`WORLD JNTELLECfUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 99/43706
`
`(11) International Publication Number:
`
`(51) International Patent Classification 6 :
`C07K 14/605, A61K 38/26
`
`Al
`
`(43) International Publ.ication Date:
`
`2 September 1999 (02.09.99)
`
`(21) International Application Number:
`
`PCT/DK99l00082
`
`(22) International Filing Date:
`
`25 February 1999 (25.02.99)
`
`(30) Priority Data:
`0268/98
`
`27 February 1998 (27.02.98)
`
`DK
`
`(71) Applicant: NOVO NORDISK NS [DK/DK); Novo Alie,
`DK-2880 Bagsvaerd (DK).
`
`(72) Inventors: KNUDSEN, Liselotte, Bjerre; Valby Langgade
`49A, I.
`tv., DK-2500 Valby (DK). HUUSFELDT,
`Per, Olaf; Applebys Plads 27,5. mf., DK-1411 Copen(cid:173)
`hagen K {DK). NIELSEN, Per, Franklin; Dals0 Park 59,
`DK-3500 V.erwse (DK). PEDERSEN, Freddy, Zimmer(cid:173)
`dahl; T11mh0jgArdvej 26, DK-3500 Vrerl0se (DK).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY,CA,CH,CN,CU,CZ,DE, DK,EE,ES,FI,GB,GD,
`GE, GH, GM, HR, HU, ID, IL, IN, 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, TI, UA, UG, UZ. VN, YU, ZW,
`ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
`ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European patent (AT, BE. CH, CY, DE, DK, ES, FI,
`FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`SN, TD, TG).
`
`Published
`With international search report.
`
`(54) Title: DERIVATIVES OF GLP-1 ANALOGS
`
`(57) Abstract
`
`The present invention relates to derivatives of GLP- 1 analogs having a lipophilic substituenL The derivatives of GLP-1 analogs of
`the present invention have a protracted profile of action.
`
`FRESENIUS EXHIBIT 1033
`Page 1 of 270
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`Cl
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Annenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Bwkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Swit.zerland
`Cate d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Gennany
`Denmark
`Estonia
`
`ES
`Fl
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`Uniled Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Tceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republ.ic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`M.L
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`lcso<bo
`Lithuania
`LW<embourg
`Latvia
`Monaco
`Republic of Moldova
`MJldagascar
`The fonner Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauri1ania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turk.men is tan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
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`FRESENIUS EXHIBIT 1033
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`WO99/43706
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`PCT/DK99/00082
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`1
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`DERIVATIVES OF GLP-1 ANALOGS
`
`FIELD OF THE INVENTION
`The present invention relates to novel derivatives of human glucagon-like peptide-1
`
`5
`
`(GLP-1 ) and fragments thereof and analogues of such fragments which have a protracted pro(cid:173)
`
`file of action and to methods of making and using them.
`
`BACKGROUND OF THE INVENTION
`
`Peptides are widely used in medical practice, and since they can be produced by re-
`
`1 o
`
`combinant 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 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
`
`15
`
`high clearance are: ACTH, corticotropin-releasing factor, angiotensin, calcitonin, insulin, gluca(cid:173)
`
`gon, glucagon-like peptide-1, glucagon-like peptide-2, insulin-like growth, factor-1 , insulin-like
`
`growth factor-2, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate
`
`cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin,
`
`parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin,
`
`20
`
`thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opiods and
`
`analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deami(cid:173)
`
`nase, 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.
`
`25
`
`The hormones regulating insulin secretion belong to the so-called enteroinsular a-
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`xis, 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 potentia(cid:173)
`
`ted 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 gastrointestinal hormones,
`
`30
`
`including gastrin and secretin (cholecystokinin is not insulinotropic in man), are insulinotro(cid:173)
`
`pic, 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 attrac(cid:173)
`
`ted considerable interest among diabetologists. However, numerous investigations carried
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`out during the following years clearly indicated that a defective secretion of GIP was not in(cid:173)
`
`volved in the pathogenesis of insulin dependent diabetes mellitus (IDDM) or non insulin(cid:173)
`
`dependent diabetes mellitus (NIDDM) (2). Furthermore, as an insulinotropic hormone, GIP
`
`was found to be almost ineffective in NIDDM (2). The other incretin hormone, GLP-1 is the
`
`5 most potent insulinotropic substance known (3). Unlike GIP, it is surprisingly effective in sti(cid:173)
`
`mulating insulin secretion in NIDDM patients. In addition, and in contrast to the other insuli(cid:173)
`
`notropic hormones (perhaps with the exception of secretin) it also potently inhibits glucagon
`
`secretion. Because of these actions it has pronounced blood glucose lowering effects parti(cid:173)
`
`cularly in patients with NIDDM.
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`10
`
`GLP-1 , a product of the proglucagon (4), is one of the youngest members of these-
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`cretin-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 pan(cid:173)
`
`creas (9), the processing leads to the formation and parallel secretion of 1) glucagon itself,
`
`15
`
`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 hexapepti(cid:173)
`
`de 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 mucosa, it is glucagon
`
`20
`
`that is buried in a larger molecule, while the two glucagon-like peptides are formed separa(cid:173)
`
`tely (8). The following products are formed and secreted in parallel: 1) glicentin, correspon(cid:173)
`
`ding 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 glycine-extended but equally bioactive
`
`25 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 (126-158)) (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.
`
`Being secreted in parallel with glicentin/enteroglucagon, it follows that the many
`
`30
`
`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). Carbohy(cid:173)
`
`drate 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. En-
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`docrine or neural mechanisms promoting GLP-1 secretion may exist but have not yet been
`
`demonstrated in humans.
`
`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
`
`5
`
`effect elicited by oral glucose in rats (21 , 22). The hormone interacts directly with the !}cells
`
`via the GLP-1 receptor (23) which belongs to the glucagonNIP/calcitonin family of G-protein(cid:173)
`
`coupled 7-transmembrane spanning receptors. The importance of the GLP-1 receptor in re(cid:173)
`
`gulating 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
`
`10
`
`had greatly deteriorated glucose tolerance and fasting hyperglycaemia, and even hete(cid:173)
`
`rozygous animals were glucose intolerant (24). The signal transduction mechanism (25) pri-
`
`marily 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 stimula-
`
`15
`
`tion is not known. It may involve a calcium-induced calcium release (26, 27). As already
`
`mentioned, the insulinotropic action of GLP-1 is preserved in diabetic 13-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
`
`20
`
`secretion (29). The mechanism is not known, but seems to be paracrine, via neighbouring
`
`insulin or somatostatin cells (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
`
`25
`
`circulation is greatly increased, whereby hepatic glucose production decreases (30). As a
`
`result blood 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
`
`preserved in patients with diabetes mellitus (32), in whom infusions of slightly supraphysiolo-
`
`30
`
`gical doses of GLP-1 may completely normalise blood glucose values in spite of poor meta(cid:173)
`
`bolic control and secondary failure to sulphonylurea (33). The importance of the glucagono(cid:173)
`
`static effect is illustrated by the finding that GLP-1 also lowers blood glucose in type-1 diabe(cid:173)
`
`tic patients without residual !}cell secretory capacity (34).
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`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(cid:173)
`
`induced as well as meal-induced gastric acid secretion (35, 36). It also inhibits gastric emp(cid:173)
`
`tying rate and pancreatic enzyme secretion (36). Similar inhibitory effects on gastric and
`
`5
`
`pancreatic secretion and motility may be elicited in humans upon perfusion of the ileum with
`
`carbohydrate- or lipid-containing solutions (37, 38). Concomitantly, GLP-1 secretion is gre(cid:173)
`
`atly 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
`
`Heal-brake effects of GLP-1 may be more important than its effects on the pancreatic islets.
`
`10
`
`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).
`
`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
`
`15
`
`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 re(cid:173)
`
`mains possible that GLP-1 secreted from the intestinal L-cells may also act as a satiety sig(cid:173)
`
`nal.
`
`Not only the insulinotropic effects but also the effects of GLP-1 on the gastrointesti-
`
`20
`
`nal tract are preserved in diabetic patients (43), and may help curtailing meal-induced gluco(cid:173)
`
`se excursions, but, more importantly, may also influence food intake. Administered intra(cid:173)
`
`venously, continuously for one week, GLP-1 at 4 ng/kg/min has been demonstrated to dra(cid:173)
`
`matically improve glycaemic control in NIDDM patients without significant side effects (44).
`
`The peptide is fully active after subcutaneous administration (45), but is rapidly degraded
`
`25 mainly due to degradation by dipeptidyl peptidase IV-like enzymes (46, 47).
`
`The amino acid sequence of GLP-1 is given i.a. by Schmidt et al. (Diabetologia 28
`
`704-707 (1985). Human GLP-1 is a 37 amino acid residue peptide originating from preprog(cid:173)
`
`lucagon which is synthesised, i.a. in the L-cells in the distal ileum, in the pancreas and in the
`
`brain. Processing of preproglucagon to GLP-1 (7-36)amide, GLP-1 (7-37) and GLP-2 occurs
`
`30 mainly in the L-cells. 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 de(cid:173)
`
`scribed by Thorton et al. (Biochemistry 33 3532-3539 (1994)), but in normal solution, GLP-1
`
`is considered a very flexible molecule. Surprisingly, we found that derivatisation of this relati-
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`vely small and very flexible molecule resulted in compounds whose plasma profile were
`
`highly protracted and still had retained activity.
`GLP-1 and analogues of GLP-1 and fragments thereof are useful i.a. in the treatment
`
`of Type 1 and Type 2 diabetes and obesity.
`
`5
`
`WO 87/06941 discloses GLP-1 fragments, including GLP-1(7-37), and functional deri(cid:173)
`
`vatives thereof and to their use as an insulinotropic agent.
`
`WO. 90/11296 discloses GLP-1 fragments, including GLP-1 (7-36), and functional de(cid:173)
`
`rivatives thereof which have an insulinotropic activity which exceeds the insulinotropic activity
`
`of GLP-1 (1-36) or GLP-1 (1-37) and to their use as insulinotropic agents.
`
`10
`
`The amino acid sequence of GLP-1 (7-36) and GLP-1 (7-37) is:
`
`7
`
`8
`
`9
`
`10 11 12 13 14 1 5 16 17
`
`His-Ala-Gl u- Gly- Thr-Phe -Th r-Ser-Asp-Val - Ser-
`
`18
`
`1 9 20 21 22
`
`23
`
`24 25 26 27 28
`
`15
`
`Ser-Tyr-Leu-Glu-Gl y-Gln -Ala-Ala-Lys-Glu-Phe-
`
`29 30 31 32
`
`33 34 35 36
`
`Ile-Ala-Trp - Leu-Val-Lys - Gly-Arg-X
`
`(I)
`
`20 wherein X is NH2 for GLP-1 (7-36) and X is Gly for GLP-1 (7-37).
`WO 91/11457 discloses analogues of the active GLP-1 peptides 7-34, 7-35, 7-36, and
`
`7-37 which can also be useful as GLP-1 moieties.
`
`Unfortunately, the high clearance limits the usefulness of these compounds. Thus the(cid:173)
`
`re still is a need for improvements in this field. Accordingly, it is an object of the present inven-
`
`25
`
`tion 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 composition comprising a compound of the
`
`invention and to use a compound of the invention to provide such a composition. Also, it is an
`
`30
`
`object of the present invention to provide a method of treating insulin dependent and non(cid:173)
`
`insulin dependent diabetes mellitus.
`
`References.
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`FRESENIUS EXHIBIT 1033
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`6
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`1.
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`Pederson RA. Gastric Inhibitory Polypeptide. In Walsh JH, Dockray GJ (eds) Gut pep(cid:173)
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`tides: Biochemistry and Physiology. Raven Press, New York 1994, pp. 217259.
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`2.
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`3.
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`Krarup T. lmmunoreactive gastric inhibitory polypeptide. Endocr Rev 1988;9:122-134.
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`0rskov C. Glucagon-like peptide-1 , a new hormone of the enteroinsular axis. Diabeto-
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`5
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`logia 1992; 35:701-711 .
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`4.
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`Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC. Exon duplication and diver(cid:173)
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`gence in the human preproglucagon gene. Nature 1983; 304: 368-371.
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`5.
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`Holst JJ. Glucagon-like peptide-1 (GLP-1) - a newly discovered GI hormone. Gastro(cid:173)
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`enterology 1994; 107: 1848-1855.
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`10
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`6.
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`Holst JJ. Gut glucagon, enteroglucagon, gut GU, glicentin - current status. Gastroen(cid:173)
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`terology 1983;84:1602-1613.
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`7. Holst JJ, 0rskov C. Glucagon and other proglucagon-derived peptides. In Walsh JH,
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`Dockray GJ, eds. Gut peptides: Biochemistry and Physiology. Raven Press, New York,
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`pp. 305-340, 1993.
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`15
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`8.
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`0rskov C, Holst JJ, Knuhtsen S, Baldissera FGA, Poulsen SS, Nielsen OV. Gluca(cid:173)
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`gon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are se(cid:173)
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`creted separately from the pig small intestine, but not pancreas. Endocrinology
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`1986;119:1467-1475.
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`9.
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`Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, 0rskov C. Proglucagon pro-
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`20
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`cessing in porcine and human pancreas. J Biol Chem, 1994; 269: 18827-1883.
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`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.
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`11. Thim L, Moody AJ , Purification and chemical characterisation of a glicentin-related
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`pancreatic peptide (proglucagon fragment) from porcine pancreas. Biochim Biophys
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`25
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`Acta 1982;703:1 34-141 .
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`12. Thim L, Moody AJ . The primary structure of glicentin (proglucagon). Regul Pept
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`1981;2:139-151.
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`13. 0rskov C, Bersani M, Johnsen AH, H0jrup P, Holst JJ. Complete sequences of gluca(cid:173)
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`gon-like peptide-1 (GLP-1) from human and pig small intestine. J. Biol. Chem.
`
`30
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`1989;264:12826-12829.
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`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
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`1991 ; 43: 535-539.
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`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 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
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`5
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`of the C-terminus of glucagon-like peptide-2 in pig and human small intestine. FEBS
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`letters, 1989;247:193-106.
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`17. Holst JJ. Evidence that enteroglucagon (II) is identical with the C-terminal sequence
`
`(residues 33-69) of glicentin. Biochem J. 1980;187:337-343.
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`18. Bataille D, Tatemoto K, Gespach C, Jornvall H, Rosselin G, Mutt V. Isolation of gluca-
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`gon-37 (bioactive enteroglucagon/oxyntomodulin) from porcine jejuno-ileum. Characte(cid:173)
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`risation of the peptide. FEBS Lett 1982;146:79-86.
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`19. 0 rskov C, Wettergren A, Holst JJ. The metabolic rate and the biological effects of
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`GLP-1 7-36amide and GLP-1 7-37 in healthy volunteers are identical. Diabetes
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`1993;42:658-661.
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`15
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`20. Elliott RM , Morgan LM, Tredger JA, Deacon S, Wright J, Marks V. Glucagon-like pepti-
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`de-1 (7-36)amide and glucose-dependent insulinotropic polypeptide secretion in re(cid:173)
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`sponse to nutrient ingestion in man: acute post-prandial and 24-h secretion patterns. J
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`Endocrinol 1993; 138: 159-166.
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`21 . Kolligs F, Fehmann HC, Goke R, Goke B. Reduction of the incretin effect in rats by the
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`20
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`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 OM, 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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`23. Thorens B. Expression cloning of the pancreatic b cell receptor for the gluco-incretin
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`25
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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 dis(cid:173)
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`ruption 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
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`(GIP). Endocrine Reviews, 1995; 16: 390-410.
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`26. Gromada J, Dissing S, Bokvist K, RenstrOm E, Fr0kj~r-Jensen J, Wulff BS, Rorsman
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`P. Glucagon-like peptide
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`I
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`increases cytoplasmic calcium
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`in
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`insulin-secreting
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`bTC3-cells by enhancement of intracellular calcium mobilisation. Diabetes 1995; 44:
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`767-774.
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`27. Holz GG, Leech CA, Habener JF. Activation of a cAMP-regulated Ca2•-signaling pa(cid:173)
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`thway in pancreatic ~-cells by the insulinotropic hormone glucagon-like peptide-1. J Bi(cid:173)
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`ol Chem, 1996; 270: 17749-17759.
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`28. Holz GG, KOhltreiber WM , Habener JF. Pancreatic beta-cells are rendered glucose
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`s
`
`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
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`(proglucagon 78-107 amide) on endocrine secretion from pig pancreas, antrum and
`
`stomach. Endocrinology 1988; 123:2009-2013.
`
`10
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`30. Hvidberg A, Toft Nielsen M, Hilsted J, 0 rskov C, Holst JJ. Effect of glucagon-like pep-
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`tide- 1 (proglucagon 78-107amide) on hepatic glucose production in healthy man. Me(cid:173)
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`tabolism 1994;43: 104-108.
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`31. Qualmann C, Nauck M, Holst JJ, 0rskov C, Creutzfeldt W. lnsulinotropic actions of in(cid:173)
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`travenous glucagon-like peptide-1 [7-36 amide] in the fasting state in healthy subjects.
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`15
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`Acta Diabetologica, 1995; 32: 13-16.
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`32. Nauck MA, Heimesaat MM, 0rskov C, Holst JJ, Ebert R, Creutzfeldt W . Preserved in(cid:173)
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`cretin activity of GLP-1 (7-36amide) but not of synthetic human GIP in patients with ty(cid:173)
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`pe 2-diabetes mellitus. J Clin Invest 1993;91:301-307.
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`33. Nauck MA, Kleine N, 0rskov C, Holst JJ, Willms B, Creutzfeldt W. Normalisation of
`
`20
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`fasting hyperglycaemia by exogenous GLP-1 (7-36amide) in type 2-diabetic patients.
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`Diabetologia 1993;36:741-744.
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`34. Creutzfeldt W , Kleine N, Willms B, 0rskov C, Holst JJ, Nauck MA. Glucagonostatic ac(cid:173)
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`tions and reduction of fasting hyperglycaemia by exogenous glucagon-liem, pepti(cid:173)
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`de-1 (7-36amide) in type I diabetic patients. Diabetes Care 1996; 19: 580-586.
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`25
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`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, inhi(cid:173)
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`bit gastric acid secretion in man. Dig. Dis. Sci. 1989; 35:703-708.
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`36. Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ. Trun(cid:173)
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`cated GLP-1 (proglucagon 72-107amide) inhibits gastric and pancreatic functions in
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`30
`
`man. Dig Dis Sci 1993;38:665-673.
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`37. Layer P, Holst JJ, Grandt D, Goebell H: lleal release of glucagon-like peptide-1
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`(GLP-1): association with inhibition of gastric acid in humans. Dig Dis Sci 1995; 40:
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`1074-1082.
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`38. Layer P, Holst JJ. GLP-1: A humoral mediator of the ileal brake in humans? Digestion
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`1993; 54: 385-386.
`39. Nauck M, Ettler R, Niedereichholz U, 0rskov C, Holst JJ, Schmiege! W. Inhibition of
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`gastric emptying by GLP-1(7-36 amide) or (7-37): effects on postprandial glycaemia
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`5
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`and insulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A124.
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`40. Schick RR, vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M. Gluca(cid:173)
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`gon-like peptide 1 - a novel brain peptide involved in feeding regulation. in Ditschuneit
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`H, Gries FA, Hauner H, Schusdziarra V, Wechsler JG (eds.) Obesity in Europe. John
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`Libbey & Company ltd, 1994; pp. 363-367.
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`10
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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
`
`intake. Am. J. Physiol., 1996, in press.
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`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.
`
`15
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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 mel(cid:173)
`
`litus. 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.
`
`20
`
`45. Ritzel R, 0rskov C, Holst JJ, Nauck MA. Pharmacokinetic, insulinotropic, and gluca-
`
`gonostatic properties of GLP-1 (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
`
`25
`
`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 de(cid:173)
`
`graded from the amino terminus in type II diabetic patients and in healthy subjects.
`
`Diabetes 44: 1126-1 131.
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`30
`
`SUMMARY OF THE INVENTION
`
`The present invention relates to derivatives of GLP-1 analogues of formula I:
`
`7
`
`8
`
`9 10 11 12 13 14 15 16
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`17
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`His-xaa- xaa-Gly-Xaa-Phe-Thr-Xaa -Asp- xaa -xaa -
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`18 19
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`20
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`21
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`22
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`23 24 25 26 27 28
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`xaa-xaa -xaa -xaa-xaa-xaa -Xaa- Xaa -xaa-Xaa - Phe -
`
`5
`
`2 9 30 31 32 33 34 35 36 37 38
`
`Ile-Xaa-Xaa-xaa -xaa- Xaa-xaa- Xaa-Xaa-xaa
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`39 40 41 42 43
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`44 45
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`Xaa-Xaa -Xaa -Xaa-Xaa-Xaa -Xaa
`
`10
`
`(I)
`
`wherein
`
`Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 9 is Glu, Asp, or Lys,
`
`Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lie, Val, Glu, Asp, or Lys,
`
`15
`
`Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, lie, Tyr, Glu, Asp, or Lys,
`
`Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, or Lys,
`
`20
`
`Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`Xaa at position 21 is Glu, Asp, or Lys,
`
`Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys,
`
`Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, lie, Val, Arg, Glu, Asp, or Lys,
`
`25
`
`Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His,
`
`Xaa at position 27 is Glu, Asp, or Lys,
`
`Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys,
`
`30
`
`Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, lie, Glu, Asp, or Lys,
`
`Xaa at position 34 is Lys, Arg, Glu, Asp, or His,
`
`Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 36 is Arg, Lys, Glu, Asp, or His,
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`Xaa at position 37 is Gly, Ala, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys, or is deleted,
`
`11
`
`Xaa at position 38 is Arg, Lys, Glu, Asp, or His, or is deleted,
`
`Xaa at position 39 is Arg, Lys, Glu, Asp, or His, or is deleted,
`
`Xaa at position 40 is Asp, Glu, or Lys, or is deleted,
`Xaa at position 41 is Phe, Trp, Tyr, Glu, Asp, or Lys, or is deleted,
`
`5
`
`Xaa at position 42 is Pro, Lys, Glu, or Asp, or is deleted,
`Xaa at position 43 is Glu, Asp, or Lys, or is deleted,
`
`Xaa at position 44 is Glu, Asp, or Lys, or is deleted, and
`
`Xaa at position 45 is Val, Glu, Asp, or Lys, or is deleted, or
`
`10
`
`(a) a C-1-6-ester thereof, (b) amide, C-1-6-alkylamide, or C-1-6-dialkylamide thereof and/or (c)
`
`a pharmaceutically acceptable salt thereof,
`
`provided that
`(i) when the amino acid at position 37, 38, 39, 40, 41 , 42, 43 or 44 is deleted, then e(cid:173)
`
`ach amino acid downstream of the amino acid is also deleted,
`(ii) the derivative of the GLP-1 analog contains only one or two Lys,
`
`(iii) the e-amino group of one or both Lys is substituted with a lipophilic substituent opti(cid:173)
`
`onally via a spacer,
`(iv) the total number of different amino acids between the derivative of the GLP-1 ana(cid:173)
`
`log and the corresponding native form of GLP-1 does not exceed six.
`
`15
`
`20
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`A simple system is used to describe fragments and analogues of GLP-1 . For exam(cid:173)
`
`ple, Gly8-GLP-1(7-37) designates a fragment of GLP-1 formally derived from GLP-1 by deleting
`
`the amino acid residues Nos. 1 to 6 and substituting the naturally occurring amino acid residue
`
`25
`
`in position 8 (Ala) by Gly. Similarly, Lys34(N'-tetradecanoyl)-GLP-1(7-37) designates GLP-1(7-
`
`37) wherein the e-amino group of the Lys residue in position 34 has been tetradecanoylated.
`
`Where reference in this text is made to C-terminally extended GLP-1 analogues, the amino
`
`acid residue in position 38 is Arg unless otherwise indicated, the optional amino acid residue in
`
`position 39 is also Arg unless otherwise indicated and the optional amino acid residue in positi-
`
`30
`
`on 40 is Asp unless otherwise indicated. Also, if a C-terminally extended analogue extends to
`
`position 41 , 42, 43, 44 or 45, the amino acid sequence of this extension is as in the correspon(cid:173)
`
`ding sequence in human preproglucagon unless otherwise indicated.
`
`GLP-1 Analogs
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`The present invention relates to derivatives of GLP-1 analogues. The derivatives of
`
`the invention have interesting pharmacological properties, in particular they have a more pro(cid:173)
`
`tracte