Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/26—Glucagons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons

Definitions

• the present invention relates to novel compounds that are agonists of the glucagon-like peptide 1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor with a protracted profile of action, suitable for oral administration to humans.
• GLP-1 glucagon-like peptide 1
• GIP glucose-dependent insulinotropic polypeptide
• Glucagon-like peptide 1 is a gut enteroendocrine cell-derived hormone and one of two prominent endogenous physiological incretins. GLP-1 improves glycemic control by stimulating glucose-dependent insulin secretion in response to nutrients (glucose), inhibits glucagon secretion from the pancreatic alpha-cells, slows gastric emptying, and induces body weight loss primary by decreasing food consumption.
• Glucose-dependent insulinotropic polypeptide GIP
• GIP the other prominent incretin, improves glycemic control by stimulation of insulin secretion in response to nutrients (fat, glucose). Furthermore, GIP appears to improve plasma lipid profile and to stimulate calcium accumulation in bones.
• GIP analogs have been shown to lower body weight and improve glycemic control, though comparatively less potent than GLP-1 analogs to lower body weight in rodent models (Mroz et al, Mol Metab, 2019, 20: 51-62). Moreover, GIP analogs induce body weight loss by additive/synergistic action with GLP-1 analogs in dual administration (Finan et al, Sci Transl Med, 2013, 5 (209): 209ra151; N ⁇ rregaard et al, Diabetes Obes Metab, 2018, 20 (1): 60-68), and as such represent suitable candidates for amplification of GLP-1-based pharmacology.
• GIPR agonism can be recruited as a non-redundant partner to GLP-1R agonism as a single molecule co-agonist to amplify GLP-1 metabolic benefits, as has been shown in preclinical animal models, most notably body weight loss and glycemic control (Finan et al, Sci Transl Med, 2013, 5 (209): 209ra151; Coskun et al, Mol Metab, 2018, 18: 3-14). Two different peptides with high potency dual incretin receptor agonism have advanced to multi-dose clinical studies.
• GLP-1 derivatives have been investigated clinically in the form of a once-daily tablet of semaglutide and the permeation enhancer sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) (Hedrington & Davis, Exp. Opin. Pharmacother. 2019, 20 (2): 133-141) to improve the very low typical exposure and bioavailability of GLP-1 derivatives following oral administration.
• SNAC sodium N-[8-(2-hydroxybenzoyl)amino]caprylate
• GLP-1/GIP co-agonists and their potential medical uses are described in several patent applications such as WO 2010/011439, WO 2013/164483, WO 2014/192284, WO 2015/067715, WO 2015/022420, WO 2015/086728, WO 2015/086729, WO 2016/111971, WO 2020/023386, U.S. Pat. No. 9,745,360, US 2014/162945, and US 2014/0357552.
• Patent applications disclosing oral delivery of GLP-1 derivatives are described in e.g. WO 2011/080103, WO 2012/080471, WO 2013/189988, and WO 2019/149880.
• the present invention relates to single molecule co-agonists comprising a peptide and a substituent, which react with both the human GLP-1 and GIP receptors with high potency and are suitable for daily oral administration to humans. This is achieved by the combination of certain peptide sequence variants with substituents via a single site acylation with a diacid based fatty acid.
• An aspect of the invention relates to a peptide having the amino acid sequence
• the invention relates to a peptide having the amino acid sequence
• a further aspect of the invention relates to a method for preparing the GLP-1/GIP co-agonists described herein.
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the GLP-1/GIP co-agonists compounds described herein.
• a further aspect of the invention relates to medical use of the GLP-1/GIP co-agonists described herein.
• the invention relates to use of the GLP-1/GIP co-agonists described herein for prevention or treatment of diabetes, obesity, and/or liver diseases.
• the present invention relates to compounds that are GLP-1 receptor and the GIP receptor agonists, also referred to as GLP-1/GIP receptor co-agonists or simply co-agonists.
• compound is used herein to refer to a molecular entity, and “compounds” may thus have different structural elements besides the minimum element defined for each compound or group of compounds. It follows that a compound may be a peptide or a derivative thereof, as long as the compound comprises the defined structural and/or functional elements.
• compound is also meant to cover pharmaceutically relevant forms hereof, i.e. a compound as defined herein or a pharmaceutically acceptable salt, amide, or ester thereof.
• analogue generally refers to a peptide, the sequence of which has one or more amino acid changes when compared to a reference amino acid sequence.
• An “analogue” may also include amino acid elongations in the N-terminal and/or C-terminal positions and/or truncations in the N-terminal and/or C-terminal positions.
• amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code. These three ways are fully equivalent.
• Amino acids are molecules containing an amino group and a carboxylic acid group, and, optionally, one or more additional groups, often referred to as a side chain.
• amino acid includes proteinogenic (or natural) amino acids (amongst those the 20 standard amino acids), as well as non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those which are naturally incorporated into proteins.
• Non-proteinogenic amino acids are either not found in proteins, or not produced by standard cellular machinery (e.g., they may have been subject to post-translational modification).
• Non-limiting examples of non-proteinogenic amino acids are Aib ( ⁇ -aminoisobutyric acid, or 2-aminoisobutyric acid), norleucine, norvaline as well as the D-isomers of the proteinogenic amino acids.
• the GLP-1/GIP receptor co-agonists described herein comprise or consist of a peptide and a substituent as defined below.
• the peptide is a synthetic peptide created to optimize the activity at the GLP-1 and GIP receptors.
• Compounds having a suitable receptor binding activity towards both the GLP-1 receptor and the GIP receptor have been identified as demonstrated in the examples herein.
• the compounds further display an extended half-life gained by the substituent comprising a fatty acid group.
• the carboxy terminus of a peptide holds a —COOH group.
• the compounds may optionally include an amide group (C( ⁇ O)—NH 2 ) at the C-terminus, which is a naturally occurring modification substituting —OH with —NH 2 , such as seen with native Exendin-4.
• the GLP-1/GIP receptor co-agonists described herein comprise a peptide and a substituent as defined below, in which the substituent is attached to the peptide backbone via an amino acid residue.
• the amino acid sequence of the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
• the amino acid sequence of the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
• X 39 is absent. In one embodiment, X 38 and X 39 are absent. In one embodiment, X 37 , X 38 and X 39 are absent. In one embodiment, X 36 , X 37 , X 38 and X 39 are absent. In one embodiment, X 35 , X 36 , X 37 , X 38 and X 39 are absent. In one embodiment, X 34 , X 35 , X 36 , X 37 , X 38 and X 39 are absent. In one embodiment, X 33 , X 34 , X 35 , X 36 , X 37 , X 38 and X 39 are absent.
• X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 and X 39 are absent.
• X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 and X 39 are absent.
• X 30 , X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 and X 39 are absent.
• X 32 X 33 X 34 X 35 is SSGA.
• X 32 X 33 X 34 X 35 is ESGA. In further such embodiments, X 32 X 33 X 34 X 35 is SKGA. In a further embodiment thereof, the peptide has an amide modification of the C-terminus.
• amino acid sequence of the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
• amino acid sequence of the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
• amino acid sequence of the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
• X 2 is Aib. In one embodiment, X 2 is A. In one embodiment, X 6 is F. In one embodiment, X 6 is V. In one embodiment, X 12 is I. In one embodiment, X 12 is Y. In one embodiment, X 13 is Y. In one embodiment, X 13 is A. In one embodiment, X 13 is L. In one embodiment, X 13 is I. In one embodiment, X 16 is K. In one embodiment, X 16 is E. In one embodiment, X 20 is Q. In one embodiment, X 20 is R. In one embodiment, X 20 is E. In one embodiment, X 20 is H. In one embodiment, X 21 is A. In one embodiment, X 21 is E.
• X 23 is I. In one embodiment, X 23 is V. In one embodiment, X 24 is E. In one embodiment, X 24 is Q. In one embodiment, X 24 is N. In one embodiment, X 28 is A. In one embodiment, X 28 is R. In one embodiment, X 30 is G. In one embodiment, X 31 is P.
• X 13 LEX 16 QAAX 20 X 21 FX 23 X 24 is selected from the group consisting of: LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and LLEEQAAREFIE.
• X 13 LEX 16 QAAX 20 X 21 FX 23 X 24 is LLEKQAAREFIN.
• X 13 LEX 16 QAAX 20 X 21 FX 23 X 24 is LLEKQAAREFIE.
• X 13 LEX 16 QAAX 20 X 21 FX 23 X 24 is LLEKQAAQEFIE.
• X 13 LEX 16 QAAX 20 X 21 FX 23 X 24 is LLEEQAAREFIE.
• amino acid sequence of the peptide is any one of SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 33, 34 and 35.
• the peptide has an amide modification of the C-terminus.
• the GLP-1 and GIP receptor agonists comprise or consist of a substituent as defined below covalently linked to a peptide.
• Such compounds may be referred to as derivatives of the peptide, as they are obtained by covalently linking a substituent to a peptide backbone.
• An aspect of the invention relates to a compound comprising a peptide and a substituent; wherein the amino acid sequence of the peptide is:
• An embodiment of the invention relates to a compound comprising a peptide and a substituent; wherein the amino acid sequence of the peptide is:
• the peptide may be defined as described herein above.
• the substituents described herein are attached to the peptide described herein via a lysine (K) residue in position 16 or 33.
• the substituent is attached to the peptide via the epsilon-amino group of a Lysine (K) when said Lysine is included at position 16 or 33.
• the substituent is a chemical structure covalently attached to the peptide that is capable of forming non-covalent complexes with plasma albumin, thereby promoting the circulation of the co-agonist with the blood stream, and also having the effect of protracting the time of action of the co-agonist, due to the fact that the complex of the co-agonist and albumin is only slowly removed by renal clearance.
• the substituent comprises a fatty acid group.
• the fatty acid group comprises a carbon chain which contains at least 8 consecutive —CH 2 — groups.
• the fatty acid group comprises at least 10 consecutive —CH 2 — groups, such as least 12 consecutive —CH 2 — groups, at least 14 consecutive —CH 2 — groups, at least 16 consecutive —CH 2 — groups, or such as at least 18 consecutive —CH 2 — groups.
• the fatty acid group comprises 8-20 consecutive —CH 2 — groups. In one embodiment, the fatty acid group comprises 10-18 consecutive —CH 2 — groups. In one embodiment, the fatty acid group comprises 12-18 consecutive —CH 2 — groups. In one embodiment, the fatty acid group comprises 14-18 consecutive —CH 2 — groups.
• the substituent consists of several elements, such as a protractor element and one or more linker elements.
• the term “protractor” is used to describe the fatty acid group which is the terminal part of the substituent responsible for extending half-life of the compound.
• the protractor may be defined by:
• Chem. 1 HOOC—(CH 2 ) n —CO—* wherein n is an integer in the range of 8-20, which may also be referred to as a C(n+2) diacid or as
• n is an integer in the range of 8-20.
• the substituent further comprises one or more linker elements.
• the linker elements are linked to each other and the protractor by amide bonds and referred to as “Z” (see further below).
• the number of linker elements may be at most 3, referred to as —Z1-Z2-Z3- where Z1 is connected with the protractor (Prot) and the last Z element is connected with the peptide, in which case the substituent may be referred to as Prot-Z1-Z2-Z3-.
• the symbol * above thus indicates the attachment point to Z1, which when bound via an amide bond is a nitrogen.
• the symbol * indicates the attachment point to the nitrogen of the neighbouring Z element.
• the substituent is defined by: Prot-Z1-Z2-Z3- wherein Prot- is selected from Chem1, Chem 1b, and wherein n is an integer in the range of 16-20.
• n 14, 15, 16, 17, or 18 in Chem. 1 or Chem. 1b.
• n 14, 16, or 18 in Chem. 1 or Chem. 1b.
• n 16, 17, 18, 19 or 20 in Chem. 1 or Chem. 1b.
• n is 16, 18 or 20 in Chem. 1 or Chem. 1b.
• n is 18 or 20 in Chem. 1 or Chem. 1b.
• the protractor (Prot) is a C16 diacid or a C18 diacid.
• the protractor (Prot) is a C18 diacid or a C20 diacid.
• the protractor (Prot) is a C16, C18 diacid or a C20 diacid.
• bond means a covalent bond.
• linker element of Z1-Z3 is defined as a bond, it is equivalent to a situation wherein said linker element is absent.
• the indication herein below that any of Z1-Z3 is a bond may also be read as any of Z1-Z3 being absent, so that the previous Z element is covalently linked to the next Z element that is not “a bond” (or absent).
• the linker elements Z1-Z3 are individually selected from chemical moieties capable of forming amide bonds, including amino acid like moieties, such as Glu, ⁇ Glu (also termed gamma Glu or gGlu and defined by *—NH—CH—(COOH)—CH 2 —CH 2 —CO—*), ⁇ -Lys (also termed epsilon Lys or eLys and defined by *—NH—(CH 2 ) 4 —CH(NH 2 )—CO—*), Ser, Ala, Thr, Ado, Aeep and Aeeep and further moieties as described below.
• amino acid like moieties such as Glu, ⁇ Glu (also termed gamma Glu or gGlu and defined by *—NH—CH—(COOH)—CH 2 —CH 2 —CO—*), ⁇ -Lys (also termed epsilon Lys or eLys and defined by *—NH—(CH 2 ) 4
• Z1 is selected from ⁇ Glu, Glu, or a bond.
• Z1 is ⁇ Glu
• Z2 and Z3, are selected, independently of each other, from Glu, ⁇ -Lys, ⁇ Glu, Gly, Ser, Ala, Thr, Ado, Aeep, Aeeep and a bond.
• Glu, Gly, Ser, Ala, Thr are amino acid residues well known in the art.
• ⁇ -Lys is defined by Chem. 2: *—NH—(CH 2 ) 4 —CH(NH 2 )—CO—* which may also be described by
• ⁇ Glu is defined by Chem. 3: *—NH—CH(COOH)—(CH 2 ) 2 —CO—* which may also be described by
• Ado is defined by Chem. 4: *—NH—(CH 2 ) 2 —O—(CH 2 ) 2 O—CH 2 —CO—* may also be referred to as 8-amino-3,6-dioxaoctanoic acid and which may also be described by
• Aeep is defined by Chem. 5: *NH—CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 CO*, which may also be described by
• Aeeep is defined of Chem. 6: *NH—CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 CO*, which may also be described by
• Z2 and Z3 are selected, independently of each other, from Glu, ⁇ -Lys, ⁇ Glu, Gly, Ala, Ado, Aeep, Aeeep and a bond.
• Z2 and Z3 are selected, independently of each other, from Glu, ⁇ -Lys, ⁇ Glu, Gly, Ala, Ado and a bond.
• Z2 and Z3 are selected, independently of each other, from Glu, ⁇ -Lys, ⁇ Glu, Gly, Ado and a bond.
• Z2 and Z3 are selected, independently of each other, from ⁇ -Lys, ⁇ Glu, Gly, Ado and a bond.
• Z2 and Z3 are selected, independently of each other, from ⁇ -Lys, ⁇ Glu, Ado and a bond.
• Z2 and Z3 are ⁇ -Lys or Ado.
• Z2 and Z3 are Ado.
• Z2 and Z3 are ⁇ -Lys.
• the substituent is selected from substituents A, B, C, D, and E defined as below.
• the substituent is selected from substituents A, B and C defined as follows:
• the substituent is covalently attached to a lysine residue of the co-agonist by acylation, i.e. via an amide bond formed between a carboxylic acid group of the substituent and the epsilon-amino group of the lysine residue.
• the substituent is covalently attached to a lysine residue in position 16 of the peptide backbone by acylation, i.e., via an amide bond formed between a carboxylic acid group of the substituent and the epsilon amino group of the lysine residue.
• the substituent is covalently attached to a lysine residue in position 33 of the peptide backbone by acylation, i.e., via an amide bond formed between a carboxylic acid group of the substituent and the epsilon amino group of the lysine residue.
• the co-agonists may exist in different stereoisomeric forms having the same molecular formula and sequence of bonded atoms but differing only in the three-dimensional orientation of their atoms in space.
• the stereoisomerism of the exemplified co-agonists is indicated in the experimental section, in the names as well as the structures, using standard nomenclature. Unless otherwise stated the invention relates to all stereoisomeric forms of the embodied derivative.
• the functional activity of the GLP-1/GIP receptor agonists as described herein can be tested in vitro as described herein in Example 2.
• EC 50 half maximal effective concentration
• the in vitro potency of compounds may thus be determined as described herein and the EC 50 determined.
• the in vitro potency may, e.g., be determined in a medium containing membranes expressing the appropriate GLP-1 and/or GIP receptor, and/or in an assay with whole cells expressing the appropriate GLP-1 and/or GIP receptor.
• the functional response of the human GLP-1 and/or GIP receptor may be measured in a reporter gene assay, e.g. in a stably transfected BHK cell line that expresses the human GLP-1 and/or GIP receptor and contains the DNA for the cAMP response element (CRE) coupled to a promoter and the gene for firefly luciferase (CRE luciferase).
• CRE cAMP response element
• Luciferase may be determined by adding luciferin, which by the enzyme is converted to oxyluciferin and produces bioluminescence, which is measured as a reporter of the in vitro potency.
• luciferin which by the enzyme is converted to oxyluciferin and produces bioluminescence, which is measured as a reporter of the in vitro potency.
• One example of such an assay is described in Example 2 as described herein.
• the compounds may include a substituent designed to bind albumin, it is also important to note that the receptor activity may be affected by the presence or absence of human serum albumin (HSA) in the assay medium.
• HSA human serum albumin
• the compounds have potent in vitro effects to activate the human GLP-1 and GIP receptors.
• the compounds are capable of activating the human GLP-1 and GIP receptors in vitro with an EC 50 of less than 20 pM in CRE luciferase reporter assays as described in Example 2 herein, when performed without HSA.
• the compounds have an in vitro potency at the human GLP-1 and GIP receptors determined using the method of Example 2 corresponding to an EC 50 at or below 100 pM, more preferably below 50 pM, or most preferably below 20 pM.
• the EC 50 in human GLP-1 and GIP receptors assays are both 1-25 pM, such as 1-20 pM, such as 1-15 pM or such as 1-10 pM.
• the pharmacokinetic properties of the co-agonistic compounds may further be determined in vivo via pharmacokinetic (PK) studies.
• Animal models such as the mouse, rat, monkey, dog, or pig may be used to perform this characterization.
• animals are typically administered with a single dose of the drug, either intravenously, subcutaneously (s.c.), or orally (p.o.) in a relevant formulation.
• Blood samples are drawn at predefined time points after dosing, and samples are analysed for concentration of drug with a relevant quantitative assay. Based on these measurements, time-plasma concentration profiles for the compound of study are plotted and a so-called non-compartmental pharmacokinetic analysis of the data is performed.
• An important parameter is the terminal half-life as a long half-life indicates that less frequent administration of a compound may be possible.
• the terminal half-life (t 1/2 ) in vivo may be measured using a suitable model, such as after i.v. administration in minipigs described in Example 3; or after p.o administration in dogs described in Example 4.
• the terminal half-life is half-life (t 1/2 ) in vivo in minipigs after iv. administration, e.g. as described in Example 3 herein.
• the terminal half-life in minipigs is at least 24 hours, such as at least 30 hours, or such as at least 40 hours.
• the terminal half-life is half-life (t 1/2 ) in vivo in dogs after p.o. administration, e.g. as described in Example 4 herein.
• the terminal half-life in dogs is at least 24 hours, such as at least 40 hours, or such as at least 50 hours.
• the co-agonists as described herein are in the form of a pharmaceutically acceptable salt.
• Salts are e.g. formed by a chemical reaction between a base and an acid, e.g.: 2NH 3 +H 2 SO 4 ⁇ (NH 4 ) 2 SO 4 .
• the salt may be a basic salt, an acid salt, or it may be neither (i.e. a neutral salt).
• Basic salts produce hydroxide ions and acid salts hydronium ions in water.
• the salts of the co-agonists may be formed with added cations or anions between anionic or cationic groups, respectively. These groups may be situated in the peptide and/or in the substituent of the derivatives.
• anionic groups include any free carboxylic acid groups in the substituent, if any, as well as in the peptide.
• the peptide may include a free carboxylic acid group at the C-terminus, if present, as well as any free carboxylic acid group of internal acidic amino acid residues such as Asp and Glu.
• Non-limiting examples of cationic groups include any free amino groups in the substituent, if any, as well as in the peptide.
• the peptide may include a free amino group at the N-terminus, if present, as well as any free imidazole or amino group of internal basic amino acid residues such as His, Arg, and Lys.
• the peptide or derivative is in the form of a pharmaceutically acceptable salt.
• the co-agonists may for instance be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see e.g. Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999; Florencio Zaragoza Dörwald, “Organic Synthesis on Solid Phase”, Wiley-VCH Verlag GmbH, 2000; and “Fmoc Solid Phase Peptide Synthesis”, Edited by W. C. Chan and P. D. White, Oxford University Press, 2000.
• the compounds may be produced by recombinant methods, e.g. by culturing a host cell containing a DNA sequence encoding the peptide sequence and capable of expressing the peptide, in a suitable nutrient medium under conditions permitting the expression of the peptide.
• host cells suitable for expression of these peptides are: Escherichia coli, Saccharomyces cerevisiae , as well as mammalian BHK or CHO cell lines.
• co-agonists that include non-natural amino acids and/or covalently attached substituents may be produced as described in the experimental part.
• a further aspect of the invention relates to a method for preparing the peptides described herein.
• a further aspect of the invention relates to a method for preparing the GLP-1/GIP co-agonists described herein.
• the method for preparing a compound as described herein comprises a step of solid phase peptide synthesis.
• the substituent may be built sequentially as part of the solid phase peptide synthesis or produced separately and attached via the lysine residue after peptide synthesis.
• the compounds are produced by a two-step process whereby two peptide fragments are ligated after attachment of the substituent to one of the peptide fragments.
• compositions comprising a GLP-1/GIP receptor co-agonist as described herein.
• Compositions comprising the compound or a pharmaceutically acceptable salt hereof, and optionally one or more a pharmaceutically acceptable excipients may be prepared as is known in the art.
• excipient broadly refers to any component other than the active therapeutic ingredient(s).
• the excipient may be an inert substance, an inactive substance, and/or a not medicinally active substance.
• the excipient may serve various purposes, e.g. as a carrier, vehicle, filler, binder, lubricant, glidant, disintegrant, flow control agent, crystallization inhibitor, solubilizer, stabilizer, coloring agent, flavoring agent, surfactant, emulsifier or combinations thereof and/or to improve administration, and/or to improve absorption of the active substance.
• the amount of each excipient used may vary within ranges conventional in the art.
• the pharmaceutical composition may be a solid formulation, e.g. a freeze-dried or spray-dried composition, which may be used as is, or whereto the physician or the patient adds solvents, and/or diluents prior to use.
• the pharmaceutical composition may be a solid formulation consisting of the active ingredient, a salt of N-[8-(2-hydroxybenzoyl)amino]caprylate, and one or more further excipients as is known in the art, e.g. using any one or more of the formulations described in WO 2012/080471, WO 2013/189988, or WO 2019/149880.
• the pharmaceutical composition is a liquid formulation, such as an aqueous formulation.
• Liquid compositions suitable for injection, can be prepared using conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product.
• a GLP-1/GIP co-agonist as described herein is dissolved in a suitable buffer at a suitable pH.
• the composition may be sterilized, for example, by sterile filtration.
• a further aspect of the invention relates to the use of GLP-1/GIP receptor co-agonist compounds as described herein as a medicament.
• the compounds described herein are for use in the following medical treatments:
• the compounds are for use in a method for prevention and/or treatment of diabetes and/or obesity.
• the compounds are for use in a method for treatment of diabetes and/or obesity.
• the compounds are for use in a method for treatment or prevention of type 2 diabetes.
• the compounds are for use in a method for treatment of type 2 diabetes.
• the compounds are for use in a method for treatment or prevention of obesity.
• the compounds are for use in a method for treatment of obesity.
• the compounds are for use in a method for weight management. In one embodiment, the compounds are for use in a method for reduction of appetite. In one embodiment, the compounds are for use in a method for reduction of food intake.
• LLEKQAAREFIN LLEKQAAREFIE
• LLEKQAAQEFIE LLEKQAAQEFIE
• LLEEQAAREFIE LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and LLEEQAAREFIE.
• LLEKQAAREFIN LLEKQAAREFIE
• LLEKQAAQEFIE LLEKQAAQEFIE
• LLEEQAAREFIE LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and LLEEQAAREFIE.
• Ado also called OEG: 8-amino-3,6-dioxaoctanoic acid
• API active pharmaceutical ingredient
• DIPEA N,N-diisopropylethylamine
• DMEM Dulbecco's Modified Eagle's Medium
• DPBS Dulbecco's phosphate buffered saline
• EDTA ethylenediaminetetraacetic acid
• FBS fetal bovine serum
• GIP glucose-dependent insulinotropic polypeptide
• GIPR glucose-dependent insulinotropic polypeptide receptor
• GLP-1 glucagon-like peptide 1
• GLP-1R glucagon-like peptide 1 receptor
• HSA human serum albumin
• IPGTT intraperitoneal glucose tolerance test
• OEG 8-amino-3,6-dioxaoctanoic acid (also called Ado)
• PBS phosphate buffered saline
• TIS triisopropylsilane
• Trt triphenylmethyl or trityl
• Trx tranexamic acid
• SPPS methods including methods for de-protection of amino acids, methods for cleaving the peptide from the resin, and for its purification), as well as methods for detecting and characterising the resulting peptide (LCMS methods) are described here below.
• Resins employed for the preparation of C-terminal peptide amides were H-Rink Amide-ChemMatrix resin (loading e.g. 0.5 mmol/g).
• the Fmoc-protected amino acid derivatives used were the standard recommended: 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-
• N-terminal amino acid was Boc protected at the alpha-amino group, either by using a reagent with the Boc group pre-installed (e.g. Boc-Tyr(tBu)-OH for peptides with Tyr at the N-terminus) or by exchanging the N-terminal Fmoc protective group for the Boc protective group after installation of the amino acid at the peptide N-terminus.
• Boc-Tyr(tBu)-OH for peptides with Tyr at the N-terminus
• Fmoc protective group for the Boc protective group after installation of the amino acid at the peptide N-terminus.
• the following suitably protected building blocks such as but not limited to Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH), Boc-Lys(Fmoc)-OH, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedioic acid mono-tert-butyl ester, nonadecanedioic acid mono-tert-butyl ester, eicosanedioic acid mono-tert-butyl ester, tetradecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy) benzoic acid tert-butyl ester were used. All operations stated below were performed within a 0.1-0.2 mmol synthesis scale range.
• SPPS was performed using Fmoc based chemistry on a Protein Technologies SymphonyX solid phase peptide synthesizer, using the manufacturer supplied protocols with minor modifications. Mixing was accomplished by occasional bubbling with nitrogen.
• the step-wise assembly was performed using the following steps: 1) pre-swelling of resin in DMF; 2) Fmoc-deprotection by the use of 20% (v/v) piperidine in DMF for two treatments of 10 min each; 3) washes with DMF to remove piperidine; 4) coupling of Fmoc-amino acid by the addition of Fmoc-amino acid (12 equiv) and Oxyma Pure® (12 equiv) as a 0.6 M solution each in DMF, followed by addition of DIC (12 equiv) as a 1.2 M solution in DMF, followed by the addition of DMF to reduce the final concentration of each component to 0.3 M, then mixing for 0.5-4 h; 4) washes with DMF to remove excess reagents; 5) final was
• the protected peptidyl resin was synthesized according to the Fmoc strategy on an Applied Biosystems 431A solid-phase peptide synthesizer using the manufacturer supplied general Fmoc protocols. Mixing was accomplished by vortexing and occasional bubbling with nitrogen.
• the step-wise assembly was done using the following steps: 1) activation of Fmoc-amino acid by dissolution of solid Fmoc-acid acid (10 equiv) in Cl-HOBt (10 equiv) as a 1 M solution in NMP, then addition of DIC (10 equiv) as a 1 M solution in NMP, then mixing simultaneous to steps 2-3; 2) Fmoc-deprotection by the use of 20% (v/v) piperidine in NMP for one treatment of 3 min then a second treatment of 15 min; 3) washes with NMP to remove piperidine; 4) addition of activated Fmoc-amino acid solution to resin, then mixing for 45-90 min; 4) washes with NMP to remove excess reagents; 5) final washes with DCM at the completion of the assembly.
• the standard protected amino acid derivatives listed above were supplied in pre-weighed cartridges (from e.g. Midwest Biotech), and non-standard derivatives were weighed by hand.
• Some amino acids such as, but not limited to, those following a sterically hindered amino acid (e.g. Aib) were “double coupled” to ensure reaction completion, meaning that after the first coupling (e.g. 45 min) the resin is drained, more reagents are added (Fmoc-amino acid, DIC, Cl-HOBt), and the mixture allowed to react again (e.g. 45 min).
• SPPS was performed using Fmoc based chemistry on a PreludeX solid phase peptide synthesizer, using the manufacturer supplied protocols with minor modifications. Mixing was accomplished by shaking at 350 rpm and occasional bubbling with nitrogen.
• the step-wise assembly was performed using the following steps: 1) pre-swelling of resin in DMF; 2) Fmoc-deprotection by the use of 20% (v/v) piperidine in DMF for one treatment of 3 min at 70° C.; 3) washes with DMF to remove piperidine; 4) coupling of Fmoc-amino acid by the addition of Fmoc-amino acid (12 equiv) and Oxyma Pure® (12 equiv) as a 0.4 M solution each in DMF, followed by addition of DIC (12 equiv) as a 1.2 M solution in DMF, then mixing for 5 min at 70° C.; 4) washes with DMF to remove excess reagents; 5) final washes with DCM at the completion
• N-epsilon-lysine protection Mtt protection group was removed by washing the resin with 30% HFIP in DCM for two treatments of 45 min each, following by washing with DCM and DMF.
• Acylation was performed on a Protein Technologies SymphonyX solid phase peptide synthesizer using the protocols described in method SPPS_A using stepwise addition of building blocks, such as, but not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedioic acid mono-tert-butyl ester, and eicosanedioic acid mono-tert-butyl ester.
• building blocks such as, but not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino
• N-epsilon-lysine protection Mtt protection group was removed by washing the resin with 30% HFIP in DCM for two treatments of 45 min each, following by washing with DCM and DMF.
• Acylation was performed on an Applied Biosystems 431A solid-phase peptide synthesizer using the protocols described in method SPPS_B using stepwise addition of building blocks, such as, but not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedioic acid mono-tert-butyl ester, and eicosanedioic acid mono-tert-butyl ester.
• building blocks such as, but not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8
• N-epsilon-lysine protection Mtt protection group was removed by washing the resin with 30% HFIP in DCM for two treatments of 45 min each, following by washing with DCM and DMF.
• Acylation was performed on a Protein Technologies PreludeX solid phase peptide synthesizer using the protocols described in method SPPS_C using stepwise addition of building blocks, such as, but are not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedioic acid mono-tert-butyl ester, and eicosanedioic acid mono-tert-butyl ester.
• building blocks such as, but are not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-
• the peptidyl resin was washed with DCM and dried, then treated with TFA/water/TIS (95:2.5:2.5 v/v/v) for approximately 2 h, followed by precipitation with diethyl ether.
• the precipitate was washed with diethyl ether, dissolved in a suitable solvent (e.g. 2:1 water/MeCN), and let stand until all labile adducts decomposed.
• Purification was performed by reversed-phase preparative HPLC (Waters 2545 binary gradient module, Waters 2489 UV/Visible detector, Waters fraction collector Ill) on a Phenomenex Luna C8(2) column (10 pM particle size, 100 ⁇ pore size, 250 ⁇ 21.2 mm dimensions).
• the freeze-dried peptide isolated from method CP_A was dissolved to 5-20 mg/mL in an appropriate aqueous buffer (e.g. 4:1 water/MeCN, 0.2 M sodium acetate) and adjusted to pH 7-8 with 1 M NaOH if necessary to achieve full solubility.
• the buffered solutions containing the peptide were salt-exchanged using a Sep-Pak C18 cartridge (0.5-2 g): The cartridge was first equilibrated with 4 column volumes of isopropanol, then 4 column volumes of MeCN, then 8 column volumes of water. The peptide solution was applied to the cartridge, and the flow through was reapplied to ensure complete retention of peptide.
• the cartridge was washed with 4 column volumes of water, then 10 column volumes of a buffer solution (e.g. pH 7.5) containing such as, but not limited to, NaHCO 3 , NaOAc, or Na 2 HPO 4 .
• a buffer solution e.g. pH 7.5
• the column was washed with 4 column volumes of water, and the peptide was eluted with 5-10 column volumes of 50-80% MeCN in water.
• the peptide-containing eluent was freeze-dried to afford the peptide sodium salt as a white solid, which was used as such.
• LCMS_A was performed on a setup consisting of an Agilent 1260 Infinity series HPLC system and an Agilent Technologies 6120 Quadrupole MS. Eluents: A: 0.05% TFA in water; B: 0.05% TFA in 9:1 MeCN/water.
• LCMS_B was performed on a setup consisting of an Agilent 1260 Infinity series HPLC system and an Agilent Technologies 6120 Quadrupole MS. Eluents: A: 0.05% TFA in water; B: 0.05% TFA in 9:1 MeCN/water.
• the compounds are in the following described using single letter amino acid codes, except for Aib.
• the substituent is included after the lysine (K) residue to which it is attached.
• the purpose of this example is to test the functional activity, or potency, of the compounds in vitro at the human GLP-1 and GIP receptors.
• the in vitro functional potency is the measure of target receptor activation in a whole cell assay.
• the potencies of derivatives of Example 1 were determined as described below. Human GLP-1(7-37) and human GIP were included in appropriate assays for comparison.
• In vitro functional potency was determined by measuring the response of the target receptor in a reporter gene assay in individual cell lines.
• the assay was performed in stably transfected BHK cell lines that expresses one of the following G-protein coupled receptors: human GLP-1 receptor or human GIP receptor; and where each cell line contains the DNA for the cAMP response element (CRE) coupled to a promoter and the gene for firefly luciferase (CRE luciferase).
• CRE cAMP response element
• luciferase substrate luciferin
• luciferin luciferase substrate
• the cells lines used in these assays were BHK cells with BHKTS13 as a parent cell line.
• the cell lines were derived from a clone containing the CRE luciferase element and were established by further transfection with the respective human receptor to obtain the relevant cell line: BHK CRE luc2P hGLP-1R or BHK CRE luc2P hGIPR.
• the cells were cultured at 37° C. with 5% CO 2 in Cell Culture Medium. They were aliquoted and stored in liquid nitrogen. The cells were kept in continuous culture and were seeded out the day before each assay.
• Pluronic F-68 10% (Gibco 2404), human serum albumin (HSA; Sigma A9511), 10% fetal bovine serum (FBS; Invitrogen 16140-071), chicken egg white ovalbumin (Sigma A5503), DMEM w/o phenol red (Gibco 21063-029), DMEM (Gibco 12430-054), 1 M Hepes (Gibco 15630), Glutamax 100 ⁇ (Gibco 35050), G418 (Invitrogen 10131-027), hygromycin (Invitrogen 10687-010), and steadylite plus (PerkinElmer 6016757).
• Pluronic F-68 10% (Gibco 2404), human serum albumin (HSA; Sigma A9511), 10% fetal bovine serum (FBS; Invitrogen 16140-071), chicken egg white ovalbumin (Sigma A5503), DMEM w/o phenol red (Gibco 21063-029), DMEM (Gibco 12
• GLP-1R Cell Culture Medium consisted of DMEM medium with 10% FBS, 500 pg/mL G418, and 300 pg/mL hygromycin.
• GIPR Cell Culture Medium consisted of DMEM medium with 10% FBS, 400 pg/mL G418, and 300 pg/mL hygromycin.
• Assay Buffer consisted of DMEM w/o phenol red, 10 mM Hepes, 1 ⁇ Glutamax. 1% ovalbumin, and 0.1% Pluronic F-68 with the addition of HSA at twice the final assay concentration. The Assay Buffer was mixed 1:1 with an equal volume of the test compound in Assay Buffer to give the final assay concentration of HSA.
• the assay plate was incubated for 3 h in a 5% CO 2 incubator at 37° C. 7) The cells were washed once with DPBS. 8) A 100 ⁇ l aliquot of DPBS was added to each well of the assay plate. 9) A 100 ⁇ l aliquot of steadylite plus reagent (light sensitive) was added to each well of the assay plate. 10) Each assay plate was covered with aluminum foil to protect it from light and shaken at 250 RPM for 30 min at room temperature. 11) Each assay plate was read in a microtiter plate reader.
• the data from the microtiter plate reader was first regressed in an Excel in order to calculate the x-axis, log scale concentrations based on the individual test compound's stock concentration and the dilutions of the assay. This data was then transferred to GraphPad Prism software for graphing and statistical analysis. The software performs a non-linear regression (log(agonist) vs response). EC 50 values which were calculated by the software and reported in pM are shown in Tables 1-3 below. A minimum of two replicates was measured for each sample. The reported values are averages of the replicates.
• the compounds of the present invention display potent functional activation of the human GLP-1R and human GIPR receptors under the given conditions. Alteration of the peptide and/or the substituent resulted in unpredictable changes in the measured potencies at each of the two receptors, as well as the ratio of potencies between the two receptors, for each compound.
• the purpose of this example is to determine the half-life in vivo of the derivatives of the present invention after i.v. administration to minipigs, i.e. the prolongation of their time in the body and thereby their time of action. This is done in a pharmacokinetic (PK) study, where the terminal half-life of the derivative in question is determined.
• terminal half-life is generally meant the period of time it takes to halve a certain plasma concentration, measured after the initial distribution phase.
• mice Female Göttingen minipigs were obtained from Ellegaard Göttingen Minipigs (Dalmose, Denmark) approximately 7-14 months of age and weighing from approximately 16-35 kg were used in the studies. The minipigs were housed individually and fed restrictedly once daily with SDS minipig diet (Special Diets Services, Essex, UK).
• the animals were fasted for approximately 18 hours before dosing and from 0 to 4 hours after dosing but had adlibitum access to water during the whole period.
• the sodium salts of compounds of Examples 1 were dissolved to a concentration of 20-40 nmol/mL in a buffer containing 0.007% polysorbate 20, 50 mM sodium phosphate, 70 mM sodium chloride, pH 7.4.
• Intravenous injections (the volume corresponding to usually 1.5-2 nmol/kg, for example 0.1 mL/kg) of the compounds were given through one catheter, and blood was sampled at predefined time points for up to 14 days post dosing (preferably through the other catheter). Blood samples (for example 0.8 mL) were collected in 8 mM EDTA buffer and then centrifuged at 4° C. and 1942 g for 10 minutes.
• the tested compounds of the present invention have very long half-lives as compared to the half-lives of hGLP-1 and hGIP measured in man to be approximately 2-4 min and 5-7 min, respectively (Meier et al., Diabetes, 2004, 53(3): 654-662).
• the measured half-lives in minipigs predict half-lives in humans sufficient for at least once-weekly administration via liquid injection or at least once-daily administration via oral tablet.
• alterations to the peptide sequence led to appreciable differences in half-lives between co-agonists tested herein even though these co-agonists incorporate the same substituent.
• the purpose of this example is to determine the half-life and plasma exposure in vivo of the compounds of the present invention after p.o. administration to beagle dogs, i.e. the terminal half-life and concentration of test substance that reaches circulation with time. This is done in a pharmacokinetic (PK) study, where these parameters of the compound in question are determined.
• terminal half-life is generally meant the period of time it takes to halve a certain plasma concentration, measured after the initial distribution phase.
• Tablet compositions comprising the test substance and SNAC (sodium N-(8-(2-hydroxybenzoyl)amino)caprylate) were prepared according to methods known to the person skilled in the art by mixing test substance with roller compacted SNAC and magnesium stearate as e.g. described in WO 2019/149880.
• the amount of SNAC in the tablet composition was 100-300 mg
• the amount of magnesium stearate in the tablet composition was 7.7 mg
• the target amount of each test substance in the tablet composition was 3-4 mg.
• the dogs were dosed in a fasting state.
• the dogs were group housed in pens (12 hours light: 12 hours dark), and fed individually and restrictedly once daily with Royal Canin Medium Adult dog food (Royal Canin Products, China Branch, or Brogaarden A/S, Denmark).
• the dogs were used for repeated PK studies with a suitable wash-out period between successive dosing. An appropriate acclimatization period was given prior to initiation of the first PK study. All handling, dosing, and blood sampling of the animals were performed by trained and skilled staff.
• the dogs were fasted overnight and from 0 to 4 hours after dosing.
• the dogs were restricted to water 1 hour before dosing until 4 hours after dosing, but otherwise had ad libitum access to water during the whole period.
• compositions were administered by a single oral dosing to the dogs in groups of 6-8 dogs.
• the tablets were administered in the following manner: 10 min prior to tablet administration, the dogs may be dosed subcutaneously with approximately 3 nmol/kg of SEQ ID NO: 48, then tablets were placed in the back of the mouth of the dog to prevent chewing. The mouth was then closed, and 10 mL of tap water was given by syringe or gavage to facilitate swallowing of the tablet.
• One blood sample was drawn before dosing, and additional samples were drawn at predefined time points after dosing for up to 240 hours, such as up to 10 hours, to adequately cover the full plasma concentration-time absorption profile of the test substance.
• For each blood sample time point approximately 0.8 mL of whole blood was collected in a 1.5 mL EDTA-coated tube, which was gently turned to mix the sample with EDTA.
• Blood samples were collected in EDTA buffer (8 mM) and then centrifuged at 4° C. 2000 g for 10 minutes. Plasma was pipetted into Micronic tubes on dry ice, and kept at ⁇ 20° C. or lower until analysis.
• Blood samples were taken as appropriate, for example from a venflon in the cephalic vein in the front leg for the first 2 hours and then with syringe from the jugular vein for the rest of the time points. The first few drops were allowed to drain from the venflon to avoid heparin saline from the venflon in the sample.
• the plasma was analysed for test substance using LC-MS (Liquid Chromatography-Mass Spectrometry) as known to the person skilled in the art.
• the system consisted of either: a Thermo Fisher QExactive mass spectrometer equipped with a 10-valve interface module TurboFlow system, CTC HTS PAL autosampler, Accela 1250 pumps, and Hot Pocket column oven; or a Thermo Fisher QExactive Plus mass spectrometer equipped with a valve interface module TurboFlow system, TriPlus RSI autosampler, Dionex UltiMate 3000 pumps, and Hot Pocket column oven.
• RP-HPLC separation was achieved using a linear gradient of 1:1 acetonitrile/methanol in 1% aqueous formic acid using either: a Phenomenex Onyx Monolithic C18 column (50 ⁇ 2.0 mm) and a flow rate of 0.8 mL/min at 30° C.; or an Agilent Poroshell 120 SB-C18 column (50 ⁇ 2.1 mm, 2.7 ⁇ m) at a flow rate of 0.4 mL/min at 60° C.
• the mass spectrometer was operated in either positive ionization SIM mode or positive ionization PRM mode.
• a plasma concentration-time profile was analysed by a non-compartmental model in Pharsight Phoenix WinNonLin ver. 6.4 software or other relevant software for PK analysis, and the resulting terminal half-life (t 1/2 ), maximum plasma concentration per dose (C max /D), time for maximum plasma concentration (t max ), and area under the curve to infinity per dose (AUC/D) were determined.
• Summary statistics of pharmacokinetic results were presented as median (for t max ), hormonic mean (t 1/2 ), or arithmetic mean (C max , AUC).
• the tested compounds of the present invention demonstrate oral bioavailability in this model, as concentrations of the compound in plasma were detected (C max /D>0 and AUC/D>0) following oral administration. Furthermore, the tested compounds of the present invention further have very long half-lives as compared to the half-lives of hGLP-1 and hGIP measured in man to be approximately 2-4 min and 5-7 min, respectively (Meier et al., Diabetes, 2004, 53(3): 654-662). Alterations to the peptide sequence led to unpredictable differences in t 1/2 , C max /D and AUC/D between co-agonists even when the same substituent was incorporated. Additionally, an unexpectedly large difference in t 1/2 was observed depending on the position of the amino acid attached to the substituent, such as eg., Compound No. 43 compared to all other compounds tested herein.

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