ZA200602000B

ZA200602000B - Albumin-binding derivatives of therapeutic peptides

Info

Publication number: ZA200602000B
Application number: ZA200602000A
Authority: ZA
Inventors: Lau Jesper; Hansen Thomas Kruse; Madsen Kjeld; Bloch Paw; Doerwald Florencio Zaragoza; Johansen Nils Langeland
Original assignee: Novo Nordisk As
Priority date: 2003-09-19
Filing date: 2006-03-09
Publication date: 2007-05-30
Also published as: CN1867360A; CN100444898C; CN101380476A

Description

£692 20MaWO . 20 06/ 02000 e Novel GLP-1 derivatives

FIELD OF THE INVENTION

The present invention relates to novel derivatives of glucagon-like-peptide=-1 (GLP-1) and fragments thereof and analogues of such fragments which have a protracted profile of action and methods of making and using them. The invention furthermore relate sto novel deriva- tives of exendin and the use of such derivatives.

BACKGROUND OF THE INVENTION

Peptides are widely used in medical practice, and since they can be produced by recombinant

DNA technology it can be expected that their importance will increase also in the years to come. When native peptides or analogues thereof are used in therapy it is generally found that they have a high clearance. A high dearance of a therapeutic agent is inco nvenient in cases where it is desired to maintain a high blood level thereof over a prolonged oeriod of time since repeated administrations will then be necessary. Examples of peptides whiach have a high clearance are: ACTH, corticotropin-releasing factor, angiotensin, calcitonin _, insulin, glucagon, glucagon-like peptide-1, glucagon-like peptide-2, insulin-like growth factor- 1, insulin-like growth factor-2, gastric inhibitory peptide. growth hormane-releasing factor, pituita ry adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, scrratomedin, parathy- roid hormone, thrombopaoietin, erythropoietin, hypothalamic releasing facto rs, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opi ods and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, argininez deaminase, adenosine deaminase and ribonuclease. In some cases itis possible to inf uence the release profile of peptides by applying suitable pharmaceutical compositions, but this approach has various shortcomings and is not generatly applicable.

The number of known endogenous peptides and proteins with interesting biological activities is growing rapidly. also as a result of the ongoing exploration of the human genome. Due to their biological activities, many of these polypeptides could in principle bez used as therapeu- tic agents. Endogenous peptides are, however, not always suitable as drug candidates be- cause these peptides often have half-lives of few minutes due to rapid dezgradation by pepti- dases and/or due to renal filtration and excretion in the urine. The half-life of polypeptides in human plasma varies strongly (from a few minutes to more than one week). Similarly, the half-life of small molecule drugs is also highly variable. The reason for this strong vanability

6692.204-NO ® of plasma half-lives off peptides, proteins, or ather cormpounds is, however, n ot well un- derstood. Thus, there is a need to modify therapeutic compounds to provide longer duration of action in vivo while maintaining low toxicity and the rapeutic advantages.

Serum albumin has a half-life of more than one week , and one approach to increasing the

S plasma half-life of peptides has been to derivatize them peptides with a chemical entity that binds to serum album in.

Knudsen et al. (J. Mead. Chem. 2000, 43, 1664-1669) have shown that acylat ed GLP-1 pepti- des exhibit high recepmtor potency and a tenfold increaase of plasma half-life ir pigs.

Zobel et al. (Bioorg. A~fed. Chem. Lett. 2003, 13, 1513-1515) have shown tha t the plasma 10» half-life of an anticoagulant peptide in rabbits increase=d by 10-50 fold on deri vatization of the amine terminus with pehosphate ester based small mo-lecules binding to serurm albumin.

SUMMARY OF THE IENVENTION

The present imnvention relates to a compound which comprises a theram peutic polypep- tide linked to an alburmein binding residue via a hydrophi lic spacer.

The presenti nvention also relates to a compeound which comprises =3 therapeutic polypeptide linked to an albumin binding residue via a hydrophilic spacer that= separates the polypeptide and the al bumin binding residue with a ch emical moiety comprisi ng at least 5 non-hydrogen atoms where 30-50% of these atoms amre either N or O.

In one embodiment of this invention the spacer is defi_ned as -{CH2ID[(CH2)nE]m(CH,),Qq-, wherein

I, m and n inc3ependently are 1-20 and p is 0- 10,

Q is -Z-(CH3;) .(D[(CH2)aGlm(CH2)s, q is an intege=rin the range from 0 to 5, each D, E, amd G independently are selected from -O-, -NR>-, -N(COOR®)-, -PR*(0)-, and -P(OR®)(+Q)-, wherein R%, R*, R®, and R® mndependently represermt hydrogen or

C,.-alkyi,

Zis selected from -C(O)NH-, -C(O)NHCH,-, —OC(O)NH -, -C{O)NHCZ=H,CH,-. -C(O)CH;-, -C(O)CH=CH-, -(CH,),-. -C(O)-, -=C(O)O- ar -NHC(Q)}-, wherein sis 0 or 1 or a pharmaceutically acceptable salt or prodrug thereof.

The present in—vention also relates to a compound which has the formcla (I).

6692 204-WO ® A—W —B-—Y—therapeutic polypeptide (n wherein

Ais an albumin binding residue,

B is a hydrophilic spacer being ~(CH)D[{CH;).E]m(CH2),Qq-, wherein [. m and n independently are 1-20 and pis 0-10,

Q is -Z-(CH2)D[(CH2)nG]m(CHa),-, q is an integer in the range from 0 to 5. each D, E, and G independently are selected from -O-, -NR®- , -N(COR"}-, -PR(O)-. and -P(OR®)(O)-, wherein R? R*, R®, and R® independently represent hydrogen or

C,¢-alkyl,

Z is selected from -C(O)NH-, -C(O)NHCH;-, -OC(O)NH -, -C{ Q)NHCH;CH,-, -C(O)CH2-, -C(O)CH=CH-, -(CH,)s-, -C(Q)-, -C{0)O- or -NHC=(0)-, wherein s is 0 or 1,

Y is a chemical group linking B and the therapeutic agent, and

Wis a chemical group linking A and 8.

The present invention also relates to a compound which has thes formula (11)

A—-W—B—Y -therapeutic polypeptide —y’'-g"-W —A * (i) wherein

A and A’ are albumin binding residues.

B and B’ are hydrophilic spacers independently selected from -(CH,)[D ((CH2)AE]m(CH2)p-Qq-. wherein

I. m and n independently are 1-20 and p 1s 0-10,

Q is -Z-(CH2:D{(CH2)G]m{CHa),-, q is an integer in the range from 0 to 5, each D, E, and G independently are selected from -O-, -NR>_, -N(COR*)-, -PR%0)-, and -P(OR®%)(0)-, wherein R®, R*, R®, and R® independently re=present hydrogen or

C,s-alkyl,

Z is selected from -C(O)NH-, -C{(O)NHCH,-, -OC(O}NH -, -C(-OQ)NHCH,CH,-, -C(O)CH;-, -C(O)CH=CH-, -(CH3)s-, -C(O)-, -C(O)O- or -NHC (0)-, wherein s is 0 or 1

Y is a chemical group linking B and the therapeutic agent, and

Y' Is a chemical group linking B™ and the therapeutic agent, and

W is a chemical group linking A and B, and

W's a chemical group linking A” and 8B".

66592.204-WO

PY 4

In anothe=r aspect the present invention rel ates to a compound which as the formula (nm

A. -W'-B—Y -therapeutic polypeptide (th) ; A wherein

A and A’ are albmumin binding residues,

Bis a hydrophilic spacer selected from -(CH;}D[(CH2)\Elm{CH2)p-Qq- wherein [, m andl n independently are 1-20 and p i s 0-10,

Q is -Z-«(CH2)D[(CH;):Glm( CHa), q is ani nteger in the range from 0 to 5, each D, E, and G are independently selected from -O-, -NR*-, -N(CCOR®)-, -PR%(O)-. and -P(COR®)(0)-, wherein R®, RY, R®, and R® independently represent hydrogen or

Cis-alkyyl,

Z is sele=ctled from -C(ONH-, -C(O)NHCH,-, -OC(OINH -, -C(OINHC=H,CH,-, -C(O)CH;-, -C(O)CH=CH-, -(CHz)s-, -C(O }-, -C(O)O- or -NHC{O)-, w~herein s is 0 or 1,

Y is a chermucal croup linking B and the therapeutixc agent, and

W" is a chemical group linking B with A and A’.

In anotler aspect the present invention relates to a compound comprising a hydro- philic spacer between a therapeutic peptide and ome or more albumin binding residue(s), said compound taving a protracted profile of action relative to the therapeutics polypeptide, where the alburmnin binding fraction as well as the Free fraction of said compowL_ind are both able to bind to ttne receptor mediating the effect of" the therapeutic polypeptides.

In one ermbodiment the hydrophilic spacer is an unbranched oligo eth=ylene glycol moiety with appr-opiate funtional groups at both terminals that forms a bridge between an amino group of che therapeutic polypeptide and a funtional group of the alburmin binding residue.

In anothezr aspect of the present invention time therapeutic polypeptide is a GLP-1 pep- tice.

6692 204- WO ® DEFINITIONS

In the present specification, the following terms have time indicated meaning .

The term “albumin binding residue” as used herein me=ans a residue which binds non- covalently to human serum albumin. The albumin binding residllue attached to the therapeutic 5 polypeptide typically has an affinity below 10 pM to human sercam albumin and preferably be- low 1 uM. A range of aitoumin binding residues are known amoeng linear and branched lipoho- phillic moieties containirg 4-40 carbon atoms, compounds witha a cyclopentanophenanthrene skeleton, peptides havirg 10-30 amino acid residues etc.

The term “hydro philic spacer” as used herein means a spacer that separates a peptides and an albumin binding residue with a chemical moiety which comprises at least 5 non- hydrogen atoms where 30-50% of these are either N or O.

The term “therapeutic polypeptide” as used herein measns a polypeptide which is being developed for therapeutic use, or which has been developed fcor therapeutic use.

The term “polypeptide” and “peptide” as used herein means a compound composed of atleast five constituent amino acids connected by peptide boneds. The constituent amino acids may be from the group of the amino acids encoded by the gen e€lic code and they may be natu - ral 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 cod=e are e.g. hydroxyproline, y- carboxyglutamate, ornit hine, phosphaserine, D-alanine and D-gglutamine. Synthetic amino ac- ids comprise amino acids manufactured by chemical synthesis , i.e. D-isomers of the amino ac — ids encoded by the genetic code such as D-alanine and D-leucsine, Aib (a-aminoisobutyric acid), Abu (a-aminobutysric acid), Tle (tert-butylglycine), B-alanisae, 3-aminomethyl benzoic acid, anthranilic acid.

The term “analo que” as used herein referring to a polypeptide means a modified pep- lide wherein one or more amino acid residues of the peptide h=ave been substituted by other amino acid residues and/or wherein one or more amino acid re=sidues have been deleted from the peptide and/or wherein one or more amino acid residues h-ave been deleted from the pep- tide and or wherein one or more amino acid residues have bee=n added to the peptide. Such addition or deletion of a mino acid residues can take place at trme N-terminal of the peptide and/or at the C-terminal of the peptide. A simple system is use d to describe analogues : For example [Arg*|GLP-1(Z-37)Lys designates a GLP-1 analogue wherein the naturally occuring lysine at pasition 34 has been substituted with arginine and a h=ysine residue has been added te the C-terminal (positiort 38). Formulae of peptide analogs and derivatives thereof are drawn using standard single [etter abbreviation for amino acids used «according to IUPAC-IUB no- menclature.

6692 20=1.WQ ® The term “derivative” as used herein in relation to a peptide me=ars a chemically modified p=ep- tide or an analogue thereof. wherein at least one substituent is mot present in the unmodified peptide or an analogue thereof, i.e. a peptide which has been covalently modified. Typical modifications are amides, carbohydrates, alky! groups, acyl growaps, esters and the like. An ex- ample of a derivative of GLIP-1(7-37} 1s N**-(y-Glu(N“-hexadecaaroy!)))- [Arg Lys*])GLP- 1(7- 37).

The term “GLP-1 peptide” &s used herein means GLP-1(7-37) ( SEQ ID No. 1), a GLP-1 arma- logue, a GLP-1 derivative or a derivative of a GLP-1 analogue. 8n one embodiment the GLFP-1 peptide is an insulinotropic agent.

The term “insulinotropic agent” as used herein means a compeound which is an agonist oefthe human GLP-1 receptor, i.e-. a compound which stimulates the formation of cAMP in a suit able medium containing thee human GLP-1 receptor. The potency of an insulinotropic agent is determined by calculatin g the ECgp value from the dose-resgoonse curve as described Boe- low.

Purified plasma membrane s from a stable transfected cell fine, BHK467-12A {tk-1513), ex- pressing the human GLP-1 receptor was stimulated with GLP-1 and peptide analogues, ard the potency of cAMP produaclion was measured using the AlphzaScreen™ cAMP Assay Kit from

Perkin Eimer Life Sciences .

A stable transfected cell line has been prepared at NN and a hicgh expressing clone was se- lected for screening. The cells were grown at 5% CO, in OMEN, 5% FCS, 1% Pen/Strep aand 0.5 mg/ml G418.

Cells at approximate 80% confluence were washed 2X with PBS and harvested with Versene, centrifuged 5 min at 1000 rpm and the supernatant removed. T he additional steps were all made on ice. The cell pelle t was homogenized by the Ultrathuraax for 20-30 sec. in 10 ml of

Buffer 1 (20 mM Na-HEPE 8, 10 mM EDTA, pH=7.4), centrifugead 15 min at 20.000 rpm arad the pellet resuspended in 1 0 mi of Buffer 2 (20 mM Na-HEPES , 0.1 mM EDTA, pH=7.4). Whe suspension was homogeni zed for 20-30 sec and centrifuged 15 min at 20.000 rpm. Suspe=n- sion in Buffer 2, homaogeni=ation and centrifugation was repeate=d once and the membranes were resuspended in Buffear 2 and ready for further analysis or =stored at -80°C.

The functional receptor asssay was carried out by measurering the peptide induced cAMP gpro- duction by The AlphaScreean Technology. The basic principle off The AlphaScreen Techno logy 1s a competition between e=ndogenous cAMP and exagenously added biotin-cAMP. The c=p- ture of CAMP is achieved boy using a specific antibody conjugated to acceptor beads. Formed cAMP was counted and measured at a AlphaFusion Microplate= Analyzer. The ECg values was calculated using the Grapi-Pad Prisme software.

6692.24€04 WO ® The term “GLP-2 peptide” as used herein means GLP-2(1-33), a GLP-2 analogue, a GLPP-2 derivative or a derivative of a GLP-2 analogue.

The term “exendin-4 peptide™ as used herein means exendin-4(1 -39), an exendin-4 analcogue. an exendin-4 derivative or a «derivative of an exendin-4 analogue. In one embodiment the= ex-

S endin-4 peptide is an insulinOtropic agent.

The terms “stable exendin-4 peptide” and “stable GLP-1 peptides” as used herein mean= chemically modified peptides derived from exendin-4(1-39) or GL_P-1(7-37), i.e. an analo-gue or a derivative which exhibits ar in vivo plasma elimination half-life of at least 10 hours in m an, as determined by the following rmethod. The method for determination of plasma eliminatior— half- life of an exendin-4 peptide ©r a GLP-1 peptide in man is : The peptide is dissolved in an iso- tonic buffer, pH 7.4, PBS or any other suitable buffer. The dose is injected peripherally, foref- erably in the abdominal or upper thigh. Blood samples for determination of active peptide are taken at frequent intervals, a nd for a sufficient duration to cover t he terminal elimination goarn {e.g. Pre-dose, 1, 2,3. 4, 5, ©, 7, 8, 10, 12, 24 (day 2), 36 (day 2), 48 (day 3), 60 (day 3)., 72 {day 4) and 84 (day 4) hours post dose). Determination of the co ncentration of active pe gptide is performed as described in Wilken et al., Diabetologia 43(51).A14-3, 2000. Derived pharmacoki- netic parameteres are calcu! ated from the concentration-time data for each individual su-bject by use of non-compartmentaxl methods. using the commercially available software WinN-onlin

Version 2.1 (Pharsight, Cary , NC, USA). The terminal eliminatior rate constant is estimaated by log-linear regression on the kerminal log-linear part of the concentration-time curve, and used for calculating the eliminatior half-life.

The term “DPP-IV protected” as used herein referring to a polypeptide means a gooly- peptide which has been chexmically modified in order to render said compound resistant tothe plasma peptidase dipeptidyl aminopeptidase-4 (DPP-1V). The DFPP-IV enzyme in plasma is known to be involved in the degradation of several peptide hormones, e.g. GLP-1, GLP--2, Ex- endin-4 etc. Thus, a conside=rable effort is being made to develop analogues and derivatives of the polypeptides susceptible to DPP-IV mediated hydrolysis in order to reduce the rate cf deg- radation by DPP-IV.

Resistance of a peptide to cdegradation by dipeptidyl aminopep tidase IV is determined by the following degradation assay :

Aliquots of the pep tides are incubated at 37 °C with am aliquot of purified dipe=ptidyl aminopeptidase IV for 4-22 hours in an appropriate buffer at pH 7-8 (buffer not being albu- min). Enzymatic reactions are terminated by the addition of trif uoroacetic acid, and th e pep- tide degradation products a re separated and quantified using HPLC or LC-MS analyssis. One method for performing this analysis is: The mixtures are applie d onto a Zorbax 300SES-C18 (30 nm pores, 5 pm particless) 150 x 2.1 mm column and eluted at a flow rate of 0.5 ml/min

6692.203--WO ® with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid (0% -100% acetonitrile o-ver 30 min). Peptides and th eir degradation products may be monitored by their absorbancez at 214 nm (peptide bonds) or 280 nm (aromatic amino acids), and are quantified by integration of their peak areas. The degradation pattern can be determined by using LC-MS where MS spectra of the separated peak can be determined. Percentaage intact/degraded compoumnd at a given time is used for eastimation of the peptides DPPIV s=tability.

A peptide is defined as [DPPIV stabilised when it is 10 time=s more stable than the natural peptide based on perceratage intact compound at a given ti me. Thus, a DPPIV stabilised

GLP-1 compound is at {east 10 times more stable than GL=>-1(7-37).

The term “C,s-alkzyl” as used herein means a saturated, branched, straight or cyclic hydrocarbon group havirg from 1 to 6 carbon atoms. Repressentative examples include, but are not limited to, methyB, ethyl, n-propy!, isopropyl, butyl, isobutyl, sec-butyl, fert-butyi, m- pentyl, isopenty!, neoperityl, tert-pentyl, n-hexyl, isohexyl, czyclohexane and the like. a5

DETAILED DESCRIPTION OF THE INVENTION

The present inveention relates to a compound which comprises a therapeutic poly pep- tide linked to an albumin binding residue via a hydrophilic spacer.

The present inv ention also relates to a compound which comprises a therapeutic polypeptide linked to an albumin binding residue via a hydr ophilic spacer that separates the polypeptide and the albumin binding residue with a chemical moiety comprising at least $ non-hydrogen atoms wh ere 30-50% of these atoms are either N or O.

In one embodiment of the is invention the spacer is defined &as -(CH)D(CH AE) (CH 2) Qq-. wherein

I, m and n indegoendently are 1-20 and p 1s 0-10,

Q is -Z-(CH2)DT(CHZ)sGlm(CH2)p-, q is an integer 1 n the range from 0 to 5, each D, E, and G independently are selected fron -O-, -NR>, -N(COR®)-, -PRZ(0)-, and -P(OR®O»-, wherein R*, R*, R®, and R°® independently represent hydroger or

Ci.s-alkyl,

Z is selected from -C(O)NH-, -C(O)NHCH,-, -OC(&D)NH -, -C(OC)NHCH,CH-, -C(O)CHyg-, -C(O)CH=CH-, -(CHy).-. -C(O)-. -C(O) O- or -NHC(O)-, wherein s 1s 0 or }

6692 Z2R04-WO @ or a pharmaceutically acceptable salt or prodrug theareof

The present irwvention also relates to a compmound which has the formula { I):

A—VVN—B—Y —therapeutic polypeptide (n wherein

A js an albumin bindimg residue,

B is a hydrophilic spa«cer being -(CH2)D[(CH2)nElm(CH2),Qq-. wherein

I, m and n independently are 1-20 and pis 0-10,

Q is -Z-(CH, > D[(CH,)Glm(CHz)p-, q is an intege=rin the range from 0 to 5, each D, E, amd G independently are selecte=d from -O-, -NR>-, -N(COR*D-, -PR*(0)-, and -P(OR®)C O)-. wherein R?, R*, R®, and R=° independently represent hy~drogen or

Ci.s-alkyl,

Zis selected from -C(O)NH-, -C(O)NHCH,;—, -OC({O)NH -, -C(O)NHCH,>CH,-, -C(O)CH,-, -C(0)CH=CH-, -(CH_)s-, -C(O}- , -C(O)O- or -NHC(O)-, wher=ein s is 0 or 1,

Y is a chemical group linking B and the therapeutic agent, and

W is a chemical group linking A and B.

The present irmvention also relates to a comp ound which has the formula ( 11)

A—W—B—¥-therapeutic polypeptide —vy -g’'-W'—A" (Hm wherein

A and A" are albumin binding residues,

B and B’ are hydroph ilic spacers independently sele=cted from -(CH )D {(CH WE] «{CH2)-Qq- wherein

I. m and n incdependently are 1-20 and p is =0-10,

Q is -Z-{CH2DD[(CH_).Glm(CHz)o-, q is an integexrin the range from 0 to 5, each D, E, amd G independently are selecte=d from -O-, -NR*-, -N(COR*D-, -PR*(0)-, and -P(OR®)C0)-. wherein R*, R*, R®, and R=? independently represent hydrogen or

C,s-alkyl,

Z is selected from -C(O)NH-, -C(O)NHCH,—, -OC(O)NH -, -C(O)NHCH,CH-, -C(O)CH,-, -«C{O)CH=CH-, -(CH;)s-, -C{O)-_. -C({O)O- or -NHC(O)-, wherein sis 0 or 1.

Y is a chemical groups linking B and the therapeutic =agent, and

6692.204-WQ

Y' is a chemical group linking B” and the therapeutic agent, and

W is a chemical group inking A and B, and

Wis a chemical gr oup linking A" and B".

In one embodiment of the invention Y' is selected from tlhe group consisting of -C(O)NRH-, -

NHC(O)-, -C{O)NHCH;-, -CH;NHC(O)-, -OC(O)NH -, -NEHC(0)O-, -C{(O)NHCH,-,

CH,NHC(O)-, -C(O JCH,-, -CH,C(0)-, -C(O)CH=CH-, -CH{=CHC(O)-, ~(CH,}s-, -C(O}-, -C(0)0-, -0C(Q)}-, —NHC(O)- and —C(O)NH-, wherein s iss 0 or 1.

In a further embodiment of the invention W' is selected f rom the group consisting of -

C(O)NH-, -NHC(O) —, -C(O)NHCH,-, -CH,NHC(O)-, -OC(®O}NH -, -NHC(O)O-, -C(O)CHS,-, -

CH,C(O)-, -C{O)CH4=CH-, -CH=CHC(O)-, -(CHjy)s~-, -C(O)-, -C(O)O-, -OC(Q)-, -NHC(O»)- and —C{O)NH-, wherein sis 0 or 1.

In another aa spect the present invention relates tos a compound which has the fo rmula {ny

A=\A"-B—Y~therapeutic polypeptide (111)

A wherein ’

A and A’ are albumin binding residues,

Bis a hydrophilic spacer selected from (CH D[(CH2)E Jn{CH2),-Qq- wherein

I, mand n independently are 1-20 and p is 0-10

Q is -Z-(CH2)iD[(CH2)aGlm(CHz)p~. gis an integer in the range from 0 to 5, each D, E. and G are independently selected from -O-, -NR>-, -N(COR®)-, -PR¥0)-, and -P(ORRY)(Q)-. wherein R®, R®, R®, and R® independently represent hydrogesn or

Cis-alkyl,

Z is select ed from -C(O)NH-, -C(O)NHCH,-, -OCT(O)NH -, -C(O)NHCH,CH,-, -C(O)CH,—, -C(O)CH=CH-, -(CH,),-, -C(O)-, -C(=0)O- or -NHC(Q)-, whereins isQor 1.

Ys a chemical gras up linking B and the therapeutic agert, and

Wis a chemical group linking B with Aand A".

In another aspect the present invention relates to a compound comprising a Faydro- philic spacer between a therapeutic peptide and one or rmore albumin binding residue (s), said compound hawing a protracted profile of action relattive to the therapeutic polypeotide,

6692 204-WO

Cy 1 where the albumin binding fraction as well as the faree fraction of said compot.nd are both able to bind to the receptor mediating the effect of the therapeutic polypeptid e.

In one embodiment the hydrophilic spacer 1 s an unbranched oligo ethylene glycol moiety with appropiate funtional groups at both ter minals that forms a bridge between an amino group of the therapeutic polypeptide and a Funtional group of the alburmin binding residue. in oree embodiment Y is selected from the= group consisting of -C{O)s NH-, -NHC(O)-, -C(O)NHCH,—, -CH,NHC(Q)-, -OC(QO)NH -, -NHC()O-, -C(O)NHCH,-, CH.NRHC(O)-, -C(O)CHz-, -CH,C(0)-, -C(O)CH=CH-, -CH=CHC(&))-, -(CH_),-, -C(O)-, -C{O )O-, -OC(O}-, -NHC(O)- and -C(O)NH-, wherein sis 0 or 1.

In armother embodiment W is selected frorm the group consisting of of -C{ONH-, -

NHC(O)-, -C{(-Q)NHCH,-, -CH,NHC(Q)-, -OC{O)NH -, -NHC(O)O-, -C{O)CH,—, -CH,C(0)-, -C(O)CH=CH -, -CH=CHC(Q)-, -(CH,j),-. -C{Q}-, -C (0)O-, -0C(0)-, -NHC(O)-- and —-C(O)NH-, wherein s is CO or 1.

In armother embodiment W'" is selected from the group consisting of —CONHEH— . —ClojeH— . (CHa), CH= , ansd TNHCIOIGNHC(O)CH O(CH,),0(CH,),NH— .whereinsis 0, 1o0r2.

In araother embodiment lis 1 or 2, n and =m are independently 1-10 and p is 0-10.

In aracother embodiment D is —O-. in armother embodiment of the invention E i 5 -0O-.

In ya=t another embodiment of the inventiom the hydrophilic spacer is -CH,O{(CH3;)} 20]lm(CH.2),Qq-, where mis 1-10, pis 1-3, and Q is -Z-CH,O{(CH;)20]m{CHa),-. in arother embodiment q is 1. in armother embodiment G is -O-.

In ye=t another embodiment of the inventior Z is selected from the gro up consisting of -

C(O)NH-, -C(«O)NHCH,-, and ~CC{O)NH-.

In ye=t another embodiment q is 0.

In armother embodiment | is 2.

In amother embodiment nis 2.

In yest ancther embodiment the hydrophilic spacer B is -[CH;CH Om. wm (CH2)oQq--

In ye=t another embodiment the hydrophilic spacer B is (CH )-O-[(CtH2)a-Olm-(CH2)o-{C(O)NH-(CH3)-O-[{ Ct2)a-Olm-(CHa2)p lo.

6692 204-WO ® y where |, m, n, and p independently are 1-5, and q is 0-5.

In yet anothe r embodiment =W-B-Y- is selected freom the group consisting of i 0 : MH

N oC N

A oc : w B Yo, 0] H oO

Agog Hamgom Ay

H H o ’ o) H o

H romero homo Famed

H H lo] oO

A y i i J

ASE No~ ~0 ~~ Noo hive eye RY , y o]

H y ©

SSO ~s Nao ~_o_AAl ~

N oY o N

H H o . 0 H oO

H

Mongo Lnsong Angad

H H

H

Og Foo Icom mg oh-

H H H o] o

In yet anothear embodiment >W"-B-Y- is

6692.204-WO 0 i 3 o =

NTT NS N ~~"

H H ! 0 O o !

Tr Oo

H

©

We B Y

In yet &another embodiment the athumun bicding residue A is selected frorra the group consisting of

Ho. 2

AS NEN NG NG o , © 1

ONT SS

© .

ONT TS TS TS TT

Ho rr

HO 0} where the chiral carbon atom is either R or S,

H

BOS SS SSS

I yh

HO 0 where the chiral carbon atom is either R or S,

6692 204- WO ® h

H

BESS SSN

I )

HO 0 where the chiral carbon atom is eith=er R or S, 0] } 0 PO

H,C i 3 NSS NSN le]

HO 0] where the two chiral carbon atoms i ndependently are either R or S,

Ss 0 0 OH

H

H,C N hedge aid gh ¢ N 0

HO [¢] where the two chiral carbon atoms i ndependently are either R or S, 0} H

H 2 ©

H,C N hdd di adh X

O

HO [o} where the two chiral carbon atoms sndependently are either L or D, 0

H

N

Sa Od

Co N0 where the chiral carbon atom is either Ror S, 0

R a ) fo]

HO” SO where the chiral carbon atom is eter Ror S,

Oo OH

O H Q ON

N a RR 3 H

Ho” So

6692 =204-WO ® where the two chiral casrbon atoms independently are either R or S, 0 , o ae oP ~ A, ~ : H

HO Yo where the two chiral ca rbon atoms independently are either R or S,

HC TSN SN NS

HC TS SN NS a a NE re

H,C :

TR Ca a re

I a NV A ae a Ne ™C : 0 wo As Fa aa Na Wa Ud

N

H

© 1

[0] os As Pe Ut Waa re Na rg

N

H o jo] " NT a a

H o .

HOS Seg

6692.204-"WO0O @® a "Ao

SN , =

OC N N N

HAL TT

Hos) TY

HN

HO

So 0

O pe ou

CH,

Sa

H,c-© ~ H :

H.C ! . ENN NNN

I ENS N

BOE

= = ’ : : fr fe Br ? Qo .

AA AAA Sao

FL El F HN io

FF F }

F_ F_F_F_F

F F F F F :

PIAA A

F F F

F Fe F F Fe 0 O oO ~~ AN ¥

H,C N’ ~~ 0 O Oo eA Ry

H,C N° ~~ 10 . 0 QO ~~ RY 0 .

Oo O ©

NY)

HOA Ss

H

0 .. and

6692 204-WO ® pa S

OI

H

HO o

In yet another embodiment the molar weight of the hydrophilic spacer is in the range from 80D to ‘#@ 000D or in the range from 80D to 300D.

In another embodiment of thes invention, the albumin binding residue is a lipophilic resi- due. in another embodiment the albumin binding residue is negatively charged at physio- logical pH. In another embodiment the albumin binding residue comprises a group which can be negatively charged. One preferre d group which can be negatively charged is a carboxylic acicd group. in another embodiment of the invention, the albumin binding residue binds non- covalently to albumin. In another em bodiment the albumin binding residue has a binding affinity towwards human serum albumin that &s below about 10 uM or below about 1 x M.

In yet another embodiment of the invention the albumin binding residue is selected fror a straight chain alkyl group, a t»ranched alkyl group, a group which has an w-carboxylic acied group, a partially or completely hydrogenated cyclopentanophenanthren e skeleton. in another embodiment the albumin binding residue is a cibacronyl residue.

In another embodiment the albumin binding residue has from 6 to 4 © carbon atoms, frorm 8 to 26 carbon atoms or from 8 to 20 carbon atoms.

In another embodiment the albumin binding residue is an acyl group selected from the grooup comprising CH,(CH)CO-, wh erein ris an integer from 4 to 38, preferably an integer fromm 4 to 24, more preferred selecte d from the group comprising CHa(CH;)sCO-,

CHR3{(CH;)sCO-, CH3(CH2)10CO-, CH=a{(CH,)1;,C0O-, CH;{CH1.C0-, CH3{CH;),eCO-, CHE5(CH,)1sCO-, CH3(CH3)2CO- and CH43(CH;)2CO-.

In another embodiment the albumin binding residue is an acyl group of a straight-chain or Branched alkane co, w-dicarboxylic acid.

In another embodiment the albumin binding residue is an acyl group selected from the greoup comprising HOOC(CH,),CO-, wherein s is an integer from 4 to 38, preferably an integer fromm 4 to 24, more preferred selected from the group comprising HOOC(CH 2),CO-,

HCO OC(CH,)1sC0O-, HOOC(CH2)1sCOO-, HOOC(CH,)2CO- and HOOC(CH;)2> CO-.

In another embodiment the =lbumin binding residue is a group of the formula

CH43(CH,),CO-NHCH(COOH)(CH;)>«CO-, wherein v is an integer of from 10 t.© 24.

6692.204-WO ® In anctter embodiment the albumin binding residue ts a group of the feormula

CHa(CH2)WCO-INHCH((CH,),COOH)CO-, where inw is an integer of from 8to 24. in another embodiment the albumin bincding residue is a group of the feormufa

COOH(CH,),CCO- wherein x is an integer of fron 8 to 24.

In anoter embodiment the albumin binding residue is a group of the { ormula -NHCH(COOH D){CH,)NH-CO(CH,),CH;, wherei ny is an integer of from 81to 1 8

In anotier embodiment of the invention the albumin binding residue is - a peptide, such as a peptide cosmprising less than 40 amino acicd residues. A number of small peptides which are albumin bimding residues as well as a methcd for their identification is fousnd in J. Biol

Chem. 277, 38 (2002) 35035-35043. in ancether embodiment of the inventior the albumin binding residue via spacer and linkers is attached to said therapeutic polypepticie via the e-amino group of a | ysine residue.

In ancether embodiment the albumin birding residue via spacer and {|inkers is attached to said therape utic polypeptide via an amino acid residue selected from cyste ine, glutamate and aspartate.

In one= embodiment of the present inve=ntion the therapeutic polypeptide is a GLP-1 peptide. in another embodiment of the invention the therapeutic polypeptide is a GLP-1 pep- tide comprising the amino acid sequence of thee formula (IV):

Xaar-Xaas-Glu -Gly-Thr-Phe-Thr-Ser-Asp-Xaa, g-5er-Xaa;s-Xaa,g-Xaa-Glu—Xaa,,-Xaag-Ala-

Xaazs-Xaas-Xaaz;-Phe-lle-Xaaz-Trp-Leu-Xaas;-Xaag-Xaass-Xaass-Xaas,- XZ aase-Xaasg-

Xaaso-Xaa, -Xcaa,2-Xa3,3-Xaa,-Xaa.s-Xaa.g

Formula (IV) (SEQ | ID No: 2) wherein

Xaay is L-histicBine, D-histidine, desamino-histicine, 2-amino-histidine, B-hyd roxy-histidine, homobhistidine, N°-acetyl-histidine, a-flugrometinyl-histidine, a-methyl-histidimae, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;

Xaag is Ala, Glmy, Val, Leu, lle, Lys, Aib, (1-amirocyclopropyl) carboxylic acid, (1- aminocyclobutwul) carboxylic acid, (t-aminocycicapentyl) carboxylic acid, (1-arminocyclohexyl) carboxylic acid , (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid:

Xaasis Valor Leu;

Xaa sis Ser, mys or Arg;

Xaasgis Tyr or Gin; Xaap is Leu or Met,

Xaaz is Gly, G {uorAib;

6692.204-WO ® Xaaps is Gln, Glu, Lys or Arg;

Xaays is Ala or Val;

Xaays is Lys, Glu or Arg;

Xaayr is Glu or Leu;

Xaagg is Ala, Glu or Arg;

Xaas; is Val or Lys;

Xaay, is Lys, Glu, Asn or Arg;

Xaazs is Gly or Aib;

Xaagg is Arg, Gly or Lys;

Xaayy is Gly, Ala, Glu, Pro, Lys, amide or is absent;

Xaajg is Lys, Ser, amide or is absent.

Xaayg is Ser, Lys, amide or is absent;

Xaayo is Gly, amide or is absent;

Xaay, is Ala, amide cr is absent;

Xaas, is Pro, amide or is absent;

Xaasz is Pro, amide or is absent;

Xaays is Pro, amide or is absent;

Xaass is Ser, amide or is absent;

Xaass is amide or is absent ; provided that if Xaazs. Xaaa, Xaas, Xaas, Xaas,, Xaad, Xaas,, Xaass or Xadss i1=s absent then each amino acid residue downstream is also absent. in another embodiment of the invention the polypeptide is a GLP-1 peptide compris- ing the amino acid sequence of formula (V): :

Xaa;-Xaas-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaag-Tyr-Leu-Glu-Xaag,-Xaa _x;-Ala-Ala-

Xaays-Glu-Phe-lle-Xaaae-Trp-Leu-Val-Xaay-Xaass-Xaadze-Xaas;-Xaass

Formula (V) (SEQ ID No: 3) wherein

Xaay is L-histidine, D-histidine, desaminao-histidine, 2-amino-histidine, f-hydroxy=-histidine, homonhistidine, N°-acetyl-histidine, a-flucromethy!-histidine, a-methyl-histidine, 3 - pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;

Xaag 1s Ala, Gly, Val, Leu, lle, Lys, Aib, {1-aminocyclopropyl) carboxylic acid, {1— aminocyclobutyl) carboxylic acid, (1-aminocyclopentyt) carboxylic acid, (1-aminccyclohexyl) carboxylic acid, (1-aminocycloheptyl} carboxylic acid, or (1-ammnocyclooctyl) car boxylic acid;

Xaag is Ser, Lys or Arg;

Xaag; is Gly, Giu or Aib;

6692.2C24.WO 2 0 0 9) / 0 2000 @ Xaaq is Gin, Glu, Lys or Arg:

Xaags is Lys. Glu or Arg;

Xaay is Ala, Glu or Arg;

Xaass 1s Lys, Glu or Arg; § Xaassis Gly or Aib;

Xaass is Arg or Lys;

Xaay; is Gly, Ala, Glu or Lys;

Xaass is Lys, amide or 1s absent.

In yet another embodirment of the invention the GLP-1 oeptide is selected from GLF- 1(7-35), GLP-1(7-36), GLP-1(7-36)-amide, GLP-1(7-37), GLP-1{7-38), GLP-1(7-39), GLP-1(7- 40), GLP-1{7-41) or an analogu e thereof.

In another embodiment the GLP-1 peptide is a fragment of a peptide selected from tlhe group comprising GLP-1(7-35), GLP-1(7-36), GLP-1(7-36)amide=, GLP-1(7-37), GLP-1(7-38®,

GLP-1(7-39), GLP-1(740) and GLP-1(7-41) or an analogue ther—eof.

In another embodiment of the invention the GLP-1 pept ide is GLP-1(A-B) wherein Ais an integer from 1 to 7 and B is an integer from 38 to 45 or an an -alogue thereof comprising o ne albumin binding residue attached via a hydrophilic spacer to the C-terminal amino acid residmue and, optionally, a second albumin binding residue attached to orme of the other amino acid re si dues. {n another embodiment the GLP-1 peptide comprises n-o more than fifteen amino ascd residues which have been exchanged, added or deleted as com pared to GLP-1(7-37) (SEQ ID

No. 1), or no more than ten amino acid residues which have bee n exchanged, added or de- leted as compared to GLP-1(7-37) (SEQ ID No. 1).

In another embodiment the GLP-1 peptide comprises n © more than six amino acid residues which have been exchanged, added or deleted as com pared to GLP-1(7-37) (SEQ ID

Ne. 1).

In another embodiment the GLP-1 peptide comprises n 0 more than 4 amino acid reasi- dues which are not encoded by the genetic code.

In another embodiment the GLP-1 peptide is a DPPIV protected GLP-1 peptide.

In another embodiment the compound according to thiss invention is DPPIV stabilised.

In another embodiment the GLP-1 peptide comprises a n Aib residue 1n position 8.

In another embodiment the amino acid residue in positieon 7 of said GLP-1 peptide ms selected from the group consisting of D-histidine, desamino-histi dine, 2-amino-histidine. B- 38 hydroxy-histidine, homohistidine, N%-acetyl-histidine , a-fluorome=thyl-histidine, a-methyl- histidine, 3-pyridylalanine, 2-py ridylalanine and 4-pyndylalanine=

6692.204-W © ® In anather embodiment the GLP-1 peptide is selected from thoe group consisting of

Arg>GLP-1(7-37),

Lys®Arg®3GLP-1(7-38), Lys ®*Arg® *GLP-1(7-38)-OH, Lys*Arg*>G LP-1(7-36),

Aib># GLP-1(7-37), Aib®* GLIP-1(7-37), Ab*Z GLP-1(7-37),

ABP Arg? ¥Lys®GLP-1(7-38), Aib®* Arg® Lys GLP-1(7-38),

Ab? Arg®HLys®GLP-1(7-38), Aib*#2* Arg®*Lys*GLP-1(7-38),

Aib®® Arg Lys ®GLP-1(7-38), AD? Arg®Lys*GLP-1(7-38),

Aib®® Arg®Lys®GLP-1(7-38), Aib** Arg®Lys®GLP-1(7-38),

Aib*#* Arg®Lys¥GLP-1(7-38). Aib>**Arg*Lys®GLP-1(7-38), Aib*?Amrg*Lys*GLP-1(7-38).

Ab*ZFAIaYLys®GLP-1(7-38), Aib**Ala¥Lys®GLP-1(7-38), Aib* “Ala” Lys*GLP-1(7-38),

Ab Lys GLP-1(7-37), Aib>3SLysGLP-1(7-37) and Aib**#Lys” GL P-1(7-38). in another embodiment the GLP-1 peptide is attached to said hydrophilic spacer via the amino acid residue in position 23, 26, 34, 36 or 38 relative to the asminao acid sequence

SEQ ID Nott.

In another embodiment the GLP-1 peptide is exendin-4 (SEQ 1D NO 4).

In another embodiment the GLP-1 peptide is ZP-10, i.e.

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-=mide (SEQ ID NO 5) in another embodiment the GLP-1 peptide is

HGEGTFTSDLSKQMEEEAVRL FIEWLKNGGX, wherein X = P or Y, ©r a fragment or an ana- logue thereof.

In another embodiment of the invention the GLP-1 peptide is

Arg", Leu®, GIn®, Lys® (N°-(y-aminobutyroyl(N®-hexadecanoyl))) Eendin-4-(7-45)-amide or

Arg®, Leu®, GIn*, Lys'® (N*-(y-aminobutyroyl(N“-hexadecanoyl))) Exendin-4-(7-45)-amide.

In another embodiment of the invention one albumin binding wesidue is attached to the

C-terminal amino acid residue of the GLP-1 peptide via the hydrophilicc spacer.

In another embaediment of the invention a second albumin birading residue is attached to an amino acid residue which is not the C-terminal amino acid residue of the GLP-1 peptide.

In another embodiment, the lipophilic substituent is attached tc the GLP-1 peptide by means of a hydrophilic spacer in such a way that a carboxyl group of tthe spacer forms an am- ide bond with an amino group of the GLP-1 peptide. {in another embodiment of the invention the compound is selected from the group consisting of

N°'7-(2-(2-(2-(dodecylamino)ethoxy)ethoxy)acetyl)-[Aib*?2**Lys*"|GL ¥P-1(7-37)amide

6692 204-WO ®

La gn § o} ER LH 2

I ten Pg ng oh

N : eee)

N®7.(2-(2-(2-(17-sulphohexad ecanoylamino)ethoxy)ethoxy)acetyl)-{Aib® #23 Lys®] GLP-& (7- 37)amide it

NE

[@]

Hy L = oF H [o] HC CH, iN

H~N" olf E-G-T-F-T-S-D— V-§-S-Y-L-E-N Q-A-A-K-E~F—i~m8-W-L-V-K-N" J-R-N NH,

H o on en 0 H gg H go pu

Hom Sy TTT

N=Y.(2-[2-(2-(15-carboxypent=adecanoylamino)ethoxy)ethoxy]-acetyl}-[Aib® 22% | ys] GLF=- 1(7-37)amide 0

HNN

- fo)

HNN 1

HC, CH lo]

HN N i E-G-T-F-T S-v-L-E-N 2 qa -K-E-F—|-A-W -L-V-K- Xo . N

NT Ge -T+5-D-V—S§- Na A-A N RN x NH, de

HC CH, HC CH, HOD mmm meme 0 0 . : N=V7-(2-(2-(2-(17-carboxyhept=adecanoylamino)ethoxy)ethoxy) acety)[Aib®?*** Lys*'|GLP- 1(7- 37)amide

Q

0)

HN ox . J [] o R, " tit Leos rmsmony ~s=s-v-i-e-d, Joma -A~K- I SN pa. reo ne’ en, "oe Xoo J

TINS

° k}

NEY-(2-(2-(2-(19-carboxynona decanoylamino)ethoxy)ethoxy)=acetyl)[Aib®**¥ Lys” |GLP-—1(7- 37)amide

6692.204-WO @ .

HN

[e

H NY Ie

I” AN 0 H,C_ CH, %

Ww @ - ES A

H-N""y——nN_Je-G-T-F-T-5-D-V-S5-5-Y-L-€-N= Q-A~A-K-E-F—I=a-W-L-v-K-N" S~R-IN NH,

TS uo, en Ho Yoo NH

ISSO, © 1 [Aib® = Arg®*GLP-1-(7-37)Lys(4-(Hexadecanoylamino)-4(S)-carboxy bs utyryl)-OH

NT

= pw 5

H H O _

H-N { Jeerrrsovss yey Sonanerawiva th nay

H © H oO H

HC CH, He CH, ) o]

A ~~~ AH NH

H.C Pa

HO

0 5s [Aib® 2% Arg®*GLP-1-(7-37)Lys(2-(2-C 2-(hexadecanoylamino)ethoxy)etoxy)acetyl)}-OH

Sa oy"

HC CH, HC CH, H Bo)

HN’ - NrE-Ge1--1-5-0-v--5v-t- Say

HN r° ° 0

H

N®T~(2-[2-(2,6-(S)-Bis-{2-[2-(2- (dode. canoylamino)ethoxy)ethoxylacetylammino}hexanoylamino)ethoxyjetho xy}) acetyM-[Aib***®|GLP-1(7-37)amide fo) wo ao

HN LE

N o HC CH, ° NH, HOR pat i g-mr=r --5-0-v=5 =v tot f IB gmnoamicgor omar ovonmy NE [ 7% "oo Fer Ho H NH

He” CH, ne’ cH, oJ — ) meg 0/0 [o] 0 hn SP 0 § ad ash a HC

§692,204-WO ® N™7-(2-[2-(2 . 6-(S)-Bis-{2-[2-(2- {tetradecanoylamino)ethoxy)ethoxyjacetylamino}exanoylamina)ethoxylethosy}) acetyl-{Aib® ZX IGLP-1(7-37)amide 9

HNN

HN al = [+] 0} HC CH, ?

HN" Ny JoE-GT-F-T-5-0-v-5-5-v-1-£-N, La-A-mmk-E-Foi-Aw-L-v-koN rR N NH, N "Oo neon HC CH, oc "oH 0, Mn aS Shae aed Yo

J

§ [o]

IPSN

8.22.35 26 34 . (Aib Arg” TIGLP-1-(7-37)Lys(2-(2-(2-(4-(He xadecanoylamino)-4(S)- carboxybuty rylamino)ethoxy)ethoxy)acetyl)-OH

HN oH (we, CH, HC Cher, .

H-N I A ES

H.C CH,

[0] [=e} NH ) NIN ne SSSA oN Tr ha

HO

© 1] (AIb*#¥IGL P-1(7-37)Lys((2-{2-[4-[4-(4-Amino-9, ~10-dioxo-3-sulfo-9,10-dihydr—o-anthracen-1- ylamino)-2-s ulfo-phenylaminol-6-(2-sulfo-phenyla mino)-[1,3,5]triazin-2-ylamirmo}-ethoxy}- : ethoxy)-acetyl))amide

HN all - © ES) HC CH nw ©

H—N a E-G=T—-F-T-$-D-V-5~-S-Y~L-E~-N CO -A-A-K-E—F—I-A-W-L-V-K-N R~-G-N MH,

Hoo ps , en or a rr Lo LS

COs ox

S N _u N nT 0g 5° Lo

HO o he son ° .

6692.208-WO ® y [AIb® FIG L_P-1(7-37)Lys(({2-[2-(2-{2-[2-(2-{2-[2-(1 5 ~carboxypentadecanoylamino 3 - ethoxy]ethoxzylacetylamino)ethoxylethoxylacetyl amimo)ethoxy]ethoxylacety!))amid «e [¢] wy

HN 1

HN N € —G-T-F~T-5-D-V-§-S-Y—L-E-N Q-A-A-K~E-F —I—A-W-L—-V—K-N R—A—N NSH, 3 b] or 0 fo] ~~

Ho ASA LAS ° Hh ~~ Og

NY ([2-(2-(3 -[2.5-dioxo-3-(15-carboxypentadecylsulfanyl)-pyrrolidin- 1-yl]- propionylami no}ethoxy)ethoxy)acetyl}-[D-Ala® Lys*’]-GLP-1-(7- 37Jamide oo eS [o]

HN

N

Cys 4 4 _ H IN Sa

H-N E-G-T=F-T-S~D-V-S=$-Y=L~E-G-Q-A-A~I —E~F—]=A-W-L~V~K-G-R-N ot

Ox Nz0 ro mri

Q

[ADP AIa=IGLP-1(7-37)Lys((2-(2-(2-(11- (oxalylamino Jundecanoylamino)ethoxy)ethoxy)acety!-}))amide [o] +N yy 1

HC CH, °

I ND FIP 1) Sv. GA NH, ’

H Oo ne’ Cy HET Tew "o o RH aQ- NH

A

Q

[AIb®%2 3° Alam ™]-GLP-1(7-37)Lys({2-[2-(2-{2-[2-(2-(15- carboxy-pentadecanoylamino )- ethoxylethox-ylacetylamino)ethoxy]ethoxy}acetyl)amid e

66292 204-WO ® q

HNN

2 Rt — WO HC. CM o]

H-N -N, J-E-G-T -F-T-$-D-V-S-S-Y-L-E-N Q-A-A-K-E-EF-1-A'W Lover SERA NH, N

H o /\ H oO Bo © HN_o

H,C CH, HC CH, J ’ [a]

[0]

PIONS W

0 H [AIb>#2* Ala¥)-GLP -1(7-37)Lys((2-{2-[11-(5-Dimethyl=aminonaphthalene-1- sulfonylamino)undecsanoylaminojethoxy}ethoxy)acetyl Damide ° _— “, Je "1 fo] [eo] He CH, 9

Baty SEPT 1) PUPS SIN Nriy / © He’ ch, nc’ em, [+] 0 0 NH \ 7 0 Nor

HCN er, [Aib%22%5 Ala¥}-GLP -1(7-37)Lys({[2-(2-{2-[1-(4-Chiorotoenzoyi)-5-methoxy-2-nmethyl-1H-indol- 3-yllacetylamino}ethm oxy)ethoxylacetyl))amide 0 nS - lo}

Hy 1 no uo H,C_ CH, 0

H-N Ve a r—ers:0v-ss-v-Le dl, Longmans Sprang NH, ’ © he CH, RC” TCH, 0 © 5 NH

Rag HC.

N 7 ) 0 Who 0 [Aib® Arg®* Glu EP RIGLP-1 H(7-37)Lys(2-(2-(2- (octadecanoylaminco ethoxy )ethoxy)acetyl)amide

8692 —204-WO @ . aw lo] p- \ [¢

SES SER, Mi, N

H [le] id H 0 NH

HC” "CH, [o]

ME ee (Aib® Arg? GluBP3NGLP-1( 7-37)Lys(2-(2-(2- (eicosanoylamino)ethoxy)ethoxy)acetyl}amide

[0] or [¢]

HN

” 14 1 [ Wo @ N

HN Ny JEG TF T=5-D-\/=§-§-Y-L-E-E~E-A-A REF ~~E-W-L-V-R-G= -R-G NH, 0 ne” om, o os"

HC eee eee [Gly® Arg®®*] GLP-1 H-(7-37)L ys(2-(2-(2-(2-(2-(2-(4-(octadecanoylamino)-4(S)- carboxybutyrylamino)ethoxy)et hoxy)acetyl)ethoxy)ethoxy)acetyl)-OH wr a " 0

GE E—F—l—A-W-L—V—R—G-R~G~N OH AN 0 ° [o} [s] NH [_ol a Ee ° - OH " 10 . ) [AIb® Arg®**|GLP-1 (7-37)Lys{ 2-(2-(2-(2-[2-(2- (octadecanoylamino)ethoxy)ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-OH

HN

3

H H ©

H-N fs SER TET IDSs LES Q-AA-RE-F-I-AW L-V R-GRGN_ A, © :

H,C CH, he u o OH

HC Nom gO A N~~Q<~g oO H

6692 204-vVO ® [AIb®] -GLP-1-(7-37)Lys (2- €2-(2-(4-(Hexadecanoylamino)-4(S)— carboxybutyrylamino)ethoxy? Jethoxy)acetyl)-OH

HNN ox" —

Q ~~ mal E-G-T-F-T-S-0-V-S-5-Y-L~E-G~Q-A-A-K-E-F—i-A~\wV-L-V-K-G-R-G-N Eds

H H o oO (eo) —

HC “cH, Ho a i. H

HESS \ oc H 0 5 . [Aib® Arg®3*} GLP-1(7-37) Luys{2-(2-(2-(2-[2-(2-(4-(octadecanoy-lamino)-4- carboxybutyrylamino)ethoxy Jethoxylacetyl)ethoxy)ethoxy)acetyl )}-OH

HNN

{ ho H &

H-N ; I JECT T So VS SLE saa ARE F-l-AW-L-V-R-GRGN Ho, 0 >

H,C CH, ~ 0. OH 0) NH

Oo H O

WSOP 1 SSN SENN Wy -Se

H 0 H [Aib® Arg?) GLP-1 (7-37) ys{2-(2-(2-(2-[2-(2-(17- carboxyheptanoylamino)eth oxy )ethoxy)acetylamino)ethoxy)ethoxy)acetyl)}-OH

HNN

A

[o] Qo

SE ES —I§-0V=5-5=Y—L-€-0-0-A-A-R-E~F —i—m-w-L~v-R-6-R-c-R, J o ne’ CH, ht [+ Q ° ne

CSS GNSS SN

H

[a] (Gly®, Arg?**] GLP1-(7-37) Lys{2-(2-(2-(2-[2-(2-(17- carboxyheptadecanoylamino)ethoxy)ethaxylacety!)ethoxy)ethory)acetyl)}-OH o 7

HN ©

AN 1

GET F——5D 8-59 E00 AAA Enr At ve eG-a-6oy Non N nd ol fe} Oy NH

PPPS rN 15. 0 "

£692.204-WO ® N [AIBYGLP—1-(7-37)Lys(2-(2-(2-(2-(2-(2-(4-(He=xadecanoylamino)-4(S)- carboxybutyrylamino)ethoxy)ethoxy)acetylamino) ethaxy)etheoxy)acetyi)-OH

HN

LN OH

- Hy-C_ CH, x ~"NH

H-N rt E61 F=T-§-0-V-$-5-Y~L-E~G-Q-A_ -A-K-E ~F~] ~A-W-L-V-K-G-R-G—N" " 00

H 0 4 Fe) H H I~

N.__/"0O

HENAN i oo

X ~~

HO © .

N*-(2-(2-( 2-(dodecanoylamino)ethoxy)ethoxw)acetyl)-[Aib® 2 *Lys*’] GL_P-1 H(7-37)-amide

Io} aN

HNTY pr 1 = HC. C™, 0 0 0

SEY OS J Wea o N on ne oe oT

H,C

N®7-(2-(2-( 2(tetradecanoylaminoethoxy)ethcaxy Jacetyl)-{Aib® 35 Lys] C3LP-1 H(7-37)- amide 0

HNN

HN al = MC_ CH fo lo] (om 3 :

Ecorse Eonner sanity NH, N [o]

HC Ch, HC Hs Jel s)

H,C

N“7-(2-(2-( 2-(hexadecanoylamino)ethoxy)ethoxy)acety!)-[Aib®#*Lys®"] GLP-1 (7-37)-amide

Q

HN

HN [0] =I } oo \

H-N Rbeorrrsovssiel, Toanreriawivcn rey NH, AN © H,C CH; R,C CH, ISAS

H,C [o]

NY" (2-(2-(_ 2-(octadecanoylamino)ethoxy Jetho=xy)acetyl)-[Aib® 22°Lys™) CSLP-1 (7-37)-amide

6662.204-WO ® ”

Q

DY

HENS

<M HC. CH, [88

H O H © > N

H-N NEG T-F-T-S:D-V-§-S-Y-L-EN_JQAAKEF-IFAW-L-V-KN SRN NH,

H oo Na yo Ho H o NH

H,C CH, H,C CH, HC eee

N®7.(=2-(2-(2-(eicosanoylamino)ethoxy Jethoxy)acetyl)-[Aib**2 Lys] GLP- | (7-37)-amide 5S 0

HNN

HN 1 ~ HC. CH o

H O H 0 Xo \

HN NEG T-FT-S DVS SY-LE NaF OAAKEFIAW LVR RN YNH,

H «© Ho Ho NH

HC CH, HC CH, H1C ema eee

N28 (2-(2-(2-(2-(2-(2-(octadecanoylamino) ethoxy ethoxy )acetylamino)etho. xy)ethoxy)acetyl))- [AiD®, Arg*®* Lys®|GLP-1-(7-37)-OH

Io o ? f ! l HN .. Vole

HINT Hy Is — N =. - _ H CGT —F—T—&— _ _ CR=F=F —— A—\W— _R=G-N— Hoon

H—N N E-G-T-F-T-§-D-V-5-S-Y-L—E-CG-0-A-A-R-E-F—|—A-W-L~-V-R-G-N No OH

ERAN "8

HC cr,

N90-(2+(2-(2-(2-(2-(2- (octacdecanoylamino)ethoxy)ethoxy)acetyla mino)ethoxy)ethoxy)acety))(Arcg®™ > Lys*|GLP- : 1(7-377)-OH 0 0 vf ) 8}

HN JN

“~

Lag

HN cH — N jal CS

HN 3 A=E=G-T—F~T-$-D-V-5-5-Y-L—E-G-0-A-A-R-E-F —I=A-W-L—V—F=-G-N Ao

NT°-{ 2.(2-(2-(2-[2-(2-(octadecanoylamino)ethoxy)ethoxylacetylamino)ethoxy)ethoxy)acetyl)}- . (Gly®. Arg®>* Lys®|GLP-1-(7-37)-OH

6692 204-WOQ : [o] lo]

NH / ~~ 1 lo] mT 0 hey ) od Ey o tf Jette eA Aah i TL

N-(2-(2-€2-(4-4(4,4,5,5.6.6.7.7.8.8.9.9.9-tradecafluorononanoylisulfam-oyl- butyrylamimno)ethoxy)ethoxy)acety))[Aib*?>= Lys*} GLP-1-(7-37)-OH

FF = 9 go fo}

AIA SI lo)

FlLel #l H I

HN"

H 2 wo =< 4

H—N ~N E-G-T-F-T-$-D-V~§=-S-Y-L-E-N Q-A~A~K-E=F—I—A-W-L-V-X-~N R-N OH - "oo =o HC” TCH, H o "oo ca,

N*'-(2-(2-¢2-(3.3.4,4.5,5.6.6,7.7,8.8.9.9,10.710,11,11,12,12, 1 2-Heneico =safluoro- dodecyloxyyacetylamino)ethoxy) ethoxy)aceatyl)[Aib®?*¥ LysY|GLP-1-(7-37)-OH

F FF _F_F “ATATAAAA n [od

NN

Yah @y@y a a 00 A

FF F FF fo} A

HY — o o HC CH,

Yr TE TUTOR v3 OH : Fic” CH, HC” CH, 0 0

N"-(2-(2-(2-(4-(hexadecanoyisulfamoyl)but yrylamino)ethoxyethoxy)acetyl)[Aib® 23 Lys”)

GLP-1-(7-237)-OH 9 AL TE

N a aa Too gL

HN"

Cs

H Q H 9 HEC 4

H=N N, _ME-G-T-F-T-5-D-v-§-5-v-L-E-N Q-A-A-K- EF == &-W-L VK = R-N oH

N Ou em, HC “CH, "oo Moa (Arg™® > |GGLP-1(7-37)Lys({2-(2-(2-(2-[2-(2- (octadecamnoytamino)ethoxy)ethoxylacetylarmuno)ethoxy)ethoxy)acetyl)}-)-OH

6632.204-WO * pos “A otf

H—N [A ETO ETS TD YES TS STL TE SATA AT ATE TET ATW SVR 2G R-G-N__ ~oH ° YT

H I

HANA ee Ag OA 0

H

[le] [Arg®3] GLP-1(7-37)Lys{2-(2-(2-(2-[2-(2-(4- octadecanoylamino)-4- carboxybu tyrylamino)ethoxy)ethoxy]acetylamino)ethoxy)ethoxy}acetyl)}-OH on

H-N T A~E~G-T-F-T-3-D-V-$-S-Y-L—E— G-Q-A-A-R-E-F—I—A-W-L-V-R-G Ren Ao "ye , 0 oy

TY ve Ao OA Ong [oo | H 0 ©

N®0-{2-(2-( 2-(2-[2-(2-(4-(hexadecancylamino }-4- carboxybu tyrylamino)ethoxy)ethoxylacetylarmiino)ethoxy)ethoxy)acetyl)}-exen din(1-39)

HI o

Cc 9

HN GEGTFTSOLSKAME—EEAVN I —LF-I-EWLKNGGPS—SCAP-PPN NH, lo} : 0

Oy CH O NH 0 H fo}

IPODS § SN SNS -N

H 0 Ht } [Al2°, Arg?™® *JGLP-1(7-37)Lys((2-[2-((2-oxaly lamino-3-carboxy-2-4.5.6,7-tetraahydro- benzo[b]jthsiophen-8-yl-acetylamino))ethoxy]e thoxyacetyl) amide

6692 204-WO ° ;

HN

=N al J

H-N I A=E-G-T-F—-T-S5-D-V-§~S-Y—L—E~— G-0-A-A-R-EF IAW Lv-R—G-RG-N A

Os NH ; igo y

HOP SSS Ne pO NT O0 0] [AiIb®#2 SIGLP-1(7-37)Lys((2-[2-({2-0xalylam @ino-3-carboxy-2-4,5,6,7-tetrarmydro- benzo[b-]Jthiophen-6-yl-acetylamino))ethoxyleathoxyacetyl) amide ry 2 nC ox ne ex ’ a, ©, OM B

OT BE Sl Io ackmg-emima-wotou-w-f Saeco “, Se: on

NLR Hoo new NO N ST y= ~~ hh t] 0 0 nr

N“-(2- 2-(2-(2-(2-(2-(4-(octadecanoylamince )-4(S)- carboxy butyrylamino)ethoxy)ethoxy)acetylarmino)ethoxy)ethoxy)acetyl)- [Aib® Arag®* Lys™|GLP-1-(7-37)-OH 0

H

N lo} alll SR

HOT 0 \__0 A

NTON—O 0) =o . CH,

H Tw

HC. CH, fo]

To eran n NJ om © Qo 0 ,

N*®.(2-¢ 2-(2-(2-(2-(2-(4-(octadecanoylamincm )-4(S)- carboxy-butyrylamino)ethoxy)ethoxy)acetylarmino)ethoxy)ethoxy)acetyl)- [Gly® Arg™* Lys®]GLP-1-(7-37)-OH

6692 204-WO ® 0 — 7 ° be NI

HO 0 Oo _[]

HN—o

OT

HN

] CH,

HN

= H OH

Ng

HN G=E= G-T—F=T-§-D-V=§-§-Y~-L-E~G-Q—A-A-R— E-F—I=A-W-L—V-R-G-N I lo]

N®7-2-(2-(2-(4-(4-(Feptadecanoylamino)-4-(S)-carboxybu tyrylamino)-4-(S)- carboxybutyrylamiro)ethoxy)ethoxy) acetyl-[Aib®*?* Lys¥Y|GLP-1-(7-37)-NH,

HN

HN EG T FT-S0V SS Y-LEN, SOA AKE- F-1-AW vn SRN 0

H oo H 5 J

H,c CH, NH,

H,C CH, 3 2

Q N a o SH

HC SS SN al

N-{ O H

H =0 OH

HO

N®-2-(2-{2-(2-{2-(4-[4-(Heptadecanoylamino)-4-(S) carboxybutyrylamiro]-4-(S)-carboxybutyrylamino)ethoxy] ethoxy)acetylamino)ethoxy]ethoxy)acetyl-[Aib®%2 * Lys* | GLP-1-(7-37)-NH,

HN

=. HC CH;

HNN Reo rrs0v-ssy-LeN Pa Anke F-1AW vk Or RN 0

Ho << I H oO $ \

A,c CH, HC CH, NH, 9 N oN ° (@) H 0 Rand

H,C TT NTT Cad

N oO H

H (o] OH

HO

N=6-(2-(2-(2-(4-(He xadecanoylamino)-4(S)-carboxybutyryl amino) ethoxy)ethoxy)acet yl)-[Aib® Arg™|GLP-1-(7-37)- -OH

6692 204-WO ® }

HY

! ne i E-G-T-F-T-S-D-V-S-S-Y-L —e-Geg-a-Al I e-pmimaw-L ~v-R-G-R—N" OH : "oo x : "oo . H,C CH,

MASS SAD tir Daas tt 3 Ao H s)

HO

N®-2—(2-2-(2-(2-(2-(4-(Octadecanoylamino)-4(S)- carboxxybutyrytamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)acetyl- [Ab Ag*|GLP-1-(7-37)-OH

ANT

[eo ag V5 5Y 8-6-0 AA Iftar vor-G-R on

HC on \ © [o] 0 bs H 0 ne SAAS ASA, YT OOo [Gly®, Arg? *JGLP-1(7-37)Lys(2-(2-(19-(C arboxy)nonadecanoylamino)ettioxy)ethoxy)acetyl)-

OH

-0

HOG ee Ogg © 0 H NH

AY

I V-R-G-R-G-N OH

H d H ¢ [GlY® Arg? GLP-1(7-37)Lys((2-(2-(17- (carbooxy)heptadecanoylamino)ethoxy)ethoxy)acetyl)}-OH oO

HOG ees Om gO fo H NH ie ie

Hp GE GT T-80m 5 L-E-G-0-A AREF 1A = RGR yom

6692.204-WO ® (Gly® Arg®® ¥|GLP-1(7-37)Lys(_2-(2-(2-(4-(19-(carboxy)nonadecamoylamino)-4- carboxybutyrylamino)ethoxy)e thoxy)acetyl)-OH 0 i lo] o

H

Hy

RE CU CmH

H J H o {Gly® Arg? *|GLP-1(7-37)Lys(=(2-(2-(2-(2-(2-(2-(2-(2-(2- {hexadecanoylamino)ethoxy)e thoxy)acetyl)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)- acetyl)-OH 0 H 0 H ?

HO Rm eee N mgm 0 Ay Og Nem ~ 0 LX 0} H 0 NH

Hn

HN N——G-E-G-T-F~T-8-D-V—8-5-¥-L-E-G-Q-A-A-R-E-F~I-A-W-L rene 0 HOT [Gly®, Arg®**]GLP-1 (7-37)LysS (2-(2-(2-(2-(2-(2-(octadecanoylarnino)ethoxy)ethoxy)- =acetylamino)ethoxy)ethoxy)acetyl) NH, 3,

NY © . pe, s 1,

VSI (Ot AN [o] [o] yr [a]

Se

NU NN

N0(2-(2-(2-(2-(2-(2-(2-(2-(2-(2=-(2-(2-(4-(17-(carboxy }heptadecaroylamino)-4- carboxybutyrylamino)ethoxy)et hoxy)acetylamino)ethoxy)ethoxy)a cetylamno) e=thoxy)ethoxy)acetylamino)eth oxy)ethoxy)acetyl) [Lys*°lexendin-=4 (1-39)-NH,

692.204 ~WO ° 3

HN

NN OH f 0

H-N G E-G-T-F-T-S-D-L-8-K-Q M-E-E-E-AV-R_IL-F-1-E W-L-KN-G SSE 0 : 0 ~N 0 H 0 yn

OA GAO gy Norge OA ~ 0g 6 H fe) H

N HOO

DONS Of: Gr" lo] H lo]

N*.(2-(2+(2-(2-(2~(2-(17-Carboxyheptadecanoylamino)ethoxy)ethoxy) acetylamino)ethoxy)ethoxy)acetyl) [Aib® Arg? Lys*] GLP-1 (7-37)

H

0 Ne o

MN

NTTN—-0 “To

HNO

HN HO

— N

H.C CH, 0 H [eo]

HN NEGF 7-5 D-V-5-S YL 5-5-0 —A~A-R-E-F—IAW-L VRC} N _A-0H

Ss © 0 fo

N-%.(2-(2-(2-(2-(2-(2-(17-Carbo xyheptadecanoylamino)ethoxy ethoxy) acetylamino)ethoxy)ethoxy)acetyl) [Arg?®™, Lys®] GLP-1 (7-37) 0 N-

SL}

NTN_-0

EN

HN ©

HNN HO

— N

Oo

H—N A—E-G-T-F-T=S5-D=V-§-5-Y~L-E-G-Q—A-A-R-E=F=I=A-W-L-~-R~G-N nJrox

Ie} [o] 10 .

N¥(2-(2-(2-(2-(2-(2-(1 7-Carbox yheptadecanoylamino)ethoxy)ethoxy) acetylamino)ethoxy)ethoxy)acetyil) (Gly? Arg” .Lys*®] GLP-1 (7-37)

6692.204-WO ® ' 0 Me fo] °No J

NTTN\_-0

MN

Seo

HN

HNN HO

— N 0

OH

H—N G—E-G-T—F~-T-§-D-V-§-SB-Y~L-E-G-0—A-A-R-E-F~I-A-Ws~L-V-R-G-N nv) 0 0

N=2(2-(2-(2-(2-(2-(2-(2-(2-(2- (Octadecanoylamino)ethoxy)ethoxy)aacetylamino)ethoxy )ethoxy)ace=tylamino)ethoxy)- et hoxy)acetyl)[Lys®] Exendin-4 (1-39= amide

[0] ~~~ A

TIO a a Ni ps . o Jo) [HTN 0 fo 0

Loo oA

HN" =~ N OH

HN G-E-GTFTSOL SKOMEEEAUN ce rremencerssosssy 0 0 bo)

N=¥.(2-(2-(2-(2-(2-(2-(4-(octadecanoywlaming)-4(S)- camrboxybutyrylamino)ethoxy)ethoxy)amcetylaminojethoxy)ethoxy)ace tyl)-[Arg® Lys™|GLP-1- (7 -37) 0]

H ball o N

HNN

HO o oof

NN

0" 0

HN

HN" CH — N as L-E-G-Q—A-A-R-E~F—I—A-W-L-V -R-G-N Nok

H o H [@] [0]

66532.204-W0 ® }

N-(2-[2-(2-[2-(2-[2--(17-Carboxyheptadecanoylamino ethoxy] ethoxy)acetylamino] ethoxy ethoxylacety!){Arg>'|GLP-—1 -(7-37)-OH

HN Tw 0 Q

NE CHS SUT I c AN

I SOUP ONPON. SUN SP ONE o H 0

N= {2-(2-[2-(2-[2-(2—[4-( 17-Carboxyheptadecanoylam ino)-4(S)- carboxybutyryla mince lethoxy)ethoxylace tylamino)ethom<ylethoxy)acetyl][Arg*]GLP-1-(7-37)-

OH

HN

LN

( HQ H q

H—N A-E-G-T-F-T-S-D-V-5-S-Y-L-E-G- aaa e-rimawot-v-rGr-N on

Q N

CT . \

HO eA ego OA On ° H 0 H 0 ;

NT0-(2-(2-(2-(2-(2-(2—(2-(2-(2-(17-

Carboxyheptadecancoylamino)ethoxy)ethoxy)acetylam&Zno)ethoxy)ethoxy)acetyl- amina)ethoxy)ethoxy)acetyl)[Lys’’] Exendin-4 (1-39) a mide

H lo]

Or ses

NH

0

Ss

LH TNS

-0Q lo) lo}

Oyo oH ge i > CH

IN EGTFTSDLSKQMEE CN FIEWL wcerssonssa ga

H H H i © © °o [Gly®, GIUP® Arg! 8% 3GLP1 (7-37) Lys(2-(2-(2-(2-C2-(2-(17- carboxyheptadecancaylamino)ethoxy)ethoxy)acetylamirio)ethoxy))ethoxy)acetyl)-NH-

6692. 2204-WO @ HS

H O

.H-N G-E-G-T- F-T-§-D-V-S-R-Y-L-E-E-E-A-A-R-E—F~I-E-W-L-V-R-G-R-G-N J

H 0 ] PH,

HO Ieee Noe OA AO gH 0 H lo] [imidazolytpropionic acid’, Asp'®, Aib®?*® |GLP1(7-37)l_ys NH({2-{[4-(17- carboxyheptadecancoylamino)butylcarbamoyl]methoxy}=ethoxy)ethoxy))

NH

HN

N o H,C CH H ee 0 H,C CH, no "oo ~~ at

Q

0 : NH 0 J

HOA AAA AAA

0 i [Imidazolylpropionic acid’, Aib%*® JGLP1(7-37)Lys NHC (2-{[4-(17- carboxyheptadecanylamino)butyticarbamoyljmethoxy} ethoxy)ethoxy))

NF,

HNN . ~~ N

H,C CH, H

Td [eo] HC CH, Ho H lo} —~ 0,

L, “po

NH o Id

HO ppm 0 N , and [3-(5-Imidazoyl)prop~ionyt’. Aib®, Arg®®™>* | GLP-1 (7-37) Lys{2-(2-(2-(2-[2-(2-(17- carboxyheptanoylanmino)ethoxy)ethoxylacetylamino)ettoxy)ethoxy)acetyl)}-OH

6692.20-4-WO ® 41 i SOOO SS ° } 6] { 0

Yeo

HTN

HT J

NN E-G-T-F-T-&-D-V-8-5-Y-L-E-G-Q-A-A-R-E-F~ 8 -A-W-L-V-R-G-R-G-N —OH of o Ho

In another embodiment the therapeutic polypeptide is a GLP-2 peptide. in another emb»odiment the GLP-2 peptide is a DP PIV-protected GLP-2 peplisde. in another embmodiment the GLP-2 peptide is Gly’- GLP-2(1-33).

In yet another embodiment the GLP-2 peptide is L ys' Arg®-GLP-2(1-33).

In another emt=odiment of the invention the therap eutic palypeptide is human mnsulin or an analogue thereof.

In another emimodiment of the invention the therap eutic polypeptide is selecte=d from the group consisting of AAsp®2-human insulin, Lys®*,Pre®®—human insulin, Lys®®,Giu®?= human insulin, Gly*?' Arg®*' Arcg®*-human insulin and des(B30) hu man insulin. in another emt=odiment of the invention the therapreutic polypeptide is human growth . hormone or an analogue thereof.

In another emitoodiment of the invention the therapeutic polypeptide is parathwyroid hormone or an analogues thereof,

In another emioodiment of the invention the therapeutic polypeptide is humane follicle stimulating hormone or an analogue thereof.

In another emtoodiment of the invention the therapeutic polypeptide has a malar weight of less than 100 kDa, less than 50 kDa, or less than 10 kDa.

In another emtmadiment of the invention the therapeutic polypeptide is selected from the group consisting of a growth factor such as platelet-derived growth factor (POGF) , trans- forming growth factor a (TGF-a), transforming growth factor 8 (TGF-8), epidermal graowih fac- tor (EGF), vascular end cthelial growth factor (VEGF), a soimatomedin such as insulin growth factor | (IGF-i). insulin growth factor Il (IFG-Il), erythropoietin (EPO), thrombopoietin (TTPO) or

6692.2 04-WQO @ angiopoietin, interferon, pro-usrokinase. urokinase, tissue plasmiriogen activator (t-PA), plasrmi- nogen activator inhibitor 1. plasminogen activator inhibitor 2, vor Willebrandt factor, a cytokirme, e.g. an interleukin such as intearleukin (IL) 1, IL-1Ra, IL-2, [L-4, IL-5, IL-6, IL-9, iL-11, |L-12, IL_- 13, 1L-15, IL-16, 1L-17, 1L-18, 1L-20 or {L-21, a colony stimulatingg factor (CFS) such as GM-

CSF, stem cell factor, a tumor necrosis factor such as TNF-a, ly mphotoxin-a, lymphotoxin-B,

CD40L, or CD30L, a protease= inhibitor e.g. aprotinin, an enzyme such as superoxide dismu- tase, asparaginase, arginase, arginine deaminase, adenosine deeaminase, ribonuclease, cat=- lase, uricase, bilirubin oxidases, trypsin, papain, alkaline phosphamtase, -glucoronidase, purines nucleoside phosphorylase or batroxobin, an ogioid, e.g. endorphins, enkephalins or non-natural opioids, a hormone or neurop eptide, e.g. calcitonin, glucagon, g astrins, adrenocorticotropic hormone (ACTH). cholecystokinins, lutenizing hormone, gonadaatropin-releassing hormone, chorionic gonadotropin, corticotrophin-releasing factor, vasopressin, oxytocin, antidiuretic hor—- mones, thyroid-stimulating ho rmone, thyrotropin-releasing hormeone, relaxin, prolactin, peptid e

YY, neuropeptide Y, pancreasstic polypeptide, leptin, CART (coc=aine and amphetamine regu— lated transcript), 8 CART related peptide, perilipin, melanocortin=s (melanocyte-stimulating ho r- mones) such as MC-4, melan in-concentrating hormones, natriur—etic peptides, adrenomedullimn, endothelin, secretin, amylin, vasoactive intestinal peptide (VIP), pituary adenylate cyclase ac®i- vating polypeptide (PACAP), bbombesin, bombesin-like peptides,. thymosin, heparin-binding protein, soluble CD4, hypotha Imic releasing factor, melanotoninss and analogues thereof.

In another aspect the present invention relates to a ph&rmaceutical composition corm. prising a compound according to the invention, and a pharmaceutically acceptable excipient.

In one embodiment the pharmaceutical composition is suited for parenteral admini- stration.

In another aspect the present invention relates to the us=se of a compound according to the invention for the preparation of a medicament. {in one embodiment <f the invention a compound according to the invention wherein the therapeutic polypeptide is a GLP-1 peptide is used for the preparation of a medicament faor the treatment or prevention off hyperglycemia, type 2 diabetes, irmpaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X. dyslipidemi.a, cognitive disorders, atherco- schlerosis, myocardial infarcti on, coronary heart disease and other cardiovascular disorders, stroke. inflammatory bowel syndrome, dyspepsia and gastric ulcers.

In another embodimeent of the invention a compound according to the invention wherein the therapeutic polyp eptide is a GLP-1 peptide is used ¥or the preparation of a me- dicament for delaying or prevsenting disease progression In type 2 diabetes.

6592 204-WO @® in another embodiment of the invention a compound according tzo the invention wherein the therapeutic polypeptide is & GLP-1 peptide is used for the presparation of a me- diesament for decreasing food intake, decreasing f3-cell apoptosis, increassing B-cell funtion and

B—celt mass, andlor for restoring glucos sensitivity to B-cells.

In another embodiment of the invention a compound according tto the invention wherein the therapeutic polypeptide is & GLP-2 peptide is used for the pre=paration of a me- di-cament for the treatment of small bovwel syndrome, inflammatory bowel syndrome or Crohns di sease. in another embodiment of the invention a compound according fo the invention wiherein the therapeutic polypeptide is &n insulin peptide is used for the p reparation of a me- di cament for the treatment or preventio n of hyperglycemia, type 1 diabetess, type 2 diabetes or

B—cell deficiency. TEne therapeutic polypeptide can be produced by a method which comprisses culturing a host cell containing a ONA sequence encod ing the polypeptide and capable o f expressing the paly- peptide in a suitable nutrient medium u nder conditions permitting the exp=ression of the peptide, af~ter which the resulting peptide is recovered from the culture.

The medium used to culture the cells may be any conventional medium suitable for growing tke host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared acco rding to published recipes (e.g. in catalogues of the Amer ican Type Culture Collection). The peptide produced by the cells may then be recovered from the culture medium by conventional procedures including seeparating the host cells from the meds um by centrifugation or filtration, orecipitating the pro- tesinaceous components of the supemaatant or filtrate by means of a salt, e.g. ammonium sul- p hate, purification by a variety of chromatographic procedures, e.g. ion e=xchange chromatog- raphy, gel filtration chromatography, affinity chromatography, or the like, dependent on the type o f peptide in question.

The DNA sequence encoding the therapeutic polypeptide may suitably bee of genomic or cDNA o rigin, for instance obtained by prepari ng a genomic or cDNA library andi screening for DNA s equences coding for all or part of the polypeptide by hybridisation usingg synthetic oligonucleo- ti de probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch,

ESF and Maniatis, T, Molecular Cloning = A Laboratory Manual, Cold Sprirmg Harbor Laboratory

Press, New York, 1989). The DNA secyuence encoding the polypeptide rmay also be prepared

6892.2204-WO ® synthetically by established standard methods, e.g. the p hosphoamidite method desscribed by

Beaucage and Caruthews, Tetrahedron Letters 22 (1981) , 1858 - 1869, or the methcad de- scribed by Matthes et ad. EMBO Journal 3 (1984), 801 - -805. The DNA sequence nay also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683.202 or Saiki et al.. , Science 239 (1988), 487 - 491.

The DNA sequence mamy be inserted into any vector which may conveniently be sutojected to recombinant DNA procedures, and the choice of vector vill often depend on the hosst cell into which it is to be introduced. Thus, the vector may be an &utonomously replicating vector, ie. a vector which exists as &an extrachromosomal entity, the reaplication of which is indepesndent of chromosomal replication, e.g. a plasmid. Alternatively, thee vector may be one which , when in- troduced into a host cel 1, is integrated into the host cell ge=nome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably~ an expression vector in which thee DNA sequence encoding the peptide is operably linked to ad=ditional segments required for trarscription of the DNA, suck as a pro- moter. The promoter m ay be any DNA sequence which SShows transcriptional activily in the host cell of choice and mmay be derived from genes encocling proteins either homolcagous or heterologous to the hosst ceil. Examples of suitable promeoters for directing the transcription of the DNA encoding the foeptide of the invention in a variet y of host cells are well kno=wn in the art, cf. for instance Sarrbrook et al., supra.

The DNA sequence en=coding the peptide may also, if ne=cessary, be operably conns ected to a suitable terminator, polmyadenyiation signals, transcriptiormal enhancer sequences, amnd transia- tional enhancer sequerces. The recombinant vectar of the invention may further comprise a

DNA sequence enablin g the vector to replicate in the hosst cell in question.

The vector may also ccomprise a selectable marker, e.g. a gene the product of whic h comple- ments a defect in the h ost cell or one which confers resistance tc a drug, e.g. ampicillin, kana- mycin, tetracyclin, chlomramphenicol, neomycin, hygromyc=in or methotrexate.

To direct a parent pept ide of the present invention into time secretory pathway of the host cells, a secretory signal sequuence (alsc known as a leader secquence, prepro sequence or pre se- quence) may be provided in the recombinant vector. Thea secretory signal sequenc: € is joined to the DNA sequence encoding the peptide in the correct reaading frame. Secretory siegnal se- quences are commonly positioned 5' to the DNA sequermce encoding the peptide. “The secre-

6392 204-WO <P tory signal se2quence may be that normally asso«ciated with the peptide or mmay be from a gene encoding ancother secreted protein.

The procedu res used to ligale the DNA sequences coding for the present p eplide, the pro- moter and optionally the terminator and/or secre tory signal sequence, respectively, and to in- sert them inteo suitable vectors containing the information necessary for repl ication, are well known to per—sons skilled in the art (cf., for instarmce, Sambrook ef al.., supram).

The host cellt into which the DNA sequence or thee recombinant vector is intr—oduced may be any cell which is -capable of producing the present peaptide and includes bacteriam, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitamtion, E£. coli, Saccharomyces cerevisiae, or mammalian BHK omr CHO cell lines.

Examples of compounds which can be useful as GLP-1 moieties according to the present in- vention are described in International Patent Aplication No. WO 87/06941 (The General Hos- pital Corporamtion) which relates to a peptide frag ment which comprises GLF-1(7-37) and func- tional derivat ives thereof and to its use as an ins ulinotropic agent.

Further GLP—1 analogues are described in Interrational Patent Application No. 90/11296 (The

General Hos pital Corporation) which relates to p eptide fragments which cormprise GLP-1(7-36) and function=al derivatives thereof and have an irssulinotropic activity which e2xceeds the insuli- notropic activity of GLP-1(1-36) or GLP-1(1-37} &=nd to their use as insulinot ropic agents.

International Patent Application No. 91/11457 (B uckley et al..) discloses analogues of the ac- tive GLP-1 p-eptides 7-34, 7-35, 7-36, and 7-37 v=vhich can also be useful as GLP-1 moieties according to the present invention.

Pharmaceutical compositions

Pharmaceuti cal compositions containing a compe ound according to the pres ent invention may be prepared by conventional techniques, e.g. as described in Remington's mPharmaceutical

Sciences, 1385 or in Remington: The Science ard Practice of Pharmacy, 1 =o" edition, 1995.

One object of the present invention is to provide= a pharmaceutical formulation comprising a compound as ccording to the present invention wehich is present in a concertration from about 0.1 mg/ml tco about 25 mg/ml, and wherein said formulation has a pH from 2.0 to 10 0. The

6692 204-WO ® pharmaceutical formulation may comprise a compoun d according to the present inve ntion which is present in a concentration from about 0.1 mg. /ml to about 50 mg/ml, and whezrein said formulation has a pH from 2.0 to 10.0. The formumlation may further comprise a buffer system, preserv ative(s), isotonicity agent(s), chelating ageni(s). stabilizers and surfactants.

In one embodiment of the invention the pharmaceuticaal formulation is an aqueous fosmula- tion, i.e. formulation comprising water. Such formulaticon is typically a solution or a su spen- sion. In a further embodiment of the invention the pha rmaceutical formutation is an aqueous solution. The term "aqueous formulation” is defined ass a formulation comprising at le ast 50 %w/w water. Likcewise, the term "aqueous solution” is «defined as a solution comprisire g at least 50 %w/w vwvater, and the term “aqueous suspens ion” is defined as a suspensiora com- prising at least 50 %w/w water. in another embodiment the pharmaceutical formulation is a freeze-dried forrmula- lion, whereto the physician or the patient adds sclvent-s and/or diluents prior to use.

In another embodiment the pharmaceutical formulation is a dried formulatior (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.

In a further aspect the invention relates to a sharmaceutical formulation conprising an aqueous solution of a compound according to the goresent invention, and a buffer, wherein said coamnpound is present in a concentration ffrom 0.1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0.

In a further aspect the invention relates to a ppharmaceutical formulation comprising an agueous solution of a compound according to the poresent invention, and a buffer, wherein said compound is present in a concentration #rom 0.1 mg/mi or above, and v=vherein said formulation has a pH from about 7.0 to about 8.5

In a another embodiment of the inventicn the pH of the formulation is selected from thelist consisting of 2.0, 2.1, 2.2,2.3,24,25,26,2.°7,2.8,29, 3.0.3.1, 3.2,3.3, 3. 4,35. 36.37,38,39,40,41,42,43,44,45,46,47,38,49,50.5.1,5.2,53,5.4. 55,586, 57,58,59,6.0,6.1,6.2,6.3,64,65,66,67,68,6.9,7.0,7.1,7.2,73,74,7.5 76,77, 78,79,80,8.1,828.3,84,85,86,87,88,89,90,9.1,92,93,94,95,96, 97,938, 9.9, and 10.0. Preferably, the pH of the formulation is at least 1 pH unit from the isoe=lectric point of the compound according to the present invention, even more preferable the pH of the formulation is at least 2 pH unit from the isoelectriac point of the compound according to the present invention.

In a further emi>odiment of the invention the buffer is sselected from the group consis ting of sodium acetate, sodium carbonate, citrate, giycylglycimne, histidine, glycine, lysine, ar ginine, sodium dihydrogen phosphate, disodium hydrogen pha osphate, sodium phosphate, a nd

6692 204-WO ® tris(hydroxymethyl )-aminomethane, hepes, bicine, triccing, malic acid, succinate, rmnaleic acid, fumaric acid, tartawic acid, aspartic acid or mixtures trereof Each one of these specific buff- ers constitutes an alternative embodiment of the inve _ntion.

Ina further embodiment of the invention the formulatit on further comprises a pharmaceutically acceptable preservative. In a furthesr embodiment of the inventicon the preservative is selected from the group consisting of gohenol, o-cresol, m-cresol, o-cresol, methyl p-hydroxyb enzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p— hydroxybenzoate, 2-phenylethanol, benzyl alcohol, et hanol, chlorobutanol, and th iomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sod ium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoates, benzethonium chloride, chiorphe nesine (3p-chlorphenoxypro pane-1,2- diol) or mixtures thereof. in a further embodiment of t he invention the preservatives is present in a concentration from 0.1 mg/ml to 30 mg/ml. In a further embodiment of the invention the preservative is pre- sent in a concentration from 0.1 mg/ml to 20 mg/ml. In a furthe-r embodiment of thea invention the preservative is present in a concentration from Os 1 mg/m! to 5 mg/ml. In a furth er emhadiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a further sembodiment of the inventior— the preservative is pre=sent in a concentration from 10 mg=/ml to 20 mg/ml. Each one Of these specific preservatives constitutes an alternative embosdiment of the invention. The use of a preservative in pharmaceutical compositions is well-krown to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmaacy, 197 edition, 19985,

In a further embodiment of the invention the formulation further comprisess an isotonic agent. in a further embodiment of the invention the is-ctonic agent is selected frorm the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino aci-d (e.g. L- glycine, L-histidinez, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonire), an alditol (e.g. glycerol (glycerine), 1,2-propanediol (peropyleneglycol), 1,3-prapan ediol, 1,3- butanediol) polyetkylenegiycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono- |, di-, or polysaccharides, or water-soluble glucans, including for example fructose=, glucose, mannose, scrbosez, xylose, maltose, iactose, sucrose , trehalose, dextran, puilutam, dextrin, cyclodextrin, solutole starch, hydroxyethyl starch and ccarboxymethylcetiulose-Na rmay be used. In one embodiment the sugar additive is sucrosse. Sugar alcohol is defined as a C4-C8 hydrocarbon havirg at least one --OH group and inclides, for example, mannitol , sorbitol, inositol, galacititol , dulcitol, xylitol, and arabitol. In one= embodiment the sugar alcohol additive is mannitol. The swgars or sugar alcohols mentioned above may be used individually or in

6652 .204-WO @ combination. There is no fixed limit to the amount used, ass long as the sugar or sugar alcohol is soluble in thie liquid preparation and does not ad versely effect the stabilizzing effects achieved usincy the methods of the invention. In on € embodiment, the sugar or sugar alcohol concentration 1s belween about 1 mg/ml and abou t 150 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a further embodiment of the invention the Isa tonic agent is present in .a concentration from 1 rg/ml to 7 mg/ml. In a further embodiment of the invention th e isotonic agent is present in a concentration from 8 mg/ml to 24 mag /ml. In a further embodiment of the invention the isotonic —agent is present in a concentration from 25 mg/ml to 50 mg/rmni. Each one of these specific issotonic agents constitutes an alterna tive embodiment of the invention.

The use of an isotonic agent in pharmaceutical compositio as is well-known to the si=illed person. For convenierice reference is made to Remington= The Science and Practic=e of

Pharmacy, 19" edition , 1995. in a further emm bodiment of the invention the formulation further comprises = chelating agent. In a further em bodiment of the invention the chelati ng agent is selected from salts of ethylenediaminetetrazacetic acid (EDTA), citric acid, and asspartic acid, and mixtures thereof.

In a further embodime=nt of the invention the chelating ages nt is present in a concen tration from 0.1mg/mi to Smgg/mi. in a further embodiment of the invention the chelating agent is present in a concentration from 0.1mg/mi to 2mg/mi. In a Further embodiment of thee invention the chelating agent is present in a concentration from 2mg/ml to Smg/mi. Each one of these specific chelating agents constitutes an alternatives embodiment of the inveantion. The use of a chelating age=nt in pharmaceutical compositions Is well-known to the skiliec person.

For convenience refer—ence is made to Remington: The Sc#ence and Practice of Pharmacy, ' 19" edition, 1995.

In a further e mbodiment of the invention the form ulation further comprisess a stabi- liser. The use of a sta bilizer in pharmaceutical compositiorus is well-known to the skilled per- son. For convenience reference is made to Remington: THe Science and Practice caf Phar- macy. 19" edition, 1935.

More particularly, compositions of the invention a re stabilized liquid pharmaceutical compositions whose t_herapeutically active components ineclude a polypeptide that possibly exhibits aggregate for~mation during storage in liquid pharrmaceutical formulations. By “ag- gregate formation" is intended a physical interaction between the polypeptide molezcules that results in formation off oligomers, which may remain solub ie, or large visible aggregates that precipitate from the seolution. By “during storage” is intend ed a liquid pharmaceutic al compo-

6692 204-WQO ® sition or fo rmulation once prepared, is not immediately administered to a subject. Rather, following pareparation, it is packaged for storage, either in a liquid form, in a frozer state, or in a dned form for later reconstitution into a liquid form or other form suitable for adrmninistration to a subjec=t. By "dried form" is intended the liquid pharmaceutical composition or formulation is dried eit_her by freeze drying (i.e.. lyophilization; see, for example, Williams and Poli (1984) J. FParenteral Sci. Technol. 38:48-59), spray drying (see Masters {1991) in Spray-

Drying Hardbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 4231-676;

Broadhea et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; and Mumenthale ret al. (1894) Ph=arm. Res. 11.12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-47%0; and Roser (1991) Biopharm. 4:47-53). Aggregate formation by a polypeptide during stor—age of a liquid pharmaceutical composition can adversely affect biologi cal activity of that pohwypeptide, resulting in loss of therapeutic efficacy of the pharmaceutical ecomposi- tion. Furtheermore, aggregate formation may cause other problems such as block=ge of tub- ing, memb-ranes, or pumps when the polypeptide-containing pharmaceutical comgoosition is administer ed using an infusion system.

THe pharmaceutical compositions of the invention may further comprise =n amount of an amin o acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition. By "aming acid base” is intended an amino acid or a combination of amino a cids, where any given amino acid is present either in its free base form orinits salt form. WNVhere a combination of amino acids is used, all of the amino acids may be pre- sent in the ir free base forms, all may be present in their salt forms, or some may toe present in their free= base forms while others are present in their salt forms. In one embod®Ement, amino acicds used for preparing the compositions of the invention are those carryirg a charged si de chain, such as arginine, lysine, aspartic acid, and glutamic acid. In cone em- bodiment, the amino acid used for preparing the compositions of the invention is clycine.

Any sterecisomer (i.e. L or D) of a particular amino acid {e.g. methionine, histidine, imida- zole, argin ine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixturess thereof) or combinations of these stereoisomers, may be present in the pharmaceutical ccompaositions of the inve-ntion so long as the particular amino acid is present either in its free basse form or its salt forrm In one embodiment the L-sterecisomer is used. Compositions of the invention may also toe formulated with analogues of these amino acids. By "amino acid ana_logue” is intended am derivative of the naturally occurring amino acid that brings about the d-esired ef- fect of decsreasing aggregate formation by the polypeptide during storage of the li quid phar- maceutica | compositions of the invention. Suitable arginine analogues include, fom example, aminoguamidine, ornithine and N-monoethyl L-arginine, suitable methionine analcegues in- clude ethionine and buthionine and suitable cystein analogues include S-methyl-L cystein.

6692.204-WQO ® As: with the other amino acids. the amino acid analogues are incor-porated into the composi- tio ns in either their free base form or their salt form. In a further ermnbodiment of the invention thes amina acids or amino acid analogues are used in a concentrat ion, which is sufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention methionine (or omther sulphuric amino acids or amino acid analogous) may be added to inhibit oxidation of met hionine residues to me- thi onine sulfoxide when the polypeptide acting as the therapeutic &@gent is a polypeptide co mprising at least one methionine residue susceptible to such ox=dation. By "inhibit" is in- ternded minimal accumulation of methionine oxidized species over time. Inhibiting methionine ox idation resuits in greater retention of the polypeptide in its prope=r molecular form. Any ste=recisomer of methionine (L, D or a mixture thereof} can be usecd. The amount to be added sh ould be an amount sufficient to irvhibit oxidation of the methionirme residues such that the armount of methionine sulfoxide is acceptable to regulatory agenciess. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Ge=nerally, this can be achieved by adding methionine such that th e ratio of methicnine adil ded to methionine residues ranges from about 1:1 to about 100®0:1, such as 10:1 to about 1080:1.

In a further embodiment of the invention the formulation fur—ther comprises a stabiliser— se-lected from the group of high moiecular weight polymers or low molecular compounds. In a further embodiment of the invention the stabilizer is selected fror polyethylene glycol (e.g.

PE=G 3350). polyvinylalcohal (PVA), polyvinylpyrrolidone, carboxy-~hydroxycelluiose or de=rivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing suzbstances as monothioglycerol, thioglycolic acid and 2-methylthicOethanol, and different sad lts (e.g. sodium chloride). Each one of these specific stabilizers constitutes an alternative emmbodiment of the invention.

The pharmaceutical compositions may also comprise addit ional stabilizing agents, which further enhance stability of a therapeutically active polypeptide therein. Stabilizing aggents of particular interest to the present invention include, but ame not limited to, maeethionine and EDTA, which protect the polypeptide against mettionine oxidation, and a noonionic surfactant, which protects the polypeptide against aggregation associated with freeeze-thawing or mechanical shea ring.

In a further embodiment of the invention the formulation f urther comprises a swarfactant. In a further embodiment of the invention the surfactant is selected from a destergent, ethoxylated castor oil, polyglycolyzed glycerides, acetyl. ated monoglycerides,

6692.2204-WO ® sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers such as Pluronic® F68. poloxarmer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, starshaped PEO, polyoxyethylene and polyethylene cderivatives such as alkylated and alkoxylated derivatives {tweens, e.g. Tween-20, Tween-<30, Tween-80 and Brij- 35), polyoxyethylene hydroxystearate, monoglycerides or ethoxylated cdenvatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lecitins and phospholipids (eg. phosphatidy! serine, phosphatidyl chaline, phospha®idy! ethanolamine, phosphatidyl! inositol, diphosph atidy! glycerol and sphingomyelin), derivwates of phospholipids (eg. dipalmitoy! phosphatidic acid) and lysophosphoiipids (eg. palmitoy~| lysophosphatidyl-L- serine and 1-acyl-sn-glycero-3—phosphate esters of ethanolamine, cho line, serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)- derivativees of lysophosphatidyl and phosphatidyicholines, e.g. lauroyl and myristoy! derivatives of lyso. phosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositcal, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine ancd lysophosphatidylthreonine, and glycerophospholipids {eg. cephalins), cglyceroglycolipids (eg. galactopyransoide), sphingogiycolipids (eg. ceramides, gangliosides), dodecylphosphochaline, hen egg lysolecithin, fusidic acid derivatives- e.g. sodium tauro- dihydrofusidate elc.), long-chain fatty acids and salts thereof C6-C12 (-eg. oleic acid and caprylic acid), acylcarnitines and derivatives, N®-acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, N®-ac-ylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N*-acylated deriva tive of a tripeptide comprising any combmination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CASS registry no [577-11- 7). docusate calcium, CAS registry no [128-438-4]}, docusate potassiurm, CAS registry no [7491-09-0}), SDS (sodium dodecyl! sulfate or sodium lauryl sulfate), scodium caprylate, cholic acid or derivatives thereof, bile acids and salts thereof and glycine or taaurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium t&aurochelate, sodium glycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonaate, anionic (alkyl-aryl- sulphonates) monovalent surfactants, zwitterionic surfactants (e.g. N-=alkyl-N,N- dimethylammeonio-1-propanesulfonates, 3-cholamido-1-propyldimethylzammaonio-1- propanesulfonate, cationic surfactants (quarternary ammonium basessz) (e.g. cetyl- trimethylammonium bromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecy! 3-

D-glucapyranoside), poloxamimes (eg. Tetronic's), which are letrafuncitional block ’ 35 copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant may be selected from the group of imidazoline

6692 204-WO ® derivatives, or mixtures thereof. Each one of these specific surfactants constitutes an alternative embodiment of the inventi on.

The use of a surfactant in pharmaceutical compositions is well-known to the skilled person . For convenience reference 1s made to Remington: The Science and Practice of Pharmasacy, 19" edition, 1995.

A composition for parenteral a dministration of GLP-1 com pounds may, for example, Ee prepare=d as described in WO 03/002 136.

It is possible that other ingre=dients may be present in the peptide pharmaceutical formulation of the present invention. Such additional ingredients= may include wetting agen ts, emulsiffiers, antioxidants, bulking age=nts, tonicity modifiers, chel ating agents, metal ions, oleagirmous vehicles, proteins (e.g., h uman serum albumin, gelatin or proteins) and a zwit- terion ((eg., an amino acid such as beetaine, taurine, arginine, glwycine, lysine and histidine).

Such a dditional ingredients, of course, should not adversely affeact the overall stability of thee pharmaceutical formulation of the preesent invention.

Pharmaceutical compositiorm s containing a compound =ccording to the present in— ventior— may be administered to a pakient in need of such treatment at several sites, for ex— ample, at topical sites, for example, skin and mucosal sites, at ssites which bypass absorp- tion, fo rexample, administration in ar artery, in a vein, in the he-art, and at sites which in- volve ambsorption, for example, admin istration in the skin, under —the skin, in a muscle or in the abdom en.

Administration of pharmaceutical compositions accordimng lo the invention may be througkn several routes of administrat ion, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the brom- chioless and alveoli or a combination ®&hereof, epidermal, dermal, transdermal, vaginal, rectal, ocular. for examples through the con junctiva, uretal, and parent eral to patients in need of such a treatment.

Compositions of the current invention may be administeered in several dosage forms, for example, as solutions, sus-pensions, emulsions, microemulsions, multiple emul- sion, foams, salves, pastes, plasters _, ointments, tablets, coated tablets, rinses, capsules, “for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsuless, drops. gels, sprays, powder, aerosolss, inhalants, eye drops. oprithalmic ointments, ophtha |- mic rin ses, vaginal pessaries, vaginal rings, vaginal ointments, i njection solution, in situ transfcorming solutions, for example i n situ geliing, in situ setting , in situ precipitating, in sit u crystal lization, infusion solution, and implants.

6692.204-WO ® Compositions of the irvention may further be compounde din, or attached to, for example through covalent, hycrophobic and electrostatic interactiosns, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the compound, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof. Exampless of carriers, drug delivery systems &nd advanced drug deliv- ery systems include, but are not limited to, polymers, for example eellulose and derivatives, polysaccharides, for example clextran and derivatives, starch and derivatives, poly(vinyl al- cohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co- polymers thereof, polyethylene= glycols, carrier proteins, for examp le albumin, gels, for ex- ample, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticula tes, liquid crystals and dispersions thereof, L2 phase -and dispersions there of, well knowr to those skilled in the art of phase behaviour in lipid-wat er systems, polymeric micelles, mul tiple emulsions, self- emulsifying, self-microemulsify=ing, cyclodextrins and derivatives th. ereof, and dendrimers.

Compositions of the current invention are useful in the for mulation of solids, semi- solids, powder and solutions for pulmonary administration of the compound, using, for ex- ample a metered dose inhaler, dry powder inhaler and a nebulizer. all being devices well known to those skilled in the art.

Compositions of the current invention are specifically useful in the formulation of controlled, sustained, protractimng, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions are useful in formulation of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), weall known to those skilled in the art. Even more preferably, are ’ controlled release and sustainead release systems administered su bcutaneous. Without limit- ing the scope of the invention, examples of useful controlled releasse system and composi- tions are hydrogels, oleaginouss gels, liquid crystals, polymeric mic=eiles, microspheres, nanoparticles,

Methods to produce c ontrolled release systems useful for compositions of the cur- rent invention include, but are not limited to, crystallization, conderisation, co-cystallization, precipitation, co-precipitation, eemulsification, dispersion, high pres=sure homogenization, en- capsulation, spray drying, microencapsulation, coacervation. phas«e separation, solvent evaporation to produce micros pheres, extrusion and supercritical fluid processes. General reference is made to Handboo k of Pharmaceutical Controlled Release (Wise, D.L., ed. Mar- cel Dekker, New York, 2000) aand Drug and the Pharmaceutical Sciences vol. 99: Protein

Formulation and Dehvery (MaccNally, E.J., ed. Marcel Dekker. News York. 2000).

6692 204-WO @® Parenteral administration may be performed by subcutaneouwus, intramuscular, in- traaperitoneal or intravenous injection by means of a syringe, optionalBy a pen-like syringe.

ARternatively, parenteral administration can be performed by means <f an infusion pump. A fun rther option 1s a composition which may be a solution or suspensiosn for the administration off the compound according to the present invention in the form of a rasal or pulmonal spray.

Ass a still further option, the pharmaceutical compositions containing tthe compound of the in vention can also be adapted to transdermal administration, e.g. by needle-free injection or fraom a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.

The term “stabilized formulation” refers to a formulation with ircreased physical stabil- ity, increased chemical stability or increased physical and chemical sta bility.

The term “physical stability" of the protein formulation as used herein refers to the te ndency of the protein to form biologically inactive and/or insoluble aggregates of the protein ass a result of exposure of the protein to thermo-mechanical stresses amnd/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic sur—faces and interfaces.

Physical stability of the aqueous protein formulations 1s evaluated by means of visual inspec- tion and/or turbidity measurements after exposing the formulation filled in suitable containers (e .g. cartridges or vials) to mechanical/ physical stress (e.g. agitation) amt different temperatures fo r various time periods. Visual inspection of the formulations is perforrmed in a sharp focused ligght with a dark background. The turbidity of the formulation is charact erized by a visual score ra nking the degree of turbidity for instance on a scale from G to 3 (a formulation showing no tu rbidity corresponds to a visual score ©, and a formulation showing vissual turbidity in daylight corresponds to visual score 3). A formulation is classified physical unstzable with respect to pro- te in aggregation, when it shows visual turbidity in daylight. Alternativelyw, the turbidity of the for- mm ulation can be evaluated by simple turbidity measurements well-knov=vn to the skilled person.

Physical stability of the aqueous protein formulations can also be evaluated by using a spec- trcoscopic agent or probe of the conformational status of the protein. THe probe is preferably a srnalt molecule that preferentially binds to a non-native conformer of th_e protein. One example of a small molecular spectroscopic probe of protein structure is Thiofla=vin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibriis. In the presence of fitorils, and perhaps other protein configurations as well, Thioflavin T gisves rise to a new excita- ticon maximum at about 450 nm and enhanced emission at about 482 rm when bound to a fibri protein form. Unbound Thioflavin T is essentially non-fluorescent at thes wavelengths.

Other small molecules can be used as probes of the change=s in protein structure from native to non-native states. For instance the “hydrophobic patch” probes that bind pref-

6692.204-WO o erentially to exposed hydrophabic patches of a protein. “The hydrophobic patches are gener- ally buried within the= tertiary structure of a protein in its mative state, but become exposed as a protein begins to wunfolg or denature. Examples of thesse small molecular, spectr-oscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthrooline or the like. Other spectros «opic probes are metal-amino acid ccomplexes, such as cobalt metal complexes of hydro gohobic amino acids, such as phenyl=lanine, leucine, 1soleucin-e, methion- ine, and vatine, or the like.

The term “chemical stability” of the protein formulation as used herein re fers to chemical covalent c hanges in the protein structure leadi ng to formation of chemical degrada- tion products with potential less biological potency and/or potential increased immunogenic properties compare« to the native protein structure. Var ious chemical degradatior products can be formed depeanding on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoid ed and increasing amounts of chermmmical degradation products is often seen during storage= and use of the protein formulation &as well-known by the pers- on skilled in the art. Most proteims are prone to deamidation, a proce=ss in which the side chair amide group in glutaminyl or asparaginyl residues is hydrolyse-d to form a free carboxylic acid.

Other degradations pathways involves formation of higha molecular weight transfom rmation products where two or more protein molecules are cova lently bound to each othe r through transamidation and/or disulfide interactions leading to fcarmation of covalently bowand dimer, oligomer and polyrms er degradation products (Stability of Protein Pharmaceuticals. Ahern.

T.J. & Manning M.C., Plenum Press, New York 1992). Oxidation (of for instance smethionine residues) can be meentioned as another variant of chemical degradation. The che mical stabil- : ity of the protein for mulation can be evaluated by measuring the amount of the chemical degradation produc ts at various time-points after exposwure to different environme=ntal condi- tions (the formation of degradation products can often bee accelerated by for instaance in- creasing temperatu re}. The amount of each individual d egradation product is ofteen deter- mined by separatiomn of the degradation products depen ding on molecule size and/or charge using various chrorsatography techniques (e.g. SEC-HLC and/or RP-HPLC).

Hence, as outlined above, a “stabilized formulamtion” refers to a formulatison with in- creased physical stability, increased chemical stability o=r increased physical and chemical stability. In general, a formulation must be stable during use and storage (in com pliance with recommended use and storage conditions) until the expiration date is reached.

6692.204-WO o In one embodiment of the invention the pharmaceutical formulation comprising the compound according to the pressent invention is stable for more tha n 6 weeks of usage and fo r more than 3 years of storages.

In another embadimen t of the invention the pharmaceutica| formulation comprising th e compound according to the present invention is stable for more than 4 weeks of usage ard for more than 3 years of storage.

In a further embodimert of the invention the pharmaceutical formulation comprising th e compound according to the present invention is stable for more than 4 weeks of usage ard for more than two years of storage. in an even further emb» odiment of the invention the pharmaceutical formulation comprising the compound is stable for more than 2 weeks of usage= and for more than two years of storage.

Pharmaceutical compositions containing a GLP-1 derivative according to the present in=vention may be administered parenterally to patients in need of such a treatment. Parenteral acddministration may be performed by subcutaneous, intramuscular or intravenous injection by m eans of a syringe, optionally a pen-like syringe. Alternatively, paren teral administration can be performed by means of an in fusion pump. A further option is a cornposition which may be a powder or a liquid for the admini stration of the GLP-1 derivative in thea form of a nasal or pul- m onal spray As a still further option, the GLP-1 derivatives of the inv-ention can also be admin- istered transdermally, e.g. from =a patch, optionally a sontophoretic pa tch, or transmucosally, e. g. bucally.

Thus, the injectable cormpositions of the GLP-1 derivative of the invention can be pre- pared using the conventional techniques of the pharmaceutical indusstry which involves dissolv- in g and mixing the ingredients a= appropriate to give the desired end product.

According to one proce=dure, the GLP-1 derivative is dissolved in an amount of water w hich is somewhat less than the final volume of the composition to b e prepared. An isotonic acgent, a preservative and a buffer is added as required and the pH value of the solution is ad- jui sted - if necessary - using an acid, e.g. hydrochioric acid, or a base, e.g. aqueous sodium hwydroxide as needed. Finally, th € volume of the solution is adjusted with water to give the de- si red concentration of the ingredients.

6E6592.204-WQ

C Further to the above-mentioned components, solutions containing a GLRP-1 derivative according to the preseritinvention may also contain a surfactant in order to improve the solubil- ity and/or the stability os f the GLP-1 derivative.

A compositiora for nasal administration of certaain peptides may, for example, be pre- pared as described in European Patent No. 272097 (1 Novo Nordisk A/S) or in WwVO 93/18785.

According to one preferred embodiment of thes present invention, the GL_.P-1 derivative is provided in the form of a composition suitable for administration by injection. Smich a compo- sition can either be an injectable solution ready for use or it can be an amount of a solid com- position, e.g. a lyophilissed product, which has to be dis=solved in a solvent before it can be mn- jected. The injectable solution preferably contains not |=ess than about 2 mg/ml, pmreferably not less than about 5 mg/renl, more preferred not less than about 10 mg/ml of the GLP-1 derivative and, preferably, not more than about 100 mg/ml of the GLP-1 derivative.

The GLP-1 de=rivatives of this invention can be used in the treatment of wsarious dis- eases. The particular GLP-1 derivative to be used and the optimal dose level for —any patient will depend on the dise ase to be treated and on a varie=ty of factors including the =efficacy of the specific peptide derivat ive employed, the age, body we ight, physical activity, and diet of the pa- tient, on a possible corrbination with other drugs, and ©n the severity of the case _ It is recom- mended that the dosage of the GLP-1 derivative of thiss invention be determined For each indi- vidual patient by those skilled in the art.

In particular, itt is envisaged that the GLP-1 de=rivative will be useful for the preparation : of a medicament with & protracted profile of action for tthe treatment of non-insulir dependent diabetes mellitus and/or for the treatment of obesity.

In another aspect the present invention relate=s to the use of a compoland according to the invention for thex preparation of a medicament.

In one embodiment the present invention relaates to the use of a compeound accord- ing to the invention for the preparation of a medicame=nt for the treatment of hy perglycemia. type 2 diabetes, impaired glucose tolerance, type 1 dmabetes, obesity, hyperten-sion, syn- drome X, dyslipidemia , B-cell apoptosis, B-cell deficie ncy, myocardial infarction , inflammatory bowel syndrome, dyspepsia, cognitive disorders, e.g. cognitive enhancing, neuroprotection, atheroschlerosis, coro nary heart disease and other cardiovascular disorders.

6692 20M4.WO 4 In another embodimeent the present invention relates to the use of a compound ac- cording to the invention for the preparation of a medicament for the treatment of small bowel syndrome, inflammatory bowel syndrome or Crohns disease.

In another embodimaent the present invention relates to the use of a compound ac- cording to the invention for the preparation of a medicament for the treatment of hypergly- cemia, type 1 diabetes, type 2 diabetes or B-cell deficiency.

The treatment with = compound according to the present irvention may also be combined with combined with a second or more pharmacologically &ctive substances, e.g. selected from antidiabetic ag ents, antiobesity agents, appetite regulating agents, antihyper- tensive agents, agents for th e treatment and/or prevention of compl ications resulting from or associated with diabetes and agents for the treatment and/or preve ation of complications and disorders resulting from or associated with obesity. In the prese=nt context the expression "antidiabetic agent” includes compounds for the treatment and/or praaphylaxis of insulin resis- tance and diseases wherein insulin resistance is the pathophysiological mechanism.

Examples of these pharmacologically active substances ar e: Insulin, GLP-1 ago- nists, sulphonylureas (e.g. tolbutamide, glibenclamide, glipizide and «gliclazide), biguanides e.g. metformin, meglitinides, glucosidase inhibitors (e.g. acorbose), glucagon antagonists,

OPP-IV (dipeptidyl peptidases -IV) inhibitors. inhibitors of hepatic enzymes involved in stimula- tion of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, thiazolidin- ediones such as troglitazone and ciglitazone, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists amd agents acting on the ATP-dependerut potassium channel of the B-cells, e.g. glibenclamide, glipizide, gliclazide and repaglinide; C=holestyramine, colestipal, clofibrate, gemfibrozil, lovast atin, pravastatin, simvastatin, probucol | dextrothyroxine, neteglinide, repaglinide; B-blackers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors succh as benazepril, capto- pril, enalapril, fosinopril, lisinopril, alatriopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, di ltiazem and verapamil, and a-blockers such as doxaszosin, urapidil, prazosin and terazosin, CART (cocaine am- phetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4 (melano- cortin 4) agonists, orexin ant agonists, TNF (tumor necrosis factor) =agorusts, CRF (corticotro- pin releasing factor) agonistss, CRF BP (corticotropin releasing factor binding protein) an- tagonists, urocortin agonists , 33 agonists, MSH {melanocyte-stimul ating hormone) agonists,

MCH (melanocyte-concentraating hormone) antagonists, CCK (choleacystokinin) agonists, se- rotonin re-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors, mixed sero-

£692 2M3TLWO ® ” tonin and noradrenergic compounds, SHT (serotonirm} agonists, bombesin ago nists, galanin antagonists. growth he ormone, growth hormone releamsing compounds, TRH (thayreotropin re- leasing hormone! agonists, UCP 2 or 3 (uncoupling gorote n 2 or 3) modulators |, leptin zgo- nists, DA agonists (br-cmacriptin, doprexin). lipase/armylase inhibitors, RXR (re tinoid X recep- tor) modulators. TR {3 agonists, histamine H3 antagonists.

It should be wnderstoad that any suitable ccombination of the compournds according ta the invertion with cane or more of the above-menti oned campounds and optisonally one or more further pharmaccologically active substances are considered to be within the scope of the present invention _

The present invention is further illustrated b vy the following examples hich, how- ever, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing descrip tion and in the following examp les may, both separately &and in any combination thereof, be material for realising the inveantion in diverse forms thereof.

BEG2 204-WC ® 6 C

EXAMPLES

The following acronyms for commercially avail=ase chemicals are used:

DMF : N.N-Dimethylforman—de.

BCC : N,N-Dicyclohexylcar bodimide

NMP : N-Methyl-2-pyrrchido=ne.

TFA : Trifluoroacetic acid.

THF : Tetrahydrofuran

DIEA : diisopropylethylamin e

HO : water

CH,RCN acetonitrile

HBTU 2-(1H-Benzotriazol- RW -yl)-1.1,3.3 tetramethyluroniLum hexatluoro- phosphate

Fmoc 9 H-fluoren-9-ylmettoxycarbonyl

Bac : tert butyloxycarbony

OtBu : tert butyl ester tBu : tert butyl

Trt : _ triphenyimethyi

Pmc : 2.2,5,7.8-Pentametmyl-chroman-6-sulfony!

Dde : 1-(4,4-Dimethyl-2 6- dioxocyclohexylidene)ethyi

DCM : dichloromethane

TIS : trilsopropylsilane) ELO: diethylether

H-Glu(OH)-OBu": : L-Glutamic acid «-te=r-buty! ester

HOOC-(CH,),-COONSuU: w-Carboxy=tridecanoic acid 2,5-dioxopyrwolidin-1-yl ester,

HOOC-(CH;)1,-COONSu: w-Carboxy _pentadecanoic acid 2,5-dioxcapyrrolidin- 1-yi ester.

HOOC-(CH;),,-COONSu: w~Carboxy heptadecanoic acid 2 5-dioxcoopyrrolidin-1-yi ester.

HOOC-{CH;):s-COONSu: w-Carboxy nonadecanoic acid 2,5-dioxa pyrrolidin-1-yl ester.

662 202.WO

G1 ® Abbreviations: rt Room temperature

PDMS: Plasma Desorption Mass Spectrometry

MALDI-MS: Matrix Assisted Laser Desorption/lonisation Mass Spectrometry

HPLC: High Perfarreaance Liquid Chromatography amu: atomic mass Lins

Analytical:

Resistance of a pep-tide to degradation by dipeptidyl amin opeptidase IV is determined by the following degradation assay :

Aliquots of the peptides are incubated at 37 °C with an aliquot of purified dipe ptidy! aminopeptidase IV For 4-22 hours in an appropriate buffer at pH 7-8 (buffer not being =lbu- min). Enzymatic rea clions are terminated by the addition of trifluoroacetic acid, and the= pep- lide degradation proeducis are separated and quantified using HPLC or LC-MS analysis. One method for performi ng this analysis is: The mixtures are a pphed onto a Zorbax 300SB—C18 {30 nm pores. 5 pm particles) 150 x 2.1 mm column and eluted at a flow rate of 0.5 ml/min with a linear gradiermt of acetonitrile in 0.1% trifluoroacetic acid (0% -100% acetonitrile over 30 min). Peptides ard their degradation products may be monitored by their absorbance at 214 nm (peptide bords) or 280 nm (aromatic amino acids ), and are quantified by integ rauon of their peak areas. The degradation pattern can be deter amined by using LC-MS wheres MS spectra of the separ ated peak can be determined. Percen tage intactdegraded compowund at a given time is used for estimation of the peptides DPPIV stability.

A peptide is defined as DPPIV stabilised when it is 10 times more stable than the natural peptide based on pesrcentage intact compound at a given time. Thus, a DPPIV stabilisead

GLP-1 compound is al least 10 times more stable than GL_P-1(7-37).

General Synthetic methods

The peptides may toe synthesized on Fmoc protected R ink amide resin (Novabiocheem) or chlorotrityl resin or & similar resin suitable for solid phase peptide synthesis. Boc cheamistry may be used but more conveinient is using Fmoc strategy eventually on an Applied Biosys. tems 433A peptide synthesizer in 0.25 mmol scale using the FastMoc UV protocols which employ HBTU (2-( TH-Benzotriazol-1-yl)-1,1.3.3 tetrame: thyluronium hexafluorophos- phate) mediated couplings in N-methyl pyrrolidone (N-methyl pyr rolidone) (HATU is better suit ted far hindered couplings) and UV monitoring of the deprotect ion of the Fmoc protection group.

Other coupling reag ents besides from HBTU and HATU a s described in e.g. Current Opinion

£6692 2C4-WO ® in Chemical Bi clogy. 2004. 8211-221 may also © e used The protected amino acid deriva- lives used mayw de stancard Fmoc-amino acids s uppliea in preweighed cartridges {Applied

Biosystems) swuitable for the ABI433A synthesizer with the exception of ure natural aminoacids such as Fmoc:=-Aib-OH (Fmoc-aminoisobutyric ac id) which are purchase=d from a supplier such as Bacheam ard transferred to empty cartridges. The last amino ac=id coupled may be

Boc protected.

The attachmert of sidechains and linkers to specific lysine residues «on the crude resin bound protectead peptide may eventually beintrodu ced in a specific positicon by incorporation of Fmoc-Lys(I>®de)-OH during automated synthesi s followed by selectives deprotection with hydrazine. Otheer orthogonal protecting groups may~ be used on Lysine.

Procedure for aremoval of Dde-protection. The resi n (0.25 mmol) may be placed placed in a manual shaker filtration apparatus and treated with 2% hydrazine in N-me#thyl pyrrolidone (20 ml, 2x12 min) t-o remove the DDE group and subse=quently washed with N—methyi pyrrolidone (4x20 ml).

Procedure for attachment of sidechains to Lysine ressidues. }

The amino acid (4 molar equivalents relative to resin) may be dissolved mn N-methyl pyrroli- done/methyienss chloride (1:1, 10 mi). Hydroxybenzotriazole (HOB) (4 molar equivalents relative to resi) and disopropylcarbodiimide (4 molar equivalents relative lo resin) is added and the solution was stirred for 15 min. The solution is added to the resin and diisopro- pyethylamine (~4 molar equivalents relative to resirm) is added. The resin i=s shaken 24 hours at room temperature. The resin is washed with N-rmethyl pyrrolidone (2x20= ml), N-methy! pyr- rolidone/MethyEene chloride (1:1) (2x20mi) and met hylene chloride (2x20 ral).

Procedure for removal of Fmaoc-protection: The ressin (0.25 mmol) is place=d in a filter flask in a manual shak ing apparatus and treated with N-m ethyl pyrrolidone/methy~lene chloride (1:1) (2x20 ml) and =with N-methyl pyrrolidone (1x20 ml) , a solution of 20% pipeeridine in N-methyl pyrrolidone (3x.20 ml, 10 min each). The resin i1s wae shed with N-methyl pyrrolidone (2x20 ml).

N-methyl pyrroBidone/methylene chloride (1:1) (2x20ml) and methylene chi- oride (2x20 ml).

Procedure for «cleaving the peptide off the resin:

The peptide is cleaved from the resin by stirring for 180 min at room temp - erature with a mix- ture of tnfluoro acetic acid, water and tnisopropylsil ane (35:2.5:2.5). The czleavage mixture is filtered and the- filtrate is concentrated to an oil by & stream of nitrogen. THe crude peptide is

6632 204-vO ® orecipitated from this oi with «45 mil diethyl ether and washed 3 times with 45 ml diethyi ether.

Purification: The crude peplid e may be purified by semipreparative HPLC on a 20 mm x 250 mm column packed with 7u C-18 silica. Depending on the peptid e one or two purification systems may used.

Ammonium sulphate: The ceolumn is equilibrated with 40% CH3;CCN in 0.05M (NH,);SO,. which is adjusted to pH 2.5 with concentrated H,SC,. After drying the crude peptide is dis- solved mn 5 ml 50% acetic acid H,0 and diluted to 20 ml with H,O ard injected on the column which then is eluted with a gr-adient of 40% - 60% CH;CN in 0.05MB (NH,),SO,, pH 2.5 at 10 ml/min during 50 min at 40 °CC. The peptide containing fractions is «allected and diluted with 3 volumes of H,O and passed through a Sep-Pak® C18 cartridge (Waters part. #:51910 ) which has been equilibrated with 0.1% TFA. It is then eluted wittw 70% CH,CN containing 0.1% TFA and the purified pe=ptide is isolated by Iyophilisation after dilution of the eluate with’ water

TFA: After drying the crude goeptide is dissolved in 5 ml 50% acetic acid H,O and diluted to ml with H,O and injected -on the column which then is eluted wi th a gradient of 40-60 %

CH;CN in 0.1% TFA 10 mi/min during 50 min al 40 °C. The pepticie containing fractions is collected. The purified peptide is lyophilized after dilution of the elua te with water. 20 The final product obtained may be characterised by analytical RP-HEPLC (retention time) and by LCMS

The RP-HPLC analysis performed in these in the experimental section was performed using

UV detection at 214 nm and & Vydac 218TP54 4.6mm x 250mm Six C-18 silica column {The

Separations Group, Hesperia , USA) which was eluted al 1 m/min at 42 °C. The different elu- tion conditions were:

Al: Equilibration of t he column with in a buffer consisting &f 3. 1M (NH,),S0., which was adjusted to pH 2.5 wilh concentrated H,SO, and efution by a gradient of 0% to 60% CH3CN in the same buffer during 50 min. 81: Equilibration of %he column with 0.1% TFA / HO and elution by a gradient of 0% CH3CN/0.1%% TFA / HO to 60% CHLCN / 0.1% TFA / H,0 during 50 min.

B6: Equilibration of &he column with 0.1% TFA / H,O and elution by a gradient of 0% CH;CN/ 0.1% TFA / HO 10 30% CH3CN/ 0.1% TFA / H,0 during 50 min.

An alternative system was:

B4 : The RP-analyses was pezrformed using a Alliance Waters 2695 system fitted with a Wa- ters 2487 dualb and detector. UV detections at 214n#n and 254nm were col-

A632 204-WO c4 ® lected using a Symmetr=y30C C18 , 5 um. 3.9 mm x 150 mm column, 42 °C.

Eluted with a linear gradient of 5-85% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.02%) in water over 15 mintates at a flow-rate of 1. 0 min/min.

LCMAS was performed on a selup conssisting of Hewlett Packard se=ries 1100 G1312A Bin

Purmp, Hewlett Packard series 1100 Column compartment, Hewlett Packard series 1100

G1315A DAD diode array detector, Hewlelt Packard series 1100 MASD and Sedere 75

Evamporative Light Scattering detectorcontrolled by HP Chemstatio n software. The HPLC pump is connected to two eluent reservoirs containing:

A: 0.05% TFA/water

B: O.05%TFA/acetonitrile

Or alternatively the two systems may te:

A: 1 OmM NH,OH in water

B: 1 OmM NH,OH in 80% acetonitrile

The= analysis was performed at 23° C toy injecting an appropriate volume of the sample (pref — eraboly 20 pl) onto the column which is eluted with a gradient of A =and B.

The= HPLC conditions. detector setting s and mass spectrometer seattings used are giving in the following table.

Column Waters Xterra MS C-18 (C50 X 3 mm id 5 um)

Gra. dient 5% - 100% acetanitrile lirmear during 6.5 min at 1.5Smi/~“min

Detection 210 nm (analogue output from DAD)

ELS (analogue output from ELS)

MS ionisation mode API-ES. Scan 550-1500 amu step 0.1 amu

Alte=rnatively, LC-MS analysis could bez performed on a PE-Sciex AP] 100 mass spectrome- ter equipped with two Perkin Elmer Se ries 200 Micropumps, a Per kin Elmer Series 200 autosampler, a Applied Biosystems 785A UV detector and a Sede x 75 Evaporative Light sca ttering deteclor. A Waters Xterra 3 .0 mm x 50 mm 5; C-18 silicza column was eluted at 1.5 ml/min at room temperature. It wass equilibrated with 5 % CH,CTN / 0.05% TFA / H,0 and eluted for 1.0 min with 5% CH;CN / 0.005% TFA / H,0 and then wit h a near gradient to 90%

CH=SCN/ 0.05% TFA / HO over 7 min. Detection was by UV detec=tion at 214nm and Evapo— rativve light Scattering. A fraction of the= column eluate was introduczed into the ionspray inter-

8692 204- WED ® 65 face of a PE-Sciex API 100 mass spectrometer The mass range -300 - 2000 amu was scanned every 2 seconds during the rin

MALDI-TOF MS analysis was carne d cut using a Voyager RP inst rument (PerSeptive Bio— systems Inc. Framingham, MA) equipped with delayed extraction and operated in finear mode. Alpha-cyano-4-hydroxy-cinnamic acic was used as matrix, and mass assignments were based on external calibration

Example 1

N®7.(2-(2-(2-(dodecylamino)elhoxy) ethoxy)acetyl)-[Aib* #2 Lys? |CSLP-1(7-37)amide

WA

Jo . PTE I

I . N

A resin (Rink amide, 0.68 mmol/g Novabiochem 0.25 mmole) weas used to produce the pii- mary sequence on an ABI433A ma chine according to manufactumrers guidelines. All protect- ing groups were acid labile with the exception of the residue u sed In position 37 (Frmo- clys(ivDde)-OH, Novabiochem) aliowing specific deprotection of this lysine rather than any other lysine.

Procedure

The above prepared resin (0.25 mmocle) containing the GLP-1 analogue amino acid se- quence was placed in a manual shaker/filtration apparatus and treated with 2% hydrazine in

N-methyl pyrrolidone in (2x12 mir. 2x20 mi) to remove the Dmde group. The resin vevas washed with N-methyi pyrrolidone (4x20 ml). Fmoc-8-amino-3,6-d ioxaoctanoic acid (Neos ys- tem FA03202) (4 molar equivalents relative to resin} was disssolved in N-methyl pyrroli- done/methylene chloride (1:1, 20 rl). Hydroxybenzotriazole (H OB) (4 molar equivale nis relative to resin) and diisopropylcarbodiimide (4 molar equivale=nis relative to resin) was added and the solution was stirred for 15 min. The solution was added lo the resin and diisopropylethylamine (4 molar equivalents relative to resin) waas added. The resin was shaken 24 hours at room temperature. The resin was washed with N-methyl pyrrolidone (4x20 ml}. A solution of 20% piperidine in N-methyl pyrrolidone (3x20 ml. 10 min each) was added to the resin while shaking. The resin was washed with N-me=thyl pyrrolidone (4x20 rn).

66532 204.WO ® Dodecanoic acid {43 molar equivaients relative to resi} was dissclved in N-methyl pyrroli- done/methylene chlonde (1°11 20 ml} Hydroxybenzotrrazole hydrate (HOBt: H,O) (<3 molar equivalents relatives to resin) and diisopropyicarbodiimide (4 molar equivalents relative to resin) were added and the solution was stirred for 15 min. The solution was added to the resin and diisopropylethylamine (4 molar equivalents realative to resin) was added. The resin was shaker 24 hou rs at room temperature. The resin wwas washed with N-methyl pyrr clidone (2x20 ml), N-methy/| pyrrolidone/methylene chloride (1 :1) {2x20ml) and methylene chloride (2x20 ml). The pep tide was cleaved from the resin by =strring for 180 min at room te mpera- ture with a mixture of trifluoroacetic acid. water and triis=opropylsilane (95:2.5:2.5 15 mil). The 0 cleavage mixture wes filtered and the filtrate was con centrated to an oil in vaccuusm. The crude peptide was precipitated from this oil with 45 ml Hiethyl ether and washed 3 tims es with 45 mi diethyl ether. The crude peptide was purified by preparative HPLC on a 20 mmm x 250 mm column packed with 7u C-18 silica. The crude peptide was dissolved in 5 ml 50% acetic acid in water and diluted to 20 mi with H,O and inje=Cled on the column which then was eluted with a gradient of 40-60 % (CH;CN in water wilt 0.1% TFA) 10 ml/min during 50 min at 40 °C The peptide containing fractions were collec®ed. The punfied peptide was Ilyophi- lized after dilution o f the eluate with waler.

HPLC: (method B86»: RT=32.8 min

HPLC: (method A1>»: RT=43.6 min

LCMS: miz= 765.0 (M+5H)>", 857.0 (M+4H)"". 1275.0 {M+3H)*". Calculated (M+H) = 3825.0

Example 2

N®7-(2-(2-(2-(17-su I phohexadecanoylamino)ethoxy)ethcxy)acetyl)-[Aib® #2 3 Lys®’] GLP-1 (7- 37)amide

Oo hn

HN A on o ° HC CH, pe weg ptt, Lert 1-5 pmmssvoiell, Roaacee Uo N

O RC CH HC CH, fo) © lo} NH ae

The compound wass prepared according to the methods in Example 1 and in "Genera Syn- thetic methods”.

HPLC: (method A1): RT=45.5 min

LCMS: mz = 792.9 (M+5H)°", 890.9 (M+4H)*". 1320.9 (M+3H)>" Calculated (M+H)" = 3959.9

6692 20 4.WO ® i

Example 3

N'7-{2-[2-(2-{15-carbeoxypentacecanoylamino)ethox-ylethoxyjacetyl}-[Aib® 22° Lys”| GLP- 1(7-37)amide 0 an on Jo = HC CH fe} oO lo} : ) et fear sovesviel Toran a Sa N

H o H 5 H ee NH

HC cH, HC CH, HO ~~ NPP lo}

The compound was psrepared according to the meth ods in Example 1 and in "Genera! Syn- thetic methods”.

HPLC: (method B1): ERT=43.8 min

HPLC: (method A1): BRT=42.0 min

LCMS: m/z = 978.3 ( M+4H)*". 1303.8 (M+3H)"" Cal culated (M+H)" = 3909.6

Example 4

N%-(2-(2+(2-(17-carb oxyheptadecanoylamino)ethoxylethoxy)acetyl)Aib®% 3? Lys” |GLP-1(7- 37)amide 1

FEN

! Q c p ol Id ? b— N. ffm Got FT =5=D-V=§~§-y=L=E-N Q-A-A—K-E—F—l—A-w/-L—V~K- R- — NH, y ¥ 3 HO 'S . ii i i i 5 [e]

Prepared according {eo the methods in Example 1 ard in "General Synthetic rnethods”.

HPLC: {method B1): 3RT=46.4 min

HPLC: (method A1): RT=44.4 min

LCMS: m/z = 985.5 (C M+4H)™", 1313.4 (M+3H)>" Cal culated (M+H)® = 3937.6

Example 5

N"-{2-(2-(2-(19-carb oxynonadecanoylamino)ethoxyw Jethoxy)acety!)[Aio®?%® I_ys¥|GLP-1(7- 37)amide

6692 204-WO . ® > i an ~

ENTS C.. lo L oC G 6 mC CH, c

NTT ETF 1-5 DoV-5-8-V1 men SF e@AAK- EE mim A WeLmV mo N NRA NN ~

HN I . Fol D 5-5 oS A-A-K 1—A-W M 5 DS NH 1

HO ANAS

0

The <ompound was prepared according to the methods in Example 1 and in "Ge=neral Syn- theticc methods”.

HPLC: (method B1): RT=43.5 min

HPL®C: (method A1): RT=47.1 min

LCM S: miz = 992.5 (M+4H)*" 1322.6 (M+3H)"" Calculated (M+H)" = 3965.7

Exarmple 6 [Ab #3 Arg?» |GLP-1-(7-37)Lys(4-(Hexade canoylamino)-4(S)-carboxybutyryl) —OH

HN

= N o H.C CH, 0. OH

H > H © 3

H-N" >—N E-G-T-F-T-S-D-V-S-S-Y-L-E-N Q-A-A-R-E-F-I-A-W-L-V-R-N R-G-N~

H 0 H oO Sl H

H,C CH, H,C CH, (o») bal:

HO oO : The compound was prepared according to the methods in Example 1 and in "Ge=neral Syn- thetiac methods”.

HPL C: (method B6): RT= 36.28min

LCNMIS: m/z = 995 (M+4H)*", 1326 (M+3H)*" Calculated (M+H)' = 3977.6

Exammple 7 [AIb-272% Arg” >*|GLP-1-(7-37)Lys(2-(2-(2-(hexadecanoylamino)ethoxy)ethoxy)a cetyl)-OH

8 ©92.204-WO ® :

HNTS

( HC, CH, me Cr, y o vy

HN NN "NE G-T-F 1 S$ 0-Y-8-S-¥-L-E-N “p= Q-A-A-R-E-F=1—A-w-(-V-R-N__LR-G-N" . rr +N k=0 o 4

Pe ep NO

H,C Te

H

The compouncd was prepared according to the ranethods in Example 1 and #n "Generali Syn- thetic methods". ’

HPLC: (methoed B86): RT=37.1min .

LCMS: m/z= 999 (M+4H)*', 1332 (M+3H)** Calculated (M+H)" = 3993.7

Example 8

N7~(2-12-(2,6—(S)-Bis-{2-[2-(2- (dodecanoylarmino)ethoxy)ethoxylacetylamino}h exanoylamino)ethoxyletho=xy}) acetyl-[Aib®***GLP-1(7-37)amide a °\,

HN — 0}

HN 'N— ( 0 0 "OM 0 : wr, HR

Hen B Je -omr-ro1-5-0-v-s-s-v-t-e—N, J—0-a-a- A 1B © Ea

Ho MC Cry He CH, ° rH ) an roc soo ]

SN 0 { ne O Naan 0 Y et real

HC nC

The compoun d was prepared according to the rmetheds in Example 1 and sn "General Syn- thelic methodss".

HPLC: (methcod B6): RT=38.2 min

LCMS: m/z = 1106.7 (M+4H)*", 1475.3 (M+3H)™ Calculated (M+H)" = 443-3.0

Example 9 N¥7.(2-[2-(2.6 -(S)-Bis-{2-[2-(2- (tetradecanoy-lamino)ethoxy)ethoxylacetylamincihexanoylamino)ethoxyletioxy}) acetyl-{Aib® #2 > |GLP-1(7-37)amide

£692 204-W0 ® .

EN

PNT rr ih x vio Cw el : RN PGT To8 3m 5-5 LEN, JC Aman EoF iA WL wi LL ~

Hoo Eon, HC’ CH, fo) To "oo u HNC

H TS CONLIN ENA

? H a ¥° c” id o) — 2S

I NN

HC N

The compound was prepared according to the methods in Example 1and in "General Syn- thelic methods”.

HPLC: (method B6): RT=42.9 min

LCMS: m/z = 1120.9 (M+4H)*", 1494.2 (M+3H)>" Calculated (M+H)™ = 4480.4

Example 10 [AIB%#2% Arg” |GLP-1-(7-37 )Lys(2-(2-(2-(4-(Hexadecanoylamino) —4(S)- carboxybutyrylamino)ethoxy)e thoxy)acetyl}-OH { H,C_ CH, HC CH, =

TENE NE le SER SINUS ;

H 0 H 0 H fo) H )

HC CH, a o NH

LN ~0

SU SV Y

) c

HO lo}

The compound was prepared according to the methods in Example 1and in "General Syn- thetic methods”.

HPLC: (method 86): RT=36.0 min

LCMS: m/z = 1032.0 (M+4H)?", 1374.0 (M+3H)"" Calculated (M+H)™ = 4122.8

Example 11 [ADP PIGLP-1(7-37)Lys((2-{2-(4-[4-(4-Amino-9.10-diox0-3-sulf0-9, 10-dihydro-anthracen-1- ylamino)-2-sulfo-phenylamino}-6-(2-sulfo-phenylamino)-{1.3,5]triazin. -2-ylamino]-ethoxy}- ethoxy)-acetyl))amide

6592 204-WO @

HAN

H N a4 { z < "x 4 hn” Ne EG TeFeTeS DoveS-S=¥=t-F-N_ Ig a acKef-FmimAvee Lov Ken SeG-N_ ir, kT nee 0 en 5g ~ :

[0] NH, 2 ht

Pee Xx 5=%

CIOy °- Ne

H RA Ne Nd

Ah

MoS TY

HN

®

HCO

=o

[0]

Prepared by | oading DdelLys{(Fmoc)-OH onto Rink resin. The resin wa s then treated with piperidine as mn “Synthetic methods” to remove Fmoc selectively. 2-(2-(=2-(Fmoc- amino ethoxy )-ethoxy)acetic acid was coupled o nto the epsilon amingrowup of lysine and

Fmoc was removed. DMSO and Cibacron Blue 3GA (17 equivalents) (Sigma C-9534) was added and thea mixture was heated at 60 °C for 15 hours, washed with v=vater (3 times), methanol (2 tirmes), THF (2 umes) and diethyl e ther (2 times). The Dde protecting group was removed and t=he remaining amino acids were added as in "Synthetic methods”

HPLC: (metho d A1) RT=38.1 min

LCMS: m/z = 1110.4 (M+4H)* 1436.4 (M+3H) > Calculated (M+H)" = =4435.9

Example 12 [A> FIGLP -1(7-37)Lys(({2-(2-(2-{2-[2-(2-{2-[ 2-(1 5-carboxypentadecamnoylaming)- ethoxy]ethoxy)acetylamino)ethoxylethoxy}acety | amino)ethoxylethoxy}a cetyi))amide i

IES

HN Os

MN ] ] we. cr Ss

HN Hoh ermmmrrms oss vel Lous tA Wve Roa NH,

H CHAS ne’ on, "oo "oo NH en, EK 2 o aa mn"

I "Ha oe OS

The compouncd was prepared according to the raethods In Example 1 ard in "Genera Syn- thetic methods”

HPLC: (metho dA1) RT=41.2 min

HPLC: (metho dB6): RT=30.7 min

LCMS: m/z = 1069.1 (M+4H)". 1424.6 (M+3H)™" Calculated (M+H) = £3271

£692 204-WO ® ’

Example 13

N“-([2-(2-{(3-[ 2.5-diox0-3-{15-carboxypentadecyl=sulfanyl)-pyrrolidin-1-yl]- ] propionylamin ojethoxy)etnoxy)acetyl]-[D-Ala® Lys .GLP-1-[7- 37)Jamice 0 no

HN J 1

IN jl . o : o

HN" SN E-G=T=F-T-$-0-V=5~$-Y-L-E-G-Q~A —4~K E-F—I~A~W-L-V-K-G-R- CH

Hoo YO ’ O W 0

CH, J

N oxy wo

HO een fo)

The compound was prepared according to the mesthads in Example 1 and in "G eneral Syn- 10 thetic methods".

HPLC: (methoed At): RT=45.2 min

LCMS: m/z = 1004.0 (M+4H)*", 1338.2 (M+3H)>" Calculated (M+H)' = 4010.7

Example 14 15 [Aib®?2%A1a* 1GLP-1(7-37)Lys((2-(2-(2-(11- (oxalylamino)indecanoylamino)ethoxy)ethoxy)acesiyl-)))amide x .

HN o [que] fo} H, N I)

H-N jf E—GuTmfml=S—O=yvy—5—S—v—L—E~N Ced—i—K—€ F—l1—A-N—(—V—K~-N" A—A—N nm,

Hoo nc’ Cm, oe ‘ i 2 r = ¢ 0 Hpac,

H

The compounsd was prepared according to the meathods in Example 1 and in "G eneral Syn- thetic methodss”.

HPLC (method A1): RT=37.9 min

HPLC (method B11); RT=39.5 min

LCMS: mfz = 993.3 (M+4H)*", 1323 9 (M+3H)’" (Calculated (M+H)™ = 3967.6

Example 15

BES2.204-WO ® y (Aib® ZF 27° Ala¥)-GLP-1(7-37)Lys({2-[2-(2-{2-[2-(2-(15-carboxy-pen tadecancylamino)- ethoxy ethoxy)acetylamino)ethoxylettoxyjacetyl)amide

Q any

HAY ~ 5

I. 5 o Hy, C Pact : "0

HN ~——N_ EG TF T-50-v-55v-L Rosa eta I ~

H — Pas Hog H o - HN_ 0

HC CH H,C CH, TY lo] a 6] lo}

FENN 4p 0 H

The compound was prepared accord ing to the methods in Exampl € 1 and in "General Syn- thetic methods”

HPLC (method B6). RT=31.1 min

HPLC (method At): RT=41.9 min

LCMS<: m/z = 1376.3 (M+3H)* Calculated (M+H)" = 4125.8

Exam ple 16 [AIB®?23 Ala T|-GLP-1(7-37)Lys((2-{2-[1 1-(5-Dimethylaminonaphtalene-1- sulfon ylamino)undecanoylamino]ethCxy}ethoxy)acetyl)amide i,

Blan VRE Is o ~ c ° XR Y

H=N —, lL E-G-T—F—T=S—D—V=S—5—v —L—E-n C-A—A—K—E—F—l—A -W-L—V-K-N NV NE " <= on pe %: H 6 YT o LNNGENP NEN

SE s.

GO=Ce

SO

=,

The ¢ ompound was prepared accorcling to the methods in Examp le 1 and in "General Syn- thetic methods”.

HPLCZ (method A1): RT=42.6 min

HPLC= (method Bb): RT=30.4 min

LCMSS:miz = 1377 3 (M+3H)>" Calculated (M+H) = 4128.8

Exanwmple 17

5692 204-WQ ® 74 [AIb®?2 3 Ala¥"]-GLP-1(7-37)Lys(([2-(2-{2-[1-(- 4-Chicrobenzoyl)-5-methoxy- = -melhyl- H-indol- 3-yllac etylaminojethoxy)ethoxylacetyl))amide

T pl

HN = =

N : . HC. CH, he

H O H [eo]

H=NT N_AE G-T-F-T-§-D-Vv-5-S-Y-L-E-N 2 CA AREF A WL VR RA -N NH, ’

Ho IN Pra H 0 H NH

HC CH, H.C Cw, 0 or

Sm.)

SAND

77 : le} CL es

The compound was prepared according to th e methods in Example 1 and | n "General Syn- thetic rmethods”.

HPLC (method A1): RT=41.1 min

HPLC (method B6): RT=31.1 min

LCMS = m/z = 1351.8 (M+3H)" Calculated (MA+H)" = 4052.0

Example 18 [Aib®, Arg? GIL? 2 *GLP-1 H(7-37)Lys(2-C 2-(2- (octade=canoylamino)ethoxy)ethoxy)acetyl)an ide 0

VN

Pi Q tn 1 ay %

NT Hy AEG TF =To5-0-V 5-5 Y= LEE ~E-A= AREF —I=E-V/-L-V-R-G-R-G-N 1 )

Oc CH, H Om oy"

NNN PPP.

The compound was prepared according to th e methods in Example 1 and i n "General Syn- thetic rmethods”.

HPLC (method B6): RT=39.3 min

LCMS =: m/z= 1366.6 (M+3H)> Calculated (NA+H) = 4095.6

Exampple 19 (Aib® Ag? Glu? GLP-1(7-37)Lys(2-(2—(2- (eicosaanoylamino)ethoxy)ethoxy)acetyl)amidee

6692 204-WO ® 75 g

S

ANY AT

Ae Pr “0 ( 0 hon —N E-G-T—F—T=5-0=\" ~5:-5-¥ =: E~E~E~A—A=R-E=F —1—E-Wd-L V-R-G-R-G-N" > no, — = < 44 no NH hed CH, ~ =

PNP

The ccmpound was prepared according to the methods in Exampole 1 and in "General Syn- thetic methods”.

HPLC {method B6) RT=42.6 mim

LCMS: m/z = 1375.7 (M+3H)> Calculated (M+H)" = 4123.7

Example 20 [Gly® Arg®¥] GLP-1 H-(7-37)Lys (2-(2-(2-(2-(2-(2-(4-(octadecanowlamino)-4(S)- carboxybutyrylamino)ethoxy)ethos xy)acetyl)ethoxy)ethoxy)acetyl)-€OH [3 nS 5

HN or JENN

H—N i G—E=G—T =F =T=-S—D—V—S —§—Y=L—E—G-Q0—A—A—R~-t —fF —1—A—-W— imamcmnmom Aon —

H G y o H [e} Ox—"

ER i SS Gh Te

H

° Q oH "The compound was prepared according to the methods in Examp le 1 and in "General Syn- “thetic methods”.

HPLC (method B6): RT=38.0 mir (39 9%)

HPLC (method Al): RT=49.0 mim

LCMS: m/z = 1054 6 (M+4H)*" 1405 3(M+3H)> Calculated (M+) = 4211 8

Example 21 [AID Arg®®>)GLP-1 (7-37)Lys{2-C 2-(2-(2-[2-(2- {octadecanoylamino)ethoxy)etho=ylacetyllethoxy)ethoxy)acetyl)}-cOH

8692.2 C1 WC ® i

WNT

Q ne 0 EGTA T-S-DV-SSYLSGLAAREF-I-AWLVRG RGN z fr 3 x > FERRO Wo BER H 2 ' ~ Ot

H,C CH, AN " o 04 NH

HCN en ge LOA (AO

Ie) H

The compound was prepared according to the methods in Excmmple 1 and in "General Sy’ n- thetic metneds”.

HPLC (method BG): RT=38.7 min

LCMS: m/z = 1029.2 (M+4H)}™" 13714 (M+3H)* Calculated ( M+H) = 4110.8

Example 22 [AibY] -GLP-1-(7-37)Lys (2-(2 -(2-(4-(Hexadecanaoylamino)-4(S )- carboxybutyrylamino)ethoxy)esthoxy)acety!)-OH

HN OH

PE oO H

Q nN

I \ org £-6-0 A AEF 6 PO 7 ko

H o C H ~~

H,C cH HO : RY Ya

Re ee DY N o H o

The compound was prepared according to the methods in Example 1 and in "General Syn- thetic methods”.

HPLC (method B68): RT= 34.77 min

LCMS: m/z = 1000.3 (M+4H»* 1337.4 (M+3H)> Calculated ( M+H) = 4110.8

Example 23 [Aib® Arg? >] GLP-1(7-37) Ly s{2-(2-(2-(2-[2-(2-(4-(octadecancaylamino)-4- carboxybutyrylamino)ethoxy)ezthoxy)acetyllethoxy)ethoxy}acet—vl)}-OH

6837 204.WO © 77

HN

AN ° — HQ yy GT-F-T-S-D-V-S-S-Y- L-E-GQ-A-A-R-E-F-1-a W | VoRGRGN HA

O :

H,C=7 "CH, AN 0 “ye H 0 Py

H 0) i

The compourmd was prepared according to the methods in Example 1 and in "General Syn- thetic method s”

HPLC (metho-dB6): RT= 37.5 min

LCMS: m/z = 1414.9 (M+3H)>" Calculated (M+H) = 4239.8

Example 24 [AIB® Arg? ¥] GLP-1 (7-37)Lys{2-(2-(2-(2-[2-(2—(17- carboxyhepta noylamino)ethoxy)ethoxylacetylaFnino)jethoxy)ethoxy)acetyl)}-O+

TN,

As e 0 rT I aa —6-o-aasp-e-fFoimawt—vorg-sg—i lL o mc Ch, AS ° c Oy roe mg ne)

The compound was prepared according to the methods in Example 1 and in * ‘General Syn- thetic method=s".

HPLC (methoed B6): RT= 32.4 min

HPLC (methoed A1) RT= 43.8 min

LCMS: m/z = 1381 3 (M+3H)* Calculated (M+ H)' = 4140.0

Example 25 [GI°. Acg®*] GLP1-(7-37) Lys{2-(2-(2-(2-[2-(2 -(17- carboxyheptacdecanoylamino)ethoxy)ethoxylaceatyl)ethoxy)ethoxy)acetyl)}-OH

5697 204-WO ® ”

AN

HN . Cs (7 i So

HNN GE 3 TF = To 5-0 V §-5-Y-L—E-G-Q-A-A-R L-F—'-AW | V R-G-R-G-N Nom ~

H o o H [s} a Nk

CNP Sr a HH

The compouncid was prepared according to the me=thods in Example 1 an din "General Syn- thetic methodss"

HPLC (methocd At) RT= 42 3 min

LCMS:m/z= 1372.3 (M+3H)" Calculated (M+H) = 41127

Example 26 [AIDY)GLP-1-(T7-37)Lys(2-(2-(2-(2-(2-(2-(4-(Hexadecanoylamino)-4(S)- carboxybutyry#®amino)ethoxy)ethoxy)acetylamino) ethoxy)ethoxy Jacetyl}-OH

A Oo CH

HCH, NH

LT rsa ann etna o aes

Oo (a) N___~o

H.eC (e) 0 oro

ITAA TNT TN

TY Pa nl ri

HO =o

The compoun=d was prepared according to the maethods in Example 1 and in "General Syn- thetic method=s". ’

HPLC (method BE): RT= 33.5 min

LCMS: miz = 1040.3 (M+4H)* 1388.6 (M+3H)*" Calculated (M+H)" = 4-155 8

Example 27

NE -(2-(2-(2-(sdodecanoylamino)ethoxy)ethoxy)acetyi)-[Aib® Lys} GL_P-1 H(7-37)-amide

Q hv

HN al = I. o HC CH, lo

MeN rH Teor ns ove svell Lon anes awivny erp o N

H,C cr, H,C™ CH, J

H,C

6692 2=0¢.WO ®

The compound was prepared accerding to the methcocs in Example 1 ard in "Geeneral Syr- thetic methcds”

HPLC: (method B6): RT=32.8 min

LC-MS m/z = 765.7 (M+H)”" 957 0 (M+H)*". 1275.7 (M+H)>" = Calculated (M++1)" = 3822.9

Example 28

N®7-(2-(2-(2-(tetrade=canoylamino)ethoxy)ethoxy Jace=tyi)-[Aib> ys} GLP-1 H (7-37)- amide

It

ES

HAS ” o o HC CH, 0] eet Beem rss rel Sonn rere LotR TT NH, N

MH fo) “ J H Ie) H a) NH

H,C CH, H.C Chi,

Pai ” H.C

The compound was prepared according to the methcods in Example 1 and in "Geeneral Syn- thetic methods”.

HPLC: {method B6)= RT= 34,6 min

LC-MS: m/z =771,4 (M+5H)>". 964. 1(M+4H)" 128 4.9(M+H)> Calculated (M+)" = 3851.5

Example 29

N“"-(2-(2-(2-(hexad~ecanoylamino)ethoxy)ethoxy)acetyl)-[Aib® 2 Lys¥] GLP-1 (7-37)-amide : g

Hn

HC CH, 0

I HO H 9 >

H-N [NEC T-F-T-5-D-V-$-S-Y-L-E-N_ J} Qh A EF 1 AW-L-VK rR NH, N

Hoo ee Ho H o NH

H,C CH, H,C CH, Re a NG NN NES b le}

Prepared according to the methods in Example 1 an din "General Synthetic mewh-ods".

HPLC: (method 86) : RT= 36.8 min

LC-MS: m/z = 970.77 (M+4H)"", 1294.3 (M+3H)’" Cal culated (M+H) = 3879.6

Example 30

6692 204-W(C

J “0

N®7-(2-(2-(2-(~cctadecanaylamine)eihaxy Jetho xy )acetyl)-[Aib® 2 *Lys®’] GL_P-1 (7-37)-amide lo}

ANS

HNN ’ al =~ HC CH, d o

H CO H O XX Gg :

SEY T—— Noh Q AACE FAW LY KN RNTYNH, OY

H oO \ Ho H o ° NH ne CoH H,C CH, HC esa og

The compoun dwas prepared according to the methods in Example 1 and in "General Syn- thetic meth-ods".

HPLC: (method B6): RT= 39,4 min

LC-MS: m/z = 977.9 (M+4H)*", 1303,7(M+H)>’ Calculated (M+H) = 3907.6

Example 31

N®7-(2-(2-(2-( eicosanaylamino)ethoxy)ethoxy Jacelyl)-[Aib%% *Lys*’} GLP- 1(7-37)-amide 0

HNN

HN “1 = H,C CH, 0

H-N N i E-GT-FT-5DVSSYLEN ra O-AAK-E-F-i-AWL-V-K nara NH aN

NTN = HE HS 7 ww

H,C CH, H,C <H, HC enone

The compoun dwas prepared according to the methods in Example 1 and in "General Syn- thetic method s"

HPLC: (method B6): RT=42.7min

LC-MS’ m/z = 984.8 (M+4H)"", 1312.8 (M+3H)’" Calculated (M+H)* = 3935.7

Example 32

NE (2-(2-(2-( 2-(2-(2-{octadecanoylamino)elh oxy)ethoxyjacetylamino)ethcxyethoxy)acelyl))- [AIb® Arg? > 1_ys™*|GLP-1-(7-37)-OH

5692 204-W © ® 8: [@] 0] - ~ AR f I! a SE GN ~~ 0 Ao HN Ag

NA Rap NC Je oo Q 0

BE energy A on

HC CH,

The compound was prepared according to the methods in Example 1 and in "Genera! Svn- thetic methods”.

HPLC: (method B6) RT= 40.7min

LC-MS: m/z = 792.3(M+5H)>", ©89.8(M+4H)"", 1319 2(M+3H)’* Calculated (M+H)" = 3855.5

Example 33

N™*(2-(2-(2-(2-(2-(2- (octadecanoylamino)ethoxy)ettaoxy)acetylamino)ethoxylethoxyacety I) Arg? Lys*}GLP- 1(7-37)-OH

Pe f "

Lm ) oi Le i” 0 oe CH, = IN

H-N i ATE-GTT—F~T=5-0—V-5-5=¥~L-E~G-0"A~A~R-E-F—I=A-W-—L-V-R-G-N Aon

HPLC: (method B6) RT= 40.5min

LC-MS: m/z = 789.5 (M+5H)*", 986.3 (M+4H)™, 1314.8 (M+3H)>* Callculated (M+H) = 3941.5

Example 34

N'°.{2-(2-(2-(2-[2-(2-(octadeca noylamino)ethoxy)ethoxylacetylamino Jethoxy)ethoxy)acetyl)}- [Gly® Arg? Lys™|GLP-1-(7-37)-OH

6682.204-W 0 ® 0 [e} Jo) ~h ~ ! [oS AN a ~~ “0 Ag A

C A { HN

HN" NL NO pe

Pia J

A 0 9

HoN eG -E-G-T—F=T-5-D-V-S-S-Y-i~E-G-Q-A- APU EY [e] eo]

The ccmp=cund was prepared according to the methods in Example 1 and im "Gereral Syn- thetic met hods”.

HPLC: (m ethod B6) RT= 38,3min

LC-MS:m /z=7868 (M+5H)>", 982.8 (M+4H)*". 1310.1 (M+3H)* Calculate=d (M+H)" = 3927.5

Example 35

N*7-(2-(2—(2-(4-4(4,4,5.5,6,6,7,7,8,8.9.9,9-tndec afluorononanoylsulfamoyl- butyrylami no}ethoxy)ethoxy)acetyl)){Aib®?** Lys *] GLP-1-(7-37)-OH

Pe Te Te © 00 a

AAA A AS ASE 0

EET RT Ed H o™\—o__[

F F F N NH

NT J c c HC CMs ne : E-G-T-F-T=5-D-V-S~S-y—L—%-H Bo Q-A~— AK-E~F—I—A-W-L-V-K-N R—N OH "oo on, ween, TY "oo

CH

The comp «und was prepared according to the methods in Example 1 and ir "General Syn- thetic methods”.

HPLC: (meethod B6) RT= 32 4min

LC-MS: m./z = 1042.7(M+4H)"", 1389.9 (M+3H)’" Calculated (M+H)' = 4166. 4

Example 36

N™7-(2-(2- (2-(3.3.4,4,5.5.6.6,7.7.8,8.9,9.10,10,1% .11,12,12,12-Heneicosaflusoio- dodecylox—yacetylamino)ethoxy) ethoxy)acezty)[Aib®?* > Lys*|GLP-1-(7-37)-OH

6692 203-WO ® .

SPP : ~~ C

IAAL AAA “oof

F F F F 3 0 NE

PNT 7

Ae c o 0] H,C ak yd J SE G-T-Fo1-§-D-V-§~5-Y-L-E-N_ Jo C-A-A-K E~F —I=A-W-L-V-K Syren OH

H > H H

Oy ac” CH, HC Cr, = o)

The compound was prepared according tc the methods in Exampte 1 and in "General Syn- thetic methods”.

HPLC: (rmethod B68) RT= 36.7 min

LC-MS: rr/z = 1062.8 (M+4H)*", 1416.9 (M+3H) ~ Calculated (M+H)" = 4247.3

Example= 37

NV_(2-(2-(2 (4-(hexadecanoylsulfamoytbutyryla mino)ethoxy)ethoxy)acetyl)[Aib™= 2° Lys*)

GLP-1-(77-37)-OH

I Wo

PPP PN -S Mam 4

H,C N oo AL

PNT

A= on

Oo a H,C 3

EN E-G-T-F~T-$-D-V-5-5-Y-L-E-N__\ QA AREF mim A WL vk ag RN OH ) 3 on A Ho " =

The com pound was prepared according to the methods in Example 1 and in "General Syn- thetic me=thods™.

HPLC: (rmethod BG): RT= 37.4min }

LC-MS: rz = 1008.8 (M+4H)* 1344.3 (M+3H)>" Calculated (M+H)" = 4030.7

Example 38 (Arg®®¥* J GLP-1(7-37)Lys({2-(2-(2-(2-{2-(2- (octadec=anoylamino)ethoxy)ethoxylacetylamino ) ethoxy)ethoxy)acetyl)})-OH

5892 204-W0 @ o

HNN

M at

AN TTT ATECG TOF TeSeDovoSoSovel~i-G-0-A 4 R-E=F—i—A w= —V-R=G-3-«G-N_ “om

Cc i h

Ng 0 Cart

H .

EI FS Ta a ae oro Ce SN ° ol

The compound! was prepared according to the methods in Example 1 and in "CSeneral Syn- thetic methods™".

HPLC (method B86). RT= 38.5 min

LCMS: m/z = CM+4H)* 1025.1 (M+3H)> 1366 7 Calculated (M+H)" = 4096.0

Example 39 [Arg?® >] GLP- —1(7-37)Lys{2-(2-(2-(2-[2-(2-(4-(octad ecanoylamino)-4- carboxybutyrylzamino)ethoxy)ethoxylacetylamino)et hoxy)ethoxy)acetyt)}-OH

HN

— N yw 9

H-N A- E-G-T-F-T-$-0-V-$-S-Y-L-E-G-Q-A —A-R-E-F—I-A-W-L-V-R-G-R -G-N_A, 0 : "ope y o OM

I I peo Aon

H H

0] 0 '

The compoundcd was prepared according to the met hods in Example 1 and in "CSeneral Syn- thetic methods”.

HPLC (method BB}: RT= 37.7 min LCMS: m/z = -(M+4H)" 1057.8 (M+3H)>" 1410.2 Calculated (M+H)" = 4235.9

Example 40

NEO {2-(2-(2-(2-[2-(2-(4-(hexadecanaylaming}-4- carboxybutyryl amino)ethoxy)ethoxylacetylamino)ezhoxy)ethoxy)acetyl)}-exendi n{1-39)

£692. 204-WO ® 85

To feu ! le ( "ar CECT TS DUSKAMEEEAVN Sl Fo EWING OP S SOAR PRN Ty ™ ~N gO . 0 bil

HESS YIN

HPLC (method B8): RT= 33.€ mun

LCMS: m/z = (M+4H)*" 1205.3 (M+3H)" 1606.9 Calculated (M+H) * = 4816.5

Example 41 (Ala? Arg? IGLP-1(7-37)Lys({2-[2-({2-oxalylamino-3-carboxy-2-4,5 6,7 -tetrahydro- benzo[blthiophen-6-yl-acetylamino))ethoxylethoxyacetyl) amide

HN

- 3

HN’ T A-E-G=T~F-T—S-D-V—5§-8-Y-L-E-G-Q~A-A-R-E-F—I—A=W-~- EE 9 u 0 >

HOT SS I gn OA Og fo}

The compound was prepared according to the methods in Exampte 1 and in "General Syn- thetic methods”.

HPLC (method BB): RT= 32.1 min

HPLC (method Al): RT= 42.2 min

LCMS: m/z = 1033.3 (M+4H)* 1376.6(M+3H)*" Calculated (M+H)"™ = 4126.7

Example 42 [Aib* #2 3IGLP-1(7-37)Lys((2-[2- ((2-oxalylamino-3-carboxy-2-4.5.6.7 -tetrahydro- benzo{b]thiophen-6-yl-acetylamino))ethoxylethoxyacetyl) amide

SEG 204-WO ® 86 ?

Lc me on, “C, RON ] wo Ie fe o Pad

BRN =x emo FoTo5aD-veS STYTUL-ESN Tyee PE I To SRC BE pany La i Te

NI = Tey H Je

The compound was prepared according to the meth =ocs in Example 1 and in "Ge=neral Syn- thetic methods”.

HPLC (method B1): RT= 37.4 min

HPLC (method At): RT= 35.5 min

LCMS. m/z = 1002 .5 (M+4H)" 1336.7 (M+3H)>” Calculated (M+H)™ = 4007.5

Example 43

N°.(2-(2-(2-(2-(2-( 2-(4-(ocladecanoylamino}-4(S)- carboxybutyrylamin o)ethoxy)ethoxy)acetylamino)ettoxy)eihoxy)acetyl)- [AIB® Arg®®** Lys**)CSLP-1-(7-37)-OH o]

H / 7 N 0 N al 0 ; fo) SN

NTN\-0

To . ine 0

CH, py

HNN iN (

H,C_ CH, lo) - H carson cas TTA-A-R-E-F=I-A-W-L-V-R-G-NE i Non 0

The compound wass prepared according to the mettods in Example 1 and in "General Syn- thetic methods”.

HPLC: (method B6 ). RT= 39.0 min

LC-MS: m/z = 1022.3 (M+4H)"", 1362.3 (M+3H)*", Calculated (M+H)'= 4084.6

Example 44

NEP (2-(2-(2-(2-(2- & 2-(4-(octadecanoylamino)-4(S)- carboxybutyrylamiro)ethoxy)ethoxy)acetylamino)et Froxy)ethoxy)acetyt)- [Gly®. Arg™™ Lys’®] GLP-1-(7-37)-OH

HO ° 0" N\__o 2

NN

< NTN—c.

TN

— oe

HN

CH,

HN" ’ — N

Ne_kg

H—N Sr —G—E-G-T~F-T- $-D-V-5-5-vV—L— E-G-C—A-A-R-E-F—~1 ‘A4-W-__-v-R-G-n~ [I

H i Fr 0

The compound was prepared according to the methods in Example 1 and in "General Syn- thetisc methods”.

HPL C: (method B6): RT= 38.6 min

LC-MS: m/z = 1015.2 (M+4H)"", 1353.4 (M +3H)>", Calculated (M+H) "= 4056.6

Exarmple 45

N=7_ 2.(2-(2-(4-(4-(Heptadecanoylamino)-4- {S)-carboxybutyrylamino)- 4-(S)- carb oxybutyrylaminojethoxy)ethoxy) acetyl-[Aib® Lys¥|GLP-1-(7-37)-NH,

HN

N

= H,C CH,

H O HO H

Ho HC CH Ho IN

H,C CH, : : ! NH, 0 N . a o oO H 0 a

HC TT Il /

N 0 H

H =0 OH

HO

The compound was prepared according to the methods in Example 1 and in "General Syn- theti ¢ methods”.

HPL C: (method B4): RT=10.72min LCMIS: m/z = 1039.0 (M+4H)*", 1385.0 (M#3H)*" Calculated (M+H)™ ==4152.0

Exammnple 46

NT 2.(2-[2-(2-[2-(4-[4-(Heptadecanoylamirio)-4-(S) carb oxybutyrylamino]-4-(S)-carboxybutyrylaamino)ethoxy]

55232 27 04-W0 ° ethoxy)acelylamino ethoxyletnoxylacetyl-[Aib™ 22 > &_ys¥|GLP-1-(7-37)-NH,

HN

H,C CH

H O H O 3 Ss 2 ne E-GT -F-7-5DV'S-S-Y-L-EN QA AKEF-I-AWLV-KN SRT O

HA x NT

B,C CH, H,C CH, \ NH, oO

Roi Ce Cot? ’ nN 0 H = =O OH

HO

The compound wasa prepared according tc the methods in Example 1 and in "Ge=neral Syn- thetic methods”.

HPLC: (method B4p: RT=10.74 min

LCMS: m/z = 1074 (M+4H)*", 1433 (M+3H)’" CalcL slated (M+H)™ = 4297

Example 47

N%-(2-(2-(2-(4-(He xadecanoylamino}-4(S)-carboxy butyrylamino) ethoxy)ethoxy)acetsyl)-{Aib® Arg™|GLP-1-(7-37)- -OH

HN

LG i GF T-5-D-V-§-§-Y~L-E-G-Q-A- mak e-Foi_a-w -R-G-F rid Ga

H,C CH, ©

H lo)

H,C N ~ 0 NH b) hdd dd 0 oY o o 0

HO

The compound wass prepared according to the mettods in Example 1 and in "Ge=neral Syn- thetic methods”.

HPLC: (method B47): RT=10.71min

LCMS: m/z = 979.0) (M+4H)*", 1304.0 (M+3H)*" Calculated (M+H)" = 3910.0

Example 48

N¥-2-(2-2-(2-(2-(2—(4-(Octadecanoylamino)-4(S)- carboxybutyrylamin o)ethoxy)ethoxy)acetylamino)et hoxy)ethoxy)acetyl- [Aib®. Arg™]GLP-1-<(7-37)-OH

6692 204-WQ ® =

PeN TN

NE h 0) \

ROTTS SAS YT TS Oey

The ccompound was prepared according (0 the methods in Example 1 &nd in "General Syn- thetic rmethods".

HPLC: (method B4): RT=11.32 min

LCMS : m/z = 1021 (M+4aH)*", 1362 (M+3H )" Calculated (M+H)" = 40634

The pe=ptide was synthesized on a chlorotrityl resin (Novabiocchem) ussing the Fmoc strategy on an Advanced Chemtech 348 peptide synthesizer (0.5 mmol/g, 103 mg resin/hole and 10 holes were used). The couplings were mediated in Diisopropylcarbcodiimide (DIC) (Fluka) and 1-hydroxybenzotriazo! (HOBt)/1-hydroxy-7-aza-benzolriazole (HOA (2:1) (Senn

Chemii cals) in 1-methyl-pyrrolidin-2-one (NMP) and 10 molar equivaleants of amino acids and couplirhg reagents were applied. The used prolected amino acid deri. vatives were standard

Fmoc- amino acids (Advanced Chemtech) with the exception of th e amino acids Fmoc-

Lys(ivi2de) (Novabiochem) and Fmoc-Glu-O!Bu (Bachem). The resin wwas afterwards divided into 5 portions (0.1 mmo!) and the N-terrrunal was then treated with (Boc),O and DIEA (5 molar equivalent) in NMP.

The a ttachment of sidechains and linkers to specific lysine residuess an the crude resin bound protected peptide was carried out in a specific position by imncorporation of Fmoc-

Lys(iviDde)-OH during automated synthesis followed by selective deprotection with hydra- zine. Procecdure for removal of Dde-protection. The resin (0.1 mmol) was golaced in a syringe and treated with 3% hydrazine and 3% piperidine in NMP (50 min at r. t) to remove the Dde group and wash with NMP (4x5 mi).

Proceedure for attachment of sidechains to Lysine residues.

The C2EG or amino acid (7 molar equivale nts relative to resin) was disssolved in NMP. HOAt (7 mo lar equivalents relative to resin) and diisopropylcarbodiimide (7 molar equivalents rela- live to resin) was added and the solutiom was stirred for 15 min. T hen, the solution was

6692 204-WO ® 90 added to the resin. The resin was shakemn overnight at room temperature. The res n was washed with NMP (3x5 ml).

Procedur—e for removal of Fmoc-protectiorn: The resin (0.1 mmol) was placed in a syringe treated with a solution of 30% piperidine in NMP (5ml in 20 min). The resin weas washed with

NMP (2x 5 ml} and methylene chloride (2x5 mi)

Procedur—e for cleaving the peptide off the r esin:

The peptide was cieaved from the resin b y stirring for 120 min at room tenperature with a mixture of trifluoroacetic acid, water and trisopropylsilane (94-3:3). The cl eavage mixture was filter~ed and the filtrate was concentramted to an oil by a stream of nitrosgen. The crude peptide wvas precipitated from this oil with “10 ml diethy! ether and washed 2 &imes with 10 mi diethyl ether.

Example 49 [Gly® Arg ®¥|GLP-1(7-37)Lys(2-(2-(19-(carBoxy)nonadecanoylamino)ethoxy)esthoxy)acetyl)-

OH oO a a fo} H NH ", Jo

IP X OH

A chlorotzrtyl resin (0.5 mmaol/g Novabiocheam, 0.1 mmole) was used lo prod uce the primary sequencse on an Advanced Chemtech 348 machine. All protecting groups wwere acid labile with the exception of the residue used in position 37 (FmoclLys(ivDde)-OH , Novabiochem) allowing specific deprotection of this lysine rather than any other lysine.

Procedure

The above prepared resin (0.1 mmole) corwtaining the GLP-1 analogue amino acid sequence was placsed in a syringe and treated with 23% hydrazine and 3% piperidine i n N-methyl pyr- rolidone (50 min) to remove the Dde group. The resin was washed with NMP (4x5 mi).

Fmoc-8-zamino-3,6-dioxaoctanocic acid (Necsystem FAQ3202) (7 molar equivalents relative to resin) waaas dissolved in NMP. HOAL (7 m olar equivalents relative to resin) and diisopropyl-

6692 204-WA” (0 ® carbediimide (7 molar equivalents relative to resin) was added and the solution was sured for 15 min. The solution wa=s then added to the resin. The= resin was shaken overnicght at room temperature. The resin was washed with NMP {4x5 miJ). A solution of 30% pipericine in

NMP (5S mi, 20min) was addeed ta the resin The resin was washed with NMP (4x5 mi). The

N-hydroxysuccinimide ester «of C20 {6 molar equivalents relative to resin, KJ. Ross-Pet ersen

A/S) and DIEA was dissolve d in NMP and added to the resin. The resin was shaken over- night at roem temperature. T he resin was washed with NMP (3x5 ml} and methylene ch loride {2x5 mi). The peptide was cleaved from the resin by stirring For 120 min at room tempemrature with a mixture of triflucroacet ic acid, water and triisopropylsilaane (94:3:3, 3 ml}. The cle=avage mixture was filtered and the filtrate was concentrated to an oil in vacuum. The crude peptide was precipitated from this oil with 10 mi diethyl ether and washed 2 times with 10 ml Hiethyl ether

Purification

The crude peptide dissolved &n DMSO at a concentration of 5-10 mg/200 pl and applied to a 7.8 x 300 mm X-Terra Prep NAS C18 10 ym column running &xt 40°C. After 5 minutes at =30%

CH;CN, 0.08% TFA, 4 mi/mir, the column was eluted with a Jinear gradient of 30 to 65%%

CH,CN over 35 minutes. The main UV peaks were collected manually and the desired Peak identified by MALDI-MS.

The concentration of the peptide in the eluate was determine < by measurement of the {JV absorption at 280 nm assumi ng molar extinction coefficients sof 1280 and 3690 for tyrosi ne and tryptophan respectively.

After the concentration deterrmination the eluate was aliquottead into vials containing the de- sired amount and dried by va cuum centrifugation.

HPLC: elutes at 27.9 min = 5.29% CH,CN

MALDI-MS: 3996 (MH")

Example 50 [Gly® Arg? *IGLP-1(7-37)Lys ((2-(2-(17- (carboxy)heptadecanoytlaminen)ethoxy)ethoxy)acety!)}-OH

5692 .204-W0O ® : i I SP NG NPN 0 ‘

HOS Sa ~~ YY

Ie} i NH

HT r ol ons SUVS GN

MH ro H ls)

The compound was prepared 3s in previous example and according to "Synthetic metheads” except that octadecanedioic acid C18 was attached as a monos protected tert-butyl ester (3 molar equivalents relative to resin) and the coupling was medi=ated with HOAL and DIC {also 3 molar equivalents relative tc resin) in NMP. The crude peptid e was dissolved in 22.5%%

CH3CN, 0.1 N NaOH for purificcation.

HPLC: elutes at 25.4 min = 50_4% CH;CN

MALDI-MS: 3969 (MH")

Example 51 [Gly® Arg? *|GLP-1(7-37)Lys(2-(2-(2-(4-(19-(carboxy)nonadec=anoytamino)-4- carboxybutyrylamino)ethoxy)et hoxyjacety!)-OH o H ol

HOP es se 0 [eo] OP 0H Nir

HN, 7 1 GE

Hoo Ho

The compound was prepared as in the two previous examples and according to "Synthealic methods”. The amino acid Fmoc-Glu(OtBu) (6 molar equivalen ts relative to resin) was c—ou- pled to the resin with HOAt and DIC (6 molar equivalents relative to resin). The crude pep- tide was dissolved in 22.5% CH,CN, 0.1 N NaOH for purificatio n.

HPLC: elutes at 27 2 min = 52. 2% CH,CN MALDI-MS: 4124 (MH")

Exampte 52

6€92 204-WO ® > (Sly Arg” “JGLP-1(7-37)Lys ((2-(2-(2 -(2-(2-(2-{2-(2-(2- (h exadecanoylamino)etnoxylethoxy)a cetyllethoxylethoxylacetylamino Jethoxy)ethoxy)- aczetyl)-OH o i. o H ©

HO A eee Norge OA Ou gn Ng ALO A

NS 154 H fo] pa i

H I GE ceon ane riamvrasno, {on ° Se

The compound was prepared as in the three previous examples and &=according to "Synthetic methods” except that additional two OEG was coupled to the side chamn of Lys.

H PLC: elutes at 25.0 min = 50.0% CH;CN

MEALDI-MS: 42538 (MH")

Example 53 [S3ly®, Arg®® *|GLP-1 (7-37)Lys (2-(2- {2-(2-(2-(2-(octadecanoylamino)e2thoxy)ethoxy)- accetylamino)ethoxy)ethoxy)acetyl) NH, i

HEN ©

PA Je 1,

RN NN “R—€—F— —A—N—L—V—R—-G— = nN aN [+] " fo] Ox 0” o

TS SS AO

T he compound was prepared as in Example 1 and in accord with "Syrathetic methods”

HPLC (method B6): RT=38.8 min

L<CMS: m/z = 1022.3 (M+4H)’" Calculated (M+H)™ = 4081.7

E xample 54

NE “2(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2- {(4-(17-(carboxy}heptadecanoylarmino)-4- c=arboxybutyrylamino)ethoxy)ethoxy)ae cetylamino)ethoxy)ethoxyacetylamino) e thoxy)ethoxy)acetyliamino)ethoxy)etbioxy)acetyl) [Lys®Jexendin-4 (1-39)-NH,

6692. 204-WO

Q4 = or fo}

Herd Gok G FTI DLS KO MEE E AH ILE -EWLKNGGP 55GAPPP ro Ne tt Q : HO ~~

N

0 9 0 0 NH e-} ANON gO A ye Qory” " H o H o> ~ HO LO 4

SNP UF GRP

0 E oO

Thee compound was prepared according to the methods in Example 1 &and in "General Syn- thetic meth-ods™.

HPLC (method A1): RT= 41.9 min

HPLC (method B6): RT= 31.3 min

LC-MS: m/z = 1722.7 (M+3H)*> Calculated (M+H)" = 5164.9

Ex ample §5

N*%(2-(2-(2-(2-(2-(2-(17-Carboxyheptade canoylamino)ethoxy)ethoxy) acs=tylamino)ethoxy)ethoxy)acetyl) [Aib® Arg? Lys™] GLP-1 (7-37)

N lo]

Ne

NN

NTO

No 0

HN" HO

N

—

HC. CH © Q 7 H

The compound was prepared according tothe methods in Example 1 and in "General Syn- thetic methods”.

HPLC: (method B6): RT= 34,2 min

LC-MS: m/z = 997.2 (M+4H)*", 1329.4 (NM+3H)*", 1993.2 (M+2H)>", Calculated (M+H)'= 3385.5

Ex<ample 56

859 2 204WO @ ”

N-8-(2-(2-(2-(2-(2- (z-(17-Carboxyheptadecanoylaranino)ethoxy)ethoxy) acelylamino)ethoxy Jethoxylacetyl) fArg™ * Lys™] (SLP-1 (7-37)

SN iY 0 / EAN ul NG

NTN_-0 0

HNN HO _ oD p — N ain 0}

H—N A- E-CS5-T-F-T-§ D-V-$~S-Y-L-E-G-Q——A-A-R-E—F—I—A-W/ -L-V-R-G- Love ! He

The compound wass prepared according to the met—eds in Example 1 and in "General Syn- thetic methods”.

HPLC: (method B6 ) RT= 34,2 min

LC-MS' m/z = 993. 8 (M+4H)*", 1324.6 (M+3H)>", 1 887.2 (M+2H)*", Calculated (M+H)'= 3971.5

Example 57

NE (2-(2-(2-(2-(2-a2-(17-Carboxyheptadecanoylamruno)ethoxy Jethoxy) acetylamino)ethoxw Jethoxy)acetyl) (Gly® Arg? Ly=™] GLP-1 (7-37)

H o=_N

Ne oO

SN

UA

=

HN ©

NTR HOD

— heigl 0

OH

H—N G—E- G-T—F-T~$-D-V-5-8-Y-L-E-G-Q—A-A-R~E-F~I-A-W-L-V-R-G -N NY

H H I HH 0 0 0

The compound wams prepared according to the methods in Example 1 and in "General Syn- thetic methods”.

HPLC: (method BE): RT= 34,2 min

LC-MS: m/z = 890 .3 (M+4H)*", 1320.3(M+3H)>". Calculated (M+H) = 3957 4

6692.2%04 WO £15) @ Example 58

N0-(2:(2-(2+(2-(2-(2-42+(2-(2- (Octadecanoylamined)ethoxy)ethoxyjacetylamino) €ithoxylethoxylacetylam ino)ethoxy)- ethoxy)acetyl)[Lys*®"] Exendin-4 (1-39)amide 0

WENN NNN cr a a 3 7 ? oo

HNN dN OH pene FTSDLSKOMEEE I KNGGP- cxemsnn ym

H il Ho H i 0 0 eo}

The compound wass prepared according to the m ethods in Example 1 and in "General Syn- thetic methods”.

HPLC: (method B68: RT= 37.7 min

LC-MS: m/z = 12168.6 (M+4H)*", 1621.4 (M+3H)>>", Calculated (M+H)'= 4-861.5

Example 59

N28. (2-(2-(2-(2-(2-( 2-(4-(octadecanoylamino)-4(S)- carboxybutyrylamina olethoxy)ethoxy)acetytamino Dethoxy)ethoxylacetyl)-[Arg®> Lys |GLP-1- i5 (7-37) 0]

H

= [eo] —

HO Ng oo J

NNO o =o

HN CH,

JN

NT OH

HN A—E-G-T—F-T-5-D-V-5-S-¥-L~-E-G-Q—A—A-R-E-F—I-A-W-L~V-R-G- hoo

Ho H © 0 o

The compound wa:s prepared according to the amethods in Example 1 armnd in "General Syn- thetic methods”.

6602.204-WQ a7 @® FPLC: (method 86) RT= 36.1 mim

E_C-MS. miz = 1018 8 (M+4H)"". 1 357 6 (M+3H)*", CaicLiated (M+H&)'= £070 6

Example 60

NO (2-[2-(2-[2-(2-[2-(17-Carboxyheptadecanoylamino)ethoxy] ethoxy)acetylarminclethoxy)ethoxy Jacetyl)[Arg>|GLP-1-(7-37)-CH

HN to. N

Hn © i

Hoy 3 A-E-G-T1-F-7-5-D-V-5-§ YUEGOA AN AogEoiaw verano ~ o y 0 ~N

HONS OA AO

0 H lo}

The compound was prepared according to the methods in Example 1 ard in "Genera! Syn- thetic methods”.

HPLC: (method B4). RT= 12.1 mim

LCMS: m/z = 993.0 (M+4H)*", 13 25.0 (M+3H)*" Calculated (M+H)™ =3970.0 13

Example 61

Ne¢.[2-(2-[2-(2-[2-(2-[4-(17-Carbo xyheptadecanoylamino)-4(S)- carboxybutyrylaminojethoxy Jethoxylacetylamino)ethoxylethoxy)ace tylJ{Arg¥|GLP-1-(7-37)-

OH

RN

AM o

Q pace Trrsovss ies onnd Ler t-a-wi-L-v-r-G-r-R. Jhon 0 OO 0 \

RO AAA Nam oO A Omg o H 0 H o

The compound was prepared according to the methods in Examplez 1 and in "General Syn- thetic methods”.

HPLC: (method B4): RT= 11.8 min

LCMS: miz = 1026 (M+4H)*". 13 68 (M+3H)" Calculated (M+H)™ =4100

6692.204-WC

C G8

Example 62

NT —(2:2-(2-{2-(2-(2-(2-(2-2-(17-

Car boxyheptadecanoylamino)ethoxy)e= thoxylacetylamino)ethoxy ethoxy yacelyl- ami rojethoxy)etnoxy)acetyl)jLys?) Exeandin-4 {1-39) amide

H Q

{

C I Ne Ne Ne Nea

TY Ne o 0 4g 0 0 / wT \ Ph 0 o 0

A CONN oN

HN / — Ot

The compound was prepared accordirng to the methods in Example ¥ and in "General Syn- thet ic methods”.

HPL_C: (method B56): RT= 32.3 min

LC- MS: m/z = 1223.9 (M+4H)"", 1630 .8 (M+3H)>, Calculated (M+H) “= 4891.5

Example 63 (Gly? Glu Arg’ ®3GLP1 (7-37) Lys(2-(2-(2-(2-(2-(2-(17- cartooxyheptadecanoylamino)ethoxy)e®hoxy)acetylamino)ethoxy))ethoxy)acelyl)-NH,

HN

Io .

H-N" y———G-E-G-T-F-T-§-D-V-S -R-Y-L_-E-E-E-A-A-R-E-F~I-E-W-L-V-R-G-R-G-N_}A

HH Ie) : NH, . . \

HOT esse Nem gO AO NH lo H 0

Thee compound was prepared accordirng to the methods in Example 1 and in "General Syn- thetic methods".

RHPE.C: (method BE): RT= 32.0 min

HPL.C: (method A1): RT=43.4 min

LCAMIS: m/z = 1438.7 (M+3H)" Calcul.ated (M+H) = 4311.8

$632 204.WO ® =

Exampl-e 64 [Imidazoolylpropionic acid’. Asp'®. Ab JGLP1(7-37)Lys NH((2-{[4-(17- carboxyBeptadecanoylamino)butylcarbamoyllnethoxylethoxy)ethoxy))

NH,

HN

As ig fe) H.C CH, H

N— AEG T-F-T-SD-D-§-S-Y-L-E-N_A CAA RE F-1-A WL V-RNSER GN TN fo] H,C™ CH, Hoc H o N al 0] 0 - lo)

HO poe I o ¥

The cormpound was prepared according to the methods in Example 1 and in "General Syn- thetic m ethods”

HPLC: (_ method B1): RT=32.5 min

HPLC: ( method A1): RT=43 5 min

LCMS: en/z = 1028.8 (MH+aH)*" Calculated (M—+H) = 4108.7

Example 65 [Imidazolylpropionic acid’, Aib™> |GLP1(7-37 JLys NH( (2-{[4-(17- carboxy~heptadecanoylamino)butylcarbamoyl]rmnethoxy}ethoxy)ethoxy))

£692 20 ~<-WO ® 100 [a

ENT re i MN ‘ xs ; a I) H.C CH, ad H

EAN

L

0

Lo

NH

Pe

Ny

HO ~~~ nA 0 H

The compound was prepared according to the methosds in Example 1 and in "(Seneral Syn- thetic methods".

S

HPLC: (method B86): R"T1=33.7 min

HPLC: (method A1). R™T=44.8 min

LCMS: m/z = 1024.8 (BM+4H)"", 1365.4 (M+3H)>" Calculated (M+H) = 4092.8

Example 66 [3-(5-Imidazoyi)propiormyl’, Aib®, Arg®™* | GLP-1 (7-3 )Lys{2-(2-(2-(2-{2-(2-(17- carboxyheptanoylamincjethoxy)ethoxylacetylamino)e=thoxy)ethoxy)acetyl)}-OH 7 : i

HOPS ees ses Nem gn JOA ’ ) 0

O

So

HN

HNT

—

H,C_ CH,

NEG T-F-T- S-D-V-5-5-Y-L-E-G-Q-A-A-R-E=-F—I-A-W-L-V-R-G-R-G-N “Noh of o Ho

The compound was prezpared according to the methcads in Example 1 and in "CSeneral Syn- thetic methods".

£2402 202.00 ® 101

MALDI-MS 24127 (hAH+)

HPLC: elutes: 25.5 min = 50 6% CH3CN

BIOLOGICAL FIND®INGS

Protraction of GLF-1 derivatives after i.v. or s.c. amdministration

The protraction of a number GLP-1 derivatives of the invention was dete=rmined by monitor- ing the concertratio nthereof in plasma after sc administration to health—y pigs. using the methods described below. For comparison also the ¢ oncentration in pla sma of GLP-1(7-37) after sc. administration was followed. The protraction of other GLP-1 derivatives of the inven- tion can be determirned in the same way.

Pharmacokinetic testing of GLP-1 analogues in miniroigs

The test substances were dissolved in a vehicle suita: ble for subcutanecaus or intravenous administration. The concentration was adjusted so th=e dosing volume was approximately 1 ml.

The study was perfCrmed in 12 male Gottingen minip igs from Ellegaard Gottingen Minipigs

ApS. An acclimatisamtion period of approximately 10 d-ays was allowed be=fore the animals en- tered the study. At start of the acclimatisation period “the minipigs were &bout 5 months old and in the weight ra nge of 8-10 kg.

The study was conducted in a suitable animal room with a room temper ature set at 21-23°C and the relative humidity to > 50%. The room was illu minated to give a cycle of 12 hours light and 12 hours darkneess. Light was from 06.00 to 18.0-Ch.

The animals were h oused in pens with straw as bedd ing. six together in each pen.

The animals had frese access to domestic quality drinling water during tlhe study, but were fasted from approxirmately 4 pm the day before dosinsg until approximately 12 hours after dosing.

The animals were weighed on arrival and on the days of dosing.

The animals received a single intravenous or subcutaneous injection. THe subcutaneous in- jection was given or the right side of the neck, approximately 5-7 cm frcom the ear and 7-9

£692.204-% © [Y 102 cm from the middie of the neack. The injections were given witty a stopper on the needle. al- lowing 0 5 cm of the needle to be introduced.

Each test substance was given to three animats Each animal received a dose of 2 nmol/kea body weight

Six ammals were dosed per week while the remaining six were rested.

A full plasma concentration-time profile was obtained from each animal. Blood samples wemre collected according to the fo llowing schedule:

After intravenous administra tion:

Predose (0), 0.17 (10 minutes), 0.5, 1,2. 4,6, 8, 12, 24,48, 7 2, 96, and 120 hours after in- jection. :

After subcutaneous adminis ¥ration:

Predose (0), 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 hou rs after injection.

At each sampling time, 2 ml of blood was drawn from each an imal. The blood samples were= taken from a jugular vein.

The blood samples were col lected into test tubes containing a buffer for stabilisation in order to prevent enzymatic degradl ation of the GLP-1 analogues.

Plasma was immediately tra nsferred to Micronic-tubes. Approximately 200 ui plasma was transferred to each Micronic —tube. The plasma was stored at - 20°C until assayed. The plasma samples were assay ed for the content of GLP-1 analosgues using a immunoassay.

The plasma concentration-tirme profiles were analysed by a ncon-compartmental pharmacok i- netic analysis. The following pharmacokinetic parameters wer «€ calculated at each occasior:

AUC, AUCI/Dose, AUC zap « Crass tax. Az, La, CL, CUS, V,, V7 fand MRT.

Selected compounds of the invention were tested in Danish Laandrace pugs.

Pharmacokinetic testing of CGLP-1 analogues in pigs

Pigs (50% Duroc. 25% York shire, 25% Danish Landrace, app 40 kg) were fasted from the beginning of the experiment _ To each pig 0.5 nmol of test compound per kg body weight was administered in a 30 pM isotonic solution {5 mM phosphate, p H 7.4, 0.02% Tween®-20 {Merck), 45 mg/m! mannitol (pyrogen free, Novo Nordisk). Blo od samples were drawn from a calheter in vena jugularis. 5 ml of the blood samples were pol_ired into chilled glasses con- taining 175 pl of the followin g solution: 0.18 M EDTA, 15000 <IE/mi aprotinin (Novo Nordisk) and 0.30 mM Valine-Pyrrolictide (Novo Nordisk). pH 7.4. Withi 1 30 min, the samples were

£602 204. WO ® 103 centrifuged for 10 min at 5-600 C'g. Temperature was kept a t 4°C. The supernatant was pi- petted into different glasses an d kept at minus 20°C until use

The plasma concentrations of t he peptides were determined in a sandwich ELISA or by RIA using different mono- or polyclconal antibodies. Choice of antibodies depends of the SLP-1 cerivatives. The time at which t he peak concentration in plasma is achieved varies wmthin wide limits, depending on the p articular GLP-1 derivalive sel ected.

General assay protocol for smandwich ELISA in 96-wells mmicrotiterplate

Coating buffer (PBS) Phosphate buffered saBine, pH7 2

Wash-buffer (PBS-wash) : Phosphate buffered saline, 0.05 % viv Tween 20, pH 7.2

Assay-buffer (BSA-buffer): Phosphate buffered saline. 10 g/t Bovin Serum Albumin

C Fluka 05477), 0.05 % viv Tw een 20, pH 7.2

Streptavidin-buffer : Phosphate buffered sali ne, 0.5 M NaCl, 0.05 % viv

Tween 20, pH 7.2

Standard : Individual compounds 1m a plasma-matrix

A-TNP : Nonsens antibody

AMDEX : Strezptavin-horseradish-peroxodase (Amersham RPN440 1V)

TMB-substrate : 3.3 ".5,5'tetramethyibenzidine {(<C».02 %), hydrogen peroxiede

The assay was carried out as f ollows (volumen/well): 1.) coat with 100 ul catching armtibody 5 ug/ml in PBS-buffer — incubate o/n , 4 °C — 5x PBS-wash — blocked with last wash in minimum 30 minute —then empty the plate 2.) 20 pl sample + 100 pl biotirylated detecting antibody 1 2 g/ml in BSA-buffer with 10 pg/mi A-TNP — incubate 2 h, room temp erature, on a shaker — 5x PE3S-wash, then empty the plate 3.) 100 ul AMDEX 1.8000 in SStreptavidin-buffer — incubate 45-60 minute, room temperature, on a shak=er —» 5x PBS-wash, then emoty the plate 4.) 100 ui TMB-subslrate — incubate x minute at ro om temperature on 3 shaker — stop the reaction with 1 00 yl 4 M HPO,

£692 264-0 ® 104

Read the absorbance at 450 nm with 620 nm as reference

The concentration mn the samples was calculaled from standard curves

Sl

General assay protocol for RIA

DB-buffer : 80 mM phosphate buffer, 0.1 % Human serum albumin, #0 mM EDTA, 0.6 mM thiomersal, pH 7.5

FAM-buffer 40 mM phosphate buffer. 0.1 % Human Serum Albumin, 0.6 mM thiomersal, pH 7.5

Charcoal : 40 mM phosphate buffer, 0.6 mM thiomersal, 16.7 % bovine plasma, g/t activated carbon, pH 7.5 {mix the suspension minimum 1 h before use at 4 °C)

Standard : Individual compounds in a plasma-matrix 15

Day 1 (Tota | [1 0] 100 pL

NSB [sop | | leo

Sample | 3004 | 30p | 100 [| 00a

Mix, incubate o/n at 4 °C

Day 2

Total | rr rr 15m rl Sm

Sample

Mix - incubate 30 min at 4 °C — centrifuge at 3000 rpm, 30 min ~ immediate=ly after transfer supernatants to new tubes, close with stapper and count on gamma-counter— for 1 minute.

The concentration in the samples was calculated from individual standard curves.

GLP-1 radio receptor assay (RRA):

The method is a radiometric-ligand binding assay using LEADseeker imag ing particles. The assay is composed of membrane fragments containing the GLP-1 receptor, unlabeled GLP-1 analogues, human GLP-1 labelled with '23 and PS LEADseeker particles czoated with wheat germ agglutinin (WGA) Cold and '“l-labelled GLP-1 will compete for tie binding to the

5692 204- WO

C $05 r-eceptor. When the LEADse eker particles are added they will bind to carbohydrates residues or the membrane fragments via the WGA-residues. The preoximity between the Bl molecules and the LEADSseeker particlees causes light emission from tte particles. The LEADseek er will iTmage the emitted light and it will be reversibly correlated tco the amount of GLP-1 analogue {oresent in the sample.

Reagents & Materials:

Pre treatment of animal plasma: Animal plasma was heal tr—eated for 4 hrs at 56°C and cen- t rifuged at 10.000 rpm for 10 minutes. Afterwards, Val-F—yr (10 pM) and aprotenin (500

IE/mL) was added and stored at <-18°C until use.

CSLP-1 analogues calibrators: GLP-1 analogues were spifked into heat-trealed plasma to oroduce dilution lines rangin g from approximately 1 gM to 1 pM.

CSLP-1 RRA assay buffer: 25 mM Na-HEPES (pH=7.5), 2.5 mM CaCl, 1 mM MgCl, 5 0 mM

MNaCl, 0.1% ovalbumin, 0.00 3% tween 20, 0.005% bacitracir, 0.05% NaN.

CSLP-1 receptor suspensiort . GLP-1 receptor membrane fragments were purified from baby

Famster kidney (BHK) cells expressing the human pancreestic GLP-1 receptor. Stor-ed «<- 80°C until use.

LAVGA-coupled polystyrene |_EADseeker imaging beads (RPNQ0260, Amersham): The toeads wvere reconstituted with GL P-1 RRA assay buffer to a co ncentration of 13.3 mg/mL_. The

CSLP-1 receptor membrane suspension was then added armd incubated cold (2-8°C) at end- over-end for at least 1 hr pri or to use. ['P1)-GLP-1(7-36)amide (No vo Nordisk A/S). Stored <-18°C until use. £=thanol 39.9% vol (De Danssk Spritfabrikker A/S): Stored <-718°C until use.

MAultiScreen® Solvinert 0.45 um hydrophobic PTFE plates (AMSRPN0450, Millipore Corp)

Moly propylene plates (cat. rio. 650201. Greiner Bio-One)

RVhite polystyrene 384-well lates (cat. no. 781075, Greiner Bio-One)

Apparatus:

E-orizontal plate mixer «Centrifuge with a standard sswinging-bucket microtitre plate mrotor assembly ®traVap - Drydown Sample Concentrator (Porvair)

R.EADseeker™ Multimodality Imaging System (Amersham)

6622 204-WO 9 106

Assay Procesdure:

Sample prep =aration:

Mount the M utiScreen® Solvinert filter plate on a chemical-comparable recewve=r plate (ie. poly propylernie plates) to collect the filtrate.

Add 150 pl. ice-cold ethanol 98 8% into the empty wells of the MultiScreen® Scolvinert filter plate followed by 50 ub calibrator or plasma sample. Place the storage lid on the filter plate.

Incubate 15 rmnutes at 18-22°C on a horizontai plate mixer.

Place the as sembled filter and receiver plate, with the lid, into a standard swin-ging-bucket microtitre pla te rotor assembly. The filtrate is then collected in the empty wells of the receiver plate at 1500» rpm for 2 minutes.

Dry down the filtrate by using the UltraVap with heated (40°C) N; for duration of 15 miuntes.

Reconstitute the dry material by adding 100 pb GL P-1 RRA assay buffer into ech well. In- cubate for 5 «minutes on a horizontal mixer.

GLP-1 radio receptor assay:

Use the following pipetting scheme and white polysty rene 384-well plates: e 35 ul_ GLP-1 RRA assay buffer

J S yl reconstituted filtrate. e 10 pl ['®1]-GLP-1(7-36)amide. The stock solution was diluted in GLP-1 RRA assay buffer to 20.000 cpm/well prior to use. eo 15 py L GLP-1 receptor membrane fragments (=0.5 pg/well) pre-coate-d to WGA. polystyrene LEADseeker imaging beads (0.2 mg/well)

Seal the plat-es and incubate over night at 18-22°C

The light em:ission from each wells are detected by using the LEADseeker™ MultZimodality

Imaging System for duration of 10 minutes.

Stimulation of cAMP formation in a cell line expressing the cloned human GLP-1 receptor.

Purified plas uma membranes from a stable transfected cell line, BHK467-12A (tk-t=s13), ex- pressing the human GLP-1 receptor was stimulated with GLP-1 and peptide anal ogues. and

§662.204-WO the potency of CAMP production was measured using the AlphaScreer * cAMP Assay Kit from Ferkin Eimer Life Sciences.

A stable transfected cell line has been Prepared a: NN and a high expressing clone was se- lecteat for screening. The cells were gro=wn at 5% CO; in DMEM, 5% Fe&CS, 1% Pen/Strep and 0.5 mgiml G418.

Cells at approximate 80% confluence weere washed 2X with PBS and hearvested with

Verse ne, centrifuged 5 min at 1000 rpm and the supernatant remaved _ The additional steps were all made on ice. The cell pellet wa=s homogenized by the Ultrathusax for 20-30 sec. in mi of Buffer 1 (20 mM Na-HEPES, 1&0 mM EDTA, pH=7.4), centrifucged 15 min at 20.000 10 rpm a nd the pellet resuspended in 10 my | of Buffer 2 (20 mM Na-HEPE-S, 0.1 mM EDTA, pH=7_4}. The suspension was homogenized for 20-30 sec and centrifu ged 15 min at 20.000 rpm. Suspension in Buffer 2, homogenization and centrifugalion was rezpeated once and the membranes were resuspended in Buffer 2 and ready for further analys is or stored at -80°C.

The franctional receptor assay was carrieed out by measurering the peptide induced cAMP produ ction by The AlphaScreen Techno logy. The basic principle of The AlphaScreen Tech- nology is a competition between endoge=nous cAMP and exogenously added biotin-cAMP.

The capture of cAMP is achieved by usimng a specific antibody conjugat ed to acceptor beads.

Formed cAMP was counted and measur—ed at a AlphafFusion Microplate Analyzer. The ECs, values was calculated using the Graph-Fad Prisme software.

Claims

6692 204. WO ® 108 CLAIMS

1. A compound which comprises a therapeutic polyperotde inked lo an albumin bir ding residue via a hydrophilic s pacer

2. A compound which comprises a therapeutic polyperotide linked to an albumin bir ding residue via a hydrophilic s pacer -(CH;3D{(CH)sEln(CH2),Qq-. wherein mand n independently are 1-20 and pis 0—10, Q is -Z-(<THD[{CH3)aGlm(CH2)p-. q is an imleger in the range from 0 to 5, each D, E, and G independently are selected from -O-, -NR’-, -N(COR*} -, -PR*(0)-, and -P(OR®)(0)-. wherein R®, R*, R®, and R® independently represent hy drogen or Cis-alky t, Z is selected from -C(O)NH-, -C(O)NHCH,-, -OC({O)NH -, -C(O)NHCH,(H,-, -C(OYCH 5, -C{Q)CH=CH-, -(CH,)s-, -C(O)-, —C(O)O- or -NHC(O)-, wheresin sis 0 or 1 or a pharmaceutically acceptable salt or prodrug therseof.

3. A compound according to claim 2, which has the formula (1) : A —W—B—Y—therapeutic polypeptide (1) wherein Ais an albumin binding residue, Bis a hydrophilic spacer being -{CH,)D{(CH)nE]m{CHH,),Qq-. wherein , mand nindependently are 1-20 and pis 0—10, Q is -Z-( CHID[(CH2)aGlm(CH2)o-. gis an irmteger in the range from 0 to 5, each D, E. and G independently are selected from -O-, -NR’-, -N(COR*} -, -PR*(O)-, and -P(QOR®)(0)-, wherein R?, R*, R®, and R® independently represent hy drogen or

Ci.s-alky |, Z is sele cted from -C{O)NH-, -C{OINHCH,-, -OC{O)NH -, -C(O)NHCH,CH;-, -C(O)CH,-, -C{O)CH=CH-, -(CH;)s-. -C(O})-. -C(O)O- or -NHC(O)}-, wherein sis Q or 1, Y is a chemical g roup linking B and the therapeutic amgent. and W is a chemical group linking A and B.

8692 204-WO 7) 109

4. A compound accorcing to claim 2, which has the formula (il) A—\W--3- Y-therapeutic polypepti de —vy' -g'-W —A" (in wherein A and A’ ares albumin binding residues, Band B’ ares hydrophilic spacers independentiy~ selected from -(CH2)D [(CHH2),E]m(CH;)o-Qc-. wherein

[. mand n independently are 1-20 and pis C-10C. Q i= -Z-(CH2)D[(CH2)1Glm( CH2)s-, q is an integer in the range from 0 to 5, each D, E, and G independently are selected from -O-, -NR>-, -N(CSORY)-, -PR(O)-, and -P(OR®)(0)-, wherein R®, R*, R®, ard R® independently represent hydrogen or C, es-alkyl, Z iss selected from -C(O)NH-, -C(O)NH CH,-, -OC(O)NH -, -C(O)NH CH,CH,-, -C( O)CHg-, -C(O)CH=CH-, -(CH;)s-. -C(0)-, -C(O)O- or -NHC(O)-. wherein sis 0 or 1, Y is a chem ical group linking B and the therape=utic agent, and Y is a chermical group linking B* and the therapeutic agent, and Wis a chemical group linking A and B, and Wis a chermical group linking A” and B".

3. A compouund according to claim 4, wherein Y "is selected from the group consisting of - C(OINH-, -NHC(O)-, -C(O)NHCH,-, -CH,NHC(O)-, -OC(O)NH -, -NHC(O)O—. -C(O)NHCH,-, CHNHC(OX»-, -C(O)CH;-, -CH,C(O}-, -C(O)CH=CH-, -CH=CHC(O)-, ~(CH;)=-, -C(O)-, -C(O)O-. -O C(O), -NHC(0)- and —~C{O)NH-, wherein sis 0 or 1.

6. A compound according to any one of claims 4-5, wherein W' is selected From the group consisting o f-C(O)NH-, -NHC(O)-, -C(O)NHCH 2, -CH,NHC(O)-, -OC(Q)NHE -, -NHC(0)O-, -C(O)CH;-, —CH,C(0)-. -C(O)CH=CH-, -CH=CHIC(O)-, -(CH,),-, -C(O)-, -C(CD)O-, -OC(O}-. -NHC(O)- agnd —C(O)NH-, wherein sis 0 or 1. 7 A compowand according to claim 2, which has t he formula (111) A—W"-B—Y-therapeutic po lypeptide (hn A wherein A and A" are= albumin binding residues.

£692 264.WO ® ro B 1s @ hydrophilic spacer selected from -(CH;) D[{CH;)E]~(C F;).-Qq- wherein l,m and n independemrtly are 1-20 and p's C-10, Q is -Z-(CH;)D[(CHz) nC] m(CH3)p-, q is an integer in the #ange from 0 to 5. each D, E, and G are independently selected fram - O-, -NR*- -N(COR"}-, —PR*(0)-, and -P(OR®)(O)-, whezrein R*. R*, R®, and R® independently represent hydrogen or C,s-alkyl, Z is selected from -CCO)NH-, -C(O}NHCH;-, -OC{ODNH -, -C{O)NHCH,CH_=-, -C(O)CHay-, -C(O)CH=CH-, -(CH,)-. -C(O)-. -C{O)O - or -NHC(O)-, wherein sis 0or 1, Y is a chemical group linking E3 and the therapeutic agent, ard Wis a chemical group linking B with A and A".

8. A compound according to cBaim 7, wherein Wis selected from the group consis ting of —C(O)NHCH— . —C(O)CH— (Cr CH= and ~~ NHC(CIENHC(O)CH,0(CH,), (CoH) NH— i” , wherein s is 0, 1 or 2.

9. A compound according to amy one of claims 3-8, wherein ~Y is selected from the group consisting of -C(O)NH-, -NHCC Q)-, -C{O)NHCH,-, -CH,NHC( O)-, -OC(O)NH -, -NHC(0Q)0-, -C(O)NHCH,-, CH;NHC(Q)-, -€C(O)CH,-, -CH,C(O}-, -C{O)CHA=CH-, -CH=CHC(QO)-. -(CHy),-, -C(0)-, -C(O)O-, -OC(0Q)-, -NFH4C(O)- and —-C(O)NH-, whereirm sis 0 or 1.

10. A compound according to any one of claims 3-9, wherein: W is selected from the= group consisting of -C(O)NH-, -NHCQ)}-, -C(O)NHCH;-, -CH:NHC( O)-, -OC(O)NH -, -NH(C(0)0-, -C(O)CH;-, -CH,C(O)-, -C(O)CH=CH-, -CH=CHC(O)-, -(CH;3,-. -C(O)~, -C(O)O-, -O=C(O)-, -NHC(O)}- and -C(O)NH-, whe reinsisQor 1.

11. A compound according to zany one of claims 2-10, wheret nl is 1 or 2, n and m ar—e inde- pendently 1-10 and p is 0-10.

12. A compound according to zany one of claims 2-11, wherein D is -O-.

13. A compound according to any one of claims 2-12. wherein Eis ~O-. 3s 14 A compound according to @any one of claims 2-10, wherei nthe hydrophilic space-ris

8692 204-WO ® a -CHO[( CH,)20]m(CH23.Q.-, where mis 1-10 pis 1-3, and Q 1s -Z-CH,O[{{ZH,1,0]m(CH>)p-

15. A co empound according to any of the prev~icus claims, wherein q is 0 or “1.

16. A co mpound according to any of the previous claims. wherein q is 1.

17. A corrpound according to any one of claims 2-10 and 12-15, wherein G iss -O-.

18. A carmpound according to any of the previeous claims, wherein Z is selecte=d from the group 180 consistin g of -C(O)NH-, -C(O)NHCH,-, and ~OC(O)NH-.

19. A cormpound according to any one of clainms 2-15, wherein q is 0.

20. A cormpound according to any one of clairms 2-13, wherein i is 2.

21. A cormpound accerding to any of the previeous claims, wherein nis 2.

22. A cormpound according to any one of claims 2-15, wherein the hydrophilic spacer Bis - [CH;CHz Om. (CH), Qq-.

23. A cormpound according to any one of claims 2-15, wherein the hydrophilic spacer B is «{(CH2)-O-[(CH2)a-Olm-(CH2)o-[C{OINH-(CH_)-O-[(CHz)0-Olm-(CHz) e-, where |, m, n, and p independently are 1-5, armd gq is 0-5.

24. A cormipound according to any one of the receding claims, wherein —“W-E=2-Y- is selected from the group consisting of

5592,204-WO ® 112 0 oO z : N oA A I a i NT J H H le) C 0 la) 0 fl H | 3 lL ASNT SCS gS TPL H 7 [=] al 0 lo] . o AN EN H y fo) ~ a0 No_-~ oA N 07 fo) H \ H 0 : o) , Q H ] Agro A momo Ago A H ! S , and lo} Hu i H I H 5 ! oO

25. A com pound according to claim 7, wherein >W "-B-Y- is 0 o] 0 N o N NI ~ ~o + | HH H Oo oO 9 NT Oo ; H oO we 3 Y

26. A compound according to any one of the p receding claims, wherein A i=s selected from the group corusisting of

S

H.C N LN NP Ga NN ~TN - IN HO So where the chiral carbon atom is either Ror S, H Rr I ) HO 0 where the chiral carbon a tom is either Ror S, H a rr HO O where the chiral carbon atom is either Ror S, 0] 0 OH H,C Ro ~~ 3 heii NE a ok H HO 0 ’ where the two chiral carb on atoms independently are either R or S, O OH " (@) oe N 6 k H HO [e} where the two chiral carbbon atoms independently are either R or S,

coud 204-WQO o Cia Cite HL C 5 I 1 x NNT INT NT NEY ~_ TN { H a Hog were the two chiral carbon atams ingeperudently are either L or D, 0 : [= = MA TC NN hh ' Crap where the chiral carbon atom is either R or S. 0 Lo ; HO 7 hdd i ¢ ~~ 0 HO YO wh ere the chiral carbon atom is either Ror S, o Co 0 Oay-On HO I a o H HC» 7 Yo wh-ere the two chiral carbon atoms independently are either R or 8, 0 o Oxy OH By Hod] HO TNT TN NS Qh NT STN HS 9 nO” Yo wheere the two chiral carbon atoms indepencdently are either R or 3.

HS TN NSN ERS I Ta HET TT SN NSN HC IRE re ie Ne Sa NN Rei i I NN

5692 204- WWD =O AN Pe a WG a ; N oN

© . i 0 ; PEW ra a Ve = NN 1 H 0 + @] [ Pa ae O ~N le} =O HO eee hi S 0 t Oo NHQO 520 (OL 0) on O N 5 N H ox ng . N_ oN oS, HN HO Sx 1-0 © of] / jo °=, CH, i A N— vi H,c~0 Pe 0 H Cc | PANS PID Ta CZ N H Ea ] N o CH, Fe fe fr © oo FAA AS FL FL Fe H dasdfos-Wo oof (- A416 ef fe fe fr Te § PRR RA F F F F F eee WP H,C NOS H,C Nd ~~ HH Q AP HOLA Ay ~~ > : Oo 00 NY in a 0 , and QO Oo ’ 0% Ss ET HO o

27. A compound according to any of the previcous claims, wherein the molar weight of said hy- drophilic spacer is in the range from 80D to 10O00D.

28. A compound according to any of the previeous claims, wherein the molar weight of said hy- drophilic spacer is in the range from 80D to 30D.

29. A compound according to any of the previeous claims, wherein said alb umin binding residue is a lipophilic residue.

30. A comporund according to any of the previsous claims, wherein said alb umin binding residue binds non-covalently to albumin.

31. A compound according to any of the previ-ous claims, wherein said albumin binding residue is negatively charged at physiological pH. AMENDED SHEET

669%204-WO kJ

32. A compound according to any of the previous claims, wherein said albumin inding residue has a bindirg affinity towards human serum albumin that is below about 10 pM.

33. A compeound according to any of the previous claims, wherein said albumin kinding residue has a bindirg affinity towards human serum albumin that is below about 1 pM.

34. A compeound according to any of the previous claims, wherein said albumin Ioinding residue is selected #From a straight chain alkyl group, a branched alkyl group, a group whisich has an w- carboxylic amcid group, a partially or completely hydrogenated cyclopentanophen=anthrene skele-

ton.

35. A compeound according to any of the previous claims, wherein said albumin Ioinding residue is a cibacromnyl residue.

36. A compe=ound according to any of the previous claims, wherein said albumin [oinding residue has from 6 ®&o 40 carbon atoms.

37. A comp=ound according to any of the previous claims, wherein said albumin toinding residue has from 8 #0 26 carbon atoms.

38. A comp-ound according to any of the previous claims, wherein said albumin ISinding residue has from 8 #®to 20 carbon atoms.

39. A comp ound according to any of the previous claims, wherein said albumin toinding residue is a peptide , such as a peptide comprising less than 40 amino acid residues.

40. A comp ound according to any one of the previous claims, wherein the albumin binding residue via spacer and linkers is attached to said therapeutic polypeptide via the ¢-amino group of a lysine residue.

41. A comp ound according to any one of the previous claims, wherein the alburmin binding residue via spacer and linkers is attached to said therapeutic polypeptide via a lirker to an amino acid residue selected from cysteine, glutamate and aspartate. ANTIENDED SHEET

6694204-WO > 118

42. A compound ac=cording to any of the previous cla ims, wherein said therapeutic polypeptide is a GLP-1 peptide.

43. A compound ac-cording to claim 36, wherein said polypeptide is a GLP—1 peptide compris- ing the amino acid sequence of the formula (IV): Xaa;-Xaag-Glu-Gly—Thr-Phe-Thr-Ser-Asp-Xaaie-Ser—Xaa,s-Xaa:¢-Xaa-Glu-Xaa,-Xaazs-Ala- Xaays-Xaaz-Xaay,- Phe-lle-Xaas-Trp-Leu-Xaass-Xaa;,-Xaass-Xaazs-Xaas 7-Xadas-Xaadsg- Xaayp-Xaas-Xaas- Xaa,;-Xaasu-Xaas-Xaaus Formula (IV) (SEQ ID Noz 2) wherein Xaa; is L-histidine, D-histidine, desamino-histidine, 22-amino-histidine, B-tydroxy-histidine, homohistidine, N°-=acetyl-histidine, a-fluoromethyl-hisstidine, a-methyl-hist idine, 3- pyridylalanine, 2-py=ridylalanine or 4-pyridylalanine; Xaag is Ala, Gly, val, Leu, lle, Lys, Aib, (1-aminocyclopropy!) carboxylic acid, (1- 156 aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (~1-aminocyciohexyl) carboxylic acid, (1-aaminocycloheptyl) carboxylic acid, or (1-aminocyclooc=tyl) carboxylic acid; Xaass is Valor Leuz Xaa,g is Ser, Lys or Arg; Xaayg is Tyr or Gin; Xaay is Leu or Met; Xaay; is Gly, Glu or Aib; Xaay; is Gln, Glu, L_ys or Arg; Xaags is Ala or Val; Xaagg is Lys, Glu or Arg; Xaay is Gluor Leu ; Xaay, is Ala, Glu or= Arg; Xaaaj is Val or Lys; ’ Xaay, is Lys, Glu, Asn or Arg; Xaas is Gly or Aib; Xaaasg is Arg, Gly or— Lys; Xaas; is Gly, Ala, Glu, Pro, Lys, amide or is absent; Xaasg is Lys, Ser, ammide or is absent. Xaas is Ser, Lys, aamide or is absent; Xaau, is Gly, amide= or is absent; Xaay, is Ala, amide or is absent; AMENDED SHEET

669%.204-WO Xaa,; is Pro, &amide or is absent; Xaay; is Pro, =2:amide or is absent; Xaay, is Pro, amide or is absent; Xaays is Ser, =}mide or is absent; Xaa is amide= or is absent ; provided that &f Xaaag, Xaase, Xaasn, Xaas;, Xaas, Xaas, Xaas, Xaass or XXaa,s is absent then each amino accid residue downstream is also abmsent.

44. A compoumnd according to claim 43, wherein ssaid polypeptide is a GLP- 1 peptide compris- ing the amino acid sequence of formula (V): Xaar;-Xaag-GlLu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Se=r-Xaa o-Tyr-Leu-Glu-Xaa ,,-Xaags-Ala-Ala- Xaay-Glu-Phea-lle-Xaas,-Trp-Leu-Val-Xaaa,-Xaaa:s-Xaass-Xaaz-Xaaas Formula (V) (SEQ ID No: 3) wherein Xaa is L-histiedine, D-histidine, desamino-histidi ne, 2-amino-histidine, -h ydroxy-histidine, homohistidine , N°-acety!-histidine, a-fluorometh-yl-histidine, a-methyl-histicdine, 3- pyridylalanine , 2-pyridylalanine or 4-pyridylalani me; Xaag is Ala, G ly, Val, Leu, lle, Lys, Aib, (1-amincocyclopropyl) carboxylic acid, (1- aminocyclobu-tyl) carboxylic acid, (1-aminocyclo pentyl) carboxylic acid, (1—aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic- acid, or (1-aminocyclooctyl) carboxylic acid; Xaa,s is Ser, L_ys or Arg; Xaa,, is Gly, Glu or Aib; Xaa,; is Gln, Glu, Lys or Arg; Xaaygg is Lys, CSlu or Arg; Xaasjp is Ala, Glu or Arg; Xaaas is Lys, Clu or Arg; Xaass is Gly o r Aib; Xaags is Arg o rLys; Xaas is Gly, Ala, Glu or Lys; Xaasg is Lys, &amide or is absent.

45. A compoumnd according to any one of claims =42-44, wherein said GLP-W peptide is selected from GLP-1(7~35), GLP-1(7-36), GLP-1(7-36)-armnide, GLP-1(7-37), GLP-1(7-38), GLP-1(7-39), GLP-1(7-40), «GLP-1(7-41) or an analogue thereof. AMENDED SHEET

6694204-WO

46. A commpound according to any one of claims 42-45, wherein saicd GLP-1 peptide comprises no more than fifteen amino acid residues which have been exchange=d, added or deleted as comparecd to GLP-1(7-37) (SEQ ID No. 1).

47. A commpound according to any one of claims 42-45, wherein saic GLP-1 peptide comprises no more *than ten amino acid residues which have been exchanged, added or deleted as com- pared to «GLP-1(7-37) (SEQ ID No. 1).

48. A cormpound according to claim 46 or 47, wherein said GLP-1 peptide comprises no more than six amino acid residues which have been exchanged, added or deleted as compared to GLP-1(7—37) (SEQ ID No. 1).

49. A cormpound according to any one of claims 46-48, wherein said GLP-1 peptide comprises no more —than 4 amino acid residues which are not encoded by the g enetic code.

50. A cormpound according to claim 42, vwherein said GLP-1 peptide is a DPPIV protected GLP-1 peptide.

51. A cormpound according to claim 42, vwvherein said compound is D®PPIV stabilised.

52. A cormpound according to any one of claims 42-51, wherein said GLP-1 peptide comprises an Aib re-sidue in position 8.

53. A cormpound according to any one of claims 42-52, wherein the samino acid residue in posi- tion 7 of =said GLP-1 peptide is selected from the group consisting of D-histidine, desamino- histidine, 2-amino-histidine, B-hydroxy-histidine, homohistidine, N°-a~cetyl-histidine , a- fluorome-thyl-histidine, a-methyl-histidine , 3-pyridylalanine, 2-pyridyl=alanine and 4- pyridylalanine.

54. A cormpound according to any one of claims 42-53, wherein saidll GLP-1 peptide is selected from the group consisting of Arg*'GLP-1 (7-37), Lys®Arg=>*GLP-1(7-38), Lys¥Arg?®*GL_P-1(7-38)-OH, Lys**Arg®*>*“*GLP-1(7-36), Aib®223 53] P-1(7-37), Aib®* GLP-1(7-37), Aib**# GLP-1(7-37), Aib®22% ang? ys®GLP-1(7-38), Aib®*° Arg”*Lys**GLP-1(7-38), Ab? Areg®™*Lys®GLP-1(7-38), Aib®*?*® Arg®*Lys**GLP-1(7-38), AMENDED SHEET

669H204-WO n 121 AI Arg®*Lys**GLP-1(7-38), Aib*?**° Arg”Lys**GLP-1(7-38), AI? Arg®Lys* GLP-1(7-38), Aib®? Arg®Lys*GLP-1(7-38), AI? 3 Arg®Lys*® GLP-1(7-38), Aib**’Arg*Lys®GLP-1(7-38), Aib*#Arg*Lys*GLP-1 (7-38), AI 231% Lys®BGLP-1(7-38), A ib**°Ala* Lys®*GLP-1(7-38), »Aib**Ala*Lys**GLP-1(7-38), AI? Lys GLP-1(7-37), Aib®*® Lys GLP-1(7-37) and Aib®**=Lys* GLP-1(7-38).

55_. A compound according to any one of claims 42-54, where in said GLP-1 peptide iss attached to =said hydrophilic spacer via the amino acid residue in positiosn 23, 26, 34, 36 or 38 reelative to thea amino acid sequence SEQ 10D No:1.

56 . A compound according to any one of claims 42-45, where in said GLP-1 peptide is exendin- 4,

57 . A compound according to any one of claims 42-45, where in said GLP-1 peptide iss ZP-10,

i.e HGEGTFTSDLSKQMEEEAVRL_FIEWLKNGGPSSGAPPSKKK_KKK-amide.

58 . A compound according to any one of claims 42-57, where-in one albumin binding residue viam said hydrophilic spacer is atta ched to the C-terminal amino acid residue of said GLP-1 pep- tidee,

59 . A compound according to cla im 58, wherein a second albmumin binding residue is attached to an amino acid residue which iss not the C-terminal amino acid residue.

60 . A compound according to any one of the previous claims, wherein said compoun«d is se- lected from the group consisting ©f N==".(2-(2-(2-(dodecylamino)ethoxy)ethoxy)acety!)»-[Aib****°L_ys*|GLP-1(7-37)amide Lo} EN o HC CH, o ¢ TY, web TTT mT HC

N7.(2-(2-(2-(17-sulphohexadec anoylamino)ethoxy)ethoxy)=acetyl)-[Aib®*** Lys] (GLP-1 (7- 37 Jamide AMEND=~ED SHEET

6691204-WO

[o] No HINT "1 Co 0 pr % H—-N T N E-G-T-F-T-8-D-V~S~S-Y—L —E-N Q-A-A~-K-E~-F—I—A-W-L-V—K-N R-N NH, ’ ° on Wet Q ee Ben Ho % 0 N®7-{ 2-[2-(2-(15-carboxypentadecanoylamino)ethoxy)ethoxylacety IF [Aib®#% Lys*'] GLP- 1(7-3=7)amide lo] HNN HINT al HN X No HCTF TS DVS SYLEN, FQOAAKEFAWLV KY IRN r NH, N, HC CH, H.C CH, HO cme © : N“7-( 2-(2-(2-(17-carboxyheptadecanoylamino)ethoxy)ethoxy)acetyl)[A ib>#*° Lys* |GLP-1(7- 37)armide wl ay "L ~ HC i$ N H H—N N e-a-r-r-1-s-0-v-s-s-v-t-= Hl, Jaa akeroawivy R-N NH, "oo oe HC CH, oo "oo NH a TS CaS 0 0 N="-( 2-(2-(2-(19-carboxynonadecanoyla mino)ethoxy)ethoxy)acetyl)[Ailb*??* Lys*' |GLP-1(7- 37)armide [¢] HN (¢] HINT 1 wn 9 <l ea S Bar -G~T-F-T-§-D-V-§-5-Y-L-E- ~A-A-K—E—F—I-A-W-L—V~K- ~ 3 H-N z a STE BYE Sg AE JRE Th Th een 0 ’ [AIb=%* Arg®**IGLP-1-(7-37)Lys(4-(Hexadecanoylamino)-4(S)-carboxybutyryl)-OH AMENDED SHEET

669. 2004-WO 1 9 HN =" o H,C C H, ox" yt Seo TEs o vss Y-E Rg Qa AREF aw wad, Srey o H oO H H,C~ CH, HC “CH, ) 2 NH HC SAH ’ bad: HO 0 [Aib®*#*° Arg®**|GL P-1-(7-37)Lys(2-(2-(2-(hexadeacanoylamino)ethoxy)e=thoxy)acetyl}-OH HNTS o =, OH HC CH, H,C. CH, lo} I GL SURO: SURO JOS § H fo) H fe} H o H.c=" "CH 3 3 HN r° Om HC ere ST Oo sy H N27(2-[2-(2,6-(S)-Bis-{2-[2-(2- (dodecanoylamino)ef®hoxy)ethoxylacetylamino}hexaanoylamino)ethoxyletrmoxy}) acetyl-[Aib*?**)|GLP—1(7-37)amide

[0] am HN oN, ply HC { ha & o o &) cr 0 NH, H N IY GATS SPOS SU. § 'o) BAN Ho He” CH, He’ CH, ° Lm 0 HN~ a o [o] NH QQ — 0) § et at HC HC N%7-(2-[2-(2,6-(S)-Biss{2-[2-(2- (tetradecanoylamino Dethoxy)ethoxylacetylamino}the xanoylamino)ethoxyje=thoxy}) acetyl-[Aib®?2*)GLP—1(7-37)amide AMENDED SHEET

665 £2.204-WO

[0] nS (o] HN 1 H 9 H © HC CH, ? H-N" i Remrrrsovesvich Sonn K-E-F—I—A-W-L-V-K-N"—R-N NH, N Ho ncn, HC CH, co HH >» H Pe HENNA Ono Lo [o}

[0]

SOP. [AIb*?2%° Arg®®*']- GLP-1-(7-37)Lys(2-(2-(2-(4-(Hex adecanoylamino)-4(S- )- carboxybutyrylam ino)ethoxy)ethoxy)acetyl}OH HN, OH H,C CH, H,C_ CHa © GL IU. ST i or H go H oO H o he - H ) H,C~ CH, FPP | pan Oo H,C ¥ Oo [a=] HO Oo [AIb*Z*PIGLP-1(7 -37)Lys((2-{2-[4-[4-(4-Amino-9,10O-dioxo-3-sulfo-9,10-dihydro-anthracen-1- ylamino)-2-suifo-pohenylamino}-6-(2-sulfo-phenylanmino)-[1,3,5]triazin-2-y=tamino]-ethoxy}- ethoxy)-acetyl))armide HN H [0] H © EN gil H—N’ al J—e-G-1-F-T-5-0-v-5-5-v-L-E-N Q-A-A-K-E-F—I—A-W- L-V-K-N R-G-N NH Ho uncer, pe H o ! )

[0] O NH, Lo hs CIT on °y™ 0 N NN N00 H rH 57S Np Hoo HN 0 jo ° [AIb>22*%)GLP-1 (7 -37)Lys(({2-[2-(2-{2-[2-(2-{2-[2-(-15-carboxypentadecamnoylamino)- ethoxy]ethoxy}aceatylamino)ethoxylethoxy}acetyl armino)ethoxylethoxy}a cetyl))amide AMENDED SHEET

669%204-WO ‘o 125

[0] nN 0] Hy ng

[0] [o] H,C CH, ? a No Jme-eT=rr-s-0-v-s-s rete fl, Ja-aa-k-epimaaw-t vor Roa NH, 3. Oc CH, HC CH, ° 0 k oo [0]

[0] ~ I HNO o NPN IN" ([2-(2-{3-[2,5-dioxo-3-(15-carboxypentadecylsulfanyl)-pyrrolidin-1-yl}- poropionylaminojethoxy)ethoxy » acetyl}[D-Ala® Lys*)-GLP-1-[7"- =37]amide [¢] HN HN oO N Ho 1, m-i-N N he 0m ToF T-5-D-\ 5-5 v-L-E-0-0-A-AK-E-Fi-A NS AL -V-K-G-R- OH N Ho I, H 9 hg ON 0 0 mmm 0 i) [Aib® 2 Ala* |GLP-1(7-37)Lys ((2-(2-(2-(11- Coxalylamino)undecanoylamino)ethoxy)ethoxy)acetyl-)))amide ry HN lo) N = [«] ° Hc. CH, 1, HN T a Sy I NH, ¢ a Ca , [Aib®?2% Ala*]-GLP-1(7-37)Lyrs({2-[2-(2-{2-[2-(2-(15-carboxy -pentadecanoylamino)- ethoxylethoxy}acetylamino)eth oxylethoxy}acetyl)amide AMENDED SHEET

660ipw»204-WO “»

[0] HN A lo) HNN 1 H [e] H [e) H,C CH, Oo H-N’ Scorer sovosvich Fanner AWK SR-A-N Ne H, N H o H © H o HN_ oO HC CH, HC CH, Y [of

[0] [¢} IPSS

[0] H [Aib®*%3° Ala®}-GLP-1(7-37)Lys((2-{2-[11-(5-Dimethylam inonaphthalene-1- sulfonylamino)undecano ylaminolethoxy}ethoxy)acetyl)armide [e] nN lo] MN 1 [eo] [eo] HC CH, ? HN T OR SA EE EE I Ne ¢ Sl

0. N aw th, [AIb®#2*® Ala*]-GLP-1(7-37)Lys(([2-(2-{2-[1-(4-Chlorobermzoy)-5-methoxy-2-methy I-1H-indol- 3-ylJacetylamino}ethoxy)ethoxy]acetyl))amide a») an 0 HN 1 0 4 0 H,C_ CH, 0 HN 1 Le orrr-s0v-s-sv-ted, Lannreri—antvicy ray NH, % © ne” en, HC CH, © © 0 a cl = N d oC , [Aib®, Arg®®™* GIuZ#* JG LP-1 H(7-37)Lys(2-(2-(2- (octadecanoylamino)etho xy)ethoxy)acetyl)amide AMEND ED SHEET

669%=.204-WO £S [o] ON [s} HN H-N Ho e-o-1- F-T—-5~D-V-§-S-V—L—E--E-E-A-A-R—-E~-F" ——E-W-L-V-R-G-R-G-N WH, N Oc cH, o oN HE nme [Aib® Arg? GIu*= 2 2GLP-1(7-37)Lys(2-(2-(2- (eicosanoylamino)esthoxy)ethoxy)acetyl)amide [¢] w HN 1 1 t, H-N Ny JE GT -F=T-§-DV=§-5 YL —E-E-E-A=_A-R-E—F—I—E-W-L-V-R-G-RE-G-} NH, © HC” “cH, "oo os eee eee ’ [Gly® Arg”) GLP—1 H-(7-37)Lys(2-(2-(2-(2-(2-(2-(4--(octadecanoylamino)-4S)- carboxybutyrylamiro)ethoxy)ethoxy)acetyl)ethoxy)e thoxy)acetyl}-OH [o] wl HN © ON 1, H—N G-E—G—T —F—T—§-D-V—5—§—Y—L—E~G—Q—A—A—R —E—F—]—A-W~-L—V—R—G—R—G—N OH \ H 0 0 H o) lo] NH a ad © oon " [Aib® Arg” *IGLP-1 (7-37)Lys{2-(2-(2-(2-[2-(2- (octadecanoylamirmo)ethoxy)ethoxy]acetyl)ethoxy)et-hoxy)acetyl)}OH HN H H H-N hoe G-T-F-T-5-D-V-8-§-Y-L-E-G-Q-A-AR-E-F-I-FAW-L-V-R-G-R—G-N_N 5,

[0] H HC CH, \ H 0 oy HESS Neg OA 0 ~0 oOo H ’ AMENDED SHEET

€5693-204-WO ES 128 [Aib®] -GLP-1 ~(7-37)Lys (2-(2-(2-(4-(Hexaadecanoylamino)-4(S)- carboxybutyry- lamino)ethoxy)ethoxy)acetwyl)-OH HN “N 00H H (aw) N Se PRR. egy I 0} — HC “cH, HO of NSN i = Nr 3 o H fo) ’ [Aib® Arg] CGLP-1(7-37) Lys{2-(2-(2-(2—[2-(2-(4-(octadecanoylamimno)-4- carboxybutyry lamino)ethoxy)ethoxylacetyijethoxy)ethoxy)acetyl)}O BH HNN Cys ¢ H H H-N i Jeerrrsove-svissanareriawivaerei lo, H o : H,C™ “CH, hG (6) H Oy MH 2 TH 2 y HES SSSA Ne No 0 N"~O~"0 oO [Aib®Arg®®*] GLP-1 (7-37)Lys{2-(2-(2-(2—[2-(2-(17- carboxyheptaroylamino)ethoxy)ethoxy]acetylamino)ethoxy)ethoxy)amcetyl)} OH ENTRY e 0 HH I Hemomrorr—s-oves-s—vii-e-o-aaaa-eroawe—enono dL ls} H,C CH, A < " o De a Th aT PL NTO [¢] [Gly®, Arg®®**] GLP1-(7-37) Lys{2-(2-(2-(2 -[2-(2-(17- carboxyheptadllecanoylamino)ethoxy)ethoxylacetyl)ethoxy)ethoxy)ac-etyl)}OH [o] nN [e] HN On 1 H-N G-E— G-T-F-T-S$-D-V-$-8-Y—-L—E-G-CQ-A—A-R—-E—F—I—A-W-L-V-R-G-R -G-N OH N H H ¢] 0 [o] [o} NH ENP A ° " AMENDED SHEET

6692.204-WO [AL®JGLP-1-(7-37)Lys(2-(2-(2-( 2-(2-(2-(4-(Hexadecanoylanino)-4(S)- carboxybutyrylamino)ethoxy)eth oxy)acetylamino) et hoxy)ethoxy)acetyl)-OH HN OH =, HC CH, ° ~~" IN H—N NS E-G-T-F-T-5-D-v-S -§-Y-L-E-G-Q-A-A-K-E-F—~I-A-W -L-V-K-G-R-G-N~ 0 A0 Ho Ho H HH ~~ N_0 H,C 0 Q or} 3 NNN NTN HN N Ho” ~° N=¥-(2-(2-(2-(dodecanoylamino »ethoxy)ethoxy)acetyl)-[Aib®=**Lys¥] GLP-1 H(7-37D)-amide Oo HN HN" °

= H.C. CH 0) nar % e-GT-F-T-5-D-v-S—s-v-L-E-N P o-a-A-k-E-F-i-A- Wyk Se NH N Ho Ix pe Ho Ho °° oOuN H,C CH, H,C CH, eS

NE.(2-(2-(2-(tetradecanoylamirmo)ethoxy)ethoxy)acetyl)- [AID *2*Lys*] GLP-1 H(7-337)- amide (@] HN HN al

C H.C. CH fo H © H Q : } H—N N E-G-T-F-T-S-D-V-S-S-Y-L-E-N Q-A-A-K-E-F—1— AW-L-V-K-N "RN NH4, AN Hg hl Nl Ho H o NH HC CH, H,c™ CH, Pal H,C NE ¥-(2-(2-(2-(hexadecanoylamiro)ethoxy)ethoxy)acetyl)-[Aito****Lys*') GLP-1 (7-377 )-amide OD ANN HN "1 = HC. CH, fo} H—N N Be GT-F-T-5-D-v-S-5-v-L-E-} RA A-K-E-F lm AW-L-V-KNR-N NH N HJ lo No” Ho Ho ° HC TCH, H,C™ CH, I ea UPN ° © AMENDESD SHEET

6692.204-WO N®%"-(2-(2—(2-(octadecanoylamino)ethoxy)ethoxy)acetyl)}-[Aib®?***Lys*"] GLP=-1 (7-37)-amide fo} PEN fo} HN N HC. CH, Lo H © H © > H-N NSECTFTSOVS SY LEN FQAAKEFIAWLVKY RN NH, N H oO Ho HO NH he” CH, HC CH, H Cae N*-(2-(2—(2-(eicosanoylamino)ethoxy)ethoxy) acetyl} [Aib®****Lys®’] GLP-1( 7-37)-amide lo} HNN 0} HN, 1 H,C CH, fe) HO HO Xo N H-N NS EGTFTSDVSSYLEN FQAAKEFI-AWLYKY RN" -NH, H oO Ho HQ NH HC CH, H,C® CH, HyC ae es

Nt®.(2-(2—(2-(2-(2-(2-(octadecanoylamino)etho xy)ethoxy)acetylamino)ethox y)ethoxy)acetyl))- [Aib%Arg®®* Lys*|GLP-1-(7-37)-OH [lo] Q i f HM 1 2 Lo wr HN = CHa © ~ N HN I | Jere P ToS Sloe 0A AR EFA WLR I NA —on H,C=" CH,

Ne. (2-(2—(2-(2-(2-(2- (octadeca noylamino)ethoxy)ethoxy)acetylamin o)ethoxy)ethoxy)acetyl))[Arg™ >> Lys*|GLP- 1(7-37)-O H AMENDED SHEET

6692.204-WO N 131 0 o) 8 { 1 HN JN 0} So 0 HN HN CH, Lo AA 0 rp hop T-8-0-y-55-¥ iE AA ro1—Ac-t-v-rm-o-y HT on lo} 0 N®®_{2-(2-(2-(2 -[2-(2-(octadecanoylamino)ethoxy)esthoxylacetylamino)ethox y)ethoxy)acetyl)} [Gly® Arg? Lys |GLP-1-(7-37)-OH e} [0] i J HN JN wey HR, CH, H 2 ee errno N_JL—on o © 1] N*"-(2-(2-(2-(4—4(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecaflLiorononanoylsulfamoyl- butyrylamino)e®thoxy)ethoxy)acety))[Aib®**% Lys*] GLP-1-(7-37)-OH F F F le] 00 [o} F |F |F OR H RN HH Sono Hy [e] 0 H,C CH, CS TUT TS—— OH © CH, H,C” “CH, oo 0 CH, N™-(2-(2-(2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,1 1,12,12,12-Heneicosaflu cro- dodecyloxyace®ylamino)ethoxy) ethoxy)acety [AILS Lys*'|GLP-1-(7-37)-OH FF _F F_F Fl FIL FT ELF oY 0-0 I F F F F F 0) NH [@] [eo] Hy C CH, H H H-N N E.-G-T-F-T-8-D-V-S-S-Y-L-E-N Q— AA K-E~F ~~ A-W-L-V—K—R-N OH H La, Fen, "o "oo N7-(2-(2-(2-(4—(hexadecanoylsulfamoyl)butyrylami no)ethoxy)ethoxy)acety| m[Aib>#** Lys®] GLP-1-(7-37)-COH AMENDED SHEET

6692.204-WO o Oo [0] NY iY BP Moo fT NH HN &

[0] [o} HC CH, Ne E-G-T-—F~T-§-D-V-S-S-Y—L-E-N Q-A-A-K~-E=-F—1-A-W-L-V-K-N R-N OH : rs on CY i; od [Arg*®**IGLP-1(7- 37)Lys({2-(2-(2-(2-[2-(2- (octadecanoylamimo)ethoxy)ethoxylacetylamino)ethoxxy)ethoxy)acetyl)}} OH HN

[0] HN T A—E~G—T—F—T—§-D—V~8—8—Y—L—E-G-Q-A—A— R-E—F—i—A-w-L-V-R-G-R-G—H_J oN if > HC a a a o! H [Arg®®*] GLP-1(7-37)Lys{2-(2-(2-(2-[2-(2-(4-(octadec -anoylamino)-4- carboxybutyrylami no)ethoxy)ethoxylacetylamino)ethomxy)ethoxy)acetyl)}-OH HN nH © I GEIS 3 0] :

HO._.O Og NH JIE ; J HC SS SSS NN OA Og H H lo) oO N2-{2-(2-(2-(2-[2—(2-(4-(hexadecanoylamino)-4- carboxybutyrylami no)ethoxy)ethoxylacetylamino)ethcaxy)ethoxy)acetyl)}exendin€ 1-39) AME NDED SHEET

6692204-WO

Hy o sp - HN EGTFT-SOLSKAMEEEAVN J —L-F-EWL-KNGCCP SS CAFPPH fo] H lo} Oy OH o oN" my 0 T™ AAA NN ~~ OA AOA HC N “Y 0 N 0 [mala Arg? *GLP-1(7-37)Lys((=2-[2-((2-oxalylamino-3-carbomxy-2-4,5,6, 7-tetrahydrom- be=enzo[b]thiophen-6-yl-acetylam Eno))ethoxylethoxyacetyl) armide HI uw © HN A-E-G-T~F=T~S§-D-V—$-8-Y-L~E-G-Q-A~A—R-E—F —I—-A-W-L-V-R-G-R-G-N__J lo} : 2 «QO NH 0 H lo) EL cocoons) 0 [A\ib® #2 *)GLP-1(7-37)Lys((2-[2-( (2-oxalylamino-3-carboxy-2—4,5,6,7-tetrahydro- be=nzo[b]thiophen-6-yl-acetylamimno))ethoxylethoxyacetyl) anmiide ny ® He cH, HC CH, I 1, Son A 1h A tine TN, CEH Ld N=%-(2-(2-(2-(2-(2-(2-(4-(octadec=anoylamino)-4(S)- carboxybutyrylamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoOxy)acetyl) [A_ib® Arg? Lys®*|GLP-1-(7-37)—0H AMENDEZ=D SHEET

6692.204-WO ~ 135 pe H,C= CH H © H © > iy ne E-G-T-F-T-S-D-V-5-S-Y-L-E-N_J-Q-A-A-K-E-F-1-AW-L-V-K-Na YR NO H o pol ay Raa H,C' ~CH, H,C CH, { NH, SN ol H o SN ° HC SS SSS Co N 0 H H =0 OH HO

N=.(2-(2-(2—(4-(Hexadecanoylamino)-4(S)-c arboxybutyrylamino) ethoxy)ethox<y)acetyl)-[Aib® Arg*|GLP-1-(7-3 7)- -OH HNN CG 9 PR) H-N T A a a SH H,C CH, H Q EE a th a Ea NH 0 Ag HM TY HO NE26.2.(2-2-(2-(2-(2-(4-(Octadecanoylamino)—4(S)- carboxybuty mylamino)ethoxy)ethoxy)acetylarmno)ethoxy)ethoxy)acetyl- [Aib®, Arg*]CGLP-1-(7-37)-OH Hy ef TOTES ESO—— "Ero AN lo} Q H worm I Tomo Tyo [Gly® Arg®*>*]GLP-1(7-37)Lys(2-(2-(19-(carboxy)nonadecanoylamino)ethcoxy)ethoxy)acetyl)- OH AMENDED SHEET

6692.204-WO « [e] I i a lo} H NH HIN I Ci G-Q-A-A-R—E—-F—I—A-W-L-V-R—G-R-G-N OH fo) H o [Gly®, Arg®**|GLP-1(7-37)Lys((2-(2-(17- (carboxy)heptadecanoylamino)ethoxy)ethox y)acetyl)-OH

[0] fo) H NH HN IS SO OH Ho H o [Gly®, Arg®®**|GLP-1(7-37)Lys(2-(2-(2-(4-(19—(carboxy)nonadecanoylamineo)-4- carboxybutyrylamino)ethoxy)ethoxy)acetyl}CH

[0] H [0] o eC Ga A [o] o OH NH Hy IS SO OH H 9 Ho [Gly®,Arg®®*IGLP-1(7-37)Lys((2-(2-(2-(2-(2- (2-(2-(2-(2- (hexadecanoylamino)ethoxy)ethoxy)acetyl)erthoxy)ethoxy)acetylamino)etBhoxy)ethoxy) acetyl )-OH Q H Q fo) N R HOPS Ng 0 ~o Yr oo A HN © o _N eer irsavesyice ernmeromsvnsme Com 0 NT [Gly®, Arg®*|GLP-1 (7-37)Lys (2-(2-(2-(2-(2-(2-(octadecanoylamino)etheoxy)ethoxy)- acetyl amino)ethoxy)ethoxy)acetyl) NH, AMENDED SHEET

6692.204-WO lo} ns SE ™L [o] EG R—E—F—I—A=W-L—V—R-G—R~G-N hk N [o] o bal J ern AL N=20(2-(2-(2-(2—(2-(2-(2-(2-(2-(2-(2-(2-(4-(1 7-(cart>oxy)heptadecanoylamino)-<3- carboxybutyryl-amino)ethoxy)ethoxy)acetylamino®»ethoxy)ethoxy)acetylamino) ethoxy)ethoxy)- acetylamino)ethoxy)ethoxy)acetyl® [Lys’’lexendin-4 (1-39)-NHI, HN N OH H-N G-E--GT-F-T-5-D-L-S-K-QM-E-E-E-A-V-N IE ~F~I-E-W-L-KN-GG-P-§-5-G FE Ho i H oO o 9’ o OH (ONO oto O00 o H fo) H N HOO NP 0 SSP lo} H lo] N=%-(2-(2-(2-(2=-(2-(2-(17-Carboxyheptadecanoylaamino)ethoxy)ethoxy) acetylamino)et hoxy)ethoxy)acetyl) [Aib® Arg®®**, E.ys*] GLP-1 (7-37) N lo} Ne 07 N\_-0 A NTN—o lo MN HO 0 H,C CH, Hof on remem Non Hoe MS 0 ,

N-%.(2-(2-(2-(2-(2-(2-(17-Carboxyheptadecanoy Bamino)ethoxy)ethoxy) acetylamino)et hoxy)ethoxy)acetyl) [Arg”>*, Lys®*® ] GLP-1 (7-37) AMENDED SHEET

6692.204-WO N 0 Ne oo J NTN ENG Li) VE. HN HNN HO — N

[0] H—N A—E-G~T-F-T-$-D-V-5—-S-Y—L-E- G-Q—A-A-R-E-F—I—A-W-L-V-FR-G-N nro H a H 0 NE (2-(2-(2=-(2-(2-(2-(17-Carboxyheptadecanoylamino)ethoxy)ethoxy) acetylaminos )ethoxy)ethoxy)acetyl) [Gly? Arg®® =“ Lys*] GLP-1 (7-37) H o Me— SN { NTN\-o0 NN Seo HN HN"™\ HO — N oO OH H—N G—E-G-T-F-T-§-D-V-§-§-Y—L-E-G~ Q—A-A-R-E-F—I-A-W-L-V-R-G -N vn o 0 N20-(2-(2-(2=-(2-(2-(2-(2-(2-(2- (Octadecan-oylamino)ethoxy)ethoxy)acetylamimo)ethoxy)ethoxy)acetylamirmo)ethoxy) ethoxy)acet—yl)[Lys*’] Exendin-4 (1-39)amide oO [o] LWT [o} (¢} [o] Loo o—A\, HN" =~ N OH HN G-E-GTF TSDLSKOMEEEAVN L-F-I—-EWL KNGGPSSGA FP om 0 © o AMENDED SHEET

6692.204-V\/O

N%.(2-(2-(2-(2-(2-(2-(4-(coctadecanoylamino)-4(S)- carboxybutyrylamino)ethoxy)ethoxy)acetylamino)ethox y)ethoxy)acetyl}[Arg?®*, Lys*|GLP-1- (7-37) 0 bell 0 N HN HO, o_o A A 0 =o HN CH, J SI ee veers nnnetannivany NOH Ho H O 0} N®2°-(2-[2-(2-[2-(2-[2-(17-Carboxyheptadecanoylamino) ethoxy] ethoxy)acetylamino]ethoxzy)ethoxy]acetyl)[Arg*|GLP-1—(7-37)-OH HNN C H 9 H i H-N I A—E-eG-T-F-T-§-D-V-§-S-Y-L-E-G-Q-A-A-N Sereawivrenf Jon

0 . \ [ROC

[0] [0] Ne26-[2-(2-[2-(2-[2-(2-[4-(1 7-Carboxyheptadecanoylamiro)-4(S)- carboxybutyrylamino]ethoxy)ethoxylacetylamino)ethoxwyjethoxy)acetylJ[Arg*]GL P-1-(7-37)- OH HN { HQ Ce ry! H-N x A-E-G-T-F-T-§-D-V-§-8-Y-L-E-G-Q aA JE FAW R-G- R-N OH or . \ I SN 0 OA AD mg NH 0 H 0 H fo)

N20. (2-(2-(2-(2-(2-(2-(2-(2-(2-(17- Carboxyheptadecanoylamino)ethoxy)ethoxy)acetylamiro)ethoxy)ethoxy)acetyl amino)ethoxy)ethoxy)acetyl)[Lys*’] Exendin-4 (1-39) armide AMEENDED SHEET

6652 .204-WO in Oo

[0] a le] a I 0 [0] [0] Oyo oO — AN HN" </ OH HN GEGTFTSDLS KOMEEEAVN LF-I-BWHL I o) S 0 , [Gly®, Glu?? “2% Arg'®?¥|GLP1 (7-37) Lys(2-(2-2-(2-(2-(2-(17- carboxyheptadecanoylamino)ethoxy)ethoxy)acet ylamino)ethoxy))ethoxy)am cetyl} NH, HN HO H-N G-E-G-T-F-T-§-D-V-S-R-Y-L-E-E-E-A-A-R-E-F-1-E-W-L-V-R-G-R—G-N_J H 0 H 2 . y : \ HoT esses Ng AO A AO ng NH 0 H 0 [Imidazolylpropionic acid’, Asp'®, Aib?* JGLP1(7-37)Lys NH((2-{[4-(17- carboxyheptadecanoylamino)butylcarbamoyljmethoxy}ethoxy)ethoxy)) NH, On H O H,C CH, H A-E=-G-T-F-T-8-D-D-8-S-Y-L-E-N_-Q-A-A—R-E-F-I~AW-L-V-R-N"J-FR-G-N o H,C CH, HO H 5 ~ Rl 0] “© NH 0 J HO ~~~ A 0 N 0 [Imidazolylpsropionic acid’, Aib***® JGLP1(7-37)Ly"s NH( (2-{[4-(17- carboxyhepotadecanoylamino)butylcarbamoyl]methoxy}ethoxy)ethoxy)) AMENDED SHEET

6692.204-WO NH, MT H,C CH H Creer ~F-T-8-D-v-5-5-Y-L-E-H, J-a-A-A- R-E-F—I-A-W-L V-R-N"-R-G —N N H o H o) H,C CH, fo} \ RY o) “po NH lo] J HO ~~~ ~~~ 3 N , and [3-(5-Imidazoyl)- propionyl”, Aib®, Arg?®** ] GLP-1 (7-37)Lys{2-(2-(2-(2-[2-(2-( 17- carboxyheptancylamino)ethoxy)ethoxylacetylamuino)ethoxy)ethoxy)acetyl)-COH 0] H 0] HOP esses Ne gO A ° } ?

[0] So A HN HN" H,C_ CH, NN E-G— T-F~T-§-D-V-8-§-Y-L-E-G-Q-A- A-R-E-F—I-A-W-L-V-R-G-R--G-N OH oH o H o

61. A compouncd according to any one of claims 1-41, wherein said therapeutic polypeptide is a GLP-2 peptide.

62. A compouncd according to claim 61, wherein said GLP-2 peptide is a DPP IV-protected GLP-2 peptide.

63. A compouncd according to claim 61, wherein said GLP-2 peptide is Gly>-G LP-2(1-33). AMENDED SHEET

6690.204-WO

64. A compound according to claim 61, wherein said GLP-2 peptide is Lys'’Arg®-GLP-2(1-

33).

65. A compound according to any one of claims 1-4 1, wherein said therapeutic polypeptide is human insulin or an analogue thereof.

66. A compound according to claim 65, wherein sai therapeutic polypeptide is selected from the group consist ing of Asp®®-human insulin, Lys®*® Prc®®-human insulin, Lys™2 GIu®??-human insulin, GY?" Arg®' Arg®®?-human insulin and des(E330) human insulin.

67. A compound according to any one of claims 1-4 1, wherein said therapeutic polypeptide is human growth hoesrmone or an analogue thereof.

68. A compound according to any one of claims 1-4 1, wherein said therapeutic polypeptide is parathyroid hormeone or an analogue thereof.

69. A compound =according to any one of claims 1-4 1, wherein said therapeutic polypeptide is human follicle stirmulating hormone or an analogue thereof.

70. A compound according to any one of claims 1-4 1, wherein said therapeutic polypeptide has a molar weight of less than 100 kDa.

71. A compound =according to any one of claims 1-4 1, wherein said therapeutic polypeptide has a molar weight of less than 50 kDa.

72. A compound =according to any one of claims 1-4 1, wherein said therapeutic polypeptide has a molar weight of less than 10 kDa.

73. A compound saccording to any one of claims 1-4 1, wherein said therapeutic polypeptide is selected from the group consisting of a platelet-dermved growth factor (PDGF), a transforming growth factor a (T~GF-a), a transforming growth factcor 8 (TGF-g), an epidermal growth factor (EGF), a vascular endothelial growth factor (VEGF), a somatomedin, insulin greowth factor (IGF-I), insulin greowth factor 11 (IFG-I1), erythropoietimn (EPO), thrombopoietin (TPO) or angio- poietin, interferon, pro-urokinase, urokinase, tissue golasminogen activator (t-PA), plasminogen activator inhibitor 1, plasminogen activator inhibitor 22, von Willebrandt factor, a cytokine, an AMENDED SHEET

6692.204-WO Lt 143 interleukin, interleukin (IL) 1, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-9, IL-11 , IL-12, IL-13, IL-15, IL-16, IL-17, IL.-18, IL-20 or IL-21, a colony stinnulating factor (CFS), GM-CSF, stem cell factor, a tu- mor nec rosis factor, TNF-a, lymphotoxir-a, lymphotoxin-g, CD40L, or CD30L, a protease in- hibitor, =aprotinin, an enzyme,, superoxicie dismutase, asparaginase, arginase, arginine deami- nase, acdenosine deaminase, ribonuclease, catalase, uricase, bilirubir oxidase, trypsin, papain, alkaline phosphatase, B-glucoronidase, gourine nucleoside phosphory lase or batroxobin, an opioid, e=ndorphins, enkephalins or non-ratural opioids, a hormone or neuropeptide, calcitonin, glucagom, gastrins, adrenocorticotropic hormone (ACTH), cholecysto kinins, lutenizing hor- mone, g-onadotropin-releassing hormone, chorionic gonadotropin, comrticotrophin-releasing fac- tor, vasopressin, oxytocin, antidiuretic homones, thyroid-stimulating Bhormone, thyrotropin- releasing hormone, relaxin, prolactin, pe ptide YY, neuropeptide Y, pamncreastic polypeptide, leptin, C_ART (cocaine and amphetamine regulated transcript), a CART related peptide, perili- pin, melanocortins (melanocyte-stimulati ng hormones), melanin-concentrating hormones, na- triuretic poeptides, adrenomedullin, endot helin, secretin, amylin, vasoamctive intestinal peptide (VIP), pituary adenylate cyclase activatimg polypeptide (PACAP), bombesin, bombesin-like peptidess, thymosin, heparin-binding prot ein, soluble CD4, hypothalmi c releasing factor, melanotconins and analogues thereof.

74. A ph armaceutical composition comp rising a compound accordings to any one of claims 1- 73, and & pharmaceutically acceptable e=xcipient.

75. The pharmaceutical composition according to claim 74, which is suited for parenteral ad- ministrattion.

76. Use of a compound according to any one of the claims 1-73 for tie preparation of a me- dicamen t.

77. Use of a compound according to anys one of the claims 42-60 for —the preparation of a me- dicamen t for the treatment or prevention of hyperglycemia, type 2 dia_betes, impaired glucose tolerancez, type 1 diabetes, obesity, hype rtension, syndrome X, dyslipmdemia, cognitive disor- ders, attmeroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorderss, stroke, inflammatory bowel sy ndrome, dyspepsia and gast ric ulcers.

78. Use of a compound according to any one of the claims 42-60 for —the preparation of a me- dicamen t for delaying or preventing disease progression in type 2 dia betes. AMENDED SHEET

6694 204-WO o 1443

79. Use of a «compound according to any one of t he claims 42-60 for the preparation of a me- dicament for decreasing food intake, decreasing B-cell apoptosis, increasing B—cell funtion and B-cell mass, and/or for restoring glucose sensitivi ty to B-cells.

80. Use of a ecompound according to any one of claims 61-64 for the preparaticon of a medica- ment for the freatment of small bowel syndrome, inflammatory bowel syndromes or Crohns dis- ease.

81. Use of a «compound according to any one of claims 65-66 for the preparation of a medica- ment for the ®treatment or prevention of hyperglycemia, type 1 diabetes, type 2 diabetes or 3- cell deficiency. AMENDED SHEET