`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`(51) International Patent Classification 6 :
`
`A61K 38/26, CO7K 14/605
`(43) International Publication Date:
`
`2 September 1999 (02.09.99)
`
`
`(21) International Application Number:
`PCT/DK99/00084
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, 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, TR, 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,TI,
`TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI,
`FR, GB, GR,IE, IT, LU, MC, NL, PT, SE), OAPIpatent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`SN, TD, TG).
`
` (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
`
`
`
`
`
`PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(11) International Publication Number:
`
`WO 99/43341
`
`
`
`(71) Applicant: NOVO NORDISK A/S [DK/DK]; Novo Allé,
`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-1411 Copenhagen K
`(DK). NIELSEN,Per, Franklin; Dalsg Park 59, DK-3500
`Verlgse (DK). KAARSHOLM,Niels, C.; Clausholmvej 38,
`DK-~2720 Vanlgse (DK). OLSEN,Helle, Birk; Skolelodden
`23, DK-3450 Allerad (DK). BJGRN, Sgren, Erik; Marie
`Grubbes Allé 47, DK-2800 Lyngby (DK).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event ofthe receipt of
`amendments.
`
`(54) Title: GLP-1 DERIVATIVES WITH HELIX-CONTENT EXCEEDING 25 %, FORMING PARTIALLY STRUCTURED MICEL-
`LAR-LIKE AGGREGATES
`
`(57) Abstract
`
`‘The present invention relates to a pharmaceutical composition comprising a GLP-1 derivative of improved solubility and/orstability,
`and to a method for improving the solubility and/or stability of GLP-1 or a fragment or an analoguethereof.
`
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`Zimbabwe
`
`Codes usedto identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`Albania
`ES
`Lesotho
`LS
`SI
`Slovenia
`Armenia
`FI
`LT
`Lithuania
`SK
`Slovakia
`Austria
`FR
`LU
`SN
`Luxembourg
`Senegal
`Australia
`GA
`LV
`Latvia
`SZ
`Swaziland
`GB
`MC
`Monaco
`TD
`Chad
`Azerbaijan
`GE
`MD
`Bosnia and Herzegovina
`Republic of Moldova
`Togo
`Barbados
`GH
`MG
`Madagascar
`Tajikistan
`GN
`MK
`Belgium
`Turkmenistan
`The former Yugoslav
`Burkina Faso
`GR
`Republic of Macedonia
`Turkey
`HU
`Mali
`Bulgaria
`Trinidad and Tobago
`Benin
`IE
`Ukraine
`Mongolia
`Brazil
`IL
`Mauritania
`Uganda
`Belarus
`IS
`Malawi
`United States of America
`IT
`Canada
`Mexico
`Uzbekistan
`JP
`Viet Nam
`Central African Republic
`Niger
`KE
`Netherlands
`Congo
`Yugoslavia
`KG
`Switzerland
`Norway
`KP
`Céte d'Ivoire
`New Zealand
`Cameroon
`Poland
`China
`Portugal
`Cuba
`Romania
`Russian Federation
`Czech Republic
`Sudan
`Germany
`Denmark
`Sweden
`Estonia
`Singapore
`
`TJ
`
`™T
`
`R
`TT
`VA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Treland
`Tsrael
`Iceland
`Tealy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`sD
`SE
`SG
`
`KR
`
`Le
`LI
`LK
`LR
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`WO 99/43341
`
`PCT/DK99/00084
`
`GLP--1 DERIVATIVES WITH HELIX-CONTENT EXCEEDING 25 %, FORMING PARTIALLY STRUCTURED MICEL-
`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 methodfor improving the solubility and/or stabi-
`
`lity of GLP-1 or a fragment or an analoguethereof.
`
`10
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`Backgroundofthe invention
`
`Peptides are widely used in medicalpractice, and since they can be produced by recombinant
`DNAtechnologyit can be expected that their importancewill increase also in the years to co-
`me.
`
`The hormonesregulating insulin secretion belong to the so-called enteroinsular axis, desig-
`nating a group of hormones,released from the gastrointestinal mucosain responseto the
`presence and absorption ofnutrients in the gut, which promote an early and potentiated re-
`leaseof insulin. The enhancing effect on insulin secretion, the so-called incretin effect, is
`probably essential for a normal glucose tolerance. Manyof the gastrointestinal hormones,
`including gastrin and secretin (cholecystokinin is not insulinotropic in man), are insulinotro-
`pic, but the only physiologically important ones, those that are responsible for the incretin
`effect, are the glucose-dependentinsulinotropic polypeptide, GIP, and glucagon-like peptide-
`1 (GLP-1). Becauseofits insulinotropic effect, GIP, isolated in 1973 (1) immediately attrac-
`ted considerable interest among diabetologists. However, numerousinvestigations carried
`out during the following years clearly indicated that a defective secretion of GIP was notin-
`volved 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 almostineffective in NIDDM (2). The other incretin hormone, GLP-1 is the
`mostpotent insulinotropic substance known(3). Unlike GIP,it is surprisingly effective in sti-
`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
`secretion. Becauseofthese actions it has pronounced blood glucose lowering effects parti-
`cularly in patients with NIDDM.
`
`GLP-1, a product of the progiucagon(4), is one of the youngest membersofthe secretin-VIP
`family of peptides, but is already established as an important gut hormone with regulatory
`function in glucose metabolism and gastrointestinal secretion and metabolism (5). The glu-
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`cagon geneis processeddifferently in the pancreas and in theintestine. 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-
`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 seemsto be the
`only biologically active product. In contrast, in the intestinal mucosa, it is glucagon that is bu-
`ried in a larger moiecule, while the two glucagon-like peptides are formed separately (8). The
`following products are formed and secretedin 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, whichis inac-
`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
`conspicuousbiological activities.
`
`The amino acid sequence of GLP-1 is given i.a. by Schmidt ef al. (Diabetologia 28 704-707
`(1985). Although the interesting pharmacological properties of GLP-1(7-37) and analogues
`thereof have attracted muchattention in recent years onlylittle is known about the structure
`of these molecules. The secondary structure of GLP-1 in micelles has been described by
`Thorton efal. (Biochemistry 33 3532-3539 (1994)), but in normal solution, GLP-1 is conside-
`red a very flexible molecule. Surprisingly, we found that derivatisation of this relatively small
`and very flexible molecule resulted in compounds whose plasmaprofile were highly protrac-
`ted andstill had retained activity (PCT application No. DK97/00340).
`
`While muchattention has been focused on the pharmacological properties of acylated GLP-
`1 derivatives, hithertolittle is known about their physico-chemical and solution structural pro-
`perties. Such knowledgeis a prerequisite for rational handling during e.g. production, purifi-
`cation and formulation work and is eventually important for understanding ofthe structural
`basis for the protraction mechanism.
`
`GLP-1 and analogues of GLP-1 and fragments thereofare potentially useful i.a. in the treat-
`mentof type 1 and type 2 diabetes. However, solubility limitations and the low stability against
`the actions of endogenous diaminopeptidyl peptidase limits the usefulness of these com-
`pounds, and thustherestill is a need for improvementsin this field. Accordingly,it is one object
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`of the present invention to provide pharmaceutical solutions comprising GLP-1 derivatives with
`
`improved solubility and stability.
`
`References.
`
`1. Pederson RA. Gastric Inhibitory Polypeptide. In Walsh JH, Dockray GJ (eds) Gut pepti-
`des: Biochemistry and Physiology. Raven Press, New York 1994, pp. 217259.
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`2. Krarup T. Immunoreactive gastric inhibitory polypeptide. Endocr Rev 1988:9:122-134.
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`3. Srskov C. Glucagon-like peptide-1, a new hormoneofthe enteroinsular axis. Diabetologia
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`4. Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC. Exon duplication and divergen-
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`5. Holst JJ. Glucagon-like peptide-1 (GLP-1) - a newly discovered GI hormone. Gastroen-
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`6. Holst JJ. Gut glucagon, enteroglucagon, gut GLI, glicentin - current status. Gastroentero-
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`7. Holst JJ, @rskov C. Glucagon and other proglucagon-derived peptides. In Walsh JH,
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`17. Holst JJ. Evidence that enteroglucagon(I!) is identical with the C-terminal sequence
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`18. Bataille D, Tatemoto K, Gespach C, Jérnvall H, Rosselin G, Mutt V. Isolation of gluca-
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`7-36amide and GLP-1 7-37 in healthy volunteers are identical. Diabetes
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`Endocrine Reviews, 1995; 16: 390-410.
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`26. Gromada J, Dissing S, Bokvist K, Renstrom E, Frekjaer-Jensen J, Wulff BS, RorsmanP.
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`27. Holz GG, Leech CA, HabenerJF. Activation of a cAMP-regulated Ca’*-signaling pathway
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`28. Holz GG, Kinhltreiber WM, Habener JF. Pancreatic beta-cells are rendered glucose com-
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`petent by the insulinotropic hormone glucagon-like peptide-1(7-37). Nature
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`1993;361:362-365.
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`29. Orskov C, Holst JJ, Nielsen OV: Effect of truncated glucagon-like peptide-1 (proglucagon
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`nology 1988; 123:2009-2013.
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`30. Hvidberg A, Toft Nielsen M, Hilsted J, @rskov C, Holst JJ. Effect of glucagon-like pepti-
`de-1 (proglucagon 78-107amide) on hepatic glucose production in healthy man. Metabo-
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`lism 1994;43:104-108.
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`31. Qualmann C, Nauck M, Holst JJ, Srskov C, Creutzfeldt W. Insulinotropic actions ofintra-
`venous glucagon-like peptide-1 [7-36 amide] in the fasting state in healthy subjects. Acta
`Diabetologica, 1995; 32: 13-16.
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`32. Nauck MA, Heimesaat MM, @rskov C, Holst JJ, Ebert R, Creutzfeldt W. Preserved incre-
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`tin 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.
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`33. Nauck MA, Kleine N, @rskov C, Holst JJ, Willms B, Creutzfeldt W. Normalisation of
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`fasting hyperglycaemia by exogenous GLP-1(7-3Gamide)in type 2-diabetic patients. Dia-
`betologia 1993:36:741-744.
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`34. Creutzfeldt W, Kleine N, Willms B, @rskov C, Holst JJ, Nauck MA. Glucagonostatic acti-
`ons and reduction of fasting hyperglycaemia by exogenous glucagon-liem, pepti-
`de-1(7-36amide)in type | diabetic patients. Diabetes Care 1996; 19: 580-586.
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`35. Schjoldager BTG, Mortensen PE, Christiansen J, @rskov C, Holst JJ. GLP-1
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`gastric acid secretion in man. Dig. Dis. Sci. 1989; 35:703-708.
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`ted GLP-1 (proglucagon 72-107amide) inhibits gastric and pancreatic functions in man.
`Dig Dis Sci 1993;38:665-673.
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`37. Layer P, Holst JJ, Grandt D, Goebell H: Ileal release of glucagon-like peptide-1 (GLP-1):
`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 mediatorofthe ileal brake in humans? Digestion
`1993; 54: 385-386.
`
`39. Nauck M, Ettler R, Niedereichholz U, @rskov C, Holst JJ, Schmiegel W.Inhibition of ga-
`stric emptying by GLP-1(7-36 amide) or(7-37): effects on postprandial glycaemia and in-
`sulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A124.
`
`40. Schick RR, vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M. Glucagon-like
`peptide 1 - a novelbrain peptide involved in feeding regulation. in Ditschuneit H, Gries
`FA, Hauner H, Schusdziarra V, Wechsler JG (eds.) Obesity in Europe. John Libbey &
`Companyltd, 1994: pp. 363-367.
`
`41. Tang-Christensen M, Larsen PJ, Géke R, Fink-Jensen A, Jessop DS, Maller M, Sheikh
`S. Brain GLP-1(7-36) amide receptors play a major role in regulation of food and water
`intake. Am. J. Physiol., 1996, in press.
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`42. Turton MD, O'Shea D, Gunn |, Beak SA, Edwards CMB, MeeranK,et al. A role for glu-
`cagon-like peptide-1 in the regulation of feeding. Nature 1996; 379: 69-72.
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`25
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`43. Willms B, WernerJ, Creutzfeldt W, @rskov C, Holst JJ, Nauck M. Inhibition of gastric
`emptying by glucagon-like peptide-1 (7-36 amide) in patients with type-2-diabetes melli-
`tus. Diabetologia 1994; 37, suppl.1: A118.
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`44. LarsenJ, Jallad N, Damsbo P. One-week continuous infusion of GLP-1(7-37) improves
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`45. Ritzel R, Orskov C, Holst JJ, Nauck MA. Pharmacokinetic, insulinotropic, and gluca-
`gonostatic properties of GLP-1 [7-36 amide] after subcutaneousinjection in healthy vo-
`lunteers. Dose-responserelationships. Diabetologia 1995; 38: 720-725.
`
`46. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human
`plasmain vitro yields an N-terminally truncated peptide that is a major endogenous meta-
`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-
`taneous and intravenously administered glucagon-like peptide-1 are rapidly degraded
`from the amino terminusin type II diabetic patients and in healthy subjects. Diabetes 44:
`1126-1131.
`
`Summary of the invention
`
`Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is
`synthesised /.a. in the L-cells in the distal ileum, in the pancreasandin the brain. Processing of
`preproglucagon to give GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs mainly in the L-
`cells. A simple system is used to describe fragments and analoguesofthis peptide. Thus, for
`example, Gly*-GLP-1(7-37) designates a fragment of GLP-1 formally derived from GLP-1 by
`deleting the aminoacid residues Nos.1 to 6 and substituting the naturally occurring amino acid
`residue in position 8 (Ala) by Gly. Similarly, Lys*(N*-tetradecanoyl)-GLP-1(7-37) designates
`GLP-1(7-37) wherein the s-amino group of the Lys residue in position 34 has been tetradeca-
`noylated. Wherereferencein this text is made to C-terminally extended GLP-1 analogues, the
`amino acid residue in position 38 is Arg unless otherwiseindicated, the optional amino acid
`residuein position 39 is also Arg unless otherwise indicated and the optional amino acid resi-
`duein position 40 is Asp unless otherwiseindicated. Also,if a C-terminally extended analogue
`extends to position 41, 42, 43, 44 or 45, the amino acid sequenceofthis extensionis as in the
`corresponding sequence in human preproglucagon unless otherwise indicated.
`
`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-
`lution structure can be ascribed, we found that someof these protracted GLP-1 derivatives
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
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`PCT/DK99/00084
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`9
`
`may exist in a partially structured micellar-like aggregated form whichis stable over a wide
`
`concentration range.
`
`Circular Dichroism (CD) can be used to show that the GLP-1 derivatives have a certain par-
`
`§_tially structured conformation independentof their concentration. In contrast, for normal GLP-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1(7-37) an increase in the helix contentis seen with increasing concentration, from 10-15% to
`30-35% (at 500 uM concentration) in parallel with peptide self-association. For the GLP-1 deri-
`
`vatives forming partially structured micellar-like aggregates in aqueous solution the helix con-
`tent remains constant above 30% at concentrations of 10 uM. The aggregated structured con-
`formation is an inherent property of the derivative present in wateror dilute aqueous buffer wit-
`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 H,O
`at 22 +2 °C exceeding 25%, preferably in the range of 25% to 50%, at a peptide concentration
`of about 10 uM.
`
`Thesize of the partially helical, micelle-like aggregates may be estimated by size-exclusion
`chromatography. Similarly, the apparent(critical micelle concentrations) CMC’sof the pepti-
`des may be estimated from the concentration dependentfluorescencein the presenceof
`appropriate dyes (e.g. Brito, R. & Vaz, W. (1986) Anal. Biochem. 152, 250-255).
`
`That the derivatives havea partially structured micellar-like aggregate conformation in aqueous
`solutions makes them more soluble and stable in solution as compared to the native peptide.
`‘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
`of more aminoacid 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 aminoacid residues have been added to the parent pepti-
`de. Such addition can take placeeither at the N-terminal end or at the C-terminal end of the
`
`parent peptide or both.
`
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`10
`
`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-
`on, acylation, ester formation or amide formation.
`
`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 someplacesreferred to as the “GLP-1 moiety’of the derivative.
`
`10
`
`In a preferred embodiment, the present invention relates to pharmaceutical composition accor-
`ding to claim 1, wherein the concentration of GLP-1 derivative is not less than 0.5 mg/ml, prefe-
`rably not less than about 5 mg/ml, more preferred not less than about 10 mg/miand, prefe-
`rably, not more than about 100 mg/ml.
`
`The pharmaceutical composition of the invention preferably comprises a GLP-1 derivative whe-
`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 havingalipophilic-substituent
`which is attached to any one of the amino acid residuesin position 18-38, preferably 26-34.
`
`The pharmaceutical composition according to the invention, preferably further comprises one
`or more ofthe following substances:
`
`20
`
`*« a pharmaceutically acceptable vehicle or carrier:
`
`¢
`
`anisotonic 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-
`hydroxybenzoate, butyl p-hydroxybenzoate and benzyl alcohol;
`
`25
`
`¢ a buffer, preferably selected from the group consisting of sodium acetate,citrate, glycylgly-
`cine,histidine, 2-phenylethanol and sodium phosphate; and
`
`e 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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`PCT/DK99/00084
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`11
`
`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.
`
`Thelipophilic substituentis preferably attached to an amino acid residue in such a way that a
`carboxyl groupofthe lipophilic substituent forms an amide bond with an amino groupof the
`amino acid residue, or, the lipophilic substituent is attached to an amino acid residue in such a
`
`way that an amino group ofthe 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-
`ses aGLP-1 derivative wherein thelipophilic substituent is attached to the parent peptide by
`meansof a spacer.
`
`The spaceris preferably, in one embodiment, an unbranched alkane c,«-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 groupofthelipophilic sub-
`stituent.
`
`5
`
`10
`
`15
`
`The spaceris preferably, in another embodiment, an aminoacid residue except Cys, or a di-
`peptide such as Gly-Lys or any unbranched alkane «,«-aminoacid having from 1 to 7 methyle-
`ne groups, preferably 2-4 methylene groups, which form a bridge between an amino group of
`the parent peptide and an amino group ofthe lipophilic substituent.
`
`20
`
`Ina preferred embodiment, the lipophilic substituent comprises a partially or completely hydro-
`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 groupofa straight-chain or branchedfatty acid,
`the acyl group morepreferably being:
`
`25
`
`
`
`e selected from the group comprising CH;(CH,),CO-, whereinnis 4 to 38, preferably
`CH,(CH,),CO-, CH3(CH,)eCO-, CH3(CH2)19CO-, CH,(CH,),2CO-, CH3(CH,),4CO-,
`CH3(CH,),,CO-, CH,(CH,),sCO-, CH,;(CH,).,CO- and CH,(CH,)2,CO-; or
`
`* an acyl groupof a straight-chain or branched alkane o.,@-dicarboxylic acid; or
`
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`PCT/DK99/00084
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`12
`
`
`
`e selected from the group comprising HOOC(CH,),,CO-, whereinmis from 4 to 38, preferably
`from 4 to 24, more preferred selected from the group comprising HOOC(CH,),,CO-,
`HOOC(CH,),.CO-, HOOC(CH,),,CO-, HOOC(CH,)..CO- and HOOC(CH,)..CO-.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`CH3(CH2)_((CH2){COOH)CHNH-CO(CH,),CO-, wherein p and q are integers and p+qis an in-
`teger of from 8 to 33, preferably from 12 to 28.
`
`in anotherpreferred embodiment, the lipophilic substituent is a group of the formula
`CH,(CH,),CO-NHCH(COOH)(CH,),CO-, wherein r is an integer of from 10 to 24.
`
`in anotherpreferred embodiment, the lipophilic substituent is a group of the formula
`10
`CH3(CH,),CO-NHCH((CH,),COOH)CO-, whereinsis an integerof from 8 to 24.
`
`In another preferred embodiment,the lipophilic substituent is a group of the formula
`-NHCH(COOH)(CH,),NH-CO(CH,),CHs, wherein u is an integer of from 8 to 18.
`
`In another preferred embodiment, the lipophilic substituent is a groupof the formula
`-NHCH(COOH)(CH,),NH-COCH((CH,),>COOH)NH-CO(CH,),CH;, wherein w is an integerof
`from 10 to 16.
`
`15
`
`In another preferred embodiment,thelipophilic substituent is a group of the formula
`-NHCH(COOH)(CH,),NH-CO(CH,),CH(COOH)NH-CO(CH,),CH,, 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)(CH,),NH-CO(CH,),CH(COOH)NH-CO(CH,),CHs, wherein y is zero or an inte-
`ger of from 1 to 22.
`
`In a preferred embodimentthe pharmaceutical composition according to the invention, compri-
`ses a GLP-1 derivative wherein the parent peptide is GLP-1(A-B) wherein A is an integer from
`1 to 7 andBis an integer from 38 to 45, or an analoguethereof.
`
`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 analoguesthereof.
`
`In yet another embodiment, the parent peptide is a GLP-1 analogue of formula|:
`
`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-Kaa-Xaa-~-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`#35
`
`36
`
`37
`
`38
`
`ITle-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
`
`16
`
`39
`
`40
`
`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,lle, Val, Glu, Asp, or Lys,
`Xaaat position 9 is Glu, Asp, or Lys,
`
`Xaaatposition 11 is Thr, Ala, Gly, Ser, Leu,lle, Val, Glu, Asp, or Lys,
`Xaaat position 14 is Ser, Ala, Gly, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`Xaaat position 16 is Val, Ala, Gly, Ser, Thr, Leu, lle, Tyr, Glu, Asp, or Lys,
`25=Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`Xaaat position 18 is Ser, Ala, Gly, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, or Lys,
`Xaaatposition 20 is Leu, Ala, Gly, Ser, Thr, Leu,lle, 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, ile, Val, Glu, Asp, or Lys,
`Xaaat position 23 is Gin, Asn, Arg, Glu, Asp, or Lys,
`Xaaat position 24 is Ala, Gly, Ser, Thr, Leu, lle, Val, Arg, Glu, Asp, or Lys,
`Xaaat position 25 is Ala, Gly, Ser, Thr, Leu,lle, Val, Glu, Asp, or Lys,
`Xaa at position 26 is Lys, Arg, Gln, Glu, Asp, or His,
`
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`WO 99/43341
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`PCT/DK99/00084
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`Xaa at position 27 is Glu, Asp, or Lys,
`Xaa atposition 30 is Ala, Gly, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`Xaaat position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys,
`Xaaat position 32 is Leu, Gly, Ala, Ser, Thr, lle, Val, Glu, Asp, or Lys,
`Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Met, Leu,lle, Glu, Asp, or Lys,
`Xaa at position 34 is Lys, Arg, Glu, Asp, or His,
`
`Xaaat position 35 is Gly, Ala, Ser, Thr, Leu,lle, Val, Glu, Asp, or Lys,
`Xaaat position 36 is Arg, Lys, Glu, Asp, or His, or
`Xaa at position 37 is Gly, Ala, Ser, Thr, Leu,ile, Val, Glu, Asp, or Lys,or is deleted,
`Xaa at position 38 is Arg, Lys, Glu, Asp, or His, or is deleted,
`Xaaat position 39 is Arg, Lys, Glu, Asp, or His, or is deleted,
`Xaaat position 40 is Asp, Glu, or Lys, or is deleted,
`
`Xaaat position 41 is Phe, Trp, Tyr, Glu, Asp, or Lys, oris deleted,
`Xaaat position 42 is Pro, Lys, Glu, or Asp, or is deleted,
`Xaa at position 43 is Glu, Asp, or Lys, oris deleted,
`Xaa atposition 44 is Giu, Asp, or Lys, or is deleted, and
`Xaaatposition 45 is Val,