EA043950B1 - INCRETIN ANALOGUES AND THEIR APPLICATION - Google Patents

Description

The invention relates to incretin analogues having activity towards each of the following receptors: glucose-dependent insulinotropic polypeptide receptor (GIP), glucagon-like peptide-1 receptor (GLP-1 -glucagon-like peptide-1) and glucagon receptor. The incretin analogues described herein have structural characteristics that provide balanced activity and have an extended duration of action at each of these receptors. Such incretin analogues may be useful for the treatment of disorders such as type 2 diabetes mellitus (T2DM), dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and/or obesity.

Over the past few decades, the prevalence of diabetes has continued to rise. T2DM is the most common form of diabetes, accounting for about 90% of all forms of diabetes. T2DM is characterized by high blood glucose levels caused by insulin resistance. The current standard of care for T2DM includes diet and exercise, as well as treatment with oral and injectable glucose-lowering medications, including incretin-based drugs such as GLP-1 receptor agonists.

GLP-1 is a 36 amino acid peptide, the main biologically active fragment of which is produced as a C-terminal amidated 30 amino acid peptide (GLP-17.36; SEQ ID NO: 2), which stimulates glucose-dependent insulin secretion and prevents hyperglycemia in patients with diabetes. . Various GLP-1 analogs are currently available for the treatment of T2DM, including dulaglutide, exenatide, and liraglutide. However, many currently marketed GLP-1 receptor agonists are dose-limited due to gastrointestinal side effects such as nausea and vomiting. When treatment with oral drugs and incretin-based drugs is insufficient, the use of insulin is considered. Despite available treatment options, a significant number of patients receiving approved therapies do not achieve glycemic control goals (see, for example, Casagrande et al. (2013), Diabetes Care, 36:2271-2279).

Uncontrolled diabetes can lead to one or more conditions that affect the morbidity and mortality of such individuals. One of the major risk factors for developing T2DM is obesity, and the majority of individuals with T2DM (~90%) are overweight or obese. Reducing body adiposity has been documented to have beneficial effects on obesity-associated comorbidities, including hyperglycemia and cardiovascular complications. Thus, to optimize treatment strategy, a therapy that is effective for glucose control and weight loss is needed.

Therefore, new treatments under investigation include compounds having not only activity at the GLP-1 receptor, but also activity at one or more other receptors, such as GIP receptors and/or glucagon receptors. In fact, some compounds have been described as having triple agonist activity (ie, activity at each of the GIP, GLP-1 and glucagon receptors). For example, international patent application publication No. WO 2015/067716 describes glucagon analogues having triple agonist activity. Similarly, international patent application WO 2016/198624 describes analogs of exendin-4, which itself is a GLP-1 analog and has triple agonist activity. Similarly, international patent applications No. WO 2014/049610 and WO 2017/116204 describe a number of analogs with triple agonist activity. In addition, international patent application No. WO 2017/153375 describes glucagon and GLP-1 co-agonists that also have GIP activity.

However, there remains a need to develop treatments, particularly for T2DM, that are capable of providing effective glucose control with the added benefit of weight loss and a favorable side effect profile. There is also a need for therapeutic agents available for use with a sufficiently extended duration of action to allow dosing to be administered less frequently, such as once daily, thrice weekly, twice weekly, or once weekly.

The incretin analogues described herein seek to meet the needs identified above. Accordingly, this document describes incretin analogues with activity at each of the GIP, GLP-1 and glucagon receptors. Therefore, the incretin analogues described herein provide the advantage of balanced activity, allowing dosages to be administered that provide sufficient activity at each receptor to obtain the benefit of agonism at that receptor, while avoiding the unwanted side effects associated with too much activity. In addition, the incretin analogues described herein have an extended duration of action at each of the GIP, GLP-1, and glucagon receptors, allowing for less frequent dosing, such as once daily, thrice weekly, twice weekly, or once daily. once a week. Thus, incretin analogues lead to enhanced glucose control, beneficial metabolic effects such as weight loss and/or

- 1 043950 improved body composition, beneficial effects on lipid metabolism, such as decreased levels of proprotein convertase subtilisin/kexin type 9 (PCSK9), and/or other benefits, such as increased bone mass or bone formation or decreased bone resorption. This document also describes effective treatments for other disorders or conditions, including obesity, NAFLD, NASH, dyslipidemia and/or metabolic disorders.

In one embodiment of the present invention, there is provided an incretin analogue that includes the formula

YX2Q GTFTS D YSI Xi з LD ΚΧι 7АХ19X20AFИЕ YL LX28X29G Ρ S SX34AP Ρ Ρ S, where X2 represents Aib,

X13 is L or aMeL;

X ₁₇ is any amino acid with a functional group available for conjugation, wherein the functional group is conjugated to a C16-C22 fatty acid;

X ₁₉ represents Q or A;

X ₂₀ represents Aib, aMeK, Q or H;

X ₂₈ represents E or A;

X ₂₉ represents G or Aib;

X ₃₄ is G or Aib (SEQ ID NO: 5); And

The C-terminal amino acid is optionally amidated, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a method for treating a disease such as dyslipidemia, fatty liver disease, metabolic syndrome, NASH, obesity and T2DM. Such methods may include at least the step of administering to an individual in need thereof an effective amount of an incretin analog described herein. In some cases, the disease in question is fatty liver disease, obesity, NASH, or T2DM.

In another embodiment of the present invention, an incretin analogue, as described herein, is provided for use in therapy. For example, an incretin analogue, as described herein, is proposed for use in the treatment of a disease such as dyslipidemia, fatty liver disease, metabolic syndrome, NASH, obesity and T2DM. In some cases, the disease in question is fatty liver disease, obesity, NASH, or T2DM.

In another embodiment of the present invention, an incretin analog as described herein is provided for use in the manufacture of a medicament for the treatment of dyslipidemia, fatty liver disease, metabolic syndrome, NASH, obesity and T2DM. In some cases, the disease in question is fatty liver disease, obesity, NASH, or T2DM.

In another embodiment, the present invention provides a pharmaceutical composition that includes an incretin analogue, as described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of incretin analogues, pharmaceutical compositions and methods, preferred methods and materials are described herein.

In addition, mentioning an element in the singular does not exclude the possibility of the presence of more than one of the elements unless the context clearly suggests the presence of one and only one of the elements. Accordingly, singular terms usually mean at least one.

GIP is a 42 amino acid peptide (SEQ ID NO: 4) and is an incretin that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the presence of glucose.

GLP-1 is a 36 amino acid peptide (SEQ ID NO: 2) and is also an incretin that stimulates glucose-dependent insulin secretion and has been demonstrated to prevent hyperglycemia in diabetic patients.

Glucagon is a 29 amino acid peptide (SEQ ID NO: 1) that helps maintain blood glucose levels by binding to and activating glucagon receptors on hepatocytes, stimulating the liver to release glucose stored in the form of glycogen through a process called glycogenolysis.

Oxyntomodulin (OXM) is a 37 amino acid peptide comprising not only the 29 amino acid sequence of glucagon, but also a carboxy-terminal octapeptide extension (SEQ ID NO: 3) that activates both glucagon and GLP-1 receptors, with a slightly higher activity towards the glucagon receptor compared to the GLP-1 receptor.

- 2 043950

In addition to T2DM, incretins and their analogues with activity at one or more of the GIP, GLP-1 and/or glucagon receptors have been described as having potential therapeutic value in a number of other pathological conditions, diseases or disorders, including e.g. , obesity, NAFLD and NASH, dyslipidemia, metabolic syndrome, bone disorders, Alzheimer's disease and Parkinson's disease. See, for example, Jall et al. (2017), Mol. 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 novel long-acting GLP-1/Glucagon/GIP triple-agonist (HM15211), ADA Poster 1139-P; Ding (2008), J. Bone Miner. Res., 23:536-543; Tai et al. (2018), Brain Res., 1678:64-74; Muller et al. (2017), Physiol. Rev. 97:721-766; Finan et al. (2013), Sci. Transl. Med., 5:209; Holscher (2014), Biochem. Soc. Trans., 42:593–600.

As used herein, the term about means a value within a statistically significant range of value or values, such as, for example, the reported concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such value or range may be within the order of, typically within 20%, more typically within 10%, and even more typically within 5% of the given value or range. The tolerance covered by the term about will depend on the particular system under study and can be readily estimated by one skilled in the art.

As used herein and with respect to one or more receptors: GIP, GLP-1 or glucagon, the term activity, activate, activating and the like. means the activity of a compound, such as the incretin analogues described herein, to bind and induce a response at the receptor(s), as measured by assays known in the art, such as the in vitro assays described below.

As used herein, the term amino acid with a functional group available for conjugation means any natural or unnatural amino acid with a functional group that can be conjugated to a fatty acid through, for example, a linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chlorine, iodine and thiol groups. Examples of naturally occurring amino acids containing such functional groups include K (amino), C (thiol), E (carboxyl), and D (carboxyl).

As used herein, the term C16-C22 fatty acid means a carboxylic acid having 16 to 22 carbon atoms. The C ₁₆ -C ₂ 2 fatty acid suitable for use in this invention may be a saturated monoacid or a saturated diacid. In this context, the term saturated means that the fatty acid does not contain carbon-carbon double or triple bonds.

As used herein, the term effective amount means the amount, concentration or dose of one or more incretin analogues described herein, or a pharmaceutically acceptable salt thereof, which, when administered in one or more doses to an individual in need thereof, provides the desired effect in such individual in diagnostic or treatment settings. The effective amount can be readily determined by one skilled in the art using known methods and by observing results obtained under similar circumstances. In determining the effective amount for an individual, a number of factors are considered, including, but not limited to, the species of mammal; its size, age and general health; specific disease or disorder; the extent or involvement or severity of the disease or disorder; individual patient response; a specific incretin analogue administered; method of administration; characteristics of the bioavailability of the administered drug; selected administration regimen; use of concomitant medications; and other relevant circumstances.

As used herein, the term extended duration of action means that the binding affinity and activity of the incretin analogue is maintained for a period of time greater than that of native human GIP, GLP-1, and glucagon peptides, allowing for less frequent dosing, e.g., once daily, three times weekly , twice a week or once a week. The temporal action profile of an incretin analogue can be measured using known pharmacokinetic assay methods, such as those used in the examples below.

As used herein, the term incretin analog means a compound having structural similarity to GIP, GLP-1 and glucagon, but with multiple differences from each of GIP, GLP-1 and glucagon, especially GIP (SEQ ID NO: 4), GLP- 1 (SEQ ID NO: 2) and human glucagon (SEQ ID NO: 1). The incretin analogues described herein include amino acid sequences resulting in compounds having affinity and activity for each of the GIP, GLP-1 and glucagon receptors (ie, triple agonist activity).

As used herein, the term "individual in need" means a mammal, such as a human, having a condition, disease, disorder or symptom requiring treatment or therapy, including, for example, those listed herein.

In this context, the term treat, treatment, influence, etc. means to control, slow, stop, or modify the progression or severity of an existing pathological condition, disease, disorder, or symptom.

- 3 043950

As used herein and with respect to an incretin analogue, triple agonist activity refers to an incretin analogue with activity at each of the GIP, GLP-1 and glucagon receptors, especially an analogue having balanced and sufficient activity at each receptor to benefit from its agonism receptor, while avoiding the unwanted side effects associated with too much activity. In addition, incretin analogues with triple agonist activity have an extended duration of action at each of the GIP, GLP-1, and glucagon receptors, giving them the advantage of allowing dosing to be administered less frequently, e.g., once daily, three times weekly, twice weekly. once a week or once a week.

The structural features of the incretin analogs described herein result in the analogs having sufficient activity at each of the GIP, GLP-1, and glucagon receptors to produce the beneficial effects of activity at each receptor (i.e., triple agonist activity ), but do not have such a degree of activity at any one receptor that they either inhibit the activity of the other two receptors or lead to undesirable side effects when administered in a dose sufficient to be active at all three receptors. Non-limiting examples of such structural features in certain embodiments of this invention and with reference to SEQ ID NO: 5 include L or aMeL at position 13, which have been found to promote optimal glucagon and GIP activity; Aib at position 20, which has been found to promote optimal GIP activity; acylation at position 17, which has been found to promote optimal glucagon activity; and Y at position 25, which has been found to promote optimal glucagon and/or GIP activity. Other examples of such structural features include the amino acids described herein at positions 22, 24 and 28-39, which have been found to promote optimal binding and effectiveness of all three receptors.

The structural features of the incretin analogs described herein also result in the analogs having many other beneficial properties relevant to their potential use as therapeutic treatments, including improving the solubility of the analogs in aqueous solutions, improving the stability of the chemical and physical composition , broadening the pharmacokinetic profile and minimizing the potential for immunogenicity. Non-limiting examples of specific structural features that lead to such features include acylation at position 17 with a C20 fatty acid, which promotes optimal pharmacokinetic (PK) profiles and manifestation; Aib, aMeK, Q or H at position 20, which promote optimal PK profiles and manifestation; and amino acid positions 22, 24 and 28-39, which promote optimal PK, immunogenicity, expression and stability.

It should be noted that the above lists of structural features are exemplary and not exhaustive, and that the combination of beneficial characteristics of the exemplary analogs described herein is not the result of any individual modification, but rather is achieved by the new combinations of structural features described in this document. In addition, the above-described effects of the above lists of modifications are not specific, since many of these modifications also have other effects important to the characteristics of the compounds described herein, as described below.

The amino acid sequences of incretin analogues described herein include naturally occurring amino acids typically depicted herein using standard single-letter codes (e.g., L=leucine), as well as alpha-methyl-substituted naturally occurring amino acid residues (e.g., α-methylleucine (aMeL ) and α-methyllysine (aMeK)) and some other unnatural amino acids such as alpha-aminoisobutyric acid (Aib). The structures of these amino acids are depicted below.

As noted above, the incretin analogs described herein have structural similarities to any of the native human peptides, but have many structural differences. For example, when compared to native human GIP (SEQ ID NO: 4), the incretin analogs described herein include modifications at one or more positions: 2, 3, 7, 13, 14, 17, 18-21, 23- 25, 28-29 and 30-42. In some cases, the incretin analogues described herein include amino acid modifications of native human GIP (SEQ ID NO: 4) at each of positions 2, 3, 7, 13, 14, 17, 18,

- 4 043950

20, 21, 23-25, 29 and 30-42. In some cases, the incretin analogues described herein include the following amino acid modifications: Aib at position 2; Q in position 3; T in position 7; L or aMeL at position 13; L in position 14; a modified K residue at position 17 that is modified by conjugation to the epsilon amino group of the K side chain with a C ₁₆ -C ₂₂ fatty acid, optionally using a linker; A in position 18; A in position 21; I in position 23; E in position 24; Y at position 25; G or Aib in position 29; and replacing amino acids at positions 30-42 with an amino acid sequence selected from GPSSGAPPPS (SEQ ID NO: 26) and GPSS-Aib-APPPS (SEQ ID NO: 27) (and truncated tail analogues). In other cases, the incretin analogs described herein also include modifications at one or more elements: A at position 19; aMeK, Aib or H at position 20; and E at position 28. In some cases, the incretin analogues described herein are amidated. In addition to the modifications described herein, the incretin analogs described herein may include one or more additional amino acid modifications, provided that the analogs remain capable of binding to and activating each of the GIP, GLP-1, and glucagon receptors.

As noted above, the incretin analogs described herein include a fatty acid moiety conjugated, for example, through a linker to a natural or non-natural amino acid with a functional group available for conjugation. This conjugation is sometimes called acylation. In some cases, the amino acid with a functional group available for conjugation may be K, C, E and D. In certain cases, the amino acid with a functional group available for conjugation is K, where the conjugation occurs to the epsilonamino group of the K side chain.

The acylation of the incretin analogues described herein occurs at position 17 in SEQ ID NO: 5, which has been identified as the optimal location for inclusion of this structure. The fatty acid and, in some embodiments of this invention, the linker act as albumin binding agents and provide the potential to form long-acting compounds.

The incretin analogues described herein utilize a C ₁₆ -C ₂₂ fatty acid chemically conjugated to an amino acid functional group either through a direct bond or a linker. The length and composition of fatty acids influences the half-life of incretin analogues, their efficacy in n vivo animal models, and their solubility and stability. Conjugation with a saturated fatty monoacid or C ₁₆ -C ₂₂ diacid results in incretin analogues that exhibit the desired half-life, desired activity in in vivo animal models, and desirable solubility and stability characteristics.

Examples of _C16 - _C22 saturated fatty acids for use herein include, but are not limited to _, palmitic acid (hexadecanoic acid) (C16 monoacid), hexadecanedionic acid ( _C16 diacid), margaric acid (heptadecanoic acid) (C16 monoacid) ₁₇ ), heptadecanedionic acid ( _C17 diacid), stearic acid ( _C18 monoacid), octadecanedioic acid ( _C18 diacid), nonadecanedic acid ( _C19 monoacid), nonadecanedioic acid ( _C19 diacid), arachidic acid ( eicosanoic acid) ( _C20 monoacid), eicosanodic acid (C20 diacid), heneicosanic acid ( _C21 monoacid ₎ , heneicosanodic acid ( _C21 diacid), behenic acid (docosanoic acid) (C22 monoacid), docosanodic acid (C22 diacid), including their branched and substituted derivatives.

In some cases, the _C16 - _C22 fatty acid may be a saturated _C18 monoacid, a saturated _C18 diacid, a saturated _C19 monoacid, a saturated _C19 diacid, a saturated C20 monoacid, a saturated C20 diacid, and branched and substituted derivatives thereof. In more specific cases, the C ₁₆ -C ₂₂ fatty acid may be stearic acid, arachidic acid and eicosandioic acid, especially arachidic acid.

In some cases, the linker may contain one to four amino acids, aminopolyethylene glycol carboxylate, or mixtures thereof. In some cases, aminopolyethylene glycol carboxylate has the following structure:

H-{NH-CH2-CH2-[O-CH2-CH2]mO-(CH ₂ )p-CO}n-OH, where m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2.

In some cases, the linker may have one or more moieties (2-[2-(2amino-ethoxy)ethoxy]acetyl), optionally in combination with one to four amino acids.

Where the linker includes at least one amino acid, said amino acid may be one to four Glu or yGIu amino acid residues. In some cases, the linker may include one or two Glu or yGlu amino acid residues, including their D forms. For example, the linker may include one or two yGIu amino acid residues. Alternatively, the linker may include one to four amino acid residues (such as, for example, Glu or yGIu amino acids) used in combination with up to thirty-six moieties (2-[2-(2-amino-ethoxy) ethoxy]acetyl). In particular, the linker may be combinations of one to four amino acids Glu or yGIu and one to four moieties (2-[2-(2-aminoethoxy)ethoxy]acetyl). In other cases, the linker may be combinations of one or two amino acids yGIu and one or two moieties (2-[2-(2-amino-ethoxy)ethoxy]acetyl).

Specifically, the incretin analogues described herein contain a linker and fatty acid components having the structure of the following formula:

(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(yClu)b-CO-(CH2)c-CO2H, where a is 0, 1 or 2, b is 1 or 2 and c is equal to 16 or 18. In the specific case, a is equal to 2, b is equal to 1 and c is equal to 18, the structure of which is depicted below.

In another specific case, a is equal to 1, b is equal to 2 and c is equal to 18, the structure of which is depicted below.

In another specific case, a is 0, b is 2, and c is 18, the structure of which is depicted below.

In another specific case, a is equal to 1, b is equal to 1, and c is equal to 18, the structure of which is depicted below.

As shown in the chemical structures of Examples 1-20 below, the linker-fatty acid moieties described above can be linked to the epsilon β-amino group of the lysine (K) side chain.

The affinity of the incretin analogs described herein for each of the GIP, GLP-1, and glucagon receptors can be measured using techniques known in the art to measure receptor binding levels, including, for example, techniques described in examples below, and is usually expressed as the value of the inhibitory constant (Ki). The activity of the incretin analogues described herein for each of the receptors can also be measured using techniques known in the art, including, for example, in vitro activity assays described below, and is usually expressed as an effective concentration 50 value (EC50 ), which is the concentration of the compound that produces the half-maximal simulation on the dose-response curve.

The incretin analogs described herein can be formulated into pharmaceutical compositions that can be administered by parenteral routes (eg, subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). Such pharmaceutical compositions and methods for their preparation are well known in the art. See, for example, Remington: the Science and Practice of Pharmacy (D.B. Troy, editor, 21st edition, Lippincott, Williams & Wilkins, 2006). In some cases, incretin analogues are administered subcutaneously.

The incretin analogues described herein can react with any of a number of inorganic and organic acids/bases to form pharmaceutically acceptable acid/base addition salts. Pharmaceutically acceptable salts and conventional methods for preparing them are well known in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd revised edition (Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use in this invention include sodium salts, trifluoroacetates, hydrochlorides and/or acetates.

This invention also provides and therefore covers new intermediates and methods for synthesizing the incretin analogs described herein, or pharmaceutically acceptable salts thereof. The incretin intermediates and analogs described herein can be prepared by various methods known in the art. For example, the examples below illustrate a method using chemical synthesis. The specific synthetic steps for each of the described pathways can be combined in various ways to produce the incretin analogues described herein. Reagents and starting materials are readily available to one skilled in the art.

- 6 043950

The specific incretin analogues described herein are generally effective over a wide range of dosages. For example, dosages for once weekly administration may range from about 0.01 mg/person/week to about 30 mg/person/week, ranging from about 0.1 mg/person/week to about 10 mg/person /week or even in the range from about 0.1 mg/person/week to about 3 mg/person/week. Thus, the incretin analogs described herein can be administered daily, three times a week, twice a week, or once a week, in particular once a week.

The incretin analogues described herein can be used to treat various pathological conditions, disorders, diseases or symptoms. In particular, methods are provided for treating T2DM in an individual, such methods comprising at least the step of administering to an individual in need thereof an effective amount of an incretin analog described herein, or a pharmaceutically acceptable salt thereof.

Also provided are methods of treating obesity in an individual, such methods comprising at least the step of administering to an individual in need thereof an effective amount of an incretin analog described herein, or a pharmaceutically acceptable salt thereof.

In addition, methods are provided for inducing non-therapeutic weight loss in an individual, such methods comprising at least the step of administering to an individual in need thereof an effective amount of an incretin analog described herein, or a pharmaceutically acceptable salt thereof.

Also provided are methods of treating metabolic syndrome in an individual, such methods comprising at least the step of administering to an individual in need thereof an effective amount of an incretin analog described herein, or a pharmaceutically acceptable salt thereof.

Also provided are methods of treating NASH in an individual, such methods comprising at least the step of administering to an individual in need thereof an effective amount of an incretin analog described herein, or a pharmaceutically acceptable salt thereof.

Also provided are methods of treating NAFLD in an individual, such methods comprising at least the step of administering to an individual in need thereof an effective amount of an incretin analog described herein, or a pharmaceutically acceptable salt thereof.

In these methods, the effectiveness of incretin analogues can be assessed, for example, by observing a significant decrease in blood glucose levels, observing a significant increase in insulin levels, observing a significant decrease in HbA1c, and/or observing a significant decrease in body weight.

Alternatively, the incretin analogs described herein or pharmaceutically acceptable salts thereof may be used to improve bone strength in an individual in need thereof. In some cases, the individual in need of this has hypoostosis or hypoosteidosis, or is being treated for a bone fracture, orthopedic procedures, prosthetics, 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.

Peptide synthesis.

Example 1.

Example 1 is a compound represented by the following description:

Y-A|b-aSTRT5OU51-aMeE-EOKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yClu)CO-(CH2)i8-CO2H)AQ-aMeK -AFIEYLLA-Aib-GPSSGAPPPS-NH2 (SEQ ID NO:7).

Below is a description of the structure of the compound of Example 1 using standard single-letter amino acid codes with the exception of the residues Aib2, aMeL13, K17, aMeK20 and Aib29, where the structures of these amino acid residues have been expanded:

The peptide backbone of Example 1 was synthesized using fluorenimethyloxycarbonyl (Fmoc)/tert-butyl (t-Bu) chemistry on a Sympony Multiplex 12-channel peptide synthesizer (Protein Technologies, Inc. Tucson, AZ).

The resin consists of 1% cross-linked polystyrene DVB (Fmoc-Rink-MBHA Low Load Resin, 100-200 mesh, EMD Millipore) at a displacement of 0.3-0.4 meq/g. Standard side chain protecting groups were used. Fmoc-Lys(Mtt)-OH was used for lysine at position 17, and Boc-Tyr(tBu)-OH) was used for tyrosine at position 1. Fmoc groups were removed before each coupling step (2x7 min), using

- 7 043950 using 20% piperidine in DMF. All standard amino acid combinations were carried out for 1 hour with the primary amine and for 3 hours with the secondary amine using an equal molar ratio of Fmoc amino acid (0.3 mM), diisopropylcarbodiimide (0.9 mM) and Oxyma (0.9 mM) at a 9-fold molar excess compared to the theoretical peptide load. The exception was combinations with Ca-methylated amino acids, the combinations with which were carried out for 3 hours. After completion of the synthesis of the peptide framework, the resin was thoroughly washed 6 times with dichloromethane (DCM) to remove residual DMF. The Mtt protecting group on lysine at position 17 was selectively removed from the peptide resin using two treatments with 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (2 x 40 minute treatment).

Subsequent addition of the fatty acid-linker moiety was accomplished by coupling 2-[2-(2-Fmoc-amino-ethoxy)ethoxy]acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic α-tert-butyl ester acid (Fmoc-Glu-OtBu, Ark Pharm, Inc.), mono-OtBu-eicosanodic acid (WuXi AppTec, Shanghai, China). A 3-fold excess of reagents (AA: PyAOP: DIPEA=1:1:1 mol/mol) was used for each coupling, which lasted for 1 h.

After completion of the synthesis, the peptide resin was washed with DEM and then thoroughly air dried. The dry resin was treated with 10 ml of digestion cocktail (trifluoroacetic acid: water: triisopropylsilane, 95: 2.5: 2.5 v/v) for 2 h at room temperature. The resin was filtered, washed twice with pure trifluoroacetic acid (TFA), and the combined filtrates were treated with cold diethyl ether (-20°C) to precipitate the crude peptide. The peptide/ester suspension was then centrifuged at 3500 rpm to form a solid, the supernatant was decanted, and the solid was triturated with ether two (2) additional times and dried under vacuum. The crude peptide was dissolved in 20% acetonitrile/20% acetic acid/60% water and purified by RP-HPLC on a Luna 5 μm phenylhexyl preparative column (21x250 mm, Phenomenex) with linear gradients of 100% acetonitrile and a 0.1% buffer system. TFA/water (30-50% acetonitrile for 60 min). Peptide purity was assessed by analytical RP-HPLC, and the pooling criterion was >95%. The purity of the main pool in Example 1 was found to be 98.8%. Subsequent lyophilization of the final bulk product pool resulted in a lyophilized TFA salt of the peptide. The molecular weight was determined by LC-MS (observed: M+4H+/4 =1226.8; calculated: M+4H+/4=1226.9).

Example 2.

Example 2 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHn)-(YGIu)-CO(CH2)i8-CO2H)AQHAFIEYLLA-Aib-GPSSGAPPPS- NH2 (SEQ ID NO: 6).

Below is an image of the structure of Example 2 using the standard single letter amino acid codes except for the residues Aib2, aMeL13, K17 and Aib29, where the structures of these amino acid residues have been expanded.

O

To synthesize the peptide scaffold for fatty acid-linker moiety conjugation, verify purity, and confirm the molecular weight of the compound of Example 2, processes similar to those described above for Example 1 were used.

Example 3.

Example 3 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMMHO-3TOKCM)-3TOKCM]-aL(eTMn)2-(YGIu)-CO(CH ₂ )i8-CO ₂ H)AQ- aMeK-AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 8).

Below is an image of the structure of the compound of Example 3 using the standard single-letter amino acid codes with the exception of residues Aib2, aMeL13, K17 and aMeK20, where the structures of these amino acid residues have been expanded.

- 8 043950

To synthesize the peptide scaffold for fatty acid-linker moiety conjugation to verify purity and to confirm the molecular weight of the compound of Example 3, processes similar to those described above for Example 1 were used.

Example 4.

Example 4 is a compound represented by the following description:

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aneTHn)-(YGIu)-CO(CH ₂ )i8-CO ₂ H)AQ-Aib-AFIEYLLA-Aib-GPSSGAPPPS -NH2 (SEQ ID NO:9).

Below is an image of the structure of the compound of Example 4 using the standard single letter amino acid codes except for residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 4, processes similar to those described above for Example 1 were used.

Example 5.

Example 5 is a compound represented by the following description:

Y-A|b-OSTPT8OU81-aMebOKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yClu)-CO(CH2)i8-CO2H)AAQAFIEYLLE-Aib-GPSSGAPPPS-NH2 (SEQ ID NO: 10).

Below is an image of the structure of the compound of Example 5 using the standard single-letter amino acid codes with the exception of residues Aib2, aMeL13, K17 and Aib29, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 5, processes similar to those described above for Example 1 were used.

Example 6.

Example 6 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMMHO-3TOKCM)-3TOKCM]-aL(eTMn)2-(YGIu)-CO(CH2)i8-CQ ₂ H)AAQAFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 11).

Below is an image of the structure of the compound of Example 6 using the standard single letter amino acid codes with the exception of residues Aib2, aMeL13 and K17, where the structures of these amino acid residues have been expanded.

- 9 043950

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 6, processes similar to those described above for Example 1 were used.

Example 7.

Example 7 is a compound represented by the following description:

Y-A|b-OSTPT8OU81-aMeb-LOCK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yClu)-CO(CH2)i8-CO2H)AQHAFIEYLLEGGPSSGAPPPS-NH2(SEQ ID NO: 12).

Below is an image of the structure of the compound of Example 7 using the standard single-letter amino acid codes with the exception of residues Aib2, aMeL13 and K17, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 7, processes similar to those described above for Example 1 were used.

Example 8.

Example 8 is a compound represented by the following description:

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMMHO-3TOKcn)-3TOKCM]-aneTMn)2-(YGIu)-CO(CH ₂ )i8-CO2H)AQ-aMeK-AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 13).

Below is an image of the structure of the compound of Example 8 using the standard single letter amino acid codes Aib2, K17 and aMeK20, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 8, processes similar to those described above for Example 1 were used.

Example 9.

Example 9 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((YGIu) ₂ -CO-(CH ₂ )i8CO2H)AQHAFIEYLLEGGPSSGAPPPS-NH ₂ (SEQ ID NO: 14).

Below is an image of the structure of the compound of Example 9 using the standard single letter amino acid codes, with the exception of residues Aib2, aMeL13 and K17, where the structures of these amino acid residues have been expanded.

- 10 043950

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 9, processes similar to those described above for Example 1 were used.

Example 10.

Example 10 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMMHO-3TOKCM)-3TOKCM]-aneTHn)-(YGIu)-CO(CH2)i8-CO2H)AQHAFIEYLLEGGPSSGAPPPS-NH ₂ (SEQ ID NO : 15).

Below is an image of the structure of the compound of Example 10 using the standard single-letter amino acid codes with the exception of residues Aib2, aMeL13 and K17, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 10, processes similar to those described above for Example 1 were used.

Example 11.

Example 11 is a compound represented by the following description:

Y-A|b-OSTPT8OU81-aMeb-LOCK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yClu)-CO(CH2)i8-CO2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS -NH2 (SEQ ID NO: 16).

Below is an image of the structure of Example 11 using the standard single letter amino acid codes except for residues Aib2, aMeL13, K17 and Aib20, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 11, processes similar to those described above for Example 1 were used.

Example 12.

Example 12 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMMHO-3TOKCM)-3TOKCM]-ai_(eTMn)-(YGIu)-CO(CH ₂ )i8-CO2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS -NH ₂ (SEQ ID NO: 17).

Below is an image of the structure of Example 12 using the standard single letter amino acid codes except for the residues Aib2, aMeL13, K17 and Aib20, where the structures of these amino acid residues have been expanded.

- 11 043950

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 12, processes similar to those described above for Example 1 were used.

Example 13.

Example 13 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMMHO-3TOKCM)-3TOKcn]-aL(eTMH)-(YGIu)-CO(CH2)i8-CO ₂ H)AQ-Aib- AFIEYLLE-Aib-GPSSGAPPPS-NH2 (SEQ ID NO: 18).

Below is an image of the structure of Example 13 using the standard single letter amino acid codes except for the residues Aib2, aMeL13, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 13, processes similar to those described above for Example 1 were used.

Example 14.

Example 14 is a compound represented by the following description:

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHH)-(YGIu)-CO(CH2)i8-CO2H)AQ-Aib-AFIEYLLE- Aib-GPSS-Aib-APPPS-NH2 (SEQ ID NO: 19).

Below is an image of the structure of Example 14 using the standard single letter amino acid codes except for the residues Aib2, aMeL13, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 14, processes similar to those described above for Example 1 were used.

Example 15.

Example 15 is a compound represented by the following description:

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKCM)-3TOKCM]-aL(e™H)2-(YGIu)-CO(CH ₂ )i8-CO ₂ H)AQ-Aib- AFIEYLLA-Aib-GPSSGAPPPS-NH2 (SEQ ID NO: 20).

Below is an image of the structure of the compound of Example 15 using the standard single letter amino acid codes except for residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded.

- 12 043950

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 15, processes similar to those described above for Example 1 were used.

Example 16.

Example 16 is a compound represented by the following description:

Y-A|b-OSTPT8OU81bOKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yClu)-CO(CH2)i8-CO2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 21).

Below is an image of the structure of Example 16 using the standard single letter amino acid codes except for residues Aib2, K17 and Aib20, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 16, processes similar to those described above for Example 1 were used.

Example 17.

Example 17 is a compound represented by the following description:

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTMn)2-(YGIu)-CO(CH ₂ )i8-CO2H)AQ-Aib-AFIEYLLE-Aib- GPSSGAPPPS-NH2 (SEQ ID NO: 22).

Below is an image of the structure of the compound of Example 17 using the standard single letter amino acid codes except for residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 17, processes similar to those described above for Example 1 were used.

Example 18.

Example 18 is a compound represented by the following description:

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKCM)-3TOKCM]-ai_(eTHn)-(YGIu)-CO(CH ₂ )i8-CO ₂ H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 23).

Below is a picture of the structure of Example 18 using the standard single letter amino acid codes except for residues Aib2, K17 and Aib20, where the structures of these amino acid residues have been expanded.

- 13 043950

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 18, processes similar to those described above for Example 1 were used.

Example 19.

Example 19 is a compound represented by the following description:

Y-Aib-QGTFTSDYSILLDKK((242-(2-aMHHO-3TOKCM)-3TOKCM]-apeTi/in)-(YGIu)2-CO(CH2)i8-CO ₂ H)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS- NH2 (SEQ ID NO: 24).

Below is an image of the structure of the compound of Example 19 using the standard single letter amino acid codes except for residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 19, processes similar to those described above for Example 1 were used.

Example 20.

Example 20 is a compound represented by the following description:

Y-A|b-OSTPT8OU81bOKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(yClu)-CO(CH2)i8-CO2H)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS- NH2 (SEQ ID NO: 25).

Below is an image of the structure of the compound of Example 20 using the standard single letter amino acid codes except for residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded.

To synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to check the purity, and to confirm the molecular weight of the compound of Example 20, processes similar to those described above for Example 1 were used.

Function in vitro.

Binding affinity.

A competitive radioligand binding assay was performed to determine the equilibrium dissociation constant for representative compounds and comparator molecules. This analysis used scintillation proximity assay (SPA) techniques and membranes obtained from transfected HEK293 cells overexpressing human GIP receptor (GIPR), GLP-1 receptor (GLP-1R) or human glucagon receptor (GcgR).

Assays were performed in the presence of bacitracin as a nonspecific blocking agent to prevent binding of acylated moieties of test analogues to protein components used in standard assay buffers (eg, albumin).

- 14 043950

Competition curves are plotted as percent specific inhibition (y-axis) versus log compound concentration (x-axis) and analyzed using a four-parameter nonlinear variable slope regression fit (ABase or Genedata). Ki values were calculated according to the equation

Ki=IC ₅ o/(l+(D/K _d )), where 1C ₅ o represents the concentration of the compound leading to 50% inhibition of binding;

D is the concentration of the radioligand used in the assay; And

_Kd is the equilibrium dissociation constant for the receptor and radioligand determined from saturation binding assay (shown in Table 1 below).

Table 1

Equilibrium dissociation constants (CD determined from saturation binding analysis

Kd, nM GLP-1R GcgR GIPR 1.2 3.9 0.14

The Ki values of typical analogues and comparator molecules are given in Table. 2.

table 2

In vitro binding affinity (Ki) of compounds by examples and comparators for human GIPR, GLP-1R and GcgR

K, nM (SEM, n) Molecule GcgR GIPR GLP-1 R hGcg 3.1 (0.5, 4) hGIP 0.12(0.02, 4) hGLP-1 1.2 (0.2, 4) Example 1 8.41 (2.71.5) 0.0469 (0.00558, 4/5) 2.64 (0.501.5) Example 2 3.71 (1.49, 2) 0.0665 (0.0377, 2) 4.50 (0.735, 2) Example 3 12.0(2.74, 4) 0.0446 (0.00838, 4) 6.06 (0.849, 4) Example 4 2.55 (0.411.4) 0.0374 (0.0137, 4) 3.54 (0.503, 6) Example 5 0.422 (0.0887, 3) 0.179 (0.0466, 3) 36.7 (8.99, 3) Example 6 0.835 (0.369, 3) 0.249 (0.0369, 3) 36.7 (10.5, 3) Example 7 16.3(2.37, 5) 0.110 (0.0206, 5) 21.4(3.53, 5) Example 8 30.4 (38.9, 3) 0.0958 (0.0295, 3) 29.4 (29.8, 3) Example 9 8.27 (0.855, 4) 0.126 (0.0274, 4) 11.5(1.85, 4) Example 10 7.37(1.38, 4) 0.118(0.0363, 4) 12.0(2.75, 4) Example 11 10.8(1.11.4) 0.0890 (0.0369, 3/4) 9.97(1.53, 4) Example 12 5.60 (0.796, 4) 0.0570 (0.00322, 4) 7.17(1.68, 4) Example 13 1.91 (0.128, 3) 0.0452 (0.00297, 3) 6.43(1.89, 5) Example 14 2.64 (0.231.4) 0.0350 (0.00326, 4) 6.27(1.12, 6) Example 15 4.56 (2.68, 2) 0.0972 (η ⁼ 1/2) 5.80(1.80, 3) Example 16 11.5(1.97, 5) 0.106 (0.0182, 5) 16.1 (2.30, 6) Example 17 5.81 (0.875, 3) 0.0895 (0.0290, 3) 10.2 (1.74, 3) Example 18 5.71 (0.588, 4) 0.0835 (0.0128, 4) 8.06 (2.04, 4) Example 19 5.20 (0.572, 3) 0.0789 (0.0261.3) 12.0(2.07, 3) Example 20 3.76 (0.397, 3) 0.109 (0.0247, 3) 9.23 (2.14, 3)

Note.

The qualifier (>) indicates that the data did not achieve 50% inhibition relative to maximum binding, resulting in Ki calculated using the highest concentration tested in the assay, n=1/x meaning only one value from the total number of replicates ( x) is used to express the average value. SEM was calculated only if there were n=2 or more unqualified results.

As can be seen from table. 2, exemplary analogs have receptor binding affinities for each of GIP, GLP-1, and glucagon.

- 15043950

Functional activity.

Functional activity was determined in clonal HEK-293 cell lines expressing GIP-R-GLP-1R- and GcgR. A cell line overexpressing each receptor was treated with peptide (20-point CRC, 2.75x Labcyte Echo direct dilution) in DMEM (Gibco Cat #31053) supplemented with 1X GlutaMAX™ (Gibco Cat #35050), 0.25% FBS (fetal bovine serum, Gibco Cat #26400), 0.05% BSA Fraction V (bovine serum albumin, Gibco Cat #15260), 250 µM IBMX and 20 mM HEPES (Gibco Cat #15630) in a 20 µL assay volume.

After 60 min incubation at room temperature, the resulting increase in intracellular cAMP was quantified using the CisBio cAMP Dynamic 2 HTRF Assay Kit (62AM4PEJ). Briefly, cellular cAMP levels were detected by adding cAMP-d2 conjugate to cell lysis buffer followed by the addition of anti-cAMP-Eu ³⁺ -cryptate antibody, also in cell lysis buffer. The resulting competition assay was incubated for at least 60 min at room temperature and then detected using a PerkinElmer Envision® instrument with excitation at 320 nm and emission at 665 and 620 nm. Envision units (emission at 665/620 nm x 10,000) were inversely proportional to the amount of cAMP present and were converted to nM cAMP per well using the cAMP standard curve.

The amount of cAMP produced (nM) in each well was converted to a percentage of the maximum response observed with either human GLP-1(7-36)NH2, human _Gcg , or human GIP(1-42)NH2. The relative EC ₅₀ and top percentage (E _max ) were obtained by nonlinear regression analysis using the percentage maximum response versus added peptide concentration fitted to a four-parameter logistic equation.

Data for typical analogues and hGIP(1-42)NH2, hGLP-1(7-36)NH ₂ and hGcg are given in table. 3 below.

Table 3

Functional efficiency of cAMP (EC ₅₀ ) for typical analogues and comparators in the presence of FBS and BSA

cAMP EC ₅ o, nM (SEM, p) GcgR GIPR GLP-1 R hGcg 0.0125 (0.000280014, 112) gGIP amide 0.133 (0.0082, 60) hGLP-1 amide 0.0591 (0.00091, 113) Example 1 2.54 (0.199, n ⁼ 6) 0.914(0.0915, 6) 7.49(1.02, 6) Example 2 2.66 (0.175.5) 2.19 (0.392.5) 12.8 (2.50.5) Example 3 8.03 (0.997, 6) 1.91 (0.205, 6) 12.9 (1.58.6) Example 4 2.49 (0.371, 7) 1.55 (0.245, 7) 10.4(1.80, 7) Example 5 1.47 (0.171.6) 4.86 (0.682, 6) 22.3 (3.78, 6) Example 6 1.99 (0.201.6) 7.41 (0.667, 6) 21.2 (2.02, 6) Example 7 14.2(2.24, 6) 4.38 (0.750, 6) 15.5(2.40, 6) Example 8 6.24 (0.673, 6) 2.23 (0.164, 6) 9.39 (0.959, 6) Example 9 6.32 (0.290, 4) 4.17 (0.695, 4) 9.76 (1.98, 4) Example 10 8.42 (1.17, 4) 4.30 (0.987, 4) 19.3 (1.52, 4) Example 11 11.9(0.727, 6) 1.50 (0.124, 6) 10.3 (0.808, 6) Example 12 6.61 (0.512, 6) 2.24 (0.303, 6) 12.4(1.41.6) Example 13 3.61 (0.197, 8) 1.76 (0.126, 8) 12.2 (1.00, 8) Example 14 4.05 (0.255, 7) 1.55 (0.165, 7) 14.4(1.71, 7) Example 15 5.92 (1.10, 4) 1.47 (0.264, 4) 10.7 (1.85.4) Example 16 13.2(1.93, 6) 4.37 (0.589, 6) 19.0(2.39, 6) Example 17 8.05(1.26, 3) 2.38 (0.212, 3) 18.4(3.75, 3) Example 18 5.71 (0.256, 4) 5.89 (1.05, 4) 16.1 (2.61.4) Example 19 8.45 (0.828, 3) 3.13(0.179, 3) 24.4 (2.85, 3) Example 20 3.97 (0.284, 3) 3.70(1.02, 3) 20.6(5.10, 3)

Note.

Determination of EC ₅₀ of human GLP-1(7-36) _NH2 with human GLP-1R, human Gcg with human GcgR and human GIP(1-42)NH2 with human GIP-R:

- 16 043950 peptide concentration ranges were from 448 to 99.5 nM.

Determination of EC ₅₀ compounds as exemplified in human GLP-1R, human GcgR and human GIP-R: peptide concentration ranges were from 51.5 fM to 11.4 μM.

As can be seen from table. 3, in the presence of FBS and BSA, the generic analogues have agonist activity, as determined by the GIP-R, GLP-1R and GcgR cAMP assays, which are lower than those of the native ligands.

An additional set of cAMP assays was performed in HEK293 cells expressing human GLP-1, GIP, and glucagon receptors. Time-resolved homogeneous fluorescence assays were used to determine the intrinsic activity of typical analogues and comparator molecules in the presence of casein (instead of serum albumin) as a nonspecific blocker that does not interact with the fatty acid moieties of the molecules analyzed.

Intracellular cAMP levels were determined by extrapolation using a standard curve. Dose-response curves for compounds were presented as percentage stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four-parameter nonlinear variable slope regression fit (Genedata Screener 13). EC ₅₀ is the concentration of a compound that produces a half-maximal simulation on the dose-response curve.

The data is shown in the table below. 4.

Table 4

Functional activation of hGLP-1R, hGIPR, hGcgR in the presence of 0.1% casein

cAMP EC50, nM (SEM, p) GcGR GIPR GLP-1 R hGcg 0.0119(0.00356, 163) hGIP amide 0.154 (0.037, 118) gGLP-1 amide 0.063 (0.022, 197) Example 1 0.114(0.0203, 5) 0.0523 (0.0112.5) 0.153 (0.0132, 12) Example 2 0.0553 (0.00975, 4) 0.0474 (0.00485, 4) 0.207 (0.0213, 6) Example 3 0.152 (0.0147, 7) 0.0376 (0.00284, 7) 0.107 (0.0108, 7) Example 4 0.0674 (0.00532, 15) 0.0648 (0.00507, 14) 0.180 (0.0144, 17) Example 5 0.0226 (0.00304, 10) 0.0757 (0.0127.5) 0.147 (0.0204, 7) Example 6 0.0282 (0.00409.7) 0.274 (0.0377, 7) 0.142 (0.0127, 10) Example 7 0.180 (0.0190, 6) 0.0798 (0.0111.6) 0.109 (0.0134.5) Example 8 0.120 (0.0210, 5) 0.114(0.0101.4) 0.117 (0.0151.7) Example 9 0.139 (0.0281.5) 0.0522 (0.00816, 4) 0.0931 (0.00852.8) Example 10 0.123 (0.00784, 15) 0.0928 (0.00721, 16) 0.143 (0.0103, 12) Example 11 0.205 (0.0175, 11) 0.0425 (0.00744, 12) 0.123 (0.0119, 13) Example 12 0.122 (0.00931, 15) 0.0529 (0.00394, 18) 0.162 (0.0100, 18) Example 13 0.0815(0.00835, 12) 0.0391 (0.00315, 14) 0.125 (0.00961, 13) Example 14 0.0876 (0.00687, 17/18) 0.0356 (0.00242, 20) 0.146 (0.0108, 17) Example 15 0.131 (0.0141, 10) 0.0689 (0.00730.9) 0.253 (0.0197, 9) Example 16 0.174 (0.00882, 22) 0.114(0.0100, 20) 0.157 (0.0105, 20) Example 17 0.135 (0.00643, 12) 0.0439 (0.00457, 11) 0.153 (0.0135, 10) Example 18 0.0861 (0.00631, 16) 0.123 (0.00954, 13) 0.141 (0.00862, 13) Example 19 0.0874 (0.0317, 2) 0.0455 (0.00516, 2) 0.143 (0.0187, 3) Example 20 0.0641 (0.00369, 12) 0.0572 (0.00527, 11) 0.149 (0.00937, 11)

As can be seen from table. 4, exemplary analogues stimulate cAMP from human GIP, GLP-1 and glucagon receptors in the presence of 0.1% casein.

In vivo studies.

Pharmacokinetics in male Sprague Dawley rats.

The pharmacokinetics of typical analogues were assessed after a single subcutaneous administration of 100 nM/kg to male Sprague Dawley rats. Blood samples were collected over 120 hours and individual plasma concentrations were used to calculate pharmacokinetic parameters. Plasma peptide concentrations (K ₃ EDTA) were determined using a qualified LC/MS method that measures the intact mass of the analogue. Each peptide and internal standard analogue was extracted from 100% indicated plasma using methanol with 0.1% formic acid. A Thermo Q-Exactive, high resolution instrument and PepMap Thermo Easy Spray were combined for LC/MS determination. Average pharmacokinetic parameters are given in table. 5.

Table 5

Average pharmacokinetic parameters of peptides after a single subcutaneous injection of 100 nmol/kg to male Sprague Dawley rats

Tl/2 (h) Tmax (h) Cmax/D (KG * nmol/l/nmol) AUCINF_D_Ha6n (hour * kg * nmol/l/nmol) CL/F (ml/h/kg) Example 1 11.7 8 3.5 95.7 10.4 Example 3 19.2 16 2.5 146 6.9 Example 4 19.9 16 2.9 140 7.2 Example 10 23.4 24 3.1 203.5 4.9 Example 11 24.3 20 3.7 215.1 4.7 Example 12 26.5 24 3.7 197.1 5.1 Example 13 21.7 20 3.8 205.7 4.9 Example 16 29.1 20 3.8 274.9 3.6 Example 18 34.8 24 5.0 284.3 3.6

Abbreviations.

T _1/2 = half-life;

T _max = time to reach maximum concentration;

C _max =maximum plasma concentration;

AUCINF_D_observed=AUCinf divided by dose;

CL/F = clearance/bioavailability.

Note.

Data are means where n=3/group.

As can be seen from table. 5, exemplary analogues demonstrate an enhanced pharmacokinetic profile.

In vivo effects on insulin secretion in male Wistar rats.

To evaluate the insulinotropic activity of the typical analogues, an intravenous glucose tolerance test (ivGTT) was performed in rats (male Wistar line). Semaglutide GLP-1 RA was used as a positive control. Rats surgically implanted with jugular vein and carotid artery cannulas (Envigo, Indianapolis, IN; 280–320 g) were housed, one per cage, in polycarbonate cages with a filter at the top. Rats were maintained on a 12-h light-dark cycle at 21°C and administered 2014 Teklad Global diet (Envigo, Indianapolis) and deionized water ad libitum. Rats were randomized by body weight and injected subcutaneously (s.c.) with 1.5 ml/kg of standard analogues 16 hours before glucose administration and then left without food. Initial concentrations of 211 nM typical analogs were diluted in Tris buffer, pH 8.0 to 6.667 nmol/ml, 2.0, 0.667, 0.2 nmol/ml; The doses tested 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 insulinotropic effects were measured both in the assay on its own (vehicle and doses of 1, 3, 10 and 30 nmol/kg) and in assays with each reference analog (dose of 10 nmol/kg).

A blood sample was collected at time 0 into EDTA tubes, followed by glucose (0.5, 5 ml/kg). Blood samples were collected for analysis of glucose and insulin levels at 2, 4, 6, 10, 20, and 30 min after intravenous glucose administration. Plasma insulin was determined using an electrochemiluminescence assay (Meso Scale, Gaithersburg, MD). Insulin area under the curve (AUC) was examined and compared with vehicle control, with n=6 animals per group.

Statistical analysis was performed using JMP using one-way ANOVA followed by comparison using Dunnett's test with vehicle control. The results are presented in table. 6.

- 18 043950

Table 6

Effect of vehicle, semaglutide, and generic analogues on insulin secretion during an intravenous glucose tolerance test in anesthetized Wistar rats

Insulin aiSzomin after an IV bolus dose of glucose Dose (nmol/kg)

Semaglutide (10 nmol/kg)

Note 1

Note 3

Note 4

Note eleven

Note 12

Note 13

Note 16

Note 18

11.1 ±1.3

12.6 ± 2.1

13.7 ±2.3

11.3 ± 2.1

9.3 ± 1.8

8.9 ± 1.2

14.4 ± 2.1

12.3 ±2.0

60.1 ± 10.3

34.4 ±2.9

27.4 ±3.0

43.0 ±6.8

32.4 +/- 2.4

25.8 ±2.7

18.9 ±3.5

27.6 ±4.6

53.8 ±5.1

71.5 ± 6.1

51.86 +/-6.7

47.5 ±4.0

52.9 ±3.0

59.2 ±6.3

44.8 ±2.3

47.3 ±8.0

50.3 ±4.2

44.8 ±8.3

51.6 ±5.4

70.8 ±8.4

62.0 ±4.6

53.6 ±6.0

70.6 ±4.8

50.1 ±4.2

57.3 ± 10.0

45.6 +/- 6.6

44.7 +/- 3.7

55.1 +/-9.0

40.2 +/- 2.5

38.4 +/- 6.0

56.3 +/- 7.7

48.4 +/- 6.6

Note.

Results are expressed as mean ± standard error of the mean (SEM) for 6 rats per group. Statistical test was one-way ANOVA followed by Dunnett's test * p < 0.05 vs. vehicle; +p<0.05 compared with semaglutide.

As can be seen from table. 6, typical analogues demonstrate a dose-dependent increase in insulin secretion.

Studies in C57BL/6 mice with diet-induced obesity.

The exemplary incretin analogs described herein are proposed as a treatment not only for diabetes, but also for metabolic syndrome, which is a collection of comorbidities (dyslipidemia, obesity, hepatic steatosis, etc.) associated with insulin resistance and diabetes. To investigate the effects of generic analogues on parameters such as weight loss, metabolism, body composition and hepatic steatosis, they were administered to C57BL/6 mice with diet-induced obesity (DIO). These animals, although not diabetic, had insulin resistance, dyslipidemia and hepatic steatosis, all signs of metabolic syndrome, after being kept on a high-fat diet for 18 weeks.

Specifically, the following studies used 24- to 25-week-old male C57BL/6 DIO mice maintained on a high-calorie diet. Mice were individually housed in a room at (24°C) with a 12-hour light-dark cycle (lights at 10:00 p.m.) and free access to food (TD95217) (Teklad) and water. After a minimum of 2 weeks of indoor acclimation, mice were randomized according to their body weight so that each experimental group of animals had a similar body weight. Body weight varied from 40 to 51 g.

All groups consisted of 5-6 mice. Vehicle, generic analogues dissolved in vehicle (40 mM Tris-HCl at pH 8.0), and semaglutide dissolved in vehicle were administered by subcutaneous (SC) injection (10 ml/kg) to ad-fed DIO mice libitum, 30-90 minutes before the start of the dark cycle every 3 days for 15 days. SC injections were given on days 1, 4, 7, 10, and 13. Body weight and food intake were measured daily throughout the study.

Absolute changes in body weight are calculated by subtracting the body weight of the same animal before the first injection of vehicle, analogue, or semaglutide. On days 0 and 15, total fat mass was measured by nuclear magnetic resonance (NMR) using the Echo Medical System (Houston, TX). On day 15, animals were sacrificed before the dark photoperiod, and the livers were removed and frozen. Liver triglyceride levels in liver homogenates collected at sacrifice and plasma cholesterol levels were measured on a Hitachi Modular P clinical analyzer.

Data are presented as mean ± SEM for 5-6 animals per group in table. 7 and 8 below. Statistical analysis was performed using repeated measures ANOVA followed by Dunnett's test for multiple comparisons. Significant differences were identified at p<0.05.

- 19 043950

Table 7

Change . body weight fifteen days after the introduction of standard analogues

Processing group (3 nmol/kg) (10 nmol/kg) (30 nmol/kg) Δ from carrier (g) % change Δ from carrier (O % change Δ from carrier (g) % change Note 1 -5.42 ± 0.54 -10.04 ± 1.14 -9.26 ± 0.36 -20.20 ± 1.51 -21.36 ± 1.08 -44.82 ± 1.54 Note 3 -10.14 ± 0.72 -21.18 ± 1.98 Note 4 -11.58 ± 0.85 -23.64 ± 1.70 -19.98 ± 1.63 -43.88 ± 3.71 Note 5 -12.30 ± 2.20 -25.40 ± 4.56 Note 6 -11.72 ± 1.78 -24.02 ± 3.31 Note 7 -5.26 ± 0.49 -12.22 ± 1.19 -8.38 ± 0.50 -19.65 ± 1.27 -16.26 ± 2.23 Note 10 -5.4 ± 0.49 -12.70 ± 1.17 -10.98 ± 0.76 -24.95 ± 1.99 -17.08 ± 1.43 -39.51 ± 3.84 Note eleven -7.22 ± 0.38 -16.58 ± 0.88 -11.82 ± 1.72 -26.83 ± 4.04 -16.48 ± 1.98 -37.35 ± 3.91 Note 12 -8.40 ± 0.77 -19.33 ± 1.66 -10.34 ± 0.69 -23.37 ± 1.40 -17.44 ± 1.37 -46.60 ± 3.78 Note 13 -4.92 ± 0.86 -10.14 ± 1.87 -11.02 ± 0.77 -23.80 ± 1.25 -21.12 ± 2.09 Note 14 -6.12 ± 0.80 -13.38 ± 1.80 -14.76 ± 1.06 -32.32 ± 2.49 -20.04 ± 2.40 -45.26 ± 4.79 Note 16 -7.44 ± 0.74 -16.78 ± 1.88 -12.24 ± 1.66 -28.08 ± 4.23 -16.70 ± 1.67 -37.42 ± 3.58 Note 18 -13.16 ± 0.82 -28.48 ± 2.05 -19.54 ± 2.37 -43.02 ± 4.59 Note 20 -4.18 ± 0.50 -8.98 ± 1.26 -10.76 ± 1.57 -24.42 ± 3.74 -23.00 ± 0.59 -52.14 ± 1.74 Sema -2.62 ± 0.49 -10.31 ± 1.25 -15.49 ± 2.44

Note.

Δ from vehicle refers to the difference between body weight at day 15 between the study group and the vehicle group.

% change refers to the percentage reduction in body weight between days 1 and 15 in the study groups.

Sema means semaglutide.

The percentage reduction in body weight in vehicle-treated animals was recorded and was less than 1% in each study.

Δ from vehicle and % change were statistically significantly different (p<0.05) from control for compounds of all examples at all doses tested.

As can be seen from the table above. 7, typical analogues demonstrate a dose-dependent reduction in body weight.

Data on metabolic parameters when using a dose of 3 nmol/kg are given in the table below. 8.

- 20 043950

Table 8

Effect of typical analogues at a dose of 3 nmol/kg on blood glucose, insulin, cholesterol, alanine aminotransferase (ALT) and liver triglyceride levels after fifteen days of treatment

Metabolic parameters (mean ± SEM) Treatment Glucose (mg/dl) Plasma insulin (pg/ml) Cholesterol (mg/dl) ALT (IU/l) Liver triglycerides (mg/g tissue) Carrier 149.7 ± 3.09 8549±1265 234.6 ± 3.42 141.8± 11.5 290.4 ± 13.3 Semaglutide 137.8 ± 7.34 4196 ± 1014* 218.0 ±9.23 99.8 ± 14.84 226.4 ± 23.8 Example 1 107.4 ± 4.38* 3439 ± 936.4* 191.2 ±7.55* 118.0±20.73 150.6 ±37.0* Example 7 109.3 ± 5.78* 2656 ± 949 * 169.2 ±11.79 * 51.0 ± 7.78* 97.9 ± 18.2* Example 10 109.6 ± 4.81* 571.4 ± 158.4* 181.0±6.20* 77.2 ± 9.35* 126.8 ±17.0* Example 11 114.7 ± 3,051* 1569± 318.3* 178.2 ±7.95* 56.2 ± 4.51* 93.8 ±21.8* Example 12 114.9 ± 2.12* 1691 ± 231.3* 169.0 ±8.15* 58.6 ± 6.03* 98.2 ± 13.2* Example 13 119.7 ± 4.19* 2903 ± 737.4 * 192.2 ± 11.44 * 54.4 ± 6.82* 94.5 ±22.0* Example 14 111.5 ± 1.77* 1971 ± 499.8* 164.8 ±5.85* 31.4 ± 2.79* 63.1 ±7.3* Example 16 110.1 ± 3.61* 3227 ± 1070* 173.8 ±9.32 * 20.6 ± 4.43* 40.2 ± 13.2* Example 18 102.9 ± 5.37* 1958± 460.5 * 190.0 ± 13.71 * 66.0 ± 10.21* 109.5 ±22.9*

Note.

* p<0.05 compared with vehicle group; one-way ANOVA analysis, Dunnett's test.

In addition to significant weight loss, as can be seen in Table. 8, typical analogues reduce blood glucose, plasma insulin (as a sign of increased insulin sensitivity), and plasma cholesterol, and also improve liver health, as evidenced by a decrease in plasma ALT and liver triglyceride levels.

SEQ ID NO: 1 - human glucagon

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

SEQ ID NO: 2 - Human GLP-1 (7-36) amide

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

SEQ ID NO: 3 - Human OXM (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA)

SEQ ID NO: 4 - Human GIP

YAE GTFIS DYSI AM D KI H Q Q D FV N WLLAQ KG KKN D WKH NITQ

SEQ ID NO: 5 - incretin analog

YX ₂ Q GTFTS DYS IXi з LD KXi 7 AXi ₉ X ₂ oAF IE YL LX ₂₈ X ₂₉ GPS SX34 AR Ρ PS where X ₂ represents Aib;

X ₁₃ represents L or aMeL;

X ₁₇ represents any amino acid with a functional group available for conjugation, wherein said functional group is conjugated to a C16-C22 fatty acid;

- 21 043950

X ₁ 9 represents Q or A;

X20 is Aib, aMeK, Q or H;

X ₂₈ represents E or A;

X ₂₉ represents G or Aib; And

X ₃₄ represents G or Aib.

SEQ ID NO: 6 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL|eTHn)-(YGIu)-CO(CH2)i8-CO2H)AQHAFIEYLLA-Aib-GPSSGAPPPS- NH2

SEQ ID NO: 7 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL|eTHP)2-(YGIu)-CO· (CH2)i8-CO ₂ H)AQ- aMeK-AFIEYLLA-Aib-GPSSGAPPPS-NH2

SEQ ID NO: 8 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL|eTHP)2-(YGIu)-CO· (CH2)i8-CO ₂ H)AQ- aMeK-AFIEYLLEGGPSSGAPPPS-NH2

SEQ ID NO: 9 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL[eTHP)-(YGIu)-CO(CH2)i8-CO ₂ H)AQ-Aib-AFIEYLLA-Aib- GPSSGAPPPS-NH2

SEQ ID NO: 10 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKCH)-3TOKcn]-aneTHP)2-(YGIu)-CO (CH ₂ )i8-CO ₂ H)AAQAFIEYLLE-Aib- GPSSGAPPPS-NH2

SEQ ID NO: 11 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKCH)-3TOKcn]-aneTHP)2-(YGIu)-CO (CH2)i8-CO ₂ H)AAQAFIEYLLEGGPSSGAPPPS-NH2

SEQ ID NO: 12 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKCH)-3TOKcn]-aneTHP)2-(YGIu)-CO (CH2)i8-CO ₂ H)AQHAFIEYLLEGGPSSGAPPPS-NH2

SEQ ID NO: 13 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL[eTHP)2-(YGIu)-CO(CH ₂ )i8-CO ₂ H)AQ-aMeK-AFIEYLLEGGPSSGAPPPS- NH2

SEQ ID NO: 14 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((YGIu)2-CO-(CH ₂ )i8CO ₂ H)AQHAFIEYLLEGGPSSGAPPPS-NH ₂

- 22 043950

SEQ ID NO: 15 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHn)-(YGIu)-CO(CH2)i8-CO2H)AQHAFIEYLLEGGPSSGAPPPS-NH2

SEQ ID NO: 16 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHo)2-(YGIu)-CO (CH ₂ )i8-CO ₂ H)AQ -Aib-AFIEYLLEGGPSSGAPPPS-NH2

SEQ ID NO: 17 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHo)-(YGIu)-CO(CH2)i8-CO ₂ H)AQ-Aib- AFIEYLLEGGPSSGAPPPS-NH2

SEQ ID NO: 18 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHH0-3T0Kcn)-3T0Kcn]-aL(eTHP)-(YGIu)-C0(CH ₂ )i8-CO2H)AQ-Aib-AFIEYLLE -Aib-GPSSGAPPPS-NH ₂

SEQ ID NO: 19 - incretin analog

Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHo)-(YGIu)-CO(CH2)i8-CO ₂ H)AQ-Aib- AFIEYLLE-Aib-GPSS-Aib-APPPS-NH2

SEQ ID NO: 20 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(e™p)2-(YGIu)-CO(CH2)i8-CO ₂ H)AQ-Aib-AFIEYLLA -Aib-GPSSGAPPPS-NH2

SEQ ID NO: 21 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(e™o)2-(YGIu)-CO(CH2)i8-CO ₂ H)AQ-Aib-AFIEYLLEGGPSSGAPPPS -NH2

SEQ ID NO: 22 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKCH)-3TOKCH]-aneTHo)2-(YGIu)-00(CH2)i8-CO ₂ H)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS -NH2

SEQ ID NO: 23 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(e™o)-(YGIu)-CO(CH2)i8-CO ₂ H)AQ-Aib-AFIEYLLEGGPSSGAPPPS- NH2

SEQ ID NO: 24 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHo)-(YGIu)2-CO(CH2)i8-CO ₂ H)AQ-Aib-AFIEYLLE-Aib -GPSSGAPPPS-NH2

SEQ ID NO: 25 - incretin analog

Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-aMHHO-3TOKcn)-3TOKcn]-aL(eTHo)-(YGIu)-CO(CH ₂ )i8-CO ₂ H)AQ-Aib-AFIEYLLE-Aib -GPSSGAPPPS-NH2

SEQ ID NO: 26 - Artificial Sequence

GPSSGAPPPS

SEQ ID NO: 27 - Artificial Sequence

GPSS-Aib-APPPS

List of sequences < 110> Eli Lilly and Company < 120> INCRETIN ANALOGUES AND THEIR USE

- 23 043950 <130> X21606 <150> US 62/608,613 <151> 2017-12-21 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 29 <212> PROTEIN <213> Homo sapiens < 400>1

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp 1 5 1015

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 2025 <210>2 <211> 30 <212> PROTEIN <213> Homo sapiens <400> 2

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu 1 5 10 15

Gln Ala Ala Lys Glu 20

Phe Ile Ala Trp Leu 25

Val Lys Gly Arg 30 <210>3 <211>37 <212> PROTEIN <213> Homo sapiens <400>3

Ser

Gly

His Ser Gln Gly Thr Phe Thr 1 5

Ser Asp Tyr Ser Lys Tyr Leu Asp

15

Ser

Arg Arg Ala Gln Asp Phe Val 20

Gln Trp Leu Met Asn Thr Lys Arg

thirty

Asn

Arg Asn Asn Ile Ala 35 <210> 4 <211> 42 <212> PROTEIN <213> Homo sapiens

- 24 043950 <400> 4

Tyr Ala 1

Glu Gly Thr Phe Ile Ser Asp 5

Tyr Ser Ile Ala Met Asp Lys

15

Ile His

Gln Gln Asp Phe Val Asn Trp

25

Leu Leu Ala Gln Lys Gly Lys 30

Lys Asn Asp Trp Lys His Asn 35

Ile Thr Gln 40 <210> 5 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa in position 2 is Aib <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is Leu or alpha-methyl-Leu <220>

<221> OTHER_FEATURE <222> (17) .. (17) <223> Xaa at position 17 is any amino acid with a functional group available for conjugation, where the functional group is conjugated to a C16-C22 fatty acid <220>

< 221> OTHER_CHARACTER < 222> (19)..(19) < 223> Haa at position 19 is Gln or Ala <220>

< 221> OTHER_FEATURE < 222> (20)..(20) < 223> Xaa at position 20 is Aib, alpha-methyl-Lys, Gln or His <220>

< 221> OTHER_FEATURE < 222> (28).. (28) < 223> Haa at position 28 is Glu or Ala <220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Haa at position 29 is Gly or Aib

- 25 043950 <220>

< 221> OTHER_CHARACTER < 222> (34).. (34) < 223> Haa at position 34 is Gly or Aib < 400> 5

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser 1 5 10

Ile Xaa Leu Asp Lys

Xaa Ala Xaa

Xaa Ala Phe 20

Ile Glu Tyr Leu Leu

Xaa Xaa Gly Pro Ser

Ser Xaa Ala Pro Pro Pro Ser <210> 6 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

< 221> OTHER_CHARACTER < 222> (17) .. (17) < 223> Lys at position 17 is modified (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(gamma-Glu)-CO -(CH2)18-HCO 2H <220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400> 6

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys

5 10 15

Lys Ala Gln His Ala Phe Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser

- 26 043950

Ser Gly Ala Pro Pro Pro Ser 35 <210> 7 <211> 39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Xaa at position 2 is alpha-amino-isobutyric acid < 220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu < 220>

< 221> MOD_RES < 222> (17)..(17) < 223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain with (2-[2-(2-amino-ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H) <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Haa at position 20 is alpha-methyl-Lys < 220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid < 220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400> 7

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys

5 10 15

Lys Ala Gln Xaa Ala Phe Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser 20 25 30

Ser Gly Ala Pro Pro Pro Ser 35

- 27 043950 <210> 8 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic construction <220>

<221> OTHER_CHARACTER <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H) <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Haa at position 20 is alpha-methyl-Lys <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400>8

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 1015

Lys Ala Gln Xaa Ala Phe Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>9 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

<221> OTHER_CHARACTERISTIC

- 28 043950 <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino-ethoxy)-ethoxy]- acetyl)-(gamma-Glu)-CO-(CH2)18-CO2

H<220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (29).. (29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400> 9

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Asp Lys

5 1015

Lys Ala Gln Xaa Ala Phe 20

Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35 < 210>10 < 211>39 < 212> PROTEIN < 213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

<221>MOD_RES

- 29 043950 <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain with (2-[2-(2-amino)ethoxy)-ethoxy]-acetyl )2-(gamma-Glu)-CO-(CH2)18-CO

2H <220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400>10

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 1015

Lys Ala Ala Gln Ala Phe 20

Ile Glu Tyr Leu Leu Glu Xaa Gly Pro Ser

2530

Ser Gly Ala Pro

Pro Pro Ser <210> 11 <211> 39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

< 221> MOD_RES < 222> (17)..(17) < 223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO 2H <220>

< 221> MOD_RES < 222> (39)..(39) <223> Ser at position 39 is optionally amidated

- 30 043950 <400>11

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys

5 1015

Lys Ala Ala Gln Ala Phe Ile Glu Tyr Leu Leu Glu Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>12 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400>12

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys

5 1015

Lys Ala Gln His Ala Phe Ile Glu Tyr Leu Leu Glu Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210> 13 <211> 39 <212> PROTEIN

- 31 043950 <213> Artificial sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Haa at position 20 is alpha-methyl-Lys <220>

<221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400>13

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser 1 510

Ile Leu Leu Asp Lys 15

Lys Ala Gln Xaa Ala Phe 20

Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>14 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

- 32 043950 <221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (gamma-Glu)2-CO-(CH2)18- CO2H <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated <400>14

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser 1 510

Ile Xaa Leu Asp Lys

Lys Ala Gln His Ala Phe 20

Ile Glu Tyr Leu Leu Glu Gly Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>15 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

< 221> MOD_RES < 222> (17)..(17) < 223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain with (2-[2-(2-amino-ethoxy)-ethoxy]- acetyl)-(gamma-Glu)-CO-(CH2)18-CO2

H<220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated <400> 15

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15

- 33 043950

Lys Ala Gln His Ala Phe Ile Glu Tyr Leu Leu Glu Gly Gly Pro Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 16 <211> 39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400>16

Tyr Xaa

Lys Ala

Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 5 1015

Gln Xaa Ala Phe Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210> 17 <211> 39 <212> PROTEIN

- 34 043950 <213> Artificial sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Xaa at position 2 is alpha-methyl-leucine <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino-ethoxy)-ethoxy]- acetyl)-(gamma-Glu)-CO-(CH2)18-CO2

H<220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400>17

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 1015

Lys Ala Gln Xaa Ala Phe Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>18 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

<221> OTHER_CHARACTER <222> (2)..(2) <223> Xaa at position 2 is alpha-amino-isobutyric acid

- 35 043950 <220>

< 221> OTHER_CHARACTER < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino-ethoxy)-ethoxy]- acetyl)-(gamma-Glu)-CO-(CH2)18-CO2

H<220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (29).. (29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400> 18

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys

5 1015

Lys Ala Gln Xaa Ala Phe 20

Ile Glu Tyr Leu Leu Glu Xaa Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>19 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> OTHER_FEATURE < 222> (13)..(13) < 223> Haa at position 13 is alpha-methyl-Leu

- 36 043950 <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino-ethoxy)-ethoxy]- acetyl)-(gamma-Glu)-CO-(CH2)18-CO2H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (29).. (29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (34).. (34) < 223> Xaa at position 34 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400>19

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys

5 1015

Lys Ala Gln Xaa Ala Phe 20

Ile Glu Tyr Leu Leu Glu Xaa Gly Pro Ser

2530

Ser Xaa Ala Pro Pro Pro Ser 35 <210>20 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (17)..(17) < 223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to

- 37 043950 (2-[2-(2-amino)ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)-CO-(CH2)18-CO 2H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400>20

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Asp Lys

5 1015

Lys Ala Gln Xaa Ala Phe Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>21 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) <223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

- 38 043950

Ile Leu Leu Asp Lys <221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400>21

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser 1 510

Lys Ala Gln Xaa Ala Phe 20

Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>22 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400> 22

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Asp Lys 1 5 10 15

- 39 043950

Lys Ala Gln Xaa Ala Phe Ile Glu Tyr Leu Leu Glu Xaa Gly Pro Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 23 <211> 39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino-ethoxy)-ethoxy]- acetyl)-(gamma-Glu)-CO-(CH2)18-CO2

H<220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (39)..(39) < 223> Ser at position 39 is optionally amidated < 400> 23

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Asp Lys

5 1015

Lys Ala Gln Xaa Ala Phe Ile Glu Tyr Leu Leu Ala Xaa Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210> 24 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction

- 40 043950 <220>

<221> OTHER_CHARACTER <222> (2)..(2) <223> Xaa at position 2 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (17) .. (17) <223> Lys at position 17 is chemically modified by conjugating the epsilon amino group of the Lys side chain to (2-[2-(2-amino)ethoxy)-ethoxy]- acetyl)2-(gamma-Glu)-CO-(CH2)18-CO

2H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400>24

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Asp Lys 1 5 1015

Lys Ala Gln Xaa Ala Phe 20

Ile Glu Tyr Leu Leu Glu Xaa Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>25 <211>39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic construction <220>

< 221> OTHER_CHARACTER < 222> (2)..(2) < 223> Haa at position 2 is alpha-amino-isobutyric acid <220>

< 221> MOD_RES < 222> (17)..(17) < 223> Lys at position 17 is chemically modified by conjugation

- 41 043950 epsilon amino group of the Lys side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(gamma-Glu)-CO-(CH2)18-CO2 H <220>

< 221> OTHER_CHARACTER < 222> (20)..(20) < 223> Xaa at position 20 is alpha-amino-isobutyric acid <220>

< 221> OTHER_CHARACTER < 222> (29)..(29) < 223> Xaa at position 29 is alpha-amino-isobutyric acid <220>

<221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is optionally amidated <400>25

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Asp Lys

5 1015

Lys Ala Gln Xaa Ala Phe 20

Ile Glu Tyr Leu Leu Glu Xaa Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>26 <211>10 <212> PROTEIN <213> Artificial Sequence <220>

< 223> Synthetic construction < 400>26

Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 510 <210>27 <211>10 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic construction <220>

<221> OTHER_CHARACTER <222> (5) .. (5) <223> Haa at position 5 is alpha-amino-isobutyric acid

- 42 043950 <400> 27

Gly Pro Ser Ser Xaa Ala Pro Pro Pro Ser

5 10

Claims (35)

CLAIM 1. An incretin analog containing yx ₂ qgtftsdysix ₁₃ ldkx ₁₇ ax ₁₉ x ₂₀ afieyllx ₂₈ x ₂₉ gpssx ₃₄ appps where X2 is Aib; X _{1 3} represents L or aMeL; X _{1 7} represents any amino acid with a functional group available for conjugation, wherein said functional group is conjugated to a C ₁₆ -C ₂₂ fatty acid; X _{1 9} represents Q or A; X _{2 0} represents Aib, aMeK, Q or H; X _{2 8} represents E or A; X2 9 represents G or Aib; X ₃₄ is G or Aib (SEQ ID NO: 5); And The C-terminal amino acid is optionally amidated, or a pharmaceutically acceptable salt thereof. 2. An incretin analog according to claim 1, characterized in that said amino acid with a functional group available for conjugation at position _X17 is selected from the group consisting of K, C, E and D. 3. An incretin analog according to claim 1, characterized in that said amino acid with a functional group available for conjugation at position X17 is K. 4. An incretin analog according to any one of claims 1 to 3, characterized in that said amino acid with a functional group available for conjugation at position X ₁₇ and a C ₁₆ -C ₂₂ fatty acid are conjugated by a linker between the amino acid and the fatty acid. 5. An incretin analog according to claim 4, characterized in that said linker contains from one to four amino acids. 6. An incretin analog according to claim 5, characterized in that said amino acids are Glu or yGlu. 7. An incretin analogue according to any one of claims 4-6, characterized in that said linker additionally contains the structure H-{NH-CH ₂ -CH ₂ -[O-CH ₂ -CH ₂ ] _m -O-(CH ₂ ) _p -CO}n-OH, where m is any integer from 1 to 12; n is any integer from 1 to 12; and p is 1 or 2. 8. An incretin analogue according to any one of claims 4-7, characterized in that said linker additionally contains from one to four fragments (2-[2-(2-amino-ethoxy)ethoxy]acetyl). 9. An incretin analog according to claim 1, characterized in that X ₁₇ is K chemically modified by conjugation with the epsilon amino group of the K side chain with the following structure: (2-[2-(2-amino-ethoxy)ethoxy]acetyl) _a -(γGly) _b -CO-(CH ₂ ) _c -CO ₂ H, where a is 0, 1 or 2; b is equal to 1 or 2; and c is an integer between 16 and 18. 10. An incretin analog according to claim 9, characterized in that a is equal to 1. 11. An incretin analogue according to claim 9, characterized in that a is equal to 2. 12. An incretin analogue according to any one of claims 9-11, characterized in that b is equal to 1. 13. An incretin analogue according to any one of claims 9-11, characterized in that b is equal to 2. 14. An incretin analogue according to any one of claims 9-13, characterized in that c is equal to 18. 15. An incretin analog according to any one of claims 1 to 14, characterized in that X ₁₃ is aMeL. 16. An incretin analogue according to any one of claims 1 to 14, characterized in that X ₁₃ represents L. 17. An incretin analogue according to any one of claims 1 to 16, characterized in that X ₂₀ represents Aib. 18. An incretin analogue according to any one of claims 1 to 17, characterized in that X ₂₈ represents E. 19. An incretin analogue according to any one of claims 1 to 18, characterized in that X ₂₉ represents G. 20. An incretin analog according to any one of claims 1 to 19, characterized in that X ₃₄ is G. 21. An incretin analogue according to any one of claims 1 to 20, characterized in that X ₁₉ represents Q. 22. An incretin analogue according to claim 9, characterized in that a is equal to 1; b is equal to 1; c equals 18; X ₁₃ represents aMeL; - 43 043950 X19 represents Q; X20 represents Aib; X ₂₈ represents E; X ₂ 9 represents G; And X34 represents G. 23. The incretin analogue according to claim 1, characterized in that the incretin analogue has a formula selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25. 24. The incretin analogue according to claim 23, wherein the incretin analogue has 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. 25. Incretin analog according to π.24, characterized in that the incretin analog has the formula SEQIDNO: 17. 26. A method of treating a disease selected from the group consisting of diabetes mellitus, obesity, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia and metabolic syndrome, the method comprising the step of administering to an individual in need thereof an effective amount of an incretin analog according to any one of claims 1 -25. 27. A method of treating type II diabetes mellitus, the method comprising the step of administering to an individual in need thereof an effective amount of an incretin analog according to any one of claims 1 to 25. 28. Pharmaceutical composition containing an incretin analogue according to any one of claims 1-25; and a pharmaceutically acceptable carrier, diluent or excipient. 29. Use of an incretin analogue according to any one of claims 1 to 25 for the treatment of a disease selected from the group consisting of diabetes mellitus, dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic steatohepatitis and obesity. 30. Use of an incretin analogue according to any one of claims 1 to 25 for the treatment of type II diabetes mellitus. 31. Use of an incretin analogue according to any one of claims 1 to 25 in the manufacture of a medicament for the treatment of a disease selected from the group consisting of diabetes mellitus, dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic steatohepatitis and obesity. 32. Use of an incretin analogue according to any one of claims 1 to 25 in the manufacture of a medicinal product for the treatment of type II diabetes mellitus. 33. A method of treating obesity, the method comprising the step of administering to an individual in need thereof an effective amount of an incretin analog according to any one of claims. 1-25. 34. Use of an incretin analogue according to any one of claims 1 to 25 for the treatment of obesity. 35. Use of an incretin analogue according to any one of claims 1 to 25 in the manufacture of a medicinal product for the treatment of obesity.