`
`(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
`
`(51) International Patent Classification?
`
`C07K 14/00
`
`(21) International Application Number:
`
`PCT/USOI/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)
`13 October 2000 (13.10.2000)
`
`US
`US
`
`(71) Applicant (for all 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 Fieldstone Court, Indianapolis, IN
`46254 (US). MILLICAN, Rohn, Lee [US/US]; 5319 Deer
`Creek Avenue, Indianapolis, IN 46254 (US).
`
`(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, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
`SL, T], 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, 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 of that report
`with sequence listing part of description published sepa-
`rately in electronic form and available upon request from
`the International Bureau
`
`(74) Agents: STEWART, Mark, J., et a1.; 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 Gazette.
`
`O01/98331A2
`
`(54) Title: GLUCAGON—LIKE PEPTIDE—1 ANALOGS
`
`(57) Abstract: Disclosed are glucagon—like peptide—1 (GLP—l) 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—l compounds are also disclosed.
`
`MYLAN INST. EXHIBIT 1045 PAGE 1
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`MYLAN INST. EXHIBIT 1045 PAGE 1
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`PCT/US01/16474
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`GLUCAGON-LIKE 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.
`'
`
`Glucagon-Like Peptide l
`
`(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, 8., Int.
`
`J1 Peptide Protein Research,
`
`15
`
`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-GLP—l(7—37)OH.
`
`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
`1 water at pH 7.4. Unfortunately,
`the inactive form is
`
`readily produced when aqueous GLP-1 solutions are agitated,
`
`30
`
`exposed to hydrophobic surfaces or have large air/water
`
`MYLAN INST. EXHIBIT 1045 PAGE 2
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`MYLAN INST. EXHIBIT 1045 PAGE 2
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`
`-2-
`
`interfaces.
`
`The tendency to convert to the insoluble form
`
`considerably complicates the production of commercial
`
`quantities of active GLP—l 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—l analogs which convert less readily to the insoluble
`
`form than currently available GLP—l 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—l(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—l(7-37)OH and ValB—GLP—l(7—37)OH.
`
`For example,
`
`the aggregation time of ValB—GluZZ—GLP(7—37)OH
`
`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-l compounds and methods of
`
`treatment using the novel GLP-l compounds are disclosed
`
`‘herein.
`
`One embodiment of the present invention is a
`
`polypeptide having the amino acid sequence of formula I
`
`(SEQ
`
`ID NO: 1):
`
`’
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1045 PAGE 3
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`MYLAN INST. EXHIBIT 1045 PAGE 3
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`-3...
`
`His—Xaa8—Glu-Gly—Xaau—Xaan—Thr—Ser—Asp—Xaam—Ser-
`
`Ser—Tyr-Leu—Glu—Xaan—XaaB—Xaam-Ala—Xaa%—Xaam—Phe-
`
`Ile—Ala~Xaan—Leu—Xaaw—Xaam—Xaafi—Xaa%—R
`
`formula I
`
`(SEQ ID NO: 1)
`
`wherein:
`
`Xaag is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`
`Xaau_is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
`
`Xaan is: His, Trp, Phe, or Tyr;
`
`Xaam is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu,
`
`‘
`or Ala;
`Xaan is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, Cys, or Cysteic
`Acid;
`
`Xaam is: His, Asp, Lys, Glu, or Gln;
`
`Xaam is: Glu, His, Ala, or Lys;
`Xaam is: Asp, Lys, Glu, or His;
`
`10
`
`15
`
`Xaam is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys;
`
`Xaaw is: Ala, Glu, Asp, Ser, or His;
`
`20
`
`Xaafi is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
`XaaM is: Glu, Lys, or Asp;
`
`Xaag is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro,
`His, or Glu;
`
`Xaaw is: Arg, Glu, or His;
`
`25
`
`R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His,
`—NH2, Gly, Gly-Pro, or Gly—Pro—NH; or is deleted.
`
`provided that the polypeptide does not have the
`
`' sequence of GLP-l(7—37)OH or GLP-l(7—36)-NH2 and provided
`
`30
`
`that the polypeptide is not Gly8—GLP-l(7-37)OH, Glya—GLP—
`l(7—36)NH2, ValB-GLP-l(7—37)OH, Vale—GLP-l(7—36)NH2, Leus-
`GLP—l(7-37)OH, LeuB—GLP—l(7—36)NH2,
`IleB-GLP-l(7-37)OH,
`Ilea—
`GLP—l(7-36)NH2, Sere—GLP-l(7—37)OH, SerB-GLP-l(7—36)NH2,
`Thre—GLP4-1(7—37)OH, or ThrB—GLP—l(7—36)NH2, Alall—Glp—ln—
`37)0H, Alall-Glp-1(7—36)NH2, Ala16—Glp—l(7-37)OH, Alals—Glp-
`
`MYLAN INST. EXHIBIT 1045 PAGE 4
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`MYLAN INST. EXHIBIT 1045 PAGE 4
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`
`-4-
`
`l(7—36)NH2, Ala27-Glp-l(7—37)OH, Ala27—Glp—l(7—36)NH2, G1u27~
`
`Glp—l(7—37)OH, Glum—Glp-l(7—36flflh, Ala”—Glp—1(7—37)0H, or
`
`Ala”—G1p—1(7—36)NH2.
`
`Another embodiment of the present invention is a
`
`5
`
`polypeptide having the amino acid sequence of formula II
`
`(SEQ ID NO: 2):
`
`His—Xaa8—Glu—Gly-Thr—Xaan—Thr—Ser—Asp—Xaam-Ser—
`
`Ser—Tyr-Leu-Glu—Xaam—XaaB—Ala—Ala—Xaa%—Glu~Phe-
`
`lO
`
`Ile—Xaaw-Trp—Leu—Val-Lys-Xaa%—Arg-R
`
`formula II (SEQ ID NO: 2)
`
`wherein:
`
`Xaag is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`
`15 Xaan is: His, Trp, Phe, or Tyr;
`
`Xaam is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu,
`or Ala;
`
`Xaan is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, Cys, or Cysteic
`Acid;
`
`20
`
`Xaam is: His, Asp, Lys, Glu, or Gln;
`
`XaaM is: Asp, Lys, Glu, or His;
`
`Xaaw is: Ala, Glu, Asp, Ser, or His;
`
`Xaafl is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro,
`
`His, or Glu;
`
`25
`
`R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His,
`—NH2, Gly, Gly—Pro, or Gly—Pro-NHL or is deleted.
`
`provided that the polypeptide does not have the sequence of
`
`. GLP-l(7-37)OH or GLP—l(7—36)—NH2 and provided that the
`
`30
`
`35
`
`polypeptide is not GlyB—GLP—l(7—37)OH, GlyS—GLP—l(7—36)NH2,
`ValB—GLP—l(7—37)OH, Vale—GLP—l(7-36)NH2, LeuB—GLP—l(7-37)OH,
`LeuS—GLP-l(7—36)NH2,
`Iles—GLP-l(7-37)OH,
`Ile8-GLP—l(7—36)NH2,
`SerS-GLP—l(7—37)OH, Sera—GLp—1(7—365NH2, ThrB—GLP—l(7-37)OH,
`ThrB—GLP—l(7—36)NHL Alan—GLP(7—37)OH, or A1a”—G1p—1(7—
`36)NH2.
`
`MYLAN INST. EXHIBIT 1045 PAGE 5
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`MYLAN INST. EXHIBIT 1045 PAGE 5
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`WO 01/98331
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`
`-5—
`
`Another embodiment of the present invention is a
`
`polypeptide having the amino acid sequence of formula III
`
`(SEQ ID NO: 3):
`
`5
`
`His—Xaag-Glu—Gly—Thr—Phe—Thr—Ser—Asp—Val—Ser—Ser—
`
`Tyr—Leu—Glu—Xaan—Xaam-Ala-Ala—Lys—Xaay—Phe—Ile—
`
`Xaaw—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;
`
`Xaam is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, Cys, or Cysteic
`Acid;
`
`Xaan is: His, Asp, Lys, Glu, or Gln;
`
`15 Xaam is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys
`
`Xaam 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—NHL or is deleted.
`
`20
`
`provided that the polypeptide does not have the
`sequence of GLP—l(7—37yOH or GLP—l(7—36)—NH2 and provided
`
`that the polypeptide is not Glya-GLP—l(7—37)OH, Gly8—GLP—
`1(7—36)NH2, ValB-GLP—l(7-37)OH, Vale—GLP—l(7-36)NH2, Leus-
`
`GLP—l(7—37)OH, LeuB—GLP—1(7—36)NH2,
`
`IleB-GLP—l(7-37)OH, Ilea-
`
`25
`
`GLP—l(7—36)NH2, SerB—GLP-l(7—37)OH, Sers—GLP-l(7-36)NH2,
`ThrB—GLP-l (7—37)OH, Thrs—GLP-1(7-3'6)NH2, AlalG—Glp—l(7—37)OH,
`Ala16-Glp-l(7-36)NH2, Glu27-Glp—l(7—37)OH, or GiuZVlep-im—
`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-XaaB—Glu—Gly-Thr—Phe-Thr—Ser—Asp-Val—Ser—
`
`Ser—Tyr-Leu-Glu—Xaam—Gln—Ala-Ala—Lys—Glu—Phe—
`
`Ile—Ala—Trp—Leu—Val-Lys~Gly—Arg—R
`
`35
`
`(SEQ ID NO: 4)
`
`MYLAN INST. EXHIBIT 1045 PAGE 6
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`MYLAN INST. EXHIBIT 1045 PAGE 6
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`PCT/US01/16474
`
`wherein:
`
`Xaa7 is L—histidine, D—histidine, desamino—histidine,
`
`2amino—histidine, B—hydroxy—histidine, homohistidine,
`
`a—
`
`fluoromethyl—histidine or a—methyl—histidine;
`
`Xaag is glycine, alanine, valine,
`
`leucine,
`
`isoleucine,
`
`serine or threonine. Preferably, Xaag is glycine, valine,
`
`leucine,
`
`isoleucine, serine or threonine;
`
`Xaan is aspartic acid, glutamic acid, glutamine,
`
`10
`
`asparagine,
`
`lysine, arginine, cysteine, or cysteic acid.
`
`R is —NH2 or Gly(OH).
`
`Another embodiment of the present invention is a
`
`glucagon-like peptide—I
`
`(GLP—l) compound having an amino
`
`acid other than alanine at position 8 and an amino acid
`other than glycine at position 22.
`‘
`
`15
`
`20
`
`25
`
`30
`
`Another embodiment of the present invention is a method
`
`of stimulating the GLP—1 receptor in a subject in need of
`
`GLP—l receptor stimulation.
`
`The method comprises the step
`
`of administering to the subject an effective amount of the
`
`GLP—l 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—l compounds described herein or the polypeptide having
`
`the amino acid sequence of SEQ ID NO: I, 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—l compounds of the present invention retain
`
`GLP—1 receptor activation ability and,
`
`in addition, have
`
`decreased propensity to aggregate compared with other GLP-l
`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
`
`MYLAN INST. EXHIBIT 1045 PAGE 7
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`_7_
`
`vivo aggregation will occur after administration to
`
`patients,
`
`thereby increasing activity and minimizing the
`
`potential for adverse reactions.
`
`In addition,
`
`these GLP-l
`
`compounds are resistant to diaminopeptidase IV degradation
`
`and bind zinc and are therefore believed to provide extended
`
`time action in vivo.
`
`Figure 1 shows the amino acid sequences of Val8—Glu22-
`
`GLP—1(7—37)OH (SEQ ID NO: 5), VaIB—AspZZ—GLp—1(7-37)QH (SEQ
`
`10
`
`ID NO: 6), VaIB—ArgZZ—GLP—1(7—37)0H (SEQ ID NO: 7) and Va18—
`
`LysZZ—GLP—1(7—37)0H (SEQ ID NO: 8).
`
`Figure 2 shows the amino acid sequences of Gly8—Glu22—
`
`GLP—l(7—37)OH (SEQ ID NO: 9), GIy8—Asp22—GLP—1(7—37)QH (SEQ
`
`ID NO: 10), G1y8—Arg22—GLP—1(7-37)QH (SEQ ID NO: 11) and
`
`15
`
`Gly8—Lys22—GLP—1(7—37)0H (SEQ ID NO: 12).
`
`20
`
`25
`
`30
`
`Figure 3 shows the amino acid sequence of Vale-Gluw-
`
`GLP—1(7-37)OH (SEQ ID NO: 13), Gly8-Gluw—GLP—l(7—37)OH (SEQ
`
`ID NO: 14), ValB—Hisfl—GLP—l(7—37)OH (SEQ ID NO: 15), and
`
`GlyB—Hisfl—GLP—l(7-37)OH (SEQ ID NO: 16).
`
`Figure 4 shows the amino acid sequence of Vale—Glun—
`A1a”—GLP~1(7-37)0H (SEQ ID NO: 17)and Vals—Lysn—Glum—GLP—
`
`1(7—37)OH (SEQ ID NO: 18).
`
`A GLP-l 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—l(7—37)OH such that it exhibits insulinotropic
`
`activity.
`
`Examples of non—naturally occurring amino acids‘
`
`include d—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
`
`MYLAN INST. EXHIBIT 1045 PAGE 8
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`
`—8—
`
`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-l
`
`compounds of the present invention comprise only naturally
`
`occurring amino acids except as otherwise specifically
`
`provided herein.
`
`A GLP—l compound typically comprises a polypeptide
`
`having the amino acid sequence of GLP-l(7—37)OH, an analog
`
`of GLP—1
`
`(7—37)OH, a fragment of GLP—l(7-37)OH or a fragment
`
`of a GLP—1(7—37)OH analog. GLP—l(7—37)OH has the amino acid
`
`sequence of SEQ ID NO: 19:
`
`7His-Ala—Glu—loGly—Thr—Phe—Thr—Ser—15Asp-Val-Ser-Ser-
`
`Tyr-mLeu-Glu—Gly—Gln—Ala-mAla—Lys—Glu-Phe—Ile—wAla-
`
`Trp—Leu—Val-Lys—%Gly—Arg—WGly
`(SEQ ID NO: 19)
`'
`
`10
`
`15
`
`By custom in the art,
`
`the amino terminus of GLP-l(7-37)OH
`
`2O
`
`has been assigned number residue 7 and the carboxy—terminus,
`number 37.
`The other amino acids in the polypeptide are
`
`25
`
`30
`
`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—l fragment" is a polypeptide obtained after
`
`truncation of one or more amino acids from the Neterminus
`
`and/or C—terminus of GLP-1(7-37)OH or a GLP-l(7-37)OH
`
`analog.
`
`The nomenclature used to describe GLP-l
`
`(7—37)OH
`
`carries over to GLP-l fragments.
`
`For example, GLP—l(9-36)OH
`
`denotes a GLP—l fragment obtained by truncating two amino
`
`acids from the NFterminus 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 Neterminal glutamic acid in GLP—
`
`MYLAN INST. EXHIBIT 1045 PAGE 9
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`-9-
`
`l(9—36)OH is at position 9; position 12 is occupied by
`
`phenylalanine; and position 22 is occupied by glycine, as in
`
`GLP—l(7—37)OH.
`
`"GLP—l compound" also includes polypeptides in which
`
`one or more amino acids have been added to the Neterminus
`
`and/or C—terminus of GLP—l(7~37)OH or fragments thereof.
`
`GLP—l compounds of this type have up to about thirty—nine
`
`amino acids.
`
`The amino acids in the “extended” GLP-l
`
`10
`
`compound are denoted by the same number as the corresponding
`amino acid in GLP-l(7—37)OH.
`For example,
`the Neterminus
`
`amino acid of a GLP-l compound obtained by adding two amino
`
`acids to the NLterminal of GLP—1(7—37)OH is at position 5;
`
`and the C-terminus amino acid of a GLP—l compound obtained
`
`15
`
`20
`
`by adding one amino acids to the C—terminal of GLP—l(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—l compounds, as in GLP—l(7-37)OH.
`
`Amino acids l—6 of an extended GLP—l compound are preferably
`
`the same as or a conservative substitution of the amino acid
`
`at the corresponding position of GLP—l(l—37)OH. Amino acids
`
`38—45 of an extended GLP-l 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—l analog” has sufficient homology to GLP-l(7—
`
`'25
`
`37)OH or a fragment of GLP-l(7-37)OH such that the analog
`
`has insulinotropic activity. Preferably, a GLP—l analog has
`
`the amino acid sequence of GLP—l(7—37)OH or a fragment
`
`thereof, modified so that from one,
`
`two,
`
`three,
`
`four or five
`
`amino acids differ from the amino acid in corresponding
`
`30
`
`position of GLP—l(7-37)OH or the fragment of GLP-l(7-37)QH.
`
`In the nonmenclature used herein to designate GLP—l
`
`compounds,
`
`the substituting amino acid and its position is
`
`indicated prior to the parent structure.
`For example,
`Gluzz-GLP-l(7-37)OH designates a carp—1 compound in which the
`
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`
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`
`_10_
`
`glycine normally found at position 22 of GLP—l(7—37)OH has
`
`been replaced with glutamic acid; ValB—GluZZ'GLP-l(7—37)OH
`
`designates a GLP-1 compound in which alanine normally found
`
`at position 8 and glycine normally found at position 22 of
`
`GLP—l(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—terminus can be unsubstituted,
`as is the case with GLP—l(7—37)OH, amidated with ~NH2,
`-NHR
`
`or NRR’ or esterified with —OR”. R and R' are independently
`
`alkyl or acyl groups (preferably Cl—C20). R” is an alkyl
`
`(Cl—C20).
`
`GLP—l(7—36)NH2 is an example of an “amidated GLP
`
`compound”. Preferably,
`
`the GLP—l 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-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
`
`ppositions 11, 12, 16, 22, 23, 24, 26, 27, 3o, 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—l(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,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1045 PAGE 11
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`_ll._.
`
`and 37 compared to the corresponding amino acid of native
`
`GLP-l(7—37)OH.
`
`It is also preferable that these analogs have 6 or
`
`fewer changes compared to the corresponding amino acids in
`
`native GLP-l(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—l(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—l(7—37)OH.
`
`It is most preferable that these analogs
`
`have 2 or fewer changes compared to the corresponding amino
`
`acids in native GLP—l(7-37)OH.
`
`It has been found that these substitutions reduce the
`
`propensity of GLP—1 compounds to aggregate and generate the
`insoluble form.
`The GLP-l 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—l compounds described herein
`
`are analogs of GLP—1(7—36)NH2 or GLP—l(7—37)OH.
`
`In a preferred embodiment,
`
`the amino acid at position
`
`22 of the GLP—l 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
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1045 PAGE 12
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`
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`
`-12-
`
`acid group.
`
`Thus, examples of preferred amino acids at
`
`position 22 include 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
`
`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(CHZSQ3)—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
`
`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 acid, serine, or histidine and more
`
`preferably glutamatic acid.
`
`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 37 is histidine,
`
`lysine, arginine,
`
`threonine, serine, glutamic acid, aspartic
`
`acid,
`
`tryptophan,
`
`tyrosine, phenylalanine and more
`
`10
`
`15
`
`20
`
`25
`
`preferably histidine.
`
`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
`
`30
`
`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.
`
`MYLAN INST. EXHIBIT 1045 PAGE 13
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`
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`
`-13-
`
`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.
`
`In another preferred embodiment,
`
`the GLP—l compounds of
`
`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—l(7—37)OH.
`
`In another preferred embodiment,
`
`the GLP-l compounds of
`
`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-l(7—37)OH.
`
`As described above,
`
`the GLP—l compounds of the present
`
`invention can have amino acids in addition to those at
`‘ position 8, ll, 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—l(7~37) or a fragment of GLP-
`1(7—37).
`The amino acids other than those at positions 8,
`
`ll, 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—l(7—37)OH are preferably
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1045 PAGE 14
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`
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`
`~14—
`
`conservative substitutions and, more preferably, are highly
`
`conservative substitutions.
`
`Preferably,
`
`the GLP—l 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—l compound differ from the corresponding 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-l compounds with reduced aggregating properties can be
`obtained from known, biologically active GLP-l 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—l 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., J3 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 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
`
`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 substituted phenvl groups in their side chains are
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1045 PAGE 15
`
`MYLAN INST. EXHIBIT 1045 PAGE 15
`
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`
`_15_
`
`considered to have about the same size and shape. Listed
`
`below are five groups of amino acids. Replacing an amino
`
`acid in a GLP—l compound with another amino acid from the
`
`same groups results in a conservative substitution:
`
`Group I: glycine, alanine, valine,
`
`leucine,
`
`isoleucine, serine,
`
`threonine, cysteine, and non-
`
`naturally occurring amino acids with C1—C4 aliphatic
`
`or Cl—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 Cl—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 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.
`
`10
`
`15
`
`20
`
`25
`
`Except as otherwise specifically provided herein,
`
`30
`
`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
`
`MYLAN INST. EXHIBIT 1045 PAGE 16
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`
`_16_
`
`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 highly conservative
`
`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—l compound of the present
`
`invention is a polypeptide having the amino acid sequence of
`
`SEQ ID NO:1.
`
`In a preferred example,
`
`the GLP—l compound is
`
`GLP—l(7—37)OH except that Xaag is Gly or Val, Xaaw is Glu or
`Lys, and Xaam is Glu or Lys.‘
`In another example,
`the GLP-l
`
`compound is GLP-l(7—37)OH except that Xaag is Gly or Val and
`
`Xaam is Glu.
`
`An additional example is a GLP—l compound
`
`10
`
`15
`
`which is GLP—l(7—37)OH except that XaaB is Gly or Val and
`Xaay is His.
`
`20
`
`25
`
`30
`
`Another example of a GLP—l compound of the present
`
`invention is a polypeptide having the amino acid sequence of
`
`SEQ ID. No. 4.
`
`In a preferred example, Xaa7 is L—histidine,
`
`D—histidine, desamino—histidine, 2amino—histidine,
`
`B—
`
`hydroxy—histidine, homohistidine, a—fluoromethyl—histidine
`
`and a-methyl-histidine, Xaag 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 Xaan is lysine, glutamic acid,
`
`aspartic acid or arginine in SEQ ID NO:4.
`In a more
`preferred example, Xaa7 is L-histidine, Xaag is glycine or
`valine, Xaafl is lysine, glutamic acid, aspartic acid or
`
`arginine and R is Gly(OH). Alternatively, Xaa7, Xaag and R
`
`in SEQ ID NO:
`
`4 are as described above and Xaan is an amino
`
`MYLAN INST. EXHIBIT 1045 PAGE 17
`
`MYLAN INST. EXHIBIT 1045 PAGE 17
`
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`
`-17-
`
`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 Cl-C4 alkyl
`
`side chain, a C1—C4 hydroxylated alkyl side chain or a C1—C4
`
`thiolated alkyl chain.
`In another example of the GLP—l 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,
`
`