Improved peptide drugs for the treatment of NASH and other disorders

Abstract

To provide the use of a peptide product which have improved properties, including increased duration of action and bioavailability.SOLUTION: The disclosure provides the use of a peptide product of GLP-1 / glucagon or a pharmaceutically acceptable salt thereof, comprising a peptide covalently attached to a surfactant moiety of glucuronic acid or the like, in the production of pharmaceuticals for the treatment of fatty liver disease.SELECTED DRAWING: 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 with the potential for epidemic spread, which is a leading cause of patient morbidity and mortality as well as a major 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 or / and 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 diseases associated with insulin resistance or / and obesity, such as type 2 diabetes, metabolic syndrome, cardiovascular disease (including coronary artery disease such as atherosclerosis and myocardial infarction), hypertension, NASH, and PCOS, and diseases associated with such disorders. The peptide products include a peptide covalently attached to a surfactant moiety that contains 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 enhanced 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 description of the drawings]

[0005] [Figure 1] Specific peptide products are shown, all of which are incorporated into this disclosure. [Diagram 2] Specific peptide products are shown, all of which are incorporated into this disclosure. [Diagram 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. [Diagram 5] Illustrated is the X-ray crystal structure of the GLP-1 receptor N-terminal region / extracellular domain binding site, showing the key hydrophobic ligand-binding region (Val19*, Phe22*, Trp25*, and Leu26* of the ligand exendin-4, and sequences of exendin-4 beyond Glu15 interact with this region as amphipathic helices). [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] FIG. 1 shows the concentration 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]Glycemic responses of db / db mice following subcutaneous injection of EU-A992, EU-A1167, and EU-A1168 (250 nmol / kg each) at t=0 and 7 hours are shown. [Figure 10] FIG. 1 shows weight loss in diet-induced obese (DIO) mice following 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 changes in whole body adipose and lean tissue mass in treated DIO mice as measured by NMR at days 1 (pre-dose) and 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] FIG. 1 shows the effect of test compounds on plasma triglyceride (TG) and total cholesterol (TC) levels in DIO-NASH mice. [Figure 15] FIG. 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 test compounds on fatty liver in DIO-NASH mice using hematoxylin-eosin staining is shown (the large oval structures in FIG. 17C are veins). [Figure 18A] FIG. 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] FIG. 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] FIG. 1 shows the effect of test compounds on hepatic triglyceride (TG) and total cholesterol (TC) levels in DIO-NASH mice. [Figure 20] FIG. 1 shows the effect of test compounds on total hepatic collagen type 1 alpha 1 (col1a1) content as measured by immunohistochemistry in DIO-NASH mice. [Figure 21] FIG. 1 shows the effect of test compounds on total hepatic galectin-3 content as measured by immunohistochemistry in DIO-NASH mice. [Figure 22] 1 shows the effect of a test compound on fatty liver score 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 hepatocyte ballooning scores in DIO-NASH mice. [Diagram 25] 1 shows the effect of test compounds on NAS (non-alcoholic fatty liver disease activity score) scores in DIO-NASH mice. [Figure 26] FIG. 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 fibrosis. [Diagram 30] FIG. 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. [Diagram 31]1 shows the effect of test compounds in DIO-NASH mice on hepatic expression of mRNA involved in pyroptosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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" stands for "also known as."

[0009] The term "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" is applied to every single one of the numbers in the series or range of numbers.

[0011] A "peptide" comprises two or more naturally occurring and / or non-naturally occurring amino acid residues, usually linked via peptide bonds. Such amino acids may include naturally occurring structural variants, naturally occurring non-proteinogenic amino acids, or / and synthetic non-naturally occurring analogs of naturally occurring amino acids. The terms "peptide" and "polypeptide" are used synonymously. Peptides include short peptides (about 2-20 amino acids), medium length peptides (about 21-50 amino acids), and long peptides (greater than about 50 amino acids, which may also be referred to as "proteins"). In some embodiments, the peptide product comprises a surfactant moiety covalently attached to a peptide of about 50, 40, or 30 or less 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. The abbreviations used in the amino acid sequences disclosed herein represent L-amino acids unless otherwise specified as D- or DL-, or the amino acid is achiral, but the equivalent D isomer 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 Nal(1) = 3-(1-naphthyl)alanine; Na1 or Nal(2) = 3-(2-naphthyl)alanine; Nle = 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 reduced amide bonds between the residues (designated as Tic-Ψ[CH2-NH]-Ψ-Phe) have the following structure:

[0013] [ka]

[0014] Unless specifically stated 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 be made 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 have two or more different types of modifications. 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., acetylation of the N-terminus), amidation (e.g., amidation of the C-terminus), 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, a peptide is acetylated at the N-terminus and / or a carboxamide (-CONH) at the C-terminus. 2 ) group, which can increase the stability of the peptide.

[0015] Possible modifications of the peptides also include the deletion of one or more amino acids, the addition / insertion of one or more natural and / or non-natural amino acids, or the substitution with one or more natural and / or non-natural amino acids, or any or all combinations thereof. The 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 through errors due to PCR amplification. The non-natural amino acids may have the same chemical structure as the corresponding natural amino acids, but with a D-stereochemical configuration, or may have a different chemical structure and a D- or L-stereochemical configuration. Non-natural amino acids can be used, for example, to promote α-helix formation and / or to increase the stability of the peptide (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 replacement of an amino acid in a peptide with a functionally, structurally, or chemically similar natural or non-natural amino acid. In certain embodiments, each of the following groups contains 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 includes 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 non-naturally occurring 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), Na1, 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 constraint or / and α-helix stabilization. Reduction of the amide bond between two residues (such as in Tic-Ψ[CH2-NH]-Ψ-Phe) can increase protease resistance and may, for example, modify receptor binding.

[0021] The present disclosure includes all pharma- ceutically acceptable salts of the peptides, including those that carry a positive overall charge, those that carry a negative overall charge, and those that have no overall charge.

[0022] Sugars include monosaccharides, disaccharides, and oligosaccharides (e.g., trisaccharides, tetrasaccharides, etc.). Reducing sugars exist in equilibrium in cyclic and open-chain forms, which usually favor 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. The alkyl group may be saturated or unsaturated and may be straight-chained (linear), branched, or cyclic. In some embodiments, the alkyl group is not cyclic. In some embodiments, the 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 chain containing acyl groups, heteroalkyl, heteroalicyclic, aryl, alkoxyaryl, heteroaryl, hydrophobic natural compounds (e.g., steroids), and the like.

[0024] In some embodiments, the alkyl group as a substituent is a linear or branched C alkyl group, referred to as a "lower alkyl." 1 -C 6 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 tertbutyl), 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 N-atom of a residue of the peptide (e.g., Tyr or Dmt). In certain embodiments, the N-alkyl group is a straight or branched C 1 -C 10 It is an 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 1 -C 30 , C 6 -C 30 , C 6 -C 20 , or C 8 -C 20 It is an alkyl group.

[0027] In some embodiments, the alkyl group (e.g., 1-alkyl group) is an aryl, -OH, -OR 1 , -SH, -SR 1 , -NH 2 , -NHR 1 , -N(R 1 ) 2 , oxo(=O), -C(=O)R 2 , carboxyl (CO 2 H), carboxylate (CO 2 - ), -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 4C(=O)N(R 1 ) 2 and wherein R 1 is independently at each occurrence hydrogen, alkyl, or aryl; R 1 and 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, the alkyl group (e.g., 1-alkyl group) is substituted internally or / and terminally with a carboxyl / carboxylate group, an aryl group, or an -O-aryl group. In some embodiments, the alkyl group (e.g., 1-alkyl group) is substituted with a carboxyl or carboxylate group at the distal end of the alkyl group. In further embodiments, the alkyl group (e.g., 1-alkyl group) is substituted with an aryl group at the distal end of the alkyl group. In other embodiments, the alkyl group (e.g., 1-alkyl group) is substituted with an -O-aryl group 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 may be saturated or unsaturated and may be linear, branched, or cyclic. In certain embodiments, R contains 1-20, 1-10, or 1-6 carbon atoms. Acyl groups may 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, where the 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, the at least one aromatic ring containing one or more heteroatoms independently selected from O, N, and S. The heteroaromatic ring may be bonded to or fused to one or more saturated, partially unsaturated, or aromatic rings, which may contain only carbon atoms or may contain 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 the arrangement of four fused rings, designated A, B, C, and D, as shown below:

[0035] [ka] The steroid nucleus can bear 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 "pharmaceutical 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 "pharmaceutical 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 disease being treated, or to alleviate to some extent the medical disease or one or more symptoms or complications thereof, in at least a portion 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. Reference to "treatment" of a medical disease includes prevention thereof. The terms "prevent," "preventing," and "prevention" include eliminating, reducing the risk of progression, 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 causes insulin resistance, which is the impaired ability of cells in the body to respond to insulin stimulation through a reduced number of insulin receptors and reduced binding of insulin receptors to key intracellular signaling systems. The obese state also causes metabolic syndrome, which is a collection of disorders such as 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 health care burden worldwide.

[0041] Metabolic Hormones Incretins are metabolic hormones that induce a decrease in blood glucose levels. Incretins are released after a meal and stimulate insulin production and secretion from pancreatic β cells in the islets of Langerhans through a blood glucose-dependent mechanism. Incretins also slow the rate of absorption of nutrients into the bloodstream by slowing gastric emptying, which induces satiety and suppresses appetite. In addition, incretins inhibit glucagon release from α 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 the presence of type 2 diabetes, insulin secretion from pancreatic β cells in response to GLP-1 is impaired.

[0042] GLP-1 has other beneficial effects, such as stimulating pancreatic β-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 a C-terminal extension of eight amino acid residues. 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 causes weight loss by reducing food intake and body fat.

[0044] Glucagon is produced from preproglucagon in the pancreatic α 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): His 1 -Ala 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -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: His 1 -Ser 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 -Ser 16 -Arg 17 -Arg 18 -Ala 19 -Gln 20 -Asp 21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -Met 27 -Asn 28 -Thr 29 (SEQ.ID.NO.2) Oxyntomodulin: His 1 -Ser 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 -Ser 16 -Arg 17 -Arg 18 -Ala 19 -Gln 20 -Asp21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -Met 27 -Asn 28 -Thr 29 -Lys 30 -Arg 31 -Asn 32 -Arg 33 -Asn 34 -Asn 35 -Ile 36 -Ala 37 (SEQ.ID.NO.3)

[0046] Amino acid residues throughout the sequences of GLP-1 and glucagon may be substituted while retaining biological activity. For example, substitution with Ala is widely accepted in the N-terminal region of GLP-1, especially 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 strongly bind 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 peptide gut hormones (e.g., GLP-1, GIP, glucagon, oxyntomodulin, or exendins (e.g., exendin-4)), or analogs or variants 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, 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 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 pharmaceutical agents, 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 include peptides covalently attached to surfactant moieties that contain hydrophilic sugar groups and hydrophobic groups. In some embodiments, the peptides are attached to sugar groups, which are subsequently attached to hydrophobic groups. In some embodiments, the peptides are metabolic hormones, such as GLP-1, glucagon, oxyntomodulin, or exendins (e.g., exendin-4), or analogs or variants thereof. The peptide products are useful, for example, for the treatment of insulin resistance, diabetes, obesity, metabolic syndrome, and cardiovascular disease, and diseases associated therewith, 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 a 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 a 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 alkyl glycoside, such as a 1-alkyl glycoside). 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 one embodiment, the alkyl glycoside 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 a 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, an unsubstituted or substituted C 1 -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, an unsubstituted or substituted C 1 -C 30 an alkyl group, an unsubstituted or substituted aralkyl group, an unsubstituted or substituted alkyloxyaryl group, or a steroid nucleus-containing group; R 2is independently at each occurrence a single bond, a single bond to U, H, unsubstituted or substituted C 1 -C 30 Alkyl groups, unsubstituted or substituted aralkyl groups, unsubstituted or substituted alkyloxyaryl groups, steroid nucleus-containing groups, -NH-, -S-, -O-NH-, spacers, -triazolo-, -NH(C=O)-CH 2 - or -(CH 2 ) m -maleimide-; W 1 independently at each occurrence, -CH 2 -, -CH 2 -O-, -CH 2 -S-, -CH 2 -NH-, -(C=O)-, -(C=O)-NH-, -(C=S)-, or -(C=S)-NH-; W 2 is independently at each occurrence -O-, -S-, -NH-, or -CH 2 - and; R 1a , R 1b , R 1c , R 1d , or R 2 At least one occurrence of 1 -C 30 is an alkyl, aralkyl, alkyloxyaryl, or steroid nucleus-containing group; m is an integer from 1 to 10; and n is 1, 2, or 3; and The peptide has the formula II: aa 1 -aa 2 -aa 3 -aa 4 -aa 5 -aa 6 -aa 7 -aa 8 -aa 9 -aa 10 -aa 11 -aa 12 -aa 13 -aa 14 -aa 15 -aa 16 -aa17 -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 C 1 -C 12 alkyl, or a PEG-containing group of less than 10 Da; and R 4 , H, C 2 -C 10 acyl (e.g., acetyl), or -C(=O)-aryl (e.g., benzoyl); aa 1 is His, N(R 3 )His, N(R 4 )His, or pGlu-His; aa 2 is Ser, D-Ser, Ala, Gly, Pro, MePro, Aib, Ac4c, or Ac5c; aa 3 is Gln or Cit; aa 4 is Gly, or D-Ala; aa 5 is Thr, or Ser; aa 6is Phe, Trp, 2FPhe, MePhe, 2FMePhe, or NaI2; aa 7 is Thr, or Ser; aa 8 is Ser, or Asp; aa 9 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, 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 19 is 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, 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, 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 34 is 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 not present or is U; U is a natural or unnatural amino acid containing a functional group used for covalent attachment to surfactant X; aa 1 -aa 37 any two of which can be optionally cyclized through their side chains to form a lactam bond; However, aa 10 -aa 37 is a linker amino acid U covalently attached to the surfactant X.

[0056] The surfactant moiety may also be R 1a , R 1b , R 1c , or R 1d The peptide can be linked to the linker amino acid U via, for example, 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 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 may be an amino-containing natural or unnatural amino acid (e.g., lysine or ornithine). 1a U is an unnatural amino acid that is attached to the surfactant moiety via 1a The 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 a glycan having a W 2 and the second glycoside -OR 1b , -OR 1c -OR 1dIn 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 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 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 C 1 -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 1 -C 30 is an alkyl group; R 2 is a single bond to U, -NH-, -S-, -triazolo-, -NH(C=O)-CH 2 - or -(CH 2 ) m -maleimide-, W 1 is -CH 2 -, -CH 2 -O-, -CH 2 -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 C 1 -C 30 It is an alkyl group, or a group containing a steroid nucleus.

[0060] In one embodiment, the surfactant X is R 2 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 (eg, the side chain amino group of a lysine residue).

[0062] In some embodiments, surfactant X is

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

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

[0065] [ka] During the ceremony, R1a is H, a protecting group, a sugar, unsubstituted or substituted C 1 -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 unsubstituted or substituted C 1 -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 C 1 -C 30 It is an alkyl group, or a group containing a steroid nucleus.

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

[0067] [ka] During the ceremony, R 1a is unsubstituted or substituted C 1 -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, the surfactant X is a 1-O-alkyl β-D-glucuronyl moiety:

[0069] [ka] In the formula, R 1a is unsubstituted or substituted C 1 -C 30 , C 1 -C 20 , or C 6 -C 20 and R' is a peptide that is 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 an unsubstituted or substituted "C 8 -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 1 -C 30 , C 1 -C 20 , C 6 -C 30 , or C 6 -C 20 In one embodiment, the alkyl group attached to the sugar is an unsubstituted or substituted C 8 -C 20 , C 6 -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 C 1 -C 30 In one embodiment, the alkyl group is linked to R via an ether bond (-O-alkyl).1b , R 1c , or R 1d In a further embodiment, 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 C 1 -C 30 , C 6 -C 30 , C 6 -C 20 , or C 8 -C 20 In one embodiment, R 1a At least one occurrence or a single occurrence of is unsubstituted or substituted C 12 -C 20 It is an alkyl group.

[0073] In some embodiments, the alkyl group (e.g., 1-alkyl group) is internally and / or terminally substituted with a carboxyl or carboxylate group. In certain embodiments, the alkyl group (e.g., 1-alkyl group) is substituted with a carboxyl or carboxylate group 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 some 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 some 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 some embodiments, the disaccharide is diglucuronic acid or appropriately functionalized kentiobiose or lactose, such as melibiose, maltose, isomaltose, melibiouronic acid, maltouronic acid, isomaltouronic acid, gentiobiouronic acid, or lacturonic acid. The terms "-uronic acid" and "-uronyl" are used interchangeably herein with respect to the sugar group of the surfactant moiety.

[0076] In some embodiments, the surfactant X is a 1-alkyl glycoside, in which 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 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-isomalturonic acid, 1-octadecyl β-D-isomalturonic acid, 1-hexadecyl β-D-isomalturonic acid, 1-tetradecyl β-D-isomalturonic acid, 1-dodecyl β-D-isomalturonic acid, 1-decyl β-D-isomalturonic acid, 1-octyl β-D-isomalturonic acid, 1-eicosyl β-D-gentiobiouronic acid, 1-octadecyl β-D-gentiobiouronic acid, 1-hexadecyl β-D-gentiobiour ... 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 6-carboxyl or 6,6'-dicarboxyl groups, the corresponding 1-alkyl α-anomers, etc. In a particular embodiment, the 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 of the carboxyl groups can be attached to the peptide), and a 1-O-tetradecyl β-D-isomaltouronyl moiety (bottom right) that is attached to an amino group of the 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 linked 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 -N 3 Unnatural amino acids with acetyl groups, acetylene groups, or haloacetyl groups (e.g., -NHC(=O)CH 2 Br) or -(CH 2 ) m -maleimido group), where m is 1-10. A haloacetyl or maleimido group of the linker amino acid can be coupled, for example, with 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, for example, using copper catalysis to form a triazole. In certain embodiments, U is a residue of lysine, ornithine (Orn), cysteine, or glutamic acid. Like any amino acid residue of a peptide, U can be an L- or D-amino acid.

[0083] The linker amino acid U may be an internal or / and terminal amino acid residue of the peptide. The functional group of U which forms the covalent bond with the surfactant moiety may 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 (noted 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 non-natural 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] ). Additionally, 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 has at least amino acid residues aa of SEQ.ID.NO. 1 -aa 17 , aa 1 -aa 18 , aa 1 -aa 19 , or aa 1 -aa 20 In a further embodiment, the peptide product of formula IA comprises at least amino acid residues aa of SEQ.ID.NO.1108. 1 -aa 27 , aa 1 -aa 28 , aa 1 -aa 29 , or aa 1 -aa 30 Includes.

[0086] In some embodiments, the peptide product of formula IA has formula III-A: aa 1 -aa 2 -aa 3 -aa 4 -aa 5 -aa 6 -aa 7 -aa 8 -aa 9 -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.1109) During the ceremony, Z is -OH or -NHR 3 where R 3 is H, unsubstituted or substituted C 1 -C 12 alkyl, or a PEG-containing group less than 10 Da; aa 1 is His, N(R 3 )His, N(Ac)His, or pGlu-His; aa 2 is Ser, Ala, Gly, MePro, Aib, Ac4c, or Ac5c; aa 3 is Gln or Cit; aa 4 is Gly, or D-Ala; aa 5 is Thr, or Ser; aa 6is Phe, Trp, 2FPhe, MePhe, 2FMePhe, or NaI2; aa 7 is Thr, or Ser; aa 8 is Ser, or Asp; aa 9 is Asp, or Glu; aa 10 is Tyr, Leu, Met, NaI2, Bip, Bip2EtMeO, Glu, Lys, or U(X); aa 11 is not present or is Ser, Asn, Bip, or U(X); aa 12 is 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 21is 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, 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 26 is 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); Here, aa 1 -aa 29 any two of which can be optionally cyclized through 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 that is covalently attached to the surfactant X.

[0087] In one embodiment, the peptide product of formula III-A has the structure: aa 1 -aa 2 -aa 3 -aa 4 -aa 5 -aa 6 -aa 7 -aa8 -aa 9 -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 3 is H, unsubstituted or substituted C 1 -C 12 alkyl, or a PEG-containing group less than 10 Da; aa 1 is His; aa 2 is Aib; aa 3 is Gln; aa 4 is Gly; aa 5 is Thr; aa 6 is Phe; aa 7 is Thr; aa 8 is Ser; aa 9 is Asp; aa 10 is Tyr, Glu, Lys, or U(X); aa 11 is Ser; aa 12 is Lys, or Glu; aa13 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; aa 27 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 a surfactant X.

[0088] In a further embodiment, the peptide product of formula IA has the formula III-B: His 1 -aa 2 -aa 3 -Gly 4 -Thr 5 -aa 6 -Thr 7 -Ser 8 -Asp 9 -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 C 1 -C 12 alkyl, or a PEG-containing group less than 10 Da; aa 2 is Gly, MePro, or Aib; aa 3 is Gln or Cit; aa 6 is Phe, 2FPhe, MePhe, 2FMePhe, or NaI2; aa 10 is Tyr, NaI2, Bip, Bip2EtMeO, Glu, Lys, or U(X); aa 11 is absent, 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, 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 not present 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 27is 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, Arg, or U(X); Here, aa 1 -aa 23 any two of which can be optionally cyclized through 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 that is covalently attached to the surfactant X.

[0089] In some embodiments of the peptide product of formula IA, III-A, or III-B, aa 2 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 , aa 18 , aa 20 , aa 24 , or aa 28 or any combination thereof is a lysine bound to surfactant X; or aa 16 and aa 20 is cyclized through its side chain to form a lactam bond; or The 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, the peptides of the peptide products described herein contain one or more Aib residues at or near the N-terminus, at or near the C-terminus, internally, or any or all combinations thereof. The Aib residues can protect the peptide from degradation by proteases such as dipeptidyl peptidase 4 (DPP-4). In certain embodiments, the aa 2 is Aib.

[0091] In some embodiments, the peptide product of formula IA, III-A, or III-B has the structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.1126) During the ceremony, aa 2is 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 structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.1127) During the ceremony, aa 2 is Gly or Aib; aa 16 is 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 structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.774) During the ceremony, aa 2 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 structure: His 1 -aa 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 -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 -NH 2(SEQ.ID.NO.775) During the ceremony, aa 2 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 structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.776) During the ceremony, aa 2 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 structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.777) During the ceremony, aa 2 is Gly or Aib; aa 16 is Glu, Ser, Ala, or Aib; aa 17is 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 structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.778) During the ceremony, aa 2 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, the surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C 8 -C 20 It is an alkyl.

[0098] In other embodiments, the peptide product of formula IA, III-A, or III-B has the structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.779) During the ceremony, aa 2 is Aib or Gly, aa 16 is Glu, Ala, or Aib; aa 17 is Lys(N-omega-X). In some embodiments, the surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C 8 -C 20 It is an alkyl.

[0099] In other embodiments, the peptide product of formula IA, III-A, or III-B has the structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.780) During the ceremony, aa 2 is Gly or Aib; aa 16 is Glu, Ala, or Aib; aa 17is Arg or hArg; aa 20 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, the surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C 8 -C 20 It is an alkyl.

[0100] In other embodiments, the peptide product of formula IA, III-A, or III-B has the structure: His 1 -aa 2 -Gln 3 -Gly 4 -Thr 5 -aa 6 -Thr 7 -Ser 8 -Asp 9 -aa 10 -aa 11 -Z (SEQ.ID.NO.781) During the ceremony, aa 2 is Gly, Aib, or MePro, aa 6 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, the surfactant X is a 1'-alkyl β-D-glucuronyl. In some embodiments, the alkyl group is a linear C 8 -C 20It is an alkyl.

[0101] In other embodiments, the peptide product of formula IA, III-A, or III-B has the structure: His 1 -aa 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 -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 -NH 2 (SEQ.ID.NO.795) During the ceremony, aa 2 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 structure: His 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr10 -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 -NH 2 (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 comprises a 1-alkyl β-D-glucoside, a 1-alkyl β-D-maltoside, a 1-alkyl β-D-melibioside, etc., or the corresponding α-anomer, where the alkyl is a linear C 8 -C 20 It is an alkyl.

[0103] In other embodiments, the peptide product of formula IA, III-A, or III-B has the structure: 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 * -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 -NH 2 (SEQ.ID.NO.797) During the ceremony, Glu 16 * and Lys 20 * is cyclized through its side chain to form a lactam bond; and The surfactant X comprises a 1-alkyl β-D-glucoside, a 1-alkyl β-D-maltoside, a 1-alkyl β-D-melibioside, etc., or the corresponding α-anomer, where the alkyl is a linear C 8 -C 20 It is an alkyl.

[0104] In other embodiments, the peptide product of formula IA, III-A, or III-B has the structure: His 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -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 structure: 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 -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 structure: His 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -aa 10 * -Ser 11 -Lys 12 -Tyr 13 -aa 14 * -Asp 15 -Ser 16 -aa 17 -Ala 18 -Ala19 -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 structure: His 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -aa 12 * -Tyr 13 -Gln 14 -Asp 15 -aa 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.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 structure: 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 -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 comprises a 1-alkyl β-D-glucoside, a 1-alkyl β-D-maltoside, a 1-alkyl β-D-melibioside, etc., or the corresponding α-anomer, where the alkyl is a linear C 8 -C 20 It is an alkyl.

[0109] In other embodiments, the peptide product of formula IA, III-A, or III-B has the structure: 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 * -Gln 17 -Ala18 -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 -Z (SEQ.ID.NO.1113) Where: Z is -OH or -NHR 3 and Glu 16 * and Lys 20 * is cyclized through its side chain to form a lactam bond; and The surfactant X comprises a 1-alkyl β-D-glucoside, a 1-alkyl β-D-maltoside, a 1-alkyl β-D-melibioside, etc., or the corresponding α-anomer, where the alkyl is a linear C 8 -C 20 It is an alkyl.

[0110] In some embodiments, the peptide product has the following structure: 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 * -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 -NH 2 (SEQ.ID.NO.601); 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 * -Gln 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 -NH 2 (SEQ.ID.NO.602); 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 * -Gln 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 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.603); 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 * -Gln 17 -Ala 18 -Ala 19 -Lys 20 * -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-octadecyl β-D-glucuronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.604); His1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leo 14 -Asp 15 -Glu * 16 -Gln 17 -No 18 -No 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-Lys(1-β-D-Lys]) 24 -Trp 25 -Leo 26 -Leo 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.630); 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 -Leo 14 -Asp 15 -Glu * 16 -Gln 17 -No 18 -No 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23-Lys(N-omega[1-dodecyl β-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.631); 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 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-tetradecyl β-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.632); 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 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-hexadecyl β-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.633); 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 -Gln 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Lys(N-omega[1-octadecyl β-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.634); His 1 -Aib 2 -Gln 3 -Gly4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -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-hexadecyl α-D-melibiouronyl]) 24 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.805); 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-tetradecyl α-D-melibiouronyl]) 17 -Ala 18 -Ala 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24-Trp 25 -Leo 26 -Leo 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.819); 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 -Leo 14 -Asp 15 -Glu * 16 -Lys(N-オメガ[1-ヘキサデオルα-D-メリビオオロニル]) 17 -No 18 -No 19 -Lys * 20 -Glu 21 -Phe 22 -Ile 23 -Gln 24 -Trp 25 -Leo 26 -Leo 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.820); 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 -Leo 14 -Asp 15 -Glu *16 -Lys(N-omega[1-octadecyl 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 -NH 2 (SEQ.ID.NO.821); 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-dodecyl α-D-melibiouronyl]) 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 -NH 2 (SEQ.ID.NO.1114); 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-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 -NH 2 (SEQ.ID.NO.1115); 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-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 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.1116); 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-(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 -NH 2 (SEQ.ID.NO.1117); 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-(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 -NH 2 (SEQ.ID.NO.1118); 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 -Trp 25 -Leu 26 -Leu 27 -Gln 28 -Thr 29 -NH 2 (SEQ.ID.NO.1119); His1 -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 -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 -NH 2 (SEQ.ID.NO.1120); 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 -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 -NH 2 (SEQ.ID.NO.1121); 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 -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 -NH 2 (SEQ.ID.NO.1122); 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 -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 -NH 2 (SEQ.ID.NO.1123); 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 -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 -NH 2(SEQ.ID.NO.1124);or 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 -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 -NH 2 (SEQ.ID.NO.1125);or and pharma- ceutically acceptable salts thereof, wherein Glu 16 * and Lys 20 * represents a residue that can be cyclized through 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] FIG. 5 illustrates the X-ray crystal structure of the GLP-1 receptor N-terminal region / extracellular domain binding site, 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, which exceeds 1, interacts with this region as an amphipathic helix.) The 1-alkyl group of the surfactant moiety of the peptide product may participate 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 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 may be

[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 of 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, 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., C-terminus, C-aminopropyl ... 8 -C 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 the peptide products described herein includes all pharma- ceutically 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 the binding of the peptide surfactant conjugate to a hydrophobic carrier protein such as human serum albumin (HSA), which increases the half-life and duration of action of the peptide product through a number of mechanisms, including increased resistance to proteases and other types of hydrolytic enzymes, 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 surfaces 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, so that the peptide product avoids 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 amphipathic 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 the tight junctions between cells in the mucosal cell membrane, 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 the membrane. Furthermore, the process of breaking down the micelles can extend the duration of action of the peptide product. Surfactants form micelles when the concentration is greater than the critical micelle concentration (CMC), and alkyl glycosides with the same sugar group tend to have a lower CMC for longer alkyl chains.

[0122] The peptide product becomes a drug in itself due to the significantly increased plasma and elimination half-life compared to pellets not bound to the surfactant moiety. 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 the receptors of the basic class of peptides. 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 Compositions The present disclosure also provides a pharmaceutical composition comprising a peptide product described herein, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers or excipients. The pharmaceutical composition comprises a therapeutically effective amount of the peptide product, or a suitable fraction thereof. The composition can optionally include 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 pharma-ceutically 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, thickening agents, solvents, isotonicity agents, buffers, pH adjusters, absorption retarders, 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 includes the use of conventional excipients and carriers in formulations containing 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] Suitable formulations may depend on various factors, such as the route of administration selected. Possible routes of administration of pharmaceutical compositions containing 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., by nasal spray or drops], ocular [e.g., by eye drops], pulmonary [e.g., by oral or nasal inhalation], buccal, sublingual, rectal [e.g., by suppository], and vaginal [e.g., by suppository]. In some 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 that may contain submicron anhydrous α-lactose or / and other excipients, such as in formulations for administration by oral or nasal inhalation or insufflation.

[0127] In some embodiments, the peptide product is administered parenterally (e.g., subcutaneously, intravenously, or intramuscularly) by injection. Parenteral administration 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 solutions, buffered saline [e.g., PBS], balanced salt solutions [e.g., Ringer's BSS], and aqueous dextrose), isotonic / isoosmotic agents (e.g., salts [e.g., NaCl, KCl, and CaCl 2], and sugars [e.g., sucrose]), buffers and pH adjusters (e.g., sodium dihydrogen phosphate [monobasic sodium phosphate] / disodium hydrogen phosphate [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] Excipients 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] linked 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 variants thereof)}. 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, which is where the volume of the bolus administration is limited (e.g., about 1.5 mL or more). In addition, such substances can be used to stabilize the peptide during preparation, storage, and reconstitution of lyophilized peptides.

[0129] Examples of excipients for aqueous formulations, whether pre-manufactured with the peptide therapeutic for parenteral (e.g., subcutaneous or intravenous) administration or prepared following reconstitution of the 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 specifically 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 provided, for example, in a pre-filled syringe of a disposable pen, or in a pen with a dose counter. Alternatively, the peptide product can be dissolved or suspended in an aqueous solvent, which may optionally contain one or more excipients, before lyophilization (freeze-drying). Just before parenteral administration, the lyophilized peptide product stored in a suitable container (e.g., a vial) can be reconstituted, for example, with sterile water, which may optionally contain one or more excipients.

[0133] In other embodiments, the peptide product is administered intranasally. The nasal mucosa provides a large surface area, a highly porous endothelium, a high degree of vascular epithelial layer, and a high rate of absorption, thus allowing for a high degree of bioavailability. Intranasal formulations may include the peptide product 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 shows typical excipients for nasal spray formulations.

[0134] [Table 2]

[0135] In further embodiments, the peptide product is administered pulmonary, such as by oral or nasal inhalation. Pulmonarily administered drugs can treat lung diseases or / and systemic disorders, since the lungs act 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) large surface area in the alveolar region for absorption, high permeability of the lungs (thin air-blood barrier), and sufficient vasculature of the airways; and 4) low extracellular enzyme levels compared to the GI tract due to the large alveolar surface area. Advantages of oral administration over nasal inhalation include deep penetration / deposition of drugs in the lungs, while nasal inhalation can deliver drugs to the oral mucosa in the nasal cavity and lungs to the systemic circulation.

[0136] Oral or nasal inhalation can be accomplished, for example, by a metered dose inhaler (MDI), nebulizer, or dry powder inhaler (DPI). For example, the peptide product may be formulated for aerosol administration to the respiratory system by oral or nasal inhalation. The drug may be delivered in small particle sizes (e.g., about 0.5 microns to about 5 microns) available through micronization to improve, for example, drug deposition in the lungs and stability of the drug suspension. The drug may be provided in a pressurized pack 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). The drug in the aerosol formulation is dissolved or often suspended in the propellant for delivery to the lungs. Aerosols contain excipients such as surfactants (which enhance lung 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 can be, for example, phospholipids such as lecithin) or / and stabilizers, although the surfactant portion of the peptide product can perform the surfactant function. For example, an MDI formulation can contain the peptide product, a propellant (e.g., an HFA such as 1,1,1,2-tetrafluoroethane), and a co-solvent (e.g., an alcohol such as ethanol), and optionally, a surfactant (e.g., a fatty acid such as oleic acid). MDI formulations can optionally contain dissolved gases (e.g., CO 2 After activation of the device, the CO in the released aerosol droplets may 2The bursting of the gas bubbles causes the droplets to break up into smaller droplets, thereby increasing the respirable fraction of the drug. As another example, a nebulizer formulation may contain a peptide product, a chelating agent or preservative (e.g., disodium edetate), 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). The drug 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., about 20-100 μL) per actuation of the device. MDIs usually generate aerosol faster than the user can inhale, which can result in a lot of 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 coordinated devices. Breath-actuated MDIs (e.g., Easibreathe®) are actuated when the device senses the user's inhalation and expels a drug 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 allows for evaporation of the propellant from the larger droplets by slowing the rate at which the aerosol enters the mouth. The spacer simplifies the use of the inhaler and increases the amount of drug deposited in the lungs instead of the upper airways. Spacers are sometimes made of antistatic polymers to minimize electrostatic adhesion of released drug particles to the inner walls of the spacer.

[0138] Nebulizers generate aerosol droplets of approximately 1-5 microns. Nebulizers do not require user coordination between actuation of the device and inhalation, which can greatly 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 can aerosolize a fixed amount of drug per pulse and may include a photoacoustic trigger that 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), for example, to improve 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 area of ​​the terminal bronchioles and alveoli. In contrast, the majority of larger particles (>5 microns) do not follow the airflow to the many branches of the airways and are deposited by impaction in the upper airways, including the oropharyngeal region of the throat. DPI formulations can contain drug particles alone or mixed with larger suitable base / carrier powders such as lactose, starch, starch derivatives (e.g., hydroxypropyl methylcellulose), or polyvinylpyrrolidine. Carrier particles enhance flow, reduce aggregation, improve dose uniformity, and aid in the dispersion of drug particles. DPI formulations may optionally contain excipients such as magnesium stearate or / and leucine, which improve the performance of the formulation by preventing interparticle binding (due to anti-adhesive effects). Powder formulations may be provided in unit dosage forms such as capsules (e.g., gelatin capsules) or cartridges in blister packs that can be manually loaded or preloaded into the inhaler. Drug particles can be drawn into the lungs by placing the mouthpiece or nosepiece of the inhaler into the mouth or nose, inhaling hard and deep 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, the airflow through the device creates shear and turbulence, and the inhaled air is directed into the powder bed, causing the electrostatic powder mixture to become fluid and enter the user's airways. There, the drug particles are separated from the carrier particles by the turbulent airflow and are carried deep into the lungs, while the larger carrier particles impact the surfaces of 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 drug and larger size drugs (e.g., macromolecules) to the lungs because they create turbulence and high velocities that increase drug deposition due to impaction 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., about 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 that has a ≦5 micron aerodynamic particle size in the inspired air. A high FPF, and therefore good DPI performance, can be obtained from a DPI formulation, for example, comprising 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 almost 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-activated ("passive"), relying on the user's inhalation to generate 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 used 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), which 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 unit dose), Exubera® (single unit dose), MicroDose® (multiple unit dose and electronically activated), Omnihaler® (single unit dose), Pfeiffer DPI (single unit dose), and Spiros® (multiple unit dose).

[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 enhances GI absorption by protecting against enzymatic degradation through local buffering. Oral dosage forms (e.g., tablets, capsules, or pills) can optionally be enteric coated to protect the contents from the strong acid 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, extended, extended, delayed, slow-release, and controlled release compositions, systems, and devices. In some embodiments, the sustained release composition delivers the peptide product for 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 composed of a biodegradable polymer and incorporating the peptide product. In some embodiments, the biodegradable polymer comprises lactic acid or / and glycolic acid (e.g., L-lactic acid-based copolymers such as poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid)).

[0148] In further embodiments, 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. In 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 need to be mixed to form an administered composition. Unit dosage forms generally contain a therapeutically effective dose of drug, but can contain an appropriate fraction thereof, so that a therapeutically effective dose can be achieved by presenting multiple unit dosage forms. Examples of unit dosage forms include tablets, capsules, or pills for oral ingestion; solutions in pre-filled syringes, 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 an inhaler.

[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 may contain the active and non-active ingredients all in unit dosage form, or in two or more separate containers, and may include instructions for administration or use of 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 the peptide product or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising same, and instructions for administration or use of the peptide product or composition to treat a medical disease disclosed herein, such as insulin resistance, diabetes, obesity, metabolic syndrome, or cardiovascular disease, or a disease 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 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, 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 a variety of mechanisms, including stimulating blood glucose-dependent insulin secretion, increasing insulin sensitivity, stimulating fat burning, and reducing body weight. 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), liver disorders (e.g., alcoholism), and the like. These include fatty liver disease [FLD], including alcoholic and nonalcoholic FLD, neurological disorders (e.g., carpal tunnel syndrome, dementia [e.g., Alzheimer's disease and vascular dementia], paresthesia neuralgia, 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, esophageal, colorectum, gallbladder, kidney, liver [e.g., hepatocellular carcinoma], pancreatic, and skin cancers [e.g., melanoma], and leukemia).

[0156] In some embodiments, the peptide products described herein are used to treat polycystic ovarian 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, FLD is non-alcoholic fatty liver disease (NAFLD). In some embodiments, NAFLD is non-alcoholic steatohepatitis (NASH). FLD, also known as fatty liver, is characterized by excess 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 may progress to cirrhosis or liver cancer (e.g., hepatocellular carcinoma [HCC]). Less than about 10% of people with AFLD with cirrhosis will develop HCC, while up to about 45% of people with NASH without cirrhosis may 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, especially free fatty acids and triglycerides, accumulate in liver cells (fatty liver) due to causes other than excessive alcohol consumption, such as nutrient overload, high caloric intake, and metabolic dysfunction (e.g., dyslipidemia and defective glucose control). The liver may remain fat-laden without interfering with liver function, but fatty liver may progress to NASH, a disease in which adiposity is accompanied by inflammation, hepatocyte ballooning, and cellular damage with or without liver fibrosis. Fibrosis is the strongest predictor of death from NASH. NAFLD is characterized by adiposity alone; adiposity with lobular or portal venous inflammation but without ballooning; adiposity with ballooning but without inflammation; or adiposity with inflammation and ballooning.

[0159] NASH is the most extreme form of NAFLD. NASH is a progressive disease, with approximately 20% of patients progressing to cirrhosis and approximately 10% dying 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 pharma- ceutically acceptable salts thereof.

[0162] The peptide product may be administered by any route suitable for the treatment of 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 mucosal, intranasal (e.g., by nasal spray or drops), ocular (e.g., by eye drops), pulmonary (e.g., by oral or nasal inhalation), buccal, sublingual, rectal (e.g., by suppository), and vaginal (e.g., by 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 the 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 a day, 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 some 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 of a medical disease with a peptide product can be determined by the treating physician based, for example, on the nature and severity of the disease and the subject's response to treatment. In some embodiments, the peptide product is administered to treat a disease 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 disease disappear or a clinical target is achieved, such as blood glucose levels, blood pressure, blood levels of lipids, body weight or body mass index, waist-to-hip ratio, body fat percentage, or any combination thereof. If clinical symptoms of the disease return or clinical targets are not maintained, administration of the peptide product can be resumed.

[0168] The present disclosure provides a method of treating a medical disease disclosed herein, comprising administering to a subject a therapeutically effective amount of a peptide product described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. The present disclosure further provides a peptide product described herein or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof in the preparation of a medicament. A medicament comprising a peptide product can be used to treat any medical disease described herein. The peptide product can be optionally 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, glycilamide [tolhexamide], metahexamide, tolazamide, and tolbutamide), and second generation (e.g., glibenclamide [glyburide], glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, and glyclopyramide)} that inhibit ATP-dependent K+ receptor activation 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] Antiobesity drugs include, but are not limited to: Appetite suppressants (appetite reducers), including amphetamine, dextroamphetamine, amfepramone, clobenzorex, matindol, 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, everactone A and B, estellastin, lipstatin, orlistat, persikinin, panclicin AE, valilactone, and vibralactone; Antihyperlipidemic agents; and their analogs, derivatives, and salts;

[0173] Antihyperlipidemic agents include, but are not limited to, the following: 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); Thiazolidinediones (mentioned 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 J 2 [15d-PGJ2 ]), and PPAR-γ agonists, including 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, terpendol 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'-piperidine]-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, aka stearoyl-CoA delta-9 desaturase) activity or expression, including the cis-9, trans-11 isomer, and the trans-10, cis-12 isomer of stearoyl-CoA delta-9 desaturase, 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 transfer 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 (above); Fibroblast growth factor 21 (FGF21), including BMS-986036 (pegylated FGF21), and its analogs and derivatives; 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 PCSK9, PCSK9-binding adnectins (e.g., BMS-962476), antisense polynucleotides and PNAs that target mRNA for PCSK9, and PCSK9-targeting siRNAs (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-CH 1, 5A-CH2, 5A-H1, 18A, 37pA [18AP-18A], ELK [name], ELK-1A, ELK-1F, ELK-1K1A1E, ELK-1L1K, ELK-1W, ELK-2 A, 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 apoE mimetics (e.g., Ac-hE18A-NH 2[AEM-28], Ac-[R]hE18A-NH 2 apolipoprotein mimetic peptides, including 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); Alpha- 2 -Adrenergic receptor agonists; Alpha-amycin, including doxazosin, indoramin, nicergoline, phenoxybenzamine, phentolamine, prazosin, terazosin, and tolazoline 1 -Adrenergic receptor antagonists (alpha-blockers); β-adrenergic receptor (β ) antagonists, including atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, and timolol 1 or / and β 2 ) antagonists (beta-blockers); mixed 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 analogues (e.g., limaprost and misoprostol), prostacyclin and its analogues (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, taprostenyl, 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, the following: 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 activation and migration of inflammatory cells (e.g., macrophages) to the liver and reduce 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 conjunction 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 or / and 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. EXAMPLES

[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, N-ε-(1-octyl β-D-glucuronide-6-yl)L-lysine Place 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) in an oven-dried 250 mL Erlenmeyer flask. Add a cooled (4 °C) solution of N,N'-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in 50 mL of DMF with stirring and allow the reaction to proceed for 5 min. Filter the copious white precipitate of N,N'-dicyclohexylurea on a fritted glass funnel and add the filtrate to a solution of N-α-Fmoc-L-lysine (3.68 g, 10 mmol) in 25 mL of anhydrous DMF. Allow the reaction to proceed for 25 min while warming to room temperature or until the color of the ninhydrin has faded considerably. Filter the reaction mixture, remove to dryness, and eluate by dissolution in MeOH and Et 2 Slow dilution with 200 to the cloud point resulted in the formation of MeOH / Et 2 Crystallize from O followed by freezing. 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 incorporation and cleavage at the N-terminus 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 incorporation at the N-terminus of 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. A similar reaction using other N-mono-protected diamino acids gave the corresponding reagents. Alternatively, a transient Me-protection during coupling and without prior activation of 1-octyl β-D-glucuronic acid was performed. 3 The use of Si-ester protecting groups provides a facile route to the formation of the reagent. 3Si esters are prepared by dissolving in dichloromethane (CH 2 Cl 2 The organic layer is prepared by reaction of Fmoc-Lys-OH with an equimolar amount of N,O-bis(trimethylsilyl)acetamide in CH 2 Cl 2 . The organic layer is then purified by HPLC, ready for coupling with 1-alkyl glucuronides as described above. 2 Cl 2 The filtered reaction mixture contains the desired reagents as a solution in NaHSO 4 Wash with aqueous solution of Me 3 The Si ester is hydrolyzed, dried over MgSO4 and the solvent removed.

[0184] Similarly, the use of peracetyl or perbenzoyl 1-octyl β-D-glucuronic acid allows the synthesis of Ac- or Bz-protected forms of the reagent (e.g., Ac 2 Such reagents increase the stability during acidic cleavage from the resin and are used to detect instability during deprotection. Final deprotection of such products can be achieved using MeOH / NH 3 , MeOH / NaOMe, or MeOH / NH 2 NH 2 With the use of , after cleavage, this is carried out by base-catalyzed transesterification.

[0185] Example 2. Synthetic peptides In general, peptide synthesis methods require the successive addition of protected amino acids to a growing peptide chain. Usually, either the amino or carboxyl group of the first amino acid and any reactive side groups are protected. This protected amino acid is then coupled 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 the formation of an amide bond. After all the 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 less than about 50 amino acids involves solid-phase peptide synthesis. In this method, the α-amino (Nα) group, and any reactive side chain functional groups, are protected by 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-dimethoxybenzyloxy-carbonyl, 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 the synthesis of compounds is Fmoc. Preferred side chain protecting groups include -Ot-butyl group 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 modifications of the Lys side chain 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 schemes are 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 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-di-isopropylethylamine (DIEA) using standard protocols in the art.

[0188] In solid-phase synthesis, the C-terminal amino acid is first coupled to a suitable resin support. A suitable resin support is a material that is inert to the reagents and reaction conditions of the stepwise condensation and deprotection reactions and is insoluble in the medium used. Examples of commercially available resins include styrene / divinylbenzene resins modified with reactive groups, such as chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylated co-poly-(styrene-divinylbenzene), etc. Benzylated, hydroxymethylated, phenylacetamidomethyl (PAM) resins, 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 resins ("Rink amide"). Preferred supports for the synthesis of larger peptides are commercially available resins that contain 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. Thus, in some cases, it may be desirable to have an amide bond to the PEG chain. In such cases, it is preferred to couple N-Fmoc-amino-PEG-carboxylic acid to an amide-forming resin, such as Rink amide resin. The first amino acid of the chain can be coupled to the amino function of the PEG chain as an N-Fmoc-amino acid. Final deprotection provides the desired peptide-NH-PEG-CO-NH 2 The product is obtained.

[0189] Coupling to PAM or HMPA resin can be accomplished 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 12-72 hours, preferably about 48 hours, at elevated temperatures, e.g., about 40-60° C., preferably about 50° C. This ultimately results in the product of a peptide acid after acidolysis, or an amide after aminolysis.

[0190] Nα-Boc-amino acids are, for example, 2 Cl 2 or DMF, preferably CH 2 Cl 2 or the like, at a temperature of about 10-50° C., preferably 25° C., for about 2-24 hours, preferably about 2 hours, via DIC / HOBt mediated coupling to the benzhydrylamine resin.

[0191] For Boc-based protocols, the sequential coupling of protected amino acids can be carried out by methods known in the art, usually in 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 about a 1.5- to 2.5-fold molar excess and reacted in CH at ambient temperature. 2 Cl 2The coupling is carried out in an inert, non-aqueous, polar solvent, such as 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 usually carried out in DMF, NMP, DMA, or mixed solvents, preferably DMF. Representative coupling agents are N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropyl-carbodiimide (DIC), and other carbodiimides, 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 coupling agents are 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), etc.

[0192] A preferred method of coupling 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 in 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 coupling 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. Thus, they are essentially unnatural amino acids and are treated similarly to other amino acids for synthesis. For example, Fmoc-amidooxy-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 provides the corresponding short dPEG C-terminal modified peptides 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 and then 3 minutes at a 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 75° C. for 5 minutes.

[0195] At the end of the solid phase synthesis, the fully protected peptide is removed from the resin. If the bond to the resin support is of the benzyl ester type, cleavage can be achieved by aminolysis with alkylamines or fluoroalkylamines for peptides with alkylamide C-termini, or by ammonolysis, for example with ammonia / methanol or ammonia / ethanol for peptides with unsubstituted amide C-termini, for about 12-24 hours, preferably about 18 hours, at temperatures of about -10 to 50°C, preferably about 25°C. Peptides with hydroxy C-termini 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 peptides by, for example, treatment of 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 on 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 utilizing liquid FH and anisole as described above, or preferably through the use of a milder cleavage cocktail. For example, for PAL-PEG-PS resin, the 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-iso-propylsilane / 3,6-dioxa-1,8-octanedithiol (DODT) (92.5 / 2.5 / 2.5 / 2.5) for 18 minutes each time at 38°C. Cleavage of alkyl glycolide-containing material showed 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% H 2 HO; 2% TIS. Typically, the fully deprotected product is precipitated and purified by elution with cold (-70 to 4 °C) Et 2 Wash with 0, dissolve in deionized water, and freeze-dry.

[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 the 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 packed.

[0199] Provided herein is a process for preparing the covalently modified peptides described herein and their pharma- ceutically acceptable salts, which comprises sequentially condensing protected amino acids on a suitable resin support to obtain a 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 coupled to an alkyl glycoside.

[0200] Example 3. Oxidation Method to Make 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) and upon completion, the reaction mixture was diluted with water and lyophilized to dryness to obtain 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 acid is prepared, which is used to make the products and reagents described herein.

[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 with a C-terminal amide (EU-A387) 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 N-Fmoc-protected amino acids as described in Example 1 and incubated in DMF / CH4 overnight at room temperature in the dark. 2 Cl 2 (1:1), Pd(PPh 3 ) 4 (0.5 equiv.) and DMBA (20 equiv.). 2 Cl 2 After washing with , the Lys side chain was cleaved with DMF / CH using DIC / HOBt. 2 Cl 2 The product was acylated with 1'-dodecyl β-D-glucuronic acid in 1,2-dichloro-1,2-diphenyl-2,3-tetrahydrofuran. The completion of the coupling was confirmed by ninhydrin and the product was purified by CH 2 Cl 2 It was washed extensively with.

[0203] The product resin was incubated at room temperature for 240 min with a final deprotection and cleavage cocktail (94% TFA:2% EDT; 2% H 2The mixture was subjected to cleavage from the resin by treatment with Et 2 2H to precipitate the product, and 2 Extensive washing with O gave the crude title peptide product after drying in vacuum.

[0204] Purification was carried out in two batches by reversed-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 in 60 min at 50 °C. The product fractions were lyophilized and purified by analytical HPLC (18.6 min; 30-60% CH in 0.1% TFA). 3 The title peptide product was obtained with a purity of 98% by NMR / mass spectrometry (M+1 peak=2382.14). In a similar manner, other peptide products of the present disclosure were prepared and characterized as follows:

[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-alkyl glucuronyl, or other uronic acid acylated analogs can be prepared by first purification of 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 Glu and Lys residues were deprotected in DMF / CH overnight at room temperature in the dark. 2 Cl 2 (1:1), Pd(PPh 3 ) 4 (0.5 equiv.) and DMBA (20 equiv.). The resin was deprotected by incubation with 0.5% DIEA in DMF (twice), 0.5% sodium diethyldithiocarbamate in DMF (twice), and DMF / CH 2 Cl 2 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 if necessary. DMF / CH 2 Cl 2 After washing with 5% hydrazine hydrate in DMF (10 equiv.) for 15 min each, the Lys side chain was deprotected twice. 2 Cl 2 After washing with 1000 mM NaCl, the side chain amino groups of the deprotected Lys residues were deprotected with DMF / CH using DIC / HOBt. 2 Cl 2 The reaction was completed with 1'-tetradecyl β-D-melibiouronic acid in 10 mL of 1000 mM NaCl. The coupling was confirmed by ninhydrin and the product was purified by CH 2 Cl 2The resulting mixture was washed extensively with ninhydrin. Any couplings that were 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 incubated at room temperature for 240 min with a final deprotection and cleavage cocktail (94% TFA:2% EDT; 2% H 2 The mixture was subjected to cleavage from the resin by treatment with Et 2 2H to precipitate the product, and 2 The crude title peptide product was obtained after extensive washing with O and drying in vacuum. Typically, 5-8 g of crude peptide product was obtained.

[0208] Purification was carried out in two batches by reversed-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 in 70 min at room temperature. Repurification of less pure fractions was performed on fractions with purity >70%. Product fractions were lyophilized and purified by analytical HPLC (10.3 min, 45-75% CH in 0.1% TFA). 3 EU-A1077 was obtained with a purity of 98.7% by CN / mass spectrometry (1317.67, +3 charged; 1976.13, +2 charged; molecular weight 3950.44). In a similar manner, other peptide products of the present disclosure were prepared and their characteristics are shown 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 purification of 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 carried out 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% CH for 30 minutes 3 CN [b] 30% to 60% CH for 20 minutes 3 CN [c] 35% to 65% CH for 20 minutes 3 CN [d] 25% to 55% CH for 20 minutes 3 CN [e] 40% to 70% CH for 20 minutes 3 CN [f] 45% to 75% CH for 20 minutes 3 CN 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% CH for 30 minutes 3 CN [b] 30% to 60% CH for 20 minutes 3 CN [c] 35% to 65% CH for 20 minutes 3 CN [d] 25% to 55% CH for 20 minutes 3 CN [e] 40% to 70% CH for 20 minutes 3 CN [f] 45% to 75% CH for 20 minutes 3 CN [g] 50% to 80% CH for 20 minutes 3 CN [h] 10% to 40% CH for 20 minutes 3 CN [i] 30% to 90% CH for 20 minutes 3 CN [j] 10% to 90% CH for 20 minutes3 CN [k] 30% to 95% CH for 20 minutes 3 CN [l] 30% to 60% CH for 30 minutes 3 CN [m] 20% to 100% CH for 20 minutes 3 CN [n] 20% to 80% CH for 20 minutes 3 CN [o] 30% to 50% CH for 20 minutes 3 CN 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 stock solution of 1 mg / mL was prepared in DMSO / CH 3 Prepared in 50% CHCN (1 / 1). 3 A standard working solution in CN was prepared 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 working solution was spiked into 90 μL of blank plasma and the mixture was vortexed. 300 μL of internal standard solution (verapamil, 100% CH 3 20 ng / mL in CN) was added and the mixture was vortexed and centrifuged. The supernatant was transferred to an HPLC injection plate and loaded onto the HPLC column. 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 quantification) and sampled at time points t=0, 0.5, 1, 2, 4, and 8 hours during incubation at 30° C. Samples were diluted with internal standard solution (100% CH) as described above. 3CN) to precipitate the peptides, and the supernatant was loaded onto an injection plate and then onto an HPLC column for quantification 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 and 5 mM NHOAc in mobile phase B:CH. 3 0.1% formic acid in CN, and 10 μL of sample was 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 showed protection from proteolytic degradation in in vitro assays, and long plasma half-lives.

[0220] Example 7. Cellular assays of activity of peptide products at GLP-1 and glucagon receptors Compounds were accurately weighed in amounts of approximately 1 mg and assayed in a standard cell assay (Cerep SA). The readout was the amount of cAMP produced in cells treated with test compounds in either 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 (murine 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 and altered GLP1R cellular responses with an EC 50 rose sharply due to

[0222] [Table 7]

[0223] Further series of cell assays are carried out by standard cell assays (DiscoveRx, LeadHunter assay) using cAMP stimulation or arrestin activation readout.Compounds are accurately weighed in approximately 1mg amounts and shipped to DiscoveRx for dilution and assay.Cell assays use human glucagon receptor cloned into CHO cells and 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 body 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 as vehicle in saline, pH 7.4. The other group of eight female mice received liraglutide as a positive control compound at a dose level of 50 nmoles / kg. Test compounds, vehicle, and positive control compounds 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 prior to randomization, and daily on days 1 through 5. Body weights were measured and recorded at receipt, prior to randomization, and daily on days 1 through 5. Food intake was measured and recorded daily on days 1 through 5. Blood samples for glucose analysis were collected prior to the 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 low dose EU-A1026 on days 3 and 4. With regard to food intake, animals treated with liraglutide were significantly different from vehicle on days 1 and 2, and high dose EU-A994 on day 1, and 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, from liraglutide. Glucose levels for liraglutide at 10 hours, and high dose EU-A994 at 10 hours 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] FIG. 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 5 mice per cage. The animal room was illuminated throughout by artificial fluorescent lighting with a controlled 12-h light / dark cycle. Standard temperature and relative humidity ranges in 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 each group (n=10): Group 1: vehicle treatment; 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 dosed on days 1 (0 and 7 hours), 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 following 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 (GLP-1 agonist) and elafibranor (PPAR-α / δ agonist).Nonalcoholic steatohepatitis (NASH) was induced in male C57BL / 6JRj mice (approximately 20 g, 5 weeks old at time of arrival and 37 weeks old at study initiation) by feeding an AMLN diet containing 40% fat (18% trans fat), 2% cholesterol, and 40% carbohydrate (20% fructose) for 32 weeks prior to study initiation and for the duration of the study.

[0236] All mice entering the study were stratified based on pre-biopsy liver 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. Groups in the NASH study were: 1) vehicle (0.05% Tween 80, 50 mM NaCl, 0.01% Tween 80 ... 2 HPO 4, pH8) (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) elafibranor 78 μmole / kg administered orally (PO) QD for 12 weeks (n=12); and 5) semaglutide 10 nmole / kg 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 commercial kits (Roche Diagnostics, Germany) and a Cobas™ C-501 automated analyzer according to the manufacturer's instructions.

[0239] For liver tissue assay, the triglyceride and cholesterol contents in 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 by 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) to histology. The cut surface of the liver was immediately electrocoagulated using a bipolar coagulator (ERBE VIO 100 electrocautery). After prebiopsy, the liver was replaced into the abdominal cavity, the abdominal wall was sutured, and the skin was closed with staples. For postoperative recovery, mice were given carprofen (5 mg / kg) subcutaneously 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 in Pertex, 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 slides were dehydrated in three changes of 99% ethanol, removed with xylene, mounted in Pertex, and then allowed to dry before scanning.

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

[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 low normal range, whereas semaglutide and elafibranor caused less weight loss (Figures 12A and 12B [high dose SP-1373 was inadvertently administered to the vehicle group on day 62]). SP-1373 significantly reduced total body adipose tissue mass (Figures 13A and 13B) with minimal effect on total body lean tissue mass. Total cholesterol (TC, Figure 14B), alanine transaminase (ALT, Figure 15A), and aspartate transaminase (AST, Figure 15B) plasma levels were also significantly reduced in animals treated with SP-1373. Plasma ALT and AST levels, and the ratio of AST / ALT, 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, lowers blood glucose levels, suppresses appetite, and induces weight loss. Glucagon receptor agonism has antihyperlipidemic and satiety effects, stimulates fat burning and energy expenditure, and reduces 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 doses of SP-1373 (Figure 17C [large white oval structures are veins]) in contrast to animals treated with semaglutide (Figure 17E), indicating a moderate reduction in hepatic steatosis. Figures 18A and 18B show post-biopsy hepatic steatosis in terms of % area affected and total hepatic lipid content in the different groups of animals, as determined by quantitative evaluation of histology, confirming the almost complete absence of hepatic steatosis in animals treated with high doses of SP-1373. SP-1373 reduced hepatic triglyceride (TG) and total cholesterol (TC) levels to a greater extent than semaglutide, whereas elafibranor had little or no effect on hepatic TG and TC levels (Figures 19A and 19B). SP-1373 also reduced total hepatic collagen type 1 alpha 1 by anti-col1alpha1 antibodies (a measure of liver fibrosis) to a greater extent than semaglutide, whereas elafibranor had a modest effect (Figure 20). Similarly, SP-1373 reduced total hepatic galectin-3 by 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 0-3 in terms of surface area harboring steatosis. The majority of treated animals showed little 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 0-3 in terms of severity. Nearly all of the 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 plotted as a line, and hepatocyte ballooning was scored for severity from 0-2. The scores for fatty liver, inflammation, and ballooning are set forth in Table 9.

[0248] NAS (non-alcoholic fatty liver disease [NAFLD] activity score), a clinical measure of NASH activity, improved in the majority of treated animals, with the highest extent of reduction achieved by high doses of SP-1373 (Figures 25A-25E). In Figures 25A-25E, a line shows the change in biopsy from pre-study to post-study for each animal. The NAS score is the sum of the scores for fatty liver (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 as having 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 doses of 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. In Figures 26A-26E, for each animal, a line is drawn depicting the change in biopsy from pre-study to post-study, and liver fibrosis was scored from 0-4 as outlined in Table 9.

[0250] [Table 10]

[0251] SP-1373 has been shown to inhibit several key mechanisms of inflammation, including 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, into, for example, macrophages {e.g., GAL3, EGF-like module-containing mucin-like hormone receptor-like 1 (EGF-1, aka GAL3, ... SP-1373 reduced the expression of NASH-related mRNAs in the liver, including mRNAs encoding proteins involved in fibrosis {EMR1, aka F4 / 80), CD14, CD68, and CD86} (Figures 27A-27F and Figures 28A-28F). More specifically, with respect to fibrosis, SP-1373 reduced hepatic expression of mRNAs encoding proteins involved in fibrosis fibrogenesis (Figures 29A-29I), and activation of hepatic stellate cells (the main cell type involved in liver fibrosis) and myofibroblast proliferation (Figures 30A-30F). In addition, 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 respect to the figures referred to in this paragraph, the expression levels of specific mRNAs are mean + SEM and compared to vehicle after gene-wise multiple test 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 NAS scores in a well-recognized conversion mouse model of NASH, while also addressing metabolic and glucose regulatory defects. 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, reduces body weight, adipose tissue mass, and food intake to the greatest extent, and has a longer duration of action. In DIO-NASH mouse model, treatment with SP-1373 resulted in significant reductions in 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 alpha 1 content (a measure of hepatic fibrosis), and hepatic galectin-3 content (a measure of hepatitis 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 diseases related thereto, such as NAFLD (including NASH).

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

Claims

1. 2. Use of a peptide product of GLP-1 / glucagon, or a pharma- ceutical acceptable salt thereof, in the manufacture of a medicament for the treatment of fatty liver disease, comprising: 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 １ ５ -Glu １６ ＊ -Lys(N-omega[1-(17-carboxyl-heptadecyloxy) β-D-glucuronyl １７ -Ala １８ -Ala １９ -Lys ２０ ＊ -Glu ２ １ -Phe ２２ -Ile ２３ -Gln ２４ -Trp ２５ -Leu ２６ -Leu ２７ -Gl n ２８ -Thr ２９ -NH ２ (SEQ.ID.NO. 1119), and pharma- ceutically acceptable salts thereof; Here, Glu １６ ＊ and Lys ２０ ＊ represents a residue which is cyclized through its side chain to form a lactam bond, or a pharma- ceutically acceptable salt thereof.

2. The use of a peptide product of GLP-1 / Glucagon according to claim 1, wherein said fatty liver disease is non-alcoholic fatty liver disease (NAFLD).

3. The use of a peptide product of GLP-1 / Glucagon according to claim 1, wherein said fatty liver disease is non-alcoholic steatohepatitis (NASH).

4. The use of a GLP-1 / Glucagon peptide product according to claim 1, wherein said treatment comprises administration of the peptide product via subcutaneous, intravenous or intramuscular injection.

5. The use of a GLP-1 / Glucagon peptide product according to claim 1, wherein said treatment comprises administration of the peptide product by oral inhalation, nasal inhalation or insufflation.

6. The use of a GLP-1 / Glucagon peptide product according to claim 4, wherein said treatment is administration of the peptide product at a dosage of 0.1 mg to 1, 5, or 10 mg, 0.1-1 mg, or 1-10 mg, for a period of about one week.

7. 7. The use of a GLP-1 / Glucagon peptide product according to claim 6, wherein the administration of said peptide product is at a dosage of 0.1-1 mg, 0.1-0.5 mg, or 0.5-1 mg for a period of about one week.

8. The use of a GLP-1 / Glucagon peptide product according to claim 4, wherein said treatment comprises administering said peptide product once per week.

9. The use of a GLP-1 / Glucagon peptide product according to claim 1, wherein said treatment further comprises the administration of one or more additional therapeutic agents.

10. 10. The use of a GLP-1 / Glucagon peptide product according to claim 9, wherein the one or more additional therapeutic agents are selected from diabetes agents, anti-obesity agents, anti-inflammatory agents, anti-fibrotic agents, antioxidants, anti-hypertensive agents, and combinations thereof.

11. 10. The use of a GLP-1 / Glucagon peptide product of claim 9, wherein the fatty liver disease is NAFLD or NASH, and the one or more additional therapeutic agents are selected from a peroxisome proliferator-activated receptor (PPAR) agonist, an HMG-CoA reductase inhibitor, a farnesoid X receptor (FXR) agonist, or an antioxidant, or any combination thereof.

12. 1. Use of a peptide product or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for the treatment of fatty liver disease, said treatment comprising administering said peptide product or a pharma- ceutically acceptable salt thereof to a subject in need thereof, wherein the peptide product is a) 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 -Trp ２５ -Leu ２６ -Leu ２７ -Gln ２８ -Thr ２９ -NH ２ (SEQ.ID.NO.1119), wherein Glu １６ ＊ and Lys ２０ ＊ represents a residue that is cyclized via its side chain to form a lactam bond, and b) a pharma- ceutically acceptable diluent or buffer; Wherein the fatty liver disease is nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH); Use of peptide products.

13. 13. Use of the peptide product according to claim 12, wherein said treatment comprises administering said peptide product via subcutaneous, intravenous or intramuscular injection.

14. 13. Use of a peptide product according to claim 12, wherein said treatment comprises administering said peptide product once per week.

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