HK1238162A1 - Use of a long acting glp-1/glucagon receptor dual agonist for the treatment of non-alcoholic fatty liver disease - Google Patents

Description

Application of long-acting GLP-1/glucagon receptor double agonist to treatment of non-alcoholic fatty liver disease

Technical Field

The present invention relates to a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease comprising a long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist; and a method for preventing or treating a non-alcoholic fatty liver disease, which comprises administering the composition.

Background

Nonalcoholic fatty liver disease (NAFLD) is a type of disease that, although not associated with alcohol consumption, exhibits a similar histological architecture (histopathological organization) as alcoholic liver disease, and is a metabolic syndrome associated with nonalcoholic fatty liver disease (NAFL), nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. The incidence of nonalcoholic fatty liver disease increases with the increase in the population of obesity and diabetes. In korea, the annual incidence has reached about 16%.

Non-alcoholic fatty liver disease is known to be caused by various etiologies such as insulin resistance, lipotoxicity and inflammatory reactions. Among them, the most common cause is insulin resistance.

Much work has been done to improve insulin resistance to prevent/treat non-alcoholic fatty liver disease. For example, clinical trials of Thiazolidinone (TZD) or metformin, an insulin sensitizer, have been actively conducted (see, Hepatology (2003)38:1008-17, J Clin Invest (2001)108: 1167-74).

However, in the case of treatment with TZD-based drugs, there are disadvantages of large weight gain and slow fluid flow, and thus it is known that the application of this treatment cannot be applied to patients with heart disease. In addition to TZD-based drugs, clinical trials of GLP-1 receptor agonists such as vicoza or Byetta for nonalcoholic fatty liver disease have been actively conducted. However, in these cases, the half-life in vivo is very short and therefore, like other polypeptide hormones, repeated administration must be carried out once or at least twice a day. Therefore, there is a disadvantage in that inconvenience is caused to the patient. Such frequent administration causes a great deal of pain and discomfort to the patient. That is, the use of only general therapeutic agents for diabetes as therapeutic agents for non-alcoholic fatty liver disease by improving insulin resistance mechanism has disadvantages such as various side effects or patient inconvenience. Based on these factors, various factors that may cause problems such as side effects when drugs known to be effective in treating diabetes, such as drugs that improve insulin resistance, are directly used as therapeutic agents for nonalcoholic fatty liver disease are known in the art. Therefore, it is controversial whether a drug effective for the treatment of diabetes (e.g., a drug for improving insulin resistance) can be certainly used as a therapeutic agent for non-alcoholic fatty liver disease. Therefore, there is still a need to develop a drug capable of treating non-alcoholic fatty liver disease while ensuring patient convenience without side effects.

Disclosure of Invention

Technical problem

The present inventors have made much work to develop a drug for preventing or treating non-alcoholic fatty liver disease that maximizes patient compliance while increasing half-life and has no side effects such as weight gain. As a result, the inventors have found that long-acting GLP-1/glucagon receptor dual agonists linked to an Fc fragment have a greatly increased half-life in vivo, and also have effective results in weight loss, as well as further reduction of hepatic triglycerides and blood cholesterol. The present invention has been completed on the basis of such findings.

Solution to the problem

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease comprising a long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist.

It is another object of the present invention to provide a method for preventing or treating non-alcoholic fatty liver disease, said method comprising administering the composition to a subject suspected of having or suffering from non-alcoholic liver disease.

Advantageous effects of the invention

The long-acting GLP-1/glucagon receptor dual agonists according to the present invention may broaden patient's choice by expanding the class of drugs that have been so far adapted for non-alcoholic fatty liver disease, and increase patient convenience by significantly increasing blood half-life. Furthermore, the present invention provides a new alternative that can be administered without risk to patients suffering from diseases other than non-alcoholic fatty liver disease by reducing side effects such as weight gain.

Brief Description of Drawings

Figure 1 is a graph showing changes in body weight and liver weight of long-acting GLP-1/glucagon receptor dual agonists in a high trans fat feed intake ob/ob mouse model containing high fat, fructose, and cholesterol.

FIG. 2 is a graph showing the results of measuring collagen-1 a, TNF- α, SREBP-1c mRNA of long-acting GLP-1/glucagon receptor dual agonists in a high trans-fat feed intake ob/ob mouse model containing high fat, fructose, and cholesterol.

Figure 3 is a graph showing a reduction in hepatic triglyceride and serum cholesterol levels of long-acting GLP-1/glucagon receptor dual agonists in a high trans fat feed intake DI0 mouse model.

Best Mode for Carrying Out The Invention

To achieve the object, the present invention provides, in one aspect, a pharmaceutical composition for preventing or treating a non-alcoholic fatty liver disease, which includes a long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist.

The long-acting GLP-1/glucagon receptor dual agonist may be a long-acting GLP-1/glucagon receptor dual agonist in the form of a conjugate, wherein a biocompatible substance or carrier capable of increasing the duration of activity of the dual agonist is linked to the agonist by a covalent bond or a linker.

In the case of treatment with TZD-based drugs, which are drugs that improve insulin response as a mechanism for improving insulin resistance and are conventional therapeutic agents for nonalcoholic fatty liver disease, there is a disadvantage in that the treatment cannot be applied to patients with heart disease due to side effects such as large weight gain and slow fluid flow. In the case of protein drugs such as peptide hormones, there is a disadvantage that the half-life in vivo is short and thus repeated administration is required. The present inventors have discovered that long-acting GLP-1/glucagon receptor dual agonists do not have or reduce the side effects of weight gain in various animal models of non-alcoholic liver disease, and that long-acting GLP-1/glucagon receptor dual agonists can treat non-alcoholic fatty liver disease in a form that is significantly increased in persistence in the blood. Accordingly, the present invention has been accomplished to provide the use of a long-acting GLP-1/glucagon receptor dual agonist for the prevention or treatment of non-alcoholic fatty liver disease.

The compositions of the present invention are characterized by having no or reduced weight gain side effects.

In addition, the composition of the present invention can prevent or treat non-alcoholic fatty liver disease by performing at least one of the following functions: (a) reducing the expression or activity of collagen-1 a as a marker of fibrosis; (b) reducing the expression or activity of tumor necrosis factor-alpha (TNF-a) as a pro-inflammatory marker; (c) reducing the expression or activity of sterol regulatory element binding protein-1 c (SREBP-1c) as a lipogenesis marker; (d) reducing hepatic triglycerides; and (e) reducing blood cholesterol.

In one embodiment of the invention, the long-acting GLP-1/glucagon receptor dual agonists of the invention are administered to various animal models of non-alcoholic fatty liver disease. The results demonstrate that body and liver weights are significantly reduced compared to untreated group (figure 1) and there are no side effects such as weight gain as in conventional therapeutic applications. In addition, it was confirmed that the expression of collagen-1 a, TNF- α, SREBP-1c was significantly reduced as compared to the untreated group, thereby preventing fibrosis, i.e., liver fibrosis, inhibiting inflammation, and inhibiting fat accumulation inhibition (FIG. 2). Therefore, the long-acting GLP-1/glucagon receptor double agonist of the invention is proved to be used as a medicament for preventing and treating various nonalcoholic liver diseases. In addition, it was confirmed that liver triglycerides and blood cholesterol were significantly reduced compared to the untreated group, and that they were significantly reduced to normal animal levels (fig. 3). Therefore, the long-acting GLP-1/glucagon receptor double agonist of the invention is proved to be used as an outstanding medicament for preventing and treating various nonalcoholic liver diseases.

As used herein, the term "GLP-1/glucagon receptor dual agonist" is used interchangeably with "GLP-1/glucagon dual agonist". GLP-1/glucagon receptor dual agonists include, but are not limited to, all peptides or fragments, precursors, derivatives or variants thereof having GLP-1/glucagon dual activity, such as oxyntomodulin, native GLP-1/glucagon receptor dual agonists, and substances that simultaneously activate GLP-1 and glucagon receptors. In the present invention, the GLP-1/glucagon receptor agonist may be a receptor double-double (dual-dual) agonist that utilizes long-acting techniques to overcome a short half-life, and is preferably a long-acting receptor double agonist that may be administered once a week, but is not so limited. Specific examples of GLP-1/glucagon receptor dual agonists according to the invention can include, for example, the GLP-1/glucagon receptor dual agonists described in Korean patent application publication Nos. 10-2012-0137271 and 10-2012-0139579 (the entire contents of which are incorporated herein by reference), derivatives thereof, and long-acting forms thereof.

In one embodiment of the invention, the long-acting GLP-1/glucagon receptor dual agonist may be in the form of a conjugate in which a biocompatible substance or carrier is linked to the agonist via a covalent bond or linker. In another embodiment, the long-acting form may be in the form of: wherein the biocompatible substance or vector can be directly linked to the GLP-1/glucagon receptor dual agonist by covalent bonds by known genetic recombination techniques. The long acting form of the GLP-1/glucagon receptor dual agonist may have an increased half-life or bioavailability compared to a form in which the sequence of the GLP-1/glucagon receptor dual agonist is not long acting but is otherwise identical. According to an embodiment of the present invention, as an example of the long-acting GLP-1/glucagon receptor dual agonist, a composition in which an immunoglobulin Fc region is linked to the amino acid at position 30 of the GLP-1/glucagon receptor dual agonist through a non-peptide polymer linker (preferably PEG) may be used, but is not limited thereto.

As used herein, the term "biocompatible agent" or "carrier" refers to an agent that can increase the duration of activity of a GLP-1/glucagon receptor dual agonist when the biocompatible agent and carrier are directly or indirectly, covalently or non-covalently linked to a GLP-1/glucagon receptor dual agonist of the present invention to form a conjugate. For example, a substance that can increase the in vivo half-life of a GLP-1/glucagon receptor dual agonist when forming a conjugate can be a biocompatible substance or carrier according to the present invention. Examples of biocompatible substances or carriers that can be used to increase half-life vary, and can include polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, Fc neonatal receptor (FcRn) binding substances, connective tissue in vivo, nucleotides, fibronectin, transferrin, sugars, polymers, and the like. Of course, at least two of the carrier or the biocompatible substance may be used in combination. Biocompatible materials or carriers include biocompatible materials that extend half-life in vivo through covalent or non-covalent bonds.

In the present invention, methods for linking a biocompatible substance or carrier to the GLP-1/glucagon receptor dual agonist include a gene recombination method and in vitro linking using a polymer or a low molecular chemical, but are not limited thereto. The FcRn binding substance may be an immunoglobulin Fc region. For example, when polyethylene glycol is used as the carrier, the Recode technology of Ambrx inc, which can be site-specifically linked to polyethylene glycol, can be included. The methods may include the Neose sugar pegylation (glycosylation) technique, which may be specifically linked to a glycosylation moiety. Additionally, the method may include releasable PEG technology, wherein the polyethylene glycol is removed, but is not limited thereto. The methods may include techniques that may utilize PEG to increase bioavailability. In addition, polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin or hyaluronic acid may be included.

When albumin is used as the carrier, the method may include techniques in which albumin or an albumin fragment may be covalently linked directly to a peptide of the GLP-1/glucagon receptor dual agonist to increase stability in vivo. Even if albumin is not directly linked, a technique in which an albumin-binding substance (e.g., albumin-specific binding antibody or antibody fragment) is bound to the peptide to bind albumin, and a technique in which a certain peptide/protein having binding affinity for albumin is bound to the peptide may be included. Further, the method may include a technique in which a fatty acid or the like having a binding affinity for albumin is bound to the peptide, but is not limited thereto. Any technique or binding method that can utilize albumin to increase in vivo stability can be included herein.

Techniques for increasing the half-life in vivo by using antibodies or antibody fragments as carriers in conjunction with peptides may also be included in the present invention. Antibodies or antibody fragments having an FcRn binding site may be used, and any antibody fragment that does not contain an FcRn binding site, such as Fab, may be used. The CovX body (CovX-body) technology of CovX company using a catalytic antibody may be included herein, and the present invention may include a technology of increasing in vivo half-life using an Fc fragment. When the Fc fragment is used, the linker bound to the Fc fragment and the peptide and the binding method thereof may include, but are not limited to, a peptide bond or polyethylene glycol or the like, and any chemical binding method may be applicable. Further, the binding ratio of the GLP-1/glucagon receptor agonist dual agonists of the present invention may be 1:1 or 1:2, but is not limited thereto, and may include any ratio that increases half-life in vivo without limitation.

In addition, the carrier for increasing the half-life in vivo may be a non-peptidyl substance such as a polysaccharide or a fatty acid.

The linker bound to the carrier for increasing the half-life in vivo may include peptides, polyethylene glycol, fatty acids, sugars, polymers, low molecular compounds, nucleotides, and combinations thereof, and may be any chemical bond such as a non-covalent chemical bond, a covalent chemical bond, and the like, without limitation.

The preparation capable of increasing bioavailability or continuously maintaining activity may include sustained-release preparation using PLGA, hyaluronic acid, chitin, etc., by microparticles, nanoparticles, and the like.

In addition, formulations that can increase bioavailability or sustain different aspects of activity can be formulations such as implants, inhalants, nasal formulations, or patches.

In an exemplary embodiment of the invention, examples of GLP-1/glucagon receptor dual agonists can include native GLP-1/glucagon receptor dual agonists, such as oxyntomodulin and derivatives thereof, as well as long acting formulations and analogs thereof.

As used herein, the term "oxyntomodulin" means a peptide derived from glucagon precursor, pre-glucagon (pre-glucagon), and includes native oxyntomodulin, precursors, derivatives, fragments, and variants thereof. Preferably, it may have the amino acid sequence of SEQ ID NO.1 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).

The term "oxyntomodulin variant" is a peptide having one or more amino acid sequences different from that of a natural oxyntomodulin, and means a peptide retaining a function of activating GLP-1 and glucagon receptors, and it can be prepared by any one of substitution, addition, deletion, and modification or a combination thereof in a partial amino acid sequence of a natural oxyntomodulin.

The term "oxyntomodulin derivative" includes a peptide, peptide derivative or peptidomimetic prepared by the addition, deletion or substitution of an amino acid of oxyntomodulin to activate the GLP-1 receptor and glucagon receptor 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, the 16 th and 20 th amino acids thereof form a loop.

The term "oxyntomodulin fragment" means a fragment having one or more amino acid additions or deletions to the N-terminus or C-terminus of a natural oxyntomodulin, to which a non-naturally occurring amino acid (e.g., a D-type amino acid) may be added, and which has a function of activating the GLP-1 receptor and the glucagon receptor.

The respective methods for preparing the variants, derivatives and fragments of oxyntomodulin may be used alone or in combination. For example, the invention includes peptides having one or more amino acids different from the native peptide and having the N-terminal amino acid residue deamidated and having the function of activating the GLP-1 receptor and the glucagon receptor.

The C-terminus of the variants, derivatives and fragments of oxyntomodulin of the invention may be amidated.

The carrier material useful in the present invention may be selected from the group consisting of antibodies, immunoglobulin Fc regions, albumin, fatty acids, carbohydrates, polymers with peptide repeat units, transferrin, and PEG, and is preferably an immunoglobulin Fc region. In an exemplary embodiment of the invention, the long-acting GLP-1/glucagon receptor dual agonist is linked to the carrier by a non-peptidyl polymer as a linker. In yet another exemplary embodiment, the carrier linked to the non-peptidyl polymer is an immunoglobulin Fc fragment.

In the present invention, the long-acting GLP-1/glucagon receptor dual agonist is a form in which GLP-1/glucagon receptor dual agonists are each linked to an immunoglobulin Fc region, and exhibits persistence and safety. The binding of the immunoglobulin Fc region and the GLP-1/glucagon receptor dual agonist can be an in-frame fusion (infra fusion) without a linker, or can be linked using a non-peptidic polymeric linker. In the present invention, the immunoglobulin Fc may be used interchangeably with the immunoglobulin fragment.

As used herein, the term "non-peptidyl polymer" refers to a biocompatible polymer comprising at least two repeating units linked to each other by any covalent bond excluding peptide bonds. In the present invention, a non-peptidyl polymer may be used interchangeably with a non-peptidyl linker.

The non-peptidyl polymer useful in the present invention may be selected from biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyethylene ethyl ether, polylactic acid (PLA) or polylactic-glycolic acid (PLGA), lipopolymer, chitin, hyaluronic acid, and a combination thereof, and preferably, the biodegradable polymer is polyethylene glycol. In addition, derivatives thereof known in the art and derivatives that are readily prepared by methods known in the art are included within the scope of the present invention.

The peptide linker used in the fusion protein obtained by the conventional in-frame fusion method has a disadvantage of being easily cleaved by a protease in vivo, and thus a sufficient effect of increasing the serum half-life of the active drug by the carrier cannot be obtained as expected. However, in the present invention, a polymer resistant to protease can be used, similar to a carrier, to maintain the serum half-life of the peptide. Therefore, any non-peptidyl polymer can be used without limitation as long as it is a polymer having the above-described function, i.e., a polymer having resistance to in vivo protease. The non-peptidyl polymer has a molecular weight in the range of 1 to 100kDa, preferably 1 to 20 kDa. In addition, the non-peptidyl polymer of the present invention linked to the immunoglobulin Fc region may be one polymer or a combination of different types of polymers.

The non-peptidyl polymer used in the present invention has a reactive group capable of binding to an immunoglobulin Fc region 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, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative. The succinimide derivative may be a succinimide propionate, a hydroxysuccinimide group, a succinimidyl carboxymethyl group or a succinimide carbonate. Specifically, when the non-peptidyl polymer has reactive groups of reactive aldehyde groups at both ends thereof, it is effective to minimize non-specific reactions and link the physiologically active polypeptide and the immunoglobulin at both ends of the non-peptidyl polymer. The final product produced by reductive alkylation through an aldehyde linkage is far more stable than the product linked through an amide linkage. The reactive aldehyde group selectively binds to the N-terminus at low pH and to the lysine residue at high pH (e.g., pH9.0) to form a covalent bond. The reactive groups at both ends of the non-peptidyl polymer may be the same as or different from each other. For example, the non-peptidyl polymer may have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. When polyethylene glycol having reactive hydroxyl groups at both ends is used as the non-peptidyl polymer, the hydroxyl groups can be activated into various reactive groups by known chemical reactions, or commercially available polyethylene glycol having modified reactive groups can be used to prepare the long-acting GLP-1/glucagon receptor dual agonist conjugates of the present invention.

In addition, the immunoglobulin Fc region is advantageous in preparation, purification and yield of the conjugate because not only the molecular weight is relatively small compared to the whole molecule, but also material homogeneity is greatly increased and the possibility of inducing antigenicity in blood is reduced because the amino acid sequences in the respective antibodies are different, thus removing Fab portions showing high heterogeneity.

As used herein, the term "immunoglobulin Fc region" refers to the heavy chain constant region 2(CH2) and heavy chain constant region 3(CH3) of an immunoglobulin, excluding the heavy and light chain variable regions, heavy chain constant region 1(CH1), and light chain constant region 1(CL1) of an immunoglobulin. It may further comprise a hinge region at the heavy chain constant region. Furthermore, the immunoglobulin Fc region of the present invention may contain a part or all of the Fc region including heavy chain constant region 1(CH1) and/or light chain constant region 1(CL1) in addition to the heavy and light chain variable regions of the immunoglobulin, so long as it has substantially the same or better effect than the native protein. Furthermore, the immunoglobulin Fc region may be a fragment in which the amino acid sequence corresponding to a relatively long portion of CH2 and/or CH3 is deleted. That is, the immunoglobulin Fc region 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 of one or more domains and an immunoglobulin hinge region (or partial hinge region); and 6) dimers of the respective domains of the heavy and light chain constant regions.

In addition, the immunoglobulin Fc region of the present invention includes natural amino acid sequences and sequence derivatives (mutants) thereof. Amino acid sequence derivatives have different sequences due to deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues of the native amino acid sequence, or a combination thereof. For example, in IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important in binding, can be used as appropriate modification targets.

In addition, various derivatives are possible, including the following: in which a region capable of forming a disulfide bond is deleted, some amino acid residues at the N-terminus of the natural Fc are eliminated, a methionine residue is added to the N-terminus of the natural Fc, etc. In addition, to remove effector functions, complement binding sites such as Clq binding sites and antibody-dependent cell-mediated cytotoxicity (ADCC) sites may be deleted. Techniques for preparing such sequence derivatives of immunoglobulin Fc regions are disclosed in International publication Nos. WO 97/34631, WO 96/32478, and the like. Amino acid exchanges in Proteins and peptides that do not alter The activity of The molecule as a whole are known in The art (h.neurath, r.l.hill, The Proteins, Academic Press, new york, 1979). The most commonly occurring exchanges 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 bi-directional. In addition, the Fc region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like, if desired.

An Fc derivative is a derivative that has the same biological activity as the Fc region of the present invention and has improved structural stability (e.g., with respect to heat, pH, or the like) of the Fc region.

In addition, these Fc regions may be obtained from native forms isolated from humans and other animals (including cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc.), or may be recombinant or derivative thereof, obtained from transformed animal cells or microorganisms. Here, it can be obtained in native form by isolating intact immunoglobulins from human or animal organisms, which are then treated with proteases. Papain digests native immunoglobulins into Fab and Fc regions when treated with papain, and the native immunoglobulins are cleaved into pF' c and f (ab)2 when treated with pepsin. Fc or pF' c can be separated by size exclusion chromatography or the like. Preferably, the human Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

Furthermore, the immunoglobulin Fc region may be in a form having native sugar chains, or sugar chains that are increased or decreased compared to the native form, or may be in a deglycosylated form. The addition, reduction or removal of immunoglobulin Fc sugar chains can be achieved by means of microorganisms by methods commonly used in the art, such as chemical, enzymatic and genetic engineering methods. Removal of the sugar chain from the Fc region results in a significant reduction in binding affinity to the C1q moiety and a reduction or loss of antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, thereby not inducing an unnecessary immune response in vivo. In this regard, deglycosylated or non-glycosylated forms of immunoglobulin Fc regions may be more suitable for the purposes of the present invention as pharmaceutical carriers.

As used herein, the term "deglycosylation" refers to the enzymatic removal of sugar moieties from an Fc region, and the term "aglycosylation" refers to an Fc region produced in prokaryotes, preferably e.

Meanwhile, the immunoglobulin Fc region may be derived from a human or other animals including cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and is preferably derived from a human.

Furthermore, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, a combination thereof, or a hybrid thereof. Preferably, it is derived from IgG or IgM, which are most abundant in human blood, and most preferably from IgG, which is known to enhance the half-life of ligand binding proteins, but is not limited thereto.

As used herein, the term "combination" refers to polypeptides encoding single chain immunoglobulin Fc regions of the same origin linked to single chain polypeptides of different origin to form dimers or multimers. That is, the dimer or multimer may be formed of two or more fragments selected from IgG Fc, IgAFc, IgM Fc, IgD Fc, and IgE Fc fragments.

As used herein, the term "hybrid" refers to the presence of sequences corresponding to at least two Fc fragments of different origin in a single chain immunoglobulin Fc region. In the present invention, different types of hybrids are possible. That is, hybrids consisting of 1 to 4 domains of CH1, CH2, CH3 and CH4 selected from IgG Fc, IgMFc, IgA Fc, IgE Fc and IgD Fc are possible and may include a hinge.

On the other hand, IgG may be further classified into IgG1, IgG2, IgG3 and IgG4 subclasses, and combination or hybridization thereof is possible in the present invention. The IgG2 and IgG4 subclasses are preferred, and the Fc region of IgG4 is most preferred, which has substantially no effector function such as Complement Dependent Cytotoxicity (CDC).

That is, the immunoglobulin Fc region of the carrier for the drug of the present invention may be, for example, a human IgG 4-derived aglycosylated Fc region, but is not limited thereto. More preferably, the Fc region of human origin is than the Fc region of non-human origin, which may elicit an undesirable immune response, e.g., it may act as an antigen in humans to produce new antibodies.

Methods of preparing the long-acting GLP-1/glucagon receptor dual agonists of the present invention are not particularly limited. For example, the details of the preparation method and the effects thereof are described in Korean patent application laid-open No. 10-2012-0139579.

The use of long-acting GLP-1/glucagon receptor dual agonists is of great advantage in that the number of administrations to chronic patients requiring daily administration can be significantly reduced due to the increased blood half-life and persistence in the body, thereby improving the quality of life of the patients. Therefore, it is very helpful in the treatment of non-alcoholic fatty liver disease.

As used herein, the term "non-alcoholic fatty liver disease" refers to a case of fatty liver that has no history of alcohol consumption or has no association with alcohol consumption. Fatty liver refers to the phenomenon of abnormal accumulation of triglycerides in hepatocytes compared to normal triglyceride levels. Normal liver is approximately 5% composed of adipose tissue, and the main components of fat are triglycerides, fatty acids, phospholipids, cholesterol and cholesterol esters. However, once fatty liver occurs, most of the components are replaced by triglycerides. If the amount of triglycerides is more than 5% of the liver weight, it is diagnosed as fatty liver. Fatty liver is caused by a disorder of lipid metabolism or a defect in the process of carrying excessive fat in hepatocytes, and is mainly caused by a disorder of lipid metabolism of the liver. The majority of fat accumulated in fatty liver may be triglycerides. Non-alcoholic fatty liver disease includes non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, liver cancer and the like, but fatty liver disease to be prevented or treated using the composition of the present invention is included without limitation.

As used herein, the term "prevention" refers to the prevention or delay of all behaviors of non-alcoholic fatty liver disease by administering the composition of the present invention. "treating" refers to all activities that result in the reduction or positive alteration of the symptoms of non-alcoholic fatty liver disease. The treatment of non-alcoholic fatty liver disease is applicable to any mammal that may experience non-alcoholic fatty liver disease, and examples thereof include not only humans and primates, but also without limitation cows (e.g., cows), pigs, sheep, horses, dogs, and cats, but are preferably humans.

As used herein, the term "administering" refers to introducing an amount of a predetermined substance into a patient by an appropriate method. The composition of the present invention may be administered via any common route as long as it is able to reach the desired tissue. For example, it may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonary, or rectally, but is not limited thereto. However, since peptides are digested upon oral administration, the active ingredients of orally administered compositions should be coated or formulated to prevent degradation in the stomach. Preferably, the composition can be administered in the form of an injection. Furthermore, the depot may be administered by any device that can deliver the active agent into the target cells.

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

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate an organism and inhibit the biological activity or properties of the administered compound. For oral administration, the carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents and flavoring agents. For injectable formulations, the carrier may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers and the like. For topical formulations, carriers may include bases (bases), excipients, lubricants, preservatives, and the like.

The composition of the present invention can be formulated into various dosage forms in combination with the above pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical compositions may be formulated as tablets, troches, capsules, elixirs, suspensions, syrups or wafers (wafers). For injectable preparations, the pharmaceutical compositions may be formulated in ampoules, either as single-dose or multi-dose containers. The pharmaceutical compositions may also be formulated as solutions, suspensions, tablets, pills, capsules and depot preparations.

On the other hand, examples of carriers, excipients and diluents suitable for pharmaceutical preparations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber (acacia rubber), alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical preparation may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, and bactericides.

In another aspect, the invention provides a method of preventing or treating non-alcoholic liver disease, comprising the steps of: administering a composition comprising a long-acting GLP-1/glucagon receptor dual agonist to a subject at high risk or suffering from non-alcoholic liver disease, excluding humans.

The composition and non-alcoholic fatty liver disease are described above.

MODE OF THE INVENTION

The present invention will be described in more detail below by way of examples. These examples are intended only to illustrate the present invention, and the scope of the present invention is not construed as being limited to these examples.

Example 1: synthesis of oxyntomodulin derivatives

In the examples, oxyntomodulin derivatives having the following amino acid sequences were synthesized (table 1).

[ Table 1]

[ Table 1]

Oxyntomodulin and oxyntomodulin derivatives

In table 1, amino acids represented in bold and underline represent a loop, and amino acids represented by X mean an unnatural amino acid, α -methylglutamic acid. Further, CA represents 4-imidazoleacetyl group, and DA represents desaminohistidyl group.

Hereinafter, a representative long-acting GLP-1/glucagon receptor dual agonist, i.e., a long-acting GLP-1/glucagon receptor dual agonist in which Fc is linked to the 30 th amino acid of the GLP-1/glucagon receptor dual agonist by a non-peptidyl polymer, PEG (polyethylene glycol), was prepared and used in examples 2 to 3 below.

Example 2: in a high trans-fat feed intake ob/ob mouse model containing high fat, fructose and cholesterol, growth Validation of Effect of GLP-1/glucagon receptor Dual agonists on non-Alcoholic fatty liver disease

To verify the effect of a long-acting GLP-1/glucagon receptor dual agonist on non-alcoholic fatty liver disease, an ob/ob mouse model was given for 8 weeks using a high trans-fat diet (HTF diet) containing high fat (40% kcal), fructose (22%) and cholesterol (2%) to prepare an animal model of non-alcoholic fatty liver disease. Then, long-acting GLP-1/glucagon receptor dual agonist was administered subcutaneously to mice at 0.7 and 1.4nmol/kg once every two days (Q2D) and the administration was repeated for 4 weeks. Animal weights were compared to vehicle treated groups during the 4 week test period. After the 4-week test was completed, liver weights were measured and compared. In addition, after the 4-week test was completed, mRNA of collagen-1 a as a fibrosis marker, TNF-. alpha.as a proinflammatory marker, and SREBP-1c as a lipogenesis marker was verified.

As a result, body and liver weight measurements after 4 weeks of administration showed a significant reduction in weight in the long acting GLP-1/glucagon receptor dual agonist compared to the vehicle treated group (figure 1). Such results indicate that the long-acting GLP-1/glucagon receptor dual agonists of the present invention can inhibit weight gain occurring in animal models of non-alcoholic fatty liver disease, and that they can reduce the side effects of conventional drugs that improve insulin resistance.

In addition, comparison of mRNA for collagen-1 a, TNF- α, SREBP-1c showed a significant reduction in these mRNAs in the long acting GLP-1/glucagon receptor dual agonist treated group (FIG. 2). Such results indicate that the long-acting GLP-1/glucagon receptor dual agonist of the present invention reduces fibrosis, proinflammation and the like in an animal model of non-alcoholic fatty liver disease and inhibits lipogenesis, thus being effective for the prevention and treatment of non-alcoholic fatty liver disease.

Example 3: long-acting GLP-1/glucagon receptor doublets in a high trans-fat feed intake DI0 mouse model Validation of Effect of agonists on non-alcoholic fatty liver disease

To verify the effect of a long-acting GLP-1/glucagon receptor dual 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. Then, 3nmol/kg of the long-acting GLP-1/glucagon receptor dual agonist was subcutaneously administered once a week (QW) to the mice, and the administration was repeated for 4 weeks. After completion of the 4-week test, liver triglycerides (liver TG) and serum cholesterol were measured.

As a result, measurements of hepatic triglycerides and serum cholesterol after 4 weeks of administration showed a significant reduction in the long-acting GLP-1/glucagon receptor dual agonist in comparison to the vehicle-treated group, and a significant reduction to the level of normal animals undergoing a normal diet (chow diet) (fig. 3). Such results indicate that the long-acting GLP-1/glucagon receptor dual agonist of the present invention can reduce hepatic triglycerides and serum cholesterol to normal animal levels in animal models of non-alcoholic fatty liver disease, and thus is effective for the prevention and treatment of non-alcoholic fatty liver disease.

From the above description, those skilled in the art will appreciate that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics, and in this respect, the above embodiments should be construed as illustrative in all aspects and not restrictive. The scope of the invention should be construed in such a way that the meaning and scope of the appended claims (rather than the detailed description) and all changes or modifications that come within the meaning and range of equivalency of the claims are to be embraced therein.

Sequence listing

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<120> use of long-acting GLP-1/glucagon receptor dual agonists for the treatment of non-alcoholic fatty liver disease

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu Glu Glu

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Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Met Asn Lys

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Xaa Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 5 10 15

Glu Ala Val Xaa Leu Phe Ile Glu Trp Leu Met Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

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<223> Xaa = 4-imidazoleacetyl

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Met Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

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<223> Xaa = 4-imidazoleacetyl

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Xaa Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Ala Ala His Ser Gln Gly Thr

20 25 30

Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Lys

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Gly

1 5 10 15

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Glu Glu Glu Ala Val Lys

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Xaa

1 5 10 15

Glu Ala Val Xaa Leu Phe Ile Glu Trp Leu Met Asn Thr Lys

20 25 30

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<212>PRT

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Xaa Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 5 10 15

Glu Ala Val Xaa Leu Phe Ile Xaa Trp Leu Met Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

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Xaa Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

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Glu Ala Val Arg Leu Phe Ile Xaa Trp Leu Met Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu Glu Gly

1 5 10 15

Gly Gly His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu

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Glu Lys

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Ile Arg Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

35

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Ile Arg Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 5 10 15

Glu Ala Val Lys Leu Phe Ile Glu Trp Ile Arg Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

35

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<212>PRT

<213> Artificial sequence

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<223> oxyntomodulin derivatives

<220>

<221> variants

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<223> Xaa = 4-imidazoleacetyl

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 5 10 15

Glu Ala Val Lys Leu Phe Ile Glu Trp Ile Arg Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

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<212>PRT

<213> Artificial sequence

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<223> oxyntomodulin derivatives

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<221> variants

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<223> Xaa = 4-imidazoleacetyl

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Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Val Arg Asn Thr Lys Arg Asn

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Arg Asn Asn Ile Ala

35

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Lys Arg Ala Lys Glu Phe Val Cys Trp Leu Met Asn Thr

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<223> oxyntomodulin derivatives

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His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

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<212>PRT

<213> Artificial sequence

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<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

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His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

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<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

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His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>27

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>27

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Met Asn Thr

20 25

<210>28

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>28

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210>29

<211>37

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(2)

<223> Xaa = d-serine

<400>29

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

35

<210>30

<211>37

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(1)

<223> Xaa = 4-imidazoleacetyl

<400>30

Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

35

<210>31

<211>37

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(1)

<223> Xaa = 4-imidazoleacetyl

<220>

<221> variants

<222>(2)

<223> Xaa = d-serine

<400>31

Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn

20 25 30

Arg Asn Asn Ile Ala

35

<210>32

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(1)

<223> Xaa = 4-imidazoleacetyl

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>32

Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>33

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>33

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>34

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivatives

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>34

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

Claims (19)

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease comprising a long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist.

2. The pharmaceutical composition of claim 1, wherein the composition is characterized by having no or reduced weight gain side effects.

3. The pharmaceutical composition according to claim 1, wherein the composition exhibits at least one of the following characteristics:

a) reducing the expression or activity of collagen-1 a as a marker of fibrosis;

b) reducing the expression or activity of tumor necrosis factor-alpha (TNF-a) as a pro-inflammatory marker;

c) reducing the expression or activity of sterol regulatory element binding protein-1 c (SREBP-1c) as a lipogenesis marker;

d) reducing hepatic triglycerides; and

e) reducing blood cholesterol.

4. The pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is at least one disease selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis and liver cancer.

5. The pharmaceutical composition of claim 1, wherein the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist simultaneously activates the GLP-1 receptor and the glucagon receptor.

6. The pharmaceutical composition of claim 1, wherein the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist is in the form of a conjugate wherein a biocompatible substance or carrier capable of increasing the duration of activity of the dual agonist is linked to the agonist by a covalent bond or a linker.

7. The pharmaceutical composition of claim 1, wherein the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist has the amino acid sequence of SEQ ID No.25 and the amino acids at positions 16 and 20 are looped.

8. The pharmaceutical composition of claim 1, wherein the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist is linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, and combinations thereof.

9. The pharmaceutical composition of claim 8, wherein the immunoglobulin Fc region is non-glycosylated.

10. The pharmaceutical composition of claim 9, wherein the immunoglobulin Fc region comprises 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 domains.

11. The pharmaceutical composition of claim 10, wherein the immunoglobulin Fc region further comprises a hinge region.

12. The pharmaceutical composition of claim 8, wherein the immunoglobulin Fc region is an Fc region derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

13. The pharmaceutical composition of claim 12, wherein each domain on the immunoglobulin Fc region is a hybrid of domains having different origins selected from IgG, IgA, IgD, IgE, and IgM.

14. The pharmaceutical composition of claim 12, wherein the immunoglobulin Fc region is a dimer or multimer consisting of single chain immunoglobulins consisting of domains of the same origin.

15. The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 1, wherein the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist is a conjugate wherein the GLP-1/glucagon dual agonist represented by SEQ ID NO:25 and the immunoglobulin Fc region are linked by a non-peptidyl polymeric linker.

17. The pharmaceutical composition of claim 16, wherein amino acids at positions 16 and 20 of the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist represented by SEQ ID No.25 are cyclized.

18. The pharmaceutical composition according to claim 16, wherein the nonpeptidyl polymer linker is PEG.

19. A method for the prevention or treatment of non-alcoholic liver disease comprising administering to a subject, not including a human, at high risk or suffering from non-alcoholic liver disease the pharmaceutical composition of any one of claims 1 to 18.