(12) United States Patent
`Knudsen et al.
`
`I 1111111111111111 11111 lllll lllll lllll lllll lllll lllll 111111111111111 11111111
`US006268343Bl
`US 6,268,343 Bl
`Jul. 31, 2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) DERIVATIVES OF GLP-1 ANALOGS
`
`(75)
`
`Inventors: Liselotte Bjerre Knudsen, Valby; Per
`Olaf Huusfeldt, K0benhavn K; Per
`Franklin Nielsen, V1erl0se; Niels C.
`Kaarsholm, Vanl0se; Helle Birk Olsen,
`Aller0d; Soren Erik Bjorn, Lyngby;
`Freddy Zimmerdahl Pedersen; Kjeld
`Madsen, both of V1erl0se, all of (DK)
`
`(73) Assignee: Novo Nordisk NS, Bagsvaerd (DK)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`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(cid:173)
`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
`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
`
`(DK) ................................................... 0931/96
`(DK) ................................................... 1259/96
`(DK) ................................................... 1470/96
`(DK) ................................................... 0263/98
`(DK) ................................................... 0264/98
`(DK) ................................................... 0268/98
`(DK) ................................................... 0272/98
`(DK) ................................................... 0274/98
`(DK) ................................................... 0508/98
`(DK) ................................................... 0509/98
`
`(51)
`
`Int. Cl.7 .......................... A61K 39/16; A61K 38/26;
`C07K 14/00; C07K 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
`
`5,120,712
`5,512,549
`5,545,618
`5,614,492
`
`6/1992 Habener ................................. 514/12
`4/1996 Chen et al.
`............................ 514/12
`8/1996 Buckley et al.
`....................... 514/12
`3/1997 Habener ................................. 514/12
`
`FOREIGN PATENT DOCUMENTS
`
`0 708 179
`WO 90/11296
`WO 91/11457
`WO 95/07931
`WO 95/31214
`WO 96/29342
`WO 96/29344
`WO 87/06941
`WO 98/08531
`WO 98/08871
`WO 98/08873
`WO 98/19698
`
`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.
`
`(57)
`
`ABSTRACT
`
`The present invention relates to GLP-1 derivatives having a
`lipophilic substituent, pharmaceutical compositions com(cid:173)
`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
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1034-0001
`
`

`

`U.S. Patent
`
`Jul. 31, 2001
`
`US 6,268,343 Bl
`
`Fig. 1
`
`-+- a
`
`-- O -- b
`
`-.A- C
`
`- • - d
`
`- v -
`
`glp-1(7-37)
`
`-o- e
`
`-□- f
`
`1
`
`10
`
`100
`
`[peptide] (µM)
`
`-■- h
`1000
`
`.. ~------+-..ip--o ..
`
`..,...__
`
`· ...
`
`0
`
`-1
`
`-2
`
`-3
`
`-4
`
`-5
`0.1
`
`u -
`
`MPI EXHIBIT 1034 PAGE 2
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1034-0002
`
`

`

`US 6,268,343 Bl
`
`5
`
`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 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
`
`20
`
`25
`
`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(cid:173)
`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(cid:173)
`dependent diabetes mellitus (NIDDM)(2). Furthermore, as
`an insulinotropic hormone, GIP was found to be almost
`10 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
`15 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(cid:173)
`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-
`30 creatic peptide, GRPP (10, 11); 3) a hexapeptide correspond(cid:173)
`ing to PG (64-69); and, finally, the so-called major proglu(cid:173)
`cagon fragment (PG (72-158)), in which the two glucagon(cid:173)
`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
`55 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 ~-cells via the GLP-1
`receptor (23) which belongs to the glucagon/VIP/calcitonin
`family of G-protein-coupled- 7-transmembrane spanning
`
`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 35
`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, 40
`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, 45
`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, 50
`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 60
`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(cid:173)
`notropic in man), are insulinotropic, but the only physiologi- 65
`cally important ones, those that are responsible for the
`incretin effect, are the glucose-dependent insulinotropic
`
`MPI EXHIBIT 1034 PAGE 3
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1034-0003
`
`

`

`US 6,268,343 Bl
`
`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(cid:173)
`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
`45 have an insulinotropic activity which exceeds the insulino(cid:173)
`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):
`
`17
`16
`15
`14
`12
`10
`9
`11
`13
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
`
`(I)
`
`28
`27
`26
`24
`23
`22
`21
`20
`19
`18
`25
`55 Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-
`
`3
`receptors. The importance of the GLP-1 receptor in regu(cid:173)
`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 5
`fasting hyperglycaemia, and even heterozygous animals
`were glucose intolerant (24). The signal transduction mecha(cid:173)
`nism (25) primarily involves activation of adenylate cyclase,
`but elevations of intracellular Ca2 + are also essential (25,
`26). The action of the hormone is best described as a 10
`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 ~-cells. The relation 15
`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). 20
`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- 25
`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. 30
`Because of the glucose dependency of the insulinotropic and
`glucagonostatic actions, the glucose lowering effect is self(cid:173)
`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 35
`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(cid:173)
`lurea (33). The importance of the glucagonostatic effect is
`illustrated by the finding that GLP-1 also lowers blood 40
`glucose in type-1 diabetic patients without residual ~-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(cid:173)
`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 50
`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 60
`low as those required to influence islet secretion (39).
`GLP-1 seems to have an effect on food intake. Intraven(cid:173)
`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).
`
`36
`35
`34
`33
`32
`31
`30
`29
`Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-X
`
`wherein Xis H2 for GLP-1 (7-36) and Xis 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-
`65 logues and derivatives that include an N-terminal imidazole
`group and optionally an unbranched C6-C10 acyl group in
`attached to the lysine residue in position 34.
`
`MPI EXHIBIT 1034 PAGE 4
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1034-0004
`
`

`

`US 6,268,343 Bl
`
`5
`EP 0699686-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(cid:173)
`ance than GLP-1 (7-37).
`It is a further object of the invention to provide a phar(cid:173)
`maceutical composition with improved solubility and sta(cid:173)
`bility.
`
`References
`
`5
`
`6
`15. Buhl T, Thim L, Kofod H, Orskov C, Harling H, &
`Holst JJ: 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, Rabenhoj L, Kofod H, Holst JJ:
`Carboxypeptidase-B-like processing of the C-terminus of
`glucagon-like peptide-2 in pig and human small intestine.
`FEES letters, 1989; 247:193-106.
`17. Holst JJ. Evidence that enteroglucagon (II) is identical
`10 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
`15 enteroglucagon/oxyntomodulin) from porcine jejuna-ileum.
`Characterisation of the peptide. FEES Lett 1982;
`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
`25 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, 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: 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(cid:173)
`tide 1. Proc Natl Acad Sci 1992; 89:8641-4645.
`24. Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ.
`40 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 hormones glucagon-like peptide-I
`( GLP-1) and glucose-dependent insulin releasing polypep-
`45 tide (GIP). Endocrine Reviews, 1995; 16: 390-410.
`26. Gromada J, Dissing S, Bokvist K, Renstrom 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(cid:173)
`sation. Diabetes 1995; 44:767-774.
`27. Holz GG, Leech CA, Habener JF, Activation of a
`cAMP-regulated Ca2+-signaling pathway in pancreatic
`~-cells by the insulinotropic hormone glucagon-like
`peptide-1. J. Biol Chem, 1996; 270: 17749-17759.
`28. Holz GG, Kuhltreiber WM, Habener JF, Pancreatic
`beta-cells are rendered glucose competent by the insulino(cid:173)
`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,
`65 Holst JJ. Effect of glucagon-like peptide-l(proglucagon
`78-107amide) on hepatic glucose production in healthy
`man. Metabolism 1994; 43:104-108.
`
`35
`
`1. Pederson RA Gastric Inhibitory Polypeptide. In Walsh
`JH, Dockray GJ (eds) Gut peptides: Biochemistry and 20
`Physiology. Raven Press, New York 1994, pp. 217259.
`2. Kramp T. Immunoreactive gastric inhibitory polypep(cid:173)
`tide. Endocr Rev 1988;9: 122-134.
`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(cid:173)
`glucagon gene. Nature 1983; 304: 368-371.
`5. Holst JJ. Glucagon-like peptide-1 (GLP-1)-a newly 30
`discovered GI hormone. Gastroenterolgoy 1994; 107:
`1848-1855.
`6. Holst JJ. Gut glucagon, enteroglucagon, gut GLI,
`glicentin-current status. Gastroenterology 1983;
`84:1602-1613.
`7. Holst JJ, Orskov C. Glucagon and other proglucagon(cid:173)
`derived peptides. In Walsh JH, Dockray GJ, eds. Gut pep(cid:173)
`tides: Biochemistry and Physiology, Raven Press, New
`York, pp. 305-340, 1993.
`8. Orskov C, Holst JJ. 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 50
`of glicentin to proglucagon and glucagon in the porcine
`pancreas. Nature 1981; 289: 514-516.
`11. Thim L, Moody AJ, Purification and chemical char(cid:173)
`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 JJ. 60
`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, Rabenhoj L, Kofod H, WettergrenA, Holst
`JJ. Production and secretion of amidated and glycine(cid:173)
`extended glucagon-like peptide-1 (GLP-1) in man. Diabetes
`1991; 43: 535-539.
`
`55
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`MPI EXHIBIT 1034 PAGE 5
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`Petitioner Apotex Exhibit 1034-0005
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`US 6,268,343 Bl
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`8
`46. Deacon CF, Johnson AH, Holst JJ. Degradation of
`glucagon-like peptide-1 by human plasma in vitro yields an
`N-terminally truncated peptide that is a major endogenous
`metabolite in vivo. J Clin Endocrinol Metab 1995; 80:
`5 952-957.
`47. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L,
`Willms B, Holst JJ, 1995. Both subcutaneous and intrave(cid:173)
`nously administered glucagon-like peptide-1 are rapidly
`degraded from the amino terminus in type II diabetic
`10 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
`15 derivatives of the present invention have interesting phar(cid:173)
`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(cid:173)
`notropic activity, ability to decrease glucagon, ability to
`20 suppress gastric motility, ability to restore glucose compe(cid:173)
`tency to beta-cells, and/or ability to suppress appetite/reduce
`weight.
`
`7
`31. Qualmann C, Nauck M, Holst JJ, Orskov C.
`Creutzfeldt W. Insulinotropic actions of intravenous
`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 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 JJ. 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. WettergrenA, Schjoldager B, Mortensen PE, Myhre J, 25
`Christiansen J, Holst JJ. 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: Heal release
`of glucagon-like peptide-1 (GLP-1): association with inhi- 30
`bition of gastric acid in humans. Dig Dis Sci 1995; 40:
`1074-1082.
`38. Layer P, Holst JJ. 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, Schmiege! W. Inhibition of gastric emptying by
`GLP-1(7-36 amide) or (7-37); effects on postprandial gly(cid:173)
`caemia and insulin secretion. Abstract. Gut 1995; 37 (suppl.
`2); A124.
`40. Schick RR, vorm Walde T, Zimmermann JP, Schus(cid:173)
`dziarra V, Classen M Glucagon-like peptide 1-a novel
`brain peptide involved in feeding regulation. in Ditschuneit
`H, Gries FA, Rauner H, Schusdziarra V, Wechsler JG (eds.)
`Obesity in Europe. John Libbey & Company ltd, 1994; pp. 45
`363-367.
`41. Tang-Christensen M, Larsen PJ, Goke 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 50
`water intake. Am. J. Physiol., 1996, in press.
`42. Turton MD, O'Shea D, Gunn I, Beak SA, Edwards
`CME, 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(cid:173)
`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 60
`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(cid:173)
`ties of GLP-1 [7-36 amide] after subcutaneous injection in
`healthy volunteers. Dose-response relationships. Diabetolo(cid:173)
`gia 1995; 38: 720---725.
`
`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(cid:173)
`logues of GLP-1. For example, Gly8-GLP-1(7-37) desig-
`35 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, Lys34(W-tetradecanoyl)-GLP-1(7-37) des(cid:173)
`ignates GLP-1 (7-37) wherein the E-amino group of the Lys
`40 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(cid:173)
`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(cid:173)
`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,
`65 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.
`
`55
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`MPI EXHIBIT 1034 PAGE 6
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1034-0006
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`US 6,268,343 Bl
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`5
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`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 Gly8 Arg26Lys34(Nc -(7-
`deoxycholoyl)-GLP-1(7-40) and the corresponding native
`GLP-1 (i.e., GLP-1(7-40)). The differences are located at 10
`positions 8 and 26. Similarly, there is only one different
`amino acid between the derivative Lys 26 (Nc-(7-
`deoxycholoyl))Arg34-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 15
`to a GLP-1 d