Treatment of liver and cardiovascular disorders

Abstract

The present invention relates to peptides and their use as medicaments, in particular for the treatment of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD) or cholestatic liver disease.

Description

Background of the Invention

[0002] Metabolic syndrome is a cluster of at least three of the following five medical conditions: central obesity, hypertension, hyperglycemia, high serum triglycerides, and low serum high-density lipoprotein (HDL).

[0003] According to the American Heart Association, metabolic syndrome occurs when a person has three or more of the following measurements:

[0004] - Abdominal obesity (waist circumference greater than 40 inches for men and greater than 35 inches for women).

[0005] - A triglyceride level of 150 milligrams per deciliter of blood (mg / dL) or higher.

[0006] -HDL cholesterol less than 40 mg / dL for men or less than 50 mg / dL for women.

[0007] - A systolic blood pressure (top number) of 130 millimeters of mercury (mm Hg) or higher, or a diastolic blood pressure (bottom number) of 85 mm Hg or higher.

[0008] -Fasting blood sugar of 100 mg / dL or higher.

[0009] Metabolic syndrome is associated with the risk of developing cardiovascular disease (CVD), type 2 diabetes, and / or fatty liver disease, such as nonalcoholic fatty liver disease (NAFLD).

[0010] NAFLD generally refers to a range of liver lipid disorders characterized by liver fat accumulation (steatosis) in people who consume little or no alcohol. NAFLD is also defined as a range of progressive liver diseases ranging from liver fat accumulation (simple steatosis) to nonalcoholic steatohepatitis (NASH).

[0011] NASH is a progressive disease of the liver characterized histologically by hepatic lipid accumulation, hepatocellular damage, and inflammation similar to alcoholic hepatitis. NASH is a critical stage in a process that may lead to advanced fibrosis (also known as "NASH-associated fibrosis"), cirrhosis, liver failure, and / or HCC (hepatocellular carcinoma). A detailed history without significant alcohol intake is crucial to confirm this diagnosis. NASH is one of the most common reasons for elevated transaminases in patients referred to a hepatologist. NASH is often associated with pathologies of energy metabolism, including obesity, dyslipidemia, diabetes, and metabolic syndrome.

[0012] NASH is the hepatic expression of metabolic syndrome.

[0013] A widespread dysregulation of hepatic cholesterol homeostasis drives progressive liver inflammation and fibrosis and has been demonstrated in NASH. This dysregulation occurs at multiple levels, including decreased excretion of cholesterol in the bile, either as cholesterol or bile acids

[13] .

[0014] Therefore, circulating cholesterol levels are thought to play an important role in metabolic syndrome, particularly in the dyslipidemia pattern in this syndrome.

[0015] The cholesterol that circulates in the human body is carried by plasma lipoproteins, which are particles of complex lipid and protein compositions that transport lipids in the blood. The two plasma lipoproteins that carry cholesterol are low-density lipoproteins ("LDL") and high-density lipoproteins ("HDL"). The LDL particles are considered to be responsible for transporting cholesterol from the liver (wherein cholesterol is synthesized or obtained from dietary sources) to the extrahepatic tissues in the body. On the other hand, the HDL particles are considered to be responsible for transporting cholesterol from the extrahepatic tissues to the liver, where cholesterol is catabolized and eliminated. This cholesterol transport from extrahepatic tissues to the liver is referred to as "reverse cholesterol transport."

[0016] The reverse cholesterol transport ("RCT") pathway is a multistep process that includes: (i) HDL-mediated efflux, the initial removal of cholesterol from various peripheral cellular pools; (ii) esterification of cholesterol by the action of phosphatidylcholine:cholesterol acyltransferase ("LCAT"), thereby preventing the re-entry of effluxed cholesterol into cells, (iii) endocytosis of HDL by hepatocytes, and (iv) excretion of cholesterol from HDL into bile either directly or after conversion to bile acids.

[0017] The RCT pathway is mediated by HDL particles. [1] describes a pathway for HDL endocytosis in the liver that involves "cell surface F1Fo-ATPase" (also known as "ecto F1Fo-ATPase") and the P2Y13 receptor that regulates HDL-cholesterol removal. [2] describes the nuclease activity of the F1-ATPase subunit present on the cell surface of hepatocytes (hereinafter referred to as "F1-ATPase activity") (hereinafter referred to as "ecto-F1-ATPase activity"), which allows ATP to be hydrolyzed to ADP, which in turn stimulates the P2Y13 receptor activity that leads to cellular uptake of HDL. References [3] and

[14] confirm the relationship between the P2Y13 receptor and reverse cholesterol transport and atherogenesis in mice.

[0018] On the cell surface of endothelial cells, F1-ATPase activity hydrolyzes extracellular ATP to ADP, which in turn stimulates P2Y1 receptor activity, leading to endothelial nitric oxide synthase (eNOS) producing nitric oxide and promoting signal transduction survival pathways

[15] [5].

[0019] In light of the above, F1-ATPase activators are a promising therapeutic class for the treatment of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD), or cholestatic liver diseases.

[0020] Apolipoprotein AI (apoA-I) is known to be an F1-ATPase activator [1]. ApoA-I binding to cell surface F1Fo-ATPase stimulates its ATPase activity (F1-ATPase activity), which produces extracellular adenosine diphosphate (ADP), a process that is blocked by F1-ATPase inhibitors called inhibitory factor 1 (IF1) [1]. ApoA-I and its mimetics have been shown to have excellent potential for clinical use in the treatment of metabolic syndromes, such as cardiovascular disease (CVD) [4]. However, even though clinical proof of principle has long been established, apoA-I and its mimetics have never reached drug status.

[0021] There is therefore a need to identify new F1-ATPase activators that can be used as drugs, in particular for the treatment of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver diseases. SUMMARY OF THE INVENTION

[0023] The inventors have surprisingly found that peptides 10-40 of mature human IF1 (SEQ ID NO: 1) are activators of Fi-ATPase and can be used as drugs, in particular for the treatment of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver diseases.

[0024] Therefore, the present invention relates to a peptide comprising a peptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 (RGAGSIREAGGAFGKREQAEEERYFRAQSRE), wherein the peptide is an F1-ATPase activator.

[0025] The present invention also relates to a pharmaceutical composition comprising a therapeutically active amount of a peptide according to the present invention and a pharmaceutically acceptable vehicle or carrier.

[0026] The present invention also relates to a peptide according to the invention or a pharmaceutical composition according to the invention for use as a medicament, in particular for the treatment of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD) or cholestatic liver disease.

[0027] The present invention also relates to a nucleotide sequence encoding a peptide according to the invention, a vector comprising a nucleotide sequence according to the invention and a cell comprising a nucleotide sequence according to the invention. Detailed Description of the Invention

[0029] definition

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] The articles "a," "an," and "the" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0032] Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "an embodiment," "an additional embodiment," "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.

[0033] The term "peptide" refers to an amino acid sequence, i.e., a chain of amino acids linked by peptide bonds, and may include modifications such as glycosylation, acetylation, phosphorylation, amidation, N- and / or C-terminal modifications, and other modifications known in the art to increase the stability of the peptide, its in vivo half-life, and / or its cell permeability compared to a peptide without such modifications. Such modifications may be, for example, at least one covalent attachment of the peptide to at least one long-acting molecule, e.g., at the N- or C-terminus.

[0034] As used herein, the term "amino acid" is intended to include natural and synthetic amino acids, as well as D and L amino acids.

[0035] For the purposes of the present invention, "identity" or "homology" is calculated by comparing two aligned sequences in a comparison window. The alignment of the sequences makes it possible to determine the number of positions (nucleotides or amino acids) that are common to the two sequences in the comparison window. The number of common positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the homology percentage. The determination of sequence identity percentage can be completed manually or by using a well-known computer program. In a specific embodiment of the present invention, identity or homology corresponds to 1 to 8 substitutions, such as substitutions of 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residues. Preferably, at least one substitution is a conservative amino acid substitution. "Conservative amino acid substitution" refers to that an amino acid is replaced by another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0036] The term "long-acting molecule" means a molecule that can be attached to a peptide and increases the in vivo half-life of the peptide compared to a peptide not attached to the long-acting molecule. In particular, the in vivo half-life of the peptide attached to the long-acting molecule is at least 2 times higher, preferably at least 5 times higher, such as at least 10 times higher, such as at least 20 times higher, compared to the in vivo half-life of the peptide not attached to the long-acting molecule. According to the present invention, the long-acting molecule can be a fatty acid, albumin, polyethylene glycol (PEG) and / or the Fc portion of immunoglobulin G.

[0037] Term " fatty acid " according to the present invention refers to the carboxylic acid consisting of hydrocarbon chain and terminal carboxyl group, especially any those existing in fat and oil as ester.According to the present invention, fatty acid improves the half-life of peptide.On the one hand, fatty acid is palmitic acid.On the other hand, fatty acid includes but is not limited to pentadecanoic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, ω-carboxyl pentadecanoyl (C16-dioic acid), ω-carboxyl heptadecanoyl (C18-dioic acid) or any other saturated fatty acid.In another aspect, fatty acid includes but is not limited to linoleic acid, arachidonic acid, stearic acid, palmitoleic acid, vaccenic acid, eicosenoic acid, oleic acid or any other unsaturated fatty acid.

[0038] The term "albumin" means a plasma protein that is produced in the liver and forms the majority of all plasma proteins, such as human serum albumin (HSA, CAS No.: 70024-90-7).

[0039] The term "polyethylene glycol" or "PEG" refers to any water-soluble poly(ethylene glycol) or poly(ethylene oxide). The representation PEG will contain the structure (CH2CH2O) n -, where n is an integer from 2 to about 1000. Commonly used PEGs are end-capped PEGs, where one end of the PEG is capped with a relatively inactive group, such as an alkoxy group, and the other end is a hydroxyl group, which can be further modified with a linker. A common end-capping group is a methoxy group, and the corresponding end-capped PEG is usually expressed as mPEG. Thus, mPEG is CHO (CHCHO) n -, where n is an integer from 2 to about 1000, which is sufficient to give an indicated average molecular weight for the entire PEG portion, e.g., for mPEG Mw 2,000, n is about 44 (a number that varies with batch). The concept of PEG is often used instead of mPEG. "PEG" followed by a number (not a subscript) indicates a PEG portion with an approximate molecular weight equal to that number. Thus, "PEG 2000" is a PEG portion with a molecular weight of approximately 2000.

[0040] The term "Fc portion of immunoglobulin G" refers to a pair of antibody heavy chain domains, each domain having a C H 3 Fusion C H 2, which forms a structure of approximately 50 kDa.

[0041] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and humans.

[0042] "Pharmaceutical composition" means a composition comprising a pharmaceutically acceptable carrier. For example, a carrier can be a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. When the pharmaceutical composition is suitable for oral administration, tablets or capsules can be prepared by conventional methods using pharmaceutically acceptable carriers, such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as dry products for formulation with water or other suitable excipients (vehicles) before use. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethanol, or fractionated vegetable oils); and preservatives (e.g., methylparaben or propylparaben or sorbic acid). The preparations may also contain buffer salts, flavorings, coloring agents, and sweeteners as appropriate.

[0043] The term "vector" is used herein to refer to a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmids and non-viral compounds that promote nucleic acid transfer or delivery to cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, recombinant viral vectors, etc. Examples of non-viral vectors include but are not limited to liposomes, polyamine derivatives of DNA, etc. According to the present invention, the vector can be an expression vector.

[0044] The term "expression vector" refers to a vector comprising a polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses that incorporate polynucleotides.

[0045] An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0046] The term "administering" includes administering a peptide or composition of the present invention by any number of routes and means, including but not limited to topical, oral, buccal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ocular, pulmonary, or rectal routes, preferably intravenous.

[0047] As used herein, the terms "subject," "patient," or "individual" refer to a human or non-human mammal (e.g., a rodent (mouse, rat), feline, canine, or primate) that is or may be affected by metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD), or cholestatic liver disease. Preferably, the subject is a human, male or female.

[0048] The terms "treating" or "treatment" mean to reverse, alleviate, inhibit the progress of, or prevent the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0049] Peptides and compositions

[0050] The present invention relates to peptides comprising a peptide having at least 70%, such as at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, for example at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, for example at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. Accordingly, the present invention relates to peptides comprising a peptide having 1 to 8 substitutions, such as 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residue substitutions, compared to the amino acid sequence of SEQ ID NO: 1.

[0051] The peptides of the present invention are Fi-ATPase activators. Fi-ATPase activation can be measured as described in detail in the Examples. The Fi-ATPase can be human Fi-ATPase or non-human mammalian Fi-ATPase (e.g., rodent (mouse, rat), feline, canine, or primate). Preferably, the peptides of the present invention are human Fi-ATPase activators.

[0052] In some embodiments, the peptides of the present invention are peptides having at least 70%, such as at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, such as at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, such as at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. Thus, the present invention relates to peptides having 1 to 8 substitutions, such as 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residue substitutions, compared to the amino acid sequence of SEQ ID NO: 1.

[0053] The peptide according to the present invention can be modified and / or have internal modifications at the N-terminus, C-terminus to increase the stability, efficacy and / or half-life of the peptide. The N-terminal modification can be, for example, acetylation, biotinylation, dansylation, 2,4-dinitrophenylation and / or the attachment of a long-acting molecule. The C-terminal modification can be, for example, the attachment of an amidation and / or a long-acting molecule. Internal modifications can be the attachment of cysteine ​​carbamidomethylation, amino acid replacement, amino acid replacement with aminoisobutyric acid (Aib), phosphorylation and / or a long-acting molecule. The peptide can include one or more modifications.

[0054] In one embodiment, the peptides according to the present invention have N-terminal acetylation. N-terminal acetylation removes the positive charge at the N-terminus of the peptide. This modification increases the stability of the peptide by preventing N-terminal degradation.

[0055] In another embodiment, the peptide according to the present invention has a C-terminal amidation, ie the C-terminus of the peptide is synthesized as an amide to neutralize the negative charge generated by the C-terminal COOH. This modification is added to prevent enzymatic degradation.

[0056] Thus, the present invention includes peptides having N-terminal acetylation and C-terminal amidation, ie, peptides of the present invention have the formula: CH3CO-[peptide comprising a peptide having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1]-NH2.

[0057] In a specific embodiment, the peptide of the present invention may have the formula: CH3CO-[peptide having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1]-NH2. Thus, the peptide of the present invention may have the formula: CH3CO-[peptide having 1 to 8 substitutions compared to the amino acid sequence of SEQ ID NO: 1]-NH2.

[0058] In a preferred embodiment, the peptide of the present invention has the formula:

[0059] CH3CO-[peptide comprising a peptide having the sequence of SEQ ID NO: 1]-NH2(I).

[0060] In a particularly preferred embodiment, when the peptide of the present invention is a peptide having the amino acid sequence of SEQ ID NO: 1, the peptide of the present invention has the following formula (I):

[0061] CH3CO-[SEQ ID NO: 1]-NH2 (I).

[0062] exist Figure 1 A represents formula (I).

[0063] In another embodiment, the peptide according to the present invention is modified by attaching at least one long-acting molecule to one or more amino acid residues of the amino acid sequence, wherein the long-acting molecule is selected from the group consisting of albumin, albumin-binding small molecules (such as myristic acid, naphthoylsulfonamide, diphenylcyclohexanol phosphate and 6-(4-(4-iodophenyl)butyramido)hexanoate), fatty acids, fatty diacids, Fc portion of immunoglobulin G, polyethylene glycol (PEG), natural polymers such as polysialic acid (PSA or hydroxyethyl starch (HES), recombinant PEG mimetics based on long unstructured peptides, such as homotypic amino acid polymers (HAPs) composed of Gly4Ser repeats and polypeptides XTEN. The long-acting molecule is attached to the peptide to increase the serum half-life of the peptide. Amino acids and / or linkers such as 2-aminoethoxy-2-ethoxyacetyl (AEEA) and oligoethylene glycol (OEG) linkers can be inserted between SEQD NO: 1 and the long-acting molecule to avoid steric hindrance.

[0064] In some embodiments, the peptide of the present invention is modified by attaching at least one long-acting molecule to the C-terminus of the amino acid sequence. The long-acting molecule can be attached to the C-terminus of the amino acid sequence directly or through a linker. The linker is preferably one or more amino acids, such as 1 to 10 amino acids, such as 1 to 5 amino acids, which connect the peptide to the long-acting molecule. In some embodiments, the linker is an amino acid, such as alanine (A) or lysine (K).

[0065] The attachment of PEG to a peptide is called PEGylation. Short bifunctional PEG (poly(ethylene glycol)) can be used as a spacer for bioconjugation of peptides to other molecules. PEG bioconjugation is used to improve the proteolytic stability, biodistribution, and solubility of peptides. In the prior art, the technique of PEGylation is described in detail, for example, in

[18] .

[0066] In a specific embodiment, the long-acting molecule is a fatty acid molecule, such as palmitic acid. In particular, the peptides of the present invention are modified by attaching at least one fatty acid molecule to one or more amino acid residues of the amino acid sequence, preferably the peptides are modified by attaching a fatty acid molecule, such as palmitic acid, to the C-terminus of the amino acid sequence.

[0067] Palmitic acid (also referred to as "palmitoyl" in this specification, particularly in the following formula) is a 16-carbon fatty acid having the following formula:

[0068]

[0069] Palmitic acid is conjugated to the peptides of the present invention to increase their cell permeability and to facilitate binding of the peptides to cell membranes.

[0070] Fatty acids can be attached to the peptides of the present invention by chemical cycloadditions. In one aspect, the chemical cycloadditions include copper-catalyzed alkyne-azide cycloadditions. In another aspect, cycloadditions include, but are not limited to, transition metal-catalyzed or mediated [5+1] cycloadditions, formal [3+3] cycloadditions, and ring cleavage reactions.

[0071] In a preferred embodiment, the fatty acid is attached to the C-terminus of the amino acid sequence directly or through a linker. The linker is preferably one or more amino acids, such as 1 to 10 amino acids, such as 1 to 5 amino acids, which connect the peptide to the long-acting molecule. In some embodiments, the linker is an amino acid, such as alanine (A or Ala) or lysine (K or Lys).

[0072] Thus, the present invention includes peptides having the formula: CH3CO-[peptide comprising a peptide having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1]-K-[palmitoyl]-NH2, such as the following formula: CH3CO-[peptide having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1]-K-[palmitoyl]-NH2. In a preferred embodiment, the peptide of the present invention has the formula CH3CO-[peptide comprising a peptide having the sequence of SEQ ID NO: 1]-K-[palmitoyl]-NH2, such as the peptide of formula (II):

[0073] CH3CO-[SEQ ID NO: 1]-K-[palmitoyl]-NH2(II).

[0074] exist Figure 1 B represents formula (II).

[0075] In the present specification, the term "at least 70% sequence identity" includes "at least 70%, such as at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, for example at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, for example at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity".

[0076] The present invention also relates to a pharmaceutical composition comprising a therapeutically active amount of a peptide according to the invention and a pharmaceutically acceptable vehicle or carrier.

[0077] Therapeutic uses

[0078] The present invention relates to a peptide according to the invention or a pharmaceutical composition according to the invention for use as a medicament, in particular for the treatment of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD) or cholestatic liver disease.

[0079] Non-limiting examples of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD), or cholestatic liver disease that can be treated by administering the peptides of the present invention or compositions thereof include:

[0080] (i) metabolic syndrome, which includes disorders of lipoprotein metabolism, dyslipoproteinemia, lipoprotein overproduction or deficiency, elevated total cholesterol, elevated low-density lipoprotein concentrations, elevated triglyceride concentrations, decreased high-density lipoprotein cholesterol, bile and fecal lipid elimination, bile and fecal phospholipid elimination, bile and fecal oxysterol elimination, bile and fecal bile acid elimination, and peroxisome proliferator-activated receptor-related disorders;

[0081] (ii) metabolic syndrome, which includes impaired glucose metabolism, insulin resistance, impaired glucose tolerance, impaired fasting blood glucose levels, diabetes mellitus, lipodystrophy, central obesity, peripheral lipoatrophy, diabetic nephropathy, diabetic retinopathy, nephropathy, and sepsis;

[0082] (iii) cardiovascular disease or related vascular diseases, including hypertension, coronary artery disease, myocardial infarction, stroke, arrhythmia, atrial fibrillation, valvular heart disease, heart failure, cardiomyopathy, pericarditis and impotence;

[0083] (iv) non-alcoholic fatty liver disease (NAFLD), which includes hepatic steatosis and non-alcoholic steatohepatitis (NASH);

[0084] (v) Cholestatic liver diseases, which include primary biliary cholangitis (PBC, formerly known as primary biliary cirrhosis) and primary sclerosing cholangitis (PSC).

[0085] As used herein, the term "lipoprotein metabolism disorder" refers to "dyslipidemia". Dyslipidemia includes, but is not limited to, high blood lipids and low blood levels of high-density lipoprotein (HDL) cholesterol. Therefore, the peptides or compositions thereof according to the present invention can also change the lipid metabolism of the subject, such as increasing HDL cholesterol and / or the number of HDL particles in the subject's blood, reducing LDL in the subject's blood, improving HDL metabolism, improving HDL function in the subject, reducing free triglycerides in the subject's blood and / or increasing the ratio of HDL to LDL in the subject's blood.

[0086] As used herein, the term "disturbance of glucose metabolism" or "glucose metabolism disorder" relates to abnormal glucose storage and / or utilization. To the extent that one or more glucose metabolism indicators (i.e., blood insulin, blood glucose) are abnormally high, the peptide of the present invention or its composition is administered to the subject to restore normal levels. Conversely, to the extent that one or more glucose metabolism indicators are abnormally low, the peptide of the present invention or its composition is administered to the subject to restore normal levels. Normal indicators of glucose metabolism are well known to those skilled in the art. Glucose metabolism disorders include, but are not limited to, impaired glucose tolerance; diabetic retinopathy, diabetic nephropathy, insulin resistance; cancers associated with insulin resistance, such as breast cancer, colon cancer, or prostate cancer; diabetes, including but not limited to non-insulin-dependent diabetes mellitus (NIDDM), insulin-dependent diabetes mellitus (IDDM), gestational diabetes (GDM), and maturity-onset diabetes of the young (MODY); pancreatitis; hypertension; polycystic ovary disease; and high levels of blood insulin or glucose, or both.

[0087] As used herein, the term "cardiovascular disease" or "CVD" refers to a disease of the heart or circulatory system. Cardiovascular disease may be associated with dyslipoproteinemia or dyslipidemia, or both. Cardiovascular disease includes, but is not limited to, arteriosclerosis; atherosclerosis; stroke; ischemia; perivascular disease (PVD); transient ischemic attack (TIA), fulgurant atherosclerosis; organ transplant atherosclerosis; endothelial dysfunction, particularly those affecting vascular elasticity; peripheral vascular disease; coronary heart disease; myocardial infarction; cerebral infarction and restenosis. Non-limiting examples of cardiovascular disease symptoms include angina, shortness of breath, dizziness, nausea, fatigue, arrhythmia, and impotence. In some embodiments, treatment of cardiovascular disease treats one or more symptoms of cardiovascular disease. In some embodiments, treatment of cardiovascular disease treats impotence.

[0088] In a preferred embodiment, NAFLD is nonalcoholic steatohepatitis (NASH).

[0089] The peptides of the present invention and their pharmaceutical compositions can be administered by any convenient route, for example, orally, by intravenous infusion or bolus injection, by adsorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another bioactive agent. Administration can be systemic or local. A variety of delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer the peptides of the present invention. In certain embodiments, more than one peptide of the present invention is administered to a subject. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topically, particularly to the ear, nose, eye, or skin. The mode of administration can be determined at the discretion of the practitioner and depends in part on the site of the medical condition. In most cases, administration results in the release of the compound of the present invention into the bloodstream.

[0090] Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or by infusion of a fluorocarbon or synthetic lung surfactant. In certain embodiments, the compounds of the invention can be formulated as suppositories with traditional binders and vehicles such as triglycerides.

[0091] Advantageously, the peptide or pharmaceutical composition according to the invention is administered intravenously or subcutaneously.

[0092] Depending on the mode of administration, the dosage form will be adjusted. Those skilled in the art will know how to adjust the dosage form to suit the chosen route of administration. For example, for oral administration, the dosage form can be selected from tablets, including orodispersible tablets, capsules, beverages, or syrups. For pulmonary administration, the dosage form can be in the form of a spray or inhalation product. For intravenous administration, the dosage form can be a sterile solution for injection.

[0093] The peptide or pharmaceutical composition according to the present invention can be administered in one or more doses. The dosage administered to a subject in need thereof will vary according to several factors, including but not limited to the route of administration, the disease being treated, or the age of the subject. One skilled in the art can readily determine the dosage range required based on these and other factors based on their knowledge in the art.

[0094] The amount of the peptides of the present invention that is effective in treating a particular disease disclosed herein may depend on the nature of the disease and may be determined by standard clinical techniques. In vitro or in vivo assays may be used to help determine the optimal dosage range. The precise dosage to be used in the composition may also depend on the route of administration or the severity of the condition and may be determined according to the judgment of the practitioner and the circumstances of each subject.

[0095] Other objects of the present invention

[0096] The present invention relates to a nucleotide sequence encoding a peptide comprising a peptide having at least 70%, such as at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, for example at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, for example at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ D NO: 1.

[0097] In some embodiments, the present invention relates to a nucleotide sequence encoding a peptide having at least 70%, such as at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, for example at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, for example at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0098] The nucleotide sequence can be a ribonucleic acid (RNA) sequence or a deoxyribonucleic acid (DNA) sequence, and preferably, the nucleotide sequence is a DNA sequence. The nucleotide sequence can include one or more introns to increase the stability of the corresponding RNA. The selection of introns and their positioning in the nucleotide sequence are within the capabilities of those skilled in the art. Advantageously, the nucleotide sequence of the peptide according to the present invention is optimized to improve the translation efficiency of the peptide. The optimization of the nucleotide sequence does not have any special obstacles for those skilled in the art who can easily implement the teachings of the prior art.

[0099] The present invention also relates to a vector comprising a nucleotide sequence according to the present invention. Preferably, the vector is an expression vector for expressing a peptide comprising a peptide having a sequence identity of at least 70%, such as at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, for example at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, for example at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the vector is an expression vector for expressing a peptide having at least 70%, such as at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, for example at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, for example at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0100] The present invention also relates to a cell (host cell) comprising a nucleotide sequence according to the invention or a vector according to the invention. In particular, the cell according to the invention has been transfected, infected or transformed with a nucleotide sequence and / or vector according to the invention.

[0101] Any transfection method known to those skilled in the art can be used to prepare cells according to the invention, such as lipofection or calcium phosphate cell transfection or electroporation.

[0102] For the purposes of the present invention, the term "transformation" refers to the introduction of a nucleotide sequence into a host cell so that the host cell is able to express the introduced nucleotide sequence to produce the desired peptide. In particular, the host cell according to the present invention is able to express the peptide of the present invention.

[0103] The example of host cell includes but is not limited to prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli (E. coli), Kluyveromyces (Kluyveromyces) or yeast (Saccharomyces), mammalian cell lines (such as Vero, CHO, 3T3, BHK, COS, Huh-7, HEK, etc.) and primary or established mammalian cell cultures (such as lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neurons, adipocytes, etc.). Cell lines such as SP2 / O-Agl4 (ATCC CRL1581), P3X63-Ag8.653 (ATCC CRL1580), CHO DHFR, YB2 / 0 (ATCC CRL1 662) or Huh-7 (ATCC CCL-185) can also be mentioned. It can also be a stem cell taken from a patient, such as a mesenchymal cell. Stem cells are particularly useful in gene therapy or cell therapy, whether autologous or allogeneic.

[0104] The nucleic acid sequences, vectors or cells according to the invention may be used as medicaments, eg for the treatment of the diseases disclosed above in the section "Therapeutic uses".

[0105] Treatment

[0106] The present invention relates to a method for treating metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD) or cholestatic liver disease, which comprises administering a peptide according to the present invention or a pharmaceutical composition according to the present invention to a subject.

[0107] Specific embodiments of the methods of treatment are derived from the above description.

[0108] The present invention is further defined by reference to the following examples.

[0109] Description of the drawings

[0110] Figure 1 The formula of Fmoc-Lys-[palmitoyl]-OH and formulas (I), (II), (III) and (IV) are represented.

[0111] Figure 2 Surface plasmon resonance analysis showing the interaction of the peptide of formula (I) and the peptide of formula (II) with purified human FiFo-ATPase. o Dose-dependent binding of -ATPase to the peptide of formula (I) (A) and the peptide of formula (II) (B) immobilized on a sensor chip. All sensorgrams represent RU as a function of time.

[0112] Figure 3 Figure 2 shows the effect of human IF1, peptides derived from mature human IF1 (1-60, 10-56, 10-47), peptides of formula (I), and peptides of formula (II) on the ATPase activity of human F1Fo-ATPase (F1-ATPase activity). The F1-ATPase activity assay was measured as described in Materials and Methods in the presence of IF1 (1 μM), IF1-derived peptides (1 μM each), peptides of formula (I) (1 μM), peptides of formula (II) (1 μM), or scrambled peptides (SCR and SCR-K-C16, 1 μM each). Results are expressed as a percentage of control (PBS). n = 3 independent experiments per condition. Unless otherwise indicated, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett's multiple comparison test relative to control. ***p < 0.001, ns: not statistically significant.

[0113] Figure 4 Figure 5 shows the effect of peptides of formula (I) and formula (II) on ecto-F1-ATPase activity, which was analyzed by measuring extracellular ADP content. As described in Materials and Methods, extracellular ADP concentration was measured by luciferin-luciferase assay. The contribution of ecto-F1-ATPase activity to extracellular ADP concentration was assessed using the F1-ATPase inhibitor IF1. (A) HepG2 cells were incubated with increasing concentrations of peptides of formula (I) for 5 minutes, and extracellular ADP concentration was measured. Scrambled peptide (SCR, 1 μM) and apoA-I (10 μg / mL) were used as negative and positive controls, respectively (n=3-7 per condition). (B) HepG2 cells were preincubated with or without IF1 (1 μM) for 10 minutes and then treated with apoA-I (10 μg / mL, positive control), peptides of formula (I) (1 μM), peptides of formula (II) (1 μM), or scrambled peptides (SCR and SCR-K-C16, 1 μM) for 5 minutes and extracellular ADP concentration was measured. n = 3-7 independent experiments per condition. Unless otherwise indicated, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett's multiple comparison test relative to control (PBS). *p < 0.05, **p < 0.01, ***p < 0.001, ns: not statistically significant.

[0114] Figure 5The effects of peptides of formula (I) and formula (II) on HDL endocytosis in human hepatocytes are shown. HepG2 cells were pre-incubated with apoA-I (10 μg / mL), peptides of formula (I) (1 μM), peptides of formula (II) (1 μM), or scrambled peptides (SCR and SCR-K-C16, 1 μM) in the presence or absence of IF1 (1 μM), followed by incubation with HDL-Alexa568 (50 μg / mL) for 25 minutes, and cell fluorescence content was quantified as described in Materials and Methods. n = 3-7 independent experiments per group. Unless otherwise noted, data are expressed as percentages (± SEM) above or below control values ​​(PBS) analyzed by one-way ANOVA followed by Dunnett's multiple comparison test relative to control (PBS). ***p < 0.001.

[0115] Figure 6 The effects of peptides of formula (I) and formula (II) on nitric oxide (NO) production in human endothelial cells are shown. As described in Materials and Methods, NO production in HUVEC was measured using the NO-sensitive fluorescent probe DAF-FM-DA. Scrambled peptide (SCR, 1 μM) and apoA-I (10 μg / mL) were used as negative and positive controls, respectively. (A) NO production for 10 minutes under basal conditions (PBS) or in the presence of increasing concentrations of peptides of formula (I). n = 3-6 independent experiments per group. (B) NO production for 10 minutes under basal conditions (PBS) or in the presence of peptides of formula (I) (1 μM) or peptides of formula (II) (1 μM), with or without IF1 (1 μM) pre-treatment for 10 minutes. n = 3-6 independent experiments per group. Unless otherwise indicated, data are expressed as percentage (± SEM) above or below control values ​​(PBS) analyzed by one-way ANOVA followed by Sudak's (A) or Dunnett's (B) multiple comparison test relative to control (PBS). *p<0.05, **p<0.01, ***p<0.001.

[0116] Figure 7Figures show the effects of peptides of formula (I) and (II) on oleic acid / palmitic acid-induced steatosis in human hepatocytes (HepG2 cells) and primary mouse hepatocytes. (A) HepG2 cells were cultured for 48 hours in a medium containing 1% BSA (vehicle) or oleic acid (OA, 0.33 mM) and palmitic acid (PA, 0.16 mM) (OA:PA, 2:1) to induce steatosis. 24 hours after induction of steatosis, the cells were incubated with a peptide of formula (I) (1 μM) or a peptide of formula (II) (1 μM) for 24 hours. The cells were then scraped in 5% NP-40 buffer for quantification of intracellular triglyceride levels. (B) Primary mouse hepatocytes isolated from C57BL / 6J mice fed a Western diet were incubated with apoA-I (10 μg / mL), a peptide of Formula (I) (1 μM), or a peptide of Formula (II) (1 μM) for 24 hours. n = 7 independent experiments per group. Data are presented as means (± SEM) relative to control (PBS) analyzed by one-way ANOVA with Dunnett's multiple comparison test. *p < 0.05, **p < 0.005, ***p < 0.001.

[0117] Figure 8 Figure 2 shows the effects of peptides of formula (I) and formula (II) on cytotoxicity in human hepatocytes (HepG2 cells). The MTT assay was used to detect cell growth rate and toxicity in HepG2 cells. (A) Cells were treated with PBS (vehicle), scrambled peptide (SCR, 1 μM) or a single dose of a peptide of formula (I) at increasing concentrations for 24 h (A), 48 h (B) or 72 h (C) and then subjected to the MTT assay. (D) Cells were treated with scrambled peptide (SCR, 1 μM) or a peptide of formula (I) at increasing concentrations for 48 h, the dose was repeated within 24 h, and then subjected to the MTT assay. n = 3 independent experiments per group. Data are expressed as percentages (± SEM) above or below the control value (PBS) analyzed by Kruskal-Wallis test and Dunn's post hoc relative to the control (PBS). No significant differences were observed.

[0118] Figure 9 Degradation of the peptide of formula (I) (circles) and the peptide of formula (II) (squares) over time at 4° C. (open shapes) and 37° C. (full shapes) in PBS (A, B), human plasma (C, D), human serum (E, F), mouse plasma (G, H), and mouse serum (I, J) is shown. The amount of peptide is calculated relative to the amount determined at time point zero.

[0119] Figure 10Figure 1 shows the pharmacokinetic properties of the peptides of Formula (I) and Formula (II) in mice. (A, B, C) Mean plasma concentration-time curves of the peptides of Formula (I) (AB) and Formula (II) (C) in mouse plasma following intravenous (iv, A) or subcutaneous (sc, BC) administration at 25 mg / kg (n = 3 mice per time point per condition). Closed squares: mean concentration + / - SD; open circles: data points generated from the fitted curves.

[0120] Figure 11 Figure 2 shows the in vivo efficacy of peptides of formula (I) and (II) on bile lipid secretion in wild-type C57B / L6J and dyslipidemic LDLR KO mice. Bile flow (A), bile cholesterol (B), and bile acid (C) secretion were measured in C57B / L6J mice 2, 4, and 6 hours after a single intraperitoneal (ip) administration of peptides of formula (I) (12.5 mg / kg or 25 mg / kg), scrambled peptide (SCR, 25 mg / kg), or vehicle (PBS). N = 5-8 mice per group. Bile flow (D), bile cholesterol (E), and bile acid (F) secretion were measured in C57B / L6J and LDLR KO mice 2 hours after a single intraperitoneal (ip) administration of a peptide of formula (I) (25 mg / kg), a peptide of formula (II) (25 mg / kg), SCR (25 mg / kg), or vehicle (PBS). n = 4-6 per C57BL / 6 mouse group, n = 7-15 per LDLR KO mouse group. Bile flow (G) and bile cholesterol (H) and bile acid (I) secretion were measured in C57BL / 6J mice 14 days after subcutaneous placement of an alzet osmotic pump to ensure that the peptide of formula (I) was released at an estimated rate of 0.5 μL / h, which corresponds to an estimated delivery of 5 mg of the peptide of formula (I) per kilogram of body weight per day (5 mg / kg BW / day). n = 3-4 mice per group. Data are expressed as mean (± SEM) and analyzed by Mann Whitney test relative to control (PBS). *p<0.05, **p<0.01.

[0121] Figure 12Figure 5 shows the effect of the peptide of formula (II) in Western diet-induced hepatic steatosis. Mice were administered intraperitoneally with the peptide of formula (I) or PBS (control group) at 1 mg / kg / day daily for 2 weeks. OGTT was performed 10 days after the start of peptide administration, and other measurements were performed at the end of the treatment period. (A) Body weight, (B) liver to body weight ratio, (C) liver triglyceride content, (DEF) plasma triglyceride, cholesterol and HDL-C concentrations, (GH) plasma levels of AST and ALT, (IJ) OGTT and plasma insulin concentrations at -15 and +30 minutes after OGTT. n = 5 mice per group. Data are expressed as mean (± SEM) and analyzed using an unpaired t-test.

[0122] Figure 13 Figure 5 shows the effect of the peptide of formula (I) in CDAHFD-induced liver fibrosis. The peptide of formula (I) was infused subcutaneously for 2 weeks using an alzet osmotic pump to ensure an estimated delivery of 5 mg of the peptide of formula (I) per kilogram of body weight per day (5 mg / kg BW / day). (A) Representative images of liver histological analysis of mice fed CDAHFD for 6 weeks with or without (sham) treatment with the peptide of formula (I) in the last two weeks of diet by staining with Sirius red. (B) Quantification of collagen deposition, which was assessed from the percentage of Sirius red area. (C) Quantification of hydroxyproline (μg / g) in liver tissue from mice fed CDAHFD for 6 weeks with or without (sham) treatment with the peptide of formula (I) in the last two weeks of diet. Data are expressed as mean (±SEM) and analyzed by Wilcoxon-Mann Whitney test. **p<0.01. n=6 mice per group Example

[0123] Example 1: Preparation of peptides

[0124] The following peptides were produced by Bachem AG (Bubendorf, Switzerland) with a purity of >90% in acetate and dissolved in phosphate-buffered saline (PBS) solution before use:

[0125] -Formula (I): CH3CO-[SEQ ID NO: 1]-NH2, hereinafter referred to as "peptide of formula (I)" or "formula (I)" ( Figure 1 A);

[0126] -Formula (II): CH3CO-[SEQ D NO: 1]-K-[palmitoyl]-NH2, hereinafter referred to as "peptide of formula (II)" or "formula (II)" ( Figure 1 B);

[0127] -SEQ ID N°2: GEAKSYAEKGEARGERGTKGEFRIFKREATD

[0128] - Formula (III): CH3CO-[SEQ ID NO: 2]-NH2, hereinafter referred to as "scrambled peptide" or "SCR" ( Figure 1 C); and

[0129] -Formula (IV): CH3CO-[SEQ ID NO: 2]-K-[palmitoyl]-NH2, hereinafter referred to as "scrambled peptide K-C16" or "SCR-K-C16" ( Figure 1 D).

[0130] Palmitic acid was prepared by coupling a preformed derivative Fmoc-K-[palmitoyl]-OH( Figure 1 E) introduced.

[0131] Other labeled and unlabeled peptides are SEQ ID NO: 3 (EAGGAFGK) and SEQ ID NO: 4 (EAGGAFG-[ 13 C6, 15 N4]-K) was purchased from ThermoFisher Scientific with a purity of >90% and dissolved in 50% acetonitrile at 1 mM.

[0132] Example 2: Surface plasmon resonance analysis of the interaction between the peptide of formula (I) and the peptide of formula (II) and human F1Fo-ATPase.

[0133] Materials and methods :

[0134] Surface plasmon resonance (SPR) assay. Binding studies based on SPR technology were performed on a BIAcore T200 optical biosensor instrument (GE The peptide of formula (I) with a C-terminal 6xHis-tag (Formula (I)-His-tag: CH3CO-[SEQ ID NO: 1]-HHHHHH) and the peptide of formula (II) with a C-terminal biotin (Formula (II)-biotin: CH3CO-[SEQ ID NO: 1]-K-[palmitoyl]-AEEAc-K-[biotin]-NH2) were custom synthesized by BACHEM AG (Bubendorf, Switzerland) with a purity of >90% in trifluoroacetate. Human FiFo-ATPase was purified from HepG2 cells by immunocapture using a mouse monoclonal anti-ATP synthase antibody (12F4AD8AF8, #ab109867, Abcam) according to the manufacturer's instructions.

[0135] The immobilization of the peptide of formula (I)-6His-tagged was performed on a nitrilotriacetic acid (NTA) sensor chip (GE Healthcare) in HBS-P+ buffer (10 mM Hepes pH 7.4, 150 mM NaCl and 0.05% surfactant P20). + The NTA surface was saturated and the flow cell (Fc) was loaded with a 0.5 mM NiCl solution. Channel Fc1 was left empty and used as a reference surface for nonspecific binding measurements. Formula (I)-6xHis was injected into channel Fc2 at a flow rate of 5 μL / min and stabilized by amine coupling (Laboratory Guide 29-0057-17AB). The total amount of immobilized Formula (I)-His-tag was 300-350 resonance units (RU): the final concentration was 25 μg / mL.

[0136] Immobilization of a C-terminally biotinylated peptide of formula (II)-biotin was performed on a streptavidin-coated (SA) sensor chip (GE Healthcare) in HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20). Channel Fc1 was left empty and used as a reference surface for nonspecific binding measurements. Formula (II)-biotin was injected into channel Fc2 at a flow rate of 5 μL / min. The total amount of immobilized formula (II)-biotin was 350-380 RU: final concentration 100 ng / mL. For the binding analysis, F1F0 analyte (584 kDa) was sequentially injected onto the immobilized peptide in a single cycle at increasing concentrations (3.125 nM-6.25 nM-12.5 nM-25 nM-50 nM) without regenerating the sensor between injections. Allows single cycle kinetic (SCK) analysis to determine the association, dissociation, and affinity constants (Ka, Kd, ​​and K, respectively) D Binding parameters were obtained by fitting the superimposed sensorgrams using a 1:1 Langmuir binding model or a steady-state constant Rmax model in BIAevaluation software version 3.0.

[0137] result :

[0138] The results are shown in Figure 2 middle. Figure 2 The sensorgram in FIG shows the interaction between purified c-ATPase used as analyte and the peptide of formula (I) coated on a BIAcore sensor chip ( Figure 2 A) and a peptide of formula (II) ( Figure 2B) direct interaction between the F1Fo-ATPase and the immobilized peptide of formula (I) and the peptide of formula (II) in a dose-dependent manner (31.25 nM-500 nM), which allowed us to determine the affinity (K) between the multi-subunit complex and the peptide of formula (I). D =18.97 nM) and the affinity (K D =4.45nM)

[0139] in conclusion :

[0140] Direct high affinity interactions between F1Fo-ATPase and the peptide of formula (I) and between F1Fo-ATPase and the peptide of formula (II) were measured.

[0141] Example 3: Effects of the Peptide of Formula (I) and the Peptide of Formula (II) on F1-ATPase Activity

[0142] Materials and methods :

[0143] F1-ATPase activity assay.

[0144] The mature human IF1 protein (SEQ ID NO: 5) was chemically synthesized by GenScript (Piscataway, NJ, USA) with a purity of >80%. Peptides derived from the mature human IF1 sequence (IF1-1-60, IF1-10-56, IF1-10-47) were produced by Bachem AG (Bubendorf, Switzerland) with a purity of >90%.

[0145] Human F1Fo-ATPase was purified from HepG2 cells by immunocapture using a mouse monoclonal anti-ATP synthase antibody (12F4AD8AF8, #ab109867, Abcam) according to the manufacturer's instructions.

[0146] F1-ATPase activity was measured as previously described

[20] . Briefly, 10 μg of F1Fo-ATPase was prepared into 50 μL of activity assay buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.5 mM phosphoenolpyruvate, 250 μM NADH, 100 μM ATP, 20 U lactate dehydrogenase, 120 U pyruvate kinase). The mixture was incubated at 37°C for 30 minutes. F1-ATPase activity was then measured in a 96-well microplate by adding 5 μL of the mixture / well (1 μg of F1Fo-ATPase per spot) to 200 μL of activity assay buffer at 37°C and adding 5 μL of buffer with or without peptide (1 μM each). The decrease in NADH absorbance was measured at 340 nm for 5 minutes using a Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific). The slope of each well was calculated and the results were expressed as a percentage of the control slope.

[0147] result :

[0148] The results are shown in Figure 3 Human IF1, IF1-1-60, IF1-10-56, and IF1-10-47 (1 μM each) strongly inhibited F1-ATPase activity. As expected, IF1 showed the strongest inhibitory activity (96% inhibition compared to the control), followed by IF1-10-60 (94%), then IF1-10-56 (88%), and IF1-10-47 (75%). In contrast, the peptide of formula (I) and the peptide of formula (II) stimulated F1-ATPase activity by 36% and 43%, respectively, while their respective scrambled peptides SCR and SCR-K16 had no effect.

[0149] in conclusion :

[0150] Unlike IF1 and other peptides derived from the IF1 sequence, the peptides of formula (I) and formula (II) do not inhibit but stimulate F1-ATPase activity. Therefore, these peptides are F1-ATPase activators.

[0151] Example 4: In vitro activity of the peptide of formula (I) and the peptide of formula (II): F1-ATPase activation.

[0152] Materials and methods :

[0153] The human hepatocyte cell line HepG2 was obtained from the American Type Culture Collection (#HB-8065). HepG2 was cultured in Dulbecco's Modified Eagle's Medium (DMEM)-high glucose (D0822, Sigma-Aldrich) supplemented with 10% fetal bovine serum (10270098, Life Technologies), 1% penicillin-streptomycin solution (P0781, Sigma-Aldrich). HepG2 cells were seeded on 24-well plates at 75,000 cells / well (Day 0). After 24 hours of growth, the cells were serum starved for 24 hours to synchronize the cell cycle (Day 1), and then replaced in complete cell growth medium for another 24 hours (Day 2). On Day 3, the cells were washed and balanced for 1 hour in fresh DMEM-high glucose (D1145, Sigma-Aldrich) without red phenol.

[0154] The cells were then treated for 5 minutes with different concentrations of the peptide of formula (I) (0.1 to 5 μM), the peptide of formula (II) (1 μM), SCR (1 μM), SCR-K-16 (1 μM), or apoA-I purified from human plasma (10 μg / mL) [5].

[0155] Specific ecto-F1-ATPase activation was assessed in the presence of IF1 protein (1 μM), a natural F1-ATPase inhibitor that interacts with the β-subunit to inhibit ATP hydrolysis activity [1][5].

[0156] The supernatant was then collected and centrifuged (10,000 g, 5 minutes, 4 ° C) and processed for ADP and ATP measurement. For ADP measurement, ADP was converted into ATP in 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, pH 7.5 buffer containing 0.5 mM phosphoenolpyruvate (PEP) and pyruvate kinase (PK, 6 U per point, 15 minutes at 37 ° C). For ATP measurement, 100 μl of sample was analyzed using the ATP bioluminescence assay kit CLS II (Roche Diagnostics). The sample was added to the ATP assay mixture and luminescence was measured for 1000 ms in a microplate reader Infinite F500 (Tecan, Switzerland). The ATP standard curve was generated in the same culture medium as the sample and the 10 -5 to 10 -10 The ADP concentration was then calculated as the ATP concentration after ADP conversion minus the basal ATP concentration. Data are expressed as nanomoles ADP generated.

[0157] result :

[0158] The results are shown in Figure 4 Under physiological conditions, ecto-F1Fo-ATPase works catalytically in the opposite direction to that described in functional mitochondria. In fact, the binding of apoA-I to ecto-F1Fo-ATPase stimulates the hydrolysis of extracellular ATP to ADP and phosphate, a process that is inhibited by the natural inhibitor of F1-ATPase, IF1 [1]. Here, we used IF1 to inhibit ecto-F1-ATPase activity [5]. As expected, incubation of HepG2 cells with apoA-I increased the extracellular ADP concentration ( Figure 4 A), while inhibition of F1-ATPase activity by IF1 attenuated this effect ( Figure 4 B), which reflects the ability of apoA-I to stimulate the hydrolysis activity of ecto-F1-ATPase. The peptide of formula (I) increases the extracellular ADP concentration in a dose-dependent manner, reaching a maximum efficacy at 1 μM ( Figure 4 A), whereas inhibition with IF1 attenuated this effect ( Figure 4 B). Similar results were observed with 1 μM of the peptide of formula (I) ( Figure 4 B) Those results indicate that both the peptide of formula (I) and the peptide of formula (II) stimulate the ecto-F1-ATPase hydrolysis activity.

[0159] in conclusion :

[0160] The peptides of formula (I) and formula (II) stimulate ecto-F1-ATPase activity in hepatocytes and compete with IF1 for binding to cell surface F1Fo-ATPase. Therefore, these peptides are good candidates for activating cell surface pore Fo-ATPase.

[0161] Example 5: In vitro activity: HDL endocytosis in hepatocytes

[0162] Materials and methods :

[0163] HDL endocytosis assay.

[0164] HepG2 cells were seeded in 96-well plates at 50,000 cells / well. HDL3 (d 1.12-1.21) was isolated from the plasma of healthy human donors

[12] and designated as HDL. HDL was fluorescently labeled with 568 dye (A10238, Thermofisher Scientific). 1 h30 before the assay, cells were serum starved for 1 h30 to stabilize nucleotide secretion. Cells were incubated with an inhibitor (H49K, 1 μM) for 10 min before treatment with different peptides (1 μM) or apoA-I purified from human plasma (10 μg / ml) [5]. Five minutes after peptide treatment, endocytosis was initiated by 50 μg / mL Labeled HDL was used to start. The same experiment was performed using a 25-fold excess of unlabeled HDL (2.5 mg / mL) to determine the nonspecific fluorescence signal. After 25 minutes at 37°C, the cells were then washed in serum-free DMEM and the extracellular membrane-bound HDL was dissociated by incubating the cells at 4°C for 90 minutes in serum-free DMEM. After washing, the cells were lysed in NaOH 0.1 M SDS 1% for 2 hours, the lysate was transferred to a black 96-well plate, and fluorescence was recorded at 568 nm (Varioscan flash). The fluorescence for each condition was a substrate with the value obtained under unlabeled HDL conditions, and the results were expressed as a fold change compared to the basal condition (untreated cells).

[0165] result :

[0166] The results are shown in Figure 5 The F1Fo-ATPase-mediated HDL endocytosis pathway depends on the activation of cell surface F1Fo-ATPase by apoA-I and the production of extracellular ADP and P2Y receptor activation [6]. As previously reported in [7], because preincubation with IF1 abolished the effect of apoA-I on HDL endocytosis, apoA-I (10 μg / mL) significantly stimulated HDL endocytosis by approximately 45% ( ) compared to unstimulated cells in a process strictly dependent on ecto-F1-ATPase activity. Figure 5 ). Similarly, the peptide of formula (I) (1 μM) and the peptide of formula (II) (1 μM) stimulated HDL endocytosis, and pretreatment with IF1 (1 μM) completely abolished this effect. SCR (1 μM) and SCR-K-C16 (1 μM) had no effect on HDL endocytosis, which remained at the level of PBS treatment.

[0167] in conclusion :

[0168] When the peptide of formula (I) and the peptide of formula (II) pharmacologically stimulate F1-ATPase activity, the pore Fo-ATPase-mediated HDL endocytosis in hepatocytes is significantly increased. Given that HDL endocytosis in hepatocytes is the key last step in reverse cholesterol transport to remove excess cholesterol [6], the peptides of formula (I) and formula (II) are good candidates for improving reverse cholesterol transport and removing excess cholesterol from the body.

[0169] Example 6: In vitro activity: Endothelial nitric oxide (NO) production

[0170] Materials and methods :

[0171] Production of nitric oxide.

[0172] Nitric oxide (NO) was detected using the DAF-FM-DA probe (D2321, Sigma-Aldrich), which forms a fluorescent benzotriazole upon reaction with NO. HUVEC cells (PromoCell #C-12203) were seeded in 96-well plates (10,000 cells per well) and cultured in endothelial cell basal medium 2 (PromoCell #C-22211) supplemented with GM2 supplement mix (PromoCell #C-39211) until 80-90% confluence. The medium was then replaced with serum-free M-199 for 4 hours, and the cells were incubated with DAF-FM-DA (5 μM) diluted in PBS for 45 minutes. The cells were treated with increasing concentrations of the different peptides or apoA-I purified from human plasma (10 μM) [5] or histamine (1 mM) as a positive control. In another set of experiments, cells were incubated with inhibitors (IF1, 1 μM) for 10 minutes and then treated with peptides or apoA-I. Fluorescence was recorded for 30 minutes (λex = 495 nm, λem = 515 nm) using a Tecan Flash multimode reader (Thermo Fisher Scientific). The fluorescence of each condition was compared with the value obtained in untreated cells, and the results were expressed as the fold change compared to the basal condition (untreated cells).

[0173] result :

[0174] The results are shown in Figure 6 Ecto-F1Fo-ATPase is expressed on the plasma membrane of endothelial cells and is involved in the production of NO [5]. As described in [5], apoA-I activates ecto-F1Fo-ATPase to stimulate endothelial cells to produce NO ( Figure 6 A), and this effect was abolished when cells were pretreated with IF1 ( Figure 6B). Similarly, the peptide of formula (I) (1 μM) and the peptide of formula (II) (1 μM) stimulated NO production by approximately 50% of endothelial cells, and this effect was completely abolished by IF1 ( Figure 6 B).

[0175] According to the protocol disclosed in [5], the peptide of formula (I) increased femoral artery blood flow in conscious wild-type C57B / L6J mice in a process strictly dependent on endothelial NO production (data not shown).

[0176] in conclusion :

[0177] In human endothelial cells, when the peptides of formula (I) and formula (II) pharmacologically stimulated F1-ATPase activity, NO production mediated by F1Fo-ATPase of eNOS was significantly increased. Given that NO production by eNOS maintains vascular homeostasis

[16] and maintains quiescence of hepatic stellate cells involved in liver fibrosis and Kupffer cells involved in liver inflammation [8], the peptides of formula (I) and formula (II) are therefore good candidates for the treatment of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver disease.

[0178] Example 7: In vitro activity: hepatic steatosis

[0179] Materials and methods :

[0180] Preparation of oleate and palmitate solutions.

[0181] A solution containing 250 mM palmitate (P0500, Sigma-Aldrich) was first prepared in 0.1 M NaOH at 70°C for 30 minutes, then diluted in DMEM low glucose (D5546, Sigma-Aldrich) containing 10% fatty acid-free BSA (A7030, Sigma-Aldrich) to produce a 10 mM palmitate solution and dissolved at 37°C for 30 minutes, filter sterilized and stored in glass vials at -20°C until use. This palmitate stock solution and ready-to-use oleate solution (O3008, Sigma-Aldrich) were used to prepare a 0.5 mM solution with a 2:1 ratio of oleate to palmitate in complete culture medium containing DMEM low glucose, 10% fetal bovine serum, 1% penicillin-streptomycin and 1% fatty acid-free BSA.

[0182] In vitro evaluation of steatosis.

[0183] HepG2 cells were grown to 60-70% confluence in 12-well plates and then exposed to culture medium alone (DMEM low glucose, 10% fetal bovine serum, 1% penicillin-streptomycin and 1% fatty acid-free BSA) or culture medium containing a 0.5 mM oleate / palmitate mixture (2:1) for 48 hours to induce steatosis. During the last 24 hours of the 48 hours, cells were treated with 1 μM of the peptide of formula (I) or the peptide of formula (II).

[0184] Primary mouse hepatocytes were isolated from mice fed a Western diet for 11 weeks (Envigo #TD.88137, containing 0.2% cholesterol, 42% kcal from fat, 34% sucrose by weight) as described in Example 3. Primary mouse hepatocytes were seeded at a density of 600,000 cells / well in growth medium in 12-well plates and treated with apoA-I (10 μg / mL), a peptide of formula (I) (1 μM), or a peptide of formula (II) (1 μM) for 24 hours.

[0185] To measure intracellular triglyceride content, cells were washed with PBS, scraped in 5% NP-40 lysis buffer and heated at 85°C for 10 minutes. Triglycerides were then quantified using a commercial triglyceride kit (Biolabo #87319). Values ​​were normalized to the protein concentration in the cell lysate.

[0186] result :

[0187] The results are shown in Figure 7 Compared with untreated cells (vehicle, BSA 1%), cells exposed to fatty acids (oleate and palmitate in a ratio of 2 ： 1 in a 0.5 mM solution) showed more than 300% higher intracellular triglyceride content ( Figure 8 A). When HepG2 cells were treated with the peptide of formula (I), the fatty acid-induced intracellular triglyceride accumulation was significantly reduced by 18% (p < 0.05, Figure 7 A), which was reduced by 32% (p<0.001, Figure 7 Furthermore, treatment with the peptide of formula (I) and the peptide of formula (II) for 24 hours significantly reduced the intracellular accumulation of triglycerides in steatotic primary mouse hepatocytes compared to the PBS control (p < 0.05 and p < 0.005, respectively). Figure 7 B) Similar effects were observed when primary mouse hepatocytes were treated with apoA-I (p < 0.005 compared to PBS, Figure 7 B).

[0188] in conclusion:

[0189] The peptides of formula (I) and (II) did reduce steatosis in a steatotic human hepatocyte model and in steatotic primary mouse hepatocytes. Given that steatotic hepatocytes are a key driver of the pathogenic process in NAFLD / NASH

[11] , the peptides of formula (I) and (II) are good candidates for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0190] Example 8: In vitro toxicity assay (hepatocytes)

[0191] Materials and methods :

[0192] Detailed toxicity determination.

[0193] HepG2 cells were seeded in growth medium (DMEM high glucose, 10% fetal bovine serum) in 96-well plates at a density of 10,000 cells / well. The next day, the growth medium was replaced and the peptide of formula (I) was added once in an increasing dose for 24h, 48 or 72h or 48h, and repeated once within 24h. Similarly, the peptide of formula (II) was added once in an increasing dose for 24h or 48h or for 48h, and repeated once within 24h. The cells were incubated for 4h with 5mg / L MTT, and 100 μL DMSO was added to the wells. Absorbance was recorded at 570 and 660nm using a microplate spectrophotometer system (Varioscan flash). Cell viability was calculated by subtracting the background measured at 660nm from the 570nm absorbance.

[0194] result :

[0195] The results are shown in Figure 8 Treatment of hepatocytes with a single increasing dose of the peptide of formula (I) from 0.1 to 50 μM for 24, 48, or 72 hours had no effect on cell viability, nor did treatment with multiple increasing doses for 24 and 48 hours. Treatment of hepatocytes with a single increasing dose of the peptide of formula (II) from 0.1 to 25 μM for 24 or 48 hours had no effect on cell viability, nor did treatment with multiple increasing doses for 24 and 48 hours.

[0196] in conclusion :

[0197] The peptides of formula (I) and formula (II) did not exhibit any cytotoxicity over time, either with increasing single or repeated doses.

[0198] Example 9: Stability of the peptide of formula (I) and the peptide of formula (II) in PBS, plasma and serum

[0199] Materials and methods :

[0200] Reagents.

[0201] UPLC / MS grade acetonitrile and water, phosphate buffered saline (PBS) and formic acid were purchased from Biosolve (Valkenswaard, The Netherlands).

[0202] Stability determination.

[0203] The peptide of formula (I) or the peptide of formula (II) was prepared in PBS at a concentration of 200 μg / mL or mixed with the peptide from human (Etablissement Du Sang, EFS) or EDTA plasma or serum from mice (C57BL / 6J, cardiac puncture) were mixed. Aliquots from PBS samples (40 μL) were incubated at 4°C or 37°C for 0, 1, 2, and 4 weeks. Aliquots from plasma and serum samples (50 μL) were incubated at 4°C or 37°C for 0, 1, 2, 4, 6, 12, and 24 hours.

[0204] Sample analysis.

[0205] A mixed solution of peptides of formula (I) and (II) was prepared and serially diluted in PBS to obtain seven standard solutions ranging from 200 μg / mL to 0.2 μg / mL. In parallel, a mixed solution of labeled peptides of formula (I) and (II) was prepared at 100 μg / mL in PBS (Thermo Scientific, Biopolymers Darmstadt, Germany). The mixed solution of labeled peptides (25 μL) was added to 25 μL of standard solution and PBS, plasma and serum samples. Acetonitrile (150 μL) was added to each sample to precipitate plasma / serum proteins. After centrifugation (10,000 × g, 4 ° C, 10 minutes), the clarified supernatant (150 μL) was dried under a gentle nitrogen flow (45 ° C), reconstituted with 10% acetonitrile containing 0.1% formic acid (100 μL), and injected into a liquid chromatography-high resolution mass spectrometry (LC-HRMS) system. The samples were analyzed by injecting 10 μL of the sample into the H-Class UPLC system (Waters Corporation, Milford, MA, USA). Peptide CSH C 18LC-HRMS analysis was performed on a chromatographic column (2.1 mm × 150 mm, 1.7 μm; Waters Corporation) maintained at 60°C. The mobile phase consisted of 5% acetonitrile as solvent A and 100% acetonitrile as solvent B, each containing 0.1% formic acid. Elution was performed using a gradient of solvent B in solvent A at a constant flow rate of 250 μL / min for 20 minutes. Mobile phase B was kept constant at 1% for 1 minute, increased linearly from 1% to 80% for 15 minutes, kept constant for 1 minute, returned to the initial state for more than 1 minute, and kept constant again for 2 minutes before the next injection. HRMS detection was performed by a Synapt G2 HRMS Q-TOF mass spectrometer (Waters Corporation) equipped with a Z-Spray interface for electrospray ionization. In positive ionization mode, the resolution mode was applied at a mass resolution of 25,000 full width at half maximum over a mass-to-charge ratio (m / z) range of 200 to 4,000. The ionization parameters were as follows: capillary voltage 3 kV, cone voltage 30 V, desolvation gas flow 900 L / hr, source temperature 120°C, desolvation temperature 450°C, and nitrogen as desolvation gas. Data were collected in continuous mode at a rate of four spectra per second. A leucine enkephalin solution prepared at 2 μg / mL in an acetonitrile / water (50 / 50, v / v) mixture was injected into the locked spray channel at a constant flow rate (10 μL / min). Spectra were acquired for 1 second every 20 seconds, allowing for mass correction during the experiment. The leucine enkephalin solution was analyzed based on its primary accurate m / z (±5 ppm, Table 1 ) were used to analyze peptides and normalize each peptide signal to the signal of its labeled internal standard. Peptide concentrations were calculated using a calibration curve drawn from the standard solutions (linear regression, 1 / x weighting, excluding the origin).

[0206] Table 1: Mass spectrometry parameters used for peptide detection by LC-HRMS.

[0207]

[0208] IS: Internal Standard

[0209] result :

[0210] The results are shown in Figure 9 In, it represents the stability of the peptides over time in different matrices (PBS, human and mouse serum, human and mouse plasma) at 4°C and 37°C. The peptides of formula (I) and formula (II) are stable in PBS at 4°C and 37°C for 4 weeks ( Figure 9 AB). Both the peptide of formula (I) and the peptide of formula (II) are degraded faster in human plasma than in human serum ( Figure 9 CF). The same observations were observed for the peptide of formula (I) in mouse plasma and serum ( Figure 9G and 9I), while the peptide of formula (II) was as stable in mouse serum as in mouse plasma ( Figure 9 H and 9J). When comparing peptide stability at 37°C versus 4°C, the peptide of formula (I) degraded faster at 37°C than at 4°C in any of the matrices tested (serum and plasma) and species (human and mouse), while no significant difference in the stability of the peptide of formula (II) was observed between 37°C and 4°C. At 37°C, the degradation of the peptide of formula (II) in serum and plasma was much lower than that of the peptide of formula (I): after 24 hours at 37°C, the recovery of the peptide of formula (II) was 100% in serum and 50% in plasma, while the recovery of the peptide of formula (I) was only 30% and 10% respectively.

[0211] in conclusion : The peptides of formula (I) and formula (II) can be stored in PBS at 4°C and up to 37°C for at least 4 weeks without being degraded. The peptides of formula (II) show minimal degradation in human and mouse biological matrices at 37°C and up to 24 hours, and are therefore more suitable for long-term injection than the peptides of formula (I).

[0212] Example 10: In vivo pharmacokinetics of the peptide of formula (I) and the peptide of formula (II)

[0213] Materials and methods :

[0214] LC-MS / MS peptide quantification.

[0215] The peptides of formula (I) and formula (II) were analyzed in mouse EDTA plasma using a validated assay involving trypsin proteolysis and subsequent analysis of the signature peptide (SEQ ID NO: 3) by liquid chromatography-tandem mass spectrometry (LC-MS / MS). A working solution of the unlabeled peptide (SEQ ID NO: 3, 1 mM) was serially diluted in water to yield seven standard solutions ranging from 0.05 to 5 μM. ProteinWorks was used according to the manufacturer's instructions (except for the trypsin incubation time, which was optimized to 7 hours). TM Plasma and standard samples (40 μL) were reduced, alkylated, and trypsinized overnight using the ready-to-use solution of the eXpress kit (Waters Corporation, Milford, MA, USA). The labeled proteolytic peptide ([SEQ ID NO: 4]-[ 13 C6, 15A working solution of N4]-K, 1 mM) was used as an internal standard (IS) and added to the digestion buffer to a final concentration of 0.5 μM. After digestion, the samples were washed using 30 mg Oasis HLB 1cc Cartridges (Waters Corporation). The cartridges were conditioned, equilibrated, loaded, washed, and eluted using methanol (1 mL), water (1 mL), sample (~200 μL), 5% methanol containing 0.1% TFA (1 mL), and 60% methanol containing 0.1% TFA (1 mL), respectively. The eluate was dried under a stream of nitrogen, reconstituted with 100 μL of 10% acetonitrile containing 0.1% formic acid, and 10 μL was injected into the LC-MS / MS system. LC-MS / MS analysis was performed on a 100 μL Oasis HLB 1cc Cartridge with an electrospray ionization (ESI) interface and an Acquity UPLCT M Equipment The mass spectrometer was used on a TQD mass spectrometer (Waters Corporation). A linear gradient of mobile phase B (100% acetonitrile) in mobile phase A (5% acetonitrile) was maintained at 60°C. BEH C 18 The proteolytic peptides were separated on a column (2.1×100 mm, 1.7 μm, Waters Corporation) at a flow rate of 600 μL / min, each mobile phase containing 0.1% formic acid. Mobile phase B was linearly increased from 1% to 50% for 5 minutes, kept constant for 1 minute, returned to the initial state for more than 1 minute, and kept constant for 2 minutes before the next injection. The proteolytic peptides were then detected by a mass spectrometer with an ESI interface operating in positive ion mode (capillary voltage, 3 kV; desolvation gas (N2) flow and temperature, 900 L / h and 400°C; source temperature, 150°C). Multiple reaction monitoring mode was applied to MS / MS detection (SEQ ID NO: 3, m / z 368.8→536.5, y6 + ; [SEQ ID NO: 4]-[ 13 C6, 15 N4]-K, m / z 372.8→544.4, y6 + ), the cone voltage and collision voltage were set to 20 and 14 V respectively. and Data acquisition and analysis were performed using ELISA software (version 4.1, Waters Corporation). The chromatographic peak area ratio between the unlabeled peptide and IS constituted the detector response. The standard solution was used to construct the calibration curve for peptide quantification. Linearity was determined by an average r greater than 0.998. 2The concentrations of the peptides of formula (I) and (II) are expressed as μM, assuming that 1 mole of peptide is equivalent to 1 mole of the peptide of formula (I) and (II), respectively. The concentrations are converted to their standard units (ng / mL) assuming that the molecular weights of the peptides of formula (I) and (II) are 3540 Da and 3917 Da, respectively.

[0216] animal.

[0217] Wild-type C57B / L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). Mice were housed in an animal room under specific pathogen-free conditions at the animal facility in Rangueil (Anexplo platform, US006, Toulouse, France) with a 12-hour light / dark schedule and fed ad libitum with normal chow (#V1535R / MH, Ssniff, Germany). All animal experimental procedures were performed in accordance with the guidelines of the animal experimentation institution approved by the local animal care ethics committee and in accordance with the guidelines of Directive 2010 / 63 / EU of the European Parliament on the protection of animals used for scientific purposes or the NIH guidelines.

[0218] Pharmacokinetic (PK) studies.

[0219] Eight-week-old C57B / L6J male mice weighing 24.5±1.3 g were used for the following pharmacokinetic studies. During the experiment, all animals were allowed free access to food and water. The peptide of formula (I) was administered intravenously (iv) or subcutaneously (sc) in the tail vein at 25 mg / kg. The peptide of formula (II) was administered subcutaneously at 25 mg / kg. Three different animals were used for each time point. After administration, for the peptide of formula (I), intracardiac blood was collected at 0.03, 0.117, 0.25, 0.5, 0.75, 1, 1.5, 2, 4h, and for the peptide of formula (II), it was collected at 0.03, 1, 4, 6, 8, 10, 12, 16, 20, 24, 30, 48 hours. EDTA was used as an anticoagulant, and plasma was separated by centrifugation at 4,000 rpm for 10 minutes at 4°C. Plasma samples were placed on wet ice and stored at -80°C within 1 hour of collection until analysis by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) for quantification. Table 2 reports the pharmacokinetic parameters calculated in plasma, namely, distribution and elimination half-lives (t1 / 2lbd1 and t1 / 2lbdz), maximum concentration (Cmax), time to Cmax (Tmax), area under the curve (AUC), total plasma clearance (Cl), volume of distribution (Vd), and mean residence time (MRT).

[0220] Table 2: Pharmacokinetic characteristics of the peptide of formula (I) and the peptide of formula (II)

[0221]

[0222] result:

[0223] The results are shown in Figure 10 and Table 2. After one dose of 25 mg / kg intravenously (iv) and subcutaneously (sc), the peptide of formula (I) Figure 10 A (iv) and 10B (sc) are shown to be rapidly distributed and eliminated. Under these conditions, the elimination half-life (t1 / 2lbdz) of the peptide of Formula (I) was 0.26 hours and 0.21 hours for iv and sc administration, respectively (Table 2). The peptide of Formula (I) exhibited intermediate clearance (Cl = 1.9 L / hour / kg for both routes of administration) and volume of distribution (Vd = 0.7 L / kg and 0.5 L / kg for iv and sc administration, respectively).

[0224] Compared to the peptide of formula (I), the distribution and elimination rate of the peptide of formula (II) administered subcutaneously at 25 mg / L was slower ( Figure 10 C), which has an elimination half-life more than 50 times longer than that of the peptide of formula (I) (t1 / 21bdz=12.54 hours) and a lower clearance rate (Cl=0.05 L / hour / kg).

[0225] in conclusion :

[0226] The peptides of formula (II) exhibit improved pharmacokinetic properties compared to the peptides of formula (I).

[0227] Example 11: In vivo efficacy of the peptide of formula (I) and the peptide of formula (II) on bile lipid secretion.

[0228] Materials and methods :

[0229] animal.

[0230] Wild-type C57B / L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). LDLR knockout mice (male, C57B / L6J background) were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). Mice were housed in an animal room under specific pathogen-free conditions at the animal facility of Rangueil (Anexplo platform, US006, Toulouse, France) with a 12-hour light / dark schedule and ad libitum feeding of normal chow (#V1535 R / MH, Ssniff, Germany). All animal experimental procedures were performed in accordance with the guidelines of the animal experimentation institution approved by the local animal care ethics committee and in accordance with the guidelines of the European Parliament's Directive 2010 / 63 / EU on the protection of animals used for scientific purposes or the NIH guidelines.

[0231] Gallbladder cannulation and bile collection.

[0232] According to the experiment, 8-week-old mice were injected intraperitoneally with PBS, peptide of formula (I), peptide of formula (II) and SCR. Figure 11 Details of the use of the peptide, the dosage and mode of administration, and the time course are described in the description of . Given the short elimination half-life of the peptide of formula (I), an osmotic pump was also used to ensure continuous delivery for 14 days. Briefly, a 200 μL osmotic pump was filled with 10 mg / mL of the peptide of formula (I) in PBS and the pump was operated according to the manufacturer's instructions ( Model pump #2002) was implanted subcutaneously into mice to ensure that the peptide of formula (I) was released at an estimated rate of 0.5 vL / hour, which corresponds to an estimated delivery of 5 mg of the peptide of formula (I) per kilogram of body weight per day (5 mg / kg BW / day). After treatment, mice were fasted for 2 hours and then anesthetized by intraperitoneal injection of ketamine and xylazine hydrochloride. The common bile duct was ligated near the duodenum, the gallbladder was punctured and cannulated with a polyethylene 10 catheter. After stabilization for 30 minutes, newly secreted bile was collected for 30 minutes. During bile collection, a temperature mattress was used to stabilize body temperature. Assuming the density of bile is 1 g / mL, bile flow rate was determined by weight (expressed in μL / minute / 100 g body weight). At the end of the experiment, blood was collected and mice were killed by cervical dislocation.

[0233] Bile lipid analysis.

[0234] For bile acid analysis, 1 μL bile sample was diluted with 99 μL milliQ water and then incubated with working reagent (6 mg NAD, 0.5 M hydrazine hydrate buffer, 0.05 M sodium pyrophosphate) for 4 minutes. The mixture was then incubated with starting reagent (0.03 M Tris-EDTA; 0.3 U / mL 3-α-OH steroid dehydrogenase) and measured at 340 / 330 nm excitation and 440 / 420 nm emission for 30 minutes. For phospholipid analysis, 1 μL bile sample was diluted with 49 μL milliQ water and then incubated with working reagent (100 mM MOPS, pH 8; 0.55 mM HVA; 20 mM CaCl2; 11 U / mL phospholipase-D; 1.66 U / mL peroxidase; 0.1% Triton X-100) for 4 minutes. The mixture is then incubated with starting reagent (1M MOPS, pH 8, 50U / mL choline oxidase), and measured at 340 / 330 excitation and 440 / 40 emission for 67.5 minutes. For cholesterol analysis, 1 μL bile sample is diluted with 29 μL milliQ water, then with working reagent (100mM MOPS, pH 8, 0.25mM HVA; 0.1% Triton X-100) incubation for 4 minutes. The mixture is then incubated with starting reagent (0.1M MOPS, pH 8, 0.06U / mL cholesterol oxidase, 0.15U / mL cholesterol esterase, 0.45U / mL peroxidase, 0.06mM taurocholate), and measured at 340 / 330nm excitation and 440 / 420nm emission for 45 minutes. The secretion values ​​of bile acids, phospholipids, and cholesterol were calculated by multiplying the concentrations by the bile flow values ​​and expressed as nmol / min / 100 g body weight (BW).

[0235] result :

[0236] The results are shown in Figure 11 Compared with mice injected with PBS or 25 mg / kg SCR, C57BL / 6 mice treated with 25 mg / kg of the peptide of formula (I) by intraperitoneal bolus injection showed a significant increase in bile flow and bile secretion of cholesterol and bile acid. This effect of the peptide of formula (I) was maintained for up to 4 hours after injection ( Figure 11 AC). In comparison, a 12 mg / kg bolus injection of the peptide of formula (I) was less effective than a 25 mg / kg dose on bile flow and bile lipid secretion ( Figure 11 AC). Intraperitoneal bolus injection of 25 mg / kg of the peptide of formula (II) in C57BL / 6 mice also stimulated bile flow and bile secretion of cholesterol and bile acids to the same extent as similarly treated mice with the peptide of formula (I) ( Figure 11DF). Those effects of the peptide of formula (I) and the peptide of formula (II) on stimulating bile flow and bile lipid secretion were also maintained in dyslipidemic LDL KO mice ( Figure 11 DF). When delivered continuously for 14 days at 5 mg / kg BW / day, the peptide of formula (I) also stimulated bile flow and bile cholesterol secretion ( Figure 11 GI).

[0237] in conclusion :

[0238] The peptides of formula (I) and (II) stimulate bile flow and bile secretion of cholesterol and bile acids in wild-type and dyslipidemic mice. Hepatic excretion of cholesterol in bile, whether bile acids or cholesterol, represents a major pathway for removing excess cholesterol that contributes to the development of atherosclerosis [6]. Similarly, downregulation of bile flow and bile lipid secretion leads to hepatic lipotoxicity and has been demonstrated in NASH

[13] and cholestatic liver disorders

[17] . Therefore, the peptides of formula (I) and (II) are good candidates for preventing the development of metabolic syndrome, cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) or cholestatic liver disease.

[0239] Example 12: In vivo efficacy of the peptide of formula (II) on the development of NASH-associated hepatic steatosis.

[0240] Materials and methods:

[0241] animal.

[0242] Wild-type C57B / L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). Mice were housed in an animal room under specific pathogen-free conditions at the animal facility of Rangueil (Anexplo platform, US006, Toulouse, France) with a light / dark schedule of 12 hours / 12 hours. At the start of the dietary intervention, all animals were 8 weeks old and fed ad libitum with normal chow (#V1535 R / MH, Ssniff, Germany). All animal experimental procedures were performed in accordance with the guidelines of the animal experimentation institution approved by the local animal care ethics committee and in accordance with the guidelines of the European Parliament's Directive 2010 / 63 / EU on the protection of animals used for scientific purposes or the NIH guidelines.

[0243] A mouse model of diet-induced hepatic steatosis.

[0244] Eight-week-old mice were fed a Western diet for 4 weeks (Envigo #TD.88137, containing 0.2% cholesterol, 42% kcal from fat, and 34% sucrose by weight). During the last 2 weeks of the 4-week period, mice were intraperitoneally administered with a peptide of Formula (I) or PBS (control group) at 1 mg / kg / day. Following the treatment period, mice were fasted overnight, anesthetized with an intraperitoneal injection of ketamine and xylazine hydrochloride, and then sacrificed by exsanguination. Body weight, liver triglyceride content, plasma lipids, and transaminases were measured at the time of sacrifice.

[0245] Liver triglyceride content.

[0246] 100 mg of liver tissue was homogenized in 900 μL of phosphate buffered saline (pH 7.4) until the tissue was completely lysed. Lipids were extracted by mixing 125 μL of the lysate with 1 mL of CHCl3: MeOH (2: 1). After centrifugation, the chloroform phase was evaporated under a stream of nitrogen and the dried residue was dissolved in 200 μL of isopropanol. Triglycerides were measured using a commercial kit based on the GPO-PAP assay (Biolabo SA, Maizy, France). Results were expressed as mg triglycerides / g liver.

[0247] Analysis of plasma lipid and transaminase levels

[0248] Triglyceride and cholesterol levels were measured using commercial colorimetric kits (Biolabo SA, Maizy, France), based on the CHOD-PAP and GPO-PAP assays, coupled enzymatic reactions, and spectrophotometric detection of the reaction end products. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a COBAS-MIRA+ biochemical analyzer (Anexplofacility, Toulouse, France).

[0249] Oral glucose tolerance test (OGTT)

[0250] After the diet of 8 weeks, after overnight fasting period, mice were treated with oral gavage glucose load (3mg / g body weight). 0, 15, 30, 45, 60, 90 and 120 minutes after oral glucose load and oral glucose load, blood glucose level was measured by tail vein sampling with a portable glucometer (Accu-check, Roche). According to the explanation of the manufacturer, ELISA test kit (Mercodia, Uppsala, Sweden) was used to load glucose in 5 μ L blood plasma for 30 minutes before and after loading for 15 minutes to measure plasma insulin concentration.

[0251] result :

[0252] The results are shown in Figure 12Two weeks of intraperitoneal injection of the peptide of formula (II) significantly reduced hepatic steatosis, as shown by a decrease in the liver / body weight ratio ( Figure 12 B, p < 0.01)) and reduction in liver triglyceride concentration ( Figure 12 C, p < 0.05 compared to PBS). Treatment with the peptide of formula (II) had no effect on plasma triglyceride and HDL-cholesterol (HDL-C) levels ( Figure 12 D and 12F), but significantly reduced plasma levels of total cholesterol ( Figure 12 E, p < 0.05 compared to PBS), indicating the beneficial effect of the peptide of formula (II) in reducing hypercholesterolemia. Treatment with the peptide of formula (II) significantly reduced plasma ALT levels ( Figure 12 H, p < 0.05 compared with PBS), indicating a potential improvement in liver function.

[0253] Regarding glucose metabolism, the peptide of formula (II) improves oral glucose tolerance ( Figure 12 I) and reduced basal insulin levels ( Figure 12 J, p < 0.05 compared with PBS PBS).

[0254] Treatment with the peptide of formula (I) demonstrated benefits on NASH-associated steatosis and glucose metabolism. Therefore, the peptide of formula (I) is a good candidate for treating and reversing hepatic steatosis and resolving glucose metabolism disorders, particularly in non-alcoholic steatohepatitis (NASH).

[0255] Example 13: In vivo efficacy of the peptide of formula (I) on the progression of NASH-associated liver fibrosis.

[0256] Materials and methods :

[0257] animal.

[0258] Wild-type C57B / L6J male mice were purchased from Janvier Labs (Le Genest Saint Isle, France). Under specific pathogen-free conditions at the animal facility of Rangueil (Anexplo platform, US006, Toulouse, France), mice were kept in an animal room with a light / dark schedule of 12 hours / 12 hours. At the start of the dietary intervention, all animals were 8 weeks old and fed ad libitum with normal chow (#V1535R / MH, Ssniff, Germany). All animal experimental procedures were performed in accordance with the guidelines of the animal experimentation institution approved by the local animal care ethics committee and in accordance with the guidelines of the European Parliament's Directive 2010 / 63 / EU on the protection of animals used for scientific purposes or the NIH guidelines.

[0259] A mouse model of diet-induced NASH-related liver fibrosis.

[0260] Eight-week-old mice were fed a choline-deficient, L-amino acid defined high-fat diet (CDAHFD#A06071302, Research Diet, USA) consisting of 60 kcal% fat and 0.1% methionine by weight for 6 weeks

[11] . During the last 2 weeks of the 6-week period, one group of mice was implanted with an osmotic pump containing the peptide of formula (I). Briefly, a 200 μL osmotic pump was filled with 10 mg / mL of the peptide of formula (I) in PBS and the pump was placed in the diaphragm according to the manufacturer's instructions ( Model pump #2002) was implanted subcutaneously into mice to ensure release of the peptide of formula (I) at an estimated rate of 0.5 μL / hour, which corresponds to an estimated delivery of 5 mg of the peptide of formula (I) per kilogram of body weight per day (5 mg / kg BW / day). A control group consisted of mice that underwent the same surgical procedure for osmotic pump implantation (sham-operated mice).

[0261] Liver Histology. After the treatment period, mice were fasted overnight, anesthetized by intraperitoneal injection of ketamine and xylazine hydrochloride, and then sacrificed by exsanguination. Liver main lobe samples were fixed with paraformaldehyde, embedded in paraffin, and then cut into 5 μm sections, which were then dewaxed and rehydrated. Fibrosis was assessed by Sirius red staining. Briefly, sections were incubated in 1% Sirius red (Sigma-Aldrich) dissolved in saturated picric acid for 10 minutes and then rinsed with distilled water. The sections were then dehydrated with anhydrous ethanol for 15 minutes and washed with water. The slides were incubated with a clearing agent (Euromedex, France), then mounted with distyrene plasticizer xylene (DPX) and covered with a cover slip. After staining, the slides were scanned using a NanoZoomer 2.0RS (Hamamatsu, Japan).

[0262] Hepatic hydroxyproline quantification.

[0263] Liver hydroxyproline was determined by hydrolyzing 80-140 mg of liver in 6N HCl solution overnight at 110°C. The sample was diluted in citrate-acetate buffer and treated with chloramine T (Sigma-Aldrich-Aldrich) and 4-(dimethyl)aminobenzaldehyde (Sigma-Aldrich-Aldrich). The absorbance was measured at 550 nm and the results were expressed as μg liver hydroxyproline / mg tissue.

[0264] result :

[0265] The results are shown in Figure 13Two weeks of subcutaneous infusion of the peptide of formula (I) significantly attenuated the increase in liver fibrosis induced by CDAHFD diet in mice. First, histological examination of the mouse liver by Sirius red staining ( Figure 13 A, representative images) show that mice treated with the peptide of formula (I) had less collagen deposition than untreated sham-operated mice (p<0.01 compared to sham-operated mice, Figure 13 B). Second, liver fibrosis was assessed by measuring the liver content of the fibrosis marker hydroxyproline. As reported in Figure 14C, mice treated with the peptide of formula (I) had a greater than 35% reduction in the concentration of hydroxyproline per mg of liver (p < 0.05 compared to sham-operated mice).

[0266] in conclusion :

[0267] Treatment with the peptides of formula (I) demonstrated benefits on NASH-associated fibrosis.Thus, the peptides of formula (I) are good candidates for treating and reversing liver fibrosis, particularly in non-alcoholic steatohepatitis (NASH).

[0268] Sequence Listing

[0269]

[0270] References

[0271] [1]Martinez et al.2003.Nature421;75-79

[0272] [2]Jacquet et al.2005Cell Mol Life Sci 62;2508-2515

[0273] [3]Fabre et al.Hepatology 52;1477-1483

[0274] [4]Smith et al.Curr Opin Investig Drugs.2010Sep;11(9):989-996

[0275] [5]Cabou et al.2019.Acta Physiol(Oxf).;226(3):e13268

[0276] [6]Martinez et al.2015.Atherosclerosis.Jan;238(1):89-100

[0277] [7]Martinez et al.2003.Nature 421;75-79

[0278] [8]Iwakiri Y.et al.Trends Pharmacol Sc.2015Aug;36(8):524-36

[0279] [9]Musso,G.et al.2013.Prog.Lipid Res.52,175-191

[0280]

[10] Min,H.K.et al.2012.Cell Metab.15,665-674

[0281]

[11] Matsumoto et al.2013.Int.J.Exp.Path.94,93-103

[0282]

[12] Havel RJ et al.(1955)J.Clin.Invest.34,1345-1353

[0283]

[13] Ioannou G.N.Trends in Endocrinology&Metabolism,February2016,Vol.27,No.2

[0284]

[14] Lichtenstein et al.Cardiovasc Res.2015May 1;106(2):314-23

[0285]

[15] Castaing-Berthou A et al.Cell Physiol Biochem.2017;42(2):579-593

[0286]

[16] Farah C et al.Nat Rev Cardiol.2018;15(5):292-316.

[0287]

[17] Corpechot et al.Clin Res Hepatol Gastroenterol.2012

[0288]

[18] Pasut,G.;Veronese,F.M.(2012).″State of the art in PEGylation:Thegreat versatility achieved after forty years of research″.Journal ofControlled Release.161(2):461-472

[0289]

[19] Rai,AK(2013)J.Bioenerg.Biomembr.45,569e579

[0290]

[20] Cardouat et al.Biochim Biophys Acta Mol Cell Biol Lipids.2017Sep;1862(9):832-841

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the peptide is an activator of F1-ATPase. 2 . The peptide according to claim 1 , wherein the peptide has N-terminal acetylation and / or C-terminal amidation.

3. The peptide according to claim 1 or 2, wherein the peptide is modified by attaching a palmitic acid at the C-terminus of the amino acid sequence.

4. The peptide according to any one of claims 1 to 3, wherein the peptide is of formula (I) or (II): CH3CO-[SEQ ID NO: 1]-NH2 (I) CH3CO-[SEQ ID NO: 1]-K-[palmitoyl]-NH2 (II).

5. A pharmaceutical composition comprising a therapeutically effective amount of the peptide according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient or carrier.

6. Use of the peptide according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating non-alcoholic steatohepatitis (NASH), metabolic syndrome or cholestatic liver disease.

7. A nucleic acid encoding the peptide according to claim 1.

8. A vector comprising the nucleic acid according to claim 7.

9. A cell comprising the nucleic acid according to claim 7 or the vector according to claim 8.

Images