PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6;
`C07K 14/605, A61K 38/26
`
`(11) International Publication Number:
`
`2 September 1999 (02.09.99)
`
`
`
`WO 99/43706
` (43) International Publication Date:
`
`
`(21) International Application Number:
` PCT/DK99/00082|(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,
`
`
`
` (22) International Filing Date:
`GE, GH, GM, HR, HU,ID, IL, IN, IS, JP, KE, KG, KP,
`25 February 1999 (25.02.99)
`
`
`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, UZ, VN, YU, ZW,
`
`(30) Priority Data:
`ARIPO patent (GH, GM, KE, LS, MW,SD,SL, SZ, UG,
`DK
`0268/98
`27 February 1998 (27.02.98)
`
`
`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
`(71) Applicant: NOVO NORDISK A/S [DK/DK]; Novo Allé,
`
`
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`DK~-2880 Bagsvaerd (DK).
`
`
`
`SN, TD, TG).
`
`(72) Inventors: KNUDSEN,Liselotte, Bjerre; Valby Langgade
`
`
`49A,
`1.
`tv., DK-2500 Valby (DK).
`HUUSFELDT,
`
`
`Published
`
`Per, Olaf; Applebys Plads 27,5. mf, DK-1411 Copen-
`
`
`
`
`With international search report.
`hagen K (DK). NIELSEN,Per, Franklin; Dalsg Park 59,
`
`
`DK-3500 Verlgse (DK). PEDERSEN, Freddy, Zimmer-
`dahl; Tarmhgjgardvej 26, DK~3500 Verlgse (DK).
`
`
`
`(57) Abstract
`
`The present invention relates to derivatives of GLP--1 analogs having a lipophilic substituent. The derivatives of GLP-1 analogs of
`the present invention have a protracted profile of action.
`
`PFIZER, INC. v. NOVO NORDISK AJS - IPR2020-01252, Ex. 1033, p. 1 of 270
`
`(54) Title: DERIVATIVES OF GLP-1 ANALOGS
`
`
`
`
`
`
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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
`Albania
`ES
`Spain
`LS
`Lesotho
`SI
`Slovenia
`AM
`Armenia
`FI
`Finland
`LT
`Lithuania
`SK
`Slovakia
`
`
`
`
`AT
`Austria
`FR
`France
`LU
`Luxembourg
`SN
`Senegal
`AU
`Australia
`GA
`Gabon
`LV
`Latvia
`SZ
`Swaziland
`AZ
`Azerbaijan
`GB
`United Kingdom
`MC
`Monaco
`TD
`Chad
`
`
`BA
`Bosnia and Herzegovina
`GE
`Georgia
`MD
`Republic of Moldova
`TG
`Togo
`BB
`Barbados
`GH
`Ghana
`MG
`Madagascar
`TJ
`Tajikistan
`
`
`BE
`Belgium
`GN
`Guinea
`MK
`The former Yugoslav
`T™
`Turkmenistan
`BF
`Burkina Faso
`GR
`Greece
`Republic of Macedonia
`TR
`Turkey
`
`
`BG
`Bulgaria
`HU
`Hungary
`Mali
`TT
`Trinidad and Tobago
`
`
`BJ
`Benin
`IE
`Ireland
`Mongolia
`UA
`Ukraine
`BR
`Brazil
`IL
`Israel
`Mauritania
`UG
`Uganda
`
`BY
`Belarus
`Is
`Iceland
`Malawi
`us
`United States of America
`
`CA
`Canada
`IT
`Ttaly
`Mexico
`UZ
`Uzbekistan
`CF
`Central African Republic
`JP
`Japan
`Niger
`VN
`Viet Nam
`cG
`Congo
`KE
`Kenya
`Netherlands
`YU
`Yugoslavia
`CH
`Switzerland
`KG
`Kyrgyzstan
`Norway
`Zw
`Zimbabwe
`cl
`Céte d'Ivoire
`KP
`Democratic People’s
`New Zealand
`CM
`Cameroon
`Republic of Korea
`Poland
`CN
`China
`Republic of Korea
`Portugal
`cu
`Cuba
`Kazakstan
`Romania
`CZ
`Czech Republic
`Saint Lucia
`Russian Federation
`DE
`Germany
`Liechtenstein
`Sudan
`DK
`Denmark
`Sri Lanka
`Sweden
`EE
`Estonia
`Liberia
`Singapore
`
`
`
`
`
`
`
`
`
`
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`sD
`SE
`SG
`
`KR
`KZ
`Le
`ui
`LK
`LR
`
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`WO 99/43706
`
`PCT/DK99/00082
`
`1
`
`DERIVATIVES OF GLP-1 ANALOGS
`
`FIELD OF THE INVENTION
`
`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 pro-
`
`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-
`
`10
`
`combinant DNA technology it can be expected that their importance will increase also in the
`years to come. When native peptides or analogues therec? are used in therapy it is generally
`found that they have a high clearance. A high clearance ci a therapeutic agentis inconvenient
`
`in cases whereit 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, gluca-
`
`gon, glucagon-like peptide-1, glucagon-like peptide-2,
`
`insulin-like growthfactor-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-
`
`nase, adenosine deaminase and ribonuclease.
`
`in some casesit is possible to influence the
`
`release profile of peptides by applying suitable pharmaceutical compositions, but this approach
`
`has various shortcomings andis not generally applicable.
`
`25
`
`The hormones regulating insulin secretion belong to the so-called enteroinsular a-
`
`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-
`
`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-
`
`pic, but the only physiologically important ones, those that are responsible for the incretin
`
`effect, are the glucose-dependentinsulinotropic polypeptide, GIP, and glucagon-like peptide-
`
`1 (GLP-1). Becauseofits insulinotropic effect, GIP, isolated in 1973 (1) immediately attrac-
`
`ted considerable interest among diabetologists. However, numerousinvestigations carried
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`2
`
`out during the following years clearly indicated that a defective secretion of GIP was not in-
`
`voived in the pathogenesis of insulin dependent diabetes mellitus (IDDM) or non insulin-
`
`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
`
`most potent insulinotropic substance known (3). Unlike GIP,it is surprisingly effective in sti-
`
`mulating insulin secretion in NIDDM patients. In addition, and in contrast to the other insuli-
`
`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-
`
`cularly in patients with NIDDM.
`
`GLP-1, a product of the proglucagon (4), is one of the youngest membersof the se-
`
`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 differentiy in the pancreas andin the intestine. In the pan-
`
`creas (9), the processing leads to the formation and parallel secretion of 1) glucagonitself,
`
`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-
`
`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 seemsto
`
`be the only biologically active product.
`
`In contrast,
`
`in the intestinal mucosa,
`
`it is glucagon
`
`that is buried in a larger molecule, while the two glucagon-like peptides are formed separa-
`
`tely (8). The following products are formed and secreted in parallel: 1) glicentin, correspon-
`
`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
`
`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 conspicuousbiological activities.
`
`10
`
`15
`
`20
`
`25
`
`Being secreted in parallel with glicentin/enteroglucagon,
`
`it follows that the many
`
`30
`
`studies of enterogiucagon 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-
`
`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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`3
`
`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
`
`effect elicited by oral glucose in rats (21, 22). The hormoneinteracts directly with the B-cells
`
`via the GLP-1 receptor (23) which belongs to the glucagon/VIP/calcitonin family of G-protein-
`
`coupled 7-transmembrane spanning receptors. The importance of the GLP-1 receptorin re-
`
`gulating insulin secretion wasillustrated in recent experiments in which a targeted disruption
`
`of the GLP-1 receptor gene was carried out in mice. Animals homozygousfor the disruption
`
`10
`
`had greatly deteriorated glucose tolerance and fasting hyperglycaemia, and even hete-
`
`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 hormoneis best described as a potentiation of glucose
`
`stimulated insulin release (25), but the mechanism that couples glucose and GLP-1 stimula-
`
`tion is not known.
`
`{ft may involve a calcium-induced calcium release (26, 27). As already
`
`mentioned, the insulinotropic action of GLP-1 is preserved in diabetic 6-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 hormonealso potently inhibits glucagon
`
`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. Becauseofthis dualeffect,
`
`if the plasma GLP-1 concentrations increase either by increased secretion or by exogenous
`
`infusion the molarratio of insulin to glucagon in the blood that reachestheliver via the portal
`
`circulation is greatly increased, whereby hepatic glucose production decreases (30). As a
`result blood glucose concentrations decrease. Because of the glucose dependencyof 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
`
`20
`
`25
`
`preservedin patients with diabetes mellitus (32), in whom infusions of slightly supraphysiolo-
`
`30
`
`gical doses of GLP-1 may completely normalise blood glucose valuesin spite of poor meta-
`
`bolic control and secondary failure to sulphonylurea (33). The importance of the glucagono-
`
`Static effect is illustrated by the finding that GLP-1 also lowers blood glucosein type-1 diabe-
`
`tic patients without residual B-cell secretory capacity (34).
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`4
`
`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 emp-
`tying rate and pancreatic enzyme secretion (36). Similar inhibitory effects on gastric and
`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-
`atly stimulated, and it has been speculated that GLP-1 maybeat 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 iow as those required to influence islet secretion (39).
`
`GLP-1 seems to have an effect on food intake. Intraventricular administration of
`
`GLP-1 profoundlyinhibits 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 notinhibit food intake acutely in rats (41, 42). However,it re-
`
`mains possible that GLP-1 secreted from theintestinal L-cells may also act as a satiety sig-
`
`nal.
`
`Not only the insulinotropic effects but also the effects of GLP-1 on the gastrointesti-
`
`20
`
`nal tract are preservedin diabetic patients (43), and may help curtailing meal-induced gluco-
`
`se excursions, but, more importantly, may also influence food intake. Administered intra-
`
`venously, continuously for one week, GLP-1 at 4 ng/kg/min has been demonstrated to dra-
`
`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 a/. (Diabetologia 28
`
`704-707 (1985). Human GLP-1 is a 37 amino acid residue peptide originating from preprog-
`
`lucagon whichis 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
`
`mainlyin the L-cells. Although the interesting pharmacological properties of GLP-1(7-37) and
`
`analogues thereof have attracted much attention in recent years onlylittle is known about
`
`the structure of these molecules. The secondary structure of GLP-1 in micelles has been de-
`
`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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`5
`
`vely small and very flexible molecule resulted in compounds whose plasma profile were
`
`highly protracted andstill had retained activity.
`
`GLP-1 and analogues of GLP-1 and fragments thereof are useful /.a. in the treatment
`
`of Type 1 and Type 2 diabetes and obesity.
`
`WO 87/06941 discloses GLP-1 fragments, including GLP-1(7-37), and functional deri-
`
`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-
`
`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.
`
`The amino acid sequence of GLP-1 (7-36) and GLP-1 (7-37)is:
`7
`8
`9
`10
`11
`12
`#13
`14
`15
`#16
`«#197
`
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`#35
`
`36
`
`Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-X
`
`(I)
`
`10
`
`15
`
`20
`
`wherein X is NH, for GLP-1(7-36) and X is Gly for GLP-1(7-37).
`
`WO 91/11457 discloses analoguesof 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. Thusthe-
`
`re still is a need for improvementsin 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-
`
`insulin dependent diabetes mellitus.
`
`References.
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`6
`
`Pederson RA. Gastric Inhibitory Polypeptide. In Walsh JH, Dockray GJ (eds) Gut pep-
`
`tides: Biochemistry and Physiology. Raven Press, New York 1994, pp. 217259.
`Krarup T. immunoreactive gastric inhibitory polypeptide. Endocr Rev 1988;9:122-134.
`@rskov C. Glucagon-like peptide-1, a new hormoneof the enteroinsular axis. Diabeto-
`
`logia 1992; 35:701-711.
`
`Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC. Exon duplication and diver-
`
`gencein the human preproglucagon gene. Nature 1983; 304: 368-371.
`
`Holst JJ. Glucagon-like peptide-1 (GLP-1) - a newly discovered Gi hormone. Gastro-
`
`enterology 1994; 107: 1848-1855.
`
`Holst JJ. Gut glucagon, enteroglucagon, gut GLI, glicentin - current status. Gastroen-
`
`terology 1983:84:1602-1613.
`
`Holst JJ, @rskov C. Glucagon and other progiucagon-derived peptides.
`
`In Walsh JH,
`
`Dockray GJ, eds. Gut peptides: Biochemistry and Physiology. Raven Press, New York,
`
`pp. 305-340, 1993.
`
`@rskov C, Holst JJ, Knuhtsen S, Baldissera FGA, Poulsen SS, Nielsen OV. Gluca-
`
`gon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are se-
`
`creted separately from the pig small
`
`intestine, but not pancreas. Endocrinology
`
`1986; 119:1467-1475.
`
`10
`
`15
`
`Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, @rskov C. Proglucagon pro-
`
`20
`
`cessing 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
`
`glucagonin 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
`
`25
`
`Acta 1982;703:134-141.
`
`12.
`
`Thim L, Moody AJ. The primary structure of glicentin (proglucagon). Regul Pept
`
`1981;2:139-151.
`
`@rskov C, Bersani M, Johnsen AH, Hajrup P, Holst JJ. Complete sequences of giuca-
`
`gon-like peptide-1 (GLP-1)
`
`from human and pig small
`
`intestine. J. Biol. Chem.
`
`30
`
`1989;264: 12826-12829.
`
`14.
`
`@rskov C, Rabenhgj 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.
`
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`PCT/DK99/00082
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`7
`
`15.
`
`Buhl T, Thim L, Kofod H, @rskov C, Harling H, & Holst JJ: Naturally occurring products
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`of proglucagon 111-160 in the porcine and human small
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`intestine. J. Biol. Chem.
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`1988;263:8621-8624.
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`16.
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`@rskov C, Buhl T, Rabenhaj L, Kofod H, Holst JJ: Carboxypeptidase-B-like processing
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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.
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`Hoist JJ. Evidence that enteroglucagon(Il) is identical with the C-terminal sequence
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`(residues 33-69) of glicentin. Biochem J. 1980;187:337-343.
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`18.
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`Bataille D, Tatemoto K, Gespach C, Jérnvaill H, Rosselin G, Mutt V. Isolation of gluca-
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`10
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`gon-37 (bioactive enteroglucagon/oxyntomodulin) from porcine jejuno-ileum. Characte-
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`risation of the peptide. FEBS Lett 1982:146:79-86.
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`19.
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`@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.
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`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-
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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,
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`Kolligs F, Fenmann HC, Géke R, Goke B. Reduction of the incretin effect in rats by the
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`20
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`22.
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`23.
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`glucagon-like peptide-1 receptor antagonist exendin (9-39)amide. Diabetes 1995; 44:
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`Wang Z, Wang RM, Owji AA, Smith DM, Ghatei M, Bloom SR. Glucagon-like peptide-1
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`is a physiologicalincretin in rat. J. Clin. Invest. 1995; 95: 417-421.
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`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.
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`Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ. Diabetes in mice with targeted dis-
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`ruption of the GLP-1 receptor gene. Diabetes 1996; 45: 21A.
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`25.
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`Fehmann HC, Goke R, Géke B. Cell and molecular biology of the incretin hormones
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`glucagon-like peptide-| (GLP-1) and glucose-dependent insulin releasing polypeptide
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`(GIP). Endocrine Reviews, 1995; 16: 390-410.
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`26.
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`Gromada J, Dissing S, Bokvist K, Renstrém E, Frekjeer-Jensen J, Wulff BS, Rorsman
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`P. Glucagon-like peptide
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`|
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`increases
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`cytoplasmic calcium in
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`insulin-secreting
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`bTC3-cells by enhancementofintracellular calcium mobilisation. Diabetes 1995; 44:
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`767-774.
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`Holz GG, Leech CA, HabenerJF. Activation of a cAMP-regulated Ca?'-signaling pa-
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`thway in pancreatic B-cells by the insulinotropic hormone glucagon-like peptide-1. J Bi-
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`ol Chem, 1996; 270: 17749-17759.
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`28.
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`Holz GG, Kuhitreiber WM, Habener JF. Pancreatic beta-cells are rendered glucose
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`competent by the insulinotropic hormone glucagon-like peptide-1(7-37). Nature
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`1993;361:362-365.
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`29.
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`@rskov C, Holst JJ, Nielsen OV: Effect of
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`truncated glucagon-like peptide-1
`
`(proglucagon 78-107 amide) on endocrine secretion from pig pancreas, antrum and
`
`stomach. Endocrinology 1988;123:2009-2013.
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`30.
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`Hvidberg A, Toft Nielsen M, Hilsted J, @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-
`
`tabolism 1994;43:104-108.
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`31.
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`Qualmann C, Nauck M, Holst JJ, @rskov C, Creutzfeldt W. Insulinotropic actions of in-
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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.
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`Nauck MA, Heimesaat MM, @rskov C, Holst JJ, Ebert R, Creutzfeldt W. Preserved in-
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`cretin activity of GLP-1(7-36amide) but not of synthetic human GIP in patients with ty-
`
`pe 2-diabetes mellitus. J Clin Invest 1993;91:301-307.
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`33.
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`Nauck MA, Kleine N, @rskov 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.
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`Creutzfeldt W, Kleine N, Willms B, @rskov C, Holst JJ, Nauck MA. Glucagonostatic ac-
`
`tions and reduction of fasting hyperglycaemia by exogenous glucagon-liem, pepti-
`
`de-1(7-36amide)in type | diabetic patients. Diabetes Care 1996; 19: 580-586.
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`25
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`35.
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`Schjoldager BTG, Mortensen PE, Christiansen J, @rskov C, Holst JJ. GLP-1
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`(glucagon-like peptide-1) and truncated GLP-1, fragments of human proglucagon, inhi-
`
`bit 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. Trun-
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`cated GLP-1 (proglucagon 72-107amide) inhibits gastric and pancreatic functions in
`
`30
`
`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:
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`1074-1082.
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`Layer P, Holst JJ. GLP-1: A humoral mediator of the ileal brake in humans? Digestion
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`1993; 54: 385-386.
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`39.
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`Nauck M, Ettler R, Niedereichholz U, @rskov C, Holst JJ, Schmiegel 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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`and insulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A124.
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`40.
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`Schick RR, vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M. Gluca-
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`gon-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 & CompanyItd, 1994; pp. 363-367.
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`41.
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`Tang-Christensen M, Larsen PJ, Géke R, Fink-Jensen A, Jessop DS, Maller M, Sheikh
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`S. Brain GLP-1(7-36) amide receptors play a majorrole in regulation of food and water
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`intake. Am. J. Physiol., 1996, in press.
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`42.
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`Turton MD, O'Shea D, Gunn |, 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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`15
`
`43.
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`Willms B, Werner J, Creutzfeldt W, @rskov C, Holst JJ, Nauck M. Inhibition of gastric
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`emptying by glucagon-like peptide-1 (7-36 amide) in patients with type-2-diabetes mel-
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`litus. Diabetologia 1994; 37, suppl.1: A118.
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`44.
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`Larsen J, Jallad N, Damsbo P. One-week continuous infusion of GLP-1(7-37) improves
`
`glycaemic contro/ in NIDDM. Diabetes 1996; 45, suppl. 2: 233A.
`
`20
`
`45.
`
`Ritzel R, @rskov C, Holst JJ, Nauck MA. Pharmacokinetic, insulinotropic, and gluca-
`
`gonostatic properties of GLP-1 [7-36 amide] after subcutaneous injection in healthy
`
`volunteers. Dose-responserelationships. 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 de-
`
`graded from the amino terminusin type Hl diabetic patients and in healthy subjects.
`
`Diabetes 44: 1126-1131.
`
`SUMMARYOF THE INVENTION
`
`25
`
`30
`
`The presentinvention relates to derivatives of GLP-1 analogues of formula I:
`7
`8
`9
`10
`11
`12
`13
`#14
`#15
`16
`#417
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`His-Xaa-Xaa-Gly-Xaa-Phe-Thr-Xaa-Asp-Xaa-Xaa-
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`#19
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`20
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`21
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`22
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`23
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`24
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`25
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`26
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`27
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`28
`
`Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`38
`
`Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
`
`39
`
`40
`
`41
`
`42
`
`43
`
`44
`
`45
`
`Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
`
`(I)
`
`wherein
`
`Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 9 is Glu, Asp, or Lys,
`
`Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, lle, Tyr, Glu, Asp, or Lys,
`
`Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, or Lys,
`
`Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 21 is Glu, Asp, or Lys,
`
`Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lle, 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, lle, Val, Arg, Glu, Asp, or Lys,
`
`Xaaat position 25 is Ala, Gly, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 26 is Lys, Arg, Gln, Glu, Asp, or His,
`
`Xaa at position 27 is Glu, Asp, or Lys,
`
`Xaa at position 30is Ala, Gly, Ser, Thr, Leu, fle, Val, Glu, Asp, or Lys,
`
`Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys,
`
`Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, lle, Val, Glu, Asp, or Lys,
`
`Xaaat position 33 is Val, Gly, Ala, Ser, Thr, Leu, lle, Glu, Asp, or Lys,
`
`Xaa at position 34 is Lys, Arg, Glu, Asp, or His,
`
`Xaaat position 35 is Gly, Ala, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaaat position 36 is Arg, Lys, Glu, Asp, or His,
`
`20
`
`25
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`30
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`Xaaat position 37 is Gly, Ala, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys, oris deleted,
`
`11
`
`Xaa at position 38 is Arg, Lys, Glu, Asp, orHis, 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,
`
`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 Giu, Asp, or Lys, or is deleted, and
`
`Xaa at position 45 is Val, Glu, Asp, or Lys, oris deleted, or
`
`(a) a C-1-6-ester thereof, (b) amide, C-1-6-alkylamide, or C-1-6-dialkylamide thereof and/or (c)
`
`a pharmaceutically acceptabie salt thereof,
`
`provided that
`
`(i) when the amino acid at position 37, 38, 39, 40, 41, 42, 43 or 44 is deleted, then e-
`
`ach amino acid downstream of the amino acid is also deleted,
`
`(il) the derivative of the GLP-1 analog contains only one or two Lys,
`
`(iii) the s-amino group of one or both Lys is substituted with a lipophilic substituent opti-
`
`onally via a spacer,
`
`(iv) the total numberof different amino acids between the derivative of the GLP-1 ana-
`
`log and the corresponding native form of GLP-1 does not exceed six.
`
`20
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`A simple system is used to describe fragments and analogues of GLP-1. For exam-
`ple, Gly*-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
`in position 8 (Ala) by Gly. Similarly, Lys™(N*-tetradecanoyl)-GLP-1(7-37) designates GLP-1(7-
`
`25
`
`37) wherein the c-amino groupof 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 sequenceof this extension is as in the correspon-
`
`ding sequence in human preproglucagonunless otherwiseindicated.
`
`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-
`
`tracted profile of action than the parent peptides.
`
`In the present text, the designation “an analogue’is used to designate a peptide whe-
`
`rein one or more amino acid residues of the parent peptide have been substituted by another
`
`amino acid residue.
`
`The total numberof different amino acids between the derivative of the GLP-1 analog
`
`and the corresponding native form of GLP-1 does not exceed six. Preferably, the number of
`
`different amino acids is five. More preferably, the number of different amino acids is four.
`
`Even more preferably, the numberof different amino acids is three. Even more preferably, the
`
`numberofdifferent amino acids is two. Mos