Incretin analogs and uses thereof

Abstract

To provide a therapeutic agent that can provide effective glucose control with the benefit of weight loss and a favorable side effect profile, and that has a sufficiently long duration of action to allow for less frequent dosing. The present invention provides an incretin analog comprising: YX2QGTFTSDYSIX 13 LDKX 17 AX 19 X 20 AFIEYLLX 28 X 29 GPSSX 34 APPPS In the formula, X2 is Aib, and X 13 is L or αMeL, and X 17 is K chemically modified through conjugation to the epsilon-amino group of the K side chain, and X 19 is Q or A, and X 20 is Aib, αMeK, Q, or H, and X 28 is E or A, and X 29 is G or Aib, and X 34 is an incretin analog, or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is G or Aib, and is optionally amidated.

Description

[Technical field]

[0001] The present disclosure relates to glucose-dependent insulinotropic polypeptide (GIP), a glucagon-like It is a compound that has activity at the insulin receptors, insulin receptor 1 (GLP-1), and glucagon receptors. The incretin analogs described herein are directed to the incretin receptors They have structural features that provide balanced activity in various therapeutic areas and have a long duration of action. Such incretin analogs are useful in treating type 2 diabetes mellitus (T2DM), dyslipidemia, and metabolic syndrome. Drome, Nonalcoholic Fatty Liver Disease (NAFLD), Nonalcoholic Steatohepatitis (N These may be useful for treating disorders such as ASH, and / or obesity. [Background technology]

[0002] The prevalence of diabetes has been increasing for the past few decades. T2DM accounts for approximately 90% of all diabetes cases. T2DM is the most common form of diabetes, accounting for 100% of all cases. The current standard treatment for T2DM is dietary restriction. and exercise, as well as treatment with oral medications and incremental medications such as GLP-1 receptor agonists. Treatment with injectable hypoglycemic agents, including thiamine-based therapy, is recommended.

[0003] GLP-1 is a 36 amino acid peptide, the major biologically active fragment of which is glucoside. Stimulate glucose-dependent insulin secretion and prevent hyperglycemia in diabetic patients. Amino acid, C-terminal amidated peptide (GLP-1 7-36 , SEQ ID NO:2) Currently, there are various GLP-1 inhibitors available, including dulaglutide, exenatide, and liraglutide. Analogs are available to treat T2DM. However, currently Many GLP-1 receptor agonists are dose-dependent due to gastrointestinal side effects such as nausea and vomiting. When treatment with oral agents and incretin-based therapies is insufficient, Insulin is considered. A significant number of individuals receiving glycemic control do not achieve glycemic control goals (e.g., Casagrande et al., 2013). et al. (2013) Diabetes Care 36:2271-2279 Please refer to.

[0004] Uncontrolled diabetes is one factor that contributes to morbidity and mortality in such individuals. One of the main risk factors for T2DM is obesity, which can cause T2 The majority of individuals with DM (approximately 90%) are overweight or obese. may result in improvements in obesity-related comorbidities, including hyperglycemia and cardiovascular events. Therefore, glucose control and weight loss are important for better disease management. There is a need for effective therapies in

[0005] From that perspective, the new therapies being investigated should not only have activity at the GLP-1 receptor, but also Compounds that also have activity at one or more other receptors, such as the GIP and / or glucagon receptors. Indeed, certain compounds have triple agonist activity (i.e., GIP, GL P-1, and glucagon receptor activity. International Patent Application Publication No. WO2015 / 067716 describes a compound that has triple agonist activity. Glucagon analogues are also described in International Patent Application No. WO2016 / 19862. No. 4 is an exogenous GLP-1 analogue with triple agonist activity. Analogs of Gin-4 are also described in International Patent Application No. WO2014 / 04961. No. 0 and WO2017 / 116204 each have triple agonist activity Various analogues are described. International Patent Application No. WO2017 / 153375 Regarding glucagon and GLP-1 coagonists, which are also said to have GIP activity, Please write it down.

[0006] Nevertheless, it is an effective treatment with weight loss benefits and a favorable side effect profile. There remains a need for treatments, particularly for T2DM, that can provide adequate glucose control. They also allow for less frequent dosing, such as once daily, three times a week, twice a week, or once a week. There is a need for therapeutic agents available for use with sufficient long-term action to achieve this. Summary of the Invention

[0007] The incretin analogs described herein aim to meet the above needs. Thus, the present disclosure provides compounds that have activity at each of the GIP, GLP-1, and glucagon receptors. Advantageously, the incretin analogs described herein are The aim of this study was to develop a novel agonist analogue that can activate the receptor while avoiding the undesirable side effects associated with excessive activity. Allows administration of doses that provide sufficient activity at each receptor to provide agonistic benefit The incretin analogs described herein also have a balanced activity. G, which allows for less frequent dosing, such as once daily, three times a week, twice a week, or once a week It has long-lasting effects at each of the IP, GLP-1, and glucagon receptors. Incretin analogs have been shown to have therapeutic effects such as enhanced glucose control, weight loss, and / or improved body composition. What metabolic benefits does Proprotein Convertase Subtilisin / Kexin Type 9 (PCSK9) Lowering? and / or increased bone mass or bone formation or decreased bone resorption. The present disclosure also provides methods for treating obesity, NAFLD, NASH, dyslipidemia, and other conditions. and / or describe effective treatments for other disorders or conditions, including metabolic disorders. .

[0008] In one embodiment, an incretin analog comprises the formula: YX2QGTFTSDYSIX 13 LDKX 17 AX 19 X 20 AFIEYLLX 28 X 29 GPSSX 34 APPPS, wherein X2 is Aib and X 13 is L or α MeL and X 17 is any amino acid with a functional group available for conjugation, The group is C 16 -C 22 Conjugated to fatty acids, X 19 is Q or A, and X 20 Ai b, αMeK, Q, or H, and X 28 is E or A, and X 29 is G or Aib and X 34 is G or Aib (SEQ ID NO: 5), and the C-terminal amino acid is any In one embodiment, an incretin analog, or a pharma- ceutically acceptable salt thereof, is provided. do.

[0009] In another embodiment, the present invention relates to a method for treating dyslipidemia, fatty liver disease, metabolic syndrome, NASH, or the like. Methods for treating diseases such as osteoporosis, obesity, and T2DM are provided. administering to an individual in need thereof an effective amount of an incretin analog described herein. In some examples, the disease is fatty liver disease. , obesity, NASH, or T2DM.

[0010] In another embodiment, the incretin analogs described herein are useful in therapy. For example, the incretin analogs described herein are useful for treating dyslipidemia, Diseases such as fatty liver disease, metabolic syndrome, NASH, obesity, and T2DM In some examples, the disease is fatty liver disease, obesity have chronic obstructive pulmonary disease, NASH, or T2DM.

[0011] In another embodiment, the incretin analogs described herein are used to treat dyslipidemia, lipid For treating liver disease, metabolic syndrome, NASH, obesity, and T2DM In some examples, the disease is fatty liver disease. , obesity, NASH, or T2DM.

[0012] In another embodiment, the incretin analogs described herein and a pharma- ceutically acceptable A pharmaceutical composition is provided that includes a carrier, diluent, or excipient.

[0013] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the disclosure pertains. Any methods and materials similar or equivalent to those described herein may be used to treat incretin analogs. can be used in the practice or testing of methods, pharmaceutical compositions, and methods, but preferred methods and and materials are described herein.

[0014] Also, reference to an element by the indefinite article "a" or "an" means that the context is one and only one Unless the document explicitly requires that only one element be present, it does not exclude the possibility that more than one element may be present. Thus, the indefinite article "a" or "an" usually means "at least one."

[0015] GIP stimulates insulin secretion from pancreatic beta cells in the presence of glucose. A 42 amino acid peptide (SEQ ID NO: 1) that plays a physiological role in glucose homeostasis No. 4) and is an incretin.

[0016] GLP-1 stimulates glucose-dependent insulin secretion and reduces hyperglycemia in diabetic patients. It is a 36 amino acid peptide (SEQ ID NO:2) that has been shown to prevent infection. He is a cretin.

[0017] Glucagon binds to and activates glucagon receptors on liver cells, It helps maintain blood sugar levels and helps the liver break down glycogen through a process called glycogenolysis. It is a 29 amino acid peptide (SEQ ID NO: 1) that releases glucose stored in the form of acetylcholine. be.

[0018] Oxyntomodulin (OXM) is a 29-amino acid sequence that is identical to that of glucagon. Octapeptide Carboxy-Terminal Extension Activates Both Kagon and GLP-1 Receptors (SEQ ID NO:3), which has slightly higher potency at the glucagon receptor than at the GLP-1 receptor , a 37 amino acid peptide.

[0019] T2DM plus one of the GIP, GLP-1, and / or glucagon receptors Incretins and analogs thereof having activity in one or more of these diseases are useful for treating, for example, obesity, NAFLD, and and NASH, dyslipidemia, metabolic syndrome, bone-related disorders, and Alzheimer's disease , as well as therapeutic value in many other conditions, diseases, or disorders, including Parkinson's disease. For example, Jall et al. (2017) l.Metab.6:440-446;Carbone et al.(2016)J. Gastroenterol.Hepatol.31:23-31;Finan et al.(2016)Trends Mol.Med.22:359-376;Choi et al. (2017) Potent body weight loss and efficacy in a NASH animal model by a nov el long-acting GLP-1 / Glucagon / GIP triple -agonist (HM15211),ADA Poster 1139-P;Din g(2008)J.Bone Miner.Res.23:536-543;Tai e t al.(2018)Brain Res.1678:64-74;Muller e t al.(2017)Physiol.Rev.97:721-766;Finan et al.(2013)Sci.Transl.Med.5:209;Holsche See (2014) Biochem. Soc. Trans. 42:593-600 I want to.

[0020] As used herein, "about" refers to, for example, a described concentration, length, molecular weight, p Within a statistically significant range of a value or values, such as H, sequence identity, time frame, temperature, or volume Such values ​​or ranges are typically within 20% of the given value or range. More typically, it will be within 10% and even more typically within 5%. The permissible variations encompassed by "will depend on the particular system under study and may be determined by one of ordinary skill in the art. This can be easily understood.

[0021] As used herein, one of the GIP, GLP-1, or glucagon receptors With respect to one or more of the above, "activity," "activate," "activating," and the like are described below. The amount of acetylcholine in the blood is measured using assays known in the art, such as in vitro assays. bind to a receptor(s) and induce a response at the receptor(s) such that This refers to the ability of compounds such as the incretin analogs described herein.

[0022] As used herein, an "amino acid having a functional group available for conjugation" refers to, e.g., For example, any natural or unnatural amine having a functional group that can be conjugated to a fatty acid by a linker. Examples of such functional groups include alkynyl, alkenyl, amino, azido, These include, but are not limited to, bromo, carboxyl, chloro, iodo, and thiol groups. Examples of natural amino acids containing such functional groups are K (amino), C (thiol), and C (thiol). Examples include aryl, E (carboxyl), and D (carboxyl).

[0023] As used herein, "C 16 -C 22 "Fatty acids" are those with 16 to 22 carbon atoms The C 16 -C 22 Fatty acids are saturated As used herein, "saturated" means that the fatty acid is It is meant that it does not contain any carbon-carbon double or triple bonds.

[0024] As used herein, an "effective amount" refers to a single or multiple dose administered to an individual in need thereof. The present invention provides a method for treating a subject, comprising administering to a subject a desired effect in such an individual under diagnosis or treatment after several doses. an amount of one or more incretin analogs, or pharma- ceutically acceptable salts thereof, as described herein; The effective amount can be determined by using known techniques and under similar circumstances. This can be easily determined by one skilled in the art by observing the results obtained in the In determining an effective amount for a mammal, the species, its size, age, and general health, The specific disease or disorder involved; the extent, involvement, or severity of the disease or disorder; the subject's response, the particular incretin analog administered, the mode of administration, the biology of the preparation administered bioavailability characteristics, selected dosing regimen, use of concomitant medications, and other relevant circumstances. A number of factors are taken into consideration, including but not limited to:

[0025] As used herein, "long-term action" refers to the binding affinity of an incretin analog and activity for a longer period than native human GIP, GLP-1, and glucagon peptides On an ongoing basis, at least once a day, or more infrequently, such as three times a week, twice a week, or once a week Time-action profile of incretin analogues can be measured using known pharmacokinetic testing methods, such as those utilized in the examples below.

[0026] As used herein, "incretin analogs" refers to GIP, GLP-1, and and glucagon, in particular human GIP (SEQ ID NO: 4), GLP-1 (SEQ ID NO: 2), and and a compound with several differences but structural similarities to glucagon (SEQ ID NO: 1). The incretin analogs described herein are GIP, GLP-1, and Glucagon receptor affinity and activity at each of them (i.e., triple agonist activity) ) is a sequence of amino acids that results in a compound having the

[0027] As used herein, an "individual in need thereof" refers to, for example, any of the subjects listed herein. having a condition, disease, disorder, or symptom in need of treatment or therapy, including those " refers to mammals, such as humans.

[0028] As used herein, "treat," "treatment," "treating," and the like refer to any of the preceding terms. inhibit, slow, halt, or reverse the progression or severity of a condition, disease, disorder, or symptom of It means.

[0029] As used herein, with reference to an incretin analog, the term "triple agonist activity" refers to an incretin analog that is "Inhibitory activity" refers to an ink that has activity at each of the GIP, GLP-1, and glucagon receptors. Retin analogs, particularly those that are able to enhance the receptor activity while avoiding the undesirable side effects associated with excessive activity, are have balanced and sufficient activity at each receptor to provide the benefits of agonism of both receptors In addition, incretin analogs with triple agonist activity are It has a long-lasting effect at each of the IP, GLP-1, and glucagon receptors, which can be achieved by administering 1 dose per day. This advantageously allows for administration as infrequently as once a week, three times a week, twice a week, or once a week.

[0030] The structural features of the incretin analogs described herein favor activity at each receptor. To achieve optimal efficacy, sufficient activity at each of the GIP, GLP-1, and glucagon receptors is required. (i.e., triple agonist activity) but dominates activity at the other two receptors or when administered in a dose sufficient to produce activity at all three receptors. There is little or no evidence that any one receptor is sufficient to induce undesirable side effects. In certain embodiments, such structural features result in analogs that are free of any of the above activities. A non-limiting example sees SEQ ID NO:5, which contributes to optimal glucagon and GIP activity. It was found that L or αMeL at position 13 contributes to optimal GIP activity. Aib at position 20 was found to contribute to optimal glucagon activity; acylation, and have been found to contribute to optimal glucagon and / or GIP activity. Other examples of such structural features include the Y at position 25, which is the most abundant cytochrome P450 in all three receptors. The 22, 24, and 28-39 positions were found to contribute to optimal binding and efficacy. The amino acids described herein are included.

[0031] The structural features of the incretin analogs described herein also improve the viability of the analogs in aqueous solution. Improved solubility, improved chemical and physical formulation stability, broadened pharmacokinetic profile, and their potential development as therapeutic treatments, including minimizing potential immunogenicity. The specific structures that give rise to such attributes provide analogs with many other beneficial attributes. Non-limiting examples of therapeutic features include optimal pharmacokinetic (PK) profiles and Give, C 20 Acylation at position 17 with fatty acids, optimal PK profile and developability Contributing to, Aib, αMeK, Q, or H at position 20, as well as optimal PK, immunogenicity The amino acids at positions 22, 24, and 28-39 contribute to the development potential and stability of the peptide. Included.

[0032] The foregoing list of structural features is illustrative rather than comprehensive and is not intended to be limiting. The combination of beneficial properties of the exemplary analogs described is not the result of any one modification alone. Instead, this is achieved through novel combinations of structural features described herein. In addition, the above effects of the preceding list of modifications may vary with the number of these modifications. However, as described below, other properties important to the properties of the compounds described herein may be It is not exclusive, since it also has an effect.

[0033] The amino acid sequences of the incretin analogs described herein are typically Naturally occurring amino acids, designated using the letter code (e.g., L=leucine), and Alpha-methyl substituted residues of natural amino acids (e.g., α-methylleucine (αMeL) and and α-methyllysine (αMeK), and alpha-aminoisobutyric acid (Aib). , and incorporates certain other unnatural amino acids, the structures of which are shown below. [ka]

[0034] As noted above, the incretin analogs described herein are analogs of any of the naturally occurring human peptides. It has structural similarities to both but also many structural differences. When compared to IP (SEQ ID NO: 4), the incretin analogs described herein have a 2 , 3, 7, 13, 14, 17, 18-21, 23-25, 28-29, and 30-42 In some examples, the compound comprises a modification at one or more of the positions in the Chin analogues are 2, 3, 7, 13, 14, 17, 18, 20, 21, 23-25, 29, and comprising modifications to the amino acids of native human GIP (SEQ ID NO: 4) at each of positions 30 to 42. In certain examples, the incretin analogs described herein have the following amino acid modifications: Aib at position 1, Q at position 3, T at position 7, L or αMeL at position 13, L at position 14, optionally phosphorus Through the use of the car, 16 -C 22 To the epsilon-amino group of the K side chain with fatty acid Modified through conjugation: modified K residue at position 17, A at position 18, A at position 21, I at position 23; E at position 24, Y at position 25, G or Aib at position 29, and GPSSGAPPPS (alteration Sequence No. 26) and GPSS-Aib-APPPS (Sequence No. 27) (and tail truncations and a substitution of amino acids at positions 30 to 42 with an amino acid sequence selected from the group consisting of: In other examples, the incretin analogs described herein also contain an A at position 19, an α at position 20, MeK, Aib, or H, and one or more of the following modifications at position 28: E. In the present invention, the incretin analogs described herein are amidated. In addition to the changes described herein, the incretin analogs described herein further include those in which the analog inhibits GIP, It is capable of binding to and activating the GLP-1 and glucagon receptors. The amino acid sequence may include one or more additional amino acid modifications, provided that:

[0035] As noted above, the incretin analogs described herein contain functional groups available for conjugation. A fatty acid moiety conjugated, e.g., by a linker, to a natural or unnatural amino acid having Such conjugation may also be referred to as acylation. In certain instances, the conjugation may be beneficial. The amino acids having available functional groups can be K, C, E, and D. In particular examples, Amino acids with functional groups available for conjugation are those in which conjugation occurs to the epsilon-amino group of the K side chain. It is K that does this.

[0036] The acylation of the incretin analogs described herein is optimal for containing this structure. It is located at position 17 in SEQ ID NO:5, which was determined to be the most important site for the In embodiments, the linker acts as an albumin binder to generate a long-acting compound. Offer possibilities.

[0037] The incretin analogs described herein are linked to the incretin receptor by either a direct bond or a linker. C, chemically conjugated to the functional group of the amino acid 16 -C 22 Use fatty acids. The length and composition of the incretin analogs will determine their half-life, their function in in vivo animal models, and their overall efficacy in treating incretin-related disorders. It affects the efficacy of the C 16 -C 22 saturated fat Conjugation to mono- or di-acids provides desirable half-lives, desirable potency in in vivo animal models, This results in incretin analogs that exhibit a potent agonist, as well as desirable solubility and stability properties.

[0038] Saturated C for purposes of this specification16 -C 22 Examples of fatty acids include those with branched and Palmitic acid (hexadecanoic acid) (C 16 Monoacid), Hexadecane diacid Acid (C 16 diacid), margaric acid (heptadecanoic acid) (C 17 monoacid), heptadecanedioic acid (C 17 diacid), stearic acid (C 18 monoacid), octadecanedioic acid (C 18 diacid), Nona Decylic acid (nonadecanoic acid) (C 19 monoacid), nonadecanedioic acid (C 19 diacid), alakadin Acid (eicosanoic acid) (C 20 monoacid), eicosanedioic acid (C 20 diacid), heneicosylic acid (Heneicosanoic acid) (C 21 monoacid), heneicosanoic acid (C 21 diacid), behenic acid (do Cosanoic acid) (C 22 monoacid), docosanedioic acid (C 22 Diacids) but are not limited to Not done.

[0039] In a specific example, C 16 -C 22 Fatty acids are saturated C 18 monoacid, saturated C 18 Diacid, saturated C1 9 monoacid, saturated C 19 Diacid, saturated C 20 monoacid, saturated C 20 Diacids, and their branched and In a more specific example, C 16 -C 22 Fatty acids include stearic acid, It may be alachadic acid, and eicosanedioic acid, especially alachadic acid.

[0040] In some examples, the linker is a 1-4 amino acid, aminopolyethylene glycol carboxylate, or mixtures thereof. Polyethylene glycol carboxylate has the following structure: [ka] In the formula, m is an integer of 1 to 12, n is an integer of 1 to 12, and p is It is either 1 or 2.

[0041] In certain examples, the linker can be one or more amino acids, optionally in combination with one to four amino acids. (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moiety. Cut.

[0042] In the example where the linker comprises at least one amino acid, the amino acid may be from 1 to 4 Glu. or γGlu amino acid residues. In some examples, the linker includes the D form thereof. For example, The linker may contain either one or two γGlu amino acid residues. Alternatively, the linker may contain up to 36 (2-[2-(2-amino-ethoxy)-ethoxy]- 1 to 4 amino acid residues (e.g., Glu or acetyl) used in combination with a Specifically, the linker may include 1 to 4 amino acids. Glu or γGlu amino acid and one to four (2-[2-(2-amino-ethoxy)- In another embodiment, the linker may be one or more or two γGlu amino acids and one or two (2-[2-(2-amino-ethoxy) -ethoxy]-acetyl) moiety.

[0043] In certain instances, the incretin analogs described herein have the structure of the formula: having a linker and a fatty acid moiety, (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γGlu)b -CO-(CH2)c-CO2H, In the formula, a is 0, 1, or 2; b is 1 or 2; and c is 16 or 18. In a particular example, a is 2, b is 1, and c is 18, and the structure is shown below. [ka] .

[0044] In another particular example, a is 1, b is 2, and c is 18, and the structure As shown below. [ka]

[0045] In another particular example, a is 0, b is 2, and c is 18, and the structure As shown below. [ka]

[0046] In another particular example, a is 1, b is 1, and c is 18, and the structure As shown below. [ka]

[0047] As shown in the chemical structures of Examples 1-20 below, the linker-fatty acid moiety is It can be linked to the epsilon (ε)-amino group of a lysine (K) side chain.

[0048] The GIP, GLP-1, and glucagon receptors are each as described herein. The affinity of an incretin analog can be determined, for example, by receptor binding assays, including those described in the examples below. The levels of erythrocytes may be measured using techniques known in the art, generally The inhibitory constants (Ki) are expressed as values. The activity of the amine analogs can also be assayed using in vitro activity assays, including, for example, the in vitro activity assays described below. This can be measured using techniques known in the art and is generally known as the effective concentration 50 (EC 50 )value This is expressed as the chemical compound that causes half-maximal simulation in a dose-response curve. is the concentration of the compound.

[0049] The incretin analogs described herein can be administered parenterally (e.g., subcutaneously, intravenously, intraperitoneally, Intracavitary, intramuscular, or transdermal administration Such pharmaceutical compositions and techniques for their preparation are well known in the art. For example, see Remington: The Science and Practice of Pharmacy(Troy,Ed.,21 st Edit (See Lippincott, Williams & Wilkins, 2006). In certain instances, the incretin analog is administered subcutaneously.

[0050] The incretin analogs described herein can be synthesized using a number of inorganic and organic acids / bases. It can react with either to form a pharma- ceutically acceptable acid / base addition salt. Such salts and common techniques for preparing them are well known in the art (e.g., , Stahl et al., Handbook of Pharmaceuticals Salts:Properties,Selection and Use,2 nd Revised Edition (Wiley-VCH, 2011). Pharmaceutically acceptable salts for use herein include sodium salts, trifluoroacetate salts, Salts include, hydrochloride and / or acetate salts.

[0051] The present disclosure also relates to an incretin analog as described herein, or a pharma- ceutically acceptable derivative thereof. The present invention provides and therefore encompasses novel intermediates and methods for synthesizing the salts described herein. The intermediates and incretin analogs that can be synthesized can be prepared by a variety of techniques known in the art. For example, methods using chemical synthesis are illustrated in the examples below. The specific synthetic steps for each of the described pathways are described in detail herein. The reagents and starting materials can be combined in different ways to prepare the cretin analogues. , are readily available to one of skill in the art.

[0052] The specific incretin analogs described herein generally are administered over a wide dosage range. For example, the dosage for once-weekly administration is within the range of about 0.01 to about 30 mg / person / week. , within the range of about 0.1 to about 10 mg / person / week, or even within the range of about 0.1 to about 3 mg / person / week Thus, the incretin analogs described herein may be administered once daily, It may be administered three times a week, twice a week, or once a week, particularly once a week.

[0053] The incretin analogs described herein are useful in treating a variety of conditions, disorders, diseases, or symptoms. In particular, methods for treating T2DM in an individual are provided. Such methods include administering to an individual in need of such treatment an effective amount of a compound as described herein. The method further comprises administering an incretin analog as described above, or a pharma- ceutical acceptable salt thereof. At least includes.

[0054] Additionally, methods are provided for treating obesity in an individual, such methods comprising: To an individual in need of such treatment, an effective amount of an incretin analog as described herein, or a pharma- ceutically acceptable salt thereof.

[0055] Additionally, methods are provided for inducing non-therapeutic weight loss in an individual, such methods administering to an individual in need of such treatment an effective amount of an incretin analogue as described herein The method includes at least the step of administering an analogue, or a pharma- ceutically acceptable salt thereof.

[0056] Additionally, a method for treating metabolic syndrome in an individual is provided, Such methods include administering to an individual in need of such treatment an effective amount of a Administering an incretin analog, or a pharma- ceutical acceptable salt thereof, include.

[0057] Additionally, methods are provided for treating NASH in an individual, such methods comprising: An individual in need of such treatment is administered an effective amount of an incretin mimetic as described herein. The method includes at least the step of administering to the subject a therapeutically effective amount of the compound, or a pharma- ceutically acceptable salt thereof, to the subject.

[0058] Additionally, methods are provided for treating NAFLD in an individual, such methods comprising: and administering to an individual in need of such treatment an effective amount of the incretins described herein. The method includes administering to a subject a therapeutically effective amount of a compound, such as a medicament for treating a disease, comprising administering to the subject a therapeutically effective amount of a compound, such as a medicament for treating a disease, a subject for a disease, or a medicament for treating a disease.

[0059] In these methods, the effectiveness of the incretin analog can be determined, for example, by the effect of increasing blood glucose levels. Observe a significant decrease in insulin, observe a significant increase in HbA1c, The test is assessed by observing a decrease in body weight and / or a significant decrease in body mass. It is possible.

[0060] Alternatively, the incretin analogs described herein or their pharmacologic acceptable salts can be used to improve bone strength in individuals in need thereof.In some instances, the individuals in need thereof have hypoossification or osteoid hypoplasia or are recovering from fractures, joint muscle repair surgery, prosthetic implants, dental implants, and / or spinal fusion.The incretin analogs described herein can also be used to treat other disorders, such as Parkinson's disease or Alzheimer's disease. Various embodiments of the present invention are presented below. 1. An incretin analog comprising: YX 2 QGTFTSDYSIX 13 LDKX 17 AX 19 X 20 AFIEYLLX 28 X 29 GPSSX 34 APPPS During the ceremony, X 2 is Aib, X 13 is L or αMeL, X17 is any amino acid having a functional group available for conjugation, said functional group being C 16 -C 22 Conjugated to fatty acids X 19 is Q or A, X 20 is Aib, αMeK, Q, or H; X 28 is E or A, X 29 is G or Aib, X 34 is G or Aib, (SEQ ID NO:5) incretin analogs, wherein the C-terminal amino acid is optionally amidated; or a pharma- ceutically acceptable salt thereof. 2.X 17 2. The incretin analog of claim 1, wherein the amino acid having the functional group available for conjugation at position 1 is selected from the group consisting of K, C, E, and D. 3.X 17 2. The incretin analog of claim 1, wherein the amino acid having the functional group available for conjugation at position is K. 4.X 17 The amino acid having the functional group available for conjugation at the C position and 16 -C 22 4. The incretin analogue according to any one of 1 to 3 above, wherein the fatty acid is conjugated by a linker between the amino acid and the fatty acid. 5. The incretin analog of claim 4, wherein the linker comprises 1 to 4 amino acids. 6. The incretin analog of claim 5, wherein the amino acid is Glu or γGlu. 7. The linker is H-{NH-CH 2 -CH 2 -[O-CH 2 -CH 2 ] m -O-(CH 2 ) p -CO} n 7. The incretin analogue according to any one of 4 to 6 above, further comprising an -OH structure, wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2. 8. The incretin analogue according to any one of 4 to 7 above, wherein the linker further comprises one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties. 9.X 17 is K chemically modified through conjugation to the epsilon-amino group of the K side chain having the structure (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γGlu) b -CO-(CH 2 ) c -CO 2 H, the incretin analog according to claim 1, wherein a is 0, 1 or 2, b is 1 or 2, and c is an integer from 16 to 18. 10. The incretin analogue according to claim 9, wherein a is 1. 11. The incretin analogue according to claim 9, wherein a is 2. 12. An incretin analogue according to any one of 9 to 11 above, wherein b is 1. 13. An incretin analogue according to any one of 9 to 11 above, wherein b is 2. 14. An incretin analog according to any one of 9 to 13 above, wherein c is 18. 15.X 13 The incretin analogue according to any one of 1 to 14 above, wherein is αMeL. 13 15. The incretin analogue according to any one of 1 to 14 above, wherein is L. 17.X 20 The incretin analogue according to any one of 1 to 16 above, wherein is Aib. 28 The incretin analogue according to any one of 1 to 17 above, wherein is E. 19.X 29 19. The incretin analogue according to any one of 1 to 18 above, wherein is G. 20.X 34 20. The incretin analogue according to any one of 1 to 19 above, wherein is G. 21.X 19 The incretin analogue according to any one of 1 to 20 above, wherein 22. a is 1, b is 1, c is 18, and X 13 But, αMeL, X 19 is A and X 20 But Aib and X 28 But E and X 29 But G and X 34 10. The incretin analogue according to claim 9, wherein is G. 23. An incretin analog having a formula selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:23. 24. A method for treating a disease selected from the group consisting of diabetes, obesity, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, and metabolic syndrome, said method comprising: A method comprising the step of administering to an individual in need thereof an effective amount of an incretin analogue described in any one of 1 to 23 above. 25. A method for treating type II diabetes, comprising: A method comprising the step of administering to an individual in need thereof an effective amount of an incretin analogue described in any one of 1 to 23 above. 26. An incretin analogue according to any one of 1 to 23 above, A pharmaceutical composition comprising a pharma- ceutically acceptable carrier, diluent, or excipient. 27. An incretin analogue according to any one of 1 to 23 above for use in the treatment of a disease selected from the group consisting of diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic steatohepatitis, and obesity. 28. An incretin analogue according to any one of 1 to 23 above for use in the treatment of type II diabetes. 29. Use of an incretin analogue according to any one of 1 to 23 above in the manufacture of a medicament for treating a disease selected from the group consisting of diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic steatohepatitis, and obesity. 30. Use of an incretin analogue as defined in any one of 1 to 23 above in the manufacture of a medicament for treating type II diabetes. EXAMPLES

[0061] Peptide synthesis Example 1: Example 1 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-Amino No-ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -C O2H)AQ-αMeK-AFIEYLLA-Aib-GPSSGAPPPS-NH2( Sequence number 7).

[0062] The following are the residues except for Aib2, αMeL13, K17, αMeK20, and Aib29: 1 is a representation of the structure of Example 1 using standard one-letter amino acid codes for these The structure of the amino acid residues of is extended. [ka]

[0063] The peptide backbone of Example 1 was synthesized using a Symphony 12-Channel Multiplier lex Peptide Synthesizer(Protein Technolo) Fluorenylmethyloxycarbonyl (Fm It is synthesized using tert-butyl (t-Bu) / octyl chemistry.

[0064] The resin was 1% DVB cross-linked polystyrene (Fmoc) with substitution of 0.3–0.4 meq / g. -Rink-MBHA Low Loading Resin, 100-200 Mesh, EMD Millipore). Standard side chain protecting groups are used. Fmoc-Lys (Mtt)-OH is used for lysine at position 17, and Boc-Tyr(tBu)-OH) is Used for tyrosine at position 1. Prior to each coupling step (7 min × 2), DM The Fmoc group is removed using 20% ​​piperidine in F. All standard amino acid carboxylates are The coupling consisted of Fmoc amino acid (0.3 mM), diisopropylcarbodiimide (0. The theoretical peptide loading was achieved using equimolar ratios of 0.9 mM of 1000 mM glycerol (0.9 mM), and 0.9 mM of Oxyma. The reaction is carried out in a 9-fold molar excess over the primary amine for 1 hour and the secondary amine for 3 hours. The exception is the coupling to a Cα-methylated amino acid, which is coupled for 3 hours. After the synthesis of the peptide backbone was completed, the resin was thoroughly washed six times with DCM to remove residual DMF. The Mtt protecting group of lysine 17 was removed by 30% hexafluoroisopropanol in DCM. Treated twice with Nol (Oakwood Chemicals) (40 minutes x 2 times) , is selectively removed from the peptide resin.

[0065] Subsequent attachment of the fatty acid-linker moiety is accomplished by 2-[2-(2-Fmoc-amino-ethoxy]phenyl]- F)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), moc-Glutamic acid α-t-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.), and mono-OtBu-eicosanedioic acid (WuXi AppT This was achieved by coupling with 3-fold excess of reagents (AA:PyAO P:DIPEA = 1:1:1 mol / mol) was used for each coupling of 1 h duration. do.

[0066] Once synthesis is complete, wash the peptide resin with DCM and air dry thoroughly. The mixture was incubated at room temperature for 2 h in 10 mL of cleavage cocktail (trifluoroacetic acid:water:triisopropyl The resin was filtered off and each was treated with 2 mL of NiCl. The extract was washed twice with concentrated TFA, and the combined filtrates were treated five times with cold diethyl ether (-20°C). The crude peptide is precipitated by centrifugation at 3500 rpm for 2 h. The mixture was centrifuged for 1 min to form a solid pellet, the supernatant was decanted, and the solid pellet was washed with ether. The crude peptide was triturated twice with 20% acetonitrile / 20% acetic acid / The solution was solubilized in 60% water and buffered in 100% acetonitrile and 0.1% TFA / water. Luna 5 μM phenylalanine with a linear gradient of acetonitrile (30 to 50% acetonitrile in 60 min) RP-HP with ethyl-hexyl preparative column (21×250 mm, Phenomenex) The purity of the peptides was assessed using analytical RP-HPLC and the peptides were purified by HPLC. The purity of the main pool in Example 1 was 98.8%. Subsequent lyophilization of the final major product pool yields the lyophilized peptide TFA salt. The molecular weight is determined by LC-MS (obsd: M+4H+ / 4=1226. 8, Calc M+4H+ / 4=1226.9).

[0067] Example 2: Example 2 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H ) AQHAFIEYLLA-Aib-GPSSGAPPPS-NH2 (sequence number 6).

[0068] The following are standard sequences, except for residues Aib2, αMeL13, K17, and Aib29: 1 is a representation of the structure of Example 2 using the one-letter amino acid code, and these amino acid residues The structure has been expanded. [ka]

[0069] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight as in Example 2.

[0070] Example 3: Example 3 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H) AQ-αMeK-AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 8) .

[0071] The following are canonical except for residues Aib2, αMeL13, K17, and αMeK20: FIG. 1 is a representation of the structure of Example 3 using the standard one-letter amino acid code; The structure of the group is expanded. [ka]

[0072] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight as in Example 3.

[0073] Example 4: Example 4 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-A ib-AFIEYLLA-Aib-GPSSGAPPPS-NH2 (sequence number 9).

[0074] The following are standard 1 sequences, except for residues Aib2, K17, Aib20, and Aib29: 1 is a representation of the structure of Example 4 using the letter amino acid code, with these amino acid residues The structure is expanded. [ka]

[0075] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight as in Example 4.

[0076] Example 5: Example 5 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H) AAQAFIEYLLE-Aib-GPSSGAPPPS-NH2 (SEQ ID NO: 10) .

[0077] The following are standard sequences, except for residues Aib2, αMeL13, K17, and Aib29: 1 is a representation of the structure of Example 5 using the one-letter amino acid code, and these amino acid residues The structure has been expanded. [ka]

[0078] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight as in Example 5.

[0079] Example 6: Example 6 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H) AAQAFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 11).

[0080] The following are standard one-letter amino acids, except for residues Aib2, αMeL13, and K17: 1 is a representation of the structure of Example 6 using the acid code. The structures of these amino acid residues are shown in the expanded form. It is stretched. [ka]

[0081] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight as in Example 6.

[0082] Example 7: Example 7 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H) AQHAFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 12).

[0083] The following are standard one-letter amino acids, except for residues Aib2, αMeL13, and K17: 1 is a representation of the structure of Example 7 using the acid code. The structures of these amino acid residues are shown in the expanded form. It is stretched. [ka]

[0084] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 7.

[0085] Example 8: Example 8 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- αMeK-AFIEYLLEGGPSSGAPPPS-NH2 (sequence number 13).

[0086] The following are standard one-letter amino acids, except for residues Aib2, K17, and αMeK20: 1 is a representation of the structure of Example 8 using the acid code. The structures of these amino acid residues are shown in the expanded form. It is stretched. [ka]

[0087] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 8.

[0088] Example 9: Example 9 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((γGlu)2-CO-(C H2) 18 -CO2H)AQHAFIEYLLEGGPSSGAPPPS-NH2 (sequence Number 14).

[0089] The following are standard one-letter amino acids, except for residues Aib2, αMeL13, and K17: 1 is a representation of the structure of Example 9 using the acid code. The structures of these amino acid residues are shown in the expanded form. It is stretched. [ka]

[0090] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight as in Example 9.

[0091] Example 10: Example 10 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H ) AQHAFIEYLLEGGPSSGAPPPS-NH2 (sequence number 15).

[0092] The following are standard one-letter amino acids, except for residues Aib2, αMeL13, and K17: 1 is a representation of the structure of Example 10 using the acid code, where the structures of these amino acid residues are: It has been expanded. [ka]

[0093] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 10.

[0094] Example 11: Example 11 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H) AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 16) .

[0095] The following are standard sequences, except for residues Aib2, αMeL13, K17, and Aib20: 1 is a representation of the structure of Example 11 using the one-letter amino acid code; The structure of the group is expanded. [ka]

[0096] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 11.

[0097] Example 12: Example 12 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H ) AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 (sequence number 17).

[0098] The following are standard sequences, except for residues Aib2, αMeL13, K17, and Aib20: A representation of the structure of Example 12 using the one-letter amino acid code. The structure of the group is expanded. [ka]

[0099] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 12.

[0100] Example 13: Example 13 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H ) AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 (SEQ ID NO: 18).

[0101] The following are the residues except for Aib2, αMeL13, K17, Aib20, and Aib29: 1 is a representation of the structure of Example 13 using standard one-letter amino acid codes; The structure of the amino acid residues of is extended. [ka]

[0102] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 13.

[0103] Example 14: Example 14 is a compound represented by the following description: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H )AQ-Aib-AFIEYLLE-Aib-GPSS-Aib-APPPS-NH2( Sequence number 19).

[0104] The following are residues Aib2, αMeL13, K17, Aib20, Aib29, and Ai Representation of the structure of Example 14 using standard one-letter amino acid codes, except for b34. and the structures of these amino acid residues are extended. [ka]

[0105] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 14.

[0106] Example 15: Example 15 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLA-Aib-GPSSGAPPPS-NH2 (sequence number 20).

[0107] The following are standard 1 sequences, except for residues Aib2, K17, Aib20, and Aib29: 1 is a representation of the structure of Example 15 using the letter amino acid code, and these amino acid residues The structure has been expanded. [ka]

[0108] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 15.

[0109] Example 16: Example 16 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLEGGPSSGAPPPS-NH2 (sequence number 21).

[0110] The following are standard one-letter amino acids, except for residues Aib2, K17, and Aib20: 1 is a representation of the structure of Example 16 using the code. The structure of these amino acid residues is It is stretched. [ka]

[0111] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 16.

[0112] Example 17: Example 17 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 (sequence number 22).

[0113] The following are standard 1 sequences, except for residues Aib2, K17, Aib20, and Aib29: 1 is a representation of the structure of Example 17 using the letter amino acid code, and these amino acid residues The structure has been expanded. [ka]

[0114] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 17.

[0115] Example 18: Example 18 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-A ib-AFIEYLLEGGPSSGAPPPS-NH2 (sequence number 23).

[0116] The following are standard one-letter amino acids, except for residues Aib2, K17, and Aib20: 1 is a representation of the structure of Example 18 using the code. The structure of these amino acid residues is It is stretched. [ka]

[0117] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 18.

[0118] Example 19: Example 19 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)2-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 (sequence number 24).

[0119] The following are standard 1 sequences, except for residues Aib2, K17, Aib20, and Aib29: 1 is a representation of the structure of Example 19 using the letter amino acid code, and these amino acid residues The structure has been expanded. [ka]

[0120] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 19.

[0121] Example 20: Example 20 is a compound represented by the following description: Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-A ib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 (sequence number 25).

[0122] The following are standard 1 sequences, except for residues Aib2, K17, Aib20, and Aib29: 2 is a representation of the structure of Example 20 using the letter amino acid code, and these amino acid residues The structure has been expanded. [ka]

[0123] The peptide backbone was synthesized using a process similar to that described above for Example 1. , conjugate to a fatty acid-linker moiety, check for purity, and confirm molecular weight of Example 20.

[0124] In vitro function Binding affinity: Radioligand competitive binding assays were performed to compare the activity of exemplary compounds and comparator molecules. Determine the equilibrium dissociation constant of the molecule. Such an assay is known as the scintillation proximity assay (SCA). SPA) method and human GIP receptor (GIPR), GLP-1 receptor (GLP-1R), or transfected HEK293 cells overexpressing the human glucagon receptor (GcgR) Membranes prepared from the cells are used.

[0125] The assay was performed in the presence of bacitracin as a nonspecific blocking agent to measure the activity of the test analogs. The silyl moiety is a protein component (e.g., albumin) that is used in standard assay buffers. This prevents the binding of

[0126] Competition curves are plotted as percent specific inhibition (y-axis) versus log concentration of compound (x-axis). 4-parameter nonlinear regression function with variable slope (ABase or Genedata) The data are analyzed using the K i The value is given by the formula K i =IC 50 / (1+(D / K d )) where IC 50 is the concentration of compound that results in 50% inhibition of binding, and D is the concentration of radioligand used in the assay, and K d is determined from saturation binding analysis is the equilibrium dissociation constant of the receptor and the radioligand, as determined by the NMR spectroscopy (as shown in Table 1 below). [Table 1] [Table 2]

[0127] As seen in Table 2, exemplary analogs are GIP, GLP-1, and glucagon receptor agonists. The receptors each have a binding affinity.

[0128] Functional activity: Functional activity was determined in GIP-R-GLP-1R- and GcgR-expressing HEK-293 clones. The assay was performed using 1× GlutaMAX (commercially available) in a 20 μl assay volume. (Gibco Cat. No. 35050), 0.25% FBS (fetal bovine serum, Gib co Catalog No. 26400), 0.05% Fraction V BSA (Bovine Serum Albumin, Gi bco Catalog No. 15260), 250 μM IBMX, and 20 mM HEPES DMEM (Gibco Cat. No. 15630) supplemented with 31053) (20-point CRC, 2.75x Labcyte Echo Each receptor-overexpressing cell line was treated with 100 mM MgCl2 (direct dilution).

[0129] After 60 min of incubation at room temperature, the resulting increase in intracellular cAMP was measured using CisBio cAMP Dynamic 2 HTRF Assay Kit (62AM4PEJ) was used. Briefly, cAMP-d2 conjugate in cell lysis buffer was used, followed by Similarly, the antibody anti-cAMP-cAMP-Eu in cell lysis buffer 3+ -Cryptates The resulting competitive assay detects intracellular cAMP levels by adding Incubate at room temperature for at least 60 minutes and then measure with excitation at 320 nm and 66 PerkinElmer Envision (registered trademark) with emission at 5 nm and 620 nm Detect using the Envision unit (emission at 665 nm / 620 nm). *10,000) is inversely proportional to the amount of cAMP present and can be calculated using a cAMP standard curve. Converted to nM cAMP per well.

[0130] The amount of cAMP (nM) produced in each well was determined using human GLP-1(7-36)NH2, The maximal response observed with either human Gcg or human GIP(1-42)NH2 was The maximum response parameter is calculated by fitting a four-parameter logistic equation. Relative EC values ​​were calculated by nonlinear regression analysis using the concentration of each compound versus the concentration of added peptide. 50 Inducing values do.

[0131] Exemplary Analogs and hGIP(1-42)NH2, hGLP-1(7-36)NH2 , and hGcg data are shown in Table 3 below. [Table 3]

[0132] As can be seen in Table 3, in the presence of FBS and BSA, the exemplary analogs exhibited the same activity as the natural Human GIP-R, GLP-1R, and GcgR cAMP cA lower than ligand It has agonist activity as determined by MP assay.

[0133] An additional set of cAMP assays was performed to assay human GLP-1, GIP, and glucagon receptors. The assay was carried out in HEK293 cells expressing . Using homogeneous time-resolved fluorometry, The assay was performed with a nonspecific blocker that does not interact with the fatty acid portion of the analyzed molecule. Exemplary analogs and conjugates performed in the presence of casein (instead of serum albumin) are The intrinsic potency of the modulator molecule is determined.

[0134] Intracellular cAMP levels are determined by extrapolation using a standard curve. Response curves were normalized to the minimum (buffer only) and maximum (maximum concentration of each control ligand) values. Plotted as percentage of stimulation received and fitted with a variable slope, four-parameter Analyze using nonlinear regression fit (Genedata Screener 13) . E.C. 50 is the concentration of compound that induces half-maximal simulation in the dose-response curve. degrees.

[0135] The data is provided below in Table 4. [Table 4]

[0136] As can be seen in Table 4, the exemplary analogs inhibited the activity of human GIP in the presence of 0.1% casein. , GLP-1, and glucagon receptors to stimulate cAMP release.

[0137] In vivo studies Pharmacokinetics in male Sprague Dawley rats: Pharmacokinetics of exemplary analogs were determined using 100 nM dosing in male Sprague Dawley rats. Blood samples were collected over 120 hours and the individual Calculate the pharmacokinetic parameters using the plasma concentration of peptide plasma (K3EDTA) Concentrations are determined using a qualified LC / MS method that measures the intact mass of the analogues. Each peptide and analogue was analyzed using methanol containing 0.1% formic acid as a standard. Extracted from 100% specific plasma. Thermo Q-Exacti for LC / MS detection. ve, high resolution instruments, and Thermo Easy Spray PepMap The mean pharmacokinetic parameters are shown in Table 5. [Table 5]

[0138] As can be seen in Table 5, the exemplary analogs exhibit extended pharmacokinetic profiles.

[0139] In vivo effects on insulin secretion in male Wistar rats: Using an intravenous glucose tolerance test (ivGTT) in rats (male Wistar) , and estimate the insulin secretory potential of exemplary analogs. Used as a control. The jugular vein and carotid artery (Envigo, Indianapolis, Rats were surgically implanted with cannulas in the peritoneal cavity (IN, 280-320 g) and then Rats were housed one per cage in polycarbonate cages with filter tops. The cells were maintained at 21°C with a 12-h light / dark cycle and were incubated at 2014 Teklad Global Diet (Envigo, Indianapolis) and deionized water ad libitum Rats were randomized by body weight and administered an exemplary similar dose 16 hours prior to glucose administration. The subjects were administered 1.5 mL / kg subcutaneously (sc) and then fasted. A typical analogue of this degree was measured at 6.667 nMol / mL in Tris buffer pH 8.0 and 2. The samples were diluted to 0 nMol / mL, 0.667 nMol / mL, and 0.2 nMol / mL and tested. The doses administered were vehicle, 1, 3, and 10 nMol / kg, and in some cases 0.3 and 30 nMol / kg. Semaglutide was used as a positive control and its insulin The phosphorus secretory effect was evaluated in its own test (vehicle and 1, 3, 10, and 30 nMol / kg dose) and an exemplary analog (10 nMol / kg dose) in each run. can be.

[0140] Blood samples at time 0 were collected in EDTA tubes, followed by glucose administration (0.5 Blood samples were collected 2, 4, 6, and 10 days after intravenous glucose administration. Glucose and insulin levels are collected at 1, 20, and 30 minutes. Insulin was measured using an electrochemiluminescence assay (Meso Scale, Gaithersburg, Massachusetts). g,MD) was used to determine the area under the insulin curve (AUC), n = 6 animals per group. The results are compared with vehicle controls.

[0141] Statistical analysis was performed using JMP with one-way ANOVA followed by Dunnett's comparison with vehicle control. The data is provided below in Table 6. [Table 6]

[0142] As can be seen in Table 6, the exemplary analogs demonstrate a dose-dependent increase in insulin secretion.

[0143] Studies in diet-induced obese C57BL / 6 mice: The exemplary incretin analogs described herein are useful in treating not only diabetes, but also insulin A collection of resistance and diabetes-related comorbidities (dyslipidemia, obesity, hepatic steatosis, etc.) It is also proposed as a treatment for metabolic syndrome. To investigate the effect of exemplary analogs on parameters such as hepatic steatosis and was administered to C57BL / 6 diet-induced obese (DIO) mice. These animals However, after 18 weeks of high-fat diet, metabolic syndrome was not observed. These patients exhibit insulin resistance, dyslipidemia, and hepatic steatosis, all characteristics of the idiopathic pulmonary disease.

[0144] Specifically, 24-25 week-old DIO male C57BL mice maintained on a calorie-rich diet were / 6 mice are used in the following studies. 12-hour light / dark cycle (lights on at 22:00) and a temperature-controlled (24°C) diet with free access to food (TD95217) and water. After a minimum of 2 weeks of acclimation to the facility, each experimental group of animals was housed in a similar Mice are randomized according to their body weight so that they have a body weight of 40-51 kg. The range is g.

[0145] All groups contained 5-6 mice. analogue (40 mM Tris-HCl, pH 8.0), and Semaglu dissolved in vehicle Tide was administered subcutaneously (SC) (10 mL / kg) every 3 days for 15 days in the dark. Administered to ad libitum-fed DIO mice 30–90 min prior to the start of the cycle. SC injections are administered on days 4, 7, 10, and 13. Body weight and food intake are monitored throughout the study. Measure.

[0146] Absolute changes in body weight were measured using the same animal model before the first injection of vehicle, analogue, or semaglutide. On days 0 and 15, Echo Med Nuclear magnetic resonance imaging was performed using the CAL System Instrument (Houston, TX). Total fat mass was measured by magnetic resonance (NMR). On day 15, animals were sacrificed before the dark period. The liver is removed and frozen. The liver triglycerides and plasma cholesterol were measured using the Hitachi Modula r P is measured on a clinical analyzer.

[0147] Data are presented in Tables 7 and 8 below as mean ± SEM of 5-6 animals per group. Statistical analysis was performed using repeated measures ANOVA followed by Dunnett's method for multiple comparisons. Significant differences were defined as p<0.05. [Table 7]

[0148] As can be seen in Table 7 above, the exemplary analogs demonstrate a dose-dependent decrease in body weight.

[0149] Metabolic parameter data at the 3 nmol / kg dose are provided below in Table 8. [Table 8]

[0150] In addition to substantial weight loss, as seen in Table 8, the exemplary analogs significantly reduced blood glucose levels. increased plasma insulin (as a sign of increased insulin sensitivity), and plasma cholesterol. and improved liver health as indicated by reduced plasma ALT and hepatic triglycerides Improve.

[0151] SEQ ID NO:1 - Human glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEQ ID NO:2-Human GLP-1(7-36)amide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 SEQ ID NO:3 - Human OXM HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO:4 - Human GIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNI TQ SEQ ID NO:5 - Incretin analogs YX2QGTFTSDYSIX 13 LDKX 17 AX 19 X 20 AFIEYLLX 28 X 29 GPSSX 34 APPPS During the ceremony, X2 is Aib, X 13 is L or αMeL, X 17 is any amino acid having a functional group available for conjugation, the functional group being C1 6-C 22 Conjugated to fatty acids X 19 is Q or A, X 20 is Aib, αMeK, Q, or H; X 28 is E or A, X 29 is G or Aib, X 34 is G or Aib SEQ ID NO:6 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H )AQHAFIEYLLA-Aib-GPSSGAPPPS-NH2 SEQ ID NO:7 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H)AQ-αMeK-AFIEYLLA-Aib-GPSSGAPPPS-NH2 SEQ ID NO:8 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H)AQ-αMeK-AFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:9 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-A ib-AFIEYLLA-Aib-GPSSGAPPPS-NH2 SEQ ID NO:10 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H)AAQAFIEYLLE-Aib-GPSSGAPPPS-NH2 SEQ ID NO:11 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H)AAQAFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:12 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H)AQHAFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:13 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- αMeK-AFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:14 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((γGlu)2-CO-(C H2) 18 -CO2H)AQHAFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:15 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H )AQHAFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:16 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2 H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:17 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H )AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:18 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H )AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 SEQ ID NO:19 - Incretin analogs Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- Ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H )AQ-Aib-AFIEYLLE-Aib-GPSS-Aib-APPPS-NH2 SEQ ID NO:20 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLA-Aib-GPSSGAPPPS-NH2 SEQ ID NO:21 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:22 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 SEQ ID NO:23 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-A ib-AFIEYLLEGGPSSGAPPPS-NH2 SEQ ID NO:24 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)2-CO-(CH2) 18 -CO2H)AQ- Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 SEQ ID NO:25 - Incretin analogs Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy) -ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-A ib-AFIEYLLE-Aib-GPSSGAPPPS-NH2 SEQ ID NO:26 - Artificial sequence GPS SGA PPS SEQ ID NO:27 - Artificial sequence GPSS-Aib-APPPS

Claims

1. below: <h2 style=";text-align:left;direction:ltr">(If you don't mind, I'll call you back.)<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; <h2 style=";text-align:left;direction:ltr">I am sorry, I did not receive any reply.<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; <h2 style=";text-align:left;direction:ltr">(If you don't mind, I'll call you back.)<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; Boc-Tyr(tBu)-Aib-QGTFTSDYSILLDKK(Mtt)AQ-Aib-AFIEYLLA-Aib-GPSSGAPPPS-NH ２ ; Boc-Tyr(tBu)-Aib-QGTFTSDYSI-αMeL-LDKK(Mtt)AAQAFIEYLLE-Aib-GPSSGAPPPS-NH ２ ; <h2 style=";text-align:left;direction:ltr">I am sorry, I did not find this information.<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; <h2 style=";text-align:left;direction:ltr">I am sorry, I did not find this information.<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; <h2 style=";text-align:left;direction:ltr">I am sorry, I did not find this information.<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; <h2 style=";text-align:left;direction:ltr">(If you don't mind, I'll call you back.)<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; <h2 style=";text-align:left;direction:ltr">(If you don't mind, I'll call you back.)<h2 style=";text-align:left;direction:ltr"> ２ <h2 style=";text-align:left;direction:ltr"> ; Boc-Tyr(tBu)-Aib-QGTFTSDYSI-αMeL-LDKK(Mtt)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH ２ ; Boc-Tyr(tBu)-Aib-QGTFTSDYSI-αMeL-LDKK(Mtt)AQ-Aib-AFIEYLLE-Aib-GPSS-Aib-APPPS-NH ２ ; Boc-Tyr(tBu)-Ab-QGTFTSDYSILLDKKMttAQ-Am-AFIEILLEAAAH ２ ; or Boc-Tyr(tBu)-Aib-QGTFTSDYSILLDKK(Mtt)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH ２ A resin-bound peptide comprising:

2. A composition comprising the peptide of claim 1 for use in the manufacture of an incretin analogue.

3. 13. A composition comprising the peptide of claim 1 for use in the manufacture of a medicament for use in the treatment of a disease selected from the group consisting of diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic steatohepatitis, and obesity.

4. 13. A composition comprising the peptide of claim 1 for use in the manufacture of a medicament for use in the treatment of type II diabetes.

5. 13. A composition comprising the peptide of claim 1 for use in the manufacture of a medicament for treating obesity.

Images