Improved peptide drugs for the treatment of NASH and other disorders

Abstract

The present disclosure provides peptide products, including peptides covalently attached to surfactant moieties, that have improved properties, including increased duration of action and bioavailability, and are useful for treating insulin resistance, diabetes, obesity, metabolic syndrome, and cardiovascular disease and their associated conditions, such as NASH and PCOS. [Selected figure] Figure 18A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 613,396, filed January 3, 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Field of Disclosure The increasing prevalence of diabetes mellitus is a global health crisis threatening pandemic status, representing a major cause of patient morbidity and mortality as well as a significant economic burden. Obesity is a significant risk factor for type 2 diabetes, with approximately 90% of type 2 diabetes patients being overweight or obese. Obesity is a rapidly growing problem worldwide, with more than 65% of adults in the United States being overweight. The present disclosure provides improved peptide agents for the treatment of obesity- and / or diabetes-related disorders, such as nonalcoholic steatohepatitis (NASH) and polycystic ovary syndrome (PCOS). Summary of the Invention

[0003] Described herein are peptide products and their uses in the treatment of conditions associated with insulin resistance and / or obesity, such as type 2 diabetes, metabolic syndrome, cardiovascular disease (including coronary artery disease such as atherosclerosis and myocardial infarction), hypertension, NASH, and PCOS, as well as diseases associated with such disorders. The peptide products comprise a peptide covalently attached to a surfactant moiety containing a hydrophilic sugar group and a hydrophobic group. In some embodiments, the peptide is a metabolic hormone, such as GLP-1, glucagon, oxyntomodulin, or exendin (e.g., exendin-4), or an analog or variant thereof. The peptide products have improved properties, including increased duration of action and bioavailability.

[0004] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0005] [Figure 1] Specific peptide products are shown, all of which are incorporated into this disclosure. [Figure 2] Specific peptide products are shown, all of which are incorporated into this disclosure. [Figure 3] Specific peptide products are shown, all of which are incorporated into this disclosure. [Figure 4] Specific peptide products are shown, all of which are incorporated into this disclosure. [Figure 5] Illustrated is the X-ray crystal structure of the binding site of the N-terminal region / extracellular domain of the GLP-1 receptor, showing the key hydrophobic ligand-binding region (Val19*, Phe22*, Trp25*, and Leu26* of the ligand exendin-4, and the sequence of exendin-4 beyond Glu15 interact with this region as an amphipathic helix). [Figure 6] The structures of three representative peptide products, EU-A992, EU-A1022, and EU-A1169, are illustrated, in which the epsilon (side chain) amino group of the lysine residue at position 24 of the peptide forms an amide bond with 1-O-octyl β-D-glucuronic acid, 1-O-octyl β-D-melibiouronic acid, or 1-O-octyl α-D-melibiouronic acid, respectively. [Figure 7] 1 is a more detailed illustration of the structure of EU-A992. [Figure 8] 1 shows the concentrations of peptide products EU-A993 and EU-A1023, and the native hormone GLP-1(7-36), over time during incubation in human plasma at 37° C. [Figure 9]1 shows the glycemic response of db / db mice after subcutaneous injection of EU-A992, EU-A1167, and EU-A1168 (250 nmol / kg each) at t=0 and 7 hours. [Figure 10] Figure 1 shows weight loss in diet-induced obese (DIO) mice after subcutaneous injection of the peptide product EU-A1024 at two different doses on days 1 (T = 0 and 7 hours), 3, 6, 9, 12, 15, 18, 21, and 24. [Figure 11] 1 shows the change in whole body adipose and lean tissue mass in treated DIO mice as measured by NMR at day 1 (pre-dose) and day 26. [Figure 12] 1 shows the effect of test compounds on body weight in a diet-induced obese (DIO) mouse model of NASH. [Figure 13] 1 shows the effect of test compounds on whole body adipose tissue mass in DIO-NASH mice. [Figure 14] 1 shows the effect of test compounds on plasma triglyceride (TG) and total cholesterol (TC) levels in DIO-NASH mice. [Figure 15] 1 shows the effect of test compounds on plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels in DIO-NASH mice. [Figure 16] 1 shows the effect of test compounds on liver weight in DIO-NASH mice. [Figure 17] The effect of the test compound on fatty liver in DIO-NASH mice using hematoxylin-eosin staining is shown (the large oval structures in FIG. 17C are veins). [Figure 18A] 1 shows the effect of test compounds on post-biopsy hepatic steatosis in terms of % affected area and total hepatic lipid content as determined by histological quantitative measurements in DIO-NASH mice. [Figure 18B] 1 shows the effect of test compounds on post-biopsy hepatic steatosis in terms of % affected area and total hepatic lipid content as determined by histological quantitative measurements in DIO-NASH mice. [Figure 19] 1 shows the effect of test compounds on liver triglyceride (TG) and total cholesterol (TC) levels in DIO-NASH mice. [Figure 20] 1 shows the effect of test compounds on total liver collagen type 1 alpha 1 (col1a1) content as measured immunohistochemically in DIO-NASH mice. [Figure 21] 1 shows the effect of test compounds on total liver galectin-3 content as measured by immunohistochemistry in DIO-NASH mice. [Figure 22] 1 shows the effect of a test compound on fatty liver scores in DIO-NASH mice. [Figure 23] 1 shows the effect of test compounds on hepatitis scores in DIO-NASH mice. [Figure 24] 1 shows the effect of test compounds on hepatocellular ballooning scores in DIO-NASH mice. [Figure 25] 1 shows the effect of test compounds on NAS (non-alcoholic fatty liver disease activity score) scores in DIO-NASH mice. [Figure 26] 1 shows the effect of test compounds on liver fibrosis scores in DIO-NASH mice, where liver fibrosis was assessed by picrosirius red staining. [Figure 27] 1 shows the effect of test compounds in DIO-NASH mice on hepatic expression of mRNAs involved in fibrosis, inflammation, and monocyte recruitment and differentiation. [Figure 28] 1 shows the effect of test compounds in DIO-NASH mice on hepatic expression of mRNA involved in monocyte recruitment and differentiation. [Figure 29] 1 shows the effect of test compounds in DIO-NASH mice on hepatic expression of mRNA involved in fibrosis fibrogenesis. [Figure 30] 1 shows the effect of test compounds in DIO-NASH mice on hepatic expression of mRNA involved in hepatic stellate cell activation and myofibroblast proliferation. [Figure 31]1 shows the effect of test compounds in DIO-NASH mice on hepatic expression of mRNA involved in pyroptosis. DETAILED DESCRIPTION OF THE INVENTION

[0006] definition Unless otherwise defined or clearly indicated by usage herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0007] As used in this specification and the appended claims, the terms "a" or "an" mean one or more. As used herein, the term "another" means two or more.

[0008] The acronym "aka" means "also known as."

[0009] The word "exemplary" as used herein means "serving as an example, instance, or illustration." Any embodiment or feature characterized herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments or features.

[0010] In some embodiments, the term "about" or "approximately" means within ±10% or 5% of a particular value. Whenever the term "about" or "approximately" precedes the first number in a series of two or more numbers, or in a range of two or more numbers, the term "about" or "approximately" applies to every single one of the numbers in the series or range.

[0011] A "peptide" comprises two or more natural and / or unnatural amino acid residues, usually linked via a peptide bond. Such amino acids may include naturally occurring structural variants, naturally occurring non-proteinogenic amino acids, or / and synthetic, non-naturally occurring analogs of natural amino acids. The terms "peptide" and "polypeptide" are used synonymously. Peptides include short peptides (about 2-20 amino acids), intermediate-length peptides (about 21-50 amino acids), and long peptides (more than about 50 amino acids, sometimes also referred to as "proteins"). In some embodiments, peptide products contain a surfactant moiety covalently attached to a peptide of about 50, 40, or 30 or fewer amino acids. Synthetic peptides can be synthesized, for example, using an automated peptide synthesizer. Peptides can also be produced recombinantly in cells expressing a nucleic acid sequence encoding the peptide. Conventional notation is used herein to depict peptide sequences. The left-hand end of a peptide sequence is the amino (N)-terminus, and the right-hand end of a peptide sequence is the carboxyl (C)-terminus.

[0012] Standard one-letter and three-letter abbreviations for common amino acids are used herein. Unless otherwise specified as D- or DL-, the abbreviations used in the amino acid sequences disclosed herein represent L-amino acids, or amino acids are achiral, although the equivalent D isomers can generally be used at any position (e.g., to resist proteolysis). Other amino acid abbreviations used herein include: Ac3c = 1-aminocyclopropane-1-carboxylic acid; Ac4c = 1-aminocyclobutane-1-carboxylic acid; Ac5c = 1-aminocyclopentane-1-carboxylic acid; Ac6c = 1-aminocyclohexane-1-carboxylic acid; Aib = α-aminoisobutyric acid (or 2-methylalanine or Cα-methylalanine); Bip = 3-(biphenyl-4-yl)alanine; Bip2Et = 3-(2'-ethylbiphenyl-4-yl)alanine; Bip2EtMeO = 3-(2'-ethyl-4'-methoxybiphenyl-4-yl)alanine; Cit = citrulline; Deg = 2,2-diethylglycine; Dmt = (2,6-dimethyl)tyrosine; 2FPhe = (2-fluorophenyl)alanine; 2FMePhe or 2Fα MePhe = Cα-methyl-(2-fluorophenyl)alanine; hArg = homoarginine; MeLys or αMeLys = Cα-methyllysine; MePhe or αMePhe = Cα-methylphenylalanine; MePro or αMePro = Cα-methylproline; Na1 or Na1(1) = 3-(1-naphthyl)alanine; Na1 or Na1(2) = 3-(2-naphthyl)alanine; N1e = norleucine; Orn = ornithine; and Tmp = (2,4,6-trimethylphenyl)alanine. 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and the Tic-Phe dipeptide moiety with a reduced interresidue amide bond (designated as Tic-Ψ[CH2-NH]-Ψ-Phe) have the following structures:

[0013] [ka]

[0014] Unless specifically stated otherwise or unless the context clearly dictates otherwise, the present disclosure encompasses any and all forms of peptides that may be produced, whether the peptide is produced synthetically (e.g., using a peptide synthesizer) or cellularly (e.g., by recombinant production). Such forms of peptides may include one or more modifications that may occur during the synthetic or cellular production of the peptide, such as one or more post-translational modifications, whether or not the one or more modifications are intentional. A peptide can have the same type of modification in two or more different locations and / or two or more different types of modification. Modifications that may occur during synthetic or cellular production of peptides, including chemical and post-translational modifications, include, but are not limited to, glycosylation (e.g., N-linked glycosylation and O-linked glycosylation), lipid modification, phosphorylation, sulfation, acetylation (e.g., N-terminal acetylation), amidation (e.g., C-terminal amidation), hydroxylation, methylation, intramolecular or intermolecular disulfide bond formation, lactam formation between two side chains, pyroglutamate formation, and ubiquitination. A peptide can have one or more modifications anywhere, such as at the N-terminus, C-terminus, one or more amino acid side chains, or peptide backbone, or any combination thereof. In some embodiments, the peptide is acetylated at the N-terminus and / or has a carboxamide (-CONH) group at the C-terminus, which can increase the stability of the peptide.

[0015] Possible modifications of peptides also include deletion of one or more amino acids, addition / insertion of one or more natural and / or non-natural amino acids, or substitution with one or more natural and / or non-natural amino acids, or any or all combinations thereof. Substitutions may be conservative or non-conservative. Such modifications may be deliberate, such as through site-directed mutagenesis or in the chemical synthesis of the peptide, or may be accidental, such as through mutations occurring in the host cell producing the peptide or errors during PCR amplification. Non-natural amino acids may have the same chemical structure as the corresponding natural amino acid but with a D-stereochemical configuration, or may have a different chemical structure and D- or L-stereochemical configuration. Non-natural amino acids can be used, for example, to promote α-helix formation and / or increase peptide stability (e.g., to resist proteolysis).

[0016] A peptide having one or more modifications relative to a reference peptide may be referred to as an "analog" or "variant" of the reference peptide, as appropriate. An "analog" typically retains one or more essential properties of the reference peptide (e.g., receptor binding, receptor or enzyme activation, receptor or enzyme inhibition, or other biological activity). A "variant" may or may not retain the biological activity of the reference peptide, and / or may have a different biological activity. In some embodiments, an analog or variant of a reference peptide has an amino acid sequence that differs from the reference peptide.

[0017] The term "conservative substitution" refers to the substitution of an amino acid in a peptide with a functionally, structurally, or chemically similar natural or non-natural amino acid. In certain embodiments, the following groups each contain natural amino acids that are conservative substitutions for one another: 1) glycine (Gly / g), alanine (Ala / A); 2) isoleucine (Ile / I), leucine (Leu / L), methionine (Met / M), valine (Val / V); 3) phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W); 4) serine (Ser / S), threonine (Thr / T), cysteine ​​(Cys / C); 5) asparagine (Asn / N), glutamine (Gln / Q); 6) aspartic acid (Asp / D), glutamic acid (Glu / E); and 7) Arginine (Arg / R), Lysine (Lys / K), Histidine (His / H).

[0018] In further embodiments, each of the following groups comprises natural amino acids that are conservative substitutions for one another: 1) Non-polar: Ala, Val, Leu, Ile, Met, Pro (proline / P), Phe, Trp; 2) Hydrophobicity: Val, Leu, Ile, Phe, Trp; 3) Aliphatic: Ala, Val, Leu, Ile; 4) Aromatic: Phe, Tyr, Trp, His; 5) Uncharged polar or hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln, Tyr; 6) Aliphatic hydroxyl or sulfhydryl containing: Ser, Thr, Cys; 7) Amide-containing: Asn, Gln; 8) Acidic: Asp, Glu; 9) Basic: Lys, Arg, His; and 10) Small size: Gly, Ala, Ser, Cys.

[0019] In other embodiments, the amino acids can be grouped as follows: 1) Hydrophobicity: Val, Leu, Ile, Met, Phe, Trp; 2) Aromatic: Phe, Tyr, Trp, His; 3) Neutral hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln; 4) Acidic: Asp, Glu; 5) Basic: Lys, Arg, His; and 6) Residues that affect backbone orientation: Pro.

[0020] Examples of unnatural or non-proteinogenic amino acids include, but are not limited to, alanine analogs (e.g., α-ethyl Gly [α-aminobutyric acid or Abu], α-n-propyl Gly [norvaline or Nva], α-tert-butyl Gly [Tbg], α-vinyl Gly [Vg or Vlg], α-allyl Gly [Alg], α-propargyl Gly [Prg], 3-cyclopropyl Ala [Cpa], and Aib), leucine analogs (e.g., Nle), proline analogs (e.g., α-MePro), phenylalanine analogs {e.g., Phe(2-F), Phe(2-Me), Tmp, Bip, Bip(2'-Et-4'), -OMe), Na11, Na12, Tic, α-MePhe, α-MePhe(2-F), and α-MePhe(2-Me)}, tyrosine analogs (e.g., Dmt and α-MeTyr), serine analogs (e.g., homoserine [isothreonine or hSer]), glutamine analogs (e.g., Cit), arginine analogs (e.g., hArg), lysine analogs (e.g., homolysine [hLys], Orn, and α-MeLys), α,α-disubstituted amino acids (e.g., Aib, α,α-diethylGly[Deg], α-cyclohexylAla[2-Cha], Ac3c, Ac4c, Ac5c, and Ac6c), and other unnatural amino acids disclosed in A. Santoprete et al., J. Pept. Sci., 17:270-280 (2011). α,α-disubstituted amino acids can confer conformational constraints and / or α-helix stabilization. Reduction of the amide bond between two residues (e.g., in Tic-Ψ[CH2-NH]-Ψ-Phe) can increase protease resistance and may also, for example, modify receptor binding.

[0021] The present disclosure includes all pharmaceutically acceptable salts of the peptides, including those with a positive overall charge, those with a negative overall charge, and those with no overall charge.

[0022] Sugars include monosaccharides, disaccharides, and oligosaccharides (e.g., trisaccharides, tetrasaccharides, etc.). Reducing sugars exist in equilibrium between cyclic and open-chain forms, usually favoring the cyclic form. The functionalized sugars of the surfactant moiety have functional groups suitable for forming covalent bonds with amino acids of peptides.

[0023] An "alkyl" group refers to an aliphatic hydrocarbon group. Alkyl groups can be saturated or unsaturated and can be straight-chained (linear), branched-chained, or cyclic. In some embodiments, an alkyl group is not cyclic. In some embodiments, an alkyl group contains 1-30, 6-30, 6-20, or 8-20 carbon atoms. A "substituted" alkyl group is substituted with one or more substituents. In some embodiments, the one or more substituents are independently selected from halogen, nitro, cyano, hydroxy, alkoxy, haloalkoxy, aryloxy, thiol, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, amino, alkylamino, dialkylamino, arylamino, alkoyl, carboxyl, carboxylate, ester, amide, carbonate, carbamate, urea, alkyl, haloalkyl, fluoroalkyl, aralkyl, alkyl chains containing acyl groups, heteroalkyl, heteroalicyclic, aryl, alkoxyaryl, heteroaryl, hydrophobic natural compounds (e.g., steroids), and the like.

[0024] In some embodiments, an alkyl group as a substituent is a linear or branched C1-C6 alkyl, referred to as a “lower alkyl.” Non-limiting examples of lower alkyl groups include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including all isomeric forms such as n-butyl, isobutyl, sec-butyl, and tert-butyl), pentyl (including all isomeric forms such as n-pentyl), and hexyl (including all isomeric forms such as n-hexyl).

[0025] In some embodiments, the alkyl group is attached to the Nα-atom of the residue of the peptide (e.g., Tyr or Dmt). In certain embodiments, the N-alkyl group is a straight or branched C1-C 10 It is alkyl or aryl-substituted alkyl such as benzyl or phenylethyl. One or two alkyl groups may be attached to the N-terminal Nα-atom.

[0026] In some embodiments, the alkyl group is a 1-alkyl group that is attached to the C-1 position of a sugar (e.g., glucose) via a glycosidic bond (e.g., an O-, S-, N-, or C-glycosidic bond). In some embodiments, such 1-alkyl groups are unsubstituted or substituted C-C 30 , C6-C 30 , C6-C 20 , or C8-C 20 It is an alkyl group.

[0027] In some embodiments, the alkyl group (e.g., 1-alkyl group) is selected from the group consisting of aryl, —OH, —OR 1 , -SH, -SR 1 , -NH2, -NHR 1 , -N(R 1 )2, oxo(=O), -C(=O)R 2 , carboxyl (CO2H), carboxylate (CO2 - ), -C(=O)OR 1 , -OC(=O)R 3 , -C(=O)N(R 1 )2, -NR 4 C(=O)R 3 , -OC(=O)OR 5 , -OC(=O)N(R 1 )2, -NR 4 C(=O)OR 5 , and -NR 4 C(=O)N(R 1 )2 is substituted with one or more (e.g., two or three) groups independently selected from R 1 is, independently at each occurrence, hydrogen, alkyl, or aryl; R 1and the nitrogen atom to which they are attached, both occurrences form a heterocyclyl or heteroaryl ring; R 2 is independently at each occurrence alkyl, heterocyclyl, aryl, or heteroaryl; R 3 is independently at each occurrence hydrogen, alkyl, heterocyclyl, aryl, or heteroaryl; R 4 is, independently at each occurrence, hydrogen or alkyl; and R 5 is independently at each occurrence alkyl or aryl.

[0028] In some embodiments, alkyl groups (e.g., 1-alkyl groups) are substituted internally or / and terminally with carboxyl / carboxylate groups, aryl groups, or -O-aryl groups. In certain embodiments, alkyl groups (e.g., 1-alkyl groups) are substituted with carboxyl or carboxylate groups at the distal end of the alkyl group. In further embodiments, alkyl groups (e.g., 1-alkyl groups) are substituted with aryl groups at the distal end of the alkyl group. In other embodiments, alkyl groups (e.g., 1-alkyl groups) are substituted with -O-aryl groups at the distal end of the alkyl group.

[0029] The terms "halogen," "halide," and "halo" refer to fluoride, chloride, bromide, and iodide.

[0030] The term "acyl" refers to -C(=O)R, where R can be saturated or unsaturated and can be linear, branched, or cyclic. In certain embodiments, R contains 1-20, 1-10, or 1-6 carbon atoms. The acyl group can be optionally substituted with one or more groups such as halogen, hydroxyl, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cycloalkyl, aryl, acyl, carboxyl, ester, amide, hydrophobic natural compounds (e.g., steroids), and the like.

[0031] The terms "heterocyclyl" and "heterocyclic" refer to a monocyclic non-aromatic or polycyclic group containing at least one non-aromatic ring, wherein at least one non-aromatic ring contains one or more heteroatoms independently selected from O, N, and S. The non-aromatic ring containing one or more heteroatoms may be attached to or fused to one or more saturated, partially unsaturated, or aromatic rings. In certain embodiments, a heterocyclyl or heterocyclic group has 3-15, 3-12, 3-10, 3-8, or 3-6 ring atoms. Heterocyclyl or heterocyclic groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, azepanyl, azocanyl, oxiranyl, oxetanyl, tetrahydrofuranyl (oxolanyl), tetrahydropyranyl, oxepanyl, and oxocanyl.

[0032] The term "aryl" refers to a monocyclic aromatic hydrocarbon group or a polycyclic group containing at least one aromatic hydrocarbon ring. In certain embodiments, an aryl group has 6-15, 6-12, or 6-10 ring atoms. Aryl groups include, but are not limited to, phenyl, naphthalenyl (naphthyl), fluorenyl, azulenyl, anthryl, phenanthryl, biphenyl, and terphenyl. The aromatic hydrocarbon ring of an aryl group may be bonded to or fused to one or more saturated, partially unsaturated, or aromatic rings, such as dihydronaphthyl, indenyl, indanyl, and tetrahydronaphthyl (tetralinyl). Aryl groups may be optionally substituted with one or more (e.g., two or three) substituents independently selected from halogen (including -F and -Cl), cyano, nitro, hydroxyl, alkoxy, thiol, alkylthio, alkylsulfoxide, alkylsulfone, amino, alkylamino, dialkylamino, alkyl, haloalkyl (including fluoroalkyl such as trifluoromethyl), acyl, carboxyl, ester, amide, and the like.

[0033] The term "heteroaryl" refers to a monocyclic aromatic or polycyclic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, N, and S. The heteroaromatic ring may be attached to or fused to one or more saturated, partially unsaturated, or aromatic rings, which may contain only carbon atoms or one or more heteroatoms. In certain embodiments, heteroaryl groups have 5-15, 5-12, or 5-10 ring atoms. Monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl (thiophenyl), oxadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridonyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridazinonyl, and triazinyl. Non-limiting examples of bicyclic heteroaryl groups include indolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzothienyl, benzothiophenyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzotriazolyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinazolinyl, quinoxalinyl, indazolyl, naphthyridinyl, phthalazinyl, quinazolinyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl.

[0034] The term "steroid nucleus" refers to the steroid core containing an arrangement of four fused rings, designated A, B, C, and D, as shown below:

[0035] [ka] The steroid nucleus can have one or more groups such as halogen, hydroxyl, oxo, alkyl, acyl, carboxyl, ester, amide, etc. Steroid nucleus-containing moieties include, but are not limited to, cholesterol, and the like.

[0036] The term "pharmaceutically acceptable" refers to a substance (e.g., an active ingredient or excipient) that is suitable for use in contact with the tissues and organs of a subject without undue irritation, allergic response, immunogenicity, and toxicity, commensurate with a reasonable benefit-risk ratio, and that is effective for its intended use. A "pharmaceutically acceptable" excipient or carrier of a pharmaceutical composition is also compatible with the other ingredients of the composition.

[0037] The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to prevent, reduce the risk of development of, delay the onset of, slow the progression of, or cause regression of the medical condition being treated, or to alleviate to some extent the medical condition or one or more symptoms or complications thereof, in at least some of the subjects receiving the compound. The term "therapeutically effective amount" also refers to an amount of a compound sufficient to elicit the biological or medical response of a cell, tissue, organ, or human that is desired by a physician or clinician.

[0038] The terms "treat," "treating," and "treatment" include alleviating, ameliorating, inhibiting the progression of, reversing, or suppressing a medical disease, or one or more symptoms or complications associated therewith, and alleviating, ameliorating, or eradicating one or more causes of a medical disease. A reference to "treating" a medical disease includes its prevention. The terms "prevent," "preventing," and "prevention" include eliminating, reducing the risk of its development, and delaying the onset of a medical disease, or one or more symptoms or complications associated therewith.

[0039] The term "medical illness" (or for brevity, "disease") includes diseases and disorders. The terms "disease" and "disorder" are used synonymously herein.

[0040] Obesity and insulin resistance Obesity leads to insulin resistance, which is a reduced ability of cells throughout the body to respond to insulin stimulation through a reduction in the number of insulin receptors and their binding to key intracellular signaling pathways. The obese state also leads to metabolic syndrome, a constellation of disorders including insulin resistance, hypertension, and atherosclerosis. Insulin resistance often leads to type 2 diabetes. Long-term hyperglycemia increases the risk of micro- and macrovascular complications, sensory neuropathy, myocardial infarction, stroke, macrovascular mortality, and all-cause mortality. Type 2 diabetes, metabolic syndrome, and other diseases associated with obesity and insulin resistance represent a significant financial and healthcare burden worldwide.

[0041] metabolic hormones Incretins are metabolic hormones that induce a decrease in blood glucose levels. They are released after a meal and stimulate insulin production and secretion from pancreatic beta cells in the islets of Langerhans through a glucose-dependent mechanism. Incretins also slow the rate of nutrient absorption into the bloodstream by slowing gastric emptying, thereby inducing satiety and suppressing appetite. Furthermore, incretins inhibit glucagon release from alpha cells in the islets of Langerhans. The two main incretins are glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP, also known as glucose-dependent insulinotropic polypeptide), which are secreted by intestinal L cells and are members of the glucagon peptide superfamily. GLP-1 and GIP are rapidly downregulated by dipeptidyl peptidase 4 (DPP-4). In patients with type 2 diabetes, insulin secretion from pancreatic beta cells in response to GLP-1 is impaired.

[0042] GLP-1 has other beneficial effects, such as stimulating pancreatic beta cell proliferation, increasing peripheral insulin sensitivity, hepatic glucose uptake and glycogen production, and improving cardiac function (e.g., left ventricular function).

[0043] Like GLP-1, oxyntomodulin (OXM) is derived from preproglucagon and is secreted from intestinal L-cells in response to a meal. OXM is essentially glucagon with an eight-amino acid C-terminal extension. OXM activates both the glucagon receptor (GCGR) and the GLP-1 receptor (GLP1R), but with 10- to 100-fold lower potency than glucagon and GLP-1. OXM induces satiety and reduces body fat (e.g., white adipose tissue). OXM reduces food intake and body fat, thereby causing weight loss.

[0044] Glucagon is produced from preproglucagon in the pancreatic alpha cells of the islets of Langerhans. Glucagon induces satiety, stimulates lipolysis in adipose tissue, and increases energy expenditure. Glucagon reduces weight by reducing food intake and stimulating fat burning.

[0045] The amino acid sequences of the biologically active forms of human GLP-1, glucagon, and oxyntomodulin are shown below: GLP-1 (using glucagon numbering): His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 -Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Lys 28 -Gly 29 -Arg 30 (SEQ.ID.NO.1) Glucagon: His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Dear 16 -Arg 17 -Arg 18 -God 19 -Gln 20 -Asp 21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -But 27 -Asn 28 -Thr 29 (SEQ.ID.NO.2) オキシントモジュリン: His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Dear 16 -Arg 17 -Arg 18 -God 19 -Gln 20 -Asp 21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -But 27 -Asn 28 -Thr 29 -Lys 30 -Arg 31 -Asn 32 -Arg 33 -Asn 34 -Asn 35 -Ile 36 -God 37 (SEQ.ID.NO.3)

[0046] Amino acid residues throughout the GLP-1 and glucagon sequences may be substituted while retaining biological activity. For example, substitution with Ala is widely accepted in the N-terminal region of GLP-1, particularly at positions 2, 3, 5, 8, 11, and 12 (Adelhorst et al., J. Biol. Chem., 269:6275-6278

[1994] ). Furthermore, short N-terminal analogs of GLP-1 can potently bind to and activate GLP1R (Mapelli et al., J. Med. Chem., 52:7788-7799

[2009] ; and Haque et al., Peptides, 31:950-955 and 1353-1360

[2010] ). A chimeric analog capable of binding to both GLP1R and GCGR can be generated by grafting the C-terminal residues of GLP-1 onto the N-terminus of glucagon (Hjorth et al., J. Biol. Chem., 269:30121-30124

[1994] ).

[0047] The present disclosure provides peptide products comprising a peptide gut hormone (e.g., GLP-1, GIP, glucagon, oxyntomodulin, or exendin [e.g., exendin-4]), or an analog or variant thereof, covalently attached to a surfactant moiety. The peptide products are potent agonists of basal peptide receptors, promote metabolic homeostasis, and have long duration of action and enhanced bioavailability. In some embodiments, the peptide products are agonists of the GLP-1 receptor (GLP1R). GLP1R agonists stimulate blood glucose-dependent insulin secretion, increase insulin sensitivity with reduced risk of hypoglycemia, and induce satiety, thereby reducing food intake and thereby reducing weight gain or causing weight loss. In further embodiments, the peptide products are dual agonists of the GLP1R and glucagon receptor (GCGR), providing the added benefits of GCGR activation, including stimulation of fat burning, loss of body fat mass, and prevention of hypoglycemia.

[0048] Peptide Products Peptides and proteins often suffer from deficiencies in their use as pharmaceuticals, including formulation instability, aggregation, short duration of action, and poor bioavailability. Disclosed herein are peptide products with improved pharmaceutical properties, including increased stability, half-life, duration of action, and bioavailability, and reduced immunogenicity. The peptide products comprise a peptide covalently attached to a surfactant moiety containing a hydrophilic sugar group and a hydrophobic group. In some embodiments, the peptide is attached to a sugar group, which is subsequently attached to a hydrophobic group. In some embodiments, the peptide is a metabolic hormone, such as GLP-1, glucagon, oxyntomodulin, or exendin (e.g., exendin-4), or an analog or variant thereof. The peptide products are useful, for example, for the treatment of insulin resistance, diabetes, obesity, metabolic syndrome, and cardiovascular disease, and their associated conditions, such as NASH and PCOS.

[0049] In some embodiments, the peptide product comprises GLP-1, or an analog or variant thereof, covalently attached to a surfactant moiety (e.g., an alkyl glycoside, such as an 1-alkyl glycoside). The GLP-1 peptide may have a native or non-native amino acid sequence or structure. The alkyl group may be unsubstituted or substituted (e.g., with a carboxyl / carboxylate, aryl, or oxyaryl group). In one embodiment, the alkyl glycoside is a 1-O-alkyl β-D-glucuronic acid.

[0050] In further embodiments, the peptide product comprises glucagon, or an analog or variant thereof, covalently attached to a surfactant moiety (e.g., an alkyl glycoside, such as an 1-alkyl glycoside). The glucagon peptide may have a native or non-native amino acid sequence or structure. The alkyl group may be unsubstituted or substituted (e.g., with a carboxyl / carboxylate, aryl, or oxyaryl group). In one embodiment, the alkyl glycoside is a 1-O-alkyl β-D-glucuronic acid.

[0051] In additional embodiments, the peptide product comprises oxyntomodulin, or an analog or variant thereof, covalently attached to a surfactant moiety (e.g., an alkylglycoside, such as an 1-alkylglycoside). The oxyntomodulin peptide may have a native or non-native amino acid sequence or structure. The alkyl group may be unsubstituted or substituted (e.g., with a carboxyl / carboxylate, aryl, or oxyaryl group). In certain embodiments, the alkylglycoside is a 1-O-alkyl β-D-glucuronic acid.

[0052] In other embodiments, the peptide product comprises an exendin (e.g., exendin-4), or an analog or variant thereof, covalently attached to a surfactant moiety (e.g., an alkyl glycoside, such as an 1-alkyl glycoside). The exendin peptide may have a native or non-native amino acid sequence or structure. The alkyl group may be unsubstituted or substituted (e.g., with a carboxyl / carboxylate, aryl, or oxyaryl group). In one embodiment, the alkyl glycoside is a 1-O-alkyl β-D-glucuronic acid.

[0053] Some embodiments relate to a peptide product of formula IA comprising a surfactant X covalently attached to a peptide, the peptide comprising a linker amino acid U and at least one other amino acid:

[0054] [ka] wherein surfactant X is a moiety of formula I:

[0055] [ka] During the ceremony, R 1a is independently at each occurrence a single bond, H, a protecting group, a sugar, or an unsubstituted or substituted C1-C 30 an alkyl group, an unsubstituted or substituted aralkyl (-alkylaryl) group, an unsubstituted or substituted alkyloxyaryl group, or a steroid nucleus-containing group; R 1b , R 1c , and R 1d is independently at each occurrence a single bond, H, a protecting group, a sugar, or an unsubstituted or substituted C1-C 30 an alkyl group, an unsubstituted or substituted aralkyl group, an unsubstituted or substituted alkyloxyaryl group, or a steroid nucleus-containing group; R 2 is independently at each occurrence a single bond, a single bond to U, H, or unsubstituted or substituted C1-C 30 alkyl group, unsubstituted or substituted aralkyl group, unsubstituted or substituted alkyloxyaryl group, steroid nucleus-containing group, -NH-, -S-, -O-NH-, spacer, -triazolo-, -NH(C=O)-CH2-, or -(CH2) m -maleimide-; W 1 is independently at each occurrence -CH2-, -CH2-O-, -CH2-S-, -CH2-NH-, -(C=O)-, -(C=O)-NH-, -(C=S)-, or -(C=S)-NH-; W 2 is independently at each occurrence -O-, -S-, -NH-, or -CH2-; R 1a , R 1b , R 1c , R 1d , or R 2At least one occurrence of is unsubstituted or substituted C1-C 30 alkyl, aralkyl, alkyloxyaryl, or steroid nucleus-containing groups; m is an integer from 1 to 10; and n is 1, 2, or 3; and The peptide has formula II: aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9-aa 10 -aa 11 -aa 12 -aa 13 -aa 14 -aa 15 -aa 16 -aa 17 -aa 18 -aa 19 -aa 20 -aa 21 -aa 22 -aa 23 -aa 24 -aa 25 -aa 26 -aa 27 -aa 28 -aa 29 -aa 30 -aa 31 -aa 32 -aa 33 -aa 34 -aa 35 -aa 36 -aa 37 -Z Formula II (SEQ.ID.NO.1108) During the ceremony, Z is -OH, -NHR 3 , or -N(R 4 )His, where: R 3 is H, unsubstituted or substituted C1-C 12 alkyl, or a PEG-containing group of less than 10 Da; and R 4 is H, C2-C 10 acyl (e.g., acetyl), or -C(=O)-aryl (e.g., benzoyl); aa1 is His, N(R 3 )His, N(R 4)His, or pGlu-His; aa2 is Ser, D-Ser, Ala, Gly, Pro, MePro, Aib, Ac4c, or Ac5c; aa3 is Gln or Cit; aa4 is Gly, or D-Ala; aa5 is Thr or Ser; aa6 is Phe, Trp, 2FPhe, MePhe, 2FMePhe, or Na12; aa7 is Thr or Ser; aa8 is Ser or Asp; aa9 is Asp or Glu; aa 10 is Tyr, Leu, Met, NaI2, Bip, Bip2EtMeO, Glu, Lys, or U; aa 11 is absent, Ser, Asn, Bip, or U; aa 12 is absent, Lys, Glu, Ser, Arg, or U; aa 13 is absent, Tyr, Gln, Cit, or U; aa 14 is absent, Leu, Met, Nle, Glu, Lys, or U; aa 15 is absent, Asp, Glu, or U; aa 16 is absent or is Ser, Gly, Glu, Ala, Aib, Ac5c, Lys, Arg, or U; aa 17 is absent, Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, Lys, or U; aa 18 is absent, Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U; aa 19is absent, Ala, Val, Aib, Ac4c, Ac5c, or U; aa 20 is absent, Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or U; aa 21 is absent, Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U; aa 22 is absent, Phe, Trp, NaI2, Aib, Ac4c, Ac5c, or U; aa 23 is absent or is Val, Ile, Aib, Ac4c, Ac5c, or U; aa 24 is absent, Ala, Gln, Glu, Cit, or U; aa 25 is absent, Trp, NaI2, or U; aa 26 is absent, Leu, or U; aa 27 is absent or is Met, Val, Leu, Nle, Lys, or U; aa 28 is absent, Asn, Lys, Glu, Gln, Cit, or U; aa 29 is absent, Thr, Gly, Aib, Ac4c, Ac5c, or U; aa 30 is absent, Lys, Aib, Ac4c, Ac5c, Arg, or U; aa 31 is absent, Arg, Aib, Ac4c, Ac5c, or U; aa 32 is absent, Asn, Aib, Ac4c, Ac5c, or U; aa 33 is absent, Arg, Aib, Ac4c, Ac5c, or U; aa 34is absent, Asn, Aib, Ac4c, Ac5c, or U; aa 35 is absent, Asn, Aib, Ac4c, Ac5c, or U; aa 36 is absent, Ala, Ile, Aib, Ac4c, Ac5c, or U; aa 37 is absent or is U; U is a natural or unnatural amino acid containing a functional group used for covalent attachment to surfactant X; aa1-aa 37 any two of which can be optionally cyclized via their side chains to form a lactam bond; However, aa 10 -aa 37 is a linker amino acid U covalently attached to surfactant X.

[0056] The surfactant moiety may also be R 1a , R 1b , R 1c , or R 1d The linker amino acid U of the peptide can be attached via a W 2 is -O- and R 1a One occurrence of W may be a single bond to a hydroxyl-containing natural or unnatural amino acid (e.g., serine, homoserine, or threonine), or W 2 is -S- and R 1a One occurrence of may be a thiol / sulfhydryl-containing natural or unnatural amino acid (e.g., cysteine ​​or homocysteine), or 2 is -NH- and R 1a One occurrence of R may be an amino-containing natural or unnatural amino acid (e.g., lysine or ornithine). 1a U is an unnatural amino acid attached to the surfactant moiety via R 1aThe linker amino acid U, which is attached to the surfactant moiety via may be the L or D isomer. In certain embodiments, U is the D isomer.

[0057] In some embodiments, n is 1. In other embodiments, n is 2 and the first glycoside is 2 and the second glycoside -OR 1b , -OR 1c , or -OR 1d In yet another embodiment, n is 3 and the first glycoside is linked to the second glycoside via a single bond between the W 2 and the second glycoside -OR 1b , -OR 1c , or -OR 1d and the second glycoside is linked to the second glycoside via a single bond between the W 2 and the third glycoside -OR 1b , -OR 1c , or -OR 1d It is linked to a third glycoside via a single bond between

[0058] In some embodiments of the peptide product of formula IA, surfactant X is:

[0059] [ka] During the ceremony, R 1a is H, a protecting group, a sugar, unsubstituted or substituted C1-C 30 an alkyl group, or a steroid nucleus-containing group; R 1b , R 1c , and R 1d are independently H, a protecting group, a sugar, or an unsubstituted or substituted C-C 30 is an alkyl group; R 2 is a single bond to U, -NH-, -S-, -triazolo-, -NH(C=O)-CH2-, or -(CH2) m-maleimide-, W 1 is -CH2-, -CH2-O-, -CH2-S-, -(C=O)-, -(C=O)-NH-, -(C=S)-, or -(C=S)-NH-; W 2 is -O-, or -S-; m is 1-10; and R 1a , R 1b , R 1c , and R 1d At least one of the following is unsubstituted or substituted C1-C 30 It is an alkyl group or a group containing a steroid nucleus.

[0060] In one embodiment, surfactant X is R 2 It has the following structure where is bonded to U.

[0061] [ka] In one embodiment, W 1 is -C(=O)NH- and R 2 is a single bond between W1 and an amino acid residue U of the peptide (for example, the side chain amino group of a lysine residue).

[0062] In some embodiments, surfactant X is

[0063] [ka] In one embodiment, W 1 is -CH2-O-, -CH2-S-, or -(C=O)-NH-, and R 2 forms a bond with the appropriate functional group of the amino acid residue U of the peptide -(CH2) m - a maleimide group (for example, the thiol group of a cysteine ​​residue forms a thioether bond with the maleimide group).

[0064] In a further embodiment, surfactant X has the structure:

[0065] [ka] During the ceremony, R 1a is H, a protecting group, a sugar, unsubstituted or substituted C1-C 30 an alkyl group, or a steroid nucleus-containing group; R 1b , R 1c , and R 1d are independently H, a protecting group, or an unsubstituted or substituted C-C 30 is an alkyl group; R 2 is a single bond to U; W 1 is —(C═O)—NH—; W 2 is -O-; and R 1a , R 1b , R 1c , and R 1d At least one of the following is unsubstituted or substituted C1-C 30 It is an alkyl group or a group containing a steroid nucleus.

[0066] In one embodiment, surfactant X has the structure:

[0067] [ka] During the ceremony, R 1a is unsubstituted or substituted C1-C 30 is an alkyl group; R 1b , R 1c , and R 1d is H; R 2 is a single bond to U; W 1 is —(C═O)—NH—; and W 2 is -O-.

[0068] In some embodiments, surfactant X is a 1-O-alkyl β-D-glucuronyl moiety:

[0069] [ka] In the formula, R 1a is unsubstituted or substituted C1-C 30 , C1-C 20 , or C6-C 20 and R' is a peptide attached to the surfactant moiety through the N-terminal alpha amino group or the side chain amino acid of a natural or unnatural amino acid (e.g., lysine or ornithine). 1a is unsubstituted or substituted "C8-C 20 , C 12 -C 20 , or C 12 -C 16 is an alkyl group.

[0070] In some embodiments, the alkyl group attached to the sugar is unsubstituted or substituted C-C 30 , C1-C 20 , C6-C 30 , or C6-C 20 In one embodiment, the alkyl group attached to the sugar is an unsubstituted or substituted C8-C 20 , C6-C 18 , or C 12 -C 18 is an alkyl group.

[0071] In some embodiments, R 1a , R 1b , R 1c , R 1d , or R 2 At least one occurrence of is unsubstituted or substituted C1-C 30 In some embodiments, the alkyl group is connected to R via an ether bond (—O-alkyl). 1b , R 1c , or R 1dIn further embodiments, the alkyl group is bonded to at least one occurrence of R through an ether bond (—O-alkyl) or an amide bond [—C(═O)NH-alkyl]. 2 is bound to at least one occurrence of

[0072] In some embodiments, R 1a At least one occurrence, or a single occurrence, of is unsubstituted or substituted C1-C 30 , C6-C 30 , C6-C 20 , or C8-C 20 In one embodiment, R 1a At least one occurrence, or a single occurrence, of 12 -C 20 It is an alkyl group.

[0073] In some embodiments, alkyl groups (e.g., 1-alkyl groups) are internally and / or terminally substituted with carboxyl or carboxylate groups. In certain embodiments, alkyl groups (e.g., 1-alkyl groups) are substituted with carboxyl or carboxylate groups at the distal end of the alkyl group.

[0074] In a further embodiment, R 1a At least one occurrence, or a single occurrence, of is a sugar. In certain embodiments, the sugar is galactose. The galactose may be α- or β-linked. In some embodiments, the galactose is α-linked galactopyranose, β-linked galactopyranose, α-linked galactofuranose, or β-linked galactofuranose.

[0075] In some embodiments, the sugar group of the surfactant moiety is a monosaccharide. In certain embodiments, the monosaccharide is appropriately functionalized glucose, galactose, or mannose, such as glucuronic acid, galacturonic acid, or mannouronic acid. In other embodiments, the sugar is a disaccharide. In some embodiments, the disaccharide comprises two glucose molecules, or one glucose molecule and one galactose molecule. In certain embodiments, the disaccharide is diglucuronic acid or kentiobiose or lactose, such as appropriately functionalized melibiose, maltose, isomaltose, melibiouronic acid, malturonic acid, isomalturonic acid, gentiobiouronic acid, or lacturonic acid. The terms "-uronic acid" and "-uronyl" are used interchangeably herein in reference to the sugar group of the surfactant moiety.

[0076] In some embodiments, surfactant X is an 1-alkyl glycoside, and the unsubstituted or substituted alkyl group is attached to the C-1 position of the sugar via a glycosidic bond. The glycosidic bond may be an O-, S-, N-, or C-glycosidic bond. In some embodiments, the glycosidic bond is an O-glycosidic bond. The 1-alkyl group may be a β-anomer or an α-anomer. In some embodiments, the 1-alkyl group is a β-anomer.

[0077] In some embodiments, surfactant X is 1-eicosyl β-D-glucuronic acid, 1-octadecyl β-D-glucuronic acid, 1-hexadecyl β-D-glucuronic acid, 1-tetradecyl β-D-glucuronic acid, 1-dodecyl β-D-glucuronic acid, 1-decyl β-D-glucuronic acid, 1-octyl β-D-glucuronic acid, 1-eicosyl β-D-diglucuronic acid, 1-octadecyl β-D-diglucuronic acid, 1-hexadecyl β-D-diglucuronic acid, 1-tetradecyl β-D-diglucuronic acid, 1-dodecyl β-D-diglucuronic acid, 1- ...decyl β-D-diglucuronic acid, 1-decyl β-D-diglucuronic acid, 1-decyl β-D-diglucuronic acid, 1-decyl β-D-diglucuronic acid, 1-decyl β-D-diglucuronic acid, 1-decyl β-D-diglucuronic 1-octyl β-D-diglucuronic acid, 1-eicosyl β-D-isomalturonate, 1-octadecyl β-D-isomalturonate, 1-hexadecyl β-D-isomalturonate, 1-tetradecyl β-D-isomalturonate, 1-dodecyl β-D-isomalturonate, 1-decyl β-D-isomalturonate, 1-octyl β-D-isomalturonate, 1-eicosyl β-D-gentiobiouronic acid, 1-octadecyl β-D-gentiobiouronic acid, 1-hexadecyl β-D-gentiobiouronic acid, 1-tetradecyl β-D-isomalturonate Tradecyl β-D-gentiobiouronic acid, 1-dodecyl β-D-gentiobiouronic acid, 1-decyl β-D-gentiobiouronic acid, 1-octyl β-D-gentiobiouronic acid, 1-eicosyl β-D-melibiouronic acid, 1-octadecyl β-D-melibiouronic acid, 1-hexadecyl β-D-melibiouronic acid, 1-tetradecyl β-D-melibiouronic acid, 1-dodecyl β-D-melibiouronic acid, 1-decyl β-D-melibiouronic acid, 1-octyl β-D-melibiouronic acid, functionalized 1-eicosyl β-D-glucose, 1-octadecyl 1-hexadecyl β-D-glucose, 1-tetradecyl β-D-glucose, 1-dodecyl β-D-glucose, 1-decyl β-D-glucose, 1-octyl β-D-glucose, 1-eicosyl β-D-maltoside, 1-octadecyl β-D-maltoside, 1-hexadecyl β-D-maltoside, 1-tetradecyl β-D-maltoside, 1-dodecyl β-D-maltoside, 1-decyl β-D-maltoside, 1-octyl β-D-maltoside, 1-eicosyl β-D-melibioside, 1-octadecyl β-D-melibioside,The surfactant X is selected from 1-hexadecyl β-D-melibioside, 1-tetradecyl β-D-melibioside, 1-dodecyl β-D-melibioside, 1-decyl β-D-melibioside, 1-octyl β-D-melibioside, the corresponding 1-alkyl glycosides with a 6-carboxyl or 6,6'-dicarboxyl group, the corresponding 1-alkyl α-anomers, etc. In a particular embodiment, surfactant X is a 1-alkyl β-D-glucuronic acid.

[0078] Illustrative examples shown below are a 1-O-octyl β-D-melibiouronyl moiety (top left), a 1-O-hexadecyl β-D-gentiobiouronyl moiety (top right), a 1-O-dodecyl β-D-maltouronyl moiety (bottom left, where either or both carboxyl groups can be attached to the peptide), and a 1-O-tetradecyl β-D-isomaltouronyl moiety (bottom right) attached to an amino group of a peptide (e.g., the side chain amino group of a lysine residue).

[0079] [ka]

[0080] In some embodiments, the surfactant moiety is attached to the linker amino acid U of the peptide via an amide bond.

[0081] The peptide of the peptide product can be covalently attached to one or more surfactant moieties. In one embodiment, the peptide product has one surfactant moiety. In a further embodiment, the peptide product has two surfactant moieties, which may be the same or different. In another embodiment, the peptide product has three surfactant moieties, which may be the same or different.

[0082] The linker amino acid U can be any natural or unnatural amino acid having a functional group suitable for covalent attachment to a surfactant moiety. In some embodiments, U is a dibasic natural or unnatural amino acid (e.g., one with a side chain amino group such as lysine or ornithine), a diacidic natural or unnatural amino acid (e.g., one with a side chain carboxyl group such as glutamic acid or aspartic acid), a natural or unnatural amino acid with a thiol / mercapto group (e.g., cysteine), a natural or unnatural amino acid with a hydroxyl group (e.g., serine or threonine), or an unnatural amino acid with an -N3 group, an acetylene group, a haloacetyl group (e.g., -NHC(=O)CH2Br) or -(CH2) m -maleimide group), where m is 1-10. A haloacetyl or maleimide group of the linker amino acid can be coupled, for example, to a thiol group of a sugar group of the surfactant moiety, or vice versa. An azide or alkyne group of the linker amino acid can undergo 1,3-dipolar cycloaddition with an alkyne or azide group, respectively, of a sugar group of the surfactant moiety using a copper catalyst, for example, to form a triazole. In certain embodiments, U is a residue of lysine, ornithine (Orn), cysteine, or glutamic acid. Like any amino acid residue in a peptide, U can be an L- or D-amino acid.

[0083] The linker amino acid U can be an internal or / and terminal amino acid residue of the peptide. The functional group of U that forms the covalent bond with the surfactant moiety can be a side chain functional group of an internal or terminal amino acid residue, or the alpha amino group of the N-terminal amino acid residue, or the alpha carboxyl group of the C-terminal amino acid residue.

[0084] In some embodiments, the side chain functional groups of two amino acid residues (annotated with an asterisk), such as a lysine residue and a glutamic acid residue, of a peptide are linked to form a cyclic structure, such as a lactam. The lactam formed between the side chain functional groups of a lysine residue and a glutamic acid residue can stabilize the α-helical structure of the peptide. A cyclic structure can also be formed by a disulfide bond between the thiol groups of two cysteine ​​residues, which may restrict conformation and promote α-helix formation. A "click reaction" between the side chain azide and alkyne groups of two unnatural amino acid residues can also form a heterocyclic structure (triazole) that restricts conformation and stabilizes α-helices (Le Chevalier et al., J. Peptide Sci., 15:451-454

[2009] ). Furthermore, the side chain alkene groups of two unnatural amino acid residues can be cyclized by olefin transformation to form a C=C double bond, which can then be reduced to a C-C single bond (Verdine and Hilinski, Meth. Enzymol., 503:3-33

[2011] ).

[0085] In some embodiments, the peptide product of formula IA comprises at least amino acid residues aa1-aa of SEQ.ID.NO. 1108. 17 , aa1-aa 18 , aa1-aa 19 , or aa1-aa 20 In a further embodiment, the peptide product of formula IA comprises at least amino acid residues aa1-aa of SEQ.ID.NO.1108. 27 , aa1-aa 28 , aa1-aa 29 , or aa1-aa 30 Includes:

[0086] In some embodiments, the peptide product of formula IA has formula III-A: aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9-aa 10 -aa 11 -aa12 -aa 13 -aa 14 -aa 15 -aa 16 -aa 17 -aa 18 -aa 19 -aa 20 -aa 21 -aa 22 -aa 23 -aa 24 -aa 25 -aa 26 -aa 27 -aa 28 -aa 29 -Z Formula III-A (SEQ.ID.NO.1109) During the ceremony, Z is -OH or -NHR 3 where R 3 is H, unsubstituted or substituted C1-C 12 alkyl, or a PEG-containing group less than 10 Da; aa1 is His, N(R 3 )His, N(Ac)His, or pGlu-His; aa2 is Ser, Ala, Gly, MePro, Aib, Ac4c, or Ac5c; aa3 is Gln or Cit; aa4 is Gly, or D-Ala; aa5 is Thr or Ser; aa6 is Phe, Trp, 2FPhe, MePhe, 2FMePhe, or Na12; aa7 is Thr or Ser; aa8 is Ser or Asp; aa9 is Asp or Glu; aa 10 is Tyr, Leu, Met, NaI2, Bip, Bip2EtMeO, Glu, Lys, or U(X); aa 11 is absent or is Ser, Asn, Bip, or U(X); aa 12is absent, Lys, Glu, Ser, Arg, or U(X); aa 13 is absent, Tyr, Gln, Cit, or U(X); aa 14 is absent, Leu, Met, Nle, Glu, Lys, or U(X); aa 15 is absent, Asp, Glu, or U(X); aa 16 is absent, Ser, Gly, Glu, Ala, Aib, Ac5c, Lys, Arg, or U(X); aa 17 is absent, Arg, hArg, Gln, Glu, Lys, Cit, Aib, Ac4c, Ac5c, or U(X); aa 18 is absent, Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X); aa 19 is absent, Ala, Val, Aib, Ac4c, Ac5c, or U(X); aa 20 is absent, Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or U(X); aa 21 is absent, Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X); aa 22 is absent, Phe, Trp, NaI2, Aib, Ac4c, Ac5c, or U(X); aa 23 is absent or is Val, Ile, Aib, Ac4c, Ac5c, or U(X); aa 24 is absent, Ala, Gln, Glu, Cit, or U(X); aa 25 is absent, Trp, NaI2, or U(X); aa 26is absent, Leu, or U(X); aa 27 is absent, Met, Val, Leu, Nle, Lys, or U(X); aa 28 is absent, Asn, Lys, Glu, Gln, or U(X); aa 29 is absent, Thr, Gly, Aib, Ac4c, Ac5c, or U(X); where aa1-aa 29 any two of which can be optionally cyclized via their side chains to form a lactam bond; However, aa 10 -aa 12 and aa 16 -aa 29 is a natural or unnatural amino acid U covalently attached to surfactant X.

[0087] In one embodiment, the peptide product of formula III-A has the following structure: aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9-aa 10 -aa 11 -aa 12 -aa 13 -aa 14 -aa 15 -aa 16 -aa 17 -aa 18 -aa 19 -aa 20 -aa 21 -aa 22 -aa 23 -aa 24 -aa 25 -aa 26 -aa 27 -aa 28 -aa 29 -Z Formula III-A (SEQ.ID.NO.1110) During the ceremony, Z is -OH or -NHR 3 where R 3is H, unsubstituted or substituted C1-C 12 alkyl, or a PEG-containing group less than 10 Da; aa1 is His; aa2 is Aib; aa3 is Gln; aa4 is Gly; aa5 is Thr; aa6 is Phe; aa7 is Thr; aa8 is Ser; aa9 is Asp; aa 10 is Tyr, Glu, Lys, or U(X); aa 11 is Ser; aa 12 is Lys, or Glu; aa 13 is Tyr; aa 14 is Leu, Glu, or Lys; aa 15 is Asp; aa 16 is Glu, or Lys; aa 17 is Gln, Glu, or U(X); aa 18 is Ala; aa 19 is Ala; aa 20 is Glu, Lys, or U(X); aa 21 is Glu; aa 22 is Phe; aa 23 is Ile; aa 24 is Gln, Glu, or U(X); aa 25 is Trp; aa 26 is Leu; aa27 is Leu; aa 28 is Glu or Gln; aa 29 is Thr; aa 10 and aa 14 , aa 12 and aa 16 , or aa 16 and aa 20 can be optionally cyclized via its side chain to form a lactam; However, aa 10 , aa 17 , aa 20 , and aa 24 is a natural or unnatural amino acid U covalently attached to surfactant X.

[0088] In a further embodiment, the peptide product of formula IA has formula III-B: His1-aa2-aa3-Gly4-Thr5-aa6-Thr7-Ser8-Asp9-aa 10 -aa 11 -aa 12 -aa 13 -aa 14 -aa 15 -aa 16 -aa 17 -aa 18 -aa 19 -aa 20 -aa 21 -aa 22 -aa 23 -aa 24 -aa 25 -aa 26 -aa 27 -aa 28 -aa 29 -aa 30 -Z Formula III-B (SEQ.ID.NO.1111) During the ceremony, Z is -OH or -NHR 3 where R 3 is H, unsubstituted or substituted C1-C 12alkyl, or a PEG-containing group less than 10 Da; aa2 is Gly, MePro, or Aib; aa3 is Gln or Cit; aa6 is Phe, 2FPhe, MePhe, 2FMePhe, or Na12; aa 10 is Tyr, NaI2, Bip, Bip2EtMeO, Glu, Lys, or U(X); aa 11 is absent or is Ser, Asn, Bip, or U(X); aa 12 is absent, Lys, Glu, Ser, or U(X); aa 13 is absent, Tyr, Gln, Cit, or U(X); aa 14 is absent, Leu, Nle, Glu, Lys, or U(X); aa 15 is absent, Asp, Glu, or U(X); aa 16 is absent or is Ser, Gly, Glu, Ala, Aib, Lys, Arg, or U(X); aa 17 is absent, Arg, hArg, Gln, Glu, Lys, Cit, Aib, or U(X); aa 18 is absent, Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X); aa 19 is absent, Ala, Aib, or U(X); aa 20 is absent, Gln, Lys, Arg, Cit, Glu, Aib, or U(X); aa 21 is absent, Asp, Glu, Leu, Aib, or U(X); aa 22 is absent, Phe, or U(X); aa 23 is absent or is Val, Ile, Aib, or U(X); aa 24 is absent, Ala, Glu, Gln, or U(X); aa 25 is absent, Trp, or U(X); aa 26 is absent, Leu, or U(X); aa 27 is absent, Met, Val, Leu, Nle, Lys, or U(X); aa 28 is absent, Asn, Glu, Gln, Cit, or U(X); aa 29 is absent, Thr, Aib, or U(X); aa 30 is absent or is Arg or U(X); where aa1-aa 23 any two of which can be optionally cyclized via their side chains to form a lactam bond; However, aa 10 -aa 12 , aa 16 -aa 24 , and aa 28 is a natural or unnatural amino acid U covalently attached to surfactant X.

[0089] In some embodiments of the peptide product of Formula IA, III-A, or III-B, aa2 is Gly, Aib, or Ac4c; or aa 12 is lysine; or aa 14 is leucine; or aa 17 is glycine or homoarginine (hArg); or aa 17 , aa18 , aa 20 , aa 24 , or aa 28 or any combination thereof is lysine bound to surfactant X; or aa 16 and aa 20 is cyclized via its side chain to form a lactam bond; or surfactant X comprises an unsubstituted or substituted dodecyl, tetradecyl, hexadecyl, or octadecyl alkyl group; or Any combination or all of the above.

[0090] In some embodiments, a peptide of a peptide product described herein contains one or more Aib residues at or near the N-terminus, at or near the C-terminus, internally, or any or all combinations thereof. Aib residues can protect the peptide from degradation by proteases such as dipeptidyl peptidase 4 (DPP-4). In certain embodiments, aa2 is Aib.

[0091] In some embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 -aa 17 -Ala 18 -Ala 19 -aa 20 -Glu 21 -Phe 22 -aa 23 -aa 24 -Trp 25 -Leu 26 -aa 27 -aa 28 -Thr 29-NH2 (SEQ.ID.NO.1126) During the ceremony, aa2 is Gly or Aib; aa 16 is Glu, Ser, Ala, Lys, or Aib; aa 17 is Gln, Lys, or U(X); aa 20 is Lys, Glu, or Arg; aa 23 is Ile, or Val; aa 24 is Ala, Gln, or U(X); aa 27 is Met, Val, or Leu; and aa 28 is Asn, Gln, or U(X).

[0092] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 -aa 17 -Arg 18 -Ala 19 -aa 20 -Asp 21 -Phe 22 -aa 23 -aa 24 -Trp 25 -Leu 26 -aa 27 -aa 28 -Thr 29 -NH2 (SEQ.ID.NO.1127) During the ceremony, aa2 is Gly or Aib; aa 16is Glu, Ala, or Aib; aa 17 is Arg, hArg, or Gln; aa 20 is U(X) [e.g., Lys(X)]; aa 23 is Ile, or Val; aa 24 is Gln, or Ala; aa 27 is Leu, or Val; and aa 28 is Asn or Gln.

[0093] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 -aa 17 -Ala 18 -Ala 19 -aa 20 -Glu 21 -Phe 22 -aa 23 -aa 24 -Trp 25 -Leu 26 -aa 27 -aa 28 -Thr 29 -NH2 (SEQ.ID.NO.774) During the ceremony, aa2 is Gly or Aib; aa 16 is Glu, Ser, Ala, Lys, or Aib; aa 17 is Gln, Glu, Lys, or U(X); aa 20 is Lys, Glu, or Arg; aa 23 is Ile, or Val; aa 24 is Ala, Gln, or U(X); aa 27 is Met, Val, or Leu, and aa 28 is Asn, Gln, or U(X).

[0094] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 -aa 17 -Ala 18 -Ala 19 -Lys 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leu 26 -aa 27 -Asn 28 -Thr 29 -NH2 (SEQ.ID.NO.775) During the ceremony, aa2 is Gly or Aib; aa 16 is Glu, Ala, or Aib; aa 17 is U(X) [e.g., Lys(X)]; and aa 27 is Leu or Val.

[0095] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 -aa 17 -Arg 18 -Ala 19 -aa 20 -Asp 21 -Phe 22 -aa 23 -aa 24 -Trp 25 -Leu 26 -aa 27 -aa 28 -Thr 29 -NH2 (SEQ.ID.NO.776) During the ceremony, aa2 is Gly or Aib; aa 16 is Glu, Ser, Ala, or Aib; aa 17 is Arg, hArg, or Gln; aa 20 is Lys or U(X); aa 23 is Ile, or Val; aa 24 is Ala, Gln, or U(X); aa 27 is Leu, or Val; and aa 28 is Asn, Gln, or U(X).

[0096] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu14 -Asp 15 -aa 16 -aa 17 -Arg 18 -Ala 19 -aa 20 -Asp 21 -Phe 22 -aa 23 -aa 24 -Trp 25 -Leu 26 -aa 27 -aa 28 -Thr 29 -NH2(SEQ.ID.NO.777) During the ceremony, aa2 is Gly or Aib; aa 16 is Glu, Ser, Ala, or Aib; aa 17 is Arg, hArg, or Gln; aa 20 is Lys or U(X); aa 23 is Ile, or Val; aa 24 is Gln, Ala, or U(X); aa 27 is Leu, or Val; and aa 28 is Asn, Gln, or U(X).

[0097] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 * -aa 17 -Ala 18 -Ala 19 -aa 20* -Glu 21 -Phe 22 -Ile 23 -Lys(N-Omega-X) 24 -Trp 25 -Leu 26 -aa 27 -aa 28 -Thr 29 -NH2(SEQ.ID.NO.778) During the ceremony, aa2 is Aib or Gly, aa 16 * and aa 20 * is independently Lys or Glu and is cyclized via its side chain to form a lactam bond; aa 17 is Arg, hArg, or Gln; aa 27 is Met, Val, Leu, or Nle; and aa 28 is Asn or Gln. In some embodiments, surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C8-C 20 It is alkyl.

[0098] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 -aa 17 -Ala 18 -Ala 19 -Lys 20 -Glu 21 -Phe 22 -Ile 23 -Ala 24-Trp 25 -Leu 26 -Leu 27 -Asn 28 -Thr 29 -NH2(SEQ.ID.NO.779) During the ceremony, aa2 is Aib or Gly, aa 16 is Glu, Ala, or Aib; aa 17 is Lys(N-omega-X). In some embodiments, surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C8-C 20 It is alkyl.

[0099] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 -aa 17 -Arg 18 -Ala 19 -aa 20 -Asp 21 -Phe 22 -aa 23 -aa 24 -Trp 25 -Leu 26 -aa 27 -aa 28 -Thr 29 -NH2 (SEQ.ID.NO.780) During the ceremony, aa2 is Gly or Aib; aa 16 is Glu, Ala, or Aib; aa 17 is Arg or hArg; aa20 is Lys(N-omega-X); aa 23 is Ile, or Val; aa 24 is Gln, or Ala; aa 27 is Leu, or Val; and aa 28 is Asn or Gln. In some embodiments, surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C8-C 20 It is alkyl.

[0100] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-aa6-Thr7-Ser8-Asp9-aa 10 -aa 11 -Z (SEQ.ID.NO.781) During the ceremony, aa2 is Gly, Aib, or MePro; aa6 is Phe, 2FPhe, MePhe, or 2FMePhe; aa 10 is Tyr, Nal2, Bip, Bip2Et, or Bip2EtMeO, and aa 11 is Lys(N-omega-X). In some embodiments, surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C8-C 20 It is alkyl.

[0101] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-aa2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys12 -Tyr 13 -Leu 14 -Asp 15 -Glu 16 -U(X) 17 -Ala 18 -Ala 19 -Lys 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leu 26 -Leu 27 -aa 28 -Thr 29 -NH2 (SEQ.ID.NO.795) During the ceremony, aa2 is Gly or Aib; and aa 28 is Asn or Gln.

[0102] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 * -Gln 17 -Ala 18 -Ala 19 -aa 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-Omega-X) 24 -Trp 25 -Leu 26 -Leu 27 -aa 28 -Thr 29 -NH2 (SEQ.ID.NO.796) During the ceremony, aa 16* and aa 20 * is independently Lys or Glu and is cyclized via its side chain to form a lactam bond; aa 28 is Asn or Gln; and The surfactant X includes 1-alkyl β-D-glucosides, 1-alkyl β-D-maltosides, 1-alkyl β-D-melibiosides, etc., or the corresponding α-anomers, where alkyl is a linear C8-C 20 It is alkyl.

[0103] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu 16 * -Gln 17 -Ala 18 -Ala 19 -Lys 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-Omega-X) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2 (SEQ.ID.NO.797) During the ceremony, Glu 16 * and Lys 20 * is cyclized via its side chain to form a lactam bond; and The surfactant X includes 1-alkyl β-D-glucosides, 1-alkyl β-D-maltosides, 1-alkyl β-D-melibiosides, etc., or the corresponding α-anomers, where alkyl is a linear C8-C 20 It is alkyl.

[0104] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-U(X)10-Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 * -aa 17 -Ala 18 -Ala 19 -aa 20 * -Glu 21 -Phe 22 -Ile 23 -aa 24 -Trp 25 -Leu 26 -Leu 27 -aa 28 -Thr 29 -Z (SEQ.ID.NO.1025) During the ceremony, Z is -OH or -NHR 3 where R 3 is H or a PEG-containing group of less than 10 Da; aa 16 * and aa 20 * is independently Lys or Glu and is cyclized via its side chain to form a lactam bond; aa 17 is Glu or Gln; aa 24 is Ala, Glu, or Gln; and aa 28 is Asn or Gln.

[0105] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 * -U(X) 17 -Ala 18 -Ala 19 -aa 20 * -Glu 21 -Phe 22 -Ile 23 -aa 24 -Trp 25 -Leu 26 -Leu 27 -aa 28 -Thr 29 -Z (SEQ.ID.NO.1026) During the ceremony, Z is -OH or -NHR 3 where R 3 is H or a PEG-containing group of less than 10 Da; aa 16 * and aa 20 * is independently Lys or Glu and is cyclized via its side chain to form a lactam bond; aa 24 is Ala, Glu, or Gln; and aa 28 is Asn or Gln.

[0106] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-aa 10 *-Ser 11 -Lys 12 -Tyr 13 -aa 14 * -Asp 15 -Ser 16 -aa 17 -Ala 18 -Ala 19 -U(X) 20 -Glu 21 -Phe 22 -Ile 23 -aa 24 -Trp 25 -Leu 26 -Leu 27 -aa 28 -Thr 29 -Z (SEQ.ID.NO.1027) During the ceremony, Z is -OH or -NHR 3 where R 3 is H or a PEG-containing group of less than 10 Da; aa 10 * and aa 14 * is independently Lys or Glu and is cyclized via its side chain to form a lactam bond; aa 17 is Glu or Gln; aa 24 is Ala, Glu, or Gln; and aa 28 is Asn or Gln.

[0107] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -aa 12 * -Tyr 13 -Gln 14 -Asp 15 -aa 16 * -aa17 -Ala 18 -Ala 19 -U(X) 20 -Glu 21 -Phe 22 -Ile 23 -aa 24 -Trp 25 -Leu 26 -Leu 27 -aa 28 -Thr 29 -Z (SEQ.ID.NO.1028) During the ceremony, Z is -OH or -NHR 3 where R 3 is H or a PEG-containing group of less than 10 Da; aa 12 * and aa 16 * is independently Lys or Glu and is cyclized via its side chain to form a lactam bond; aa 17 is Glu or Gln; aa 24 is Ala, Glu, or Gln; and aa 28 is Asn or Gln.

[0108] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -aa 16 * -Gln 17 -Ala 18 -Ala 19 -aa 20 * -Glu 21 -Phe 22 -Ile 23 -U(X)24 -Trp 25 -Leu 26 -Leu 27 -aa 28 -Thr 29 -Z (SEQ.ID.NO.1029) During the ceremony, Z is -OH or -NHR 3 where R 3 is H or a PEG-containing group of less than 10 Da; aa 16 * and aa 20 * is independently Lys or Glu and is cyclized via its side chain to form a lactam bond; aa 28 is Asn or Gln; and The surfactant X includes 1-alkyl β-D-glucosides, 1-alkyl β-D-maltosides, 1-alkyl β-D-melibiosides, etc., or the corresponding α-anomers, where alkyl is a linear C8-C 20 It is alkyl.

[0109] In other embodiments, the peptide product of formula IA, III-A, or III-B has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu 16 * -Gln 17 -Ala 18 -Ala 19 -Lys 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-Omega-X) 24 -Trp 25 -Leu 26 -Leu 27 -Gln28 -Thr 29 -Z (SEQ.ID.NO.1113) where: Z is -OH or -NHR 3 and Glu 16 * and Lys 20 * is cyclized via its side chain to form a lactam bond; and The surfactant X includes 1-alkyl β-D-glucosides, 1-alkyl β-D-maltosides, 1-alkyl β-D-melibiosides, etc., or the corresponding α-anomers, where alkyl is a linear C8-C 20 It is alkyl.

[0110] In some embodiments, the peptide product has the following structure: His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu 16 * -Gln 17 -Ala 18 -Ala 19 -Lys 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-dodecyl β-D-glucuronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.601); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr13 -Leu 14 -Asp 15 -Glu 16 * -Gln 17 -God 18 -God 19 -Lys 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-オメガ[1-テトラデシルβ-D-グルクロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.602); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu 16 * -Gln 17 -God 18 -God 19 -Lys 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-オメガ[1-ヘキサデシルβ-D-グルクロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.603); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu 16* -Gln 17 -God 18 -God 19 -Lys 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-オオメガ[1-オクタデシルβ-D-グルクロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.604); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -God 18 -God 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-オメガ[1-オクチルβ-D-メリビオウロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.630); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -God 18 -God19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-オメガ[1-ドデシルβ-D-メリビオウロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.631); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -God 18 -God 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-オメガ[1-テトラデシルβ-D-メリビオウロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.632); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -God 18 -God 19 -Lys * 20 -Glu 21-Phe 22 -Ile 23 -Lys(N-オメガ[1-ヘキサデシルβ-D-メリビオウロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.633); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -God 18 -God 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-オオメガ[1-オクタデシルβ-D-メリビオウロニル]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.634); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -God 18 -God 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23-Lys(N-omega[1-hexadecyl α-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.805); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-omega[1-tetradecyl α-D-melibiouronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.819); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-omega[1-hexadecyl α-D-melibiouronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.820); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-オオメガ[1-オクタデシルα-D-メリビオウロニル]) 17 -God 18 -God 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.821); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Dear 11 -Lys 12 -Taurus 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-オメガ[1-ドデシルα-D-メリビオウロニル]) 17 -God 18 -God 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-オメガ[1-ドデシルβ-D-グルコウロニル]) 24 -Trp 25 -Leu 26 -Leu27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1114); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-omega[1-tetradecyl α-D-melibiouronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-tetradecyl β-D-glucuronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1115); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-omega[1-hexadecyl α-D-melibiouronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-hexadecyl β-D-glucuronyl]) 24 -Trp 25 -Leu 26 -Leu27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1116); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-omega[1-(13-carboxyl-tridecyloxy)β-D-glucuronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1117); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-omega[1-(15-carboxyl-pentadecyloxy)β-D-glucuronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28-Thr 29 -NH2(SEQ.ID.NO.1118); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Lys(N-omega[1-(17-carboxyl-heptadecyloxy)β-D-glucuronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1119); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-(13-carboxyl-tridecyloxy)β-D-glucuronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29-NH2(SEQ.ID.NO.1120); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-(15-carboxyl-pentadecyloxy)β-D-glucuronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1121); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-(17-carboxyl-heptadecyloxy)β-D-glucuronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1122); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-(13-carboxyl-tridecyloxy)β-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2(SEQ.ID.NO.1123); His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-(15-carboxyl-pentadecyloxy)β-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2 (SEQ.ID.NO.1124); or His1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Glu * 16 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-(17-carboxyl-heptadecyloxy)β-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH2 (SEQ.ID.NO.1125); or and pharmaceutically acceptable salts thereof, wherein Glu 16 * and Lys 20 * represents a residue that can be cyclized via its side chain to form a lactam.

[0111] Additional peptide products are listed in Table 1 of FIG. 1, Table 2 of FIG. 2, Table 3 of FIG. 3, and Table 4 of FIG.

[0112] Figure 5 illustrates the X-ray crystal structure of the N-terminal region / extracellular domain binding site of the GLP-1 receptor, showing the key hydrophobic ligand-binding region (Val of the ligand exendin-4). 19* , Phe 22* , Trp 25* , and Leu 26* , and Glu 15 The sequence of exendin-4 exceeding 100 interacts with this region as an amphipathic helix.) The 1-alkyl group of the surfactant moiety of the peptide product may be involved in hydrophobic interactions with the GLP-1 receptor.

[0113] In some embodiments, the surfactant moiety is connected to the linker amino acid U of the peptide via a spacer. The spacer can increase the aqueous solubility of the peptide product and increase the flexibility of the attachment point of the peptide surfactant. In certain embodiments, the spacer is an amino acid spacer containing one or more amino acids. In some embodiments, the R of surfactant X is 2 One occurrence of is an amino acid spacer used for attachment to the peptide. In one embodiment, the amino acid spacer is Gly m , Glu m , or Lys m where m is an integer from 1 to 10. For example, W 1 is -(C=O)- and R 2 is attached to the side chain amino group of a lysine residue of a peptide or to the N-terminus of a peptide via the carboxyl group of a terminal glycine residue. m Spacer, Glu linked via the α or γ carboxyl group of the terminal glutamic acid residue m is a spacer, or R 2 is a Lys bonded to the side chain amino group of a lysine residue of the peptide or to the N-terminus via the carboxyl group of a terminal lysine residue m It may be a spacer.

[0114] In other embodiments, the spacer is a hydrophilic spacer that does not contain an amino acid residue. For example, such a spacer is

[0115] [ka] where n is 1, 2, or 3. The amino group of such a spacer may be attached, for example, to a carboxyl group at the C-6 position of a sugar in a surfactant moiety, and the carboxyl group of such a spacer may be attached, for example, to the side chain amino group of an amino acid (e.g., lysine) residue or the N-terminus of a peptide.

[0116] Three examples of attachment of a peptide to a surfactant moiety via a spacer are shown below: the peptide is on top, the spacer is in the middle, and C 12 The surfactant containing an alkyl group (represented as "C12") is at the bottom. In the example on the left, the spacer is a single ornithine residue, whose carboxyl group forms an amide bond with the side chain amino group of a lysine residue of the peptide. In the example in the middle, the spacer is a single glutamic acid residue, whose gamma carboxyl group forms an amide bond with the side chain amino group of a lysine residue of the peptide. In the example on the right, the spacer contains a single glutamic acid residue.

[0117] [ka]

[0118] The peptide product can optionally have one or more additional modifications, including, but not limited to, acylation (e.g., acetylation) at the N-terminus, attachment of a natural or synthetic polymer (e.g., a hydrophilic synthetic polymer such as polyethylene glycol [PEG], which can increase the aqueous solubility of the peptide product), and / or lipid modifications (e.g., C8-C10) at the N-terminus, C-terminus, or / and one or more side chains. 20 acylation with saturated or unsaturated fatty acids / diacids, or conjugation to a steroid nucleus-containing group such as cholesterol).

[0119] The present disclosure includes analogs and variants of the peptide products described herein. Such analogs and variants include, but are not limited to, those with conservative substitutions at one or more positions of the amino acid sequence of a particular peptide product disclosed herein. Reference to a peptide product described herein includes all pharmaceutically acceptable salts thereof.

[0120] The covalent attachment of a surfactant moiety to a peptide confers a unique combination of beneficial properties to the peptide product. The hydrophobic nature of the surfactant moiety facilitates binding of the peptide-surfactant conjugate to a hydrophobic carrier protein such as human serum albumin (HSA), thereby increasing the half-life and duration of action of the peptide product through multiple mechanisms, including increased resistance to proteases and other types of hydrolases, reduced renal clearance of the peptide product from the carrier protein, and slowing its release rate. The strong but reversible non-covalent binding of the hydrophobic group of the surfactant moiety of the peptide product to the hydrophobic pocket of a carrier protein such as HSA (MW of approximately 67 kDa) protects the peptide product from proteases (e.g., DPP-4) and other types of degradative enzymes circulating in the blood and expressed on the surface of cells, making the peptide product less immunogenic, and increases the molecular weight of the peptide product above the filtration threshold of approximately 60-70 kDa, thereby allowing the peptide product to avoid renal clearance into the urine via glomerular filtration. Furthermore, binding of peptide products to HSA increases their half-life through pH-dependent HSA recycling mediated by the neonatal Fc receptor (FcRn). In some embodiments, the peptide products have a half-life (e.g., circulation half-life or / and elimination half-life) of at least about 3 days, 1 week, 10 days, or 2 weeks (e.g., at least about 1 week).

[0121] In addition, the amphiphilic nature of the surfactant moiety, which contains a hydrophilic sugar group and a hydrophobic group, increases the bioavailability of the peptide product and its duration of action. The surfactant moiety can disrupt tight junctions between cells in mucosal cell membranes, thereby facilitating the penetration of the peptide product across the mucosal barrier. The surfactant moiety can also form micelles, which can facilitate the penetration of the peptide product through membranes. Furthermore, the micelle breakdown process can extend the duration of action of the peptide product. Surfactants form micelles when their concentration is greater than the critical micelle concentration (CMC), and alkyl glycosides with the same sugar group tend to have a lower CMC as the alkyl chain length increases.

[0122] The significantly increased plasma and elimination half-lives compared to pellets not bound to the surfactant moiety make the peptide product a drug in its own right. In other words, the stable surfactant moiety remains bound to the peptide in vivo, and the intact peptide-surfactant conjugate can exert its biological activity by binding to receptors of the basic peptide classes. For example, the peptide products disclosed herein can exert their biological activity by binding to and activating the GLP-1 receptor (GLP1R) or / and the glucagon receptor (GCGR). In some embodiments, the peptide product is a dual GLP1R / GCGR agonist. The potency or / and selectivity of receptor binding or activation can be fine-tuned by varying the surfactant component or / and the peptide component.

[0123] Pharmaceutical Composition The present disclosure also provides pharmaceutical compositions comprising a peptide product described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition contains a therapeutically effective amount of the peptide product, or a suitable fraction thereof. The composition can optionally contain an additional therapeutic agent. In some embodiments, the purity of the peptide product is at least about 90%, 95%, or 98%.

[0124] Pharmaceutically acceptable excipients and carriers include pharmaceutically acceptable substances, materials and vehicles.Non-limiting examples of excipient types include liquid and solid fillers, diluents, binders, lubricants, glidants, surface active agents, dispersants, disintegrants, emulsifiers, wetting agents, suspending agents, thickeners, solvents, isotonicity agents, buffer solutions, pH adjusters, absorption retardants, stabilizers, antioxidants, preservatives, antimicrobial agents, antibacterial agents, antifungal agents, chelating agents, adjuvants, sweeteners, flavoring agents, coloring agents, encapsulating materials and coating materials.The use of such excipients in pharmaceutical preparations is known in the art. For example, conventional vehicles and carriers include, but are not limited to, oils (e.g., vegetable oils such as olive oil or sesame oil), aqueous solvents {e.g., saline, buffered saline (e.g., phosphate-buffered saline [PBS]), isotonic solutions (e.g., Ringer's solution)}, and organic solvents (e.g., dimethyl sulfoxide, and alcohols [e.g., ethanol, glycerol, and propylene glycol]). Except to the extent that any conventional excipient or carrier is incompatible with the peptide product, the present disclosure encompasses the use of conventional excipients and carriers in formulations comprising the peptide product. For example, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania) (2005); Handbook of Pharmaceutical Excipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); and Pharmaceutical Pre-formulation and Formulation, Gibson, Ed., CRC Press (Boca Raton, Florida) (2004).

[0125]

[0013] Suitable formulations may depend on various factors, such as the route of administration selected. Possible routes of administration of pharmaceutical compositions comprising peptide products include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intracavity, and topical), and topical (transdermal, oral mucosal, intranasal [e.g., via nasal spray or drops], ophthalmic [e.g., via eye drops], pulmonary [e.g., via oral or nasal inhalation], buccal, sublingual, rectal [e.g., via suppository], and vaginal [e.g., via suppository]. In certain embodiments, the peptide product is administered parenterally (e.g., subcutaneously, intravenously, or intramuscularly). In other embodiments, the peptide product is administered by oral or nasal inhalation, or by insufflation.

[0126] In some embodiments, the carrier is an aqueous-based carrier, such as in parenteral (e.g., subcutaneous, intravenous, intramuscular) formulations. In other embodiments, the carrier is a non-aqueous-based carrier. In certain embodiments, the non-aqueous-based carrier is a hydrofluoroalkane (HFA) or HFA-like solvent, which may contain submicron anhydrous α-lactose and / or other excipients, such as in formulations for administration by oral or nasal inhalation or insufflation.

[0127] In some embodiments, peptide products are administered parenterally by injection (e.g., subcutaneously, intravenously, or intramuscularly), which avoids the strongly acidic environment of the stomach, gastrointestinal (GI) absorption, and first-pass metabolism. Excipients and carriers that can be used to prepare parenteral formulations include, but are not limited to, solvents (e.g., aqueous solvents such as water, saline, physiological salt solution, buffered saline [e.g., PBS], balanced salt solutions [e.g., Ringer's BSS], and aqueous dextrose), isotonic / iso-osmotic agents (e.g., salts [e.g., NaCl, KCl, and CaCl2] and sugars [e.g., sucrose]), buffers, and pH adjusters (e.g., sodium phosphate dibasic [monobasic sodium phosphate] / disodium phosphate dibasic [dibasic sodium phosphate], citric acid / sodium citrate, and L-histidine / L-histidine HCl), and emulsifiers (e.g., non-ionic surfactants such as polysorbates [e.g., polysorbate 20 and 80] and poloxamers [e.g., poloxamer 188]). Peptide formulations and delivery systems are discussed, for example, in AJ Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd Ed., CRC Press (Boca Raton, Florida) (2015).

[0128] The excipient may optionally include one or more substances that increase peptide stability, increase peptide solubility, inhibit peptide aggregation, reduce solution viscosity, or any or all combinations thereof, including, but not limited to, hydrophilic amino acids (e.g., arginine and histidine), polyols (e.g., myo-inositol, mannitol, and sorbitol), sugars {e.g., glucose (including D-glucose [dextrose]), lactose, sucrose, and trehalose}, osmolytes (e.g., trehalose, taurine, amino acids [e.g., glycine, sarcosine, alanine, proline, serine, β-alanine, and γ-aminobutyric acid], and betaines [e.g., trimethylglycine and trimethylamine]). N-oxides]), and non-ionic surfactants (e.g., alkyl polyglycosides, ProTek® alkyl saccharides (e.g., monosaccharides [e.g., glucose] or disaccharides [e.g., maltose or sucrose] conjugated to long-chain fatty acids or corresponding long-chain alcohols), and polypropylene glycol / polyethylene glycol block copolymers (e.g., poloxamers [e.g., Pluronic® F-68] and Genapol® PF-10, and their variants)). Such substances increase peptide solubility and can be used to increase peptide concentration in the formulation. Higher peptide concentrations in the formulation are particularly effective for subcutaneous administration, where bolus volumes are limited (e.g., approximately 1.5 mL or greater). In addition, such substances can be used to stabilize peptides during preparation, storage, and reconstitution of lyophilized peptides.

[0129] Examples of excipients for aqueous formulations, whether pre-manufactured with a peptide therapeutic for parenteral (e.g., subcutaneous or intravenous) administration or prepared after reconstitution of a peptide therapeutic in lyophilized or powder form, include, but are not limited to, those shown in Table 5. Any formulation in Table 5 in which sodium chloride (NaCl) is not explicitly listed may optionally include NaCl. A typical parenteral formulation includes the peptide product, mannitol, methionine, sodium thioglycolate, polysorbate 20, a pH adjuster (e.g., NaOH or / and HCl), and deionized water.

[0130] [Table 1-1]

[0131] [Table 1-2]

[0132] For parenteral (e.g., subcutaneous, intravenous, or intramuscular) administration, a sterile solution or suspension of the peptide product in an aqueous solvent containing one or more excipients can be prepared in advance and may be provided, for example, in a pre-filled syringe of a disposable pen or a pen with a dose counter. Alternatively, the peptide product may be dissolved or suspended in an aqueous solvent, optionally containing one or more excipients, before lyophilization (freeze-drying). Just prior to parenteral administration, the lyophilized peptide product stored in a suitable container (e.g., a vial) can be reconstituted, for example, with sterile water, optionally containing one or more excipients.

[0133] In other embodiments, the peptide product is administered intranasally. The nasal mucosa offers a large surface area, a highly porous endothelium, a highly vascular epithelial layer, and a high absorption rate, thus allowing for a high level of bioavailability. Intranasal formulations may contain the peptide product along with excipients such as a solubility enhancer (e.g., propylene glycol), a humectant (e.g., mannitol or sorbitol), a buffer and water, and optionally a preservative (e.g., benzalkonium chloride), a mucoadhesive (e.g., hydroxyethylcellulose), or / and a penetration enhancer. Intranasal solution or suspension formulations can be administered to the nasal cavity by any suitable means, including, but not limited to, a dropper, pipette, or spray, for example, using a metered atomizing spray pump. Table 6 lists typical excipients for nasal spray formulations.

[0134] [Table 2]

[0135] In a further embodiment, the peptide product is administered via the lungs, such as by oral or nasal inhalation. Pulmonarily administered drugs can treat lung diseases or / and systemic disorders because the lungs serve as a gateway to the systemic circulation. Advantages of pulmonary drug delivery include, for example: 1) avoidance of first-pass metabolism; 2) rapid drug action; 3) a large surface area in the alveolar region for absorption, high lung permeability (thin air-blood barrier), and ample vasculature in the airways; and 4) lower extracellular enzyme levels compared to the GI tract due to the large alveolar surface area. Advantages of oral administration over nasal inhalation include deeper penetration / deposition of drugs in the lungs, while nasal inhalation can deliver drugs via the oral mucosa into the systemic circulation within the nasal cavity and lungs.

[0136] Oral or nasal inhalation can be achieved, for example, by a metered dose inhaler (MDI), nebulizer, or dry powder inhaler (DPI). For example, peptide products may be formulated for aerosol administration to the respiratory system via oral or nasal inhalation. Drugs may be delivered in small particle sizes (e.g., about 0.5 microns to about 5 microns), obtainable through micronization, to improve, for example, drug deposition in the lungs and drug suspension stability. Drugs may be provided in pressurized packs with a suitable propellant, such as hydrofluoroalkanes (HFAs, e.g., 1,1,1,2-tetrafluoroethane [HFA-134a]), chlorofluorocarbons (CFCs, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane), or a suitable gas (e.g., oxygen, compressed air, or carbon dioxide). In aerosol formulations, the drug is dissolved, or often suspended, in the propellant for pulmonary delivery. Although the aerosol contains excipients such as surfactants (which enhance pulmonary penetration by reducing the high surface tension at the air-water interface in the alveoli and can emulsify, solubilize, or / and stabilize the drug, and which may be, for example, a phospholipid such as lecithin) or / and stabilizers, the surfactant portion of the peptide product can perform the surfactant function. For example, an MDI formulation may contain a peptide product, a propellant (e.g., an HFA such as 1,1,1,2-tetrafluoroethane), and a cosolvent (e.g., an alcohol such as ethanol), and optionally, a surfactant (e.g., a fatty acid such as oleic acid). The MDI formulation may optionally contain a dissolved gas (e.g., CO2). After actuation of the device, the bursting of CO2 bubbles within the released aerosol droplets causes the droplets to break down into smaller droplets, thereby increasing the respirable fraction of the drug. As another example, a nebulizer formulation may include the peptide product, a chelating agent or preservative (e.g., edetate disodium), an isotonicity agent (e.g., NaCl), a pH buffer (e.g., citric acid / sodium citrate) and water, and optionally, a surfactant (e.g., Tween® such as polysorbate 80).Drugs can be delivered, for example, by a nebulizer or an MDI with or without a spacer, and the delivered drug dose can be controlled by a metering chamber (nebulizer) or a metering valve (MDI). Table 6 shows typical MDI, nebulizer, and DPI formulations.

[0137] Metered-dose inhalers (also called pressurized metered-dose inhalers [pMDIs]) are the most widely used inhalation devices. A metered valve delivers a precise amount of aerosol (e.g., approximately 20-100 μL) with each actuation of the device. MDIs typically generate aerosol faster than the user can inhale, which can result in significant aerosol deposition in the mouth and throat. The problem of poor coordination between device actuation and inhalation can be addressed by the use of breath-actuated MDIs or linked devices. Breath-actuated MDIs (e.g., Easibreathe®) are activated when the device senses the user's inhalation and expels a medication dose accordingly. The inhalation flow rate is regulated via an actuator, allowing the user time to reliably actuate the device during inhalation. In a linked device, the spacer (or valved holding chamber) is a tube attached to the mouthpiece end of the inhaler that acts as a reservoir or chamber to hold the drug being aerosolized by the inhaler and slows the rate at which the aerosol enters the mouth, allowing for evaporation of the propellant from larger droplets. The spacer simplifies the use of the inhaler and increases the amount of drug deposited in the lungs instead of the upper airways. The spacer may be made of an antistatic polymer to minimize electrostatic adhesion of released drug particles to the interior walls of the spacer.

[0138] Nebulizers generate aerosol droplets of approximately 1-5 microns. Nebulizers do not require user coordination between device actuation and inhalation, which can significantly affect the amount of drug deposited in the lungs. Compared to MDIs and DPIs, nebulizers can deliver larger amounts of drug despite longer administration times. Examples of nebulizers include, but are not limited to, manual nebulizers, jet nebulizers (e.g., AeroEclipse® II BAN [breath-actuated], CompAIR™ NE-C801 [virtual valve], PARI LC® Plus [breath-enhanced], and SideStream Plus [breath-enhanced]), ultrasonic nebulizers, and vibrating mesh nebulizers (e.g., Akita2® Apixneb, I-neb AAD System with metering chamber, MicroAir® NE-U22, Omron U22, and PARI eFlow® rapid). As an example, a pulsed ultrasonic nebulizer may include a photoacoustic trigger that can aerosolize a fixed amount of drug per pulse and allows the user to synchronize each breath to each pulse.

[0139] For oral or nasal inhalation using a powder inhaler (DPI), the peptide product can be provided in the form of a micronized dry powder, where the drug particles are of a small size (e.g., about 0.5 microns to about 5 microns) to improve, for example, the aerodynamic properties of the dispersed powder and drug deposition in the lungs. Particles of about 0.5 microns to about 5 microns are deposited by sedimentation in the terminal bronchioles and alveolar regions. In contrast, the majority of larger particles (>5 microns) do not follow airflow to the many branches of the respiratory tract and are deposited by impaction in the upper respiratory tract, including the oropharyngeal region of the throat. DPI formulations can contain drug particles alone or mixed with larger powders of a suitable base / carrier, such as lactose, starch, starch derivatives (e.g., hydroxypropylmethylcellulose), or polyvinylpyrrolidine. Carrier particles enhance flow, reduce agglomeration, improve dose uniformity, and aid in the dispersion of drug particles. DPI formulations may optionally contain excipients such as magnesium stearate and / or leucine, which improve formulation performance by preventing interparticle bonding (due to anti-adhesion effects). Powder formulations can be provided in unit-dose forms, such as capsules (e.g., gelatin capsules) or cartridges in blister packs, which can be manually or pre-filled into an inhaler. Drug particles can be drawn into the lungs by placing the inhaler mouthpiece or nosepiece over the mouth or nose, inhaling forcefully and deeply to create turbulent airflow, and maintaining the breath for a period of time (e.g., about 5-10 seconds), allowing the drug particles to settle in the bronchiolar and alveolar regions. When a user activates the DPI and inhales, airflow through the device creates shear and turbulence, directing the inhaled air into the powder bed, causing the electrostatic powder mixture to become fluid and enter the user's respiratory tract. There, the drug particles are separated from the carrier particles by the turbulent airflow and are transported deep into the lungs, while the larger carrier particles impact the oropharynx and are removed. Thus, the user's inspiratory flow achieves powder deagglomeration and air ionization, determining drug deposition in the lungs. (Passive DPIs require rapid inspiration to deagglomerate drug particles, whereas rapid inspiration is not recommended for MDIs or nebulizers.)Compared to MDIs, DPIs (including breath-actuated DPIs) can deliver larger doses of drugs and larger size drugs (e.g., macromolecules) to the lungs because they create turbulence and high velocities that increase drug deposition due to impact on the upper airway.

[0140] Lactose (e.g., alpha-lactose monohydrate) is the most commonly used carrier in DPI formulations. Examples of lactose monohydrate grades / types for DPI formulations include, but are not limited to, DCL 11, Flowlac® 100, Inhalac® 230, Lactohale® 300, Lactopress® SD 250 (spray-dried lactose), Respitose® SV003, and Sorbolac® 400. DPI formulations may contain a single lactose grade or a combination of different lactose grades. For example, fine lactose grades such as Lactohale® 300 or Sorbolac® 400 may not be suitable DPI carriers and may need to be mixed with coarse lactose such as DCL 11, Flowlac® 100, Inhalac® 230, or Respitose® SV003 to improve flow (e.g., approximately a 1:9 ratio of fine to coarse lactose). Tables 7 and 8 provide non-limiting examples of lactose grades / types that can be used in DPI formulations. The carrier particle size distribution influences the fine particle fraction / dose (FPF or FPD) of the drug, with a high FPF being desirable for drug delivery to the lungs. The FPF / FPD is the respirable fraction / dose mass from a DPI device with an aerodynamic particle diameter of ≤5 microns in the inspired air. High FPF, and therefore good DPI performance, can be obtained from a DPI formulation comprising, for example, fine lactose (e.g., Lactohale® 300) and coarse lactose (e.g., Respitose® SV003) in a ratio of about 1:9 and with an overage of about 20% w / w, which avoids drug deposition in the capsule shell or DPI device and allows nearly all of the drug to be delivered to the airways.

[0141] [Table 3]

[0142] [Table 4]

[0143] Other carriers for DPI formulations include, but are not limited to, glucose, mannitol (e.g., crystallized mannitol [Pearlitol 110C] and spray-dried mannitol [Pearlitol 100 SD]), maltitol (e.g., crystallized maltitol [Maltisorb P90]), sorbitol, and xylitol.

[0144] Most DPIs are breath-actuated ("passive"), relying on the user's inhalation to generate the aerosol. Examples of passive DPIs include, but are not limited to, Airmax®, Novolizer®, and Otsuka DPI (compact cake). Air classifier technology (ACT) is an efficient passive powder dispersion mechanism utilized in DPIs. In ACT, multiple feed channels generate tangential airflow, which creates a cyclone within the device during inhalation. There are also power-assisted ("active") DPIs (e.g., based on aerodynamics, impact force, or vibration) that use energy to assist in, for example, particle deagglomeration. For example, the active mechanism of the Exubera® inhaler utilizes mechanical energy stored in a spring or compressed air chamber. Examples of active DPIs include, but are not limited to, Actispire® (single unit dose), Aspirair® (multiple doses), Exubera® (single unit dose), MicroDose® (multiple unit doses and electronically activated), Omnihaler® (single unit dose), Pfeiffer DPI (single unit dose), and Spiros® (multiple unit doses).

[0145] The peptide product can also be administered by other routes, such as orally. Oral formulations may contain the peptide product, conventional excipients known in the art, and optionally, an absorption enhancer such as sodium N-[8-(2-hydroxybenzoyl)aminocaprylate] (SNAC). SNAC protects against enzymatic degradation through local buffering and enhances GI absorption. Oral dosage forms (e.g., tablets, capsules, or pills) can optionally be enteric coated to protect their contents from the strong acidity and proteolytic enzymes of the stomach.

[0146] In some embodiments, the peptide product is delivered from a sustained release composition. As used herein, the term "sustained release composition" includes sustained-release, extended-release, extended-release, delayed-release, slow-release, and controlled-release compositions, systems, and devices. In some embodiments, the sustained release composition delivers the peptide product for a period of at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or more.

[0147] In some embodiments, the sustained release composition is formulated as nanoparticles or microparticles comprised of a biodegradable polymer and incorporating the peptide product. In certain embodiments, the biodegradable polymer comprises lactic acid and / or glycolic acid (e.g., an L-lactic acid-based copolymer, such as poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid)).

[0148] In a further embodiment, the sustained release product is in the form of a depot that is generated when a mixture of the peptide product and a polymer is injected intramuscularly or subcutaneously into a subject, hi certain embodiments, the polymer is or comprises PEG, polylactic acid (PLA), polyglycolic acid (PGA), or copolymers thereof (e.g., PLGA or PLA-PEG).

[0149] Pharmaceutical compositions can be provided in unit dosage forms as single doses, where all active and inactive ingredients are combined in a suitable system, and the components do not necessarily need to be mixed to form an administered composition. Unit dosage forms generally contain a therapeutically effective dose of a drug, but can contain an appropriate fraction thereof so that a therapeutically effective dose can be achieved by providing multiple unit dosage forms. Examples of unit dosage forms include tablets, capsules, or pills for oral ingestion; solutions in pre-filled syringes of disposable pens or pens with dose counters for parenteral (e.g., intravenous, subcutaneous, intramuscular) injection; and capsules, cartridges, or blisters that are pre-filled or manually filled into inhalers.

[0150] Alternatively, the pharmaceutical composition can be provided as a kit, in which the active ingredient, excipients, and carriers (e.g., solvents) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles, or syringes) and must be combined to form the composition to be administered. The kit may include instructions for storing, preparing, and administering the composition (e.g., a solution to be parenterally injected).

[0151] The kit can contain the active and inactive ingredients all in unit dosage form, or in two or more separate containers, and can include instructions for administering or using the pharmaceutical composition to treat a medical condition disclosed herein. The kit may further include a device for delivering the composition, such as an injection pen or inhaler.

[0152] In some embodiments, the kit comprises a peptide product or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, and instructions for administering or using the peptide product or composition to treat a medical condition disclosed herein, such as insulin resistance, diabetes, obesity, metabolic syndrome, or cardiovascular disease, or a condition related thereto (e.g., NASH or PCOS). In certain embodiments, the kit further comprises a device for delivering the peptide product or composition, such as an injection pen or inhaler.

[0153] Therapeutic uses of peptide products The present disclosure further provides uses of the peptide products described herein for treating, for example, insulin resistance, diabetes, obesity, metabolic syndrome, and cardiovascular disease, and diseases related thereto, such as NASH and PCOS.

[0154] In some embodiments, the peptide products are used to treat hyperglycemia, insulin resistance, hyperinsulinemia, prediabetes, diabetes (including type 1 and type 2, fetal and juvenile diabetes), diabetic complications, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, elevated blood levels of free fatty acids, obesity, metabolic syndrome, syndrome X, cardiovascular disease (including coronary artery disease), atherosclerosis, acute cardiovascular syndrome, ischemia (including cardiac ischemia and cerebral ischemia / stroke), ischemic reperfusion injury (including myocardial and cerebral IRI), infarction (including myocardial infarction and cerebral infarction), angina pectoris, heart failure (e.g., congestive heart failure), peripheral vascular disease, thrombosis (e.g., deep vein thrombosis), embolism (e.g., pulmonary embolism), systemic inflammation (e.g., characterized by elevated blood levels of C-reactive protein), and hypertension. Peptide products can achieve therapeutic effects through various mechanisms, including stimulation of blood glucose-dependent insulin secretion, increasing insulin sensitivity, stimulating fat burning, and weight loss. Peptide products can also promote, for example, pancreatic beta cell protection, cardioprotection, and wound healing.

[0155] The peptide products described herein can be used to treat other diseases associated with insulin resistance and / or obesity, including, but not limited to, arthritis (e.g., osteoarthritis), back pain, respiratory disorders (e.g., asthma, obesity hypoventilation syndrome [Pickwickian syndrome], and obstructive sleep apnea), dermatological disorders (e.g., diabetic ulcers, acanthosis nigricans, cellulitis, hirsutism, intertrigo, and lymphedema), gastroenterological disorders (e.g., cholelithiasis [gallstones], gastroesophageal reflux disease [GERD], and gastroparesis), gout, hypercortisolism (e.g., Cushing's syndrome), renal disorders (e.g., chronic kidney disease), and liver disorders (e.g., alcoholism). These include fatty liver disease (FLD), including alcoholic and non-alcoholic FLD, neurological disorders (e.g., carpal tunnel syndrome, dementia [e.g., Alzheimer's disease and vascular dementia], paresthesia, migraine, and multiple sclerosis), urinary tract disorders (e.g., erectile dysfunction, hypogonadism, and urinary incontinence), polycystic ovary syndrome, infertility, menstrual irregularities, mood disorders (e.g., depression), and cancer (e.g., endometrial cancer, esophageal cancer, colorectal cancer, gallbladder cancer, kidney cancer, liver cancer [e.g., hepatocellular carcinoma], pancreatic cancer, and skin cancer [e.g., melanoma], and leukemia).

[0156] In some embodiments, the peptide products described herein are used to treat polycystic ovary syndrome (PCOS). In other embodiments, the peptide products are used to treat chronic kidney disease (CKD), also known as chronic kidney / renal failure (CKF / CRF). The most common causes of CKD are diabetes and long-term uncontrolled hypertension.

[0157] In further embodiments, the peptide products described herein are used to treat fatty liver disease (FLD). In some embodiments, the FLD is nonalcoholic fatty liver disease (NAFLD). In certain embodiments, the NAFLD is nonalcoholic steatohepatitis (NASH). FLD, also known as fatty liver, is characterized by excessive fat accumulation in the liver. FLD includes alcoholic fatty liver disease (AFLD) and NAFLD. Chronic alcoholism causes fatty liver due to the production of toxic metabolites, such as aldehydes, during alcohol metabolism in the liver. NAFLD is described below. FLD is associated with diabetes, obesity, and metabolic syndrome. Fatty liver can progress to cirrhosis or liver cancer (e.g., hepatocellular carcinoma [HCC]). Fewer than 10% of people with cirrhotic AFLD will develop HCC, while up to 45% of people with NASH without cirrhosis will develop HCC. HCC is the most common type of primary liver cancer in adults and arises in conditions of chronic hepatitis.

[0158] NAFLD is characterized by fatty liver, which occurs when fat, particularly free fatty acids and triglycerides, accumulate in hepatocytes (fatty liver) due to causes other than excessive alcohol consumption, such as nutrient overload, high calorie intake, and metabolic dysfunction (e.g., dyslipidemia and impaired glucose control). While the liver may remain fat-laden without interfering with liver function, fatty liver may progress to NASH, a condition characterized by steatosis accompanied by inflammation, hepatocellular ballooning, and cellular damage with or without liver fibrosis. Fibrosis is the strongest predictor of death from NASH. NAFLD is characterized by steatosis alone; steatosis with lobular or portal venous inflammation but without ballooning; steatosis with ballooning but without inflammation; or steatosis with inflammation and ballooning.

[0159] NASH is the most extreme form of NAFLD. NASH is a progressive disease; approximately 20% of patients will develop cirrhosis, and approximately 10% will die from liver disease, such as cirrhosis or liver cancer (e.g., HCC). NAFLD is the most common liver disorder in developed countries, and NASH is expected to replace hepatitis C as the leading cause of liver transplants in the United States by 2020. Approximately 12-25% of people in the United States have NAFLD, and NASH affects approximately 2-5% of people in the United States.

[0160] NAFLD, including NASH, is associated with insulin resistance, obesity and metabolic syndrome.For example, insulin resistance contributes to the progression of fatty liver to liver inflammation and fibrosis, and thus to NASH.In addition, obesity induces and aggravates NASH, and weight loss can alleviate NASH.Therefore, the peptide products described herein, including GLP-1 receptor (GLP1R) agonist, glucagon receptor (GCGR) agonist and dual GLP1R / GCGR agonist, can be used to treat NAFLD, including NASH.

[0161] In some embodiments, the peptide products used to treat diseases associated with insulin resistance and / or obesity disclosed herein, such as NAFLD (e.g., NASH) or PCOS, are selected from peptide products of SEQ.ID.NO.601, 602, 603, 604, 630, 631, 632, 633, 634, 805, 819, 820, 821, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, and pharmaceutically acceptable salts thereof.

[0162] The peptide product can be administered by any route suitable for treating the diseases disclosed herein. Possible routes of administration of the peptide product include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intracavity, and topical), and topical (transdermal, oral intramucosal, intranasal [e.g., via nasal spray or drops], ophthalmic [e.g., via eye drops], pulmonary [e.g., via oral or nasal inhalation], buccal, sublingual, rectal [e.g., via suppository], and vaginal [e.g., via suppository]. In some embodiments, the peptide product is administered parenterally, such as subcutaneously, intravenously, or intramuscularly. In other embodiments, the peptide product is administered by oral or nasal inhalation, or by insufflation.

[0163] The therapeutically effective amount of a peptide product for treating a disease disclosed herein, the frequency of administration, and the length of treatment therewith may depend on a variety of factors, including the nature and severity of the disease, the potency of the compound, the route of administration, the age, weight, health, sex, and diet of the subject, and the subject's response to treatment, and can be determined by the treating physician.

[0164] In some embodiments, the peptide product is administered parenterally (e.g., subcutaneously [sc], intravenously [iv], or intramuscularly [im]) at a dosage of about 0.1 mg to about 1, 5, 10 mg, or about 0.1-1 mg or 1-10 mg for a period of about one week for the treatment of a disease disclosed herein (e.g., one associated with insulin resistance and / or obesity, such as NASH or PCOS). In further embodiments, the peptide product is administered parenterally (e.g., sc, iv, or im) at a dosage of about 0.1-0.5 mg, 0.5-1 mg, 1-5 mg, or 5-10 mg for a period of about one week. In certain embodiments, the peptide product is administered parenterally (e.g., sc, iv, im) at a dosage of about 0.1-1 mg, 0.1-0.5 mg, or 0.5-1 mg for a period of about one week.

[0165] The peptide product can be administered at a frequency suitable for treating a disease disclosed herein (e.g., one associated with insulin resistance and / or obesity, such as NASH or PCOS). In some embodiments, the peptide product is administered once daily, once every two days, once every three days, twice a week, once a week, or once every two weeks, e.g., sc or iv. In certain embodiments, the peptide product is administered once a week, e.g., sc or iv.

[0166] The peptide product can be administered at any time convenient for the patient. The peptide product can be taken substantially with food (e.g., with a meal or within about 1 hour or 30 minutes before or after a meal), or substantially without food (e.g., at least about 1 hour or 2 hours before or after a meal).

[0167] The length of treatment for a medical condition with a peptide product can be determined by the treating physician based on, for example, the nature and severity of the condition and the subject's response to treatment. In some embodiments, the peptide product is administered to treat a condition disclosed herein chronically, such as for at least about 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 5 years, 10 years, or more. The peptide product can also be taken ad libitum (as needed) until clinical symptoms of the condition disappear or a clinical target, such as blood glucose level, blood pressure, blood lipid level, body weight or body mass index, waist-to-hip ratio, body fat percentage, or any combination thereof, is achieved. If clinical symptoms of the condition reappear or clinical targets are not maintained, administration of the peptide product can be resumed.

[0168] The present disclosure provides a method for treating a medical disease disclosed herein, the method comprising administering to a subject a therapeutically effective amount of a peptide product described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. The present disclosure further provides a peptide product described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use as a medicament. In addition, the present disclosure provides the use of a peptide product described herein, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament. A medicament comprising the peptide product can be used to treat any medical disease described herein. The peptide product can optionally be used in combination with one or more additional therapeutic agents.

[0169] Combination therapy The peptide products described herein can optionally be used in combination with one or more additional therapeutic agents to treat any of the disorders disclosed herein, such as insulin resistance, diabetes, obesity, metabolic syndrome or cardiovascular disease, or any disease related thereto, NASH or PCOS, etc. In some embodiments, the one or more additional therapeutic agents are selected from diabetes agents, anti-obesity agents (including lipid-lowering agents and pro-satiety agents), anti-atherosclerotic agents, anti-inflammatory agents, antioxidants, anti-fibrotic agents, anti-hypertensive agents, and combinations thereof.

[0170] Diabetes medications include, but are not limited to: AMP-activated protein kinase (AMPK) agonists, including biguanides (e.g., buformin and metformin); peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, including thiazolidinediones (e.g., balaglitazone, ciglitazone, daraglitazone, englitazone, lobeglitazone, netoglitazone, pioglitazone, rivoglitazone, rosiglitazone, and troglitazone) and saroglitazar (a dual PPAR-α / γ agonist); glucagon-like peptide-1 (GLP-1) receptor agonists, including exendin-4, albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, taspoglutide, CNTO736, CNTO3649, HM11260C (LAPS-Exendin), NN9926 (OG9S7GT), TT401, and ZY0G1; dipeptidyl peptidase 4 (DPP-4) inhibitors, including alogliptin, anagliptin, dutogliptin, evogliptin, gemigliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, septagliptin, sitagliptin, teneligliptin, trelagliptin, and vildagliptin; sodium-glucose transport protein 2 (SGLT2) inhibitors, including canagliflozin (which also inhibits SGLT1), dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, remogliflozin etabonate, sotagliflozin (which also inhibits SGLT1), and tofogliflozin; Meglitinides (e.g., mitiglinide, nateglinide, and repaglinide) and sulfonylureas {including first-generation (e.g., acetohexamide, carbutamide, chlorpropamide, glycirilamide [tolhexamide], metahexamide, tolazamide, and tolbutamide), and second-generation (e.g., glibenclamide [glyburide], glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, and glyclopyramide)} inhibit ATP-dependent K+ receptor agonists on pancreatic β cells + (K ATP ) channel blockers; Insulin and its analogs, including rapid-acting insulins (e.g., insulin aspart, insulin glulisine, and insulin slipro), intermediate-acting insulins (e.g., NPH insulin), and long-acting insulins (e.g., insulin degludec, insulin detemir, and insulin glargine); and their analogs, derivatives, and salts;

[0171] In certain embodiments, the diabetes drug is or includes a biguanide (e.g., metformin), a thiazolidinedione (e.g., pioglitazone or rosiglitazone), or an SGLT2 inhibitor (e.g., empagliflozin or tofogliflozin), or any combination thereof.

[0172] Anti-obesity medications include, but are not limited to: appetite suppressants (drugs that reduce appetite), including amphetamine, dextroamphetamine, amfepramone, clobenzorex, matindole, phentermine (with or without topiramate), and lorcaserin; satiety promoters, including ciliary neurotrophic factor (e.g., axoxoquin) and longer-acting analogs of amylin, calcitonin, cholecystokinin (CCK), GLP-1, leptin, oxyntomodulin, pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY); Lipase inhibitors, including caulerpinine, cetilistat, ebelactone A and B, estellastin, lipstatin, orlistat, persikinin, panclicin AE, valilactone, and vibralactone; antihyperlipidemic drugs; and their analogs, derivatives, and salts;

[0173] Antihyperlipidemic agents include, but are not limited to: HMG-CoA reductase inhibitors, including statins {(e.g., atorvastatin, cerivastatin, fluvastatin, mevastatin, monacolins (e.g., monacolin K [lovastatin]), pitavastatin, pravastatin, rosuvastatin, and simvastatin} and flavanones (e.g., naringenin); squalene synthase inhibitors, including lapaquistat, zaragozic acid, and RPR-107393; Anthocyanins, avenaciolide, chloroacetylated biotin, cyclodium, diclofop, haloxyfop, soraphen (e.g., soraphen A 1α), 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), CP-640186, GS-0976, NDI-010976; 7-(4-propyloxy-phenylethynyl)-3,3-dimethyl-3,4-dihydro-2H-benzo[b][1,4]dioxepin; N-ethyl-N'-(3-{[4-(3,3-dimethyl-1-oxo-2-oxa-7-azaspiro[4.5]dec-7-yl)piperidin-1-yl]-carbo acetyl-CoA carboxylase (ACC) inhibitors, including 1-(3-{[4-(3,3-dimethyl-1-oxo-2-oxa-7-azaspiro[4.5]dec-7-yl)piperidin-1-yl]-carbonyl}-5-(pyridin-2-yl)-2-thienyl)-3-ethylurea; 5-(3-acetamidobut-1-ynyl)-2-(4-propyloxyphenoxy)thiazole; and 1-(3-{[4-(3,3-dimethyl-1-oxo-2-oxa-7-azaspiro[4.5]dec-7-yl)piperidin-1-yl]-carbonyl}-5-(pyridin-2-yl)-2-thienyl)-3-ethylurea; PPAR-α agonists, including fibrates (e.g., bezafibrate, ciprofibrate, clinofibrate, clofibric acid, clofibrate, aluminum clofibrate [alfibrate], clofibrate, etofibrate, fenofibric acid, fenofibrate, gemfibrozil, lonifibrate, and simfibrate), isoflavones (e.g., daidzein and genistein), and perfluoroalkanoic acids (e.g., perfluorooctanoic acid and perfluorononanoic acid); PPAR-δ agonists, including elafibranor (dual PPAR-α / δ agonist), GFT505 (dual PPAR-α / δ agonist), GW0742, GW501516 (dual PPAR-β / δ agonist), soderglitazar (GW677954), MBX-8025, and isoflavones (e.g., daidzein and genistein); PPAR-γ agonists, including thiazolidinediones (described above), saroglitazar (a dual PPAR-α / γ agonist), 4-oxo-2-thioxothiazolinones (e.g., rhodanine), berberine, honokiol, perfluorononanoic acid, cyclopentenone prostaglandins (e.g., cyclopentenone 15-deoxy-Δ-prostaglandin J2 [15d-PGJ2]), and isoflavones (e.g., daidzein and genistein); liver X receptor (LXR) agonists, including endogenous ligands (e.g., oxysterols such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid) and synthetic agonists (e.g., acetylpodocarpic acid dimer, hypocholamide, N,N-dimethyl-3β-hydroxy-cholenamide [DMHCA], GW3965, and T0901317); retinoid X receptor (RXR) agonists, including endogenous ligands (e.g., 9-cis-retinoic acid) and synthetic agonists (e.g., bexarotene, AGN191659, AGN191701, AGN192849, BMS649, LG100268, LG100754, and LGD346); inhibitors of acyl-CoA cholesterol acyltransferase (ACAT, aka sterol O-acyltransferase [SOAT], including ACAT1 [SOAT1] and ACAT2 [SOAT2]), including avasimide, pactimibe, pellitorin, terpendole C, and flavanones (e.g., naringenin); Aramchol, CAY-10566, CVT-11127, SAR-224, SAR-707, XEN-103; 3-(2-hydroxyethoxy)-4-methoxy-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide and 4-ethylamino-3-(2-hydroxyethoxy)-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide; 1'-{6-[5-(pyridin-3-ylmethyl )-1,3,4-Oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,4'-piperidine];5-Fluoro-1'-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-3,4-dihydrospiro[chromene-2,4'-piperidine];6-[5-(cyclopropylmethyl)-4,5-dihydro-1'H,3H- Spiro[1,5-benzoxazepine-2,4'-piperidin]-1'-yl]-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-carboxamide; 6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine-3-carboxylic acid (2-hydroxy-2-pyridin-3-ylethyl)amide; 4-(2-chlorophenoxy)-N-[3-(methylcarbamoyl)phenyl]piperidine-1-carboxamide; Conjugated linoleic acid inhibitors of stearoyl-CoA desaturase-1 (SCD-1, also known as stearoyl-CoA delta-9 desaturase) activity or expression, including the cis-9, trans-11 isomer and the trans-10, cis-12 isomer of carboxylic acid, substituted heteroaromatic compounds disclosed in WO2009 / 129625A1, antisense polynucleotides and peptide nucleic acids (PNAs) targeting mRNA for SCD-1, and SCD-1-targeting siRNA; cholesteryl ester transport protein (CETP) inhibitors, including anacetrapib, dalcetrapib, evacetrapib, torcetrapib, and AMG 899 (TA-8995); inhibitors of microsomal triglyceride transfer protein (MTTP) activity or expression, including implitapide, lomitapide, dirlotapide, mitratapide, CP-346086, JTT-130, SLx-4090, antisense polynucleotides and PNAs targeting mRNA for MTTP, MTTP-targeting microRNAs (e.g., miRNA-30c), and MTTP-targeting siRNAs; GLP-1 receptor agonists (mentioned above); fibroblast growth factor 21 (FGF21) and its analogs and derivatives, including BMS-986036 (pegylated FGF21); inhibitors of proprotein convertase subtilisin / kexin type 9 (PCSK9) activity or expression, including berberine (which reduces PCSK9 levels), annexin A2 (which inhibits PCSK9 activity), anti-PCSK9 antibodies (e.g., alirocumab, bococizumab, evolocumab, LGT-209, LY3015014, and RG7652), peptides that mimic the epidermal growth factor-A (EGF-A) domain of the LDL receptor that binds to PCSK9, PCSK9-binding adnectins (e.g., BMS-962476), antisense polynucleotides and PNAs that target mRNA for PCSK9, and PCSK9-targeting siRNA (e.g., inclisiran [ALN-PCS] and ALN-PCS02); apoA-I mimetics (e.g. 2F, 3F, 3F-1, 3F-2, 3F-14, 4F, 4F-P-4F, 4F-IHS-4F, 4F2, 5F, 6F, 7F, 18F, 5A, 5A-C1, 5A-CH1, 5A-CH2, 5A-H1, 18A, 37pA[1 8AP-18A], ELK[Name], ELK-1A, ELK-1F, ELK-1K1A1E, ELK-1L1K, ELK-1W, ELK-2A, ELK-2A2K2E, ELK-2E2K, ELK-2F, ELK-3E3EK, ELK-3E3K3A , ELK-3E3LK, ELK-PA, ELK-P2A, ELKA[name], ELKA-CH2, ATI-5261, CS-6253, ETC-642, FAMP[name], FREL[name], and KRES[name]), and apolipoprotein mimetic peptides, including apoE mimetics (e.g., Ac-hE18A-NH2[AEM-28], Ac-[R]hE18A-NH2, AEM-28-14, EpK, hEp, mR18L, COG-112, COG-133, and COG-1410); Omega-3 fatty acids, including docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA), fish oils (e.g., containing DHA and EPA), and their esters (e.g., glyceryl and ethyl esters); and their analogs, derivatives, and salts;

[0174] In certain embodiments, the antiobesity agent is or includes a lipase inhibitor (eg, orlistat) or / and an antihyperlipidemic agent (eg, a statin, such as atorvastatin, or / and a fibrate, such as fenofibrate).

[0175] Antihypertensive drugs include, but are not limited to: antagonists of the renin-angiotensin-aldosterone system (RAAS), including renin inhibitors (e.g., aliskiren), angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril), angiotensin II receptor type 1 (AT1) antagonists (e.g., azilsartan, candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan medoxomil, olmesartan, telmisartan, and valsartan), and aldosterone receptor antagonists (e.g., eplerenone and spironolactone); Diuretics, including loop diuretics (e.g., bumetanide, ethacrynic acid, furosemide, and torsemide), thiazide diuretics (e.g., bendroflumethiazide, chlorothiazide, hydrochlorothiazide, epitizide, methyclothiazide, and polythiazide), thiazide-like diuretics (e.g., chlorthalidone, indapamide, and metolazone), cicletanine (an early distal tubular diuretic), potassium-sparing diuretics (e.g., amiloride, eplerenone, spironolactone, and triamterene), and theobromine; calcium channel blockers, including dihydropyridines (e.g., amlodipine, levamlodipine, cilnidipine, clevidipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, and nitrendipine), and non-dihydropyridines (e.g., diltiazem and verapamil); alpha2-adrenergic receptor agonists, including clonidine, guanabenz, guanfacine, methyldopa, and moxonidine; alpha1-adrenergic receptor antagonists (alpha-blockers), including doxazosin, indoramin, nicergoline, phenoxybenzamine, phentolamine, prazosin, terazosin, and tolazoline; beta-adrenergic receptor (beta1 or / and beta2) antagonists (beta-blockers), including atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, and timolol; combined alpha / beta blockers, including bucindolol, carvedilol, and labetalol; endothelin receptor antagonists, including selective ETA receptor antagonists (e.g., ambrisentan, atrasentan, edonentan, sitaxsentan, zibotentan, and BQ-123) and dual ETA / ETB antagonists (e.g., bosentan, macitentan, and tezosentan); Hydralazine, minoxidil, theobromine, sodium nitroprusside, organic nitrates (e.g., isosorbide mononitrate, isosorbide dinitrate, and nitroglycerin, which are converted to nitric oxide in the body), endothelial nitric oxide synthase (eNOS) stimulators (e.g., cicletanine), activators of soluble guanylate cyclase (e.g., cinaciguat and riociguat), phosphodiesterase type 5 (PDE5) inhibitors (e.g., avanafil, benzamidonam), fil, dasantafil, dynafil, lodenafil, mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, dipyridamole, papaverine, propentofylline, zaprinast, and T-1032), prostaglandin E1 (alprostadil) and its analogs (e.g., limaprost and misoprostol), prostacyclin and its analogs (e.g., ataprost, beraprost [e.g., esveraprost]) , 5,6,7-triol-4,8-inter-m-phenylene-9-fluoro-PGI2, carbacyclin, isocarbacyclin, clinprost, ciprostene, eptaloprost, cicaprost, iloprost, pimilprost, SM-10906 (des-methylpimilprost), naxaprostene, taprosten, treprostinil, CS-570, OP-2507, and TY-11223), and non-prostanoid prostacyclin receptors other vasodilators, including agonists (e.g., 1-phthalazinol, larinepag, selexipag, ACT-333679 [MRE-269, the active metabolite of selexipag], and TRA-418), phospholipase C (PLC) inhibitors, and protein kinase C (PKC) inhibitors (e.g., BIM-1, BIM-2, BIM-3, BIM-8, chelerythrine, cicletanine, gossypol, myabenol C, myricitrin, rubaxistaurin, and verbascoside); minerals, including magnesium and magnesium sulfate; and their analogs, derivatives, and salts;

[0176] In certain embodiments, the antihypertensive agent is or includes a thiazide or thiazide-like diuretic (e.g., hydrochlorothiazide or chlorthalidone), a calcium channel blocker (e.g., amlodipine or nifedipine), an ACE inhibitor (e.g., benazepril, captopril, or perindopril), or an angiotensin II receptor antagonist (e.g., olmesartan medoxomil, olmesartan, telmisartan, or valsartan), or any combination thereof.

[0177] In some embodiments, the peptide products described herein are used in combination with one or more additional therapeutic agents to treat NAFLD, such as NASH. In some embodiments, the one or more additional therapeutic agents are selected from diabetes drugs, anti-obesity drugs, anti-inflammatory drugs, anti-fibrotic drugs, antioxidants, antihypertensive drugs, and combinations thereof.

[0178] Therapeutic agents that can be used to treat NAFLD (e.g., NASH) include, but are not limited to: PPAR agonists (PPAR-δ and -γ agonism increases insulin sensitivity, PPAR-α agonism reduces hepatic steatosis, and PPAR-δ agonism inhibits macrophage and Kupffer cell activation), including PPAR-δ agonists (e.g., MBX-8025, elafibranor [dual PPAR-α / δ agonist], and GW501516 [dual PPAR-β / δ agonist]) and PPAR-γ agonists (e.g., thiazolidinediones such as pioglitazone and saroglitazar [dual PPAR-α / γ agonist]); farnesoid X receptor (FXR) agonists, such as obeticholic acid and GS-9674 (FXR agonists reduce hepatic gluconeogenesis, lipogenesis, adiposity, and fibrosis); fibroblast growth factor 19 (FGF19), its analogs and derivatives, such as NGM-282 (FGF19 analogs reduce hepatic gluconeogenesis and adiposity); Fibroblast growth factor 21 (FGF21), its analogs and derivatives, such as BMS-986036 (pegylated FGF21) (FGF21 reduces hepatic steatosis, cellular injury, and fibrosis); HMG-CoA reductase inhibitors, including statins (e.g., rosuvastatin) (statins reduce steatohepatitis and fibrosis); ACC inhibitors such as NDI-010976 (liver targeted) and GS-0976 (ACC inhibitors reduce de novo lipogenesis, and hepatic steatosis); SCD-1 inhibitors such as aramchol (SCD-1 inhibitors reduce liver steatosis and increase insulin sensitivity); SGLT2 inhibitors such as canagliflozin, ipragliflozin, and luseogliflozin (SGLT2 inhibitors reduce body weight, hepatic ALT levels, and fibrosis); CCR2 or / and CCR5 antagonists, such as cenicriviroc (CCR2 (binding to CCL2 [MCP1]) and CCR5 (binding to CCL5 [RANTES]) inhibit the activation and migration of inflammatory cells (e.g., macrophages) into the liver, reducing liver fibrosis); apoptosis inhibitors (apoptosis inhibitors reduce hepatic steatosis and fibrosis), including apoptosis signal-regulating kinase 1 (ASK1) inhibitors (e.g., selonsertib) and caspase inhibitors (e.g., emricasan [pan-caspase inhibitor]); lysyl oxidase-like 2 (LOXL2) inhibitors, such as simtuzumab (LOXL2 is a key matrix enzyme in collagen formation and is highly expressed in the liver); galectin-3 inhibitors such as GR-MD-02 and TD139 (galectin-3 inhibitors are important in the progression of liver fibrosis); Antioxidants, including vitamin E (e.g., α-tocopherol) and scavengers of reactive oxygen species (ROS) and free radicals (e.g., cysteamine, glutathione, melatonin, and pentoxifylline [also anti-inflammatory via inhibition of TNF-α and phosphodiesterase]) (vitamin E reduces hepatic steatosis, hepatocyte ballooning, and lobular inflammation); and their analogs, derivatives, and salts;

[0179] In some embodiments, the peptide products described herein are used in combination with a PPAR agonist (e.g., a PPAR-δ agonist such as elafibranor, or / and a PPAR-γ agonist such as pioglitazone), an HMG-CoA reductase inhibitor (e.g., a statin such as rosuvastatin), an FXR agonist (e.g., obeticholic acid), or an antioxidant (e.g., vitamin E), or any combination thereof, to treat NAFLD (e.g., NASH). In certain embodiments, the one or more additional therapeutic agents for the treatment of NAFLD (e.g., NASH) are or include vitamin E and / or pioglitazone.

[0180] Preparation of peptide products WO2015 / 184177A1 describes the synthesis of peptide products. The Examples below also describe the synthesis of representative peptide products. [Example]

[0181] The following examples are intended only to illustrate the present disclosure. Other processes, assays, tests, protocols, procedures, methods, reagents, and conditions may alternatively be used, if desired.

[0182] Example 1. Reagents such as N-α-Fmoc and N-ε-(1-octyl β-D-glucuronide-6-yl)L-lysine An oven-dried 250 mL Erlenmeyer flask was charged with 1-octyl β-D-glucuronic acid (3.06 g, 10 mmol, Carbosynth Ltd.), 50 mL of anhydrous DMF, and anhydrous 1-hydroxybenzotriazole (1.62 g, 12 mmol). A cooled (4 °C) solution of N,N'-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in 50 mL of DMF was added with stirring, and the reaction was allowed to proceed for 5 min. The large amount of white precipitate of N,N'-dicyclohexylurea was filtered on a fritted glass funnel, and the filtrate was added to a solution of N-α-Fmoc-L-lysine (3.68 g, 10 mmol) in 25 mL of anhydrous DMF. The reaction was allowed to warm to room temperature for 25 min, or until the color of the ninhydrin had faded significantly. The reaction mixture is filtered, stripped to dryness, and crystallized from MeOH / EtO by dissolution in MeOH and slow dilution with EtO to the cloud point, followed by refrigeration. Further purification can be achieved by silica gel chromatography using a solvent gradient from EtOAc to EtOAc / EtOH / AcOH.

[0183] A similar reaction using N-α-Boc-L-lysine gave N-α-Boc,N-ε-(1-octyl β-D-glucuronid-6-yl)-L-lysine, which is suitable for N-terminal incorporation and cleavage relative to the free N-terminus. A similar reaction using N-α-Ac-L-lysine gave N-α-Ac,N-ε-(1-octyl β-D-glucuronid-6-yl)-L-lysine, which is suitable for N-terminal incorporation into peptides with a blocked N-terminus. A similar reaction using an appropriate amount of N-α-Fmoc-L-ornithine gave N-α-Fmoc,N-δ-(1-octyl β-D-glucuronid-6-yl)-L-ornithine. Similar reactions using other N-mono-protected diamino acids gave the corresponding reagents. Alternatively, the use of a temporary Me3Si ester protecting group during coupling and without prior activation of 1-octyl β-D-glucuronic acid provides a facile route to the formation of the reagent. The temporary Me3Si ester is generated by reaction of Fmoc-Lys-OH with an equimolar amount of N,O-bis(trimethylsilyl)acetamide in dichloromethane (CHCl). The organic layer contains the desired reagent as a solution in CHCl, ready for coupling with the 1-alkyl glucuronide as described above. The filtered reaction mixture is washed with aqueous NaHSO to hydrolyze the Me3Si ester, dried over MgSO, and the solvent is removed.

[0184] The use of peracetyl or perbenzoyl 1-octyl β-D-glucuronic acid, although similar, affords Ac- or Bz-protected forms of the reagent (e.g., 2,3,4-trisacetyl 1-octyl β-D-glucuronic acid, formed by treatment with AcO). Such reagents offer increased stability during acidic cleavage from the resin and are used to detect instability during deprotection. Final deprotection of such products is carried out by base-catalyzed transesterification after cleavage using MeOH / NH, MeOH / NaOMe, or MeOH / NHNH, as described above.

[0185] Example 2. Synthetic peptides In general, peptide synthesis methods require the sequential addition of protected amino acids to a growing peptide chain. Typically, either the amino or carboxyl group of the first amino acid, as well as any reactive side groups, are protected. This protected amino acid is then attached to an inert solid support or utilized in solution, and the next amino acid in the sequence, also appropriately protected, is added under conditions applicable to amide bond formation. After all desired amino acids have been coupled in the appropriate sequence, the protecting groups and any solid support are removed to yield the crude peptide. The peptide is desalted and purified by chromatography.

[0186] One method for preparing peptides with fewer than about 50 amino acids involves solid-phase peptide synthesis. In this method, the α-amino (Nα) group and any reactive side chain functionalities are protected with acid- or base-sensitive groups. The protecting groups must be stable to the conditions of peptide bond formation while being easily removable without affecting the remaining peptide chain. Suitable α-amino protecting groups include, but are not limited to, t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc), isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenyl-methoxycarbonyl (Fmoc), and the like, preferably Boc or Fmoc. Suitable side chain protecting groups include, but are not limited to, acetyl, benzyl (Bzl or Bn), benzyloxymethyl (Bom), Boc, t-butyl, o-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trimethylsilyl, and trityl. The preferred Nα-protecting group for compound synthesis is Fmoc. Preferred side chain protecting groups include -Ot-butyl for Glu, Tyr, Thr, Asp, and Ser; Boc for the side chains of Lys and Trp; Pbf for Arg; and Trt for Asn, Gln, and His. For selective modification of Lys residues, orthogonal protection with protecting groups that are not removed by reagents that cleave Fmoc or t-butyl-based protecting groups is preferred.Preferred examples of Lys side chain modifications include, but are not limited to, those that are removed by hydrazine but not piperidine, such as 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), and allyloxycarbonyl (Alloc).

[0187] The Fmoc-Lys(ivDde) ​​or Fmoc-Lys(Dde) protecting group scheme is preferred when side-chain lactam formation is desired because Fmoc-Glu(Oallyl) and Fmoc-Lys(Alloc) can be incorporated and used to provide temporary protection, and then deprotected to form the lactam, while the Lys(Dde) protecting group remains for later removal and reaction with a functionalized surfactant. Side chain lactams between acidic and basic residues (e.g., Glu and Lys) are achieved after removal of the allyl-based protecting groups by activation of the carboxyl side chain functions with N,N'-diisopropylcarbodiimide (DIC) / 1-hydroxybenzotriazole (HOBt) or 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) / N,N-diisopropylethylamine (DIEA) using standard protocols in the art.

[0188] In solid-phase synthesis, the C-terminal amino acid is first attached to a suitable resin support. Suitable resin supports are materials that are inert to the reagents and reaction conditions of the stepwise condensation and deprotection reactions and insoluble in the media used. Examples of commercially available resins include styrene / divinylbenzene resins modified with reactive groups, such as chloromethylated co-poly(styrene-divinylbenzene) and hydroxymethylated co-poly(styrene-divinylbenzene). Benzylated, hydroxymethylated, phenylacetamidomethyl (PAM) resin, and hydroxymethylphenoxyacetylamidomethyl (HMPA) are preferred for the preparation of peptide C-terminal acids. When the C-terminus of the compound is an amide, preferred resins include p-methylbenzhydrylamino-co-poly(styrene-divinylbenzene) resin and 2,4-dimethoxybenzhydrylamino-based resin ("Rink amide"). Preferred supports for the synthesis of larger peptides are commercially available resins containing PEG sequences grafted onto other polymer matrices, such as Rink amide-PEG and PAL-PEG-PS resins (Applied Biosystems) and similar resins designed for peptide amide synthesis using the Fmoc protocol. Therefore, in some cases, it may be desirable to have an amide bond to the PEG chain. In such cases, it is preferred to attach N-Fmoc-amino-PEG-carboxylic acid to an amide-forming resin (such as Rink amide resin). The first amino acid in the chain can be attached to the amino function of the PEG chain as an N-Fmoc-amino acid. Final deprotection yields the desired peptide-NH-PEG-CO-NH2 product.

[0189] Coupling to PAM or HMPA resin can be achieved by reaction of the resin with an Nα-protected amino acid, e.g., a Boc amino acid, such as its ammonium, cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene, tetramethylammonium, or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF), etc., preferably the cesium salt in DMF, for about 12 to 72 hours, preferably about 48 hours, at elevated temperatures, e.g., about 40 to 60° C., preferably about 50° C. This ultimately leads to the product peptide acid after acidolysis, or amide after aminolysis.

[0190] Nα-Boc-amino acids can be coupled to benzhydrylamine resins, for example, by DIC / HOBt-mediated coupling in a solvent such as CH2Cl2 or DMF, preferably CH2Cl2, at a temperature of about 10-50°C, preferably 25°C, for about 2-24 hours, preferably about 2 hours.

[0191] For Boc-based protocols, the sequential coupling of protected amino acids can be carried out by methods known in the art, typically on an automated peptide synthesizer. After neutralization with triethylamine, DIEA, N-methylmorpholine (NMM), collidine, or a similar base, the protected amino acids are each introduced in approximately a 1.5- to 2.5-fold molar excess, and the coupling is carried out at ambient temperature in an inert, non-aqueous, polar solvent such as CHCl, DMF, N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), or mixtures thereof, preferably dichloromethane. For Fmoc-based protocols, no acid is used for deprotection, but a base, preferably DIEA or NMM, is usually incorporated into the coupling mixture. The coupling is typically carried out in DMF, NMP, DMA, or a mixed solvent, preferably DMF. Representative coupling agents are N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropyl-carbodiimide (DIC), and other carbodiimides, either alone or in the presence of HOBt, O-acylurea, benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBop), N-hydroxysuccinimide, other N-hydroxyimides, or oximes. Alternatively, protected amino acid active esters (e.g., p-nitrophenyl, pentafluorophenyl, etc.) or symmetrical anhydrides may be used. Preferred binding agents are from the aminium / uronium class, such as HBTU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU).

[0192] A preferred method of attachment to Fmoc-PAL-PEG-PS resin can be achieved by deprotection of the resin linker with 20% piperidine in DMF, followed by reaction of the N-α-Fmoc-protected amino acid with approximately a 5-fold molar excess over the N-α-Fmoc-amino acid, using HBTU:di-isopropylethylamine (DIEA) (1:2) in DMF in a microwave-assisted peptide synthesizer with a maximum attachment cycle of 5 min at 75°C.

[0193] The peptides of the present disclosure may contain a PEG group (dPEG) at the C-terminus. Such PEG groups may be short chains of polyethylene glycol with amino and carboxyl termini. Therefore, they are essentially unnatural amino acids and are treated similarly to other amino acids for synthesis. For example, Fmoc-amidoxy-dPEG4-acid is commercially available from Quanta Biodesign (#10213) and is coupled to Rink or HMPA resin in the first step of synthesis in a manner similar to that used for the N-Fmoc or N-Boc amino acids described above. Deprotection under standard strong acidic conditions yields the corresponding short dPEG C-terminally modified peptide with the corresponding acid or amide C-terminus.

[0194] For Fmoc-based protocols in a microwave-assisted peptide synthesizer, N-α-Fmoc amino acid protecting groups are removed with 20% piperidine in DMF containing 0.1 M 1-hydroxybenzotriazole (HOBt) in a double deprotection protocol for 30 seconds, then 3 minutes, with the maximum temperature set at 75 °C. HOBt is added to the deprotection solution to reduce aspartimide formation. Coupling of the next amino acid then utilizes a 5-fold molar excess using HBTU:DIEA (1:2) with a maximum double coupling cycle of 5 minutes at 75 °C.

[0195] At the end of solid-phase synthesis, the fully protected peptide is removed from the resin. If the bond to the resin support is in the form of a benzyl ester, cleavage can be achieved by aminolysis with alkylamines or fluoroalkylamines for peptides with alkylamide C-termini, or by ammonolysis with, for example, ammonia / methanol or ammonia / ethanol for peptides with unsubstituted amide C-termini, for about 12 to 24 hours, preferably about 18 hours, at temperatures of about -10 to 50°C, preferably about 25°C. Peptides with a hydroxy C-terminus can be cleaved by HF or other strongly acidic deprotection regimes, or by saponification. Alternatively, the peptide can be removed from the resin by transesterification, for example with methanol, followed by aminolysis or saponification. The protected peptide can be purified by silica gel or reverse-phase HPLC.

[0196] Side chain protecting groups can be removed from the peptide by, for example, treating the aminolysis product with anhydrous liquid HF in the presence of anisole or other carbonium ion scavenger for about 15 minutes to 2 hours, preferably about 1.5 hours, at a temperature of about −10 to 10° C., preferably about 0° C.; by treatment with HF / pyridine complex; by treatment with tris(trifluoroacetyl)boron and trifluoroacetic acid; by reduction with hydrogen and palladium over carbon or polyvinylpyrrolidone; or by reduction with sodium in liquid ammonia, preferably liquid FH, and anisole.

[0197] For peptides on benzhydrylamine-type resins, the resin cleavage and deprotection steps can be combined in a single step using liquid FH and anisole as described above, or, preferably, through the use of a milder cleavage cocktail. For example, for PAL-PEG-PS resin, a preferred method is the use of a dual deprotection protocol in a microwave-assisted peptide synthesizer using one of the milder cleavage cocktails known in the art, such as TFA / water / tri-isopropylsilane / 3,6-dioxa-1,8-octanedithiol (DODT) (92.5 / 2.5 / 2.5 / 2.5), for 18 minutes each at 38°C. Cleavage of alkyl glycolide-containing materials has demonstrated survival of the alkyl glycoside bond using protocols with TFA / water ratios ranging from 9 / 1 to 19 / 1. A typical cocktail is 94% TFA: 2% EDT; 2% HO; 2% TIS. Typically, the fully deprotected product is precipitated, washed with cold (-70 to 4 °C) EtO, dissolved in deionized water, and lyophilized.

[0198] The peptide solution is desalted (e.g., with BioRad AG-3® anion exchange resin), and the peptide can be purified by a series of chromatographic steps utilizing any or all of the following types: ion exchange on a weakly basic resin in acetate form; hydrophobic adsorption chromatography on underivatized co-poly(styrene-divinylbenzene) such as Amberlite® XAD; silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g., on Sephadex® G-25; countercurrent distribution; supercritical fluid chromatography; and HPLC, particularly reverse-phase HPLC on octyl- or octadecylsilylsilica (ODS) bonded phase columns.

[0199] Provided herein is a process for preparing the covalently modified peptides described herein and their pharmaceutically acceptable salts, comprising sequentially condensing protected amino acids onto a suitable resin support to obtain the covalently modified peptide, removing the protecting groups and the resin support, and purifying the product. The process can utilize any protocol for peptide synthesis known in the art, such as microwave-assisted solid-phase synthesis. In some embodiments, the peptide is conjugated to an alkyl glycoside.

[0200] Example 3. Oxidation Methods for Making Uronic Acids To a solution of 1-dodecyl β-D-glucopyranoside (2.0 g, 5.74 mmol, Carbosynth) in 20 mL of acetonitrile and 20 mL of deionized (DI) water was added (diacetoxyiodo)benzene (4.4 g, 13.7 mmol, Fluka) and TEMPO (0.180 g, 1.15 mmol). The resulting mixture was stirred at room temperature for 20 hours. The reaction mixture was then subjected to mass spectrometry (e.g., LCQ ESI). Upon completion, the reaction mixture was diluted with water and lyophilized to dryness to give 1.52 g (73% crude yield) of crude product, 1-dodecyl β-D-glucuronic acid, as a white powder, which was used directly in solid-phase synthesis without further purification. For longer alkyl groups, 1,4-dioxane was used instead of acetonitrile, and the temperature was increased to 30 °C. In a similar manner, the desired alkyl sugar uronic acids used to make the products and reagents described herein were prepared.

[0201] The desired 1-alkyl sugar uronic acids used to make the products and reagents described herein were prepared in a similar manner, but using, for example, the corresponding 1-octyl, 1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecyl glycosides (Anatrace, Maumee, Ohio).The desired 1-alkyl disaccharide uronic acids used to make the products and reagents described herein were prepared in a similar manner, but using, for example, the corresponding 1-octyl, 1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecyl β-D-melibiosides or β-D-maltosides (Anatrace).

[0202] Example 4. Preparation of a peptide product (EU-A387) with a C-terminal amide A sample of Fmoc-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Bip-Ser-Lys-Tyr-Leu-Glu-Ser-Lys(Alloc)-Rink amide resin (SEQ ID NO:782) was prepared by sequential addition of Nα-Fmoc-protected amino acids as described in Example 1 and deprotected at the Lys-N-ε position by incubation with Pd(PPh3)4 (0.5 equiv.) and DMBA (20 equiv.) in DMF / CHCl2 (1:1) overnight at room temperature in the dark. After washing with DMF / CHCl2, the Lys side chain was acylated with 1'-dodecyl β-D-glucuronic acid in DMF / CHCl2 using DIC / HOBt. Completion of the coupling was confirmed with ninhydrin, and the product was washed extensively with CHCl2.

[0203] The product resin was subjected to final deprotection and cleavage from the resin by treatment with a cleavage cocktail (94% TFA:2% EDT; 2% HO; 2% TIS) at room temperature for 240 min. The mixture was treated with EtO, and the product was precipitated and washed extensively with EtO to give the crude title peptide product after drying in vacuo.

[0204] Purification was carried out in two batches by reverse-phase (C18) HPLC. The crude peptide was loaded onto a 4.1 x 25 cm HPLC column (15% organic modifier, acetate buffer) at a flow rate of 15 mL / min and eluted with a gradient from 15-45% Buffer B at 50°C for 60 min. The product fractions were lyophilized to give the title peptide product with a purity of 98% by analytical HPLC (18.6 min; 30-60% CHCN in 0.1% TFA) / mass spectrometry (M+1 peak = 2382.14). Other peptide products of this disclosure were prepared in a similar manner and are characterized below.

[0205] The corresponding 1-methyl and 1-octyl analogs of the title compound are prepared in a similar manner, but using the reagents 1'-methyl β-D-glucuronic acid and 1'-octyl β-D-glucuronic acid (Carbosynth). The corresponding 1-decyl, 1-dodecyl, 1-tetradecyl, 1-hexadecyl, 1-octadecyl, 1-eicosyl, and higher analogs are prepared using the mono- and disaccharide uronic acids prepared as described above. Alternatively, 1-alkylglucuronyl, or other uronic acid acylated analogs, can be prepared by first purifying the deprotected or partially deprotected peptide, followed by acylation with the desired uronic acid.

[0206] Example 5. Preparation of peptide products with C-terminal acids A sample of Boc-His(Trt)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Glu(O-Allyl)-Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(O-tBu)-Phe-Ile-Lys(Dde)-Trp(Boc)-Leu-Leu-Gln(Trt)-Thr(tBu)-HMPA (SEQ ID NO: 783) resin (starting from Fmoc-Thr(tBu)-HMPA resin, substitution 0.45 mmol / g) was prepared with N-Fmoc-protected amino acids as described in Example 1. The allyl-protected side chains of the Glu and Lys residues were deprotected by incubation with Pd(PPh3)4 (0.5 equiv.) and DMBA (20 equiv.) in DMF / CHCl2 (1:1) overnight at room temperature in the dark. The resin was washed with 0.5% DIEA in DMF (twice), 0.5% sodium diethyldithiocarbamate in DMF (twice), and DMF / CHCl2 to yield a pale yellow resin. The side-chain lactam bond was formed by coupling the Glu and Lys residues with DIC / HOBT (5 equiv.) in DMF. The reaction was checked for completeness with ninhydrin and recoupled as necessary. After washing with DMF / CHCl2, the Lys side chain was deprotected twice by incubation with 5% hydrazine hydrate in DMF (10 equiv.) for 15 min each. After washing with DMF / CHCl, the side chain amino group of the deprotected Lys residue was reacted with 1'-tetradecyl β-D-melibiouronic acid in DMF / CHCl using DIC / HOBt. Completion of the coupling was confirmed with ninhydrin, and the product was washed extensively with CHCl. ​​Any couplings not completed by ninhydrin were rerun. Typically, 10-12 g of peptide product resin was obtained from a 2 mmole synthesis.

[0207] The product resin was subjected to final deprotection and cleavage from the resin by treatment with a cleavage cocktail (94% TFA:2% EDT;2% HO;2% TIS) at room temperature for 240 minutes. The mixture was treated with EtO, and the product was precipitated and washed extensively with EtO to give the crude title peptide product after drying in vacuo. Typically, 5-8 g of crude peptide product was obtained.

[0208] Purification was performed in two batches by reverse-phase (C18) HPLC. The crude peptide (1-1.5 g) was loaded onto a 4.1 x 25 cm HPLC column (15% organic modifier, 0.1% TFA buffer) at a flow rate of 15 mL / min and eluted with a gradient from 35-55% Buffer B over 70 min at room temperature. Repurification of less pure fractions was performed on fractions with a purity of >70%. The product fractions were lyophilized, and EU-A1077 was obtained with a purity of 98.7% by analytical HPLC (10.3 min, 45-75% CH3CN in 0.1% TFA) / mass spectrometry (1317.67, +3 charged; 1976.13, +2 charged; molecular weight 3950.44). Other peptide products of this disclosure were prepared in a similar manner and are characterized below.

[0209] The corresponding 1-methyl and 1-octyl analogs of the title compound are prepared in a similar manner, but using the reagents 1'-methyl β-D-glucuronic acid and 1'-octyl β-D-glucuronic acid (Carbosynth). The peptide products of the present disclosure were prepared in a similar manner, but using the corresponding 1-octyl, 1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecyl β-D-glucuronic acids (prepared as described above). The peptide products of the present disclosure were prepared in a similar manner, but using the corresponding 1-octyl, 1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecyl β-D-melibiouronic acid or β-D-maltouronic acid (prepared as described above). Alternatively, 1-alkylglucuronyl or other uronic acid acylated analogs can be prepared by first purifying the deprotected or partially deprotected peptide, followed by acylation with the desired uronic acid reagent. Alternatively, ammonolysis of the HMPA resin-bound intermediate gave the corresponding C-terminal amide.

[0210] Analysis and characterization was performed by HPLC / mass spectrometry in positive ion mode using the elution gradient provided in the table below.

[0211] [Table 5-1]

[0212] [Table 5-2] HPLC gradient in 0.1% TFA: [a] 35% to 65% CH3CN for 30 minutes [b] 30% to 60% CH3CN for 20 minutes [c] 35% to 65% CH3CN for 20 minutes [d] 25% to 55% CH3CN for 20 minutes [e] 40% to 70% CH3CN for 20 minutes [f] 45% to 75% CH3CN for 20 minutes HPLC was performed on a Phenomenex Luna C18 5 micron 250 x 4.6 mm analytical column.

[0213] The following table lists other compounds that were synthesized and analyzed as described above:

[0214] [Table 6-1]

[0215] [Table 6-2]

[0216] [Table 6-3] HPLC gradient in 0.1% TFA: [a] 35% to 65% CH3CN for 30 minutes [b] 30% to 60% CH3CN for 20 minutes [c] 35% to 65% CH3CN for 20 minutes [d] 25% to 55% CH3CN for 20 minutes [e] 40% to 70% CH3CN for 20 minutes [f] 45% to 75% CH3CN for 20 minutes [g] 50% to 80% CH3CN for 20 minutes [h] 10% to 40% CH3CN over 20 minutes [i] 30% to 90% CH3CN for 20 minutes [j] 10% to 90% CH3CN for 20 minutes [k] 30% to 95% CH3CN over 20 minutes [l] 30% to 60% CH3CN for 30 minutes [m] 20% to 100% CH3CN for 20 min [n] 20% to 80% CH3CN for 20 minutes [o] 30% to 50% CH3CN for 20 minutes HPLC was performed on a Phenomenex Luna C18 5 micron 250 x 4.6 mm analytical column.

[0217] Example 6. In vitro stability of peptide products in human plasma A 1 mg / mL stock solution was prepared in DMSO / CH3CN (1 / 1). A standard working solution in 50% CH3CN was prepared along with the stock solution. The concentrations of the working solutions were 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, and 20000 ng / mL. 10 μL of the working solution was spiked into 90 μL of blank plasma, and the mixture was vortexed. 300 μL of internal standard solution (verapamil, 20 ng / mL in 100% CH3CN) was added, and the mixture was vortexed and centrifuged. The supernatant was transferred to an HPLC injection plate and loaded onto the HPLC column. The standard samples were 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng / mL. QC samples were 5 (LQC), 50 (MQC), and 500 (HQC) ng / mL. For plasma stability studies, samples of EU-A993, EU-A1023, and human GLP-1(7-36) (Bachem) were prepared in human plasma (approximately 6-20 ng / mL or similar concentrations, above the limit of quantitation) and sampled at t = 0, 0.5, 1, 2, 4, and 8 hours during incubation at 30°C. Samples were treated with internal standard solution (100% CH3CN) as described above to precipitate the peptides, and the supernatant was loaded onto an injection plate and then onto an HPLC column for quantitation by mass spectrometry.

[0218] The instruments used were an API-4000 mass spectrometer, ESI positive, MRM scan; and a Shimadzu HPLC / CTC autosampler with an ACE C8 column (2.1 × 50 mm, 5 μm), mobile phase A: water, 0.1% formic acid in mobile phase B and 5 mM NHOAc: 0.1% formic acid in CHCN, with 10 μL of sample injected.

[0219] Plots of compound signal (amount) versus time show nearly complete cleavage of GLP-1(7-36) at 4 hours, while no change in the amount of intact peptide product was detected in human plasma over a period of at least 8 hours (Figure 8). Both peptide products exhibited protection from proteolysis and long plasma half-lives in in vitro assays.

[0220] Example 7. Cellular assay of activity of peptide products at GLP-1 and glucagon receptors Compounds were accurately weighed in amounts of approximately 1 mg and assayed in standard cell assays (Cerep SA). The readout was the amount of cAMP produced in cells treated with test compounds, either in agonist or antagonist mode. The assay used was the stimulation of cAMP levels in cell assays of glucagon (human, cloned into CHO cells) and GLP-1 (mouse cell line). The assay is described in Chicchi et al., J. Biol. Chem., 272:7765-7769 (1997) and Runge et al., Br. J. Pharmacol., 138:787-794 (2003).

[0221] EU-A391 did not alter GCGR cellular responses, but altered GLP1R cellular responses with an EC of 420 nM. 50 rose sharply due to

[0222] [Table 7]

[0223] Further cell assays were carried out by standard cell assays (DiscoveRx, LeadHunter assay) using cAMP stimulation or arrestin activation as a readout. Compounds were accurately weighed in approximately 1 mg amounts and transported to DiscoveRx for dilution and assay. Cell assays used the human glucagon receptor cloned into CHO cells and the human GLP-1 receptor cloned into CHO cells.

[0224] [Table 8-1]

[0225] [Table 8-2]

[0226] [Table 8-3]

[0227] Example 8. Glucose-lowering activity of peptide products in db / db mice Sixty female db / db B6BKS(D)Leprdb / J (strain 000697) mice for this study were approximately 8-9 weeks old upon arrival (Jackson Laboratory, Bar Harbor, Maine). Mice were randomized by weight, and two treatment groups of eight female mice were administered test compounds EU-A994, EU-A995, or EU-A1026 at dose levels of 100 or 300 nmoles / kg, respectively. One group of eight female mice served as a vehicle control and received 0.2% BSA in pH 7.4 saline as a vehicle. The other group of eight female mice received liraglutide as a positive control compound at a dose level of 50 nmoles / kg. The test compounds, vehicle, and positive control compound were injected subcutaneously at approximately 0, 7, and 24 hours on Day 1 in a dose volume of 6 mL / kg.

[0228] Clinical observations were performed on the day of receipt before randomization and daily from Days 1 to 5. Body weights were measured and recorded at the day of receipt, before randomization, and daily from Days 1 to 5. Food intake was measured and recorded daily from Days 1 to 5. Blood samples for glucose analysis were collected pre-study (Day -3) and at 0, 1, 2, 4, 8, 10, 24, 48, 72, and 96 hours after the first dose on Day 1. At the end of the study, all animals were sacrificed and carcasses were discarded without further evaluation.

[0229] Significant changes in body weight relative to vehicle were evident for liraglutide, high-dose EU-A994, and high-dose EU-A1026 on days 2 and 3, and for low-dose EU-A1026 on days 3 and 4. With regard to food intake, animals treated with liraglutide differed significantly from vehicle on days 1 and 2 and high-dose EU-A994 on day 1, and from liraglutide for low-dose EU-A995 on days 1 and 2, high-dose EU-A994 on day 1, and low- and high-dose EU-A1026 on day 2. Glucose levels for liraglutide at 10 hours and for high-dose EU-A994 at 10 and 24 hours were significantly different from vehicle. In a similar manner, other peptide products were examined for their effects on blood glucose, body weight, and food intake.

[0230] [Table 9]

[0231] Figure 9 shows that EU-A992 (250 nmol / kg), EU-A1167 (250 nmol / kg), and EU-A1168 (250 nmol / kg) injected subcutaneously at T = 0 and 7 hours reduced elevated blood glucose levels in mice to the normal range for approximately 72 hours in a db / db mouse model of diabetes.

[0232] Example 9. Activity of peptide products in DIO mice Fifty diet-induced obese (DIO) C57BL / 6J male mice (JAX Labs) were received at 6 weeks of age. Mice were ear-notched for identification and individually housed in positive-pressure ventilated polycarbonate cages containing HEPA-filtered air at a density of five mice per cage. The animal room was illuminated throughout with artificial fluorescent lighting on a controlled 12-hour light / dark cycle. The standard temperature and relative humidity ranges for the animal room were 22 ± 4°C and 50 ± 15%, respectively. Filtered tap water acidified to a pH of 2.8-3.1 and a high-fat diet (Research Diets D12492, 60 kcal%) were provided ad libitum.

[0233] After 2 weeks of acclimation, 50 mice were randomized into groups (n = 10): Group 1: vehicle-treated; Group 2: low-dose EU-A994; Group 3: high-dose EU-A594; Group 4: low-dose EU-A1024; and Group 5: high-dose EU-A1024. Mice were subcutaneously administered on days 1 (0 and 7 h), 3, 6, 9, 12, 15, 18, 21, and 24. Body weight and cage-side observations were recorded daily. Food and water intake was recorded weekly. Mice underwent NMR measurements to determine total body fat and lean composition on days 1 (pre-dose) and 26. On day 26, mice were fasted overnight and an oral glucose tolerance test was performed. The next day, the first blood sample was collected via tail nick (t = 0). Mice were then administered a bolus of 1.0 g / kg glucose. Blood samples were obtained via tail nick at 0, 15, 30, 60, 90, and 120 min after glucose administration, and plasma glucose was immediately determined using a glucometer.

[0234] Mice were sacrificed on day 28. Terminal blood was processed to serum / plasma, and aliquots were sent for analysis of glucose, insulin, and lipid profiles. Body composition was determined by NMR. EU-A1024 reduced glucose excursion in the oral glucose tolerance test (OGTT), reduced basal insulin secretion, but increased glucose-dependent insulin secretion, reduced body weight gain (Figure 10), and reduced fat mass, with little effect on lean mass (Figure 11).

[0235] Example 10: Effect of SP-1373 in a mouse model of NASH In a biopsy-confirmed DIO-NASH mouse model, SP-1373, a peptide-surfactant conjugate and dual GLP-1 receptor / glucagon receptor agonist demonstrated superior liver histopathological and metabolic endpoints compared with semaglutide (a GLP-1 agonist) and elafibranor (a PPAR-α / δ agonist). Nonalcoholic steatohepatitis (NASH) was induced in male C57BL / 6JRj mice (approximately 20 g, 5 weeks old at the time of arrival, 37 weeks old at the start of the study) by feeding them an AMLN diet containing 40% fat (18% trans fat), 2% cholesterol, and 40% carbohydrate (20% fructose) for 32 weeks prior to and during the study period.

[0236] All mice entering the study were stratified based on their pre-biopsy livers at week -3. Mice with a fibrosis score of ≥1 and adiposity score of ≥2 were included in the study. Stratified mice were randomized into treatment groups based on quantification of hepatic collagen type 1 alpha 1 (col1α1) at week -3. The arms in the NASH study were: 1) vehicle (0.05% Tween 80, 50 mM NaHPO, pH 8) administered subcutaneously (SC) once daily (QD) for 12 weeks (n=12); 2) low-dose (5 nmole / kg) SP-1373 administered SC QD for 12 weeks (n=11); 3) high-dose (10 nmole / kg) SP-1373 administered SC QD for 12 weeks (n=11); 4) 78 μmol / kg elafibranor administered orally (PO) QD for 12 weeks (n=12); and 5) 10 nmole / kg semaglutide administered SC QD for 2 weeks (n=12).

[0237] Body weight was measured daily throughout the study. Food intake was measured daily for the first 14 days and then weekly until the end of the study. EchoMRI whole-body scans were performed at week 11. At the end of the study, plasma levels of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyltransferase (GGT), triglycerides (TG), and total cholesterol (TC); hepatic levels of TG, TC, col1α1, and galectin-3; expression levels of various hepatic mRNAs; and liver weight were measured, and liver biopsies / necropsies were performed to assess hepatic steatosis, inflammation, ballooning, and fibrosis.

[0238] For blood and plasma assays, blood samples were collected in heparin tubes, and plasma was separated and stored at −80°C until analysis. ALT, AST, GGT, TG, and TC levels were measured using commercially available kits (Roche Diagnostics, Germany) and a Cobas™ C-501 automated analyzer according to the manufacturer's instructions.

[0239] For liver tissue assays, the triglyceride and cholesterol contents in the liver were measured using triglyceride and esterol reagents (Cat. No. 22-045-795 and Cat. No. 22-045-780, respectively, Roche Diagnostics, Germany) and a Cobas™ C-501 automated analyzer. Homogenized liver tissue was heated twice to 80-100°C and centrifuged in a microcentrifuge, and the triglyceride and cholesterol contents in the resulting supernatant were measured.

[0240] Body composition of mice was assessed using the EchoMRI3-1 body composition analyzer (EchoMRI, US). Unanesthetized mice were placed in a plastic tube inside the MRI scanner for approximately 80 seconds. Body composition was expressed as adipose tissue mass, fat-free tissue mass (lean tissue mass), and water.

[0241] For liver biopsy, mice were anesthetized by inhalation of isoflurane (2-3%). A small abdominal incision was made in the midline to expose the left lateral lobe of the liver. A conical wedge of liver tissue (approximately 50 mg) was excised from the distal portion of the left lateral lobe and fixed in 10% neutral buffered formalin (4% formaldehyde) for histological analysis. The cut surface of the liver was immediately electrocoagulated using a bipolar coagulator (ERBE VIO 100 electrocautery). After pre-biopsy, the liver was returned to the abdominal cavity, the abdominal wall was sutured, and the skin was closed with staples. For postoperative recovery, mice were given subcutaneous carprofen (5 mg / kg) on ​​the day of surgery and on postoperative days 1 and 2.

[0242] For histological staining, paraffin-embedded sections were briefly deparaffinized in xylene and then rehydrated in a graded series of ethanol. For hematoxylin and eosin (H&E) staining, slides were incubated in Mayer's hematoxylin (Dako), washed with tap water, stained with Eosin Y solution (Sigma-Aldrich), hydrated, mounted on Pertex gel, and allowed to dry before scanning. For picrosirius red (PSR) staining, slides were incubated in Weigert's iron hematoxylin (Sigma-Aldrich), washed with tap water, stained with picrosirius red (Sigma-Aldrich), and then washed twice in acidified water. Excess water was removed by shaking the slides, and the slides were dehydrated in three sets of 99% ethanol, removed with xylene, mounted on Pertex gel, and then allowed to dry before scanning.

[0243] For immunohistochemical (IHC) staining of collagen type 1 α1 (col1α1) and galectin-3 (GAL3), slides were incubated with primary antibodies (Southern Biotech Cat. No. 1310-01 for col1α1 or Biolegend Cat. No. 125402 for galectin-3) after antigen retrieval and inhibition of endogenous peroxidase activity. The anti-col1α1 primary antibody was detected using a polymeric horseradish peroxidase (HRP)-linker antibody conjugate, and the anti-GAL3 primary antibody was detected using a linker secondary antibody followed by amplification with a polymeric HRP-linker antibody conjugate. The primary antibodies were then visualized with 3,3'-diaminobenzidine (DAB) as a chromogen. Finally, sections were counterstained in hematoxylin and coverslipped.

[0244] To assess hepatic steatosis, inflammation, ballooning, and fibrosis, liver samples were fixed in formalin and embedded in paraffin, and sections were stained with hematoxylin-eosin or picrosirius red. Samples were scored for steatosis, inflammation, ballooning, and fibrosis as summarized in Table 9.

[0245] SP-1373 (high dose) significantly reduced body weight in obese mice to the lower normal range, whereas semaglutide and elafibranor did not significantly reduce body weight (Figures 12A and 12B [high dose SP-1373 was inadvertently administered to the vehicle group on day 62]). SP-1373 significantly reduced whole-body adipose tissue mass (Figures 13A and 13B) with minimal effect on whole-body lean tissue mass. Plasma levels of total cholesterol (TC, Figure 14B), alanine transaminase (ALT, Figure 15A), and aspartate transaminase (AST, Figure 15B) were also significantly reduced in SP-1373-treated animals. Plasma ALT and AST levels, and the AST / ALT ratio, are clinical biomarkers of liver health. The beneficial effects of SP-1373 demonstrate the benefits of dual GLP-1 receptor / glucagon receptor agonism. GLP-1 receptor agonism stimulates insulin production and blood glucose-dependent insulin secretion, lowering blood glucose levels, suppressing appetite, and causing weight loss. Glucagon receptor agonism has antihyperlipidemic and satiety effects, stimulating fat burning and energy expenditure, and reducing body weight.

[0246] SP-1373 reduced liver weight to a greater extent than semaglutide, whereas elafibranor actually increased liver weight (Figure 16). Liver histology using hematoxylin-eosin staining revealed an almost complete absence of hepatic steatosis in animals treated with high-dose SP-1373 (Figure 17C [large white oval structures are veins]), in contrast to animals treated with semaglutide (Figure 17E), indicating a modest reduction in hepatic steatosis. Figures 18A and 18B show post-biopsy hepatic steatosis in terms of % area affected and total hepatic lipid content in animals from different groups, as determined by quantitative histology evaluation, confirming the almost complete absence of hepatic steatosis in animals treated with high-dose SP-1373. SP-1373 reduced liver triglyceride (TG) and total cholesterol (TC) levels to a greater extent than semaglutide, whereas elafibranor had little or no effect on liver TG and TC levels (Figures 19A and 19B). SP-1373 also reduced total liver collagen type 1 alpha 1 (anti-col1alpha1 antibodies, a measure of liver fibrosis) to a greater extent than semaglutide, whereas elafibranor had only a modest effect (Figure 20). Similarly, SP-1373 reduced total liver galectin-3 (anti-GAL3 antibodies, a biomarker of hepatitis and fibrosis) to a greater extent than semaglutide, whereas elafibranor had virtually no effect (Figure 21).

[0247] High doses of SP-1373 significantly reduced or eliminated hepatic steatosis in all treated animals (Figure 22C). In Figures 22A-22E, the change in biopsy from pre-study to post-study for each animal is shown as a line, and hepatic steatosis was scored from 0 to 3 in terms of surface area bearing steatosis. The majority of treated animals showed minimal hepatitis (Figures 23A-23E). In Figures 22A-23E, the change in biopsy from pre-study to post-study for each animal is shown as a line, and hepatitis was scored from 0 to 3 in terms of severity. Nearly all animals that initially showed hepatocyte ballooning did not show ballooning after treatment with SP-1373, elafibranor, or semaglutide (Figures 24A-24E). 22A-24E, for each animal, the change in biopsy from pre-study to post-study is shown as a line, and hepatocyte ballooning was scored for severity from 0 to 2. The scores for hepatic steatosis, inflammation, and ballooning are set forth in Table 9.

[0248] NAS (nonalcoholic fatty liver disease [NAFLD] activity score) scores, a clinical measure of NASH activity, improved in the majority of treated animals, with the greatest extent of reduction achieved by high-dose SP-1373 (Figures 25A-25E). In Figures 25A-25E, a line represents the change in biopsy from pre-study to post-study for each animal. The NAS score is the sum of scores for steatosis (0-3), inflammation (0-3), and ballooning (0-2), with a maximum score of 8. Individuals with an NAS score of 5 or higher are diagnosed with NASH, while those with an NAS score of 3 or lower are diagnosed as not having NASH. When translated to a mouse model of NASH, high-dose SP-1373 reversed NASH in all treated animals (Figure 25C).

[0249] SP-1373 and elafibranor reduced liver fibrosis in the majority of animals (Figures 26B-26D). Liver fibrosis was assessed with picrosirius red, which stains collagen types 1 and 3. For each animal in Figures 26A-26E, a line represents the change in biopsy from pre-study to post-study, and liver fibrosis was scored from 0 to 4 as described in Table 9.

[0250] [Table 10]

[0251] SP-1373 inhibits fibrosis {e.g., collagen type 1 alpha 1 (col1α1), transforming growth factor-beta 1 (TGFβ1), and galectin-3 (GAL3, aka MAC2)}, inflammation {e.g., interleukin-1α (IL-1α), IL-1β, tumor necrosis factor-alpha (TNF-α), GAL3, CD14, toll-like receptor 4 (TLR4), p38 mitogen-activated protein (MAP) kinase 11, and NF-κB}, monocyte recruitment {e.g., chemokine (CC motif) ligand 5 (CCL5, aka RANTES), monocyte chemoattractant protein 1 (MCP1, aka CCL2), chemokine (CC motif) receptor 1 (CCR1, and CCR2)}, and monocyte differentiation, e.g., into macrophages {e.g., GAL3, EGF-like module-containing mucin-like hormone receptor-like 1 (EGF ... SP-1373 reduced the expression of NASH-related mRNAs in the liver, including mRNAs encoding proteins involved in fibrosis (Figures 27A-27F and 28A-28F), including those encoding proteins involved in fibrosis (EMR1, aka F4 / 80), CD14, CD68, and CD86. More specifically, with regard to fibrosis, SP-1373 reduced the hepatic expression of mRNAs encoding proteins involved in fibrosis fibrogenesis (Figures 29A-29I) and activation of hepatic stellate cells (the primary cell type involved in liver fibrosis) and myofibroblast proliferation (Figures 30A-30F). Furthermore, SP-1373 reduced the hepatic expression of mRNAs encoding proteins involved in pyroptosis, a highly inflammatory form of programmed cell death (Figures 31A-31F). With regard to the figures referred to in this paragraph, the expression levels of specific mRNAs are mean + SEM and are compared to vehicle after gene-wise replicate calibration. * P<0.05, ** P<0.01, and ***P<0.001. SP-1373 also reduced hepatic expression of mRNAs encoding proteins involved in lipid synthesis (e.g., fatty acid synthase [FAS], stearoyl-CoA desaturase-1 [SCD1], and 3-hydroxy-3-methylglutaryl-CoA synthase 1 [HMGCS1]) and lipid uptake (e.g., CD36). Furthermore, SP-1373 increased hepatic expression of farnesoid X receptor (FXR) mRNA. In addition, SP-1373 modulated the expression of genes encoding proteins involved in, for example, insulin signaling and glucose homeostasis.

[0252] In summary, SP-1373 reversed hepatic steatosis, reduced hepatitis and fibrosis, and significantly improved NASH scores in a well-recognized conversion mouse model of NASH, while also addressing metabolic and glucose control deficits. SP-1373 is a potent, balanced, full agonist at GLP-1 and glucagon receptors, with antidiabetic and antiobesity effects. Compared to the GLP-1 agonist semaglutide, SP-1373 has a more potent antihyperglycemic effect, the greatest reduction in body weight, adipose tissue mass, and food intake, and a longer duration of action. In a DIO-NASH mouse model, treatment with SP-1373 significantly reduced body weight, plasma total cholesterol (TC) and alanine transaminase (ALT) levels, NAS score, hepatic steatosis, hepatic triglyceride (TG) and TC levels, liver weight, hepatic collagen type I α1 content (a measure of hepatic fibrosis), and hepatic galectin-3 content (a measure of hepatic inflammation and fibrosis) compared with treatment with semaglutide or elafibranor (a dual PPAR-α / δ agonist). Therefore, SP-1373 is useful for treating, for example, insulin resistance, diabetes (including type 1 and type 2), hyperlipidemia, obesity, metabolic syndrome, cardiovascular disease, and related diseases, such as NAFLD (including NASH).

[0253] While various embodiments of the present disclosure have been described herein, such embodiments are provided by way of illustration and example only. Many variations and modifications thereto will be apparent to those skilled in the art and are encompassed by this disclosure. It will be understood that various alternatives to the embodiments of the present disclosure may be utilized in practicing the present disclosure and are encompassed by the present disclosure.

Claims

1. 1. A pharmaceutical composition or medicament comprising a GLP-1 / glucagon peptide product or a pharmaceutically acceptable salt thereof for use in treating fatty liver disease, which is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), wherein the GLP-1 / glucagon peptide product is His １ -Aib ２ -Gln ３ -Gly ４ -Thr ５ -Phe ６ -Thr ７ -Ser ８ -Asp ９ -Tyr １０ -Ser １１ -Lys １２ -Tyr １３ -Leu １４ -Asp １５ -Glu ＊ １６ -Lys(N-omega[1-(17-carboxyl-heptadecyloxy)β-D-glucuronyl]) １７ -Ala １８ -Ala １９ -Lys ＊ ２０ -Glu ２１ -Phe ２２ -Ile ２３ -Gln ２４ -Trump ２５ -Leu ２６ -Leu ２７ -Gln ２８ -Thr ２９ -NH ２ (SEQ.ID.NO.1119), Here, Glu １６ ＊ and Lys ２０ ＊ represents a residue that is cyclized via its side chain to form a lactam, A pharmaceutical composition or a drug thereof, characterized in that:

2. 10. The pharmaceutical composition or medicament thereof of claim 1, wherein the treatment comprises parenteral administration (e.g., subcutaneous, intravenous, or intramuscular) of the peptide product.

3. The pharmaceutical composition or medicament thereof according to claim 1, wherein said treatment comprises administering the peptide product by oral inhalation, or nasal inhalation, or insufflation.

4. 2. The pharmaceutical composition or medicament thereof of claim 1, wherein the treatment comprises parenteral administration (e.g., subcutaneous [sc], intravenous [iv], or intramuscular [im]) of the peptide product at a dosage of about 0.1 mg to 10 mg for a period of about one week.

5. 10. The pharmaceutical composition or medicament thereof of claim 1, wherein the treatment comprises parenteral administration (e.g., subcutaneous [sc], intravenous [iv], or intramuscular [im]) of the peptide product at a dosage of about 0.1-1 mg for a period of about one week.

6. 10. The pharmaceutical composition or medicament thereof of claim 1, wherein the treatment comprises parenteral administration (e.g., subcutaneous [sc], intravenous [iv], or intramuscular [im]) of the peptide product once a week.

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