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`US008129343B2
`
`c12) United States Patent
`Lau et al.
`
`(IO) 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 NordiskA/S, Bagsvaerd (DK)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 682 days.
`
`WO 00/34331
`WO
`WO 02/46227
`WO
`WO0246227
`WO
`WO 02/098446
`WO
`WO 03/040309
`WO
`WO WO 2004/065621
`WO WO 2004/099246
`WO WO 2005/014049
`WO
`W02005014049
`WO WO 2005/027978
`WO
`W02005027978
`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
`
`(21) Appl. No.:
`
`11/908,834
`
`(22) PCT Filed:
`
`Mar. 20, 2006
`
`(86) PCT No.:
`
`PCT /EP2006/060855
`
`§ 371 (c)(l),
`(2), ( 4) Date:
`
`Sep.17,2008
`
`(87) PCT Pub. No.: WO2006/097537
`
`PCT Pub. Date: Sep. 21, 2006
`
`(65)
`
`Prior Publication Data
`
`US 2009/0156478 Al
`
`Jun. 18,2009
`
`Related U.S. Application Data
`
`(60)
`
`Provisional application No. 60/664,497, filed on Mar.
`23, 2005.
`
`(30)
`
`Foreign Application Priority Data
`
`Mar. 18, 2005
`
`(EP) ..................................... 05102171
`
`(51)
`
`Int. Cl.
`A61K 38/26
`(2006.01)
`A61P 3/10
`(2006.01)
`A61P 7112
`(2006.01)
`C07K 141605
`(2006.01)
`(52) U.S. Cl. ...................................................... 514/11.7
`(58) Field of Classification Search ........................ None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
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`8/ 1996 Buckley et al.
`6,268,343 Bl *
`7/2001 Knudsen et al.
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`3/2003 Larsen et al.
`2007/0203058 Al*
`8/2007 Lau et al.
`
`514/4.8
`514/6.8
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`WO
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`05102171
`3/2005
`1704165
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`2006107600
`10/2007
`WO 91/11457
`8/1991
`WO 96/29342
`9/1996
`WO 9629342
`9/1996
`WO 98/08871
`3/1998
`WO9808871
`3/1998
`WO 99/43708
`9/1999
`WO9943708
`9/1999
`WO0034331
`6/2000
`WO 00/69911
`11/2000
`WO0069911
`11/2000
`
`OTHER PUBLICATIONS
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`Simonovsky et al. Poly( ether urethane )s incorporating long alkyl
`side-chains with terminal carboxy groups as fatty acid mimics: syn(cid:173)
`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(cid:173)
`ential Adsorption ofAlbnmin," Biomaterials, 2001, vol. 22, p. 3015-
`3023.
`Kurtzhals, P, et al., "Albumin Binding oflnsulins Acy lated With Fatty
`Acids: Characterization of the Ligand-Protein Interaction and Cor(cid:173)
`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(cid:173)
`lems and Solutions," Biomaterials, 2001, vol. 22, p. 405-417.
`Watanabe et al., "Structure-Activity Relationships ofGlucagon-Like
`Peptide-1 (7-36) Amide: Insulinotropic Activities in Perfused Rat
`Pancreases, and Receptor Binding and Cyclic Amp Production in
`RINm5F 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 1068 PAGE 1
`
`MPI EXHIBIT 1068 PAGE 1
`
`MPI EXHIBIT 1068 PAGE 1
`
`MPI EXHIBIT 1068 PAGE 1
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0001
`
`

`

`US 8,129,343 B2
`
`1
`ACYLATED GLP-1 COMPOUNDS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`2
`The invention provides a method for increasing the time of
`action in a patient of a GLP-1 analog, characterised in acy(cid:173)
`lating said GLP-1 analog with a moiety B-U' as disclosed in
`any of the preceding claims, on the lysine residue in position
`5 26 of said GLP-1 analog.
`
`This application is a 35 U.S.C. §371 national stage appli(cid:173)
`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 10
`claims priority under 35 U.S.C. §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
`
`A range of different approaches have been used for modi(cid:173)
`fying the structure of glucagon-like peptide 1 (GLP-1) com(cid:173)
`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.
`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.
`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.
`Many diabetes patients particularly in the type 2 diabetes
`segment are subject to so-called "needle-phobia", i.e. a sub(cid:173)
`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(cid:173)
`pounds. Thus, there is a need to develop new GLP-1 com(cid:173)
`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.
`
`SUMMARY OF THE INVENTION
`
`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
`15 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,
`omithine, phosphoserine, D-alanine and D-glutamine. Syn-
`20 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 (a-aminoisobutyric acid), Abu (a-aminobutyric acid),
`Tie (tert-butylglycine), ~-alanine, 3-aminomethyl benzoic
`25 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,
`30 Phenylalanine, 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,
`35 omithine, phosphoserine, the D-amino acids such as D-ala(cid:173)
`nine and D-glutamine, Synthetic non-proteogenic amino
`acids comprise amino acids manufactured by chemical syn(cid:173)
`thesis, i.e. D-isomers of the amino acids encoded by the
`genetic code such as D-alanine and D-leucine, Aib (a-ami-
`40 noisobutyric acid), Abu (a-aminobutyric acid), Tie (tert-bu(cid:173)
`tylglycine), 3-aminomethyl benzoic acid, anthranilic acid,
`des-amino-Histidine, the beta analogs of amino acids such as
`~-alanine etc. D-histidine, desamino-histidine, 2-amino-his(cid:173)
`tidine, ~-hydroxy-histidine, homohistidine, N"'-acetyl-histi-
`45 dine, a-fluoromethyl-histidine, a-methyl-histidine, 3-py(cid:173)
`ridylalanine,
`2-pyridylalanine
`or
`4-pyridylalanine,
`(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl)
`carboxylic acid, (1-aminocyclopentyl) carboxylic acid,
`(1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl)
`50 carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;
`The term "analogue" as used herein referring to a polypep(cid:173)
`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
`55 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 [Arg34]GLP-
`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 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
`
`The invention provides a GLP-1 analog having a modifi(cid:173)
`cation 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 60
`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(cid:173)
`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.
`
`MPI EXHIBIT 1068 PAGE 2
`
`MPI EXHIBIT 1068 PAGE 2
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`MPI EXHIBIT 1068 PAGE 2
`
`MPI EXHIBIT 1068 PAGE 2
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0002
`
`

`

`US 8,129,343 B2
`
`3
`maximum of 1 7 amino acids have been modified. In embodi(cid:173)
`ments 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 5
`embodiments of the invention a maximum of 7 amino acids
`have been modified. In embodiments of the invention a maxi(cid:173)
`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 10
`amino acids have been modified. In embodiments of the
`invention a maximum of3 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 or an analogue thereof,
`wherein at least one substituent is not present in the unmodi(cid:173)
`fied peptide or an analogue thereof, i.e. a peptide which has
`been covalently modified. Typical modifications are amides, 20
`carbohydrates, alkyl groups, acyl groups, esters and the like.
`An example of a derivative of GLP-1(7-37) is NE26
`-(( 4S)-4-
`(hexadecanoylamino )-carboxy-butanoyl)[ Arg34
`Lys 26
`,
`]
`GLP-1-(7-37).
`The term "GLP-1 peptide" as used herein means GLP-1(7- 25
`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 agonist of the human GLP-1 receptor, 30
`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(cid:173)
`tropic agent is determined by calculating the EC50 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
`µg/mL streptomycin, 5% fetal calf serum and 0.5 mg/mL
`Geneticin G-418 (Life Technologies). The cells were washed 40
`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(cid:173)
`ing cyclic AMP ( cAMP) as 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-area 96-well microtiter
`plates ina total volume of50 µL buffer3 (50mMTris-HCl, 5
`mM HEPES, 10 mM MgC12 , 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 preparation, 15 µg/mL acceptor beads, 20 µg/mL 60
`donor beads preincubated with 6 nM biotinyl-cAMP. Com(cid:173)
`pounds to be tested for agonist activity were dissolved and
`diluted in buffer 3. GTP was freshly prepared for each experi(cid:173)
`ment. The plate was incubated in the dark with slow agitation
`for three hours at room temperature followed by counting in 65
`the Fusion™ instrument (Perkin Elmer Life Sciences). Con(cid:173)
`centration-response curves were plotted for the individual
`
`4
`compounds and EC50 values estimated using a four-param(cid:173)
`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(cid:173)
`ceptible to DPP-IV mediated hydrolysis in order to reduce the
`rate of degradation by DPP-IV. In one embodiment a DPP-IV
`15 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(cid:173)
`nopeptidase IV is determined by the following degradation
`assay:
`Aliquots of the peptide (5 nmol) are incubated at 37° C.
`with 1 µL of purified dipeptidyl aminopeptidase IV corre(cid:173)
`sponding to an enzymatic activity of 5 m U for 10-180 minutes
`in 100 µL of0.1 M triethylamine-HCl buffer, pH 7.4. Enzy(cid:173)
`matic reactions are terminated by the addition of 5 µL 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
`a Vydac C18 widepore (30 nm pores, 5 µm particles) 250x4.6
`mm colunm 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
`35 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 "C 1_calkyl" 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
`55 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(cid:173)
`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
`
`50
`
`MPI EXHIBIT 1068 PAGE 3
`
`MPI EXHIBIT 1068 PAGE 3
`
`MPI EXHIBIT 1068 PAGE 3
`
`MPI EXHIBIT 1068 PAGE 3
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0003
`
`

`

`US 8,129,343 B2
`
`In the formulas below
`
`6
`
`5
`control the disease, condition or disorder as well as to allevi(cid:173)
`ate the symptoms or complications associated with the dis(cid:173)
`ease, condition or disorder.
`In another aspect the present invention relates to an acy-
`lated GLP-1 analogue that can bind to albumin and the GLP-1
`receptor simultaneously.
`In another aspect the present invention relates to an acy(cid:173)
`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
`ence of 2% albumin.
`In another aspect the present invention relates to an acy(cid:173)
`lated 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, preferable below 30 fold and more pref(cid:173)
`erable below 10 fold.
`The term "albumin binding moiety" as used herein means
`a residue which binds non-covalently to human serum albu(cid:173)
`min. 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 range of albu(cid:173)
`min 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 resi(cid:173)
`due with a chemical moiety which comprises at least 5 non(cid:173)
`hydrogen atoms where 30-50% of these are either Nor 0.
`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 car(cid:173)
`boxylic acids, sulphonic acids, phosphoric acids or heterocy(cid:173)
`clic ring systems which are fully or partly negatively charged
`at physiological pH.
`In the below structural formula II the moiety U is a di(cid:173)
`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 45
`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 attachment bonds and not ending
`in methylene groups unless stated.
`
`5
`
`15
`
`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 lipophilic albumin binding moiety containing
`at least two free acidic chemical groups attached via a non
`natural 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
`20 GLP-1 analog where said GLP-1 analog is stabilised against
`DPP-IV by modification of at least one amino acid residue in
`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
`25 amino acid hydrophilic linker.
`In an embodiment of the invention 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 withamoietytothe
`30 lysine residue in position 26, and where said moiety com(cid:173)
`prises 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
`35 26 comprises a hydrophilic linker.
`An embodiment provides a GLP-1 analog according to the
`above embodiments, wherein the hydrophilic linker com(cid:173)
`prises at least 5 non-hydrogen atoms where 30-50% of these
`40 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 separated
`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, wherein the acylated moiety is
`B-U', where U' is selected from
`
`MPI EXHIBIT 1068 PAGE 4
`
`MPI EXHIBIT 1068 PAGE 4
`
`MPI EXHIBIT 1068 PAGE 4
`
`MPI EXHIBIT 1068 PAGE 4
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0004
`
`

`

`7
`
`US 8,129,343 B2
`
`-continued
`
`8
`
`/'--. . , o~ ~
`........._ ~~~ /'--..._/o
`' J
`~~- ' - /
`ooy
`
`O
`
`o
`
`~\
`
`0
`
`,
`
`o
`
`0
`
`N
`H
`
`'OqOH i j~ ~o~ ~ , o~ ~ ,
`
`0
`
`, N'
`H
`
`......._,,,,
`
`0
`
`n
`
`0
`
`O
`
`0~ ~
`
`~I/
`............ /S
`N
`H
`
`H
`N~ /'--..._/0
`0
`
`m
`
`/'--. ~ ,
`.,0.._
`/'--.
`'o
`, N'
`......._,,,,
`......._,,,,
`n
`H
`
`0
`
`O
`
`0
`0
`H ~O~
`~I/
`........._,,,,s~N~
`N
`0
`m
`H
`
`P
`
`n
`
`,
`
`0
`
`0
`
`MPI EXHIBIT 1068 PAGE 5
`
`MPI EXHIBIT 1068 PAGE 5
`
`MPI EXHIBIT 1068 PAGE 5
`
`MPI EXHIBIT 1068 PAGE 5
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0005
`
`

`

`US 8,129,343 B2
`
`9
`
`mis 0, 1, 2, 3, 4, 5, or 6,
`n is 1, 2 or 3
`s is 0, 1, 2, or 3,
`tis 0, 1, 2, 3, or4
`p~l,2,3,4,5,6,7,~9, 10, 11,12, 13, 1~ 15, 16, 1~ 18, 5
`19, 20, 21, 22, or 23;
`and where B is an acidic group selected from
`
`HO~ ,
`
`HO /N~
`
`H
`
`and
`
`10
`
`0
`
`0
`
`0
`
`0
`
`HO~~~ 0
`
`0
`
`O
`
`HO
`
`0
`
`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 (SEQ ID No. 2):
`
`15
`
`20
`
`10
`N"'-formyl-histidine, a-fluoromethyl-histidine, a-methyl(cid:173)
`histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylala(cid:173)
`mne
`Xaa8 is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocy(cid:173)
`clopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic
`acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclo(cid:173)
`hexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic
`acid, or (1-aminocyclooctyl) carboxylic acid;
`Xaa16 is Val or Leu;
`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;
`Xaa25 is Ala or Val;
`Xaa27 is Glu or Leu;
`Xaa30 is Ala, Glu or Arg;
`Xaa33 is Val or Lys;
`Xaa34 is Lys, Glu, Asn or Arg;
`Xaa35 is Gly or Aib;
`
`Xaar Xaas-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa 16-Ser-Xaa 1s-Xaa 19-Xaa20-Glu-Xaa22-
`
`Formula!
`
`0
`
`Xaa23-Ala-Xaa25-~ ~ Xaa27-Phe-Ile-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa33
`
`\ B-U'-NH
`
`wherein
`Xaa7 is L-histidine, imidazopropionyl, a-hydroxy-histidine,
`D-histidine, desamino-histidine, 2-amino-histidine, ~-hy(cid:173)
`droxy-histidine,
`homohistidine,
`N"' -acetyl-histidine,
`
`Xaa36 is Arg, Gly or Lys, or is absent;
`65 Xaa37 is Gly, Ala, Glu, Pro, Lys, or is absent;
`and B and U' together is the acylated moiety, where U' is
`selected from
`
`MPI EXHIBIT 1068 PAGE 6
`
`MPI EXHIBIT 1068 PAGE 6
`
`MPI EXHIBIT 1068 PAGE 6
`
`MPI EXHIBIT 1068 PAGE 6
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0006
`
`

`

`US 8,129,343 B2
`
`11
`
`12
`
`O~OH
`
`'-._N~, '-f_N
`H
`lH
`
`,
`
`p
`
`0
`
`'--OqOH i j~ ~o~ ~ , o~ ~ ,
`
`O
`
`, N'
`H
`
`.....,_,,,,,,
`
`0
`
`n
`
`N
`H
`
`0
`
`O
`
`0~ ~
`
`.,,,o~ ~ ,
`/"-.,._
`, N'
`.....,_,,,,,,
`0
`n
`H
`
`~f
`'--- /s
`N
`H
`
`H
`N~ ~o
`O
`
`m
`
`0
`
`0
`
`0
`0
`~f
`'-._ /S~N~
`N
`O
`m
`H
`
`H ~O~
`
`0
`
`P
`
`n
`
`,
`
`0
`
`MPI EXHIBIT 1068 PAGE 7
`
`MPI EXHIBIT 1068 PAGE 7
`
`MPI EXHIBIT 1068 PAGE 7
`
`MPI EXHIBIT 1068 PAGE 7
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0007
`
`

`

`US 8,129,343 B2
`
`13
`
`mis 0, 1, 2, 3, 4, 5, or 6,
`n is 1, 2 or 3
`s is 0, 1, 2, or 3,
`tis 0, 1, 2, 3, or4
`p~l,2,3,4,5,6,7,~9, 10, 11,12, 13, 1~ 15, 16, 1~ 18, 5
`19, 20, 21, 22, or 23;
`and where B is an acidic group selected from
`
`14
`acetyl-histidine, a-fluoromethyl-histidine, a-methyl-histi-
`dine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
`
`Xaa8 is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopro(cid:173)
`pyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid,
`(1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)
`carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or
`(1-aminocyclooctyl) carboxylic acid;
`
`HO~ ,
`
`HO /N~
`
`H
`
`and
`
`10
`
`0
`
`0
`
`0
`
`0
`
`HOD~~
`0
`0
`
`O
`
`HO
`
`0
`
`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 (SEQ ID No. 3):
`
`15
`
`20
`
`Xaa16 is Val or Leu;
`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;
`Xaa25 is Ala or Val;
`Xaa27 is Glu or Leu;
`
`Xaar Xaas-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa 16-Ser-Xaa 1s-Xaa 19-Xaa20-Glu-Xaa22-
`
`Formula II
`
`0
`
`Xaa23-Ala-Xaa25-~ ~ Xaa27-Phe-Ile-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa33
`
`\ B-UyNH
`
`0
`
`The formula II is identical to formula I as stated in an
`embodiment above, where the moiety 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:
`is L-histidine, D-histidine, desamino-histidine,
`Xaa7
`2-amino-histidine, ~-hydroxy-histidine, homohistidine, N"'-
`
`Xaa30 is Ala, Glu or Arg;
`60 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;
`65 Xaa37 is Gly, Ala, Glu, Pro, Lys, or is absent;
`Xaa38 is Lys, Ser, amide or is absent;
`and where U is a spacer selected from
`
`MPI EXHIBIT 1068 PAGE 8
`
`MPI EXHIBIT 1068 PAGE 8
`
`MPI EXHIBIT 1068 PAGE 8
`
`MPI EXHIBIT 1068 PAGE 8
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0008
`
`

`

`US 8,129,343 B2
`
`15
`
`16
`
`wherenis 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,
`
`mis 0, 1, 2, 3, 4, 5, or 6,
`
`s is 0, 1, 2, or 3,
`
`30
`
`35
`
`and
`
`pis3,4,5,6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
`20, 21, 22, or 23;
`
`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
`
`MPI EXHIBIT 1068 PAGE 9
`
`MPI EXHIBIT 1068 PAGE 9
`
`MPI EXHIBIT 1068 PAGE 9
`
`MPI EXHIBIT 1068 PAGE 9
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0009
`
`

`

`US 8,129,343 B2
`
`17
`
`18
`
`'O qOH i j~ ~o~ ~ , o~ ~ ,
`
`N
`H
`
`O
`
`, N'
`H
`
`-.....,,,,-
`
`0
`
`n
`
`-continued
`
`0
`
`O
`
`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
`pis 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
`
`·
`IT
`Hooe
`'M ( " - -N~
`o
`oo~
`
`( i j~ ~o TT\ ~o~ / ' - - /
`
`0
`
`~N
`
`H
`
`O
`
`II
`o
`
`0
`
`MPI EXHIBIT 1068 PAGE 10
`
`MPI EXHIBIT 1068 PAGE 10
`
`MPI EXHIBIT 1068 PAGE 10
`
`MPI EXHIBIT 1068 PAGE 10
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0010
`
`

`

`19
`
`US 8,129,343 B2
`
`-continued
`
`20
`
`~ oqoH i j~ ~o TT\ ~o~ IT'
`
`Hooe
`~N
`H
`
`0
`
`~N
`H
`
`0
`
`0
`
`0
`
`~ O~OH i j~ ~O~
`
`0
`
`p
`
`H00C
`~N
`H
`
`O
`
`0
`
`~ /o.._ ~ IT'
`0 0 4 ~
`~f
`II
`_,,...s
`Hooe
`~N
`H
`
`, N/
`H
`
`-...._,,,,-
`
`-...._,,,,-
`
`"o
`
`H
`N~ ~o
`O
`
`m
`
`0
`
`0
`
`MPI EXHIBIT 1068 PAGE 11
`
`MPI EXHIBIT 1068 PAGE 11
`
`MPI EXHIBIT 1068 PAGE 11
`
`MPI EXHIBIT 1068 PAGE 11
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0011
`
`

`

`21
`where I is 14, 15, 16, 17, 18, 19or20;
`
`pis 1, 2, 3, 4, 7, 8, 9, 10, 11 or 12.
`
`sis 0, 1 or 2
`
`tis 0 or 1;
`
`US 8,129,343 B2
`
`22
`An embodiment according to the above wherein
`where] is 14, 15, 16, 17 or 18
`p is 1, 2, 3, 4 or 11;
`sis 0, 1 or 2;
`th
`d.
`t is O or 1;
`An embodiment provides a GLP-1 analog accor mg to e
`embodiment above, wherein B-U' is
`
`Hooe
`
`IT
`
`.
`
`( ij ~o~o~N~o~o~
`~f
`~
`
`'H
`
`0
`
`HO
`
`i j~~o ~\ ~ ,o~~
`
`0
`
`~N ' -....._,,,,-
`H
`
`0
`
`0
`
`MPI EXHIBIT 1068 PAGE 12
`
`MPI EXHIBIT 1068 PAGE 12
`
`MPI EXHIBIT 1068 PAGE 12
`
`MPI EXHIBIT 1068 PAGE 12
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1068-0012
`
`

`

`US 8,129,343 B2
`
`23
`wherelis 14, 15, 16, 17, 18, 19or20;
`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 embodi-
`ments is wherein B is
`
`5
`
`24
`-continued
`
`HO - - f>=~YY
`0
`0
`
`O
`
`HO
`
`0
`
`10
`
`15
`
`where 1 is 14, 15, or 16.
`An embodiment provides a GLP-1 analog according to any
`of the embodiments above, w