash cycles and a distilled

DMF wash cycle, with each cycle of 15 minutes. The amino group was treated with Fmoc-amino acid (4 equivalent), preactivated with TBTU (3.9 equivalent) in the presence of DIPEA (8 equivalent) and recycled for 120 minutes. The excess amino acid and soluble by-products were removed from column and loop by four auxiliary wash cycles and distilled DMF wash cycles, with each cycle of 10 minutes. Furthermore, synthetic cycles (deprotection, wash, acylation and wash) were repeated for complete assembly of linear peptide.

Final deprotection cycle was performed with 20% piperidine in DMF for 15 minutes to remove the terminal Fmoc group, followed by wash cycle (10 X 4 minutes). Completed peptide-resin was filtered through sintered glass filter, washed three times successively with DMF, DCM, methanol, DMF and diethyl ether (100 mL each). Peptide-resin was dried in vacuo over P₂0₅ (2 hr) and stored at -20 °C.

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKEFICWLM

(Seq. ID. No.80)

Scheme 2: SPPS of Seq. ID. No. 80 ' Ninhydrin resin test was carried out to check the N-terminal free amino group of resin bound peptide. Appearance of blue-purple colouration of the solution and the resin beads indicates the presence of free amino group on resin bound peptide and was considered to be a positive test.

Small-scale cleavage was carried out to assess the purity of resin bound peptide.

The dried Peptide-resin (ca 10-mg) was treated with mixture (1 mL) of TFA, water, triisopropylsilane (95: 2.5: 2.5 v/v), for 90 minutes at room temperature with gentle occasional swirling. The resin was filtered, washed thoroughly with neat TFA (1 mL) and the entire filtrate was evaporated under reduced pressure. Residual TFA was azeotroped three times with diethyl ether (2 mL). Residue obtained was suspended in distilled water (2 mL) and the aqueous layer was extracted three times with diethyl ether (3 mL). The aqueous layer was separated and freeze-dried to yield the crude peptide H-Aib-QGT-(2F-Phe)-TSDYS YLDEQAAKEFICWLM The lyophilised peptide H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKEFICWLM was dissolved in 0.1% aqueous TFA (ca lmg /l mL) and its purity was analyzed by analytical RP-HPLC and characterized by electrospray ionisation mass spectrometry (ESI-MS). Percent purity: 76 % (crude peptide). ESI-MS; Calcd. For H-Aib-QGT-(2F-Phe)- TSDYSKYLDEQAAKEFICWLM; 3214.6 (M⁺), 3237.6 (M+Na⁺) and 3253.6 (M+K⁺); Found (m/z): 3214.6 (M⁺), 3237.6 (M+Na⁺) and 3253.6 (M+K⁺);

Using above protocol and suitable variations thereof which are within the scope of a person skilled in the art, the novel peptides designed in the present invention were prepared, using Fmoc-SPPS approach. Furthermore, resin bound novel peptides were cleaved and deprotected, purified and characterized using following protocol.

Cleavage and deprotection:

The desired peptides were cleaved and deprotected from their respective peptidylrresins by treatment with TFA cleavage mixture as follows. A solution of TFA / Water / Triisopropylsilane (95: 2.5: 2.5) (10 ml / 100 mg of peptidyl-resin) was added to peptidyl-resins and the mixture was kept at room temperature with occasional starring. The resin was filtered, washed with a cleavage mixture and the combined filtrate was evaporated to dryness. Residue obtained was dissolved in 10 ml of water and the aqueous layer was extracted 3 times with ether (20 ml each) and finally the aqueous layer was freeze-dried. Crude peptide obtained after freeze-drying was purified by preparative HPLC as follows:

Preparative HPLC purification of the crude peptides:

Preparative HPLC was carried out on a Shimadzu LC-8A liquid chromatograph. A solution of crude peptide dissolved in DMF or water was injected into a semi-Prep column (Luna 10μ C_\ ; 100 A^D), dimension 250 X 50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1 % TFA, using a flow rate of 15 -50 ml / min, with effluent monitoring by PDA detector at 220 nm. A typical gradient of 20 % to 70 % of water- ACN mixture, buffered with 0.1 % TFA was used, over a period of 50 minutes, with 1% gradient change per minute. The desired product eluted were collected in a single 10-20 ml fraction and pure peptides were obtained as amorphous white powders by lyophilisation of respective HPLC fractions.

HPLC analysis of the purified peptides

After purification by preparative HPLC as described above, each peptide was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD analytical HPLC system. For analytic HPLC analysis of peptides, Luna 5μ; C ₈; 100 A^°, dimension 250 X 4.6 mm column was used, with a linear gradient of 0.1% TFA and ACN buffer and the acquisition of chromatogram was carried out at 220 nm, using a PDA detector.

Characterization by Mass Spectrometry

Each peptide was characterized by electrospray ionisation mass spectrometry

(ESI-MS), either in flow injection or LC/MS mode. Triple quadrupole mass spectrometers (API-3000 (MDS-SCIES, Canada) was used in all analyses in positive and negative ion electrospray mode. Full scan data was acquired over the mass range of quadrupole, operated at unit resolution. In all cases, the experimentally measured molecular weight was within 0.5 Daltons of the calculated monoisotopic molecular weight. Quantification of the mass chromatogram was done using Analyst 1.4.1 software.

Utilizing the synthetic methods described herein along with other commonly known techniques and suitable variations thereof, the following novel peptides were prepared. This list is indicative of the various groups of peptides, which can be prepared according to the present invention, and are expected to at least include obvious variations of these peptides. However, such disclosure should not be construed as limiting the scope of the invention in any way. In Table 2-4, novel peptides of present invention are listed along with their corresponding Seq. ID. No.

Table 2: List of novel peptides prepared

25

82 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDEQAAKEFICWLM

83 H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKE-(2F-Phe)-ICWLM

84 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAAKE-(a-Me-2,6-diF-Phe)- ICWLM

85 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDEQAAKE-(a-Me-2F-Phe)-ICWLM

86 H-Aib-QGT-(2F-Phe)-TSDYS YLD-Aib-QAAKEFICWLM

87 H-Aib-QGT-(2F-Phe)-TSDYSKYLD-Aib-QAAKE-(2F-Phe)-ICWLM

88 H-Aib-QGT-(2F-Phe)-TSDYS-Arg( 0₂)-YLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle

89 H-Aib-QGT-(2F-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(2F-Phe)-ICWL-Nle

90 H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(2F-Phe)-ICWL-Nle

91 H-Aib-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-Arg(N0₂)-AAKE-(2F-Phe)-ICWL- Nle

92 H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKEFICWLM

93 H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(2F-Phe)-ICWLM

94 H-(ACP)-QGT-(2F-Phe)-TSDYS YLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle

95' H-(ACP)-QGT-(2F-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle

96 H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle

97 H-(ACP)-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(2F-Phe)-ICWL- Nle

98 H-(ACP)-QGT-(2F-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(2F-Phe)-ICWL-Nle

99 H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(2F-Phe)-ICWL-Nle

100 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(a-Me-2,6-diF-Phe)- ICWL-Nle

101 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle

102 H-Aib-QGT-( -Me-2,6-diF-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(a-Me-2,6-diF-Phe)- ICWL-Nle

103 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle

104 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-Arg(N0₂)-AAKE-(a- Me-2,6-diF-Phe)-ICWL-Nle

105 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle

106 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle

107 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle

108 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(a-Me- 2,6-diF-Phe)-ICWL-Nle

109 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle

110 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle

111 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg( 0₂)-YLD-(ACP)-Arg(N0₂)-AAKE- (a-Me-2,6-diF-Phe)-ICWL-Nle

112 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLD-Aib-QAAKEFICWLM

113 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWLM 114 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL-Nle

115 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-Aib-QAAKE-(a-Me-2F-Phe)-IC L-Nle

116 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle

117 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(a-Me-2F-Phe)- ICWL-Nle

118 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(a-Me-2F-Phe)-ICWL-Nle

119 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(a-Me-2F-Phe)- ICWL-Nle

12b H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-Arg(N0₂)-AAKE-(a-Me-2F- Phe)-ICWL-Nle

121 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAAKEFICWLM

122 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWLM

123 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS YLD-(ACP)-QAAKE-(a-Me-2F-Phe)-ICWL- Nle

124 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle

125 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle

126 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(a-Me-2F- Phe)-ICWL-Nle

127 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(a-Me-2F-Phe)- ICWL-Nle

128 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me-2F-Phe)- ICWL-Nle

129 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-Arg(N0₂)-AAKE-(a- Me-2F-Phe)-ICWL-Nle

130 H-Aib-QGTFTSDYSKYLDEQAAKEFICWLMT

131 H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKEFICWLMT

132 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAA EFICWLMT

133 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDEQAAKEFICWLMT

134 H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKE-(2F-Phe)-ICWLMT

135 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAA E-(a-Me-2,6-diF-Phe)- ICWLMT

136 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDEQAAKE-(a-Me-2F-Phe)-ICWLMT

137 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWLMT

138 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL-Nle-T

139 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2F-Phe)-ICWL- Nle-T

140 H-Aib-QGT-(2F-Phe)-TSDYS YLDEQAAKE-(2F-Phe)-ICWLMT (El 6-K20 Lactam)

Table 3: List of peptides prepared

142 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQDFVQWLMNT

143 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDSRRAQDFVQWLMNT

144 H-Aib-QGT-(2F-Phe)- TSDYSKYLDSRRAQD-(2F-Phe)- VQWLMNT

145 H-Aib-QGT-(a-Me-2F-P e)-TSDYS YLDSRRAQD-(a-Me-2F-Phe)- VQWLMNT

146 H-Aib-QGT-(2F-Phe)-TSDYS YLDSRRAQDFV-C-WLMNT

147 H-Aib-QGT-(2F-Phe)- TSDYS YLDSRRAQD-(2F-Phe)-V-C-WLMNT

148 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQD-(a-Me-2,6-diF-Phe)- V- C-WLMNT

149 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSPvPvAQD-(a-Me-2F-Phe)- V-C- WLMNT

150 H-Aib-QGT-(2F-Phe)-TSDYSKYLDSRRAQDFV-C-WL-Nle-NT

151 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQDFV-C-WL-Nle-NT

152 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDSRRAQDFV-C-WL-Nle-NT

153 H-Aib-QGT-(2F-Phe)- TSDYS YLDSRRAQD-(2F-Phe)-V-C-WL-Nle-NT

154 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSRRAQD-(a-Me-2F-Phe)-V-C-WL- Nle-NT

155 H-Aib-QGT-(2F-Phe)- TSDYSKYLD-Aib-RRAQD-(2F-Phe)-V-C-WLMNT

156 H-Aib-QGT-(2F-Phe)- TSDYSKYLD-Aib-RRAQD-(2F-Phe)-V-C-WL-Nle-NT

157 H-(ACP)-QGT-(2F-Phe)- TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WL-Nle- NT

158 H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WLMN

159 H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-PvPvAQDFV-C-WL-Nle-N

160 H-(ACP)-QGT-(2F-Phe)- TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WLMN

161 H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WLM

162 H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WL-Nle

163 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-Aib-R AQDFV-C-WLMNT

164 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WL-Nle-NT

165 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-Aib-RRAQD-(a-Me-2,6-diF-Phe)- V-C-WLMNT

166 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-Aib-RRAQD-(a-Me-2,6-diF-Phe)- V-C-WL-Nle-NT

167 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2,6-diF- Phe)-V-C-WL-Nle-NT

168 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WLMN

169 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-R AQDFV-C-WL-Nle- N

170 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WL-Nle

171 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2,6-diF- Phe)-V-C-WLM

172 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2,6-diF- Phe)-V-C-WL-Nle

173 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RPvAQDFV-C-WLMNT

174 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WL-Nle-NT

175 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-R AQD-(a-Me-2F-Phe)-V-C- WLMNT

176 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-R AQD-(a-Me-2F-Phe)-V-C- WL-Nle-NT 177 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS YLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WLMNT

178 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WL-Nle-NT

179 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WLMN

180 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS YLD-(ACP)-RRAQDFV-C-WL-Nle-N

181 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WL N

182 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WL-Nle-N

183 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WLM

184 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WL-Nle

185 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WLM

186 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WL-Nle

187 H-Aib-QGT-(2F-Phe)-TSDYS YLDSRRAQDFVQWLMT

188 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQDFVQWLMT

189 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSR AQDFVQWLMT

190 H-Aib-QGT-(2F-Phe)- TSDYSKYLDSRRAQD-(2F-Phe)-VQWLMT

191 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQD-(a-Me-2,6-diF-Phe)- VQWLMT

192 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQDFVQWLMT

193 H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WLMT

194 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFVQWLMT

195 H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WLMT

Table 4: List of peptides prepared

Seq. ID.

No. Peptide Sequence

196 H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFVQWLMNT

197 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har- AQDFVQWLMNT

198 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFVQWLMNT

199 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2F-Phe)- VQWLMNT

200 H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WLMNT

201 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C- WLMNT

202 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WLMNT

203 H-Aib-QGT-(2F-Phe)- TSDYS-Har-YLDS-Har-Har-AQD-(2F-Phe)-V-C- WLM T

204 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2F-Phe)- V-C-WLMNT

205 H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C- L-Nle-NT

206 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WL- Nle-NT

207 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WL-Nle- NT

208 H-Aib-QGT-(a-Me-2,6-diF-P e)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2,6- diF-Phe)-V-C-WL-Nle-NT

209 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2F-Phe)- V-C-WL-Nle-NT

210 H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C-WLMNT

211 H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C-WL-Nle-NT

212 H-Aib-QGT-(2F-Phe)- TSDYS-Har-YLD-Aib-Har-Har-AQD-(2F-Phe)-V-C- WLMNT

213 H-Aib-QGT-(2F-Phe)- TSDYS-Har-YLD-Aib-Har-Har-AQD-(2F-Phe)-V-C-WL- Nle-NT

214 H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(2F-Phe)-V-C- WL-Nle-NT

215 H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C-WLMN

216 H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C-WLM

217 H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C-WL-Nle

218 H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(2F-Phe)-V-C- WLM

21? H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(2F-Phe)-V-C- WL-Nle

220 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C- WLMNT

221 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C- WL-Nle-NT

222 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(a-Me- 2,6-diF-Phe)-V-C-WLMNT

223 H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(a-Me- 2,6-diF-Phe)-V-C-WL-Nle-NT

224 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV- C-WLMNT

225 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a- Me-2,6-diF-Phe)-V-C-WL-Nle-NT

226 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV- C-WLMN

227 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV- C-WL-Nle-N

228 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a- Me-2,6-diF-Phe)-V-C-WLMN

229 H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a- Me-2,6-diF-Phe)-V-C-WL-Nle

230 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C- WLMNT

231 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C-WL- Nle-NT

232 H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(a-Me-2F-

In a preferred embodiment, the present invention provides a method of making a peptides, that function as triple agonist of GLP-1, glucagon & GIP receptors having different degree of affinity/selectivity towards both the receptors and useful for reducing circulating glucose levels, feed intake, body weight for the treatment of diabetes and obesity.

The synthetic peptides described in the present embodiment exhibit desirable in vitro GCGR, GLP-IR and GIPR agonistic activities in CHO cells transfected with human glucagon or HGLP-IR or HGIPR, in the range of 1- 100 nM concentration, and in vivo, some of the peptides showed blood glucose reduction, feed intake, body weight, when tested in different diabetic and obese animal models, such as hyperglycemic C57 mice, db / db and DIO mice.

The novel peptides of the present invention can be formulated into suitable pharmaceutically acceptable compositions by combining with suitable excipients as are well known.

The pharmaceutical composition is provided by employing conventional techniques. Preferably the composition is in unit dosage form containing an effective amount of the active component, that is, the compounds of formula (I) either alone or combination, according to this invention.

The quantity of active component, that is, the compounds of formula (I) according to this invention, in the pharmaceutical composition and unit dosage form thereof may be varied or adjusted widely depending upon the particular application method, the potency of the particular compound and the desired concentration. Generally, the quantity of active component will range between 0.5 % to 90 % by Weight of the composition.

IN VITRO AND IN VIVO STUDIES OF NOVEL PEPTIDES:

The peptides prepared as described above were tested for

a) In vitro Human GLP-1R agonistic activity (Cyclic AMP determination);

b) In vitro human glucagon receptor (GCGR) agonistic activity (Cyclic AMP determination);

c) In vitro human GIP receptor agonistic activity (Cyclic AMP determination);

d) Stability of peptides against DPP IV enzyme, human plasma, and liver microsomes; and

e) Demonstration of in vivo efficacy of test compounds (peptides) in C57BL/6J mice (in vivo), as described below.

a) In vitro Human GLP-1 R agonist activity (Cyclic AMP determination)

The novel peptides were screened for Human GLP-1 receptor (HGLP-1 R) agonist activity (in vitro), using the cAMP cell-based assay, in stably transfected CHO/ human GLP1R cells. The CHO-K1 cells (CRL 9618) were obtained from American Type Culture Collection (Rockville, MD). CHO cells were grown in Ham's F12 medium containing L-Glutamine (2mM), HEPES (25 mM), NaHC0₃ (1.1 g/L) and supplemented with New Born Calf Serum (NBCS; 10%), Penicillin (50 U /ml (v/v)) and Streptomycin (50 ug/ml (v/v)). Cells were split every 3 days 1:8.

Production of Stable CHO Cell Lines expressing the human GLP-1 Receptor.

The cDNA encoding the human GLP-1 receptor was isolated by RT- PCR according to standard protocol. The full-length cDNA was cloned in pcDNA3.1(+). For the production of CHO cell lines expressing the GLP-1 receptor, CHO cells were transfected with 10 μg of the expression plasmid pcDNA hGLP-lR using CaP0₄ according to the standard protocol (Wheeler, M.B., et al., Endocrinology 1993, 133, 57.). Clones expressing the receptor were generated by G418 (800 μg/ml active, Sigma) selection. The stable clones were thereafter maintained at 500 ug/ml (G418). The selected clone was used between passages 9-25 for cAMP assays.

Determination of cAMP generation.

The CHO cells stably transfected with human GLP-1 R were maintained in Ham's F12 + 10% NBCS + 500 ug/ml G418 upto a confluency of 70-75%. The cells were trypsinized using 2 ml of TPVG (0.25% trypsin, 0.53 mM EDTA, 1.38-mM glucose). The trypsin was inactivated using Ham's F12 medium containing 10% NBCS and the cells were suspended in 2 ml of complete medium. 2 X 10⁵ cells /well were then seeded in 12 well plate and the plates were incubated in humidified atmosphere at 37°C for 16 -18 h (Fehmann, H.C., et al., Peptides 1994, 15, 453). The next day the assay was preceded, when the cells showed 90-95% confluency. The medium was aspirated off from the 12 well plate and the cells were washed once using Ham's F12 (plain). The cells were incubated at 37°C with 500 ul of Ham's F12 + 1% BSA+ 0.125 mM RO-20 for 30 min. After the incubation, the medium was aspirated off and fresh medium (plain Ham's F12 + 1% BSA+ 0.25 mM RO-20) was added with 5ul of test peptides that has been dissolved i water (MilliQ). The cells were incubated with the test compounds for 30 min in humidified atmosphere and 37°C. After the incubation, the medium was removed and cells were washed once with plain Ham's F12. Subsequently, the cells were lysed by adding 500 ul of ice cold 0.1 N HC1 to each well and shaking for 30 minutes at 200 rpm. The cells were then scrapped; the lysate was collected in micro centrifuge tubes and centrifuged at 12000 rpm for 10 min to remove the debris. 300 ul of supernatant from each micro-centrifuge tube was then removed into a glass tube and dried under N₂ for 30 min, for cAMP estimation. The total cAMP was estimated from the sample according to the manufacturer's protocol using Cyclic AMP immunoassay kit (R&D systems, Minneapolis. MN). The remaining supernatant is used to determine the protein concentration using micro BCA (Sigma). Data is calculated as percent of control (Vehicle: water) and expressed as Mean ± SD. The in- vitro human GLP-1 receptor agonistic activities of representative peptides are listed in Table 5.

Table 5: In vitro Human GLP-1 R activity (cAMP release) of representative test peptides, shown as % activity with respect to control

Compounds InM 10 nM 100 nM

EX-4 90±0.12 101+0.1 1 1 10+0.16 Seq. ID. 18 38±0.13 93+0.15 112±0.12 Seq. ID. 35 36±0.11 110+0.14 1 10+0.11

Seq. ID. 86 56+0.12 94+0.1 1 1 15±0.10 Seq. ID. 118 66±0.15 101±0.14 1 13+0.12

Seq. ID. 155 88+0.17 113±0.15 121+0.10 Seq. ID. 188 93±0.09 120±0.14 12510.12

Seq. ID. 190 51+0.11 7710.10 9210.1 1 Seq. ID. 199 81+0.16 10910.13 12110.09

Seq. ID. 210 37+0.13 9210.15 11110.12 Seq. ID. 222 63+0.1 1 8910.14 99+0.14

Seq. ID. 230 9110.13 1 19+0.10 130+0.12 Seq. ID. 246 56+0.11 90±0.16 102+0.10

b) In vitro human glucagon agonistic activity (measurement of amount of Cyclic AMP production, with test peptides).

The novel peptides were screened for human glucagon receptor (H-GCGR) agonistic activity (in vitro), using the cAMP cell-based assay, in stably transfected CHO/ human glucagon R cells. The CHO-K1 cells (CRL 9618) were obtained from American Type Culture Collection (Rockville, MD). CHO cells were grown in Ham's F12 medium containing L-Glutamine (2mM), HEPES (25 mM), NaHC0₃ (1.1 g/L) and supplemented with newborn Calf Serum (NBCS; 10%), Penicillin (50 U /ml (v/v)) and Streptomycin (50 ug/ml (v/v)). Cells were split every 3 days 1 :8.

Production of Stable CHO Cell Lines expressing the human glucagon Receptor.

The cDNA encoding the human glucagon receptor was isolated by RT- PCR according to standard protocol. The full-length cDNA was cloned in pcDNA3.1 (Invitrogen). For the production of CHO cell lines expressing the glucagon receptor, CHO cells were transfected with 10 μg of the expression plasmid pcDNA/ H-glucagon- R using CaP0₄ according to the standard protocol. Clones expressing the receptor were generated by G418 (800 μ ηή active, Sigma) selection. The stable clones were thereafter maintained at 500 ug/ml (G418). The selected clone was used between passages 9-25 for cAMP assays.

Determination of glucagon agonistic activity bv measuring amount of cAMP production after addition of test peptides

The CHO cells stably transfected with human glucagon R were maintained in Ham's F12 + 10% NBCS + 500 ug/ml G418 upto a confluency of 70-75%. The cells were trypsinized using 2 ml of TPVG (0.25% trypsin, 0.53 mM EDTA, 1.38-mM glucose). The trypsin was inactivated using Ham's F12 medium containing 10% NBCS and the cells were suspended in 2 ml of complete medium. 2 X 10^s cells /well were then seeded in 12 well plate and the plates were incubated in humidified atmosphere at 37°C for 16 -18 h. The next day the assay was proceeded, when the cells showed 90-95% confluency. The medium was aspirated off from the 12 well plate and the cells were washed once using Ham's F12 (plain). The cells were incubated at 37°C with 500 ul of Ham's F12 + 1% BSA+ 0.125 mM RO-20 for 30 min. After the incubation, the medium was aspirated off and fresh medium (plain Ham's F12 + 1% BSA+ 0.25 mM RO-20) was added with 5ul of test peptides that has been dissolved in water (MilliQ). The cells were incubated with the peptides for 30 min in humidified atmosphere and 37 °C. After the incubation, the medium was removed and cells were washed once with plain Ham's F12. Subsequently, the cells were lysed by adding 500 μΐ of ice cold 0.1 N HCl to each well and shaking for 30 minutes at 200 rpm. The cells were then scrapped, the lysate was collected in micro centrifuge ,tubes and centrifuged at 12000 rpm for 10 min to remove the debris. 300 μΐ of supernatant from each micro-centrifuge tube was then removed into a glass tube and dried under N₂ for 30 min, for cAMP estimation. The total cAMP was estimated from the sample according to the manufacturer's protocol using Cyclic AMP immunoassay kit (R&D systems, Minneapolis. MN). The remaining supernatant is used to determine the protein concentration using micro BCA (Sigma). Data is calculated as percent of control (Vehicle: water) and expressed as Mean ± SD. The in-vitro human GCGR receptor agonistic activities of representative peptides are listed in Table 6.

Table 6: In vitro Human GCGR activity (cAMP release) of representative test peptides, shown as % activity with respect to control

Glucagon 90±0.11 100±0.12 130±0.15

Seq. ID. 15 66+0.14 102±0.09 110+0.08

Seq. ID. 37 87±0.12 101+0.12 107+0.11

Seq. ID. 42 99±0.11 122+0.14 136±0.12

Seq. ID. 55 _. 57±0.18 96±0.16 109±0.13

Seq. ID. 90 85±0.12 108+0.13 110±0.19

Seq. ID. 133 36+0.11 88±0.12 99±0.14

Seq. ID. 147 77±0.18 99±0.16 102±0.13

Seq. ID. 166 5610.11 93+0.14 10010.12 Seq. ID. 170 88±0.13 103+0.15 110±0.16

Seq. ID. 190 93±0.14 107+0.12 116±0.15

Seq. ID. 233 89±0.11 102+0.12 110+0.11

Seq. ID. 240 47±0.18 87+0.15 94±0.17

Seq. ID. 247 66±0.12 87+0.13 101+0.11

Seq. ID. 246 77+0.14 101±0.15 107±0.12

c) In vitro Human GIPR agonist activity (Cyclic AMP determination)

The novel peptides were screened for Human GIP receptor (GIP R) agonist activity (in vitro), using the cAMP cell-based assay, in stably transfected CHO/ human GIP cells. The CHO-K1 cells (CRL 9618) were obtained from American Type Culture Collection (Rockville, MD). CHO cells were grown in Ham's F12 medium containing L-Glutamine (2mM), HEPES (25 mM), NaHC0₃ (1.1 g/L) and supplemented with New orn Calf Serum (NBCS; 10%), Penicillin (50 U /ml (v/v)) and Streptomycin (50 ug/ml (v/v)). Cells were split every 3 days 1:8.

Production of Stable CHO Cell Lines expressing the human GIP Receptor.

The cDNA encoding the human GIP receptor was isolated by RT- PCR according to standard protocol. The full-length cD A was cloned in pcDNA3.1(+). For the production of CHO cell lines expressing the GIP receptor, CHO cells were transfected with 10 μg of the expression plasmid pcDNAThGLP-lR using CaP0₄ according to the standard protocol (Gremlich et al., Diabetes, 1995, 44:1202-1208; Yamada et al., Genomics 29:773-776, 1995) Clones expressing the receptor were generated by G418 (800 μg/ml active, Sigma) selection. The stable clones were thereafter maintained at 500 ug/ml (G418). The selected clone was used between passages 9-25 for cAMP assays.

Determination of cAMP generation.

The CHO cells stably transfected with human GIP R were maintained in Ham's F12 + 10% NBCS + 500 ug/ml G418 upto a confluency of 70-75%. The cells were trypsinized using 2 ml of TPVG (0.25% trypsin, 0.53 mM EDTA, 1.38-mM glucose). The trypsin was inactivated using Ham's F12 medium containing 10% NBCS and the cells were suspended in 2 ml of complete medium. 2 X 10⁵ cells /well were then seeded in 12 well plate and the plates were incubated in humidified atmosphere at 37°C for 16 -18 h (Fehmann, H.C., et al., Peptides 1994, 15, 453). The next day the assay was preceded, when the cells showed 90-95% confluency. The medium was aspirated off from the 12 well plate and the cells were washed once using Ham's F12 (plain). The cells were incubated at 37°C with 500 ul of Hani's F12 + 1% BSA+ 0.125 mM RO-20 for 30 min. After the incubation, the medium was aspirated off and fresh medium (plain Ham's F12 + 1% BSA+ 0.25 mM RO-20) was added with 5ul of test peptides that has been dissolved in water (MilliQ). The cells were incubated with the test compounds for 30 min in humidified atmosphere and 37°C. After the incubation, the medium was removed and cells were washed once with plain Ham's F12. Subsequently, the cells were lysed by adding 500 ul of ice cold 0.1 N HC1 to each well and shaking for 30 minutes at 200 rpm. The cells were then scrapped; the lysate was collected in micro centrifuge tubes and centrifuged at 12000 rpm for 0 min to remove the debris. 300 ul of supernatant from each micro-centrifuge tube was then removed into a glass tube and dried under N₂ for 30 min, for cAMP estimation. The total cAMP was estimated from the sample according to the manufacturer's protocol using Cyclic AMP immunoassay kit (R&D systems, Minneapolis. MN). The remaining supernatant is used to determine the protein concentration using micro BCA (Sigma). Data is calculated as percent of control (Vehicle: water) and expressed as Mean ± SD. The in-vitro human GIPreceptor agonistic activities of representative peptides are listed in Table 7.

Table 7: In vitro Human GIP activity (cAMP release) of representative test peptides, shown as % activity with respect to control

Seq. NO lnM 10 nM 100 nM

GIP 88±0.10 109±0.12 120±0.19

Seq. ID. 10 41±0.12 93±0.15 119±0.10

Seq. ID. 22 44±0.13 119±0.13 126+0.15

Seq. ID. 29 51±0.17 80±0.15 117±0.10

Seq. ID. 59 70±0.15 100+0.11 120+0.12 Seq. ID. 109 66+0.16 116+0.14 122+0.13 Seq. ID. 139 98±0.09 12210.10 130+0.11

Seq. ID. 171 49±0.11 70+0.17 99+0.12 Seq. ID. 189 88±0.20 11110.23 13110.10

Seq. ID. 219 31+0.13 99+0.12 119+0.19 Seq. ID. 222 65+0.11 7110.14 9810.12 Seq. ID. 233 6910.10 7710.19 101+0.15

Seq. ID. 239 9510.20 12310.14 13310.09 Seq. ID. 250 6010.12 81+0.22 108+0.19

d) Stability of peptides against DPP IV enzyme, human plasma, and liver microsomes:

Different peptides (final concentration 2 μΜ) were incubated with either DPP IV (1 : 25 mU) or pooled human plasma (7.5 μί) or human liver microsomes, for 0, 2, 4, 6, 12 and 24 h (37 °C; 50 mM triethanolamine-HCl buffer; pH 7.8). Concentrations of DPP IV enzyme/ human plasma/ human liver microsomes were selected in preliminary experiments to provide degradation of approximately 50% of Exendin within 2-4 h, therefore allowing time-dependent degradation to be viewed over 24 h. Reactions were terminated by the addition of TFA/H₂0 (15 mL, 10% (v/v)). The reaction products were then applied to a Vydac C₁₈ analytical column (4.6 x 250-mm) and the major degradation fragment separated from intact peptides. The column was equilibrated with TFA/H₂0, at a flow rate of 1 mL/min. Using 0.1% (v/v) TFA in 70% acetonitrile/H₂0, the concentration of acetonitrile in the eluting solvent was raised from 0% to 28% over 10 min and from 28% to 42% over 30 min. The absorbance was monitored at 206 nm using UV detector and peaks were collected manually prior to ESI-MS analysis. Area under the curve was measured for test peptides and their metabolites and percentage degradation were calculated at each time point over a period of 24 h. Stability study results of selected peptides, against DPP IV enzyme, human plasma and liver microsomes (in vitro) are listed in Table 8.

e) In vivo efficacy studies:

Demonstration of in vivo efficacy (antihyperglycaemic/ antidiabetic activity of test compounds (peptides) in C57BL/6J or db/db mice, both by parenteral dp) and oral routes of administration.

Animals

Acute single dose 120-min time-course experiments were carried out in male C57BL/6J or db/db mice, age 8-12 weeks, bred in-house. Animals were housed in groups of 6 animals per cage, for a week, in order to habituate them to vivarium conditions (25 + 4 °C, 60-65 % relative humidity, 12: 12 h light: dark cycle, with lights on at 7.30 am). All the animal experiments were carried out according to the internationally valid guidelines following approval by the 'Zydus Research Center animal ethical committee'.

Procedure

The in- vivo glucose lowering properties of some of the test compounds (peptides) and Exendin-4 were evaluated in C57BL/6J (mild hyperglycemic) or db/db animal models as described below. Two days prior to the study, the animals were randomised and divided into 5 groups (n = 6), based upon their fed glucose levels. On the day of experiment, food was withdrawn from all the cages, water was given ad- libitum and were kept for overnight fasting. Vehicle (normal saline) / test / standard compounds were administered intraperitoneally (i.p.), on a body weight basis. Soon after the 0 min. blood collection from each animal, the subsequent blood collections were done at 30, 60 and 120 or upto 240 min., via retro-orbital route, under light ether anesthesia (Chen, D., et al., Diabetes Obesity Metabolism, 2005, 7, 307. Kim, J. G. et al., Diabetes, 2003, 52, 751).

Blood samples were centrifuged and the separated serum was immediately subjected for the glucose estimation. Serum for insulin estimation was stored at -70 °C until used for the insulin estimation. The glucose estimation was carried out with DPEC- GOD/POD method (Ranbaxy Fine Chemicals Limited, Diagnostic division, India), using Spectramax-190, in 96-microwell plate reader (Molecular devices Corporation, Sunnyvale, California). Mean values of duplicate samples were calculated using Microsoft excel and the Graph Pad Prism software (Ver 4.0) was used to plot a 0 min base line corrected line graph, area under the curve (0-120 min AUC) and base line corrected area under the curve (0 min BCAUC). The AUC and BCAUC obtained from graphs were analyzed for one way ANOVA, followed by Dunnett's post test, using Graph Pad prism software. Furthermore, the insulin estimation was carried out using rat / mouse insulin ELISA kit (Linco research, Missouri USA). Changes in the blood glucose levels, at 0, 30, 60 and 120 min, with selected peptides are shown in Table 9 (Via ip route of administration) and Table 8 (via oral route of administration) respectively.

i.p)

Seq. ID.121 (5 nM/kg, i.p) 180 ± 5.2 120 ± 2.9 138 ± 2.4 149 ± 2.0

Seq. ID. 147 (50 nM/kg, 181 ± 5.3 119 ± 3.0 128 ± 2.6 147 ± 1.9 i.p)

Seq. ID. 161 (20 nM/kg, 144 ± 1.1 113 -t 2.9 149 ± 3.1 141 ± 3.4 i.p)

Seq. ID. 177 (30 nM/kg, 165 ± 3.1 122 ± 1.8 121 ± 2.0 139 ± 1.5 i.p)

Seq. ID. 211 (30 nM/kg, 177 ± 1.2 122 ± 1.6 128 ± 2.0 130 ± 2.3 i.p)

Seq. ID. 222 (20 nM/kg, 166 ± 2.0 144 ± 2.2 144 . ± 1.4 142 ± 1.9 'i.p)

Seq. ID. 233 (5 nM/kg, 167 ± 2.3 139 ± 2.9 131 ± 1.7 156 ± 1.4 i.p)

Seq. ID. 237 (50 nM/kg, 177 ± 4.2 133 2.2 137 ± 2.6 159 ± 1.9 i.p)

Claims

We claim:

1. Peptides having sequence of Formula (I), including their tautomers, solvates

A-Z₁-Z2-Z₃-Z₄-Z5-Z₆-Z₇-Z₈-Z9-Z₁₀-Z₁₁-Z₁2-Z₁3-Z₁₄-Z₁₅-Z₁₆-Z₁₇-Z₁₈-Z₁9-Z20-Z₂₁-

Z22-Z₂₃-Z₂₄-Z2₅-Z2₆-Z₂7-Z₂8-Z₂9-B

(I)

wherein,

'A' represents the groups -NH-Ri or R₃-CO-NH- wherein Ri represents hydrogen or optionally substituted linear or branched (Cng) alkyl chain; R₃ is selected from optionally substituted linear or branched (^-₁₈) alkyl chain, ( - 6)alkoxy, (C₃-C₆) cycloalkyl, aryl, heteroaryl or arylalkyl groups; 'B' represents -COOR₂, -CONHR₂ or CH₂OR2, wherein R₂ represents H; Z_\ represents Histidine (H); Each of Z₂, Z₃, Z₄, Z₁₆ & Z₁₉ independently represents a naturally or unnaturally occurring amino acids selected from the group comprising of Glycine, L-Serine, D-Serine, L-alanine, D-alanine, a-amino-isobutyric acid (Aib), 1 -amino cyclopropane carboxylic acid (ACP), 1-amino- cyclopentanecarboxylic acid (AC₅C), 1-amino-cyclohexanecarboxylic acid (AC₆C), Glutamic acid (Glu; E), Glutamine (Glu; Q); Each of Z₅, Z_7, Z₈ & Z_u independently represents a naturally or non-naturally occurring amino acid comprising a hydroxyl side chain; Each of Z₆ & Z₂₂ independently represents a naturally or unnaturally occurring amino acid having a disubstituted alpha carbon having two side chains, wherein each of them may independently be an optionally substituted alkyl or aryl or an aralkyl group; Each of Z₉, Z₁₅, Z₂₀ & Z₂₁ independently represent a naturally or non-naturally occurring amino acid having an amino acid side chain comprising an acidic or amide group;

Each of Z₁₀ & Z₁₃ & Z₂₅ independently represents a naturally or unnaturally occurring amino acid selected from the group comprising of Tryptophan (W), D-Tryptophan (dW), alpha-methyl-tryptophan (a-Me-Trp), N-methyl tryptophan (N-Me-Trp), Tyrosine (Y), D-Tyrosine (dY), alpha-methy-tyrosine (a-Me-Tyr), N-methyl tyrosine (N-Me-Tyr), alpha-methyl-phenylalanine (a- Me-Phe), alpha-methyl-2-fluorophenylalanine (a-Me-2F-Phe), alpha-methyl- 2,6-diflurophenylalanine (a-Me-2,6-F-Phe), 2-fluorophenylalanine (2F-Phe) groups;

Each of Z₁₂ & Z]7 & Zi8 independently represents a naturally or non-naturally occurring amino acid selected from the group comprising of Lysine (K), D- Lysine (dK), Lysine(Octyl), Lysine(Decyl), Lysine(Dodecyl), Lysine(Myristyl), Lysine(Palmityl), D-Lysine(Octyl), D-Lysine(Decyl), D-Lysine(Dodecyl), D- Lysine(Myristyl), D-Lysine(Palmityl), N-Lysine (N-Lys), N-Arginine (N-Arg), Arginine (R), Arginine(Nitro); (Arg(N0₂)), N-Homoarginine (N-Har), Homoarginine (Har), Homoarginine(Nitro); (Har(N0₂), D-Homoarginine (dHar), beta-homoarginine (β-Har), Ornithine (Orn), Citrulline (Cit), Homocitrulline (HoCit), L-alanine, D-alanine, a-amino-isobutyric acid (Aib), 1- amino cyclopropane carboxylic acid (ACP), 1-amino-cyclopentanecarboxylic acid (AC₅C), 1-amino-cyclohexanecarboxylic acid (AC₆C); Each of Z₁₄ , Z₂₃ & Z₂₆ represents a naturally or unnaturally occurring amino acid selected from the group comprising of Leu (L), He (I), alpha-methyl-isoleucine (a-Me-Ile), Val (V), Nle (Norleucine), HoLeu (Homoleucine); Z₂₄ represents a naturally or unnaturally occurring amino acid selected from the group comprising of Cystine (C), D-Cystine (dC), alpha-methyl-cystine (a-Me-Cys), N-methyl Cystine (N- Me-Cys), Glutamic acid (Glu; E), Glutamine (Glu; Q); Z₂₇ represents a naturally or unnaturally occurring amino acid selected from the group comprising of Methonine (M), D- Methonine (dM), alpha-methyl-Methonine (a-Me-Met), N-methyl Methonine (N-Me-Met), Leu (L), He (I), alpha-methyl- isoleucine (a-Me-Ile), Val (V), Nle (Norleucine), alpha-methyl-norleucine (a- Me-Nle), HoLeu (Homoleucine); Z₂₈ when present, represents a naturally or unnaturally occurring amino acid selected from the group comprising of Glutamine (Glu; Q), Aspargine (Asn; N); Z₂₉ may be present or absent, if present, it represents a naturally or nonnaturally occurring amino acid comprising a hydroxyl side chain.

The compound as claimed in claim 1 wherein each of Z₅, Z_7j Zg & Zn are independently selected from threonine or serine.

The compound as claimed in claim 1 wherein each of Z₆ & Z₂₂ independently represents the amino acids Phe (F), alpha-methyl-phenylalanine (-a-Me-Phe-), alpha-methyl-2-fluorophenylalanine (-a-Me-2F-Phe-) or alpha-methyl-2,6- diflurophenylalanine (-a-Me-2,6-F-Phe-) or 2-fluorophenylalanine (-2F-Phe-).

4. The compound as claimed in claim 1 wherein each of Z , Z_\s, Z₂₀ & Z₂i independently represents the amino acids Aspartic acid, Glutamic acid, Asparagine, Glutamine.

5. The compound as claimed in claim 1 wherein Z29 is threonine.

6. The compounds of any of the preceding claims wherein the aryl group is selected from phenyl, napthyl, indanyl, fluorenyl or biphenyl, group and the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, indolyl, benzofuranyl groups.

7. The compounds of any of the preceding claims wherein wherein the substituents on each of Z₆ & Z₂₂ are independently selected from one or more alkyl groups or one or more halogen atoms.

8. The compound of formula (I) selected from

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKEFICWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDEQAAKEFICWLMNT

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKE-(2F-Phe)-ICWLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAAKE-(a-Me-2,6-diF-Phe)- ICWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDEQAAKE-(a-Me-2F-Phe)-ICWLMNT

H-Aib-QGT-(2F-Phe)-TSDYSKYLD-Aib-QAAKEFICWLMNT

H-Aib-QGT-(2F-Phe)-TSDYS-R-YLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle-NT

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle-NT

H-Aib-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle-

NT

H-Aib-QGT-(2F-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(2F-Phe)-ICWL-Nle-NT

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(2F-Phe)-ICWL-Nle-NT

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKEFICWLMNT

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(2F-Phe)-ICWLMNT

H-(ACP)-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(2F-Phe)- ICWL-Nle-NT H-(ACP)-QGT-(2F-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(2F-Phe)-ICWL-Nle- NT

H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(2F-Phe)-ICWL- Nle-NT

H-(ACP)-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-Arg( 0₂)-AAKE-(2F- Phe)-ICWL-Nle-NT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle-NT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(a-Me-2,6- diF-P e)-ICWL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me- 2,6-diF-Phe)-ICWL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-Arg(N0₂)- AAKE-(a-Me-2,6-diF-Phe)-ICWL-Nle-NT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKEFICWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)- ICWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle-NT

H-Aib-QGT-( -Me-2F-Phe)-TSDYS-R-YLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle-NT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(a-Me-2F-Phe)- ICWL-Nle-NT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(a-Me-2F- Phe)-ICWL-Nle-NT H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(a-Me-2F-Phe)-ICWL- Nle-NT ·

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(a-Me-2F-Phe)- ICWL-Nle-NT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Arg( 0₂)-YLD-Aib-Arg(N0₂)-AAKE-(a- Me-2F-Phe)-IQWL-Nle-NT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(a-Me- 2F-Phe)-ICWL-Nle-NT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(a-Me-2F-Phe)- ICWL-Nle-NT

H-(ACP)-QGT-(a-Me-2F-P e)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me-2F- Phe)-ICWL-Nle-NT

H-Aib-QGTFTSDYSKYLDEQAAKEFICWLMN

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAA EFICWLMN

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDEQAAKEFICWLMN

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAA E-(2F-Phe)-ICWLM

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDEQAAKE-(a-Me-2F-Phe)-ICWLMN

H-Aib-QGT-(2F-Phe)-TSDYSKYLD-Aib-QAAKEFICWLMN

H-Aib-QGT-(2F-Phe)-TSDYSKYLD-Aib-QAAKE-(2F-Phe)-ICWLMN

H-Aib-QGT-(2F-Phe)-TSDYSKYLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle-N

H-Aib-QGT-(2F-Phe)-TSDYS-R-YLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle-N

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle-N

H-Aib-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-Arg(N0₂)-AAKE-(2F-Phe)-

ICWL-Nle-N

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKEFICWLMN

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(2F-Phe)-ICWLMN

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(2F-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle-

N ^·

H-(ACP)-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(2F-Phe)- ICWL-Nle-N H-(ACP)-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-Arg(N0₂)-AAKE-(2F- Phe)-ICWL-Nle-N

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2,6-diF-Phe)- ICWL-Nle-N

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-Aib-QAAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle-N

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle-N

H-Aib-QGT-(a-Me-2₅6-diF-Phe)-TSDYS-Arg(N02)-YLD-Aib-Arg(N0₂)-AAKE- (a-Me-2,6-diF-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-QAA E-(a-Me-2,6-diF- Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(a- Me-2,6-diF-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me- 2,6-diF-Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N02)-YLD-(ACP)-Arg(N0₂)- AAKE-(a-Me-2,6-diF-Phe)-ICWL-Nle-N

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLD-Aib-QAAKEFICWLMN

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)-

ICWLMN

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle-N

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle-N

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(a-Me-2F-Phe)- ICWL-Nle-N

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(a-Me-2F- Phe)-ICWL-Nle-N

' H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(a-Me-2F-Phe)-ICWL- Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(a-Me- 2F-Phe)-ICWL-Nle-N

, H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(a-Me-2F-Phe)- ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me-2F- Phe)-ICWL-Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-Arg(N0₂)-AAKE- (a-Me-2F-Phe)-ICWL-Nle-N

H-Aib-QGT-(2F-P e)-TSDYSKYLDEQAAKEFICWLM

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAA EFICWLM

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDEQAAKEFICWLM

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKE-(2F-Phe)-ICWLM

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAAKE-(a-Me-2,6-diF-Phe)- ' ICWLM

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDEQAAKE-(a-Me-2F-Phe)-ICWLM

H-Aib-QGT-(2F-Phe)-TSDYSKYLD-Aib-QAAKEFICWLM

H-Aib-QGT-(2F-Phe)-TSDYSKYLD-Aib-QAAKE-(2F-Phe)-ICWLM

H-Aib-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(2F-Phe)-ICWL-Nle

H-Aib-QGT-(2F-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(2F-Phe)-ICWL-Nle

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(2F-Phe)-ICWL-Nle

H-Aib-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-Arg(N0₂)-AAKE-(2F-Phe)- ICWL-Nle

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKEFICWLM

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(2F-Phe)-ICWLM

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle

H-(ACP)-QGT-(2F-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(2F-Phe)-ICWL-Nle

H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(2F-Phe)-ICWL^Nle

H-(ACP)-QGT-(2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(2F-Phe)-

ICWL-Nle

H-(ACP)-QGT-(2F-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(2F-Phe)-ICWL-Nle , H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(2F-Phe)-ICWL- Nle

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-QAA E-(a-Me-2,6-diF- Phe)-ICWL-Nle

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-Arg(N0₂)-AAKE- (a-Me-2,6-diF-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2,6-diF- Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(a-Me-2,6- diF-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(a- Me-2,6-diF-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(a-Me-2,6- ■ diF-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me- 2,6-diF-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-Arg(N0₂)- AA E-(a-Me-2,6-diF-Phe)-ICWL-Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKEFICWLM

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWLM ' H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-QAAKE-(a-Me-2F-Phe)- ICWL-Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-QAAKE-(a-Me-2F- Phe)-ICWL-Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-Aib-R-AAKE-(a-Me-2F-Phe)-ICWL- Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-AAKE-(a-Me-2F-Phe)- ICWL-Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-Aib-Arg(N0₂)-AAKE-(a- Me-2F-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAAKEFICWLM

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2F-Phe)-

ICWLM

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAA E-(a-Me-2F-Phe)- ICWL-Nle

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle

' H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Arg(N0₂)-YLD-(ACP)-QAAKE-(a-Me- 2F-Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-R-YLD-(ACP)-R-AAKE-(a-Me-2F-Phe)- ICWL-Nle H-(ACP)-QGT-( -Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-AAKE-(a-Me-2F- Phe)-ICWL-Nle

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Arg( 0₂)-YLD-(ACP)-Arg(N0₂)-AAKE-

(a-Me-2F-Phe)-ICWL-Nle

H-Aib-QGTFTSDYS YLDEQAAKEFICWLMT

H-Aib-QGT-(2F-Phe)-TSDYS YLDEQAA EFICWLMT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAAKEFICWLMT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDEQAAKEFICWLMT

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKE-(2F-Phe)-ICWLMT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDEQAAKE-(a-Me-2,6-diF-Phe)-

ICWLMT

, H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDEQAAKE-(a-Me-2F-Phe)-ICWLMT H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)- ICWLMT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-QAAKE-(a-Me-2F-Phe)-ICWL- Nle-T

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-QAAKE-(a-Me-2F-Phe)- ICWL-Nle-T

H-Aib-QGT-(2F-Phe)-TSDYSKYLDEQAAKE-(2F-Phe)-ICWLMT (E₁₆-K₂₀ Lactam)

H-Aib-QGT-(2F-Phe)-TSDYSKYLDSRRAQDFVQWLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQDFVQWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSRRAQDFVQWLMNT

H-Aib-QGT-(2F-Phe)- TSDYSKYLDSRRAQD-(2F-Phe)-VQWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSRPvAQD-(a-Me-2F-Phe)- VQWLMNT H-Aib-QGT-(2F-Phe)-TSDYSKYLDSRRAQDFV-C-WLMNT

H-Aib-QGT-(2F-Phe)- TSDYSKYLDSR AQD-(2F-Phe)-V-C-WLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQD-(a-Me-2,6-diF-Phe)- V- C-WLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSRRAQD-(a-Me-2F-Phe)- V-C- WLMNT

H-Aib-QGT-(2F-Phe)-TSDYSKYLDSRRAQDFV-C-WL-Nle-NT H-Aib-QGT-( -Me-2,6-diF-Phe)-TSDYSKYLDSRRAQDFV-C-WL-Nle-NT H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLDSPvRAQDFV-C-WL-Nle-NT

H-Aib-QGT-(2F-Phe)- TSDYSKYLDSRRAQD-(2F-Phe)-V-C-WL-Nle-NT H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSRRAQD-(a-Me-2F-Phe)-V-C-WL- Nle-NT

H-Aib-QGT-(2F-Phe)- TSDYSKYLD-Aib-RPvAQD-(2F-Phe)-V-C-WLMNT H-Aib-QGT-(2F-Phe)- TSDYSKYLD-Aib-RRAQD-(2F-Phe)-V-C-WL-Nle-NT H-(ACP)-QGT-(2F-Phe)- TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WL-Nle- NT

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-RPvAQDFV-C-WLMN

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WL-Nle-N

H-(ACP)-QGT-(2F-Phe)- TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WLMN

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WLM

H-(ACP)-QGT-(2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(2F-Phe)-V-C-WL-Nle

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WL-NIe-NT

H-Aib-QG.T-(a-Me-2,6-diF-Phe)-TSDYS YLD-Aib-RRAQD-(a-Me-2,6-diF-Phe)-

V-C-WLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS YLD-Aib-RRAQD-(a-Me-2,6-diF-Phe)- V-C-WL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS YLD-(ACP)-RRAQD-(a-Me-2,6-diF- Phe)-V-C-WL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WLMN

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS YLD-(ACP)-RRAQDFV-C-WL-Nle-

N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C,WL-Nle

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS YLD-(ACP)-RRAQD-(a-Me-2,6-diF-

Phe)-V-C-WLM

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2,6-diF- Phe)-V-C-WL-Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WL-Nle-NT H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQD-(a-Me-2F-Phe)-V-C- WLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQD-(a-Me-2F-Phe)-V-C- WL-Nle-NT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WLMNT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WL-Nle-NT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV^C-WLMN H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WL-Nle-N H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-PvRAQD-(a-Me-2F-Phe)-V- C-WLMN

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQD-(a-Me-2F-Phe)-V- C-WL-Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-RRAQDFV-C-WLM H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)- RAQDFV-C-WL-Nle H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-PvRAQD-(a-Me-2F-Phe)-V- C-WLM

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)-PvRAQD-(a-Me-2F-Phe)-V- C-WL-Nle

H-Aib-QGT-(2F-Phe)-TSDYSKYLDSRRAQDFVQWLMT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQDFVQWLMT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLDSRRA DFVQWLMT

H-Aib-QGT-(2F-Phe)- TSDYSKYLDSRRAQD-(2F-Phe)-VQWLMT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYSKYLDSRRAQD-(a-Me-2,6-diF-Phe)- VQWLMT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS YLD-Aib-RRAQDFVQWL T

H-Aib-QGT-(a-Me-2F-Phe)-TSDYSKYLD-Aib-RRAQDFV-C-WLMT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)- RAQDFVQWLMT H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYSKYLD-(ACP)- RAQDFV-C-WLMT H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFVQWLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har- AQDFVQWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFVQWLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2F-Phe)-

VQWLMNT

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C- WLMNT .

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har YLDS-Har-Har-AQDFV-C-WLMNT H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(2F-Phe)-V-C- , WLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2F-Phe)- V-C-WLMNT

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WL-Nle-NT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WL-

Nle-NT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFV-C-WL-Nle- Γ

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2,6- diF-Phe)-V-C-WL-Nle-NT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2F-Phe)- V-C-WL-Nle-NT

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C-WLMNT H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C-WL-Nle-NT H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(2F-Phe)-V-C- WLMNT

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLD Aib-Har-Har-AQD-(2F-Phe)-V-C-WL- Nle-NT

H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(2F-Phe)-V-C- WL-Nle-NT

H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C-WLMN ' H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C-WLM H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C-WL-Nle H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(2F-Phe)-V-C- WLM

H-(ACP)-QGT-(2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(2F-Phe)-V-C- WL-Nle

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C- WLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C- WL-Nle-NT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(a-Me- 2,6-diF-Phe)-V-C-WLMNT

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(a-Me- 2,6-diF-Phe)-V-C-WL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV- C-WLMNT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLP-(ACP)-Har-Har-AQD-(a- Me-2,6-diF-Phe)-V-C-WL-Nle-NT

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV- C-WLMN

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV- C-WL-Nle-N

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a- Me-2,6-diF-Phe)-V-C-WLMN

H-(ACP)-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a- Me-2,6-diF-Phe)-V-C-WL-Nle

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C- WLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C-WL- Nle-NT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(a-Me-2F- Phe)-V-C-WLMNT

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQD-(a-Me-2F- Phe)-V-C-WL-Nle-NT H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C- WLMNT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C- WL-Nle-NT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a-Me- 2F-Phe)-V-C-WLMNT

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a-Me- 2F-Phe)-V-C-WL-Nle-N

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C- WLM

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C- WL-Nle

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a-Me- 2F-Phe)-V-C-WLM

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQD-(a-Me- 2F-Phe)-V-C-WL-Nle

H-Aib-QGT-(2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFVQWL-Nle-T

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFVQWL-

Nle-T

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLDS-Har-Har-AQDFVQWL-Nle-T H-Aib-QGT-(2F-Phe)- TSDYS-Har-YLDS-Har-Har-AQD-(2F-Phe)-VQWL-Nle- T

H-Aib-QGT-(a-Me-2,6-diF-Phe)-TSDYS-Har-YLDS-Har-Har-AQD-(a-Me-2,6- diF-Phe)- VQWL-Nle-T

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFVQWL-Nle- T

H-Aib-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-Aib-Har-Har-AQDFV-C-WL- Nle-T

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFVQWL- Nle-T

H-(ACP)-QGT-(a-Me-2F-Phe)-TSDYS-Har-YLD-(ACP)-Har-Har-AQDFV-C- WL-Nle-T

9. A pharmaceutical composition comprising compounds of formula (I) as defined in claim 1 and optionally one or more pharmaceutically acceptable carriers, excipients or diluents.

10. The compounds of formula (I) as defined in claim 1 or their pharmaceutical compositions containing them, which act as an agonist of the GLP-1, GIP & glucagon receptors.

11. The compounds of formula (I) as defined in claim 1 or their pharmaceutical compositions containing them for the treatment or prevention of diseases caused by hyperlipidaemia, hypercholesteremia, hyperglycemia, hyperinsulinemia, elevated blood levels of free fatty acids or glycerol, hypertriglyceridemia, wound healing, impaired glucose tolerance, leptin resistance, insulin resistance or other diabetic complications.

12. A method of preventing or treating diseases caused by hyperlipidaemia, hypercholesteremia, hyperglycemia, hyperinsulinemia, elevated blood levels of free fatty acids or glycerol, hypertriglyceridemia, wound healing, impaired glucose tolerance, leptin resistance, insulin resistance or other diabetic complications comprising administering an effective, non-toxic amount of compound of formula (I) as defined in claim no 1 or their suitable pharmaceutical composition to a patient in need of such treatment.

13. A medicine for treating/reducing any of the disease conditions described in any preceding claims which comprises administering a compound of formula (I), as claimed in any preceding claims and a pharmaceutically acceptable carrier, diluent, excipients or solvate to a patient in need thereof.

14. Use of compounds of formula (I) or their pharmaceutical composition as defined in claim 1 for the preparation of medicines suitable for the treatment of diseases mentioned in any of the aforesaid claims.