GLP-1 derivatives

Description of the invention

The invention relates to a GLP-1 (7-37) derivative or GLP-1 (7-37) analogue, a pharmaceutical agent and the use of a GLP- (7-37) derivative or GLP-1 (7-37) analog.

Peptides are widely used in medicine and, since their production using recombinant DNA techniques, it can be expected that their importance will also increase in the coming years. When native peptides or their analogs are used in therapy, it is generally found that they exhibit a high level of clearance. The high level of clearance of a therapeutic agent may be inconvenient in cases where it is required to maintain high levels in the blood for prolonged periods of time, as multiple administrations may then be necessary.

Examples of high clearance peptides are: ACTH, corticotropin releasing factor, angiotensin, calcitonin, insulin, glucagon, glucagon-like peptide-1, glucagon-like peptide-2, insulin-like growth factor-1, insulin-like growth factor-2, gastrin inhibitory peptide, growth hormone releasing factor, pituitary adenylate cyclase activating peptide , secretin, enterogastrin, somatostatin, somatropin, parathyroid gland hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyrotropic hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and their analogues, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase and ribonuclease. In some cases, it is possible to influence the release profile of the peptides by the use of appropriate pharmaceutical agents, however, this solution has many different drawbacks and is not generally used.

The hormones that regulate insulin secretion belong to the so-called enteroinsular axis, that is, a group of hormones released from the gastrointestinal mucosa in response to the presence and absorption in the gut of nutrients that induce early and increased insulin release. The insulin secretion-enhancing effect, the so-called incretin effect, is possibly crucial for normal glucose tolerance. Many gastrointestinal hormones, including gastrin and secretin (cholecystokinin is not an insulinotropic hormone in humans), are insulinotropic hormones, but the only physiologically significant glucose-dependent polypeptide, GIP, and glucagon-like peptide-1 (GIP-) are the only physiologically relevant hormones responsible for the incretin effect. 1). GIP, isolated in 1973 (1), immediately aroused considerable interest among diabetologists due to its insulinotropic properties. However, many studies conducted over the years clearly showed that the disturbance of GIP secretion is not associated with the pathogenesis of insulin-dependent diabetes (IDDM) and non-insulin-dependent diabetes (NIDDM) (2).

Moreover, it has been found that the insulinotropic hormone, GIP, is almost ineffective in NIDDM (2). Another incretin hormone, GLP-1, is the most potent insulinotropic substance known (3). Unlike GIP, it is surprisingly effective in stimulating insulin secretion in NIDDM patients. Moreover, and unlike other insulinotropic hormones (possibly with the exception of secretin), it strongly inhibits the secretion of glucagon. Due to this action, it has a potentiated blood glucose lowering effect, especially in patients with NIDDM.

GLP-1, a product of proglucagon (4), is one of the youngest members of the secretin-VIP peptide family, but has now been established to be an important gut hormone with a regulatory function in glucose metabolism and gastrointestinal secretion and metabolism (5). The glucagon gene is processed differently in the pancreas and intestine. In the pancreas (9) the treatment leads to the formation and simultaneous secretion of 1) glucagon itself, comprising positions 33-61 of proglucagon (PG); 2) an N-terminal peptide of 30 amino acids (PG 1-30), often called glycentin-related peptide, GRPP (10, 11); 3) the hexapeptide corresponding to PG (64-69); 4) and the so-called major fragment of proglucagon (PG (72-158)) carrying two glucagon-like sequences (9). Glucagon appears to be the only biologically active product. In contrast, in the intestinal mucosa, glucagon is found in the larger molecule, with the two glucagon-like peptides being formed separately (8).

The following products are formed and secreted simultaneously: 1) glycentin, corresponding to PG (1-69), with the glucagon sequence occupying positions Nos. 33-61 (12); 2) GLP-1 (7-36) amide (PG (78-107)) amide (13) (not as originally thought PG (72-107) amide or 108 which is inactive). There are also small amounts of glycine-extended but equally biologically active GLP-1 (7-37) peptides (PG (78-108)) (14); 3) linker peptide-2 (PG (111-122) amide) (15); and 4) GLP-2

PL 192 359 B1 (PG (126-158)) (15, 16). The glycentin fraction is then cleaved into GRPP (PG 1-30)) and oxyntomodulin (PG (33-69)) (17, 18). Of these peptides, GLP-1 has the most prominent biological properties.

Due to the simultaneous secretion of GLP-1 with glycentin / enteroglucagon, many studies on the secretion of enteroglucagon (6, 7) also relate to some extent to GLP-1 secretion, but GLP-1 is metabolized much faster, with a plasma half-life in humans of 2 minutes (19). Carbohydrate or fat-rich meals stimulate secretion (20), possibly as a result of direct interaction of as yet unabsorbed nutrients with the open-type L-cell microvilli of the intestinal mucosa. There may be endocrine or neural mechanisms to produce GLP-1 but have not been shown to exist in humans.

The incretin function of GLP-1 (29-31) was clearly illustrated in experiments with the GLP-1 receptor antagonist, exendin 9-39, which significantly reduces the incretin effect induced by oral administration of glucose in rats (21, 22). The hormone interacts directly with β cells via the GLP-1 receptor (23) belonging to the family of G-protein-linked glucagon / VIP / calcitonin receptors having 7 transmembrane domains.

The important role of the GLP-1 receptor in the regulation of insulin secretion has been illustrated in recent experiments in which targeted disruption of the GLP-1 receptor gene was performed in mice. Animals homozygous for this violation showed a significant deterioration of glucose tolerance and hyperglycemia during fasting, and even heterozygous animals showed glucose intolerance (24). The signal transduction mechanism (25) is initially associated with the activation of adenylate cyclase, but the elevation of the intracellular Ca level²⁺it is also important (25, 26). The action of the hormone is best described as enhancement of glucose-stimulated insulin release (25), but the linking mechanism between glucose and GLP-1 stimulation is unknown. It may be related to calcium-induced release of calcium (26, 27).

As already mentioned, the insulinotropic effect of GLP-1 is preserved in diabetic β cells. The association of the latter with their ability to confer "glucose competence" to isolated insulin secreting cells (26, 28), which respond poorly to either glucose alone or GLP-1, but fully to a combination of both, is also unknown.

However, it is equally important that this hormone also strongly inhibits the secretion of glucagon (29). The mechanism of this action is unknown, but it appears to be paracrine, via adjacent insulin or somatostatin cells (25). Moreover, the glucagonostatic effect is glucose dependent, so that the inhibitory effect decreases as the blood glucose level decreases. Due to this dual effect, if the plasma concentration of GLP-1 is increased, either by increased secretion or by exogenous administration, the molar ratio of insulin to glucagon in the blood reaching the liver via the portal circulation is significantly increased, thereby reducing hepatic glucose production. (thirty).

As a result, blood glucose levels are lowered. Due to the glucose-dependence of insulinotropic and glucagonostatic effects, the glucose-lowering effect is self-limiting, and therefore this hormone does not cause hypoglycaemia regardless of the dose (31). This effect is preserved in patients with diabetes mellitus (32), in whom administration of slightly above-physiological doses of GLP-1 can completely normalize blood glucose levels, despite poor metabolic control and secondary damage to the sulfonylurea response (33). The role of glucagonostatic action is illustrated by the finding that GLP-1 also lowers blood glucose levels in patients with type 1 diabetes without residual β-cell secretory capacity (34).

In addition to its action on pancreatic islets, GLP-1 has a strong effect in the gastrointestinal tract. When administered in physiological amounts, GLP-1 strongly inhibits pentagastrin-induced gastric acid secretion, as well as food (35, 36). It also inhibits the rate of gastric emptying and the secretion of pancreatic enzymes (36).

A similar effect of inhibiting gastric and pancreatic secretion and motility can be induced in humans by perfusion of the ileum with solutions containing carbohydrates or lipids (37, 38). At the same time, GLP-1 secretion is strongly stimulated, and it was speculated that GLP-1 might be at least partially responsible for the so-called “ileal puncture effect (38). In fact, recent studies have suggested that the physiological puncture effect of GLP-1 in the ileum may be more significant than the effect on pancreatic islets. Thus, according to the results of dose-response studies, GLP-1 affects gastric emptying when administered in amounts at least as low as required to affect islet secretion (39).

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GLP-1 appears to have an effect on food intake. The intraventricular administration of GLP-1 significantly inhibits food intake in rats (40, 42). This effect appears to be highly specific. Thus, the N-terminally extended GLP-1 (PG 72-107) amide is inactive and appropriate dosages of the GLP-1 antagonist Extendin 9-39 abolish the effects of GLP-1 (41). Acute, peripheral administration of GLP-1 did not acutely inhibit food intake in rats (41, 42). However, it is still possible that GLP-1 secreted from intestinal L cells may act as a satiety signal.

Not only the insulinotropic effects, but also the gastrointestinal effects of GLP-1 persist in patients with diabetes mellitus (43) and may help reduce food-induced increases in glucose levels, and more importantly, may affect food intake. It has been shown that intravenous administration of GLP-1 at 4 ng / kg / min, over one week, significantly improves blood glucose control in NIDDM patients without significant side effects (44). The peptide is fully active after subcutaneous administration (45), but is rapidly degraded mainly by dipeptidyl peptidyl IV-like enzymes (46, 47).

Literature cited above

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2. Krarup T. Immunoreactive gastric inhibitors of polypep tide. Endocr Rev 1988; 9: 122-134.

3. 0rskov C. Glucagon-like peptide-1, a new hormone of the enteroinsular axis. Diabetology

1992; 35:701-711.

4. Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC. Exon duplication and divergence in the human preproglucagon gene. Nature 1983; 304: 368-371.

5. Holst J J. Glucagon-like peptide-1 (GLP-1) - a newly discovered GI hormone. Gastroenterology 1994; 107: 1848-1855.

6. Holst JJ. Gut glucagon, enteroglucagon, gut GLI, glicentin-current status. Gastroenterology

1983;84:1602-1613.

7. Holst JJ, 0rskov C. Glucagon and other proglucagon-derived peptides. In Walsh JH, Dockray GJ, eds. Gut peptides: Biochemistry and Physiology. Raven Press, New York, pp. 305-340,1993.

8. 0rskov C, Holst JJ, Knuhtsen S, Baldissera FGA, Poulsen SS, Nielsen OV. Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separately from the pig smali intestine, but not pancreas. Endocrinology 1986; 119: 1467-1475.

9. Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, 0rskov C. Proglucagon processing in porcine and human pancreas. J Biol Chem, 1994; 269: 18827-1883.

10. Moody AJ, Holst JJ, Thim L, Jensen SL. Relationship of glicentin to proglucagon and glucagon in the porcine pancreas. Nature 1981; 289: 514-516.

11. Thim L, Moody AJ, Purification and chemical characterization of a glicentin-related pancreatic peptide (proglucagon fragment) from porcine pancreas. Biochim Biophys Acta 1982; 703: 134-141.

12. Thim L, Moody AJ. The primary structure of glicentin (proglucagon). Regul Pept 1981; 2: 139-151.

13. 0rskov C, Bersani M, Johnsen AH, H0jrup P, Holst JJ. Complete sequences of glucagon-like peptide-1 (GLP-1) from human and pig smali intestine. J. Biol. Chem. 1989; 264: 12826-12829.

14. 0rskov C, Rabenh0j L, Kofod H, Wettergren A, Holst JJ. Production and secretion of amidated and glycine-extended glucagon-like peptide-1 (GLP-1) in man. Diabetes 1991; 43: 535-539.

15. Buhl T, Thim L, Kofod H, 0rskov C, Harling H, & Holst JJ: Naturally occurring products of proglucagon 111-160 in the porcine and human smali intestine. J. Biol. Chem. 1988; 263: 8621-8624.

16. 0rskov C, Buhl T, Rabenh0j L, Kofod H, Holst JJ: Carboxypeptidase-B-like processing of the C-terminus of glucagon-like peptide-2 in pig and human smali intestine. FEBS Letters, 1989; 247: 193-106.

17. Holst JJ. Evidence that enteroglucagon (II) is iden tical with the C-terminal sequence (residues 33-69) of glicentin. Biochem. J. 1980; 187: 337-343.

18. Bataille D, Tatemoto K, Gespach C, Jornvall H, Rosselin G, Mutt V. Isolation of glucagon-37 (bioactive enteroglucagon / oxyntomodulin) from porcine jejuno-ileum. Characterization of the peptide. FEBS Lett. 1982; 146: 79-86.

19. 0rskov C, Wettergren A, Holst JJ. The metabolic rate and the biological effects of GLP-1 7-36 amide and GLP-1 7-37 in healthy volunteers are identical. Diabetes 1993; 42: 658-661.

twenty. Elliott RM, Morgan LM, Tredger JA, Deacon S, Wright J, Marks V. Glucagon-like peptide-1 (7-36) amide and glucose-dependent insulinotropic polypeptide secretion in response to

Nutrient ingestion in man: acute post-prandial and 24-h secretion patterns. J. Endocrinol. 1993; 138: 159-166.

21. Kolligs F, Fehmann HC, Goke R, Goke B. Reduction of the incretin effect in rats by the glucagon-like peptide-1 receptor antagonist exendin (9-39) amide. Diabetes 1995; 44: 16-19.

22. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei M, Bloom SR. Glucagon-like peptide-1 is a physiological incretin in rat. J. Clin. Invest. 1995; 95: 417-421.

23. Thorens B. Expression cloning of the pancreatic b cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc. Natl. Acad. Sci. 1992; 89: 8641-4645.

24. Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ. Diabetes in mice with targeted disruption of the GLP-1 receptor gene. Diabetes 1996; 45:21 A.

25. Fehmann HC, Goke R, Goke B. Cell and molecular biology of the incretin hormones glucagon-like peptide-I (GLP-1) and glucose-dependent insulin releasing polypeptide (GIP). Endocrine Reviews, 1995; 16: 390-410.

26. Gromada J, Dissing S, Bokvist K, Renstrom E, Fr0kjćr-Jensen J, Wulff BS, Rorsman P. Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting bTC3-cells by enhancement of intracellular calcium mobilization. Diabetes 1995; 44: 767-774.

27. Holz GG, Leech CA, Habener JF. Activation of a cAMP-regulated Ca²⁺-signaling pathway in pancreatic β-cells by the insulinotropic hormone glucagon-like peptide-1. J. Biol. Chem. 1996; 270: 17749-17759.

28. Holz GG, Kijhltreiber WM, Habener JF.- Pancreatic beta-cells are rendered glucose competent by the insulinotropic hormone glucagon-like peptide-1 (7-37). Nature 1993, 361: 362-365.

29. 0rskov C, Holst JJ, Nielsen OV: Effect of truncated glucagon-like peptide-1 (proglucagon 78-107 amide) on endocrine secretion from pig pancreas, antrum and stomach. Endocrinology 1988; 123: 2009-2013.

thirty. Hvidberg A, Toft Nielsen M, Hilsted J, Orskov C, Holst JJ. Effect of glucagon-like peptide-1 (proglucagon 78107 amide) on hepatic glucose production in healthy man. Metabolism 1994; 43: 104-108.

31. Qualmann C, Nauck M, Holst JJ, 0rskov C, Creutzfeldt W. Insulinotropic actions of intravenous glucagon-like peptide-1 [7-36 amide] in the fasting state in healthy subjects. Acta Diabetologica, 1995; 32: 13-16.

32. Nauck MA, Heimesaat MM, 0rskov C, Holst JJ, Ebert R, Creutzfeldt W. Preserved incretin activity of GLP-1 (7-36 amide) but not of synthetic human GIP in patients with type 2-diabetes mellitus. J. Clin. Invest. 1993; 91: 301-307.

33. Nauck MA, Kleine N, 0rskov C, Holst JJ, Willms B, Creutzfeldt W. Normalization of fasting hyperglycaemia by exogenous GLP-1 (7-36 amide) in type 2-diabetic patients. Diabetologia 1993; 36: 741-744.

34. Creutzfeldt W, Kleine N, Willms B, 0rskov C, Holst JJ, Nauck MA. Glucagonostatic actions and reduction of fasting hyperglycaemia by exogenous glucagon-liem, peptide-1 (7-36 amide) in type I diabetic patients. Diabetes Care 1996; 19: 580-586.

35. Schjoldager BTG, Mortensen PE, Christiansen J, 0rskov C, Holst JJ. GLP-1 (glucagon-like peptide-1) and truncated GLP-1, fragments of human proglucagon, inhibit gastric acid secretion in man. Dig. Dis. Sci. 1989; 35: 703-708.

36. Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ. Truncated GLP-1 (proglucagon 72-107amide) inhibits gastric and pancreatic functions in man. Dig. Dis. Sci. 1993, 38: 665-673.

37. Layer P, Holst JJ, Grandt D, Goebell H: Ileal release of glucagon-like peptide-1 (GLP-1): association with inhibition of gastric acid in humans. Dig. Dis. Sci. 1995; 40: 1074-1082.

38. Layer P, Holst JJ. GLP-1: A humoral mediator of the ileal brake in humans? Digestion 1993; 54: 385-386.

39. Nauck M, Ettler R, Niedereichholz U, 0rskov C, Holst JJ, Schmiegel W. Inhibition of gastric emptying by GLP-1 (7-36 amide) or (7-37): effects on postprandial glycaemia and insulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A124.

40. Schick RR, vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M. Glucagon-like peptide 1-a novel brain peptide involved in feeding regulation. In Ditschuneit H, Gries FA, Hauner H, Schusdziarra V, Wechsler JG (eds.) Obesity in Europe. John Libbey & Company Etc., 1994; pp. 363-367.

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41. Tang-Christensen M, Larsen PJ, Goke R, Fink-Jensen A, Jessop DS, M0ller M, Sheikh S. Brain GLP-1 (7-36) amide receptors play a major role in regulation of food and water intake. Am. J. Physiol., 1996, in press.

42. Turton MD, O 'Shea D, Gunn I, Beak SA, Edwards CMB, Meeran K, et al. A role for glucagon-like peptide-1 in the regulation of feeding. Nature 1996; 379: 69-72.

43. Willms B, Werner J, Creutzfeldt W, 0rskov C, Holst JJ, Nauck M. Inhibition of gastric emptying by glucagon-like peptide-1 (7-36 amide) in patients with type-2-diabetes mellitus. Diabetologia 1994; 37, suppl. 1: A118.

44. Larsen J, Jallad N, Damsbo P. One-week continuous infusion of GLP-1 (7-37) improves glycaemic control in NIDDM. Diabetes 1996; 45, suppl. 2: 233A.

45. Ritzel R, 0rskov C, Holst JJ, Nauck MA. Pharmacokinetic, insulinotropic, and glucagonostatic properties of GLP1 [7-36 amide] after subcutaneous injection in healthy volunteers. Dose-response relationships. Diabetologia 1995; 38: 720-725.

46. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J. Clin. Endocrinol. Metab. 1995; 80: 952-957.

47. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L, Willms B, Holst JJ. 1995. Both subcutaneous and intravenously administered glucagon-like peptide-1 are rapidly degraded from the amino terminus in type II diabetic patients and in healthy subjects. Diabetes 44: 1126-1131.

The amino acid sequence of GLP-1 was reported e.g. by Schmidt et al (Diabetologia 28, 704-707 (1985)). Although the interesting pharmacological properties of GLP-1 (7-37) and its analogues have attracted a lot of attention in recent years, little is known about the structure of these molecules. The secondary structure of GLP-1 in micelles has been described by Thorton et al (Biochemistry 33, 3532-3539 (1994)), but GLP-1 is considered a very flexible molecule in normal solutions. It was surprisingly found that derivatization of this relatively small and very flexible molecule provided compounds whose plasma levels are maintained for a prolonged period of time while remaining active.

GLP-1 and GLP-1 analogs and fragments thereof are potentially useful, eg, in the treatment of type 1 and type 2 diabetes. However, the high level of clearance limits the utility of these compounds, and there is still a need for improvements in the art. Thus, one of the objectives of the invention is to provide GLP-1 derivatives and analogues thereof showing a prolonged profile of action compared to GLP-1 (7-37). It is a further object of the invention to provide GLP-1 derivatives and analogues thereof with lower clearance than GLP-1 (7-37). Another object of the invention is to provide a pharmaceutical agent containing a compound of the invention and the use of a compound of the invention to deliver such agent.

Human GLP-1 is a 37 amino acid peptide derived from a preproglucagon synthesized in, inter alia, L cells in the distal ileum, pancreas and brain. The processing of preproglucagon to GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2 occurs mainly in L cells. A simple system was used to describe the fragments and analogs of this peptide. So for example Gly⁸-GLP-1 (7-37) is a GLP-1 fragment formally derived from GLP-1 by deleting amino acid residues 1-6 and replacing the naturally occurring amino acid residue at position 8 (Ala) with Gly.

Likewise, Lys³⁴(NMetradecanoyl) -GLP-1 (7-37) means GLP-1 (7-37) in which the ε-amino group at the Lys residue at position 34 has been tetradecanoylated. When C-terminally extended GLP-1 analogs are mentioned in the text, the amino acid residue at position 38 is Arg, unless otherwise specified, the optional amino acid residue at position 39 is also Arg, unless otherwise stated, and the optional amino acid residue at position 39 is position 40 is Asp, unless otherwise stated. Moreover, in the case where the C-terminal extended analog is extended to position 41, 42, 43, 44, or 45, the amino acid sequence in that extension corresponds to that of the human preproglucagon, unless otherwise stated.

Thus, the invention relates to GLP-1 derivatives and their analogs. The derivatives according to the invention show interesting pharmacological properties, in particular an extended activity profile compared to the parent peptide.

As used herein, the term "analog is used to denote a peptide in which one or more amino acid residues of the parent peptide have been replaced with a different amino acid residue, and / or in which one

One or more amino acid residues of the parent peptide have been deleted and / or wherein one or more amino acid residues have been added to the parent peptide. Such additions can take place at the N-terminus and / or C-terminus of the parent peptide.

The term "derivative" as used herein denotes a peptide in which one or more amino acid residues of the parent peptide have been chemically modified, e.g., by alkylation, acylation, ester formation, or amide formation.

The term "derivative of GLP-1 (7-37) as used herein means a derivative of GLP-1 (7-37) or an analog thereof. In the specification, the parent peptide from which such a derivative is formally derived is at some places referred to as the "GLP-1 (7-37) moiety of the derivative."

Thus, the invention relates to a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP receptor -1 having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein said lipophilic substituent is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue.

Furthermore, the invention relates to a GLP-1 derivative (7-37) or a GLP-1 analog (7-37) in which up to 6 amino acid residues have been altered from GLP-1 (7-37), an agonist of the human GLP-1 receptor. having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl. wherein one of said lipophilic substituents is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue.

Furthermore, the invention relates to a GLP-1 derivative (7-37) or a GLP-1 analog (7-37) in which up to 6 amino acid residues have been altered from GLP-1 (7-37), an agonist of the human GLP-1 receptor. having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl. wherein said lipophilic substituents are attached to amino acid residues other than the N-terminal or C-terminal amino acid residues.

Furthermore, the invention relates to a derivative of a GLP-1 (7-37) analogue in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor, the analog being GLP-1 ( 7-C) where the value of C is 38-45 having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein said lipophilic substituent is attached to the C-terminal amino acid residue.

Furthermore, the invention relates to a derivative of a GLP-1 (7-37) analogue in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor, said analog being GLP-1. (AB) where A is an integer from 1 to 7 and B is an integer from 38 to 45 containing one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO-, where m is 4-38, and lithocholyl, and attached to a C-terminal amino acid residue, and a second optional lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, and attached to one of the other amino acid residues.

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In the above-defined GLP-1 (7-37) derivatives according to the invention, the GLP-1 (7-37) analogue is preferably selected from the group consisting of:

Arg²⁶-GLP-1 (7-37); Arg³⁴-GLP-1 (7-37);

Lys³⁶-GLP-1 (7-37); Arg²⁶'³⁴Lys³⁶-GLP-1 (7-37);

Arg²⁶'34_Lys38_GLP_₁(7_₃₈). _Arg26,34_Lys39__GLP_₁(7-39)_;

Arg²⁶'³⁴Lys⁴⁰-GLP-1 (7-40); Arg²⁶Lys³⁶-GLP-1 (7-37);

Arg³⁴Lys³⁶-GLP-1 (7-37); Arg²⁶Lys³⁹-GLP-1 (7-39);

Arg³⁴Lys⁴⁰-GLP-1 (7-40); Arg²⁶'³⁴Lys³⁶'³⁹-GLP-1 (7-39);

Arg²⁶'34_Lys36,40__glp_₁(7-40); Gly⁸Arg²⁶-GLP-1 (7-37);

Gly⁸Arg³⁴-GLP-1 (7-37); Gly⁸Lys³⁶-GLP-1 (7-37);

Gly⁸Arg²⁶'³⁴Lys³⁶-GLP-1 (7-37); Gly⁸Arg²⁶'³⁴Lys³⁹-GLP-1 (7-39); Gly⁸Arg²⁶'³⁴Lys⁴⁰-GLP-1 (7-40); Gly⁸Arg²⁶Lys³⁶-GLP-1 (7-37); Gly⁸Arg³⁴Lys³⁶-GLP-1 (7-37); Gly⁸Arg²⁶Lys³⁹-GLP-1 (7-39); Gly⁸Arg³⁴Lys⁴⁰-GLP-1 (7-40); Gly⁸Arg²⁶'³⁴Lys³⁶'³⁹-GLP-I (7-39) and Gly⁸Arg²⁶'³⁴Lys³⁶'⁴⁰-GLP-l (7-40).

In the above-defined GLP-1 (7-37) derivatives according to the invention, the GLP-1 (7-37) analogue is preferably selected from the group consisting of:

_Arg26,34_Lys38_GLP_₁(7-38)_; Arg²⁶'³⁴Lys³⁹GLP-1 (7-39);

Arg²⁶'34_Lys40_GLP_₁(7-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (7-41);

_Arg26,34_Lys42_GLP_₁(7-42); Arg²⁶'³⁴Lys⁴³GLP-1 (7-43);

Arg²⁶'³⁴Lys⁴⁴GLP-1 (7-44); Arg²⁶'³⁴Lys⁴⁵GLP-1 (7-45);

Arg²⁶'34_Lys38_GLP_₁(i_38); Arg²⁶'³⁴Lys³⁹GLP-I (1-39);

Arg²⁶'34_Lys40_GLP_₁(i-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (1-41);

Arg²⁶'³⁴Lys⁴²GLP-I (1-42); Arg²⁶'³⁴Lys⁴³GLP-I (1-43);

Arg²⁶'³⁴Lys⁴⁴GLP-1 (1-44); Arg²⁶'³⁴Lys⁴⁵GLP-1 (1-45);

Arg²⁶'³⁴Lys³⁸GLP-1 (2-38); Arg²⁶'³⁴Lys³⁹GLP-1 (2-39);

Arg²⁶'34_Lys40_GLP_₁(2-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (2-41);

Arg²⁶'34_Lys42_GLP_₁(2-42); Arg²⁶'³⁴Lys⁴³GLP-1 (2-43);

PL 192 359 B1 _Arg26,34_Lys44_GLP_i (2-44); Arg²⁶'³⁴Lys⁴⁵GLP-I (2-45);

Arg²⁶'34_Lys38_GLP_₁(3-38); Arg²⁶'³⁴Lys³⁹GLP-1 (3-39);

Arg²⁶'34_Lys40_GLP_i (3-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (3-41);

Arg²⁶'34Lys42GLP_1 (3-42); Arg²⁶'³⁴Lys⁴³GLP-1 (3-43); Arg26.34_Lys44_GLP_₁(3-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (3-45);

Arg²⁶'34_Lys38_GLp _! (4_38)_; Arg²⁶'³⁴Lys³⁹GLP-1 (4-39);

Arg²⁶'34_Lys40_GLp_₁(4-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (4-41);

Arg²⁶'34_Lys42_GLP_₁(4-42); Arg²⁶'³⁴Lys⁴³GLP-1 (4-43);

Arg²⁶'34_Lys44_GLp _! (4-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (4-45);

Arg²⁶'34_Lys38_GLp_i (5-38); Arg²⁶'³⁴Lys³⁹GLP-1 (5-39);

Arg²⁶'34£_γ340_ΟΕρ _! (5-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (5-41);

Arg²⁶'34_Lys42_GLP_₁(5-42); Arg²⁶'³⁴Lys⁴³GLP-1 (5-43);

Arg²⁶'34_Lys44_GLP_₁(5-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (5-45);

Arg²⁶'34_Lys38_GLp_₁(6-38); Arg²⁶'³⁴Lys³⁹GLP-1 (6-39);

Arg²⁶'34Lys40GLp_i (6-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (6-41); Arg26.34_Lys42_GLp_i (6-42); Arg²⁶'³⁴Lys⁴³GLP-1 (6-43);

Arg²⁶'34_Lys44_GLp_₁(6-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (6-45); Arg²⁶Lys³⁸GLP-I (1-38); Arg³⁴Lys³⁸GLP-I (1-38);

Arg²⁶'34_Lys36,38_GLp_x (i_38)_; Arg²⁶Lys³⁸GLP-1 (7-38); Arg³⁴Lys³⁸GLP-1 (7-38); Arg²⁶'³⁴Lys³⁶'³⁸GLP-1 (7-38);

Arg²⁶'34Lys38GLP_1 (7-38); Arg²⁶Lys³⁹GLP-I (1-39); Arg³⁴Lys³⁹GLP-I (1-39) and Arg²⁶'³⁴Lys³⁶'³⁹GLP-1 (1-39); Arg²⁶Lys³⁹GLP-1 (7-39); Arg³⁴Lys³⁹GLP-1 (7-39) and Arg²⁶'34 Lys36, 39_glp_i (7-39).

Furthermore, the invention relates to a derivative of a GLP-1 (7-37) analogue in which up to 6 amino acid residues have been altered from GLP-1 (7-37), which is an agonist of the human GLP-1 receptor, in which at least one amino acid residue is a lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl wherein said analog is selected from the group consisting of :

PL 192 359 B1

Arg²⁶'34Lys38GLP_1 (7-38); Arg²⁶'³⁴Lys³⁹GLP-1 (7-39); Arg²⁶'34Lys40_GLp _! (7-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (7-41); Arg²⁶'34Lys42GLp_1 (7-42); Arg²⁶'³⁴Lys⁴³GLP-1 (7-43); Arg²⁶'34Lys44_GLP_₁(7-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (7-45); Arg26, 34Lys38GLp_i (1-38); Arg²⁶'³⁴Lys³⁹GLP-I (1-39); Arg26.34_Lys40_GLp_₁(1-40)_; Arg²⁶'³⁴Lys⁴¹GLP-1 (1-41); Arg26.34Lys42GLP_1 (1-42); Arg²⁶'³⁴Lys⁴³GLP-I (1-43); Arg²⁶'34Lys44_GLp_i (i-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (1-45); Arg26, 34Lys38GLp_i (2-38); Arg²⁶'³⁴Lys³⁹GLP-1 (2-39); Arg²⁶'34Lys40_GLp_i (2-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (2-41); Arg²⁶'34Lys42GLp_i (2-42); Arg²⁶'³⁴Lys⁴³GLP-1 (2-43); Arg²⁶'34Lys44_GLp_i (2-44); Arg²⁶'³⁴Lys⁴⁵GLP-I (2-45); Arg²⁶'34Lys38GLp_i (3-38); Arg²⁶'³⁴Lys³⁹GLP-1 (3-39); Arg²⁶'34Lys40_GLp_i (3_40)_; Arg²⁶'³⁴Lys⁴¹GLP-1 (3-41); Arg26, 34Lys42GLp_i (3-42); Arg²⁶'³⁴Lys⁴³GLP-1 (3-43); Arg²⁶'34Lys44_GLP_i (3-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (3-45); Arg²⁶'34Lys38GLp_i (4-38); Arg²⁶'³⁴Lys³⁹GLP-1 (4-39); Arg²⁶'34Lys40_GLp_i (4-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (4-41); Arg²⁶'34Lys42GLP_i (4-42); Arg²⁶'³⁴Lys⁴³GLP-1 (4-43); Arg²⁶'34Lys44_GLp_i (4-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (4-45); Arg²⁶'34Lys38GLp_i (5-38); Arg²⁶'³⁴Lys³⁹GLP-1 (5-39); Arg²⁶'34Lys40_GLp_i (5-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (5-41); Arg²⁶'34Lys42GLp_i (5-42); Arg²⁶'³⁴Lys⁴³GLP-1 (5-43); Arg²⁶'34Lys44_GLP_i (5-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (5-45); Arg²⁶'34Lys38GLP_i (6_38); Arg²⁶'³⁴Lys³⁹GLP-1 (6-39); Arg²⁶'34Lys40_GLp_i (6-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (6-41); Arg²⁶'34Lys42GLp_i (6-42); Arg²⁶'³⁴Lys⁴³GLP-1 (6-43); Arg²⁶'34Lys44_GLp_i (6-44); Arg²⁶'³⁴Lys⁴⁵GLP-1 (6-45); Arg²⁶Lys³⁸GLP-I (1-38); Arg³⁴Lys³⁸GLP-I (1-38);

PL 192 359 B1

Arg²⁶'34_L.y_S.36,38_GLp _! (1-38)_; Arg²⁶Lys³⁸GLP-1 (7-38); Arg³⁴Lys³⁸GLP-1 (7-38); Arg²⁶'³⁴Lys³⁶'³⁸GLP-1 (7-38);

Arg²⁶'³⁴Lys³⁸GLP-1 (7-38); Arg²⁶Lys³⁹GLP-I (1-39); Arg³⁴Lys³⁹GLP-I (1-39); Arg²⁶'34_Lys36,39_glp_and₍and_₃₉)_;Arg²⁶Lys³⁹GLP-1 (7-39); Arg³⁴Lys³⁹GLP-1 (7-39) and Arg²⁶'³⁴Lys³⁶'³⁹GLP-1 (7-39).

In a preferred embodiment, the invention relates to a GLP-1 derivative (7-37) in which the lipophilic substituent contains 8-25 carbon atoms.

In a further preferred embodiment, the invention relates to a GLP-1 derivative (7-7) wherein a lipophilic substituent is attached to an amino acid residue such that the carboxyl group of the lipophilic substituent forms an amide bond with the amino group of that amino acid residue.

In a further preferred embodiment, the invention relates to a GLP-1 derivative (7-37) wherein the lipophilic substituent is attached to the amino acid residue via a linker, more preferably the linker is an amino acid residue except Cys, or a dipeptide such as Gly-Lys.

As used herein, the phrase "a dipeptide such as Gly-Lys is used to denote a dipeptide where the C-terminal amino acid residue is Lys, His, or Trp, preferably Lys, and the N-terminal amino acid residue is selected from the group consisting of Ala, Arg, Asp. , Asn, Gly, Glu, Gin, Ile, Leu, Val, Phe, and Pro.

In a further preferred embodiment, the invention relates to a GLP-1 derivative (7-37) wherein the carboxyl group of the parent peptide forms an amide bond with the amino group of a Lys residue or a dipeptide containing a Lys residue, and a second amino group of a Lys linker or dipeptide linker containing a Lys residue forms the bond amide with a carboxyl group of a lipophilic substituent.

In a further preferred embodiment, the invention relates to a GLP-1 derivative (7-37), in which the amino group of the peptide forms an amide bond with the carboxyl group of that amino acid residue or dipeptide linker, and the amino group of the amino acid residue or dipeptide linker forms an amide bond with the carboxyl group of the lipophilic substituent. .

In a further preferred embodiment, the invention relates to a GLP-1 derivative (7-37) having a lipophilic substituent which is an acyl group selected from CH3 (CH2) nCO- groups, where n is 4-38, preferably 4-24, more preferably from CH3 (CH2) ) 6CO-, CH3 (CH2) 8CO-, CH3 (CH2) 10CO-, CH3 (CH2) 12CO-, CH3 (CH2) 14CO-, CH3 (CH2) 16CO-, CH3 (CH2) 18CO-, CH3 (CH2) 20CO- and CH3 (CH2) 22CO-.

In a further preferred embodiment, the invention relates to a GLP-1 derivative (7-37) having a lipophilic substituent which is an acyl group selected from HOOC (CH2) mCO-, where m is 4-38, preferably 4-24, and more preferably from HOOC (CH2) ) 14CO-, HOOC (CH2) 16CO-, HOOC (CH2) 18CO-, HOOC (CH2) 20CO- and HOOC- (CH2) 22CO-.

In a further preferred embodiment, the invention relates to a GLP-1 (7-37) derivative in which up to 6 amino acid residues of GLP-1 (7-37) have been replaced with any α-amino acid residues which can be encoded by the genetic code.

In a particularly preferred embodiment, the invention relates to GLP-1 (7-37) derivatives selected from the group consisting of:

PL 192 359 B1

Lys ^ S (N^e-tetradecanoyl) -GLP-1 (7-37);

Lys34 (N^e-tetradecanoyl) -GLP-1 (7-37);

Lys ^ S (N^e-tetradecanoyl) Arg34-GLP-1 (7-37);

Gly ^ Arg ^ Sz 34Ly_S.36 (No.^e-tetradecanoyl) -GLP-1 (7-37);

Arg²6.34Ly_S.36 (No.^e-tetradecanoyl) -GLP-1 (7-37);

Arg34Lys26 (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37);

Arg34Lys26 (Ν^ε- (ω-carboxyheptadecanoyl)) - GLP-1 (7-37);

Arg26.34Ly_S.36 (1 E- (ω-carboxyheptadecanoyl)) - GLP-1 (7- 37); Arg ^ Sf 34Ly_S.36 (nE_ (ω-carboxyundecanoyl)) -GLP-1 (7-37); Arg34Lys26 (n^e- (ω-carboxyundecanoyl)) - GLP-1 (7-37);

Arg34Lys26 (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37);

Arg26.34Ly_S.36 (ne_ (ω-carboxyheptanoyl)) - GLP-1 (7-37); Arg³⁴Lys²6 (Ν^ε- (ω-carboxypentadecanoyl)) - GLP-1 (7-37); Arg³⁴Lys²⁶(No.^s-litocholyl) -GLP-1 (7-37);

Arg ^ Lys²^ (Ν^ε- (γ-glutamyl- (N^and-hexadecanoyl))) -GLP- 1 (7-37); Arg34Lys²6 (Ν^ε- (γ-glutamyl- (N^and-tetradecanoyl))) -GLP-1 (7-37); Arg26, 34LyS36 (jsjE- (ω-carboxynoperecanoyl)) - GLP-1 (7-36) - OH; Arg²^, 34LyS36 (^ ε_ (ω-carboxyheptadecanoyl)) - GLP-1 (7-36) -OH; Arg²^, 34Ly_S.36 (iqE_ (ω-carboxyundecanoyl)) -GLP-1 (7-36) -OH; Arg26.34Ly_S.36 (n10 (ω-carboxyheptanoyl)) - GLP-1 (7-36) -OH; Arg26r34Ly_S.36 (nE- (ω-carboxypentadecanoyl)) - GLP-1 (7- 36) -OH; Lys²^ / 34) -. 13 (N^e-tetradecanoyl) -GLP-1 (7-37);

Lys²⁶'34) -, i_s (nE- (ω-carboxynecanoyl)) -GLP-1 (7-37);

Lys²⁶'34) -. 13 (] sjE_ (ω-carboxyundecanoyl)) -GLP-1 (7-37);

Lys²⁶'34j2, is (ne_ (ω-carboxyheptanoyl)) -GLP-1 (7-37);

Ly_S.26.34_) 2, is (ne_ (ω-carboxytridecanoyl)) -GLP-1 (7-37);

Lys²⁶'34j2, is (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37);

Lys²⁶'34) 2.13 (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP- 1 (7-37);

Arg²⁶'34Ly_S.38 _ (^ ε_ (ω-carboxynoperecanoyl)) - GLP-1 (7-38);

PL 192 359 B1

Arg²®'34LyS38_ (nE_ (ω-carboxyheptadecanoyl)) - GLP-1 (7-38); Arg²®'34 £ yS38_ (n^e- {β-carboxyundecanoyl)) - GLP-1 (7-38);

Arg²®'34 £ y_S.38 (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-38);

Glu²²'²³'3C> Arg²⁶'34Ly_S.38 (^^ε- (γ-glutamyl (N^and-tetradecanoyl))) GLP-1 (7-38);

Glu²²'²⁶Arg3⁴Lys²⁸ (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-38);

Arg²®'34Ly_S.38 (n^k- (ω-carboxypentadecanoyl)) - GLP-1 (7-38);

Arg²® '34LyS38 (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-38); Arg²® '34LyS38 (γ-glutamyl (N^and-hexadecanoyl)}) -GLP-1 (7-38); Argl® / 23.26,30.34Ly_S.38 (No.^e-hexadecanoyl) -GLP-1 (7-38);

Arg²® '34Lys38 (Ν^ε- (ω-carboxytridecanoyl)) - GLP-1 (7-38) i

Arg²® '34Lys38 (Ν^ε- (γ-glutamyl (N^and-octadecanoyl))) -GLP- 1 (7-38).

The invention furthermore relates to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a GLP-1 derivative (7-37) or a GLP-1 analog (7-37), in which up to 6 amino acid residues changed from GLP-1 (7-37), an agonist of the human GLP-1 receptor, having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, the group HOOC (CH2) mCO-, wherein m is 4-38, and lithocholyl, wherein said lipophilic substituent is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue.

Furthermore, the invention relates to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a GLP-1 (7-37) derivative selected from the group consisting of:

PL 192 359 B1

Lys26 (N^e-tetradecanoyl) -GLP-1 (7-37),

Lys34 (N^e-tetradecanoyl) -GLP-1 (7-37),

Lys ^ S (N^e-tetradecanoyl) Arg34-GLP-1 (7-37),

Gly8Arg26 <34Ly_S.36 (No.^e-tetradecanoyl) -GLP-1 (7-37),

Arg26.34 £ ys36 (N^e-tetradecanoyl) -GLP-1 (7-37),

Arg34Lys26 (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37), Arg34Lys26 (Ν^ε- (ω-carboxyheptadecanoyl)) - GLP-1 (7-37),

Arg26.34Ly_S.36 (Ν^ε- (ω-carboxyheptadecanoyl)) - GLP-1 (7-37), Arg26 / 34Ly_S.36 (^ ε_ (ω-carboxyundecanoyl)) - GLP-1 (7-37), Arg34Lys26 (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Arg34Lys26 (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Arg26.34Ly_S.36 (^ ε_ (ω-carboxyheptanoyl)) - GLP-1 (7-37), Arg34Lys26 (Ν^ε- (ω-carboxypentadecanoyl)) - GLP-1 (7-37), Arg34Lys26 (N^e-litocholyl) -GLP-1 (7-37),

Arg34Lys26 (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP-1 (7- 37) and Arg34Lys26 (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37).

The invention furthermore relates to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a GLP-1 derivative (7-37) or a GLP-1 analog (7-37), in which up to amino acid residues have been changed from GLP-1 (7-37), an agonist of the human GLP-1 receptor having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl wherein one of these lipophilic substituents is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue.

The invention furthermore relates to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a GLP-1 derivative (7-37) or a GLP-1 analog (7-37), in which up to amino acid residues have been changed from GLP-1 (7-37), an agonist of the human GLP-1 receptor having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO-, in which m is 4-38, and lithocholyl, wherein said lipophilic substituents are attached to amino acid residues other than the N-terminal or C-terminal amino acid residues.

Furthermore, the invention relates to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a GLP-1 (7-37) derivative selected from the group consisting of:

PL 192 359 B1

Lys²⁶'³⁴bis (N^s-tetradecanoyl) -GLP-1 (7-37),

Lys²⁶'³⁴bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37),

Lys²⁶'³⁴bis (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Lys²⁶'³⁴bis (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxytridecanoyl)) - GLP-1 (7-37),

Lys²⁶' ³⁴bis (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37) i

Lys²⁶' ³⁴bis (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP- 1 (7-37).

Furthermore, the invention relates to a pharmaceutical composition containing an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a derivative of a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered relative to GLP- 1 (7-37), which is an agonist of the human GLP-1 receptor, where the analog is GLP-1 (7-C) with a C value of 38-45, having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) nCO- in which m is 4-38, and lithocholyl wherein said lipophilic substituent is attached to the C-terminal amino acid residue.

Furthermore, the invention relates to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a derivative of a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered relative to GLP- 1 (7-37), which is an agonist of the human GLP-1 receptor, wherein the analog is GLP-1 (AB), where A is an integer from 1 to 7 and B is an integer from 36 to 45, containing one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, and attached to the C-terminal residue and a second optional lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, and attached to one from other amino acid residues.

Furthermore, the invention relates to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a GLP-1 (7-37) derivative selected from the group consisting of

Arg^26:'⁴Lys^:'⁸(No. ^:- (i '<- carboxynecanoyl)) - GLP-1 (7-38),

Arg^26:'' Lys^:'⁸(No. ^:- (i '<- carboxyheptadecanoyl)) - GLP-1 (7-38),

Arg²⁶²⁴Lys²⁸(No. ^:- (i '<- carboxyundecanoyl)) - GLP-1 (7-28),

Arg²⁶²⁴Lys²⁸(N '- (i' <- carboxyheptanoyl)) - GLP-1 (7-28),

Glu ^^^ Arg ^^ Lys ^ / N ^ Y-glutamyl ^ -tetradecanoyl))) - GLP-1 (7-28),

Glu ²²⁶Arg²⁴Lys²⁸(N L'-glutamyl (N -tetradecanoyl))) - GLP-1 (7-28),

Arg²⁶²⁴Lys²⁸(No. ^:- (i '<- carboxypentadecanoyl)) - GLP-1 (7-38),

Arg²⁶²⁴Lys²⁸(N '- (- / - glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-28),

Arg²⁶²⁴Lys²⁸(N '- (- / - glutamyl (N^and-hexadecanoyl))) - GLP-1 (7-28),

Arg¹⁸'²²'²⁶'²⁰'²⁴Lys²⁸(N = -hexadecanoyl) -GLP-1 (7-28),

Arg²⁶²⁴Lys²⁸(No. ^:- (i '<- carboxytndecanoyl)) - GLP-1 (7-28) i

Arg^26,24Lys²⁸(N '- (Y-glutamyl (N^and-octadecanoyl))) - GLP-1 (7-28).

Furthermore, the invention relates to a pharmaceutical composition containing the active ingredient and a pharmaceutically acceptable excipient or carrier, the feature of which is that the active ingredient is a derivative of a GLP-1 (7-27) analog in which up to 6 amino acid residues have been altered relative to GLP- 1 (7-27), which is an agonist of the human GLP-1 receptor, wherein at least one amino acid residue has a lipophilic substituent selected from the group consisting of CH2 (CH2) nCO- in which n is 4-28, HOOC (CH2) mCO- in which m is 4-28 and lithocholyl wherein said analog is selected from the group consisting of:

PL 192 359 B1

Arg²⁶'34Lys38GLP_i (7-38); Arg²⁶'³⁴Lys³⁹GLP-1 (7-39); Arg26.34_Lys40_GLP_i (7-40); Arg²⁶'34Lys⁴¹GLP-1 (7-41); Arg²⁶'³⁴Lys⁴²GLP-1 (7-42); Arg²⁶'³⁴Lys⁴³GLP-1 (7-43); Arg²⁶'34Lys44_GLp _! (7-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (7-45); Arg26, 34Lys38GLp_i (1-38); Arg²⁶'³⁴Lys³⁹GLP-I (1-39); Arg²⁶'34Lys40_GLp_i (1-40); Arg²⁶'3⁴Lys⁴¹GLP-1 (1-41); Arg²⁶'34Lys42GLP_1 (1-42); Arg²⁶'³⁴Lys⁴³GLP-I (1-43); Arg²⁶'34Lys44_GLP_₁(1-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (1-45); Arg²⁶'34Lys38GLp _! (2-38); Arg²⁶'³⁴Lys³⁹GLP-1 (2-39); Arg²⁶'34Lys40_GLP_i (2-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (2-41); Arg²⁶'34Lys42GLp _! (2-42); Arg²⁶'³⁴Lys⁴³GLP-1 (2-43); Arg²⁶'34Lys44_GLP_₁(2-44); Arg²⁶'³⁴Lys⁴⁵GLP-I (2-45); Arg²⁶'34Lys38GLP_i (3-38); Arg²⁶'³⁴Lys³⁹GLP-1 (3-39); Arg26.34_Lys40_GLp_₁(3-40)_; Arg²⁶'³⁴Lys⁴¹GLP-1 (3-41); Arg²⁶'34Lys42GLp_1 (3-42); Arg²⁶'³⁴Lys⁴³GLP-1 (3-43); Arg²⁶'34Lys44_GLp_i (3-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (3-45); Arg²⁶'34Lys38GLp_i (4-38); Arg²⁶'³⁴Lys³⁹GLP-1 (4-39); Arg²⁶'34Lys40_GLp_₁(4-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (4-41); Arg²⁶'34Lys42GLp_1 (4-42); Arg²⁶'³⁴Lys⁴³GLP-1 (4-43); Arg²⁶'34Lys44_GLp_₁(4-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (4-45); Arg²⁶'34Lys38GLp_1 (5-38); Arg²⁶'³⁴Lys³⁹GLP-1 (5-39); Arg²⁶'34Lys40_GLP_₁(5-40) Arg²⁶'³⁴Lys⁴¹GLP-1 (5-41); Arg²⁶'34Lys42GLP_i (5-42); Arg²⁶'³⁴Lys⁴³GLP-1 (5-43); Arg²⁶'34Lys44_GLP_i (5-44); Arg²⁶'³⁴Lys⁴⁵GLP-1 (5-45); Arg²⁶'34Lys38GLp_i (6-38); Arg²⁶'³⁴Lys³⁹GLP-1 (6-39); Arg26.34_Lys40_GLp_i (6-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (6-41); Arg²⁶'34_Lys42_GLP_₁(6-42); Arg²⁶'³⁴Lys⁴³GLP-1 (6-43);

Arg²⁶' 34_Lys44_GLp _! (6_44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (6-45); Arg²⁶Lys³⁸GLP-I (1-38); Arg³⁴Lys³⁸GLP-I (1-38);

PL 192 359 B1 _Arg26,34_Lys36.38g_L.p_i (1-38); Arg²⁶Lys³8GLP-1 (7-38); Arg34Lys38GLP-1 (7-38); Arg²^ 34 Lys36, 38_GLP_i (7-38); _Arg26,34_Lys38GLP-1 (7-38); Arg²⁶Lys³9GLP-1 (1-39); Arg34LyS39GLP-1 (1-39); Arg²^, 34 Lys 36.39_GLP-1₍and_₃₉); Arg²⁶Lys³9GLP-1 (7-39); Arg3⁴Lys39_G.LP-1 (7-39) i _Arg26,34_Lys36,39_G.LP-1 (7-39).

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP receptor. -1 having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein said lipophilic substituent is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue, for the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of:

Lys²^ (N^e-tetradecanoyl) -GLP-1 (7-37),

Lys34 (N^s-tetradecanoyl) -GLP-1 (7-37),

Lys²^ (N^e-tetradecanoyl) Arg34-GLP-1 (7-37),

Gly8Arg²6.34Ly_S.36 (No.^s-tetradecanoyl) -GLP-1 (7-37),

Arg²^ / 34Ly_S.36 (No.^e-tetradecanoyl) -GLP-1 (7-37),

Arg34Lys²8 (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37),

Arg34Lys²8 (Ν^ε- (ω-carboxyheptadecanoyl)) - GLP-1 (7-37),

Arg²^'34_ANDy_S.36 (^ ε_ (ω-carboxyheptadecanoyl)) - GLP-1 (7-37),

Arg²^'34_ANDys36 (^ ε_ (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Arg34Lys²® (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37), Arg34Lys²^ (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Arg²^'34_ANDys36 (Nt_ (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Arg³⁴Lys²⁶(No.^:- (i '<- carboxypentadecanoyl)) - GLP-1 (7-37),

Arg³⁴Lys²⁶(NMitocholyl) -GLP-1 (7-37),

Arg³⁴Lys²⁶(No. ^:- (? -glutamyl (N-hexadecanoyl))) - GLP-1 (7-37) and

Arg³⁴Lys²⁶(No. ^:- (? -glutamyl (N-tetradecanoyl))) - GLP-1 (7-37), for the manufacture of a medicament for the treatment of non-insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP- receptor. 1 having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl wherein one of these lipophilic substituents it is attached to

Any other amino acid residue other than the N-terminal or C-terminal amino acid residue, in the manufacture of a medicament for the treatment of non-insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP- receptor. 1 having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein said lipophilic substituents are attached to amino acid residues other than the N-terminal or C-terminal amino acid residues, in the manufacture of a medicament for the treatment of non-insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of: Lys²⁶'34j-, i_s (No.^e-tetradecanoyl) -GLP-1 (7-37),

Lys²⁶'34bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37),

Lys²⁶'34bis (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Lys²⁶'34fc, is (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Lys²⁶'34 -bis (nE_ (ω-carboxytridecanoyl)) - GLP-1 (7-37),

Lys²⁶'34fc> is (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37) and Lys²⁶'34) 2.13 (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP-1 (7-37), for the manufacture of a medicament for the treatment of non-insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) analog derivative in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor, the analog being GLP-1. (7-C) where the value of C is 38-45 having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein said lipophilic substituent is attached to the C-terminal amino acid residue, in the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

Further, the invention relates to the use of a GLP-1 (7-37) analog derivative in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor, said analog being GLP- 1 (AB) where A is an integer from 1 to 7 and B is an integer from 38 to 45 containing one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO-, where m is 4-38, and lithocholyl, and attached to a C-terminal amino acid residue, and a second optional lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, and attached to one of the other amino acid residues, in the manufacture of a medicament for the treatment of non-insulin dependent diabetes mellitus.

PL 192 359 B1

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of:

Arg²⁶'³⁴Lys³⁸- (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38),

Arg²⁶'³⁴Lys³⁸- (Ν^ε- (ω-carboxyheptadecanoyl)) - GLP-1 (7-38),

Arg²⁶'³⁴Lys³⁸- (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-38),

Arg²⁶'³⁴Lys³⁸ (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-38),

Glu²²'²³'³⁹Arg²⁶'³⁴Lys³⁸(Ν^ε- (γ-glutamyl (Ν^α-tetradecanoyl))) GLP-1 (7-38),

Glu²³'²⁶Arg³⁴Lys³⁸ (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-38),

Arg²⁶'34Ly_S.38 (^ ε_ (_ω_ carboxypentadecanoyl)) - GLP-1 (7-38),

Arg²⁶'³⁴Lys³⁸ (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-38), Arg²⁶'34_Lys38 _(Νε_ (γ-giutamylo (N^and-hexadecanoyl))) - GLP-1 (7-38), Argl-θ ' ²³'²⁶' ³θ ' ³⁴Lys³⁸ (No.^s-hexadecanoyl) -GLP-1 (7-38),

Arg²⁶' ³⁴Lys³⁸(Ν^ε- (ω-carboxytridecanoyl)) - GLP-1 (7-38) and Arg²⁶'³⁴Lys³⁸ (Ν^ε- (γ-glutamyl (N^and-octadecanoyl))) - GLP-1 (7-38), for the manufacture of a medicament for the treatment of non-insulin dependent diabetes.

Furthermore, the invention relates to the use of a GLP-1 (7-37) analog derivative in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor in which at least one amino acid residue is has a lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl wherein said analog is selected from the group include:

PL 192 359 B1

Arg²⁶'34Lys38GLP_i (7-38); Arg²⁶'34Lys40_GLP_i (7-40); Arg²⁶'34Lys42GLp _! (7-42); Arg²⁶'34Lys44_GLp_₁(7-44)_;Arg²⁶'34Lys38GLp_i (1-38); Arg²⁶'34Lys40_GLp_i (1-40); Arg²⁶'34Lys42GLp_1 (1-42); Arg²⁶'34Lys44_GLP_₁(1-44)_;Arg²⁶'34Lys38GLp _! (2-38); Arg²⁶'34Lys40_GLp_₁(2-40); Arg²⁶'34Lys42GLp_i (2-42); Arg²⁶'34Lys44_GLp _! (2-44); Arg²⁶'34Lys38GLP_i (3-38); Arg²⁶'34Lys40_GLp_i (3-40); Arg²⁶'34Lys42GLp_i (3-42); Arg26.34Lys44GLp_1 (3-44); Arg²⁶'34Lys38_GLp _! (4_38); Arg²⁶'34Lys40GLp_i (4-40); Arg²⁶'34Lys42_GLp_i (4-42); Arg²⁶'34Lys44GLp_i (4-44); Arg²⁶'34Lys38_GLP_i (5-38); Arg²⁶'34Lys40GLp_i (5-40); Arg²⁶'34Lys42_GLp_₁(5-42); Arg²⁶'34Lys44GLP_1 (5-44); Arg²⁶'34Lys38_GLp_i (6-38); Arg²⁶'34Lys40GLp_i (6-40); Arg²⁶'34Lys42_GLP_₁(6-42);

Arg²⁶'34Lys39GLP_i (7-39); Arg²⁶'34Lys41_GLP_i (7-41) _;Arg²⁶'34Lys43GLp-i (7-43); Arg²⁶'34Lys45_GLP_₁(7-45); Arg²⁶'34Lys39GLp_i (1-39); Arg²⁶'34Lys41_GLp_i (1-41); Arg²⁶'34Lys43GLp_i (1-43); Arg²⁶'34Lys45_GLp_i (i_45)_;Arg²⁶'34Lys39GLp_i (2-39); Arg²⁶'34Lys41_GLp_i (2-41); Arg²⁶'34Lys43GLp_i (2-43); Arg²⁶'34Lys45_GLP_i (2-45); _Arg26,34_Lys39_GLP_i (3-39)_;Arg²⁶'34Lys41GLp_i (3-41); Arg²⁶'34Lys43_GLp_i (3-43); Arg²⁶'34Lys45GLp_i (3-45); Arg²⁶'34Lys39_GLp_i (4-39)_;Arg²⁶'34Lys41GLp_i (4-4i); Arg²⁶'34Lys43_GLp_i (4-43)_;Arg²⁶'34Lys45GLP_i (4-45); Arg²⁶'³⁴Lys³⁹GLP-1 (5-39); Arg²⁶'34Lys41_GLp_i (5-41); Arg²⁶'³⁴Lys⁴³GLP-1 (5-43); Arg²⁶'³⁴Lys⁴³GLP-1 (5-45); Arg²⁶'34Lys39GLP_i (6-39); Arg²⁶'34Lys41_GLP_i (6-41)_;Arg²⁶'34_Lys43_GLp_i (6-43)_;

PL 192 359 B1 _Arg26,34_Lys44_GLP_₁(₆_44); Arg²⁶'³⁴Lys⁴⁵GLP-1 (6-45); Arg²⁶Lys³⁸GLP-I (1-38); Arg³⁴Lys³⁸GLP-I (1-38);

Arg²⁶'34Lys36,38GLP -! (1-38); Arg²⁶Lys³⁸GLP-1 (7-38); Arg³⁴Lys³⁸GLP-1 (7-38); Arg²⁶'³⁴Lys³⁶'³⁸GLP-1 (7-38); Arg26.34_Lys38_GLP_i (7-38); Arg²⁶Lys³⁹GLP-I (1-39); Arg³⁴Lys³⁹GLP-I (1-39); Arg²⁶'³⁴Lys³⁶'³⁹GLP-1 (1-39); Arg²⁶Lys³⁹GLP-1 (7-39); Arg³⁴Lys³⁹GLP-1 (7-39) and Arg²⁶'34_Lys36,39_glp_i (7-39), for the manufacture of a medicament for the treatment of non-insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP- receptor. 1, having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl. wherein said lipophilic substituent is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue, in the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of:

Lys²⁶ (No.^s-tetradecanoyl) -GLP-1 (7-37),

Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-37),

Lys²⁶ (No.^e-tetradecanoyl) Arg³⁴-GLP-1 (7-37),

Gly⁸Arg²⁶'³⁴Lys³⁶(No.^s-tetradecanoyl) -GLP-1 (7-37),

Arg²⁶'³⁴Lys³⁶(No.^e-tetradecanoyl) -GLP-1 (7-37),

Arg³⁴Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37),

Arg³⁴Lys²⁶(Ν^ε- (ω-carboxyheptadecanoyl)) - GLP-1 (7-37),

Arg²⁶'³⁴Lys³⁶ (Ν^ε- (ω-carboxyheptadecanoyl)) - GLP-1 (7-37),

Arg²⁶'³⁴Lys³⁶(Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Arg³⁴Lys²⁶(Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Arg³⁴Lys²⁶ (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Arg²⁶'³⁴Lys³⁶(Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Arg³⁴Lys²⁶(Ν^ε- (ω-carboxypentadecanoyl)) - GLP-1 (7-37),

Arg³⁴Lys²⁶(No.^e-litocholyl) -GLP-1 (7-37),

Arg³⁴Lys²⁶ (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP-1 (7-37) i

Arg³⁴Lys²⁶ (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37), for the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

PL 192 359 B1

Furthermore, the invention relates to the use of a GLP-1 (7-27) derivative or a GLP-1 (7-27) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-27) agonist of the human GLP- receptor. 1 having only two lipophilic substituents independently selected from the group consisting of CH2 (CH2) nCO- in which n is 4-28, HOOC (CH2) mCO- in which m is 4-26, and lithocholyl, wherein one of said lipophilic substituents is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue, for the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-27) derivative or a GLP-1 (7-27) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-27) agonist of the human GLP- receptor. 1 having only two lipophilic substituents independently selected from the group consisting of CH2 (CH2) nCO- in which n is 4-28, HOOC (CH2) mCO- in which m is 4-28, and lithocholyl, wherein said lipophilic substituents are attached to amino acid residues other than the N-terminal or C-terminal amino acid residues, in the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus

Furthermore, the invention relates to the use of a GLP-1 (7-27) derivative selected from the group consisting of: Lys²⁶'³⁴bis (N^E.-tetradecanoyl) -GLP-1 (7-37),

Lys²⁶'³⁴bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37),

Lys²⁶'³⁴bis (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Lys²⁶'³⁴bis (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxytridecanoyl)) - GLP-1 (7-37),

Lys²⁶' ³⁴bis (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37) and Lys²⁶' ³⁴bis (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP-1 (7-37), for the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-27) analog derivative wherein up to 6 amino acid residues have been altered from a GLP-1 (7-27) agonist of the human GLP-1 receptor, the analog being GLP-1. (7-C) where the value of C is 28-45 having only one lipophilic substituent selected from the group consisting of CH2 (CH2) nCO- in which n is 4-28, HOOC (CH2) mCO- in which m is 4-28, and lithocholyl, wherein said lipophilic substituent is attached to the C-terminal amino acid residue, in the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-27) analog derivative in which up to 6 amino acid residues have been altered from a GLP-1 (7-27) agonist of the human GLP-1 receptor, said analog being GLP- 1 (AB), where A is an integer from 1 to 7 and B is an integer from 28 to 45, containing one lipophilic substituent selected from the group consisting of CH2 (CH2) nCO- in which n is 4-28, the group HOOC (CH2) mCO-, where m is 4-28, and lithocholyl, and attached to a C-terminal amino acid residue, and a second optional lipophilic substituent selected from the group consisting of CH2 (CH2) nCO- in which n is 4-28, HOOC (CH2) mCO- in which m is 4-28, and lithocholyl, and linked to one of the other amino acid residues, in the manufacture of a medicament for the treatment of insulin dependent diabetes.

PL 192 359 B1

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of: Arg² 6, 34Ly_S.38_ (ne_ (ω-carboxynecanoyl)) - GLP-1 (7-38),

Arg²^, £ 34 y_S.38_ (^ ε_ (ω-carboxyheptadecanoyl)) - GLP-1 (7-38), Arg26, 34Ly_S.38_ (nE_ (ω-carboxyundecanoyl)) -GLP-1 (7-38),

Arg²^ / 34Ly_S.38 (fjE_ (ω-carboxyheptanoyl)) - GLP-1 (7-38),

Gi_at22,23,30_Arg26, 34Ly_S.38 (ne_ (γ-glutamyl (N^and-tetradecanoyl))) GLP-1 (7-38),

G1u²3,26_Arg34Ly_S.38 (^ ε_ (γ-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-38),

Arg²^ / 34Ly_S.38 (ne_ (ω-carboxypentadecanoyl)) - GLP-1 (7-38),

Arg26 / 34Ly_S.38 (ne_ (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-38), Arg26, 34LyS38 (^ ε_ (γ-glutamyl (N^and-hexadecanoyl))) -GLP- 1 (7-38), Arg ^ -θ> 23.26, 30.34LyS38 (N^e-hexadecanoyl) -GLP-1 (7-38),

Arg26 <34Ly_S.38 (nE_ (ω-carboxytridecanoyl)) -GLP-1 (7-38) and Arg2 6 <34Ly_S.38 (ne_ (γ-glutamyl (N^and-octadecanoyl))) -GLP-1 (7-38), for the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus.

Furthermore, the invention relates to the use of a GLP-1 (7-37) analog derivative in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor in which at least one amino acid residue is has a lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl wherein said analog is selected from the group including:

PL 192 359 B1

Arg²⁶'34Lys38GLP_1 (7-38); Arg²⁶'34Lys40_GLp_i (7-40); Arg²⁶'34Lys42GLp_i (7-42); Arg²⁶'34Lys44_GLP_₁(7-44)_;Arg²⁶'34Lys38GLp_i (1-38); Arg²⁶'34Lys40_GLP_i (1-40); Arg²⁶'34Lys42GLp_i (1-42); Arg²⁶'34Lys44_GLp_i (1-44)_;Arg²⁶'34Lys38GLp_i (2-38); Arg²⁶'34Lys40_GLP_i (2-40); Arg²⁶'34Lys42GLp_i (2-42); Arg26, 34Lys44GLP_i (2-44); Arg²⁶'34Lys38_GLp_i (3-38); Arg²⁶'34Lys40GLp_i (3-40); Arg²⁶'34Lys42_GLP_i (3-42); Arg²⁶'34Lys44GLP_i (3-44); Arg²⁶'34Lys38_GLp_i (4_38)_;Arg²⁶'34Lys40GLp_i (4-40); Arg²⁶'34Lys42_GLP_i (4-42); _Arg26,34_Lys44_GLp_i (4-44)_;Arg²⁶'34Lys38GLp_i (5-38); Arg²⁶'34Lys40_GLP_i (5-40); Arg²⁶'34_Lys42_GLP_i (5-42);

Arg²⁶'34Lys39GLp_i (7-39); Arg26, 34Lys41GLP_i (7-41); Arg²⁶'34Lys43_GLp_i (7-43); Arg²⁶'³⁴Lys⁴³GLP-1 (7-45); Arg²⁶'34Lys39GLp_i (i_39); Arg²⁶'34Lys41_GLP_i (1-41); Arg²⁶'34Lys43GLp_i (1-43); Arg²⁶'³⁴Lys⁴³GLP-1 (1-45); Arg²⁶'34Lys39_GLp_i (2-39); _Arg26,34_Lys41_GLp_i (2-41); Arg²⁶'34Lys43GLp_i (2-43); Arg²⁶'34Lys45_GLp_i (2-45); Arg²⁶'³⁴Lys³⁹GLP-1 (3-39); Arg²⁶'34Lys41GLp_i (3_4i); Arg²⁶'34Lys43_GLp_i (3-43)_;Arg²⁶'34Lys45GLP_i (3-45); Arg²⁶'34Lys39_GLp_i (4-39)_;Arg²⁶'34Lys41GLp_i (4-4i); Arg²⁶'34Lys43_GLp_i (4_43)_;Arg²⁶'³⁴Lys⁴³GLP-1 (4-45); Arg²⁶'34Lys39GLp_i (5-39); Arg²⁶'34Lys41_GLP_i (5-41); Arg²⁶'³⁴Lys⁴³GLP-1 (5-43);

PL 192 359 B1

Arg²⁶'³⁴Lys⁴⁴GLP-1 (5-44) Arg²⁶'34Lys38GLP_1 (6-38) Arg²⁶'34Lys40_GLp_i (6-40) Arg²⁶'34Lys42GLP_1 (6-42) Arg²⁶'34Lys44_GLP_₁(6-44); Arg²⁶'³⁴Lys⁴⁵GLP-1 (5-45); and Arg²⁶'³⁴Lys³⁹GLP-1 (6-39); ; Arg²⁶'³⁴Lys⁴¹GLP-1 (6-41); ; Arg²⁶'³⁴Lys⁴³GLP-1 (6-43); ; Arg²⁶'³⁴Lys⁴⁵GLP-1 (6-45);

Arg²⁶Lys³⁸GLP-I (1-38); Arg³⁴Lys³⁸GLP-I (1-38);

Arg²⁶'34Lys36,38CLP -! (I_38). Arg²⁶Lys³⁸GLP-1 (7-38); Arg³⁴Lys³⁸GLP-1 (7-38); Arg²⁶'34Lys36, 38 (^^ (7-38); Arg²⁶'34_Lys38_GLP_₁(7-38); Arg²⁶Lys³⁹GLP-I (1-39); Arg³⁴Lys³⁹GLP-I (1-39); Arg²⁶'³⁴Lys³⁶'³⁹GLP-1 (1-39); Arg²⁶Lys³⁹GLP-1 (7-39); Arg³⁴Lys³⁹GLP-I (7-39) i

<img file="PL192359B1_D0001.tif" />

for the manufacture of a medicament for the treatment of insulin dependent diabetes mellitus

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP receptor. -1 having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein said lipophilic substituent is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue, in the manufacture of a medicament for the treatment of obesity.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of:

Lys²⁶(No.^E.-tetradecanoyl) -GLP-1 (7-37),

Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-37),

Lys²⁶(No.^e-tetradecanoyl) Arg³⁴-GLP-1 (7-37),

Gly⁸Arg²⁶'³⁴Lys³⁶(No.^e-tetradecanoyl) -GLP-1 (7-37),

Arg²⁶'³⁴Lys³⁶(No.^e-tetradecanoyl) -GLP-1 (7-37),

Arg³⁴Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37),

PL 192 359 B1

Arg ^ Lys²^ (Ν^ε- (ω-carboxyheptadecanoyl)) -GLP-1 (7-37),

Arg26.34L_ys36 (n £ - (ω-carboxyheptadecanoyl)) - GLP-1 (7-37), Arg26.34_Αγ₃36 (nE_ (ω-carboxyundecanoyl)) - GLP-1 (7-37), Arg34Lys²6 (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Arg34Lys26 (Ν^ε- (ω-carboxyheptanoyl)) - GLP-1 (7-37),

Arg²⁶,34_Lys36(_Νε_ (ω-carboxyheptanoyl)) - GLP-1 (7-37), Arg34Lys²6 (Ν^ε- (ω-carboxypentadecanoyl)) - GLP-1 (7-37), Arg34Lys²⁶(No.^e-litocholyl) -GLP-1 (7-37),

Arg³⁴Lys²⁶ (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP-1 (7-37) and Arg34Lys²⁶ (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37), for the manufacture of a medicament for the treatment of obesity.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP- receptor. 1 having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein one of said lipophilic substituents is attached to an amino acid residue other than the N-terminal or C-terminal amino acid residue, in the manufacture of a medicament for the treatment of obesity.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative or a GLP-1 (7-37) analog in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP- receptor. 1 having only two lipophilic substituents independently selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, wherein said lipophilic substituents are attached to amino acid residues other than the N-terminal or C-terminal amino acid residues, in the manufacture of a medicament for the treatment of obesity.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of Ly_S.26, 34) 3.13 (N^e-tetradecanoyl) -GLP-1 (7-37),

Lys²6, 34) -, i_s (Νε_ (ω-carboxynecanoyl)) -GLP-1 (7-37),

Lys²^ '34bi_s (Ν^ε- (ω-carboxyundecanoyl)) - GLP-1 (7-37),

Lys²^ '34j-, i_s (ω-carboxyheptanoyl)) -GLP-1 (7-37),

Lyg26, 34 _] -, i_s (ω-carboxytridecanoyl)) -GLP-1 (7-37),

Lys²θ / 34] -, i_s (^ ε_ (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-37) and Lys²^ '34fc, is (Ν^ε- (γ-glutamyl (N^and-hexadecanoyl))) -GLP-1 (7-37), for the manufacture of a medicament for the treatment of obesity.

Further, the invention relates to the use of a GLP-1 (7-37) analog derivative wherein up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor, the analog being GLP-1 (7-C), where the value of C is 36-45, having only one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO-, in which n is 4-38, HOOC (CH2) mCO- where m is 4-38 and lithocholyl, wherein said lipophilic substituent is attached to the C-terminal amino acid residue, for the manufacture of a medicament for the treatment of obesity.

Further, the invention relates to the use of a GLP-1 (7-37) analog derivative in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor, said analog being GLP- 1 (AB) where A is an integer from 1 to 7 and B is an integer from 38 to 45 containing one lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO-, where m is 4-38, and lithocholyl, and attached to a C-terminal amino acid residue, and a second optional lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl, and linked to one of the other amino acid residues, in the manufacture of a medicament for the treatment of obesity.

Furthermore, the invention relates to the use of a GLP-1 (7-37) derivative selected from the group consisting of: Arg²⁶'34Ly_S.38_ (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38),

Arg²⁶'34LyS38_ (^ ε_ (ω-carboxyheptadecanoyl)) - GLP-1 (7-38), Arg²⁶'34LyS38_ (^ ε_ (ω-carboxyundecanoyl)) - GLP-1 (7-38),

Arg²⁶'34Ly_S.38 (^ ε_ (ω-carboxyheptanoyl)) - GLP-1 (7-38),

Glu²²'²³'³0Arg²⁶' ³4Lys³⁶ (Nt_ (γ-glutamyl (N^and-tetradecanoyl))) GLP-1 (7-38),

Glu²²r 26Arg34Lys38 (Ν^ε- (γ-glutamyl (N^and-tetradecanoyl))) -GLP-1 (7-38),

Arg²⁶'34Ly_S.38 (Nt_ (ω-carboxypentadecanoyl)) - GLP-1 (7-38),

Arg²⁶'34LyS38 (jqt_ (γ-glutamyl (N^and-tetradecanoyl))) -GLP- 1 (7-38), Arg²⁶'34LyS38 (^ ε_ (γ-glutamyl (N^and-hexadecanoyl))) -GLP-1 (7-38), Argl⁶'²²z 26,30,34i, y_S.38 (No.^e-hexadecanoyl) -GLP-1 (7-38),

Arg²⁶'34LyS38 (^ ε_ (ω-carboxytridecanoyl)) - GLP-1 (7-38) and Arg²⁶'34LyS38 (jqs_ (γ-glutamyl (N^and-octadecanoyl))) -GLP-1 (7-38), for the manufacture of a medicament for the treatment of obesity.

Furthermore, the invention relates to the use of a GLP-1 (7-37) analog derivative in which up to 6 amino acid residues have been altered from a GLP-1 (7-37) agonist of the human GLP-1 receptor in which at least one amino acid residue is has a lipophilic substituent selected from the group consisting of CH3 (CH2) nCO- in which n is 4-38, HOOC (CH2) mCO- in which m is 4-38, and lithocholyl wherein said analog is selected from the group including:

PL 192 359 B1

Arg²⁶'34Lys38GLP_1 (7-38); Arg²⁶'34Lys40_GLP_₁ (7-40); Arg²⁶'34Lys42GLP_1 (7-42); Arg²⁶'34Lys44_GLp_₁(7-44)_;Arg²⁶'34Lys38GLp_1 (1-38); Arg26, 34Lys40GLp_1 (i-40); Arg²⁶'34Lys42_GLp_i (1-42); Arg²⁶'34_Lys44_GLp_i (i_44)_;Arg²⁶'34Lys38GLp_i (2-38); Arg²⁶'34Lys40_GLp_i (2-40); Arg²⁶'34_Lys42_GLP_i (2-42); Arg²⁶'34Lys44GLp_i (2-44); Arg²⁶'34Lys38_GLP_i (3-38); Arg²⁶'34Lys40GLp_i (3-40); Arg²⁶'34Lys42_GLP_i (3-42); Arg²⁶'34Lys44GLp_i (3-44); Arg²⁶'34Lys38_GLp_i (4-38); Arg²⁶'34Lys40GLp_i (4-40); Arg²⁶'34Lys42_GLP_i (4-42); Arg²⁶'34_Lys44_GLP_i (4-44)_;Arg²⁶'34Lys38GLp_i (5-38); Arg²⁶'34Lys40_GLp_i (5_40)_;

Arg²⁶'34Lys39GLp_i (7-39); Arg²⁶'34Lys41_GLP_i (7-41)_;Arg²⁶'34Lys43GLP_i (7-43); Arg²⁶'34Lys45_GLp_i (7-45); Arg²⁶'34Lys39GLP_i (i_39); Arg²⁶'34Lys41_GLP_i (i-4i)_;Arg²⁶'34Lys43GLp_i (1-43); Arg²⁶'34Lys45_GLP_i (1-45)_;Arg²⁶'34Lys39GLp_i (2-39); Arg²⁶'34Lys41_GLp_i (2-41); Arg²⁶'34Lys43GLp_i (2-43); Arg²⁶'34Lys45_GLp_i (2-45); Arg²⁶'34Lys39GLp_i (3-39); Arg²⁶'34Lys41_GLp_i (3_4i). Arg²⁶'34Lys43GLp_i (3-43); Arg26, 34Lys45GLp_i (3-45); Arg²⁶'34Lys39_GLp_i (4-39)_{; Arg}26,34_Lys41_GLp_i (4_4i)_;Arg²⁶'34Lys43GLp_i (4-43); Arg²⁶'34Lys45_GLP_i (4-45)_;Arg²⁶'34Lys39GLp_i (5-39); Arg²⁶'34Lys41_GLp_i (5-41)_;

PL 192 359 B1

Arg²⁶'34_Lys42_GLP_₁(5-42); Arg²⁶'³⁴Lys⁴³GLP-1 (5-43);

Arg²⁶'34_Lys44_GLP_₁(5-44)_; Arg²⁶'³⁴Lys⁴⁵GLP-1 (5-45);

Arg²⁶/³⁴Lys³⁸GLP-1 (6-38); Arg²⁶'³⁴Lys³⁹GLP-1 (6-39);

Arg²⁶'34_Lys40_GLP_i (6-40); Arg²⁶'³⁴Lys⁴¹GLP-1 (6-41);

Arg²⁶'34_Lys42_GLP_i (6-42); Arg²⁶'³⁴Lys⁴³GLP-1 (6-43);

Arg26.34_Lys44_GLp_₁(6-44); Arg²⁶'³⁴Lys⁴⁵GLP-1 (6-45); Arg²⁶Lys³⁸GLP-I (1-38); Arg³⁴Lys³⁸GLP-I (1-38);

Arg²⁶'34Lys36.38glp_i (i_38); Arg²⁶Lys³⁸GLP-1 (7-38); Arg³⁴Lys³⁸GLP-1 (7-38); Arg²⁶'34 Lys36.38_GLP_i (7-38);

Arg²⁶'34Lys38GLp_1 (7-38); Arg²⁶Lys³⁹GLP-I (1-39); Arg³⁴Lys³⁹GLP-I (1-39); Arg²⁶'34 Lys36.39_GLp _! (1-39)_;Arg²⁶Lys³⁹GLP-1 (7-39); Arg³⁴Lys³⁹GLP-1 (7-39) and Arg²⁶'34_Lys36,39_GLp_i (7-39), for the manufacture of a medicament for the treatment of obesity.

Moreover, derivatives of GLP-1 (7-37) are preferred, wherein the parent peptide is selected from the group consisting of:

Arg²⁶-GLP-1 (7-37), Arg³⁴-GLP-1 (7-37), Lys³⁶-GLP-1 (7-37), _Arg26,34_Lys36__GLp_₁(7-37)_f Arg²⁶Lys³⁶-GLP-1 (7-37), Arg³⁴Lys³⁶-GLP-l (7-37), Gly⁸Arg²⁶-GLP-l (7-37), Gly⁸Arg³⁴-GLP1 (7-37), Gly⁸Lys³⁶-GLP-l (7-37), Gly⁸Arg²⁶'³⁴Lys³⁶-GLP-1 (737), Gly⁸Arg²⁶Lys³⁶-GLP-l (7-37) and Gly⁸Arg³⁴Lys³⁶-GLP-l (7-37).

Moreover, derivatives of GLP-1 (7-37) are preferred, wherein the parent peptide is selected from the group consisting of:

Arg²⁶Lys³⁸-GLP-1 (7-38), Arg²⁶'³⁴Lys³⁸-GLP-l (7-38),

Arg²⁶'³⁴Lys³⁶'³⁸-GLP-l (7-38), Gly⁸Arg²⁶Lys³⁸-GLP-l (7-38) and Gly⁸Arg²⁶'³4Lys³⁶'³⁸-GLP-l (7-38).

Moreover, derivatives of GLP-1 (7-37) are preferred, wherein the parent peptide is selected from the group consisting of:

Arg²⁶Lys³⁹-GLP-1 (7-39), Arg²⁶'34Lys³⁶'³⁹-GLP-l (7-39),

Gly⁸Arg²⁶Lys³⁹-GLP-I (7-39) and Gly⁸Arg²⁶'³⁴Lys³⁶'³⁹-GLP-l (7-39).

PL 192 359 B1

Moreover, derivatives of GLP-1 (7-37) are preferred, wherein the parent peptide is selected from the group consisting of:

Arg³⁴Lys⁴⁰-GLP-1 (7-40), Arg²⁶/34_Lys36,40__glp_i (7-40),

Gly⁸Arg³⁴Lys⁴⁰-GLP-l (7-40) and Gly⁸Arg²⁶'³⁴Lys³⁶'40__GLP_₁(7-40).

In addition to the above-mentioned derivatives, preferred are:

Gly⁸Lys²⁶(No.^s-tetradecanoyl) -GLP-1 (7-37);

Gly⁸Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-37);

Gly⁸Lys²⁶'³⁴-bis (N^e-tetradecanoyl) -GLP-1 (7-37);

Arg²⁶Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-37);

Lys²⁶(No.^e-tetradecanoyl) -GLP-1 (7-38);

Lys³⁴(No.^£-tetradecanoyl) -GLP-1 (7-38);

Lys²⁶'³⁴-bis (N^e-tetradecanoyl) -GLP-1 (7-38);

Gly⁸Lys²⁶(No.^s-tetradecanoyl) -GLP-1 (7-38);

Gly⁸Lys³⁴ (No.^e-tetradecanoyl) -GLP-1 (7-38);

Gly⁸Lys²⁶'³⁴-bis (N^e-tetradecanoyl) -GLP-1 (7-38);

Arg²⁶Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-38);

Lys²⁶(No.^e-tetradecanoyl) -GLP-1 (7-39);

Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-39);

Lys²⁶' ³⁴-bis (N^e-tetradecanoyl) -GLP-1 (7-39);

Gly⁸Lys²⁶ (No.^e-tetradecanoyl) -GLP-1 (7-39);

Gly⁸Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-39);

Gly⁸Lys²⁶'³⁴-bis (N^e-tetradecanoyl) -GLP-1 (7-39);

Arg²⁶Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-39);

Lys²⁶ (No.^e-tetradecanoyl) -GLP-1 (7-40);

Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-40);

Lys²⁶'³⁴-bis (N^e-tetradecanoyl) -GLP-1 (7-40);

Gly⁸Lys²⁶(No.^s-tetradecanoyl) -GLP-1 (7-40);

Gly⁸Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-40);

Gly⁸Lys²⁶'³⁴-bis (N^s-tetradecanoyl) -GLP-1 (7-40);

Arg²⁶Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-40);

PL 192 359 B1

Lys²⁶(No.^E.-tetradecanoyl) -GLP-1 (7-36);

Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-36);

Lys²⁶'³⁴-bis (N^E.-tetradecanoyl) -GLP-1 (7-36);

Gly⁸Lys²⁶(No.^E.-tetradecanoyl) -GLP-1 (7-36);

Gly⁸Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-36);

Gly⁸Lys²⁶'³⁴-bis (N^E.-tetradecanoyl) -GLP-1 (7-36); Arg²⁶Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-36);

Lys²⁶(No.^E.-tetradecanoyl) -GLP-1 (7-35);

Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-35);

Lys²⁶'³⁴-bis (N^E.-tetradecanoyl) -GLP-1 (7-35); Gly⁸Lys²⁶(No.^E.-tetradecanoyl) -GLP-1 (7-35);

Gly⁸Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-35);

Gly⁸Lys²⁶'³⁴-bis (N^E.-tetradecanoyl) -GLP-1 (7-35); Arg²⁶Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-35);

Lys²⁶(No.^E.-tetradecanoyl) -GLP-1 (7-36) amide;

Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-36) amide;

Lys²⁶'³⁴-bis (N^E.-tetradecanoyl) -GLP-1 (7-36) amide; Gly⁸Lys²⁶(No.^E.-tetradecanoyl) -GLP-1 (7-36) amide; Gly⁸Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-36) amide; Gly⁸Lys²⁶'³⁴-bis (N^E.-tetradecanoyl) -GLP-1 (7-36) amide; Arg²⁶Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-36) amide; Gly⁸Arg²⁶Lys³⁴(No.^E.-tetradecanoyl) -GLP-1 (7-37); Gly⁸Lys²⁶(No.^E.-tetradecanoyl) Arg³⁴-GLP-1 (7-37);

Arg²⁶'³⁴Lys³⁶(No.^E.-tetradecanoyl) -GLP-1 (7-37); Gly⁸Arg²⁶Lys³⁴ (No.^E.-tetradecanoyl) -GLP-1 (7-38);

Lys²⁶(No.^E.-tetradecanoyl) Arg³⁴-GLP-1 (7-38);

Gly⁸Lys²⁶(No.^E.-tetradecanoyl) Arg³⁴-GLP-1 (7-38);

Arg²⁶'³⁴Lys³⁶(No.^E.-tetradecanoyl) -GLP-1 (7-38);

Arg²⁶'³⁴Lys³⁸(No.^E.-tetradecanoyl) -GLP-1 (7-38); Gly⁸Arg²⁶'³⁴Lys³⁶(No.^E.-tetradecanoyl) -GLP-1 (7-38); Gly⁸Arg²⁶Lys³⁴ (No.^E.-tetradecanoyl) -GLP-1 (7-39);

PL 192 359 B1

Lys²⁶(No.^e-tetradecanoyl) Arg³⁴-GLP-1 (7-39);

Gly⁸Lys²⁶(No.^e-tetradecanoyl) Arg³⁴-GLP-1 (7-39);

Arg²⁶'³⁴Lys³⁶(No.^e-tetradecanoyl) -GLP-1 (7-39);

Gly⁸Arg²⁶'³⁴Lys³⁶(No.^e-tetradecanoyl) -GLP-1 (7-39);

Gly⁸Arg²⁶Lys³⁴(No.^e-tetradecanoyl) -GLP-1 (7-40);

Lys²⁶(No.^e-tetradecanoyl) Arg³⁴-GLP-1 (7-40);

Gly⁸Lys²⁶(No.^e-tetradecanoyl) Arg³⁴-GLP-1 (7-40);

Arg²⁶'³⁴Lys³⁶(No.^e-tetradecanoyl) -GLP-1 (7-40);

Gly⁸Arg²⁶'³⁴Lys³⁶ (No.^e-tetradecanoyl) -GLP-1 (7-40);

Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37);

Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37);

Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37); Gly⁸Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37); Gly⁸Lys²⁶' ³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) -GLP-1 (7- 37); Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38);

Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38);

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Gly⁸Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39);

Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39);

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39); Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39); Gly⁸Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39);

Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40);

Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40);

PL 192 359 B1

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40);

Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40);

Gly⁸Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40);

Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36);

Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36);

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36); Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36);

Gly⁸Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36); Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36) amide;

Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36) amide;

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36) amide; Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36) amide; Gly⁸Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36) amide; Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-36) amide; Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-35);

Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-35);

Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynoperecanoyl)) - GLP-1 (7-35); Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-35);

Gly⁸Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-35);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (ω-carboxynoperecanoyl)) - GLP-1 (7-35); Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37); Gly⁸Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37);

Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-37);

PL 192 359 B1

Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-37); Arg²⁶'³⁴Lys³⁶ (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37);

Gly⁸Arg²⁶'³⁴Lys³⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-37); Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Gly⁸Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Lys²⁶ (Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-38); Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-38); Arg²⁶'³⁴Lys³⁶ (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Arg²⁶'³⁴Lys³⁸(Ν^ε- (ω-carboxynoperecanoyl)) - GLP-1 (7-38);

Gly⁸Arg^{2 6}'³⁴Lys³⁶(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-38); Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39); Gly⁸Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39); Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-39); Gly⁸Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-39); Arg²⁶'³⁴Lys³⁶ (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39); Gly⁸Arg²⁶'³⁴Lys³⁶ (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-39); Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40); Gly⁸Arg²⁶Lys³⁴(Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40); Lys²⁶(Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-40); Gly⁸Lys²⁶ (Ν^ε- (ω-carboxynecanoyl)) Arg³⁴-GLP-1 (7-40); Arg²⁶'³⁴Lys³⁶ (Ν^ε- (ω-carboxynecanoyl)) - GLP-1 (7-40);

PL 192 359 B1

Gly⁸Arg²⁶' ³⁴Lys³⁶ (ε_ (ω-carboxynoperecanoyl)) -GLP-1 (7-40);

Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37);

Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37);

Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37);

Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37);

Gly⁸Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37); Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37);

Lys²⁶(Ν^ε- (7-deoxycholoyl) -GLP-1 (7-38);

Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38);

Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38);

Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38);

Gly⁸Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38); Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38);

Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39);

Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39);

Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39);

Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39);

Gly⁸Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39); Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39);

Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40);

Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40);

Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40);

Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40);

Gly⁸Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40); Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40);

Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36);

PL 192 359 B1

Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36);

Lys²⁶'34_bi_s(nE_ (7-deoxycholoyl)) - GLP-1 (7-36);

Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36);

Gly⁸Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36); Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36);

Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-35);

Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-35);

Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-35); Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-35);

Gly⁸Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-35);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-35); Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-35);

Lys²⁶(Ν^ε- (7-deoxycholoyl)) -GLP-1 (7-36) amide;

Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36) amide;

Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36) amide; Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36) amide; Gly⁸Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36) amide; Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36) amide; Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-36) amide; Gly⁸Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-37);

Lys²⁶(Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-37);

Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-37);

Arg²⁶' ³⁴Lys³⁶ (7-deoxycholoyl)) -GLP-1 (7-37); Gly⁸Arg²⁶'³⁴Lys³⁶ (^ ε_ (7-deoxycholoyl)) - GLP-1 (7-37); Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-37);

Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-37);

Lys²⁶' 34__{bis (Ν}ε_ (choloyl)) - GLP-1 (7-37);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-37);

Gly⁸Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-37);

PL 192 359 B1

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-37);

Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-37);

Gly⁸Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38); Lys²⁶(Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-38); Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-38); Arg²⁶'³⁴Lys³⁶ (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38); Arg²⁶'³⁴Lys³⁸(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38); Gly⁸Arg²⁶'³⁴Lys³⁶ (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-38); Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-38);

Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-38);

Lys²⁶'34__{bis (Ν}ε_ (choloyl)) - GLP-1 (7-38);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-38);

Gly⁸Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-38);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-38);

Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-38);

Gly⁸Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39); Lys²⁶(Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-39); Gly⁸Lys²⁶ (Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-39); Arg²⁶'³⁴Lys³⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39); Gly⁸Arg²⁶'³⁴Lys³⁶ (Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-39); Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-39);

Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-39);

Lys²⁶'34__{bis (Ν}ε_ (choloyl)) - GLP-1 (7-39);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-39);

Gly⁸Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-39);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-39);

Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-39);

Gly⁸Arg²⁶Lys³⁴(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40); Lys²⁶(Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-40); Gly⁸Lys²⁶(Ν^ε- (7-deoxycholoyl)) Arg³⁴-GLP-1 (7-40); Arg²⁶'³⁴Lys³⁶(Ν^ε- (7-deoxycholoyl)) - GLP-1 (7-40);

PL 192 359 B1

Gly⁸Arg²⁶'³⁴Lys³⁶(nS_ (7-deoxycholoyl)) - GLP-1 (7-40);

Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-40);

Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-40);

Lys²⁶'34__{bis (Ν}ε_ (choloyl)) - GLP-1 (7-40);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-40);

Gly⁸Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-40);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-40);

Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-40);

Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-36);

Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-36);

Lys²⁶'34__band_{s (Ν}ε_ (choloyl)) - GLP-1 (7-36);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-36);

Gly⁸Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-36);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-36); Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-36);

Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-35);

Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-35);

Lys²⁶'34__{bis (Ν}ε_ (choloyl)) - GLP-1 (7-35);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-35);

Gly⁸Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-35);

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-35); Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-35);

Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-36) amide;

Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-36) amide;

Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-36) amide;

Gly⁸Lys²⁶(Ν^ε- (choloyl)) - GLP-1 (7-36) amide;

Gly⁸Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-36) amide;

Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (choloyl)) - GLP-1 (7-36) amide; Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-36) amide; Gly⁸Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-37);

Lys²⁶(Ν^ε- (choloyl)) Arg³⁴-GLP-1 (7-37);

PL 192 359 B1

Gly⁸Lys²⁶(Ν^ε- (choloyl)} Arg³⁴-GLP-1 (7-37);

Arg²⁶'³⁴Lys³⁶ (Ν^ε- (choloyl)) - GLP-1 (7-37); Gly⁸Arg²⁶'³⁴Lys³⁶ (Ν^ε- (choloyl)) -GLP-1 (7-37); Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-37);

Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-37);

Lys²6, 34 -) ^ 13 (N^s- (lithocholoyl)) -GLP-1 (7-37); Gly⁸Lys²⁶(Ν^ε- (lithocholoyl)) -GLP-1 (7-37); Gly⁸Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-37); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-37); Arg²⁶Lys³⁴ (Ν^ε- (lithocholoyl)) - GLP-1 (7-37); Gly⁸Arg²⁶Lys³⁴ (Ν^ε- (choloyl)) -GLP-1 (7-38);

Lys^{2 6}(Ν^ε- (choloyl)) Arg³⁴-GLP-1 (7-38);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) Arg³⁴-GLP-1 (7-38);

Arg²⁶'³⁴Lys³⁶ (No.^s- (choloyl)) - GLP-1 (7-38);

Arg²⁶'34_Lys38 (_Νε_ (choloyl)) -GLP-1 (7-38); Gly⁸Arg²⁶'³⁴Lys³⁶ (Ν^ε- (choloyl)) - GLP-1 (7-38); Lys²⁶(Ν^ε- (lithocholoyl)} - GLP-1 (7-38);

Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-38);

Lys²⁶'³⁴-bis (N^s- (lithocholoyl)) - GLP-1 (7-38); Gly⁸Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-38);

Gly⁸Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-38); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-38); Arg²⁶Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-38);

Gly⁸Arg²⁶Lys³⁴ (Ν^ε- (choloyl)) - GLP-1 (7-39);

Lys²⁶(Ν^ε- (choloyl)) Arg³⁴-GLP-1 (7-39);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) Arg³⁴-GLP-1 (7-39);

Arg²⁶'³⁴Lys³⁶(Ν^ε- (choloyl)) - GLP-1 (7-39); Gly⁸Arg²⁶'³⁴Lys³⁶ (Ν^ε- (choloyl)) -GLP-1 (7-39); Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-39);

Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-39);

Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-39);

PL 192 359 B1

Gly⁸Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-39); Gly⁸Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-39); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-39); Arg²⁶Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-39); Gly⁸Arg²⁶Lys³⁴(Ν^ε- (choloyl)) - GLP-1 (7-40);

Lys²⁶(Ν^ε- (choloyl)) Arg³⁴-GLP-1 (7-40);

Gly⁸Lys²⁶(Ν^ε- (choloyl)) Arg³⁴-GLP-1 (7-40);

Arg²⁶'34_Lys36_(Νε_ (choloyl)) - GLP-1 (7-40); Gly⁸Arg²⁶'⁸⁴Lys³⁶(Ν^ε- (choloyl)) - GLP-1 (7-40); Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-40);

Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-40);

Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-40); Gly⁸Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-40); Gly⁸Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-40); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-40); Arg²⁶Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-37);

Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-36);

Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-36);

Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-36); Gly⁸Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-36); Gly⁸Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-36); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-36); Arg²⁶Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-36);

Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-35);

Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-35);

Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-35); Gly⁸Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-35); Gly⁸Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-35); Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-35); Arg²⁶Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-35);

Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-36) amide;

PL 192 359 B1

Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-36) amide;

_Lys26,34__{bis (Ν}ε_ (lithocholoyl)) -GLP-1 (7-36) amide; Gly⁸Lys²⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-36) amide; Gly⁸Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-36) amide; Gly⁸Lys²⁶'³⁴-bis (Ν^ε- (lithocholoyl)) - GLP-1 (7-36) amide; Arg²⁶Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-36) amide; Gly⁸Arg²⁶Lys³⁴ (Ν^ε- (lithocholoyl)) -GLP-1 (7-37);

Lys²⁶(Ν^ε- (litocholoyl)) Arg³⁴-GLP-1 (7-37);

Gly⁸Lys²⁶(Ν^ε- (litocholoyl)) Arg³⁴-GLP-1 (7-37);

Arg²⁶'34_Lys36 (Ν^ε- (lithocholoyl)) -GLP-1 (7-37);

Arg²⁶'³⁴Lys³⁸(Ν^ε- (lithocholoyl)) - GLP-1 (7-37); Gly⁸Arg²⁶'³⁴Lys³⁶ (Ν^ε- (lithocholoyl)) - GLP-1 (7-37); Gly⁸Arg²⁶Lys³⁴ (No.^s- (lithocholoyl)) - GLP-1 (7-38);

Lys²⁶(Ν^ε- (litocholoyl)) Arg³⁴-GLP-1 (7-38);

Gly⁸Lys²⁶(Ν^ε- (litocholoyl)) Arg³⁴-GLP-1 (7-38);

Arg²⁶'³⁴Lys³⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-38);

Arg²⁶'³⁴Lys³⁸(Ν^ε- (lithocholoyl)) - GLP-1 (7-38); Gly⁸Arg²⁶'³⁴Lys³⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-38); Gly⁸Arg²⁶Lys³⁴ (Ν^ε- (lithocholoyl)) - GLP-1 (7-39);

Lys²⁵ (Ν^ε- (litocholoyl)) Arg³⁴-GLP-1 (7-39);

Gly⁸Lys²⁶(Ν^ε- (litocholoyl)) Arg³⁴-GLP-1 (7-39);

Arg²⁶'³⁴Lys³⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-39); Gly⁸Arg²⁶'³⁴Lys³⁶N^E.- (lithocholoyl)) - GLP-1 (7-39) Gly⁸Arg²⁶Lys³⁴(Ν^ε- (lithocholoyl)) - GLP-1 (7-40)

Lys²⁶(Ν^ε- (litocholoyl)} Arg³⁴-GLP-1 (7-40);

Gly⁸Lys²⁶(Ν^ε- (litocholoyl)) Arg³⁴-GLP-1 (7-40);

Arg²⁶, 34_Lys36 (Ν^ε- (lithocholoyl)) -GLP-1 (7-40) and Gly⁸Arg²⁶'³⁴Lys³⁶(Ν^ε- (lithocholoyl)) - GLP-1 (7-40).

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To achieve a satisfactory extended action profile of the GLP-1 derivative, the lipophilic substituent attached to the GLP-1 moiety preferably contains 4-40 carbon atoms, in particular 8-25 carbon atoms. Further, a lipophilic substituent may be attached to the amino group of the GLP-1 moiety through the carboxyl group of the lipophilic substituent forming an amide bond with the amino group of the amino acid residue to which the substituent is attached.

Further, a lipophilic substituent may be attached to this amino acid residue such that the amino group of the lipophilic substituent forms an amide bond with the carboxyl group of that amino acid residue. According to another possibility, a lipophilic substituent may be linked to the GLP-1 moiety via an ester bond. The ester may be formed either by reaction between the carboxyl group of the GLP-1 moiety and the hydroxyl group of the future substituent or by reaction between the hydroxyl group of the GLP-1 moiety and the carboxyl group of the future substituent. Further, the lipophilic substituent may be an alkyl group introduced into the primary amino group of the GLP-1 moiety.

According to one preferred embodiment of the invention, a lipophilic substituent is attached to the GLP-1 moiety through a linker such that the carboxyl group of the linker forms an amide bond with the amino group of the GLP-1 moiety. Examples of suitable linkers are succinic acid, Lys, Glu or Asp, or a dipeptide such as Gly-Lys. When the linker is succinic acid, one of its carboxyl groups may form an amide bond with the amine group of the amino acid residue, and the other its carboxyl group may form an amide bond with the amine group of the lipophilic substituent. When the linker is Lys, Glu, or Asp, their carboxyl groups can form an amide bond with the amino group of the amino acid residue, and their amino groups can form an amide bond with the carboxyl group of the lipophilic substituent. When the linker is Lys, in some instances there may be an additional linker inserted between the ε-amino group of the Lys and the lipophilic substituent.

According to one preferred embodiment, the additional linker is succinic acid forming an amide bond with the ε-amino group of Lys and with the amino group present on the lipophilic substituent. According to another preferred embodiment, the additional linker is Glu or Asp which forms an amide bond with the ε-amino group of Lys and a second amide bond with the carboxyl group present on the lipophilic substituent, i.e. the lipophilic substituent is N^E.-acylated lysine.

According to another preferred embodiment of the invention, the lipophilic substituent has a moiety that may be negatively charged. One of the preferred groups that can be negatively charged is a carboxylic acid group.

The parent peptide may be produced by a method of culturing a host cell containing a DNA sequence encoding a polypeptide and capable of expressing the polypeptide in a suitable medium under conditions that permit expression of the peptide, and the resulting peptide is then isolated from the culture.

The medium used for culture may be standard medium suitable for culturing the host cells, such as minimal or complex medium containing appropriate additives. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogs of the American Type Culture Collection). The peptide produced by the host cells can be isolated from the medium by standard methods, including separation of the host cells from the medium by centrifugation or filtration, precipitation of the proteinaceous components from the supernatant, or filtration with a salt, e.g., ammonium sulfate, purification using various chromatographic techniques, e.g., chromatography. ion exchange, gel permeation chromatography, affinity chromatography, etc. depending on the type of peptide being isolated.

The DNA sequence encoding the parent peptide may be of genomic or cDNA origin, respectively, and may be obtained by preparing a genomic or cDNA library and screening it for the presence of the sequence encoding the entire peptide or a fragment thereof by hybridization with synthetic oligonucleotide probes according to standard techniques (see for example, Sambrook, J., Fritsch, EF. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the peptide can also be produced synthetically by known methods, e.g., the phosphoamidine method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805 . The DNA sequence can also be obtained by the polymerase chain reaction using specific primers as described, for example, in US Patent No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

PL 192 359 B1

The DNA sequence can be inserted into any vector which can be suitably used in recombinant DNA techniques, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, e.g. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Furthermore, the vector may also be of a type that, when introduced into a host cell, it integrates into the genome of the host cell and replicates together with the chromosome (s) into which it has integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to additional segments required for DNA transcription, such as a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice, and may be derived from genes encoding proteins both homologous and heterologous to the host cell. Examples of suitable promoters to direct the transcription of DNA encoding a peptide of the invention in a variety of cell types are well known to those skilled in the art, see, for example, Sambrook et al., Supra.

The DNA sequence encoding the peptide may also be operably linked, if necessary, to an appropriate terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell of choice.

The vector may further comprise a selectable marker, e.g. a gene whose product complements a defect in the host cell or conveys drug resistance, e.g. ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate.

In order to direct the parent peptide of the invention into the secretory pathway of host cells, the recombinant vector can be equipped with a signal sequence (also known as leader sequence, prepro sequence or pre sequence). The secretory signal sequence is added to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are typically located at the 5 'end of the DNA sequence encoding the peptide. The secretory signal sequence may be normally associated with the peptide or may be derived from a gene encoding another secreted protein.

The techniques used to ligate the DNA sequences encoding the present peptide, promoter and optionally terminator and / or secretory signal sequence, respectively, and inserting them into appropriate vectors containing the information necessary for replication, are well known to those skilled in the art (see, for example, Sambrook et al., Supra. ).

The host cell into which the DNA sequence or the recombinant vector is introduced can be any cell capable of producing the present peptide, including bacteria, yeast, fungi, and higher eukaryotic cells. Examples of suitable host cells well known and used by those skilled in the art include, but are not limited to, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

Examples of compounds that may be useful as GLP-1 moieties of the invention are described in WO 87/06941 (The General Hospital Corporation) which relates to a peptide fragment containing GLP-1 (7-37) and functional derivatives thereof and its use as an insulinotropic agent.

Further GLP-1 analogs are described in International Patent Application No. 90/11296 (The Genenral Hospital Corporation), which relates to peptide fragments containing GLP-1 (7-36) and its functional derivatives having insulinotropic activity higher than the insulinotropic activity of GLP-1 (1-36) or GLP-1 (1-37) and their use as insulinotropic agents.

International Patent Application No. 91/11457 (Buckley et al.) Discloses analogs of the active GLP-1 peptides 7-34, 7-35, 7-36 and 7-37, which also may be useful as GLP-1 moieties of the invention.

Pharmaceutical compositions containing the GLP-1 (7-37) derivatives of the invention can be administered parenterally to patients in need of such treatment. These agents can be administered parenterally by subcutaneous injection, intramuscular injection or intravenous injection using a syringe or a pen-shaped syringe. In addition, these agents can be administered parenterally using an infusion pump. Another embodiment is a powder or liquid agent for administering a GLP-1 (7-37) derivative in a nasal or pulmonary spray. Another solution is the administration of GLP-1 (7-37) derivatives transdermally, e.g. from patches, possibly iontophoretic patches, or through the mucosa, e.g. buccal.

PL 192 359 B1

Pharmaceuticals containing a GLP-1 (7-37) derivative of the invention can be prepared by standard techniques, e.g. as described in Remington^,s Pharmaceutical Sciences, 1985 or in Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

Thus, injectables containing a GLP-1 (7-37) derivative of the invention can be prepared by standard pharmaceutical industry techniques for dissolving and mixing the ingredients as needed to obtain the desired end product.

According to one procedure, the GLP-1 (7-37) derivative is dissolved in the water used in an amount that is slightly less than the final volume of the formulation made. Then, an isotonic agent, preservative and buffer are added if required, and the pH of the solution is adjusted, if necessary, with an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide. Finally, the final volume of the solution is obtained with the addition of water so as to obtain the required concentration of the components.

Examples of isotonic agents are sodium chloride, mannitol and glycerin.

Examples of preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate and sodium phosphate.

In addition to the above-mentioned ingredients, solutions containing a GLP-1 (7-37) derivative according to the invention may also contain a surfactant to improve the solubility and / or stability of the GLP-1 derivative.

An agent for intranasal administration of certain peptides can be prepared, for example, as described in European Patent No. 272097 (for Novo Nordisk A / S) or in WO 93/18785.

According to a preferred embodiment of the invention, the GLP-1 (7-37) derivative is provided in the form of an injectable agent. Such an agent may be either in the form of a ready-to-use injectable solution or in the form of a solid agent such as a lyophilized product, which must be dissolved in a solvent prior to injection. The solution for injection preferably contains not less than about 2 mg / ml, preferably not less than about 5 mg / ml, more preferably not less than about 10 mg / ml of the GLP-1 (7-37) derivative and preferably not more than about 100 mg / ml of the GLP-1 derivative (7-37).

The GLP-1 (7-37) derivatives according to the invention can be used in the treatment of various diseases. The particular GLP-1 derivative used and the optimal dose level for the patient will depend on the nature of the disease being treated and on various factors, including the effectiveness of the particular peptide derivative used, age, weight, physical activity, diet of the patient, and possible combination with other drugs. and on the severity of the case. It is recommended that the dosage of the GLP-1 (7-37) derivative according to the invention be adjusted to the individual patient by specialists.

In particular, it is expected that the GLP-1 (7-37) derivative will find application in the preparation of drugs with a prolonged action profile for the treatment of non-insulin dependent diabetes mellitus and / or in the treatment of obesity.

The invention is illustrated in more detail by the following non-limiting examples.

The following abbreviations for commercially available chemicals are used:

DMF: N, N-Dimethylformamide

NMP: N-Methyl-2-pyrrolidone

EDPA: N-Ethyl-N, N-diisopropylamine

EGTA: Ethylene glycol bis (e-aminoethyl ether) -N, N, N ', N'-tetraacetic acid

Guanosine-5-triphosphate GTP

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

Myr-ONSu: Tetradecanoic acid 2,5-Dioxopyrrolidin-1-yl ester

Pal-ONSu: Hexadecanoic acid 2,5-Dioxopyrrolidin-1-yl ester

Ste-ONSu: Octadecanoic acid 2,5-Dioxopyrrolidin-1-yl ester

HOOC- (CH₂)₆-COONSu: ω-carboxyheptanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC- (CH₂)₁₀-COONSu: ω-carboxyundecanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC- (CH₂)₁₂-COONSu: ω-carboxytridecanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC- (CH₂)₁₄-COONSu: ω-carboxypentadecanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC- (CH₂)₁₆-COONSu: ω-carboxyheptadecanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC- (CH₂)₁₈-COONSu: ω-carboxynoperecanoic acid 2,5-dioxopyrrolidin-1-yl ester

PL 192 359 B1

Shortcuts:

PDMS: Plasma Desorption Mass Spectrometry

MALDI-MS: Mass spectrometry with matrix-assisted laser desorption / ionization

HPLC: High performance liquid chromatography amu: atomic mass units

Analytical part

Plasma desorption mass spectrometry

Preparation of samples:

The sample is dissolved in 0.1% TFA / EtOH (1: 1) at a concentration of 1 µg / µl. The sample solution (5-10 μL is placed on a nitrocellulose target (Bio-ion AB, Uppsala, Sweden) and allowed to be absorbed into the target for 2 minutes, then the target is washed with 2x25 μL 0.1% TFA and dried by centrifugation. Finally, the target is placed in the target carousel and placed in the mass spectrometer. Mass Spectrum Analysis:

PDMS analysis was performed using a Bio-ion 20 flight time measuring apparatus (Bio-ion Nordic AB, Uppsala, Sweden). An accelerating voltage of 15 kV was applied and the molecular ions formed by bombardment of the nitrocellulose surface with 252-Cf decay fragments were accelerated towards the stop detector. The obtained time-of-flight spectra were converted into the true mass spectrum using H ions⁺ and NO⁺ ohm / z 1 and 30, respectively. Mass spectra were usually collected for 1.0x10⁶ decay events, which corresponded to a time of 15-20 minutes. The resulting assigned weights correspond to isotopically average molecular weights. The accuracy of the mass assignment usually exceeded 0.1%

MALDI-MS

MALDI-TOF MS analysis was performed on a Voyager RP apparatus (PerSeptive Biosystems Inc., Framingham, MA) with a delayed extraction device operating in an in-line fashion. A-cyano-4-hydroxycinnamic acid was used as a matrix and weights were assigned based on external calibration.

Example 1

Lys synthesis²⁶ N -tetradecanoyl) -GLP-1 (7-37)

The title compound was synthesized from GLP-1 (7-37). A mixture of GLP-1 (7-37) (25 mg, 7.45 μm), EDPA (26.7 mg, 208 μm), NMP (520 μθ, and water (260 μθ) was gently shaken for 5 minutes at room temperature. to the mixture, a solution of Myr-ONSu (2.5 mg, 7.67 µm) in NMP (62.5 µM) was added, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for 20 minutes. An additional amount of Myr-ONSu (2.5 mg, 7.67 µm) in NMP (62.5 µM) was added and the resulting mixture was shaken gently for 5 minutes. After a total reaction time of 40 minutes, the reaction was quenched with the addition of glycine solution (12.5 mg). , 166 µl) in 50% ethanol in water (12.5 ml).

The title compound was isolated from the reaction mixture by HPLC using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system; 1.3 mg (corresponding to 4.9% of theory) were obtained. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The isolated product was analyzed by PDMS and the m / z value for the protonated molecular ion was found to be 3567.9 3. Thus, the resulting molecular weight is 3566.9 3 amu (theoretical value: 3565.9 amu). The acylation position (Lys²⁶) was verified by enzymatic cleavage of the title compound with Staphylococcus aureus V8 protease, and then determination of the mass of the peptide fragments by PDMS.

In addition to the title compound, two other GLP-1 derivatives were isolated from the reaction mixture using the same chromatography column and a milder gradient (35-38% acetonitrile over 60 minutes), see examples 2 and 3.

Example 2

Lys synthesis³⁴(NMetradecanoyl) -GLP-1 (7-37)

The title compound was isolated by HPLC from the reaction mixture described in Example 1. PDMS analysis showed a protonated molecular ion at m / z 3567.7 ± 3. The resulting molecular weight is therefore 3566.7 ± 3 amu (theoretical value: 3565.9 amu) . The acylation site was determined on the basis of the fragment arrangement.

PL 192 359 B1

Example 3

Lys synthesis^26,34-bis (NMetradecanoyl) -GLP-1 (7-37)

The title compound was isolated by HPLC from the reaction mixture described in Example 1. PDMS analysis showed a protonated molecular ion at m / z 3778.4 ± 3. The resulting molecular weight is therefore 3777.4 ± 3 amu (theoretical value: 3776.1 amu) .

Example 4

Lys synthesis²⁶(NMetradecanoyl) Arg³⁴-GLP-1 (7-37)

The title compound was synthesized from Arg³⁴-GLP-1 (7-37). A mixture of Arg³⁴-GLP-1 (7-37) (5 mg, 1.47 μm), EDPA (5.3 mg, 41.1 μm), NMP (105 μΓ&gt; and water (50 μΓ&gt; gently shaken for 5 minutes at A solution of Myr-ONSu (0.71mg, 2.2μm in NMP (17.8μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for 20 minutes) was added to the resulting mixture. After a total reaction time of 30 minutes. the reaction was quenched by the addition of a solution of glycine (25 mg, 33.3 µm) in 50% ethanol in water (2.5 mL). The reaction mixture was purified by HPLC as described in Example 1. PDMS analysis showed a protonated molecular ion at m / z 3594.9 ± 3. The resulting molecular weight is thus 3593.9 3 amu (theoretical value: 3593.9 amu).

Example 5

Gly synthesis⁸Arg²⁶'³⁴Lys³⁶(NMetradecanoyl) -GLP-1 (7-37)

The title compound was synthesized from Gly⁸Arg^26,34Lys³⁶-GLP-1 (7-37), which was obtained from QCB. Gly mixture⁸Arg^26,34Lys³⁶-GLP-1 (7-37) (1.3 mg, 0.39 pm), EDPA (1.3 mg, 10 pm), NMP (125 μ!) And water (30 μ μ shaken gently for 5 minutes in A solution of Myr-ONSu (0.14 mg, 0.44 μm) in NMP (3.6 ml) was added to the resulting mixture, the reaction mixture was gently shaken for 15 minutes at room temperature. , 1 mg, 1.33 µm) in 50% ethanol in water (10 µm. The reaction mixture was purified by HPLC and the title compound was isolated (60 µg, 4%).

Example 6

Synthesis of Arg^26,34Lys³⁶(NMetradecanoyl) -GLP-1 (7-37) -OH

A mixture of Arg^26,34Lys³⁶-GLP-1 (7-37) -OH (5.0 mg, 1.477 pmol), EDPA (5.4 mg, 41.78 μmol), NMP (105 μθ and water (50 μθ gently shaken for 5 minutes at A solution of Myr-ONSu (0.721 mg, 2.215 µmol) in NMP (18 µL) was added to the resulting mixture. The reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 45 minutes at room temperature. 2.5 mg, 33.3 µmol) in 50% ethanol in water (250 µm. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (1.49 mg, 28%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3595 3. The resulting molecular weight is thus 3594 3 amu (theoretical value 3594 amu).

Example 7

Lys synthesis²⁶’³⁴-bis (N^e- (α-carboxynoperecanoyl)) - GLP-1 (7-37) -OH

A mixture of GLP-1 (7-37) -OH (70 mg, 20.85 μmol), EDPA (75.71 mg, 585.8 μmol), NMP (1.47 ml) and water (700 μθ) was gently shaken for 10 minutes at room temperature A solution of HOOC (CH₂)₁₈-COONSu (27.44 mg, 62.42 pmol) in NMP (686 μθ), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 50 minutes at room temperature. , 458.7 pmol) in 50% ethanol in water (3.44 ml) The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes.

The title compound (8.6 mg, 10%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 4006 3. The resulting molecular weight is thus 4005 3 amu (theoretical value 4005 amu).

Example 8

Synthesis of Arg²⁶’³⁴Lys³⁶(No.^e- (α-carboxynoperecanoyl)) - GLP-1 (7-36) -OH

A mixture of Arg^26,34Lys³⁶-GLP-1 (7-36) -OH (5.06 mg, 1.52 pmol), EDPA (5.5 mg, 42.58 μmol), NMP (106 μθ and water (100 μθ shaken gently for 5 minutes) in room temperature.

PL 192 359 B1

A solution of HOOC- (CH₂)₁₈-COONSu (1.33 mg, 3.04 μmol) in NMP (33.2 μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 2.5 hours at room temperature. The reaction was quenched by the addition of glycine solution ( 2.50 mg, 33.34 µmol) in 50% ethanol in water (250 µΐ) The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.46 mg, 8) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3652 3. The resulting molecular weight is thus 3651 3 amu (theoretical value 3651 amu).

Example 9

Synthesis of Arg^26:'Lys^:'⁸(N T '&lt;- carboxynoperecanoyl)) - GLP-1 (7-38) -OH

A mixture of Arg^2–34Lys³⁸-GLP-1 (7-38) -OH (5.556 mg, 1.57 μola), EDPA (5.68 mg, 43.96 μola), NMP (116.6 μθ and water (50 μθ gently shaken for 10 minutes at room temperature A solution of HOOC- (CH₂)₁₈-COONSu (1.38 mg, 3.14 μmol) in NMP (34.5 μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 2.5 hours at room temperature. The reaction was quenched by the addition of glycine solution ( 2.5 mg, 33.3 µmol) in 50% ethanol in water (250 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.7 mg, 12%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3866 3. The resulting molecular weight is thus 3865 3 amu (theoretical value 3865 amu).

Example 10

Synthesis of Arg³⁴Lys²⁶(No.^and!- ((O-carboxynecanoyl)) - GLP-1 (7-37) -OH

A mixture of Arg³⁴-GLP-1 (7-37) -OH (5.04 mg, 1.489 (gmol), EDPA (5.39 mg, 41.70 μmol), NMP (105 μθ and water (50 μθ shaken gently for 10 minutes in HOOC- (CH₂)₁₈-COONSu (1.31 mg, 2.97 μmol) in NMP (32.8 μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 30 minutes at room temperature. 46 mg, 32.75 µmol) in 50% ethanol in water (246 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (1.2 mg, 22%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3709 3. The resulting molecular weight is thus 3708 3 amu (theoretical value 3708 amu).

Example 11

Synthesis of Arg³⁴Lys²⁶(No.^and!- ((O-carboxyheptadecanoyl)) - GLP-1 (7-37) -OH

A mixture of Arg³⁴-GLP-1 (7-37) -OH (5.8 mg, 1.714 gmol), EDPA (6.20 mg, 47.99 gmol), NMP (121.8 μθ and water (58 μθ gently shaken for 10 minutes) at room temperature A solution of HOOC- (CH₂)₁₆-COONSu (2.11 mg, 5.142 µ ^) in NMP (52.8 µ]), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 2 hours at room temperature. The reaction was quenched by the addition of a solution of glycine (2.83 mg, 37.70 µmol) in 50% ethanol in water (283 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. . The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.81 mg, 13%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3681 3. The resulting molecular weight is thus 3680 3 amu (theoretical value 3680 amu).

Example 12

Synthesis of Arg^26,34Lys³⁶(No.^and!((O-carboxyheptadecanoyl)) - GLP-1 (7-37) -OH

A mixture of Arg^2–34Lys³⁶-GLP-1 (7-37) -OH (3.51 mg, 1.036 μola), EDPA (3.75 mg, 29.03 μola), NMP (73.8 μθ and water (35 μθ gently shaken for 10 minutes at room temperature A solution of HOOC- (CH₂)₁₆-COONSu (1.27 mg, 3.10 μmol) in NMP (31.8 μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 2 hours and 10 minutes at room temperature. (1.71 mg, 22.79 gmol) in 50% ethanol in water (171 μθ. Reaction mixture 48

The solution was purified by column chromatography using a cyanopropyl column (Zorbax 200SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.8 mg, 21%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 2682 ± 2. The resulting molecular weight is thus 2681 ± 2 amu (theoretical value 2681 amu).

Example 12

Synthesis of Arg²⁶²⁴Lys²⁸(N '- (and' &lt;- carboxyheptadecanoyl)) - GLP-1 (7-28) -OH

A mixture of Arg^2–24Lys²⁸-GLP-1 (7-28) -OH (5.168 mg, 1.459 µmol), EDPA (5.28 mg, 40.85 µmol), NMP (108.6 µL), and water (51.8 µL) was gently shaken for 10 minutes at room temperature. A solution of HOOC- (CH₂)₁₆ COONSu (1.80 mg, 4.27 µmol) in NMP (45 µL), the reaction mixture was gently shaken for 10 minutes at room temperature and allowed to stand for an additional 2 hours and 15 minutes at room temperature. The reaction was quenched by adding a solution of glycine (2.41 mg, 22.09 µmol) in 50% ethanol in water (241 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 200SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.6 mg, 14%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 2828 2. The resulting molecular weight is thus 2827 2 amu (theoretical value 2827 amu).

Example 14

Synthesis of Arg^26,24Lys²⁶(No.^and!- ((O-carboxyheptadecanoyl)) - GLP-1 (7-26) -OH

A mixture of Arg^26,24Lys²⁶-GLP-1 (7-26) -OH (24.44 mg, 7.24 µm), EDPA (26.56 mg, 205.52 µm), NMP (512 µL) and water (244.4 µL) gently shake for 5 minutes at room temperature. A solution of HOOC- (CH2) 16-COONSu (9.06 mg, 22.02 µmol) in NMP (1.21 ml) was added to the resulting mixture, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 20 minutes at room temperature. room temperature. The reaction was quenched by the addition of a solution of glycine (12.12 mg, 161.46 µmol) in 50% ethanol in water (1.21 mL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 200SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (7.5 mg, 28%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 2625 2. The resulting molecular weight is thus 2624 2 amu (theoretical value 2624 amu).

Example 15

Synthesis of Arg^26,24Lys²⁶(No.^and!- ((O-carboxyundecanoyl)) - GLP-1 (7-27) -OH

A mixture of Arg^26,24Lys²⁶-GLP-1 (7-27) -OH (4.2 mg, 1.24 µm), EDPA (4.49 mg, 24.72 µm), NMP (88.2 µL) and water (42 µL) gently shaken for 10 minutes at room temperature. A solution of HOOC- (CH2) 10-COONSu (1.21 mg, 2.72 µmol) in NMP (20.25 µL) was added to the resulting mixture, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand an additional 40 minutes at room temperature. room temperature. The reaction was quenched by the addition of a solution of glycine (2.04 mg, 27.28 µmol) in 50% ethanol in water (204 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 200SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.8 mg, 18%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 2598 2. The resulting molecular weight is thus 2597 2 amu (theoretical value 2597 amu).

Example 16

Synthesis of Arg^26,24Lys²⁸(No.^and!- ((O-carboxyundecanoyl)) - GLP-1 (7-28) -OH

A mixture of Arg^26,24Lys²⁸-GLP-1 (7-28) -OH (5.168 mg, 1.46 µmol), EDPA (5.28 mg, 40.88 µmol), NMP (108.6 µL) and water (51.7 µL) gently shaken for 10 minutes at room temperature. A solution of HOOC- (CH2) 10-COONSu (1.42 mg, 4.28 µmol) in NMP (25.8 µL) was added to the resulting mixture, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 50 minutes at room temperature. room temperature. The reaction was quenched by the addition of a solution of glycine (2.41 mg, 22.12 µmol) in 50% ethanol in water (241 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.85 mg, 16%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3753 3. The resulting molecular weight is thus 3752 3 amu (theoretical value 3752 amu).

Example 17

Lys synthesis^26,34bis (N^and!- (? -Carboxyundecanoyl)) - GLP-1 (7-37) -OH

The mixture of GLP-1 (7-37) -OH (10.0 mg, 2.98 Lmol), EDPA (10.8 mg, 83.43 gmol), NMP (210 μ!) And water (100 μΓ) was gently shaken for 10 minutes at room temperature. A solution of HOOC- (CH₂)]₁₀-COONSu (2.92 mg, 8.94 μmol) in NMP (73 μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 50 minutes at room temperature. ,

65.56 μmol) in 50% ethanol in water (492 μm!). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (1.0 mg, 9%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3781 3. Thus, the resulting molecular weight is 3780 3 amu (theoretical value 3780 amu).

Example 18

Synthesis of Arg^26,34Lys³⁶(No.^and!- (? -Carboxyundecanoyl)) - GLP-1 (7-36) -OH

A mixture of Arg^26,34Lys³⁶-GLP-1 (7-36) -OH (15.04 mg, 4.52 gmol), EDPA (16.35 mg,

126.56 μmol), NMP (315.6 μ) and water (150.4 μΡ) were gently shaken for 10 minutes at room temperature. A solution of HOOC- (CH₂)₁₀-COONSu (4.44 mg, 13.56 Limol) in NMP (111 µL), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand an additional 40 minutes at room temperature. The reaction was quenched by the addition of a solution of glycine (7.5 mg, 99.44 Lmol) in 50% ethanol in water (750 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (3.45 mg, 22%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3540 3. The resulting molecular weight is thus 3539 3 amu (theoretical value 3539 amu).

Example 19

Synthesis of Arg³⁴Lys²⁶(No.^and!- (? -Carboxyundecanoyl)) - GLP-1 (7-37) -OH

A mixture of Arg³⁴-GLP-1 (7-37) -OH (5.87 mg, 1.73 gmol), EDPA (6.27 mg, 48.57 gmol), NMP (123.3 μ!) And water (53.7 µ!) was gently shaken for 10 minutes at room temperature. A solution of HOOC- (CH₂)₁₀-COONSu (1.70 mg, 5.20 Lmol) in NMP (42.5 µL), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand an additional 40 minutes at room temperature. The reaction was quenched by the addition of a solution of glycine (2.86 mg, 286 Lmol) in 50% ethanol in water (286 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (1.27 mg, 20%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3597 3. The resulting molecular weight is thus 3596 3 amu (theoretical value 3596 amu).

Example 20

Synthesis of Arg³⁴Lys²⁶(No.^and!- (? -Carboxyheptanoyl)) - GLP-1 (7-37) -OH

A mixture of Arg³⁴-GLP-1 (7-37) -OH (4.47 mg, 1.32 gmol), EDPA (4.78 mg, 36.96 gmol), NMP (94 μ!) And water (44.8 μ!) Gently shake for 5 minutes at room temperature. A solution of HOOC (CH₂)₆-COONSu (1.07 mg, 3.96 Lmol) in NMP (26.8 µL), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand an additional 1 hour and 50 minutes at room temperature. The reaction was quenched by adding a solution of glycine (2.18 mg, 29.04 Lmol) in 50% ethanol in water (218 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and gra50 was used

Acetonitrile gradient 0-100% in 60 minutes. The title compound (0.5 mg, 11%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3540 3. The resulting molecular weight is thus 3539 3 amu (theoretical value 3539 amu).

Example 21

Synthesis of Arg^26,34Lys³⁸(No.^and!- ((O-carboxyheptanoyl)) - GLP-1 (7-38) -OH

A mixture of Arg^2–34Lys³⁸-GLP-1 (7-38) -OH (5.168 mg, 1.459 µmol), EDPA (5.28 mg, 40.85 µmol), NMP (108.6 µθ, and water (51.6 µL) gently shaken for 10 minutes at room temperature A solution of HOOC- (CH₂)₆-COONSu (1.18 mg, 4.37 µmol) in NMP (29.5 µL), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand an additional 1 hour and 50 minutes at room temperature. The reaction was quenched by the addition of a solution of glycine (2.40 mg, 32.09 µmol) in 50% ethanol in water (240 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.5 mg, 9%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3697 3. The resulting molecular weight is thus 3695 3 amu (theoretical value 3695 amu).

Example 22

Synthesis of Arg^26,34Lys³⁶- (N^and!- ((O-carboxyheptanoyl)) - GLP-1 (7-37) -OH

A mixture of Arg^26,34Lys³⁶-GLP-1 (7-37) -OH (5.00 mg, 1.47 µmol), EDPA (5.32 mg, 41.16 µmol), NMP (105 µL) and water (50 µL) shaken gently for 5 minutes at room temperature A solution of HOOC- (CH₂)₆-COONSu (1.19 mg, 4.41 µmol) in NMP (29.8 µL), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 2 hours at room temperature. The reaction was quenched by the addition of a solution of glycine (2.42 mg, 32.34 µmol) in 50% ethanol in water (242 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.78 mg, 15%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3542 3. The resulting molecular weight is thus 3541 3 amu (theoretical value 3541 amu).

Example 23

Synthesis of Arg^26,34Lys³⁶(No.^and!- ((O-carboxyheptanoyl)) - GLP-1 (7-36) -OH

A mixture of Arg^26,34Lys³⁶-GLP-1 (7-36) -OH (5.00 mg, 1.50 µmol), EDPA (5.44 mg, 42.08 µmol), NMP (210 µL), and water (50 µL shaken gently for 5 minutes at room temperature To the resulting mixture was added a solution of HOOC- (CH2) 6-COONSu (1.22 mg, 4.5 µmol) in NMP (30.5 µL), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand aside. for an additional 2 hours at room temperature The reaction was quenched by the addition of a solution of glycine (2.47 mg, 33.0 µmol) in 50% ethanol in water (247 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.71 mg, 14%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3484 3. The resulting molecular weight is thus 3483 3 amu (theoretical value 3483 amu).

Example 24

Lys synthesis^26,34bis (N^and!- ((O-carboxyheptanoyl)) - GLP-1 (7-37) -OH

A mixture of GLP-1 (7-37) -OH (10 mg, 2.5 µmol), EDPA (10.8 mg, 83.56 µmol), NMP (210 µL) and water (100 µL) was gently shaken for 10 minutes. in room temperature. A solution of HOOC- (CH2) 6-COONSu (2.42 mg, 8.92 µmol) in NMP (60.5 µL) was added to the resulting mixture, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 2 hours. 35 minutes at room temperature. The reaction was quenched by the addition of a solution of glycine (4.92 mg, 65.54 µm) in 50% ethanol in water (492 µL). The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (2.16 mg, 24%) was isolated, and the product was analyzed by PDMS.

PL 192 359 B1

The m / z value for the protonated molecular ion was found to be 3669 3. The resulting molecular weight is thus 3668 3 amu (theoretical value 3668 amu).

Example 25

Synthesis of Arg³⁴Lys²⁶(No.^and!- ((O-carboxypentadecanoyl)) - GLP-1 (7-37) -OH

A mixture of Arg³⁴-GAP-1 (7-37) -OH (4.472 mg, 1.321 gmol), EDPA (4.78 mg, 36.99 μ olola), NMP (93.9 μθ and water (44.7 μθ) shaken gently for 10 minutes at room temperature.

A solution of HOOC- (CH₂)₁₄-COONSu (1.519 mg, 3.963 μmol) in NMP (38 μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand an additional 1 hour at room temperature. The reaction was quenched by the addition of glycine solution (2.18 mg, 29.06 µmol) in 50% ethanol in water (218 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.58 mg, 12%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3654 3. The resulting molecular weight is thus 3653 3 amu (theoretical value 3653 amu).

Example 26

Synthesis of Arg^26,34Lys³⁶(No.^and!- ((O-carboxypentadecanoyl)) - GLP-1 (7-36) -OH

A mixture of Arg^26,34Lys³⁶-GLP-1 (7-36) -OH (5.00 mg, 1.50 gmol), EDPA (5.44 mg, 42.08 μmol), NMP (210 μθ and water (50 μθ gently shaken for 5 minutes) at room temperature A solution of HOOC- (CH₂)₁₄-COONSu (1.72 mg, 4.5 μmol) in NMP (43 μθ, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 1 hour at room temperature. , 33 gmol) in 50% ethanol in water (248 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.58 mg, 11%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3596 3. The resulting molecular weight is thus 3595 3 amu (theoretical value 3595 amu).

Example 27

Synthesis of lithocholic acid 2,5-dioxopyrrolidin-1-yl ester

To a mixture of lithocholic acid (5.44 g, 14.34 mmol), N-hydroxysuccinimide (1.78 g, 15.0 mmol), anhydrous THF (120 ml) and anhydrous acetonitrile (30 ml), kept at 10 ° C , a solution of N, N'-dicyclohexylcarbodiimide (3.44 g, 16.67 mmol) in anhydrous THF was added. The reaction mixture was stirred at ambient temperature for 16 hours, filtered and concentrated in vacuo. The residue was dissolved in dichloromethane (450 ml), washed with 10% aq. Na2CO3 (2x150 ml) and water (2x150 ml) then dried (MgSO4). After filtration and concentration of the filtrate in vacuo, a crystalline residue was obtained. The residue was recrystallized from a mixture of dichloromethane (30 ml) and n-heptane (30 ml) to give the title compound (3.46 g, 51%) as a crystalline solid.

Example 28

Synthesis of Arg³⁴Lys²⁶(NMitocholyl) -GLP-1 (7-37) -OH

A mixture of Arg³⁴-GLP-1 (7-37) -OH (4.472 mg, 1.32 gmol), EDPA (4.78 mg, 36.96 gmol), NMP (94 μ) and water (44.8 μ) were gently shaken by 10 minutes at room temperature. A solution of lithocholic acid 2,5-dioxopyrrolidin-1-yl ester (1.87 mg, 3.96 µmol) in NMP (46.8 µM) was added to the resulting mixture, the reaction mixture was gently shaken for 5 minutes at room temperature and set aside for additional 1 hour at room temperature. The reaction was quenched by the addition of a solution of glycine (2.183 mg, 29.04 µmol) in 50% ethanol in water (218 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system. heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes The title compound (1.25 mg, 25%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3744 3. The resulting molecular weight is thus 3743 3 amu (theoretical value 3743 amu).

PL 192 359 B1

Example 29

Synthesis of N^and-tetradecanoyl-Glu (ONSu) -Ot-Bu

To a suspension of H-Glu (OH) -Ot-Bu (2.5 g, 12.3 mmol), DMF (283 ml) and EDPA (1.58 g, 12.3 mmol), a solution of Myr-ONSu (4. 0 g, 12.3 mmol) in DMF (59 ml). The reaction mixture was stirred for 16 hours at room temperature, then concentrated in vacuo to a volume of 20 ml. The residue was partitioned between 5% aqueous citric acid (250 ml) and ethyl acetate (150 ml) and the phases were separated. The organic phase was concentrated in vacuo and the residue was dissolved in DMF (40 ml). The resulting solution was added dropwise to a 10% aqueous citric acid solution (300 ml) kept at 0 ° C. The precipitated compound was separated, washed with ice water and dried in a vacuum oven. The dried compound was dissolved in DMF (23 ml) and HONSu (1.5 g, 13 mmol) was added. A solution of N, N-dicyclohexylcarbodiimide (2.44 g, 11.9 mmol) in dichloromethane (47 mL) was added to the resulting mixture. The reaction mixture was stirred for 16 hours at room temperature, then the precipitated compound was filtered off. The precipitate was recrystallized from n-heptane / 2-propanol to give the title compound (3.03 g, 50%).

Example 30

Synthesis of Glu ^^ Arg ^ Lys ^ CNHy-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-38) -OH

A mixture of Glu^22,23,30Arg^26,34Lys³⁸-GLP-1 (7-38) -OH (1.0 mg, 0.272 gmol), EDPA (0.98 mg, 7.62 gmol), NMP (70 μθ and water (70 μΓ&gt; gently shaken for 5 minutes in A solution of N was added to the resulting mixture^and-tetradecanoyl-Glu (ONSu) -O-t-Bu, prepared as described in example 29, (0.41 mg, 0.816 gmol) in NMP (10.4 μΓ&gt;, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 45 minutes at room temperature The reaction was quenched by the addition of a solution of glycine (0.448 mg, 5.98 µmol in 50% ethanol in water (45 µM). A 0.5% aqueous ammonium acetate solution (0.9 ml) was added and the resulting mixture was bound to a Varian 500 mg C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 5% acetonitrile in water (10 ml) and finally released from the cartridge by elution. TFA (10 ml). The eluate was concentrated in vacuo and the reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.35 mg, 32%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 4012 3. The resulting molecular weight is thus 4011 3 amu (theoretical value 4011 amu).

Example 31

Gliu synthesis^3;6Arg³⁴Lys³⁸(N '- (Y-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-38) -OH

A mixture of Glu^23,26Arg³⁴Lys³⁸-GLP-1 (7-38) -OH (6.07 mg, 1.727 pmol), EDPA (6.25 mg, 48.36 pmol), NMP (425 μΓ&gt; and water (425 μΓ&gt; shaken gently for 5 minutes at room temperature A solution of N was added to the resulting mixture^and-tetradecanoyl-Glu (ONSu) -Ot-Bu, prepared as described in example 29, (2.65 mg, 5.18 μmol) in NMP (66.3 μΓ&gt;, the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 45 minutes at room temperature The reaction was quenched by the addition of a solution of glycine (2.85 mg, 38.0 µmol) in 50% ethanol in water (285 µM). A 0.5% aqueous ammonium acetate solution (5.4 ml) was added and the resulting mixture was bound to a Varian 500 mg C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 5% acetonitrile in water (10 ml) and finally released from the cartridge by elution. TFA (10 ml). The eluate was concentrated in vacuo and the reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.78 mg, 12%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3854 3. The resulting molecular weight is thus 3853 3 amu (theoretical value 3853 amu).

Example 32

Lys synthesis^26,34-bis (N^and!- ((O-carboxytridecanoyl)) - GLP-1 (7-37) -OH

A mixture of GLP-1 (7-37) -OH (30 mg, 8.9 gmol), EDPA (32.3 mg, 250 µl), NMP (2.1 ml) and water (2.1 ml) was gently shaken for 5 minutes at room temperature. A solution of HOOC- (CH₂)₁₂-COONSu (12.7 mg, 35.8 μmol) in NMP (318 μM), the reaction mixture was gently shaken for 1 hour and 40 minutes at room temperature. ) in 50% ethanol in water (335 μΓ&gt;.) The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (10 mg, 29%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3840 3. The resulting molecular weight is thus 3839 3 amu (theoretical value 3839 amu).

Example 33

Lys synthesis²⁶'³⁴-bis (N^and!- (Y-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-37) -OH (NNC 90-1167)

A mixture of GLP-1 (7-37) -OH (300 mg, 79.8 gmol), EDPA (288.9 mg, 2.24 mmol), NMP (21 ml) and water (21 ml) was gently shaken for 5 minutes in room temperature. A solution of N was added to the resulting mixture^and-tetradecanoyl-Glu (ONSu) -Ot-Bu, prepared as described in example 29, (163 mg, 319.3 gmol) in NMP (4.08 ml), the reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand. for an additional 1 hour at room temperature. The reaction was quenched by the addition of a solution of glycine (131.8 mg, 1.76 mmol) in 50% ethanol in water (13.2 mL). A 0.5% aqueous ammonium acetate solution (250 ml) was added and the resulting mixture was divided into 4 equal portions. Each aliquot was loaded onto a Varian 500 mg C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 0.1% aqueous TFA (3.5 ml) and finally released from the cartridge by elution with 70% aqueous acetonitrile (4 ml). The combined eluates were diluted with 0.1% aqueous TFA (300 ml). The precipitated compound was centrifuged, washed with 0.1% aqueous TFA (50 ml) and finally centrifuged. TFA (60 ml) was added to the precipitate and the reaction mixture was stirred for 1 hour and 30 minutes at room temperature. Excess TFA was removed in vacuo and the residue poured into water (50 ml). The precipitated compound was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (27.3 mg, 8%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 4036 3. Thus, the resulting molecular weight is 4035 3 amu (theoretical value 4035 amu).

Example 34

Synthesis of Arg^26,34Lys³⁸(No.^and!- ((O-carboxypentadecanoyl)) - GLP-1 (7-38) -OH

A mixture of Arg^26,34Lys³⁸-GLP-1 (7-38) -OH (30 mg, 8.9 gmol), EDPA (32.3 mg, 250 gmol) NMP (2.1 ml) and water (2.1 ml) gently shaken for 5 minutes at room temperature. A solution of HOOC- (CH₂)₁₄-COONSu (13.7 mg, 35.8 gmol) in NMP (343 μθ, the reaction mixture was gently shaken for 1 hour at room temperature. The reaction was quenched by adding a 50% glycine solution (3.4 mg, 44.7 μmol) ethanol in water (335 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system. The column was heated to 65 ° C and an acetonitrile gradient was applied 0-100% in 60 minutes. The title compound (4.8 mg, 14%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3894 3. The resulting molecular weight is thus 3893 3 amu (theoretical value 3893 amu).

Example 35

Synthesis of N^and-hexadecanoyl-Glu (ONSu) -Ot-Bu

To a suspension of H-Glu (OH) -Ot-Bu (4.2 g, 20.6 mmol), DMF (500 mL), and EDPA (2.65 g, 20.6 mmol) was added a solution of Pal-ONSu (7. 3 g, 20.6 mmol) in DMF (100 mL). The reaction mixture was stirred for 64 hours at room temperature, then concentrated in vacuo to a volume of 20 ml. The residue was partitioned between 10% aqueous citric acid (300 ml) and ethyl acetate (250 ml) and the phases were separated. The organic phase was concentrated in vacuo and the residue was dissolved in DMF (50 ml). The resulting solution was added dropwise to a 10% aqueous citric acid solution (500 ml) kept at 0 ° C. The precipitated compound was separated, washed with ice water and dried in a vacuum oven. The dried compound was dissolved in DMF (45 ml) and HONSu (2.15 g, 18.7 mmol) was added. A solution of N, N'-dicyclohexylcarbodiimide (3.5 g, 17 mmol) in dichloromethane (67 mL) was added to the resulting mixture. The reaction mixture was stirred for 16 hours at room temperature and the precipitated compound was filtered off. The precipitate was recrystallized from n-heptane / 2-propanol to give the title compound (6.6 g, 72%).

PL 192 359 B1

Example 36

Lys synthesis^26,34-bis (N^and!- (Y-glutamylglutamyl (N^and-hexadecanoyl))) - GLP-1 (7-37) -OH

A mixture of GLP-1 (7-37) -OH (10 mg, 2.9 gmol), EDPA (10.8 mg, 83.4 gmol), NMP (0.7 ml) and water (0.7 ml) shake for 5 minutes at room temperature. A solution of N was added to the resulting mixture^and-hexadecanoyl-Glu (ONSu) -Ot-Bu, prepared as described in Example 33, (163 mg, 319.3 Lmol) in NMP (4.08 ml), the reaction mixture was gently shaken for 1 hour and 20 minutes at room. The reaction was quenched by the addition of a solution of glycine (4.9 mg, 65.6 Lmol) in 50% ethanol in water (492 µL). A 0.5% aqueous ammonium acetate solution (9 ml) was added and the resulting mixture was applied to a Varian 1 g C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (10 ml) and finally released from the cartridge by eluting with TFA ( 10 ml). The eluate was concentrated in vacuo and the residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (2.4 mg, 20%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 4092 3. The resulting molecular weight is thus 4091 3 amu (theoretical value 4091 amu).

Example 37

Synthesis of Arg³⁴Lys²⁶(No.^and!- (Y-glutamyl (N^and-hexadecanoyl))) - GLP-1 (7-37) -OH

A mixture of Arg³⁴-GLP-1 (7-37) -OH (3.7 mg, 1.1 Lmol), EDPA (4.0 mg, 30.8 Lmol), acetonitrile (260 μ!) And water (260 μ!) Gently shake for 5 minutes at room temperature. A solution of N was added to the resulting mixture^and-hexadecanoyl-Glu (ONSu-Ot-Bu, prepared as described in Example 35) (1.8 mg, 3.3 Lmol) in acetonitrile (44.2 μL) and the reaction mixture was gently shaken for 1 hour and 20 minutes at room temperature The reaction was quenched by the addition of a solution of glycine (1.8 mg, 24.2 Lmol) in 50% ethanol in water (181 µL). 0.5% aqueous ammonium acetate (12 ml) and NMP (300 μL) were added and the resulting mixture was applied to a Varian 1 g C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (10 ml) and finally was released from the cartridge by eluting with TFA (6 ml). The eluate was allowed to stand for 2 hours at room temperature then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.23 mg, 6%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3752 3. The resulting molecular weight is thus 3751 3 amu (theoretical value 3751 amu).

Example 38

Synthesis of Arg^26,34Lys³³(No.^and!- (Y-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-38) -OH

A mixture of Arg^26,34Lys³⁸-GLP-1 (7-38) -OH (14 mg, 4.0 Lmol), EDPA (14.3 mg, 110.6 Lmol), NMP (980 μ!) And water (980 μ!) Were gently shaken for 5 minutes at room temperature. A solution of N was added to the resulting mixture^and-tetradecanoyl-Glu (ONSu) -Ot-Bu, prepared as described in Example 29, (12.1 mg, 23.7 Lmol) in NMP (303 µL) and the reaction mixture was gently shaken for 2 hours at room temperature. The reaction was quenched by the addition of a solution of glycine (6.5 mg, 86.9 mmol) in 50% ethanol in water (652 µL). A 0.5% aqueous ammonium acetate solution (50 ml) was added and the resulting mixture was applied to a Varian 1 g C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (15 ml) and finally released from the cartridge by eluting with TFA ( 6 ml). The eluate was allowed to stand for 1 hour and 45 minutes at room temperature then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes.

The title compound (3.9 mg, 26%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3881 3. The resulting molecular weight is thus 3830 3 amu (theoretical value 3680 amu).

Example 39

Synthesis of Arg^26,34Lys³⁸(No.^and!- (α-carboxypentadecanoyl)) - GLP-1 (7-38) -OH

A mixture of Arg^26,34Lys³⁸-GLP-1 (7-38) -OH (14 mg, 4.0 Lmol), EDPA (14.3 mg, 111 Lmol), NMP (980 μ!) And water (980 μ!) Were gently shaken for 5 minutes in room temperature. A solution of HOOC- (CH₂)₁₄-COONS (4.5 mg, 11.9 Lmol) in NMP (114 μ!),

The reaction mixture was gently shaken for 1 hour and 45 minutes at room temperature. More HOOC- (CH₂)₁₄-COONSu (4.0 mg, 10.4 μmol) in NMP (100 μθ) and the resulting mixture was gently shaken for an additional 1 hour and 30 minutes at room temperature. µmol) in 50% ethanol in water (148 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (3.9 mg, 26%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3809 3. The resulting molecular weight is thus 3808 3 amu (theoretical value 3808 amu).

Example 40

Synthesis of Arg^26: '' Lys^:'⁸(N '- (- / - glutamyl (N^and-hexadecanoyl))) - GLP-1 (7-38) -OH

A mixture of Arg^26,34Lys³⁸-GLP-1 (7-38) -OH (14 mg, 4.0 μmol), EDPA (14.3 mg, 110.6 μmol), NMP (980 μθ and water (980 μθ) shaken gently for 5 minutes at A solution of N was added to the resulting mixture^and-hexadecanoyl-Glu (ONSu) -Ot-Bu, prepared as described in Example 35, (6.4 mg, 11.9 μmol) in NMP (160 μθ and the reaction mixture gently shaken for 1 hour and 20 minutes at room temperature The reaction was quenched by the addition of a solution of glycine (6.5 mg, 87 mmol) in 50% ethanol in water (653 μθ. 0.5% aqueous ammonium acetate (50 ml) was added and the resulting mixture was applied to a Varian 1 g C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (10 ml) and finally released from the cartridge by eluting with TFA ( 6 ml). The eluate was allowed to stand for 1 hour and 30 minutes at room temperature then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes.

The title compound (7.2 mg, 47%) was isolated, and the product was analyzed by PDMS. The m / z value for the protonated molecular ion was found to be 3881 3. The resulting molecular weight is thus 3880 3 amu (theoretical value 3880 amu).

Example 41

Synthesis of Arg¹⁸’²³’²⁶’³⁰’³⁴Lys³⁸(No.^and!-hexadecanoyl) -GLP-1 (7-38) -OH

A mixture of Arg¹⁸²³’²⁶’³⁰’³⁴Lys³⁸-GLP-1 (7-38) -OH (1.0 mg, 0.27 gmol), EDPA (0.34 mg, 2.7 μmol) and DMSO (600 μθ was gently shaken for 5 minutes at room temperature. of the resulting mixture, a solution of Pal-ONSu (0.28 mg, 0.8 µmol) in NMP (7 µM) was added. The reaction mixture was gently shaken for 5 minutes at room temperature and allowed to stand for an additional 6 hours at room temperature. glycine (1.6 mg, 21.7 gmol) in 50% ethanol in water (163 μ. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (0.17 mg, 16%) was isolated, and the product was analyzed by MALDI-MS. The m / z value for the protonated molecular ion was found to be 3961 3. The resulting molecular weight is thus 3960 3 amu (theoretical value 3960 amu).

Example 42

Synthesis of Arg^26,34Lys³⁸(No.^and!- ((O-carboxytridecanoyl)) - GLP-1 (7-38) -OH

A mixture of Arg^26,34Lys³⁸-GLP-1 (7-38) -OH (14 mg, 4.0 μmol), EDPA (14.3 mg, 111 μmol), NMP (980 μθ and water (980 μ) was gently shaken for 5 minutes at room temperature. A solution of HOOC- (CH₂)₁₂-COONSu (4.2 mg, 11.9 μmol) in NMP (105 μθ, the reaction mixture was gently shaken for 1 hour and 50 minutes at room temperature. The reaction was quenched by the addition of a glycine solution (6.5 mg, 87 μmol) in 50 μmol. % ethanol in water (652 µC. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and standard acetonitrile / TFA system. The column was heated to 65 ° C and an acetonitrile gradient was applied 0-100% in 60 minutes. . The title compound (5.8 mg, 39%) was isolated, and the product was analyzed by MALDI-MS. The m / z value for the protonated molecular ion was found to be 3780 3. The resulting molecular weight is thus 3779 3 amu (theoretical value 3781 amu).

PL 192 359 B1

Example 43

Synthesis of Arg³⁴Lys²⁶(No.^E.- (Y-glutamyl (N^and-tetradecanoyl))) - GLP-1 (7-37) -OH

A mixture of Arg³⁴-GLP-1 (7-37) -OH (15 mg, 4.4 gmol), EDPA (16 mg, 124 gmol), NMP (2 ml) and water (4.8 ml) were gently shaken for 5 minutes at temperature room. A solution of N was added to the resulting mixture^and-tetradecanoyl-Glu (ONSu) -Ot-Bu, prepared as described in Example 29, (12.1 mg, 23.7 µmol) in NMP (303 µM and the reaction was gently shaken for 2 hours at room temperature. by adding a solution of glycine (6.5 mg, 86.9 μmol) in 50% ethanol in water (652 μθ. A 0.5% aqueous ammonium acetate solution (50 ml) was added and the resulting mixture was applied to a Varian 1 g CS Mega Bond Elut® cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (15 ml) and finally released from the cartridge by eluting with TFA ( 6 ml). The eluate was allowed to stand for 1 hour and 45 minutes at room temperature then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (3.9 mg, 26%) was isolated, and the product was analyzed by MALDI-MS. The m / z value for the protonated molecular ion was found to be 3723 3. The resulting molecular weight is thus 3722 3 amu (theoretical value 3723 amu).

Example 44

Synthesis of N^and-octadecanoyl-Glu (ONSu) -Ot-Bu

To a suspension of H-Glu (OH) -Ot-Bu (2.82 g, 13.9 mmol), DMF (370 ml) and EDPA (1.79 g, 13.9 mmol), a solution of Ste-ONSu (5 3 g, 13.9 mmol) in DMF (60 ml). Dichloromethane (35 ml) was added and the reaction mixture was stirred for 24 hours at room temperature then concentrated in vacuo. The residue was partitioned between 10% aqueous citric acid (330 ml) and ethyl acetate (200 ml) and the phases were separated. The organic phase was concentrated in vacuo and the residue was dissolved in DMF (60 ml). The resulting solution was added dropwise to a 10% aqueous citric acid solution (400 ml) kept at 0 ° C. The precipitated compound was separated, washed with ice water and dried in a vacuum oven. The dried compound was dissolved in DMF (40 ml) and HONSu (1.63 g, 14.2 mmol) was added. A solution of DCC (2.66 g, 12.9 mmol) in dichloromethane (51 mL) was added to the resulting mixture. The reaction mixture was stirred for 64 hours at room temperature and the precipitated compound was filtered off. The precipitate was recrystallized from n-heptane / 2-propanol to give the title compound (4.96 g, 66%).

Example 45

Synthesis of Arg^26,34Lys³⁸(No.^E.- (Y-glutamyl (N^and-octadecanoyl))) - GLP-1 (7-38) -OH

A mixture of Arg^26,34-GLP-1 (7-38) -OH (28 mg, 7.9 gmol), EDPA (28.6 mg, 221.5 gmol), NMP (1.96 ml) and water (1.96 ml) gently shake for 5 minutes at room temperature. A solution of N was added to the resulting mixture^and-octadecanoyl-Glu (ONSu) -Ot-Bu (17.93 g, 31.6 gmol), prepared as described in Example 44, in NMP (448 μθ and the reaction mixture was gently shaken for 2 hours at room temperature. by adding a solution of glycine (13.1 mg, 174 µmol) in 50% ethanol in water (1.3 ml) 0.5% aqueous ammonium acetate (120 ml) was added and the resulting mixture was divided into 2 equal portions. Each aliquot was applied to a Varian 5 g C8 Mega Bond Elut® cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (25 ml) and finally released from the cartridge by eluting with TFA (25 ml). The combined eluates were allowed to stand for 1 hour and 25 minutes at room temperature then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The column was heated to 65 ° C and the acetonitrile gradient was 0-100% in 60 minutes. The title compound (3.6 mg, 11%) was isolated, and the product was analyzed by MALDI-MS. The m / z value for the protonated molecular ion was found to be 3940 3. The resulting molecular weight is thus 3939 3 amu (theoretical value 3937 amu).

Biological research

Prolonged action of GLP-1 derivatives after subcutaneous administration.

The prolonged effect of several GLP-1 derivatives according to the invention was determined by monitoring their plasma concentration after subcutaneous administration to healthy pigs by the method described below. For comparison, the plasma GLP-1 (7-37) concentration was also monitored after subcutaneous administration. The results are shown in Table 1. The prolonged effect of other GLP-1 derivatives according to the invention can be determined in the same manner.

PL 192 359 B1

Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, approximately 40 kg) were fasted from the start of the experiment. Each pig was administered 0.5 nmol of test compound per kilogram of body weight in 50 μM isotonic solution (5 mM phosphate, pH 7.4, 0.02% Tween^®-20 (Merck), 45 mg / ml mannitol (pyrogen free, Novo Nordisk)). Blood samples were collected through a jugular vein catheter at the times shown in Table 1. 5 ml of blood samples were poured into chilled glass tubes containing 175 μl of the following solution: 0.18M EDTA, 1500 KlE / ml aprotinin (Novo Nordisk) and 2% bacitracin ( Sigma), pH 7.4. The samples were centrifuged for 10 minutes at 5-6,000 xg within 20 minutes. The temperature was kept at 4 ° C. The supernatant was transferred to other glass tubes and stored at 20 ° C until use.

Plasma peptide concentration was determined by RIA using a monoclonal antibody specific for the N-terminal region of GLP-1 (7-27). Cross-reactivity was less than 1% with GLP-1 (7-27) and GLP-1 (8-26) amide and less than 0.1% with GLP-1 (9-27), GLP-1 (10-26 ) amide and GLP-1 (11-26) amide. The entire experiment was performed at 4 ° C.

The test was performed as follows: 100 µl of plasma was mixed with 271 µl of 96% ethanol by vortex and centrifuged at 2600 xg for 20 minutes. The supernatant was decanted into Minisorp tubes and evaporated completely (Savant Speedvac AS290). The dry residue was resuspended in assay buffer containing 80 mM NaH2PO4 / Na2HPO4, 0.1% HSA (Orpha 20/21, Behring), 10 mM EDTA, 0.6 mM thiomersal (Sigma), pH 7.5. The samples were suspended in volumes appropriate to their expected concentrations and allowed to suspend for 20 minutes. To 200 µl of the sample was added 100 µl of the antibody solution in a dilution buffer containing 40 mM NaH2PO4 / Na2HPO4, 0.1% HSA, 0.6 mM thiomersal, pH 7.5. A nonspecific sample was prepared by mixing 200 µl of buffer with 100 µl of dilution buffer. Individual standards were prepared from frozen dried stocks dissolved in 200 µl of assay buffer.

All samples were preincubated with antibody Minisorp tubes as described above for 72 hours. 200 µl of tracer in dilution buffer containing 6-7,000 CPM was added, samples were mixed and incubated for 48 hours. 1.5 ml of a suspension of 200 ml / l of heparin stabilized bovine plasma and 18 g / l of activated charcoal (Merck) in 40 mM NaH2PO4, 0.6 mM thiomersal, pH 7.5 was added to each tube. Before use, the suspension was stirred and allowed to stand for 2 hours at 4 ° C. All samples were incubated for 1 hour at 4 ° C and then centrifuged at 2400 xg for 25 minutes. Immediately after centrifugation, the supernatant was decanted and the activity was counted in a counter /. The concentration in the samples was calculated from the individual standard curves. The following plasma concentrations, calculated as% of the maximum concentration, were found for each compound (n = 2):

Table 1

<td rowspan="2">Examined relationship*)</td><td colspan="9">Number of hours after subcutaneous administration</td>

<td> 0,75</td><td> 1</td><td> 2</td><td> 4</td><td> 6</td><td> 8</td><td> 10</td><td> 12</td><td> 24</td>

<td> 1</td><td> 2</td><td> 2</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>GLP-1 (7-27)</td><td></td><td> 100</td><td> 9</td><td> 1</td><td></td><td></td><td></td><td></td><td></td>

<td>Example 25</td><td> 72</td><td> 92</td><td> 100</td><td> 98</td><td> 82</td><td> 24</td><td> 16</td><td> 16</td><td> 16</td>

<td>Example 17</td><td> 76</td><td> 71</td><td> 91</td><td> 100</td><td> 84</td><td> 68</td><td> 20</td><td></td><td> 9</td>

<td>Example 42</td><td></td><td> 29</td><td> 71</td><td> 92</td><td> 100</td><td> 91</td><td> 59</td><td> 50</td><td> 17</td>

<td>Example 27</td><td></td><td> 26</td><td> 28</td><td> 97</td><td> 100</td><td> 71</td><td> 81</td><td> 80</td><td> 45</td>

<td>Example 11</td><td> 24</td><td> 47</td><td> 59</td><td> 71</td><td> 100</td><td> 94</td><td> 100</td><td></td><td> 94</td>

<td>Example 12</td><td> 26</td><td> 54</td><td> 65</td><td> 94</td><td> 80</td><td> 100</td><td> 85</td><td></td><td> 92</td>

<td>Example 22</td><td> 55</td><td> 52</td><td> 90</td><td> 82</td><td> 88</td><td> 70</td><td> 98</td><td> 100</td><td> 100</td>

<td>Example 14</td><td> 18</td><td> 25</td><td> 22</td><td> 47</td><td> 98</td><td> 82</td><td> 97</td><td></td><td> 100</td>

<td>Example 12</td><td> 15</td><td> 22</td><td> 28</td><td> 59</td><td> 97</td><td> 85</td><td> 100</td><td></td><td> 76</td>

<td>Example 28</td><td> 60</td><td> 52</td><td> 100</td><td> 66</td><td> 48</td><td> 29</td><td> 25</td><td> 29</td><td> 0</td>

<td>Example 29</td><td> 28</td><td> 100</td><td> 70</td><td> 47</td><td> 22</td><td> 22</td><td> 18</td><td> 27</td><td> 14</td>

PL 192 359 B1 cont. table 1

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>Example 40</td><td> 47</td><td> 19</td><td> 50</td><td> 100</td><td> 51</td><td> 56</td><td> 34</td><td> 14</td><td> 0</td>

<td>Example 34</td><td> 19</td><td> 32</td><td> 44</td><td> 84</td><td> 59</td><td> 66</td><td> 83</td><td> 84</td><td> 100</td>

*) Test compounds are compounds numbered according to the examples

As shown in Table 1, the GLP-1 derivatives of the invention have a prolonged profile of action relative to GLP-1 (7-37) and are much more stable in plasma than GLP-1 (7-37). Table 1 also shows that the time to reach the maximum plasma concentration varies widely depending on the particular GLP-1 derivative selected.

Stimulation of cAMP formation in a cell line expressing the cloned human GLP-1 receptor.

In order to demonstrate the efficiency of GLP-1 derivatives, the ability to stimulate cAMP formation in a cell line expressing the cloned human GLP-1 receptor was tested. From the dose-response curve, the EC50 was determined.

Baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1 receptor (Knudsen and Pridal, 1996, Eur. J. Pharm. 318, 429-435) were used. Plasma membranes (Adelhorst et al., 1994, J. Biol. Chem. 269, 6275) were prepared by homogenization in a buffer (10 mmol / L Tris-HCl and 40 mmol / L NaCl, pH 7.4, additionally containing 1 mmol / L). dithiothreitol, 5 mg / l leupeptin (Sigma, St. Louis, Mo, USA), 5 mg / l pepstatin (Sigma, St. Louis, Mo, USA) 100 mg / l bacitracin (Sigma, St. Louis, USA) Louis, Mo, USA) and 16 mg / l aprotinin (Novo Nordisk A / S, Bagsvaerd, Denmark). The homogenate was centrifuged on the surface of the layer 41% w / v. sucrose. The white band lying between the two layers was diluted in buffer and centrifuged. Plasma membranes were stored at -80 ° C until use.

The assay was performed in 96-well microtiter plates in a total volume of 140 µl. Buffer 50mmol / L Tris-HCl, pH 7.4 with the addition of 1mmol / L EGTA, 1.5mmol / L MgSO4, 1.7mmol / L ATP, 20mM GTP, 2mmol / L 3-isobutyl-1 was used. -methylxanthines, 0.01% Tween-20 and 0.1% human serum albumin (Reinst, Behringwerke AG, Marburg, Germany). Test compounds for agonist activity were dissolved and diluted in buffer, added to the membrane preparation, and the mixture was incubated for 2 hours at 37 ° C. The reaction was stopped by adding 25 µL 0.05 mol / L HCl. Samples were diluted 10-fold prior to cAMP determination by scintillation proximity assay (RPA 538, Amersham, UK). The following results were obtained:

<td>Test compound *)</td><td>EC50, pM</td><td>Test compound *)</td><td>EC50, pM</td>

<td>GLP-1 (7-37)</td><td> 61</td><td>Example 31</td><td> 96</td>

<td>Example 45</td><td> 120</td><td>Example 30</td><td> 41</td>

<td>Example 43</td><td> 24</td><td>Example 26</td><td> 8,8</td>

<td>Example 40</td><td> 55</td><td>Example 25</td><td> 99</td>

<td>Example 39</td><td> 5,1</td><td>Example 19</td><td> 79</td>

<td>Example 38</td><td> 54</td><td>Example 16</td><td> 3,5</td>

<td>Example 37</td><td> 60</td><td></td><td></td>

*) Test compounds are the title compounds numbered according to the examples.

PL 192 359 B1