USOO8129343B2
`
`(12) United States Patent
`Lau et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,129,343 B2
`Mar. 6, 2012
`
`(54) ACYLATED GLP-1 COMPOUNDS
`(75) Inventors: Jesper Lau, Farum (DK); Paw Bloch,
`Taastrup (DK); Thomas Kruse Hansen,
`Herlev (DK)
`(73) Assignee: Novo Nordisk A/S, Bagsvaerd (DK)
`(*) Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 682 days.
`11/908,834
`
`(21) Appl. No.:
`
`Mar. 20, 2006
`PCT/EP2006/060855
`
`(22) PCT Filed:
`(86). PCT No.:
`S371 (c)(1),
`Sep. 17, 2008
`(2), (4) Date:
`(87) PCT Pub. No.: WO2006/097537
`PCT Pub. Date: Sep. 21, 2006
`
`(65)
`
`Prior Publication Data
`US 2009/O156478 A1
`Jun. 18, 2009
`
`Related U.S. Application Data
`(60) Provisional application No. 60/664,497, filed on Mar.
`23, 2005.
`Foreign Application Priority Data
`
`(30)
`
`Mar. 18, 2005 (EP) ..................................... 05102171
`(51) Int. Cl.
`(2006.01)
`A6 IK38/26
`(2006.01)
`A6IP3/10
`(2006.01)
`A6IP 7/2
`(2006.01)
`C07K I4/605
`(52) U.S. Cl. ...................................................... S14/117
`(58) Field of Classification Search ........................ None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,545,618 A
`8/1996 Buckley et al.
`6,268,343 B1* 7/2001 Knudsen et al. ............... 514/4.8
`6,528,486 B1* 3/2003 Larsen et al. .................. 514f6.8
`2007/02O3058 A1* 8, 2007 Lau et al.
`
`EP
`EP
`RU
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`05102171
`3, 2005
`1704165
`9, 2006
`2006 107600
`10/2007
`WO91f11457
`8, 1991
`WO96,29342
`9, 1996
`WO9629342
`9, 1996
`WO 98.08871
`3, 1998
`WO98O8871
`3, 1998
`WO99.43708
`9, 1999
`WO994.3708
`9, 1999
`WOOO34331
`6, 2000
`WOOOf 69911
`11, 2000
`WOOO699.11
`11, 2000
`
`WOOO,34331
`WO
`WOO2,46227
`WO
`WOO246227
`WO
`WO O2/O98446
`WO
`WO 03/040309
`WO
`WO WO 2004/065621
`WO WO 2004/099246
`WO WO 2005/O14049
`WO
`WO2005O14049
`WO WO 2005/027978
`WO
`WO2O05O27978
`WO WO 2006/097537
`
`6, 2002
`6, 2002
`6, 2002
`12/2002
`5, 2003
`8, 2004
`11, 2004
`2, 2005
`2, 2005
`3, 2005
`3, 2005
`9, 2006
`
`OTHER PUBLICATIONS
`Simonovsky et al. Poly(ether urethane)s incorporating long alkyl
`side-chains with terminal carboxy groups as fatty acid mimics: Syn
`thesis, structural characterization and protein adsorption. Journal of
`Biomaterials Science, Polymer Edition, 2005, vol. 16, No. 12, pp.
`1463-1483.
`Green, Brian D., Biological Chemistry (2004), vol. 385, No. 2, pp.
`169-177.
`Knudsen, L.B. et al., Journal of Medicinal Chemistry, vol. 43, pp.
`1664-1669 (2000).
`Knudsen, L.B. et al., Journal of Medicinal Chemistry, vol. 47, pp.
`4128-4134 (2004).
`Deacon, C.F. et al., Diabetologia, vol. 41, pp. 271-278 (1998).
`Greenwald RB, “Peg Drugs: An Overview.” Journal of Controlled
`Release, 2001, vol. 74, p. 159-171.
`Ji et al., “Stearyl Poly(Ethylene Oxide) Grafted Surfaces for Prefer
`ential Adsorption of Albmnin.” Biomaterials, 2001, vol. 22, p. 3015
`3O23.
`Kurtzhals, P. et al., “Albumin Binding of Insulins Acylated With Fatty
`Acids: Characterization of the Ligand-Protein Interaction and Cor
`relation Between Binding Affinity and Timing of the Insulin Effect in
`Vivo.” Biochem J, 1995, vol. 312, p. 725-731.
`Simonovsky et al., “Poly(Etherurethane)s Incorporating Long Alkyl
`Side-Chains With Terminal Carboxyl Groups as Fatty Acid Mimics:
`Synthesis, Structural Characterization and Protein Adsorption.” J
`Biomat Sei Polymer EDN, 2005, vol. 16, p. 1463-1483.
`Soltero et al., “The Oral Delivery of Protein and Peptide Drugs.”
`Innovations in Pharmaceutical Technology, 2001, vol. 1, No. 9, p.
`106-110.
`Still JG, “Development of Oral Insulin: Progress and Curent Status.”
`Diabetes/Metab Res Rev, 2002, vol. 18, Suppl 1, p. S29-S37.
`Veronese FM, "Peptide and Protein Pegylation: A Review of Prob
`lems and Solutions.” Biomaterials, 2001, vol. 22, p. 405-417.
`Watanabe et al., “Structure-Activity Relationships of Glucagon-Like
`Peptide-1 (7-36) Amide: Insulinotropic Activities in Perfused Rat
`Pancreases, and Receptor Binding and Cyclic Amp Production in
`RINm.5F Cells,” Journal of Endocrinology, 1994, vol. 140, p. 45-52.
`* cited by examiner
`
`Primary Examiner — Marcela M Cordero Garcia
`(74) Attorney, Agent, or Firm — Richard W. Bork
`
`(57)
`ABSTRACT
`Protracted GLP-1 compounds and therapeutic uses thereof.
`
`6 Claims, No Drawings
`
`MPI EXHIBIT 1002 PAGE 1
`
`MPI EXHIBIT 1002 PAGE 1
`
`

`

`1.
`ACYLATED GLP-1 COMPOUNDS
`
`US 8,129,343 B2
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a 35 U.S.C. S371 national stage appli
`cation of International Patent Application PCT/EP2006/
`060855 (published as WO 2006/097537), filed Mar. 20, 2006,
`which claimed priority of European Patent Application
`05102171.5, filed Mar. 18, 2005; this application further
`claims priority under 35 U.S.C. S 119 of U.S. Provisional
`Application 60/664,497, filed Mar. 23, 2005.
`
`FIELD OF THE INVENTION
`
`This invention relates to the field of therapeutic peptides,
`i.e. to new protracted GLP-1 compounds.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`15
`
`25
`
`30
`
`35
`
`A range of different approaches have been used for modi
`fying the structure of glucagon-like peptide 1 (GLP-1) com
`pounds 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.
`WO98/08871 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.
`WO 00/699.11 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.
`Many diabetes patients particularly in the type 2 diabetes
`segment are subject to so-called “needle-phobia’, i.e. a Sub
`stantial 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 administered, the
`fear of injections may become a serious obstacle for the
`widespread use of the clinically very promising GLP-1 com
`pounds. Thus, there is a need to develop new GLP-1 com
`50
`pounds 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.
`
`40
`
`45
`
`SUMMARY OF THE INVENTION
`
`55
`
`The invention provides a GLP-1 analog having a modifi
`cation of at least one non-proteogenic amino acid residue in
`positions 7 and/or 8 relative to the sequence GLP-1 (7-37)
`(SEQID 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.
`The present invention also provides pharmaceutical com
`positions comprising a compound according to the present
`65
`invention and the use of compounds according to the present
`invention for preparing medicaments for treating disease.
`
`60
`
`2
`The invention provides a method for increasing the time of
`action in a patient of a GLP-1 analog, characterised in acy
`lating 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.
`
`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 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-carboxyglutamate,
`ornithine, phosphoserine, D-alanine and D-glutamine. Syn
`thetic amino acids comprise amino acids manufactured by
`chemical synthesis, i.e. D-isomers of the amino acids
`encoded by the genetic code such as D-alanine and D-leucine,
`Aib (CL-aminoisobutyric acid), Abu (CL-aminobutyric acid),
`Tle (tert-butylglycine), B-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, Isoleucine, Leucine, Lysine, Methionine,
`Phenylalanine, Proline, 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-ala
`nine and D-glutamine, Synthetic non-proteogenic amino
`acids comprise amino acids manufactured by chemical Syn
`thesis, i.e. D-isomers of the amino acids encoded by the
`genetic code such as D-alanine and D-leucine, Aib (CL-ami
`noisobutyric acid), Abu (CL-aminobutyric acid), Tle (tert-bu
`tylglycine), 3-aminomethyl benzoic acid, anthranilic acid,
`des-amino-Histidine, the beta analogs of amino acids such as
`B-alanine etc. D-histidine, desamino-histidine, 2-amino-his
`tidine, B-hydroxy-histidine, homohistidine, N-acetyl-histi
`dine, C-fluoromethyl-histidine, C.-methyl-histidine, 3-py
`ridylalanine,
`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 polypep
`tide means a modified peptide wherein one or more amino
`acid residues of the peptide have been substituted by 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'IGLP
`1 (7-37)Lys designates a GLP-1 (7-37) analogue wherein 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 Gly. Allamino acids
`for which the optical isomer is not stated is to be understood
`to mean the L-isomer. In embodiments of the invention a
`
`MPI EXHIBIT 1002 PAGE 2
`
`MPI EXHIBIT 1002 PAGE 2
`
`

`

`3
`maximum of 17 amino acids have been modified. In embodi
`ments of the invention a maximum of 15 amino acids have
`been modified. In embodiments of the invention a maximum
`of 10amino acids have been modified. In embodiments of the
`invention a maximum of 8amino acids have been modified. In
`embodiments of the invention a maximum of 7 amino acids
`have been modified. In embodiments of the invention a maxi
`mum of 6 amino acids have been modified. In 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.
`The term "derivative' as used herein in relation to a peptide
`means a chemically modified peptide oran analogue thereof,
`wherein at least one Substituent is not present in the unmodi
`fied 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 N'-((4S)-4-
`(hexadecanoylamino)-carboxy-butanoyl)|Arg,
`Lys?
`GLP-1-(7-37).
`The term “GLP-1 peptide' as used herein means GLP-1 (7-
`37) (SEQID 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 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 insulino
`tropic 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 (BHK-467-12A) were grown in
`DMEM media with the addition of 100 IU/mL penicillin, 100
`ug/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
`centrifuged at 48,000xg for 15 min at 4°C. The pellet was
`45
`suspended by homogenization in buffer 2 (20 mM HEPES
`Na, 0.1 mM EDTA, pH 7.4), then centrifuged at 48,000xg 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 measur
`ing cyclic AMP (cAMP) as a response to stimulation by the
`insulinotropic agent. cAMP formed was quantified by the
`AlphaScreenTM cAMP Kit (Perkin Elmer Life Sciences).
`Incubations were carried out in half-area 96-well microtiter
`plates in a total volume of 50 uL buffer 3 (50 mM Tris-HCl, 5
`mM HEPES, 10 mM MgCl, pH 7.4) and with the following
`addiditions: 1 mM ATP, 1 uM GTP, 0.5 mM 3-isobutyl-1-
`methylxanthine (IBMX), 0.01% Tween-20, 0.1% BSA, 6 ug
`membrane preparation, 15 ug/mL acceptor beads, 20 g/mL
`60
`donor beads preincubated with 6 nM biotinyl-cAMP. Com
`pounds to be tested for agonist activity were dissolved and
`diluted in buffer 3. GTP was freshly prepared for each experi
`ment. The plate was incubated in the dark with slow agitation
`for three hours at room temperature followed by counting in
`65
`the FusionTM instrument (PerkinElmer Life Sciences). Con
`centration-response curves were plotted for the individual
`
`55
`
`35
`
`40
`
`50
`
`US 8,129,343 B2
`
`10
`
`15
`
`25
`
`30
`
`4
`compounds and ECso values estimated using a four-param
`eter logistic model with Prism v. 4.0 (GraphPad, Carlsbad,
`Calif.).
`The term “DPP-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 (DPP-IV).
`The DPP-IV enzyme in plasma is 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 of the polypeptides Sus
`ceptible to DPP-IV mediated hydrolysis in order to reduce the
`rate of degradation by DPP-IV. In one embodiment a DPP-IV
`protected peptide is more resistant to DPP-IV than GLP-1 (7-
`37) or Exendin-4(1-39).
`Resistance of a peptide to degradation by dipeptidyl ami
`nopeptidase IV is determined by the following degradation
`assay:
`Aliquots of the peptide (5 nmol) are incubated at 37° C.
`with 1 uL of purified dipeptidyl aminopeptidase IV corre
`sponding to an enzymatic activity of 5 mU for 10-180 minutes
`in 100 uL of 0.1 M triethylamine-HCl buffer, pH 7.4. Enzy
`matic reactions are terminated by the addition of 5 uL of 10%
`trifluoroacetic acid, and the peptide degradation products are
`separated and quantified using HPLC analysis. One method
`for performing this analysis is: The mixtures are applied onto
`aVydac C18 widepore (30 nm pores, 5um particles) 250x4.6
`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 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.
`The term "Ce-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-pentyl, isopentyl, neopentyl,
`tert-pentyl, n-hexyl, isohexyl, cyclohexane and the like.
`The term “pharmaceutically acceptable' as used herein
`means Suited for normal 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.
`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 composition is also known
`in the art as a pharmaceutical formulation.
`The term “treatment of a disease' as used herein means the
`management and care of a patient having developed the dis
`ease, 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
`
`MPI EXHIBIT 1002 PAGE 3
`
`MPI EXHIBIT 1002 PAGE 3
`
`

`

`US 8,129,343 B2
`
`6
`
`5
`control the disease, condition or disorder as well as to allevi-
`ate the symptoms or complications associated with the dis
`ease, condition or disorder.
`In another aspect the present invention relates to an acy- 5
`lated GLP-1 analogue that can bind to albuminand the GLP-1
`receptor simultaneously.
`In another aspect the present invention relates to an acy-
`HC CH
`lated GLP-1 analogue that bind to the GLP-1 receptor with an
`affinity below 100 nM, preferable below 30 nM in the pres- 10 means the HN-His-Aib-N-terminal of the GLP-1 analogue.
`ence of 2% albumin.
`In an embodiment the invention provides a GLP-1 analog
`In another aspect the present invention relates to an acy-
`acylated with a lipophilic albumin binding moiety containing
`lated GLP-1 analogue which affinity to the GLP-1 receptor is
`at least two free acidic chemical groups attached via a non
`only partly decreased when comparing the affinity in the
`natural amino acid linker to the lysine residue in position 26.
`presence of very low concentration (e.g. 0.005% to 0.2%) of
`In an embodiment, the term free acidic chemical groups is
`human albumin to the affinity in the presence of 2% human
`to be understood as having the same meaning as "acidic
`albumin. The shift in binding affinity under these conditions
`groups' as used herein.
`is less than 50 fold, preferable below 30 fold and more pref-
`In an embodiment the invention provides an acylated
`erable below 10 fold.
`GLP-1 analog where said GLP-1 analog is stabilised against
`DPP-IV by modification of at least one amino acid residue in
`The term “albumin binding moiety” as used herein means
`positions 7 and 8 relative to the sequence GLP-1 (7-37) (SEQ
`a residue which binds non-covalently to human serum albu
`ID No 1), and where said acylation is a diacid attached to the
`min. The albumin binding residue attached to the therapeutic
`lysine residue in position 26 optionally via a non natural
`polypeptide typically has an affinity below 10 uM to human
`amino acid hydrophilic linker.
`serum albumin and preferably below 1 uM. A range of albu- ?
`In an embodiment of the invention a GLP-1 analog having
`min binding residues are known among linear and branched
`a modification of at least one non-proteogenic amino acid
`lipohophillic moieties containing 4-40 carbonatoms having a
`residue in positions 7 and/or 8 relative to the sequence GLP
`distal acidic group.
`1(7-37) (SEQID No 1), which is acylated with a moiety to the
`The term “hydrophilic linker as used herein means a 30
`lysine residue in position 26, and where said moiety com
`spacer that separates a peptide and an albumin binding resi
`prises at least two acidic groups, wherein one acidic group is
`due with a chemical moiety which comprises at least 5 non
`attached terminally.
`hydrogen atoms where 30-50% of these are either N or O.
`An embodiment provides a GLP-1 analog according to the
`above embodiment, wherein the moiety attached in position
`The term "acidic groups' as used herein means organic
`chemical groups which are fully or partly negatively charged 35 26 comprises a hydrophilic linker.
`at physiological pH. The pKa value of such groups is below 7.
`An embodiment provides a GLP-1 analog according to the
`preferable below 5. This includes but is not limited to car-
`above embodiments, wherein the hydrophilic linker com
`boxylic acids, Sulphonic acids, phosphoric acids or heterocy-
`prises at least 5 non-hydrogen atoms where 30-50% of these
`clic ring systems which are fully or partly negatively charged
`are either N or O.
`at physiological pH.
`An embodiment provides a GLP-1 analog according to any
`of the above embodiments, wherein the moiety attached in
`In the below structural formula II the moiety U is a di
`position 26 comprises an albumin binding moiety separated
`radical may be attached to the terminal groups B and the
`from the peptide by the hydrophilic linker.
`aminogroup of the lysine amino acid in the peptide in two
`An embodiment provides a GLP-1 analog according to the
`different ways. In embodiments of the invention the U in 45
`above embodiment, wherein the albumin binding moiety is a
`formula II is attached with the group Battached at the end of
`linear or branched lipophilic moiety containing 4-40 carbon
`the alkyl chain and the peptide at the other end.
`atoms having a distal acidic group.
`An embodiment provides a GLP-1 analog according to any
`In the formulas below the terminal bonds from the attached
`groups are to be regarded as attachment bonds and not ending
`of the above embodiments, wherein the acylated moiety is
`in methylene groups unless stated.
`B U", where U is selected from
`
`In the formulas below
`
`O
`
`H
`NH-H-N
`
`O
`
`OH
`
`O
`
`N- in
`
`H
`
`H
`
`MPI EXHIBIT 1002 PAGE 4
`
`MPI EXHIBIT 1002 PAGE 4
`
`

`

`US 8,129,343 B2
`
`-continued
`
`OH
`
`OH
`
`O
`
`O
`
`N
`
`N
`
`N-1-1-9
`
`N
`H
`
`H
`
`N-1N-N- p
`
`O
`
`H
`
`1Ns
`MV
`O O
`
`MPI EXHIBIT 1002 PAGE 5
`
`MPI EXHIBIT 1002 PAGE 5
`
`

`

`US 8,129,343 B2
`
`9
`
`HO
`
`H
`HO-N
`
`10
`
`d
`
`8.
`
`O
`
`O
`
`O
`
`O
`
`H
`N
`
`o
`
`O
`
`HO
`
`O
`
`HO
`
`10
`N-formyl-histidine, C-fluoromethyl-histidine, C.-methyl
`m is 0, 1, 2, 3, 4, 5, or 6,
`histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylala
`n is 1, 2 or 3
`nine
`s is 0, 1, 2, or 3,
`Xaas is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocy
`t is 0, 1, 2, 3, or 4
`p is 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 5 clopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic
`19, 20, 21, 22, or 23;
`acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclo
`and where B is an acidic group selected from
`hexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic
`acid, or (1-aminocyclooctyl) carboxylic acid;
`Xaa is Val or Leu:
`Xaas is Ser, Lys or Arg;
`Xaao is Tyr or Gln;
`Xaao is Leu or Met;
`15 Xaa- is Gly, Glu or Aib;
`Xaa- is Gln, Glu, Lys or Arg;
`Xaas is Ala or Val;
`Xaa, is Glu or Leu:
`Xaao is Ala, Glu or Arg;
`2O Xaa- is Val or Lys;
`Xaa34 is Lys. Glu, ASnor Arg:
`Xaas is Gly or Aib;
`
`where 1 is 12, 13, 14, 15, 16, 17, 18, 19 or 20;
`An embodiment provides a GLP-1 analog according to any
`of the above embodiments, which is a compound of formula
`I (SEQID No. 2):
`
`Xaa-7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa16-Ser-Xaa18-Xaa19-Xaa20-Glu-Xaa22
`
`Formula I
`
`Xs usu-sur-issurplexexuxa 5-Xaa36-Xaa37-Xaa38
`
`H
`
`B-U-NH
`
`Xaa- is Arg, Gly or Lys, or is absent;
`wherein
`Xaa, is L-histidine, imidazopropionyl, c-hydroxy-histidine, 6s Xaa37 is Gly, Ala, Glu, Pro, Lys, or is absent:
`D-histidine, desamino-histidine, 2-amino-histidine, B-hy-
`and B and U" together is the acylated moiety, where U is
`droxy-histidine,
`homohistidine,
`N-acetyl-histidine,
`selected from
`
`MPI EXHIBIT 1002 PAGE 6
`
`MPI EXHIBIT 1002 PAGE 6
`
`

`

`US 8,129,343 B2
`
`12
`
`A™
`
`11
`
`0.
`
`OH
`
`oO
`
`oO
`HOY||
`“So
`
`°
`
`oO
`
`oO
`
`oO
`
`AL
`
`oO
`VNON , WONOR
`H
`
`HN
`
`I
`
`oO
`
`0.
`
`OH
`
`Ny
`
`0.
`
`OH
`
`NY N\A AAA
`
`H
`
`A
`oO
`
`oO
`
`ol)
`
`NTN } NONOON™
`
`H
`
`H
`
`\/
`
`7
`
`oO
`
`oO
`
`&
`
`HAIAH
`HteeeyH
`me oO
`6
`
`oO
`
`oO
`
`oO
`
`oO
`
`0
`
`oO
`0
`\
`
`Sy
`
`COOH
`
`Sy
`
`oO
`
`OH
`
`~
`
`N
`
`COOH
`
`Oo
`
`H
`
`oO
`
`oO
`
`O
`
`0
`
`COOH
`
`HSyetet
`- Rtyy5
`D.
`TOpyN
`°
`onhak
`
`,
`
`O
`
`6
`
`4
`
`i
`
`OT,
`
`MPI EXHIBIT 1002 PAGE 7
`
`MPI EXHIBIT 1002 PAGE 7
`
`MPI EXHIBIT 1002 PAGE 7
`
`

`

`US 8,129,343 B2
`
`13
`
`14
`acetyl-histidine, C-fluoromethyl-histidine, C.-methyl-histi
`m is 0, 1, 2, 3, 4, 5, or 6,
`dine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
`n is 1, 2 or 3
`s is 0, 1, 2, or 3,
`Xaas is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopro
`t is 0, 1, 2, 3, or 4
`p is 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 5 pyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid,
`19, 20, 21, 22, or 23;
`(1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)
`and where B is an acidic group selected from
`carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or
`(1-aminocyclooctyl) carboxylic acid;
`
`HO
`
`HO
`
`s
`
`N
`
`HO
`
`and
`
`10
`
`O
`
`O
`
`O
`
`O
`
`H
`
`St.
`O
`O
`
`O
`
`O HO
`
`where 1 is 12, 13, 14, 15, 16, 17, 18, 19 or 20,
`In an embodiment the invention provides a compound
`which is a compound of formula II (SEQID No. 3):
`
`2O
`
`Xaa is Val or Leu;
`Xaas is Ser, Lys or Arg:
`Xaa, is Tyr or Gln;
`Xaao is Leu or Met;
`Xaa- is Gly, Glu or Aib;
`Xaa is Gln, Glu, Lys or Arg;
`Xaas is Ala or Val;
`Xaa, is Glu or Leu:
`
`Xaa-7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa16-Ser-Xaa18-Xaa19-Xaa20-Glu-Xaa22
`
`Formula II
`
`Xs usu-sur-issurplexexuxa 5-Xaa36-Xaa37-Xaa38
`
`H
`
`i-st O
`
`Xaao is Ala, Glu or Arg;
`The formula II is identical to formula I as stated in an
`embodiment above, where the moiety B. U is replaced by 60 Xaass is Val or Lys,
`B U". The difference being only the incorporation of the
`Xaa- is Lys, Glu, ASnor Arg;
`carboxy group in the U' relative to U, which is without the
`Xaas is Gly or Aib;
`attaching carboxy group.
`Xaa- is Arg, Gly or Lys, or is absent;
`Informula II each of the Xaas has the following meaning:
`Xaas, is Gly, Ala, Glu, Pro, Lys, or is absent;
`Xaa, is L-histidine, D-histidine, desamino-histidine,
`Xaass is Lys, Ser, amide or is absent;
`2-amino-histidine, B-hydroxy-histidine, homohistidine, N'-
`and where U is a spacer selected from
`
`MPI EXHIBIT 1002 PAGE 8
`
`MPI EXHIBIT 1002 PAGE 8
`
`

`

`15
`
`O
`
`O
`
`O
`
`OH
`
`O
`
`N-
`
`O
`
`N-
`
`O
`
`OH
`
`Q O O
`\/
`N1
`
`US 8,129,343 B2
`
`16
`
`O
`
`H
`
`N-1-1-9 S -n-n-n-n
`
`H
`
`N-1-(N-N-N 1.-1.
`
`H
`
`/\,
`
`O
`
`O
`N-1N1\-1
`
`iii.
`
`O
`
`S 1n-N-1-1-
`H
`
`where n is 12, 13, 14, 15, 16, 17 or 18
`
`and where B is an acidic group selected from
`
`1 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;
`
`30
`
`35
`
`and
`HO
`1.
`Y HO Y
`O
`O
`
`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" is selected from
`
`O
`
`O
`
`HOS so
`YN
`
`O
`N-1\o1N1
`
`O
`
`O
`
`O
`
`N
`
`N-1 no-1N1
`
`O
`
`1N1'N-1-
`
`HO
`
`O
`
`N 1n-N-1- pi
`
`O
`
`O
`
`MPI EXHIBIT 1002 PAGE 9
`
`MPI EXHIBIT 1002 PAGE 9
`
`

`

`17
`
`O
`
`OH
`
`US 8,129,343 B2
`
`-continued
`
`O
`
`18
`
`YN
`
`O
`N-1N1\-1
`
`O
`
`OH
`
`N
`H
`
`O. O
`\/
`N- iii.
`
`O
`
`O
`
`O
`
`H
`N
`O
`N-1--6\- p
`
`O
`N-1 no-1S-1
`
`S 1n-N-1 no
`H
`
`pi
`
`s
`
`O
`
`H
`N
`1Ns
`/\
`O O
`
`O
`
`iii.
`
`O
`
`pi
`
`O
`
`1n-'N-1-
`H
`
`s
`
`O
`
`O O
`V/
`S
`
`H
`N
`
`N- N-> N-1--6\- S
`
`O
`
`O
`
`O
`
`O
`
`O
`
`N
`
`r
`
`COOH
`
`N
`
`OH
`
`O
`n
`
`N
`
`COOH
`
`O
`
`m is 2, 3, 4 or 5,
`
`n is 1 or 2
`S is 0, 1, or 2,
`
`H
`
`O
`
`s
`
`N ~ ----
`
`O
`
`S
`
`~ O N-1N
`
`COOH
`
`O
`
`S
`
`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 U' is
`
`45
`
`O
`
`Ho S.
`
`loos-
`
`N
`
`O
`N-1 no-1N1
`
`N 1-'N-1-~-
`
`O
`
`loos---
`
`H
`
`N-1-1-9 S ~~~~
`
`O
`
`MPI EXHIBIT 1002 PAGE 10
`
`MPI EXHIBIT 1002 PAGE 10
`
`

`

`19
`
`O
`
`OH
`
`O
`
`loos-
`
`N
`
`O
`
`OH
`
`O
`
`loos-
`
`N
`H
`
`HOOC
`
`Ns
`MV
`O O
`
`3
`
`HOOC
`
`O O O
`VW
`-S
`N
`
`US 8,129,343 B2
`
`-continued
`
`O
`
`20
`
`N- -) O
`s
`S 1- N-1 no r
`O
`
`O
`
`O
`N-1s-ha- p
`
`O
`
`H
`N-1N1S-19
`O
`
`O
`
`O
`S 1- N-1\o
`
`O
`
`O
`
`O
`
`iii.
`
`iii.
`
`O
`
`9
`
`HOOC
`
`O
`p
`
`O O
`\/
`H N
`N1 >1. N1-a-ha
`H
`O ---,
`
`O
`
`O
`
`O
`
`O
`O
`
`loos--- N
`
`COOH
`
`O
`
`O
`
`O
`
`O
`
`OH
`
`O
`
`O
`
`O
`
`H
`N
`
`O
`
`?
`O HO
`O
`
`N
`
`N-1N1-1
`
`O
`
`O
`S 1N1 N-1\o
`H
`
`O
`
`O
`
`O
`
`O
`
`MPI EXHIBIT 1002 PAGE 11
`
`MPI EXHIBIT 1002 PAGE 11
`
`

`

`21
`where 1 is 14, 15, 16, 17, 18, 19 or 20;
`p is 1, 2, 3, 4, 7, 8, 9, 10, 11 or 12.
`s is 0, 1 or 2
`t is 0 or 1:
`
`US 8,129,343 B2
`
`22
`An embodiment according to the above wherein
`where 1 is 14, 15, 16, 17 or 18
`p is 1, 2, 3, 4 or 11,
`s is 0, 1 or 2;
`t is 0 or 1:
`An embodiment provides a GLP-1 analog according to the
`embodiment above, wherein B U" is
`
`5
`
`O
`
`Hos
`
`so
`
`O
`
`H
`N
`
`N-1 no-1-1
`
`O
`
`O
`
`HOOC
`
`N-
`
`S 1N1'N-1N pi
`
`H
`
`O
`
`1N-1'N-1 no
`
`O
`
`H
`
`O
`
`O
`
`O
`
`O
`
`H
`N
`
`N-1 no-1N1
`
`O
`
`HOOC
`
`---,
`s
`
`O
`
`O
`O
`
`loos---
`N
`
`H
`NN-1N1-19
`O
`
`O
`
`O
`S 1- N-1\o pi
`
`O
`
`HO
`
`O
`
`H
`N
`
`O
`
`O
`
`u).
`N-1 no1N1
`s N 1N1 N-1\o
`
`O
`O
`
`H
`
`O
`
`O os N
`O
`N-
`
`HOOC
`
`OH
`
`p
`O
`
`OH
`
`O
`
`O
`
`H
`N
`
`N-n-hn
`
`O
`
`O
`
`OH
`
`O
`
`HOOC
`
`O
`
`N-
`O ---,
`
`()
`
`OH
`
`O
`
`HOOC Y& Sai YO N
`
`COOH
`
`O
`
`H
`
`O
`
`()
`
`s
`
`O
`
`MPI EXHIBIT 1002 PAGE 12
`
`MPI EXHIBIT 1002 PAGE 12
`
`

`

`US 8,129,343 B2
`
`23
`where 1 is 14, 15, 16, 17, 18, 19 or 20;
`p is 1, 2, 3, or 4.
`s is 0, 1 or 2
`n is 0, 1 or 2
`An embodiment according to any of the above embodi
`ments is wherein B is
`
`HO
`
`5
`
`24
`-continued
`
`HO
`
`HO
`
`s
`
`H
`-N
`HO
`
`and
`
`O
`
`O
`
`O
`
`O
`
`H
`
`N 11
`
`O
`O
`
`O
`
`O HO
`
`15
`
`and 1 is 14, 16, 18 or 20;
`An embodiment provides a GLP-1 analog according to any
`of the embodiments above, wherein B is
`
`HO
`
`s
`
`H
`N
`HO1
`
`and
`
`25
`
`O
`
`O
`
`O
`
`O
`
`where 1 is 14, 15, or 16.
`An embodiment provides a GLP-1 analog according to any
`of the embodiments above, whereins is 1.
`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, whereinl is 14, 15 or 16; In embodi
`ments 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 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.
`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 abov