`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`ACYLATED GLP-1 COMPOUNDS
`
`FIELD OF THE INVENTION
`
`5
`
`This invention relates to the field of therapeutic peptides, i.e. to new protracted GLP-1
`
`compounds.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`A range of different approaches have been used for modifying the structure of glucagon-like
`
`peptide 1 (GLP-1) compounds in order to provide a longer duration of action in vivo.
`
`WO 96/29342 discloses peptide hormone derivatives wherein the parent peptide hormone
`
`has been modified by introducing a lipophilic substituent in the C-terminal amino acid residue
`
`or in the N-terminal amino acid residue.
`
`15 WO 98/08871 discloses GLP-1 derivatives wherein at least one amino acid residue of the
`
`parent peptide has a lipophilic substituent attached.
`
`WO 99/43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives which have a lipophilic
`
`substituent attached to the C-terminal amino acid residue.
`
`WO 00/34331 discloses acylated GLP-1 analogs.
`
`20 WO 00/69911 discloses activated insulinotropic peptides to be injected into patients where
`
`they are supposed to react with blood components to form conjugates and thereby alledgedly
`
`providing longer duration of action in vivo.
`
`WO 02/46227 discloses GLP-1 and exendin-4 analogs fused to human serum albumin in
`
`order to extend in vivo half-life.
`
`25
`
`Many diabetes patients particularly in the type 2 diabetes segment are subject to so-called
`
`"needle-phobia", i.e. a substantial fear of injecting themselves. In the type 2 diabetes
`
`segment most patients are treated with oral hypoglycaemic agents, and since GLP-1
`
`compounds are expected to be the first injectable product these patients will be
`
`30
`
`administered, the fear of injections may become a serious obstacle for the widespread use of
`
`the clinically very promising GLP-1 compounds. Thus, there is a need to develop new GLP-1
`
`compounds which can be administered less than once daily, e.g. once every second or third
`
`day preferably once weekly, while retaining an acceptable clinical profile.
`
`35
`
`SUMMARY OF THE INVENTION
`
`MPI EXHIBIT 1015 PAGE 2
`
`MPI EXHIBIT 1015 PAGE 2
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`2
`
`The invention provides a GLP-1 analog having a modification of at least one non-proteogenic
`
`amino acid residue in positions 7 and/or 8 relative to the sequence GLP-1 (7-37) (SEQ ID No
`
`1 ), which is acylated with a moiety to the lysine residue in position 26, and where said moiety
`
`comprises at least two acidic groups, wherein one acidic group is attached terminally.
`
`5
`
`The present invention also provides pharmaceutical compositions comprising a compound
`
`according to the present invention and the use of compounds according to the present
`
`invention for preparing medicaments for treating disease.
`
`10
`
`The invention provides a method for increasing the time of action in a patient of a GLP-1
`
`analog, characterised in acylating said GLP-1 analog with a moiety B-U' as disclosed in any
`
`of the preceding claims, on the lysine residue in position 26 of said GLP-1 analog.
`
`15
`
`DESCRIPTION OF THE INVENTION
`
`In the present specification, the following terms have the indicated meaning :
`
`The term "polypeptide" and "peptide" as used herein means a compound composed
`
`of at least five constituent amino acids connected by peptide bonds. The constituent amino
`
`acids may be from the group of the amino acids encoded by the genetic code and they may
`
`20
`
`be natural amino acids which are not encoded by the genetic code, as well as synthetic
`
`amino acids. Natural amino acids which are not encoded by the genetic code are e.g., y(cid:173)
`
`carboxyglutamate, ornithine, phosphoserine, D-alanine and O-glutamine. Synthetic amino
`
`acids comprise amino acids manufactured by chemical synthesis, i.e. O-isomers of the amino
`
`acids encoded by the genetic code such as D-alanine and O-leucine, Aib (a-aminoisobutyric
`
`25
`
`acid), Abu (a-aminobutyric acid), Tie (tert-butylglycine), ~-alanine, 3-aminomethyl benzoic
`
`acid, anthranilic acid.
`
`The 22 proteogenic amino acids are:
`
`Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Cystine, Glutamine, Glutamic acid,
`
`Glycine, Histidine, Hydroxyproline, lsoleucine, Leucine, Lysine, Methionine, Phenylalanine,
`
`30
`
`Praline, Serine, Threonine, Tryptophan, Tyrosine, Valine.
`
`Thus a non-proteogenic amino acid is a moiety which can be incorporated into a peptide via
`
`peptide bonds but is not a proteogenic amino acid. Examples are y-carboxyglutamate,
`
`ornithine, phosphoserine, the D-amino acids such as D-alanine and D-glutamine, Synthetic
`
`non-proteogenic amino acids comprise amino acids manufactured by chemical synthesis, i.e.
`
`35
`
`D-isomers of the amino acids encoded by the genetic code such as D-alanine and O-leucine,
`
`MPI EXHIBIT 1015 PAGE 3
`
`MPI EXHIBIT 1015 PAGE 3
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`3
`
`Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid), Tie (tert-butylglycine), 3-
`
`aminomethyl benzoic acid, anthranilic acid, des-amino-Histidine, the beta analogs of amino
`
`acids such as [3-alanine etc. D-histidine, desamino-histidine, 2-amino-histidine, f3-hydroxy(cid:173)
`
`histidine, homohistidine, Na-acetyl-histidine, a-fluoromethyl-histidine, a-methyl-histidine, 3-
`
`5
`
`pyridylalanine, 2-pyridylalanine or 4-pyridylalanine, (1-aminocyclopropyl) carboxylic acid, (1-
`
`aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)
`
`carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;
`
`The term "analogue" as used herein referring to a polypeptide means a modified
`
`peptide wherein one or more amino acid residues of the peptide have been substituted by
`
`10
`
`other amino acid residues and/or wherein one or more amino acid residues have been
`
`deleted from the peptide and/or wherein one or more amino acid residues have been deleted
`
`from the peptide and or wherein one or more amino acid residues have been added to the
`
`peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of
`
`the peptide and/or at the C-terminal of the peptide. A simple system is often used to describe
`analogues : For example [Arg 34]GLP-1 (7-37)Lys designates a GLP-1 (7-37) analogue wherein
`
`15
`
`the naturally occurring lysine at position 34 has been substituted with arginine and wherein a
`lysine has been added to the terminal amino acid residue, i.e. to the Gly37
`
`. All amino acids for
`
`which the optical isomer is not stated is to be understood to mean the L-isomer. In
`
`embodiments of the invention a maximum of 17 amino acids have been modified. In
`
`20
`
`embodiments of the invention a maximum of 15 amino acids have been modified. In
`
`embodiments of the invention a maximum of 10 amino acids have been modified. In
`
`embodiments of the invention a maximum of 8 amino acids have been modified. In
`
`embodiments of the invention a maximum of 7 amino acids have been modified. In
`
`embodiments of the invention a maximum of 6 amino acids have been modified. In
`
`25
`
`embodiments of the invention a maximum of 5 amino acids have been modified. In
`
`embodiments of the invention a maximum of 4 amino acids have been modified. In
`
`embodiments of the invention a maximum of 3 amino acids have been modified. In
`
`embodiments of the invention a maximum of 2 amino acids have been modified. In
`
`embodiments of the invention 1 amino acid has been modified.
`
`30
`
`The term "derivative" as used herein in relation to a peptide means a chemically modified
`
`peptide or an analogue thereof, wherein at least one substituent is not present in the
`
`unmodified peptide or an analogue thereof, i.e. a peptide which has been covalently
`
`modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters
`and the like. An example of a derivative of GLP-1 (7-37) is NE26-((4S)-4-(hexadecanoylamino)-
`, Lys26]G LP-1-(7-37).
`carboxy-butanoyl)[ Arg 34
`
`35
`
`MPI EXHIBIT 1015 PAGE 4
`
`MPI EXHIBIT 1015 PAGE 4
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`4
`
`The term "GLP-1 peptide" as used herein means GLP-1 (7-37) (SEQ ID No 1 ), a
`
`GLP-1 (7-37) analogue, a GLP-1 (7-37) derivative or a derivative of a GLP-1 (7-37) analogue.
`
`In one embodiment the GLP-1 peptide is an insulinotropic agent.
`
`The term "insulinotropic agent" as used herein means a compound which is an
`
`5
`
`agonist of the human GLP-1 receptor, i.e. a compound which stimulates the formation of
`
`cAMP in a suitable medium containing the human GLP-1 receptor (one such medium
`
`disclosed below). The potency of an insulinotropic agent is determined by calculating the
`
`ECso value from the dose-response curve as described below.
`
`Baby hamster kidney (BHK) cells expressing the cloned human GLP-1 receptor
`
`10
`
`(BHK- 467-12A) were grown in DMEM media with the addition of 100 IU/ml penicillin, 100
`
`µg/ml streptomycin, 5% fetal calf serum and 0.5 mg/ml Geneticin G-418 (Life
`
`Technologies). The cells were washed twice in phosphate buffered saline and harvested with
`
`Versene. Plasma membranes were prepared from the cells by homogenisation with an
`
`Ultraturrax in buffer 1 (20 mM HEPES-Na, 10 mM EDTA, pH 7.4). The homogenate was
`
`15
`
`centrifuged at 48,000 x g for 15 min at 4 ° C. The pellet was suspended by homogenization in
`
`buffer 2 (20 mM HEPES-Na, 0.1 mM EDTA, pH 7.4), then centrifuged at 48,000 x g for 15
`
`min at 4°C. The washing procedure was repeated one more time. The final pellet was
`
`suspended in buffer 2 and used immediately for assays or stored at -80 ° C.
`
`The functional receptor assay was carried out by measuring cyclic AMP (cAMP) as
`
`20
`
`a response to stimulation by the insulinotropic agent. cAMP formed was quantified by the
`
`AlphaScreen™ cAMP Kit (Perkin Elmer Life Sciences). Incubations were carried out in half(cid:173)
`
`area 96-well microtiter plates in a total volume of 50 µL buffer 3 (50 mM Tris-HCI, 5 mM
`
`HEPES, 10 mM MgCl2, pH 7.4) and with the following addiditions: 1 mM ATP, 1 µM GTP, 0.5
`
`mM 3-isobutyl-1-methylxanthine (IBMX), 0.01 % Tween-20, 0.1 % BSA, 6 µg membrane
`
`25
`
`preparation, 15 µg/ml acceptor beads, 20µg/ml donor beads preincubated with 6 nM
`
`biotinyl-cAMP. Compounds to be tested for agonist activity were dissolved and diluted in
`
`buffer 3. GTP was freshly prepared for each experiment. The plate was incubated in the dark
`
`with slow agitation for three hours at room temperature followed by counting in the Fusion™
`
`instrument (Perkin Elmer Life Sciences). Concentration-response curves were plotted for the
`
`30
`
`individual compounds and ECso values estimated using a four-parameter logistic model with
`
`Prism v. 4.0 (GraphPad, Carlsbad, CA).
`The term "OPP-IV protected" as used herein referring to a polypeptide means a
`
`polypeptide which has been chemically modified in order to render said compound resistant to
`
`the plasma peptidase dipeptidyl aminopeptidase-4 (OPP-IV). The OPP-IV enzyme in plasma is
`
`35
`
`known to be involved in the degradation of several peptide hormones, e.g. GLP-1, GLP-2,
`
`Exendin-4 etc. Thus, a considerable effort is being made to develop analogues and derivatives
`
`MPI EXHIBIT 1015 PAGE 5
`
`MPI EXHIBIT 1015 PAGE 5
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`5
`
`of the polypeptides susceptible to OPP-IV mediated hydrolysis in order to reduce the rate of
`
`degradation by OPP-IV. In one embodiment a OPP-IV protected peptide is more resistant to
`
`OPP-IV than GLP-1 (7-37) or Exendin-4(1-39).
`
`Resistance of a peptide to degradation by dipeptidyl aminopeptidase IV is determined by the
`
`5
`
`following degradation assay :
`
`Aliquots of the peptide (5 nmol) are incubated at 37 QC with 1 µL of purified dipeptidyl
`
`aminopeptidase IV corresponding to an enzymatic activity of 5 mU for 10-180 minutes in 100
`
`µL of 0.1 M triethylamine-HCI buffer, pH 7.4. Enzymatic reactions are terminated by the
`
`addition of 5 µL of 10% trifluoroacetic acid, and the peptide degradation products are
`
`10
`
`separated and quantified using HPLC analysis. One method for performing this analysis is :
`
`The mixtures are applied onto a Vydac C18 widepore (30 nm pores, 5 µm particles) 250 x
`
`4.6 mm column and eluted at a flow rate of 1 ml/min with linear stepwise gradients of
`
`acetonitrile in 0.1 % trifluoroacetic acid (0% acetonitrile for 3 min, 0-24% acetonitrile for 17
`
`min, 24-48% acetonitrile for 1 min) according to Siegel et al., Regul. Pept. 1999;79:93-102
`
`15
`
`and Mentlein et al. Eur. J. Biochem. 1993;214:829-35. Peptides and their degradation
`
`products may be monitored by their absorbance at 220 nm (peptide bonds) or 280 nm
`
`(aromatic amino acids), and are quantified by integration of their peak areas related to those
`
`of standards. The rate of hydrolysis of a peptide by dipeptidyl aminopeptidase IV is estimated
`
`at incubation times which result in less than 10% of the peptide being hydrolysed.
`
`20
`
`The term "C1_5-alkyl" as used herein means a saturated, branched, straight or cyclic
`
`hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but
`
`are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n(cid:173)
`
`pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, cyclohexane and the like.
`
`The term "pharmaceutically acceptable" as used herein means suited for normal
`
`25
`
`pharmaceutical applications, i.e. giving rise to no adverse events in patients etc.
`
`The term "excipient" as used herein means the chemical compounds which are normally
`
`added to pharmaceutical compositions, e.g. buffers, tonicity agents, preservatives and the
`
`like.
`
`30
`
`The term "effective amount" as used herein means a dosage which is sufficient to be
`
`effective for the treatment of the patient compared with no treatment.
`
`The term "pharmaceutical composition" as used herein means a product comprising an
`
`active compound or a salt thereof together with pharmaceutical excipients such as buffer,
`
`preservative, and optionally a tonicity modifier and/or a stabilizer. Thus a pharmaceutical
`
`35
`
`composition is also known in the art as a pharmaceutical formulation.
`
`MPI EXHIBIT 1015 PAGE 6
`
`MPI EXHIBIT 1015 PAGE 6
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`6
`
`The term "treatment of a disease" as used herein means the management and care of a
`
`patient having developed the disease, condition or disorder. The purpose of treatment is to
`
`combat the disease, condition or disorder. Treatment includes the administration of the active
`
`compounds to eliminate or control the disease, condition or disorder as well as to alleviate
`
`5
`
`the symptoms or complications associated with the disease, condition or disorder.
`
`In another aspect the present invention relates to an acylated GLP-1 analogue that can bind
`
`to albumin and the GLP-1 receptor simultaneously.
`
`In another aspect the present invention relates to an acylated GLP-1 analogue that bind to
`
`the GLP-1 receptor with an affinity below 1 00nM, preferable below 30 nM in the presence of
`
`10
`
`2% albumin.
`
`In another aspect the present invention relates to an acylated GLP-1 analogue which affinity
`
`to the GLP-1 receptor is only partly decreased when comparing the affinity in the presence of
`
`very low concentration (e.g. 0.005% to 0.2%) of human albumin to the affinity in the presence
`
`of 2% human albumin. The shift in binding affinity under these conditions is less than 50 fold,
`
`15
`
`preferable below 30 fold and more preferable below 10 fold.
`
`The term "albumin binding moiety" as used herein means a residue which binds non-covalently
`
`to human serum albumin. The albumin binding residue attached to the therapeutic polypeptide
`
`typically has an affinity below 10 µM to human serum albumin and preferably below 1 µM. A
`
`20
`
`range of albumin binding residues are known among linear and branched lipohophillic moieties
`
`containing 4-40 carbon atoms having a distal acidic group.
`
`The term "hydrophilic linker" as used herein means a spacer that separates a peptide and an
`
`albumin binding residue with a chemical moiety which comprises at least 5 non-hydrogen
`atoms where 30-50% of these are either Nor 0.
`
`25
`
`The term "acidic groups" as used herein means organic chemical groups which are fully or
`
`partly negatively charged at physiological pH. The pKa value of such groups is below 7,
`
`preferable below 5. This includes but is not limited to carboxylic acids, sulphonic acids,
`
`phosphoric acids or heterocyclic ring systems which are fully or partly negatively charged at
`
`physiological pH.
`
`30
`
`In the below structural formula II the moiety U is a di-radical may be attached to the terminal
`
`groups B and the aminogroup of the lysine amino acid in the peptide in two different ways. In
`
`embodiments of the invention the U in formula II is attached with the group B attached at the
`
`end of the alkyl chain and the peptide at the other end.
`
`In the formulas below the terminal bonds from the attached groups are to be regarded as
`
`35
`
`attachment bonds and not ending in methylene groups unless stated.
`
`In the formulas below
`
`MPI EXHIBIT 1015 PAGE 7
`
`MPI EXHIBIT 1015 PAGE 7
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`7
`
`0
`H
`NH2-H-N>!-
`
`H3C
`
`CH3 means the H2N-His-Aib- N-terminal of the GLP-1 analogue.
`
`In an embodiment the invention provides a GLP-1 analog acylated with a lipophillic albumin
`
`binding moiety containing at least two free acidic chemical groups attached via a non natural
`
`5
`
`amino acid linker to the lysine residue in position 26.
`
`In an embodiment, the term free acidic chemical groups is to be understood as having the
`
`same meaning as "acidic groups" as used herein.
`
`In an embodiment the invention provides an acylated GLP-1 analog where said GLP-1
`
`analog is stabilised against OPP-IV by modification of at least one amino acid residue in
`
`10
`
`positions 7 and 8 relative to the sequence GLP-1 (7-37) (SEQ ID No 1 ), and where said
`
`acylation is a diacid attached to the lysine residue in position 26 optionally via a non natural
`
`amino acid hydrophilic linker.
`
`In an embodiment of the invention a GLP-1 analog having a modification of at least one non(cid:173)
`
`proteogenic amino acid residue in positions 7 and/or 8 relative to the sequence GLP-1 (7-37)
`
`15
`
`(SEQ ID No 1 ), which is acylated with a moiety to the lysine residue in position 26, and
`
`where said moiety comprises at least two acidic groups, wherein one acidic group is attached
`
`terminally.
`
`An embodiment provides a GLP-1 analog according to the above embodiment, wherein the
`
`moiety attached in position 26 comprises a hydrophilic linker.
`
`20
`
`An embodiment provides a GLP-1 analog according to the above embodiments, wherein the
`
`hydrophilic linker comprises at least 5 non-hydrogen atoms where 30-50% of these are either
`Nor 0.
`
`An embodiment provides a GLP-1 analog according to any of the above embodiments,
`
`wherein the moiety attached in position 26 comprises an albumin binding moiety seperated
`
`25
`
`from the peptide by the hydrophilic linker.
`
`An embodiment provides a GLP-1 analog according to the above embodiment, wherein the
`
`albumin binding moiety is a linear or branched lipophilic moiety containing 4-40 carbon atoms
`
`having a distal acidic group.
`
`An embodiment provides a GLP-1 analog according to any of the above embodiments,
`
`30
`
`wherein the acylated moiety is B-U', where U' is selected from
`
`'-- ~ ['Noya"
`
`,
`N H~p
`o
`H
`
`MPI EXHIBIT 1015 PAGE 8
`
`MPI EXHIBIT 1015 PAGE 8
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`8
`
`5
`
`10
`
`m is 0, 1, 2, 3, 4, 5, or 6,
`
`n is 1, 2 or 3
`
`s is 0, 1, 2, or 3,
`t is 0, 1, 2, 3, or 4
`pis 1,2,3,4,5,6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19,20,21,22,or23;
`
`and where B is an acidic group selected from
`
`HO~ / H~ /
`n i TI
`Ho_N n
`i TI
`o
`0
`O
`O
`
`,
`
`d
`an
`
`H
`HO~NY 'h r
`
`o Ao o
`
`HO
`
`O
`
`15 where I is 12, 13, 14, 15, 16, 17, 18, 19 or20;
`
`An embodiment provides a GLP-1 analog according to any of the above embodiments, which
`
`is a compound of formula I (SEQ ID No. 2) :
`
`MPI EXHIBIT 1015 PAGE 9
`
`MPI EXHIBIT 1015 PAGE 9
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`9
`
`Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa16-Ser-Xaa18-Xaa19-Xaa20-Glu-Xaa22-
`o
`Xaa23-Ala-Xaa25-~yL-xaa27-Phe-lle-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37
`
`\
`
`B--U '------NH
`
`Formula I
`
`wherein
`Xaa1 is L-histidine, imidazopropionyl, a-hydroxy-histidine, D-histidine, desamino-histidine, 2-
`
`5
`
`amino-histidine, f3-hydroxy-histidine, homohistidine, Na-acetyl-histidine, Na-formyl-histidine, a(cid:173)
`
`fluoromethyl-histidine, a-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-
`
`pyridylalanine
`
`Xaaa is Ala, Gly, Val, Leu, lie, Thr, Ser, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-
`
`aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)
`
`10
`
`carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;
`
`Xaa15 is Val or Leu;
`
`Xaa18 is Ser, Lys or Arg;
`
`Xaa19 is Tyr or Gin;
`
`Xaa20 is Leu or Met;
`
`15
`
`Xaa22 is Gly, Glu or Aib;
`
`Xaa23 is Gin, Glu, Lys or Arg;
`
`Xaa25 is Ala or Val;
`
`Xaa21 is Glu or Leu;
`
`Xaa30 is Ala, Glu or Arg;
`
`20
`
`Xaa33 is Val or Lys;
`
`Xaa34 is Lys, Glu, Asn or Arg;
`
`Xaa35 is Gly or Aib;
`
`Xaa36 is Arg, Gly or Lys, or is absent;
`
`25
`
`Xaa31 is Gly, Ala, Glu, Pro, Lys, or is absent;
`and B and U' together is the acylated moiety, where U' is selected from
`O½H
`~
`HO-s:::o
`H
`0
`'N~N-../'o,,...___,,0Ji-N,,...____,o..../'o¾
`H
`O
`H
`0
`
`p
`
`0
`
`~
`L "N
`H
`
`MPI EXHIBIT 1015 PAGE 10
`
`MPI EXHIBIT 1015 PAGE 10
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`10
`
`5
`
`10
`
`m is 0, 1, 2, 3, 4, 5, or 6,
`
`n is 1, 2 or 3
`
`s is 0, 1, 2, or 3,
`t is 0, 1, 2, 3, or 4
`
`pis 1,2,3,4,5,6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19,20,21,22,or23;
`
`and where B is an acidic group selected from
`
`HO~ / H~ /
`n i TI
`Ho_N n
`i TI
`o
`0
`O
`O
`
`,
`
`d
`an
`
`H
`HO~NY 'h r
`
`o Ao o
`
`HO
`
`O
`
`15 where I is 12, 13, 14, 15, 16, 17, 18, 19 or20;
`
`In an embodiment the invention provides a compound which is a compound of formula II
`
`(SEQ ID No. 3) :
`
`MPI EXHIBIT 1015 PAGE 11
`
`MPI EXHIBIT 1015 PAGE 11
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`11
`
`Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa16-Ser-Xaa18-Xaa19-Xaa20-Glu-Xaa22-
`o
`Xaa23-Ala-Xaa25-~r-Xaa27-Phe-lle-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38
`
`\
`
`B--U--U--NH
`
`0
`
`Formula II
`
`The formula II is identical to formula I as stated in an embodiment above, where the moiety
`
`5
`
`B-U is replaced by B-U'. The difference being only the incorporation of the carboxy group in
`
`the U' relative to U, which is without the attaching carboxy group.
`
`In formula II each of the Xaa's has the following meaning:
`Xaa1 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ~-hydroxy-histidine,
`
`homohistidine, Na-acetyl-histidine, a-fluoromethyl-histidine, a-methyl-histidine, 3-
`
`10
`
`pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
`
`Xaas is Ala, Gly, Val, Leu, lie, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-
`
`aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)
`
`carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;
`
`Xaa15 is Val or Leu;
`
`15
`
`Xaa18 is Ser, Lys or Arg;
`Xaa19 is Tyr or Gin;
`
`Xaa20 is Leu or Met;
`
`Xaa22 is Gly, Glu or Aib;
`
`Xaa23 is Gin, Glu, Lys or Arg;
`
`20
`
`Xaa25 is Ala or Val;
`
`Xaa21 is Glu or Leu;
`
`Xaa30 is Ala, Glu or Arg;
`
`Xaa33 is Val or Lys;
`
`Xaa34 is Lys, Glu, Asn or Arg;
`
`25
`
`Xaa35 is Gly or Aib;
`
`Xaa36 is Arg, Gly or Lys, or is absent;
`
`Xaa31 is Gly, Ala, Glu, Pro, Lys, or is absent;
`
`Xaa3s is Lys, Ser, amide or is absent;
`
`and where U is a spacer selected from
`
`MPI EXHIBIT 1015 PAGE 12
`
`MPI EXHIBIT 1015 PAGE 12
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`12
`
`where n is 12, 13, 14, 15, 16, 17 or 18
`
`5
`
`I is 12, 13, 14, 15, 16, 17 or 18,
`
`m is 0, 1, 2, 3, 4, 5, or 6,
`
`s is 0, 1, 2, or 3,
`
`p is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23;
`
`and where B is an acidic group selected from
`
`HOY
`0
`
`and
`
`10
`
`H
`,N.......,.......
`HO
`11
`0
`
`In the embodiments below when referring to U' in formula I it is to be understood as also
`
`referring to formula II and U, with the only difference being the carboxy group.
`
`An embodiment provides a GLP-1 analog according to the embodiments above, wherein U'
`
`15
`
`is selected from
`
`MPI EXHIBIT 1015 PAGE 13
`
`MPI EXHIBIT 1015 PAGE 13
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`13
`
`5
`
`10
`
`m is 2, 3, 4 or 5,
`
`n is 1 or 2
`
`s is 0, 1, or 2,
`t is 0, 1, 2, or 3
`p is 1, 2, 3, 4, 7, 11 or 23
`
`An embodiment provides a GLP-1 analog according to the embodiments above, wherein B(cid:173)
`
`U'- is
`
`0
`
`0 H0_/1 ¼S:::::o
`
`N
`H
`
`O
`
`0
`~'-./'- ~O Jlj__ ~0--./'- ~
`II
`O
`...._,,, T;"N
`O
`H
`0
`
`HOOC
`
`I
`
`15
`
`MPI EXHIBIT 1015 PAGE 14
`
`MPI EXHIBIT 1015 PAGE 14
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`14
`
`0 ~\l'/o
`H
`0
`Hooc.J ULN.s: l-l
`/_N....,,,....O----------o JlL N _________ o....,,,....O...-......,,...
`' 90
`__, 1-;- H
`~
`\'"/1 H
`
`H
`0 ~\l'/o
`HOOC-+--t-1'-~-S~N..._,,,....O- r - , -o~
`o
`0
`
`where I is 14, 15, 16, 17, 18, 19 or 20;
`
`pis 1, 2, 3, 4, 7, 8, 9, 10, 11 or 12.
`
`sis 0, 1 or 2
`
`tis 0 or 1;
`
`An embodiment according to the above wherein
`
`where I is 14, 15, 16, 17 or 18
`
`p is 1, 2, 3, 4 or 11;
`
`sis 0, 1 or 2;
`
`tis 0 or 1;
`
`An embodiment provides a GLP-1 analog according to the embodiment above, wherein B-U'
`
`is
`
`5
`
`10
`
`15
`
`20
`
`MPI EXHIBIT 1015 PAGE 15
`
`MPI EXHIBIT 1015 PAGE 15
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`15
`
`0
`H
`H
`0
`.,,...,....,,,0-../' k-l ,,,
`Hooe--'
`'t.JL_~N-../' ~o )l.\.__
`\"/1
`-
`.._,,.
`0
`/;-N
`O' 'nl(
`HO
`O
`H
`O
`0
`
`HOOC~NOrOH
`
`,
`
`I H~p
`0
`
`5
`
`10
`
`where I is 14, 15, 16, 17, 18, 19 or 20;
`
`p is 1, 2, 3, or 4.
`
`sis 0, 1 or 2
`
`n is 0, 1 or 2
`
`An embodiment according to any of the above embodiments is wherein B is
`
`HO~ / H~ /
`n i TI
`Ho_N n
`i TI
`o
`0
`O
`O
`
`,
`
`d
`an
`
`HO-/'YNY'hr
`o Ao o
`
`H
`
`HO
`
`O
`
`15
`
`and I is 14,16, 18 or 20;
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein Bis
`
`HO~ / H~ /
`n
`Ho_N n
`i n
`i TI
`o
`0
`O
`O
`,
`
`d
`an
`
`20
`
`HO-/'YNY'hr
`o Ao o
`
`H
`
`HO
`
`O
`
`MPI EXHIBIT 1015 PAGE 16
`
`MPI EXHIBIT 1015 PAGE 16
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`16
`
`where I is 14, 15, or 16.
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein s is 1 .
`
`5
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein n is 1.
`
`An embodiment provides a GLP-1 analog according any of the embodiments above, wherein
`
`I is 14, 15 or 16; In embodiments I is 17, 18, 19 or 20. In embodiments I is 15, 16 or 17. In
`
`embodiments I is 18, 19 or 20. In embodiments I is 14. In embodiments I is 16. In
`
`10
`
`embodiments I is 18. In embodiments I is 20.
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein p is 1.
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein p is 2.
`
`15
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein p is 3.
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein p is 4.
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`20
`
`wherein B-U' is
`
`o O~OH
`N~~~O~o~O~o~O~o~O~o~
`
`H
`
`O
`
`0
`
`o OIOH
`N~~~O~o~O~o~O~o~O~o~
`
`H
`
`O
`
`0
`
`OyOH
`
`0
`
`~~~~o~or(
`0
`0
`
`25
`
`HO
`
`HO
`
`0
`
`0
`
`0
`
`0
`
`OH
`
`HO
`
`0
`
`MPI EXHIBIT 1015 PAGE 17
`
`MPI EXHIBIT 1015 PAGE 17
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`17
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein B-U' is
`
`0
`H
`OO"f'OH
`HOOC~N~N-../'o""'-'o._)l.__N....._____o-../'o"'y'
`o
`16 H
`H
`O
`
`5
`
`10
`
`An embodiment provides a GLP-1 analog according to any of the embodiments above,
`
`wherein B-U' is
`
`o
`O o~OH
`HOOC~N~~-../'o""'-'o._)l.__N....._____o-../'o"'y'
`o
`o
`1s H
`H
`
`An embodiment provides a GLP-1 analog according to formula I above, wherein
`
`Xaa1 is His or desamino-histidine;
`
`Xaaa is Ala, Gly, Val, Leu, lie, Lys or Aib;
`
`Xaa16 is Val;
`
`15
`
`Xaa1a is Ser;
`
`Xaa19 is Tyr;
`
`Xaa20 is Leu;
`
`Xaa22 is Gly, Glu or Aib;
`
`Xaa23 is Gin or Glu;
`
`20
`
`Xaa2s is Ala;
`
`Xaa21 is Glu;
`
`Xaa30 is Ala or Glu;
`
`Xaa33 is Val;
`
`Xaa34 is Lys or Arg;
`
`25
`
`Xaa3s is Gly or Aib;
`
`Xaa36 is Arg or Lys
`
`Xaa31 is Gly, amide or is absent;
`
`An embodiment provides a GLP-1 analog according to formula I above, wherein
`
`30
`
`Xaa1 is His
`
`Xaas is Gly, or Aib;
`
`Xaa16 is Val;
`
`Xaa1a is Ser;
`
`MPI EXHIBIT 1015 PAGE 18
`
`MPI EXHIBIT 1015 PAGE 18
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`18
`
`Xaa19 is Tyr;
`
`Xaa20 is Leu;
`
`Xaa22 is Glu or Aib;
`
`Xaa23 is Gin;
`
`5
`
`Xaa2s is Ala;
`
`Xaa21 is Glu;
`
`Xaa30 is Ala;
`
`Xaa33 is Val;
`
`Xaa34 is Lys or Arg;
`
`10
`
`Xaa3s is Gly or Aib;
`
`Xaa36 is Arg
`
`Xaa31 is Gly
`
`15
`
`An embodiment provides a GLP-1 analog according to any one of the above embodiments,
`
`wherein said GLP-1 analog comprises a modification of the N-terminal L-histidine in position
`
`7 of the GLP-1 (7-37) sequence.
`
`An embodiment provides a GLP-1 analog according to the embodiment above, wherein said
`20 GLP-1 analog comprises imidazopropionyl7, a-hydroxy-histidine7 or N-methyl-histidine7, D(cid:173)
`histidine7, desamino-histidine7, 2-amino-histidine7, f3-hydroxy-histidine7, homohistidine7, Na(cid:173)
`acetyl-histidine7, a-fluoromethyl-histidine7, a-methyl-histidine7, 3-pyridylalanine7, 2-
`pyridylalanine7 or 4-pyridylalanine7.
`
`25
`
`An embodiment provides a GLP-1 analog according to any one of the embodiments above,
`
`wherein said GLP-1 analog comprises a substitution of the L-alanine in position 8 of the
`
`GLP-1 (7-37) sequence for another amino acid residue.
`
`An embodiment provides a GLP-1 analog according to the embodiment above, wherein said
`
`30 GLP-1 analog comprises Aibs, Gly8, Vais, lies, Leus, Sers, Thrs, (1-aminocyclopropyl)
`
`carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-
`
`aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-
`
`aminocyclooctyl) carboxylic acid.
`
`35
`
`An embodiment provides a GLP-1 analog according to any of the the embodiment above,
`
`wherein said GLP-1 analog comprises Aibs;
`
`MPI EXHIBIT 1015 PAGE 19
`
`MPI EXHIBIT 1015 PAGE 19
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`19
`
`In one embodiment of the invention said GLP-1 analog is Aib8,Arg34-GLP-1 (7-37) or
`22,Arg34-GLP-1 (7-37).
`Aib8
`
`•
`
`An embodiment provides a GLP-1 analog according to any of the above embodiments, wherein
`
`5
`
`said GLP-1 analog comprises no more than fifteen amino acid residues which have been
`
`exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ),
`
`An embodiment provides a GLP-1 analog according to the embodiment above, wherein no
`
`more than ten amino acid residues which have been exchanged, added or deleted as compared
`
`10
`
`to GLP-1 (7-37) (SEQ ID No. 1 ).
`
`An embodiment provides a GLP-1 analog according to the embodiment above, wherein said
`
`GLP-1 analog comprises no more than six amino acid residues which have been exchanged,
`
`added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ).
`
`15
`
`An embodiment provides a GLP-1 analog according to any of the above embodiments, wherein
`
`said GLP-1 analog comprises no more than 3 amino acid residues which are not encoded by
`
`the genetic code.
`
`20
`
`An embodiment provides a GLP-1 analog according to any of the above embodiments,
`
`wherein said GLP-1 analog comprises only one lysine residue.
`
`An embodiment provides a GLP-1 analog according to any of the above embodiments, which
`
`25
`
`30
`
`•
`
`is
`Aib8,Arg34-GLP-1 (7-37)
`22,Arg34-GLP-1 (7-37).
`Aib8
`Arg34-GLP-1 (7-37).
`[3-(4-lmidazolyl)Propionyl7,Arg34]GLP-1-(7-37)peptide
`Gly8,Arg34-GLP-1 (7-37)
`Aib8,Arg34, Pro37-GLP-1 (7-37)
`Aibs,22,21,3o,3s,Arg34,Pro31_ GLP-1 (7-37)amide,
`
`all of which are substituted by B-U' in position 26.
`
`An embodiment provides a GLP-1 analog according to any one of the preceding
`
`35
`
`embodiments, which is selected from
`
`MPI EXHIBIT 1015 PAGE 20
`
`MPI EXHIBIT 1015 PAGE 20
`
`

`

`WO 2006/097537
`
`PCT /EP2006/060855
`
`20
`
`H 0
`H O
`NH,-H-N£E G T F T s D V s s y L E G Q A A-NrE F I Aw L V R G R G-cOOH
`H3C CH 3
`
`\
`
`HO~NH
`0
`
`N-£26-(17-carboxyheptadecanoyl)-[Aib8,Arg34 ]GLP-1-(7-37)-peptide,
`
`NH,-H-~J-E G T F T S D V S S Y L E G QA A-~_)L-E F I AW L V R G R G-ooOH
`
`H,C CH
`
`3
`
`O
`
`\
`
`HO~NH
`0
`
`N-£26-(19-carboxynonadecanoyl)-[A