(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2011/0166321 A1
`(43) Pub. Date:
`Jul. 7, 2011
`GARBAY et al.
`
`US 2011 0166321A1
`
`(54) DOUBLE-ACYLATED GLP-1 DERIVATIVES
`
`(75) Inventors:
`
`PATRICK WILLIAM GARIBAY,
`HOLTE (DK); JANE SPETZLER,
`BRONSHOJ (DK); JANOS
`TIBOR KODRA, KOBENHAVN
`O (DK); LARS LINDEROTH,
`ALLEROD (DK); JESPER LAU,
`FARUM (DK); PER
`SAUERBERG, FARUM (DK)
`
`(73) Assignee:
`
`NOVO NORDISKA/S,
`BAGSVAERD (DK)
`
`(21) Appl. No.:
`
`12/970,196
`
`(22) Filed:
`
`Dec. 16, 2010
`
`Related U.S. Application Data
`(60) Provisional application No. 61/288,601, filed on Dec.
`21, 2009.
`
`(30)
`
`Foreign Application Priority Data
`
`Dec. 16, 2009 (EP) .................................. 09 179390.1
`
`Publication Classification
`
`(51) Int. Cl.
`C07K I4/435
`
`(2006.01)
`
`(52) U.S. Cl. ......................................... 530/323: 530/324
`(57)
`ABSTRACT
`The invention relates to a derivative of a GLP-1 analogue,
`which analogue comprises a first K residue at a position
`corresponding to position 37 of GLP-1 (7-37) (SEQ ID NO:
`1), a second Kresidue at a position corresponding to position
`26 of GLP-1 (7-37), and a maximum often amino acid modi
`fications as compared to GLP-1 (7-37), wherein the first K
`residue is designated K. and the second Kresidue is desig
`nated K, which derivative comprises two albumin binding
`moieties attached to K and K7, respectively, wherein the
`albumin binding moiety comprises a protracting moiety
`selected from:
`Chem. 1:
`HOOC-(CH2). CO *
`Chem. 2:
`HOOC-CH-O-(CH2), CO *
`Chem. 3:
`R'—CH-(CH2)—CO *
`Chem. 4:
`HOOC-CSH2-(CH2), CO *
`in which X is an integer in the range of 6-18, y is an integer in
`the range of 3-17, Z is an integer in the range of 1-5, R' is a
`group having a molar mass not higher than 150 Da, and w is
`an integer in the range of 6-18; with the proviso that when the
`protracting moiety is Chem. 1, the albumin binding moiety
`further comprises a linker of formula Chem. 5: * NH
`(CH2) (O-(CH2)). O—(CH), CO-*, whereinkis
`an integer in the range of 1-5, and n is an integer in the range
`of 1-5; or a pharmaceutically acceptable salt, amide, or ester
`thereof.
`The invention also relates to the pharmaceutical use thereof,
`for example in the treatment and/or prevention of all forms of
`diabetes and related diseases, as well as to corresponding
`novel peptides and side chain intermediates. The derivatives
`are suitable for oral administration.
`
`MPI EXHIBIT 1061 PAGE 1
`
`

`

`US 2011/0166321 A1
`
`Jul. 7, 2011
`
`DOUBLE-ACYLATED GLP-1 DERVATIVES
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`0001. The present application claims the benefit under 35
`U.S.C. 119 of Danish application EP09179390.1 filed Dec.
`16, 2009, and of U.S. provisional application 61/288,601,
`filed Dec. 21, 2009, the contents of which are hereby incor
`porated by reference.
`
`FIELD OF THE INVENTION
`0002 The present invention relates to derivatives of Glu
`cagon-Like Peptide 1 (GLP-1) and their pharmaceutical use,
`viz. to double-acylated GLP-1 derivatives acylated at position
`26 and 37, and their pharmaceutical use.
`
`INCORPORATION-BY-REFERENCE OF THE
`SEQUENCE LISTING
`0003. The Sequence Listing, entitled “SEQUENCE LIST
`ING”, is 1 kilobyte, was created on Dec. 16, 2010, and is
`incorporated herein by reference.
`
`BACKGROUND OF THE INVENTION
`0004 Journal of Medicinal Chemistry (2000), vol.43, no.
`9, p. 1664-669 discloses derivatives of GLP-1 (7-37) that are
`double-acylated at K
`see Table 1.
`0005 WO98/08871 discloses a number of GLP-1 deriva
`tives including some that are double-acylated at K, see
`Examples 3, 7, 17, 24, 32, 33, and 36. Liraglutide, a mono
`acylated GLP-1 derivative for once daily administration
`which is marketed as of 2009 by Novo Nordisk A/S, is also
`disclosed in WO98/08871 (Example 37).
`0006 WO 99/43706 discloses a number of mono- and
`double-acylated GLP-1 derivatives including some K7
`derivatives (see p. 148-178).
`0007 WO 2005/027978 discloses a number of GLP-1
`derivatives including a few that are double-acylated at one
`and the same residue, K, see Examples 8 and 9.
`0008 WO 2009/030738 discloses a number of GLP-1
`derivatives including one double-acylated at K', Dap, see
`Example 37.
`0009 Journal of Controlled Release (2010), vol. 144, p.
`10-16 relates to acylated exendin-4 analogs and discloses,
`among others, a double-acylated exendin-4 (K'7-diLUA
`Exendin-4) is disclosed (LUA is lauric acid, C12).
`0010 WO 06/097537 discloses a number of GLP-1
`derivatives including Semaglutide (Example 4), a mono-acy
`lated GLP-1 derivative for once weekly administration which
`is under development by Novo Nordisk A/S.
`0011 Angewandte Chemie International Edition 2008,
`vol. 47, p. 3196-3201 reports the discovery and characterisa
`tion of a class of 4-(p-iodophenyl)butyric acid derivatives
`which purportedly display a stable noncovalent binding inter
`action with both mouse serum albumin (MSA) and human
`serum albumin (HSA).
`
`SUMMARY OF THE INVENTION
`0012. The invention relates to derivatives of GLP-1 pep
`tides.
`0013 The derivatives are acylated at the native lysine at
`position 26, as well as at a lysine substituted for the native
`glycine at position 37. The side chains are albumin binding
`
`moieties. They comprise a protracting moiety, preferably
`selected from fatty diacids, and fatty acids with a distal phe
`nyl, phenoxy, or thiophene group, all optionally Substituted.
`A carboxy group of the fatty acid or fatty diacid is acylated,
`optionally via a linker, to a lysine residue of the GLP-1
`peptide, preferably at the epsilon-amino group thereof. The
`GLP-1 peptide may be an analogue of GLP-1 (7-37) (SEQID
`NO: 1) having a total of up to ten amino acid differences as
`compared to GLP-1 (7-37), for example one or more addi
`tions, one or more deletions, and/or one or more Substitutions.
`0014 More in particular, the invention relates to a deriva
`tive of a GLP-1 analogue, which analogue comprises a first K
`residue at a position corresponding to position 37 of GLP-1
`(7-37) (SEQ ID NO: 1), a second K residue at a position
`corresponding to position 26 of GLP-1 (7-37), and a maxi
`mum often amino acid modifications as compared to GLP-1
`(7-37), wherein the first Kresidue is designated K, and the
`second K residue is designated K'; which derivative com
`prises two albumin binding moieties attached to K and K7.
`respectively, wherein each albumin binding moiety com
`prises a protracting moiety selected from Chem. 1. Chem. 2.
`Chem. 3, and Chem. 4:
`HOOC-(CH2). CO *
`
`Chem. 1:
`
`HOOC-CH-O-(CH2), CO *
`
`R'—CH-(CH2)—CO *
`
`Chem. 2:
`
`Chem. 3:
`
`Chem. 4:
`HOOC-CSH-(CH2). CO *,
`in which X is an integer in the range of 6-18, y is an integer in
`the range of 3-17, Z is an integer in the range of 1-5, R is a
`group having a molar mass not higher than 150 Da, and w is
`an integer in the range of 6-18; with the proviso that when the
`protracting moiety is Chem. 1, the albumin binding moiety
`further comprises a linker of formula Chem. 5:
`
`Chem.5:
`
`:
`
`whereink is an integer in the range of 1-5, and n is an integer
`in the range of 1-5; or a pharmaceutically acceptable salt,
`amide, or ester thereof.
`0015 The invention also relates to such derivative for use
`as a medicament, in particular for use in the treatment and/or
`prevention of all forms of diabetes and related diseases, such
`as eating disorders, cardiovascular diseases, gastrointestinal
`diseases, diabetic complications, critical illness, and/or poly
`cystic ovary syndrome; and/or for improving lipid param
`eters, improving B-cell function, and/or for delaying or pre
`venting diabetic disease progression.
`0016. The invention furthermore relates to intermediate
`products in the form of GLP-1 peptides and side chains,
`which are relevant for the preparation of certain GLP-1 pep
`tides and derivatives of the invention.
`0017. The derivatives of the invention are biologically
`active. Also, or alternatively, they have a protracted pharma
`cokinetic profile. Also, or alternatively, they are stable against
`degradation by gastro intestinal enzymes. Also, or alterna
`tively, they have a high oral bioavailability. These properties
`
`MPI EXHIBIT 1061 PAGE 2
`
`

`

`US 2011/0166321 A1
`
`Jul. 7, 2011
`
`are of importance in the development of next generation
`GLP-1 compounds for Subcutaneous, intravenous, and/or in
`particular oral administration.
`
`DESCRIPTION OF THE INVENTION
`0018. The invention relates to derivatives of GLP-1 pep
`tides. The derivatives are acylated at the native lysine at
`position 26, as well as at a lysine substituted for the native
`glycine at position 37. The side chains are albumin binding
`moieties. They comprise a protracting moiety, preferably
`selected from fatty diacids, and fatty acids with a distal, or
`terminal, phenyl, thiophene, or phenoxy group, all optionally
`substituted. A carboxy group of the fatty acid or fatty diacid is
`acylated, optionally via a linker, to a lysine residue of the
`GLP-1 peptide, preferably at the epsilon-amino group
`thereof. The GLP-1 peptide may be an analogue of GLP-1 (7-
`37) (SEQ ID NO: 1) having a total of up to ten amino acid
`differences as compared to GLP-1 (7-37), for example one or
`more additions, one or more deletions, and/or one or more
`Substitutions.
`0019 More in particular, in a first aspect, the invention
`relates to a derivative of a GLP-1 analogue, which analogue
`comprises a first K residue at a position corresponding to
`position 37 of GLP-1 (7-37) (SEQ ID NO: 1), a second K
`residue at a position corresponding to position 26 of GLP-1
`(7-37), and a maximum often amino acid modifications as
`compared to GLP-1 (7-37), wherein the first K residue is
`designated K, and the second Kresidue is designated K.
`which derivative comprises two albumin binding moieties
`attached to K and K7, respectively, wherein the albumin
`binding moiety comprises a protracting moiety selected from
`Chem. 1, Chem. 2, Chem. 3, and Chem. 4:
`HOOC-(CH), CO *
`
`Chem. 1:
`
`HOOC-CH-O-(CH), CO *
`
`R'—CH-(CH2)-CO-
`
`Chem. 2:
`
`Chem. 3:
`
`Chem. 4:
`HOOC-CSH2-(CH2), CO *
`in which X is an integer in the range of 6-18, y is an integer in
`the range of 3-17, Z is an integer in the range of 1-5, R' is a
`group having a molar mass not higher than 150 Da, and w is
`an integer in the range of 6-18; with the proviso that when the
`protracting moiety is Chem. 1, the albumin binding moiety
`further comprises a linker of formula Chem. 5:
`
`Chem.5:
`
`: N-N-N- --- :
`wherein k is an integer in the range of 1-5, and n is an integer
`in the range of 1-5; or a pharmaceutically acceptable salt,
`amide, or ester thereof.
`0020. Thus, in a first aspect, the invention relates to a
`derivative of a GLP-1 analogue, wherein the GLP-1 analogue
`comprises a first K residue at a position corresponding to
`position 37 of GLP-1 (7-37) (SEQ ID NO: 1), a second K
`residue at a position corresponding to position 26 of GLP-1
`(7-37), and a maximum often amino acid modifications as
`compared to GLP-1 (7-37), wherein the first K residue is
`
`designated K', and the second Kresidue is designated K.
`which derivative comprises two albumin binding moieties
`attached to K and K", respectively, wherein the albumin
`binding moiety comprises a protracting moiety selected from
`Chem. 2, Chem. 3, and Chem. 4:
`HOOC-CH-O-(CH2), CO *
`
`Chem. 2:
`
`R'—CH-(CH2)—CO *
`
`Chem. 3:
`
`Chem. 4:
`HOOC-CSH2-(CH2), CO *
`in whichy is an integer in the range of 3-17, Z is an integer in
`the range of 1-5, R' is a group having a molar mass not higher
`than 150 Da, and w is an integer in the range of 6-18; or a
`pharmaceutically acceptable salt, amide, or ester thereof.
`0021. In a second aspect, the invention relates to a deriva
`tive of a GLP-1 analogue, wherein the GLP-1 analogue com
`prises a first Kresidue at a position corresponding to position
`37 of GLP-1 (7-37) (SEQID NO: 1), a second Kresidue at a
`position corresponding to position 26 of GLP-1 (7-37), and a
`maximum of ten amino acid modifications as compared to
`GLP-1 (7-37), wherein the first K residue is designated K7.
`and the second Kresidue is designated K, which derivative
`comprises two albumin binding moieties attached to K and
`K", respectively, wherein the albumin binding moiety com
`prises i) a protracting moiety of formula Chem. 1:
`Chem. 1:
`HOOC-(CH), CO *
`in which X is an integer in the range of 6-18; and ii) a linker of
`formula Chem. 5:
`
`Chem.5:
`
`: N-N-N- --- :
`whereink is an integer in the range of 1-5, and n is an integer
`in the range of 1-5:
`or a pharmaceutically acceptable salt, amide, or ester thereof.
`0022. In a third aspect, the invention relates to a derivative
`of a GLP-1 analogue, wherein the GLP-1 analogue comprises
`a first K residue at a position corresponding to position 37 of
`GLP-1 (7-37) (SEQID NO: 1), a second Kresidue at a posi
`tion corresponding to position 26 of GLP-1 (7-37), and a
`maximum of ten amino acid modifications as compared to
`GLP-1 (7-37), wherein the first K residue is designated K7.
`and the second Kresidue is designated K'; which derivative
`comprises two protracting moieties attached to K and K7.
`respectively, via a linker, wherein the protracting moiety is
`selected from Chem. 1, Chem. 2, Chem. 3, and Chem. 4:
`HOOC-(CH2). CO *
`Chem. 1:
`
`HOOC-CH-O-(CH), CO *
`
`R'—CH-(CH2)—CO *
`
`Chem. 2:
`
`Chem. 3:
`
`Chem. 4:
`HOOC-CSH2-(CH2), CO *
`in which X is an integer in the range of 6-18, y is an integer in
`the range of 3-17, Z is an integer in the range of 1-5, R' is a
`group having a molar mass not higher than 150 Da, and w is
`an integer in the range of 6-18; and the linker comprises
`Chem. 5:
`
`MPI EXHIBIT 1061 PAGE 3
`
`

`

`US 2011/0166321 A1
`
`Jul. 7, 2011
`
`Chem.5:
`
`:
`
`wherein k is an integer in the range of 1-5, and n is an integer
`in the range of 1-5; or a pharmaceutically acceptable salt,
`amide, or ester thereof.
`0023 The invention also relates to an intermediate product
`in the form of a GLP-1 analogue which comprises the follow
`ing modifications as compared to GLP-1 (7-37) (SEQID NO:
`1): (i) (8Aib,31H, 34Q, 37K); (ii) (des7-8, 34R, 37K, 38E):
`(iii) (des7-8,34R,37K); (iv) (8Aib,9G,34R,37K); (v) (8Aib,
`23R, 34R,37K); (vi) (31H,34Q,37K); (vii) (9Q,34R, 37K);
`(iix) (30E, 34R, 37K); (ix) (34R, 37K, 38G); (x) (34R, 36G,
`37K); or (xi) (34R, 37K, 38E); or a pharmaceutically accept
`able salt, amide, or ester of any of the analogues thereof.
`0024. The invention also relates to an intermediate product
`comprising a protracting moiety selected from Chem. 2c,
`Chem. 3b, and Chem. 4b:
`HOOC-CH-O-(CH2), CO-PG
`
`Chem. 2c:
`
`R—CH-(CH2)—CO-PG
`
`Chem. 3b:
`
`Chem. 4b:
`HOOC-CSH2-(CH2), CO-PG
`0025 in which y is an integer in the range of 3-17, Z is an
`integer in the range of 1-5, R' is a group having a molar mass
`not higher than 150 Da, w is an integer in the range of 6-18,
`and *—CO-PG is an activated ester; wherein, optionally, the
`distal *—COOH group of the protracting moiety, if present, is
`functionalised as a non-reactive ester, or a pharmaceutically
`acceptable salt, amide, or ester thereof.
`0026. And finally the invention also relates to the pharma
`ceutical use of the analogues and derivatives of the invention,
`in particular for use in the treatment and/or prevention of all
`forms of diabetes and related diseases, such as eating disor
`ders, cardiovascular diseases, gastrointestinal diseases, dia
`betic complications, critical illness, and/or polycystic ovary
`syndrome; and/or for improving lipid parameters, improving
`B-cell function, and/or for delaying or preventing diabetic
`disease progression.
`0027. In what follows, Greek letters may be represented
`by their symbol or the corresponding written name, for
`example: C. alpha; B-beta; e-epsilon; Y-gamma; () omega;
`etc. Also, the Greek letter of my be represented by 'u', e.g.
`in ul-ul, or in uMuM.
`0028. Anasterisk (*) in a chemical formula designates i) a
`point of attachment, ii) a radical, and/or iii) an unshared
`electron.
`
`GLP-1 Analogues
`0029. The term “GLP-1 analogue' or “analogue of GLP
`1 as used herein refers to a peptide, or a compound, which is
`a variant of the human Glucagon-Like Peptide-1 (GLP-1 (7-
`37)), the sequence of which is included in the sequence listing
`as SEQID NO: 1. The peptide having the sequence of SEQID
`NO: 1 may also be designated “native' GLP-1.
`0030. In the sequence listing, the first amino acid residue
`of SEQ ID NO: 1 (histidine) is assigned no. 1. However, in
`what follows—according to established practice in the art—
`
`this histidine residue is referred to as no. 7, and subsequent
`amino acid residues are numbered accordingly, ending with
`glycine no. 37. Therefore, generally, any reference herein to
`an amino acid residue number or a position number of the
`GLP-1 (7-37) sequence is to the sequence starting with His at
`position 7 and ending with Gly at position 37.
`0031 GLP-1 analogues of the derivatives of the invention
`may be described by reference to i) the number of the amino
`acid residue in native GLP-1 (7-37) which corresponds to the
`amino acid residue which is modified (i.e., the corresponding
`position in native GLP-1), and to ii) the actual modification.
`The following are non-limiting examples of suitable analogue
`nomenclature.
`0032. A non-limiting example of a GLP-1 analogue of the
`derivative of the invention is an analogue that only is modified
`So as to comprise a first lysine residue at a position corre
`sponding to position 37 of GLP-1 (7-37). The amino acid
`sequence of this analogue is otherwise identical to that of
`native GLP-1, and this analogue may be designated K7
`GLP-1 (7-37). This designation represents the amino acid
`sequence of native GLP-1 where glycine at position 37 has
`been substituted with lysine.
`0033. This GLP-1 analogue of the derivative of the inven
`tion furthermore comprises a second lysine residue at a posi
`tion corresponding to position 26 of GLP-1 (7-37). As the
`amino acid sequence of this analogue is otherwise identical to
`that of native GLP-1, such analogue is, still, designated K
`GLP-1 (7-37), as K is implied by the reference to native
`GLP-1 (7-37), SEQID NO: 1.
`0034. Accordingly, K-GLP-1 (7-37) designates a GLP-1
`(7-37) analogue wherein the naturally occurring glycine at
`position 37 has been substituted with lysine.
`0035. The term “analogue of K7-GLP-1 (7-37) refers to
`an analogue of GLP-1 (7-37) which comprises the modifica
`tion K7 and at least one additional modification, as compared
`to GLP-1 (7-37).
`0036. The GLP-1 analogue forming part of the derivative
`of the invention comprises a first K residue at a position
`corresponding to position 37 of GLP-1 (7-37) (SEQ ID NO:
`1), a second Kresidue at a position corresponding to position
`26 of GLP-1 (7-37), and a maximum often amino acid modi
`fications as compared to GLP-1 (7-37), wherein the first K
`residue is designated K. and the second Kresidue is desig
`nated K. In other words, it is a modified GLP-1 (7-37) pep
`tide in which a number of amino acid residues have been
`changed when compared to native GLP-1 (7-37) (SEQ ID
`NO: 1). These changes, or modifications, may represent,
`independently, one or more amino acid Substitutions, addi
`tions, and/or deletions.
`0037 Another non-limiting example of an analogue of a
`derivative of the invention is Aib.Arg, Lys7GLP-1 (7-
`37), which designates a GLP-1 (7-37) analogue, in which the
`alanine at position 8 has been Substituted with O-aminoisobu
`tyric acid (Aib), the lysine at position 34 has been substituted
`with arginine, and the glycine at position 37 has been Substi
`tuted with lysine. This analogue may also be designated
`(8Aib, R34, K37) GLP-1 (7-37).
`0038 An additional non-limiting example of an analogue
`of a derivative of the invention is an analogue “which com
`prises 34E, 34Q, or 34R which refers to a GLP-1 analogue
`which has either a glutamic acid (E), a glutamine (Q), or an
`arginine (R) at a position corresponding to position 34 of
`native GLP-1 (SEQ ID NO: 1), and which may comprise
`further modifications as compared to SEQID NO: 1.
`
`MPI EXHIBIT 1061 PAGE 4
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`

`US 2011/0166321 A1
`
`Jul. 7, 2011
`
`0039. A still further non-limiting example of an analogue
`of a derivative of the invention is the analogue of GLP-1 (7-
`37) (SEQID NO: 1) which is simply designated “(8Aib,31H,
`34Q, 37K)'. This designation refers to an analogue which is
`identical to SEQID NO: 1 except for these four substitutions,
`i.e. an analogue in which the alanine at position 8 has been
`substituted with O-aminoisobutyric acid (Aib), the tryp
`tophan at position 31 has been substituted with histidine, the
`lysine at position 34 has been Substituted with glutamine, and
`the glycine at position 37 has been substituted with lysine.
`This analogue does not comprise further modifications as
`compared to SEQID NO: 1.
`0040. A still further non-limiting example of an analogue
`of a derivative of the invention is an analogue comprising
`des7 (or Des'), which refers to an analogue of GLP-1 (7-37) in
`which the N-terminal amino acid, histidine, has been deleted.
`This analogue may also be designated GLP-1 (8-37).
`0041. Similarly, (des7+des8); (des7, des8); (des7-8); or
`(Des'. Des) in relation to an analogue of GLP-1 (7-37),
`where the reference to GLP-1 (7-37) may be implied, refers to
`an analogue in which the amino acids corresponding to the
`two N-terminal amino acids of native GLP-1, histidine and
`alanine, have been deleted. This analogue may also be desig
`nated GLP-1 (9–37).
`0042. A still further non-limiting example of an analogue
`of a derivative of the invention is an analogue comprising
`Imp', and/or (Aib or S), which refers to a GLP-1 (7-37)
`analogue, which, when compared to native GLP-1, comprises
`a substitution of histidine at position 7 with imidazopropionic
`acid (Imp); and/or a Substitution of alanine at position 8 with
`C.-aminoisobutyric acid (Aib), or with serine.
`0043 Analogues “comprising certain specified modifi
`cations may comprise further modifications, when compared
`to SEQID NO: 1. Two examples, non-limiting, of analogues
`comprising Imp, and/or (Aib or S), and forming part of
`derivatives of the invention are the peptide parts of Chem. 47
`and Chem. 58.
`0044) Non-limiting examples of an analogue of GLP-1 (7-
`37) comprising (des7+des8), Arg34, Lys37, and Glu38 are the
`following:
`Des". DesArg, Lys7IGLP-1 (7-37)-Glu
`peptide; and N'-(2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl
`2-methyl-propionylArg, Lys7GLP-1 (9-37)Glu-pep
`tide. In the latter compound a dipeptide mimetic of the N-ter
`minus of native GLP-1 (His-Ala) is attached to the new
`N-terminus, Glu 9, via an amide bond.
`0045 Suitable His- or His-Ala mimetics that may be used
`as a kind of a substitute for the deleted N-terminal amino
`acids, if any, comprise a heterocyclic, nitrogen-containing,
`aromatic ring structure, e.g. pyridine or imidazole. Preferred
`His- or His-Ala mimetics are derivatives of an imidazole or a
`pyridine, other than His and His-Ala, in one embodiment
`having a Substituent with a free carboylic acid group, which
`can form an amide bond with an amino group of the N-ter
`minal amino acid of the peptide. The term imidazole refers to
`imidazoles as a class of heterocycles with similar ring struc
`ture but varying Substituents, and Vice-versa for pyridine.
`0046. As is apparent from the above examples, amino acid
`residues may be identified by their full name, their one-letter
`code, and/or their three-letter code. These three ways are fully
`equivalent.
`0047. The expressions “a position equivalent to or “cor
`responding position” may be used to characterise the site of
`modification in a modified GLP-1 (7-37) sequence by refer
`ence to native GLP-1 (7-37) (SEQ ID NO: 1). Equivalent or
`
`corresponding positions, as well as the number of modifica
`tions, are easily deduced, e.g. by simple handwriting and
`eyeballing; and/or a standard protein or peptide alignment
`program may be used, such as “align' which is a Needleman
`Wunsch alignment. The algorithm is described in Needle
`man, S. B. and Wunsch, C. D., (1970), Journal of Molecular
`Biology, 48: 443-453, and the align program by Myers and W.
`Miller in "Optimal Alignments in Linear Space’ CABIOS
`(computer applications in the biosciences) (1988) 4:11-17.
`For the alignment, the default scoring matrix BLOSUM50
`and the default identity matrix may be used, and the penalty
`for the first residue in a gap may be set at -12, or preferably at
`-10, and the penalties for additional residues in a gap at -2, or
`preferably at -0.5.
`0048. An example of such alignment is inserted hereinbe
`low, in which sequence no. 1 is SEQID NO: 1, and sequence
`no. 2 is the analogue (des7-8, 34R, 37K, 38E) thereof:
`
`1: GLP-1 (7-37)
`2 : GLP-1 (7-37) Analogue
`Matrix: EBLOSUM62
`Gap penalty: 10. O
`Extend penalty: 0.5
`Length: 32
`Identity: 27/32 (84.4%)
`Similarity: 28/32 (87.5%)
`Gaps: 3/32 (9.4%)
`Score : 138. O
`
`1.
`
`2
`
`1 HAEGTFTSDWSSYLEGOAAKEFIAWLWKGRG - 31
`
`1 - - EGTFTSDWSSYLEGOAAKEFIAWLVRGRKE 3O
`
`0049. In case of non-natural amino acids such as Imp
`and/or Aib being included in the sequence, or in case of
`His-Ala mimetics, these may, for alignment purposes, be
`replaced with X. If desired, X can later be manually corrected.
`0050. The term "peptide', as e.g. used in the context of the
`GLP-1 analogues of the derivatives of the invention, refers to
`a compound which comprises a series of amino acids inter
`econnected by amide (or peptide) bonds.
`0051. In a particular embodiment the peptide is to a large
`extent, or predominantly, composed of amino acids intercon
`nected by amide bonds (e.g., at least 50%, 60%, 70%, 80%, or
`at least 90%, by molar mass). In another particular embodi
`ment the peptide consists of amino acids interconnected by
`peptide bonds.
`0.052 The peptides of the invention comprise at least five
`constituent amino acids connected by peptide bonds. In par
`ticular embodiments the peptide comprises at least 10, pref
`erably at least 15, more preferably at least 20, even more
`preferably at least 25, or most preferably at least 28 amino
`acids.
`0053. In particular embodiments, the peptide is composed
`of at least five constituent amino acids, preferably composed
`of at least 10, at least 15, at least 20, at least 25, or most
`preferably composed of at least 28 amino acids.
`0054. In additional particular embodiments, the peptide is
`a) composed of, or b) consists of i) 29, ii)30, iii) 31, or iv) 32
`amino acids.
`0055. In a still further particular embodiment the peptide
`consists of amino acids interconnected by peptide bonds.
`0056 Amino acids are molecules containing an amine
`group and a carboxylic acid group, and, optionally, one or
`more additional groups, often referred to as a side chain.
`
`MPI EXHIBIT 1061 PAGE 5
`
`

`

`US 2011/0166321 A1
`
`Jul. 7, 2011
`
`0057 The term "amino acid includes proteogenic amino
`acids (encoded by the genetic code, including natural amino
`acids, and standard amino acids), as well as non-proteogenic
`(not found in proteins, and/or not coded for in the standard
`genetic code), and synthetic amino acids. Thus, the amino
`acids may be selected from the group of proteinogenic amino
`acids, non-proteinogenic amino acids, and/or synthetic
`amino acids.
`0058. Non-limiting examples of amino acids which are
`not encoded by the genetic code are gamma-carboxy
`glutamate, ornithine, and phosphoserine. Non-limiting
`examples of synthetic amino acids are the D-isomers of the
`amino acids such as D-alanine (in what follows sometimes
`abbreviated “a” as f.ex. in “a8, which accordingly refers to
`D-Ala) and D-leucine, Aib (CL-aminoisobutyric acid), 3-ala
`nine, and des-amino-histidine (desh, alternative name imida
`Zopropionic acid, abbreviated Imp).
`0059. In what follows, all amino acids for which the opti
`cal isomer is not stated is to be understood to mean the
`L-isomer (unless otherwise specified).
`0060. The GLP-1 derivatives and analogues of the inven
`tion have GLP-1 activity. This term refers to the ability to bind
`to the GLP-1 receptor and initiate a signal transduction path
`way resulting in insulinotropic action or other physiological
`effects as is known in the art. For example, the analogues and
`derivatives of the invention can be tested for GLP-1 activity
`using the assay described in Example 50 herein.
`
`GLP-1 Derivatives
`0061. The term "derivative” as used herein in the context
`of a GLP-1 peptide or analogue means a chemically modified
`GLP-1 peptide or analogue, in which one or more Substituents
`have been covalently attached to the peptide. The substituent
`may also be referred to as a side chain.
`0062. In a particular embodiment, the side chain is capable
`of forming non-covalent aggregates with albumin, thereby
`promoting the circulation of the derivative with the blood
`stream, and also having the effect of protracting the time of
`action of the derivative, due to the fact that the aggregate of
`the GLP-1-derivative and albumin is only slowly disinte
`grated to release the active pharmaceutical ingredient. Thus,
`the substituent, or side chain, as a whole is preferably referred
`to as an albumin binding moiety.
`0063. In particular embodiments, the side chain has at
`least 10 carbonatoms, or at least 15, 20, 25, 30, 35, or at least
`40 carbon atoms. In further particular embodiments, the side
`chain may further include at least 5 hetero atoms, in particular
`O and N. for example at least 7, 9, 10, 12, 15, 17, or at least 20
`hetero atoms. Such as at least 1, 2, or 3 N-atoms, and/or at least
`3, 6, 9, 12, or 15 O-atoms.
`0064. In another particular embodiment the albuminbind
`ing moiety comprises a portion which is particularly relevant
`for the albumin binding and thereby the protraction, which
`portion may accordingly be referred to as a protracting moi
`ety. The protracting moiety may beat, or near, the opposite
`end of the albumin binding moiety, relative to its point of
`attachment to the peptide.
`0065. In a still further particular embodiment the albumin
`binding moiety comprises a portioninbetween the protracting
`moiety and the point of attachment to the peptide, which
`portion may be referred to as a linker, linker moiety, spacer, or
`the like. The linker may be optional, and hence in that case the
`albumin binding moiety may be identical to the protracting
`moiety.
`0066. In particular embodiments, the albumin binding
`moiety and/or the protracting moiety is lipophilic, and/or
`negatively charged at physiological pH (7.4).
`
`0067. The albuminbinding moiety, the protracting moiety,
`or the linker may be covalently attached to a lysine residue of
`the GLP-1 peptide by acylation. Additional or alternative
`conjugation chemistry includes alkylation, ester formation,
`oramide formation, or coupling to a cysteine residue, such as
`by maleimide or haloacetamide (such as bromo-/fluoro-/
`iodo-) coupling.
`0068. In a preferred embodiment, an active ester of the
`albumin binding moiety, preferably comprising a protracting
`moiety and a linker, is covalently linked to an amino group of
`alysine residue, preferably the epsilon amino group thereof,
`underformation of an amide bond (this process being referred
`to as acylation).
`0069. Unless otherwise stated, when reference is made to
`an acylation of a lysine residue, it is understood to be to the
`epsilon-amino group thereof.
`0070 A derivative comprising two protracting moieties
`attached to K and K7, optionally via a linker, may be
`referred to as a derivative which has been acylated twice,
`double-acylated, or dual acylated at the epsilon-amino groups
`of the lysine residues at positions corresponding to position
`26 and 37, respectively, of GLP-1 (7-37).
`0071. For the present purposes, the terms “albumin bind
`ing moiety”, “protracting moiety', and “linker may include
`the unreacted as well as the reacted forms of these molecules.
`Whether or not one or the otherform is meant is clear from the
`context in which the term is used.
`0072. In one aspect, each protracting moiety comprises, or
`consists of a protracting moiety independently selected from
`Chem. 1, Chem. 2, Chem. 3, and Chem. 4:
`HOOC-(CH2). CO *
`
`Chem. 1:
`
`HOOC-CH-O-(CH2), CO *
`
`R'—CH-(CH2)—CO *
`
`Chem. 2:
`
`Chem. 3:
`
`Chem. 4:
`HOOC-CSH2-(CH2), CO *
`0073 in which X is an integer in the range of 6-18, y is an
`integer in the range of 3-17, Z is an integer in the range of 1-5,
`R" is a group having a molar mass not higher than 150 Da, and
`w is an integer in the range of 6-18.
`0074. In one embodiment, *—(CH), * refers to straight
`or branched, preferably straight, alkylene in which X is an
`integer in the range of 6-18.
`(0075.