`(10) Patent N0.:
`US 6,268,343 Bl
`
`Knudsen et al.
`(45) Date of Patent:
`Jul. 31, 2001
`
`U3006268343B1
`
`(54) DERIVATIVES 0F GLP-l ANALOGS
`
`(75)
`
`Inventors: Liselotte Bjerre Knudsen, Valby; Per
`Olaf Hyusffldt’ KV’bCHhaVH K} Per
`Franklin Nlelsen, Vaerlgase; Nlels C.
`
`Kaarsholm, Vanl¢sc; Helle Birk Olsen,
`Allerod; Saren Erik Bjarn, Lyngby;
`Freddy Zimmerdahl Pedersen; Kjeld
`Madsen, both of Vaerlsztse, all of (DK)'
`,
`.,
`.
`.
`(73) A551gnee. Now Nordisk A/S, 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) APP1~ NO-i 09/258,750
`'7
`.
`.
`(2‘) Flled'
`
`(51)
`
`Int. Cl.7 .......................... A61K 39/16; A61K 38/26;
`C07K 14/00; C07K 14/605
`............................................... 514/12; 530/324
`(52) US. Cl.
`(58) Field of Search ................................ 530/324; 514/12
`
`(56)
`’
`
`References Cited
`U.S. PATENT DOCUMENTS
`6/1992 Habener
`5,120,712
`..
`4/1990 Chen et al.
`5,512,549
`8/1996 Buckley et a1.
`5,545,618
`3/1997 Habener
`5’014’492
`FOREIGN PATENT DOCUMENTS
`
`..... 514/12
`. 514/12
`
`. 514/12
`""" 514/12
`
`,
`,
`0 708 179
`W0 90/11296
`W0 91/11457
`VVO 95/07931
`WO 95/31214
`WO 96/29342
`WO 9609344
`WO 87/06941
`“70 98/08531
`' 5
`$8 33823;:
`7
`V‘ 0 98/19698
`
`4/1990 (EP).
`10/1990 (W0) .
`8/1991 (W0) .
`3/1995 (W0) .
`11/1995 (W0) .
`9/1996 WO .
`9/1996 Ewog '
`11/1997 (W0) .
`3/1998 (W0)
`5
`-
`3/133: ($8) '
`_/
`(
`) ’
`3/1998 (W0) -
`
`OTHER PUBLICATIONS
`_
`_
`Klm Ct 31-; (1994) J- 0f Pharm a, 891011995 83(8);1175—1180-
`Clodfelter et al., (1998) Pharmaceutical Res. 15(2):254—262.
`
`1 B -
`- h
`.
`~
`Primal , F ,
`orin
`3 aammei—Mic ac
`(74) Attorney, Agent, 01‘Firm%teve T. Zelson, Esq.; Elias
`J. Lambiris, Esq.
`
`(57)
`
`ABSTRACT
`
`The present invention relates to GLP-l derivatives having a
`lipophilic substituent, pharmaceutical compositions com—
`prising same, and methods of making an using same. The
`‘
`GLP-l derivatives of the present invention have a protracted
`pmfile 0f “no”
`
`40 Claims, 1 Drawing Sheet
`
`FBI" 26’ 1999
`.
`.
`Related US. Application Data
`,
`,
`,
`,
`,
`,
`,
`(63) Continuation -in—part of application No. 09/038,432, filed on
`Mai: 11, 1998, now abandoned, which is a continiiation—in-
`part of application No. 08/918,810, filed on Aug. 26, 1997,
`110W abandoned, and a continuation-in-pa1't of application
`No. PCT/DK97/00340, filed 011 Aug. 22, 1997
`Provisional application No. 60/035,904, filed on Jan. 24,
`1997, provisional application No. 60/036,226, filed on Jan.
`25, 1997, provisional application No. 60/036,255, filed on
`Jan. 24, 1997, provisional application No. 60/082,478, filed
`011 Am 21, 1998, PI‘OViSiOIlill application N0. 60/082,480,
`filed on Apr. 21, 1998, provisional application No. 60/082,
`/
`802, filed on Apr. 23, 1998, and provisional application No.
`,
`,
`00‘084’357’ filed on May 5’ 1998'
`Foreign Application Priority Data
`
`
`.. 0931/96
`(DK) .....................
`1259/96
`(DK) .....................
`
`1470/96
`(DK) .....................
`0293/98
`(D1?)
`0204/:98
`(DIE)
`/
`3332/3:
`$113
`0274/98
`(DK)
`0508/98
`(DK)
`
`(DK) ........................................ 0509/98
`
`(60)
`
`(30)
`
`Aug. 30, 1996
`Nov. 8, 1996
`Dec. 20, 1996
`Feb- 27; 1998
`Feb~ 27, 1998
`e .
`,
`:63 3;? 133:
`Feb. 27, 1998
`Apr. 8, 1998
`Apr. 8, 1998
`
`
`
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`
`
`
`US. Patent
`
`Jul. 31, 2001
`
`US 6,268,343 B1
`
`Fig. 1
`
`
`
`
`_ y _. g
`
`_ . _ h
`
`0.1
`
`1
`
`10
`
`100
`
`1000
`
`[peptide] (11M)
`
`‘ V-
`
`glp-1(7-37)
`
`_- 0 - e
`
`— :1 — f
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1006, p. 2 of 138
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`
`
`US 6,268,343 B1
`
`1
`DERIVATIVES 0F GLP-l 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 US. 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
`
`invention relates to novel derivatives of
`The present
`human glucagon-like peptide-1 (GLP-1) and fragments and/
`or analogues thereof which have a protracted profile of
`action and to methods of making and using them.
`BACKGROUND OF THE INVENTION
`
`Peptides are widely used in medical practice, and since
`they can be produced by recombinant DNA 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 e ect, are the glucose-dependent
`insulinotropic
`
`15
`
`35
`
`40
`
`_
`
`55
`
`60
`
`65
`
`2
`(GLP-l).
`polypeptide, GIP, and glucagon-like peptide-l
`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
`33461 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 maior 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(7436)amide (PG(784107)amide (13), not as origi—
`nally believed PG (724107)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 (3469)) (17, 18). Of these peptides, GLP—l, 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-l 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 GIP—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 (2|, 22). The
`hormone interacts directly with the [3—eells via the GLP—1
`receptor (23) which belongs to the glucagon/VIP/calcitonin
`family of G-protein-couplecl- 7-transmenibrane spanning
`
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`US 6,268,343 B1
`
`15
`
`-
`
`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 Ca2+ 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 [3—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-l 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—l 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 o ‘ slightly supraphysiological doses of GLP-1
`
`may comple ely 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—l also lowers blood
`glucose in type-1 diabetic patients without residual [S-cell
`secretory capacity (34).
`In addition to its effects on the pancreatic islets, GLP-l
`has powerful actions on the gastrointestinal tract. Infused in
`physiological amounts GLP-l 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-l
`secretion is greatly stimulated, and it has been speculated
`that GLP—l 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 e ects on the
`pancreatic islets. Thus,
`in dose response studies GLP-l
`influences gastric emptying rate at infusion rates at least as
`low as those required to influence islet secretion (39).
`GLP-l seems to have an ellect 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—l (PG 724107)
`amide is inactive and appropriate doses of the GLP-l
`antagonist, exendin 9—39, abolish the effects of GLP-1 (41).
`
`35
`
`4O
`
`55
`
`60
`
`65
`
`4
`Acute, peripheral administration of GLP-1 does not inhibit
`food intake acutely in rats (41, 42). However,
`it remains
`possible that GLP-l secreted from the intestinal L-cells may
`
`
`also act as a satiety signal.
`
`
`
`
`
`
`Not only the insulinotropic e ects but also the e ects 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-l 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. (Diabetulugia 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 GIP-l
`(7—36)amide, GLP-l
`(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.
`including
`fragments,
`WO 87/06941 discloses GLP—l
`GLP-l
`(7—37), and functional derivatives thereof and to
`their use as an insulinotropic agent.
`including
`fragments,
`W0 90/11296 discloses GLP-l
`GLP—l
`(7—36), and functional derivatives thereof which
`have an insulinotropic activity which exceeds the insulino-
`tropic activity of GLP-1 (1—36) or GLP-l (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
`l3
`12
`ll
`10
`9
`8
`7
`His—Ala—Glu—Gly—Thr—Phe-Thr—Ser—Asp-Val—Ser—
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`Ser—Tyr—Leu—Glu—Gly—Gln-Ala—Ala—Lys-Glu—Phe—
`29
`30
`31
`32
`33
`34
`35
`36
`I1e—Ala—Trp—Leu—Val—Lys—Gly—Arg—X
`
`(I)
`
`wherein X is 112 for GLP-l (7—36) and X is Gly for GLP-l
`(7—37).
`WO 91/11457 discloses analogues of the active GLP—l
`peptides 7—34, 7—35, 7—36, and 7—37 which can also be
`useful as GLP-l moieties.
`
`EP 0708179-A2 (Eli Lilly & Co.) discloses GIP-l ana-
`logues and derivatives that include an N—terminal imidazole
`group and optionally an unbranched C6—Cm acyl group in
`attached to the lysine residue in position 34.
`
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`US 6,268,343 B1
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`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.
`'Ihus 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-l (7—37).
`It is a further object of the invention to provide a phar-
`maceutical composition with improved solubility and sta—
`bilitv.
`
`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
`with the C-terminal sequence (residues 33—69) of glicentin.
`Biochem J. 1980; 187:337—343,
`
`15
`
`18. Bataille D, Tatemoto K, Gespach C, Jornvall II,
`Rosselin G, Mutt V. Isolation of glucagon—37 (bioactive
`enteroglucagon/oxyntomodulin) from porcine jejuno—ileum.
`Characterisation of the peptide. FEBS Lett 1982;
`146279—86,
`
`1. Pederson RA. Gastric Inhibitory Polypeptide. In Walsh
`JH, Dockray GJ (eds) Gut peptides: Biochemistry and -
`Physiology. Raven Press, New York 1994, pp. 217259.
`2. Krarup T. Immunoreactive gastric inhibitory polypep-
`tide. Endocr Rev 1988;9z 122—134.
`3. Orskov C. Glucagon-like peptide-1, a new hormone of
`the enteroinsular axis. Diabetologia 1992; 35:7014711.
`4. Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian
`RC. Exon duplication and divergence in the human prepro-
`glncagon gene. Nature 1983; 304: 368—371.
`5. Holst JJ. Glucagon—like peptide—1 (GLP—1)ia newly
`discovered GI hormone. Gastroenterolgoy 1994; 107:
`1848—1855.
`
`3O
`
`.lJ. Gut glucagon, enteroglucagon, gut GLI,
`6. Holst
`glicentin—current status. Gastroenterology 1983;
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`
`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.
`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
`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; 2:1394151.
`13. Orskov C, Bersani M, Johnsen AH, Hojrup P, Holst 1].
`Complete sequences of glucagon-like peptide-1 (GLP-1)
`from human and pig small intestine. J. Biol. Chem. 1989,
`264:12826—12829.
`
`l4. Orskov C, Rabenhoj L, Kofod H, W'ettergren A, Holst
`JJ. Production and secretion of amidated and glycine—
`extended glucagon-like peptide-1 (GLP-1) in man. Diabetes
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`20. Elliott RM, Morgan LM, Tredger JA, Deacon S,
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`and 24—h secretion patterns. J Endocrinol 1993; 138:
`159—166.
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`21. Kolligs F, Fehmann HC, Goke R, Goke B. Reduction
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`22. Wang Z, Wang RM, ()wji AA, Smith DM, Ghatei M,
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`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, Goke R, Goke G. Cell and molecular
`biology of the incretin hormones glucagon-like peptide-I
`(GLP—1) and glucose—dependent insulin releasing polypep—
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`26. Gromada J, Dissing S, Bokvist K, Renstrom E,
`Frokjaer-Jensen J, Wulff BS, Rorsman P. Glucagon-like
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`bTC3-cells by enhancement of intracellular calcium mobili-
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`CAMP-regulated Ca2+-signaling pathway in pancreatic
`[S-cells by the insulinotropic hormone glucagon-like
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`28. Holz GG, Kiihltreiber WM, Habener JF, Pancreatic
`beta-cells are rendered glucose competent by the insulino-
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`1993; 361:362—365.
`29. Orskov C, Holst JJ, Nielsen 0V: Effect of truncated
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`endocrine secretion from pig pancreas, antrum and stomach.
`Endocrinology 1988; 123200942013.
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`Creutzfeldt W.
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`glucagon-like peptide-1 [7—36 amide] in the fasting state in
`healthy subjects. Acta Diabetologica, 1995; 32:13—16.
`32. Nauck MA, Heimesaat MM, Orskov C, Holst JJ, Ebert
`R, Creutzfeldt W. Preserved incretin activity of GLP-1
`(7—36amide) but not of synthetic human 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,
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`truncated GIP-l , fragments of human proglucagon, inhibit
`gastric acid secretion in man. Dig. Dis. Sci. 1989;
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`Christiansen J, Holst JJ. Truncated GLP-l (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 II: IIeaI release
`of glucagon-like peptide-l (GLP-l): association with inhi-
`bition of gastric acid in humans. Dig Dis Sci 1995; 40:
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`38. Layer P, Holst JJ. GLP—l: 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
`GIP-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
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`Obesity in Europe. John Libbey & Company ltd, 1994; pp.
`363—367.
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`41. Tang-Christensen M, Larsen PJ, Goke R, Fink-Jensen
`A, Jessop DS, Moller M, Sheikh S. Brian GLP—1(7436)
`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—l (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.
`Pharmacoldnetic, insulinotropic, and glucagonstatic proper-
`ties of GLP—1 [7436 amide] after subcutaneous injection in
`healthy volunteers. Dose-response relationships. Diabetolo-
`gia 1995; 38: 720—725.
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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:
`952—957.
`47. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L,
`Willms B, Holst JJ, 1995. Both subcutaneous and intrave-
`nously administered glucagon-Iike 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—l 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-l (7—37) and various GLP-l derivatives of the present
`invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`35
`
`4O
`
`55
`
`60
`
`65
`
`A simple system is used to describe fragments and ana—
`logues of GLP—1. For example, GlysiGLP—l(7i37) desig—
`nates a peptide which relates to GI .P-l 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(N‘-tetradecanoyl)-GLP-1(7—37) des-
`ignates GLP—l (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-l 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-l 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-l derivative and the corre-
`sponding native form of GLP-1 is up to fifteen, preferably 11p
`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.
`
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`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-l derivative of the present invention with the corre-
`sponding native GLP—l. For example, there are two di erent
`amino acids between the derivative GlygArg26Lys34(\I‘—(7—
`deoxycholoyl)-GIP-l (7—40) and the corresponding native
`GLP-l (i.e., GLP-1(7—40)). The differences are located at
`positions 8 and 26. Similarly, there is only one di erent
`amino acid between the derivative Lys25(N‘-(7-
`deoxycholoyl))Arg34—GLP—1(7—40) and the corresponding
`native GLP—l. The difference is located at position 34.
`In a preferred embodiment, the present invention relates
`to a GLP—l derivative wherein the parent peptide is GLP—l
`(1—45) or an analogue thereof.
`In a further preferred
`embodiment,
`the parent peptide is GLP-1(1—35), GLP-l
`(1—36), GLP—1(1—36)amide, GLP—1(1—37), GLP-1(1—38),
`GLP—l(li39), GLP—l(140), GLP—l(li4l) or an analogue ~
`thereof.
`In a preferred embodiment, the present invention relates
`to derivatives of GLP-1 analogues of formula I (SEQ ID
`N02):
`
`15
`
`17
`16
`15
`14
`13
`12
`11
`10
`9
`8
`7
`Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—
`
`(I)
`
`28
`27
`26
`25
`24
`23
`22
`21
`20
`19
`18
`Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Phe—
`
`38
`37
`36
`35
`34
`33
`32
`31
`3O
`29
`Ile—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa
`
`45
`44
`43
`42
`41
`4O
`39
`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 position 11 is Thr, Ala, Gly, Ser, Len, Ile, Val, Glu,
`Asp, or Lys or is deleted,
`Xaa at position 12 is Phe or is deleted,
`Xaa at position 13 is Thr or is deleted,
`Xaa at aosition I4 is Ser, Ala, Gly, Thr, Len, Ile, Val, Glu,
`Asp, or Lys or is deleted,
`Xaa at position 15 is Asp or is deleted,
`Xaa at osition 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Glu,
`Asp, or Lys or is deleted,
`Xaa at osition 17 is Ser, Ala, Gly, Thr, Len, Ile, Val, Glu,
`Asp, or Lys, or is deleted,
`Xaa at aosition 18 is Ser, Ala, Gly, Thr, Len, Ile, Val, Glu,
`Asp, or Lys,
`Xaa at position 19 is 'I'yr, I’he, Trp, Glu, Asp, or Lys,
`Xaa at osition 20 is Leu, Ala, Gly, Ser, Thr, Leu, Ile, Val,
`Glu, Asp, or Lys,
`Xaa at posit