Patent Application
20240247080
The present invention relates to hybrid polypeptides that agonize the GIP, GLP-1 and neuropeptide Y2 (NPY2) receptors and to their medical use in the treatment of a variety of diseases, conditions or disorders, such as obesity, diabetes, and/or NASH.
Overweight and obesity are defined as abnormal or excessive fat accumulation that presents a risk to health. A body mass index (BMI) over 27 kg/m2 is considered as overweight, and a BMI over 30 kg/m2 is considered as obese. The BMI is calculated based on body weight and height. Obesity has in the past 50 years reached pandemic levels. Worldwide obesity has nearly tripled since 1975. In 2016, more than 1.9 billion adults and more than 340 million children and adolescents were overweight or obese. Both overweight and obesity are a major risk factor for a number of chronic diseases, including type 2 diabetes, cardiovascular diseases and cancer which are the leading causes of morbidity and death in the United States. Obesity is thus a serious condition and associated with poorer mental health outcomes, reduced quality of life, and contributes to a decline in life expectancy (Abdelaal 2017). According to the WHO overweight, and obesity are no longer considered a problem limited to high income countries but are now dramatically on the rise in low- and middle-income countries. WHO's Global Health Observatory indicate that, in 2016, 39% of women or men aged 18 and over were overweight and 11% of men and 15% of women were obese.
Peptide YY (PYY) is a 36-amino acid peptide with the sequence YPIKPEAPREDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 308), found in endocrine L cells in the mucosa of the gastrointestinal tract, especially in the ileum and colon. PYY belongs to the pancreatic polypeptide (PP) family together with neuropeptide Y (NPY) and pancreatic polypeptide (PP). This family of peptides act upon the NPY receptors designated NPY1R (Y1), NPY2R (Y2), NPY4R (Y4), and NPY5R (Y5). The receptor family belongs to a class of G protein-coupled receptors (GPCRs) that are expressed in the CNS, especially in regions of the hypothalamus. The receptors NPY1R-NPY5R exhibit both anorectic (NPY2R, NPY4R) and orexigenic effects (NPY5R, NPY1R). The two major forms of peptide YY are PYY1-36 and PYY3-36. PYY1-36 is released postprandially from intestinal L cells in proportion to energy intake and in part truncated to PYY3-36, which is the main circulating form of PYY and a relatively selective Y2 receptor agonist. PYY1-36 and PYY3-36 inhibit gastric acid secretion, gastrointestinal transit and food intake. Food intake is inhibited both via a stimulant effect on Y2 receptors on vagal afferent neurons and an interaction with Y2 receptors in the hypothalamus, which is consistent with the ability of PYY to gain access to the brain via circumventricular organs such as the area postrema and subfornical organ. Furthermore, it is known that PYY concentrations in the blood of people with obesity are lower than those of healthy individuals. Thus, NPY2R and/or NPY4R agonists may hold potential in the treatment of obesity.
Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), belong to the family of incretins. GLP-1(7-37) is a 31-amino acid peptide, with the sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 309) and GIP is a 42-amino acid peptide, with the sequence YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ (SEQ ID NO: 310). GLP-1 and GIP are secreted from small intestinal L cells and K cells, respectively. GLP-1 acts via GLP-1 receptors and is known to have a sugar-dependent insulinotropic action (i.e. stimulate insulin release) and a feeding suppressive action. GIP acts via GIP receptors and is also known to have a sugar-dependent insulinotropic action, though its influence on feeding is less clear. GLP-1 receptor/GIP receptor coagonist peptide has been reported to show a stronger hypoglycemic action and body weight-lowering action than those of a GLP-1 receptor agonist alone. Therefore, research efforts have been made to develop GLP-1/GIP receptor co-agonists for the treatment of obesity and/or diabetes, based on the structure of natural glucagon, GIP, or GLP-1.
The most noteworthy effects of GLP-1 agonists are their ability to promote insulin secretion in a glucose-dependent manner by binding to GLP-1 receptors expressed on the pancreatic p cells. Almost as importantly, GLP-1 agonists have been shown to inhibit glucagon secretion at glucose levels above fasting levels. Critically, this does not affect the glucagon response to hypoglycemia, making GLP-1 agonists a safe anti-diabetic drug with very low incidence of hypoglycemia compared to insulin. In June 2021, Semaglutide, a GLP-1 agonist, was approved by the FDA for chronic weight management in adults with obesity or overweight with at least one weight-related condition (such as high blood pressure, type 2 diabetes, or high cholesterol). As an anti-obesity drug, GLP-1 agonists work by binding to GLP-1 receptors in hypothalamus thereby supressing appetite. Furthermore, GLP-1 agonists bind to GLP-1 receptors in the stomach, inhibiting gastric emptying, acid secretion, and motility, which collectively promote satiety. Consequently, diabetic subjects treated with GLP-1 receptor agonists often also experience a beneficial weight loss in addition to a control of their blood sugar levels.
However, despite long-standing efforts, the number of overweight and obese patients is still growing. First line therapy for overweight and obese patients comprises diet and exercise but often are not sufficiently efficacious. Second line treatment options are bariatric surgery and pharmacotherapy. Available pharmacological treatments, seem to lack in efficacy and/or safety, and only a limited number of approved therapies, such as Semaglutide, are available in the US and in Europe. Therefore, there is still a high medical need for more efficacious and safe treatment options. Future obesity treatments may benefit from targeting GLP1-R, GIPR and NPY2R simultaneously in view of the biological role of PYY, GIP, or GLP-1. Indeed, recent research efforts in the obesity field have aimed at developing hybrid polypeptides as triple agonists for these receptors (see e.g. EP3467106 A1). Hybrid polypeptides are highly desirable compared to combination therapy with individual polypeptides for a number of reasons. First, hybrid polypeptides are easier to formulate into a single dosing unit as compared to mixtures of different polypeptides. This is primarily because different peptides may possess different physiochemical properties, e.g., isoelectric point, solubility, or chemical stability at a given pH, which means that one formulation developed for one polypeptide may be less optimal for or incompatible with a different polypeptide. Therefore, combination therapy with individual polypeptides may require individual dosing units. Secondly, hybrid polypeptides are cheaper to manufacture compared to individual polypeptides and also reduce the burden of regulatory approval.
The present invention set out to provide hybrid polypeptides that agonize all of the receptors GLP1-R, GIPR and NPY2R, in order to provide improved treatments for e.g. obesity, diabetes, and/or metabolic syndrome.
Further, it is an aim of the present invention to provide triple agonists that are soluble at or around physiological pH (e.g pH 7).
A further aim of the present invention is to provide triple agonists that have a long duration of action in the body, i.e. a long in-vivo half life.
A further aim of the present invention is to provide triple agonists that agonize all the receptors GLP1-R, GIPR and NPY2R and are soluble at or around physiological pH (e.g pH 7).
A further aim of the present invention is to provide triple agonists that agonize all the receptors GLP1-R, GIPR and NPY2R, are soluble at or around physiological pH (e.g pH 7) and have a long duration of action in the body.
A further aim of the present invention is to provide triple agonists that show good selectivity for the GLP-1, GIP and NPY2 receptors against other incretin or related receptors. For example, the agonists of the invention might not activate other receptors from the NPY receptor family, such as NPY1, NPY4 or NPY5 receptor, and/or might not activate GLP-2 or glucagon receptors.
In a first aspect, the invention provides a polypeptide according to the general structure of Formula (I) or a pharmaceutically acceptable salt thereof,
In a second aspect, the invention provides a polypeptide comprising the general structure of Formula (I) or a pharmaceutically acceptable salt thereof,
In a third aspect, the invention relates to a polypeptide according to the first or second aspect, for use as a medicament.
In a fourth aspect, the invention relates to a method for the treatment of a disease, disorder and/or condition selected from the list consisting of excessive weight, obesity, type 1 and type 2 diabetes, eating disorders, hyperlipidemia, metabolic syndrome, NAFLD/NASH, and/or cardiovascular diseases, said method comprising administering a therapeutic effective amount of a polypeptide or a pharmaceutically acceptable salt thereof, according to first or second aspect, to an individual in need thereof.
Terms not specifically defined herein should be given the meanings that would be given to them by one of skill in the art in light of the disclosure and the context. As used in the specification, however, unless specified to the contrary, the following terms have the meaning indicated and the following conventions are adhered to.
According to the present invention, unless otherwise stated, the amino acids are all L-amino acids (L-stereoisomer, natural amino acids). In the present context, substitutions in an analogue/derivative may be substitutions to natural amino acids as well as unnatural amino acids, including L- and D-stereoisomers. A substitution in a derivative may be a conservative substitution with a conservative amino acid. The groups of conservative amino acids may be defined as:
Common unnatural amino acids include NMeG, which denotes the amino acid methylglycine (also referred to as N-Methylglycine, NMeGly, MeGly or sarcosine); NMeP, which denotes the amino acid methyl-L-proline (also referred to as N-Methyl-L-proline, NMePro or (S)-1-methylpyrrolidine-2-carboxylic acid); Dpr, which denotes the amino acid (S)-2,3-diaminopropionic acid (also referred to as L-2,3-diaminopropionic acid); Aib, which denotes the amino acid 2-amino-2-methylpropanoic acid (also referred to as 2-aminoisobutyric acid); NMeQ, which denotes the amino acid N-methyl-L-glutamine (also referred to as N-Methylglutamine, NMeGIn, or MeGln); NMeAla, which denotes the amino acid N-methyl-L-alanine (also referred to as N-Methylalanine, NMeA, or MeAla); Cha, which denotes the amino acid (S)-2-amino-3-cyclohexylpropanoic acid (also referred to as L-cyclohexylalanine); Tle, which denotes the amino acid (S)-2-amino-3,3-dimethylbutanoic acid (also referred to as L-2-(tert-butyl)glycine); 1-Nal denotes the amino acid (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (also referred to as 3-(1-napthyl)-L-alanine); Pip, which denotes the amino acid (S)-piperidine-2-carboxylic acid (also referred to as L-pipecolic acid); Nip, which denotes the amino acid (S)-piperidine-3-carboxylic acid (also referred to as L-nipecotic acid); Nle, which denotes the amino acid (2S)-2-aminohexanoic acid (also referred to as L-norleucine); Phe(4F), which denotes the amino acid (S)-2-amino-3-(4-fluorophenyl)propanoic acid (also referred to as 4-fluoro-L-phenylalanine).
According to the present invention, a polypeptide or a derivative thereof may be in the form of a salt. According to the present invention, a polypeptide or a derivative thereof may be in the form of a pharmaceutically acceptable salt. Thus, pharmaceutically acceptable salts are intended to include any salts that are commonly used in formulations of peptides. Such salts include both acid addition salts and basic salts, and examples may be found in e.g., Remington's pharmaceutical sciences, 17th edition. Likewise, a polypeptide or a pharmaceutically acceptable salt may be in the form of a solvate (e.g., a hydrate).
Compounds are represented in the Boehringer Ingelheim Line Notation (BILN), which describes complex peptides in a human-readable format (Fox et al, J. Chem. Inf Model. 2022, 62, 17, 3942-3947,). In BILN, chains are built up of monomers, e.g. A represents alanine, Aib represents isobutyric acid, and two monomers are linked by a hyphen “-”. Different chains are separated by a dot “.” and connections between chains are explicitly defined in brackets following the monomer, which includes the link ID number and the R-group number, e.g. K(1,3) represents a lysine residue that is linked to another monomer carrying the same link ID “1” by its R″3″-group (E-amino group). As an example, Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-E-K-Q-A-Q-Aib-A-F-V-E-W-L-I-K(1,3)-G-G-P—S-S-G-A-S-L-R-H-Y-L-N-W-L-T-R-Q-R—Y-NH2.C20DA-gGlu-gGlu-gGlu-gGlu-gGlu-gGlu-eLys(1,2) (SEQ ID NO: 412), wherein C20DA represents 19-carboxynonadecanoyl, gGlu ([γE]) represents L-y-glutamyl, connected via its amino-group to C20DA and via its y-carboxy-group to the amino-group of the next gGlu, which is repeated 4 times resulting in 6 gGlu residues total, connected via the final y-carboxy-group to the E-amino-group of lysine (eLys), connected via is carboxy-group to the E-amino-group of lysine (K) in the peptide backbone, completely defines the following structure:
In the present context, lipidation refers to the covalent attachment of a lipid (L), such as C18DA (octadecanedioic acid), C20DA (icosanedioic acid) or other half-life extending moieties, to a hybrid polypeptide according to the invention, optionally through a linker/spacer (—(Y—)1-8). A linker/spacer may consist of one or more covalently connected monomers commonly used in the art, such as [γE], [OEG] or [AHX] illustrated below.
Lipidation may be performed at a lysine residue (i.e. at the E (epsilon) amino group) in the polypeptide or at the N-terminal, preferably at a lysine residue as exemplified herein. Without wishing to be bound by any theory, it is thought that such lipophilic substituents bind albumin and other plasma components in the blood stream, thereby shielding the compound of the invention from renal filtration as well as enzymatic degradation. Thus, lipidation is typically performed to improve the pharmacokinetic profile of a polypeptide by e.g. improving metabolic stability, reducing enzymatic degradation, lowering excretion and metabolism, all in all resulting in a prolonged in vivo half-life (t1/2). The polypeptides according to the invention may be lipidated or non-lipidated depending on the desired half-life. If a linker/spacer is present, the lipid (L) is covalently attached to linker/spacer (—(Y—)1-8) in one end and the polypeptide is covalently attached to the linker/spacer (—(Y—)1-8) in the opposite end (i.e. lipid-linker-peptide). Alternatively, when no linker/spacer is present, the lipid (L) is directly covalently attached to polypeptide (i.e. Lipid-polypeptide). The linker/spacer may consist of 1 up to and including 8 covalently connected monomers (i.e. —Y1—; —Y1—Y2—; —Y1—Y2—Y3—; —Y1—Y2—Y3—Y4—; —Y1—Y2—Y3—Y4—Y5—; —Y1-Y2—Y3—Y4—Y5—Y6—; —Y1—Y2—Y3—Y4—Y5—Y6—Y7—; or —Y1—Y2—Y3—Y4—Y5—Y6—Y7—Y8-), wherein each unit is independently selected from e.g. [γE], [OEG] or [AHX], or, alternatively, from e.g. [γE], [E], [OEG], [eLys], or [AHX]. Most preferably, the polypeptide is lipidated with a structure of general formula L-Y1—Y2—Y3—Y4—Y5—Y6—Y7—Y8-, wherein L is a lipid selected from C18DA or C20DA and further wherein each of Y1—Y8 are independently selected from being, not present, [γE], [OEG], [eLys], or [AHX] or, alternatively, from being, not present, [γE], [E], [OEG], [eLys], or [AHX].
The lipid (L) may be attached to the linker/spacer (—(Y—)1-8) via an ester, ether, a sulfonyl ester, a thioester, an amide, an amine, triazole or a sulfonamide, preferably via an amide or ester. Accordingly, it will be understood that preferably the lipid (L) includes an acyl group, a sulfonyl group, an alkyne, an azide, an N atom, an O atom or an S atom, which forms part of the ester, sulfonyl ester, thioester, triazole, amide, amine or sulfonamide. Preferably, an acyl group, or an 0 or N atom in the lipophilic substituent (L) forms part of an amide or ester with the linker/spacer (—(Y—)1-8).
Likewise, the linker/spacer (—(Y—)1-8) (if present) is attached to an amino acid residue of the hybrid polypeptides according to the invention via an ester, a sulfonyl ester, a thioester, an amide, an amine or a sulfonamide. Accordingly, it will be understood that preferably the linker/spacer (if present) includes an acyl group, a sulfonyl group, an N atom, an O atom or an S atom which forms part of the ester, sulfonyl ester, thioester, amide, amine or sulfonamide. Preferably, an acyl group, or an O or N atom in the linker (—(Y—)1-8) forms part of an amide or ester with the amino acid residue.
The lipophilic substituent (L) may comprise a hydrocarbon chain having from 10 to 24 carbon atoms, e.g. from 14 to 22 carbon atoms, e.g. from 16 to 20 carbon atoms. Preferably, it has at least 14 carbon atoms, and preferably has 20 carbon atoms or fewer. For example, the hydrocarbon chain may contain 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The hydrocarbon chain may be linear or branched, and may be saturated or unsaturated. Furthermore, it can include a functional group at the end of the hydrocarbon chain, e.g. a carboxylic acid group, a sulphonic acid group, or a tetrazole group. From the discussion above it will also be understood that the hydrocarbon chain is preferably substituted with a moiety, which forms part of the attachment to an amino acid residue of the hybrid polypeptides according to the invention or to the linker (—(Y—)1-8), for example an acyl group, a sulfonyl group, an N atom, an O atom or an S atom. Most preferably, the hydrocarbon chain is substituted with an acyl group (for the attachment to the linker/spacer), and accordingly the hydrocarbon chain may be part of an alkanoyl group, for example a dodecanoyl, 2-butyloctanoyl, tetradecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl or eicosanoyl group. These hydrocarbon chains substituted with an acyl group at one end may further be functionalized with a carboxylic acid group at the other end of the chain. Examples of functionalized hydrocarbon chains (e.g. lipophilic substituents L) are 15-carboxy-pentadecanoyl (briefly C16DA), 17-carboxy-heptadecanoyl (briefly C18DA) and 19-carboxy-nonadecanoyl (briefly C20DA). Preferred lipid or lipid/linkers in the present disclosure are C18DA, C18DA[E][E][E][E]- (SEQ ID NO: 640), C18DA[E][E][E][E][E]- (SEQ ID NO:636), C18DA[E][E][E][E][E][E]-(SEQ ID NO: 637), C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][γE][γE]-, C18DA[γE][γE][γE][γE]-, C18DA[γE][γE][γE][γE][γE]-, C18DA[γE][γE][γE][γE][γE][γE]-, C18DA[γE][γE][γE][γE][γE][γE][γE]-, C20DA, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][γE][γE]-, C20DA[γE][γE][γE][γE]-, C20DA[γE][γE][γE][γE][γE]-, C20DA[γE][γE][γE][γE][γE][γE]-, C20DA[γE][γE][γE][γE][γE][γE][γE]-, C18DA[eLys], C18DA[E][E][E][E][E][eLys]-(SEQ ID NO: 635), C18DA[E][E][E][E][E][E][eLys]- (SEQ ID NO: 643), C18DA[γE][eLys]-, C18DA[γE][γE][eLys]-C18DA[γE][γE][γE][eLys]-, C18DA[γE][γE][γE][γE][eLys]-, C18DA[γE][γE][γE][γE][γE][eLys]-, C18DA[γE][γE][γE][γE][γE][γE][eLys]-, C20DA[eLys], C20DA[E][E][E][E][E][eLys]- (SEQ ID NO: 639), C20DA[γE][eLys]-, C20DA[γE][γE][eLys]-, C20DA[γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][γE][eLys][eLys]-, C18DA[OEG][OEG]-, C18DA[E][E][E][OEG][OEG]-, C18DA[E][E][E][E][OEG][OEG]- (SEQ ID NO: 634), C18DA[γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][γE][γE][OEG][OEG]-, C18DA[γE][γE][γE][γE][OEG][OEG]-, C18DA[γE][γE][γE][γE][γE][OEG][OEG]-, C20DA[OEG][OEG]-, C20DA[E][E][E][E][OEG][OEG]- (SEQ ID NO: 638), C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][γE][γE][OEG][OEG]-, C20DA[γE][γE][γE][γE][OEG][OEG]-, C20DA[γE][γE][γE][γE][γE][OEG][OEG]-, C18DA[OEG]-, C18DA[γE][OEG]-, C18DA[γE][γE][OEG]-, C18DA[γE][γE][γE][OEG]-, C18DA[γE][γE][γE][γE][OEG]-, C18DA[γE][γE][γE][γE][γE][OEG]-, C18DA[γE][γE][γE][γE][γE][γE][OEG]-, C20DA[OEG]-, C20DA[γE][OEG]-, C20DA[γE][γE][OEG]-, C20DA[γE][γE][γE][OEG]-, C20DA[γE][γE][γE][γE][OEG]-, C20DA[γE][γE][γE][γE][γE][OEG]-, C20DA[γE][γE][γE][γE][γE][γE][OEG]-, C18DA[OEG][eLys]-, C18DA[γE][OEG][eLys]-, C18DA[γE][γE][OEG][eLys]-, C18DA[γE][γE][γE][OEG][eLys]-, C18DA[γE][γE][γE][γE][OEG][eLys]-, C18DA[γE][γE][γE][γE][γE][OEG][eLys]-, C20DA[OEG][eLys]-, C20DA[γE][OEG][eLys]-, C20DA[γE][γE][OEG][eLys]-, C20DA[γE][γE][γE][OEG][eLys]-, C20DA[γE][γE][γE][γE][OEG][eLys]-, C20DA[γE][γE][γE][γE][γE][OEG][eLys]-, C18DA[OEG][OEG][eLys]-, C18DA[γE][OEG][OEG][eLys]-, C18DA[γE][γE][OEG][OEG][eLys]-, C18DA[γE][γE][γE][OEG][OEG][eLys]-, C18DA[γE][γE][γE][γE][OEG][OEG][eLys]-, C20DA[OEG][OEG][eLys]-, C20DA[γE][OEG][OEG][eLys]-, C20DA[γE][γE][OEG][OEG][eLys]-, C20DA[γE][γE][γE][OEG][OEG][eLys]-, C20DA[γE][γE][γE][γE][OEG][OEG][eLys]-, C18DA[AHX], C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C18DA[γE][γE][γE][AHX]-, C18DA[γE][γE][γE][γE][AHX]-, C18DA[γE][γE][γE][γE][γE][AHX]-, C18DA[γE][γE][γE][γE][γE][γE][AHX]-, C20DA[AHX], C20DA[γE][AHX]-, C20DA[γE][γE][AHX]-, C20DA[γE][γE][γE][AHX]-, C20DA[γE][γE][γE][γE][AHX]-, C20DA[γE][γE][γE][γE][γE][AHX]-, or C20DA[γE][γE][γE][γE][γE][γE][AHX]-. Preferably the polypeptide is lipidated at the lysine (K) residue in amino acid position X28.
In the present context, the polypeptides are generally amidated at the C-terminal (—CONH2), like the native peptides. However, the polypeptides of the present invention may also have either a free carboxylic acid (—COOH) or another post-translational modification, such as a methyl ester (—COOMe). In a highly preferred embodiment of the invention, the polypeptides are amidated at the C-terminal. The polypeptides according to the present invention may have a free amine (—NH2), be N-acylated (—NHCOR), N-methylated (—NHCH3 or -N(CH3)2), deaminated at the N-terminal, or N-lipidated.
In the present context it should be understood that the polypeptides according to the invention, or pharmaceutically acceptable salts thereof, may be in the form of a pharmaceutical composition. A pharmaceutical composition may comprise a pharmaceutically acceptable carrier and/or one or more excipients. The pharmaceutical composition (i.e. formulation) includes but are not limited to tablets, pills, capsules, emulsions, suspensions, sustained release formulations, solutions, or freeze-dried powder intended for dissolution prior to administration. In some embodiments, the formulation may be a depot formulation providing slow release. It should be appreciated that different routes of administration may be used depending on the choice of formulation and chemical and/or metabolic stability of the polypeptides. Such administration routes may include but are not limited to oral administration, parenteral administration (intravenous (IV), subcutaneous (SC), intradermal (ID) and intramuscular (IM)), or inhalation. In a preferred embodiment of the invention, the administration route is parenteral administration. In an even more preferred embodiment, the administration route is subcutaneous.
Peptide therapeutics are usually provided as pharmaceutical liquid formulation in a pre-filled ready-to-use injection device. These peptide formulations for subcutaneous administration have limited application volumes. Therefore, good solubility of the peptides at or around physiological pH (e.g pH 7) is a requirement for the application in a ready-to-use injection device.
In a first aspect, the invention provides a polypeptide according to the general structure of Formula (I) or a salt or a pharmaceutically acceptable salt thereof,
The compounds of the present invention bind to and/or activate the GLP-1, the GIP and the hY2 (NPY2) receptors.
The peptide fragments Z1 and Z3 are linked through a linker Z2. It should be appreciated that Z1 and Z3 may be linked by various linkers commonly used in the art of e.g. fusion protein. Preferably the linker comprises or consists of a short peptide consisting of 1-10 amino acid residues, such as 2-9 amino acids, preferably 3-8 amino acids, more preferably, 4-7 amino acids, even more preferably 5-7 amino acids, most preferably 6 amino acid residues. Thus, in a preferred embodiment, the linker Z2 consists of 6 amino acid residues (i.e. amino acids X29-X34). In a more preferred embodiment, Z2 has or comprises the amino acid sequence GGPSEG (SEQ ID NO: 311, i.e. amino acids X29-X34) or is a derivative thereof having 1, 2, 3, or 4 amino acid substitution(s). Preferably, the 1, 2, 3, or 4 amino acid substitution(s) in Z2 is/are present in any of the positions X31, X32, X33, or X34 (i.e. in the amino acid sequence PSEG (SEQ ID NO: 644)). Therefore, said Z2 derivatives are Z2 peptide analogues of the amino acid sequence SEQ ID NO: 311 having one or more amino acid substitution(s) compared to SEQ ID NO: 311.
In a highly preferred embodiment (Z2-Emb1), Z2 (i.e. amino acids X29-X34) has or comprises the amino acid sequence GGX31X32X33X34, wherein X31 is selected as P, G, or Q; X32 is selected as S, E, or R; X33 is selected as S, E, or Y, or, preferably, as S, E, Y, or T; X34 is selected as G, P, Y, or A; or is a derivative thereof having 1 amino acid substitution.
In further highly preferred embodiments, Z2 has the amino acid sequence of any one of the Z2 embodiments as disclosed hereinafter in the second aspect of the present invention (e.g. Z2-Emb2, Z2-Emb3).
Z1 (i.e. amino acids X1-X28) is a GIP/GLP1 hybrid polypeptide comprising or consisting of the amino acid sequence, Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-E-K-Q-A-Q-Aib-A-F-V-E-W-L-I-K (SEQ ID NO: 306), or is a derivative thereof having 1, 2, or 3 amino acid substitution(s). The sequence Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-E-K-Q-A-Q-Aib-A-F-V-E-W-L-I-K (SEQ ID NO: 306) (i.e. Z1 in SEQ ID NO: 286) was chosen as reference sequence. Table 1 summarizes (see page 21ff), the number of substitutions in Z1, the position(s) of Z1 that was/were substituted, and the exemplified amino acids in the substitutions in comparison with the reference (i.e. SEQ ID NO: 286). As shown in table 1, the amino acid positions X3, X10, X13, X15, X16, X17, X19, X20, X21, X23, X24, X25, or X27 were found to tolerate substitutions. Thus, in a preferred embodiment, the 1, 2 or 3 substitution(s) in the derivate of Z1 is/are present in any of the amino acid positions X3, X10, X13, X15, X16, X17, X19, X20, X21, X23, X24, X25, or X27. Preferably, the derivate of Z1 has 1, or 2 substitution(s). Most preferably, the derivate of Z1 has 1 substitution. Therefore, said Z1 derivatives are Z1 peptide analogues of the amino acid sequence SEQ ID NO: 306 (i.e. Z1 in SEQ ID NO: 286) having one or more amino acid substitution(s) compared to SEQ ID NO: 306.
In a highly preferred embodiment, the substitution(s), in the derivate of Z1, is/are selected as follows: X3 is selected as D; X10 is selected as L; X13 is selected as L; X15 is selected as D or A; X16 is selected as E, D, Aib, G, LysAc, A, L, S, Q, or R; X17 is selected as E, K, or A; X19 is selected as E; X20 is selected as D-Asp, or D-Arg; X21 is selected as E or, preferably, as E or K; X23 is selected as I; X24 is selected as Q; X25 is selected as Y; X27 is selected as L.
Z1 may consist of or comprise the amino acid sequence Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-E-K-Q-A-Q-Aib-A-F-V-E-W-L-I-K (SEQ ID NO: 306) or may be a derivative thereof having 1, 2 or 3 amino acid substitution(s) at any of the amino acid positions X3, X10, X13, X15, X16, X17, X19, X21, X23, X24, X25, or X27, wherein the substitution(s) is/are selected as follows: X3 is selected as D; X10 is selected as L; X13 is selected as L; X15 is selected as D; X16 is selected as E, D, G, A, S, Q, or R; X17 is selected as E, or A; X19 is selected as E; X21 is selected as E or K; X23 is selected as I; X24 is selected as Q; X25 is selected as Y; X27 is selected as L.
Z1 may consist of or comprise the amino acid sequence Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-E-K-Q-A-Q-Aib-A-F-V-E-W-L-I-K (SEQ ID NO: 306) or may be a derivative thereof having 1, 2 or 3 amino acid substitution(s) at any of the amino acid positions X3, X10, X13, X15, X16, X17, X21, X23, X24, or X25, wherein the substitution(s) is/are selected as follows: X3 is selected as D; X10 is selected as L; X13 is selected as L; X15 is selected as D; X16 is selected as E, D, G, A, S, Q, or R; X17 is selected as E, or A; X21 is selected as E; X23 is selected as I; X24 is selected as Q; X25 is selected as Y.
Z1 may consist of or comprise the amino acid sequence Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-E-K-Q-A-Q-Aib-A-F-V-E-W-L-I-K (SEQ ID NO: 306) or may be a derivative thereof having 1 or 2 amino acid substitution(s), preferably 1, at any of the amino acid positions X21, or X24, wherein the substitution(s) is/are selected as follows: X21 is selected as A or E; X24 is selected as E or Q.
Z3 (i.e. amino acids X35-X49) is a polypeptide providing hY2R agonism comprising or consisting of the amino acid sequence, A-S-L-R-H-Y-Y-N-W-L-T-R-Q-R-Y (SEQ ID NO: 307) or a derivative thereof having 1, 2, 3, 4, or 5 amino acid substitution(s). The sequence A-S-L-R-H-Y-Y-N-W-L-T-R-Q-R-Y (SEQ ID NO: 307) (i.e. Z3 in SEQ ID NO: 286) was chosen as reference sequence. Table 1 summarizes (see page 39ff) the number of substitutions in Z3, the positions of Z3 that can be substituted, and the exemplified amino acids in the substitutions in comparison with the reference (i.e. SEQ ID NO: 286). As shown in table 1, the amino acid positions X35, X36, X37, X38, X39, X41, X42, X43, X44, X46, X47, X48, or X49 were found to tolerate substitutions. Thus, in a preferred embodiment, the 1, 2, 3, 4, or 5 substitution(s) in the derivate of Z3 is/are present in any of the amino acid positions X35, X36, X37, X38, X39, X41, X42, X43, X44, X46, X47, X48, or X49. In a preferred embodiment, Z3 has 1, 2, or 3 substitution(s). More preferably, Z3 has 1 or 2 substitution(s). Most preferably Z3 has 1 substitution. Therefore, said Z3 derivatives are Z3 peptide analogues of the amino acid sequence SEQ ID NO: 307 (i.e. Z3 in SEQ ID NO: 286) having one or more amino acid substitution(s) compared to SEQ ID NO: 307.
In a highly preferred embodiment, the substitution(s), in the derivate of Z3, is selected as follows: X35 is selected as Q, I, V, E, or L; X36 is selected as E, T, D, or L; X37 is selected as Aib, V, D-Leu, or I, or, preferably, as Aib, V, D-Leu, I, or Tle; X38 is selected as L or Aib; X39 is selected as Aib, D-His, or Tle; X41 is selected as L, Aib, I, or Tle; X42 is selected as Aib; X43 is selected as H, L, R, or Aib; X44 is selected as Aib, D-Arg, or D-Asp, or, preferably, as Aib, D-Arg, D-Asp, or Tle; X46 is selected as hArg or D-Arg; X47 is selected as bh-Gln or NMeQ; X48 is selected as NMeR; X49 is selected as Tle, Chg, D-Tyr, Phg.
Z3 may consist of or comprise the amino acid sequence A-S-L-R-H-Y-Y-N-W-L-T-R-Q-R-Y (SEQ ID NO: 307) or may be a derivative thereof having 1, 2 or 3 amino acid substitution(s) at any of the amino acid positions X35, X36, X37, X38, X39, X41, X44, X47, or X48, wherein the substitution(s) is/are selected as follows: X35 is selected as Q, I, V, E, or L; X36 is selected as T; X37 is selected as Aib, V, I or Tle; X38 is selected as Aib; X39 is selected as Tle; X41 is selected as I, L, or Tle; X44 is selected as Tle; X47 is selected as NMeQ; X48 is selected as NMeR.
Z3 may consist of or comprise the amino acid sequence A-S-L-R-H-Y-Y-N-W-L-T-R-Q-R-Y (SEQ ID NO: 307) or may be a derivative thereof having 1, or 2 amino acid substitution(s), preferably 1, at any of the amino acid positions X35, X37, X47, or X48, wherein the substitution(s) is/are selected as follows: X35 is selected as Q, V; X37 is selected as I; X47 is selected as NMeQ; X48 is selected as NMeR.
Overall, the polypeptides Z1-Z2-Z3 according to the first aspect of the invention, and according to any or more embodiments disclosed in the present invention, preferably have 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, more preferably 0, 1, 2, 3, or 4 amino acid substitutions, highly preferably 0, 1, 2, or 3 substitutions, compared to SEQ ID NO: 286.
In a second aspect, the invention provides a polypeptide according to the general structure of Formula (I) or a salt or a pharmaceutically acceptable salt thereof,
In a preferred embodiment (Z1-Emb1), X3 is selected as E or D;
In a preferred embodiment (Z1-Emb2), X3 is selected as E or D;
In a preferred embodiment (Z1-Emb3), X3 is selected as E;
In a preferred embodiment, X3 in Z1 is selected as E. In a preferred embodiment, X10 in Z1 is selected as Y. In a preferred embodiment, X13 in Z1 is selected as Aib. In a preferred embodiment, X15 in Z1 is selected as E. In a preferred embodiment, X16 in Z1 is selected as K. In a preferred embodiment, X17 in Z1 is selected as Q. In a preferred embodiment, X19 in Z1 is selected as Q. In a preferred embodiment, X20 in Z1 is selected as Aib. In a preferred embodiment, X21 in Z1 is selected as A. In a preferred embodiment, X23 in Z1 is selected as V. In a preferred embodiment, X24 in Z1 is selected as E. In a preferred embodiment, X25 in Z1 is selected as W. In a preferred embodiment, X27 in Z1 is selected as I.
In a highly preferred embodiment, X3 in Z1 is selected as E; X10 in Z1 is selected as Y; and X13 in Z1 is selected as Aib. In a highly preferred embodiment, X15 in Z1 is selected as E; X16 in Z1 is selected as K; and X17 in Z1 is selected as Q. In a highly preferred embodiment, X19 in Z1 is selected as Q; X20 in Z1 is selected as Aib; and X21 in Z1 is selected as A. In a highly preferred embodiment, X23 in Z1 is selected as V; X24 in Z1 is selected as E; X25 in Z1 is selected as W; and X27 in Z1 is selected as I.
Preferred amino acids in Z2 In a preferred embodiment (Z2-Emb1), Z2 comprises or consist of the amino acid sequence GGX31X32X33X34, wherein X31 is selected as P, G, or Q; X32 is selected as S, E, or R; X33 is selected as S, E, Y, or T; X34 is selected as G, P, Y, or A; or Z2 is a derivative thereof having 1 amino acid substitution.
In a preferred embodiment (Z2-Emb2), Z2 comprises or consists of the amino acid sequence G-G-X31-X32-X33—X34 wherein X31 is selected as P or Q; X32 is selected as S or E; X33 is selected as S, E, Y or T; X34 is selected as G, P, Y, or A; or Z2 is a derivative thereof having 1 amino acid substitution.
In a highly preferred embodiment (Z2-Emb3), Z2 comprises or consists of the amino acid sequence GGPSEG (SEQ ID NO: 311) or GGPSSG (SEQ ID NO: 320); or Z2 is a derivative thereof having 1 amino acid substitution.
In a preferred embodiment, X31 in Z2 is selected as P. In a preferred embodiment, X32 in Z2 is selected as S. In a preferred embodiment, X33 in Z2 is selected as S. In a preferred embodiment, X34 in Z2 is selected as G.
In a further embodiment (Z2-Emb4) Z2 is selected from the group consisting of GGPEEG (SEQ ID NO: 313), GGPEEP (SEQ ID NO: 314), GGPESG (SEQ ID NO: 315), GGPESP (SEQ ID NO: 316), GGPSEG (SEQ ID NO: 311), GGPSEP (SEQ ID NO: 317), GGPSEY (SEQ ID NO: 318), GGPSSA (SEQ ID NO: 319), GGPSSG (SEQ ID NO: 320), GGPSSP (SEQ ID NO: 321), GGPSSY (SEQ ID NO: 322), GGPSTG (SEQ ID NO: 323), GGPSYG (SEQ ID NO: 324), GGQSSG (SEQ ID NO: 325), GGGSEY (SEQ ID NO:648), GGPEYY (SEQ ID NO: 649), GGPRSG (SEQ ID NO: 650), GGPRYY (SEQ ID NO: 651), GGPSYY (SEQ ID NO: 652), GGQRSY (SEQ ID NO: 653), GGQRYY (SEQ ID NO: 654), GGQSSY (SEQ ID NO: 655).
In a further embodiment (Z2-Emb5) Z2 is selected from the group consisting of GGPEEG (SEQ ID NO: 313), GGPEEP (SEQ ID NO: 314), GGPESG (SEQ ID NO: 315), GGPESP (SEQ ID NO: 316), GGPSEG (SEQ ID NO: 311), GGPSEP (SEQ ID NO: 317), GGPSEY (SEQ ID NO: 318), GGPSSA (SEQ ID NO: 319), GGPSSG (SEQ ID NO: 320), GGPSSP (SEQ ID NO: 321), GGPSSY (SEQ ID NO: 322), GGPSTG (SEQ ID NO: 323), GGPSYG (SEQ ID NO: 324), GGQSSG (SEQ ID NO: 325), GGPRSG (SEQ ID NO: 650), GGPRYY (SEQ ID NO: 651), GGPSYY (SEQ ID NO: 652).
In a further embodiment (Z2-Emb6) Z2 is selected from the group consisting of GGPEEG (SEQ ID NO: 313), GGPEEP (SEQ ID NO: 314), GGPESG (SEQ ID NO: 315), GGPESP (SEQ ID NO: 316), GGPSEG (SEQ ID NO: 311), GGPSEP (SEQ ID NO: 317), GGPSEY (SEQ ID NO: 318), GGPSSA (SEQ ID NO: 319), GGPSSG (SEQ ID NO: 320), GGPSSP (SEQ ID NO: 321), GGPSSY (SEQ ID NO: 322), GGPSTG (SEQ ID NO: 323), GGPSYG (SEQ ID NO: 324), GGQSSG (SEQ ID NO: 325).
In a further embodiment (Z2-Emb7) Z2 is selected from the group consisting of GGPEEG (SEQ ID NO: 313), GGPESG (SEQ ID NO: 315), GGPESP (SEQ ID NO: 316), GGPSEG (SEQ ID NO: 311), GGPSEP (SEQ ID NO: 317), GGPSEY (SEQ ID NO: 318), GGPSSA (SEQ ID NO: 319), GGPSSG (SEQ ID NO: 320), GGPSSP (SEQ ID NO: 321), GGPSSY (SEQ ID NO: 322), GGPSTG (SEQ ID NO: 323), GGPSYG (SEQ ID NO: 324), GGQSSG (SEQ ID NO: 325).
In a further embodiment (Z3-Emb1) X35 is selected as A, E, I, L, Q, and V;
In a preferred embodiment (Z3-Emb2) X35 is selected as A, Q or V;
In a preferred embodiment, X35 in Z3 is selected as A. In a preferred embodiment, X36 in Z3 is selected as S. In a preferred embodiment, X37 in Z3 is selected as L. In a preferred embodiment, X38 in Z3 is selected as R. In a preferred embodiment, X39 in Z3 is selected as H. In a preferred embodiment, X41 in Z3 is selected as Y. In a preferred embodiment, X42 in Z3 is selected as N. In a preferred embodiment, X43 in Z3 is selected as W. In a preferred embodiment, X44 in Z3 is selected as L. In a preferred embodiment, X46 in Z3 is selected as R. In a preferred embodiment, X47 in Z3 is selected as Q. In a preferred embodiment, X48 in Z3 is selected as R. In a preferred embodiment, X49 in Z3 is selected as Y.
In a highly preferred embodiment, X35X36X37X38X39 is selected as ASLRH (SEQ ID NO: 645). In a highly preferred embodiment, X41X42X43X44 are selected as YNWL (SEQ ID NO: 326). In a highly preferred embodiment, X46X47X48X49 are selected as RQRY (SEQ ID NO: 327).
It should be appreciated that the second aspect of the invention and/or any relative embodiment hereinbefore and hereinafter disclosed may be dependent from the first aspect of the invention and/or any relative embodiment hereinbefore and hereinafter disclosed.
It should be appreciated that one or more of the embodiments, according to either the first or second aspect of the invention, describing preferred amino acids in the positions of Z1 may be combined with one or more of the embodiments describing preferred amino acids in the positions of Z2 and/or Z3 and vice versa. Thus, any combination of one or more of the embodiments mentioned under “Preferred amino acids in Z1” may be combined with one or more of the embodiments mentioned under “Preferred amino acids in Z2” and/or “Preferred amino acids in Z3” and vice versa. Any such combination of embodiments is to be understood as a directly and unambiguously part of the disclosure.
With reference to the second aspect of the invention, examples of combinations of embodiments mentioned hereinbefore are:
The polypeptide according to the first and/or second aspect of the invention, may be lipidated at a lysine (K) residue, preferably at the lysine (K) residue in amino acid position X28, with a structure (i.e lipid/linker) of general formula L-Y1—Y2—Y3—Y4—Y5—Y6—Y7—Y8, wherein L is a lipid selected from 17-carboxy-heptadecanoyl (briefly C18DA) and 19-carboxy-nonadecanoyl (briefly C20DA) and further wherein each of Y1—Y8 are independently selected from: not present, [γE], [E], [OEG], [eLys], or [AHX].
Preferably, the lipid/linker has a structure of general formula L-Y1—Y2—Y3—Y4—Y5—Y6—Y7—Y8, wherein L is a lipid selected from C18DA or C20DA, wherein each of Y1—Y8 are independently selected from: not present, [γE], [E], [OEG], [eLys], or [AHX], wherein each of Y1—Y3 are [γE] or each of Y1—Y3 are [E], wherein Y4 is selected from: [γE], [E], or [OEG], and wherein each of Y5—Y8 are independently selected from: not present, [γE], [E], [OEG], [eLys], or [AHX].
Preferably, the lipid/linker has a structure of general formula L-Y1—Y2—Y3—Y4—Y5—Y6—Y7—Y8, wherein L is C20DA, wherein each of Y1—Y8 are independently selected from: not present, [γE], [E], [OEG], or [eLys], wherein each of Y1—Y4 are [γE] or each of Y1—Y4 are [E], wherein Y5 is selected from: [γE], [E], [OEG], or [eLys], and wherein each Y6—Y8 are independently selected from: not present, [γE], [E], [OEG], or [eLys].
The polypeptide of the invention, for example, is lipidated at the lysine (K) residue in amino acid position X28, and the lipid/linker is selected from the group consisting of:
The polypeptide of the invention, for example, is lipidated at the lysine (K) residue in amino acid position X28, and the lipid/linker is selected from the group consisting of:
The triple agonists of the invention are generally soluble around pH 7. There are several techniques known to the skilled person in the art how to determine solubility. Preferably the solubility of the peptide of the invention is determined as disclosed in Example 2 hereinafter.
In some embodiments, the solubility of the compounds of the invention is greater than or equal to 1.0 mg/ml around pH 7.
In some embodiments, the solubility of the compounds of the invention is greater than 3.0 mg/ml around pH 7.
In some embodiments, the solubility of the compounds of the invention is greater than 5.0 mg/ml around pH 7.
In some embodiments, the solubility of the compounds of the invention is equal or greater than 6.0 mg/ml, 7.0 mg/ml, 8.0 mg/ml or 9.0 mg/ml around pH 7.
The hybrid polypeptides of the present invention are able to agonize the hGLP1R, the GIPR and the hNPY2R in the presence of human serum albumin (see table 3), the main protein present in human plasma. Activity of peptides of the invention in the presence of human plasma is an important prerequisite for viable compounds useful for the treatment of the disclosed diseases. The skilled person will be aware of suitable assay formats, and an example is provided hereinafter. For instance, for peptides of the present invention the EC50 values to the hGLP1R, the GIPR and the hNPY2R in the presence of 100% human plasma (100% hP) was evaluated by the assay as described in Example 1 hereinafter.
In some embodiments of peptides of the present invention, the EC50 towards hGLP1R 100% hP and GIPR 100% hP is below 100 nM (e.g. 0.01 nM to 100 nM), and towards hNPY2R 100% hP is below 1000 nM (e.g. 0.01 nM to 1000 nM).
In some embodiments of peptides of the present invention, the EC50 towards hGLP1R 100% hP and GIPR 100% hP is below or equal to 30 nM (e.g. 0.01 nM to 50 nM), and towards hNPY2R 100% hP is below 300 nM (e.g. 0.01 nM to 300 nM).
In some embodiments of peptides of the present invention, the EC50 towards hGLP1R 100% hP and GIPR 100% hP is below or equal to 10 nM (e.g. 0.01 nM to 10 nM), and towards hNPY2R 100% hP is below 100 nM (e.g. 0.01 nM to 100 nM).
In a preferred embodiment of the peptides of the present invention, the EC50 towards hGLP1R 100% hP and GIPR 100% hP is below or equal to 10 nM (e.g. 0.01 nM to 10 nM), and towards hNPY2R 100% hP is below 100 nM (e.g. 0.01 nM to 100 nM), and the solubility is greater than or equal to 1.0 mg/ml around pH 7.
In some embodiments the peptides of the invention have favourable pharmacokinetic properties. In this regard, the in-vivo half-life of the peptides of the invention may be greater than 6 hours or greater than 8 hours or greater than 10 hours in the mouse (NMRI mice, see measurement described in Example 3).
In a preferred embodiment of the peptides of the present invention, the EC50 towards hGLP1R 100% hP and GIPR 100% hP is below or equal to 10 nM (e.g. 0.01 nM to 10 nM), and towards hNPY2R 100% hP is below 100 nM (e.g. 0.01 nM to 100 nM), the solubility is greater than or equal to 1.0 mg/ml around pH 7, and the in-vivo half-life is greater than 8 hours.
In a third aspect, the invention provides a polypeptide for use as a medicament. Since Y2 receptors have a feeding suppressive action, a polypeptide having Y2 agonistic activity may be useful in the treatment of symptoms associated with eating disorder, obesity (e.g. simple obesity or symptomatic obesity) and/or diabetes. Furthermore, the gut hormones GLP-1 and GIP (called incretins), promote insulin secretion from the pancreas. Since incretins are closely related to glucose metabolism, a polypeptide having GLP-1 receptor agonistic activity and GIP receptor agonistic activity may be useful in the treatment of symptoms associated with a glucose metabolism disorder, including diabetes and obesity. Thus, the polypeptides of the present invention may have a feeding suppressive action, and/or a body weight-lowering action. In a preferred embodiment the invention provides a polypeptide for use in the prevention, treatment, and/or remission of a disease, disorder, or condition selected from the list consisting of excessive weight (overweight), obesity (e.g. simple obesity (chronic weight management) or symptomatic obesity), insulin resistance, diabetes (e.g. type 1 diabetes or type 2 diabetes) or prediabetes, eating disorders, hyperlipidemia (e.g., hypertriglyceridemia, hypercholesterolemia, high LDL-cholesterolemia, low HDL-cholesterolemia, postprandial hyperlipemia), metabolic syndrome (three of the following five medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein (HDL)), liver diseases such as metabolic associated fatty liver disease (MAFLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steato-hepatitis (NASH), portal hypertension; cardiovascular diseases (e.g. hypertension, atherosclerosis, stroke or cardiac failure); kidney diseases such as diabetic kidney disease (DKD), and chronic kidney disease (CKD); and neurodegenerative diseases (Alzheimer's or Parkinson's disease), but also other obesity related diseases such as osteoporosis, sleep apnoe, obesity associated cancers, and obesity associated asthma. Examples of the symptomatic obesity include endocrine obesity (e.g., Cushing syndrome, hypothyroidism, insulinoma, obese type 2 diabetes, pseudohypoparathyroidism, hypogonadism and associated endocrine disorders, such as polycystic ovary syndrome (PCOS)), central obesity (e.g., hypothalamic obesity, frontal lobe syndrome, Kleine-Levin syndrome), hereditary obesity (e.g., Prader-Willi syndrome, Laurence-Moon-Biedl syndrome), drug-induced obesity (e.g., steroid, phenothiazine, insulin, sulfonylurea agent, P-blocker-induced obesity).
The polypeptides of the invention may also be used for the prevention of obesity, or prevention or reversal of co-morbidities of obesity and/or overweight, such as type 2 diabetes, high blood pressure, NAFLD, NASH, DKD, CKD, sleep apnoe, obesity associated cancers, or obesity associated asthma.
In a fourth aspect, the invention provides a method for the treatment of a disease, disorder and/or condition said method comprising administering a therapeutic effective amount of a polypeptide or a pharmaceutically acceptable salt thereof, according to any of the aspects and embodiments disclosed herein, to an individual in need thereof. Preferably, the disease, disorder and/or condition is selected from the list consisting of excessive weight, obesity, diabetes (type 1 or type 2) or prediabetes, eating disorders, hyperlipidemia, metabolic syndrome, NAFLD, NASH, and/or cardiovascular diseases. Most preferably the disease, disorder and/or condition is obesity, prediabetes and/or diabetes (type 1 or type 2).
Furthermore, the invention provides a method of binding to and/or activating the GLP-1, the GIP and the hNPY2 receptors in an individual in need thereof, said method comprising administering a therapeutic effective amount of a polypeptide or a pharmaceutically acceptable salt thereof, according to any of the aspects and embodiments disclosed herein.
Likewise, the present invention relates to the use of a polypeptide or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a disease, disorder and/or condition, according to any of the aspects and embodiments disclosed herein.
The dose range of the polypeptide according to the general structure of Formula (I) applicable per week is usually from 0.01 to 100 mg for humans (subcutaneous administration).
The polypeptide of the invention may be administered subcutaneously using a suitable device such as a pre-filled ready-to-use injection device, a syringe, a pen injector, or an auto-injector.
The actual pharmaceutically effective amount or therapeutic dosage will usually depend on factors known by those skilled in the art such as age and weight of the patient, route of administration and severity of disease.
In any case, the compounds will be administered at dosages and in a manner, which allows a pharmaceutically effective amount to be delivered based upon patient's unique condition.
All peptides were synthesized by standard Fmoc-based solid phase peptide chemistry on a Tentagel S RAM resin (loading 0.23-0.25 mmol/g, bead size 90 μm) supplied by Iris Biotech GmbH or Rapp Polymere GmbH.
The following protected amino acids were used: Fmoc-Ala-OH, Fmoc-Aib-OH, Fmoc-Arg(Pbf)-OH, Fmoc-NMeArg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NMeGln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu-OtBu, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Boc-Tyr(tBu)-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-Val-OH. The L-form of the amino acid building blocks was utilized if not specified otherwise.
The modular half-life extending group was built up by solid-phase peptide synthesis (SPPS) using protected building blocks such as, but not limited to, C18DA(tBu), C20DA(tBu), Fmoc-OEG-OEG-OH, Fmoc-OEG-OH, Boc-Lys(Fmoc)-OH, Fmoc-Glu-OtBu, Fmoc-Glu(tBu)-OH, Fmoc-Ahx-OH, Fmoc-Trx-OH and Fmoc-Sar-OH.
The amino acids, Fmoc-Glu-OtBu, Oxyma and DIC, were purchased from standard suppliers, e.g. Bachem, Novabiochem, ABCR GmbH & CO. KG., Iris Biotech GmbH, Sigma-Aldrich. C18DA(tBu) and C20DA(tBu) were supplied by Cool Pharm Ltd. or AstraTech, Fmoc-OEG-OEG-OH was supplied by ABCR GmbH & CO. KG or Iris Biotech GmbH, Fmoc-OEG-OH was supplied by Angene International Limited, Combi Blocks Inc., Iris Biotech GmbH or Hangzhou APIChem Technology Co., Ltd. Fmoc-Ahx-OH was purchased from Activate Scientific GmbH, Fmoc-Trx-OH was purchased from ABCR GmbH & CO. KG.
Assembly of peptides started from the C-terminus by stepwise chain elongation towards the N-terminus according to the respective sequences until the N-terminal amino acid was reached. Deprotection of the side chain of the branching amino acid, e.g. Lys(Dde), was followed by assembly of the half-life extending group. The peptides were obtained as TFA salts from cleavage/deprotection or from HPLC purification. The trifluoroacetate can be exchanged by common procedures, such as resin-ion exchange procedures, e.g. as disclosed in Roux, St. et al. J. Pept. Sci. 2008; 14: 354-359.
Synthesis Method 1 (501) Peptides were synthesized by microwave-assisted solid-phase peptide synthesis (SPPS) on a CEM Liberty Blue Peptide Synthesizer at 0.25 mmol scale on Tentagel S RAM resin using the Fmoc strategy. The stoichiometry and concentrations for peptide coupling reactions were 4 eq of suitably protected amino acid in DMF (0.2 mol/l, 5 ml), 4 eq of Oxyma in DMF (1 mol/l, 1 ml) and 8 eq DIC in DMF (1 mol/l, 2 ml).
The reaction times and temperatures were different for the amino acids. Single coupling 4 min at 90° C. was used for all standard amino acids except those mentioned below. Single coupling 20 min at 75° C. was used for Fmoc-Aib-OH, the amino acid after Aib was coupled twice. Double coupling 4 min at 90° C. was used for Fmoc-Arg(Pbf)-OH, C18DA(tBu), C20DA(tBu), Fmoc-OEG-OH, FmocNMeArg(Pbf)-OH and Fmoc-NMeGln(Trt)-OH. The amino acid after NMeArg and NMeGln was coupled twice. The last 8 standard amino acids in the main chain were coupled at 90° C. for 8 min.
A capping step with 20% acetic anhydride in DMF (10 ml) for 5 min at 65° C. was performed prior and after the coupling of Fmoc-Glu-OtBu and prior the coupling of C18DA(tBu) and C20DA(tBu). Na Fmoc deprotection was performed with 10% piperidine/DMF (10 ml) for 1 min at 90° C. Deprotection of the Lys(Dde)-group was carried out 2 times with 5% hydrazine hydrate in DMF (10 ml) for 3 min at 90° C. Raw products were washed on resin with DCM and dried prior to cleavage. Cleavage from resin and deprotection was performed with a mixture of 95% TFA/water (10 ml) and triisopropylsilane (250 μl) for 45 min at 40° C. Crude peptides were precipitated with cold tert.butyl-methyl ether, dissolved in 50% acetonitrile/water and purified by preparative HPLC.
Peptides were synthesized by SPPS on a MultiSynTech SYRO II at 0.2 mmol scale. Standard coupling of amino acids was achieved by using 4 eq of suitably protected amino acids dissolved in 0.5 mol/l Oxyma-DMF solution (0.5 mol/1) and 4.5 eq DIC (1 mol/1) in DMF. Fmoc-Phe-OH was dissolved in 0.5 mol/l Oxyma-NMP and 4 eq were used for coupling (0.5 mol/1). Coupling time of the first 23 amino acids starting from the C-terminus was 15 min at 75° C. (Cys and His were coupled at 50° C.). Further elongation was done by double coupling (2×15 min at 75° C.). Deprotection of Lys(Mtt)-group was carried out selectively using hexafluoroisopropanol (10×10 min at RT, 10 ml). Derivatisation of the deprotected lysine intermediate was done by double coupling 4 eq. of the relevant linker building blocks (Fmoc-OEG-OEG-OH, Fmoc-Glu-OtBu, Boc-Lys(Fmoc)-OH, C18DA(tBu), C20DA(tBu) for 15 min at 75° C. Na Fmoc deprotections were performed using 40% piperidine in NMP (4 ml) for 3 min followed by 20% piperidine in NMP (4 ml) for 15 min at 45° C. Suitably protected Fmoc-Asp, Fmoc-Cys and Fmoc-His intermediates were deprotected at room temperature. Peptides were cleaved from resin and side-chains deprotected by adding 15 ml 95:2:1:2 TFA/DODT/TES/water for 4 h at RT or 60 min at 45° C. The peptides were precipitated with cold diethyl ether, dissolved in acetonitrile/water and purified by preparative HPLC (Purification Method 3, P02).
Crude peptides were dissolved in DMF/acetonitrile/water and purified by reversed phase chromatography using an Agilent preparative HPLC-MS System with preparative pumps G7161B, G7111B and G7110B, a diode array detector G7115A, a mass-spectrometer G6135B and a fraction collector G7158B. A Waters Luna Prep C8(3) column (100 Å, 10 μm, 300 g, self-filled steel column) served as stationary phase. The mobile phase was run with a gradient of buffer A (ACN) and buffer (H2O+0.1% TFA) as described in the table below at a flow rate of 150 ml/min at 40° C. The relevant fractions were pooled and lyophilized. The final product was characterized by analytical HPLC-MS (U046_001 and U046_006).
Crude peptides were dissolved in DMF/acetonitrile/water and purified by reversed phase chromatography using a GILSON preparative HPLC System with preparative pumps AP-MOD (max. flow rate: 200 ml/min), a diode array detector ECOM Flash 10 and a fraction collector GILSON GX 281. Stationary phase was a Phenomenex LUNA C8 10 μm Prep Column (50×250 mm). The peptides were eluted with a focused gradient using water (eluent A) and acetonitrile (eluent B) at a flow rate of 120 ml/min and 40° C.; modifier solution was added in ‘at-column dilution’ mode to maintain a constant amount of 0.1% TFA within the mobile phase. Homogeneous fractions were pooled and lyophilized. The final product was characterized by HPLC-MS (U046_001 and U046_006).
Crude peptides were purified by reverse phase HPLC using a Waters preparative HPLC system with C8 column (Reprosil Gold 200 Å, 5 μm, 40 mm×250 mm), preparative pumps (waters 2545), UV/VIS detector (Waters 2489) and a Waters fraction collector III. The mobile phase was run with a gradient of buffer A (0.1% TFA in H2O) and buffer B (0.1% TFA in ACN, gradient: 35-45% B over 20 min) at a flow rate of 50 ml/min at RT. Relevant fractions were analysed, pooled and lyophilized. The final product was characterized by analytical UPLC-MS (A02).
Peptide purity and mass were estimated by analytical HPLC-MS on a Kinetex C8 column (4.6 mm×150 mm, 2.6 um, Phenomenex) using a Agilent 1260 HPLC system equipped with Mass Detector G6135. Analysis was performed by gradient elution with buffer A (0.3% TFA in H2O) and buffer B (0.24% TFA in ACN) at a temperature of 40° C. Details of the gradient and flow rates are summarized in the tables below. Retention times and masses were recorded.
Peptide purities (relative peak areas @214 nm) were in the range from 80 to 99%, preferably greater than 95%.
Peptide purity and mass were determined by analytical HPLC-MS on a Kinetex C8 column (Phenomenex, 100 2.6 μm, 4.6 mm×150 mm) using a Waters Acquity HPLC System equipped with 3100 Mass Detector. Analysis was performed by gradient elution with buffer A (0.3% TFA in H2O) and buffer B (0.3% TFA in ACN) at a temperature of 40° C. Details of the gradient and flow rates are summarized in the table below. Retention times and masses were recorded.
The following compounds were synthesised. All compounds were obtained as TFA salts:
and 637, respectively)
and 641, respectively)
and 642, respectively)
The following examples demonstrate certain specific embodiments of the present invention. The following examples were carried out using standard techniques that are well known and routine to those of skill in the art, except where otherwise described in detail.
CHO and HEK-293 recombinant cell lines express a luciferase reporter gene under control of the cAMP responsive element (CRE). As a second recombinant protein the GLP-1 and NPY2 receptors were expressed in the reporter gene HEK cells, and the GIP receptor was expressed in the reporter gene CHO cells, respectively. Stimulation of these recombinant cells with an agonist leads to an increase of intracellular cAMP levels. In the presence of cAMP, the transcription factor CRE binding protein (CREB) binds to the CRE and to the CREB binding protein (CBP), which leads to transcription of the luciferase reporter gene. Peptides upon serial-dilution in 100% DMSO were transferred into 384-well assay plates with 5 μl pre-dispensed assay buffer (1×HBSS, 20 mM HEPES, pH7.4 supplemented with 0.5% human plasma) using acoustic dispensing on a Labcyte ECHO. Cryo-preserved transgenic reporter gene cells were thawed in assay buffer. 20 μl (10.000 cells/well) were added to the plate with the peptides and incubated for 4 hours at 37° C. in a humidified incubator. Following incubation, assay plates are equilibrated to room temperature, followed by addition of 25 μl of Bright-GIo™ Luciferase reagent, incubation at room temperature for 10 minutes and analysis of luminescence (Envision Reader). Concentration response evaluation of compounds was performed with 8 concentrations of peptides (covering four decades). EC50 values were calculated by non-linear regression using sigmoid concentration-response with variable slope. The same assays were run in the presence of 100% plasma with no additional buffer constituents.
The results are summarized in Table 3, below.
Peptides (as TFA-salts) were weighed out in a filter unit (Mini-UniPrep Syringieless Filter 0.45 prn. Whatman PVDF), and 0.1 M phosphate buffer at pH 7 was added to achieve 10 mg/mL final peptide concentration. The peptides were dissolved by shaking the filter units horizontally at 600 rpm for 2 hours at room temperature. Samples were then filtered to remove any insoluble particles. Controls were prepared by weighing out the corresponding peptide solid material and dissolving it in an appropriate vehicle (e.g. ACN:H2O) to a final concentration of 1 mg/mL. Both, the control and sample were analyzed by reversed phase chromatography. The area under the peak of the sample was compared to the control and the solubility was calculated based on that ratio. The pH was measured and recorded for each sample.
Pharmacokinetic parameters of the peptides were determined after intravenous application to NMRI mice. Male NMRI mice were obtained from Janvier Laboratories (France) weighing 30 to 40 g. Mice were housed in standard cages with a 12:12 hour light:dark cycle and standard food and water access ad libitum. Each test peptide was dissolved in 50 mM phosphate buffer (pH 7.4)/3.5% mannitol. Intravenous application of 30-60 nmol/kg was performed via the tail vein. Serial blood samples from conscious mice were collected from the saphenous vein in EDTA-containing vials at different time points up to 56 hours post-dosing. Subsequently, plasma was prepared by centrifugation for 5 min at approximately 5000 rpm and stored at −20° C. until quantification of the peptide plasma levels by liquid chromatography mass spectrometry (LC-MS). Individual plasma concentration—time profiles were analysed by a non-compartmental approach and the resulting pharmacokinetic parameters were calculated. Mouse mean residence times (MRT) of GGY peptides according to the invention have been measured and summarized in table 4 (below).
Male NMRI mice were obtained from Janvier (Janvier Labs, France) or from Charles River (Charles River Research Models & Services Germany GmbH) at the age of 3 weeks. The animals get a microchip (Datamars, Slim Microchip T-SL) for individual identification after delivery. They were housed with 4 mice per cage under a 12/12 dark-light cycle, light off at 3 p.m. Room temperature was controlled to 21° C. +/−1° C. and humidity 60%+/−20%. Mice had ad libitum access to regular rodent chow (KLIBA Nafag 3040 or Altromin 1324, Brogaarden, Denmark) and tap water.
Latest transfer into the HM2-System of MBRose, Denmark (real-time monitoring system of food- and water intake) of the animals 5 days before study start, to allow acclimatization to experimental conditions. As the animals were uniquely identified with the microchips, each individual animal was identified by its own microchip upon entry and exit from the food channel via antennae. Randomization of the mice for each study group (n=8; age earliest 6 weeks old) was based on food intake (middle of last three days 24 h-Intervall) and bodyweight directly before start of the study. A vehicle treated (50 mM phosphate buffer pH7.4 with 3.5% Mannitol) group was included in each experiment. To have the same nutrition standard for each animal the food access was locked eight hours before dark phase. One hour before night, the animals were once treated subcutaneously with the test peptide. Food intake was reported hourly for a period of 24 h. The food intake was normalized [%] to the average food intake of the vehicle group and the value are summarized in table 5 (below).
1. A polypeptide according to the general structure of Formula (I) or a pharmaceutically acceptable salt thereof,
2. A polypeptide according to item 1, wherein X3 in Z1 is selected as E.
3. A polypeptide according to any of the preceding items, wherein X10 in Z1 is selected as Y.
4. A polypeptide according to any of the preceding items, wherein X13 in Z1 is selected as Aib.
5. A polypeptide according to any of the preceding items, wherein X15 in Z1 is selected as E.
6. A polypeptide according to any of the preceding items, wherein X16 in Z1 is selected as K.
7. A polypeptide according to any of the preceding items, wherein X17 in Z1 is selected as Q.
8. A polypeptide according to any of the preceding items, wherein X19 in Z1 is selected as Q.
9. A polypeptide according to any of the preceding items, wherein X20 in Z1 is selected as Aib.
10. A polypeptide according to any of the preceding items, wherein X21 in Z1 is selected as A or E, preferably A.
11. A polypeptide according to any of the preceding items, wherein X23 in Z1 is selected as V.
12. A polypeptide according to any of the preceding items, wherein X24 in Z1 is selected as E or Q, preferably E.
13. A polypeptide according to any of the preceding items, wherein X25 in Z1 is selected as W.
14. A polypeptide according to any of the preceding items, wherein X27 in Z1 is selected as I.
15. A polypeptide according to any of the preceding items, wherein X31 in Z2 is selected as P.
16. A polypeptide according to any of the preceding items, wherein X32 in Z2 is selected as S.
17. A polypeptide according to any of the preceding items, wherein X33 in Z2 is selected as E or S, preferably S.
18. A polypeptide according to any of the preceding items, wherein X34 in Z2 is selected as G.
19. A polypeptide according to any of the preceding items, wherein X35 in Z3 is selected as A, Q or V, preferably A.
20. A polypeptide according to any of the preceding items, wherein X36 in Z3 is selected as S.
21. A polypeptide according to any of the preceding items, wherein X37 in Z3 is selected as I or L, preferably L.
22. A polypeptide according to any of the preceding items, wherein X38 in Z3 is selected as R.
23. A polypeptide according to any of the preceding items, wherein X39 in Z3 is selected as H.
24. A polypeptide according to any of the preceding items, wherein X41 in Z3 is selected as Y.
25. A polypeptide according to any of the preceding items, wherein X42 in Z3 is selected as N.
26. A polypeptide according to any of the preceding items, wherein X43 in Z3 is selected as W.
27. A polypeptide according to any of the preceding items, wherein X44 in Z3 is selected as L.
28. A polypeptide according to any of the preceding items, wherein X46 in Z3 is selected as R.
29. A polypeptide according to any of the preceding items, wherein X47 in Z3 is selected as Q or NMeQ, preferably Q.
30. A polypeptide according to any of the preceding items, wherein X48 in Z3 is selected as R or NMeR, preferably R.
31. A polypeptide according to any of the preceding items, wherein X49 in Z3 is selected as Y.
32. A polypeptide according to any of the preceding items, wherein X35X36X37X38X39 are selected as ASLRH (SEQ ID NO: 645).
33. A polypeptide according to any of the preceding items, wherein X41X42X43X44 are selected as YNWL (SEQ ID NO: 326).
34. A polypeptide according to any of the preceding items, wherein X46X47X48X49 are selected as RQRY (SEQ ID NO: 327).
35. A polypeptide according to any of the preceding items, wherein the polypeptide is able to bind and/or activate the GLP-1, GIP and hY2 (hNPY2) receptors.
1. A polypeptide according to the general structure of Formula (I) or a pharmaceutically acceptable salt thereof,
2. A polypeptide according to claim 1, wherein the linker consists of 1-10 amino acid residues.
3. A polypeptide according to any of the preceding claims, wherein Z2 has the amino acid sequence GGPSEG (SEQ ID NO: 311) or is a derivative thereof having 1, 2, 3, or 4 amino acid substitution(s).
4. A polypeptide according to claim 3, wherein the 1, 2, 3, or 4 amino acid substitution(s) in Z2 is/are present in any of the positions X31, X32, X33, or X34.
5. A polypeptide according to any of the preceding claims, wherein Z2 has the amino acid sequence GGX31X32X33X34, wherein X31 is selected as P, G, or Q; X32 is selected as S, E, or R; X33 is selected as S, E, or Y; X34 is selected as G, P, Y, or A; or is a derivative thereof having 1 amino acid substitution.
6. A polypeptide according to any of the preceding claims, wherein the 1, 2 or 3 substitution(s) in the derivate of Z1 is present in any of the amino acid positions X3, X10, X13, X15, X16, X17, X19, X20, X21, X23, X24, X25, or X27.
7. A polypeptide according to any of the preceding claims, wherein the 1, 2, 3, 4, or 5 substitution(s) in the derivate of Z3 is/are present in any of the amino acid positions X35, X36, X37, X38, X39, X41, X42, X43, X44, X46, X47, X48, or X49.
8. A polypeptide according to any of the preceding claims, wherein the derivate of Z3 has 1, 2, or 3 substitution(s), preferably 1 or 2 substitution(s), most preferably 1 substitution.
9. A polypeptide according to any of the preceding claims, wherein the derivate of Z1 has 1, or 2 substitution(s), preferably 1 substitution.
10. A polypeptide according to any of the preceding claims, wherein the substitution(s), in the derivate of Z1, is selected as follows:
11. A polypeptide according to any of the preceding claims, wherein the substitution(s), in the derivate of Z3, is selected as follows: X35 is selected as Q, I, V, E, or L; X36 is selected as E, T, D, or L; X37 is selected as Aib, V, D-Leu, or I; X38 is selected as L or Aib; X39 is selected as Aib, D-His, or Tle; X41 is selected as L, Aib, I, or Tle; X42 is selected as Aib; X43 is selected as H, L, R, or Aib; X44 is selected as Aib, D-Arg, or D-Asp; X46 is selected as hArg or D-Arg; X47 is selected as bh-Gln or NMeQ; X48 is selected as NMeR; X49 is selected as Tle, Chg, D-Tyr, Phg.
12. A polypeptide according to any of the preceding claims, wherein the polypeptide is lipidated at a lysine (K) residue, preferably at the lysine (K) residue in amino acid position X28.
13. A polypeptide according to any of the preceding claims, wherein the polypeptide is lipidated with a structure of general formula L-Y1—Y2—Y3—Y4—Y5—Y6—Y7—Y8, wherein L is a lipid selected from C18DA or C20DA and further wherein each of Y1—Y8 are independently selected from: not present, [γE], [OEG], [eLys], or [AHX].
14. A polypeptide according to any of the preceding claims, wherein the lipid is selected from a list consisting of C18DA, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][γE][γE]-, C18DA[γE][γE][γE][γE]-, C18DA[γE][γE][γE][γE][γE]-, C18DA[γE][γE][γE][γE][γE][γE]-, C18DA[γE][γE][γE][γE][γE][γE][γE]-, C20DA, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][γE][γE]-, C20DA[γE][γE][γE][γE]-, C20DA[γE][γE][γE][γE][γE]-, C20DA[γE][γE][γE][γE][γE][γE]-, C20DA[γE][γE][γE][γE][γE][γE][γE]-, C18DA[eLys], C18DA[γE][eLys]-, C18DA[γE][γE][eLys]-, C18DA[γE][γE][γE][eLys]-, C18DA[γE][γE][γE][γE][eLys]-, C18DA[γE][γE][γE][γE][γE][eLys]-, C18DA[γE][γE][γE][γE][γE][γE][eLys]-, C20DA[eLys], C20DA[γE][eLys]-, C20DA[γE][γE][eLys]-, C20DA[γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][γE][eLys]-, C20DA[γE][γE][γE][γE][γE][γE][eLys]-, C18DA[OEG][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][γE][γE][OEG][OEG]-, C18DA[γE][γE][γE][γE][OEG][OEG]-, C18DA[γE][γE][γE][γE][γE][OEG][OEG]- , C20DA[OEG][OEG]-, C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][γE][γE][OEG][OEG]-, C20DA[γE][γE][γE][γE][OEG][OEG]-, C20DA[γE][γE][γE][γE][γE][OEG][OEG]-, C18DA[OEG]-, C18DA[γE][OEG]-, C18DA[γE][γE][OEG]-, C18DA[γE][γE][γE][OEG]-, C18DA[γE][γE][γE][γE][OEG]-, C18DA[γE][γE][γE][γE][γE][OEG]-, C18DA[γE][γE][γE][γE][γE][γE][OEG]-, C20DA[OEG]-, C20DA[γE][OEG]-, C20DA[γE][γE][OEG]-, C20DA[γE][γE][γE][OEG]-, C20DA[γE][γE][γE][γE][OEG]-, C20DA[γE][γE][γE][γE][γE][OEG]-, C20DA[γE][γE][γE][γE][γE][γE][OEG]-, C18DA[OEG][eLys]-, C18DA[γE][OEG][eLys]-, C18DA[γE][γE][OEG][eLys]-, C18DA[γE][γE][γE][OEG][eLys]-, C18DA[γE][γE][γE][γE][OEG][eLys]-, C18DA[γE][γE][γE][γE][γE][OEG][eLys]-, C20DA[OEG][eLys]-, C20DA[γE][OEG][eLys]-, C20DA[γE][γE][OEG][eLys]-, C20DA[γE][γE][γE][OEG][eLys]-, C20DA[γE][γE][γE][γE][OEG][eLys]-, C20DA[γE][γE][γE][γE][γE][OEG][eLys]-, C18DA[OEG][OEG][eLys]-, C18DA[γE][OEG][OEG][eLys]-, C18DA[γE][γE][OEG][OEG][eLys]-, C18DA[γE][γE][γE][OEG][OEG][eLys]-, C18DA[γE][γE][γE][γE][OEG][OEG][eLys]-, C20DA[OEG][OEG][eLys]-, C20DA[γE][OEG][OEG][eLys]-, C20DA[γE][γE][OEG][OEG][eLys]-, C20DA[γE][γE][γE][OEG][OEG][eLys]-, C20DA[γE][γE][γE][γE][OEG][OEG][eLys]-, C18DA[AHX], C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C18DA[γE][γE][γE][AHX]-, C18DA[γE][γE][γE][γE][AHX]-, C18DA[γE][γE][γE][γE][γE][AHX]-, C18DA[γE][γE][γE][γE][γE][γE][AHX]-, C20DA[AHX], C20DA[γE][AHX]-, C20DA[γE][γE][AHX]-, C20DA[γE][γE][γE][AHX]-, C20DA[γE][γE][γE][γE][AHX]-, C20DA[γE][γE][γE][γE][γE][AHX]-, C20DA[γE][γE][γE][γE][γE][γE][AHX]-.
15. A polypeptide according to any of the preceding claims, wherein the polypeptide is selected from group consisting of Compound 1 to Compound 305.
1. A polypeptide according to the general structure of Formula (I) or a salt or a pharmaceutically acceptable salt thereof,
2. A polypeptide according to item 1,
3. A polypeptide according to any one of the preceding items,
4. A polypeptide according to any one of the preceding items,
5. A polypeptide according to any one of the preceding items,
| Number | Date | Country | Kind |
|---|---|---|---|
| 22215510.3 | Dec 2022 | EP | regional |
