Oral peptides and uses thereof for weight loss, weight maintenance, and management of metabolic conditions

Abstract

The present disclosure provides peptides for reducing weight and / or preventing weight gain comprising a GLP-1R agonist. As described herein, the GLP-1R agonist can be administered orally to a subject.

Description

[0001] Atty Ref. METS-030 / 01 WO 350242-2268

[0002] ORAL PEPTIDES AND USES THEREOF FOR WEIGHT LOSS, WEIGHT MAINTENANCE, AND MANAGEMENT OF METABOLIC CONDITIONS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004]

[0001] The present application claims priority to U. S. Provisional Application 63 / 721,870, filed November 18, 2024, the content of which is incorporated herein by reference in its entireties.

[0005] REFERENCE TO THE ELECTRONIC SEQUENCE FILE

[0006]

[0002] The contents of the electronic sequence listing (METS 030 01WO SeqList ST26.xml; Size: 82,936 bytes; and Date of Creation: November 11, 2025) are herein incorporated by reference in their entirety.

[0007] BACKGROUND

[0008]

[0003] Advances in peptide technologies for weight loss, weight maintenance, and the treatment / prevention of metabolic conditions have resulted in the development of incretin / hormone mimics that act as single and / or multi-receptor agonists to relevant target receptors, such as G-protein coupled receptors (e.g., glucagon-like peptide-1 receptor (GLP-1R) and gastric inhibitory polypeptide receptor (GIP)). The current standard for weight loss, weight maintenance, and metabolic disorder management are injectable peptides (e.g, liraglutide, semaglutide, tirzepatide), which, in some instances, have drawbacks such as frequency of injection and side effects that create issues with individual compliance.

[0009] SUMMARY

[0010]

[0004] The present disclosure, in some aspects, relates to peptides, such as oral peptides, that offer significant advantages over current peptide agents, particularly traditional injectable peptide forms. Peptides of the disclosure, in some embodiments, exhibit enhanced stability' and bioavailability, reduce the risk of side effects, and improve individual (e.g, patient, subject, etc.) compliance. Peptides described herein exhibit a biased and extended activity at the human glucagon-like peptide-1 (GLP-1) receptor, which make them ideal compounds for weight loss, weight maintenance (e.g., preventing weight gain), and / or for treating metabolic conditions such as obesity, type 2 diabetes mellitus, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, metabolic dysfunction-associated steatohepatitis, and / or non-alcoholic steatohepatitis. Atty Ref. METS-030 / 01 WO 350242-2268

[0011] Peptides described herein can also be used for the treatment of neurodegenerative diseases (e.g., Alzheimer’s disease, dementia, and Parkinson’s disease).

[0012]

[0005] In some embodiments, a peptide is orally bioavailable (j.e., modified or formulated for oral administration). For example, provided herein are peptides comprising one or more amino acid modifications, biotin moieties, and / or lipid moieties that improve oral bioavailability and / or pharmacokinetics of the peptides, as described herein.

[0013]

[0006] In other aspects, the present disclosure relates to pharmaceutical compositions comprising a peptide described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0014]

[0007] Also provided herein are methods of reducing weight of a subject, the method comprising administering to the subject an effective amount of a peptide described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0015]

[0008] Also provided herein are methods of maintaining weight, for example, preventing weight gain, in a subject, the method comprising administering to the subject an effective amount of a peptide described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0016]

[0009] Also provided herein are methods of stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, or reducing caloric intake in a subject in need thereof, the method comprising administering to the subject an effective amount of a peptide described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0017]

[0010] Also provided herein are methods of modulating β-arrestin signaling and / or GLP-1 receptor internalization, the method comprising contacting a cell with a peptide described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0018] [Oil] In yet other aspects, the present disclosure relates to a peptide described here, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in: (a) reducing weight of a subject; (b) preventing weight gain in a subject; and / or (c) stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, and / or reducing caloric intake in a subject. Atty Ref. METS-030 / 01 WO 350242-2268

[0019]

[0012] In still other aspects, provided herein is use of a peptide described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as a medicament.

[0020]

[0013] The details of certain embodiments of the disclosure are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

[0014] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the disclosure and together with the description, provide non-limiting examples.

[0023]

[0015] FIGs. 1A-1B show line graphs of GLP-1 R activation of cAMP release and P-Arrestin-2 recruitment of peptides semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0024]

[0016] FIG. 2 shows a bar graph of GLP-1 R activation and P-Arrestin-2 recruitment bias for peptides semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0025]

[0017] FIGs. 3A-3B show line graphs of GLP-1R activation of cAMP release (FIG. 3A) and P~ Arrestin-2 recruitment (FIG. 3B) of peptide SEQ ID NO: 6 compared to exendin-4 (Ex-4).

[0026]

[0018] FIG. 4 shows a bar graph of GLP-1R activation and P-Arrestin-2 recruitment bias for peptides exendin-4 (Ex -4), SEQ ID NO:6.

[0027]

[0019] FIGs. 5A-5B show line graphs of GLP-1R activation of cAMP release (FIG. 5A) and P-Arrestin-2 recruitment (FIG. 5B) of pepti de SEQ ID NO:7 compared to Exendin-4 (Ex-4).

[0028]

[0020] FIG. 6 shows a graph of GLP-1R activation of cAMP release of peptides GLP-1, SEQ ID NO:1, SEQ ID NO:32, and SEQ ID NO:33.

[0029]

[0021] FIGs. 7A-7B shows graphs of GLP-1R activation of cAMP release (FIG. 7A) and P- Arrestin-2 recruitment (FIG. 7B) of peptides GLP-1, SEQ ID NO: 6, SEQ ID NO:22, and SEQ ID NO:23.

[0030]

[0022] FIGs. 8A-8D show graphs showing half-lives determined by remaining amount of peptides SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 against FaSSIF / P (FIG. 8A and FIG. 8C) and against trypsin (FIG. 8B and FIG. 8D).

[0031]

[0023] FIG. 9 is a graph showing half-lives determined by remaining amount of tested peptides against trypsin.

[0032]

[0024] FIGs. 10A-10D show graphs of blood glucose concentrations and amounts in mice at 0, 20, 40, and 50 minutes after glucose dosing with peptides SEQ ID NO:3, SEQ ID NON, and SEQ ID Atty Ref. METS-030 / 01 WO 350242-2268

[0033] N0:5 administered 4 hours (FIGs. 10A-10B) and 72 hours (FIGs. 10C-10D) prior to glucose injection. Vehicle was administered as control.

[0034]

[0025] FIGs. 11A-11D show graphs of blood glucose concentrations and amounts in mice at 0, 20, 40, 60, 90 and 120 minutes after glucose dosing with peptides SEQ ID NO.3, SEQ ID NO:4, SEQ ID NO:6 and semaglutide administered 4 hours (FIGs. 11A-11B) and 72 hours (FIGs. IIC-I ID) prior to glucose injection. Vehicle was administered as control.

[0035]

[0026] FIG. 12 shows a line graph of plasma concentration in dogs of peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 from 0 to 96 hours after oral administration.

[0036]

[0027] FIG. 13 shows a line graph of plasma concentration in dogs of peptides SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:7 from 0 to 96 hours after oral administration.

[0037]

[0028] FIG. 14 shows a line graph of plasma concentration in rats of peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 from 0 to 72 hours after IV administration.

[0038]

[0029] FIG. 15 shows a line graph of plasma concentration in mice of peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 from 0 to 72 hours after SC administration.

[0039]

[0030] FIG. 16 is a line graph showing plasma concentration in rats of peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 after from 0 to 48 hours ID administration.

[0040]

[0031] FIG. 17 is a line graph showing plasma concentration in rats of peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 from 0 to 48 hours ID administration.

[0041]

[0032] FIG. 18 shows a line graph of plasma concentration in dogs of peptides SEQ ID NO:22, and SEQ ID NO:23 from 0 to 96 hours after IV administration. The formulation included two unspecified peptides, along with SEQ ID NO:22 and SEQ ID NO:23.

[0042]

[0033] FIG. 19 shows a line graph of plasma concentration in dogs of peptides SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:23 from 0 to 96 hours after oral administration.

[0043]

[0034] FIG. 20 show's a line graph of body weight change an in DIO mice of peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 and semaglutide for 28 days after SC daily dosing.

[0044]

[0035] FIG. 21 show's a line graph of accumulative food intake in DIO mice of peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 and semaglutide for 28 days after SC daily dosing.

[0045]

[0036] FIG. 22 show's a line graph of body weight change an in DIO mice of peptides tirzepatide, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:6 for 19 days after SC daily dosing.

[0046]

[0037] FIG. 23 show's a line graph of accumulative food intake in DIO mice of peptides tirzepatide, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:6 for 19 days after SC daily dosing. Atty Ref. METS-030 / 01 WO 350242-2268

[0047] DEFINITIONS

[0048]

[0038] The following definitions are general terms used throughout the present disclosure.

[0049]

[0039] The term “peptide” includes a polymer of amino acid residues linked together by peptide bonds. Typically, a peptide will be at least the length required by an amino acid sequence provided herein. Peptides provided herein can include natural amino acids and / or unnatural amino acids (z.e., compounds that do not occur in nature but that can be incorporated into a peptide chain) in any combination. One or more of the amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a lipid moiety, a linker for conjugation or functionalization, or other modification. A peptide may be naturally occurring, recombinant, synthetic, or any combination of these. Peptide derivatives are also encompassed herein. A peptide derivative can, for example, comprise one or more derivatizations selected from amidation, glycosylation, carb amyl ati on, acylation, sulfation, phosphorylation, cyclization, lipidization, pegylation, biotinylation, and fusion to another peptide or protein to form a fusion protein. A peptide structure can be modified at random positions within the molecule, or at predetermined positions within the molecule and can include one, two, three or more attached chemical moieties.

[0050]

[0040] Pepti des described herein include those with an ami dated C -terminus. “Amidated C-terminus” refers to peptides wherein the traditional C -terminus of the peptide (-C(=O)OH) is replaced with an amide (-C(=O)NH2).

[0051]

[0041] A peptide provided herein can be of any length. In certain embodiments, a peptide is 50 amino acids or fewer in length. In certain embodiments, a peptide is 45 amino acids or fewer in length. In certain embodiments, a peptide is 41 amino acids or fewer in length. In certain embodiments, a peptide is 40 amino acids or fewer in length. In certain embodiments, a peptide is at least the length of an amino acid sequence provided herein. In certain embodiments, a peptide is the length of an amino acid sequence provided herein.

[0052]

[0042] The term “amino acid” includes a molecule containing both an amino group and a carboxyl group. Unless otherwise indicated, an amino acid is an alpha-amino acid (a-amino acid), the generic structure of which is depicted below (wherein each R is independently II or an amino acid sidechain, z.e., an “a-sidechain”). Unless otherwise indicated, reference to a particular amino acid implies the L-isomer of the amino acid. Each amino acid referred to herein may be denoted by a Atty Ref. METS-030 / 01 WO 350242-2268

[0053] 1- to 4-letter code (e.g., L and Lys represent L-Lysine, etc.).

[0054] R R

[0055]

[0056] a amino acid

[0057]

[0043] The term “iso-aspartic acid” or “isoD” refers to a structural isomer of the amino acid aspartic acid (D), wherein the peptide bond is formed through the P-carboxyl group of the side chain rather than the a-carboxyl group of the main chain.

[0058]

[0044] Suitable amino acids include, without limitation, natural a-amino acids such as D- and L-isomers of the 20 common naturally occurring a-amino acids found in peptides (e.g. A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as provided below), and unnatural a-amino acids. Unless

[0059]

[0045] Exemplary natural a-amino acids (with one-letter code provided in parentheses) include L-alanine (A), L-arginine (R), L-asparagine (N), L-aspartic acid (D), L-cysteine (C), L -glutamic acid (E), L-glutamine (Q), glycine (G), L-histidine (H), L-isoleucine (I), L-leucine (L), L-lysine (K), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), and L- valine (V).

[0060]

[0046] Exemplar} / unnatural a-amino acids include, without limitation, D-arginine, D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid, D-glutamine, D-histidine, D-isoleucine, D-leucine, D-lysine, D-methionine, D-phenylalanine, D-proline, D-serine, D-threonine, D-tryptophan, D-tyrosine, D-valine, Di-vinyl, a-methyl-alanine (Aib), a-methyl-arginine, a-methyl-asparagine, a-methyl-aspartic acid, a-methyl-cysteine, a-methyl-glutamic acid, a-methyl-glutamine, a-methyl-histidine, a-methyl -isoleucine, a-methyl-leucine, a-methyl-lysine, a-methyl-methionine, a-methyl-phenylalanine, a-methyl-proline, a-methyl-serine, a-methyl-threonine, a-methyl-tryptophan, a-methyl-tyrosine, a-methyl -valine, norleucine, and terminally unsaturated a-amino acids. There are many known unnatural amino acids, any of which may be included in the peptides of the present disclosure. See for example, S. Hunt, The Non-Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, edited by G. C. Barrett, Chapman and Hall, 1985.

[0061]

[0047] Unnatural amino acids also include amino acids comprising a substituent (i.e., non-hydrogen group) on the peptide nitrogen. For example, any amino acid described herein can comprise a Ci-6 alkyl group on the peptide nitrogen (“N-alkyl”-amino acid). In certain embodiments, any amino acid described herein can comprise a methyl group on the peptide nitrogen (“N-methyl”-amino Atty Ref. METS-030 / 01 WO 350242-2268

[0062] acid).

[0063]

[0048] The term “amino acid substitution” when used in reference to an amino acid sequence refers to an amino acid of the amino acid sequence being replaced by a different amino acid (e.g, replaced by a natural or unnatural amino acid described herein). An amino acid sequence provided herein may include one or more amino acid substitutions. In certain embodiments, an amino acid sequence provided herein includes 0, 1, 2, 3, 4, or 5 amino acid substitutions.

[0064]

[0049] The term “amino acid addition” when used in reference to an amino acid sequence refers to an amino acid (e.g, a natural or unnatural amino acid described herein) being inserted between two amino acids of the amino acid sequence or added at either end of the sequence. In certain embodiments, an amino acid sequence herein includes 0, 1, 2, 3, 4, or 5 amino acid additions.

[0065]

[0050] The term “amino acid deletion” when used in reference to an amino acid sequence refers to an amino acid of the amino acid sequence being deleted from the amino acid sequence. In certain embodiments, an amino acid sequence herein includes 0, 1, 2, 3, 4, or 5 amino acid deletions.

[0066]

[0051] Throughout the present disclosure, references to “the peptide,” “a peptide,” “the compound,” or “a compound” provided herein are intended to encompass peptides comprising any amino acid sequence provided herein (including any disclosed amino acid substitutions and / or modifications), and pharmaceutically acceptable salts, stereoisomers, tautomers, isotopically labeled derivatives, solvates, hydrates, polymorphs, co-crystals, and prodrugs thereof as described herein.

[0067]

[0052] As is clear to the skilled artisan, the peptide sequences disclosed herein are shown proceeding from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end of the sequence being the C -terminus of the peptide. In some cases, a “-OH” or an “-NH2” moiety at the C-terminus of the sequence indicates a carboxylic acid (COOH) or an amido (CONH2) group at the C-terminus, respectively. In each sequence of the disclosure, a C-terminal “-OH” moiety may be substituted for a C-terminal “-NH2” moiety, and vice-versa.

[0068]

[0053] As used herein, the term “salt” includes any and all salts, and encompasses pharmaceutically acceptable salts. Salts include ionic compounds that result from the neutralization reaction of an acid and a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions) so that the salt is electrically neutral (without a net charge). Salts of the peptides of this invention include those deri ved from inorganic and organi c acids and bases.

[0069]

[0054] The term “pharmaceutically acceptable salt” includes those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower Atty Ref. METS-030 / 01 WO 350242-2268

[0070] animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the peptides of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bi sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenyl propionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(CI-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary' ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aiyl sulfonate.

[0071]

[0055] The terms “biotinylation” and “biotinylated” refer to the process and product of both covalent attachment of one or more biotin moieties or derivatives thereof to molecules and macrostructures, such as a therapeutic protein.

[0072]

[0056] The terms “lipidation” and “lipidated” refer to the process and product of both covalent attachment of one or more fatty acid moieties or derivatives thereof to molecules and macrostructures, such as a therapeutic protein.

[0073]

[0057] The term “neurological disease” includes any disease of the nervous system, including diseases that involve the central nervous system (brain, brainstem and cerebellum), the peripheral Atty Ref. METS-030 / 01 WO 350242-2268

[0074] nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both central and peripheral nervous system). Neurodegenerative diseases refer to a type of neurological disease marked by the loss of nerve cells, including, but not limited to, Alzheimer’ s disease, Parkinson’s disease, amyotrophic lateral sclerosis, tauopathies (including frontotemporal dementia), and Huntington’s disease.

[0075]

[0058] A “subject” to which administration is contemplated includes a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal.

[0076]

[0059] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0077]

[0060] The term “prevent,” “preventing,” or “prevention” includes a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

[0078]

[0061] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0079]

[0062] An “effective amount” of a peptide or composition described herein includes an amount sufficient to elicit the desired biological response. An effective amount of a peptide or composition described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular peptide or composition, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the Atty Ref. METS-030 / 01 WO 350242-2268

[0080] subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a peptide or composition described herein in a single dose or multiple doses.

[0081]

[0063] The term “therapeutically effective amount” refers to an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent.

[0082]

[0064] A “prophylactically effective amount” of a peptide described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a peptide means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0083] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0084] Peptides

[0085]

[0065] The present disclosure provides peptides and pharmaceutical compositions for reducing weight and / or preventing weight gain in a subject (e.g., an obese subject), among other uses. In some embodiments, the peptide is orally bioavailable (i.e., for oral administration). For example, provided herein are peptides comprising one or more biotin moieties and / or one or more lipid moi eties. Peptides having one or more biotin moieties and / or lipid moieties conjugated thereto can have improved bioavailability and / or pharmacokinetics as described herein.

[0086]

[0066] In some embodiments, provided here are peptides comprising an amino acid sequence of X1(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKX28GGPSSGX35PPPX39KX41 (SEQ ID NO: 50), or a pharmaceutically acceptable salt thereof,

[0087] wherein Atty Ref. METS-030 / 01 WO 350242-2268

[0088] Xi is H, F, Y, or D-H;

[0089] Aib is 2-aminoisobutyric acid;

[0090] X28 is D, isoD, N, or Q;

[0091] X35 is A or K;

[0092] X39is G or S; and

[0093] X41 is K or absent; and

[0094] wherein the C -terminus of the amino acid sequence is optionally amidated.

[0095]

[0067] Table A provides exemplary sequences of the peptides of the present disclosure.

[0096] Table A. Exemplary sequences of the peptides of the present disclosure

[0097] SEQ ID NO Sequence

[0098] 1 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK

[0099] 2 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLK(isoD)GGPSSGAPPPSK

[0100] 8 F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSK

[0101] 10 F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSKK

[0102] 12 F(Aib)EGTFTSDLSKQMEEEAVRLFTEWLKQGGPSSGAPPPGKK

[0103] 13 F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGKPPPSKK

[0104] 16 Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK

[0105] 17 F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK

[0106] 18 Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK

[0107] 19 F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK

[0108] 20 F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLK(isoD)GGPSSGAPPPSK

[0109] 45 F(Aib)EGTFTSDLSKQMEEEAVRLFIEWIXNGGPSSGAPPPSK-NH2

[0110]

[0111] 46 Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK-NH2

[0112]

[0068] In some embodiments, the peptides of the present disclosure comprise a C -terminal carboxylic acid. In some embodiments, the peptides of the present disclosure comprise a C-terminal carboxamide, wherein the C -terminal carboxylic acid (-COOH) is amidated to form carboxamide (--CONH2).

[0113]

[0069] In some embodiments, the peptides of the present disclosure comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in Table A. In some embodiments, the peptide of the present disclosure comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO. 10.

[0114]

[0070] In some embodiments, the peptides of the present disclosure comprise one or more biotin moieties. In some embodiments, the lysine (K) residues at positions 12 and / or 27 of the amino acid Atty Ref. METS-030 / 01 WO 350242-2268

[0115] sequence independently comprise a biotin moiety attached to their epsilon nitrogens. In some embodiments, the biotin moiety is of the formula:

[0116] . In some embodiments, the biotin moiety are of the formula:

[0117] S— \ H

[0118]

[0119]

[0071] In some embodiments, the lysine residues at positions 12 and / or 27 of the amino acid sequence comprise the following:

[0120]

[0121] , wherein * indicates attachment to the epsilon nitrogen of the lysine residue. In some embodiments, the lysine residues at positions 12 and / or 27 of the amino acid

[0122] NH

[0123] sequence are of the following structure:

[0124]

[0125]

[0072] In certain embodiments, a peptide described herein comprises a lipid moiety. In some embodiments, the lipid moiety is attached to the C-temiinal amino acid of the peptide. In certain embodiments, the lipid moiety is attached to the epsilon nitrogen of a C-terminal lysine of the peptide.

[0126]

[0073] In some embodiments, the lipid moiety comprises a) one or more groups selected from OH N

[0127] H

[0128] g

[0129]

[0130] roups selected from Ci-20 alkyl, C2-20 alkenyl, or C2-20 alkynyl. In some embodiments, n is 1 or 2. In some embodiments, Atty Ref. METS-030 / 01 WO 350242-2268

[0131] p is 14, 16, 18, or 20.

[0132]

[0074] In some embodiments, the lipid moiety comprises a)

[0133]

[0134]

[0135] is 10-20. In some embodiments, n is 1 or 2. In some embodiments, p is 14, 16, 18, or 20.

[0136]

[0075] In some embodiments, the lipid moiety comprises a)

[0137]

[0138] and b) O O O O JL YOH

[0139] "OH

[0140]

[0141] or 'p, wherein p is 10-20. In some embodiments, p is 14, 16, 18, or 20.

[0142]

[0076] In some embodiments, the lipid moiety comprises glutamic acid. In some embodiments, the lipid moiety comprises a dicarboxylic acid (e.g., eicosanedioic acid). In some embodiments, the lipid moiety comprises glutamic acid and a dicarboxylic acid (e.g., eicosanedioic acid).

[0143]

[0077] In some embodiments, the lipid moiety comprises the following formula:

[0144] or

[0145]

[0146] wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

[0147]

[0078] In some embodiments, the lipid moiety comprises the following formula: Atty Ref. METS-030 / 01 WO 350242-2268

[0148]

[0149] wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

[0150]

[0079] In some embodiments, the lipid moiety comprises one of the following formulae:

[0151]

[0152]

[0081] In some embodiments, the lipid moiety is of one of the following formulae:

[0153]

[0154] Atty Ref. METS-030 / 01 WO 350242-2268

[0155]

[0156]

[0082] In certain embodiments, the lipid moiety is of one of the following formulae:

[0157]

[0158]

[0083] In some embodiments, provided here are peptides comprising an amino acid sequence of Xi(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKX28GGPSSGX35PPPX39KX41(SEQ ID NO: 50), or a pharmaceutically acceptable salt thereof,

[0159] wherein

[0160] Xi is H, F, Y, or D-H;

[0161] Aib is 2-aminoisobutyric acid;

[0162] X28 is D, isoD, N, or Q;

[0163] X35 is A or K;

[0164] X39 is G or S; and

[0165] X41 is K or absent; Atty Ref. METS-030 / 01 WO 350242-2268

[0166] wherein the K residues at positions 12 and / or 27 of the amino acid sequence independently comprise a biotin moiety;

[0167] wherein the C-terminal K of the amino acid sequence comprises a lipid moiety; and wherein the C -terminus of the amino acid sequence is optionally amidated.

[0168]

[0084] Table B provides exemplary peptides of the present disclosure that comprise a biotin moiety and a lipid moiety.

[0169] Table B. Exemplary sequences of the peptides of the present disclosure

[0170] SEQ ID Sequence

[0171] NO:

[0172] 3 H(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK ((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)i6-CO2H)-NH2 4 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK ((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH2 5 Y(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK ((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2~(Y-Glu)-CO-(CH2)i6-CO2H)-NH2 6 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK K((Y-G1U)-CO-(CH2)18-CO2H)-OH

[0173] 7 Y(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK K((y-Glu)-CO-(CFl2)i8-CO2H)-OFI

[0174] 21 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK ((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)i6-CO2H)-NH2 22 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK K((Y-G1U)-CO-(CH2)I8-CO2H)-OH

[0175] 23 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPGK K((Y-G1U)-CO-(CH2)18-CO2H)-OH

[0176] 30 H(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)DGGPSSGAPPPSK ((2-[’2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH2 31 H(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)(isoD)GGPSSGAPP PSK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH2 32 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)DGGPSSGAPPPSK ((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH2 33 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)(isoD)GGPSSGAPP PSK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)16-CO2H)-NH235 (o-His)- (Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK(( 2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH2 39 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGKPPPSK K((Y-G1U)-CO-(CH2)18-CO2H)-OH

[0177] 40 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-phosphonate))-OH

[0178] 41 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK

[0179]

[0180] K((Y-Glu)-(Trx)-CO-(CH2)i8-CO2H)-OH Atty Ref. METS-030 / 01 WO 350242-2268

[0181] 42 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK K((PEG)8-(Malonate)-CO(CH2)2o)-C02H)-OH

[0182] 43 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)-(Urea)-(DAB)-(y-Glu)-CO-(CH2)i8- CO2H)-OH

[0183] 44 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK K((glucuronicacid-CO-(CH2)i8-CO2H)-OH

[0184] 47 H(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)(isoD)GGPSSGAPP PSK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetvl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH248 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)(isoD)GGPSSGAPP

[0185]

[0186] PSK((2-[2-(2-7Xmino-ethoxy)-ethoxy]-acetyl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH2

[0187]

[0085] In some embodiments, the peptide of the present disclosure comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in Table B. In some embodiments, the peptide of the present disclosure comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 22.

[0188]

[0086] In one aspect, provided herein are peptides comprising the amino acid sequence:

[0189] H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK (SEQ ID NO: 1), or pharmaceutically acceptable salts thereof, wherein Aib is 2-aminoisobutyric acid.

[0190]

[0087] In some embodiments, the peptide comprises one or more biotin or biotin derivatives. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin or biotin derivatives attached to their epsilon nitrogens. In some embodiments, the one or more biotins or biotin derivatives are of the formula:

[0191] S— \

[0192]

[0193] °. In some embodiments, the one or more biotins or biotin derivativesS'A>H

[0194] J 'H

[0195] are of the formula:

[0196]

[0197] °.

[0198]

[0088] In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following: Atty Ref. METS-030 / 01 WO 350242-2268

[0199]

[0200] , wherein * indicates attachment to the epsilon nitrogen of the lysine residue. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid

[0201] sequence are of the following structure:

[0202]

[0203]

[0089] In certain embodiments, a peptide described herein comprises a lipid moiety. In some embodiments, the lipid moiety is attached to the C -terminal amino acid of the peptide. In certain embodiments, the lipid moiety is attached to the epsilon nitrogen of a C-terminal lysine of the peptide. In certain embodiments, the lipid moiety is of the formula:

[0204]

[0205]

[0090] In certain embodiments, the peptide comprises an amidated C -terminus.

[0206]

[0091] In some embodiments, the peptide is:

[0207] H OH

[0208]

[0209] Atty Ref. METS-030 / 01 WO 350242-2268

[0210] (SEQ IDNO: 30),

[0211] or a pharmaceutically acceptable salt thereof.

[0212]

[0092] In another aspect, provided herein are peptides comprising the amino acid sequence:

[0213] H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLK(isoD)GGPSSGAPPPSK (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof, wherein:

[0214] Aib is 2-aminoisobutyric acid; and

[0215] isoD is iso-aspartic acid.

[0216]

[0093] In certain embodiments, isoD is L-isoD. In certain embodiments, isoD is D-isoD.

[0217]

[0094] In some embodiments, the peptide comprises one or more biotin or biotin derivatives. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin or biotin derivatives attached to their epsilon nitrogens. In some embodiments, the one or more biotins or biotin derivatives are of the formula:

[0218] s

[0219] NH

[0220]

[0221] some embodiments, the one or more biotins or biotin derivatives

[0222] are of the formula:

[0223]

[0224]

[0095] In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following:

[0225]

[0226] , wherein * indicates attachment to the epsilon nitrogen of the lysine residue. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid H N

[0227] NH

[0228] sequence are of the following structure:

[0229]

[0230]

[0096] In certain embodiments, a peptide described herein comprises a lipid moiety. In some embodiments, the lipid moiety is attached to the C -terminal amino acid of the peptide. In certain Atty Ref. METS-030 / 01 WO 350242-2268

[0231] embodiments, the lipid moiety is attached to the epsilon nitrogen of a C -terminal lysine of the peptide. In certain embodiments, the lipid moiety is of the formula:

[0232]

[0233]

[0097] In certain embodiments, the peptide comprises an amidated C -terminus.

[0234]

[0098] In certain embodiments, the peptide is:

[0235]

[0236] (SEQ IDNO: 47),

[0237] or a pharmaceutically acceptable salt thereof.

[0238]

[0099] In certain embodiments, the peptide is: Atty Ref. METS-030 / 01 WO 350242-2268

[0239]

[0240]

[0100] In another aspect, provided herein are peptides, or pharmaceutically acceptable salts thereof, comprising one of the following amino acid sequences:

[0241] F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSKK (SEQ ID NO: 10), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSK (SEQ ID NO: 8), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPGKK (SEQ ID NO: 12), or F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGKPPPSKK (SEQ ID NO: 13).

[0242]

[0101] In some embodiments, the peptide comprises one or more biotin or biotin derivatives. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin or biotin derivatives attached to their epsilon nitrogens. In some embodiments, the one or more biotins or biotin derivatives are of the formula:

[0243] s—x

[0244] J HN-A

[0245]

[0246] °. In some embodiments, the one or more biotins or biotin derivatives

[0247] .-A / 'NH

[0248] J ' 1

[0249] are of the formula:

[0250]

[0251] °.

[0252]

[0102] In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following: Atty Ref. METS-030 / 01 WO 350242-2268

[0253]

[0254] , wherein * indicates attachment to the epsilon nitrogen of the lysine residue. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid

[0255] sequence are of the following structure:

[0256]

[0257]

[0103] In certain embodiments, the lipid moiety is of the formula:

[0258]

[0259]

[0104] In certain embodiments, the lipid moiety is of the formula:

[0260]

[0261] certain embodiments, the lipid moiety is of the formula:

[0262]

[0263]

[0105] In certain embodiments, the lipid moiety is of the following formula:

[0264] OH O H

[0265] N -OH

[0266] N

[0267] H

[0268]

[0269] . In certain embodiments, the lipid moiety is of the following formula: Atty Ref METS-030 / 01 WO 350242-2268

[0270]

[0271]

[0106] In certain embodiments, the lipid moiety is of the following formula:

[0272] In certain embodiments, the lipid moiety is of the

[0273]

[0274]

[0107] In certain embodiments, the lipid moiety is of the following formula:

[0275]

[0276]

[0108] In certain embodiments, the lipid moiety is of the following formula:

[0277]

[0278]

[0109] In certain embodiments, the lipid moiety is of the following formula:

[0279] 3 / 127 Atty Ref. METS-030 / 01 WO 350242-2268

[0280] In certain embodiments, the lipid moiety is of the following formula:

[0281]

[0282]

[0110] In certain embodiments, the peptide comprises an amidated C -terminus.

[0283]

[0111] In certain embodiments, the peptide is selected from:

[0284] (SEQ ID NO: 21),

[0285]

[0286] (SEQ ID NO: 23), Atty Ref. METS-030 / 01 WO 350242-2268 H o H

[0287] (SEQ ID NO: 40),

[0288]

[0289] (SEQ ID NO: 41),

[0290] 5 / 127 Atty Ref. METS-030 / 01 WO 350242-2268

[0291]

[0292] (SEQ ID NO: 44), Atty Ref. METS-030 / 01 WO 350242-2268

[0293]

[0294] (SEQ ID NO: 39)

[0295] or pharmaceutically acceptable salts thereof.

[0296]

[0112] In another aspect, provided herein are peptides, or pharmaceutically acceptable salts thereof, comprising one of the following amino acid sequences:

[0297] Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK (SEQ ID NO: 16), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK (SEQ ID NO: 17), Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK (SEQ ID NO: 18), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK (SEQ ID NO: 19), or F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLK(isoD)GGPSSGAPPPSK (SEQ ID NO: 20).

[0298]

[0113] In certain embodiments, isoD is L-isoD. In certain embodiments, isoD is D-isoD.

[0299]

[0114] In some embodiments, the peptide comprises one or more biotin or biotin derivatives. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin or biotin derivatives attached to their epsilon nitrogens. In some embodiments, the one or more biotins or biotin derivatives are of the formula:

[0300] s--\

[0301]

[0302] J

[0303] °. In some embodiments, the one or more biotins or biotin derivatives S-w H

[0304] ,-L / ^ NH

[0305] J '

[0306] are of the formula:

[0307]

[0308] °.

[0309]

[0115] In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following:

[0310] Xi Tl Atty Ref. METS-030 / 01 WO 350242-2268

[0311]

[0312] , wherein * indicates attachment to the epsilon nitrogen of the lysine residue. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid

[0313] sequence are of the following structure:

[0314]

[0315]

[0116] In certain embodiments, the peptide comprises an amidated C-terminus.

[0316]

[0117] In certain embodiments, a peptide described herein comprises a lipid moiety. In some embodiments, the lipid moiety is attached to the C-terminal amino acid of the peptide. In certain embodiments, the lipid moiety is attached to the epsilon nitrogen of a C-terminal lysine of the peptide. In certain embodiments, the lipid moiety is of the formula:

[0317]

[0318] . In certain embodiments, the

[0319] In certain embodiments, the lipid

[0320]

[0321]

[0118] In some embodiments, provided herein is a peptide having the structure: Atty Ref. METS-030 / 01 WO 350242-2268

[0322]

[0323] (SEQ ID NO: 35),

[0324] or a pharmaceutically acceptable salt thereof.

[0325]

[0119] In some embodiments, the peptide is:

[0326] F-HK,i{--E--€;-T--S--T-S-»--L-S-l-< ' \-Q-M-E-E-E-A-V-R-L-F- MA.E°

[0327]

[0328] (SEQ ID NO: 48),

[0329] or a pharmaceutically acceptable salt thereof.

[0330]

[0120] In some embodiments, the peptide is selected from: Atty Ref. METS-030 / 01 WO 350242-2268

[0331] (SEQ ID NO: 5),

[0332] (SEQ ID NO: 6),

[0333]

[0334] (SEQ ID NO: 7), Atty Ref METS-030 / 01 WO 350242-2268

[0335]

[0336] (SEQ IDNO: 33),

[0337] or pharmaceutically acceptable salts thereof.

[0338]

[0121] In another aspect, provided herein is a peptide having the structure: Atty Ref. METS-030 / 01 WO 350242-2268

[0339] H OH

[0340]

[0341] (SEQ ID NO: 3),

[0342] or a pharmaceutically acceptable salt thereof.

[0343]

[0122] In another aspect, provided herein are peptides comprising an amino acid sequence having greater than 95% sequence identity with the amino acid sequence:

[0344] F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSKK (SEQ ID NO: 10), or pharmaceutically acceptable salts thereof, wherein Aib is 2-aminoisobutyric acid.

[0345]

[0123] In certain embodiments, the amino acid sequence includes 1 amino acid substitution (or 1 amino acid addition or deletion) relative to SEQ ID NO: 10. In certain embodiments, the amino acid sequence has 100% sequence identity with SEQ ID NO: 10.

[0346]

[0124] In another aspect, provided herein are peptides comprising the amino acid sequence:

[0347] F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGX35PPPX39KX41(SEQ ID NO: 9), or pharmaceutically acceptable salts thereof, wherein:

[0348] Aib is 2-aminoisobutyric acid;

[0349] X35 is alanine or lysine;

[0350] X39 is serine or glycine; and

[0351] X41 is lysine or absent.

[0352]

[0125] In some embodiments, X35 is alanine. In some embodiments, X39 is serine. In some embodiments, X41 is lysine.

[0353]

[0126] In some embodiments, the peptide comprises one or more biotin or biotin derivatives. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin or biotin derivatives attached to their epsilon nitrogens. In some embodiments, the one or more biotins or biotin derivatives are of the formul: Atty Ref. METS-030 / 01 WO 350242-2268

[0354] NH

[0355]

[0356] °. In some embodiments, the one or more biotins or biotin derivatives S— \, H

[0357] NH

[0358] are of the formula:

[0359]

[0360] o

[0361]

[0127] In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following:

[0362]

[0363] wherein indicates attachment to the epsilon nitrogen of the lysine residue. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid

[0364] sequence are of the following structure:

[0365]

[0366]

[0128] In certain embodiments, a peptide described herein comprises a lipid moiety. In some embodiments, the lipid moiety is attached to the C -terminal amino acid of the peptide. In certain embodiments, the lipid moiety is attached to the epsilon nitrogen of a C -terminal lysine of the

[0367] peptide. In certain embodiments, the lipid moiety is of the formula:

[0368]

[0369] In certain embodiments, the lipid moiety is of the formula:

[0370]

[0371]

[0129] In certain embodiments, the peptide is: Atty Ref. METS-030 / 01 WO 350242-2268

[0372]

[0373] (SEQ ID NO: 22),

[0374] or a pharmaceutically acceptable salt thereof.

[0375]

[0130] In another aspect, provided herein are peptides comprising the amino acid sequence:

[0376] X1(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKX28GGPSSGAPPPSKX41(SEQ ID NO: 14), or a pharmaceutically acceptable salt thereof, wherein:

[0377] X1is phenylalanine or tyrosine;

[0378] Aib is 2-aminoisobutyric acid;

[0379] X28 is asparagine, aspartic acid, or iso-aspartic acid (isoD); and

[0380] X41 is lysine or absent.

[0381]

[0131] In some embodiments, X1is phenylalanine. In certain embodiments, X28 is asparagine. In certain embodiments, X41 is absent.

[0382]

[0132] In some embodiments, the peptide comprises one or more biotin or biotin derivatives. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin or biotin derivatives attached to their epsilon nitrogens. In some embodiments, the one or more biotins or biotin derivatives are of the formula:

[0383] s— \

[0384] J

[0385]

[0386] 0. In some embodiments, the one or more biotins or biotin derivatives S— X H

[0387] are of the for ula:

[0388]

[0389] 0.

[0390]

[0133] In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following: Atty Ref. METS-030 / 01 WO 350242-2268

[0391]

[0392] , wherein * indicates attachment to the epsilon nitrogen of the lysine residue. In some embodiments, the lysine residues at positions 12 and 27 of the amino acid

[0393] sequence are of the following structure:

[0394]

[0395]

[0134] In certain embodiments, a peptide described herein comprises a lipid moiety. In some embodiments, the lipid moiety is attached to the C -terminal amino acid of the peptide. In certain embodiments, the lipid moiety is attached to the epsilon nitrogen of a C-terminal lysine of the peptide. In certain embodiments, the lipid moiety is of the formula:

[0396]

[0397]

[0135] In certain embodiments, the peptide comprises an amidated C -terminus.

[0398]

[0136] In certain embodiments, the peptide has the structure: Atty Ref. METS-030 / 01 WO 350242-2268

[0399] o H OH

[0400]

[0401] (SEQ ID NO; 4),

[0402] or a pharmaceutically acceptable salt thereof.

[0403] Biotinylation and Lipidation

[0404]

[0137] Provided herein are peptides comprising one or more biotin moieties and / or one or more lipid moieties. Any amino acid sequence provided herein can comprise one or more biotin and / or lipid moieties as described herein. Peptides having one or more biotin moieties and / or lipid moieties conjugated thereto can have improved bioavailability and / or pharmacokinetics as described herein.

[0405] Biotinylation

[0406]

[0138] In certain embodiments, a peptide described herein comprises one or more biotin or biotin derivatives. Biotin modifications to exendin derivatives are described in, for example, International Publication Nos. WO 2009 / 107900, WO 2020 / 242268, WO 2022 / 112849, WO 2022 / 114908, WO 2023 / 038450, and WO 2021 / 107519, the entire contents of each of which relating to biotin modifications are incorporated herein by reference.

[0407]

[0139] In some embodiments, peptides have an improved in vivo oral bioavailability compared to the same peptides without the one or more biotin moieties conjugated thereto. In preferred embodiments, the biotin-conjugated peptides retain most of the activity of the same peptides without the one or more biotin moieties conjugated thereto.

[0408]

[0140] In some embodiments, the one or more biotins or biotin derivatives are independently selected from: Atty Ref. METS-030 / 01 WO 350242-2268

[0409]

[0410] wherein * indicates the point of attachment to the peptide.

[0411]

[0141] In some embodiments, the one or more biotins or biotin derivatives are:

[0412] *S'A>H

[0413] Y d ' H*X HN^ i,

[0414]

[0415] O

[0416]

[0142] In some embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin or biotin derivatives attached to their epsilon nitrogens. In certain embodiments, the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following:

[0417]

[0418] °, wherein * indicates attachment to the epsilon nitrogen of the lysine residue. In certain embodiments, the lysine residues at positions 12 and 27 of the amino acid

[0419] sequence are of the following structure:

[0420]

[0421] Lipidation

[0422]

[0143] Lipidated peptides can have an increased lipophilicity, an increased in vivo half-life Atty Ref. METS-030 / 01 WO 350242-2268

[0423] (enabling once daily oral administration), and a reduced variability in pharmacokinetics at steady state. In some embodiments, an additional amino acid is added to the C -terminus of the peptide to enable the conjugation of one or more fatty acid molecules with increased linker stability. In preferred embodiments, the amino acid is lysine. In some embodiments, the fatty acid moiety is conjugated to the peptides via one or more additional lysine residues added to the C-terminus of the peptide.

[0424]

[0144] In certain embodiments, a peptide described herein comprises a lipid moiety. In some embodiments, the lipid moiety is attached to the C-terminal amino acid of the peptide. In certain embodiments, the lipid moiety is attached to the epsilon nitrogen of a C-terminal lysine of the peptide.

[0425]

[0145] In certain embodiments, the lipid moiety comprises glutamic acid. In certain embodiments, the lipid moiety comprises a dicarboxylic acid (e.g., eicosanedioic acid). In certain embodiments, the lipid moiety comprises glutamic acid and a dicarboxylic acid (e.g., eicosanedioic acid).

[0426]

[0146] In some embodiments, the lipid moiety comprises the following formula:

[0427] □H

[0428] O O

[0429]

[0430] H '

[0431] wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

[0432]

[0147] In some embodiments, the lipid moiety comprises the following formula:

[0433]

[0434] wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

[0435]

[0148] In certain embodiments, the lipid moiety comprises one of the following formulae:

[0436]

[0437]

[0149] In certain embodiments, the lipid moiety comprises one of the following formulae: Atty Ref. METS-030 / 01 WO 350242-2268

[0438]

[0439]

[0150] In certain embodiments, the lipid moiety is of one of the following formulae:

[0440]

[0441]

[0151] In certain embodiments, the lipid moiety is of one of the following formulae:

[0442]

[0443] Atty Ref. METS-030 / 01 WO 350242-2268

[0444]

[0445] Pharmaceutical Compositions, Administration, and Kits

[0446]

[0152] The present disclosure provides pharmaceutical compositions comprising peptide or a pharmaceutically acceptable salt thereof described herein (“peptide” or “compound”). The pharmaceutical composition can comprise one or more pharmaceutically acceptable carriers and / or excipients. In certain embodiments, the peptide is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount.

[0447]

[0153] The terms “composition” and “formulation” are used interchangeably herein.

[0448]

[0154] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the peptide described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0449]

[0155] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising predetermined amounts of the active ingredients. The amount of the active ingredients is generally equal to the dosage of the active ingredients which would be administered to a subject and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage. Atty Ref. METS-030 / 01 WO 350242-2268

[0450]

[0156] Relative amounts of the active ingredient, the pharmaceutically acceptable carrier or excipient, and / or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered.

[0451]

[0157] Pharmaceutically acceptable carriers / excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, solvents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, oils, butters, and / or waxes. Excipients such as coloring agents, coating agents, sweetening agents, flavoring agents, and fragrances may also be present in the composition.

[0452]

[0158] The peptides and compositions provided herein can be administered by any route, including parenteral, enteral (e.g., oral), intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agents (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g,, whether the subject is able to tolerate oral administration).

[0453]

[0159] A specifically contemplated route is oral administration. In some embodiments, a pharmaceutical composition or peptide thereof is administered orally. In some embodiments, the peptides or analogs thereof are administered orally. For oral administration, suitable formulations include tablets, pellets, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc.

[0454]

[0160] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier.

[0455]

[0161] Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and Atty Ref. METS-030 / 01 WO 350242-2268

[0456] granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology.

[0457]

[0162] In some embodiments, one or more absorption or permeation enhancers are used for oral formulation. Exemplary permeation enhancers include bile acid, cholic acid, deoxycholic acid, glycocholic acid, glycochonodeoxy cholic acid, taurochenodeoxy cholic acid, taurocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, Labrasol(Caprylocaproyl Polyoxyl-8 glycerides), SNAC(sodium N-(8-[2-hydroxybenzoyl] amino) caprylate, propyl gallate and their salt forms.

[0458]

[0163] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with ordinary experimentation.

[0459]

[0164] Peptides provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific effective or therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the condition being treated and / or the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0460]

[0165] The exact amount of peptides required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular peptide, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject, any two doses of the multiple doses include different or substantially the same amounts Atty Ref. METS-030 / 01 WO 350242-2268

[0461] of the peptides described herein.

[0462]

[0166] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or peptide described herein and a container (e.g., a vial, ampule, bottle, syringe, and / or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or peptide described herein. In some embodiments, the pharmaceutical composition or peptide described herein provided in the first container and the second container are combined to form a single unit dosage form.

[0463]

[0167] In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U. S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition. In certain embodiments, the kits provide instructions for reducing weight. In certain embodiments, the kits provide instructions for preventing weight gain.

[0464] Methods of Treatment and Uses

[0465]

[0168] Over the past several decades, the prevalence of diabetes has continued to rise. Type 2 Diabetes Mellitus (T2DM) is the most common form of diabetes, accounting for approximately 90% of all recognized diabetes cases. T2DM is characterized by high blood glucose levels associated mainly with insulin resistance. The current standard of care for T2DM includes diet and exercise, treatment with oral medications, and injectable glucose lowering drugs, including incretin-based therapies, such as GLP-1 receptor agonists. A variety of GLP-1 receptor agonists are currently available for treatment of T2DM, although currently marketed GLP-1 receptor agonists are generally dose-limited by gastrointestinal side effects, such as nausea and vomiting. Subcutaneous injection is the typical route of administration for the available GLP-1 receptor agonists. When treatment with oral medications and incretin-based therapies are insufficient, insulin treatment is considered. Despite the advances in treatment available today, many patients with T2DM are unable to reach their glycemic control goals. Uncontrolled diabetes leads to several conditions associated with increased morbidity and mortality of patients. There is a need for a treatment to enable more patients with T2DM to reach their glycemic treatment goal. Atty Ref. METS-030 / 01 WO 350242-2268

[0466]

[0169] Obesity is a complex medical disorder resulting in excessive accumulation of adipose tissue mass. Today obesity is a global public health concern that is associated with undesired health outcomes and morbidities. Desired treatments for patients with obesity' strive to reduce excess body weight, improve obesity -related co-morbidities, and maintain long-term weight reduction. Available treatments for obesity are particularly unsatisfactoiy for patients with severe obesity. There is a need for alternative treatment options to induce therapeutic weight loss in patients in need of such treatment.

[0467]

[0170] Recently, with economic development and medical advancement, aging populations are rapidly growing. Aging increases the risk of chronic diseases such as dementias, heart disease, T2DM, arthritis, cancer, and neurodegenerative diseases. Mortality rates of heart and cerebrovascular diseases, which are complications accompanying obesity, are ranked as first and second. Obesity is suggested as a cause of various adult diseases such as diabetes mellitus, and non-alcoholic fatty liver disease.

[0468]

[0171] Non-alcoholic fatty liver diseases (NAFLD) includes a series of diseases including simple steatosis with excessive accumulation of fat in the liver cells independent of alcohol consumption, non-alcoholic steatohepatitis (NASH) including hepatocellular injury (hepatocellular ballooning), metabolic dysfunction-associated steatohepatitis (MASH), inflammation, fibrosis, and, in more advanced cases, cirrhosis. The prevalence rate of non-alcoholic fatty liver disease is rapidly increasing with the increase in the prevalence rate of obesity all over the world, and although the prevalence rate of diabetes varies from country to country, it accounts for about 20% to 30% of the total populations in Western countries, and the incidence rate thereof reaches about 16% in South Korea.

[0469]

[0172] Glucagon-like peptide- 1 (GLP-1) is a hormone secreted by the small intestine stimulated by food intake. GLP-1 promotes insulin secretion in the pancreas in a blood glucose-dependent manner and inhibits the secretion of glucagon, thus helping the action of lowering blood glucose levels. Additionally, GLP-1 slows digestive action in the gastrointestinal tract by acting as a satiety factor and reduces the amount of food intake by delaying the time for emptying digested food in the gastrointestinal tract. Administration of GLP-1 to rats was reported to have effects of inhibiting food intake and reducing body weight, and these effects were confirmed to occur equally both in normal and obese states, thus showing the potential of GLP-1 as an agent for treating obesity.

[0470]

[0173] G-protein coupled receptors including GLP-1R can exhibit signaling “bias” in favor of G Atty Ref. METS-030 / 01 WO 350242-2268

[0471] protein-dependent signaling. A consistent effect of G protein-biased GLP-1R agonists is their prolonged GLP-1R signaling ability, circumventing natural mechanisms that prevent receptor overstimulation such as steric hinderance by P-arrestins and receptor internalization (Jones, et al., Nat Commun. 2018;9: 1602.). The importance of biased agonism has been highlighted by the realization that the GLP-1R / GIPR co-agonist tirzepatide, shows pronounced G protein bias at the GLP-1R (Willard, et al. JCI Insight. 2020;5:1202). Biased signaling may increase the duration of efficacy. Further, biased signaling may decrease side effects such as nausea and vomiting, lower oral BA, and reduce intervals of administration compared to current unbiased GLP-1R agonists (e.g., semaglutide).

[0472]

[0174] There is a need for T2DM treatments capable of providing effective glucose control for a larger portion of the patients in need of such treatment. There is a further need for T2DM treatments capable of providing effective glucose control and with a favorable side effect profile. There is also a need for more convenient routes of administration including oral therapies. There is also a need for alternate treatment options to provide therapeutic weight loss in a patient in need of such treatment. There is also a need for an alternate treatment option for a patient in need of treatment for severe obesity.

[0473]

[0175] Therefore, it is an object of the disclosure to provide compositions and methods for treating or preventing one or more sympto s of diseases including type 2 diabetes mellitus, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, metabolic dysfunction-associated steatohepatitis, non-alcoholic steatohepatitis, obesity, and / or neurodegenerative diseases.

[0474]

[0176] It is also an object of the disclosure to provide peptides, compositions and methods suitable for activating GLP-1 receptors to control blood glucose levels and reduce body weight without causing side effects.

[0475]

[0177] It is a further object of the disclosure to provide peptides, compositions and methods that provide effective glucose control.

[0476]

[0178] It is a further object of the disclosure to provide peptides, compositions and methods that provide weight loss benefits.

[0477]

[0179] It is a further object of the disclosure to provide peptides, compositions and methods have a favorable side effect profile.

[0478]

[0180] It is a further object of the present disclosure to provide peptides and compositions with extended duration of action. Atty Ref. METS-030 / 01 WO 350242-2268

[0479]

[0181] It is a further object of the present disclosure to modify the in-vivo oral absorption of peptides and compositions.

[0480]

[0182] It is a further object of the present disclosure to modify the pharmacokinetic profile of peptides and compositions.

[0481]

[0183] It is a further object of the present disclosure to provide peptides compositions for biased GLP1-R activity.

[0482]

[0184] It is a further object of the present disclosure to provide peptides and compositions that give rise to decreased undesirable side effects, such as nausea and vomiting.

[0483]

[0185] It is a further object of the present disclosure to increase the oral bioavailability of peptides and compositions.

[0484]

[0186] It is a further object of the present disclosure to increase the minimum interval for oral readministration of peptides and compositions.

[0485]

[0187] It is a further object of the present disclosure to provide peptides and compositions that have cAMP biased GLP1R signaling.

[0486]

[0188] It is a further object of the present disclosure to provide peptides and compositions that has reduced beta-arrestin bias signaling.

[0487]

[0189] It is a further object of the present disclosure to provide peptides and compositions that have cAMP biased GLP1R signaling and reduced beta-arrestin bias signaling.

[0488]

[0190] In some embodiments, the present disclosure provides methods of inducing or maintaining weight loss or managing body weight in a subject, wherein the method comprises oral administration of a peptide of the present disclosure (e.g., SEQ ID NO: 50 or SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is obese, overweight or normal weight. Some embodiments provide a method of inducing or maintaining weight loss or managing body weight in a subject with obesity' or with overweight and at least one weight-related comorbidity, wherein the method comprises oral administration of a peptide of the present disclosure (e.g., SEQ ID NO: 50 or SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof. In some embodiments, the weight-related comorbidity includes hypertension, dyslipidemia, obstructive sleep apnea, cardiovascular disease, Type 2 diabetes (T2DM), or nonalcoholic fatty liver disease (NAFLD). In some embodiments, the comorbid condition is T2DM In some embodiments, the subject has a body mass index (B T) of at least 27 kg / m2. In some embodiments, the subject has a body mass index (BMI) of at least 27 kg / m2and one or more weight-related Atty Ref. METS-030 / 01 WO 350242-2268

[0489] comorbid conditions. In some embodiments, the comorbid condition is type 2 diabetes. In some embodiments, the subject has a body mass index (BMI) of at least 27 kg / m2and hypertension. In some embodiments, the subject has a body mass index (BMI) of at least 27 kg / m2and dyslipidemia. In some embodiments, the subject has a body mass index (BMI) of at least 27 kg / m2and low HDL-C. In some embodiments, the subject has a body mass index (BMI) of at least 27 kg / m2and T2DM.

[0490]

[0191] In some embodiments, the present disclosure provides methods of treating type 2 diabetes mellitus, wherein the method comprises oral administration of a peptide of the present disclosure (e.g., SEQ ID NO: 50 or SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof. Some embodiments provide a method of improving glycemic control, wherein the method comprises oral administration of a peptide of the present disclosure (e.g., SEQ ID NO: 50 or SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure provides methods of reducing the risk of a major cardiovascular event, wherein the method comprises oral administration of a peptide of the present disclosure (e.g., SEQ ID NO: 50 or SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure provides methods of reducing the risk of eGFR decline or end-stage kidney disease, wherein the method comprises oral administration of a peptide of the present disclosure (e.g., SEQ ID NO: 50 or SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure provides methods of treating obstructive sleep apnea, wherein the method comprises oral administration of a peptide of the present disclosure (e.g., SEQ ID NO: 50 or SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof

[0491]

[0192] In some embodiments, the peptides or pharmaceutically acceptable salts thereof are administered in an amount and with a dosing regimen effective to prevent, inhibit, or reduce one or more symptoms associated with one or more metabolic disorders such as overweight, dyslipidemia, fatty liver disease, metabolic syndrome, NAFLD, obesity and T2DM in the subject. Examples of metabolic disorders include, but are not limited to, diabetes (e.g., Type I diabetes, Type II diabetes, gestational diabetes), hyperglycemia, hyperinsulinemia, insulin resistance, and obesity. In certain embodiments, the metabolic disorder is a diabetic condition. In some embodiments, the metabolic disorder is selected from obesity, prediabetes, diabetes, non-alcoholic fatty liver disease (NAFLD), and polycystic ovary syndrome (PCOS).

[0492]

[0193] Methods of treating one or more diseases selected from the group including obesity, diabetes, non-alcoholic fatty liver disease, metabolic dysfunction-associated steatohepatitis, and Atty Ref. METS-030 / 01 WO 350242-2268

[0493] neurodegenerative disease in a subject in need thereof are provided. The methods include administering an effective amount of a peptide or composition described herein. In preferred embodiments, the pharmaceutical formulation is administered in an amount effective to induce weight loss, reduce the body fat, reduce food intake, improve glucose homeostasis, or combinations thereof, in a normal or obese patient. In some embodiments, the subject is suffering from non-alcoholic fatty liver disease (NAFLD), for example, non-alcoholic fatty liver, non¬ alcoholic steatohepatitis, metabolic dysfunction-associated steatohepatitis, liver cirrhosis, and liver cancer. In the case of NAFLD, the pharmaceutical formulation is administered in an amount effective to inhibit or reduce serum levels of one or more of alanine aminotransferase, aspartate aminotransferase, triglyceride, gamma-glutamyl transferase, total cholesterol, low density lipoprotein, fasting blood sugar or combinations thereof. In preferred embodiments, the pharmaceutical formulation is administered in an amount effective to reduce one or more of steatosis, inflammation, ballooning, fibrosis, cirrhosis, or combinations thereof, in a subject with NAFLD.

[0494]

[0194] In some embodiments, the peptides described herein agonize GLP-1 receptor and exhibit a value of cAMP EC50 of less than about 10 nM, 1 nM or 0.1 nM when measured in an cAMP assay where the EC50 value of the native ligand of GLP-1R, GLP-l(7-36) or GLP-l(7-37), is less than 0.1 nM.

[0495]

[0195] In some embodiments, the peptides described herein modulate (e.g., reduce, attenuate, disrupt, inhibit) P-Arrestin coupling and / or P-Arrestin signaling, and GLP-1 receptor internalization. Reduction of P-Arrestin signaling and GLP-1 receptor internalization can prolong the treatment effect and allowing for increased dosing intervals by effect by days and preferably weeks and months. The effects of the peptides described herein on P-Arrestin signaling can be assessed using conventional methods, e.g., PathHunter P-Arrestin Assay (see Examples section).

[0496]

[0196] In some embodiments, the peptides described herein reduce P-Arrestin recruitment to GLP-1R and exhibit an Emax value of P-Arrestin recruitment of less than 20% when measured in a P-Arrestin assay where the Emax value of the exenatide or semaglutide is 100%,

[0497]

[0197] In some embodiments, the peptides described herein demonstrate resistance to degradation by digestive enzymes in gastro-intestinal tract (e.g., pancreatin, trypsin). Most peptides are susceptible to enzyme digestion, which poses challenges for enteral administration. Consequently, enhancing the stability of peptides against digestive enzymes is crucial for improving their Atty Ref. METS-030 / 01 WO 350242-2268

[0498] absorption following oral administration. The enzymatic stability of these peptides can be assessed by measuring their half-life in an artificial intestinal environment (see Examples section). By way of example, a value of half-life in FaSSIF / P > 20 min and Trypsin > 2 min in the assay indicates a peptide that sufficient amount of peptide can retain for absorption after oral administration.

[0499]

[0198] In some embodiments, the peptides described herein stimulate glucose clearance in vivo (ipGTT test). Glucose clearance is representative marker for diabetes, with poor glucose clearance typically resulting from limited insulin secretion and / or increased insulin resistance. The peptides described herein exhibit a value of AUC Score of 0-50% of vehicle after 4 hours from administration and 0-80% of vehicle after 72 hours from administration. The peptides described herein exhibit a value of blood glucose of less than 50% of vehicle at 20 min after glucose challenging at day 0 and less than 80% of vehicle at 20 min after glucose challenging at day 3.

[0500]

[0199] In some embodiments, the peptides described herein demonstrate increased in vivo oral absorption. After oral administration, several PK parameters, such as Cmax and AUC can be obtained. These parameters are representative for assessing the oral absorption of peptides. To demonstrate efficacy, the concentration of peptide in the blood should remain above a specific threshold, which varies for each peptide. The peptides described herein exhibit a value of AUClast or AUCinf > 3000 ng·hr / mL in beagle dog after oral administration of 10 mg of peptide.

[0501]

[0200] In some embodiments, the peptides described herein demonstrate modified pharmacokinetic profile (e.g., half-life). Longer in vivo half-life can prolong the treatment effect and allow for increased dosing interval. The peptides described herein exhibit a value of half-life > 9 hours after IV dosing in rats.

[0502]

[0201] In some embodiments, the peptides described herein reduce body weight and food intake. The efficacy of peptides observed in obese mouse model translates well to humans, suggesting that similar results can be expected. For example, semaglutide, a GLP- 1 receptor agonistic peptide, demonstrated approximately 15% body weight loss in obese / overweight humans. The peptides described herein demonstrate body weight reduction of greater than 10% in diet-induced obese mice following treatment, compared to their weight prior to peptide administration. The peptides described herein demonstrate food intake of 0-80% of vehicle control in diet-induced obese mice following treatment.

[0503]

[0202] As described, provided herein are methods of reducing weight of a subject, the method comprising administering to the subject an effective amount of a peptide described herein, or a Atty Ref. METS-030 / 01WO 350242-2268

[0504] pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0505]

[0203] Also provided herein are methods of preventing weight gain in a subject, the method comprising administering to the subject an effective amount of a peptide described herein (e.g., SEQ ID NO: 50 or SEQ ID NO: 22), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0506]

[0204] In certain embodiments, the subject has a metabolic disorder. In some embodiments, the metabolic disorder is selected from obesity, prediabetes, diabetes, non-alcoholic fatty liver disease (NAFLD), cardiometabolic disease, polycystic ovary' syndrome (PCOS), chronic kidney disease, peripheral arterial disease, heart failure, Alzheimer’s disease, sleep apnea, or knee osteoarthritis.

[0507]

[0205] In certain embodiments, the subject has a reduction in weight of about 5% to about 40%. In some embodiments, the subject has a reduction in weight of about 1% to about 5%. In some embodiments, the subject has a reduction in weight of about 5%. In some embodiments, the subject has a reduction in weight of about 10%. In some embodiments, the subject has a reduction in weight of about 15%. In some embodiments, the subject has a reduction in weight of about 20%. In some embodiments, the subject has a reduction in weight of about 25%. In some embodiments, the subject has a reduction in weight of about 30%. In some embodiments, the subject has a reduction in weight of about 35%. In some embodiments, the subject has a reduction in weight of about 40%. In some embodiments, the subject has a reduction in weight of about 40% or more.

[0508]

[0206] Also provided herein are methods of stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, or reducing caloric intake in a subject in need thereof, the method comprising administering to the subject an effective amount of a peptide described herein (e.g., SEQ ID NO: 50 or SEQ ID NO: 22), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0509]

[0207] Also provided herein are methods of modulating P-arrestin signaling and / or GLP-1 receptor internalization, the method comprising contacting a cell with a peptide described herein (e.g., SEQ ID NO: 50 or SEQ ID NO: 22), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0510]

[0208] In another aspect, provided is a peptide described here, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in:

[0511] (a) reducing weight of a subject; Atty Ref. METS-030 / 01 WO 350242-2268

[0512] (b) preventing weight gain in a subject; and / or

[0513] (c) stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, and / or reducing caloric intake in a subject.

[0514]

[0209] In another aspect, provided herein is use of a peptide described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as a medicament.

[0515]

[0210] The term “metabolic disorder” includes any disorder that involves an alteration in the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or a combination thereof. A metabolic disorder is associated with either a deficiency or excess in a metabolic pathway resulting in an imbalance in metabolism of nucleic acids, proteins, lipids, and / or carbohydrates. Factors affecting metabolism include, and are not limited to, the endocrine (hormonal) control system (e.g., the insulin pathway, the enteroendocrine hormones including GLP-1, PYY or the like), the neural control system (e g., GLP-1 in the brain), or the like. Examples of metabolic disorders include, but are not limited to, diabetes (e.g., Type I diabetes, Type II diabetes, gestational diabetes), hyperglycemi, hyperinsulinemia, insulin resistance, and obesity. In certain embodiments, the metabolic disorder is a diabetic condition. In certain embodiments, the metabolic disorder is selected from obesity, prediabetes, diabetes, non-alcoholic fatty liver disease (NAFLD), and polycystic ovary syndrome (PCOS).

[0516]

[0211] A “diabetic condition” includes diabetes and pre-diabetes. Diabetes includes a group of metabolic disorders in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger). There are several types of diabetes. Type I diabetes results from the body’s failure to produce insulin, and presently requires the person to inject insulin or wear an insulin pump. Type II diabetes results from insulin resistance a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency. Gestational diabetes occurs when pregnant women without a previous diagnosis of diabetes develop a high blood glucose level. Other forms of diabetes include congenital diabetes, which is due to genetic defects of insulin secretion, cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several forms of monogenic diabetes, e.g., mature onset Atty Ref. METS-030 / 01 WO 350242-2268

[0517] diabetes of the young (e.g., MODY 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Pre-diabetes indicates a condition that occurs when a person’s blood glucose levels are higher than normal but not high enough for a diagnosis of diabetes. All forms of diabetes increase the risk of long-term complications. These typically develop after many years but may be the first symptom in those who have otherwise not received a diagnosis before that time. The major long-term complications relate to damage to blood vessels. Diabetes doubles the risk of cardiovascular disease and macrovascular diseases such as ischemic heart disease (angina, myocardial infarction), stroke, peripheral vascular disease, and peripheral arterial disease. Diabetes also causes microvascular complications, e.g., damage to the small blood vessels. Diabetic retinopathy, which affects blood vessel formation in the retina of the eye, can lead to visual symptoms, reduced vision, and potentially blindness. Diabetic nephropathy, the impact of diabetes on the kidneys, can lead to scarring changes in the kidney tissue, loss of small or progressively larger amounts of protein in the urine, and eventually chronic kidney disease requiring dialysis. Diabetic neuropathy is the impact of diabetes on the nervous system, most commonly causing numbness, tingling and pain in the feet and also increasing the risk of skin damage due to altered sensation. Together with vascular disease in the legs, neuropathy contributes to the risk of diabetes-related foot problems, e.g., diabetic foot ulcers, that can be difficult to treat and occasionally require amputation.

[0518]

[0212] In certain embodiments, the metabolic disorder is an obesity -related condition or complication thereof. An “obesity-related condition” includes, but is not limited to, obesity, undesired weight gain (e.g., from medication-induced weight gain, from cessation of smoking) and an over-eating disorder (e.g., binge eating, bulimia, compulsive eating, or a lack of appetite control each of which can optionally lead to undesired weight gain or obesity). “Obesity” and “obese” includes class I obesity, class II obesity, class III obesity and pre-obesity (e.g., being “over-weight”) as defined by the World Health Organization.

[0519]

[0213] Reduction of storage fat is expected to provide various primary and / or secondary benefits in a subject (e.g., in a subject diagnosed with a complication associated with obesity) such as, for example, an increased insulin responsiveness (e.g., in a subject diagnosed with Type II diabetes mellitus); a reduction in elevated blood pressure; a reduction in elevated cholesterol levels; and / or a reduction (or a reduced risk or progression) of ischemic heart disease, arterial vascular disease, angina, myocardial infarction, stroke, migraines, congestive heart failure, deep vein thrombosis, pulmonary embolism, gall stones, gastroesophagael reflux disease, obstructive sleep apnea, Atty Ref. METS-030 / 01 WO 350242-2268

[0520] obesity hypoventilation syndrome, asthma, gout, poor mobility, back pain, erectile dysfunction, urinary incontinence, liver injury (e.g., fatty liver disease, liver cirrhosis, alcoholic cirrhosis, endotoxin mediated liver injury) or chronic renal failure. Thus, the compositions and methods described herein are applicable to these conditions.

[0521] NUMBERED EMBODIMENTS OF THE DISCLOSURE

[0522]

[0214] In addition to the disclosure above, the Examples below, and the appended claims, the disclosure sets forth the following numbered embodiments.

[0523] 1. A peptide comprising an amino acid sequence of X1(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKX28GGPSSGX35PPPX39KX41 (SEQ ID NO: 50), or a pharmaceutically acceptable salt thereof,

[0524] wherein

[0525] Xi is FI, F, Y, or D-H;

[0526] Aib is 2-aminoisobutyric acid;

[0527] X28 is D, isoD, N, or Q;

[0528] X35 is A or K;

[0529] X39is G or S; and

[0530] X41 is K or absent; and

[0531] wherein the C -terminus of the amino acid sequence is optionally amidated.

[0532] 2. The peptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises a biotin moiety.

[0533] 3. The peptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises two biotin moieties.

[0534] 4. The peptide of embodiment 1 or 2, or a pharm ceutically acceptable salt thereof, wherein the K residues at positions 12 and / or 27 of the amino acid sequence independently comprise a biotin moiety.

[0535] 5. The peptide of embodiment 3 or 4, or a pharmaceutically acceptable salt thereof, wherein the biotin moiety is attached to the epsilon nitrogens of the K residues at positions 12 and / or 27. Atty Ref. METS-030 / 01 WO 350242-2268

[0536] 6. The peptide of any one of embodiments 2-5, or a pharmaceutically acceptable salt thereof, wherein the biotin moiety comprises the formula:

[0537]

[0538] 7. The peptide of any one of embodiments 2-5, or a pharmaceutically acceptable salt thereof, wherein the biotin moiety comprises the formula:

[0539]

[0540] 8. The peptide of any one of embodiments 1-7, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises a lipid moiety.

[0541] 9. The peptide of embodiment 8, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to a C-terminal lysine of the peptide.

[0542] 10. The peptide of embodiment 9, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to the epsilon nitrogen of the C-terminal lysine of the peptide.

[0543] 11. The peptide of any one of embodiments 8-10, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula:

[0544]

[0545] wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

[0546] 12. The peptide of any one of embodiment 11, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula: Atty Ref. METS-030 / 01 WO 350242-2268

[0547]

[0548] 13. The peptide of any one of embodiment 11, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula:

[0549]

[0550] 14. The peptide of any one of embodiment 11, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the formula selected from

[0551]

[0552] Atty Ref. METS-030 / 01 WO 350242-2268

[0553]

[0554] 15. A peptide comprising the amino acid sequence:

[0555] H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, wherein Aib is 2-aminoisobutyric acid.

[0556] 16. The peptide of embodiment 15, wherein the peptide comprises

[0557]

[0558] (SEQ ID NO: 30),

[0559] or a pharmaceutically acceptable salt thereof.

[0560] 17. A peptide comprising the amino acid sequence:

[0561] H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLK(isoD)GGPSSGAPPPSK (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof, wherein:

[0562] Aib is 2-aminoisobutyric acid; and

[0563] isoD is iso-aspartic acid. Atty Ref. METS-030 / 01 WO 350242-2268

[0564] 18. The peptide of embodiment 17, wherein the peptide comprises

[0565]

[0566] (SEQ ID NO: 31),

[0567] or a pharmaceutically acceptable salt thereof.

[0568] 19. The peptide of embodiment 17, wherein the peptide comprises

[0569]

[0570] (SEQ ID NO: 47),

[0571] or a pharmaceutically acceptable salt thereof.

[0572] 20. A peptide comprising Atty Ref. METS-030 / 01 WO 350242-2268

[0573]

[0574] (SEQ ID NO: 35),

[0575] or a pharmaceutically acceptable salt thereof.

[0576] 21. A peptide, or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence selected from the group consisting of F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSKK (SEQ ID NO: 10), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSK (SEQ ID NO. 8), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPGKK (SEQ ID NO: 12), and F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGKPPPSKK (SEQ ID NO: 13).

[0577] 22. The peptide of embodiment 21, wherein the peptide comprises

[0578]

[0579] Atty Ref. METS-030 / 01 WO 350242-2268

[0580] (SEQ ID NO: 40),

[0581]

[0582] (SEQ ID NO: 41), Atty Ref. METS-030 / 01 WO 350242-2268

[0583]

[0584] (SEQ ID NO: 44), or

[0585] f>o / ni Atty Ref. METS-030 / 01 WO 350242-2268

[0586]

[0587] (SEQ ID NO: 39),

[0588] or pharmaceutically acceptable salts thereof.

[0589] 23. A peptide, or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence selected from the group consisting of:

[0590] Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK (SEQ ID NO. 16), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK (SEQ ID NO: 17), Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK (SEQ ID NO: 18), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK (SEQ ID NO: 19), and F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLK(isoD)GGPSSGAPPPSK (SEQ ID NO: 20).

[0591] 24. The peptide of embodiment 23, wherein the peptide comprises

[0592]

[0593] (SEQ ID NO: 5), Atty Ref. METS-030 / 01 WO 350242-2268

[0594] (SEQ ID NO: 6), (SEQ ID NO: 7), H O

[0595]

[0596] Atty Ref. METS-030 / 01 WO 350242-2268

[0597] (SEQ ID NO: 33), or

[0598]

[0599] (SEQ ID NO: 48),

[0600] or pharmaceutically acceptable salts thereof.

[0601] 25. A peptide comprising Atty Ref. METS-030 / 01 WO 350242-2268

[0602] H OH

[0603]

[0604] (SEQ ID NO: 3),

[0605] or a pharmaceutically acceptable salt thereof.

[0606] 26. A peptide comprising an amino acid sequence having greater than 95% sequence identity with the amino acid sequence:

[0607] F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSKK (SEQ ID NO: 10), or a pharmaceutically acceptable salt thereof, wherein Aib is 2-aminoisobutyric acid.

[0608] 27. The peptide of embodiment 26, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 amino acid substitution relative to SEQ ID NO: 10.

[0609] 28, The peptide of embodiment 26, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence has 100% sequence identity with SEQ ID NO: 10.

[0610] 29. A peptide comprising the amino acid sequence:

[0611] F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGX35PPPX39KX41(SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof, wherein:

[0612] Aib is 2-aminoisobutyric acid;

[0613] X35 is alanine or lysine;

[0614] X39 is serine or glycine; and

[0615] X41 is lysine or absent. Atty Ref. METS-030 / 01 WO 350242-2268

[0616] 30. The peptide of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein X35 is alanine,

[0617] 31. The peptide of embodiment 29 or 30, or a pharmaceutically acceptable salt thereof, wherein X39 is serine.

[0618] 32. The peptide of any one of embodiments 29-31, or a pharmaceutically acceptable salt thereof, wherein X41 is lysine.

[0619] 33. The peptide of any one of embodiments 26-32, wherein the peptide comprises

[0620]

[0621] (SEQ ID NO: 22),

[0622] or a pharmaceutically acceptable salt thereof.

[0623] 34. A peptide comprising the amino acid sequence:

[0624] Xi(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKX28GGPSSGAPPPSKX4i (SEQ ID NO: 14), or a pharmaceutically acceptable salt thereof, wherein:

[0625] X1is phenylalanine or tyrosine;

[0626] Aib is 2-aminoisobutyric acid;

[0627] X28 is asparagine, aspartic acid, or iso-aspartic acid (isoD); and

[0628] X41 is lysine or absent.

[0629] 35. The peptide of embodiment 34, or a pharmaceutically acceptable salt thereof, wherein Xi is phenylalanine. Atty Ref. METS-030 / 01 WO 350242-2268

[0630] 36. The peptide of embodiment 34 or 35, or a pharmaceutically acceptable salt thereof, wherein X28 is asparagine.

[0631] 37. The peptide of any one of embodiments 33-36, or a pharmaceutically acceptable salt thereof, wherein X41 is absent.

[0632] 38. The peptide of any one of embodiments 33-37, wherein the peptide comprises:

[0633] o H O H

[0634]

[0635] (SEQ ID NO: 4),

[0636] or a pharmaceutically acceptable salt thereof.

[0637] 39. The peptide of any one of embodiments 15, 17, 20, 22, 25-31, and 33-36, or a pharmaceutically acceptable salt thereof, comprising an amidated C-terminus.

[0638] 40. The peptide of any one of embodiments 15, 17, 20, 22, 25-31, and 33-36, or a pharmaceutically acceptable salt thereof, comprising one or more biotin moieties.

[0639] 41. The peptide of embodiment 40, or a pharmaceutically acceptable salt thereof, wherein the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin moieties attached to their epsilon nitrogens,

[0640] 42. The peptide of embodiment 41, or a pharmaceutically acceptable salt thereof, wherein the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following: Atty Ref. METS-030 / 01 WO 350242-2268

[0641]

[0642] wherein * indicates attachment to the epsilon nitrogen of the lysine residue.

[0643] 43, The peptide of embodiment 42, or a pharmaceutically acceptable salt thereof, wherein the lysine residues at positions 12 and 27 of the amino acid sequence are of the following structure:

[0644]

[0645] 44, The peptide of any one of embodiments 15, 17, 20, 22, 25-31, and 33-36, or a pharmaceutically acceptable salt thereof, comprising a lipid moiety.

[0646] 45. The peptide of embodiment 44, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to the C-terminal amino acid of the peptide.

[0647] 46. The peptide of embodiment 44 or 45, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to the epsilon nitrogen of a C-terminal lysine of the peptide.

[0648] 47. The peptide of any one of embodiments 44-46, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula:

[0649]

[0650] wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

[0651] 48. The peptide of any one of embodiments 44-46, or a pharmaceutically acceptable salt Atty Ref. METS-030 / 01 WO 350242-2268

[0652] thereof, wherein the lipid moiety comprises one of the following formulae:

[0653]

[0654] 49. The peptide of any one of embodiments 44-46, wherein the lipid moiety comprises one of the following formulae:

[0655]

[0656] Atty Ref. METS-030 / 01 WO 350242-2268

[0657]

[0658] 50. A pharmaceutical composition comprising the peptide of any one of the preceding embodiments, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0659] 51, A method of inducing or maintaining weight loss or managing body weight in a subject, the method comprising administering to the subject an effective amount of the peptide of any one of embodiments 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0660] 52, A method of preventing weight gain in a subject, the method comprising administering to the subject an effective amount of a peptide of any one of embodiments 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0661] 53, The method of embodiment 51 or 52, wherein the subject has a metabolic disorder.

[0662] 54. The method of any one of embodiments 51-53, wherein the subject has obesity, prediabetes, diabetes, non-alcoholic fatty liver disease (NAFLD), cardiometabolic disease, polycystic ovary syndrome (PCOS), chronic kidney disease, peripheral arterial disease, heart failure, Alzheimer’s disease, sleep apnea, or knee osteoarthritis.

[0663] 55. A method of stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, or reducing caloric intake in a Atty Ref. METS-030 / 01 WO 350242-2268

[0664] subject in need thereof, the method comprising administering to the subject an effective amount of a peptide of any one of embodiments 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0665] 56. The method of any one of embodiments 51-55, wherein the peptide is administered to the subject orally.

[0666] 57. A method of modulating P-arrestin signaling and / or GLP-1 receptor internalization, the method comprising contacting a cell with the peptide of any one of embodiments 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0667] 58. The method of any one of embodiments 51-57, wherein the peptide comprises

[0668]

[0669] (SEQ ID NO: 22),

[0670] or a pharmaceutically acceptable salt thereof.

[0671] 59. The peptide of any one of embodiments 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in:

[0672] (a) inducing or maintaining weight loss or managing body weight in a subject;

[0673] (b) preventing weight gain in a subject; and / or

[0674] (c) stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, and / or reducing caloric intake in a subject. Atty Ref. METS-030 / 01 WO 350242-2268

[0675] 60. Use of the peptide of any one of embodiments 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as a medicament.

[0676] EXAMPLES

[0677] R15] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the peptides, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting in their scope.

[0678] Example A: Methods of Making Peptides

[0679]

[0216] Peptide analogs can be prepared and modified via a variety of chemical reaction steps, that may be solid-phase-peptide synthesis or solution-phase-peptide synthesis, or recombinant techniques. Typically, methods for modifying the peptides include biotinylation and / or lipidation. In some embodiments, biotinylation and lipidation can be applied during or after peptide synthesis using biotin conjugated amino acids or lipid conjugated amino acids.

[0680] 1. Biotinylation

[0681]

[0217] Biotin modifications to exendin derivatives have been previously described, for example, in International Publication Nos. WO 2009 / 107900 Al, WO 2020 / 242268 Al, WO 2022 / 112849 Al, WO 2022 / 114908 Al, WO 2023 / 038450 Al, and WO 2021 / 107519 Al.

[0682] 2. Lipidation

[0683]

[0218] In one embodiment, the methods include dissolving the peptide and C20-yGlu-NHS in the polar aprotic solvent (e.g., dimethyl sulfoxide), under weakly basic condition. In one embodiment, the peptide and C20-yGlu-NHS are mixed at a volume ratio of 1: 1 to 1:5. In one embodiment, the concentration of peptide is 2 to 10 mg / mL, and the molar ratio is 1:1 to 1:5 (peptide: lipid). The mixture then reacts at room temperature for 30 to 180 min with gently shaking. In one embodiment, lipidated peptides is purified by Prep-LC and the eluate is collected in individual fractions. In one embodiment, the ACN contained in the fractionated solution is evaporated using the centrifugal evaporator at 45°C for 30 to 60 min. The solvent can be changed to water by ultrafiltration. The purified samples can then be analyzed by reversed phase-HPLC for purity check. In one embodiment, the samples are lyophilized at -88°C for 18hr and then stored at -20°C.

[0684]

[0219] In some embodiments, in addition to NHS, other amine-reactive reagents, e.g. 4-nitrophenyl Atty Ref. METS-030 / 01 WO 350242-2268

[0685] carbonate-ester (NPC), 2,3,5,6-tetrafluorophenyl-ester (TFP), and additional equivalents, may also be used.

[0686]

[0220] In one embodiment, the methods include dissolving the peptide and C20-yGlu-NPC in the polar aprotic solvent (e.g., dimethyl sulfoxide), under weakly basic condition. In one embodiment, the peptide and C20-yGlu-NPC are mixed at a volume ratio of 1: 1 to 1:5. In one embodiment, the concentration of peptide is 2 to 10 mg / mL, and the molar ratio is 1:1 to 1:5 (peptidedipid). The mixture then reacts at room temperature for 30 to 180 min with gently shaking. In one embodiment, lipidated peptides is purified by Prep-LC and the eluate is collected in individual fractions. In one embodiment, the ACN contained in the fractionated solution is evaporated using the centrifugal evaporator at 45°C for 30 to 60 min. The solvent can be changed to water by ultrafiltration. The purified samples can then be analyzed by reversed phase-HPLC for purity check. In one embodiment, the samples are lyophilized at -88°C for 18hr and then stored at -20°C.

[0687]

[0221] In one embodiment, the methods include dissolving the peptide and C20-yGlu-TFP in the polar aprotic solvent (e.g., dimethyl sulfoxide), under weakly basic condition. In one embodiment, the peptide and C20-yGlu-TFP are mixed at a volume ratio of 1:1 to 1:5. In one embodiment, the concentration of peptide is 2 to 10 mg / mL, and the molar ratio is 1:1 to 1:5 (peptidedipid). The mixture then reacts at room temperature for 30 to 180 min with gently shaking. In one embodiment, lipidated peptides is purified by Prep-LC and the eluate is collected in individual fractions. In one embodiment, the ACN contained in the fractionated solution is evaporated using the centrifugal evaporator at 45°C for 30 to 60 min. The solvent can be changed to water by ultrafiltration. The purified samples can then be analyzed by reversed phase-HPLC for purity check. In one embodiment, the samples are lyophilized at -88°C for 18hr and then stored at -20°C.

[0688] 3. Synthesis of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:35

[0689] 1) Peptide Synthesis

[0690]

[0222] Rink Amide MBHA resin in DMF was agitated with Ns at 20 °C or 25 °C for 0.5 h, 1 h, 2 h or 4 h. The mixture was filtered to get the resin. For deprotection, 20% piperidine in DMF was added and agitated the resin with Ns at 25 °C for 15 min, 20 min or 30 min. The resin was washed with DMF for 5 times and filtered to get the resin. For coupling, a solution of HATU and Fmoc-Lys(Dde)-OH or Fmoc-Lys(Ivdde)-OH in DMF was added to the resin, then the DIEA was added, the mixture was agitated with Ns at 20 °C or 25 °C for 30 min. For this coupling of SEQ ID NO:35, Atty Ref. METS-030 / 01 WO 350242-2268

[0691] a solution of Oxyma and Fmoc-Lys(Dde)-OH in DMF was added to the resin, then the DIG was added, the mixture was agitated at 25 °C for 5 min. The resin was washed with DMF for 5 times.

[0692]

[0223] Repeat above steps for the coupling of following amino acids: (2-40).

[0693] # Materials Coupling reagents

[0694] HATU and DIE A, HBTU and DIEAor 2 F moc- Ser(tBu)-OH

[0695] Oxima and DIG

[0696] 3 Fmoc-Pro-OH HATU and DIEA or Oxima and DIC 4 Fmoc-Pro-OH HATU and DIEA or Oxima and DIC 5 Fmoc-Pro-OH HATU and DIEA or Oxima and DIC 4&5 Fmoc-Pro-Pro-OH HATU and DIEA

[0697] HATU and DIEA, HBTU and DIEA or 6 Fmoc-Ala-OH

[0698] Oxima and DIC

[0699] HATU and DIEA, HBTU and DIEA or 7 Fmoc-Gly-OH

[0700] Oxima and DIG

[0701] HATU and DIEA, HBTU and DIEA or 8 F m oc- Ser(tBu)-0 FI

[0702] Oxima and DIC

[0703] HATU and DIEA, HBTU and DIEA or 9 Fmoc-Ser(tBu)-OH

[0704] Oxima and DIC

[0705] Fnioc- S er(tBu)- S er(P si (Me, Me)pro)- 8&9 HATU and DIEA

[0706] OH

[0707] 10 Fmoc-Pro-OH HATU and DIEA or Oxima and DIC 11 Fmoc-Gly-OH HATU and DIEA or HBTU and DIEA Fmoc-Gly-OH HATU and DIEA or HBTU and DIEA 12

[0708] or Fmoc-Gly(Dmb)-OH HATU and DIEA

[0709] 11&12 Fmoc-Gly-Gly-OH Oxyma and DIC

[0710] Fmoc-Asn(Trt)-OH, HBTU and DIEA or Oxima and DIC 13 Fmoc- Asp(OtBu)-OH, HATU and DIEA

[0711] or Fmoc-Gln(Trt)-OH HATU and DIEA

[0712] 14 Fmoc-Lys(Biotin)-OH HATU and DIEA or HBTU and DIEA HATU and DIEA, HBTU and DIEA or 15 Fmoc-Leu-OH

[0713] Oxima and DIC

[0714] HATU and DIEA, HBTU and DIEA or 16 Fmoc-Trp(Boc)-OH

[0715] Oxima and DIC

[0716] HATU and DIEA, HBTU and DIEA or 17 Fmoc-Glu(OtBu)-OH

[0717] Oxima and DIC

[0718] HATU and DIEA, HBTU and DIEA or 18 Fmoc-Ile-OH

[0719] Oxima and DIC

[0720] HATU and DIEA, HBTU and DIEA or 19 Fmoc-Phe-OH

[0721] Oxima and DIC

[0722] HATU and DIEA, HBTU and DIEA or 20 Fmoc-Leu-OH

[0723] Oxima and DIC

[0724] HATU and DIEA, HATU and NMM or 21 F moc- Arg(Pbf)-OH

[0725]

[0726] HBTU and DIEA Atty Ref. METS-030 / 01 WO 350242-2268

[0727] HATU and DIEA, HATU and NMM or 22 Fmoc-Val-OH

[0728] HBTU and DIEA

[0729] HATU and DIEA, HBTU and DIEA, 23 Fmoc-Ala-OH

[0730] Oxyma and DIG or TOTU and NMM HATU and DIEA, HBTU and DIEA or 24 Fmoc-Glu(OtBu)-OH

[0731] TOTU and NMM

[0732] HATU and DIEA, HBTU and DIEA or 25 Fmoc-Glu(OtBu)-OH

[0733] Oxyma and DIG

[0734] HATU and DIEA, HBTU and DIEA or 26 Fmoc-Glu(OtBu)-OH

[0735] Oxyma and DIC

[0736] HATU and DIEA, HBTU and DIEA or 27 Fmoc-Met-OH

[0737] COMU and NMM

[0738] HATU and DIEA, HBTU and DIEA, 28 Fmoc-Gln(Trt)-OH

[0739] Oxyma and DIC or COMU and NMM 29 Fmoc-Lys(Biotin)-OH HATU and DIEA or HBTU and DIEA HATU and DIEA, HBTU and DIEA or 30 Fmoc- S er(tB u)-OH

[0740] Oxima and DIG

[0741] HATU and DIEA, HBTU and DIEA or 31 Fmoc-Leu-OH

[0742] Oxima and DIC

[0743] 30&31 Fmoc-Leu-Ser(Psi(Me, Me)pro)-OH HATU and DIEA

[0744] HATU and DIEA, HBTU and DIEA or 32 Fmoc-Asp(OtBu)-OH

[0745] Oxima and DIC

[0746] HATU and DIEA, HBTU and DIEA or 33 F moc- Ser(tBu)-OH

[0747] Oxima and DIG

[0748] HATU and DIEA, HBTU and DIEA or 34 Fmoc-Thr(tBu)-OH

[0749] Oxima and DIC

[0750] HATU and DIEA, HBTU and DIEA or 35 Fmoc-Phe-OFI

[0751] Oxima and DIC

[0752] 34&35 Fmoc-Phe-Thr(Psi(M e, M e)pro )-OH Oxyma and DIC

[0753] HATU and DIEA, HBTU and DIEA or 36 Fmoc-Thr(tBu)-OH

[0754] Oxima and DIC

[0755] HATU and DIEA, HBTU and DIEA or 37 Fmoc-Gly-OH

[0756] Oxima and DIC

[0757] 38 Fmoc-Glu(OtBu)-OH HATU and DIEA or Oxima and DIC

[0758] 39 Fmoc-Aib-OH HATU and DIEA or Oxima and DIC Boc-Phe-OH, H AFU and DIEA

[0759] Boc-Tyr(tBu)-OH, H AFU and DIEA

[0760] 40

[0761] Boc-His(Trt)-OH Oxyma and DIC

[0762]

[0763] or Boc-D-His(Trt)-OH DEPBT and DIEA or HATU and DIEA

[0764]

[0224] After the coupling of Boc-Phe-OH, Boc-Tyr(tBu)-OH, Boc-His(Trt)-OH or Boc D-His(Trt)-OH, add 3% FEN NH2 / DMF and react on 30 min and then repeat it for one more time. Drain and wash with DMF for 5 times. Atty Ref. METS-030 / 01 WO 350242-2268

[0765]

[0225] Repeat above steps for the coupling of following amino acids: (41-44).

[0766] # Materials Coupling reagents

[0767] 41 Fmoc-AEEA-OH HATU and DIEA or Oxyma and DIC

[0768] 42 Fmoc-AEEA-OH HAITI and DIEA or Oxyma and DIC

[0769] 43 Fmoc-Glu-OtBu HAITI and DIEA or Oxyma and DIC

[0770] 44 C18Diacid(tBu) HAITI and DIEA

[0771]

[0772]

[0226] Drain and wash with DMF for 5 times and then drain and wash with MEOH for 3 times. For SEQ ID NO:35, drain and wash with DMF for 5 times, and then drain and wash with DCM for 3 times, IPA for 3 times and DMF for 3 times. Then filtered to get the resin.

[0773] 2) Peptide Cleavage and Purification

[0774]

[0227] ?\dd cleavage solution (92.5% TFA / 2.50% TIS / 2.50% H2O / 2.50% 3 -Mercaptopropionic acid) to the flask containing resin at room temperature and stirred for 2 h. Precipitated the peptide with cold isopropyl ether. Filter and collect the filter cake. The filter cake was washed with isopropyl ether for 2 times. Dry the crude peptide under vacuum 2 h to get the crude peptide, the crude peptide was confirmed via LCMS. The crude peptide was purified by prep-HPLC (TFA condition: A: 0.075 % TFA in H2O, B: ACN) and (HC1 condition: A: 0.01 % HC1 in H2O, B: ACN) to give the final product as a white solid and confirmed via LCMS and IIPLC.

[0775] 4. Synthesis of SEQ ID NO:6 and SEQ ID NO: 7

[0776] 1) Peptide Synthesis

[0777]

[0228] To a solution of 2-CTC resin, Dde-Lys(Fmoc)-OH and DIEA in DCM was agitated with N2 at 25 °C for 2 h. Then added MeOH and agitated with N2 at 25CC for 0.5 h. The resin was washed with DMF for 5 times to get the resin. For deprotection, 20% piperidine in DMF was added and agitated the resin with N2 at 25 °C for 20 min. The resin was washed with DMF for 5 times and filtered to get the resin. For coupling, a solution of Fmoc-Glu-OTBU and HBTU in DMF was added DIEA to the resin and agitated with N2 at 25 °C for 40 min. The resin was then washed with DMF for 5 times.

[0778]

[0229] Repeat above steps for the coupling of following amino acids: (1-39).

[0779] # Materials Coupling reagents

[0780] 1 C20-tBu HBTU and DIEA

[0781]

[0782] De-Dde 3% Hydrazine hydrate / DMF Atty Ref. METS-030 / 01 WO 350242-2268

[0783] Fmoc-Lys (Boc)-OH HBTLT and DIE A

[0784] Fmoc-Ser (tBu)-OH

[0785] HBTU and DIEA

[0786] or Fmoc-Gly (tBu)-OH

[0787] Fmoc-Pro-OH HBTU and DIEA

[0788] Fmoc-Pro-OH HBTU and DIEA

[0789] Fmoc-Pro-OH HBTU and DIEA

[0790] Fmoc-Ala-OH

[0791] HBTU and DIEA

[0792] or Fmoc-Lys (tBu)-OH

[0793] Fmoc-Gly-OH HBTLT and DIEA

[0794] Fmoc-Ser (tBu)-OH HBTU and DIEA

[0795] Fmoc-Ser (tBu)-OH HBTU and DIEA

[0796] Fmoc-Pro-OH HBTU and DIEA

[0797] Fmoc-Gly-Gly-OH HBTU and DIEA

[0798] Fnioc-Asn (Trt)-OH

[0799] HBTLT and DIEA

[0800] or Fmoc-GIn (Trt)-OH

[0801] Fmoc-Lys (Dde)-OH HBTU and DIEA

[0802] Fmoc-Leu-OH HBTU and DIEA

[0803] Fmoc-Trp (Boc)-OH HBTU and DIEA

[0804] Fmoc-Glu (tBu)-OH HBTU and DIEA

[0805] Fmoc-Ile-OH HBTU and DIEA

[0806] Fmoc-Phe-OH HBTLT and DIEA

[0807] Fmoc-Leu-OH

[0808] HBTU and DIEA

[0809] or Fmoc-Glu-OH

[0810] Fmoc-Arg (Pbf)-OH HBTU and DIEA

[0811] Fmoc-Val-OH HBTU and DIEA

[0812] Fmoc-Ala-OH

[0813] HBTU and DIEA

[0814] or Fmoc-Arg-OH

[0815] Fmoc-Glu (tBu)-OH

[0816] HBTU and DIEA

[0817] or Fmoc-Lys (tBu)-OH

[0818] Fmoc-Glu (tBu)-OH HBTU and DIEA

[0819] Fmoc-Glu (tBu)-OH HBTU and DIEA

[0820] Fmoc-Met-OH

[0821] HBTLT and DIEA

[0822] or Fmoc-Leu-OH

[0823] Fmoc-GIn (Trt)-OH HBTU and DIEA

[0824] Fmoc-Lys (Dde)-OH HBTU and DIEA

[0825] Fmoc-Ser (tBu)-OH HBTU and DIEA

[0826] Fmoc-Leu-OH

[0827] HBTU and DIEA

[0828] or Fmoc-Val-OH

[0829] Fmoc-Asp (tBu)-OH HBTU and DIEA

[0830] Fmoc-Thr(tBu)-SerPsi(Me, Me)Pro-OH HATH and DIEA

[0831] Fmoc-Phe-OH HBTU and DIEA

[0832] Fmoc-Gly-ThrPsi(Me, Me)Pro-OH HAITI and DIEA

[0833]

[0834] Fmoc-Glu (tBu)-OH HBTU and DIEA Atty Ref. METS-030 / 01 WO 350242-2268

[0835] 37 Fmoc-Aib-OH HBTLT and DIEA

[0836] Fmoc-Phe-OH

[0837] 38 HBTU and DIEA

[0838] or Fmoc-Tyr-OH

[0839] # Di- TERT-BUTYL DICARBONATE DIEA

[0840] # De-Dde 3% Hydrazine hydrate / DMF

[0841]

[0842] 39 D-Biotin HBTU and DIEA

[0843] 2) Peptide Cleavage and Purification

[0844]

[0230] ?\dd cleavage solution (90.0% TFA / 2.50% H2O / 2.50% 3-Mercapto-propionicacid / 2.50% TIS) to the flask containing resin at 25 °C and stirred for 3 h. Precipitated the peptide with cold isopropyl ether. Filter and collect the filter cake. The filter cake was washed with isopropyl ether. Dry' the crude peptide under vacuum 2 h to get the crude peptide, the crude peptide was confirmed via LCMS. The crude peptide was purified by prep-HPLC (TFA condition: A: 0.075 % TFA in H2O, B: ACN) and prep-HPLC (HC1 condition: A: 0.01 % HO in H2O, B: ACN) to give the final product as a white solid and confirmed via LCMS and HPLC

[0845] 5. Synthesis ofSEQ ID NO:22 and SEQ ID NO:23

[0846] 1) Peptide Synthesis

[0847]

[0231] To the 2-CTC resin was added Dde-Lys(Fmoc)-OH and DIEA in DCM. The mixture was agitated with N2 for 2 h at 20 °C, then added MeOH and agitated with N2 for another 30 min. The resin was then washed with DMF for 6 times.

[0848]

[0232] For deprotection, 20% piperidine in DMF was added and agitated the resin with N2 at 25 °C for 15 min. The resin was washed with DMF for 5 times and filtered to get the resin. A solution of Fmoc-Glu-OtBu, HBTU and DIEA in DMF was added to the resin and agitated with N2 at 20 °C for 30 min. The resin was then washed with DMF for 5 times.

[0849]

[0233] For deprotection, 20% piperidine in DMF was added and agitated the resin with N2 at 25 °C for 15 min. The resin was washed with DMF for 5 times and filtered to get the resin. A solution of C20Diacid(OtBu), HBTU and DIEA in DMF was added to the resin and agitated with N2 at 20CC for 30 min. The resin was then washed with DMF for 5 times. After the coupling C20Diacid(OtBu), add 3% H2N NH2 / DMF and react on 30 min and then repeat it for one more time. Drain and wash with DMF for 5 times.

[0850]

[0234] Repeat above steps for the coupling of following amino acids: (4-43). Atty Ref. METS-030 / 01 WO 350242-2268

[0851] Materials Coupling reagents

[0852] Fmoc-Lys(Boc)-OH (3. OOeq) HATU and DIEA

[0853] Fmoc-Ser(tBu)-OH (3. OOeq) HATU and DIEA

[0854] Fmoc-Pro-OH (3. OOeq) HATU and DIEA

[0855] Fmoc-Pro-OH (3. OOeq) HATU and DIEA

[0856] Fmoc-Pro-OH (3. OOeq) HATU and DIEA

[0857] Fmoc-Ala-OH (3. OOeq) HATU and DIEA

[0858] Fmoc-Gly-OH (3. OOeq) HATU and DIEA

[0859] Fmoc-Ser(tBu)-OH (3. OOeq) HATU and DIEA

[0860] Fmoc-Ser(tBu)-OH (3. OOeq) HATU and DIEA

[0861] Fmoc-Pro-OH (3. OOeq) HATU and DIEA

[0862] Fmoc-Gly-OH (3. OOeq) HATU and DIEA

[0863] Fmoc-Gly-OH (3. OOeq) HATU and DIEA

[0864] Fmoc-Gln(Trt)-OH (3. OOeq) HATU and DIEA

[0865] Fmoc-Lys(Biotin)-OH (3. OOeq) HATU and DIEA

[0866] Fmoc-Leu-OH (3. OOeq) HATU and DIEA

[0867] Fmoc-Trp(Boc)-OH (3. OOeq) HATU and DIEA

[0868] Fmoc-Glu(OtBu)-OH (3. OOeq) HATU and DIEA

[0869] Fnioc-Ile-OH (3. OOeq) HATU and DIEA

[0870] Fmoc-Phe-OH (3. OOeq) HATU and DIEA

[0871] Fmoc-Leu-OH (3. OOeq) HATU and DIEA

[0872] Fmoc-Arg(Pbf)-OH (3. OOeq) HATU and DIEA

[0873] Fmoc-Val-OH (3. OOeq) HATU and DIEA

[0874] Fmoc-Ala-OH (3. OOeq) HATU and DIEA

[0875] Fmoc-Glu(OtBu)-OH (3, OOeq) HATU and DIEA

[0876] Fmoc-Glu(OtBu)-OH (3, OOeq) HATU and DIEA

[0877] Fmoc-Glu(OtBu)-OH (3, OOeq) HATU and DIEA

[0878] Fmoc-Met-OH (3. OOeq) HATU and DIEA

[0879] Fmoc-Gln(Trt)-OH (3. OOeq) HATU and DIEA

[0880] Fmoc-Lys(Biotin)-OH (3. OOeq) HATU and DIEA

[0881]

[0882] Atty Ref. METS-030 / 01 WO 350242-2268

[0883] 33 Fmoc-Ser(tBu)-OH (3.00eq) HATU and DIEA

[0884] 34 Fmoc-Leu-OH (3.00eq) HATU and DIEA

[0885] 35 Fmoc-Asp(OtBu)-OH (3.00eq) HATU and DIEA

[0886] 36 Fmoc-Ser(tBu)-OH (3.00eq) HATU and DIEA

[0887] 37 Fmoc-Thr(tBu)-OH (3.00eq) HATU and DIEA

[0888] 38 Fmoc-Phe-OH (3.00eq) HATU and DIEA

[0889] 39 Fmoc-Thr(tBu)-OH (3.00eq) HATU and DIEA

[0890] 40 Fmoc-Gly-OH (3.00eq) HATU and DIEA

[0891] 41 Fmoc-Glu(OtBu)-OH (3.00eq) HATU and DIEA

[0892] 42 Fmoc-Aib-OH (3.00eq) HATU and DIEA

[0893] 43 Boc-Phe-OH (3.00eq) HATU and DIEA

[0894]

[0895] 2) Peptide Cleavage and Purification

[0896]

[0235] After last step, the resin was washed with MeOH for 3 times, and dried under vacuum. Then the peptide resin was treated with the cleavage cocktail (92.5%TFA / 2.5% 3-MPA / 2.5%Tis / 2.5%H2O) for 2.0 hours. 2) Precipitated the peptide with cold isopropyl ether. Filter and collect the filter cake. The filter cake was washed with isopropyl ether for 2 times. Dry the crude peptide under vacuum 2 h to get the crude peptide. The crude peptide was confirmed by LCMS. The crude peptide was purified by prep-HPLC (TFA condition: A: 0.075 % TFA in H2O, B: ACN) and (HC1 condition: A: 0.01 % HC1 in H2O, B: ACN) to give the final product as a white solid.

[0897] Example Peptides and Peptide Analogs

[0898]

[0236] Peptides SEQ ID NOs: 45 and 46 are modified to form peptides SEQ ID NO: 6 and SEQ ID NO: 7, respectively, for possible extended half-life. Amino acid sequences of the peptides are listed in Table 4 with biotinylation at a Lysine residue and lipidation at a Lysine residue with the fatty acid derivative 2OEG-yGlu-C18.

[0899] Table 4. Peptide sequences, unmodified and modified

[0900] SEQ ID

[0901] Amino Acid Sequence

[0902] NO:

[0903] F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK-NH245 Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK-NH246 H(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK((2-[2-(2- 3

[0904]

[0905] Atty Ref. METS-030 / 01 WO 350242-2268

[0906] Amino-ethoxyO-ethoxy] -acetyrl)2-(Y-Ghj)-CO-(CH2) 16-COzH)-NH2 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSK((2-[2-(2- Amino-etlioxy)-ethoxy] -acetyl)2-(y-Glu)-CO-(CH2)i 6-CO2H)-NH2

[0907] Y(Aib)EGTFTSD^

[0908] Amino-ethoxy)-ethoxjr]-acetyl)2-(y-Glu)-CO-(CH2))6-CO2H)-NH2

[0909]

[0910] O H OH

[0911]

[0912] (SEQ ID NO: 3)

[0913]

[0237] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5511.33; observed molecular weight [M+3H]3+: 1838.2.

[0914] H

[0915]

[0916] (SEQ IDNO: 4)

[0917]

[0238] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: Atty Ref. METS-030 / 01 WO 350242-2268

[0918] 5521.36; observed molecular weight [M+3H]3+: 1841.2589.

[0919] o H o H

[0920]

[0921] (SEQ ID NO: 5)

[0922]

[0239] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5537.36; observed molecular weight [M+3H]3+: 1846.5911.

[0923]

[0240] Amino acid sequences of the peptides are listed in Table 5. Biotinylation is at a Lysine residue. Lipidation is at a Lysine residue. SEQ ID NOs: 6 and 7 are amidated at C -terminus. Table 5. Peptide sequences, unmodified and modified

[0924] Amino Acid Sequence SEQ ID NO: F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFlEWLK(Biotin)NGGPSSGAPPPSKK((y-Glu)-CO- 6 (CH2)18-CO2H)-OH

[0925] Y(Aib)EGTFTSDLSK(Biotm)QMEEEAVRLFIEWLK(Biotin)NGGPSSGAPPPSKK((y-Glu)-CO- 7 (CH2)I8-CO2H)-OH

[0926] F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSK((2-[2-(2-Amino- 21 ethoxy)-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)16-CO2H)-NH2F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIFA\AK(Biotin)QGGPSSGAPPPSKK((y-Glu)-CO- 22 (CH2)18-CO2H)-OH

[0927] F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPGKK((y-Glu)-CO- 23 (CH2)18-CO2H)-OH

[0928] H(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEVvLK(Biotin)DGGPSSGAPPPSK((2-[2-(2-Amino- 30 ethoxy)-etboxy] -acetyl)2-(y-Ghi)-CO-(CH2)i6-CO2H)-NH2H(Aib)EGrlTTSDLSK(Biotin)QMEEEAVRLF]EWLK(Biodn)-(isoD)-GGPSSGAPPPSK((2-[2-(2- 31 Amino-ethoxy )-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)i6-CO2H)-NH2

[0929]

[0930] Atty Ref. METS-030 / 01 WO 350242-2268

[0931] F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIE^K(Biotin)DGGPSSGAPPPSK((2-[2-(2-Aroino- 32 ethoxy)-ethoxy]-acetyl)2-(Y-Glu)-CO-(CH2)i6-CO2H)-NH2 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)-(isoD)-GGPSSGAPPPSK((2-[2-(2- 33 Amino-etlioxy)-ethoxy] -acetyl)2-(y-Glu)-CO-(CH2)i 6-CO2H)-NH2

[0932] ()> His)-AibEGTFTSDI.. SK(B!otin)QAffiEEAV7RI, FIEWI. K(Biotin)NGGPSSGAPPPSK((2-[2-(2- 35 Amino-ethoxy)-ethoxjr] -acetyl)2-(y-Glu)-CO-(CH2)) 6-CO2H)-NH2 F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGKPPPSKK((y-Glu)-CO- 39 (CH2)i8-CO2H)-OH

[0933] F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGKPPPSKK((2-[2-(2-Amino- 40 ethoxy)-ethoxy;]-acetyl)2-(y-Glu)-CO-(CH2)i8-phosphonate)-OH F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGG PSSGAPPPSKK((y-Glu)-(Trx)- 41 CO-(CH2)i8-CO2l-I)-OH F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSKK((PEG)s- 42 (Malonate)-CO(CH2)2o)-C02H)-OH F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotiii)QGGPSSGAPPPSKK((2-[2-(2-Aniino- 43 ethoxy)-ethoxy]-acetyl)-(Urea)-(diaminobutane)-(Y-Glu)-CO-(CH2)i8-CO2H)-OH F(Aib)EGTFTSDLSK(Biotin)QMEEEAVRLFIEWLK(Biotin)QGGPSSGAPPPSKK((glucuronic 44 acid-CO-(CH2)i8-CO2H)-OH

[0934]

[0935]

[0936] (SEQ IDNO: 6)

[0937]

[0241] The molecular weight is determined by LCMS and HPLC Calculated molecular weight: 5388.26; observed molecular weight [M+3H]3+: 1796.9115. Atty Ref. METS-030 / 01 WO 350242-2268

[0938]

[0939] (SEQ ID NO: 7)

[0940]

[0242] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5406,26; observed molecular weight [M+3H]3+: 1802.2435.

[0941]

[0942] (SEQ ID NO: 21)

[0943]

[0243] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5535.39; observed molecular weight [M+3H]3+: 1845.9337. Atty Ref. METS-030 / 01 WO 350242-2268

[0944]

[0945] (SEQ ID NO: 22)

[0946]

[0244] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5402.29; observed molecular weight [M+3H]3: 1801.5827.

[0947]

[0948] (SEQ ID NO:23)

[0949]

[0245] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5372.26; observed molecular weight [M+3H]3+: 1791.5774.

[0950]

[0951] Atty Ref. METS-030 / 01 WO 350242-2268

[0952] (SEQ ID NO: 30)

[0953]

[0246] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5512.31; observed molecular weight [M+3H]3+: 1838.2061.

[0954] o H O H

[0955]

[0956] (SEQ ID NO: 31)

[0957]

[0247] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5512.31; observed molecular weight [M+3H]3+: 1838.3101.

[0958] H OH

[0959]

[0960] (SEQ ID NO: 32)

[0961]

[0248] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5522.35; observed molecular weight [M+3H]3+: 1841.5535. Atty Ref. METS-030 / 01 WO 350242-2268

[0962]

[0963] (SEQ ID NO: 33)

[0964]

[0249] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5522.35; observed molecular weight [M+3H]3+: 1841.5811.

[0965]

[0966] (SEQ ID NO: 35)

[0967]

[0250] The molecular weight is determined by LCMS and HPLC. Calculated molecular weight: 5511.33; observed molecular weight [M+3H]3+: 1838.2. Atty Ref. METS-030 / 01 WO 350242-2268 (SEQ ID NO: 39)

[0968] (SEQ ID NO: 40)

[0969]

[0970] Atty Ref. METS-030 / 01 WO 350242-2268

[0971]

[0972] (SEQ ID NO: 44) Atty Ref. METS-030 / 01 WO 350242-2268

[0973] Example 1: In vitro activity of SEQ ID NO: 4 and SEQ ID NO: 5

[0974] Activity of cAMP for GLP-1 R and fl-arrestin-2 recruitment

[0975]

[0251] To determine the activity of peptides in the GLP-1 receptor (GLP-1 R)-expressing CH0-K1 cell line, cell lines were purchased from Eurofins. HITHUNTER® cAMP Assay for small molecules (DiscoverX) was used and P-arrestin-2 recruitment was measured using a PathHunter® express GLP1R CH0-K1 P-Arrestin-2 GPCR Assay.

[0976]

[0252] In cAMP assay, the cells were seeded in a 96-well plate at 7 x 103cells / well using cell plate reagent and cells were incubated overnight at 37°C and 5% CO2. To perform the cAMP detection assay, cell plating reagent was replaced with the cAMP Assay buffer. Each of the wells was then treated with the diluted peptide and incubated for 30 minutes under conditions of a temperature of 37°C in a CO2 incubator.

[0977]

[0253] After the incubation, cAMP Antibody reagent and working cAMP detection solution was added to all wells. The plate was incubated for 1 hour at room temperature in the dark. Then, cAMP Solution A was added and incubated for 3 hours at room temperature in the dark. Subsequently, the luminescence was measured using the microplate reader device.

[0978]

[0254] In the p-arrestin-2 recruitment assay, the cells were seeded in a 96-well plate at 1 x 104cells / well using cell plate reagent and cells were incubated for 48h at 37°C and 5% CO2. To perform the P-arrestin-2 recruitment detection assay, each of the wells was then treated with the diluted peptide and incubated for 90 minutes under conditions of a temperature of 37°C in a CO2 incubator.

[0979]

[0255] After the incubation, working detection solution was added to all wells. The plate was incubated for 1 hour at room temperature in the dark. Subsequently, the luminescence was measured using the microplate reader device.

[0980]

[0256] Peptides SEQ ID NOs:45 and 46 were modified to form peptides SEQ ID NOs:4 and 5, respectively. GLP-1 R activation and p-Arrestin-2 activation comparisons among semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 are shown in FIG. 1A and FIG. IB. Data points are the mean ± SD of duplicate measurements. cAMP assay conducted with HitHunter® cAMP Assay for Small Molecules (Eurofins, #90-0075 SM 10). P-arrestin-2 recruitment measured using a PathHunter® eXpress GLP1R CHO-K1 P-Arrestin GPCR Assay (Eurofins, #93-0300E2CP0M).

[0981]

[0257] For GLP-1R cAMP activity, EC50 (nM) of semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 were 0.093, 0.051, 0.131, and 0.109, respectively, shown in Table 6. Atty Ref. METS-030 / 01 WO 350242-2268

[0982] Table 6. GLP-1R activation with cAMP assay for SEQ ID NO:4 and SEQ ID NO:5

[0983] SEQ ID NO: EC50(nM) Emax (%)

[0984] semaglutide 0.09315 100%

[0985] 3 0.05083 90%

[0986] 4 0.1306 84%

[0987] 5 0.1094 79%

[0988]

[0989]

[0258] For P-Arrestin-2 activity, ECso (nM) of semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 were 7.16, 9.31, 132, and 106, respectively, shown in Table 7.

[0990] Table 7. p-Arrestin-2 recruitment assay for SEQ ID NO:4 and SEQ ID NO:5

[0991] SEQ ID NO: ECso (nM) Emax (%)

[0992] semaglutide 7.158 100%

[0993] 3 9.314 55%

[0994] 4 132.3 8%

[0995] 5 106 8%

[0996]

[0997]

[0259] Signaling bias to preferentially activate cAMP while having minimal to B-arrestin-2 recruitment results in decreased receptor internalization (endocytosis) and desensitization, both of which may lead to prolonged pharmacological activity. We measured G protein-dependent cAMP generation and P-arrestin-2 recruitment with peptides and calculated the bias by fitting response data. P-arrestin-2 recruitment response for SEQ ID NO:4 and SEQ I D NO: 5 were barely detectable. This indicated enhanced cAMP signaling and reduced P-arrestin-2 recruitment for SEQ ID NO:4 and SEQ ID NO:5 compared with Semaglutide and SEQ ID NO:3. GLP-1R and P-Arrestin-2 activation bias is represented in FIG.2 as bias factors AAlog(r / KA) are calculated for semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. Bias between cAMP and p-arrestin-2 responses was determined using a modified operational model of agonism (Westphal et al., 2013). Bias factors could not be calculated for SEQ ID NO:4 and SEQ ID NO:5, because the P-arrestin-2 recruitment assay showed no quantifiable activity for SEQ ID NO:4 and SEQ ID NO:5. The significant difference determined using an unpaired r-test between semaglutide and SEQ ID NO:3, **,p < 0.01. Atty Ref. METS-030 / 01 WO 350242-2268

[0998] Example 2: In vitro activity of SEQ ID NO:6

[0999]

[0260] GLP-1R activation and P-Arrestin-2 activation were performed as described above for Example 1 and comparisons among exendin-4 and SEQ ID NO:6 are shown in FIG.3A and FIG.

[1000] 3B. Data points are the mean ± SD of duplicate measurements. cAMP assay conducted with HitHunter® cAMP Assay for Small Molecules (Eurofins, #90-0075 SAHO). P-arrestin-2 recruitment measured using a PathHunter® eXpress GLP1R CHO-K1 P-Arrestin GPCR Assay (Eurofins, #93-0300E2CP0M).

[1001]

[0261] For GLP-1R cAMP activity, ECso (nM) of exendin-4 and SEQ ID NO:6 were 0.0203, and 0.0809 respectively, shown in Table 8.

[1002] Table 8. GLP-1R activation with cAMP assay for SEQ ID NO:6

[1003] ECso (nM) Emax(%)

[1004] exendin-4 0.02032 100%

[1005] SEQ ID NO: 6 0.08086 80%

[1006]

[1007]

[0262] For P-Arrestin-2 activity, ECso (nM) of exendin-4 and SEQ ID NO:6 were 1.67 and 180, respectively, shown in Table 9.

[1008] Table 9. p-Arrestin-2 recruitment assay for SEQ ID NO:6

[1009] ECso (nM) Emax(%)

[1010] exendin-4 1.673 100%

[1011] SEQ ID NO: 6 179.9 11%

[1012]

[1013]

[0263] GLP-1R and P-Arrestin-2 activation bias for exendin-4 and SEQ ID NO:6 were calculated and represented in FIG. 4. Bias factors AAlogQ / KA) were calculated for exendin-4 and SEQ ID NO:6. Bias between cAMP and p-arrestin-2 responses was determined using a modified operational model of agonism (Westphal et al., 2013).

[1014] Example 3: In vitro activity of SEQ ID NO:7

[1015]

[0264] GLP-1R activation and P-Arrestin-2 activation were performed as described above for Example 1 and comparisons among exendin-4 and SEQ ID NO:7 are shown in FIG. 5A and FIG.

[1016] 5B. Data points are the mean ± SD of duplicate measurements. cAMP assay conducted with HitHunter® cAMP Assay for Small Molecules (Eurofins, #90-0075 SM 10). P-arrestin-2 Atty Ref. METS-030 / 01 WO 350242-2268

[1017] recruitment measured using a PathHunter® eXpress GLP1R CH0-K1 P-Arrestin GPCR Assay (Eurofin s, #93-0300E2CP0M).

[1018]

[0265] For GLP-1R cAMP activity, ECso (nM) of exendin-4 and SEQ ID NO:7, were 0.0203 and 0.0977, respectively, shown in Table 10.

[1019] Table 10. GLP-1R activation with cAMP assay for SEQ ID NO:7

[1020] ECso (nM) Emax(%)

[1021] exendin-4 0.02032 100%

[1022] SEQ ID NO: 7 0.09771 72%

[1023]

[1024]

[0266] For p-Arrestin-2 activity, ECso (nM) of exendin-4 and SEQ ID NO:7 were 3.466 and 84.1, respectively, shown in Table 11.

[1025] Table 11. P-Arrestin-2 recruitment assay for SEQ ID NO:7

[1026] ECso (nM) Emax(%)

[1027] exendin-4 3.466 100%

[1028] SEQ ID NO: 7 84.1 10%

[1029]

[1030] Example 4: In vitro activity of SEQ ID NO:4, SEQ ID NO:32, and SEQ ID NO:33

[1031]

[0267] GLP-1 R activation were performed as described above for Example I and comparisons among GLP-1, SEQ ID NO:4, SEQ ID NO:32, and SEQ ID NO:33 are shown in FIG. 6. Data points are the mean ± SD of duplicate measurements. c / XMP assay conducted with HitHunter® cAMP Assay for Small Molecules (Eurofins, #90-0075SM10).

[1032]

[0268] For GLP-1 R cAMP activity, ECso (nM) of GLP-1, SEQ ID NO:4, SEQ ID NO:32, and SEQ IDNO:33 were 0.02962, 0.132, 0.2151, and 10.73, respectively, shown in Table 12.

[1033] Table 12. GLP-1R activation with cAMP assay for SEQ ID NO:4, SEQ ID NO:32, and SEQ ID NO:33

[1034] ECso (nM) Emax(%)

[1035] GLP-1 0.02962 100%

[1036] SEQ ID NO: 4 0.132 94%

[1037] SEQ ID NO: 32 0.2151 77%

[1038] SEQ ID NO: 33 10.73 68%

[1039]

[1040] Atty Ref. METS-030 / 01 WO 350242-2268

[1041] Example 5: In vitro activity of SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:23

[1042]

[0269] GLP-1R activation and P-Arrestin-2 activation were performed as described above for Example 1 and comparisons among GLP-1, SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:23 are shown in FIG. 7 A and FIG. 7B. Data points are the mean ± SD of duplicate measurements. cAMP assay conducted with HitHunter® cAMP Assay for Small Molecules (Eurofins, #90-0075SM10). p-arrestin-2 recruitment measured using a PathHunter® eXpress GLP1R CHO-K1 P-Arrestin GPCR Assay (Eurofins, #93-0300E2CP0M).

[1043]

[0270] For GLP-1R cAMP activity, ECso (nM) of GLP-1, SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:23 were 0.05677, 0.1338, 0.1893, and 0.1509, respectively, shown in Table 13.

[1044] Table 13. GLP-1R activation with cAMP assay for SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:23

[1045] ECso (nM) Emax(%)

[1046] GLP-1 0.05677 100%

[1047] SEQ ID NO: 6 0.1338 101%

[1048] SEQ ID NO: 22 0.1893 102%

[1049] SEQ ID NO: 23 0.1509 100%

[1050]

[1051]

[0271] For P-Arrestin-2 activity, ECso (nM) of GLP-1, SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:23 were 5.947, 45.09, 112, and 122.6, respectively, shown in Table 14.

[1052] Table 14. p-Arrestin-2 recruitment assay for SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:23

[1053] ECso (nM) Emax(%)

[1054] GLP-1 5.947 100%

[1055] SEQ ID NO: 6 45.09 9%

[1056] SEQ ID NO: 22 112 7%

[1057] SEQ ID NO: 23 122.6 6%

[1058]

[1059] Example 6: In vitro stability study with intestinal enzymes of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7

[1060]

[0272] This study was conducted to determine the enzymatic stability of SEQ ID NO:3, SEQ ID Atty Ref. METS-030 / 01 WO 350242-2268

[1061] N0:4, SEQ ID N0:5, SEQ ID N0:6, and SEQ ID N0:7 in artificial intestinal environment. Methods

[1062]

[0273] For the stability against FaSSIF / P, the test samples were prepared by mixing stock solutions of peptide and pancreatin dissolved in FaSSIF solution (pH 6.5) in a weight ratio of 19: 1. The test samples were incubated at 37°C for 120 min. The sampling from test samples was proceeded at the predetermined time points (10, 30, 60, and 120 min) and stopped by 1% TFA solution.

[1063]

[0274] For the stability against trypsin, the test samples were prepared by mixing stock solutions of peptide and trypsin dissolved in 50 niM PBS (pH 7.8) containing 0.02% PS80 in a weight ratio of 5:1. The test samples were incubated at 37°C for 180 min. The sampling from test samples was proceeded at the predetermined time points (10, 30, 60, 90, 120, and 180 min) and stopped by 0.1% TFA solution.

[1064]

[0275] Analytical HPLC was performed to determine the remaining amount on an Infinity II 1290 (Agilent, USA) with an ACQUITY CSH C18 column (Waters, USA) at 35°C using 0.1% TFA in water and acetonitrile. The remaining amount was determined as a percentage of the peak area at the sampling time point relative to the peak area of the initial sample.

[1065] Results

[1066]

[0276] The enzymatic stability was determined in artificial intestinal environment. The half-lives determined by remaining amount in FaSSIF / P and trypsin are summarized in Table 15 and Table 16. As a result of this study, peptide constructs had similar stability against FaSSIF / P and SEQ ID NO:6 and SEQ ID NO.7 showed greater stability against trypsin (FIGs. 8A-8D) while exenatide and semaglutide had low stability (Table 17).

[1067] Table 15. In vitro stability study of SEQ ID NO:4 and SEQ ID NO:5 against FaSSIF / P and trypsin.

[1068] SEQ ID Half-life (min)

[1069] NO: FaSSIF / P Trypsin

[1070] 3 37.8 73.5

[1071] 4 29.4 97.3

[1072] 5 31.7 83.9

[1073]

[1074] Table 16. In vitro stability study of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, against FaSSIF / P and trypsin. Atty Ref. METS-030 / 01 WO 350242-2268

[1075] SEQ ID Half-life (min)

[1076] NO: FaSSIF / P Trypsin

[1077] 3 77.3 102.4

[1078] 4 55.0 150.4

[1079] 6 48.1 422.5

[1080] 7 50.5 367.2

[1081]

[1082] Table 17. In vitro stability study of exenatide and semaglutide against FaSSIF / P and trypsin.

[1083] Compound Half-life (min)

[1084] FaSSIF / P Trypsin

[1085] Exenatide 10.0 Less than 1 min

[1086] Semaglutide 7.9 Less than 1 min

[1087]

[1088] Example 7: In vitro stability study of SEQ ID NO: 22 against Trypsin Methods

[1089]

[0277] The enzymatic stability of the test compounds was evaluated using trypsin. Stock solutions of test compounds and trypsin (trypsin from bovine pancreas, Sigma Aldrich, Cat No. T1426) were prepared in phosphate buffer (pH 7.8). The mixtures were prepared to achieve a final peptide concentration and trypsin was 500 pg / mL and 100 pg / mL, respectively. The reaction mixtures were incubated at 37°C.

[1090]

[0278] ?\t predetermined points, samples were taken from the reaction mixture, and the enzymatic reactions were terminated by addition of an appropriate acidic organic solvent. The remaining amounts of the test compounds were quantified by L. C / UV. The results were expressed as percentages relative to the initial value, with the initial value defined as 100%.

[1091]

[0279] The half-live (T1 / 2) of each compound was determined by linear regression of In (remaining amount, %) versus incubation time. The rate constant (k) was obtained from the slope of the regression line, and T1 / 2 was calculated using the first-order kinetics equation (T1 / 2 = 0.693 / k).

[1092]

[0280] Table 18 provides half-live (T1 / 2) of semaglutide, SEQ ID NO: 3, and SEQ ID NO: 22 in trypsin.

[1093] Table 18. Half-live (T1 / 2) of semaglutide, SEQ ID NO: 3, and SEQ ID NO: 22 against trypsin Atty Ref. METS-030 / 01 WO 350242-2268

[1094] Test Compounds Half Life

[1095] Semaglutide <5 min

[1096] SEQ ID NO: 3 53.3 min

[1097] SEQ ID NO: 22 153.0 min

[1098]

[1099]

[0281] FIG. 9 shows the percentage of the remaining amount of the test compounds over 180 minutes.

[1100] Example 8: In vivo blood glucose regulation of SEQ ID NO:4 and SEQ ID NO:5

[1101]

[0282] To observe glucose regulation, peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 were administered to mice, and an intraperitoneal glucose tolerance test (ipGTT) was carried out to measure blood glucose regulating efficacy.

[1102] Methods

[1103]

[0283] To measure intraperitoneal glucose tolerance in an animal model, dosing volume 10 mL / kg of peptide (3 or 1 nmol / kg) was administered subcutaneously at 4 hours prior to glucose injection to male mice (C57BL / 6) at 7 weeks old, followed by intraperitoneal injection of 10 mL / kg dose of glucose (2g / kg). Changes in blood glucose in blood samples collected from the tail vein at 0, 20, 40, and 60 minutes after glucose dosing were observed. Vehicle was used as control. ipGTTs were repeated at 4, 24, 48 and 72 hours after peptide construct administrations.

[1104] Results

[1105]

[0284] Glycemic effects via ipGTT performed at 4 h and 72 h after a single SC dose are shown in FIGs. 10A-10D 3 nmol / kg of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 were highly effective at 4 h (FIGs. 10A-10B) and their glucose control abilities were maintained by 72 h (FIGs.

[1106] 10C-10D). When compared to vehicle, SEQ ID NO:4 and SEQ ID NO:5 seemed to be slightly improved than SEQ ID NO:3. ipGTT at 4 h and 72 h appeared similar between SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[1107] Example 9: In vivo blood glucose regulation of SEQ ID NO:4 and SEQ ID NO:6 Methods

[1108]

[0285] To observe glucose regulation, peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 were administered to mice, and an intraperitoneal glucose tolerance test (ipGTT) was carried out to measure blood glucose regulating efficacy. Atty Ref. METS-030 / 01 WO 350242-2268

[1109]

[0286] To measure glucose tolerance in an animal model, dosing volume 10 mL / kg of peptide (3 or 1 nmol / kg) was administered subcutaneously at 4 hours prior to glucose injection to male mice (C57BL / 6) at 8 weeks old, followed by intraperitoneal injection of 10 mL / kg dose of glucose (2g / kg). Changes in blood glucose in blood samples collected from the tail vein at 0, 20, 40, 60, 90 and 120 minutes after glucose dosing were observed. Vehicle was used as control. Semaglutide and SEQ ID NOG were observed for comparison. ipGTTs were repeated at 4, 24, 48 and 72 hours after peptide construct administrations.

[1110] Results

[1111]

[0287] Glycemic effects via ipGTT performed at 4 h and 72 h after a single SC dose are shown in FIGs. 11A-11D At 1 nmol / kg of semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 were highly effective at 4 h (FIGs. 11A-11B) and their glucose control abilities were maintained by 72 h (FIGs. 11C-11D). When compared to vehicle, SEQ ID NOG, and semaglutide, SEQ ID NO:4 and SEQ ID NO:6 seemed to be slightly improved. ipGTT at 4 h and 72 h appeared similar between SEQ ID NOG, SEQ ID NO:4, and SEQ ID NO:6

[1112] Example 10: In vivo oral pharmacokinetics of SEQ ID NO:4 and SEQ ID NO:5

[1113]

[0288] To observe oral pharmacokinetics, peptides SEQ ID NOG, SEQ IDN0:4, and SEQ ID NOG were orally (PO) administered to beagle dogs with a 3-in-l capsule and single capsule with SEQ ID NOG was used as reference. Pharmacokinetics were measured after oral administration.

[1114] Methods

[1115]

[0289] To measure pharmacokinetics, male beagle dogs fasted over-night at least 16 hrs and returned to food 4 hrs post dosing. Pentagastrin was administered to adjust the constant gastric-pH of dogs. Pentagastrin was administered IM at 0.006 mg / kg (0.25 mg / mL and 0.024 mL / kg) before 30 min capsule administration. Dogs were dosed PO with either 3-in-l capsule or single capsule.

[1116]

[0290] The blood samples were collected over 96 hrs and the individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma (K2-EDTA) concentrations were determined using a qualified LC-MS method.

[1117]

[0291] 3-in-l capsule contained 10 mg / capsule each of SEQ ID NOG, SEQ ID NO:4, and SEQ ID NO:5. Single capsule contained 10 mg / capsule of only SEQ ID NOG and same capsule composition as 3-in-l capsule. Enteric capsules were enterosoluble vacant gelatin capsule (Guangsheng, China). Enteric capsules further contained chenodeoxycholic acid, ursodeoxycholic acid, and propyl gallate. Atty Ref. METS-030 / 01 WO 350242-2268

[1118] Results

[1119]

[0292] Similar PK profiles of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO.5 were obtained in 3-in-1 capsules (10 mg per compound) which indicates no differences in oral absorption between the tested compounds shown in FIG. 12. Cmax and AUG of SEQ ID NO.3 from 3 -in- 1 capsules were lower than those of SEQ ID NO:3 10 mg capsule (single capsule) shown in Table 19, and the comparison of oral absorption is possible in cassette PK using 3-in-l capsules shown in Table 19 and FIG. 12

[1120] Table 19. Mean pharmacokinetic parameters of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 following a single oral administration to male beagle dogs

[1121] SEQ ID tl / 2 Tmas(>niax AUCiast AUCtof Formulation

[1122] NO: (hr) (hr) (ng / mL) (ng-hr / mL) (ng-hr / mL)

[1123] 4.6

[1124] 3 59.7 (22%) 82.6 (87%) 3349.9 (83%) 5265.4 (87%) (26%)

[1125] 89.8 5.0

[1126] 3-in-l capsule 4 67.1 (88%) 3331.5 (84%) 6362.1 (83%) (40%) (35%)

[1127] 4.7

[1128] 5 52.2 (42%) 81.1 (90%) 3384.4 (84%) 4769.6 (86%) (39%)

[1129] Single capsule 5.9 315.0 21239.8

[1130] 3 75.0 (41%) 12900.8 (60%) (reference) (39%) (63%) (64%) t.1 / 2, Tmax, Cmax, AUCiast, AUCinf are shown as “mean (CV%)”

[1131] The normality or homogeneity of variances were not verified of half-life values of SEQ ID NO: 5, the significant differences of half-life was evaluated using Kruskal-Wallis test with Dunn’s

[1132]

[1133] multiple comparisons test.

[1134] Example 11: In vivo oral pharmacokinetics of SEQ ID NO:6 and SEQ ID NO:7

[1135]

[0293] To observe oral pharmacokinetics, peptides SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:7 were orally (PO) administered to beagle dogs with a 3-in-l capsule and single capsule with SEQ ID NO:3 was used as reference. Pharmacokinetics were measured after oral administration.

[1136] Methods

[1137]

[0294] To measure pharmacokinetics, male beagle dogs fasted over-night at least 16 hrs and returned to food 4 hrs post dosing. Pentagastrin was administered to adjust the constant gastric-pH of dogs. Pentagastrin was administered IM at 0.006 mg / kg (0.25 mg / mL and 0.024 mL / kg) before 30 min capsule administration. Dogs were dosed PO with either 3-in-l capsule or single capsule. The blood samples were collected over 96 hrs and the individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma (K2-EDTA) concentrations were determined Atty Ref. METS-030 / 01 WO 350242-2268

[1138] using a qualified LC-MS method.

[1139]

[0295] 3-in- 1 capsule contained 10 mg / capsule each of SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:7. Single capsule contained 10 mg / capsule of only SEQ ID NO:3 and same capsule composition as 3-in-l capsule. Enteric capsules were enterosoluble vacant gelatin capsule (SHAOXING ZHONGYA, China).

[1140] Results

[1141]

[0296] Similar PK profiles of SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:7 were obtained in 3- in-1 capsules (10 mg per compound) which indicates no differences in oral absorption between the tested compounds shown in FIG. 13. The comparison of oral absorption is possible in cassette PK using 3-in-l capsules shown in Table 20 and FIG. 13.

[1142] Table 20. Mean pharmacokinetic parameters of SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:7 following a single oral administration to male beagle dogs

[1143] SEQ ID tl / 2 TmaCmax AUCiast AUCinf Formulationx

[1144] NO: (hr) (hr) (ng / mL) (ng-hr / mL) (ng-hr / mL)

[1145] 102.4 5652.3 9224.7 3 73.8 (50%) 5.5 (39%)

[1146] (201%) (213%) (208%) 3-in-l 106.1 7100.2 13383.2

[1147] 6 92.0 (55%) 8.2 (111%)

[1148] capsule (197%) (208%) (202%)

[1149] 109.8 6856.0 11701.6 7 106.9 (46%) 5.8 (39%)

[1150] ( 196%) (206%) (194%) ti / 2, Tmax, Cmax, AUCiast, and AUCinf are shown as “mean (CV%)”.

[1151]

[1152] Example 12: In vivo intravenous pharmacokinetics of SEQ ID NO:4 and SEQ ID NO:6

[0297] To observe pharmacokinetics, peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 were intravenously (IV) administered to fasted male Sprague Dawley rats. Pharmacokinetics were measured at 10 points from 0 to 72 hours after IV administration.

[1153] Methods

[1154]

[0298] To measure pharmacokinetics, male Sprague Dawley rats were fasted over-night at least 16 hrs and returned to food 4 hrs post dosing. Rats were dosed IV at 50 nmol / kg. Plasma was sampled and measured at 0, 0.17, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours. The individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma (K2-EDTA) concentrations were determined using a qualified LC-MS method.

[1155] Results Atty Ref. METS-030 / 01 WO 350242-2268

[1156]

[0299] PK profiles of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 were obtained after IV administration that shows time-plasma concentration between the tested compounds shown in FIG.

[1157] 14. Mean PK parameters are shown in Table 21, and the comparison of IV PK profile and parameters is shown in Table 21 and FIG. 14.

[1158] Table 21. Mean pharmacokinetic parameters of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 following a single IV administration to male Sprague Dawley rats

[1159] SEQ ID Dose tl / 2 Co Co / dose AUClast AUCiast / dose AUCinf AUCtnf / dose NO: (mg / kg) (hr) (ng / mL) (kg / L) (ng-hr / mL) (hr -kg / L) (ng-hr / mL) (hr-kg / L) 0.276 9.8 6164.8 22.3 35719.3

[1160] 3 35510 (6%) 128.7 (6%) 129.4 (6%) (0%) (11%) (13%) (13%) (6%)

[1161] 0.276 15.1 6888.3 25.0 59368.0 61157.8

[1162] 4 215.1 (11%) 221.6 (12%) (0%) (16%) (21%) (21%) (11%) (12%)

[1163] 0.269 16.0 6437.3 23.9 88381.5 92640.0

[1164] 6 328.6 (9%) 344.4 (8%)

[1165]

[1166] (0%) (14%) (12%) (12%) (9%) (8%)

[1167] Example 13: In vivo subcutaneous pharmacokinetics of SEQ ID NO:4 and SEQ ID NO:6

[0300] To observe pharmacokinetics, peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 were subcutaneously (SC) administered to mice. Pharmacokinetics were measured at 7 points from 2 to 72 hours after SC administration.

[1168] Methods

[1169]

[0301] To measure pharmacokinetics, male mice were fasted over-night at least 16 hrs and returned to food 4 hrs post dosing. Mice were dosed SC at 100 nmol / kg. Plasma was sampled and measured at 2, 4, 8, 12, 24, 48, and 72 hours. The individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma (K2-EDTA) concentrations were determined using a qualified LC-MS method.

[1170] Results

[1171]

[0302] PK profiles of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 were obtained after SC administration that shows time-plasma concentration between the tested compounds shown in FIG.

[1172] 15. Mean PK parameters are shown in Table 22, and the comparison of SC PK profile and parameters is shown in Table 22 and FIG. 15.

[1173] Table 22. Mean pharmacokinetic parameters of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 following a single SC administration to male mice Atty Ref. METS-030 / 01 WO 350242-2268

[1174] tl / 2 Cmax / dOS6 AUCiast / dose Fold vs. SEQ. ID. NO. 3 SEQ ID NO:

[1175] (hr) (kg / L) (hr kg / L) (AUCiast / dose)

[1176] 3 3.98 (4%) 3.06 (6%) 28.74 (7%) 1.0

[1177] 4 4.97 (7%) 3.73 (3%) 37.1 (3%) 1.3

[1178] 6 16.15 (8%) 4.7 (7%) 145.76 (6%) 5.1

[1179]

[1180] Example 14: In vivo intraduodenal pharmacokinetics of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:22

[1181]

[0303] To observe pharmacokinetics, peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 were intraduodenal (ID) administered to fasted male Sprague Dawley rats. Pharmacokinetics were measured at 9 points from 0 to 48 hours after ID administration.

[1182] Methods

[1183]

[0304] To measure pharmacokinetics, male Sprague Dawley rats were fasted over-night at least 16 hrs and returned to food 4 hrs post dosing. Rats were dosed ID at 300 nmol / kg of each peptide with formulation. Formulation included of 68 mg / kg sodium chenodeoxycholate, 34 mg / kg sodium ursodeoxy cholate, and 68 mg / kg propyl gallate in 0.02% (v / v) polysorbate 80 in 10 mM PBS (pH 7.4). Plasma was sampled and measured at 0, 0.17, 0.5, 1, 2, 4, 8, 24 and 48 hours. The individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma (K2-EDTA) concentrations were determined using a qualified LC-MS method.

[1184] Results

[1185]

[0305] PK profiles of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 were obtained after ID administration that shows time-plasma concentration between the tested compounds shown in FIG.

[1186] 16. Mean PK parameters are shown in Table 23. The comparison of intestinal absorption is possible in cassette PK using 3-in-l formulation shown in Table 23 and FIG. 16.

[1187] Table 23. Mean pharmacokinetic parameters of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 following a single ID administration to male Sprague Dawley rats

[1188] SEQ TO Dose tl / 2 Cmax Cmax / dose AUG Iasi AUCiasi / dose AUCmf AUCmf / dose NO: (mg / kg) (hr) (ng / mL) (kg / L) (ng-hr / mL) (hr kg / L) (ng hr / mL) (hr kg / L) 3 658.4 3349.6

[1189] 1.7 (0%) 7 (5%) 0.4 (33%) 3104 (27%) 1.9 (27%) 2 (27%) (33%) (27%)

[1190]

[1191] Atty Ref. METS-030 / 01 WO 350242-2268

[1192] 10.7 7043.3 7382.9

[1193] 4 1.7 (0%) 755 (34%) 0.5 (34%) 4.3 (36%) 4.5 (37%)

[1194] (10%) (36%) (37%)

[1195] 12.8 330.8 4698.9 5101.2

[1196] 22 1.6 (0%) 0.2 (29%) 2.9 (30%) 3.1 (31%)

[1197] (9%) (29%) (30%) (31%)

[1198]

[1199] Example 15: In vivo intraduodenal pharmacokinetics of SEQ ID NO:3, SEQ. ID NO.4 and SEQ ID NO:22

[1200]

[0306] To observe pharmacokinetics, peptides SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 were intraduodenal (ID) administered to fasted male Sprague Dawley rats. Pharmacokinetics were measured at 9 points from 0 to 48 hours after ID administration.

[1201] Methods

[1202]

[0307] To measure pharmacokinetics, male Sprague Dawley rats were fasted over-night at least 16 hrs and returned to food 4 hrs post dosing. Rats were dosed ID at 300 nmol / kg of each peptide with formulation. Formulation included of 94 mg / kg sodium caprate and 68 mg / kg propyl gallate in 0.02% (v / v) polysorbate 80 in 10 mM PBS (pH 7.4). Plasma was sampled and measured at 0, 0.17, 0.5, 1, 2, 4, 8, 24 and 48 hours. The individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma (K2-EDTA) concentrations were determined using a qualified LC-MS method.

[1203] Results

[1204]

[0308] PK profiles of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 were obtained after ID administration that shows time-plasma concentration between the tested compounds shown in FIG.

[1205] 17. Mean PK parameters are shown in Table 24. The comparison of intestinal absorption is possible in cassette PK using 3-in-l formulation shown in Table 24 and FIG. 17.

[1206] Table 24. Mean pharmacokinetic parameters of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:22 following a single ID administration to male Sprague Dawley rats

[1207] SEQ ID Dose tl / 2 Cmax Cmax / dOSC AUCi^t AUCtast / dose AUCmf AUCint / dose NO: (mg / kg) (hr) (ng / mL) (kg / L) (ng-hr / mL) (hr-kg / L) (ng-hr / mL) (hr-kg / L)

[1208] 3227.9 3476.1

[1209] 3 1.7 (0%) 8.2 (7%) 660.3 (30%) 0.4 (30%) 2.0 (46%) 2.1 (41%)

[1210] (46%) (41%)

[1211] 6088.2 6426.3

[1212] 4 1.7 (0%) 11.2 (10%) 663.5 (37%) 0.4 (37%) 3.7 (38%) 3.9 (40%)

[1213] (38%) (40%)

[1214] 3989.3 4555.9

[1215] 22 1.6 (0%) 15.4 (20%) 319.1 (37%) 0.2 (37%) 2.5 (42%) 2.8 (41%)

[1216] (42%) (41%)

[1217]

[1218] Atty Ref. METS-030 / 01 WO 350242-2268

[1219] Example 16: In vivo intravenous pharmacokinetics of SEQ ID NO: 22 and SEQ ID NO: 23 Methods

[1220]

[0309] To observe pharmacokinetics, peptides SEQ ID NO:22 and SEQ ID NO:23 were intravenously (IV) administered to beagle dogs in a 4-in-l formulation which included two unspecified compounds, along with SEQ ID NO:22 and SEQ ID NO:23. Pharmacokinetics were measured at 12 points from 0 to 96 hours after IV administration. Dogs were dosed IV at 10 nmol / kg / compound. The Plasma (K2-EDTA) concentrations were determined using a qualified LC-MS method.

[1221] Results

[1222]

[0310] PK profiles of SEQ ID NO:22, and SEQ ID NO:23 were obtained after IV administration that shows mean time-plasma concentration and PK parameters between the tested compounds shown in FIG. 18 and Table 25. The formulation included two unspecified compounds, along with SEQ ID NO:22 and SEQ ID NO:23.

[1223] Table 25. Mean pharmacokinetic parameters of SEQ ID NO:22 and SEQ ID NO:23 following a single IV administration to male beagle dogs

[1224] CoAUC,astAUC

[1225] SEQ ID NO: tl / 25'*inf

[1226] (hr) (ng / mL) (ng hr / mL) (ng hr / mL)

[1227] 22 108.3 (21%) 1034.8 (5%) 43086.3 (4%) 91526.5 (16%) 23 117.5 (21%) 974.0 (8%) 43583.3 (4%) 98249.0 (15%) ti / 2, Cmax, AUCiast, and AUCinf are shown as “mean (CV%)”.

[1228] 1}When calculated the half-life some individual values were calculated with the adjusted linear regression coefficient of the concentration value on the terminal phase with less than 0.9. Thus,

[1229]

[1230] ti / 2 might not be accurately estimated.

[1231] Example 17: Oral pharmacokinetics of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:23

[0311] To observe oral pharmacokinetics, peptides SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:23 were orally (PO) administered to beagle dogs with a 3-in-l capsule and single capsule with SEQ ID NO:3 was used as reference. Pharmacokinetics were measured after oral administration. Methods

[1232]

[0312] To measure pharmacokinetics, male beagle dogs fasted over-night at least 16 hrs and returned to food 4 hrs post dosing. Pentagastrin was administered to adjust the constant gastric-pH of dogs. Pentagastrin was administered IM at 0.006 mg / kg (0.25 mg / mL and 0.024 mL / kg) before Atty Ref. METS-030 / 01 WO 350242-2268

[1233] 30 min capsule administration. Dogs were dosed PO with either 3-in-l capsule or single capsule. The blood samples were collected over 96 hrs and the individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma (K2-EDTA) concentrations were determined using a qualified LC-MS method.

[1234]

[0313] 3-in-l capsule contained 10 mg / capsule each of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:23. Single capsule contained 10 mg / capsule of only SEQ ID NO:3 and same capsule composition as 3-in-l capsule. Enteric capsules were enterosoluble vacant gelatin capsule (SHANXI GUANGSHENG, China).

[1235] Results

[1236]

[0314] The comparison of oral absorption is possible in cassette PK using 3-in-l capsules shown in Table 26 and FIG. 19. AUCinf is high due to longer half-life of SEQ ID NO:22 and SEQ ID NO:23 compared to the SEQ ID NO:3 despite of lower Cmax-of those compounds.

[1237] Table 26. Mean pharmacokinetic parameters of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:23 following a single oral administration to male beagle dogs

[1238] SEQ ID tl / 2 Tmax(-max AUClastAUCinf Formulation

[1239] NO: (hr) (hr) (ng / mL) (ng-hr / mL) (ng-hr / mL) 3 36.4 (10%) 3.2 (41%) 560.6 (84%) 19252.6 (80%) 23218 (82%) 3-in-l capsule 22 70.9 (12%) 3.4 (26%) 418.8 (89%) 20762.2 (85%) 35293.2 (83%)

[1240] 23 79.4 (34%) 2.9 (43%) 405.2 (96%) 19037 (93%) 34011.4 (92%)

[1241]

[1242] tl / 2, T max, Crnax, AUCiast, and AUCinf are shown as “mean (CV%)”.

[1243]

[1244] Example 18: In vivo efficacy of SEQ ID NO:4 and SEQ ID NO:6

[1245] Methods

[1246]

[0315] To observe in vivo efficacy, peptides semaglutide, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6 were subcutaneously (SC) administered to diet-induced obesity (DIO) mice. A DIO mouse model was developed by feeding six-weeks old C57BL / 6 male mice with high-fat diet (60% kcal from fat, TD6414) for 17 weeks. Drug administration was carried out with daily subcutaneous injections, increasing the dose of all test articles from 1 nmol / kg to 5 nmol / kg over 19 days. Body weight and food intake was measured for 28 days after SC daily dosing. Vehicle was administered to a control group.

[1247] Results

[1248]

[0316] Body weight change and accumulative food intake of mice administered with vehicle, Atty Ref. METS-030 / 01 WO 350242-2268

[1249] semaglutide, SEQ ID N0:3, SEQ ID N0:4, and SEQ ID N0:6 were obtained after SC administration shown in FIG. 20 and FIG. 21.

[1250]

[0317] The body weight was slightly increased after 28 days administration in DIO control groups. Semaglutide, SEQ ID NO.3, SEQ ID NO:4, and SEQ ID NO:6 were led to body weight loss. In particular, SEQ ID NO: 6 showed the most body weight loss. All treated groups had markedly decreased mean body weight compared to DIO control mean at Day 28. Amount of cumulative food intake was reduced in all treated groups during treatment.

[1251] Example 19: In vivo efficacy of SEQ I D NO:22 and SEQ ID NO:23

[1252] Methods

[1253]

[0318] To observe in vivo efficacy, peptides tirzepatide, SEQ ID NO:22, SEQ ID NO.23, and SEQ ID NO:6 were subcutaneously (SC) administered to diet-induced obesity (DIO) mice. A DIO mouse model was developed by feeding six-weeks old C57BL / 6 male mice with high-fat diet (60% kcal from fat, TD6414) for 24 weeks. Drug administration was carried out with daily subcutaneous injections, increasing the dose of all test articles from 1 nmol / kg to 2.5 nmol / kg over 14 days. Body weight and food intake was measured for 28 days after SC daily dosing. Vehicle was administered to a control group.

[1254] Results

[1255]

[0319] Body weight change and accumulative food intake of mice administered with vehicle, tirzepatide, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:6 were obtained after SC administration shown in FIG. 22 and FIG. 23.

[1256]

[0320] The body weight was slightly increased after 19 days administration in DIO control groups. Tirzepatide, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:6 led to body weight loss. In particular, SEQ ID NO:22 showed the most body weight loss. All treated groups had markedly decreased mean body weight compared to DIO control mean at Day 19. Amount of cumulative food intake was reduced in all treated groups during treatment.

[1257]

[0321] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[1258]

[0322] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Atty Ref. METS-030 / 01 WO 350242-2268

[1259] Such equivalents are intended to be encompassed by the following claims.

[1260] EQUIVALENTS AND SCOPE

[1261]

[0323] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[1262]

[0324] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elem ents is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[1263]

[0325] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification Atty Ref. METS-030 / 01 WO 350242-2268

[1264] shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[1265]

[0326] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

Atty Ref. METS-030 / 01 WO 350242-2268CLAIMSWhat is claimed is:

1. A peptide comprising an amino acid sequence of Xi(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKX28GGPSSGX35PPPX39KX41 (SEQ ID NO: 50), or a pharmaceutically acceptable salt thereof,whereinXi is H, F, Y, or D-H;Aib is 2-aminoisobutyric acid;X28 is D, isoD, N, or Q;X35 is A or K;X39 is G or S; andX41 is K or absent; andwherein the C-terminus of the amino acid sequence is optionally amidated.

2. The peptide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises a biotin moiety.

3. The peptide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises two biotin moieties.

4. The peptide of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the K residues at positions 12 and / or 27 of the amino acid sequence independently comprise a biotin moiety.

5. The peptide of claim 3 or 4, or a pharmaceutically acceptable salt thereof, wherein the biotin moiety is attached to the epsilon nitrogens of the K residues at positions 12 and / or 27.

6. The peptide of any one of claims 2-5, or a pharmaceutically acceptable salt thereof, wherein the biotin moiety comprises the formula:Atty Ref. METS-030 / 01 WO 350242-22687. The peptide of any one of claims 2-5, or a pharmaceutically acceptable salt thereof, wherein the biotin moiety comprises the formula:

8. The peptide of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises a lipid moiety.

9. The peptide of claim 8, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to a C -terminal lysine of the peptide.

10. The peptide of claim 9, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to the epsilon nitrogen of the C-terminal lysine of the peptide.

11. The peptide of any one of claims 8-10, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula:wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

12. The peptide of any one of claim 11, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula:Atty Ref. METS-030 / 01 WO 350242-226813. The peptide of any one of claim 11, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula:

14. The peptide of any one of claim 11, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the formula selected fromAtty Ref. METS-030 / 01 WO 350242-226815. A peptide comprising the amino acid sequence:H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, wherein Aib is 2-aminoisobutyric acid.

16. The peptide of claim 15, wherein the peptide comprises(SEQ ID NO: 30),or a pharmaceutically acceptable salt thereof.

17. A peptide comprising the amino acid sequence:H(Aib)EGTFTSDLSKQMEEEAVRLFlEWLK(isoD)GGPSSGAPPPSK (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof, wherein:Aib is 2-aminoisobutyric acid; andisoD is iso-aspartic acid.Atty Ref. METS-030 / 01 WO 350242-226818. The peptide of claim 17, wherein the peptide comprises(SEQ ID NO: 31),or a pharmaceutically acceptable salt thereof.

19. The peptide of claim 17, wherein the peptide comprises(SEQ ID NO: 47),or a pharmaceutically acceptable salt thereof.

20. A peptide comprisingAtty Ref. METS-030 / 01 WO 350242-2268(SEQ ID NO: 35),or a pharmaceutically acceptable salt thereof.

21. A peptide, or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence selected from the group consisting of F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSKK (SEQ ID NO: 10), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSK (SEQ ID NO. 8), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPGKK (SEQ ID NO: 12), and F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGKPPPSKK (SEQ ID NO: 13).

22. The peptide of claim 21, wherein the peptide comprisesAtty Ref. METS-030 / 01 WO 350242-2268(SEQ ID NO: 40),(SEQ ID NO: 41),Atty Ref. METS-030 / 01 WO 350242-2268(SEQ ID NO: 44), orAtty Ref. METS-030 / 01 WO 350242-2268(SEQ ID NO: 39),or pharmaceutically acceptable salts thereof.

23. A peptide, or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence selected from the group consisting of:Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK (SEQ ID NO. 16), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK (SEQ ID NO: 17), Y(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKK (SEQ ID NO: 18), F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPSK (SEQ ID NO: 19), and F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLK(isoD)GGPSSGAPPPSK (SEQ ID NO: 20).

24. The peptide of claim 23, wherein the peptide comprises(SEQ ID NO: 5),Atty Ref. METS-030 / 01 WO 350242-2268(SEQ ID NO: 6), (SEQ ID NO: 7), H OAtty Ref. METS-030 / 01 WO 350242-2268o H O H(SEQ ID NO: 33), or(SEQ ID NO: 48),or pharmaceutically acceptable salts thereof.

25. A peptide comprisingAtty Ref. METS-030 / 01 WO 350242-2268H OH(SEQ ID NO: 3),or a pharmaceutically acceptable salt thereof.

26. A peptide comprising an amino acid sequence having greater than 95% sequence identity with the amino acid sequence:F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPSKK (SEQ ID NO: 10), or a pharmaceutically acceptable salt thereof, wherein Aib is 2-aminoisobutyric acid.

27. The peptide of claim 26, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 amino acid substitution relative to SEQ ID NO: 10.28, The peptide of claim 26, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence has 100% sequence identity with SEQ ID NO: 10.

29. A peptide comprising the amino acid sequence:F(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGX35PPPX39KX41(SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof, wherein:Aib is 2-aminoisobutyric acid;X35 is alanine or lysine;X39 is serine or glycine; andX41 is lysine or absent.Atty Ref. METS-030 / 01WO 350242-226830. The peptide of claim 29, or a pharmaceutically acceptable salt thereof, wherein X35 is alanine.

31. The peptide of claim 29 or 30, or a pharmaceutically acceptable salt thereof, wherein X39 is serine.

32. The peptide of any one of claims 29-31, or a pharmaceutically acceptable salt thereof, wherein X41 is lysine.

33. The peptide of any one of claims 26-32, wherein the peptide comprises(SEQ ID NO: 22),or a pharmaceutically acceptable salt thereof.

34. A peptide comprising the amino acid sequence:Xi(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKX28GGPSSGAPPPSKX4i (SEQ ID NO: 14), or a pharmaceutically acceptable salt thereof, wherein:X1is phenylalanine or tyrosine;Aib is 2-aminoisobutyric acid;X28 is asparagine, aspartic acid, or iso-aspartic acid (isoD); andX41 is lysine or absent.

35. The peptide of claim 34, or a pharmaceutically acceptable salt thereof, wherein Xi is phenylalanine.Atty Ref. METS-030 / 01 WO 350242-226836. The peptide of claim 34 or 35, or a pharmaceutically acceptable salt thereof, wherein X 8 is asparagine.

37. The peptide of any one of claims 33-36, or a pharmaceutically acceptable salt thereof, wherein X41 is absent.

38. The peptide of any one of claims 33-37, wherein the peptide comprises:o H O H(SEQ ID NO: 4),or a pharmaceutically acceptable salt thereof.

39. The peptide of any one of claims 15, 17, 20, 22, 25-31, and 33-36, or a pharmaceutically acceptable salt thereof, comprising an amidated C-terminus.

40. The peptide of any one of claims 15, 17, 20, 22, 25-31, and 33-36, or a pharmaceutically acceptable salt thereof, comprising one or more biotin moieties.

41. The peptide of claim 40, or a pharmaceutically acceptable salt thereof, wherein the lysine residues at positions 12 and 27 of the amino acid sequence independently comprise one or more biotin moieties attached to their epsilon nitrogens.

42. The peptide of claim 41, or a pharmaceutically acceptable salt thereof, wherein the lysine residues at positions 12 and 27 of the amino acid sequence comprise the following:Atty Ref. METS-030 / 01 WO 350242-2268wherein * indicates attachment to the epsilon nitrogen of the lysine residue.

43. The peptide of claim 42, or a pharmaceutically acceptable salt thereof, wherein the lysine residues at positions 12 and 27 of the amino acid sequence are of the following structure:44, The peptide of any one of claims 15, 17, 20, 22, 25-31, and 33-36, or a pharmaceutically acceptable salt thereof, comprising a lipid moiety.

45. The peptide of claim 44, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to the C-terminal amino acid of the peptide.

46. The peptide of claim 44 or 45, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety is attached to the epsilon nitrogen of a C-terminal lysine of the peptide.

47. The peptide of any one of claims 44-46, or a pharmaceutically acceptable salt thereof, wherein the lipid moiety comprises the following formula:wherein n is 0, 1, or 2; and p is an integer from 1-20, inclusive.

48. The peptide of any one of claims 44-46, or a pharmaceutically acceptable salt thereof, Atty Ref. METS-030 / 01 WO 350242-2268wherein the lipid moiety comprises one of the following formulae:

49. The peptide of any one of claims 44-46, wherein the lipid moiety comprises one of the following formulae:Atty Ref. METS-030 / 01 WO 350242-226850. A pharmaceutical composition comprising the peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

51. A method of inducing or maintaining weight loss or managing body weight in a subject, the method comprising administering to the subject an effective amount of the peptide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof,52. A method of preventing weight gain in a subject, the method comprising administering to the subject an effective amount of a peptide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

53. The method of claim 51 or 52, wherein the subject has a metabolic disorder.

54. The method of any one of claims 51-53, wherein the subject has obesity, prediabetes, diabetes, non-alcoholic fatty liver disease (NAFLD), cardiometabolic disease, polycystic ovary syndrome (PCOS), chronic kidney disease, peripheral arterial disease, heart failure, Alzheimer’s disease, sleep apnea, or knee osteoarthritis.

55. A method of stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, or reducing caloric intake in a subject in need thereof, the method comprising administering to the subject an effective amountAtty Ref. METS-030 / 01 WO 350242-2268of a peptide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

56. The method of any one of claims 51-55, wherein the peptide is administered to the subject orally.

57. A method of modulating P-arrestin signaling and / or GLP-1 receptor internalization, the method comprising contacting a cell with the peptide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.58, The method of any one of claims 51-57, wherein the peptide comprises(SEQ ID NO: 22),or a pharmaceutically acceptable salt thereof.

59. The peptide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in:(a) inducing or maintaining weight loss or managing body weight in a subject;(b) preventing weight gain in a subject; and / or(c) stimulating glucose clearance, stimulating glucose clearance, stimulating insulin release, stimulating carbohydrate metabolism, stimulating lipid metabolism, improving carbohydrate tolerance, reducing appetite, reducing food intake, and / or reducing caloric intake in a subject.

60. Use of the peptide of any one of claims 1-49, or a pharmaceutically acceptable saltAtty Ref. METS-030 / 01 WO 350242-2268thereof, or a pharmaceutical composition thereof, as a medicament.

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