PCT
`
`Intemattonal Bureau
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER-THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 6 =
`(11) International Publication Number:
`WO 98/08871 I
`C07K 14/605, AGIK 38/26
`‘
`
`(43) International Publication Date:
`
`5 March 1998 (05.03.98)
`
`AI
`
`(2]) International Application Number:
`
`PC’I/DK97/00340
`
`(22) International Filing Date:
`
`22 August 1997 (22.08.97)
`
`(30) Priority Data:
`0931/96
`1259/96
`1470/96
`
`30 August 1996 (30.08.96)
`8 November 1996 (08.11.96)
`20 December 1996 (20.12.96)
`
`DK
`DK
`DK
`
`(71) Applicant (for all designated States except US): NOVO
`NORDISK A/S [DK/DK]; Novo A116, DK-2880 Bagsvaerd
`(DK).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KNUDSEN, Liselotte,
`Bjcrre [DK/DK]; Valby Langgade 49A, 1.tv., DK-ZSOO
`Valby (DK). SQRENSEN, Per, Olaf [DK/DK]; Applebys
`Plads 27. 5. mf., DK-141 1 Copenhagen K (DK). NIELSEN,
`Per, Franklin [DK/DK]; Dalstzt Park 59, DK-3500 Vwrlose
`(0K).
`
`(74) Common Representative: NOVO NORDISK A/S; Corporate
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE. ES, Fl, GB, GE,
`GH, HU, IL, 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,
`
`TI‘, UA, UG, US, UZ, VN, YU, ZW, ARIPO patent (GH.
`KE, LS, MW, SD, 52, U0, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE.
`CH, DE, DK, ES, Fl, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`ML, MR, NE, SN, TD. T6).
`
`Published
`With international search report.
`
`Patents, Novo A116, DK-2880 Bagsvaard (DK).
`
`(54) Title: GLP-l DERIVATIVES
`
`(57) Abstract
`
`Derivatives of GLP-1 and analogues thereof having a lipophilic substituent have interesting pharmacological properties, in particular
`they have a more protracted profile of action than GLP-1(7-37).
`
`
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Hemgovma
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cbtc d’lvoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`Fl
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`[E
`[L
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`Ll
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`ltaly
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kmkstan
`Saint Lucia
`Liechtenstein
`Sri Lanlra
`Liberia
`
`
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`R0
`RU
`SD
`SE
`SG
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`TR
`"IT
`UA
`UG
`US
`UZ
`VN
`Y U
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`
`
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`WO 98/08871
`
`PCT/DK97/00340
`
`GLP-1 DERIVATIVES
`
`FIELD OF THE INVENTION
`
`The present invention relates to novel derivatives of human glucagon-like peptide-1 (GLP-1)
`
`and fragments thereof and analogues of such fragments which have a protracted profile of
`
`action and to methods of making and using them.
`
`1O
`
`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 anatogues thereof are used in therapy it
`
`is
`
`generally found that they have a high clearance. A high clearance of a therapeutic agent is
`
`inconvenient in cases where it
`
`is desired to maintain a high blood level thereof over a
`
`prolonged period of time since repeated administrations will then be necessary. Examples of
`
`peptides which have a high clearance are: ACTH, corticotropin-releasing factor, angiotensin,
`
`calcitonin,
`
`insulin, glucagon, glucagon-like peptide-1, glucagon—like peptide-2,
`
`insulin—like
`
`growth factor-1,
`
`insulin-like growth factor—2, gastric inhibitory peptide. growth hormone-
`
`releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin,
`
`somatostatin,
`
`somatotropin.
`
`somatomedin,
`
`parathyroid
`
`hormone,
`
`thrombopoietin,
`
`erythropoietin, hypothalamic releasing factors, prolactin,
`
`thyroid stimulating hormones,
`
`endorphins, enkephalins. vasopressin, oxytocin. opiods and analogues thereof, superoxide
`
`dismutase,
`
`interferon, asparaginase, arginase, arginine deaminase. adenosine deaminase
`
`and ribonuclease. in some cases it is possible to influence the release profile of peptides by
`
`applying suitable pharmaceutical compositions, but this approach has various shortcomings
`
`and is not generally applicable.
`
`The hormones regulating insulin secretion belong to the so-called enteroinsular axis,
`
`designating a group of hormones, released from the gastrointestinal mucosa in response
`
`to the presence and absorption of nutrients in the gut, which promote an early and
`
`potentiated release of insulin. The enhancing effect on insulin secretion,
`
`the so-called
`
`incretin effect,
`
`is probably essential
`
`for a normal glucose tolerance. Many of
`
`the
`
`gastrointestinal hormones.
`
`including oastrin and secretin
`
`(cholecystokinin is
`
`not
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`W0 98/0887!
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`PCT/DK97/00340
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`insulinotropic in man), are insulinotropic, but the only physiologically important ones, those
`
`that are responsible for the incretin effect, are the glucose—dependent
`
`insulinotropic
`
`polypeptide, GlP, and glucagon-like peptide-1 (GLP-1). Because of its insulinotropic effect,
`
`GlP, isolated in 1973 (1) immediately attracted considerable interest among diabetologists.
`
`However, numerous investigations carried out during the following years clearly indicated
`
`that a defective secretion of GlP was not involved in the pathogenesis of insulin dependent
`
`diabetes mellitus
`
`(IDDM) or non insulin-dependent diabetes mellitus
`
`(NlDDM)
`
`(2).
`
`Furthermore, as an insulinotropic hormone, GlP was found to be almost
`
`ineffective in
`
`NlDDM (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 NlDDM
`
`patients. in addition, and in contrast to the other insulinotropic hormones (perhaps with the
`
`exception of secretin) it also potently inhibits glucagon secretion. Because of these actions
`
`it has pronounced blood glucose lowering effects particularly in patients with NlDDM.
`
`GLP-1, a product of the proglucagon (4), is one of the youngest members of the secretin—
`
`VlP 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 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 pancreatic peptide. GRPP (10. 11);
`
`3) a hexapeptide corresponding 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 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-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-l, has the most conspicuous biological activities.
`
`Being secreted in parallel with glicentin/enteroglucagon, it follows that the many studies of
`
`enteroglucagon secretion (6, 7) to some extent also apply to GLP-1 secretion, but GLP—1
`
`is metabolised more quickly with a plasma half-life in humans of 2 min (19). Carbohydrate
`
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`PCT/DK97/00340
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`or fat-rich meals stimulate secretion (20), presumably as a result of direct interaction of yet
`
`unabsorbed nutrients with the microvilli of the open-type L-cells of the gut mucosa.
`
`Endocrine or neural mechanisms promoting GLP—1 secretion may exist but have not yet
`
`been demonstrated in humans.
`
`The incretin function of GLP-1(29-31) has been clearly illustrated in experiments with the
`
`GLP-1 receptor antagonist. exendin 9-39. which dramatically reduces the incretin effect
`
`elicited by oral glucose in rats (21. 22). The hormone interacts directly with the B—cells via
`
`the GLP-1 receptor (23) which belongs to the glucagoanP/calcitonin family of G-protein-
`
`coupled 7-transrnembrane spanning receptors. The importance of the GLP-1 receptor in
`
`regulating 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
`
`mechanism (25) primarily involves activation of adenylate cyclase. but elevations of
`
`intracellular 032+ 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 B—cells. The relation of the latter to its ability to convey "glucose
`
`competence" to 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 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
`
`concentrations increase either by increased secretion or by exogenous infusion the molar
`
`ratio of insulin to glucagon in the blood that reaches the liver via the portal circulation is
`
`greatly increased. whereby hepatic glucose production decreases (30). As a result blood
`
`glucose concentrations decrease. Because of the glucose dependency of the insulinotropic
`
`and glucagonostatic actions, the glucose lowering effect is self-limiting, and the hormone.
`
`therefore. does not cause hypoglycaen'iia regardless of dose (31). The effects are
`
`preserved in patients with diabetes mellitus
`
`(32),
`
`in whom infusions of
`
`slightly
`
`supraphysiological doses of GLP-1 may completely normalise blood glucose values in
`
`spite of poor metabolic control and secondary failure to sulphonyiurea (33). The
`
`importance of the glucagonostatic effect is illustrated by the finding that GLP-1 also lowers
`
`blood glucose in type-1 diabetic patients without residual B—cell secretory capacity (34).
`
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`WO 98/08871
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`PCT/DK97/00340
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`in addition to its effects on the pancreatic islets, GLP—i has powerful actions on the
`
`gastrointestinal
`
`tract.
`
`Infused
`
`in
`
`physiological
`
`amounts. GLP-1
`
`potently
`
`inhibits
`
`pentagastrin-induced as well as meal-induced gastric acid secretion (35, 36).
`
`it also
`
`inhibits gastric emptying rate and pancreatic enzyme secretion (36). Similar inhibitory
`
`effects on gastric and pancreatic secretion and motility may be elicited in humans upon
`
`perfusion of
`
`the
`
`ileum with carbohydrate- or
`
`lipid-containing solutions
`
`(37, 38).
`
`Concomitantly, GLP-l secretion is greatly stimulated, and it has been speculated that
`
`GLP-1 may be at least partly responsible for this so-called "Heal-brake" effect (38). in fact,
`
`10
`
`recent studies suggest that. physiologically, the Heal-brake effects of GLP-1 may be more
`
`important than its effects on the pancreatic islets. Thus, in dose response studies GLP-‘l
`
`influences gastric emptying rate at infusion rates at least as low as those required to
`
`influence islet secretion (39).
`
`15
`
`GLP-1 seems to have an effect on food intake.
`
`lntraventricular administration of GLP-1
`
`profoundly inhibits food intake in rats (40. 42). This effect seems to be highly specific.
`
`Thus, N-terminally extended GLP-i (PG 72-107)amide is inactive and appropriate doses
`
`of the GLP—i antagonist. exendin 9-39, abolish the effects of GLP-1 (41). Acute, peripheral
`
`administration of GLP-1 does not inhibit food intake acutely in rats (41, 42). However,
`
`it
`
`remains possible that GLP-i secreted from the intestinal L—cells may also act as a satiety
`
`signal.
`
`Not only the insulinotropic effects but also the effects of GLP-1 on the gastrointestinal tract
`
`are preserved in diabetic patients (43), and may help curtailing meal-induced glucose
`
`excursions, but. more importantly, may also influence food intake. Administered
`
`intravenously. continuously for one week, GLP-l at 4 ng/kg/min has been demonstrated to
`
`dramatically improve glycaemic control in NlDDM patients without significant side effects
`
`(44). The peptide is fully active after subcutaneous administration (45), but
`
`is
`
`rapidly
`
`degraded mainly due to degradation by dipeptidyl peptidase lV-Iike enzymes (46, 47).
`
`The amino acid sequence of GLP~1 is given La. by Schmidt et al. (Diabetologia 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 considered a very flexible molecule. Surprisingly, we found that
`
`derivatisation of this relatively small and very flexible molecule resulted in compounds
`
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`WO 98/08871
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`PCT/DK97/00340
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`whose plasma profile were highly protracted and still had retained activity.
`
`GLP-1 and analogues of GLP—1 and fragments thereof are potentially useful La.
`
`in the
`
`treatment of type 1 and type 2 diabetes. However, the high clearance limits the usefulness of
`
`these compounds, and thus there still is a need for improvements in this field. Accordingly, it
`
`is one 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
`
`clearance than GLP-1(7—37). It is a further object of the invention to provide a pharmaceutical
`
`10
`
`composition comprising a compound according to the invention and to use a compound of
`
`the invention to provide such a composition. Also, it is an object of the present invention to
`
`provide a method of treating insulin dependent and non—insulin dependent diabetes mellitus.
`
`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. Krarup T. lmmunoreactive gastric inhibitory polypeptide. Endocr Rev 1988;91122—134.
`
`3.
`
`iZJrskov C. Glucagon-like peptide-1,
`
`a new hormone of
`
`the enteroinsular axis.
`
`Diabetologia 1992; 35:701-71 1.
`
`4. Bell Gl. Sanchez-Pescador R. Laybourn PJ, Najarian RC. Exon duplication and
`
`divergence in the human preproglucagon gene. Nature 1983; 304: 368-371.
`
`5. Holst JJ. Glucagon-like peptide-1
`
`(GLP-1)
`
`— a newly discovered GI hormone.
`
`Gastroenterology 1994; 107: 1848-1855.
`
`6. Holst
`
`JJ. Gut glucagon.
`
`enteroglucagon, gut GLI.
`
`glicentin - current
`
`status.
`
`Gastroenterology 1 983:8411602-1613.
`
`7. Holst JJ, Qrskov C. Glucagon and other proglucagon-derived peptides.
`
`In Walsh JH,
`
`Dockray GJ, eds. Gut peptides: Biochemistry and Physiology. Raven Press, New York,
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`pp. 305-340, 1993.
`
`8. Erskov C, Holst JJ, Knuhtsen S, Baldissera FGA, Poulsen SS, Nielsen OV.
`
`Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are
`
`secreted separately from the pig small
`
`intestine, but not pancreas. Endocrinology
`
`1986;119:1467-1475.
`
`9. Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, erkov C. Proglucagon
`
`processing in porcine and human pancreas. J Biol Chem, 1994; 269: 18827-1883.
`
`10. Moody AJ, Holst JJ, Thim L, Jensen SL. Relationship of glicentin to proglucagon and
`
`glucagon in the porcine pancreas Nature 1981; 289: 514-516.
`
`11. Thim L. Moody AJ, Purification and chemical characterisation of a glicentin-related
`
`pancreatic peptide (proglucagon fragment) from porcine pancreas. Biochim Biophys
`
`Acta 1982;703:134-141.
`
`12. Thim L. Moody AJ. The primary structure of glicentin (proglucagon). Regul Pept
`
`1981;22139-151.
`
`13. erkov C. Bersani M. Johnson AH. Hojrup P, Holst JJ. Complete sequences of
`
`glucagon-like peptide-1 (GLP-1) from human and pig small
`
`intestine. J. Biol. Chem.
`
`1989;264:12826—12829.
`
`14. erskov C, Rabenhoj L, Kofod H, Wettergren A, Holst JJ. Production and secretion of
`
`amidated and glycine-extended glucagon-like peptide-1 (GLP-l) in man. Diabetes 1991;
`43: 535-539.
`
`15. Buhl T, Thim L. Kofod H, Qrskov 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. Qrskov C, Buhl T, Rabenhoj L. Kofod H, Holst JJ: Carboxypeptidase-B-like processing
`
`of the C-terminus of glucagon-like peptide—2 in pig and human small intestine. FEBS
`
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`letters, 1989;247:193-106.
`
`17. Holst JJ. Evidence that enteroglucagon (II) is identical with the C-terminal sequence
`
`(residues 33—69) of glicentin. Biochem J. 1980;187:337-343.
`
`18. Bataille D, Tatemoto K, Gespach C, Jernvall H, Rosselin G, Mutt V.
`
`isolation of
`
`glucagon-37 (bioactive enteroglucagon/oxyntomodulin)
`
`from porcine jejuno—ileum.
`
`Characterisation of the peptide. FEBS Lett 1982;146:79-86.
`
`19. erkov C, Wettergren A, Holst JJ. The metabolic rate and the biological effects of
`
`GLP-1 7-363mide 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-1 (7-36)amide and glucose-dependent insulinotropic polypeptide secretion in
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`response to nutrient ingestion in man: acute post-prandial and 24-h secretion patterns
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`J Endocrinol 1993; 138: 159-166.
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`21. Kolligs F, Fehmann HC, Goke R, G6ke B. Reduction of the incretin effect in rats by the
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`glucagon-like peptide-1 receptor antagonist exendin (9-39)amide. Diabetes 1995; 44:
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`22. Wang Z, Wang RM, Owji AA. Smith DM, Ghatei M, Bloom SR. Glucagon-like peptide-1
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`is a physiological incretin in rat. J. Clin. Invest. 1995; 95: 417-421.
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`23. Thorens B. Expression cloning of the pancreatic b cell receptor for the gluco-incretin
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`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
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`disruption 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
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`(GlP). Endocrine Reviews. 1995; 16: 390-410,
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`26. Gromada J, Dissing S, Bokvist K, Renstrbm E, Frokjaer-Jensen J, Wulff BS, Rorsman
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`P. Glucagon-like peptide l increases cytoplasmic calcium in insulin-secreting bTC3~cells
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`by 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
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`pathway in pancreatic B-cells by the insulinotropic hormone glucagon-like peptide-1. J
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`Biol Chem, 1996; 270: 17749-17759.
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`28. Holz GG, Ktihltreiber WM, Habener JF. Pancreatic beta-cells are rendered glucose
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`competent by the
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`insulinotropic hormone glucagon-like peptide-1(7-37). Nature
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`1993;361:362-365.
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`29. @rskov C. Hoist JJ. Nielsen OV: Effect of
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`stomach. Endocrinology 1988;123:2009-2013.
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`30. Hvidberg A, Toft Nielsen M, Hilsted J, Erskov C, Holst JJ. Effect of glucagon-like
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`peptide-1 (proglucagon 78-107amide) on hepatic glucose production in healthy man.
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`Metabolism 1994;43:104-108.
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`31. Qualmann C, Nauck M, Holst JJ, @rskov C, Creutzfeldt W.
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`lnsulinotropic actions of
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`intravenous glucagon-like peptide-1 [7-36 amide] in the fasting state in healthy subjects.
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`Acta Diabetologica, 1995; 32: 13-16.
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`32. Nauck MA, Heimesaat MM, Qrskov C, Holst JJ, Ebert R, Creutzfeldt W. Preserved
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`incretin activity of GLP-1(7-36amide) but not of synthetic human GIP in patients with
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`type 2-dlabetes mellitus. J Clin invest 1993;91:301-307.
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`33. Nauck MA, Kleine N, Erskov 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.
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`Diabetologia 1993;36:741-744.
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`34. Creutzfeldt W, Kleine N, Willms B, Qrskov C, Holst .JJ, Nauck MA. Glucagonostatic
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`fasting hyperglycaemia by exogenous glucagon-liem,
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`peptide-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, @rskov C, Holst JJ. GLP-1
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`(glucagon-like peptide-1) and truncated GLP-1, fragments of human proglucagon,
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`inhibit gastric acid secretion in man. Dig. Dis. Sci. 1989; 35:703-708.
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`36. Wettergren A, Schjoidager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ.
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`Truncated 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:
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`ileal release of glucagon—like peptide-1
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`(GLP-1): association with inhibition of gastric acid in humans. Dig Dis Sci 1995; 40:
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`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, Qrskov C, Holst JJ, Schmiegel W. Inhibition of
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`gastric emptying by GLP-1(7-36 amide) or (7-37): effects on postprandial glycaemia
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`and insulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A124.
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`40. Schick RR.
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`vorm Walde T, Zimmermann JP, Schusdziarra V, Classen M.
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`Glucagon‘like peptide 1
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`- a novel brain peptide involved in feeding regulation.
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`in
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`Europe. John Libbey & Company ltd. 1994; pp. 363-387.
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`41. Tang~Christensen M, Larsen PJ, G'c'>ke R, Fink-Jensen A, Jessop DS, Moller M, Sheikh
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`8. 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 l, Beak SA, Edwards CMB, Meeran K, et al. A role for
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`glucagon-iike peptide-1 in the regulation of feeding. Nature 1996; 379: 69-72.
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`43. Willms B, Werner J. 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
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`mellitus. Diabetologia 1994; 37. suppl.1: A118.
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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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`45.Ritzel R,
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`inskov C. Holst JJ, Nauck MA. Pharmacokinetic,
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`insulinotropic, and
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`glucagonostatic properties of GLP-1 [7-36 amide] after subcutaneous injection in
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`healthy volunteers. Dose-response relationships. Diabetologia 1995; 38: 720-725.
`
`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
`
`metabolite in vivo. J Clin Endocrinol Metab 1995; 80: 952—957.
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`47. Deacon CF, Nauck MA, Taft-Nielsen M, Pridal L, Willms B, Holst JJ. 1995. Both
`
`subcutaneous 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.
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`15
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`20
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`SUMMARY OF THE INVENTION
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`Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is
`
`25
`
`synthesised la. 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-cells. A simple system is used to describe fragments and analogues of this peptide. Thus.
`
`for example, Gly”~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
`
`30
`
`acid residue in position 8 (Ala) by Gly. Similarly, Lys3‘(N‘-tetradecanoyl)-GLP-1(7-37)
`
`designates GLP-1(7-37) wherein the a-amino group of the Lys residue in position 34 has
`
`been tetradecanoylated. Where reference in this text is made to C-tenninally extended GLP—
`
`1 analogues, the amino acid residue in position 38 is Arg unless otherwise indicated, the
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`optional amino acid residue in position 39 is also Arg unless otherwise indicated and the
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`optional amino acid residue 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.
`
`In its broadest aspect, the present invention relates to derivatives of GLP-1 and analogues
`
`thereof. The derivatives according to the invention have interesting pharmacological
`
`properties,
`
`in particular they have a more protracted profile of action than the parent
`
`peptides.
`
`In the present text, the designation “an analogue" is used to designate a peptide wherein one
`
`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 peptide. Such addition can take place either at the N-terminal end or at the C-terminal
`
`end of the parent peptide or both.
`
`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
`
`alkylation, acylation, ester formation or amide formation.
`
`The term “a GLP—i 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-l moiety" of the derivative.
`
`in a preferred embodiment, as described in Claim 1, the present invention relates to a GLP-1
`
`derivative wherein at least one amino acid residue of the parent peptide has a lipophilic
`
`substituent attached with the proviso that if only one lipophilic substituent is present and this
`
`substituent is attached to the N-terminal or to the C-terminal amino acid residue of the parent
`
`peptide then this substituent is an alkyl group or a group which has an m-carboxylic acid
`
`group.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`In another preferred embodiment, as described in Claim 2, the present invention relates to a
`
`GLP-1 derivative having only one lipophilic substituent.
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`In another preferred embodiment. as described in Claim 3. the present invention relates to a
`
`GLP-1 derivative having only one lipophilic substituent which substituent is an alkyl group or
`
`a group which has an m-carboxylic acid group and is attached to the N-terminal amino acid
`
`residue of the parent peptide.
`
`in another preferred embodiment. as described in Claim 4, the present invention relates to a
`
`GLP-1 derivative having only one lipophilic substituent which substituent is an alkyl group or
`
`a group which has an w-carboxylic acid group and is attached to the C-terminal amino acid
`
`10
`
`residue of the parent peptide.
`
`ln another preferred embodiment, as described in Claim 5, the present invention relates to a
`
`GLP-i derivative having only one lipophilic substituent which substituent can be attached to
`
`any one amino acid residue which is not the N-terminal or C-terminal amino acid residue of
`
`15
`
`the parent peptide.
`
`20
`
`25
`
`30
`
`In another preferred embodiment. as described in Claim 6. the present invention relates to a
`
`GLP-1 derivative wherein two lipophilic substituents are present.
`
`In another preferred embodiment, as described in Claim 7, the present invention relates to a
`
`GLP~1 derivative wherein two lipophilic substituents are present, one being attached to the
`
`N-terminal amino acid residue while the other is attached to the C-terminal amino acid
`
`residue.
`
`In another preferred embodiment. as described in Claim 8. the present invention relates to a
`
`GLP-1 derivative wherein two lipophilic substituents are present, one being attached to the
`
`N-terminal amino acid residue while the other is attached to an amino acid residue which is
`
`not N-terminal or the C-terminal amino acid residue.
`
`In another preferred embodiment. as described in Claim 9. the present invention relates to a
`
`GLP-1 derivative wherein two lipophilic substituents are present, one being attached to the
`
`C-terminal amino acid residue while the other is attached to an amino acid residue which is
`
`not the N-terminal or the C-terminal amino acid residue.
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`In further preferred embodiment. as described in Claim 10, the present invention relates to a
`
`derivative of GLP-1(7-C), wherein C is selected from the group comprising 38, 39, 40, 41, 42.
`
`43, 44 and 45 which derivative has just one lipophilic substituent which is attached to the C-
`
`terminal amino acid residue of the parent peptide.
`
`in a further preferred embodiment.
`
`the present invention relates to a GLP-1 derivative
`
`wherein the lipophilic substituent comprises from 4 to 40 carbon atoms, more preferred from
`
`8 to 25 carbon atoms.
`
`in a further preferred embodiment,
`
`the present
`
`invention relates to a GLP-1 derivative
`
`wherein a lipophilic substituent is attached to an amino acid residue in such a way that a
`
`carboxyl group of the lipophilic substituent forms an amide bond with an amino group of the
`amino acid residue.
`
`in a furth