Glucagon-like peptide 1 (GLP-1) fusion peptide conjugated cyclic peptide tyrosine tyrosine conjugates and uses thereof

Abstract

The present invention includes conjugates comprising a glucagon-like peptide 1 (GLP-1) fusion peptide linked to a cyclic PYY peptide. The present invention also relates to pharmaceutical compositions and methods of use thereof. The novel conjugates are useful for preventing, treating, or ameliorating the diseases and disorders disclosed herein.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to International Application No. PCT / US2018 / 029284, filed April 25, 2018, and U.S. Provisional Application No. 62 / 662,313, filed April 25, 2018, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION The present invention is generally directed to novel glucagon-like peptide-1 (GLP-1) fusion peptide-coupled cyclic peptide tyrosine tyrosine (PYY) conjugates that are modulators of the neuropeptide Y2 receptor and the GLP-1 receptor. The present invention also relates to pharmaceutical compositions and methods of use thereof. The novel GLP-1 fusion peptide-coupled cyclic PYY conjugates are useful for preventing, treating, or ameliorating diseases and disorders such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance, and dyslipidemia, among others.

[0003] (Reference to electronically submitted sequence listing) This application includes a sequence listing, submitted electronically via EFS-Web as an ASCII-formatted sequence listing under the file name "PRD3465 Sequence Listing", with a creation date of April 2, 2019, and having a size of 409 kb. The sequence listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. In the event of any inconsistency between the information set forth herein and the sequence listing submitted electronically via EFS-Web under the file name "PRD3465 Sequence Listing", regarding the structure of SEQ ID NOs: 225-262, the information herein shall prevail. [Background technology]

[0004] Neuropeptide Y (NPY) receptors are activated by a group of closely related peptide agonists, called the "NPY family," with different affinities for each receptor subtype. NPY, peptide tyrosine-tyrosine (PYY), and pancreatic polypeptide (PP), all 36 amino acids in length, are agonists of the NPY receptor family. NPY is a neurotransmitter that is synthesized, co-stored, and released with norepinephrine and epinephrine. NPY is one of the most abundant and widely distributed peptides in the central nervous system (CNS) of humans and rodents, and is expressed in areas of the brain related to feeding and stress. In the peripheral nervous system, NPY-containing neurons are primarily sympathetic. PYY is primarily synthesized and released by enteroendocrine cells. Cleavage of NPY and PYY by the endothelial serine-protease, dipeptidyl peptidase IV (DPP-IV), results in the selective ligand of the Y2 and Y5 subtypes of the NPY receptor family, NPY 3-36 and PYY 3-36 PP is found primarily in pancreatic islet cells distinct from those that store insulin, glucagon, or somatostatin.

[0005] Five different NPY receptors have been identified so far, four of which are understood to be relevant in human physiology: Receptors Y1, Y2, and Y5 preferentially bind NPY and PYY, whereas the Y4 receptor preferentially binds PP. The Y2 and Y5 receptors also bind NPY 3-36 and PYY 3-36 In general, the NPY family of ligands has variable selectivity for each of the NPY receptor isoforms, and PYY 3-36 have previously been reported to have moderate to strong selectivity for the Y2 isoform. Each of these receptors is coupled to the inhibition of adenylyl cyclase via a pertussis toxin-sensitive Gαi.

[0006] PYY is secreted from endocrine L-cells in response to food, especially after fat ingestion. 1-36is predominant in the fasting state, and PYY 3-36 PYY is the predominant form found after a meal in humans, and plasma concentrations are inversely correlated with the number of calories consumed. 3-36 PYY has been demonstrated to reduce food intake in humans, monkeys, rats, rabbits, and mice (Batterham et al., Nature 418(6898):650-4(2002); Batterham et al., N Engl J Med 349(10):941-8(2003); Challis et al., Biochem Biophys Res Commun 311(4):915-9(2003)). 3-36 The anorectic effect of PYY is thought to be mediated by Y2, based on preferential binding at this receptor and loss of feeding efficacy in Y2-deficient mice (Batterham et al., Nature 418(6898):650-4(2002)). 3-36 Intraarch injection of PYY reduces food intake in rats and mice (Batterham et al., Nature 418(6898):650-4(2002)), suggesting that hypothalamic Y2 receptor engagement may mediate these effects. It has also been shown that the acute effects on feeding translate into dose-dependent effects on body weight in ob / ob, DIO, and Zucker fa / fa mice (Pittner et al., Int J Obes Relat Metab Disord 28(8):963-71(2004)). In addition, PYY 3-36 has also been shown to improve insulin-mediated glucose disposal and insulin sensitivity in DIO rodents (Vrang et al., Am J Physiol Regul Integr Comp Physiol 291(2):R367-75(2006)). Bariatric surgery results in increased circulating PYY immunoreactivity (le Roux et al., Ann Surg 243(1):108-14(2006)), which appears to play a role in postoperative weight loss.

[0007] Given its role in regulating appetite and food intake, as well as its antisecretory and pro-absorptive effects in the mammalian gastrointestinal tract, PYY 3-36 may be effective in treating obesity and related conditions, as well as a number of gastrointestinal disorders. 3-36 Its therapeutic utility as a therapeutic agent is limited by its rapid metabolism and the resulting short circulatory half-life (Torang et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 310:R866-R874 (2016)).

[0008] Activation of the incretin hormone GLP-1 and its receptor (GLP1R) has many beneficial effects on glucose metabolism and energy balance in humans, including stimulation of glucose-dependent insulin secretion (Kreymann et al., Lancet 2:1300-1304 (1987)), as well as inhibition of glucagon secretion (Gutniak et al., N Engl J Med 326:1316-1322 (1992)), gastric emptying (Wettergren et al. Digestive Diseases Sciences 38:665-673 (1993)), and food intake (Flint et al., JCI 101:515-520 (1998)). GLP1R agonists have also been shown to significantly reduce the risk of cardiovascular and microvascular outcomes (Marso et al., N Engl J Med 375(4):311-322(2016)) and kidney disease (Mann et al., N Engl J Med 377(9):839-848(2017)) in patients with type 2 diabetes. GLP-1 is characterized by a short half-life, which makes its use as a potential therapeutic agent impractical. The circulating half-life is approximately 10 times longer than that of dipeptidyl peptidase IV (Zhu, L. et al., JBC 278:22418-22423(2003)) and neutral endopeptidase (Hupe-Sodmann et al., Regul Pept 58(3):149-156 (1995)) as well as by renal filtration (Ruiz-Grande et al., Can J Physiol Pharmacol 68(12):1568-1573 (1990)).

[0009] Therefore, PYY 3-36It would be desirable to have PYY analogs or derivatives thereof and / or GLP-1 analogs or derivatives thereof that have improved metabolic stability and pharmacokinetic profiles compared to GLP-1 and / or PYY analogs or derivatives thereof that have improved half-lives in vivo and provide modulation of the Y2 and / or GLP-1 receptors with a longer duration of action, making them suitable as therapeutic agents for subjects in need of such modulation.

[0010] The preceding discussion has been presented merely to provide a better understanding of the nature of the problems faced in the art and should not be construed as an admission of prior art in any manner, and citation of any reference herein should not be construed as an admission that such reference constitutes "prior art" to the present application. Summary of the Invention [Means for solving the problem]

[0011] In one general aspect, the present invention relates to novel glucagon-like peptide-1 (GLP-1) fused cyclic peptide tyrosine tyrosine (PYY) conjugates that are modulators of the neuropeptide Y2 receptor and the GLP-1 receptor.

[0012] Provided herein is a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide linked to a cyclic PYY peptide, the GLP-1 fusion peptide comprising a GLP-1 or GLP-1 variant peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, the first linker optionally being absent.

[0013] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative or pharma- ceutically acceptable salt thereof:

[0014] [ka] During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if does not exist.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K; Z9 is G or K; Z 11 is D or K, Z 22 is A or K, Z 23 is S or K, Z 26 is A or H, Z 30 is L, W or absent, (However, Z 30 does not exist if and only if q is 1.) Z 34 teeth,

[0015] [ka] and Z 35 teeth,

[0016] [ka] and The derivatives are compounds of formula I that have been modified by one or more processes selected from the group consisting of amidation, acylation, and PEGylation.

[0017] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative or pharma- ceutically acceptable salt thereof, wherein: p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if does not exist.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH2-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K, the amino side chain of which is optionally

[0018] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z9 is G or K, the amino side chain of K is optionally

[0019] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 11 is D or K, the amino side chain of K is optionally

[0020] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 22 is A or K, the amino side chain of K is optionally

[0021] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 23 is S or K, the amino side chain of K is optionally

[0022] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 26 is A or H, Z 30 is L, Z 34 teeth,

[0023] [ka] and Z 35 teeth,

[0024] [ka] It is.

[0025] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative or pharma- ceutically acceptable salt thereof, wherein: p is 0 or 1; m is 0, 1, 2, 3, or 5; n is 1, 2, or 4; q is 0 or 1 (wherein q is Z 30 ) can be 1 if and only if is absent. BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z9 is G or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 11 is D or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 22 is A or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 23 is S or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 26 is A or H, Z 30 is L, Z 34 teeth,

[0026] [ka] and Z 35 teeth,

[0027] [ka] It is.

[0028] In certain embodiments, the cyclic PYY peptide is selected from the group consisting of SEQ ID NOs: 1-54, or a pharma- ceutically acceptable salt thereof. In certain embodiments, the cyclic PYY peptide is selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34.

[0029] In certain embodiments, the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide at a lysine residue of the cyclic PYY peptide. 11 , Z 22 , and Z 23 Exactly one of them is a lysine, which is covalently linked to a cysteine ​​residue in the second linker peptide of the GLP-1 fusion peptide.

[0030] In certain embodiments, the GLP-1 peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59. In certain embodiments, the first linker peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-83. In certain embodiments, the hinge-Fc region peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-90. In certain embodiments, the second linker peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 91-112. In certain embodiments, the second linker peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106, or 111.

[0031] Also provided is a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide conjugated to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274, and the cyclic PYY peptide comprises an amino acid sequence selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34. In certain embodiments, the GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 136. In certain embodiments, a cysteine ​​residue between amino acid residues 287-289 of SEQ ID NO: 113 or SEQ ID NO: 136, preferably cysteine ​​residue 288 of SEQ ID NO: 113 or SEQ ID NO: 136, is covalently linked directly or via a chemical linker to a lysine residue at residues 7, 9, 11, 22, or 23 of the cyclic PYY peptide, preferably lysine residue 11 of the cyclic PYY peptide.

[0032] Also provided is a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide conjugated to a cyclic PYY peptide, the conjugate comprising a sequence selected from the group consisting of SEQ ID NOs: 225-262, or a pharma- ceutically acceptable salt thereof.

[0033] Also provided is a method for producing a conjugate of the invention, comprising reacting an electrophile, preferably bromoacetamide or maleimide, introduced into the side chain of a cyclic PYY peptide, preferably into the side chain of a lysine residue of the cyclic PYY peptide, with a sulfhydryl group of a cysteine ​​residue of a second linker peptide (e.g., a carboxy-terminal linker peptide) of a GLP-1 fusion peptide, thereby generating a covalent linkage between the cyclic PYY peptide and the GLP-1 fusion peptide.

[0034] Also provided is a pharmaceutical composition comprising a conjugate of the invention and a pharma- ceutically acceptable carrier.

[0035] Also provided is a method of treating or preventing a disease or disorder in a subject in need thereof, the disease or disorder being obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors, such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and / or eczema. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present invention.

[0036] Also provided is a method of reducing at least one of food intake or body weight in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention.

[0037] Also provided is a method of modulating Y2 receptor activity or GLP-1 receptor activity in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention.

[0038] In certain embodiments, the pharmaceutical composition is administered by injection.

[0039] Also provided is a kit comprising a compound of the invention, preferably further comprising liraglutide and an injection device.

[0040] Also provided is a method of producing a pharmaceutical composition of the invention, the method comprising combining a conjugate with a pharma- ceutically acceptable carrier to obtain a pharmaceutical composition.

[0041] Further aspects, features, and advantages of the present invention will be better understood by reading the detailed description and claims. [Brief description of the drawings]

[0042] The above summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1A] Figure 1A shows the ex vivo human plasma stability of the GLP-1 fusion peptide moiety. GLP-1 fusion peptides GF32 (SEQ ID NO: 144) (▲), GF36 (148) (△), GF33 (145) (▼), GF39 (151) (▽), and GF34 (146) were incubated in human plasma at 37°C for 7 days.

number

[0043] Various publications, articles and patents are cited or described in the Background and throughout the specification. Each of these references is incorporated herein by reference in its entirety. Discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention(s) disclosed or claimed.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0045] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0046] Unless otherwise indicated, any numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases as being modified by the term "about". Thus, numerical values ​​typically include ±10% of the described value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within the range, unless the context clearly indicates otherwise.

[0047] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. 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. Such equivalents are intended to be encompassed by the present invention.

[0048] As used herein, it will be understood that the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to include a stated integer or group of integers, but not to exclude other integers or groups of integers other than those, and are intended to be non-exclusive or non-limiting. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or not inherent in such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0049] It should also be understood that the terms "about," "approximately," "generally," "substantially," and the like, used herein when referring to dimensions or features of preferred inventive components, indicate that the described dimensions / features are not precise boundaries or parameters, and do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, and the like).

[0050] Two or more nucleic acid or polypeptide sequences (e.g., a GLP-1 peptide, a linker peptide, a hinge-Fc region peptide, a cyclic PYY 3-36 The terms "identical" or percent "identity" in the context of a peptide sequence refer to two or more sequences or subsequences that have identical or a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below, or by visual inspection using methods known in the art in light of this disclosure.

[0051] For sequence comparison, typically, one sequence acts as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on designated program parameters.

[0052] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally using the methods described in Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 This can be done by the method of the Supplement (Ausubel).

[0053] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described, respectively, in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0054] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by a first nucleic acid is immunologically cross-reactive with the polypeptide encoded by a second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0055] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0056] The term "administration" with respect to the methods of the invention refers to a method of therapeutically or prophylactically preventing, treating, or ameliorating a syndrome, disorder, or disease described herein by using a conjugate or compound of the invention, or a form, composition, or medicament thereof. Such methods include administering effective amounts of the conjugate, compound, form, composition, or medicament thereof, in a combined form, at different times during the course of treatment, or simultaneously. The methods of the invention are to be understood as embracing all known therapeutic treatment regimes.

[0057] The term "effective amount" refers to the amount of active conjugate, compound, or agent that elicits the biological or medical response sought by a researcher, veterinarian, physician, or other clinician in a tissue system, animal, or human (including preventing, treating, or ameliorating the syndrome, disorder, or disease being treated, or a symptom of the syndrome, disorder, or disease being treated).

[0058] As used herein, the term "composition" is intended to encompass a product containing specified ingredients in specified amounts, as well as any product that results directly or indirectly from combining the specified ingredients in specified amounts.

[0059] As used herein, the term "conjugated / linked" refers to joining or connecting two or more entities together. When referring to a chemical or biological compound, conjugated / linked may refer to a covalent connection between two or more chemical or biological compounds. As a non-limiting example, the glucagon-like peptide-1 (GLP-1) fusion peptide of the present invention can be linked to a cyclic PYY peptide of interest to form a GLP-1 fusion peptide-linked cyclic PYY peptide conjugate. In certain embodiments, the GLP-1 fusion peptide of the present invention can be covalently linked to the cyclic PYY peptide of the present invention through at least one linker. The GLP-1 fusion peptide-linked cyclic PYY peptide conjugate can be formed through a specific chemical reaction designed to conjugate the GLP-1 fusion peptide to the cyclic PYY peptide. As an example, the GLP-1 fusion peptide-linked cyclic PYY peptide conjugate can be formed through a conjugation reaction. The conjugation reaction may involve, for example, reacting an electrophilic group (e.g., bromoacetamide or maleimide) with a sulfhydryl group of a cysteine ​​residue in a peptide of interest (e.g., a GLP-1 fusion peptide). The electrophilic group may, for example, be introduced into the side chain of an amino acid residue of a cyclic PYY peptide. The reaction of the electrophilic group with the sulfhydryl group results in the formation of a covalent thioether bond.

[0060] As used herein, the term "peptide linker" or "linker peptide" refers to a chemical module containing one or more amino acids that links a GLP-1 peptide to a hinge-Fc region peptide or a hinge-Fc region peptide to a cyclic PYY peptide to form a GLP-1 fusion peptide-linked cyclic PYY peptide conjugate. The GLP-1 fusion peptide can include, for example, a first linker peptide and a second linker peptide.

[0061] As used herein, the term "chemical linker" refers to a chemical module that does not contain any amino acid that connects the cyclic PYY peptide to the GLP-1 fusion peptide. In certain embodiments, the cyclic PYY peptide comprises a chemical linker. The chemical linker for the cyclic PYY peptide can include, but is not limited to, for example, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a linker containing an acyl group, a hybrid linker consisting of PEG and an embedded heterocycle, and a hydrocarbon chain. For example, the chemical linker can be first covalently connected to the cyclic PYY peptide and then covalently connected to the second linker peptide of the GLP-1 fusion peptide, preferably the GLP-1 fusion peptide.

[0062] As used herein, the term "conjugate" refers to a peptide (e.g., a GLP-1 fusion peptide) covalently bound to another pharma- ceutical active moiety (e.g., a cyclic PYY peptide). The term "conjugated" refers to a peptide of the invention that is covalently linked or covalently connected, either directly or indirectly through a linker, to another pharma- ceutical active moiety, preferably a therapeutic peptide. As a non-limiting example, the peptide may be a GLP-1 fusion peptide of the invention and the other pharma- ceutical active moiety may be a therapeutic peptide, such as a cyclic PYY peptide of interest.

[0063] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right, although isomeric forms of amino acids are known, unless expressly indicated otherwise, it is the L-form of the amino acid that is shown.

[0064] Glucagon-like peptide-1 (GLP-1) fusion peptide In one general aspect, the present invention relates to a glucagon-like peptide 1 (GLP-1) fusion peptide comprising a GLP-1 or GLP-1 variant peptide, a first linker peptide (e.g., an amino (N)-terminal linker), a hinge-Fc region peptide, and a second linker peptide (e.g., a carboxy (C)-terminal linker).

[0065] Glucagon-like peptide-1 or GLP-1 variant peptides Glucagon-like peptide 1 (GLP-1) is an insulin secretagogue that is synthesized in the intestine and released in response to food intake. It is secreted primarily in two forms, GLP-1-(7-37) and GLP-1-(7-36)NH2, both of which bind to specific GLP-1 receptors (GLP-1R) on pancreatic β-cells and enhance glucose-stimulated insulin secretion.

[0066] Many GLP-1 analogs and derivatives are known and may be referred to herein as "GLP-1 variants." These GLP-1 variant peptides may include exendins, peptides found in the venom of the Gila monster. These exendins share sequence homology with native GLP-1 and are capable of binding to the GLP-1 receptor and initiating a signaling cascade response to activity due to GLP-1(7-37).

[0067] GLP-1 and GLP-1 variant peptides have been shown to act in a variety of ways, including, but not limited to, stimulating insulin release, reducing glucagon secretion, inhibiting gastric emptying, and enhancing glucose utilization.

[0068] GLP-1R belongs to the class B family of seven-transmembrane heterotrimeric G protein-coupled receptors and is expressed in a wide range of tissues, including but not limited to α-, β-, and δ-cells of pancreatic islets, heart, kidney, stomach, intestine, ganglionic nerves of the vagus nerve, and several regions of the central nervous system (CNS), including the hypothalamus and brainstem. GLP-1R increases intracellular calcium, adenylate cyclase, and phospholipase C, and activates PKA, PKC, PI-3K, Epac2, and MAPK signaling pathways. s , Gα q , Gα i , and Gα o (Montrose-Rafizadeh et al., Endocrinology 140:1132-40(1999); Hallbrink et al., Biochim Biophys Acta 1546:79-86(2001)). 133:57-62 (1993); and Holz et al., JBC 270:17749-57 (1995)).

[0069] Provided herein is a GLP-1 fusion peptide comprising a first component, the first component being a GLP-1 or GLP-1 variant peptide. As used herein, the terms "GLP-1 peptide", "GLP-1 variant peptide", "GLP-1 peptide variant" and "GLP-1 or GLP-1 variant peptide" are used interchangeably. The GLP-1 or GLP-1 variant peptide may comprise one of the sequences provided in Table 1. The GLP-1 or GLP-1 variant peptide sequence may be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro stability, (iii) in vitro potency, (iv) retention of in vitro potency after chemical conjugation with the cyclic PYY peptide, (v) serine xylosylation or lack of potential for serine xylosylation, and (vi) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate, such as in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide can have agonist activity at the GLP-1 and Y2 receptors, respectively).

[0070] The GLP-1 or GLP-1 variant peptide constituting the first component of the GLP-1 fusion peptide is intended to encompass peptides having sufficient homology and functionality to natural GLP-1. The GLP-1 or GLP-1 variant peptide is designed to be capable of binding to the GLP-1 receptor in pancreatic β-cells, resulting in the same signal transduction pathway as when natural GLP-1 binds to the GLP-1 receptor on pancreatic β-cells, and exhibiting the same or similar insulin secretion activity.

[0071] [Table 1]

[0072] First linker peptide: amino-terminal linker (N-terminal linker) Provided herein is a GLP-1 fusion peptide comprising a second component, the second component being a first linker peptide (i.e., an amino-terminal linker peptide). The first linker peptide may comprise one of the sequences provided in Table 2. The first linker peptide sequence may be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro potency, (iii) in vitro stability, (iv) serine xylosylation or lack of potential for serine xylosylation, and (v) the properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate, such as in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide can have agonist activity against the GLP-1 and Y2 receptors, respectively).

[0073] In certain embodiments, the GLP-1 fusion peptide does not include a second component that is a first linker peptide.

[0074] [Table 2]

[0075] Hinge-Fc region peptide In a particular embodiment, the GLP-1 fusion peptide comprises a third component, which is a hinge-Fc region peptide. The hinge-Fc region peptide can increase the circulating half-life of the therapeutic peptide by increasing the molecular weight and reducing glomerular filtration, in addition to regenerating the FcRn receptor. In a particular embodiment, the hinge-Fc region peptide can be derived from a human IgG4 Fc region. The human IgG4 Fc region has a reduced ability to bind to FcγR and complement factors compared to other IgG subtypes. Preferably, the Fc region contains a human IgG4 Fc region with substitutions that eliminate effector functions. Thus, the GLP-1 fusion peptide further comprises an Fc region having a modified human IgG4 Fc region containing one or more of the following substitutions: proline instead of glutamic acid at residue 233, alanine or valine instead of phenylalanine at residue 234, and alanine or glutamic acid instead of leucine at residue 235 (EU numbering, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. US Dept. of Health and Human Services, Bethesda, Md., NIH Publication no. 91-3242 (1991)). Removal of the N-linked glycosylation site in the IgG4 Fc region by using Ala instead of Asn at residue 297 (EU numbering) is another way to ensure that residual effector activity is eliminated.

[0076] In a particular embodiment, the GLP-1 fusion peptide of the invention exists as a monomer or a dimer. In a preferred embodiment, the GLP-1 fusion peptide exists as a dimer. In a particular embodiment, the GLP-1 fusion peptide exists as a dimer, said dimer being a homodimer, i.e. said dimer comprising two GLP-1 fusion peptides with the same sequence. In a particular embodiment, the GLP-1 fusion peptide exists as a dimer, said dimer being a heterodimer, i.e. said dimer comprising two GLP-1 fusion peptides with different sequences.

[0077] Preferably, the GLP-1 fusion peptide of the present invention exists as a dimer joined together by disulfide bonds and various non-covalent interactions. Thus, the Fc portion useful for the antibody of the present invention may be a human IgG4 Fc region containing a substitution such as a serine for proline at position 228 (EU numbering) that stabilizes heavy chain dimer formation and prevents the formation of half IgG4 Fc chains. In certain embodiments, the N-terminus of the hinge disulfide may include a natural sequence of amino acids. The x-ray crystal structure of human IgG4 suggests that these amino acids may form a structure that tends to reverse the orientation of the upstream structure in space. Such a tendency is advantageous for maintaining spatial separation of upstream elements such as the GLP-1 fusion peptide. In other embodiments, the natural amino acids N-terminus of the hinge disulfide may be omitted.

[0078] In a specific embodiment, a hybrid human IgG2a hinge fused to human PAA Fc was used to examine its effect on the potency and stability of GLP-1 fusion peptides.

[0079] In another embodiment, the C-terminal Lys residue of the heavy chain is removed, as is commonly found in recombinantly produced monoclonal antibodies.

[0080] The hinge-Fc region peptide may comprise one of the sequences provided in Table 3. The hinge-Fc region peptide sequence may be selected based on at least one of the following criteria: (i) in vitro stability, (ii) in vitro potency, (iii) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate, such as in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide can have agonist activity at the GLP-1 and Y2 receptors, respectively).

[0081] [Table 3]

[0082] Provided herein is a hinge-Fc region platform peptide comprising a hinge-Fc region peptide and at least one of a first linker peptide connected to the amino terminus of the hinge-Fc region peptide and a second linker peptide connected to the carboxy terminus of the hinge-Fc region peptide. The hinge-Fc region peptide may be, for example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 90, the first linker peptide may be, for example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 60 to 83, and the second linker peptide may be, for example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112. In a specific embodiment, the hinge-Fc region peptide comprises SEQ ID NO: 84, and the first linker peptide comprises SEQ ID NO: 60. In a specific embodiment, the hinge-Fc region peptide comprises SEQ ID NO: 84, and the second linker peptide comprises SEQ ID NO: 93, 94, 95, 106, or 111. In a specific embodiment, the hinge-Fc region peptide comprises SEQ ID NO:84, the first linker peptide comprises SEQ ID NO:60, and the second linker peptide comprises SEQ ID NO:93, 94, 95, 106, or 111.

[0083] Second linker peptide: Carboxy-terminal linker (C-terminal linker) Provided herein is a GLP-1 fusion peptide comprising a fourth component, the fourth component being a second linker peptide (i.e., a carboxy-terminal linker peptide). The second linker peptide may comprise one of the sequences provided in Table 4. The second linker peptide sequence may be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro potency, (iii) in vitro stability, (iv) serine xylosylation or lack of potential for serine xylosylation, (v) conjugation yield, and (vi) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate, such as in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide can have agonist activity against the GLP-1 and Y2 receptors, respectively).

[0084] In addition, the second linker peptide sequence may be selected based on its ability to specifically and effectively conjugate to the cyclic PYY peptide. In this context, specificity refers to preferential conjugation at the engineered cysteine ​​residue in the C-terminal region of the second linker over conjugation at any other part of the fusion protein. Examples of strategies to maximize specificity include (i) introducing amino acids adjacent to the engineered cysteine ​​residue to increase the nucleophilicity of the sulfhydryl side chain and enhance its reactivity, (ii) including the engineered cysteine ​​residue in an amino acid sequence that has already demonstrated enhanced selectivity in thiol-electrophilic conjugation, and (iii) incorporating amino acids with anionic or cationic side chains that electrostatically attract cationic or anionic disulfide reducing agents, respectively, to specifically liberate reactive thiols for conjugation. Exemplary strategies for maximizing conjugation efficiency include (i) introducing the same charge, either anionic or cationic, into any portion of the second linker peptide to create mutual electrostatic repulsion of the second linker peptide, thereby reducing disulfide formation between engineered cysteine ​​residues, (ii) incorporating amino acids that enhance the rigidity of the linker, (iii) incorporating amino acids that increase the flexibility of the linker, and (iv) incorporating amino acids with anionic or cationic side chains that electrostatically allow efficient conjugation of cyclic PYY peptides.

[0085] [Table 4]

[0086] GLP-1 fusion peptide Provided herein is a GLP-1 fusion peptide comprising a first, second, third and fourth component as described above, wherein the second component is optionally absent. The first component is a GLP-1 or GLP-1 variant peptide, the second component is a first linker peptide, the third component is a hinge-Fc region peptide and the fourth component is a second linker peptide. The GLP-1 fusion peptide may comprise one of the sequences provided in Table 5. The GLP-1 fusion peptide sequence may be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation, (v) conjugation yield, and (vi) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate, such as in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide can have agonist activity at the GLP-1 and Y2 receptors, respectively).

[0087] [Table 5-1]

[0088] [Table 5-2]

[0089] [Table 5-3]

[0090] [Table 5-4]

[0091] [Table 5-5]

[0092] [Table 5-6]

[0093] [Table 5-7]

[0094] [Table 5-8]

[0095] [Table 5-9]

[0096] [Table 5-10]

[0097] [Table 5-11]

[0098] [Table 5-12]

[0099] Cyclic PYY peptides PYY 3-36 PYY is an endogenous hormone secreted by L-cells in the distal intestine that acts as an agonist of the Y2 receptor to inhibit food intake. Given its role in the control of appetite and food intake, as well as its antisecretory and proabsorptive effects in the mammalian gastrointestinal tract, PYY 3-36 may be effective in treating obesity and related conditions, as well as many gastrointestinal disorders. 3-36 Its therapeutic utility as a therapeutic agent is limited by its rapid metabolism and short circulatory half-life. Thus, the present invention generally relates to a method for the treatment of PYY. 3-36Modified PYY that extends peptide half-life and reduces peptide metabolism in vivo 3-36 Conjugates are of interest.

[0100] In certain embodiments of the present invention, modified PYY 3-36 The peptide is a cyclic PYY peptide. 3-36 Analogues, and Cyclic PYY 3-36 The terms "peptide analog" and "cyclic PYY peptides" may be used interchangeably. Examples of cyclic PYY peptides that can be used in the conjugates of the present invention are described in U.S. Patent Application No. 15 / 794,231, filed October 26, 2017, and U.S. Patent Application No. 15 / 794,171, filed October 26, 2017, the contents of both applications being incorporated herein by reference in their entireties.

[0101] As used herein, the term "NTSC-PYY" is meant to describe an N-terminal to side chain cyclic analog of PYY.

[0102] The peptide sequences described herein are written according to the usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, unless expressly indicated otherwise, it is the L-form of the amino acid that is shown. For convenience in describing the molecules of the invention, conventional and non-conventional abbreviations (both single and three letter codes) of the various amino acids and functional moieties are used. These abbreviations are well known to those of skill in the art, but for clarity, are listed below: A=Ala=alanine; R=Arg=arginine; N=Asn=asparagine; D=Asp=aspartic acid; βA=βAla=beta-alanine; C=Cys=cysteine; hC=hCys=homocysteine; E=Glu=glutamic acid; Q=Gln=glutamine; G=Gly=glycine; H=His=histidine; I=Ile=isoleucine; L=Leu=leucine; K=Lys=lysine; Nle=norleucine; F=Phe=phenylalanine; P=Pro=proline; S=Ser=serine; T=Thr=threonine; W=Trp=tryptophan; Y=Tyr=tyrosine, and V=Val=valine.

[0103] For convenience, the numbering convention for amino acid residues used in naming the NTSC-PYY peptides of the present invention is: hPYY 3-36 Follow the instructions in hPYY. 3-36 The specific amino acid substitutions introduced into the NTSC-PYY peptides relative to the native residue at the corresponding position in are indicated by the appropriate amino acid code followed by the position of the substitution. Thus, "S4" in the NTSC-PYY peptide is 3-36 In the NTSC-PYY peptide, "hC31" refers to a peptide in which the corresponding natural lys4 residue is replaced by serine. 3-36 NTSC-PYY peptides refer to peptides in which the corresponding naturally occurring val31 residue is replaced by homocysteine. Further amino acid substitutions which occur within the NTSC-PYY peptides are described according to this rule and will be recognized as such by one of skill in the art.

[0104] Also for convenience, the nomenclature used for the NTSC-PYY peptides of the present invention incorporates, starting from the N-terminal residue contained in the cycle, the amino acid residues contained in the cycle from left to right along with the linking groups between them. In all cases, the N-terminal amino acid residue of the cycle is linked via its α-amino functionality to a linking group, which in turn connects to the side chain residue of the amino acid at position 31 of the NTSC-PYY peptide. Thus, "cyclo-(I3-m-COPhCH2-hC31)" is used to describe a cycle of the NTSC-PYY peptide in which the α-amino functionality of Ile3 is acylated with a meta-toluic acid residue, the methyl group of which is further linked to the side chain of the hCys31 residue via a thioether bond. Similarly, "cyclo-(K4-CO(CH2)2NHCOCH2-hC31)" is used to describe the ring of the NTSC-PYY peptide in which the native Ile3 residue is missing and the (now N-terminal) α-amino function of lys4 is acylated with a 3-acetamidopropanoyl group, the acetamido methylene carbon of which is connected to the side chain of the hCys31 residue via a thioether bond.

[0105] To provide a convenient functional handle for further derivatization, hPYY 3-36 Lysine residues may be incorporated at various positions in the sequence. Lysine residues may be modified to be indirectly linked to GLP-1 fusion peptide. Lysine residues may be modified to include a chemical linker that links cyclic PYY peptide to GLP-1 fusion peptide in indirect conjugation to GLP-1 fusion peptide. Those skilled in the art will recognize that related orthologs can also be effectively used in this way and are contemplated herein.

[0106] The term "K(PEG24-AcBr)" refers to a lysinyl residue whose side chain ε-amino group is acylated via its 1-carboxylic acid functionality with N-bromoacetyl-75-amino-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73-tetracosaoxapentaheptacontanoic acid.

[0107] The term "K(PEG12-AcBr)" refers to a lysinyl residue whose side chain ε-amino group is acylated via its 1-carboxylic acid functionality with N-bromoacetyl-39-amino-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxanonatriacantanoic acid.

[0108] The term "K(PEG6-AcBr)" refers to a lysinyl residue whose side chain ε-amino group is acylated via its 1-carboxylic acid functionality with N-bromoacetyl-3-[(17-amino-3,6,9,12,15-pentaoxaheptadec-1-yl)oxy]-propanoic acid.

[0109] The term "K(PEG-triazolyl-CH2CH2CO-PEG4-AcBr)" refers to a lysinyl residue whose side chain ε-amino group is acylated via its 1-carboxylic acid functionality with 27-[4-[2-[3-[2-[2-[3-(N-bromoacetylamino)propoxy]ethoxy]ethoxy]propylaminocarbonyl]ethyl]tetrazol-1-yl]-4,7,10,13,16,19,22,25-octaoxaheptacosanoic acid.

[0110] Many of the compounds / conjugates of the invention incorporate a reduced amide bond between the C-terminal residue of the sequence and the adjacent residue, Y36, R35. This reduced amide linkage is represented by the term "psi-(R35,Y36)".

[0111] Various amino acid residues comprising certain sequences of the invention contain an α-amino group that is methylated. Thus, the terms "N-Me-Q34" or "N-Me-R35" refer to an α-N-methylated glutamine at position 34 of the sequence and an α-N-methylated arginine at position 35 of the sequence, respectively.

[0112] The term "N-Me-Q34,psi-(R35,Y36)" in the sequence description refers to a sequence that includes both an α-methylglutamine residue at position 34 as well as a reduced amide bond between residues R35 and Y36.

[0113] Similarly, the term "N-Me-R35,psi-(R35,Y36)" in the sequence description refers to a sequence that includes both an α-methylarginine residue at position 35 as well as a reduced amide bond between this residue and Y36.

[0114] Examples of cyclic PYY peptides are provided in Table 6.

[0115] [Table 6-1]

[0116] [Table 6-2]

[0117] As used herein, human PYY 3-36 (hPYY 3-36 Also provided are side chain cyclic analogs of N-terminal to PYY that exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the peptide 100 of the present invention.

[0118] [ka] (SEQ ID NO:1) and hPYY 3-36

[0119] [ka] (SEQ ID NO: 55). 3-36 The sequence identity of an analog to hPYY is calculated by subtracting the number of residues that differ from the total number of aligned residues (i.e., the number of identical aligned residues):3-36 The sequence identity is calculated by dividing the total number of residues in the sequence. In this example, the differing residues are D11 replaced with substitution K11, followed by V31 replaced with hC31, and finally R35 which is decarbonylated. Thus, in this example, the sequence identity is (34-3) / 34×100.

[0120] Conjugates In another general aspect, the present invention relates to a conjugate comprising a GLP-1 fusion peptide of the invention covalently conjugated in a site-specific manner to a cyclic PYY peptide such that the GLP-1 fusion peptide-conjugated cyclic PYY peptide has an extended / increased half-life compared to the GLP-1 or GLP-1 variant peptide or the cyclic PYY peptide alone. The present invention also relates to pharmaceutical compositions and methods of use thereof. The conjugates are useful for preventing, treating, or ameliorating diseases or disorders such as obesity, type 2 diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, glucose intolerance (e.g., impaired glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors, such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, and eczema, among others.

[0121] In a particular embodiment, the GLP-1 fusion peptide of the invention comprises at least one cysteine ​​residue that can be conjugated to a cyclic PYY peptide. In a particular embodiment, the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274. In a particular embodiment, the GLP-1 fusion comprises an amino acid sequence selected from SEQ ID NOs: 113 or 136. In a particular embodiment, the at least one cysteine ​​residue is comprised in the second linker peptide of the GLP-1 fusion peptide. In a particular embodiment, the cysteine ​​residue is located between residues 287 and 289 of the GLP-1 fusion peptide of SEQ ID NO: 113 or SEQ ID NO: 136, preferably the cysteine ​​residue is located at residue 288 of the GLP-1 fusion. In a particular embodiment, the GLP-1 fusion peptide is covalently linked to a lysine residue at residues 7, 9, 11, 22 or 23 of the cyclic PYY peptide, preferably lysine residue 11. In certain embodiments, the GLP-1 fusion peptide is covalently linked directly to the cyclic PYY peptide or indirectly to the cyclic PYY peptide via a chemical linker on the cyclic PYY peptide.

[0122] In certain embodiments, the cyclic PYY peptide may include a chemical linker. The chemical linker may be chemically modified (e.g., an electrophilic group may be added to the linker) to conjugate the GLP-1 fusion peptide to the cyclic PYY chemical linker. Chemical linkers may include, but are not limited to, peptide linkers, hydrocarbon linkers, polyethylene glycol (PEG) linkers, polypropylene glycol (PPG) linkers, polysaccharide linkers, polyester linkers, linkers containing acyl groups, hybrid linkers consisting of PEG and an embedded heterocycle, or a hydrocarbon chain. The PEG linker may, for example, include 2-24 PEG units.

[0123] Methods for conjugating the GLP-1 fusion peptide of the present invention with the cyclic PYY peptide of the present invention are known in the art. Briefly, the GLP-1 fusion peptide of the present invention can be reduced with a reducing agent (e.g., 1,3,5-triaza-7-phosphaadamantane (PTA)), purified (e.g., by desalting chromatography), and conjugated with the cyclic PYY peptide (e.g., by providing the reduced GLP-1 fusion peptide under conditions that allow conjugation). During the conjugation reaction, the electrophilic leaving group of the cyclic PYY peptide is displaced and a covalent bond is formed between the GLP-1 fusion peptide and the cyclic PYY peptide to form a GLP-1 fusion peptide-linked cyclic PYY peptide conjugate. After the conjugation reaction, the conjugate can be purified by ion exchange chromatography or hydrophobic interaction chromatography (HIC) with a final purification step of Protein A adsorption. In certain embodiments, the GLP-1 fusion peptide of the present invention can be purified before reduction using HIC methods. See, eg, Example 2 for a more detailed description of conjugation methods.

[0124] Provided herein is a conjugate comprising a glucagon-like peptide 1 (GLP-1)-fusion peptide linked to a cyclic PYY peptide, the GLP-1 fusion peptide comprising a GLP-1 peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, the first linker being, optionally, absent.

[0125] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative or pharma- ceutically acceptable salt thereof:

[0126] [ka] During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if does not exist.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K; Z9 is G or K; Z 11 is D or K, Z 22 is A or K, Z 23 is S or K, Z 26 is A or H, Z 30 L, W, does not exist, (However, Z 30 does not exist if and only if q is 1.) Z 34 teeth,

[0127] [ka] and Z 35 teeth,

[0128] [ka] and The derivatives are compounds of formula I that have been modified by one or more processes selected from the group consisting of amidation, acylation, and PEGylation.

[0129] In certain embodiments, the cyclic PYY peptide is represented by formula I, or a derivative or pharma- ceutically acceptable salt thereof: During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if does not exist.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K, the amino side chain of which is optionally

[0130] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z9 is G or K, the amino side chain of K is optionally

[0131] [ka] (wherein i is an integer from 0 to 24, and X=Br, I, or Cl), is substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 11 is D or K, the amino side chain of K is optionally

[0132] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CH2I, -C(O)CH2Cl, or -C(O)CH2Br; Z 22 is A or K, the amino side chain of K is optionally

[0133] [ka] (wherein i is an integer from 0 to 24 and X=Br, I, or Cl), substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 23 is S or K, the amino side chain of K is optionally

[0134] [ka] (wherein i is an integer from 0 to 24 and X=Br, I, or Cl), substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 26 is A or H, Z 30 L, W, does not exist, (However, Z 30 does not exist if and only if q is 1.) Z 34 teeth,

[0135] [ka] and Z 35 teeth,

[0136] [ka] and X is an electrophilic group, and the Br, Cl, or I of the X electrophilic group is displaced in the conjugation reaction to form a GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate.

[0137] In certain embodiments, the cyclic PYY peptide is represented by formula I, or a derivative or pharma- ceutically acceptable salt thereof: During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, or 5; n is 1, 2, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if absent.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of K is

[0138] [ka] is replaced by Z9 is G or K; Z 11 is D or K, the amino side chain of K is optionally

[0139] [ka] -C(O)CH2Br, or Z 22 is A or K, and the amino side chain of K is

[0140] [ka] is replaced by Z 23 is S or K, and the amino side chain of K is

[0141] [ka] is replaced by Z 26 is A or H, Z 30 is L, Z 34 teeth,

[0142] [ka] and Z 35 teeth,

[0143] [ka] and Br is replaced in the conjugation reaction to form the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate.

[0144] In certain embodiments, the conjugate comprises a GLP-1 fusion peptide conjugated to a cyclic PYY peptide, the cyclic PYY peptide being selected from the group consisting of SEQ ID NOs: 1-54. In preferred embodiments, the conjugate comprises a GLP-1 fusion peptide conjugated to a cyclic PYY peptide, the cyclic PYY peptide being selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34.

[0145] In certain embodiments, the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide at a lysine residue of the cyclic PYY peptide via a chemical linker on the lysine residue, which may include, for example, a linker selected from C(O)CH2, polyethylene glycol (PEG)8-triazolyl-CH2CH2CO-PEG4, a PEG chain of 2-24 PEG units, a linker containing an acyl group, or an alkyl chain containing 2-10 carbon atoms.

[0146] The GLP-1 fusion peptides according to embodiments of the invention may be conjugated to a cyclic PYY peptide at one or more amino acid positions of the cyclic PYY peptide, such as amino acid residues 4, 7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 30, or 31 of the cyclic PYY peptide, using methods known in the art. The numbering of the amino acid residues is as follows: hPYY 3-36 In certain embodiments, Z, Z, Z in formula I are 11 , Z 22 , and Z 23 is lysine, which is covalently linked to a cysteine ​​residue in the second linker peptide of the GLP-1 fusion peptide. In a preferred embodiment, the GLP-1 fusion peptide according to the present invention is conjugated to a cyclic PYY peptide at residue 11 of the cyclic PYY peptide, where residue 11 is lysine. In another preferred embodiment, an electrophile such as bromoacetamide or maleimide is introduced into the side chain of the cyclic PYY peptide, such as the amino side chain of lysine at residue 11 of the cyclic PYY peptide, where the reactive site of the electrophile specifically reacts with the sulfhydryl group of a cysteine ​​residue in the second linker peptide of the GLP-1 fusion peptide, preferably the second linker peptide of the GLP-1 fusion peptide is SEQ ID NO: 93, 94, 95, 106, or 111, whereby a covalent linkage is created between the cyclic PYY peptide and the GLP-1 fusion peptide. More preferably, the cyclic PYY peptide is selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34. In one embodiment, the electrophile is introduced directly onto the side chain of cyclic PYY. In another embodiment, the electrophile is introduced indirectly onto the side chain of cyclic PYY via a chemical linker.

[0147] In a particular embodiment, the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide is reduced by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent, whereby the reduced cysteine ​​residue reacts with an electrophile. The azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) or a derivative thereof.

[0148] Also provided is a pharmaceutical composition comprising a conjugate of the invention and further comprising a pharma- ceutically acceptable carrier.

[0149] Non-limiting examples of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates are provided in Table 7.

[0150] [Table 7]

[0151] Half-life extension moiety In addition to the GLP-1 fusion peptide, the conjugates of the present invention may incorporate one or more other moieties to extend the half-life of the pharma- ceutical active moiety (e.g., cyclic PYY peptide), for example, via covalent interactions. Exemplary other half-life extending moieties include, but are not limited to, albumin, albumin variants, albumin binding proteins and / or domains, transferrin, and fragments and analogs thereof. Additional half-life extending moieties that can be incorporated into the conjugates of the present invention include, for example, polyethylene glycol (PEG) molecules such as PEG5000 or PEG20,000, polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides) for desired properties. These moieties may be direct fusions with protein scaffold coding sequences or may be produced by standard cloning and expression techniques. Alternatively, well-known chemical conjugation methods can be used to attach the moieties to recombinantly and chemically produced conjugates of the present invention.

[0152] A PEG moiety may, for example, be added to a peptide molecule of the invention by incorporating a cysteine ​​residue at the C-terminus of the molecule and attaching a PEG group to the cysteine ​​using well-known methods.

[0153] Peptide molecules of the invention incorporating further moieties can be compared for functionality by a number of well-known assays, for example, the biological or pharmacokinetic activity of therapeutic peptides of interest, alone or in conjugates according to the invention, can be assayed and compared using known in vitro or in vivo assays.

[0154] Pharmaceutical Compositions In another general aspect, the present invention relates to a pharmaceutical composition comprising the conjugates and compounds of the present invention and a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a product comprising the conjugates of the present invention together with a pharma- ceutically acceptable carrier. The conjugates and compounds of the present invention and compositions comprising them are also useful for the manufacture of medicaments for the therapeutic applications mentioned herein.

[0155] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy of the composition according to the invention or the biological activity of the composition according to the invention. According to certain embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in conjugated peptide pharmaceutical compositions may be used in the present invention.

[0156] Pharmaceutically acceptable acid / anionic salts for use in the present invention include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estrus, esylate, fumarate, grisepate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthalene ... The acid salts include, but are not limited to, phthalate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucoate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, basic acetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide.The organic or inorganic acid includes, but is not limited to, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, saccharic acid, or trifluoroacetic acid.

[0157] Pharmaceutically acceptable basic / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethane, or "TRIS"), ammonia, benzathine, t-butylamine, calcium, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0158] In some embodiments of the present invention, a pharmaceutical formulation is provided comprising a conjugate of the present invention in an amount of about 0.001 mg / mL to about 100 mg / mL, about 0.01 mg / mL to about 50 mg / mL, or about 0.1 mg / mL to about 25 mg / mL. The pharmaceutical formulation may have a pH of about 3.0 to about 10, e.g., about 3 to about 7, or about 5 to about 9. The formulation may further comprise at least one component selected from a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, and a surfactant.

[0159] Formulation of pharma- ceutical active ingredients with pharma- ceutical acceptable carriers is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent revisions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity adjusters, preservatives, stabilizers, and chelating agents. One or more pharma- ceutical acceptable carriers can be used in formulating the pharmaceutical composition of the present invention.

[0160] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e. a formulation that contains water. The liquid formulation may include a solution, a suspension, an emulsion, a microemulsion, a gel, etc. An aqueous formulation typically contains at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0161] In one embodiment, the pharmaceutical composition can be formulated as an injectable that can be injected, for example, via an injection device (e.g., a syringe or an infusion pump). By injection, it can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0162] In another embodiment, the pharmaceutical composition is a solid formulation, for example a freeze-dried or spray-dried composition, which can be used as is or to which the physician or patient adds solvents and / or diluents before use.Solid dosage forms can include tablets, such as compressed tablets and / or coated tablets, and capsules (e.g., hard or soft gelatin capsules).The pharmaceutical composition can also be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstitution.

[0163] The dosage form may be immediate release, in which case it may include a water soluble or dispersible carrier, or it may be delayed release, sustained release, or modified release, in which case it may include a water insoluble polymer that controls the dissolution rate of the dosage form in the gastrointestinal tract.

[0164] In other embodiments, the pharmaceutical compositions may be delivered intranasally, bucally, or sublingually.

[0165] The pH of the aqueous formulation may be from pH 3 to pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0166] In another embodiment of the invention, the pharmaceutical composition comprises a buffering agent. Non-limiting examples of buffering agents include arginine, aspartic acid, bicine, citrate, disodium monohydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffers may be present individually or in aggregates at a concentration of about 0.01 mg / mL to about 50 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of these specific buffering agents constitute alternative embodiments of the invention.

[0167] In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservatives may be present individually or in aggregates in a concentration of about 0.01 mg / mL to about 50 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of these specific preservatives constitute alternative embodiments of the invention.

[0168] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonicity agent. Non-limiting examples of isotonicity agents include salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), alditols (such as glycerol, 1,2-propanediol propylene glycol, 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (e.g., PEG400), and mixtures thereof. Another example of an isotonicity agent includes sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonicity agent is a sugar alcohol, the term "sugar alcohol" being defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. The isotonicity agent may be present individually or in aggregates at a concentration of about 0.01 mg / mL to about 50 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of these specific isotonicity agents constitute alternative embodiments of the invention.

[0169] In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agents may be present individually or in aggregates at a concentration of about 0.01 mg / mL to about 50 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of these specific chelating agents constitute alternative embodiments of the invention.

[0170] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, non-limiting examples of which include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0171] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, which is carboxy- / hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances, such as monothioglycerol, or thioglycolic acid. The stabilizers may be present individually or in aggregates at a concentration of about 0.01 mg / mL to about 50 mg / mL, for example about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of these specific stabilizers constitute alternative embodiments of the invention.

[0172] In a further embodiment of the invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion composed of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant may be selected, for example, from anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactants may be present, individually or in aggregates, in a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of these specific surfactants constitute alternative embodiments of the invention.

[0173] In further embodiments of the invention, the pharmaceutical compositions include one or more protease inhibitors, such as EDTA, and / or benzamidine hydrochloride (HCl). The protease inhibitors may be present, individually or in aggregates, in a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of these specific protease inhibitors constitute alternative embodiments of the invention.

[0174] The pharmaceutical compositions of the invention may contain an amount of an amino acid base sufficient to reduce the formation of polypeptide aggregates during storage of the composition. The term "amino acid base" refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine, etc.), or analogs thereof. Any amino acid may be present in either its free base form or its salt form. Any stereoisomer of the amino acid base may be present (i.e., L, D, or mixtures thereof). The amino acid base may be present, individually or in combination with other amino acid bases, in a concentration of about 0.01 mg / mL to about 50 mg / mL, e.g., about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific amino acid bases constitute alternative embodiments of the invention.

[0175] Pharmaceutically acceptable salts of the conjugates of the present invention include conventional non-toxic salts or quaternary ammonium salts formed from inorganic or organic acids or bases. Examples of such acid addition salts include acetates, adipates, benzoates, benzenesulfonates, citrates, camphorates, dodecylsulfates, hydrochlorides, hydrobromides, lactates, maleates, methanesulfonates, nitrates, oxalates, pivalates, propionates, succinates, sulfates and tartrates. Basic salts include ammonium salts, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts with organic bases such as dicyclohexylamino salts, and salts with amino acids such as arginine. In addition, basic nitrogen-containing groups may be quaternized, for example, by alkyl halides.

[0176] The pharmaceutical composition of the present invention can be administered by any means that achieves its intended purpose. Examples include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral or intraocular administration. It may be administered by oral route. Suitable formulations for parenteral administration include aqueous solutions of the active conjugate in water-soluble form, such as water-soluble salts, acidic solutions, alkaline solutions, aqueous dextrose solutions, isotonic carbohydrate solutions, and cyclodextrin inclusion complexes. In certain embodiments, the conjugate of the present invention is administered peripherally.

[0177] The invention also encompasses methods of making pharmaceutical compositions comprising mixing a pharma- ceutically acceptable carrier with any of the conjugates of the invention.In addition, the invention includes pharmaceutical compositions made by mixing one or more pharma- ceutically acceptable carriers with any of the conjugates of the invention.

[0178] Furthermore, the conjugates of the present invention may have one or more crystalline polymorphs or amorphous crystalline forms, which are also intended to be included within the scope of the present invention. In addition, the conjugates may form solvates, for example with water (i.e., hydrates) or common organic solvents. As used herein, the term "solvate" refers to a physical association of the conjugates of the present invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, such as hydrogen bonding. In certain cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be isolated. The term "solvate" is intended to encompass both solution-phase solvates and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.

[0179] The present invention is intended to encompass within its scope polymorphs and solvates of the conjugates of the present invention. Thus, the term "administration" in the treatment methods of the present invention includes means of treating, ameliorating, or preventing the syndromes, disorders, or diseases described herein with the conjugates of the present invention, or crystalline polymorphs or solvates thereof that are not specifically disclosed but are clearly included within the scope of the present invention.

[0180] In another embodiment, the present invention relates to a conjugate of the present invention for use as a medicament.

[0181] The scope of the present invention includes prodrugs of the conjugates of the present invention. In general, such prodrugs are functional derivatives of the conjugates that can be easily converted in vivo into the required conjugate. Thus, in the treatment methods of the present invention, the term "administration" is intended to encompass the treatment of the various disorders described with a specifically disclosed conjugate or with a conjugate that may not be specifically disclosed but that converts to a particular conjugate in vivo after administration to a patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs" (Ed. H. Bundgaard, Elsevier, 1985).

[0182] Furthermore, within the scope of the present invention, any element, particularly when referred to with respect to the conjugates of the present invention, is intended to include all isotopes and mixtures of isotopes of that element, whether naturally occurring or synthetically produced, and either in natural abundance or in isotopically enriched form. For example, a reference to hydrogen includes within its scope 1 H, 2 H(D), and 3 H(T). Similarly, references to carbon and oxygen include within their scope 12C, 13 C and 14 C, and 16 O and 18O. The isotope may be a radioactive isotope or a non-radioactive isotope. The radiolabeled conjugates of the present invention include 3 H, 11 C. 18 F, 122 I, 123 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, and 82 Preferably, the radioisotope is selected from the group consisting of: 3 H, 11 C, and 18 F.

[0183] Some conjugates of the present invention may exist as atropisomers, which are stereoisomers resulting from hindered rotation around a single bond, where the steric strain barrier to rotation is high enough to allow for the isolation of conformers. It is understood that all such conformers and mixtures thereof are encompassed within the scope of the present invention.

[0184] Where the conjugate according to the invention has at least one stereocenter, the conjugate may accordingly exist as enantiomers or diastereomers, and it is understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0185] When the process for preparing the conjugates according to the invention results in a mixture of stereoisomers, these isomers can be separated by conventional techniques such as preparative chromatography. The conjugates can be prepared as racemates or the individual enantiomers can be prepared either by enantiospecific synthesis or by resolution. The conjugates can be resolved into their constituent enantiomers by standard methods such as, for example, forming diastereomeric pairs by salt formation with optically active acids such as (-)-di-p-toluoyl-D-tartaric acid and / or (+)-di-p-toluoyl-L-tartaric acid, followed by fractional crystallization and regeneration of the free base. The conjugates can also be resolved by forming diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the conjugates can be resolved using chiral columns via high performance liquid chromatography (HPLC) or SFC. In some cases, there may be rotamers of the conjugate observable by 1H NMR, resulting in complex multiplets and peak integrals in the 1H NMR spectrum.

[0186] During any of the processes for preparing the conjugates of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved. This can be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. JFW MacOmie, Plenum Press, 1973, and TW Greene & PG M Huts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, each of which is incorporated herein by reference in its entirety for all purposes. The protecting groups can be removed at a convenient subsequent stage using methods known in the art.

[0187] How to use The present invention is directed to a method of preventing, treating, or ameliorating a Y2 receptor mediated syndrome and / or a GLP-1 receptor mediated syndrome, disorder, or disease in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the invention.

[0188] The present invention also provides a method of preventing, treating, delaying the onset of, or ameliorating a disorder, disease, or condition, or any one or more symptoms of said disorder, disease, or condition, in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention.

[0189] According to certain embodiments, the disease disorder, or condition is selected from obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., impaired glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and / or eczema.

[0190] According to certain embodiments, a therapeutically effective amount refers to a therapeutic amount that is sufficient to achieve one, two, three, four or more of the following effects: (i) reducing the severity or ameliorating the disease, disorder, or condition being treated, or the symptoms associated therewith; (ii) shortening the duration of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iv) causing regression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (v) preventing the progression or onset of the disease, disorder, or condition being treated, or the symptoms associated therewith. (vi) preventing the recurrence of the disease, disorder or condition being treated, or a symptom associated therewith; (vii) reducing hospitalization of a subject having the disease, disorder or condition being treated, or a symptom associated therewith; (viii) reducing the length of hospitalization of a subject having the disease, disorder or condition being treated, or a symptom associated therewith; (ix) increasing the survival rate of a subject having the disease, disorder or condition being treated, or a symptom associated therewith; (xi) inhibiting or alleviating the disease, disorder or condition being treated, or a symptom associated therewith in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic efficacy of another treatment.

[0191] The therapeutically effective amount or dose can vary depending on a variety of factors, such as the disease, disorder or condition being treated, the means of administration, the target site, the physiological state of the subject (including, for example, age, weight, health status), whether the subject is human or animal, other agents being administered, and whether the treatment is prophylactic or therapeutic. Treatment doses are optimally titrated to optimize safety and efficacy.

[0192] As used herein, the terms "treat", "treating" and "treatment" all refer to an improvement or reversal of at least one measurable physical parameter associated with a disease, disorder or condition, which may, but is not necessarily discernible in the subject. The terms "treat", "treating" and "treatment" may also refer to causing regression, preventing the progression or at least slowing the progression of a disease, disorder or condition. In certain embodiments, "treat", "treating" and "treatment" refer to alleviating, preventing the progression or onset of, or shortening the duration of, one or more symptoms associated with a disease, disorder or condition. In specific embodiments, "treat", "treating" and "treatment" refer to preventing the recurrence of a disease, disorder or condition. In specific embodiments, "treat", "treating" and "treatment" refer to improving the survival rate of a subject having a disease, disorder or condition. In specific embodiments, "treat", "treat" and "treatment" refer to the elimination of a disease, disorder or condition in a subject.

[0193] In one embodiment, the present invention provides a method of preventing, treating, delaying the onset of, or ameliorating obesity, or any one or more symptoms of obesity, in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention.

[0194] In one embodiment, the present invention provides a method of reducing weight in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention.

[0195] In some embodiments, the subject's body weight is reduced, for example, by about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 2% to about 3%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50%, relative to the subject's body weight prior to administration of any of the conjugates, compounds, pharmaceutical compositions, forms, or medicaments of the invention described herein, or compared to a control subject not administered any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein.

[0196] In some embodiments, the weight reduction is maintained for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.

[0197] The present invention provides a method of preventing, treating, delaying the onset of, or ameliorating a syndrome, disorder, or disease, or any one or more symptoms of said syndrome, disorder, or disease, in a subject in need thereof, wherein said syndrome, disorder, or disease is selected from obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., impaired glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors, such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and eczema, the method comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention.

[0198] As used herein, metabolic syndrome refers to a subject having any one or more of the following: hyperglycemia (e.g., high fasting blood glucose), high blood pressure, abnormal cholesterol levels (e.g., low HDL levels), abnormal triglyceride levels (e.g., high triglycerides), a large waistline (i.e., waist circumference), increased fat in the abdominal area, insulin resistance, impaired glucose tolerance, high C-reactive protein levels (i.e., a proinflammatory state), and plasma plasminogen activator inhibitor-1 and fibrinogen levels (i.e., a thrombotic state).

[0199] The present invention provides a method of reducing food intake in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention. In some embodiments, the food intake of the subject is reduced by, for example, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 2% to about 3%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% relative to the food intake of the subject prior to administration of any of the conjugates, compounds, compositions, forms, agents, or combinations of the present invention described herein, or compared to a control subject not receiving any of the conjugates, compounds, compositions, forms, agents, or combinations of the present invention described herein.

[0200] In some embodiments, the reduction in food intake is maintained for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.

[0201] The present invention provides a method for reducing glycated hemoglobin (A1C) in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention. In some embodiments, the subject's A1C is reduced by about 0.001% to about 0.01%, e.g., about 0.001% to about 0.01%, ... The concentration is reduced by about 0.01% to about 0.1%, about 0.1% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.4% to about 0.5%, about 0.5% to about 1%, about 1% to about 1.5%, about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, or about 9% to about 10%.

[0202] In another embodiment, a method of reducing fasting blood glucose levels in a subject in need thereof is provided, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the invention. The fasting blood glucose level may be reduced to less than about 140 to about 150 mg / dL, less than about 140 to about 130 mg / dL, less than about 130 to about 120 mg / dL, less than about 120 to about 110 mg / dL, less than about 110 to about 100 mg / dL, less than about 100 to about 90 mg / dL, or less than about 90 to about 80 mg / dL relative to the subject's fasting blood glucose level prior to administration of any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described herein, or compared to a control subject not receiving any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described herein.

[0203] The present invention provides a method of modulating Y2 receptor activity and GLP-1 receptor activity in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention. As used herein, "modulate" refers to increasing or decreasing receptor activity.

[0204] In some embodiments, an effective amount of the conjugate or compound of the invention, or a form, composition, or medicament thereof, is administered to a subject in need thereof once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, or eight times a day. In other embodiments, an effective amount of the conjugate or compound of the invention, or a form, composition, or medicament thereof, is administered to a subject in need thereof once every other day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, twice a month, three times a month, or four times a month.

[0205] Another embodiment of the present invention provides a method of preventing, treating, delaying the onset of, or ameliorating a disease, disorder, or syndrome, or one or more symptoms of any of said diseases, disorders, or syndromes, in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention in combination therapy. In certain embodiments, the combination therapy is a second therapeutic agent. In certain embodiments, the combination therapy is a surgical therapy.

[0206] As used herein, the term "combined" refers to the use of multiple therapeutic agents in the context of the administration of two or more therapeutic agents to a subject.

[0207] As used herein, combination therapy refers to administering to a subject in need thereof an effective amount of a conjugate or compound of the invention, or a form, composition, or medicament thereof, along with one or more additional therapeutic agents or one or more surgical therapies. In some embodiments, the one or more additional therapeutic agents or surgical therapies may be administered on the same day as an effective amount of a conjugate of the invention, while in other embodiments, the one or more additional therapeutic agents or surgical therapies may be administered the same week or month as an effective amount of a conjugate or compound of the invention.

[0208] The present invention also contemplates preventing, treating, delaying the onset of, or ameliorating any of the diseases, disorders, syndromes, or conditions described herein in a subject in need thereof using a combination therapy, which comprises administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention in combination with any one or more of the following therapeutic agents: a dipeptidyl peptidase-4 (DPP-4) inhibitor (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); GLP-1 receptor agonists (e.g., short-acting GLP-1 receptor agonists, such as exenatide and lixisenatide; intermediate-acting GLP-1 receptor agonists (e.g., liraglutide); long-acting GLP-1 receptor agonists, such as extended-release exenatide, albiglutide, dulaglutide; sodium-glucose cotransporter-2 (SGLT-2) inhibitors (e.g., canaglifozin, dapaglifozin, englifozin, paglifozin, etc.); bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor agonists (e.g., bromocriptine rapid release); biguanides (e.g., metformin, etc.); insulin; oxyntomodulin; sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glyburide, glibenclamide, glibornuride, glisoxepide, glyclopyramide, tolazamide, tolbutamide, acetohexamide, cabutamide, etc.); and thiazolidinediones ( (e.g., pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone, etc.). In some embodiments, the dose of the additional therapeutic agent is reduced when given in combination with a conjugate or compound of the invention. In some embodiments, when used in combination with a conjugate or compound of the invention, the additional therapeutic agent may be used in a lower dose than when each is used alone.

[0209] The present invention contemplates preventing, treating, delaying the onset of, or ameliorating any of the diseases, disorders, syndromes, or conditions described herein in a subject in need thereof using a combination therapy, which comprises administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention in combination with a surgical therapy. In certain embodiments, the surgical therapy can be bariatric surgery (e.g., gastric bypass surgery, such as Roux-en-Y gastric bypass surgery; sleeve gastrectomy; adjustable gastric band surgery; biliary diversion with duodenal switch; intragastric balloon; gastric plication, and combinations thereof).

[0210] In embodiments in which one or more additional therapeutic or surgical therapies are administered on the same day as an effective amount of a conjugate or compound of the invention, the conjugate or compound of the invention can be administered before, after, or simultaneously with the additional therapeutic or surgical therapy. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. For example, a first therapeutic agent (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second therapeutic agent to a subject.

[0211] Embodiment The present invention also provides the following non-limiting embodiments.

[0212] Embodiment 1 is a conjugate comprising a glucagon-like peptide (GLP-1) fusion peptide linked to a cyclic PYY peptide, the GLP-1 fusion peptide comprising a GLP-1 peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, the first linker being optionally absent.

[0213] In embodiment 2, the cyclic PYY peptide is represented by formula I, or a derivative or pharma- ceutically acceptable salt thereof:

[0214] [ka] During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if does not exist.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K; Z9 is G or K; Z 11 is D or K, Z 22 is A or K, Z 23 is S or K, Z 26 is A or H, Z 30 is L, W or absent, (However, Z 30 does not exist if and only if q is 1.) Z 34 teeth,

[0215] [ka] and Z 35 teeth,

[0216] [ka] and The conjugate according to embodiment 1, wherein the derivative is a compound of formula I modified by one or more processes selected from the group consisting of amidation, acylation, and PEGylation.

[0217] In embodiment 3, the cyclic PYY peptide is represented by formula I, or a derivative or pharma- ceutically acceptable salt thereof: During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if does not exist.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH2-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K, the amino side chain of which is optionally

[0218] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z9 is G or K, the amino side chain of K is optionally

[0219] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 11 is D or K, the amino side chain of K is optionally

[0220] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 22 is A or K, the amino side chain of K is optionally

[0221] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 23 is S or K, the amino side chain of K is optionally

[0222] [ka] (In the formula, i is an integer of 0 to 24, and X=Br, I, or Cl.) substituted with -C(O)CHBr, -C(O)CHI, or -C(O)CHCl; Z 26 is A or H, Z 30 is L, Z 34 teeth,

[0223] [ka] and Z 35 teeth,

[0224] [ka] and A conjugate according to embodiment 2, wherein X is an electrophilic group, and the Br, Cl, or I of the X electrophilic group is displaced in the conjugation reaction to form a GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate.

[0225] In embodiment 4, the cyclic PYY peptide is represented by formula I, or a derivative or pharma- ceutically acceptable salt thereof: During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, or 5; n is 1, 2, or 4; q is 0 or 1 (wherein q is Z 30 ) can be 1 if and only if is absent. BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH2-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z9 is G or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 11 is D or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 22is A or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 23 is S or K, and the amino side chain of K is -C(O)CH2Br is substituted, Z 26 is A or H, Z 30 is L, Z 34 teeth,

[0226] [ka] and Z 35 teeth,

[0227] [ka] and The conjugate according to embodiment 2, in which Br is replaced in the conjugation reaction to form a GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate.

[0228] Embodiment 5 is a conjugate according to embodiment 1 or 2, wherein the cyclic PYY peptide is selected from the group consisting of SEQ ID NOs: 1-54, or a pharma- ceutically acceptable salt thereof.

[0229] Embodiment 6 is a conjugate according to embodiment 5, wherein the cyclic PYY peptide is selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33, or 34, or a pharma- ceutically acceptable salt thereof.

[0230] Embodiment 7 is a conjugate according to any one of embodiments 1 to 6, wherein the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide at a lysine residue of the cyclic PYY peptide.

[0231] Embodiment 8 is a conjugate according to embodiment 7, wherein the cyclic PYY peptide comprises a chemical linker.

[0232] Embodiment 9 is a conjugate according to embodiment 8, wherein the chemical linker comprises one selected from the group consisting of C(O)CH2, polyethylene glycol (PEG)8-triazolyl-CH2CH2CO-PEG4, a PEG chain of 2 to 24 PEG units, a linker containing an acyl group, and an alkyl chain containing 2 to 10 carbon atoms.

[0233] Embodiment 10 is a compound represented by the formula I in which Z7, Z9, Z 11 , Z 22 , and Z 23 A conjugate according to any one of embodiments 7 to 9, wherein only one of the linker peptides is a lysine, and the lysine is covalently linked to a cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide.

[0234] Embodiment 11 is a compound represented by the formula I, 11 is lysine.

[0235] Embodiment 12 is a conjugate according to any one of embodiments 1 to 11, wherein said GLP-1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59.

[0236] Embodiment 13 is a conjugate according to embodiment 12, wherein the GLP-1 peptide comprises the amino acid sequence of SEQ ID NO:57.

[0237] Embodiment 14 is a conjugate according to any one of embodiments 1 to 13, wherein the first linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-83.

[0238] Embodiment 15 is a conjugate according to embodiment 14, wherein the first linker peptide comprises the amino acid sequence of SEQ ID NO:60.

[0239] Embodiment 16 is a conjugate according to any one of embodiments 1 to 15, wherein the hinge-Fc region peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-90.

[0240] Embodiment 17 is a conjugate according to embodiment 16, wherein the hinge-Fc region peptide comprises the amino acid sequence of SEQ ID NO:84 or SEQ ID NO:85.

[0241] Embodiment 18 is a conjugate according to any one of embodiments 1 to 17, wherein the second linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-112.

[0242] Embodiment 19 is a conjugate according to embodiment 18, wherein the second linker peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106, or 111.

[0243] Embodiment 20 is a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide conjugated to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274, and the cyclic PYY peptide comprises an amino acid sequence selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34.

[0244] Embodiment 21 is a conjugate according to embodiment 20, wherein said GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO:113.

[0245] Embodiment 22 is a conjugate according to embodiment 20, wherein said GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO: 136.

[0246] Embodiment 23 is a conjugate according to embodiment 21 or 22, wherein a cysteine ​​residue at residues 287-289 of the GLP-1 fusion peptide is covalently linked to a lysine residue at residues 7, 9, 11, 22 or 23 of the cyclic PYY peptide.

[0247] Embodiment 24 is a conjugate according to embodiment 23, wherein said cysteine ​​residue is present at residue 288 of said GLP-1 fusion peptide.

[0248] Embodiment 25 is a conjugate according to any one of embodiments 20 to 24, wherein the lysine residue is present at residue 11 of the cyclic PYY peptide.

[0249] Embodiment 26 is a conjugate according to any one of embodiments 20 to 25, wherein the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide via a chemical linker in the cyclic PYY peptide.

[0250] Embodiment 27 is a conjugate according to embodiment 26, wherein the chemical linker is selected from the group consisting of C(O)CH2, polyethylene glycol (PEG)8-triazolyl-CH2CH2CO-PEG4, a PEG chain of 2 to 24 PEG units, a linker containing an acyl group, or an alkyl chain containing 2 to 10 carbon atoms.

[0251] Embodiment 28 is a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide conjugated to a cyclic PYY peptide, the conjugate comprising a sequence selected from the group consisting of SEQ ID NOs: 225-262, or a pharma- ceutically acceptable salt thereof.

[0252] Embodiment 29 is a conjugate according to any one of embodiments 1 to 28, wherein the GLP-1 fusion peptide is a monomer.

[0253] Embodiment 30 is a conjugate according to any one of embodiments 1 to 28, wherein the GLP-1 fusion peptide is a dimer.

[0254] Embodiment 31 is a method of producing a conjugate according to any one of embodiments 1 to 30, comprising reacting an electrophile, preferably bromoacetamide or maleimide, introduced into a side chain of the cyclic PYY peptide, preferably into the amino side chain of a lysine residue of the cyclic PYY peptide, with a sulfhydryl group of a cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide, thereby generating a covalent linkage between the cyclic PYY peptide and the GLP-1 fusion peptide.

[0255] Embodiment 32 is the method of embodiment 31, wherein a cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide is reduced by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent, whereby the reduced cysteine ​​residue reacts with the electrophile.

[0256] Embodiment 33 is the method of embodiment 32, wherein the azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) or a derivative thereof.

[0257] Embodiment 34 is a pharmaceutical composition comprising a conjugate according to any one of embodiments 1 to 30 and a pharma- ceutically acceptable carrier.

[0258] Embodiment 35 is a method for treating or preventing obesity in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of embodiment 34.

[0259] Embodiment 36 is the method described in embodiment 35, wherein administering an effective amount of the pharmaceutical composition to a subject in need thereof results in a reduction in body weight of about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 25% compared to the body weight of the subject before administration of the pharmaceutical composition.

[0260] Embodiment 37 is a method of treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and / or eczema, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of embodiment 34.

[0261] Embodiment 38 is the method of embodiment 37, wherein the disease or disorder is obesity.

[0262] Embodiment 39 is the method of embodiment 37, wherein the disease or disorder is type I diabetes.

[0263] Embodiment 40 is the method of embodiment 37, wherein the disease or disorder is type II diabetes.

[0264] Embodiment 41 is the method of embodiment 37, wherein the disease or disorder is metabolic syndrome.

[0265] Embodiment 42 is the method of embodiment 37, wherein the disease or disorder is a renal disease.

[0266] Embodiment 43 is the method of embodiment 37, wherein the disease or disorder is nonalcoholic steatohepatitis (NASH).

[0267] Embodiment 44 is the method of embodiment 37, wherein the disease or disorder is nonalcoholic fatty liver disease (NAFLD).

[0268] Embodiment 45 is a method of reducing at least one of food intake or body weight in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of embodiment 34.

[0269] Embodiment 46 is the method of embodiment 45, wherein administering an effective amount of the pharmaceutical composition to a subject in need thereof reduces food intake by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% compared to the food intake of the subject before administration of the pharmaceutical composition.

[0270] Embodiment 47 is a method for modulating Y2 receptor activity and GLP-1 receptor activity in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of embodiment 34.

[0271] Embodiment 48 is the method of any one of embodiments 35 to 47, wherein the pharmaceutical composition is administered by injection.

[0272] Embodiment 49 is the method of embodiment 48, wherein the injection is delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0273] Embodiment 50 is the method of any one of embodiments 35-49, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

[0274] Embodiment 51 is the method of any one of embodiments 35 to 50, wherein the pharmaceutical composition is administered daily, weekly, or monthly to a subject in need thereof.

[0275] Embodiment 52 is the method of embodiment 51, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times, or six times daily.

[0276] Embodiment 53 is the method of embodiment 51, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times, or six times per week.

[0277] Embodiment 54 is the method of embodiment 51, wherein the pharmaceutical composition is administered once, twice, three times, or four times a month.

[0278] Embodiment 55 is a kit comprising the conjugate according to any one of embodiments 1 to 30 or the pharmaceutical composition of embodiment 34, preferably further comprising an injection device.

[0279] Embodiment 56 is a method of producing a pharmaceutical composition comprising a conjugate according to any one of embodiments 1 to 30, comprising combining the conjugate with a pharma- ceutically acceptable carrier to obtain a pharmaceutical composition.

[0280] Embodiment 57 is an isolated hinge-Fc region platform peptide, comprising: a hinge-Fc region peptide comprising an amino acid sequence preferably selected from the group consisting of SEQ ID NOs: 84-90; a first linker peptide comprising an amino acid sequence preferably selected from the group consisting of SEQ ID NOs: 60-83, which is optionally missing; and a second linker peptide comprising an amino acid sequence selected from SEQ ID NOs: 91-112, wherein the first linker peptide is connected to the amino terminus of the hinge-Fc region peptide and the second linker peptide is connected to the carboxy terminus of the hinge-Fc region peptide.

[0281] Embodiment 58 is an isolated hinge-Fc region platform peptide of embodiment 57, wherein the hinge-Fc region peptide comprises the amino acid sequence of SEQ ID NO:84.

[0282] Embodiment 59 is an isolated hinge-Fc region platform peptide of embodiment 57 or 58, wherein the first linker peptide comprises the amino acid sequence of SEQ ID NO:60.

[0283] Embodiment 60 is an isolated hinge-Fc region platform peptide according to any one of embodiments 57 to 59, wherein the second linker peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106, or 111.

[0284] Embodiment 61 is a conjugate comprising the hinge-Fc region platform peptide covalently linked to a target peptide at the second linker peptide.

[0285] In embodiment 62, the targeting peptide is a cyclic PYY peptide represented by formula I, or a derivative or pharma- ceutically acceptable salt thereof:

[0286] [ka] During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (wherein q is Z 30 1 if and only if does not exist.) BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S-; Z4 is K, A, E, S, or R; Z7 is A or K; Z9 is G or K; Z 11 is D or K, Z 22 is A or K, Z 23 is S or K, Z 26 is A or H, Z 30 is L, W or absent, (However, Z 30 does not exist if and only if q is 1.) Z 34 teeth,

[0287] [ka] and Z 35 teeth,

[0288] [ka] and The conjugate according to embodiment 61, wherein the derivative is a compound of formula I modified by one or more processes selected from the group consisting of amidation, acylation, and PEGylation.

[0289] Embodiment 63 is a method of producing a conjugate of a hinge-Fc region platform peptide and a target peptide, comprising creating a covalent linkage between a second linker peptide and the target peptide. EXAMPLES

[0290] synthesis The compounds or conjugates of the present invention can be synthesized according to general synthetic methods known to those skilled in the art. The synthetic descriptions below are for illustrative purposes and are not intended to limit the invention in any way.

[0291] The NTSC cyclic PYY (NTSC-PYY) analogs or derivatives of the present invention can be synthesized by a variety of known conventional procedures for forming successive peptide linkages between amino acids, preferentially by solid phase peptide synthesis (SPPS), as outlined by Merrifield (J. Am. Chem. Soc., 85:2149-2154 (1963)), using an automated peptide synthesizer, conventional bench synthesis, or a combination of both approaches. Conventional procedures for peptide synthesis involve condensation between the free amino group of one amino acid residue (with other reactive functionalities suitably protected) and the free carboxyl group of another amino acid (with its reactive functionalities also suitably protected). Examples of condensing agents typically utilized for peptide bond formation include 1-hydroxybenztriazole (HOBT) or ethyl cyano(hydroxyimino)acetate (Oxyma). Pure), diisopropylcarbodiimide (DIC), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU), 2-(1H-7-azabenztriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HATU), 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium ammonium hexafluorophosphate (HCTU), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOxim), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU) bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) and the like.

[0292] Automated peptide synthesis methods, as described by Yu (J. Org. Chem., 57:4781-4784 (1992)) and more recently elaborated by Palasek (J. Pept. Sci., 13:143-148 (2007)), may be carried out at room temperature (rt) or at elevated temperatures, preferably through the application of microwave heating.

[0293] The compounds of the invention (C-terminal amides) can be conveniently prepared using N-α-FMOC (9-fluoroenylmethyloxycarbonyl) protected amino acid methodology, in which the carboxy terminus of a suitable protected N-α-FMOC-protected amino acid is coupled onto a conventional solid phase resin using a suitable coupling agent. Suitable conventional commercially available solid phase resins include Rink amide MBHA resin, Rink amide AM resin, Tentagel S RAM Resin, FMOC-PAL-PEG PS resin, SpheriTide Rink amide resin, ChemMatrix Rink resin, Sieber amide resin, TG Sieber resin, and the like. The resin-bound FMOC-amino acid may then be deprotected by exposure to 20% piperidine in either N,N-dimethylformamide (DMF) or 1-methyl-2-pyrrolidone (NMP), which treatment serves to selectively remove the FMOC protecting group. Additional FMOC-protected amino acids are then subsequently coupled and deprotected in sequence, thereby generating the desired resin-bound, protected peptide. In certain cases, it may be necessary to utilize orthogonally reactive protecting groups for other amines in the peptide sequence that can withstand FMOC deprotection conditions. Protecting groups such as 4-methyltrityl (Mtt) or 4-methoxytrityl (Mmt), both of which can be removed by treatment with 1% trifluoroacetic acid (TFA) / dichloromethane (DCM), or preferably allyloxycarbonyl (alloc; removable by treatment with Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium(0)) / PhSiH3 (phenylsilane), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde; removable by treatment with 2-3% hydrazine / DMF), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde; removable by treatment with 2-3% hydrazine / DMF) can be effectively used in such cases.

[0294] In conventional peptide synthesis methodologies, the reactive side chains of alpha amino acids are generally protected throughout synthesis with suitable protecting groups to render them inert to coupling and deprotection protocols. Although several protecting groups for amino acid side chains are known in the art, the following protecting groups are most preferred herein: tert-butyl (t-Bu) for serine, threonine, glutamic acid, aspartic acid, and tyrosine; trityl (Trt) for asparagine, glutamine, cysteine, homocysteine, and histidine; tert-butyloxycarbonyl (Boc) for the ε-amino groups of tryptophan and lysine; and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine. These protecting groups are removed upon treatment with strong acids, such as high concentrations of trifluoroacetic acid (TFA).

[0295] Upon completion of SPPS, the resin-bound and side-chain protected peptide is deprotected and simultaneously cleaved from the resin using a cleavage cocktail consisting mainly of (TFA) with various combinations of carbocation scavengers such as triisopropylsilane (TIPS), water, phenol, and anisole. The crude solid peptide is then isolated by precipitating the peptide / cocktail filtrate with cold ether. In the special case of protected peptides bound to Sieber resin, cleavage of the protected peptide from the resin can be advantageously performed upon repeated treatment with 1-2% TFA in DCM without causing side-chain deprotection. Once isolated, further manipulation of the protected peptide may be performed in solution-phase reactions. Finally, the protected peptide may be globally deprotected and precipitated as described above using a separate treatment with the cleavage cocktail. The crude peptide thus obtained is then dissolved at low concentration (approximately <4 mg / mL) in a primarily aqueous solvent system containing an organic co-solvent such as acetonitrile or ethanol. Upon raising the pH of the solution to >5, the peptide then undergoes an intramolecular cyclization reaction to form the corresponding crude NTSC PYY analog of the present invention. The NTSC PYY analog thus formed can be purified using purification techniques generally known in the art. The preferred method of peptide purification used herein is reversed-phase high performance liquid chromatography (HPLC). The purified peptide is then characterized by liquid chromatography / mass spectrometry (LC / MS).

[0296] General schemes and methods for producing cyclic PYY peptides of the invention are described in U.S. Patent Application No. 15 / 794,231, filed October 26, 2017, and U.S. Patent Application No. 15 / 794,171, filed October 26, 2017, the contents of both applications being incorporated herein by reference in their entireties.

[0297] It is understood that the following examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light of them will be suggested to those skilled in the art and are within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0298] Example 1: Expression and purification of glucagon-like peptide 1 (GLP-1) fusion peptides from transiently transfected mammalian cells DNA constructs for GLP-1 fusion peptides were generated by cloning the synthesized gene fragments into pCDNA3.1-derived mammalian expression vectors using endonuclease restriction enzymes of various designs. The synthesized gene fragments were designed to contain DNA sequences encoding one or all of the following components of the fusion protein (from N-terminus to C-terminus): a GLP-1 peptide or a GLP-1 variant peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide containing Cys for conjugation to a cyclic PYY peptide.

[0299] GLP-1 fusion proteins were expressed in ExpiCHO-S™ cells (ThermoFisher Scientific, Waltham, Mass., Catalog No. A29127) by transiently transfecting the cells with purified plasmid DNA of the fusion protein expression construct according to the manufacturer's recommendations. Briefly, ExpiCHO-S™ cells were maintained in suspension in ExpiCHO™ Expression Medium (ThermoFisher Scientific, Catalog No. A29100) in a shaking incubator set at 37° C., 8% CO2 and 125 RPM. Cells were passaged and cultured at a concentration of 6.0×10 6The transfection was performed using the ExpiFectamine™ CHO Transfection Kit (ThermoFisher Scientific Catalog No. A29131). One microgram of plasmid DNA was used for every mL of diluted cells to be transfected, diluted in OptiPRO™ SFM complexing medium. ExpiFectamine™ CHO Reagent was used in a 1:3 ratio (v / v, DNA:reagent), also diluted in OptiPRO™. The diluted DNA and transfection reagent were combined for 1 minute to form DNA / lipid complexes, and then added to the cells. After overnight incubation, ExpiCHO™ Feed and ExpiFectamine™ CHO Enhancer were added to the cells. The cells were cultured at 37°C with shaking for 5 days, after which the culture supernatant was collected.

[0300] Culture supernatants from transiently transfected ExpiCHO-S™ cells were clarified through centrifugation (30 min, 6000 rpm) and then harvested by filtration (0.2μ PES membrane, Corning (Corning, NY)). Large-scale transfections (5-20 liters) were first concentrated 10-fold using a Pall Centramate Tangential Flow Filtration system. 10× DPBS (pH 7.2) was added to the supernatant to a 1× final concentration before loading onto an equilibrated (DPBS, pH 7.2) HiTrap MabSelect Sure Protein A column (GE Healthcare; Little Chalfont, United Kingdom) at a relative concentration of approximately 20 mg protein per mL of resin using an AKTA FPLC chromatography system. After loading, the column was washed sequentially with 10 column volumes of DPBS (pH 7.2). In some cases, additional washes of 20 column volumes of 100 mM Tris, 2.5 M NaCl, 50 mM sodium caprylate (pH 9) and 10 column volumes of DPBS (pH 7.2) were included. Protein was eluted with 10 column volumes of 0.1 M sodium acetate (Na) (pH 3). Protein fractions were immediately neutralized by elution into tubes containing 20% ​​of the elution fraction volume of 2.0 M Tris (pH 7). Peak fractions were pooled and the pH was adjusted to approximately 7 with additional Tris, if necessary. Purified proteins were filtered (0.2μ) and their concentrations determined by absorbance at 280 nm on a BioTek Synergy™ HTM spectrophotometer. The quality of the purified proteins was assessed by SDS-PAGE and analytical size exclusion HPLC (Dionex™ HPLC system). Endotoxin levels were measured using a turbidimetric LAL assay (Pyrotell®-T, Associates of Cape Cod; FALMOUTH, Mass.).

[0301] Alternatively, GLP-1 fusion proteins were expressed in Expi293F™ cells (ThermoFisher Scientific, Catalog No. A14527) by transiently transfecting the cells with purified plasmid DNA of the fusion protein expression constructs according to the manufacturer's recommendations. Briefly, Expi293F™ cells were maintained in suspension in Expi293F™ Expression Medium (ThermoFisher Scientific, Catalog No. A1435101) in a shaking incubator set at 37°C, 8% CO2 and 125 RPM. Cells were passaged and cultured at 2.5 x 10 6 The transfection was performed using the ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific Catalog No. A14525). One microgram of plasmid DNA was used for every 1 mL of diluted cells to be transfected, diluted in OptiMEM™ SFM complexing medium. ExpiFectamine™ 293 Reagent was used in a 1:2.6 ratio (v / v, DNA: Reagent), diluted in OptiMEM™, and incubated at room temperature for 5 minutes. The diluted DNA and transfection reagent were combined for 20 minutes to form DNA / lipid complexes, and then added to the cells. After overnight incubation, Expi293™ Feed and ExpiFectamine™ 293 Enhancer were added to the cells. The cells were cultured at 37°C for 4 days with shaking, after which the culture supernatant was collected.

[0302] Culture supernatant from transiently transfected Expi293™ cells was clarified through centrifugation (30 min, 6000 rpm) and then collected by filtration (0.2μ PES membrane, Corning). 10×DPBS (pH 7.2) was added to the supernatant to a final concentration of 1×, and then loaded onto an equilibrated (DPBS, pH 7.2) HiTrap MabSelect Sure Protein A column (GE Healthcare) at a relative concentration of approximately 20 mg protein per mL of resin using an AKTA FPLC chromatography system. After loading, the column was washed with 10 column volumes of DPBS, pH 7.2. Protein was eluted with 10 column volumes of 0.1 M Na acetate (pH 3). Protein fractions were immediately neutralized by elution into tubes containing 20% ​​elution fraction volume of 2.0 M Tris (pH 7). Peak fractions were pooled and the pH adjusted to approximately 5.5 with additional Tris, if necessary. The purified protein was filtered (0.2μ) and its concentration determined by absorbance at 280 nm on a BioTek Synergy™ HTM spectrophotometer. The quality of the purified protein was assessed by SDS-PAGE and analytical size-exclusion HPLC (Dionex™ HPLC system). Endotoxin levels were measured using a turbidimetric LAL assay (Pyrotell®-T, Associates of Cape Cod).

[0303] The GLP-1 fusion protein having a first linker peptide containing the amino acid sequence of SEQ ID NO: 61, which is a polyglycine linker, was expressed at a low level and had a low purification yield. Therefore, the use of polyglycine in the first linker peptide when genetically engineering the GLP-1 fusion protein was not preferred in this experiment.

[0304] GLP-1 fusion proteins having a second linker peptide comprising the amino acid sequence of SEQ ID NO: 100 were determined to be cleaved and not of the expected molecular weight when analyzed by mass spectrometry. Therefore, the use of the second linker peptide of SEQ ID NO: 100 in engineering GLP-1 fusion proteins was not preferred in this experiment.

[0305] Furthermore, when preparing the GLP-1 fusion peptide construct, the carboxy-terminal lysine of the hinge-Fc region peptide (i.e., human IgG4) was deleted from the final construct to avoid potential proteolytic burden.

[0306] Example 2: Generation of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates Reduction of GLP-1 fusion protein Method A Purified GLP-1 fusion proteins are a mixture of recombinant protein and a genetically engineered second linker peptide containing at least one cysteine ​​residue in each linker, either disulfide-linked to another cysteine ​​or intramolecularly disulfide-linked to an exogenous cysteine ​​residue in the cytosol or growth medium. To such a heterologous GLP-1 fusion protein solution (5-12 mg / mL in TrisOAc, pH 5.4-6.4), an excess of the phosphine reducing reagent 1,3,5-triaza-7-phosphaadamantane (PTA, 15-40 equivalents) was added, followed by the addition of 100 mM EDTA (final concentration 1-2 mM). The resulting reaction mixture was gently stirred at room temperature until reduction of the targeted cysteine ​​residues was complete (4-16 h). Free cysteines and remaining PTA were removed by desalting chromatography.

[0307] Method B To the heterologous GLP-1 fusion protein solution (5-15 mg / mL in TrisOAc, pH 5.0-6.5), an excess of the phosphine reducing reagent tris(2-carboxyethyl)phosphine (TCEP, 4-25 equivalents) was added, followed by the addition of 100 mM EDTA (final concentration 1 mM). The resulting reaction mixture was gently stirred overnight at room temperature and then applied to a desalting column equilibrated with TrisOAc (pH 7.0). The fully reduced GLP-1 fusion protein (with the hinge disulfide bond cleaved) was treated with dehydroascorbic acid (DHAA, 10 equivalents) for 2 h, after which the reduced GLP-1 fusion peptide was oxidized to obtain the complete GLP-1 fusion peptide with the engineered cysteine ​​in the second linker peptide, which was completely decapped.

[0308] Preparation of GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugates A solution of reduced GLP-1 fused peptide in TrisOAc ((3-10 mg / mL, pH 5.5) was added to a solution of cyclic PYY peptide (2.6-3.0 equiv., 10-15 mg / mL) in deionized water. To protect reactive thiols from metal-catalyzed oxidation, 100 mM EDTA solution was added to the reaction mixture until the concentration of EDTA was 1 mM. The pH of the reaction solution was raised to pH 8.0-8.2 by dropwise addition of Tris buffer (1 M, pH 9.1). The reaction was allowed to proceed for 3-7 h at room temperature and then for 18 h at 5 °C. LCMS showed that the conjugation was complete and the formation of GLP-1 fused cyclic PYY peptide conjugate was more than 95%. This The reaction was either directly subjected to Protein A purification to remove excess cyclic PYY peptide or quenched by adding 5 equivalents of cysteine ​​(to cap remaining reactive cyclic PYY peptide). The resulting crude conjugate was purified by hydrophobic interaction chromatography (HIC) immediately followed by adsorption with Protein A. The conjugate was eluted (sodium acetate, pH 3.5) and subsequently neutralized with Tris buffer to pH 7-7.5 to obtain the final product. Alternatively, the HIC purified conjugate can be exchanged into the desired buffer by a PD-10 column without further Protein A polishing if performed prior to HIC purification.

[0309] The conjugation reaction may also be carried out at lower temperature (5° C.) and monitored by LCMS (approximately 48 hours).

[0310] In some cases, reactive cysteines in the second linker peptide of the GLP-1 fusion protein were capped by bromoacetamide to form stable thioether bonds. These acetamide-modified GLP-1 fusion proteins were prepared as follows: To a GLP-1 fusion protein solution (~12 mg / mL in TrisOAc, pH 5.4-6.4), an excess of the phosphine reducing reagent 1,3,5-triaza-7-phosphaadamantane (PTA, 15-20 equivalents) was added, followed by 100 mM EDTA (final concentration 1-2 mM). The resulting reaction mixture was gently stirred for 18 h at room temperature. 15-20 equivalents of bromoacetamide were added to the GLP-1 fusion peptide solution, and the pH value of the reaction mixture was adjusted to pH 7.5-8.1 by Tris buffer (1 M, pH 9.1). After 2 h, the reaction mixture was purified by hydrophobic interaction chromatography (HIC) immediately followed by Protein A polishing.

[0311] Table 8 provides the isolated yield of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates. It was observed that the isolated yield of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates was poor when the first linker peptide was an AP linker, as in GLP-1 fusion peptide 64 (GF64), and the second linker peptide was a G4A linker, as in GLP-1 fusion peptide 8 (GF8).

[0312] [Table 8]

[0313] Example 3: Characterization of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates Analytical characterization of the GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugates was performed using (i) hydrophobic interaction chromatography (HIC), (ii) intact mass measurement by LC-ESIMS, and (iii) size exclusion chromatography (SEC). The analytical characterization results and conjugation method of the GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugates are shown in Table 9.

[0314] [Table 9]

[0315] Example 4: In vitro assay Conjugates were screened for functional activity and in vitro potency in a cell-based assay measuring intracellular cAMP using the Lance competitive cAMP immunoassay (Perkin Elmer, Waltham, Massachusetts) according to the kit instructions. Clonal HEK293 cells stably expressing mouse or human GLP-1R or NPY2 receptor (Y2R) were used in the assay. Cells expressing GLP1R were thawed and suspended in HBSS, 5 mM HEPES, 0.1% BSA, 0.5 mM IBMX. Cells were mixed with the anti-cAMP antibody from the kit and added to serially diluted conjugates in HBSS, 5 mM HEPES, 0.1% BSA in 384-well white opti-plates. After 10 minutes of incubation at room temperature, the immunoassay detection mixture was added. The assay plate was read on an Envision plate reader (excitation 320 nm, emission 615 nm and 665 nm) as a TR-FRET assay and the data was used to calculate the EC values ​​of compounds using Prism statistical software (GraphPad Software San Diego). 50 values ​​were calculated.

[0316] To determine the activity of compounds at the Y2R receptor, HEK293 cells expressing Y2R were cultured in DMEM high glucose medium (Cellgro) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 1% penicillin / streptomycin, and 600ug / mL G418, and plated in 384-well plates overnight in medium without G418. On the day of the assay, cell growth medium was removed from the cells and 6μL of conjugate (2x) in 5mM HEPES, 500uM IBMX, and 0.1% BSA in HBSS was added. Then, 6μL of stimulation buffer containing forskolin (2x, 5uM final concentration) and LANCE cAMP antibody (1:100) was added to the cells. After 25 minutes of incubation at room temperature, 12μL of detection mix was added and cAMP concentration was quantified in a LANCE cAMP immunoassay.

[0317] 4. Data Analysis Data from the Envision plate reader were expressed as relative fluorescence units (RFU) calculated as (615 nm / 665 nm) x 10,000. All samples were measured in triplicate. The unknown cAMP concentrations in each well were interpolated from reference standards of known cAMP concentrations included in each plate. EC 50 , Log(EC 50 Parameters such as ΔcAMP, HillSlope (nH), maximum, and minimum were obtained by plotting cAMP concentration values ​​against log compound concentration fitted with the 4-P model using a nonlinear weighted least squares application.

[0318] [Table 10]

[0319] [Table 11]

[0320] Comparison of the GLP-1R potency of SEQ ID NOs: 135, 134, 146, and 176 with SEQ ID NOs: 238, 242, 262, 253, and 241 showed that the GLP-1 fusion peptide with exendin 4 (1-39) (SEQ ID NO: 58) lost GLP-1R potency after conjugation with the PYY peptide. Based on these results, testing with the GLP-1 fusion peptide with exendin 4 (1-39) (SEQ ID NO: 58) was not preferred in this experiment.

[0321] Human NPY2R Primary Screening Assay: The method used to screen the NPY2R potency of GLP-1 fusion peptide-linked cyclic PYY peptide conjugates in vitro was a cell-based assay designed to measure inhibition of forskolin-induced cAMP generated by adenylate cyclase through modulation of the human NPY2R Gi-protein coupled receptor. Forskolin-induced cAMP generation in human NPY2R transfected CHO-K1 cells (DiscoverX) was dose-dependently reduced by activation of NPY2R with PYY analogs and controls, as measured in a FRET-based competitive cAMP immunoassay.

[0322] Cells were removed from frozen storage, thawed in a 37°C water bath, added to 40 mL of 1x DPBS (pH 7.2) (Gibco), and filtered through a cell strainer. The cells were centrifuged at 450 x g for 5 min and the supernatant was discarded. The cell pellet was diluted to 0.125 x 10 6Cells were resuspended in DMEM / high glucose, 10% HIFBS, 1% Pen / Strep, 1% L-glutamine, 1% Na-pyruvate at a density of 1000 cells / mL. Cells were dispensed at 40 μL / well into collagen-coated white 384-well plates for a final 5000 cells / well and incubated at 37°C, 5% CO2 for 16-24 hours. Cells in the assay plate were washed twice by adding 80 μL / well of 1x DPBS and the supernatant was decanted. Sample and control dilutions were prepared in 1x HBSS, 5 mM HEPES, 0.002 mM forskolin, 0.1% BSA, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 20 μL / well of each sample was added to the designated well and incubated at room temperature for 30 minutes with shaking. 20 μL / well of cAMP detection reagent mix was then added to each assay plate and incubated at room temperature for 2-24 hours with shaking. Plates were read on a plate reader. All samples were measured in quadruplicate. Data were analyzed by plotting raw LANCE cAMP values ​​over log compound concentration, fitted with a 4-P model using a nonlinear weighted least squares application within the R environment.

[0323] Human GLP-1R Primary Screening Assay: The method used to screen the GLP-1R potency of GLP-1 fusion proteins and GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates in vitro was a cell-based assay designed to measure cAMP production through modulation of the human GLP-1R Gs-protein coupled receptor. Following concentration-dependent activation of GLP-1R by GLP-1 fusion peptides and GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates, cAMP accumulation in HEK cells transfected with human GLP-1R was measured in a TRFRET-based competitive cAMP assay.

[0324] HEK cells transfected with human GLP-1R were thawed on the day of assay and diluted to 0.5 x 10 in 1x HBSS, 5mM HEPES, 0.1% BSA, and 1mM IBMX.6 Cells were resuspended at 1000 cells / mL and 10 μL was added to each well of a 384-well plate (5000 cells / well). Samples were diluted in 1×HBSS, 5 mM HEPES, 0.1% BSA and added to the assay plate at 10 μL / well and incubated at room temperature for 30 minutes. cAMP detection reagent was added at 20 μL / well. Plates were incubated at room temperature for 2 hours before being read on a plate reader. All samples were measured in quadruplicate. Data were analyzed by plotting raw LANCE cAMP values ​​over log compound concentration fitted with a 4-P model using the nonlinear weighted least squares application within the R environment.

[0325] Example 5: Xylose analysis in GLP-1 fusion peptides and GLP-1 fusion peptide-coupled cyclic PYY conjugates Sample preparation: To prepare samples for peptide mapping, each protein molecule (1 mg / mL): GLP-1 fusion peptide-coupled cyclic PYY conjugate (SEQ ID NO:244), GF40 (SEQ ID NO:152), and GF34 (SEQ ID NO:146) was diluted 1:4 with 8M guanidine / HCl buffered at pH 8.0. To these samples, an aliquot of 1M DTT (Sigma 43816-10ML, BioUltra) was added until a final concentration of 25 mM was reached, and the samples were then incubated at 37°C for 1 hour. Alkylation was performed using either iodoacetamide (Sigma, A3221-10VL) or N-methylmaleimide (NEM) (Sigma, E3876). Freshly prepared 1M alkylating agent was added until approximately 50 mM was reached, and the samples were incubated at room temperature in the dark for 60 minutes. The alkylation reaction was then quenched by adding 15 μL of 1M DTT per 400 uL of reaction mixture (Sigma 43816-10ML, BioUltra). Samples were desalted using Zeba Spin desalting columns (Thermo, catalog #89833) following the manufacturer's protocol in 50 mM Tris, 1 mM CaCl2 (pH=8.0). Samples were digested with trypsin (Promega sequencing grade, V511A) for 4 hours at 37°C after adding 1 μL of 1 μg / μL trypsin reconstituted in 50 mM acetic acid (supplied with lyophilized trypsin). Trypsin digestion was quenched by adding 0.6 μL of 100% TFA to a 60 μL aliquot of digested protein. Samples were resuspended in microvials and placed in an autosampler set at 4°C for LC / MS analysis.

[0326] Liquid chromatography and mass spectrometry: 2 μg (~10 μL) of digested protein was injected onto an Agilent AdvanceBio Peptide Map Micro Bore Rapid Resolution Column (1 × 150 mm, 2.7 μm, part number 863600-911) using an Agilent Infinity 1290 UHPLC (Agilent Technologies, part numbers: G1330B, G4226A, G4220A, G4212A) at a flow rate of 0.1 mL / min. The column temperature was maintained at 65 °C. Mass spectrometry grade HPLC solvents (A: 0.1% formic acid and B: 100% ACN in 0.1% formic acid) were purchased from VWR (part numbers: LC452-1, LC441-1). Proteolytic peptides were eluted from the column using a 50 min gradient of ACN from 2% to 40% in 0.1% FA. The column eluate was introduced into an Orbitrap Q-Exactive mass spectrometer (Thermofisher Scientific) via a heated electrospray ionization probe (HESI) with a positive spray voltage of 3.5 kV, sheath gas of 20 (arbitrary units), auxiliary gas of 7 (arbitrary units), ion transfer tube at 299 °C, and evaporator at 100 °C. A data-dependent acquisition method was performed by sequentially dissociating the top five abundant peptide ions observed in the full MS scan. Mass analysis was performed with Orbitrap detection, precursor scans set at 70,000 resolution, mass range 150-2000 m / z, automatic gain control (AGC) target 1.0e6, and maximum injection time 50 ms. One microscan spectrum was acquired in profile mode. Precursor determination criteria were monoisotopic precursor selected peptides, charge state: 2-7, dynamic exclusion: 6.0 s, and precursor intensity threshold 5e4. The precursor peptides were isolated by a quadrupole with an isolation width of 1.6 m / z sent to the collision cell. A normalized collision energy of 28 (arbitrary units) was used for higher energy collision dissociation (HCD) of the peptide ions.Product ions were transferred to the orbitrap for mass analysis using the orbitrap settings: isolation power of 17,500, 200-2000 m / z range, AGC target 5e5, maximum injection time 100 ms, and spectra acquired in centroid mode 1 microscan.

[0327] Data analysis: LC-MS / MS raw data were subjected to database searching using Byonic software (version 2.15.7) (Protein Metrics). The following parameters were used for data searching: precursor ion mass tolerance, 8 ppm; product ion mass tolerance, 20 ppm for HCD spectra; variable modifications included cysteine ​​carbamidomethylation, or N-methylmaleimide (NEM), cysteine ​​DTT adduct formation, cysteine ​​peptide YY (PYY) conjugates when present, asparagine, serine xylose deamination, and serine and threonine O-glycosylation of 78 mammalian O-linked glycans obtained from the Byonic glycan library. For all searches, a full trypsin specificity search with a maximum of two missing cleavages was selected. The protein false discovery rate (FDR) was set to 1%. The protein database contained the amino acid sequence of each protein. All Byonic search results were processed with Bylogic software (version 2.15.296) (Protein Metrics) and peptides were filtered with a minimum Byonic score of 15, a maximum Altranscore / PrimaryRank score of 0.99, with a maximum precursor m / z error of 8 ppm and an XIC area window of 2 min. Serine xylosylation levels were reported by considering all peptide XIC peak areas of the isoforms that make up the linker region. Xylosylation at each serine residue was then calculated as a sum of fractions of the total XIC peak area. PTM site localization probabilities were also obtained by performing database searches using the Andromeda search engine of MaxQuant software version 1.6.1 (MaxPlank Institute). Masses corresponding to fully cyclized PYY and trypsin-cleaved PYY structures were obtained using BIOVIA drawing software. Data are reported in Table 12.

[0328] [Table 12] Xylose modification at serine in the linker: Site identification and modification levels ND = not detected by MS / MS, NQ = not quantified; NA = the serine position of the linker is not applicable to the molecule. ** Number: SEQ ID NO: The brackets indicate the position numbers of SEQ ID NO:152.

[0329] Table 12 summarizes the site-specific xylosylations identified and quantified using peak integration in Bylogic, where the XIC% based on the peak / peak of interest (putative modification, i.e., serine xylosylation) was normalized to the sum of all species contributing to site-specific xylosylation according to the following equation:

[0330] XIC% of modification = (XIC of modified peptide / ΣXIC of peptide counterpart) × 100 All three molecules examined consist of the same first linker peptide AS(G4S)2 connecting the GLP-1 peptide and the hinge-Fc region peptide. However, due to the difference between the GLP-1 fusion peptide found in SEQ ID NO:152 (GF40) and both SEQ ID NO:244 and 146 (GF34), the positions of three serine residues in SEQ ID NO:152 are different from SEQ ID NO:244 and 146, as shown in Table 12. Analysis of the peptides demonstrated three site-specific serine residue xylosylation events at Ser-41, Ser-46, and Ser-51 in SEQ ID NO:244 and 146, and at Ser-33 in SEQ ID NO:152. Quantification of site-specific xylosylation was only limitedly achieved because most xylosylated peptide species co-eluted during reversed-phase liquid chromatography. For example, in SEQ ID NO:244, Ser-41 was about 9%, while Ser-46 and Ser-51 were about 7%. Similarly, all three sites in SEQ ID NO:146 were xylosylated at about 5%. Xylosylation of SEQ ID NO:152 was found to occur to a lesser extent, at about 0.1%.

[0331] It has been shown that the G4S linker region of therapeutic proteins is susceptible to xylosylation, and the level of xylosylation varies based on the linker length (Wen et al., "The propensity for xylosylation in (G4S) n linkers occur when n>2," Anal. Chem. 85:4805-12 (2013); Sphar et al., Protein Sci. 22:1739-53 (2013), Sphar et al., mAbs 6:904-14 (2014)). The first linker peptide found in all three molecules, the AS(G4S)2 linker, exhibited xylosylation at all or some of the serine residues, and based on these data, the use of serine-containing first linker peptides in preparing GLP-1 fusion peptide constructs was not preferred in this experiment.

[0332] Example 6: Ex vivo human plasma stability of GLP-1 fusion peptides Fresh whole blood from four healthy human donors with sodium heparin anticoagulant was received on the day of preparing stability samples. Fresh whole blood was processed for plasma by centrifugation at 3500 RPM for 15 minutes, and then plasma was isolated. Plasma from all four donors was combined, filtered through a 0.2μ filter, and warmed to 37°C. Samples were spiked with 20nM GLP-1 fusion peptide and GLP-1 fusion peptide-coupled cyclic PYY conjugate dual agonist (DA) analog, and zero-time samples were collected immediately and stored at -80°C until analysis. Samples were incubated at 37°C, collected at the designated time points, and stored at -80°C until analysis. On the day of analysis, zero-time and subsequent time point samples were thawed and analyzed together in a functional cell-based bioassay.

[0333] The method used to detect and quantitate the levels of bioactive GLP-1 fusion peptides and GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugate DA analogs in human plasma samples was a cell-based bioassay designed to measure cAMP production through modulation of the human GLP-1R G protein-coupled receptor. Following concentration-dependent activation of GLP-1R by GLP-1 fusion proteins and GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugates, cAMP accumulation in human GLP-1R-transfected HEK cells was measured with a TRFRET-based competitive cAMP assay. Human GLP-1R-transfected HEK cells were thawed on the day of the assay and diluted with 0.5×10 IgG in 1×HBSS, 5 mM HEPES, 0.1% BSA, and 1 mM IBMX. 6 Cells were resuspended at 1000 cells / mL and 10 μL was added to each well of a 384-well plate (5000 cells / well). Stability samples were thawed and diluted to 20% plasma in assay buffer consisting of 1×HBSS, 5 mM HEPES, 0.1% BSA, 5 mM EDTA, protease inhibitors, with subsequent dilutions in assay buffer containing 20% ​​normal human plasma. Reference standards of known concentrations were prepared for each compound in assay buffer containing 20% ​​normal human plasma. Standards and samples were added to each assay plate at 10 μL / well and incubated at room temperature for 30 minutes. cAMP detection reagent was added at 20 μL / well. Plates were incubated at room temperature for 2 hours before being read on a plate reader.

[0334] All samples were measured in quadruplicate. Reference standard curves for compounds were constructed using log-transformed concentrations, nonlinear fitting, and Log v. response (variable slope) to obtain EC 10 and E.C. 90 Values ​​were determined as the upper and lower limits of the quantitative assay. Concentrations of stability samples for each compound were extrapolated from the corresponding reference standard curve. The remaining bioactivity of each stability sample was determined relative to the corresponding time zero sample and reported as % of starting plasma concentration over the 168 hour time course. % starting plasma concentration = [mean of stability sample] / [mean of time zero] x 100.

[0335] Results: GLP-1 fusion peptides SEQ ID NO: 144 (GF32), 148 (GF36), 145 (GF33), 151 (GF39), 146 (GF34), 147 (GF35), 149 (GF37), 152 (GF40), and dulaglutide control were incubated ex vivo in human plasma for 7 days at 37° C. and functional stability was measured in an in vitro GLP-1R cAMP functional assay ( FIG. 1A ). GLP-1 fusion protein with GLP-1 peptide SEQ ID NO: 58 (Exendin 4(1-39)) showed the highest stability, followed by SEQ ID NO: 57 ((A8G, G22E, R36G) GLP-1(7-37)) and dulaglutide control, with SEQ ID NO: 56 ((A8S, A30E) GLP-1(7-36)) being the least stable. Based on these results, testing with an Fc fusion protein bearing the GLP-1 peptide SEQ ID NO:56 was not preferred in this experiment.

[0336] GLP-1 fusion peptides SEQ ID NO: 152 (GF40) and 146 (GF34), and their corresponding GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates SEQ ID NO: 248 and 262, and another GLP-1 fusion peptide SEQ ID NO: 153 (GF41), were incubated ex vivo in human plasma with dulaglutide control for 7 days at 37° C. Functional stability was measured in an in vitro GLP-1R cAMP functional assay (FIG. 1B). The stability of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates was comparable to the corresponding unconjugated fusion proteins, indicating that the conjugation of PYY peptide did not affect the stability of the GLP-1 peptide. The GLP-1 fusion peptide SEQ ID NO:146 (GF34) and the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate SEQ ID NO:262 had the highest stability, while SEQ ID NOs:152 (GF40), 248, and 153 (GF41) had similar stability to the dulaglutide control.

[0337] Example 7: In vivo mouse stability assay method LCMS method: Plasma samples were processed by immunoaffinity capture using anti-human Fc antibody, followed by digestion with trypsin and analysis by reversed-phase LC-MS / MS analysis on a triple quadrupole mass spectrometer. Peptides at the N-terminus of GLP-1, i.e., HGE (HGEGTFTSDVSSYLEEQAAK (SEQ ID NO: 263) for GLPD30 and HGEGTFTSDLSK (SEQ ID NO: 264) for GLPD31, were monitored as a surrogate for molecules containing active GLP1. A peptide located in the Fc, i.e., VVS (VVSVLTVLHQDWLNGK (SEQ ID NO: 265)), was monitored as a surrogate for total Fc levels. A peptide located in the cyclic PYY peptide, YYA (YYASLR (SEQ ID NO: 266)), was monitored as a surrogate for PYY levels. Calibration standard curves and quality control samples were prepared by spiking the reference standards in plasma and processed using the same procedure at the same time as the test samples.

[0338] result Plasma exposure levels of intact GLP-1 fusion peptide moiety N-terminal 72 hours after administration in mice were measured by LCMS assay and reported as percentage of HGE N-terminal GLP-1 fusion peptide moiety level relative to Fc level (HGE% of Fc). The results in Table 13 show that the first linker peptides of SEQ ID NO:62 and 66 containing AP repeats were less abundant than the first linker peptide of SEQ ID NO:60 containing G4A repeats. Based on these results, testing with GLP-1 fusion peptides with first linker peptides containing AP repeats was not preferred in this experiment.

[0339] [Table 13] BQL = below quantification limit

[0340] Acute and subchronic pharmacodynamic studies in DIO mice All rodents used in these studies were maintained at Janssen R&D (Spring House, PA) according to protocols approved by the Institutional Animal Care & Use Committee (IACUC). Animals were housed under standard temperature and humidity conditions with free access to food and water, with a 12-hour light / 12-hour dark cycle (unless otherwise noted). Individually housed 20-week-old male DIO (60% kcal% high fat diet for 15 weeks) C57BL / 6T mice (Taconic Laboratory) were used. Animals were randomized into groups based on body weight. For intraperitoneal (IP) glucose tolerance tests (IPGTT), mice were administered vehicle, dulaglutide (purchased from Eli Lilly; Myoderm (Norristown, PA)), or compounds 1-4 (SEQ ID NO:113, SEQ ID NO:225, SEQ ID NO:136, SEQ ID NO:229, respectively) (n=8 / group). Twenty-four hours later (after an overnight fast), mice were administered dextrose (1 g / kg) IP. Blood glucose was measured at the indicated time points using a One Touch Ultra glucometer (LifeScan) and plasma insulin (Meso Scale Discovery) was measured at 0 and 10 min. For acute food intake (FI) and weight loss (WL) studies, individually housed DIO mice were administered vehicle, dulaglutide, or compound and food and weight were measured over 3 days (n=8 / group). Data are shown in Figures 2A-2B, 3A-3B, and 4A-4B. The data in Figures 2A-2B, 3A-3B, and 4A-4B show that the GLP-1 fusion peptide has desirable GLP-1R potency and that potency is improved when conjugated to the cyclic PYY peptide SEQ ID NO:27. From the data in Figures 5A-5B and 6A-6B, it was determined that the upper hinge of the GLP-1 fusion peptide contributes to GLP-1R potency.

[0341] Example 8: In vivo study using cynomolgus monkeys method Efficacy Testing Monkeys were selected and randomized into groups for compound treatment based on baseline (average of 3 days prior to day 0) food intake (primary, weighted 80%) and body weight (secondary, weighted 20%) measured during run-in and vehicle treatment. The study spanned 2 weeks of acclimation / training, 3 weeks of vehicle treatment every 3 days (Q3D), and 9 weeks of baseline food intake measurements, 12 days of treatment (5 times, Q3D), and 2 weeks of rest. Animals received 7 doses of vehicle and 5 doses of one of the test articles (dosing volume 0.5 mL / kg) Q3D at approximately 8 am. The doses in each group were as follows: dulaglutide (0.0125 mg / kg, n=11), compound 4 (high dose; 0.0148 mg / kg, n=11), compound 4 (low dose; 0.0074 mg / kg, n=6). Caloric intake was measured daily and body weight was measured Q3D.

[0342] Exposure-response analysis Two analytical methods were used to measure concentrations of compound 4 and dulaglutide from in vivo plasma samples from cynomolgus monkeys.

[0343] Ligand binding assay (LBA) exposure analysis of compound 4 and dulaglutide in cynomolgus monkeys was performed by a purpose-built electrochemiluminescence immunoassay (ECLIA) method using a Meso Scale Discovery (MSD) Sector Imager S600 (Meso Scale Diagnostics, Rockville, MD USA). The format for measuring active GLP-1 for both compound 4 and dulaglutide is described as follows: Analytes were captured with a GLP-1 N-terminus (7-17) specific mAb (biotin-anti-GLP1 (7-37, 7-36, amide, free NT) mAb) and detected with an anti-human Fc specific mAb (SulfoTag-R10). Raw data regression was performed in Watson LIMS™ software (Thermo Fischer Scientific, Waltham, MA USA) using a 5-parameter logistic fit with 1 / F2 standard curve weighting.

[0344] LC-MS / MS analysis of compound 4 and dulaglutide in cynomolgus monkeys was also performed by bottom-up trypsin assay for quantification of individual parts of the molecule. In the trypsin assay, analytes were purified from plasma samples using immunoaffinity capture with anti-human Fc antibody, followed by digestion with trypsin and analysis by reversed-phase LC-MS / MS on a SCIEX Triple Quad™ 5500 LC-MS / MS System (CIEX, Concord, Ontario, Canada). Stable isotope [13C6, 15N4-arginine] and [13C6, 15N2-lysine] labeled human IgG4 (Sigma, Cat. No. MSQC7) was used as an internal standard and was added at the beginning of the immunoaffinity capture step. Since the HGE peptide is essential for its activity, a peptide from the N-terminus of GLP-1, ie, HGE(HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 263), was monitored as a surrogate for molecules containing active GLP-1.

[0345] result The dual agonist according to the embodiment of the present application contains a GLP-1 fusion (GF) peptide covalently linked to a cyclic PYY peptide. The dual agonist is composed of a recombinant glucagon-like peptide-1 (GLP-1) peptide or a GLP-1 variant peptide fused to a hinge-Fc region through a first linker peptide (N-terminal linker peptide), and a cyclic PYY peptide chemically conjugated to a target cysteine ​​contained within a second linker peptide (C-terminal linker peptide) of the recombinant GLP-1 fusion (GF) peptide. Each component of the dual agonist is empirically selected based on several important parameters, including but not limited to potency at each receptor, stability, post-translational modification, conjugation product yield, and biophysical properties.

[0346] GLP-1 peptide variant SEQ ID NO:57 was selected as the GLP-1 peptide variant. GLP-1 peptide-1 variant SEQ ID NO:56 was not preferred in this experiment because it was less potent in vivo and less stable in ex vivo human plasma stability test (Figure 1A). GLP-1 peptide variant SEQ ID NO:58 unexpectedly lost GLP-1R potency when conjugated to cyclic PYY peptide (Table 11) (see, for example, GLP-1R potency of SEQ ID NO:135, 134, 146, and 176), and SEQ ID NO:238, 242, 262, 253, and 241 showed that GLP-1 fusion peptide with exendin 4 (1-39) (SEQ ID NO:58) lost or significantly reduced GLP-1R potency after conjugation with some PYY peptides. Based on these results, testing with a GLP-1 fusion peptide with exendin 4(1-39) (SEQ ID NO:58) was not preferred in this experiment. The GLP-1 peptide variant SEQ ID NO:58 further contains a NG-prone deamidation motif. SEQ ID NO:59 formed a NG-prone deamidation motif at the GLP-1 peptide / first linker peptide interface when the first amino acid in the first linker peptide was glycine. Furthermore, GF28 (SEQ ID NO:140), which contains the GLP-1 peptide SEQ ID NO:59, was less potent than dulaglutide for both % change in food intake (FI) and body weight (BW) (Figures 10A and 10B, respectively).

[0347] SEQ ID NO: 60 was selected as the first linker peptide (N-terminal linker peptide). GLP-1 fusion proteins containing the first peptide linker, SEQ ID NO: 61, had low expression and purification yields and were not preferred in this experiment for generating the desired amount of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates. First linker peptides containing AP repeats had poor yields when the second terminal peptide was a G4A repeat (see, e.g., Example 2 and Table 8) and were less stable in vivo compared to SEQ ID NO: 60 (see, e.g., Table 13). Based on these results, testing with AP repeat linkers was not preferred in this experiment. Shortening the linker resulted in reduced potency (see, e.g., SEQ ID NO: 152 in Figure 5, SEQ ID NO: 140 in Figures 10A-10B, and Figure 12, which compare the potency of GLP-1 fusion peptides containing the same GLP-1 peptide (SEQ ID NO: 58) with first linker peptides of different lengths). Therefore, based on these results, testing with shorter linkers was not preferred in this experiment. Based on the nature of the serine residue that is xylosylated, the use of serine-containing linkers was not preferred in this experiment (see, e.g., Table 12).

[0348] A GLP-1 fusion peptide, GF19 (SEQ ID NO: 131), which contained a GLP-1 variant peptide, SEQ ID NO: 57, a first linker peptide, SEQ ID NO: 60, and a hinge-Fc region peptide, SEQ ID NO: 84, showed comparable efficacy to dulaglutide in terms of both food intake (FI) and % change in body weight (BW) (see, e.g., Figures 6A and 6B, respectively).

[0349] The GLP-1 fusion peptides GF1 (SEQ ID NO:113, Compound 1), GF19 (SEQ ID NO:131), and GF24 (SEQ ID NO:136, Compound 3) all demonstrated comparable efficacy to dulaglutide for food intake and % body weight change (see, e.g., Figures 2A-2B, 6A-6B, and 3A-3B, respectively). GF1 (SEQ ID NO:113, Compound 1) and GF24 (SEQ ID NO:136, Compound 3) also demonstrated comparable efficacy to dulaglutide for glucose tolerance (Figures 4A-4B and 7A-7B).

[0350] However, when a GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate, SEQ ID NO:251, was made by conjugating a cyclic PYY peptide (SEQ ID NO:27) to a second linker peptide of GF19 (SEQ ID NO:131), SEQ ID NO:92, there was no additional pharmacological effect on food intake or % body weight change compared to dulaglutide (Figures 11A and 11B, respectively). Additional second linker peptides were tested but were not preferred in this experiment for at least the following reasons: (a) SEQ ID NO:95 had low production yields when the first linker peptide was an AP repeat, (b) SEQ ID NO:100 was cleaved during recombinant expression, (c) the hinge Fc region peptide containing the target cysteine ​​for PYY peptide conjugation at the C-terminus of the hinge Fc region peptide and without the second linker peptide was cleaved during protein expression, indicating that the second linker peptide was necessary to generate an intact protein amenable to PYY peptide conjugation, (d) the serine containing second linker peptide had the potential to be xylosylated, and (e) shorter second linker peptides were conjugated with lower purity and lower efficiency.

[0351] The GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate with the second linker peptide, SEQ ID NO: 94, SEQ ID NO: 225 (Compound 2), showed significantly higher potency than dulaglutide for both FI and % BW change (see, e.g., Figures 2A and 2B, respectively), but surprisingly resulted in significant protein loss during 2 weeks of storage at 4°C in high concentration liquid formulations. The equivalent GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate with the second linker peptide, SEQ ID NO: 229, SEQ ID NO: 93, was stable in high concentration liquid formulations during 2 weeks of storage at 4°C and 40°C, maintaining protein levels (Table 14). The second linker peptide was the only difference between these two molecules, and therefore SEQ ID NO: 93 was selected as the second linker peptide in this experiment.

[0352] [Table 14]

[0353] Several modifications are required to the functional activity of the cyclic PYY peptides to confer the necessary in vivo stability for a long half-life agent and to allow linkage to a GLP-1 fusion protein while retaining potency. Using synthetic peptide chemistry, cyclic PYY peptides incorporating these modifications were made and the cyclic PYY peptides were chemically conjugated to a GLP-1 fusion peptide to generate a GLP-1 fusion peptide-linked cyclic PYY peptide conjugate.

[0354] Cyclic PYY peptides were selected and conjugated to GLP-1 fusion peptides to investigate and compare the modification of the potency and stability of the PYY peptides. The PYY peptides were cyclized to stabilize multiple sites of proteolysis along the peptide backbone.

[0355] Cyclic PYY peptides were selected and conjugated to GLP-1 fusion peptides to investigate the spacing and size of the N-terminal to C-terminal linkage of the cyclic PYY peptides and their effect on potency and stability. These cyclic PYY peptides varied in the number of carbons in the ring and in the position of the cysteine ​​and homocysteine ​​used to selectively link the N-terminus to the C-terminus. See, for example: a) βA2 SEQ ID NO:25 linked to hCys31; b) βA2 SEQ ID NO: 28 and 29 linked to Cys30; c) G2 SEQ ID NO: 27 and 30 linked to Cys30; d) γAba2 linked to hCys31 SEQ ID NO:34.

[0356] N-Me arginine substituted at position 35 (N-MeR35) (SEQ ID NOs:27, 28, 33, and 34) and reduced amide bond between positions 35 and 36 (psi35,36) (SEQ ID NOs:24, 25, 29, and 30) were compared for their ability to stabilize an amidated C-terminal tyrosine at position 36 in cyclic PYY peptides and retain potency (see, e.g., U.S. Patent Publication No. 2018 / 0117170, which is incorporated by reference in its entirety). In certain cases, the N-Me35 modification resulted in reduced potency compared to the psi35,36 modification (see, e.g., U.S. Patent Publication No. 2018 / 0117170, Table 3, which shows that SEQ ID NO:102 with the psi35,36 modification was approximately 12-fold more potent against the human Y2 receptor than SEQ ID NO:122 with the N-Me35 modification).

[0357] Unexpectedly, the potency of the N-MeR35 modification in the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate, SEQ ID NO:229, containing the cyclic PYY peptide, SEQ ID NO:27, was equal to or greater than the psi35,36 modification of SEQ ID NO:232, containing the cyclic PYY peptide, SEQ ID NO:30 (Table 11).

[0358] Cyclic PYY peptide, SEQ ID NO:27, was selected as the PYY peptide in this experiment based on potency and stability, in addition to the fact that the combination of components was determined to reduce the cost of goods and facilitate the procurement of the reagent. The natural amino acid glycine at the N-terminus linked to a cysteine ​​at position 30 near the C-terminus had potency and stability equivalent to the highest level of potency and stability achieved for any cyclic PYY peptide tested when the cyclic PYY peptide was conjugated to a GLP-1 fusion peptide. Cysteine ​​at position 30 was chosen over homocysteine ​​at position 31 because the cyclic PYY peptide SEQ ID NO:27 had sufficient potency and stability. Thus, non-natural amino acids, which are more expensive and less readily available, were not required in this experiment. N-MeR35, used to stabilize the C-terminal amidated tyrosine at position 36, is a less expensive and more readily available reagent than the costly psi35,36 modification. This eliminates the need for special components and provides the same stability and potency.

[0359] In addition, it was determined that the PEG spacer was not necessary for efficacy, and unexpectedly, the PEG spacer was not necessary for PYY stability in vivo. This was unexpected, since it had been determined that mAb-cyclic PYY peptide conjugates with a PEG spacer had increased in vivo stability (see, e.g., US Patent Application Publication No. 2018 / 0117170, Table 4, which shows the percentage of intact compound remaining relative to the amount of total human mAb levels in blood samples from mice taken 48 hours after administration. Compound 1, containing a 12×PEG spacer, remained 90.8% at 48 hours, compound 2, containing a 6×PEG spacer, remained 65.6% at 48 hours, and compound 3, without a PEG spacer, remained only 51% at 48 hours). This eliminated the need for a PEG component, which would increase the manufacturing cost of the product.

[0360] SEQ ID NO:229, which contained the GLP-1 fusion peptide SEQ ID NO:136 with two stable cyclic PYY peptides SEQ ID NO:27 appended, was selected as the GLP-1 fusion peptide-linked cyclic PYY peptide conjugate in this experiment. SEQ ID NO:229 showed additional pharmacodynamic potency from Y2R association compared to SEQ ID NO:136 (GLP1R only association) for both FI and BW (Figures 3A-3B), along with comparable GLP1R potency to dulaglutide and SEQ ID NO:136 on glucose tolerance (Figures 4A-4B and 7A-7B).

[0361] The effects of dulaglutide and SEQ ID NO: 229 (compound 4) on FI and BW were compared in DIO mice. Based on integrated exposure-response nonlinear regression analysis of available studies (6 studies with compound 4 and 7 studies with dulaglutide; doses 0.03-1.0 nmol / kg), at doses that resulted in clinically relevant active GLP-1 (HGE) exposure on day 3 (single SC dose of 0.3 nmol / kg), compound 4 showed additional pharmacological action to dulaglutide through Y2R association with the conjugated cyclic PYY peptide in reducing FI and BW at equivalent active GGLP1 (HGE) exposure (Figures 8A-8B). At the same HGE concentration of dulaglutide at 0.3 nmol / kg (single SC dose), the calculated FI and BW % changes were approximately 2.1 and 1.5 times greater than dulaglutide, respectively, based on the exposure-response nonlinear regression with compound 4 (sequence number 229).

[0362] In overweight cynomolgus monkeys, individual PK and caloric intake-based exposure-response analyses (Figures 9A-9B) demonstrated clinically relevant dulaglutide exposure (steady-state C trough and C maxAt GLP-1 exposures of similar activity (1–2 nM [Geiser et al., Clin Pharmacokinet 55(5):(2016)]), compound 4 reduced caloric intake by approximately 1.7- to 2-fold (95% CI: 1.3-3.3, ligand binding assay [LBA]-based PK, Figure 9A) or approximately 1.5- to 1.6-fold (95% CI: 1.0-2.8, LC-MS / MS HE-based PK, Figure 9B) compared with dulaglutide, demonstrating additional pharmacological effects from the cyclic PYY peptide of compound 4.

[0363] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this description.

[0364] All documents cited are incorporated herein by reference.

[0365] Exemplary GLP-1 fused cyclic PYY peptide conjugates of the invention include:

[0366] SEQ ID NO:225 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0367] [ka]

[0368] SEQ ID NO:226 Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0369] [ka]

[0370] SEQ ID NO:227 Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30),K(Ac)11,psi-(R35,Y36)]-PYY2-36 homodimer conjugate structure:

[0371] [ka]

[0372] SEQ ID NO:228 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,psi-(R35,Y36)]-PYY2-36 homodimer conjugate structure:

[0373] [ka]

[0374] SEQ ID NO:229 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0375] [ka]

[0376] SEQ ID NO:230 Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0377] [ka]

[0378] SEQ ID NO:231 Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30),K(Ac)11,psi-(R35,Y36)]-PYY2-36 homodimer conjugate structure:

[0379] [ka]

[0380] SEQ ID NO:232 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,psi-(R35,Y36)]-PYY2-36 homodimer conjugate structure:

[0381] [ka]

[0382] SEQ ID NO:233 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0383] [ka]

[0384] SEQ ID NO:234 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0385] [ka]

[0386] SEQ ID NO:235 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0387] [ka]

[0388] SEQ ID NO:236 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0389] [ka]

[0390] SEQ ID NO:237 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0391] [ka]

[0392] SEQ ID NO:238 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0393] [ka]

[0394] SEQ ID NO:239 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0395] [ka]

[0396] SEQ ID NO:240 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0397] [ka]

[0398] SEQ ID NO:241 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0399] [ka]

[0400] SEQ ID NO:242 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0401] [ka]

[0402] SEQ ID NO:243 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0403] [ka]

[0404] SEQ ID NO:244 Name: GLP-1 fusion-[cyclo-(G2-COCH2-hC31),K(PEG24Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0405] [ka]

[0406] SEQ ID NO:245 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0407] [ka]

[0408] SEQ ID NO:246 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0409] [ka]

[0410] SEQ ID NO:247 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0411] [ka]

[0412] SEQ ID NO:248 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0413] [ka]

[0414] SEQ ID NO:249 Name: GLP-1 fusion-[cyclo-(G2-E30),S4,K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0415] [ka]

[0416] SEQ ID NO:250 Name: GLP-1 fusion-[cyclo-(G2-E30),S4,K(Ac)11,psi-(R35,Y36)]-PYY2-36 homodimer conjugate structure:

[0417] [ka]

[0418] SEQ ID NO:251 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0419] [ka]

[0420] SEQ ID NO:252 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0421] [ka]

[0422] SEQ ID NO:253 Name: GLP-1 fusion-[cyclo-(gAba2-COCH2-hC30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0423] [ka]

[0424] SEQ ID NO:254 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0425] [ka]

[0426] SEQ ID NO:255 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0427] [ka]

[0428] SEQ ID NO:256 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0429] [ka]

[0430] SEQ ID NO:257 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0431] [ka]

[0432] SEQ ID NO:258 Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0433] [ka]

[0434] SEQ ID NO:259 Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30),K(Ac)11,psi-(R35,Y36)]-PYY2-36 homodimer conjugate structure:

[0435] [ka]

[0436] SEQ ID NO:260 Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0437] [ka]

[0438] SEQ ID NO:261 Name: GLP-1 fusion - [cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 monomer conjugate structure:

[0439] [ka]

[0440] SEQ ID NO:262 Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30),K(Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0441] [ka] The following aspects may be included. [1] A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide linked to a cyclic PYY peptide, the GLP-1 fusion peptide comprising a GLP-1 peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, the first linker optionally being absent. [2] The cyclic PYY peptide is represented by formula I, or a derivative or a pharma- ceutically acceptable salt thereof:

change

change

change

change

change

change

change

change

change

change

change

change

[10] The conjugate according to any one of the above [1] to [9], wherein the GLP-1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56 to 59.

[11] The conjugate according to any one of the above [1] to

[10] , wherein the first linker peptide is present and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60 to 83.

[12] The conjugate according to any one of [1] to

[11] above, wherein the hinge-Fc region peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 90.

[13] The conjugate according to any one of [1] to

[12] above, wherein the second linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93 to 112.

[14] The conjugate according to any one of the above [1] to

[12] , wherein the second linker peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106 or 111.

[15] A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide conjugated to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274, and the cyclic PYY peptide comprises an amino acid sequence selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34, or a pharma- ceutically acceptable salt thereof.

[16] The conjugate according to

[15] above, wherein a cysteine ​​residue between amino acid residues 287 and 289 of the GLP-1 fusion peptide, preferably cysteine ​​residue 288, is covalently linked via a chemical linker to a lysine residue at residues 7, 9, 11, 22 or 23 of the cyclic PYY peptide, preferably lysine residue 11 of the cyclic PYY peptide.

[17] A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide conjugated to a cyclic PYY peptide, the conjugate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 225-262, or a pharma- ceutically acceptable salt thereof.

[18] A method for producing a conjugate according to any one of the above [1] to

[17] , comprising reacting an electrophile, preferably bromoacetamide or maleimide, introduced into a side chain of the cyclic PYY peptide, preferably into a side chain of a lysine residue of the cyclic PYY peptide, with a sulfhydryl group of a cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide, thereby generating a covalent bond between the cyclic PYY peptide and the GLP-1 fusion peptide.

[19] The method according to

[18] above, wherein the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide is reduced by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent, and the reduced cysteine ​​residue is reacted with the electrophile.

[20] The method according to

[19] above, wherein the azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) or a derivative thereof.

[21] A pharmaceutical composition comprising the conjugate according to any one of [1] to

[17] above and a pharma- ceutically acceptable carrier.

[22] A method for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and / or eczema, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described in

[21] above.

[23] A method for reducing at least one of food intake or body weight in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described in

[21] above.

[24] A method for regulating Y2 receptor activity and / or GLP-1 receptor activity in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described in

[21] above.

[25] The method according to any one of

[22] to

[24] above, wherein the pharmaceutical composition is administered by injection.

[26] A kit comprising the conjugate according to any one of the above [1] to

[17] , preferably further comprising an injection device.

[27] A method for producing a pharmaceutical composition comprising the conjugate according to any one of [1] to

[17] above, comprising combining the conjugate with a pharma- ceutically acceptable carrier to obtain the pharmaceutical composition.

Claims

1. 1. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide linked to a cyclic PYY peptide, said GLP-1 fusion peptide comprising a GLP-1 peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59, an optional first linker peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-83, a hinge-Fc region peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-90, and a second linker peptide comprising a cysteine ​​residue linked to the cyclic PYY peptide and an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-112; The cyclic PYY peptide is represented by Formula I, or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is 0 or 1 (provided that q is Z ３０ is 1 if and only if does not exist), BRIDGE is -Ph-CH ２ -S-, -triazolyl-, -NHC(O)CH ２ S-, -SCH ２ C(O)NH-, - (OCH ２ CH ２ ) ２ NHC(O)CH ２ S, -NHC(O)-, or -CH ２ S-, Z ４ is K, A, E, S, or R; Z ７ is A or K, the amino side chain of said K is optionally 【Chemistry 2】 where i is an integer from 0 to 24 and X=Br, I, or Cl. -C(O)CH ２ Br, -C(O)CH ２ I, or -C(O)CH ２ Cl is acylated, Z ９ is G or K, the amino side chain of said K is optionally 【Chemistry 3】 where i is an integer from 0 to 24 and X=Br, I, or Cl. -C(O)CH ２ Br, -C(O)CH ２ I, or -C(O)CH ２ Cl is acylated, Z １１ is D or K, the amino side chain of said K is optionally 【Chemistry 4】 where i is an integer from 0 to 24 and X=Br, I, or Cl. -C(O)CH ２ Br, -C(O)CH ２ I, or -C(O)CH ２ Cl is acylated, Z ２２ is A or K, the amino side chain of said K is optionally 【Chemistry 5】 where i is an integer from 0 to 24 and X=Br, I, or Cl. -C(O)CH ２ Br, -C(O)CH ２ I, or -C(O)CH ２ Cl is acylated, Z ２３ is S or K, the amino side chain of said K is optionally 【Chemistry 6】 where i is an integer from 0 to 24 and X=Br, I, or Cl. -C(O)CH ２ Br, -C(O)CH ２ I, or -C(O)CH ２ Cl is acylated, Z ２６ is A or H, Z ３０ is L, W or absent; (However, Z ３０ does not exist if and only if q is 1), Z ３４ teeth, 【Chemistry 7】 and Z ３５ teeth, 【Chemistry 8】 and Formula I is Z ７ , Z ９ , Z １１ , Z ２２ , or Z ２３ wherein at least one of said acylated lysine (K) residues is covalently and reactively conjugated to said cysteine ​​residue of said second linker peptide.

2. The cyclic PYY peptide is represented by Formula I, or a pharma- ceutically acceptable salt thereof: During the ceremony, p is 0 or 1; m is 0, 1, 2, 3, or 5; n is 1, 2, or 4; q is 0 or 1 (provided that q is Z ３０ can be 1 if and only if BRIDGE is -Ph-CH ２ -S-, -triazolyl-, -NHC(O)CH ２ S-, -(OCH ２ CH ２ ) ２ NHC(O)CH ２ S, -NHC(O)-, or -CH ２ S-, Z ４ is K, A, E, S, or R; Z ７ is A or K, and the amino side chain of K is -C(O)CH ２ is optionally acylated with Br; Z ９ is G or K, and the amino side chain of K is -C(O)CH ２ is optionally acylated with Br; Z １１ is D or K, and the amino side chain of K is -C(O)CH ２ is optionally acylated with Br; Z ２２ is A or K, and the amino side chain of K is -C(O)CH ２ is optionally acylated with Br; Z ２３ is S or K, and the amino side chain of K is -C(O)CH ２ is optionally acylated with Br; Z ２６ is A or H, Z ３０ is L, Z ３４ teeth, 【Chemistry 9】 and Z ３５ teeth, 【Chemistry 10】 The conjugate of claim 1 ,

3. 2. The conjugate of claim 1, wherein the cyclic PYY peptide is selected from the group consisting of SEQ ID NOs: 1-54, or a pharma- ceutically acceptable salt thereof.

4. 2. The conjugate of claim 1, wherein the cyclic PYY peptide is selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33, or 34, or a pharma- ceutically acceptable salt thereof.

5. Z in formula I １１ The conjugate of claim 1 , wherein is lysine.

6. The conjugate according to any one of claims 1 to 5, wherein the second linker peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106 or 111.

7. 1. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide linked to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274, and the cyclic PYY peptide comprises an amino acid sequence selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34, or a pharma- ceutically acceptable salt thereof, and wherein a cysteine ​​residue between amino acid residues 287-289 of the GLP-1 fusion peptide is covalently linked via a chemical linker to a lysine residue at amino acid residue 11 (K11) of the cyclic PYY peptide.

8. 1. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide (GF) linked to a cyclic PYY peptide, the conjugate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 225-262, or a pharma- ceutically acceptable salt thereof, wherein the GLP-1 fusion peptide comprises a GLP-1 peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59, an optional first linker peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-83, a hinge-Fc region peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-90, and a second linker peptide comprising a cysteine ​​residue linked to the cyclic PYY peptide and an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-112.

9. 7. A method for producing a conjugate according to any one of claims 1 to 6, comprising reacting an electrophile introduced into the side chain of a lysine residue of the cyclic PYY peptide with a sulfhydryl group of a cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide, thereby generating a covalent linkage between the cyclic PYY peptide and the GLP-1 fusion peptide.

10. 10. The method of claim 9, wherein the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide is reduced by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent, and reacting the reduced cysteine ​​residue with the electrophile.

11. 11. The method of claim 10, wherein the azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) or a derivative thereof.

12. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 8 and a pharma- ceutically acceptable carrier.

13. 13. The pharmaceutical composition of claim 12, the pharmaceutical composition is for use in a method for treating or preventing a disease or disorder in a subject in need thereof; the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and / or eczema, The method comprises administering to a subject in need thereof an effective amount of the pharmaceutical composition.

14. 13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is for use in a method of reducing at least one of food intake or body weight in a subject in need thereof; The method comprises administering to a subject in need thereof an effective amount of the pharmaceutical composition.

15. 13. The pharmaceutical composition of claim 12, the pharmaceutical composition is for use in a method of modulating Y2 receptor activity and / or GLP-1 receptor activity in a subject in need thereof; The method comprises administering to a subject in need thereof an effective amount of the pharmaceutical composition.

16. The pharmaceutical composition according to any one of claims 13 to 15, wherein the pharmaceutical composition is administered by injection.

17. A kit comprising the conjugate of any one of claims 1 to 8, further comprising an injection device.

18. 9. A method of producing a pharmaceutical composition comprising a conjugate according to any one of claims 1 to 8, comprising combining said conjugate with a pharma- ceutically acceptable carrier to obtain said pharmaceutical composition.

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