HK40005973A - Pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease - Google Patents

Description

Pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease

The application is a divisional application, and the application date of the original application is 3 and 8 in 2013, application number is 2013800131410, and the invention is named as 'a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease'.

Technical Field

The present invention relates to a pharmaceutical composition comprising a long-acting insulinotropic peptide conjugate, which is useful for preventing or treating non-alcoholic fatty liver disease. In particular, the present invention relates to an insulinotropic peptide conjugate in which an insulinotropic peptide, a non-peptidyl polymer, and an immunoglobulin Fc region are covalently linked to each other so as to significantly increase a blood half-life, effectively prevent triglyceride accumulation, and to the use thereof in the prevention or treatment of non-alcoholic fatty liver disease.

Background

Non-alcoholic fatty liver disease refers to a wide range of diseases ranging from simple steatosis, which does not accompany an inflammatory response in patients who do not excessively take alcohol, to liver fibrosis and cirrhosis, which is caused by the progression of simple steatosis and shows hepatocellular inflammation.

Non-alcoholic fatty liver disease can be classified into primary and secondary non-alcoholic fatty liver disease depending on the cause of the pathology. Primary fatty liver diseases are caused by hyperlipidemia, diabetes, obesity, etc., which are characteristic of metabolic syndrome. Secondary fatty liver disease is the result of nutritional causes (sudden weight loss, hunger, intestinal diversion surgery), various medications, toxic substances (poisonous mushrooms, bacterial toxins), metabolic causes, and other factors.

The incidence of primary nonalcoholic fatty liver disease, in which diabetes and obesity, which are important features of metabolic syndrome, are known to be primary factors, is about 50% in diabetic patients, about 76% in obese patients, and mostly obese diabetic patients (Gupte P et al, 2004). Furthermore, when liver biopsies are taken from diabetic and obese patients with increased levels of alanine Aminotransferase (ALT), the incidence of steatohepatitis ranges from 18% to 36% (Braillon A et al, 1985).

Currently, there is no established method for determining the cause of non-alcoholic fatty liver disease. This is because the incidence of nonalcoholic fatty liver disease is associated with various factors such as diabetes, obesity, coronary artery disease, and lifestyle habits. There are some reports on the effects of antidiabetic or obese drugs on fatty liver disease. Orlistat (orlistat) used as an oral antiobesity drug shows histological improvement of the liver in patients with steatohepatitis (steatohepatitis) (hussei et al, 2007), and metformin shows a decrease in liver enzyme blood levels and a decrease in hepatonecrotizing inflammation and fibrosis in non-alcoholic fatty liver disease patients who do not show diabetes (Bugianesi et al, 2005). In addition, Thiazolidinediones (TZD) class of drugs, which are PPAR (peroxisome proliferator-activated receptor) agonists, inhibit the accumulation of fat in the liver and muscle, and exhibit direct anti-fibrotic effects on the liver in animal models of non-alcoholic fatty liver disease (Galli A et al, 2002).

GLP-1 receptor expression has recently been shown in hepatocytes and GLP-1 via the GLP-1 receptor of hepatocytes has shown good effects on the treatment of non-alcoholic fatty liver diseases by activating phosphoinositide-dependent kinase-1 (PDK-1) and protein kinase C- (PKC), which are major proteins in the insulin signaling pathway, through activation of phosphoinositide-dependent kinase-1 (PDK-1) and protein kinase C- (PKC), which are major proteins in the insulin signaling pathway (Gupta et al, 2010) GLP-1 also used to reduce fatty acid accumulation or prevent death of hepatocytes by endoplasmic reticulum stress (sham S et al, GLP-1) by both activation of chaperone protein, which promotes fatty acid accumulation or promotes death of hepatocytes by endoplasmic reticulum stress (sarma) and which are recently reported as important liver disease inhibitors and lipid-oxidizing inhibitors (sara S et al).

However, the main obstacle to the use of GLP-1 as a therapeutic agent for nonalcoholic fatty liver is its short blood half-life (maximum half-life: 2 minutes). This is due to the loss of GLP-1 titre via cleavage in the body between amino acid 8 (Ala) and amino acid 9 (Asp) by dipeptidyl peptidase iv (dpp iv). Thus, various studies have been conducted on GLP-1 analogs having DPP IV resistance, and experiments have been conducted for substituting Ala with Gly⁸(Deacon et al, 1998; Burchelin et al, 1999) for Ala⁸Or replacement of Ala with Leu or D-Ala⁸(Xiao et al, 2001), thereby increasing resistance to DPP IV while maintaining activity. N-terminal amino acid His of GLP-1⁷Thus, U.S. Pat. No. 5,545,618 describes N-terminal modification with alkyl or acyl groups, and Gallwitz et al describes N-methylation of His at position 7, or α -methylation, or substitution of the entire His with imidazole to increase resistance to DPP IV and maintain physiological activity.

In addition to these modifications, exendin-4, which is a GLP-1 analog purified from the salivary gland of the gila monster (U.S. patent No. 5,424,686), has resistance to DPP IV and higher physiological activity than GLP-1. As a result, the half-life in vivo is 2 to 4 hours, which is a longer period of time than GLP-1. However, with methods only for increasing resistance to DPP IV, maintaining physiological activity is not sufficient, and for example, in the case of commercially available exendin-4 (exenatide), it requires twice daily injections into a patient. This frequency remains difficult for the patient. The peptide prepared to ameliorate this problem is the DPP IV resistant exendin-4, which has a blood half-life of 2 to 4 hours. Although its blood half-life is longer than that of GLP-1, it also requires daily infusion.

Disclosure of Invention

Technical problem

Accordingly, the present inventors used a method of site-specifically linking the immunoglobulin Fc region, the non-peptidyl polymer and the insulinotropic peptide by a covalent bond, in order to maximize the effect of increasing the blood half-life of the insulinotropic peptide and maintaining the activity in vivo. As a result, the present inventors found that this method significantly increases the blood half-life of the peptide conjugate and provides a blood half-life much longer than known in-frame fusion methods. The present inventors also found that a conjugate prepared by site-specifically linking an immunoglobulin Fc to an amino acid residue of insulinotropic peptide other than an amino or thiol group occurring at the N-terminus maintains higher titer than a conjugate prepared by linking at the N-terminus of insulinotropic peptide. Thus, it was confirmed that the conjugate shows excellent therapeutic effects on non-alcoholic fatty liver disease even though it is administered less frequently than the known exendin-4 dosage form, thereby completing the present invention.

Technical scheme

It is an object of the present invention to provide a long-acting insulinotropic peptide conjugate, which maintains an extended half-life in vivo and effectively prevents triglyceride accumulation, and thus is useful for preventing or treating non-alcoholic fatty liver disease.

Advantageous effects

The insulinotropic peptide conjugate according to the present invention maintains the in vivo activity of the peptide at a relatively high level, has a significantly increased blood half-life, and effectively activates major proteins involved in lipolysis to prevent triglyceride accumulation, thereby being useful for the prevention and treatment of non-alcoholic fatty liver disease.

Drawings

Figure 1 shows an image of liver tissue of ob/ob mice administered with a long-acting exendin-4 conjugate according to one embodiment of the present invention (hematoxylin & eosin staining, H & E staining, purple stained area: normal liver tissue, white stained area: lipid droplets); and

figure 2 shows a graph of intrahepatic triglyceride accumulation in high fat induced-hypertrophic mice administered a long-acting exendin-4 conjugate according to one embodiment of the present invention (#: significantly increased at 99% confidence (p <0.01) compared to the normal diet group and significantly decreased at 99% confidence (p <0.01) compared to the high fat diet group).

Detailed Description

In one aspect, to achieve the above objects, one embodiment relates to a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, which comprises, as an active ingredient, an insulinotropic peptide drug conjugate prepared by covalently linking an insulinotropic peptide and an immunoglobulin Fc region via a non-peptidyl polymer.

In the pharmaceutical composition of the invention the insulinotropic peptide is selected from the group consisting of exendin-4, exendin-4 derivatives prepared by deletion of the N-terminal amino group of exendin-4, exendin-4 derivatives prepared by substitution of the N-terminal amino group of exendin-4 with a hydroxyl group, exendin-4 derivatives prepared by modification of the N-terminal amino group of exendin-4 with a dimethyl group, and exendin-4 derivatives prepared by deletion of the α -carbon of the N-terminal histidine residue of exendin-4 and the N-terminal amino group attached to the α -carbon.

The non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol-propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, and combinations thereof.

The insulinotropic peptide of the present invention is a peptide having an insulinotropic function to promote synthesis and expression of insulin in cells of pancreas β these peptides include precursors, derivatives, fragments, variants or analogs, and preferably GLP (glucagon-like peptide) -1, Exendin-3, Exendin-4 or analogs.

GLP-1 is a hormone secreted by the small intestine. Generally, it promotes biosynthesis and secretion of insulin, inhibits secretion of glucagon, and promotes glucose uptake in cells. In the small intestine, the glucagon precursor is broken down into three peptides, glucagon, GLP-1 and GLP-2. Here, GLP-1 means GLP-1(1-37), which is originally in a form having no insulinotropic action. But it is then processed and converted to the activated GLP-1(7-37) form. The amino acid sequence of GLP-1(7-37) is as follows:

GLP-1(7-37)(SEQ ID NO:1)

HAEGT FTSDV SSYLE GQAAK EPIAW LVKGR G

GLP-1 derivatives means peptides exhibiting at least 80% amino acid sequence homology to GLP-1, which may be in chemically modified form, and which exhibit an insulinotropic release function at least equal to or greater than GLP-1.

A GLP-1 fragment means a form in which one or more amino acids are added or deleted at the N-terminus or C-terminus of native GLP-1 and the added amino acid may be a non-naturally occurring amino acid (e.g., a D-form amino acid).

GLP-1 variant means a peptide having an insulinotropic action, which has one or more amino acid sequences different from native GLP-1.

Exendin-3 and Exendin-4 are insulinotropic peptides consisting of 39 amino acids with 53% amino acid sequence homology to GLP-1. The amino acid sequences of exendin-3 and exendin-4 are as follows:

exendin-3 (SEQ ID NO:2)

HSDGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS

Exendin-4 (SEQ ID NO:3)

HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS

An exendin derivative means a peptide having at least 80% amino acid sequence homology to a native exendin, which may have some groups chemically substituted on the amino acid residues and which exhibits an insulinotropic release function at least equal to or greater than that of the native exendin.

An exendin fragment means a fragment having one or more added or deleted amino acids at the N-terminus or C-terminus of a natural exendin, and the added amino acids may be non-naturally occurring amino acids (e.g. D-type amino acids).

An exendin variant means a peptide with an insulinotropic release function having one or more amino acid sequences different from the native exendin.

In particular embodiments, the native insulinotropic peptides and modified insulinotropic peptides used in the present invention can be synthesized using solid phase synthesis methods and most native peptides, including native insulinotropic peptides, can be produced by recombinant techniques.

In addition, the insulinotropic peptide used in the present invention may be bound to the non-peptidyl polymer at various sites.

The conjugate prepared in the present invention may have an activity that varies depending on the binding site of the insulinotropic peptide.

For example, they may be coupled to the N-terminus, and other termini, including the C-terminus, respectively, which exhibit different in vitro activities. Aldehyde reactive groups are selectively bound to the N-terminus at low pH and can bind to lysine residues to form covalent bonds at high pH, e.g., pH 9.0. The pegylation reaction was allowed to proceed at different pH and an ion exchange column could then be used to separate the positional isomers from the reaction mixture.

If the insulinotropic peptide is coupled at a site other than the N-terminus, which is a site important for in vivo activity, a reactive thiol group may be introduced to the site of the amino acid residue to be modified in the natural amino acid sequence, so as to form a covalent bond using a maleimide linker at the non-peptidyl polymer.

If the insulinotropic peptide is coupled at a site other than the N-terminus, which is important for in vivo activity, a reactive amine group may be introduced to the site of the amino acid residue to be modified in the natural amino acid sequence, so that a covalent bond is formed using an aldehyde linker at the non-peptidyl polymer.

When an aldehyde linker at the non-peptidyl polymer is used, it reacts with an amine group and a lysine residue at the N-terminal, and the modified form of insulinotropic peptide can be used to selectively increase the reaction yield. For example, only one amine group to be reacted may remain at a desired site using an N-terminal blocking method, a lysine residue substitution method, a method for introducing an amine group at a carboxyl terminal, etc., thereby increasing the yield of the pegylation reaction and the coupling reaction. Methods for protecting the N-terminus include dimethylation, and methylation, deamination, acetylation, etc., but are not limited to such alkylation methods.

In a preferred embodiment, the insulinotropic peptide conjugate of the present invention is an insulinotropic peptide conjugate in which an immunoglobulin Fc region specifically binds to an amine group other than the N-terminus of the insulinotropic peptide.

In a specific embodiment, the inventors caused pegylation of native exendin-4 at pH 9.0 to selectively couple PEG to the lysine residue of the insulinotropic peptide alternatively exendin-4 derivatives with N-terminal deletions or protected could be synthesized to be coupled, pegylation at the N-terminus could be blocked by deletion of the α amine group of the N-terminal histidine or by modification of the nitrogen terminal histidine with two methyl groups this N-terminal modification did not affect in vitro activity (table 1).

Unlike the N-terminal coupling of exendin-4, the coupling at the lysine residue maintained approximately 6% activity in vitro (table 1). Furthermore, the exendin-4-PEG-immunoglobulin Fc conjugates prepared in the present invention exhibit a significantly increased blood half-life of 60-70 hours, indicating an unexpectedly high persistence efficacy. Therefore, also the titer reduction is minimized by coupling to lysine residues which do not affect the activity and therefore a new long acting exendin-4 formulation can be prepared which is able to maintain its activity in vivo.

The immunoglobulin Fc region is safe for use as a drug carrier because it is a biodegradable polypeptide that is metabolized in vivo. Furthermore, the immunoglobulin Fc region has a relatively low molecular weight compared to the entire immunoglobulin molecule, and thus it is advantageous for the preparation, purification and yield of the conjugate. Since the immunoglobulin Fc region does not contain Fab fragments whose amino acid sequences differ according to the antibody subclasses and thus are highly nonhomologous, it is predicted that the immunoglobulin Fc region can greatly increase the homogeneity of substances and is less antigenic.

The term "immunoglobulin Fc region" as used herein refers to the heavy chain constant region 2 (C) of an immunoglobulin_H2) And heavy chain constant region 3 (C)_H3) And does not include the variable regions of the heavy and light chains, heavy chain constant region 1 (C)_H1) And light chain constant region 1 (C)_L1). It may further comprise a hinge region at the heavy chain constant region. Furthermore, in addition to the variable regions of the heavy and light chains, the immunoglobulin Fc region of the present invention may comprise part or all of the Fc region, including heavy chain constant region 1 (C)_H1) And/or light chain constant region 1 (C)_L1) As long as it has substantially similar or better effects than the native protein. WhileAnd, the immunoglobulin Fc region may be at C_H2 and/or C_H3 has a deleted fragment in a relatively long portion of the amino acid sequence. That is, the immunoglobulin Fc region of the present invention may include 1) C_H1 domain, C_H2 domain, C_H3 domains and C_H4 domain, 2) C_H1 domain and C_H2 domain, 3) C_H1 domain and C_H3 domain, 4) C_H2 domain and C_H3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or a portion of a hinge region), and 6) a dimer of each of the domains of the heavy and light chain constant regions.

The immunoglobulin Fc region of the present invention includes native amino acid sequences and sequence derivatives (mutants) thereof. Amino acid sequence derivatives are sequences that differ from the native amino acid sequence by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof. For example, in IgG Fc, amino acid residues known to be important for binding at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 can be used as appropriate targets for modification. Also, various other derivatives are possible, including those in which a region capable of forming a disulfide bond is deleted or certain amino acid residues are removed at the N-terminus of the native Fc form, or a methionine residue is added thereto. In addition, to remove effector function, deletions may occur at complement-binding sites, such as the C1 q-binding site and ADCC site. International patent publication Nos. WO97/34631 and WO96/32478 disclose techniques for preparing such sequence derivatives of immunoglobulin Fc regions.

Amino acid exchanges in Proteins and peptides are known in The art, which do not generally alter molecular activity (h.neurath, r.l.hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are the bi-directional 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, Asp/Gly.

If desired, the Fc region may be modified by phosphorylation, sulfation, acrylation (acrylation), glycosylation, methylation, farnesylation (farnesylation), acetylation, amidation, and the like.

The aforementioned Fc derivatives are derivatives having the same biological activity as the Fc region of the present invention or improved structural stability against heat, pH, etc.

In addition, these Fc regions may be obtained from natural forms isolated from humans and other animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof obtained from transformed animal cells or microorganisms. Herein, they may be obtained from native immunoglobulins by isolating whole immunoglobulins from human or animal organisms and treating them with proteolytic enzymes. Papain digests native immunoglobulins into Fab and Fc regions, and pepsin treatment results in the production of pF' c and f (ab)2 fragments. These fragments can be subjected to size exclusion chromatography to separate Fc or pF' c.

Preferably, the Fc region of human origin is a recombinant immunoglobulin Fc region obtained from a microorganism.

In addition, the immunoglobulin Fc region may be in the form of having a natural sugar chain, an increased sugar chain as compared to the natural form, or a decreased sugar chain as compared to the natural form, or may be in a deglycosylated form. The addition, reduction or removal of immunoglobulin Fc sugar chains can be achieved by methods common in the art, such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc region results in a drastic decrease in binding affinity to complement (c1q) and a decrease or loss in antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, thereby not inducing an unnecessary immune response in vivo. In this regard, immunoglobulin Fc regions in deglycosylated or unglycosylated form may be more suitable as drug carriers for the purposes of the present invention.

As used herein, the term "deglycosylation" refers to the enzymatic removal of the sugar moiety from the Fc region, and the term "aglycosylation" means the production of the Fc region in an aglycosylated form by a prokaryote, preferably e.

Although the immunoglobulin Fc region may preferably be derived from a human, it may also be derived from other animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, or an Fc region manufactured by a composition thereof or a mixture thereof. Preferably, it is derived from IgG or IgM, which is one of the most abundant proteins in human blood, and most preferably from IgG, which is known to enhance the half-life of ligand-binding proteins.

On the other hand, the term "combination", as used herein, means that polypeptides encoding single-chain immunoglobulin Fc regions of the same origin are linked to single-chain polypeptides of different origin to form dimers or multimers. That is, the dimer or multimer may be formed from two or more fragments selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

As used herein, the term "hybrid" means that sequences encoding two or more immunoglobulin Fc regions of different origins occur in a single chain immunoglobulin Fc region. In the present invention, various types of hybrids are possible. That is, the domain mixture may consist of one to four domains selected from CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc, and may include a hinge region.

On the other hand, IgG may be classified into subclasses IgG1, IgG2, IgG3, and IgG4, and the present invention may include compositions and mixtures thereof. Preferred are the IgG2 and IgG4 subclasses, and most preferred is the Fc region of IgG4, which has little effector function, such as CDC (complement dependent cytotoxicity).

That is, the most preferred immunoglobulin Fc region as a drug carrier of the present invention is an aglycosylated Fc region derived from human IgG 4. The Fc region of human origin is more preferred than the Fc region of non-human origin, which can act as an antigen in humans and cause an undesirable immune response, such as the production of new antibodies against the antigen.

As used herein, the term "non-peptidyl polymer" refers to a biocompatible polymer comprising two or more repeating units linked to each other by any covalent bond other than a peptide bond.

The non-peptidyl polymer useful in the present invention may be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipopolymers, chitin, hyaluronic acid, and combinations thereof, and it is preferably polyethylene glycol. Also, derivatives thereof which are well known in the art and are readily prepared by those skilled in the art are included within the scope of the present invention.

The peptide linker for the fusion protein obtained by the conventional in-frame fusion method has a disadvantage in that it is easily cleaved by a proteolytic enzyme in vivo, and thus a sufficient effect of increasing the blood half-life of the active drug by the carrier cannot be obtained as desired. However, in the present invention, a polymer having resistance to proteolytic enzymes can be used to maintain the blood half-life of the peptide similar to that of the carrier. Therefore, any non-peptidyl polymer used in the present invention can be used without any limitation as long as it is a polymer having the aforementioned function, i.e., having resistance to proteolytic enzymes in vivo. Preferably, the non-peptidyl polymer has a molecular weight ranging from 1 to 100kDa, and preferably from 1 to 20 kDa. Further, 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 non-peptidyl polymer has a reactive group at both ends, which is preferably selected from the group consisting of a reactive glyoxal group (aldehydegroup), a propionaldehyde group, a butyraldehyde group, a maleimide group, and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. In particular, when the nonpeptidyl polymer has reactive aldehyde groups at both terminals, it is effective to link it at both terminals with the physiologically active polypeptide and the immunoglobulin having minimal nonspecific reactions. The final product produced by reductive alkylation via an aldehyde linkage is much more stable than when linked by an amide linkage. The aldehyde reactive group selectively binds to the N-terminus at low pH and may bind to a lysine residue to form a covalent bond at high pH, e.g., at pH 9.0.

The reactive groups at both ends of the non-peptidyl polymer may be the same or different. For example, the non-peptidic polymer may have a maleimide group at one end and it may have an glyoxal group, a propionaldehyde group, or a butyraldehyde group at the other end. When polyethylene glycol having reactive hydroxyl groups at both ends thereof is used as the non-peptidyl polymer, the hydroxyl groups can be activated into various reactive groups by known chemical reactions, or polyethylene glycol having commercially available modified reactive groups can be used in order to prepare the insulinotropic peptide conjugate of the present invention.

In another embodiment, the present invention provides a method for preparing an insulinotropic peptide conjugate, which comprises the steps of:

(1) covalently linking a non-peptidyl polymer having a reactive group of aldehyde, maleimide or succinimide derivative at both ends thereof with an amine group or thiol group of insulinotropic peptide;

(2) isolating a conjugate comprising an insulinotropic peptide from the reaction mixture of (1), wherein the non-peptidyl polymer is covalently attached to a site other than the amino terminus; and

(3) covalently linking the immunoglobulin Fc region to the other end of the non-peptidyl polymer of the isolated conjugate, thereby producing a peptide conjugate having the immunoglobulin Fc region and insulinotropic peptide linked to the respective ends of the non-peptidyl polymer.

As used herein, the term "conjugate" refers to an intermediate prepared by covalently linking a non-peptidyl polymer and an insulinotropic peptide, and then an immunoglobulin Fc region is linked to the other end of the non-peptidyl polymer in the conjugate.

In a preferred embodiment, the present invention provides a method of preparation comprising the steps of:

(1) covalently linking a non-peptidyl polymer having aldehyde-reactive groups at both terminals thereof with a lysine residue of exendin-4;

(2) isolating a conjugate comprising exendin-4 from the reaction mixture of (1), wherein the nonpeptidyl polymer is covalently attached to a lysine residue; and

(3) covalently linking an immunoglobulin Fc region to the other end of the non-peptidyl polymer of the isolated conjugate, thereby producing a protein conjugate comprising the immunoglobulin Fc region and exendin-4 linked to the respective ends of the non-peptidyl polymer. More preferably, the nonpeptidyl polymer in (1) and the lysine residue of exendin-4 are linked at pH 9.0 or higher.

The insulinotropic peptide conjugate of the present invention activates a major protein of an insulin signaling pathway via a GLP-1 receptor, and thus can be used for preventing or treating non-alcoholic fatty liver disease. In particular, the insulinotropic peptide conjugate of the present invention increases the activity of PKC- ζ (protein kinase C- ζ), which modulates the enzyme activity involved in lipolysis and maintains the in vivo activity of insulinotropic peptide known to increase the expression of Glut2 (glucose carrier protein-2), and increases the blood half-life of insulinotropic peptide, thereby significantly increasing the duration of in vivo efficacy. Therefore, superior therapeutic effects on non-alcoholic fatty liver disease can be obtained with lower administration frequency than known dosage forms.

In the present invention, non-alcoholic fatty liver disease (NAFLD) includes primary and secondary non-alcoholic fatty liver diseases, and more specifically means non-alcoholic fatty liver disease caused by primary hyperlipidemia, diabetes, or obesity. For example, nonalcoholic fatty liver disease includes simple steatosis; fatty liver disease caused by malnutrition, hunger, obesity, and diabetes; steatohepatitis; and liver fibrosis and cirrhosis that occur as a result of the progression of these diseases.

The pharmaceutical composition comprising the insulinotropic peptide conjugate of the present invention may further comprise a pharmaceutically acceptable carrier. For oral administration, pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, and flavors. For injectable formulations, pharmaceutically acceptable carriers can include buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers. For formulations for topical administration, pharmaceutically acceptable carriers can include bases, excipients, lubricants, and preservatives. The pharmaceutical compositions of the present invention may be formulated in a variety of pharmaceutical dosage forms in combination with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical compositions may be formulated as tablets, troches, capsules, elixirs, suspensions, syrups or wafers. For injectable preparations, the pharmaceutical compositions may be formulated in ampoules, such as in multi-dose containers, either as single-dose administration forms or as unit administration forms. 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, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical preparation may further include a filler, an anti-agglomerating agent, a lubricant, a humectant, a fragrance, and an antibacterial agent.

The conjugate according to the present invention is used for preventing or treating non-alcoholic fatty liver disease. Thus, pharmaceutical compositions comprising the conjugates can be administered for the treatment of the disease.

As used herein, the term "administering" means introducing a predetermined substance into a patient by some suitable method. The conjugate of the invention may be administered via any common route, provided that it is capable of reaching the desired tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and intrarectal, but the invention is not limited to these exemplary modes of administration. However, because peptides are digested when administered orally, the active ingredients of compositions for oral administration should be coated or formulated to prevent degradation in the stomach. Preferably, the present compositions may be administered in injectable form. In addition, the pharmaceutical compositions can be administered using certain devices capable of transporting the active ingredient to the target cell.

The pharmaceutical composition of the present invention can be determined by several relevant factors including the type of disease to be treated, the administration route, the age, sex, body weight and severity of the disease of the patient, and the type of drug as an active ingredient. Because the pharmaceutical compositions of the present invention have superior in vivo efficacy durations and titers, they can significantly reduce the frequency and dosage of administration of the pharmaceutical agents of the present invention.

In addition, the pharmaceutical composition of the present invention can be used alone or in combination with surgical procedures, hormonal therapies, drug therapies and biological response modifiers in order to prevent and treat non-alcoholic fatty liver disease.

In one aspect the invention relates to the use of a pharmaceutical composition in the manufacture of a medicament for the prevention or treatment of non-alcoholic liver disease.

Examples

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples.

Example 1 testing of the in vitro Activity of Long-acting Exendin-4

Various long-acting exendin-4 derivatives used in this experiment were prepared in the same manner as in korean patent No. 10-1058315 of the present inventors.

A method for measuring cell activity in vitro was used in order to measure the efficacy of long acting formulations of exendin-4. In vitro activity measurements, RIN-m5F, known as rat insulinoma cells, was used. Because the cell has a GLP-1 receptor, it is typically used in methods of measuring the activity of the GLP-1 family in vitro. RIN-m5F was treated with different concentrations of GLP-1, Exendin-4 and test material. EC50 values were determined by measuring the occurrence of the signaling molecule cAMP in cells caused by the test material and compared to each other. The results are summarized in table 1.

TABLE 1

Test material	Blood half-life (hr)	In vitro Titers (%)
			Exendin-4	0.7	100
Exendin-4 (N) -PEG-Fc	61.5	<0.2
			Exendin-4 (Lys27) -PEG-Fc	70.5	6.3

Exendin-4 (N) -PEG-Fc: a conjugate prepared by attaching the N-terminus of exendin-4 and the Fc region via PEG.

Exendin-4 (Lys27) -PEG-Fc: a conjugate prepared by attaching a lysine residue at position 27 of exendin-4 and an Fc region via PEG.

As shown in table 1, when the non-peptidyl polymer was attached to a lysine residue other than the N-terminus of native exendin-4, the in vitro titer remained at 6.3% and the blood half-life increased significantly to about 70 hours.

Example 2 Effect on fatty liver formation in ob/ob mice in a hypertrophic animal model

<2-1> grouping of Experimental animals

Female 5-week old ob/ob mice (C57BL/6JHAMSLC-ob/ob, 24-34g) were purchased from Japanese Slc. ob/ob mice are animal models commonly used in efficacy testing of anti-obesity and anti-diabetic dosage forms. They were free-feeding solid feed for experimental animals (manufacturer: Picolab Rodent Diet, product name: 5053) sterilized by radiation, and free-feeding tap water filtered, UV-irradiated in water bottles. They were kept in a box system that met GLP standard requirements with 12 hour dark-light cycles (lights on at 6:00 am and off at 6:00 pm) according to animal care standards guidelines (animal carestandard guidelines). Thereafter, healthy ob/ob mice were selected and acclimated to laboratory conditions for 1 week. Then, drug administration was performed, and the mice were divided into 4 groups and administered as follows.

Group 1 (negative control): subcutaneously injecting PHOSPHATE BUFFEREDSALINE (Sigma) of DULBECCO once or more times a week in an administration volume of 5ml/kg

Group 2 (positive control): byetta was injected subcutaneously at 10.8nmol/kg per day in an administration volume of 5ml/kg

Group 3(3.7nmol/kg of long acting exendin-4 derivative-treated group): subcutaneous injection of 3.7nmol/kg of a long acting exendin-4 derivative (HM11260C) once weekly in an administration volume of 5ml/kg

Group 4(8.2nmol/kg of long acting exendin-4 derivative-treated group): subcutaneous injection of 8.2nmol/kg of a long acting exendin-4 derivative (HM11260C) once weekly in an administration volume of 5ml/kg

Byettta (eli lilly) is native exendin-4, and the long-acting exendin-4 derivative (HM11260C) is a CA exendin-4-PEG-Fc conjugate prepared by attaching an imidazoleacetyl-exendin-4 with α carbons from which the first amino acid histidine is removed to the Fc region via PEG, as described in korean patent No. 10-1058315.

Saline solution or drug was administered to each group for 7 weeks and their effect on fatty liver formation was analyzed.

<2-2> Effect of Long-acting Exendin-4 derivatives on fatty liver formation

To examine the effect of the long-acting exendin-4 derivatives according to the present invention on fatty liver formation in ob/ob mice, the following experiments were performed. Drugs were administered to the groups divided in example <2-1>, and livers were taken from ob/ob mice, and a part thereof was fixed in 4% formaldehyde and embedded in paraffin, followed by H & E staining. The results are shown in FIG. 1.

As shown in fig. 1, the pathological features of fatty liver were clearly observed in the negative control group treated with vehicle, whereas a significant dose-dependent decrease in the pathological features of fatty liver disease was observed in the experimental group treated with the long-acting exendin-4 derivative of the present invention. It was also found that the long acting exendin-4 derivatives of the present invention show superior efficacy against fatty liver even with lower doses compared to the positive control BYETTA.

Example 3 Effect on high fat induced intrahepatic triglyceride accumulation in hypertrophic mice

<3-1> grouping of Experimental animals

6-week-old C57BL/6 mice were stabilized and divided into two groups and received a normal diet containing 10% fat and a high fat diet containing 60% fat for 12 weeks (manufacturer: Research diets Inc., product name: D12492). Thus, normal mice and high-fat induced-hypertrophic mice were prepared and used for experiments. According to the standard guidelines for animal care, they were kept in a box system that met GLP standard requirements with 12 hour dark-light cycles (lights on at 6:00 am and off at 6:00 pm). Thereafter, healthy high fat induced-hypertrophic mice were selected and acclimated to laboratory conditions for 1 week. Then, drug administration was performed, and the mice were divided into 4 groups and administered as follows.

Group 1 (normal diet group): subcutaneously injecting PHOSPHATEBUFFERED SALINE (Sigma) of DULBECCO once or more times a week at an administration volume of 5ml/kg

Group 2 (high fat diet group): subcutaneously injecting PHOSPHATEBUFFERED SALINE (Sigma) of DULBECCO once or more times a week at an administration volume of 5ml/kg

Group 3 (high fat diet group treated with 3nmol/kg of long acting exendin-4 derivative): subcutaneous injection of 3nmol/kg of a long acting exendin-4 derivative (HM11260C) once weekly at an administration volume of 5ml/kg

Group 4 (high fat diet group treated with 10nmol/kg of long acting exendin-4 derivative): subcutaneous injection of 10nmol/kg of a long acting exendin-4 derivative (HM11260C) once weekly in an administration volume of 5ml/kg

Saline solution or drug was administered to each group for 2 weeks, and the amount of accumulated triglycerides in liver tissue was analyzed.

<3-2> measurement of intrahepatic triglyceride accumulation in high-fat-induced-hypertrophic mice

Livers were removed from the group divided in example <3-1> and were high fat induced-hypertrophic mice with or without the administration of long-acting exendin-4 derivatives, and intrahepatic triglyceride concentrations were determined. As shown in fig. 2, the intrahepatic triglyceride concentration of the high-fat diet group was 172.3mg/g, which was higher than that of the low-fat diet group (114.0mg/g), but the high-fat diet group treated with 3nmol/kg of the long-acting exendin-4 derivative showed a triglyceride level of 93mg/g, showing a 46% decrease compared to the high-fat diet group. These results suggest that the long-acting exendin-4 derivatives of the present invention have therapeutic effects on non-alcoholic fatty liver disease.

<110> Korea scientific Co., Ltd

<120> pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease

<130>OPA13019

<150>KR 10-2012-0024632

<151>2012-03-09

<160>3

<170>KopatentIn 2.0

<210>1

<211>31

<212>PRT

<213> Artificial sequence

<220>

<223>GLP-1

<400>1

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Pro Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210>2

<211>39

<212>PRT

<213> Artificial sequence

<220>

<223> Exendin-3

<400>2

His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

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

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210>3

<211>39

<212>PRT

<213> Artificial sequence

<220>

<223> Exendin-4

<400>3

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

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

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

Claims (15)

1. Use of an insulinotropic peptide drug conjugate in which an insulinotropic peptide is covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer,

wherein the insulinotropic peptide is selected from the group consisting of exendin-4, an exendin-4 derivative prepared by deletion of the N-terminal amine group of exendin-4, an exendin-4 derivative prepared by substitution of the N-terminal amine group of exendin-4 with a hydroxyl group, an exendin-4 derivative prepared by modification of the N-terminal amine group of exendin-4 with a dimethyl group, and an exendin-4 derivative prepared by deletion of the α -carbon of the N-terminal histidine residue of exendin-4 and the N-terminal amine group attached to said α -carbon,

and the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol-propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipopolymer, chitin, hyaluronic acid, and a combination thereof,

the non-peptidyl polymer is linked to a lysine residue at position 27 of the insulinotropic peptide, and

the non-alcoholic fatty liver disease is selected from simple steatosis, fatty liver disease caused by malnutrition or hunger, hepatic fibrosis and liver cirrhosis.

2. The use according to claim 1, wherein the immunoglobulin Fc region and the amine group or thiol group of the insulinotropic peptide are linked to both ends of the non-peptidyl polymer, respectively.

3. The use according to claim 1, wherein the nonpeptidyl polymer is polyethylene glycol.

4. The use of claim 1, wherein the immunoglobulin Fc region is aglycosylated.

5. The use of claim 1, wherein the immunoglobulin Fc region consists of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 domains.

6. The use of claim 5, wherein the immunoglobulin Fc region further comprises a hinge region.

7. The use of claim 1, 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.

8. The use of claim 7, wherein the immunoglobulin Fc region is an IgG4 Fc region.

9. The use of claim 8, wherein the immunoglobulin Fc region is a human aglycosylated IgG4 Fc region.

10. The use according to claim 1, wherein the reactive group of the nonpeptidyl polymer is selected from the group consisting of an glyoxal group, a malonyl group, a butyraldehyde group, a maleimide group and a succinimide derivative.

11. The use of claim 10, wherein the succinimide derivative is selected from succinimidyl propionic acid, succinimidyl carboxymethyl, hydroxysuccinimidyl, and succinimidyl carbonate.

12. The use according to claim 1, wherein the nonpeptidyl polymer has reactive aldehyde groups at both ends thereof.

13. The use of claim 1, wherein the insulinotropic peptide drug conjugate increases the activity of PKC-zeta (protein kinase C-zeta) which modulates the activity of enzymes involved in lipolysis.

14. The use of claim 1, wherein the insulinotropic peptide drug conjugate increases the expression of Glut2 (glucose carrier protein-2) involved in lipolysis.

15. The use according to claim 1, wherein the insulinotropic peptide is 4-imidazoacetyl exendin-4 and the nonpeptidyl polymer is polyethylene glycol.