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

Abstract

To provide novel glucagon like peptide-1 (GLP-1) fusion peptide coupled cyclic peptide tyrosine tyrosine (PYY) conjugates, serving as modulators of the neuropeptide Y2 receptor and the GLP-1 receptor, which are useful for preventing, treating or ameliorating diseases and disorders, such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance, and dyslipidemia.SOLUTION: A conjugate comprises a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises a GLP-1 peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, wherein the first linker is optionally missing.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of International Application No. PCT / US2018 / 02, filed April 25, 2018. No. 9284 and U.S. Provisional Patent Application No. 62 / 662,31 filed April 25, 2018 No. 6,293,793, the disclosures of which are incorporated herein by reference in their entireties. be absorbed.

[0002] FIELD OF THE INVENTION The present invention generally relates to modulators of the neuropeptide Y2 receptor and the GLP-1 receptor, A novel glucagon-like peptide-1 (GLP-1) fusion peptide conjugated cyclic peptide tyrosine The present invention also relates to pharmaceutical compositions and uses thereof. The novel GLP-1 fusion peptide-coupled cyclic PYY conjugates are, inter alia, , obesity, type 2 diabetes, metabolic syndrome, insulin resistance, and dyslipidemia These compounds are useful in preventing, treating, or ameliorating any of the diseases and disorders.

[0003] (Reference to electronically submitted sequence listing) This application is based on the AS filed under "PRD3465 Sequence Listing". Submitted electronically via EFS-Web as a CII format sequence listing, April 2019 It contains a sequence listing with a size of 409kb, created on the 2nd. The sequence listing provided is a part of the present specification and is incorporated herein by reference in its entirety. The information provided herein and the filename "PRD3465 Sequence L Sequence IDs between the sequence listing submitted electronically via EFS-Web and the In the event of any discrepancy regarding the structure of 225-262, the information herein shall prevail. do. [Background technology]

[0004] Neuropeptide Y (NPY) receptors have different affinities for each receptor subtype. Activated by a group of closely related peptide agonists called the "NPY family" NPY, peptide tyrosine-tyrosine (PYY), and pancreatic polypeptide (P P), all 36 amino acids in length, are agonists of the NPY receptor family. PY is a neurotransmitter that is synthesized, co-stored, and acted upon by norepinephrine. NPY is released along with epinephrine and steroid hormones in the central nervous system (CNS) of humans and rodents. It is one of the most abundant and widely distributed peptides in the brain, and is involved in feeding and stress. In the peripheral nervous system, NPY-containing neurons are primarily sympathetic PYY is synthesized and released primarily by enteroendocrine cells. The enzyme dipeptidyl peptidase IV (DPP-IV) inhibits NPY and PYY By cleavage, it is a selective ligand for the Y2 and Y5 subtypes of the NPY receptor family. NPY 3-36 and PYY 3-36 PP is mainly used to produce insulin, glucan, and It is found in pancreatic islet cells that are different from those that store gonadotropin, or somatostatin.

[0005] Five different NPY receptors have been identified to date, four of which are present in human biology. Receptors Y1, Y2, and Y5 are understood to be involved in the physiological function of NPY and P YY receptors bind preferentially to YY, while Y4 receptors bind preferentially to PP. Y2 and Y5 receptors The body also uses NPY 3-36 and PYY 3-36 Generally, N Ligands of the PY family have variable selectivity for each of the NPY receptor isoforms. , PYY 3-36 has previously been shown to have moderate to strong selectivity for the Y2 isoform. Each of these receptors mediates adenosine triphosphate (ATT) signaling via pertussis toxin-sensitive Gαi receptors. It is linked to the inhibition of acetylcholine cyclase.

[0006] PYY is secreted from endocrine L-cells in response to food, particularly after fat ingestion. . PYY 1-36 is predominant in the fasting state, and PYY 3-36 is seen in humans after eating It is the primary form of serotonin consumed, and plasma concentrations are inversely correlated with the number of calories consumed. Y 3-36 reduces food intake in humans, monkeys, rats, rabbits, and mice It has been demonstrated that (Batterham et al., Nature 418(6 898):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)). PYY 3-36 The anorectic effect of is thought to be due to preferential binding and Based on the loss of feeding efficacy in Y2-deficient mice, this is thought to be mediated by Y2. (Batterham et al., Nature 418(6898):650- 4(2002)). PYY 3-36 Intrafornix injection of α-glucan significantly increased food intake in rats and mice. The intake decreased (Batterham et al., Nature 418(6898): 650-4 (2002)), which suggests that hypothalamic Y2 receptor engagement mediates these effects. The acute effects on feeding were observed in ob / ob mice, The dose-dependent effect on the body weight of DIO mice and Zucker fa / fa mice was observed. It has also been shown that the α-glucan is converted into α-glucan (Pittner et al., Int J Obes Relat Metab Disord 28(8):963-71(2004)). Canada And PYY 3-36 inhibits insulin-mediated glucose disposal and insulin secretion in DIO rodents It has also been shown to improve insulin sensitivity (Vrang et al., Am J Physiol Regul Integr Comp Physiol 291(2) :R367-75(2006)). Bariatric surgery results in increased circulating PYY immunoreactivity (l e Roux et al., Ann Surg 243(1):108-14(200 6)), which appears to play a role in postoperative weight loss.

[0007] Its role in the regulation of appetite and food intake, and its antagonists in the mammalian gastrointestinal tract Considering the secretion and absorption promoting effects, PYY 3-36 obesity and related conditions, and many However, PYY may be effective in treating gastrointestinal disorders. 3-36 as a therapeutic agent The therapeutic utility of is limited by its rapid metabolism and resulting short circulatory half-life. (Torang et al.,Am.J.Physiol.Regul.Inte gr.Comp.Physiol.310:R866-R874(2016)).

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

[0009] Therefore, PYY 3-36 and / or improved metabolic stability compared to GLP-1 PYY analogs or derivatives thereof and / or G having qualitative and pharmacokinetic profiles It is desirable to obtain an LP-1 analog or derivative thereof that has a long in vivo half-life. Such derivatives may modulate the Y2 and / or GLP-1 receptors with a longer duration of action. whereby the derivatives can be used as therapeutic agents in subjects in need of such modulation. It will be suitable.

[0010] The foregoing discussion is presented merely to provide a better understanding of the nature of the problems facing the art. is presented for the purpose of this application and should not be construed as an admission of prior art in any way. Citation of any reference herein without prior written consent is to be construed as an admission that such reference is a "prior art" of this application. This document should not be construed as an admission that any technology constitutes a "technology." Summary of the Invention [Means for solving the problem]

[0011] In one general aspect, the present invention provides a method for modulating the neuropeptide Y2 receptor and the GLP-1 receptor. A novel glucagon-like peptide-1 (GLP-1) fused cyclic peptide tyrosine conjugate is a factor Concerning tyrosine (PYY) conjugates.

[0012] Glucagon-like peptide 1 (GLP-1) fusion peptide linked to a cyclic PYY peptide A conjugate comprising the GLP-1 fusion peptide, A first variant peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide. a conjugate comprising a linker peptide, the first linker being optionally absent; The present invention provides a method for the preparation of a medicament for the treatment of malaria.

[0013] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative thereof: or a pharmaceutically 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 (provided that 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- and 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 derivative may be one or more selected from the group consisting of amidation, acylation, and PEGylation. Compounds of formula I that have been modified by more than one process.

[0017] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative thereof: or a pharmaceutically 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 (provided that 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- and Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of said K is optionally

[0018] [ka] (wherein 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, and the amino side chain of said K is optionally

[0019] [ka] (wherein 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, and the amino side chain of said K is optionally

[0020] [ka] (wherein 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, and the amino side chain of K is optionally

[0021] [ka] (wherein 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, and the amino side chain of said K is optionally

[0022] [ka] (wherein 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, Z34 teeth,

[0023] [ka] and Z 35 teeth,

[0024] [ka] is.

[0025] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative thereof: or a pharmaceutically 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 (provided that 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 - and Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of said K is is substituted with -C(O)CHBr, Z9 is G or K, and the amino side chain of the K is is substituted with -C(O)CHBr, Z 11 is D or K, and the amino side chain of K is is substituted with -C(O)CHBr, Z 22 is A or K, and the amino side chain of K is is substituted with -C(O)CHBr, Z 23 is S or K, and the amino side chain of K is is substituted with -C(O)CHBr, Z 26 is A or H, Z 30 is L, Z 34 teeth,

[0026] [ka] and Z 35 teeth,

[0027] [ka] is.

[0028] In certain embodiments, the cyclic PYY peptide is selected from the group consisting of SEQ ID NOs: 1-54. or a pharmaceutically acceptable salt thereof. In certain embodiments, cyclic PYY peptides The code 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 substituted with a lysine residue of the cyclic PYY peptide. In certain embodiments, the cyclic PYY peptide is covalently linked to the cyclic PYY peptide. Z7, Z9, Z 11 , Z 22 , and Z 23 Only one of the ricin is ricin, is covalently linked to a cysteine residue in the second linker peptide of the GLP-1 fusion peptide. They are connected together.

[0030] In a particular embodiment, the GLP-1 peptide of the GLP-1 fusion peptide is SEQ ID NO: 56 In certain embodiments, the amino acid sequence comprises an amino acid sequence selected from the group consisting of GLP-1 The first linker peptide of the fusion peptide is selected from the group consisting of SEQ ID NOs: 60 to 83. In a specific embodiment, the hinge-Fc region of the GLP-1 fusion peptide comprises an amino acid sequence The specific peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 90. In this embodiment, the second linker peptide of the GLP-1 fusion peptide is selected from the group consisting of SEQ ID NOs: 91 to 96. 112. In certain embodiments, the second phosphorylase comprises an amino acid sequence selected from the group consisting of: The car peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106, or 111. .

[0031] and glucagon-like peptide 1 (GLP-1) fusions conjugated to cyclic PYY peptides. A conjugate comprising a peptide, wherein the GLP-1 fusion peptide is represented by SEQ ID NO: 11. the cyclic amino acid sequence selected from the group consisting of 3 to 224 and 267 to 274; The PYY peptide is selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34. Also provided are conjugates comprising the selected amino acid sequence. The LP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 136. In certain embodiments, the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 136 is selected from amino acid residues 287 to 289. a cysteine residue between, preferably cysteine residue 2 of SEQ ID NO: 113 or SEQ ID NO: 136 88 is a lysine residue at residue 7, 9, 11, 22, or 23 of a cyclic PYY peptide; Preferably, the cyclic PYY peptide is covalently attached to lysine residue 11 either directly or via a chemical linker. They are connected together.

[0032] and glucagon-like peptide 1 (GLP-1) fusions conjugated to cyclic PYY peptides. A conjugate comprising a peptide selected from the group consisting of SEQ ID NOs: 225 to 262. Also provided are conjugates comprising the sequence or a pharmaceutically acceptable salt thereof.

[0033] Also provided are methods for producing the conjugates of the invention, the methods comprising: an electrophile introduced into the side chain of a lysine residue of a cyclic PYY peptide, preferably Preferably, bromoacetamide or maleimide is attached to the second phosphoryl group of the GLP-1 fusion peptide. The sulfhydryl group of a cysteine residue in a linker peptide (e.g., a carboxy-terminal linker peptide) and reacting the cyclic PYY peptide with the hydroxyl group, thereby forming a cyclic PYY peptide and a GLP-1 fusion peptide. This involves creating a covalent bond between the

[0034] Also provided is a pharmaceutical composition comprising a conjugate of the invention and a pharmaceutically acceptable carrier. It is served.

[0035] Also, 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, Congenital hyperinsulinism (CHI) causes hypoglycemia, dyslipidemia, atherosclerosis, and diabetes. Diabetic nephropathy and other conditions associated with uncontrolled cholesterol and / or lipid levels Other cardiovascular risk factors, such as hypertension and cardiovascular risk factors, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and and / or eczema. The method comprises administering to a subject in need thereof an effective The method comprises administering an amount of a pharmaceutical composition of the present invention.

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

[0037] Also, Y2 receptor activity or GLP-1 receptor activity in a subject in need thereof. Also provided is a method for regulating the activity of a steroid hormone, comprising administering to a subject in need thereof an effective amount of the compound of the present invention. The method includes administering the pharmaceutical composition of the present invention.

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

[0039] Also provided is a kit comprising the compound of the present invention, preferably liraglutide and an injectable device. A kit is also provided, further comprising a chair.

[0040] Also provided is a method for producing the pharmaceutical composition of the invention, the method comprising: and combining it with a physiologically acceptable carrier to obtain a pharmaceutical composition.

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

[0042] The above summary and the following detailed description of preferred embodiments of the present application should be taken in conjunction with the accompanying drawings. However, the present application will be better understood by reading the accompanying drawings. It should be understood that the present invention is not limited to the precise embodiment. [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 referenced in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which is included in the specification is indicative of the context of this invention. Such discussion is intended to provide a basis for understanding whether any or all of these matters are disclosed or implied. is admitted to constitute prior art to any claimed invention. isn't it.

[0044] Unless otherwise defined, all technical and scientific terms used herein refer to the It has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. Certain terms used herein have the meanings set forth herein.

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

[0046] Unless otherwise specified, any numerical values, such as concentrations or concentration ranges, described herein are In all cases, the terms "about" and "about" should be understood as being modified. Therefore, numerical values typically include ±10% of the stated value. For example, A concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, 1% to 10 The concentration range of % (w / v) includes 0.9% (w / v) to 11% (w / v). When used, the use of a numerical range shall be interpreted as meaning that the range is true unless the context clearly indicates otherwise. All possible subranges, including integers and fractions of values, and all individual numbers within that range Explicitly included.

[0047] Unless otherwise stated, the term "at least" preceding a series of elements refers to every element in the series. It should be understood that the present invention refers to elements of the present invention. The reader will recognize many equivalents to the specific embodiments described herein, or Such equivalents are intended to be encompassed by this invention. can be.

[0048] As used herein, the terms "comprises" and "comprising" "includes," "including," "has," "having" "ing," "contains," or "containing" or any of these Any other variations are intended to include the stated integer or groups of integers, but this It does not exclude any other integer or group of integers other than these, and is non-exclusive or non-limiting. It will be understood that the term "composition, mixture, process" is intended to include, for example, a series of elements. The terms "method, article, or apparatus" are not necessarily limited to only those elements, and should not be construed as limiting the scope of the invention. Any composition, mixture, process, method, article, or apparatus not specifically listed or related to such It may contain other elements not inherently present. Furthermore, unless expressly stated to the contrary, "is" refers to an inclusive "or" and not an exclusive "or." For example, For example, condition A or B is true if A is true (or exists) and B is false (or does not exist). ), if A is false (or does not exist) and B is true (or exists), then If both A and B are true (or exist), then it is satisfied by one of .

[0049] The term "about" as used herein when referring to dimensions or characteristics of components of the preferred invention The terms "approximately," "generally," "substantially," and the like, as will be understood by those skilled in the art, are intended to The dimensions / features listed are not strict boundaries or parameters and are functionally the same or similar. It should also be understood that this does not exclude slight variations therefrom. Such references, including value parameters, are based on mathematical formulas accepted in the art. and using industrial principles (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, etc.) The least significant figure will then include the variant that does not change.

[0050] Two or more nucleic acid or polypeptide sequences (e.g., a GLP-1 peptide, a linker peptide) PYY, hinge-Fc region peptide, cyclic PYY 3-36 peptide sequence) The terms "percent identity" or "identity" are used in conjunction with one of the following sequence comparison algorithms: or by visual inspection using methods known in the art in light of this disclosure. Therefore, when compared and aligned for maximum agreement, or two that have a specified percentage of identical amino acid residues or nucleotides. It refers to the above sequence or subsequence.

[0051] For sequence comparison, typically one sequence serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer If necessary, subsequence coordinates are specified and the sequence algorithm program parameters are entered. The sequence comparison algorithm is then run using the designated program parameters. Based on the data, the percent sequence identity of the test sequence relative to the reference sequence is calculated.

[0052] Optimal alignment of sequences for comparison can be performed using, for example, Smith & Waterman's n, Adv. Appl. Math. 2:482 (1981) local homology algorithm; Needleman & Wunsch, J. Mol. Biol. 48:443(197 0) Homology Alignment Algorithm, Pearson & Lipman, Proc. Similarity search method in Nat'l Acad Sci USA 85:2444(1988) , computerized implementations of these algorithms (Wisconsin Genet ics Software Package,Genetics Computer G GAP at 575 Science Dr., Madison, WI ESTFIT, FASTA, and TFASTA), or visual inspection (generally nt Protocols in Molecular Biology,FMAu Subel et al., eds., Current Protocols, a jo int venture between Greene Publishing As sociates,Inc.and John Wiley & Sons,Inc., (1995 Supplement) (see Ausubel) It is possible.

[0053] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is BLA BLAST and BLAST 2.0 algorithms, respectively. et al., J. Mol. Biol. 215:403-410 (1990) and Al tschul et al., Nucleic Acids Res.25:3389- 3402 (1997). The software for BLAST analysis is available from the National Institute of Biological Sciences. Publicly available through the National Center for Biotechnology Information is available at

[0054] Further indications that two nucleic acid sequences or polypeptides are substantially identical include the following: As described above, the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid. Therefore, the polypeptide is immunologically cross-reactive with the polypeptide to be administered. A peptide is typically a second peptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two polypeptides The ability of two molecules to hybridize to each other under stringent conditions.

[0055] As used herein, a "subject" refers to any animal, preferably a mammal, most preferably a As used herein, the term "mammal" refers to any mammalian animal or human. Examples of mammals include, but are not limited to, cows, Ma, sheep, pig, cat, dog, mouse, rat, rabbit, guinea pig, monkey, human, etc. and more preferably humans.

[0056] The term "administering" with respect to the methods of the present invention refers to administering a conjugate or compound of the present invention, or The use of the formulations, compositions, or medicaments may be used to treat the syndromes, disorders, or conditions described herein. or a method of therapeutically or prophylactically preventing, treating, or ameliorating a disease. Such methods include administering an effective amount of the conjugate, compound, form, composition, or This includes administering drugs in combination form, at different times during the course of treatment, or simultaneously. The methods of the present invention are understood to encompass all known therapeutic treatment regimens. That is why.

[0057] The term "effective amount" refers to the biologically effective amount sought by a researcher, veterinarian, physician, or other clinician. eliciting a therapeutic or medical response in a tissue system, animal, or human (the syndrome, disorder being treated) or to prevent or treat the disease or symptoms of the syndrome, disorder or disease being treated. "Among the active conjugates, compounds, or agents that are capable of inhibiting or ameliorating the effects of a disease, including inflammatory bowel disease, is the amount of active conjugate, compound, or agent that inhibits or ameliorates the effects of a disease, including inflammatory bowel ...

[0058] As used herein, the term "composition" refers to a product containing specified ingredients in specified amounts, as well as and any product resulting directly or indirectly from the combination of specific ingredients in specific amounts. This includes:

[0059] As used herein, the term "conjugated" refers to the joining or bonding of two or more entities together. When referring to chemical or biological compounds, conjugated / bonded , may refer to a covalent bond between two or more chemical or biological compounds. As a typical example, the glucagon-like peptide-1 (GLP-1) fusion peptide of the present invention can be used as a target The GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugate is then combined with a cyclic PYY peptide. In certain embodiments, the GLP-1 fusion peptides of the present invention can form conjugates. The peptide is covalently linked to a cyclic PYY peptide of the invention through at least one linker. The GLP-1 fusion peptide-linked cyclic PYY peptide conjugate can be used in combination with GLP-1. A specific engineered fusion peptide conjugated to a cyclic PYY peptide These can be formed through chemical reactions. For example, GLP-1 fusion peptide conjugated cyclic PYY peptides can be used. The tide conjugates can be formed through a conjugation reaction. Conjugation reactions can be carried out, for example, by coupling electrophilic groups (e.g., bromoacetamide or maleimide) The sulphuric acid is added to the cysteine residue in the peptide of interest (e.g., a GLP-1 fusion peptide). The electrophilic group may include reacting with a hydryl group of, for example, a cyclic PYY peptide. It can be introduced into the side chain of an amino acid residue by reaction of an electrophilic group with a sulfhydryl group. This results in the formation of a covalent thioether bond.

[0060] As used herein, the term "peptide linker" or "linker peptide" refers to a peptide having a G The LP-1 peptide is linked to a hinge-Fc region peptide or The peptide is linked to a cyclic PYY peptide to form a GLP-1 fusion peptide-conjugated cyclic PYY peptide. Chemical modules containing one or more amino acids that form tidoconjugates The GLP-1 fusion peptide may be, for example, a first linker peptide and a second linker peptide. The peptide may be included.

[0061] As used herein, the term "chemical linker" refers to a linker that connects a cyclic PYY peptide to GLP-1. It refers to a chemical module that does not contain any amino acids to be linked to a fusion peptide. In some embodiments, the cyclic PYY peptides include a chemical linker. Examples of the chemical linker include a hydrocarbon linker, a polyethylene glycol (PEG) linker, and the like. Linker, Polypropylene Glycol (PPG) Linker, Polysaccharide Linker, Polyester A hybrid consisting of a linker, an acyl group-containing linker, PEG, and an embedded heterocycle. These include, but are not limited to, lid linkers, as well as hydrocarbon chains. The chemical linker may, for example, first be covalently attached to a cyclic PYY peptide and then attached to a GLP -1 fusion peptide, preferably a GLP-1 fusion peptide, covalently linked to a second linker peptide They may be connected by bonds.

[0062] As used herein, the term "conjugate" refers to a compound that is bonded to another pharmaceutically active moiety, e.g., Peptides (e.g., GLP-1 fusion peptides) covalently linked to cyclic PYY peptides The term "conjugated" refers to a peptide of the present invention that is directly or Indirectly, via a linker, covalently attached to another pharmaceutically active moiety, preferably a therapeutic peptide As a non-limiting example, a peptide may be linked or covalently connected by The other pharmaceutically active moiety may be a GLP-1 fusion peptide of the present invention, and the cyclic PY It may be a therapeutic peptide such as the Y peptide.

[0063] The peptide sequences described herein are written according to the usual convention and include the N-terminal region of the peptide. The C-terminal region is on the left and the C-terminal region is on the right. The isomeric forms of the amino acids are known, but are not shown separately. Unless explicitly stated, 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 invention provides glucagon-like peptide 1 (GLP-1) fusion peptides. The GLP-1 fusion peptide is a GLP-1 or GLP-1 variant peptide, linker peptides (e.g., amino (N)-terminal linkers), hinge-Fc region peptides, and a second linker peptide (eg, a carboxy (C)-terminal linker).

[0065] Glucagon-like peptide-1 or GLP-1 variant peptides Glucagon-like peptide 1 (GLP-1) is synthesized in the intestine and released in response to food intake. It is an insulin secretagogue that is mainly produced by GLP-1-(7-37) and G LP-1-(7-36)NH2 is secreted in two forms, both of which are expressed on pancreatic β cells. It binds to the specific GLP-1 receptor (GLP-1R) of To enhance.

[0066] Many GLP-1 analogs and derivatives are known and are referred to herein as "GLP-1 variants." These GLP-1 variant peptides are sometimes called "Gila monster" peptides. These exendins are peptides found in natural It has sequence homology with GLP-1, binds to the GLP-1 receptor, and binds to the GLP-1(7- 37) can initiate a signaling cascade response to activity caused by the

[0067] GLP-1 and GLP-1 variant peptides have been shown to act in a variety of ways. These include stimulating insulin release, reducing glucagon secretion, inhibiting gastric emptying, and These may include, but are not limited to, increased course utilization.

[0068] GLP-1R is a seven-transmembrane heterotrimeric G protein-coupled receptor of the class B family. They belong to the α-, β-, and δ-cells of the pancreatic islets, the heart, kidneys, stomach, intestines, and the ganglionic nerves of the vagus nerve. It includes the nervous system, the nervous system, and several regions of the central nervous system (CNS), including the hypothalamus and brainstem. GLP-1R is expressed in a wide range of tissues, including but not limited to intracellular calcium, adenosine triphosphate (AGT), and thrombopoietin (TH). Increases phospholipase C, PKA, PKC, PI-3K, and Ep ac2 and Gα, which activates the MAPK signaling pathway s , Gα q , Gα i , and G α o (Montrose-Rafizadeh et al., Endocrinolo Gy 140:1132-40(1999);Hallbrink et al.,Bi ochim Biophys Acta 1546:79-86(2001)) (Drucker et al., PNAS 84:3434-8 (1987); W heeler et al., Endrocrinology 133:57-62(1 993); and Holz et al., JBC 270:17749-57 (1995 )).

[0069] A GLP-1 fusion peptide comprising a first component, wherein the first component is GLP Provided herein are fusion peptides which are GLP-1 or GLP-1 variant peptides. As used herein, the terms "GLP-1 peptide," "GLP-1 variant peptide," and " "GLP-1 peptide variant" and "GLP-1 or GLP-1 variant peptide" are interchangeable terms The GLP-1 or GLP-1 variant peptides may be of the sequence provided in Table 1. The GLP-1 or GLP-1 variant peptide sequence may comprise one of the following criteria: (i) Expression yield, (ii) In vitro stability, (iii) In vitro potency, (iv) Cyclicity (v) retention of in vitro potency after chemical conjugation with PYY peptides; (vi) the lack of potential for silyl or serine xylosylation; and (vi) the GLP-1 fusion peptide. The properties of the deconjugated cyclic PYY peptide conjugates (e.g., in vivo stability and in vivo Potency (i.e., the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide Whether or not they can have agonist activity against GLP-1 and Y2 receptors, respectively. The selection may be based on at least one of the following:

[0070] GLP-1 or a GLP-1 variant constituting the first component of the GLP-1 fusion peptide The peptides include peptides having sufficient homology and functionality to natural GLP-1. It is intended that GLP-1 or GLP-1 variant peptides act in the beta-cells of the pancreas. It is designed to be able to bind to the GLP-1 receptor, thereby mimicking the natural GLP-1 The same signaling pathway occurs when GLP-1 binds to the GLP-1 receptor on the beta cells of the pancreas, They exhibit the same or similar insulinotropic activity.

[0071] [Table 1]

[0072] First linker peptide: amino-terminal linker (N-terminal linker) A GLP-1 fusion peptide comprising a second component, wherein the second component is a first a fusion peptide that is a linker peptide (i.e., an amino-terminal linker peptide), The first linker peptide has one of the sequences provided in Table 2. The first linker peptide sequence may be selected based on the following criteria: (i) expression yield, (ii) (iii) in vitro potency; (iv) serine xylosylation or serine xylosylation; (v) lack of silosylation potential, and (v) GLP-1 fusion peptide-conjugated cyclic PYY peptides The properties of the conjugate (e.g., in vivo stability and in vivo efficacy (i.e., GLP- 1 or GLP-1 variant peptides and cyclic PYY peptides are GLP-1 and At least one of the following is true: whether or not the compound has agonist activity against the Y2 receptor The selection may be based on one of the following:

[0073] In certain embodiments, the GLP-1 fusion peptide comprises a first linker peptide, a second linker peptide, and a does not contain the components of

[0074] [Table 2]

[0075] Hinge-Fc region peptide In certain embodiments, the GLP-1 fusion peptide comprises a third component, The component is a hinge-Fc region peptide. The hinge-Fc region peptide binds to FcRn In addition to receptor regeneration, increased molecular weight and reduced glomerular filtration may also contribute to the release of therapeutic peptides into the circulation. In certain embodiments, the hinge-Fc region peptide may have a longer half-life. The human IgG4 Fc region may be derived from a human IgG4 Fc region. Compared to the G subtype, it has a reduced ability to bind to FcγR and complement factors. Alternatively, the Fc region is a human IgG4 Fc region with substitutions that eliminate effector function. Thus, the GLP-1 fusion peptide contains one or two of the following substitutions: and further comprising an Fc region having a modified human IgG4 Fc region containing residue 2 or more. Proline was used in place of glutamic acid at residue 33, and phenylalanine at residue 234. alanine or valine instead of thiamin, and leucine at residue 235 Alanine or glutamic acid was used (EU numbering, Kabat et al., Sequ ences of Proteins of Immunological Inter est,5th Ed.USDept.of Health and Human Services,Bethesda,Md.,NIH Publication no. 91-3242 (1991)). At residue 297 (EU numbering) Asn is substituted. N-linked glycosylation sites in the IgG4 Fc region are eliminated by using Ala at the Removal of ATP is another way to ensure that residual effector activity is eliminated. .

[0076] In certain embodiments, the GLP-1 fusion peptides of the present invention exist as monomers or dimers. In a preferred embodiment, the GLP-1 fusion peptide exists as a dimer. In certain embodiments, the GLP-1 fusion peptide exists as a dimer, and the dimer is a homodimer. The dimer is a dimer, i.e., the dimer is composed of two GLP-1 fusion peptides with the same sequence. In certain embodiments, the GLP-1 fusion peptide exists as a dimer, and the dimer The dimer is a heterodimer, i.e., it is composed of two GLP- Contains 1 fusion peptide.

[0077] Preferably, the GLP-1 fusion peptides of the present invention are bonded via disulfide bonds and various non-covalent bonds. They exist as dimers joined together by binding interactions. The Fc portion useful to the human body stabilizes heavy chain dimer formation and prevents the formation of half IgG4 Fc chains. containing a substitution such as serine for proline at position 228 (EU numbering) In certain embodiments, the N-terminal end of the hinge disulfide may be a human IgG4 Fc region. The termini may contain naturally occurring sequences of amino acids. The x-ray crystal structure of human IgG4 shows these amino acids. It has been shown that amino acids can form structures that tend to reverse the orientation of upstream structures in space. This tendency suggests that the spatial location of upstream elements such as the GLP-1 fusion peptide In another embodiment, the amino acid sequence of the N-terminal hinge disulfide is advantageous for maintaining the structural separation. Natural amino acids may be omitted.

[0078] In a specific embodiment, a hybrid human IgG2a human fused to a human PAA Fc is The effect of GLP-1 fusion peptides on potency and stability was investigated using a range of

[0079] In another embodiment, the C-terminal Lys residue of the heavy chain is a recombinantly produced monoclonal antibody. This is typically seen with monoclonal antibodies.

[0080] The hinge-Fc region peptide may comprise one of the sequences provided in Table 3. - The Fc region peptide sequence was determined based on the following criteria: (i) in vitro stability, (ii) in vitro (iii) Properties of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates (e.g., in vivo stability and in vivo efficacy (i.e., GLP-1 or GLP-1 variants) The cyclic PYY peptides and cyclic PYY peptides act as receptors for GLP-1 and Y2 receptors, respectively. Selection based on at least one of the following: You may.

[0081] [Table 3]

[0082] a hinge-Fc region platform peptide, the hinge-Fc region peptide; a first linker peptide connected to the amino terminus of the hinge-Fc region peptide and the A second linker peptide is attached to the carboxy terminus of the hinge-Fc region peptide. and at least one of the following. The hinge-Fc region peptide is, for example, 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 SEQ ID NO: 60 to 83, and the second linker peptide The code may be, for example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 112. In certain embodiments, the hinge-Fc region peptide comprises SEQ ID NO: 84 and the first linker The anchor peptide comprises SEQ ID NO: 60. In certain embodiments, the hinge-Fc region peptide The first linker peptide comprises SEQ ID NO: 84, and the second linker peptide comprises SEQ ID NO: 93, 94, 95, 1 06, or 111. In certain embodiments, 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: comprises SEQ ID NO: 93, 94, 95, 106, or 111.

[0083] Second linker peptide: carboxy-terminal linker (C-terminal linker) A GLP-1 fusion peptide comprising a fourth component, wherein the fourth component is a second A fusion peptide that is a linker peptide (i.e., a carboxy-terminal linker peptide). The second linker peptide may be any of the sequences provided in Table 4. The second linker peptide sequence may comprise one of the following criteria: (i) expression yield; (iii) in vitro potency, (iv) serine xylosylation or serine xylosylation (v) lack of potential for xylosylation, (v) conjugation yield, and (vi) GLP- 1. Characterization of Fusion Peptide-Conjugated Cyclic PYY Peptide Conjugates (e.g., In Vivo Stability) and in vivo efficacy (i.e., GLP-1 or GLP-1 variant peptides and cyclic PYY The peptides have agonist activity for the GLP-1 and Y2 receptors, respectively. The selection may be based on at least one of the following:

[0084] In addition, the second linker peptide sequence is specific for and effectively conjugates the cyclic PYY peptide. In this context, specificity refers to the ability of the fusion protein to conjugate. The C-terminal region of the second linker is preferred over conjugation at any other part of the protein. Preferential conjugation at engineered cysteine residues in Examples of strategies to maximize specificity include (i) nucleophilic deprotection of sulfhydryl side chains; The cysteine residues adjacent to the cysteine residues were genetically engineered to increase the affinity and enhance its reactivity. (ii) introducing an amino acid that is capable of reacting with the thiol-electrophilic conjugation Genetically engineered cysteine residues into amino acid sequences that have previously demonstrated enhanced selectivity and (iii) specifically hosting reactive thiols for conjugation. Electrostatic attraction of cationic or anionic disulfide reducing agents, respectively, to separate the Examples of such methods include incorporating amino acids with anionic or cationic side chains. Examples of strategies to maximize ligation efficiency include: (i) adding a second linker peptide; In order to generate mutual electrostatic repulsion between the peptides, an amino acid sequence may be added to any part of the second linker peptide. The same charge, either anionic or cationic, is introduced, thereby allowing the molecules to be genetically engineered. (ii) reducing disulfide formation between the cysteine residues in the linker; and (iii) enhancing the rigidity of the linker. (iii) incorporating amino acids that increase the flexibility of the linker. and (iv) an approach that efficiently conjugates cyclic PYY peptides electrostatically. This includes incorporating amino acids with anionic or cationic side chains.

[0085] [Table 4]

[0086] GLP-1 fusion peptide As described above, the GLP-1 fusion peptide contains the first, second, third, and fourth components. 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, and the third component is The first 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. GLP-1 The fusion peptide sequence was selected based on the following criteria: (i) expression yield, (ii) in vitro potency, (iii) (iv) in vitro stability, (iv) lack of serine xylosylation, (v) conjugation yield (vi) the properties of the GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugates (e.g., in vivo stability and in vivo efficacy (i.e., GLP-1 or GLP-1 variants) The cyclic PYY peptides and cyclic PYY peptides act as receptors for GLP-1 and Y2 receptors, respectively. Selection based on at least one of the following: You may.

[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 acts as a Y2 receptor agonist to inhibit food intake , an endogenous hormone secreted by L cells in the distal intestine. Control of appetite and food intake Considering its role in the Then, PYY 3-36 is effective in treating obesity and related conditions, as well as many gastrointestinal disorders. However, PYY 3-36 Its therapeutic utility as a therapeutic agent is based on its rapid Therefore, the present invention generally relates to the use of PYY inhibitors in the treatment of psoriasis and rheumatoid arthritis. 3-36 Modified peptides that increase the half-life of peptides and reduce metabolism of peptides in vivo PYY 3-36 Conjugates are intended.

[0100] In certain embodiments of the present invention, modified PYY 3-36 The peptide is a cyclic PYY peptide. "Cyclic PYY peptides," "Cyclic PYY 3-36 Analogue" and "Cyclic P YY 3-36 The terms "peptide analog" and "peptide analog" may be used interchangeably. Examples of cyclic PYY peptides that can be used in injectates are listed in the October 2017 issue of U.S. Patent Application No. 15 / 794,231 filed on October 6 and October 26, 2017 No. 15 / 794,171 filed on Dec. 15, 2002, and the contents of both applications are incorporated herein by reference. The entirety of which is incorporated herein by reference.

[0101] As used herein, the term "NTSC-PYY" refers to the N-terminal end of PYY. It is intended to illustrate cyclic analogs.

[0102] The peptide sequences described herein are written according to the usual convention and include the N-terminal region of the peptide. The C-terminal region is on the left and the C-terminal region is on the right. The isomeric forms of the amino acids are known, but are not shown separately. Unless explicitly stated, it is the L-form of the amino acids that is shown. For convenience, conventional and non-conventional abbreviations (both single and three letter codes) for the various amino acids are provided. ) and functional moieties are used. These abbreviations are well known to those skilled in the art, but for clarity For this purpose, the following is listed: A = Ala = alanine; R = Arg = arginine; N = As n = 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=Il e = isoleucine; L = Leu = leucine; K = Lys = lysine; Nle = norleucine ;F=Phe=phenylalanine; P=Pro=proline; S=Ser=serine; T=T hr = threonine; W = Trp = tryptophan; Y = Tyr = tyrosine, and V = Va l=valine.

[0103] For convenience, the amino acid residues used in naming the NTSC-PYY peptides of the present invention are: The numbering convention is hPYY 3-36 Follow the instructions of hPYY 3-36 At the corresponding position of The specific amino acid substitutions introduced into the NTSC-PYY peptides relative to the natural residues in , indicated by the appropriate amino acid code followed by the position of the substitution. "S4" in the TSC-PYY peptide is hPYY 3-36 The corresponding natural lys This refers to a peptide in which four residues have been replaced by serine. Similarly, the NTSC-PYY peptide "hC31" in hPYY 3-36 The corresponding natural val31 residue of This refers to peptides in which an additional amino acid occurs within the NTSC-PYY peptide. Acid substitutions are described according to this rule and are recognized as such by those skilled in the art. It will be.

[0104] Also for convenience, the nomenclature used for the NTSC-PYY peptides of the present invention is cycle Starting from the N-terminal residue contained in the ring, from left to right, In all cases, the N-terminal amino acid residue of the ring is The α-amino functional group is then linked to a linking group, which is then linked to the 3′-amino functional group of the NTSC-PYY peptide. It is connected to the side chain residue of the amino acid at position 1. Therefore, it is called "cyclo-(I3-m-COPhC H2-hC31) is a cylated α-amino functional group of Ile3 with a meta-toluic acid residue. The methyl group is further linked to the side chain of the hCys31 residue via a thioether bond. It is used to describe the ring of the NTSC-PYY peptide, which is rho-(K4-CO(CH2)2NHCOCH2-hC31)" is a natural Ile3 residue. The α-amino functional group of lys4 (here at the N-terminus) is replaced by 3-acetamido. acylated with a methylpropanoyl group, and the acetamido methylene carbon is converted to a thioether The ring of the NTSC-PYY peptide is connected to the side chain of the hCys31 residue via a bond. is used to explain.

[0105] hPYY to provide a convenient functional handle for further derivatization. 3-36 Distribution Lysine residues may be incorporated at various positions in the sequence. The lysine residue may be modified so that the lysine residue is and containing a chemical linker that connects the cyclic PYY peptide to the GLP-1 fusion peptide. The lysine residues may be modified as described above. Those skilled in the art will recognize that related orthologues may also be modified in this manner. It will be appreciated that such techniques can be used effectively and are contemplated herein.

[0106] The term "K(PEG24-AcBr)" refers to a compound in which the side chain ε-amino group is N-Bromoacetyl-75-amino-4,7,10,13,1 via the carboxylic acid functional group 6,19,22,25,28,31,34,37,40,43,46,49,52,55 ,58,61,64,67,70,73-tetracosaoxapentaheptaconanoic acid represents a lysinyl residue acylated with

[0107] The term "K(PEG12-AcBr)" refers to a compound in which the side chain ε-amino group is N-Bromoacetyl-39-amino-4,7,10,13,1 6,19,22,25,28,31,34,37-dodecaoxanonatriacontanoic acid It therefore represents an acylated lysinyl residue.

[0108] The term "K(PEG6-AcBr)" refers to a PEG whose side chain ε-amino group is N-Bromoacetyl-3-[(17-amino-3,6,9,12 ,15-pentaoxaheptadec-1-yl)oxy]-propanoic acid represents a lysinyl residue.

[0109] The compound "K(PEG8-triazolyl-CH2CH2CO-PEG4-AcBr)" is used. The term refers to the 27-[4-[2 -[3-[2-[2-[3-(N-bromoacetylamino)propoxy]ethoxy]ethoxy oxy]propylaminocarbonyl]ethyl]tetrazol-1-yl]-4,7,10, Acylated with 13,16,19,22,25-octaoxaheptacosanoic acid represents a lysinyl residue.

[0110] Many of the compounds / conjugates of the present invention have a C-terminal residue in the sequence, Y36 and its adjacent A reduced amide bond is incorporated between the residue R35 and the corresponding residue R4. This reduced amide bond is called "psi- (R35,Y36)”.

[0111] Various amino acid residues comprising the specific sequences of the present invention have α-amino groups that are methylated. Therefore, the terms "N-Me-Q34" or "N-Me-R35" are α-N-methylated glutamine at position 34 of the sequence and α-N-methylated glutamine at position 35 of the sequence, respectively. Represents N-methylated arginine.

[0112] In the sequence description, the term "N-Me-Q34,psi-(R35,Y36)" , an α-methylglutamine residue at position 34, and a reduced amino acid residue between residues R35 and Y36. It refers to a sequence containing both amide bonds.

[0113] Similarly, in the description of the sequence, "N-Me-R35,psi-(R35,Y36)" The term refers to the α-methylarginine residue at position 35 and the residue between this residue and Y36. It refers to a sequence containing both a reduced amide bond and a cyclic amide bond.

[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 (hPYY3-36 ) for at least 70 %, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the N-terminus Also provided are side chain cyclic analogs of PYY. Methods for determining sequence identity between two analogs. As an example, two peptides

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

[0119] [ka] (SEQ ID NO: 55). 3-36 ) analog to Sequence identity is calculated by subtracting the number of different residues from the total number of aligned residues (i.e. , number of aligned identical residues), hPYY 3-36 Divide by the total number of residues in In this example, the different residue is D11, which is replaced with K11. Then V31 exchanged with hC31, and finally R35 was decarbonylated. Continued. Thus, in this example, the sequence identity is (34-3) / 34 x 100.

[0120] Conjugates In another general aspect, the present invention provides a GLP-1 fusion peptide conjugated cyclic PYY peptide. , compared to GLP-1 or GLP-1 variant peptides or cyclic PYY peptides alone site-specifically covalently linked to cyclic PYY peptides to have enhanced / increased half-lives The present invention relates to a conjugate comprising a conjugated GLP-1 fusion peptide of the present invention. The present invention also relates to pharmaceutical compositions and methods of use thereof. The conjugates are, inter alia, , obesity, type 2 diabetes, metabolic syndrome (i.e., syndrome X), insulin Phosphate resistance, glucose intolerance (e.g., impaired glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, Hyperglycemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis atherosclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or Other cardiovascular risk factors such as hypertension and cardiovascular risk factors related to lipid levels, osteoporosis, Inflammation, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) ), kidney disease, and eczema, and other diseases or disorders. It is useful.

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

[0122] In certain embodiments, the cyclic PYY peptides may include a chemical linker. To conjugate a GLP-1 fusion peptide to a cyclic PYY chemical linker, It may be chemically modified (e.g., electrophilic groups may be added to the linker). Examples of the linker include a peptide linker, a hydrocarbon linker, and a polyethylene glycol linker. Polyethylene glycol (PEG) linker, polypropylene glycol (PPG) linker, polysaccharide linker , polyester linker, acyl group-containing linker, PEG and embedded heterocycle or a hydrocarbon chain. The PEG linker can contain, for example, 2 to 24 PEG units.

[0123] The GLP-1 fusion peptides of the present invention are conjugated to the cyclic PYY peptides of the present invention. Briefly, the GLP-1 fusions of the present invention are Peptides are reacted with a reducing agent (e.g., 1,3,5-triaza-7-phosphaadamantane (PTA )), purified (e.g., by desalting chromatography), and by providing the reduced GLP-1 fusion peptide under conditions that allow gating It can be conjugated to a cyclic PYY peptide. During the conjugation reaction, The electrophilic leaving group of the cyclic PYY peptide is displaced to form the GLP-1 fusion peptide and the cyclic PYY peptide. A covalent bond is formed between the GLP-1 fusion peptide and the cyclic PYY peptide. After the conjugation reaction, the conjugate is Proteins can be isolated by exchange chromatography or hydrophobic interaction chromatography (HIC). In certain embodiments, the present invention can be purified by a final purification step of protein A adsorption. The GLP-1 fusion peptide can be purified prior to reduction using the HIC method. See, eg, Example 2, for a more detailed description of the jugation method.

[0124] Glucagon-like peptide 1 (GLP-1)-fusion peptides linked to cyclic PYY peptides wherein the GLP-1 fusion peptide is a GLP-1 peptide, A first linker peptide, a hinge-Fc region peptide, and a second linker peptide. Conjugates are provided herein in which the first linker is optionally absent. can be.

[0125] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative thereof: or a pharmaceutically 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 (provided that 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- and 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 derivative may be one or more selected from the group consisting of amidation, acylation, and PEGylation. Compounds of formula I that have been modified by more than one process.

[0129] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative thereof: or a pharmaceutically 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 (provided that 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- and Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of said K is optionally

[0130] [ka] (wherein 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, and the amino side chain of said K is optionally

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

[0132] [ka] (wherein 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, and 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), —C(O) substituted with —CHBr, —C(O)CHI, or —C(O)CHCl; Z 23 is S or K, and the amino side chain of said K is optionally

[0134] [ka] (wherein i is an integer from 0 to 24 and X=Br, I, or Cl), —C(O) substituted with —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 X electrophilic group Br, Cl, or I is a and the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate is substituted with Form.

[0137] In certain embodiments, the cyclic PYY peptide is represented by Formula I, or a derivative thereof: or a pharmaceutically 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 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- and 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, and the amino side chain of said K is optionally

[0139] [ka] -C(O)CHBr, 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 substituted in the conjugation reaction to form the GLP-1 fusion peptide conjugate ring. to form a PYY peptide conjugate.

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

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

[0146] GLP-1 fusion peptides according to embodiments of the present invention can be prepared by methods known in the art. using amino acid residues 4, 7, 9, 10, 11, 13, 14, and 15 of the cyclic PYY peptide. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 30, or 31 to the cyclic PYY peptide at one or more amino acid positions of the cyclic PYY peptide, such as The amino acid residue numbering is as follows: hPYY 3-36 Follow the one in. In certain embodiments, Z, Z, Z in Formula I 11 , Z 22 , and Z 23 It's one of The lysine is a lysine, and the lysine is attached to the second linker peptide of the GLP-1 fusion peptide. In a preferred embodiment, the present invention is The GLP-1 fusion peptides in this form contain cyclic PYY at residue 11 of the cyclic PYY peptide. In another preferred embodiment, the Y peptide is conjugated to a Y peptide, and residue 11 is a lysine. Electrophiles such as bromoacetamide or maleimide are used to cleave residue 11 of the cyclic PYY peptide. The electrophile is introduced into the side chain of a cyclic PYY peptide, such as the amino side chain of a lysine in The reactive site of the cysteine residue of the second linker peptide of the GLP-1 fusion peptide is and preferably reacts specifically with the alkylhydryl group of the second linker of the GLP-1 fusion peptide. The peptide is SEQ ID NO: 93, 94, 95, 106, or 111, thereby forming a ring A covalent bond is formed between the PYY peptide and the GLP-1 fusion peptide. Alternatively, the cyclic PYY peptide may be any of 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 indirectly attached to the side of cyclic PYY via a chemical linker. is introduced into the chain.

[0147] In certain embodiments, the cysteine of the second linker peptide of the GLP-1 fusion peptide The residues are converted by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent. The cysteine residue is then reduced by hydroxybenzoates, which allows the reduced cysteine residue to react with electrophiles. The reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]deca It is polytetrafluoroethylene (PTA) or its derivatives.

[0148] Also provided is a pharmaceutical composition comprising the conjugate of the present invention and further comprising a pharmaceutically acceptable carrier. Goods are also provided.

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

[0150] [Table 7]

[0151] Half-life extension moiety In addition to GLP-1 fusion peptides, the conjugates of the invention may also include, for example, covalently linked to extend the half-life of a pharmaceutically active moiety (e.g., a cyclic PYY peptide) through its action Exemplary other half-life extending moieties include: 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 invention include: For example, polyethylene such as PEG 5000 or PEG 20,000 for desired properties. Polyethylene glycol (PEG) molecules, polylysine, octane, carbohydrates (dextran, cellulose These moieties include the protein scaffold coding sequence. It may be a direct fusion with a sequence or may be generated by standard cloning and expression techniques. Alternatively, recombinantly and chemically produced vectors may be used, using well-known chemical conjugation methods. The moiety can then be attached to a conjugate of the invention.

[0152] Using well-known methods, a cysteine residue was incorporated at the C-terminus of the molecule and a PEG group was attached to the cysteine. PEG moieties can be added to peptide molecules of the invention, for example, by conjugating them to That's fine.

[0153] The peptide molecules of the invention incorporating additional moieties can be assayed by several well-known assays. For example, the biological or functional properties of a therapeutic peptide of interest can be compared. Pharmacokinetic activity can be measured by known in vitro assays, either alone or in the conjugates according to the invention. Alternatively, in vivo assays can be used to assay and compare.

[0154] Pharmaceutical Composition In another general aspect, the present invention provides conjugates and compounds of the invention, and pharmaceutical As used herein, "pharmaceutical composition" refers to a pharmaceutical composition comprising an acceptable carrier. " refers to a product comprising a conjugate of the present invention together with a pharmaceutically acceptable carrier. The conjugates and compounds of the present invention, and compositions containing them, are They are also useful in the manufacture of medicaments for the therapeutic applications mentioned therein.

[0155] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, base, or the like. buffers, stabilizers, solubilizers, oils, lipids, lipid-containing vesicles, microspheres, liposomes Carriers, excipients, or other materials known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the diluent or diluent will depend on the route of administration for a particular application. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutical composition that is effective or refers to a non-toxic material that does not interfere with the biological activity of the composition according to the invention. In view of the present disclosure, any peptide suitable for use in a conjugated peptide pharmaceutical composition may be Any of the following pharmaceutically acceptable carriers may be used in the present invention.

[0156] Pharmaceutically acceptable acidic / anionic salts for use in the present invention include acetic acid, Acid salts, benzenesulfonates, benzoates, bicarbonates, bitartrates, bromides, edetic acid Calcium, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edi Silates, estolates, esylates, fumarates, gliceptates, gluconates, gluconates glutamate, glycolyl arsanilate, hexylresorcinol, hydrabamine, odor Hydrochloride, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lauryl ctobionate, malate, maleate, mandelate, mesylate, methyl bromide , methyl nitrate, methyl sulfate, mucoate, napsylate, nitrate, pamoate, pantothenate Phosphate, Phosphate / Diphosphate, Polygalacturonate, Salicylate, Stearate , basic acetate, succinate, sulfate, tannate, tartrate, tetradecyl, tosyl Also, organic or inorganic acids include, but are not limited to, triethoxysilanes, triethoxysilanes, and triethoxysilanes. Organic acids include 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, saccharinic acid or triflic acid These include, but are not limited to, oroacetic acid.

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

[0158] In some embodiments of the present invention, from about 0.001 mg / mL to about 100 mg / mL, 0.01 mg / mL to about 50 mg / mL, or about 0.1 mg / mL to about 25 mg / mL The pharmaceutical formulation comprises a conjugate of the present invention in an amount of about 3.0 to about 1. The formulation may have a pH of about 0, for example, about 3 to about 7, or about 5 to about 9. The formulation may also contain a buffer system, preservatives, At least one component selected from a tonicity agent, a chelating agent, a stabilizer, and a surfactant It may further include:

[0159] Formulations of pharmaceutically active ingredients with pharmaceutically acceptable carriers are described, for example, in Remingto n:The Science and Practice of Pharmacy (e.g. For example, in the 21st edition (2005) and any subsequent revisions , which are known in the art. Non-limiting examples of additional components include buffers, diluents, , solvents, tonicity adjusting agents, preservatives, stabilizers, and chelating agents. The following pharmaceutically 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 a water-soluble Liquid formulations include solutions, suspensions, emulsions, and emulsions. The aqueous formulation may comprise a liquid emulsion, a microemulsion, a gel, etc. The aqueous formulation typically comprises 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 is administered, for example, via an injection device (e.g., a syringe or an infusion pump). ) can be formulated as an injection that can be injected via the It may be delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously.

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

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

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

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

[0166] In another embodiment of the present invention, the pharmaceutical composition comprises a buffering agent. Non-limiting examples of buffering agents include Contains arginine, aspartic acid, bicine, citrate, disodium monohydrogen phosphate, Malic acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, vinegar Sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinic acid salts, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and The buffer solution may contain, individually or in aggregate, from about 0.01 mg / mL to about 50 mg / mL. mg / mL, for example, from about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific buffering agents constitute alternative embodiments of the invention.

[0167] In another embodiment of the present invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include Benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, Ruphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate Dibenzoate, imidourea, methyl 4-hydroxybenzoate, phenol, 2-hydroxybenzoate Phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, Preservatives include sodium dehydroacetate, thiomersal, and mixtures thereof. , individually or in aggregates, from about 0.01 mg / mL to about 50 mg / mL, for example about 0.1 mg These preservatives may be present in concentrations of 100 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising the compound constitute an alternative embodiment 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: For example, salt (sodium chloride, etc.), amino acids (glycine, histidine, arginine, lysine amine, isoleucine, aspartic acid, tryptophan, and threonine), alditol alcohols (glycerol, 1,2-propanediol, propylene glycol, etc.), 1,3- propanediol, and 1,3-butanediol), polyethylene glycol (e.g. Other examples of isotonicity agents include sugars, Non-limiting examples of sugars include, for example, fructose, glucose, mannose, Sorbose, xylose, maltose, lactose, sucrose, trehalose, dextrose Tolan, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. The isotonicity agent may be a monosaccharide, disaccharide, or polysaccharide, including a sugar alcohol. The term "sugar alcohol" refers to a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, Examples include inositol, galactitol, dulcitol, xylitol, and arabitol. The isotonicity agent may be present individually or in aggregate at a concentration of about 0.01 mg / mL to about 50 mg / mL. For example, it may be present at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising each one of the isotonic agents constitute alternative embodiments of the invention.

[0169] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Examples include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and and mixtures thereof. The chelating agent may be present individually or in aggregate in an amount 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 containing each one of these specific chelating agents are considered alternative embodiments of the present invention. It constitutes a state.

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

[0171] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy- / Hydroxycellulose and its derivatives (HPC, HPC-SL, HPC-L, and HPMC, cyclodextrin, 2-methylthioethanol, polyethylene glycol (e.g., PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolide salts (such as sodium chloride), sulfur-containing substances, e.g., monothioglycerol, or The stabilizer is glycolic acid. The stabilizer may be present individually or in aggregates at a concentration of from about 0.01 mg / mL to about 5 mg / mL. It may be present at a concentration of about 0.0 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing 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 or surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to a surfactant that consists of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. Surfactants refer to any molecule or ion that can be dissolved in water. Examples of surfactants include anionic surfactants, cationic surfactants, and the like. Selected from ionic surfactants, nonionic surfactants, and / or zwitterionic surfactants The surfactants may be present individually or in aggregates at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific surfactants may be present in a concentration of 1 L. This constitutes a clear alternative embodiment.

[0173] In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors. Protease inhibitors include, for example, EDTA, and / or benzamidine hydrochloride (HCl). The inhibitors are present individually or in aggregates at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific protease inhibitors are also available as alternatives of the present invention. This constitutes an embodiment.

[0174] The pharmaceutical compositions of the present invention are useful in reducing the formation of polypeptide aggregates during storage of the compositions. The term "amino acid base" refers to one or more amino acid bases. Amino acids (methionine, histidine, imidazole, arginine, lysine, isoleucine) amino acids, aspartic acid, tryptophan, threonine, etc.), or their analogs Any amino acid may be present in either its free base form or its salt form. Any stereoisomer of the acid or base may be present (i.e., L, D, or mixtures thereof). Amino acid bases, individually or in combination with other amino acid bases, are present in a concentration of about 0.01 mg / mL to about It may be present at a concentration of 50 mg / mL, for example, from 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. do.

[0175] Pharmaceutically acceptable salts of the conjugates of the present invention include salts of inorganic or organic acids or bases. Examples of such acid salts include conventional non-toxic salts or quaternary ammonium salts formed from Examples of salts include acetate, adipate, benzoate, benzenesulfonate, and citrate. Acid salt, camphorate, dodecyl sulfate, hydrochloride, hydrobromide, lactate, maleate, meta Sulfonate, nitrate, oxalate, pivalate, propionate, succinate, sulfate Basic salts include ammonium salts, sodium salts and potassium salts. alkali metal salts such as sodium salts, alkaline earth metal salts such as calcium salts and magnesium salts salts with organic bases such as metal salts, dicyclohexylamino salts, and amino salts such as arginine Furthermore, basic nitrogen-containing groups can be converted to salts with, for example, alkyl halides. It may be quaternized by

[0176] The pharmaceutical compositions of the present invention may be administered by any means that achieves their intended purpose. Examples include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral or intraocular administration. Administration may be by oral route. Suitable formulations for parenteral administration include: Aqueous solutions of the active conjugate in water-soluble form, e.g., water-soluble salts, acidic solutions, alkaline solutions solutions, aqueous dextrose solutions, isotonic carbohydrate solutions, and cyclodextrin inclusion complexes. In certain embodiments, the conjugates of the invention are administered peripherally.

[0177] The present invention also provides a method for the preparation of a pharmaceutical composition comprising mixing a pharmaceutically acceptable carrier with any of the conjugates of the present invention. Additionally, the present invention also encompasses a method for making a pharmaceutical composition, comprising: by mixing a pharmaceutically acceptable carrier of The pharmaceutical compositions produced include:

[0178] Furthermore, the conjugates of the present invention may have one or more crystalline polymorphs or amorphous crystalline forms. Conjugates may also be used, which are also intended to be within the scope of the present invention. The salts may form solvates, for example with water (i.e., hydrates) or common organic solvents. As used herein, the term "solvate" refers to a compound or compounds that combine one or more of the conjugates of the present invention. refers to a physical association with two or more solvent molecules. This physical association can be achieved by means of an inert solvent, such as hydrogen bonding. with varying degrees of covalent and ionic bonding. In certain cases, e.g., one or more It is possible to isolate the solvate when the solvent molecule is incorporated into the crystal lattice of the crystalline solid. The term "solvate" refers to both solution-phase solvates and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methyl ... Examples include thanolate.

[0179] The present invention includes within its scope polymorphs and solvates of the conjugates of the invention. Therefore, the term "administration" in the treatment methods of the present invention includes the Conjugates, or compounds not specifically disclosed, are clearly included within the scope of the present invention. The crystalline polymorphs or solvates thereof are used to treat the syndromes, disorders, or diseases described herein. This includes means to prevent, ameliorate, or prevent a disease.

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

[0181] Included within the scope of the present invention are prodrugs of the conjugates of the present invention. Such prodrugs are compounds that are readily convertible in vivo into the required conjugate. Therefore, in the treatment methods of the present invention, the term "administration" is used. "Administered" refers to administration of a conjugate, whether or not specifically disclosed. However, conjugates that are converted to specific conjugates in vivo after administration to a patient are used. The present invention is intended to encompass the treatment of a variety of disorders described above. Conventional procedures for selection and preparation are described, for example, in "Design of Prodrugs (Ed. H. Bundgaard, Elsevier, 1985).

[0182] Furthermore, within the scope of the present invention, any element may be mentioned specifically with respect to the conjugates of the present invention. If present, they may be either naturally occurring or synthetically produced and in their natural abundance. All isotopes and mixtures of isotopes of the element in question, either in pure or isotopically enriched form For example, a reference to hydrogen includes within its scope 1 H, 2 H (D), and 3 Similarly, references to carbon and oxygen include their ranges. 12C inside, 13 C and 14 C, and 16 O and 18 O is contained in each of the isotopes. The radioactive label of the present invention may be a radioactive isotope or a non-radioactive isotope. The cognitive conjugate is 3 H, 11 C. 18 F, 122 I, 123 I, 125 I, 131 I , 75 Br, 76 Br, 77 Br, and 82 a radioisotope selected from the group consisting of Br Preferably, the radioisotope comprises: 3 H, 11 C, and 18 Is it a group consisting of F? are selected.

[0183] Some conjugates of the present invention may exist as atropisomers. Tropic isomers are stereoisomers resulting from hindered rotation around a single bond, The steric 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. can be.

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

[0185] If the process for preparing the conjugate according to the invention results in a mixture of stereoisomers, These isomers can be separated by conventional techniques such as preparative chromatography. The conjugates may be prepared as racemates, or the individual enantiomers may be enantiomerically separated. Conjugates may be prepared either by thiospecific synthesis or by resolution. For example, (-)-di-p-toluoyl-D-tartaric acid and / or (+)-di-p-toluoyl- Formation of diastereomeric pairs by forming salts with optically active acids such as L-tartaric acid and then its constituents can be purified by standard methods such as fractional crystallization and regeneration of the free base. The conjugates can also be separated into diastereomers. After formation of the ester or amide of the chiral auxiliaries, chromatographic separation is performed to remove the chiral auxiliary. Alternatively, separation can be achieved by high performance liquid chromatography (HPLC). Conjugates can also be resolved using chiral columns via LC (LC) or SFC. In some cases, complex multiplets and peak integrals in the 1H NMR spectrum can be observed. There may be rotamers of the conjugate observable by 1H NMR, resulting in

[0186] During any of the processes for preparing the conjugates of the present invention, any of the molecules involved It may be necessary and / or desirable to protect sensitive or reactive groups in the This is the Protective Groups in Organic Chemistry stry, ed. JFW McOmie, Plenum Press, 1973, and TWGreene & PGMWuts, Protective Group ps in Organic Synthesis,John Wiley & Son s., 1991 (each of which is incorporated herein by reference in its entirety for all purposes). This can be achieved by conventional protecting group means, such as those described in (incorporated herein). The protecting groups may be removed at a convenient subsequent stage using methods known in the art. can be done.

[0187] How to use The present invention relates to a method for treating Y2 receptor-mediated syndrome and / or GL in a subject in need thereof. Methods for preventing, treating, or ameliorating P-1 receptor-mediated syndromes, disorders, or diseases administering to a subject in need thereof an effective amount of a conjugate, compound, or a method comprising administering the pharmaceutical composition.

[0188] The present invention relates to a method for treating a disorder, disease, or condition, or a method for treating said disorder, disease, or condition, in a subject in need thereof. Preventing, treating, or preventing the onset of any one or more symptoms of a disease or condition A method for delaying or ameliorating inflammatory bowel disease comprising administering to a subject in need thereof an effective amount of a compound of the present invention. Also provided are methods comprising administering a conjugate, compound, or pharmaceutical composition of

[0189] In certain embodiments, the disease disorder or condition is obesity, type I or II diabetes, metastasis, or a combination of these. cerebrovascular syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance ( For example, impaired glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, congenital hyperlipidemia, Insulinosis (CHI)-induced hypoglycemia, dyslipidemia, atherosclerosis, diabetic Nephropathy and hypertension associated with uncontrolled cholesterol and / or lipid levels Other cardiovascular risk factors such as blood pressure, osteoporosis, inflammation, and non-alcoholic fat liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and / or Selected from eczema.

[0190] According to certain embodiments, a therapeutically effective amount is one, two, three, or four of the following effects: A therapeutic amount refers to a therapeutic dose that is sufficient to achieve or exceed the therapeutic dose for the disease, disorder, or condition being treated. or reducing the severity of or ameliorating a disease state or symptoms associated therewith; i) shortening the duration of the disease, disorder or condition being treated or its associated symptoms; (iii) prevent the progression of the disease, disorder, or condition being treated, or symptoms associated therewith. (iv) regression of the disease, disorder, or condition being treated or symptoms associated therewith. (v) causing the progression of the disease, disorder, or condition being treated or symptoms associated therewith. (vi) to prevent the onset or progression of the disease, disorder, or condition being treated, or related thereto; (vii) to prevent the recurrence of the disease, disorder, or condition being treated or its (viii) reducing hospitalization of subjects with symptoms associated with the disease being treated; reducing the length of hospital stay for subjects with a disorder or condition, or symptoms associated therewith; (ix) the survival rate of subjects with the disease, disorder, or condition being treated, or symptoms associated therewith; (xi) enhancing the ability of, or relating to, the disease, disorder, or condition being treated; (xii) inhibiting or alleviating symptoms, and / or (xiii) enhancing the prophylactic or therapeutic effect of another treatment. Or to improve.

[0191] The therapeutically effective amount or dose depends on the disease, disorder or condition being treated, the means of administration, the target site, and the subject's Physiological status (including, for example, age, weight, and health status), whether the subject is human or animal other medications being administered, and whether the treatment is prophylactic or therapeutic, etc. Treatment doses may vary depending on a variety of factors, including: The dose is increased gradually as needed.

[0192] As used herein, "treat," "treating," and and "treatment" all refer to at least one condition related to a disease, disorder, or condition. refers to the improvement or reversal of at least one measurable physical parameter in a subject. It may be recognizable in the subject, but not necessarily in the individual. The terms "treat," "treating," and "treatment" are also used interchangeably. or causing regression of, preventing the progression of, or at least slowing the progression of, a disease, disorder, or condition. In certain embodiments, the term "treat" or "delay" may refer to the progression of a disease. "Treating" and "treatment" refer to the treatment of a disease, disorder, or condition. Alleviating, preventing the onset or progression of, or shortening the duration of, one or more associated symptoms In specific embodiments, "treat," "treating," and "treatment" refer to a disease, In a specific embodiment, "treating" refers to preventing the recurrence of a disorder or condition. " and "treatment" refers to improving the survival rate of a subject with a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the treatment of a disease, disorder, or condition in a subject. It refers to the disappearance of harm or pathology.

[0193] In one embodiment, the present invention provides a method for treating obesity, or any of the symptoms of obesity, in a subject in need thereof. Prevent, treat, delay the onset of, or ameliorate one or more symptoms The method comprises administering to a subject in need thereof an effective amount of a conjugate, compound, or provides a method comprising administering a pharmaceutical composition.

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

[0195] In some embodiments, the conjugates, compounds, pharmaceutical compositions of the invention described herein the body weight of the subject prior to administration of any of the compositions, forms, or medicaments, or Any of the conjugates, compounds, compositions, formulations, medicaments, or combinations of the present invention described above The body weight of the subject may be reduced, for example, by about 0.01% to about 0.01% compared to a control subject not receiving the drug. 0.1%, approximately 0.1% to approximately 0.5%, approximately 0.5% to approximately 1%, approximately 1% to approximately 5%, approximately 2% to approximately 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 reduced by about 45% to about 50%.

[0196] In some embodiments, weight loss is observed over a period of about 1 week, about 2 weeks, about 3 weeks, about 1 month, or more. , about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months Months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, Approximately 2.5 years, approximately 3 years, approximately 3.5 years, approximately 4 years, approximately 4.5 years, approximately 5 years, approximately 6 years , maintained for about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years .

[0197] The present invention relates to a method for treating a syndrome, disorder, or disease, or a related condition, in a subject in need thereof. Preventing or treating any one or more symptoms of said syndrome, disorder, or disease a method for delaying the onset of, or ameliorating, a syndrome, disorder, or disease, wherein the syndrome, disorder, or disease is , 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 Hyperglycemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia Diabetes, atherosclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or other cardiovascular risk factors, such as hypertension and cardiovascular risk factors related to lipid levels; Osteoporosis, inflammation, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and eczema, administering to the animal an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention. , a method is provided.

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

[0199] The present invention provides a method for reducing food intake in a subject in need thereof, comprising: Administering an effective amount of the conjugate, compound, or pharmaceutical composition of the present invention to a subject in need thereof. In some embodiments, the subject's food intake is measured using a method as described herein. The conjugate, compound, composition, formulation, agent or combination of the present invention as described in with respect to the subject's food intake prior to any administration or administration of the conjugates of the invention described herein. Control subjects received no steroid, compound, composition, formulation, agent, or combination. Compared to the elephant, for example, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5 % to approximately 1%, approximately 1% to approximately 5%, approximately 2% to approximately 3%, approximately 5% to approximately 10%, approximately 10% to approximately 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%.

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

[0201] The present invention provides a method for reducing glycated hemoglobin (A1C) in a subject in need thereof. administering to a subject in need thereof an effective amount of a conjugate, compound, or drug of the present invention In some embodiments, the method comprises administering a pharmaceutical composition to a subject. C is a conjugate, compound, composition, formulation, agent, or is based on the subject's A1C prior to administration of any of the combinations or the methods of the present invention described herein. any conjugate, compound, composition, form, agent, or combination of For example, about 0.001% to about 0.01%, about 0.01% to about 0.1%, approximately 0.1% to approximately 0.2%, approximately 0.2% to approximately 0.3%, approximately 0.3% to approximately 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% % reduction.

[0202] In other embodiments, the method for reducing fasting blood glucose levels in a subject in need thereof includes administering to a subject in need thereof an effective amount of a conjugate, compound, or medicament of the present invention The fasting blood glucose level is measured by administering a composition described herein. Any of the conjugates, compounds, compositions, formulations, agents, or combinations of the present invention or to the fasting blood glucose level of the subject prior to administration of the conjugates of the invention described herein. Control subjects received no steroid, compound, composition, formulation, agent, or combination. Compared to elephants, approximately 140 to less than 150 mg / dL, approximately 140 to less than 130 mg / dL , about 130 to less than about 120 mg / dL, about 120 to less than about 110 mg / dL, about 110 to less than about 120 mg / dL Less than about 100 mg / dL, about 100 to about 90 mg / dL, or about 90 to about 80 mg / It can be reduced to less than 300 dL.

[0203] The present invention relates to a method for treating Y2 receptor activity and GLP-1 receptor activity in a subject in need thereof. A method for regulating sexual activity comprises administering to a subject in need thereof an effective amount of a conjugate of the present invention. The present invention provides methods for treating a patient suffering from atopic dermatitis, the method comprising administering a compound, a steroid, a compound, or a pharmaceutical composition to a patient. When used herein, "modulate" refers to increasing or decreasing receptor activity.

[0204] In some embodiments, an effective amount of a conjugate or compound of the invention, or The form, composition, or medicament may be administered once daily, twice daily, three times daily, or both to a subject in need thereof. The dose is administered once a day, four times a day, five times a day, six times a day, seven times a day, or eight times a day. In this case, an effective amount of the conjugate or compound of the present invention, or a form, composition, or The drug may be administered to subjects who need it once every two days, once a week, twice a week, three times a week, or once a week. Administered 4 times a week, 5 times a week, 6 times a week, 2 times a month, 3 times a month, or 4 times a month .

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

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

[0207] As used herein, combination therapy refers to administering to a subject in need thereof an effective amount of a compound of the present invention. Conjugates or compounds, or forms, compositions, or drugs thereof, together with one or more This refers to the administration of one or more additional therapeutic agents or one or more surgical treatments. In some embodiments, one or more additional therapeutic or surgical therapies are effective. In other embodiments, one or more of the The above additional therapeutic or surgical therapy may be administered in the same week as an effective amount of a conjugate or compound of the invention. Or it can be administered in the same month.

[0208] The present invention also provides a method for treating a disease, disorder, or syndrome described herein in a subject in need thereof. or any of the symptoms using combination therapy to prevent, treat, or delay the onset of The combination therapy is also intended to improve or alleviate the symptoms of the disease, and the combination therapy comprises administering to a subject in need thereof an effective amount of the The conjugate, compound, or pharmaceutical composition of the invention may be administered in combination with any one or more of the following therapeutic agents: includes administration in combination with two or more of: dipeptidyl peptidase-4 (DPP -4) inhibitors (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, GLP-1 receptor agonists (e.g., exenatide and lixisenatide) Short-acting GLP-1 receptor agonists, such as; intermediate-acting GLP-1 receptor agonists extended-release exenatide, albiglutide, duraglutide Long-acting GLP-1 receptor agonists such as benzodiazepines; sodium-glucose cotransporters SGLT-2 inhibitors (e.g., canaglifozin, dapaglifozin, empaglifozin) rifozin, etc.); bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor antagonists nitrates (e.g., bromocriptine rapid release); biguanides (e.g., metformin, etc.) ); insulin; oxyntomodulin; sulfonylureas (e.g., chlorpropamide, Glimepiride, glipizide, glyburide, glibenclamide, glibornuride, glisoxin Sepid, glyclopyramide, tolazamide, tolbutamide, acetohexamide, cabutamide etc.); and thiazolidinediones (e.g., pioglitazone, rosiglitazone, lobeglitazone, etc.) dazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone In some embodiments, the dose of the additional therapeutic agent is In some cases, the effect is reduced when given in combination with a conjugate or compound of In embodiments, when used in combination with a conjugate or compound of the invention, additional The therapeutic agents may be used in lower doses than when each is used alone.

[0209] The present invention relates to a method for treating a disease, disorder, syndrome, or condition described herein in a subject in need thereof. or any of the symptoms of the disease, using combination therapy to prevent, treat, delay the onset of, or The combination therapy is intended to provide relief from the symptoms of rheumatoid arthritis, and the combination therapy comprises administering to a subject in need thereof an effective amount of the compound of the present invention. in combination with surgical therapy. In certain embodiments, the surgical treatment is bariatric surgery (e.g., Roux-en-Y gastric bypass). Surgeries such as gastric bypass surgery; sleeve gastrectomy; adjustable gastric band surgery; duodenal bypass gastric diversion; intragastric balloon; gastric plication, and combinations thereof) It is possible.

[0210] One or more additional therapeutic or surgical therapies may be administered in combination with an effective amount of a conjugate of the invention. In embodiments where the conjugate or compound is administered on the same day as the conjugate or compound of the invention, It may be administered before, after, or simultaneously with the additional therapeutic or surgical therapy. The use of the terms does not restrict the order in which treatments are administered to a subject. For example, a first therapeutic agent (e.g., , a composition described herein) prior to administration of a second therapeutic agent to the subject (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours 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), at the same time, or after (e.g., 5 minutes, 15 minutes, 3 0 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 The administration can be made 12 weeks after the initial administration of the steroid hormone.

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

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

[0213] In embodiment 2, the cyclic PYY peptide is represented by Formula I, or a derivative thereof: or a pharmaceutically 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 (provided that 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- and 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 derivative is one or more selected from the group consisting of amidation, acylation, and PEGylation. The conjugates of embodiment 1, which are compounds of formula I modified by more than one process. It is a gate.

[0217] Embodiment 3 is a cyclic PYY peptide represented by Formula I, or a derivative thereof: or a pharmaceutically 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 (provided that 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- and Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of said K is optionally

[0218] [ka] (wherein 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, and the amino side chain of said K is optionally

[0219] [ka] (wherein 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, and the amino side chain of said K is optionally

[0220] [ka] (wherein 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; Z22 is A or K, and the amino side chain of K is optionally

[0221] [ka] (wherein 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, and the amino side chain of said K is optionally

[0222] [ka] (wherein 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 X is an electrophilic group, and the X electrophilic group Br, Cl, or I is a and the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate is substituted with 3. The conjugate of embodiment 2, wherein

[0225] Embodiment 4 is a method for treating a cyclic PYY peptide having a cyclic structure represented by Formula I or a derivative thereof. or a pharmaceutically 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 is absent. BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S -, -SCH2C(O)NH2-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)-, or -CH2S- and Z4 is K, A, E, S, or R; Z7 is A or K, and the amino side chain of said K is is substituted with -C(O)CHBr, Z9 is G or K, and the amino side chain of the K is is substituted with -C(O)CHBr, Z 11 is D or K, and the amino side chain of K is is substituted with -C(O)CHBr, Z 22 is A or K, and the amino side chain of K is is substituted with -C(O)CHBr, Z 23 is S or K, and the amino side chain of K is is substituted with -C(O)CHBr, Z 26 is A or H, Z 30 is L, Z 34 teeth,

[0226] [ka] and Z 35 teeth,

[0227] [ka] and Br is substituted in the conjugation reaction to form the GLP-1 fusion peptide conjugate ring. 3. The conjugate of embodiment 2, forming a PYY peptide conjugate as described in embodiment 2. .

[0228] In a fifth embodiment, the cyclic PYY peptide is selected from the group consisting of SEQ ID NOs: 1-54. or a pharmaceutically acceptable salt thereof. is.

[0229] Embodiment 6 is a method for producing a cyclic PYY peptide comprising administering to a subject a cyclic PYY peptide selected from the group consisting of SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34, or a pharmaceutically acceptable salt thereof. The conjugate according to embodiment 5.

[0230] Embodiment 7 is a method for preparing a cyclic PYY peptide comprising administering to a patient a fusion protein ... 7. Any of embodiments 1 to 6, wherein The conjugate is described in one of the above.

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

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

[0233] Embodiment 10 is directed to Z7, Z9, Z in Formula I 11 , Z 22 , and Z 23 Only one of is a lysine, and the lysine is located between the second linker peptide of the GLP-1 fusion peptide and the 10. The method of any one of embodiments 7 to 9, wherein the cysteine residue of the ribonucleotide is covalently linked to the cysteine residue of the ribonucleotide. It is a conjugate of

[0234] Embodiment 11 is directed to Z in Formula I 11 The conjugate of embodiment 10, wherein It is.

[0235] Embodiment 12 is a method for treating a GLP-1 peptide comprising administering to a subject a subject therapies comprising administering to a subject a subject a GLP-1 peptide selected from the group consisting of SEQ ID NOs: 56 to 59. 12. The conjugate according to any one of embodiments 1 to 11, comprising an amino acid sequence be.

[0236] Embodiment 13 is a method for producing a GLP-1 peptide comprising administering to a subject a therapeutically effective amount of the GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 57. 13. The conjugate according to embodiment 12.

[0237] Embodiment 14 is a method for preparing a peptide comprising administering to a subject the steps of: 14. The conjugate of any one of embodiments 1 to 13, comprising a selected amino acid sequence. It is.

[0238] Embodiment 15 is a method for producing a medicament comprising the steps of: , a conjugate according to embodiment 14.

[0239] In embodiment 16, the hinge-Fc region peptide is selected from the group consisting of SEQ ID NOs: 84 to 90. 16. The conjugate of any one of embodiments 1 to 15, comprising an amino acid sequence selected from It is a route.

[0240] Embodiment 17 is a method for producing a hinge-Fc region peptide comprising administering to a mammalian subject the present invention ... 17. The conjugate of embodiment 16, comprising the amino acid sequence

[0241] Embodiment 18 is a method for preparing a peptide comprising administering to a subject the steps of: 18. The conjugate of any one of embodiments 1 to 17, comprising an amino acid sequence selected from It is a route.

[0242] Embodiment 19 is a method for preparing a peptide comprising administering to a subject the steps of: or 111 amino acid sequence.

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

[0244] Embodiment 21 is a method for treating a GLP-1 fusion peptide comprising administering to a subject a therapeutically effective amount of the ... 21. The conjugate of embodiment 20.

[0245] Embodiment 22 is a method for treating a GLP-1 fusion peptide comprising administering to a subject a therapeutically effective amount of the ... 21. The conjugate of embodiment 20.

[0246] Embodiment 23 is a method for treating a GLP-1 fusion peptide comprising administering to a patient a cysteine residue at residues 287-289 of the GLP-1 fusion peptide. to a lysine residue at residue 7, 9, 11, 22, or 23 of the cyclic PYY peptide. 23. The conjugate of embodiment 21 or 22, which is covalently linked.

[0247] Embodiment 24 is a method for treating a cysteine residue in a fusion peptide comprising administering to a subject a subject the cysteine residue being at residue 288 of the GLP-1 fusion peptide. 24. The conjugate of embodiment 23, wherein

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

[0249] Embodiment 26 is a method for preparing a cyclic PYY peptide comprising administering to a subject the GLP-1 fusion peptide of the present invention a compound comprising: 20 to 23, wherein the cyclic PYY peptide is covalently linked to the cyclic PYY peptide via an organic linker. 25. A conjugate according to any one of claims 1 to 25.

[0250] Embodiment 27 is a method for preparing a hydroxylase comprising administering to a subject the chemical linker is selected from the group consisting of C(O)CH2, polyethylene glycol (P EG) 8-Triazolyl-CH2CH2CO-PEG4, PE with 2 to 24 PEG units G chain, a linker containing an acyl group, or an alkyl chain containing 2 to 10 carbon atoms 27. The conjugate of embodiment 26, wherein the conjugate is selected from the group consisting of:

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

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

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

[0254] Embodiment 31 produces a conjugate according to any one of embodiments 1 to 30. The method further comprises: An electrophile, preferably bromoacetamide or maleic acid, is introduced into the amino side chain of the amino group of the amino acid residue. a cysteine residue of the second linker peptide of the GLP-1 fusion peptide; reacting with the sulfhydryl groups, thereby forming a fusion protein between the cyclic PYY peptide and the GLP-1. and forming a covalent bond between the peptide and the nucleotide.

[0255] Embodiment 32 is directed to a system of the second linker peptide of the GLP-1 fusion peptide. The amino acid residues are then contacted with an excess of an azaphosphine reducing agent to form the GLP-1 fusion peptide. and the reduced cysteine residue reacts with the electrophile. 32. The method of claim 31.

[0256] Embodiment 33 is an embodiment in which the azaphosphine reducing agent is 1,3,5-triaza-7-phosphine 32. The compound according to claim 32, wherein the compound is tricyclo[3.3.1.1]decane (PTA) or a derivative thereof. This is the method described.

[0257] Embodiment 34 relates to a method for treating a conjugate according to any one of embodiments 1 to 30 with a pharmaceutical and a carrier acceptable thereto.

[0258] Embodiment 35 is a method of treating or preventing obesity in a subject in need thereof. and administering to said subject in need thereof an effective amount of the pharmaceutical composition of embodiment 34. The method includes:

[0259] Embodiment 36 includes administering to a subject in need thereof an effective amount of a pharmaceutical composition. By administering the pharmaceutical composition, the subject's body weight may be increased by about 5% to about 10%, or by about 10% to about 15%, about 15% to about 20%, or about 20% to about 25% weight loss, The method according to embodiment 35.

[0260] Embodiment 37 is a method for treating or preventing a disease or disorder in a subject in need thereof. The method, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, ROHM, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia Hypertension, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerotic arteries sclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or lipid levels Other cardiovascular risk factors, such as hypertension and cardiovascular risk factors associated with steroids, osteoporosis, inflammation, Alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and / or eczema, wherein the method comprises administering to the subject in need thereof an effective amount of 35. A method comprising administering 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 1 diabetes. is.

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

[0264] Embodiment 41 is a method according to embodiment 3, wherein the disease or disorder is metabolic syndrome. 7. The method according to claim 7.

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

[0266] In embodiment 43, the disease or disorder is nonalcoholic steatohepatitis (NASH). 38. The method of embodiment 37.

[0267] Embodiment 44 is a method for treating a patient in which the disease or disorder is non-alcoholic fatty liver disease (NAFLD). 38. The method of embodiment 37, wherein

[0268] Embodiment 45 is directed to a method for reducing at least one of food intake or body weight in a subject in need thereof. an embodiment of a method for reducing both of the above-mentioned symptoms by administering an effective amount of 34. A method for treating a rheumatoid arthritis comprising administering a pharmaceutical composition according to claim 34.

[0269] Embodiment 46 includes administering to a subject in need thereof an effective amount of a pharmaceutical composition. by about 5% to about 10% compared to the food intake of the subject before administration of the pharmaceutical composition. , 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 5 46. The method of embodiment 45, wherein food intake is reduced by 0%.

[0270] Embodiment 47 is a method for improving Y2 receptor activity and GLP-1 receptor activity in a subject in need thereof. 10. A method of regulating receptor activity, comprising administering to a subject in need thereof an effective amount of 34. A method for treating a rheumatoid arthritis comprising administering a pharmaceutical composition according to claim 34.

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

[0272] Embodiment 49 is an embodiment in which the injection is delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously. 49. The method according to claim 48.

[0273] Embodiment 50 is an embodiment in which the pharmaceutical composition is administered in combination with a second therapeutic agent. The method according to any one of aspects 35 to 49.

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

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

[0276] Embodiment 53 is a method for administering the pharmaceutical composition once, twice, three times, four times, five times, or six times per week. 52. The method of embodiment 51, wherein the method is administered once.

[0277] Embodiment 54 is a method for administering the pharmaceutical composition once, twice, three times, or four times per month. , the method according to embodiment 51.

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

[0279] Embodiment 56 is a pharmaceutical comprising a conjugate according to any one of embodiments 1 to 30. A method of producing a composition comprising combining the conjugate with a pharmaceutically acceptable carrier. and combining the compounds to obtain a pharmaceutical composition.

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

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

[0282] Embodiment 59 is a method for producing a medicament comprising administering to a subject the method of claim 1, wherein the first linker peptide comprises the amino acid sequence of SEQ ID NO: 60. 59. The isolated hinge-Fc region platform peptide of embodiment 57 or 58. is.

[0283] Embodiment 60 is a method for preparing a peptide comprising administering to a subject the steps of: 60. The isolated polypeptide of any one of embodiments 57 to 59, comprising the amino acid sequence of It is a hinge-Fc region platform peptide.

[0284] Embodiment 61 is a method for preparing a peptide comprising the steps of: and a conjugate comprising the hinge-Fc region platform peptide.

[0285] Embodiment 62 is a method for treating a cyclic PYY peptide according to the present invention, wherein the targeting peptide is a cyclic PYY peptide represented by Formula I or or a derivative or pharmaceutically 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 (provided that 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- and Z4 is K, A, E, S, or R; Z7 is A or K; Z9 is G or K; Z 11 is D or K, Z22 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 derivative is one or more selected from the group consisting of amidation, acylation, and PEGylation. The compound according to embodiment 61, which is a compound of formula I modified by more than one process. It's Jugate.

[0289] Embodiment 63 is a conjugation of a hinge-Fc region platform peptide with a target peptide. A method for producing a conjugate comprising: forming a conjugate between a second linker peptide and the target peptide; and forming a covalent bond between the [Example]

[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 following synthetic descriptions are for illustrative purposes and in no way limit the scope of the invention. It is not intended to define

[0291] The NTSC cyclic PYY (NTSC-PYY) analogs or derivatives of the present invention can be used in automated using a peptide synthesizer, traditional bench synthesis, or a combination of both approaches , Merrifield(J.Am.Chem.Soc.,85:2149-2154( As outlined in

[1963] , the amino acids are linked together to form successive peptide linkages between amino acids. They can be synthesized by various known conventional procedures, preferentially by solid phase peptide synthesis ( The conventional procedure for peptide synthesis is to synthesize a single amino acid residue. A free amino group (other reactive functional groups are suitably protected) and a free cap of another amino acid The reaction involves condensation between a carboxyl group (whose reactive functional group is also suitably protected) and a peptide. An example of a condensing agent typically used for the formation of the benzoyl bond is 1-hydroxybenzotriazole. hydroxyimino)acetate (HOBT) or ethyl cyano(hydroxyimino)acetate (Oxyma Pur Diisopropylcarbodiimide (DIC), 2-(1H-benzotriazole) (1-isotriazolyl)-1,1,3,3-tetramethylaminium hexafluoro Phosphate (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-tetramethyl Aminium hexafluorophosphate (HCTU), 1-cyano-2-ethoxy-2- Oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophos Phate (PyOxim), 2-(1H-benzotriazol-1-yl)-1,1,3 ,3-Tetramethylaminium tetrafluoroborate (TBTU) bromo-tris-pyridinium lolidino-phosphonium hexafluorophosphate (PyBroP) .

[0292] The automated peptide synthesis method was described by Yu (J. Org. Chem., 57:4781- 4784 (1992)) and more recently by Palasek (J. Pept. S ci.,13:143-148(2007)), room temperature (rt ) or at elevated temperatures, preferably through the application of microwave heating.

[0293] The compounds of the present invention (C-terminal amides) can be prepared by reacting a suitable N-α-FMOC protected amide with The carboxy terminus of the amino acid is coupled to a conventional solid phase resin using a suitable coupling agent. -α-FMOC (9-fluoroenylmethyloxycarbonyl) protected amino acid method Suitable conventional, commercially available solid phase resins include Ri Rink Amide MBHA Resin, Rink Amide AM Resin, Tentagel S RAM R esin, FMOC-PAL-PEG PS resin, SpheriTide Rink mesh Resin, ChemMatrix Rink Resin, Sieber Amide Resin, TG Sie The resin-bound FMOC-amino acid is then converted to N,N- Either dimethylformamide (DMF) or 1-methyl-2-pyrrolidone (NMP) The FMOC-protected amine may be deprotected by exposure to 20% piperidine in water, which treatment results in The additional FMOC-protected amino acid is then subsequently , which are sequentially coupled and deprotected to give the desired resin-bound, protected peptide. In certain cases, another amine in the peptide sequence that withstands the FMOC deprotection conditions is generated. It may be necessary to utilize an orthogonal reactive protecting group for 4-methyltrityl (M The protecting groups, 4-methoxytrityl (Mmt) and 4-methoxytrityl (tt), were both prepared using 1% trifluoride. It can be removed by trichloroacetic acid (TFA) / dichloromethane (DCM) treatment, or it is preferred. or allyloxycarbonyl (alloc; Pd(PPh3)4 (tetrakis(trifluoromethyl) (phenylphosphine)palladium(0) / PhSiH3 (phenylsilane) treatment Removable), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethylene Chill (Dde; removable by treatment with 2-3% hydrazine / DMF), and 1 -(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutanol Chill (ivDde; can be removed by treatment with 2-3% hydrazine / DMF) It can be used effectively in such cases.

[0294] In conventional peptide synthesis methodologies, the reactive side chains of the alpha amino acids are generally linked and Protected throughout the synthesis with suitable protecting groups to render them inert to the deprotection protocol. Although several protecting groups for amino acid side chains are known in the art, In the present specification, the following protecting groups are most preferred: serine, threonine, glutamic acid, aspartic acid, tert-Butyl (t-Bu) for glutamic acid and tyrosine; asparagine, glutamine trityl (Trt) for cysteine, homocysteine, and histidine; tryptophan (Tf); tert-butyloxycarbonyl (Boc) for the ε-amino groups of methylamino and lysine; and Arginine is substituted with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl These protecting groups can be removed by treatment with strong acids such as concentrated trifluoroacetic acid (TFA). It is removed when

[0295] Upon completion of SPPS, the resin-bound, side-chain protected peptide is deprotected and triisopropylated. Various combinations of isopropylsilane (TIPS), water, phenol, and anisole were used. A cleavage cocktail consisting primarily of (TFA) with a carbocation scavenger was used. The peptide / cocktail filtrate was then precipitated with cold ether. The crude solid peptide is isolated by cleaving the protected peptide attached to the Sieber resin. In the particular case of protected peptides, cleavage of the protected peptide from the resin involves deprotection of the side chains. This can be advantageously achieved by repeated treatment with 1-2% TFA in DCM without causing any degradation. Once isolated, further manipulation of the protected peptide may be carried out in a solution phase reaction. Finally, the protected peptide can be purified using a separate treatment with a cleavage cocktail. The crude product thus obtained may then be optionally deprotected and precipitated as described above. The peptides are dissolved in a predominantly aqueous solvent system containing an organic co-solvent such as acetonitrile or ethanol. It dissolves in water at low concentrations (approximately <4 mg / mL). When the pH of the solution is increased to >5, The peptide undergoes an intramolecular cyclization reaction to form the corresponding crude NTSC PYY analog of the present invention. The NTSC PYY analogs thus formed are generally known in the art. The preferred methods of peptide purification used herein are: A suitable method is reversed-phase high performance liquid chromatography (HPLC). The peptides are characterized by liquid chromatography / mass spectrometry (LC / MS).

[0296] The general scheme and method for producing the cyclic PYY peptides of the present invention is U.S. Patent Application No. 15 / 794,231 filed October 26, 2017 and U.S. Patent Application No. 15 / 794,231 filed October 26, 2017 The invention is described in U.S. patent application Ser. No. 15 / 794,171, filed on May 26, 2015. The contents of which are incorporated herein by reference in their entirety.

[0297] The following examples and embodiments described herein are for illustrative purposes only and should not be considered as such. Various modifications or variations thereto will be suggested to those skilled in the art and will fall within the spirit and scope of the present application and the accompanying patents. It is understood that all publications cited herein are within the scope of the claims. The patents and patent applications are incorporated herein by reference in their entirety for all purposes. It can be enjoyed.

[0298] Example 1: Glucagon-like peptides from transiently transfected mammalian cells Expression and purification of 1 (GLP-1) fusion peptide The synthesized gene fragments were then transduced into pCD using various restriction endonucleases. GLP-1 fusion by cloning into NA3.1-derived mammalian expression vector The DNA construct for the peptide was generated. The synthesized gene fragment was used to produce the fusion protein. The following components of the substance (from N-terminus to C-terminus): GLP-1 peptide or GLP-1 variant a first linker peptide, a hinge-Fc region peptide, and a cyclic PYY peptide; one of the second linker peptides containing Cys for conjugation to the tide or designed to contain the entire coding DNA sequence.

[0299] Purify plasmid DNA of the fusion protein expression construct according to the manufacturer's recommendations. ExpiCHO-S(TM) Cells (ThermoFisher Scientific( Transiently transfect the cells with 100% ribosomal RNA (Waltham, MA, Cat. No. A29127) The GLP-1 fusion protein was expressed in the cells. xpiCHO-S™ cells were cultured at 37°C, 8% CO2 and a shaking table set at 125 RPM. 2. In an incubator, add ExpiCHO™ Expression Medium (ThermoFisher Cells were maintained in suspension in PBS (Chemical Biosciences, Cat. No. A29100). , 6.0 × 10 6 The cells were diluted to 1000 cells / mL and maintained at a cell viability of 98% or higher. Transfection was performed using ExpiFectamine™ CHO transfection. Kit (ThermoFisher Scientific catalog number A29131 ) for every 1 mL of diluted cells to be transfected. Plasmid DNA was used and diluted in OptiPRO™ SFM complexed medium. xpiFectamine™ CHO Reagent in a 1:3 ratio (v / v, DNA:reagent) The diluted DNA and transfection The reagents were combined for 1 minute to allow the DNA / lipid complex to form, and then added to the cells. After overnight incubation, ExpiCHO™ feed and ExpiFectami The ne™ CHO enhancer was added to the cells. The cells were cultured at 37°C with shaking for 5 days. After culturing, the culture supernatant was collected.

[0300] Culture supernatant from transiently transfected ExpiCHO-S™ cells was After clarification through centrifugation (30 min, 6000 rpm), the mixture was filtered (0.2 μ PES membrane). , Corning (Corning, NY). First, Pall C Entramate Tangential Flow Filtration System Large-scale transfections (5-20 liters) were concentrated 10-fold using 10 PBS (pH 7.2) was added to the supernatant to a final concentration of 1x, and then AKTA FPL Using a C chromatography system, approximately 20 mg of protein per mL of resin was obtained. Equilibrated (DPBS, pH 7.2) HiTrap MabSelect S at paired concentrations ure Protein A column (GE Healthcare; Little Chalfo After packing, the column was loaded into a 10-column column. In some cases, the column was washed sequentially with 20 column volumes of 100 mL of DPBS (pH 7.2). 10 mM Tris, 2.5 M NaCl, 50 mM sodium caprylate (pH 9) and 10 mM An additional wash of 1000 volumes of DPBS (pH 7.2) was included. Protein was then transferred to the column via a 1000 sieve. The protein fraction was eluted with 0.1 M sodium acetate (Na) (pH 3). The elution fraction volume was 0.01 mg / ml into a tube containing 2.0 M Tris (pH 7). Peak fractions were pooled and, if necessary, the pH was adjusted with additional Tris. The pH was adjusted to approximately 7. The purified protein was filtered (0.2 μm) and purified using a BioTek Syner The concentration was determined by absorbance at 280 nm on a gy™ HTM spectrophotometer. The quality of the purified protein was confirmed by SDS-PAGE and analytical size exclusion HPLC (Dionex Endotoxin levels were assessed by turbidimetric LAL assay (Py rotell®-T, Associates of Cape Cod; FA Measurements were performed using a 3000kJ / s ion exchange membrane (EMM, MA).

[0301] Alternatively, purify the fusion protein expression construct using the purified plasmid according to the manufacturer's recommendations. Expi293F™ cells (ThermoFisher Scientific) were cultured with 100% lysed DNA. fic, Cat. No. A14527) to transiently transfect GL The P-1 fusion protein was expressed in the cells. ™) cells in a shaking incubator set at 37°C, 8% CO2 and 125 RPM. and Expi293F™ Expression Medium (ThermoFisher Scientific Cells were maintained in suspension in 100% PBS (Cat. No. A1435101). Cells were passaged and maintained at 2.5× 10 6 The transfection was performed by diluting the solution to 1000 cells / mL and maintaining cell viability above 95%. The transfection was performed using the ExpiFectamine™ 293 transfection kit (T HermoFisher Scientific Catalog Number A14525 One microgram of plasmid D was added to every 1 mL of diluted cells to be transfected. NA was used and diluted in OptiMEM™ SFM complex medium. tamine™ 293 reagent was used in a 1:2.6 ratio (v / v, DNA:reagent), The diluted DNA and and transfection reagent are combined for 20 minutes to allow DNA / lipid complexes to form, and then After overnight incubation, Expi293™ feed and E xpiFectamine™ 293 enhancer was added to the cells. The cells were incubated at 37°C. After culturing with shaking for 4 days, the culture supernatant was collected.

[0302] Culture supernatant from transiently transfected Expi293™ cells was centrifuged After clarification through separation (30 min, 6000 rpm), the mixture was filtered (0.2 μ PES membrane, C The supernatant was collected by filtration. 10x DPBS (pH 7.2) was added to the supernatant. After bringing the concentration to 1x, the chromatograms were analyzed using an AKTA FPLC chromatography system. The lipid was equilibrated at a relative concentration of approximately 20 mg of protein per mL of lipid (DPBS, pH 7. 2) HiTrap MabSelect Sure Protein A column (GE Heal After packing, the column was washed with 10 column volumes of DPBS, pH 7.2. The protein was eluted with 10 column volumes of 0.1 M Na-acetate (pH 3). The protein fraction was collected in a tube containing 20% of the elution volume of 2.0 M Tris (pH 7). The peak fractions were pooled and, if necessary, neutralized immediately by elution with PBS. The pH was adjusted to approximately 5.5 with Tris. The purified protein was filtered (0.2 μl). , absorbance at 280 nm on a BioTek Synergy™ H™ spectrophotometer The quality of the purified protein was determined by SDS-PAGE and analytical size analysis. Endotoxin levels were assessed by exclusion HPLC (Dionex™ HPLC system). The turbidimetric LAL assay (Pyrotell®-T, Associates Inc.) was used. f Cape Cod).

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

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

[0305] Furthermore, when preparing GLP-1 fusion peptide constructs, the burden of proteolysis is important. To avoid the possibility of cloning, we used a hinge-Fc region peptide (i.e., human IgG4). The carboxy-terminal lysine was deleted from the final construct.

[0306] Example 2: Production of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates Reduction of GLP-1 fusion proteins Method A The purified GLP-1 fusion protein is prepared by combining the recombinant protein with at least one GLP-1 fusion protein per linker. and a genetically engineered second linker peptide containing a cysteine residue. The cysteine residue is either disulfide bonded to another cysteine or is Is it intramolecularly disulfide bonded to an exogenous cysteine residue in the sol or growth medium? Such a heterologous GLP-1 fusion protein solution (5 to 100 ml of Tris-OAc) 12 mg / mL, pH 5.4-6.4), an excess of phosphine reducing reagent 1,3,5-trimethyl-2-propanol Addition of riaza-7-phosphaadamantane (PTA, 15-40 equivalents) followed by 10 0 mM EDTA (final concentration 1-2 mM) was added. The resulting reaction mixture was The mixture was stirred gently at room temperature until the reduction of the methyl group was complete (4-16 hours). The amine and residual PTA were removed by desalting chromatography.

[0307] Method B Heterologous GLP-1 fusion protein solution (5–15 mg / mL in Tris-OAc, pH 5.0 ~6.5), an excess of the phosphine reducing reagent tris(2-carboxyethyl)phosphine (TCEP, 4-25 equivalents) was added, followed by 100 mM EDTA (final concentration 1 mM The resulting reaction mixture was gently stirred at room temperature overnight, and then TrisOAc was added. The fully reduced GLP-1 fusion protein was applied to a desalting column equilibrated with HCl (pH 7.0). Protein (the hinge disulfide bond is broken) is converted to dehydroascorbic acid (DHAA, After treatment with 10 equivalents of HCl for 2 hours, the reduced GLP-1 fusion peptide was oxidized to complete The decapped second linker peptide has an engineered cysteine. The complete GLP-1 fusion peptide was obtained.

[0308] Preparation of GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugates Solution of reduced GLP-1 fusion peptide in TrisOAc (3-10 mg / mL, pH 5. 5) Dissolve cyclic PYY peptide (2.6-3.0 equivalents, 10-15 mg / mL) in deionized water. To protect reactive thiols from metal-catalyzed oxidation, a concentrated solution of EDTA was added. 100 mM EDTA solution was added to the reaction mixture until the concentration reached 1 mM. The pH of the reaction solution was adjusted to pH 8.0-8.2 by adding dropwise 1M of HCl (pH 9.1). The reaction was allowed to proceed for 3-7 hours at room temperature, then at 5°C for 18 hours. S is the conjugation completion, GLP-1 fused cyclic PYY peptide conjugate This reaction was directly subjected to Protein A purification to obtain Remove excess cyclic PYY peptide or (remaining reactive cyclic PYY peptide) The reaction was quenched by adding 5 equivalents of cysteine (to cap the reaction mixture). The crude conjugate was directly purified by hydrophobic interaction chromatography (HIC). Afterwards, the conjugate was adsorbed with Protein A. The conjugate was eluted (sodium acetate, pH 3.5). The final product was obtained by neutralizing the mixture with Tris buffer to a pH of 7 to 7.5. However, HIC-purified conjugates may not exhibit additional protein synthesis if performed prior to HIC purification. A PD-10 column can be used to exchange the buffer into the desired one without polishing. do.

[0309] The conjugation reaction may also be carried out at low temperature (5°C) and monitored by LCMS. You can also leave it to dry for about 48 hours.

[0310] In some cases, reactivity in the second linker peptide of the GLP-1 fusion protein The cysteines were capped with bromoacetamide to form stable thioether bonds. These acetamide-modified GLP-1 fusion proteins were synthesized as follows: Prepared: GLP-1 fusion protein solution (approximately 12 mg / mL in Tris-OAc, pH 5 4-6.4), an excess of phosphine reducing reagent 1,3,5-triaza-7-phosphatase Damantane (PTA, 15-20 equivalents) was added, followed by 100 mM EDTA (final The resulting reaction mixture was gently stirred at room temperature for 18 hours. 15-20 equivalents of bromoacetamide was added to the GLP-1 fusion peptide solution, and the reaction mixture was The pH value of the mixture was adjusted to pH 7.5-8.1 with Tris buffer (1 M, pH 9.1). After 2 hours, the reaction mixture was purified by hydrophobic interaction chromatography (HIC). Immediately afterwards, Protein A polishing was performed.

[0311] Table 8 shows the isolated yields of the GLP-1 fusion peptide-linked cyclic PYY peptide conjugates. The first linker peptide is the same as GLP-1 fusion peptide 64 (GF64). AP linker, and the second linker peptide is similar to GLP-1 fusion peptide 8 (GF8). GLP-1-conjugated cyclic PYY peptide conjugates when the peptide was a G4A linker It was observed that the isolated yield of was poor.

[0312] [Table 8]

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

[0314] [Table 9]

[0315] Example 4: In vitro assay Lance competitive cAMP immunoassay (Perkin E) was performed according to the kit instructions. Intracellular cAMP was measured using a chemiluminescence assay (Chimer, Waltham, Massachusetts). Conjugates were tested for functional activity and in vitro potency in cell-based assays measuring The nucleotides were screened against mouse or human GLP-1R or NPY2 receptors (Y2 Clonal HEK293 cells stably expressing GLP1R were used in the assay. Thaw the expressing cells and resuspend them in HBSS, 5 mM HEPES, 0.1% BSA, 0.5 mM The cells were suspended in IBMX. The cells were mixed with the anti-cAMP antibody provided in the kit and plated in a 384-well white plate. In an opti-plate, HBSS, 5mM HEPES, 0.1% BSA was added continuously. The diluted conjugate was added to the immunoassay after 10 minutes of incubation at room temperature. The assay plate was read by an Envision plate reader (excitation mix). as a TR-FRET assay (at 320 nm, emission at 615 nm and 665 nm) Read and analyze data using Prism statistical software (GraphPad Software) EC of compounds using the software San Diego 50 values were calculated.

[0316] To determine the activity of compounds at the Y2R receptor, HEK29 expressing Y2R was used. 3. Cells were cultured in 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 1% DMEM supplemented with penicillin / streptomycin and 600ug / mL G418 Cultured in high glucose medium (Cellgro) and grown in medium without G418 for 384 days. On the day of the assay, the cell growth medium was removed from the cells. Extracted in 5mM HEPES, 500uM IBMX, and 0.1% BSA in HBSS 6 μL of the conjugate (2×) was added. Next, forskolin (2×, 5 μM maximum) was added. 6 μL of stimulation buffer containing 1:100 ATP (final concentration) and LANCE cAMP antibody was added to the cells. After incubation at room temperature for 25 minutes, 12 μL of detection mixture was added. , cAMP concentrations were quantified in a LANCE cAMP immunoassay.

[0317] Data analysis Data from the Envision plate reader is (615nm / 665nm) x1 The results were expressed as relative fluorescence units (RFU), calculated as 0,000. All samples were run in triplicate. The unknown cAMP concentration in each well was measured using the known cAMP concentration in each plate. MP concentrations were interpolated from reference standards. 50 , Log(EC 50 ), HillSlop Parameters such as e(nH), maximum and minimum values are used in the nonlinear weighted least squares application. The cAMP concentration values were plotted against the logarithmic compound concentration fitted with a 4-P model using the This was obtained by testing.

[0318] [Table 10]

[0319] [Table 11]

[0320] SEQ ID NO: 23 for the GLP-1R potency of SEQ ID NO: 135, 134, 146, and 176 8, 242, 262, 253, and 241, exendin 4(1-39)( A GLP-1 fusion peptide having the sequence SEQ ID NO: 58 is conjugated to a PYY peptide. Based on these results, we have demonstrated that exendin lost GLP-1R potency after administration. Tests using a GLP-1 fusion peptide with phenylalanine 4(1-39) (SEQ ID NO: 58) This was not desirable in this experiment.

[0321] Human NPY2R primary screening assay: In vitro co-administration with GLP-1 fusion peptide Used to screen NPY2R potency of cyclic PYY peptide conjugates The method described here targets adenylate synthase through modulation of the human NPY2R Gi-protein coupled receptor. Designed to measure inhibition of forskolin-induced cAMP produced by ATPase The cell-based assay was performed using CHO cells transfected with human NPY2R. Forskolin-induced cAMP production in PY-K1 cells (DiscoverX) Activation of NPY2R by Y analogs and controls resulted in a dose-dependent decrease in FRET-based cAMP levels were measured in a competitive cAMP immunoassay.

[0322] Cells were removed from frozen storage, thawed in a 37°C water bath, and resuspended in 40 mL of 1x DPBS (p The cells were added to a 1000 ml PBS containing 100% ethanol (H7.2) (Gibco) and filtered through a cell strainer. The cells were centrifuged for 5 minutes and the supernatant was discarded. The cell pellet was collected at 0.125 x 10 6cells / mL At density, DMEM / high glucose, 10% HIFBS, 1% Pen / Strep, 1 The cells were resuspended in 1% L-glutamine and 1% sodium pyruvate at a final concentration of 5,000 cells / well. Add 40 μL / well to a collagen-coated white 384-well plate. The cells were then aliquoted into wells and incubated at 37°C and 5% CO2 for 16 to 24 hours. Repeat the incubation of cells in the plate twice by adding 80 μL / well of 1x DPBS. Wash and decant the supernatant. Glucolin, 0.1% BSA, 0.5 mM 3-isobutyl-1-methylxanthine (I Prepare sample and control dilutions in PBS (BMX) and add 20 μL / well of each sample to the designated well. The mixture was added to the wells and incubated at room temperature for 30 minutes with shaking. Add the cAMP detection reagent mixture to each well of the assay plate and incubate at room temperature for 2-24 hours. Incubate with shaking. Read the plate on a plate reader. The samples were measured in quadruplicate. A nonlinear weighted least squares application was used within the R environment. Raw LANCE cAMP values over log compound concentrations fitted with a 4-P model were plotted. Data were analyzed by quantification.

[0323] Human GLP-1R Primary Screening Assay: In Vitro GLP-1 Fusion Proteins and GLP-1R activity of GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugates The method used to screen for the potency of human GLP-1R Gs-protein coactivators was A cell-based assay designed to measure cAMP production through modulation of ATP receptors GLP-1 fusion peptide and GLP-1 fusion peptide-conjugated cyclic PYY peptide Concentration-dependent activation of GLP-1R by the conjugate was followed by a transcription factor 1 (TNF-α) at the human GLP-1R. TRFRET-based competitive cAMP suppression of cAMP accumulation in transfected HEK cells was measured by assay.

[0324] HEK cells transfected with human GLP-1R were thawed on the day of the assay and diluted to 1x 0.5 x 10 in HBSS, 5 mM HEPES, 0.1% BSA, and 1 mM IBMX 6 The cells were resuspended at 500 cells / mL and 10 μL was added to each well of a 384-well plate (5 000 cells / well). Samples were diluted in 1x HBSS, 5mM HEPES, 0.1% BSA Dilute with 10 μL / well of the assay plate and incubate at room temperature for 30 minutes. cAMP detection reagent was added at 20 μL / well. The plate was incubated at room temperature for 2 hours. After incubation, the plates were read on a plate reader. All samples were measured in quadruplicate. The 4-P model was fitted using the nonlinear weighted least squares application in the R environment. Data were obtained by plotting raw LANCE cAMP values across log compound concentrations. The data was analyzed.

[0325] Example 5: GLP-1 fusion peptides and GLP-1 fusion peptide-conjugated cyclic PYY conjugates Xylose analysis in dugate Sample preparation: To prepare samples for peptide mapping, each protein molecule is (1 mg / mL): GLP-1 fusion peptide-coupled cyclic PYY conjugate (SEQ ID NO: 2 44), GF40 (SEQ ID NO: 152), and GF34 (SEQ ID NO: 146) were added to a 100 ml PBS containing 100 ml of GF40 (SEQ ID NO: 152) at pH 8.0. These samples were diluted 1:4 with 8M guanidine / HCl buffered at a final concentration of Add 1M DTT (Sigma 43816-10ML, BioUl) until the concentration reaches 25 mM. An aliquot of 1000 mg ... Iodoacetamide (Sigma, A3221-10VL) or N-methylmaleimide Alkylation was carried out using either (NEM) (Sigma, E3876). Freshly prepared 1M alkylating agent was added to reach approximately 50 mM, and the sample was stored in the dark. The reaction mixture (Sigma 43816- (10ML, BioUltra) Add 15µL of 1M DTT for every 400µL The alkylation reaction was quenched by 50 mM Tris, 1 mM CaCl (pH The solution was desalted in a Zeba Spin desalting column (Thermo Fisher Scientific) according to the manufacturer's protocol. Samples were desalted using 50 mM acetic acid (lyophilized). After adding 1 μL of 1 μg / μL trypsin reconstituted in PBS (supplied with dried trypsin), , 37°C for 4 hours, trypsin (Promega sequencing grade, V511A ) and a 60 μL aliquot of the digested protein was diluted with 100% T The trypsin digestion was quenched by adding 0.6 μL of FA. The mixture was resuspended in a microvial and placed in an autosampler set at 4°C for LC / MS analysis. I put it in.

[0326] Liquid chromatography and mass spectrometry: Agilent Inf Inity 1290 UHPLC (Agilent Technologies, parts Numbers: G1330B, G4226A, G4220A, G4212A) to Agi lent AdvanceBio Peptide Map Micro Bore R apid Resolution Column(1×150mm, 2.7μm, part number Two micrograms (~10 μL) of digested protein was injected into a tube (No. 863600-911). The column temperature was maintained at 65° C. Mass spectrometry grade HPLC solvent (0.1% formic acid and B: 100% ACN in 0.1% formic acid) in VWR (Part Numbers: LC452-1, LC441- Proteolytic peptides were purchased from 1). The column was eluted with a 50 min gradient of CN. A positive spray voltage of 3.5 kV, 20 ( (arbitrary units) sheath gas, 7 (arbitrary units) auxiliary gas, 299°C ion transfer tube, and 1 via a heated electrospray ionization probe (HESI) using a 00°C evaporator. The column eluate was analyzed by an Orbitrap Q-Exactive mass spectrometer (Thermofisher Science The top five abundant peptides observed in the full MS scan were introduced into the entific Data-dependent acquisition was performed by sequentially dissociating the doped ions. Trap detection, 70,000 resolution, mass range 150-2000 m / z, automatic gain control ( AGC) target 1.0e6, maximum injection time 50 ms, precursor scan mass The analysis was performed. One microscan spectrum was acquired in profile mode. The criteria for determining the sequence are monoisotopic precursor-selected peptides, with charge states of 2 to 10. 7, Dynamic Exclusion: 6.0 seconds, and precursor intensity threshold 5e4. The precursor was separated by a quadrupole with an isolation width of 1.6 m / z sent to the collision cell. The peptides were isolated. The results of the normalized collision energy 28 (arbitrary units) were used to calculate the peptide The mass spectrometer was used for high-energy collisional dissociation (HCD) of ions using an orbitrap setup. Product ions transferred to the Orbitrap for analysis were analyzed at an isolation power of 17,500 and 2 00-2000 m / z range, AGC target 5e5, maximum injection time 100 ms, centroid mode The spectrum is one microscan acquired at 1000 Hz.

[0327] Data analysis: The raw LC-MS / MS data were analyzed using Byonic software (version 2.15.7) (Protein Metrics) to perform database searches. The following parameters were used for data retrieval: precursor ion mass tolerance, 8 ppm; product ion mass tolerance, 20 ppm for HCD spectra; variable modification Examples include cysteine carbamidomethylation, or N-methylmaleimide (NEM), cysteine cysteine-DTT adduct formation, and, if present, cysteine-peptide YY (PYY) conjugates It contains asparagine, serine, xylose deamination, and byonic glycol Serine and threonine O-linked glycans of 78 mammalian O-linked glycans obtained from a glycan library -glycosylation. For all searches, complete sequences with up to two truncations were included. A total trypsin specificity search was selected, with the protein false discovery rate (FDR) set at 1%. The protein database contained the amino acid sequence of each protein. The nic search results were analyzed using Bylogic software (version 2.15.296) (Pr Protein Metrics) and peptides were analyzed with a maximum precursor m / z error of 8ppm. m and XIC area window 2 minutes, minimum Byonic score 15, maximum Altransco Filtering was performed based on a primary rank score of 0.99. By considering all peptide XIC peak areas of the isoforms, serine xylose The xylosylation level at each serine residue was then calculated based on the total XIC peaks. The area was calculated as a fraction of the total area of the sample. 6.1 (MaxPlank Institute) Andromeda search engine A database search was performed using the following to obtain PTM site localization probabilities: BIOVIA plots the masses corresponding to PYY and trypsin-cleaved PYY structures. The data was obtained using the software and is reported in Table 12.

[0328] [Table 12] Xylose modification at linker serine: Site identification and modification level ND = not detected by MS / MS, NQ = not quantified; NA = serine position in the linker does not apply to molecules, ** Number: SEQ ID NO: The brackets indicate the position number of SEQ ID NO: 152 vinegar.

[0329] Table 12 shows the peaks / peaks of interest based on putative modifications, i.e., serine xylosylation. <XIC%> is the sum of all species contributing to site-specific xylosylation according to the following equation: Identified and quantified using peak integration in Bylogic, normalized to Site-specific xylosylation is summarized.

[0330] XIC% of modification = (XIC of modified peptide / ΣXIC of peptide counterpart) × 100 All three molecules examined contain a GLP-1 peptide and a hinge-Fc region peptide. However, SEQ ID NO: 15 is composed of the same first linker peptide AS(G4S)2. The GLP-1 fusion peptide found in GF40 and SEQ ID NOs: 244 and 146 (GF3 4) As shown in Table 12, due to the difference between the GLP-1 fusion peptide and the The positions of the three serine residues in SEQ ID NO: 152 differ from SEQ ID NOs: 244 and 146. Analysis of the peptides identified Ser-41 and Ser-46 of SEQ ID NOs: 244 and 146. , three site-specific serine residue xylosylation events at Ser-51, and SEQ ID NO:1 A serine residue xylosylation event at Ser-33 of 52 was demonstrated. Quantification of xylosylation was performed by reversed-phase liquid chromatography, which revealed that most xylosylated peptide species were For example, in SEQ ID NO: 244, Se Ser-41 was about 9%, while Ser-46 and Ser-51 were about 7%. In total, all three sites of SEQ ID NO: 146 were xylosylated at approximately 5%. Xylosylation of was found to occur to a lesser extent, about 0.1%.

[0331] The G4S linker region of therapeutic proteins is susceptible to xylosylation, and Levels have been shown to vary based on linker length (Wen et al., " The propensity for xylosylation in(G4S) n linkers occur when n>2,” Anal.Chem.85:4 805-12(2013);Sphar et al.,Protein Sci.22 :1739-53(2013),Sphar et al.,mAbs 6:904-1 4 (2014)). The first linker peptide, AS(G4S), is found in all three molecules. The two linkers exhibit xylosylation at all or some of the serine residues, and these derivates Based on the data, when preparing the GLP-1 fusion peptide construct, a serine-containing The use of a first linker peptide that does not bind to the nucleotide sequence 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 containing sodium heparin anticoagulant was analyzed for stability. Fresh whole blood received on the day of sample preparation was centrifuged at 3500 RPM for 15 minutes. The blood was processed for plasma by filtration and then plasma was isolated. The plasma was combined, filtered through a 0.2 μ filter, and warmed to 37°C. LP-1 fusion peptide and GLP-1 fusion peptide-conjugated cyclic PYY conjugate dual agonist Spike the sample with an agonist (DA) analog and immediately collect a time zero sample. Samples were incubated at 37°C and stored at -80°C until analysis. On the day of analysis, samples from time zero and subsequent time points were collected and stored at -80°C until analysis. The samples were thawed and analyzed together in a functional cell-based bioassay.

[0333] Bioactive GLP-1 fusion peptides and GLP-1 fusion peptide co-reactivity in human plasma samples Methods used to detect and quantify levels of cyclic PYY-conjugated DA analogs The method measures cAMP production through modulation of the human GLP-1R G protein-coupled receptor. The GLP-1 fusion protein and GLP-1R activation by cyclic PYY peptide conjugates and GLP-1 fusion peptides Following concentration-dependent activation, in HEK cells transfected with human GLP-1R cAMP accumulation was measured using a TRFRET-based competitive cAMP assay. Thaw transfected HEK cells on the day of the assay and resuspend them in 1x HBSS, 5 mM 0.5 x 10 in HEPES, 0.1% BSA, and 1 mM IBMX 6 Reassembly in cells / mL The cells were suspended and 10 μL was added to each well of a 384-well plate (5000 cells / well). Stability samples were thawed and diluted in 1x HBSS, 5mM HEPES, 0.1% BSA, 20% plasma in assay buffer consisting of 5 mM EDTA, protease inhibitors Subsequent dilutions were made in assay buffer containing 20% normal human plasma. Reference standards of known concentrations were prepared for each compound in assay buffer containing normal human plasma. Standards and samples were added to each assay plate at 10 μL / well and incubated at room temperature for 30 minutes. The plate was incubated for 1 hour. 20 μL / well of cAMP detection reagent was added. After incubation at room temperature for 2 hours, the plate was read on a plate reader.

[0334] All samples were measured in quadruplicate. Log-transformed concentrations, nonlinear fitting, Log v Response (variable slope) was used to generate a reference standard curve for the compound, and EC 10 and EC 90 Value The concentrations of the stability samples for each compound were determined as the upper and lower limits of the quantitative assay. The residual bioactivity of each stability sample was calculated by extrapolating the corresponding reference standard curve. The starting plasma concentrations were calculated for the zero-time sample over the 168-hour time course. Reported as %. % Starting Plasma Concentration = [Stability Sample Mean] / [Time Zero Mean] x 100.

[0335] Results: GLP-1 fusion peptides SEQ ID NOs: 144 (GF32), 148 (GF36), 1 45(GF33), 151(GF39), 146(GF34), 147(GF35), 1 49 (GF37), 152 (GF40), and dulaglutide control were incubated at 37°C for 7 days. Ex vivo incubation in human plasma and in vitro GLP-1R cAMP Functional stability was measured in a functional assay (Figure 1A). GLP-1 peptide SEQ ID NO: 58 ( The GLP-1 fusion protein with exendin 4(1-39) showed the highest stability. followed by SEQ ID NO: 57 ((A8G, G22E, R36G) GLP-1(7-37) ) and dulaglutide control, SEQ ID NO: 56 ((A8S, A30E) GLP-1(7 Based on these results, the GLP-1 peptide sequence Testing with the Fc fusion protein with number 56 was not preferred in this experiment.

[0336] GLP-1 fusion peptides SEQ ID NOs: 152 (GF40) and 146 (GF34), and These corresponding GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate sequence numbers Nos. 248 and 262, and another GLP-1 fusion peptide SEQ ID NO: 153 (GF41) , along with a dulaglutide control, were tested ex vivo in human plasma at 37°C for 7 days. Functional stability was measured using an in vitro GLP-1R cAMP functional assay. (Figure 1B). The stability of the GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugate was , equivalent to the corresponding unconjugated fusion protein, which is The results showed that the conjugation of tide did not affect the stability of the GLP-1 peptide. GLP-1 fusion peptide SEQ ID NO: 146 (GF34) and GLP-1 fusion peptide The cyclic PYY peptide conjugate SEQ ID NO: 262 had the highest stability, and SEQ ID NO: 152 (GF40), 248, and 153 (GF41) were similar to the dulaglutide control. It had stability.

[0337] Example 7: In vivo mouse stability assay method LCMS method: Plasma samples processed by immunoaffinity capture using anti-human Fc antibodies Afterwards, the samples were digested with trypsin and analyzed by reversed-phase LC-MS / MS on a triple quadrupole mass spectrometer. The peptide at the N-terminus of GLP-1, i.e., GLPD30, was analyzed. HGE (HGEGTFTSDVSSYLEEQAAK (SEQ ID NO: 263)), GLPD3 HGEGTFTSDLSK (SEQ ID NO: 264) for 1, containing active GLP1 The peptide located in the Fc region, i.e., VVS (VV SVLTVLHQDWLNGK (SEQ ID NO: 265)) was monitored as a surrogate for total Fc levels. The peptides located in the cyclic PYY peptide, YYA (YYASLR (SEQ ID NO: 1)), No. 266)) was monitored as a surrogate for PYY levels. Calibration standard curve and quality control Control samples were prepared by spiking the reference standard in plasma and were analyzed in the same manner as the test samples. were treated using the same procedure.

[0338] result The plasma concentration of intact GLP-1 fusion peptide N-terminal fragments 72 hours after administration in mice Plasma exposure levels were measured by LCMS assay and HGE N-terminal GLP- 1 fusion peptide subsequence level (HGE% of Fc). Table 13 The results show that the first linker peptides of SEQ ID NOs: 62 and 66 containing AP repeats are effective against the G4A reaction. These results show that the number of linker peptides was less than that of the first linker peptide of SEQ ID NO: 60, which contains two repeats. Based on the results, a GLP-1 fusion peptide with a first linker peptide containing AP repeats was developed. Testing with the ion beam was not preferred in this experiment.

[0339] [Table 13] BQL = below limit of quantitation

[0340] Acute and subchronic pharmacodynamic studies in DIO mice All rodents used in these studies were obtained from Janssen R&D (Spring, Institutional Animal Care (Institutional Animal Care House, PA) According to a protocol approved by the Institutional Use Committee (IACUC) Animals were maintained at standard temperature and humidity conditions with free access to food and water for 1 They were housed under a 2-hour light / 12-hour dark cycle (unless otherwise noted). Male DIO (60% kcal / 15-week high-fat diet) C57BL / 6T mice (Ta Animals were randomized into groups based on body weight. For the intraperitoneal (IP) glucose tolerance test (IPGTT), vehicle, dulaglutide (purchased from Eli Lilly; Myoderm (Norristown, PA)), or Compounds 1 to 4 (SEQ ID NO: 113, SEQ ID NO: 225, SEQ ID NO: 136, SEQ ID NO: 229) was administered to mice (n=8 / group). 24 hours later (after overnight fasting), the mice One Touch Ultra glucose (1 g / kg) was administered IP. Blood glucose was measured at designated time points using a LifeScan meter, and plasma Insulin (Meso Scale Discovery) was measured at 0 and 10 min. Acute Food Intake (FI) For the weight loss (WL) test, individually housed DIO mice were given vehicle, duo Raglutide or compound was administered, and food intake and body weight were measured over a 3-day period (n=8 / The data are shown in Figures 2A-2B, 3A-3B, and 4A-4B. The data in Figures 3A-3B and 4A-4B show that the GLP-1 fusion peptide is preferably GLP-1. R, and has improved efficacy when conjugated to the cyclic PYY peptide SEQ ID NO:27 The data in Figures 5A to 5B and Figures 6A to 6B show that the GLP-1 fusion peptide It was determined that the upper hinge of the dome contributes to GLP-1R efficacy.

[0341] Example 8: In vivo testing using cynomolgus monkeys method Efficacy Testing Baseline (average of 3 days before day 0) measured during run-in and vehicle treatment Based on average food intake (primary, weighted 80%) and body weight (secondary, weighted 20%), monkeys The animals were selected and randomized into groups for compound treatment. The study consisted of 2 weeks of acclimation / training, Vehicle treatment every 3 days (Q3D) for 3 weeks, and baseline food intake measurements, treatment (5 The animals were given 7 doses of vehicle (Q3D) for 12 days followed by a 2-week rest period over a 9-week period. One of the test articles (dosing volume 0.5 mL / kg) was administered Q3D at approximately 8:00 AM for 5 days. 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). Calorie intake was measured daily, and body weight was measured in Q3. Measured at D.

[0342] Exposure-response analysis Compound 4 and Dextromethorphan were analyzed from in vivo plasma samples of cynomolgus monkeys using two analytical methods. Tulaglutide concentrations were measured.

[0343] Ligand Binding Assay (LBA) of Compound 4 and Dulaglutide in Cynomolgus Monkeys Exposure analysis was performed by Meso Scale Discovery (MSD) Sector Im ager S600(Meso Scale Diagnostics(Rockvil) A purposeful electrochemiluminescence immunoassay (EC) was used. The active GLP- The format for measuring 1 is described as follows: the analyte is measured using GLP- 1. N-terminal (7-17) specific mAb (biotin-anti-GLP1 (7-37, 7-36, The antibody was captured with anti-human Fc specific mAb (SulfoTag-R1) and then purified with IgG. 0). Five-parameter logistic fit with 1 / F2 standard curve weighting Watson LIMS™ software (Thermo Fisc) was used to Raw data are available from Her Scientific (Waltham, MA USA). Regression was performed.

[0344] For quantification of individual parts of the molecule, trypsin-mediated bottom-up assay was performed. LC-MS / MS analysis of compound 4 and dulaglutide in trypsin-resistant monkeys was also performed. The assay uses immunoaffinity capture with an anti-human Fc antibody to extract analytes from plasma samples. The light was purified and subsequently digested with trypsin and purified using a SCIEX Triple Quad ( Trademark)5500 LC-MS / MS System(CIEX(Concord, Ont. The compounds were analyzed by reversed-phase LC-MS / MS at Ario, Canada. 3C6, 15N4-arginine] and [13C6, 15N2-lysine] labeled human IgG4 (Sigma, Cat. No. MSQC7) was used as an internal standard during the immunoaffinity capture step. The HGE peptide is essential for its activity, so the HGE peptide was added at the N-terminus of GLP-1. The end peptide, i.e., HGE(HGEGTFTSDVSSYLEEQAAK) (sequence No. 263) was monitored as a surrogate for molecules containing active GLP-1.

[0345] result Dual agonists according to embodiments of the present application are covalently linked to a cyclic PYY peptide. The dual agonist contains a GLP-1 fusion (GF) peptide containing the first linker peptide. A recombinant IgG1A-Fc region fused to a hinge-Fc region via a peptide (N-terminal linker peptide) Lucagon-like peptide-1 (GLP-1) peptide or GLP-1 variant peptide, and recombinant The second linker peptide (C-terminal linker peptide) of the GLP-1 fusion (GF) peptide cyclic PYY peptides chemically conjugated to target cysteines contained within Each component of a dual agonist has different potency, stability, and agonist activity at each receptor. These include post-translational modifications, conjugation yields, and biophysical properties. The empirical selection was based on several important parameters, including but not limited to:

[0346] GLP-1 peptide variant SEQ ID NO: 57 was selected as the GLP-1 peptide variant. The GLP-1 peptide-1 variant SEQ ID NO: 56 was less potent in vivo However, because of the poor stability in ex vivo human plasma stability studies (Fig. 1A), The GLP-1 peptide variant SEQ ID NO: 58 unexpectedly In addition, they lost potency on GLP-1R when conjugated to cyclic PYY peptides (Table 11 ) (See, e.g., GLP-1R potency of SEQ ID NOs: 135, 134, 146, and 176 ), SEQ ID NOs: 238, 242, 262, 253, and 241 are exendin 4 (1- 39) (SEQ ID NO: 58) GLP-1 fusion peptide with some PYY peptides It was shown that the potency of GLP-1R was lost or significantly reduced after conjugation. Based on these results, GL with exendin-4(1-39) (SEQ ID NO: 58) Testing with the GLP-1 fusion peptide was not preferred in this study. The mutant SEQ ID NO: 58 further contains a NG deamidation-prone motif. When the first amino acid in the first linker peptide was glycine, the GLP-1 peptide A NG deamidation-prone motif was formed at the tide / first linker peptide interface. GF28 (SEQ ID NO: 140) containing the P-1 peptide SEQ ID NO: 59 increased food intake (FI It was less effective than dulaglutide in terms of both % change in body weight (BW) and % change in serotonin (S). 10A and 10B, respectively).

[0347] SEQ ID NO: 60 was selected as the first linker peptide (N-terminal linker peptide). The GLP-1 fusion protein containing the first peptide linker, SEQ ID NO: 61, showed low The present invention has a high expression and purification yield and can deliver a desired amount of GLP-1 fusion peptide-conjugated cyclic PYY peptide. This was not preferred in this experiment for generating peptide conjugates containing AP repeats. The first linker peptide is insufficient when the second terminal peptide is a G4A repeat. yields comparable to SEQ ID NO: 60 (see, e.g., Example 2 and Table 8), Based on these results, the AP reaction Testing with a longer linker was not preferred in this experiment. reduced potency (e.g., the same GLP-1 with a first linker peptide of different length) The sequence of FIG. 5 compares the potency of GLP-1 fusion peptides containing the peptide (SEQ ID NO: 58). (See SEQ ID NO: 152, SEQ ID NO: 140 in Figures 10A-10B, and Figure 12). 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 a serine-containing linker is In some experiments, this was not favorable (see, for example, Table 12).

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

[0349] GLP-1 fusion peptide GF1 (SEQ ID NO: 113, Compound 1), GF19 (SEQ ID NO: 1 31), and GF24 (SEQ ID NO: 136, Compound 3) all had a significant effect on food intake and % body weight change. showed efficacy equivalent to that of dulaglutide (e.g., Figures 2A-2B and 6A-6C, respectively). 6B, and Figures 3A-3B). GF1 (SEQ ID NO: 113, Compound 1) and GF24 ( SEQ ID NO: 136, Compound 3) also showed efficacy comparable to dulaglutide on glucose tolerance ( 4A-4B and 7A-7B).

[0350] However, the cyclic PYY peptide (SEQ ID NO: 27) was GLP- 1. Fusion peptide-coupled cyclic PYY peptide conjugate, SEQ ID NO: 251 There were no additional pharmacological effects on food intake or % change in body weight compared to dulaglutide. (Figures 11A and 11B, respectively). Additional second linker peptides were tested. However, it was not preferred in this experiment for at least the following reasons: (a) SEQ ID NO: 95 , had a low production yield when the first linker peptide was an AP repeat, and (b) SEQ ID NO: No. 100 is cleaved during recombinant expression, and (c) PY is added to the C-terminus of the hinge Fc region peptide. Y peptide with a second linker peptide containing the target cysteine for conjugation The hinge Fc region peptide is cleaved during protein expression, which is why the PYY peptide A second linker peptide was added to generate an intact protein that is amenable to conjugation. (d) a serine containing second linker peptide was required. (e) the shorter the second linker peptide, the lower the likelihood of being xylosylated. It was conjugated with lower efficiency at higher purity.

[0351] GLP-1 fusion peptide conjugated cyclic PY with second linker peptide, SEQ ID NO: 94 The Y peptide conjugate, SEQ ID NO: 225 (Compound 2), significantly increased both FI and % BW change. showed significantly higher efficacy than dulaglutide for B), which surprisingly allowed the drug to be stored for 2 weeks at 4°C in a highly concentrated liquid formulation. There was significant protein loss during the incubation. an equivalent GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate having SEQ ID NO: 229 is stable in high-concentration liquid formulations during storage at 4°C and 40°C for 2 weeks. The protein levels were maintained (Table 14). A second linker peptide was added to connect these two molecules. This was the only difference between the two children, and therefore SEQ ID NO: 93 was used as the second linker in this experiment. was selected as a peptide.

[0352] [Table 14]

[0353] GLP-compliant drugs with long half-lives can be formulated to provide the necessary in vivo stability while retaining efficacy. 1. The functional activity of the cyclic PYY peptide must be such that it can be linked to a fusion protein. Several modifications are required. Using synthetic peptide chemistry, cyclic PYs incorporating these modifications can be synthesized. A cyclic PYY peptide is prepared and chemically linked to a GLP-1 fusion peptide. to form a GLP-1 fusion peptide-linked cyclic PYY peptide conjugate. Generated.

[0354] Cyclic PYY peptides were selected and conjugated to GLP-1 fusion peptides to form PY The potency and stability of the Y peptide were investigated and compared. The PYY peptide was cyclized to Multiple sites of proteolysis along the peptide backbone were stabilized.

[0355] A cyclic PYY peptide is selected and conjugated to a GLP-1 fusion peptide to form a cyclic The spacing and size of the N-terminal to C-terminal bonds of PYY peptides, as well as their potency and safety These cyclic PYY peptides have various carbon numbers in the ring. and cysteine and homocysteine used to selectively link the N-terminus to the C-terminus. The location of the inn was also different. For example, see: a) βA2 SEQ ID NO: 25 linked to hCys31; b) βA2 SEQ ID NOs: 28 and 29 linked to Cys30; c) G2 SEQ ID NOs: 27 and 30 linked to Cys30; d) γAba2 SEQ ID NO:34 linked to hCys31.

[0356] N-Me arginine substituted at position 35 (N-MeR35) (SEQ ID NOs: 27, 28, 3 3, and 34) and a reduced amide bond between positions 35 and 36 (psi35,36) (sequence Nos. 24, 25, 29, and 30) were amidated at position 36 in the cyclic PYY peptide. The ability of the modified C-terminal tyrosine to stabilize and retain potency was compared (see, e.g., See U.S. Patent Publication No. 2018 / 0117170, which is incorporated herein by reference in its entirety. In certain cases, N-Me35 modifications result in increased potency compared to psi35,36 modifications. (e.g., U.S. Patent Publication No. 2018 / 0117170), see Table 3. SEQ ID NO: 102 with a psi35,36 modification is SEQ ID NO: 103 with an N-Me35 modification. (The data show that it was approximately 12-fold more potent at the human Y2 receptor than No. 122.)

[0357] Unexpectedly, a GLP-1 fusion peptide containing the cyclic PYY peptide, SEQ ID NO:27, was N-MeR35 modification in SEQ ID NO: 229. The efficacy of the cyclic PYY peptide, psi35 of SEQ ID NO:232, containing SEQ ID NO:30, was , which was equivalent to or better than 36 modifications (Table 11).

[0358] The combination of components reduces product costs and makes reagent procurement easier. In addition, based on potency and stability, the cyclic PYY peptide, SEQ ID NO: 27, The PYY peptide selected for this experiment was a cysteine-linked 30-mer peptide near the C-terminus. The natural amino acid glycine at the N-terminus of the cyclic PYY peptide binds to GLP-1. The results were obtained for any cyclic PYY peptide tested when conjugated to the synthetic peptide. The efficacy and stability of cyclic PYY were comparable to the highest levels achieved. Since peptide SEQ ID NO:27 had sufficient potency and stability, the homocysteine at position 31 was Cysteine at position 30 was chosen over cysteine, which is therefore more expensive and less readily available. Unnatural amino acids that cannot be synthesized were not required in this experiment. N-MeR35, used to stabilize terminal amidated tyrosines, is an expensive ps It is a cheaper and more readily available reagent than the i35,36 modification. This eliminates the need for components while providing the same stability and efficacy.

[0359] In addition, the PEG spacer is not required for efficacy and, unexpectedly, It was also determined that the PEG spacer was not required for PYY stability in vivo. mAb-cyclic PYY peptide conjugates with a cyclic PYY peptide activator have increased in vivo stability. This was unexpected, since it had been determined that the See Table 4 of No. 8 / 0117170, which shows the results of blood samples taken from mice 48 hours after administration. The percentage of intact compound remaining relative to the amount of total human mAb levels in the fluid sample is shown. Compound 1 containing a 2×PEG spacer remained at 90.8% at 48 hours, while 6×P Compound 2, which contains an EG spacer, remained at 65.6% at 48 hours, while the PEG spacer Compound 3, which does not have a saturant, remained at only 51% for 48 hours. The need for a PEG component, which would increase the manufacturing cost of the product, was eliminated.

[0360] GLP-1 fusion peptide sequence with two stable cyclic PYY peptides SEQ ID NO: 27 added SEQ ID NO: 229, which contained sequence number 136, was used as the GLP-1 fusion peptide in this experiment. SEQ ID NO: 229 was selected as the FI and cyclic PYY peptide conjugate. For both BW, the Y2R association was increased compared to SEQ ID NO: 136 (GLP1R only association). The drug showed a significant pharmacodynamic effect (Figures 3A-3B), and also demonstrated a significant effect on glucose tolerance. The GLP1R potency was comparable to that of SEQ ID NO: 136 (Figures 4A-4B and 7A-7B). ).

[0361] The effects of dulaglutide and SEQ ID NO: 229 (Compound 4) on FI and BW were measured DIO. Based on an integrated exposure-response nonlinear regression analysis of available studies (compound 4) Six trials of rituximab and seven trials of dulaglutide; doses 0.03 to 1.0 nmol / kg), resulting in clinically relevant active GLP-1 (HGE) exposure on day 3 At a dose of 0.3 nmol / kg (single subcutaneous administration), compound 4 exhibited the same activity as GGL In the reduction of FI and BW with P1(HGE) exposure (Figures 8A-8B), the conjugate Further pharmacological effects of dulaglutide may be observed through Y2R association with cyclic PYY peptides. The same HGE concentration of 0.3 nmol / kg (single SC administration) of dulaglutide was Based on the nonlinear exposure-response regression with Compound 4 (SEQ ID NO: 229), the calculated F The % changes in I and BW were approximately 2.1 and 1.5 times greater than dulaglutide, respectively. .

[0362] Individual PK and calorie intake-based exposure-response analyses were performed in overweight cynomolgus monkeys (Figure 9A-9B) demonstrated clinically relevant dulaglutide exposure (1.5 m in patients with T2DM). Steady-state C after QW SC administration of g trough and C max (surrounding 1-2 nM) [Geiser et al., Clin Pharmacokinet 55(5): (2016)]), compound 4 showed similar activity in duraglutide at the same GLP-1 exposure. Approximately 1.7 to 2 times higher (95% confidence interval: 1.3 to 3.3, ligand binding increase) than PK based on Sei [LBA], Figure 9A) or approximately 1.5 to 1.6 times (95% confidence interval: 1. 0-2.8, PK based on LC-MS / MS HE, Figure 9B) significantly reduced calorie intake This suggests that compound 4, a cyclic PYY peptide, exerts additional pharmacological effects. It was a testimony.

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

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

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

[0366] SEQ ID NO: 225 Name: GLP-1 fusion complex - [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 complex - [cyclo-(βA2-COCH2-C30),K(Ac)1 1,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0369] [ka]

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

[0371] [ka]

[0372] SEQ ID NO: 228 Name: GLP-1 fusion complex - [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 complex - [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 complex - [cyclo-(βA2-COCH2-C30),K(Ac)1 1,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0377] [ka]

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

[0379] [ka]

[0380] SEQ ID NO: 232 Name: GLP-1 fusion complex - [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 complex - [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 complex - [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 complex - [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 complex - [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 complex - [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 complex - [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 complex - [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 complex - [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 complex - [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 complex - [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 complex - [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(PEG2 4Ac)11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0405] [ka]

[0406] SEQ ID NO: 245 Name: GLP-1 fusion complex - [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 complex - [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 complex - [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 complex - [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,ps i-(R35,Y36)]-PYY2-36 homodimer conjugate structure:

[0417] [ka]

[0418] SEQ ID NO: 251 Name: GLP-1 fusion complex - [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 complex - [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 complex - [cyclo-(gAba2-COCH2-hC30), K(A c) 11,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0423] [ka]

[0424] SEQ ID NO: 254 Name: GLP-1 fusion complex - [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 complex - [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 complex - [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 complex - [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 complex - [cyclo-(βA2-COCH2-C30),K(Ac)1 1,N-Me-R35]-PYY2-36 homodimer conjugate structure:

[0433] [ka]

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

[0435] [ka]

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

[0437] [ka]

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

[0441] [ka]

Claims

1. Glucagon-like peptide 1 (GLP-1) fusion peptide linked to a cyclic PYY peptide a conjugate comprising a GLP-1 fusion peptide, a linker peptide, a hinge-Fc region peptide, and a second linker peptide. , wherein said first linker is optionally missing.

2. The cyclic PYY peptide is represented by Formula I, or a derivative or pharmaceutical is a salt acceptable to 【Chemical 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 there is no BRIDGE is -Ph-CH ２ -S-, -triazolyl-, -NHC(O)CH ２ S -、-SCH ２ C(O)NH-、 -(OCH ２ CH ２ ) ２ NH C(O)CH ２ S, -NH C(O)-, or -CH ２ S- and Z ４ is K, A, E, S, or R; Z ７ is A or K, Z ９ 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, 【Chemistry 2】 and Z ３５ teeth, 【Chemistry 3】 the law of nature The derivative is one or more selected from the group consisting of amidation, acylation, and PEGylation.

10. The conjugate of claim 1, which is a compound of formula I modified by more than one process. gate.

3. The cyclic PYY peptide is represented by Formula I, or a derivative or pharmaceutical is a salt acceptable for 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 there is no BRIDGE is -Ph-CH ２ -S-, -triazolyl-, -NHC(O)CH ２ S -、-SCH ２ C(O)NH ２ -、-(OCH ２ CH ２ ) ２ I'm sorry ２ S、-N HC(O)- or -CH ２ S- and Z ４ is K, A, E, S, or R; Z ７ is A or K, and the amino side chain of said K is optionally 【Chemistry 4】 wherein 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 is substituted with Cl, Z ９ is G or K, and the amino side chain of said K is optionally 【Chemistry 5】 wherein 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 is substituted with Cl, Z １１ is D or K, and the amino side chain of said K is optionally 【Chemistry 6】 wherein 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 is substituted with Cl, Z ２２ is A or K, and the amino side chain of said K is optionally 【Chemistry 7】 wherein 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 is substituted with Cl, Z ２３ is S or K, and the amino side chain of said K is optionally 【Chemistry 8】 wherein 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 is substituted with Cl, 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 2, wherein

4. The cyclic PYY peptide is represented by Formula I, or a derivative or pharmaceutical is a salt acceptable to 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 is absent), BRIDGE is -Ph-CH ２ -S-, -triazolyl-, -NHC(O)CH ２ S -, -(OCH ２ CH ２ ), ２ NH C(O)CH ２ S, -NH C(O)-, or -CH ２ S - and Z ４ is K, A, E, S, or R; Z ７ is A or K, and the amino side chain of said K is —C(O)CH ２ substituted with Br And, Z ９ is G or K, and the amino side chain of K is —C(O)CH ２ substituted with Br And, Z １１ is D or K, and the amino side chain of said K is —C(O)CH ２ substituted with Br And, Z ２２ is A or K, and the amino side chain of said K is —C(O)CH ２ Br is substituted And, Z ２３ is S or K, and the amino side chain of said K is —C(O)CH ２ substituted with Br And, Z ２６ is A or H, Z ３０ is L, Z ３４ teeth, 【Chemistry 11】 and Z ３５ teeth, 【Chemistry 12】 The conjugate of claim 2, wherein

5. The cyclic PYY peptide is selected from the group consisting of SEQ ID NOs: 1-54, or The conjugate of claim 2 , which is a pharmaceutically acceptable salt.

6. The cyclic PYY peptide is selected from the group consisting of SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, 34 or a pharmaceutically acceptable salt thereof. Conjugates.

7. The GLP-1 fusion peptide comprises a cyclic PYY peptide at a lysine residue of the cyclic PYY peptide. The conjugate of any one of claims 1 to 6, covalently linked to a PYY peptide. Jugate.

8. Z in Formula I ７ , Z ９ , Z １１ , Z ２２ , and Z ２３ only one of which is lysine, The lysine is a cis-linked lysine in the second linker peptide of the GLP-1 fusion peptide.

8. The conjugate of claim 7, wherein the conjugate is covalently linked to a tein residue.

9. Z in Formula I １１ The conjugate of claim 8, wherein is lysine.

10. The GLP-1 peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59. The conjugate of any one of claims 1 to 9, comprising a sequence.

11. the first linker peptide is present and selected from the group consisting of SEQ ID NOs: 60-83 The conjugate according to any one of claims 1 to 10, comprising an amino acid sequence as defined above.

12. The hinge-Fc region peptide is an amino acid selected from the group consisting of SEQ ID NOs: 84-90. The conjugate of any one of claims 1 to 11, comprising a nucleotide sequence.

13. The second linker peptide is an amino acid selected from the group consisting of SEQ ID NOs: 93-112. The conjugate of any one of claims 1 to 12, comprising a nucleotide sequence.

14. The second linker peptide is an amino acid sequence of SEQ ID NO: 93, 94, 95, 106 or 111. The conjugate of any one of claims 1 to 12, comprising a nucleotide sequence.

15. Glucagon-like peptide 1 (GLP-1) fusion peptide conjugated to a cyclic PYY peptide a conjugate comprising a GLP-1 fusion peptide selected from the group consisting of SEQ ID NOs: 113-2; 24 and 267-274, The peptide is selected from SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34. or a pharmaceutically acceptable salt thereof. 。

16. a cysteine residue between amino acid residues 287 and 289 of the GLP-1 fusion peptide, preferably Preferably, cysteine residue 288 is linked to the remainder of the cyclic PYY peptide via a chemical linker. Lysine residues at groups 7, 9, 11, 22, or 23, preferably 16. The conjugate of claim 15, wherein the conjugate is covalently linked to lysine residue 11 of the amino acid sequence of hydroxybenzoate.

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

18. the side chains of the cyclic PYY peptide, preferably the side chains of the lysine residues of the cyclic PYY peptide The GLP-1000 is preferably a hydroxybenzoate or a hydroxybenzoate. -1 fusion peptide and the sulfhydryl group of the cysteine residue of the second linker peptide reacting the cyclic PYY peptide with the GLP-1 fusion peptide, thereby forming a bond between the cyclic PYY peptide and the GLP-1 fusion peptide.

18. The conjugate of claim 1, wherein the conjugate is a hydroxyl group or a hydroxyl group. How to generate a tugate.

19. the cysteine residue of the second linker peptide of the GLP-1 fusion peptide is The GLP-1 fusion peptide is reduced by contacting it with an excess of an azaphosphine reducing agent. and the reduced cysteine residue is reacted with the electrophile. method.

20. The azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3. 3.1.1]decane (PTA) or a derivative thereof.

21. A conjugate according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:

22. 1. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising: The disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, or insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, congenital hyperlipidemia Insulinism (CHI)-induced hypoglycemia, dyslipidemia, atherosclerosis, diabetic Nephropathy and hypertension associated with uncontrolled cholesterol and / or lipid levels Other cardiovascular risk factors such as blood pressure, osteoporosis, inflammation, and non-alcoholic fat liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and / or 22. The method of claim 21, wherein the subject is suffering from eczema, and the method comprises administering to the subject in need thereof an effective amount of the A method comprising administering a pharmaceutical composition.

23. Reducing at least one of food intake or body weight in a subject in need thereof.

22. A method of administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 21. The method comprises administering a composition.

24. Y2 receptor activity and / or GLP-1 receptor activity in a subject in need thereof 22. A method of modulating the immune response of a subject in need thereof, comprising administering to said subject an effective amount of the pharmaceutical composition of claim 21. A method comprising administering a pharmaceutical composition.

25. The pharmaceutical composition according to any one of claims 22 to 24, wherein the pharmaceutical composition is administered by injection. method.

26. A kit comprising the conjugate according to any one of claims 1 to 17, preferably or a kit further comprising an injection device.

27. A method for producing a pharmaceutical composition comprising a conjugate according to any one of claims 1 to 17. The method comprises combining the conjugate with a pharmaceutically acceptable carrier to form the pharmaceutical composition. A method involving obtaining something.

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