`c12) Reissued Patent
`Larsen et al.
`
`I IIIII
`
`11111111
`
`1111111111111111111111111111111111111111111111
`USOORE45313E
`
`US RE45,313 E
`(10) Patent Number:
`(45) Date of Reissued Patent:
`Dec. 30, 2014
`
`(54) EXENDIN VARIANT PEPTIDES
`
`(71) Applicant: Zealand PharmaA/S, Glostrup (DK)
`
`(72)
`
`Inventors: Bjarne Due Larsen, Roskilde (DK);
`Jens Damsgaard Mikkelsen, Kgs.
`Lyngby (DK); Soren Neve, Kgs. Lyngby
`(DK)
`
`(73) Assignee: Zealand PharmaA/S, Glostrup (DK)
`
`(21) Appl. No.: 13/944,735
`
`(22) Filed:
`
`Jul. 17, 2013
`
`Related U.S. Patent Documents
`
`Reissue of:
`(64) Patent No.:
`Issued:
`Appl. No.:
`Filed:
`U.S. Applications:
`(60) Provisional application No. 60/143,591, filed on Jul.
`12, 1999.
`
`6,528,486
`Mar. 4, 2003
`09/614,847
`Jul. 12, 2000
`
`7,223,725 Bl
`7,226,990 B2
`7,235,627 B2
`7,297,761 B2
`7,348,404 B2
`7,399,489 B2
`7,407,932 B2
`7,414,107 B2
`7,419,952 B2
`7,442,680 B2
`7,452,858 B2
`7,521,423 B2
`7,544,657 B2
`7,601,691 B2
`7,608,692 B2
`7,623,530 B2
`7,683,030 B2
`7,691,963 B2
`7,696,161 B2
`7,700,549 B2
`7,741,269 B2
`7,858,740 B2
`7,928,065 B2
`7,935,786 B2
`8,026,210 B2
`8,057,822 B2
`8,097,698 B2
`8,263,550 B2
`
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`6/2007 Knudson et al.
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`7/2008 Kolterman et al.
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`10/2008 Young eta!.
`1112008 Hiles eta!.
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`10/2009 Bridon eta!.
`10/2009 Prickett et a!.
`1112009 Hurtta
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`1112011 Prickett et a!.
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`(Continued)
`
`(51)
`
`Int. Cl.
`A61K 38100
`(52) U.S. Cl.
`USPC ............ 514/7.2; 514/1.1; 514/11.7; 530/300;
`530/303
`
`(2006.01)
`
`(58) Field of Classification Search
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`(Continued)
`
`Primary Examiner- Dong Jiang
`(74) Attorney, Agent, or Firm- Kristina Bieker-Brady;
`Clark & Elbing LLP
`
`(57)
`
`ABSTRACT
`
`The present invention relates to novel peptide conjugates
`which have increased stability and are useful in the treatment
`of excess levels of blood glucose.
`
`2 Claims, 8 Drawing Sheets
`
`SANOFI-AVENTIS Exhibit 1012 - Page 1
`
`IPR for Patent No. 8,951,962
`
`
`
`US RE45,313 E
`Page 2
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`(56)
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`IPR for Patent No. 8,951,962
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`
`
`US RE45,313 E
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`* cited by examiner
`
`SANOFI-AVENTIS Exhibit 1012 - Page 3
`
`IPR for Patent No. 8,951,962
`
`
`
`U.S. Patent
`
`Dec. 30, 2014
`
`Sheet 1 of8
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`SANOFI-AVENTIS Exhibit 1012 - Page 6
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`Dec. 30, 2014
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`SANOFI-AVENTIS Exhibit 1012 - Page 7
`IPR for Patent No. 8,951,962
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`
`U.S. Patent
`
`Dec. 30, 2014
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`Sheet 5 of8
`
`Us RE45,313 E
`
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`VEHICLE 0.001
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`10
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`DOSE (nmol/kg i.p.)
`
`FIG. 5
`
`SANOFI-AVENTIS Exhibit 1012 - Page 8
`IPR for Patent No. 8,951,962
`
`
`
`U.S. Patent
`
`4102093oneD
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`Sheet 6 of 8
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`US RE45,3l3 E
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`Dec. 30, 2014
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`Sheet 7 of8
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`SANOFI-AVENTIS Exhibit 1012 - Page 10
`IPR for Patent No. 8,951,962
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`
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`U.S. Patent
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`Dec. 30, 2014
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`Sheet 8 of8
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`US RE45,313 E
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`
`SANOFI-AVENTIS Exhibit 1012 - Page 11
`IPR for Patent No. 8,951,962
`
`
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`US RE45,313 E
`
`1
`EXENDINVARIANT PEPTIDES
`
`Matter enclosed in heavy brackets [ ] appears in the
`original patent but forms no part of this reissue specifica-
`tion; matter printed in italics indicates the additions
`made by reissue.
`
`5
`
`2
`era! strategies circumventing this problem have been pro(cid:173)
`posed, some using inhibitors of DPP-IV and other DPP-IV
`resistant analogues of GLP-1 (7-36)amide (Deacon, 1998,
`Diabetologia 41:271-287; Deacon et a!., 1998, Diabetes
`47:764-769; Ritzel, 1998, J. Endocrinol. 159:93-102; U.S.
`Pat. No. 5,545,618; Pederson, 1998, Diabetes 47:1253-
`1258).
`Exendins, another group of peptides that lower blood glu(cid:173)
`cose levels have some sequence similarity (53%) to GLP-1
`[7-36]NH2 (Goke eta!., 1993, J. Bioi. Chern. 268: 19650-55).
`The exendins are found in the venom of Helodermatidae or
`beaded lizards (Raufman, 1996, Reg. Peptides 61:1-18).
`Exendin-3 is present in the venom of Heloderma horridum,
`the Mexican beaded lizard and exendin-4 is present in the
`15 venom of Heloderma suspectum, the Gila monster. Exen(cid:173)
`din-4 differs from exendin-3 at just positions two and three.
`The eDNA encoding the exendin-4 precursor protein, a 47
`amino acid peptide fused to the amino terminus of exendin-4
`has been cloned and sequenced (Pohl et a!., 1998, J. Bioi.
`20 Chern. 273:9778-9784 and W098/35033). Both exendin-3
`and exendin-4 stimulate an increase in cellular cAMP pro(cid:173)
`duction in guinea pig pancreatic acinar cells by interacting
`with exendin receptors (Raufman, 1996, Reg. Peptides 61:1-
`18). Exendin-3 causes a biphasic increase in cellular cAMP
`25 production, but a monophasic increase in amylase release in
`pancreatic acinar cells. In contrast, exendin-4 causes a
`monophasic increase in cAMP production and does not alter
`amylase release.
`Exendin-4 is a strong GLP-1 receptor agonist on isolated
`30 rat insulinoma cells (Goke eta!., 1993, J. Bioi. Chern. 268:
`19650-55). This is expected as the (His Ala) domainofGLP-1
`recognised by DPP-IV is not present in exendin-4 (Goke et
`a!., 1993, J. Bioi Chern. 268:19650-55). Binding of [125I]
`GLP-1 to the nucleus of the solitary tract was inhibited con-
`35 centration-dependently by unlabelled GLP-1 and [Tyr39]ex(cid:173)
`endin-4 with Ki values of 3.5 and 9.4 nM respectively, and
`similar values are found in cell lines (Goke eta!., 1995, Eur.
`J. Neurosci. 7:2294-2300 and Goke et a!., 1993, J. Bioi.
`Chern. 268: 19650-55). Further, exendin-4 given systemically
`40 lowers blood glucose levels by 40% in diabetic db/db mice
`(W0/99/07404). Recently, Grieg et a!. (1999, Diabetologia
`42:45-50) has shown a long lasting blood glucose lowering
`effect of once daily intraperitoneal injection of exendin-4 to
`diabetic ob/ob mice). U.S. Pat. No. 5,424,286 discloses that a
`45 considerable portion of theN-terminal sequence is essential
`in order to preserve insulinotropic activity ( exendin-4(1-31)
`andY31 -exendin-4 (1-31 )) whereas anN-terminallytruncated
`exendin ( exendin-4(9-39) has inhibitory properties.
`The use of exendin-3, exendin-4 and exendin agonists has
`50 been proposed for the treatment of diabetes mellitus, reducing
`gastric motility and delaying gastric emptying and the pre(cid:173)
`vention of hyperglycemia (U.S. Pat. No. 5,424,286, W098/
`0535) as well as for the reduction of food intake (W098/
`30231). There has been proposed ways of obtaining novel
`55 compounds by modifYing the native exendin sequences. One
`way is to attach lipophilic substituents to the molecule, e.g. as
`described in WO 99/43708 which discloses derivatives of
`exendin with just one lipophilic substituent attached to the
`C-terminal amino acid residue.
`A major approach has been to devise exendin analogues
`characterised by amino acid substitutions and/or C-terminal
`truncation of the native exendin-4 sequence. This approach is
`represented by the compounds of W099/07404, WO
`99/25727 and WO 99/22728. W099/07404 discloses exendin
`65 agonists having a general formula I that defines a peptide
`sequence of39 amino acid residues with Gly Thr in positions
`4-5, Ser Lys GLn in positions 11-13, Glu Glu GluA!a Val Arg
`
`This application is a reissue of U.S. Pat. No. 6,528,486, 10
`which claims benefit ofU.S. Provisional application Ser. No.
`60/143,591, filed on Jul. 12, 1999.
`
`FIELD OF THE INVENTION
`
`The present invention relates to novel peptide agonists of
`GLP-1 activity. More specifically the invention relates to
`novel peptides that lower blood glucose levels comprising
`variants of the exendin-4 polypeptide sequence and peptide
`conjugates comprising variants of the GLP-1 or the exendin-4
`polypeptide sequences which are pharmacologically active
`and stable, and as agonists of GLP-1 activity are useful in the
`treatment of diseases that benefit from regulation or excess
`levels ofblood glucose and/or regulation of gastric emptying,
`such as diabetes and eating disorders. The present invention
`also relates to methods of preparing said novel peptides, a
`composition, e.g., a pharmaceutical composition, comprising
`a peptide of the invention and a physiologically acceptable
`carrier, to said peptide for use in therapy, a method of treating
`a disorder and to the use of said peptide for the manufacture
`of a pharmaceutical composition for use in therapy.
`
`BACKGROUND OF THE INVENTION
`
`A number of hormones that lower blood glucose levels are
`released from the gastrointestinal mucosa in response to the
`presence and absorption of nutrients in the gut. These include
`gastrin, secretin, glucose-dependent insulinotropic polypep(cid:173)
`tide (GIP) and glucagon-like peptide-! (GLP-1). The most
`potent substance known is GLP-1 (0orskov, 1992, Diabeto(cid:173)
`logia 35:701-711). Glucagon-like peptide 1 (GLP-1) is a
`product of pro glucagon, a 180 amino acid peptide (Drucker,
`1998 Diabetes 47: 159-169). The overall sequence of pro glu(cid:173)
`cagon contains the 29-amino acid sequences of glucagon, the
`36 or 37 amino acid sequence ofGLP-1 and the 34 amino acid
`sequence of glucagon-like peptide-2 (GLP-2), an intesti(cid:173)
`notrophic peptide. GLP-1 has a number of functions. It is a
`physiological hormone that enhances the effect on insulin
`secretion in normal humans and is therefore an incretin hor(cid:173)
`mone. In addition, GLP-1 also lowers glucagon concentra(cid:173)
`tions, slows gastric emptying, stimulates (pro) insulin biosyn(cid:173)
`thesis, and enhances insulin sensitivity (Nauck, 1997, Harm.
`Metab. Res. 47:1253-1258). The peptide also enhances the
`ability for the ~-cells to sense and respond to glucose in
`subjects with imparted glucose tolerance (Byrne, 1998, Eur.
`J. Clin. Invest. 28:72-78). The insulinotropic effect of the
`GLP-1 in humans increases the rate of glucose disappearance
`partly because of increased insulin levels and partly because
`of enhanced insulin sensitivity (D' Alessio, 1994, Eur. J. Clin.
`Invest. 28:72-78). This has placed GLP-1 as a promising 60
`agent for treatment of type II diabetes. Active fragments of
`GLP-1 have been found to be GLP-1(7-36) andGLP-1(7-37).
`However, a major pharmacological problem with native
`GLP-1 is its short half-life. In humans and rats, GLP-1 is
`rapidly degraded by dipeptidyl peptidase-IV (DPP-IV) into
`GLP-1 (9-36)amide, acting as an endogenous GLP-1 receptor
`antagonist (Deacon, 1998, Diabetologia 41:271-278). Sev-
`
`SANOFI-AVENTIS Exhibit 1012 - Page 12
`
`IPR for Patent No. 8,951,962
`
`
`
`US RE45,313 E
`
`3
`Leu in positions 15-21, Leu Lys Asn Gly Gly in positions
`26-30, Ser Ser Gly Ala in positions 32-35, and wherein the
`remaining positions may be occupied by wild-type exendin
`amino acid residues or may be occupied by specified amino
`acid substitutions. The formula I does not cover any exendin 5
`agonists or analogues having specific amino acid deletions
`and/or being conjugates as described herein, such as the novel
`compounds desPro36-exendin-4 (1-39), exendin-4(1-39)-K6
`or desPro36 -exendin-4(1-39)-K6. WO 99/25727 discloses
`exendin agonists having a general formula I that defines a 10
`peptide sequence of from 28 to 38 amino acid residues with
`Gly in position 4 and Ala in position 18, and wherein the
`remaining positions may be occupied by wild-type exendin
`amino acid residues or may be occupied by specified amino
`acid substitutions. Formula I does not comprise a peptide 15
`sequence having Ser as the C-terminal amino acid and exen(cid:173)
`din agonists or analogues having specific amino acid dele(cid:173)
`tions and/or being conjugates as described herein, such as the
`novel compounds desPro36-exendin-4(1-39), exendin-4(1-
`39)-K6 or desPro36-exendin-4)1-39)-K6. Further, formula II 20
`of WO 99/25727 defines a peptide sequence similar to for(cid:173)
`mula I, but including exendin derivatives having a C(l-10)
`alkanoyl or cycloalkylalkanoyl substituent on lysine in posi(cid:173)
`tion 27 or 28. When treating inappropriate post-prandial
`blood glucose levels the compounds are administered fre- 25
`quently, for example one, two or three times a day. WO
`99/25728 discloses exendin agonists having a general for(cid:173)
`mula I that defines a peptide sequence offrom 28 to 39 amino
`acid residues with fixed Ala in position 18, and wherein the
`remaining positions may be occupied by wild-type exendin 30
`amino acid residues or may be occupied by specified amino
`acid substitutions. Said exendin agonists all correspond to a
`truncated exendin analogues having a varying degree of
`amino acid substitutions. Peptide sequences of from 34 to 38
`amino acid residues do not have Ser C-terminally. A peptide 35
`sequence of39 amino acid residues may have either Ser orTyr
`C-terminally, but no further residues. Exendin agonist or ana(cid:173)
`logues having specific amino acid deletions and/or being
`conjugates according to the invention described herein are not
`comprised by formula I. Further, formula II defines a peptide 40
`sequence similar to formula I, but including exendin deriva(cid:173)
`tives having a C(1-10)alkanoyl or cycloalkylalkanoyl sub(cid:173)
`stituent on lysine in position 27 or 28.
`WO 99/46283 (published 16.09.99) discloses peptide con(cid:173)
`jugates comprising a pharmacologically active peptide X and 45
`a stabilising peptide sequence Z of 4-20 amino acid residues
`covalently bound to X, where said conjugates are character(cid:173)
`ised in having an increased half-life compared to the half-life
`of X. X may be exendin-4 or exendin-3.
`
`4
`(a) an exendin having at least 90% homology to exendin -4;
`(b) a variant of said exendin wherein said variant comprises
`a modification selected from the group consisting of
`between one and five deletions at positions 34-39 and
`contains a Lys at position 40 having a lipophilic substitu(cid:173)
`ent; or
`(c) GLP-1 (7-36) or GLP-1 (7-37) having at least one
`modification selected from the group consisting of:
`(i) substitution of D-alanine, glycine or alpha-amino
`isobutyric acid for alanine at position 8 and
`(ii) a lipophilic substituent;
`and Z, a peptide sequence of 4-20 amino acid units covalently
`bound to said variant, wherein each amino acid unit in said
`peptide sequence, Z is selected from the group consisting of
`Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His,
`Met, Om, and amino acid units of the general formula I
`
`(I)
`wherein R 1 and R 2 are selected from the group consisting of
`hydrogen, cl-6-alkyl, phenyl, and phenyl-methyl, wherein
`C 1 - 6-alkyl is optionally substituted with from one to three
`substituents selected from halogen, hydroxy, amino, cyano,
`nitro, sulfono, and carboxy, and phenyl and phenyl-methyl is
`optionally substituted with from one to three substituents
`selected from cl-6-alkyl, c2-6-alkenyl, halogen, hydroxy,
`amino, cyano, nitro, sulfono, and carboxy, or R 1 and R2
`together with the carbon atom to which they are bound form
`a cyclopentyl, cyclohexyl, or cycloheptyl ring, e.g., 2,4-di(cid:173)
`aminobutanoic acid and 2,3-diaminopropanoic acid, with the
`proviso that X is not exendin-4 or exendin-3.
`The peptide X is further characterised in being effective in
`improving glucose tolerance in a diabetic mammal.
`Furthermore, the invention is directed to a novel variant of
`a parent exendin, wherein said parent exendin has an amino
`acid sequence having at least an 90% homology to exendin-4
`and wherein said variant lowers the blood glucose level in a
`mammal, binds to a GLP-1 receptor and has at least one
`modification selected from the group consisting of (a)
`between one and five deletions at positions 34-38, and (b)
`contains a Lys at position 40 having a lipophilic substituent
`attached to the epsilon amino group of said lysine.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`OBJECTIVE OF THE INVENTION
`
`There is a need for compounds that lower blood glucose
`levels in mammals, and are stable and effective. Therefore, it
`is an objective of the invention to provide novel compounds
`that lower blood glucose levels in mmals. Ideally, the