Treatment of nonalcoholic fatty liver disease

Abstract

The applicant discovered that the polypeptide SEQUENCE ID NO: 1 (WKDEAGKPLVK) mediates changes in the activity of key biomarkers associated with NASH (Table 1), and that the polypeptide is able to penetrate HepG2 hepatocytes in a hepatocyte penetration assay (Figure 1). In addition, the applicant demonstrated that treatment with pep_260 (SEQ ID 1) for 44 days significantly reduced macrovesicular steatosis in obese diabetic KKAy mice (Figure 2). In a first aspect, the present invention relates to a polypeptide comprising SEQUENCE ID NO: 1, or a functional (or therapeutically effective) variant or functional fragment of SEQUENCE ID NO: 1 (referred to as a "polypeptide active agent" or "polypeptide of the present invention"), for use in treating or preventing non-alcoholic fatty liver disease (NAFLD), particularly non-alcoholic steatohepatitis (NASH), in mammals.

Description

Technical Field

[0001] The present invention relates to the treatment of non-alcoholic fatty liver disease (NAFLD) in mammals, in particular to the treatment of non-alcoholic steatohepatitis (NASH). Background Art

[0002] NASH is a widespread, serious and "silent" liver disease that can lead to cirrhosis or cancer and affects millions of people worldwide - including children.

[0003] NASH is an imminent public health threat not only because of the high cost of liver transplantation (approximately $800,000 per patient in the United States) and related complications, but also because NASH is closely associated with non-liver diseases such as cardiovascular events, which are the leading cause of death in NASH patients. NASH remains little known to the public because it is a silent disease that does not show symptoms, which means that most NASH patients are not diagnosed. NASH is underdiagnosed because it is difficult to detect due to the lack of simple, accurate and cost-effective diagnostic solutions. People with NASH must overcome the stigma and misconceptions associated with the disease. They also lack easy-to-digest information to help them explain their condition to relatives, friends and colleagues, who often struggle to understand the nature and consequences of the condition;

[0004] Due to limited access to information and educational resources about the disease, the medical community – including diabetologists, endocrinologists, obesity specialists, cardiologists, obstetricians and gynecologists, general practitioners and nurses – remains largely uninformed about the disease, with the exception of a small number of international experts.

[0005] While there are still no approved treatments, several cutting-edge development programs are underway, offering hope to millions of clinicians and patients worldwide.

[0006] The present invention aims to solve at least one of the above-mentioned problems. Summary of the Invention

[0007] The applicant found that the peptide SEQUENCE ID NO: 1 (WKDEAGKPLVK) mediated changes in the activity of key biomarkers associated with NASH (Table 1), and that the peptide was able to penetrate HepG2 hepatocytes in a hepatocyte penetration assay ( Figure 1 ). In addition, the applicant demonstrated that treatment with SEQ ID 1 for 44 days significantly reduced macrovesicular steatosis in obese diabetic KKAy mice ( Figure 2 ).

[0008] The applicant also provides a variant of SEQ ID 1, {d}W{d}KDE{d}AGKPL{d}V{d}K (SEQUENCE ID NO: 2), which has 5 modified residues compared to SEQ ID 1 (residues 1, 2, 5, 10, and 11 are provided in the form of D-amino acids). The variant polypeptide SEQ ID 2 significantly enhances glucose uptake in skeletal muscle cells compared to insulin ( Figure 3 ), significantly reduced HbA1c% (the amount of glucose attached to human red blood cells) compared to liraglutide ( Figure 4 ), and showed T 1 / 2 = 93min optimized PK curve.

[0009] Also provided is a cyclized variant of SEQ ID 1, (1(clac)wKE(Me)EC1GK(Me)PLVk-OH) (SEQUENCE ID NO: 3), which exhibits enhanced in vivo stability and significantly increases glucose uptake in human skeletal muscle cells in vitro ( Figure 5 ).

[0010] Bioactive peptides have a favorable safety profile and possess target-targeting capabilities that can attenuate a wide range of pathways.

[0011] In the first aspect, the present invention relates to a polypeptide comprising SEQUENCE ID NO: 1, or a functional (or therapeutically effective) variant or functional fragment of SEQUENCE ID NO: 1 (hereinafter referred to as "polypeptide active agent" or "polypeptide of the present invention"), for use in treating or preventing non-alcoholic fatty liver disease (NAFLD) in mammals, in particular non-alcoholic steatohepatitis (NASH).

[0012] In another aspect, the present invention relates to a method for treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a polypeptide comprising SEQUENCE ID NO: 1, or a functional (or therapeutically effective) variant or functional fragment of SEQUENCE ID NO: 1 (hereinafter referred to as a "polypeptide active agent").

[0013] The polypeptide (or functional variant or fragment) can be administered alone or in combination with other co-drugs that provide enhanced therapeutic effects, including but not limited to drugs with proven efficacy in the treatment of diabetes or obesity.

[0014] In one embodiment, the polypeptide has up to 40, 35, 30, 25, 20, or 15 amino acids. In one embodiment, the polypeptide has 11-15 amino acids. In one embodiment, the polypeptide consists essentially of SEQUENCE ID NO: 1.

[0015] In one embodiment, the polypeptide variant has 1-6 modifications compared to SEQUENCE ID NO: 1, each modification generally independently selected from insertions, additions, deletions and substitutions (ideally conservative substitutions). In one embodiment, one or more amino acids (e.g., 1-5, 1-4, -1-3, or 1-2) are replaced by D-amino acids. In one embodiment, one or more of residues 1, 2, 5, 10, 11 are replaced by D-amino acids, e.g., 2, 3, 4, or 5 residues are replaced. In one embodiment, one or more amino acids are replaced by conservative amino acid substitutions. In one embodiment, the functional variant has the sequence {d}W{d}KDE{d}AGKPL{d}V{d}K (SEQUENCE ID NO: 2), which is identical to SEQ ID 1 except that amino acids 1, 2, 5, 10, and 11 are replaced by D-amino acids.

[0016] In one embodiment, the polypeptide is modified. In one embodiment, the polypeptide is a recombinant polypeptide. In one embodiment, the polypeptide is cyclized. An example of a cyclized polypeptide is (1(clac)wKE(Me)EC1GK(Me)PLVk-OH) - SEQ ID 3. In this variant, residues "w" and "k" are D-amino acids, residues "E" and "P" are methylated, and the polypeptide includes a thioether cyclization between the n-terminus and the cysteine ​​residue, where "1(clac)" and "C1" refer to the ends of the ring.

[0017] In another embodiment, the polypeptides can be administered alone or in combination with other co-drugs that provide enhanced therapeutic effects, including but not limited to drugs with established efficacy in the treatment of diabetes or obesity.

[0018] In another aspect, the present invention provides a polypeptide having the sequence {d}W{d}KDE{d}AGKPL{d}V{d}K (SEQ ID 2). In another embodiment, the present invention provides a cyclized polypeptide (1(clac)wKE(Me)EC1GK(Me)PLVk-OH) - SEQ ID 3.

[0019] The polypeptide of the present invention may be a composition or a pharmaceutical composition comprising the polypeptide of the present invention.

[0020] In another aspect, the present invention provides nucleic acids encoding the polypeptides of the present invention.

[0021] In another aspect, the present invention provides an expression vector comprising a DNA encoding the polypeptide of the present invention, wherein the vector is configured to heterologously express the polypeptide of the present invention in a host cell (hereinafter referred to as "the expression vector of the present invention").

[0022] In another aspect, the present invention provides a host cell, particularly a bacterial or mammalian production cell, which is engineered to heterologously express a polypeptide of the present invention (hereinafter referred to as a "transformed cell of the present invention"). In one embodiment, the transformed host cell comprises an expression vector of the present invention.

[0023] Further aspects and preferred embodiments of the invention are defined and described in the further claims listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Cell permeabilization in hepatocytes HepG2 hepatocytes were treated with Cy5-labeled pep_260 (SEQ ID 1) or treated with Cy5 alone (untreated) for 60 min.

[0025] Figure 2 : Effect of pep_260 (SEQ ID 1) treatment on hepatic steatosis in KKAy mice. Liver tissues from each treatment group were scored for NALFD.

[0026] Figure 3 : Effect of the variant polypeptide (pep_yxww2f=SEQ ID 2) on glucose uptake in skeletal muscle cells compared to insulin and the polypeptide of the present invention (pep_1E99R5=SEQ ID 1).

[0027] Figure 4 (A) Effect of the variant peptide (pep_yxww2f = SEQ ID 2) on glycosylated hemoglobin (HbA1c%) in skeletal muscle cells compared to liraglutide. (B) Signatures were scored using the rat liver scoring system designed by Sherwani SI et al. (Sherwani SI, et al., Significance of HbA1c Test in Diagnosis and Prognosis of Diabetic Patients. Biomark Insights. 2016; 11:95-104). Data are mean ± SEM (n = 6 per group; baseline = 12 weeks of age) and were analyzed by Dunnett's test to compare differences between the two peptide-treated groups, the vehicle control group, and the liraglutide group (*p < 0.05, **p < 0.01, ***p < 0.001).

[0028] Figure 5: Effects of the variant cyclized polypeptide (pep_DJKTTM=SEQ ID 3) on glucose uptake in skeletal muscle cells compared to a control group and the polypeptide of the present invention (pep_1E99R5=SEQ ID 1).

[0029] Figure 6 HepG2 cells treated with pep_1E99R5 (pep_1E99R5 = SEQ ID 1) or 10PANX reduced IL-8 secretion (measured by sandwich ELISA). Cells were treated with peptide (5 ng / mL), 10PANX (μg / mL), or PBS for 24 h and then stimulated with 100 ng / mL LPS for an additional 24 h. Data are presented as mean ± SD of three independent experiments. ***p ≤ 0.001 between groups.

[0030] Figure 7 pep_1E99R5 exhibits antifibrotic activity in stimulated primary human hepatic stellate cells. (A) Confocal imaging of human stellate cells treated with TGF-β to stimulate α-SMA expression and then treated with elafibranor (10 μM) or pep_1E99R5 (5 nM). (B) Cell size is quantified in pixels for each treatment condition; all conditions include cells from three independent replicates. Untreated: × cells, untreated + TGF-β × cells, elafibranor × cells. (A) *p ≤ 0.05 between groups, **p ≤ 0.01 between groups, ***p ≤ 0.001 between groups.

[0031] Figure 8 : Liver enzyme changes in a mouse model of acute liver injury induced by acetaminophen (APAP). APAP was administered by IP (intraperitoneal) injection at 0 hours. Pep_1E99R5, 10PANX, or saline control treatment was administered by IV (intravenous injection) 1.5 hours later. Each group included 5 animals. ALT and AST levels were measured in peripheral blood of all surviving mice at 2.25 and 6 hours. At 2.25 hours, administration of pep_1E99R5 significantly reduced ALT levels compared to APAP / saline treatment (p < 0.05) and was superior to 10PANX. The APAP / saline group showed increases in both ALT and AST compared to the saline / saline group. Data are mean ± SEM and analyzed by T-test, followed by multiple comparison tests as appropriate. DETAILED DESCRIPTION

[0032] All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and were set forth in its entirety.

[0033] Definition and general preferences

[0034] Unless expressly stated otherwise, the following terms used herein are intended to have the following meanings, as well as any broader (or narrower) meanings that these terms may have in the art:

[0035] Unless the context requires otherwise, as used herein, the singular shall be understood to include the plural, and vice versa. The term "a" or "an" in relation to an entity shall be understood to refer to one or more of that entity. Thus, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

[0036] As used herein, the term "comprise" or variations thereof such as "comprises" or "comprising" will be understood to mean the inclusion of any recited integer (e.g., features, elements, characteristics, properties, method / process steps, or limitations) or group of integers (e.g., features, elements, characteristics, properties, method / process steps, or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprises" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps.

[0037] As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to include any disorder, disease, abnormality, pathology, discomfort, condition, or syndrome in which physiological function is impaired, regardless of the nature of its cause (or whether an etiological basis for the disease is actually established). Thus, it includes conditions caused by infection, trauma, injury, surgery, radiation ablation, age, toxicity, or nutritional deficiency.

[0038] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administering a pharmaceutical agent to a subject) that cures, ameliorates, or alleviates the symptoms of a disease or eliminates its cause (or alleviates the effects of its cause) (e.g., reducing the pathological level of a lysosomal enzyme). In this case, the term is used synonymously with the term "therapy."

[0039] In addition, the term "treatment" or "treating" refers to an intervention (e.g., administering a pharmaceutical agent to a subject) that prevents or delays the onset or progression of a disease or reduces (or eliminates) its incidence in a treated population. In this context, the term treatment is used synonymously with the term "prophylaxis."

[0040] As used herein, an effective amount or therapeutically effective amount of a medicament defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio, but sufficient to provide the desired effect, such as treatment or prevention manifested as permanent or temporary improvement of the subject's condition. The dosage varies from subject to subject, depending on the subject's size, age and general condition of the individual, the mode of administration and other factors. Therefore, although it is impossible to specify an exact effective amount, those skilled in the art will be able to determine the appropriate "effective" amount in any individual case using routine experiments and background knowledge. In this case, the therapeutic outcome includes elimination or alleviation of symptoms, relief of pain or discomfort, prolonged survival, improved mobility and other clinical signs of improvement. The therapeutic outcome is not necessarily a complete cure. Improvement may be observed in biological / molecular markers, clinical or observational improvement. In a preferred embodiment, the method of the present invention is applicable to humans, large racing animals (horses, camels, dogs) and domestic companion animals (cats and dogs).

[0041] In the context of the treatment and effective amount defined above, the term subject (which will be interpreted as including "individual," "animal," "patient," or "mammal" where the context permits) defines any subject, particularly a mammalian subject for whom treatment is to be given. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sports animals, pet animals (such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, domestic cattle, cows), primates (such as apes, monkeys, gorillas, and chimpanzees), canines (such as dogs and wolves), felines (such as cats, lions, and tigers), equines (such as horses, donkeys, and zebras), food animals (such as cows, pigs, and sheep), ungulates (such as deer and giraffes), and rodents (such as mice, rats, hamsters, and guinea pigs). In a preferred embodiment, the subject is a human. As used herein, the term "equine" refers to mammals of the equine family, including horses, donkeys, asses, wild donkeys, and zebras.

[0042] "Pharmaceutical composition": Another aspect of the present invention relates to a pharmaceutical composition comprising a polypeptide active agent, mixed with one or more pharmaceutically acceptable diluents, excipients or carriers, or administered in combination with other drugs that enhance the therapeutic effect. Even though the polypeptide active agent can be administered alone, it is typically administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human treatment. The pharmaceutical composition can be used for human or animal use in human medicine and veterinary medicine. Examples of suitable excipients for various forms of pharmaceutical compositions described herein can be found in the Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), edited by A Wade and PJ Weller. Specifically, formulations for topical delivery are described in Topical drug delivery formulations, edited by David Osborne and Antonio Aman, Taylor & Francis, the entire contents of which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in "Remington's Pharmaceutical Sciences" (Remington's Pharmaceutical Sciences, Mack Publishing Co., edited by A.R. Gennaro, 1985). Suitable carrier examples include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Suitable diluent examples include ethanol, glycerol, and water. The selection of pharmaceutical carriers, excipients, or diluents can be selected according to the intended route of administration and standard pharmaceutical practice. In addition to carriers, excipients, or diluents, pharmaceutical compositions may include, for example, any suitable adhesive, lubricant, suspending agent, coating agent, solubilizing agent. Suitable adhesive examples include starch, gelatin, natural sugars (such as glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweeteners), natural and synthetic gums (such as gum arabic, tragacanth, or sodium alginate), carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, dyes, and even flavorings may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and parahydroxybenzoates. Antioxidants and suspending agents may also be used.

[0043] As used herein, an "effective amount" or "therapeutically effective amount" of a polypeptide active agent is defined as an amount that can be administered to a subject without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio, but sufficient to provide the desired effect, such as treatment or prevention manifested as permanent or temporary improvement of the subject's condition. The dosage varies from subject to subject, depending on the age and general condition of the individual, the mode of administration and other factors. Therefore, while it is not possible to specify an exact effective amount, a person skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background common sense. In this context, therapeutic outcomes include elimination or alleviation of symptoms, relief of pain or discomfort, prolonged survival, improved mobility and other signs of clinical improvement. The therapeutic outcome is not necessarily a complete cure.

[0044] The term "polypeptide" as used herein refers to a polymer consisting of up to 50 amino acids, for example, 5 to 50 amino acid monomers are typically connected by peptide bonds. The polypeptide of the present invention and used for the present invention (including its fragments and variants) can be produced in whole or in part by chemical synthesis or by expression of nucleic acids. For example, according to the standard liquid phase or preferred solid phase polypeptide synthesis method recognized in the art, the polypeptide of the present invention and used for the present invention can be easily prepared, for example, referring to "Solid Phase Peptide Synthesis" (JM Stewart and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984)), and referring to "The Practice of Peptide Synthesis" (M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984)). If necessary, any polypeptide used in the present invention can be chemically modified to increase its stability. Chemically modified polypeptides or polypeptide analogs include any functional chemical equivalents of the polypeptide, characterized in that: with respect to the implementation of the present invention, its stability and / or efficacy in vivo or in vitro increase. The term polypeptide analog also refers to any amino acid derivative of a polypeptide described herein. Polypeptide analogs can be prepared by, but are not limited to, modification of the side chains, incorporation of non-natural amino acids and / or their derivatives during polypeptide synthesis, and the use of cross-linking agents and other methods to impose conformational constraints on the polypeptide or its analogs. Examples of side chain modifications include amino modifications, such as reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methyl acetimidate; acetylation with acetic anhydride; carbamylation of the amino group with a cyanate; trinitrobenzylation of the amino group with 2,4,6, trinitrobenzenesulfonic acid (TNBS); aminoalkylation with succinic anhydride and tetrahydrophthalic anhydride; and pyroxylation of lysine with 5'-pyridoxal phosphate followed by reduction with NABH4. The guanidine group of an arginine residue can be modified by forming a heterocyclic condensation product with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal. Carboxyl groups can be modified by activation with carbodiimides to form o-acylisoureas followed by derivatization (e.g., to the corresponding amides). Sulfhydryl groups can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation with performic acid to cysteine; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; formation of mercury derivatives using 4-chloromercuric benzoate, 4-chloromercuric benzenesulfonic acid, phenylmercuric chloride, 2-chloromercuric-4-nitrophenol, and other mercury compounds; and carbamylation with cyanates at alkaline pH.Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides. Tyrosine residues can be modified by nitration with tetranitromethane to form 3-nitrotyrosine derivatives. Modification of the imidazole ring of histidine residues can be accomplished by alkylation with iodoacetic acid derivatives or N-ethoxylation with diethyl pyrocarbonate. Examples of incorporation of unnatural amino acids and derivatives during polypeptide synthesis include, but are not limited to, the use of norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, tert-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids. Modifications of polypeptide structure include the generation of retro-inverso peptides containing reverse sequences encoded by D-amino acids. The changes may be to reduce susceptibility to proteolysis, reduce susceptibility to oxidation, alter the binding affinity of the variant sequence (usually desirably to increase affinity) and / or confer or modify other physicochemical or functional properties of the variant / polypeptide analog in question.

[0045] The term "therapeutically effective variant" as applied to a reference polypeptide refers to a polypeptide having substantially the same amino acid sequence as the reference polypeptide and having therapeutic effectiveness as defined below. Thus, for example, the term should be understood to include variants that have been altered in one or more amino acid residues. Preferably, such alterations include insertions, additions, deletions, and / or substitutions of 6 or fewer amino acids, preferably 5 or fewer, 4 or fewer, even more preferably 3 or fewer, and most preferably 1 or 2 amino acids. Insertions, additions, and substitutions with both natural and modified amino acids are contemplated. Variants may have conservative amino acid changes, wherein the introduced amino acid is structurally, chemically, or functionally similar to the substituted amino acid. Typically, a variant will have at least 50%, 60%, or 70% amino acid sequence identity to the parent sequence, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and ideally at least 95%, 96%, 97%, 98%, or 99% sequence identity. It should be noted that any variant will have essentially the same therapeutic effect, or possibly an enhanced effect, when tested in an in vitro or in vivo disease model.An exemplary variant in which 5 amino acids are replaced with D-amino acids is provided as SEQUENCE ID NO:2.

[0046] "Therapeutically effective" as applied to the polypeptides of the present invention means that the polypeptide is able to penetrate HepG2 hepatocytes in the hepatocyte penetration assay described herein and significantly alleviate macrovesicular steatosis in obese diabetic KKAy mice in the chronic diabetic mouse model described below. In one embodiment, the therapeutically effective polypeptide is able to mediate changes in the activity of all or most of the biomarkers listed in Table 1.

[0047] The term variant is also intended to include the term "fragment," which refers to an amino acid fragment of SEQUENCE ID NO: 1. Typically, the fragment is 3 to 13 consecutive amino acids in length. Typically, the fragment has a charge of -5 to +3. The charge of a polypeptide, fragment, or region is determined using the following method: "Derivation and use of a formula to calculate the net charge of acid-base compounds. Its application to amino acids, proteins and nucleotides" (Cameselle, JC, Ribeiro, JM, and Sillero, A. (1986). Derivation and use of a formula to calculate the net charge of acid-base compounds. Its application to amino acids, proteins and nucleotides. Biochem. Educ. 14, 131–136.).

[0048] In this specification, the term "sequence identity" should be understood to include sequence identity and similarity, that is, a variant (or homolog) having 70% sequence identity with a reference sequence means that any 70% of the aligned residues in the variant (or homolog) are identical or conservatively substituted with the corresponding residues in the reference sequence over the entire sequence length. Sequence identity is the number of characters that completely match between two different sequences. Therefore, gaps are not calculated, and the measurement is performed relative to the shorter of the two sequences.

[0049] With respect to "sequence homology", when the percentage of aligned residues of the variant (or homolog) is identical or conservatively substituted with the corresponding residues in the reference sequence, and the variant (or homolog) has the same function as the reference sequence, the term should be understood as a variant (or homolog) having a certain percentage of similarity or identity with the reference sequence.

[0050] Alignment and percent homology or sequence identity can be determined using software programs known in the art, for example, one alignment program is BLAST using default parameters. Detailed information on these programs can be found on the Internet at: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi.

[0051] "C-terminal domain" as applied to a fragment refers to the first three amino acids at the C-terminus of the fragment.

[0052] "N-terminal domain" as applied to a fragment refers to the last three amino acids at the n-terminus of the fragment.

[0053] "Homologs" of a reference protein are understood to be proteins from different plant species that have at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the reference protein.

[0054] "Pharmaceutical composition": Another aspect of the present invention relates to a pharmaceutical composition comprising a polypeptide active agent, mixed with one or more pharmaceutically acceptable diluents, excipients or carriers, or administered in combination with other drugs that enhance the therapeutic effect. Although the polypeptides and compositions of the present invention can be administered alone, they are typically administered in admixture with pharmaceutical carriers, excipients or diluents, particularly for human treatment. Pharmaceutical compositions can be used for human or animal use in human medicine and veterinary medicine. Examples of suitable excipients for various forms of pharmaceutical compositions of this type described herein can be found in the Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), edited by A. W. De and P. J. Weller. Specifically, formulations for topical delivery are described in Topical drug delivery formulations, edited by David Osborne and Antonio Aman, Taylor & Francis, the entire contents of which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in "Remington's Pharmaceutical Sciences" (Remington's Pharmaceutical Sciences, Mack Publishing Co., edited by A.R. Gennaro, 1985). Suitable carrier examples include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Suitable diluent examples include ethanol, glycerol, and water. The selection of pharmaceutical carriers, excipients, or diluents can be selected according to the intended route of administration and standard pharmaceutical practice. In addition to carriers, excipients, or diluents, pharmaceutical compositions can include, for example, any suitable adhesive, lubricant, suspending agent, coating agent, solubilizing agent. Suitable adhesive examples include starch, gelatin, natural sugars (such as glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweeteners), natural and synthetic gums (such as gum arabic, tragacanth, or sodium alginate), carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, dyes, and even flavorings may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and parahydroxybenzoates. Antioxidants and suspending agents may also be used.

[0055] The polypeptide or composition is suitable for and can be administered by topical, oral, rectal, parenteral, intramuscular, abdominal, arterial, intrabronchial, subcutaneous, intradermal, intravenous, nasal, vaginal, oral or sublingual routes of administration. For oral administration, compressed tablets, pills, tablets, gels, drops and capsules are particularly used. Preferably, these compositions contain 1 to 250 mg, more preferably 10-100 mg, of active ingredient per dose. Other forms of administration include solutions or emulsions, which can be injected intravenously, arterially, subcutaneously, intradermally, intraperitoneally or intramuscularly and are prepared from sterile or sterilizable solutions. The pharmaceutical composition of the present invention can also be in the form of a suppository, vaginal ring, vaginal suppository, suspension, emulsion, lotion, ointment, cream, gel, spray, solution or powder. The composition of the present invention can be formulated for local delivery. Local delivery usually refers to delivery to the skin, but can also refer to delivery to body cavities composed of epithelial cells, such as the lungs or airways, gastrointestinal tract, oral cavity. Particularly, preparations for local delivery are described in "Topical drug delivery formulations" edited by David Osborne and Antonio Aman, Taylor & Francis, the entire contents of which are incorporated herein by reference. Compositions or preparations for delivery to the airways are described in O'Riordan et al. (Respir Care, 2002, Nov. 47), EP2050437, WO2005023290, US2010098660 and US20070053845. Compositions and preparations for delivering active agents to the ileum, especially the proximal ileum, include microparticles and microcapsules, wherein the active agent is encapsulated in a protective matrix formed by a polymer or milk protein, which has acid resistance but is easy to dissolve in the more alkaline environment of the ileum. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1. Another transdermal administration method is to use a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycol or liquid paraffin. The active ingredient can also be incorporated into an ointment at a concentration of 1 to 10% by weight, which ointment consists of a white wax or white soft paraffin base and, if necessary, stabilizers and preservatives.

[0056] Each injection dosage form may contain 10-1000 mg, preferably 10-250 mg, of the active ingredient.

[0057] The compositions may be formulated in unit dosage form, ie, in discrete portions comprising a unit dose or containing multiples or subunits of a unit dose.

[0058] Those of ordinary skill in the art can easily determine the appropriate dosage of a present composition to be applied to a subject without excessive experimentation. Typically, a doctor will determine the actual dosage that best suits an individual patient, depending on a variety of factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, body weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, severity of a specific condition, and the individual being treated. The dosages disclosed herein are examples of average conditions. Of course, there may be individual cases where a higher or lower dosage range is required, and these fall within the scope of the present invention. As needed, the medicament can be administered in a dosage range of 0.01 to 30 mg / kg body weight, for example 0.1 to 10 mg / kg, more preferably 0.1 to 1 mg / kg body weight. In an exemplary embodiment, one or more dosages of 10 to 300 mg / day or more preferably 10 to 150 mg / day are administered to the patient to treat inflammatory diseases.

[0059] In a specific preferred embodiment, the methods and uses of the present invention include administering a polypeptide or composition in combination with one or more other active agents, such as a commercially available NAFLD drug or pharmacological enhancer. In this case, the compound of the present invention can be administered continuously, simultaneously, or sequentially with the one or more other active agents.

[0060] In one embodiment of the present invention, the polypeptide active agent can be administered in the form of a conjugate comprising a polypeptide, a linker, and an antibody molecule (or antibody fragment) intended to increase the half-life of the conjugate in vivo.

[0061] "Modified polypeptide": In one embodiment, a polypeptide of the present invention (including polypeptide variants) can be a modified polypeptide. The term "modified polypeptide" is used interchangeably with the term polypeptide derivative. In one embodiment, the term "modified polypeptide" refers to a polypeptide that has been modified to exhibit one or more of the following properties compared to an unmodified polypeptide: increased plasma half-life; increased lipophilicity of the polypeptide; increased renal clearance of the modified polypeptide; and increased resistance of the modified polypeptide to proteolytic degradation, typically while retaining rpS6 phosphorylation activity. Disclosed herein are various methods for modifying the polypeptides of the present invention to exhibit these properties, including conjugation of the polypeptide to a binding partner (e.g., an albumin-binding small molecule, a large polymer, a long-lived plasma protein, or an antibody or antibody fragment), cyclization, addition of N- or C-termini or side chains, addition of protecting groups, replacement of L-amino acids with D-isomers, amino acid modification, increased plasma protein binding, and increased albumin binding. Modified polypeptides include, but are not limited to, polypeptides that have been substituted with one or more groups as defined herein, or conjugated to a binding partner, or cyclized. Typically, the polypeptide is modified to increase its half-life in an animal. Various modification methods are provided below.

[0062] In one embodiment, the modification can be any modification that provides an increased ability to penetrate cells for a polypeptide and / or composition of the present invention. In one embodiment, the modification can be any modification that increases the half-life of a composition of the present invention or polypeptide. In one embodiment, the modification can be any modification that increases the activity of a composition of the present invention or polypeptide. In one embodiment, the modification can be any modification that increases the selectivity of a composition of the present invention or polypeptide.

[0063] In one embodiment, the group is a protecting group. The protecting group can be an N-terminal protecting group, a C-terminal protecting group, or a side chain protecting group. The polypeptide can have one or more of these protecting groups.

[0064] Those skilled in the art are aware of suitable techniques for reacting amino acids with these protecting groups. These groups can be added by methods known in the art, such as those outlined in paragraphs

[0104] to

[0107] of US2014120141. These groups can remain on the polypeptide or can be removed. Protecting groups can be added during the synthesis process.

[0065] In embodiments of the present invention, the polypeptide may be substituted with one or more linear or branched, long or short chain, saturated or unsaturated, substituted with hydroxyl, amino, aminoacyl, sulfate or sulfide groups, or unsubstituted chains having 1 to 29 carbon atoms. N-acyl derivatives include acyl groups derived from acetic acid, capric acid, lauric acid, myristic acid, caprylic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid, isostearic acid, elaidic acid, 2-ethylhexanoic acid, coconut fatty acid, tallow fatty acid, hardened tallow fatty acid, palm kernel fatty acid, lanolin fatty acid, or similar acids. These may be substituted or unsubstituted. When substituted, they are preferably substituted with hydroxyl or sulfur-containing groups, such as, but not limited to, SO3H, SH, or SS.

[0066] In an embodiment of the invention, the polypeptide is R1-X-R2.

[0067] The R1 and / or R2 groups are bound to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the polypeptide sequence, respectively.

[0068] In one embodiment, the polypeptide is R1-X. Alternatively, the polypeptide is X-R2.

[0069] Preferably, R1 is H, C1-4 alkyl, acetyl, benzoyl or trifluoroacetyl;

[0070] X is a polypeptide of the present invention;

[0071] R2 is OH or NH2.

[0072] In an embodiment, R1 is selected from the group consisting of H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroaralkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, tert-butyloxycarbonyl groups, 9-fluorenylmethoxycarbonyl groups (Fmoc), and R5-CO-, wherein R5 is selected from the group consisting of H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, substituted or unsubstituted heterocyclic groups, and substituted or unsubstituted heteroaralkyl groups;

[0073] R2 is selected from the group consisting of: -NR3R4, -OR3 and -SR3, wherein R3 and R4 are independently selected from the group consisting of: H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroarylalkyl groups, substituted or unsubstituted aryl groups and substituted or unsubstituted arylalkyl groups; and under the condition that R1 and R2 are not α-amino acids.

[0074] According to another preferred embodiment, R2 is -NR3R4, -OR3 or -SR3, wherein R3 and R4 are independently selected from the group consisting of H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C24 cycloalkyl, substituted or unsubstituted C5-C24 cycloalkenyl, substituted or unsubstituted C8-C24 cycloalkynyl, substituted or unsubstituted C6-C30 aryl, substituted or unsubstituted C7-C24 aralkyl, substituted or unsubstituted 3-10 membered heterocyclyl ring, and substituted or unsubstituted heteroaralkyl of 2 to 24 carbon atoms and 1 to 3 atoms other than carbon, wherein the alkyl chain has 1 to 6 carbon atoms. Optionally, R3 and R4 can be combined through a saturated or unsaturated carbon-carbon bond to form a ring with the nitrogen atom. More preferably, R2 is -NR3R4 or -OR3, wherein R3 and R4 are independently selected from the group consisting of: H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C15 aryl and substituted or unsubstituted 3-10 membered heterocyclyl, substituted or unsubstituted heteroarylalkyl with a 3 to 10 membered ring and an alkyl chain of 1 to 6 carbon atoms. More preferably, R3 and R4 are selected from the group consisting of: H, methyl, ethyl, hexyl, dodecyl or hexadecyl. Even more preferably, R3 is H and R4 is selected from the group consisting of: H, methyl, ethyl, hexyl, dodecyl or hexadecyl. According to an even more preferred embodiment, R2 is selected from -OH and -NH2.

[0075] According to another embodiment of the present invention, R1 is selected from the group consisting of: H, acetyl, lauroyl, myristoyl or palmitoyl, and R2 is -NR3R4 or -OR3, wherein R3 and R4 are independently selected from the group consisting of: H, methyl, ethyl, hexyl, dodecyl and hexadecyl, preferably R2 is -OH or -NH2. More preferably, R1 is acetyl or palmitoyl, and R2 is -NH2.

[0076] In a preferred embodiment, the acyl group is bound to at least one amino acid at the N-terminus of the polypeptide.

[0077] In an embodiment of the present invention, the polypeptide is modified to include a side chain protecting group. The side chain protecting group can be one or more of the following groups, including: benzyl or benzyl-based groups, tert-butyl-based groups, benzyloxy-carbonyl (Z) groups, and allyloxycarbonyl (alloc) protecting groups. The side chain protecting group can be derived from an achiral amino acid, such as achiral glycine. The use of achiral amino acids helps to stabilize the polypeptide thus prepared and also helps the simple synthetic route of the present invention. Preferably, the polypeptide also includes a modified C-terminus, preferably an amidated C-terminus. The achiral residue can be α-aminoisobutyric acid (methylalanine). It should be understood that the specific side chain protecting group used will depend on the sequence of the polypeptide and the type of N-terminal protecting group used.

[0078] In one embodiment of the present invention, the polypeptide is conjugated, linked or fused to one or more polyethylene glycol (PEG) polymers or other compounds (e.g., molecular weight-increasing compounds). A molecular weight-increasing compound is any compound that can increase the molecular weight of the resulting conjugate, typically by 10% to 90%, or 20% to 50%, and the resulting conjugate can have a molecular weight of 200 to 20,000, preferably 500 to 10,000. The molecular weight-increasing compound can be PEG, any water-soluble (amphiphilic or hydrophilic) polymer moiety, a homopolymer or copolymer of PEG, a monomethyl-substituted polymer of PEG (mPEG) and polyoxyethylene glycerol (POG), polyamino acids (e.g., polylysine, polyglutamic acid, polyaspartic acid, particularly polyamino acids in the L conformation), pharmacologically inactive proteins (e.g., albumin), gelatin, fatty acids, oligosaccharides, lipoamino acids, and dextran. The polymer portion may be linear or branched, and may have a molecular weight of 500 to 40,000 Da, 5,000 to 10,000 Da, or 10,000 to 5,000 Da. The compound may be any suitable cell penetrating compound, such as a tat polypeptide, a penetratin, or pep-1. The compound may be an antibody molecule. The compound may be a lipophilic group or a polymeric group.

[0079] Lipophilic substituents and polymeric substituents are known in the art. Lipophilic substituents include acyl groups, sulfonyl groups, N atoms, O atoms, or S atoms that form a portion of an ester, sulfonyl ester, thioester, amide, or sulfonamide. The lipophilic portion may include a hydrocarbon chain having 4 to 30 carbon atoms, preferably 8 to 12 carbon atoms. It may be straight or branched, saturated or unsaturated. The hydrocarbon chain may be further substituted. It may be a cycloalkane or a heterocycloalkane.

[0080] The polypeptide may be modified at the N-terminus, the C-terminus, or both. The polymer or compound is preferably linked to an amino, carboxyl, or sulfhydryl group and may be linked via the N-terminus or C-terminus of any amino acid residue side chain. The polymer or compound may be conjugated to the side chain of any suitable residue.

[0081] The polymer or compound can be conjugated via a spacer. The spacer can be a natural or non-natural amino acid, succinic acid, lysyl, glutamyl, aspartyl, glycyl, β-alanyl, γ-aminobutyryl. The polymer or compound can be conjugated via an ester, sulfonyl ester, thioester, amide, carbamate, urea, or sulfonamide. Those skilled in the art will appreciate suitable methods for preparing the conjugate.

[0082] For example, polypeptides can be chemically modified by covalently conjugating to polymers to increase their circulation half-life. Exemplary polymers and methods for attaching such polymers to polypeptides are described, for example, in U.S. Patent Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546. Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) moieties.

[0083] The polypeptide of the present invention can be subjected to one or more modifications to control any aspect of the storage stability, pharmacokinetics and / or biological activity of the polypeptide, such as efficacy, selectivity, interaction with drugs. Acceptable chemical modifications of polypeptides include, but are not limited to, polypeptides conjugated to one or more of the following compounds: polyethylene glycol (PEG), methoxy-polyethylene glycol, dextran, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polypropylene glycol, polyoxyethylated polyols (such as glycerol) and polyvinyl alcohol, colonic acid or other carbohydrate-based polymers, amino acid polymers and biotin derivatives. Proteins conjugated to PEG at Cys residues have been disclosed, for example, as disclosed in Goodson, RJ & Katre, NV (1990) Bio / Technology 8, 343, and Kogan, TP (1992) Synthetic Comm. 22, 2417.

[0084] The polypeptide of modification can also include a sequence in which one or more residues are modified (i.e., by phosphorylation, sulfation, acylation, pegylation, etc.), and a mutant comprising one or more modified residues relative to the parental sequence. Amino acid sequence can also be modified with a marker that can directly or indirectly provide a detectable signal, including but not limited to radioisotopes, fluorescent markers, and enzyme labels. Fluorescent markers include, for example, Cy3, Cy5, Alexa, BODIPY, fluorescein (such as FluorX, DTAF, and FITC), rhodamine (such as TRITC), auramine, Texas Red (Texas Red), AMCA Blue, and Lucifer Yellow. Preferred isotope labels include 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 286Re. Preferred enzyme labels include peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase, and alkaline phosphatase (see, e.g., U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043). Enzymes can be conjugated by reacting with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde, and the like. Enzyme labels can be detected visually or measured by calorimetry, spectrophotometry, fluorescence spectrophotometry, amperometry, or gasometric techniques. Other labeling systems, such as avidin / biotin, tyramide signal amplification (TSA), and the like, can be used to identify the enzymes that are present in the sample. TM ), are known in the art and commercially available, (see, for example, ABC kit, Vector Laboratories, Inc., Burlingame, Calif.; NEN Life Science Products, Inc., Boston, Mass.).

[0085] In an embodiment, polypeptides, variants and / or compositions are modified to increase the performance capabilities of the drug. In an embodiment, polypeptides, variants and / or compositions are modified to increase stability, permeability, maintain efficacy, avoid toxicity and / or increase half-life. Modification can be performed in the manner described above. For example, modification can be to protect the N and C-termini, can be modified amino acids, cyclization, replacement amino acids, and / or conjugated to macromolecules or large polymers or long-lived plasma proteins. Strategies for extending half-life can be described by Strohl et al. (BioDrugs, 2015), Schlapschy et al. (Protein Eng Des Sel. 2013), Podust, VN et al. (Protein Eng Des Sel. 2013), Zhang, L et al. (Curr Med Chem. 2012), Gaberc-Porekar, V et al. (Curr Opin Drug Discov Devel. 2008). Examples include the use of PEGylation (polyethylene glycolation), lipidation (covalent attachment of fatty acids to polypeptide side chains), fusion to Fc domains and human serum albumin, fusion to hydrophilic amino acid polymers (e.g., XTEN or PAS), and / or fusion to half-life extending proteins.

[0086] Modifications of polypeptides to extend their in vivo half-life are described, for example, in:

[0087] "Strategies to improve plasma half-life of peptide and protein drugs." Werle M, Bernkop-Schnürch A. Amino Acids. 2006 Jun; 30(4): 351-67.

[0088] Given the significant advantages of long-acting peptide and protein drugs, strategies to extend the plasma half-life of such compounds are highly desirable. Plasma half-lives are typically short due to rapid renal clearance and enzymatic degradation during systemic circulation. Modification of peptides / proteins can lead to extended plasma half-lives. The derivative octreotide, developed by shortening the total amino acid sequence of somatostatin and replacing L-amino acids with D-amino acids, has a plasma half-life of 1.5 hours, compared to only a few minutes for somatostatin. A PEG(2,40K) conjugate of INF-α-2b has a plasma half-life 330-fold longer than that of the native protein. This review aims to outline potential strategies for extending plasma half-life, such as modification of the N- and C-termini or PEGylation, and to outline methods for evaluating the effectiveness of drug modifications. Furthermore, basic data on the most important proteolytic enzymes in human blood, liver, and kidney, their cleavage specificities, and inhibitors are provided to predict enzymatic cleavage of peptide and protein drugs during systemic circulation.

[0089] “Strategic Approaches to Optimizing Peptide ADME Properties.” Li Di AAPS J. 2015 Jan;17(1):134–143.

[0090] Strategies for stabilizing peptides from proteolysis

[0091] Numerous approaches are available to enhance peptide stability through structural modification. Some methods not only improve stability but also enhance other ADME (absorption, distribution, metabolism, excretion) properties. For example, cyclization can improve stability and permeability, while conjugation to macromolecules can improve stability and reduce renal clearance. Maintaining potency and avoiding toxicity while improving peptide stability and ADME properties is crucial.

[0092] Protect N-terminus and C-terminus

[0093] Many proteolytic enzymes found in blood / plasma, liver, or kidneys are exopeptidases, aminopeptidases, and carboxypeptidases, which cleave peptide sequences at the N- and C-termini. Modification of the N- or C-termini can often improve peptide stability. Numerous examples of N-acetylation and C-amidation increasing the resistance of proteins to hydrolysis have been reported.

[0094] Substituting D-amino acids for L-amino acids

[0095] Substituting natural L-amino acids with unnatural D-amino acids can reduce substrate recognition and binding affinity of proteolytic enzymes and increase stability. An example is vasopressin, which contains L-Arg, which has a half-life of 10–35 minutes in humans. Its D-Arg analog, desmopressin, has a half-life of 3.7 hours in healthy human volunteers. In studies of bicyclic peptide inhibitors of the cancer-associated protease urokinase-type plasminogen activator (uPA), replacing a specific glycine with D-serine not only increased its potency by 1.8-fold in mouse plasma but also improved its stability by 4-fold.

[0096] Amino acid modification

[0097] Modification of natural amino acids can improve the stability of polypeptides by introducing steric hindrance or disrupting enzyme recognition. For example, gonadotropin-releasing hormone has a very short half-life (a few minutes), while buserelin, in which one Gly is replaced by tert-butyl-D-Ser and another Gly is replaced by acetamide, has a significantly longer half-life in humans.

[0098] Cyclization

[0099] The polypeptides of the present invention can be cyclized. Cyclization introduces conformational constraints, reduces the flexibility of the polypeptide, and increases stability and permeability. Depending on the functional groups, the polypeptide can be cyclized to head to tail, head / tail to side chain, or side chain to side chain. Cyclization is usually accomplished by lactamization, lactonization, and sulfide bridging. Disulfide bonds produce folding and conformational constraints, which can improve efficacy, selectivity, and stability. Many disulfide-rich polypeptides are already on the market or in preclinical or clinical development, such as linaclotide, lepirudin, and ziconotide. In one embodiment, the polypeptide is cyclized between the amino and carboxyl termini of the polypeptide. In one embodiment, the polypeptide is cyclized between the amino terminus and the side chain. In one embodiment, the polypeptide is cyclized between the carboxyl terminus and the side chain. In one embodiment, the polypeptide is cyclized between the side chains. In one embodiment, the cyclized polypeptide is selected from pure cyclic peptides, cyclized isomeric peptides, cyclized decapeptides, or monocyclic or bicyclic peptides. The cyclization method of the polypeptide is described as follows:

[0100] "Peptide and Protein Design for Biopharmaceutical Applications" (Jensen, Knud (2009-09-01). Peptide and Protein Design for Biopharmaceutical Applications. John Wiley & Sons. ISBN 9780470749715.);

[0101] Wenyan, Xu; Jun, Tang; Changjiu, Ji; Wenjun, He; Ninghua, Tan (2008). "Application of a TLC chemical method to detection of cyclotides in plants". Science Bulletin. 53(11): 1671–1674. doi: 10.1007 / s11434-008-0178-8.

[0102] Borthwick AD (May 2012). "2,5-Diketopiperazines: Synthesis, Reactions, Medicinal Chemistry, and Bioactive Natural Products". Chemical Reviews. 112(7):3641–3716. doi:10.1021 / cr200398y. PMID 22575049.

[0103] Barber, Carla JS; Pujara, Pareshkumar T.; Reed, Darwin W.; Chiwocha, Shiela; Zhang, Haixia; Covello, Patrick S. (2013). "The Two-step Biosynthesis of Cyclic Peptides from Linear Precursors in a Member of the Plant Family Caryophyllaceae Involves Cyclization by a Serine Protease-like Enzyme". Journal of Biological Chemistry. 288(18):12500–12510. doi:10.1074 / jbc.M112.437947. PMC 3642298. PMID 23486480.

[0104] "Various mechanisms in cyclopeptide production from precursors synthesized independently of non-ribosomal peptide synthetases" (Wenyan Xu et al. (2011). "Various mechanisms in cyclopeptide production from precursors synthesized independently of non-ribosomal peptide synthetases". Acta Biochimica et Biophysica Sinica. 43(10):757–762. doi:10.1093 / abbs / gmr062.PMC3180235. PMID 21764803.);

[0105] "Plant Cyclopeptides and Possible Biosynthetic Mechanisms" (Wenyan Xu et al. "Plant Cyclopeptides and Possible Biosynthetic Mechanisms");

[0106] "Seamless proteins tie up their loose ends" (David J. Craik (17 March 2006). "Seamless Proteins Tie Up Their Loose Ends". Science. 311(5767): 1563–7. doi: 10.1126 / science.1125248. PMID 16543448.).

[0107] Conjugation with macromolecules

[0108] Conjugation with macromolecules (such as polyethylene glycol (PEG) and albumin) is an effective strategy to improve peptide stability and reduce renal clearance.

[0109] Renal clearance

[0110] Many peptides show good pharmacological activity in vitro, but their in vivo effectiveness has not been demonstrated due to their very short half-life in vivo (a few minutes). The rapid clearance and short half-life of peptides have hindered their development into successful drugs. The main reason for the rapid clearance of peptides from the systemic circulation is enzymatic proteolysis and / or renal clearance. The pore size of the glomerulus is about 8nm, and hydrophilic peptides with MW <2-25kDa are easily filtered rapidly through the glomeruli of the kidney. Since peptides are not easily reabsorbed through the renal tubules, they usually have high renal clearance and short half-life. Other minor pathways of peptide clearance are endocytosis and degradation through the proteasome and liver. Comparison between systemic clearance and renal clearance in animal models provides useful information on whether renal clearance may be the main clearance pathway.

[0111] In patients with renal impairment, the dose of peptide drugs may need to be adjusted to avoid drug accumulation and higher drug exposure, as inappropriate dosing in patients with renal impairment may lead to toxicity or ineffective treatment. Several strategies have been developed to reduce the renal clearance and prolong the half-life of peptides. These strategies are reviewed below.

[0112] Increased plasma protein binding

[0113] When peptides are bound to membrane or serum proteins, their renal clearance is reduced. An example is the cyclic peptide drug octreotide, a drug for the treatment of endocrine tumors, which has a half-life of approximately 100 minutes in humans (compared to 0.65 when unbound) due to its lipoprotein binding.

[0114] Covalent attachment of small molecules to albumin

[0115] Covalent attachment of albumin-binding small molecules to peptides can reduce glomerular filtration, increase proteolytic stability, and prolong half-life through indirect interactions of the highly bound small molecules with albumin.

[0116] Conjugation to large polymers

[0117] Conjugation of polypeptides to large synthetic or natural polymers or carbohydrates can increase the molecular weight and hydrodynamic volume of the polypeptide, thereby reducing its renal clearance. Commonly used polymers for polypeptide conjugation are PEG, polysialic acid (PSA) and hydroxyethyl starch (HES).

[0118] Fusion with long-lived plasma proteins

[0119] Plasma proteins, such as albumin and immunoglobulin (IgG) fragments, have a half-life of 19-21 days in humans. These proteins have low renal clearance due to their high MW (67-150 kDa), and their binding to the neonatal Fc receptor (FcRn) reduces their elimination by vascular epithelial cells via pinocytosis. Covalent attachment of peptides to albumin or IgG fragments can reduce renal clearance and extend their half-life.

[0120] Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters (William R. Strohl BioDrugs. 2015; 29(4): 215–239.)

[0121] PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng Des Sel. 2013; 26(8): 489-501.

[0122] Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymers (Podust, VN, Sim, BC, Kothari, D et al. Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer. Protein Eng DesSel. 2013; 26(11):743-53.)

[0123] Converting peptides into drug leads by lipidation (Zhang, L, Bulaj, G. Converting peptides into drug leads by lipidation. Curr Med Chem. 2012; 19(11): 1602-18.)

[0124] Obstacles and pitfalls in the PEGylation of therapeutic proteins (Gaberc-Porekar, V, Zore, I, Podobnik, B et al. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Devel. 2008; 11(2): 242-50.)

[0125] Long-lived peptides evolution of peptide half-life extension technologies and emerging hybrid approaches (By Dr. Ronald V. Swanson - Long-lived peptides evolution of peptide half-life extension technologies and emerging hybrid approaches. From Drug Discovery World online. Spring 2014.)

[0126] PEGylation

[0127] PEGylation, which attaches long chains of the hydrophilic polymer polyethylene glycol to a molecule of interest, was initially conceived as a modification to prevent the immune system from recognizing foreign proteins, thereby enabling their use as therapeutic agents. Once formed, antibodies against the unmodified drug can rapidly neutralize and clear the protein drug. Unexpectedly, PEGylation improves the pharmacokinetics of proteins even in the absence of anti-drug antibodies. 1 . Simply by making the drug molecule larger, PEGylation can cause the drug to be filtered more slowly by the kidneys. The empirical observation that increasing the size or hydrodynamic radius leads to reduced renal clearance and increased half-life has become the main rationale for PEGylation of protein and peptide drugs. PEGylation can have a variety of effects on molecules, including making the protein or peptide more water-soluble and protecting them from degradation by proteolytic enzymes. PEGylation can also affect the binding of therapeutic proteins to their cognate cellular receptors, generally reducing affinity. Changes in the size, structure, and attachment pattern of the PEG polymer can affect the biological activity of the conjugated drug.

[0128] The first generation of PEGylation methods were challenging. However, the chemistry of PEGylation is very simple. It involves the covalent attachment of polyethylene glycol chains to reactive side chains on proteins or peptides. For example, PEG is easily attached to the β-amino groups of lysine on the surface of proteins or peptides. 2 The reaction is pH dependent. At high pH (8.0 or higher), the lysine side chain amino group is covalently linked to the PEG via N-hydroxysuccinimide. This approach typically produces a series of products containing different numbers of PEG chains attached to different sites on the protein, rather than a single discrete product. 3 The first approved PEGylated drugs were bovine pegademase (bovine PEGylated adenosine deaminase) for enzyme replacement therapy in severe combined immunodeficiency and pegaspargase (PEGylated asparaginase) for the treatment of acute lymphoblastic leukemia. 1These drugs are complex mixtures of various PEGylated species but have improved therapeutic properties compared to the native enzyme, including increased serum half-life and reduced immunogenicity of the protein. Due to the inherent polydispersity of PEG, quality and batch-to-batch reproducibility are difficult to ensure. Despite this limitation, two PEGylated interferons (pegylated interferon alfa-2b and peginterferon alfa-2a), which are heterogeneous mixtures composed of numerous monoPEGylated positional isomers, have received FDA approval for the treatment of hepatitis C. These two drugs were launched in 2001 and 2002, respectively.

[0129] Various improvements and variations have been made to the basic PEGylation technology. Second-generation PEGylation processes introduced the use of branched structures and alternative chemistries for PEG attachment. Specifically, PEG with cysteine ​​reactive groups (e.g., maleimide or iodoacetamide) allows PEGylation to target single residues within a polypeptide or protein, thereby reducing the heterogeneity of the final product, but due to the polydispersity of PEG itself, it cannot eliminate heterogeneity.

[0130] Although the original rationale for PEGylation was to reduce immunogenicity, there are some examples of PEGylated proteins that are immunogenic. One example is PEGylated uricase, an enzyme that reduces plasma uric acid levels in gout patients. In clinical trials, a high proportion of gout patients did not respond to treatment and developed antibodies specific to PEG, but not to the uricase protein. 2 PEGylated liposomes, which are generally considered non-immunogenic, have been found to be immunogenic in some studies. PEGylated liposomes elicit a strong anti-PEG immunoglobulin M (IgM) response. Furthermore, multiple injections of PEG-glucuronidase can trigger the production of specific anti-PEG IgM antibodies, thereby accelerating the clearance of PEG-modified proteins from the body.

[0131] A major potential disadvantage of using PEG as a modifier is that it is not biodegradable. The U.S. Food and Drug Administration (FDA) has approved PEG for use as a carrier for drugs, including parenteral, topical, rectal, and nasal formulations. PEG has little toxicity and is eliminated intact from the body through the kidneys (for PEG < 30 kDa) or feces (for PEG > 20 kDa). 1Repeated administration of certain PEGylated proteins to animals has been observed to result in vacuolation of renal tubular cells. Recently, vacuolation of choroid plexus epithelial cells has also been observed in toxicity studies of large (≥40 kDa) PEG-conjugated proteins. Choroid plexus epithelial cells produce cerebrospinal fluid and form the cerebrospinal fluid (CSF) barrier. The long-term negative consequences of cellular vacuolation are unknown, but it does represent an undesirable consequence of some potential therapies. One possible alternative is to replace PEG with a biodegradable polymer. Polymers such as hydroxyethyl starch (HES) are one possible alternative. HES is nontoxic, biodegradable, and acts as a blood dilator. The HESylation process acts similarly to PEGylation, reducing renal clearance by increasing the hydrodynamic radius of the peptide, but the biodegradability of HES may reduce its propensity to accumulate. However, HES and other proposed biodegradable polymer PEG alternatives, like PEG, are polydisperse, making characterization of the final product and metabolites difficult. An emerging solution to alleviate both of these concerns is to use a defined peptide as the polymer component; this approach will be discussed later in this article.

[0132] lipidation

[0133] The second major chemical modification method to increase peptide half-life is lipidation, which involves the covalent attachment of fatty acids to peptide side chains. 4 . Lipidation was originally conceived and developed as a method to extend the half-life of insulin, with the same basic mechanism of half-life extension as PEGylation, namely increasing the hydrodynamic radius to reduce renal filtration. However, the lipid portion itself is relatively small, and the effect is indirectly mediated by non-covalent binding of the lipid portion to circulating albumin. Albumin is a large (67KDa) and highly abundant protein in human serum (35-50g / L) whose natural function is to transport molecules, including lipids, throughout the body. Binding to plasma proteins can also protect the polypeptide from attack by peptidases through steric hindrance, which is similar to what is observed with PEGylation. One consequence of lipidation is reduced aqueous solubility of the polypeptide, but engineering of the linker between the polypeptide and the fatty acid can regulate this, for example by using glutamic acid or small molecule PEG within the linker. Linker engineering and changes in the lipid portion can affect self-aggregation, which can extend half-life by slowing biodistribution and can be independent of albumin. 5 .

[0134] As pioneering work on insulin began 6Lipidation of various peptides has been explored, particularly in the field of diabetes, including human glucagon-like peptide-1 (GLP-1) analogs, glucose-dependent insulinotropic peptides, and GLP-1R / glucagon receptor co-agonists. Currently, two lipidated peptide drugs have received FDA approval for human use. The GLP-1 analogs liraglutide and insulin detemir are both long-acting antidiabetic drugs.

[0135] A potentially pharmacologically relevant difference between PEGylation and lipidation is that the therapeutically active peptide is covalently linked to the larger PEG, whereas the smaller fatty acyl-peptide conjugate is non-covalently bound to the larger albumin, existing in equilibrium as bound and unbound forms. This can lead to differences in biodistribution and, consequently, to different pharmacology, as reaching receptors located in different tissues may elicit different effects. In some cases, a more stringent biodistribution may be desired, while in other cases, greater tissue penetration may be important. An interesting variation of the PEG approach to address this issue was developed by Santi et al., in which a releasable PEG conjugate with a predictable cleavage rate was used. 7 .

[0136] Both PEGylation and lipidation confer protection to the modified substance against proteases and peptidases through steric shielding and extend the circulation half-life directly or indirectly by increasing the hydrodynamic radius. Both methods utilize chemical conjugation and are flexible because both modifications have no restrictions on the preparation method of the polypeptide they are modifying, whether by biological or synthetic methods. The advantage of using synthetic polypeptides is that they can incorporate non-natural amino acids designed to solve many specific problems, including solving the instability caused by known proteolytic cleavage tendencies. If activity or potency is highly dependent on free ends or modified residues (e.g., C-terminal amides), synthetic polypeptides are also more flexible in the selection of important attachment sites.

[0137] Classic gene fusion: Fc and HAS

[0138] Classical genetic fusions with long-lived serum proteins offer an alternative approach to extending half-life, distinct from chemical conjugation with PEG or lipids. Two main proteins have traditionally been used as fusion partners: the antibody Fc domain and human serum albumin (HSA). Fc fusions involve the fusion of a peptide, protein, or receptor extradomain to the Fc portion of an antibody. Both Fc and albumin fusions extend half-life not only by increasing the size of the peptide drug but also by leveraging the body's natural recycling mechanism: the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn protects the fusion protein from degradation within endosomal compartments. Fusions based on these proteins have half-lives of 3-16 days, significantly longer than typical PEGylated or lipidated peptides. Fusion to the antibody Fc domain can improve the solubility and stability of peptide or protein drugs. An example of a peptide Fc fusion is the GLP-1 receptor agonist dulaglutide, currently in late-stage clinical trials. Human serum albumin, the same protein used for fatty-acylated peptides, is another commonly used fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. The main difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HAS, resulting in the fusion polypeptide being presented as a dimer or monomer, depending on the choice of fusion partner. If the target receptors are close enough to each other or are themselves dimers, the dimeric nature of the polypeptide Fc fusion can produce an avidity effect. Depending on the target, this may be desirable or undesirable.

[0139] Designed Peptide Fusions: XTEN and PAS

[0140] An interesting variation on the recombinant fusion concept is the development of engineered low-complexity sequences as fusion partners, essentially unstructured, hydrophilic amino acid polymers that serve as functional analogs of PEG. The inherent biodegradability of peptide platforms has made them attractive as potentially more benign alternatives to PEG. Another advantage, compared to the polydispersity of PEG, is the precise molecular structure of the recombinant molecule. Unlike HSA and Fc peptide fusions, which require maintenance of the three-dimensional folding of the fusion partner, recombinant fusions with unstructured partners can, in many cases, withstand higher temperatures or harsh conditions, such as high-performance liquid chromatography (HPLC) purification.

[0141] The most advanced peptide of this type, called XTEN (Amunix), is 864 amino acids long and composed of six amino acids (A, E, G, P, S, and T). Due to the biodegradable nature of the polymer, this is much larger than the commonly used 40 kDa PEG and confers a superior half-life extension. The fusion of XTEN with peptide drugs results in a half-life that is 60 to 130 times longer than that of the native molecule. Two fully recombinantly produced XTEN-based products have entered the clinic: VRS-859 (Exenatide-XTEN) and VRS-317 (human growth hormone-XTEN). In a Phase Ia study, VRS-859 was found to be well tolerated and effective in patients with type 2 diabetes. Compared to previously studied rhGH products, VRS-317 reports superior pharmacokinetic and pharmacodynamic properties and has the potential for once-monthly dosing.

[0142] A second polymer considered based on a similar concept is PAS (XL-Protein GmbH) 9 It is a random coil polymer composed of a more restricted set of only three small, uncharged amino acids: proline, alanine, and serine. Whether differences in the biophysical properties of PAS and the highly negatively charged XTEN may lead to differences in biodistribution and / or in vivo activity is unknown, but these differences will be revealed as these peptides are combined with more therapeutics and the behavior of the fusions is characterized.

[0143] All peptide-protein fusions, regardless of whether their partner is Fc, HSA, XTEN, or PAS, are genetically encoded and therefore subject to similar constraints. One limitation is that only naturally occurring amino acids can be incorporated, unlike approaches using chemical conjugation, which allow the incorporation of unnatural amino acids into synthetic peptides. Although companies such as Ambrx or Sutro are developing approaches to overcome this problem by expanding the genetic code, they are not yet widely used. A second limitation is that either the N-terminus or the C-terminus of the peptide needs to be fused to the partner. Typically, the peptide termini are involved in receptor interactions, and genetic fusion to one or both termini greatly diminishes their activity. Because PEG or lipid binding sites can be located anywhere on the peptide, they can be optimized to maximize the biological activity of the resulting therapeutic.

[0144] A hybrid approach to conjugating synthetic peptides to half-life-extending proteins.

[0145] While gene fusions have historically offered superior half-life extension potential, they lack the advantages offered by approaches utilizing chemical conjugation, PEGylation, and lipidation in terms of flexibility in attachment sites and incorporation of unnatural amino acids or modifications to the peptide backbone. Researchers at the Scripps Research Institute in LaJolla were the first to combine the advantages of gene fusion with chemical conjugation to extend half-life, a technology that later became the basis for the biotechnology company CovX. 10,11 Using a catalytic aldolase antibody, the researchers developed a platform whereby the antibody's active site lysine forms a reversible covalent enamine bond with a β-diketone conjugated to a peptide or small molecule. The resulting complex is called CovXBody TM This approach combines the functional properties of peptide drugs or small molecules with the long serum half-life of antibodies, not through genetic fusion but through chemical linkage. After the initial demonstration of the technology, the researchers expanded the prototype CovX-Body based on integrin-targeting peptide pharmacophores. TM At least three molecules based on this architecture have entered clinical development: the Glp-1R agonist CVX-096; the angiopoietin-2 binding peptide CVX-060; and the thrombospondin mimetic CVX-045.

[0146] Recently, XTEN polypeptides have also been used in chemical conjugation formats. 12 , making it more directly analogous to PEG. The first example of an XTENed peptide created using this approach is GLP2-2G-XTEN, in which the peptide was chemically conjugated to the XTEN protein polymer using maleimide-thiol chemistry. The chemically conjugated GLP2-2GXTEN molecule exhibited similar in vitro activity, in vitro plasma stability, and rat pharmacokinetics to the recombinant fused GLP2-2G-XTEN.

[0147] In fully designed XTEN or PAS polypeptide sequences, the number and spacing of reactive groups (such as lysine or cysteine ​​side chains) can be precisely controlled through targeted site-specific changes because they are composed of a restricted set of amino acids. This provides an additional degree of flexibility for approaches that might utilize Fc or albumin, as their sequences naturally contain many reactive groups, in contrast to CovX technology, which relies on reactive residues in highly specific active sites. In addition, the lack of tertiary structure in XTEN or PAS provides greater flexibility in the conditions and chemistries used in coupling and conjugate purification.

[0148] In summary, hybrid peptide half-life extension methods are emerging that combine the advantages and overcome the limitations of chemical conjugation and gene fusion methods. These methods can produce molecules based on recombinant peptide partners that confer longer half-lives, but free the therapeutic peptide portion from the limitations of linear, unidirectional peptides composed only of natural L-amino acids or configured only as fusions at the N- or C-terminus, thereby opening the door to a wide range of long-acting peptide drugs.

[0149] As used herein, the term "expression vector of the present invention" can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (including a nucleic acid sequence of a suitable set of expression control elements) suitable for expressing the polypeptide of the present invention in cells. Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from plasmid and phage DNA combinations, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid molecule encoding the polypeptide is contained in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), a compact nucleic acid vector (such as described in U.S. Patent No. 6,077,835 and / or WO 00 / 70087), or a plasmid vector, such as pBR322, pUC19 / 18 or pUC118 / 119. Such nucleic acid vectors and their uses are well known in the art (see, eg, US Patent No. 5,589,466 and US Patent No. 5,973,972). In one embodiment, the DNA includes expression control sequences.

[0150] In one embodiment, the vector is suitable for expressing the polyamino acid sequence of the present invention in bacterial cells. Examples of such vectors include expression vectors, such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, 1989, J Biol Chem 264, 5503-5509), pET vectors (Novagen, Madison, Wis.) and the like. In one embodiment, the expression vector may also be or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters (such as yeast alpha factor, alcohol oxidase and PGH) (reviewed in: F.Ausubel et al., ed., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York; and Grant et al., 1987, Methods in Enzymol 153, 516-544). In other embodiments, the expression vector is suitable for expression in baculovirus-infected insect cells (Kost, T; and Condreay, JP, 1999, Current Opinion in Biotechnology 10(5):428-33.).

[0151] Expression control sequences are engineered to control and drive transcription of a gene of interest, and subsequently protein expression, in various cell systems. Plasmids combine the expressible gene of interest with expression control sequences (i.e., expression cassettes) that include required elements such as promoters, enhancers, selection markers, operators, and the like. In the expression vectors of the present invention, the nucleic acid molecule encoding the polyamino acid sequence can include or be combined with any suitable promoter, enhancer, selection marker, operator, repressor protein, polyA termination sequence, and other elements that promote expression.

[0152] As used herein, " promoter " refers to DNA sequence dna, it is enough to guide the transcription of the DNA sequence dna operably connected thereto, i.e., connected in this way, when suitable signal occurs, allows the nucleotide sequence encoding the polyamino acid sequence to be transcribed. The expression of the nucleotide sequence encoding the polyamino acid sequence can be placed under the control of any promoter known in the art or enhancer element. The example of this type of element includes strong expression promoter (for example, people CMV IE promoter / enhancer or CMV main IE (CMV-MIE) promoter, and RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC) and HIV LTR promoter). In certain embodiments, the promoter included in the vector is selected from the group consisting of: SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC and HIV LTR.

[0153] The nucleic acid molecules of the present invention can also be operably linked to effective poly (A) termination sequences, the replication origin of E. coli plasmid products, antibiotic resistance genes as screening markers and / or convenient cloning sites (e.g., polylinkers). In contrast to constitutive promoters (e.g., CMV IE), nucleic acids can also contain regulatable inducible (inducible, repressible, developmentally regulated) promoters (those skilled in the art will recognize that these terms are actually descriptions of the extent of gene expression under specific conditions).

[0154] Screening markers are elements well known in the art. Under the screening conditions, only cells expressing the appropriate screening marker can survive. Typically, screening marker genes express proteins, usually enzymes, which confer resistance to various antibiotics in cell culture. Under other screening conditions, cells expressing fluorescent protein markers are visible and therefore screenable. Examples include β-lactamase (bla) (β-lactam antibiotic resistance or ampicillin resistance gene or ampR), bls (blasticidin resistance acetyltransferase gene), bsd (blasticidin-S deaminase resistance gene), bsr (blasticidin-S resistance gene), Sh ble (Zeocin resistance gene), hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM (tetracycline resistance gene or tetR), neomycin phosphotransferase II (npt) (neomycin resistance gene or neoR), kanR (kanamycin resistance gene), and pac (puromycin resistance gene).

[0155] In certain embodiments, the vector comprises one or more selection marker genes selected from the group consisting of bla, bls, BSD, bsr, Sh ble, hpt, tetR, tetM, npt, kanR, and pac. In other embodiments, the vector comprises one or more selection marker genes encoding the following proteins: green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyanine fluorescent protein (CFP), enhanced cyanine fluorescent protein (eCFP), or yellow fluorescent protein (YFP).

[0156] For purposes of the present invention, gene expression in eukaryotic cells can be strictly regulated using a strong promoter controlled by an operator that is in turn regulated by a regulatory protein that can be a recombinant "regulatory fusion protein" (RFP). RFP consists essentially of a transcription blocking domain and a ligand binding domain that regulates its activity. Examples of such expression systems are described in US20090162901A1, which is incorporated herein by reference in its entirety.

[0157] As used herein, "operon" refers to a DNA sequence introduced into or near a gene in such a way that the gene can be regulated by binding of RFP to the operator, thereby preventing or allowing transcription of the gene of interest (i.e., the nucleotide encoding the polypeptide of the present invention). Operators in many prokaryotes and phages have been well characterized (Neidhardt, ed., Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol 2 ASM Press, Washington DC 1996). These include, but are not limited to, the Escherichia coli LexA gene operon region that binds to the LexA polypeptide, and the lactose and tryptophan operons encoded by the Escherichia coli Lad and trpR genes that bind to the repressor protein. Such operons also include phage operators from lambda PR and phage P22 ant / mnt genes that bind to the repressor protein encoded by lambda cI and P22 arc. In some embodiments, when the transcription blocking domain of RFP is a restriction enzyme, such as NotI, the operator is the recognition sequence of the enzyme. Those skilled in the art will recognize that the operator must be located near or 3' of the promoter to enable transcription to be controlled by the promoter. For example, U.S. Patent No. 5,972,650, incorporated herein by reference, specifies that the tetO sequence is located within a specific distance from the TATA box. In specific embodiments, the operator is preferably placed immediately downstream of the promoter. In other embodiments, the operator is located within 10 base pairs of the promoter.

[0158] In an exemplary cell expression system, cells are engineered to express tetracycline repressor protein (TetR), and the protein of interest is placed under the transcriptional control of a promoter, the activity of which is regulated by TetR. Two series-connected TetR operons (tetO) are placed in the immediate downstream of the CMV-MIE promoter / enhancer in the vector. In the absence of tetracycline or other suitable inducing agents (such as doxycycline, doxycycline), the transcription of the coding protein of interest gene directed by the CMV-MIE promoter in such vectors can be blocked by TetR. In the presence of inducing agents, TetR protein can not bind tetO, and therefore transcription and translation (expression) of the protein of interest can occur. (See, for example, U.S. Patent No. 7,435,553, the entire contents of which are incorporated herein by reference.)

[0159] The vectors of the present invention can also use the Cre-lox recombination tool to promote the integration of the gene of interest into the host genome. The Cre-lox strategy requires at least two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites; 2) loxP sites (e.g., a specific 34 base pair sequence consisting of an 8-bp core sequence (in which recombination occurs) and two flanking 13bp inverted repeats) or mutated lox sites. (For example, see Araki et al., 1995, PNAS 92: 160-4; Nagy, A. et al., 2000, Genesis 26: 99-109; Araki et al., 2002, Nuc Acids Res 30 (19): e103; and US20100291626A1, all of which are incorporated herein by reference). In another recombination strategy, yeast-derived FLP recombinase can be used together with the consensus sequence FRT (eg, see Dymecki, SM, 1996, PNAS 93(12):6191-6196).

[0160] As used herein, the term "host cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotes and eukaryotes (single or multicellular), bacterial cells (such as strains of Escherichia coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Methyloblasts (P. methylica), etc.), plant cells, insect cells (such as SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni cells, etc.), non-human animal cells, mammalian cells, human cells, or cell fusions (such as hybridomas or quadromas). In specific embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell is a eukaryotic cell and is selected from the group consisting of CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the above. In some embodiments, the cell comprises one or more viral genes, such as retinal cells (e.g., PER.C6) expressing viral genes. In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

[0161] As used herein, the term "transformed cell of the present invention" refers to a host cell comprising a nucleic acid stably integrated into the cell genome, the nucleic acid comprising a nucleotide sequence encoding for expression of a polypeptide of the present invention. In another embodiment, the cell provided by the present invention comprises a non-integrated (i.e., episomal) nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence encoding for expression of a polypeptide of the present invention. In other embodiments, the present invention provides a cell line generated by stably transfecting a host cell with a plasmid comprising an expression vector of the present invention.

[0162] As used herein, the term "engineered" as applied to cells refers to genetic engineering using recombinant DNA technology, typically comprising the steps of synthesizing a suitable expression vector (see above) and then transfecting the expression vector into a host cell (typically a stable transfection).

[0163] As used herein, the term "heterologous expression" refers to the expression of a nucleic acid in a host cell that does not naturally harbor the nucleic acid. The nucleic acid is inserted into a heterologous host by recombinant DNA technology.

[0164] Example

[0165] The present invention will now be described with reference to specific embodiments. These are exemplary only and are for illustrative purposes only: they are not intended to limit the exclusive scope of the claimed invention or the invention described in any way. These examples constitute the best mode presently contemplated for practicing the invention.

[0166] Hepatocyte penetration

[0167] method

[0168] HepG2 (3×10 4 hole -1 ) cells were seeded on 18 mm glass coverslips (Paul Marienfeld GmbH & Co. KG, Lauda- , Germany), plated in 24-well plates, and allowed to adhere overnight. Cells were treated with 0.5 μg / mL CY5-labeled pep-260 for 1 hour and then fixed with 4% paraformaldehyde (Sigma, Arklow, Ireland) for 20 minutes at room temperature. Subsequently, the coverslips were washed in 1× PBS (Sigma, Arklow, Ireland) and incubated in 0.1% Tween 20 (Sigma, Arklow, Ireland) for 30 minutes at room temperature. The coverslips were then washed three times in 1× PBS for 5 minutes each and then treated with rabbit anti-human early endosomal antigen 1 (EEA1) monoclonal antibody (Cell Signaling Technology, Danvers, Massachusetts, USA) diluted 1:500 in PBS at 4°C overnight. The coverslips were then washed three times in 1× PBS for 5 minutes each. The 1× PBS was aspirated and the coverslips were treated with AlexaFluor 546 goat anti-rabbit IgG (Life Technologies, Eugene, USA) diluted 1:100 in PBS for 2 h at room temperature. The coverslips were then washed three times in 1× PBS for 5 min each and then treated with Hoechst 33342 solution (Thermo Scientific, Waltham, USA). The coverslips were then washed three times in PBS, removed from the 24-well plate with forceps, and mounted on Superfrost Plus microscope slides (Thermo Scientific, Waltham, USA) with 5 μL of polyvinyl alcohol (mowiol) mounting medium [6 g glycerol (Sigma Aldrich), 2.4 g polyvinyl alcohol (mowiol 4-88) (Sigma Aldrich), and 0.026 g 1,4-diazabicyclo[2.2.2]octane (DABCO) (Sigma Aldrich) dissolved in 18 mL 0.2 M Tris buffer (pH 8.5) (Sigma Aldrich)].

[0169] Confocal images were captured using an Olympus Fluoview FV1000 confocal laser scanning biological microscope (Shinjuku, Tokyo, Japan) with a 60× oil-immersion objective. Hoechst nuclear stain was detected with a 405 nm laser diode, AlexaFlour 546 fluorophore was detected with a diode-pumped solid-state laser, and Cy5-labeled pep_260 was detected with a red diode-pumped solid-state laser.

[0170] result

[0171] Confocal imaging showed that pep_260 (SEQ ID 1) (red staining) was able to penetrate HepG2 hepatocytes. This effect was observed after 5 min of pep_260 treatment, and more intracellular localization was observed in HepG2 cells at 60 min (see Figure 1 ).

[0172] In vitro studies in primary human cell systems

[0173] method

[0174] Early passage human primary cells (passage 4 or earlier) were used to minimize adaptation to cell culture conditions and maintain physiological signaling responses. All cells were obtained from a pool of multiple donors (n = 2-6), purchased commercially and processed according to the manufacturer's recommendations. Human blood-derived CD14+ monocytes were differentiated into macrophages in vitro and then added to the lmphg system. The following abbreviations are used: human umbilical vein endothelial cells (HUVEC), peripheral blood mononuclear cells (PBMC), human neonatal dermal fibroblasts (HDFn), B cell receptor (BCR), T cell receptor (TCR), and Toll-like receptor (TLR).

[0175] The cell types and stimuli used in each system were as follows: 3C system [HUVEC+(IL-1β, TNFα, IFNγ)], 4H system [HUVEC+(IL-4 and histamine)], LPS system [PBMC and HUVEC+LPS(TLR4 ligand)], SAg system [PBMC and HUVEC+TCR ligand], BT system [CD19+B cells and PBMC+(α-IgM and TCR ligand)], BF4T system [bronchial Epithelial cells and HDFn+(TNFα and IL-4)], BE3C system [bronchial epithelial cells+(IL-1β, TNFα and IFNγ)], CASM3C system [coronary artery smooth muscle cells+(IL-1β, TNFα, IFNγ)], HDF3CGF system [HDFn+(IL-1β, TNFα, IFNγ, EGF, bFGF, PDGF-BB)], KF3CT system [keratinocytes and HDFn+(IL 1β, TNFα, IFNγ, TGFβ)], MyoF system [differentiated lung myofibroblasts+(TNFα and TGFβ)], lMphg system [HUVEC and M1 macrophages+zymosan (TLR2 ligand)].

[0176] Systems are available as single cell types or co-culture systems. Adherent cells are cultured in 96- or 384-well plates until confluence, then PBMCs are added (SAg and LPS systems). BT systems consist of CD19+ B cells co-cultured with PBMCs, which are stimulated with BCR activators and low levels of TCR. Test reagents are prepared in DMSO (small molecules; final concentration ≤0.1%) or PBS (biologicals), added at the specified concentration 1 hour before stimulation, and cultured for 24 hours or as otherwise indicated (48 hours, MyoF system; 72 hours, BT system (soluble readout); 168 hours, BT system (secreted IgG)). Each well plate contains a drug control appropriate for each system (e.g., 1.1 μM of the traditional control test agent colchicine), a negative control (e.g., non-stimulated conditions), and a vehicle control (e.g., 0.1% DMSO). Direct ELISA is used to measure biomarker levels of cell-associated and cell membrane targets. Using HTRF Soluble factors from the supernatant are quantified by detection, bead-based multiplex immunoassays, or capture ELISAs. Significant adverse effects of the test agents on cell proliferation and viability (cytotoxicity) are detected by sulforhodamine B (SRB) staining (for adherent cells) and alamarBlue. Reduction (for suspension cells) was used for detection. For proliferation assays, single cell types were cultured at subconfluence and measured at the optimized time points for each system (48 h: 3C and CASM3C systems; 72 h: BT and HDF3CGF systems; 96 h: SAg system). Cytotoxicity in adherent cells was assessed by SRB at the indicated time points (24 h: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, 1Mphg systems; 48 h: MyoF system), and cytotoxicity in suspension cells was assessed by alamarBlue staining at the indicated time points (24 h: SAg system; 42 h: BT system).

[0177] result

[0178] Pep_260 (SEQ ID 1)-mediated changes in the activity of key biomarkers are listed by biological and disease category (see Table 1). Pep_260 was not cytotoxic at the concentrations tested in this study. Pep_260 exhibited antiproliferative effects on primary human endothelial cells.

[0179]

[0180] Table 1: In vitro biomarker profiles of primary human cell lines in response to pep_260 treatment

[0181] Hepatic effects of pep_260 (SEQ ID 1) in KKAy mice

[0182] method

[0183] The liver effects of pep_260 (SEQ ID 1) and liraglutide were evaluated in the KKAy obese diabetic mouse model. 12-week-old male mice (n=11 per group) were treated with pep_260 (12.7 and 63.5 mg / kg), liraglutide (250 μg / mL) or vehicle by subcutaneous administration every day for 44 days. On the 44th day, the animals were killed by cervical dislocation 1 hour after the administration. For all animals, the whole liver was collected and fixed in formalin. Liver tissue was sliced ​​and stained with hematoxylin and eosin. Histopathology experts who were blinded to the study grouping evaluated histological fatty degeneration according to the NALFD scoring system. Gene expression profiling in the liver was also performed.

[0184] result

[0185] Expert scores indicated that 44-day treatment with pep_260 (SEQ ID 1) significantly alleviated macrovesicular steatosis in obese diabetic mice. Gene expression analysis of liver tissue revealed that treatment with pep_260 (SEQ ID 1) induced a decrease in SREBF1, FASN, and Caspase-3 genes. These results suggest that pep_260 (SEQ ID 1) inhibits hepatic cholesterol production, suppresses hepatic lipogenesis, and promotes hepatocyte survival.

[0186] Vehicle control and liraglutide-treated mice showed signs of NAFLD, however these signs were significantly reduced in 50 μM pep_1E99R5 and trended towards reduction in 10 μM pep_1E99R5 treatment, suggesting a dose-dependent effect ( Figure 2 B).

[0187] Immortalized hepatocytes HepG2 cells and primary lung fibroblasts WI-38 cells stimulated by LPS and TGFβ IL-8 secretion

[0188] method

[0189] IL-8 enzyme-linked immunosorbent assay (ELISA):

[0190] HepG2 hepatocytes (density of 1×10 4 Cells were incubated with 100 ng / mL of LPS for 24 h and then stimulated with LPS (100 ng / mL) for 24 h to further increase IL-8 expression. Lipopolysaccharide (LPS) is a known pro-inflammatory signal that increases IL-8 expression in vitro. IL-8 levels in culture supernatants were measured using a human IL-8-specific sandwich ELISA (Perkin Elmar, Waltham, MA, USA) according to the manufacturer's instructions.

[0191] result

[0192] The results showed that the expression of the proinflammatory cytokine IL-8 was significantly reduced in cells pretreated with pep_1E99R5 (p < 0.001). This data further confirms the beneficial effects of inflammation, such as in NAFLD / NASH. The proinflammatory cytokine IL-8 is strongly activated in NASH and leads to liver inflammation and fibrosis ( Figure 6 ).

[0193] Anti-fibrotic effect of pep_1E99R5 in primary hepatic stellate cells stimulated by TGF-β

[0194] A robust initiation model of hepatic stellate cell fibrosis, a key inflammatory driver of fibrosis induction in liver fibrosis and NASH / NAFLD, can be established using primary human stellate cells incubated with varying concentrations of transforming growth factor β (TGF-β).

[0195] method

[0196] Imaging of CY5-labeled peptides in HSkMC was performed according to the above procedure with some minor modifications. Primary hepatic stellate cells were studied because they are considered to be the most relevant profibrotic cells in acute and chronic liver diseases. Stellate cells were seeded directly onto glass coverslips at a density of 5,000 cells / mL. After starvation, cells were treated with pep_1E99R5 (5 ng / mL) or Elafibranor (10 μM) for 6 h. The cells were washed and subsequently treated with 5 ng / mL TGF-β (Bio Techne, Minneapolis, MN, United States) for 24 h to induce fibrosis. The cells were fixed and stained with ACTA2 antibody (Assay Genie, Dublin, Ireland), a marker of fibroblast activation.

[0197] Confocal images were analyzed using a custom Python script from the scikit-image library, version 0.15. Cells and nuclei were segmented using a simple threshold calculated from the image mean. Using nuclei as seeds, we separated cells within the cell mask using watershed segmentation. Cell size was then quantified in pixels. [Stéfan van der Walt, Johannes L. ,Juan Nunez-Iglesias, Boulogne, Joshua D. Warner, Neil Yager, Emmanuelle Gouillart, Tony Yu and the scikit-imagecontributors. scikit-image: Image processing in Python. PeerJ 2:e453 (2014).

[0198] result

[0199] Stimulated primary hepatic stellate cells were used as an acute liver injury model. Fluorescence images of untreated primary stellate cells, TGFβ-stimulated cells (5 nM), Elafibranor-treated (10 μM), and pep_1E99R5 (5 nM)-treated cells are shown in Figure 4 Figure A. TGF-β stimulation effectively induced cell fibrosis (p < 0.05). Increased ACTA2 expression, fibrosis, proliferation, and morphological changes were observed in stimulated cells, but not in control cells. Preincubation with pep_1E99R5 significantly reduced ACTA2 expression in astrocytes, as shown by the yellow fibers ( Figure 7 A). When the expression of fibrotic markers was quantified in pixels using image analysis, this reduction was significant (p<0.01) and comparable to the antifibrotic effect of 10 μM elafibranor ( Figure 7 B).

[0200] Interestingly, the changes observed with pep_1E99R5 at 5 nM were comparable to those observed with elafibranor (a leading clinical candidate that acts on astrocytes in vitro and in vivo) at 10 μM, suggesting that the therapeutic effect of pep_1E99R5 may be greater because a significantly lower concentration is required to achieve the same effect.

[0201] This evidence, combined with the unique protein target and biomarker fingerprint, underscores the potential of this peptide entity in preclinical development programs targeting NASH / NAFLD.

[0202] APAP model

[0203] method

[0204] Acetaminophen (APAP)-induced acute liver injury mouse model: All animal procedures were performed by Melior Discovery (Exton, PA, USA) in accordance with Institutional Animal Care and Use (IACUC) guidelines in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility. A pilot study was performed before the APAP-induced hepatotoxicity study to establish the optimal APAP dose for subsequent liver injury studies (Supplementary Table 1). Figure 2 ). The studies were conducted using 8-week-old male C57BL / 6 mice obtained from Charles River Laboratory and randomly assigned to treatment groups based on body weight. The mice were acclimated for 7 days, housed 4 per cage, on a 12-h light / dark cycle, with free access to standard rodent chow and water. For the preliminary study, the mice were fasted overnight and then given a single dose of 200 mpk APAP, 300 mpk APAP, or saline as a control group, intraperitoneally. A total of 18 mice were included in the preliminary study, divided into 3 groups of 6 animals each. Mice were tail-bleeded at the first time point to reduce stress and improve mouse survival. Blood samples from the 6-h time point were used to generate serum samples for ALT and AST analysis. When evaluating the efficacy of the peptides in the APAP-induced acute liver injury mouse model, APAP (200 mpk) was administered intraperitoneally, while Pep_1E99R5 and 10PANX were both administered intravenously at 10 mg / kg. Each group consisted of 5 animals, and vehicle or polypeptide was administered 1.5 hours after APAP injection. Survival bleeding was performed at 2.25 and 6 hours for ALT / AST analysis. Terminal blood samples were collected by cardiac puncture under isoflurane anesthesia and used to generate serum samples for full panel clinical chemistry analysis (data not shown).

[0205] result

[0206] The screening results of DiscoverX BioMap and Retrogenix, combined with in vitro studies of Hep-G2 and hepatic stellate cells, strongly suggested the anti-inflammatory and anti-fibrotic effects of pep_1E99R5, so the N-acetyl-p-aminophenol (APAP) model was used to evaluate the effect of the peptide in an in vivo liver injury model. Preliminary studies have shown that 200mpk of APAP significantly increased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), biochemical markers of APAP-induced liver injury in mice compared to saline controls. Therefore, it is considered to be the optimal concentration for liver injury testing. 1.5h after the initial APAP administration, the peptides pep_1E99R5 and 10PANX were administered intravenously (IV), and the same liver enzyme levels were measured at 2.25h and 6h ( Figure 8At an earlier time point, pep_1E99R5 significantly reduced ALT levels by 80% compared to the APAP / saline control (p < 0.05) and was significantly superior to 10PANX, which resulted in a 41% reduction in ALT. A similar trend was observed in AST levels at 2.25 hours, however, this may not have reached significance due to the small number of animals included in the study. After 6 hours, ALT and AST levels in the APAP / saline group had increased 4-fold and 2-fold, respectively. 10PANX activity remained largely stable at this time point, resulting in non-significant reductions of both liver enzymes between 45% and 52%. AST and ALT levels measured in pep_1E99R5-treated mice were elevated at 6 hours, consistent with those in the APAP control group. This suggests that the pharmacokinetic profile of pep_1E99R5 appears to exhibit effective function within the first 2 hours and is then likely metabolized, unlike 10PANX, which persists at least 3-fold longer in mice.

[0207] equivalent

[0208] The foregoing description describes in detail the presently preferred embodiments of the present invention. In view of these descriptions, it is expected that those skilled in the art will make numerous modifications and variations thereto in practice. These modifications and variations are intended to be encompassed by the appended claims. Sequence Listing <110> Nuritas Ltd. <120> Treatment of nonalcoholic fatty liver disease <130> P22JM1WN00288IE <140> <141> <150> EP19204536.7 <151> 2019-10-22 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> / comment="Description of artificial sequence: synthetic polypeptide" <400> 1 Trp Lys Asp Glu Ala Gly Lys Pro Leu Val Lys 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> / comment="Description of artificial sequence: synthetic polypeptide" <220> <221> MOD_RES <222> (1)..(1) <223> D-Trp <220> <221> MOD_RES <222> (2)..(2) <223> D-Lys <220> <221> MOD_RES <222> (5)..(5) <223> D-Ala <220> <221> MOD_RES <222> (10)..(10) <223> D-Val <220> <221> MOD_RES <222> (11)..(11) <223> D-Lys <400> 2 Xaa Xaa Asp Glu Xaa Gly Lys Pro Leu Xaa Xaa 1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> / comment="Description of artificial sequence: synthetic polypeptide" <220> <221> SITE <222> (1)..(5) <223> / Comment="This sequence contains a thioether cyclization between the N-terminus and the Cys residue at position 5" <220> <221> MOD_RES <222> (1)..(1) <223> D-Trp <220> <221> MOD_RES <222> (4)..(4) <223> Glu(Me) <220> <221> MOD_RES <222> (8) <223> Pro(Me) <220> <221> MOD_RES <222> (11)..(11) <223> D-Lys <220> <221> source <223> / annotation="C-terminal OH" <400> 3 Xaa Lys Glu Xaa Cys Gly Lys Xaa Leu Val Xaa 1 5 10

Claims

1. Use of the polypeptide of SEQUENCE ID NO: 1 in the preparation of a medicament for treating or preventing non-alcoholic fatty liver disease in mammals.

2. The use according to claim 1, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.

3. The use according to claim 1 or 2, wherein the polypeptide is a modified polypeptide SEQUENCE ID NO:

2.

4. The use according to claim 1 or 2, wherein the polypeptide is cyclized, and the cyclized polypeptide is polypeptide SEQUENCE ID NO: 3.

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