Glucagon analogs as long-acting GLP-1 receptor / glucagon receptor agonists in the treatment of fatty liver disease and steatohepatitis

Abstract

The present invention relates to the medical use of certain long-acting glucagon analogues with dual GLP-1 receptor / glucagon receptor agonist activity in the prevention and / or treatment of metabolic liver diseases, in particular non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), liver fibrosis and / or cirrhosis associated with NAFLD.

Description

[Technical Field] 【0001】 Field of Invention The present invention relates to the medical use of certain long-acting glucagon analogs having GLP-1 receptor / glucagon receptor dual agonist activity in the prevention and / or treatment of metabolic liver diseases, particularly non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic fibrosis and / or cirrhosis associated with NAFLD. 【0002】 Background of the Invention Fatty liver disease is a chronic condition characterized by excessive triglyceride deposition in the liver. It can be caused by multiple factors, with two main forms associated with excessive alcohol consumption or, in the absence of excessive alcohol intake, metabolic dysregulation. The latter is called non-alcoholic fatty liver disease (NAFLD). It is generally associated with metabolic syndromes and their individual components (obesity, type 2 diabetes, dyslipidemia, and hypertension). The range of NAFLD extends from isolated hepatic steatohepatitis, also called non-alcoholic fatty liver (NAFL), to non-alcoholic steatohepatitis (NASH), characterized by triglyceride deposition in the liver, hepatocyte damage, and inflammation of the hepatic lobules, and to hepatic fibrosis. In particular, fibrosis can be present to varying degrees in patients with NASH. The presence of non-alcoholic steatohepatitis (NASH) with fibrosis is a strong risk factor for the development of cirrhosis and potentially hepatocellular carcinoma (Hagstrom et al., Journal of Hepatology 2017, vol. 67, pp. 1265-1273). 【0003】 NAFLD is a common condition; its prevalence worldwide is estimated to be as high as 25% (Younossi et al, Hepatology 2016, vol. 64, pp. 73-84). The transition from NAFL, a relatively benign condition, to NASH, particularly the progression of fibrosis, is associated with an increased risk of all-cause mortality (see, for example, Dulai et al, Hepatology 2017, vol. 65, pp. 1557-1565). 【0004】 NAFLD is known to be caused by a variety of etiologies, including insulin resistance, lipotoxicity, and inflammatory responses. Of these, insulin resistance is the most common etiology. 【0005】 Much effort has been made to improve insulin resistance and prevent / treat non-alcoholic fatty liver disease. For example, clinical trials of thiazolidinedione (TZD) or metformin, which are types of insulin sensitizers, have been conducted (see Hepatology (2003) 38: 1008-17, J Clin Invest (2001) 108: 1167-74). 【0006】 However, treatment with TZD-type drugs has the drawbacks of significant weight gain and fluid retention, and therefore, it is known that such treatment is not feasible for patients with heart disease. In addition to TZD-type drugs, clinical trials have been conducted using GLP-1 receptor agonists such as Victoza (liraglutide) or Byetta for NAFLD. 【0007】 Liraglutide is a chemically modified analog of the commercially available glucagon-like peptide-1 (GLP-1). The amino acid sequence of liraglutide is shown as Sequence ID No. 1. 【0008】 HAEGTFTSDVSSYLEGQAAK((S)-4-carboxy-4-hexadecanoyl-amino-butyryl-)EFlAWLVRGRG(Sequence ID 1). 【0009】 Liraglutide acts as a GLP-1 receptor (GLP1R) agonist. Such GLP1R agonists have been shown to lower blood glucose and reduce body weight. Furthermore, treatment with the GLP1R agonist liraglutide in overweight or obese patients diagnosed with NASH by biopsy resulted in recovery of NASH in 39% of patients, compared to 9% with placebo (Armstrong et al., BMJ Open 2013, vol. 3, e003995; Armstrong et al, Lancet 2016, vol. 387, pp.679-690). 【0010】 A PEGylated oxytomodulin synthetic analog, a dual agonist of the GLP-1 receptor and glucagon receptor, exhibits reduced affinity compared to single agonists of GLP-1 and glucagon, which was tested in a rodent model of NASH (Valdecantos et al., Hepatology 2017, vol. 65, pp. 950-968). The analog, named G49 and having a length of 29 amino acids, was analyzed in microarrays and in liver regeneration after partial hepatectomy. 【0011】 International Publication No. 2014 / 091316 concerns glucagon and GLP-1 coagonists. International Publication No. 2017 / 153575 discloses further data on these coagonists, including clinical trial data for G933. The geometric mean half-life of G933 appears to be approximately 10-12 hours. 【0012】 International Publication Nos. 2014 / 056872A1 and 2018 / 100174A1 disclose exendin-4 derivatives that activate GLP-1 and glucagon receptors. In these exendin-4 derivatives, the methionine at position 14 is replaced, among other substituents, by an amino acid having an NH2 group in its side chain, which is further replaced by a nonpolar residue (e.g., a fatty acid sometimes linked to a linker). International Publication No. 2014 / 056872 reports the half-lives of several exendin-4 derivatives in mice (Example 10, Table 6). All reported values ​​are less than 4 hours. Further information on several compounds, for example from a diet-induced NASH mouse model, is provided in International Publication No. 2019 / 030268. 【0013】 International Publication Nos. 2015 / 055801 and 2015 / 055802 disclose glucagon analog peptides that exhibit enhanced selectivity for the GLP-1 receptor compared to human glucagon. Methods for the treatment of obesity, overweight, and diabetes based on these peptides are also disclosed. 【0014】 Simultaneous activation of GLP-1 and glucagon receptors is expected to reduce food intake and increase energy expenditure, leading to weight loss in obese or overweight patients. While glycemic control is achieved through the GLP-1 receptor agonist properties, food intake is reduced by both receptors, and energy expenditure is increased by the agonist effect on the glucagon receptor. The combination of effects on food intake and energy expenditure is expected to result in a longer-lasting negative energy balance than pure GLP-1 receptor agonists, leading to robust weight loss and improvement in NASH. It is hypothesized that the balance of GLP-1 and glucagon receptor activation is a crucial factor not only for improving NASH but also for achieving weight loss and maintenance in the presence of a favorable benefit-risk profile. 【0015】 However, for the above compounds, the half-life in vivo is short, and thus administration has to be repeated frequently, for example, once a day. Since these administrations are usually performed subcutaneously, there is a drawback due to inconvenience to the patient. Such frequent administrations cause pain and discomfort to the patient. 【0016】 No pharmacological treatment of any condition falling within the scope of NAFLD has been approved by medical authorities to date. Thus, there is a high need to identify novel, safe and effective compounds that can slow, halt or reverse the time course of progression of NAFLD (including NASH), or progression in the direction of advanced fibrosis and / or cirrhosis. 【0017】 Furthermore, there is a need for the treatment of NAFLD (including NASH) that includes administration of pharmaceuticals that need to be administered less frequently (e.g., once a week), i.e., long-acting. At the same time, the treatment should still be effective as determined, for example, by the NAFLD activity score or other relevant biomarkers (e.g., reduction of liver fat content, or change in liver enzymes). 【0018】 Compounds for use in the treatment as disclosed herein have the potential to be administered less frequently and still be effective. 【0019】 Summary of the Invention Disclosed herein is the medical use of certain long-acting GLP-1 receptor / glucagon receptor dual agonists in the prevention and / or treatment of metabolic liver diseases such as non-alcoholic fatty liver disease (NAFLD), particularly non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), liver fibrosis and / or cirrhosis associated with NAFLD. 【0020】 WO 2015 / 055801 discloses peptidic compounds (different from native glucagon) derived from glucagon that have the potential to activate both the GLP-1 and glucagon receptors. 【0021】 This study revealed that glucagon analogs of formula I, possessing dual agonist activity, may be effectively used to treat NAFLD, particularly certain liver diseases such as NASH. All compounds of formula I are 29 amino acid long, have an amidated C-terminus, share 22 amino acid identity in total, and are structurally closely related. 【0022】 Therefore, the present invention relates to a method for preventing and / or treating metabolic liver diseases, particularly NAFLD, NASH, and / or cirrhosis, using General Formula I RH-X2-QGTFTSDYSKYL-X15-X16-X17-X18-AKDFI-X24-WLE-X28-A-NH2(I) (In the formula, R is H, C 1~4 Selected from alkyl and acetyl, X2 is selected from Aib and Ac4c; X15 is selected from aspartic acid and glutamic acid; X16 is selected from glutamate and Ψ; X17 is selected from arginine and Ψ; X18 is selected from alanine and arginine; X24 is selected from glutamate and Ψ; X28 is selected from serine and Ψ. Regarding compounds (including salts) having, The compound here contains only Ψ, where Ψ is a lysine residue, and the amino group of its side chain is HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-, and HOOC-(CH2) 16 It is conjugated to a substituent selected from the group consisting of -(CO)-isoGlu-GSGSGG-. 【0023】 In more specific embodiments, the present invention is for use in methods for the prevention and / or treatment of NAFLD, NASH, and / or cirrhosis of the liver. H-H-Aib-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2 (Compound 1, SEQ ID NO: 3), H-H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 (Compound 2, SEQ ID NO: 4), H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2 (Compound 3, SEQ ID NO: 5), H-H-Aib-QGTFTSDYSKYLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 (Compound 4, SEQ ID NO: 6), H-H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 (Compound 5, SEQ ID NO: 7), H-H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2(Compound 6, SEQ ID NO: 8) Regarding compounds selected from. 【0024】 Further aspects and embodiments of the present invention will become apparent from the following disclosure. 【0025】 Detailed description of the invention As described above, the present invention relates to a method for preventing or treating metabolic liver diseases, particularly NAFLD, NASH, and / or cirrhosis, using general formula I: RH-X2-QGTFTSDYSKYL-X15-X16-X17-X18-AKDFI-X24-WLE-X28-A-NH2(I) (In the formula, R is H, C 1~4 Selected from alkyl and acetyl, X2 is selected from Aib and Ac4c; X15 is selected from aspartic acid and glutamic acid; X16 is selected from glutamate and Ψ; X17 is selected from arginine and Ψ; X18 is selected from alanine and arginine; X24 is selected from glutamate and Ψ; X28 is selected from serine and Ψ. Regarding compounds having, The compound here contains only Ψ, where Ψ is a lysine residue, and the amino group of its side chain is HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-, and HOOC-(CH2) 16 It is conjugated to a substituent selected from the group consisting of -(CO)-isoGlu-GSGSGG-. 【0026】 Unless otherwise defined herein, scientific and technical terms used herein will have meanings that are generally understood by those skilled in the art. 【0027】 Throughout this specification, the word “comprise” or its variations such as “comprises” or “comprising” is intended to include one integer or component, or a group of integers or components, as described, but not to exclude any other integer or component, or a group of integers or components. 【0028】 The singular forms of "a," "an," and "the" also include the plural form unless otherwise specified. 【0029】 The term "includes" is used to mean "includes but not limited to these." "Includes" and "includes but not limited to the following" are used as synonyms. 【0030】 The terms “patient”, “subject” and “individual” may be used synonymously and refer to a human or animal. 【0031】 The above definition of Formula I also includes the corresponding compounds that exist in a neutral state or a charged state. Compounds in a charged state exist, for example, in the form of salts, such as pharmaceutically acceptable salts, or in solutions, specifically aqueous solutions. 【0032】 The term “pharmaceutically acceptable salt” as used herein is intended to indicate a salt that is not harmful to the patient or subject to whom the salt in question is administered. It may suitably be, for example, a salt selected from among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts. Examples of basic salts include salts in which the cation is an alkali metal cation, such as a sodium ion or a potassium ion, an alkaline earth metal cation, such as a calcium ion or a magnesium ion, and a substituted ammonium ion, such as N(R 1 )(R 2 )(R 3 )(R 4 ) + of the type of ion (where R 1 , R 2 , R 3 and R 4 are each independently typically hydrogen, optionally substituted C 1~6 alkyl, or optionally substituted C 2~6 alkenyl), and salts include those selected therefrom. Examples of suitable C 1~6 alkyl groups include a methyl group, an ethyl group, a 1-propyl group, and a 2-propyl group. Potentially suitable C 2~6Examples of alkenyl groups include the ethenyl, 1-propenyl, and 2-propenyl groups. Other examples of pharmaceutically acceptable salts are listed in "Remington's Scientific Pharmacy," 17th edition, edited by Alfonso R. Gennaro, Mark Publishing Company, Easton, Pennsylvania, USA, 1985 (and more recent editions), "Encyclopedia of Pharmacological Technologies," 3rd edition, edited by James Swarbrick, Informa Healthcare USA, New York, USA, 2007, and J. Pharm. Sci. 66: 2 (1977). 【0033】 As used in the context of this invention, the term "agonist" typically refers to a substance that activates a receptor of the type in question by binding to it (i.e., acting as a ligand). 【0034】 Throughout this specification, the customary one- and three-letter codons for natural amino acids, as well as generally accepted abbreviations for other amino acids, such as Aib (α-aminoisobutyric acid) and Ac4c (1-amino-cyclobutanecarboxylic acid), are used. The term "isoGlu" refers to the gamma-glutamic acid unit. 【0035】 Unless otherwise specified, the L-isomer of the amino acid in question will also be mentioned. 【0036】 Additional abbreviations include: NAFL: Non-alcoholic fatty liver disease NAFLD: Non-alcoholic fatty liver disease NAS: NAFLD Activity Score NASH: Non-alcoholic steatohepatitis MRI-PDEF: Proton Density-Derived Fat Fractionation by Magnetic Resonance Imaging. 【0037】 In the context of the methods described herein for the treatment or other therapeutic intervention according to the present invention, the term “therapeutic effective dose” as used herein means an amount sufficient to cure, remit, alleviate or partially cessate the clinical signs of a particular disease, disorder, or condition that is the target of the treatment or other therapeutic intervention in question, for example, an established clinical endpoint or other biomarker (established or experimental), such as measured by liver biopsy. A therapeutically appropriate dose may be empirically determined by those skilled in the art based on the indication being treated or prevented and the subject to whom the therapeutically appropriate dose is administered. For example, those skilled in the art may measure one or more clinically appropriate indicators of the physiological activity described herein, such as hepatic fat content via MRI-PDEF, body weight, or NAS (NAFLD activity score). Those skilled in the art may determine a clinically appropriate dose through in vitro or in vivo measurements. Other exemplary measures include fibrosis markers (serum or plasma), weight loss, changes in the histological score of NASH or fibrosis, a decrease in hepatic fat content, and changes in liver enzymes. 【0038】 The amount appropriate to achieve any or all of these effects is defined as the therapeutically effective dose. Dosage and administration methods may be tailor-made to achieve optimal efficacy. The effective dose for a given purpose will depend, among other factors, on the severity of the disease, disorder, or condition that is the target of the particular treatment or other therapeutic intervention, the body weight and general condition of the subject in question, diet, possible concomitant medications, and other factors well known to technicians in the medical field. The determination of the appropriate dose size and dosing regimen best suited for the administration of the peptides or pharmaceutically acceptable salts thereof described in the present invention to humans may be guided by the results obtained by the present invention and confirmed in appropriately designed clinical trials. Effective doses and treatment protocols may be determined by conventional means, starting with low doses in experimental animals, then increasing the dose while monitoring the effect, and similarly systematically changing the dose regimen. When determining the optimal dose for a given subject, numerous factors may be considered by the clinician. Such considerations are well known to those skilled in the art. 【0039】 In this context, the term “treatment” and its grammatical variations (e.g., “treated,” “treating,” “to treat”) refer to an approach to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes for the purposes of this invention include, but are not limited to, symptom relief, reduction of disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, remission or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” also means an extension of survival compared to the survival expected without treatment. Thus, subjects (e.g., humans) requiring treatment may be subjects already suffering from the disease or disorder in question. The term “treatment” includes suppression or reduction of the increase in the severity of the pathological condition or symptoms (e.g., progression to cirrhosis, progression of fibrosis, or worsening of NASH, e.g., increase in NAS) compared to no treatment, and does not necessarily mean the complete cessation of the associated disease, disorder, or condition. 【0040】 In this context, the term “prevention” and its grammatical variations (e.g., “prevented,” “preventing,” “preventing”) refer to approaches aimed at interfering with or preventing the onset of a condition, disease, or disorder, or at altering the pathological state. Thus, “prevention” may refer to preventive or deterrent measures. Beneficial or desired clinical outcomes for the purposes of this invention include, but are not limited to, the prevention or slowing of the symptoms, progression, or onset of the disease, whether detectable or undetectable. Thus, subjects (e.g., humans) requiring “prevention” may be subjects who do not yet have the disease or disorder in question. Accordingly, the term “prevention” includes inhibiting or slowing the onset of the disease compared to no treatment, and does not necessarily imply permanent prevention of the disease, disorder, or condition in question. In more specific embodiments, prevention refers to the prevention of progression of liver disease (e.g., progression to cirrhosis, progression of fibrosis, or worsening of NASH, e.g., an increase in NAS). 【0041】 C may exist as an R group in the context of the compound of the present invention. 1~4 As an alkyl group, C 1~3 Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, or 2-propyl. 【0042】 Peg3 refers to the following structural units, which include ethylene glycol units: -NH(CH2)2O(CH2)2OCH2C(O)NH(CH2)2O(CH2)2OCH2C(O)-. 【0043】 All publications, patents, and published patent applications referenced in this application are incorporated herein by reference, specifically the contents of International Publication No. 2015 / 055801. In case of any conflict, this specification shall prevail, including its specific definitions. 【0044】 Each embodiment of the present invention described herein may be employed alone or in combination with one or more other embodiments of the present invention. 【0045】 All compounds described in Formula I share sequence identity in at least 22 of the 29 positions. Due to the constraint that each compound contains only one Ψ, the possible variations are further limited, so effectively there are only 4 variable positions, resulting in at least 86% sequence identity. Furthermore, they share the same amidated C-terminus, and the substituents at the lysine residue (Ψ at positions 16, 17, 24, or 28) are two distinctly different substitutes, namely HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-and HOOC-(CH2) 16 Selected from -(CO)-isoGlu-GSGSGG-. Their structures are shown here (in each case, ... indicates the attachment site of the amino acid component (lysine) of Ψ to the side chain): 【0046】 HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-, or [17-carboxyheptadecanoyl]-isoGlu-Peg3-Peg3-: [ka] 【0047】 HOOC-(CH2) 16 -(CO)-isoGlu-GSGSGG-, or [17-carboxyheptadecanoyl]-isoGlu-GSGSGG: [ka] 【0048】 Compounds 1-6 are GLP-1 and glucagon receptor agonists, as determined by their ability to stimulate intracellular cAMP formation in appropriate assays (e.g., as disclosed in International Publication No. 2015 / 055801, Example 2, p. 36, Table 1, and Examples 3 and 4, pp. 37-40, Tables 2 and 3). 【0049】 Use in treatment In a first aspect, the compounds of the present invention may provide treatment and / or prophylactic options for metabolic diseases, including, in particular, non-alcoholic steatohepatitis (NASH) with and without fibrosis / cirrhosis, as well as obesity and type 2 diabetes, as discussed below. 【0050】 Metabolic syndromes are characterized by a group of metabolic risk factors in a single individual. These include abdominal obesity (excess adipose tissue around the abdominal viscera), pro-atherosclerotic dyslipidemia (abnormal blood lipid levels, including high triglycerides, low HDL cholesterol, and / or high LDL cholesterol, which promote the development of atherosclerotic plaques in the arterial walls), elevated blood pressure (hypertension), insulin resistance, and impaired glucose tolerance, pro-thrombotic conditions (e.g., high levels of fibrinogen or plasminogen activator inhibitor-1 in the blood), pro-inflammatory conditions (e.g., elevated C-reactive protein in the blood), and non-alcoholic fatty liver disease (NAFLD, including NASH with or without fibrosis / cirrhosis). 【0051】 While we do not wish to adhere to any particular theory, the compounds of the present invention are thought to act as dual agonists on both the human glucagon receptor and the human GLP1 receptor, abbreviated herein as GLP-1 / glucagon dual agonists. Dual agonists may combine, for example, the effects of glucagon on fat metabolism and, for example, the effects of GLP-1 on blood glucose levels and food intake. Therefore, they may act to accelerate the elimination of excess adipose tissue, including the oxidation of fatty acids in the liver, induce sustainable weight loss, and improve hepatic steatosis and inflammation. Thus, the compounds of the present invention may be used to treat NAFLD by reducing hepatic fat, for example, by increasing lipid oxidation. GLP-1 / glucagon dual agonists may also act to reduce cardiovascular risk factors such as high cholesterol, high LDL cholesterol, or a low HDL / LDL cholesterol ratio. 【0052】 Therefore, the compounds of the present invention may be used as pharmaceuticals to treat NASH and subsequent fibrosis / cirrhosis, promote weight loss, treat obesity, and related diseases and health conditions, including but not limited to metabolic syndrome-related inflammation and NASH-related hepatocellular carcinoma, in subjects who require them. The compounds of the present invention may also be used to treat conditions caused by or related to impaired glycemic control, including insulin resistance, impaired glucose tolerance, prediabetes, fasting hyperglycemia, type 2 diabetes, hypertension, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, and stroke, in subjects who require them. Some of these conditions may be associated with metabolic syndromes and NASH / NAFLD. However, the effects of the compounds of the present invention on these conditions may be mediated, either entirely or partially, through or independently of their effects on body weight. 【0053】 Accordingly, the present invention provides the use of the compounds of the present invention in the treatment of the above-described conditions in individuals who require it. For example, the compounds described may be used to improve NASH and / or fibrosis, to prevent progression to cirrhosis, to reverse cirrhosis, and to promote weight loss. 【0054】 In specific embodiments, the present invention includes the use of compounds in methods for treating and / or preventing conditions in individuals requiring such treatment, such as non-alcoholic fatty liver disease, metabolic and alcoholic fatty liver disease, and / or metabolic syndromes, for example, non-alcoholic fatty liver (NAFL), non-fibrotic NASH, fibrotic NASH, NASH-associated cirrhosis, NASH-associated inflammation, overweight and obesity, prediabetes, diabetes, particularly type 2 diabetes, hypertension, atherosclerotic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, or microvascular disease. 【0055】 In another aspect, the present invention relates to a compound having general formula I (as defined above) for use in a method for preventing or treating metabolic liver disease. 【0056】 In a related aspect, the present invention relates to a pharmaceutical composition comprising a compound having general formula I (as defined above) for use in a method for preventing or treating metabolic liver disease. 【0057】 In another aspect, the present invention relates to a method for preventing or treating metabolic liver disease in a patient, comprising the step of administering a therapeutically effective dose of a compound having general formula I (as defined above) to a patient in need thereof. 【0058】 In a related aspect, the present invention relates to a method for preventing or treating metabolic liver disease in a patient, comprising the step of administering to a patient in need a pharmaceutical composition containing a therapeutically effective amount of a compound having general formula I (as defined above) or a salt thereof. 【0059】 As used herein, the term “metabolic liver disease” refers to alcohol-induced liver disease (also known as alcoholic liver disease or ALD), non-alcoholic liver disease, and combinations thereof. Metabolic liver disease encompasses a range of conditions characterized by fatty deposits in the liver, such as excessive fatty deposits in the liver, including non-alcoholic fatty liver disease (NAFLD), more specifically non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD-associated hepatic fibrosis, and NAFLD-associated cirrhosis. 【0060】 The range of ALD (Alcoholic Liver Disease) is from alcoholic fatty liver (steatosis) through alcoholic steatohepatitis (ASH) to liver fibrosis and cirrhosis. Steatohepatosis is the earliest stage of alcoholic liver disease and the most common alcohol-induced liver injury. Steatohepatosis is reversible if excessive alcohol consumption is stopped in time. ASH is defined by the presence of fatty liver, inflammatory infiltrates mainly composed of polymorphonuclear leukocytes, and hepatocellular damage. 【0061】 Non-alcoholic liver disease (NAFLD) relates to metabolic dysregulation in the absence of excessive alcohol consumption. Of particular interest in the context of this invention is non-alcoholic fatty liver disease (NAFLD). NAFLD is the presence of hepatic steatosis in the absence of other causes for secondary hepatic fat accumulation (e.g., heavy alcohol consumption). Patients with NAFLD have hepatic steatosis with or without inflammation, hepatocellular injury, or fibrosis / cirrhosis. 【0062】 In the context of this invention, NAFLD is further classified into non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD-associated hepatic fibrosis, and NAFLD-associated cirrhosis. Therefore, patients with NAFLD according to this invention are diagnosed with NAFL or NASH and / or NAFLD-associated hepatic fibrosis / cirrhosis. In particular, fibrosis can typically be present in individuals with NASH to varying degrees, from mild (histological fibrosis stage F1, Kleiner et al.) to cirrhosis (histological fibrosis stage F4). In NAFL, hepatic steatohepatitis is present without evidence of ballooning hepatocytes and advanced inflammation, whereas in NASH, hepatic steatohepatitis is accompanied by hepatitis that may be histologically indistinguishable from alcoholic steatohepatitis. According to Ratziu et al (Gastroenterology 2016, vol. 150, pp. 147-159) and Kleiner and Bedossa (Gastroenterology 2015, vol. 149, pp. 1305-1308), NASH is distinguished from NAFL by the presence of ballooning hepatocytes with some degree of inflammation, in addition to steatosis. Other terms used to describe NASH include pseudoalcoholic hepatitis, alcoholic hepatitis, fatty liver hepatitis, steatonecrosis, and diabetic hepatitis. 【0063】 Hepatic fibrosis arises from chronic damage to the liver, along with the accumulation of extracellular matrix proteins, which is characteristic of most types of chronic liver disease. Major causes of hepatic fibrosis in industrialized countries include chronic HCV (hepatitis C virus) infection, alcohol abuse, and non-alcoholic steatohepatitis (NASH). The accumulation of extracellular matrix proteins distorts the liver structure by forming fibrous scars, and the subsequent development of nodules of regenerating hepatocytes defines cirrhosis (Bataller and Brenner, J Clin Invest. 2005, vol. 1 15, pp. 209-218). 【0064】 Non-alcoholic steatohepatitis (NASH) is a clinical entity characterized by liver biopsy findings identical to those seen in alcoholic hepatitis; however, NASH patients do not consume the amount of alcohol known to cause liver damage. NASH patients are typically middle-aged or elderly, with asymptomatic hepatomegaly, diabetes or hyperlipidemia, overweight or obese, and present with unrelated medical problems. Analysis of liver biopsy specimens is the basis of the diagnosis; morphological findings of the liver range from mild steatosis and inflammation to cytodegeneration, fibrosis, and cirrhosis with or without Mallory vitreous humor. 【0065】 Patients with NASH may experience no specific symptoms for a long period but may progress to cirrhosis or hepatocellular carcinoma (HCC). Therefore, NAFLD-associated cirrhosis and NAFLD-associated hepatocellular carcinoma are also referred to herein as sequelae of NASH. The risk of progression from NASH to cirrhosis is particularly high in patients with advanced fibrosis. NAFLD-associated cirrhosis is a significant risk factor for further disease development to NAFLD-associated HCC. However, NAFLD-associated HCC can also develop in NASH patients without cirrhosis. 【0066】 Non-alcoholic fatty liver disease (NAFLD) is found worldwide and is the most common metabolic liver disorder in Western industrialized countries. NAFLD, central obesity, type 2 diabetes, dyslipidemia, and metabolic syndromes are common risk factors in Western industrialized countries. In the United States, studies report a NAFLD prevalence of 10–46%, and most biopsy-based studies report a NASH prevalence of 3–5%. Globally, NAFLD is reported to have a prevalence of 6–35% (median 20%) (Williams CD et al., Gastroenterology. 2011:140(1):124-31; Vernon G et al., Alimwent Pharmacol Ther. 2011:34(3):274-85; Lazo M et al., Am J Epidemiol. 2013;178(1):38-45). 【0067】 While most patients with NAFLD are asymptomatic, some patients with NASH may complain of non-liver-specific symptoms such as fatigue, malaise, and vague right upper abdominal discomfort. Patients are more likely to receive attention if elevated liver aminotransferase levels are found in clinical trials or if liver fibrosis is incidentally detected on abdominal imaging. Once fatty liver disease develops, the risk of cirrhosis increases compared to simple fatty liver. According to the publication by Bertot and Adams (Int J Mol Sci. 2016;17(5):774-85), the progression rates of patients from NAFLD to NASH, from NAFLD to NASH with fibrosis, from NASH to NAFLD-associated cirrhosis, and from NASH with fibrosis to hepatocellular carcinoma are analyzed. The risk of progression to cirrhosis is clearly increased in NASH patients, and approximately 25% of NAFLD patients may progress to NASH within a three-year period. Once NASH is established, up to 38% of patients will develop NAFLD-associated cirrhosis over time, depending on additional risk factors. 【0068】 As described above, one aspect of the present invention relates to the use of a compound of formula I or a pharmaceutical composition containing such a compound in a method for treating non-alcoholic fatty liver disease (NAFLD). More precisely, the disease conditions to be treated are non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD-associated hepatic fibrosis, or NAFLD-associated cirrhosis. 【0069】 In more specific embodiments, the compound of formula I or a pharmaceutical composition containing such a compound is intended for use in methods for the prevention or treatment of non-alcoholic steatohepatitis (NASH). NASH may or may not be accompanied by various stages of hepatic fibrosis, including cirrhosis. 【0070】 In 2005, the Pathology Committee of the NASH Clinical Research Network (CRN) developed the so-called "NAFLD Activity Score (NAS)" for use in clinical trials (Kleiner et al., Hepatology 2005, vol. 41, pp. 1313-1321). Other scoring systems may also be used to diagnose the severity of NAFLD and its components. 【0071】 NAS specifically includes features of active damage that may be reversible in the short term. NAS is defined as a simple sum of subscores for (i) steatosis, (ii) hepatic lobular inflammation, and (iii) ballooning hepatocytes. Each subscore is semi-quantitatively graded as shown in Table 1 below. 【0072】 [Table 1] 【0073】 Typically, NAFLD is defined by the presence of steatosis in more than 5% of hepatocytes, while NASH is defined in addition by the presence of ballooning hepatocytes of any degree and any amount of inflammatory infiltrates of the hepatic lobules (Bedossa et al., Hepatology 2015, vol. 56, pp. 1751-1759). 【0074】 The diagnosis of NAFLD and NASH according to the present invention is described in Table 2 below: 【0075】 [Table 2] 【0076】 Therefore, in the context of this invention, a patient is diagnosed with NAFLD if their steatosis subscore (sometimes also referred to herein as the “steatosis subscore” or simply the “steatosis score”) is greater than 1. NASH can be distinguished from NAFL or mere steatosis by the presence of ballooning hepatocytes (sometimes referred to herein as the “ballooning subscore” or simply the “ballooning subscore”) with or without some degree of inflammation (sometimes also referred to herein as the “inflammation subscore” or simply the “inflammation score”). In this context, recovery from NASH is defined as the disappearance of ballooning hepatocytes (subscore = 0), accompanied by either the disappearance of hepatic lobular inflammation or the persistence of only mild hepatic lobular inflammation (subscore = 0 or 1) (Kleiner and Bedossa, Gastroenterology 2015, vol 149, pp. 1305-1308). This definition was used for the diagnostic algorithm described in Table 2. 【0077】 To evaluate the efficacy of the compounds of the present invention, not only the NAS but also the fibrosis score is determined. The fibrosis score can be determined, for example, according to Kleiner et al. (Hepatology 2005, vol. 41, pp. 1313-1321; also referred to herein as the "Kleiner fibrosis score"), which is summarized in Table 3 below. 【0078】 [Table 3] 【0079】 The NAS determines the degree of NAFL and NASH (higher scores indicate higher disease activity), while the Kleiner fibrosis score determines the degree of fibrosis progression. If fibrosis does not progress further, only a decrease in the NAS is relevant. Therefore, a positive response to treatment is present when there is no worsening (i.e., increase) of the NAS or improvement (lower score), particularly the disappearance of ballooning hepatocytes (ballooning enlargement score = 0), without a worsening (i.e., increase) of the Kleiner fibrosis score. 【0080】 Pharmaceutical composition The present invention also extends to compositions such as pharmaceutical compositions containing the compound of Formula I for use in methods for the prevention or treatment of metabolic liver diseases, particularly for the treatment of NAFLD and / or NASH. As with all aspects of the present invention, it should be understood that references to the compound of Formula I also include references to the compound in the form of pharmaceutically acceptable salts. 【0081】 The compounds of Formula I can be formulated as pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of the present invention together with a carrier, excipient, or vehicle that is suitable for administration and is typically pharmaceutically acceptable. 【0082】 The term “pharmaceutically acceptable carrier” includes any standard pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers for use in treatment are well known in the pharmaceutical field and are described, for example, in Remington’s Pharmacological Science, 17th edition, edited by Alfonso R. Gennaro, Mark Publishing Company, Easton, Pennsylvania, USA, 1985. For example, slightly acidic or physiological pH sterile salines and phosphate-buffered salines may be used. Suitable pH buffering agents may be, for example, phosphates, citrates, acetates, tris(hydroxymethyl)aminomethane (Tris), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine, or acetates (e.g., sodium acetate), or mixtures thereof. The term further includes any carrier substances listed in the United States Pharmacopeia for use in humans. 【0083】 The pharmaceutical compositions of the present invention may be in unit dose forms. In such dosage forms, the composition is divided into unit doses containing an appropriate amount of the active ingredient or group of ingredients. The unit dose form may also be presented as a packaged formulation, the packaging containing distinctly different amounts of the formulation, e.g., packaged tablets, capsules, or powders, in vials or ampoules. The unit dose form may also be, for example, a capsule, cachet, or tablet itself, or it may be an appropriate number of packaged dosage forms of any of these. The unit dose form may also be an injectable dosage form for application, e.g., in the form of a pen-type device or auto-injector containing a liquid-phase (typically aqueous) composition. 【0084】 Subcutaneous administration is generally the most common route of application for therapeutic peptides and appears suitable for compounds of formula I. In this case, single-use (providing an amount of the compound for a single dose unit) or multi-use (providing an amount of the compound for more than one dose unit) devices can be used. Suitable devices include auto-injectors (e.g., for single use) or pens (e.g., for multi-use) that contain a cartridge along with a liquid (e.g., aqueous) formulation of the compound. 【0085】 dose Typical doses of the compound described in Formula I may range from approximately 0.0005 to approximately 5 mg / kg(body weight) / week, for example, approximately 0.001 to approximately 0.5 mg / kg(body weight) / week. The exact dose used may depend, among other factors, on the nature and severity of the disease or disorder to be treated, the sex, age, weight and general condition of the subject to be treated, any other co-existing diseases or disorders that may be present or planned to be treated, and other factors that would be known to a skilled medical professional in the art. 【0086】 Combination therapy The compound described in Formula I may be administered as part of a combination therapy with another active substance for the treatment of liver diseases, such as NAFLD, particularly NAFL, NASH, or NAFLD-associated hepatic fibrosis. In such cases, the two active substances may be administered together or separately, for example, as components in the same pharmaceutical composition or pharmaceutical preparation, or as separate preparations. 【0087】 Therefore, the peptides of the present invention are amine oxidase copper-containing 3 (AOC3) inhibitors, soluble guanylate cyclase (sGC) activators, fibroblast growth factor (FGF) 21 agonists, growth and differentiation factor (GDF) 15 agonists, HSD17B13 (17β-hydroxysteroid dehydrogenase 13) inhibitors, ketohexokinase (KHK) inhibitors, retinoic acid receptor-associated orphan receptor c (RORc) inhibitors, cyclic GMP-AMP synthase (cGAS) inhibitors, interferon gene-stimulating factor (STING) inhibitors, and acetyl-CoA carboxylase inhibitors. These compounds may be used in combination with other pharmaceutically active compounds, including but not limited to compounds selected from the group consisting of (ACC) inhibitors, farnesoid X receptor (FXR) agonists, thyroid hormone receptor (THR) β agonists, FGF19 agonists, Nod-like receptor protein (NLRP) 3 inhibitors, Klotho-β (KLB) / FGFR1c inhibitors, patatin-like phospholipase-encoding protein (PNPLA) 3 inhibitors, αVβ integrin inhibitors, leukotriene inhibitors, and sodium / glucose cotransporter (SGLT) 2 inhibitors. 【0088】 In some embodiments, the present invention relates to an apparatus for delivering the compound to a subject, comprising the compound described in Formula I or a combination as defined above, or a pharmaceutical composition of the present invention. 【0089】 Specific Embodiments Further embodiments of the present invention are described below: 【0090】 1. General formula I for use in methods for preventing or treating metabolic liver diseases RH-X2-QGTFTSDYSKYL-X15-X16-X17-X18-AKDFI-X24-WLE-X28-A-NH2(I) (In the formula, R is H, C 1~4 Selected from alkyl and acetyl, X2 is selected from Aib and Ac4c; X15 is selected from aspartic acid and glutamic acid; X16 is selected from glutamate and Ψ; X17 is selected from arginine and Ψ; X18 is selected from alanine and arginine; X24 is selected from glutamate and Ψ; X28 is selected from serine and Ψ. A compound having, The compound here contains only Ψ, where Ψ is a lysine residue, and the amino group of its side chain is HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-, and HOOC-(CH2) 16 -(CO)-isoGlu-GSGSGG- A compound that is conjugated to a substituent selected from the group consisting of the following. 【0091】 2. The compound according to Embodiment 1 for use according to Embodiment 1, wherein X2 is Aib. 【0092】 3. The compound according to Embodiment 1 for use according to Embodiment 1, wherein X2 is Ac4c. 【0093】 4. The compound according to any one of Embodiments 1 to 3 for use according to Embodiment 1, wherein X15 is aspartic acid. 【0094】 5. The compound according to any one of Embodiments 1 to 4 for use according to Embodiment 1, wherein X16 is glutamic acid. 【0095】 6. A compound according to any one of Embodiments 1 to 4, for use according to Embodiment 1, wherein X16 is Ψ. 【0096】 7. The compound according to any one of Embodiments 1 to 6 for use according to Embodiment 1, wherein X17 is arginine. 【0097】 8. A compound according to any one of Embodiments 1 to 6 for use according to Embodiment 1, wherein X17 is Ψ. 【0098】 9. The compound according to any one of Embodiments 1 to 8 for use according to Embodiment 1, wherein X18 is alanine. 【0099】 The compound according to any one of Embodiments 1 to 8 for use according to Embodiment 1, wherein 10.X18 is arginine. 【0100】 11. The compound according to any one of Embodiments 1 to 10 for use according to Embodiment 1, wherein X24 is glutamic acid. 【0101】 A compound according to any one of Embodiments 1 to 10, for use according to Embodiment 1, wherein 12.X24 is Ψ. 【0102】 The compound according to any one of Embodiments 1 to 12 for use according to Embodiment 1, wherein 13.X28 is serine. 【0103】 A compound according to any one of Embodiments 1 to 12, for use according to Embodiment 1, wherein 14.X28 is Ψ. 【0104】 15.Ψ is Lys(-Peg3-Peg3-isoGlu-(CO)-(CH2) 16 A compound according to any one of Embodiments 1 to 14, which is -COOH) for use as described in Embodiment 1. 【0105】 16.Ψ is lysine (-GSGSGG-isoGlu-(CO)-(CH2) 16 A compound according to any one of Embodiments 1 to 14, which is -COOH) for use as described in Embodiment 1. 【0106】 17. Embodiment 1, wherein the compound is H-H-Aib-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2 (compound 1). 【0107】 18. Embodiment 1, wherein the compound is H-H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 (compound 2). 【0108】 19. Embodiment 1, wherein the compound is H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2 (compound 3). 【0109】 20. Embodiment 1, in which the compound is H-H-Aib-QGTFTSDYSKYLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 (compound 4). 【0110】 21. Embodiment 1, wherein the compound is H-H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 (compound 5). 【0111】 22. Embodiment 1, wherein the compound is H-H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2 (compound 6). 【0112】 23. The compound according to any one of Embodiments 1 to 22, wherein the compound is in the form of a salt, more specifically in the form of a pharmaceutically acceptable salt. 【0113】 24. A compound according to any one of Embodiments 1 to 23 for use in methods of preventing or treating non-alcoholic fatty liver disease (NAFLD) (including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), or NAFLD-associated hepatic fibrosis and / or cirrhosis). 【0114】 25. Embodiment 24, in which the metabolic liver disease is NAFL. 【0115】 26. Embodiment 24, in which the metabolic liver disease is NASH, and possibly NASH with hepatic fibrosis (e.g., advanced hepatic fibrosis). 【0116】 27. Embodiment 24, in which the metabolic liver disease is NAFLD-associated hepatic fibrosis, for example, advanced hepatic fibrosis (moderate (F2) to severe (F3) fibrosis stage). 【0117】 28. Embodiment 24 for use in patients having at least 2 NAS scores. 【0118】 29. Embodiment 25 for use in patients having at least 2 NAS scores. 【0119】 30. Embodiment 26 for use in patients having at least 2 NAS scores. 【0120】 31. Embodiment 24 for use in patients having at least a NAS score of 3. 【0121】 32. Embodiment 25 for use in patients with at least 3 NAS scores . 【0122】 33. Embodiment 26 for use in patients having at least a NAS score of 3. 【0123】 34. Embodiment 24 for use in patients having at least a NAS score of 4. 【0124】 35. Embodiment 25 for use in patients having at least a NAS score of 4. 【0125】 36. Embodiment 26 for use in patients having at least a NAS score of 4. 【0126】 37. An embodiment according to any one of 28 to 36, wherein at least one NAS score is derived from the subscore for ballooning enlargement. 【0127】 38. An embodiment according to any one of 28, 30, 31, 33, 34, and 36, wherein at least one NAS score is derived from the subscores for ballooning swelling and inflammation, respectively. 【0128】 39. An embodiment according to any one of 28-38, wherein the NAS score is confirmed by biopsy. 【0129】 40. A compound according to any one of Embodiments 1 to 23 for use in a method for preventing the progression of NAFL, NASH, or NAFLD-associated hepatic fibrosis. 【0130】 41. Any one of embodiments 24 to 40, wherein the method includes a step of preventing the worsening of one of the NAS subscores (lipidemia, inflammation, or ballooning). 【0131】 42. Any one of embodiments 24 to 41, wherein the method includes a step of preventing deterioration of the NAS score. 【0132】 43. Any one of embodiments 24 to 42, wherein the method includes a step of improving a subscore of steatosis. 【0133】 44. Any one of embodiments 24 to 43, wherein the method includes a step of improving the subscore of balloon-like swelling. 【0134】 45. Any one of embodiments 24 to 44 for use in patients who are overweight or obese. 【0135】 46. ​​Any one of embodiments 24 to 44 for use in obese patients. 【0136】 47.27 kg / m 2 Any one of embodiments 24 to 44 for use in patients with the above BMI. 【0137】 48. Embodiment 47, in which the patient has an additional obesity-related comorbidity. 【0138】 49. Embodiment 48, in which comorbidities are selected from the group consisting of type 2 diabetes, hypertension, dyslipidemia, sleep apnea, and cardiovascular disease. 【0139】 50.30 kg / m 2 Any one of embodiments 24 to 44 for use in patients with the above BMI. 【0140】 51. A pharmaceutical composition comprising a compound according to any one of Embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in a method for preventing or treating metabolic liver disease. 【0141】 52. A pharmaceutical composition comprising a compound according to any one of Embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in a method of treating NAFLD (including NASH, NAFL, and NAFLD-associated hepatic fibrosis and / or cirrhosis). 【0142】 53. A pharmaceutical composition comprising a compound according to any one of Embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in a method of treating NAFL, NASH, or NAFLD-associated hepatic fibrosis. 【0143】 54. A pharmaceutical composition comprising a compound according to any one of Embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in a method for treating NASH. 【0144】 55. A pharmaceutical composition comprising a compound described in any one of Embodiments 1 to 23 and a pharmaceutically acceptable carrier for use as described in any one of Embodiments 28 to 39. 【0145】 56. A pharmaceutical composition comprising a compound according to any one of Embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in a method of preventing or treating NASH, optionally accompanied by hepatic fibrosis (e.g., advanced hepatic fibrosis), in patients having at least a NAS score of 4. 【0146】 57. A method for preventing or treating metabolic liver disease in a patient, comprising the step of administering a therapeutically effective amount of a compound described in any one of Embodiments 1 to 23 to a patient in need thereof. 【0147】 58. A method for treating NASH, optionally accompanied by hepatic fibrosis, in a patient, comprising the step of administering a therapeutically effective amount of a compound described in any one of Embodiments 1 to 23 to a patient in need thereof. [Brief explanation of the drawing] 【0148】 [Figure 1a] a) Reduction of hepatic steatosis / hepatic triglycerides in diet-induced obesity (DIO) mice (Example 1). b) Reduction of plasma ALT (alanine aminotransferase) / ALT in diet-induced mice. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test. Significant differences are indicated by asterisks (**, p<0.01; ***, p<0.001). [Figure 1b] a) Reduction of hepatic steatosis / hepatic triglycerides in diet-induced obesity (DIO) mice (Example 1). b) Reduction of plasma ALT (alanine aminotransferase) / ALT in diet-induced mice. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test. Significant differences are indicated by asterisks (**, p<0.01; ***, p<0.001). [Figure 2a]Efficacy of 8 weeks of treatment with compound 5 at doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg (4 times daily), and liraglutide at a dose of 400 μg / kg 4 times daily, in mice with AMLN-induced NASH compared to control animals fed the same diet and treated with a vehicle (Example 2). a) Body weight at baseline and after 55 days of treatment. b) Overall NAFLD activity score at 56 days after the end of treatment. c) Change in NAFLD activity score (pre-biopsy before treatment vs. post-biopsy after treatment). d) Change in NAFLD activity score (adjusted for vehicle). e) Components of the NAFLD activity score at the end of the study (hepatic steatohepatic dysplasia, hepatic lobular inflammation, hepatocyte ballooning enlargement). f) Change in fibrosis score. [Figure 2b] Efficacy of 8 weeks of treatment with compound 5 at doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg (4 times daily), and liraglutide at a dose of 400 μg / kg 4 times daily, in mice with AMLN-induced NASH compared to control animals fed the same diet and treated with a vehicle (Example 2). a) Body weight at baseline and after 55 days of treatment. b) Overall NAFLD activity score at 56 days after the end of treatment. c) Change in NAFLD activity score (pre-biopsy before treatment vs. post-biopsy after treatment). d) Change in NAFLD activity score (adjusted for vehicle). e) Components of the NAFLD activity score at the end of the study (hepatic steatohepatic dysplasia, hepatic lobular inflammation, hepatocyte ballooning enlargement). f) Change in fibrosis score. [Figure 2c]Efficacy of 8 weeks of treatment with compound 5 at doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg (4 times daily), and liraglutide at a dose of 400 μg / kg 4 times daily, in mice with AMLN-induced NASH compared to control animals fed the same diet and treated with a vehicle (Example 2). a) Body weight at baseline and after 55 days of treatment. b) Overall NAFLD activity score at 56 days after the end of treatment. c) Change in NAFLD activity score (pre-biopsy before treatment vs. post-biopsy after treatment). d) Change in NAFLD activity score (adjusted for vehicle). e) Components of the NAFLD activity score at the end of the study (hepatic steatohepatic dysplasia, hepatic lobular inflammation, hepatocyte ballooning enlargement). f) Change in fibrosis score. [Figure 2d] Efficacy of 8 weeks of treatment with compound 5 at doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg (4 times daily), and liraglutide at a dose of 400 μg / kg 4 times daily, in mice with AMLN-induced NASH compared to control animals fed the same diet and treated with a vehicle (Example 2). a) Body weight at baseline and after 55 days of treatment. b) Overall NAFLD activity score at 56 days after the end of treatment. c) Change in NAFLD activity score (pre-biopsy before treatment vs. post-biopsy after treatment). d) Change in NAFLD activity score (adjusted for vehicle). e) Components of the NAFLD activity score at the end of the study (hepatic steatohepatic dysplasia, hepatic lobular inflammation, hepatocyte ballooning enlargement). f) Change in fibrosis score. [Figure 2e]Efficacy of 8 weeks of treatment with compound 5 at doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg (4 times daily), and liraglutide at a dose of 400 μg / kg 4 times daily, in mice with AMLN-induced NASH compared to control animals fed the same diet and treated with a vehicle (Example 2). a) Body weight at baseline and after 55 days of treatment. b) Overall NAFLD activity score at 56 days after the end of treatment. c) Change in NAFLD activity score (pre-biopsy before treatment vs. post-biopsy after treatment). d) Change in NAFLD activity score (adjusted for vehicle). e) Components of the NAFLD activity score at the end of the study (hepatic steatohepatic dysplasia, hepatic lobular inflammation, hepatocyte ballooning enlargement). f) Change in fibrosis score. [Figure 2f] Efficacy of 8 weeks of treatment with compound 5 at doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg (4 times daily), and liraglutide at a dose of 400 μg / kg 4 times daily, in mice with AMLN-induced NASH compared to control animals fed the same diet and treated with a vehicle (Example 2). a) Body weight at baseline and after 55 days of treatment. b) Overall NAFLD activity score at 56 days after the end of treatment. c) Change in NAFLD activity score (pre-biopsy before treatment vs. post-biopsy after treatment). d) Change in NAFLD activity score (adjusted for vehicle). e) Components of the NAFLD activity score at the end of the study (hepatic steatohepatic dysplasia, hepatic lobular inflammation, hepatocyte ballooning enlargement). f) Change in fibrosis score. 【0149】 Examples Example 1: Effect on fatty liver in diet-induced obese mice method Six-to-seven-week-old male C57BL / 6J mice were obtained from Charles River (Sulzfeld, Germany). Upon arrival at the animal facility, they were given free access to Provimi Kliba (Kaiserauchst, Switzerland) pelletized diet number 3808 and tap water. After one week, the animals were fed a high-fat diet in which 45% of their metabolizable energy came from fat (ssniff EF R / M acc. D12451 (I) mod.; ssniff Spezialdiaten, Soest, Germany). After 25 weeks on the high-fat diet, the animals were randomized into groups with similar average body weights (day 0). An additional control group (lean controls) was used in the study, consisting of animals maintained on a standard low-fat diet (Provimi Kliba diet number 3808). The age of the mice at the start of compound treatment was 32-33 weeks. 【0150】 All animals received subcutaneous injections twice daily, the first at 7:00 AM and the second eight hours later at 3:00 PM. Compound 5 was administered once daily by subcutaneous injection at 7:00 AM, followed by an additional vehicle injection eight hours later. Liraglutide was administered twice daily by subcutaneous injection at 7:00 AM and 3:00 PM. Control animals (either high-fat diet or lean control) received vehicle injections (25 mM PBS) twice daily. Body weight was measured once daily before the morning administration. The first dose was administered on day 1, and the last dose on day 28. Animals were sacrificed by neck dislocation immediately after the final blood collection on day 29. Approximately 100 mg of aliquots were taken from the lateral left lobe for measurement of hepatic triglycerides. 【0151】 Plasma ALT (alanine aminotransferase) was measured using an automated clinical analyzer (Cobas Integra 400 plus; Roche Diagnostics, Mannheim, Germany). To determine hepatic triglycerides, 100 mg of tissue from the lateral left lobe was extracted using a Fastprep tube (Mp Biomedicals) with 1 ml of isopropanol. Triglycerides in the supernatant were measured using an automated clinical analyzer (Cobas Integra 400 plus; Roche Diagnostics, Mannheim, Germany). 【0152】 result Liver fat was measured on day 29, the end of the study, after a 12-hour fast (Figure 1a). The mean liver triglyceride content of animals on a high-fat diet treated with the vehicle showed hepatic steatodystrophy (203 mg per gram of liver) compared to lean controls (53 mg per gram of liver). Liver triglycerides decreased significantly by 63 mg / g and -146 mg / g after treatment with compound 5 at 10 nmol / kg and 30 nmol / kg four times daily (40 μg / kg and 120 μg / kg, respectively). Liraglutide (10 nmol / kg twice daily and 40 μg / kg twice daily) reduced liver triglycerides by -27 mg / g, which was significantly lower than compound 5 at a dose of 10 nmol / kg four times daily. Overall, compound 5 showed a greater effect on reducing liver triglycerides compared to liraglutide. 【0153】 Transaminases, primarily expressed in the liver, showed significantly elevated mean levels in the plasma of vehicles treated with a high-fat diet (404 U / L; Figure 1b) compared to lean controls (38 U / L), indicating hepatocyte damage induced by steatosis in this diet-induced obesity mouse model. ALT decreased significantly by 250 U / L and -347 U / L after treatment with compound 5 at 10 nmol / kg four times daily, respectively. Liraglutide reduced ALT by -188 U / L, which was significantly lower than compound 5 at a dose of 10 nmol / kg four times daily. The more pronounced decrease in plasma ALT with compound 5 compared to liraglutide occurred simultaneously with its greater decrease in hepatic triglycerides. 【0154】 Example 2: Efficacy in a mouse diet-induced NASH model method Compound 5 and liraglutide were studied in a NASH mouse model (diet-induced NASH mouse model) as described by Kristiansen et al. (World Journal of Hepatology 2016, vol. 8, pp. 673-684). C57BL / 6J mice were freely fed a diet (D09100301, Research Diet, USA) that was previously described as an AMRN diet (Clapper et al., Am J Physiol Gastrointest Liver Physiol 2013, vol. 305, pp. G483-G495), high in fat (40%, of which 18% is trans fat), carbohydrates (40%, of which 20% is fructose), and cholesterol (2%). The control group continued to receive a standard rodent diet (Altromin 1324, Brogaarden, Denmark). After 26 weeks of dietary feeding, liver biopsies were performed for histological evaluation of baseline NASH and fibrosis. For this purpose, mice were anesthetized by inhalation anesthesia using isoflurane (2-3%). A small midline abdominal incision was made to expose the lateral left lobe of the liver. A conical wedge of liver tissue (approximately 50 mg) was excised from the distal portion of the liver lobe and fixed with 10% neutral buffered formalin (4% formaldehyde) for histological examination. The excised surface of the liver was immediately electrocoagulated using bipolar coagulation (ERBE VIO 100 electrosurgical unit). The liver was returned to the abdominal cavity, the abdominal wall was sutured, and the skin was stapled closed. For postoperative recovery, mice received subcutaneous administration of capprofen (5 mg / kg) on ​​the day of surgery and on the first and second postoperative days. 【0155】 For histological evaluation, slides containing paraffin-embedded sections were deparaffinized in xylene and rehydrated in a series of graded ethanols. For staining with hematoxylin and eosin (H&E), slides were incubated in Meyer hematoxylin (Dako), washed with tap water, stained with eosin Y solution (Sigma-Aldrich), hydrated, mounted on a Pertex mount, dried, and then scanned. For Sirius Red staining, slides were incubated in Weigert iron hematoxylin (Sigma-Aldrich), washed with tap water, stained with picrosirius Red (Sigma-Aldrich), and washed twice with acidified water. Excess water was removed by shaking the slides, then the slides were hydrated three times with 100% ethanol, clarified in xylene, mounted on a Pertex mount, dried, and then scanned. Histological scoring was performed by pathologists who were blinded to the study. NAFLD activity scores and fibrosis scores were determined according to the clinical criteria outlined by Kleiner et al (Hepatology 2005, vol. 14, pp. 1313-1321). 【0156】 Animals were randomized to various treatment groups according to body weight and the degree of fibrosis. The treatment period was 8 weeks, during which time the animals were maintained on an AMLN diet. The compound was administered by subcutaneous injection once daily. Animals on an AMLN diet treated on a vehicle and untreated animals on a standard rodent diet were included as controls. Group sizes ranged from 10 to 14 animals. 【0157】 After the procedure, terminal liver samples were collected and analyzed for pre-biopsy findings. 【0158】 result Compound 5 (doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg once daily) and liraglutide (dosage of 400 μg / kg once daily) were studied in a diet-induced NASH model. At baseline, after 26 weeks on the AMLN diet, animal body weight was 38–40 g, significantly higher than the lean controls, but not significantly different between treatment groups. After 8 weeks of treatment, a similarly significant weight loss relative to the vehicle was achieved with all doses of compound 5 and liraglutide. Body weight differed significantly between compound 5 at 120 μg / kg (4 times daily) (32.0 g) or 160 μg / kg (4 times daily) (31.2 g) and liraglutide (33.8 g) (Figure 2a). 【0159】 After 8 weeks of treatment, the NAFLD activity score (NAS) was significantly lower in all doses of compound 5 and liraglutide than in the vehicle group (6.2). The mean rank of compound 5 at 120 μg / kg (4 times daily) (3.3) or 160 μg / kg (4 times daily) (3.2) was significantly lower than the mean rank of the liraglutide group (4.1) (Figure 2b). Treatment with all doses of compound 5 or liraglutide also resulted in a significant improvement in NAS compared to animals treated with the vehicle (Figure 2c). NAS increased by 0.9 points in the vehicle group, but decreased by 2.0 points, 2.5 points, and 2.4 points in the 80 μg / kg, 120 μg / kg, and 160 μg / kg (4 times daily) doses of compound 5, respectively. The 1.8-point reduction achieved with liraglutide was not significantly different from the reduction in NAS relative to baseline achieved with compound 5. The vehicle-adjusted change in NAS relative to baseline is shown in Figure 2d. 【0160】 The scores for the individual components of the NAS (steatosis, inflammation, and ballooning hepatocytes) are shown in Figure 2e. After 8 weeks of treatment, the steatosis score was 3 in vehicles-treated animals, while it was significantly reduced to 1.5, 1.3, and 1.2 with compound 5 at doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg (four times daily). Liraglutide significantly reduced the steatosis score to 2.1. In particular, all reductions achieved with compound 5 were statistically significant compared to the treatment effect of liraglutide. The inflammation score was 2.3 in vehicles-treated animals, and the inflammation score was moderately and not statistically significant reduced to 2 with all doses of compound 5. Liraglutide significantly reduced the inflammation score to 1.9. However, the effects of compound 5 and liraglutide on the inflammation score were not statistically different from each other. The mean rank of the ballooning cell enlargement score was 0.9 in animals treated with the vehicle. Ballooning cells were not detected at all in animals treated with any dose of compound 5 (ballooning cell enlargement score 0), and were detected only in some animals treated with liraglutide (ballooning cell enlargement score 0.1), with no statistically significant difference between compound 5 and liraglutide. 【0161】 The fibrosis score increased moderately by only 0.2 in animals treated with the vehicle, but remained unchanged with compound 5 at 80 μg / kg (four times daily). It decreased significantly by 0.1 and 0.2 with compound 5 at 120 μg / kg and 160 μg / kg (four times daily), respectively. The change in the fibrosis score relative to baseline with liraglutide was 0.1, which was not significantly different from the decrease observed with compound 5 (Figure 2f). 【0162】 The results demonstrate that representative examples of the GLP-1 receptor / glucagon receptor dual agonists of the present invention significantly reduce body weight and NAFLD in DIO-NASH mice. The improvement in NAFLD activity score is primarily driven by the improvement in hepatic steatosis. The reduction in body weight and hepatic steatosis is more pronounced than with liraglutide. 【0163】 Example 3: Estimation of pharmacokinetic parameters The pharmacokinetic parameters of the test compound were determined after intravenous administration to Han / Wistar rats. The acylated GLP-1 analog, semaglutide, was also tested for comparative purposes. 【0164】 Male Wistar rats were obtained from Charles River, Germany, and weighed approximately 180–210 g upon arrival at the laboratory. The rats were placed in standard European rat cages (Type IV) with a photoperiod of 12 hours of darkness and 12 hours of light. During the experiment, the rats were housed in standard rat cages (Type III). Both the diet Altromin 1324 (Altromin, Germany) and water were freely administered throughout the entire experimental period. The animals were housed in the laboratory for at least 4 days to ensure proper acclimatization. 【0165】 The compound was first dissolved in 0.1% aqueous ammonia to a nominal concentration of 2 mg / ml, and then diluted to the desired dose strength (10 μM) with sterile PBS containing 25 mM phosphate buffer (pH 7.4). An intravenous injection equivalent to 20 nmol / kg was administered via the lateral tail vein. 【0166】 Blood samples (200 μl) were collected from the periorbital reticular tissue into K3 EDTA tubes at 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 32, and 48 hours after administration, and centrifuged at 4°C for 5 minutes within 20 minutes of sample collection. Plasma samples (over 100 μl) were transferred to a 96-well PCR plate, immediately frozen, and kept at -20°C until analysis of the plasma concentrations of each GLP-1-glucagon compound was performed using LC-MS / MS (liquid chromatography-mass spectrometry). Individual plasma concentration-time profiles were analyzed using a non-compartmental approach with ToxKin™ version 3.2 (Unilog IT Services) to determine the resulting pharmacokinetic parameters. See Table 4. 【0167】 [Table 4] 【0168】 Example 4: Outline of a clinical trial protocol to evaluate the efficacy, safety, and tolerability of multiple subcutaneous (sc) doses of compound 5 in patients with NASH and fibrosis. Test evaluation items The primary endpoint was improvement (with / without) in histological findings of the liver based on liver biopsy from baseline after 48 weeks of treatment in patients with NASH (NAS ≥ 4, fibrosis F1-F3). 【0169】 Improvement in histological findings Improvement of NASH: A decrease of at least 2 points in the NAS is accompanied by a decrease of at least 1 point in the NAS subscore for inflammation or ballooning hepatic lobules, and This is defined as having no progression of fibrosis stages, meaning there is no worsening of fibrosis. It is defined as a composite of the two. 【0170】 The second effectiveness evaluation criterion is: -Improvement in hepatic fat content (with / without), defined as a relative reduction of at least 30% in hepatic fat content after 48 weeks of treatment compared to baseline, as assessed by magnetic resonance imaging proton density lipid fractionation (MRI-PDFF). - Absolute and relative changes in liver fat content from baseline after 48 weeks of treatment, as assessed by MRI-PDFF. -Improvement of fibrosis (with / without), defined as a reduction of at least one stage in the fibrosis stage after 48 weeks of treatment, as assessed by liver biopsy. - Absolute change from baseline in NAS after 48 weeks of treatment, as assessed by liver biopsy. These are some examples. 【0171】 Test design Multicenter, randomized, dose-range exploration, double-blind, placebo-controlled, parallel-group trial. 【0172】 Total number of randomized patients 240 patients 【0173】 Number of patients in each procedure Compound 5: 2.4 mg for 60 patients (Group 1) Compound 5: 4.8 mg for 60 patients (Group 2) Compound 5:6.0 mg in 60 patients (Group 3) 60 patients receiving a placebo (Group 4) 【0174】 diagnosis Non-alcoholic steatohepatitis (NASH) and fibrosis 【0175】 Main selection and exclusion criteria Selection criteria: - Male or female patients who are 18 years of age or older (or the legal age in countries where the legal age is higher than 18) and 80 years of age or younger at the time of consent. - Diagnosis of NASH (NAS ≥ 4, at least 1 point each in inflammation and ballooning) and fibrosis stages F1–F3, determined by biopsy performed during the screening period, or by a stable body weight defined as a self-reported weight change of less than 5% between the biopsy and randomization, if a previous biopsy is used. - Liver fat fraction ≥8% as measured by MRI-PDFF and liver stiffness >6.0kPa as measured by FibroScan® at the time of the screening visit (If a biopsy is planned during the screening period, MRI-PDFF and FibroScan® evaluations must be performed before the biopsy). - Patients are willing and able to undergo protocol-related liver biopsies, as determined by the clinical trial staff. -BMI ≥ 25 kg / m² at the first visit 2 And weight ≥ 70kg. 【0176】 Exclusion criteria: - Significant current or past alcohol consumption (defined as an average intake exceeding 210g per week for men and 140g per week for women over a period exceeding 3 months) or inability to reliably quantify alcohol consumption within the past 5 years based on the judgment of the clinical trial staff. - Ingestion of any medications in the 12 weeks prior to the first visit that have a history of liver injury, hepatic steatohepatitis, or fatty liver disease. Ingestion of any restricted medications or any medications that may interfere with the safe conduct of the study. - A history of other forms of chronic liver disease (e.g., viral hepatitis, autoimmune liver disease, primary biliary sclerosis, primary sclerosing cholangitis, Wilson's disease, hemochromatosis, α1-antitrypsin deficiency, history of liver transplantation). - Suspected, diagnosed, or history of hepatocellular carcinoma (UCC), or any recorded active or suspected malignancy, or a history of malignancy within the five years prior to screening, excluding appropriately treated basal cell carcinoma of the skin or cervical neoplasia in situ. - Diagnosis of serious or unstable diseases, including liver (other than NASH), kidney, gastrointestinal, respiratory, cardiovascular (including ischemic heart disease), endocrine, neurological, psychiatric, immunological, or hematological disorders and other conditions, which may interfere with the safety and efficacy analysis of this trial, as determined by the clinical investigator. Patients with a history of organ transplantation other than corneal transplantation, and patients with an expected life expectancy of less than two years will also be excluded. 【0177】 Products (groups) under test Injectable solutions of compound 5: 0.6 mg / mL, 1.8 mg / mL, 3.6 mg / mL, 4.8 mg / mL, and 6.0 mg / mL. Pre-filled syringe, 0.5 mL filling capacity 【0178】 dose Group 1: Starting dose of 0.3 mg, followed by dose escalation to a maintenance dose of 2.4 mg, once weekly using two pre-filled syringes. Group 2: Starting dose of 0.3 mg, followed by dose escalation to a maintenance dose of 4.8 mg, once weekly using two pre-filled syringes. Group 3: Starting dose of 0.3 mg, followed by dose escalation to a maintenance dose of 6.0 mg, once weekly using two pre-filled syringes. 【0179】 Administration method: subcutaneous, sc 【0180】 Comparison product: Group 4: Placebo Dose: Match Method of administration: subcutaneous, sc. 【0181】 Treatment period A 48-week treatment plan consisting of a dose escalation period of up to 24 weeks and a maintenance period of at least 24 weeks.

Claims

[Claim 1] A formula for preventing or treating NASH (non-alcoholic steatohepatitis) H-H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxyheptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2(Compound 5) A pharmaceutical composition containing the compound. [Claim 2] The pharmaceutical composition according to claim 1 for treating NASH accompanied by hepatic fibrosis. [Claim 3] The pharmaceutical composition according to claim 1 for treating NASH accompanied by moderate (F2) and severe (F3) hepatic fibrosis. [Claim 4] The patient has been diagnosed with having at least a NAS (NAFLD activity score) of 4. The pharmaceutical composition according to claim 1. [Claim 5] The pharmaceutical composition according to claim 4, wherein the patient has been diagnosed with hepatic fibrosis. [Claim 6] The pharmaceutical composition according to claim 4, wherein at least one NAS point each derives from the subscores for ballooning swelling and inflammation. [Claim 7] The pharmaceutical composition according to claim 1, wherein compound 5 is in the form of a pharmaceutically acceptable salt. [Claim 8] The pharmaceutical composition according to claim 1 or 2, further comprising a pharmaceutically acceptable carrier. [Claim 9] The pharmaceutical composition according to any one of claims 1 to 8, wherein the compound is used to be administered to a human once a week. [Claim 10] The pharmaceutical composition according to claim 1, wherein the compound is used in a patient diagnosed with NASH and hepatic fibrosis.

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