(12) United States Patent
`Knudsen et al.
`
`USOO6268343B1
`(10) Patent No.:
`US 6,268,343 B1
`(45) Date of Patent:
`Jul. 31, 2001
`
`(54) DERIVATIVES OF GLP-1ANALOGS
`(75) Inventors: Liselotte Bjerre Knudsen, Valby; Per
`Olaf Huusfeldt, Kobenhavn K; Per
`Franklin Nielsen, Vaerlose; Niels C.
`Kaarsholm, Vanløse; Helle Birk Olsen,
`Allerød; Søren Erik Bjørn, Lyngby;
`Freddy Zimmerdahl Pedersen; Kjeld
`Madsen, both of Vaerløse, all of (DK)
`
`(73) Assignee: Novo Nordisk A/S, Bagsvaerd (DK)
`Subject to any disclaimer, the term of this
`(*) Notice:
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/258,750
`(22) Filed:
`Feb. 26, 1999
`Related U.S. Application Data
`(63) Continuation-in-part of application No. 09/038,432, filed on
`Mar. 11, 1998, now abandoned, which is 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
`(60) 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 (DK) ................................................... O931/96
`Nov. 8, 1996 (DK) ................................................... 1259/96
`Dec. 20, 1996 (DK) ................................................... 1470/96
`Feb. 27, 1998 (DK).
`... 0263/98
`Feb. 27, 1998 (DK).
`... O264/98
`Feb. 27, 1998 (DK).
`... 0268/98
`Feb. 27, 1998 (DK)
`O272/98
`O274/98
`Feb. 27, 1998 (DK)
`... 0508/98
`Apr. 8, 1998 (DK) ...
`Apr. 8, 1998 (DK) ................................................... 0509/98
`
`
`
`(51) Int. Cl." .......................... A61K 39/16; A61K 38/26;
`C07K 14/00; CO7K 14/605
`(52) U.S. Cl. ............................................... 514/12; 530/324
`(58) Field of Search ................................ 530/324; 514/12
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`6/1992 Habener ................................. 514/12
`4/1996 Chen et al. ......
`... 514/12
`8/1996 Buckley et al. .
`... 514/12
`3/1997 Habener ................................. 514/12
`
`5,120,712
`5,512,549
`5,545,618
`5,614,492
`
`O 708 179
`WO 90/11296
`WO 91/11457
`WO95/07931
`WO95/31214
`WO 96/29342
`WO 96/29344
`WO 87/06941
`WO 98/08531
`WO 98/O8871
`WO 98/O8873
`WO 98/19698
`
`FOREIGN PATENT DOCUMENTS
`4/1996 (EP).
`10/1990 (WO).
`8/1991 (WO).
`3/1995 (WO).
`11/1995 (WO).
`9/1996 (WO).
`9/1996 (WO).
`11/1997 (WO).
`3/1998 (WO).
`3/1998 (WO).
`3/1998 (WO).
`5/1998 (WO).
`OTHER PUBLICATIONS
`Kim et al., (1994) J. of Pharma, Sciences 83(8); 1175–1180.
`Clodfelter et al., (1998) Pharmaceutical Res. 15(2):254-262.
`Primary Examiner Michael Borin
`(74) Attorney, Agent, or Firm-Steve T. Zelson, Esq.; Elias
`J. Lambiris, Esq.
`ABSTRACT
`(57)
`The present invention relates to GLP-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
`
`MPI EXHIBIT 1034 PAGE 1
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1034, p. 1 of 137
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`

`

`U.S. Patent
`
`Jul. 31, 2001
`
`US 6,268,343 B1
`
`Fig. 1
`
`
`
`O.1
`
`1
`
`10
`peptide (uM)
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`100
`
`1OOO
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
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`

`

`1
`DERVATIVES 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 priority
`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 60/084,357 filed Jan. 24, 1997, Jan. 25, 1997, Jan.
`24, 1997, Apr. 21, 1998, Apr. 21, 1998, Apr. 23, 1998, and
`May 5, 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 herein by reference.
`FIELD OF THE INVENTION
`The present invention relates to novel derivatives of
`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 technology it
`can be expected that their importance will increase also in
`the years to come. When native peptides or analogues
`thereof are used in therapy it is generally found that they
`have a high clearance. A high clearance of a therapeutic
`agent is inconvenient in cases where it is desired to maintain
`a high blood level thereof over a prolonged period of time
`Since repeated administrations will then be necessary.
`Examples of peptides which have a 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 hormones,
`endorphins, enkephalins, vasopressin, oxytocin, opiods and
`analogues thereof, Superoxide dismutase, interferon,
`asparaginase, arginase, arginine deaminase, adenosine
`deaminase and ribonuclease. In Some cases it is possible to
`influence the release profile of peptides by applying Suitable
`pharmaceutical compositions, but this approach has various
`Shortcomings and is not generally applicable.
`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 (choleystokinin 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
`
`5
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`40
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`US 6,268,343 B1
`
`2
`polypeptide, GIP, and glucagon-like peptide-1 (GLP-1).
`Because of its insulinotropic effect, GIP, isolated in 1973 (1)
`immediately attracted considerable interest among diabe
`tologist. However, numerous investigations carried out dur
`ing the following years clearly indicated that a defective
`secretion of GIP was not involved 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 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,
`but is already established as an important gut hormone with
`regulatory function in glucose metabolism and gastrointes
`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) glucagon itself, occupying positions
`33-61 of proglucagon (PG); 2) an N-terminal peptide of 30
`amino acids (PG (1-30)) often called glicentin-related 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 seems to 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-extended but equally
`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 activities.
`Being Secreted in parallel with glicentin/enteroglucagon,
`it follows that the many Studies of enteroglucagon Secretion
`(6, 7) to some extent also apply to GLP-1 secretion, but
`GLP-1 is metabolised more quickly with a plasma half-life
`in humans of 2 min (19). Carbohydrate or fat-rich meals
`Stimulate (20), presumably as a result of direct interaction of
`yet unabsorbed nutrients with the microVilli of the open-type
`L-cells of the gut mucosa. Endocrine or neural mechanisms
`promoting GLP-1. Secretion may exist but have not yet been
`demonstrated in humans.
`The incretin function of GLP-1 (29-31) has been clearly
`illustrated in experiments with the GLP-1 receptor
`antagonist, exendin 9-39, which dramatically reduces the
`incretin effect elicited by oral glucose in rats (21, 22). The
`hormone interacts directly with the f-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
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`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 deteriorated glucose 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 f-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, but fully to a combination
`of the two, is also not known. Equally importantly, however,
`the hormone also potently inhibits glucagon Secretion (29).
`The mechanism is not known, but seems to be paracrine, Via
`neighbouring insulin or Somatostatin cells (25). Also the
`glucagonostatic action is glucose-dependent, So that the
`inhibitory effect decreases as blood glucose decreases.
`Because of this dual effect, if the plasma GLP-1 concentra
`tions increase either by increased Secretion or by exogenous
`infusion the molar ratio of insulin to glucagon in the blood
`that reaches the liver via the portal circulation is greatly
`increased, whereby hepatic glucose production decreases
`(30). As a result blood glucose concentrations decrease.
`Because of the glucose dependency of the insulinotropic and
`glucagonostatic actions, the glucose lowering effect is Self
`limiting, and the hormone, therefore, does not cause
`hypoglycaemia regardless of dose (31). The effects are
`preserved in patients with diabetes mellitus (32), in whom
`infusions off slightly Supraphysiological doses of GLP-1
`may completely normalise blood glucose values in Spite of
`poor metabolic control and Secondary failure 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 B-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 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 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 effect seems to be highly
`specific. Thus, N-terminally extended GLP-1 (PG 72-107)
`amide is inactive and appropriate doses of the GLP-1
`antagonist, exendin 9-39, abolish the effects of GLP-1 (41).
`
`15
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`25
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`35
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`40
`
`45
`
`50
`
`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.
`Not only the insulinotropic effects but also the effects of
`GLP-1 on the gastrointestinal tract are preserved in diabetic
`patients (43), and may help curtailing meal-induced glucose
`excursions, but, more importantly, may also influence food
`intake. Administered intravenously, continuously for one
`week, GLP-1 at 4 ng/kg/min has been demonstrated to
`dramatically improve glycaemic control in NIDDM patients
`without significant side effects (44). The peptide is fully
`active after Subcutaneous administration (45), but is rapidly
`degraded mainly due to degradation by dipeptidyl peptidase
`IV-like enzymes (46, 47).
`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 and in the brain. Processing
`of preproglucagon to GLP-1 (7-36)amide, GLP-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 et 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 plasma profile were
`highly protracted and Still had retained activity.
`GLP-1 and analogues of GLP-1 and fragments thereof are
`useful i.a. in the treatment of Type 1 and Type 2 diabetes and
`obesity.
`WO 87/06941 discloses GLP-1 fragments, including
`GLP-1 (7-37), and functional derivatives thereof and to
`their use as an insulinotropic agent.
`WO 90/11296 discloses GLP-1 fragments, including
`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) and to their
`use as insulinotropic agents.
`The amino acid sequence of GLP-1 (7-36) and GLP-1
`(7–37) is (SEQ ID NO:1):
`
`9 10 11 12 13 14 15 16 17
`8
`7
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser
`
`(I)
`
`18 19 20 21 22 23 24 25 26 27 28
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe
`
`55
`
`29 30 31. 32 33 34 35 36
`Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-X
`wherein X is H 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 Co-Co acyl group in
`attached to the lysine residue in position 34.
`
`60
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`65
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`IPR2024-00009
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`

`US 6,268,343 B1
`
`S
`EP 06996.86-A2 (Eli Lilly & Co.) discloses certain
`N-terminal truncated fragments of GLP-1 that are reported
`to be biologically active.
`Unfortunately, the high clearance limits the usefulness of
`these compounds. Thus there still is a need for improve
`ments in this field.
`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 lower clear
`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.
`
`5
`
`15
`
`6
`15. Buhl T, Thim L., Kofod H, Orskov C, Harling H, &
`Holst J.J. Naturally occurring products of proglucagon
`111-160 in the porcine and human Small intestine. J. Biol.
`Chem. 1988; 263:8621-8624.
`16. Orskov C, Buhl T, Rabenho. L, Kofod H, Holst JJ:
`Carboxypeptidase-B-like processing of the C-terminus of
`glucagon-like peptide-2 in pig and human Small intestine.
`FEBS letters, 1989; 247: 193-106.
`17. Holst J.J. Evidence that enteroglucagon (II) is identical
`with the C-terminal sequence (residues 33-69) of glicentin.
`Biochem J. 1980; 187:337-343.
`18. Bataille D, Tatemoto K, Gespach C, Jornvall H,
`Rosselin G, Mutt V. Isolation of glucagon-37 (bioactive
`enteroglucagon/oxyntomodulin) from porcine jejuno-ileum.
`Characterisation of the peptide. FEBS Lett 1982;
`146:79-86.
`19. Orskov C, Wettergren A, Holst J.J. 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
`in response to nutrient ingestion in man: acute post-prandial
`and 24-h secretion patterns. J Endocrinol 1993; 138:
`159-166.
`21. Kolligs F, Fehmann HC, Göke R, Göke B. Reduction
`of the incretin effect in rats by the glucagon-like peptide-1
`receptor antagonist exendin (9–39)amide. Diabetes 1995;
`44: 16-19.
`22. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei M.,
`Bloom SR. Glucagon-like peptide-1 is a physiological incre
`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. Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ.
`Diabetes in mice with targeted disruption of the GLP-1
`receptor gene. Diabetes 1996; 45, 21A.
`25. Fehmann HC, Göke R, Göke G. Cell and molecular
`biology of the incretin hormones 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, Renström E,
`Frokjaer-Jensen J, Wulff BS, Rorsman P. Glucagon-like
`pepide I increases cytoplasmic calcium in insulin-Secreting
`bTC3-cells by enhancement of 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
`peptide-1. J. Biol Chem, 1996; 270: 17749–17759.
`28. Holz GG, Kühltreiber 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. Hvidberg A, Toft Nielsen M, Hilsted J, Orskov C,
`Holst J.J. Effect of glucagon-like peptide-1 (proglucagon
`78-107amide) on hepatic glucose production in healthy
`man. Metabolism 1994; 43:104-108.
`
`25
`
`35
`
`References
`1. Pederson RA. Gastric Inhibitory Polypeptide. In Walsh
`JH, Dockray GJ (eds) Gut peptides: Biochemistry and
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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. EXon duplication and divergence in the human prepro
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`5. Holst J.J. Glucagon-like peptide-1 (GLP-1)-a newly
`discovered GI hormone. Gastroenterolgoy 1994; 107:
`1848-1855.
`6. Holst J.J. Gut glucagon, enteroglucagon, gut GLI,
`glicentin-current Status. Gastroenterology 1983;
`84:1602-1613.
`7. Holst JJ, Orskov C. Glucagon and other proglucagon
`derived peptides. In Walsh JH, Dockray GJ, eds. Gut pep
`tides: Biochemistry and Physiology, Raven Press, New
`York, pp. 305-340, 1993.
`40
`8. Orskov C, Holst J.J. Knuhtsen S, Baldissera FGA,
`Poulsen SS, Nielsen OV. Glucagon-like peptides GLP-1 and
`GLP-2, predicted products of the glucagon gene, are
`Secreted Separately from the pig Small intestine, but not
`pancreas. Endocrinology 1986; 119:1467–1475.
`9. Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann
`B, Orskov C. Proglucagon processing 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 glucagon in the porcine
`pancreas. Nature 1981; 289: 514-516.
`11. Thim L, Moody AJ, Purification and chemical char
`acterisation of a glicentin-related pancreatic peptide
`(proglucagon fragment) from porcine pancreas. Biochim
`Biophys Acta 1982; 703:134-141.
`12. Thim L. Moody AJ. The primary structure of glicentin
`(proglucagon). Regul Pept 1981; 2:139-151.
`13. Orskov C, Bersani M, Johnsen AH, Hojrup P. Holst J.J.
`Complete Sequences of glucagon-like peptide-1 (GLP-1)
`from human and pig Small intestine. J. Biol. Chem. 1989;
`264:12826-12829.
`14. Orskov C, Rabenho. L, Kofod H, Wettergren A, Holst
`J.J. Production and Secretion of amidated and glycine
`extended glucagon-like peptide-1 (GLP-1) in man. Diabetes
`1991; 43: 535-539.
`
`45
`
`50
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`55
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`60
`
`65
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`IPR2024-00009
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`7
`31. Qualmann C, Nauck M, Holst JJ, Orskov C.
`Creutzfeldt W. Insulinotropic actions of intravenous
`glucagon-like peptide-17-36 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 GIP in patients with
`type 2-diabetes mellitus. J Clin Invest 1993; 91:301-307.
`33. Nauck MA, Kleine N, Orskov C. Holst JJ, Willms B,
`Creutzfeldt W. Normalisation of fasting hyperglycaemia by
`exogenous GLP-1(7-36amide) in type 2-diabetic patients.
`Diabetologia 1993; 36:741-744.
`34. Creutzfeldt W. Kleine N, Willms B, Orskov C, Holst
`JJ, Nauck MA. Glucagonostatic actions and reduction of
`fasting hyperglycaemia by exogenous glucagon-liem,
`peptide-1 (7-36amide) in type 1 diabetic patients. Diabetic
`Care 1996; 19: 580-586.
`35. Schjoldager BTG, Mortensen PE, Christiansen J,
`Orskov C, Holst J.J. GLP-1 (glucagon-like peptide-1) and
`truncated GLP-1, fragments of human proglucagon, inhibit
`gastric acid secretion in man. Dig. Dis. Sci. 1989;
`35:703-708.
`36. Wettergren A, Schjoldager B, Mortensen PE, Myhre J,
`Christiansen J, Holst J.J. Truncated GLP-1 (proglucagon
`72-107amide) inhibits gastric and pancreatic functions in
`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 inhi
`bition of gastric acid in humans. Dig Dis Sci 1995; 40:
`1074-1082.
`38. Layer P, Holst J.J. GLP-1: A humoral mediator of the
`ileal brake in humans? Digestion 1993; 54:385–386.
`39. Nauck M, Ettler R, Niedereichholz U, Orskov C,
`Holst JJ, Schmiegel W. Inhibition of gastric emptying by
`GLP-1 (7-36 amide) or (7–37); effects on postprandial gly
`caemia and insulin secretion. Abstract. Gut 1995; 37 (suppl.
`2); A124.
`40. Schick RR, vorm Walde T, Zimmermann JP, Schus
`dZiarra V, Classen M Glucagon-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 & Company ltd, 1994; pp.
`363-367.
`41. Tang-Christensen M, Larsen PJ, Göke R, Fink-Jensen
`A, Jessop DS, Moller M, Sheikh S. Brian GLP-1(7–36)
`amide receptors play a major role in regulation of food and
`water intake. Am. J. Physiol., 1996, in press.
`42. Turton MD, O'Shea D, Gunn I, Beak SA, Edwards
`CMB, Meeran K, et al. A role for glucagon-like peptide-1 in
`the regulation of feeding. Nature 1996; 379; 69–72.
`43. Willms B, Werner J, Creutzfeldt W, Orskov C, Holst
`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.
`44. Larsen J, Jallad N, Damsbo P. One-week continuous
`infusion of GLP-1 (7-37) improves glycaemic control in
`NIDDM. Diabetes 1996; 45, suppl. 2: 233A.
`45. Ritzel R, Orskov C, Holst JJ, Nauck MA.
`Pharmacokinetic, insulinotropic, and glucagonstatic proper
`ties of GLP-17-36 amide after subcutaneous injection in
`healthy Volunteers. Dose-response relationships. Diabetolo
`gia 1995; 38: 720–725.
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`US 6,268,343 B1
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`8
`46. Deacon CF, Johnson AH, Holst J.J. 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 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.
`SUMMARY OF 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
`notropic activity, ability to decrease glucagon, ability to
`SuppreSS gastric motility, ability to restore glucose compe
`tency to beta-cells, and/or ability to SuppreSS appetite/reduce
`weight.
`BRIEF DESCRIPTION OF THE FIGURES
`FIG. 1 shows the results of Circular Dichroism (CD) at
`222 nm as a function of peptide concentration for native
`GLP-1 (7-37) and various GLP-1 derivatives of the present
`invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`A simple System is used to describe fragments and ana
`logues of GLP-1. For example, Gly-GLP-1(7–37) desig
`nates a peptide which relates to GLP-1 by the deletion of the
`amino acid residues at positions. 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) des
`ignates GLP-1 (7-37) wherein the e-amino group of the Lys
`residue in position 34 has been tetradecanoylated. Where
`reference in this text is made to C-terminally extended
`GLP-1 analogues, the amino acid residue in position 38 is
`Arg unless otherwise indicated, the amino acid residue in
`position 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 extends to 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 amino acid 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 up to fifteen, preferably up
`to ten amino acid residues, 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 number of different amino acids is five. More preferably,
`the number of different amino acids is four. Even more
`preferably, the number of different amino acids is three.
`
`MPI EXHIBIT 1034 PAGE 6
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1034, p. 6 of 137
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`US 6,268,343 B1
`
`9
`Even more preferably, the number of different amino acids
`is two. Most preferably, the number of different amino acids
`is one. In order to determine the number of 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 GlyArg-Lys-(N-(7-
`deoxycholoyl)-GLP-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))Arg-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 I (SEQ ID
`NO:2):
`
`9 10 11 12 13 14 15 16 17
`8
`7
`Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
`
`(I)
`
`18, 19 20 21 22 23 24 25 26 27 28
`Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe
`
`32 33 34 35 36 37 38
`29 30 31.
`Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
`
`42 4 3 44 45
`39 40 41.
`Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
`
`wherein
`Xaa at position 7 is His, a modified amino acid or is
`deleted,
`Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
`Asp, or LyS, or is deleted,
`Xaa at position 9 is Glu, Asp, or LyS, or is deleted,
`Xaa at position 10 is Gly or is deleted,
`Xaa at po