(12) United States Patent
`Knudsen et al.
`
`I IIIII IIIIIIII Ill 11111 IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIIII IIII IIII IIII
`US006268343B1
`US 6,268,343 Bl
`Jul. 31, 2001
`
`(l0) Patent No.:
`(45) Date of Patent:
`
`(54) DERIVATIVES OF GLP·lA ALOGS
`
`(75)
`
`Inventors: Liselotte Bjerre Knudsen, Valby; Per
`Olaf Huusfeldt, K¢benhavn K; Per
`Franklin
`ielsen, Vrerl¢se;
`iels C.
`Kaarsholm, Vanl¢se; Helle Birk Olsen,
`Allerl'.ld; ~ren Erik Bj0rn, Lyngby;
`Freddy Zimmerdahl Pedersen; Kjeld
`Madsen , both of Vrerl0se, all of (DK)
`
`(73) A5.5ignee: Novo Nordisk A/S, Bagsvaerd (DK)
`
`( * )
`
`orice:
`
`Subject lo any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. o.: 09/258,750
`
`(22) Filed:
`
`Feb. 26, 1999
`
`Related U.S. Application Data
`
`(63)
`
`(60)
`
`Continuation-in -part of application No. 09/038,432, fi led on
`Mar. 11, 1998, now abandoned, which is a con ti nuation -ii1-
`part of application No. 08/918,810, filed on A ug. 26, 1997,
`110w abandoned_. aad a cootiuuation-in -part of appl ication
`No . PCT;DK97/00340, filed 011 Aug. 22, 1997
`Provisional application No. 60ft)35,904, filed on Jan. 24,
`1997, provisional application No . 60/036,226, filed on Jan.
`25, 1997, provisional application o. 60/036,255, filed on
`Jan. 24, 1997, provisional application No. 60/082,478, filed
`011 Apr. 21 , 1998, provisio nal application No. 60/082,480,
`filed on Apr. 21, 1998, provisional application No. 60/082,
`802, filed on Apr. 23, 1998, and pwvisional application No.
`60/084,357, filed on May 5, 1998.
`Foreign Application Priority Data
`
`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) ................. ................ ... ... ... ... ...... 093 1196
`(DK) ....................................... ............ 1259/96
`(DK) .. .. .... .. ......................................... 1470196
`(DK) ................................................... 0263/98
`(DK) ................................. ... ...... ... ...... 0264/98
`(DK) ....................................... ............ 0268/98
`(DK) .. .. .... .. ......................................... 0272198
`(DK) ................................................... 0274/98
`(DK) ... ... ... ... .. ... . .. ... ... ... . ... ...... ...... ... ... 0508198
`(DK) ....................................... ............ 0509198
`
`(S l )
`
`Int. Cl.7
`
`. .. . ..• ... .. • .. . ......... . A61K 39/16; A61K 38/26;
`C07K 14/00; C07K 14/60S
`(S2) U.S. Cl . ............................................... 514/12; 530/324
`(S8) Field of Search ..... ··- ·· ... ..... .. .. .. 530/324; 514/12
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUME TS
`
`5,120,712
`5,512,549
`5,54 ,618
`5,614,492
`
`6/1992 Habener ................................ . 514/ 12
`4/1996 Che.n et al.
`........................... . 514/ 12
`8/1996 Buckley et al. ...................... . 14/ 12
`3/1997 Habener ...................... ... ...... .. 514/ 12
`
`FOREIG N PATENT DOCUME TS
`
`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) Pha.rmaceutical Res, 15(2):254--262.
`
`Primary Examine,·--Michael Borio
`teve T, Zelsoa, Esq.; Elias
`(74) Attorney, Agent, or Firm
`J. Lambiris, Esq.
`
`(57)
`
`ABSTRACT
`
`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 deriv atives of tbe present invention have a protracted
`profile of action.
`
`40 Claims, 1 Drawing Sheet
`
`FRESENIUS EXHIBIT 1006
`Page 1 of 138
`
`

`

`U.S. Patent
`
`Jul. 31, 2001
`
`US 6,268,343 Bl
`
`Fig. 1
`
`1
`
`10
`
`100
`
`[peptide] (µM)
`
`0
`
`-1
`
`-2
`
`-3
`
`-4
`
`-5
`0.1
`
`-+- a
`
`-· 0 -- b
`
`-A.- C
`
`- • - d
`
`- v -
`
`gJp-1(7-37)
`
`-o- e
`
`- □ - f
`
`-'Y- g
`
`-■- h
`1000
`
`FRESENIUS EXHIBIT 1006
`Page 2 of 138
`
`

`

`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-pari 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 Dani5h 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-! (GLP-1) and fragments and/
`or analogues thereof which have a protracted profile of
`action and io methods of making and using them.
`
`BACKGROUND OF THE INVENTION
`
`20
`
`25
`
`2
`polypeptide, GIP, and glucagon-like peptide-I (GLP-1).
`Because of its insulinotropic effect, GIP, isolated in 1973 (1)
`immediately attracted considerable in terest 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 (NIDDMX2) . 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 tbe 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 iL5elf, occupying positions
`33-61 of proglucagon (PG); 2) an N-terminal peptide of 30
`amino acids (PG (1- 30)) often called glicentin-related pan(cid:173)
`creatic peptide, GRPP (10, 11); 3) a hexapeptide correspond(cid:173)
`ing to PG (64- 69); and, finally, the so-called major proglu-
`cagon fragment (PG (72- 158)), in which the two glucagon(cid:173)
`like sequences are buried (9). Giucagon seems to be the only
`biologically active product. In contrast, in the intestinal
`mucosa, it L5 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: L) glicentin, corresponding to PG (1 -69), with the
`glucagon sequence occupying residues Nos. 33-61 (12); 2)
`GLP-1(7- 36)antide (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(l26-158))(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-cell5 of the gut mucosa. Endocrine or neural mechanisn15
`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 30
`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 tLsed in therapy it is generally found that they
`have a high clearance. A higb 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 admini5trations 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-I,
`glucago n-like peptide-2, insulin-like growth factor-1 ,
`insulin-like growth factor-2, gastric inhibitory peptide,
`growth hormone-releasing factor, pituitary adenylate
`cyclase activa ting pep tide, secretin, enterogastrin, 45
`somatostai n, somatotropin, somatomedin, parathyroid
`hormone, thrombopoietin, erythropoietin, hypothalamic
`releasing factors, prolactin, thyroid stimulating hormones,
`endorphins, enkephalins, vasopressin, oxytocin, opiods and
`analogues thereof, superoxide disnmtase, interferon, so
`asparaginase, arginase, arginine deaminase, adenosine
`deamioase 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
`in5ulin. The enhancing effect on insulin secretion, the
`so-called incretin effect, is probably essential for a normal
`glucose tolerance. Many of tbe 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
`
`FRESENIUS EXHIBIT 1006
`Page 3 of 138
`
`

`

`US 6,268,343 Bl
`
`10
`
`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 sa tiery 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 glycaernic control in NIDDM patients
`without significant side effects ( 44). The peptide i5 fully
`active after subcutaneous administration (45), but is rapidly
`15 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 attract1:1d 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 thi5 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 l aod 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
`bave an insulinotropic activity which exceeds the insulino(cid:173)
`tropic activity of GLP-1 (l- 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 N0:1):
`
`7
`15 16 17
`14
`11 12 13
`10
`9
`8
`Hi s-Ala-Gl u-Gl y -Thr-Phe-Thr-Ser-Asp-Val -Ser-
`
`(I)
`
`3
`receptors. The importance of the GLP-1 re<.."eptor 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
`di5ruption bad greatly deteriorated glucose tolerance and s
`fa5ting hyperglycaemia, and even heterozygous animals
`were glucose intolerant (24). The signal transduction mecha(cid:173)
`nism (25) primarily involves activation of adenylate cyclase,
`+ are also essential (25,
`but elevations of intracellular Ca2
`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 ~-cells. The relation
`of the latter to its ability to convey "glucose competance" lo
`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
`glucagooostatic 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). A5 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 p atients with dia betes mellit.us (32), in wbom 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 sulpbony(cid:173)
`lurea (33). The importance of the glucagonostatic effect is
`illustrated by the finding that GLP-1 also Jowers blood 40
`glucose in type-1 diabetic patients without residual P-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- 45
`induced as well as meal-induced gastric acid secretion (35,
`36). ll 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).
`
`26 27 28
`25
`18 19 20 21 22 23 24
`55 Ser-Ty r-Leu-Glu-Gly-Gln -Ala-Al a -Lys-Glu -Phe-
`
`34 35
`32 3 3
`3 1
`30
`29
`36
`Ile-Ala-Trp-Leu-Va l-Ly s-Gl y-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.
`
`FRESENIUS EXHIBIT 1006
`Page 4 of 138
`
`

`

`US 6,268,343 Bl
`
`5
`EP 0699686-Al (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- s
`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
`
`6
`15. Buhl T, Thim L, Kofocl 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.
`FEBS letters, 1989; 247:193-106.
`17. Holst JJ. Evidence that enteroglucagon (U) is identical
`10 with the C-terminal sequence (residues 33- 69) of glicentin.
`Biochem .T. 1980; 187:337- 343.
`18. Bataille D, Taternoto K, Gespach C, Jornvall H,
`Rosselin G, Mull V. Isolation of glucagon-37 (bioactive
`15 enteroglucagon/oxyntomodulin) from porcine jejuno-ileum.
`Characterisation of the peptide . FEBS 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. Elliou RM, M organ LM, Tredger JA, Deacon S,
`Wright J, Marks V. Glucagon-like peptide-! (7-36)amide
`and glucose-dependen t insulinotropic polypeptide secretion
`2s in response to nutrient ingestion in man: acute post-prandial
`and 24-h secretion patterns. J Endocrinol 1993; 138:
`159- 166.
`21. Ko!Jigs F, Fehm ann HC, Goke R, Goke B. Reduction
`of the incretin effect in rats by the glucagon-like peptide-1
`receptor antagonist exendin (9-39)arnide. Diabetes 1995;
`44: 16-19.
`22. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei M,
`Bloom S R. Glucagon-like peptide-1 is a physiological incre(cid:173)
`tin in rat. J. Clio. Invest. 1995; 95:417-421.
`23. Thorens B. Expression cloning of the pancreatic b ce!J
`receptor for the gluco-incretin hormone glucagon-like pep(cid:173)
`tide l. Proc Natl Acad Sci 1992; 89:8641- 4645.
`24. Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ.
`40 D iabetes in mice with targeted dismption of the GLP-1
`receptor gene. D iabetes 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, Bokvi5t K, Renstrom E,
`Frokjaer-Jensen J, Wulff BS, Rorsman P. Glucagon-like
`pepicle I increases cytoplasmic calcium in insulin-secreting
`bTC3-cells by enbancemen1 of intracellular calcium mobili(cid:173)
`sation. Diabetes 1995; 44:767-774.
`27. Holz GG, Leech CA, H abeuer JF, Activation of a
`cAMP-regulated Ca2 +-signaling pathway in pancreatic
`~-cell5 by the insulinotropic hormone glucagon-like
`peptide-1. J. Biol Chem, 1996; 270: 17749- 17759.
`28. Holz GG, Kuhltreiber WM, H abener JF, Pancreatic
`beta-cells are rendered glucose competent by the insulino(cid:173)
`tropic hormone glucagon-like pepticle-1(7- 37). Nature
`1993; 361:362- 365.
`29. Orskov C, Ho lst JJ, NieL<;en OV: Effect of truncated
`glucagon-like peptide 1 (Proglucagon 78-107 amide) on
`endocrine secretion from pig pancreas, anirum and stomach.
`Endocrinology 1988; 123:2009- 2013.
`30. Hvidberg A, Toft Nielsen M, Hilsted .T, Orskov C,
`65 Holst JJ. Effect of glucagon-like pepticle-l(proglucagoo
`78-107arnide) 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. Irnrnunoreactive 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-! (GLP-1)- a newly 30
`discovered GI hormone. Gastroenterolgoy 1994; 107:
`1848-1855.
`'·st JJ.
`6. Ho
`Gut glucagon, enteroglucagon, gut GLI,
`"
`glicentin- current status. Gastroenterology 1983;
`· 60
`6
`4:1 2- 1 13.
`8
`7. Holst JJ, Orskov C. Glucagon and other proglucagon(cid:173)
`derived peptides. lo 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 Chern, 1994; 269; 18827- 1883.
`10. Moody AT, Holst.TJ, 111im L, Jensen S L. Relationship so
`of glicentin to proglucagon and glucagon in the porcine
`pancreas. Nature 1981; 289: 514-516.
`11 . Thim L, Moody AT, 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 AT . The primary structure of glicentin
`(proglucagou). 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 intesti.ne. J. Biol. Chem. 1989;
`264:12826- 12829.
`14. Orskov C, Rabenboj L, Kofod H, Wettergren A, 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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`US 6,268,343 Bl
`
`8
`46. Deacon CF, Johnson AH, Holst JJ. Degradation of
`glucagon-like peptide-1 by human plasma in vitro yields an
`N-termioally truncated peptide that is a major endogenous
`metabolite in vivo. J Clio E ndocriool Metab 1995; 80:
`s 952- 957.
`47. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L,
`Willms B, HoL,;1 JJ, 1995. Both subcutaneous and intra.ve(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-ll31.
`
`SUMMARY OF THE INVENTION
`The present invention relates to derivatives of GLP-1
`(1- 45) and analogs and/ or fragments thereof. lbe 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 tbe 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.
`
`BRIEF DESCRfPTION 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 derivarives of the present
`invention.
`
`7
`31. Qualmann C, Nauck M, Holst JJ, Orskov C.
`Creutzfeldt W. l nsuliootropic actions of intravenous
`glucagoo-like peptide-1 [7- 36 amide] in the fasting state in
`healthy subjects. Acta Diabetologica, 1995; 32:13- 16.
`32 Nauck MA, Heimesaai MM, Orskov C, Holst JJ, Ebert
`R, Creutzfeldt W. Preserved incretin activity of GLP-1
`(7- 36amide) bui noi of synthetic human GIP in paiients with
`type 2-diabetes mellitw;. J Clio 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
`J.T, 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.T. GLP-1 (glucagon-like pepride-1) and
`truncated GLP-1, fragments of human proglucagon, inhibit
`gastric acid secretio n in man. Dig. Dis. Sci. 1989;
`35:703-708.
`36 Wettergren A, Schjoldager B, Mortensen PE, M yhre J, 25
`Christiansen J, Holst JJ. Truncated GLP-1 (proglucagon
`72-107 amide) inhibits gastric and pancreatic functions in
`man. Dig Dis Sci 1993; 38:665-673.
`37. Layer P, Holst JJ, Grandt D, Goebell H: Ilea! release
`of glucagon-like peptide-1 (GLP-1) : association with inbi- 30
`bitioo 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 posiprandial gly(cid:173)
`caemia and insulin secretion. Abstract. Gui 1995; 37 (suppl.
`2); Al24.
`40. Schick RR, vorm Walde T, Zimmermann JP, Schus(cid:173)
`dziarra V, Classen M Glucagon-like peptide 1- a novel
`brain pep tide involved in feeding regulation. in Ditschuneit
`H, Gries FA, Hauner H, Schusdziarra V, Wechsler JG (eds.)
`Obesit y in Europe. John Libbey & Company ltd, 1994; pp.
`363-367.
`
`DETAJLED DESCRIPTION OF THE
`INVENTION
`A simple system is used to describe fragments and ana(cid:173)
`logues of GLP-1. For examp le, 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)
`ignate.<; GLP-1 (7- 37) wherein the E-amino group of the Lys
`40 residue in position 34 ha,; been tetradecanoylated. Where
`reference in this text i,; made to C-tenninally extended
`GLP-1 analogues, tbe amino acid residue io position 38 is
`Arg unless otherwise indicated, the amino acid residue in
`45 position 39 is also Arg unless otherwise indicated and the
`optional amino acid residue in position 40 is Asp un less
`otherwise indicated. Also, if a C-rerminaUy 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
`so sequence in human preproglucagon unless otherwi,;e 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 corre.spooding 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.
`
`60
`
`55
`
`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
`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(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
`infusion of GLP-1(7- 37) improves glycaemic control in
`NIDDM. Diabetes 1996; 45, suppl. 2: 233A
`45. Ritzel R, Orskov C, Holst JJ, Na uck 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.
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`US 6,268,343 Bl
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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- 5
`sponding native GLP-1. For example, there are two different
`amino acids between the derivative Gly8 Arg26Lys34(N'-(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 diffe rent
`amino acid between !he derivative Lys26(N£•(7-
`dcoxycholoyl))Arg34-GLP-1(7-40) and the corresponding
`native GLP-1. TI1e difference is located al position 34.
`In a preferred embodiment, the present invention relates 15
`to a GLP-1 derivative wherein the parent peptide is GLP-1
`(1-45) or an analogue thereof. In a further preferred
`embodiment, the