EA042870B1 - LONG-LASTING GLP-1/GLUCAGON RECEPTOR AGONIST USE FOR THE TREATMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE - Google Patents

Description

Technical field

The invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a dual long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor agonist, as well as a method for the prevention or treatment of non-alcoholic fatty liver disease, including the administration of the composition.

State of the art

Non-alcoholic fatty liver disease (NAFLD) is a disease in which histological changes similar to those observed in alcoholic liver disease develop, although they are not associated with alcohol consumption, and this is a kind of metabolic syndrome associated with non-alcoholic fatty liver disease. liver dystrophy (NAFL), non-alcoholic steatohepatitis (NASH), cirrhosis of the liver and hepatocellular carcinoma. The incidence of non-alcoholic fatty liver disease is increasing in parallel with the increase in obesity and diabetes in the population. In Korea, the annual development rate reaches about 16%.

There are several known causes of NAFLD, such as insulin resistance, lipotoxicity, and inflammatory responses. Among them, the most common cause is insulin resistance.

Much effort has been put into improving insulin resistance in order to prevent/treat non-alcoholic fatty liver disease. For example, clinical studies of thiazolidinedione (TZD) or metformin, insulin receptor sensitivity enhancers, have recently been actively pursued (see Hepatology (2003) 38: 1008-17, J Clin Invest (2001) 108: 1167-74).

However, in the case of drug treatment based on TZD, there are disadvantages in the form of large weight gain and slow fluid flow, and thus, the use of such therapy is not possible for patients with heart diseases. In addition to TZD-based drugs, clinical trials are being actively pursued using GLP-1 receptor agonists such as Victoza or Byetta for the treatment of non-alcoholic fatty liver disease. However, in these cases, the in vivo half-life is extremely short and thus repeated administrations once or at least twice a day are necessary, as with other polypeptide hormones. Thus, there is a disadvantage in terms of inconvenience to patients. Such frequent injections cause marked pain and discomfort in patients. In other words, the mere use of standard drugs for the treatment of diabetes mellitus as drugs for the treatment of non-alcoholic fatty liver disease through the mechanism of improving insulin resistance has disadvantages such as various side effects or inconvenience to the patient. Because of these factors, and in the case where a drug that is known to be effective in treating diabetes mellitus, such as a drug for improving insulin resistance, is used directly as a drug for treating non-alcoholic fatty liver disease, various factors may lead to problems such as as side effects known in the art. So that a drug that is effective in the treatment of diabetes mellitus, such as a drug for improving insulin resistance, can be used as a drug in the treatment of non-alcoholic fatty liver disease is debatable. Thus, there is still a need to develop drugs capable of treating non-alcoholic fatty liver disease while being comfortable for the patient and free of side effects.

Disclosure of invention Technical problem

The present inventors have made great efforts to develop a drug for the prevention or treatment of non-alcoholic fatty liver disease that maximizes patient compliance while increasing half-life without side effects such as weight gain. As a result, the inventors have found that the in vivo half-life of the long-acting dual GLP-1 receptor agonist/glucagon associated with the Fc region is significantly increased, and an effective result in weight loss and further reduction in liver triglycerides and blood cholesterol is observed. The present invention has been completed based on this discovery.

Solution

The purpose of the present invention is to provide a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a long-acting dual GLP-1/glucagon receptor agonist.

Another object of the present invention is to provide a method for preventing or treating non-alcoholic fatty liver disease, comprising administering the composition to a patient suspected of having or having non-alcoholic fatty liver disease.

Benefits of the Invention

The long-acting dual GLP-1/glucagon receptor agonist of the present invention can increase patient choice by expanding the category of drugs that are available and used for the treatment of non-alcoholic fatty liver disease, and can also improve patient comfort by extending the period half-life. Additionally, the present invention provides a new alternative that can be used safely for patients with diseases other than non-alcoholic fatty liver disease by reducing side effects such as weight gain.

Brief description of the drawings

Fig. 1 is a graph showing changes in body weight and liver weight with a long acting dual GLP-1/glucagon receptor agonist in an ob/ob mouse model with a diet high in fat, fructose and cholesterol, high in trans fat.

Fig. 2 is a graph showing the results of changes in the mRNA concentration of collagen-1a, TNF-α, sterol-regulating element-binding protein-1c (SREBP-1c) with a long-acting dual GLP-1/glucagon receptor agonist in an ob/ob mouse model with consumption of foods rich in fats, fructose and cholesterol, containing large amounts of trans fats.

Fig. 3 is a graph showing the reduction in liver triglycerides and serum cholesterol with a long-acting dual GLP1/glucagon receptor agonist in a DI0 mouse model with a diet rich in trans fats.

The best embodiment of the invention

To achieve the objectives, one aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a long-acting dual GLP-1/glucagon receptor agonist.

A long acting GLP-1/glucagon receptor dual agonist may be a long acting GLP-1/glucagon receptor dual agonist which is in a conjugated form wherein a biocompatible material or carrier capable of prolonging the duration of the dual agonist's activity is linked to the agonist via a covalent bond. or linker.

In the case of treatment with TZD-based drugs, which are drugs for improving insulin response, which is a mechanism for improving insulin resistance, and standard drugs for the treatment of non-alcoholic fatty liver disease, there are disadvantages that make it impossible to use them in patients with heart disease. due to the development of side effects such as large weight gain and slow fluid flow. In the case of protein-based drugs such as peptide hormones, there are disadvantages such as a short in vivo half-life, which necessitates repeated administration. The present inventors have found that a long acting dual GLP-1/glucagon receptor agonist either has no side effects of weight gain or reduces the side effect of weight gain in various animal models of non-alcoholic fatty liver disease, as well as a long acting dual GLP-1/glucagon receptor agonist can treat non-alcoholic fatty liver disease in a form in which the stability of the drug in the blood is greatly increased. Accordingly, the present invention has been made to provide the use of a long acting dual GLP-1/glucagon receptor agonist for the prevention or treatment of non-alcoholic fatty liver disease.

The composition of the present invention is characterized by both the absence of the side effect of weight gain and the reduction of the side effect of weight gain.

Moreover, the composition of the present invention can prevent or treat non-alcoholic fatty liver disease by doing one of the following: (a) reducing the expression or activity of collagen-1a, which is a marker of fibrosis; (b) reducing the expression or activity of tumor necrosis factor-α (TNF-α), which is a pro-inflammatory marker; (c) reducing the expression or activity of sterol-regulating element-1c binding protein (SREBP-1c), which is a marker of lipogenesis; (d) lowering the concentration of liver triglycerides; and (e) lowering the concentration of cholesterol in the blood.

In one embodiment of the present invention, the long-acting GLP1/glucagon receptor dual agonist of the present invention is administered to various animal models of non-alcoholic fatty liver disease. As a result, it is confirmed that the body weight and liver weight are significantly reduced compared to those in the untreated group (Fig. 1), and side effects such as weight gain are not observed, as in the case of the standard drug. Moreover, the expression of collagen-1a, TNF-α, SREBP-1c was confirmed to be significantly reduced compared to the untreated group, thus preventing the development of fibrosis, i.e. liver fibrosis, inhibiting inflammation and inhibiting the deceleration of fat accumulation (Fig. 2). Thus, it was confirmed that the long-acting GLP-1/glucagon receptor dual agonist of the present invention can be used as a drug for

- 2 042870 prevention and treatment of various non-alcoholic liver diseases. In addition, it was confirmed that liver triglycerides and blood cholesterol were significantly reduced compared to those in the untreated group, and that they were significantly reduced to normal levels for animals (FIG. 3). Thus, it has been confirmed that the long-acting GLP-1/glucagon receptor dual agonist of the present invention can be used as a first-class drug for the prevention and treatment of various non-alcoholic liver diseases.

As used herein, the term GLP-1/glucagon dual agonist can be used interchangeably with the term GLP-1/glucagon dual agonist. A dual GLP-1/glucagon receptor agonist includes all peptides or fragments, precursors, derivatives, or variants thereof that have dual GLP-1/glucagon activity, such as oxyntomodulin, a naturally occurring dual GLP-1/glucagon receptor agonist, and materials , which can activate GLP-1 and glucagon receptors simultaneously, but are not limited to the above. In the present invention, the GLP-1/glucagon receptor agonist may be a dual receptor agonist that solves the problem of a short half-life by prolonged action, and preferably may be a long-acting dual receptor agonist that can be administered once a week, but at it is not limited to the above. Specific examples of the dual GLP1/glucagon receptor agonist according to the present invention may partially include, for example, the dual GLP-1/glucagon receptor agonist, its derivative, and its sustained-release type as described in Korean Published Patent Applications No. 10-2012-0137271 and 10-2012-0139579, which are incorporated herein in their entirety by reference.

In one embodiment of the present invention, the long acting GLP-1/glucagon dual agonist may be in a conjugated form in which a biocompatible material or carrier is linked to the agonist via a covalent bond or linker. In another embodiment, this type of sustained release may be in a form in which the biocompatible material or carrier can be linked directly to a dual GLP-1/glucagon receptor agonist via a covalent bond through known genetic recombination techniques. A prolonged type of said dual GLP-1/glucagon receptor agonist may increase half-life or bioavailability compared to a form in which the sequence of the dual GLP-1/glucagon receptor agonist is not prolonged but is otherwise similar. According to one embodiment of the present invention, as an example of a long-acting GLP-1/glucagon receptor dual agonist, a composition can be used wherein the immunoglobulin Fc region is linked to the thirtieth amino acid of the dual GLP-1/glucagon receptor agonist via a non-peptide polymeric linker, preferably polyethylene glycol (PEG), but is not limited to the above.

As used herein, the terms biocompatible material or carrier refers to materials that can increase the duration of action of a dual GLP1/glucagon receptor agonist when the biocompatible material or carrier is covalently or non-covalently linked to the dual GLP-1/glucagon receptor agonist of the present invention, either directly or indirectly. way to form a conjugate. For example, when forming a conjugate, a material that can increase the half-life of a dual GLP-1 receptor/glucagon agonist in vivo can be a biocompatible material or carrier that can be used to increase half-life variations, and examples can include polyethylene glycol, fatty acid, cholesterol, albumin or fragment thereof, albumin-binding substance, polymer consisting of repeating regions of a certain amino acid sequence, antibody, antibody fragment, binding substance for neonatal Fc receptor (FcRn), in vivo connective tissue, nucleotide, fibronectin, transferrin, saccharide , polymer, etc. Of course, carriers or biocompatible materials can be used in combination of at least two of the above substances. The biocompatible material or carrier includes a biocompatible material that prolongs the in vivo half-life through covalent or non-covalent bonds.

In the present invention, methods by which a biocompatible material or carrier binds to a dual GLP-1/glucagon receptor agonist include, but are not limited to, genetic recombination, and in vitro binding with polymers or small molecular weight chemicals. The FcRn binding material may be an Fc region of an immunoglobulin. For example, when using polyethylene glycol as a carrier, the Ambrx Inc. transcoding technique can be used, which can facilitate accurate positional attachment to the polyethylene glycol. The methods may include Neose's glycopegylation technique, which promotes specific attachment to the glycosylated moiety. Moreover, the methods may include, but are not limited to, a PEG removal technique that removes polyethylene glycol. The methods may include techniques that can enhance bioavailability with PEG. In addition, polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin or hyaluronic acid may also be included.

When albumin is used as a carrier, the methods may include technology whereby albumins or albumin fragments can be directly covalently linked to GLP-1/glucagon dual agonist peptides to improve in vivo stability. Even if albumin does not bind directly, a technique where albumin-binding substances, e.g., an albumin-specific binding antibody or antibody fragment, binds to peptides to bind to albumin, as well as a technique where a particular peptide/protein having a binding affinity for albumin, binds to peptides. In addition, the methods may include, but are not limited to, a technique in which fatty acids, etc., having a binding affinity for albumin, bind to peptides. Any binding technique or method that can enhance in vivo stability using albumin may be included in this application.

The technique of coupling to a peptide by using an antibody or antibody fragment as a carrier to increase the in vivo half-life can also be included in this invention. An antibody or antibody fragment having an FcRn binding site can be used, and any antibody fragment that does not contain an FcRn binding site, such as a Fab, can also be used. CovX's CovX-body technique, which uses a catalytic antibody, as well as a technique that increases the in vivo half-life with Fc fragments, may be included in this application. When using an Fc fragment, the linker connecting the Fc fragment and the peptide, as well as their linking method, may include, but are not limited to, a peptide bond or polyethylene glycol, and the like, any chemical linkage method can also be used. In addition, the binding ratio of the dual GLP-1 receptor agonist/glucagon of the present invention can be 1:1 or 1:2, but is not limited to this, and any ratio that can increase the in vivo half-life can be included without limitation.

Further, the carrier which is used to increase the in vivo half-life may be a non-peptidyl material such as a polysaccharide or a fatty acid.

A carrier-binding linker that is used to increase the in vivo half-life may include peptides, polyethylene glycols, fatty acids, sugars, polymers, small molecules, nucleotides, and combinations of the foregoing, and may be any chemical bond, such as a non-covalent chemical bond, covalent chemical bond, etc. no limits.

A formulation that may enhance bioavailability or sustained activity may include a sustained release formulation in the form of microparticles, nanoparticles, and the like. with the use of a copolymer of lactic and glycolic acids (PLGA), hyaluronic acid, chitosan, etc.

Moreover, the formulation in various aspects that can enhance bioavailability or maintain long-term activity may be a formulation such as implants, inhalants, transnasal formulations, or patches.

In one exemplary embodiment of the invention, examples of a dual GLP-1/glucagon receptor agonist may include a native GLP-1/glucagon dual agonist such as oxyntomodulin and its derivatives, its sustained release formulation, and the like. may also be included.

As used herein, the term oxyntomodulin refers to a peptide derived from the precursor of glucagon, pre-glucagon, and includes native oxyntomodulin, its precursors, derivatives, fragments, as well as variants thereof. It may also preferably have the amino acid sequence SEQ ID NO.

1(HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNIA).

The term variant oxyntomodulin is a peptide having one or more amino acid sequences different from those found in native oxyntomodulin, and refers to a peptide that retains the activation function of GLP-1 receptors and glucagon, and which can be obtained by any one method of substitution, addition , deletions and modifications, or by combining the above methods with respect to the amino acid sequences of native oxyntomodulin.

The term oxyntomodulin derivative includes peptides, peptide derivatives or peptidomimetics that can be obtained by adding, deleting or substituting amino acids of oxyntomodulin so that both the GLP-1 receptor and the glucagon receptor are activated at a high level compared to native oxyntomodulin. Preferably, the oxyntomodulin derivative has the amino acid sequence of SEQ ID NO: 25, and more preferably, its sixteenth and twentieth amino acids form a ring.

The term oxyntomodulin fragment refers to a fragment having one or more amino acids added or removed at the N-terminus or C-terminus of native oxyntomodulin, to which non-naturally occurring amino acids (e.g., a D-type amino acid) may be added, and which performs an activation function both the GLP-1 receptor and the glucagon receptor.

- 4 042870

Each of the manufacturing methods for variants, derivatives and fragments of oxyntomodulin can be used alone or in combination. For example, the present invention includes a peptide that has one or more amino acids different from those found in the native peptide and deamination of the N-terminal amino acid residue, as well as the function of activating both the GLP-1 receptor and the glucagon receptor.

The C-terminal amino acids of variants, derivatives and fragments of oxyntomodulin of the present invention may be amidated.

The carrier material that can be used in the present invention can be selected from the group consisting of an antibody, an immunoglobulin Fc region, an albumin, a fatty acid, a carbohydrate, a polymer having a peptide repeating unit, a transferrin, and a PEG, and is preferably an immunoglobulin Fc region. . In one exemplary embodiment of the present invention, a long acting GLP-1/glucagon dual agonist is linked to the carrier using a non-peptidyl polymer as a linker. In another exemplary embodiment, the non-peptidyl polymer-linked carrier is an immunoglobulin Fc region.

In the present invention, the long-acting GLP-1/glucagon receptor dual agonist is in a form in which the GLP-1/glucagon receptor dual agonist is bound to the Fc region of an immunoglobulin and exhibits reliability and safety. The binding of the immunoglobulin Fc region and the dual GLP-1/glucagon receptor agonist may be in-frame or they may be linked via a non-peptide polymeric linker. In the present invention, an immunoglobulin Fc can be used interchangeably with immunoglobulin fragments.

As used in this application, the term non-peptidyl polymer refers to a biocompatible polymer comprising at least two repeating units linked to each other by a covalent bond, with the exception of the peptide bond. In the present invention, a non-peptidyl polymer can be used interchangeably with a non-peptidyl linker.

The non-peptidyl polymer that can be used in the present invention can be selected from the group consisting of a biodegradable polymer, e.g. polylactic-glycolic acid (PLGA), a lipid polymer, chitin, hyaluronic acid and a combination of the above, and preferably the biodegradable polymer is polyethylene glycol. In addition, derivatives thereof known in the art and derivatives readily produced by methods known in the art may be included within the scope of the invention.

The peptide linker which is used in the fusion protein obtained by the conventional in-frame coupling method has the disadvantage that it is easily degraded in vivo by the protease, and thus the effect of increasing the serum half-life of the active drug is sufficient. carrier cannot be reached as expected. However, in the present invention, a protease-resistant polymer can be used to maintain the serum half-life of a peptide in a similar manner to a carrier. Thus, any non-peptidyl polymer can be used without limitation, as long as the polymer fulfills the above function, hence being a protease resistant polymer in vivo. The non-peptidyl polymer has a molecular weight in the range of 1 to 100 kDa, preferably 1 to 20 kDa. Additionally, the non-peptidyl polymer of the present invention linked to the Fc region of an immunoglobulin may be a single polymer or a combination of different types of polymers.

The non-peptidedyl polymer used in the present invention has a reactive group capable of binding to the Fc region of an immunoglobulin and a protein drug. The reactive group at both ends of the non-peptidyl polymer is preferably selected from the group consisting of a reactive aldehyde group, propionaldehyde, butylaldehyde group, maleimide group, and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxysuccinimidyl, succinimidylcarboxymethyl or succinimidyl carbonate. In particular, in the case where the non-peptidyl polymer has a reactive group represented by a reactive aldehyde group at both ends thereof, it is effective to minimize non-specific reactions and bind the physiologically active polypeptide and immunoglobulin at both ends of the non-peptidyl polymer. The end product formed by reductive alkylation with an aldehyde bond is much more stable than that with an amide bond. The aldehyde reactive group selectively binds to the N-terminus at low pH and binds to the lysine residue to form a covalent bond at high pH, such as 9.0. The reactive groups at both ends of the non-peptidyl polymer may be the same or different from each other. For example, a non-peptidyl polymer may contain a maleimide group at one end and

- 5 042870 aldehyde group, propionaldehyde group or butyraldehyde group at the other end. When polyethylene glycol having reactive hydroxyl groups at both ends is used, the hydroxyl group can be activated to different reactive groups by known chemical reactions, or commercially available polyethylene glycol having a modified reactive group can be used to make the dual GLP-1 receptor agonist/glucagon conjugate of the present invention .

In addition, the Fc region of an immunoglobulin is favorable with respect to the manufacture, purification, and yield of the conjugate, not only because its molecular weight is relatively small compared to the whole molecule, but also because the homogeneity of the materials is also greatly improved, and the ability to induce antigenicity in the blood decreases because the amino acid sequences are different in each antibody and thus the Fab fragment showing high inhomogeneity is removed.

As used herein, the term immunoglobulin Fc region refers to the heavy chain constant domain 2 (CH2) and the heavy chain constant domain 3 (CH3) of an immunoglobulin, excluding the heavy and light chain variable domains, the heavy chain constant domain 1 (CH1) and the constant domain light chain 1 (CL1) immunoglobulin. It may further include a heavy chain constant domain hinge region.

Also, the Fc region of an immunoglobulin of the present invention may comprise part or all of an Fc region comprising a heavy chain constant domain 1 (CH1) and/or a light chain constant domain 1 (CL1) excluding the immunoglobulin heavy and light chain variable domains, since it has an effect that is practically equivalent or greater than that of the native protein. Moreover, the Fc region of an immunoglobulin may be a fragment having a deletion in a relatively long portion of the amino acid sequence that corresponds to CH2 and/or CH3. Thus, the Fc region of an immunoglobulin of the present invention may include 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination one or more domains and an immunoglobulin hinge region (or part of a hinge region); and 6) a dimer of each heavy chain constant domain and light chain constant domain.

Additionally, the Fc region of an immunoglobulin of the present invention includes a native amino acid sequence as well as a derivative of its sequence (mutant). An amino acid sequence derivative has a different sequence due to deletion, insertion, non-conservative or conservative substitutions. For example, in IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 that are considered important for binding can be used as a suitable target for modification.

Additionally, various types of derivatives are possible, including one in which a site capable of forming a disulfide bond has been removed, certain amino acid residues have been removed at the N-terminus of the native Fc region, a methionine residue has been added to the N-terminus of the native Fc region, and so on. Additionally, to remove effector functions, a complement binding site, such as a C1q binding site, and an antibody-dependent cell-mediated cytotoxicity (ADCC) site can be removed. Techniques for the preparation of such derived immunoglobulin Fc region sequences are disclosed in International Publication Nos. WO 97/34631, WO 96/32478, etc. Amino acid substitutions in proteins and peptides that do not completely change the activity of the molecules are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly observed substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly in both directions. In addition, the Fc region can optionally be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

Fc derivatives are derivatives that have identical biological activity to the Fc region of the present invention, with improved structural stability of the Fc region, eg, with respect to heat, pH, and the like.

Moreover, these Fc regions can be obtained from native forms isolated from humans and other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or they can be their recombinants or derivatives obtained from transformed cells of animals or microorganisms. In this application, they can be obtained from the native form by isolating whole immunoglobulins from human or animal bodies and then processing them with a protease. When treated with papain, it digests native immunoglobulin into Fab and Fc regions; when treated with pepsin, native immunoglobulin is degraded into pF'c and F(ab)2. Fc and pF'c can be isolated by size exclusion chromatography, etc. Preferably, the human-derived Fc region is the Fc region of a recombinant immunoglobulin that is derived from microorganisms.

In addition, the Fc region of the immunoglobulin may be in a form having native sugars.

- 6 042870 sugar chains or an increased or decreased number of sugar chains compared to the native form, or may be in a deglycosylated form. Increasing, reducing or removing immunoglobulin Fc sugar chains can be achieved by methods well known in the art, such as chemical, enzymatic and genetic engineering methods using microorganisms. Removal of sugar chains from the Fc region results in a significant reduction in binding affinity for the C1q moiety and in a reduction or loss of antibody-dependent cell-mediated or complement-dependent cytotoxicity, thus not inducing unnecessary immune responses in vivo. In this regard, the Fc region of an immunoglobulin in deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a drug carrier.

As used herein, the term deglycosylation refers to the enzymatic removal of the sugar components of the Fc region, and the term aglycosylation refers to the Fc region that is present in prokaryotes, preferably E. coli, and is not glycosylated.

Meanwhile, the immunoglobulin Fc region can be obtained from humans or other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and preferably from humans.

Also, the Fc region of an immunoglobulin may be an Fc region that is derived from IgG, IgA, IgD, IgE, and IgM, a combination of the above, or hybrids thereof. It is preferably derived from, but not limited to, IgG or IgM, which are found in excess in human blood, and most preferably from IgG, which is known to increase the half-lives of ligand-binding proteins.

As used herein, the term combination refers to those polypeptides that encode single chain Fc regions of an immunoglobulin of the same origin linked to a single chain polypeptide of different origin to form a dimer or multimer. Thus, a dimer or multimer can be formed from two or more fragments selected from the group consisting of Fc IgG, Fc IgA, Fc IgM, Fc IgD and Fc IgE fragments.

As used herein, the term hybrid refers to a sequence that corresponds to at least two Fc regions of different origin and is present in a single chain Fc region of an immunoglobulin. Various types of hybrids are possible in the present invention. Thus, it is possible to use a hybrid consisting of 1-4 domains selected from the group consisting of CH1, CH2, CH3 and CH4 Fc IgG, Fc IgA, Fc IgM, Fc IgE and Fc IgD, and optionally containing a hinge region.

On the one hand, IgG can also be classified into subclasses IgGl, IgG2, IgG3 and IgG4, and combination or hybridization is possible in the present invention. Preferably, they are IgG2 and IgG4 subclasses, and most preferably, they are an IgG4 Fc region that has little to no effector function such as complement-dependent cytotoxicity (CDC).

Thus, the Fc region of an immunoglobulin for a drug carrier of the present invention may be, for example, a human IgG4 derived aglycosylated Fc region, but is not limited thereto. A human-derived Fc region is preferred over a non-human Fc region that can elicit immediate type immune responses, for example, that can act as an antigen in the human body and induce new antibodies.

The method for preparing the long-acting GLP-1/glucagon receptor dual agonist of the present invention is not particularly limited. For example, detailed information on the preparation method and effects is described in, for example, Korean Published Patent Application No. 10-2012-0139579.

The use of a long-acting dual GLP-1/glucagon receptor agonist has enormous advantages in terms of the number of doses to a chronically ill patient requiring daily administration, in the form of a significant reduction in their number due to increased blood half-life and in vivo stability, thus improves the patient's quality of life. Thus, it is extremely useful in the treatment of non-alcoholic fatty liver disease.

As used in this application, the term non-alcoholic fatty liver disease refers to cases of fatty liver disease in which there is no history of alcohol consumption or in which alcohol consumption is not associated with the development of the disease. Fatty liver refers to a phenomenon in which there is an abnormal accumulation of triglycerides in liver cells compared to normal triglyceride concentrations. Approximately 5% of normal liver tissue is adipose tissue, and the major components of fat are triglycerides, fatty acids, phospholipids, cholesterol, and cholesterol esters. However, with the development of fatty degeneration of the liver, most of the components are replaced by triglycerides. If the amount of triglycerides exceeds 5% of the weight of the liver, a diagnosis of fatty liver is made. The reason for the development of fatty

- 7 042870 liver degeneration is a violation of lipid metabolism or a defect in the process of transferring excess fat to the liver cells, and is mainly caused by a violation of lipid metabolism in the liver. Most of the fat that accumulates in fatty liver disease may be in the form of triglycerides. Non-alcoholic fatty liver disease includes non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver cancer, and the like, but fatty liver disease to be prevented or treated with the composition of the present invention is included without limitation.

As used herein, the term prophylaxis refers to all actions that prevent or delay the development of non-alcoholic fatty liver disease using the administration of a composition of the present invention. The term treatment refers to all activities that provide relief or improvement in the symptoms of non-alcoholic fatty liver disease. Treatment of non-alcoholic fatty liver disease is applicable to all mammals that can develop non-alcoholic fatty liver disease, and examples include not only humans and primates, but also cattle, such as cows, pigs, sheep, horses, dogs and cats, without limitation , but preferably applicable to humans.

As used in this application, the term administration refers to the introduction of a quantity of a predetermined substance to a patient using a suitable method. The composition of the present invention can be administered by any standard route of administration so that it can reach the desired tissues. For example, it can be intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary or rectal administration, but not limited to the above. However, since the peptides are digested upon oral administration, the active ingredients of the oral composition must be coated or dispensed with protection from degradation in the stomach. Preferably the composition can be administered in the form of injections. In addition, sustained release formulations can be administered by any device in which the active material can be transported into the target cell.

The dosage for administration and the frequency of administration of the pharmaceutical composition of the present invention is determined by the type of active ingredient together with various factors such as the disease to be treated, the route of administration, the age of the patient, sex and body weight, and the severity of the disease.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent. As used in this application, the term pharmaceutically acceptable carrier refers to a carrier or solvent that does not stimulate the body and inhibit the biological activity or properties of the administered compound. For oral administration, the carrier may include a binder, an emollient, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a coloring agent, and a flavoring agent. In formulations for injection, the carrier may include a buffering agent, a preservative, an analgesic agent, a solubilizer, an isotonic agent, a stabilizer, and the like. In topical formulations, the carrier may include a base, an excipient, an emollient, a preservative, and the like.

The composition of the present invention may be formulated into various dosage forms in combination with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition may be in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, or cachets. For injectable formulations, the pharmaceutical composition may be in the form of an ampule as a single dose form or a multi-dose container. The pharmaceutical composition may also be formulated as solutions, suspensions, tablets, dragees, capsules and sustained release formulations.

On the other hand, examples of carrier, excipient and solvent suitable for pharmaceutical formulations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oils. In addition, pharmaceutical formulations may further include excipients, anticoagulants, emollients, moisturizers, flavorings, and antiseptics.

In another aspect, the present invention provides a method for the prevention or treatment of non-alcoholic liver disease, comprising the step of administering a composition comprising a long acting dual GLP-1/glucagon receptor agonist to a patient, excluding humans, at high risk of developing or having non-alcoholic liver disease.

The description of the composition and non-alcoholic fatty liver disease is the same as above.

Variant of the invention

Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention should not be construed as limited by these examples.

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Example 1 Synthesis of oxyntomodulin derivatives.

In the examples synthesized derivatives of oxyntomodulin, which have the following amino acid sequences (table).

Oxyntomodulin and oxyntomodulin derivatives

SEQID No. Amino acid sequence SEQ ID No. 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNIA SEQ ID No. 2 CA-SQGT FT S DYSKYLDEEAVRLFIEWLMNTKRNRNNIA SEQ ID No. 3 CA-SQGTFTSDYSKYLDERRAQDFVAWLKNTGPSSGAPPPS SEQ ID No. 4 CA-GQGTFTSDYSRYLEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID No. 5 CA-GQGTFTSDYSRQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID No. 6 CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGTFTSDYSKYLD SEQ ID No. 7 CA-S QGT FT S DYSRYLDEEAVRL FIEWLMNTK SEQ ID No. 8 CA-S QGT FT S DLSRQLEEEAVRL FIEWLMNK SEQ ID No. 9 CA-GQGTFTSDYSRYLDEEAVXLFIEWLMNTKRNRNNIA SEQ ID No. 10 CA-SQGTFTSDYSRQMEEEAVRLFIEWLMNGGPSSGAPPPSK SEQ ID No. 11 CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGTFTSDYSRYLDK SEQ ID No. 12 CA-SQGTFTSDYSRYLDGGGHGEGTFTSDLSKQMEEEAVK SEQ ID No. 13 CA-SQGTFTSDYSRYLDXEAVXLFIEWLMNTK SEQ ID No. 14 CA-GQGTFTSDYSRYLDEEAVXLFIXWLMNTKRNRNNIA SEQ ID No. 15 CA-GQGTFTSDYSRYLDEEAVRLFIXWLMNTKRNRMMIA SEQ ID No. 16 CA-SQGTFTSDLSRQLEGGGHSQGTFTSDLSRQLEK SEQ ID No. 17 CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNTKRNRNIA SEQ ID No. 18 CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNGGPSSGAPPPSK SEQ ID No. 19 CA-SQGTFTSDYSRYLDEEAVKLFIEWIRNTKRNRNIA SEQ ID No. 20 CA-SQGTFTSDYSRYLDEEAVKLFIEWIRNGGPSSGAPPPSK SEQ ID No. 21 CA-SQGT FT S DYSRQLEEEAVRLFIEWVRNTKRNRNNIA SEQ ID No. 22 DA-S QGT FT S DYSKYLDEKRAKE FVQWLMNTK SEQ ID No. 23 HAibQGTFTSDYSKYLDEKRAKEFVCWLMNT SEQ ID No. 24 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID No. 25 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID No. 26 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID No. 27 HAibQGTFTSDYSKYLDEQAAKEFICWLMNT SEQ ID No. 28 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNT SEQ ID No. 29 H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID No. 30 CA-SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID No. 31 CA-(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID No. 32 CA-AibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID No. 33 HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC SEQ ID No. 34 YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC

In the table, amino acids in bold and underlined form a ring, and amino acids marked as X represent the non-native amino acid, alpha-methylglutamic acid. In addition, CA is 4-imidazolacetyl and DA is deaminohistidil.

Hereinafter, a typical long-acting dual GLP-1/glucagon receptor agonist, i.e. A long-acting GLP-1/glucagon receptor dual agonist in which Fc is linked to the thirtieth amino acid of a dual GLP-1/glucagon receptor agonist via a non-peptidyl polymer, PEG, was made and used in Examples 2 and 3 below.

Example 2 Confirmation of the effect of a long-acting dual GLP-1/glucagon receptor agonist on non-alcoholic fatty liver disease in an ob/ob mouse model with diet rich in fats, fructose and cholesterol containing high levels of trans fats.

In order to confirm the effect of a long-acting dual GLP-1/glucagon receptor agonist on non-alcoholic fatty liver disease, a diet high in fat (40% kcal), fructose (22%), and cholesterol (2%) is chosen to introduce the ob/ob mouse model, high in trans fat (HTF diet) for 8 weeks to prepare a mouse model for non-alcoholic fatty liver disease. Next, a long-acting dual GLP-1/glucagon receptor agonist was subcutaneously administered to mice once every 2 days (Q2D) at a dose of 0.7 and 1.4 nmol/kg, and the administration was repeated for 4 weeks. The weight of the animals is compared with that of the group receiving the base, not containing the active substance, during a four-week test. After four weeks of testing, liver weights are measured and compared. Additionally, after completion of the four-week test, the presence of collagen-1a mRNA, which is a marker of fibrosis, is confirmed; TNF-α, which is a pro-inflammatory marker; and SREBP-1c, which is a marker of lipogenesis.

As a result, measurement of body weight and liver weight after 4 weeks of administration demonstrates that in the case of the long-acting dual GLP-1 receptor/glucagon agonist, the weight is significantly reduced compared to the base group receiving no active substance (Fig. 1). These results suggest that the long-acting GLP-1/glucagon receptor dual agonist of the present invention can suppress the weight gain that is observed in an animal model of non-alcoholic fatty liver disease and that it can reduce the side effects of standard drugs to improve insulin resistance.

Additionally, comparison of collagen-1a, TNF-α, and SREBP-1c mRNA demonstrates that in the long-acting dual GLP-1/glucagon receptor agonist group, the amount of these mRNAs is significantly reduced (FIG. 2). These results suggest that the long-acting dual GLP1/glucagon receptor agonist of the present invention reduces fibrosis, inflammation, and the like.

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Claims (10)

in an animal model of non-alcoholic fatty liver disease and inhibits fat formation, thus being effective for the prevention and treatment of non-alcoholic fatty liver disease. Example 3 Confirmation of the effect of a long-acting dual GLP-1 receptor agonist/glucagon on non-alcoholic fatty liver disease in a DI0 mouse model with high trans fat intake. To confirm the effect of a long acting dual GLP-1/glucagon receptor agonist on non-alcoholic fatty liver disease, a 60% high trans fat diet was administered to a normal mouse model (C57BL/6) for 12 weeks to prepare an animal model of non-alcoholic fatty liver disease. Next, mice are injected subcutaneously with 3 nmol/kg of a dual GLP-1 receptor agonist/glucagon long-acting once a week (QW), and the introduction is repeated for 4 weeks. After completion of the four-week test, liver triglycerides (liver TG) and serum cholesterol are measured. As a result, the measurement of liver triglycerides and serum cholesterol after administration for 4 weeks demonstrates that in the case of administration of a long-acting dual GLP-1/glucagon receptor agonist, their concentrations are significantly reduced compared to that observed in the base group without active substance, and they are significantly reduced compared with the concentrations in a normal animal on a balanced diet (Fig. 3). These results suggest that the long-acting GLP-1/glucagon receptor dual agonist of the present invention can reduce liver triglycerides and serum cholesterol to normal levels in an animal model of non-alcoholic fatty liver disease, thus being effective for the prevention and treatment of non-alcoholic fatty liver disease. liver disease. From the foregoing description, those skilled in the art will appreciate that the invention may be embodied in other specific forms without changing the technical nature or essential characteristics. In this regard, the embodiments described above are to be understood as illustrative and not restrictive in every respect. The scope of the invention is to be construed so that the meaning and scope of the appended claims, rather than the detailed description, and all changes or variations derived from equivalent approaches, fall within the scope of the present invention. CLAIM 1. Use of a pharmaceutical composition containing a long-acting glucagon-like peptide-1 (GLP-1)/glucagon dual agonist conjugate, wherein said conjugate comprises a GLP-1/glucagon dual receptor agonist comprising the amino acid sequence of any of SEQ ID NOs: 2-34 ; an immunoglobulin Fc region and a non-peptidyl polymer, wherein said non-peptidyl polymer covalently binds a dual GLP-1/glucagon receptor agonist and an immunoglobulin Fc region, to prevent or treat liver inflammation. 2. The use of a pharmaceutical composition according to claim 1, wherein the composition is characterized by either no weight gain side effect or reduced weight gain side effect. 3. The use of a pharmaceutical composition according to claim 1 or 2, wherein the composition reduces the expression or activity of tumor necrosis factor-α (TNF-α), which is a pro-inflammatory marker. 4. The use of a pharmaceutical composition according to any one of the preceding claims, wherein the long acting dual GLP-1/glucagon receptor agonist simultaneously activates the GLP-1 receptor and the glucagon receptor. 5. The use of a pharmaceutical composition according to any of the preceding claims, wherein the long acting dual GLP-1/glucagon receptor agonist comprises the amino acid sequence of SEQ ID NO: 24, 25 or 26. 6. Use of a pharmaceutical composition according to claim 5, wherein the dual GLP-1/glucagon receptor agonist comprises the amino acid sequence of SEQ ID NO: 24. 7. Use of a pharmaceutical composition according to claim 5, wherein the amino acids at positions 12 and 16 or 16 and 20 of the GLP-1/glucagon dual receptor agonist form a ring. 8. Use of a pharmaceutical composition according to claim 7, wherein the dual receptor agonist GLP-1/glucagon contains the amino acid sequence of SEQ ID NO: 25. 9. Use of a pharmaceutical composition according to claim 7, wherein the dual receptor agonist GLP-1/glucagon contains the amino acid sequence of SEQ ID NO: 26. 10. The use of a pharmaceutical composition according to any one of the preceding claims, wherein the non-peptidyl polymer is polyethylene glycol, polypropylene glycol, ethyl copolymer -