PCT
`
`WORLDINTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6;
`C07K 14/605, A61K 38/26
`
`(22) International Filing Date:
`
`25 February 1999 (25.02.99)
`
`
`
`(11) International Publication Number:
`WO 99/43708
`
`(43) International Publication Date:
`2 September 1999 (02.09.99)
`
`
`PCT/DK99/00086|(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`(21) International Application Number:
`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, SL,
`
`SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW,
`(30) Priority Data:
`
`
`ARIPOpatent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
`DK
`27 February 1998 (27.02.98)
`0274/98
`
`
`ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU,TJ,
`
`
`5 May 1998 (05.05.98)60/084,357 US
`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,
`
`
`(71) Applicant
`(for all designated States except US): NOVO
`SN, TD, TG).
`NORDISK A/S [DK/DK]; Novo Allé, DK-2880 Bagsvaerd
`(DK).
`
`
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KNUDSEN,Liselotte,
`
`
`Bjerre [DK/DK]; Valby Langgade 49A, 1.
`tv., DK-2500
`Valby (DK). HUUSFELDT,Per, Olaf [DK/DK]; Applebys
`Plads 27,5. mf., DK-1411 Copenhagen K (DK). NIELSEN,
`Per, Franklin [DK/DK]; Dalsg Park 59, DK-3500 Vzrlgse
`(DK). MADSEN, Kjeld [DK/DK]; Nyvestergaérdsvej 3,
`
`DK-3500 Verlgse (DK).
`
`(74) CommonRepresentative: NOVO NORDISK A/S; Novo Allé,
`
`DK-2880 Bagsvaerd (DK).
`
`
`
`Published
`With international search report.
`
`
`
`
`
`
`
`(54) Title: GLP-1 DERIVATIVES OF GLP-1 AND EXENDIN WITH PROTRACTED PROFILE OF ACTION
`
`(57) Abstract
`
`
`
`
`The present invention relates to derivatives exendin and of GLP-1(7-C), wherein C is 35 or 36, which derivatives have just one
`lipophilic substituent which is attached to the C-terminal aminoacid residue.
`
`
`
`
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Albania
`Armenia.
`Austria
`Australia
`Azerbaijan
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`Barbados
`Belgium
`Burkina Faso
`Bulgaria
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`Brazil
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`Céte d'Ivoire
`Cameroon
`China
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`Czech Republic
`Germany
`Denmark
`Estonia
`
`ML
`MN
`MR
`MW
`MX
`NE
`
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`
`SG
`
`Zimbabwe
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`Lesotho
`SI
`Slovenia
`LS
`LT
`SK
`Slovakia
`Lithuania
`SN
`LU
`Senegal
`Luxembourg
`LV
`SZ
`Swaziland
`Latvia
`TD
`MC
`Monaco
`Chad
`MD
`TG
`Togo
`Republic of Moldova
`MG
`TJ
`Tajikistan
`Madagascar
`T™
`MK
`Turkmenistan
`The former Yugoslav
`TR
`Turkey
`Republic of Macedonia
`TT
`Mali
`Trinidad and Tobago
`UA
`Ukraine
`Mongolia
`UG
`Mauritania
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`US
`United States of America
`Malawi
`Mexico
`UZ
`Uzbekistan
`VN
`Viet Nam
`Niger
`YU
`Netherlands
`‘Yugoslavia
`ZW
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`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
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Treland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
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`Liberia
`
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`WO 99/43708
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`PCT/DK99/00086
`
`GLP-1 DERIVATIVES OF GLP-1 AND EXENDIN WITH PROTRACTED PROFILE
`OF ACTION
`
`FIELD OF THE INVENTION
`
`The present invention relates to novel derivatives of human glucagon-like peptide-1 (GLP-7)
`
`and fragments thereof and analogues of such fragments which have a protracted profile of ac-
`
`tion and to methods of making and using them. The invention furthermore relates to novel deri-
`
`vatives of exendin and the uses of such derivatives.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`Peptides are widely used in medical practice, and since they can be produced by recombinant
`
`DNAtechnologyit can be expectedthat their importancewill increase also in the years to co-
`me. Whennative peptides or analogues thereof are usedin therapyit is generally found that
`
`15
`
`they have a high clearance. A high clearance of a therapeutic agent is inconvenient in cases
`
`whereit is desired to maintain a high bloodlevel thereof over a prolonged period of time since
`repeated administrations will then be necessary. Examples of peptides which have a high clea-
`rance are: ACTH, corticotropin-releasing factor, angiotensin, calcitonin, insulin, glucagon, glu-
`cagon-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, parathy-
`roid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid
`stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opiods and analogues
`thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, aden-
`osine deaminase and ribonuclease. in somecasesit is possible to influence the release profile
`of peptides by applying suitable pharmaceutical compositions, but this approach has various
`shortcomings andis not generally applicable.
`
`The hormonesregulating insulin secretion belong to the so-called enteroinsular axis, desig-
`nating a group of hormones,released from the gastrointestinal mucosa in responseto the
`presence and absorption of nutrients in the gut, which promote an early and potentiated re-
`lease ofinsulin. The enhancing effect on insulin secretion, the so-called incretin effect, is
`probably essential for a normal glucose tolerance. Manyof the gastrointestinal hormones,
`including gastrin and secretin (cholecystokinin is not insulinotropic in man), are insulinotro-
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`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
`out during the following years clearly indicated that a defective secretion of GIP wasnotin-
`volved in the pathogenesis of insulin dependent diabetes mellitus (IDDM) or non insulin-
`dependentdiabetes mellitus (NIDDM) (2). Furthermore, as an insulinotropic hormone, GIP
`was found to be almostineffective in NIDDM (2). The otherincretin 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(perhapswith 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 secretin-VIP
`family of peptides, but is already established as an important gut hormonewith regulatory
`function in glucose metabolism and gastrointestinal secretion and metabolism (5). The glu-
`cagon geneis processeddifferently in the pancreas andin the intestine. In the pancreas(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 hexapeptide corre-
`sponding to PG (64-69); 4) and, finally, the so-called major proglucagon fragment (PG (72-
`158)), in which the two glucagon-like sequencesare buried (9). Glucagon seemsto be the
`only biologically active product. In contrast, in the intestinal mucosa,it is glucagonthat is bu-
`ried in a larger molecule, while the two glucagon-like peptides are formed separately (8). The
`following products are formed andsecretedin 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, whichis inac-
`tive). 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.
`
`Being secreted in parallel with glicentin/enteroglucagon,it follows that the many studiesof
`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 or
`fat-rich meals stimulate secretion (20), presumably as a result of direct interaction of yet
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`unabsorbed nutrients with the microvilli of the open-type L-celis of the gut mucosa. Endocri-
`ne or neural mechanisms promoting GLP-1 secretion may exist but have not yet been de-
`monstrated in humans.
`
`Theincretin function of GLP-1(29-31) has been clearlyillustrated in experiments with the
`GLP-1 receptor antagonist, exendin 9-39, which dramatically reduces the incretin effect eli-
`cited by oral glucosein 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
`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 elevationsofintracellular 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. It may involve a calcium-induced calcium release (26, 27). As already
`mentioned, the insulinotropic action of GLP-1 is preserved in diabetic B-cells. The relation of
`thelatter to its ability to convey "glucose competence"to isolated insulin-secreting cells (26,
`28), which respond poorly to glucose or GLP-1 alone, butfully 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 dual effect,
`if the plasma GLP-1 concentrations increase either by increased secretion or by exogenous
`infusion the molar ratio of insulin to glucagonin 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 effectis 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 ofslightly supraphysiolo-
`gical doses of GLP-1 may completely normalise blood glucose values in spite of poor meta-
`bolic control and secondary failure to sulphonylurea (33). The importance of the glucagono-
`static effectis illustrated by the finding that GLP-1 also lowers blood glucose in type-1 diabe-
`tic patients without residual B-cell secretory capacity (34).
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`In addition to its effects on the pancreatic islets, GLP-1 has powerful actions on the gastro-
`intestinal tract. Infused in physiological amounts, GLP-1 potently inhibits pentagastrin-
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`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- orlipid-containing solutions (37, 38). Concomitantly, GLP-1 secretion is gre-
`atly stimulated, and it has been speculated that GLP-1 may beat 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 thanits effects on the pancreatic islets.
`Thus, in dose responsestudies GLP-1 influences gastric emptying rate at infusion rates at
`least as low as those required to influence islet secretion (39).
`
`GLP-1 seemsto have an effect on food intake. Intraventricular administration of GLP-1 pro-
`foundly inhibits food intake in rats (40, 42). This effect seemsto be highly specific. Thus, N-
`terminally extended GLP-1 (PG 72-107)amideis inactive and appropriate doses of the GLP-
`1 antagonist, exendin 9-39, abolish the effects of GLP-1 (41). Acute, peripheral administrati-
`on of GLP-1 doesnotinhibit food intake acutely in rats (41, 42). However,it remains possi-
`ble that GLP-1 secreted from theintestinal L-cells may also act as a satiety signal.
`
`Not only the insulinotropic effects but also the effects of GLP-1 on the gastrointestinal tract
`are preservedin diabetic patients (43), and may help curtailing meal-induced glucose excur-
`sions, but, more importantly, mayalso influence food intake. Administered intravenously,
`continuously for one week, GLP-1 at 4 ng/kg/min has been demonstrated to dramatically im-
`prove glycaemic control in NIDDM patients withoutsignificant side effects (44). The peptide
`is fully active after subcutaneous administration (45), but is rapidly degraded mainly dueto
`degradation by dipeptidy! 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). 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 described by
`Thorton et al. (Biochemistry 33 3532-3539 (1994)), but in normal solution,GLP-1 is conside-
`red a very flexible molecule. Surprisingly, we found that derivatisation ofthis relatively small
`and very flexible molecule resulted in compounds whoseplasmaprofile were highly protrac-
`ted andstill had retained activity.
`
`GLP-1 and analogues of GLP-1 and fragments thereof are potentially useful /.a.in the treat-
`ment of type 1 and type 2 diabetes. However,the high clearancelimits the usefulness of these
`compounds,and thustherestill is a need for improvementsin this field. Accordingly,it is one
`object of the present invention to provide derivatives of GLP-1 and analoguesthereof which
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`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 lowerclearance than
`
`GLP-1(7-37). It is a further object of the invention to provide a pharmaceutical composition
`
`comprising a compound according to the invention and to use a compoundofthe 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.
`
`References.
`
`1. Pederson RA. Gastric Inhibitory Polypeptide. In Walsh JH, Dockray GJ (eds) Gut pepti-
`
`des: Biochemistry and Physiology. Raven Press, New York 1994, pp. 217259.
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`2. Krarup T. Immunoreactive gastric inhibitory polypeptide. Endocr Rev 1988;9:122-134.
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`3. @rskov C. Glucagon-like peptide-1, a new hormoneofthe enteroinsular axis. Diabetologia
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`1992: 35:701-711.
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`4. Bell Gl, Sanchez-Pescador R, Laybourn PJ, Najarian RC. Exon duplication and divergen-
`ce in the human preproglucagon gene. Nature 1983: 304: 368-371.
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`5. Holst JJ. Glucagon-like peptide-1 (GLP-1) - a newly discovered GI hormone. Gastroen-
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`6. Holst JJ. Gut glucagon, enteroglucagon, gut GLI, glicentin - current status. Gastroentero-
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`7. Halst JJ, @rskov C. Glucagon and other proglucagon-derived peptides. In Walsh JH,
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`11. Thim L, Moody AJ, Purification and chemical characterisation of a glicentin-related pan-
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`12. Thim L, Moody AJ. The primary structure ofglicentin (proglucagon). Regul Pept
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`1981;2:139-151.
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`1989;264:12826-12829.
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`14. @rskov C, Rabenhgj L, Kofod H, Wettergren A, Holst JJ. Production and secretion of
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`43: 535-539.
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`15. Buhl T, Thim L, Kofod H, @rskov C, Harling H, & Holst JJ: Naturally occurring products of
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`16. @rskov C, Buhl T, Rabenhgj L, Kofod H, Holst JJ: Carboxypeptidase-B-like processing of
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`18. Bataille D, Tatemoto K, Gespach C, Jérnvall H, Rosselin G, Mutt V. Isolation of gluca-
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`gon-3/ (bioactive enteroglucagon/oxyntomodulin) from porcine jejuno-ileum. Characteri-
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`sation of the peptide. FEBS Lett 1982;146:79-86.
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`19. Orskov C, Wettergren A, Holst JJ. The metabolic rate and the biological effects of GLP-1
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`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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`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-dependentinsulinotropic polypeptide secretion in response
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`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
`glucagon-like peptide-1 receptor antagonist exendin (9-39)amide. Diabetes 1995; 44:
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`22. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei M, Bloom SR. Glucagon-like peptide-1 is
`a physiological incretin in rat. J. Clin. Invest. 1995; 95: 417-421.
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`20
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`23. Thorens B. Expressioncloning of the pancreatic b cell receptor for the gluco-incretin
`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 disrup-
`tion of the GLP-1 receptor gene. Diabetes 1996; 45: 21A.
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`25. Fehmann HC, Goke R, Goke B. Cell and molecularbiology of the incretin hormonesglu-
`cagon-like peptide-| (GLP-1) and glucose-dependentinsulin releasing polypeptide (GIP).
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`26. Gromada J, Dissing S, Bokvist K, Renstrém E, Frakjeer-Jensen J, Wulff BS, Rorsman P.
`Glucagon-like peptide | increases cytoplasmiccalcium in insulin-secreting bTC3-cells by
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`27. Holz GG, Leech CA, Habener JF. Activation of a cAMP-reguiated Ca*signaling pathway
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`in pancreatic B-cells by the insulinotropic hormone glucagon-like peptide-1. J Biol Chem,
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`1996; 270: 17749-17759.
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`28. Holz GG, Kuhltreiber WM, Habener JF. Pancreatic beta-cells are rendered glucose com-
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`petent by the insulinotropic hormone glucagon-like peptide-1(7-37). Nature
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`1993;361:362-365.
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`29. Orskov C, Holst JJ, Nielsen OV: Effect of truncated glucagon-like peptide-1 (proglucagon
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`78-107 amide) on endocrine secretion from pig pancreas, antrum and stomach. Endocri-
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`nology 1988; 123:2009-2013.
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`30. Hvidberg A, Toft Nielsen M, Hilsted J, @rskov C, Holst JJ. Effect of glucagon-like pepti-
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`de-1 (proglucagon 78-107amide) on hepatic glucose production in healthy man. Metabo-
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`15
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`lism 1994:43:104-108.
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`31. Qualmann C, Nauck M, Holst JJ, @rskov C, Creutzfeldt W. Insulinotropic actionsof intra-
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`venous glucagon-like peptide-1 [7-36 amide] in the fasting state in healthy subjects. Acta
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`Diabetologica, 1995; 32: 13-16.
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`32. Nauck MA, Heimesaat MM, @rskov C, Holst JJ, Ebert R, Creutzfeldt W. Preserved incre-
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`tin activity of GLP-1(7-36amide) but not of synthetic human GIP in patients with type
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`2-diabetes mellitus. J Clin Invest 1993:91:301-307.
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`33. Nauck MA,Kleine N, @rskov C, Holst JJ, Willms B, Creutzfeldt W. Normalisation of
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`fasting hyperglycaemia by exogenous GLP-1(7-36amide) in type 2-diabetic patients. Dia-
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`betologia 1993;36:741-744.
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`34. Creutzfeldt W, Kleine N, Willms B, @rskov C, Holst JJ, Nauck MA. Glucagonostatic acti-
`ons 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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`35. 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, inhibit
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`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. Trunca-
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`ted GLP-1 (proglucagon 72-107amide) inhibits gastric and pancreatic functions in man.
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`Dig Dis Sci 1993;38:665-673.
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`37. Layer P, Holst JJ, Grandt D, Goebell H: Ileal release of glucagon-like peptide-1 (GLP-1):
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`association with inhibition of gastric acid in humans. Dig Dis Sci 1995: 40: 1074-1082.
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`38. Layer P, Holst JJ. GLP-1: A humoral mediator ofthe ileal brake in humans? Digestion
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`1993; 54: 385-386.
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`39. Nauck M, Ettler R, Niedereichholz U, @rskov C, Holst JJ, Schmiegel W.Inhibition of ga-
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`stric emptying by GLP-1(7-36 amide) or (7-37): effects on postprandial glycaemia andin-
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`sulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A124.
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`40. Schick RR, vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M. Glucagon-like
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`peptide 1 - a novel brain peptide involved in feeding regulation. in Ditschuneit H, Gries
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`FA, Hauner H, Schusdziarra V, Wechsler JG (eds.) Obesity in Europe. John Libbey &
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`CompanyItd, 1994; pp. 363-367.
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`41. Tang-Christensen M, Larsen PJ, Géke R, Fink-Jensen A, Jessop DS, Maller M, Sheikh
`S. Brain GLP-1(7-36) amide receptors play a major role in regulation of food and water
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`intake. Am. J. Physiol., 1996,in press.
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`42. Turton MD, O'Shea D, Gunn |, Beak SA, Edwards CMB, MeeranK,et al. A role for glu-
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`cagon-like peptide-1 in the regulation of feeding. Nature 1996; 379: 69-72.
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`43. Willms B, WernerJ, Creutzfeldt W, @rskov C, Holst JJ, Nauck M. Inhibition of gastric
`emptying by glucagon-like peptide-1 (7-36 amide) in patients with type-2-diabetes melli-
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`tus. Diabetologia 1994; 37, suppl.1: A118.
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`44, Larsen J, Jallad N, Damsbo P. One-week continuous infusion of GLP-1(7-37) improves
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`glycaemic control in NIDDM. Diabetes 1996; 45, suppl. 2: 233A.
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`45. Ritzel R, @rskov C, Holst JJ, Nauck MA. Pharmacokinetic, insulinotropic, and gluca-
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`gonostatic properties of GLP-1 [7-36 amide] after subcutaneousinjection in healthy vo-
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`lunteers. Dose-response relationships. Diabetologia 1995; 38: 720-725.
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`46. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human
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`plasma in vitro yields an N-terminally truncated peptide that is a major endogenous meta-
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`bolite 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 B, Holst JJ. 1995. Both subcu-
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`taneous and intravenously administered glucagon-like peptide-1 are rapidly degraded
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`from the amino terminusin type II diabetic patients and in healthy subjects. Diabetes 44:
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`1126-1131.
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`SUMMARYOF THE INVENTION
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`20
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`Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon whichis
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`synthesised/.a. in the L-cells in the distal ileum, in the pancreas andin the brain. Processing of
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`preproglucagon to give GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs mainly in the L-
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`cells. A simple system is used to describe fragments and analoguesof this peptide. Thus, for
`example, 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
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`GLP-1(7-37) wherein the e-amino group of the Lys residue in position 34 has been tetradeca-
`
`noylated. Where reference in this text is made to C-terminally extended GLP-1 analogues, the
`aminoacid residuein 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 resi-
`due in position 40 is Asp unless otherwiseindicated. Also, if a C-terminally extended analogue
`extendsto position 41, 42, 43, 44 or 45, the amino acid sequenceof this extensionis as in the
`
`corresponding sequence in human preproglucagon unless otherwiseindicated.
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`25
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`30
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`In its broadest aspect, the present invention relates to derivatives of GLP-1 and analoguesthe-
`
`reof. The derivatives according to the invention have interesting pharmacological properties, in
`
`particular they have a more protracted profile of action than the parent peptides.
`
`In the present text, the designation “an analogue” is used to designate a peptide wherein one
`
`or more amino acid residues of the parent peptide have been substituted by another amino
`
`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 addedto the parent pepti-
`
`de. Such addition can take place either at the N-terminal end or at the C-terminal end of the
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`10
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`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 moreof
`
`the amino acid residues of the parent peptide have been chemically modified, e.g. by alkylati-
`
`on, acylation, ester formation or amide formation.
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`The term “a GLP-1 derivative” is used in the present text to designate a derivative of GLP-1 or
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`an analogue thereof. In the present text, the parent peptide from which such a derivativeis
`
`formally derived is in some places referred to as the “GLP-1 moiety”of the derivative.
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`20
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`In a preferred embodiment, the present invention relates to a GLP-1 derivative wherein atleast
`
`one amino acid residue of the parent peptide hasalipophilic substituent attached with the pro-
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`viso that if only one lipophilic substituent is present and this substituent is attached to the N-
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`terminalor to the C-terminal amino acid residue of the parent peptide then this substituentis an
`
`alkyl group or a group which has an w-carboxylic acid group.
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`25
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`In another preferred embodiment, the present invention relates to a GLP-1 derivative having
`
`only onelipophilic substituent.
`
`In another preferred embodiment, the presentinvention relates to a GLP-1 derivative having
`only onelipophilic substituent which substituent is an alkyl group or a group which has an -
`
`30
`
`carboxylic acid group and is attached to the N-terminal amino acid residue of the parent pepti-
`de.
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`In another preferred embodiment, the present invention relates to a GLP-1 derivative having
`
`only onelipophilic substituent which substituent is an alkyl group or a group which has an w-
`
`carboxylic acid group and is attached to the C-terminal amino acid residue of the parent pepti-
`
`de.
`
`in another preferred embodiment, the present invention relates to a GLP-1 derivative having
`
`only onelipophilic substituent which substituent can be attached to any one aminoacid residue
`
`which is not the N-terminal or C-terminal amino acid residue of the parent peptide.
`
`10
`
`In another preferred embodiment, the present invention relates to a GLP-1 derivative wherein
`
`twolipophilic substituents are present.
`
`In another preferred embodiment, the present invention relates to a GLP-1 derivative wherein
`
`twolipophilic substituents are present, one being attached to the N-terminal amino acid residue
`
`15
`
`while the other is attached to the C-terminal amino acid residue.
`
`In another preferred embodiment, 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 otheris attached to an amino acid residue which is not N-terminal or the C-terminal
`
`20
`
`amino acid residue.
`
`In another preferred embodiment, the present invention relates to a GLP-1 derivative wherein
`
`twolipophilic substituents are present, one being attached to the C-terminal amino acid residue
`
`while the other is attached to an aminoacid residue which is not the N-terminal or the C-
`
`25
`
`terminal amino acid residue.
`
`In a further preferred embodiment, 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 deri-
`
`vative hasjust onelipophilic substituent which is attached to the C-terminal amino acid residue
`
`30
`
`of the parent peptide.
`
`In a further preferred embodiment, the present invention relates to a GLP-1 derivative, being a
`derivative of GLP-1(7-C), wherein C is 35 or 36 which derivative has just one lipophilic substi-
`
`tuent which is attached to the C-terminal amino acid residue.
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`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.
`
`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
`
`ofthelipophilic substituent forms an amide bond with an amino group of the a