(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll 111111111111111 IIIII IIII I 1111111111 11111 lllll lllll llll 1111111111111111111
`
`(43) International Publication Date
`27 December 2001 (27.12.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/98331 A2
`
`(81) Designated States (national) : AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, IX, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
`SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA,
`zw.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZVf), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`with sequence listing part of description published sepa(cid:173)
`rately in electronic form and available upon request from
`the International Bureau
`
`For two-letter codes and other abbreviations, refer lo the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazelle.
`
`(51) International Patent Classification 7:
`
`C07K 14/00
`
`(21) International Application Number: PCT/US0 l/16474
`
`(22) International Filing Date:
`
`1 June 2001 (01.06.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/212,171
`60/240,349
`
`16 June 2000 (16.06.2000) US
`13 October 2000 (13.10.2000) US
`
`(71) Applicant (for all designated States except US): ELI
`LILLY AND COMPANY [US/US); LiJiy Corporate
`Center, Indianapolis, IN 46285 (US).
`
`_
`-
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): GLAESNER, Wolf(cid:173)
`
`46254 (US). MILLICAN, Rohn, Lee [US/US); 5319 Deer
`
`= gang [DE/US]; 7512 Fieldstone Court, lndianapolis, IN
`= Creek Avenue, Indianapolis, IN 46254 (US).
`==
`--== == --=
`
`iiiiiiii
`
`(74) Agents: STEWART, Mark, J., et al.; Eli Lilly and Com-
`pany, Lilly Corporate Center, Indianapolis, IN 46285 (US).
`
`-
`
`iiiiiiii -iiiiiiii
`
`iiiiiiii
`
`iiiiiiii
`
`~
`~
`~
`00
`...._
`0',
`~ ----------------------------------------
`= (54) Title: GLUCAGON-LIKE PEPTIDE-I ANALOGS
`0 > (57) Abstract: Disclosed are glucagon-like peptide-I (GLP-1) compounds with modifications at one or more of the following posi-
`
`~ tions: 11, 12, 16, 22, 23, 24, 25, 27, 30, 33, 34, 35, 36, or 37. Methods of treating these GLP-1 compounds are also disclosed.
`
`FRESENIUS EXHIBIT 1045
`Page 1 of 69
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`

`

`WO 01/98331
`
`PCT/US0l/16474
`
`GLOCAGON-LII<E PEPTIDE-1 ANALOGS
`
`This application claims the benefit of U.S . Provisional
`5 Application Number 60/212,171, filed June 16, 2000 and U.S.
`Provisional Application Number 60/240,349, filed October 13,
`2000.
`
`15
`
`Glucagon- Like Peptide 1 (GLP- 1) is a 37 amino acid
`10 peptide that is secreted by the L-cells of the intestine in
`response to food ingestion .
`It has been found to stimulate
`insulin secretion (insulinotropic action), thereby causing
`glucose uptake by cells and decreased serum glucose levels
`(see, e g., Mojsov, S. , Int . J. Peptide Protein Research,
`.!Q:333-343 (1992)). Howeverl GLP- 1(1-37) is poorly active
`and attention has been focused on truncated analogs,
`referred to as GLP compounds, which are biologically much
`more potent than GLP-1. Examples include GLP- 1(7- 37), GLP-
`1(7-36)NH2, Gly8- GLP- 1(7-37)0H and Ser34 - GLP-1(7- 37)0H.
`20 Because of their ability to stimulate insulin secretion, GLP
`compounds show great promise as agents for the treatment of
`diabetes, obesity, and related conditions.
`GLP-1 compounds can exist in at least two different
`forms. The first form is physiologically active and
`25 dissolves readily in. aqueous solution at physiological pH
`(7.4).
`In contrast, the second form has little or no
`insulinotropic activity and is substantially insoluble in
`water at pH 7 . 4. Unfortunately, the inactive form is
`readily produced when aqueous GLP-1 solutions are agitated,
`exposed to hydrophobic _ surfaces or have large air/water
`
`30
`
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`

`

`WO 01/98331
`
`PCT/US0l/16474
`
`-2-
`
`interfaces . The tendency to convert to the insoluble form
`considerably complicates the production of commercial
`quantities of active GLP- 1 compounds ; mixing operations or
`continuous movement through a pump are common operations in
`bulk manufacturing processes and these operations cause the
`agitation, air/water interfaces and/or contact with
`hydrophobic surfaces that results in the insoluble form .
`Conversion to the inactive form may also occur during
`storage or after administration to a patient , further
`complicating the use of these compounds as drugs .
`Therefore, there is a great need for biological ly active
`GLP- 1 a nalogs which convert less readily to· the insoluble
`form than currently available GLP- 1 compounds .
`
`It has now been found that a number of GLP- 1 analogs
`with modifications at one or more of the following
`positions : 11 , 12, 16 , 22, 23 , 24 , 26, 27, 30 , 33, 34 , 35 ,
`36 or 37 , show markedl y decreased propensity to aggregate
`compared with GLP-1 (7 - 3:)OH .
`Many of these analogs retain GLP- 1 recept or activation
`that is comparable and in some cases- greater than known GLP-
`1 compounds such as GLP- 1(_7- 37}OH and ValB- GLP- 1(7- 37)OH .
`For exampl e ,
`t he aggregation t i me of va1B - G1u22- GLP(7 - 37)0H
`is over t wenty fold greater and its GLP- 1 receptor
`activation is about 25% greater than GLP- 1(7 - 37)OH. Based on
`. t hese discover ies , novel GLP- 1 compound s and methods of
`treatment using t he novel GLP- 1 compounds are di sclosed
`herein .
`One embodiment of the present invention i s a
`polypeptide having the amino acid sequence of formula I
`ID NO: 1} :
`
`(SEQ
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
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`
`- 3-
`
`His- Xaaa-Glu-Gly-Xaa11-Xaa12- Thr- Ser- Asp-Xaa16- Ser(cid:173)
`Ser- Tyr- Leu-Glu-Xaa22-Xaa2rXaa24- Ala-Xaa2G-Xaa27 - Phe(cid:173)
`Ile-Ala- Xaa31-Leu-Xaa33-Xaa3rXaa3s- Xaa3G- R
`formula I
`(SEQ ID NO : 1)
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`wherein :
`Gly, Ala , Val , Leu , Ile, Ser, or Thr ;
`Xaaa
`is :
`Asp , Glu, Arg, Thr , Al a , Lys , or Hi s ;
`is :
`Xa a11
`His , Tr p, Phe , or Tyr;
`is :
`Xaa12
`is : Leu , Ser , Thr , Trp, His , Phe , Asp , Val , Glu,
`Xa a16
`or Ala ;
`Xaa22 is : Gly, Asp, Glu, Gln, Asn, Lys , Arg, Cys, or Cystei c
`Acid;
`is :
`His, Asp, Lys , Glu, or Gln ;
`is :
`Glu , His , Ala , or Lys ;
`is :
`Asp, Lys ,
`Glu , or His ;
`i s :
`Ala , Glu,
`His, Phe, Tyr , Trp, Arg, or Lys ;
`is :
`Ala, Gl u ,
`Asp, Ser , or His ;
`Asp , Arg ,
`is :
`Val , Lys , Ala , Gly, or Glu ;
`is :
`Gl u , Lys,
`or Asp;
`is : Thr , Ser, Lys , Arg, Trp, Tyr , Phe , Asp , Gl y, Pro ,
`His , or Glu ;
`Xaa3G is : Arg, Gl u , or His ;
`R is : Lys , Arg , Thr , Se r , Glu , Asp , Trp, Tyr , Phe , His ,
`-NH2, Gly, Gly- Pro , or Gly-Pro-NH2, or is deleted .
`
`Xaa23
`Xaa24
`Xaa2G
`Xaa21
`Xaa30
`Xaa33
`Xaa34
`Xaa35
`
`provided t hat the polypeptide doe s not have the
`sequence of GLP-1(7-37)0H or GLP- 1(7-36) - NH2 a nd provided
`t hat t he polypeptide is not Gl y8- GLP-1(7-37)0H, Gly8- GLP-
`1 (7 - 36) NH2 , Val 8-GLP- 1(7-37)0H , Val 8-GLP- 1(7- 36)NH2, Leu8 -
`GLP-1(7- 37)0H, Leu8-GLP-1(7-36)NH2, Ile 8-GLP-1(7-37)0H, Ile8 -
`GLP- 1 (7 - 36) NH2 , Ser8-GLP- 1(7-37)0H, Ser8- GLP-1 (7 - 36) NH 2 ~
`Thr8 -GLP.:.1 (7- 37 ) OH, o r Thr8-GLP- 1 (7 - 36) NH2, Ala11-Glp- 1 (7-
`37) OH , Ala11-Glp- 1 (7- 36) NH2 , Ala16- Glp- 1 (7- 37) OH, Ala16-Glp-
`
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`
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`
`-4-
`1{7 -36)NH2, Ala27-Glp- 1( 7- 37)OH, Ala27-Glp- 1{7-36)NH2, Glu27-
`Glp-1(7-37)OH, Glu27-Glp-1(7-36)NH2, Ala33- Glp-1{7- 37)OH, or
`Ala 33- Glp- 1 (7- 36) NH2.
`
`5
`
`invention is a
`Another embodiment of the present
`polypeptide having the amino acid sequence of formula II
`( SEQ I D NO: 2 ) :
`
`10
`
`His-Xaa8-Glu-Gly-Thr-Xaa12-Thr-Ser-Asp- Xaa16- Ser(cid:173)
`Ser-Tyr-Leu-Glu-Xaa22-Xaa23- Ala- Ala- Xaa26-Glu-Phe-
`Ile- Xaa30- Trp-Leu-Val- Lys - Xaa35-Arg- R
`formula II (SEQ ID NO: 2 )
`
`wherein:
`Xaaa is : Gly, Ala, Val, Leu, Ile, Ser , or Thr;
`15 Xaa12 is: His , Trp, Phe, or Tyr;
`Xaa16 is: Leu , Ser, Thr, Trp,· His, Phe, Asp, Val, Glu,
`or Ala;
`Xaa22 is: Gly, Asp, Glu, Gln , Asn, Lys, Arg, Cys, or Cysteic
`Acid;
`20 Xaa23 is : His, Asp, Lys, Glu, or Gln;
`Xaa26 is: Asp, Lys, Glu, or His;
`Xaa30 is: Ala, Glu, Asp, Ser, or His;
`Xaa3s is: Thr, Ser, Lys , Arg, Trp, Tyr, Phe , Asp , Gly, Pro,
`His, or Glu;
`R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His,
`~NH2 , Gly , · Gly-Pro, or Gly- Pro-NH2, or is deleted.
`
`25
`
`provided that the polypeptide does not have the sequence of
`GLP- 1{7-37)OH or GLP- 1(7- 36)-NH2 and provided that the
`30 polypeptide is not Gly8- GLP-1 {7-37) OH, Gly8-GLP- 1 {7- 36) NH2,
`Val8-GLP-1(7 - 37)OH, Val8-GLP- 1(7 -36)NH2, Leu8- GLP-1{7 - 37 )OH,
`Leu8-GLP-1(7-36)NH2 , Ile8- GLP- 1{7-37)OH, Ile8- GLP- 1(7 - 36)NH2 ,
`Ser8-GLP-1{7 - 37)OH, Ser8-GLP- 1(7- 36)NH2 , Thr8-GLP- i(7-37)0H,
`Thr8- GLP- 1(7- 36)NH2, Ala16- GLP(7- 37)OH, or Ala16-Glp-1(7-
`36)NHz.
`
`35
`
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`

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`
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`
`-5-
`
`Another embodiment of the present invention is a
`polypeptide having the amino acid sequence of formula I II
`(SEQ ID NO: 3) :
`
`5
`
`Hi s - Xaa8- Glu-Gly-Thr- Phe- Thr - Ser-Asp-Val - Ser- Ser(cid:173)
`Tyr - Leu- Glu- Xaa22- Xaa2rAla-Ala-Lys-Xaa21- Phe-Ile(cid:173)
`Xaa30- Trp-Leu- Val- Lys- Gly-Arg- R
`f ormula II I
`(SEQ ID NO: 3)
`
`10 wherein :
`Xaa 8 is : Gly , Ala , Val, Leu, Ile, Ser , or Thr ;
`Xaa22 is : Gly, Asp, Glu, Gln, Asn , Lys , Arg , Cys , or Cysteic
`Acid;
`Xaa23 is : His , Asp, Lys, Glu , or Gln ;
`Xaa21 is : Ala, Gl u, His, Phe , Tyr , Trp, Arg , o r Lys
`Xaa30 i s : Ala, Glu, Asp, Ser, or His ;
`R is: Lys , Arg, Thr , Ser, Glu , Asp, Trp, Tyr , Phe , His ,
`-NH2, Gly, Gly-Pro , or Gly-Pro- NH2, or is deleted .
`
`15
`
`20
`
`35
`
`provided that the polypeptide does not have the
`sequence of GLP-1(7 - 37)·OH or GLP-1 (7 - 36)-NH2 and provided
`that the polypeptide is not Gly8- GLP- 1(7-37)OH, Gly8- GLP-
`1(7-36)NH2 , Val8- GLP-1(7- 37)OH, Val8-GLP-1(7-36)NH2, Leu8 -
`GLP-1 (7 -37 )OH, Leu8- GLP- 1(7 - 36)NH2, Ile8-GLP- 1(7 -37)OH, Ile8
`25 GLP-1 (7- 36) NH2, Ser8- GLP- 1 (7-37) OH, Ser 8-GLP- l (7-36) NH2 ,
`Thr8- GLP-1(7 -37)OH , Thr8-GLP-1(7 - l6)NH2, Ala16-Glp-1 (7 - 37)OH,
`Ala16-Glp- 1 (7 - 36)NH2, Glu27-Glp- l (7-37)OH, or Gl u27.:.Glp - 1 (7 -
`36) NH2,
`Another embodiment of the present invention is a
`30 polypeptide having the amino acid sequence of formula IV
`(SEQ ID NO : 4):
`Xaa7- Xaa 8- Glu-Gly- Thr- Phe- Thr-Ser-Asp- Val- Ser(cid:173)
`Ser-Tyr-Leu- Glu-Xaa22-Gln-Ala- Ala-Lys -Glu-Phe(cid:173)
`Ile- Ala- Trp- Leu-Val- Lys- Gly-Arg- R
`(SEQ ID NO : 4)
`
`-
`
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`

`WO 01/98331
`
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`
`- 6-
`
`wherein:
`Xaa 7 is L-histidine , D-histidine, desamino- histidine,
`
`2amino-histidine , P-hydroxy-histidine , homohistidine, a -
`
`5
`
`10
`
`fluoromethyl-histidine or a-methyl- histidine;
`Xaa 8 is glycine, alanine , valine , leucine , isoleucine,
`serine or threonine. Preferably, Xaa8 is glycine, valine,
`leucine, isoleucine, serine or threonine;
`Xaa22 is aspartic acid, glutamic acid , glutarnine,
`asparagine, lysine, arginine, cysteine, or cysteic acid .
`R is -NH2 or Gly(OH).
`Another embodiment of the present invention is a
`glucagon-l ike peptide- 1 (GLP-1) compound having an amino
`acid other than alanine at posit.ion 8 and an amino acid
`15 other than glycine at position 22.
`Another embodiment of the present invention is a method
`of stimulating the GLP-1 receptor in a subject in need of
`GLP- 1 receptor stimulation. The method comprises the step
`of administering to the subject an effective amount of the
`20 GLP-1 compounds described herein or the polypeptide having
`the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO : 2, SEQ
`ID NO:3 , SEQ ID NO:4.
`Yet another embodiment of the present invention is the
`GLP- 1 compounds described herein or the polypeptide having
`the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ
`ID NO:3, or SEQ ID NO:4 for use in stimulating the GLP-1
`receptor in a subject in need of GLP- 1 receptor stimulation .
`The GLP- 1 compounds of the present invention retain (cid:173)
`GLP-1 receptor activation ability and, in addition, have
`30 decreased propensity to aggregate compared with other GLP- 1
`compounds. As a result, solutions of these compounds .can be
`agitated with minimal conversion ·to the insoluble , inactive
`form. This advantage greatly simplifies the manufacturing
`process. In addition, it is expected that little or no in
`
`25
`
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`

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`
`-7-
`
`vivo aggregation will occur after admini stration to
`patients , thereby increasing activity and minimizing the
`potential fo r adverse reactions .
`In addition, these GLP- 1
`compounds are resistant to diaminopeptidase IV degr adation
`and bind zinc and are therefore believed to provide extended
`time action in vivo.
`
`5
`
`10
`
`Figure 1 shows the amino acid sequences of Val8-Glu22_
`GLP- 1(7-37)OH (SEQ ID NO: 5), Val8- Asp22- GLP- 1(7 - 37)0H (SEQ
`ID NO : 6) , Val8-Arg22- GLP- 1(7- 37)0H (SEQ I D NO: 7) and Val8-
`Lys22- GLP-1(7- 37)0H (SEQ ID NO: 8).
`Figure 2 shows the ami no acid sequences of Gly8-Glu22_
`GLP- 1(7-37)OH (SEQ ID NO : 9) , Gly8- Asp22- GLP- 1(7 - 37)0H (S EQ
`ID NO : 10) , Gly8- Arg22- GLP- 1(7- 37)0H (SEQ ID NO : 11) and
`15 Gly8-Lys22-GLP- 1 (7 - 37)OH (SEQ ID NO : 12).
`Figure 3 shows the amino acid sequence of Val 8- Glu30-
`GLP- 1(7-37)OH (SEQ ID NO: 13), Gly8- Glu30- GLP- 1(7- 37)OH (SEQ
`ID NO : 14) , Val 8- His37- GLP-1(7-37)OH (SEQ ID NO : 15) , and
`Gly8-His37- GLP- 1(7- 37)OH (SEQ ID NO: 16).
`Figure 4 shows the ami no acid sequence of Val8- Glu22-
`Ala27- GLP-1(7- 37)0H (SEQ ID NO : 17)and Val 8- Lys 22- Glu23- GLP-
`1(7 - 37)OH (SEQ ID NO : 18).
`
`20
`
`25
`
`30
`
`A GLP- 1 compound is a polypeptide having from about
`twenty-five to about thirty- nine naturall y oc curring or non(cid:173)
`naturally occurri ng amino acids and has sufficient homology
`to GLP-1(7- 37)OH such that it exhibits insulinot ropic
`activity . Examples of non-naturally occurring ami no acids
`include a - methyl amino aci ds (e . g. , a-methyl a l a nine), D-
`amino acids , histidine- like amino acids (e .g., 2- amino(cid:173)
`histidine , P-hydroxy- histidine , homohistidi ne, a(cid:173)
`
`fluoromethyl - histidine and a-methyl- hi stidine) , amino acids
`having an e xtra methylene in the side chain ("homo" amino
`
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`

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`
`- 8-
`
`acids) and amino acids in which a carbox ylic acid functional
`group in the side chain is replaced with a sulfonic acid
`group (e.g. , cysteic acid). Preferably, however,
`t he GLP- 1
`compounds of the present invention comprise only naturally
`occurring amino aci ds except . as otherwise s pecifically
`provided herein .
`A GLP~l compound typically comprises a polypeptide
`having the amino acid sequence of GLP- 1(7- 37)OH, a n ana l og
`of GLP- 1 (7- 37)OH , a fragment of GLP- 1(7-37)OH o r a f r agment
`of a GLP- 1(7- 37)OH analog. GLP- 1(7 - 37)OH has the amino acid
`sequence of SEQ ID NO : 19 :
`
`7His- Ala- Gl u- 10Gly-Thr- Phe- Thr- Ser- 15Asp- Val - Ser- Ser(cid:173)
`Tyr- 20Leu- Glu- Gly-Gln- Ala - 25Ala- Lys- Gl u- Phe- I le- 30Ala(cid:173)
`Trp- Leu- Val- Lys- 35Gly- Arg- 37Gly
`(SEQ ID NO : 19)
`
`By custom in the art, the amino terminus of GLP- 1(7- 37)OH
`has been assigned number residue 7 and the carboxy- terminus ,
`number 37 . The other amino acids in t he pol ypeptide are
`numbered consecutively, as shown in SEQ ID NO : 19. For
`exampl e, position 12 is phenylalani ne and position 22 is
`glycine. When not specified, the C- terminal is i n the
`traditional car boxyl form .
`A "GLP-1 f r agment " is a polypeptide obtained. after
`truncation of one or more amino acids from the N-terminus
`and/or C-ter minus of GLP- 1(7 - 37)OH or a GLP- 1(7 - 37)OH
`analog . The nomenc lature used to describe GLP- 1 (7-37)OH
`carri es ove r to GLP-1 fragments. For example , GLP- 1(9- 36)OH
`denotes a GLP- 1 fragm~nt obtained by truncat ing two amino
`acids from the N-terminus and one amino acid from the c(cid:173)
`ter minus. The amino acids in the fragment are denoted by the
`same number as the corresponding amino acid in GLP- 1{7 -
`37)OH . For example, the N- terminal glutamic acid in GLP-
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
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`

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`
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`
`-9-
`
`5
`
`10
`
`15
`
`1(9- 36)OH is at position 9; position 12 is occupied by
`phenylalanine ; and position 22 is occupied by glycine, as in
`GLP- 1(7- 37)OH.
`"GLP-1 compound" also includes polypeptides in which
`one or more amino acids have been added to the N- terminus
`and/or C-terminus of GLP- 1(7 - 37)OH or fragments thereof.
`GLP- 1 compounds of this typ e have up to about thirty- nine
`amino aci ds. The amino acids i n t he "extended" GLP- 1
`compound are denoted by the same number as the corresponding
`amino acid in GLP- 1(7 - 37)OH. For example, the N- terminus
`amino aci d of a GLP- 1 compound obtained by adding two amino
`acids to the N- terminal of GLP- 1(7 - 37)OH is at position 5;
`and the C-terminus amino acid of a GLP- 1 compound obtained
`by adding one amino acids to the C- terminal of GLP- 1(7-37)OH
`is at position 38. Thus , position 12 is occupied by
`phenylalanine and position 22 is occupied by glycine in both
`of these "extended" GLP- 1 compounds, as in GLP- 1(7- 37)OH.
`Amino acids 1- 6 of an extended GLP- 1 compound are preferably
`the same as or a conservative substitution of the amino acid
`at the co r responding position of GLP- l (l- 37)OH . Amino acids
`38-45 of an extended GLP- 1 compound are preferably the same
`as or a conser vat ive substitution of the amino acid at the
`corresponding position of glucagon or exendin- 4 .
`A "GLP- 1 analog" has sufficient homology to GLP-1(7-
`37)OH or a fragment of GLP-1(7-37)OH s uch that the analog
`has insulinotropic activity . Preferably, a GLP- 1 analog has
`the amino acid sequence of GLP- 1(7-37)OH or a fragment
`thereof, modified so that from one, two,
`t hree , four or five
`amino acids differ from the amino acid in corresponding
`30 position of GLP- 1(7 - 37)OH or the fragment of GLP- 1(7 - 37)0.H ,
`In the nonrnencl ature used herein to designate GLP- 1
`compounds , the substituting amino ac i d and its position is
`indicated pri or to the parent structure. For e xample,
`Glu22- GLP- 1(7 - 37)OH desiqnates a GLP- 1 compound in which ~he
`
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`glycine normally found at position 22 of GLP-1(7 - 37)OH has
`been replaced with glutamic acid; Val8 - Glu22- GLP-1(7-37)OH
`designates a GLP-1 compound in which alanine normally found
`at position 8 and glycine normally found at position 22 of
`5 GLP-1(7-37)OH have been replaced with valine and glutamic
`acid, respectively.
`The N- terminus of a GLP-1 compound is generally
`unsubstituted but can also be alkylated or acylated
`(preferably Cl-C20). The C- terrninus can be unsubstituted,
`- NHR
`as is the case with GLP-1(7-37)OH , amidated with -NH2 ,
`or NRR' or esterified with -OR". Rand R' are independently
`alkyl or acyl groups (preferably Cl-C20) . R" is an alkyl
`(Cl-C20) . GLP- 1(7-36)NH2 is an example of an "amidated GLP
`compound". Preferably, the GLP-1 compounds of the present
`invention have a C-terminus that is unsubstituted or
`substituted with -NH2 •
`Preferably GLP-1 compounds of the present invention
`comprise GLP- 1 analogs or fragments of GLP-1 analogs
`wherein the backbone for such analogs or fragments contains
`an amino acid other than alanine at position 8 (position 8
`analogs) . The backbone may also include L-hi stidine , D(cid:173)
`histidine, or modified forms of histidine such as desami no-
`
`10
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`15
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`20
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`histidine, 2-amino-histidine, ~-hydroxy-histidine,
`
`homohistidine , a - fluoromethyl - his tidine , or a - methyl-
`25 histidine at position 7 .
`It is preferable that these
`position 8 analogs contain one or more additional changes at
`positions 11 , 12, 16, 22, 23, 24, 26, 27, 30 , 33, 34, .35,
`36, and 37 compared to the corresponding amino acid of
`native GLP- 1(7-37)OH .
`It is more preferable that these
`30 position 8 analogs contain one or more additional changes at
`positions 12, 16, 22 , 23 , 26 , 30 , 35, and 37 compared to the
`corresponding amino acid of native GLP- 1(7-37)OH .
`It is
`even more preferable t hat these position 8 analogs contain
`one or more additional changes at positions 22, 23 , 27, 30,
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`and 37 compared to the corresponding amino acid of nat i ve
`GLP-1(7-37)OH .
`It is also preferable that these analogs have 6 or
`fewer changes compared to the corresponding amino acids in
`5 native GLP-1(7-37)OH. More preferred analogs have 5 or fewer
`changes compared to the corresponding amino acids in native
`GLP-1(7-37)OH or have 4 or fewer changes compared to the
`corresponding amino acids in native GLP-1 (7-37)OH. It is
`even more preferable that these analogs have 3 or fewer
`changes compared to the corresponding amino acids in native
`GLP-1(7-37)OH.
`It is most preferable that these analogs
`have 2 or fewer changes compared to the corresponding amino
`acids in native GLP-1( 7- 37)OH.
`It has been found that these substitutions reduce the
`15 propensity of GLP-1 compounds to aggregate and generate the
`insoluble form. The GLP-1 compounds of the present
`invention generally aggregate at least about 5 times less
`rapidly than GLP-1(7-37)OH when assessed, for e xample , by
`the aggregation assay described in Example 3, preferably at
`least 20 times l ess rapidly, more preferably at least 40
`times less rapidly, more preferably at least about 50 times
`less rapidly, even more preferably about 60 t imes less
`rapidly, and even more preferably at least about 65 times
`less rapidly . · · Preferably, GL.P-1 compounds described herein
`are analogs of GLP-1 (7-36 ) NH2 or GLP-1 (7-37) OH.
`In a preferred embodiment, the amino acid at position
`22 of the GLP-1 compound of the present invention has a side
`chain which comprises at least two carbon atoms and a polar
`or charged functional group. Aspartic acid, wh ich has a
`30 methylene and carboxyl carbon, is included. More
`preferably, the side chain of the amino acid at position 22
`is a straight or branched chain alkyl group with from two to
`six carbon atoms and a charged functional group, e.g., a
`carboxylic acid, an amine , guanidino group or a sulfonic
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`acid group. Thus, exampl es of preferred amino acids at
`position 22 include glutamic acid , aspartic acid, arginine
`a nd lysine . When position 22 is aspartic acid, glutamic
`acid, arginine or lysine, position 8 is preferabl y glycine ,
`5 valine, leuci ne, isolecine, serine, threoni ne or methionine
`and more preferably valine or glycine . An example of an
`amino acid with a sulfonic acid group in the side chain
`cysteic acid (- NH-CH(CH2Sp3) - CO-, abbreviated as "Cya " ).
`When position 22 is a sulfonic acid such as cysteic acid,
`10 position 8 is preferably glycine , valine , leucine,
`isolecine , serine , threonine or methi onine and more
`pr eferably val ine or glycine .
`In another preferred embodiment , the amino acid at
`position 8 is preferably glycine , valine, leucine ,
`isoleucine , serine , threonine , or methionine and more
`preferably valine or glycine and position 30 is glutamic
`acid, aspartic aci d, serine , or histidine and more
`preferably glutamatic acid .
`In another preferred embodiment , the amino acid at
`position 8 is preferably glycine , valine , leucine ,
`i soleucine, s·erine, threonine , or methionine and more
`preferably valine or glycine and position 37 is histidine,
`l ysine , arginine , threonine, serine, glutamic acid, aspartic
`acid, tryptophan, tyrosine, phenylalanine and more
`25 preferably histidine.
`In another pre ferred embodiment , the amino acid at ·
`position 8 is preferably glycine , valine , leucine ,
`isoleucine, serine , t hr eonine, or methionine and more
`preferably vali ne or glycine and position 22 is gl utamic
`acid, lysine, aspartic acid, or arginine and more preferably
`glutamine acid or lysi ne and position 23 is lysine ,
`arginine , glutamic ac id , aspartic acid, and histidine and
`more preferably lysine or glutamic aci d.
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`In another preferred embodiment , the amino acid at
`position 8 is preferably glycine, va l ine, leucine ,
`isoleucine , serine , threonine , or methionine and more
`preferably valine or glycine and positi on 22 is glutamic
`acid, lysine , aspartic acid, or arginine and more preferably
`glutamine acid o r lysine and position 27 is a l anine, l ysine ,
`arginine, tryptophan , tyrosine , phenyl alanine , or histidine
`and more pr eferabl y alanine.
`In another preferred embodiment , the GLP- 1 compounds of
`the present invention have an amino aci d at position 8 and
`have one , two , or three amino acids selected from the group
`consi s t ing of position 11, position 12, posi tion 16,
`position 22 , position 23 , position 24 , position 26 , position
`27 , position 30 , position 33, positi on 34 , positi on 35 ,
`15 position 36 , and position 37, which differ from the ami no
`acid at the corresponding position of native GLP-1(7-37)OH .
`I n another preferred embodiment , the GLP-1 compounds of
`the present invention have one or two amino acids ,
`i n
`addition to t he amino acid at position 8 , selected from the
`group consisting of positi on 11 , position 12 , position 16,
`position 22 , position 23 , position 24 , position 26, posit ion
`27, position 30 , posi tion 33 , position 34 , posit ion 35 ,
`position 36, and position 37 , which differ from the amino
`acid at the corresponding position of native . GLP-1(7-37)OH .
`
`20
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`25
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`30
`
`As described above, t he GLP- 1 compounds of the pre s ent
`invention can have amino acids i n addition to those at
`position 8, 11, 12, 16, 22, 23 , 24 , 26 , 27 , 30 , 33 , 34 , 35 ,
`36, and 37 which differ from the amino acid at t he
`corresponding position of GLP- 1(7- 37 ) or a fragment of GLP-
`1(7 - 37). The amino acids other than those at position s 8,
`11, 12 , 16, 2 2 , 2 3 , 2 4, 2 6, 2 7 , 3 0 , 3 3 , 3 4 , 3 5 , 3 6, and 3 7
`in the GLP compound which differ from the amino acid in
`corresponding p osition of GLP- 1 (7- 37)OH are preferabl y
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`conservative substitutions and, more preferably, are highly
`conservative substitutions.
`Preferably, the GLP-1 compounds of the present
`invention have zero, one, two or three amino acids in
`addition to the amino acids at positions 8 and 22 which
`differ from the amino acid at the corresponding position of
`GLP-1(7 - 37)OH or a GLP-1(7-37)OH fragment.
`In one example,
`one or more of the amino acids at positions 7, 21 and 27 of
`the GLP- 1 compound differ from the ~orresponding amino acid
`in GLP- 1(7- 37)OH or a GLP-1(7- 37)OH fragment, in addition to
`the amino acids at positions 8 and 22.
`Preferably, only positions 7, 8 and 22 differ from the
`amino acid at the corresponding position of GLP- 1(7- 37)OH
`(or a fragment thereof) .
`It is expected that other improved
`GLP-1 compounds with reduced aggregating properties can be
`obtai ned from known, biologically active GLP- 1 compounds by
`replacing gl ycine at position 22 and preferabl y alanine at
`position 8 -of these compounds with a suitable amino acid, as
`described herein. Kno~n biologically active GLP- 1 compounds
`are disclosed in U.S. Patent No 5,977,071 to Hoffmann , et
`al. , U. S. Patent No. 5,545,618 to Buckley, et al.,
`Adelhorst, et al ., J. Biol. Chem. 269:6275 (1994) , the
`entire teachings of which are incorporated herein by
`reference.
`A "conservative substitution" is the replacement of an
`amino acid with another amino acid that has the same net
`electronic charge and approximately t he same size and shape.
`Amino acids with aliphatic or substituted aliphatic amino
`acid side chains have approximately the same size when the
`total number carbon and heteroatorns in their side chains
`differs by no more than about four. They have approximately
`the same shape when the number of branches ·in the their side
`chains differs by no more than one. Amino acids with phenyl
`or substitut ed ohenvl orouos in their side chains are
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`considered to have about the same size and shape. Listed
`below are five groups of amino acids. Replacing an amino
`acid in a GLP-1 compound with another amino acid from the
`same groups resu1ts in a conservative substitution:
`Group I : glycine, alanine, valine, leucine,
`isoleucine , serine, threonine, cysteine, and non(cid:173)
`naturally occurring amino acids with Cl-C4 aliphatic
`or Cl-C4 hydroxyl substituted aliphatic side chains
`(straight chained or monobranched).
`
`Group II : glutamic acid, aspartic acid and non(cid:173)
`naturally occurring amino acidp with carboxylic acid
`substituted Cl-C4 aliphatic side chains (unbranched
`or one branch point).
`
`Group III : lysine, ornithine, arginine and non(cid:173)
`naturally occurring amino acids with amine or
`guanidino substituted Cl-C4 aliphatic side chains
`(unbranched or one branch point).
`
`Group IV: glutamine, asparagine and non-naturally
`occurring amino acids with amide substituted Cl-C4
`aliphatic side chains (unbranched or one branch
`point).
`
`Group V: phenylalanine, phenylglycine, tyrosine and
`tryptophan.
`
`Except as otherwise specifically provided herein,
`conservative substitutions are preferably made with
`naturally occurring amino acids.
`A "highly conservative substitution" is the replacement
`of an amino acid with another amino acid that has the same
`functional group in the side chain and nearly the same size
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`and shape. Amino acids with aliphatic or substituted
`aliphatic amino acid side chains have nearly the same size
`when the total number carbon and heteroatoms in their side
`chains differs by no more than two. They have nearly the
`same shape when they have the same number of branches in the
`their side chains. Example of high l y conservative
`substitutions include valine for leucine, threonine for
`serine, aspartic acid for glutamic acid and phenylglycine
`for phenylalanine. Examples of substitut ions which are not
`10 highly conservative include alanine for valine, alanine for
`serine and aspartic acid for serine.
`One example of a GLP-1 compound of the present
`invention is a polypeptide having the amino acid sequence of
`SEQ ID NO:l .
`In a preferred example , the GLP-1 compound is
`15 GLP- 1(7 - 37)OH except that Xaa8 is Gly or Val, Xaa22 is Glu or
`In another example , the GLP- 1
`Lys, and Xaa23 is Glu or Lys.
`compound is GLP-1 (7- 37)OH except that Xaa 8 is Gly or Val and
`Xaa30 is Glu. An additional example is a GLP-1 compound
`which is GLP- 1(7- 37)OH except that Xaa 8 is Gly or Val and
`20 Xaa31 is His.
`Another example of a GLP-1 compound of the present
`invention is a polypeptide having the amino acid sequence of
`SEQ ID. No. 4.
`In a preferred example, Xaa 7 is L- histidine,
`D- histidine , desamino- histidine, 2amino-histidine, ~-
`
`25 hydroxy-histidine , homohistidine, a -fluorornethyl-histi dine
`
`30
`
`and a-methyl-histidine, Xaa8 is glycine, alanine, valine,
`leucine, isoleucine, serine , or threonine, and preferably ,
`glycine, valine, leucine, isoleucine, serine, or threonine,
`R is - NH2 or Gly(OH) and Xaa22 is lysine, glutarnic acid,
`aspartic acid or arginine in SEQ ID NO: 4.
`In a mor.e
`preferred example, Xaa7 is L-histidine, Xaa8 is glycine or
`valine , Xaa22 is lysine , glutamic acid, aspartic acid or
`arginine and R is Gly(OH) . Alternatively, Xaa7 , Xaa8 and R