PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER:THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 6 :
`(11) International Publication Number:
`WO 98/08871
`C07K 14/605, A61K 38/26
`
`(43) International Publication Date:
`
`5 March 1998 (05.03.98)
`
`|
`
`
`
`(22) International Filing Date:
`
`22 August 1997 (22.08.97)
`
`(21) Internationa] Application Number: PCT/DK97/00340|(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG,BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, 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,
`TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO patent (GH,
`KE, LS, MW,SD, SZ, UG, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE,
`CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, Cl, CM, GA, GN,
`ML, MR, NE, SN, TD, TG).
`
`Patents, Novo Allé, DK-2880 Bagsvzrd (DK).
`
`(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 Allé, DK-2880 Bagsverd
`(DK).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KNUDSEN, Liselotte,
`Bjerre [DK/DK]; Valby Langgade 49A,
`I.tv., DK-2500
`Valby (DK), S@RENSEN, Per, Olaf [DK/DK]; Applebys
`Plads 27, 5. mf., DK-14]1 Copenhagen K (DK). NIELSEN,
`Per, Franklin [DK/DK]; Dals@ Park 59, DK-3500 Vzrlgse
`(DK).
`
`Published
`With international search report.
`
`(74) Common Representative: NOVO NORDISK A/S; Corporate
`
`(54) Title: GLP-1 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 Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Céte d'Ivoire
`Cameroon
`China
`Cuba
`Czech Repubtic
`Germany
`Denmark
`Estonia
`
`ES
`
`KR
`KZ
`Le
`ul
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`lreland
`Tsracl
`Iceland
`Ttaly
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugostav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`
`™T
`
`R
`
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`WO 98/08871
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`GLP-1 DERIVATIVES
`
`FIELD OF THE INVENTION
`
`The presentinvention 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.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`Peptides are widely used in medical practice, and since they can be produced by
`recombinant DNA technology it can be expected that their importance will increase also in
`
`the years to come. When native peptides or analogues thereof are used in therapyit 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 administrationswill 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 casesit is possible to influence the release profile of peptides by
`applying suitable pharmaceutical compositions, but this approach has various shortcomings
`andis not generaily 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,
`includina
`aastrin and secretin
`(cholecystokinin is
`not
`
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`insulinotropic in man), are insulinotropic, but the only physiologically important ones, those
`
`insulinotropic
`that are responsible for the incretin effect, are the giucose-dependent
`polypeptide, GIP, and glucagon-like peptide-1 (GLP-1). Becauseofits insulinotropic effect,
`
`GIP, 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 G!P was not invoived in the pathogenesis of insulin dependent
`
`(IDDM) or non insulin-dependent diabetes mellitus
`diabetes mellitus
`Furthermore, as an insulinotrapic hormone, GIP was found to be almost
`
`(2).
`(NIDDM)
`ineffective in
`
`NIDDM (2). The other incretin hormone, GLP-1 is the most potent insulinotropic substance
`
`10
`
`known (3). Unlike GIP, it is surprisingly effective in stimulating insulin secretion in NIDDM
`
`patients. In addition, and in contrast to the other insulinotropic hormones (perhapswith the
`
`exception of secretin) it also potently inhibits glucagon secretion. Because of these actions
`it has pronounced blood glucose lowering effects particularly in patients with NIDDM.
`
`15
`
`20
`
`GLP-1, a product of the proglucagon (4), is one of the youngest members of the secretin-
`VIP family of peptides, but
`is already established as an important gut hormone with
`
`regulatory function in glucose metabolism and gastrointestinal secretion and metabolism
`(5). The glucagon geneis processed differently in the pancreas andin 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 cailed glicentin-related pancreatic peptide, GRPP (10, 11);
`3) a hexapeptide corresponding to PG (64-69); 4) and,
`finally,
`the so-called major
`progtucagon 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
`
`25
`
`it is glucagon that is buried in a larger molecule, while the two glucagon-like
`mucosa,
`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-1, has the most conspicuous biological activities.
`
`Being secretedin 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 haif-life in humans of 2 min (19). Carbohydrate
`
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`or fat-rich meals stimulate secretion (20), presumably as a resuit 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 glucagon/VIP/catcitonin family of G-protein-
`coupled 7-transmembrane spanning receptors. The importance of the GLP-1 receptorin
`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 homozygousfor
`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 Ca2* are also essential (25, 26). The action of the hormoneis 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 glucagonosiatic action is glucose-dependent,
`so that
`the inhibitory effect
`decreases as blood glucose decreases. Because ofthis 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 dependencyof the insulinotropic
`and glucagonostatic actions, the glucose lowering effect is self-limiting, and the hormone,
`therefore, does not cause hypoglycaemia regardless of dose (31). The effects are
`preserved in patients with diabetes mellitus
`(32),
`in whom infusions of
`slightly
`supraphysiological doses of GLP-1 may completely ncrmatise blood giucose values in
`spite of poor metabolic control and secondary failure to sulphonyiurea (33). The
`importance of the glucagonostatic effectis illustrated by the finding that GLP-1 also lowers
`blood glucosein type-1 diabetic patients without residual 6-cell secretory capacity (34).
`
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`
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`20
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`In addition to its effects on the pancreatic islets, GLP-1 has powerful actions on the
`gastrointestinal
`tract.
`Infused
`in
`physiological
`amounts, GLP-1
`potentiy
`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-1 secretion is greatly stimulated, and it has been speculated that
`GLP-1 may beat least partly responsible for this so-called "ileal-brake" effect (38). In fact,
`recent studies suggest that, physiologically, the ileal-brake effects of GLP-1 may be more
`important than its effects on the pancreatic islets. Thus, in dose response studies GLP-1
`influences gastric emptying rate at infusion rates at least as low as those required to
`influenceislet secretion (39).
`
`10
`
`15
`
`GLP-1 seems to have an effect on food intake. Intraventricular administration of GLP-1
`
`profoundly inhibits food intake in rats (40, 42). This effect seems to be highly specific.
`
`Thus, N-terminally extended GLP-1 (PG 72-107)amide is inactive and appropriate doses
`
`of the GLP-1 antagonist, exendin 9-39, abolish the effects of GLP-1 (41). Acute, peripheral
`
`administration of GLP-1 does not inhibit food intake acutely in rats (41, 42). However,it
`
`20
`
`remains possible that GLP-1 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
`
`25
`
`excursions, but, more importantly, may also influence food intake. Administered
`
`intravenously, continuously for one week, GLP-1 at 4 ng/kg/min has been demonstrated to
`dramatically improve glycaemic control in NIDDM patients without significant side effects
`(44). The peptide is fully active after subcutaneous administration (45), but
`is
`rapidly
`
`degraded mainly due to degradation by dipeptidy! peptidase {V-like enzymes (46, 47).
`
`30
`
`The amino acid sequence of GLP-1 is given ia. by Schmidt et a/. (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
`
`35
`
`in normal
`(Biochemistry 33 3532-3539 (1994)), but
`described by Thorton ef al.
`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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`whose plasma profile were highly protracted andstill had retained activity.
`
`GLP-1 and analogues of GLP-1 and fragments thereof are potentially useful ia.
`
`in the
`
`treatment of type 1 and type 2 diabetes. However, the high clearancelimits the usefulness of
`
`these compounds, and thustherestill is a need for improvementsin 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 methodof 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.
`
`20
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`2. Krarup T. Immunoreactive gastric inhibitory polypeptide. Endocr Rev 1988;9:122-134.
`
`3. Srskov C, Glucagon-like peptide-1,
`
`a new hormone of
`
`the enteroinsular axis.
`
`Diabetologia 1992; 35:701-711.
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`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
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`Gastroenterology 1994; 107: 1848-1855.
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`6. Holst
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`JJ. Gut glucagon,
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`status.
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`Gastroenterology 1983;84:1602-1613.
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`7. Holst JJ, S@rskov C. Glucagon and other proglucagon-derived peptides.
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`In Walsh JH,
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`pp. 305-340, 1993.
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`8. @rskov C, Holst JJ, Knuhtsen S, Baldissera FGA, Poulsen SS, Nielsen OV.
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`Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are
`
`secreted separately from the pig small
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`intestine, but not pancreas. Endocrinology
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`1986;119:1467-1475.
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`9. Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, @rskov C. Proglucagon
`processing in porcine and human pancreas. J Bio! Chem, 1994, 269: 18827-1883.
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`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.
`
`14. Thim L, Moody AJ, Purification and chemical characterisation of a glicentin-related
`
`pancreatic peptide (progtucagon fragment) from porcine pancreas. Biochim Biophys
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`Acta 1982;703:134-141.
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`12. Thim L, Moody AJ. The primary structure of glicentin (proglucagon). Regul Pept
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`4981;2:139-151.
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`13. Orskov C, Bersani M, Johnsen AH, Hajrup P, Holst JJ. Complete sequences of
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`glucagon-like peptide-1 (GLP-1) from human and pig smail
`
`intestine. J. Biol. Chem.
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`1989;264: 12826-12829.
`
`14. S@rskov C, Rabenhgj 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;
`43: 535-539.
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`15. Buhl T, Thim L, Kofod H, @rskov C, Harling H, & Holst JJ: Naturally occurring products
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`intestine. J. Biol. Chem.
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`1988;263:8621-8624.
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`16. Orskov C, Buhl T, Rabenhaj L, Kofod H, Holst JJ: Carboxypeptidase-B-like processing
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`letters, 1989:247:193-106.
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`17. Holst JJ. Evidence that enteroglucagon(Il) 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, Jérnvall H, Rosselin G, Mutt V. {solation of
`
`glucagon-37 (bioactive enteroglucagon/oxyntomodulin)
`
`from porcine jejuno-ileum.
`
`Characterisation of the peptide. FEBS Lett 1982;146:79-86.
`
`10
`
`19. S@rskov 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
`
`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.
`
`J Endocrinol 1993; 138: 159-166.
`
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`21. Kolligs F, Fenmann HC, Goke R, Goke B. Reduction of the incretin effect in rats by the
`
`glucagon-like peptide-1 receptor antagonist exendin (9-39)amide. Diabetes 1995; 44:
`16-19.
`
`22. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei M, Bloom SR. Glucagon-like peptide-1
`is a physiological incretin in rat. J. Clin. Invest. 1995; 95: 417-421.
`
`23. Thorens B. Expression cioning of the pancreatic b cell receptor for the gluco-incretin
`hormone glucagon-like peptide 1. Proc Nat! Acad Sci 1992;89:8641-4645.
`
`24. Scrocchi L, Auerbach AB, Joyner AL, Drucker DJ. Diabetes in mice with targeted
`disruption of the GLP-1 receptor gene. Diabetes 1996; 45: 21A.
`
`30
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`25. Fehmann HC, Géke R, Géke B. Cell and molecular biology of the incretin hormones
`
`glucagon-like peptide-| (GLP-1) and glucose-dependent insulin releasing polypeptide
`(GIP}. Endocrine Reviews, 1995; 16: 390-410.
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`26. Gromada J, Dissing S, Bokvist K, Renstrom E, Frokjeer-Jensen J, Wulff BS, Rorsman
`P. Glucagon-like peptide | increases cytoplasmic calcium in insulin-secreting bTC3-cells
`by enhancementofintracellular calcium mobilisation. Diabetes 1995; 44: 767-774.
`
`27. Holz GG, Leech CA, Habener JF. Activation of a cAMP-regulated Ca’’-signaling
`
`pathway in pancreatic f-cells by the insulinotropic hormone glucagon-like peptide-1. J
`
`Biol Chem, 1996; 270: 17749-17759.
`
`10
`
`28. Holz GG, Kihltreiber WM, Habener JF. Pancreatic beta-ceills 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, Holst JJ, Nielsen OV: Effect of
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`truncated glucagon-like peptide-1
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`(proglucagon 78-107 amide) on endocrine secretion from pig pancreas, antrum and
`stomach. Endocrinology 1988;123:2009-2013.
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`30. Hvidberg A, Toft Nielsen M, Hilsted J, @rskov C, Holst JJ. Effect of glucagon-like
`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, Hoist Ju, @rskov C, Creutzfeldt W. Insulinotropic 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, @rskov C, Holst JJ, Ebert R, Creutzfeldt W. Preserved
`incretin activity of GLP-1(7-36amide) but not of synthetic human GIP in patients with
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`type 2-diabetes mellitus. J Clin invest 1993;91:301-307.
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`33. Nauck MA, Kleine N, @rskov C, Hoist JJ, Willms B, Creutzfeidt W. Normalisation of
`fasting hyperglycaemia by exogenous GLP-1(7-36amide) in type 2-diabetic patients.
`Diabetologia 1993;36:741-744.
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`34. Creutzfeldt W, Kleine N, Willms B, @rskov C, Hoist JJ, Nauck MA. Glucagonostatic
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`peptide-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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`(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, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst Ju.
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`Truncated GLP-1 (progiucagon 72-107amide) inhibits gastric and pancreatic functions
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`in man. Dig Dis Sci 1993;38:665-673.
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`37. Layer P, Holst JJ, Grandt D, Goebel! H:
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`iteal 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, @rskov 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
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`JT, 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 & CompanyItd, 1994; pp. 363-367.
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`41. Tang-Christensen M, Larsen PJ, Gdke R, Fink-Jensen A, Jessop DS, Maller 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 |, Beak SA, Edwards CMB, Meeran K, et al. A role for
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`glucagon-like peptide-1 in the regulation of feeding. Nature 1996; 379: 69-72.
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`43. Willms B, Werner J, Creutzfeldt W, Orskov C, Holst JJ, Nauck M. Inhibition of gastric
`emptying by glucagon-like peptide-1 (7-36 amide)
`in patients with type-2-diabetes
`mellitus, Diabetologia 1994; 37, suppl.1: A118.
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`44. Larsen J, Jallad N, Damsbo P. One-weekcontinuous 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, @rskov C, Hoist 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-responserelationships. Diabetologia 1995; 38: 720-725.
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`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
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`metabolite in vivo. J Clin Endocrinol Metab 1995; 80: 952-957.
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`47. Deacon CF, Nauck MA, Toft-Nieisen M, Pridal L, Willms 8, Holst JJ. 1995. Both
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`subcutaneous and intravenously administered glucagon-like peptide-1 are rapidly
`
`degraded from the amino terminus in type || diabetic patients and in healthy subjects.
`Diabetes 44: 1126-1131.
`
`10
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`15
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`20
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`SUMMARY OF THE INVENTION
`
`Human GLP-1 is a 37 amino acid residue peptide originating from prepraglucagon whichis
`
`25
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`synthesised /.a. in the L-cells in the distal ileum, in the pancreas andin 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 analoguesof 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, Lys*(N*-tetradecanoyl}-GLP-1(7-37)
`
`designates GLP-1(7-37) wherein the e-amino group of the Lys residue in position 34 has
`
`been tetradecanoylated. Where referencein this text is made to C-terminally extended GLP-
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`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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`if a C-
`optional amino acid residue in position 40 is Asp unless otherwise indicated. Also,
`terminally extended analogue extends to position 41, 42, 43, 44 or 45, the amino acid
`sequenceof this extension is as in the corresponding sequence in human preprogiucagon
`
`unless otherwise indicated.
`
`In its broadest aspect, the present invention relates to derivatives of GLP-1 and analogues
`
`thereof. The derivatives according ta the invention have interesting pharmacological
`
`properties,
`
`in particular they have a more protracted profile of action than the parent
`
`peptides.
`
`10
`
`in the presenttext, 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
`
`20
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`alkylation, 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 presenttext, the parent peptide from which such a derivativeis
`
`formally derived is in some places referred to as the “GLP-1 moiety” of the derivative.
`
`25
`
`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 onelipophilic substituent is present and this
`
`substituent is attached to the N-terminal or to the C-terminal amino acid residue of the parent
`
`30
`
`peptide then this substituent is an alkyl group or a group which has an w-carboxylic acid
`
`group.
`
`In another preferred embodiment, as described in Claim 2, the present invention relates to a
`
`GLP-1 derivative having only onelipophilic 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 alkyt group or
`a group which has an w-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.
`
`in another preferred embodiment, as described in Claim 5, the present invention relates to a
`
`GLP-1 derivative having only onelipophilic 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
`
`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 twolipophilic 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 twolipophilic 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.
`
`30
`
`In another preferred embodiment, as described in Claim 9, the present invention relates to a
`
`GLP-1 derivative wherein twolipophilic 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 lipaphilic 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.
`
`15
`
`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 an
`
`amino group ofthe lipophilic substituent forms an amide bond with a carboxy! group of the
`amino acid residue.
`
`20
`
`In a further preferred embodiment,
`
`the present invention relates to a GLP-1 derivative
`
`wh