NZ748274B2 - Gip and glp-1 co-agonist compounds - Google Patents

Abstract

The present invention relates to dual incretin peptide mimetic compounds that agonize receptors for both human glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and may be useful for treating obesity. The present invention provides a compound of Formula: YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 11), or a pharmaceutically acceptable salt thereof and has proved to be useful in the treatment of obesity. EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 11), or a pharmaceutically acceptable salt thereof and has proved to be useful in the treatment of obesity.

Description

GIP AND GLP-1 CO-AGONIST NDS This application is a divisional of New Zealand patent application 732000, which is the national phase entry in New Zealand of PCT international application (published as are incorporated herein by reference.

The present invention relates to the field of ne. More particularly, the present ion relates to dual incretin peptide mimetic compounds that agonize receptors for both human glucose-dependent insulinotropic ptide (GIP) and glucagon-like e-1 (GLP-1), and may be useful for treating type 2 es mellitus (T2D).

T2D is the most common form of diabetes accounting for imately 90% of all diabetes. T2D is characterized by high blood glucose levels caused by insulinresistance.

The current standard of care for T2D includes diet and exercise along with available oral and injectable glucose ng drugs. Nonetheless, many patients with T2D still remain inadequately controlled. Currently marketed incretin mimetics or dipeptidyl peptidase IV (DPP-IV) inhibitors only utilize a single established mechanism of action for glycemic control. A compound for T2D is needed that utilizes a dual mechanism of action.

GIP is a 42-amino acid gastrointestinal regulatory peptide that plays a logical role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the ce of glucose and protecting pancreatic beta cells. GLP-1 is a 37- amino acid peptide that stimulates insulin ion, protects atic beta cells, and inhibits glucagon secretion, gastric emptying and food intake which leads to weight loss.

GIP and GLP-1 are known as incretins; incretin receptor signaling exerts physiologically nt action critical for glucose homeostasis. In normal physiology, GIP and GLP-1 are secreted from the gut following a meal, and these incretins enhance the physiological response to food including sensation of satiety, insulin secretion, and nutrient disposal.

T2D patients have impaired incretin responses.

Dosing of GLP-1 analogues has been found to be limited by adverse effects, such as nausea and vomiting, and as a consequence dosing most often cannot reach full efficacy for ic control and weight loss. GIP alone has very modest elowering ability in type 2 diabetic humans. Both native GIP and GLP-1 are inactivated rapidly by the ubiquitous protease, DPP IV, and therefore, can only be used for short-term metabolic control.

Glucagon is a 29-amino acid peptide produced by the pancreas, and when bound to glucagon receptor, signals the liver to e glucose leading to an increase in blood glucose. GLP-2, a peptide that like GLP-1 is produced from processing of proglucagon, is known to be associated with cellular proliferation in the gut. Thus, stimulation of glucagon and GLP-2 receptors should be minimized during chronic treatment of T2D patients in order to maximize glucose lowering and reduce potential long term carcinogenic risks.

Certain GIP analogs have been described as exhibiting both GIP and GLP-1 activity in DPP IV is in the exopeptidase class of proteolytic enzymes. The introduction of non-natural amino acids in a sequence can increase the proteolytic stability of any given peptide. While use of non-natural amino acids can help with the stability of peptides against DPP IV proteolysis and other forms of degradation, it was discovered by Applicants as part of the present invention that non-natural amino acids can have unexpected effects on the balance of agonist ty between GIP and GLP-1. Non- l amino acids also increase the likelihood that a peptide may be seen as foreign and set off undesirable immune reactions, such as human immunogenicity and injection site reactions.

Fatty acids, through their albumin binding motifs, can improve the pharmacokinetics of a peptide by extending the ife, for example. While use of fatty acids can improve peptide ife, it was ered by Applicants as part of the present invention that the length, composition, and placement of the fatty acid chain and the linker between the e and the fatty acid chain can have unexpected effects on the balancing of the GIP and GLP-1 t activity. bility of certain GLP-1 analogues has been found to prevent a dose of the GLP-1 analogue that reaches better efficacy for glycemic control and weight loss. The most common side effects ed to GLP-1 analogues are nausea and vomiting but some compounds may also impact heart rate. The HPA axis is part of a physiological stress response and GLP-1 has been found to stimulate the HPA axis in rats resulting in increased corticosterone levels providing a potential link to adverse events such as increased heart rate. As part of the present invention, Applicants unexpectedly found that a compound described herein did not lead to elevated corticosterone levels like seen with semaglutide in a rat model and so possibly can be dosed to higher efficacy levels than GLP-1R-selective agents.

There remains a need to provide a compound that is a balanced nist of GIP and GLP-1 ors, but is selective against related glucagon and GLP-2 receptors.

Also, there remains a need to provide a compound with balanced co-agonist activity of GIP and GLP-1 ors which may provide weight loss given activity found in animal models. Additionally, there s a need to provide a compound with balanced coagonist activity of GIP and GLP-1 receptors that delivers adequate stability t DPP IV and other forms of degradation, but while still ining a low immunogenicity potential. Also, there remains a need to provide a compound with balanced co-agonist activity of GIP and GLP-1 receptors that ts potential once-weekly dosing in humans.

It is an object of the present invention to go some way towards meeting these needs and/or to at least provide the public with a useful . Accordingly, certain compounds described herein have lower potential for immunogenicity and injection site reactions than certain GIP-GLP-1 co-agonist compounds in the art. Certain compounds bed herein have potential for producing weight loss in patients based on animal energy expenditure data. Furthermore, certain compounds described herein have a balanced co-agonist activity against GIP and GLP-1 receptors and selectivity against both on and GLP-2 receptors, low genicity potential, and pharmacokinetic (PK) teristics that support eekly dosing in humans.

Summary of the Invention In a first aspect the present invention provides a use, in the manufacture of a medicament for the treatment of obesity in a patient in need thereof, of a compound of Formula: YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is chemically ed through conjugation to the epsilon-amino group of the K hain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)a- CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 11), or a pharmaceutically acceptable salt thereof.

Also described is a compound of Formula I: YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is ally ed through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 11), or a pharmaceutically able salt thereof.

In a further embodiment, described is a compound of Formula I, wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]- acetyl)2-(γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 18; X3 is Phe; and the C-terminal amino acid is optionally amidated as a C-terminal primary amide, or a pharmaceutically acceptable salt thereof.

In a further ment, described is a compound of Formula I, wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified h conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2- (γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 18; X3 is 1-Nal; and the C- terminal amino acid is optionally amidated as a C-terminal primary amide, or a pharmaceutically acceptable salt thereof.

In a further ment, described is a compound of a I, wherein X1 is Aib; X2 is Aib; K at position 20 is ally modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2- (γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 14 to 18; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally ed as a C-terminal primary amide, or a pharmaceutically acceptable salt thereof. In a further embodiment, bed is a compound wherein b is 16 to 18. Additionally, described is a compound wherein b is 18.

In a further embodiment, described is a compound of Formula I, wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2- (γGlu)a-CO-(CH2)b-CO2H wherein a is 1 and b is 10 to 18; X3 is Phe or 1-Nal; and the C- terminal amino acid is optionally amidated as a C-terminal primary amide, or a pharmaceutically acceptable salt thereof.

In a further embodiment, described is a compound of Formula I, wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2- (γGlu)a-CO-(CH2)b-CO2H wherein a is 2 and b is 10 to 18; X3 is Phe or 1-Nal; and the C- al amino acid is optionally amidated as a C-terminal primary amide, or a pharmaceutically able salt thereof.

In a further embodiment, described is a compound of Formula I, wherein X1 is Aib; X2 is Aib; K at on 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with -Amino-ethoxy)-ethoxy]-acetyl)2- (γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 18; X3 is Phe or 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide, or a pharmaceutically acceptable salt f.

In an embodiment, described is a compound of the Formula: YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS; n X1 is Aib; X2 is Aib; K at position 20 is chemically modified through ation to the epsilon-amino group of the K hain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 3), or a pharmaceutically acceptable salt thereof.

In an embodiment, described is a compound of the a: YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)18-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

In an embodiment, described is a nd of the Formula: YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through ation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 5), or a ceutically acceptable salt thereof.

In an embodiment, described is a compound of the Formula: YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the n-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 6), or a pharmaceutically acceptable salt thereof.

In an embodiment, described is a compound of the Formula: YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically ed h ation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 7), or a pharmaceutically acceptable salt thereof.

In an ment, described is a compound of the Formula: YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is ally modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)16-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 8), or a pharmaceutically acceptable salt thereof.

In an embodiment, described is a compound of the a: YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with -Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)16-CO2H; X3 is 1-Nal; and the inal amino acid is amidated as a inal primary amide (SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof.

In an embodiment, described is a compound of the Formula: FTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is ally modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 10), or a pharmaceutically acceptable salt f.

In an embodiment, described is a composition comprising a compound described herein with a pharmaceutically acceptable carrier, diluent, or ent.

In an embodiment, described is a method of treating type 2 diabetes us, comprising administering to a patient in need thereof, an effective amount of a compound described herein. In a further embodiment, described is a method of treating type 2 diabetes mellitus further comprising administering simultaneously, separately, or sequentially in combination with an effective amount of one or more agents selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 tors, and sodium glucose co-transporters.

In an embodiment, described is a method to improve glycemic control in adults with type 2 diabetes mellitus, comprising administering to a patient in need thereof, an effective amount of a compound described herein as an adjunct to diet and exercise. In an embodiment, described is a method for chronic weight management in adults with an initial body mass index ≥ 27 and type 2 diabetes mellitus, comprising administering to a patient in need thereof, an effective amount of a compound described herein as an adjunct to a reduced-calorie diet and increased physical activity.

In an embodiment, described is a method to treat lic syndrome, comprising administering to a patient in need thereof, an effective amount of a compound described herein. In a further embodiment, described is a method to treat dyslipidemia, obesity, and/or hepatic steatosis associated with insulin resistance and diabetes, comprising administering to a patient in need thereof, an effective amount of a compound described herein. Additionally, described is a method to treat frailty or increase bone strength, comprising administering to a patient in need thereof, an effective amount of a compound bed herein.

In an ment, described is a compound described herein for use in therapy.

In a further embodiment, described is a compound described herein for use in the treatment of type 2 diabetes mellitus. In a further embodiment, described is a compound described herein in simultaneous, separate, or sequential combination with one or more agents selected from min, lidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters for use in the treatment of type 2 diabetes In an embodiment, described is a compound described herein for use in glycemic control in adults with type 2 diabetes us as an adjunct to diet and exercise. In an embodiment, described is a compound described herein for use in chronic weight management in adults with an initial body mass index ≥ 27 and type 2 diabetes us as an adjunct to a reduced-calorie diet and sed physical activity.

In an embodiment, described is the use of a compound described herein for the manufacture of a medicament for the treatment of type 2 diabetes mellitus. In a further embodiment, described is the use of a compound described herein in simultaneous, te, or sequential combination with one or more agents selected from metformin, lidinediones, sulfonylureas, idyl peptidase 4 inhibitors, and sodium glucose co-transporters for the manufacture of a medicament for the treatment of type 2 diabetes mellitus.

Also bed are compounds that y a balanced GIP and GLP-1 activity.

Balanced activity against GIP and GLP-1 as used herein refers to a compound that has affinity for GIP receptors and GLP-1 receptors in an in vitro binding assay at a molar ratio that is close to 1:1, such as 1:1 GLP-1/GIP, 2:1 GLP-1/GIP, 3:2 GLP-1/GIP, 1:2 GLP-1/GIP, or 2:3 GLP-1/GIP.

Also described are compounds that display selectivity for GIP and GLP-1 receptors versus receptors for glucagon and GLP-2. The term “selectivity” or “selective against” when used herein to reference GIP and GLP-1 activity in comparison to on activity, refers to a compound that displays 1000-, 500-, or about 100-fold higher potency for GIP and GLP-1 over glucagon when the data is normalized from the respective in vitro binding assays. The term “selectivity” or “selective against” when used herein to reference GIP and GLP-1 activity in comparison to GLP-2 activity, refers to a compound that displays 250-, 200-, 100-, or about 50-fold higher y for GIP and GLP-1 over GLP-2 when the data is normalized from the respective in vitro functional assays.

Also described is a method for treatment of type 2 diabetes in a patient sing administering to a t in need of such treatment an effective amount of a compound described , or a pharmaceutically acceptable salt thereof. Also bed is a method for treatment of type 2 diabetes in a patient comprising stering to a patient in need of such treatment an effective amount of a compound described herein, or a pharmaceutically able salt thereof, wherein the administration is aneous.

Also described is a method of treatment of type 2 diabetes in a patient comprising administering to a patient in need of such treatment an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, and simultaneously, tely, or sequentially an effective amount of one or more other active ingredients. In one embodiment, the other active ingredient or ingredients is currently available oral glucose lowering drugs from a class of drugs that is considered prior to administration the rd of care as determined by industry guidelines such as the American Diabetes Association.

The compounds bed herein utilize a fatty acid chemically conjugated to the epsilon-amino group of a lysine hain. The fatty acid is conjugated to the epsilon- amino group of a lysine side-chain through a linker. The linker comprises [2-(2-Aminoethoxy )-ethoxy]-acetyl)2-(γGlu)a wherein a is 1 to 2. The fatty acid and the gamma- glutamic acid in the linker act as albumin binders, and provide the potential to generate cting compounds. nds described herein comprise a lysine at position 20 that is chemically modified h conjugation to the epsilon-amino group of the K sidechain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20. As shown in the chemical structures of Examples 1 and 2, the first unit of [2-(2-Amino-ethoxy)-ethoxy]-acetyl is linked to the epsilon-amino group of the lysine side-chain. The second unit of Amino-ethoxy)-ethoxy]-acetyl is then attached to the amino-group of the first unit of [2-(2-Amino-ethoxy)-ethoxy]-acetyl.

Then, the first unit of γGlu is attached to the amino –group of the second unit of [2-(2- Amino-ethoxy)-ethoxy]-acetyl through the γ-carboxyl group of the side-chain. When a = 2, the second unit of γGlu is attached to the α-amino –group of the first unit of γGlu through the γ-carboxyl group of the side-chain. Finally, the symmetrical fatty acid is attached to the α-amino –group of the first (when a = 1) or second (when a =2) unit of γGlu.

The nds described herein are preferably formulated as pharmaceutical compositions stered by parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, uscular, or transdermal). Such pharmaceutical compositions and processes for ing same are well known in the art. (See, e.g., Remington: The Science and Practice of Pharmacy (D.B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006). The preferred route of administration is subcutaneous.

The compounds described herein may react with any of a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art.

See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: ties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011); S.M. Berge, et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.

Pharmaceutically acceptable salts described herein include trifluoroacetate, hydrochloride, and acetate salts.

As used herein, the term “effective amount” refers to the amount or dose of compound described herein, or a pharmaceutically acceptable salt thereof which, upon single or le dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment. An effective amount can be y determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for a t, a number of s are considered by the attending stician, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation stered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

As used herein, the term “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

As used , “semaglutide” refers to a chemically synthesized GLP-1 analogue that has the peptide backbone and overall compound structure of that found in CAS Registry Number 9104632.

Certain compounds described herein are generally effective over a wide dosage range. For example, dosages for once-weekly dosing may fall within the range of about 0.05 to about 30 mg per person per week. Certain compounds described herein may be dosed daily. Additionally, n compounds described herein may be dosed once- weekly.

The amino acid ces described herein n the rd single letter or three letter codes for the twenty naturally occurring amino acids. Additionally, “Aib” is alpha amino isobutyric acid, and “1-Nal” is thylalanine.

The present sure also encompasses novel intermediates and processes useful for the synthesis of compounds described herein, or a pharmaceutically acceptable salt thereof. The intermediates and compounds described herein may be prepared by a variety of procedures known in the art. In particular, the process using al synthesis is illustrated in the Examples below. The specific synthetic steps for each of the routes described may be combined in different ways to prepare compounds described herein, or salts thereof. The reagents and starting materials are readily available to one of ordinary skill in the art. It is understood that these Examples are not ed to be limiting to the scope of the invention in any way.

EXAMPLE 1 FTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically ed through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 3) trifluoroacetate salt The above structure contains the standard single letter amino acid code with exception of residues Aib2, Aib13 and K20 where the structures of these amino acid residues have been expanded.

The peptide according to SEQ ID NO: 3 described herein is generated by solidphase e synthesis using a -Bu strategy carried out on a Symphony ted peptide synthesizer (PTI Protein Technologies Inc.) starting from RAPP AM-Rink Amide resin and with couplings using 6 equivalents of amino acid activated with diisopropylcarbodiimide (DIC) and hydroxybenzotriazole (HOBt) (1:1:1 molar ratio) in dimethylformamide (DMF) for 90 min at 25°C.

Extended couplings (4h each) for Pro31, Trp25, Gln24, Val23, Phe22, Lys20, Gly4, Glu3 and Aib2 are necessary to improve the quality of the crude peptide. A Fmoc- Lys(Alloc)-OH building block is used for Lys20 coupling (orthogonal protecting group) to allow for site specific attachment of the fatty acid moiety later on in the synthetic process. The following conditions are used for the coupling of Fmoc-Ile-OH at position 12: Fmoc-Ile-OH (6 , PyBOP (6 equiv), and DIEA (12 equiv) in DMF for 24 h at 25°C. The N-terminal e is incorporated as Boc-Tyr(tBu)-OH using DIC-HOBt protocols as described above.

After finishing the elongation of the peptide-resin described above, the Alloc protecting group present in Lys20 is removed using catalytic amounts of Pd(PPh3)4 in the presence of PhSiH3 as a scavenger. Additional coupling/deprotection cycles using a Fmoc/t-Bu strategy to extend the Lys20 side-chain involved Fmoc-NH-PEG2-CH2COOH (ChemPep Catalog#280102), Fmoc-Glu(OH)-OtBu (ChemPep Catalog#100703) and HOOC-(CH2)18-COOtBu. In all couplings, 3 lents of the building block are used with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4h at 25°C.

Concomitant cleavage from the resin and side chain protecting group removal are carried out in a solution containing trifluoroacetic acid (TFA): triisopropylsilane : 1,2- ethanedithiol: water : thioanisole 90:4:2:2:2 (v/v) for 2 h at 25°C followed by precipitation with cold ether. Crude peptide is purified to > 99% purity (15-20% purified yield) by reversed-phase HPLC chromatography with water / acetonitrile (containing 0.05% v/v TFA) gradient on a C18 column, where suitable fractions are pooled and lyophilized.

In a synthesis performed essentially as bed above, the purity of Example 1 was examined by ical reversed-phase HPLC, and identity was med using LC/MS (observed: M+3H+/3 =1605.2; Calculated M+3H+/3 5; observed: 4 =1204.3; Calculated M+4H+/4 =1204.4).

EXAMPLE 2 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS n X1 is Aib; X2 is Aib; K at on 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)18-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 4) trifluoroacetate salt The above ure contains the standard single letter amino acid code with exception of residues Aib2, Aib13, K20 and 1-Nal22 where the structures of these amino acid residues have been expanded.

The peptide according to SEQ ID NO: 4 described herein is synthesized similarly as described above in e 1. The following conditions are used for the coupling of Fmoc-1Nal-OH at position 22: Nal-OH (6 equiv), PyBOP (6 equiv), and DIEA (12 equiv) in DMF for 4 h at 25°C.

EXAMPLE 3 YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified h conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 5) trifluoroacetate salt The compound according to SEQ ID NO: 5 bed herein is synthesized similarly as bed above for Example 1.

EXAMPLE 4 YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 6) trifluoroacetate salt The compound according to SEQ ID NO: 6 bed herein is synthesized rly as described above for Example 1.

EXAMPLE 5 YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS n X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 7) trifluoroacetate salt The compound according to SEQ ID NO: 7 described herein is synthesized similarly as described above for Example 1.

YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified h conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)16-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 8) trifluoroacetate salt The compound ing to SEQ ID NO: 8 described herein is synthesized similarly as described above for Example 1.

EXAMPLE 7 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the n-amino group of the K hain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)16-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 9) trifluoroacetate salt The compound according to SEQ ID NO: 9 bed herein is synthesized similarly as described above for Example 1.

EXAMPLE 8 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified h conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 10) trifluoroacetate salt The compound according to SEQ ID NO: 10 described herein is synthesized similarly as described above for Example 1.

ASSAYS Provided below are the conditions and data for Examples in several assays: in vitro function and selectivity, genicity profiling, pharmacokinetics, and in vivo type 2 diabetes models.

In vitro function and selectivity In vitro g potency to human GLP-1 and GIP Receptors The in vitro binding potency of compounds bed herein to human GIP and GLP-1 receptors is evaluated by measuring the binding affinities, as Ki, using crude cellular membranes ed from clonal cell lines over-expressing either the human GLP1R cDNA or human GIP-R cDNA.

The human glucose-dependent insulinotropic polypeptide receptor binding assay uses hGIP-R (Usdin,T.B., Gruber,C., . and ,T.I., GenBank: AAA84418.1) cloned into pcDNA3.1 (Promega)-NeoR plasmid. The hGIP-R-pcDNA3.1/Neo plasmid is transfected into Chinese Hamster Ovary cells, CHO-S, for suspension cultures and selected in the presence of 500 µg/mL Geneticin (Invitrogen).

Crude plasma membranes are prepared using cells from suspension culture. The cells are lysed on ice in hypotonic buffer ning 25 mM Tris HCl, pH 7.5, 1 mM MgCl2, DNAse1, 20 µg/mL, and Roche Complete™ Inhibitors without EDTA. The cell suspension is homogenized with a glass dounce homogenizer using a Teflon® pestle for strokes. The homogenate is centrifuged at 4°C at 1800 x g for 15 minutes. The supernatant is collected and the pellet resuspended in nic buffer and re- homogenized. The mixture is centrifuged at 1800 x g for 15 s. The second supernatant is combined with the first supernatant. The combined supernatants are recentrifuged at 1800 x g for 15 minutes to clarify. The clarified supernatant is transferred to high speed tubes and centrifuged at 25,000 x g for 30 minutes at 4°C. The membrane pellet is resuspended in homogenization buffer and stored as frozen aliquots at -80 °C r until use.

GIP is radioiodinated by the Ilactoperoxidase procedure (Markalonis, J.J., Biochem. J. 113:299 (1969)) and ed by reversed phase HPLC (Perkin-Elmer Life and Analytical Sciences NEX-402). The specific activity is 2200 Ci/mmol. KD determination is performed by homologous competition using cold hGIP instead of saturation binding. The receptor binding assay is carried out using a Scintillation Proximity Assay (SPA) with wheat germ agglutinin (WGA) beads (Perkin Elmer Life and Analytical Sciences) previously blocked with 1% fatty acid free BSA (Gibco, 7.5% BSA). The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% fatty acid free BSA, 0.003% Tween20, and Roche Complete™ Inhibitors without EDTA. hGIP and the compounds described herein are dissolved in 100% DMSO and stored at -20°C. The compounds are serially d into binding buffer. Next, 10 µL diluted compound or 100% DMSO is transferred into Corning® 3632 clear bottom assay plates containing 40 µL assay binding buffer or cold GIP (NSB at 0.1 µM final). Then, 90 µL membranes (3 l), 50 µL [125I] GIP (Perkin Elmer Life and Analytical es at 0.15 nM final in reaction), and 50 µL of WGA beads (150 µg/well) is added, , and mixed on a plate shaker for 1 . Plates are read with a MicroBeta® scintillation counter after 12 hours of settling time at room temperature.

Results are calculated as a percentage of specific IGIP binding in the presence of compound. The Absolute IC50 concentration is derived by near regression of percent specific binding of GIP versus the concentration of compound added. The IC50 concentration is converted to Ki using the Cheng-Prusoff equation.

The GLP-1 receptor binding assay uses cloned human glucagon-like peptide 1 receptor (hGLP-1R) ano MP, Hey PJ, ski D, Chicchi GG, Strader CD, Biochem Biophys Res Commun. 196(1): 141-6, 1993) isolated from 293HEK membranes.

The R cDNA is subcloned into the expression plasmid phD (Trans-activated expression of fully gamma-carboxylated recombinant human protein C, an antithrombotic factor. Grinnell, B.W., Berg, D.T., Walls, J. and Yan, S.B. Bio/Technology 5: 1189-1192, 1987). This plasmid DNA is transfected into 293HEK cells and selected with 200 µg/mL Hygromycin.

Crude plasma membranes are prepared using cells from suspension culture. The cells are lysed on ice in hypotonic buffer ning 25 mM Tris HCl, pH 7.5, 1 mM MgCl2, DNAse1, 20 µg/mL, and Roche Complete™ Inhibitors without EDTA. The cell suspension is homogenized with a glass dounce homogenizer using a ® pestle for strokes. The homogenate is centrifuged at 4°C at 1800 x g for 15 minutes. The supernatant is collected and the pellet resuspended in hypotonic buffer and re- homogenized. The mixture is centrifuged at 1800 x g for 15 minutes. The second supernatant is combined with the first supernatant. The combined supernatants are recentrifuged at 1800 x g for 15 minutes to clarify. The clarified supernatant is transferred to high speed tubes and centrifuged at 25000 x g for 30 minutes at 4°C. The ne pellet is resuspended in homogenization buffer and stored as frozen aliquots at -80 °C freezer until use.

Glucagon-like e 1 (GLP-1) is radioiodinated by the Ilactoperoxidase procedure and purified by reversed phase HPLC at -Elmer Life and Analytical Sciences (NEX308). The specific ty is 2200 Ci/mmol. KD determination is med by homologous competition instead of saturation g due to high ol content in the I-125 GLP-1 material. The KD is estimated to be 0.329 nM and is used to calculate Ki values for all compounds tested.

The receptor binding assay is carried out using a Scintillation Proximity Assay (SPA) with wheat germ agglutinin (WGA) beads previously blocked with 1% fatty acid free BSA (Gibco). The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% fatty acid free BSA, 0.003% Tween20, and Roche Complete™ tors without EDTA. Glucagon-like peptide 1 is dissolved in 100% DMSO at 1 mg/mL and stored frozen at -20 °C in 30 µL aliquots. The glucagon-like peptide 1 aliquot is diluted and used in binding assays within an hour. The peptide analog is dissolved in 100% DMSO and serially diluted in 100% DMSO. Next, 10 µL diluted compounds described herein or 100% DMSO are transferred into Corning® 3632 clear bottom assay plates containing 40 µL assay binding buffer or cold glucagon (NSB at 1 µM . Then, 90 µL membranes (0.5 µg/well), 50 µL I-125 Glucagon-like peptide 1 (0.15 nM final in reaction), and 50 µL of WGA beads (150 µg/well) is added, , and mixed on a plate shaker for 1 minute. Plates are read with a PerkinElmer Life and Analytical Sciences Trilux MicroBeta® scintillation counter after 12 hours of settling time at room temperature.

Results are calculated as a percentage of specific IGlucagon-like peptide 1 binding in the presence of compounds. The te IC50 tration of compound is derived by non-linear regression of percent specific binding of IGlucagon-like peptide 1 versus the concentration of compound added. The IC50 concentration is converted to Ki using the Cheng-Prusoff equation.

In experiments performed essentially as described in this assay, certain compounds described herein display an hGLP-1R/hGIPR ratio of approximately 0.5-4.0 (Table 1). The molar binding ratio is normalized to the corresponding molar ratio of a mixture of native GIP and GLP-1. This normalization factor is 4.53 based on binding data for GIP (Ki=0.175 nM) and GLP-1 (Ki=0.793 nM). The value of 1.48 demonstrates the balanced co-agonist activity of Example 1.

Table 1: Receptor Binding Affinity, Ki, nM (SEM, n) Absolute Molar ratio ratio of of Compound Human GIP-R Human GLP-1R hGLP- hGLP- 1R/hGIP-R P-R Example 1 34.4 (5.0, n=8) 232 (40, n=8) 6.7 1.48 GIP, 1-42 0.175 (0.022, n=8) >175 (n=14) >1000 GLP-1, 7- >100 (n=15) 0.793 (0.099, n=8) <0.008 36-NH2 Example 3 26.7 (2.3, n=17) 427 (33, n=17) 16 3.53 Example 6 44.2 (3.6, n=14) 365 (28, n=14) 8.3 1.83 e 7 46.1 (5.9, n=11) 352 (39, n=11) 7.6 1.68 e 8 67.5 (9.9, n=6) 307 (35, n=6) 4.5 0.99 Example 4 40.7 (5.1, n=7) 714 (76, n=7) 17.5 3.86 Example 2 63.9 (11.8, n=8) 344 (60, n=8) 5.4 1.19 Example 5 17.8 (3.0, n=5) 158 (32, n=5) 8.9 1.96 Means are expressed as geometric means with the standard error of the mean (SEM) and the number of replicates (n) indicated in parenthesis. A qualifier (>) indicates data did not reach 50% inhibition and the Ki is calculated using the t tration tested in the assay.

Functional hGIP-R, hGLP-1R, and hGCGR assays.

The in vitro functional activity towards human GIP, GLP-1, and on ors for compounds described herein are determined in HEK-293 clonal cell lines expressing these receptors. Each receptor over-expressing cell line is d with compounds described herein in DMEM (Gibco Cat# 31053) supplemented with 1X GlutaMAXTM (Gibco Cat# 35050), 0.25% FBS, 0.05% fraction V BSA, 250 µM IBMX and 20 mM HEPES in a 40 µl assay volume. After an incubation of 60 minutes at room temperature, the resulting increase in intracellular cAMP is quantitatively determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit (Bedford, MA). Briefly, cAMP levels within the cell are detected by adding the cAMP-d2 conjugate in cell lysis buffer (20 µl) followed by the antibody anti-cAMP-Eu3+-Cryptate, also in cell lysis buffer (20 µl). The resulting competitive assay is incubated for at least 60 minutes at room temperature, then detected using a PerkinElmer on® instrument with excitation at 320 nm and emission at 665 nm and 620 nm. Envision units (emission at 620nm*10,000) are inversely proportional to the amount of cAMP present and are converted to nM cAMP per well using a cAMP standard curve. The amount of cAMP generated (nM) in each well is ted to a percent of the maximal response observed with either human GIP(1-42)NH2, human GLP-1(7-36)NH2, or human glucagon controls.

A relative EC50 value and percent top (Emax) is derived by non-linear regression analysis using the percent maximal response versus the tration of the compound bed herein, fitted to a four-parameter logistic equation.

In experiments performed essentially as described in this assay, certain compounds described herein demonstrate activity against human GIP and GLP-1 receptors, while also demonstrating selectivity over the on receptor. In Table 2, functional potency t the receptors is shown for the native hGIP(1-42)NH2, hGLP- 1(7-36)NH2, and hGlucagon controls, and certain compounds bed herein.

Table 2. Functional Potency (EC50) against human GIP, GLP-1, and glucagon receptors.

Compound Human GIP-R Human GLP-1R Human GCGR EC50, Emax,% EC50, Emax, % EC50, Emax, % nM±SEM nM±SEM nM±SEM, (n) (n) (n) Example 1 11.0±0.9 97.9±3.0 71.2±7.2 85.2±4.4 >1000 (13) ND (17) (17) Example 3 3.15±0.34 106±3 33.9±3.2 96.2±6.2 >1000 (10) ND (14) (14) Example 6 4.40±0.71 106±3 28.4±4.2 104±4 >1000 (9) ND (13) (13) Example 7 8.07±0.70 106±3 35.5±2.6 97.2±3.4 >1000 (13) ND (17) (17) Example 8 .7 108±4 .8 88.4±2.3 >1000 (10) ND (14) (13) Example 4 3.76±0.83 102±3 66.9±14.9 100±5 >1000 (2) ND (6) (6) Example 2 .0 94.7±2.0 75.7±6.0 98.2±5.5 >1000 (9) ND (13) (13) Example 5 8.76±0.86 105±2 70.9±9.7 105±4 >1000 (10) ND (10) (10) hGLP-1(7- 0.176 102±2 36)NH2 ±0.015 hGIP(1- 0.135 100±1 42)NH2 ±0.010 hGlucagon 0.0208 115±2 ±0.0024 Means for EC50 are expressed as geometric means +/- rd error of the mean (SEM) with the number of replicates (n) indicated in parenthesis. Means for Emax are expressed as the arithmetic mean +/- standard error. ND signifies that agonist activity was not detected. A ier (>) indicates that an EC50 could not be determined. All values shown are to three significant digits Functional activation of hGIP-R cells to generate intracellular cAMP in incretin- secreting cell lines The functional ty of hGIP-R for nds described herein are demonstrated by the ability of the compounds to generate intracellular cAMP in GLUTag cells, a stable immortalized relatively differentiated murine enteroendocrine cell line that expresses the proglucagon gene and secretes the glucagon-like peptides in a regulated . The cells are maintained at 37ºC, 5% CO2, 95% humidity in DMEM medium supplemented with 5.5 mM glucose, 10% FBS, and 2 mM glutamine. Prior to assay, cells are trypsinized, pelleted, and seeded into 96-well tissue culture assay plates at a density of ,000 cells/well. Cells are allowed to attach and are incubated for 48 hours at 37ºC, 5% CO2. On the day of the assay, media is decanted from cells and 50 µl EBSS Buffer (0.1% BSA, 2 mM glucose and 0.25 mM IBMX) containing a range of compound concentrations (0.001 – 3 µM) is added to cells. The plate is incubated at 37ºC for one hour and cAMP levels determined using Cisbio Dynamic 2 cAMP HTRF kit (Bedford, MA). 25 µl of anti-cAMP cryptate and 25 µl cAMP d2 is added to each well and plates incubated for one hour at room temperature. Plates are read at 620nm and 665nm on a Tecan Genios Pro. Results are calculated from the 665nm/620nm ratio multiplied by 10000, and ted to nM cAMP per well using a cAMP standard curve. Data is analyzed with GraphPad using a 4-parameter non-linear logistic algorithm.

In experiments performed ially as bed in this assay, certain compounds described herein show a dose-dependent, enhanced cAMP accumulation of GLUTag cells (Table 3). The native GLP-1 control fails to induce any s in cAMP at all trations tested and indicates that this cell system exclusively expresses the GIP receptor; therefore, certain compounds described herein can be shown to exert an effect through the GIP receptor.

Table 3: EC50 in GLUTag cells.

Compound Average EC50, nM (n) Example 3 1610 (1) Example 7 2746 (1) Example 4 2186 (1) Example 2 2918 (1) Example 1 1494 (2) Native GIP 11.62 (3) Measurement of Intracellular cAMP in HEK293 Cells Transiently Expressing the Human GLP-2 Receptor The functional activity of hGLP-2R in the presence of compounds described herein is demonstrated by measuring intracellular cAMP in HEK293 cells. These cells are passaged in complete medium, transfected in suspension with Promega Fugene6 reagent and human full-length GLP-2R cDNA in pcDNA3.1 expression vector, and allowed to adhere to tissue culture flasks in a humidified 37°C 5% CO2 environment.

Following imately 48 hours of propagation, cells are lifted, strained, and cryopreserved with controlled rate freezing and 10% DMSO as a otectant. In subsequent assays, a single assay-ready vial from the same cell freeze is thawed to minimize inter-assay variation. On the day of the cellular assay, freezing medium is exchanged with Invitrogen 31053 DMEM containing 0.5% FBS.

Cells are counted for viability and equilibrated for approximately one to two hours at 37°C prior to ent. Compounds described herein are solubilized in DMSO and immediately diluted in DMEM medium containing 0.1% fraction V BSA and the nonspecific phosphodiesterase inhibitor, IBMX. Duration of treatment is 30 minutes at 37°C.

Final DMSO concentration does not exceed 1.1%, and final IBMX tration is 250 µM. Cyclic AMP is measured using the c 2 assay with homogenous time- ed fluorescence technology (Cisbio Bioassays, Bedford, MA). Respective cAMP concentrations are d from the ratio method of calculation and external standards. dal dose-responses of tested compounds are examined using the four parameter logistic equation and compared to the native C18-acylated ligand.

In experiments med essentially as bed in this assay, the C18-acylated human GLP-2 control has an EC50 value for receptor activation of 1.71 nM while certain compounds described herein have EC50 values from approximately 100x to 1000x higher.

The EC50 values for certain compounds described herein demonstrate selectivity against the GLP-2 receptor.

Table 4: GLP-2R onal activity measurement in HEK293 cells. nd Rel EC50 (nM) n GLP2-C18-diacid 1.857 4 Example 3 199.3 4 Example 7 1,800 4 Example 4 405 2 Example 1 238 4 Example 2 1612 2 Rodent islet insulin ion To assess action of compounds described herein in a system enting physiological GLP-1R and GIP-R sion levels insulin secretion, compounds are tested for effects on insulin secretion from wild-type rodent islets.

After common bile duct cannulation in male C57Bl/6 mice (22-26 g) or male Sprague-Dawley rats (approx. 250 g), the pancreas is distended with Hank's buffer (3 ml for mice or 10 ml for rats, containing 2% BSA and 0.75 mg/ml Clzyme collagenase (VitaCyte). Subsequently, tissues are digested in Hank's buffer at 37°C for 11–13 minutes (mice) or 14-16 minutes for rat pancreata. Purified islets (Histopaque-1100 gradient [Sigma-Aldrich], 18 min at 750x gravity) are cultured overnight in RPMI-1640 medium (Invitrogen) containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, and preconditioned by starvation in Earle’s ed Salt Solution (EBSS) supplemented with 0.1% BSA and 2.8 mM glucose. Subsequently, islets are incubated in EBSS (Invitrogen) supplemented with 0.1% BSA, 2.8–11.2 mM glucose and increasing levels of compound (6 s of 4 islets/condition). GLP-1(7-36)amide (30 nM) is used as a positive control. Insulin is measured over 90 minutes in supernatant using the MSD n Assay (Meso Scale, Gaithersburg, MD).

Certain compounds described herein dose-dependently increase insulin ion from both rat and mouse islets as depicted in Table 5.

Table 5. Rodent Islet n Secretion Rat islet insulin secretion Compound Mean ED50 (nM) N Example 3 34.9 2 Example 1 15.5 3 Mouse islet insulin secretion Compound Mean ED50 (nM) N Example 3 58.9 2 Example 6 51.4 1 Example 7 11.3 1 Example 4 3.5 1 Example 2 30.0 1 Example 1 47.2 2 Immunogenicity Profiling The risk of immunogenicity for compounds described herein is assessed using in silico prediction programs, such as Epivax in silico analysis. The risk of genicity for compounds described herein is also assessed by an ex-vivo method to e cultured T cells responses (3H-thymidine uptake and IL-2 cytokine ion) in the presence of compounds described herein.

With Epivax immune-informatic tools, an in silico assessment is performed on compounds described herein to predict an immune response following administration.

The analysis es the probability of a 9-mer frame to bind to a given human leukocyte antigen leukocyte antigen (HLA) allele and then detection of these Epi-Bars. For Example 1, a EpiMatrix score of approximately +1.13 indicates a much lower potential to induce an immune response compared to native GIP peptide backbone with a EpiMatrix score of + 15.4. A GIP / GLP-1 co-agonist Example from of + 29.5.

A measure of predicted clinical immunogenicity is also examined for compounds described herein by using the terization of CD4+ T-cell proliferation and IL-2 cytokine ion in a cohort of 50 healthy donors representative of the world HLA allotype population. n compounds described herein demonstrate a degree of T-cell stimulation and IL-2 secretion following exposure that does not exceed the threshold associated with known or positive genic compounds, indicating a low risk of producing al immunogenicity.

Pharmacokinetics Pharmacokinetics in Cynomolgus s.

The in vivo pharmacokinetic properties for compounds described herein are demonstrated using cynomolgus monkeys. The compounds are administered by a single intravenous or subcutaneous dose (0.2 mg/kg) in 20 mM citrate buffer (pH 7.0) at a volume of 0.21 ml/kg. Blood is collected from each animal at 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 204, 240, and 312 hours post-dosage. The plasma concentrations of compounds described herein are determined by a LC/MS method. Briefly, a compound bed herein is extracted from 100% Monkey plasma sample (50 µl) diluted with 1X PBS (150 µl) and mixed with N-butanol (400 µl). Three distinct liquid layers are formed with the compound located in the top layer. A volume of 200 µl is transferred to a vbottom 96-well plate, dried down using heated Nitrogen gas and reconstituted with 100 µl of 30% acetonitrile/ 0.1% formic acid. 20 µl of the tituted sample is injected onto a Supelco Analytical Discovery bio wide C5 3 µm column. The column effluent is directed into a Thermo Q-Exactive mass spectrometer for detection and quantitation.

In experiments performed essentially as described for this assay, Example 1 d a mean maximum plasma concentration approximately 8 hours post the subcutaneous dose. The mean half-life is 55 hours and the mean clearance is 0.73 mL/hr/kg. The bioavailability is approximately 83%. This data ts the potential of once-weekly dosing for Example 1. Data for other compounds described herein are summarized in Table 6.

Table 6: Mean Pharmacokinetic Parameters Following a Single Subcutaneous Dose of 0.2 mg/kg to Male Cynomolgus Monkeys Compound Mean Mean Mean Cmax Mean AUC0-inf Mean CL/F r) Tmax (hr) (µg/mL) (hr*µg/mL) (mL/hr/kg) Example 3 34 8 3.0 153 1.3 Example 7 31 6 2.9 136 1.5 Example 6 23 4 2.2 72.3 2.8 Example 8 23 10 1.0 42.8 4.7 Example 2 43 24 2.1 173 1.2 n = 2, nf = area under the curve from 0 to infinity, CL/F = clearance / bioavailability, Tmax = time to maximum concentration, Cmax = maximum plasma concentration, T1/2 = half-life.

Dose y projection The enous glucose tolerance test (ivGTT) in the rat is used to estimate relative potency of compounds described herein in comparison to semaglutide. Single subcutaneous (SC) doses of 0.1-10 nmol/kg of each compound are administered to the rats and an ivGTT is administered to each rat 16 hours osage. Exposure is measured at the time of the ivGTT, and for exposure response modeling, the insulin AUC in response to the ivGTT is used as the primary endpoint.

An Emax model is used to compare the re response profiles for Example 1 to utide. In experiments performed essentially as described for this assay, exposure is essentially the same for Example 1 and semaglutide for the dose levels that had drug levels above the limit of quantitation of the assay. Both data sets are fit simultaneously and E0 and Emax values are constrained to be the same for both nds. Only ED50 values are fit separately for the compounds. ED50 value for semaglutide is estimated as 0.6 +/- 0.2 nmol/kg. Potency of Example 1 is estimated as a relative potency to semaglutide, and is 1.7 +/- 0.6 times the potency of semaglutide. Adjusting for CL/F (apparent clearance) differences between the two molecules in monkeys and also for the difference in molecular weight, the projected mean human equivalent dose to 1 mg semaglutide is approximately 1.3 mg/week for Example 1.

Type 2 Diabetes Rat in vivo n secretion following intravenous glucose ) Male Wistar rats n Labs, Indianapolis, IN) are randomized by body weight and dosed 1.5 ml/kg s.c. 16 hour prior to glucose administration and then fasted. Doses are vehicle, 0.1, 0.3, 1, 3 and 10 nmol/kg. Animals are weighed, and then anesthetized with sodium pentobarbital (Nembutal Sodium solution; Ovation Pharmaceuticals) dosed i.p. (65 mg/kg, 30 mg/ml). A time zero blood sample is collected into EDTA tubes after which glucose is administered (0.5 mg/kg, 5 ml/kg). Blood s are collected at 2, 4, 6, 10, 20, and 30 minutes post glucose. Plasma glucose levels are determined using a Hitachi analyzer (Roche) and plasma insulin is measured by the MSD insulin assay (Meso Scale, Gaithersburg, MD).

As shown in Table 7, certain compounds described herein ependently enhance insulin secretion following i.v. injection of glucose. The ED50 for insulin and the maximal increases in insulin ion red as area under the insulin curve) are given in Table 7.

Table 7. Enhancement of insulin secretion in the rat IVGTT assay Compound ED50 (nmol/kg) % max increase of insulin AUC Example 3 1.00 314 +/- 38% Example 3 1.42 219 +/- 19% e 6 2.58 289 +/- 4% Example 7 4.33 335 +/- 35% Example 7 1.10 278 +/- 26% Example 8 6.13 324 +/- 30% semaglutide 0.70 231 +/- 13% Example 2 1.62 233 +/- 19% Example 1 0.87 298 +/- 17% Example 5 1.02 349 +/- 39% Effect on weight loss, body ition and hepatic steatosis in diet-induced obese (DIO) mice The effects on weight loss, body composition and hepatic steatosis in DIO mice for compounds described herein are ted in 6 DIO mice. These animals, although not diabetic, display insulin resistance, dyslipidemia, and hepatic steatosis, which are all characteristics of metabolic syndrome, after being placed on a high fat (60% Kcal from fat) diet for 12 weeks.

In this study, 23-24 week old male diet-induced obese (DIO) C57/Bl6 male mice are used, with each weighing 41-49 g and having an initial fat mass ranging from 10.5- 17.5 g. Animals are individually housed in a temperature-controlled (24°C) facility with a 12 hour light/dark cycle (lights on 22:00), and free access to food and water. After 2 weeks acclimation to the facility, the mice are randomized to treatment groups (n=5/group) based on body weight so each group has similar starting mean body weight.

Vehicle control, compounds described herein (with dose ranging from 10 to 100 nmol/kg), or a cting GLP1 analogue semaglutide (30 nmol/kg), dissolved in vehicle (20 mM Citrate Buffer at pH 7.0), are administered by SC injection to ad libitum fed DIO mice 30-90 minutes prior to the onset of the dark cycle every three days for 15 days. SC injections are made on Day 1, 4, 7, 10, and 13. Daily body weight and food intake are ed throughout the study. Absolute changes in body weight are calculated by subtracting the body weight of the same animal prior to the first injection of compound.

On days 0 and 14, total fat mass is measured by nuclear magnetic resonance (NMR) using an Echo Medical System (Houston, TX) instrument.

On Day 15, blood glucose is measured with Accu-Chek glucometer (Roche) from tail vein blood and then animals may be iced and livers removed and frozen. Liver triglycerides, determined from homogenates of livers collected at sacrifice, and plasma cholesterol are measured on a Hitachi Modular P clinical analyzer. tical comparisons between groups are done using one-way ANOVA followed by t’s multiple comparison test. The ED50 for weight loss lowering is determined in GraphPad Prism using the non-linear fit tool.

In experiments performed essentially as described in this assay, certain nds bed herein d body weight and fat mass in a dose-dependent manner (Table 8-13), and compared to semaglutide, may be 3-5x more efficacious in lowering body weight. The ED50 of Example 1 in percent body weight loss is 5.422 nmol/kg (95% Confidence al levels [nmol/kg] = 2.2 to 13.6). Reduced body weight is found to be primarily due to reduction in fat mass.

Table 8. Percent body weight or fat mass change in DIO mice.

Treatment Dose (nmol/kg) % Change from % Change from starting body weight starting fat mass Control 0 -3.14 ± 0.88 -4.84 ± 1.79 Semaglutide 10 -12.36 ± 1.00**** -18.21 ± 2.24** Semaglutide 30 -14.20 ± 1.01**** -21.90 ± * utide 100 -19.30 ± 1.38**** -33.51 ± 3.30*** Example 3 10 -13.38 ± 0.88**** -20.76 ± 2.42*** Example 3 30 -18.13 ± ** -30.90 ± ** Example 3 100 -25.84 ± 1.93**** -45.92 ± 2.15**** Example 6 10 -15.31 ± 1.25**** -24.75 ± 1.89**** Example 6 30 -21.62 ± 0.92**** -36.30 ± 2.47**** Example 6 100 -33.95 ± 1.93**** -64.64 ± 4.04**** **p<0.01, ***p<0.001, ****p<0.0001 from control group ay ANOVA, Dunnett’s). The results are expressed as Mean ± SEM of 5 mice per group.

Table 9. Percent body weight or fat mass change in DIO mice.

Treatment Dose (nmol/kg) % Change from % Change from starting body weight starting fat mass Control 0 -0.74 ± 1.49 3.04 ± 3.65 Semaglutide 30 -17.03 ± 0.98**** -35.94 ± 4.09**** Example 2 10 -23.27 ± 1.72**** -49.89 ± 5.62**** e 2 30 33.07 ± 1.65**** -72.80 ± 4.04**** Example 2 100 -34.66 ± 1.80**** -76.20 ± 3.78**** Example 5 10 -23.42 ± 1.43**** -51.28 ± 1.89**** Example 5 30 -26.84 ± 3.14**** -62.77 ± 5.49**** Example 5 100 -37.86 ± 2.25**** -81.08 ± 1.68**** Example 1 10 -25.18 ± .1.82**** -50.98 ± 2.87**** Example 1 30 -26.58 ± 2.49**** -59.98 ± 6.60**** e 1 100 -38.14 ± 1.67**** -79.79 ± 3.10**** ****p<0.0001 from control group ay ANOVA, Dunnett’s). The results are expressed as Mean ± SEM of 5 mice per group.

Table 10. Percent body weight or fat mass change in DIO mice.

Treatment Dose (nmol/kg) % Change from % Change from ng body weight starting fat mass Control 0 -2.43 ± 2.06 -1.49 ± 3.69 Example 6 10 -17.54 ± 1.17**** -34.30 ± 1.20**** e 6 30 -19.52 ± 1.18**** -39.52 ± ** Example 6 100 -29.36 ± 2.62**** -56.66 ± 4.96**** Example 7 10 -15.08 ± 1.22**** -26.46 ± 2.31*** Example 7 30 -20.70 ± 1.95**** -43.49 ± 5.47**** Example 7 100 -24.36 ± 2.06**** -49.92 ± 3.40**** Example 8 10 -17.13 ± ** -34.20 ± 1.62**** Example 8 30 -25.27 ± 0.70**** -54.24 ± 2.35**** Example 8 100 -29.91 ± 2.03**** -65.23 ± 6.69**** ****p<0.001 from control group (One-Way ANOVA, Dunnett’s). The results are expressed as Mean ± SEM of 5 mice per group.

Table 11. Blood glucose, plasma cholesterol and plasma triglycerides in DIO mice.

Treatment Dose Blood Glucose Plasma Plasma (nmol/kg) (mg/dl) Cholesterol Triglycerides (mg/dl) (mg/dl) Control 0 141.6 ± 5.59 303.2 ± 13.97 54.2 ± 11.14 Semaglutide 10 147.6 ± 6.13 226.8 ± 13.86** 27.36 ± 3.56* Semaglutide 30 146.8 ± 8.43 229.8 ± 10.96** 27.9 ± 6.01* utide 100 134.3 ± 9.22 218.4 ± 18.70** 36.46 ± 5.34 Example 3 10 109.5 ± 2.35*** 213.2 ± 15.54*** 30.38 ± 8.23 Example 3 30 107.6 ± 1.32*** 177.4 ± 16.58**** 21.32 ± 2.48** Example 3 100 102.00 ±0.50**** 194.00 ± 14.40*** 20.55 ± 4.60** Example 6 10 105.8 ± 2.10*** 198.4 ± 6.76**** 20.78 ± 4.40** e 6 30 100.1 ± 3.29**** 186.4 ± 17.04**** 26.12 ± 6.85* Example 6 100 103.6 ± 3.20**** 151.4 ± 14.32**** 17.26 ± 1.67*** *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from control group (One-Way ANOVA, Dunnett’s). The results are expressed as Mean ± SEM of 5 mice per group.

Table 12. Blood glucose, plasma cholesterol and liver cerides in DIO mice.

Treatment Dose Blood Glucose Plasma Liver (nmol/kg) (mg/dl) Cholesterol cerides ) (mg/g tissue) Control 0 144.30 ± 8.16 233.6 ± 12.99 206.65 ± 29.47 Semaglutide 30 136.3 ± 3.81 161.0 ±13.92*** 67.63 ± 23.40**** Example 2 10 110.8 ± 3.87** 121.8 ± 60.77 ± 13.24**** 13.64**** e 2 30 110.8 ± 3.20** 114.00 ± 65.78 ± 17.07**** 9.70**** Example 2 100 113.2 ± 4.86** 109.4 ± 56.74 ± 17.76**** 8.83**** Example 5 10 111.00 ± 6.56** 126.6 ± 48.30 ± ** 9.67**** Example 5 30 104.5 ± 5.30*** 108.2 ± 39.60 ± 4.71**** Example 5 100 105.3 ± 6.16*** 108.6 ± 67.96 ± 13.53**** 4.83**** Example 1 10 102.3 ± 120.6 ± 60.74 ± 5.33**** .59**** 8.55**** Example 1 30 110.7 ± 5.85** 118.2 ± 45.24 ± 5.87**** .11**** Example 1 100 106.7 ± 7.33*** 107.6 ± 66.98 ± 17.29**** .43**** *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from control group (One-Way ANOVA, Dunnett’s). The s are expressed as Mean ± SEM of 5 mice per group.

Table 13. Blood glucose and plasma cholesterol in DIO mice.

Treatment Dose (nmol/kg) Blood Glucose Plasma Cholesterol ) (mg/dl) Control 0 152.4 ± 3.63 243.6 ± 13.12 Example 6 10 121.4 ± 2.74*** 167.8 ± 15.59**** Example 6 30 121.9 ± 6.65** 159.8 ± ** Example 6 100 116.1 ± 4.67**** 144.2 ± 7.12**** Example 7 10 113.6 ± 4.16**** 161.8 ± 6.2**** Example 7 30 114.7 ± 4.70**** 153.6 ± 13.47**** Example 7 100 114 ± 2.36**** 145.4 ± 9.48**** e 8 10 114.7 ± 4.61**** 158.8 ± 7.57**** Example 8 30 117.1 ± 8.26*** 139.4 ± 6.83**** e 8 100 125.4 ± 6.30** 127.8 ± 6.34**** *p<0.05, 01, ***p<0.001, ****p<0.0001 from control group (One-Way ANOVA, Dunnett’s). The results are expressed as Mean ± SEM of 5 mice per group.

The effect on energy lism in DIO mice The effects on energy metabolism in DIO mice for compounds described herein are evaluated in 26 week old C57/Bl6 DIO male mice, weighing 43-50 g. Mice are individually housed in a temperature-controlled (24°C) facility with a 12 hour light/dark cycle (lights on 22:00), and with free access to food TD95217 (Teklad) and water. After 2 weeks acclimation to the facility, mice are randomized to treatment groups roup) based on body weight so each group has similar starting mean body weight. s are placed in a PhenoMaster/LabMaster calorimeter (TSE Systems, Chesterfield, MO) for 3 days of acclimation. Vehicle control (20 mM citrate buffer at pH 7.0, 10 ml/kg), compounds described herein, or a long-acting GLP1 analogue, semaglutide, (30 nmol/kg) are subcutaneously administered to ad libitum fed DIO mice 30-90 minutes prior to the onset of the dark cycle every three days for 22 days. Heat and respiratory quotient (RER) are measured by indirect calorimetry as described using an open-circuit calorimetry system. RER is the ratio of the volume of CO2 produced (Vco2) to the volume of O2 consumed (Vo2). Heat is calculated with full body weight considered: VO2= FlowML*(V1+V2)/N2Ref*Animal weight*100) VCO2=FlowML*dCO2/Animal weight*100) Heat= (CVO2*VO2+CVCO2*VCO2)/1000; where CVO2=3.941; 1.106 In experiments performed essentially as described in this assay, mice treated with Example 1 significantly increased their metabolic rate 10 to 15 % compared to the l group, starting from week 2, and sustained the effect hout the treatment period.

Semaglutide, however, had no effect on metabolic rate. The increase in metabolic rate for Example 1 lly accounts for the additional weight loss observed with Example 1 treatment in comparison with semaglutide treatment.

The effect on gastric emptying in DIO mice The s on gastric emptying in DIO mice for compounds described herein are evaluated in 23 week old diet-induced obese (DIO) male mice (Harlan). The mice are fasted for 16-17 hours. During the start of fasting period the mice are dosed subcutaneously with vehicle l (20 mM citrate buffer at pH 7.0); escalating doses of compounds described herein (3, 10, 30 and 100 nmol/kg), or a long-acting GLP1 analogue, utide, (30 nmol/kg). The next day, mice are administered 0.5 ml (0.5 gram) of freshly prepared semi-liquid diet (2 minutes apart) by oral gavage. Water is removed at this time to prevent dilution of administered diet. Two hours after diet administration, mice are euthanized two minutes apart by CO2 gas. The stomach is removed while clamped at both cardiac and pyloric openings, then clamps removed and the full stomach weighed in a weigh boat. The stomach is then incised and contents removed. The stomach is washed and dried and re-weighed to assess food contents in stomach. The % gastric emptying equals 100 x (1 - (food remaining in stomach/food orally administered)).

In experiments performed essentially as described in this assay, Example 1 slowed the gastric ng rate of the iquid diet in a dose dependent . The m inhibition of gastric emptying was observed at a dose of 10 nmol/kg +/- dose (Table 14).

Table 14. Gastric emptying of a semi-liquid diet in lean C57/BL6 DIO mice.

Treatment Dose (nmol/kg) Percent Gastric ng (Mean ± SEM) Vehicle (n=5) 0 69.50 +/- 6.60 Semaglutide (n=5) 30 30.56 +/- 7.53** Example 1 (n=4) 3 49.11 +/- 8.52 Example 1 (n=5) 10 9.76 +/- 7.69**** Example 1 (n=5) 30 26.53 +/- 8.14** Example 1 (n=5) 100 18.45 +/- 6.87*** Statistical comparisons n groups are done by using one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from control group. The results are expressed as mean +/- SEM of 4-5 mice per group.

Plasma Corticosterone Measurements in Sprague Dawley Rats As suggested in certain published studies, elevated plasma corticosterone levels are an indicator of possible reduced tolerability for GIP and GLP-1 analogues. Plasma osterone levels are evaluated using Sprague Dawley Rats (Harlan, apolis), weighing approximately 220 g and acclimated for at least 72 hours before handling. The rats are then dosed with vehicle (20 mM e buffer, pH 7), semaglutide (10 nmol/kg), or compounds described herein at 3, 10 or 30 nmol/kg s.c. with 8 rats per dose group.

The rats are decapitated 16 hours later. Blood is collected into EDTA tubes on ice, then centrifuged 5 minutes at 8000 RPM in a Eppendorf 5402 tabletop centrifuge. Plasma is stored at -80°C until analysis.

For corticosterone analysis, corticosterone rds , 27840) are prepared by serial dilutions in HPLC-grade methanol, H2O and the addition of 5% charcoal stripped rat serum (Bioreclamation, RATSRM-STRPD-HEV). Rat plasma s are diluted with PBS, precipitated with cold methanol, incubated for 20 minutes at -20° C, and then fuged at 14,000 RPM with an Eppendorf 5417R at 4°C. Supernatants are extracted, evaporated under a stream of N2 gas, and reconstituted in MeOH/H2O (1:1) solution. Samples are ed on the LC/MS equipped with a t CSH C18 3.5 μm HPLC column (2.1 mm x 30 mm) (Waters #186005254).

In experiments performed essentially as described for this assay, Example 1 demonstrated no increase in plasma corticosterone levels at any of the doses tested while semaglutide had an approximately 4x increase over control.

Table 15: Plasma corticosterone analysis in Sprague Dawley Rats Corticosterone (ng/ml) Compound Mean SEM Vehicle 60.78 8.41 nmol/kg Semaglutide 274.57 42.06 3 nmol/kg Example 1 52.21 19.39 nmol/kg Example 1 32.46 9.78 nmol/kg Example 1 31.35 5.86 Amino Acid Sequences SEQ ID NO: 1 (Human GIP) YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ SEQ ID NO: 2 (Human GLP-1) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR SEQ ID NO: 3 YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS n X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide.

SEQ ID NO: 4 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is ally modified through conjugation to the n-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)18-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide.

SEQ ID NO: 5 FTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide.

SEQ ID NO: 6 YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide.

SEQ ID NO: 7 YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS n X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K hain with -Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)18-CO2H; and the inal amino acid is amidated as a C-terminal y amide.

SEQ ID NO: 8 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is ally modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)16-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide.

SEQ ID NO: 9 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)2-CO-(CH2)16-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide.

SEQ ID NO: 10 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS n X1 is Aib; X2 is Aib; K at on 20 is chemically ed through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide.

SEQ ID NO: 11 YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS wherein X1 is Aib; X2 is Aib; K at on 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with -Amino-ethoxy)- ethoxy]-acetyl)2-(γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally amidated as a C-terminal y amide.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification, and claims which include the term “comprising”, it is to be understood that other features that are additional to the features prefaced by this term in each ent or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.

In this ication where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any iction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

The following numbered aphs define particular aspects of the present invention: 1. A compound of the a: YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS; wherein X1 is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino- ethoxy)-ethoxy]-acetyl)2-(γGlu)a-CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally amidated as a C- terminal primary amide (SEQ ID NO: 11), or a pharmaceutically acceptable salt thereof. 2. The compound of Paragraph 1, wherein X3 is Phe. 3. The compound of Paragraph 1, n X3 is 1-Nal. 4. The compound of any one of Paragraphs 1-3, wherein b is 14 to 18.

. The nd of Paragraph 4, wherein b is 16 to 18. 6. The compound of Paragraph 5, n b is 18. 7. The compound of any one of Paragraphs 1-6, wherein a is 1. 8. The compound of any one of Paragraphs 1-6, wherein a is 2. 9. The compound of any one of Paragraphs 1-8, wherein the C-terminal amino acid is amidated as a C-terminal primary amide. 10. The compound of Paragraph 1, wherein X1 is Aib X2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino- ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H; X3 is Phe; and the inal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 3), or a pharmaceutically acceptable salt thereof. 11. The compound of Paragraph 1, wherein X1 is Aib X2 is Aib; K at on 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Aminoethoxy xy]-acetyl)2-(γGlu)2-CO-(CH2)18-CO2H; X3 is 1-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof. 12. The compound of Paragraph 1, wherein the Formula is: HO H N OH HN O O O O N HN O H Y N E G T F T S D Y S I N L D K I A QN A F V Q W L I A G G P S S G A P P P S NH2 O O 13. The compound of Paragraph1, wherein the Formula is: O HO H N OH O N OH O O O N HN O O O H Y N E G T F T S D Y S I N L D K I A QN A N V Q W L I A G G P S S G A P P P S NH O O 14. A pharmaceutical composition comprising the compound of any one of Paragraphs 1-13 with a pharmaceutically acceptable carrier, diluent, or excipient.

. A method of treating type 2 diabetes us, comprising administering to a t in need thereof, an effective amount of the compound of any one of Paragraphs 1-13. 16. The method of Paragraph 13, further comprising stering simultaneously, separately, or sequentially in combination with an effective amount of one or more agents selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters. 17. The compound of any one of Paragraphs 1-13, for use in therapy. 18. The compound of any one of aphs 1-13, for use in the treatment of type 2 diabetes mellitus. 19. The compound of any one of Paragraphs 1-13 in simultaneous, separate, or sequential combination with one or more agents selected from metformin, lidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, and sodium e co-transporters.

WE

Claims (13)

CLAIM :

1. A use, in the manufacture of a medicament for the treatment of obesity in a patient in need thereof, of a compound of Formula: FTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS; 5 wherein X1 is Aib; X2 is Aib; K at on 20 is chemically modified through conjugation to the epsilon-amino group of the K hain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)a- 10 CO-(CH2)b-CO2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe or 1-Nal; and the C-terminal amino acid is optionally amidated as a inal primary amide (SEQ ID NO: 11), or a pharmaceutically acceptable salt thereof.

2. The use of Claim 1, wherein X3 is Phe.

3. The use of Claim 1, wherein X3 is 1-Nal. 20

4. The use of Claim 1, wherein b is 14 to 18.

5. The use of Claim 4, wherein b is 16 to 18.

6. The use of Claim 5, wherein b is 18.

7. The use of Claim 4, wherein a is 1.

8. The use of Claim 4, wherein a is 2. 30

9. The use of Claim 4, wherein the C-terminal amino acid is amidated as a C- terminal primary amide.

10. The use of Claim 1, wherein X1 is Aib X2 is Aib; 5 K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1- CO-(CH2)18-CO2H; X3 is Phe; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ 10 ID NO: 3), and wherein said compound is a pharmaceutically acceptable salt thereof.

11. The use of Claim 1, wherein X1 is Aib 15 X2 is Aib; K at position 20 is ally modified through ation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2- CO-(CH2)18-CO2H; X3 is 1-Nal; 20 and the C-terminal amino acid is amidated as a C-terminal y amide (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof.

12. A use as claimed in claim 1, wherein the compound is a compound of Formula: HO H N OH HN O O O O N HN O H Y N E G T F T S D Y S I N L D K I A QN A F V Q W L I A G G P S S G A P P P S NH O O or a pharmaceutically acceptable salt thereof. 5

13. Use according to any one of claims 1-12 substantially as herein bed with reference to any example thereof. 9564674_1.txt