(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`27 December 2001 (27.12.2001)
`
` (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,
`(21) International Application Number:=PCT/US01/16474
`GM, HR, HU,ID,IL,IN,IS, JP, KE, KG, KP, KR, KZ, LC,
`LK,LR, LS, LT, 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.
`
`(51) International Patent Classification’:
`
`C07K 14/00
`
`(22) International Filing Date:
`
`1 June 2001 (01.06.2001)
`
`(25) Filing Language:
`
`Lnglish
`
`(26) Publication Language:
`
`English
`
`(30) Priority Data:
`60/212,171
`60/240,349
`
`16 June 2000 (16.06.2000)
`13 October 2000 (13.10.2000)
`
`US
`US
`
`(71) Applicant (for ail designated States except US): ELI
`LILLY AND COMPANY [US/US]; Lilly Corporate
`Center, Indianapolis, IN 46285 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): GLAESNER, Wolf-
`gang [DE/US], 7512 Ficldstone Court, Indianapolis, IN
`46254 (US). MILLICAN,Rohn, Lee [US/US]; 5319 Deer
`Creck Avenue, Indianapolis, IN 46254 (US).
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), 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 ofthat report
`with sequence listing part ofdescription published sepa-
`rately in electronic form and available upon request from
`the International Bureau
`
`(74) Agents: STEWART, Mark, J., ef al.; Eli Lilly and Com-
`pany, Lilly Corporate Center, Indianapolis, IN 46285 (US).
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes andAbbreviations" appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazelle.
`
`O01/98331A2
`
`(54) Title: GLUCAGON-LIKE PEPTIDE-1 ANALOGS
`
`(57) Abstract: Disclosed are glucagon-like peptide-1 (GLP-1) compounds with modifications at one or moreof the following posi-
`tions: 11, 12, 16, 22, 23, 24, 25, 27, 30, 33, 34, 35, 36, or 37. Methodsoftreating these GLP-1 compoundsarealso disclosed.
`
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`GLUCAGON-LIKE PEPTIDE-1 ANALOGS
`
`This application claims the benefit of U.S. Provisional
`Application Number 60/212,171, filed June 16, 2000 and U.S.
`Provisional Application Number 60/240,349, filed October 13,
`2000.
`.
`
`Glucagon-Like Peptide 1
`(GLP-1)
`is a 37 amino acid
`peptide that is secreted by the L-cells of the intestine in
`
`10
`
`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,
`40:333-343 (1992)). However, 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) OH and Ser34-GEP-1(7-37) 0H.
`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
`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
`
`15
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`20
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`25
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`30
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`
`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 biologically active
`GLP-1 analogs 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 markedly decreased propensity to aggregate
`compared with GLP-1 (7-37) OH.
`Many of these analogs retain GLP-1 receptor activation
`that is comparable and in some cases. greater than known GLP-
`1 compounds such as GLP~1(7-37)0H and Val8-GLP-1 (7-37) OH.
`For example,
`the aggregation time of Val8-Glu22~GLP (7-37) 0H
`is over twenty fold greater and its GLP-1 receptor
`activation is about 25% greater than GLP-1(7-37)OH. Based on
`these discoveries, novel GLP-1 compounds and methods of
`treatment using the novel GLP-1 compounds are disclosed
`‘herein.
`
`One embodiment of the present invention is a
`polypeptide having the amino acid sequence of formula I
`ID NO: 1):
`,
`
`(SEQ
`
`10
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`15
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`20
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`25
`
`30
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`
`His-Xaag-Glu-Gly~-Xaaji~Xaajyo-Thr-Ser-Asp-Xaai¢-Ser-
`Ser-Tyr-Leu-Glu-Xaa22-Xaa23~Xaaeq-Al a-Xaazg~-Xaaz7—-Phe-
`Tle-Ala-Xaa3)-Leu-Xaa33-Xaa34-Xaaz5~Xaayze-R
`formula I
`(SEQ ID NO: 1)
`
`10
`
`15
`
`20
`
`25
`
`30
`
`wherein:
`
`Xaag is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`Xaai, is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
`Xaai2 is: His, Trp, Phe, or Tyr;
`Xaaig 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;
`
`Xaa23 is: His, Asp, Lys, Glu, or Gln;
`Xaaoq is: Glu, His, Ala, or Lys;
`Xaaz6 is: Asp, Lys, Glu, or His;
`Xaa27 1s: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys;
`Xaaz3o is: Ala, Glu, Asp, Ser, or His;
`Xaa33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
`Xaaz3, is: Glu, Lys, or Asp;
`Xaazgs is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro,
`His, or Glu;
`
`Xaaz¢ is: Arg, Glu, or His;
`R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His,
`-NH2, Gly, Gly-Pro, or Gly-Pro-NHz, or is deleted.
`
`provided that the polypeptide does not have the
`' sequénce of GLP-1(7-37)0H or GLP-1(7-36)-NH» and provided
`that the polypeptide is not Gly®-GLP-1(7-37) OH, Gly®-GLP-
`1(7-36)NH2, Val°-GLP-1(7-37)0H, Val®-GLP-1(7-36)NH>, Leu®-
`GLP-1(7-37)OH, Leu®-GLP-1(7-36)NH»,
`Ile*-GLP-1(7~37)OH, Tle®-
`GLP-1(7-36)NH2, Ser*-GLP-1(7-37)OH, Ser®-GLP-1 (7-36) NHo;
`Thr°-GLP-1(7-37)OH, or Thr®-GLP-1 (7-36) NHo, Ala'!-Glp-1(7-
`37) OH, Ala*'-Glp-1(7-36)NH, Ala?®-Glp-1 (7-37) 0H, Ala’®-Glp-
`
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`1(7-36)NH2, Ala®’~Glp-1(7-37)OH, Ala?’~Glp-1(7-36)NHo, Glu27~
`Glp-1(7-37)OH, Glu*’-Glp-1(7-36)NH2, Ala®°-Glp-1(7-37)0H, or
`Ala**-Glp-1 (7-36) NH2.
`
`Another embodiment of the present invention is a
`polypeptide having the amino acid sequence of formula II
`(SEQ ID NO: 2):
`
`His-Xaag-Glu-Gly-Thr-Xaaj.-Thr-Ser-Asp-Xaaig-Ser-
`Ser-Tyr-Leu-Glu-Xaa22—-Xaaz3-Ala-Ala-Xaaog-Glu~-Phe-
`Ile~Xaazo-Trp-Leu-Val-Lys-Xaa35-Arg-R
`formula II (SEQ ID NO: 2)
`
`wherein:
`
`Xaag is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`Xaai2 is: His, Trp, Phe, or Tyr;
`Xaaig 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;
`
`Xaa23 is: His, Asp, Lys, Glu, or Gln;
`Xaa2g is: Asp, Lys, Glu, or His;
`Xaazo is: Ala, Glu, Asp, Ser, or His;
`Xaags 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,
`-NHz, Gly, Gly-Pro, or Gly-Pro-NHz, or is deleted.
`
`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 Gly®-GLP-~1(7-37)OH, Gly®-GLP-1 (7-36) NHo,
`Val°-GLP-1(7-37) OH, Val®-GLP-1(7-36)NH2, Leu®-GLP-1 (7-37) OH,
`Leu®-GLP-1(7-36)NH2,
`Ile®-GLP-1(7-37)OH,
`Ile®-GLP-1 (7-36) NHo,
`Ser®-GLP-1(7-37) OH, Ser®-GLP-1(7-36)NH2, Thr®-GLP-1 (7-37) OH,
`Thr°-GLP-1(7-36)NHz, Ala!®-GLP (7-37) OH, or Ala’®-Glp-1(7-
`36) NHo.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
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`
`Another embodiment of the present invention is a
`polypeptide having the amino acid sequence of formula III
`(SEQ ID NO: 3):
`
`His-Xaag-Glu-Gly-Thr-Phe-Thr~-Ser-Asp-Val-Ser-Ser-
`Tyr-Leu-Glu-Xaa22-Xaaz3~Ala-Ala-Lys—Xaaz7-Phe-Ile-
`Xaa3o-Trp-Leu-Val-Lys-Gly-Arg-R
`formula III (SEQ ID NO: 3)
`
`10
`
`wherein:
`
`Xaag is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`Xaazg2 is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, Cys, or Cysteic
`Acid;
`
`Xaa23 is: His, Asp, Lys, Glu, or Gln;
`Xaa27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys
`Xaaz3o is: 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-NHz, or is deleted.
`
`provided that the polypeptide does not have the
`sequence of GLP-1(7-37)0H or GLP~1 (7-36) -NH, and provided
`that the polypeptide is not Gly®-GLP-1 (7-37) 0H, Gly®-GLP-
`1(7-36)NH2, Val°-GLP-1(7-37)0H, Val®-GLP-1(7-36)NH2, Leu®-
`GLP-1(7-37)OH, Leu®-GLP-1(7-36)NHz,
`Ile®-GLP-1(7-37)0H,
`Ile®-
`GLP-1(7-36)NH2, Ser®-GLP-1(7-37)OH, Ser®-GLP-1 (7-36) NH>,
`Thr*-GLP-1 (7-37) OH, Thr®-GLP-1(7-36)NHz, Ala!®-Glp-1 (7-37) 0H,
`Ala’®-Glp-1 (7-36) NH2, Glu?’-Glp-1(7-37) OH, or Glu?’~Glp-1 (7-
`36) NHo.
`
`Another embodiment of the present invention is a
`polypeptide having the amino acid sequence of formula IV
`(SEQ ID NO: 4):
`
`Xaa7-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
`Ser-Tyr-Leu-Glu-Xaa22~Gln-Ala-Ala-Lys-Glu-Phe-~
`Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R
`(SEQ ID NO: 4)
`
`15
`
`20
`
`25
`
`30
`
`35
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`wherein:
`
`Xaa; is L-histidine, D-histidine, desamino-histidine,
`
`Zamino-histidine, B-hydroxy-histidine, homohistidine, a-
`
`fluoromethyl-histidine or a-methyl-histidine;
`isoleucine,
`Xaag is glycine, alanine, valine,
`leucine,
`serine or threonine. Preferably, Xaag is glycine, valine,
`leucine,
`isoleucine, serine or threonine;
`
`Xaae2 is aspartic acid, glutamic acid, glutamine,
`asparagine,
`lysine, arginine, cysteine, or cysteic acid.
`R is -NHz or Gly(OH).
`
`Another embodiment of the present invention is a
`glucagon-like peptide-1 (GLP-1) compound having an amino
`acid other than alanine at position 8 and an amino acid
`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
`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
`GLP-1 receptor activation ability and,
`in addition, have
`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
`
`10
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`15
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`20
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`25
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`30
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`
`vivo aggregation will occur after administration to
`patients,
`thereby increasing activity and minimizing the
`potential for adverse reactions.
`In addition,
`these GLP-1
`compounds are resistant to diaminopeptidase IV degradation
`and bind zinc and are therefore believed to provide extended
`time action in vivo.
`
`10
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`15
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`20
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`25
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`30
`
`Figure 1 shows the amino acid sequences of Val8-Glu22-
`GLP-1(7-37)OH (SEQ ID NO: 5), Vail8-Asp22-GLP-1(7-37)OH (SEQ
`ID NO: 6), Val8-Arg22-GLP-1(7-37)OH (SEQ ID NO: 7) and val8-
`Lys22-GLP-1(7-37)0H (SEQ ID NO: 8).
`Figure 2 shows the amino acid sequences of Gly®-Glu22-
`GLP-1(7-37)OH (SEQ ID NO: 9), Gly8-Asp22-GLP-1(7-37)OH (SEQ
`ID NO: 10), Gly8-Arg22-GLP-1(7-37)OH (SEQ ID NO: 11) and
`Gly®-Lys22-GLP-1(7-37)OH (SEQ ID NO: 12).
`Figure 3 shows the amino acid sequence of Val®-Glu*-
`GLP-1(7-37)OH (SEQ ID NO: 13), Gly®-Glu°°-GLP-1(7-37)OH (SEQ
`ID NO: 14), Val®-His?’-GLP-1(7-37)0H (SEQ ID NO: 15), and
`Gly®-His*’-GLP-1(7-37)0H (SEQ ID NO: 16).
`Figure 4 shows the amino acid sequence of Val®-Glu??-
`Ala®’-GLP-~1(7-37)0H (SEQ ID NO: 17)and Val®-Lys?*~Glu??~GLP-
`1(7-37)0H (SEQ ID NO: 18).
`
`A GLP-1 compound is a polypeptide having from about
`twenty-five to about thirty-nine naturally occurring or non-
`naturally occurring amino acids and has sufficient homology
`to GLP-1(7-37)0H such that it exhibits insulinotropic
`activity.
`Examples of non-naturally occurring amino acids:
`include a-methyl amino acids (e.g., a-methyl alanine), D-
`amino acids, histidine-like amino acids (e.g., 2-amino-
`histidine, B-hydroxy-histidine, homohistidine, a-
`
`fluoromethyl-histidine and a-methyl-histidine), amino acids
`having an extra methylene in the side chain (“homo” amino
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`
`acids) and amino acids in which a carboxylic acid functional
`group in the side chain is replaced with a sulfonic acid
`group (e€.g.,
`cysteic acid). Preferably, however,
`the GLP-1
`compounds of the present invention comprise only naturally
`occurring amino acids except as otherwise specifically
`provided herein.
`
`A GLP-1 compound typically comprises a polypeptide
`having the amino acid sequence of GLP-1(7-37)0H, an analog
`of GLP-1 (7-37)0H, a fragment of GLP-1(7-37)OH or a fragment
`of a GLP-1(7-37)0H analog. GLP-1(7-37)OH has the amino acid
`
`Sequence of SEQ ID NO: 19:
`
`"His-Ala-Glu-!Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-
`Tyr-*°Leu-Glu-Gly-Gln-Ala-*Ala-Lys-Glu-Phe-Ile-"ala-
`Trp-Leu-Val-Lys-*Gly-Arg-?"Gly
`(SEQ ID NO: 19)
`|
`
`the amino terminus of GLP-1(7-37) 0H
`By custom in the art,
`has been assigned number residue 7 and the carboxy~-terminus,
`number 37.
`The other amino acids in the polypeptide are
`numbered consecutively, as shown in SEQ ID NO: 19.
`For
`example, position 12 is phenylalanine and position 22 is
`glycine. When not specified,
`the C-terminal is in the
`
`traditional carboxyl form.
`
`A "GLP-1 fragment" is a polypeptide obtained after
`truncation of one or more amino acids from the N-terminus
`and/or C-terminus of GLP-1(7-37)OH or a GLP-1(7-37)0H
`analog.
`The nomenclature used to describe GLP-1 (7-37) 0H
`carries over to GLP-1 fragments.
`For example, GLP-1(9-36)0H
`denotes a GLP-1 fragment obtained by truncating two amino
`acids from the N-terminus and one amino acid from the C-
`terminus. 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-
`
`10
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`
`1(9-36)0H is at position 9; position 12 is occupied by
`phenylalanine; and position 22 is occupied by glycine, as in
`GLP=1 (7-37) 0H.
`
`"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)0H or fragments thereof.
`GLP-1 compounds of this type have up to about thirty-nine
`amino acids.
`The amino acids in the “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 acid of a GLP-1 compound obtained by adding two amino
`acids to the N-terminal of GLP-1(7-37)0H 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)0OH
`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 corresponding position of GLP-1(1-37)0H. Amino acids
`38-45 of an extended GLP-1 compound are preferably the same
`as or a conservative 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)0H such that the analog
`has insulinotropic activity. Preferably, a GLP-1 analog has
`the amino acid sequence of GLP-1(7-37)0H or a fragment
`thereof, modified so that from one,
`two,
`three,
`four or five
`amino acids differ from the amino acid in corresponding
`position of GLP-1(7-37)0OH or the fragment of GLP-1 (7-37) OH.
`In the nonmenclature used herein to designate GLP-1
`compounds,
`the substituting amino acid and its position is
`indicated prior to the parent structure.
`For example,
`Glu22-GLP-1(7-37)OH designates a GLP-1 compound in which the
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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) 0H
`designates a GLP-1 compound in which alanine normally found
`at position 8 and glycine normally found at position 22 of
` GLP-1(7-37)0H 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 C1-C20).
`The C-terminus can be unsubstituted,
`as is the case with GLP-1(7-37)0H, amidated with -NH2,
`-NHR
`or NRR’ or esterified with -OR”. R and R’ are independently
`alkyl or acyl groups (preferably C1-C20). R” is an alkyl
`(C1-C20). GLP-1(7-36)NH, 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 -NH».
`
`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-histidine, D-
`histidine, or modified forms of histidine such as desamino~
`
`histidine, 2-amino-histidine, B-hydroxy-histidine,
`
`homohistidine, a-fluoromethyl-histidine, or a-methyl-
`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
`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 that 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 native
`
`GLP-1 (7-37) OH.
`
`It is also preferable that these analogs have 6 or
`fewer changes compared to the corresponding amino acids in
`native GLP~1(7-37)0H. 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) 0H.
`
`It has been found that these substitutions reduce the
`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 example, by
`the aggregation assay described in Example 3, preferably at
`least 20 times less rapidly, more preferably at least 40
`times less rapidly, more preferably at least about 50 times
`less rapidly, even more preferably about 60 times less
`rapidly, and even more preferably at least about 65 times
`less rapidly. “Preferably, GLP-1 compounds described herein
`are analogs of GLP-1(7-36)NH2 or GLP-1(7-37)0OH.
`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, which has a
`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, examples of preferred amino acids at
`position 22 inckude glutamic acid, aspartic acid, arginine
`and lysine. When position 22 is aspartic acid, glutamic
`acid, arginine or lysine, position 8 is preferably glycine,
`valine,
`leucine,
`isolecine, serine,
`threonine or methionine
`
`An example of an
`and more preferably valine or glycine.
`amino acid with a sulfonic acid group in the side chain
`cysteic acid (~NH-CH(CH2S03)-CO-, abbreviated as "Cya").
`When position 22 is a sulfonic acid such as cysteic acid,
`position 8 is preferably glycine, valine,
`leucine,
`isolecine, serine,
`threonine or methionine and more
`
`preferably valine or glycine.
`
`In another preferred embodiment,
`
`the amino acid at
`
`leucine,
`position 8 is preferably glycine, valine,
`isoleucine, serine,
`threonine, or methionine and more
`preferably valine or glycine and position 30 is glutamic
`acid, aspartic acid, serine, or histidine and more
`
`preferably glutamatic acid.
`
`the amino acid at
`In another preferred embodiment,
`position 8 is preferably glycine, valine,
`leucine,
`isoleucine, serine,
`threonine, or methionine and more
`preferably valine or glycine and position 37 is histidine,
`lysine, arginine,
`threonine, serine, glutamic acid, aspartic
`acid,
`tryptophan,
`tyrosine, phenylalanine and more
`preferably histidine.
`
`In another preferred embodiment,
`
`the amino acid at
`
`leucine,
`position 8 is preferably glycine, valine,
`isoleucine, serine,
`threonine, or methionine and more
`preferably valine or glycine and position 22 is glutamic
`acid,
`lysine, aspartic acid, or arginine and more preferably
`glutamine acid or lysine and position 23 is lysine,
`arginine, glutamic acid, aspartic acid, and histidine and
`
`more preferably lysine or glutamic acid.
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`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 22 is glutamic
`acid,
`lysine, aspartic acid, or arginine and more preferably
`glutamine acid or lysine and position 27 is alanine,
`lysine,
`arginine,
`tryptophan,
`tyrosine, phenylalanine, or histidine
`
`and more preferably alanine.
`
`the GLP-1 compounds of
`In another preferred embodiment,
`the present invention have an amino acid at position 8 and
`have one,
`two, or three amino acids selected from the group
`consisting of position 11, position 12, position 16,
`position 22, position 23, position 24, position 26, position
`27, position 30, position 33, position 34, position 35,
`position 36, and position 37, which differ from the amino
`acid at the corresponding position of native GLP-1(7-37) OH.
`
`the GLP-1 compounds of
`In another preferred embodiment,
`the present invention have one or two amino acids,
`in
`addition to the amino acid at position 8, selected from the
`group consisting of position 11, position 12, position 16,
`position 22, position 23, position 24, position 26, position
`27, position 30, position 33, position 34, position 35,
`position 36, and position 37, which differ from the amino
`
`acid at the corresponding position of native. GLP-1(7-37) 0H.
`
`the GLP-1 compounds of the present
`As described above,
`invention can have amino acids in 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 the
`corresponding position of GLP-1(7-37) or a fragment of GLP-
`1(7-37).
`The amino acids other than those at positions 8,
`11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36, and 37
`in the GLP compound which differ from the amino acid in
`corresponding position of GLP-1(7-37)0H are preferably
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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
`
`In one example,
`GLP-1(7-37)OH or a GLP-1(7-37)OH fragment.
`one or more of the amino acids at positions 7, 21 and 27 of
`the GLP-1 compound differ from the corresponding amino acid
`in GLP-1(7-37)OH or a GLP-1(7-37)0H 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)0H
`
`It is expected that other improved
`(or a fragment thereof).
`GLP-1 compounds with reduced aggregating properties can be
`obtained from known, biologically active GLP-1 compounds by
`replacing glycine at position 22 and preferably alanine at
`position 8-of these compounds with a suitable amino acid, as
`described herein.
`Known 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.,
`
`the
`Adelhorst, et al., J. Biol. Chem. 269:6275 (1994),
`entire teachings of which are incorporated herein by
`reference,
`no
`.
`
`A “conservative substitution” is the replacement of an
`amino acid with another amino acid that has the same net
`
`electronic charge and approximately the 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 heteroatoms in their side chains
`
`They have approximately
`differs by no more than about four.
`the same shape when the number of branches in the their side
`chains differs by no more than one. Amino acids with phenyl
`or substituted phenyl groups in their side chains are
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`the same size and shape. Listed
`considered to have about
`below are five groups of amino acids. Replacing an amino
`acid in a GLP-1 compound with another amino acid from the
`
`same groups results in a conservative substitution:
`
`leucine,
`Group I: glycine, alanine, valine,
`isoleucine, serine,
`threonine, cysteine, and non-
`naturally occurring amino acids with C1-C4 aliphatic
`or C1-C4 hydroxyl substituted aliphatic side chains
`(straight chained or monobranched) .
`
`Group II: glutamic acid, aspartic acid and non-
`naturally occurring amino acids with carboxylic acid
`substituted C1-C4 aliphatic side chains (unbranched
`or one branch point).
`
`Group III: lysine, ornithine, arginine and non-
`naturally occurring amino acids with amine or
`guanidino substituted C1-C4 aliphatic side chains
`- (unbranched or one branch point).
`
`Group IV: glutamine, asparagine and non-naturally
`occurring amino acids with amide substituted C1-c4
`aliphatic side chains (unbranched or one branch
`point).
`
`Group V: phenylalanine, phenylglycine,
`tryptophan.
`
`tyrosine and
`
`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
`
`Example of highly conservative
`their side chains.
`substitutions include valine for leucine,
`threonine for
`serine, aspartic acid for glutamic acid and phenylglycine
`for phenylalanine.
`Examples of substitutions which are not
`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:1.
`In a preferred example,
`the GLP-1 compound is
`GLP-1(7-37)OH except that Xaag is Gly or Val, Xaaz2 is Glu or
`Lys, and Xaaz3 is Glu or Lys.
`In another example,
`the GLP-1
`compound is GLP-1(7-37)OH except that Xaag is Giy or Val and
`Xaazo is Glu.
`An additional example is a GLP-1 compound
`which is GLP-1(7-37)OH except that Xaag is Gly or Val and
`Xaa37 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.
`Ina preferred example, Xaa, is L-histidine,
`D-histidine, desamino-histidine, 2amino-histidine, B-
`
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`hydroxy-histidine, homohistidine, a-fluoromethyl-histidine
`
`30
`
`.
`
`and a-methyl-histidine, Xaag is glycine, alanine, valine,
`leucine,
`isoleucine, serine, or threonine, and preferably,
`glycine, valine,
`leucine,
`isoleucine, serine, or threonine,
`‘Ris -NH2 or Gly(OH) and Xaazg2 is lysine, glutamic acid,
`aspartic acid or arginine in SEQ ID NO:4.
`Ina more
`preferred example, Xaa7 is L-histidine, Xaag is glycine or
`valine, Xaaz2 is lysine, glutamic acid, aspartic acid or
`arginine and R is Gly(OH). Alternatively, Xaaj, Xaag and R
`in SEQ ID NO:
`4 are as described above and Xaaz2 is an amino
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`acid with a side chain comprising a sulfonic acid group,
`e.g., cysteic acid.
`
`In another example of GLP-1 compounds of the present
`invention,
`the amino acid at position 8 is not a D-amino
`acid and does not have the side chain of glycine, serine,
`threonine, cysteine or beta-alanine when the amino acid at
`
`position 22 has a C1-C2 alkyl side chain,
`a C1-c2
`hydoxylated alkyl side chain or a C1-C2 thiolated alkyl
`chain (e.g., cysteine).
`In a preferred example of GLP-1
`compounds of the present invention,
`the amino acid at
`
`position 8 is not a D-amino acid and does not have the side
`chain of glycine, serine,
`threonine, cysteine or beta-
`alanine when the amino acid at position 22 has a C1-C4 alkyl
`side chain, a C1-C4 hydroxylated alkyl side chain or a C1-C4
`thiolated alkyl chain.
`In another example of the GLP-1 compounds of the
`present invention,
`the amino acid at position 8 is glycine,
`valine,
`leucine,
`isoleucine, methionine, serine,
`threonine,
`cysteine, aspartic acid, glutamic acid,
`lysine, arginine,
`asparagine, glutamine, phenylalanine,
`tyrosine, histidine or
`tryptophan; and the amino acid at position 22 is aspartic
`acid, glutamic acid,
`lysine, arginine, asparagine, glutamine
`or histidine.
`
`Specific examples of GLP-1 compounds of the pr