PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER·THE PATENT COOPERATION TREATY (PCT)
`WO 98/08871
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`C07K 14/605, A61K 38126
`
`(11) International Publication Number:
`
`Al
`
`(43) International Publication Date:
`
`5 March 1998 (05.03.98)
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB. BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, Fl, GB, GE,
`GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV. MD, MG, MK, MN, MW, MX, NO, NZ,
`PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR,
`TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO patent (OH,
`KE, LS, MW, SD, SZ. UG, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE,
`CH, DE, DK, ES, Fl, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAP! patent (BF, BJ, CP, CG, Cl, CM, GA, GN,
`ML, MR. NE, SN, TD, TO).
`
`Published
`With international search report.
`
`(21) International Application Number:
`
`PCT/DK97/00340
`
`(22) International Filing Date:
`
`22 August 1997 (22.08.97)
`
`(30) Priority Data:
`0931/96
`1259/96
`1470/96
`
`30 August 1996 (30.08.96)
`DK
`DK
`8 November 1996 (08.11.96)
`20 December 1996 (20. 12.96) DK
`
`(71) Applicant (for all designated States except US): NOVO
`NORDISK A/S [DK/DK]; Novo Alie. DK-2880 Bagsva:rd
`(DK).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KNUDSEN, Liselone,
`Bjerre [DK/DK]; Valby Langgade 49A, 1.tv., DK-2500
`Valby (DK). S0RENSEN, Per, Olaf [DK/DK]; Applebys
`Plads 27, 5. mf., DK-1411 Copenhagen K (DK). NIELSEN,
`Per, Franklin [DK/DK]; Dals0 Park 59, DK-3500 Vrerl0se
`(DK).
`
`(74) Common Representative: NOVO NORDISK A/S; Corporate
`Patents, Novo Alie, DK-2880 Bagsva:rd (DK).
`
`(54) Title: GLP-1 DERIVATIVES
`
`(57) Abstract
`
`Derivatives of GLP-1 and analogues thereof having a lipophilic substituent have interesting pharmacological properties, in particular
`they have a more protracted profile of a.ction than GLP-1 (7-37).
`
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States pany to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`B£
`BF
`BG
`BJ
`BR
`BV
`CA
`c•·
`CG
`CH
`Cl
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Annenia
`Aus1ria
`Aus1ralla
`Azerbaijan
`Bosnia and Herz.egovina
`Barbados
`Belgium
`Burtina l'aso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Cenlral African ReJ)llblic
`Congo
`Switzerland
`C«c d'Ivoire
`Cameroon
`China
`CUba
`Czech Republic
`Gennany
`Denmartc
`Estonia
`
`ES
`Fl
`FR
`GA
`GB
`GE
`GIi
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LIC
`LR
`
`Spain
`Finland
`kance
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hunglll)'
`Ireland
`bracl
`Iceland
`l1aly
`Japan
`Kenya
`Kyrgyutan
`Democra«ic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanh
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`so
`SE
`SG
`
`LeSO!ho
`Li1huania
`Luxembourg
`La1via
`Mooaco
`Republic of Moldova
`Madagascar
`The fonner Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Nethcdands
`Norway
`New Zealand
`Poland
`Ponugal
`Romania
`Russian Federa«ion
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`Tr
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Oiad
`Togo
`Tajikistan
`Turkmenistan
`Turlccy
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`UzbekiSlan
`Vici Nam
`Yugoslavia
`Zimbabwe
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`W098/08871
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`PCT/DK97/00340
`
`1
`
`GLP-1 DERIVATIVES
`
`FIELD OF THE INVENTION
`
`5 The present invention relates to novel derivatives of human glucagon-like peptide-1 (GLP-1 )
`
`and fragments thereof and analogues of such fragments which have a protracted profile of
`
`action and to methods of making and using them.
`
`10 BACKGROUND OF THE INVENTION
`
`Peptides are widely used in medical practice, and since they can be produced by
`
`recombinant DNA technology it can be expected that their importance will increase also in
`
`the years to come. When native peptides or analogues thereof are used in therapy it is
`
`15 generally found that they have a high clearance. A high clearance of a therapeutic agent is
`
`inconvenient in cases where it is desired to maintain a high blood level thereof over a
`
`prolonged period of time since repeated administrations will then be necessary. Examples of
`
`peptides which have a high clearance are: ACTH, corticotropin-releasing factor, angiotensin,
`
`calcitonin, insulin, glucagon, glucagon-like peptide-1 , glucagon-like peptide-2, insulin-like
`
`20 growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone(cid:173)
`
`releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin,
`
`somatostatin,
`
`somatotropin,
`
`somatomedin,
`
`parathyroid hormone,
`
`thrombopoietin,
`
`erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones.
`
`endorphins, enkephalins, vasopressin, oxytocin, opiods and analogues thereof, superoxide
`
`25 dismutase, interferon, asparaginase, arginase, arginine deaminase. adenosine deaminase
`
`and ribonuclease. In some cases it is possible to influence the release profile of peptides by
`
`applying suitable pharmaceutical compositions, but this approach has various shortcomings
`
`and is not generally applicable.
`
`30 The hormones regulating insulin secretion belong to the so-called enteroinsular axis.
`designating a group of hormones, released from the gastrointestinal mucosa in response
`
`to the presence and absorption of nutrients in the gut, which promote an early and
`potentiated release of insulin. The enhancing effect on insulin secretion, the so-called
`incretin effect, is probably essential
`for a normal glucose tolerance. Many of the
`35 qastrointestinal hormones.
`includina aastrin and secretin
`(cholecystokinin
`is not
`
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`WO98/08871
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`PCT/DK97/00340
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`2
`
`insulinotropic in man), are insulinotropic, but the only physiologically important ones, those
`that are responsible for the incretin effect. are the glucose-dependent insulinotropic
`polypeptide, GIP, and glucagon-like peptide-1 (GLP-1). Because of its insulinotropic effect,
`GIP, isolated in 1973 (1) immediately attracted considerable interest among diabetologists.
`5 However, numerous investigations carried out during the following years clearly indicated
`that a defective secretion of GIP was not involved in the pathogenesis of insulin dependent
`diabetes mellitus (IOOM) or non insulin-dependent diabetes mellitus (NIODM) (2).
`Furthermore. as an insulinotropic hormone, GIP was found to be almost ineffective in
`NIDDM (2). The other incretin hormone, GLP-1 is the most potent insulinotropic substance
`10 known (3). Unlike GIP, it is surprisingly effective in stimulating insulin secretion in NIDDM
`patients. In addition, and in contrast to the other insulinotropic hormones (perhaps with the
`exception of secretin) it also potently inhibits glucagon secretion. Because of these actions
`it has pronounced blood glucose lowering effects particularly in patients with NIDDM
`
`20
`
`15 GLP-1 , a product of the proglucagon (4), is one of the youngest members of the secretin(cid:173)
`VIP family of peptides, but is already established as an important gut hormone with
`regulatory function in glucose metabolism and gastrointestinal secretion and metabolism
`(5). The glucagon gene is processed differently in the pancreas and in the intestine. In the
`pancreas (9), the processing leads to the formation and parallel secretion of 1) glucagon
`itself, occupying positions 33-61 of proglucagon (PG); 2) an N-terminal peptide of 30
`amino acids (PG (1 -30)) often called glicentin-related pancreatic peptide, GRPP (10, 11);
`3) a hexapeptide corresponding to PG (64-69); 4) and, finally, the so-called major
`proglucagon fragment (PG (72-158) ). in which the two glucagon-like sequences are buried
`(9). Glucagon seems to be the only biologically active product. In contrast, in the intestinal
`25 mucosa, it is glucagon that is buried in a larger molecule, while the two glucagon-like
`peptides are formed separately (8). The following products are formed and secreted in
`parallel: 1) glicentin, corresponding to PG (1-69), with the glucagon sequence occupying
`residues Nos. 33-61 (12); 2) GLP-1(7-36)amide (PG (78-107))amide (13), not as originally
`believed PG (72-107)amide or 108, which is inactive). Small amounts of C-terminally
`30 glycine-extended but equally bioactive GLP-1 (7-37), (PG (78-108)) are also formed (14);
`3) intervening peptide-2 (PG (111-122)amide) (15); and 4) GLP-2 (PG (1 26-158)) (15, 16).
`A fraction of glicentin is cleaved further into GRPP (PG (1-30)) and oxyntomodulin (PG
`(33-69)) (17, 18). Of these peptides, GLP-1, has the most conspicuous biological activities.
`
`35 Being secreted in parallel with glicentin/enteroglucagon, it follows that the many studies of
`enteroglucagon secretion (6, 7) to some extent also apply to GLP-1 secretion, but GLP-1
`is metabolised more quickly with a plasma half-life in humans of 2 min ( 19). Carbohydrate
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`W098/08871
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`or fat-rich meals stimulate secretion (20), presumably as a result of direct interaction of yet
`unabsorbed nutrients with the microvilli of the open-type L-cells of the gut mucosa.
`Endocrine or neural mechanisms promoting GLP-1 secretion may exist but have not yet
`been demonstrated in humans.
`
`5
`
`The incretin function of GLP-1 (29-31) has been clearly illustrated in experiments with the
`GLP-1 receptor antagonist, exendin 9-39, which dramatically reduces the incretin effect
`elicited by oral glucose in rats (21 , 22). The hormone interacts directly with the f3-cells via
`the GLP-1 receptor (23) which belongs to the glucagonNIP/calcitonin family of G-protein-
`10 coupled 7 -transmembrane spanning receptors. The importance of the GLP-1 receptor in
`regulating insulin secretion was illustrated in recent experiments in which a targeted
`disruption of the GLP-1 receptor gene was carried out in mice. Animals homozygous for
`the disruption had greatly deteriorated glucose tolerance and fasting hyperglycaemia, and
`even heterozygous animals were glucose intolerant (24). The signal transduction
`15 mechanism (25) primarily involves activation of adenylate cyclase, but elevations of
`intracellular ca2+ are also essential (25, 26). The action of the hormone is best described
`as a potentiation of glucose stimulated insulin release (25), but the mechanism that
`couples glucose and GLP-1 stimulation is not known. It may involve a calcium-induced
`calcium release (26. 27). As already mentioned, the insulinotropic action of GLP-1 is
`20 preserved in diabetic r3-cells. The relation of the latter to its ability to convey "glucose
`competence" to isolated insulin-secreting cells (26, 28), which respond poorly to glucose or
`GLP-1 alone. but fully to a combination of the two, is also not known. Equally importantly,
`however, the hormone also potently inhibits glucagon secretion (29). The mechanism is
`not known. but seems to be paracrine, via neighbouring insulin or somatostatin cells (25).
`25 Also the glucagonostatic action is glucose-dependent, so that the inhibitory effect
`decreases as blood glucose decreases. Because of this dual effect, if the plasma GLP-1
`concentrations increase either by increased secretion or by exogenous infusion the molar
`ratio of insulin to glucagon in the blood that reaches the liver via the portal circulation is
`greatly increased, whereby hepatic glucose production decreases (30). As a result blood
`30 glucose concentrations decrease. Because of the glucose dependency of the insulinotropic
`and glucagonostatic actions, the glucose lowering effect is self-limiting, and the hormone.
`therefore , does not cause hypoglycaemia regardless of dose (31 ). The effects are
`in whom
`infusions of slightly
`in patients with diabetes mellitus (32),
`preserved
`supraphysiological doses of GLP-1 may completely normalise blood glucose values in
`35 spite of poor metabolic control and secondary failure to sulphonylurea (33). The
`importance of the glucagonostatic effect is illustrated by the finding that GLP-1 also lowers
`blood glucose in type-1 diabetic patients without residual p-cell secretory capacity (34).
`
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`5
`
`10
`
`In addition to its effects on the pancreatic islets, GLP-1 has powerful actions on the
`gastrointestinal
`tract.
`Infused
`in physiological amounts, GLP-1 potently
`inhibits
`pentagastrin-induced as well as meal-induced gastric acid secretion (35, 36). It also
`inhibits gastric emptying rate and pancreatic enzyme secretion (36). Similar inhibitory
`effects on gastric and pancreatic secretion and motility may be elicited in humans upon
`periusion of the
`ileum with carbohydrate- or
`lipid-containing solutions (37. 38).
`Concomitantly, GLP-1 secretion is greatly stimulated, and it has been speculated that
`GLP-1 may be at least partly responsible for this so-called ~ileal-brake" effect (38). In fact,
`recent studies suggest that, physiologically, the ileal-brake effects of GLP-1 may be more
`important than its effects on the pancreatic islets. Thus, in dose response studies GLP-1
`influences gastric emptying rate at infusion rates at least as low as those required to
`influence islet secretion (39).
`
`15 GLP-1 seems to have an effect on food intake. lntraventricular administration of GLP-1
`profoundly inhibits food intake in rats (40. 42). This effect seems to be highly specific.
`Thus, N-terminally extended GLP-1 (PG 72-107)amide is inactive and appropriate doses
`of the GLP-1 antagonist, exendin 9-39, abolish the effects of GLP-1 (41 ). Acute, peripheral
`administration of GLP-1 does not inhibit food intake acutely in rats (41 , 42). However, it
`remains possible that GLP-1 secreted from the intestinal L-cells may also act as a satiety
`signal.
`
`20
`
`Not only the insulinotropic effects but also the effects of GLP-1 on the gastrointestinal tract
`are preserved in diabetic patients (43), and may help curtailing meal-induced glucose
`25 excursions. but, more
`importantly, may also influence
`food
`intake. Administered
`intravenously, continuously for one week, GLP-1 at 4 ng/kg/min has been demonstrated to
`dramatically improve glycaemic control in NIDDM patients without significant side effects
`(44). The peptide is fully active after subcutaneous administration (45), but is rapidly
`degraded mainly due to degradation by dipeptidyl peptidase IV-like enzymes (46, 47).
`
`30
`
`The amino acid sequence of GLP-1 is given i.a. by Schmidt et al. (Diabetologia 28 704-707
`(1 985). Although the interesting pharmacological properties of GLP-1(7-37) and analogues
`thereof have attracted much attention in recent years only little is known about the
`structure of these molecules. The secondary structure of GLP-1 in micelles has been
`35 described by Thorton et al.
`(1 994)), but in normal
`(Biochemistry 33 3532-3539
`solution,GLP-1
`is considered a very flexible molecule. Surprisingly, we found that
`derivatisation of this relatively small and very flexible molecule resulted in compounds
`
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`WO98/08871
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`PCT/DK97/00340
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`5
`
`whose plasma profile were highly protracted and still had retained activity.
`
`GLP-1 and analogues of GLP-1 and fragments thereof are potentially useful i.a. in the
`
`treatment of type 1 and type 2 diabetes. However, the high clearance limits the usefulness of
`
`5
`
`these compounds. and thus there still is a need for improvements in this field. Accordingly, it
`
`is one object of the present invention to provide derivatives of GLP-1 and analogues thereof
`
`which have a protracted profile of action relative to GLP-1 (7-37). It is a further object of the
`
`invention to provide derivatives of GLP-1 and analogues thereof which have a lower
`
`clearance than GLP-1 (7-37). It is a further object of the invention to provide a pharmaceutical
`
`1 O composition comprising a compound according to the invention and to use a compound of
`
`the invention to provide such a composition. Also, it is an object of the present invention to
`
`provide a method of treating insulin dependent and non-insulin dependent diabetes mellitus.
`
`15 References.
`
`1. Pederson RA Gastric Inhibitory Polypeptide. In Walsh JH, Dockray GJ (eds) Gut
`
`peptides: Biochemistry and Physiology. Raven Press, New York 1994, pp. 217259.
`
`20 2. Krarup T. lmmunoreactive gastric inhibitory polypeptide. Endocr Rev 1988;9: 122-134.
`
`3. 0rskov C. Glucagon-like peptide-1, a new hormone of the enteroinsular axis.
`
`Diabetologia 1992; 35:701-711 .
`
`25 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 JJ. Glucagon-like peptide-1 (GLP-1) - a newly discovered GI hormone.
`
`Gastroenterology 1994; 107: 1848-1855.
`
`30
`
`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,
`
`35
`
`Dockray GJ, eds. Gut peptides: Biochemistry and Physiology. Raven Press, New York,
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`6
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`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
`
`5
`
`secreted separately from the pig small intestine, but not pancreas. Endocrinology
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`1986;119: 1467-1475.
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`9. Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, 0rskov C. Proglucagon
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`processing in porcine and human pancreas. J Biol Chem, 1994; 269: 18827-1883.
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`10
`
`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 characterisation of a glicentin-related
`pancreatic peptide (proglucagon fragment) from porcine pancreas. Biochim Biophys
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`15
`
`Acta 1982;703:134-141.
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`12. Thim L, Moody AJ. The primary structure of glicentin (proglucagon). Regul Pepi
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`1981;2:139-151 .
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`20
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`13. 0 rskov C, Bersani M. Johnsen AH. H0jrup P. Holst JJ. Complete sequences of
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`glucagon-like peptide-1 (GLP-1) from human and pig small intestine. J. Biol. Chem.
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`1989;264: 12826-12829.
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`25 14. 0rskov C, Rabenh0j L, Kofod H, Wettergren A, Holst JJ. Production and secretion of
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`amidated and glycine-extended glucagon-like peptide-1 (GLP-1) in man. Diabetes 1991;
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`43: 535-539.
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`15. Buhl T, Thim L, Kofod H, 0rskov C, Harling H, & Holst JJ: Naturally occurring products
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`30
`
`of proglucagon 111-160 in the porcine and human small intestine. J. Biol. Chem.
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`1988;263:8621-8624.
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`16. 0rskov C, Buhl T, Rabenh0j L, Kofod H, Holst JJ: Carboxypeptidase-B-like processing
`
`of the C-terminus of gtucagon-like peptide-2 in pig and human small intestine. FESS
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`letters, 1989;247:193-106.
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`17. Holst JJ. Evidence that enteroglucagon (11) is identical with the C-terminal sequence
`
`(residues 33-69) of glicentin. Biochem J. 1980;187:337-343.
`
`5
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`18. Bataille D, Tatemoto K, Gespach C, Jornvall H, Rosselin G, Mutt V. Isolation of
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`glucagon-37 (bioactive enteroglucagon/oxyntomodulin)
`
`from porcine
`
`jejuna-ileum.
`
`Characterisation of the peptide. FESS Lett 1982; 146:79-86.
`
`10 19. 0rskov C, Wettergren A, Holst JJ. The metabolic rate and the biological effects of
`GLP-1 7-36amide and GLP-1 7 -37 in healthy volunteers are identical. Diabetes
`1993;42:658-661.
`
`20. Elliott RM, Morgan LM, Tredger JA, Deacon S. Wright J, Marks V. Glucagon-like
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`15
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`peptide-1 (7-36)amide and glucose-dependent insulinotropic polypeptide secretion in
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`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
`
`20
`
`glucagon-like peptide-1 receptor antagonist exendin (9-39)amide. Diabetes 1995; 44:
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`16-19.
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`22. Wang Z, Wang RM, Owji AA, Smith DM. Ghatei M, Bloom SR. Glucagon-like peptide-1
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`is a physiological incretin in rat. J. Clin. Invest. 1995; 95: 417-421.
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`25
`
`23. Thorens B. Expression cloning of the pancreatic b cell receptor for the gluco-incretin
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`hormone glucagon-like peptide 1. Proc Natl Acad Sci 1992;89:8641-4645.
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`24. Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ. Diabetes in mice with targeted
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`30
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`disruption of the GLP-1 receptor gene. Diabetes 1996; 45: 21A.
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`25. Fehmann HC, Goke R, Goke B. Cell and molecular biology of the incretin hormones
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`glucagon-like peptide-I (GLP-1) and glucose-dependent insulin releasing polypeptide
`
`(GIP). Endocrine Reviews. 1995; 16: 390-410.
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`26. Gromada J, Dissing S, Bokvist K, Renstrbm E, Frnkjrer-Jensen J , Wulff BS, Rorsman
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`P. Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting bTC3-cells
`
`by enhancement of intracellular calcium mobilisation. Diabetes 1995; 44: 767-774.
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`5
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`27. Holz GG, Leech CA, Habener JF. Activation of a cAMP-regulated ca2·-signaling
`pathway in pancreatic (}cells by the insulinotropic hormone g!ucagon-like peptide-1. J
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`Biol Chem, 1996; 270: 17749-17759.
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`1 O 28. Holz GG, KOhltreiber WM, Habener JF. Pancreatic beta-cells are rendered glucose
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`competent by
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`the
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`insulinotropic hormone glucagon-like peptide-1(7-37). Nature
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`1993;361 :362-365.
`
`29. 0rskov C, Holst JJ, Nielsen OV: Effect of truncated glucagon-like peptide-1
`
`15
`
`(proglucagon 78-107 amide) on endocrine secretion from pig pancreas, antrum and
`
`stomach. Endocrinology 1988; 123:2009-2013.
`
`30. Hvidberg A, Toft Nielsen M, Hilstad J, 0rskov C, Holst JJ. Effect of glucagon-like
`
`peptide-1 (proglucagon 78-107amide) on hepatic glucose production in healthy man.
`
`20
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`Metabolism 1994;43:104-108.
`
`31. Qualmann C, Nauck M, Holst JJ, 0rskov C, Creutzfeldt W. lnsulinotropic actions of
`
`intravenous glucagon-like peptide-1 [7-36 amide] in the fasting state in healthy subjects.
`
`Acta Diabetologica, 1995; 32: 13-16.
`
`25
`
`32. Nauck MA, Heimesaat MM, 0rskov C, Holst JJ , Ebert R, Creutzfeldt W. Preserved
`incretin activity of GLP-1(7-36amide) but not of synthetic human GIP in patients with
`
`type 2-diabetes mellitus. J Clin Invest 1993;91 :301-307.
`
`30 33. Nauck MA. Kleine N, 0rskov C. Holst JJ, Willms B, Creutzfeldt W . Normalisation of
`
`fasting hyperglycaemia by exogenous GLP-1 (7-36amide) in type 2-diabetic patients.
`
`Diabetologia 1993;36:741-744.
`
`34. Creutzfeldt W, Kleine N, Willms B, 0rskov C, Holst JJ, Nauck MA. Glucagonostatic
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`9
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`actions and reduction of
`
`fasting hyperglycaemia by exogenous glucagon-liem,
`
`peptide-1 (7-36amide) in type I diabetic patients. Diabetes Care 1996; 19: 580-586.
`
`35. Schjoldager BTG, Mortensen PE, Christiansen J, 0rskov C, Holst JJ. GLP-1
`
`5
`
`(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.
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`Truncated GLP-1 (proglucagon 72-107amide) inhibits gastric and pancreatic functions
`
`10
`
`in man. Dig Dis Sci 1993;38:665-673.
`
`37. Layer P, Holst JJ, Grandt D, Goebell H: lleal release of glucagon-like peptide-1
`
`(GLP-1 ): association with inhibition of gastric acid in humans. Dig Dis Sci 1995; 40:
`
`1074-1082.
`
`15
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`38. Layer P. Holst JJ. GLP-1 : A humeral mediator of the ileal brake in humans? Digestion
`
`1993; 54: 385-386 .
`
`39. Nauck M, Ettler R. Niedereichholz U. 0rskov C, Holst JJ, Schmiege! W. Inhibition of
`gastric emptying by GLP-1 (7-36 amide) or (7-37): effects on postprandial glycaemia
`
`20
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`and insulin secretion . Abstract. Gut 1995; 37 (suppl. 2): A 124.
`
`40. Schick RR, vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M.
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`Glucagon-like peptide 1 - a novel brain peptide involved in feeding regulation. in
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`25
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`Ditschuneit H, Gries FA, Hauner H, Schusdziarra V, Wechsler JG (eds .) Obesity in
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`Europe. John Libbey & Company ltd, 1994; pp. 363-367.
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`41 . Tang-Christensen M. Larsen PJ, Goke R, Fink-Jensen A, Jessop OS, M0ller M. Sheikh
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`S. Brain GLP-1(7-36) amide receptors play a major role in regulation of food and water
`
`30
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`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
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`glucagon-like peptide-1 in the regulation of feeding. Nature 1996; 379: 69-72.
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`10
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`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: A 118.
`
`5 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.
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`45. Ritzel R, 0rskov C, Holst JJ, Nauck MA. Pharmacokinetic, insulinotropic, and
`
`glucagonostatic properties of GLP-1
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`[7 -36 amide] after subcutaneous injection in
`
`1 O
`
`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.
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`47. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L, Willms 8, Holst JJ. 1995. Both
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`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.
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`15
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`20
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`SUMMARY OF THE INVENTION
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`Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is
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`25 synthesised i.a. in the L-cells in the distal ileum, in the pancreas and in the brain. Processing
`
`of preproglucagon to give GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs mainly in the
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`L-cells. A simple system is used to describe fragments and analogues of this peptide. Thus.
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`for example, Gly8-GLP-1(7-37) designates a fragment of GLP-1 formally derived from GLP-1
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`by deleting the amino acid residues Nos. 1 to 6 and substituting the naturally occurring amino
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`30 acid residue in position 8 (Ala) by Gly. Similarly, Lys34(W-tetradecanoyl)-GLP-1(7-37)
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`designates GLP-1 (7-37) wherein the £-amino group of the Lys residue in position 34 has
`
`been tetradecanoylated. Where reference in this text is made to C-terminally extended GLP-
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`1 analogues, the amino acid residue in position 38 is Arg unless otherwise indicated, the
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`optional amino acid residue in position 39 is also Arg unless otherwise indicated and the
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`optional amino acid residue in position 40 is Asp unless otherwise indicated. Also, if a C(cid:173)
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`terminally extended analogue extends to position 41, 42, 43, 44 or 45, the amino acid
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`sequence of this extension is as in the corresponding sequence in human preproglucagon
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`unless otherwise indicated.
`
`In its broadest aspect, the present invention relates to derivatives of GLP-1 and analogues
`
`thereof. The derivatives according to the invention have interesting pharmacological
`
`properties. in particular they have a more protracted profile of action than the parent
`
`peptides.
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`5
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`10
`
`In the present text, the designation "an analogue" is used to designate a peptide wherein one
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`or more amino acid residues of the parent peptide have been substituted by another amino
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`acid residue and/or wherein 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
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`15 parent peptide. Such addition can take place either at the N-terminal end or at the C-terminal
`
`end of the parent peptide or both.
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`The term "derivative" is used in the present text to designate a peptide in which one or more
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`of the amino acid residues of the parent peptide have been chemically modified , e.g. by
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`20 alkylation, acylation. ester formation or amide formation.
`
`The term "a GLP-1 derivative" is used in the present text to designate a derivative of GLP-1
`
`or an analogue thereof. In the present text, the parent peptide from which such a derivative is
`
`formally derived is in some places referred to as the "GLP-1 moiety" of the derivative.
`
`25
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`In a preferred embodiment, as described in Claim 1, the present invention relates to a GLP-1
`
`derivative wherein at least one amino acid residue of the parent peptide has a lipophilic
`
`substituent attached with the proviso that if only one lipophilic substituent is present and this
`
`substituent is attached to the N-terminal or to the C-terminal amino acid residue of the parent
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`30 peptide then this substituent is an alkyl group or a group which has an w-carboxylic acid
`
`group.
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`In another preferred embodiment, as described in Claim 2, the present invention relates to a
`
`GLP-1 derivative having only one lipophilic substituent.
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`In another preferred embodiment. as described in Claim 3, the present invention relates to a
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`GLP-1 derivative having only one lipophilic substituent which substituent is an alkyl group or
`
`a group which has an ro-carboxylic acid group and is attached to the N-terminal amino acid
`
`5
`
`residue of the parent peptide.
`
`In another preferred embodiment, as described in Claim 4, the present invention relates to a
`
`GLP-1 derivative having only one lipophilic substituent which substituent is an alkyl group or
`
`a group which has an ro-carboxylic acid group and is attached to the C-terminal amino acid
`
`1 O residue of the parent peptide.
`
`In another preferred embodiment, as described in Claim 5, the present invention relates to a
`
`GLP-1 derivative having only one lipophilic substituent which substituent can be attached to
`
`any one amino acid residue which is not the N-terminal or C-terminal amino acid residue of
`
`15
`
`the parent peptide.
`
`In another preferred embodiment, as described in Claim 6, the present invention relates to a
`
`GLP-1 derivative wherein two lipophilic substituents are present.
`
`20
`
`In another preferred embodiment, as described in Claim 7, the present invention relates to a
`
`GLP-1 derivative wherein two lipophilic substituents are present, one being attached to the
`
`N-terminal amino acid residue while the other is attached to the C-terminal amino acid
`
`residue.
`
`25
`
`In another preferred embodiment. as described in Claim 8. the present invention relates to a
`
`GLP-1 derivative wherein two lipophilic substituents are present, one being attached to the
`
`N-terminal amino acid residue while the other is attached to an amino acid residue which is
`
`not N-terminal or the C-terminal amino acid residue.
`
`30
`
`In another preferred embodiment, as described in Claim 9, the present invention relates to a
`
`GLP-1 derivative wherein two lipophilic substituents are present, one being attached to the
`
`C-terminal amino acid residue while the other is attached to an amino acid residue which is
`
`not the N-terminal or the C-terminal amino acid residue.
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`In further preferred embodiment. as described in Claim 10, the present invention relates to a
`
`derivative of GLP-1(7-C), wherein C is selected from the group comprising 38, 39, 40, 41 , 42,
`
`43, 44 and 45 which derivative has just one lipophilic substituent which is attached to the C(cid:173)
`
`terminal amino acid residue of the parent peptide.
`
`5
`
`In a further preferred embodiment, the present invention relates to a GLP-1 derivative
`
`wherein the lipophilic substituent comprises from 4 to 40 carbon atoms, more preferred from
`
`8 to 25 carbon atoms.
`
`10
`
`In a further preferred embodiment, the present invention relates to a GLP-1 derivative
`
`wherein a lipophilic substituent is attached to an amino acid residue in such a way that a
`
`carboxyl group of the lipophil