IP Library › Granted Patent US 9,896,481

Granted Patent B2

US 9,896,481 · App. 15/082,158 · Granted Feb 20, 2018

Modified mini-hepcidin peptides and methods of using thereof

Inventors: Tomas Ganz (Los Angeles, CA); Elizabeta Nemeth (Sherman Oaks, CA); Piotr Ruchala (Los Angeles, CA)

Assignee: The Regents of the University of California

C07K7/06 A61K38/04 C07K14/47 C07K14/575 C07K16/00 A61K38/00

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Quick Facts

Patent No.: US 9,896,481
App. No.: 15/082,158
Granted: Feb 20, 2018
Kind: B2

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Abstract

Disclosed herein are peptides which exhibit hepcidin activity and methods of making and using thereof.

Claims (225)

1. An isolated peptide having the following structural formula IA or IB

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10 IA

A10-A9-A8-A7-A6-A5-A4-A3-A2-A1 IB

wherein

A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D-pyroglutamate, Gln, D-Gln, Asn, D-Asn, bhAsp, Ida, Ida(NHPal), or N-MeAsp;

A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile, Ala, D-Ala, Tle, Inp, Chg, bhThr, or N-MeThr;

A3 is His, D-His, Asn, D-Asn, Arg, D-Arg, L-His(π-Me), D-His(π-Me), L-His(τ-Me), or D-His(τ-Me);

A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr, D-Tyr, Phg, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine;

A5 is Pro, D-Pro, Ser, D-Ser, Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, or Idc;

A6 is Arg, L-Nω,ω-dimethyl-arginine, L-homoarginine, L-norarginine, citrulline, or N-MeArg;

A7 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, Cys(S-tBut), homoCys, Pen, (D)Pen, S-tertiary butyl-cysteine, Cys(S-S-Pal), Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys);

A8 is Arg, L-Nω,ω-dimethyl-arginine, L-homoarginine, L-norarginine, citrulline, or N-MeArg;

A9 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Tyr, D-Tyr, Trp, D-Trp, Phe-R a , D-Phe-R a , Dpa-R a , D-Dpa-R a , Trp-R a , bhPhe-R a , PheF5, N-MePhe, benzylamide, 2-aminoindane, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, or cyclohexylalanine, which may or may not have R a linked thereto, wherein R a is palmitoyl-PEG- wherein PEG is PEG11 or miniPEG3, palmitoyl-PEG-PEG11, palmitoyl-PEG-miniPEG3, butanoyl (C4)-PEG11-, octanoyl (C8, Caprylic)-PEG11-, palmitoyl (C16)-PEG11-, or tetracosanoyl (C24, Lignoceric)-PEG11-; and

A10 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, Ida, Ahx-Ida(NHPal), or Ahx-Ida(NBzl2);

wherein the carboxy-terminal amino acid is in amide or carboxy-form;

wherein at least one sulfhydryl amino acid is present as one of the amino acids in the sequence; and

wherein A1, A1 to A2, A10, or a combination thereof are optionally absent, and wherein

bhAsp is 3-aminopentanedioic acid

Ida is 2,2′-azanediyldiacetic acid;

Ida(NHPal) is a palmitylamine amide on Ida side chain;

Ida(NBzl2) is N,N′-Dibenzylamine amide on Ida side chain;

N-MeAsp is methyl-L-aspartic acid;

Ahx is aminohexanoic acid;

Tle is L-tert-leucine;

Inp is isonipecotic acid;

Chg is L-α-cyclohexylglycine;

bhThr is β-homothreonine;

N-MeThr is (2S)-3-hydroxy-2-(methylamino)butanoic acid;

His(π-Me) is N π -methylhistidine;

His(τ-Me) is N τ -methylhistidine;

Phg is L-phenylglycine;

bhPhe is β-homophenylalanine;

Dpa is 3,3-diphenyl-L-alanine;

Bip is L-biphenylalanine;

1Nal is (1-naphthyl)-L-alanine;

2Nal is (2-naphthyl)-L-alanine;

bhDpa is (S)-3-Amino-4,4-diphenylbutanoic acid;

Amc is 4-(aminomethyl)cyclohexane carboxylic acid;

PheF5 is (R)-2-amino-3-(perfluorophenyl)propanoic acid;

hPhe is (S)-2-amino-4-phenylbutanoic acid;

Igl is (S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid;

Oic is octahydroindole-2-carboxylic acid;

bhPro is L-β-homoproline;

trans-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid;

cis-4-PhPro is (2S,4R)-4-phenylpyrrolidine-2-carboxylic acid;

cis-5-PhPro is (2R,5S)-5-phenylpyrrolidine-2-carboxylic acid;

Idc is (R)-indoline-2-carboxylic acid;

Cys(S-tBut) is S-t-Butylthio-L-cysteine;

homoCys is L-homocysteine;

Pen is L-Penicillamine;

(D)Pen is D-Penicillamine;

Cys(S-S-Pal) is palmitoyl attached to Cys via a disufide bond;

Cys(S-S-cysteamine-Pal) is palmitoyl attached to Cys via SS-Cysteamine;

Cys(S-S-Cys-NHPal) is palmitylamine attached to Cys via another Cys;

Cys(S-S-Cys) is Cys attached to Cys via disulfide bond;

N-MeArg is N ω -methyl-L-arginine;

PEG11 is O-(2-aminoethyl)-O′-(2-carboxyethyl)-undecaethyleneglycol; and

miniPEG3 is 11-amino-3,6,9-trioxaundecanoic acid.

2. The peptide according to claim 1 , wherein the peptide contains at least one of the following:

a) A1=N-MeAsp, Ida, or Ida(NHPal);

b) A5=bhPro;

c) A6=Arg, or N-MeArg;

d) A7=Cys(S-S-Pal), Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys); and/or

e) A8=Arg, or N-MeArg.

3. The peptide according to claim 1 , wherein:

i) when A1 is Ida and A9 is Phe, then A10 is not Ahx-Ida(NHPal);

ii) when A1 is Ida, A9 is not bhPhe-R b , wherein R b is S-(palmityl)thioglycolic-PEG-;

iii) when A4 is D-Phe, A7 is not D-Cys(S-S-tBut) and A9 is not D-Trp-R c , wherein R c is Butanoyl-PEG11-, Octanoyl-PEG11-, Palmitoyl-PEG11-, or Tetracosanoyl-PEG11-.

4. The peptide according to claim 1 , wherein

A1 is D-Asp, N-MeAsp, Ida, or Ida(NHPal);

A2 is Thr or D-Thr;

A3 is His or D-His;

A4 is Dpa or D-Dpa;

A5 is Pro, D-Pro, bhPro, or Oic;

A6 is Arg, or N-MeArg;

A7 is Cys, D-Cys, Cys(S-S-Pal), Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys);

A8 is Arg, or N-MeArg;

A9 is Phe, D-Phe, Dpa, D-Dpa, Trp, D-Trp, bhPhe, Phe-R a , D-Phe-R a , Dpa-R a , D-Dpa-R a bhPhe-R a , where in R a is palmitoyl-PEG- wherein PEG is PEG11 or miniPEG3, palmitoyl-PEG-PEG wherein PEG is PEG11 or miniPEG3, butanoyl (C4)-PEG11-, octanoyl (C8, Caprylic)-PEG11-, palmitoyl (C16)-PEG11-, or tetracosanoyl (C24, Lignoceric)-PEG11-; and

A10, if present, is Ahx-Ida(NHPal) or Adx-Ida(NBzl2).

5. The peptide of according to claim 1 , wherein the peptide is selected from the group consisting of:

Asp-Thr-His-Dpa-Pro-Arg-Cys-Arg-Dpa (SEQ ID NO: 67),

N-MeAsp-Thr-His-Dpa-Oic-Arg-Cys-Arg-bhPhe-R14 (SEQ ID NO: 77),

N-MeAsp-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe-R14 (SEQ ID NO: 78),

Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe-R14 (SEQ ID NO: 82),

Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe-R15 (SEQ ID NO: 86),

Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe (SEQ ID NO:87),

Ida-Thr-His-Dpa-bhPro-Arg-Cys(S-S-Pal)-Arg-bhPhe (SEQ ID NO: 88),

Ida-Thr-His-Dpa-bhPro-Arg-Cys(S-S-cysteamine-Pal)-Arg-bhPhe (SEQ ID NO: 89),

Ida-Thr-His-Dpa-bhPro-Arg-Cys(S-S-Cys-NHPal)-Arg-bhPhe (SEQ ID NO: 90),

Ida-Thr-His-Dpa-bhPro-Arg-Cys(S-S-Cys)-Arg-bhPhe-R14 (SEQ ID NO: 91),

Ida(NHPal)-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe (SEQ ID NO: 92),

Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe-Ida(NHPal) (SEQ ID NO:93),

Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe-Ahx-lda(NBzl2) (SEQ ID NO: 95), and

Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-Trp-Ahx-Ida(NHPal) (SEQ ID NO: 101),

wherein

Ahx is an aminohexanoic acid spacer between peptide residue 9 and Ida residue;

R14 is Palmitoyl-PEG-miniPEG3-, and miniPEG3 is 11-amino-3,6,9-trioxaundecanoic acid; and

R15 is Palmitoyl-PEG-.

6. The peptide according to claim 1 , wherein the peptide exhibits hepcidin activity.

7. The peptide according to claim 1 , wherein the peptide binds ferroportin.

8. A composition which comprises at least one peptide according to claim 1 .

9. A method of binding a ferroportin or inducing ferroportin internalization and degradation which comprises contacting the ferroportin with at least one peptide according to claim 1 or a composition comprising the at least one peptide.

10. A method of treating a disease of iron metabolism in a subject which comprises administering at least one peptide according to claim 1 or a composition comprising the at least one peptide to the subject.

11. The method of claim 10 , wherein the disease of iron metabolism is an iron overload disease.

12. A kit comprising at least one peptide according to claim 1 or a composition comprising the at least one peptide packaged together with a reagent, a device, instructional material, or a combination thereof.

13. A complex comprising at least one peptide according to claim 1 bound to a ferroportin or an antibody.

14. The method according to claim 10 , wherein the at least one peptide is administered at an effective daily dose as a single daily dose or as divided daily doses.

15. The method according to claim 14 , wherein the effective daily dose is about 10-500 μg/kg/day and the at least one peptide is provided in a formulation for subcutaneous injection.

16. The method according to claim 14 , wherein the effective daily dose is about 10-1000 μg/kg/day and the at least one peptide is provided in a formulation for oral, pulmonary, parenteral, or mucosal administration.

17. An isolated peptide having the following structural formula IA or IB

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10 IA

A10-A9-A8-A7-A6-A5-A4-A3-A2-A1 IB

wherein

A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D-pyroglutamate, Gln, D-Gln, Asn, D-Asn, bhAsp, Ida, Ida(NHPal), or N-MeAsp;

A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile, Ala, D-Ala, Tle, Inp, Chg, bhThr, or N-MeThr;

A3 is His, D-His, Asn, D-Asn, Arg, D-Arg, L-His(π-Me), D-His(π-Me), L-His(τ-Me), or D-His(τ-Me);

A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr, D-Tyr, Phg, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine;

A5 is Pro, D-Pro, Ser, D-Ser, Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, or Idc;

A6 is Arg;

A7 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, Cys(S-tBut), homoCys, Pen, (D)Pen, S-tertiary butyl-cysteine, Cys(S-S-Pal), Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys);

A8 is Arg;

A10 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, Ida, Ahx-Ida(NHPal), or Ahx-Ida(NBzl2);

wherein the carboxy-terminal amino acid is in amide or carboxy-form;

wherein at least one sulfhydryl amino acid is present as one of the amino acids in the sequence; and

wherein A1, A1 to A2, A10, or a combination thereof are optionally absent, and wherein

bhAsp is 3-aminopentanedioic acid

Ida is 2,2′-azanediyldiacetic acid;

Ida(NHPal) is a palmitylamine amide on Ida side chain;

Ida(NBzl2) is N,N′-Dibenzylamine amide on Ida side chain;

N-MeAsp is methyl-L-aspartic acid;

Ahx is aminohexanoic acid;

Tle is L-tert-leucine;

Inp is isonipecotic acid;

Chg is L-α-cyclohexylglycine;

bhThr is β-homothreonine;

N-MeThr is (2S)-3-hydroxy-2-(methylamino)butanoic acid;

His(π-Me) is N π -methylhistidine;

His(τ-Me) is N τ -methylhistidine;

Phg is L-phenylglycine;

bhPhe is β-homophenylalanine;

Dpa is 3,3-diphenyl-L-alanine;

Bip is L-biphenylalanine;

1Nal is (1-naphthyl)-L-alanine;

2Nal is (2-naphthyl)-L-alanine;

bhDpa is (S)-3-Amino-4,4-diphenylbutanoic acid;

Amc is 4-(aminomethyl)cyclohexane carboxylic acid;

PheF5 is (R)-2-amino-3-(perfluorophenyl)propanoic acid;

hPhe is (S)-2-amino-4-phenylbutanoic acid;

Igl is (S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid;

Oic is octahydroindole-2-carboxylic acid;

bhPro is L-β-homoproline;

trans-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid;

cis-4-PhPro is (2S,4R)-4-phenylpyrrolidine-2-carboxylic acid;

cis-5-PhPro is (2R,5S)-5-phenylpyrrolidine-2-carboxylic acid;

Idc is (R)-indoline-2-carboxylic acid;

Cys(S-tBut) is S-t-Butylthio-L-cysteine;

homoCys is L-homocysteine;

Pen is L-Penicillamine;

(D)Pen is D-Penicillamine;

Cys(S-S-Pal) is palmitoyl attached to Cys via a disufide bond;

Cys(S-S-cysteamine-Pal) is palmitoyl attached to Cys via SS-Cysteamine;

Cys(S-S-Cys-NHPal) is palmitylamine attached to Cys via another Cys;

Cys(S-S-Cys) is Cys attached to Cys via disulfide bond;

N-MeArg is N ω -methyl-L-arginine;

PEG11 is O-(2-aminoethyl)-O′-(2-carboxyethyl)-undecaethyleneglycol; and

miniPEG3 is 11-amino-3,6,9-trioxaundecanoic acid.

18. An isolated peptide consisting of the following structural formula IA or IB

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10 IA

A10-A9-A8-A7-A6-A5-A4-A3-A2-A1 IB

wherein

A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D-pyroglutamate, Gln, D-Gln, Asn, D-Asn, bhAsp, Ida, Ida(NHPal), or N-MeAsp;

A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile, Ala, D-Ala, Tle, Inp, Chg, bhThr, or N-MeThr;

A3 is His, D-His, Asn, D-Asn, Arg, D-Arg, L-His(π-Me), D-His(π-Me), L-His(τ-Me), or D-His(τ-Me);

A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr, D-Tyr, Phg, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine;

A5 is Pro, D-Pro, Ser, D-Ser, Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, or Idc;

A6 is Arg;

A7 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, Cys(S-tBut), homoCys, Pen, (D)Pen, S-tertiary butyl-cysteine, Cys(S-S-Pal), Cys(S-S-cysteamine-Pal), Cys(S-S-Cys-NHPal), or Cys(S-S-Cys);

A8 is Arg;

A10 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, Ida, Ahx-Ida(NHPal), or Ahx-Ida(NBzl2);

wherein the carboxy-terminal amino acid is in amide or carboxy-form;

wherein at least one sulfhydryl amino acid is present as one of the amino acids in the sequence; and

wherein A1, A1 to A2, A10, or a combination thereof are optionally absent, and wherein

bhAsp is 3-aminopentanedioic acid

Ida is 2,2′-azanediyldiacetic acid;

Ida(NHPal) is a palmitylamine amide on Ida side chain;

Ida(NBzl2) is N,N′-Dibenzylamine amide on Ida side chain;

N-MeAsp is methyl-L-aspartic acid;

Ahx is aminohexanoic acid;

Tle is L-tert-leucine;

Inp is isonipecotic acid;

Chg is L-α-cyclohexylglycine;

bhThr is β-homothreonine;

N-MeThr is (2S)-3-hydroxy-2-(methylamino)butanoic acid;

His(π-Me) is N π -methylhistidine;

His(τ-Me) is N τ -methylhistidine;

Phg is L-phenylglycine;

bhPhe is β-homophenylalanine;

Dpa is 3,3-diphenyl-L-alanine;

Bip is L-biphenylalanine;

1Nal is (1-naphthyl)-L-alanine;

2Nal is (2-naphthyl)-L-alanine;

bhDpa is (S)-3-Amino-4,4-diphenylbutanoic acid;

Amc is 4-(aminomethyl)cyclohexane carboxylic acid;

PheF5 is (R)-2-amino-3-(perfluorophenyl)propanoic acid;

hPhe is (S)-2-amino-4-phenylbutanoic acid;

Igl is (S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid;

Oic is octahydroindole-2-carboxylic acid;

bhPro is L-β-homoproline;

trans-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid;

cis-4-PhPro is (2S,4R)-4-phenylpyrrolidine-2-carboxylic acid;

cis-5-PhPro is (2R,5S)-5-phenylpyrrolidine-2-carboxylic acid;

Idc is (R)-indoline-2-carboxylic acid;

Cys(S-tBut) is S-t-Butylthio-L-cysteine;

homoCys is L-homocysteine;

Pen is L-Penicillamine;

(D)Pen is D-Penicillamine;

Cys(S-S-Pal) is palmitoyl attached to Cys via a disufide bond;

Cys(S-S-cysteamine-Pal) is palmitoyl attached to Cys via SS-Cysteamine;

Cys(S-S-Cys-NHPal) is palmitylamine attached to Cys via another Cys;

Cys(S-S-Cys) is Cys attached to Cys via disulfide bond;

N-MeArg is N ω -methyl-L-arginine;

PEG11 is O-(2-aminoethyl)-O′-(2-carboxyethyl)-undecaethyleneglycol; and

miniPEG3 is 11-amino-3,6,9-trioxaundecanoic acid.

Continuity (3)

Continuation 14363012

Provisional Application 61568724 · Dec 9, 2011

Related Publication 20160200765A1 · Jul 14, 2016