`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Clasmfication 6 :
`A61K 38126, C07K 14/605
`
`(11) International Publication Number:
`
`WO 99/43341
`
`Al
`
`(43) International Publication Date:
`
`2 September 1999 (02.09.99)
`
`(21) International Application Number:
`
`PCT/DK99/00084
`
`(22) International Filing Date:
`
`25 February 1999 (25.02.99)
`
`(30) Priority Data:
`0268/98
`0272/98
`
`27 February 1998 (27.02.98)
`27 February 1998 (27.02.98)
`
`DK
`DK
`
`(71) Applicant: NOVO NORDISK A/S [DK/DK]; Novo Alie,
`DK- 2880 Bagsvaerd (DK).
`
`(72) Inventors: KNUDSEN, Liselotte, Bjerre; Valby Langgade
`49A, 1.
`tv., DK- 2500 Valby (DK). HUUSFELDT, Per,
`Olaf; Applebys Plads 27, 5. mf., DK-141 I Copenhagen K
`(DK). NIELSEN, Per, Franklin; Dals0 Park 59, DK-3500
`Vrerl0se (DK). KAARSHOLM, Niels, C.; Clausholmvej 38,
`DK-2720 Vanl0se (DK). OLSEN, Helle, Birk; Skolelodden
`23, DK-3450 Aller0d (DK). BJ0RN, S0ren, Erik; Marie
`Grubbes Alie 47, DK-2800 Lyngby (DK).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES. Fl, GB, GD,
`GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP,
`KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, lR, TT, UA, UG, UZ, VN, YU, ZW,
`ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
`ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European patent (AT, BE, CH, CY, DE, DK, ES, Fl,
`FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: GLP-1 DERIVATIVES WITH HELIX-CONTENT EXCEEDING 25 %, FORMING PARTIALLY SlRUCTURED MICEL(cid:173)
`LAR- LIKE AGGREGATES
`
`(57) Abstract
`
`The present invention relates to a pharmaceutical composition comprising a GLP-1 derivative of improved solubility and/or stability,
`and to a method for improving the solubility and/or stability of GLP- 1 or a fragment or an analogue thereof.
`
`FRESENIUS EXHIBIT 1038
`Page 1 of 63
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`
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`FOR THE PURPOSES OF INFOR.MATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`Cl
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Annenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Henegovina
`Brutados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COie d'Tvoire
`Can,eroon
`China
`Cuba
`Cuch Republic
`Germany
`Denmark
`Estonia
`
`ES
`Fl
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`Uni1ed Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Tsrael
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kaz.akslan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`Lesotho
`LS
`LT
`Lithuania
`Luxembourg
`LU
`Latvia
`LV
`Monaco
`MC
`MD
`Republic of Moldova
`MG Madagascar
`MK
`'The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`SI
`SK
`SN
`sz
`T D
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turlcmenislan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United Slates of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
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`FRESENIUS EXHIBIT 1038
`Page 2 of 63
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`W099/43341
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`PCT/DK99/00084
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`GLP-1 DERIVATIVES wrrn HELIX-CONTENT EXCEEDING 25 %, FORMING PARTIALLY STRUCTIJRED MICEL(cid:173)
`LAR- LIKE AGGREGATES
`
`Field of the invention
`
`5
`
`The present invention relates to a pharmaceutical composition comprising a GLP-1 derivative
`
`of improved solubility and/or stability, and to a method for improving the solubility and/or stabi(cid:173)
`
`lity of GLP-1 or a fragment or an analogue thereof.
`
`Background of the invention
`
`10
`
`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 co(cid:173)
`me.
`
`15
`
`The hormones regulating insulin secretion belong to the so-called enteroinsular axis, desig-
`nating 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 re(cid:173)
`
`20
`
`25
`
`lease 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 (cholecystokinin is not insulinotropic in man), are insulinotro-
`pie, but the only physiologically important ones, those that are responsible for the incretin
`
`effect, are the glucose-dependent insulinotropic polypeptide, GIP, and glucagon-like peptide-
`1 (GLP-1). Because of its insulinotropic effect, GIP, isolated in 1973 (1) immediately attrac(cid:173)
`
`ted considerable interest among diabetologists. However, numerous investigations carried
`out during the following years clearly indicated that a defective secretion of GIP was not in-
`volved in the pathogenesis of insulin dependent diabetes mellitus (IDDM) or non insulin(cid:173)
`
`dependent diabetes mellitus (NIDDM) (2). Furthermore, as an insulinotropic hormone, GIP
`
`was found to be almost 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 sti(cid:173)
`
`mulating insulin secretion in NIDDM patients. In addition, and in contrast to the other insuli-
`notropic hormones (perhaps with the exception of secretin) it also potently inhibits glucagon
`
`30
`
`secretion. Because of these actions it has pronounced blood glucose lowering effects parti(cid:173)
`cularly 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
`
`35
`
`function in glucose metabolism and gastrointestinal secretion and metabolism (5). The glu-
`
`FRESENIUS EXHIBIT 1038
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`2
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`cagon 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 pancreatic peptide, GRPP (10, 11); 3) a hexapeptide corre-
`
`5
`
`sponding 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 to be the
`
`only biologically active product. In contrast, in the intestinal mucosa, it is glucagon that is bu(cid:173)
`ried 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-
`
`10
`
`69), with the glucagon sequence occupying residues Nos. 33-61 (12); 2) GLP-1(7-36)amide
`(PG (78-1O7))amide (13), not as originally believed PG (72-1O7)amide or 108, which is inac(cid:173)
`
`tive). 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 (111-122)amide) (15); and
`
`4) GLP-2 (PG (126-158)) (15, 16). A fraction of glicentin is cleaved further into GRPP (PG
`(1~30)) and oxyntomodulin (PG (33-69)) (17, 18). Of these peptides, GLP-1 , has the most
`conspicuous biological activities.
`
`The amino acid sequence of GLP-1 is given i.a. by Schmidt et al. (Oiabetologia 28 704-707
`(1985). 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 conside(cid:173)
`red 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 protrac-
`ted and still had retained activity (PCT application No. DK97/OO34O).
`
`15
`
`20
`
`25
`
`While much attention has been focused on the pharmacological properties of acylated GLP-
`
`1 derivatives, hitherto little is known about their physico-chemical and solution structural pro(cid:173)
`perties. Such knowledge is a prerequisite for rational handling during e.g. production, purifi-
`
`30
`
`cation and formulation work and is eventually important for understanding of the structural
`basis for the protraction mechanism.
`
`GLP-1 and analogues of GLP-1 and fragments thereof are potentially useful i.a. in the treat(cid:173)
`
`ment of type 1 and type 2 diabetes. However, solubility limitations and the low stability against
`
`35
`
`the actions of endogenous diaminopeptidyl peptidase limits the usefulness of these com(cid:173)
`
`pounds, and thus there still is a need for improvements in this field. Accordingly, it is one object
`
`FRESENIUS EXHIBIT 1038
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`WO99/43341
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`PCT/DK99/00084
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`of the present invention to provide pharmaceutical solutions comprising GLP-1 derivatives with
`
`3
`
`improved solubility and stability.
`
`References.
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`5
`
`10
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`1. Pederson RA. Gastric Inhibitory Polypeptide. In Walsh JH, Dockray GJ (eds) Gut pepti(cid:173)
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`des: Biochemistry and Physiology. Raven Press, New York 1994, pp. 217259.
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`2. Krarup T. lmmunoreactive gastric inhibitory polypeptide. Endocr Rev 1988;9:122-134.
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`3. 0rskov C. Glucagon-like peptide-1, a new hormone of the enteroinsular axis. Diabetologia
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`1992; 35:701-711.
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`4. Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC. Exon duplication and divergen-
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`ce in the human preproglucagon gene. Nature 1983; 304: 368-371 .
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`5. Holst JJ. Glucagon-like peptide-1 (GLP-1 ) - a newly discovered GI hormone. Gastroen(cid:173)
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`terology 1994; 107: 1848-1855.
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`6. Holst JJ. Gut glucagon, enteroglucagon, gut GLI, glicentin - current status. Gastroentero-
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`logy 1983;84:1602-1613.
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`7. Holst JJ, 0rskov C. Glucagon and other proglucagon-derived peptides. In Walsh JH,
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`8. 0rskov C, Holst JJ, Knuhtsen S, Baldissera FGA, Poulsen SS, Nielsen OV. Glucagon-like
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`peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted sepa(cid:173)
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`rately from the pig small intestine, but not pancreas. Endocrinology 1986;119:1467-1475.
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`9. Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, 0rskov C. Proglucagon proces(cid:173)
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`10. Moody AJ , Holst JJ, Thim L, Jensen SL. Relationship of glicentin to proglucagon and
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`glucagon in the porcine pancreas. Nature 1981; 289: 514-516.
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`11 . Thim L, Moody AJ, Purification and chemical characterisation of a glicentin-related pan-
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`12. Thim L, Moody AJ. The primary structure of glicentin (proglucagon). Regul Pept
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`1981 ;2:139-151.
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`13. 0rskov C, Bersani M, Johnsen AH, H0jrup P, Holst JJ. Complete sequences of gluca(cid:173)
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`1989;264: 12826-12829.
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`14. 0rskov C, Rabenh0j L, Kofod H, Wettergren A, Holst JJ. Production and secretion of
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`amidated and glycine-extended glucagon-like peptide-1 (GLP-1) in man. Diabetes 1991 ;
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`43: 535-539.
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`15. Buhl T, Thim L, Kofod H, 0 rskov C, Harling H, & Holst JJ: Naturally occurring products of
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`16. 0rskov C, Buhl T, Rabenh0j L, Kofod H, Holst JJ: Carboxypeptidase-B-like processing of
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`1989;247:193-106.
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`17. Holst JJ. Evidence that enteroglucagon (II) is identical with the C-terminal sequence
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`18. Bataille D, Tatemoto K, Gespach C, Jornvall H, Rosselin G, Mutt V. Isolation of gluca-
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`19. 0rskov 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
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`20. Elliott RM, Morgan LM, Tredger JA, Deacon S, Wright J, Marks V. Glucagon-like pepti-
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`de-1 (7-36)amide and glucose-dependent insulinotropic polypeptide secretion in response
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`to nutrient ingestion in man: acute post-prandial and 24-h secretion patterns. J Endocrinol
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`21. Kolligs F, Fehmann HC, Goke R, Goke B. Reduction of the incretin effect in rats by the
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`22. Wang Z, Wang RM, Owji AA, Smith OM, Ghatei M, Bloom SR. Glucagon-like peptide-1 is
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`23. Thorens B. Expression cloning of the pancreatic b cell receptor for the gluco-incretin
`hormone glucagon-like peptide 1. Proc Natl Acad Sci 1992;89:8641-4645.
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`24. Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ. Diabetes in mice with targeted disrup-
`tion of the GLP-1 receptor gene. Diabetes 1996; 45: 21A.
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`25. Fehmann HC, Goke R, Goke B. Cell and molecular biology of the incretin hormones glu(cid:173)
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`cagon-like peptide-I (GLP-1) and glucose-dependent insulin releasing polypeptide (GIP).
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`26. Gromada J, Dissing S, Bokvist K, Renstrom E, Fnzikjer-Jensen J, Wulff BS, Rorsman P.
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`Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting bTC3-cells by
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`enhancement of intracellular calcium mobilisation. Diabetes 1995; 44: 767-774.
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`27. Holz GG, Leech CA, Habener JF. Activation of a cAMP-regulated Ca2+-signaling pathway
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`in pancreatic !3-cells by the insulinotropic hormone glucagon-like peptide-1 . J Biol Chem,
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`28. Holz GG, KOhltreiber WM , Habener JF. Pancreatic beta-cells are rendered glucose com(cid:173)
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`1993;361 :362-365.
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`29. 0rskov C, Holst JJ, Nielsen OV: Effect of truncated glucagon-like peptide-1 (proglucagon
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`78-107 amide) on endocrine secretion from pig pancreas, antrum and stomach. Endocri(cid:173)
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`nology 1988; 123:2009-2013.
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`30. Hvidberg A, Toft Nielsen M, Hilsted J, 0rskov C, Holst JJ. Effect of glucagon-like pepti-
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`de-1 (proglucagon 78-107amide) on hepatic glucose production in healthy man. Metabo(cid:173)
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`lism 1994;43:104-108.
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`31. Qualmann C, Nauck M, Holst JJ, 0rskov C, Creutzfeldt W. lnsulinotropic actions of intra(cid:173)
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`venous glucagon-like peptide-1 [7-36 amide] in the fasting state in healthy subjects. Acta
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`32. Nauck MA, Heimesaat MM, 0rskov C, Holst JJ, Ebert R. Creutzfeldt W. Pres&P/ed incre(cid:173)
`tin activity of GLP-1(7-36amide) but not of synthetic human GIP in patients with type
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`2-diabetes mellitus. J Clin Invest 1993;91:301-307.
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`33. Nauck MA, Kleine N, 0rskov C, Holst JJ, Willms B, Creutzfeldt W. Normalisation of
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`fasting hyperglycaemia by exogenous GLP-1(7-36amide) in type 2-diabetic patients. Dia(cid:173)
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`betologia 1993;36:741-744.
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`34. Creutzfeldt W, Kleine N, Willms B, 0rskov C, Holst JJ, Nauck MA. Glucagonostatic acti-
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`ons and reduction of fasting hyperglycaemia by exogenous glucagon-liem, pepti-
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`de-1 (7-36amide) in type I diabetic patients. Diabetes Care 1996; 19: 580-586.
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`35. Schjoldager BTG, Mortensen PE, Christiansen J, 0rskov C, Holst JJ. GLP-1
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`(glucagon-like peptide-1) and truncated GLP-1 , fragments of human proglucagon, inhibit
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`gastric acid secretion in man. Dig. Dis. Sci. 1989; 35:703-708.
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`36. Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ. Trunca(cid:173)
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`ted GLP-1 (proglucagon 72-107amide) inhibits gastric and pancreatic functions in man.
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`Dig Dis Sci 1993;38:665-673.
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`5
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`37. Layer P, Holst JJ, Grandt D, Goebell H: lleal release of glucagon-like peptide-1 (GLP-1 ):
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`association with inhibition of gastric acid in humans. Dig Dis Sci 1995; 40: 1074-1082.
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`38. Layer P, Holst JJ. GLP-1 : A humoral mediator of the ileal brake in humans? Digestion
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`1993; 54: 385-386.
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`39. Nauck M, Ettler R, Niedereichholz U, 0rskov C, Holst JJ, Schmiege! W. Inhibition of ga(cid:173)
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`stric emptying by GLP-1(7-36 amide) or (7-37): effects on postprandial glycaemia and in(cid:173)
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`sulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A 124.
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`40. Schick RR. vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M. Glucagon-like
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`peptide 1 - a novel brain peptide involved in feeding regulation. in Ditschuneit H, Gries
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`FA, Hauner H, Schusdziarra V, Wechsler JG (eds.) Obesity in Europe. John Libbey &
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`Company ltd, 1994; pp. 363-367.
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`41. Tang-Christensen M, Larsen PJ, Goke R, Fink-Jensen A, Jessop OS, M0ller M, Sheikh
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`S. Brain GLP-1 (7-36) amide receptors play a major role in regulation of food and water
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`intake. Am. J. Physiol., 1996, in press.
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`42. Turton MD, O'Shea D, Gunn I, Beak SA, Edwards CMB, Meeran K, et al. A role for glu-
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`cagon-like peptide-1 in the regulation of feeding. Nature 1996; 379: 69-72.
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`43. Willms B, Werner J, Creutzfeldt W , 0rskov C, Holst JJ, Nauck M. Inhibition of gastric
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`emptying by glucagon-like peptide-1 (7-36 amide) in patients with type-2-diabetes melli(cid:173)
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`tus. Diabetologia 1994; 37, suppl.1: A 118.
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`44. Larsen J, Jallad N, Damsbo P. One-week continuous infusion of GLP-1 (7-37) improves
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`glycaemic control in NIDDM. Diabetes 1996; 45, suppl. 2: 233A.
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`8
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`45. Ritzel R, 0rskov C, Holst JJ, Nauck MA. Pharmacokinetic, insulinotropic, and gluca(cid:173)
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`gonostatic properties of GLP-1 [7-36 amide] after subcutaneous injection in healthy vo(cid:173)
`lunteers. Dose-response relationships. Diabetologia 1995; 38: 720-725.
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`5
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`46. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human
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`plasma in vitro yields an N-terminally truncated peptide that is a major endogenous meta(cid:173)
`bolite 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 subcu-
`
`10
`
`taneous and intravenously administered glucagon-like peptide-1 are rapidly degraded
`
`from the amino terminus in type II diabetic patients and in healthy subjects. Diabetes 44:
`1126-1131.
`
`Summary of the invention
`
`15
`
`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 give GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs mainly in the L(cid:173)
`
`cells. A simple system is used to describe fragments and analogues of this peptide. Thus, for
`
`20
`
`example, Gly8-GLP-1(7-37) designates a fragment of GLP-1 formally derived from GLP-1 by
`
`deleting the amino acid residues Nos. 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) designates
`
`GLP-1 (7-37) wherein the &-amino group of the Lys residue in position 34 has been tetradeca(cid:173)
`
`noylated. Where reference in this text is made to C-terminally extended GLP-1 analogues, the
`
`25
`
`amino acid residue in position 38 is Arg unless otherwise indicated, the optional amino acid
`
`residue in position 39 is also Arg unless otherwise indicated and the optional amino acid resi(cid:173)
`
`due in position 40 is Asp unless otherwise indicated. Also, if a C-terminally extended analogue
`
`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 indicated.
`
`30
`
`PCT application No. DK97/00340 describes various GLP-1 derivatives that are found to be
`
`very protracted. Whereas GLP-1 and GLP-1 analogues are molecules to which no defined so(cid:173)
`
`lution structure can be ascribed, we found that some of these protracted GLP-1 derivatives
`
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`may exist in a partially structured micellar-like aggregated form which is stable over a wide
`concentration range.
`
`9
`
`Circular Dichroism (CD) can be used to show that the GLP-1 derivatives have a certain par-
`
`5
`
`tially structured conformation independent of their concentration. In contrast, for normal GLP-
`
`1 (7-37) an increase in the helix content is seen with increasing concentration, from 10-1 5% to
`
`30-35% (at 500 µM concentration) in parallel with peptide self-association. For the GLP-1 deri(cid:173)
`
`vatives forming partially structured micellar-like aggregates in aqueous solution the helix con(cid:173)
`
`tent remains constant above 30% at concentrations of 10 µM. The aggregated structured con-
`
`10
`
`formation is an inherent property of the derivative present in water or dilute aqueous buffer wit(cid:173)
`
`hout the need for any additional structure-inducing components.
`
`Thus, in its broadest aspect, the present invention relates to a pharmaceutical composition
`comprising a GLP-1 derivative which has a helix content as measured by CD at 222 nm in H20
`at 22 ± 2 °C exceeding 25%, preferably in the range of 25% to 50%, at a peptide concentration
`of about 1 O µM.
`
`15
`
`The size of the partially helical, micelle-like aggregates may be estimated by size-exclusion
`
`chromatography. Similarly, the apparent (critical micelle concentrations) CMC's of the pepti-
`
`20
`
`des may be estimated from the concentration dependent fluorescence in the presence of
`
`appropriate dyes (e.g. Brito, R. & Vaz, W. (1986) Anal. Biochem. 152, 250-255).
`
`That the derivatives have a partially structured micellar-like aggregate conformation in aqueous
`
`solutions makes them more soluble and stable in solution as compared to the native peptide.
`
`25
`
`The increased solubility and stability can be seen by comparing the solubility after 9 days of
`
`standing for a derivative and normal GLP-1(7-37) in a pharmaceutical formulation, e.g. 5 mM
`
`phosphate buffer, pH 6.9 added 0.1 M NaCl.
`
`In the present text, the designation "an analogue" is used to designate a peptide wherein one
`
`30
`
`or more amino acid residues of the parent peptide have been substituted by another amino
`
`acid residue and/or wherein one or more amino acid residues of the parent peptide have been
`
`deleted and/or wherein one or more amino acid residues have been added to the parent pepti(cid:173)
`
`de. Such addition can take place either at the N-terminal end or at the C-terminal end of the
`parent peptide or both.
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`10
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`The term "derivative" is used in the present text to designate a peptide in which one or more of
`
`the amino acid residues of the parent peptide have been chemically modified, e.g. by alkylati(cid:173)
`on, acylation, ester formation or amide formation.
`
`5
`
`The term "a GLP-1 derivative" is used in the present text to designate a derivative of GLP-1 or
`
`an analogue thereof. In the present text, the parent peptide from which such a derivative is
`
`formally derived is in some places referred to as the "GLP-1 moiety" of the derivative.
`
`10
`
`In a preferred embodiment, the present invention relates to pharmaceutical composition accor(cid:173)
`
`ding to claim 1, wherein the concentration of GLP-1 derivative is not less than 0.5 mg/ml, prefe(cid:173)
`rably not less than about 5 mg/ml, more preferred not less than about 10 mg/ml and, prefe(cid:173)
`rably, not more than about 100 mg/ml.
`
`15
`
`The pharmaceutical composition of the invention preferably comprises a GLP-1 derivative whe(cid:173)
`
`rein at least one amino acid residue of the parent peptide has a lipophilic substituent attached.
`
`More preferred are compositions comprising a GLP-1 derivative having a lipophilic-substituent
`
`which is attached to any one of the amino acid residues in position 18-38, preferably 26-34.
`
`The pharmaceutical composition according to the invention, preferably further comprises one
`or more of the following substances:
`
`20
`
`• a pharmaceutically acceptable vehicle or carrier;
`
`• an isotonic agent, preferably selected from the group consisting of sodium chloride, mannitol
`and glycerol;
`
`• a preservative, preferably selected from the group consisting of phenol, m-cresol, methyl p-
`
`25
`
`hydroxybenzoate, butyl p-hydroxybenzoate and benzyl alcohol;
`
`• a buffer, preferably selected from the group consisting of sodium acetate, citrate, glycylgly(cid:173)
`cine, histidine, 2-phenylethanol and sodium phosphate; and
`
`• a surfactant capable of improving the solubility and/or the stability of the GLP-1 derivative,
`preferable selected from poloxymer 188, tween 20 and tween 80.
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`11
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`In a preferred embodiment, the pharmaceutical composition of the invention comprises a GLP-
`
`1 derivative wherein the lipophilic substituent comprises from 4 to 40 carbon atoms, preferably
`from 8 to 25 carbon atoms.
`
`The lipophilic substituent is preferably attached to an amino acid residue in such a way that a
`
`5
`
`carboxyl group of the lipophilic substituent forms an amide bond with an amino group of the
`
`amino acid residue, or, the lipophilic substituent is attached to an amino acid residue in such a
`
`way that an amino group of the lipophilic substituent forms an amide bond with a carboxyl
`
`group of the amino acid residue.
`
`In a preferred embodiment the pharmaceutical composition according to the invention compri-
`
`1 o
`
`ses a GLP-1 derivative wherein the lipophilic substituent is attached to the parent peptide by
`means of a spacer.
`
`The spacer is preferably, in one embodiment, an unbranched alkane a ,ro-dicarboxylic acid
`
`group having from 1 to 7 methylene groups, preferably two methylene groups, which form a
`
`bridge between an amino group of the parent peptide and an amino group of the lipophilic sub-
`stituent.
`
`15
`
`The spacer is preferably, in another embodiment, an amino acid residue except Cys, or a di(cid:173)
`
`peptide such as Gly-Lys or any unbranched alkane a.,ro-aminoacid having from 1 to 7 methyle(cid:173)
`
`ne groups, preferably 2-4 methylene groups, which form a bridge between an amino group of
`the parent peptide and an amino group of the lipophilic substituent.
`
`20
`
`In a preferred embodiment, the lipophilic substituent comprises a partially or completely hydro(cid:173)
`
`genated cyclopentanophenathrene skeleton.
`
`In another preferred embodiment, the lipophilic substituent is a straight-chain or branched alkyl
`group.
`
`The lipophilic substituent is preferably the acyl group of a straight-chain or branched fatty acid,
`
`25
`
`the acyl group more preferably being:
`
`• selected from the group comprising CHiCH2)nCO-, wherein n is 4 to 38, preferably
`CH3(CH2)sCO-, CH3(CH2)8CO-, CH3(CH2)10CO-, CHiCH2)12CO-, CH3(CH2),4CO-,
`CH3(CH2)1sCO-, CH3(CH2)1aCO-, CH3(CH2}i0CO- and CH3(CH2bCO-; or
`
`• an acyl group of a straight-chain or branched alkane a,ro-dicarboxylic acid; or
`
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`12
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`• selected from the group comprising HOOC(CH2)mCO-, wherein m is from 4 to 38, preferably
`from 4 to 24, more preferred selected from the group comprising HOOC(CH2) 14CO-,
`HOOC(CH2) 16CO-, HOOC(CH2)18CO-, HOOC(CH2h0CO- and HOOC(CH2h2CO-.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`5 CH3(CH2)p((CH2)qCOOH)CHNH-CO(CH2hCO-, wherein p and q are integers and p+q is an in(cid:173)
`teger of from 8 to 33, preferably from 12 to 28.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`CH3(CH2)rCO-NHCH(COOH)(CH2hCO-, wherein r is an integer of from 10 to 24.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`10 CHi CH2)sCO-NHCH((CH2hCOOH)CO-, wherein sis an integer of from 8 to 24.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`-NHCH(COOH)(CH2) 4NH-CO(CH2)uCH3, wherein u is an integer of from 8 to 18.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`-NHCH(COOH)(CH2)4NH-COCH((CH2hCOOH)NH-CO(CH2)wCH3, wherein w is an integer of
`from 10 to 16.
`
`15
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`-NHCH(COOH)(CH2)4NH-CO(CH2) 2CH(COOH)NH-CO(CHJxCH3, wherein x is an integer of
`from 10 to 16.
`
`20
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`-NHCH(COOH)(CH2)4NH-CO(CH2hCH(COOH)NH-CO(CH2}yCH3, wherein y is zero or an inte(cid:173)
`ger offrom 1 to 22.
`
`In a preferred embodiment the pharmaceutical composition according to the invention, compri(cid:173)
`
`ses a GLP-1 derivative wherein the parent peptide is GLP-1 (A-B) wherein A is an integer from
`
`1 to 7 and B is an integer from 38 to 45, or an analogue thereof.
`
`25 The parent peptide is preferably, in one embodiment, selected from the group comprising GLP-
`
`1(7-35); GLP-1 (7-36); GLP-1(7-36)amide; GLP-1(7-37); GLP-1(7-38); GLP-1 (7-39); GLP-1(7-
`40) and GLP-1 (7-41 ); and analogues thereof.
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`13
`
`The parent peptide is preferably, in another embodiment, selected from the group comprising
`
`GLP-1(1-35); GLP-1(1-36); GLP-1(1-36)amide; GLP-1(1-37); GLP-1(1-38); GLP-1(1-39); GLP-
`1(1-40); GLP-1(1-41); and an analogues thereof.
`
`In yet another embodiment, the parent peptide is a GLP-1 analogue of formula I:
`
`5
`
`7
`
`8
`
`9
`
`10 11 12 13
`
`14
`
`15 16 17
`
`His-Xaa-xaa-Gly-Xaa-Phe-Thr-Xaa- Asp-Xaa - Xaa-
`
`18 19 20 21 22 23
`
`24 25 26 27 28
`
`10
`
`Xaa- Xaa- Xaa-Xaa - Xaa-Xaa -Xaa-Xaa-Xaa-Xaa-Phe-
`
`29 30 31 32 33 34 35 36 37 38
`
`Ile-Xaa-Xaa - Xaa-Xaa-Xaa -Xaa - Xaa-Xaa-Xaa
`
`15
`
`39 4 0
`
`41 42 43 44 45
`
`Xaa -Xaa-Xaa-Xaa- Xaa - Xaa- Xaa
`
`(I)
`
`wherein
`
`20 Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`Xaa at position 9 is Glu, Asp, or Lys,
`
`Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, lie, Tyr, Glu, Asp, or Lys,
`
`25 Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, or Lys,
`
`Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 21 is Glu, Asp, or Lys,
`
`30 Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys,
`
`Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, lie, Val, Arg, Glu, Asp, or Lys,
`
`Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His,
`
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`
`Xaa at position 27 is Glu, Asp, or Lys,
`Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys,
`
`Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys,