WO2020225781A1 - Agonistes d'amyline conjugués peptidiques et leurs utilisations - Google Patents

Agonistes d'amyline conjugués peptidiques et leurs utilisations Download PDF

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Publication number
WO2020225781A1
WO2020225781A1 PCT/IB2020/054364 IB2020054364W WO2020225781A1 WO 2020225781 A1 WO2020225781 A1 WO 2020225781A1 IB 2020054364 W IB2020054364 W IB 2020054364W WO 2020225781 A1 WO2020225781 A1 WO 2020225781A1
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WIPO (PCT)
Prior art keywords
independently selected
cys
xaa
hcy
peptide
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PCT/IB2020/054364
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English (en)
Inventor
Christopher Stuart WALKER
Kerry Martin Loomes
Debbie Lucy HAY
Margaret Anne Brimble
Lauren Rose YULE
Alexander Tups
Paul William Richard Harris
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Auckland Uniservices Limited
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Publication of WO2020225781A1 publication Critical patent/WO2020225781A1/fr

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    • 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
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally relates to peptide conjugates that are amylin agonists, pharmaceutical compositions and kits comprising such conjugates, and uses of such agonists.
  • BACKGROUND OF THE INVENTION Human amylin is a 37 amino acid peptide hormone that is co-secreted with insulin from pancreatic b-cells following nutrient intake and a corresponding rise in blood glucose.
  • Amylin displays glucoregulatory effects, and is also involved in the reduction of body weight through inducing satiety, inhibiting postprandial glucagon secretion, and slowing gastric emptying.
  • amylin offers significant potential for the treatment of obesity and diabetes.
  • the physiological effects of amylin are thought to be mediated by stimulation of amylin- responsive receptors in the area postrema of the brainstem.
  • Amylin receptors are heterodimers formed by the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMP-1, -2 and -3).
  • CTR calcitonin receptor
  • RAMP-1, -2 and -3 receptor activity-modifying proteins
  • Human amylin (hAmylin) (SEQ ID NO: 1) readily forms amyloid fibrils, making it unsuitable as a therapeutic drug.
  • Pramlintide (SEQ ID NO: 2) is a FDA-approved adjunctive treatment for diabetes, with further scope as an anti-obesity therapy. Despite this, pramlintide has shortcomings as a drug. Pramlintide is formulated as a
  • the present invention broadly consists in a peptide conjugate comprising an amylin peptide comprising or consisting of an amino acid sequence that is an analogue of SEQ ID NO: 1, wherein the peptide comprises a proline residue at positions 25, 28 and 29, wherein at least one amino acid of the peptide is covalently conjugated to a lipid-containing moiety via a sulfur atom of a sulfhydryl group, and wherein the peptide conjugate is a amylin-responsive receptor agonist.
  • the following embodiments and preferences may relate alone or in any combination of any two or more to any of the aspects herein.
  • the at least one amino acid is cysteine or homocysteine. In exemplary embodiments, the at least one amino acid is cysteine. In exemplary embodiments, the peptide conjugate comprises only one amino acid conjugated to a lipid-containing moiety. In other embodiments, the peptide conjugate comprises two or more amino acids each conjugated to a lipid-containing moiety. In some embodiments, the lipid-containing moiety comprises one or more straight or branched aliphatic or heteroaliphatic chains each containing at least 4 or at least 6 chain-linked atoms. In certain embodiments, the lipid-containing moiety comprises one or more saturated or unsaturated fatty acid esters. In various embodiments, the fatty acid is saturated. In some embodiments, the lipid-containing moiety is of the formula (A):
  • * represents a bond to the sulfur atom of the sulfhydryl group of the amino acid to which the lipid-containing moiety is conjugated;
  • Z and Z 1 are each independently selected from the group consisting of–O–,–NR–,–S–, –S(O)–,–SO 2 –,–C(O)O–,–OC(O)–,–C(O)NR–,–NRC(O)–,–C(O)S–,–SC(O)–,– OC(O)O–,–NRC(O)O–,–OC(O)NR–, and–NRC(O)NR–;
  • R is hydrogen or C1-6aliphatic
  • n is an integer from 0 to 4.
  • n 1 or 2;
  • R 1 and R 2 at each instance of m are each independently hydrogen, C1-6aliphatic; or R 1 is L 2 –Z 1 –C 1-6 alkyl;
  • R 3 , R 4 , and R 5 are each independently hydrogen or C1-6aliphatic; or R 3 is L 2 –Z 1 –C1- 6alkyl;
  • L 1 and L 2 are each independently C 5-21 aliphatic or C 4-20 heteroaliphatic;
  • R 3 is L 2 –Z 1 –C1-6alkyl
  • R 1 is not L 2 –Z 1 –C1-6alkyl
  • R 1 when m is an integer from 2 to 4, no more than one R 1 is L 2 –Z 1 –C1-6alkyl; and wherein any aliphatic, alkyl, or heteroaliphatic present in any of R, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , and L 2 is optionally substituted with one or more independently selected optional substituents.
  • R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , and L 2 is optionally substituted with one or more independently selected optional substituents.
  • R is hydrogen, C1-6alkyl, or C3-6cycloalkyl
  • n is an integer from 0 to 4.
  • n 1 or 2;
  • R 1 and R 2 at each instance of m are each independently hydrogen, C 1-6 alkyl, or C 3- 6cycloalkyl; or R 1 is L 2 –Z 1 –C1-6alkyl;
  • R 3 , R 4 , and R 5 are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R 3 is L 2 – Z 1 –C 1-6 alkyl;
  • L 1 and L 2 are each independently C5-21alkyl, C5-21alkenyl, or C4-20heteroalkyl;
  • any alkyl, alkenyl, cycloalkyl, or heteroalkyl present in any of R, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , and L 2 is optionally substituted with one or more independently selected optional substituents.
  • R is hydrogen or C 1-6 alkyl
  • n is an integer from 0 to 4.
  • n 1 or 2;
  • R 1 and R 2 at each instance of m are each independently hydrogen or C1-6alkyl; or R 1 is L 2 –Z 1 –C 1-6 alkyl;
  • R 3 , R 4 , and R 5 are each independently hydrogen or C1-6alkyl; or R 3 is L 2 –Z 1 –C1-6alkyl; L 1 and L 2 are each independently C5-21alkyl, C5-21alkenyl, or C4-20heteroalkyl;
  • any alkyl, alkenyl, or heteroalkyl present in any of R, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , and L 2 is optionally substituted with one or more independently selected optional substituents.
  • Z and Z 1 are each independently selected from–C(O)O–,–C(O)NR–, and–C(O)S–, preferably–C(O)O–.
  • the lipid-containing moiety is of the formula (I)
  • L 1 , R 1 , R 2 , R 3 , R 4 , and R 5 are as defined in any of the embodiments herein;
  • Z 1 when present is–C(O)O–.
  • m is an integer from 0 to 2. In certain embodiments, m is 0 or 1. In exemplary embodiments, m is 0.
  • R 1 and R 2 at each instance of m are each independently hydrogen.
  • R 4 and R 5 are each hydrogen.
  • R 3 is hydrogen or C1-6alkyl.
  • the lipid-containing moiety is of the formula (IV):
  • R 3 is hydrogen, L 2 –C(O)–OCH 2 , or L 2 –C(O)–OCH 2 CH 2 ;
  • L 1 and L 2 are each independently C 5-21 alkyl, C 5-21 alkenyl, or C 4-20 heteroalkyl.
  • L 1 and L 2 are each independently C5-21aliphatic, for example C9- 21 alihpatic, C 11-21 aliphatic, or C 9 -, C 11 -, C 13 -, C 15 -, C 17 -, or C 19 -aliphatic.
  • L 1 and L 2 are each independently C 5-21 alkyl.
  • L 1 and L 2 are each independently C9-21alkyl.
  • L 1 and L 2 are each independently is C11-21alkyl.
  • L 1 and L 2 are each independently C 9 , C 11 , C 13 , C 15 , C 17 , or C19alkyl, preferably n-alkyl. In various specifically contemplated embodiments, L 1 and L 2 are each independently C15alkyl. In certain embodiments, L 1 and L 2 are each independently linear C 15 alkyl. In various embodiments, L 1 and L 2 each independently comprise a linear chain of 9-21 carbon atoms. In some embodiments, R 3 is L 2 –C(O)–OCH2CH2. In some embodiments, R 3 is L 2 –C(O)–OCH2. In exemplary embodiments, R 3 is hydrogen.
  • L 1 is C5-21alkyl; m is 0; R 3 is hydrogen, L 2 –C(O)–OCH2, or L 2 –C(O)– OCH2CH2; L 2 is C11-21alkyl; and R 4 and R 5 are each hydrogen.
  • L 1 is C 5-21 alkyl; m is 0; R 3 is hydrogen; L 2 is C 11-21 alkyl; and R 4 and R 5 are each hydrogen.
  • L 1 is C5-21alkyl; m is 0; R 3 is L 2 –C(O)–OCH2; L 2 is C11-21alkyl; and R 4 and R 5 are In one embodiment, L 1 is C 5-21 alkyl; m is 0; R 3 is L 2 –C(O)–OCH 2 CH 2 ; L 2 is C 11-21 alkyl; and R 4 and R 5 are each hydrogen.
  • the moieties L 1 –Z 1 – and L 2 – Z 2 – may be fatty acid groups, for example fatty acid esters.
  • the moieties L 1 –Z 1 – and L 2 –Z 2 – may be saturated or unsaturated fatty acid esters.
  • the fatty acid is saturated.
  • the fatty acid is a C 4-22 fatty acid.
  • the fatty acid is a C6-22 fatty acid.
  • the fatty acid is a C10-22 fatty acid.
  • the fatty acid is a C12-22 fatty acid.
  • the fatty acid is a C 10 , C 12 , C 14 , C 16 , C 18 , or C 20 fatty acid.
  • the fatty acid is decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachic acid, palmitoleic acid, oleic acid, elaidic acid, linoleic acid, a-linolenic acid, and arachidonic acid.
  • the fatty acid is decanoic acid, lauric acid, myristic acid, palmitic acid, or stearic acid.
  • the fatty acid is palmitic acid (and the moieties L 1 –Z 1 – and L 2 –Z 2 – are each palmitoyl groups).
  • the one or more independently selected optional substituents are selected from halo, CN, NO 2 , OH, NH 2 , NHR x , NR x R y , C 1-6 haloalkyl, C 1-6 haloalkoxy, C(O)NH 2 , C(O)NHR x , C(O)NR x R y , SO2R x , OR y , SR x , S(O)R x , C(O)R x , and C1-6aliphatic; wherein R x and R y are each independently C 1-6 aliphatic, for example C 1-6 alkyl.
  • the N-terminal group of the peptide is–NR a R b , wherein R a and R b are each independently hydrogen, alkyl, cycloalkyl, acyl, aryl, or arylalkyl; and/or the C-terminal group of the peptide is–CH 2 OR c ,–C(O)OR c or–C(O)NR c R d , wherein R c and R d are each independently hydrogen, alkyl, cycloalkyl, aryl, or arylalkyl.
  • the N-terminal group of the peptide is–NR a R b , wherein R a and R b are each independently hydrogen, alkyl, cycloalkyl, acyl, aryl, or arylalkyl; and/or the C-terminal group of the peptide is–C(O)OR c or–C(O)NR c R d , wherein R c and R d are each independently hydrogen alkyl cycloalkyl aryl or arylalkyl
  • the N-terminal group of the peptide is aboutNH 2 or–NH(acyl), for example –NHAc; and/or the C-terminal group of the peptide is–C(O)NH2.
  • the N-terminal group of the peptide is–NH2.
  • the C-terminal group of the peptide is–C(O)NR c R d .
  • the C-terminal group of the peptide is–C(O)NH2.
  • the peptide conjugate is a lipopeptide.
  • the amylin peptide comprises or consists of an amino acid sequence: Xaa 1 -Cys-Xaa 3 -Xaa 4 -Xaa 5 -Thr-Cys-Ala-Thr-Xaa 10 -Xaa 11 -Leu-Ala-Xaa 14 - Xaa 15 - Xaa 16 -Xaa 17 - Xaa18-Ser-Xaa20-Xaa21-Xaa22- Xaa23- Xaa24-Pro-Xaa26- Xaa27-Pro-Pro-Thr-Xaa31-Xaa32-Gly- Xaa 34 -Xaa 35 -Xaa 36 - Xaa 37 [SEQ ID NO: 3];
  • Xaa 3 is independently selected from Gly, Pro, His, Arg, Ser, Asn, Gln, Cys, Hcy and Lys;
  • Xaa4 is independently selected from Met; Leu, Phe, Ile, Thr, Cys, Hcy and Ser;
  • Xaa5 is independently selected from Ser, Thr, Ala, Val, Leu, Cys, Hcy and Ile;
  • Xaa 10 is independently selected from Ala, Val, Leu, Ile, Gln, Cys, Hcy and Asn;
  • Xaa 11 is independently selected from His, Asn, Gln, Lys, Cys, Hcy and Arg;
  • Xaa14 is independently selected from Ala, Val, Leu, Ile, Glu, Asn, Gln, Lys, His, Cys, Hcy and Arg;
  • Xaa 15 is independently selected from Ala, Met, Leu, Tyr, Trp, Cys, Hcy and Phe;
  • Xaa16 is independently selected from Ala, Ile, Val, Met, Phe, Cys, Hcy and Leu;
  • Xaa17 is independently selected from Ala, Asp, Val, Leu, Ile, His, Arg, Lys, Val, Met, Cys, Hcy and Phe;
  • Xaa18 is independently selected from Arg, Pro, Lys, Gln, Asn, Cys, Hcy and His;
  • Xaa20 is independently selected from Arg, Lys, Gln, Asn, Ser, Cys, Hcy and Thr;
  • Xaa 21 is independently selected from Ala, Lys, Gln, Ser, Asn, His, Cys, Hcy and Arg;
  • Xaa 22 is independently selected from Lys, Arg, Glu, Gln, Ser, Thr, Asn, His, Cys, Hcy and Arg;
  • Xaa23 is independently selected from Leu, Ile, Val, Met, Ala, Cys, Hcy and Phe;
  • Xaa 24 is independently selected from Ala, Gly, Cys, Hcy and Pro;
  • Xaa 26 is independently selected from Ala, Val, Ile, Leu, Met, Phe, Pro, Arg, Cys, Hcy and Ile;
  • Xaa 27 is independently selected from Phe, Val, Ile, Ala, Met, Cys, Hcy and Leu;
  • Xaa31 is independently selected from Lys, Arg, Gln, Ser, Glu, Asp, Asn, Cys, Hcy and His;
  • Xaa32 is independently selected from Met, Leu, Phe, Ile, Ala, Cys, Hcy and Val;
  • Xaa 34 is independently selected from Ala, Val, Leu, Ile, Cys, Hcy, Thr, Tyr, Asp, Gln and Ser;
  • Xaa35 is independently selected from His, Arg, Lys, Asp, Gln, Glu, Cys, Hcy and Asn;
  • Xaa36 is independently selected from Ala, Cys, Hcy, Ser, Tyr, Asp, Gln and Thr;
  • Xaa 37 is independently selected from Pro and Tyr;
  • N-terminal is optionally acetylated
  • Xaa1, Xaa3-Xaa5, Xaa10, Xaa11, Xaa14-Xaa18, Xaa20-Xaa24, Xaa26, Xaa27, Xaa 31 , Xaa 32 , and Xaa 34 to Xaa 36 is cysteine or homocysteine and is covently conjugated to a lipid-containing moiety.
  • the amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein
  • Xaa1 is deleted or independently selected from Ala, Cys, Hcy, Glu, Gly, His, Arg, Ser, Ile and Lys;
  • Xaa 3 is independently selected from Gly, His, Arg, Ser, Cys, Hcy and Asn;
  • Xaa 4 is independently selected from Met, Cys, Hcy and Thr;
  • Xaa5 is independently selected from Ser, Cys, Hcy or Ala;
  • Xaa 10 is independently selected from Ala, Cys, Hcy and Gln;
  • Xaa 11 is independently selected from His, Cys, Hcy and Arg;
  • Xaa14 is independently selected from Ala, Glu, Cys, Hcy and Asn;
  • Xaa15 is independently selected from Ala, Leu, Tyr, Cys, Hcy and Phe;
  • Xaa 16 is independently selected from Ala, Ile, Val, Cys, Hcy and Leu;
  • Xaa17 is independently selected from Ala, Asp, His, Arg, Lys, Cys, Hcy and Val;
  • Xaa18 is independently selected from Arg, Pro, Lys, Cys, Hcy and His;
  • Xaa 20 is independently selected from Arg, Cys, Hcy and Ser;
  • Xaa 21 is independently selected from Ala, Lys, Gln, Ser, Cys, Hcy and Asn;
  • Xaa22 is independently selected from Lys, Glu, Gln, Ser, Thr, Cys, Hcy and Asn;
  • Xaa23 is independently selected from Leu, Ala, Cys, Hcy and Phe;
  • Xaa 24 is independently selected from Ala, Cys, Hcy and Gly;
  • Xaa26 is independently selected from Ala, Val, Pro, Arg, Cys, Hcy and Ile;
  • Xaa27 is independently selected from Phe, Cys, Hcy and Leu;
  • Xaa 31 is independently selected from Lys, Ser, Glu, Asp, Cys, Hcy and Asn;
  • Xaa 32 is independently selected from Met, Cys, Hcy and Val;
  • Xaa34 is independently selected from Ala, Cys, Hcy and Ser;
  • Xaa35 is independently selected from His, Arg, Lys, Asp, Gln, Glu, Cys, Hcy and Asn;
  • Xaa 36 is independently selected from Ala, Cys, Hcy and Thr;
  • Xaa37 is independently selected from Pro and Tyr;
  • N-terminal is optionally acetylated
  • Xaa 1 , Xaa 3 -Xaa 5 , Xaa 10 , Xaa 11 , Xaa 14 -Xaa 18 , Xaa 20 -Xaa 24 , Xaa 26 , Xaa 27 , Xaa31, Xaa32, and Xaa34 to Xaa36 is cysteine or homocysteine and is covently conjugated to a lipid-containing moiety.
  • one or more of Xaa 1 , Xaa 3 , Xaa 14 , Xaa 21 , Xaa 22 , Xaa 31 and Xaa 35 is cysteine or homocysteine and is covently conjugated to a lipid-containing moiety.
  • Xaa14 is Ala or Glu.
  • Xaa17 is Ala, His or Arg; more preferably Arg.
  • Xaa 35 is Asn or Gln; more preferably Asn.
  • Xaa14 is Glu and Xaa17 is Arg or His.
  • the amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein
  • Xaa 1 is deleted or independently selected from Ile, Cys, Hcy and Lys;
  • Xaa3 is independently selected from Gly, Cys, Hcy and Asn;
  • Xaa 4 is independently selected from Met and Thr;
  • Xaa 5 is Ala
  • Xaa10 is Gln
  • Xaa11 is independently selected from His and Arg;
  • Xaa 14 is Asn
  • Xaa17 is independently selected from Ala, Asp and Val;
  • Xaa18 is independently selected from Arg, Pro and His;
  • Xaa 20 is independently selected from Arg and Ser;
  • Xaa 21 is independently selected from Asn, Cys and Hcy;
  • Xaa22 is independently selected from Lys, Cys, Hcy and Asn;
  • Xaa 23 is independently selected from Leu and Phe;
  • Xaa 24 is Gly; Xaa 26 is independently selected from Val and Ile;
  • Xaa27 is independently selected from Phe and Leu;
  • Xaa31 is independently selected from Lys, Cys, Hcy and Asn;
  • Xaa 32 is Val
  • Xaa34 is Ser
  • Xaa35 is independently selected from Asp, Cys, Hcy and Asn;
  • Xaa 36 is Thr
  • Xaa37 is Tyr
  • N-terminal is optionally acetylated
  • amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein:
  • Xaa1 is Lys
  • Xaa 3 is Asn
  • Xaa 14 is Glu
  • Xaa15 is Phe
  • Xaa 16 is Leu
  • Xaa 17 is His or Arg
  • Xaa18 is His
  • Xaa21 is Cys, Hcy or Asn
  • Xaa 22 is Asn
  • Xaa26 is Ile
  • Xaa31 is Asn
  • Xaa 35 is Cys, Hcy or Asn.
  • the amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3 wherein:
  • Xaa 1 is deleted or independently selected from Cys, Hcy, His, Arg and Lys;
  • Xaa3 is independently selected from Cys, Hcy, Gly, His and Asn;
  • Xaa10 is independently selected from Ala and Gln;
  • Xaa 14 is independently selected from Cys, Hcy, Ala, Glu and Asn;
  • Xaa17 is independently selected from Ala, His, Arg and Val;
  • Xaa18 is independently selected from Arg and His;
  • Xaa 21 is independently selected from Cys, Hcy, Ser and Asn;
  • Xaa22 is independently selected from Cys, Hcy, Asn;
  • Xaa23 is independently selected from Ala and Phe;
  • Xaa 24 is independently selected from Ala and Gly;
  • Xaa26 is independently selected from Pro, Arg and Ile;
  • Xaa31 is independently selected from Cys, Hcy, Glu and Asn;
  • Xaa 35 is independently selected from Cys, Hcy, His, Arg, Lys, Asp, Gln and Glu;
  • Xaa 37 is independently selected from Pro and Tyr;
  • N-terminal is optionally acetylated
  • amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein:
  • Xaa 1 is deleted or independently selected from Cys, Hcy, Gly, His, Arg, Ser and Lys;
  • Xaa 3 is independently selected from Cys, Hcy, His and Asn;
  • Xaa10 is independently selected from Ala and Gln;
  • Xaa 14 is independently selected from Cys, Hcy, Ala, Glu and Asn;
  • Xaa 17 is independently selected from Ala, His, Arg and Val;
  • Xaa18 is independently selected from Arg and His;
  • Xaa21 is independently selected from Cys, Hcy, Gln, Ser and Asn;
  • Xaa 22 is independently selected from Cys, Hcy and Asn;
  • Xaa23 is independently selected from Ala and Phe;
  • Xaa24 is independently selected from Ala and Gly;
  • Xaa 26 is independently selected from Pro and Ile;
  • Xaa 31 is independently selected from Cys, Hcy and Asn;
  • Xaa35 is independently selected from Cys, Hcy and Asn;
  • Xaa37 is Pro
  • N-terminal is optionally acetylated
  • amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein:
  • Xaa1 is deleted or independently selected from Cys, Hcy, His, Arg and Lys;
  • Xaa 3 is independently selected from Cys, Hcy, Gly, His and Asn;
  • Xaa10 is independently selected from Ala and Gln;
  • Xaa14 is independently selected from Cys, Hcy, Ala, Glu and Asn;
  • Xaa 17 is independently selected from Ala, His, Arg and Val;
  • Xaa18 is independently selected from Arg and His
  • Xaa21 is independently selected from Cys, Hcy, Ser and Asn;
  • Xaa 22 is independently selected from Cys, Hcy and Asn;
  • Xaa 23 is independently selected from Ala and Phe;
  • Xaa24 is independently selected from Ala and Gly;
  • Xaa26 is Ile
  • Xaa 31 is independently selected from Cys, Hcy, Glu and Asn;
  • Xaa35 is independently selected from Cys, Hcy, His, Arg, Lys, Asp, Gln and Glu;
  • N-terminal is optionally acetylated
  • amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein:
  • Xaa 1 is deleted or independently selected from Cys, Hcy, Gly, His, Arg, Ser and Lys;
  • Xaa3 is independently selected from Cys, Hcy, His and Asn;
  • Xaa10 is independently selected from Ala and Gln;
  • Xaa 14 is independently selected from Cys, Hcy, Ala, Glu and Asn;
  • Xaa17 is independently selected from Ala, His, Arg and Val;
  • Xaa18 is independently selected from Arg and His;
  • Xaa 21 is independently selected from Cys, Hcy, Gln, Ser and Asn;
  • Xaa 22 is independently selected from Cys, Hcy and Asn;
  • Xaa23 is independently selected from Ala and Phe;
  • Xaa24 is independently selected from Ala and Gly;
  • Xaa 26 is independently selected from Pro and Ile;
  • Xaa31 is independently selected from Cys, Hcy and Asn;
  • Xaa35 is independently selected from Cys, Hcy and Asn; wherein one or more of Xaa 1 , Xaa 3 , Xaa 14 , Xaa 21 , Xaa 22 , Xaa 31 and Xaa 35 is cysteine or homocysteine and is covently conjugated to a lipid-containing moiety.
  • the amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein:
  • Xaa1 is deleted or independently selected from Ala, Cys, Hcy, Glu, Gly, His, Arg, Ser and Lys;
  • Xaa3 is independently selected from Cys, Hcy and Asn;
  • Xaa 10 is independently selected from Ala and Gln;
  • Xaa14 is independently selected from Cys, Hcy, Ala and Glu;
  • Xaa17 is independently selected from Ala, His, Arg and Val;
  • Xaa 18 is independently selected from Arg, Lys and His;
  • Xaa 21 is independently selected from Cys, Hcy, Gln and Asn;
  • Xaa22 is independently selected from Cys, Hcy, Thr and Asn;
  • Xaa26 is Ile
  • Xaa 31 is independently selected from Cys, Hcy and Asn;
  • Xaa35 is independently selected from Cys, Hcy, Gln, Gly and Asn;
  • N-terminal is optionally acetylated
  • amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein:
  • Xaa3 is independently selected from Cys, Hcy, Gly and Asn
  • Xaa10 is independently selected from Ala and Gln;
  • Xaa 14 is independently selected from Cys, Hcy, Ala and Glu;
  • Xaa17 is independently selected from Ala, His, Arg and Val;
  • Xaa18 is independently selected from Arg and His;
  • Xaa 21 is independently selected from Cys, Hcy, Gln and Asn;
  • Xaa 22 is independently selected from Cys, Hcy, Gln and Asn;
  • Xaa26 is Ile
  • Xaa31 is independently selected from Cys, Hcy, Glu and Asn;
  • Xaa 35 is independently selected from Cys, Hcy, Asn, Gln and Glu;
  • the N-terminal is optionally acetylated; wherein one or more of Xaa 3 , Xaa 14 , Xaa 21 , Xaa 22 , Xaa 31 and Xaa 35 is cysteine or homocysteine and is covently conjugated to a lipid-containing moiety.
  • the amylin peptide comprises or consists of an amino acid sequence which is an analogue of SEQ ID No: 3, wherein:
  • Xaa1 is independently selected from Lys, Cys and Hcy;
  • Xaa3 is independently selected from Asn, Cys and Hcy;
  • Xaa 10 is independently selected from Ala and Gln;
  • Xaa14 is independently selected from Cys, Hcy, Ala and Glu;
  • Xaa17 is independently selected from Ala, His, Lys, Arg and Val;
  • Xaa 18 is independently selected from Arg and His;
  • Xaa 21 is independently selected from Cys, Hcy, Ala, Lys, Gln and Ser;
  • Xaa22 is independently selected from Cys, Hcy, Glu, Gln, Ser, Thr and Asn;
  • Xaa23 is independently selected from Ala and Phe;
  • Xaa 24 is independently selected from Ala and Gly;
  • Xaa26 is independently selected from Pro and Ile;
  • Xaa31 is independently selected from Cys, Hcy, Ser, Glu, Asp and Asn;
  • Xaa 35 is independently selected from Cys, Hcy, Gln, Glu and Asn; wherein the N-terminal is optionally acetylated;
  • amylin peptide comprises or consists of an amino acid sequence selected from:
  • amylin peptide comprises or consists of a functional variant of any amylin peptide amino acid sequence of the embodiments above wherein the amino acid sequence of the functional variant has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least about 99% sequence identity to the amylin peptide amino sequence of the embodiments above.
  • the peptide comprises from about 1 to about 5 amino acids covalently conjugated to a lipid-containing moiety. In some embodiments, the peptide comprises from about 1 to about 3 amino acids covalently conjugated to a lipid-containing moiety. In some embodiments, the peptide comprises 1 or 2 amino acids covalently conjugated to a lipid- containing moiety. In some embodiments, Xaa 21 and/or Xaa 35 is covalently conjugated to a lipid-containing moiety. In some embodiments, the peptide comprises a C-terminal amide (that is, the C-terminal amino acid is amidated).
  • the peptide comprises an N-terminal acyl group, for example an acetyl group (that is, the N-terminal amino acid is acetylated).
  • a pharmaceutical composition comprising a peptide conjugate of the present invention; and a pharmaceutically acceptable carrier.
  • a kit comprising a peptide conjugate as herein disclosed; and instructions for use.
  • a method of agonising an amylin-responsive receptor in a subject in need thereof comprising administering to the subject an effective amount of a peptide conjugate as herein disclosed.
  • a method of treating a disease or condition mediated by or modulated by an amylin-responsive receptor in a subject comprising administering to the subject a therapeutically effective amount of a peptide conjugate as herein disclosed.
  • a method of treating, inhibiting or reducing weight gain, promoting weight loss, reducing food intake, and/or reducing excess body weight in a subject comprising administering to the subject a therapeutically effective amount of a peptide conjugate as herein disclosed.
  • the peptide conjugates are used in a method of treating obesity as well as associated diseases, disorders and health conditions, including, but not limited to, morbid obesity, obesity prior to surgery, obesity-linked inflammation, obesity-linked gallbladder disease and obesity-induced sleep apnea and respiratory problems, degeneration of cartilage, osteoarthritis, and reproductive health complications of obesity or overweight such as infertility in a subject.
  • the subject may be affected by obesity accompanied by at least one weight-related co-morbid condition, such as diabetes (e.g. type 2 diabetes), hypertension, dyslipidemia, sleep apnea and cardiovascular disease.
  • Atherosclerosis macrovascular disease, microvascular disease, diabetic heart disease (including diabetic cardiomyopathy and heart failure as a diabetic complication) coronary heart disease, peripheral artery disease or stroke, myocardial infarction, syndrome X, cognitive disorders, inflammatory bowel disease, dyspepsia, gastric ulcers, depression, anxiety, psychosis, schizophrenia, pain, osteoporosis, in a subject, comprising administering to the subject a therapeutically effective amount of a peptide conjugate as herein disclosed.
  • a method of lowering circulating LDL levels and/or increasing HDL/LDL ratio in a subject comprising administering a therapeutically effective amount of a peptide conjugate as herein disclosed to the subject.
  • a peptide conjugate as herein disclosed for use in agonising an amylin-responsive receptor In another embodiment, there is provided a peptide conjugate as herein disclosed for use in treating a disease or condition mediated by or modulated by an amylin-responsive receptor.
  • the peptide conjugates are useful, inter alia, in the reduction of food intake, promotion of weight loss, and inhibition or reduction of weight gain. As a result, they may be used for treatment of a variety of conditions, diseases, or disorders in a subject, including, but not limited to, obesity and various obesity-related conditions, diseases, or disorders, such as diabetes (e.g. type 2 diabetes), hypertension, dyslipidemia, sleep apnea and cardiovascular disease.
  • the subject may be affected by obesity accompanied by at least one weight-related co-morbid condition, such as diabetes (e.g. type 2 diabetes), hypertension, dyslipidemia, sleep apnea and cardiovascular disease.
  • diabetes e.g. type 2 diabetes
  • hypertension e.g. hypertension
  • dyslipidemia e.g., hypertension
  • sleep apnea e.g., hypertension
  • hypertension e.g. type 2 diabetes
  • dyslipidemia e.g. type 2 diabetes
  • sleep apnea e.g. type 2 diabetes
  • cardiovascular disease e.g. type 2 diabetes
  • the peptide conjugates may thus be administered to subjects affected by conditions characterised by inadequate control of appetite or otherwise over-feeding, such as binge- eating disorder and Prader-Willi syndrome.
  • the peptide conjugates can be used for treatment of combinations of the conditions described.
  • Treatment may be achieved, for example, by control of appetite, feeding, food intake, calorie intake and/or energy expenditure.
  • the subject may be affected by obesity accompanied by at least one weight-related co-morbid condition, such as diabetes (e.g. type 2 diabetes), hypertension, dyslipidemia, sleep apnea and cardiovascular disease.
  • diabetes e.g. type 2 diabetes
  • a peptide conjugate as herein disclosed for use in a method of treating, reducing or decreasing (e.g., in a statistically significant manner relative to an appropriate control) the severity or likelihood of occurrence of Alzheimer's disease, diabetes, type 1 diabetes, type 2 diabetes, pre-diabetes, insulin resistance syndrome, impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic disease including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, hepatic steatosis ("fatty liver”; including non alcoholic fatty liver disease (NAFLD) which itself includes non-alcoholic steatohepatitis (NASH)), kidney failure, arteriosclerosis (e.g.
  • a peptide conjugate as herein disclosed for use in a method of lowering circulating LDL levels and/or increasing HDL/LDL ratio. Effects of peptide conjugates as herein disclosed on these conditions may be mediated in whole or in part via an effect on body weight, or may be independent thereof.
  • a peptide conjugate as herein disclosed in the manufacture of a medicament for treating, inhibiting or reducing weight gain, promoting weight loss and/or reducing excess body weight in another embodiment, there is provided use of a peptide conjugate as herein disclosed in the manufacture of a medicament for treating obesity as well as associated diseases, disorders and health conditions, including, but not limited to, morbid obesity, obesity prior to surgery, obesity- linked inflammation, obesity-linked gallbladder disease and obesity-induced sleep apnea and respiratory problems, degeneration of cartilage, osteoarthritis, and reproductive health complications of obesity or overweight such as infertility.
  • the subject may be affected by obesity accompanied by at least one weight-related co-morbid condition, such as diabetes (e.g.
  • a peptide conjugate as herein disclosed in the manufacture of a medicament for treating, reducing or decreasing (e.g., in a statistically significant manner relative to an appropriate control) the severity or likelihood of occurrence of Alzheimer's disease, diabetes, type 1 diabetes, type 2 diabetes, pre-diabetes, insulin resistance syndrome, impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic disease including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, hepatic steatosis ("fatty liver”; including non-alcoholic fatty liver disease (NAFLD), which itself includes non-alcoholic steatohepatitis (NASH)), kidney failure, arteriosclerosis (e.g.
  • agonising the amylin-responsive receptor comprises treating a disease or condition mediated by or modulated by the amylin-responsive receptor.
  • agonising the amylin-responsive receptor comprises contacting a cell and a peptide conjugate as herein disclosed in an amount effective agonise the amylin- responsive receptor.
  • a method for preparing a peptide conjugate as herein disclosed comprising
  • the amino acid conjugate or the peptide conjugate comprising the peptide fragment is bound to a solid phase support; or the amino acid conjugate or the peptide conjugate is coupled to an amino acid or peptide bound to a solid phase. In various embodiments, the amino acid conjugate or the peptide conjugate comprising the peptide fragment is bound to a solid phase support.
  • a method for preparing a peptide conjugate as herein disclosed comprising reacting a lipid-containing conjugation partner comprising a carbon carbon double bond, and an amino acid-comprising conjugation partner comprising at least one amino acid comprising a thiol
  • a peptide conjugate as herein disclosed made by a method as herein disclosed. It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed.
  • Figures 1 to 6 show concentration-response curves for cyclic adenosine monophosphate (cAMP) production by unmodified pramlintide 1 compared with lipidated analogues 2 to 7, respectively, at the AMY1 receptor, as an example of amylin-responsive receptor activity. Curves are plotted as a percentage of maximal pramlintide-stimulated cAMP production. Data points represent the mean +/- S.E.M of at least three independent experiments performed in triplicate.
  • cAMP cyclic adenosine monophosphate
  • Figure 7 shows the change in bodyweight of C57BL/6J male mice exposed to either low- or high-fat diet followed by daily treatment of vehicle, pramlintide or lipidated analogue 3 (HFD- Lip Pram) for 4 weeks.
  • Figure 8 shows total food intake by C57BL/6J male mice exposed to either low- or high-fat diet and daily treatment of vehicle, pramlintide or lipidated pramlintide analogue 3.
  • LFD-V low-fat diet vehicle
  • HFD-V high-fat diet vehicle
  • HFD-P HFD pramlintide
  • HFD-LP HFD lipidated pramlintide analogue 3.
  • Figure 9 shows blood glucose concentrations of mice on the 8th day following treatment with vehicle, pramlintide, or lipidated pramlintide analogue 3, during intraperitoneal glucose tolerance testing (ipGTT).
  • Figure 10 shows area under curve analyses corresponding to Figure 9.
  • Figure 11 shows concentration-response curves for cyclic adenosine monophosphate (cAMP) production by unmodified pramlintide 1 compared with lipidated analogue 16 at the AMY1 receptor, as an example of amylin-responsive receptor activity. Data points represent the mean +/- S.E.M of five independent experiments.
  • cAMP cyclic adenosine monophosphate
  • Figure 12 shows concentration-response curves for cyclic adenosine monophosphate (cAMP) production by unmodified pramlintide 1 compared with lipidated analogue 17 at the AMY1 receptor, as an example of amylin-responsive receptor activity. Data points represent the mean +/- S.E.M of five independent experiments.
  • Figure 13 shows body weight (% starting) in mice treated with pramlintide 1 or analogues 3, 16 or 17, up to day 10 of treatment. Data are mean +/- S.E.M.
  • Figure 14 shows shows cumulative food intake over the same 10 days as figure 13. Data are mean +/- S.E.M.
  • Figure 15 shows total food intake over the same 10 day period as figures 13 and 14.
  • Asymmetric centers may exist in the compounds described herein.
  • the asymmetric centers may be designated as (R) or (S), depending on the configuration of substituents in three dimensional space at the chiral carbon atom.
  • stereochemical isomeric forms of the compounds including diastereomeric, enantiomeric, and epimeric forms, as well as d-isomers and l-isomers, and mixtures thereof, including enantiomerically enriched and diastereomerically enriched mixtures of stereochemical isomers, are within the scope of the invention.
  • Individual enantiomers can be prepared synthetically from commercially available enantiopure starting materials or by preparing enantiomeric mixtures and resolving the mixture into individual enantiomers. Resolution methods include conversion of the enantiomeric mixture into a mixture of diastereomers and separation of the diastereomers by, for example,
  • the compounds described herein may also exist as isotopologues and isotopomers, wherein one or more atoms in the compounds are replaced with different isotopes.
  • Suitable isotopes include, for example, 1 H, 2 H (D), 3 H (T), 12 C, 13 C, 14 C, 16 O, and 18 O. Procedures for incorporating such isotopes into the compounds described herein will be apparent to those skilled in the art.
  • Isotopologues and isotopomers of the compounds described herein are also within the scope of the invention.
  • salts of the compounds described herein including pharmaceutically acceptable salts.
  • Such salts include, acid addition salts, base addition salts, and quaternary salts of basic nitrogen-containing groups.
  • Acid addition salts can be prepared by reacting compounds, in free base form, with inorganic or organic acids. Examples of inorganic acids include, but are not limited to, hydrochloric, hydrobromic, nitric, sulfuric, and phosphoric acid.
  • organic acids include, but are not limited to, acetic, trifluoroacetic, propionic, succinic, glycolic, lactic, malic, tartaric, citric, ascorbic, maleic, fumaric, pyruvic, aspartic, glutamic, stearic, salicylic, methanesulfonic, benzenesulfonic, isethionic, sulfanilic, adipic, butyric, and pivalic.
  • Base addition salts can be prepared by reacting compounds, in free acid form, with inorganic or organic bases.
  • inorganic base addition salts include alkali metal salts, alkaline earth metal salts, and other physiologically acceptable metal salts, for example, aluminium, calcium, lithium, magnesium, potassium, sodium, or zinc salts.
  • organic base addition salts include amine salts, for example, salts of trimethylamine, diethylamine, ethanolamine, diethanolamine, and ethylenediamine.
  • Quaternary salts of basic nitrogen-containing groups in the compounds may be may be prepared by, for example, reacting the compounds with alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides, dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfates, and the like.
  • alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides
  • dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfates, and the like.
  • the compounds described herein may form or exist as solvates with various solvents. If the solvent is water, the solvate may be referred to as a hydrate, for ea e, a oo-y ae, - hydrate, or a
  • aliphatic is intended to include saturated and unsaturated, nonaromatic, straight chain, branched, acyclic, and cyclic hydrocarbons.
  • aliphatic groups include, for example, alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl and
  • aliphatic groups comprise from 1-12, 1-8, 1-6, or 1-4 carbon atoms. In some embodiments, aliphatic groups comprise 5-21, from 9-21, or from 11-21 carbon atoms, such as from 11, 13, 15, 17, or 19 carbon atoms. In some
  • the aliphatic group is saturated.
  • heteroaliphatic is intended to include aliphatic groups, wherein one or more chain and/or ring carbon atoms are independently replaced with a heteroatom, preferably a heteroatom selected from oxygen, nitrogen and sulfur.
  • the heteroaliphatic is saturated.
  • heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl groups.
  • alkyl is intended to include saturated straight chain and branched chain hydrocarbon groups. In some embodiments, alkyl groups have from 1 to 12, 1 to 10, 1 to 8, 1 to 6, or from 1 to 4 carbon atoms.
  • alkyl groups have from 5-21, from 9-21, or from 11- 21 carbon atoms, such as from 11, 13, 15, 17, or 19 carbon atoms.
  • straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n- heptyl, and n-octyl.
  • Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl.
  • alkenyl is intended to include straight and branched chain alkyl groups having at least one double bond between two carbon atoms.
  • alkenyl groups have from 2 to 12, from 2 to 10, from 2 to 8, from 2 to 6, or from 2 to 4 carbon atoms.
  • alkenyl groups have from 5-21, from 9-21, or from 11-21 carbon atoms, such as from 11, 13, 15, 17, or 19 carbon atoms.
  • alkenyl groups have one, two, or three carbon-carbon double bonds.
  • alkynyl is intended to include straight and branched chain alkyl groups having at least one triple bond between two carbon atoms. In some embodiments, the alkynyl group have from 2 to 12, from 2 to 10, from 2 to 8, from 2 to 6, or from 2 to 4 carbon atoms. In some embodiments, alkynyl groups have one, two, or three carbon-carbon triple bonds.
  • heteroalkyl is intended to include alkyl groups, wherein one or more chain carbon atoms are replaced with a heteroatom, preferably a heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur. Heteroalkyl groups include, for example,
  • cycloalkyl is intended to include mono-, bi- or tricyclic alkyl groups.
  • cycloalkyl groups have from 3 to 12, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5 carbon atoms in the ring(s).
  • cycloalkyl groups have 5 or 6 ring carbon atoms.
  • monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • the cycloalkyl group has from 3 to 8, from 3 to 7, from 3 to 6, from 4 to 6, from 3 to 5, or from 4 to 5 ring carbon atoms.
  • Bi- and tricyclic ring systems include bridged, spiro, and fused cycloalkyl ring systems. Examples of bi- and tricyclic ring cycloalkyl systems include, but are not limited to, bicyclo[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, adamantyl, and decalinyl.
  • the term“cycloalkenyl” is intended to include non-aromatic cycloalkyl groups having at least one double bond between two carbon atoms.
  • cycloalkenyl groups have one, two or three double bonds. In some embodiments, cycloalkenyl groups have from 4 to 14, from 5 to 14, from 5 to 10, from 5 to 8, or from 5 to 6 carbon atoms in the ring(s). In some embodiments, cycloalkenyl groups have 5, 6, 7, or 8 ring carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl.
  • the term“aryl” is intended to include cyclic aromatic hydrocarbon groups that do not contain any ring heteroatoms.
  • Aryl groups include monocyclic, bicyclic and tricyclic ring systems.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl.
  • aryl groups have from 6 to 14, from 6 to 12, or from 6 to 10 carbon atoms in the ring(s).
  • the aryl groups are phenyl or naphthyl.
  • Aryl groups include aromatic-aliphatic fused ring systems. Examples include, but are not limited to, indanyl and tetrahydronaphthyl.
  • arylalkyl refers to an alkyl group, as defined herein, substituted with an aryl group, as defined herein.
  • Arylalkyl groups are attached to the parent molecular moiety via the alkyl group. Examples of arylalkyl groups include, but are not limited to, benzyl, 2- phenylethyl, 3- phenylpropyl, 2-naphth-2-ylethyl, and the like.
  • acyl is intended to include R n -C(0)- groups, wherein R n is an aliphatic, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl group as defined herein.
  • R n is an alkyl group as defined herein, for example an acetyl group.
  • halo or“halogen” is intended to include F, Cl, Br, and I.
  • heteroatom is intended to include oxygen, nitrogen, sulfur, selenium, or phosphorus.
  • the heteroatom is selected from the group consisting of oxygen, nitrogen, and sulfur.
  • substituents in the compounds described herein include but are not limited to halo, CN, NO2, OH, NH 2 , NHR x , NR x R y , Ci- 6 haloalkyl, Ci-ehaloalkoxy, C(0)NH 2 , C(0)NHR x ,
  • the present invention relates to a peptide conjugate comprising an amylin peptide comprising or consisting of an amino acid sequence that is an analogue of SEQ ID NO: 1, wherein the peptide comprises a proline residue at positions 25, 28 and 29, wherein at least one amino acid of the peptide is covalently conjugated to a lipid-containing moiety via a sulfur atom of a sulfhydryl group.
  • the peptide conjugates as herein disclosed are amylin-responsive receptor agonists, i.e. they are capable of binding to, and inducing signaling by, one or more receptors or receptor complexes regarded as receptors responsive to human amylin.
  • These include the human calcitonin receptor hCTR, as well as complexes comprising the human calcitonin receptor hCTR and at least one of the human receptor activity-modifying proteins designated hRAMP1, hRAMP2 and hRAMP3.
  • Complexes between hCTR and hRAMP1, hRAMP2 and hRAMP3 are designated hAMY1, hAMY 2 and hAMY 3 (i.e. human amylin receptors 1, 2 and 3) respectively.
  • a peptide conjugate may be considered an amylin- responsive receptor agonist if it has agonist activity at one or more of hCTR, hAMY 1 , hAMY 2 and hAMY3, e.g. against hAMY1 and/or hAMY3, e.g. at hAMY3.
  • the peptide conjugate has an agonist potency value (pEC50) at an amylin- responsive receptor more than a value about 20-fold less than, 10-fold less than, 5-fold less than, 3-fold less than, 2-fold less than, 1-fold less than the agonist potency value (pEC 50 ) of pramlintide (SEQ ID No:2) at an amylin-responsive receptor or an agonist potency value (pEC50) at an amylin-responsive receptor more than a value equal to the agonist potency value (pEC 50 ) of pramlintide (SEQ ID No:2) at an amylin-responsive receptor, for example as measured by a cAMP assay as described in the Examples herein.
  • pEC50 agonist potency value
  • an amylin-responsive receptor agonist will also have agonist activity at hCTR when expressed in the absence of hRAMP1, hRAMP2 and hRAMP3.
  • the agonist will have activity at hCTR (when expressed in the absence of hRAMP1, hRAMP2 and hRAMP3) which is approximately less than 10-fold higher than its activity at any one of hAMY1, hAMY2 and hAMY 3 (i.e. its activity at all of these receptors) in a comparable assay.
  • Agonist activity at hCTR may be less than 5-fold higher than agonist activity at hAMY 1 , hAMY 2 and hAMY 3 , substantially equal to (e.g.
  • the agonist potency value (pEC 50 ) at an amylin-responsive receptor is measured by a cAMP assay, for example as described in the Examples herein, optionally wherein the amylin-responsive receptor is a CTR/RAMP1 amylin-responsive receptor or a CTR/RAMP3 amylin-responsive receptor.
  • a cAMP assay for example as described in the Examples herein, optionally wherein the amylin-responsive receptor is a CTR/RAMP1 amylin-responsive receptor or a CTR/RAMP3 amylin-responsive receptor.
  • Other suitable assays will be apparent to those skilled in the art.
  • the ability to induce cAMP formation i.e.
  • adenylate cyclase activity as a result of binding to the relevant receptor or receptor complex is typically regarded as indicative of agonist activity.
  • Other intracellular signaling pathways or events may also be used as read-outs for amylin-responsive receptor agonist activity. These may include calcium release, b- arrestin recruitment, receptor internalisation, kinase activation or inactivation, lipase activation, inositol phosphate release, diacylglycerol release or nuclear transcription factor translocation.
  • Peptide conjugates as herein disclosed may exhibit a number of advantageous properties in relation to human amylin and existing analogues thereof, such as pramlintide, and analogues described in WO2012/168430, WO2012/168431 and WO2012/168432.
  • peptide conjugates as herein disclosed may, for example, exhibit improved efficacy (e.g., in the form of improved in vitro activity or potency at one or more of the receptors hCTR, hAMY1, hAMY2 or hAMY3.
  • certain peptide conjugates as herein disclosed may have improved pharmokinetic properties such as increased half life, compared to human amylin and existing analogues thereof such as pramlintide or traditional N- acylated lipopeptides that are amylin-responsive receptor agonists lacking the covalently conjugated lipid-containing moiety or moieties present in the peptide conjugates as herein disclosed.
  • the peptide conjugate as herein disclosed in some embodiments may have a half life that is at least 2-fold (i.e.2-times) longer than the half life of human amylin and existing analogues thereof such as pramlintide (SEQ ID No: 2).
  • the half life may be measured by any suitable method known in the art, for example in a suitable rodent model, preferably a rat model.
  • the peptide conjugate has a half life at least 2-, 3-, 4-, 5-, 10-, 20-, 30-, 40-, or -50-fold longer than the half life of pramlintide, for example as measured in a suitable rodent model, for example a rat model.
  • peptide conjugates as herein disclosed may be longer acting, for example, it may have an extended receptor residence time.
  • the peptide conjugate may, for example, have a slower receptor dissociation rate compared to human amylin and existing analogues thereof such as pramlintide, maintaining functional cAMP activity.
  • Peptide conjugates that have an extended receptor residence time and/or a slower receptor dissociation rate may display superior in vivo efficacy.
  • peptide conjugates as herein disclosed may have an earlier onset of effect, for example in treating, inhibiting or reducing weight gain, promoting weight loss, reducing food intake, and/or reducing excess body weight, compared to human amylin or existing analogues thereof such as pramlintide.
  • peptide conjugates as herein disclosed may display reduced in vitro potency compared to human amylin or existing analogues thereof such as pramlintide, yet may retain useful biological properties, for example in vivo activity and longevity.
  • peptide conjugates as herein disclosed may exhibit improved solubility in aqueous media, especially at pH values in the range from 4 to 7.5, or at a range of pH values across that range. Moreover, peptide conjugates as herein disclosed may additionally or alternatively exhibit reduced tendency to undergo fibrillation in pharmaceutically relevant aqueous media, especially at pH values in the range from 4 to 7, or at a range of pH values across that range. Furthermore, peptide conjugates as herein disclosed may additionally or alternatively exhibit improved chemical stability (i.e. reduced tendency to undergo chemical degradation) in aqueous media, especially at pH values in the range from 4 to 9, or at a range of pH values across that range.
  • Peptide conjugates as herein disclosed may thus be well suited for formulation in acidic media (e.g. pH 4) and in neutral or near-neutral media (e.g. pH 7 or 7.4).
  • acidic media e.g. pH 4
  • neutral or near-neutral media e.g. pH 7 or 7.4
  • pramlintide for example, which generally exhibits poor chemical stability and rapid fibrillation in
  • peptide conjugates as herein disclosed may be thus well suited for co-formulation with, for example, insulin, various insulin analogues and/or other therapeutic (e.g. anti-diabetic or anti-obesity) agents that require a neutral or near- neutral formulation pH.
  • therapeutic agents e.g. anti-diabetic or anti-obesity
  • Activation of the calcitonin/amylin-responsive receptor by peptide conjugates as herein disclosed induces cAMP formation and activation of other intracellular signaling pathways and events.
  • cAMP cAMP or any other suitable parameter in suitable cells expressing the receptor
  • production of cAMP or any other suitable parameter in suitable cells expressing the receptor can be used to monitor agonist activity towards the receptor.
  • the assays may make use of the human calcitonin receptor (hCTR, e.g. isoform 2 of the hCTR) or the hAMY 1 receptor (see the examples below).
  • hCTR human calcitonin receptor
  • hAMY 1 receptor see the examples below.
  • sequences of precursor proteins are referred to, it should be understood that assays may make use of the mature protein, lacking the signal sequence.
  • pEC 50 values may also be used as a numerical measure of agonist potency at a given receptor.
  • the pEC50 towards hCTR is below 9 nM (e.g.0.001 to 9.0 nM). In some embodiments of peptide conjugates as herein disclosed, the pEC 50 towards hCTR is below 10 nM (e.g.0.001 to 10 nM). Additionally or alternatively, peptide conjugates as herein disclosed may show excellent resistance to fibrillation. For example, they may show no detectable fibrillation after 96 hours at pH 4.0 and/or pH 7.0, e.g. at 40°C, e.g. under the conditions described in Example 4 of WO 2018/046719, which is incorporated herein by reference in its entirety.
  • peptide conjugates as herein disclosed may show excellent chemical stability, i.e. resistance to degradation in solution. For example, they may retain at least 70% purity, at least 75% purity, at least 80% purity, at least 85% purity, at least 90% purity, or at least 95% purity after incubation at pH 4, pH 6, and/or pH 7 at 40°C for 72 hours, or for 14 days, e.g. under the conditions described in Example 5 of WO 2018/046719, which is incorporated herein by reference in its entirety.
  • Various amylin analogues including amylin analogues that are amylin-responsive receptor agonists, have been characterised and are suitable for use in the present invention.
  • amylin-responsive receptors include pramlintide (SEQ ID NO: 2).
  • the amylin peptides comprise or consist of an amino acid sequence that is an analogue of SEQ ID NO: 2 or a functional variant thereof, wherein at least one amino acid is or is substituted with an amino acid coavalently conjugated to a lipid-containing moiety.
  • the amylin peptides comprise full length pramlintide or a functional variant thereof wherein at least one amino acid is or is substituted with an amino acid coavalently conjugated to a lipid-containing moiety.
  • amylin peptides comprise an N-terminally truncated amylin peptide or functional variant thereof.
  • examples include peptides comprising or consisting of an amino acid sequence of SEQ ID No: 8.
  • amylin peptides including amylin peptides that are amylin-responsive receptor agonists, comprising one or more amino acid substitutions, such as one or more conservative amino acid substitutions.
  • A“conservative amino acid substitution” is one in which an amino acid residue is replaced with another residue having a chemically similar or derivatised side chain. Families of amino acid residues having similar side chains, for example, have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine,
  • Amino acid analogues e.g., phosphorylated or glycosylated amino acids
  • amino acid analogues are also contemplated in the present invention, as are peptides substituted with non-naturally occurring amino acids, including but not limited to N-alkylated amino acids (e.g. N-methyl amino acids), D-amino acids, b-amino acids, and g-amino acids.
  • amylin peptides including amylin peptides that are amylin agonists, are also specifically contemplated.
  • A“fragment” of a peptide is a sub-sequence of the peptide.
  • a“fragment” of a peptide is typically a sub-sequence of the peptide that performs a function that is required for the enzymatic or binding activity and/or provides three dimensional structure of the peptide, such as the three dimensional structure of a polypeptide.
  • a“fragment” of a peptide refers to any subsequence of the peptide, whether or not that sub-sequence performs a biological function.
  • variant refers to peptide sequences, including for example peptide sequences different from the specifically identified sequences, wherein one or more amino acid residues is deleted, substituted, or added. Variants are naturally-occurring variants, or non- naturally occurring variants. Variants are from the same or from other species and may encompass homologues, paralogues and orthologues.
  • functional variant refers to variants of peptides that possess biological activities that are the same or similar to those of the wild type peptides.
  • a functional variant of an amylin peptide for example pramlintide
  • the term“variant” with reference to peptides encompasses all forms of peptides as defined herein.
  • the degree of sequence identity between a variant and the sequence of a peptide described herein can be determined by comparing a candidate amino acid sequence to a sequence described herein, such as full length amylin or pramlintide using the BLAST suite of programs (version 2.2.12; 28 August 2005) that is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the term“a-amino acid” or“amino acid” refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the a-carbon.
  • Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogues. In certain embodiments the peptide-congugates as herein disclosed comprise only naturally occurring amino acids.
  • naturally occurring amino acid refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
  • amylin peptides including amylin peptides that are amylin-responsive receptor agonists, comprising one or more amino acid substitutions with a non-canonical amino acid.
  • Non-canonical amino acid includes naturally rare (in peptides or proteins) amino acids or non-naturally occurring amino acid residues.
  • Non-canonical amino acid inlcudes amino acids in D- or L-form that are not among the 20 naturally occurring amino acids.
  • “Non-canonical amino acids” include molecules which are structurally similar to an amino acid and which can be substituted for an amino acid, including without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g., a-amino b-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine (e.g.
  • N-methyl- amino acids N-methyl- amino acids
  • substitution or the carboxy group with an ester or carboxamide peptoids, wherein the amino acid side chain is appended to the nitrogen atom of the Na-amino group, rather than the a-carbon
  • a,a-disubstituted amino acids for example a- alkyl amino acids, wherein the a-carbon is substituted with an alkyl group in addition to the side chain of the amino acid
  • a,a-diamino acids wherein the a-carbon is substituted with two amino groups.
  • Other examples of non-canonical amino acids may include, for example conformationally constrained amino acids.
  • non-canonical amino acids include, without limitation (in the L-form or D-form; abbreviated as in parentheses): citrulline (Cit), homocitrulline (hCit), Na-methylcitmlline (NMeCit), Na-methylhomocitrulline (Na-MeHoCit), ornithine (Orn), Na-Methylornithine (Na- MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Na-methylarginine (NMeR), Na-methylleucine (Na-MeL or NMeL), N- methylhomolysine (NMeHoK), Na-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), Octahydro
  • aminophenylalanine aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (benzylphe), g- carboxyglutamic acid (g-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), a-aminoadipic acid (Aad), Na-methyl valine (NMeVal), N-a-methyl leucine (NMeLeu), Na-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), a,b-diaminopropionoic acid (Dpr), a,g-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (
  • the one or more amino acid substitutions with a non-naturally occurring amino acid is made to reduce the susceptibility of the amylin peptide to enzymatic proteolysis. This reduced susceptibility may be due to an effect on a protease binding site or cleavage site for an exopeptidase or endopeptidase.
  • substitutions include the substitution of one or more arginines or one or more lysines for a D-arginine, N-methylarginine, citrulline, dimethylarginine, homoarginine, N-methyl-citrulline, homocitrulline, 4-guanidino
  • polypeptide and“peptide” and the like are used herein interchangeably to refer to any polymer of amino acid residues of any length.
  • the polymer can be linear or non-linear (e.g., branched), it can comprise modified amino acids or amino acid analogues.
  • the term also encompasses amino acid polymers that have been modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other modification or manipulation, for example conjugation with labeling or bioactive components.
  • a lipid containing moiety is covalently conjugated (that is, covalenly bound) to at least one amino acid of the amylin peptide of the peptide conjugate as herein disclosed, for example via a heteroatom of a side chain of the amino acid, such as a sulfur atom of a sulfhydryl group.
  • Lipid containing moieties of various structures are contemplated for use herein.
  • a lipid containing moiety comprises a lipid and may comprise one or more other moiety, for example through which the lipid is attached to the amino acid.
  • the term“lipid” as used herein unless indicated otherwise refers to substances that are soluble in organic solvents, including, but not limited to, oils, fats, fatty acids and esters thereof, and the like.
  • the lipid or lipid containing moiety is lipophilic and/or hydrophobic.
  • Methods of preparation The peptide conjugates as herein disclosed may be prepared using the methods and procedures described herein. Other suitable methods for preparing peptide conjugates as herein disclosed will be apparent to those skilled in the art.
  • the peptide conjugates as herein disclosed may be prepared from readily available starting materials using the methods and procedures described herein. It will be appreciated that where typical or preferred process conditions (for example, reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are indicated, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants used. Conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art (see, for example, T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999).
  • the starting materials useful in the methods and reactions are commercially available or can be prepared by known procedures or modifications thereof, for example those described in in standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1- 15 (John Wiley and Sons, 1991), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4 th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
  • the various starting materials, intermediates, and compounds may be isolated and purified where appropriate using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography. Characterization of the compounds may be performed using conventional methods such as by melting point, mass spectrum, nuclear magnetic resonance, and various other spectroscopic analyses.
  • the present invention relates in certain embodiments to a method for preparing a peptide conjugate as herein disclosed comprising
  • lipid-containing conjugation partner comprising a carbon carbon double bond
  • amino acid-comprising conjugation partner comprising at least one amino acid comprising a thiol
  • the present invention also relates in certain embodiments to a peptide conjugate as herein disclosed made by a method as herein disclosed.
  • the amino acid acid conjugate comprising an amino acid of an amylin peptide of (A) or peptide- conjugate comprising a peptide fragment of an amylin peptide of (B) may be provided by methods known in the art or analogous thereto. Such methods include the conjugation methods described in WO 2014/207708 A2, WO 2016/103192 A1, and WO 2017/145097 A2, each of which are incorporated herein by reference in their entirety.
  • Such methods also include the thiol-ene reaction based conjugation method described in PCT/IB2018/058684 (published as WO 2019/087161 A1), the contents of which are also incorporated herein by reference in their entirety.
  • the thiol-ene reaction involves the addition of a thiol across a non-aromatic carbon-carbon double bond (i.e. hydrothiolation of the carbon-carbon double bond).
  • the amino acid-comprising conjugation partner comprises the thiol and the lipid- containing conjugation partner comprises the carbon carbon double bond.
  • the lipid-containing conjugation partner and amino acid-comprising conjugation partner in the reaction are as defined in any of the embodiments described herein.
  • the reaction proceeds via a free radical mechanism.
  • initiation There are three distinct phases in the reaction: initiation, polymerisation or coupling, and termination.
  • Radical generation gives rise to an electrophilic thiyl radical which propagates across the ene group, forming a carbon-centred radical.
  • the carbon-centred radical may be quenched by chain transfer from additional thiol molecule to give the final hydrothiolation product.
  • the carbon-centred radical may react with a carbon carbon double bond of a second molecule of a lipid-containing conjugation partner to provide a bis- addition product (or bis-adduct) in which the sulfur atom from the thiol is conjugated to a carbon atom from the carbon-carbon double bond of a first lipid-containing conjugation partner, and a carbon atom from the carbon-carbon double bond of the first lipid-containing conjugation partner is conjugated to a carbon atom from the carbon-carbon double bond of a second lipid- containing conjugation partner.
  • the first lipid-containing conjugation partner and second lipid-containing conjugation partner are identical. The two pathways are believed to be competitive.
  • the amino acid comprising conjuation parter is a peptide-containing conjugation partner.
  • the amino acid comprising conjugation partner comprises, consists essentially of, or consists of an amino acid (as opposed to a peptide).
  • the amino acid comprising conjugation partner comprises a peptide of an amylin peptide, wherein at least one amino acid of the peptide comprises a thiol.
  • reaction with the lipid-containing conjugation partner provides a peptide conjugate as herein disclosed.
  • the amino acid comprising conjugation partner comprises a peptide fragment of an amylin peptide, wherein at least one amino acid of the peptide fragment comprises a thiol.
  • the amino acid comprising conjugation partner comprises an amino acid of an amylin peptide, wherein the amino acid comprises a thiol.
  • reaction with the lipid containing conjugation partner provides an amino acid conjugate or peptide conjugate, which may be coupled to one or more amino acids and/or one or more peptides to provide a peptide conjugate as herein disclosed.
  • One or more free radicals may be generated in the reaction by any method known in the art. The free radicals may be generated thermally and/or photochemically.
  • One or more free radical initiators may be used to initiate the generation of free radicals.
  • Suitable free radical initiators include thermal initiators and photoinitiators.
  • the lipid-containing conjugation partner and amino acid-comprising conjugation partner may be prepared using known synthetic chemistry techniques or modifications thereof (for example, the methods generally described in Louis F Fieser and Mary F, Reagents for Organic Synthesis v. 1- 19, Wiley, New York (1967-1999 ed.) or Beilsteins Handbuch der organischen Chemie , 4, Aufl. Ed.
  • the lipid-containing conjugation partner may be a compound of the formula (A-l):
  • lipid-containing conjugation partner compounds of the formula (II) For example, lipid-containing conjugation partner compounds of the formula (II)
  • Y is H, a metal or metalloid, or acyl (for example, alkylcarbonyl) under conditions effective for esterification (or transesterification where Y is an acyl group).
  • Methods for esterification are well known in the art.
  • X is chloro
  • the reaction may be carried out in the presence of a base, such as pyridine or triethylamine, in a suitable solvent.
  • the acid chloride may be converted in situ to a more reactive species (e.g. to the corresponding iodide, using sodium iodide).
  • the temperature at which the reaction is carried out depends on the reactivity of the acid species and the solvent used.
  • vinyl esters of the formula (II) may be produced by transesterification with vinyl acetate (itself produced industrially by the reaction of acetic acid and acetylene or acetic acid and ethylene over a suitable catalyst) using an acid or metal catalyst. See, for example, EP 0376075 A2 and S. K. Karmee, J. Oil Palm Res., 2012, 1518-1523. Vinyl esters of the formula (II) may also be prepared by the addition a carboxylic acid to a terminal acetylene in the presence of a catalyst (usually a palladium or ruthenium complex). See, for example, V. Cadierno, J. Francos, J. Gimeno Organometallics, 2011, 30, 852-862; S.
  • the peptide conjugates as herein disclosed produced by, the peptide conjugates used in, the amino acid-comprising conjugation partners used in, and/or the peptides coupled in the methods as herein disclosed may comprise a synthetic peptide.
  • Synthetic peptides may be prepared using solid phase peptide synthesis (SPPS).
  • the coupling of one or more amino acids and/or one or more peptides to provide peptides or peptide conjugates may also be carried out by SPPS.
  • Synthetic peptides may also be prepared by liquid phase peptide synthesis.
  • the methods as herein disclosed may comprise coupling one or more amino acid and/or one or more peptide.
  • the one or more amino acid and/or one or more peptide may be coupled by SPPS. In some embodiments, all of the one or more amino acid and/or one or more peptides are coupled by SPPS .
  • the methods comprise coupling the amino acid of the amino acid conjugate to one or more amino acid and/or one or more peptide to provide the peptide conjugate as herein disclosed.
  • the method comprises coupling the amino acid of the amino acid conjugate to an amino acid or peptide bound to a solid phase support by SPPS .
  • the method comprises coupling the amino acid of the amino acid conjugate to a peptide bound to a solid phase support by SPPS.
  • the method may comprise synthesising the peptide bound to the solid phase support by SPPS.
  • the method comprises coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conjugate to one or more amino acid and/or one or more peptide to provide the peptide conjugate as herein disclosed.
  • the coupling may be carried out by SPPS as described herein.
  • the peptide of the peptide conjugate to be coupled is bound to a solid phase support, and the method comprises coupling an amino acid of the peptide conjugate to be coupled to one or more amino acid and/or one or more peptide to provide a solid phase bound peptide conjugate.
  • the coupling may be carried out by SPPS as described herein.
  • the method comprises coupling an amino acid of the peptide conjugate to an amino acid or peptide bound to a solid phase support by SPPS to provide a solid phase bound peptide conjugate.
  • coupling an amino acid or a peptide to another amino acid or peptide as described herein typically comprises forming a peptide bond between the Na-terminus of the amino acid or an amino acid of the peptide of one coupling partner and the C-terminus of the amino acid or an amino acid of the peptide of the other coupling partner.
  • the method as herein disclosed comprises synthesising the amino acid sequence of the peptide of the peptide-containing conjugation partner by SPPS; and reacting the peptide-containing conjugation partner with the lipid containing conjugation partner.
  • synthesising the amino acid sequence of the peptide of the peptide- containing conjugation partner by SPPS comprises coupling one or more amino acid and/or one or more peptide to an amino acid or peptide bound to a solid phase support to provide the amino acid sequence of the peptide or a portion thereof.
  • the amino acid sequence of the entire peptide of the peptide-containing conjugation partner is synthesised by SPPS.
  • the amino acid comprising conjugation partner may be reacted with the lipid-containing conjugation partner while bound to a solid phase support.
  • the peptide containing conjugation partner may be cleaved from the solid phase support, and optionally purified, prior to reaction, for example with the lipid- containing conjugation partner. Confirmation of the identity of the peptides synthesized may be conveniently achieved by, for example, amino acid analysis, mass spectrometry, Edman degradation, and the like.
  • the method as herein disclosed may further comprise separating the peptide conjugate as herein disclosed from the liquid reaction medium. Any suitable separation methods known in the art may be used, for example, precipitation and filtration.
  • the conjugate may be subsequently purified, for example, by HPLC using one or more suitable solvents.
  • the peptide conjugates thus, may be pure or purified, or substantially pure or purified.
  • purified does not require absolute purity; rather, it is intended as a relative term where the material in question is more pure than in the environment it was in previously. In practice the material has typically, for example, been subjected to fractionation to remove various other components, and the resultant material has substantially retained its desired biological activity or activities.
  • substantially purified refers to materials that are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free, at least about 95% free, at least about 98% free, or more, from other components with which they may be associated during manufacture.
  • the present invention relates to a method of agonising an amylin-responsive receptor in a subject in need thereof, comprising administering to the subject an effective amount of a peptide conjugate as herein disclosed.
  • the present invention also relates to a method of treating a disease or condition mediated by or modulated by an amylin-responsive receptor in a subject in need thereof, comprising
  • peptide conjugates as herein disclosed are useful, inter alia, in the reduction of food intake, promotion of weight loss and inhibition or reduction of weight gain They may therefore provide an attractive treatment option for, inter alia, obesity and metabolic diseases caused, characterised by, or associated with, excess body weight.
  • the peptide conjugates may be used in a method of treating, inhibiting or reducing weight gain, promoting weight loss, reducing food intake, and/or reducing excess body weight.
  • Treatment may be achieved, for example, by control of appetite, feeding, food intake, calorie intake and/or energy expenditure.
  • the compounds may be used in a method of treating obesity as well as associated diseases, disorders and health conditions, including, but not limited to, morbid obesity, obesity prior to surgery, obesity-linked inflammation, obesity-linked gallbladder disease and obesity-induced sleep apnea and respiratory problems, degeneration of cartilage, osteoarthritis, and reproductive health complications of obesity or overweight such as infertility.
  • the peptide conjugates may also be used in in a method of prevention or treatment of
  • Alzheimer's disease diabetes, type 1 diabetes, type 2 diabetes, pre-diabetes, insulin resistance syndrome, impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic disease including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, hepatic steatosis ("fatty liver”; including non-alcoholic fatty liver disease (NAFLD), which itself includes non-alcoholic steatohepatitis (NASH)), kidney failure, arteriosclerosis (e.g. atherosclerosis), macrovascular disease, microvascular disease, diabetic heart disease (including diabetic cardiomyopathy and heart failure as a diabetic complication) coronary heart disease, peripheral artery disease or stroke.
  • metabolic disease including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, hepatic steatosis ("fatty liver”; including non-alcoholic fatty liver disease (NAFLD), which itself includes non-alcoholic steatohepatitis (NASH)
  • arteriosclerosis e.g. athe
  • the peptide conjugates may also be useful in lowering circulating LDL levels and/or increasing HDL/LDL ratio.
  • the effects of the peptide conjugates described above may be mediated in whole or in part via an effect on body weight, or may be independent thereof.
  • Metabolic syndrome is characterized by a group of metabolic risk factors in one person. They include abdominal obesity (excessive fat tissue around the abdominal internal organs), atherogenic dyslipidemia (blood fat disorders including high triglycerides, low HDL cholesterol and/or high LDL cholesterol, which foster plaque buildup in artery walls), elevated blood pressure (hypertension), insulin resistance and glucose intolerance, prothrombotic state (e.g.
  • A“subject” refers to a human or a non-human animal, preferably a vertebrate that is a mammal, preferably a human.
  • Non-human mammals include, but are not limited to, farm animals, such as, cattle, sheep, swine, deer, and goats; sport and companion animals, such as, dogs, cats, and horses; and research animals, such as, mice, rats, rabbits, and guinea pigs.
  • the subject is a human.
  • treatment and related terms such as“treating” and“treat”, as used herein, unless indicated otherwise, relates generally to treatment, of a human or a non-human subject, in which some desired therapeutic effect is achieved.
  • the therapeutic effect may, for example, be inhibition, reduction, amelioration, halt, or prevention of the disease or condition.
  • A“therapeutically effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results.
  • a therapeutically effective amount can be administered in one or more administrations by various routes of administration.
  • the therapeutically effective amount to be administered to a subject depends on, for example, the purpose for administeration, mode of administration, nature and dosage of any co-administered compounds, and characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs. A person skilled in the art will be able to determine appropriate dosages having regard to these any other relevant factors.
  • the efficacy of a peptide conjugate can be evaluated both in vitro and in vivo.
  • the peptide conjugate can be tested in vitro or in vivo for its ability to act as an agonist of an amylin- responsive receptor.
  • the peptide conjugate can be administered to an animal (e.g., a rat or a mouse), for example by injection, and its effects evaluated.
  • the peptide conjugates as herein disclosed may be injected into mice and the effects on glucose homeostasis, measured using intraperitoneal glucose tolerance tests. Based on the results, an appropriate dosage range and administration route can be determined.
  • the peptide conjugate is typically administered in the form of a pharmaceutical composition as described herein.
  • the composition may be administered as a single dose or a multiple dose schedule.
  • the peptide conjugate can be used or administered as the sole therapeutic agent or in
  • antidiabetic agents such as antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
  • these pharmacologically active substances are: insulin, insulin derivative, insulin analogues, GLP-1 , GLP-1 derivatives, GLP-1 analogues, oxyntomodulin derivatives, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the b-cells; Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine
  • peptide conjugates as herein disclosed can be used or administered in combination with leptin agonists or leptin antagonists for treating or preventing a disease, disorder or condition in which aberrant leptin signaling is implicated, such as obesity, hyperphagia-related syndromes, type 1 diabetes, type 2 diabetes, metabolic syndrome, hypertriglyceridemia, atherosclerosis, lipodystrophy or infertility; or promotion of angiogenesis.
  • the peptide conjugate and one or more additional therapeutic agents may be used or
  • the one or more additional therapeutic agents will depend on the disease or condition to be treated or other desired therapeutic benefit.
  • the one or more additional therapeutic agents can be used in therapeutic amounts indicated or approved for the particular agent, as would be known to those skilled in the art.
  • two or more peptide conjugates as herein disclosed are used or administered in combination.
  • the two or more peptide conjugates may be used or administered simultaneously, sequentially, or separately.
  • the present invention also relates to a method of agonising an amylin-responsive receptor comprising contacting a cell with a peptide conjugate as herein disclosed in an amount effective to agonise the receptor.
  • the cell may be in vivo, in vitro, or ex vivo.
  • the cell may be contacted with the peptide conjugate by administering the peptide conjugate to a subject.
  • Methods of agonising amylin-responsive receptors in a cell in vitro or ex vivo may be useful, for example, in a variety of diagnostic tests or laboratory research.
  • Pharmaceutical compositions The present invention further relates to a pharmaceutical composition comprising a peptide conjugate as herein disclosed; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an effective amount of the peptide conjugate.
  • the pharmaceutical compositions may comprise two or more peptide conjugates as herein disclosed.
  • pharmaceutically acceptable carrier refers to a carrier (e.g.
  • compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate
  • Cyclodextrins such as a-, b-, and g-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-3- cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery.
  • Oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
  • compositions are formulated to allow for administration to a subject by any chosen route, including but not limited to oral or parenteral (including topical, subcutaneous, intramuscular and intravenous) administration.
  • the compositions may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, diluents, auxiliaries, and combinations thereof) selected with regard to the intended route of admini tration and standard pharmaceutical practice.
  • the compositions may be administered orally as a powder, liquid, tablet or capsule, or topically as an ointment, cream or lotion.
  • Suitable formulations may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents, and may be adapted for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release.
  • compositions may be administered via the parenteral route.
  • parenteral dosage forms include aqueous solutions, isotonic saline of the active agent, or other well-known pharmaceutically acceptable excipients.
  • Cyclodextrins for example, or other solubilising agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent.
  • dosage forms suitable for oral administration include, but are not limited to tablets, capsules, lozenges, or like forms, or any liquid forms such as syrups, aqueous solutions, emulsions and the like, capable of providing a therapeutically effective amount of the composition.
  • Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose.
  • Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the active ingredients with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. Active ingredients can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tabletting agent.
  • Examples of dosage forms suitable for transdermal administration include, but are not limited, to transdermal patches, transdermal bandages, and the like.
  • Examples of dosage forms suitable for topical administration of the compositions include any lotion, stick, spray, ointment, paste, cream, gel, etc., whether applied directly to the skin or via an intermediary such as a pad, patch or the like.
  • Examples of dosage forms suitable for suppository administration of the compositions include any solid dosage form inserted into a bodily orifice particularly those inserted rectally, vaginally and urethrally.
  • Examples of dosage of forms suitable for injection of the compositions include delivery via bolus such as single or multiple administrations by intravenous injection, subcutaneous, subdermal, and intramuscular administration or oral administration.
  • Examples of dosage forms suitable for depot administration of the compositions include pellets or solid forms wherein the active(s) are entrapped in a matrix of biodegradable polymers, microemulsions, liposomes or are microencapsulated.
  • Examples of infusion devices for the compositions include infusion pumps for providing a desired number of doses or steady state administration, and include implantable drug pumps.
  • Examples of implantable infusion devices for compositions include any solid form in which the active(s) are encapsulated within or dispersed throughout a biodegradable polymer or synthetic, polymer such as silicone, silicone rubber, silastic or similar polymer.
  • dosage forms suitable for transmucosal delivery of the compositions include depositories solutions for enemas, pessaries, tampons, creams, gels, pastes, foams, nebulised solutions, powders and similar formulations containing in addition to the active ingredients such carriers as are known in the art to be appropriate.
  • dosage forms include forms suitable for inhalation or insufflation of the compositions, including compositions comprising solutions and/or suspensions in pharmaceutically acceptable, aqueous, or organic solvents, or mixture thereof and/or powders.
  • Transmucosal administration of the compositions may utilize any mucosal membrane but commonly utilizes the nasal, buccal, vaginal and rectal tissues.
  • Formulations suitable for nasal administration of the compositions may be administered in a liquid form, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, including aqueous or oily solutions of the polymer particles.
  • Formulations may be prepared as aqueous solutions for example in saline, solutions employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bio-availability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.
  • Examples of dosage forms suitable for buccal or sublingual administration of the compositions include lozenges, tablets and the like.
  • dosage forms suitable for opthalmic administration of the compositions include inserts and/or compositions comprising solutions and/or suspensions in pharmaceutically acceptable, aqueous, or organic solvents.
  • compositions examples include, for example, Sweetman, S. C. (Ed.). Martindale. The Complete Drug Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, 2483 pp.; Aulton, M. E. (Ed.) Pharmaceutics. The Science of Dosage Form Design. Churchill Livingstone, Edinburgh, 2000, 734 pp.; and, Ansel, H. C, Allen, L. V. and Popovich, N. G. Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott 1999, 676 pp. Excipients employed in the manufacture of drug delivery systems are described in various publications known to those skilled in the art including, for example, Kibbe, E. H.
  • the USP which also describes specific tests to determine the drug release capabilities of extended-release and delayed-release tablets and capsules.
  • the USP test for drug release for extended-release and delayed-release articles is based on drug dissolution from the dosage unit against elapsed test time. Descriptions of various test apparatus and procedures may be found in the USP. Further guidance concerning the analysis of extended release dosage forms has been provided by the F.D.A. (See Guidance for Industry. Extended release oral dosage forms: development, evaluation, and application of in vitro/in vivo correlations. Rockville, MD: Center for Drug Evaluation and Research, Food and Drug Administration, 1997).
  • the dosage forms described herein can be in the form of physically discrete units suitable for use as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect. Dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to provide an amount of the active ingredient which is effective to achieve the desired therapeutic effect for a particular patient, composition, and mode of administration, without being toxic to the patient (an effective amount).
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, the route of administration, the time of administration, the rate of excretion of the particular peptide conjugate being employed, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the daily amount or regimen should be in the range of about 1 to about 10,000 micrograms (mg) of the amylin peptide per kilogram (kg) of body mass, preferably about 1 to about 5000 mg per kilogram of body mass, and most preferably about 1 to about 1000 mg per kilogram of body mass.
  • Kits In certain embodiments the present invention also provides a kit comprising a peptide conjugate as herein disclosed; and instructions for use.
  • the peptide conjugate is typically in the form of a pharmaceutical composition, and contained within a container.
  • the instructions for use may describe the method(s) of treatment in which the peptide conjugates are administered. In various embodiments, the instructions for use describe methods of treating the diseases and conditions indicated herein.
  • the container may be any vessel or other sealed or sealable apparatus that can hold the pharmaceutical composition. Examples include bottles, ampules, divided or multi-chambered holders, bottles, wherein each division or chamber comprises a single dose of said composition, a divided foil packet wherein each division comprises a single dose of said composition, or a dispenser that dispenses single doses of said composition.
  • the container can be in any conventional shape or form and is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag, or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule.
  • the container employed typically depends on the dosage form involved. More than one container can be used together in a single package for a single dosage form.
  • the kits may also comprise a device to administer or to measure out a unit dose of the pharmaceutical composition.
  • the device may include, for example, an inhaler if the
  • kits may comprise, for example in a separate vessel or container, one or more additional therapeutic agent, typically in the form of a pharmaceutical composition comprising the additional therapeutic agent and a pharmaceutically acceptable carrier.
  • Fmoc-Cys(tBu)-OH was purchased from CS Bio (Menlo Park, California). N-Methylmorpholine (NMM), piperidine, 2,2’-dithiobis(5-nitropyridine) (DTNP), triisopropylsilane (TIS), guanidine hydrochloride (Gu ⁇ HCl), vinyl decanoate, vinyl palmitate, vinyl stearate, 2,2-dimethoxy-2-phenylacetophenone (DMPA), N,N’-diisopropylcarbodiimide (DIC), 2,2’-(ethylenedioxy)diethanethiol (DODT), and deuterochloroform (CDCl3) were purchased from Sigma-Aldrich (St.
  • HATU O-(7- Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate
  • 6-chloro- 1-hydroxybenzotriazole dihydrate 6-Cl-HOBt
  • Fmoc-Ser(tBu)-Ser( ⁇ Me,Me Pro)-OH were purchased from Aapptec (Louisville, Kentucky).
  • Dimethylformamide (DMF) and trifluoroacetic acid (TFA) were purchased from Scharlau (Barcelona, Spain).
  • Disodium hydrogen phosphate (Na 2 HPO 4 ) and dichloromethane (DCM) were purchased from ECP Ltd (Auckland, New Zealand).
  • Dimethyl sulfoxide (DMSO) was purchased from Avantor (Center Valley,
  • Analytical reverse-phase HPLC was performed on a Dionex (Sunnyvale, CA) Ultimate 3000 or a Waters (Milford, MA) 2690 Alliance analytical HPLC using an Agilent (Santa Clara, CA) Zorbax C 18 , 5 ⁇ M, 4.6 x 250 mm column (1 mL/min).
  • LCMS Analytical liquid chromatography mass spectrometry
  • HP Hewlett-Packard
  • HP Hewlett-Packard
  • Semi-preparative reverse- phase HPLC was performed using either a Waters 600E System with a Waters 2487 dual wavelength absorbance detector, or a Dionex Ultimate 3000 using a Phenomenex (Torrance, CA) Gemini C18110 ⁇ , 5 ⁇ M, 10.0 x 250 mm column (5 mL/min).
  • TMS tetramethylsilane
  • J Coupling constants (J) are in Hertz (Hz).
  • 13 C NMR values are reported as the chemical shift d, the degree of hybridisation and assignment.
  • Fmoc-Rink amide-ChemMatrix® resin was used as the solid support for synthesis of peptides.
  • Fmoc-Rink amide linker (270 mg, 0.5 mmol) was coupled to aminomethyl-ChemMatrix® resin (145 mg, 0.1 mmol) using 6-Cl-HOBt (84.8 mg, 0.5 mmol) and DIC (77.4 ⁇ L, 0.5 mmol) in DMF (5 mL) at room temperature for 2 h.
  • the peptides were cleaved from the resin with simultaneous removal of side-chain protecting groups using TFA/iPr3SiH/H2O/DODT (94/1/2.5/2.5, v/v/v/v, 5 mL) for 2.5 h, precipitated with cold diethyl ether, isolated by centrifugation, dissolved in 50% aqueous acetonitrile containing 0.1% TFA and lyophilised.
  • TFA/iPr3SiH/H2O/DODT 94/1/2.5/2.5, v/v/v/v, 5 mL
  • N a -Fmoc removal was carried out using 20% piperidine in DMF (v/v, 2 x 5 min) at rt, while Fmoc-AA-OH (5 eq.), HATU (4.5 eq.) and 2 M NMM in DMF (10 eq.) were used for subsequent amino acid couplings (5 min) at 72 °C, 110 W.
  • Coupling of histidine and cysteine residues was carried out for 20 min at rt, followed by a second coupling for 5 min at 40 °C, 110 W.
  • Coupling of arginine residues was carried out for 20 min at rt, followed by a second coupling for 5 min at 72 °C, 110W.
  • N-Terminal capping was carried out after every coupling using 20% Ac2O and NMM (2.0 M, 0.1 mmol) in DMF (v/v) at rt for 5 min.
  • the peptides were cleaved from the resin with simultaneous removal of side-chain protecting groups using TFA/iPr3SiH/H2O/DODT (94/1/2.5/2.5, v/v/v/v, 5 mL) at rt for 2.5 h, precipitated with cold diethyl ether, isolated by centrifugation, dissolved in 50% aqueous acetonitrile containing 0.1% TFA and lyophilised.
  • Fmoc-Cys-OH 1.1 (295 mg, 0.86 mmol) was subjected to CLipPA lipidation with vinyl stearate (401 mg, 1.29 mmol) using DMPA (220 mg, 0.86 mmol) as the photoinitiator in DCM (3 mL) under UV irradiation at 365 nm until consumption of starting material 1.1 was confirmed by TLC.
  • the reaction mixture was then concentrated in vacuo and purified by flash column chromatography (SiO2, 5:1 petroleum ether/EtOAc; followed by 95:5 DCM/MeOH). The organic solvent was evaporated under reduced pressure and the resulting yellow oil was lyophilised to afford the title compound 1c as a white amorphous solid (223 mg, 40% yield); [a] 24
  • the disulfide- containing peptide (16.4 mg) then underwent tBu group removal as described in section 1.5 and was purified by semi-preparative RP-HPLC using a Waters 600E on a Phenomenex Gemini C 18 column, using a gradient of 0%B to 18%B over 9 min (ca.2% B/min) then 18%B to 60%B over 210 min (ca.0.2%B/min). Fractions (2.5 mL) were collected at 0.5 min intervals and analysed by ESI-MS and RP-HPLC.
  • the crude linear peptide 9 (50 mg) underwent disulfide bond formation (Cys-2/Cys-7) as described in section 1.4 and was purified by semi-preparative RP- HPLC using a Waters 600E on a Phenomenex Gemini C18 column using a gradient of 0%B to 36%B over 18 min (ca.2% B/min) then 36%B to 60%B over 160 min (ca.0.15%B/min).
  • the crude linear peptide 10 (50 mg) underwent disulfide bond formation (Cys-2/Cys-7) as described in section 1.4 and was purified by semi-preparative RP- HPLC using a Dionex Ultimate 3000 on a Phenomenex Gemini C18 column using a gradient of 0%B to 36%B over 18 min (ca.2% B/min) then 36%B to 60%B over 160 min (ca.
  • the crude linear peptide 11 (50 mg) underwent disulfide bond formation (Cys-2/Cys-7) as described in section 1.4 and was purified by semi-preparative RP- HPLC using a Dionex Ultimate 3000 on a Phenomenex Gemini C 18 column using a gradient of 0%B to 30%B over 15 min (ca.2% B/min) then 30%B to 60%B over 200 min (ca.
  • the crude linear peptide 12 (50 mg) underwent disulfide bond formation (Cys-2/Cys-7) as described in section 1.4 and was purified by semi-preparative RP- HPLC using a Dionex Ultimate 3000 on a Phenomenex Gemini C 18 column using a gradient of 0%B to 36%B over 18 min (ca.2% B/min) then 36%B to 60%B over 160 min (ca.
  • the crude linear peptide 13 (50 mg) underwent disulfide bond formation (Cys-2/Cys-7) as described in section 1.4 and was purified by semi-preparative RP- HPLC using a Dionex Ultimate 3000 on a Phenomenex Gemini C18 column using a gradient of 0%B to 36%B over 18 min (ca.2% B/min) then 36%B to 60%B over 160 min (ca.
  • lipidated pramlintide analogues against human receptors Lipidated pramlintide analogues 2-7 were tested at the CTR, AMY 1 , and AMY 3 receptors as well as the related receptors CGRP, AM 1 and AM 2 for cyclic adenosine monophosphate (cAMP) accumulation, and were directly compared to native pramlintide 1 (control) in each experiment (see tables 1 and 2).
  • Biological activity was tested by transfecting Cos7 cells with the necessary receptor DNA components as described in section 2.1, and cAMP production or receptor binding (AMY1 only) was measured as described in sections 2.3 and 2.4 respectively.
  • COS-7 cells were used to study receptor activity and binding, and are a cell line derived from African Green Monkey kidney cells. These cells were sourced from the School of Biological Sciences, University of Auckland, and originally sourced from the American Type Culture Collection. COS-7 cells were used as functional cAMP responses in these cells transfected with CTR or AMY receptors show pharmacology consistent with amylin binding responses, and because they do not contain endogenous CTR, CLR or RAMPs so that receptor pharmacology can be accurately attributed to only the introduced receptor. COS-7 cells were grown in complete Gibco® Dulbecco’s Modified Eagle’s Medium
  • DMEM Fetal Bovine Serum
  • FBS Fetal Bovine Serum
  • PEI was added drop-wise to the DNA/glucose mixture, gently agitated, and left to incubate at rt for 10 min, followed by addition of DMEM. Plated cells were aspirated of old media, and 100 mL of transfection mix was added per well. The transfected plates were returned to the 37 °C humidified 95% air/5% CO2 incubator for approximately 36-48 h. These transfections gave rise to the AMY 1(a) , AMY 3(a) , and the CT (a) receptor, respectively. This protocol was also used to express the CGRP, AM 1 and AM 2 receptors in COS-7 cells.
  • the calcitonin-like receptor was transfected together with either RAMP1, RAMP2 or RAMP3 to obtain the desired receptor.
  • CLR calcitonin-like receptor
  • Each peptide was serially diluted in this same serum-free media in a 10-fold dilution series to give final concentrations ranging from 1 mM to 10 pM, or if the peptide was expected to have lower activity, from 10 mM to 10 pM.50 mM forskolin was used as a positive control and was prepared in the serum-free media. Following the 30 min serum-starve, 10 mL of the serially diluted agonists, or 10 mL media only were added to each well to give a final volume of 100 mL/well.50 mL of media were removed from forskolin control wells, and 50 mL forskolin was added to two wells per plate.
  • LANCE® TR-FRET assay is based on competition between endogenous cAMP and exogenous cAMP (biotinylated-cAMP bound to a streptavidin europium chelate (Eu chelate)) for binding to an Alexafluor®647 antibody.
  • a cAMP standard curve was prepared using LANCE cAMP detection buffer in concentrations ranging from 1 mM to 10 pM.
  • the cell lysates (5 mL) and serially diluted cAMP standards (5 mL) were then transferred to a 384-well opti-plate and sealed with a Sealplate®-A-384-well microplate adhesive film. Plates were then centrifuged (10 s, 500 rpm, 23 °C), and 5 mL of Alexa Fluor® 647 anti-cAMP antibody (1:200 dilution) was added to each well.
  • I-CGRP Radiolabelled [125] was chosen as the competitor peptide due to a lack of availability of a functional radiolabelled human or rat amylin, and because it binds and activates the AMY 1 receptor with equal potency to human amylin.36-48 hours after transfection, cells were washed once in binding media. Binding media, radiolabelled [125] I-CGRP (30,000 cpm/well), and decreasing concentrations of lipidated pramlintide, or the control pramlintide were added. Total binding was obtained from 4 wells per plate in the absence of cold peptide and non-specific binding was attained from 2 wells per plate (3 ⁇ M human amylin).
  • LFD low-fat diet
  • HFD high-fat diet
  • D12492 Research Diets 60% fat by energy
  • LFD fed mice received vehicle, and the three groups fed HFD either received vehicle, pramlintide (1 mg/kg; 253 nmol/kg) or lipidated pramlintide analogue 3 (1 mg/kg; 237 nmol/kg) for 1 week.
  • Daily body weight (in grams) was recorded throughout the experiment.
  • the vehicle treatment was sterile phosphate-buffered saline (PBS), and the volume varied depending on the animal’s bodyweight e.g.30g mouse would get 150 ⁇ L, to match the volume that was given to the pramlintide and lipidated pramlintide groups.
  • PBS sterile phosphate-buffered saline
  • mice fed HFD attained a significantly higher body weight compared to mice on LFD within the two weeks.
  • a significant difference in body weight was observed between mice treated daily with lipidated pramlintide compared to vehicle treated mice (figure 7; results are mean ⁇ SEM, *P ⁇ 0.05, **P ⁇ 0.01).
  • Mice treated with lipidated pramlintide did not gain as much body weight as mice treated with vehicle. This significant difference was observed after 21 d of treatment.
  • mice treated with pramlintide also displayed restriction in gaining body weight compared to vehicle treated mice (figure 7; p ⁇ 0.05), but after 26 d of treatment.
  • Example 4 In vivo food intake and glucose tolerance 4.1 Animals A similar cohort of male mice was used as described for Example 3. Diet-induced obesity was initiated as described for Example 3, except for a change in the duration of HFD feeding before the initiation of treatment. Mice were fed the new diet for three weeks. 4.2 Glucose tolerance assay To investigate whether lipidated pramlintide affects glucose homeostasis, intraperitoneal glucose tolerance tests (ipGTTs) in diet-induced obese (DIO) mice were performed as shown in Example 1. Prior to the ipGTT, mice were fasted for 16h. Glucose (1.5 g/kg body weight; dissolved in 0.9% NaCl) was injected intraperitoneally (i.p.) 60 minutes after the last injection of
  • Table 3 Summary of potency (pEC50) in cAMP assays of lipidated pramlintide analogues 3, 16, and 17. Summary of potency in cAMP (pEC50) of lipidated pramlintide analogues 3, 16 and 17 at the human and mouse CTR, AMY 1 , AMY 3 , CGRP, and AM 2 receptors. Values are mean ⁇ S.E.M. of at least 3 independent experiments. Data not shown for AM1 as responses were too weak to fit a curve to the data for pramlntide or analogues.
  • LFD low-fat diet
  • HFD high-fat diet
  • D12492 Research Diets D12492 Research Diets
  • mice in each of the groups including the LFD were then treated once daily subcutaneously; the LFD group was treated with vehicle, and the HFD groups were treated with vehicle, pramlintide, lipidated analogue 3, lipidated analogue 16, or lipidated analogue 17 (300 ⁇ g/kg; 253 nmol/kg) for 19 days.
  • the vehicle treatment was sterile 0.1M Acetic Acid Sodium Acetate buffer (pH 4.0), containing 2% DMSO and the volume varied depending on the animal’s body weight e.g. a 30g mouse would receive 90 ⁇ L, to match the volume that was given to the peptide-treated groups.
  • Results Figure 13 shows that, as expected, vehicle-treated animals continued to gain weight on the HFD.
  • the data in figure 14 were analysed by two-way ANOVA with post hoc Dunnett’ s test, comparing all treatments to the vehicle control. Significance (P ⁇ 0.05) was achieved for pramlintide 1 (days 5-10), analogue 3 (days 6-10), analogue 16 (days 4-10) and analogue 17 (days 5-10).
  • the data in figure 15 were analysed by one-way ANOVA with post hoc Dunnett’ s tests comparing all treatments to the vehicle control. Significance ( P ⁇ 0.05) was achieved for pramlintide 1, analogue 3, analogue 16 and analogue 17.
  • LFD low-fat diet
  • HFD high-fat diet
  • D12492 Research Diets D12492 Research Diets
  • mice in each of the groups including the LFD were then treated once daily subcutaneously; the LFD group was treated with vehicle, and the HFD groups were treated with vehicle, pramlintide (100 mg/kg, 300 mg/kg and 1000 mg/kg; 253 nmol/kg) or lipidated analogue 3 (100 mg/kg, 300 mg/kg and 1000 mg/kg; 237 nmol/kg) for 10 days.
  • Daily body weight and food intake measurements (both in grams) were recorded throughout the experiment. On the 11 th day (one day after the final injection), final body weight and food weight was measured, followed by euthanasia of the animals. Blood, brain, liver was collected and stored.
  • Liver weight and epidydimal white adipose tissue (eWAT) weights were recorded.
  • the vehicle treatment was sterile 0.1M PBS (pH 8.0), containing 2% DMSO, and the volume varied depending on the animal’s body weight e.g. a 30g mouse would get 30 ⁇ L, to match the volume that was given to the pramlintide and lipidated analogue 3 groups.
  • 7.3 Results Figure 16 shows that vehicle-treated animals continued to gain weight on the HFD. In contrast, body weight gain was attenuated in the analogue 3-treated groups at 300 and 1000 ⁇ g/kg.
  • Example 8 Continued in vivo research on half-life and dose-frequency has been delayed due to COVID-19 shut-down of key facilities. We expect this data to be available soon after laboratory operations are allowed to resume. 8.
  • IV intravenous
  • SC subcutaneous
  • Rats will be allowed one week to acclimate to experimental conditions prior to entering an experiment. All manipulations will be performed under anaesthesia with monitoring for heart rate, blood pressure, and colonic body temperature for the duration of the procedure. Briefly, after administration of anaesthetic, a tracheostomy will be performed for mechanical ventilation. For IV administration, rats will be cannulised in the right jugular vein for drug delivery. For SC administration, rats will receive an injection in their lower abdominal region. For blood collection, all rats will be cannulised in the right femoral artery.
  • All rats will receive SC or IV administration of pramlintide 1 or lipidated analogues 3, 16 or 17 at doses between 0.1-1 mg/kg.
  • the starting dose for SC will be 300 ⁇ g/kg, in line with the body weight studies in Example 6.
  • Blood will be collected at times 0, 2, 5, 10, 15, 30, 60, 120, and 180 minutes, taking no more than 10% of the total blood volume. At 180 minutes, a final bleed of approximately 7-15ml will be collected and the rats will be euthanised.
  • Whole blood will be collected into EDTA-treated tubes and kept on ice. Cells will be removed from plasma by centrifugation for 10 minutes at 2,000 ⁇ g in a refrigerated centrifuge.
  • Plasma will be aspirated and put into fresh tubes and stored at -80°C until analysis. 8.3 Expected results Collected blood will be analysed for the amount of pramlintide 1 or analogue present. We expect that after an initial increase from baseline, the amount of pramlintide 1, analogue 3, analogue 16 and analogue 17 present will decrease over time, and that analogue 3, analogue 16 and analogue 17 will have an increased circulatory half-life in vivo compared to pramlintide 1.
  • Example 9 9.
  • LFD low-fat diet
  • HFD high-fat diet
  • mice fed HFD for 3 weeks will subsequently be treated with equimilolar concentrations of pramlintide 1, analogue 3, analogue 16 or analogue 17, once daily, or every second, third, fourth, fifth, sixth or seventh day.
  • Mice fed LFD will serve as a control and receive the same number of injections with vehicle. Body weight will be recorded daily.
  • 9.3 Expected results We expect that analogue 3, analogue 16 and analogue 17 will require less frequent dosing to achieve a reduction in weight gain, or an increase in weight loss, compared with pramlintide 1.
  • Such diseases and conditions include, for example Alzheimer's disease, diabetes, type 1 diabetes, type 2 diabetes, pre-diabetes, insulin resistance syndrome, impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic disease including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, hepatic steatosis ("fatty liver”; including non-alcoholic fatty liver disease (NAFLD), which itself includes non-alcoholic steatohepatitis (NASH)), kidney failure, arteriosclerosis (e.g.atherosclerosis), macrovascular disease, microvascular disease, diabetic heart disease (including diabetic cardiomyopathy and heart failure as a diabetic complication), coronary heart disease, peripheral artery disease or stroke, myocardial infarction, syndrome X, cognitive disorders, inflammatory bowel disease, dyspepsia, gastric ulcers, depression, anxiety, psychosis, schizophrenia, pain, osteoporosis, in a subject in need thereof.
  • ITT impaired glucose tolerance
  • metabolic disease including metabolic syndrome,

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Abstract

L'invention concerne des conjugués peptidiques qui sont des agonistes d'amyline comprenant un peptide d'amyline comprenant ou consistant en une séquence d'acides aminés qui est un analogue de SEQ ID NO : 1, le peptide comprenant un résidu proline aux positions 25, 28 et 29, au moins un acide aminé du peptide étant conjugué de manière covalente à une fraction contenant des lipides par l'intermédiaire d'un atome de soufre d'un groupe sulfhydryle. L'invention concerne également des compositions pharmaceutiques et des kits comprenant de tels conjugués, des procédés de préparation de tels conjugués, et des procédés et des utilisations de ces agonistes.
PCT/IB2020/054364 2019-05-08 2020-05-08 Agonistes d'amyline conjugués peptidiques et leurs utilisations WO2020225781A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022129254A1 (fr) * 2020-12-16 2022-06-23 Medimmune Limited Polypeptides et leurs utilisations
WO2022187305A1 (fr) * 2021-03-03 2022-09-09 Eli Lilly And Company Agonistes du récepteur de l'amyline à action prolongée et leurs utilisations

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007104789A2 (fr) * 2006-03-15 2007-09-20 Novo Nordisk A/S Dérivés d'amyline
WO2009034119A1 (fr) * 2007-09-11 2009-03-19 Novo Nordisk A/S Dérivés améliorés de l'amyline
WO2010046357A1 (fr) * 2008-10-21 2010-04-29 Novo Nordisk A/S Dérivés de l’amyline
WO2013059336A1 (fr) * 2011-10-18 2013-04-25 Amylin Pharmaceuticals, Llc Peptides chimériques d'amyline-calcitonines conjugués à des radicaux augmentant la durée de l'activité biologique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007104789A2 (fr) * 2006-03-15 2007-09-20 Novo Nordisk A/S Dérivés d'amyline
WO2009034119A1 (fr) * 2007-09-11 2009-03-19 Novo Nordisk A/S Dérivés améliorés de l'amyline
WO2010046357A1 (fr) * 2008-10-21 2010-04-29 Novo Nordisk A/S Dérivés de l’amyline
WO2013059336A1 (fr) * 2011-10-18 2013-04-25 Amylin Pharmaceuticals, Llc Peptides chimériques d'amyline-calcitonines conjugués à des radicaux augmentant la durée de l'activité biologique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG, S. -H. ET AL.: "Lipidation of Cysteine or Cystein-Containing Peptides Using the Thiol-Ene Reaction (CLipPA", EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, vol. 15, 2016, pages 2608 - 16, XP055426738, DOI: 10.1002/ejoc.201501375 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022129254A1 (fr) * 2020-12-16 2022-06-23 Medimmune Limited Polypeptides et leurs utilisations
WO2022187305A1 (fr) * 2021-03-03 2022-09-09 Eli Lilly And Company Agonistes du récepteur de l'amyline à action prolongée et leurs utilisations
US12115210B2 (en) 2021-03-03 2024-10-15 Eli Lilly And Company Long acting amylin receptor agonists and uses thereof

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