Treatment of non-alcoholic fatty liver disease

Abstract

The present applicant discovered that the peptide of SEQ ID NO: 1 (WKDEAGKPLVK) mediates changes in the activity of key biomarkers associated with NASH (Table 1) and can penetrate HepG2 hepatocytes in a hepatocyte permeability assay (Figure 1). Furthermore, the present applicant demonstrated that administration of pep_260 (SEQ ID NO: 1) for 44 days significantly alleviated macrovesicular steatosis in obese diabetic KKAy mice (Figure 2). In a first aspect, the present invention relates to the use of a peptide consisting of SEQ ID NO: 1, or a functional (or therapeutically effective) variant or functional fragment of SEQ ID NO: 1, in a method for the treatment or prevention of non-alcoholic fatty liver disease (NAFLD), in particular non-alcoholic steatohepatitis (NASH), in a mammal.

Description

Technical Field

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

Background Art

[0002] NASH is a widespread and serious "silent" liver disease that can lead to cirrhosis or cancer and affects millions of people worldwide, including children. Studies proven by biopsy have shown that 12% of adults in the United States have NASH, and an increase of 63% is expected by 2030. The NAFLD rate warns that there is already a 10% concern in children, 31% in South America, 32% in the Middle East, and 23% in Europe. Due to the rising prevalence and its close association with the modern lifestyle related to the diabetes后备军, type 2 diabetes, and obesity epidemic, NASH is expected to become the leading cause of liver transplantation in the United States by 2020;

[0003] NASH is not only associated with the high cost of liver transplantation (about $800,000 per patient in the United States) and related complications, but also a looming public health threat because NASH is closely associated with non-liver disorders such as cardiovascular events, which are the leading causes of death in NASH patients. NASH is a silent disease with no symptoms, so it is generally little known, and most NASH patients are currently undiagnosed. NASH is also a disease that is difficult to detect and has insufficient diagnosis because there is no simple, accurate, and cost-effective diagnostic solution. NASH patients have to fight against disease-related prejudices and misunderstandings. There is also a lack of access to easy-to-understand information to explain their condition to relatives, friends, and colleagues who find it difficult to understand the nature and consequences of NASH;

[0004] It should be noted that there is an unclear expression "糖尿病予備軍" in the original text. I translated it as "diabetes后备军" as it is, but it might need to be further clarified in the original context.With the exception of a few international experts, healthcare professionals, including diabetes specialists, endocrinologists, obstetricians, cardiologists, obstetricians, gynecologists, general practitioners, and nurses, remain largely uninformed, with limited opportunities to learn about this disease and limited relevant educational resources.

[0005] While there are currently no approved treatments, several advanced development programs are underway, offering hope to millions of clinicians and patients worldwide (only 10% of patients succeed in fighting the disease through challenging lifestyle changes). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of the present invention is to overcome at least one of the above-mentioned problems. [Means for solving the problem]

[0007] The applicant discovered that the peptide of Sequence ID No. 1 (WKDEAGKPLVK) mediates changes in the activity of key biomarkers associated with NASH (Table 1), and found that the peptide can permeate HepG2 hepatocytes in a hepatocyte permeability assay (Figure 1). In addition, the applicant demonstrated that treatment with Sequence ID No. 1 for 44 days significantly alleviates macrodroplet steatosis in obese diabetic KKAy mice (Figure 2).

[0008] The applicant also provides a variant of SEQ ID NO: 1, {d}W{d}KDE{d}AGKPL{d}V{d}K (SEQ ID NO: 2), which has five modified residues compared to SEQ ID NO: 1 (residues 1, 2, 5, 10, and 11 are provided as D-amino acids). The variant peptide of SEQ ID NO: 2 significantly enhances glucose uptake into skeletal muscle cells compared to insulin (Figure 3) and significantly reduces HbA1c% (amount of glucose attached to red blood cells in the body) compared to liraglutide (Figure 4). 1 / 2 = Shows an optimized PK profile for 93 minutes.

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

[0010] Bioactive peptides possess a favorable safety profile and the ability to engage targets that can attenuate numerous pathways.

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

[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 mammals, comprising the step of administering to a mammal a therapeutically effective amount of a peptide containing SEQ ID NO: 1, or a functional (or therapeutically effective) variant or functional fragment of SEQ ID NO: 1 (hereinafter referred to as the "peptide activator").

[0013] The peptide (or functional variant or fragment) may be administered alone or in combination with other co-drugs that provide an enhanced therapeutic effect, including, but not limited to, drugs that have been shown to have effects in the treatment of diabetes or obesity.

[0014] In one embodiment, the peptide has up to 40, 35, 30, 25, 20, or 15 amino acids. In one embodiment, the peptide has 11 to 15 amino acids. In one embodiment, the peptide essentially consists of Sequence ID No. 1.

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

[0016] In one embodiment, the peptide is modified. In one embodiment, the peptide is a recombinant peptide. In one embodiment, the peptide is cyclized. An example of a cyclized peptide is (1(clac)wKE(Me)EC1GK(Me)PLVk-OH)-SEQ ID NO: 3. In this variant, residues "w" and "k" are D-amino acids, residues "E" and "P" are methylated, and the peptide includes a thioether cyclization between the N-terminus and a cysteine ​​residue, with "1(clac)" and "C1" indicating the two ends of the ring.

[0017] In other embodiments, the peptide may be administered alone or in combination with other co-drugs that provide an enhanced therapeutic effect, including, but not limited to, drugs that have been identified as having effects in the treatment of diabetes or obesity.

[0018] In another embodiment, the present invention provides a peptide having the sequence {d}W{d}KDE{d}AGKPL{d}V{d}K (SEQ ID NO: 2). In another embodiment, the present invention provides a cyclized peptide of (1(clac)wKE(Me)EC1GK(Me)PLVk-OH)-SEQ ID NO: 3.

[0019] The peptide of the present invention may be a composition or a pharmaceutical composition containing the peptide of the present invention.

[0020] In another aspect, the present invention provides a nucleic acid encoding the peptide of the present invention.

[0021] In another aspect, the present invention provides an expression vector containing DNA encoding the peptide of the present invention, and this vector is constructed for heterologous expression of the peptide 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 that has been engineered to heterologously express the peptide of the present invention, particularly a bacterial or mammalian producer cell (hereinafter referred to as "the transformed cell of the present invention"). In one embodiment, the transformed host cell contains the expression vector of the present invention.

[0023] Other aspects and preferred embodiments of the present invention are described in the definitions and other claims set forth below.

Brief Description of the Drawings

[0024] <N [Figure 1] Figure 1 shows cell membrane permeability in hepatocytes. HepG2 liver cells were treated with Cy5-labeled pep_260 (SEQ ID NO: 1) or Cy5 alone (untreated) for 60 minutes. [Figure 2] Figure 2 shows the effect of pep_260 (SEQ ID NO: 1) treatment on hepatic steatosis in KKAy mice. NALFD scoring was performed on liver tissues of each treatment group. [Figure 3] Figure 3 shows the effect of the mutant peptide (pep_yxww2f = SEQ ID NO: 2) on glucose turnover in skeletal muscle cells compared to insulin and the peptide of the present invention (pep_1E99R5 = SEQ ID NO: 1). [Figure 4]Figure 4 shows the effect of the mutant peptide (pep_yxww2f = SEQ ID NO: 2) on glycosylated hemoglobin (HbA1c%) in skeletal muscle cells compared to liraglutide. (B) Features were scored according to the mouse liver scoring system devised by 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; 12 weeks old at baseline) and were analyzed by Dunnett's test to compare the differences between the two peptide treatment groups, the vehicle control group, and the liraglutide group (*p < 0.05 **p < 0.01 ***p < 0.001). [Figure 5] Figure 5 shows the effect of the mutant cyclic peptide (pep_DJKTTM = SEQ ID NO: 3) on glucose turnover in skeletal muscle cells compared to the control and the peptide of the present invention (pep_1E99R5 = SEQ ID NO: 1). [Figure 6] Figure 6 shows the decrease in IL-8 secretion measured using sandwich ELISA in HepG2 cells treated with pep_1E99R5 (pep_1E99R5 = SEQ ID NO: 1) or 10PANX. Cells were treated with either peptide (5 ng / ml), 10PANX (μg / mL), or PBS for 24 hours and then further stimulated with 100 ng / ml of LPS for 24 hours. Data are presented as mean ± SD of three independent experiments. ***p ≤ 0.001 between replicates. [Figure 7]Figure 7 shows the anti-fibrotic activity of pep_1E99R5 in stimulated primary human hepatic stellate cells. (A) Confocal imaging of human stellate cells treated with TGF-β to stimulate α-SMA expression before treatment with ellafibranol (10 μM) or pep_1E99R5 (5 nM). (B) Quantification of cell size in pixels for each treatment condition. All conditions contain cells from three independent replicates: untreated: x cells, untreated + TGFβ x cells, ellafibranol x cells. (A).* p ≤ 0.05 between replicates, ** p ≤ 0.01 between replicates, *** p ≤ 0.001 between replicates. [Figure 8] Figure 8 shows changes in liver enzymes in an APAP-treated acute liver injury mouse model study. APAP was administered via IP injection at 0 hours. Pep_1E99R5, 10PANX, or saline control treatments were administered IV at 1.5 hours. Five animals were included in each group. ALT and AST levels in all mice were measured from survival and terminal bleeding at 2.25 and 6 hours. At 2.25 hours, administration of pep_1E99R5 significantly reduced ALT levels compared to the APAP / saline treatment (p<0.05) and surpassed 10PANX. The APAP / saline group showed increased ALT and AST compared to the saline group. Data are mean ± SEM and analyzed by t-tests, with multiple comparative tests performed as needed. [Modes for carrying out the invention]

[0025] All publications, patents, patent applications, and other references described herein are incorporated herein by reference in whole for any purpose as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference and their contents were fully enumerated.

[0026] Definitions and general settings Where used herein, unless otherwise indicated, the following terms are intended to have the following meanings in addition to the broader (or narrower) meanings that the terms may enjoy in the art:

[0027] Unless otherwise specified in the context, the use of the singular form here is to be interpreted as including the plural form, and vice versa. The terms "a" or "an" used in relation to things are to be interpreted as referring to one or more of those things. That is, the terms "a" (or "an"), "one or more," and "at least one" are used synonymously in this specification.

[0028] Where used herein, the term “comprise,” or its variations such as “comprises” or “comprising,” should be read to indicate the inclusion of an enumerated integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature, element, characteristic, property, method / process step, or limitation), but not the exclusion of other integers or groups of integers. Thus, where used herein, the term “comprises” is inclusive or open-ended and does not exclude additional, unenumerated integers or methods / process steps.

[0029] As used herein, the term “disease” is used to define an abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, condition, disease, state, or syndrome in which physiological function is impaired, regardless of the nature of the etiology (or whether the etiological basis of the disease is actually established). Thus, it includes conditions resulting from infection, trauma, injury, surgery, radiation therapy, poisoning, or malnutrition.

[0030] As used herein, the terms “treatment” or “treating” refer to an intervention (e.g., the administration of a drug to a subject) that cures, improves or alleviates the symptoms of a disease or eliminates its cause (or reduces its effects). In this context, the term is used synonymously with the term “therapy.”

[0031] Furthermore, the terms "treatment" or "treating" refer to interventions (e.g., the administration of drugs to subjects) that prevent or delay the onset or progression of a disease, or reduce (or eliminate) its incidence within a treated population. In this case, the term treatment is used synonymously with the term "prevention."

[0032] As used herein, an effective dose or therapeutic dose of a drug is defined as 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, e.g., treatment or prevention manifested by a permanent or transient improvement of the subject's condition. The amount varies from subject to subject depending on the subject's size, age, and general condition, method of administration, and other factors. Therefore, it is impossible to determine an exact effective dose, but those skilled in the art can determine an appropriate “effective” dose in any individual case using routine experimentation and background general knowledge. Therapeutic outcomes in this context include elimination or reduction of symptoms, reduction of pain or discomfort, extension of survival time, improvement of mobility, and other markers of clinical improvement. Therapeutic outcomes do not need to be complete cures. Improvements may be observed in biological / molecular markers, clinical or observational improvements. In preferred embodiments, the methods of the present invention are applicable to humans, large racing animals (horses, camels, dogs), and domestic companion animals (cats and dogs).

[0033] In the context of the treatment and effective dose as defined above, the term "subject" (which should be read as including "individual," "animal," "patient," or "mammal," where the context allows) is defined as any subject in need of treatment, in particular a mammalian subject. Mammalian subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sporting animals, pet animals, e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felines such as cats, lions, and tigers; horses such as horses, donkeys, and zebras; edible animals such as cattle, 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 human. As used herein, the term “horse” refers to mammals of the family Equidae, including horses, donkeys, asses, muntjacs, and zebras.

[0034] "Pharmaceutical Compositions": Further aspects of the present invention relate to pharmaceutical compositions comprising a peptide activator, which are mixed with one or more pharmaceutically acceptable diluents, excipients, or carriers, or administered co-administered with other drugs that enhance therapeutic effects. Peptide activators can be administered alone, but for human therapy in particular, they are generally administered in mixture with pharmaceutical carriers, excipients, or diluents. Pharmaceutical compositions may be for human or animal use in human and veterinary medicine. Examples of such suitable excipients for various different forms of pharmaceutical compositions described herein can be found in "Handbook of Pharmaceutical Excipients, 2nd Edition, (1994)" edited by A Wade and PJ Weller. Formulations for topical delivery in particular are described in "Topical drug delivery formulations," Taylor & Francis, edited by David Osborne and Antonio Aman, the complete 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, Mack Publishing Co. (edited by AR Gennaro, 1985). Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol. Examples of suitable diluents include ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent can be made considering the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may include, or in addition to, any suitable binders, lubricants, suspending agents, coatings, and solubilizers as carriers, excipients, or diluents. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol.Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, dyes, and even flavorings may be provided in pharmaceutical compositions. Examples of preservatives include esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0035] As used herein, the “effective dose” or “therapeutic effective dose” of a peptide activator defines an amount sufficient to provide the desired effect, e.g., treatment or prevention indicated by a permanent or transient improvement of the subject’s condition, without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio. The amount varies from subject to subject depending on the individual’s age and general condition, method of administration, and other factors. Therefore, it is impossible to specify an exact effective dose; however, those skilled in the art can determine an appropriate “effective” dose in any individual case using routine experimentation and background general knowledge. Therapeutic outcomes in this context include elimination or reduction of symptoms, reduction of pain or discomfort, extension of survival time, improvement of mobility, and other markers of clinical improvement. Therapeutic outcomes do not necessarily have to be a complete cure.

[0036] As used herein, the term “peptide” refers to a polymer consisting of up to 50 amino acids, for example, 5 to 50 amino acid monomers typically linked via peptide bonds. The peptides of the present invention and for use in the present invention (including their fragments and variants) can be produced whole or partially by chemical synthesis or by expression from nucleic acids. For example, the peptides of the present invention and for use in the present invention can be readily prepared in well-established standard liquids or, preferably, according to solid-phase peptide synthesis methods known in the art (see, for example, J.M. Stewart and J.D. Young, Solid-Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Company, Rockford, Illinois (1984); M. Bodanzky and A. Bodanzky, The Practice of Peptide Synthesis, Springerberg, New York (1984)). If necessary, any of the peptides used in the present invention can be chemically modified to increase their stability. Chemically modified peptides or peptide analogs include any functional chemical equivalents of the peptides characterized by their increased stability and / or efficacy in vivo or in vitro with respect to the implementation of the present invention. The term peptide analog also refers to any amino acid derivative of a peptide as described herein. Peptide analogs can be produced by procedures including, but not limited to, side-chain modification, incorporation of non-natural amino acids and / or their derivatives during peptide synthesis, and the use of crosslinking agents, and other methods that impose conformational constraints on peptides or their analogs. Examples of side-chain modification include reductive alkylation by reaction with aldehydes followed by reduction with NaBH4; amidation with methyl acetimidate; acetylation with acetic anhydride; carbamylation of the amino group with cyanates; trinitrobenzylation of the amino group with 2,4,6,trinitrobenzenesulfonic acid (TNBS); alkylation of the amino group with succinic anhydride and tetrahydrophthalic anhydride; and modification of the amino group by pyridoxylation of lysine with pyridoxa-5'-phosphate followed by reduction with NABH4.The guanidino group of the arginine residue may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal. The carboxyl group can be modified by carbodiimide activation via o-acyl isourea formation, followed by derivatization to, for example, the corresponding amide. The sulfhydryl group can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performation to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; formation of mercury derivatives using 4-chloromercuric benzoic acid, 4-chloromercuric phenylsulfonic acid, phenylmercuric chloride, 2-chloromercuric-4-nitrophenol, and other mercury compounds; and carbamylation with cyanates at an alkaline pH. Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or halogenated sulfonyl. Tyrosine residues may be converted to form 3-nitrotyrosine derivatives by nitration with tetranitromethane. Modification of the imidazole ring of histidine residues can be achieved by alkylation with iodoacetic acid derivatives or N-carbetoxylation with diethyl pyrocarbonate. Examples of incorporating non-natural amino acids and derivatives during peptide synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and / or D-isomers of amino acids. Peptide structure modification involves the creation of retroinversopeptides containing the reverse sequence encoded by the D-amino acids.The changes may involve reducing susceptibility to proteolysis, reducing susceptibility to oxidation, altering the binding affinity of the mutant sequence (typically and preferably increasing the affinity), and / or conferring or modifying other physicochemical or functional properties to the associated mutant / analog peptide.

[0037] The term “therapeutically effective variant” applied to a reference peptide means a peptide having an amino acid sequence substantially identical to the reference peptide, such that it is therapeutically effective as defined below. Therefore, for example, this term should be interpreted to include variants modified with respect to one or more amino acid residues. Preferably, such modifications include insertions, additions, deletions, and / or substitutions of 6 or fewer, more preferably 5 or fewer, 4 or fewer, even more preferably 3 or fewer, and most preferably only 1 or 2 amino acids. Insertions, additions, and substitutions of native and modified amino acids are assumed. The variant may have conservative amino acid changes, and the introduced amino acid is structurally, chemically, or functionally similar to the one being substituted. Generally, the variant has at least 50%, 60%, or 70% amino acid sequence identity with the parent sequence, preferably at least 80%, more preferably at least 90%, and ideally at least 95%, 96%, 97%, 98%, or 99% sequence identity. It should be noted that any variant may have essentially the same therapeutic effect, or an enhanced effect, when tested in an in vitro or in vivo model of the disease. An exemplary variant in which five amino acids are replaced with the D-form of the amino acids is provided as SEQ ID NO: 2.

[0038] The term "therapeutably effective" as applied to the peptides of the present invention means a peptide that can penetrate HepG2 hepatocytes in the hepatocyte penetration assay described herein and that can significantly alleviate macrodroplet steatosis in obese diabetic KKAy mice in the chronic diabetic mouse model described later. In one embodiment, a therapeutically effective peptide can mediate changes in all or most of the biomarker activities listed in Table 1.

[0039] The term "mutant" is also understood to encompass the term "fragment," and therefore refers to the segment of amino acid sequence number 1. Typically, fragments have a length of 3 to 13 consecutive amino acids. Generally, fragments have a charge ranging from -5 to +3. The charge of a peptide, fragment, or region is determined using the method described in Cameselle, JC, Ribeiro, JM, and Sillero, A. Biochem Educ. 14, 131-136 (1986), “Derivation and use of a formula to calculate the net charge of acid-base compounds. Its application to amino acids, proteins and nucleotides.”

[0040] In this specification, the term “sequence identity” should be understood to include both sequence identity and similarity. That is, a variant (or homolog) that shares 70% sequence identity with a reference sequence is a variant (or homolog) in which any 70% of the aligned residues of the variant (or homolog) over the entire length of the sequence are identical or conserved substitutions of the corresponding residues in the reference sequence. Sequence identity is the amount of letters that exactly match between two different sequences. Here, gaps are not counted, and the measurement relates to the shorter of the two sequences.

[0041] Regarding the term "sequence homology," it should be understood that this term means sharing a defined percentage similarity or identity with a reference sequence when the percentage of aligned residues in a mutant (or homolog) is identical to or conserved to the corresponding residues in the reference sequence, and the mutant (or homolog) shares the same function as the reference sequence.

[0042] This alignment and homology or sequence identity percentage can be determined using software programs known in the art; for example, one alignment program is BLAST, which uses default parameters. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi.

[0043] The term "C-terminal domain" applied to a fragment refers to the first three amino acids at the C-terminus of the fragment.

[0044] The "N-terminal domain" applied to a fragment refers to the last three amino acids at the N-terminus of the fragment.

[0045] The "homolog" of a reference protein should be understood to mean a protein derived from a different plant species that has at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to the reference protein.

[0046] "Pharmaceutical Compositions": Further aspects of the present invention relate to pharmaceutical compositions comprising a peptide activator, which are mixed with one or more pharmaceutically acceptable diluents, excipients, or carriers, or co-administered with other drugs that enhance therapeutic effects. While the peptides and compositions of the present invention may be administered alone, they will generally be administered in mixture with pharmaceutical carriers, excipients, or diluents, particularly for human treatment. Pharmaceutical compositions may be for human or animal use in human and veterinary medicine. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein can be found in "Handbook of Pharmaceutical Excipients, 2nd Edition (1994)" edited by A Wade and PJ Weller. Formulations for topical delivery, in particular, are described in "Topical drug delivery formulations," Taylor & Francis, edited by David Osborne and Antonio Aman, the complete 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," Mack Publishing Co. (AR Gennaro ed., 1985). Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent can be made considering the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may include, or in addition to, any suitable binders, lubricants, suspending agents, coatings, or solubilizers as carriers, excipients, or diluents. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol.Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, dyes, and even flavorings may be provided in pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters. Antioxidants and suspending agents may also be used.

[0047] Peptides or compositions may be adapted to and administered via topical, oral, rectal, parenteral, intramuscular, intraperitoneal, intra-arterial, intra-bronchial, subcutaneous, intradermal, intravenous, nasal, vaginal, buccal, 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 to 100 mg, of the active ingredient per dose. Other forms of administration may include solutions or emulsions prepared from sterile or buccal solutions and may be injected intravenously, intra-arterial, subcutaneous, intradermal, intraperitoneal, or intramuscularly. The pharmaceutical compositions of the present invention may also be in the form of suppositories, vaginal rings, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions, or dusting powders. The compositions of the present invention may be formulated for topical delivery. Topical delivery generally means delivery to the skin, but may also mean delivery to epithelial cells, e.g., to body cavities lined with the lungs or airways, gastrointestinal tract, or buccal cavity. In particular, formulations for topical delivery are described in Topical drug delivery formulations, Taylor & Francis, edited by David Osborne and Antonio Aman, the complete contents of which are incorporated herein by reference. Compositions or formulations for delivery to the airway are described by O'Riordan et al. in Respir Care, 2002, Nov. 47, EP2050437, WO2005023290, US2010098660, and US20070053845. Compositions and formulations for delivery of active ingredients to the ileum, particularly the proximal ileum, include microparticles and microencapsulated products in which the activator is encapsulated within a protective matrix formed of a polymer or milk protein that is acid-resistant but readily soluble in the more alkaline environment of the ileum. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1. An alternative to transdermal administration is by the use of 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 may also be incorporated, as needed, along with stabilizers and preservatives, into an ointment consisting of a white wax or white soft paraffin base at a concentration of 1 to 10% by weight.

[0048] The injectable form may contain 10 to 1000 mg, preferably 10 to 250 mg, of the active ingredient per dose.

[0049] The composition may be formulated in unit dosage forms, i.e., in the form of separate parts containing a unit dose, or in the form of multiple or subunits of a unit dose.

[0050] Those skilled in the art can readily determine one appropriate dose of the present composition for administration to a subject without excessive experimentation. Typically, a physician will determine the most appropriate actual dose for an individual patient, which will depend on various factors including the activity of the particular compound used, the metabolic stability and duration of action of that compound, age, weight, general health, sex, diet, mode and timing of administration, excretion rate, drug combination, severity of the particular condition, and the individual being treated. The dosages disclosed herein are illustrative examples for average cases. Of course, there may be individual examples where higher or lower dosage ranges are beneficial, and such are within the scope of the present invention. Where necessary, the drug may be administered in doses of 0.01 to 30 mg / kg body weight, e.g., 0.1 to 10 mg / kg, more preferably 0.1 to 1 mg / kg body weight. In exemplary embodiments, one or more doses of 10 to 300 mg / day or more preferably 10 to 150 mg / day are administered to a patient for the treatment of an inflammatory disorder.

[0051] In particularly preferred embodiments, the methods and uses of the present invention involve administering the peptide or composition in combination with one or more other activators, such as commercially available existing NAFLD drugs or pharmacological enhancers. In such cases, the compounds of the present invention may be administered sequentially, simultaneously, or in sequence with one or more other activators.

[0052] In one embodiment of the present invention, the peptide activator may be administered in the form of a conjugate comprising a peptide, a linker, and an antibody molecule (or antibody fragment) intended to increase the in vivo half-life of the conjugate.

[0053] "Modified peptide": In one embodiment, the peptides of the present invention (including peptide variants) may be modified peptides. The term "modified peptide" is used interchangeably with the term "derived peptide." In one embodiment, the term "modified peptide" means a peptide modified to exhibit one or more of the following properties compared to an unmodified peptide: extended plasma half-life; increased lipophilicity of the peptide; increased renal clearance of the modified peptide; and improved resistance of the modified peptide to proteolysis, typically while retaining rpS6 phosphorylation activity. Various methods for modifying the peptides of the present invention to exhibit these properties are disclosed herein and include binding of the peptide to a binding partner (e.g., albumin-binding small molecule, large polymer, long-lived plasma protein, or antibody or antibody fragment), cyclization, addition of an N-terminus or C-terminus, or side chain, protecting group, substitution of an L-amino acid with a D-isomer, amino acid modification, increased plasma protein binding, and increased albumin binding. Modified peptides include, but are not limited to, peptides that are substituted with, bound to, or cyclized with one or more groups as defined herein. Generally, peptides are modified to extend their half-life in living animals. Various modification methods are described below.

[0054] In one embodiment, the modification may be any modification that provides the peptide and / or composition of the present invention with an increased ability to penetrate cells. In one embodiment, the modification may be any modification that increases the half-life of the composition or peptide of the present invention. In one embodiment, the modification may be any modification that increases the activity of the composition or peptide of the present invention. In one embodiment, the modification may be any modification that increases the selectivity of the composition or peptide of the present invention.

[0055] In one embodiment, the group is a protecting group. The protecting group may be an N-terminal protecting group, a C-terminal protecting group, or a side-chain protecting group. The peptide may have one or more of these protecting groups.

[0056] Those skilled in the art know appropriate techniques for reacting amino acids with these protecting groups. These groups can be added by preparation methods known in the art, for example, by the methods outlined in paragraphs

[0104] to

[0107] of US2014120141. The groups may remain on the peptide or be removed. Protecting groups may be added during synthesis.

[0057] In embodiments of the present invention, the peptide may be substituted with one or more linear or branched, long or short, saturated or unsaturated groups having 1 to 29 carbon atoms, substituted with hydroxyl, amino, aminoacyl, sulfate or sulfide groups, or unsubstituted. N-acyl derivatives include acyl groups derived from acetic acid, capric acid, lauric acid, myristic acid, octanoic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid, isosteric acid, elidoic acid, 2-ethylhexanoic acid, coconut oil fatty acids, tallow fatty acids, hydrogenated tallow fatty acids, palm kernel fatty acids, lanolin fatty acids, or similar acids. These may be substituted or unsubstituted. If substituted, they are preferably substituted with hydroxyl, or SO3H, SH, or SS, etc., but are not limited to these.

[0058] In one embodiment of the present invention, the peptide is R1-X-R2. The R1 and / or R2 groups are attached to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the peptide sequence, respectively.

[0059] In one embodiment, the peptide is R1-X, or the peptide is X-R2. Preferably, R1 is H, C1-4 alkyl, acetyl, benzoyl, or trifluoroacetyl.

[0060] X is the peptide of the present invention.

[0061] R2 is either OH or NH2.

[0062] In one embodiment, R1 is selected from a group formed by H, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and R5-CO-, where R5 is selected from a group formed by H, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocyclyl, and a substituted or unsubstituted heteroarylalkyl;

[0063] R2 is selected from the group formed by -NR3R4, -OR3 and -SR3, where R3 and R4 are independently selected from the group formed by H, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl; and R1 and R2 are not α-amino acids.

[0064] According to another preferred embodiment, R2 is -NR3R4, -OR3 or -SR3, and R3 and R4 are independently selected from the group formed by H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, Tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (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, 3- to 10 membered substituted or unsubstituted heterocyclyl rings, and substituted or unsubstituted heteroarylalkyls having 2 to 24 carbon atoms and 1 to 3 non-carbon atoms, with the alkyl chain having 1 to 6 carbon atoms. Optionally, R3 and R4 may be linked by saturated or unsaturated carbon-carbon bonds and form a ring with a nitrogen atom. More preferably, R2 is -NR3R4 or -OR3, and R3 and R4 are independently selected from the group formed by 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 3- to 10-membered substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl having a 3- to 10-membered ring and an alkyl chain of 1-6 carbon atoms. More preferably, R3 and R4 are selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. More preferably, R3 is H, and R4 is selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. According to a more preferred embodiment, R2 is selected from -OH and -NH2.

[0065] According to another embodiment of the present invention, R1 is selected from the group formed by H, acetyl, lauroyl, myristoyl, or palmitoyl, R2 is -NR3R4 or -OR3, and R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl, and hexadecyl, preferably R2 is -OH or -NH2. More preferably, R1 is acetyl or palmitoyl and R2 is -NH2.

[0066] In a preferred embodiment, the acyl group is bonded to the N-terminus of at least one amino acid of the peptide.

[0067] In one embodiment of the present invention, the peptide is modified to include a side-chain protecting group. The side-chain protecting group may be one or more groups including a benzyl or benzyl-based group, a t-butyl-based group, a benzyloxycarbonyl (Z) group, and an allyloxycarbonyl (alloc) protecting group. The side-chain protecting group may be derived from an achiral amino acid such as achiralglycine. The use of an achiral amino acid helps to stabilize the resulting peptide and also facilitates the simple synthetic route of the present invention. Preferably, the peptide further includes a modified C-terminus, preferably an amidated C-terminus. The achiral residue may be α-aminoisobutyric acid (methylalanine). It will be understood that the specific side-chain protecting group used depends on the sequence of the peptide and the type of N-terminal protecting group used.

[0068] In one embodiment of the present invention, a peptide is conjugated, linked, or fused to one or more polyethylene glycol polymers or other compounds such as molecular weight increasing compounds. The molecular weight increasing compound is any compound that increases the molecular weight, typically by 10% to 90% or 20% to 50% of the resulting conjugate, and may have a molecular weight between 200 and 20,000, preferably between 500 and 10,000. The molecular weight increasing compound may be PEG, any water-soluble (amphiphilic or hydrophilic) polymer moiety, homo or copolymer of PEG, monomethyl-substituted polymer of PEG (mPEG) and polyoxyethylene glycerol (POG), polyamino acids such as polylysine, polyglutamic acid, and polyaspartic acid, particularly those in their L-conformations, pharmacologically inactive proteins such as albumin and gelatin, fatty acids, oligosaccharides, lipid amino acids and dextran. The polymer portion may be linear or branched and may have molecular weights 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-permeable compound such as tat peptide, penetratin, or pep-1. The compound may be an antibody molecule. The compound may be a lipophilic portion or a polymer portion.

[0069] Lipophilic substituents and polymeric substituents are known in the art. Lipophilic substituents include N, O, or S atoms that form part of an acyl group, sulfonyl group, 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 linear or branched, saturated or unsaturated. The hydrocarbon chain may be further substituted. It may be a cycloalkane or heterocycloalkane.

[0070] The peptide may be modified at its N-terminus, C-terminus, or both. The polymer or compound is preferably linked to an amino, carboxyl, or thio group, and may be linked by the N-terminus or C-terminus of the side chain of any amino acid residue. The polymer or compound may be conjugated to the side chain of any suitable residue.

[0071] Polymers or compounds can be conjugated via spacers. Spacers may be natural or non-natural amino acids, succinic acid, lysyl, glutamyl, asparagyl, glycyl, beta-alanyl, or gamma-aminobutanoyl. Polymers or compounds can also be conjugated via esters, sulfonyl esters, thioesters, amides, carbamates, urea, or sulfonamides. Those skilled in the art will recognize appropriate means for preparing the conjugates described.

[0072] Peptides can have their cyclic half-life extended by chemical modification, for example, by covalent bonding to a polymer. Exemplary polymers and methods for attaching such polymers to peptides are shown, for example, in U.S. Patents 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) moieties.

[0073] The peptides of the present invention may undergo one or more modifications to manipulate any aspect of the peptide's biological activity, such as storage stability, pharmacokinetics, and / or efficacy, selectivity, and drug interactions. Possible chemical modifications to the peptide include, but are not limited to, conjugations to the peptide of one or more of the following: polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polypropylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, colomic acid or other carbohydrate-based polymers, amino acid polymers, and biotin derivatives. PEG conjugation of proteins at Cys residues is disclosed, for example, in Goodson, RJ & Katre, NV (1990) Bio / Technology 8, 343 and Kogan, TP (1992) SyntheticCom. 22, 2417.

[0074] Modified peptides may also include sequences in which one or more residues are modified (i.e., by phosphorylation, sulfation, acylation, PEGylation, etc.), and variants containing one or more modified residues relative to the parent sequence. Amino acid sequences may also be modified with labels that can provide a directly or indirectly detectable signal, including but not limited to radioisotopes, fluorescence, and enzyme labels. Fluorescent labels include, for example, Cy3, Cy5, Alexa, BODIPY, fluorescein (e.g., FluorX, DTAF, and FITC), rhodamine (e.g., TRITC), auramine, Texas Red, AMCA Blue, and Lucifer Yellow. Preferred isotopic 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 and peroxidase, and alkaline phosphatase (see, for example, U.S. Patent No. 3,654,090; U.S. Patent No. 3,850,752 and U.S. Patent No. 4,016,043). The enzymes can be conjugated by reaction with cross-linking molecules such as carbodiimide, diisocyanate, and glutaraldehyde. Enzyme labels can be detected visually or measured by calorimetry, spectrophotometry, fluorescence spectrophotometry, amperometry, or gas assays. Other labeling systems, such as avidin / biotin and tyramide signal amplification (TSA®), are known in the art and are commercially available (see, for example, ABC Kits, Vector Laboratories, Inc., Burlingame, California; NEN® Life Science Products, Inc., Boston, Massachusetts).

[0075] In one embodiment, peptides, variants, and / or compositions are modified to enhance their drug performance capabilities. In one embodiment, peptides, variants, and / or compositions are modified to increase stability, permeability, maintain potency, avoid toxicity, and / or increase half-life. Modifications may be as described above. For example, modifications may involve protecting the N and C termini, which may be modified amino acids, cyclization, amino acid substitution, and / or binding to high molecular weight or large polymers or long-lived plasma proteins. Strategies for extending half-life may be those described by Strohl et al. (BioDrugs, 2015), Schlapschy et al. (Protein Eng DesSel. 2013), Podust, V.N. et al. (Protein Eng DesSel. 2013), Zhang, L. et al. (Curr MedChem. 2012), and Gaberc-Porekar, V. et al. (Curr Opin Drug DiscovDevel. 2008). Examples include PEGylation, lipidation (covalent bonding of fatty acids to peptide side chains), fusion to Fc domains and human serum albumin, fusion with hydrophilic amino acid polymers (such as XTEN or PAS), and / or fusion with half-life extension proteins.

[0076] Peptide modifications to extend the in vivo half-life of peptides are described in the literature, for example: Strategies to improve the plasma half-life of peptide and protein drugs. Werle M, Bernkop-Schnurch A. Amino Acids. 2006 Jun; 30(4): 351-67.

[0077] Due to the obvious advantages of long-acting peptide and protein drugs, strategies for extending the plasma half-life of such compounds are highly sought after. Short plasma half-lives are generally due to rapid renal clearance and enzymatic degradation occurring in systemic circulation. Peptide / protein modifications can lead to extension of plasma half-life. By shortening the overall amino acid content of somatostatin and replacing L:-analog amino acids with D:-amino acids, the plasma half-life of the derivative octreotide was 1.5 hours, compared to only a few minutes for somatostatin. The PEG(2,40 K) conjugate of INF-alpha-2b showed a 330-fold extension of plasma half-life compared to the native protein. The aim of this review was to provide an overview of possible strategies for extending plasma half-life, such as N-terminal and C-terminal modifications and PEGylation, and methods for evaluating the effectiveness of drug modifications. Furthermore, basic data on the most important proteolytic enzymes in human blood, liver, and kidneys, as well as their cleavage specificity and their inhibitors, are provided to predict the enzymatic cleavage of peptide and protein drugs in systemic circulation.

[0078] Strategic Approaches to Optimizing Peptide ADME Properties. Li Di AAPS J. 2015 Jan; 17(1):134-143.

[0079] Strategies for stabilizing peptides from proteolysis Many approaches are available to enhance peptide stability through structural modification. Some approaches not only improve stability but also enhance other ADME properties. For example, cyclization can increase stability and permeability, while conjugation to polymers can improve stability and decrease renal clearance. It is important to maintain potency and avoid toxicity while improving peptide stability and ADME properties.

[0080] • Protection of the N-terminus and C-terminus Many proteolytic enzymes in blood / plasma, liver, or kidneys are exopeptidases, aminopeptidases, and carboxypeptidases, which degrade peptide sequences from the N-terminus and C-terminus. Modifications to the N-terminus and / or C-terminus often improve peptide stability. In many cases, N-acetylation and C-amidation have been reported to increase resistance to proteolysis.

[0081] • Substitution of L-amino acids with D-amino acids Substituting a natural L-amino acid with a non-natural D-amino acid reduces the substrate recognition and binding affinity of proteolytic enzymes, while improving stability. One example is vasopressin, which contains L-arginine and has a half-life of 10-35 minutes in humans. The half-life of desmopressin, a D-Arg analog, is 3.7 hours in healthy human volunteers. In studies of bicyclic peptide inhibitors of cancer-related proteaseurokinase-type plasminogen activators (uPAs), substituting a specific glycine with D-serine not only increased potency by 1.8 times but also improved stability in mouse plasma by 4 times.

[0082] • Amino acid modification Modification of natural amino acids can improve peptide stability by introducing steric hindrance or interfering with enzymatic recognition. For example, while gonadotropin-releasing hormone has a very short half-life (a few minutes), buserelin in humans, in which one glycerin molecule is replaced with t-butyl-D-Ser and another with ethylamide, has a much longer half-life.

[0083] ·Cyclization The peptides of the present invention may be cyclized. Cyclization imposes conformational constraints, reducing the flexibility of the peptide and increasing its stability and permeability. Depending on the functional group, the peptide can be cyclized from head to tail, head / tail to side chain, or side chain to side chain. Cyclization is usually carried out via lactamization, lactonization, and sulfide-based bridging. Disulfide crosslinking can form folding and conformational constraints and improve potency, selectivity, and stability. Several disulfide-rich peptides are commercially available or in preclinical or clinical development, e.g., linaclotide, repiridine, and diconotide. In one embodiment, the peptide is cyclized between the amino terminus and the carboxyl terminus. In one embodiment, the peptide is cyclized between the amino terminus and the side chain. In one embodiment, the peptide is cyclized between the carboxyl terminus and the side chain. In one embodiment, the peptide is cyclized between side chains. In one embodiment, the cyclic peptide is selected from homodetic cyclic peptides, cyclic isopeptides, cyclic depsipeptides, or monocyclic or bicyclic peptides. The method for cyclizing the peptide is described below: Jensen, Knud (2009-09-01). Peptide and Protein Design for Biopharmaceutical Applications. John Wiley & Sons. ISBN 9780470749715. 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. 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. 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.M112.43794. PMC 3642298.PMID 23486480. 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.PMC 3180235. PMID 21764803. Wenyan Xu; et al."Plant Cyclopeptides and Possible Biosynthetic Mechanisms". David J. Craik (March 17, 2006). "Seamless Proteins Tie Up Their Loose Ends". Science. 311 (5767):1563-7. doi: 10.1126 / science.1125248.PMID 16543448.

[0084] • Conjugate to polymers Conjugation to polymers (e.g., polyethylene glycol (PEG), albumin) is an effective strategy to improve peptide stability and reduce renal clearance.

[0085] Renal clearance Many peptides exhibit promising pharmacological activity in vitro, but their very short half-lives (several minutes) prevent them from demonstrating in vivo efficacy. Rapid peptide clearance and short half-lives hinder their development into successful drugs. The primary causes of rapid peptide clearance from the systemic circulation are enzymatic proteolysis and / or renal clearance. Glomerular pore size is approximately 8 nm, making hydrophilic peptides with MW < 2–25 kDa susceptible to rapid filtration via the renal glomeruli. Because peptides are not readily reabsorbed from the renal tubules, they often have high renal clearance and short half-lives. Other minor pathways of peptide clearance include endocytosis and degradation by the proteasome and liver. Comparing systemic and renal clearance in animal models provides useful information regarding whether renal clearance is likely the primary excretion pathway.

[0086] In patients with renal impairment, inappropriate administration of peptide drugs can lead to toxicity or ineffective treatment; therefore, it may be necessary to adjust the dosage of peptide drugs to avoid drug accumulation and exposure to high concentrations. Several strategies have been developed to reduce peptide renal clearance and extend half-life. These are outlined below.

[0087] • Improved plasma protein binding When peptides bind to membrane proteins or serum proteins, their renal clearance decreases. An example is octreotide, a cyclic peptide drug used to treat endocrine tumors. Because it binds to lipoproteins, its half-life in humans is approximately 100 minutes (unbound fraction 0.65).

[0088] • Covalent bonding to albumin-binding small molecules When albumin-binding small molecules are covalently bonded to peptides, they indirectly interact with albumin through the highly bound small molecules, thereby reducing glomerular filtration, improving the stability of proteolysis, and extending the half-life.

[0089] • Conjugate to large polymers Conjugating peptides into large synthetic or natural polymers or carbohydrates can increase molecular weight and hydrodynamic volume, potentially reducing renal clearance. Common polymers used in peptide conjugation include PEG, polysialic acid (PSA), and hydroxyethyl starch (HES).

[0090] • Fusion to long-lived plasma proteins Plasma proteins such as albumin and immunoglobulin (IgG) fragments have long half-lives of 19-21 days in humans. Due to their high molecular weight (MW) (67-150 kDa), these proteins have low renal clearance, and when they bind to the neonatal Fc receptor (FcRn), their excretion by vascular epithelium is reduced. Covalently binding peptides to albumin or IgG fragments can reduce renal clearance and extend their half-lives.

[0091] Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. William R. Strohl. BioDrugs. 2015; 29(4):215-239. Schlapschy, M, Binder, U, Borger, C et al. 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. 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 Des Sel. 2013; 26(11):743-53.

[0092] Zhang, L, Bulaj, G. Converting Peptides into Drug Leads by Lipidation. Curr Med Chem. 2012; 19(11):1602-18. Gaberc-Porekar, V, Zore, I, Podobnik, B et al. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug DiscovDevel. 2008; 11(2):242-50. Dr. Ronald V. Swanson - Evolution of Long-Lived Peptides and Novel Hybrid Approaches to Peptide Half-Life Extension Technology. From DrugDiscoveryWorld online, Spring 2014.

[0093] PEGylation PEGylation, which involves attaching long chains of the hydrophilic polymer polyethylene glycol to a target molecule, was originally conceived as a modification that would prevent the immune system from recognizing foreign proteins, thereby enabling their usefulness as therapeutic agents. Once antibodies against unmodified drugs are formed, protein drugs are rapidly neutralized and removed. Unexpectedly, PEGylation improved the pharmacokinetics of proteins even in the absence of anti-drug antibodies.1 By simply increasing the size of the drug molecule, PEGylation slowed the filtration of the drug by the kidney. The empirical observation that increased size or hydrodynamic radius leads to decreased renal clearance and increased half-life subsequently became the main rationale for PEGylation of protein and peptide drugs. PEGylation can have various effects on molecules, such as making proteins and peptides more water-soluble or protecting them from degradation by proteolytic enzymes. PEGylation also affects the binding of therapeutic proteins to their homologous cell receptors, which can usually decrease affinity. Changes in the size, structure, and binding mode of PEG polymers can affect the biological activity of the bound drug.

[0094] First-generation PEGylation methods were fraught with challenges. However, the chemistry of PEGylation is quite simple. The process involves the covalent bonding of polyethylene glycol chains to the reactive side chains of a protein or peptide. For example, PEG readily binds to the lysine-amino groups on the surface of proteins and peptides.2 The reaction is pH-dependent. At high pH (above 8.0), the amino groups of the lysine side chains covalently bond to PEG via N-hydroxysuccinimide. This method typically yields a family of products in which varying numbers of PEG chains are attached to different sites on the protein, rather than a single, distinct product.3 The first PEGylated drugs approved were Pegademase bovine (PEGylated bovine siadenosine deamidase) as enzyme replacement therapy for severe combined immunodeficiency and Pegaspargase (PEGylated asparaginase) for the treatment of acute lymphoblastic leukemia.1 These drugs were complex mixtures of various PEGylated species, but they offered improved therapeutic properties compared to natural enzymes, such as extended serum half-life and reduced immunogenicity of the protein. Due to the inherent polydispersity of PEG, quality and batch-to-batch reproducibility were difficult. Despite these limitations, two PEGylated interferons (pegylated interferon alpha-2b and pegylated interferon alpha-2a), which are a heterogeneous population of numerous monoPEGylated site isomers, have been FDA approved for the treatment of hepatitis C. These drugs were launched in 2001 and 2002, respectively.

[0095] Basic PEGylation techniques have undergone various improvements and variations. Second-generation PEGylation processes have introduced the use of branched structures and alternative chemicals for PEG attachment. In particular, PEGs with cysteine-reactive groups, such as maleimide and iodoacetamide, allow for targeting of PEGylation to single residues within peptides or proteins, reducing heterogeneity in the final product, but they do not eliminate heterogeneity due to the polydispersity of PEG itself.

[0096] The initial rationale for PEGylation was to reduce immunogenicity; however, there are several examples of immunogenic PEGylated proteins. One example is PEGylated urate oxidase, an enzyme that lowers plasma uric acid levels in gout patients. In clinical trials, a relatively high percentage of gout patients did not respond to treatment, and an antibody specific to PEG but not to the uricase protein was developed.2 PEGylated liposomes are generally considered non-immunogenic, but several studies have shown them to be immunogenic. PEGylated liposomes induce a potent anti-PEG immunoglobulin M (IgM) response. Furthermore, it has been shown that multiple injections of PEG-glucuronidase induce the production of specific anti-PEG IgM antibodies and promote the clearance of PEG-modified proteins from the body.

[0097] The main potential drawback of using PEG as a modifier is that it is non-biodegradable. The U.S. Food and Drug Administration (FDA) has approved PEG for use as a vehicle in pharmaceuticals, including injectable, topical, rectal, and nasal formulations. PEG is largely non-toxic and is excreted intact from the body either by the kidney (for PEG < 30 kDa) or feces (for PEG > 20 kDa)¹. Repeated administration of several PEGylated proteins to animals has resulted in observation of renal tubular cell vacuolation. More recently, vacuolation of choroid plexus epithelial cells has also been observed in toxicity studies using proteins conjugated with large (≧40 kDa) PEG. Choroid plexus epithelial cells produce cerebrospinal fluid and form the blood CSF barrier. While the long-term adverse effects of cell vacuolation are unknown, it is certainly an undesirable outcome for some potential therapeutics. One possible alternative is to use a biodegradable polymer instead of PEG. Polymers such as hydroxyethyl starch (HES) are considered alternatives. HES is non-toxic and biodegradable and is used as a plasma substitute. The HESation process functions similarly to PEGylation in that it reduces renal clearance by increasing the hydrodynamic radius of the peptide, but it may have a lower tendency to accumulate due to its biodegradability. However, HES and other proposed biodegradable polymer PEG alternatives are polydispersible, similar to PEG, making characterization of the end product and metabolites difficult. One novel solution that mitigates both concerns is to use polypeptides defined as polymer components. This approach will be discussed later.

[0098] Lipidization A second major chemical modification method for extending the half-life of peptides is lipidization, which involves the covalent bonding of fatty acids to the peptide side chain.4 Originally conceived and developed as a method to extend the half-life of insulin, lipidization shares the same basic mechanism as PEGylation for half-life extension: increasing the hydrodynamic radius and reducing renal filtration. However, the lipid portion itself is relatively small, and its effect is indirectly mediated through non-covalent bonding of the lipid portion to circulating albumin. Albumin is a large (67 kDa) protein that is very abundant in human serum (35-50 g / L) and naturally possesses the function of transporting lipid-containing molecules throughout the body. Binding to plasma proteins can also protect the peptide from peptidase attack through steric hindrance, similar to that seen in PEGylation. As a result of lipidization, the water solubility of the peptide decreases, but this can be mitigated by manipulating the linker between the peptide and the fatty acid, for example, by using glutamate or mini-PEG in the linker. Linker manipulation and changes in the lipid portion affect self-aggregation and may contribute to extending the half-life by slowing down in vivo distribution independently of albumin.

[0099] Following pioneering research on insulin, the lipidization of various peptides, particularly those in the diabetes field such as human glucagon-like peptide-1 (GLP-1) analogs, glucose-dependent insulin-secreting polypeptides, and especially GLP-1R / glucagon receptor coagonists, has been studied. Two lipidized peptide drugs are currently approved by the FDA for human use. These are both long-acting antidiabetic drugs: the GLP-1 analog liraglutide and insulin detemir.

[0100] The pharmacologically relevant difference between PEGylation and lipidation is that therapeutically active peptides covalently bind to much larger PEGs, while smaller lipid acyl peptide conjugates uncovalently bind to larger albumin, and it is thought that bound and unbound forms exist in equilibrium. This can lead to differences in biodistribution and potentially different pharmacologies, as access to receptors localized in different tissues may induce different effects. In some cases, more restricted biodistribution is desirable, while in others, greater tissue penetration is important. An interesting variation of the PEG approach addressing this problem was developed by Santi et al., utilizing a release-type PEG conjugate with a predictable cleavage rate.7

[0101] PEGylation and lipidation confer protection to proteases and peptidases by shielding through steric hindrance, and directly or indirectly extend the circulating half-life through an increased hydrodynamic radius. Both methods are flexible in that they utilize chemical conjugations and are independent of the means used to produce the peptides to which they are modified, whether biologically or synthetically produced. The advantage of using synthetic peptides is that they can incorporate non-natural amino acids designed to address several specific issues, including instability due to known proteolytic cleavage loads. They can also be more flexible in terms of the selection of binding sites, which is important when activity or potency depends heavily on modified residues such as free-terminus or C-terminal amides.

[0102] Classical gene fusion: Fc and HSA Classical gene fusion to long-lived serum proteins offers an alternative method for half-life extension, distinct from chemical conjugation to PEG or lipids. Two key proteins: antibody Fc domains and human serum albumin (HAS) have traditionally been used as fusion partners. Fc fusion involves the fusion of a peptide, protein, or receptor exodomain to the Fc portion of an antibody. Both Fc and albumin fusions achieve half-life extension not only by increasing the size of peptide drugs but also by utilizing the body's natural recycling mechanism, FcRn, the neonatal Fc receptor. pH-dependent binding of these proteins to FcRn prevents the degradation of the fusion protein within endosomes. Fusions based on these proteins can have half-lives ranging from 3 to 16 days, which is much longer than typical PEGylated or lipid-conjugated peptides. Fusion to antibody Fc can improve the solubility and stability of peptide or protein drugs. An example of peptide Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin is the same protein utilized by lipid acylated peptides and is another common fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. The main difference between Fc and albumin is that Fc is dimeric while HAS is monomeric, and depending on the choice of fusion partner, the fusion peptide is presented as either dimeric or monomeric. The dimeric nature of peptide Fc fusions can lead to avidity effects if the target receptor is sufficiently close or is itself dimeric. This may or may not be desirable depending on the target.

[0103] Designed polypeptide fusion: XTEN and PAS An interesting variation of the recombinant fusion concept is the development of low-complexity sequences designed as fusion partners for essentially unstructured, hydrophilic amino acid polymers, which are functional analogues of PEG. The inherent biodegradability of the polypeptide platform makes it an attractive, potentially more benevolent alternative to PEG. Another advantage is the precise molecular structure of the recombinant molecule, in contrast to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, which require maintaining the three-dimensional folding of the fusion partner, recombinant fusion to unstructured partners can often be subjected to harsher conditions such as higher temperatures or HPLC purification.

[0104] The most advanced polypeptide in this class is called XTEN (Amunix), with an amino acid length of 864 and consisting of six amino acids (A, E, G, P, S, and T). The polymer's biodegradability allows it to be much larger than the commonly used 40 kDa PEG, simultaneously resulting in a greater extension of its half-life. Fusion of XTEN with peptide drugs extends the half-life by 60 to 130 times compared to the natural molecule. Two fully recombinant XTEN products, namely VRS-859 (Exenatide-XTEN) and VRS-317 (Human Growth Hormone-XTEN), have entered clinical trials. In Phase Ia trials, VRS-859 was found to have good tolerability and efficacy in patients with type 2 diabetes. VRS-317 has reported superior pharmacokinetic and pharmacodynamic properties compared to previously studied rhGH products and may be administered monthly.

[0105] A second polymer based on a similar conceptual consideration is PAS (XL-Protein GmbH). It is a random coil polymer consisting of an even more restricted set of only three small uncharged amino acids: proline, alanine, and serine. It is unknown whether the biophysical properties of PAS and the differences from the highly negatively charged XTEN may contribute to differences in in vivo distribution and / or in vivo activity, but this will become clear as these polypeptides are incorporated into more therapeutics and the behavior of the fusions is characterized.

[0106] Regardless of whether the partner is Fc, HSA, XTEN, or PAS, all peptide-protein fusions are genetically encoded and consequently suffer from similar constraints. One limitation is that only naturally occurring amino acids are incorporated, unlike methods using chemical conjugations which allow the use of synthetic peptides incorporating non-natural amino acids. Methods to overcome this by extending the genetic code have been developed by companies such as Ambrx and Sutro, but are not yet widely used. A second limitation is that either the N-terminus or C-terminus of the peptide must be fused to the partner. Often, the peptide ends are involved in receptor interactions, and gene fusion to one or both ends can significantly impair activity. The site of the PEG or lipid conjugation can be anywhere on the peptide, so it can be optimized to maximize the biological activity of the resulting therapeutic agent.

[0107] Hybrid method for fusing synthetic peptides with half-life extension proteins While gene fusion has offered the potential for longer half-live extensions, it lacks the advantages of methods utilizing chemical conjugation, PEGylation, and lipidation in terms of binding site flexibility and the incorporation of non-natural amino acids or modifications to the peptide backbone. One of the first attempts to combine the advantages of gene fusion and chemical conjugation for half-life extension was made by researchers at the Scripps Research Institute in La Jolla, whose technology later formed the basis of the biotechnology company CovX. These researchers developed a platform using catalytic aldolase antibodies in which the lysine active site of the antibody forms a reversible covalent enamine bond with a beta-diketone incorporated into a peptide or small molecule. The resulting complex is called CovXBody®. This approach combines the functional properties of peptide drugs or small molecules with the long serum half-life of antibodies, not through gene fusion, but rather through chemical bonding. Following the initial demonstration of this technology, researchers expanded the use of the CovX-Body™ prototype, which is based on integrins targeting peptide-mimicking pharmacophores. At least three molecules based on this configuration are now in clinical development: CVX-096, a Glp-1R agonist; CVX-060, an angiopoietin-2 binding peptide; and CVX-04, a thrombospongin mimic.

[0108] Recently, XTEN polypeptides have also been used in a chemical conjugation mode, which is even more directly analogous to PEG. The first example of an XTENized peptide produced using this method is GLP2-2G-XTEN, which is chemically conjugated to an XTEN protein polymer using maleimide-thiol chemistry. The chemically conjugated GLP2-2GXTEN molecule exhibited in vitro activity, in vitro plasma stability, and pharmacokinetics in rats comparable to recombinant fusion GLP2-2GXTEN.

[0109] The number and spacing of reactive groups, such as lysine or cysteine ​​side chains, in the fully designed sequences of XTEN or PAS polypeptides can be precisely controlled through site-directed modifications by the limited set of amino acids that comprise them. This offers a greater degree of flexibility compared to methods that can utilize Fc or albumin, which naturally contain many reactive groups, and stands in contrast to CovX technology, which relies on reactive residues in highly specialized active sites. In addition, the lack of tertiary structure in XTEN or PAS should provide greater flexibility in the conditions and chemicals used for the purification of coupling and conjugates.

[0110] In summary, hybrid peptide half-life extension methods are emerging that combine the advantages of chemical conjugation and gene fusion techniques, overcoming their individual limitations. These methods confer longer half-lives while freeing the therapeutic peptide portion from the limitations of being composed solely of native L-amino acids or linear, unidirectional polypeptides fused at either the N-terminus or C-terminus. This enables the creation of molecules based on recombinant polypeptide-based partners, thus opening the door to a wide range of long-acting peptide-based drugs.

[0111] As used herein, the term “expression vector of the present invention” may mean any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression regulatory elements), suitable for the expression of the peptide of the present invention in cells. Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid molecule encoding the peptide is contained in a naked DNA or RNA vector, and includes, for example, a linear expression element (such as those described in, e.g., Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), a compactified nucleic acid vector (such as those described in, e.g., 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 (e.g., U.S. Patents 5,589,466 and 5,973,972). In one embodiment, the DNA includes an expression control sequence.

[0112] In one embodiment, the vector is suitable for the expression of the polyamino acid sequence of the present invention in bacterial cells. Examples of such vectors include BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, 1989, J Biol Chem 264, 5503-5509), and pET vectors (Novagen, Madison, Wis.). In one embodiment, the expression vector may also, 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, yeast alpha factor, alcohol oxidase, and vectors containing constitutive or inducible promoters such as PGH (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 another embodiment, 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.)

[0113] Expression regulatory sequences are engineered to control and drive the transcription of a gene of interest and the subsequent expression of the protein in various cell lines. A plasmid combines an expressible gene of interest with an expression regulatory sequence (i.e., an expression cassette) containing desired elements such as a promoter, enhancer, selectable marker, or operator. In the expression vector of the present invention, the nucleic acid molecule encoding the polyamino acid sequence may contain, or be associated with, any suitable promoter, enhancer, selectable marker, operator, repressor protein, poly(A) termination sequence, and other expression promoters.

[0114] As used herein, “promoter” refers to a DNA sequence sufficient to direct the transcription of a DNA sequence, such that it is operably ligated, i.e., ligated in such a manner that it enables the transcription of a nucleotide sequence encoding a polyamino acid sequence when an appropriate signal is present. The expression of the nucleotide sequence encoding a polyamino acid sequence may be controlled by any promoter or enhancer element known in the art. Examples of such elements include potent expression promoters (e.g., the human CMV IE promoter / enhancer or the CMV major IE (CMV-MIE) promoter, as well as the RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (Ubc), and HIV LTR promoters). In some embodiments, the vector includes a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC, and HIV LTR.

[0115] The nucleic acid molecules of the present invention may also be operably ligated to an effective poly(A) termination sequence, a replication origin of plasmid products in E. coli, an antibiotic resistance gene as a selection marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also contain a moduloable inductive promoter (inductive, repressible, developmentally moduloable) as opposed to a constitutive promoter such as CMV IE (those skilled in the art will recognize that such terms are descriptors of the degree of gene expression under certain conditions).

[0116] Selectable markers are a well-known element in the art. Under selective conditions, only cells expressing the appropriate selectable marker can survive. Generally, selectable marker genes express proteins, usually enzymes, that confer resistance to various antibiotics in cell culture. Under other selective conditions, cells expressing fluorescent protein markers are visible and therefore selectable. Embodiments include beta-lactamase (bla) (beta-lactam antibiotic resistance or ampicillin resistance gene or ampR), bls (blastidine resistance acetyltransferase gene), bsd (blastidine-S deaminase resistance gene), bsr (blastidine-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).

[0117] In one particular embodiment, the vector comprises one or more selectable 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 selectable marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyanofluorescent protein (CFP), enhanced cyanofluorescent protein (eCFP), or yellow fluorescent protein (YFP).

[0118] For the purposes of the present invention, gene expression in eukaryotic cells may be tightly regulated using a strong promoter controlled by an operator which is sequentially regulated by a regulatory protein that may be a recombinant "regulatory fusion protein" (RFP). An RFP essentially consists of a transcription-blocking domain and a ligand-binding domain which modulates its activity. An example of such an expression system is described in US20090162901 A1, which is incorporated herein by reference in its entirety.

[0119] As used herein, “operator” refers to a DNA sequence introduced into or near a gene, which may consequently regulate the gene by binding of RFP to the operator, thereby preventing or enabling the transcription of the gene of interest, i.e., the nucleotide encoding the peptide of the present invention. Numerous operators in prokaryotic cells and bacteriophages 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 operator region of the E. coli LexA gene that binds to the LexA peptide, as well as lactose and tryptophan operators that bind to repressor proteins encoded by the E. coli Lad and trpR genes. These also include bacteriophage operators derived from the lambda PR and phage P22 ant / mnt genes that bind to repressor proteins encoded by lambda cI and P22 arc. In some embodiments, if the transcriptional blocking domain of RFP is a restriction enzyme such as NotI, the operator is the recognition sequence of that enzyme. Those skilled in the art will recognize that the operator must be located adjacent to the promoter or 3' to the promoter so that transcription can be controlled by the promoter. For example, Patent No. 5,972,650, incorporated herein by U.S. Reference, specifies that the tetO sequence is within a certain distance from the TATA box. In certain embodiments, the operator is preferably located immediately downstream of the promoter. In other embodiments, the operator is located within 10 base pairs of the promoter.

[0120] In an exemplary cell expression system, cells are engineered to express a tetracycline repressor protein (TetR), and the protein of interest is placed under the transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operators (tetO) are positioned immediately downstream of the CMV-MIE promoter / enhancer in the vector. Transcription of the gene encoding the protein of interest, directed by the CMV-MIE promoter in such a vector, can be blocked by TetR in the absence of tetracycline or several other suitable inducers (e.g., doxycycline). In the presence of the inducer, the TetR protein cannot bind to tetO, thus allowing transcription, and subsequently translation (expression), of the protein of interest. (See, for example, U.S. Patent No. 7,435,553, which is incorporated herein by reference in its entirety.)

[0121] The vectors of the present invention may also utilize Cre-lox recombination tools to facilitate 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; and 2) a loxP site (e.g., a specific 34 base pairs consisting of an 8-bp core sequence where recombination occurs and two adjacent 13-bp inverted repeats) or a mutant lox site. (See, for example, 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 in conjunction with the consensus sequence FRT (see, for example, Dymecki, SM, 1996, PNAS 93 (12): 6191-6196).

[0122] As used herein, the term “host cell” includes any cell suitable for expressing recombinant nucleic acid sequences. Cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of Escherichia coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisia, S. pombe, P. partris, P. metanorica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Tricoplusia ni, etc.), non-human animal cells, mammalian cells, human cells, or cell fusions (e.g., cells such as hybridomas or quadromas). In certain embodiments, cells are human, monkey, ape, hamster, rat, or mouse cells. In other embodiments, cells are eukaryotes and are selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS -7), retinal cells, Vero, CV1, kidney (e.g., HEK 293, 293 EBNA, MSR 293, MDCK, HaK, BHK 21), HeLa, HepG2, WI 38, MRC 5, Colo 25, 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 aforementioned cells. In some embodiments, the cells include one or more viral genes, e.g., retinal cells expressing a viral gene (e.g., PER.C6® cells). In some embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0123] As used herein, the term "transformed cells of the present invention" refers to host cells comprising nucleic acids stably incorporated into a cell genome containing a nucleotide sequence encoding the expression of the peptide of the present invention. In another embodiment, the present invention provides cells comprising non-incorporated (i.e., episomal) nucleic acids, such as plasmids, cosmids, phagemids, or linear expression elements, containing a sequence encoding the expression of the peptide of the present invention. In yet another embodiment, the present invention provides cell lines produced by stably transfecting host cells with a plasmid containing the expression vector of the present invention.

[0124] As used herein, the term “engineered” as applied to cells means that they are genetically engineered using recombinant DNA technology, and generally comprises the steps of synthesizing a suitable expression vector (see above), and then transfecting the expression vector into host cells (generally stable transfection).

[0125] As used herein, the term "heterogeneous expression" refers to the expression of nucleic acids in host cells that do not naturally possess the nucleic acid. Insertion of nucleic acids into heterogeneous hosts is performed by recombinant DNA technology. [Examples]

[0126] Next, the present invention will be described with reference to specific embodiments. These are merely illustrative and for illustrative purposes only. They are not intended to limit the scope of any claimed exclusivity or the invention described. These examples constitute the best possible mode currently considered for carrying out the present invention.

[0127] hepatocyte permeation method HepG2(3×10 4 Well -1The cells were placed in 18 mm glass coverslips (Paul Marienfeld GmbH&Co.KG, Lauda-Koenigshofen, Germany) in a 24-well plate and allowed to adhere overnight in the culture medium. The cells were treated with 0.5 μg / mL CY5-tagged pep_260 for 1 hour, and then fixed in 4% paraformaldehyde (Sigma, Arklow, Ireland) at room temperature for 20 minutes. Subsequently, the coverslips were washed in 1× PBS (Sigma, Arklow, Ireland) and incubated in 0.1% Tween20 (Sigma, Arklow, Ireland) at room temperature for 30 minutes. The coverslips were then washed three times in 1× PBS for 5 minutes each, and then treated overnight at 4°C in PBS at a 1:500 dilution with anti-human early endosomal antigen 1 (EEA1) rabbit monoclonal antibody (Cell Signalling Technology, Danvers, Massachusetts, USA). The coverslips were then washed three times in 1× PBS for 5 minutes each. 1×PBS was aspirated, and the coverslips were treated in 1:100 dilution of PBS with AlexaFluor 546 goat anti-rabbit IgG (Life Technologies, Eugene, USA) for 2 hours at room temperature. The coverslips were then washed three times in 1×PBS for 5 minutes each, and treated with Hoechst 33342 solution (Thermo Scientific, Waltham, USA). Next, the coverslip was washed three times in PBS, removed from the 24-well plate using tweezers, and mounted onto a Superfrost Plus microscope slide (Thermo Scientific, Waltham, USA) using 5 μL of Mowiol-mounted medium dissolved in 18 mL of 0.2 M Tris-buffer (pH 8.5) (Sigma Aldrich) with 6 g glycerol (Sigma Aldrich), 2.4 g Mowiol 4-88 (Sigma Aldrich), and 0.026 g 1,4-diazabicyclo[2.2.2]octane (DABCO) (Sigma Aldrich).

[0128] Confocal images were acquired using an Olympus Fluoview FV 1000 confocal laser scanning biological microscope (Shinjuku, Tokyo, Japan) with a 60× oil immersion material. Hoechst nucleus staining was detected with a 405nm laser diode, AlexaFlour 546 fluorophores were detected with a diode pump solid-state laser, and Cy5-tagged pep_260 was detected with a solid-state red diode laser.

[0129] result Confocal imaging demonstrates that pep_260 (SEQ ID NO: 1) (red staining) can penetrate HepG2 hepatocytes. This effect is observed after 5 minutes of pep_260 treatment, with greater localization within HepG2 cells observed after 60 minutes (see Figure 1).

[0130] In vitro studies in the primary human cell system method To minimize adaptation to cell culture conditions and preserve physiological signaling responses, primary human cells were used in early passage (passage 4 or earlier). All cells originated from a pool of multiple donors (n=2-6), were commercially purchased, and handled according to manufacturer recommendations. Human blood-derived CD14+ monocytes were differentiated into macrophages in vitro before being added to the lMphg system. Abbreviations used include human umbilical vein endothelial cells (HUVECs), peripheral blood mononuclear cells (PBMCs), human neonatal dermal fibroblasts (HDFn), B cell receptor (BCR), T cell receptor (TCR), and Toll-like receptor (TLR).

[0131] The types of cells and stimuli used in each system are as follows: 3C system [HUVEC+ (IL-1β, TNFα and 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 and IFNγ)], HDF3CG system [HDFn+ (IL-1β, TNFα, IFNγ, EGF, bFGF and PDGF-BB)], KF3CT system [keratinocytes and HDFn + (IL 1β, TNFα, IFNγ, and TGFβ)], MyoF system [differentiated pulmonary myofibroblasts + (TNFα and TGFβ)], IMphg system [HUVEC and M1 macrophages + zymosan (TLR2 ligand)].

[0132] The systems are derived from either single-cell types or co-culture systems. Adherent cell types are cultured in 96 or 384-well plates until confluence, followed by the addition of PBMCs (SAg and LPS systems). The BT system consists of CD19+ B cells co-cultured with PBMCs and stimulated with a BCR activator and low levels of TCR stimulation. The test drug, prepared in either DMSO (small molecule; final concentration ≤0.1%) or PBS (biological agent), is added at the indicated concentration one hour before stimulation, and the culture is maintained for 24 hours or as otherwise indicated (48 hours for the MyoF system; 72 hours for the BT system (soluble readout); 168 hours for the BT system (secreted IgG)). Each plate contains an appropriate drug control for each system (e.g., 1.1 μM conventional control test drug colchicine), a negative control (e.g., non-stimulation conditions), and a vehicle control (e.g., 0.1% DMSO). Direct ELISA is used to measure biomarker levels of cell-associated targets and cell membrane targets. Soluble factors from the supernatant are quantified using either HTRF® detection, a bead-based multiplex immunoassay, or a capture ELISA. Any apparent adverse effects of the test reagents on cell proliferation and viability (cytotoxicity) are detected by sulfodamine B (SRB) staining for adherent cells and Alamer Blue® reduction for suspension cells. For proliferation assays, individual cell types are cultured in subconfluence and measured at time points optimized for each system (48 hours: 3C and CASM3C systems; 72 hours: BT and HDF3CGF systems; 96 hours: SAg system). Cytotoxicity of adherent cells is measured at the indicated time points by SRB (24 hours: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, and lMphg systems; 48 hours: MyoF system) and Alamer Blue staining of cells in suspension (24 hours: SAg system; 42 hours: BT system).

[0133] result The pep_260 (SEQ ID NO: 1)-mediated changes in major biomarker activities are listed by biological and disease classification (see Table 1). At the concentrations tested in this study, pep_260 is non-cytotoxic. Pep_260 is antiproliferative against human primary endothelial cells.

[0134] [Table 1]

[0135] Liver effects of pep_260 (SEQ ID NO: 1) in KKAy mice method The effects of pep_260 (SEQ ID NO: 1) and liraglutide on the liver were evaluated in the KKAy obese diabetic mouse model. 12-week-old male mice (n=11 / group) were subcutaneously administered pep_260 (12.7 and 63.5 mg / kg), liraglutide (250 μg / ml), or vehicle daily for 44 days. On day 44, animals were sacrificed by neck dislocation one hour after treatment administration. The whole liver was collected from all animals and fixed in formalin. Liver tissue was sectioned and stained with hematoxylin and eosin. Histological steatosis was assessed by a specialist histopathologist blinded to the research group, according to the NAFLD scoring system. Gene expression profiling in the liver was additionally performed.

[0136] result Expert grading demonstrates that 44-day treatment with pep_260 (SEQ ID NO: 1) significantly alleviates macrodroplet steatosis in obese diabetic mice. Analysis of gene expression in liver tissue shows that treatment with pep_260 (SEQ ID NO: 1) induces a decrease in the SREBF1, FASN, and caspase-3 genes. These results indicate that pep_260 (SEQ ID NO: 1) inhibits hepatic cholesterol production, inhibits hepatic lipid synthesis, and promotes hepatocyte survival.

[0137] Vehicle-controlled and liraglutide-treated mice showed signs of NAFLD, which were significantly reduced with 50 μM pep_1E99R5 and tended to decrease with 10 μM pep_1E99R5 treatment, suggesting a dose-dependent effect (Figure 2B).

[0138] IL-8 secretion in immortal hepatocytes (HepG2 cells) and primary pulmonary fibroblasts (WI-38 cells) stimulated with LPS and TGFβ. method IL-8 enzyme-linked immunosorbent assay (ELISA): HepG2 hepatocytes (density 1 × 10 4 Cells (1 / mL) were treated with 5 ng / mL of Pep_1E99R5, 10PANX (PANX1 mimetic), or PBS for 24 hours, followed by LPS stimulation (100 ng / mL) for a further 24 hours to increase IL-8 expression. Lipopolysaccharide (LPS) is a known pro-inflammatory signal that increases IL-8 expression in vitro. IL-8 levels in the culture supernatant were determined using human IL-8 specific sandwich ELISA (Perkin Elmar, Waltham, Ma, USA) according to the manufacturer's instructions.

[0139] result The results revealed a significant decrease in the expression of the pro-inflammatory cytokine IL-8 in cells pretreated with pep_1E99R5 (p<0.001). This data provided further evidence for beneficial effects on inflammation, such as the inflammatory phenotype in NAFLD / NASH. The pro-inflammatory cytokine IL-8 is strongly activated in NASH and contributes to hepatitis and fibrosis (Figure 6).

[0140] Antifibrotic effect of pep_1E99R5 in TGF-β stimulated primary hepatic stellate cells The onset of fibrosis in hepatic stellate cells (a major inflammatory driver for fibrosis induction in hepatic fibrosis and NASH / NAFLD) can be reliably modeled using incubation of primary human stellate cells with varying concentrations of transformed growth factor β (TGF-β).

[0141] method This procedure was carried out as described above, with some minor modifications to Cy5-labeled peptide imaging in HSkMCs. Primary hepatic stellate cells were investigated because they are considered to be the most relevant pro-fibrotic cells acting in acute and chronic liver disease. The stellate cells were directly seeded on glass coverslips at a density of 5,000 cells / mL. After starvation, the cells were treated with pep_1E99R5 (5 ng / mL) or ellafibranol (10 μM) for 6 hours. The cells were washed and subsequently treated with 5 ng / mL of TGF-β (Bio Techne, Minneapolis, MN, United States) for 24 hours to induce fibrosis. The cells were fixed and stained with ACTA2 antibody (Assay Genie, Dublin, Ireland), a fibroblast activation marker.

[0142] Confocal images were analyzed using a custom Python script with the scikit-image library version 0.15. Cells and nuclei were segmented using a simple threshold calculated using the mean value of the images. Cells within a cell mask were separated using watershed segmentation with the nucleus as the species. Cell size was then quantified in pixels. [Stefan van der Walt, Johannes L. Schoenberger, Juan Nunez - Iglesias, Fransois Boulogne, Joshua D. Warner, Neil Yager, Emmanuelle Gouillart, Tony Yu and the scikit-image contributors. scikit-image: Image processing in Python. PeerJ 2: e 453 (2014)]

[0143] result Stimulated primary hepatic stellate cells were used as a model for acute liver injury. Fluorescence images of untreated primary stellate cells, TGFβ-stimulated cells (5 nM), ellafibranol-treated cells (10 μM), and pep_1E99R5 (5 nM)-treated cells are shown in Figure 4A. TGF-β stimulation was effective in inducing intracellular fibrosis (p < 0.05). Increased ACTA2 expression, fibrogenesis, proliferation, and morphological changes were observed in stimulated cells, which were not observed in control cells. Pre-incubation with Pep_1E99R5 dramatically reduced ACTA2 expression in stellate cells, represented as yellow fibers in the image (Figure 7A). Pixel-level quantification of fibrosis marker expression using image analysis showed this reduction to be significant (p < 0.01) and comparable to the anti-fibrotic effect of 10 μM ellafibranol (Figure 7B).

[0144] Interestingly, the changes observed with pep_1E99R5 at 5 nM were comparable to those induced by ellafibranol at 10 μM (a lead clinical candidate with in vitro and in vivo effects on astrocellular cells), and the fact that the same effect was observed at a significantly lower concentration suggests that pep_1E99R5 potentially exhibits a better therapeutic profile.

[0145] This evidence, linked to unique protein targets and biomarker fingerprints, highlights the potential of this peptide entity to be deployed in preclinical development programs for NASH / NAFLD.

[0146] APAP Model method Acetaminophen (APAP)-induced acute liver injury mouse model: All animal treatments were performed by Melior Discovery (Exton, PA, USA) in accordance with the Institute Animal Care and Use (IACUC) guidelines at an AAALAC-accredited facility. A pilot study was conducted prior to the APAP-induced hepatotoxicity trail to establish the optimal APAP dose for the follow-up liver injury study (Supplementary Figure 2). The study was conducted using 8-week-old male C57BL / 6 mice obtained from Charles River Laboratory, who were randomly assigned to treatment groups based on body weight. Mice were acclimatized for 7 days, housed in cages of 4 mice each, with a 12-hour light-dark cycle and free access to standard rodent food and water. For the pilot study, mice were fasted overnight before intraperitoneal administration and received a single dose of either 200 mpk APAP, 300 mpk APAP, or saline as a control. A total of 18 mice were included in the pilot study, divided into three groups of 6 animals each. To reduce stress and improve viability in mice, tail hemorrhage was induced at the time of initial blood collection. Serum samples for ALT and AST analysis were obtained using blood samples from 6 hours onward. In evaluating the efficacy of peptides in an APAP-induced acute liver injury mouse model, APAP (200 mpk) was administered intravenously, and Pep_1E99R5 and 10PANX were administered intravenously at 10 mg / kg. Five animals were included in each group, with either the vehicle or peptide 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 via cardiac puncture under isoflurane anesthesia and used to obtain serum samples for full-panel clinical chemistry analysis (data not shown).

[0147] result Based on DiscoverX BioMap and Retrogenix screening results, as well as in vitro studies in Hep-G2 and hepatic stellate cells, the anti-inflammatory and anti-fibrotic effects of pep_1E99R5 were strongly suggested. Therefore, an N-acetyl-para-aminophenol (APAP) model was used to evaluate the effects of the peptide in an in vivo liver injury model. A pilot study showed that 200 mpk of APAP significantly increased the levels of both alanine aminotransferase (ALT) and aspartate aminotransferase (AST), biochemical features of APAP-induced liver injury in mice, compared to a saline control. This concentration was therefore considered optimal for use in liver injury studies. Peptides pep_1E99R5 and 10PANX were administered intravenously (IV) 1.5 hours after the initial APAP dose, and the levels of the same liver enzymes were measured at 2.25 and 6 hours (Figure 8). At an earlier stage, pep_1E99R5 significantly reduced ALT levels by 80% compared to APAP / saline controls (p<0.05), far surpassing 10 PANX, which induced a 41% reduction in ALT. A similar trend was observed in AST levels at 2.25 hours, but no significant difference was found, possibly due to the small number of animals included in the study. ALT and AST levels in the APAP / saline groups increased 4-fold and 2-fold, respectively, by 6 hours. The activity of 10PANX remained nearly stable at this point, inducing non-significant reductions in both liver enzymes between 45% and 52%. AST and ALT levels measured in pep_1E99R5-treated mice increased at 6 hours, similar to APAP control animals. This indicates a pharmacokinetic profile of pep_1E99R5 that appears to exhibit potent functionality for the first two hours, but is likely to be metabolized thereafter, unlike 10PANX, which lasts at least three times longer in mice.

[0148] Equivalents The foregoing description details currently preferred embodiments of the present invention. Those skilled in the art will anticipate that numerous practical modifications and variations will arise in light of these descriptions. These modifications and variations are intended to be included within the claims appended herein.

Claims

1. A pharmaceutical composition for use in a method for treating or preventing non-alcoholic fatty liver disease (NAFLD) in mammals, comprising a peptide having the amino acid sequence of SEQ ID NO: 1, 2, or 3.

2. The pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

3. The pharmaceutical composition according to claim 1 or 2, wherein the peptide is a modified peptide.

4. The pharmaceutical composition according to claim 3, wherein the peptide is modified for storage stability, pharmacokinetic manipulation, drug performance, increased permeability, maintenance of potency, reduction or avoidance of toxicity, and / or increase of half-life.

5. The pharmaceutical composition according to claim 3 or 4, wherein the modification is a chemical modification selected from conjugation, phosphorylation, sulfation, acylation, PEGylation, cyclization, lipidation, modification of side chains, introduction of protecting groups, and use of crosslinking agents, as well as other methods that impose conformational constraints on the peptide, by polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polypropylene glycol, polyoxyethylated polyol and polyvinyl alcohol, colomic acid or other carbohydrate-based polymers, amino acid polymers and biotin derivatives to one or more peptides.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the peptide is conjugated, linked, or fused to a binding partner.

7. The pharmaceutical composition according to claim 6, wherein the binding partner is selected from polyethylene glycol polymer, molecular weight increasing compound, lipophilic compound, or antibody molecule.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition is administered in combination with a drug for diabetes or a drug for obesity.

Images