Case 1:22-cv-00023-JPB Document 2-3 Filed 03/18/22 Page 1 of 71 PageID #: 124
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`Exhibit C
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`

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`Case 1:22-cv-00023-JPB Document 2-3 Filed 03/18/22 Page 2 of 71 PageID #: 125
`eeSSN TTA TATAAEA
`
`US008536122B2
`
`a2) United States Patent
`US 8,536,122 B2
`(0) Patent No.:
`*Sep. 17, 2013
`(45) Date of Patent:
`Lau et al.
`
`(54) ACYLATED GLP-1 COMPOUNDS
`
`(75)
`
`Inventors: Jesper Lau, Farum (DK); Florencio
`Zaragoza Doerwald, Smorum (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 ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 13/412,283
`
`(22)
`
`Filed:
`
`Mar.5, 2012
`
`(65)
`
`Prior Publication Data
`
`US 2012/0295847 Al
`
`Nov. 22, 2012
`
`Related U.S. Application Data
`
`(30)
`
`Foreign Application Priority Data
`
`Mar. 18, 2005)
`
`(EP) on. eeeeeeeeeeereeeens 05102171
`
`FOREIGN PATENT DOCUMENTS
`1329458 A2
`7/2003
`EP
`05102171.5
`3/2005
`EP
`1704165 Al
`9/2006
`EP
`2000-500505 A
`1/2000
`JP
`2002-504527 A
`2/2002
`JP
`2002-508162 A
`3/2002
`JP
`2003-505347
`2/2003
`JP
`2004-528014 A
`9/2004
`JP
`2004-535442 A
`11/2004
`JP
`2010-116407 A
`5/2010
`JP
`2006107600 A
`10/2007
`RU
`90/11296
`10/1990
`WO
`91/11457 Al
`8/1991
`WO
`96/29342
`9/1996
`WO
`98/0887 1
`3/1998
`WO
`98/08872 Al
`3/1998
`WO
`99/4334 1
`9/1999
`WO
`99/43361 Al
`9/1999
`WO
`99/43705 Al
`9/1999
`WO
`99/43708
`9/1999
`WO
`9943707
`9/1999
`WO
`00/3433 1
`6/2000
`WO
`00/6991 1
`11/2000
`WO
`01/04156
`1/2001
`WO
`0151071
`7/2001
`WO
`0258725
`1/2002
`WO
`02/46227 A2
`6/2002
`WO
`
`WO 02098446 Al=12/2002
`WO
`03/002 136
`1/2003
`WO
`03/013573 Al
`2/2003
`WO
`03/040309 A2
`5/2003
`(63) Continuation of application No. 11/908,834, filed as
`WO
`03/058203 A2
`7/2003
`application No. PCT/EP2006/060855 on Mar. 20,
`
`WO 03/087139 A2—10/2003
`WO
`2004/065621 Al
`8/2004
`2006, now Pat. No. 8,129,343.
`WO
`2004/074315 A2
`9/2004
`WO
`2004/093823 A2
`11/2004
`(60) Provisional application No. 60/664,497, filed on Mar.
`
`WO 2004/099246 A2=11/2004
`23, 2005.
`WO
`2005/014049 A2
`2/2005
`WO
`2005/027978
`3/2005
`WO
`2005/028516 A2
`3/2005
`WO
`2005/058958 A2
`6/2005
`WO
`2006/005667 A2
`1/2006
`WO
`2006/037810 A2
`4/2006
`WO
`2006/097536 A2
`9/2006
`WO
`2006/097537
`9/2006
`WO
`2006/097537 A2
`9/2006
`WO
`2006/097538 Al
`9/2006
`OTHER PUBLICATIONS
`
`Declaration of Per Franklin Nielsen, 2012.
`Annual Report 2003 Novo Nordisk A/S.
`Curry, Stephen, Plasma Albumin asa Fatty Acid Carrier, Advancesin
`Molecular and Cell Biology, 2004, vol. 33, pp. 29-46.
`Annual Report 2004, Novo Nordisk A/S.
`Naucketal., The Once-Weekly Human GLP-1 Analogue ..., EASD,
`2012, 49th Annual Meeting.
`International Non-Rpoprietary Names... , 2003, vol. 17(2), pp. 115,
`125,
`Table of S.C. Half-Life(Mining) and Potency Data 2011.
`Berendsen, 1998, “A Glimpse of the Holy Grail?” Science 282:642-
`643.
`Bradleyet al., 2002, “Limits of Cooperativity in a Structually Modu-
`lar Protein: Response of the Notch Ankyrin Domain to Analogous
`Alanine Substitutions in Each Repeat,” Journal of Molecular Biology
`324:373-386.
`
`(Continued)
`
`Primary Examiner — Marcela M Cordero Garcia
`(74) Attorney, Agent, or Firm — Richard W. Bork
`
`ABSTRACT
`(57)
`Protracted GLP-1 compoundsand therapeutic uses thereof.
`
`16 Claims, No Drawings
`
`(51)
`
`Int. Cl.
`A6LK 38/26
`A61K 38/28
`A6IP 3/10
`A6IP 7/12
`CO7K 14/605
`C07K 5/00
`C07K 7/00
`C07K 16/00
`C07K 17/00
`(52) U.S. Cl.
`USPC oie rece §14/7.2; 514/11.7; 530/308
`(58) Field of Classification Search
`None
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,545,618 A
`6,268,343 Bl
`6,528,486 Bl
`2001/0011071 Al
`2004/0001827 Al
`2004/0053370 Al
`2007/0203058 Al
`2009/0156478 Al
`
`8/1996 Buckley etal.
`7/2001 Knudsenetal.
`3/2003 Larsenetal.
`8/2001 Knudsenet al.
`1/2004 Dennis
`3/2004 Glaesner etal.
`8/2007 Lau etal.
`6/2009 Lauet al.
`
`

`

`Case 1:22-cv-00023-JPB Document 2-3 Filed 03/18/22 Page 3 of 71 PageID #: 126
`Case 1:22-cv-00023-JPB Document 2-3 Filed 03/18/22 Page 3 of 71 PagelD #: 126
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`US 8,536,122 B2
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`Page 2
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`(56)
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`References Cited
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`OTHER PUBLICATIONS
`Chuanget al., 2002, “Pharmaceutical Strategies Utilizing Recombi-
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`577.
`Han, 2002, “Targeted Prodrug Design to Optimize Drug Delivery,”
`AAPSPharmsci 2(1):1-11.
`Hodgsonet al., 2004, “The Synthesis of Peptides and Proteins Con-
`taining Non-Natural Amino Acids,” Chemical Reviews 33(7):422-
`430.
`Holzet al., 2003, “Glucagon-Like Peptide-1 Synthetic Analogs: New
`Therapeutic Agents for Use in the Treatment of Diabetes Mellitus,”
`Current Medicinal Chemistry 10(22):2471-2483.
`Kimetal., 2003, “Development and Characterization of a Glucagon-
`Like Peptide 1-Albumin Conjugate,” Diabetes 52:75 1-759.
`Makinoet al., 2005, “Semisynthesis of Human Ghrelin: Condensa-
`tion of a Boc-Protected Recombinant Peptide With a Synthetic
`O-Acylated Fragment,” Biopolymers 79(5):238-247.
`Okada, 2001, “Synthesis of Peptides by Solution Methods,” Current
`Organic Chemistry 5(1):1-43.
`Ostrovsky, 1975, “Comparative Characteristics of the Hydrophobic
`Nature of Certain Proteins by Their Interaction With 2-P Toluidino,”
`Ukrayins’kyi Biokhimichnyi Zhurnal 47(6):70 1-707.
`Pico, 1990, “Use of 1-Anilino-8-Naphthalene Sulfonate as a
`Reporter Molecule to Study the Bile Salts-Bovine Serum Albumin
`Binding,” Studia Biophysica 136(1):2 1-26, Abstract XP-008039734.
`Rudinger, 1976, “Characteristics of the Amino Acids as Components
`of a Peptide Hormone Sequence,” Peptides Hormones, JA Parsons
`Edition, University Park Press, Jun. 1976, pp. 1-7.
`Schinzel et al.,
`1991, “The Phosphate Recognition Site of
`Escherichia coli Maltodextrin Phosphorylase,” Federation of Euro-
`pean Biochemical Society Jul. 1991, 286(1, 2):125-128.
`Sheffield, 2001, “Modification of Clearance of Therapeutic and
`Potentially Therapeutic Proteins,” Current Drug Targets Cardiovas-
`cular & Haematological Disorders 1(1):1-22.
`SIGMAGenosys (Web Site), Designing Custom Peptides, pp. 1-2,
`Accessed Aug. 16, 2004.
`Voetet al., 1995, Biochemistry 2nd ed., John Wiley & Sons, Inc., pp.
`235-241.
`Wallace, 1995, “Peptide Ligation and Semisynthesis,” Current Opin-
`ion in Biotechnology 6(4):403-410.
`Zobel et al., 2003, “Phosphate Ester Serum Albumin Affinity Tags
`Greatly Improve Peptide Half-Life In Vivo,” Bioorganic & Medicinal
`Chemistry Letters 13:1513-1515.
`Knudsen,L.B. et al., “Potent Derivatives ofGlucagon-Like Peptide-1
`With Pharmacokinetic Properperties Suitable for Once Daily Admin-
`istration”, Journal of Medicinal Chemistry, 2000 vol. 43, pp. 1664-
`1669.
`
`Deacon, C.F.et al., “Dipeptidyl peptidase IV resistant analogues of
`glucagon-like peptide-1 which have extended metabolic stability and.
`improvedbiologicalactivity.” 1998, Diabetologia, vol. 41, pp. 271-
`278.
`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 Timingofthe Insulin Effect In
`Vivo,” Biochem J, 1995, vol. 312, pp. 725-731.
`Soltero et al., “The Oral Delivery of Protein and Peptide Drugs,”
`Innovations in Pharmaceutical Technology, 2001, vol. 1, No. 9, pp.
`106-110.
`Watanabeetal., “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
`RINmSF Cells,” Journal ofEndocrinology, 1994, vol. 140, pp. 45-52.
`Inflammatory Bowel Disease from e-Medicine, pp. 1.24, Accessed.
`Sep. 24, 2008.
`NgoJTet al., “Computational Complexity, Protein Structure Predic-
`tion, and the Levinthal Paradox,” The Protein Folding Problem and
`Tertiary Structure Prediction, K. Mere Jr. and S. LeGrand Edition,
`1994, pp. 491-495.
`Residue definition from www.dictionary.com,pp. 1-6, Accessed May
`5, 2009.
`Small Bowel Syndrome from e-Medicine, pp. 1-21, Accessed Sep.
`24, 2008.
`Green, Brian D. et al Biological Chemistry. Degradation, Receptor
`Binding, Insulin .
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`. 2004 385 2 169-177.
`Greenwald Journal of the Controlled Release Peg Drugs: An Over-
`view 2001 74-159-171.
`Ji, J. et al. Biomaterials Stearyl Poly (Ethylene Oxide) Grafted Sur-
`faces for Preferential Adsorption of Albumin. 2001 22-3015-3023.
`Knudsen, L.B. Journal of Medicinal Chemistry Glucagon-Like
`Peptide-1 .. . 2004 47-4128-4134.
`Simonovsky et al. Journal of Biomaterials Science, Polymer Edition
`Poly(Ether Urethane)S Incorporating Long Alkyl Side-Chains With
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`Soltero and Ekwurlbe Innovations in Pharmaceutical Technology the
`Oral Delivery of Protein and Peptide Drugs. 2001 1-106-110.
`Still, J. Gordon, Diabetes/Metabolism Research Reviews, Develop-
`ment of Oral Insulin: Progress and Current Status, 2002, vol. 18,
`Suppl 1, pp. S29-S37.
`Veronese F. M Biomaterials Peptide and Protein Pegylation: A
`Review of Porblems and Solutions 2001 22 5 405-417.
`English abstract of JP 2004535442, Sep. 16, 2004.
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`
`

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`US 8,536,122 B2
`
`1
`ACYLATED GLP-1 COMPOUNDS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a Continuation of copending U.S. appli-
`cation Ser. No. 11/908,834, filed Sep. 17, 2007, which is a35
`USS.C. §371 national stage application of International Patent
`Application PCT/EP2006/060855 (published as WO 2006/
`097537), filed Mar. 20, 2006, which claimedpriority of Euro-
`pean Patent Application 05102171.5, filed Mar. 18, 2005; this
`application further claims priority under 35 U.S.C. §119 of
`USS. 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-
`fying the structure of glucagon-like peptide 1 (GLP-1) com-
`poundsin order to provide a longer durationofaction in vivo.
`WO 96/29342 discloses peptide hormonederivatives wherein
`the parent peptide hormonehas 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 whereinat least
`one aminoacid 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 supposedto react
`with blood components to form conjugates and therebyalleg-
`edly 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.
`
`Manydiabetes 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 compoundsare expected to be the
`first injectable product these patients will be administered, the
`fear of injections may becomea serious obstacle for the
`widespreaduseofthe clinically very promising GLP-1 com-
`pounds. Thus, there is a need to develop new GLP-1 com-
`pounds which can be administered less than oncedaily, e.g.
`once every secondorthird day preferably once weekly, while
`retaining an acceptable clinicalprofile.
`
`SUMMARYOF THE INVENTION
`
`The invention provides a GLP-1 analog having a modifi-
`cation of at least one non-proteogenic aminoacid residue in
`positions 7 and/or 8 relative to the sequence GLP-1(7-37)
`(SEQ ID No 1), whichis acylated with a moiety to the lysine
`residue in position 26, and where said moiety comprises at
`least two acidic groups, wherein one acidic groupis attached
`terminally.
`The present invention also provides pharmaceutical com-
`positions comprising a compound according to the present
`
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`invention and the use of compoundsaccording to the present
`invention for preparing medicaments for treating disease.
`The invention provides a methodfor 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
`encodedby the genetic code such as D-alanine and D-leucine,
`Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid),
`Tle (tert-butylglycine), B-alanine, 3-aminomethyl benzoic
`acid, anthranilic acid.
`The 22 proteogenic aminoacidsare:
`Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Cys-
`tine, Glutamine, Glutamic acid, Glycine, Histidine, Hydrox-
`yproline, Isoleucine, Leucine, Lysine, Methionine, Phenyla-
`lanine, Proline, Serine, Threonine, Tryptophan, Tyrosine,
`Valine.
`
`Thusa non-proteogenic amino acid is a moiety which can
`be incorporated into a peptide via peptide bondsbutis not a
`proteogenic amino acid. Examples are y-carboxyglutamate,
`omithine, 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 (a-ami-
`noisobutyric acid), Abu (a-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, a-fluoromethyl-histidine, oa-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 aminoacid residues have been deleted from the
`peptide and or wherein one or more aminoacid residues have
`been addedto the peptide. Such addition or deletion of amino
`acid residues can take place at the N-terminalof the peptide
`and/or at the C-terminal of the peptide. A simple system is
`often used to describe analogues: For example [Arg**]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 aminoacid residue,i.e. to the Gly*’. All aminoacids
`
`

`

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`US 8,536,122 B2
`
`3
`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 embodi-
`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 embodimentsof the
`invention a maximum of 8 aminoacids have been modified. In
`embodiments of the invention a maximum of 7 amino acids
`have been modified. In embodiments ofthe 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 aminoacids have been modified. In
`embodiments of the invention a maximum of 2 amino acids
`have been modified. In embodiments ofthe invention 1 amino
`acid has been modified.
`
`Theterm “derivative”as used herein in relation to a peptide
`means a chemically modified peptide or an analoguethereof,
`whereinat 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 thelike.
`An example ofa 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) (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
`embodimentthe GLP-1 peptide is an insulinotropic agent.
`The term “insulinotropic agent” as used herein means a
`compound whichis an agonist of the homan GLP-1 receptor,
`i.e. a compound which stimulates the formation of cAMP in
`asuitable medium containing the human GLP-1 receptor (one
`such medium disclosed below). The potency of an insulino-
`tropic agent is determinedby calculating the EC.,, 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
`DMEMmedia with the addition of 100 [U/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
`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 suspendedin 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
`AlphaScreen™ cAMP Kit (Perkin Elmer Life Sciences).
`Incubations were carried out in half-area 96-well microtiter
`
`plates in a total volume of 50 wL buffer 3 (50 mM Tris-HCl, 5
`mM HEPES, 10 mM MgCl, pH 7.4) and with the following
`additions:
`1 mM ATP,
`1 uM GTP, 0.5 mM 3-isobutyl-1-
`methylxanthine (BMX), 0.01% Tween-20, 0.1% BSA,6 ug
`membranepreparation, 15 g/mL acceptor beads, 20 ug/mL
`donor beads preincubated with 6 nM biotinyl-cAMP. Com-
`pounds to be tested for agonist activity were dissolved and
`diluted in buffer 3. GTP wasfreshly prepared for each experi-
`ment. The plate was incubatedin the dark with slow agitation
`for three hours at room temperature followed by counting in
`
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`the Fusion™instrument (Perkin Elmer Life Sciences). Con-
`centration-response curves were plotted for the individual
`compounds and EC., 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 compoundresistant to the
`plasma peptidase dipeptidyl aminopeptidase-4 (DPP-IV).
`The DPP-IV enzymein plasma is knownto be involvedin 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-HClbuffer, 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 performingthis analysis is: The mixtures are applied onto
`aVydac C18 widepore (30 nm pores, 5 um particles) 250x4.6
`mm column andeluted 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 oftheir peak areasrelatedto those of
`standards. The rate of hydrolysis of a peptide by dipeptidy]
`aminopeptidase IV is estimated at incubation times which
`result in less than 10% ofthe peptide being hydrolysed.
`The term “C,_,-alkyl” as used herein meansa saturated,
`branched,straightor 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 andthe like. The
`term “pharmaceutically acceptable” as used herein means
`suited for normal pharmaceutical applications, 1.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
`meansa product comprising an active compoundora salt
`thereof together with pharmaceutical excipients such as
`buffer, preservative, and optionally a tonicity modifier and/or
`astabilizer. 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
`
`managementandcare of a patient having developedthe dis-
`ease, condition or disorder. The purpose of treatment is to
`combatthe disease, condition or disorder. Treatment includes
`
`

`

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`
`US 8,536,122 B2
`
`5
`the administration of the active compounds to eliminate or
`control the disease, condition or disorderas wellas to allevi-
`ate the symptomsor complications associated with the dis-
`ease, condition or disorder.
`In another aspect the present invention relates to an acy-
`lated GLP-1 analoguethat can bind to albumin and the GLP-1
`receptor simultaneously.
`In another aspect the present invention relates to an acy-
`lated GLP-1 analoguethat bind to the GLP-1 receptor with an
`affinity below 100 nM,preferable below 30 nM in the pres-
`ence of 2% albumin.
`
`In another aspect the present invention relates to an acy-
`lated GLP-1 analogue whichaffinity 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 albuminto 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 morepref-
`erable below 10 fold.
`
`20
`
`The term “albumin binding moiety”as used herein means
`a residue which binds non-covalently to human serum albu-
`min. The albumin binding residue attached to the therapeutic
`polypeptide typically has an affinity below 10 uM to human
`serum albumin and preferably below 1 uM.A rangeof albu-
`min binding residues are known amonglinear 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 bindingresi-
`due with a chemical moiety which comprises at least 5 non-
`hydrogen atoms where 30-50% ofthese are either N or O.
`The term “acidic groups” as used herein means organic
`chemical groups whichare fully or partly negatively charged
`at physiological pH. The pKavalue of such groupsis below 7,
`preferable below 5. This includes but is not limited to car-
`boxylic acids, sulphonic acids, phosphoric acids or heterocy-
`clic ring systems whicharefully or partly negatively charged
`at physiological pH.
`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
`formulaII is attached with the group B attachedat 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 regardedas attachment bondsandnot ending
`in methylene groups unless stated.
`
`40
`
`45
`
`0.
`
`OH
`
`In the formulas below
`
`H
`NH,—H—N
`
`H;C
`
`CH;
`
`means the H,N-His-Aib-N-terminal of the GLP-1 analogue.
`In an embodimentthe invention provides a GLP-1 analog
`acylated with a lipophillic albumin binding moiety contain-
`ing at least two free acidic chemical groups attached via anon
`natural amino acid linker to the lysine residue in position 26.
`In an embodiment, the term free acidic chemical groupsis
`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 analogis 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 No1), and where said acylationis a diacid attachedto the
`lysine residue in position 26 optionally via a non natural
`amino acid hydrophilic linker.
`In an embodimentof 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 No1), whichis acylated with a moiety to the
`lysine residue in position 26, and where said moiety com-
`prises at least two acidic groups, wherein one acidic group is
`attached terminally.
`An embodiment provides a GLP-1 analog accordingto the
`above embodiment, wherein the moiety attached in position
`26 comprises a hydrophilic linker.
`An embodiment provides a GLP-1 analog accordingto the
`above embodiments, wherein the hydrophilic linker com-
`prises at least 5 non-hydrogen atoms where 30-50% of these
`are either N or O.
`An embodimentprovides 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 accordingto the
`above embodiment, wherein the albumin binding moiety is a
`linear or branchedlipophilic moiety containing 4-40 carbon
`atomshaving a distal acidic group.
`An embodimentprovides a GLP-1 analog according to any
`of the above embodiments, wherein the acylated moiety is
`B—U'," where U'is selected from
`
`

`

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`
`US 8,536,122 B2
`
`-continued
`0
`
`N
`
`WNON
`
`Oo
`
`O
`
`Ny
`
`0
`
`Oo
`
`yo Oohy
`
`O
`
`oO
`
`oO
`
`0
`
`H
`
`oO
`
`oO
`
`HRARoO
`.
`pd AA
`Wn,Fr anal
`K,
`H
`zm KARAettoO
`
`0
`
`~mrARe
`
`oO
`
`OH
`
`oO
`
`OH
`
`x
`W
`Sy
`
`eenSy
`
`Le 1yte™ NK
`
`
`
`N
`
`°
`
`
`
`oTNo
`
`>
`
`COOH
`
`COOH
`
`°
`
`a
`
`mis 0, 1, 2, 3, 4, 5, or 6,
`nis1,2o0r3
`
`sis 0, 1, 2, or 3,
`
`tis 0, 1,2,3,or4
`
`.
`pis 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
`19, 20, 21, 22, or 23;
`:
`24:
`and where B is an acidic group selected from
`
`ON so
`
`,
`
`0
`
`0
`
`H
`
`'
`
`oO
`
`oO
`
`d
`
`™
`
`-continued
`
`qt
`
`YN
`
`HO
`
`HO
`
`where lis 12, 13, 14, 15, 16, 17, 18, 19 or20;
`
`:
`:
`.
`An embodimentprovides a GLP-1 analog according to any
`of the above embodiments, which is a compound of formula
`1 (SEQ ID No. 2):
`
`55
`
`60
`
`65
`
`

`

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`
`US 8,536,122 B2
`
`10
`
`Formula I
`
`Xaa7-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa| 6-Ser-Xaajg-Xaajo-Xaao9-Glu-Xaap7-
`0
`
`Xaaz3-Ala-Xaaj5-——tJL-Xaap7-Phe-Ile-Xaazo-Trp-Leu-Xaa3z3-Xaaz4-Xaa35-Xaaz6-Xaaz7-
`
`H
`
`B—U'—NH
`
`wherein
`
`Xaa, is L-histidine, imidazopropionyl, a-hydroxy-histidine,
`D-histidine, desamino-histidine, 2-amino-histidine, B-hy-
`droxy-histidine,
`homohistidine,
`N“-acetyl-histidine,
`N®-formyl-histidine, oa-fluoromethyl-histidine, a-methyl-
`histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylala-
`nine
`
`Xaa, is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocy-
`clopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic
`acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclo-
`hexyl) carboxylic acid,
`(1-aminocycloheptyl) carboxylic
`acid, or (1-aminocyclooctyl) carboxylic acid;
`Xaa,, 1s Val or Leu;
`Xaa,, is Ser, Lys or Arg;
`
`15
`
`20
`
`25
`
`Xaa,, is Tyr or Gln;
`Xaay, is Leu or Met;
`Xaa,, 1s Gly, Glu or Aib;
`Xaa,, 1s Gln, Glu, Lys or Arg;
`Xaa,; is Ala or Val;
`Xaa,, is Glu or Leu;
`Xaa,, is Ala, Glu or Arg;
`Xaa,;, 1s Val or Lys;
`Xaa,, 1s Lys, Glu, Asn or Arg;
`Xaa,, 1s Gly or Aib;
`Xaa;, 1s Arg, Gly or Lys, or is absent;
`Xaa,, is Gly, Ala, Glu, Pro, Lys,or is absent;
`and B and U' together is the acylated moiety, where U' is
`selected from
`
`HO.
`
`oO
`
`NONON MS
`
`mz
`
`:
`
`Zi
`
`\
`
`JS
`
`

`

`Case 1:22-cv-00023-JPB Document 2-3 Filed 03/18/22 Page 9 of 71 PageID #: 132
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`
`US 8,536,122 B2
`
`11
`
`12
`
`continued
`O
`K
`WW
`x5oh
`. WNNR
`Sy
`oN
`‘
`x
`SyNIN ~~~,
`
`O
`
`0
`
`oO
`
`0
`
`ro yw eThe
`
`°
`
`Oo
`
`oO
`
`oO
`
`Oo
`
`Sy
`
`COOH
`N
`
`oO
`
`OH
`
`Sy
`
`oO
`
`oO
`
`COOH
`
`O
`
`COOH
`
`0
`
`tL™N
`
`30
`
`O
`
`,
`
`pou
`
`m is 0, 1, 2, 3, 4, 5, or 6,
`nis1,2o0r3
`sis 0, 1, 2, or 3,
`tis 0, 1,2, 3,or4
`pis 1, 2,3, 4,5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16,17, 18, 35
`19, 20, 21, 22, or 23;
`and where B is an acidic group selected from
`
`-continued
`
`H
`N
`
`O
`
`9
`
`:
`
`oO
`
`HO
`
`9
`
`HG
`
`HO
`
`!
`
`q
`
`aWN. HO SN and
`
`0
`
`0
`
`O
`
`O
`
`40
`
`where1is 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):
`
`Xaa7-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa6-Ser-Xaag-Xaaj9Xaa7o-Glu-Xaaz-
`
`Formula I
`
`0
`
`Xaaz3-Ala-Xaay5-—tJ-Xaay7-Phe-Ile-Xaa3o-Trp-Leu-Xaa33-Xaa3z4-Xaaz5-Xaaz6-Xaaz7-Naazg
`
`H
`
`

`

`Case 1:22-cv-00023-JPB Document 2-3 Filed 03/18/22 Page 10 of 71 PageID #: 133
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`
`US 8,536,122 B2
`
`13
`The formula II is identical to formula J as stated in an
`embodiment above, where the moiety B—Uis 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 formulaII each of the Xaa’s has the following meaning:
`Xaa,
`is L-histidine, D-histidine,
`desamino-histidine,
`2-amino-histidine, B-hydroxy-histidine, homohistidine, N*-
`acetyl-histidine, o-fluoromethyl]-histidine, o-methy]-histi-
`dine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
`Xaa, is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopro-
`pyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid,
`(1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)
`carboxylic acid, (1-aminocycloheptyl)