The present invention relates to conjugates comprising an GLP-1 /Glucagon dual agonist, a linker and hyaluronic acid, pharmaceutical compositions comprising said conjugates, as well as their use as a medicament for treating or preventing diseases or disorders which can be treated by a GLP-1 /Glucagon dual agonist, for example in the treatment of disorders of the metabolic syndrome, including diabetes and obesity, as well as for reduction of excess food intake.

BACKGROUND OF THE INVENTION

GLP-1 agonists

Exendin-4 is a 39-amino acid peptide, isolated from the salivary secretions of the venomous Gila monster (Heloderma suspectum). It has some sequence similarity to several members of the glucagon-like peptide family, with the highest homology of 53% being to glucagon-like peptide-1 [7-36]-amide (GLP-1 ). Exendin-4 acts as a agonist on the GLP-1 receptor and bears GLP-1 -like insulin sectretagogue action in isolated rat islets. Exendin-4 is a high potency agonist and truncated GLP-1 agonist-(9-39)-amide is an antagonist at the glucagon-like peptide 1 -(7-36)-amide receptor of insulin-secreting beta-cells. Exendin-4 ("exenatide") was approved recently in the US and EU for improving glycemic control in patients with type 2 diabetes taking metformin and/or a sulfonylurea but have not achieved adequate glycemic control.

The amino acid sequence of exendin-4 is shown as SEQ ID NO: 1

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

The amino acid sequence of GLP-1 (7-36)-amide is shown as SEQ ID NO: 2

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2

Glucagon is a 29-amino acid peptide which is released into the bloodstream when circulating glucose is low. Glucagon's amino acid sequence is shown in SEQ ID NO: 3.

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH

Liraglutide is a marketed chemically modified GLP-1 analog in which, among other modifications, a fatty acid is linked to a lysine in position 20 leading to a prolonged duration of action (Drucker DJ et al., Nature Drug Disc. Rev. 9, 267-268, 2010; Buse, J.B. et al., Lancet, 374:39-47, 2009).

The amino acid sequence of Liraglutide is shown as SEQ ID NO: 4.

HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4-hexadecanoylamino-butyryl-)

EFIAWLVRGRG-OH

During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing an increase of blood glucose levels to reach a normal level. Hypoglycemia is a common side effect of insulin treated patients with hyperglycemia (elevated blood glucose levels) due to diabetes. Thus, glucagon's most predominant role in glucose regulation is to counteract insulin action and maintain blood glucose levels.

GLP-1 receptor agonists, such as GLP-1 , liraglutide and exendin-4, have 3 major pharmacological activities to improve glycemic control in patients with T2DM by reducing fasting and postprandial glucose (FPG and PPG): (i) increased glucose-dependent insulin secretion (improved first- and second-phase), (ii) glucagon

suppressing activity under hyperglycemic conditions, (iii) delay of gastric emptying rate resulting in retarded absorption of meal-derived glucose.

GLP-1/Glucagon (Glc) agonists

Pocai et al (Obesity. 2012;20:1566-1571 ; Diabetes 2009, 58, 2258) and Day et al. (Nat Chem Biol 2009;5:749) describe that dual activation of the GLP-1 and glucagon receptors, e.g., by combining the actions of GLP-1 and glucagon in one molecule leads to a therapeutic principle with anti-diabetic action and a pronounced weight lowering effect.

Peptides which bind and activate both the glucagon and the GLP-1 receptor (Hjort et al. Journal of Biological Chemistry, 269, 30121 -30124,1994; Day JW et al, Nature Chem Biol, 5: 749-757, 2009) and suppress body weight gain and reduce food intake are described in patent applications WO 2008/071972, WO 2008/101017, WO

2009/155258, WO 2010/096052, WO 2010/096142, WO 201 1 /075393, WO

2008/152403, WO 2010/070251 , WO 2010/070252, WO 2010/070253, WO

2010/070255, WO 201 1/160630, WO 201 1/006497, WO 201 1/152181 , WO

201 1 /152182, WO201 1/1 17415, WO201 1/1 17416, and WO 2006/134340.

Bloom et al. (WO 2006/134340) disclose that peptides which bind and activate both the glucagon and the GLP-1 receptor can be constructed as hybrid molecules from glucagon and exendin-4, where the N-terminal part (e.g. residues 1 -14 or 1 -24) originate from glucagon and the C-terminal part (e.g. residues 15-39 or 25-39) originate from exendin-4.

Otzen et al (Biochemistry, 45, 14503-14512, 2006) disclose that N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon, due to the hydrophobicity and/or high β-sheet propensity of the underlying residues.

WO2014/056872 discloses peptides which bind and activate both the glucagon and the GLP-1 receptor that are derived from exendin-4 wherein at least the aminoacid at position 14 bear a side chain for a prolonged halflife which makes them appropriate as active ingredient in the present invention.

Longacting GLP-1 /Glucagon agonists

Ideally, the peptide is formulated in a fashion that provides for a sustained plasma level in human for at least one week after application to a human body resulting in a once-weekly or longer injection frequency.

Current therapy with a long acting GLP-1 agonists is Bydureon® which is exendin-4 in a depot suspension for a once weekly injection based on poly(glycol-co lactic acid) using a 23 gauge needle.

WO2012/173422 describes a GLP-1 /Glucagon agonist conjugated to the Fc region of an immunoglobulin for weekly administration wherein the peptide is derived from oxyntomodulin.

Carrier linked prodrugs

To enhance physicochemical or pharmacokinetic properties of a drug in vivo, such as its half-life, such drug can be conjugated with a carrier. If the drug is transiently bound to a carrier and/or a linker, such systems are commonly assigned as carrier-linked prodrugs. According to the definitions provided by lUPAC, a carrier-linked prodrug is a prodrug that contains a temporary linkage of a given active substance with a transient carrier group that produces improved physicochemical or pharmacokinetic properties and that can be easily removed in vivo, usually by a hydrolytic cleavage.

The linkers used in such carrier-linked prodrugs may be transient, meaning that they are non-enzymatically hydrolytically degradable (cleavable) under physiological conditions (aqueous buffer at pH 7.4, 37°C) with half-lives ranging from, for example, one hour to three months. Suitable carriers are polymers and can either be directly conjugated to the linker or via a non-cleavable spacer.

Transient polymer conjugation through traceless prodrug linkers combines the advantages of prolonged residence time due to polymer attachment, a controlled drug release via linker optimization and the recovery of the original pharmacology of the native peptide after release from the polymer conjugate.

Using polymer-linker peptide conjugates, native unchanged peptide is slowly released after application to a patient, governed only by release kinetics of the linker and pharmacokinetics of the polymer carrier. Ideally, release kinetics would be independent from the presence of enzymes like proteases or esterases in body fluids to guarantee a consistent and homogenous release pattern.

Many acute side effects of drugs may be related to the drug peak levels which thereby may limit the dose for a given drug formulation. Reducing the drug peak levels (maximal drug concentration, Cmax) for a given dose may allow those drugs to be administered in higher doses without raising the risk of acute side effects. Administration of higher

doses in turn may allow to reduce the dosing frequency eventually leading to a once-weekly or even once-monthly dosing interval.

The administration of carrier-linked prodrugs may allow to control the drug release in a manner that the drug concentration remains relatively flat for a certain time period. The drug release itself will be mainly controlled by the linker which needs to be optimized for the intended dosing interval.

A suitable polymer needs to have a clearance half-life significantly longer than the drug release half-life of the linker as otherwise a part of the polymer will be cleared whil the drug is still attached to it. A short carrier half-life would therefore lead to a loss of drug for a given dose and to a steeper drug concentration curve.

WO2008/148839, WO2009/095479 and WO2012/035139 refer to prodrugs comprising drug linker conjugates, where the linker is covalently attached via a cleavable bond to a biologically active moiety, such as the GLP 1 - agonist exendin-4. The biologically active moiety is released from the prodrug upon cyclization-activation by cyclic imide formation. The release kinetic is dependent on the pH value and is minimum for storage of the prodrug at pH values from 4.5 to 5 and reach its intended release rate at physiological pH of around 7.4 to 7.5. An GLP-1 agonist-prodrug is described in which the linker is based on L-alanine and the polymeric carrier is a PEG-lysine based hydrogel. Not described are dual GLP-1 /Glucagon agonist-prodrugs.

Hyaluronic acid (HA)

Dhal et al (Journal of Biomedical Nanotechnology, vol 9, 2013, 1 - 13) report hyaluronic acid as a suitable carrier for drug conjugates. Kong et al. (Biomaterials 31 (2010), 4121 -4128) report an exendin-4-hyaluronic acid conjugate which showed an glucose lowering effect over 3 days in mice. The used HA was a linear polymer with a drug load ranging from about 2.4 to 12.%.

Shendi et al (J. Mater. Chem B, 2016, 4, 2803-2818) discloses a hyaluronic acid which was modified with divinylsulfone wherein a part of the viylsulfone groups are used to conjugate bioactive molecules and the remaining vinylsulfone groups are used as crosslinkers to form the hyaluronic acid hydrogel.

EP1790665 A1 discloses a process for producing a water soluble modified hyaluronic acid using a condensing agent for the conjugation of a drug to the hyaluronic acid.

These conjugates enhances the residence time of the drug in the blood by i.v.

administration. Also disclosed was the crosslinking of these conjugates to form a gel.

DESCRIPTION OF THE INVENTION

GLP-1/Glucagon dual agonist peptides suitable for the conjugates of the invention have a high solubility at acidic and/or physiological pH values e.g. at pH 4.5 and/or pH 7.4 at 25°C. Also the chemical stability at pH values of 4.5 to 5 is an important criterion for the long acting prodrug product. The prodrug is preferably formulated in this pH range in order to obtain a shelflife from at least 6 month at 4°C.

In the present invention hydrogels of crosslinked hyaluronic acid were chosen due to their longer residence time as a local depot at the application site than soluble HA. Important criteria for the use of hyaluronic acid (HA) as a carrier polymer is the achievable drug load in the final drug product which is determined by the drug load on the polymer itself and the concentration of the final solution/suspension. Giving the fact that the injection volume for subcutaneous drug depots is practically limited to equal/less than 1 ml_, preferably equal/less than 0.6 ml_.

The more concentrated the polymer solutions/suspensions of HA is, the more viscous is the formulation which has a negative impact on the syringability of the conjugate formulation. Viscous solutions need injection needles of a larger diameter to limit the force on the plunger of which the syringe is pressed. Also the time for injection is longer.

It was an object of the present invention to provide a conjugate for administering as a subcutaneous depot which releases a GLP-1 /Glucagon agonist in an active form over the time period of at least 6 days after administrations and which can be injected through 26 gauge needles or even needles of smaller inner diameter for good patient compliance.

An object of the invention is a conjugate or a pharmaceutically acceptable salt thereof, comprising a

crosslinked hyaluronic acid hydrogel, in which

0.001 to 20 mol% of the monomeric disaccharide units are crosslinked by a crosslinker; and

0.2 to 20 mol% of the monomeric disaccharide units bear -L1-L2 -L-Y-R20 groups;

L1 is a C-1-20 alkyl chain, in which optionally one or more carbon atoms are replaced by a group selected from -O-, NH(R5aa) and C(O)N(R5aa), and is optionally substituted with one or more groups independently selected from OH and C(O)N(R5aaR5aaa), wherein R5aa and R5aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L1 is attached to the hydrogel via a terminal amino group forming an amide bond with the carboxy group of the beta-1 ,3-D-glucuronic acid of the hyaluronic acid

L2 is a single chemical bond or is a Ci-2o alkyl chain, in which optionally one or more carbon atoms are replaced by a group selected from -O- and C(O)N(R3aa), and is optionally substituted with one or more groups independently selected from OH and C(O)N(R3aaR3aaa), wherein R3aa and R3aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L2 is attached to L1 via a terminal group selected from the group consisting of

and HO O

wherein L is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line;

L is a linker of formula (la),

wherein the dashed line indicates the attachment to the N-Terminus of Y by forming an amide bond;

X is C(R4R4a) or N(R4);

R1, R1 a, are independently selected from the group consisting of H; and d. 4 alkyl;

R2, R2a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

R4, R4a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

wherein one of R2 , R2a, R4 or R4a is attached to L2;

Y is a peptide moiety having the formula (lb)

His- D-Ser -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Glu-Ser-Lys-Ala- Ala-Gln-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lb)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-hexadecanoylamino-butyryl;

or Y is a peptide moiety having the formula (lc)

His- dSer-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Asp-Glu-Gln-Leu- Ala-Lys-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lc)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-octadecanoylamino-butyryl;

FTU is OH or NH2.

The present invention relates to a conjugate which provides a GLP-1 /Glucagon agonist release from a subcutaneous depot in an active form over the time period of at least 6 days after administration.

This helps patients to reduce the frequency of injections, while being able to maintain optimal control the plasma levels of GLP-1 /Glucagon agonist and consequently blood glucose.

Additionally, the conjugate according to this invention may release the dual GLP- 1 /Glucagon agonist in a release profile resulting in a very flat pharmacokinetic profile of the agonist leading to a lower risk of Cmax-related side effects.

Further advantages of the conjugate of the invention are the good injectability through a 26 gauge needle or even a needle of a smaller inner diameter.

LEGENDS TO THE FIGURES

Figure 1 a: In Vivo Degradation Profile and Kinetics of Degradation of High Molecular Wight (2.5 Million Da) Hyaluronan (HA).

The implanted HA was monitored by MRI technique. Representative MRI images obtained by both Standard (top) and CEST (bottom) imaging techniques.

Figure 1 b: In Vivo Degradation Profile and Kinetics of Degradation of Divinyl Sulfone Crosslinked Hyaluronan. Kinetics of degradation was determined by plotting peak magnitude at 1 ppm.

Figure 1 c: In Vivo Degradation Profile and Kinetics of Degradation of High Molecular Wight (2.5 Million Da) Hyaluronan (HA).

Kinetics of degradation was determined by plotting peak magnitude at 1 ppm.

Figure 2: Solid state 1 H-NMR spectra of HA-hydrogel-Aib-linker conjugate of peptide of Seq. ID No. 5.

Figure 3. In vitro release kinetics of Peptide with Seq. ID No. 5 from HA-hydrogel-Aib-linker conjugate.

Figure 4: Fed blood glucose profile in male db/db mice treated with a single-dose (day 1 ) SEQ ID NO: 5 with non-crossl inked HA.

Figure 5A: Body mass change of female diet-induced obese mice treated with four doses (day 1 , 8, 15, 22) HA-hydrogel-Aib-linker conjugate with peptide of SEQ ID NO: 5.

Figure 5B: Feed consumption of female diet-induced obese mice treated with four doses (day 1 , 8, 15, 22) HA-hydrogel-Aib-linker conjugate with peptide of SEQ ID NO: 5.

Figure 6: Plasma concentrations of peptide of Seq. NO: 5 after single subcutaneous administration of 4.5 mg/kg of HA-Aib-linker-conjugate to female C57BL/6 mice.

Figure 7: Plasma concentrations of peptide of Seq. NO: 5 after single subcutaneous administration of 0.623 mg/kg in suspension (16.05%) as HA-Aib-linker-conjugate to female Gottingen minipigs.

Figure 8. Fed blood glucose profile in male db/db mice: Single s.c. treatment with 50 nmol/kg crosslinked HA conjugate of SEQ ID NO: 5 and once daily s.c. treatment with 1 .7 nmol/kg pure peptide of SEQ ID NO: 5

Detailed Description

The GLP-1 /Glucagon agonist bound to a linker-L2- is referred to as "GLP-1 /Glucagon agonist moiety".

"Protective groups" refers to a moiety which temporarily protects a chemical functional group of a molecule during synthesis to obtain chemoselectivity in subsequent chemical reactions. Protective groups for alcohols are, for example, benzyl and trityl, protective groups for amines are, for example, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and benzyl and for thiols examples of protective groups are 2,4,6-trimethoxybenzyl, phenylthiomethyl, acetamidomethyl, p-methoxybenzyloxycarbonyl, tert-butylthio, triphenylmethyl, 3-n itro-2-pyridylth io, 4-methyltrityl.

"Protected functional groups" means a chemical functional group protected by a protective group.

"Acylating agent" means a moiety of the structure R-(C=O)-, providing the acyl group in an acylation reaction, optionally connected to a leaving group, such as acid chloride, N-hydroxy succinimide, pentafluorphenol and para-nitrophenol.

"Alkyl" means a straight-chain or branched carbon chain. Each hydrogen of an alkyl carbon may be replaced by a substituent.

"Alkylene" means a straight-chain or branched carbon chain, wherein two moieties of a molecule are linked to the alkylene group. Each hydrogen of an alkylene carbon may be replaced by a substituent.

"Aryl" refers to any substituent derived from a monocyclic or polycyclic or fused aromatic ring, including heterocyclic rings, e.g. phenyl, thiophene, indolyl, napthyl, pyridyl, which may optionally be further substituted.

"Acyl" means a chemical functional group of the structure R-(C=O)-, wherein R is an alkyl or aryl.

"Ci-4 alkyl" means an alkyl chain having 1 - 4 carbon atoms, e.g. if present at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl tert-butyl, or e.g. -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-, when two moieties of a molecule are linked by the alkyl group. Each hydrogen of a Ci-4 alkyl carbon may be replaced by a substituent.

"Ci-6 alkylene" means an alkyl chain having 1 - 6 carbon atoms, wherein two moieties of a molecule are linked to the alkylene group, e.g. -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-. Each hydrogen of a Ci-6 alkyl carbon may be replaced by a substituent.

Accordingly, "CMS alkyl" means an alkyl chain having 1 to 18 carbon atoms and "Cs-is alkyl" means an alkyl chain having 8 to 18 carbon atoms. Accordingly, "Ci-5o alkyl" means an alkyl chain having 1 to 50 carbon atoms.

"Halogen" means fluoro, chloro, bromo or iodo. It is generally preferred that halogen is fluoro or chloro.

"Hyaluronic acid" means a polymer of a disaccharide composed of of beta-1 ,3-D-glucuronic acid and beta-1 ,4-N-acetyl-D-glucosamine and their respective sodium salts. These polymers are linear.

"Disaccharide unit" means the disaccharide composed of beta-1 ,3-D-glucuronic acid and beta-1 ,4-N-acetyl-D-glucosamine and their respective sodium salts and is the monomeric building block for HA.

"Crosslinked hyaluronic acid" means a polymer of hyaluronic acid" wherein different chains of HA are covalently connected by a crosslinker, forming a 3-dimensional polymer network. The degree of crosslinking refers the molar ratio of disaccharide units to crosslinker units in the polymer network.

"Crosslinker" may be a linear or branched molecule or chemical group, preferably is a linear molecule with at least chemical functional groups on each distal ends.

"Functionalized hyaluronic acid" means a polymer of hyaluronic acid" wherein HA is chemically modified with a group L1 which bears a chemical functional chemical group at its distal end. The degree of functionalization refers the molar ratio of disaccharide units to L1 units in the polymer.

The term "chemical functional group" refers to but not limited to carboxylic acid and activated derivatives, amino, maleimide, thiol and derivatives, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyi and other alpha-beta unsaturated michael acceptors, arylating agents like aryl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, oxirane, and aziridine.

If a chemical functional group is coupled to another chemical functional group, the resulting chemical structure is referred to as "linkage". For example, the reaction of an amine group with a carboxyl group results in an amide linkage.

"Reactive functional groups" are chemical functional groups of the backbone moiety, which are connected to the hyperbranched moiety.

"Functional group" is the collective term used for "reactive functional group",

"degradable interconnected functional group", or "conjugate functional group".

The terms "blocking group" or "capping group" are used synonymously and refer to moieties which are irreversibly connected to reactive functional groups to render them incapable of reacting with for example chemical functional groups.

The terms "protecting group" or "protective group" refers to a moiety which is reversibly connected to reactive functional groups to render them incapable of reacting with for example other chemical functional groups under specific conditions.

The term "activation goup" refers to chemical functional groups suitably to activate forms of a corresponding chemical functional group which are known to the person skilled in the art. For example, activated forms of carboxyl groups include but are not limited to active esters, such as succinimidyl ester, benzotriazyl ester, nitrophenyl ester, pentafluorophenyl ester, azabenzotriazyl ester, acyl halogenides, mixed or symmetrical anhydrides, acyl imidazole.

The term "non-enzymatically cleavable linker" refers to linkers that are hydrolytically degradable under physiological conditions without enzymatic activity.

The terms "spacer", "spacer group", "spacer molecule", and "spacer moiety" are used interchangeably and if used to describe a moiety present in the hydrogel carrier of the invention, refer to any moiety suitable for connecting two moieties, such as Ci-5o alkyl, which fragment is optionally interrupted by one or more groups selected from -NH-, -N(Ci-4 alkyl)-, -O-, -S-, -C(O)-, -C(O)NH-, -C(O)N(Ci-4 alkyl)-, -O-C(O)-, -S(O)-, -S(O)2-.

The terms "terminal", "terminus" or "distal end" refer to the position of a functional group or linkage within a molecule or moiety, whereby such functional group may be a chemical functional group and the linkage may be a degradable or permanent linkage, characterized by being located adjacent to or within a linkage between two moieties or at the end of an oligomeric or polymeric chain.

The phrases "in bound form" or "moiety" refer to sub-structures which are part of a larger molecule. The phrase "in bound form" is used to simplify reference to moieties by naming or listing reagents, starting materials or hypothetical starting materials well known in the art, and whereby "in bound form" means that for example one or more hydrogen radicals (-H), or one or more activating or protecting groups present in the reagents or starting materials are not present in the moiety.

It is understood that all reagents and moieties comprising polymeric moieties refer to macromolecular entities known to exhibit variabilities with respect to molecular weight, chain lengths or degree of polymerization, or the number of functional groups.

Structures shown for crosslinking reagents, and crosslinked moieties are thus only representative examples.

A reagent or moiety may be linear or branched. If the reagent or moiety has two terminal groups, it is referred to as a linear reagent or moiety. If the reagent or moiety has more than two terminal groups, it is considered to be a branched or multi-functional reagent or moiety.

The linkers used in the conjugates of the invention are transient, meaning that they are non-enzymatically hydrolytically degradable (cleavable) under physiological conditions (aqueous buffer at pH 7.4, 37°C) with half-lives ranging from, for example, one hour to three months.

The term "GLP-1 /Glucagon agonist hydrogel conjugate" refers to carrier-linked conjugates of GLP-1 /Glucagon agonist, wherein the carrier is a hydrogel. The terms "hydrogel conjugate" and "hydrogel-linked conjugate" refer to conjugates of biologically active agents transiently linked to a hydrogel and are used synonymously.

A "hydrogel" may be defined as a three-dimensional, hydrophilic or amphiphilic polymeric network capable of taking up large quantities of water. The networks are composed of homopolymers or copolymers, are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity.

Hydrogels exhibit a thermodynamic compatibility with water which allows them to swell in aqueous media. The chains of the network are connected in such a fashion that pores exist and that a substantial fraction of these pores are of dimensions between 1 nm and 1000 nm.

"Free form" of a drug refers to a drug, specifically to GLP-1 /Glucagon agonist, in its unmodified, pharmacologically active form, such as after being released from a polymer conjugate.

The terms "drug", "biologically active molecule", "biologically active moiety", "biologically active agent", "active agent", are used synonymously and refer to GLP-1 /Glucagon agonist, either in its bound or free form.

A "therapeutically effective amount" of GLP-1 /Glucagon agonist as used herein means an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which are all within the ordinary skills of a trained physician.

"Stable" and "stability" means that within the indicated storage time the hydrogel conjugates remain conjugated and do not hydrolyze to a substantial extent and exhibit an acceptable impurity profile relating to GLP-1 /Glucagon agonist. To be considered stable, the composition contains less than 5% of the drug in its free form.

The term "pharmaceutically acceptable" means approved by a regulatory agency such as the EMEA (Europe) and/or the FDA (US) and/or any other national regulatory agenciesy for use in animals, preferably in humans.

"Pharmaceutical composition" or "composition" means one or more active ingredients, and one or more inert ingredients, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the

pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable excipient (pharmaceutically acceptable carrier).

"Dry composition" means that the GLP-1 /Glucagon agonist hydrogel conjugate composition is provided in a dry form in a container. Suitable methods for drying are for example spray-drying and lyophilization (freeze-drying). Such dry composition of GLP-1 /Glucagon agonist hydrogel conjugate has a residual water content of a maximum of 10 %, preferably less than 5% and more preferably less than 2% (determined according to Karl Fischer method). The preferred method of drying is lyophilization. "Lyophilized composition" means that the GLP-1 /Glucagon agonist hydrogel conjugate composition was first frozen and subsequently subjected to water reduction by means of reduced pressure. This terminology does not exclude additional drying steps which occur in the manufacturing process prior to filling the composition into the final container.

"Lyophilization" (freeze-drying) is a dehydration process, characterized by freezing a composition and then reducing the surrounding pressure and, optionally, adding heat to allow the frozen water in the composition to sublime directly from the solid phase to gas. Typically, the sublimed water is collected by desublimation.

"Reconstitution" means the addition of a liquid to a dry composition to bring it into the form of a liquid or suspension composition. It is understood that the term "reconstitution" is not limited to the addition of water, but refers to the addition of any liquid, including for example buffers or other aqueous solutions.

"Reconstitution solution" refers to the liquid used to reconstitute the dry composition of an GLP-1 /Glucagon agonist hydrogel conjugate prior to administration to a patient in need thereof.

"Container" means any container in which the GLP-1 /Glucagon agonist hydrogel conjugate composition is comprised and can be stored until reconstitution.

"Buffer" or "buffering agent" refers to chemical compounds that maintain the pH in a desired range. Physiologically tolerated buffers are, for example, sodium phosphate, succinate, histidine, bicarbonate, citrate and acetate, pyruvate. Antacids such as

Mg(OH)2 or ZnCO3 may be also used. Buffering capacity may be adjusted to match the conditions most sensitive to pH stability.

"Excipients" refers to compounds administered together with the therapeutic agent, for example, buffering agents, isotonicity modifiers, preservatives, stabilizers, anti-adsorption agents, oxidation protection agents, or other auxiliary agents. However, in some cases, one excipient may have dual or triple functions.

A "lyoprotectant" is a molecule which, when combined with a protein of interest, significantly prevents or reduces chemical and/or physical instability of the protein upon drying in general and especially during lyophilization and subsequent storage.

Exemplary lyoprotectants include sugars, such as sucrose or trehalose; amino acids such as arginine, glycine, glutamate or histidine; methylamines such as betaine;

lyotropic salts such as magnesium sulfate; polyols such as trihydric or higher sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol;

ethylene glycol; propylene glycol; polyethylene glycol; pluronics; hydroxyalkyi starches, e.g. hydroxyethyl starch (HES), and combinations thereof.

"Surfactant" refers to wetting agents that lower the surface tension of a liquid.

"Isotonicity modifiers" refer to compounds which minimize pain that can result from cell damage due to osmotic pressure differences at the injection depot.

The term "stabilizers" refers to compouds used to stabilize the conjugate of the invention. Stabilisation is achieved by strengthening of the protein-stabilising forces, by destabilisation of the denatured state, or by direct binding of excipients to the protein.

"Anti-adsorption agents" refers to mainly ionic or non-ionic surfactants or other proteins or soluble polymers used to coat or adsorb competitively to the inner surface of the composition's container. Chosen concentration and type of excipient depends on the effect to be avoided but typically a monolayer of surfactant is formed at the interface just above the CMC value.

"Oxidation protection agents" refers to antioxidants such as ascorbic acid, ectoine, glutathione, methionine, monothioglycerol, morin, polyethylenimine (PEI), propyl gallate, vitamin E, chelating agents such aus citric acid, EDTA, hexaphosphate, thioglycolic acid.

"Antimicrobial" refers to a chemical substance that kills or inhibits the growth of microorganisms, such as bacteria, fungi, yeasts, protozoans and/or destroys viruses.

"Sealing a container" means that the container is closed in such way that it is airtight, allowing no gas exchange between the outside and the inside and keeping the content sterile.

The term "reagent" or "precursor" refers to an intermediate or starting material used in the assembly process leading to a conjugate of the present invention.

In another embodiment of the conjugate

-L1-L2-L- is a linker moiety of formula (I la),

lla),

wherein the dashed line indicates attachment to Y by forming an amide bond;

R1, R1 a, R2a are selected independently from the group consisting of H and Ci-4 alkyl; -L1-L2- is defined as described above.

In another embodiment of the conjugate

-L1-L2-L- is a linker moiety of formula (lla), wherein

R1 is CH3;

R1 a is H;

R2a is H; and

-L1-L2- is defined as described above.

In another embodiment of the conjugate

-L1-L2-L- is a linker moiety of formula (lla), wherein

R1 is H;

R1 a is CH3;

R2a is H; and

-L1-L2- is defined as described above.

In another embodiment of the conjugate

-L1-L2-L- is a linker moiety of formula (lla), wherein

R1 is CH3;

R1 a is CH3;

R2a is H; and

-L1-L2- is defined as described above.

In another embodiment of the conjugate

-L1-L2-L- is a linker moiety of formula (Mb),

' (lib)

wherein the dashed line indicates attachment to Y by forming an amide bond;

R1 is selected from H or Ci-4 alkyl, preferably H;

R1 a is selected from H or Ci-4 alkyl, preferably H;

R2, R2a are independently selected from the group consisting of H and Ci-4 alkyl;

wherein "L1-L2- is defined as described above.

In another embodiment of the conjugate

-L1-L2-L is a linker moiety of formula (Mb),

(lib)

wherein the dashed line indicates attachment to Y by forming an amide bond;

R1 and R1 a are H;

R2, R2a are independently selected from the group consisting of H and CH3;

wherein -L1-L2- is defined as described above.

In another embodiment of the conjugate

-L1-L2-L- is a linker moiety -L of formula (Mb), wherein

R1 and R1 a are H;

R2 is H and R2a is CH3;

wherein -L1-L2- is defined as described above.

In another embodiment of the conjugate

L2 is a C-1-10 alkyl chain, in which optionally one or two carbon atoms are independently replaced by a group selected from -O- and C(O)N(R3aa) and, wherein R3aa is independently selected from the group consisting of H and Ci-4 alkyl; and

L2 is attached to L1 via a terminal group selected from the group consisting of

wherein L2 is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line.

In another embodiment of the conjugate

L2 is a C-1-6 alkyl chain, in which optionally one carbon atoms is independently replaced by a group selected from -O- and C(O)N(R3aa) and, wherein R3aa is independently selected from the group consisting of H and Ci-4 alkyl; and

L2 is attached to L1 via a terminal group selected from the group consisting of

wherein L2 is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line.

In another embodiment of the conjugate

L2 is -CH2-CH2-CH2-CH2-CH2-C(O)NH- or -CH2-CH2-CH2-CH2-CH2-CH2- and

is attac

wherein wherein L2 is attached to the Sulfur atom indicated with the dashed line and and L1 is attached to nitrogen atom indicated with the dashed line.

In another embodiment of the conjugate

L2 is -CH2-CH2-CH2-CH2-CH2-C(O)NH- or -CH2-CH2-CH2-CH2-CH2-CH2- and

is attac e terminal group

wherein wherein L2 is attached to the Sulfur atom indicated with the dashed line and and L1 is attached to nitrogen atom indicated with the dashed line.

In another embodiment of the conjugate

L1 is a C-1-10 alkyl chain, with an amino group on one distal end, which is optionally interrupted by one or two groups independently selected from -O- and C(O)N(R5aa) and, wherein R5aa is independently selected from the group consisting of H and Ci-4 alkyl.

A further embodiment relates to conjugates, wherein the crosslinker is divinylsulfone.

A further embodiment relates to conjugates, wherein

0.001 to 15 mol% of the monomeric disaccharide units are crosslinked by a crosslinker in the crosslinked hyaluronic acid hydrogel.

A further embodiment relates to conjugates, wherein

0.1 to 5 mol% of the monomeric disaccharide units are crosslinked by a crosslinker in crosslinked hyaluronic acid hydrogel.

A further embodiment relates to conjugates, wherein

0.2 to 10 mol% of the monomeric disaccharide units of the crosslinked hyaluronic acid hydrogel bear -L1-L2 -L-Y-R20 groups.

A further embodiment relates to conjugates, wherein

0.5 to 7 mol% of the monomeric disaccharide units of the crosslinked hyaluronic acid hydrogel bear -L1-L2 -L-Y-R20 groups.

A further embodiment relates to conjugates, wherein

0.5 to 5 mol% of the monomeric disaccharide units of the crosslinked hyaluronic acid hydrogel bear -L1-L2 -L-Y-R20 groups.

A further embodiment relates to conjugates, wherein

1 to 3.5 mol% of the monomeric disaccharide units of the crosslinked hyaluronic acid hydrogel bear -L1-L2 -L-Y-R20 groups.

An further embodiment is a conjugate or a pharmaceutically acceptable salt thereof comprising a

crosslinked hyaluronic acid hydrogel, in which

0.001 to 20 mol% of the monomeric disaccharide units are crosslinked by a crosslinker; and

0.2 to 20 mol% of the monomeric disaccharide units bear -L1-L2 -L-Y-R20 groups; and

0.2 to 30 mol% of the monomeric disaccharide units of the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups;

L1 is a C-1-20 alkyl chain, in which optionally one or more carbon atoms are independently replaced by a group selected from -O-, NH(R5aa) and C(O)N(R5aa) and is optionally substituted with one or more groups independently selected from OH and

C(O)N(R5aaR5aaa), wherein R5aa and R5aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L1 is attached to the hydrogel via a terminal amino group forming an amide bond with the carboxy group of the beta-1 ,3-D-glucuronic acid of the hyaluronic acid

L2 is a single chemical bond or is a Ci-2o alkyl chain, in which optionally one or more carbon atoms are independently replaced by a group selected from -O- and

C(O)N(R3aa) and is optionally substituted with one or more groups independently selected from OH and C(O)N(R3aaR3aaa), wherein R3aa and R3aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L2 is attached to L1 via a terminal group selected from the group consisting of

wherein L is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line;

Z is a C-1-16 alkyl chain, in which optionally one or more carbon atoms are independently replaced by a group selected from -O- and C(O)N(R6aa); wherein R6aa is hydrogen or Ci. 4 alkyl; or

and

Z is attached to L1 via a terminal group selected from the group consisting of

O and HO O

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line;

L is a linker of formula (la),

wherein the dashed line indicates the attachment to the N-Terminus of Y by forming an amide bond;

X is C(R4R4a) or N(R4);

R1, R1 a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

R2, R2a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

R4, R4a, are independently selected from the group consisting of H; and d. 4 alkyl;

wherein one of R2 , R2a, R4 or R4a is attached to L2;

Y is a peptide moiety having the formula (lb)

His- D-Ser -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Glu-Ser-Lys-Ala- Ala-Gln-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lb)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-hexadecanoylamino-butyryl;

or Y is a peptide moiety having the formula (lc)

His- dSer-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Asp-Glu-Gln-Leu- Ala-Lys-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lc)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-octadecanoylamino-butyryl;

R^u is OH or NH2.

An further embodiment is a conjugate or a pharmaceutically acceptable salt thereof comprising a

crosslinked hyaluronic acid hydrogel as described above, wherein

0.2 to 20 mol% of the monomeric disacchahde units of the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups.

An further embodiment is a conjugate or a pharmaceutically acceptable salt thereof comprising a

crosslinked hyaluronic acid hydrogel as described above, wherein

0.2 to 10 mol% of the monomeric disacchahde units of the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups.

An further embodiment is a conjugate or a pharmaceutically acceptable salt thereof comprising a

crosslinked hyaluronic acid hydrogel as described above, wherein

the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups and wherein

Z is a C-1-16 alkyl chain, in which optionally one or more carbon atoms are independently replaced by a group selected from -O- and C(O)N(R

Z is

and

Z is attached to L1 via a terminal group selected from the group consisting of

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line.

An further embodiment is a conjugate or a pharmaceutically acceptable salt thereof comprising a

crosslinked hyaluronic acid hydrogel as described above, wherein

the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups and wherein

Z is a C-1-8 alkyl chain, in which optionally one or more carbon atoms are independently replaced by a selected from -O-; or

Z is attached to L1 via a terminal group selected from the group consisting of

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line.

An further embodiment is a conjugate or a pharmaceutically acceptable salt thereof comprising a

crosslinked hyaluronic acid hydrogel as described above, wherein

the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups and wherein

Z is -CH2-CH2- ; or

Z is attached to L1 via a terminal group selected from the group consisting of

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line.

An further embodiment is a conjugate or a pharmaceutically acceptable salt thereof comprising a

crosslinked hyaluronic acid hydrogel as described above, wherein

the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups and wherein

Z is -CH2-CH2- ; and

Z is attached to L1 via a terminal group selected from the group consisting of

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line.

In another embodiment of the conjugate the -L1-L2 -L-Y group has a structure as represented by formula (Ilia)

In another embodiment of the conjugate the -L1-L2 -L-Y group has a structure as represented by formula (1Mb)

In another embodiment of the conjugate the -L1-L2 -L-Y group has a structure as represented by formula (lllc)

In another embodiment of the conjugate the -L1-L2 -L-Y group has a structure as represented by formula (llld)

Mid).

In one embodiment of the conjugate Y refers to an GLP-1/Glucagon agonist selected from sequence ID NO: 5.

In one embodiment of the conjugate Y refers to an GLP-1/Glucagon agonist selected from sequence ID NO: 6.

Another embodiment of the invention is a conjugate or a pharmaceutically acceptable salt thereof, comprising a

crosslinked hyaluronic acid hydrogel, in which

0.001 to 20 mol% of the monomeric disacchahde units are crosslinked by divinylsulfone; and

0.5 to 5 mol% of the monomeric disacchahde units bear -L1-L2 -L-Y-R20 groups;

presented by formula (1Mb)

L1 is a NH-CH2-CH2-CH2-NH-CO-CH2-CH2- or -CH2-CH2-CH2-NH-CO-CH2-CH2-; and

L1 is attached to the hydrogel via a terminal amino group forming an amide bond with the carboxy group of the beta-1 ,3-D-glucuronic acid of the hyaluronic acid; and

Y is a peptide moiety having sequence ID NO: 5.

Another embodiment of the invention is a conjugate or a pharmaceutically acceptable salt thereof, comprising a

crosslinked hyaluronic acid hydrogel, in which

0.001 to 20 mol% of the monomeric disacchahde units are crosslinked by divinylsulfone; and

0.5 to 5 mol% of the monomeric disaccharide units bear -L1-L2 -L-Y-R20 groups;

nted by formula (1Mb)

L1 is a NH-CH2-CH2-CH2-NH-CO-CH2-CH2- or -CH2-CH2-CH2-NH-CO-CH2-CH2-; and

L1 is attached to the hydrogel via a terminal amino group forming an amide bond with the carboxy group of the beta-1 ,3-D-glucuronic acid of the hyaluronic acid; and

0.2 to 30 mol% of the monomeric disacchahde units of the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups;

Z is -CH2-CH2-; and

Z is attached to L1 via a terminal group

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line;

Y is a peptide moiety having Y is a peptide moiety having sequence ID NO: 5.

Table 1

SEQ sequence

ID

1 H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-l-E-W-L-K-N-G-G-P-S- S-G-A-P-P-P-S-NH2

2 H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-K-G-R-NH2

3 H-S-Q-G-T-F-T-S-D-Y-S-K-Y-L-D-S-R-R-A-Q-D-F-V-Q-W-L-M-N-T

H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K(YE-X53)-E-I-A-W-L-V-R-G- 4

R-G-OH

5 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(YE-x53)-E-S-K-A-A-Q-D-F-l-E-W-L-K- A-G-G-P-S-S-G-A-P-P-P-S-NH2

6 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(YE-x70)-D-E-Q-L-A-K-D-F-l-E-W-L-K- A-G-G-P-S-S-G-A-P-P-P-S-NH2

Κ(γΕ-χ53) represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-hexadecanoylamino-butyryl .

Κ(γΕ-χ70) represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-octadecanoylamino-butyryl.

In case the conjugates of the invention contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts. Thus, the conjugate of the inventionwhich contain acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. The conjugates of the invention which contain one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. If the the conjugates of the invention

simultaneously contain acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). The respective salts according to the conjugate of the inventioncan be obtained by customary methods which are known to the person skilled in the art like, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the conjugate of the invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of

pharmaceutically acceptable salts.

Process of making

Hyaluronic acid hydrogel synthesis

Crosslinked hyaluronic acid may be derived by different methods. Reaction of HA with the crossl inker, reaction of modified (activated) HA with the crossl inker, the reaction of two different modified HA with the crosslinker. Examples are described in Oh et al, Journal of Controlled Release 141 (2010), 2-12. Example 7 decribes the crosslinking of unmodified HA with divinylsulfone which is a mono bifunctional crosslinker as depicted in scheme 1 . Crosslinking of unmodified HA with a crosslinker may also achived by the hydroxyl mediated alkylation (Scheme 2), the Auto crosslinking with 1 -methyl, 2-chloro pyridinium iodide (Scheme 3), Amide formation (Scheme 4) and the diol-epoxide chemistry (Scheme 5).

Crosslinking methods starting from two different modified HA's are the Michael addition reaction of thiols with maleiimides (Scheme 6), and the Click chemistries shown in Shemes 7 and 8.

Crosslinking methods starting from modified HA are aldehyde (diol oxidation) (Scheme 9) and 2 + 2cyclo addition reactions shown in Scheme 10 and 1 1 .



Scheme 3: Auto crosslinking 

Scheme 6: Michael Addition Crosslinking (Thiol - maleimide)

Scheme 7: Click Chemistry

Scheme 8: Click Chemistry

Scheme 9: Aldehyde (diol oxidation) - amine reductive amination

Scheme 10: 2 +2 Cydoaddition

The linkers L are prepared by methods as described in the examples and as disclosed in WO2009/095479, WO201 1/012718 and WO2012/035139.

Peptide-linker conjugates synthesis

A preferred way of manufacturing peptides that contain unnatural amino acids and side-chain modifications of amino-groups like within lysine is solid phase synthesis on a suitable resin (SPPS).

Examples are given in: Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, III., 1984; Atherton and Sheppard, Solid Phase Peptide

Synthesis, a practical approach, Oxford, IRL Press, New York, 1989; Pennington and Dunn, Methods in Molecular Biology, Volume 35, Peptide Synthesis Protocols, Humana Press, Totowa, New Jersey, 1994; Jones, The Chemical Synthesis of Peptides, Clarendon Press, Oxford, 1991 .

Solid-phase synthesis of a peptide is started with a N-terminally protected amino acid derivative to a solid support-bearing linker. A solid support can be any polymer which is compatible to the solvents used in SPPS and allows coupling of an amino acid derivative with its carboxy group onto the resin (e.g. a trityl resin, a chlorotrityl resin, a Wang-resin when a peptide acid is wanted or a Rink-resin, a Sieber-resin when a peptide amide has to be obtained by using the Fmoc-strategy). Stability of the polymer support must be given under the conditions used for deprotection of the α-amino group during peptide synthesis.

After the first N-terminally protected amino acid was coupled onto the linker-resin construct the N-terminal protecting group is cleaved with bases such as with

piperidine/dimethylformamide mixtures (Fmoc-strategy). The liberated amino group is reacted with a Fmoc-protected amino acid derivative using coupling reagents such as e.g. BOP (Benzotriazole-l -yl-oxy-tris(dimethylamino)-phosphonium

hexafluorophosphate), HBTU (2-(1 H-Benzotriazole-1 -yl)-1 ,1 ,3,3-tetramethylaminium hexafluorophosphate), HATU (O-(7-Azabenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorphosphat) together with a tertiary base like DIPEA (Diisopropylethyl amine) or NMM (N-Methylmorpholine) or alternatively with DIC (N,N'-diisopropylcarbodiimide) / HOBt Hydrate (1 -hydroxybenzotriazol). This process is repeated until the desired amino acid sequence is obtained.

Reactive side-chain functions of the amino acid derivatives are usually blocked with suitable protecting groups that are stable under the conditions used for solid phase peptide synthesis. They are removed concomitantly with the cleavage of the desired product from the resin under the same conditions after the peptide has been assembled

on the solid phase. Protecting groups and the procedures for their introduction can be found in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd ed., Wiley & Sons, New York, 1999 or in Kocienski, Protecting Groups, Georg Thieme Verlag, Stuttgart, New York, 1994.

There is also a possibility to remove side-chain protecting groups selectively in SPPS in order to modify them. The conditions for the removal of such a protecting group must be as such that all other protecting groups remain intact. A lysine may be protected with the ivDde- or the Dde-group (see Chhabra et al., Tetrahedron Lett. 39, 1603, 1998) which is labile to a hydrazine-solution in DMF. Once the N-terminal protecting group as well as all the side-chain protecting groups are with acid labile protecting groups, the ivDde- or the Dde-protecting group can be cleaved with hydrazine in DMF. The liberated amino group from the lysine side-chain can be modified thereafter e.g. with other Fmoc-amino acids or fatty acids.

Modification of the Lys(14) side-chain; The peptides Y have 4 lysine amino acids in their sequence, wherein the lysine in position 14 is modified in the side chain (see formula I and II). Therefore for the peptide synthesis two different side chain protected lysines are used: As building block for the position 14, a Mmt-side chain (Monomethoxytrityl) protected lysin is used and Boc-sidechain protected lysines for the others.

The cleavage of these two different protecting groups can be selectively chosen to each other.

The peptide can be finally be cleaved from the resin concomitantly with all the side chain protecting groups with the use of trifluoroacetic acid containing cocktails e.g. the King's cocktail (King et al., Int. J. Peptide Protein Res. 36, 255-266, 1990). Such cocktails might e.g. contain trifluoroacetic acid (TFA), water, ethandithiol (EDT), thioanisol, phenol, triethylsilane (TES) or triisoproplysilane (TIPS) in variable amounts.

After a certain reaction time the resin is filtered off and the crude peptide precipitated in ether e.g. diethyl ether, methyltert. butyl ether or diisopropyl ether. The precipitate can be filtered off or separated from the solution by centrifugation.

A further object of the invention was therefore providing a process for the preparation of the linker-conjugate L2*"L-Y comprising the steps

a) Assembling of the peptide sequence of Y on a resin including the D-Ser in position 2;

b) Coupling of His as Fmoc-His(Trt)-OH at position 1 ;

c) Deprotection of Fmoc;

d) Coupling of the linker reagent reagent L2*-L- of formula laa

Claims

1 . A conjugate comprising a

crosslinked hyaluronic acid hydrogel, in which

0.001 to 20 mol% of the monomeric disaccharide units are crosslinked by a crosslinker; and

0.2 to 20 mol% of the monomeric disaccharide units bear -L1-L2 -L-Y-R20 groups

L1 is a Ci-2o alkyl chain, in which optionally one or more carbon atoms are replaced by a group selected from -O-, NH(R5aa) and C(O)N(R5aa) and is optionally substituted with one or more groups independently selected from OH and C(O)N(R5aaR5aaa), wherein R5aa and R5aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L1 is attached to the hydrogel via a terminal amino group forming an amide bond with the carboxy group of the beta-1 ,3-D-glucuronic acid of the hyaluronic acid

L2 is a single chemical bond or is a Ci-2o alkyl chain, in which optionally one or more carbon atoms are replaced by a group selected from -O- and C(O)N(R3aa) and is optionally substituted with one or more groups independently selected from OH and C(O)N(R3aaR3aaa), wherein R3aa and R3aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L2 is attached to L1 via a terminal group selected from the group consisting of

L is a linker of formula (la),

wherein the dashed line indicates the attachment to the N-Terminus of Y by forming an amide bond;

X is C(R4R4a) or N(R4);

R1, R1 a, are independently selected from the group consisting of H; and d. 4 alkyl;

R2, R2a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

R4, R4a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

wherein one of R2 , R2a, R4 or R4a is attached to L2;

Y is a peptide moiety having the formula (lb)

His- D-Ser -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Glu-Ser-Lys-Ala- Ala-Gln-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lb)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-hexadecanoylamino-butyryl;

or Y is a peptide moiety having the formula (lc)

His- dSer-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Asp-Glu-Gln-Leu- Ala-Lys-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lc)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-octadecanoylamino-butyryl;

R20 is OH or NH2,

or a salt or solvate thereof.

2. The conjugate of claim 1 comprising a

crosslinked hyaluronic acid hydrogel, in which

0.001 to 20 mol% of the monomeric disaccharide units are crosslinked by a crosslinker; and

0.2 to 20 mol% of the monomeric disaccharide units bear -L1-L2 -L-Y-R20 groups; and

0.2 to 30 mol% of the monomeric disaccharide units of the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups;

L1 is a C-1-20 alkyl chain, in which optionally one or more carbon atoms are replaced by a group selected from -O-, NH(R5aa) and C(O)N(R5aa) and is optionally substituted with one or more groups independently selected from OH and C(O)N(R5aaR5aaa), wherein R5aa and R5aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L1 is attached to the hydrogel via a terminal amino group forming an amide bond with the carboxy group of the beta-1 ,3-D-glucuronic acid of the hyaluronic acid

L2 is a single chemical bond or is a Ci-2o alkyl chain, in which optionally one or more carbon atoms are replaced by a group selected from -O- and C(O)N(R3aa) and is optionally substituted with one or more groups independently selected from OH and C(O)N(R3aaR3aaa), wherein R3aa and R3aaa are independently selected from the group consisting of H and Ci-4 alkyl; and

L2 is attached to L1 via a terminal group selected from the group consisting of

Z is a C-1-16 alkyl chain, in which optionally one or more carbon atoms are replaced by a group selected from -O- and C(O)N(R6aa); wherein R6aa is hydrogen or Ci-4 alkyl; or

and

Z is attached to L1 via a terminal group selected from the group consisting of

O and HO O

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line;

L is a linker of formula (la),

wherein the dashed line indicates the attachment to the N-Terminus of Y by forming an amide bond;

X is C(R4R4a) or N(R4);

R1, R1 a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

R2, R2a, are independently selected from the group consisting of H; and Ci- 4 alkyl;

R4, R4a, are independently selected from the group consisting of H; and d. 4 alkyl;

wherein one of R2 , R2a, R4 or R4a is attached to L2;

Y is a peptide moiety having the formula (lb)

His- D-Ser -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Glu-Ser-Lys-Ala- Ala-Gln-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lb)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-hexadecanoylamino-butyryl;

or Y is a peptide moiety having the formula (lc)

His- dSer-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Asp-Glu-Gln-Leu- Ala-Lys-Asp-Phe-lle-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser (lc)

wherein X14 represents Lys, wherein the -NH2 side chain group is functionalized by (S)-4-carboxy-4-octadecanoylamino-butyryl;

R20 is OH or NH2;

or a salt or solvate thereof.

The conjugate as claimed in claim 1 or 2, wherein

-L- is a linker moiety of formula (I la),

wherein the dashed line indicates attachment to Y by forming an amide bond;

R1 is CH3;

R1 a is CH3;

R2a is H; and wherein -L1-L2 are defined as in claim 1 or 2;

or a salt or solvate thereof.

4. The conjugate as claimed in claims 1 to 3, wherein

L2 is a C-1-6 alkyl chain, in which optionally one carbon atom is replaced by a group selected from -O- and C(O)N(R3aa) and, wherein R3aa is independently selected from the group consisting of H and Ci-4 alkyl; and

L2 is attached to L1 via a terminal group selected from the group consisting of

wherein L2 is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line.

5. The conjugate as claimed in claims 1 to 4, wherein

the crosslinker is divinylsulfone.

6. The conjugate as claimed in claims 1 to 5, wherein

Z is -CH2-CH2- ; or

Z is attached to L1 via a terminal group selected from the group consisting of

wherein Z is attached to the one position indicated with the dashed line and and L is attached to the position indicated with the other dashed line.

The conjugate as claimed in claims 1 to 6, wherein

-CH2-CH2- ; and

Z is attached to L1 via a terminal group

wherein Z is attached to the one position indicated with the dashed line and and L is attached to the position indicated with the other dashed line.

The conjugate of any of claims 1 to 7, wherein

-L2-L-Y is a moiety of formula (I I lb),

The conjugate of any of claims 1 to 7, wherein

-L-Y is a moiety of formula (Ilia),

The conjugate of any of claims 1 to 7, wherein

-L-Y is a moiety of formula (I lie),

1 1 . The conjugate of any of claims 1 to 10, wherein

Y is GLP-1/Glucagon agonist selected from sequences Seq. ID NO: 5.

12. The conjugate of any of claims 1 to 10, wherein

Y is GLP-1/Glucagon agonist selected from sequences Seq. ID NO: 6.

13. The conjugate of claim 1 comprising a

crosslinked hyaluronic acid hydrogel, in which

0.001 to 20 mol% of the monomeric disacchahde units are crosslinked by divinylsulfone; and

0.5 to 5 mol% of the monomeric disaccharide units bear -L1-L2 -L-Y-R20 groups;

wherein In the -L1-L2 -L-Y group has a structure as represented by formula (1Mb)

L1 is a NH-CH2-CH2-CH2-NH-CO-CH2-CH2- or -CH2-CH2-CH2-NH-CO-CH2-CH2-; and

L1 is attached to the hydrogel via a ternninal amino group forming an amide bond with the carboxy group of the beta-1 ,3-D-glucuronic acid of the hyaluronic acid; and

0.2 to 30 mol% of the monomeric disacchahde units of the crosslinked hyaluronic acid hydrogel bear -L1-Z-OH groups;

Z is -CH2-CH2-; and

Z is attached to L1 via a terminal group

, and;

wherein Z is attached to the one position indicated with the dashed line and and L1 is attached to the position indicated with the other dashed line;

Y is a peptide moiety having Y is a peptide moiety having sequence ID NO: 5.

14. A pharmaceutical composition comprising a conjugate of any of claims 1 to 13 or a pharmaceutical salt thereof together with at least one pharmaceutically acceptable excipient.

15. A pharmaceutical composition of claim 14 and a viscosity modifier.

16. A pharmaceutical composition as claimed in claim 15,

wherein the viscosity modifier is hyaluronic acid.

17. A pharmaceutical composition as claimed in claim 16,

wherein the viscosity modifier is hyaluronic acid in a concentration of 5 to 30 weight/volume percent.

18. A pharmaceutical composition as claimed in claims 14 to 17 in form of an

injectable formulation.

19. A pharmaceutical composition as claimed in claims 14 to 18 in form of an injectable formulation, which can be administered by injection through a needle smaller than 0.26 mm inner diameter (26 Gauge).

20. A pharmaceutical composition as claimed in claims 18 to 19 in form of an

injectable formulation, wherein a volume of 1 ml_ can be extruded at room temperature within 10 seconds by applying a force of equal/less than 20 Newton through a needle of 26 gauge.

21 . A pharmaceutical composition as claimed in claims 14 to 20 in form of a

suspension.

22. A pharmaceutical composition as claimed in claims 14 to 21 in form of a

suspension, wherein a conjugate of any of claims 1 to 13 has a concentration of 0.5 to 8 weight/volume percent.

23. A pharmaceutical composition as claimed in claims 14 to 22 in form of a

suspension, wherein a conjugate of any of claims 1 to 13 has a concentration of 1 .5 to 3 weight/volume percent.

24. A composition according to any of claims 14 to 23, wherein the conjugate is sufficiently dosed in the composition to provide a therapeutically effective amount of GLP1/Glucagon agonist for at least 6 days in one application.

25. A composition according to any of claims 14 to 24, wherein it is a single dose composition.

26. The conjugate of any of claims 1 to 13 or the pharmaceutical composition of claims 14 to 25 for use as a medicament.

27. The conjugate of any of claims 1 to 13 or the pharmaceutical composition of claims 14 to 25 for use in a method of treating or preventing diseases or disorders which can be treated by GLP-1 /Glucagon agonist.

28. The conjugate of any of claims 1 to 13 or the pharmaceutical composition of claims 14 to 25 for use in a method of treating or preventing diabetes.

29. The conjugate of any of claims 1 to 13 or the pharmaceutical composition of claims 14 to 25 for use in a method of treating or preventing dyslipemia.

30. The conjugate of any of claims 1 to 13 or the pharmaceutical composition of claims 14 to 25 for use in a method of treating or preventing metabolic syndrome.

31 . The conjugate of any of claims 1 to 13 or the pharmaceutical composition of claims 14 to 25 for use in a method of treating or preventing obesity.

32. The conjugate of any of claims 1 to 13 or the pharmaceutical composition of claims 14 to 25 for use in a method of treating or preventing hepatosteatosis.

An intermediate L2*-L-Y of formula (IVa)

wherein Y is a peptide of Seq ID NO: 5 or 6.

wherein Y is a peptide of Seq ID NO: 5 or 6.

35. An GLP-1 /Glucagon agonist-linker conjugate intermediate L -L-Y of formula (IVc)

wherein Y is a peptide of Seq ID NO: 5 or 6.

A composition comprising at least one conjugate of any of claims 1 to 13 for use in a method of treatment of Type 1 diabetes, Type 2 diabeters, obesity or hyperglycemia characterized in that the composition is subcutaneously administered via an injection device comprising a tube having a needle gauge of 26 or greater and wherein said composition is administered once weekly.

An injection device comprising a tube having a gauge of 28 or greater and further comprising a conjugate of any of claims 1 to 13 for use use in a method of treatment of Type 1 diabetes, Type 2 diabeters, obesity or hyperglycemia.