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

Abstract

The present invention relates to the medical use of specific long-acting glucagon analogs having dual GLP-1 / glucagon receptor agonist activity, which are used for 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), NAFLD-related liver fibrosis and / or cirrhosis.

Description

Technical Field

[0001] The present invention relates to the medical use of specific long-acting glucagon analogs with dual GLP-1 / glucagon receptor agonist activity, which are used to prevent and / or treat metabolic liver diseases, in particular non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD-related liver fibrosis and / or cirrhosis. Background Art

[0002] Fatty liver disease is a chronic condition characterized by excessive hepatic triglyceride deposition. It can be caused by a variety of reasons, with the two main forms being associated with excessive drinking or metabolic disorders in the absence of excessive alcohol intake. The latter is called non-alcoholic fatty liver disease (NAFLD). It is often associated with metabolic syndrome and its individual components (obesity, type 2 diabetes, dyslipidemia, and hypertension). The range of NAFLD ranges from isolated hepatic steatosis, also known as non-alcoholic fatty liver (NAFL), to non-alcoholic steatohepatitis (NASH), characterized by hepatic triglyceride accumulation, hepatocellular damage, and lobular inflammation, to liver fibrosis. It is worth noting that fibrosis can exist in various degrees in patients with NASH. The presence of NASH with fibrosis is a strong risk factor for developing cirrhosis and potential hepatocellular carcinoma (Hagstrom et al., Journal of Hepatology 2017, Vol. 67, pp. 1265-1273).

[0003] NAFLD is a common condition; the global incidence has been estimated to be as high as 25% (Younossi et al., Hepatology 2016, Vol. 64, pp. 73-84). The transition from the relatively benign condition of NAFL to NASH, and in particular the progression of fibrosis, is associated with an increased risk of overall mortality (see, e.g., Dulai et al., Hepatology 2017, Vol. 65, pp. 1557-1565).

[0004] NAFLD is known to be caused by various pathogenic factors, such as insulin resistance, lipotoxicity, and inflammatory response. Among them, insulin resistance is the most common cause.

[0005] Considerable effort has been made to improve insulin resistance to prevent / treat nonalcoholic fatty liver disease. For example, clinical trials of thiazolidinediones (TZDs) or metformin (an insulin sensitizer) have been conducted (see Hepatology (2003) 38: 1008-17, J Clin Invest (2001) 108: 1167-74).

[0006] However, treatment with TZD-based drugs has the disadvantages of significant weight gain and fluid retention, and therefore it has been found impossible to use such treatments for patients with heart disease. In addition to TZD-based drugs, clinical trials using GLP-1 receptor agonists such as Victoza (liraglutide) or Exenatide (Byetta) have also been conducted for NAFLD.

[0007] Liraglutide is a commercially available chemically modified analog of glucagon-like peptide-1 (GLP-1). The amino acid sequence of liraglutide is shown as SEQ ID NO: 1

[0008] HAEGTFTSDVSSYLEGQAAK((S)-4-carboxy-4-heptadecanoyl-amino-butyryl-)EF1AWLVRGRG (SEQ ID NO.: 1)

[0009] Liraglutide is a GLP-1 receptor (GLP1R) agonist. These GLP1R agonists have been shown to lower blood sugar and reduce weight. Furthermore, treatment with the GLP1R agonist liraglutide resulted in resolution of NASH in 39% of patients with overweight or obesity and biopsy-confirmed NASH, 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 synthetic analog of oxytocin, a dual agonist of the GLP-1 and glucagon receptors, has been tested in rodent models of NASH but has reduced affinity compared to the single agonists GLP-1 and glucagon (Valdecantos et al., Hepatology 2017, Vol. 65, pp. 950-968). A 29-amino acid analog, designated G49, was analyzed in microarrays and liver regeneration after partial hepatectomy.

[0011] WO 2014 / 091316 relates to co-agonists of glucagon and GLP-1. WO 2017 / 153575 discloses additional data on these co-agonists, including data from clinical trials of G933. The geometric mean half-life of G933 appears to be approximately 10 to 12 hours.

[0012] WO 2014 / 056872 A1 and WO 2018 / 100174 A1 disclose exendin-4 derivatives that activate GLP-1 and glucagon receptors. Among these exendin-4 derivatives, among other substitutions, the methionine at position 14 is replaced with an amino acid bearing an NH2 group in the side chain, which is further substituted with a non-polar residue (e.g., a fatty acid, optionally in combination with a linker). WO 2014 / 056872 reports the half-lives of several exendin-4 derivatives in mice (Example 10, Table 6). The reported values ​​were all below 4 hours. Additional information on some compounds (e.g., from a murine diet-induced NASH model) is provided in WO 2019 / 030268.

[0013] WO 2015 / 055801 and WO 2015 / 055802 disclose glucagon analog peptides that, compared to human glucagon, exhibit increased selectivity for the GLP-1 receptor. Methods for treating obesity, overweight, and diabetes based on these peptides have also been disclosed.

[0014] Simultaneous activation of GLP-1 and glucagon receptors is expected to reduce food intake and increase energy expenditure and is expected to lead to weight loss in patients with obesity or overweight. While providing glycemic control through the GLP-1 receptor agonistic properties, food intake is reduced through the receptor and energy expenditure is increased through glucagon receptor agonism. The combined effects on food intake and energy expenditure are expected to produce a more sustained negative energy balance than pure GLP-1 receptor agonists and lead to stable weight loss and improvement of NASH. It is hypothesized that the balance of GLP-1 and glucagon receptor activation is a key factor in achieving and maintaining weight loss and improving NASH, with a favorable benefit-risk profile.

[0015] However, the compounds mentioned above have short half-lives in vivo, necessitating frequent and repeated administration, such as once daily. Because these administrations are typically administered subcutaneously, they present the disadvantage of inconvenience to patients. This frequent administration causes pain and discomfort for the patient.

[0016] To date, no pharmacological treatments for the range of symptoms of NAFLD have been approved by a medical authority. Therefore, there is a great need to identify novel, safe, and effective compounds that can slow, halt, or reverse the progression of NAFLD (including NASH), or the progression toward advanced fibrosis and / or cirrhosis.

[0017] In addition, treatment for NAFLD (including NASH) is required, which involves the administration of (long-acting) medications that need to be administered at a lower frequency. Simultaneously, the treatment should remain effective, for example, as determined by NAFLD activity scores or other relevant biomarkers (e.g., reduced liver fat content or changes in liver enzymes).

[0018] Compounds used in treatments as disclosed herein may be administered at a lower frequency and still be effective. Summary of the Invention

[0019] Provided herein are specific long-acting dual GLP-1 / glucagon receptor agonists for the medical use of preventing and / or treating metabolic liver diseases, such as non-alcoholic fatty liver disease (NAFLD), in particular non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH); and NAFLD-associated liver fibrosis and / or cirrhosis.

[0020] WO 2015 / 055 801 discloses peptide compounds derived from glucagon, which (different from natural glucagon) effectively activates GLP-1 and glucagon receptor.

[0021] It has now been discovered that glucagon analogs of Formula I with dual agonist activity can be effectively used to treat specific liver diseases, such as NAFLD, particularly NASH. All compounds of Formula I are closely related structurally, having a length of 29 amino acids, an amidated C-terminus, and sharing an overall identity of 22 amino acids.

[0022] Therefore, the present invention relates to compounds (including salts) having general formula I.

[0023] RH-X2-QGTFTSDYSKYL-X15-X16-X17-X18-AKDFI-X24-WLE-X28-A-NH2 (I)

[0024] in

[0025] R is selected from H, C 1-4 Alkyl and acetyl groups;

[0026] X2 is selected from Aib and Ac4c;

[0027] X15 is selected from Asp and Glu;

[0028] X16 is selected from Glu and Ψ;

[0029] X17 is selected from Arg and Ψ;

[0030] X18 is selected from Ala and Arg;

[0031] X24 is selected from Glu and Ψ;

[0032] X28 is selected from Ser and Ψ;

[0033] Where the compound contains one and only one Ψ

[0034] and wherein the Ψ is a Lys residue, wherein the amino group of the side chain is conjugated with a substituent selected from the group consisting of

[0035] HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-, and

[0036] HOOC-(CH2) 16 -(CO)-isoGlu-GSGSGG-,

[0037] It is used for the prevention and / or treatment of metabolic liver diseases, in particular for use in a method for the treatment of NAFLD, NASH and / or cirrhosis.

[0038] In a more specific embodiment, the present invention relates to a compound selected from the group consisting of

[0039] HH-Aib-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2 (Compound 1, SEQ ID NO.: 3)

[0040] HH-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 (Compound 2, SEQ ID NO.: 4)

[0041] HH-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2 (Compound 3, SEQ ID NO.: 5)

[0042] HH-Aib-QGTFTSDYSKYLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 (Compound 4, SEQ ID NO.: 6)

[0043] HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 (Compound 5, SEQ ID NO.: 7)

[0044] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2 (Compound 6, SEQ ID NO.: 8)

[0045] It is used in a method for preventing and / or treating NAFLD, NASH and / or liver cirrhosis.

[0046] Other aspects and embodiments of the invention will become apparent from the following disclosure. DETAILED DESCRIPTION

[0047] As mentioned above, the present invention relates to a compound having the general formula I:

[0048] RH-X2-QGTFTSDYSKYL-X15-X16-X17-X18-AKDFI-X24-WLE-X28-A-NH2 (I)

[0049] in

[0050] R is selected from H, C 1-4 Alkyl and acetyl groups;

[0051] X2 is selected from Aib and Ac4c;

[0052] X15 is selected from Asp and Glu;

[0053] X16 is selected from Glu and Ψ;

[0054] X17 is selected from Arg and Ψ;

[0055] X18 is selected from Ala and Arg;

[0056] X24 is selected from Glu and Ψ;

[0057] X28 is selected from Ser and Ψ;

[0058] Where the compound contains one and only one Ψ

[0059] and wherein the Ψ is a Lys residue, wherein the amino group of the side chain is conjugated with a substituent selected from the group consisting of

[0060] HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-, and

[0061] HOOC-(CH2) 16 -(CO)-isoGlu-GSGSGG-,

[0062] It is used for preventing or treating metabolic liver diseases, in particular for use in a method for treating NAFLD, NASH and / or liver cirrhosis.

[0063] Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art.

[0064] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or component or group of integers or components but not the exclusion of any other integer or component or group of integers or components.

[0065] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0066] The term "including" is used to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0067] The terms "patient," "subject," and "individual" are used interchangeably and refer to a human animal.

[0068] The above definition of formula I includes the corresponding compounds in a neutral or charged state. The compounds in a charged state are, for example, in the form of salts, such as pharmaceutically acceptable salts, or in solution, in particular in aqueous solution.

[0069] As used herein, the term "pharmaceutically acceptable salt" is intended to mean a salt that is not harmful to the patient or subject to which it is administered. It may suitably be, for example, a salt selected from 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 selected from alkali metal cations (such as sodium or potassium ions), alkaline earth metal cations (such as calcium or magnesium ions), and substituted ammonium ions (such as N(R 1 )(R 2 )(R 3 )(R 4 ) + type ion) salt, wherein R 1 、R 2 、R 3 and R 4 Typically independently represents hydrogen, optionally substituted C 1-6 Alkyl or optionally substituted C 2-6 Alkenyl. Related C 1-6 Examples of alkyl groups include methyl, ethyl, 1-propyl and 2-propyl. 2-6Examples of alkenyl groups include ethenyl, 1-propenyl, and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and later editions thereof), and in J. Pharm. Sci. 66:2 (1977).

[0070] As used herein, the term "agonist" refers to a substance that activates the receptor type in question, typically by binding thereto (ie, acting as a ligand).

[0071] In this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted abbreviations for other amino acids, such as Aib (α-aminoisobutyric acid) and Ac4c (1-amino-cyclobutanecarboxylic acid). The term "isoGlu" refers to a γ-glutamic acid unit.

[0072] Unless otherwise indicated, reference is made to the L-isomeric form of the amino acids.

[0073] Other abbreviations include the following:

[0074] NAFL: Non-alcoholic fatty liver disease

[0075] NAFLD: Non-alcoholic fatty liver disease

[0076] NAS: NAFLD Activity Score

[0077] NASH: nonalcoholic steatohepatitis

[0078] MRI-PDFF: Magnetic resonance imaging proton density fat fraction

[0079] As used herein in the context of the treatments or other therapeutic interventions described above, the term "therapeuticly effective amount" refers to an amount sufficient to cure, improve, alleviate, or partially suppress the clinical expression of a particular disease, condition, or symptom that is the target of the treatment or other therapeutic intervention, such as by recognized clinical endpoints or other biomarkers (recognized or experimental), including liver biopsy. Therapeuticly relevant amounts can be determined empirically by those skilled in the art based on the condition being treated or prevented and the subject receiving the therapeutically relevant amount. For example, those skilled in the art can measure one or more clinically relevant indicators of the bioactivity described herein, such as liver fat content via MRI-PDFF, body weight, or NAS (NAFLD activity score). Clinically relevant amounts can be determined by those skilled in the art via in vitro or in vivo measurements. Other exemplary measurements include fibrosis markers (serum or plasma), weight loss, changes in histological scores for NASH or fibrosis, reduction in liver fat content, and changes in liver enzymes.

[0080] A therapeutically effective amount is defined as the amount sufficient to achieve any or all of these effects. Dosage and administration methods can be tailored to achieve optimal efficacy. The effective amount for a given purpose will depend in particular on the severity of the disease, condition, or symptom targeted as a particular treatment or other therapeutic intervention, the subject's weight and general condition, diet, possible concurrent medications, and other factors well known to those skilled in the art. The results obtained by this invention can guide the determination of suitable dosage sizes and administration regimens for the peptides of this invention or pharmaceutically acceptable salts thereof, which are most suitable for administration to humans and can be confirmed in appropriately designed clinical trials. Effective dosages and treatment regimens can be determined by conventional means, starting with low doses in laboratory animals and subsequently increasing the dose while monitoring effects, and similarly systematically changing the administration regimen. When determining the optimal dosage for a given subject, clinicians may consider many factors. Such considerations are well known to those skilled in the art.

[0081] As used herein, the terms "treat" and "treatment" and grammatical variations thereof (e.g., "treated," "treating," and "treat") refer to an approach for obtaining a favorable or desired clinical result. For purposes of this invention, favorable or desired clinical results include, but are not limited to, alleviation of symptoms, lessening of disease severity, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonging survival as compared to expected survival if not receiving treatment. Thus, a subject (e.g., a human) in need of treatment may be a subject already suffering from the disease or disorder. The term "treating" includes inhibiting or reducing the increase in severity of a pathological state or symptom (e.g., progression to cirrhosis, progression of fibrosis, or worsening of NASH, e.g., an increase in NAS) relative to no treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder, or condition.

[0082] As used herein, the term "prevent" and its grammatical variants (e.g., "prevented / preventing / prevent") refers to hindering or preventing the development of a condition, disease, or disorder or altering its pathological pathway. Thus, "prevention" may refer to a prophylactic or preventative measure. For the purposes of the present invention, favorable or desired clinical outcomes include, but are not limited to, preventing or slowing the symptoms, progression, or development of a disease, whether detectable or undetectable. Thus, a subject (e.g., a human) in need of "prevention" may be a subject that is not already suffering from the disease or disorder. Thus, the term "prevention" includes inhibiting or slowing the onset of a disease compared to no treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder, or condition. In a more specific aspect, prevention refers to preventing the progression of liver disease (e.g., progression to cirrhosis, progression of fibrosis, or worsening of NASH, e.g., an increase in NAS).

[0083] In the case of the compounds of the present invention, C can exist in the form of group R. 1-4 Alkyl groups include (but are not limited to) C 1-3 Alkyl groups, such as methyl, ethyl, 1-propyl, or 2-propyl.

[0084] Peg3 refers to the following structural units containing ethylene glycol units:

[0085] -NH(CH2)2O(CH2)2OCH2C(O)NH(CH2)2O(CH2)2OCH2C(O)-.

[0086] All publications, patents, and published patent applications mentioned in this application are incorporated herein by reference, in particular, the content of WO 2015 / 055 801 is incorporated by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0087] Each embodiment of the invention described herein may be taken alone or in combination with one or more other embodiments of the invention.

[0088] All compounds according to formula I share sequence identity at least 22 of the 29 positions. Under the restriction that the compounds contain only one Ψ, the possible variations are further restricted so that there are effectively only 4 variable positions, resulting in at least 86% sequence identity. In addition, they share the same amidated C-terminus and the substituents at the Lys residues (Ψ at positions 16, 17, 24 or 28) are selected from two different alternatives, namely HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3- and HOOC-(CH2) 16 -(CO)-isoGlu-GSGSGG-. Its structure is shown here (in each case, --- indicates the point of attachment to the side chain of the amino acid component of Ψ(Lys)):

[0089] HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-or

[0090] [17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3:

[0091]

[0092] HOOC-(CH2) 16 -(CO)-isoGlu-GSGSGG or

[0093] [17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG:

[0094]

[0095] Compounds 1 to 6 are GLP-1 and glucagon receptor agonists as determined by their ability to stimulate intracellular cAMP formation in an appropriate assay (e.g., as disclosed in WO2015 / 055801, Example 2, page 36, Table 1 and Examples 3 and 4, pages 37-40, Tables 2 and 3).

[0096] Therapeutic uses

[0097] In a first aspect, as discussed below, the compounds of the present invention can provide treatment and / or prevention options, particularly for non-alcoholic steatohepatitis (NASH) with and without fibrosis / cirrhosis, and for metabolic diseases including obesity and type 2 diabetes.

[0098] Metabolic syndrome is characterized by a group of metabolic risk factors in an individual. These risk factors include abdominal obesity (excess fat tissue around abdominal organs), atherogenic dyslipidemia (a blood fat disorder including high triglycerides, low HDL cholesterol, and / or high LDL cholesterol that promotes plaque buildup in artery walls), elevated blood pressure (hypertension), insulin resistance and glucose intolerance, a prothrombotic state (e.g., elevated fibrinogen or plasminogen activator inhibitor-1 in the blood), and a proinflammatory state (e.g., elevated C-reactive protein in the blood), and non-alcoholic fatty liver disease (NAFLD, including NASH with or without fibrosis / cirrhosis).

[0099] Without wishing to be bound by any particular theory, it is believed that the compounds of the present invention act as dual agonists at the human glucagon-receptor and the human GLP1-receptor, referred to herein as dual GLP-1 / glucagon agonists. Dual agonists can combine the effects of glucagon on, for example, fat metabolism with the effects of GLP-1 on, for example, blood glucose levels and food intake. They can therefore be used to accelerate the elimination of excess adipose tissue (including fatty acid oxidation in the liver), induce sustainable weight loss, and improve steatosis and inflammation in the liver. Therefore, the compounds of the present invention can be used to treat NAFLD by reducing liver fat, for example, by increasing lipid oxidation. Dual GLP-1 / glucagon agonists can also be used to reduce cardiovascular risk factors, such as high cholesterol, high LDL-cholesterol, or a low HDL / LDL cholesterol ratio.

[0100] Therefore, the compounds of the present invention can be used as agents for treating NASH and subsequent fibrosis / cirrhosis, promoting weight loss, treating obesity and related diseases and health conditions (including but not limited to inflammation associated with metabolic syndrome and NASH-related hepatocellular carcinoma), for subjects in need. The compounds of the present invention can also be used to treat conditions caused by or associated with abnormal glucose control in subjects in need, including insulin resistance, glucose intolerance, prediabetes, increased fasting glucose, type 2 diabetes, hypertension, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, and stroke. Some of these conditions may be associated with metabolic syndrome and NASH / NAFLD. However, the effects of the compounds of the present invention on these conditions may be mediated entirely or in part through an effect on body weight, or may be unrelated thereto.

[0101] Thus, the present invention provides the use of the compounds of the present invention for treating a condition as described above in a subject in need thereof. For example, the compounds described can be used to improve NASH and / or fibrosis, prevent progression of cirrhosis, reverse cirrhosis, and promote weight loss.

[0102] In a specific embodiment, the present invention includes the use of a compound in a method for treating non-alcoholic fatty liver disease, metabolic and alcoholic fatty liver disease and / or the disease cluster pathology of metabolic syndrome, for example, treating and / or preventing non-alcoholic fatty liver disease (NAFL), NASH without fibrosis, NASH with fibrosis, NASH-associated cirrhosis, NASH-associated inflammation, overweight and obesity, prediabetes, diabetes, especially type 2 diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, stroke or microvascular disease in a subject in need thereof.

[0103] In another aspect, the present invention relates to a compound of general formula I (as defined above) for use in a method for preventing or treating a metabolic liver disease.

[0104] In a related aspect, the invention relates to a pharmaceutical composition comprising a compound having general formula I (as defined above) for use in a method of preventing or treating a metabolic liver disease.

[0105] In another aspect, the present invention relates to a method of preventing or treating a metabolic liver disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound having general formula I (as defined above).

[0106] In a related aspect, the invention relates to a method of preventing or treating a metabolic liver disease in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I (as defined above) or a salt thereof.

[0107] As used herein, the term "metabolic liver disease" refers to alcohol-induced liver disease (also known as alcoholic liver disease, ALD), non-alcoholic liver disease, and combinations thereof. Metabolic liver disease encompasses a range of conditions characterized by hepatic fat deposition (e.g., excessive hepatic fat deposition), including non-alcoholic fatty liver disease (NAFLD), more particularly, non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD-associated liver fibrosis, and NAFLD-associated cirrhosis.

[0108] ALD ranges from alcoholic fatty liver disease (steatosis) through alcoholic steatohepatitis (ASH) to liver fibrosis and cirrhosis. Steatosis is the earliest stage of alcoholic liver disease and the most common alcohol-induced liver disorder. It is reversible with prompt cessation of excessive alcohol intake. ASH is defined by the presence of fatty liver, an inflammatory infiltrate primarily composed of polymorphonuclear leukocytes and damaged hepatocytes.

[0109] Non-alcoholic liver disease is associated with metabolic disorders in the absence of excessive alcohol intake. A non-alcoholic liver disease of particular interest in the context of the present invention is non-alcoholic fatty liver disease (NAFLD). NAFLD is a fatty degeneration of the liver that occurs in the absence of other causes, leading to secondary fat accumulation (e.g., heavy drinking). Patients with NAFLD have fatty degeneration of the liver with or without inflammation, liver cell damage, or fibrosis / cirrhosis.

[0110] In the context of the present invention, NAFLD is subdivided into non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD-associated liver fibrosis and NAFLD-associated liver cirrhosis. Therefore, a patient with NAFLD according to the present invention must be diagnosed with NAFL or NASH and / or NAFLD-associated liver fibrosis / cirrhosis. It is worth noting that in individuals with NASH, fibrosis can typically present in various degrees from mild (histological fibrosis stage F1, Kleiner et al.) to cirrhosis (histological fibrosis stage F4). In NAFL, there is hepatic steatosis without signs of hepatocellular ballooning and late-stage inflammation, while in NASH, hepatic steatosis is associated with liver inflammation, which is histologically indistinguishable from alcoholic steatohepatitis. According to Ratziu et al. (Gastroenterology 2016, Vol. 150, pp. 1147-1159) and Kleiner and Bedossa (Gastroenterology 2015, Vol. 149, pp. 1305-1308), NASH is distinguished from NAFL by the presence of hepatocellular ballooning with some degree of inflammation (in addition to steatosis). Other terms used to describe NASH include pseudoalcoholic hepatitis, alcoholic hepatitis, steatohepatitis, fatty necrosis, and diabetic hepatitis.

[0111] Liver fibrosis is caused by a combination of long-term damage to the liver and the accumulation of extracellular matrix proteins, and is a characteristic of most types of chronic liver disease. The main causes of liver 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 architecture by forming fibrous scars, and the subsequent development of nodules of regenerating liver cells defines cirrhosis (Bataller and Brenner, J Clin Invest. 2005, Vol. 115, pp. 209-218).

[0112] Nonalcoholic steatohepatitis is a clinical entity characterized by liver biopsy findings identical to those seen in alcoholic hepatitis; however, patients with NASH do not consume amounts of alcohol known to cause liver damage. Patients with NASH are typically middle-aged or elderly individuals with asymptomatic hepatomegaly, diabetes mellitus or hyperlipidemia, and are overweight or obese and present with unrelated medical problems. Analysis of a liver biopsy specimen is the basis for diagnosis; liver morphological findings range from mild steatosis and inflammation to cellular dysplasia, fibrosis, and cirrhosis, with or without the presence of Mallory hyaline bodies.

[0113] Patients with NASH do not have specific symptoms for a long time, but may progress to cirrhosis or hepatocellular carcinoma (HCC). Therefore, NAFLD-related cirrhosis and NAFLD-related hepatocellular carcinoma are also referred to as sequelae of NASH in this article. The risk of NASH progressing to cirrhosis is particularly high in patients with advanced fibrosis. NAFLD-related cirrhosis is an important risk factor for the disease to further develop into NAFLD-related HCC. However, NAFLD-related HCC can also occur in NASH patients without cirrhosis.

[0114] Non-alcoholic fatty liver disease (NAFLD) is found worldwide and is the most common metabolic liver disease in Western industrialized countries, with NAFLD, central obesity, type 2 diabetes, dyslipidemia, and the major risk factors for metabolic syndrome being prevalent. In the United States, studies have reported a prevalence of NAFLD of 10% to 46%, with most biopsy-based studies reporting a prevalence of NASH of 3% to 5%. Worldwide, the reported prevalence of NAFLD is 6% to 35% (median 20%) (Williams CD et al., Gastroenterology. 201 1: 140(1): 124-31; Vernon G et al., Alimwent Pharmacol Ther. 201 1: 34(3): 274-85; Lazo M et al., Am J Epidemiol. 2013; 178(1): 38-45).

[0115] Most patients with NAFLD are asymptomatic, but 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 come to attention because laboratory tests have revealed elevated liver aminotransferases or because of incidental detection of hepatic steatosis on abdominal imaging. After the development of steatohepatitis, the risk of cirrhosis increases compared to simple steatosis. According to a publication by Bertot and Adams (Int J Mol Sci. 2016; 17(5): 774-85), patients were analyzed for the progression rate of NAFLD to NASH, NAFLD to NASH with fibrosis, NASH to NAFLD-related cirrhosis, and NASH with fibrosis to hepatocellular carcinoma. There is a significantly increased risk of progression to cirrhosis in patients with NASH, and approximately 25% of patients with NAFLD may progress to NASH within a 3-year period. After the diagnosis of NASH, and depending on additional risk factors, up to 38% of patients will develop NAFLD-related cirrhosis over time.

[0116] As mentioned above, one aspect of the present invention relates to the use of a compound of Formula I or a pharmaceutical composition comprising such a compound in a method for treating non-alcoholic fatty liver disease (NAFLD). More specifically, the disease state to be treated is non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD-related liver fibrosis, or NAFLD-related cirrhosis.

[0117] In a more specific aspect, a compound of Formula I or a pharmaceutical composition comprising such a compound is used in a method for preventing or treating non-alcoholic steatohepatitis (NASH). NASH may or may not be associated with different stages of liver fibrosis, including cirrhosis.

[0118] 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 can also be used to diagnose the severity of NAFLD and its components.

[0119] NAS specifically includes features of active lesions that may be reversible in the short term. NAS is defined as the unweighted sum of the subscores for (i) steatosis, (ii) lobular inflammation, and (iii) hepatocellular ballooning. Each subscore is semiquantitatively graded as described in Table 1 below.

[0120] Table 1: Definition of NAS sub-scores

[0121]

[0122] Typically, NAFLD is defined by the presence of steatosis in >5% of hepatocytes, and NASH is defined by the additional presence of any degree of hepatocyte ballooning and any amount of lobular inflammatory infiltrate (Bedossa et al., Hepatology 2015, Vol. 56, pp. 1751-1759).

[0123] The diagnosis of NAFLD and NASH according to the present invention is described in Table 2 below:

[0124] Table 2: Diagnostic algorithm for NAFL relative to NASH according to NAS subscores

[0125]

[0126] Thus, in the context of the present invention, a patient is diagnosed as having NAFLD if at least the subscore for steatosis (sometimes also referred to herein as the "steatosis subscore" or simply the "steatosis score") is > 1. NASH can be distinguished from NAFL or simple steatosis by the presence of hepatocellular ballooning (sometimes also referred to herein as the "ballooning subscore" or simply the "ballooning score") with or without some degree of inflammation (sometimes also referred to herein as the "inflammation subscore" or simply the "inflammation score"). In the present context, NASH resolution has been defined as the disappearance of ballooning (subscore = 0) and the disappearance of lobular inflammation or the persistence of only mild lobular inflammation (subscore = 0 or 1) (Kleiner and Bedossa, Gastroenterology 2015, Vol. 149, pp. 1305-1308). This definition is used in the diagnostic algorithm described in Table 2.

[0127] To assess the efficacy of the compounds of the present invention, not only the NAS score 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"), as summarized in Table 3 below.

[0128] Table 3. Definition of Kleiner fibrosis score

[0129]

[0130] While the NAS determines the extent of NAFL and NASH (higher scores indicate greater disease activity), the Kleiner fibrosis score determines the extent of fibrosis progression. A reduction in the NAS is only meaningful if there is no further progression of fibrosis. Therefore, a positive response to treatment is present if there is no worsening (i.e., increase) or improvement (lower score) in the NAS, particularly if there is resolution of hepatocellular ballooning (ballooning score = 0) in the absence of worsening (i.e., increase) of the Kleiner fibrosis score.

[0131] Pharmaceutical composition

[0132] The present invention also extends to compositions, such as pharmaceutical compositions, comprising a compound of formula I for use in preventing or treating metabolic liver disease, in particular for use in a method of treating NAFLD and / or NASH. As with all aspects of the present invention, it will be understood that reference to a compound of formula I encompasses a reference to the compound in the form of a pharmaceutically acceptable salt.

[0133] The compounds of Formula I can be formulated into pharmaceutical compositions suitable for administration and which typically comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier, excipient or vehicle.

[0134] The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985. For example, sterile saline and phosphate-buffered saline can be used at slightly acidic or physiological pH. Suitable pH buffers can be, for example, phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine or acetate (e.g., in the form of sodium acetate) or a mixture thereof. The term further encompasses any carrier listed in the US Pharmacopeia for use in humans.

[0135] The pharmaceutical compositions of the present invention may be in unit dosage form. In such forms, the composition is divided into unit doses containing appropriate amounts of one or more active ingredients. The unit dosage form may be in the form of a packaged preparation containing discrete quantities of the preparation, such as encapsulated tablets, capsules, or powders in a vial or ampoule. The unit dosage form itself may also be in the form of, for example, a capsule, cachet, or tablet, or it may be an appropriate number of any of these packaged forms. The unit dosage form may also be provided in an injectable form in an application device, such as a pen-type device or an automatic injector containing a liquid (typically, aqueous) phase of the composition.

[0136] Subcutaneous administration is the most common route for administering therapeutic peptides and appears to be suitable for compounds of Formula I. For this purpose, single-use (providing a single dosage unit of compound) or multiple-use (providing more than one dosage unit of compound) devices can be used. Suitable devices include automatic injectors (e.g., for single use) or pen-type devices (e.g., for multiple use) containing a cartridge of a liquid (e.g., aqueous) formulation of the compound.

[0137] dose

[0138] Typical dosages of compounds according to Formula I may be in the range of about 0.0005 to about 5 mg / kg body weight / week, for example, about 0.001 to about 0.5 mg / kg body weight / week. The precise dosage used may depend, inter alia, on the nature and severity of the disease or condition being treated, the sex, age, weight and general condition of the subject being treated, possible other or concomitant diseases or conditions being or to be treated, and other factors known to practitioners in the art.

[0139] Combination therapy

[0140] Compounds according to Formula I can be administered as part of a combination therapy with another active agent to treat liver diseases, such as NAFLD, especially NAFL, NASH, or NAFLD-related liver fibrosis. In this case, the two active agents can be administered, for example, as components in the same pharmaceutical composition or formulation, or as separate formulations, either together or alone.

[0141] Therefore, the peptides of the present invention can be used in combination with another pharmaceutically active compound, including but not limited to a compound selected from the group consisting of: AOC3 inhibitors, sGC activators, FGF21 agonists, GDF15 agonists, HSD17B13 inhibitors, KHK inhibitors, RORc inhibitors, cGAS inhibitors, STING inhibitors, ACC inhibitors, FXR agonists, THRβ agonists, FGF19 agonists, NLRP3 inhibitors, KLB / FGFR1c inhibitors, PNPLA3 inhibitors, αVβ integrin inhibitors, leukotriene inhibitors and SGLT2 inhibitors.

[0142] In some embodiments, the present invention relates to a device comprising a compound according to formula I or a combination as defined above or a pharmaceutical composition of the present invention, for delivering the compound to a subject.

[0143] Specific implementation plans

[0144] Describing other embodiments of the present invention:

[0145] 1. A compound having the general formula I

[0146] RH-X2-QGTFTSDYSKYL-X15-X16-X17-X18-AKDFI-X24-WLE-X28-A-NH2(I),

[0147] in

[0148] R is selected from H, C 1-4 Alkyl and acetyl groups;

[0149] X2 is selected from Aib and Ac4c;

[0150] X15 is selected from Asp and Glu;

[0151] X16 is selected from Glu and Ψ;

[0152] X17 is selected from Arg and Ψ;

[0153] X18 is selected from Ala and Arg;

[0154] X24 is selected from Glu and Ψ;

[0155] X28 is selected from Ser and Ψ;

[0156] Where the compound contains one and only one Ψ

[0157] and wherein the Ψ is a Lys residue, wherein the amino group of the side chain is conjugated with a substituent selected from the group consisting of

[0158] HOOC-(CH2) 16 -(CO)-isoGlu-Peg3-Peg3-, and

[0159] HOOC-(CH2) 16 -(CO)-isoGlu-GSGSGG-,

[0160] It is for use in a method of preventing or treating metabolic liver diseases.

[0161] 2. The compound according to embodiment 1 for use according to embodiment 1, wherein X2 is Aib.

[0162] 3. The compound according to embodiment 1 is used according to embodiment 1, wherein X2 is Ac4c.

[0163] 4. The compound according to any one of embodiments 1 to 3 is used according to embodiment 1, wherein X15 is Asp.

[0164] 5. Compound according to any one of embodiments 1 to 4 for use according to embodiment 1, wherein X16 is Glu.

[0165] 6. The compound according to any one of embodiments 1 to 4 is used according to embodiment 1, wherein X16 is Ψ.

[0166] 7. Compounds according to any one of embodiments 1 to 6 for use according to embodiment 1, wherein X17 is Arg.

[0167] 8. Compounds according to any one of embodiments 1 to 6 for use according to embodiment 1, wherein X17 is Ψ.

[0168] 9. The compound according to any one of embodiments 1 to 8 is used according to embodiment 1, wherein X18 is Ala.

[0169] 10. The compound according to any one of embodiments 1 to 8 is used according to embodiment 1, wherein X18 is Arg.

[0170] 11. Compound according to any one of embodiments 1 to 10 for use according to embodiment 1, wherein X24 is Glu.

[0171] 12. Compounds according to any one of embodiments 1 to 10 for use according to embodiment 1, wherein X24 is Ψ.

[0172] 13. Compounds according to any one of embodiments 1 to 12 for use according to embodiment 1, wherein X28 is Ser.

[0173] 14. Compounds according to any one of embodiments 1 to 12 for use according to embodiment 1, wherein X28 is Ψ.

[0174] 15. The compound according to any one of embodiments 1 to 14 is used according to embodiment 1, wherein Ψ is Lys(-Peg3-Peg3-isoGlu-(CO)-(CH2) 16 -COOH).

[0175] 16. The compound according to any one of embodiments 1 to 14 is used according to embodiment 1, wherein Ψ is Lys(-GSGSGG-isoGlu-(CO)-(CH2) 16 -COOH).

[0176] 17. Embodiment 1, wherein the compound is

[0177] HH-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2 (Compound 1).

[0178] 18. Embodiment 1, wherein the compound is

[0179] HH-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxyl-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2(Compound 2).

[0180] 19. Embodiment 1, wherein the compound is

[0181] HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2 (Compound 3).

[0182] 20. Embodiment 1, wherein the compound is

[0183] HH-Aib-QGTFTSDYSKYLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 (Compound 4).

[0184] 21. Embodiment 1, wherein the compound is

[0185] HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 (Compound 5).

[0186] 22. Embodiment 1, wherein the compound is

[0187] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2 (Compound 6).

[0188] 23. The compound according to any one of embodiments 1 to 22, wherein the compound is in the form of a salt, more particularly in the form of a pharmaceutically acceptable salt.

[0189] 24. A compound as described in any one of embodiments 1 to 23, for use in a method of preventing or treating non-alcoholic fatty liver disease (NAFLD) (including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL) or NAFLD-related liver fibrosis and / or cirrhosis).

[0190] 25. Embodiment 24, wherein the metabolic liver disease is NAFL.

[0191] 26. Embodiment 24, wherein the metabolic liver disease is NASH, optionally NASH associated with liver fibrosis (eg, advanced liver fibrosis).

[0192] 27. Embodiment 24, wherein the metabolic liver disease is NAFLD-related liver fibrosis, such as advanced liver fibrosis (fibrosis stage moderate (F2) and severe (F3)).

[0193] 28. The method according to claim 24, for use in patients with a NAS score of at least 2.

[0194] 29. The method according to claim 25, for use in patients with a NAS score of at least 2.

[0195] 30. Embodiment 26, for use in patients with a NAS score of at least 2.

[0196] 31. Embodiment 24, for use in patients with a NAS score of at least 3.

[0197] 32. Embodiment 25, for use in patients with a NAS score of at least 3.

[0198] 33. Implementation scheme 26, which is used for patients with a NAS score of at least 3.

[0199] 34. Implementation scheme 24, which is used for patients with a NAS score of at least 4.

[0200] 35. Implementation method 25, which is used for patients with a NAS score of at least 4.

[0201] 36. Implementation scheme 26, which is used for patients with a NAS score of at least 4.

[0202] 37. According to any one of 28 to 36, wherein at least 1 point in the NAS score is caused by the balloon-like variational score.

[0203] 38. The embodiment of any one of 28, 30, 31, 33, 34, and 36, wherein at least 1 point of the NAS score is due to each of the ballooning and inflammation subscores.

[0204] 39. The embodiment according to any one of 28 to 38, wherein the NAS score is confirmed by biopsy.

[0205] 40. A compound according to any one of embodiments 1 to 23 for use in a method of preventing the progression of NAFL, NASH or NAFLD associated liver fibrosis.

[0206] 41. Any one of embodiments 24 to 40, wherein the method comprises preventing worsening of one of the NAS subscores (steatosis, inflammation, or ballooning).

[0207] 42. Any one of embodiments 24 to 41, wherein the method comprises preventing worsening of the NAS score.

[0208] 43. Any one of implementation schemes 24 to 42, wherein the method includes improving the fatty degeneration sub-score.

[0209] 44. Any one of implementation schemes 24 to 43, wherein the method includes improving the balloon-like variation sub-score.

[0210] 45. Implement any one of 24 to 44, for use in overweight or obese patients.

[0211] 46. ​​Implement any one of schemes 24 to 44, for use in obese patients.

[0212] 47. Any one of embodiments 24 to 44, which is used for BMI ≥ 27 kg / m 2 patients.

[0213] 48. Embodiment 47, wherein the patient suffers from other obesity-related comorbidities.

[0214] 49. Implementation scheme 48, wherein the comorbidity is selected from the group consisting of: type 2 diabetes, hypertension, dyslipidemia, sleep apnea and cardiovascular disease.

[0215] 50. Any one of implementation schemes 24 to 44, which is used for BMI ≥ 30 kg / m² 2 patients.

[0216] 51. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in the prevention or treatment of metabolic liver disease.

[0217] 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 liver fibrosis and / or cirrhosis).

[0218] 53. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in the treatment of NAFL, NASH, or NAFLD-related liver fibrosis.

[0219] 54. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier for use in the treatment of NASH.

[0220] 55. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier for use according to any one of embodiments 28 to 39.

[0221] 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 in a patient with a NAS score of at least 4, optionally NASH associated with liver fibrosis (e.g., advanced liver fibrosis).

[0222] 57. A method of treating or preventing metabolic liver disease in a patient in need, comprising administering to the patient a therapeutically effective amount of a compound according to any one of embodiments 1 to 23.

[0223] 58. A method of treating NASH, particularly NASH associated with liver fibrosis, in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound according to any one of embodiments 1 to 23. Sequence Listing <110> Boehringer Ingelheim International GmbH <120> Glucagon analogs as long-acting GLP-1 / glucagon receptor agonists for the treatment of fatty liver disease and steatohepatitis <130> 01-3420-PCT-1 <160> 8 <170> BiSSAP 1.3.6 <210> 1 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Liraglutide <220> <221> Site <222> 20 <223> The ε amino group has the following substituents: (S)-4-carboxy-4-hexadecanoyl-amino-butyryl- <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 Phe Leu Ala Trp Leu Val Arg Gly Arg Gly 20 25 30 <210> 2 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Formula I <220> <221> Variants <222> 2 <223> Xaa is selected from the group consisting of Aib and Ac4c <220> <221> Variants <222> 15 <223> Xaa is selected from the group consisting of Asp and Glu <220> <221> Variants <222> 16 <223> Xaa was selected from the group composed of Glu and Psi. <220> <221> Variants <222> 17 <223> Xaa was selected from the group composed of Arg and Psi. <220> <221> Variants <222> 18 <223> Xaa is selected from the group consisting of Ala and Arg <220> <221> Variants <222> twenty four <223> Xaa is selected from the group consisting of Glu and Psi <220> <221> Variants <222> 28 <223> Xaa is selected from the group composed of Ser and Psi. <400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Xaa Xaa 1 5 10 15 Xaa Xaa Ala Lys Asp Phe Ile Xaa Trp Leu Glu Xaa Ala 20 25 <210> 3 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon analogues <220> <221> Site <222> 2 <223> Aib <220> <221> Site <222> 16 <223> The epsilon amino group of Lys is substituted by: ([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)- <400> 3 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys 1 5 10 15 Arg Ala Ala Lys Asp Phe Ile Glu Trp Leu Glu Ser Ala 20 25 <210> 4 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon analogues <220> <221> Site <222> 2 <223> Aib <220> <221> Site <222> twenty four <223> The epsilon amino group of Lys is substituted by: ([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)- <400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Lys Asp Phe Ile Lys Trp Leu Glu Ser Ala 20 25 <210> 5 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon analogues <220> <221> Site <222> 2 <223> Ac4c <220> <221> Site <222> 17 <223> The epsilon amino group of Lys is substituted by: ([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)- <400> 5 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Asp Phe Ile Glu Trp Leu Glu Ser Ala 20 25 <210> 6 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon analogues <220> <221> Site <222> 2 <223> Aib <220> <221> Site <222> 16 <223> The epsilon amino group of Lys is substituted by: ([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)- <400> 6 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Lys 1 5 10 15 Arg Ala Ala Lys Asp Phe Ile Glu Trp Leu Glu Ser Ala 20 25 <210> 7 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon analogues <220> <221> Site <222> 2 <223> Ac4c <220> <221> Site <222> twenty four <223> The epsilon amino group of Lys is substituted by: ([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)- <400> 7 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Lys Asp Phe Ile Lys Trp Leu Glu Ser Ala 20 25 <210> 8 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon analogues <220> <221> Site <222> 2 <223> Ac4c <220> <221> Site <222> 28 <223> The epsilon amino group of Lys is substituted by: ([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)- <400> 8 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Lys Asp Phe Ile Glu Trp Leu Glu Lys Ala 20 25 BRIEF DESCRIPTION OF THE DRAWINGS

[0224] Figure 1: a) Hepatic steatosis / reduction of liver triglycerides in DIO mice (Example 1)

[0225] b) Reduction of plasma ALT / ALT in DIO mice

[0226] Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test. Significant differences are indicated by asterisks ( ** , p<0.01, *** (p<0.001).

[0227] Figure 2: Effects of eight weeks of treatment with 80 μg / kg, 120 μg / kg, and 160 μg / kg (qd) of compound 5 and 400 μg / kg qd of liraglutide in mice with AMLN diet-induced NASH compared to vehicle-treated control animals on the same diet. (Example 2)

[0228] a) Body weight at baseline and after 55 days of treatment

[0229] b) Global NAFLD activity score after treatment termination on day 56

[0230] c) Change in NAFLD activity score (pre-treatment biopsy vs post-treatment biopsy)

[0231] d) Change in NAFLD activity score (vehicle-corrected)

[0232] e) Components of the NAFLD activity score at the end of the study (hepatic steatosis, lobular inflammation, hepatocyte ballooning)

[0233] f) Changes in fibrosis score.

[0234] Example

[0235] Example 1: Effects on fatty liver in DIO mice

[0236] method

[0237] Male C57BL / 6J mice aged 6-7 weeks were obtained from Charles River (Sulzfeld, Germany). Upon arrival at the animal facility, animals were given pelleted diet No. 3808 from Provimi Kliba (Kaiseraugst, Switzerland) ad libitum and had free access to tap water. One week later, animals were placed on a high-fat diet in which 45% of metabolizable energy came from fat (ssniff EF R / M acc. D12451(I) mod.; ssniff GmbH, Soest, Germany). After 25 weeks of high-fat diet, animals were randomized into groups with similar mean body weights (day 0). An additional control group (lean control group) consisting of animals maintained on a standard low-fat diet (diet number 3808 from Provimi Kliba) was used in the study. Mice were 32 to 33 weeks old when compound treatment began.

[0238] All animals received subcutaneous injections twice a day, the first time at 7 AM in the morning and the second time at 3 pM in the afternoon after 8 hours. Compound 5 was administered once a day by subcutaneous injection at 7 AM, followed by vehicle injection after 8 hours. Liraglutide was administered twice a day by subcutaneous injection at 7 AM and 3 PM. Control animals (given a high-fat diet or a relatively thin control group) received vehicle injection (25 mM PBS) twice a day. Body weight was measured before administration in the morning every day. The first administration occurred on the 1st day, and the last administration occurred on the 28th day. Animals were sacrificed by cervical dislocation immediately after final blood sampling on the 29th day. Liver triglycerides were measured using approximately 100 mg aliquots from the left lateral lobe of the liver.

[0239] ALT (alanine aminotransferase) was measured in plasma using an automated clinical analyzer (Cobas Integra 400 plus; Roche Diagnostics, Mannheim, Germany). To determine liver triglycerides, 100 mg of tissue from the left lateral lobe was extracted with 1 ml of isopropanol using a Fastprep tube (Mp Biomedicals). Triglycerides in the supernatant were measured using an automated clinical analyzer (Cobas Integra 400 plus; Roche Diagnostics, Mannheim, Germany).

[0240] result

[0241] At the end of the study, liver fat was measured on day 29 after a 12-h fast ( Figure 1a). When compared to the lean control group (53 mg / g liver), the mean liver triglyceride content of the vehicle-treated animals given a high-fat diet indicated liver steatosis (203 mg / g liver). Following treatment with 10 and 30 nmol / kg qd of compound 5 (40 μg / kg and 120 μg / kg, respectively), liver triglycerides were significantly reduced by 63 mg / g and -146 mg / g. Liraglutide (10 nmol / kg bid, 40 μg / kg bid) reduced liver triglycerides by -27 mg / g, which was significantly less than the 10 nmol / kg qd dose of compound 5. Overall, compound 5 demonstrated a greater effect in lowering liver triglycerides than liraglutide.

[0242] When compared with a lean control group (38 U / l), the transaminase ALT, which is mainly expressed in the liver, showed a strongly elevated mean level (404 U / l) in the plasma of mediator-treated animals on a high-fat diet. Figure 1b ), indicating steatosis-induced hepatocellular damage in this DIO mouse model. Following treatment with 10 and 30 nmol / kg daily doses of Compound 5, ALT significantly decreased by 250 U / L and -347 U / L, respectively. Liraglutide reduced ALT by -188 U / L, significantly less than the 10 nmol / kg daily dose of Compound 5. Compound 5's more pronounced reduction in plasma ALT was accompanied by a greater reduction in liver triglycerides compared to liraglutide.

[0243] Example 2: Efficacy in a murine diet-induced NASH model

[0244] method

[0245] Compound 5 and liraglutide were studied in a mouse model of NASH (a murine diet-induced NASH model) as described in Kristiansen et al. (World Journal of Hepatology 2016, Vol. 8, pp. 673-684). C57BL / 6J mice were given a diet (D09100301, Research Diet, United States) containing high fat (40%, of which 18% was trans fat), carbohydrates (40%, of which 20% was fructose), and cholesterol (2%) ad libitum. This diet has been previously described as the AMLN diet (Clapper et al., Am J Physiol Gastrointest Liver Physiol 2013, Vol. 305, pp. G483-G495). A control group maintained a regular rodent chow diet (Altromin 1324, Brogaarden, Denmark). After 26 weeks of this diet, livers were examined for histological assessment of NASH and fibrosis at baseline. For this purpose, mice were anesthetized using isoflurane (2-3%) by inhalation anesthesia. A small abdominal incision was performed in the midline and the left lateral lobe of the liver was exposed. A tapered wedge of liver tissue (approximately 50 mg) was removed from the distal portion of the liver lobe and fixed in 10% neutral buffered formalin (4% formaldehyde) for histological preservation. The cut surface of the liver was immediately electrocoagulated using a bipolar coagulation (ERBEVIO 100 electrosurgery). The liver was returned to the abdominal cavity, the abdominal wall was sutured, and the skin was sutured with a stapler. For postoperative recovery, mice received subcutaneous carprofen (5 mg / kg) on ​​the OP day and on the 1st and 2nd day after OP.

[0246] For histological evaluation, slides with paraffin-embedded sections were dewaxed in xylene and rehydrated in a graded ethanol series. For hematoxylin and eosin (H&E) staining, slides were incubated in Mayer's hematoxylin (Dako), washed in tap water, stained in eosin Y solution (Sigma-Aldrich), hydrated, mounted with Pertex and then allowed to dry before scanning. For Sirius red staining, slides were incubated in Weigert's iron hematoxylin (Sigma-Aldrich), washed in tap water, stained in Sirius red (Sigma-Aldrich) and washed twice in acidified water. Excess water was removed by shaking the slides and then the slides were hydrated in three changes of 100% ethanol, cleared in xylene and mounted with Pertex and allowed to dry before scanning.

[0247] Histological scoring was performed by a pathologist 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).

[0248] Animals were randomly divided into treatment groups based on body weight and degree of fibrosis. The treatment period lasted eight weeks, during which time the animals remained on an AMLN diet. Compounds were administered subcutaneously once daily. Control groups included vehicle-treated animals on an AMLN diet and untreated animals on a regular rodent chow diet. Each group consisted of between 10 and 14 animals.

[0249] After treatment, terminal liver samples were collected and analyzed for pre-vitro testing.

[0250] result

[0251] Compound 5 (daily doses of 80 μg / kg, 120 μg / kg, and 160 μg / kg) and liraglutide (daily dose of 400 μg / kg) were investigated in a diet-induced NASH model. At baseline, after 26 weeks of administration of the AMLN diet, animal body weight was between 38 and 40 g, significantly higher than the lean control group, but not significantly different between treatment groups. After 8 weeks of treatment, significant weight loss was achieved relative to the mediator at all doses of compound 5 and liraglutide. Significant differences in body weight were observed between compound 5 (32.0 g) and liraglutide (33.8 g) at 120 μg / kg qd (31.2 g) and 160 μg / kg qd (31.2 g). Figure 2a ).

[0252] After 8 weeks of treatment, the NAFLD activity score (NAS) was significantly lower for all doses of compound 5 and liraglutide compared to the vehicle group (6.2). The average rank of compound 5 at 120 μg / kg qd (3.3) or 160 μg / kg qd (3.2) was significantly lower than the average rank of the liraglutide group (4.1) ( Figure 2b Treatment with all doses of Compound 5 or liraglutide also resulted in a significant increase in NAS relative to baseline compared to vehicle-treated animals ( Figure 2c ). Although NAS increased by 0.9 points in the vehicle group, it decreased by 2.0 points, 2.5 points, and 2.4 points at 80, 120, and 160 μg / kg qd of compound 5, respectively. The 1.8-point reduction with liraglutide was not statistically different from the baseline reduction in NAS achieved with compound 5. The vehicle-corrected change from baseline in NAS is shown in Figure 2d middle.

[0253] The individual components of NAS (steatosis, inflammation, and hepatocellular ballooning scores) are shown in Figure 2eAfter 8 weeks of treatment, the steatosis score in vehicle-treated animals was 3, while it was significantly reduced to 1.5, 1.3 and 1.2 at 80, 120 and 160 μg / kg qd doses of compound 5. Liraglutide significantly reduced the steatosis score to 2.1. It is worth noting that all reductions achieved with compound 5 were statistically significant relative to the efficacy of liraglutide. The inflammation score in vehicle-treated animals was 2.3, and it was moderately and not statistically significantly reduced to 2 by 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 average rank of the ballooning score in vehicle-treated animals was 0.9. Ballooning cells were not detected in animals treated with any dose of Compound 5 (ballooning score 0) and only in very few animals treated with liraglutide (ballooning score 0.1), with no statistical difference between Compound 5 and liraglutide.

[0254] The fibrosis score increased modestly by 0.2 in vehicle-treated animals, remained unchanged at 80 μg / kg qd of compound 5, and decreased significantly by 0.1 and 0.2 at 120 and 160 μg / kg qd of compound 5. The baseline change in fibrosis score with liraglutide was 0.1 and was not significantly different from the decrease seen with compound 5 ( Figure 2f ).

[0255] Results showed that a representative embodiment of the dual GLP-1 / glucagon receptor agonist of the present invention significantly reduced body weight and NAS in DIO-NASH mice. The improvement in NAFLD activity score was primarily driven by improvements in hepatic steatosis. The reductions in body weight and hepatic steatosis were more pronounced than with liraglutide.

[0256] Example 3: Estimation of pharmacokinetic parameters

[0257] Pharmacokinetic parameters of the test compounds were determined following intravenous administration to Han / Wistar rats.The acylated GLP-1 analogue semaglutide was also tested for comparative purposes.

[0258] Male Weiss rats were obtained from Charles River (Germany) and weighed approximately 180 to 210 g upon arrival at the testing facility. Rats were housed in Type IV European standard rat cages with a 12-hour dark and 12-hour light cycle. For the duration of the study, rats were housed in Type III standard rat cages. A diet of Altromin 1324 (Altromin, Germany) and water were administered ad libitum throughout the experimental period. Animals were housed in the testing facility for at least 4 days to ensure proper acclimatization.

[0259] The compounds were first dissolved in 0.1% ammonia water to a nominal concentration of 2 mg / ml and then diluted to the desired dosing strength (10 μM) in sterile PBS, pH 7.4 containing 25 mM phosphate buffer. Intravenous injections corresponding to 20 nmol / kg were performed via the lateral tail vein.

[0260] Blood samples (200 μl) were collected from the periorbital plexus at time points 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 32, and 48 hours post-dose into K3EDTA tubes and centrifuged at 4°C for 5 minutes within 20 minutes of sampling. Plasma samples (>100 μl) were transferred to 96-well PCR plates, immediately frozen, and kept at -20°C until analysis of plasma concentrations of each GLP-1-glucagon compound using LC-MS / MS. Individual plasma concentration-time profiles were analyzed using a non-compartmental model approach using ToxKin™ version 3.2 (Unilog IT Services) and the resulting pharmacokinetic parameters were determined. See Table 4.

[0261]

[0262] Table 4.

[0263] Example 4: Evaluation of various subcutaneous (sc) doses of Compound 5 in patients with NASH and fibrosis Clinical trial protocol outline for the efficacy, safety, and tolerability of

[0264] Test indicators

[0265] The primary endpoint was improvement in liver histological findings from baseline (yes / no) based on liver biopsy after 48 weeks of treatment in patients with NASH (NAS ≥ 4, fibrosis F1-F3).

[0266] A modified definition of histologic findings was defined as a combination of the following:

[0267] Improvement of NASH:

[0268] A decrease of at least two points in the NAS score, including a decrease of at least one point in the NAS subscore for lobular inflammation or ballooning, and

[0269] No worsening of fibrosis, defined as the absence of any increase in fibrosis stage

[0270] Secondary efficacy indicators include:

[0271] - Improvement in liver fat content (yes / no) defined as at least a 30% relative reduction in liver fat content compared to baseline after 48 weeks of treatment as assessed by magnetic resonance imaging proton density fat fraction measurement (MRI-PDFF)

[0272] - Absolute and relative changes in liver fat content from baseline after 48 weeks of treatment, as assessed by MRI-PDFF

[0273] - Improvement in fibrosis (yes / no) defined as a decrease in fibrosis stage by at least one stage after 48 weeks of treatment, as assessed by liver biopsy

[0274] - Absolute change from baseline in NAS after 48 weeks of treatment, as assessed by liver biopsy

[0275] Experimental design

[0276] Multicenter, randomized, dose-ranging, double-blind, placebo-controlled, parallel-group trial

[0277] Total number of randomized patients

[0278] 240 patients

[0279] Number of patients in each treatment group

[0280] 60 patients were given Compound 5: 2.4 mg (Group 1)

[0281] 60 patients were given Compound 5: 4.8 mg (Group 2)

[0282] 60 patients were given Compound 5: 6.0 mg (Group 3)

[0283] 60 patients were given placebo (Group 4)

[0284] diagnosis

[0285] Nonalcoholic steatohepatitis (NASH) and fibrosis

[0286] Main inclusion and exclusion criteria

[0287] Inclusion criteria:

[0288] - Male or female patients who are ≥18 years of age (or the legal age in their country of origin) and ≤80 years of age at the time of signing the consent form.

[0289] - A diagnosis of NASH (NAS ≥ 4 with at least 1 point each of inflammation and ballooning) and fibrosis stage F1-F3 confirmed by a biopsy performed during the screening period or by a historical biopsy performed within the last 6 months prior to randomization, and stable weight, defined as less than 5% self-reported weight change between the historical biopsy and randomization (when using a historical biopsy).

[0290] - Liver fat fraction ≥ 8% as measured by MRI-PDFF at the screening visit and The measured liver stiffness is >6.0 kPa (if a physical examination is scheduled during the screening period, an MRI-PDFF test must be performed before the physical examination). Evaluate).

[0291] - Patients willing and able to undergo a liver biopsy per protocol as judged by the investigator.

[0292] - BMI ≥25 kg / m at the first visit 2 And the body weight is ≥70kg.

[0293] Exclusion criteria:

[0294] - Current heavy drinking or a history of heavy drinking (defined as an average intake >210 g / week for men and >140 g / week for women over a continuous period exceeding three months) or drinking within the past five years that cannot be reliably quantified based on the investigator's judgment.

[0295] - Taking any medications historically associated with liver damage, hepatic steatosis, or steatohepatitis within the 12 weeks prior to Visit 1. Taking any restrictive medications or any medications that are considered to have the potential to interfere with the safe conduct of the trial.

[0296] - 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, A1At deficiency, history of liver transplantation).

[0297] - Suspected, diagnosed, or history of hepatocellular carcinoma (HCC) within 5 years prior to screening, or any documented active or suspected malignancy or history of malignancy, except for appropriately treated basal cell carcinoma of the skin or carcinoma in situ of the cervix.

[0298] - Diagnosis of severe or unstable diseases, including liver disease (except NASH), kidney disease, gastrointestinal disease, respiratory disease, cardiovascular disease (including ischemic heart disease), endocrine disease, neurological disease, psychiatric disease, immune disease or hematological disease, and other conditions that, in the investigator's clinical judgment, may interfere with the safety and efficacy analysis of this trial. Patients with a history of organ transplantation (except corneal transplantation) and patients with a life expectancy of less than 2 years were also excluded.

[0299] Test products

[0300] Compound 5 solution for injection: 0.6 mg / mL, 1.8 mg / mL, 3.6 mg / mL, 4.8 mg / mL, and 6.0 mg / mL pre-filled syringes, 0.5 mL fill volume

[0301] dose

[0302] Group 1: Starting dose of 0.3 mg, followed by dose escalation to a maintenance dose of 2.4 mg, two prefilled syringes once weekly

[0303] Group 2: Starting dose of 0.3 mg, followed by dose escalation to a maintenance dose of 4.8 mg, two prefilled syringes once weekly

[0304] Group 3: Starting dose of 0.3 mg, followed by dose escalation to a maintenance dose of 6.0 mg, two prefilled syringes once weekly

[0305] Mode of administration :Subcutaneous (sc)

[0306] Reference products : Group 4: Placebo

[0307] Dosage: Match

[0308] Mode of administration: Subcutaneous

[0309] Duration of treatment

[0310] The 48 weeks of treatment consisted of a dose-escalation period of up to 24 weeks and a maintenance period of at least 24 weeks.

Claims

1. Use of a compound in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease (NAFLD), wherein the compound is H-H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2, where Ac4c is 1-amino-cyclobutane carboxylic acid and isoGlu is a γ-glutamic acid unit.

2. The use according to claim 1, wherein the NAFLD is non-alcoholic fatty liver, non-alcoholic steatohepatitis, NAFLD-related liver fibrosis or NAFLD-related cirrhosis.

3. The use according to claim 1, wherein the NAFLD is non-alcoholic steatohepatitis.

4. The use according to claim 1, wherein the NAFLD is non-alcoholic steatohepatitis associated with liver fibrosis.

5. The use according to claim 4, wherein the patient with NAFLD is diagnosed as having a NAFLD activity score of at least 4.

6. The use according to claim 5, wherein the patient is diagnosed as having liver fibrosis.

7. The use according to claim 5 or 6, wherein at least 1 point in the NAFLD activity score is caused by ballooning degeneration and inflammation sub-item scores respectively.

8. The use according to any one of claims 1 to 6, wherein the compound is in the form of a pharmaceutically acceptable salt.

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