Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 1 of 73 PageID #: 51
`
`
`
`
`
`Exhibit B
`
`
`
`
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 2 of 73 PageID #: 52
`eeeAAT
`
`US008129343B2
`
`a2) United States Patent
`US 8,129,343 B2
`(0) Patent No.:
`Mar.6, 2012
`(45) Date of Patent:
`Lau et al.
`
`(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 ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 682 days.
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`WO 00/3433 1
`WO 02/46227
`W00246227
`WO 02/098446
`WO 03/040309
`WO 2004/065621
`WO 2004/099246
`WO 2005/014049
`WO20050 14049
`WO 2005/027978
`WO2005027978
`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)
`
`PCTFiled:
`
`Mar. 20, 2006
`
`(86) PCT No::
`
`PCT/EP2006/060855
`
`§ 371 (©),
`(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) oes eeeeceecteteeeseneneees 05102171
`
`(51)
`
`Int. Cl.
`(2006.01)
`AGIK 38/26
`(2006.01)
`AG6IP 3/10
`(2006.01)
`AG6IP 7/12
`(2006.01)
`CO7K 14/605
`(52) U.S. Cd ices ceenseseecneeenenseecenesenees 514/11.7
`(58) Field of Classification Search «0.0.0.0... None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,545,618 A
`6,268,343 B1*
`6,528,486 B1*
`2007/0203058 Al*
`
`8/1996 Buckley etal.
`.....0.0... 514/4.8
`7/2001 Knudsen etal.
`3/2003 Larsenetal. ou... 514/6.8
`8/2007 Lauetal.
`
`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
`WO 91/11457
`8/1991
`WO 96/29342
`9/1996
`WO9629342
`9/1996
`WO 98/08871
`3/1998
`W09808871
`3/1998
`WO 99/43708
`9/1999
`W09943708
`9/1999
`W00034331
`6/2000
`WO 00/699 11
`11/2000
`WO000699 11
`11/2000
`
`OTHER PUBLICATIONS
`
`Simonovsky et al. Poly(ether urethane)s incorporating long alkyl
`side-chains with terminal carboxy groupsasfatty 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 ofAlbmnin,” Biomaterials, 2001, vol. 22, p. 3015-
`3023.
`
`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 ofthe 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, “Developmentof Oral Insulin: Progress and CurentStatus,”
`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.
`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 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 compoundsand therapeutic uses thereof.
`
`6 Claims, No Drawings
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 3 of 73 PageID #: 53
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 3 of 73 PagelD #: 53
`
`US 8,129,343 B2
`
`1
`ACYLATED GLP-1 COMPOUNDS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a 35 U.S.C. §371 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. §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-
`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 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 aminoacidresidue.
`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 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.
`
`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
`invention and the use of compoundsaccordingto the present
`invention for preparing medicaments for treating disease.
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`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,
`Cystine, Glutamine, Glutamic acid, Glycine, Histidine,
`Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine,
`Phenylalanine, 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
`for which the optical isomer is not stated is to be understood
`to mean the L-isomer. In embodiments of the invention a
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 4 of 73 PageID #: 54
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 4 of 73 PagelD #: 54
`
`US 8,129,343 B2
`
`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 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
`addiditions:
`1 mM ATP,
`1 uM GTP, 0.5 mM 3-isobuty]-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
`the Fusion™instrument (Perkin Elmer Life Sciences). Con-
`centration-response curves were plotted for the individual
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`4
`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, isohexy], cyclohexane andthelike.
`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
`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
`the administration of the active compounds to eliminate or
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 5 of 73 PageID #: 55
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 5 of 73 PagelD #: 55
`
`US 8,129,343 B2
`
`5
`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.
`
`10
`
`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.
`
`25
`
`30
`
`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 4
`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
`
`40
`
`groups are to be regardedas attachment bondsandnot ending
`in methylene groups unless stated.
`
`0
`
`OH
`
`O
`
`VA YY
`
`H
`
`H
`
`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 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 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
`
`WONONA
`H
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 6 of 73 PageID #: 56
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 6 of 73 PagelD #: 56
`
`US 8,129,343 B2
`
`7
`
`8
`
`-continued
`Oo
`
`HSftty
`me Oo
`"
`O
`
`OH
`
`0.
`
`q
`
`O
`
`H
`
`0
`
`Ny eeeeH
`
`oO
`
`im
`
`O
`
`QO
`
`0.
`
`OH
`
`oO
`
`NH
`
`\F
`8
`
`Sy
`
`Ny
`
`COOH
`Sy
`
`O
`
`OH
`
`Sy
`
`r WNTR KW
`
`H
`
`O
`
`O
`
`6NS
`
`QO
`0J °
`x
`
`SNONO
`
`°
`
`N
`
`O
`
`O
`
`QO
`
`i
`N
`O
`N
`
`yt—™ O
`N
`oO
`O77NS
`
`COOH
`
`O
`
`COOH
`
`O
`
`s
`
`"
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 7 of 73 PageID #: 57
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 7 of 73 PagelD #: 57
`
`US 8,129,343 B2
`
`10
`N°*-formyl-histidine, o-fluoromethyl-histidine, a-methyl-
`histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylala-
`nine
`
`5
`
`15
`
`20
`
`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,, is Val or Leu;
`Xaa,, is Ser, Lys or Arg;
`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;
`Xaaz, 1s Ala, Glu or Arg;
`Xaa,;, 1s Val or Lys;
`Xaa,, 1s Lys, Glu, Asn or Arg;
`Xaa,, 1s Gly or Aib;
`
`Formula I
`
`9
`
`mis 0, 1, 2, 3, 4, 5, or 6,
`nis1,2o0r3
`sis 0, 1, 2, or 3,
`tis 0, 1, 2,3, or 4
`pis 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,
`19, 20, 21, 22, or 23;
`and where B is an acidic group selected from
`
`,
`
`H
`N
`
`HOo~
`
`and
`
`]
`
`O
`
`O
`
`oeoO
`
`oO
`
`oO
`
`HN
`
`]
`
`O
`
`O
`
`HO
`
`HO
`
`°
`
`HO
`
`where 1 is 12, 13, 14, 15, 16, 17, 18, 19 or 20;
`An embodimentprovides a GLP-1 analog according to any
`of the above embodiments, which is a compoundof formula
`1 (SEQ ID No. 2):
`
`Xaa7-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaaj6-Ser-Xaajg-Xaaj9-Xaa79-Glu-Xaaz2-
`0
`
`H
`
`KaaseXaasNXanPeleXngrLeeXanXaneXa5-Xa36-XA437-haze
`
`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,
`
`65
`
`Xaa,, is Arg, Gly or Lys, or is absent;
`Xaa,- 1s Gly, Ala, Glu, Pro, Lys, or is absent;
`and B and U' together is the acylated moiety, where U' is
`selected from
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 8 of 73 PageID #: 58
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 8 of 73 PagelD #: 58
`
`11
`
`0
`
`OH
`
`O
`
`US 8,129,343 B2
`
`12
`
`VA YN
`
`H
`
`H
`
`P
`
`oO
`
`Ho.
`
`|>!
`
`‘So
`
`Oo
`
`~y
`
`oO
`
`0.
`
`OH
`
`0.
`
`H
`
`OH
`
`O
`
`N
`
`H
`
`o
`
`oO
`
`O
`
`oO
`
`H
`
`LNON ‘aaa
`Hteeey
`me 0
`.
`5
`Sy aeH
`H
`1
`H
`i
`~y Naot ALAA
`Voy
`(i
`ol)
`yy
`' WNerr YOON
`Wa
`
`O
`
`H
`
`0
`
`oO
`
`7
`
`0
`
`H
`
`oO
`
`i \
`o
`0
`
`O
`
`oO
`
`oO
`
`SyINAh
`
`Oo
`
`Sy
`
`-
`
`COOH
`
`Ny
`
`O
`
`OH
`
`Sy
`
`Oo
`
`O
`
`H
`
`oO
`
`N mee, NoON
`
`:
`
`Oo
`
`N
`
`COOH
`
`O
`
`COOH
`
`0
`
`N
`
`N
`
`O
`
`VN
`
`N
`
`“Nr
`
`O
`
`s
`
`i
`O77NS ye
`
`oO.
`
`"
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 9 of 73 PageID #: 59
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 9 of 73 PagelD #: 59
`
`US 8,129,343 B2
`
`13
`
`mis 0, 1, 2, 3, 4, 5, or 6,
`nis 1,2o0r3
`sis 0, 1, 2, or 3,
`tis 0, 1, 2,3, or 4
`pis 1, 2,3, 4,5, 6,7, 8, 9, 10, 11,12, 13, 14, 15,16,17,18,
`19, 20, 21, 22, or 23;
`and where B is an acidic group selected from
`
`14
`acetyl-histidine, o-fluoromethyl-histidine, o-methyl-histi-
`dine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
`
`Xaa, is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopro-
`5 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
`
`!
`
`oO
`
`oO
`
`H
`
`UN
`
`HO
`
`!
`
`oO
`
`oO
`
`and
`
`10
`
`Xaa,, 1s Val or Leu;
`
`Xaa,, is Ser, Lys or Arg;
`
`HO
`
`oO
`
`oO
`
`aina15 Xaayo isTyrorGln;
`:
`Xaa5, is Leu or Met;
`9 9
`Oo
`Xaa,, is Gly, Glu or Aib;
`Xaa,, is Gln, Glu, Lys or Arg;
`Xaa,, is Ala or Val;
`
`.
`.
`where| 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):
`
`20
`
`Xaa,, is Glu or Leu;
`
`Xaa7-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaaj6-Ser-Xaajg-Xaaj9-Xaa79-Glu-Xaaz2-
`oO
`
`H
`Xaaz3-Ala-Xaags5-N
`
`Xaaz7-Phe-Ile-Xaa3-Trp-Leu-Xaa3z3-Xaa3zq4-Xaaz5-Xaaz6-Xaaz7-Naazg
`
`Formula II
`
`—oO
`
`Xaa3q is Ala, Glu or Arg;
`The formula II is identical to formula I as stated in an
`embodiment above, where the moiety B—Uis replaced by 60 Xaag, is Val or Lys;
`B—U"'. The difference being only the incorporation of the
`Xaa;, is Lys, Glu, Asn or Arg;
`carboxy group in the U' relative to U, which is without the
`Xaa,, is Gly or Aib;
`attaching carboxygroup.
`Xaa,, is Arg, Gly or Lys, or is absent;
`In formulaII each ofthe Xaa’s has the following meaning:
`65 %4437 18 Gly, Ala, Glu, Pro, Lys, or is absent;
`Xaa,
`is L-histidine, D-histidine,
`desamino-histidine,
`Xaa3x is Lys, Ser, amide oris absent,
`2-amino-histidine, B-hydroxy-histidine, homohistidine, N*-
`and where U is a spacer selected from
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 10 of 73 PageID #: 60
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 10 of 73 PagelD #: 60
`
`US 8,129,343 B2
`
`16
`
`oO
`
`H
`
`SUNNP / NOONON,
`
`15
`
`oO
`
`oO.
`
`OH
`
`oO
`
`SA,
`
`oO.
`
`OH
`
`oO
`
`H
`
`SNARRES ANJ,
`AK,
`Mn“ Nw > SNNP
`
`
`
`
`
`WONONSNON
`
`oO
`
`where n is 12, 13, 14, 15, 16,17 or 18
`
`lis 12, 13, 14, 15, 16, 17 or 18,
`
`mis 0, 1, 2,3, 4,5, or 6,
`
`sis 0, 1, 2, or 3,
`
`30
`
`35
`
`:
`P is 3,4, 5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
`20, 21, 22, or 23;
`
`0
`
`OH
`
`O
`
`VA YY
`
`H
`
`H
`
`P
`
`oO
`
`and where B is an acidic group selected from
`
`HO
`
`and
`
`q
`
`“
`
`.
`
`a HO na
`
`oO
`
`oO
`
`In the embodiments below whenreferring to U' in formula
`] it is to be understoodas also referring to formula II and U,
`with the only difference being the carboxy group.
`An embodiment provides a GLP-1 analog accordingto the
`embodiments above, wherein U'is selected from
`
`O
`10.||
`
`So
`
`y
`
`N
`
`LNON
`
`0
`
`O
`
`O
`
`xNN .
`
`oO
`
`Oo
`
`N
`
`oO
`
`Sy
`
`HO.
`
`SS0 YOONor
`
`0
`
`*
`
`#H
`
`oO
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 11 of 73 PageID #: 61
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 11 of 73 PagelD #: 61
`
`17
`
`US 8,129,343 B2
`
`-continued
`
`18
`
`OH
`
`OH
`
`0
`
`H
`
`O
`
`H
`
`Oo
`
`Oo
`
`H
`
`O
`
`O
`
`SNSoNAaH
`Sy
`H
`1
`H
`i
`Sy Nt ALAKM
`Mv
`t
`ol
`Sy
`3 NON x™ ,
`Oo
`
`0
`
`na
`
`O
`
`yn
`oO
`Oo
`
`H
`
`oO
`
`0
`
`~N oANt
`
`\W
`
`H
`
`y
`
`O
`
`i]
`
`P
`
`in
`
`Ny
`
`COOH
`N
`
`OH
`
`O
`™ Zz
`
`O
`
`O
`
`HN wT NoON
`
`O
`
`.
`
`°
`
`°
`
`COOH
`
`oO
`
`COOH
`
`m is 2, 3, 4 or 5,
`
`nis lor2
`sis 0, 1, or 2,
`
`tis 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
`
`oO
`HOW|
`
`So
`
`oO
`
`woe,
`
`x
`
`N
`
`oO
`
`Vm“NZ
`
`oO
`
`O
`
`~~“NS W™No
`
`H
`
`O
`
`woo,SL “NNNWHyout
`
`H
`
`O
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 12 of 73 PageID #: 62
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 12 of 73 PagelD #: 62
`
`US 8,129,343 B2
`
`-continued
`
`oO
`
`20
`
`woeJLToAAoO
`oOwdTA“DN
`
`Oo
`
`OH
`
`oO
`
`oO
`
`roeTN Ns
`6 Jf,
`
`oO
`
`0
`
`0
`
`wor.HL VW
`:
`na
`
`ol)
`qu
`> LNON ; ONH
`
`oO
`
`O
`
`O
`
`oO
`
`0
`
`°o
`
`0
`q
`wor,J VW
`“ a A WNANY IN
`wk,
`
`Oo
`
`Oo
`
`COOH
`
`asUnaihnry
`0AATfL.wey
`
`HO
`oO
`
`°
`
`N
`
`o
`
`HO
`
`O
`
`‘OH
`
`oT
`
`O
`
`O
`
`oO
`
`O
`
`N
`
`O
`
`OH
`
`O
`
`NNv YN,
`
`H
`
`P
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 13 of 73 PageID #: 63
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 13 of 73 PagelD #: 63
`
`21
`where| is 14, 15, 16, 17, 18, 19 or 20;
`
`pis 1, 2, 3, 4, 7, 8, 9, 10, 11 or 12.
`
`sis 0, 1 or 2
`
`tis Oor 1;
`
`HOOC )°
`
`mz,
`
`oO
`
`Ho.| S
`
`0
`
`US 8,129,343 B2
`
`22
`An embodimentaccording to the above wherein
`where | is 14, 15, 16, 17 or 18
`pis 1, 2,3, 4 or 11;
`sis 0, 1 or 2;
`tisOor 1;
`An embodiment provides a GLP-1 analog accordingto the
`embodiment above, wherein B—U'is
`
`5
`
`Oo
`
`HVever™,aH
`
`Oo
`
`HS
`
`°
`
`NON OOoO
`
`O
`
`HOOC
`
`HOOC
`
`wm3°oom
`
`qo
`
`oO
`
`NANON } oOooO
`
`Oo
`
`mz,
`
`o
`
`OH
`
`OH
`
`HOOC
`
`HOOC
`
`HOOCCot 5(
`
`Oo
`
`OH
`
`§(
`
`°
`
`°
`
`ao
`
`Zz
`
`°
`
`OH
`
`HOOC )°
`
`Zz
`
`COOH
`
`oO
`
`°
`
`

`

`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 14 of 73 PageID #: 64
`Case 1:22-cv-00023-JPB Document 2-2 Filed 03/18/22 Page 14 of 73 PagelD #: 64
`
`US 8,129,343 B2
`
`23
`where | is 14, 15, 16, 17, 18, 19 or 20;
`pis 1, 2,3, or 4.
`sis 0, 1 or 2
`
`nis 0,1 or2
`An embodiment according to any of the above embodi-
`ments is wherein B is
`
`5
`
`24
`-continued
`
`Xl
`
`,
`
`SON
`°°
`0
`
`HO
`
`d
`
`HO
`
`
`
`
`
`HO
`
`6
`
`HO
`
`oO
`
`oO
`
`oO
`
`and 1 is 14, 16, 18 or 20;
`An embodimentprovides a GLP-1 analog according to any
`of the embodiments above, wherein B is
`
`15
`
`20
`
`where | is 14, 15, or 16.
`An embodimentprovides a GLP-1 analog according to any
`io
`and
`UN
`,
`HO
`
`! of the embodiments above, whereinsis 1.HO !
`6
`6
`6
`6
`An embodimentprovides a GLP-1 analog according to any
`H
`of the embodiments above, whereinnis 1.
`N
`.
`An embodimentprovides a GLP-1 analog according any of
`the embodiments above, wherein | is 14, 15 or 16; In embodi-
`ments I is 17, 18, 19 or 20. In embodiments J is 15, 16 or 17.
`In embodiments I is 18, 19 or 20. In embodiments J is 14. In
`embodiments I is 16. In embodiments I is 18. In embodiments
`Tis 20.
`An embodimentprovides a GLP-1 analog according to any
`of the embodiments above, wherein p is 1.
`An embodimentprovides a GLP-1 analog according to any
`of the embodiments above, wherein p is 2.
`An