a2) United States Patent
`US 6,268,343 Bl
`(0) Patent No.:
`Knudsenet al.
`(45) Date of Patent:
`Jul. 31, 2001
`
`
`US006268343B1
`
`(54) DERIVATIVES OF GLP-1 ANALOGS
`
`(75)
`
`Inventors: Liselotte Bjerre Knudsen, Valby; Per
`Olaf Huusfeldt, Kobenhavn K; Per
`Franklin Nielsen, Verlgse; Niels C.
`Kaarsholm, Vanlgsc; Helle Birk Olsen,
`Alleréd; Seren Erik Bjorn, Lyngby;
`Freddy Zimmerdah] Pedersen; Kjeld
`Madsen, both of Verlgse, all of (DK)
`.
`.
`ee
`(73) Assignee: Novo Nordisk A/S, Bagsvaerd (DK)
`(*) Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`US.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/258,750
`
`(51)
`
`Int. Ch? eee A61K 39/16; A61K 38/26;
`CO7K 14/00; CO7K 14/605
`(52) US. Ch. escscccssssssssssssstsusssessssessnsie 514/12; 530/324
`(58) Field of Search ...scoscenssnne 530/324; 514/12
`
`(56)
`‘
`
`References Cited
`U.S. PATENT DOCUMENTS
`s1ay12
`6/1992 Habener
`5.100.712
`. 514/12
`4/1996 Chen etal.
`..
`5,512,549
`
`S142
`8/1996 Buckleyet al.
`5545618
`3/1997 Habener wv... eecsescssesereeeseeses 514/12
`5,614,492
`FOREIGN PATENT DOCUMENTS
`
`
`
`0 708 179
`4/1996 (EP).
`WO 90/11296
`10/1990 (WO).
`WO 91/11457
`8/1991 (WO).
`WO 95/07931
`3/1995 (WO).
`WO 95/31214
`(22) Filed:—Feb. 26, 1999
`11/1995 (WO).
`WO 96/29342
`9/1996 (WO).
`WO 96/29344
`9/1996 (WO).
`WO87/06941
`L/1997 (WO).
`WO 98/08531
`3/1998 (WO).
`WO 98/08871
`3/1998 (WO).
`WO 98/08873
`3/1998 (WO).
`WO 98/19698
`5/1998 (WO).
`OTHER PUBLICATIONS
`
`Related U.S. Application Data
`
`(60)
`
`(63) Continuation-in-part of application No. 09/038,432,filed on
`Mar. 11, 1998, now abandoned, whichis a continuation-in-
`part of application No. 08/918,810,filed on Aug. 26, 1997,
`now abandoned, and a continuation-in-part of application
`No. PCT/DK97/00340,filed on Aug. 22, 1997
`Provisional application No. 60/035,904, filed on Jan. 24,
`1997, provisional application No. 60/036,226, filed on Jan.
`25, 1997, provisional application No. 60/036,255,filed on
`Jan. 24, 1997, provisional application No. 60/082,478,filed
`on Apr. 21, 1998, provisional application No. 60/082,480,
`filed on Apr. 21, 1998, provisional application No. 60/082,
`802, filed on Apr. 23, 1998, and provisional application No.
`60/084,357, filed on May 5, 1998.
`
`
`
`Foreign Application Priority Data
`(30)
`Aug. 30, 1996
`Nov. 8, 1996
`Dec. 20, 1996
`Feb. 27, 1998
`Feb. 27, 1998
`Feb. 27,1998
`Feb. 27, 1998
`Feb. 27, 1998
`Apr. 8, 1998
`Apr. 8, 1998
`
`.. 0931/96
`1259/96
`1470/96
`0263/98
`(DK)...
`
`0264/98
`(DK)...
`
`0268/98
`(DK)...
`
`0272/98
`(DE)...
`0274/98
`(DK) ...
`0508/98
`(DK)...
`se veseneeeeseees 0509/98
`
`
`
`Kim et al., (1994) J. of Pharma, Scicnecs 83(8);1175-1180.
`Clodfelteret al., (1998) Pharmaceutical Res. 15(2):254—262.
`
`Primary Examiner—Michacl Borin
`(74) Attorney, Agent, or Firm—Steve T. Zelson, Esq.; Elias
`J. Lambiris, Esq.
`
`(57)
`
`ABSTRACT
`
`The present invention rclatcs to GT.P-1 derivatives having a
`lipophilic substituent, pharmaceutical compositions com-
`prising same, and methods of making an using same. The
`GLP-1 derivatives of the present invention have a protracted
`profile of action.
`
`40 Claims, 1 Drawing Sheet
`
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`
`

`

`U.S. Patent
`
`Jul. 31, 2001
`
`US 6,268,343 BL
`
`Fig. 1
`
`
`
`
`glp-1(7-37)
`
`"Oo @
`
`—o- f
`
`-~Y¥—g
`
`0.1
`
`1
`
`10
`
`100
`
`—m— hj
`4000
`
`[peptide] (uM)
`
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`
`

`

`US 6,268,343 B1
`
`1
`DERIVATIVES OF GLP-1 ANALOGS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation-in-part of Ser. No.
`09/038,432 filed Mar. 11, 1998, now abandoned which is a
`continuation-in-part of Ser. No. 08/918,810 filed Aug. 26,
`1997 now abandoned and of PCT application serial no.
`PCT/DK97/00340 filed Aug. 22, 1997, and claims pniorily
`of U.S. provisional application Ser. Nos. 60/035,904,
`60/036,226, 60/036,255, 60/082,478, 60/082,480, 60/082,
`802, and 60/084,357filed Jan. 24, 1997, Jan. 25, 1997, Jan.
`24, 1997, Apr. 21, 1998, Apr. 21, 1998, Apr. 23, 1998, and
`May5, 1998, respectively, and of Danish application serial
`nos. 0931/96, 1259/96, 1470/96, 0263/98, 0264/98, 0268/
`98, 0272/98, 0274/98, 0508/98, and 0509/98 filed Aug. 30,
`1996, Nov. 8, 1996, Dec. 20, 1996, Feb. 27, 1998, Feb. 27,
`1998, Feb. 27, 1998, Feb. 27, 1998, Feb. 27, 1998, Apr. 8,
`1998, and Apr. 8, 1998, respectively, the contents of each of
`which is fully incorporated hercin byreference.
`FIELD OF THE INVENTION
`
`invention relates to novel derivatives of
`The present
`human glucagon-like peptide-1 (GLP-1) and fragments and/
`or analogues thereof which have a protracted profile 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 recombinant DNA technologyit
`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 high clearance are:
`ACTH, corticotropin-releasing factor, angiotensin,
`calcitonin,
`insulin, glucagon, glucagon-like peptide-1,
`glucagon-like peptide-2,
`insulin-like growth factor-1,
`insulin-like growth factor-2, gastric inhibitory peptide,
`growth hormone-releasing factor, pituitary adenylate
`cyclase activating peptide, secretin, enterogastrin,
`somatostain, somatotropin, somatomedin, parathyroid
`hormone,
`thrombopoietin, erythropoietin, hypothalamic
`releasing factors, prolactin, thyroid stimulating hormoncs,
`endorphins, enkephalins, vasopressin, oxytocin, opiods and
`analogues thereof, superoxide dismutase,
`interferon,
`asparaginase, arginase, arginine deaminase, 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 and is not generally applicable.
`The hormones regulating insulin secretion belong to the
`so-called enteroinsular axis, designating a group of
`hormones, released from the gastrointestinal mucosa in
`response to the presence and absorption of nutrients in the
`gut, which promote an early and potentiated release of
`insulin. The enhancing effect on insulin secretion,
`the
`so-called incretin effect, is probably essential for a normal
`glucose tolerance. Many of the gastrointestinal hormones,
`including gastrin and secretin (cholecystokinin is not insuli-
`notropic in man), are insulinotropic, but the only physiologi-
`
`cally important ones,
`those that are responsible for the
`
`
`
`incretin effect, are the glucose-dependent
`insulinotropic
`
`3o
`
`35
`
`40
`
`;
`
`55
`
`6oa
`
`65
`
`2
`polypeptide, GIP, and glucagon-like peptide-1 (GLP-1).
`Because ofils insulinotropic effect, GIP, isolated in 1973 (1)
`immediately attracted considerable interest among diabe-
`tologist. However, numerousinvestigations carried out dur-
`ing the following years clearly indicated that a defective
`seerction of GIP was not involved in the pathogenesis of
`insulin dependent diabetes mellitus 7DDM)or noninsulin-
`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 stimulating
`insulin secretion in NIDDM patients. In addition, and in
`contrast to the other insulinotropic hormones (perhaps with
`the exception of secretin) it also potently inhibits glucagon
`secretion. Because of these actions it has pronounced blood
`glucose lowering effects particularly in patients with
`NIDDM.
`
`GLP-1, a product of the proglucagon (4), is one of the
`youngest members of the secretin-VIP family of peptides,
`butis alreadyestablished as an important gut hormone with
`regulatory function in glucose metabolism and gastrointcs-
`tinal secretion and metabolism (5). ‘The glucagon gene is
`processed differently in the pancreas and in the intestine. In
`the pancreas (9), the processing leads to the formation and
`parallel secretion of 1) glucagonitself, occupying positions
`33-61 of proglucagon (PG); 2) an N-terminal peptide of 30
`amino acids (PG (1-30)) often called glicentin-related pan-
`creatic peptide, GRPP (10, 11); 3) a hexapeptide correspond-
`ing to PG (64-69); and, finally, the so-called major proglu-
`cagon 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 separately
`(8). The following products are formed and secreted in
`parallel: 1) glicentin, corresponding, to PG (1-69), with the
`glucagon sequence occupying residues Nos. 33-61 (12); 2)
`GLP-1(7—36)amide (PG(78-107)amide (13), not as origi-
`nally believed PG (72-107)amide or 108, which is inactive).
`Small amounts of C-terminally glycine-cxtended but cqually
`bioactive GLP-1(7-37), (PG (78-108))are also formed (14);
`3) intervening, peptide-2(PG (111-112)amide) (15); and 4)
`GLP-2 (PG(126-158))(15, 16). A fraction of glicentin is
`claved further into GRPP (PG (1-30)) and oxyntomodulin
`(PG (33-69)) (17, 18). Of these peptides, GLP-1, has the
`most conspicuous biological activitics.
`Being secreted in parallel with glicentin/enteroglucagon,
`it follows that the many studies of enteroglucagonsecretion
`(6, 7) ta some extent also apply to GLP-1 secretion, but
`GLP-1 is metabolised more quickly with a plasmahalf-life
`in humans of 2 min (19). Carbohydrate or fat-rich meals
`stimulate (20), presumablyas a result of direct interaction of
`yet unabsorbed nutrients with the microvilli of the open-type
`L-cells of the gut mucosa. Endocrine or neural mechanisms
`promoting GLP-1 secretion mayexist 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 cffcct clicited by oral glucose in rats (21, 22). The
`hormone interacts 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,
`
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`US 6,268,343 B1
`
`3
`receptors. The importance of the GLP-1 receptor in regu-
`lating insulin secretion was illustrated in recent experiments
`in which a targeted disruption of the GLP-1 receptor gene
`was carried out
`in mice. Animals homozygous for the
`disruption had greatly detcriorated ghicose tolerance and
`fasting hyperglycaemia, and even heterozygous animals
`were glucose intolerant (24). The signal transduction mecha-
`nism(25) primarily involves activation of adenylate cyclase,
`but elevations of intracellular Ca** are also essential (25,
`26). The action of the hormone is best described as a
`potentiation of glucose stimulated insulin release (25), but
`the mechanism that couples glucose and GLP-1 stimulation
`is not known. It may involve a calcium-induced calcium
`release (26, 27). As already mentioned, the insulinotropic
`action of GLP-1 is preserved in diabetic B-cells. The relation
`of the latter to its ability to convey “glucose competance”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 hormone also potently inhibits glucagon secretion (29).
`The mechanism is not known, but seemsto be paracrine, via
`neighbouring insulin or somatostatin cells (25). Also the
`glucagonostatic action is glucosc-dependent, so that
`the
`inhibitory effect decreases as blood glucose decreases.
`Because of this dual effect, if the plasma GLP-1 concentra-
`tions increase either by increased secretion or by exogenous
`infusion the molar ratio of msulin to glucagon in the blood
`that reaches the liver via the portal circulation is greatly
`increased, whereby hepatic glucose production decreases
`(30). As a result blood 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
`
`
`preserved in patients with diabetes mellitus (32), in whom
`
`infusions off slightly supraphysiological doses of GLP-1
`
`may completcly normalise blood glucose valucs in spite of
`poor metabolic control and secondaryfailure to sulphony-
`lurea (33). The importance of the glucagonostatic effect is
`illustrated by the finding that GLP-1 also lowers blood
`glucose in type-1 diabetic patients without residual f-cell
`secretory capacity (34).
`In addition to its effects on the pancreatic islets, GLP-1
`has powerful actions on the gastrointestinal tract. Infused in
`physiological amounts GLP-1 potently inhibits pentagastrin-
`induced as well as meal-induced gastric acid secretion (35,
`36). It also inhibits gastric emptying rate and pancreatic
`enzyme scerction (36). Similar inhibitory cffects 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 greatly stimulated, and it has been speculated
`that GLP-1 may be at
`least partly responsible for this
`so-called “ileal-brake” effect (38). In fact, recent studies
`
`suggest
`that, physiologically,
`the ileal-brake effects of
`
`
`
`GLP-1 may be more important
`than its effects on the
`pancreatic islets. Thus,
`in dose response studies GLP-1
`influences gastric emptying rate at infusion rates at least as
`low as those required to influence islet secretion (39).
`GLP-1 seems to have an effect on food intake. Intraven-
`tricular administration of GLP-1 profoundly inhibits food
`intake in rats (40, 42). This cffect sccms to be highly
`specific. Thus, N-terminally extended GLP-1 (PG 72-107)
`amide is inactive and appropriate doses of the GLP-1
`antagonist, exendin 9-39, abolish the effects of GLP-1 (41).
`
`35
`
`40
`
`55
`
`60
`
`65
`
`4
`Acute, peripheral administration of GLP-1 does not inhibit
`food intake acutely in rats (41, 42). However,
`it remains
`possible that GLP-1 secreted from the intestinal L-cells may
`
`
`also act as a satiety signal.
`
`
`
`
`
`
`Notonly the insulinotropic effects but also the effects of
`GLP-1 on the gastrointestinal tract are preserved in diabetic
`patients (43), and mayhelp curtailing meal-induced glucose
`excursions, but, more importantly, may also influence food
`intake. Administered intravenously, continuously for one
`week, GLP-1 at 4 ng/kg/min has been demonstrated to
`dramatically improve glycaemic control in NIDDMpatients
`without significant side effects (44). The peptide is fully
`active after subcutancous administration (45), but is rapidly
`degraded mainly due to degradation bydipeptidyl peptidase
`TV-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
`preproglucagon which is synthesised, i.a. in the L-cells in
`the distal ileum, in the pancreas andin the brain. Processing
`of preproglucagon to GLP-1 (7-36)amide, GI.P-1 (7-37)
`and GLP-2 occurs 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
`have been described by Thorton ct al. (Biochemistry 33
`3532-3539 (1994)), but in normal solution, GLP-1 is con-
`sidered a very flexible molecule. Surprisingly, we found that
`derivatisation of this relatively small and very flexible
`molecule resulted in compounds whose plasmaprofile were
`highly protracted and still had retained activity.
`GLP-1 and analogues of GLP-1 and fragments thereof are
`useful ia. in the treatment of Type 1 and Type 2 diabetes and
`obesity.
`including
`WO 87/06941 discloses GLP-1 fragments,
`GLP-1 (7-37), and functional derivatives thereof and to
`their use as an insulinotropic agent.
`including
`WO 90/11296 discloses GLP-1 fragments,
`GLP-1 (7-36), and functional derivatives thereof which
`have an insulinotropic activity which exceeds the insulino-
`tropic activity of GLP-1 (1-36) or GLP-1 (1-37) andto their
`use as insulinotropic agents.
`The amino acid sequence of GLP-1 (7-36) and GLP-1
`(7-37) is (SEQ ID NO:1):
`
`«417
`#16
`«15
`#14
`#13
`12
`11
`10
`9
`8
`7
`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
`
`(T)
`
`wherein X is II, 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.
`
`EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-1 ana-
`logues and derivatives that include an N-terminal imidazole
`group and optionally an unbranched C,-C,, acyl group in
`attached to the lysine residue in position 34.
`
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`US 6,268,343 B1
`
`5
`EP 0699686-A2 (Eli Lilly & Co.) discloses certain
`N-terminal truncated fragments of GLP-1 that are reported
`lo be biologically active.
`Unfortunately, the high clearance limits the usefulness of
`these compounds. ‘lhus there still is a need for improve-
`ments in this ficld.
`Accordingly, it is an object of the present invention to
`provide derivatives of GLP-1 and analogues thereof which
`have a protracted profile of action relative to GLP-1 (7-37).
`It is a further object of the invention to provide derivatives
`of GLP-1 and analogues thereof which have a lowerclear-
`ance than GLP-1 (7-37).
`It is a further object of the invention to provide a phar-
`maceutical composition with improved solubility and sta-
`bility.
`
`1s
`
`References
`
`1. Pederson RA. Gastric Inhibitory Polypeptide. In Walsh
`JH, Dockray GJ (eds) Gut peptides: Biochemistry and 5,
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`3. Orskov C. Glucagon-like peptide-1, a new hormone of
`the enteroinsular axis. Diabetologia 1992; 35:701-711.
`4. Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian
`RC. Exonduplication and divergence in the humanprepro-
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`glucagon-like peptide-2 in pig and human small intestine.
`FEBSletters, 1989; 247:193-106.
`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.
`IL,
`18. Bataille D, Tatemoto K, Gespach C, Jornvall
`Rosselin G, Mutt V. Isolation of glucagon-37 (bioactive
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`146:79-86.
`
`19. Orskov C, Wettergren A, Holst JJ. The metabolic rate
`and the biological effects of GLP-1 7—-36amide and GLP-1
`7-37 in healthy volunteers are identical. Diabetes 1993;
`42:658-661.
`
`20. Elliott RM, Morgan LM, Tredger JA, Deacon S,
`Wright J, Marks V. Glucagon-like peptide-1 (7-36)amide
`and glucose-dependent insulinotropic polypeptide secretion
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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
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`22. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei M,
`Bloom SR. Glucagon-like peptide-1 is a physiological inere-
`tin in rat. J. Clin. Invest. 1995; 95:417-421.
`23. Thorens B. Expression cloning of the pancreatic b cell
`receptor for the gluco-incretin hormone glucagon-like pep-
`tide 1. Proc Natl Acad Sci 1992; 89:8641-4645.
`24. Serocchi T., Auerbach AB, Joyner AT., Drucker DJ.
`Diabetes in mice with targeted disruption of the GLP-1
`receptor gene. Diabetes 1996; 45, 21A.
`25. Fehmann HC, Goke R, Goke G. Cell and molecular
`biology of the incretin hormoncs glucagon-like peptide-I
`(GLP-1) and glucose-dependent insulin releasing polypep-
`tide (GIP). Endocrine Reviews, 1995; 16: 390-410.
`26. Gromada J, Dissing S$, Bokvist K, Renstrom E,
`Frokjacr-Jensen J, Wulff BS, Rorsman P. Glucagon-like
`pepide I increases cytoplasmic calcium in insulin-secreting
`bTC3-cells by enhancementof intracellular calcium mobili-
`sation. Diabetes 1995; 44:767-774.
`27. Holz GG, Leech CA, Habener JF, Activation of a
`cAMP-regulated Ca**-signaling pathway in pancreatic
`B-cells by the insulinotropic hormone glucagon-like
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`28. Holz GG, Kuhltreiber WM, Habener JF, Pancreatic
`beta-cells are rendered glucose competent by the insulino-
`tropic hormone glucagon-like peptide-1(7-37). Nature
`1993; 361:362-365.
`29. Orskov C, Holst JJ, Nielsen OV: Effect of truncated
`glucagon-like peptide 1 (Proglucagon 78-107 amide) on
`endocrine secretion from pig pancreas, antrum and stomach.
`Endocrinology 1988; 123:2009-2013.
`30. IIvidberg A, Toft Nielsen M, Ililsted J, Orskov C,
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`man. Metabolism 1994; 43:104-108.
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`31. Qualmann C, Nauck M, Holst JJ, Orskov C.
`Creutzfeldt W.
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`glucagon-like peptide-1 [7-36 amide] in the fasting state in
`healthy subjects. Acta Diabetologica, 1995; 32:13-16.
`32. Nauck MA, Heimesaat MM,Orskov C,Holst JJ, Ebert
`R, Creutzfeldt W. Preserved incretin activity of GLP-1
`(7-36amide) but not of synthetic human GIPin patients with
`type 2-diabetes mellitus. J Clin Invest 1993; 91:301-307.
`33. Nauck MA,Kleine N, OrskovC. Holst JJ, Willms B,
`Creutzfeldt W. Normalisation of fasting hyperglycaemia by
`exogenous GLP-1(7-36amide) in type 2-diabetic patients.
`Diabetologia 1993; 36:741-744.
`34. Creutzfeldt W, Kleine N, Willms B, Orskov C, Holst
`JJ, Nauck MA. Ghucagonostatic actions and reduction of
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`35. Schjoldager BTG, Mortensen PE, Christiansen J,
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`gastric acid secretion in man. Dig. Dis. Sci. 1989;
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`man. Dig Dis Sci 1993; 38:665-673.
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`Holst JJ, Schmiegel W. Inhibition of gastric emptying by
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`dziarra V, Classen M Glucagon-like peptide 1—a novel
`brain peptide involved in feeding regulation. in Ditschuneit
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`Obesity in Europe. John Libbey & Companyltd, 1994; pp.
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`41. Tang-Christensen M, Larsen PJ, Goke R, Fink-Jensen
`A, Jessop DS, Moller M, Sheikh S. Brian GLP-1(7-36)
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`water intake. Am. J. Physiol., 1996, in press.
`42. Turton MD, O’Shea D, Gunn I, Beak SA, Edwards
`CMB, Meeran K,et al. Arole tor glucagon-like peptide-1 in
`the regulation of feeding. Nature 1996; 379; 69-72.
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`JJ, Nauck M. Inhibition of gastric emptying by glucagon-
`like peptide-1 (7—36amide) in patients with type-2-diabetes
`mellitus. Diabetologia 1994; 37, suppl. 1: A118.
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`infusion of GLP-1(7-37) improves glycaemic control in
`NIDDM. Diabetes 1996; 45, suppl. 2: 233A.
`45. Ritzel R, Orskov C, Holst JJ, Nauck MA.
`Pharmacokinetic, insulinotropic, and glucagonstatic proper-
`ties of GLP-1 [7-36 amide] after subcutaneous injection in
`healthy volunteers. Dose-response relationships. Diabetolo-
`gia 1995; 38: 720-725.
`
`ra o
`
`5.
`
`35
`
`40
`
`55
`
`60
`
`65
`
`8
`46. Deacon CF, Johnson AH, Holst JJ. Degradation of
`glucagon-like peptide-1 by human plasmain 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 intrave-
`nously administered glucagon-like peptide-1 are rapidly
`degraded from the amino terminus in type II diabetic
`patients and in healthy subjects. Diabetes 44: 1126-1131.
`SUMMARYOF THE INVENTION
`
`The present invention relates to derivatives of GLP-1
`(1-45) and analogs and/or fragments thereof. The GLP-1
`derivatives of the present invention have interesting phar-
`macological properties,
`in particular they have a more
`protracted profile of action than the parent peptides. The
`GLP-1 derivatives of the present invention also have insuli-
`nolropic activily, abilily to decrease glucagon, abilily to
`suppress gastric motility, ability to restore glucose compe-
`tencyto beta-cclls, and/orability to suppress appctite/rcduce
`weight.
`
`BRIEF DESCRIPTION OF‘THE FIGURES
`
`FIG. 1 showsthe results of Circular Dichroism (CD) at
`222 nm as a function of peptide concentration for native
`GLP-1 (7-37) and various GLP-1 derivativesof the present
`invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`A simple system is used to describe fragments and ana-
`logues of GLP-1. For example, Gly2—GLP-1(7-37)desig-
`nates a peptide which relates to GT_P-1 bythe deletion of the
`amino acid residues at positions. 1 to 6 and substituting the
`naturally occurring, aminoacid residue in position 8 (Ala) by
`Gly. Similarly, Lys*4(N*-tetradecanoyl)-GLP-1(7-37) des-
`ignates GLP-1 (7-37) wherein the €-amino group of the Lys
`residue in position 34 has been tetradecanoylated. Where
`reference in this text
`is made to C-terminally extended
`GLP-1 analogues, the amino acid residue in position 38 is
`Arg unless otherwise indicated, the amino acid residue in
`posilion 39 is also Arg unless otherwise indicated and the
`optional amino acid residue in position 40 is Asp unless
`otherwise indicated. Also, if a C-terminally extended ana-
`logue extendsto position 41, 42, 43, 44 or 45, the amino acid
`sequence of this extension is as in the corresponding
`sequence in human preproglucagon unless otherwise indi-
`cated.
`
`GLP-1 Analogs
`The term “an analogue” is defined herein as a peptide
`wherein one or more amino acid residues of the parent
`peptide have been substituted by another aminoacid residue.
`In a preferred embodiment,
`the total number of different
`amino acids between the GLP-1 derivative and the corre-
`sponding native form of GLP-1 is upto fifteen, preferably up
`to ten amino acid residucs, and most preferably up to six
`amino acid residues.
`The total number of different amino acids between the
`
`derivative of the GLP-1 analog and the corresponding native
`form of GLP-1 preferably does not exceed six. Preferably,
`the numberof different aminoacidsis five. More preferably,
`the number of different amino acids is four. Even more
`
`preferably,
`
`the number of different amino acids is three.
`
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`US 6,268,343 B1
`
`9
`Even more preferably, the number of different amino acids
`is two. Most preferably, the numberof different amino acids
`is one. In order to determine the numberof different amino
`
`
`
`
`acids, one should compare the amino acid sequence of the
`
`GLP-1 derivative of the present invention with the corre-
`sponding native GLP-1. For example, there are two different
`amino acids between the derivative Gly*Arg?°Lys*“(N‘-(7-
`deoxycholoyl)-GT.P-1(7-40) and the corresponding native
`GLP-1 (i.e., GLP-1(7—-40)). ‘The differences are located at
`positions 8 and 26. Similarly, there is only one different
`amino acid between the derivative Lys?°(N*-(7-
`deoxycholoyl))Are**-GLP-1(7—40) and the corresponding
`native GLP-1. The difference is located at position 34.
`In a preferred embodiment, the present invention relates
`to a GLP-1 derivative wherein the parent peptide is GLP-1
`(1-45) or an analogue thereof.
`In a further preferred
`embodiment,
`the parent peptide is GLP-1(1-35), GLP-1
`(1-36), GLP-1(1-36)amide, GLP-1(1-37), GLP-1(1-38),
`GLP-1(1-39), GLP-1(1-40), GLP-1(1-41) or an analogue -
`thereof.
`In a preferred embodiment, the present invention relates
`to derivatives of GLP-1 analogues of formula | (SEQ ID
`NO:2):
`
`1s
`
`#17
`#16
`#15
`#14
`13
`12
`11
`10
`9
`8
`7
`Xaa—-Kaa-Xaa—-Xaa-KXaa-Xaa-Xaa—Xaa-Kaa-Xaa—Kaa-
`
`(1)
`
`28
`27
`26
`25
`24
`23
`22
`21
`20
`#19
`18
`Xaa-Xaa-Kaa-Xaa-Xaa-Xaa-Xaa-Xaa-Kaa-Xaa-Phe-
`
`38
`37
`36
`#%35
`34
`33
`32
`31
`30
`29
`Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
`
`45
`44
`43
`42
`41
`40
`39
`Xaa-Xaa-Xaa—-KXaa-Xaa-Xaa-Xaa
`
`wherein
`
`Xaa at position 7 is His, a modified amino acid oris
`deleted,
`Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, [e, Val, Glu,
`Asp, or Lys, or is deleted,
`Xaa al position 9 is Glu, Asp, or Lys, or is deleted,
`Xaa at position 10 is Glyoris deleted,
`Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu,
`Asp, or Lys oris deleted,
`Xaa at position 12 is Phe or is deleted,
`Xaa at position 13 is Thr or is deleted,
`Xaaat position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
`Asp, or Lys oris deleted,
`Xaa at position 15 is Asp or is deleted,
`Xaaat position 16 is Val, Ala, Gly, Ser, Thr, Leu,Ile, Glu,
`Asp, or Lys oris deleted,
`Xaaat position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
`Asp, or Lys, or is deleted,
`Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ie, Val, Glu,
`Asp, or Lys,
`Xaa at position 19 is ‘I'vr, Phe, ‘lrp, Glu, Asp, or Lys,
`Xaaat position 20 is Leu, Ala, Gly, Ser, Thr, Leu,Ile, Val,
`Glu, Asp, or Lys,
`Xaa at position 21 is Glu, Asp, or Lys,
`Xaa al position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
`Asp, or Lys,
`Xaa at position 23 is Gln, Asn, Arg, Glu, Asp, or Lys,
`Xaa at position 24 is Ala, Gly, Ser, Tr, Leu, Ile, Val, Arg,
`Glu, Asp, or Lys,
`
`
`
`35
`
`40
`
`55
`
`60
`
`65
`
`10
`