Glucagon superfamily peptides exhibiting nuclear hormone receptor activity

• US 9,127,088 B2
• Filed: 05/10/2011
• Issued: 09/08/2015
• Est. Priority Date: 05/13/2010
• Status: Active Grant

• Current Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Original Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Inventors: DiMarchi, Richard D., Yang, Bin, Finan, Brian
• Primary Examiner(s): Chandra, Gyan
• Application Number:
  US13/697,018
• Publication Number:
  US 20130123178A1
• Time in Patent Office: 1,582 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 47/55 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
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 47/64 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 peptidic linkers A61K47/65
• CPC Class Codes: A61P 25/28 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
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/605 Glucagons
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor

Abstract

Provided herein are glucagon superfamily peptides conjugated with NHR ligands that are capable of acting at a nuclear hormone receptor. Also provided herein are pharmaceutical compositions and kits of the conjugates of the invention. Further provided herein are methods of treating a disease, e.g., a metabolic disorder, such as diabetes and obesity, comprising administering the conjugates of the invention.

Claims

1. A compound comprising the structure Q-L-Y;

wherein Q is a glucagon superfamily peptide;

Y is a steroid which activates a Type I nuclear hormone receptor (NHR) with an EC₅₀ of about 1 μ

M or less, and has a molecular weight of up to about 1000 daltons; and

L is a linking group or a bond, with the proviso that Y is not a bile salt or cholesterol.

2. The compound of claim 1, wherein Q exhibits at least 0.1% of the activity of native glucagon-like peptide-1 (GLP-1) at the GLP-1 receptor, at least 0.1% of the activity of native glucagon at the glucagon receptor, or at least 0.1% of the activity of native gastric inhibitory polypeptide (GIP) at the GIP receptor, or a combination thereof.

3. The compound of claim 1, wherein Q has an EC₅₀ at the GLP-1 receptor within 10-fold of the EC₅₀ of Y at the nuclear hormone receptor, an EC₅₀ at the glucagon receptor within 10-fold of the EC₅₀ of Y at the nuclear hormone receptor, or an EC₅₀ at the GIP receptor within 10-fold of the EC₅₀ of Y at the nuclear hormone receptor, or a combination thereof.

4. The compound of claim 1, wherein Y acts at a nuclear hormone receptor selected from the group consisting of an estrogen receptor, glucocorticoid receptor, a mineralcorticoid receptor, a progesterone receptor, and an androgen receptor.

5. The compound of claim 4, wherein Y is selected from the group consisting of estradiol and derivatives thereof, estrone and derivatives thereof, testosterone and derivatives thereof, and cortisol and derivatives thereof.

6. The compound of claim 4, wherein Y is stable in vivo, hydrolyzable in vivo, or metastable in vivo.

7. The compound of claim 6, wherein L comprises an ether moiety, or an amide moiety, an ester moiety, an acid-labile moiety, a reduction-labile moiety, an enzyme-labile moiety, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety.

8. The compound of claim 4, wherein Q comprises

(I) an amino acid sequence at least 50% identical to native glucagon that retains the alpha-helix conformation of the amino acids corresponding to amino acids 12-29 of native glucagon (SEQ ID NO:

1601);

(II) an amino acid sequence at least 50% identical to native GLP-1 that retains the alpha-helix conformation of the amino acids corresponding to amino acids 12-29 of native GLP-1 (SEQ ID NO;

     1603);

or

(III) the amino acid sequence;

X₁-X₂-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Z (SEQ ID NO;

     839) with 1 to 3 amino acid modifications thereto,

wherein X₁ and/or X₂ is a non-native amino acid (relative to SEQ ID NO;

     1601) that reduces susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),

wherein Z is selected from the group consisting of —

COOH, -Asn-COOH, Asn-Thr-COOH, and W—

COOH, wherein W is 1 to 2 amino acids or GPSSGAPPPS (SEQ ID NO;

     1610),

wherein Q comprises a modification selected from the group consisting of;

(i) a lactam bridge between the side chains of amino acids at positions i and i+4, wherein i is 12, 16, 20 or 24, and

(ii) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the glucagon peptide is substituted with an α

,α

-disubstituted amino acid; and

,

wherein Q exhibits glucagon agonist activity.

9. The compound of claim 8, wherein Q comprises the amino acid sequence SEQ ID NO:

1601 and comprises;

(a) at least one amino acid modification selected from the group consisting of;

(i) substitution of Thr at position 29 with a charged amino acid;

(ii) substitution of Thr at position 29 with a charged amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid;

(iii) substitution at position 29 with Asp, Glu, or Lys;

(iv) substitution at position 29 with Glu;

(v) insertion after position 29 of 1 to 3 charged amino acids;

(vi) insertion after position 29 of Glu or Lys;

(vii) insertion after position 29 of Gly-Lys, or Lys-Lys;

(viii) substitution of Gin at position 3 with an amino acid comprising a side chain of Structure I, II, or III;

10. The compound of claim 8, wherein

(I) Q comprises a glucagon related peptide of SEQ ID NO:

1601, with the following modifications;

(a) an amino acid modification at position 1 that confers GIP agonist activity; and

,

(b) a modification selected from the group consisting of;

(i) a lactam bridge between the side chains of amino acids at positions i and i+4 or between the side chains of amino acids at positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17, and

(ii) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the analog is substituted with an α

,α

-disubstituted amino acid; and

,

(c) 1-10 further amino acid modifications; and

,

wherein Q exhibits activity at the GIP receptor;

or,

(II) Q comprises the amino acid sequence;

X₁-X₂-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X₃-Ser-X4-Tyr-Leu-X₅-X₆-X₇-X₈-Ala-X₉-X₁₀-Phe-X₁₁-X₁₂-Trp-Leu-X₁₃-X₁₄-X₁₅ (SEQ ID NO;

     55), or an analog thereof, wherein said analog differs from SEQ ID NO;

55 by 1 to 3 amino acid modifications, selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, and 25, wherein;

X₁ is His, D-His, (Des-amino)His, hydroxyl-His, acetyl-His, homo-His, or alpha, alpha-dimethyl imidazole acetic acid (DMIA), N-methyl His, alpha-methyl His, or imidazole acetic acid;

X₂ is Ser, D-Ser, Ala, D-Ala, Val, Gly, N-methyl Ser, aminoisobutyric acid (Aib) or N-methyl Ala;

X₃, X₄, X₅, X₁₀, X₁₁, and X₁₄ are, individually, any amino acid;

X₆ is Ser, Glu, Gln, homoglutamic acid or homocysteic acid;

X₇ is Arg, Gln, Lys, Cys, Orn, homocysteine or acetyl phenylalanine;

X₈ is Arg, Ala, Lys, Cys, Orn, homocysteine or acetyl phenyalanine;

X₉ is Gln, Lys, Arg, Orn or Citrulline;

X₁₂ is Ala, Gln, Glu, Lys, Cys, Orn, homocysteine or acetyl phenyalanine;

X₁₃ is Met, Leu or Nle;

X₁₅ is Thr, Gly, Lys, Cys, Orn, homocycsteine or acetyl phenyalanine; and

wherein Q optionally comprises one of the following modifications;

(a) deletion of amino acids at positions 28-29;

(b) deletion of the amino acid at positions 29;

(c) addition of the amino acid sequence GPSSGAPPPS (SEQ ID NO;

     1610) to the C-terminus; and

(d) addition of the amino acid sequence GPSSGAPPPSX (SEQ ID NO;

     1450) to the C-terminus, wherein X is any amino acid; and

(e) a combination thereof;

or,

(III) Q comprises an amino acid sequence that differs from SEQ ID NO;

1601 by no more than ten amino acid modifications, comprising;

(a) one or more amino acid substitutions with Aib at positions 16, 20, 21, and/or 24, and

(b) an amino acid modification at position 1 and/or 2 that provides reduced susceptibility to cleavage by dipeptidyl peptidase IV, and

wherein Q optionally comprising one or more of the following modifications;

(i) deletion of amino acids at positions 28 and 29,

(ii) deletion of the amino acid at position 29,

(iii) addition of the amino acid sequence GPSSGAPPPS (SEQ ID NO;

     1610) to the C-terminus,

(iv) addition of the amino acid sequence GPSSGAPPPSX (SEQ ID NO;

     1450) to the C-terminus, wherein X is any amino acid,

(v) substitution of the C-terminal carboxyl group with an amide or carboxylic ester, and

wherein Q exhibits GLP-1 agonist activity and glucagon agonist activity.

11. The compound of claim 10, wherein the 1-10 further amino acid modifications in (I) are selected from the group consisting of:

(i) substitution of serine at position 2 with D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, Aib, Val, or ε

-amino-N-butyric acid;

(ii) substitution of serine at position 16 with Glu, Gln, homoglutamic acid, homocysteic acid, Thr, Gly, or Aib;

(iii) substitution of glutamine at position 20 with Ser, Thr, Ala, Lys, Citrulline, Arg, Orn, or Aib;

(iv) substitution of methionine at position 27 with Leu;

(v) substitution of arginine at position 28 with Ala;

(vi) substitution of threonine at position 29 with Gly;

(vii) a conservative substitution at any of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 24, 27, 28, and 29;

(viii) addition of 1-21 amino acids to the C-terminus

(ix) addition of the amino acid sequence GPSSGAPPPS (SEQ ID NO;

     1610) to the C-terminus,

(x) addition of the amino acid sequence GPSSGAPPPSX (SEQ ID NO;

     1450) to the C-terminus, wherein X is any amino acid,

(xi) substitution of the C-terminal carboxyl group with an amide or carboxylic ester, and

(xii) a combination thereof.

12. The compound of claim 4, wherein L-Y is covalently conjugated to the N-terminus, C-terminus, or an amino acid side chain of Q.

13. The compound of claim 4, wherein L-Y is conjugated to an amino acid side chain of Q that corresponds to position 10, 30, 37, 38, 39, 40, 41, 42, or 43 of native glucagon (SEQ ID NO:

1601) or a sequence comprising native glucagon.

14. A prodrug of the compound of claim 4 comprising the structure A-B, wherein

A is an amino acid or a hydroxy acid;

B is an N-alkylated amino acid linked to Q through an amide bond between a carboxyl moiety of B and an amine of Q;

A, B, or the amino acid of Q to which A-B is linked is a non-coded amino acid, further wherein the chemical cleavage half-life (t_1/2) of A-B from Q is at least about 1 hour to about 1 week in PBS under physiological conditions.

15. The compound of claim 14, wherein A-B comprises the structure:

16. The compound of claim 4, further comprising an amino acid side chain on Q covalently attached to an acyl group or an alkyl group via an alkyl amine, amide, ether, ester, thioether, or thioester linkage, which acyl group or alkyl group is non-native to a naturally occurring amino acid.

17. The compound of claim 16, wherein the amino acid to which the acyl or alkyl group is attached is at a position corresponding to position 10, 20, 24, 30, 37, 38, 39, 40, 41, 32, or 43 of a sequence comprising native glucagon, or the C-terminal amino acid.

18. The compound of claim 17, wherein the acyl group or the alkyl group is attached to the side chain of the amino acid through a spacer.

19. The compound of claim 4, wherein Q is covalently attached to one or more heterologous moieties.

20. The compound of claim 4 wherein Q is selected from the group consisting of SEQ ID NOs:

1-564, 566-570, 573-575, 577, 579-580, 585-612, 616, 618-632, 634-642, 647, 657-692, 694-695, 715-718, 722, 724-725, 729, 731-760, 801-878, 883-919, 1001-1275, 1301-1371, 1401-1518, and 1601-1650.

21. A pharmaceutical composition comprising the compound of claim 4 and a pharmaceutically acceptable carrier.

22. A method for treating a disease or medical condition in a patient, wherein the disease or medical condition is selected from the group consisting of metabolic syndrome, diabetes, obesity, liver steatosis, and a neurodegenerative disease, comprising administering to the patient the pharmaceutical composition of claim 21 in an amount effective to treat the disease or medical condition.

23. The compound of claim 1, wherein the steroid activates the nuclear hormone receptor with an EC₅₀ of about 100 nM or less.