(12) United States Patent
`Knudsen et al.
`
`I 1111111111111111 11111 111111111111111 1111111111 1111111111111111 IIII IIII IIII
`US006458924B2
`US 6,458,924 B2
`*Oct. 1, 2002
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) DERIVATIVES OF GLP-1 ANALOGS
`
`(75)
`
`Inventors: Llselotte Bjerre Knudsen, Valby (DK);
`Pei· Olaf Huusfeldt, K0beohavn K
`(DK); Per Franklin Nielsen, Vrerl0se
`(DK)
`
`(73) Assignee: No,,o Nordis k 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 O days.
`
`This patent is subject to a terminal dis(cid:173)
`claimer.
`
`(21) Appl. No.: 09/398,111
`
`(22) Filed:
`
`Sep. 16, 1999
`
`Related U.S. Application Data
`
`(63)
`
`(60)
`
`(30)
`
`Continuation-in-part of application No. 09/265,141, filed on
`Mar. 8, 1999, now Pat. No. 6,384,016, and a continuation(cid:173)
`in-parl of applicalion No. 09/258,750, filed on Feb. 26,
`1999, now Pat. No. 6,268,343, which is a continuation-io(cid:173)
`part of application No. 09/038,432, filed on Mar. 11, 1998,
`now abandoned, which is a continuation-in-part of applica•
`tion No. 08/918,810, filed as application No. PCT/DK.97/
`00340 on Aug. 22, 1997, now abandoned.
`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. 60/078,422, filed
`on Mar. 18, 1998, p rovisional application No. 60/082,478,
`filed on Apr. 21, 1998, provisional application No. 60/082,
`479, 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
`
`Aug. 30, 1996
`Nov. 8, 1996
`Dec. 20, 1996
`Feb. 27, 1998
`Feb. 27, 1998
`Feb. 27, 1998
`Feb. 27, 1998
`Feb. 27, 1998
`.Mar. 13, 1998
`Apr. 8, 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
`(EP) ............................................ 98610006
`(DK) .............................................. 0508/98
`(DK) .............................................. 0509/98
`(DK) ........................................ 1998 00507
`
`(51) Int. C l.7 ......................... A61K 38/16; A61K 38/26
`(52) U.S. Cl .
`........................... 530/324; 530/345; 514/2;
`514/12
`(58) Field of Search ....................... 514/2, 12; 530/324,
`530/345
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`5,118,666 A
`5,120,712 A
`5,380,872 A
`5,512,549 A
`5,545,618 A
`
`6/1992 Habener ...................... 514/12
`6/1992 Habener ...................... 514/12
`1/1995 Sugg el al. . ................ 548/498
`4/1996 Chen et al. ................... 514/12
`8/1996 Buckley et al. ............... 514/12
`
`3/1997 Habener ...................... 514/ 12
`5,614,492 A
`5,869,602 A • 2/1999 Jonassen ..................... 530/308
`5,912,229 A
`6/1999 Thim et al. ................... 514/ 12
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`GB
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`0 619 322 A2
`0 658 568
`0 708 179
`1202607
`WO 87/06941
`WO 90/11296
`WO 91/11457
`WO 95/07931
`WO 95/31214
`WO 95/32730
`96/29342
`WO 96/29342
`WO 96/29344
`WO 97/31943
`WO 98/08871
`98/08871
`WO 98/08873
`WO 98/08531
`WO 98/19698
`
`10/1994
`6/1995
`4/1996
`8/1970
`11/1987
`10/1990
`8/1991
`3/1995
`11/1995
`12/1995
`• 9/1996
`9/1996
`9/ 1996
`9/1997
`3/ 1998
`• 3/1998
`3/ 1998
`5/1998
`5/1998
`
`OTHER PUBLICMlONS
`
`Broderick, Diabetologia (1995) vol. 38, No. Suppl. 1, pp.
`Al 71. Meeting Info.: 31st Annual Meeting of the European
`Association for the Study of Diabetes Stockholm, Sweden
`Sep. 12-16, 1995.*
`M. Gutniak et al., Antidiabetogenic Effect of Glucagoo-Like
`Peptide (7- 36) Amide in Normal Subjects and PalienLs with
`Diabetes with Diabetes Mellitus.
`M. Navarro et al., Changes in Food Intake Induced by
`GLP- 1(7- 36) Amide In the Rat, Abstracts of the 15'"
`International Diabetes Federation Congress, Nov. 6- 11,
`1194 Kobe, poster presentation 11A 5PP1295 Issued 1994.
`R. Schick et al., "Glucagon-like peptide 1-a novel brain
`peptide Involved in feeding regulation "Obesity in Europe
`1993, Chapter 53, pp. 363-367.
`P.O. Lambert et al., " A Role for GLP- 1(7- 36)NH2 in the
`Central Control Of Feeding Behavior" Digestion 1994; vol.
`54. pp. 360- 361.
`B. Willms et al., "Gastric Emptying, Glucose Responses,
`and Insulin Secretion after a Liquid Test Meal:Effects of
`Exogenous Glucagon- Like Peptide- !
`(GLP- 1) (7- 36)
`Amide
`in Type 2
`(Noninsulin- Dependent) Diabetic
`Patients" Journal of Clinical Endocrinology and Metabolism
`vol. 8 No. 1 (1996) pp. 327-332.
`
`(List continued on next page.)
`
`Primary Examiner-Christopher S . F. Low
`Assistant Examiner-David Lukton
`(74) Attorney, Agent, or Firm-Reza Green, Esq.; Richard
`Bork, Esq.
`
`(57)
`
`ABSTRACT
`
`The present invention relates to a pharmaceutical composi(cid:173)
`tion comprising a GLP-1 derivative having a liJXlpbiJic
`substituent; and a surfactant.
`
`20 Claims, 1 Drawing Sheet
`
`FRESENIUS EXHIBIT 1020
`Page 1 of 129
`
`

`

`US 6,458,924 B2
`Page 2
`
`OTI-IER PUBLICATIONS
`
`M. Tang-Christensen et al., "Central Administration of
`GLP- 1 (7- 36) Amide Inhibits Food and Water Intake in
`(1996)
`Rats", The American Physiologic.al Society
`271:R848- R856.
`Zhili Wang et al., "Glucagon- like Peptide- ! ls a Physiologi(cid:173)
`cal Incretin in Rat" J. Clio. Invest. The American Society for
`Clio vol. 95. pp. 417- 421, Jan. (1995).
`C. 0rskov, "Glucagon- like peptide- !, a new hormone of the
`entero-insular Axis" Diabetologia vol. 35:pp. 701- 711
`(1992).
`Gutzwiller et al., Abstract "Glucagon- like peptide- ! is a
`physiologic regulator of food intake in human" Gastroen(cid:173)
`terology ( 1997) vol. 12 (4, Supp.S):PA1153.
`in
`Ranganath et al., "Attenuated GLP- 1 secretion
`obesity:cause or con-Sequence" Gur vol. 38: pp. 916- 919
`(1996).
`Marx J. "Obesity gene discover may help solve weighty
`problem" [news). Science, (Dec. 2, 1994) 266 (5190) pp.
`1477- 1478.
`Zhang et al. "Positional cloning of the mouse obese gene and
`its human Homologue". Nature, (Dec. 1, 1994) 372 (6505)
`pp. 425-432.
`Rink T.J., "In search of a satiety factor''. Nature, (Dec. 1,
`1994) 372 (6505) pp. 406-407.
`Woods et al., "Signals that regulate food intake and energy
`Homeostasis". Science, 280:1378--1383, May 29, 1998.
`Thorens T. "Glucagoo--like peptide-1 and control of insulin
`secretion". Diabete & Metabol.isme (Paris). 1995, 21, pp.
`311- 318.
`Henriksen et al. Peptide amidation by chemical protein
`engineering A combination of encymic and photochemical
`synthesis. J. AM Chem. Soc. (1992), 114 (5), pp.
`1876-1877.
`Wang et al. "Glucagon- like peptide- ! is a physiological
`incretin in rat". J. Clio. Invest., (Jan 1995) (1) 417- 21.
`Bell et al. "Exon duplication and divergence in the human
`Preproglucagon gene''. Nature, (Jul. 28-Aug. 3, 1983).
`Wettergren et
`al.
`"Truncated GLP- 1
`(proglucagon
`78- 107- amide) inhibits gastric and pancreatic functions in
`man". Digest. Dis. Sci., (Apr. 1993) 38 (4) 665- 73.
`Suzuki et al. "Comparison of the effects of various C- ter(cid:173)
`minal and N- terminal fragment peptides of glucagons-like
`peptide-I on insulin and glucagons release from the isolat(cid:173)
`edcx perfused rat pancreas". Endocrinology, (Dec. 1989)
`125(6) 3109- 14.
`
`Navarro et al., Journal of Neurochemistry, vol. 67, No. 5, pp.
`1982- 1991 (Nov. 1996).
`Turton et al., Nature, vol. 379, pp. 69- 72 (Jan. 4, 1996).
`Kirn et al., (1994) J . of Pharma. Sciences 83(8):1175- 1180.
`Clodfelter et al., (1998) Pharmaceutical Res. 15(2):254- 262.
`W.B. Gratzer et al., "Relation Between Conformation and
`Association State" The Journal of Biological Chemistry,
`244, No. 24, Dec. 25, 1969, pp. 6675- 6679.
`Sasaki et al., "X- Ray Analysis of Glucagon and Its Rela(cid:173)
`tionship to Receptor Binding", Nature Vo. 257, Oct. 30,
`1975, pp. 751- 757.
`Wagman et al., "Proton NMR Studies Of Toe Association
`And Folding of Glucagon In Solution", Elsevier/North- Hol(cid:173)
`land Biomedical Press, vol. 119, No. 2, Oct. 1980, pp.
`265- 270.
`Epand et al., " Molecular Interactions In The Model Lipo(cid:173)
`protein Complex Formed Between Glucagon and Dimyris(cid:173)
`toylglycerophosphocholine", Biochemistry vol. 16, No. 20,
`1977.
`Schneider et al., " Polypeptide Hormone Interaction" (Glu(cid:173)
`cagon Binding To Lysolecithin), The Journal of Biological
`Chemistry, Vo. 247, No. 16, Aug. 25, 1972, pp. 4986-4991.
`Schneider et al., "Polypeptide Hormone Interaction" (Con(cid:173)
`formational Changes of Glucagon Bound To Lysolecithin),
`The Journal of Biological Chemistry, Vo. 247, No. 16, Aug.
`25, 1972, pp. 4992-4995.
`Robinson et al., " Lipid- Induced Conformational Changes in
`Glucagon, Secretin, and Vasoactive Intestinal Peptide",
`Biopolymers, vol. 21, 1982, pp. 1217- 1228.
`Hamed et al., "Bahavior of Ampbipathic Helices on Analysis
`Via Matrix Methods, With Application to Glucagon, Secre(cid:173)
`tin, and Vasoactive Intestinal Peptide", Biopolymers, vol.
`22, 1983, pp. 1003- 1021.
`Wu et al., "Helical Conformation of Glucagon in Surfactant
`Solutions", America! Chemical Society, 1980, pp.
`2117- 2122.
`Bosch et al., " Physicochemical Characterization of Glu(cid:173)
`cagoo--Containing Lipid Micelles" Biochimic et Biophysica
`Acta, 603 (1980) pp. 298- 312.
`Thornton et al., Structure of Glucagon- Like Peptide(7- 36)
`Amide in a Dodecylphosphocholine Micelle as Determined
`by 20 NMR, Biochemistry 1994, 33, pp. 3532- 3539.
`
`* cited by examiner
`
`FRESENIUS EXHIBIT 1020
`Page 2 of 129
`
`

`

`U.S. Patent
`
`Oct. 1, 2002
`
`US 6,458,924 B2
`
`0
`
`-+- a
`
`-- o -- b
`
`- 1 1--- - - - - - - - - - -+ - - --l -·- • -- C
`
`-2
`
`-4
`
`- 5
`0. 1
`
`-v' - gip- 1(7- 37}
`
`-◊ - e
`
`- □- -
`
`f
`
`--•-- g
`
`-- ■ -- h
`1000
`
`1
`
`10
`
`100
`
`{peptide} {µM)
`
`FIG. 1
`
`FRESENIUS EXHIBIT 1020
`Page 3 of 129
`
`

`

`US 6,458,924 B2
`
`1
`DERIVATIVES OF GLP-1 ANAWGS
`
`CROSS-REFERENCE TO RELAfED
`APPLICATIONS
`
`This application is a continuation-in-part of Ser. No.
`09/265,141 filed Mar. 8, 1999 now U.S . Pat. No. 6,384,016
`and of Ser. No. 09/258,750 filed Feb. 26, 1999 now U.S . Pat.
`No. 6,268,343 which is a cootinuatioo-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, which is a 371 and of PCT application
`Ser. 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, 60/078,422, 60/082,478,
`60/082,479, 60/082,480, 60/082,802, and 60/084,357 filed
`Jan. 24, 1997, Ja n. 25, 1997, Jan. 24, 1997, Mar. 18, 1998,
`Apr. 2 1, 1998, Apr. 2 1, 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, 0507/98, 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, Apr. 8, 1998 and Apr. 8, 1998,
`respectively, and of European application no. 98610006.3
`filed Mar. 13, 1998, the contents of each of which is fully 25
`incorporated herein by reference.
`
`30
`
`2
`so-called incretin effect, is probably essential for a normal
`glucose tolerance. Many of the gastrointestinal hormones,
`including gastrin and secretin (cholecystokinin is not insuli(cid:173)
`notropic in man), are insulinotropic, but the only physiologi-
`5 cally important ones, those that are responsible for the
`incretin effect, are the glucose-dependent insulinotropic
`polypeptide, GIP, and glucagon-like peptide-l (GLP-1).
`Because of its insulinotropic effect, GIP, isolated in 1973 (1)
`immediately attracted considerable interest among diabe-
`10 tologists. However, numerous investigations carried out
`during 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
`15 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
`20 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(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-
`35 creatic peptide, GRPP (10, 11); 3) a hexapeptide correspond(cid:173)
`ing to PG (64-69); 4) and, finally, the so-called major
`proglucagon fragment (PG (72- 158)) , in which the two
`glucagon-like sequences are buried (9) . Glucagon seems Lo
`be the only biologically active product. In contrast, in the
`intestinal mucosa, it is gJucagon 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 originally believed PG (72- 107) amide or 108,
`which is inactive). Small amounts of C-terminally glycine(cid:173)
`extended but equally bioactive GLP-1(7- 37), (PG (78- 108))
`are also formed (14) ; 3) in tervening peptide-2 (PG
`(111- 122)amide) (15) ; and 4) GLP-2 (PG (126- 158)) (15,
`16). A fraction of glicentin is cleaved further into GRPP (PG
`(1- 30)) and oxyntoroodulin (PG (33--69)) (17, 18). Of these
`peptides, GLP-1, has the most conspicuous biological activi(cid:173)
`ties.
`Being secreted in parallel with glicentin/enteroglucagon,
`it follows that the many studies of enteroglucagon secretion
`(6, 7) to some extent al'>◊ apply to GLP-1 secretion, but
`GLP-1 is metabolised more quickly with a plasma half-life
`in humans of 2 min (19). Carbohydrate or fat-rich mea15
`stimulate secretion (20) , presumably as a result of direct
`interaction of yet unabsorbed nutrients with the rnicrovilli of
`the open-type L-cel15 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 reduce.5 the
`
`FIELD OF THE INVENTION
`
`The present invention relates to novel derivatives of
`human g)ucagon-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 tLsed io therapy it is generally found that they 40
`have a high clearance. A high clearance of a therapeutic
`agent is inconvenient in cases where it is desired lo 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: 45
`ACTH, corticotropin-releasing factor, ang iotensin,
`calcitonin, insulin, glucagon, glucago n-like peptide-I,
`glucago n-like peptide-2, insulin-like growth factor-I,
`insulin-like growth factor-2, gastric inhibitory peptide,
`growth hormo ne-releasing factor, pituitary adenylate 50
`cyclase activating peptide, secretin, enterogastrin,
`somatostatin, somatotropin, somatomedin, parathyroid
`hormone, thrombopoietin, erythropoietin, hypothalamic
`releasing factors, prolactin, thyroid stimulating hormones,
`endorphins, enkephalins, vasopressin, oxytocin, opiods and 55
`analogues thereof, superoxide d ismutase, interferon,
`asparaginase, arginase, arginine deaminase, adenosine
`dearninase and ribonuclease. In some case.s it is possible to
`influence the release profile of peptides by applying suitable
`pharmaceutical compositions, but this approach bas various 60
`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
`re.spouse to the presence and absorption of nutrients in the 65
`gut, which promote an early and potentiated release of
`insulin. The enhancing effect on insulin secretion, the
`
`FRESENIUS EXHIBIT 1020
`Page 4 of 129
`
`

`

`US 6,458,924 B2
`
`4
`specific. Thus, N-terrninally extended GLP-l(PG 72- 107)
`amide is inactive and appropriate doses of the GLP-1
`antagonist, exendin 9- 39, abolish the effects of GLP-1(41).
`Acute, peripheral administration of GLP-1 does not inhibit
`s 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 ia.sulinotropic effects but also the effects of
`GLP-1 on the gastrointestinal tract are preserved in diabetic
`10 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 bas been demonstrated to
`dramatically improve glycaemic control in NIDDM patients
`15 without significant side effects ( 44). The peptide is fully
`active after subcutaneous admioistratioo ( 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. (Diaberologia 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 has been described
`by Thorton et al. (Biochemistry 33 3532- 3539 (1994)), but
`in normal solution, GLP-1 is considered a very flexible
`molecule. Surprisingly, we found that derivatisation of this
`relatively small and very flexible molecule resulted in com-
`pounds whose plasma profile were highly protracted and still
`bad 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 insu Ii notropic agent.
`WO 90/11296 discloses GLP-1 fragments, including
`GLP-1(7-36), and functional derivatives thereof which have
`an insulinotropic activity which exceeds the insulinotropic
`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):
`
`3
`incretin effect elicited by oral glucose in rats (21, 22). The
`hormone interacts directly with the Bcells via the GLP-1
`receptor (23) which belongs to the glucagonNIP/calcitonin
`family of G-protein-coupled 7-transmembrane spanning
`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
`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
`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 competence" to 20
`isolated insulin-secreting cells (26, 28), which respond
`poorly to glucose or GLP-1 alone, but fully to a combination
`of the two, is also not known. Equally importantly, however,
`the hormone also potently inhibits glucagon secretion (29).
`The mechanism is not known, but seems to be paracrine, via 25
`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(cid:173)
`tions increase either by increased secretion or by exogenous 30
`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 35
`glucagonostatic actions, the glucose lowering effect is self(cid:173)
`limiting, and the hormone, therefore, does not catLse
`hypoglycaemia regardless of dose (31) . The effects are
`preserved in patients with diabetes mellitus (32), in whom
`infusions of slightly supra physiological doses of GLP-1 may 40
`completely normalise blood glucose values in spite of poor
`metabolic control and secondary failure to sulphonylurea
`(33) . The importance of the glucagonostatic effect is illus(cid:173)
`trated by the finding that GLP-1 also Jowers blood glucose
`in type-i diabetic patients without residual ~-cell secretory 45
`capacity (34).
`In addition lo its effects on the pancreatic islets, GLP-1
`has powerful actions on the gastrointestinal tract. Infused in
`physiological amounts, GLP-1 potently inhibits
`pentagastrin-induced as well as meal-induced gastric acid 50
`secretion (35, 36). It aLso inhibiLs gastric emptying rate and
`pancreatic enzyme secretion (36). Similar inhibitory effects
`on gastric and pancreatic secretion and motility may be
`elicited in humans upon perfusion of the ileum with
`carbohydrate- or lipid-containing solutions (37, 38).
`Concomitantly, GLP-1 secretion is greatly stimulated, and it
`bas 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(cid:173)
`brake effects of GLP-1 may be more important than its 60
`effects on the pancreatic islets. Thus, in dose response
`studies GLP-1 influences gastric emptying rate at infusion
`rates at least as low as those required to influence islet
`secretion (39).
`GLP-1 seems to have an effect on food intake. Intraven- 65
`tricular administration of GLP-1 profoundly inhibits food
`intake in rats (40, 42). This effect seems to be highly
`
`15 16 17
`14
`10 11 12 13
`9
`8
`7
`Hi s-Ala-Gl u-G l y-Thr-Phe-Thr-Ser-Asp-Va l -Ser-
`
`(I)
`
`26 27 28
`25
`19 20 2 1 22 23 24
`18
`55 Ser -Tyr - Leu-G l u-Gly-G l n-Al a-Al a-Lys-G lu -Phe-
`
`29 30
`36
`31 32 33 34 35
`Ile-Ala-Tr p-Leu-Val-Lys-Gl y-Arg-X
`wherein X is NHz for GLP-1(7- 36) and X is Gly for
`GLP-1(7-37).
`WO 91/11457 discloses analogues of the active GLP-1
`peptides 7- 34, 7- 35, 7- 36, and 7- 37 which can also be
`useful as GLP-1 moieties.
`EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-1 ana(cid:173)
`logues and derivatives that include an N-terminal irnidazole
`group and optionally an unbranched C6- C10 acyl group in
`attached to the lysine residue in position 34.
`
`FRESENIUS EXHIBIT 1020
`Page 5 of 129
`
`

`

`US 6,458,924 B2
`
`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- 5
`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.
`
`10
`
`15
`
`References
`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. Kramp T. Immunoreactive gastric inhibitory polypeptide. 20
`Endocr Rev 1988; 9: 122- 134.
`3. 0 rskov C. Glucagon-like peptide-I, 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- 25
`glucagon gene. Nature 1983; 304: 368- 371.
`S. Holst JJ. Glucagon-like peptide-I (GLP-1}-a newly
`discovered GI hormone. Gastroenterology 1994; 107:
`1848-1855.
`6. Holst JJ. Gut glucagon, enteroglucagon, gut GLI, 30
`glicentin- current status. Gastroenterology 1983;
`84:1602- 1613.
`7. Hoist JJ, 0 rskov C. Glucagon and other proglucagon(cid:173)
`derived peptides. In Walsh JH, Dockray GJ, eds. Gut
`peptides: Biochemistry and Physiology. Raven Pres.s, 35
`Ne,vYork,pp. 305- 340, 1993.
`8. 0rskov C, Hoist JJ, Knuhtsen S, Baldissera FGA, Poulsen
`SS, Nielsen OV. Glucagon-li.ke peptides GLP-1 and GLP-
`2, predicted products of the glucagon gene, are secreted
`separately from the pig small intestine, but not pancreas. 40
`Endocrinology 1986; 119:1467- 1475.
`9. Hoist JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B,
`0 rskov C. Proglucagon processing in porcine and human
`pancreas. J Biol Chem, 1994; 269: 18827- 1883.
`10. Moody AJ, Hoist JJ, Thim L, Jensen SL. Relationship of 45
`glicentin to proglucagon and glucagon in the porcine
`pancreas. Nature 1981; 289: S14-S16.
`11. Thim L, Moody AJ, Purification and chemical charac(cid:173)
`teri.sation of a glicentin-related pancreatic peptide
`(proglucagon fragment) from porcine pancreas. Biochim 50
`Biophys Acta 1982; 703:134-141.
`12. Thim L, Moody AJ. The primary structure of glicentin
`(proglucagon) . Regul Pept 1981; 2:139- 151.
`13. 0rskov C, Bersani M, Johnsen AH, H12>jrup P, Holst JJ.
`Complete sequences of glucagon-like peptide-! (GLP-1) 55
`from human and pig small intestine. J . Biol. Chem. 1989;
`264:12826-12829.
`14. 0rskov C, Rabenh0j L, Kofod H, Wettergren A, Holst JJ.
`Production and secretion of amidated and glycine(cid:173)
`extended glucagon-like peptide-I (GLP-1) in man. Dia- 60
`betes 1991; 43: 535- 539.
`15. Buhl T, Thim L, Kofod H, 0rskov 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. 0 rskov C, Buhl T, Rabenh!<lj L, Kofod H, Holst JJ:
`Carboxypeptidase-B-like processing of the C-terminus of
`
`65
`
`6
`glucagon-like peptide-2 in pig and human small intestine.
`FEBS letters, 1989; 247:193-106.
`17. Holst JJ. Evidence that enteroglucagon (II) is identical
`with the C-terminal sequence (residues 33-<i9) of glicen(cid:173)
`tin. Biochem J . 1980; 187:337- 343.
`18. Bataille D, Tatemoto K, Gespach C, Jornvall H, Rosselin
`G, Mutt V. Isolation of glucagon-37 (bioactive
`enteroglucagon/oxyntomodulin) from porcine jejuno(cid:173)
`ileum. Characterisation of the peptide. FEBS Lett 1982;
`146:79-86.
`19. 0rskov C, We11ergren 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-I (7- 36)amide and
`glucose-dependent insulinotropic polypeptide secretion in
`response to nutrient ingestion in man: acute post-prandial
`and 24-h secretion patterns. J Endocrinol 1993; 138:
`159- 166.
`21. Kolligs F, Fehmann HC, Goke R, Goke B. Reduction of
`the incretin effect in rats by the glucagon-like peptide-I
`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
`incretin in rat. J. Clio. Invest. 1995; 95: 417-421.
`23. Thorens B. Expression cloning of the pancreatic b cell
`receptor for the gluco-incretin Is hormone glucagon-like
`peptide 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 B. Cell and molecular
`biology of the incretin hormones glucagon-like peptide-I
`(GLP-1) and glucose-dependent insulin releasing
`polypeptide ( GIP). Endocrine Reviews, 1995; 16:
`390-410.
`26. Gromada J, Dissing S , BoJ..."Vist K, Renstrom E, Fr0jaer(cid:173)
`Jensen J, Wulff BS, Rorsman P. Glucagon-like peptide I
`increases cytoplasmic calcium in insulin-secreting bTC3-
`cells by enhancement of intracellular calcium mobilisa-
`tion. 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 insuli(cid:173)
`notropic hormone glucagon-like peptide-1(7- 37). Nature
`1993; 361:362- 365.
`29. 0rskov C, Holst JJ, Nielsen OV: Effect of truncated
`glucagon-like peptide-] (proglucagon 78- 107 amide) on
`endocrine secretion from pig pancreas, antrum and stom(cid:173)
`ach. Endocrinology 1988; 123:2009- 2013.
`30. Hvidberg A, Toft Nielsen M, Hilsted J, 0 rskov C, Hoist
`JJ. Effect of glucagon-like peptide-I (proglucagon
`78- 107 amide) on hepatic glucose production in healthy
`man. Metabolism 1994; 43:104-108.
`31. Qualmann C, Nauck M, Hoist JJ, 0rskov C, Creutzfeldt
`W. lnsulinotropic actions of intravenous glucagon-like
`peptide-1 [7- 36 amide] in the fast ing state in healthy
`subjects. Acta Diabetologica, 1995; 32: 13- 16.
`32. Nauck MA, Heimesaat MM, 0 rskov C, Hoist JJ, Ebert
`R, Creutzfeldt W. Preserved incretin activity of GLP-1
`(7- 36 amide) but not of synthetic human GIP in patients
`with type 2-diabetes mellitus. J Clio Invest 1993;
`91:30 1- 307.
`
`FRESENIUS EXHIBIT 1020
`Page 6 of 129
`
`

`

`US 6,458,924 B2
`
`7
`33. Nauck MA, Kleine N, 0rskov C, Hoist JJ, Willms B,
`Creutzfeldt W. Normalisation of fasting hyperglycaemia
`by exogenous GLP-1(7- 36 amide) in type 2-diabetic
`patients. Diabetologia 1993; 36:741- 744.
`34. Creutzfeldt W, Kleine N, Willms B, 0rskov C, Hoist JJ, 5
`Nauck MA Glucagonostatic actions and reduction of
`fasting hyperglycaemia by exogenous glucagon-liem,
`peptide-1 (7- 36 amide) in type I diabetic patients. Dia(cid:173)
`betes Care 1996; 19: 580-586.
`35. Schjoldager BTG, Morteuseo PE, Christianseo J, 0rskov
`C, Hoist JJ. GLP-1 (glucagon-like peptide-1) and trun- 10
`cated GLP-1, fragments of human proglucagon, inhibit
`gast