`Larsen et al.
`
`US0065284.86B1
`(10) Patent No.:
`US 6,528,486 B1
`(45) Date of Patent:
`Mar. 4, 2003
`
`(54) PEPTIDEAGONISTS OF GLP-1 ACTIVITY
`(75) Inventors: Bjarne Due Larsen, Brønshøj (DK);
`Jens Damsgaard Mikkelsen, Lyngby
`(DK); Soren Neve, Lyngby (DK)
`s
`s
`(73) Assignee: Zealand Pharma A/S, Glostrup (DK)
`(*) Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O. davs.
`(b) by 0 day
`
`(21) Appl. No.: 09/614,847
`(22) Filed:
`Jul. 12, 2000
`Related U.S. Application Data
`(60) Provisional application No. 60/143.591, filed on Jul. 12,
`1999.
`(51) Int. Cl." ................................................ A61K 38/00
`(52) U.S. Cl. ............................. s14/12 514/2, 530/300.
`530/303
`(58) Field of Search
`514/12, 2:530/300
`530/303
`
`- - - - - - - - - - - - - - - - - - - - - - -
`
`s as
`
`s
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,424,286 A
`6/1995 Eng .............................. 514/2
`5,545,618 A 8/1996 Buckley et al. ............... 514/12
`FOREIGN PATENT DOCUMENTS
`W SS 3E
`WO 98/11126
`* 3/1998
`WO 98/11126 A
`3/1998
`WO 98/22577 A
`5/1998
`WO 98/30231. A
`7/1998
`W 2.g:
`2.
`WO 35.
`A.
`WO 99/43707
`9/1999
`WO 99/43708
`9/1999
`WO 99/46283
`9/1999
`
`CO7K/1/107
`
`W
`WO
`WO
`WO
`WO
`W
`WO
`WO
`WO
`WO
`
`OTHER PUBLICATIONS
`J. Eng et al. Isolation and characterization of Exendin-4, an
`Exendin-3 analogue, from Heloderma Suspectum Venom.
`1992. J. Biol. Chem., 267(11): 7402–05.*
`C. Orskov, Diabetologia, 701 (1992).
`M. Nauck et al., Horm. Metab. Res., 411 (1997).
`Jean-Pierre Raufman, Reg. Peptides 61: 1-18 (1996).
`Janet A. Meurer, et al. “Properties of Native and In Vitro
`Glycosylated Forms Of The Glucagon-Like Peptide-1
`Receptor Antagonist Exendin (9–39)', Metabolism, vol. 48,
`p
`9.
`No. 6 (Jun.), 1999, pp 716–724.
`M.M. Byrne et al., European Journal of Clinical Investiga
`tion, 72 (1998).
`C.F. Deacon et al., Diabetologia, 271 (1998).
`C. Deacon et al., Diabetes, 764 (1998).
`U. Ritzel et al., Journal of Endocrinology, 93 (1998).
`R. Pederson et al., Diabetes, 1253 (1998).
`R. Goke et al., European Journal of Neuroscience, 2294
`(1995).
`-
`R. Goke et al., The Journal of Biological Chemistry, 19650
`(1993).
`-
`M. Pohl et al., The Journal of Biological Chemistry, 9778
`N.H. Greig et al., Diabetologia, 45 (1999).
`Y. Chen et al., The Journal of Biological Chemistry, 4108
`(1997).
`J.J. Holst, Annu. Rev. Physiol., 257 (1997).
`D. A. D'Alessio et al., J. Clin. Invest. (USA) 93 (5):2263-6
`(1994)
`* cited by examiner
`Primary Examiner-Lorraine Spector
`ASSistant Examiner-Dong Jiang
`(74) Attorney, Agent, or Firm-Robert L. Buchanan;
`Edwards & Angell, LLP
`
`(1998).
`
`(57)
`ABSTRACT
`The present invention relates to novel peptide conjugates
`which have increased Stability and are useful in the treat
`ment of exceSS levels of blood glucose.
`
`2 Claims, 8 Drawing Sheets
`
`MPI EXHIBIT 1065 PAGE 1
`
`MPI EXHIBIT 1065 PAGE 1
`
`
`
`U.S. Patent
`
`Mar.4, 2003
`
`Sheet 1 of 8
`
`US 6,528,486 B1
`
` od6nOOF-LGNNOdWOD—a—O€l
`
` 3NIMVS-|GNNOdWOOo—*
`
` od6n»-|GNNOdWOO— od6nOF-|GNNOdWOO—¥—Or
`od6n$0-|GNNOdNOd
`SYNOHb€ct°pTSo09
`reoe
`
`Ov|
`
`Och
`
`|“Old
`
`OOL
`
`06
`
`TSAR ONYG-SYd AO %
`
`MPI EXHIBIT 1065 PAGE 2
`
`MPI EXHIBIT 1065 PAGE 2
`
`MPI EXHIBIT 1065 PAGE 2
`
`
`
`U.S. Patent
`
`Mar. 4, 2003
`
`Sheet 2 of 8
`
`US 6,528,486 B1
`
`
`
`
`
`ENIT\/S - Z CINDOCHWOO --?
`
`A9 SDO-9ac O %
`
`MPI EXHIBIT 1065 PAGE 3
`
`MPI EXHIBIT 1065 PAGE 3
`
`
`
`U.S. Patent
`
`Mar.4, 2003
`
`Sheet 3 of 8
`
`US 6,528,486 B1
`
`od6n4-SGNNOdWOD—fiR
`
`
`od6n0OL-S$GNNOdNWOd—F—OLL
`46;OZod6n0001-SGNNOdWOd—#—
`di6n001-$GNNOdWOD—e—09
`od6nQL-§GNNOdWOo06
`ANITVS-S$GNNOdNWOD—*—OrL
`
`O€L
`
`OOL
`
`08
`
`OL
`
`os
`
`Or
`
`‘TSAR SNUYG-3yd AO %
`
`MPI EXHIBIT 1065 PAGE 4
`
`MPI EXHIBIT 1065 PAGE 4
`
`MPI EXHIBIT 1065 PAGE 4
`
`
`
`
`
`U.S. Patent
`
`Mar. 4, 2003
`
`Sheet 4 of 8
`
`US 6,528,486 B1
`
`
`
`(!!!) CINQOdWOO -
`
`?7 CINTOCHWOO O
`
`O
`
`1.
`
`O
`O
`O
`v
`
`O
`9
`(Wu) ONOO
`
`00000||
`
`1.
`
`?-00001
`
`
`
`(u?uu) EWIL
`
`?7 ’5)|–}
`
`08||
`
`????????????????????????????º
`
`MPI EXHIBIT 1065 PAGE 5
`
`MPI EXHIBIT 1065 PAGE 5
`
`
`
`U.S. Patent
`
`Mar. 4, 2003
`
`Sheet 5 of 8
`
`US 6,528,486 B1
`
`-O- COMPOUND 2
`-O- COMPOUND 14
`- V - COMPOUND 15
`- V - COMPOUND 16
`-- COMPOUND 18
`-H COMPOUND 19
`- A - COMPOUND (i)
`
`
`
`
`
`50--------------------------------------AA-A-A-\----A---------------------------------
`
`VEHICLE 0.001
`
`10
`
`100
`
`O.O1
`0.1
`1
`DOSE (nmol/kg i.p.)
`
`F.G. 5
`
`MPI EXHIBIT 1065 PAGE 6
`
`MPI EXHIBIT 1065 PAGE 6
`
`
`
`U.S. Patent
`
`Mar. 4, 2003
`
`Sheet 6 of 8
`
`US 6,528,486 B1
`
`
`
`
`
`
`
`MPI EXHIBIT 1065 PAGE 7
`
`MPI EXHIBIT 1065 PAGE 7
`
`
`
`U.S. Patent
`
`Mar.4, 2003
`
`Sheet 7 of 8
`
`US 6,528,486 B1
`
`ZSls (dt
`GNNOdWOD—O— ‘o'd
`
`‘d'1esooni6‘|GQNNOdWOD~M@—[__--
`
`‘odasoon)B(1!)
`
`asoon|6‘}GNNOdNIOD—e—
`OOLOLLLOLOO=8AIOIHSA
`
`
`
`By/fowu)aSo0q
`
`~-------OO!
`
`(ATOIHAA OL ASNOdSAY AO %) VY OFZ0a AY
`
`MPI EXHIBIT 1065 PAGE 8
`
`MPI EXHIBIT 1065 PAGE 8
`
`MPI EXHIBIT 1065 PAGE 8
`
`
`
`U.S. Patent
`
`Mar. 4, 2003
`
`Sheet 8 of 8
`
`US 6,528,486 B1
`
`
`
`
`
`
`
`140
`
`120
`
`CC)C)
`
`20
`
`ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ
`
`*
`
`*
`
`3,
`
`
`
`
`
`(ESNO&SEH ETOIHEA %) º?º ºººººonw
`
`O
`
`120 240 360 480 600 72O 840 960 1080 1200 1320 1440
`TIME OF DRUGADMINISTRATION (min BEFORE OGTT)
`
`*: p-0.05 VERSUS VEHICLE
`
`FIG. 8
`
`MPI EXHIBIT 1065 PAGE 9
`
`MPI EXHIBIT 1065 PAGE 9
`
`
`
`1
`PEPTIDEAGONSTS OF GLP-1. ACTIVITY
`
`This application claims benefit of U.S. Provisional appli
`cation 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 pharmacologi
`cally active and Stable, and as agonists of GLP-1 activity are
`useful in the treatment of diseases that benefit from regula
`tion or exceSS levels of blood 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 composi
`tion 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
`polypeptide (GIP) and glucagon-like peptide-1 (GLP-1).
`The most potent substance known is GLP-1 (0orskov, 1992,
`Diabetologia 35:701-711). Glucagon-like peptide 1 (GLP
`1) is a product of proglucagon, a 180 amino acid peptide
`(Drucker, 1998 Diabetes 47: 159-169). The overall sequence
`of proglucagon contains the 29-amino acid Sequences of
`glucagon, the 36 or 37 amino acid Sequence of GLP-1 and
`the 34 amino acid Sequence of glucagon-like peptide-2
`(GLP-2), an intestinotrophic 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 hormone. In addition, GLP-1 also lowers gluca
`gon concentrations, Slows gastric emptying, stimulates (pro)
`insulin biosynthesis, and enhances insulin Sensitivity
`(Nauck, 1997, Horm. Metab. Res. 47: 1253-1258). The
`peptide also enhances the ability for the B-cells to Sense and
`respond to glucose in Subjects with imparted glucose toler
`ance (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 Sensi
`tivity (D’Alessio, 1994, Eur. J. Clin. Invest. 28:72–78). This
`has placed GLP-1 as a promising agent for treatment of type
`II diabetes. Active fragments of GLP-1 have been found to
`be GLP-1(7–36) and GLP-1(7–37). However, a major phar
`macological 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). Several strategies circumventing
`this problem have been proposed, 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 al., 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
`glucose levels have Some sequence similarity (53%) to
`
`2
`GLP-17–36)NH. (Goke et al., 1993, J. Biol. Chem.
`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
`5 Heloderma horridum, the Mexican beaded lizard and
`exendin-4 is present in the Venom of Heloderma Suspectum,
`the Gila monster. Exendin-4 differs from exendin-3 at just
`positions two and three. The cDNA 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 al., 1998, J. Biol. Chem. 273:9778-9784 and
`WO98/35033). Both exendin-3 and exendin-4 stimulate an
`increase in cellular cAMP production in guinea pig pancre
`atic acinar cells by interacting with eXendin receptors
`(Raufman, 1996, Reg. Peptides 61:1-18). Exendin-3 causes
`a biphasic increase in cellular cAMP 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
`rat insulinoma cells (Goke et al., 1993, J. Biol. Chem.
`268: 19650–55). This is expected as the (His Ala) domain of
`GLP-1 recognised by DPP-IV is not present in exendin-4
`(Goke et al., 1993, J. Biol Chem. 268:19650–55). Binding of
`'IIGLP-1 to the nucleus of the solitary tract was inhibited
`concentration-dependently by unlabelled GLP-1 and
`Tyr39exendin-4 with Ki values of 3.5 and 9.4 nM
`respectively, and Similar values are found in cell lines (Goke
`et al., 1995, Eur. J. Neurosci. 7:2294-2300 and Goke et al.,
`1993, J. Biol. Chem. 268: 19650–55). Further, exendin-4
`given systemically lowers blood glucose levels by 40% in
`diabetic db/db mice (WO/99/07404). Recently, Grieg et al.
`(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 considerable portion of the
`N-terminal Sequence is essential in order to preserve insuli
`notropic activity (exendin-4(1-31) and Y-exendin-4
`(1-31)) whereas an N-terminally truncated exendin
`(exendin-4(9-39) has inhibitory properties.
`The use of eXendin-3, eXendin-4 and eXendin agonistS has
`been proposed for the treatment of diabetes mellitus, reduc
`ing gastric motility and delaying gastric emptying and the
`prevention of hyperglycemia (U.S. Pat. No. 5,424,286,
`WO98/0535) as well as for the reduction of food intake
`(WO98/30231). There has been proposed ways of obtaining
`novel compounds by modifying the native eXendin
`Sequences. One way is to attach lipophilic Substituents to the
`molecule, e.g. as described in WO99/43708 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 WO99/07404, WO
`99/25727 and WO 99/22728. WO99/074.04 discloses exen
`din agonists having a general formula I that defines a peptide
`sequence of 39 amino acid residues with Gly Thr in posi
`tions 4-5, Ser Lys GLn in positions 11-13, Glu Glu Glu Ala
`Val Arg 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 agonists or analogues having specific
`amino acid deletions and/or being conjugates as described
`herein, such as the novel compounds desPro-exendin-4
`
`US 6,528,486 B1
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`MPI EXHIBIT 1065 PAGE 10
`
`MPI EXHIBIT 1065 PAGE 10
`
`
`
`3
`(1–39), exendin-4(1-39)-K or desPro-exendin-4(1-39)-
`K. WO 99/25727 discloses exendin agonists having a
`general formula I that defines a 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 Sequence having Ser as the
`C-terminal amino acid and eXendin agonists or analogues
`having Specific amino acid deletions and/or being conjugates
`as described herein, such as the novel compounds desPro
`exendin-4(1-39), exendin-4(1-39)-K or desPro-exendin
`4)1–39)-K. Further, formula II of WO99/25727 defines a
`peptide Sequence Similar to formula I, but including eXendin
`derivatives having a C(1-10)alkanoyl or cycloalkylalkanoyl
`substituent on lysine in position 27 or 28. When treating
`inappropriate post-prandial blood glucose levels the com
`pounds are administered frequently, for example one, two or
`three times a day. WO99/25728 discloses exendin agonists
`having a general formula I that defines a peptide Sequence of
`from 28 to 39 amino acid residues with fixed 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. 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 Sequence of 39 amino acid
`residues may have either Ser or Tyr C-terminally, but no
`further residues. EXendin agonist or analogues having Spe
`cific 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 sequence
`Similar to formula I, but including eXendin derivatives
`having a C(1-10)alkanoyl or cycloalkylalkanoyl Substituent
`on lysine in position 27 or 28.
`WO 99/46283 (published 16.09.99) discloses peptide
`conjugates comprising a pharmacologically active peptide X
`and a Stabilising peptide Sequence Z of 4-20 amino acid
`residues covalently bound to X, where Said conjugates are
`characterised in having an increased half-life compared to
`the half-life of X. X may be exendin-4 or exendin-3.
`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 com
`pounds that lower blood glucose levels in mammals. Ideally,
`these should be effective when administered orally. It is a
`further object of the invention to provide novel peptide
`agonists of GLP-1 activity and/or exendin-4 activity. It is a
`Still further purpose of the invention to provide peptide
`agonists of GLP-1 activity and/or exendin-4 activity having
`an increased half-life and/or a decreased clearance.
`SUMMARY OF THE INVENTION
`The invention is directed to a peptide conjugate compris
`ing a peptide X Selected from the group consisting of
`(a) an exendin having at least 90% homology to exendin
`4,
`60
`(b) a variant of Said exendin wherein said variant com
`prises 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
`Substituent; or
`(c) GLP-1 (7-36) or GLP-1 (7-37) having at least one
`modification Selected from the group consisting of:
`
`4
`(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, Gln, Asp, Glu,
`LyS, Arg, His, Met, Orn, and amino acid units of the general
`formula I
`
`wherein R and Rare selected from the group consisting of
`hydrogen, C-alkyl, phenyl, and phenyl-methyl, wherein
`Co-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 C-alkyl, C-alkenyl, halogen, hydroxy,
`amino, cyano, nitro, Sulfono, and carboxy, or R' and R'
`together with the carbon atom to which they are bound form
`a cyclopentyl, cyclohexyl, or cycloheptyl ring, e.g., 2,4-
`diaminobutanoic 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 Sub
`Stituent attached to the epsilon amino group of Said lysine.
`BRIEF DESCRIPTION OF THE FIGURES
`FIG. 1 shows the effect of Compound 1 (SEQ ID NO:101)
`(des PRO-exendin-4(1-39)-NH) on blood glucose levels
`of mice, cf. Example 25.
`FIG. 2 shows the effect of Compound 2 (SEQ ID NO:93)
`(des PRO-exendin-4(1-39)-Lys-NH on the blood glu
`cose levels of mice, cf. Example 25.
`FIG.3 shows the effect of Compound 5 (SEQ ID NO:89)
`(Gly, Lys7 (palmitoyl)-GLP1-(7–36)(Human)-(Lys),-NH
`on the blood glucose levels of mice, cf. Example 25.
`FIG. 4 shows in vivo degradation kinetics in rabbits after
`i.V. injection of 1 um/kg of Compound 4 and Compound
`(iii), cf. Example 27.
`FIG. 5 is a plot of AUC (area under the curve) values
`(meani-SEM) for Compounds, 2,14-16, 18 and 19 in an oral
`glucose tolerance test (OGTT), cf. Example 28.
`FIG. 6 shows a synthetic cDNA constructed for heterlog
`expression of Compound 2 in yeast. The new construct was
`designated pYES0010, cf. Example 20.
`FIG. 7 is a plot of dose-response on GTT in db/db mice
`based on relative AUCo-o , values (meantSEM) for
`Compound 2 and Compound (i), cf. Example 29.
`FIG. 8 shows the effects of a maximal dose of Compound
`2, i.e. 100 nmol/kg i.p., on the oral glucose tolerance test
`(OGTT) when administered up to 24 hours before the
`OGTT.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The compounds of the present invention include hitherto
`unknown deletion variants of a parent eXendin. In contrast to
`
`US 6,528,486 B1
`
`1O
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`MPI EXHIBIT 1065 PAGE 11
`
`MPI EXHIBIT 1065 PAGE 11
`
`
`
`15
`
`S
`know Substitution and/or truncation variants of eXendin-4
`(1–39) the novel compounds are believed to exhibit a
`Stabilised alpha-helix Structure with Superior Stability prop
`erties and unreduced or enhanced binding properties.
`Moreover, conjugation of the novel variants, modified GLP
`1(7–36)-NH, and modified GLP-1-(7–37) to specific short
`peptide sequences (Z) render Stability to these compounds
`without compromising the pharmacological properties.
`These conjugations confer in Vivo Stability and hydrophi
`licity to the peptide molecule. The Z is composed of
`amino-acid residues, and has alone no structural character
`istics in terms of C-helix conformation. However, from
`Studies using both circular dichroism and nuclear magnetic
`resonance (NMR) spectroscopy, addition of Z dramatically
`alters the Structural characteristics of Some peptides as
`evidenced by the increased amount of C-helix conformation
`in the peptide. For example, circular dichroism, demon
`strated that a Z-modified (Gly)-GLP-1 had much more
`C-helix conformation than (Gly)-GLP-1. Together with the
`pharmacological results, the Structural analyses Suggest that
`Z is modifying the conformation of the peptide leading to
`higher enzyme-Stability, but without losing its potency. Also
`the physical and chemical properties of peptides may be
`altered considerably by Z-modification with resulting
`impact on pharmacological formulation Strategy.
`Exendin Variants
`The eXendin variant of the present invention is a variant
`of a parent eXendin peptide having at least about 90%
`homology and most preferably at least about 95% to
`exendin-4, which have eXendin activity, e.g., lowers the
`blood glucose level in a mammal and binds to a GLP-1
`receptor. In a preferred embodiment, the parent exendin
`peptide has an amino acid Sequence which differs by five
`amino acids, preferably by four amino acids, more prefer
`ably by three amino acids, even more preferably by two
`amino acids, and Still more preferably by one amino acid
`residue from the amino acid sequence of exendin-4)1–39).
`In one embodiment, the eXendin variant comprises
`between one and five deletions at positions 34-38. Prefer
`ably the variant comprises between 1 and 4 deletions at
`positions 34-38, more preferably between 1 and 3 deletions
`at positions 36-38. Preferably the parent exendin is exendin
`4, and a preferred variant included as peptide X in the
`peptide conjugates herein has an amino acid Sequence
`wherein 1, 2 or 3 of the Pro residues in positions 36, 37 and
`38 have been deleted from the amino acid sequence of
`exendin-4 and preferably from the amino acid Sequence of
`exendin-4(1-39).
`Coupling of a Z, Sequence to the X peptide herein is
`believed to increase the stability of these compounds. Pro
`line is a rigid amino acid that may interfere with the effect
`of Z to stabilise the structure of the X peptide. Deletion of
`one, two or all of the proline amino acids in positions 36, 37
`and 38 of the exendin backbone is therefore preferred in the
`peptide conjugates comprising a variant of a parent exendin
`according to the invention, as long as the efficacy of Said
`conjugates as measured in, e.g. an oral glucose tolerance test
`(OGTT) in diabetic db/db mice, is not negatively affected.
`In another embodiment, the variant comprises an addi
`tional residue at position 40, a lysine residue which com
`60
`prises a lipophilic Substituent bound to the epsilon amino
`group of lysine via an amide bond. The lipophilic Substituent
`may be the acyl group of a Straight-chain or branched fatty
`acid or a Straight-chain or branched alkane C,c)-dicarboxylic
`acid. The acyl group may have the formula CH3(CH2),
`CO-, wherein n is a integer from 4-38 and preferably from
`4-24. In a Specific embodiment, the acyl group is
`
`45
`
`50
`
`55
`
`65
`
`US 6,528,486 B1
`
`25
`
`35
`
`40
`
`6
`Selected from the group consisting of CH3(CH2)6CO-,
`CH(CH(CO-, CH (CH)CO-, CH (CH)CO-,
`CH(CH2)CO-, CH(CH2)CO-, CH(CH2)CO-,
`CH(CH2)CO-, and CH-(CH2)CO-. The acyl group
`may have the formula HOOC(CH2)CO-, wherein n is an
`integer from 4-38 and preferably from 4-24. In a specific
`embodiment, the acyl group is Selected from the group
`consisting of HOOC(CH2)CO-, HOOC(CH2)CO-,
`HOOC(CH2)CO-, HOOC(CH2)CO- and HOOC
`(CH2)2CO-. In a more specific embodiment, the lipophilic
`Substituent is Selected from the group consisting of
`tetradecanoyl, co-carboxynonadecanoyl, 7-deoxycholoyl,
`choloyl, palmitoyl and lithocholyl. In a most Specific
`embodiment, the lipophilic Substituent is palmitoyl.
`Alternatively, the liphophilic substituent may have an NH
`group. Specific embodiments include but are not limited to
`the formulae CH(CH2)((CH),COOH)CHNHCO(CH)
`CO- wherein a and b are integers and a+b is an integer of
`from 8 to 33, preferably from 12 to 28;
`CH(CH2)CONHCH(COOH) (CH)CO- wherein c is an
`integer of from 10 to 24;
`CH(CH),CONHCH(CH) (COOH)CO- wherein d is an
`integer of from 8 to 24;
`COOH(CH2)CO- whereine is an integer of from 8 to 24;
`—NHCH(COOH)(CH.)NHCO(CH),CH, wherein f is a
`integer of from 8 to 18;
`–NHCH(COOH)(CH.)NHCOCH(CH),COOH)NHCO
`(CH2)CH, whereing is an integer of from 10 to 16; and
`-NHCH(COOH)(CH)NHCO)CH)-CH(COOH)
`NHCO(CH), CH, wherein his an integer of 0 or from 1
`to 22 and preferably from 10 to 16.
`The eXendin variants having a lysine residue at position
`40 carrying a lipophilic Substituent optionally further com
`prise between one and five deletions, preferably between one
`and three deletions, at positions 34 to 39, preferably at
`positions 34–38, such as des Ser', Lys" (palmitoyl)
`exendin-4(1-39), des Pro, Lys" (palmitoyl)exendin-4
`(1–39) and des Pro, Lys" (palmitoyl)exendin-4(1-40).
`The variant may be in a most Specific embodiment
`Selected from the group consisting of:
`Compound 1: des Pro-exendin-4(1-39)-NH. (SEQ ID
`NO:101),
`des Pro-exendin-4(1-40)-NH2,
`Compound 14: des Pro, Pro7, Profexendin-4(1-39)-
`NH,
`des Pro, Pro7, Pro-exendin-4(1-40)-NH.,
`des Pro, Pro7-exendin-4(1-39)-NH,
`des Ala-exendin-4(1-39)-NH. (SEQ ID NO:105),
`des Gly-exendin-4(1-39)-NH. (SEQ ID NO: 106),
`des Ser’-(Lys' (palmitoyl))exendin-4(1-39)-NH. (SEQ
`ID NO:107),
`des Gly-(Lys' (palmitoyl))exendin-4(1-39)-NH. (SEQ
`ID NO:108),
`des Ala-(Lys" (palmitoyl))exendin-4(1-39)-NH. (SEQ
`ID NO:109),
`des Pro-(Lys' (palmitoyl))exendin-4(1-39)-NH. (SEQ
`ID NO:110), and the free acid thereof and a pharmaceu
`tically acceptable Salt thereof.
`Modified GLP-1
`A preferred modified GLP-1 included as peptide X in the
`peptide conjugates herein has an amino acid Sequence of
`GLP-1 (7–36)-NH or GLP-1 (7–37) having a substitution
`of glycine for alanine at position 8. Alternatively, a preferred
`modified GLP-1 has an amino acid sequence of GLP-1
`(7–36) or GLP-1 (7-37) having a substitution of glycine for
`alanine at position 8 and a lipophilic Substituent, preferably
`palmitoyl, on one lysine residue at position 26, 34 or 37. The
`
`MPI EXHIBIT 1065 PAGE 12
`
`MPI EXHIBIT 1065 PAGE 12
`
`
`
`US 6,528,486 B1
`
`15
`
`25
`
`7
`lipophilic Substituent is preferably attached to the epsilon
`amino group of Said lysine and includes the Specific embodi
`ments described above for the exendin variants. The modi
`fied GLP-1(7–36) or GLP-1(7–37) used as X in the conju
`gates of the invention may be those cited in WO99/43707
`and WO 98/08871 comprising a lipophilic substituent or,
`more preferably those GLP-1 analogues having a glycine
`substitution at position 8. Preferred peptides X are
`Gly-GLP-1(7–36),
`Gly-GLP-1(*7–37), and
`Gly-GLP-1(7–36)-Lys7 (palmitoyl).
`The compounds of the invention having a lipophilic
`Substituent would have a more protracted profile of action
`than the parent peptides as demonstrated for GLP-1 deriva
`tives in WO 98/O8871.
`Peptide conjugates
`The peptide Sequence Z may be bound to the C-terminal
`or the N-terminal of the peptide Sequence, X or two peptide
`sequences may be bound individually to both the C- and
`N-terminal of X. In case the native peptide X possesses a
`free C-terminal carboxylic acid, the peptide Sequence Z may
`be attached to either the C-terminal of the peptide X or to the
`N-terminal of the peptide X, or the C- and N-terminal of X
`may both be bound to each individual peptide Sequence Z.
`Alternatively, Z may be bound to the nitrogen atom on the
`Side chain of lysine, histidine or arginine or a carbonyl
`function on the Side chain of glutamic acid or aspartic acid
`anywhere with the peptide Sequence X. In one embodiment,
`Z may be attached to X within the sequence and to the N
`and/or C-terminal of X. Whether the sequence should be
`attached to the peptide Sequence X as its C-terminal, at its
`N-terminal, or both, or within the peptide Sequence X
`depends on the Specific peptide X and can be easily deter
`mined by the person skilled in the art. Preferably, X is bound
`to Z via a peptide bond and preferably at the C-terminal of
`X.
`One aspect of the invention is directed to a peptide
`conjugate comprising a peptide X which reduces the blood
`glucose level in a mammal, wherein X is (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 between One and five
`deletions at positions 34–39 and contains a Lys at position
`40 having a lipophilic substituent; 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 isobutyrel acid (Aib) for alanine at position
`8 and (ii) a lipophilic Substituent; and Z, a peptide sequence
`of 4-20 amino acid units covalently bound to X, wherein
`each amino acid unit in Said peptide Sequence Z is Selected
`from the group consisting of Ala, Leu, Ser, Thr, Tyr, ASn,
`Gln, Asp, Glu, LyS, Arg, His, Met, Orn, and amino acid units
`of the general formula I
`
`8
`Z is typically a peptide Sequence of 4-20 amino acid
`residues, e.g., in the range of 4-15, more preferably in the
`range of 4-10 in particular in the range of 4-7 amino acid
`residues, e.g., of 4, 5, 6, 7, 8 or 10 amino acid residues,
`where 6 amino acid residues are preferred. Preferably, Z
`contains at least one Lys residue. In a preferred embodiment
`of the invention each of the amino acid residues in the
`peptide Sequence Z are independently Selected from the
`group consisting of Ala, Leu, Ser, Thr, Tyr, ASn, Gln, Asp,
`Glu, LyS, Arg, His, Met, Orn, diaminobutanoic acid and
`diaminopropanoic acid. Preferably, the amino acid residues
`are Selected from Glu, LyS, and Met, especially LyS, or the
`amino acid resides are Selected from the group consisting of
`ASn, Glu and LyS. The above-mentioned amino acids may
`have either D- or L-configuration, but preferably the above
`mentioned amino acids have an L-configuration. In a pre
`ferred embodiment of the invention Z contains at least 1
`lysine residue or when Z is attached via a peptide bond to the
`N-terminal of Said peptide X then Z has an amino acid
`Sequence Selected from the group consisting of AS-(Glu)n
`wherein n is an integer from 3 to 7.
`Thus, illustrative examples of the peptide Sequence Z are:
`Lys-Lys-Lys-Lys (SEQ ID NO:1), Xaa-Lys-Lys-Lys, Lys
`Xaa-Lys-LyS, Lys-Lys-Xaa-LyS, Lys-Lys-Lys-Xaa, Xaa
`Xaa-Lys-LyS, Xaa-Lys-Xaa-LyS, Xaa-Lys-Lys-Xaa, LyS
`Xaa-Xaa-LyS, LyS-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa, Xaa
`Xaa-Xaa-LyS, Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa, LyS
`Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa (SEQ ID NO:2), Lys
`Lys-Lys-Lys-Lys (SEQ ID NO:3), Xaa-Lys-Lys-Lys-Lys
`(SEQ ID NO:4), Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:5),
`Lys-Lys-Xaa-Lys-Lys (SEQID NO:6), Lys-Lys-Lys-Xaa
`Lys (SEQ ID NO:7), Lys-Lys-Lys-Lys-Xaa, Xaa-Xaa
`Lys-Lys-Lys, Xaa-Lys-Xaa-Lys-LyS, Xaa-Lys-Lys-Xaa
`LyS, Xaa-Lys-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys-LyS, LyS
`Xaa-Lys-Xaa-LyS, Lys-Xaa-Lys-Lys-Xaa, Lys-Lys-Xaa
`Xaa-LyS, Lys-Lys-Xaa-Lys-Xaa, Lys-Lys-Lys-Xaa-Xaa,
`Lys-Lys-Xaa-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Xaa, Lys-Xaa
`Xaa-Lys-Xaa, LyS-Xaa-Xaa-Xaa-LyS, Xaa-Lys-Lys-Xaa
`Xaa, Xaa-Lys-Xaa-Xaa-LyS, Xaa-Xaa-Lys-Lys-Xaa, Xaa
`Xaa-Lys-Xaa-LyS, Xaa-Xaa-Xaa-Lys-LyS, Lys-Xaa-Xaa
`Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa
`Xaa, Xaa-Xaa-Lys-Xaa, Xaa-Xaa-Xaa-Xaa-LyS, Xaa
`Xaa-Xaa-Xaa-Xaa (SEQID NO8), Lys-Lys-Lys-Lys-Lys
`Lys (SEQ ID NO:9), Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID
`NO:10), Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:11),
`Lys-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:12), Lys-Lys
`Lys-Xaa-Lys-Lys (SEQ ID NO:13), Lys-Lys-Lys-Lys
`Xaa-Lys (SEQ ID NO:14), Lys-Lys-Lys-Lys-Lys-Xaa
`(SEQ ID NO:15), Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID
`NO:16), Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:17),
`Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:18), Xaa, Lys
`Lys-Lys-Xaa-Lys (SEQ ID NO:19), Xaa-Lys-Lys-Lys
`Lys-Xaa (SEQ ID NO:20), Lys-Xaa-Xaa-Lys-Lys-Lys
`(SEQ ID NO:21), Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID
`NO:22), Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:23),
`Lys-Xaa-Lys-Lys-Lys-Xaa (SEQ ID NO:24), Lys-Lys
`Xaa-Xaa-Lys-Lys (SEQ ID NO:25), L