WO2013098408A1 - Conjugués peptidiques agonistes des récepteurs de la cck et du glucagon - Google Patents

Conjugués peptidiques agonistes des récepteurs de la cck et du glucagon Download PDF

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Publication number
WO2013098408A1
WO2013098408A1 PCT/EP2012/077083 EP2012077083W WO2013098408A1 WO 2013098408 A1 WO2013098408 A1 WO 2013098408A1 EP 2012077083 W EP2012077083 W EP 2012077083W WO 2013098408 A1 WO2013098408 A1 WO 2013098408A1
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amino
dioxaoctanoyl
ser
mgwmdf
lys
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PCT/EP2012/077083
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English (en)
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Jens Rosengren Daugaard
Torben ØSTERLUND
Simon Birksø LARSEN
Ditte Riber
Jakob Lind Tolborg
Oliver Demmer
Lene JESSEN
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Zealand Pharma A/S
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Publication of WO2013098408A1 publication Critical patent/WO2013098408A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/55—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 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • 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

Definitions

  • the present invention relates, inter alia, to certain peptide conjugates comprising a glucagon receptor agonist peptide moiety and a CCK-derived peptide moiety, and to the use of the conjugates in the treatment and/or prevention of a variety of diseases or disorders, including treatment and/or prevention of excess food intake, obesity and excess body weight.
  • Obesity is a globally increasing health problem that is associated with various diseases, particularly cardiovascular disease (CVD), type-2 diabetes, obstructive sleep apnea, certain types of cancer, and osteoarthritis.
  • CVD cardiovascular disease
  • type-2 diabetes type-2 diabetes
  • obstructive sleep apnea certain types of cancer
  • osteoarthritis As a result, obesity often correlates with a reduction in life expectancy.
  • WHO projections by the World Health Organization there are 400 million adults (age > 15) classified as obese worldwide. In the U.S., obesity is now believed to be the second-leading cause of preventable death after smoking.
  • Preproglucagon is a 158 amino acid precursor polypeptide that is differentially processed in the tissues to form a number of structurally related proglucagon-derived peptides, including glucagon (GCG), glucagon-like peptide-1 (GLP-1 ), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM). These molecules are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, as well as regulation of food intake.
  • GCG glucagon
  • GLP-1 glucagon-like peptide-1
  • GLP-2 glucagon-like peptide-2
  • OXM oxyntomodulin
  • Glucagon is a 29-amino acid peptide that corresponds to amino acids 53 to 81 of preproglucagon, and has the following amino acid sequence (written using conventional three-letter amino acid
  • glucagon products that are currently available commercially, and which are intended primarily for use in "rescue” situations for alleviating acute hypoglycemia in a diabetic subject who has received an excessively high dose of insulin, are available in the form of freeze-dried, solid preparations intended for reconstitution in an appropriate liquid medium (e.g.
  • CCK Cholecystokinin
  • CCK-1 and CCK-2 receptors also designated CCK-A R and CCK-B R, respectively
  • CCK-1 and CCK-2 receptors also designated CCK-A R and CCK-B R, respectively
  • CCK stimulates receptors in the intestine that activate the vagus nerve, which relays a message to the satiety centres in the hypothalamus.
  • satiety effects of CCK are eliminated in vagotomised animals, further supporting this unique proposed mechanism.
  • CCK-A R and CCK-B R have different affinities for CCK variants.
  • CCK-A R acts primarily as a receptor for sulfated CCK
  • CCK-B R acts primarily as a receptor for sulfated CCK
  • CCK-B R binds both CCK and gastrin equally well.
  • CCK-B R is considered to be the "gastrin receptor" due to the higher levels of gastrin compared to CCK in plasma (Foucaud et al. Reg. Peptides 145: 17-23 (2008)).
  • CCK-B R can initiate several intracellular pathways upon binding of ligand, which is considered to be the reason for the diverse physiological roles of CCK.
  • a key pathway downstream of CCK-B R is the MAPK (mitogen activated protein kinases) or ERK (extra-cellular regulated kinases) pathway, which is also activated by several growth hormones. Since CCK-B R is expressed in the pancreas, gastrin is able to contribute to cell proliferation and islet regeneration in this tissue.
  • the present invention relates to, inter alia, conjugates that comprise covalently coupled or linked peptide moieties derived from glucagon and CCK, respectively, and that exhibit unexpectedly high therapeutic activity in the treatment of, for example, excess food intake, obesity and excess weight, in comparison to the therapeutic activity of a combination of the two individual peptides.
  • the invention thus provides a peptide conjugate of a glucagon-derived moiety (glucagon receptor agonist) and a CCK-derived moiety, in particular CCK8.
  • One aspect of the present invention relates to a peptide conjugate having the formula I
  • R 1 is hydrogen, C-i. 4 alkyl (e.g. methyl), acetyl, formyl, benzoyl or trifluoroacetyl;
  • R 2 is NH 2 or OH
  • X is a peptide sequence having the formula la
  • X2 is selected from Aib and Ser;
  • X3 is selected from His and Gin
  • X15 is selected from Glu and Asp
  • X16 is selected from Lys, Aib and Ser;
  • X17 is selected from Lys, Ala and Arg;
  • X20 is selected from Lys, Glu and Gin;
  • X21 is selected from Glu, Ser and Asp;
  • X24 is selected from Lys, Ser and Gin;
  • X25 is selected from Trp, His, Lys, He, Leu, Ala, Met, Cys, Asn, Val, Ser, Gin, Asp, Glu, Thr and Tyr;
  • X27 is selected from Lys, Glu, Leu, Ser and Met or is absent;
  • X28 is selected from Lys, Arg, Ser and Asn or is absent;
  • X29 is Thr or is absent; wherein a Lys residue present in one or more of positions 16, 17, 20, 24 and 28, independently, is optionally conjugated, directly or via a spacer, to a lipophilic substituent;
  • L is an optional linker moiety having the formula lb L1-L2-L3-L4 (lb)
  • L1 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • L2 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • L3 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • L4 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • Y is a peptide sequence having the formula lc
  • Y1 is Asp or is absent
  • Y2 is selected from Tyr(S0 3 H), Phe(4-CH 2 S0 3 H) and isosteres thereof such as Phe(4-P0 3 H 2 ), L-(4- hydroxyphenyl)glycine, D-(4-hydroxyphenyl)-glycine, L-(4-sulfophenyl)glycine and D-(4- sulfophenyl)glycine, or is absent;
  • Y3 is selected from Met, Leu, Nle, Thr and Ser, or is absent;
  • Y4 is Gly or is absent;
  • Y6 is selected from Met, Leu, Nle, Thr, Ser, Phe and Lys(2-tolylaminocarbonyl);
  • Y7 is selected from Asp, DAsp, N-Me-Asp and N-Me-DAsp; and
  • Y8 is selected from Phe, D-Phe, N-Me-Phe and N-Me-DPhe; or a pharmaceutically acceptable salt or solvate thereof.
  • the invention provides a method of synthesis of a peptide conjugate of the invention.
  • Peptide conjugates of the invention may be manufactured by standard synthetic methods, by use of recombinant expression systems, or by any other suitable method.
  • the conjugates may be synthesized in a number of ways, including, e.g., methods comprising:
  • the method of synthesis may comprise the step of chemically modifying one of more amino acid side chains in a precursor peptide to yield a compound of the invention.
  • modification may yield a non- naturally occurring amino acid such as Tyr(S0 3 H), Phe(4-CH 2 S0 3 H), Phe(4-P0 3 H 2 ) or Lys(2- tolylaminocarbonyl), or to introduce a substituent such as a lipophilic substituent.
  • the present invention provides a composition for use in a method of medical treatment.
  • Tyr(S0 3 H) designates a Tyr residue in which a S0 3 H moiety is attached to the Tyr residue via the oxygen of the Tyr side chain
  • F(4-CH 2 S0 3 H) i.e. Phe(4-CH 2 S0 3 H)] designates Phe that is modified by attachment of a -CH 2 S0 3 H moiety to the para carbon of the phenyl ring (i.e., the ⁇ carbon of the side chain)
  • Phe(4-P0 3 H 2 ) designates Phe that is modified by attachment of a -P0 3 H 2 moiety to the para carbon of the phenyl ring (i.e., the ⁇ carbon of the side chain)
  • Lys(2- tolylaminocarbonyl) designates a Lys residue in which the ⁇ -amino group of the Lys side chain is modified by attachment of a 2-tolyl amino carbonyl moiety and has the structure:
  • the compounds described herein may be used, inter alia, in preventing weight gain or in promoting weight loss.
  • "preventing weight gain” refers to inhibiting or reducing weight gain when compared to the absence of treatment, and is not necessarily meant to imply complete cessation of weight gain.
  • the compounds may cause a decrease in food intake and/or increased energy expenditure, resulting in the observed effect on body weight.
  • the compounds of the invention may have a beneficial effect on circulating cholesterol levels by lowering circulating low-density lipoprotein (LDL) levels and increasing high-density lipoprotein/low- density lipoprotein (HDL/LDL) ratio.
  • LDL low-density lipoprotein
  • HDL/LDL high-density lipoprotein/low- density lipoprotein
  • the compounds of the invention can be used for direct or indirect therapy of any condition caused or characterized by excess body weight, such as the treatment and/or prevention of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, and/or obesity induced sleep apnea. They may also be used for the prevention or treatment of metabolic syndrome, fatty liver (hepatic steotosis), hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and/or stroke. Their effects on these conditions may be as a result of, or associated with, their effect on body weight, or may be independent thereof.
  • Figure 1 Effect of Compound 2 on food intake in normal mice: Normal chow-fed C57BL/6J male mice were kept 4 per cage in dosing groups of 8 animals. The animals were dosed once
  • the letter "A" designates Compound 3 of the present invention.
  • amino acids are referred to by their full name ' (e.g. alanine, arginine, etc.), they are designated by their conventional three-letter or single- letter abbreviations (e.g. Ala or A for alanine, Arg or R for arginine, etc.).
  • sarcosine, ornithine, etc. frequently employed three- or four-character codes are employed for residues thereof, including Nle (norleucine), Orn (ornithine, i.e.
  • 2,5-diaminopentanoic acid Sar (sarcosine, i.e. N- methylglycine), Aib (a-aminoisobutyric acid), Bal ( ⁇ -alanine, i.e. 3-aminopropanoic acid), Dab (2,4- diaminobutanoic acid), Dap (2,3-diaminopropanoic acid), ⁇ -Glu ( ⁇ -glutamic acid), Gaba ( ⁇ - aminobutanoic acid), ( ⁇ -Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid).
  • the abbreviation 8Ado (8-amino-3,6-dioxaoctanoyl; sometimes also referred to as Peg3) thus represents the following non-naturally occurring amino acid residue:
  • N-Me-Asp L-aspartic acid which is methylated at the a-nitrogen
  • N-Me-DAsp D-aspartic acid which is methylated at the a-nitrogen
  • N-Me-Phe L-phenylalanine which is methylated at the a-nitrogen
  • N-Me-DPhe D-phenylalanine which is methylated at the a-nitrogen
  • the invention provides a peptide conjugate having the formula I R 1 -X-L-Y-R 2 (I) wherein
  • R 1 is hydrogen, d. 4 alkyl (e.g. methyl), acetyl, formyi, benzoyl or trifluoroacetyl;
  • R 2 is NH 2 or OH;
  • X is a peptide sequence having the formula la His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-X15-X16-X17-Arg-Ala-X20-X21 -Phe-Val- X24-X25-Leu-X27-X28-X29 (la) wherein
  • X2 is selected from Aib and Ser; X3 is selected from His and Gin; X15 is selected from Glu and Asp;
  • X16 is selected from Lys, Aib and Ser;
  • X17 is selected from Lys, Ala and Arg, notably from Ala and Arg;
  • X20 is selected from Lys, Glu and Gin, notably from Glu and Gin;
  • X21 is selected from Glu, Ser and Asp;
  • X24 is selected from Lys, Ser and Gin;
  • X25 is selected from Trp, His, Lys, lie, Leu, Ala, Met, Cys, Asn, Val, Ser, Gin, Asp, Glu, Thr and Tyr;
  • X27 is selected from Lys, Glu, Leu, Ser and Met, notably from Glu, Leu, Ser and Met, or is absent;
  • X28 is selected from Lys, Arg, Ser and Asn, notably from Ser and Asn, or is absent; and
  • X29 is Thr or is absent; wherein a Lys residue present in one or more of positions 16, 17, 20, 24 and 28, independently, is optionally conjugated, directly or via a spacer, to a lipophilic substituent;
  • L is an optional linker moiety having the formula lb L1 -L2-L3-L4 (lb) wherein
  • L1 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • L2 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • L3 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • L4 is selected from Orn, 8Ado, Cys, Lys, Gin, Gly and 3-aminopropanoyl, or is absent;
  • Y is a peptide sequence having the formula lc
  • Y1 -Y2-Y3-Y4-Trp-Y6-Y7-Y8 (lc) wherein Y1 is Asp or is absent;
  • Y2 is selected from Tyr(S0 3 H), Phe(4-CH 2 S0 3 H) and isosteres thereof such as Phe(4-P0 3 H 2 ), L-(4- hydroxyphenyl)glycine, D-(4-hydroxyphenyl)-glycine, L-(4-sulfophenyl)glycine and D-(4- sulfophenyl)glycine, or is absent;
  • Y3 is selected from Met, Leu, Nle, Thr and Ser, or is absent;
  • Y4 is Gly or is absent;
  • Y6 is selected from Met, Leu, Nle, Thr, Ser, Phe and Lys(2-tolylaminocarbonyl); Y7 is selected from Asp, DAsp, N-Me-Asp and N-Me-DAsp; and Y8 is selected from Phe, D-Phe, N-Me-Phe and N-Me-DPhe; or a pharmaceutically acceptable salt or solvate thereof.
  • the peptide sequence Y may have the formula Id: Asp-Y2-Y3-Gly-Trp-Y6-Asp-Phe (Id) wherein
  • Y2 is selected from Tyr(S0 3 H) and Phe(4-CH 2 S0 3 H); Y3 is selected from Met, Leu, Nle, Thr and Ser; and Y6 is selected from Met, Leu, Nle, Thr, Ser and Phe.
  • X2 is Aib.
  • X15 is Glu.
  • X16 is selected from Lys, Aib and Ser.
  • X20 is Glu.
  • X21 is selected from Glu and Ser.
  • X24 is selected from Lys and Ser.
  • X27 is selected from Glu and Leu.
  • Preferred combinations of residues in the moiety X of an individual peptide conjugate of the invention include the following:
  • X2 is Aib
  • X20 is Glu
  • X24 is Ser
  • X27 is Leu
  • X28 is Ser
  • X2 is Aib, X20 is Glu, X24 is Ser, X27 is Glu, X28 is Ser; X2 is Aib, X16 is Lys*, X20 is Glu, X24 is Ser, X27 is Leu, X28 is Ser;
  • X2 is Aib, X1 7 is Lys*, X20 is Glu, X24 is Ser, X27 is Leu, X28 is Ser;
  • X2 is Aib, X16 is Lys*, X20 is Glu, X24 is Ser, X27 is Glu, X28 is Ser;
  • X2 is Aib, X17 is Lys*, X20 is Glu, X24 is Ser, X27 is Glu, X28 is Ser; X2 s Aib, X16 Lys X17 is Ala, X20 is Glu, X21 is Ser, X24 is Lys, X27 is Glu, X28 is Ser; X2 s Aib, X16 Lys*, X17 is Ala, X20 is Glu, X21 is Glu, X24 is Lys, X27 is Glu, X28 is Ser; X2 s Aib, X16 Lys*, X20 is Glu, X21 is Ser, X24 is Lys, X27 is Glu, X28 is Ser; X2 s Aib, X16 Lys*, X20 is Glu, X21 is Ser, X24 is Lys, X27 is Glu, X28 is Ser; X2
  • X16 is Lys*
  • X20 is Glu
  • X24 is Ser
  • X27 is Leu
  • X28 is Ser
  • X16 is Lys*, X20 is Glu, X24 is Ser, X27 is Glu, X28 is Ser;
  • X16 is Lys*
  • X20 is Glu
  • X21 is Glu
  • X24 is Lys
  • X27 is Glu
  • X28 is Ser
  • X16 is Lys*
  • X20 is Glu
  • X21 is Ser
  • X24 is Lys
  • X27 is Glu
  • X28 is Ser
  • X16 is Lys*
  • X20 is Glu
  • X21 is Ser
  • X24 is Lys
  • X27 is Leu
  • X28 is Ser
  • X17 is Lys*, X20 is Glu, X24 is Ser, X27 is Leu, X28 is Ser; X17 is Lys*, X20 is Glu, X24 is Ser, X27 is Glu, X28 is Ser;
  • X20 is Glu, X24 is Ser, X27 is Leu;
  • X20 is Glu, X24 is Ser, X27 is Glu; X20 is Glu, X24 is Ser, X27 is Leu, X28 is Ser;
  • X20 is Glu
  • X24 is Ser
  • X27 is Glu
  • X28 is Ser
  • X20 is Glu
  • X21 is Glu
  • X24 is Lys
  • X27 is Glu
  • X28 is Ser
  • Lys* designates a Lys residue in which the side-chain thereof is conjugated, directly or via a spacer, to a lipophilic substituent.
  • the amino acid residue at position 16 of the moiety X is a Lys residue that is conjugated, directly or via a spacer, to a lipophilic substituent.
  • Certain embodiments of a peptide conjugate of the present invention relate to a peptide conjugate within the scope of formula I as defined above, i.e.:
  • R is hydrogen, C -4 alkyl (e.g. methyl), acetyl, formyl, benzoyl or trifluoroacetyl;
  • R 2 is NH 2 or OH; wherein X is a peptide sequence having the formula le:
  • X25 is selected from Trp, His, Lys, He, Leu, Ala, Met, Cys, Asn, Val, Ser, Gin, Asp, Glu, Thr and Tyr;
  • X27 is selected from Ser and Met, or is absent;
  • X28 is Asn, or is absent; and
  • X29 is Thr, or is absent;
  • L is an optional linker moiety having the formula If L1 -L2-L3-L4 (If) wherein
  • L1 is selected from Orn, 8Ado, Cys, Lys and Gin or is absent;
  • L2 is selected from Orn, 8Ado, Cys, Lys and Gin or is absent;
  • L3 is selected from Orn, 8Ado, Cys, Lys and Gin or is absent;
  • L4 is selected from Orn, 8Ado, Cys, Lys and Gin or is absent;
  • Y is a peptide sequence having the formula lc
  • Y1 -Y2-Y3-Y4-Trp-Y6-Y7-Y8 (lc) wherein Y1 is Asp or is absent;
  • Y2 is selected from Tyr(S0 3 H), Phe(4-CH 2 S0 3 H) and isosteres thereof such as Phe(4-P0 3 H 2 ), L-(4- hydroxyphenyl)glycine, D-(4-hydroxyphenyl)-glycine, L-(4-sulfophenyl)glycine and D-(4- sulfophenyl)glycine, or is absent;
  • Y3 is selected from Met, Leu, NIe, Thr and Ser, or is absent;
  • Y4 is Gly or is absent;
  • Y6 is selected from Met, Leu, NIe, Thr, Ser, Phe and Lys(2-tolylaminocarbonyl);
  • Y7 is selected from Asp, DAsp, N-Me-Asp and N-Me-DAsp;
  • Y8 is selected from Phe, D-Phe, N-Me-Phe and N-Me-DPhe;
  • Y may have the formula le as defined above.
  • Y may have the formula Ig:
  • Y2 is selected from Tyr(S0 3 H) and Phe(4-CH 2 S0 3 H); and Y6 is selected from Met, Leu, Nle, Thr and Phe.
  • the linker moiety -8Ado-8Ado- designates the chemical moiety -NH-CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH 2 -C(0)-NH-CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH 2 -C(0)-, the -NH- moiety at the N-terminal end of the linker moiety in question being covalently attached to the C-terminal end of the Glucagon moiety of the peptide conjugate in question, and the -C(O)- moiety at the C-terminal end of the linker moiety in question being attached to the N-terminal end of the CCK- derived moiety of the peptide conjugate in question.
  • peptide conjugate in the context of the present invention refers to a molecule in which a first peptide moiety is attached (i.e. coupled or linked), either directly or via a linking (i.e. bridging or spacing) chemical moiety, to a second peptide moiety, by means of covalent chemical bonding.
  • the compounds may be named according to the name of the native compounds linked together as described above. Amino acid substitutions and/or derivatizations are indicated by their position in the native compound in question, e.g. Glucagon-8Ado-8Ado-Gln-Gln- Phe2(CH 2 S0 3 )CCK8 indicates that there is a (CH 2 S0 3 ) modification to Phe2 of the CCK8 peptide.
  • the glucagon-derived moiety (the X moiety) may have at least 60%, e.g. at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identity to native human glucagon.
  • the CCK8-derived moiety (the Y moiety) may have at least 20%, e.g. at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99% or 100%, identity to human CCK8.
  • a peptide conjugate of the invention may comprise the amino acid sequence of any one of Compound Nos: 1 , 2, 3, 4, 5, 6, 7, 8 9, 10, 1 1 , 12, 13, 14 or 15 (see below) or a functional variant thereof that is at least about 70%, e.g. at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5%, identical to one or more of the recited sequences.
  • a functional variant of a peptide conjugate of the invention may have, e.g., about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100%, of the biological activity of the original peptide conjugate of the invention (measured, for example, as an EC 50 value for activation of a relevant receptor, such as CCK-A R, CCK-B R or GCG R).
  • the X moiety (glucagon-derived moiety) and/or the Y moiety (CCK8-derived moiety) in a peptide conjugate of the invention may comprise one or more amino acid substitutions , e.g., conservative amino acid substitutions, relative to the sequence of human glucagon and CCK8, respectively.
  • substitution denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e. g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. See, for example, the table below.
  • one or more Met residues are substituted with norleucine (Nle) which is a bioisostere for Met, but which, as opposed to Met, is not readily oxidised.
  • Another example of a conservative substitution with a residue normally not found in endogenous, mammalian peptides and proteins is the conservative substitution of Arg or Lys with, for example, ornithine, canavanine, aminoethylcysteine or another basic amino acid.
  • one or more cysteines of a peptide conjugate of the invention may be substituted with another residue, such as a serine.
  • conservative substitutions of amino acids are grouped by physicochemical properties. I: neutral, hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.
  • the peptide conjugate of the invention may comprise functional fragments variants thereof that have at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to one or more of the specific sequences recited below.
  • certain embodiments provide a peptide conjugate having the sequence:
  • a compound (peptide conjugate) of the invention include a peptide conjugate having the formula:
  • certain embodiments provide a peptide conjugate having the formula:
  • the latter three embodiments of a peptide conjugate of the invention may also be designated by the formulae:
  • Glucagon-8Ado-8Ado-CCK8 (Compound 1 ); Glucagon-8Ado-8Ado-Gln-Gln-CCK8 (Compound 2);and
  • one embodiment of a compound of the invention is the compound:
  • Another embodiment of a compound of the invention is the compound: H-HSQGTFTSDYS YLDSRRAEDFVSWLEST-[8-Amino-3,6-dioxaoctanoyl]-[8-Amino-3,6- dioxaoctanoyl]-QQD-Y(S0 3 H)-MGWMDF-NH 2 (Compound 4) or a pharmaceutically acceptable salt or solvate thereof.
  • Another embodiment of a compound of the invention is the compound: H-HSQGTFTSDYSKYLDSRRAEDFVSWLLST-[8-Amino-3,6-dioxaoctanoyl]-[8-Amino-3,6- dioxaoctanoyl]-QQD-Y(S0 3 H)-TGW-Nle-DF-NH 2 (Compound 9) or a pharmaceutically acceptable salt or solvate thereof.
  • Another embodiment of a compound of the invention is the compound: H-HSQGTFTSDYSKYLDSRRAEDFVSWLLSTGGQQD-Y(S0 3 H)-MGWMDF-NH 2 (Compound 14) or a pharmaceutically acceptable salt or solvate thereof.
  • Yet another embodiment of a compound of the invention is the compound: H-HSQGTFTSDYSKYLDSRRA-K(Hexadecanoyl-isoGlu)-DFVSWLLST-[8-Amino-3,6-dioxaoctanoyl]- [8-Amino-3,6-dioxaoctanoyl]-QQD-Y(S03H)-MGWMDF-NH 2 (Compound 15) or a pharmaceutically acceptable salt or solvate thereof.
  • the invention further provides a nucleic acid encoding a peptide conjugate of the invention, an expression vector comprising such a nucleic acid, and a host cell comprising such a nucleic acid or expression vector, wherein the host cell is capable of expressing and optionally secreting the peptide conjugate of the invention.
  • the term "pharmaceutically acceptable salt” is intended to indicate a salt which is not harmful to a patient or subject to which the salt in question is administered. It may suitably be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts.
  • Examples of basic salts include salts where the cation is selected among alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R 1 )(R 2 )(R 3 )(R 4 ) + , where R 1 , R 2 , R 3 and R 4 independently will typically designate hydrogen, optionally substituted Ci -6 -alkyl or optionally substituted C 2 . 6 -alkenyl.
  • Examples of relevant C ⁇ -alky! groups include methyl, ethyl, 1 -propyl and 2-propyl groups. Examples of C 2 .
  • 6 -alkenyl groups of possible relevance include ethenyl, 1 -propenyl and 2-propenyl.
  • Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), in the
  • solvate in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a peptide conjugate or pharmaceutically acceptable salt thereof according to the invention) and a solvent.
  • the solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid.
  • a solvate is normally referred to as a hydrate.
  • the compounds of the present invention may be useful as pharmaceutical agents for the prevention of kidney failure, hypertension and/or dyslipidemia (or a combination of these metabolic and cardiovascular risk factors), atherosclerosis, arteriosclerosis, macrovascular disease, microvascular disease, coronary heart disease, peripheral artery disease and stroke. Effects of the peptide conjugates of the invention on these conditions may be mediated in whole or in part via an effect on body weight, or may be independent thereof.
  • Some embodiments of the invention relate to a peptide conjugate or pharmaceutically acceptable salt thereof according to the invention for use as or the manufacture or preparation of a medicament, or to the use of a peptide conjugate or pharmaceutically acceptable salts thereof in methods of treating or preventing a variety of diseases or conditions, for example, obesity, morbid obesity, obesity-linked inflammation, obesity-linked gall bladder disease, fatty liver (hepatic steatosis), obesity-induced sleep apnea, hypertension, atherogenic dyslipidemia, arteriosclerosis (e.g. atherosclerosis), macrovascular disease, coronary heart disease, peripheral artery disease, stroke, microvascular disease, gastric disease, metabolic syndrome, cancer (e.g. colon cancer), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, and kidney failure.
  • diseases or conditions for example, obesity, morbid obesity, obesity-linked inflammation, obesity-linked gall bladder disease, fatty liver (hepatic
  • the medicament of the invention is a medicament for inducing, in a subject in need thereof, pancreatic islet neogenesis (e.g., for promoting formation of new ⁇ -cells in the islets of the pancreas).
  • the medicament of the invention is a medicament for inducing, in a subject in need thereof, survival of ⁇ -cells in the pancreatic islets (e.g. for preventing loss of ⁇ -cells in the pancreatic islets).
  • the peptide conjugate of the invention is used as a medicament for improving survival rate of the pancreatic islet ⁇ -cells in a subject in need thereof.
  • the medicament of the invention is a medicament for use in preventing, in a subject in need thereof, ⁇ -cell apoptosis and/or necrosis in the pancreatic islets (e.g. for preventing loss of ⁇ -cells in the pancreatic islets).
  • the invention further provides use of a peptide conjugate of the invention in: a method for regulating gastric emptying in a subject in need thereof; a method for lowering plasma lipid levels in a subject in need thereof; and a method of lowering blood pressure in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.
  • a medicament of the invention is a medicament for use in treating a subject in need thereof.
  • exemplary embodiments of the invention relate to a method for treatment, in a subject in need thereof, of one or more of the diseases, disorders or conditions disclosed herein, the method comprising administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.
  • therapeutically effective amount refers to an amount that is sufficient to cure, ameliorate, alleviate or partially arrest the clinical manifestations of the particular disease, disorder or condition that is the object of the treatment or other therapeutic intervention in question e.g.
  • a therapeutically relevant amount may be determined empirically by one skilled in the art based on the indication being treated or prevented and the subject to whom the therapeutically relevant amount is being administered. For example, the skilled worker may measure one or more of the clinically relevant indicators of bioactivity described herein, e.g. blood glucose levels, Insulin release, or plasma lipid levels. The skilled worker may determine a clinically relevant amount through in vitro or in vivo measurements. Other exemplary measures include weight gain, weight loss, and change in blood pressure.
  • an amount adequate to accomplish any or all of these effects is defined as a therapeutically effective amount.
  • the administered amount and the method of administration can be tailored to achieve optimal efficacy.
  • An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject.
  • treatment and grammatical variants thereof (e.g. "treated”, “treating”, “treat") as employed in the present context refer to an approach for obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e. not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival relative to expected survival time if not receiving treatment.
  • a subject e.g.
  • a human in need of treatment may thus be a subject already afflicted with the disease or disorder in question,.
  • treatment includes inhibition or reduction of an increase in severity of a pathological state or symptoms (e.g. weight gain or hyperglycemia) relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.
  • prevention and grammatical variants thereof (e.g., “prevented”, “preventing”, “prevent”) as employed in the present context refer to an approach for hindering or preventing the development of, or altering the pathology of, a condition, disease or disorder. Accordingly, “prevention” may refer to prophylactic or preventive measures.
  • beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable.
  • a subject e.g. a human
  • prevention may thus be a subject not yet afflicted with the disease or disorder in question.
  • prevention thus includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition.
  • agonist refers to a substance (ligand) that activates the receptor type in question.
  • a subject in need of the particular treatment or other therapeutic intervention referred to in connection with the various aspects of the invention described above is a mammal.
  • the mammal is a human.
  • compositions comprising a peptide conjugate, or a pharmaceutically acceptable salt or solvate thereof, according to the invention, together with a pharmaceutically acceptable carrier, excipient or vehicle.
  • the peptide conjugates of the invention may be manufactured by standard synthetic methods, by use of recombinant expression systems, or by any other suitable method.
  • the conjugates may be synthesized in a number of ways, including, e.g., methods comprising: (a) synthesizing the peptide conjugate by standard solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the final peptide conjugate product;
  • the method of synthesis may comprise the step of chemically modifying one of more amino acid side chains in a precursor peptide to yield a compound of the invention.
  • modification may yield a non- naturally occurring amino acid such as Tyr(S0 3 H), Phe(4-CH 2 S0 3 H), Phe(4-P0 3 H 2 ) or Lys(2- tolylaminocarbonyl), or to introduce a substituent such as a lipophilic substituent.
  • the side chains of one or more amino acid residues (e.g. Lys residues) in a compound of the invention may be further conjugated (i.e. covalently attached) to a lipophilic substituent.
  • the lipophilic substituent may be covalently bonded to an atom in the amino acid side chain, or alternatively may be conjugated to the amino acid side chain via a spacer.
  • the amino acid(s) in question may be part of the peptide moiety X, or a part of the moiety L.
  • the lipophilic substituent binds to albumin in the blood stream, thereby shielding the peptide conjugate of the invention from enzymatic degradation, and thus enhancing its half-life.
  • the spacer when present, may provide spacing between the peptide conjugate and the lipophilic substituent.
  • the lipophilic substituent may be attached to the amino acid side chain or to the spacer via an ester bond, a sulfonyl ester bond, a thioester bond, an amide bond or a sulfonamide bond.
  • the lipophilic substituent may comprise an acyl group, a sulfonyl group, an N atom, an O atom or an S atom which forms part of the ester, sulfonyl ester, thioester, amide or sulfonamide bond.
  • an acyl group in a lipophilic substituent forms an amide or ester bond with the amino acid side-chain or the spacer.
  • the lipophilic substituent may comprise a hydrocarbon chain having from 4 to 30 C atoms, for example at least 8 or 12 C atoms, and preferably 24 C atoms or fewer, or 20 C atoms or fewer.
  • hydrocarbon chain may be linear or branched and may be saturated or unsaturated. It will be understood that the hydrocarbon chain is preferably substituted with a moiety which forms part of the attachment to the amino acid side chain or the spacer, for example an acyl group, a sulfonyl group, an N atom, an O atom or an S atom. In some preferred embodiments, the hydrocarbon chain is substituted with an acyl group, and accordingly the hydrocarbon chain may form part of an alkanoyl group, for example palmitoyl, caproyl, lauroyl, myristoyl or stearoyl.
  • the lipophilic substituent may have the formula shown below:
  • A may be, for example, an acyl group, a sulfonyl group, NH, N-alkyl, an O atom or an S atom, preferably acyl.
  • n is an integer from 3 to 29. In some embodiments, n is least 7 or at least 1 1 . In some embodiments, n is 23 or less. In some embodiments, n is 19 or less.
  • the hydrocarbon chain may be further substituted. For example, it may be further substituted with up to three substituents selected from NH 2l OH and COOH. If the hydrocarbon chain is further substituted, it is preferably further substituted with only one substituent. Alternatively or additionally, the hydrocarbon chain may include a cycloalkane or heterocycloalkane moiety, for example as shown below:
  • the cycloalkane or heterocycloalkane moiety is a six-membered ring, in certain preferred embodiments, it is piperidine.
  • the lipophilic substituent may be based on a cyclopentanophenanthrene skeleton, which may be partially or fully unsaturated, or saturated.
  • the carbon atoms in the skeleton may each be substituted with Me or OH.
  • the lipophilic substituent may be cholyl, deoxycholyl or lithocholyl.
  • the lipophilic substituent may be conjugated to the amino acid side-chain via a spacer.
  • the spacer is attached to the lipophilic substituent and to the amino acid side- chain.
  • the spacer may be attached to the lipophilic substituent and to the amino acid side-chain independently by means of an ester bond, a sulfonyl ester bond, a thioester bond, an amide bond or a sulfonamide bond. Accordingly, it may comprise two moieties independently selected from acyl, sulfonyl, an N atom, an 0 atom or an S atom.
  • the spacer may have the formula: wherein B and D are each independently selected from acyl, sulfonyl, NH, N-alkyl, an 0 atom or an S atom, and preferably are selected from acyl and NH.
  • n is an integer from 1 to 10, for example from 1 to 5.
  • the spacer may optionally be further substituted with one or more substituents selected from C -6 alkyl, C-i -6 alkylamino, C-,_ 6 alkylhydroxy and C 1-6 alkylcarboxy.
  • the spacer may have two or more repeat units of the formula above.
  • B, D and n are each selected independently for each repeat unit.
  • Adjacent repeat units may be covalently attached to each other via their respective B and D moieties.
  • the B and D moieties of the adjacent repeat units may together form an ester bond, a sulfonyl ester bond, a thioester bond, an amide bond or a sulfonamide bond.
  • the free B and D units at each end of the spacer are attached to the amino acid side-chain and the lipophilic substituent as described above.
  • the spacer has five or fewer, four or fewer or three or fewer repeat units. Most preferably the spacer has two repeat units, or is a single unit.
  • the spacer (or one or more of the repeat units of the spacer, if it has repeat units) may be, for example, a naturally or non-naturally occurring amino acid.
  • B and/or D may be a moiety within the side-chain of the amino acid.
  • the spacer may be any naturally occurring or non-naturally occurring amino acid.
  • the spacer (or one or more of the repeat units of the spacer, if it has repeat units) may, for example, be Gly, Pro, Ala, Val, Leu, lie, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gin, Asn, Glu, ⁇ -Glu (derived from ⁇ -glutamic acid), Asp, Ser Thr, Gaba (derived from ⁇ -aminobutyric acid), Aib, Bal, 5- aminopentanoyl, 6-aminohexanoyl, 7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl or 10- aminodecanoyl.
  • the spacer may be a single amino acid selected from ⁇ -Glu, Gaba, Bal and Gly.
  • the spacer may be 8-amino-3,6-dioxaoctanoyl (8Ado).
  • a Lys residue in a compound of the present invention (e.g. in the moiety X) is covalently attached to ⁇ -Glu (the spacer) via an amide bond. Palmitoyl is covalently attached to the ⁇ -Glu spacer via an amide bond.
  • the side-chains of one or more amino acid residues (e.g. Lys residues) in the compound of the invention may be conjugated to a biotinylic substituent.
  • the biotinylic substituent may be covalently bonded to an atom in the amino acid side-chain, or alternatively may be conjugated to the amino acid side-chain by a spacer.
  • the amino acid may be part of the moiety X, or part of the moiety L.
  • the biotinylic substituent binds to albumin in the blood stream, thereby shielding the peptide conjugate of the invention from enzymatic degradation, and thus enhancing its half-life.
  • the spacer when present, may provide spacing between the peptide conjugate and the biotinylic substituent.
  • the biotinylic substituent may be attached to the amino acid side-chain or to the spacer via an maleimide ester bond, a sulfonyl ester bond, a thioester bond, an amide bond or a sulfonamide bond.
  • the biotinylic substituent preferably comprises an maleimido group, an acyl group, a sulfonyl group, an N atom, an O atom or an S atom which forms part of the ester, sulfonyl ester, thioester, amide or sulfonamide bond.
  • the biotin moiety may suitably first be coupled to a spacer before the spacer is coupled to the peptide.
  • a number of biotin-spacer conjugates are commercially available with a spacer functionality that will allow coupling to side-chains of lysines (or cysteines).
  • the biotin-spacer conjugate may suitably contain a maleimide functionality that can couple selectively to a sulfhydryl group on a cysteine side chain.
  • biotinylic substituents may include
  • Biotin is known as Vitamin H or Coenzyme R, and is a water-soluble B-complex vitamin (vitamin B7). It has been shown to increase oral uptake of certain drugs.
  • the side-chains of one or more amino acid residues in the compound of the invention may be conjugated to a polymeric moiety, for example, in order to increase solubility and/or half-life in vivo (e.g. in plasma) and/or bioavailability. Such modifications are also known to reduce clearance (e.g. renal clearance) of therapeutic proteins and peptides.
  • the polymeric moiety is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert.
  • Suitable polymeric moieties include polyethylene glycols (PEG), homo- or copolymers of PEG, a monomethyl-substituted polymer of PEG (mPEG), or polyoxyethylene glycerol (POG). See, for example, Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992).
  • Other suitable polymeric moieties include poly-amino acids such as poly-lysine, poly-aspartic acid and poly-glutamic acid (see for example Gombotz, et al. (1995) , Bioconjugate Chem. , vol.
  • the polymeric moiety may be straight-chain or branched. It may have a molecular weight of 500-40,000 Da, for example 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, or 20,000-40,000 Da.
  • a compound of the invention may comprise two or more such polymeric moieties, in which case the total molecular weight of all such moieties will generally fall within the ranges provided above.
  • the polymeric moiety may be coupled (by covalent linkage) to an amino, carboxyl or thiol group of an amino acid side chain.
  • Preferred examples are the thiol group of Cys residues and the epsilon amino group of Lys residues, and the carboxyl groups of Asp and Glu residues may also be involved.
  • a PEG moiety bearing a methoxy group can be coupled to a Cys thiol group by a maleimido linkage using reagents commercially available from Nektar Therapeutics AL. See also WO 2008/101017, and the references cited above, for details of suitable chemistry.
  • a maleimide- functionalised PEG may also be conjugated to the side-chain sulfhydryl group of a Cys residue.
  • nucleic acids encoding entire peptides conjugates, precursors, or fragments thereof will normally be inserted in suitable vectors to form cloning or expression vectors; such novel vectors are also part of the invention.
  • the vectors can, depending on purpose and type of application, be in the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which is only expressed transiently in certain cells is an important vector.
  • Preferred cloning and expression vectors (plasmid vectors) of the invention are capable of autonomous replication, thereby enabling high copy-numbers for the purposes of high-level expression or high-level replication for subsequent cloning.
  • an expression vector comprises the following features in the 5' ⁇ 3' direction and in operable linkage: a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma), the nucleic acid fragment encoding the peptide of the invention, and optionally a nucleic acid sequence encoding a terminator. They may comprise additional features such as selectable markers and origins of replication. When operating with expression vectors in producer strains or cell lines it may be preferred that the vector is capable of integrating into the host cell genome. The skilled person is very familiar with suitable vectors and is able to design one according to their specific requirements.
  • the vectors are used to transform host cells to produce the required compound.
  • Such transformed cells can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors, or used for recombinant production of the peptides.
  • Preferred host cells are micro-organisms such as bacteria [such as the species Escherichia (e.g. E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichia pastoris), and protozoans.
  • bacteria such as the species Escherichia (e.g. E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichia pastoris), and protozoans.
  • bacteria such as the species Escherichia (e.g. E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.
  • the transformed cells may be derived from a multicellular organism, i.e. it may be fungal cell, an insect cell, an algal cell, a plant cell, or an animal cell such as a mammalian cell.
  • the transformed cell is capable of replicating the encoding nucleic acid.
  • Cells expressing the nucleic fragment are useful embodiments of the invention; they can be used for small-scale or large-scale preparation of the peptides of the invention.
  • peptide conjugates of the invention A number of therapeutic uses of peptide conjugates of the invention have already been mentioned above.
  • the use of a peptide conjugate of the invention also encompasses the use of pharmaceutically acceptable salt or solvate thereof.
  • the compounds of the invention may provide an attractive treatment option for, inter alia, obesity and metabolic diseases.
  • 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. high fibrinogen or plasminogen activator inhibitor— 1 in the blood), and proinflammatory state (e.g., elevated C-reactive protein in the blood).
  • Individuals with metabolic syndrome are at increased risk of coronary heart disease and other diseases related to other manifestations of arteriosclerosis (e.g. stroke and peripheral vascular disease). The dominant underlying risk factor for this syndrome appears to be abdominal obesity.
  • the compounds of the present invention may be used as pharmaceutical agents for preventing weight gain, promoting weight loss, reducing excess body weight or treating obesity (e.g. by control of appetite, feeding, food intake, calorie intake, and/or energy expenditure), including morbid obesity, as well as associated diseases and health conditions including but not limited to obesity linked inflammation, obesity linked gallbladder disease and obesity induced sleep apnea.
  • the compounds of the invention may also be used for treatment or prevention of metabolic syndrome, hypertension, atherogenic dyslipidemia, hepatic steatosis, atherosclerois, arteriosclerosis, coronary heart disease and stroke, all of which can be associated with obesity.
  • the effects of the compounds of the invention on these conditions may be mediated in whole or in part via an effect on body weight, or may be independent thereof.
  • the invention provides use of a compound of the invention in the treatment of a condition as described above, in an individual in need thereof.
  • the invention also provides a compound of the invention for use in a method of medical treatment, particularly for use in a method of treatment of a condition as described above.
  • the compounds described may be used in preventing weight gain or promoting weight loss.
  • the present invention comprises use of a compound for preventing weight gain or promoting weight loss in an individual in need thereof.
  • the present invention comprises use of a compound of the invention in a method of treatment of a condition caused or characterized by excess body weight, e.g. the treatment and/or prevention of obesity, morbid obesity, morbid obesity prior to surgery, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, hypertension, atherogenic dyslipidimia, atherosclerois, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in an individual in need thereof.
  • excess body weight e.g. the treatment and/or prevention of obesity, morbid obesity, morbid obesity prior to surgery, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, hypertension, atherogenic dyslipidimia, atherosclerois, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in an individual in need thereof.
  • a compound of the invention may be used in a method of lowering circulating LDL levels, and/or increasing HDL/LDL ratio.
  • the present invention comprises use of a compound of the invention in a method of lowering circulating LDL levels, and/or increasing an HDL/LDL ratio, in an individual in need thereof.
  • Dyslipidemia is associated with increased risk of cardiovascular disease.
  • High-density lipoproteins (HDL) are of clinical importance since there is apparently an inverse correlation between plasma HDL concentrations and the risk of atherosclerotic disease.
  • the majority of the cholesterol stored in atherosclerotic plaques originates from low-density lipoproteins (LDL), and elevated concentrations of LDL are thus closely associated with atherosclerosis.
  • the HDL/LDL ratio is a parameter employed to assess the clinical risk of atherosclerosis and coronary atherosclerosis in particular.
  • peptide conjugates of the invention may unexpectedly combine the physiological effects of glucagon receptor agonists with those of CCK peptides (vide supra) in a manner such that the observed activity may be significantly greater than that observed when employing a corresponding additive (non-conjugated) combination of the individual peptide components. It is consequently believed that the peptide conjugates of the invention may be of particular benefit in the prevention or treatment of, for example, obesity, morbid obesity, obesity-linked inflammation, obesity-linked gall bladder disease and obesity-induced sleep apnea.
  • Peptide conjugates of the present invention may thus be useful as pharmaceutical agents for treatment of diseases, disorders or conditions among those described herein.
  • diseases, disorders or conditions include gastric disease, metabolic syndrome, hypertension and/or dyslipidemia (or a combination of these metabolic risk factors), hepatic steatosis, atherosclerosis, arteriosclerosis, coronary heart disease, microvascular disease, macrovascular diseases, peripheral artery disease, stroke, cancer (e.g. colon cancer), inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).
  • Exemplary effects of treatment using compounds (peptide conjugates) of the invention include preventing weight gain, promoting weight loss, reducing excess body weight and/or treating obesity (e.g. by control of appetite, feeding, food intake, calorie intake, and/or energy expenditure), including morbid obesity, as well as associated diseases, disorders and health conditions, including, but not limited to, obesity-linked inflammation, obesity-linked gallbladder disease and obesity-induced sleep apnea. Effects of the peptide conjugates of the invention 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 of the invention i.e. one or more conjugates of the invention
  • a pharmaceutical composition also encompasses inclusion of a pharmaceutically acceptable salt or solvate of a peptide conjugate of the invention.
  • the peptide conjugates of the present invention may be formulated as pharmaceutical compositions which are suited for administration with or without storage, and which typically comprise a
  • pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers.
  • Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art and are described, for example, in “Remington's Pharmaceutical Sciences", 17th edition, Alfonso R.
  • Suitable pH-buffering agents may, e.g., be phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N- tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate,
  • a pharmaceutical composition of the invention may be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component or components.
  • the unit dosage form may be presented as a packaged preparation, the package containing discrete quantities of the preparation, for example, packaged tablets, capsules or powders in vials or ampoules.
  • the unit dosage form may also be, e.g., a capsule, cachet or tablet in itself, or it may be an appropriate number of any of these packaged forms.
  • a unit dosage form may also be provided in single-dose injectable form, for example in the form of a pen device containing a liquid- phase (typically aqueous) composition.
  • Compositions may be formulated for any suitable route and means of administration.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for e.g. oral, intravitreal, rectal, vaginal, nasal, topical, enteral or parenteral (including subcutaneous (SC), intramuscular (IM), intravenous (IV), intradermal and transdermal) administration or administration by inhalation.
  • SC subcutaneous
  • IM intramuscular
  • IV intravenous
  • intradermal and transdermal intradermal and transdermal
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmaceutical formulation.
  • Subcutaneous or transdermal modes of administration may be particularly suitable for the peptide conjugates of the invention.
  • FIG. 1 A block diagram illustrating an exemplary computing environment in accordance with the present invention.
  • FIG. 1 A block diagram illustrating an exemplary computing environment in accordance with the present invention.
  • FIG. 1 A block diagram illustrating an exemplary computing environment in accordance with the present invention.
  • FIG. 1 A block diagram illustrating an peptide conjugate or specified portion or variant in either the stable or preserved formulations or solutions described herein.
  • FIG. 1M injection illustrating an exemplary computing environment in accordance with the present invention.
  • FIG. 1M A block diagram illustrating an exemplary computing environment in accordance with the present invention.
  • FIG. 1M A block diagram illustrating an exemplary computing environment in accordance with the present invention.
  • Still further embodiments of the invention relate to oral formulations and oral administration.
  • Formulations for oral administration may rely on the co-administration of adjuvants (e.g. resorcinols and/or nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to artificially increase the permeability of the intestinal walls, and/or the co-administration of enzymatic inhibitors (e.g. pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) or trasylol) to inhibit enzymatic degradation.
  • adjuvants e.g. resorcinols and/or nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether
  • enzymatic inhibitors e.g. pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) or trasylol
  • the active constituent compound of a solid-type dosage form for oral administration can be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, alginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, or glyceride.
  • at least one additive such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, alginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, or glyceride.
  • These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrators, binders, thickeners, buffering agents, pH adjusting agents, sweetening agents, flavoring agents or perfuming agents.
  • additives e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrators, binders, thickeners, buffering agents, pH adjusting agents, sweetening agents, flavoring agents or perfuming agents.
  • a typical dosage of a peptide conjugate of the invention as employed in the context of the present invention may be in the range from about 0.0001 to about 100 mg/kg body weight per day, such as from about 0.0005 to about 50 mg/kg body weight per day, such as from about 0.001 to about 10 mg/kg body weight per day, e.g. from about 0.01 to about 1 mg/kg body weight per day, administered in one or more doses, such as from one to three doses.
  • the exact dosage employed will depend, inter alia, on: the nature and severity of the disease or disorder to be treated; the sex, age, body weight and general condition of the subject to be treated; possible other, concomitant, disease or disorder that is undergoing or is to undergo treatment; as well as other factors that will be known to a medical practitioner of skill in the art.
  • a peptide conjugate of the invention may be administered continuously (e.g. by intravenous administration or another continuous drug administration method), or may be administered to a subject at intervals, typically at regular time intervals, depending on the desired dosage and the
  • Regular administration dosing intervals include, e.g., once daily, twice daily, once every two, three, four, five or six days, once or twice weekly, once or twice monthly, and the like.
  • regular peptide conjugate administration regimens of the invention may, in certain circumstances such as, e.g., during chronic long-term administration, be advantageously interrupted for a period of time so that the medicated subject reduces the level of or stops taking the medication, often referred to as taking a "drug holiday.”
  • Drug holidays are useful for, e.g., maintaining or regaining sensitivity to a drug especially during long- term chronic treatment, or to reduce unwanted side-effects of long-term chronic treatment of the subject with the drug.
  • the timing of a drug holiday depends on the timing of the regular dosing regimen and the purpose for taking the drug holiday (e.g, to regain drug sensitivity and/or to reduce unwanted side effects of continuous, long- term administration).
  • the drug holiday may be a reduction in the dosage of the drug (e.g. to below the therapeutically effective amount for a certain interval of time).
  • administration of the drug is stopped for a certain interval of time before administration is started again using the same or a different dosing regimen (e.g. at a lower or higher dose and/or frequency of administration).
  • a drug holiday of the invention may thus be selected from a wide range of time-periods and dosage regimens.
  • An exemplary drug holiday is two or more days, one or more weeks, or one or more months, up to about 24 months of drug holiday.
  • a regular daily dosing regimen with a peptide conjugate of the invention may, for example, be interrupted by a drug holiday of a week, or two weeks, or four weeks, after which time the preceding, regular dosage regimen (e.g. a daily or a weekly dosing regimen) is resumed.
  • regular dosage regimen e.g. a daily or a weekly dosing regimen
  • a variety of other drug holiday regimens are envisioned to be useful for administering the peptide conjugates of the invention.
  • the peptide conjugate may be delivered via an administration regime which comprises two or more administration phases separated by respective drug holiday phases.
  • the peptide conjugate is administered to the recipient subject in a therapeutically effective amount according to a pre-determined administration pattern.
  • the administration pattern may comprise continuous administration of the drug to the recipient subject over the duration of the administration phase.
  • the administration pattern may comprise administration of a plurality of doses of the peptide conjugate to the recipient subject, wherein said doses are spaced by dosing intervals.
  • a dosing pattern may comprise at least two doses per administration phase, at least five doses per administration phase, at least 10 doses per administration phase, at least 20 doses per administration phase, at least 30 doses per administration phase, or more.
  • Said dosing intervals may be regular dosing intervals, which may be as set out above, including once daily, twice daily, once every two, three, four, five or six days, once or twice weekly, once or twice monthly, or a regular and even less frequent dosing interval, depending on the particular dosage formulation, bioavailability, and pharmacokinetic profile of the peptide conjugate.
  • An administration phase may have a duration of at least two days, at least a week, at least 2 weeks, at least 4 weeks, at least a month, at least 2 months, at least 3 months, at least 6 months, or more.
  • the duration of the following drug holiday phase is longer than the dosing interval used in that administration pattern.
  • the duration of the drug holiday phase may be greater than the mean interval between doses over the course of the administration phase.
  • the duration of the drug holiday may be longer than the longest interval between consecutive doses during the administration phase.
  • the duration of the drug holiday phase may be at least twice that of the relevant dosing interval (or mean thereof), at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 20 times that of the relevant dosing interval or mean thereof.
  • a drug holiday phase may have a duration of at least two days, at least a week, at least 2 weeks, at least 4 weeks, at least a month, at least 2 months, at least 3 months, at least 6 months, or more, depending on the administration pattern during the previous administration phase.
  • An administration regime comprises at least 2 administration phases. Consecutive administration phases are separated by respective drug holiday phases. Thus the administration regime may comprise at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 administration phases, or more, each separated by respective drug holiday phases. Consecutive administration phases may utilise the same administration pattern, although this may not always be desirable or necessary. However, if other drugs or active agents are administered in combination with the peptide conjugate of the invention, then typically the same combination of drugs or active agents is given in consecutive administration phases. In certain embodiments, the recipient subject is human.
  • a peptide conjugate of the invention also extends to a pharmaceutically acceptable salt or solvate thereof, as well as to a composition comprising more than one different peptide conjugate of the invention.
  • a peptide conjugate of the invention may be administered as part of a combination therapy together with another active agent for the treatment of the disease or disorder in question, e.g. obesity, metabolic syndrome, dyslipidemia or hypertension, and in such cases, the two active agents may be given together or separately, e.g. as constituents in the same pharmaceutical composition or formulation, or as separate formulations.
  • a peptide conjugate of the invention may thus be used in combination with an anti-obesity agent of known type, including, but not limited to, peptide YY or an analogue thereof, neuropeptide Y (NPY) or an analogue thereof, a cannabinoid receptor 1 antagonist, a lipase inhibitor, Human prolslet Peptide (HIP), a melanocortin receptor 4 agonist, liraglutide (VictozaTM), OrlistatTM, SibutramineTM, a melanin concentrating hormone receptor 1 antagonist, CCK, amylin and leptin, as well as analogues thereof.
  • an anti-obesity agent of known type, including, but not limited to, peptide YY or an analogue thereof, neuropeptide Y (NPY) or an analogue thereof, a cannabinoid receptor 1 antagonist, a lipase inhibitor, Human prolslet Peptide (HIP), a melanocort
  • a peptide conjugate of the invention may further be used in combination with an anti-hypertension agent of a known type, including, but not limited to, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor blocker, a diuretic, a beta-blocker and a calcium channel blocker.
  • an anti-hypertension agent of a known type, including, but not limited to, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor blocker, a diuretic, a beta-blocker and a calcium channel blocker.
  • a peptide conjugate of the invention may still further be used in combination with an anti-dyslipidemia agent of known type, including, but not limited to, a statin, a fibrate, a niacin and a cholesterol absorption inhibitor.
  • an anti-dyslipidemia agent of known type, including, but not limited to, a statin, a fibrate, a niacin and a cholesterol absorption inhibitor.
  • a peptide conjugate of the invention may also be used in combination with a proton pump inhibitor (i.e. a pharmaceutical agent possessing pharmacological activity as an inhibitor of H + /K + -ATPase) of known type, including, but not limited to, an agent of the benzimidazole derivative type or of the
  • imidazopyridine derivative type such as OmeprazoleTM, LansoprazoleTM, DexlansoprazoleTM, EsomeprazoleTM, PantoprazoleTM, RabeprazoleTM, ZolpidemTM, AlpidemTM, SaripidemTM or
  • a peptide conjugate of the invention may have some benefit if administered in combination with an anti-diabetic agent of known type, including, but not limited to, metformin, a sulfonylurea, a glinide, a DPP-IV inhibitor, a glitazone, a GLP-1 or a GLP-1 analogue, an exendin-4 or an exendin-4 analogue, any other GLP-1 receptor agonist, or insulin or an insulin analogue.
  • an anti-diabetic agent including, but not limited to, metformin, a sulfonylurea, a glinide, a DPP-IV inhibitor, a glitazone, a GLP-1 or a GLP-1 analogue, an exendin-4 or an exendin-4 analogue, any other GLP-1 receptor agonist, or insulin or an insulin analogue.
  • insulin analogues examples include, but are not limited to, LantusTM, NovorapidTM, HumalogTM, NovomixTM, ActraphaneTM HM, LevemirTM DegludecTM and ApidraTM.
  • GLP-1 receptor agonists such as exenatide (ByettaTM and BydureonTM exendin-4) and Byetta LARTM, lixisenatide (LyxumiaTM) and liraglutide (VictozaTM).
  • a peptide conjugate of the invention may be beneficial if administered in combination with an anti-inflammatory agent of known type, including, but not limited to: steroids and corticosteroids, such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; non-steroidal antiinflammatory agents (NSAIDs), such as propionic acid derivatives (e.g.
  • alminoprofen benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and tioxaprofen); acetic acid derivatives (e.g.
  • indomethacin acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin and zomepirac); fenamic acid derivatives (e.g. flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid); biphenylcarboxylic acid derivatives (e.g.
  • oxicams e.g. isoxicam, piroxicam, sudoxicam and tenoxicam
  • salicylates e.g. acetylsalicylic acid and sulfasalazine
  • pyrazolones e.g. apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone and phenylbutazone
  • COX II inhibitors such as rofecoxib and celecoxib
  • preparations of interferon beta e.g. interferon beta- l a or interferon beta-1 b
  • certain other compounds such as 5-aminosalicylic acid and prodrugs and pharmaceutically acceptable salts thereof.
  • Metformin has also been demonstrated to have anti-inflammatory properties (see, e.g., Haffner et al., Diabetes 54: 1566-1572 (2005)) and as such may also be useful in combination with the peptide conjugates of the invention.
  • HATU 2-(7-aza-1 H-benzotriazole-1 -yl)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate
  • DIPEA diisopropylethylamine
  • TIS triisopropylsilane
  • BSA bovine serum albumin
  • cAMP cyclic adenosine monophosphate
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • p-ERK phosphorylated extracellular regulated kinase
  • PBS phosphate-buffered saline
  • NEP N-ethylpyrroiidone
  • a CEM Liberty Peptide Synthesizer was employed, using standard Fmoc chemistry.
  • TentaGelTM S Ram resin (1 g; 0.25 mmol/g) was swelled in NEP (10 ml) prior to use and transferred between tube and reaction vessel using DCM and NEP.
  • Pseudoprolines i.e. dipeptides employed to minimize aggregation during peptide synthesis, such as Fmoc-Phe-Thr(ijj-Me,Me-Pro)-OH and Fmoc-Asp-Ser(ijj- Me,Me-Pro)-OH] were used where appropriate, and non-naturally occurring amino acids (e.g. Fmoc- 8Ado-OH) were employed without any changes to the general procedure.
  • Piperidine/NEP (1 :4, i.e. 1 part piperidine to 4 parts NEP by volume; 0 ml) was added to the resin for initial deprotection, and the mixture was microwave-heated (40°C; 30 sec). The reaction vessel was drained and a second portion of piperidine/NEP (1 :4; 10 ml) was added and heated (75°C; 3 min) again. The resin was then washed with NEP (6 x 10 ml).
  • the resin was washed with EtOH (3 x 10 ml) and Et 2 0 (3 x 10 ml) and dried to constant weight at room temperature (r.t.).
  • the crude peptide was cleaved from the resin by treatment with TFA/TIS/H 2 0 (90:5:5; 40 ml; 2 h; r.t.). Most of the TFA was removed under reduced pressure, and the crude peptide was precipitated and washed three times with Et 2 0 and dried to constant weight at room temperature.
  • the crude peptide was purified to greater than 90% purity by preparative reverse phase HPLC using a PerSeptive Biosystems VISION Workstation equipped with a suitable column and a fraction collector, and run with a gradient of buffer A (0.1 % TFA, aq.) and buffer B (0.1 % TFA, 90% MeCN, aq.). Fractions were analysed by analytical HPLC and MS, and relevant fractions were pooled and lyophilised. The final product was characterized by HPLC and MS.
  • the resin was washed with EtOH (3 x 10 ml) and Et 2 0 (3 x 10 ml) and dried to constant weight at room temperature (r.t.).
  • the crude peptide was cleaved from the resin by treatment with TFA/TIS/H 2 0 (90:5:5; 40 ml; 2 h; r.t.). Most of the TFA was removed under reduced pressure, and the crude peptide was precipitated and washed three times with Et 2 0 and dried to constant weight at room temperature.
  • the crude peptide was purified to greater than 90% purity by preparative reverse phase HPLC using a PerSeptive Biosystems VISION Workstation equipped with a suitable column and a fraction collector, and run with a gradient of buffer A (0.1 % TFA, aq.) and buffer B (0.1 % TFA, 90% MeCN, aq.).
  • Fmoc-L-Tyr(S0 3 nP)OH (commercially available from, for example, Merck Millipore/Novabiochem; Cat. No.: 852347) may be employed. After standard synthesis of the protected peptide in question using Fmoc-L-Tyr(S0 3 nP)OH and subsequent TFA cleavage the peptide is dissolved in a minimum volume of 2M ammonium acetate. Acetonitrile is added to dissolve the peptide if necessary. After overnight incubation at 40°C the peptide solution is purified directly using RP-HPLC, or in cases where a high concentration of organic solvent is present, subjected to prior evaporation of these solvents and filtered if required.
  • the peptide is purified using gradients of A and B buffers as described above, but containing 0.1 % acetic acid instead of TFA.
  • Example 2 Activation (EC 50 ) of Glucagon receptor and Gastrin CCK-B receptor in vitro by peptide conjugates of the invention
  • the cDNA encoding the human glucagon receptor (Glucagon-R; GCG-R) (primary accession number P47871 ) was cloned from the cDNA clone BC104854 (MGC:132514/IMAGE:8143857).
  • the DNA encoding the Glucagon-R was amplified by PCR using primers encoding terminal restriction sites for subcloning.
  • the 5'-end primers additionally encoded a near Kozak consensus sequence to ensure efficient translation.
  • the fidelity of the DNA encoding the Glucagon-R was confirmed by DNA sequencing.
  • the PCR products encoding the Glucagon-R were subcloned into a mammalian expression vector containing a neomycin (G418) resistance marker.
  • the mammalian expression vector encoding the Glucagon-R was transfected into HEK293 cells by a standard calcium phosphate transfection method. 48 hr after transfection, cells were seeded for limited dilution cloning and selected with 1 mg/ml G418 in the culture medium. Three weeks later 12 surviving colonies of Glucagon-R- expressing cells were picked, propagated and tested in the Glucagon-R efficacy assay as described below. One Glucagon-R expressing clone was chosen for compound profiling.
  • Glucagon receptor assay In vitro effects of peptide conjugates of the invention were assessed by measuring the induction of cAMP following stimulation of the receptor by glucagon, or conjugates of the invention using either the FlashPlateTM cAMP kit (data shown in Table 1 a) or the AlphaScreenTM cAMP kit from Perkin-Elmer (data shown in Table 1 b) according to instructions. Briefly, HEK293 cells expressing the human GCG R were seeded at 40,000 cells/well in 96-well microtiter plates coated with 0.01 % poly-L-lysine, and grown for 1 day in culture in 100 ⁇ growth medium [DMEM, 10% FCS, Penicillin (100 lU/ml),
  • the CCK receptor activity assays were performed as follows: on day 1 the CCK receptor expressing cells were seeded at 20,000 cells/well in 100 ⁇ growth medium [DMEM, 10% FCS, Penicillin (100 Ill/ml), Streptomycin (100 ⁇ g/ml)] in a 96-well plate coated with poly- D-lysine. The cells were incubated in an incubator (37°C, 5% C0 2 ) for two days. The growth medium was then changed to 80 ⁇ of serum-free medium [DMEM, Penicillin (100 Ill/ml), Streptomycin (100 ⁇ g/ml)] per well, and incubation of the cells was continued overnight in the incubator.
  • DMEM serum-free medium
  • Peptide conjugates of the invention (Compounds 1 -15; vide supra) as well as glucagon (human glucagon; synthesized and purified using a method as outlined above) were tested in the above- described assays.
  • CCK8 was used as positive control in the human CCK-A receptor (hCCK-1 R) and the CCK-B receptor (hCCK-2 R) activity assay.
  • the results (EC 50 values, in nM) are summarized in Table 1 a and Table 1 b, below. Table 1a.
  • GCG-R EC 50 value determined using AlphaScreenTM cAMP kit (vide supra)
  • Table 1b In vitro activities (EC 50 , nM) of compounds (peptide conjugates) of the invention in activation of hCCK-A R, hCCK-B R and hGCG-R.
  • GCG-R EC 50 values determined using AlphaScreenTM cA P kit (vide supra)
  • mice were given a single subcutaneous (s.c.) dose of 400 nmol/kg of each peptide to be tested. Blood samples were collected 0.25, 0.5, 1 , 2, 3, 4, 6, and 8 hours after the s.c. administration. At each time point, samples from two mice were taken. A single intravenous dose was also tested for determination of bioavailability. The mice were euthanized immediately after blood sampling by cervical dislocation. Plasma samples were analyzed after solid phase extraction (SPE) by liquid
  • mice PK after s.c. administration of 400 nmol /kg in mice
  • Example 4 Effect of peptide conjugates of the invention on food intake in normal mice
  • mice were kept in an automated food- and water-intake monitoring system (HM-2 system, MBRose, Denmark) which allows automated on-line measurements of accumulated food-intake and other parameters.
  • HM-2 system automated food- and water-intake monitoring system
  • Normal chow-fed C57BL/6J male mice were used.
  • the mice were kept 4 per cage, and each dosing group consisted of 8 animals. Prior to the experiment, the animals were stratified based on their body weight. The animals were dosed once subcutaneously with compound with the indicated amounts or PBS, just prior to the dark period in which they are active and eat. The animals were monitored by the HM-2 system up to 24 h after dosing.
  • Example 5 Sub-chronic effects of a peptide conjugate of the invention on body weight and food intake in diet-induced obese C57BL/6J mice
  • mice received vehicle injections twice-daily and was pair-fed as for animals given test compound
  • Figure 3 summarizes the body weight change data (upper part of Fig. 3) and the accumulated food intake data (lower part of Fig. 3).
  • the letter “A” designates Compound 3 of the present invention.

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Abstract

La présente invention concerne, entre autres, certains conjugués peptidiques comprenant un fraction peptidique agoniste du récepteur du glucagon et une fraction peptidique dérivée de la CCK, et l'utilisation des conjugués dans le traitement et/ou la prévention d'une variété de maladies ou de troubles, comprenant le traitement et/ou la prévention d'une absorption alimentaire excessive, de l'obésité et d'un poids corporel excessif.
PCT/EP2012/077083 2011-12-30 2012-12-28 Conjugués peptidiques agonistes des récepteurs de la cck et du glucagon WO2013098408A1 (fr)

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WO2017005765A1 (fr) * 2015-07-06 2017-01-12 Novo Nordisk A/S Nouveaux peptides et dérivés peptidiques et leurs utilisations
CN111051338A (zh) * 2017-06-29 2020-04-21 乌雷卡有限公司 具有改进的药物学性能的前药肽
EP3468569A4 (fr) * 2016-06-09 2020-05-27 AmideBio LLC Analogues du glucagon et procédés d'utilisation de ces derniers
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JP2015524419A (ja) * 2012-07-23 2015-08-24 ジーランド ファーマ アクティーゼルスカブ グルカゴン類似体
JP2019187419A (ja) * 2012-07-23 2019-10-31 ジーランド ファーマ アクティーゼルスカブ グルカゴン類似体
WO2017005765A1 (fr) * 2015-07-06 2017-01-12 Novo Nordisk A/S Nouveaux peptides et dérivés peptidiques et leurs utilisations
EP3468569A4 (fr) * 2016-06-09 2020-05-27 AmideBio LLC Analogues du glucagon et procédés d'utilisation de ces derniers
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CN111051338A (zh) * 2017-06-29 2020-04-21 乌雷卡有限公司 具有改进的药物学性能的前药肽
WO2022262837A1 (fr) * 2021-06-18 2022-12-22 北京拓界生物医药科技有限公司 Analogue du glucagon et son utilisation médicale

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