Classifications

• • 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
  • 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
• • 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
• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones

Definitions

• the invention relates to compounds that are dual acting amylin and calcitonin receptor agonists.
• the invention also relates to a pharmaceutical composition, suitable for, but not limited to, oral administration, which comprises the abovementioned compounds.
• the compounds and pharmaceutical composition comprising it may be used for the medical treatment of individuals with overweight or obesity, with or without associated comorbidities; diabetes cardiovascular diseases, non-alcoholic steatohepatitis (NASH) and cognitive impairment, such as that caused by Alzheimer’s disease.
• NASH non-alcoholic steatohepatitis
• Overweight and obesity are the abnormal or excessive accumulation of body fat that present a risk to an individual’s overall health.
• a body mass index (BMI) over 25 is classified as overweight, and a BMI of over 30 is classified obese.
• Obesity is a leading risk factor in numerous serious conditions, including type 2 diabetes and its associated co-morbidities, and cardiovascular diseases such as heart disease and stroke, which are the leading causes of death worldwide.
• Obesity is now recognized by the World Health Organization (WHO) as an issue that has grown to epidemic proportion, even in children. In 2016, 1.9 billion adults worldwide were reportedly obese; in 2019, 38.3 million children under the age of 5 worldwide were reportedly obese. According to the WHO, 422 million people worldwide have diabetes and 1.6 million deaths are directly attributed to diabetes each year. There is, therefore, a huge incentive for the individual, as well as society, for preventing and/or treating obesity.
• WHO World Health Organization
• Amylin is a 37-amino acid long polypeptide hormone that is produced in and cosecreted with insulin from the pancreatic beta (P)-cell. Endogenous amylin has a half-life of approximately 15-20 minutes. It produces its effects in several different organ systems, primarily acting via amylin receptors 1-3 (AMYR1-3). Amylin is an important regulator of energy metabolism in health and disease, inhibiting glucagon secretion, delaying gastric emptying, signaling satiety, and suppressing appetite. Other amylin actions have also been reported, such as on the cardiovascular system and on bone.
• amylin receptor agonists may be useful for the treatment of overweight, obesity, type 1 diabetes and/or type 2 diabetes.
• Symlin® which contains an amylin receptor agonist (pramlintide acetate) as the active pharmaceutical ingredient.
• Symlin® a liquid pharmaceutical formulation for subcutaneous administration, is approved for use in patients with type 1 or type 2 diabetes who use basal and mealtime insulin and have failed to achieve desired glycemic control, despite optimal insulin therapy.
• Pramlintide for use in overweight and obese patients was also investigated in the clinic. Pramlintide has a short biological half-life (less than 1 hour), necessitating administration thrice daily. Consequently, there is a large diurnal difference in the pramlintide plasma levels of patients treated with it.
• a fixed-dose combination of an amylin receptor agonist, cagrilintide, and a GLP-1 receptor agonist, semaglutide is currently under investigation for the treatment of overweight and obesity (Lancet 2021; 397: 1736-48).
• the drug products being investigated are separate liquid pharmaceutical formulations for subcutaneous use.
• a clinical trial has demonstrated that a combination of semaglutide and cagrilintide induced a greater weight loss in obese patients than the maximal approved dose of semaglutide monotherapy, without resulting in a significant worsening of the side-effect profile.
• semaglutide has successfully been formulated for oral administration, cagrilintide may not necessarily provide sufficient bioavailability for such route of administration.
• An alternative orally available amylin receptor agonist for monotherapy or combination with, but not limited to, semaglutide could therefore prove useful.
• an amylin receptor agonist comprising a peptide according to Formula I (SEQ ID NO: 36):
• X2 is S or G
• X 3 is N, H, S, Q, A or E
• X5 is A or S
• X7 is A or L
• X is Q or A
• Xig is S or absent
• X20 is S, or absent
• X21 is D, E or absent
• X22 is N, P or absent
• X25 is A, K or P
• X27 is L or P
• X28 is S or P
• X29 is S or P
• X31 is D or E
• X34 is S or P
• X 35 is N, D or E
• X 37 is Y or P, wherein the peptide comprises a C-terminal amide.
• the peptide of the amylin receptor agonist is an amylin analogue.
• the amylin receptor agonist according to Formula I may be considered a modified human amylin analogue without the disulfide bridge that normally spans from positions 2 to 7 of native human amylin. Accordingly, in a preferred embodiment the amylin receptor agonist according to the present invention does not comprise a disulfide bridge. While it has generally been believed that an intact ring structure in amylin is necessary for full bioactivity (ACS Pharmacol. Transl. Sci. 2018, 1 , 32.49) the present inventors have surprisingly found that compounds according to Formula I remain potent.
• the amylin receptor agonist comprises a peptide with alanine (Ala, A) at position 1 , leucine (Leu, L) at position 4, and threonine (Thr, T) at position 6. Furthermore, the peptide comprises serine (Ser, S) or glycine (Gly, G) at position 2, asparagine (Asn, N), histidine (His, H), serine (Ser, S), glutamine (Gin, Q), alanine (Ala, A), or glutamic acid (Glu, E) at position 3, alanine (Ala, A) or serine (Ser, S) at position 5, and alanine (Ala, A) or leucine (Leu, L) at position 7.
• the peptide comprises glutamine (Gin, Q) at position 8.
• glutamine Gin, Q
• substitutions introduced at the N-terminus sequence of the peptide allow for the entire human amylin analogue to retain potency while not having an intact ring structure.
• the human amylin analogue according to Formula I maintains a threonine (Thr, T) at position 9, arginine (Arg, R), leucine (Leu, L), and alanine (Ala, A) at positions 11 to 13, respectively, phenylalanine (Phe, F) and leucine (Leu, L) at positions 15 and 16, respectively, histidine (His, H) at position 18, phenylalanine (Phe, F) and glycine (Gly, G) at positions 23 and 24, respectively, isoleucine, (lie, I) at position 26, threonine, (Thr, T) at position 30, valine (Vai, V) and glycine (Gly, G) at positions 32 and 33, respectively, and threonine, (Thr, T) at position 36.
• Retaining full bioactivity without a disulfide bridge is considered highly advantageous as it enables a more robust production of the amylin receptor agonist. It is further believed that compounds without a disulfide bridge have improved stability in a liquid formulation for s.c. dosing. Accordingly, in an embodiment the amylin receptor agonist provides an improved stability in a liquid formulation.
• the present inventors further found that the compounds not having a disulfide bridge may be formulated at an approximate neutral pH range, which may facilitate coformulation with other compounds having the same or similar preferred pH range.
• the amylin receptor agonist according to Formula I comprises glutamine at position 8.
• the present inventors have found that amylin receptor agonists comprising this substitution are highly potent on both the amylin and calcitonin receptors (with EC50 values in the low pM range). Furthermore, the compounds comprising the substitution proved potent in reducing food intake in rats. Accordingly, in an embodiment the amylin receptor agonist according to Formula I provides an improved effect in reduction in food intake.
• the amylin receptor agonist is according to Formula I with the proviso that at least two of the amino acids at positions 21 , 31 and 35 are an aspartic acid or a glutamic acid.
• the present inventors have observed that the amylin receptor agonist in a SNAC formulation has a high bioavailability in dogs following oral dosing. Accordingly, in an embodiment the amylin receptor agonist provides an improved bioavailability in oral administration.
• the amylin receptor agonist further comprises a protraction moiety attached via an alanine (Ala, A) residue at position 1 or a lysine (Lys, K) residue at position 25.
• the compounds disclosed herein exhibit a long half-life compared to their native ligands.
• the amylin receptor agonist possesses a long biological half-life, relative to dosing interval, thus reducing the variability in steady state exposure.
• the amylin receptor agonist may exhibit a variety of properties rendering it useful as a medicament, as described herein. Accordingly, an aspect of the present invention relates to the amylin receptor agonist for use as a medicament.
• An embodiment relates to the amylin receptor agonist for use in the treatment of subjects with: an initial body mass index (BMI) of 27 or more, such as 30 or more, optionally in the presence of at least one weight-related comorbidity; diabetes, optionally in the presence of at least one comorbidity; cardiovascular disease; non- alcoholic steatohepatitis; and/or cognitive impairment, such as that caused by Alzheimer’s disease.
• BMI body mass index
• a further aspect of the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the amylin receptor agonist and pharmaceutically acceptable excipients.
• a pharmaceutical cocomposition comprising the amylin receptor agonist as disclosed herein and one or more peptide(s), such as but not limited to an insulin peptide, or a GLP-1 peptide, such as but not limited to semaglutide, liraglutide, or tirzepatide.
• SEQ ID NO: 1 represents the amino acid sequence of human amylin (1-37).
• SEQ ID NOs: 2-33 represent the amino acid sequences of the peptides in compounds 2-43.
• SEQ ID NO: 34 represents the amino acid sequence of the polypeptide backbone within cagrilintide.
• SEQ ID NO: 35 represents the amino acid sequence of the polypeptide backbone within pramlintide.
• SEQ ID NO: 36 represents the amino acid sequence of Formula I.
• the compounds disclosed herein may be a potent amylin receptor agonist with desirable properties, such as being suitable for oral administration.
• the present invention relates to an amylin receptor agonist comprising a peptide according to Formula I (SEQ ID NO: 36):
• AX2X3LX5TX7QTX10RLAEFLHHX19X20X21X22FGX25IX27X28X29TX31VGX34X35TX37 wherein X2 is S or G, X3 is N, H, S, Q, A or E, X5 is A or S, X7 is A or L, XTM is Q or A, X19 is S or absent, X20 is S, or absent, X21 is D, E or absent, X22 is N, P or absent, X25 is A, K or P, X27 is L or P, X28 is S or P, X29 is S or P, X31 is D or E, X34 is S or P, X35 is N, D or E, X37 is Y or P, wherein the peptide comprises a C-terminal amide.
• peptide or “peptide sequence”, as used herein refers to a compound which comprises a series of two or more amino acids interconnected via amide (or peptide) bonds.
• peptide is used interchangeably with the term “polypeptide” and the term “protein”.
• analogue as used herein generally refers to a polypeptide, the sequence of which has one or more amino acid changes as compared to a reference amino acid sequence. Said amino acid changes may include amino acid additions, amino acid deletions, and/or amino acid substitutions. Amino acid substitutions, deletions and/or additions may also be referred to as “mutations”.
• an analogue “comprises” specified changes.
• an analogue “consists of’ or “has” specified changes.
• the term “comprises” or “comprising” is used in relation to amino acid changes in an analogue, the analogue may have further amino acid changes as compared to its reference sequence.
• the specified amino acid mutations are the only amino acid changes in the analogue as compared to the reference sequence.
• compound refers to a molecular entity, and “compounds” may thus have different structural elements besides the minimum element defined for each compound or group of compounds.
• the term compound is used interchangeably with the term “construct”.
• the term “compound” may be used to describe an amylin receptor agonist of the invention.
• the compounds of the invention may be referred to as “compound”, and the term “compound” is also meant to cover pharmaceutically relevant forms hereof, i.e. the invention relates to a compound as defined herein or a pharmaceutically acceptable salt, amide, or ester thereof.
• derivative generally refers to a chemically modified peptide in which one or more substituents or protraction moieties are covalently linked to the amino acid sequence of the peptide, such as via a bond to the alpha position of the alanine (Ala, A) at position 1 or to the epsilon position of a lysine (Lys, K) at position 25.
• Amino acids are molecules containing an amine group and a carboxylic acid group, and, optionally, one or more additional groups, often referred to as a side chain.
• amino acid includes canonical amino acids (which are genetically encoded), and unnatural amino acids.
• unnatural amino acids are Aib (a-aminoisobutyric acid) and the D-isomers of the canonical amino acids. All amino acid residues within the polypeptide for which the optical isomer is not stated is herein to be understood to mean the L-isomer, unless otherwise specified.
• a “receptor agonist” or “agonist” is a ligand, such as a compound, that binds to and activates a biological receptor to produce a biological response.
• a full agonist may be defined as being one that elicits a response of the same magnitude as the natural ligand (see e.g. “Principles of Biochemistry “, AL Lehninger, DL Nelson, MM Cox, Second Edition, Worth Publishers, 1993, page 763).
• Receptors can be activated by either endogenous agonists, such as endogenous hormones, or exogenous agonists, such as pharmaceutical drugs.
• An amylin receptor agonist may activate or agonize the calcitonin receptors (CTRs) and/or the amylin receptors (AMYRs).
• CTRs calcitonin receptors
• AYRs amylin receptors
• Amylin receptors consist of heterodimers of two components: the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMP1- 3), resulting in three possible complexes, AMYR1-3.
• CTR calcitonin receptor
• RAMP1- 3 receptor activity-modifying proteins
• the compounds disclosed herein comprise an amylin receptor agonist.
• An “amylin receptor agonist” may be defined as a chemical entity which is capable of binding to the amylin receptor and can activate it.
• an “amylin receptor agonist” is capable of binding to and activating at least the AMYR3 complex.
• the amylin receptor agonist may also be capable of activating the calcitonin receptor and AMYR1-2.
• Examples of endogenous amylin receptor agonists are human amylin and human calcitonin.
• Human amylin (SEQ ID No: 1) is a 37 amino acid long polypeptide having an amidated C-terminus and a disulfide bridge between cysteine residues 2 and 7. Both the amidated C-terminus and the disulfide bridge appear to be necessary for the full biological activity of amylin.
• Examples of exogenous amylin receptor agonists are pramlintide and cagrilintide (disclosed in WO 2012/168432).
• the amylin receptor agonist according to the present invention is a potent amylin receptor agonist.
• the amylin receptor agonist is a potent amylin receptor agonist and a potent calcitonin receptor agonist.
• the in vitro potency of the amylin receptor agonist on the amylin-3 receptor may be measured as described in the assay of Example 4.
• the potency of the compound may be described by means of its ECso values. ECso represents the concentration of compound upon which 50% of its maximal effect is observed. The lower the ECso value, the more potent the compound.
• the in vitro potency of the amylin receptor agonist on the calcitonin receptor may be measured as described in Example 4.
• the amylin receptor agonist disclosed herein when tested as described in the assay of Example 4 for amylin-3 receptor potency, has an ECso value of less than 300 pM, such as less than 200 pM, such as less than 150 pM, preferably less than 100 pM, such as less than 75 pM, even more preferably less than 50 pM, such as less than 40 pM, such as less than 30 pM, such as less than 20 pM, such as less than 10 pM, such as less than 7 pM, such as less than 5 pM, such as less than 3 pM, such as less than 2 pM.
• the amylin receptor agonist has an ECso value of less than 10 pM, measured in an in vitro potency on the amylin-3 receptor according to the assay of Example 4.
• the amylin receptor agonist disclosed herein when tested as described in the assay Example 4 for calcitonin receptor potency, has an ECso value of less than 300 pM, such as less than 200 pM, such as less than 150 pM, preferably less than 100 pM, such as less than 75 pM, preferably less than 50 pM, such as less than 40 pM, such as less than 30 pM, such as less than 20 pM, such as less than 16 pM, such as less than 10 pM, such as less than 7 pM, such as less than 5 pM, such as less than 3 pM, such as less than 2 pM.
• the amylin receptor agonist has an ECso value of less than 20 pM, measured in an in vitro potency on the calcitonin receptor according to the assay of Example 4.
• the peptide of the amylin receptor agonist is an amylin analogue.
• the amylin receptor agonist disclosed herein comprises a peptide according to Formula I (SEQ ID NO: 36):
• AX2X3LX5TX7QTX10RLAEFLHHX19X20X21X22FGX25IX27X28X29TX31VGX34X35TX37 wherein X 2 is S or G, X 3 is N, H, S, Q, A or E, X5 is A or S, X 7 is A or L, X is Q or A, X19 is S or absent, X 2 o is S, or absent, X21 is D, E or absent, X22 is N, P or absent, X25 is A, K or P, X27 is L or P, X 2 8 is S or P, X29 is S or P, X31 is D or E, X34 is S or P, X 35 is N, D or E, X 37 is Y or P, wherein the peptide comprises a C-terminal amide.
• the amylin receptor agonist is according to Formula I, wherein X2 is S or G, X3 is H, S, Q or E, X5 is S, X7 is A, X10 is Q or A, X19 is S or absent, X20 is S or absent, X21 is D, E or absent, X22 is N, P or absent, X25 is A or P, X27 is L or P, X28 is S or P, X29 is S or P, X31 is D, X34 is S or P, X35 is N, D or E, and X37 is P.
• Formula I wherein X2 is S or G, X3 is H, S, Q or E, X5 is S, X7 is A, X10 is Q or A, X19 is S or absent, X20 is S or absent, X21 is D, E or absent, X22 is N, P or absent, X25 is A or P, X27 is L or P,
• the amylin receptor agonist is according to Formula I, wherein X2 is S or G, X3 is H, S, Q or E, X5 is S, X7 is A, X10 is Q or A, X19 is absent, X20 is absent, X21 is absent, X22 is absent, X25 is A or P, X27 is L or P, X28 is S or P, X29 is S or P, X31 is D, X34 is S or P, X35 is N, D or E, and X37 is P.
• the amylin receptor agonist is according to Formula I, wherein X2 is S or G, X3 is H, S, or E, X5 is S, X7 is A, X10 is Q or A, X19 is absent, X20 is absent, X21 is absent, X22 is absent, X25 is P, X27 is L, X28 is P, X29 is P, X31 is D, X34 is S, X35 is E, and X37 is P.
• Formula I wherein X2 is S or G, X3 is H, S, or E, X5 is S, X7 is A, X10 is Q or A, X19 is absent, X20 is absent, X21 is absent, X22 is absent, X25 is P, X27 is L, X28 is P, X29 is P, X31 is D, X34 is S, X35 is E, and X37 is P.
• amylin receptor agonist is according to Formula I, wherein X27X28X29 is selected from LPP or PSS.
• amylin receptor agonist is according to Formula I with the proviso that at least two of the amino acids at positions 21 , 31 and 35 are an aspartic acid (Asp, D) or a glutamic acid (Glu, E).
• the amino acid at position 2 is a glycine (Gly, G) or a serine (Ser, S).
• the amino acid at position 3 is a glutamic acid (Glu, E), a glutamine (Gin, Q), or a histidine (His, H).
• amino acid at position 5 is serine (Ser, S).
• the amino acid at position 7 is alanine (Ala, A).
• the amino acid at position 10 is alanine (Ala, A).
• the amino acid at position 19 is a serine (Ser, S) or absent.
• the amino acid at position 20 is a serine (Ser, S) or absent. In an embodiment, the amino acid at position 21 is an aspartic acid (Asp, D) or absent.
• the amino acid at position 22 is an asparagine (Asn, N) , a proline (Pro, P) or absent.
• the amino acid at position 25 is a proline (Pro, P) or a alanine (Ala, A).
• the amino acid at position 27 is a proline (Pro, P) or a leucine (Leu, L).
• the amino acid at position 28 is a proline (Pro, P) or a serine (Ser, S).
• the amino acid at position 29 is a proline (Pro, P) or a serine (Ser, S).
• the amino acid at position 31 is aspartic acid (Asp, D).
• the amino acid at position 34 is a serine (Ser, S) or a proline (Pro, P).
• the amino acid at position 35 is an aspartic acid (Asp, D), an asparagine (Asn, N) or a glutamic acid (Glu, E).
• the amino acid at position 37 is proline (Pro, P).
• an amylin receptor agonist comprising a peptide selected from the group consisting of SEQ ID NOs: 2-33.
• the amylin receptor agonist comprising a peptide selected from any one of the following:
• the amylin receptor agonist disclosed herein comprises a C-terminal amide, which is considered essential for bioactivity.
• the amylin receptor agonist is considered a human amylin analogue it follows that the proline substituted for tyrosine at the C-terminal comprises an amide group.
• the amine group of the C-terminal amide is NH2.
• the amylin receptor agonist does not comprise an intact ring structure.
• ring structure means as any functional group that links two or more amino acid residues to each other, such as a disulfide bridge.
• the amylin receptor agonist does not comprise a disulfide bridge.
• disulfide bridge in reference to human amylin and analogues thereof, refers to a functional group with the structure R-S-S-R' and may also be referred to as a “S S-bond”.
• amylin receptor agonist does not comprise a cysteine residue.
• the amylin receptor agonist comprises a protraction moiety, which may also be referred to as a substituent.
• protraction moiety refers to a moiety having half-life extending properties and a protraction moiety may be represented by the general formula “A- B” or (A)-(B), in which (A) is an optional linker and (B) is a protractor.
• protractor refers to a molecule which is capable of increasing the half-life of the peptide to which it is attached.
• protraction thus refers to half-life extension and a protractor or protraction moiety serves the purpose of the extending the half-life of the the amylin receptor agonists as disclosed herein.
• correction moiety refers to a moiety that is covalently attached to a peptide. If a substituent is attached to a peptide, the peptide is referred to as “substituted”. When a substituent is covalently attached to a peptide or to an amino acid residue, the peptide or amino acid is said to “carry” a substituent.
• the substituent may comprise a series of individually defined moieties; these moieties together may be referred to as “substituent elements”.
• the protraction moiety or substituent may be capable of forming a non-covalent conjugate with albumin, thereby promoting the circulation of the compound in the blood stream, and thus having the effect of protracting the time of which the compound is present in the blood stream, since the aggregate of the fusion compound and albumin is only slowly disintegrated to release the free form of the compound.
• the substituent or the protraction moiety as a whole, may also be referred to as an “albumin-binding moiety”, and the substituent or the protraction moiety may be said to have a “protracting effect”.
• the substituent may comprise a portion which is particularly relevant for the albumin binding and thereby the protraction, which portion may be referred to as a “protractor” or a “protracting moiety”. Accordingly, the term “substituent” may also be referred to as a “side-chain” or a “protraction moiety”.
• the protracting moiety may comprise a portion between the protractor (B) and the point of attachment to the polypeptide, which portion may be referred to as a “linker” (A) or “side-chain linker” (A).
• the linker (A) may comprise several “linker elements”. The linker elements may be selected so that they improve the overall properties of the molecule, e.g. so that they improve the oral bioavailability, the conversion half-life or the protracting effect, thus improving the overall exposure profile upon oral administration of the compound.
• the protractor (B) has at least 10 carbon atoms, or at least 15, 20, 25, 30, 35, or at least 40 carbon atoms.
• the protractor may further include at least 5 hetero atoms, in particular O and N, for example at least 7, 9, 10, 12, 15, 17, or at least 20 hetero atoms, such as at least 1, 2, or 3 N-atoms, and/or at least 3, 6, 9, 12, or 15 O-atoms.
• the albumin binding moiety and/or the protracting moiety is lipophilic, and/or negatively charged at physiological pH (7.4).
• the albumin binding moiety, the protracting moiety, the protractor or the linker may be covalently attached to the alanine (Ala, A) residue at position 1 or a lysine (Lys, K) residue at position 25 of the peptide by acylation, i.e. , via an amide bond formed between a carboxylic acid group thereof (of the albumin binding moiety, the protracting moiety, the protractor or the linker) and an amino group of said alanine (in alpha-position) or lysine (in epsilon-position), respectively.
• acylation i.e. , via an amide bond formed between a carboxylic acid group thereof (of the albumin binding moiety, the protracting moiety, the protractor or the linker) and an amino group of said alanine (in alpha-position) or lysine (in epsilon-position), respectively.
• Additional or alternative conjugation chemistry includes alkylation, ester formation, or amide formation, or coupling to a cysteine residue, such as by maleimide or haloacetamide (such as bromo-/fluoro-/iodo-) coupling.
• fatty acid refers to aliphatic mono- or dicarboxylic acids having from 4 to 28 carbon atoms, it is preferably un-branched, and it may be saturated or unsaturated.
• a protraction moiety may be represented by the general formula “A-B” or (A)-(B), in which (A) is an optional “linker” or “side-chain linker” and (B) is a protractor.
• Each protraction moiety attaches to the alanine residue at position 1 or a lysine residue in the peptide backbone of the amylin receptor agonist, such as a lysine residue in position 25.
• linker (A) is present, the protraction moiety attaches to the polypeptide backbone via linker (A).
• linker (A) is absent, (B) attaches to the polypeptide backbone.
• a protraction moiety represented by the formula (A)-(B) is covalently attached, via linker (A), to the alpha position of the alanine (Ala, A) at position 1 or to the epsilon position of a lysine (Lys, K) at position 25, via an amide bond formed between a carboxylic acid group of the protraction moiety and an amino group of said alanine (in alpha-position) or lysine (in epsilon-position), respectively.
• the protraction moiety is covalently attached to the alpha position (i.e. amino group) of the alanine (Ala, A) at position 1.
• the protraction moiety is covalently attached to the epsilon position (i.e. amino group) of lysine (Lys, K) at position 25.
• the amino acid at position 25 is alanine (Ala, A) or Proline (Pro, P) when the protraction moiety is attached to the alanine (Ala, A) at position 1.
• the side-chain linker (A) may comprise Ado, Aeep or Aeeep, sulfonamide, Trx, E- Lys, Ahx, Glu, yGlu, Gly, Ser, Ala, Thr, and/or a bond.
• the optional side-chain linker (A) may comprise at least a moiety which may be represented by the following chemical formula:
• Chem. 1a *-NH-(CH 2 ) 2 -(O-(CH 2 )] k -O-(CH 2 ) n -CO-*.
• Chem. 1b wherein k is an integer in the range of 1-5, and n is an integer in the range of 1-5.
• the linker element may be designated Ado, or a 8-amino-3,6- dioxaoctanoyl, which may be represented by the following chemical formula:
• Chem. 2a *-NH-(CH 2 ) 2 -O-(CH 2 ) 2 -O-CH 2 -CO-*.
• Chem. 3a *-NH-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-*, or
• Chem. 4a *-NH-(CH 2 ) 2 .O-(CH 2 ) 2 O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-*, or
• the optional side-chain linker (A) may comprise a sulfonamide-C4 moiety.
• a sulfonamide-C4 group is a sulfonamide group attached to a 4-butanoyl group and having the following chemical formula:
• Chem. 5a *NH-S(O) 2 -CH 2 -CH 2 -CH 2 -CO-*
• the optional side-chain linker (A) may comprise Trx.
• Trx is also referred to as Tranexamic acid, trans-4-(aminomethyl)cyclohexanecarboxylic acid and has the following chemical formula:
• Chem. 6a *-NH-CH 2 -(C 6 HIO)-CO-* or
• the optional side-chain linker (A) may comprise epsilon-lysine (e-Lys).
• the optional side-chain linker (A) may comprise lysine (Lys).
• the optional side-chain linker (A) may comprise Ahx.
• Ahx is also referred to as Aminocaproic acid, 6-aminohexanoic acid and is defined by
• Chem. 7a *-NH-(CH 2 ) 5 -CO-* or
• the side-chain linker (A) comprises a Glu di-radical, such as
• Chem. 8a or Chem. 8b wherein the Glu di-radical may be included p times, where p is an integer in the range of 1-3.
• Chem. 8a may also be referred to as gamma-Glu, or briefly gGlu, due to the fact that it is the gamma carboxy group of the amino acid glutamic acid which is here used for connection to another linker element, or to the epsilon-amino group of lysine, or to the alpha group of the alanine in position 1.
• the amino group of Glu in turn forms an amide bond with the carboxy group of the fatty di-acid, or with the gamma-carboxy group of, e.g., another Glu, if present.
• Chem. 8b may also be referred to as a Glu.
• the linker (A) of the protraction moiety comprises a moiety according to Chem. 8a. In some embodiments, the linker (A) of the protraction moiety comprises one or two moieties according to Chem. 8a. In a particular embodiment, the linker (A) of the protraction moiety consists of one moiety according to Chem. 8a. In another particular embodiment, the linker (A) of the protraction moiety consists of two moieties according to Chem. 8a.
• the linker (A) of the protraction moiety comprises a moiety according to Chem. 8b. In some embodiments, the linker (A) of the protraction moiety comprises one or two moieties according to Chem. 8b. In a particular embodiment, the linker (A) of the protraction moiety consists of one moiety according to Chem. 8b. In another particular embodiment, the linker (A) of the protraction moiety consists of two moieties according to Chem. 8b. In an embodiment, Glu is of the L-form.
• the protractor (B) may comprise an acyl group.
• the acyl group may be branched or unbranched.
• the acyl group may be saturated or unsaturated.
• the protractor (B) may comprise a fatty acyl group.
• the acyl group may be branched or unbranched.
• the acyl group may be saturated or unsaturated.
• the protractor (B) comprises a distal carboxylic acid group.
• the protractor (B) comprises a fatty acid group. In an embodiment, the protractor (B) comprises a fatty acid group and an amide group.
• the protractor (B) comprises a distal carboxylic acid group and an amide group.
• the protractor (B) comprises an alkyl group.
• the protractor (B) comprises an aryl group.
• the protractor (B) comprises a tetrazole group.
• the protractor (B) comprises a sulfonic acid group.
• the protractor (B) comprises a phenoxy group.
• the protractor (B) comprises a benzoic acid group.
• the protractor (B) comprises a group defined by
• Chem. 9 HOOC-(CH2)n-CO-* wherein n is an integer in the range of 8-30, which may also be referred to as a C(n+2) diacid or as
• n is an integer in the range of 8-30.
• the protractor (B) of the protraction moiety comprises a C14 diacid, a C16 diacid, a C18 diacid, a C20 diacid, a C18-tetrazole, a C14-sulfonic acid , 4-(9- carboxynonyloxy) benzoic acid , 4-(10-carboxydecyloxy) benzoic acid, or 3-(9- carboxynonyloxy) benzoic acid.
• the protractor (B) of the protraction moiety comprises a C14 diacid, C16 diacid, C18 diacid, or C20 diacid.
• the protractor (B) of the protraction moiety consists of a C18 diacid.
• the protractor (B) of the protraction moiety consists of a C20 diacid.
• the peptide comprises an N-terminal substituent.
• the substituent is covalently attached to the alpha-amino group of the amino acid residue in the N-terminus, i.e. alanine (Ala, A) at position 1.
• the N-terminal substituent is an alkanoyl or acyl group.
• the N- terminal substituent is an acetyl group.
• an N-terminal substituted amino acid is Ac-Ala at position 1.
• the compounds disclosed herein have a long half-life compared to their native ligands.
• the in vivo half-life of the amylin receptor agonist may be assessed as described in Example 5.
• the compound disclosed herein may possess a long biological half-life, relative to dosing interval, thus reducing the variability in steady state exposure.
• the half-life of the amylin receptor agonist in animal subjects may be as long as about 150 hours, or longer. In an embodiment, the half-life of the amylin receptor agonist in animal subjects is at least 4 hours. In an embodiment, the half-life of the amylin receptor agonist is more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 hours. In an embodiment, the halflife of the amylin receptor agonist is 15-160 hours, such as 50-155 hours.
• the amylin receptor agonist has a half-life [h] of at least 80 in Beagle dogs following oral administration, measured according to the assay of Example 5. More specifically, in an embodiment, the amylin receptor agonist has a half-life [h] of 80- 150 in Beagle dogs following oral administration, measured according to the assay of Example 5.
• the derivatives, analogues, and intermediate products of the invention may be in the form of a pharmaceutically acceptable salt, amide, or ester.
• An aspect of the present invention relates to a pharmaceutically acceptable salt of the amylin receptor agonist as disclosed herein.
• Another aspect of the present invention relates to a pharmaceutically acceptable ester of the amylin receptor agonist as disclosed herein.
• Yet another aspect of the present invention relates to a pharmaceutically acceptable amide of the amylin receptor agonist as disclosed herein.
• Salts are e.g. formed by a chemical reaction between a base and an acid, e.g.: 2NH 3 + H2SO4 (NH 4 ) 2 SO 4 .
• the salt may be a basic salt, an acid salt, or it may be neither nor (i.e. a neutral salt).
• Basic salts produce hydroxide ions and acid salts hydronium ions in water.
• the salts of the derivatives of the invention may be formed with added cations or anions between anionic or cationic groups, respectively. These groups may be situated in the peptide moiety, and/or in the side chain of the derivatives of the invention.
• Non-limiting examples of anionic groups of the derivatives of the invention include free carboxylic groups in the side chain, if any, as well as in the peptide moiety.
• the peptide moiety often includes a free carboxylic acid group at the C-terminus, and it may also include free carboxylic groups at internal acid amino acid residues such as Asp and Glu.
• Non-limiting examples of cationic groups in the peptide moiety include the free amino group at the N-terminus, if present, as well as any free amino group of internal basic amino acid residues such as His, Arg, and Lys.
• the ester of the derivatives of the invention may, e.g., be formed by the reaction of a free carboxylic acid group with an alcohol or a phenol, which leads to replacement of at least one hydroxyl group by an alkoxy or aryloxy group
• the ester formation may involve the free carboxylic group at the C-terminus of the peptide, and/or any free carboxylic group in the side chain.
• the amide of the derivatives of the invention may, e.g., be formed by the reaction of a free carboxylic acid group with an amine or a substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid.
• the amide formation may involve the free carboxylic group at the C-terminus of the peptide, any free carboxylic group in the side chain, the free amino group at the N-terminus of the peptide, and/or any free or substituted amino group of the peptide in the peptide and/or the side chain.
• the peptide or derivative is in the form of a pharmaceutically acceptable salt.
• the derivative is in the form of a pharmaceutically acceptable amide, preferably with an amide group at the C- terminus of the peptide.
• the peptide or derivative is in the form a pharmaceutically acceptable ester.
• amylin receptor agonist is selected from the group consisting of Compound 2-43.
• amylin receptor agonist is selected from the group consisting of Compound 16, Compound 22, and Compound 25.
• amylin receptor agonist is Compound 16 according to the formula:
• amylin receptor agonist is Compound 22 according to the formula:
• amylin receptor agonist is Compound 25 according to the formula:
• the amylin receptor agonist disclosed herein reduces food intake in a subject. In an embodiment, administration of the amylin receptor agonist disclosed herein results in an acute reduction in the intake of food. In an embodiment, the amylin receptor agonist reduces body weight. The in vivo effect of the amylin receptor agonist on food intake in rats may be assessed as described in Example 6.
• administration of the amylin receptor agonist disclosed herein results in a food intake in rats, relative to vehicle, which is 0-90%, such as 0-80%, such as 0-70%, such as 0-60%, preferably 0-50%, even more preferably 0-40%, within 0-24 hours after a single subcutaneous injection of 3 nmol/kg or 10 nmol/kg of said co-agonist, wherein a food intake of 0% relative to vehicle means that the rat does not eat.
• administration of the amylin receptor agonist disclosed herein results in a food intake in rats, relative to vehicle, which is 0-90%, such as 0- 80%, such as 0-70%, such as 0-60%, preferably 0-50%, even more preferably 0-40%, within 24-48 hours after a single subcutaneous injection of 3 nmol/kg or 10 nmol/kg of said coagonist, wherein a food intake of 0% relative to vehicle means that the rat does not eat.
• the amylin receptor agonist shows food intake reduction in rats tested according to the assay of Example 6 after administration of 3 nmol/kg. In an embodiment, the amylin receptor agonist shows reduction in food intake in rats tested according to the assay of Example 6 of which can be seen within 0-24 hours after administration of 3 nmol/kg. In an embodiment, the amylin receptor agonist shows reduction in food intake in rats tested according to the assay of Example 6 which can be seen within 0- 24 and/or 24-48 hours after administration of 3 nmol/kg.
• the amylin receptor agonist shows reduction in food intake by at least 10%, such as at least 15%, 20%, 30%, 40%, 50%, 60%, or 70%, in rats tested according to the assay of Example 6 which can be seen within 0-24 hours after administration of 3 nmol/kg. In an embodiment, the amylin receptor agonist shows reduction in food intake by at least 10% such as at least 15%, 20%, 30%, 40%, 50%, or 60%, in rats tested according to the assay of Example 6 which can be seen within 24-48 hours after administration of 3 nmol/kg.
• the amylin receptor agonist shows reduction in food intake by at least 10% such as at least 15%, 20%, 30%, 40%, 50%, or 60%, in rats tested according to the assay of Example 6 which can be seen within 48-72 hours after administration of 3 nmol/kg.
• bioavailability refers to the capability of a compound to reach systemic circulation following administration, and it may be quantified as the fractional extent of the compound dosage that reaches systemic circulation upon administration. It is desirable that a drug intended for oral administration has a high oral absorption (i.e. a high absorption form the gastrointestinal tract following oral administration) since it may reduce the dosage required to reach the intended systemic concentration of the drug, and thus e.g. reduce tablet size and manufacturing costs.
• oral bioavailability refers to the capability of a compound to reach systemic circulation following oral administration.
• the oral bioavailability reflects the extent to which a compound is absorbed in the gastrointestinal tract following oral administration. In other words a high oral bioavailability is associated with a high oral absorption.
• a high oral bioavailability of a drug is associated with a high drug exposure following oral administration.
• the oral bioavailability may be measured in a formulation with the absorption enhancer sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC) in beagle dogs as described in WO 2019/149880.
• the oral bioavailability may be measured as described in Example 5.
• the compound of the invention has a high oral bioavailability.
• the compound of the invention has an oral bioavailability that is similar to that of semaglutide.
• the compound of the invention has an oral bioavailability that is not inferior to that of semaglutide.
• the compound of the invention has an oral bioavailability that is as least as high as that of semaglutide.
• the compound of the invention has an oral bioavailability which is suitable for oral dosing in humans.
• the compound of the invention has an oral bioavailability which is determined in Beagle dogs and measured as Cmax/Dose [kg/L], Specifically, in an embodiment the oral bioavailability is determined in Beagle dogs, measured according to the assay of Example 5. In a particular embodiment, the oral exposure level is determined in Beagle dogs upon administration of tablets containing 3 mg of the compound, 300 mg sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) and 7.7 mg magnesium stearate.
• SNAC sodium N-(8-(2-hydroxybenzoyl)amino)caprylate
• the compound of the invention has an oral bioavailability which is measured as Cmax/Dose [kg/L] in Beagle dogs; wherein the Cmax/Dose [kg/L] is at least 0.1, preferably at least 0.15, and most preferably at least 0.20.
• the oral bioavailability is measured as Cmax/Dose [kg/L] in Beagle dogs; and wherein the Cmax/Dose [kg/L] is 0.1-0.5.
• the compound of the invention has an oral bioavailability which is determined in Beagle dogs and measured as AUC/Dose [kg*hr/L], In one embodiment, the compound of the invention has an oral bioavailability which is determined in Beagle dogs and measured as AUC/Dose [kg*hr/L]; wherein the AUC/Dose [kg*hr/L] is at least 2, preferably at least 5, and most preferably at least 10. In an embodiment, the oral bioavailability is measured as AUC/Dose [kg*hr/L] in Beagle dogs; and wherein the AUC/Dose [kg*hr/L] is IQ- 40.
• compositions comprising the amylin analogues disclosed herein.
• Pharmaceutical compositions comprising the amylin analogues, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, may be prepared using methods known to the person skilled in the art.
• the term "pharmaceutically acceptable excipient” refers to any ingredient in the pharmaceutical composition which is not the active pharmaceutical ingredient.
• the excipient may be functional or inert and may serve one or more purposes.
• the excipient may enhance absorption of the active substance.
• the excipient might be, amongst others, a buffer, an antimicrobial preservative, an isotonicity agent, a carrier, a vehicle, a filler, a binder, a lubricant, a glidant, a disintegrant, a flow control agent, a crystallization inhibitor, a solubilizer, a stabilizer, a coloring agent, a flavoring agent, a surfactant, an emulsifier.
• the amount of each excipient used may vary within ranges conventional in the art.
• the pharmaceutical composition may be suitable for oral administration. Techniques and excipients which may be used to formulate orally administered pharmaceutical compositions are described in Handbook of Pharmaceutical Excipients (e.g. 8 th edition, Sheskey et al., Eds., American Pharmaceuticals Association and Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2017) and later editions); and Remington: The Science and Practice of Pharmacy (e.g. 22nd edition, Remington and Allen, Eds., Pharmaceutical Press (2013), and later editions).
• the pharmaceutical composition may be a solid pharmaceutical composition (e.g. a compressed tablet or capsule) containing the active pharmaceutical ingredient, for example as a freeze-dried or spray-dried composition, and may be used as is, dissolved prior to use, or combined with excipients in the composition.
• the pharmaceutical composition comprising the compound of the invention is suitable for oral administration.
• the pharmaceutical composition comprising the compound of the invention is prepared in the form of a tablet where the compound is formulated with the absorption enhancer sodium N-(8-[2- hydroxy benzoyl] amino) caprylate (SNAC), e.g. as described in WO 2019/149880 or WO 2019/215063.
• SNAC absorption enhancer sodium N-(8-[2- hydroxy benzoyl] amino) caprylate
• the compound is further formulated with a lubricant magnesium stearate.
• the pharmaceutical composition may be a liquid formulation, such as an aqueous formulation.
• Such liquid composition may be suitable for oral administration or for parenteral administration.
• Liquid compositions that are 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.
• the compound described herein is dissolved in a suitable buffer at a suitable pH.
• the composition may be sterilized, for example, by sterile filtration. Techniques and excipients which may be used to prepare liquid formulations are described in Handbook of Pharmaceutical Excipients (e.g.
• a pharmaceutical composition comprising the amylin receptor agonist and/or pharmaceutically acceptable salt, ester, or amide thereof, as disclosed herein.
• the pharmaceutical composition is for oral administration.
• the pharmaceutical composition is a solid pharmaceutical composition. More specifically, in an embodiment the pharmaceutical composition is a tablet.
• the pharmaceutical composition is for s.c. administration.
• the pharmaceutical composition is a liquid formulation, such as an aqueous formulation.
• Such liquid composition may be suitable for oral administration or for parenteral administration.
• Liquid compositions that are 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.
• the compound described herein is dissolved in a suitable buffer at a suitable pH.
• the composition may be sterilized, for example, by sterile filtration. Techniques and excipients which may be used to prepare liquid formulations are described in Handbook of Pharmaceutical Excipients (e.g.
• the liquid formulation provides an improved stability.
• amylin receptor agonist may exhibit a variety of properties rendering it useful as a medicament, as described herein. Accordingly, in an embodiment, the amylin analogue or pharmaceutical composition thereof as disclosed herein for use as a medicament.
• the amylin analogue disclosed herein may be used for the following medical treatments:
• diabetes prevention and/or treatment of all forms of diabetes, such as hyperglycaemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reduction of HbA1c;
• diabetes such as hyperglycaemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reduction of HbA1c;
• diabetes delaying or preventing diabetic disease progression, such as progression in type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin requiring type 2 diabetes, and/or delaying the progression of non-insulin requiring type 2 diabetes to insulin requiring type 2 diabetes;
• ITT impaired glucose tolerance
• eating disorders such as obesity, e.g. by decreasing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, food cravings, bulimia nervosa and/or obesity induced by administration of an antipsychotic or a steroid; reduction of gastric motility; and/or delaying gastric emptying;
• weight maintenance after successful weight loss (either drug induced or by diet and exercise) - i.e. prevention of weight gain after successful weight loss;
• v prevention and/or treatment of cardiovascular diseases, such as delaying or reducing development of a major adverse cardiovascular event (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization for unstable angina pectoris, and hospitalization for heart failure.
• MACE major adverse cardiovascular event
• NASH non-alcoholic steatohepatitis
• the indication is (i). In some embodiments the indication is (ii). In a still further particular aspect the indication is (iii). In some embodiments the indication is (iv). In some embodiments the indication is (v). In some embodiments the indication is (vi). In some embodiments the indication is (vii). In some embodiments the indication is type 2 diabetes and/or obesity.
• treatment refers to the medical therapy of any human or other vertebrate subject in need thereof.
• Said subject is expected to have undergone physical examination by a medical practitioner, or a veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other vertebrate.
• the timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject’s health.
• said treatment may be prophylactic (preventative), palliative, symptomatic and/or curative.
• the indication is (i) and (iii). In some embodiments the indication is (ii) and (iii).
• the subject suffering from obesity is human, such as an adult human or a paediatric human (including infants, children, and adolescents).
• a human subject suffering from obesity may have a BMI of >30; this subject may also be referred to as being obese.
• the human subject suffering from obesity may have a BMI of >35 or a BMI in the range of >30 to ⁇ 40.
• the obesity is severe obesity or morbid obesity, wherein the human subject may have a BMI of >40.
• the invention relates to a method for treatment or prevention of overweight, optionally in the presence of at least one weight-related comorbidity. In some embodiments the invention relates to use of the pharmaceutical composition for treatment or prevention of overweight, optionally in the presence of at least one weight-related comorbidity.
• the subject suffering from overweight is human, such as an adult human or a paediatric human (including infants, children, and adolescents).
• a human subject suffering from overweight may have a BMI of >25, such as a BMI of >27, such as a BMI of >30, such as a BMI of >35 or a BMI of >40.
• a human subject suffering from overweight has a BMI in the range of 25 to ⁇ 30 or in the range of 27 to ⁇ 30.
• the weight-related comorbidity is selected from the group consisting of hypertension, diabetes (such as type 2 diabetes), dyslipidaemia, high cholesterol and obstructive sleep apnoea.
• reduction of body weight may include treatment or prevention of obesity and/or overweight.
• administration of the compound disclosed herein is as an adjunct to a reduced-calorie diet and increased physical activity for chronic weight management in adult or paediatric patients with an initial body mass index (BMI) of 30 kg/m 2 or greater (obesity) or 27 kg/m 2 or greater (overweight) in the presence of at least one weight-related comorbidity (e.g. hypertension, type 2 diabetes mellitus, or dyslipidemia).
• BMI body mass index
• the co-morbidity is diabetes and/or a cardiovascular disease.
• the amylin receptor agonist is for use in the treatment of subjects with diabetes, such as type II diabetes. In an embodiment, the amylin receptor agonist is for use in the treatment and/or prevention of cardiovascular disease. In an embodiment, the amylin receptor agonist is for use in the treatment of non-alcoholic steatohepatitis (NASH). In an embodiment, the amylin receptor agonist is for use in the treatment and/or prevention of cognitive impairment, such as that caused by Alzheimer’s disease.
• NASH non-alcoholic steatohepatitis
• An aspect of the present invention relates to pharmaceutical co-formulation or co-treatment comprising an amylin receptor agonist as disclosed herein, and further comprising one or more GLP-1 receptor agonist(s).
• the one or more GLP-1 receptor agonist(s) is a peptide GLP-1 receptor agonist or a small molecule GLP-1 receptor agonist.
• the at least one of the one or more GLP-1 receptor agonist(s) is for the treatment and/or prevention of diabetes, a cardiovascular disease, NASH, and/or Alzheimer’s disease.
• pharmaceutical co-formulation or co-treatment comprises one or more GLP-1-GIP receptor co-agonist(s), wherein the GLP-1-GIP receptor co-agonist(s) is a peptide GLP-1-GIP receptor co-agonist or a small molecule GLP-1-GIP receptor co-agonist.
• An aspect of the present invention relates to pharmaceutical co-formulation or cotreatment comprising an amylin receptor agonist as disclosed herein, and further comprising one or more peptide(s).
• at least one of the one or more peptide(s) is for the treatment and/or prevention of diabetes, a cardiovascular disease, NASH, and/or Alzheimer’s disease.
• at least one of the one or more peptide(s) is a GLP-1 peptide. More specifically, in an embodiment the GLP-1 peptide is a GLP-1 compound, GLP-1 analogue, or GLP-1 derivative. Even more specifically, in an embodiment the GLP-1 compound, GLP-1 analogue, or GLP-1 derivative is semaglutide or liraglutide.
• At least one of the one or more peptide(s) is a GLP-1-GIP receptor co-agonist.
• At least one of the one or more peptide(s) is an insulin peptide. More specifically, in an embodiment the insulin peptide is an insulin compound, insulin analogue, or insulin derivative.
• the amylin analogues are formulated for oral administration, such as in a tablet.
• the amylin analogues disclosed herein may be administered approximately once daily, such as once every 12-36 hours, such as once every 18-30 hours, such as approximately once every 24 hours.
• the amylin analogues disclosed herein may be administered approximately once every other day, such as once every 36-60 hours, such as once every 42-54 hours, such as approximately once every 48 hours.
• the amylin analogues disclosed herein may be administered approximately twice daily, such as once every 6-18 hours, such as once every 9-15 hours, such as approximately once every 12 hours.
• the amylin analogues are formulated for s.c. administration, such as in a liquid formulation.
• the amylin analogues disclosed herein may be administered approximately once daily, such as once every 12-36 hours, such as once every 18-30 hours, such as approximately once every 24 hours.
• the amylin analogues in a liquid formulation may be administered once weekly, once every second week, or once monthly.
• the compounds disclosed herein 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 Ddrwald, “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 and mammalian BHK or CHO. Specific examples of methods of preparing the disclosed compounds are included in the examples.
• a further aspect of the invention relates to a method for preparing the peptides described herein.
• the method for preparing a compound as described herein comprises a step of solid phase peptide synthesis.
• the substituent or protraction moiety may be built sequentially as part of the solid phase peptide synthesis or produced separately and attached via the alanine or lysine residue after peptide synthesis.
• amylin receptor agonist comprising a peptide according to Formula I (SEQ ID NO: 36):
• X2 is S or G
• X3 is N, H, S, Q, A or E
• X5 is A or S
• X 7 is A or L
• X is Q or A
• X19 is S or absent
• X 2 o is S, or absent
• X21 is D, E or absent
• X22 is N, P or absent
• X25 is A, K or P
• X27 is L or P
• X28 is S or P
• X29 is S or P
• X31 is D or E
• X34 is S or P
• X35 is N, D or E
• X3 7 is Y or P, wherein the peptide comprises a C-terminal amide.
• amylin receptor agonist according to the preceding embodiment, wherein the peptide is an amylin analogue.
• amylin receptor agonist according to any one of the preceding embodiments, with the proviso that at least two of the amino acids at positions 21, 31 and 35 are an aspartic acid (Asp, D) or a glutamic acid (Glu, E).
• AX2X3LX5TX7QTX10RLAEFLHHX19X20X21X22FGX25IX27X28X29TX31VGX34X35TX37 wherein X2 is S or G X3 is H, S, Q or E Xs is S X 7 is A X is Q or A X19 is S or absent X 2 o is S or absent X21 is D, E or absent X22 is N, P or absent X25 is A or P X27 is L or P X28 is S or P X29 is S or P X31 is D X34 is S or P X35 is N, D or E X37 is P.
• the amylin receptor agonist according to any one of the preceding embodiments, comprising a peptide according to Formula I (SEQ ID NO: 36):
• AX2X3LX5TX7QTX10RLAEFLHHX19X20X21X22FGX25IX27X28X29TX31VGX34X35TX37 wherein X2 is S or G X3 is H, S, Q or E Xs is S X 7 is A X is Q or A X19 is absent X20 is absent X21 is absent X22 is absent X25 is A or P X27 is L or P X 2 8 is S or P X29 is S or P X31 is D X34 is S or P X35 is N, D or E X37 is P.
• the amylin receptor agonist according to any one of the preceding embodiments, comprising a peptide according to Formula I (SEQ ID NO: 36):
• amylin receptor agonist according to any one of the preceding embodiments, wherein X27X28X29 is selected from LPP or PSS.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 2 is a glycine (Gly, G) or a serine (Ser, S).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 3 is a glutamic acid (Glu, E), a glutamine (Gin, Q), or a histidine (His, H).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 5 is serine (Ser, S).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 7 is alanine (Ala, A).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 10 is alanine (Ala, A).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 19 is a serine (Ser, S) or absent.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 20 is a serine (Ser, S) or absent.
• the amino acid at position 22 is an asparagine (Asn, N) , a proline (Pro, P) or absent.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 25 is a proline (Pro, P) or a alanine (Ala, A). 17. The amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 27 is a proline (Pro, P) or a leucine (Leu, L).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 28 is a proline (Pro, P) or a serine (Ser, S).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 29 is a proline (Pro, P) or a serine (Ser, S).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 21 is an aspartic acid (Asp, D) or absent.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 31 is aspartic acid (Asp, D).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 34 is a serine (Ser, S) or a proline (Pro, P).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 35 is an aspartic acid (Asp, D), an asparagine (Asn, N) or a glutamic acid (Glu, E).
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amino acid at position 37 is proline (Pro, P).
• amylin receptor agonist comprising a peptide selected from the group consisting of SEQ ID Nos: 2-33.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 2.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 3.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 4.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 5.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 6.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 7.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 8.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 9.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 10.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 11.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 12.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 13.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 14.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 15.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 16.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 17.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 18.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 19.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 20.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 21.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 22.
• An amylin receptor agonist comprising a peptide of SEQ ID NO: 23.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 24.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 25.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 26.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 27.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 28.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 29.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 30.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 31.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 32.
• amylin receptor agonist comprising a peptide of SEQ ID NO: 33.
• amylin receptor agonist comprising a peptide selected from any one of the following:
• amylin receptor agonist according to any one of embodiments 25 to 58, wherein the peptide comprises a C-terminal amide.
• amylin receptor agonist according to any one of the preceding embodiments, which does not comprise an intact ring structure.
• the amylin receptor agonist according to any one of the preceding embodiments which does not comprise a disulfide bridge.
• the amylin receptor agonist according to any one of the preceding embodiments wherein the peptide does not comprise a cysteine (Cys, C) residue.
• amylin receptor agonist according to the preceding embodiment, wherein the protraction moiety is attached to the alpha position of the alanine (Ala, A) at position 1 or to the epsilon position of a lysine (Lys, K) at position 25.
• amylin receptor agonist according to any one of preceding embodiments 64 and 65, wherein the protraction moiety is attached to the alpha position of the alanine (Ala, A) at position 1.
• amylin receptor agonist according to the preceding embodiment, wherein the N-terminal amino acid is acylated.
• alanine at position 1 is Ac-Ala.
• amylin receptor agonist according to any one of the preceding embodiments 65 to 71 , wherein the linker (A) of the protraction moiety comprises a moiety according to Chem. 8a or Chem. 8b.
• amylin receptor agonist according to the preceding embodiment, wherein the linker (A) of the protraction moiety comprises one or two moieties according to Chem. 8a or Chem. 8b.
• amylin receptor agonist according to any one of embodiments 72 and 73, wherein the linker (A) of the protraction moiety consists of one moiety according to Chem. 8a or Chem. 8b.
• amylin receptor agonist according to any one of embodiments 72 and 73, wherein the linker (A) of the protraction moiety consists of two moieties according to Chem. 8a or Chem. 8b.
• amylin receptor agonist according to any one of embodiments 72 and 73, wherein the linker (A) of the protraction moiety consists of one or two moieties according to Chem. 8a.
• amylin receptor agonist according to any one of embodiments 65 to 76, wherein the protractor (B) of the protraction moiety comprises a C14 diacid, a C16 diacid, a C18 diacid, a C20 diacid, a C 18- tetrazole, a C14-sulfonic acid , 4-(9-carboxynonyloxy) benzoic acid , 4-(10-carboxydecyloxy) benzoic acid, or 3-(9-carboxynonyloxy) benzoic acid.
• the amylin receptor agonist according to the preceding embodiment, wherein the protractor (B) of the protraction moiety comprises a C14 diacid, C16 diacid, C18 diacid, or C20 diacid. 79. The amylin receptor agonist according to the preceding embodiment, wherein the protractor (B) of the protraction moiety consists of a C18 diacid.
• amylin receptor agonist according to embodiment 78, wherein the protractor (B) of the protraction moiety consists of a C20 diacid.
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to formula:
• amylin receptor agonist according to any one of the preceding embodiments, wherein the peptide comprises a C-terminal amide.
• amylin receptor agonist according to the preceding embodiment, wherein the amine group of the C-terminal amide is NH 2 .
• amylin receptor agonist according to any one of preceding embodiments, with an ECso value of less than 10 pM, such as less than 7 pM, such as less than 5 pM, such as less than 3 pM, such as less than 2 pM, measured in an in vitro potency on the amylin-3 receptor according to the assay of Example 4.
• amylin receptor agonist according to any one of preceding embodiments, with an EC50 value of less than 20 pM, such as less than 15 pM, such as less than 10 pM, such as less than 5 pM, such as less than 3 pM, such as less than 2 pM, measured in an in vitro potency on the calcitonin receptor according to the assay of Example 4.
• a half-life [h] of at least 80 in Beagle dogs following oral administration measured according to the assay of Example 5.
• amylin receptor agonist according to the preceding embodiment, with a half-life [h] of 80-150 in Beagle dogs following oral administration, measured according to the assay of Example 5.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is suitable for oral dosing in humans.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is determined in Beagle dogs, measured according to the assay of Example 5.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is determined in Beagle dogs upon administration of tablets containing 3 mg of the compound, 300 mg sodium N-(8-(2-hydroxybenzoyl) amino)caprylate (SNAC) and 7.7 mg magnesium stearate.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is measured as Cmax/Dose [kg/L],
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is measured as AUC/Dose [kg*hr/L],
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is measured as Cmax/Dose [kg/L] in Beagle dogs; and wherein the Cmax/Dose [kg/L] is at least 0.1.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is measured as AUC/Dose [kg*hr/L] in Beagle dogs; and wherein the AUC/Dose [kg*hr/L] is at least 10.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the oral bioavailability is measured as AUC/Dose [kg*hr/L] in Beagle dogs; and wherein the AUC/Dose [kg*hr/L] is 10-40.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amylin receptor agonist reduces appetite.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amylin receptor agonist reduces food intake.
• amylin receptor agonist according to any one of the preceding embodiments, wherein the amylin receptor agonist reduces body weight.
• amylin receptor agonist according to any one of the preceding embodiments, showing food intake reduction in rats tested according to the assay of Example 6 after administration of 3 nmol/kg.
• amylin receptor agonist according to any one of the preceding embodiments, showing reduction in food intake in rats tested according to the assay of Example 6 of which can be seen within 0-24 hours after administration of 3 nmol/kg.
• amylin receptor agonist according to any one of the preceding embodiments, showing reduction in food intake in rats tested according to the assay of Example 6 which can be seen within 0-24 and/or 24-48 hours after administration of 3 nmol/kg.
• the amylin receptor agonist according to any one of the preceding embodiments showing reduction in food intake by at least 10% in rats tested according to the assay of Example 6 which can be seen within 0-24 hours after administration of 3 nmol/kg.
• amylin receptor agonist according to any one of the preceding embodiments, showing reduction in food intake by at least 10% in rats tested according to the assay of Example 6 which can be seen within 24-48 hours after administration of 3 nmol/kg.
• a pharmaceutically acceptable ester of the amylin receptor agonist according to any one of embodiments 1 to 146.
• a pharmaceutical composition comprising the amylin receptor agonist and/or pharmaceutically acceptable salt, ester, or amide thereof, according to any one of the preceding embodiments, and one or more pharmaceutically acceptable excipients.
• composition according to the preceding embodiment which is for oral administration.
• composition according to the preceding embodiment which is a solid pharmaceutical composition.
• composition according to the preceding embodiment which is a tablet.
• composition according to embodiment 150 which is a liquid formulation.
• composition according to the preceding embodiment which is for dosing approximately once weekly.
• BMI body mass index
• BMI body mass index
• BMI body mass index
• a pharmaceutical co-formulation or co-treatment comprising an amylin receptor agonist according to any one of embodiments 1 to 146, and further comprising one or more GLP-1 receptor agonist(s) and/or GLP-1-GIP receptor agonist(s).
• the one or more GLP-1 receptor agonist(s) and/or GLP-1-GIP receptor agonist(s) is a peptide GLP-1 receptor agonist or peptide GLP-1-GIP receptor agonist, or a small molecule GLP-1 receptor agonist or small molecule GLP- 1-GIP receptor agonist.
• GLP-1 peptide is a GLP-1 compound, GLP-1 analogue, or GLP-1 derivative.
• insulin peptide is an insulin compound, insulin analogue, or insulin derivative.
• the method according to the preceding embodiment comprises a step of solid phase peptide synthesis.
• This example provides the identity, materials for making and methods of synthesis of many compounds according to the current invention.
• DIPEA /V,/V-diisopropylethylamine or Hunig’s base
• EDTA ethylenediaminetetraacetic acid
• Imp imidazolepropionyl i.v.: intravenously
• NMP N-methyl pyrrolidone
• TIPS triisopropylsilane
• Trt triphenylmethyl or trityl
• Trx tranexamic acid
• This section relates to general methods for solid phase peptide synthesis (SPPS methods, including methods for cleaving the peptide from the resin and removal of the protecting groups and for its purification). Included as well are LCMS methods for detecting and characterizing the resulting peptide. acid and special amino acid building blocks
• Fmoc-Gln(Trt)-Thr(i Me Me pro)-OH was commercially available from TechnoComm Ltd.
• the preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Symphony X from Protein Technologies.
• the Fmoc-protected amino acids used in the methods were the standard recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc- Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Phe- OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-
• a Fmoc-PAL AM resin was used, which was commercially available from NovabioChem.
• the subsequent amino acids were introduced in a stepwise procedure by the Symphony X peptide synthesizer following the SPPS principles.
• Fmoc-deprotection was achieved with 20% piperidine in DMF with 0.3 M Oxyma Pure for 2 x 10 min.
• Introduction of the substituent at the alpha-position of the N-terminal amino acid was accomplished using a standard Fmoc-protected amino acid.
• the peptide couplings were performed with DIC/Oxyma Pure and collidine.
• Amino acid/Oxyma Pure solutions 0.3 M/0.3 M in DMF at a molar excess of 5-10-fold
• the same molar equivalent of DIC was added (1.5 M in DMF), followed by collidine (1.5 M in DMF). Most commonly, it was mixed for 2 hours.
• the coupling time was increased, additional DIC was added, or the coupling step was repeated.
• a capping step was performed with 1 M acetic anhydride in DMF and collidine.
• the building block Fmoc-Gln(Trt)-Thr(i Me Me Pro)-OH was introduced in the positions corresponding to 8 and 9 in the full length sequence where applicable.
• fatty acid group i.e. the fatty acid group
• suitable building block such as but not limited to, octadecanedioic acid mono-tert- butyl-ester.
• acetyl groups on the N-terminal was introduced by acetylation with 1 M acetic anhydride in DMF and collidine.
• the peptides were cleaved with TFA/TIPS/H2O/DTT (90:4:3:3) for 2 hours. Hereafter, it was drained into cold diethyl ether, and centrifuged. The ether was decanted off, and the peptide was washed with ether two additional times.
• the crude peptide was dissolved in Acetic Acid/Acetonitrile/MQ water (4:2:4) and purified by reversed-phase preparative HPLC (Waters Delta Prep 4000) on a column comprising 018-silica gel. Elution was performed with an increasing gradient of MeCN in MQ water comprising 0.1% TFA. Relevant fractions were analysed with LIPLC. Fractions containing the pure target peptide were pooled. The resulting solution was analyzed (LIPLC, LCMS) and the peptide derivative was quantified using a CAD specific HPLC detector (Thermo-Fischer Vanquish HPLC-CAD). The product was dispensed into glass vials. The vials were capped with Millipore glassfibre prefilters. Freeze-drying afforded the trifluoroacetate salt of the derivative as a white solid.
• Example 2 Amylin receptor agonists according to the invention
• LCMS analysis was performed on a set up consisting of Waters Acquity LIPLC H Class system and Waters Xevo G2-xS QTof. Eluents: A: MQ water; B: MeCN; C: 2% formic acid + 0.1% TFA in MQ water.
• the analysis was performed at RT (column temp 60 °C) by injecting an appropriate volume of the sample onto the column which was eluted with a gradient of A, B, and 5% C.
• the LIPLC conditions, detector settings, and mass spectrometer settings were: Column: Waters Acquity BEH, C-18, 1.7pm, 2.1mm x 50mm. Gradient: Linear 5% - 95% B during 4.0 min at 0.4 ml/min. Total run-time: 7.0 min. Detection: MS resolution mode, ionisation method: ES. Scan: 50-5000 amu.
• Activation of hAMYR3 leads to increased cellular concentrations of cAMP.
• a BHK cell line was stably transfected with the human calcitonin receptor (a) and a CRE-responsive luciferase (CRE-Luc) reporter gene according to methods known to the person skilled in the art (Hollex-1 cell line, obtained from Zymogenetics described in US patent 5622839).
• the cell line was further transfected with human receptor modifying protein 3 (RAMP3) using standard methods. This turns the human calcitonin receptor into a human amylin-3(a) receptor.
• RAMP3 human receptor modifying protein 3
• Cells stocks were prepared by culturing of the stably transfected BHK hAMYR3/CRE-Luc cell line in growth medium consisting of DMEM (Gibco, 31966-021) supplemented with 10% FBS (Gibco, 16140-071), 1% Pen/Strep (Gibco, 15140-122), 0.5 mg/mL Geneticin (Gibco, 10131-027), 0.4 mg/mL Hygromycin (Invitrogen, 10687010) and 250 nM Methotrexate (Sigma, A6770).
• the assay buffer consisted of DMEM without phenol red (Gibco, 11880-028) supplemented with 1X GlutaMAX (Gibco, 35050-038), 10 mM HEPES (Gibco, 15630-056) and 1% (w/v) ovalbumin (Sigma, A5503).
• BHK hAMYR3/CRE-Luc cells were thawed, washed once in PBS and seeded in 40 pL growth medium in a white 384-well culture plate (PerkinElmer, 6007688) at a cell density of 4.000 cells/well on the day before the experiment. The plate was incubated over night at 37°C in 5% CO2. On the day of the assay, cells were washed once in assay buffer. Serial dilutions (7-fold dilutions, 7 concentrations Pr.
• comparator compounds and amylin analogues were performed in assay buffer often starting from approximately 10 nM in 96-well plates and 30 pL of each concentration added to the 384-well assay plate with cells.
• the assay plate was incubated for 3 hours at 37°C in 5% CO2 after which 30 pL SteadyLite Plus (PerkinElmer, 6066759) was added to each well.
• the assay plate was sealed, incubated at room temperature with gentle shaking for 5 minutes followed by 30 minutes incubation without shaking while protected from light. Luminescence was detected on a luminescence plate reader e.g. a Synergy 2 (BioTek).
• in vitro potency assays on cells expressing the human calcitonin receptor can be performed as described below.
• Activation of hCTR leads to increased cellular concentrations of cAMP.
• CRE cAMP response element
• a BHK cell line was stably transfected with the human calcitonin receptor (a) and a CRE-responsive luciferase (CRE-Luc) reporter gene according to methods known to the person skilled in the art (Hollex-1 cell line, obtained from Zymogenetics described in US patent 5622839).
• Cells stocks were prepared by culturing of the stably transfected BHK hCTR/CRE- Luc cell line in growth medium consisting of DMEM (Gibco, 31966-021) supplemented with 10% FBS (Gibco, 16140-071), 1% Pen/Strep (Gibco, 15140-122), 0.5 mg/mL Geneticin (Gibco, 10131-027) and 250 nM Methotrexate (Sigma, A6770).
• the assay buffer consisted of DMEM without phenol red (Gibco, 11880-028) supplemented with 1X GlutaMAX (Gibco, 35050-038), 10 mM HEPES (Gibco, 15630-056) and 1% (w/v) ovalbumin (Sigma, A5503).
• BHK hCTR/CRE-Luc cells were thawed, washed once in PBS and seeded in 40 pL growth medium in a white 384-well culture plate (PerkinElmer, 6007688) at a cell density of 4.000 cells/well on the day before the experiment. The plate was incubated over night at 37°C in 5% CO2. On the day of the assay, cells were washed once in assay buffer. Serial dilutions (7-fold dilutions, 7 concentrations Pr.
• comparator compounds and amylin analogues were performed in assay buffer often starting from approximately 10 nM in 96-well plates and 30 pL of each concentration added to the 384-well assay plate with cells.
• the assay plate was incubated for 3 hours at 37°C in 5% CO2 after which 30 pL SteadyLite Plus (PerkinElmer, 6066759) was added to each well.
• the assay plate was sealed, incubated at room temperature with gentle shaking for 5 minutes followed by 30 minutes incubation without shaking while protected from light. Luminescence was detected on a luminescence plate reader e.g. a Synergy 2 (BioTek).
• the compounds listed in Table 2 are all compounds according to the invention.
• Example 5 Oral pharmacokinetic profiles in Beagle dogs
• tablet compositions comprising the test substance and SNAC (sodium N-(8-(2-hydroxybenzoyl)amino)caprylate) were prepared by mixing test substance with roller compacted SNAC and magnesium stearate as, e.g., described in WO 2019/149880.
• This assay was performed to measure the oral bioavailability of a compound.
• the assay determined the exposure of test compound following oral administration in Beagle dogs as described by relevant pharmacokinetic parameters and plasma concentration curves.
• tablets containing test compound used for the assay described herein were immediate release SNAC-based tablets.
• the test compound was spray-dried as neutral sodium salt (pH 7 - 8). Dry granulation was carried out by roller compaction on a Gerteis MINI-PACTOR.
• Tablets containing 3 mg test compound, 300 mg sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) and 7.7 mg magnesium stearate were produced on at Kilian Style One using 7.2x12 mm punches.
• Tablets containing the test compound were administered in the following manner: 10 min prior to tablet administration the dogs were dosed subcutaneously with approximately 3.2 nmol/kg of glucagon. The 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 a syringe to facilitate swallowing of the tablet. Blood was sampled at predefined time points for up till 288 hours after dosing to adequately cover the full plasma concentration-time absorption profile of the compound. For each blood sampling time point approximately 1.2 mL of whole blood was collected in a 1.5 mL EDTA coated tube, and the tube was gently turned to allowing mixing of the sample with the EDTA.
• Plasma samples were kept on ice until centrifugation (4 min, 4°C, 4000 rpm). Plasma was pipetted into Micronic tubes on dry ice and kept at -20°C 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).
• Bioanalysis was carried out as follows: plasma concentrations of test compound were assayed by plasma protein precipitation and analysed by liquid chromatography-mass spectrometry (LC-MS). Calibrators were prepared by spiking blank dog plasma with analytes to reach the final concentrations in the range typically from 0.5 to 200 nM. Calibrators, plasma blanks or study samples were prepared for LC-MS by protein precipitation by adding 3 volumes of ethanol followed by centrifugation at 4000 rpm at 4°C for 1 h. The supernatant was diluted with 2 volumes of Milli-Q water containing 0.1% formic acid before injection on the LC-MS system.
• LC-MS liquid chromatography-mass spectrometry
• the system used was a Transcend II Interface Module SRD3200 system from Thermo Scientific (Waltham, MA, USA) coupled to a Orbitrap Exploris 240 mass spectrometer from Thermo Scientific.
• the LC was equipped with a TurboFlow Cyclone Column 0.5 x 50 mm (CH-953288, Thermo Scientific) as the first dimensional trapping column and Poroshell 120 SB-C182.7 pm as the analytical column (2.1 x 50 mm from Agilent, Santa Clara, CA, USA).
• the mobile phase composition of the loading pump is as below: mobile phase A consists of 95% mill i-Q water, 2.5% acetonitrile, 2.5 % methanol and 0.1% formic acid; mobile phase B consists of 47.5% acetonitrile, 47.5% methanol, 5% milli-Q water, and 0.1% formic acid.
• the analyte of interest was loaded from the Turbo flow column at 30% B to the second dimensional analytical column.
• a gradient elution was conducted at the elution pump using mobile phase A (95% milli-Q water, 2.5% acetonitrile, 2.5 % methanol and 0.1% formic acid) and mobile phase B (47.5% acetonitrile, 47.5% methanol, 5% milli-Q water, and 0.1% formic acid) with a ramping gradient of 0% mobile phase B to 60% mobile phase B in 0.25 minute and from 60% mobile phase B to 80% mobile phase B in 1.17 min, and then from 80% mobile phase B to 100% mobile phase B in 1.17 min.
• the Orbitrap Exploris 240 was operating in positive ionization mode with the parallel reaction monitoring (PRM) scan mode.
• PRM parallel reaction monitoring
• Linear calibration curves were used for calculating the test compound concentrations in the plasma samples to determine maximal plasma concentration (Cmax). Quality control samples for analytes were included. The deviation between nominal and calculated concentration in the calibrators and quality control samples were below 15% and the LLOQ sample was below 20%.
• the plasma concentration (vs time) profile of the test compound was evaluated and standard pharmacokinetic parameters were estimated by non-compartmental analysis (NCA) using WinNonlin Phoenix 64 (version 8.3.3, CERTARA). Results were reported as dose-corrected maximum plasma concentration (Cmax/Dose) , dose-corrected area under curve (AUC/Dose) and half-life (T1/2). AUC from time of dosing extrapolated to infinity, based on the last observed concentration (_obs) or last predicted concentration (_pred). “Linear up Log down” option was chosen.
• first-order rate constant (Lambda-Z) was estimated by linear regression of time vs. log concentration. First-order rate constant associated with the terminal (log-linear) portion of the curve. The software calculated the value of Lambda-Z and provided half-life (T1/2) by the formula as below:
• the compounds in Table 4 showed improved exposure level compared to the close cagrilintide analogue compound 1 (Table 5). In addition, the compounds in Table 4 exhibit a long T 2 following oral administration.
• the compounds listed in Table 4 are all compounds according to the invention.
• Example 6 Food intake studies in the rat model
• Lyophilised compound was dissolved in vehicle, 8 mM phosphate, 250 mM glycerol, 0.007% polysorbate 20, and pH was adjusted to 7.4 with 1N NaOH.
• the final formulation was sterile filtrated and filled into penfill cartridges for dosing.
• rats Prior to study start, rats were acclimatized to the experimental settings for 14 days. During this period, the animals were handled at least 2 times. Immediately upon arrival, rats were put on a reversed light cycle (dark from 10am - 10pm or 11 am - 11 pm) and were transferred into one of three following food intake measuring systems: FeedWin, BioDaq or HM2. Rats were single housed in the FeedWin and BioDaq system and were housed three a cage in the HM2 system (rats were in this case ID-chipped). During the acclimatization period, in which the rats get used to the new light cycle and diet, the animals had free access to food and water. Since rats are nocturnal animals (e.g.
• Table 6 Acute food intake in rat after a single s.c. administration of new amylin analogues (3 nmol/kg). A negative value indicates reduced food intake relative to vehicle whereas a positive value indicates increased food intake relative to vehicle.
• Table 7 Acute food intake in rat after a single s.c. administration of amylin comparator compounds (3 nmol/kg). A negative value indicates reduced food intake relative to vehicle whereas a positive value indicates increased food intake relative to vehicle.
• Amylin comparator compound cagrilintide (Table 7) comprises a peptide according to SEQ ID NO: 34 and is further disclosed in WO 2012/168432.

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