GLP-1 derivatives and their uses

Abstract

The present invention relates to novel GLP-1 derivatives that activate GLP-1 receptors in a manner that favors the cAMP response and impairs beta-arrestin mobilization. More specifically, the present invention relates to such GLP-1 derivatives and their use for the treatment of diseases such as diabetes and obesity.

Description

Technology Field

[0001] The present invention relates to novel GLP-1 derivatives and their pharmaceutical uses. Furthermore, the present invention relates to pharmaceutical compositions comprising such GLP-1 derivatives, and the use of such compounds for the treatment of medical conditions associated with diabetes and obesity.

[0002] Integration by reference to sequence list

[0003] Sequence list

[0004] This application is filed together with a sequence list in electronic form. The entire contents of said sequence list are incorporated herein by reference. Background Technology

[0005] Glucagon-like peptide 1 (GLP-1), its analogs, and derivatives are known for their ability to stimulate insulin secretion, induce weight loss due to reduced appetite, delay gastric emptying, and improve cardiovascular, liver, and kidney health. They have been used for years, for example, in the treatment of type 2 diabetes and obesity, and one of the most recent derivatives is semaglutide, which is present in the products Ozempic® and WeGovy® (WO2006 / 097537).

[0006] GLP-1 acts by interacting with the GLP-1 receptor, a G-protein coupled receptor (GPCR) expressed in various tissues and cell types, including pancreatic beta cells (which lead to insulin secretion) and neurons in the brain (which lead to inhibition of food intake). When a GPCR is stimulated, this triggers various signaling pathways, for example, G-proteins that lead to the production of cAMP. as- It affects beta-arrestin, which leads to protein pathways, receptor internalization, and potentially receptor-mediated ligand clearance. Some GLP-1 analogs and their derivatives can activate signaling pathways that demonstrate differences in efficacy / effect between them. This concept is referred to as biased GLP-1 action. Thus, some GLP-1 analogs can activate both the cAMP and beta-arrestin pathways with maximum efficacy, whereas others will fully activate the cAMP pathway without fully activating the beta-arrestin pathway. Preclinical evidence suggests that biased GLP-1 analogs or derivatives with reduced beta-arrestin signaling may provide better weight loss and glucose control with a similar or better tolerability profile compared to balanced GLP-1 analogs or derivatives, such as semaglutide. Based on genetic evidence, low-frequency loss-of-function (LoF) variants in beta-arrestin (ARRB1) are associated with improved HbA1c control in T2D patients treated with GLP-1 receptor agonists (Dawed et al, Lancet Diabetes Endocrinol, 2023 Jan; 11(1):33-41). Therefore, it has been suggested that biased GLP-1 compounds, which are more favorable for cAMP production than for beta-arrestin recruitment, may provide greater benefits to a broader patient population by mimicking the improved GLP-1 response in patients with beta-arrestin LoF variants. While weight loss may partially explain the benefits of GLP-1 in obesity-related comorbidities (e.g., cardiovascular disease), biased GLP-1 action through other cells with reduced beta-arrestin signaling expressed in all tissues of the body may hypothetically provide additional benefits to patients with cardiometabolic disease.

[0007] GLP-1 derivatives and biased GLP-1 derivatives are disclosed, for example, in WO2009 / 030738, WO2017 / 149070, WO2016 / 161244, and EP3783014. However, there is still a need for the development of GLP-1 derivatives and analogs having biased signaling accompanied by high activation of the cAMP pathway and impaired beta-arrestin mobilization.

[0008] The present invention relates to a derivative of a GLP-1 analog having an imidazolepropionyl group at a position corresponding to position 7 of hGLP-1 (7-37) (SEQ No. 11) and a Trp or Gly group at a position corresponding to position 8 of hGLP-1 (7-37). The derivative has a substituent attached to a Lys residue of the GLP-1 analog at a position corresponding to position 37 of hGLP-1 (7-37). The present invention also relates to a corresponding GLP-1 analog having an imidazolepropionyl group at a position corresponding to position 7 of hGLP-1 (7-37) (SEQ No. 11) and a Trp or Gly group at a position corresponding to position 8 of hGLP-1 (7-37).

[0009] Additionally, the present invention relates to pharmaceutical compositions comprising such GLP-1 derivatives and pharmaceutically acceptable excipients, as well as medical uses of said derivatives.

[0010] In a first aspect, the present invention relates to a derivative of a GLP-1 analog capable of activating a GLP-1 receptor by inducing a cAMP response while exhibiting reduced beta-arrestin signaling. In a further aspect, the GLP-1 derivative of the present invention selectively activates the GLP-1 receptor compared to the GIP receptor.

[0011] Additionally or alternatively, in a second aspect, the present invention relates to a GLP-1 derivative having improved pharmacokinetic properties.

[0012] Additionally or alternatively, in a third aspect, the present invention relates to a physically stable GLP-1 derivative.

[0013] Additionally or alternatively, in a fourth aspect, the present invention relates to a GLP-1 derivative exhibiting improved chemical stability. Brief explanation of the drawing

[0014] Figure 1 shows the concentration-response curves for the recruitment of beta-arestin 2 to GLP-1R for compound 1, hGLP-1 (7-37), and semaglutide, and the efficacy (E max ) is significantly reduced for compound 1 of the present invention. Specific details for implementing the invention

[0015] In the following, Greek letters may be represented by symbols or corresponding names (e.g., α = alpha; β = beta; ε = epsilon; γ = gamma; ω = omega, etc.). Additionally, the Greek letter μ may be written as "u" (e.g., μl = ul, or μM = uM).

[0016] An asterisk (*) or dashed line in a chemical formula indicates i) an attachment point, ii) a radical, and / or iii) an unshared electron.

[0017] Any interval disclosed herein is generally a closed interval, i.e., includes an endpoint. For example, the number of consecutive -CH2- groups in the substituents attached to the lysine residue of the present invention is in the range of 14 to 18, i.e., 14 to 18, and both are included.

[0018] Unless otherwise stated in this specification, terms presented in the singular form generally include the plural form.

[0019] Additionally, derivatives, derivatives of GLP-1 analogs, methods of preparation, pharmaceutical compositions, and uses are described herein as disclosed, wherein open-ended terms such as “comprises and comprising” are replaced with closed-ended terms such as “consists of, consisting of, etc.”

[0020] The present invention relates to a derivative of a GLP-1 analog having an imidazolepropionyl group at a position corresponding to position 7 of hGLP-1 (7-37) (SEQ No. 11) and a Trp or Gly group at a position corresponding to position 8 of hGLP-1 (7-37). The derivative has a substituent attached to a Lys residue of the GLP-1 analog at a position corresponding to position 37 of hGLP-1 (7-37). The present invention also relates to a corresponding GLP-1 analog having an imidazolepropionyl group at a position corresponding to position 7 of hGLP-1 (7-37) (SEQ No. 11) and a Trp or Gly group at a position corresponding to position 8 of hGLP-1 (7-37).

[0021] Additionally, the present invention relates to pharmaceutical compositions comprising such GLP-1 derivatives and pharmaceutically acceptable excipients, as well as medical uses of said derivatives.

[0022] In a first aspect, the present invention relates to a derivative of a GLP-1 analog capable of activating a GLP-1 receptor by inducing a cAMP response while exhibiting reduced beta-arrestin signaling. In a further aspect, the GLP-1 derivative of the present invention selectively activates the GLP-1 receptor compared to the GIP receptor.

[0023] Additionally or alternatively, in a second aspect, the present invention relates to a GLP-1 derivative having improved pharmacokinetic properties.

[0024] Additionally or alternatively, in a third aspect, the present invention relates to a physically stable GLP-1 derivative.

[0025] Additionally or alternatively, in a fourth aspect, the present invention relates to a GLP-1 derivative exhibiting improved chemical stability.

[0026] compound

[0027] GLP-1 receptor agonists

[0028] Receptor agonists can be defined as compounds that bind to a receptor and induce a typical response of a natural ligand (see, for example, “Principles of Biochemistry”, AL Lehninger, DL Nelson, MM Cox, Second Edition, Worth Publishers, 1993, page 763).

[0029] As disclosed herein, a "GLP-1 receptor agonist" may be defined as a compound capable of binding to and activating a GLP-1 receptor.

[0030] GLP-1 analogue

[0031] The term "hGLP-1(7-37)" as used herein refers to human glucagon-like peptide-1, the sequence of which is included in the sequence list as SEQ ID NO. 11. The peptide having SEQ ID NO. 11 may also be designated as "natural GLP-1" or simply "GLP-1".

[0032] As used herein, the term "GLP-1 analog" refers to a peptide or compound that is a variant of hGLP-1(7-37). The term "variant" is used for a peptide comprising at least one amino acid substitution relative to hGLP-1(7-37), which can activate the GLP-1 receptor.

[0033] The GLP-1 analog of the present invention has imidazolepropionyl, e.g., 3-(imidazole-4-yl)-propionyl or 3-imidazole-2-yl, at a position corresponding to position 7 of GLP-1 (7-37). In one embodiment, imidazolepropionyl is 3-(imidazole-4-yl)propionyl, also known as deamino-histidine or abbreviated as "Imp".

[0034] In the GLP-1 analog of the present invention, the numbering of amino acid residues (e.g., "position 7") follows established conventions in the art for GLP-1, whereby the first amino acid residue is numbered or assigned as position number 7, and subsequent amino acid residues in the C-terminal direction are numbered 8, 9, 10, etc., up to the last (C-terminal) amino acid residue. Imidazolepropionyl (Imp) will be described as part of the peptide backbone, even if it is not an amino acid.

[0035] Numbering is different in the sequence list, where number 1 is assigned to the first amino acid residue (His) of sequence number 11. However, the present invention follows established numbering conventions in the art. In the sequence list, a sequence having Imp at a position corresponding to position 7 of GLP-1 (7-37) (e.g., sequence number 1), Imp is not part of the sequence list because it is not an amino acid, but is described as an N-acylation of an amino acid at a position corresponding to position 8 of GLP-1 (7-37), and thus becomes amino acid number 1 in the sequence list.

[0036] In one embodiment, the GLP-1 analog of the present invention is a peptide of Formula I (SEQ ID NO. 18):

[0037] Imp-X8EGTFTSDVSSYLEEQAAREFIAWLVRGRK,

[0038] Here, Imp is 3-(imidazole-4-yl)-propionyl, and

[0039] X8 in the formula is W or G, or a pharmaceutically acceptable salt, amide, or ester thereof.

[0040] In some embodiments, the GLP-1 analog of the present invention is the peptide of SEQ ID NO. 20 or a pharmaceutically acceptable salt, amide, or ester thereof. Additionally or alternatively, in some embodiments, the GLP-1 analog of the present invention is the peptide of SEQ ID NO. 21 or a pharmaceutically acceptable salt, amide, or ester thereof.

[0041] The GLP-1 analog of the present invention may be described by reference to i) the number of amino acid residues in hGLP-1(7-37) corresponding to the changing amino acid residues (i.e., the corresponding positions in hGLP-1(7-37)), and ii) the actual change. For example, [Imp7, Trp8, Glu22, Arg26, Arg34, Lys37]-hGLP-1(7-37) refers to a GLP-1 analog in which the positions corresponding to positions 7, 8, 22, 26, 34, and 37 of hGLP-1(7-37), respectively, are substituted with the amino acid mentioned, i.e., the peptide of SEQ ID NO. 9.

[0042] As used herein, the term “substitution” refers to the replacement of one amino acid with another amino acid or a chemical component that can be incorporated into the backbone of a peptide. In one embodiment, the amino acid may be substituted by a conservative substitution. As used herein, the term “conservative substitution” indicates the replacement of one or more amino acids with other biologically similar residues. Examples include the substitution of amino acid residues having similar characteristics (e.g., small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids). In one embodiment, a GLP-1 analog of a derivative of the present invention may comprise substituting one or more abnormal amino acids and / or non-amino acids (e.g., amino acid mimics) into the sequence of the GLP-1 analog.

[0043] An analogue that "comprising" a specific specified change may include additional changes when compared to each expression. In a specific embodiment, the analogue "has" the specified change.

[0044] As is evident from the above example, amino acid residues can be identified by their full names, one-letter codes, and / or three-letter codes. These three methods are completely equivalent.

[0045] Next, all amino acids of the GLP-1 analogs of the present invention that are not mentioned as optical isomers should be understood to mean L-isomers (unless otherwise specified).

[0046] GLP-1 derivatives

[0047] As used herein in the context of GLP-1 analogs, the term "derivative" refers to a chemically modified GLP-1 analog in which one or more substituents are covalently attached to a peptide backbone. In some embodiments, the substituents may be referred to as side chains.

[0048] In one embodiment, the GLP-1 analog comprises a substituent covalently attached to an amino acid residue corresponding to position 37 of hGLP-1 (7-37). In some embodiments, the substituent may form a non-covalent conjugate with a protein (e.g., albumin) to promote the circulation of the derivative with the bloodstream, and may have the effect of extending the duration of action of the derivative due to the fact that the aggregate of the GLP-1 derivative and albumin is only slowly degraded to release the active pharmaceutical component.

[0049] Substituents can be covalently attached to the lysine residue of a GLP-1 analog by acylation, that is, through an amide bond formed between the carboxylic acid group of the modifier and the epsilon-amino group of the lysine group. The amino group of lysine may also be attached to the aldehyde of the modifier by reductive amination.

[0050] In one embodiment, the substituent is covalently attached to the lysine residue by acylation, that is, through an amide bond formed between the carboxylic acid group of the modifier and the epsilon-amino group of the lysine residue, or at a position corresponding to position 37 of hGLP1(7-37).

[0051] In one embodiment, the substituent is defined by Equation III and:

[0052] HOOC-(CH2) c -CO-(NHC(COOH)CH2CH2CO) b -(NHCH2CH2OCH2CH2OCH2CO) a -

[0053] In the equation, a is 1 to 3, b is 1 to 2, and c is 14 to 18.

[0054] In some embodiments, the substituent is defined by Equation III, wherein a is 2, b is 1, and c is 16, i.e., Equation IIIa:

[0055] HOOC-(CH2) 16 -CO-(NHC(COOH)CH2CH2CO)-(NHCH2CH2OCH2CH2OCH2CO)2-

[0056] In some embodiments, the substituent is Chemical Formula 1:

[0057] .

[0058] Derivatives of the present invention may exist in different stereoisomer forms having the same molecular formula and sequence of bonded atoms, but differ only in the three-dimensional orientation of these atoms in space. The stereoisomer phenomena of the exemplified derivatives of the present invention are indicated in the experimental section by name and structure using standard nomenclature. Unless otherwise noted, the present invention relates to all stereoisomer forms of the claimed derivatives.

[0059] The GLP-1 derivatives and analogs of the present invention have GLP-1 activity. This term refers to the ability to bind to a GLP-1 receptor and initiate a signaling pathway to produce insulin secretion or other physiological effects known in the art. For example, the analogs and derivatives of the present invention may be tested for GLP-1 activity using assays as described in Examples 1 through 7.

[0060] Pharmaceutically acceptable salts, amides, or esters

[0061] Derivatives of the present invention may be in the form of pharmaceutically acceptable salts, amides, or esters.

[0062] Salts are formed, for example, by a chemical reaction between a base and an acid (e.g., 2NH3 + H2SO4 → (NH4)2SO4).

[0063] Salts can be basic salts or acidic salts, or neither (i.e., neutral salts). Basic salts produce hydroxide ions and acidic salt hydronium ions in water.

[0064] The salts of the derivatives of the present invention may be formed as additional cations or anions between anionic or cationic groups, respectively. These groups may be located within the peptide moiety and / or modification group of the derivatives of the present invention.

[0065] Non-limiting examples of anionic groups in derivatives of the present invention include free carboxyl groups in the side chain as well as in the peptide moiety (where present). The peptide moiety often includes a free carboxylic acid group at the C-terminus and may also include a free carboxyl group in internal acidic amino acid residues such as Asp and Glu.

[0066] Non-limiting examples of cation groups within a peptide moiety include free amino groups at the N-terminus (if present), as well as any free amino groups of internal basic amino acid residues such as His, Arg, and Lys.

[0067] The amide of the derivative of the present invention may be formed, for example, by the reaction of a free carboxylic acid group with an amine or a substituted amine, or by the reaction of a free or substituted amino group with a carboxylic acid.

[0068] The amide formation may include a free carboxyl group at the C-terminus of the peptide, any free carboxyl group in the side chain, a free amino group at the N-terminus of the peptide, and / or any free amino group or substituted amino group of the peptide in the peptide and / or the side chain.

[0069] Esters of derivatives or analogs of the present invention can be formed, for example, by the reaction of an alcohol or phenol with a free carboxylic acid group, which causes the substitution of at least one hydroxyl group by an alkoxy or aryloxy group.

[0070] Ester formation may include a free carboxylic acid group at the C-terminus of the peptide and / or any free carboxyl group in the amino acid side chain.

[0071] In one embodiment, a derivative of the present invention is in the form of a pharmaceutically acceptable salt. Additionally or alternatively, a derivative of the present invention is in the form of a pharmaceutically acceptable amide, preferably having an amide group at the C-terminus of a peptide. Additionally or alternatively, in some embodiments, a derivative of the present invention is in the form of a pharmaceutically acceptable ester.

[0072] Functional characteristics

[0073] In the first functional aspect, the derivative of the present invention has excellent efficacy at GLP-1 receptors. Additionally or alternatively, in the second functional aspect, the derivative of the present invention selectively activates GLP-1 receptors relative to GIP receptors. Additionally or alternatively, in the third functional aspect, the derivative of the present invention has low efficacy for beta-arrestin recruitment. Additionally or alternatively, in the fourth functional aspect, the derivative of the present invention has good pharmacokinetic properties. Additionally or alternatively, in the fifth functional aspect, the derivative of the present invention is physically stable. Additionally or alternatively, in the sixth functional aspect, the derivative of the present invention is chemically stable.

[0074] Biological activity - In vitro activity

[0075] According to the first functional aspect, the derivatives of the present invention as well as the constituent GLP-1 analogs are biologically active or potent at the human GLP-1 receptor.

[0076] In one embodiment, efficacy and / or activity refers to in vitro efficacy, i.e., performance in a functional GLP-1 receptor assay, and more specifically, to the ability to activate human GLP-1 receptors. In one embodiment, a derivative of the present invention can induce cAMP production upon activation of GLP-1 receptors.

[0077] In vitro efficacy can be determined, for example, in a medium containing a membrane expressing human GLP-1 receptors and / or in an assay using whole cells expressing human GLP-1 receptors.

[0078] For example, the response of the human GLP-1 receptor is measured in a reporter gene assay, for example, in a stably transfected BHK cell line, said cell line expressing the human GLP-1 receptor and containing DNA for a cAMP-responsive element (CRE) bound to a promoter and a gene for firefly luciferase (CRE luciferase). When cAMP is generated by the activation of the GLP-1 receptor, this consequently leads to the expression of luciferase. Luciferase can be determined by adding luciferin, which is converted to oxyluciferin by an enzyme and produces bioluminescence, the bioluminescence of which is measured and is a measure of in vitro efficacy. One non-limiting example of such an assay is described in Example 2 described herein.

[0079] Half-effective concentration (EC 50 ) generally refers to the concentration that induces a response corresponding to the baseline and half of the maximum value, with reference to the dose-response curve. EC 50 It is used as a measure of the efficacy of a compound and represents the concentration at which 50% of the compound's maximum effect is observed.

[0080] The in vitro efficacy of the derivative of the present invention was determined as described above, and the EC of the derivative in question 50 Can decide. EC 50 The lower the value, the better the efficacy. EC 50 It can be determined with respect to a reference compound or as an absolute value.

[0081] In a further specific embodiment, the derivative of the present invention has an absolute in vitro efficacy determined using the method of Example 2 in the absence of HSA, which is an EC of 50 pM, more preferably less than 40 pM, even more preferably less than 30 pM, or most preferably less than 25 pM. 50Corresponds to. Additionally or alternatively, in additional specific embodiments, the derivative of the present invention has an absolute in vitro efficacy determined using the method of Example 2 in the presence of 1% HSA, which is an EC of 1000 pM, more preferably less than 800 pM, or even more preferably less than 700 pM. 50 It corresponds to.

[0082] According to a second functional aspect, a derivative of the present invention can selectively activate human GLP-1 receptors relative to human GIP receptors. The term "selectively" used in relation to the activation of GLP-1 receptors relative to GIP receptors refers to when measured in an in vitro efficacy assay of receptor function (e.g., a functional efficacy assay of CRE luciferase in the absence of HSA), and EC 50 It refers to a derivative that exhibits at least 10 times, e.g., at least 50 times, at least 500 times, or at least 1,000 times better absolute efficacy with respect to the GLP-1 receptor compared to the GIP receptor when compared in terms of value. With respect to the GLP-1 receptor compared to the GIP receptor, the term "better efficacy" of the derivative of the present invention refers to the EC of the GIP receptor versus the GLP-1 receptor. 50 Each is determined by the ratio of the values.

[0083] In a third functional aspect, the derivative of the present invention exhibits reduced beta-arrestin mobilization at the GLP-1 receptor compared to a reference GLP-1 agonist such as GLP-1 or semaglutide. In one embodiment, the derivative of the present invention activates the GLP-1 receptor by activating cAMP while exhibiting reduced beta-arrestin mobilization. This has also been referred to as biased at the GLP-1 receptor.

[0084] In some embodiments, derivatives of the present invention have a lower E compared to reference compounds, such as hGLP-1 (7-37). max It has (maximum effect value). For example, Emax This can be determined in vitro in whole cells expressing a split luciferase having a Large BiT component fused to the GLP-1R C-terminus and a Small BiT fused to one of the two isoforms of beta-arrestin, beta-arrestin 1 or beta-arrestin 2, which express the human GLP-1 receptor. When GLP-1R is activated, beta-arrestin is translocated to the receptor, and subsequently, the binding of the Large BiT and Small BiT forms a functional luciferase whose activity can be detected by the addition of a substrate. One non-limiting example of such an assay is described in Example 4 described herein.

[0085] In one embodiment, the derivative of the present invention exhibits significantly reduced efficacy in the beta-arrestin assay for both isoforms 1 and 2, as in the assay described in Example 4 of this application. In a specific embodiment, the derivative of the present invention exhibits less than 50%, or more preferably less than 40%, E compared to hGLP-1 (7-37) for both beta-arrestin 1 and beta-arrestin 2. max has

[0086] Pharmacokinetic Profile - In vivo half-life in minipigs

[0087] According to the fourth functional aspect, the derivative of the present invention has improved pharmacokinetic properties, such as an increased terminal half-life.

[0088] An increase in the terminal half-life means that the compound in question is eliminated from the body more slowly. In the case of derivatives of the present invention, this entails an extension of the duration of the pharmacological effect.

[0089] The pharmacokinetic properties of the derivatives of the present invention can be appropriately determined in vivo through pharmacokinetic (PK) studies. These studies are performed to evaluate how pharmaceutical compounds are absorbed, distributed, and eliminated from the body, and how these processes affect the concentration of the compounds in the body over time.

[0090] In the preclinical stages of pharmaceutical drug discovery and pharmaceutical drug development, these characteristic analyses can be performed using animal models such as mice, rats, monkeys, dogs, or pigs. Pharmacokinetic characteristics of the derivatives of the present invention can be tested using any one of these models.

[0091] In these studies, a single dose of the drug is typically administered to animals intravenously (iv), subcutaneously (sc), or orally (po) in the associated formulation. Blood samples are collected at predefined time points after administration, and the samples are analyzed for drug concentration using associated quantitative assays. Based on these measurements, time-plasma concentration profiles for the study compound are plotted, and so-called non-compartmental pharmacokinetic analysis is performed on the data.

[0092] For most compounds, the terminal portion of the plasma concentration profile becomes linear when plotted on a semi-annual graph, reflecting that the drug is eliminated from the body at a constant fractional rate after initial absorption and distribution. Rate (lambda-Z or l z ) is equal to the negative slope of the end portion of the graph. From this velocity, the end half-life is t½ = ln(2) / λ z It can also be calculated as (for example, see the literature [Johan Gabrielsson and Daniel Weiner: Pharmacokinetics and Pharmacodynamic Data Analysis. Concepts & Applications, 3rd Ed., Swedish Pharmaceutical Press, Stockholm (2000)]).

[0093] Elimination can be determined after IV administration and is defined as the dose (D) divided by the area under the curve of the plasma concentration profile over time (AUC) (Reference [Rowland, M and Tozer TN: Clinical Pharmacokinetics: Concepts and Applications, 3 rd [See edition, 1995 Williams Wilkins]).

[0094] In the evaluation of new drug compounds, estimates of terminal half-life and / or elimination are relevant to the evaluation of the dosing regimen and are important parameters in drug development.

[0095] According to the fourth functional aspect, the derivative of the present invention has improved pharmacokinetic properties.

[0096] In a specific embodiment, the pharmacokinetic characteristics are as described in Example 5 of the present invention, with an in vivo terminal half-life (t) in a minipig after IV administration. ½ It can be determined as ).

[0097] In a specific embodiment, the terminal half-life of the minipig is at least 75 hours, preferably at least 100 hours, and more preferably at least 140 hours. The long half-life in the minipig suggests that the derivative of the present invention may be suitable for administration to humans at a frequency of once a week or less, for example, twice a month or once a month. In one embodiment, the derivative of the present invention has a long half-life in humans suitable for once-weekly administration.

[0098] Physical properties

[0099] According to the fifth functional aspect, derivatives of the present invention have improved physical stability in solution. The term “physical stability” refers to the tendency of polypeptides to form biologically inactive and / or insoluble aggregates, e.g., amyloid fibrils or gels.

[0100] In certain embodiments, improved physical stability may be determined by measuring the delay-time and / or recovery rate in a thioflavin T (ThT) microfibrosis assay, for example as described in Example 6 of the present invention.

[0101] In additional specific embodiments, derivatives of the present invention have a delay-time of more than 20 hours, preferably more than 30 hours, and more preferably more than 45 hours in the ThT microfibrosis assay, as shown in Example 6 described herein.

[0102] In additional specific embodiments, derivatives of the present invention have a recovery rate of more than 80% in the ThT microfibrosis test, as shown in Example 6 described herein. A recovery rate of more than 80% is considered a complete recovery rate.

[0103] Chemical properties

[0104] According to the sixth functional aspect, derivatives of the present invention have improved chemical stability. The term “chemical stability” refers to chemical (particularly, of covalent bonds) changes in the polypeptide structure that result in the formation of chemical degradation products, such as deamidation, isomerization, and hydrolysis products, which potentially have reduced biological efficacy and / or increased immunogenic effects compared to the intact polypeptide.

[0105] In certain embodiments, improved chemical stability can be determined by measuring the content and / or loss of purity of HMWP by measuring the amount of chemical degradation products at various times, for example by SEC-HPLC, after exposure to different environmental conditions, for example, as described in Example 7 of the present invention.

[0106] In additional specific embodiments, derivatives of the present invention have a purity loss of less than 10%, preferably less than 7%, more preferably less than 4% over four weeks, as shown in Example 7 described herein.

[0107] In additional specific embodiments, derivatives of the present invention have a 4-week HMWP formation of less than 4%, preferably less than 2%, more preferably less than 1%, as shown in Example 7 described herein.

[0108] Additional specific embodiments of derivatives of the present invention are described in the section titled “Specific Embodiments”.

[0109] production process

[0110] The production of peptides such as hGLP-1 and hGLP-1 analogs is well known in the art.

[0111] GLP analogs (or fragments thereof) of derivatives of the present invention, as well as GLP-1 analogs of the present invention, may be produced by solid-phase peptide synthesis using, for example, classical peptide synthesis, e.g., t-Boc or Fmoc chemistry, or other well-established techniques (see, for example, [Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999], [Florencio Zaragoza Dorwald, "Organic Synthesis on solid Phase", Wiley-VCH Verlag GmbH, 2000], and ["Fmoc Solid Phase Peptide Synthesis", Edited by WC Chan and PD White, Oxford University Press, 2000]).

[0112] Additionally or alternatively, in some embodiments, the entire GLP-1 analog of the present invention, or the entire GLP-1 analog portion of a derivative of the present invention, may be produced by a recombinant method, that is, by culturing a host cell capable of expressing the peptide in a suitable nutrient medium under conditions in which the peptide expression is possible, containing a DNA sequence encoding the analog. Non-limiting examples of host cells suitable for the expression of these peptides include Escherichia coli cell lines, Saccharomyces cerevisiae cell lines, as well as mammalian BHK or CHO cell lines.

[0113] Derivatives of the present invention comprising non-natural amino acids and / or covalently attached N-terminal monopeptides or dipeptide mimics may be produced, for example, as described in the experimental section. Or, for example, refer to the literature ["The synthesis of peptides and proteins containing non-natural amino acids", Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430].

[0114] Specific examples of methods for preparing derivatives of the present invention are included in the experimental part of the present invention.

[0115] Pharmaceutical composition

[0116] The present invention also relates to a pharmaceutical composition comprising a derivative of the present invention or a pharmaceutically acceptable salt, amide, or ester thereof and a pharmaceutically acceptable excipient. Such compositions can be prepared as known in the art.

[0117] The term "excipient" broadly refers to any component other than the active therapeutic ingredient(s). Excipients may be inactive substances, non-active substances, and / or non-pharmaceutical active substances.

[0118] Excipients may be used for various purposes, e.g., as carriers, vehicles, diluents, tablet aids, and / or to improve administration and / or absorption of active substances.

[0119] Formulations of pharmaceutically active ingredients having various excipients are known in the art (see, for example, the literature [Remington: The Science and Practice of Pharmacy (e.g., 19th edition (1995), and later editions)]).

[0120] Non-limiting examples of excipients are as follows: solvents, diluents, buffers, preservatives, isotonic regulators, chelating agents, and stabilizers.

[0121] Examples of formulations include liquid formulations, i.e., aqueous formulations containing water. Liquid formulations may be solutions or suspensions. Aqueous formulations generally contain at least 50% w / w of water, or at least 60%, 70%, 80%, or 90% w / w of water.

[0122] Pharmacological indications

[0123] The present invention also relates to derivatives or analogs of the present invention for use as medicine.

[0124] In some embodiments, derivatives or analogs of the present invention may be used in the following medical treatments:

[0125] (i) Prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (mature diabetes onset in young adults), gestational diabetes, and / or reduced HbA1C;

[0126] (ii) delay or prevention of progression of diabetic disease, such as progression to type 2 diabetes; delay or prevention of progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes; delay or prevention of insulin resistance; and / or delay of progression of non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes;

[0127] (iii) prevention and / or treatment of eating disorders, overweight, and / or obesity by, for example, reducing food intake, suppressing appetite, inducing satiety, and weight loss; treatment or prevention of binge eating disorders, food cravings, bulimia nervosa, and / or obesity induced by drugs such as psychotropic drugs or steroids; reduction of gastric motility; and / or delay of gastric fasting;

[0128] (iv) prevention and / or treatment of cardiovascular disease, e.g., delay or reduction of the occurrence of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization due to unstable angina, and hospitalization due to heart failure;

[0129] (v) Prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), also known as metabolic dysfunction-associated steatohepatitis (MASH) and / or alcoholic liver disease (ALD);

[0130] (vi) Prevention and / or treatment of chronic kidney disease;

[0131] (vii) Prevention and / or treatment of cognitive disorders, including dementia and Alzheimer's disease.

[0132] The indication may be (i). The indication may be (ii). The indication may be (iii). The indication may be (iv). The indication may be (v). The indication may be (vi). The indication may be (vii). The indication may be overweight or obesity. The indication may be type 2 diabetes.

[0133] Generally, all subjects suffering from obesity are considered to be overweight. Subjects suffering from obesity may be adult humans or human beings such as children, where "children" includes infants and adolescents.

[0134] The World Health Organization (WHO) defines obesity as the abnormal or excessive accumulation of fat that can impair health and considers the Body Mass Index (BMI) to be the most convenient population-level measure of overweight and obesity. The formula used to calculate BMI is weight (kg) / height (m²). 2 )am.

[0135] For adults, the WHO defines overweight and obesity as follows: overweight means having a BMI of 25 or higher; obesity means having a BMI of 30 or higher.

[0136] For children, the WHO considers age when defining overweight and obesity.

[0137] For children under 5 years of age, overweight means that the weight-to-height ratio exceeds the WHO median standard for child growth by more than 2 standard deviations; obesity means that the weight-to-height ratio exceeds the WHO median standard for child growth by more than 3 standard deviations.

[0138] Overweight and obesity are defined as follows for children aged 5 to 19 years: overweight means that the age-specific BMI exceeds the median WHO growth standard by more than one standard deviation; and obesity means that the age-specific BMI exceeds the median WHO growth standard by more than two standard deviations.

[0139] Nevertheless, for adults, as shown in Table (i) below, diagnostic criteria for underweight, normal range, pre-obesity / overweight, and obesity may vary between countries / populations.

[0140]

[0141] Guidelines for Asian populations have been published in the literature [Misra A et al. J Assoc Physicians India. 2009; 57:163-70].

[0142] Guidelines for the Chinese population are based on literature compiled by the Chinese Working Group on Obesity [ Guidelines for Prevention and Control of Overweight and Obesity in Chinese Adults It was published as the [2006 revised edition].

[0143] Guidelines for the Japanese population are based on literature by the Japanese Society for the Study of Obesity (JASSO) in 2016 [ Guidelines for the management of obesity disease It was published as ].

[0144] Guidelines for the Taiwanese population were issued in 2023 by the Health Promotion Administration (HPA) of the Ministry of Health and Welfare of the Taiwanese government [ Evidence-Based Guideline on Adult Obesity Prevention and Management It was published as the second revised edition of [].

[0145] Therefore, obese adult human subjects have 25 kg / m² 2 Above, 27 kg / m² 2 Above, 28 kg / m² 2 Above, or 30 kg / m² 2 They may have a BMI greater than or equal to 24 kg / m²; such subjects may also be referred to as obese. Obesity may be Grade I, Grade II, Grade III, or Grade IV obesity. Adult human subjects suffering from overweight have a BMI of 24 kg / m² 2 Above, 25 kg / m² 2 Above, or 27 kg / m² 2 A BMI greater than or equal to this may be present. In some embodiments, an overweight human subject has a BMI of 24 to <27 kg / m² 2 Range, 24 to <28 kg / m² 2 Range, 25 to <30 kg / m² 2Range, or 27 to <30 kg / m² 2 has a BMI within the range.

[0146] When BMI becomes higher than normal BMI, the individual's risk of developing any one of a wide range of other diseases or comorbidities increases. Weight-related comorbidities may be one of the diseases mentioned in (i), (ii), (iv), and (v) above, or a combination of any of these diseases.

[0147] The GLP-1 derivatives disclosed herein may be used for the treatment or prevention of overweight in which the patient may have at least one weight-related comorbidity. The GLP-1 derivatives disclosed herein may be used for the treatment or prevention of obesity in which the patient may have at least one weight-related comorbidity.

[0148] The GLP-1 derivative disclosed herein can be used as an adjunct to a low-calorie diet and increased physical activity for chronic weight management in subjects who are obese at the start of treatment.

[0149] The GLP-1 derivative disclosed herein can be used as an adjunct to a low-calorie diet and increased physical activity for chronic weight management in subjects who are overweight at the start of treatment and have at least one weight-related comorbidity.

[0150] The GLP-1 derivatives disclosed herein are for individuals with an initial body mass index (BMI) of 30 kg / m² 2 Above, 28 kg / m² 2 Above, 27 kg / m² 2 Above, or 25 kg / m² 2 It can be used as an adjunct to a low-calorie diet and increased physical activity for chronic weight management in adult humans.

[0151] The GLP-1 derivatives disclosed herein are for individuals with an initial body mass index (BMI) of 25 kg / m² 2 24 kg / m² or more 2It can be used as an adjunct to a low-calorie diet and increased physical activity for chronic weight management in adult humans with at least one weight-related comorbidity.

[0152] The administration of the GLP-1 derivative disclosed herein may result in a weight loss of >15%, e.g., >20%, e.g., >25%, e.g., >30%, e.g., about 15–40%, e.g., about 20–35%, e.g., about 25–30% within 26 weeks of the start of treatment.

[0153] The administration of the GLP-1 derivative disclosed herein may result in a weight loss of >15%, e.g., >20%, e.g., >25%, e.g., >30%, e.g., about 15–40%, e.g., about 20–35%, e.g., about 25–30% within 26 weeks of the start of treatment.

[0154] The GLP-1 derivatives disclosed herein may be used for the treatment or prevention of diabetes mellitus as described in (i) or (ii) above. The GLP-1 derivatives disclosed herein may be used for the treatment or prevention of diabetes mellitus and at least one diabetes-related comorbidity, such as (iv) or (vi) above.

[0155] Non-limiting implementation example

[0156] The present invention is further described by the following non-limiting embodiments:

[0157] 1. As a GLP-1 derivative, Formula I (Sequence No. 18):

[0158] It includes a GLP-1 analog of Imp-X8EGTFTSDVSSYLEEQAAREFIAWLVRGRK,

[0159] In the formula, Imp is 3-(imidazole-4-yl)-propionyl, and

[0160] X8 is W or G, and

[0161] K at the position corresponding to position 37 of GLP-1(7-37) is Equation III: HOOC-(CH2) c -CO-(NHC(COOH)CH2CH2CO) b -(NHCH2CH2OCH2CH2OCH2CO) a - is chemically modified at the epsilon-amino group by a substituent, and

[0162] In the equation, a is 1–3, b is 1–2, and c is 14–18, and

[0163] The above C-terminal amino acid is optionally a C-terminal amide

[0164] or a GLP-1 derivative that is a pharmaceutically acceptable salt thereof.

[0165] 2. As a GLP-1 derivative, Formula I (Sequence No. 18):

[0166] It consists of a GLP-1 analog of Imp-X8EGTFTSDVSSYLEEQAAREFIAWLVRGRK, and

[0167] In the formula, Imp is 3-(imidazole-4-yl)-propionyl, and

[0168] X8 is W or G, and

[0169] K at the position corresponding to position 37 of GLP-1(7-37) is Equation III: HOOC-(CH2) c -CO-(NHC(COOH)CH2CH2CO) b -(NHCH2CH2OCH2CH2OCH2CO) a - is chemically modified at the epsilon-amino group by a substituent, and

[0170] In the equation, a is 1–3, b is 1–2, and c is 14–18, and

[0171] The above C-terminal amino acid is optionally a C-terminal amide

[0172] or a GLP-1 derivative that is a pharmaceutically acceptable salt thereof.

[0173] 3. A GLP-1 derivative, wherein X8 is W, in either Embodiment 1 or 2.

[0174] 4. A GLP-1 derivative, wherein X8 is G in Embodiment 1 or 2.

[0175] 5. In any one of the embodiments described above, a is a GLP-1 derivative of 2.

[0176] 6. In any one of embodiments 1 to 4, a is a GLP-1 derivative.

[0177] 7. In any one of embodiments 1 to 4, a is a GLP-1 derivative of 3.

[0178] 8. In any one of the embodiments described above, b is a GLP-1 derivative of 1.

[0179] 9. In any one of Embodiments 1 to 7, b is a GLP-1 derivative of 2.

[0180] 10. In any one of the embodiments described above, c is a GLP-1 derivative of 16.

[0181] 11. In any one of embodiments 1 to 9, c is a GLP-1 derivative of 14.

[0182] 12. In any one of embodiments 1 to 9, c is a GLP-1 derivative of 18.

[0183] 13. In any one of the embodiments described above, the GLP-1 derivative: HOOC-(CH2), wherein the substituent is a substituent of Formula IIIa 16 -CO-(NHC(COOH)CH2CH2CO)-(NHCH2CH2OCH2CH2OCH2CO)2-.

[0184] 14. In any one of the embodiments described above, the substituent is a GLP-1 derivative having the chemical formula 1:

[0185]

[0186] 15. In any one of the embodiments described above, the derivative is a GLP-1 derivative, wherein the derivative is compound 1:

[0187] .

[0188] 16. In any one of the embodiments described above, the derivative is a GLP-1 derivative, wherein the derivative is compound 2:

[0189] .

[0190] 17. In any one of the embodiments described above, the derivative is a GLP-1 derivative that is an agonist at the human GLP-1 receptor.

[0191] 18. In any one of the embodiments described above, the derivative is capable of activating human GLP-1 receptors and inducing cAMP production.

[0192] 19. In any one of the embodiments described above, the derivative is a derivative capable of activating the human GLP-1 receptor in an assay using whole cells expressing the human GLP-1 receptor.

[0193] 20. In any one of the embodiments described above, the derivative is a derivative capable of activating the human GLP-1 receptor in the CRE luciferase assay, as in Example 2 described herein.

[0194] 21. In any one of the embodiments described above, the derivative is a GLP-1 derivative that is selective for human GLP-1 receptors compared to human GIP receptors.

[0195] 22. In any one of the embodiments described above, the derivative has a lower EC in the human GLP-1 receptor compared to the human GIP receptor. 50 A GLP-1 derivative having

[0196] 23. In any one of the embodiments described above, the derivative has a lower EC in the human GLP-1 receptor compared to the human GIP receptor in the cAMP production assay. 50A GLP-1 derivative having

[0197] 24. In any one of the embodiments described above, the derivative is an EC in human GIP receptors greater than 10,000 pM in the CRE luciferase assay, as determined in Example 3 described herein. 50 A GLP-1 derivative having

[0198] 25. In any one of the embodiments described above, the derivative is a GLP-1 derivative in which beta-arrestin mobilization is impaired compared to hGLP-1 (7-37).

[0199] 26. In any one of the embodiments described above, the derivative is <50% beta-arrestin E compared to hGLP-1 (7-37), as determined in Example 4 described herein. max A GLP-1 derivative having

[0200] 27. In any one of the embodiments described above, the derivative is <45% beta-arrestin E compared to hGLP-1 (7-37) as determined in Example 4 described herein. max A GLP-1 derivative having

[0201] 28. In any one of the embodiments described above, the derivative is <40% beta-arrestin E compared to hGLP-1 (7-37), as determined in Example 4 described herein. max A GLP-1 derivative having

[0202] 29. In any one of the embodiments described above, the derivative is a GLP-1 derivative having improved pharmacokinetic properties.

[0203] 30. In any one of the embodiments described above, the derivative is a GLP-1 derivative having an increased half-life.

[0204] 31. In any one of the embodiments described above, the derivative is a GLP-1 derivative having an increased half-life when determined in a minipig, as determined in Example 5 described herein.

[0205] 32. In any one of the embodiments described above, the GLP-1 derivative is a GLP-1 derivative having a half-life in humans suitable for once-weekly administration.

[0206] 33. In any one of the embodiments described above, the derivative is a GLP-1 derivative having improved physical stability.

[0207] 34. In any one of the embodiments described above, the derivative is a GLP-1 derivative having a recovery rate of more than 80% in the ThT microfibrosis test as determined in Example 6 described herein.

[0208] 35. In any one of the embodiments described above, the derivative is a GLP-1 derivative having 45 hours or more in the ThT microfibrosis assay as determined in Example 6 of the present invention.

[0209] 36. In any one of the embodiments described above, the derivative is a GLP-1 derivative having improved chemical stability.

[0210] 37. In any one of the embodiments described above, the derivative is a GLP-1 derivative having less than 1% HMWP formation over 4 weeks, as determined in Example 7 described herein.

[0211] 38. In any one of the embodiments described above, the derivative is a GLP-1 derivative having a purity loss of more than 1.5% over 4 weeks, as determined in Example 7 described herein.

[0212] 39. GLP-1 analog of sequence number 18.

[0213] 40. In Embodiment 39, the analog is a GLP-1 analog that is an analog of SEQ ID NO. 20 or SEQ ID NO. 21.

[0214] 41. A pharmaceutical composition comprising a GLP-1 derivative or analog according to any one of the embodiments described above, and at least one pharmaceutically acceptable excipient.

[0215] 42. A GLP-1 derivative or analog according to any one of Embodiments 1 to 40, or a composition according to Embodiment 41, for use as a medicine.

[0216] 43. A GLP-1 derivative or analog according to any one of Embodiments 1 to 40, or a composition according to Embodiment 41, for use in the prevention or treatment of the following:

[0217] (i) Prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (mature diabetes onset in young adults), gestational diabetes, and / or reduced HbA1C;

[0218] (ii) delay or prevention of progression of diabetic disease, such as progression to type 2 diabetes; delay or prevention of progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes; delay or prevention of insulin resistance; and / or delay of progression of non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes;

[0219] (iii) prevention and / or treatment of eating disorders, overweight, and / or obesity by, for example, reducing food intake, suppressing appetite, inducing satiety, and weight loss; treatment or prevention of binge eating disorders, food cravings, bulimia nervosa, and / or obesity induced by drugs such as psychotropic drugs or steroids; reduction of gastric motility; and / or delay of gastric fasting;

[0220] (iv) prevention and / or treatment of cardiovascular disease, e.g., delay or reduction of the occurrence of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization due to unstable angina, and hospitalization due to heart failure;

[0221] (v) Prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), also known as metabolic dysfunction-associated steatohepatitis (MASH) and / or alcoholic liver disease (ALD);

[0222] (vi) Prevention and / or treatment of chronic kidney disease;

[0223] (vii) Prevention and / or treatment of cognitive disorders, including dementia and Alzheimer's disease.

[0224] 44. A GLP-1 derivative or analog according to Embodiment 43, for use in the prevention or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (mature diabetes onset in young adults), gestational diabetes, and / or a decrease in HbA1C.

[0225] 45. A GLP-1 derivative or analog according to Embodiment 43, for use in delaying or preventing the progression of a diabetic disease (e.g., progression to type 2 diabetes), delaying the progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, delaying or preventing insulin resistance, and / or delaying the progression of non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes.

[0226] 46. ​​A GLP-1 derivative or analog according to Embodiment 43 for use in, for example, for the prevention or treatment of eating disorders, overweight, and / or obesity by reducing food intake, suppressing appetite, inducing satiety, and weight loss; for the treatment or prevention of binge eating disorders, food cravings, bulimia nervosa, and / or obesity induced by drugs such as psychotropic drugs or steroids; for reducing gastric motility; and / or delaying gastric fasting.

[0227] 47. A GLP-1 derivative or analog according to Embodiment 43, for use in the prevention or treatment of cardiovascular disease, e.g., delaying or reducing the occurrence of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization due to unstable angina, and hospitalization due to heart failure.

[0228] 48. A GLP-1 derivative or analog according to Embodiment 43, for use in the prevention or treatment of non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), also known as metabolic dysfunction-associated steatohepatitis (MASH) and / or alcoholic liver disease (ALD).

[0229] 49. A GLP-1 derivative or analog according to Embodiment 43, for use in the prevention or treatment of chronic kidney disease.

[0230] 50. A GLP-1 derivative or analog according to Embodiment 43, for use in the prevention or treatment of cognitive disorders including dementia and Alzheimer's disease.

[0231] 51. A GLP-1 derivative or analog according to Embodiment 43, for use in weight management, prevention and / or treatment of obesity and obesity-related disorders.

[0232] 52. A GLP-1 derivative or analog according to Embodiment 43, for use in the prevention and / or treatment of all forms of diabetes (e.g., type 2 diabetes, and diabetes-related disorders).

[0233] 53. Use of a GLP-1 derivative or analog according to any one of Embodiments 1 to 40, or a composition according to Embodiment 41, for the manufacture of the following therapeutic medicines:

[0234] (i) Prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (mature diabetes onset in young adults), gestational diabetes, and / or reduced HbA1C;

[0235] (ii) delay or prevention of progression of diabetic disease, such as progression to type 2 diabetes; delay or prevention of progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes; delay or prevention of insulin resistance; and / or delay of progression of non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes;

[0236] (iii) prevention and / or treatment of eating disorders, overweight, and / or obesity by, for example, reducing food intake, suppressing appetite, inducing satiety, and weight loss; treatment or prevention of binge eating disorders, food cravings, bulimia nervosa, and / or obesity induced by drugs such as psychotropic drugs or steroids; reduction of gastric motility; and / or delay of gastric fasting;

[0237] (iv) prevention and / or treatment of cardiovascular disease, e.g., delay or reduction of the occurrence of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization due to unstable angina, and hospitalization due to heart failure;

[0238] (v) Prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), also known as metabolic dysfunction-associated steatohepatitis (MASH) and / or alcoholic liver disease (ALD);

[0239] (vi) Prevention and / or treatment of chronic kidney disease;

[0240] (vii) Prevention and / or treatment of cognitive disorders, including dementia and Alzheimer's disease.

[0241] 54. A method for treating or preventing (i) to (vii), wherein the method comprises the step of administering a therapeutically effective amount of a GLP-1 derivative or analog according to any one of Embodiments 1 to 40, or a composition according to Embodiment 41, to a subject requiring treatment or prevention, wherein (i) to (vii) is as follows:

[0242] (i) Prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (mature diabetes onset in young adults), gestational diabetes, and / or reduced HbA1C;

[0243] (ii) delay or prevention of progression of diabetic disease, such as progression to type 2 diabetes; delay or prevention of progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes; delay or prevention of insulin resistance; and / or delay of progression of non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes;

[0244] (iii) prevention and / or treatment of eating disorders, overweight, and / or obesity by, for example, reducing food intake, suppressing appetite, inducing satiety, and weight loss; treatment or prevention of binge eating disorders, food cravings, bulimia nervosa, and / or obesity induced by drugs such as psychotropic drugs or steroids; reduction of gastric motility; and / or delay of gastric fasting;

[0245] (iv) prevention and / or treatment of cardiovascular disease, e.g., delay or reduction of the occurrence of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization due to unstable angina, and hospitalization due to heart failure;

[0246] (v) Prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), also known as metabolic dysfunction-associated steatohepatitis (MASH) and / or alcoholic liver disease (ALD);

[0247] (vi) Prevention and / or treatment of chronic kidney disease;

[0248] (vii) Prevention and / or treatment of cognitive disorders, including dementia and Alzheimer's disease.

[0249] Examples

[0250] This experimental section begins with a list of abbreviations and is followed by a section containing general methods for synthesizing and characterizing analogs and derivatives of the present invention. This is followed by numerous examples relating to the preparation of specific derivatives of the present invention, and finally, numerous examples relating to the activity and characteristics of these analogs and derivatives are included (in the section titled "Pharmacological Methods").

[0251] The examples serve to illustrate the present invention.

[0252] Materials and Methods

[0253] List of abbreviations

[0254] Ac: Acetyl

[0255] Aib: Alpha-aminoisobutyric acid

[0256] AUC: Area under the curve

[0257] Boc: t- Butyloxycarbonyl

[0258] DCM: Dichloromethane

[0259] DIC: Diisopropylcarbodiimide

[0260] DIPEA: N , N - Diisopropylethylamine or Hunig's base

[0261] DMF: Dimethylformamide

[0262] DODT: 3,6-Dioxa-1,8-Octanedithiol

[0263] DTT: Dithiothreitol

[0264] EDTA: Ethylenediaminetetraacetic acid

[0265] ELISA: Enzyme-linked immunosorbent assay

[0266] Fmoc: 9-Fluorenylmethyloxycarbonyl

[0267] HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol

[0268] HOBt: 1-Hydroxybenzotriazole

[0269] HPLC: High-performance liquid chromatography

[0270] HSA: Human serum albumin

[0271] Imp: 3-(imidazole-4-yl)-propionyl

[0272] iv: intravenous

[0273] LCMS or LC-MS: Liquid Chromatography Mass Spectrometry

[0274] MeCN: Acetonitrile

[0275] Mtt: 4-methyltrityl

[0276] NHS: N-hydroxysuccinimide

[0277] NMP: N-methylpyrrolidone

[0278] Oxyma Pure®: Ethyl cyano-hydroxyimino-acetate ester

[0279] PK: Pharmacokinetics

[0280] QTof: Quantitative Flight Time

[0281] sc: subcutaneous

[0282] SD: Standard deviation

[0283] SEC-HPLC: Size Exclusion High Performance Liquid Chromatography

[0284] SEM: Standard error of the mean

[0285] SPPS: Solid-phase peptide synthesis

[0286] tBu: t -butyl

[0287] TFA: Trifluoroacetic acid

[0288] TIS: Triisopropylsilane

[0289] Trt: Triphenylmethyl or trityl

[0290] UPLC: Ultra-high Performance Liquid Chromatography

[0291] General manufacturing method

[0292] Common methods for peptide production:

[0293] Resins used in the preparation of compounds 1 to 2 and reference compounds, e.g., pre-loaded Fmoc-Lys(Mtt)-Wang resin (0.30 mmol / g loading), or other similar pre-loaded and unpre-loaded resins suitable for SPPS.

[0294] The Fmoc-protected amino acid derivatives used in SPPS included, among other things, standard recommended building blocks as well as building blocks suitable for introducing non-natural amino acids: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, Fmoc-Aib-OH. Optionally, Fmoc-pseudoproline dipeptide building blocks, e.g., Fmoc-Ser(tBu)-Ser(ψ Me,Me pro)-OH or Fmoc-Val-Ser(ψ Me,Me pro)-OH can be used in SPPS. For N-terminal amino acids, appropriate building blocks, e.g., 3-(1 - Tritil -Imidazole-4-yl)propanoic acid (deaminoHis(Trt)-OH), Boc-His(Trt)-OH, and Boc-Tyr(tBu)-OH were used. The building blocks, as well as the Fmoc amino acids used for N-terminal insertion, were supplied from, for example, AAPPTEC, Apigenex, Anaspec, Bachem, ChemImpex, Iris Biotech, Midwest Biotech, Gyros Protein Technologies, or Novabiochem. The pseudoproline building blocks were supplied from, for example, technocomm and Iris Biotech. Unless otherwise specified, the natural L-forms of the amino acids were used. The Lys(mtt) residue was used to allow orthogonal unmasking of the epsilonamine function. Selective Lys(mtt) deprotection was achieved using a suitable orthogonal deprotectant / mixture, such as HFIP, in DCM. After that, the side chain was attached by stepwise attachment with SPPS or by directly coupling to the side chain building block of an epsilon-amino function-activated ester (e.g., NHS ester).

[0295] For stepwise side chain attachment using SPPS, appropriately protected building blocks such as Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-OEG-OH), Fmoc-Glu-OtBu, and octadecandioxane mono-tert-butyl ester are available, but are not limited to. The work described below was performed within a synthesis scale range of 50 to 450 μmol.

[0296] Peptidyl Backbone's SPPS:

[0297] SPPS was performed using Fmoc-based chemicals with a SymphonyX solid-phase peptide synthesizer from Protein Technologies (Tucson, AZ 85714 USA). Fmoc deprotection was achieved, for example, using 20% ​​piperidine in DMF containing 0–0.3 M Oxyma Pure®. Peptide coupling was performed using DIC / Oxyma Pure®. After adding an amino acid / Oxyma Pure® solution (e.g., 0.3 M / 0.3 M in 3–12 molar excess DMF) to the resin, equal molar equivalents of DIC (e.g., 0.6–1.5 M in DMF) and collidine (e.g., 1.5 M in DMF) were added. Stepwise assembly was performed using the following steps: 1) a step of pre-swelling the pre-loaded resin with DMF; 2) A step of Fmoc-deprotection by treating 1 to 5 times for 5 to 30 minutes each using 20% ​​piperidine in DMF containing 0 to 0.2 M Oxyma Pure®; 3) A step of removing trace piperidine by washing with DMF; 4) A step of coupling 3 to 12 equivalents of Fmoc-amino acids with a 0.3 M solution of 0.3 M Oxyma Pure® in DMF mixed with equimolar volumes of DIC and piperidine for 1 to 12 hours. In the case of sterically hindered amino acids, the coupling step was repeated 1 or 2 times; 5) A step of washing with DMF to remove excess reagent. 6) (Optional) A capping step performed for 20 minutes using a suitable acetic anhydride-based capping mixture, e.g., a 3 / 1 mixture of 1.0 M acetic anhydride in DMF and 1.5 M collidine in DMF (0.75 M Ac2O / 0.3 M collidine in DMF) (repeat steps 2–5, or steps 2–6, if a capping step is used, using a suitable Fmoc-amino acid, pseudoproline, or building block suitable for attaching N-terminal residues, until a peptide backbone of the desired length and sequence is obtained).7) A step of performing a final wash with DCM upon completion of assembly to prepare the resin for attaching a modulator to the ricin side chain.

[0298] Deprotection of Lys(Mtt) and attachment of side chains to a protected peptide backbone bound to a resin:

[0299] The N-epsilon-lysine Mtt protector was removed by washing the resin with a suitable orthogonal deprotection mixture such as HFIP / DCM / TIPS (22.5 / 75 / 2.5) for a number of cycles (e.g., 1 x 10 min and 2 x 20 min) before washing with DCM (3x) and DMF (4x).

[0300] Side chain attachment of lysine residues to the free epsilon-amino moiety was performed manually or on a solid-phase peptide synthesizer such as the SymphonyX solid-phase peptide synthesizer of Protein Technologies (Tucson, AZ 85714 USA). As described in the section "SPPS of Peptidyl Backbone" of this method, stepwise addition of building blocks such as Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-OEG-OH) and Fmoc-Glu-OtBu (Fmoc-gGlu(tBu)-OH) was used. The introduction of the fatty acid moiety was achieved under peptide coupling conditions using a suitable building block, such as octadecandioxane mono-tert-butyl ester, for an extended period (e.g., 2 x 6 hours).

[0301] Alternatively, the side chain moiety can be attached to the peptidyl backbone resin in a single step using a suitable side chain building block equipped with an activated ester such as NHS immediately after removing the Mtt group, as described in the first part of this section.

[0302] Cleavage and Purification of Resin-Coupled Peptides:

[0303] After synthesis, the resin was washed with DCM (4x), and the peptidyl resin was treated with a suitable TFA-based cleavage mixture such as TFA / TIPS / DTT / H2O (20 ml at 90 / 4 / 3 / 3, 0.15 mmol scale). The cleavage reaction was generally carried out at room temperature for 1 to 3 hours (e.g., 2.5 hours). Following the cleavage reaction, the mixture was filtered (using a suitable filter: e.g., P2 sintered glass filter). The unpurified peptide was precipitated from the filtrate by adding cold (e.g., 5°C) diethyl ether (e.g., 40 ml diethyl ether per 10 ml of filtrate). The precipitate was further washed with cold diethyl ether and, after a short drying period (e.g., 1 hour), dissolved in a suitable aqueous mixture such as a mixture of water, acetic acid, and acetonitrile (45 / 45 / 10), and filtered before purification by reverse-phase preparative HPLC (Waters Deltaprep 4000) using a suitable C18 preparative-HPLC column (e.g., CSH Prep C18, 5 µm, OBD, 30x150 mm selected). Elution was performed using an increasing MeCN gradient in water containing 0.1% TFA. The relevant fractions were analyzed by analytical UPLC and LC-MS. The fractions containing the pure target peptide were pooled and lyophilized.

[0304] If optional or if further purification is required, the isolated unpurified peptide or freeze-dried peptide TFA salt as described above was dissolved in a neutral (e.g., pH 7–8) aqueous buffer based on a common buffer salt (e.g., sodium hydrogen phosphate, but not limited thereto) and purified by reverse-phase preparative HPLC (Waters Deltaprep 4000) on a C18 column. Elution was performed with a progressively increasing gradient of MeCN in aqueous ammonium bicarbonate (0–10 g / l). The relevant fractions were analyzed by analytical UPLC. The fractions containing the pure target peptide were pooled and freeze-dried.

[0305] Optionally, the peptide TFA salt was converted into its sodium salt by a method known in the art, such as size exclusion chromatography, or reverse-phase chromatography methods such as C18 silica cartridges or RP-HPLC methods.

[0306] General methods for LCMS characteristic analysis

[0307] LCMS was performed in an environment consisting of a Waters Acquity UPLC H class system and a Waters Xevo G2-XS QTof. Eluent: A: 0.1% formic acid in MQ water; B: 0.1% formic acid in MeCN.

[0308] An appropriate volume of sample (e.g., 1.0 μl) was injected onto the column at a column temperature of 60°C, and analysis was performed by eluting with gradients A and B. The UPLC conditions, detector settings, and mass spectrometer settings were as follows: Column: Waters Acquity BEH, C-18, 1.7 μm, 2.1 mm x 50 mm. Gradient: Linear gradient of 5%–95% B for 4.0 min at a rate of 0.4 ml / min. Detection: Ionization method: ES, Scanning range: 50–4000 amu, Operating mode: MS, Resolution mode: Positive / Negative: Positive mode, Voltage: Capillary 3.00 kV, Sample cone 40 V, Source 80 V.

[0309] Temperature: Source 150℃, Desolvent 350℃, Scan time 0.500 sec, Inter-scan delay: 0.014 sec.

[0310] Example 1: Synthesis of a compound

[0311] The derivatives and reference compounds of the present invention were synthesized according to the general manufacturing method as described above.

[0312] Compound 1

[0313] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Imp7,Trp8,Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 1)

[0314]

[0315] C 201 H 306 N 48 O 61

[0316] Calculated molecular weight (average): 4370.87 g / mol

[0317] Single isotope mass: 4368.23 g / mol

[0318] LCMS: Measurement (M + 4H)4+ 1093.07 (single isotope)

[0319] Compound 2

[0320] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Imp7,Gly8,Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 2)

[0321]

[0322] C 192 H 299 N 47 O 61

[0323] Calculated molecular weight (average): 4241.71 g / mol

[0324] Single isotope mass: 4239.17 Da

[0325] LCMS: Measured value (M + 4H)4+ 1060.79 (single isotope)

[0326] Compound 3 ( 기준 )

[0327] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Imp7,Aib8,Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 3)

[0328]

[0329] C 194 H 303 N 47 O 61

[0330] Calculated molecular weight (average): 4269.76 g / mol

[0331] Single isotope mass: 4267.21 Da

[0332] LCMS: Measured value (M + 4H)4+ 1067.80 (single isotope)

[0333] Compound 4 (Criteria, see WO2009 / 030738 Example 29)

[0334] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Imp7,Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 4)

[0335]

[0336] C 193 H 301 N 47 O 61

[0337] Calculated molecular weight (average): 4255.73 g / mol

[0338] Single isotope mass: 4253.19 Da

[0339] LCMS: Measurement (M + 4H)4+ 1064.30 (single isotope)

[0340] Compound 5 ( 기준 )

[0341] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Trp8,Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 5)

[0342]

[0343] C 201 H 307 N 49 O 61

[0344] Calculated molecular weight (average): 4385.88 g / mol

[0345] Single isotope mass: 4383.24 Da

[0346] LCMS: Measurement (M + 4H)4+ 1096.81 (single isotope)

[0347] Compound 6 ( 기준 )

[0348] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Gly8,Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 6)

[0349]

[0350] C 192 H 300 N 48 O 61

[0351] Calculated molecular weight (average): 4256.72 g / mol

[0352] Single isotope mass: 4254.18 Da

[0353] LCMS: Measured value (M + 4H)4+ 1064.54 (single isotope)

[0354] Compound 7 (Criteria, see WO2009 / 030738 Example 27)

[0355] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib8,Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 7)

[0356]

[0357] C 194 H 304 N 48 O 61

[0358] Calculated molecular weight (average): 4284.77 g / mol

[0359] Single isotope mass: 4282.22 Da

[0360] LCMS: Measured value (M + 4H)4+ 1071.55 (single isotope)

[0361] Compound 8 ( 기준)

[0362] N{epsilon-37}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu22,Arg26,Arg34,Lys37]-GLP-1(7-37)(Sequence No. 8)

[0363]

[0364] C 193 H 302 N 48 O 61

[0365] Calculated molecular weight (average): 4270.75 g / mol

[0366] Single isotope mass: 4268.20 Da

[0367] LCMS: Measurement (M + 4H)4+ 1068.06 (single isotope)

[0368] Compound 9 ( 기준 )

[0369] [Imp7,Trp8,Glu22,Arg26,Arg34]-GLP-1(7-37)(Sequence No. 9)

[0370]

[0371] C 162 H 236 N 44 O 49

[0372] Calculated molecular weight (average): 3583.87 g / mol

[0373] Single isotope mass: 3581.73 Da

[0374] LCMS: Measurement (M + 4H)4+ 896.44 (single isotope)

[0375] Compound 10 ( 기준)

[0376] [Aib8,Arg34]-GLP-1(7-37)(Sequence No. 10)

[0377]

[0378] C 152 H 230 N 42 O 47

[0379] Calculated molecular weight (average): 3397.71 g / mol

[0380] Single isotope mass: 3395.69 Da

[0381] LCMS: Measurement (M + 4H)4+ 849.93 (single isotope)

[0382] Compound 11 ( 기준 )

[0383] hGLP-1(7-37)(Sequence No. 11)

[0384]

[0385] Compound 12 ( 기준 )

[0386] Semaglutide (Sequence No. 12) (see, for example, literature [J. Med. Chem. 2015, 58, 18, 7370-7380]); can alternatively be prepared as described in WO2006 / 097537, Example 4.

[0387]

[0388] C 187 H 291 N 45 O 59

[0389] Calculated molecular weight (average): 4113.58 g / mol

[0390] Single isotope mass: 4111.12 Da

[0391] LCMS: Measured value (M + 4H)4+ 1028.79 (single isotope)

[0392] Compound 13 ( 기준 )

[0393] N{epsilon-26}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Imp7,Aib8,Arg34]-GLP-1(7-37)(Sequence No. 13)

[0394]

[0395] C 187 H 290 N 44 O 59

[0396] Calculated molecular weight (average): 4098.56 g / mol

[0397] Single isotope mass: 4096.10 Da

[0398] LCMS: Measurement (M + 4H)4+ 1025.03 (single isotope)

[0399] Compound 14 ( 기준, refer to WO2017 / 149070 Example 2)

[0400] N{epsilon-26}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Trp8,Arg34]-GLP-1(7-37)(Sequence No. 14)

[0401]

[0402] C 194 H 294 N 46 O 59

[0403] Calculated molecular weight (average): 4214.68 g / mol

[0404] Single isotope mass: 4212.14 Da

[0405] LCMS: Measurement (M + 4H)4+ 1054.03 (single isotope)

[0406] Compound 15 ( 기준)

[0407] N{epsilon-26}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Imp7,Trp8,Arg34]-GLP-1(7-37)(Sequence No. 15)

[0408]

[0409] C 194 H 293 N 45 O 59

[0410] Calculated molecular weight (average): 4199.67 g / mol

[0411] Single isotope mass: 4197.13 Da

[0412] LCMS: Measurement (M + 4H)4+ 1050.29 (single isotope)

[0413] Compound 16 ( 기준)

[0414] Echnoglutide (SEQ No. 16) (see, e.g., reference [Mol Metab, 2023, 75, 101762])

[0415]

[0416] C 194 H 304 N 48 O 61

[0417] Calculated molecular weight (average): 4284.77 g / mol

[0418] Single isotope mass: 4282.22 Da

[0419] LCMS: Measurement (M + 4H)4+ 1071.58 (single isotope)

[0420] Compound 17 (Reference, WO2019 / 211451 Example 1 Compound 31)

[0421] N{1}-acetyl,N{epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butanoyl]amino]butanoyl]amino]butanoyl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31) amide(SEQ No. 17)

[0422]

[0423] Calculated molecular weight (average): 4551.23 g / mol

[0424] LCMS: Measured value (M + 4H)4 + 1138.12

[0425] Compound 18 ( 기준, refer to WO2017 / 149070 Example 10)

[0426] N{epsilon-37}-[(2S)-2,6-bis[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-Trp8,Glu22, Arg26, Arg34, Lys37]-GLP-1(7-37)(Sequence No. 22)

[0427]

[0428] Calculated molecular weight (average): 6725.67 g / mol

[0429] Single isotope mass: 6721.62 Da

[0430] LCMS: Measurement (M + 5H)5+ 1345.34 (single isotope)

[0431] Example 2: In vitro efficacy (hGLP-1R cAMP production)

[0432] The purpose of these studies is to determine the absolute and relative efficacy of Compound 1 and semaglutide in inducing cAMP accumulation by activating human GLP-1R. This is evaluated by a cell-based assay, in which engineered BHK-21 cells respond to cAMP accumulation by expressing the firefly luciferase reporter gene. This luciferase generates luminescence readings proportional to the amount of intracellular cAMP upon the addition of a cell-permeable substrate. After exposing cells to serial dilutions of the relevant test compounds, EC₀ from the concentration-response curve 50 The value is calculated to evaluate 'efficacy,' which corresponds to the concentration of the test compound required to produce half of the maximum possible response in the test.

[0433] Cell culture.The cell line used for this assay (BHK-FCW467-12A KZ10-1 hGLP-1R CRE-luc) is derived from a BHK-21 parental cell line stably transfected with hGLP-1R and cAMP-responsive factor (CRE)-luciferase reporter genes. Cells were grown in DMEM (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% penicillin / streptomycin (Gibco), 1x sodium pyruvate (Gibco), 0.5 mg / ml G418 (Gibco), and 240 nM methotrexate (Pfizer). Cells were maintained at 37°C in a humidified incubator. After harvesting, cells were prepared as assay preparation aliquots in a medium containing 20% ​​FBS and 10% DMSO for cryopreservation and frozen at -80°C. After freezing, the cells were transferred to liquid nitrogen for long-term storage.

[0434] Luciferi test. TestThe compound was serially diluted to three-fold the final concentration in assay buffer (DMEM (no phenol red), Glutamax, HEPES 1M, Pluronic F-68, 10% Ovalbumin (OVA)) using a Biomek liquid handler. 10 μl of assay buffer was added to each well of a 384w plate containing 0% or 3% human serum albumin (HSA), followed by 10 μl of the diluted compound, and finally 10 μl of thawed, once-washed assay-ready cells at a final concentration of 1500 cells / well. The final concentration of HSA was 0% or 1%. The plates were spun briefly and incubated in a humidified incubator at 37°C for 3 hours. After incubation, the cells were equilibrated at room temperature for 5 minutes, and then 30 μl / well of Steadylite Plus (luciferase substrate) was added. The plate was covered with foil and shaken at room temperature for 30 minutes. Then, the plate was sealed with a TopSeal film, and the signal was read using a microplate reader (Biotek Synergy2) capable of detecting luminescence. Efficacy (EC 50 The ) values ​​were derived from three-parameter concentration-response curves fitted in Graphpad prism. The results are shown in Table 1 below.

[0435]

[0436] The data in Table 1 present the absolute and relative efficacy of Compound 1 of the present invention compared with the known GLP-1 agonist semaglutide. All compounds appear to activate GLP-1R and induce a cAMP response.

[0437] Example 3: In vitro efficacy (hGIPR cAMP production)

[0438] The purpose of these studies is to determine the relative efficacy of compound 1 in inducing cAMP accumulation by activating human GIP compared to a known human GLP-1 agonist (compound 17), and to show that compound 1 is a selective GLP-1 receptor agonist.

[0439] method: The assay is performed as described above for hGLP-1R in Example 2, but cells expressing human GIPR are used instead of hGLP-1R. The results are shown in Table 2 below.

[0440]

[0441] The data suggest that compound 1 does not activate hGIPR and is selective for the GLP-1 receptor compared to the GIP receptor.

[0442] Example 4: In vitro hGLP-1R beta-arrestin 1+2

[0443] The purpose of these studies is to determine the ability of Compound 1, Compound 2, Semaglutide, various other GLP-1 receptor agonists, and Ecnoglutide to induce the recruitment of β-arrestin 1 or β-arrestin 2 to GLP-1R in HEK293 cells. This is performed in engineered cell lines expressing split nano-luciferases, where a Large BiT (LgBiT, 18 kDa) component is fused to the C-terminus of GLP-1R and a Small BiT (SmBiT, 11 amino acids) is fused to one of the two isoforms of β-arrestin. When GLP-1R is activated, β-arrestin is translocated to the receptor, and subsequently, the binding of LgBiT and SmBiT forms a functional luciferase whose activity can be detected by the addition of a substrate.

[0444] Cell culture:Cells were grown in DMEM (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% penicillin / streptomycin (Gibco), 1x sodium pyruvate (Gibco), 0.5 mg / ml G418 (Gibco), and 240 nM methotrexate (Pfizer). Cells were maintained at 37°C in a humidified incubator.

[0445] Experimental Procedure: When approximately 80% confluency was reached, cells were harvested using trypsin and resuspended in standard cell medium at a concentration of 250,000 cells / ml. 100 μl of the cell suspension was added to each well of a PDL-coated white opaque 96-well microplate to achieve a final density of 25,000 cells / well. The plates were incubated in a humidified incubator at 37°C for 2 days. On the day of the experiment, the plates were washed once with PBS and incubated at 37°C for 1 hour before adding 70 μl of phenol red-free DMEM + 1% egg albumin (test buffer). The compound was serially diluted to a final concentration of 10x in the test buffer. The Nano-Glo substrate (Promega) was diluted 100x in calibration buffer, and 20 µl was added to each well. The plate was then equilibrated in a microplate reader preheated to 37°C for 10 minutes (BMG Clariostar). Luminescence was read across the plate in kinetic mode, with values ​​per well taken every 115 seconds. After 5 cycles of baseline readings, the experiment was paused, and 10 µl of the compound was added twice using a multi-channel pipette. Luminescence was read for an additional 15 cycles. For the concentration-response curves, the area under the curve was calculated for each well after normalizing to the individual baseline luminescence and subtracting the average vehicle-treated control. E was determined as the top of the fitted concentration-response curve. max E of hGLP-1(7-37) maxIt was normalized to , and this was performed on all experimental plates as a common reference compound.

[0446]

[0447] The data show that compounds 1 and 2 of the present invention, compared to all other tested compounds, for example, even when compared to compounds 5 and 6 which differ from compounds 1 and 2 only in having Imp instead of His at the N-terminus, or compound 18 which differs from compound 1 in having His at the N-terminus and a different substituent on the C-terminal Lys, exhibit significantly reduced efficacy (E) in the beta-arrestin assay for both isoforms 1 and 2. max It is proven that it has ). The concentration-response curve of Compound 1 compared with semaglutide (Compound 12) and hGLP-(7-37) (Compound 11) is shown in Fig. 1.

[0448] In addition, compound 18 (different in that it has His at the N-terminus and a different substituent at the C-terminus Lys) and compound 15 (different mainly in that it has Imp at the N-terminus and substituents at different positions) have significantly worse efficacy in beta-arrestin recruitment (EC) compared to, for example, compounds 1 and 2 of the present invention. 50 It appears to have ).

[0449] Example 5: Pharmacokinetic study in minipigs

[0450] The purpose of this study was to determine the in vivo half-life of Compound 1 after intravenous administration to minipigs and to compare it with semaglutide, that is, to determine the extension of the residence time in the body and the resulting duration of action. This was performed in a pharmacokinetic (PK) study using Gottingen minipigs, where the terminal half-life of the compound was determined. The terminal half-life generally refers to the time it takes for a specific plasma concentration to be reduced by half, and is measured from the initial distribution phase.

[0451] Animals and breeding. Female Göttingen minipigs were obtained from Ellegaard Minipigs in Dalmos, Denmark. The minipigs were housed individually and fed a restricted diet of Altromin 9033 (Brogaarden ApS, Lynge, Denmark) twice daily. After at least two weeks of acclimatization, two permanent central venous catheters were implanted in either the caudal or cephalic vena cava of each animal under general anesthesia. The animals underwent a recovery period of approximately one week after surgery and were subsequently used in repeated pharmacokinetic studies with appropriate rest periods between consecutive administration episodes.

[0452] weight. The animals were weighed weekly, on the day of administration or the day before administration, to determine the exact dosage. The weight of the minipig was approximately 25 kg on the day of administration.

[0453] Formulation: The following formulations were used in these PK studies: Compound 1 was formulated with 8 mM phosphate, 250 mM glycerol, 0.007% polysorbate 20, pH 7.4; and semaglutide was formulated with 8 mM phosphate, 184 mM propylene glycol, 58 mM phenol, pH 7.4.

[0454] Administration of peptide and administration solution.There were no dietary restrictions during the study period, and animals had free access to water throughout the entire study period. The compounds were injected intravenously through a short central venous catheter and perfused with at least 10 ml of sterile saline after administration. Compound 1 was administered at 1.5 nmol / kg using a dosage volume of 0.1 ml / kg, and semaglutide was administered at 2 nmol / kg using a dosage volume of 0.046 ml / kg (n=3). In relation to administration, 1 to 1.3 ml of blood samples were collected via a long central venous catheter at the following time points: before administration, 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 24, 30, 48, 72, 96, 144, 168, 192, 216, 240, 264, 312, 336, 360, 384, 408, 432, and 480 hours (only at the 3-hour time point for semaglutide; only at the 336–480-hour time points for Compound 1). After each blood sample was collected, the catheter was perfused with at least 5 to 10 ml of sterile 0.9% NaCl containing 10 IE / ml heparin. Aseptic technique was required to prevent bacterial growth within the catheter; otherwise, the risk of thrombus formation within the catheter increases. Immediately after sampling, the blood was transferred to a test tube containing K3EDTA buffer (8 mM). The tube was placed on wet ice until centrifugation was performed for 10 minutes at 4°C and approximately 2000 g within ½ hour of sampling. Subsequently, plasma (at least 200 μl) was transferred to a micronic tube on dry ice and maintained at -20°C until analysis. The plasma sample was analyzed by LC / MS as described below.

[0455] Quantitative test for plasma samples.

[0456] The plasma concentration of Compound 1 was validated by plasma protein precipitation and analyzed by turboflow liquid chromatography-mass spectrometry (TF-LC-MS). A calibrator was prepared by spiked Compound 1 in the range of 0.2 to 200 nM into blank minipig plasma. Calibrators, quality control (QCs), blank plasma, and study samples for TF-LC-MS analysis by protein precipitation were prepared by adding 4 volumes of ethanol containing 20 nM internal standard to 1 volume of sample. After adding the precipitation reagent, the mixture was stirred and then centrifuged. After centrifugation, 1 volume of the supernatant was mixed with 1 volume of water (containing 1% v / v formic acid).

[0457] The mixture was analyzed by TF-LC-MS using a Cyclone turboflow column (0.5 x 50 mm, ThermoFisher Scientific) and an XBridge Peptide BEH C18 3.5 µm 300 Å analytical column (50 x 2.1 mm, obtained from Waters). Gradient elution was performed using mobile phase A (composed of milli-Q water containing 1% formic acid and 5% methanol / acetonitrile (50 / 50)) and mobile phase B (composed of methanol / acetonitrile (50 / 50) containing 1% formic acid and 5% milli-Q water). The mass spectrometer was operated in cationization mode. In Selected Reaction Monitoring (SRM) mode, a TSQ Altis mass spectrometer (ThermoFisher Scientific) was used as the detector (Compound 1: m / z 1093.55 ® 1354.72 and ISTD: m / z 1100.38 ® 1363.97). A linear calibration curve (weighted by 1 / x2) was used to calculate concentrations in plasma samples. Quality control samples were included. The acceptance criteria were as follows: the deviation between the nominal and calculated concentrations in the calibrator and quality control samples was less than 15% for 75% of all standards and quality control samples.

[0458] Plasma concentrations of semaglutide were determined by plasma protein precipitation and analyzed by turboflow liquid chromatography-mass spectrometry (TF-LC-MS). Calibrators were prepared by spiked donor minipig plasma with semaglutide concentrations ranging from 0.5 to 2000 nM. Calibrators, quality control (QCs), donor plasma, and study samples for TF-LC-MS analysis by protein precipitation were prepared by adding 4 volumes of methanol containing 20 nM of internal standard to 1 volume of plasma. After adding the precipitation reagents, the mixture was stirred and then centrifuged. After centrifugation, 1 volume of the supernatant was mixed with 1 volume of water (containing 1% v / v formic acid).

[0459] The mixture was analyzed by TF-LC-MS using a Cyclone turboflow column (0.5 x 50 mm, Thermo Fisher Scientific) and an XBridge Protein BEH C4 analysis column (50 x 2.1 mm, 3.5 μm, 300 Å, obtained from Waters). Gradient elution was performed using mobile phase A (composed of milli-Q water containing 1% formic acid and 5% methanol / acetonitrile (50 / 50)) and mobile phase B (composed of methanol / acetonitrile (50 / 50) containing 1% formic acid and 5% milli-Q water). The mass spectrometer was operated in cationization mode. A Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) was used as the detector in selected ion monitoring modes (semaglutide: 1028–1032 m / z and ISTD: 1295–1305 m / z). A linear calibration curve with a weight of 1 / x2 was used to calculate the concentration in plasma samples. The acceptance criteria were as follows: the deviation between the nominal concentration and the calculated concentration in the calibrator and quality control samples was less than 15% for 75% of all standards and quality control samples.

[0460] Data Management.Generate individual pharmacokinetic profiles of plasma concentration versus time data and Phoenix ® WinNonlin ® It was evaluated by non-compartment pharmacokinetic analysis using version 8.4 (Certara LP, Princeton, NJ, USA). The terminal elimination phase was fitted via linear regression using uniform weights. Nominal sampling time and actual dose were used in the analysis. The terminal half-lives are given in Table 4 as the harmonic mean, minimum, and maximum values.

[0461] result.

[0462]

[0463] The above results show that Compound 1 of the present invention has a half-life approximately twice that of semaglutide, which indicates that the reduction in beta-arrestin activation is achieved through a reduction in receptor-mediated clearance. It potentially indicates that it extends the half-life. Based on the half-life measured in minipigs, it is predicted that the half-life in humans will be sufficient with administration at least once a week via liquid injection. Similar PK data after subcutaneous administration were generated in mice, which show similar relative differences in half-life.

[0464] Example 6: Physical stability (fiber formation; ThT fluorescence)

[0465] The purpose of this study is to evaluate the physical stability of the compound of the present invention in an aqueous solution using the ThT assay as described below.

[0466] Peptide quantification by a charge aerosol detector (CAD).

[0467] CAD was performed on a Thermo Scientific Vanquish UPLC system equipped with UV-DAD and CAD (charge aerosol detector). The system was fitted with a Waters ACQUITY UPLC, CSH C18 column, 130 Å, 1.7 μm, 2.1 mm x 50 mm, and a column temperature of 40°C. UV detection was performed at 214 nm. Eluent A: 0.1% v / v TFA in water; Eluent B: 0.1% v / v TFA in MeCN. Appropriate volumes of samples were injected onto the column for analysis, and the column eluted a linear gradient of Eluents A and B from 0–95% B over 4 minutes at a constant flow rate of 0.45 mL / min. Run time: 6 min. Calibration was performed using a standard curve generated by injecting the standard solution Insulin Aspart at 3.5 mg / mL.

[0468] Sample manufacturing.

[0469] The freeze-dried peptide was dissolved in water and mixed with a stock solution of phosphate and d-sorbitol to obtain a nominal composition of 3 mg / ml peptide, 8 mM phosphate, and 253 mM d-sorbitol. The pH was measured and adjusted to 7.4 using diluted hydrochloric acid and sodium hydroxide. The sample was sterile filtered (0.22 μm), placed in a glass vial, and stored in a refrigerator until testing.

[0470] ThT test.

[0471] The low physical stability of peptides can lead to the formation of amyloid fibrils, which are observed in samples as well-aligned, spiral-shaped macromolecular structures, potentially resulting in gel formation. This has traditionally been measured by visual inspection of the samples. However, this type of measurement is highly subjective and varies depending on the observer. Therefore, applying small molecule marker probes is much more advantageous. Thiofluvin T (ThT) is such a probe and exhibits a distinct fluorescent signature when binding to fibrils (see literature [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 09, 274-284]).

[0472] Thioflavin T was added to the supernatant samples prepared as described above using an aqueous ThT stock solution to achieve a final concentration of approximately 1 μM. 150 μl of sample aliquots were placed into 96-well micro-titer plates (Packard OptiPlate-96, white polystyrene). Three to four copies of each sample were applied to the plates. The plates were sealed, placed in a fluorescence plate reader (Fluoroskan Ascent FL), and incubated at 37°C with orbital shaking (900 rpm; 1 mm amplitude). Fluorescence measurements were performed every 20 minutes using excitation via a 444 nm filter and emission measurements via a 485 nm filter. Between each measurement, the plates were shaken and heated as described above, and the assay was terminated after 45 hours. Fluorescence measurements for each micro-titer plate well were plotted against time, and the latency (time until an increase in ThT fluorescence was observed) was estimated. If no increase in ThT fluorescence was observed, the delay time was set to be equal to the duration of the test (45 hours). To evaluate the loss of dissolved peptides after 45 hours of incubation, the peptide recovery rate was measured as the ratio between the supernatant peptide concentration after incubation and before incubation using the CAD quantification method as described above.

[0473] Samples of compound 1 and semaglutide were prepared as described above, and ThT tests were performed as described above.

[0474] result.

[0475]

[0476] The results showed no increase in ThT fluorescence over the duration of the test (45 hours) and no loss of dissolved peptide (recovery rate ≥ 100%), suggesting high stability of compound 1 for microfibrillation.

[0477] Example 7: Chemical stability (HMWP formation; loss of purity)

[0478] The purpose of this study is to determine the chemical stability of Compound 1. As a measure of the stability of Compound 1, HMWP formation (HMWP(%)) as a function of time was analyzed by size-exclusion chromatography (SEC-MS). In addition, the loss of purity of Compound 1 was measured by RP-UPLC.

[0479] Incubation.

[0480] The sample prepared as "sample preparation" in Example 7 was incubated at 37°C, and after 2 and 4 weeks of incubation, aliquots were pooled and compared with the corresponding samples pooled before incubation. The aliquots were stored at -18°C until high molecular weight protein (HMWP) and purity were measured.

[0481] HMWP (High Molecular Weight Protein)

[0482] The relative amount of covalent HMWP was evaluated using a SEC-UPLC equipped with a Waters BEH125Å insulin column (4.6 x 150 mm, 1.7 μm) under isosolvent eluent conditions (0.3 M NaCl, 10 mM NaH2PO4, and 5 mM H3PO4, 50% (v / v) isopropanol, pH 2.4), flow rate 0.3 mL / min, column temperature 50°C, and UV detection at 215 nm. The total area of ​​the peak eluted prior to the monomer main peak was defined as HMWP (high molecular weight protein) and quantified as a percentage of the total peptide peak area. To facilitate comparison, the results were normalized by subtracting the amount of HMWP from the corresponding pooled sample prior to incubation. The results are shown in Table 6.

[0483] Loss of purity

[0484] Relative peptide purity was evaluated by RP-UPLC (Acquity, Waters Corp, Milford, MA, USA) using a BEH C4 column (2.1 x 150 mm, Waters Corp, Milford, MA, USA) with a flow rate of 0.1 ml / min, a column temperature of 60°C, and a linear gradient of acetonitrile in the trifluoroacetic acid eluent (A: 0.1% TFA, B: 0.08% TFA in ACN; 25–50% B within 55 min) at UV detection at 215 nm. Purity was calculated as the total area of ​​the monomer main peaks as a percentage of the total peptide peak area. To facilitate comparison, the purity loss was calculated below by normalizing the results by subtracting the purity of the pooled samples before incubation. The results are shown in Table 6.

[0485] result:

[0486]

[0487] As can be seen in Table 6, only a slight increase in HMWP formation was observed for both Compound 1 and Semaglutide. However, a lower loss of purity was observed for Compound 1 compared to Semaglutide.

[0488] Although specific features of the invention have been illustrated and described herein, many variations, substitutions, modifications, and equivalents will now occur to those skilled in the art. Accordingly, it should be understood that the appended claims are intended to include all such modifications and modifications that fall within the true spirit of the invention.

Claims

Claim 1 A GLP-1 derivative comprising a GLP-1 analog of Formula I (SEVENTEEN NO. 18): Imp-X8EGTFTSDVSSYLEEQAAREFIAWLVRGRK (Formula I), wherein Imp is 3-(imidazole-4-yl)-propionyl, X8 is W or G, and K at a position corresponding to position 37 of GLP-1 (7-37) is an epsilon amino group chemically modified to a substituent of Formula III: HOOC-(CH2) c -CO-(NHC(COOH)CH2CH2CO) b -(NHCH2CH2OCH2CH2OCH2CO) a -(Formula III), wherein a is 1 to 3, b is 1 to 2, and c is 14 to 18, and the C-terminal amino acid is optionally a C-terminal amide or a pharmaceutically acceptable salt thereof, a GLP-1 derivative. Claim 2 In claim 1, the GLP-1 analog of Formula I (SEQ No. 18) comprises: Imp-X8EGTFTSDVSSYLEEQAAREFIAWLVRGRK (Formula I), wherein Imp is 3-(imidazole-4-yl)-propionyl, X8 is W or G, and K at a position corresponding to position 37 of GLP-1 (7-37) is chemically modified into an epsilon amino group of Formula III as a substituent: HOOC-(CH2) c -CO-(NHC(COOH)CH2CH2CO) b -(NHCH2CH2OCH2CH2OCH2CO) a -(Formula III), wherein a is 1 to 3, b is 1 to 2, and c is 14 to 18, and the C-terminal amino acid is optionally a C-terminal amide or a pharmaceutically acceptable salt thereof, a GLP-1 derivative. Claim 3 In paragraph 1, X8 is a GLP-1 derivative, which is W. Claim 4 In paragraph 1, X8 is a GLP-1 derivative, which is G. Claim 5 In paragraph 1, a is a GLP-1 derivative of 2. Claim 6 In paragraph 1, b is a GLP-1 derivative, which is 1. Claim 7 In paragraph 1, c is a GLP-1 derivative, 16. Claim 8 In claim 1, the GLP-1 derivative: HOOC-(CH2) wherein the substituent is of formula IIIa 16 -CO-(NHC(COOH)CH2CH2CO)-(NHCH2CH2OCH2CH2OCH2CO)2- (Formula IIIa). Claim 9 In claim 1, the above substituent is a GLP-1 derivative having the chemical formula 1: . Claim 10 Compound 1: , or a GLP-1 derivative that is a pharmaceutically acceptable salt thereof. Claim 11 Compound 2: , or a GLP-1 derivative that is a pharmaceutically acceptable salt thereof. Claim 12 A pharmaceutical composition for use in preventing or treating overweight or obesity, comprising a GLP-1 derivative according to any one of claims 1 to 11 and at least one pharmaceutically acceptable excipient. Claim 13 In paragraph 12, the above GLP-1 derivative is compound 1: A pharmaceutical composition that is a pharmaceutically acceptable salt thereof. Claim 14 delete Claim 15 delete

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