U.S. patent application number 16/067568 was filed with the patent office on 2019-01-03 for analogues of hepcidin mimetics with improved in vivo half lives.
The applicant listed for this patent is Protagonist Therapeutics, Inc.. Invention is credited to Ashok Bhandari, Gregory Thomas Bourne, Brian Troy Frederick, Mark Leslie Smythe.
Application Number | 20190002503 16/067568 |
Document ID | / |
Family ID | 59225940 |
Filed Date | 2019-01-03 |
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United States Patent
Application |
20190002503 |
Kind Code |
A1 |
Bourne; Gregory Thomas ; et
al. |
January 3, 2019 |
ANALOGUES OF HEPCIDIN MIMETICS WITH IMPROVED IN VIVO HALF LIVES
Abstract
The present invention provides hepcidin analogues with improved
in vivo half-lives, and related pharmaceutical compositions and
methods of use thereof.
Inventors: |
Bourne; Gregory Thomas;
(Jindalee, AU) ; Smythe; Mark Leslie; (Bardon,
Queensland, AU) ; Frederick; Brian Troy; (Ben Lomond,
CA) ; Bhandari; Ashok; (Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Protagonist Therapeutics, Inc. |
Newark |
CA |
US |
|
|
Family ID: |
59225940 |
Appl. No.: |
16/067568 |
Filed: |
December 29, 2016 |
PCT Filed: |
December 29, 2016 |
PCT NO: |
PCT/US16/69255 |
371 Date: |
June 29, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62273265 |
Dec 30, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/575 20130101;
A61K 38/00 20130101; A61P 3/02 20180101; C07K 7/08 20130101; C07K
14/00 20130101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 14/575 20060101 C07K014/575; A61P 3/02 20060101
A61P003/02 |
Claims
1. A hepcidin analogue comprising a polypeptide sequence of Formula
(I): X--Y (I) or a pharmaceutically acceptable salt or solvate
thereof, wherein: X is a peptide sequence having the formula Xa:
TABLE-US-00022 (Xa) (SEQ ID NO: 1)
X1-Thr-His-X4-Pro-X6-X7-X8-Phe-X10
wherein X1 is Asp, isoGlu or Ida; X4 is Phe, Phe(4-F), Phe(4-CN),
4-BIP, Phe(4-OCH.sub.3), Tyr, Phe(2,3-(OCH.sub.3).sub.2),
Phe(2,3-Cl.sub.2), or Dpa; X6 is Cys or Pen; X7 is any amino acid;
X8 is Ile, Leu, Val, nLeu, Lys or Arg; and X10 is Lys, Glu or
absent; and Y is absent or present; wherein if Y is present, Y is a
peptide sequence having the formula Ya: TABLE-US-00023 (Ya) (SEQ ID
NO: 2) Y1-Y2-Y3-Y4-Y5-Y6-Y7
wherein Y1 is amino acid Y2 is any amino acid Y3 is any amino acid
Y4 is any amino acid Y5 is any amino acid; Y6 is Cys or Pen; and Y7
is Lys or absent; and wherein the hepcidin analogue comprises a
conjugated half-life extension moiety, wherein the half-life
extension moiety is optionally conjugated via a linker moiety.
2. The hepcidin analogue of claim 1, comprising one of more of the
following: X1 is Asp; X4 is Phe or Dpa; X7 is Ile, Leu, Val, nLeu,
or Lys; X7 is Ile or Lys; X8 is Lys or Arg; Y1 is Pro or hPro; Y1
is Pro; Y2 is Arg or Lys; Y2 is Arg; Y3 is Ser; Y4 is Lys, Arg or
His; Y4 is Lys; or Y5 is Gly or Sar.
3. The hepcidin analogue of claim 1, wherein: X1 is Asp; X4 is Phe
or Dpa; X7 is Ile, Leu, Val, nLeu, or Lys; X8 is Lys or Arg; Y1 is
Pro or hPro; Y2 is Arg or Lys; Y3 is Ser; Y4 is Lys, Arg or His;
and Y5 is Gly or Sar.
4. The hepcidin analogue of claim 3, wherein: X7 is Ile or Lys; Y1
is Pro; Y2 is Arg; and Y4 is Lys.
5. The hepcidin analogue of any one of claims 1-4, comprising a
structure of Formula II: R.sup.1--X-L-Y--R.sup.2 (II) or a
pharmaceutically acceptable salt or solvate thereof, wherein:
R.sup.1 is hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl
C1-C6 alkyl, or a C1-C20 alkanoyl, and including PEGylated versions
alone or as spacers of any of the foregoing; R.sup.2 is OH or
NH.sub.2; X is a peptide sequence having the formula Xa; L is
absent, a bond, or a linker moiety; and Y is absent or present;
provided that if Y is present, Y is a peptide having the formula
Ya.
6. The hepcidin analogue of any one of claims 1-5, wherein X or Y
further comprise one to three additional amino acids at the
N-terminus or C-terminus.
7. The hepcidin analogue of any one of claims 1-6, wherein Y is
present.
8. The hepcidin analogue of claim 7, comprising a disulfide bond
between X6 and Y6.
9. The hepcidin analogue of claim 7 or claim 8, wherein L is a
bond.
10. The hepcidin analogue of any one of claims 7-9, comprising a
half-life extension moiety conjugated to a Lys at X8 or X10.
11. The hepcidin analogue of any one of claims 1-10, wherein the
hepcidin analogue comprises one of the following structures:
##STR00048##
12. The hepcidin analogue of any one of claims 1-6, wherein Y is
absent.
13. The hepcidin analogue of claim 12, wherein X10 is absent.
14. A dimer comprising two hepcidin analogues of claim 12 or claim
13, wherein the two polypeptide sequence of Formula I or two
structures of Formula II are dimerized via a linker moiety.
15. The dimer of claim 14, wherein the linker moiety is bound to
the C-terminus of each hepcidin analogue.
16. The dimer of claim 14 or claim 15, wherein the half-life
extension moiety is conjugated to the linker moiety.
17. The hepcidin analogue of any one of claims 1-13 or the dimer of
any one of claims 14-16, wherein the linker moiety is selected from
IsoGlu, Dapa, PEGn where n=1 to 25, PEG11(40 atoms), OEG,
IsoGlu-Ahx, IsoGlu-OEG-OEG, IsoGlu-PEG5, IsoGlu-PEGn where n=1 to
25 .beta.Ala-PEG2, and .beta.Ala-PEG11(40 atoms).
18. The hepcidin analogue or dimer of any one of claims 1-17,
wherein the half-life extension moiety is selected from C12 (Lauric
acid), C14 (Mysteric acid), C16 (Palmitic acid), C18 (Stearic acid,
C20, C12 diacid, C14 diacid, C16 diacid, C18 diacid, C20 diacid,
biotin, and isovaleric acid.
19. The hepcidin analogue or dimer of any one of claims 1-18,
wherein the half-life extension moiety is attached to a linker
moiety that is attached to the peptide.
20. The hepcidin analogue or dimer of any one of claims 1-19,
wherein the half-life extension moiety increases the molecular
weight of the hepcidin analogue by about 50 D to about 2 KD.
21. The hepcidin analogue or dimer of any one of claims 1-20,
wherein the half-life extension moiety increases serum half-life,
enhances solubility, and/or improves bioavailability of the
hepcidin analogue.
22. The hepcidin analogue or dimer of any one of claims 1-21,
comprising an isovaleric acid moiety conjugated to the N-terminal
Asp residue.
23. The hepcidin analogue or dimer of any one of claims 1-22,
comprising an amidated C-terminal residue.
24. The hepcidin analogue or dimer of any one of claims 1-23,
comprises the sequence: TABLE-US-00024 (SEQ ID NO: 6)
Asp-Thr-His-Phe-Pro-Cys-Ile-Lys-Phe-Glu-Pro-Arg-
Ser-Lys-Gly-Cys-Lys.
25. The hepcidin analogue or dimer of any one of claims 1-23,
comprising the sequence: TABLE-US-00025 (SEQ ID NO: 5)
Asp-Thr-His-Phe-Pro-Cys-Ile-Lys-Phe-Lys-Pro-Arg-
Ser-Lys-Gly-Cys-Lys.
26. A polynucleotide encoding a peptide of the hepcidin analogue of
any one of claims 1-25.
27. A vector comprising the polynucleotide of claim 26.
28. A pharmaceutical composition comprising the hepcidin analogue
or dimer of any one of claims 1-25, the polynucleotide of claim 26,
or the vector of claim 27, and a pharmaceutically acceptable
carrier, excipient or vehicle.
29. A method of binding a ferroportin or inducing ferroportin
internalization and degradation, comprising contacting the
ferroportin with at least one hepcidin analogue or dimer of any one
of claims 1-25 or a composition of claim 28.
30. A method for treating a disease of iron metabolism in a subject
in need thereof comprising providing to the subject an effective
amount of the pharmaceutical composition of claim 28.
31. The method of claim 30, wherein the pharmaceutical composition
is provided to the subject by an oral, intravenous, peritoneal,
intradermal, subcutaneous, intramuscular, intrathecal, inhalation,
vaporization, nebulization, sublingual, buccal, parenteral, rectal,
vaginal, or topical route of administration.
32. The method of claim 31, wherein the pharmaceutical composition
is provided to the subject by an oral or subcutaneous route of
administration.
33. The method of any one of claims 30-32, wherein the disease of
iron metabolism is an iron overload disease.
34. The method of any one of claims 30-33, wherein the
pharmaceutical composition is provided to the subject at most twice
daily, at most once daily, at most once every two days, at most
once a week, or at most once a month.
35. The method of any one of claims 30-34, wherein the hepcidin
analogue is provided to the subject at a dosage of about 1 mg to
about 100 mg.
36. A device comprising the pharmaceutical composition of claim 28,
for delivery of the hepcidin analogue to a subject, optionally
orally or subcutaneously.
37. A kit comprising the pharmaceutical composition of claim 28,
packaged with a reagent, a device, or an instructional material, or
a combination thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/273,265, filed on Dec. 30, 2015, which is
incorporated by reference herein in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
PRTH_022_01WO_ST25.txt. The text file is 17 KB, was created on Dec.
29, 2016, and is being submitted electronically via EFS-Web.
FIELD OF THE INVENTION
[0003] The present invention relates, inter alia, to certain
hepcidin peptide analogues, including both peptide monomers and
peptide dimers, and conjugates and derivatives thereof, as well as
compositions comprising the peptide analogues, and to the use of
the peptide analogues in the treatment and/or prevention of a
variety of diseases, conditions or disorders, including treatment
and/or prevention of iron overload diseases such as hereditary
hemochromatosis, iron-loading anemias, and other conditions and
disorders described herein.
BACKGROUND
[0004] Hepcidin (also referred to as LEAP-1), a peptide hormone
produced by the liver, is a regulator of iron homeostasis in humans
and other mammals. Hepcidin acts by binding to its receptor, the
iron export channel ferroportin, causing its internalization and
degradation. Human hepcidin is a 25-amino acid peptide (Hep25). See
Krause et al. (2000) FEBS Lett 480:147-150, and Park et al. (2001)
J Biol Chem 276:7806-7810. The structure of the bioactive 25-amino
acid form of hepcidin is a simple hairpin with 8 cysteines that
form 4 disulfide bonds as described by Jordan et al. J Biol Chem
284:24155-67. The N terminal region is required for iron-regulatory
function, and deletion of 5 N-terminal amino acid residues results
in a loss of iron-regulatory function. See Nemeth et al. (2006)
Blood 107:328-33.
[0005] Abnormal hepcidin activity is associated with iron overload
diseases, including hereditary hemochromatosis (HH) and
iron-loading anemias. Hereditary hemochromatosis is a genetic iron
overload disease that is mainly caused by hepcidin deficiency or in
some cases by hepcidin resistance. This allows excessive absorption
of iron from the diet and development of iron overload. Clinical
manifestations of HH may include liver disease (e.g., hepatic
cirrhosis and hepatocellular carcinoma), diabetes, and heart
failure. Currently, the only treatment for HH is regular
phlebotomy, which is very burdensome for the patients. Iron-loading
anemias are hereditary anemias with ineffective erythropoiesis such
as .beta.-thalassemia, which are accompanied by severe iron
overload. Complications from iron overload are the main cause of
morbidity and mortality for these patients. Hepcidin deficiency is
the main cause of iron overload in non-transfused patients, and
contributes to iron overload in transfused patients. The current
treatment for iron overload in these patients is iron chelation
which is very burdensome, sometimes ineffective, and accompanied by
frequent side effects.
[0006] Hepcidin has a number of limitations which restrict its use
as a drug, including a difficult synthesis process due in part to
aggregation and precipitation of the protein during folding, which
in turn leads to high cost of goods. What are needed in the art are
compounds having hepcidin activity and also possessing other
beneficial physical properties such as improved solubility,
stability, and/or potency, so that hepcidin-like biologics might be
produced affordably, and used to treat hepcidin-related diseases
and disorders such as, e.g., those described herein.
[0007] The present invention addresses such needs, providing novel
peptide analogues, including both peptide monomer analogues and
peptide dimer analogues, having hepcidin activity and also having
other beneficial properties making the peptides of the present
invention suitable alternatives to hepcidin.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention generally relates to peptide
analogues, including both monomer and dimers, exhibiting hepcidin
activity and methods of using the same.
[0009] In one embodiment, the present invention includes a hepcidin
analogue comprising a polypeptide sequence of Formula (I):
X--Y (I)
or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is a peptide comprising the sequence Xa:
TABLE-US-00001 (Xa) (SEQ ID NO: 1)
X1-Thr-His-X4-Pro-X6-X7-X8-Phe-X10
[0010] wherein [0011] X1 is Asp, isoGlu or Ida; [0012] X4 is Phe,
Phe(4-F), Phe(4-CN), 4-BIP, Phe(4-OCH.sub.3), Tyr,
Phe(2,3-(OCH.sub.3).sub.2), Phe(2,3-Cl.sub.2), or Dpa; [0013] X6 is
Cys or Pen; [0014] X7 is any amino acid; [0015] X8 is Ile, Leu,
Val, nLeu, Lys or Arg; and [0016] X10 is Lys, Glu or absent; and Y
is absent or present; wherein if Y is present, Y is a peptide
comprising the sequence Ya:
TABLE-US-00002 [0016] (Ya) (SEQ ID NO: 2) Y1-Y2-Y3-Y4-Y5-Y6-Y7
[0017] wherein [0018] Y1 is amino acid [0019] Y2 is any amino acid
[0020] Y3 is any amino acid [0021] Y4 is any amino acid [0022] Y5
is any amino acid; [0023] Y6 is Cys or Pen; and [0024] Y7 is Lys or
absent; and wherein the hepcidin analogue comprises a conjugated
half-life extension moiety, wherein the half-life extension moiety
is optionally conjugated via a linker moiety. The peptides Xa and
Ya may be linked via a peptide bond to form the polypeptide of
Formula (I).
[0025] In particular embodiments of any of the hepcidin analogues,
the hepcidin analogue comprises one of more of the following: X1 is
Asp; X4 is Phe or Dpa; X7 is Ile, Leu, Val, nLeu, or Lys; X7 is Ile
or Lys; X8 is Lys or Arg; Y1 is Pro or hPro; Y1 is Pro; Y2 is Arg
or Lys; Y2 is Arg; Y3 is Ser; Y4 is Lys, Arg or His; Y4 is Lys; or
Y5 is Gly or Sar. In certain embodiments: X1 is Asp; X4 is Phe or
Dpa; X7 is Ile, Leu, Val, nLeu, or Lys; X8 is Lys or Arg; Y1 is Pro
or hPro; Y2 is Arg or Lys; Y3 is Ser; Y4 is Lys, Arg or His; and Y5
is Gly or Sar. In certain embodiments, X1 is Asp; X4 is Phe or Dpa;
X7 is Ile or Lys; X8 is Lys or Arg; Y1 is Pro; Y2 is Arg; Y3 is
Ser; and Y4 is Lys; and Y5 is Gly or Sar.
[0026] In a related embodiments, the present invention includes a
hepcidin analogue comprising a polypeptide sequence of Formula
(V):
X--Y (V)
or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is a peptide sequence having the formula Xv:
TABLE-US-00003 (Xv) (SEQ ID NO: 3)
Asp-Thr-His-X4-Pro-X6-X7-X8-Phe-X10
[0027] wherein [0028] X4 is Phe or Dpa; [0029] X6 is Cys or Pen;
[0030] X7 is Ile or Lys; [0031] X8 is Lys or Arg; and [0032] X10 is
Lys, Glu or absent; and Y is absent or present; wherein if Y is
present, Y is a peptide sequence having the formula Yv:
TABLE-US-00004 [0032] (Yv) (SEQ ID NO: 4)
Pro-Arg-Ser-Lys-Y5-Y6-Y7
[0033] wherein [0034] Y5 is Gly or Sar; [0035] Y6 is Cys or Pen;
and [0036] Y7 is Lys or absent; and wherein the hepcidin analogue
comprises a conjugated half-life extension moiety, wherein the
half-life extension moiety is optionally conjugated via a linker
moiety. The peptides Xv and Yv may be linked via a peptide bond to
form the polypeptide of Formula (V).
[0037] In certain embodiment, any of the hepcidin analogues
comprises a structure of Formula II:
R.sup.1--X-L-Y--R.sup.2 (II)
or a pharmaceutically acceptable salt or solvate thereof, wherein:
R.sup.1 is hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl
C1-C6 alkyl, or a C1-C20 alkanoyl, and including PEGylated versions
alone or as spacers of any of the foregoing; R.sup.2 is OH or
NH.sub.2; X is a peptide sequence having the formula Xa (SEQ ID
NO:1) or Xv (SEQ ID NO:3); L is absent, a bond, or a linker moiety;
and Y is absent or present; provided that if Y is present, Y is a
peptide having the formula Ya (SEQ ID NO:2) or Yv (SEQ ID
NO:4).
[0038] In particular embodiments of any of the hepcidin analogues,
X or Y further comprise one to three additional amino acids at the
N-terminus or C-terminus.
[0039] In certain embodiments, Y is present. In particular
embodiments, the hepcidin analogue comprises a disulfide bond
between X6 and Y6.
[0040] In particular embodiments, L is a bond.
[0041] In various embodiments, a hepcidin analogue comprises a
half-life extension moiety conjugated to a Lys at X8 or X10.
[0042] In certain embodiments, a hepcidin analogue comprises one of
the following structures:
##STR00001##
[0043] In particular embodiments of the hepcidin analogues, Y is
absent. In certain embodiments, X10 is absent.
[0044] In a related embodiment, the present invention includes a
dimer comprising two hepcidin analogues of the present invention,
e.g., two polypeptide sequence of Formula I or Formula V, or two
structures of Formula II, dimerized via a linker moiety. In
particular embodiments, the linker moiety is bound to the
C-terminus of each hepcidin analogue. In certain embodiments, the
half-life extension moiety is conjugated to the linker moiety.
[0045] In particular embodiments of any of the hepcidin analogues
or dimers of the present invention, the linker moiety is selected
from IsoGlu, Dapa, PEGn where n=1 to 25, PEG11 (40 atoms), OEG,
IsoGlu-Ahx, IsoGlu-OEG-OEG, IsoGlu-PEG5, IsoGlu-PEGn where n=1 to
25 .beta.Ala-PEG2, and .beta.Ala-PEG11(40 atoms). In certain
embodiments, more than one linker moiety is conjugated to a peptide
of the hepcidin analogue or dimer.
[0046] In particular embodiments of any of the hepcidin analogues
or dimers of the present invention, the half-life extension moiety
is selected from C12 (Lauric acid), C14 (Mysteric acid), C16
(Palmitic acid), C18 (Stearic acid), C20, C12 diacid, C14 diacid,
C16 diacid, C18 diacid, C20 diacid, biotin, and isovaleric acid. In
certain embodiments, the half-life extension moiety is attached to
a linker moiety that is attached to the peptide. In certain
embodiments, the half-life extension moiety increases the molecular
weight of the hepcidin analogue by about 50 D to about 2 KD. In
various embodiments, the half-life extension moiety increases serum
half-life, enhances solubility, and/or improves bioavailability of
the hepcidin analogue.
[0047] In certain embodiments, a peptide analogue or dimer of the
present invention comprises an isovaleric acid moiety conjugated to
an N-terminal Asp residue.
[0048] In certain embodiments, a peptide analogue of the present
invention comprises an amidated C-terminal residue.
[0049] In certain embodiments, a hepcidin analogue or dimer of the
present invention comprises the sequence:
Asp-Thr-His-Phe-Pro-Cys-Ile-Lys-Phe-Glu-Pro-Arg-Ser-Lys-Gly-Cys-Lys
(SEQ ID NO:6), or comprises a sequence having at least 80%, at
least 90%, or at least 94% identity to this sequence.
[0050] In certain embodiments, a hepcidin analogue or dimer of the
present invention comprises the sequence:
Asp-Thr-His-Phe-Pro-Cys-Ile-Lys-Phe-Lys-Pro-Arg-Ser-Lys-Gly-Cys-Lys
(SEQ ID NO:5), or comprises a sequence having at least 80%, at
least 90%, or at least 94% identity to this sequence.
[0051] In a related embodiment, the present invention includes a
polynucleotide that encodes a peptide of a hepcidin analogue or
dimer (or monomer subunit of a dimer) of the present invention.
[0052] In a further related embodiment, the present invention
includes a vector comprising a polynucleotide of the invention.
[0053] In another embodiment, the present invention includes a
pharmaceutical composition, comprising a hepcidin analogue, dimer,
polynucleotide, or vector of the present invention, and a
pharmaceutically acceptable carrier, excipient or vehicle.
[0054] In another embodiments, the present invention provides a
method of binding a ferroportin or inducing ferroportin
internalization and degradation, comprising contacting the
ferroportin with at least one hepcidin analogue, dimer or
composition of the present invention.
[0055] In a further embodiment, the present invention includes a
method for treating a disease of iron metabolism in a subject in
need thereof comprising providing to the subject an effective
amount of a pharmaceutical composition of the present invention. In
certain embodiments, the pharmaceutical composition is provided to
the subject by an oral, intravenous, peritoneal, intradermal,
subcutaneous, intramuscular, intrathecal, inhalation, vaporization,
nebulization, sublingual, buccal, parenteral, rectal, vaginal, or
topical route of administration. In certain embodiments, the
pharmaceutical composition is provided to the subject by an oral or
subcutaneous route of administration. In certain embodiments, the
disease of iron metabolism is an iron overload disease. In certain
embodiments, the pharmaceutical composition is provided to the
subject at most or about twice daily, at most or about once daily,
at most or about once every two days, at most or about once a week,
or at most or about once a month.
[0056] In particular embodiments, the hepcidin analogue is provided
to the subject at a dosage of about 1 mg to about 100 mg or about 1
mg to about 5 mg.
[0057] In another embodiment, the present invention provides a
device comprising pharmaceutical composition of the present
invention, for delivery of a hepcidin analogue or dimer of the
invention to a subject, optionally orally or subcutaneously.
[0058] In yet another embodiment, the present invention includes a
kit comprising a pharmaceutical composition of the invention,
packaged with a reagent, a device, or an instructional material, or
a combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIGS. 1A and 1B are graphs showing serum iron levels at the
indicated times following administration to mice of 1 uM Compound 1
either intravenously (FIG. 1A) or subcutaneously (FIG. 1B).
[0060] FIG. 2 is a graph showing serum iron levels at 30 hours or
36 hours following subcutaneous administration to healthy mice of 1
umol/kg of the indicated hepcidin analogues.
DETAILED DESCRIPTION OF THE INVENTION
[0061] The present invention relates generally to hepcidin analogue
peptides and methods of making and using the same. In certain
embodiments, the hepcidin analogues exhibit one or more hepcidin
activity. In certain embodiments, the present invention relates to
hepcidin peptide analogues comprising one or more peptide subunit
that forms a cyclized structures through an intramolecular bond,
e.g., an intramolecular disulfide bond. In particular embodiments,
the cyclized structure has increased potency and selectivity as
compared to non-cyclized hepcidin peptides and analogies thereof.
In particular embodiments, hepcidin analogue peptides of the
present invention exhibit increased half-lives, e.g., when
delivered orally, as compared to hepcidin or previous hepcidin
analogues.
Definitions and Nomenclature
[0062] Unless otherwise defined herein, scientific and technical
terms used in this application shall have the meanings that are
commonly understood by those of ordinary skill in the art.
Generally, nomenclature used in connection with, and techniques of,
chemistry, molecular biology, cell and cancer biology, immunology,
microbiology, pharmacology, and protein and nucleic acid chemistry,
described herein, are those well-known and commonly used in the
art.
[0063] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0064] Throughout this specification, the word "comprise" or
variations such as "comprises" or "comprising" will be understood
to imply the inclusion of a stated integer (or components) or group
of integers (or components), but not the exclusion of any other
integer (or components) or group of integers (or components).
[0065] The singular forms "a," "an," and "the" include the plurals
unless the context clearly dictates otherwise.
[0066] The term "including" is used to mean "including but not
limited to." "Including" and "including but not limited to" are
used interchangeably.
[0067] The terms "patient," "subject," and "individual" may be used
interchangeably and refer to either a human or a non-human animal.
These terms include mammals such as humans, primates, livestock
animals (e.g., bovines, porcines), companion animals (e.g.,
canines, felines) and rodents (e.g., mice and rats). The term
"mammal" refers to any mammalian species such as a human, mouse,
rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the
like.
[0068] The term "peptide," as used herein, refers broadly to a
sequence of two or more amino acids joined together by peptide
bonds. It should be understood that this term does not connote a
specific length of a polymer of amino acids, nor is it intended to
imply or distinguish whether the polypeptide is produced using
recombinant techniques, chemical or enzymatic synthesis, or is
naturally occurring.
[0069] The term "peptide analogue," as used herein, refers broadly
to peptide monomers and peptide dimers comprising one or more
structural features and/or functional activities in common with
hepcidin, or a functional region thereof. In certain embodiments, a
peptide analogue includes peptides sharing substantial amino acid
sequence identity with hepcidin, e.g., peptides that comprise one
or more amino acid insertions, deletions, or substitutions as
compared to a wild-type hepcidin, e.g., human hepcidin, amino acid
sequence. In certain embodiments, a peptide analogue comprises one
or more additional modification, such as, e.g., conjugation to
another compound. Encompassed by the term "peptide analogue" is any
peptide monomer or peptide dimer of the present invention. In
certain instances, a "peptide analog" may also or alternatively be
referred to herein as a "hepcidin analogue," "hepcidin peptide
analogue," or a "hepcidin analogue peptide."
[0070] The recitations "sequence identity", "percent identity",
"percent homology", or, for example, comprising a "sequence 50%
identical to," as used herein, refer to the extent that sequences
are identical on a nucleotide-by-nucleotide basis or an amino
acid-by-amino acid basis over a window of comparison. Thus, a
"percentage of sequence identity" may be calculated by comparing
two optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical nucleic
acid base (e.g., A, T, C, G, I) or the identical amino acid residue
(e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys,
Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences
to yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the window of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity.
[0071] Calculations of sequence similarity or sequence identity
between sequences (the terms are used interchangeably herein) can
be performed as follows. To determine the percent identity of two
amino acid sequences, or of two nucleic acid sequences, the
sequences can be aligned for optimal comparison purposes (e.g.,
gaps can be introduced in one or both of a first and a second amino
acid or nucleic acid sequence for optimal alignment and
non-homologous sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a reference
sequence aligned for comparison purposes is at least 30%,
preferably at least 40%, more preferably at least 50%, 60%, and
even more preferably at least 70%, 80%, 90%, 100% of the length of
the reference sequence. The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position.
[0072] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences.
[0073] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In some embodiments, the percent identity
between two amino acid sequences is determined using the Needleman
and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has
been incorporated into the GAP program in the GCG software package,
using either a Blossum 62 matrix or a PAM250 matrix, and a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,
3, 4, 5, or 6. In yet another preferred embodiment, the percent
identity between two nucleotide sequences is determined using the
GAP program in the GCG software package, using an NWSgapdna.CMP
matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters
includes a Blossum 62 scoring matrix with a gap penalty of 12, a
gap extend penalty of 4, and a frameshift gap penalty of 5. The
percent identity between two amino acid or nucleotide sequences can
also be determined using the algorithm of E. Meyers and W. Miller
(1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN
program (version 2.0), using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4.
[0074] The peptide sequences described herein can be used as a
"query sequence" to perform a search against public databases to,
for example, identify other family members or related sequences.
Such searches can be performed using the NBLAST and XBLAST programs
(version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215:
403-10). BLAST nucleotide searches can be performed with the NBLAST
program, score=100, wordlength=12 to obtain nucleotide sequences
homologous to nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) can be
used.
[0075] The term "conservative substitution" as used herein 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. In some embodiments of the invention, 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
oxidized. In some embodiments, one or more Trp residues are
substituted with Phe, or one or more Phe residues are substituted
with Trp, while in some embodiments, one or more Pro residues are
substituted with L-isonipecotic acid (NPC), or one or more NPC
residues are substituted with Pro. 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. In some
embodiments, another conservative substitution is the substitution
of one or more Pro residues with bhPro or Leu or D-isonipecotic
acid (D-NPC).
[0076] For further information concerning phenotypically silent
substitutions in peptides and proteins, see, for example, Bowie et.
al. Science 247, 1306-1310, 1990. In the scheme below, 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.
TABLE-US-00005 I II III IV V A N H M F S D R L Y T E K I W P Q V G
C
[0077] In the scheme below, conservative substitutions of amino
acids are grouped by physicochemical properties. VI: neutral or
hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic.
TABLE-US-00006 VI VII VIII IX X A E H M F L D R S Y I K T W P C G N
V Q
[0078] The term "amino acid" or "any amino acid" as used here
refers to any and all amino acids, including naturally occurring
amino acids (e.g., a-amino acids), unnatural amino acids, modified
amino acids, and non-natural amino acids. It includes both D- and
L-amino acids. Natural amino acids include those found in nature,
such as, e.g., the 23 amino acids that combine into peptide chains
to form the building-blocks of a vast array of proteins. These are
primarily L stereoisomers, although a few D-amino acids occur in
bacterial envelopes and some antibiotics. The 20 "standard,"
natural amino acids are listed in the above tables. The
"non-standard," natural amino acids are pyrrolysine (found in
methanogenic organisms and other eukaryotes), selenocysteine
(present in many noneukaryotes as well as most eukaryotes), and
N-formylmethionine (encoded by the start codon AUG in bacteria,
mitochondria and chloroplasts). "Unnatural" or "non-natural" amino
acids are non-proteinogenic amino acids (i.e., those not naturally
encoded or found in the genetic code) that either occur naturally
or are chemically synthesized. Over 140 natural amino acids are
known and thousands of more combinations are possible. Examples of
"unnatural" amino acids include .beta.-amino acids (.beta..sup.3
and .beta..sup.2), homo-amino acids, proline and pyruvic acid
derivatives, 3-substituted alanine derivatives, glycine
derivatives, ring-substituted phenylalanine and tyrosine
derivatives, linear core amino acids, diamino acids, D-amino acids,
and N-methyl amino acids. Unnatural or non-natural amino acids also
include modified amino acids. "Modified" amino acids include amino
acids (e.g., natural amino acids) that have been chemically
modified to include a group, groups, or chemical moiety not
naturally present on the amino acid.
[0079] As is clear to the skilled artisan, the peptide sequences
disclosed herein are shown proceeding from left to right, with the
left end of the sequence being the N-terminus of the peptide and
the right end of the sequence being the C-terminus of the peptide.
Among sequences disclosed herein are sequences incorporating a
"Hy-" moiety at the amino terminus (N-terminus) of the sequence,
and either an "--OH" moiety or an "--NH.sub.2" moiety at the
carboxy terminus (C-terminus) of the sequence. In such cases, and
unless otherwise indicated, a "Hy-" moiety at the N-terminus of the
sequence in question indicates a hydrogen atom, corresponding to
the presence of a free primary or secondary amino group at the
N-terminus, while an "--OH" or an "--NH.sub.2" moiety at the
C-terminus of the sequence indicates a hydroxy group or an amino
group, corresponding to the presence of an amido (CONH.sub.2) group
at the C-terminus, respectively. In each sequence of the invention,
a C-terminal "--OH" moiety may be substituted for a C-terminal
"--NH.sub.2" moiety, and vice-versa. It is further understood that
the moiety at the amino terminus or carboxy terminus may be a bond,
e.g., a covalent bond, particularly in situations where the amino
terminus or carboxy terminus is bound to a linker or to another
chemical moiety, e.g., a PEG moiety.
[0080] The term "NH.sub.2," as used herein, refers to the free
amino group present at the amino terminus of a polypeptide. The
term "OH," as used herein, refers to the free carboxy group present
at the carboxy terminus of a peptide. Further, the term "Ac," as
used herein, refers to Acetyl protection through acylation of the
C- or N-terminus of a polypeptide.
[0081] The term "carboxy," as used herein, refers to
--CO.sub.2H.
[0082] For the most part, the names of naturally occurring and
non-naturally occurring aminoacyl residues used herein follow the
naming conventions suggested by the IUPAC Commission on the
Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on
Biochemical Nomenclature as set out in "Nomenclature of
.alpha.-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2),
(1975). To the extent that the names and abbreviations of amino
acids and aminoacyl residues employed in this specification and
appended claims differ from those suggestions, they will be made
clear to the reader. Some abbreviations useful in describing the
invention are defined below in the following Table 2.
TABLE-US-00007 TABLE 2 Abbreviations of Non-Natural Amino Acids and
Chemical Moieties Abbreviation Definition DIG Diglycolic acid Dapa
Diaminopropionic acid Daba Diaminobutyric acid Pen Penicillamine
Sarc or Sar Sarcosine Cit Citroline Cav Cavanine NMe-Arg
N-Methyl-Arginine NMe-Trp N-Methyl-Tryptophan NMe-Phe
N-Methyl-Phenylalanine Ac- Acetyl 2-Nal 2-Napthylalanine 1-Nal
1-Napthylalanine Bip Biphenylalanine .beta.Ala beta-Alanine Aib
2-aminoisobutyric acid Azt azetidine-2-carboxylic acid Tic
(3S)-1,2,3,4-Tetrahydroisoquinoline- hydroxy-3-carboxylic acid
Phe(OMe) Tyrosine (4-Methyl) N-MeLys N-Methyl-Lysine N-MeLys(Ac)
N-e-Acetyl-D-lysine Dpa .beta.,.beta. diphenylalanine NH.sub.2 Free
Amine CONH.sub.2 Amide COOH Acid Phe(4-F) 4-Fluoro-Phenylalanine
PEG3
NH.sub.2CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.3CH.sub.2CH.sub.2CO.s-
ub.2H m-PEG3
CH.sub.3OCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.2CH.sub.2CH.sub.2C-
O.sub.2H m-PEG4
CH.sub.3OCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.3CH.sub.2CH.sub.2C-
O.sub.2H m-PEG8
CH.sub.3OCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.7CH.sub.2CH.sub.2C-
O.sub.2H PEG11 O-(2-aminoethyl)-O'-(2-carboxyethyl)-
undecaethyleneglycol
NH.sub.2CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.11CH.sub.2CH.sub.2CO.sub.-
2H PEG13 Bifunctional PEG linker with 13 PolyEthylene Glycol units
PEG25 Bifunctional PEG linker with 25 PolyEthylene Glycol units
PEG1K Bifunctional PEG linker with PolyEthylene Glycol Mol wt of
1000 Da PEG2K Bifunctional PEG linker with PolyEthylene Glycol Mol
wt of 2000 Da PEG3.4K Bifunctional PEG linker with PolyEthylene
Glycol Mol wt of 3400 Da PEG5K Bifunctional PEG linker with
PolyEthylene Glycol Mol wt of 5000 Da IDA or Ida Iminodiacetic acid
IDA-Palm (Palmityl)-Iminodiacetic acid hPhe homoPhenylalanine Ahx
Aminohexanoic acid DIG-OH Glycolic monoacid Triazine Amino propyl
Triazine di-acid Boc-Triazine Boc-Triazine di-acid Trifluorobutyric
acid 4,4,4-Trifluorobutyric acid 2-Methylltrifluorobutyric
2-methyl-4,4,4-Butyric acid acid Trifluorpentanoic acid
5,5,5-Trifluoropentanoic acid 1,4-Phenylenediacetic
para-Phenylenediacetic acid acid 1,3-Phenylenediacetic
meta-Phenylenediacetic acid acid DTT Dithiothreotol Nle Norleucine
.beta.hTrp or bhTrp .beta.-homoTryptophane .beta.hPhe or bhPhe
.beta.-homophenylalanine Phe(4-CF.sub.3)
4-TrifluoromethylPhenylalanine .beta.Glu or bGlu .beta.-Glutamic
acid .beta.Glu or bhGlu .beta.-homoglutamic acid 2-2-Indane
2-Aminoindane-2-carboxylic acid 1-1-Indane
1-Aminoindane-1-carboxylic acid hCha homocyclohexylalanine
Cyclobutyl Cyclobutylalanine hLeu Homoleucine Gla
.gamma.-Carboxy-glutamic acid Aep 3-(2-aminoethoxy)propanoic acid
Aea (2-aminoethoxy)acetic acid IsoGlu-octanoic acid
octanoyl-.gamma.-Glu K-octanoic acid octanoyl-.epsilon.-Lys
Dapa(Palm) Hexadecanoyl-.beta.-Diaminopropionic acid IsoGlu-Palm
hexadecanoyl-.gamma.-Glu C-StBu S-tert-butylthio-cysteine C-tBu
S-tert-butyl-cysteine Dapa(AcBr)
NY-(bromoacetyl)-2,3-diaminopropionic acid Tle tert-Leucine Phg
phenylglycine Oic octahydroindole-2-carboxylic acid Chg
.alpha.-cyclohexylglycine GP-(Hyp) Gly-Pro-HydroxyPro Inp
isonipecotic acid Amc 4-(aminomethyl)cyclohexane carboxylic acid
Betaine (CH.sub.3).sub.3NCH.sub.2CH.sub.2CO2H NPC Isonipecotic acid
D-NPC D-Isonipecotic acid
[0083] Throughout the present specification, unless naturally
occurring 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.). In the case of less common
or non-naturally occurring amino acids, unless they are referred to
by their full name (e.g. sarcosine, ornithine, etc.), frequently
employed three- or four-character codes are employed for residues
thereof, including, Sar or Sarc (sarcosine, i.e. N-methylglycine),
Aib (.alpha.-aminoisobutyric acid), Daba (2,4-diaminobutanoic
acid), Dapa (2,3-diaminopropanoic acid), .gamma.-Glu
(.gamma.-glutamic acid), pGlu (pyroglutamic acid), Gaba
(.gamma.-aminobutanoic acid), .beta.-Pro (pyrrolidine-3-carboxylic
acid), 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (4-aminobutyric
acid), bhPro (.beta.-homo-proline), bhPhe
(.beta.-homo-L-phenylalanine), bhAsp (.beta.-homo-aspartic acid]),
Dpa (.beta.,.beta. diphenylalanine), Ida (Iminodiacetic acid), hCys
(homocysteine), bhDpa
(.beta.-homo-.beta.,.beta.-diphenylalanine).
[0084] Furthermore, R.sup.1 can in all sequences be substituted
with isovaleric acids or equivalent. In some embodiments, wherein a
peptide of the present invention is conjugated to an acidic
compound such as, e.g., isovaleric acid, isobutyric acid, valeric
acid, and the like, the presence of such a conjugation is
referenced in the acid form. So, for example, but not to be limited
in any way, instead of indicating a conjugation of isovaleric acid
to a peptide by referencing isovaleroyl, in some embodiments, the
present application may reference such a conjugation as isovaleric
acid.
[0085] The term "L-amino acid," as used herein, refers to the "L"
isomeric form of a peptide, and conversely the term "D-amino acid"
refers to the "D" isomeric form of a peptide. In certain
embodiments, the amino acid residues described herein are in the
"L" isomeric form, however, residues in the "D" isomeric form can
be substituted for any L-amino acid residue, as long as the desired
functional is retained by the peptide.
[0086] Unless otherwise indicated, reference is made to the
L-isomeric forms of the natural and unnatural amino acids in
question possessing a chiral center. Where appropriate, the
D-isomeric form of an amino acid is indicated in the conventional
manner by the prefix "D" before the conventional three-letter code
(e.g. Dasp, (D)Asp or D-Asp; Dphe, (D)Phe or D-Phe).
[0087] The term "DRP," as used herein, refers to disulfide rich
peptides.
[0088] The term "dimer," as used herein, refers broadly to a
peptide comprising two or more monomer subunits. Certain dimers
comprise two DRPs. Dimers of the present invention include
homodimers and heterodimers. A monomer subunit of a dimer may be
linked at its C- or N-terminus, or it may be linked via internal
amino acid residues. Each monomer subunit of a dimer may be linked
through the same site, or each may be linked through a different
site (e.g., C-terminus, N-terminus, or internal site).
[0089] As used herein, in the context of certain peptide sequences
disclosed herein, parentheticals, e.g., (______) represent side
chain conjugations and brackets, e.g., [______], represent
unnatural amino acid substitutions or amino acids and conjugated
side chains. Generally, where a linker is shown at the N-terminus
of a peptide sequence, it indicates that the peptide is dimerized
with another peptide, wherein the linker is attached to the
N-terminus of the two peptides. Generally, where a linker is shown
at the C-terminus of a peptide sequence or structure, it indicates
that the peptide is dimerized with another peptide, wherein the
linker is attached to the C-terminus of the two peptides.
[0090] The term "isostere replacement" or "isostere substitution"
are used interchangeably herein to refer to any amino acid or other
analog moiety having chemical and/or structural properties similar
to a specified amino acid. In certain embodiments, an isostere
replacement is a conservative substitution with a natural or
unnatural amino acid.
[0091] The term "cyclized," as used herein, refers to a reaction in
which one part of a polypeptide molecule becomes linked to another
part of the polypeptide molecule to form a closed ring, such as by
forming a disulfide bridge or other similar bond.
[0092] The term "subunit," as used herein, refers to one of a pair
of polypeptide monomers that are joined to form a dimer peptide
composition.
[0093] The term "linker moiety," as used herein, refers broadly to
a chemical structure that is capable of linking or joining together
two peptide monomer subunits to form a dimer.
[0094] The term "solvate" in the context of the present invention
refers to a complex of defined stoichiometry formed between a
solute (e.g., a hepcidin analogue 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.
When the solvent in question is water, such a solvate is normally
referred to as a hydrate.
[0095] As used herein, a "disease of iron metabolism" includes
diseases where aberrant iron metabolism directly causes the
disease, or where iron blood levels are dysregulated causing
disease, or where iron dysregulation is a consequence of another
disease, or where diseases can be treated by modulating iron
levels, and the like. More specifically, a disease of iron
metabolism according to this disclosure includes iron overload
diseases, iron deficiency disorders, disorders of iron
biodistribution, other disorders of iron metabolism and other
disorders potentially related to iron metabolism, etc. Diseases of
iron metabolism include hemochromatosis, HFE mutation
hemochromatosis, ferroportin mutation hemochromatosis, transferrin
receptor 2 mutation hemochromatosis, hemojuvelin mutation
hemochromatosis, hepcidin mutation hemochromatosis, juvenile
hemochromatosis, neonatal hemochromatosis, hepcidin deficiency,
transfusional iron overload, thalassemia, thalassemia intermedia,
alpha thalassemia, sideroblastic anemia, porphyria, porphyria
cutanea tarda, African iron overload, hyperferritinemia,
ceruloplasmin deficiency, atransferrinemia, congenital
dyserythropoietic anemia, anemia of chronic disease, anemia of
inflammation, anemia of infection, hypochromic microcytic anemia,
sickle cell disease, polycythemia vera (primary and secondary),
myelodysplasia, pyruvate kinase deficiency, iron-deficiency anemia,
iron-refractory iron deficiency anemia, anemia of chronic kidney
disease, erythropoietin resistance, iron deficiency of obesity,
other anemias, benign or malignant tumors that overproduce hepcidin
or induce its overproduction, conditions with hepcidin excess,
Friedreich ataxia, gracile syndrome, Hallervorden-Spatz disease,
Wilson's disease, pulmonary hemosiderosis, hepatocellular
carcinoma, cancer, hepatitis, cirrhosis of liver, pica, chronic
renal failure, insulin resistance, diabetes, atherosclerosis,
neurodegenerative disorders, multiple sclerosis, Parkinson's
disease, Huntington's disease, and Alzheimer's disease.
[0096] In some embodiments, the disease and disorders are related
to iron overload diseases such as iron hemochromatosis, HFE
mutation hemochromatosis, ferroportin mutation hemochromatosis,
transferrin receptor 2 mutation hemochromatosis, hemojuvelin
mutation hemochromatosis, hepcidin mutation hemochromatosis,
juvenile hemochromatosis, neonatal hemochromatosis, hepcidin
deficiency, transfusional iron overload, thalassemia, thalassemia
intermedia, alpha thalassemia, sickle cell disease, polycythemia
vera (primary and secondary), mylodysplasia, and pyruvate kinase
deficiency.
[0097] In some embodiments, the hepcidin analogues of the invention
are used to treat diseases and disorders that are not typically
identified as being iron related. For example, hepcidin is highly
expressed in the murine pancreas suggesting that diabetes (Type I
or Type II), insulin resistance, glucose intolerance and other
disorders may be ameliorated by treating underlying iron metabolism
disorders. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, which
is herein incorporated by reference. As such, peptides of the
invention may be used to treat these diseases and conditions. Those
skilled in the art are readily able to determine whether a given
disease can be treated with a peptide according to the present
invention using methods known in the art, including the assays of
WO 2004092405, which is herein incorporated by reference, and
assays which monitor hepcidin, hemojuvelin, or iron levels and
expression, which are known in the art such as those described in
U.S. Pat. No. 7,534,764, which is herein incorporated by
reference.
[0098] In certain embodiments of the present invention, the
diseases of iron metabolism are iron overload diseases, which
include hereditary hemochromatosis, iron-loading anemias, alcoholic
liver diseases and chronic hepatitis C.
[0099] The term "pharmaceutically acceptable salt," as used herein,
represents salts or zwitterionic forms of the peptides or compounds
of the present invention which are water or oil-soluble or
dispersible, which are suitable for treatment of diseases without
undue toxicity, irritation, and allergic response; which are
commensurate with a reasonable benefit/risk ratio, and which are
effective for their intended use. The salts can be prepared during
the final isolation and purification of the compounds or separately
by reacting an amino group with a suitable acid. Representative
acid addition salts include acetate, adipate, alginate, citrate,
aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,
camphorate, camphorsulfonate, digluconate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, formate, fumarate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate
(isethionate), lactate, maleate, mesitylenesulfonate,
methanesulfonate, naphthylenesulfonate, nicotinate,
2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,
3-phenylproprionate, picrate, pivalate, propionate, succinate,
tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate,
bicarbonate, para-toluenesulfonate, and undecanoate. Also, amino
groups in the compounds of the present invention can be quaternized
with methyl, ethyl, propyl, and butyl chlorides, bromides, and
iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl,
lauryl, myristyl, and steryl chlorides, bromides, and iodides; and
benzyl and phenethyl bromides. Examples of acids which can be
employed to form therapeutically acceptable addition salts include
inorganic acids such as hydrochloric, hydrobromic, sulfuric, and
phosphoric, and organic acids such as oxalic, maleic, succinic, and
citric. A pharmaceutically acceptable salt 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(R1)(R2)(R3)(R4)+, where R1, R2, R3 and R4
independently will typically designate hydrogen, optionally
substituted C1-6-alkyl or optionally substituted C2-6-alkenyl.
Examples of relevant C1-6-alkyl groups include methyl, ethyl,
1-propyl and 2-propyl groups. Examples of C2-6-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 "Encyclopaedia of
Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.),
Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci.
66: 2 (1977). Also, for a review on suitable salts, see Handbook of
Pharmaceutical Salts: Properties, Selection, and Use by Stahl and
Wermuth (Wiley-VCH, 2002). Other suitable base salts are formed
from bases which form non-toxic salts. Representative examples
include the aluminum, arginine, benzathine, calcium, choline,
diethylamine, diolamine, glycine, lysine, magnesium, meglumine,
olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts
of acids and bases may also be formed, e.g., hemisulphate and
hemicalcium salts.
[0100] The term "N(alpha)Methylation", as used herein, describes
the methylation of the alpha amine of an amino acid, also generally
termed as an N-methylation.
[0101] The term "sym methylation" or "Arg-Me-sym", as used herein,
describes the symmetrical methylation of the two nitrogens of the
guanidine group of arginine. Further, the term "asym methylation"
or "Arg-Me-asym" describes the methylation of a single nitrogen of
the guanidine group of arginine.
[0102] The term "acylating organic compounds", as used herein
refers to various compounds with carboxylic acid functionality that
are used to acylate the N-terminus of an amino acid subunit prior
to forming a C-terminal dimer. Non-limiting examples of acylating
organic compounds include cyclopropylacetic acid, 4-Fluorobenzoic
acid, 4-fluorophenylacetic acid, 3-Phenylpropionic acid, Succinic
acid, Glutaric acid, Cyclopentane carboxylic acid,
3,3,3-trifluoropropeonic acid, 3-Fluoromethylbutyric acid,
Tetrahedro-2H-Pyran-4-carboxylic acid.
[0103] The term "alkyl" includes a straight chain or branched,
noncyclic or cyclic, saturated aliphatic hydrocarbon containing
from 1 to 24 carbon atoms. Representative saturated straight chain
alkyls include, but are not limited to, methyl, ethyl, n-propyl,
n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched
alkyls include, without limitation, isopropyl, sec-butyl, isobutyl,
tert-butyl, isopentyl, and the like. Representative saturated
cyclic alkyls include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and the like, while
unsaturated cyclic alkyls include, without limitation,
cyclopentenyl, cyclohexenyl, and the like.
[0104] As used herein, a "therapeutically effective amount" of the
peptide agonists of the invention is meant to describe a sufficient
amount of the peptide agonist to treat an hepcidin-related disease,
including but not limited to any of the diseases and disorders
described herein (for example, a disease of iron metabolism). In
particular embodiments, the therapeutically effective amount will
achieve a desired benefit/risk ratio applicable to any medical
treatment.
[0105] Peptide Analogues of Hepcidin
[0106] The present invention provides peptide analogues of
hepcidin, which may be monomers or dimers (collectively "hepcidin
analogues").
[0107] In some embodiments, a hepcidin analogue of the present
invention binds ferroportin, e.g., human ferroportin. In certain
embodiments, hepcidin analogues of the present invention
specifically bind human ferroportin. As used herein, "specifically
binds" refers to a specific binding agent's preferential
interaction with a given ligand over other agents in a sample. For
example, a specific binding agent that specifically binds a given
ligand, binds the given ligand, under suitable conditions, in an
amount or a degree that is observable over that of any nonspecific
interaction with other components in the sample. Suitable
conditions are those that allow interaction between a given
specific binding agent and a given ligand. These conditions include
pH, temperature, concentration, solvent, time of incubation, and
the like, and may differ among given specific binding agent and
ligand pairs, but may be readily determined by those skilled in the
art. In some embodiments, a hepcidin analogue of the present
invention binds ferroportin with greater specificity than a
hepcidin reference compound (e.g., any one of the hepcidin
reference compounds provided herein). In some embodiments, a
hepcidin analogue of the present invention exhibits ferroportin
specificity that is at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, 1000%, or
10,000% higher than a hepcidin reference compound (e.g., any one of
the hepcidin reference compounds provided herein. In some
embodiments, a hepcidin analogue of the present invention exhibits
ferroportin specificity that is at least about 5 fold, or at least
about 10, 20, 50, or 100 fold higher than a hepcidin reference
compound (e.g., any one of the hepcidin reference compounds
provided herein.
[0108] In certain embodiments, a hepcidin analogue of the present
invention exhibits a hepcidin activity. In some embodiments, the
activity is an in vitro or an in vivo activity, e.g., an in vivo or
an in vitro activity described herein. In some embodiments, a
hepcidin analogue of the present invention exhibits at least about
50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99% of
the activity exhibited by a hepcidin reference compound (e.g., any
one of the hepcidin reference compounds provided herein.
[0109] In some embodiments, a hepcidin analogue of the present
invention exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%,
97%, 98%, 99%, or greater than 99% of the ferroportin binding
ability that is exhibited by a reference hepcidin. In some
embodiments, a hepcidin analogue of the present invention has a
lower IC.sub.50 (i.e., higher binding affinity) for binding to
ferroportin, (e.g., human ferroportin) compared to a reference
hepcidin. In some embodiments, a hepcidin analogue the present
invention has an IC.sub.50 in a ferroportin competitive binding
assay which is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than a
reference hepcidin.
[0110] In certain embodiments, a hepcidin analogue of the present
invention exhibits increased hepcidin activity as compared to a
hepcidin reference peptide. In some embodiments, the activity is an
in vitro or an in vivo activity, e.g., an in vivo or an in vitro
activity described herein. In certain embodiments, the hepcidin
analogue of the present invention exhibits 1.5, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60,
70, 80, 90, 100, 120, 140, 160, 180, or 200-fold greater hepcidin
activity than a reference hepcidin. In certain embodiments, the
hepcidin analogue of the present invention exhibits at least about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or
greater than 99%, 100%, 200% 300%, 400%, 500%, 700%, or 1000%
greater activity than a reference hepcidin.
[0111] In some embodiments, a peptide analogue of the present
invention exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%,
97%, 98%, 99%, or greater than 99%, 100%, 200% 300%, 400%, 500%,
700%, or 1000% greater in vitro activity for inducing the
degradation of human ferroportin protein as that of a reference
hepcidin, wherein the activity is measured according to a method
described herein.
[0112] In some embodiments, a peptide or a peptide dimer of the
present invention exhibits at least about 50%, 60%, 70%, 80%, 90%,
95%, 97%, 98%, 99%, or greater than 99%, 100%, 200% 300%, 400%,
500%, 700%, or 1000% greater in vivo activity for inducing the
reduction of free plasma iron in an individual as does a reference
hepcidin, wherein the activity is measured according to a method
described herein.
[0113] In some embodiments, the activity is an in vitro or an in
vivo activity, e.g., an in vivo or an in vitro activity described
herein. In certain embodiments, a hepcidin analogue of the present
invention exhibits 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140,
160, 180, or 200-fold greater or at least about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or
1000% greater activity than a reference hepcidin, wherein the
activity is an in vitro activity for inducing the degradation of
ferroportin, e.g., as measured according to the Examples herein; or
wherein the activity is an in vivo activity for reducing free
plasma iron, e.g., as measured according to the Examples
herein.
[0114] In some embodiments, the hepcidin analogues of the present
invention mimic the hepcidin activity of Hep25, the bioactive human
25-amino acid form, are herein referred to as "mini-hepcidins". As
used herein, in certain embodiments, a compound (e.g., a hepcidin
analogue) having a "hepcidin activity" means that the compound has
the ability to lower plasma iron concentrations in subjects (e.g.
mice or humans), when administered thereto (e.g. parenterally
injected or orally administered), in a dose-dependent and
time-dependent manner. See e.g. as demonstrated in Rivera et al.
(2005), Blood 106:2196-9. In some embodiments, the peptides of the
present invention lower the plasma iron concentration in a subject
by at least about 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or
at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, or about 99%.
[0115] In some embodiments, the hepcidin analogues of the present
invention have in vitro activity as assayed by the ability to cause
the internalization and degradation of ferroportin in a
ferroportin-expressing cell line as taught in Nemeth et al. (2006)
Blood 107:328-33. In some embodiments, in vitro activity is
measured by the dose-dependent loss of fluorescence of cells
engineered to display ferroportin fused to green fluorescent
protein as in Nemeth et al. (2006) Blood 107:328-33. Aliquots of
cells are incubated for 24 hours with graded concentrations of a
reference preparation of Hep25 or a mini-hepcidin. As provided
herein, the EC.sub.50 values are provided as the concentration of a
given compound (e.g. a hepcidin analogue peptide or peptide dimer
of the present invention) that elicits 50% of the maximal loss of
fluorescence generated by a reference compound. The EC.sub.50 of
the Hep25 preparations in this assay range from 5 to 15 nM and in
certain embodiments, preferred hepcidin analogues of the present
invention have EC.sub.50 values in in vitro activity assays of
about 1,000 nM or less. In certain embodiments, a hepcidin analogue
of the present invention has an EC.sub.50 in an in vitro activity
assay (e.g., as described in Nemeth et al. (2006) Blood 107:328-33
or the Example herein) of less than about any one of 0.01, 0.05,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50,
60, 70, 80, 90, 100, 200 or 500 nM. In some embodiments, a hepcidin
analogue or biotherapeutic composition (e.g., any one of the
pharmaceutical compositions described herein) has an EC.sub.50
value of about 1 nM or less.
[0116] Other methods known in the art for calculating the hepcidin
activity and in vitro activity of the hepcidin analogues according
to the present invention may be used. For example, in certain
embodiments, the in vitro activity of the hepcidin analogues or the
reference peptides is measured by their ability to internalize
cellular ferroportin, which is determined by immunohistochemistry
or flow cytometry using antibodies which recognizes extracellular
epitopes of ferroportin. Alternatively, in certain embodiments, the
in vitro activity of the hepcidin analogues or the reference
peptides is measured by their dose-dependent ability to inhibit the
efflux of iron from ferroportin-expressing cells that are preloaded
with radioisotopes or stable isotopes of iron, as in Nemeth et al.
(2006) Blood 107:328-33.
[0117] In some embodiments, the hepcidin analogues of the present
invention exhibit increased stability (e.g., as measured by
half-life, rate of protein degradation) as compared to a reference
hepcidin. In certain embodiments, the stability of a hepcidin
analogue of the present invention is increased at least about 1.5,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200-fold
greater or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 200%, 300%, 400%, or 500% greater than a reference
hepcidin. In some embodiments, the stability is a stability that is
described herein. In some embodiments, the stability is a plasma
stability, e.g., as optionally measured according to the method
described herein. In some embodiments, the stability is stability
when delivered orally.
[0118] In particular embodiments, a hepcidin analogue of the
present invention exhibits a longer half-life than a reference
hepcidin. In particular embodiments, a hepcidin analogue of the
present invention has a half-life under a given set of conditions
(e.g., temperature, pH) of at least about 5 minutes, at least about
10 minutes, at least about 20 minutes, at least about 30 minutes,
at least about 45 minutes, at least about 1 hour, at least about 2
hour, at least about 3 hours, at least about 4 hours, at least
about 5 hours, at least about 6 hours, at least about 12 hours, at
least about 18 hours, at least about 1 day, at least about 2 days,
at least about 4 days, at least about 7 days, at least about 10
days, at least about two weeks, at least about three weeks, at
least about 1 month, at least about 2 months, at least about 3
months, or more, or any intervening half-life or range in between,
about 5 minutes, about 10 minutes, about 20 minutes, about 30
minutes, about 45 minutes, about 1 hour, about 2 hour, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours,
about 18 hours, about 1 day, about 2 days, about 4 days, about 7
days, about 10 days, about two weeks, about three weeks, about 1
month, about 2 months, about 3 months, or more, or any intervening
half-life or range in between. In some embodiments, the half-life
of a hepcidin analogue of the present invention is extended due to
its conjugation to one or more lipophilic substituent or half-life
extension moiety, e.g., any of the lipophilic substituents or
half-life extension moieties disclosed herein. In some embodiments,
the half-life of a hepcidin analogue of the present invention is
extended due to its conjugation to one or more polymeric moieties,
e.g., any of the polymeric moieties or half-life extension moieties
disclosed herein. In certain embodiments, a hepcidin analogue of
the present invention has a half-life as described above under the
given set of conditions wherein the temperature is about 25.degree.
C., about 4.degree. C., or about 37.degree. C., and the pH is a
physiological pH, or a pH about 7.4.
[0119] In certain embodiments, a hepcidin analogue of the present
invention, comprising a conjugated half-life extension moiety, has
an increased serum half-life following oral, intravenous or
subcutaneous administration as compared to the same analogue but
lacking the conjugated half-life extension moiety. In particular
embodiments, the serum half-life of a hepcidin analogue of the
present invention following any of oral, intravenous or
subcutaneous administration is at least 12 hours, at least 24
hours, at least 30 hours, at least 36 hours, at least 48 hours, at
least 72 hours or at least 168 h. In particular embodiments, it is
between 12 and 168 hours, between 24 and 168 hours, between 36 and
168 hours, or between 48 and 168 hours.
[0120] In certain embodiments, a hepcidin analogue of the present
invention, comprising a conjugated half-life extension moiety,
results in decreased concentration of serum iron following oral,
intravenous or subcutaneous administration to a subject. In
particular embodiments, the subject's serum iron concentration is
decreased to less than 10%, less than 20%, less than 25%, less than
30%, less than 40%, less than 50%, less than 60%, less than 70%,
less than 80%, or less than 90% of the serum iron concentration in
the absence of administration of the hepcidin analogue to the
subject. In particular embodiments, the decreased serum iron
concentration remains for a least 1 hour, at least 4 hours, at
least 10 hours, at least 12 hours, at least 24 hours, at least 36
hours, at least 48 hours, or at least 72 hours following
administration to the subject. In particular embodiments, it
remains for between 12 and 168 hours, between 24 and 168 hours,
between 36 and 168 hours, or between 48 and 168 hours. In one
embodiment, the serum iron concentration of the subject is reduced
to less than 20% at about 4 hours or about 10 hours following
administration to the subject, e.g., intravenously, orally, or
subcutaneously. In one embodiment, the serum iron concentration of
the subject is reduced to less than 50% or less than 60% for about
24 to about 30 hours following administration, e.g., intravenously,
orally, or subcutaneously.
[0121] In some embodiments, the half-life is measured in vitro
using any suitable method known in the art, e.g., in some
embodiments, the stability of a hepcidin analogue of the present
invention is determined by incubating the hepcidin analogue with
pre-warmed human serum (Sigma) at 37.degree. C. Samples are taken
at various time points, typically up to 24 hours, and the stability
of the sample is analyzed by separating the hepcidin analogue from
the serum proteins and then analyzing for the presence of the
hepcidin analogue of interest using LC-MS.
[0122] In some embodiments, the stability of the hepcidin analogue
is measured in vivo using any suitable method known in the art,
e.g., in some embodiments, the stability of a hepcidin analogue is
determined in vivo by administering the peptide or peptide dimer to
a subject such as a human or any mammal (e.g., mouse) and then
samples are taken from the subject via blood draw at various time
points, typically up to 24 hours. Samples are then analyzed as
described above in regard to the in vitro method of measuring
half-life. In some embodiments, in vivo stability of a hepcidin
analogue of the present invention is determined via the method
disclosed in the Examples herein.
[0123] In some embodiments, the present invention provides a
hepcidin analogue as described herein, wherein the hepcidin
analogue exhibits improved solubility or improved aggregation
characteristics as compared to a reference hepcidin. Solubility may
be determined via any suitable method known in the art. In some
embodiments, suitable methods known in the art for determining
solubility include incubating peptides (e.g., a hepcidin analogue
of the present invention) in various buffers (Acetate pH4.0,
Acetate pH5.0, Phos/Citrate pH5.0, Phos Citrate pH6.0, Phos pH 6.0,
Phos pH 7.0, Phos pH7.5, Strong PBS pH 7.5, Tris pH7.5, Tris pH
8.0, Glycine pH 9.0, Water, Acetic acid (pH 5.0 and other known in
the art) and testing for aggregation or solubility using standard
techniques. These include, but are not limited to, visual
precipitation, dynamic light scattering, Circular Dichroism and
fluorescent dyes to measure surface hydrophobicity, and detect
aggregation or fibrillation, for example. In some embodiments,
improved solubility means the peptide (e.g., the hepcidin analogue
of the present invention) is more soluble in a given liquid than is
a reference hepcidin.
[0124] In certain embodiments, the present invention provides a
hepcidin analogue as described herein, wherein the hepcidin
analogue exhibits a solubility that is increased at least about
1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or
200-fold greater or at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater than a
reference hepcidin in a particular solution or buffer, e.g., in
water or in a buffer known in the art or disclosed herein.
[0125] In certain embodiments, the present invention provides a
hepcidin analogue as described herein, wherein the hepcidin
analogue exhibits decreased aggregation, wherein the aggregation of
the peptide in a solution is at least about 1.5, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60,
70, 80, 90, 100, 120, 140, 160, 180, or 200-fold less or at least
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,
300%, 400%, or 500% less than a reference hepcidin in a particular
solution or buffer, e.g., in water or in a buffer known in the art
or disclosed herein.
[0126] In some embodiments, the present invention provides a
hepcidin analogue, as described herein, wherein the hepcidin
analogue exhibits less degradation (i.e., more degradation
stability), e.g., greater than or about 10% less, greater than or
about 20% less, greater than or about 30% less, greater than or
about 40 less, or greater than or about 50% less than a reference
hepcidin. In some embodiments, degradation stability is determined
via any suitable method known in the art. In some embodiments,
suitable methods known in the art for determining degradation
stability include the method described in Hawe et al J Pharm Sci,
VOL. 101, NO. 3, 2012, p 895-913, incorporated herein in its
entirety. Such methods are in some embodiments used to select
potent sequences with enhanced shelf lives.
[0127] In some embodiments, the hepcidin analogue of the present
invention is synthetically manufactured. In other embodiments, the
hepcidin analogue of the present invention is recombinantly
manufactured.
[0128] The various hepcidin analogue monomer and dimer peptides of
the invention may be constructed solely of natural amino acids.
Alternatively, these hepcidin analogues may include unnatural or
non-natural amino acids including, but not limited to, modified
amino acids. In certain embodiments, modified amino acids include
natural amino acids that have been chemically modified to include a
group, groups, or chemical moiety not naturally present on the
amino acid. The hepcidin analogues of the invention may
additionally include D-amino acids. Still further, the hepcidin
analogue peptide monomers and dimers of the invention may include
amino acid analogs. In particular embodiments, a peptide analogue
of the present invention comprises any of those described herein,
wherein one or more natural amino acid residues of the peptide
analogue is substituted with an unnatural or non-natural amino
acid, or a D-amino acid.
[0129] In certain embodiments, the hepcidin analogues of the
present invention include one or more modified or unnatural amino
acids. For example, in certain embodiments, a hepcidin analogue
includes one or more of Daba, Dapa, Pen, Sar, Cit, Cav, HLeu,
2-Nal, 1-Nal, d-1-Nal, d-2-Nal, Bip, Phe(4-OMe), Tyr(4-OMe),
.beta.hTrp, .beta.hPhe, Phe(4-CF.sub.3), 2-2-Indane, 1-1-Indane,
Cyclobutyl, .beta.hPhe, hLeu, Gla, Phe(4-NH.sub.2), hPhe, 1-Nal,
Nle, 3-3-diPhe, cyclobutyl-Ala, Cha, Bip, .beta.-Glu, Phe(4-Guan),
homo amino acids, D-amino acids, and various N-methylated amino
acids. One having skill in the art will appreciate that other
modified or unnatural amino acids, and various other substitutions
of natural amino acids with modified or unnatural amino acids, may
be made to achieve similar desired results, and that such
substitutions are within the teaching and spirit of the present
invention.
[0130] The present invention includes any of the hepcidin analogues
described herein, e.g., in a free or a salt form.
[0131] The hepcidin analogues of the present invention include any
of the peptide monomers or dimers described herein linked to a
linker moiety, including any of the specific linker moieties
described herein.
[0132] The hepcidin analogues of the present invention include
peptides, e.g., monomers or dimers, comprising a peptide monomer
subunit having at least 85%, at least 90%, at least 92%, at least
94%, at least 95%, at least 98%, or at least 99% amino acid
sequence identity to a hepcidin analogue peptide sequence described
herein (e.g., any one of the peptides disclosed herein), including
but not limited to any of the amino acid sequences shown in Tables
3 and 4.
[0133] In certain embodiments, a peptide analogue of the present
invention, or a monomer subunit of a dimer peptide analogue of the
present invention, comprises or consists of 7 to 35 amino acid
residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues,
10 to 35 amino acid residues, 7 to 25 amino acid residues, 8 to 25
amino acid residues, 9 to 25 amino acid residues, 10 to 25 amino
acid residues, 7 to 18 amino acid residues, 8 to 18 amino acid
residues, 9 to 18 amino acid residues, or 10 to 18 amino acid
residues, and, optionally, one or more additional non-amino acid
moieties, such as a conjugated chemical moiety, e.g., a half-life
extension moiety, a PEG or linker moiety. In particular
embodiments, a monomer subunit of a hepcidin analogue comprises or
consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino
acid residues. In particular embodiments, a monomer subunit of a
hepcidin analogue of the present invention comprises or consists of
10 to 18 amino acid residues and, optionally, one or more
additional non-amino acid moieties, such as a conjugated chemical
moiety, e.g., a PEG or linker moiety. In various embodiments, the
monomer subunit comprises or consists of 7 to 35 amino acid
residues, 9 to 18 amino acid residues, or 10 to 18 amino acid
residues. In particular embodiments of any of the various Formulas
described herein, X comprises or consists of 7 to 35 amino acid
residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues,
10 to 35 amino acid residues, 7 to 25 amino acid residues, 8 to 25
amino acid residues, 9 to 25 amino acid residues, 10 to 25 amino
acid residues, 7 to 18 amino acid residues, 8 to 18 amino acid
residues, 9 to 18 amino acid residues, or 10 to 18 amino acid
residues.
[0134] In particular embodiments, a hepcidin analogue or dimer of
the present invention does not include any of the compounds
described in PCT/US2014/030352 or PCT/US2015/038370.
[0135] Peptide Monomer Hepcidin Analogues
[0136] In certain embodiments, hepcidin analogues of the present
invention comprise a single peptide subunit, optionally conjugated
to a half-life extension moiety. In certain embodiments, these
hepcidin analogues form cyclized structures through intramolecular
disulfide or other bonds.
[0137] In one embodiment, the present invention includes a hepcidin
analogue comprising a polypeptide sequence of Formula (I'):
X--Y (I')
or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is a peptide comprising the sequence Xa':
TABLE-US-00008 (Xa) (SEQ ID NO: 1)
X1-Thr-His-X4-Pro-X6-X7-X8-Phe-X10
[0138] wherein [0139] X1 is Asp, isoGlu or Ida; [0140] X4 is Phe,
Phe(4-F), Phe(4-CN), 4-BIP, Phe(4-OCH.sub.3), Tyr,
Phe(2,3-(OCH.sub.3).sub.2), Phe(2,3-Cl.sub.2), or Dpa; [0141] X6 is
Cys or Pen; [0142] X7 is any amino acid; [0143] X8 is Ile, Leu,
Val, nLeu, Lys or Arg; and [0144] X10 is Lys, Glu or absent; and Y
is a peptide comprising the sequence Ya:
TABLE-US-00009 [0144] (Ya) (SEQ ID NO: 2) Y1-Y2-Y3-Y4-Y5-Y6-Y7
[0145] wherein [0146] Y1 is amino acid [0147] Y2 is any amino acid
[0148] Y3 is any amino acid [0149] Y4 is any amino acid [0150] Y5
is any amino acid; [0151] Y6 is Cys or Pen; and [0152] Y7 is Lys or
absent; and wherein the hepcidin analogue comprises a conjugated
half-life extension moiety, wherein the half-life extension moiety
is optionally conjugated via a linker moiety. The peptides Xa and
Ya may be linked via a peptide bond to form the polypeptide of
Formula (I').
[0153] In particular embodiments of any of the hepcidin analogues,
the hepcidin analogue comprises one of more of the following: X1 is
Asp; X4 is Phe or Dpa; X7 is Ile, Leu, Val, nLeu, or Lys; X7 is Ile
or Lys; X8 is Lys or Arg; Y1 is Pro or hPro; Y1 is Pro; Y2 is Arg
or Lys; Y2 is Arg; Y3 is Ser; Y4 is Lys, Arg or His; Y4 is Lys; or
Y5 is Gly or Sar. In certain embodiments: X1 is Asp; X4 is Phe or
Dpa; X7 is Ile, Leu, Val, nLeu, or Lys; X8 is Lys or Arg; Y1 is Pro
or hPro; Y2 is Arg or Lys; Y3 is Ser; Y4 is Lys, Arg or His; and Y5
is Gly or Sar. In certain embodiments, X1 is Asp; X4 is Phe or Dpa;
X7 is Ile or Lys; X8 is Lys or Arg; Y1 is Pro; Y2 is Arg; Y3 is
Ser; and Y4 is Lys; and Y5 is Gly or Sar.
[0154] In certain embodiments, a hepcidin analogue comprises a
polypeptide sequence of Formula (V'):
X--Y (V')
or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is a peptide sequence having the formula Xv:
TABLE-US-00010 (Xv) (SEQ ID NO: 3)
Asp-Thr-His-X4-Pro-X6-X7-X8-Phe-X10
[0155] wherein [0156] X4 is Phe or Dpa; [0157] X6 is Cys or Pen;
[0158] X7 is Ile or Lys; [0159] X8 is Lys or Arg; and [0160] X10 is
Lys, Glu or absent; and Y is a peptide sequence having the formula
Yv:
TABLE-US-00011 [0160] (Yv) (SEQ ID NO: 4)
Pro-Arg-Ser-Lys-Y5-Y6-Y7
[0161] wherein [0162] Y5 is Gly or Sar; [0163] Y6 is Cys or Pen;
and
[0164] Y7 is Lys or absent; and
wherein the hepcidin analogue comprises a conjugated half-life
extension moiety, and wherein the half-life extension moiety is
optionally conjugated via a linker moiety.
[0165] In particular embodiments, the hepcidin analogue comprises a
structure of Formula II':
R.sup.1--X-L-Y--R.sup.2 (II')
or a pharmaceutically acceptable salt or solvate thereof, wherein:
R.sup.1 is hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl
C1-C6 alkyl, or a C1-C20 alkanoyl, and including PEGylated versions
alone or as spacers of any of the foregoing; R.sup.2 is OH or
NH.sub.2; X is a peptide sequence having the formula Xa or Xv as
described herein; L is absent, a bond, or a linker moiety; and Y is
a peptide having the formula Ya or Yv as described herein.
[0166] In particular embodiments of hepcidin analogues of Formulaes
I', V' or II', the hepcidin analogue comprises one or more
additional amino acid residues. For example either X or Y may
further comprise one to three additional amino acids at the
N-terminus or C-terminus.
[0167] In particular embodiments of hepcidin analogues of Formulaes
I', V' or II', Y5 is Sar.
[0168] In various embodiments, the hepcidin analogues comprise a
disulfide bond between X6 and Y6.
[0169] In particular embodiments of hepcidin analogues of Formula
II', L is a bond, e.g., a peptide bond.
[0170] In particular embodiments, a half-life extension moiety is
conjugated to a Lys at X8 or X10.
[0171] Particular embodiments of hepcidin analogues comprises one
of the following illustrative structures:
##STR00002##
[0172] In certain embodiments of any of the peptide analogues
having any of the various Formulae set forth herein, R.sup.1 is
selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl,
isovaleryl, isobutyryl, octanyl, and conjugated amides of lauric
acid, hexadecanoic acid, and .gamma.-Glu-hexadecanoic acid.
[0173] In certain embodiments, the linker between the peptide and
the half-life extension moiety is PEG11, Ahx, or any of the others
described herein.
[0174] In certain embodiments, the half-life extension moiety is
Palm.
[0175] In certain embodiment, the present invention includes a
polypeptide comprising an amino acid sequence set forth in Table 3
(with or without the indicated linker moieties and half-life
extension moieties), or having any amino acid sequence with at
least 85%, at least 90%, at least 92%, at least 94%, or at least
95% identity to any of these amino acid sequences. In certain
embodiment, the present invention provides a cyclized form of any
one of the hepcidin analogues listed in Table 3, comprising a
disulfide bond between the two Cys and/or Pen residues. The
conjugated half-life extension moiety and the amino acid residue to
which it is conjugated are indicated by parentheses and brackets,
respectively.
TABLE-US-00012 TABLE 3 Illustrative Monomer Hepcidin Analogues HEK-
GFP cell based Cmpd # Sequence assay 1 Isovaleric
acid-DTHFPCI-[Lys(IsoGlu-Palm)]-FEPRSKGCK-NH.sub.2 (SEQ ID NO: 7) 5
(reference compound) 2 Isovaleric
acid-DTHFPCIKF-[Lys(IsoGlu-Palm)]-PRSKGCK-NH.sub.2 (SEQ ID NO: 8)
12 (reference compound) 3 Isovaleric
acid-DTHFP-[Pen]-IKF-[Lys(IsoGlu-Palm)]-PRSKG-[Pen]-K-NH.sub.2 (SEQ
418 ID NO: 9) 4 Isovaleric
acid-DTHFP-[Pen]-IKF-[Lys(IsoGlu-Palm)]-PRSKGCK-NH.sub.2 (SEQ ID 68
NO: 10) 5 Isovaleric
acid-DTHFPCIKF-[Lys(IsoGlu-Palm)]-PRSKG-[Pen]-K-NH.sub.2 (SEQ ID 13
NO: 11) 6 Isovaleric
acid-DTHFPCIKF-[LysIsoGlu-Palm)]-PRSK-[Sar]-CK-NH.sub.2 (SEQ ID 11
NO: 12) 7 Isovaleric
acid-DTH-[DPA]-PCIKF-[Lys(IsoGlu-Palm)]-PRSKGCK-NH.sub.2 SEQ ID 12
NO: 13) 8 Isovaleric
acid-DTH-[DPA]-PCIKF-[Lys(IsoGlu-Palm)]-PRSK[Sar]-CK-NH.sub.2 (SEQ
15 ID NO: 14) 9 Isovaleric
acid-DTHFPCIKF-[Lys(IsoGlu-Palm)]-PRSKGC-NH.sub.2 (SEQ ID NO: 15)
11 10 Isovaleric
acid-DTHFPCIKF-[Lys(IsoGlu-Palm)]-PRSK-[Sar]-C-NH.sub.2 (SEQ ID 10
NO: 16) 11 Isovaleric
acid-DTHFPCKKF-[Lys(IsoGlu-Palm)]-PRSKGCK-NH.sub.2 (SEQ ID NO: 17)
22 12 Isovaleric acid-DTHFPCIKF-[Lys(isoGlu-Lauric
acid)]-PRSKGCK-NH.sub.2 (SEQ ID 14 NO: 18) 13 Isovaleric
acid-DTHFPCIKF-Lys(isoGlu-Myristic acid)-PRSKGCK-NH.sub.2 (SEQ ID
10 NO: 19) 14 Isovaleric
acid-DTHFPCIKF-[Lys(isoGlu-Bioitin)]-PRSKGCK-NH.sub.2 (SEQ ID NO:
20) 29 15 Isovaleric acid-DTHFPCIKF-[Lys(isoGlu-Isovaleric
acid)]-PRSKGCK-NH.sub.2 (SEQ 26 ID NO: 21) 16 Isovaleric
acid-DTHFPCIKF-[Lys(PEG2-Palm)]-PRSKGCK-NH.sub.2 (SEQ ID NO: 22) 36
17 Isovaleric acid-DTHFPCIKF-[Lys(PEG11-Palm)]-PRSKGCK-NH.sub.2
(SEQ ID NO: 23) 30 18 Isovaleric acid-DTHFPCIKF-[Lys(PEG11-Lauric
acid)]-PRSKGCK-NH.sub.2 (SEQ ID 36 NO: 24) 19 Isovaleric
acid-DTHFPCIKF-[Lys(PEG11-Myristic acid)]-PRSKGCK-NH.sub.2 (SEQ ID
30 NO: 25) 20 Isovaleric
acid-DTHFPCIKF-[Lys(Palm)]-PRSKGCK-NH.sub.2 (SEQ ID NO: 26) 19 21
Isovaleric acid-DTHFPCIKF-[Lys(PEG8)]-PRSKGCK-NH.sub.2 (SEQ ID NO:
27) 32 22 Isovaleric
acid-DTHFPCIKF-[Lys(Ahx-Palm)]-PRSKGCK-NH.sub.2 (SEQ ID NO: 28) 18
23 Isovaleric acid-DTHFPCI-[Lys(Ahx-Palm)]-FEPRSK-[Sar]-CK-NH.sub.2
(SEQ ID 6 NO: 29) 24 Isovaleric
acid-DTHFPCIKF-[Lys(Ahx-Palm)]-PRSK-[Sar]-CK-NH.sub.2 (SEQ ID 10
NO: 30) 25 Isovaleric acid-DTHFPCIKF-[Lys(Ac)]-PRSKGCK-NH.sub.2
(SEQ ID NO: 31) 14 26 Isovaleric
acid-DTHFPCI-[Lys(PEG11-Palm)]-FEPRSK-[Sar]-CK-NH.sub.2 (SEQ ID 16
NO: 32) 27 Isovaleric
acid-DTHFPCIKF-[Lys(PEG11-Palm)]-PRSK-[Sar]-CK-NH.sub.2 (SEQ ID 13
NO: 33) 28 Isovaleric
acid-DTHFPCI-[Lys(isoGlu-Ahx-Palm)]-FEPRSK-[Sar]-CK-NH.sub.2 (SEQ
23 ID NO: 34 29 Isovaleric
acid-DTHFPCI-[Lys(OEG-OEG-isoGlu-(C18-diacid))]-FEPRSK-[Sar]- 98
CK-NH.sub.2 (SEQ ID NO: 35)
[0176] Peptide Dimer Hepcidin Analogues
[0177] In certain embodiments, the present invention includes dimer
hepcidin analogues, which include dimers of any of the monomer
hepcidin analogues described herein, including dimers comprising
any of the monomer peptides sequences or structures set forth in
the formulaes described herein, e.g., various embodiments of
Formulaes I, I', V, V', II, and II', and certain dimers of
sequences or structures set forth in Table 3. These dimers fall
within the scope of the general term "hepcidin analogues" as used
herein. The term "dimers," as in peptide dimers, refers to
compounds in which two peptide monomer subunits are linked. A
peptide dimer of the present invention may comprise two identical
monomer subunits, resulting in a homodimer, or two non-identical
monomer subunits, resulting in a heterodimer. A cysteine dimer
comprises two peptide monomer subunits linked through a disulfide
bond between a cysteine residue in one monomer subunit and a
cysteine residue in the other monomer subunit.
[0178] In certain embodiments, a dimer hepcidin analogue comprises
two polypeptide sequences of Formula (I''):
X--Y (I'')
or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is a peptide comprising the sequence Xa:
TABLE-US-00013 (Xa) (SEQ ID NO: 1)
X1-Thr-His-X4-Pro-X6-X7-X8-Phe-X10
[0179] wherein [0180] X1 is Asp, isoGlu or Ida; [0181] X4 is Phe,
Phe(4-F), Phe(4-CN), 4-BIP, Phe(4-OCH.sub.3), Tyr,
Phe(2,3-(OCH.sub.3).sub.2), Phe(2,3-Cl.sub.2), or Dpa; [0182] X6 is
Cys or Pen; [0183] X7 is any amino acid; [0184] X8 is Ile, Leu,
Val, nLeu, Lys or Arg; and [0185] X10 is Lys, Glu or absent; and Y
is absent, wherein the dimer hepcidin analogue comprises a
conjugated half-life extension moiety, wherein the half-life
extension moiety is optionally conjugated via a linker moiety.
[0186] In particular embodiments of any of the hepcidin analogues,
the hepcidin analogue comprises one of more of the following: X1 is
Asp; X4 is Phe or Dpa; X7 is Ile, Leu, Val, nLeu, or Lys; X7 is Ile
or Lys; or X8 is Lys or Arg. In certain embodiments: X1 is Asp; X4
is Phe or Dpa; X7 is Ile, Leu, Val, nLeu, or Lys; and X8 is Lys or
Arg. In certain embodiments, X1 is Asp; X4 is Phe or Dpa; X7 is Ile
or Lys; and X8 is Lys or Arg.
[0187] In a related embodiments, the present invention includes a
dimer hepcidin analogue comprising two polypeptide sequences of
Formula (V''):
X--Y (V'')
or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is a peptide sequence having the formula Xv:
TABLE-US-00014 (Xv) (SEQ ID NO: 3)
Asp-Thr-His-X4-Pro-X6-X7-X8-Phe-X10
[0188] wherein [0189] X4 is Phe or Dpa; [0190] X6 is Cys or Pen;
[0191] X7 is Ile or Lys; [0192] X8 is Lys or Arg; and [0193] X10 is
Lys, Glu or absent; and Y is absent, wherein the hepcidin analogue
comprises a conjugated half-life extension moiety, wherein the
half-life extension moiety is optionally conjugated via a linker
moiety.
[0194] In particular embodiments, the dimer hepcidin analogue
comprises a structure of Formula II'':
R.sup.1--X-L-Y--R.sup.2 (II'')
or a pharmaceutically acceptable salt or solvate thereof, wherein:
R.sup.1 is hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl
C1-C6 alkyl, or a C1-C20 alkanoyl, and including PEGylated versions
alone or as spacers of any of the foregoing; R.sup.2 is OH or
NH.sub.2; X is a peptide sequence having the formula Xa or Xv as
described herein; L is absent, a bond, or a linker moiety; and Y is
absent.
[0195] In particular embodiments of dimer hepcidin analogues of
Formulaes I'', V'' or II'', the hepcidin analogue comprises one or
more additional amino acid residues. For example, X may further
comprise one to three additional amino acids at the N-terminus or
C-terminus.
[0196] In particular embodiments, a dimer hepcidin analogues
comprises two polypeptide sequence of Formula I'' or Formula V'',
or two structures of Formula II'', dimerized via a linker moiety.
In particular embodiments, the linker moiety is bound to the
C-terminus of each hepcidin analogue. In particular embodiments,
the linker moiety is bound to the N-terminus of each hepcidin
analogue. In particular embodiments, the linker moiety is bound to
the N-terminus of one hepcidin analogue and the C-terminus of the
other hepcidin analogue present in the dimer.
[0197] In certain embodiments, the half-life extension moiety is
conjugated to the linker moiety.
[0198] In some embodiments, the hepcidin analogues of the present
invention are active in a dimer conformation, in particular when
free cysteine residues are present in the peptide. In certain
embodiments, this occurs either as a synthesized dimer or, in
particular, when a free cysteine monomer peptide is present and
under oxidizing conditions, dimerizes. In some embodiments, the
dimer is a homodimer. In other embodiments, the dimer is a
heterodimer.
[0199] In certain embodiments, a hepcidin analogue dimer of the
present invention is a peptide dimer comprising two hepcidin
analogue peptide monomers of the invention.
[0200] In certain embodiment, the present invention includes a
polypeptide comprising an amino acid sequence set forth in Table 4
(with or without the indicated linker moieties and half-life
extension moieties), or having any amino acid sequence with at
least 85%, at least 90%, at least 92%, at least 94%, or at least
95% identity to any of these amino acid sequences. In a related
embodiments, the present invention includes a dimer comprising two
polypeptides, each comprising an amino acid sequence set forth in
Table 4 (with or without the indicated linker moieties and
half-life extension moieties), or having any amino acid sequence
with at least 85%, at least 90%, at least 92%, at least 94%, or at
least 95% identity to any of these amino acid sequences. In
particular embodiments, a peptide dimer hepcidin analogue comprises
one or more, e.g., two, peptide monomer subunits shown in Table 4.
The conjugated half-life extension moiety and the amino acid
residue to which it is conjugated are indicated by parentheses and
brackets, respectively. Table 4 shows dimer hepcidin analogues,
each comprising a dimer of the sequences in parentheses followed by
a lower case "2", which are linked by the indicated one or more
linkers, e.g., Lys or IDA, and conjugated to the indicated
half-life extension moiety, e.g., octanoic acid or Palm.
TABLE-US-00015 TABLE 4 Illustrative Dimer Hepcidin Analogues
HEK-GFP cell based Cmpd # Sequence assay 30 (Isovaleric
acid-DTHFPCIRF).sub.2-[Lys(IsoGlu-Octanoic acid)]-NH.sub.2 18 (SEQ
ID NO: 36) 31 (Isovaleric
acid-DTHFPCIKF).sub.2-IDA-[.beta.ala]-(Peg2-Palm) 8 (SEQ ID NO: 37)
32 (Isovaleric acid-DTHFPCIKF).sub.2-IDA-[.beta.ala(Peg11-Palm)] 13
(SEQ ID NO: 38)
[0201] In particular embodiments, the monomer subunits may be
dimerized by a disulfide bridge between two cysteine residues, one
in each peptide monomer subunit, or they may be dimerized by
another suitable linker moiety, including those described herein.
Some of the monomer subunits are shown having C- and/or N-termini
that both comprise free amine. Thus, to produce a peptide dimer
inhibitor, the monomer subunit may be modified to eliminate either
the C- or N-terminal free amine, thereby permitting dimerization at
the remaining free amine. Further, in some instances, a terminal
end of one or more monomer subunits is acylated with an acylating
organic compound selected from the group consisting of
2-me-Trifluorobutyl, Trifluoropentyl, Acetyl, Octonyl, Butyl,
Pentyl, Hexyl, Palmityl, Trifluoromethyl butyric, cyclopentane
carboxylic, cyclopropylacetic, 4-fluorobenzoic, 4-fluorophenyl
acetic, 3-Phenylpropionic, tetrahedro-2H-pyran-4carboxylic,
succinic acid, and glutaric acid. In some instances, monomer
subunits comprise both a free carboxy terminal and a free amino
terminal, whereby a user may selectively modify the subunit to
achieve dimerization at a desired terminus. One having skill in the
art will, therefore, appreciate that the monomer subunits of the
instant invention may be selectively modified to achieve a single,
specific amine for a desired dimerization.
[0202] It is further understood that the C-terminal residues of the
monomer subunits disclosed herein may be amides, unless otherwise
indicated. Further, it is understood that, in certain embodiments,
dimerization at the C-terminus is facilitated by using a suitable
amino acid with a side chain having amine functionality, as is
generally understood in the art. Regarding the N-terminal residues,
it is generally understood that dimerization may be achieved
through the free amine of the terminal residue, or may be achieved
by using a suitable amino acid side chain having a free amine, as
is generally understood in the art.
[0203] Moreover, it is understood that the side chains of one or
more internal residue comprised in the hepcidin analogue peptide
monomers of the present invention can be utilized for the purpose
of dimerization. In such embodiments, the side chain is in some
embodiments a suitable natural amino acid (e.g., Lys), or
alternatively it is an unnatural amino acid comprising a side chain
suitable for conjugation, e.g., to a suitable linker moiety, as
defined herein.
[0204] The linker moieties connecting monomer subunits may include
any structure, length, and/or size that is compatible with the
teachings herein. In at least one embodiment, a linker moiety is
selected from the non-limiting group consisting of: cysteine,
lysine, DIG, PEG4, PEG4-biotin, PEG13, PEG25, PEG1K, PEG2K,
PEG3.4K, PEG4K, PEG5K, IDA, IDA-Palm, ADA, Boc-IDA, Glutaric acid,
Isophthalic acid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic
acid, 1,2-phenylenediacetic acid, Triazine, Boc-Triazine,
IDA-biotin, PEG4-Biotin, AADA, suitable aliphatics, aromatics,
heteroaromatics, and polyethylene glycol based linkers having a
molecular weight from approximately 400 Da to approximately 40,000
Da. Non-limiting examples of suitable linker moieties are provided
in Table 5. In particular embodiment, any of these linker moieties
may alternatively link a half-life extension moiety to a hepcidin
analogue.
TABLE-US-00016 TABLE 5 Illustrative Linker Moieties Abbreviation
Description Structure DIG DIGlycolic acid ##STR00003## PEG4
Bifunctional PEG linker with 4 PolyEthylene Glycol units
##STR00004## PEG13 Bifunctional PEG linker with 13 PolyEthylene
Glycol units ##STR00005## PEG25 Bifunctional PEG linker with 25
PolyEthylene Glycol units ##STR00006## PEG1K Bifunctional PEG
linker with PolyEthylene Glycol Mol wt of 1000 Da PEG2K
Bifunctional PEG linker with PolyEthylene Glycol Mol wt of 2000 Da
PEG3.4K Bifunctional PEG linker with PolyEthylene Glycol Mol wt of
3400 Da PEG5K Bifunctional PEG linker with PolyEthylene Glycol Mol
wt of 5000 Da DIG Diglycolic acid ##STR00007## .beta.-Ala-IDA
.beta.-Ala-Iminodiacetic acid ##STR00008## Boc-.beta.-Ala- IDA
Boc-.beta.-Ala-Iminodiacetic acid ##STR00009## Ac-.beta.-Ala- IDA
Ac-.beta.-Ala-Iminodiacetic acid ##STR00010## Palm-.beta.-Ala- IDA-
Palmityl-.beta.-Ala-Iminodiacetic acid ##STR00011## GTA Glutaric
acid ##STR00012## PMA Pemilic acid ##STR00013## AZA Azelaic acid
##STR00014## DDA Dodecanedioic acid ##STR00015## IPA Isopthalic
acid ##STR00016## 1,3-PDA 1,3-Phenylenediacetic acid ##STR00017##
1,4-PDA 1,4-Phenylenediacetic acid ##STR00018## 1,2-PDA
1,2-Phenylenediacetic acid ##STR00019## Triazine Amino propyl
Triazine di-acid ##STR00020## Boc-Triazine Boc-Triazine di-acid
##STR00021## IDA Iminodiacetic acid ##STR00022## AIDA n-Acetyl
imino acetic acid ##STR00023## Biotin-.beta.-ala- IDA-
N-Biotin-.beta.-Ala-Iminodiacetic acid ##STR00024## Lys Lysine
##STR00025##
[0205] One having skill in the art will appreciate that the C- and
N-terminal and internal linker moieties disclosed herein are
non-limiting examples of suitable linker moieties, and that the
present invention may include any suitable linker moiety.
[0206] In certain embodiments of any of the hepcidin analogue
peptide dimers, the N-terminus of each peptide monomer subunit is
connected by a linker moiety.
[0207] In certain embodiments of any of the hepcidin analogue
peptide dimers, the C-terminus of each peptide monomer subunit is
connected by a linker moiety.
[0208] In certain embodiments, the side chains of one or more
internal amino acid residues (e.g., Lys residues) comprised in each
peptide monomer subunit of a hepcidin analogue peptide dimer are
connected by a linker moiety.
[0209] In certain embodiments of any of the hepcidin analogue
peptide dimers, the C-terminus, the N terminus, or an internal
amino acid (e.g., a lysine sidechain) of each peptide monomer
subunit is connected by a linker moiety and at least two cysteine
or Pen residues of the hepcidin analogue peptide dimers are linked
by a disulfide bridge. In some embodiments, a peptide dimer has a
general structure shown below. Non-limiting schematic examples of
such hepcidin analogues are shown in the following
illustration:
##STR00026## ##STR00027##
[0210] Peptide Analogue Conjugates
[0211] In certain embodiments, hepcidin analogues of the present
invention, including both monomers and dimers, comprise one or more
conjugated chemical substituents, such as lipophilic substituents
and polymeric moieties, collectively referred to herein as
half-life extension moieties. Without wishing to be bound by any
particular theory, it is believed that the lipophilic substituent
binds to albumin in the bloodstream, thereby shielding the hepcidin
analogue from enzymatic degradation, and thus enhancing its
half-life. In addition, it is believed that polymeric moieties
enhance half-life and reduce clearance in the bloodstream, and in
some cases enhance permeability through the epithelium and
retention in the lamina propria. Moreover, it is also surmised that
these substituents in some cases may enhance permeability through
the epithelium and retention in the lamina propria. The skilled
person will be well aware of suitable techniques for preparing the
compounds employed in the context of the invention. For examples of
non-limiting suitable chemistry, see, e.g., WO98/08871, WO00/55184,
WO00/55119, Madsen et al (J. Med. Chem. 2007, 50, 6126-32), and
Knudsen et al. 2000 (J. Med Chem. 43, 1664-1669).
[0212] In one embodiment, the side chains of one or more amino acid
residues (e.g., Lys residues) in a hepcidin analogue of the
invention is further conjugated (e.g., covalently attached) to a
lipophilic substituent or other half-life extension moiety. 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 one or more spacers or linker moieties.
The spacer or linker moiety, when present, may provide spacing
between the hepcidin analogue and the lipophilic substituent. In
particular embodiments, the half-life extension moiety is
conjugated to the hepcidin analogue via a linker moiety, which in
certain embodiments is a linker moiety shown in Table 5 or Table 7,
or depicted in any of the illustrative compounds shown in Tables 3
and 4.
[0213] In certain embodiments, the lipophilic substituent or
half-life extension moiety comprises 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. The
hydrocarbon chain may be linear or branched and may be saturated or
unsaturated. In certain embodiments, the hydrocarbon chain is
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
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.
[0214] A lipophilic substituent may be conjugated to any amino acid
side chain in a hepcidin analogue of the invention. In certain
embodiment, the amino acid side chain includes a carboxy, hydroxyl,
thiol, amide or amine group, for forming an ester, a sulphonyl
ester, a thioester, an amide or a sulphonamide with the spacer or
lipophilic substituent. For example, the lipophilic substituent may
be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr,
Trp, Cys or Dbu, Dpr or Orn. In certain embodiments, the lipophilic
substituent is conjugated to Lys. An amino acid shown as Lys in any
of the formula provided herein may be replaced by, e.g., Dbu, Dpr
or Orn where a lipophilic substituent is added.
[0215] In further embodiments of the present invention,
alternatively or additionally, the side-chains of one or more amino
acid residues in a hepcidin analogue of the invention may be
conjugated to a polymeric moiety or other half-life extension
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.
[0216] As used herein, "Polyethylene glycol" or "PEG" is a
polyether compound of general formula H--(O--CH2-CH2)n-OH. PEGs are
also known as polyethylene oxides (PEOs) or polyoxyethylenes
(POEs), depending on their molecular weight PEO, PEE, or POG, as
used herein, refers to an oligomer or polymer of ethylene oxide.
The three names are chemically synonymous, but PEG has tended to
refer to oligomers and polymers with a molecular mass below 20,000
g/mol, PEO to polymers with a molecular mass above 20,000 g/mol,
and POE to a polymer of any molecular mass. PEG and PEO are liquids
or low-melting solids, depending on their molecular weights.
Throughout this disclosure, the 3 names are used indistinguishably.
PEGs are prepared by polymerization of ethylene oxide and are
commercially available over a wide range of molecular weights from
300 g/mol to 10,000,000 g/mol. While PEG and PEO with different
molecular weights find use in different applications, and have
different physical properties (e.g. viscosity) due to chain length
effects, their chemical properties are nearly identical. The
polymeric moiety is preferably water-soluble (amphiphilic or
hydrophilic), non-toxic, and pharmaceutically inert. Suitable
polymeric moieties include polyethylene glycols (PEG), homo- or
co-polymers 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). Also encompassed are
PEGs that are prepared for purpose of half-life extension, for
example, mono-activated, alkoxy-terminated polyalkylene oxides
(POA's) such as mono-methoxy-terminated polyethyelene glycols
(mPEG's); bis activated polyethylene oxides (glycols) or other PEG
derivatives are also contemplated. Suitable polymers will vary
substantially by weights ranging from about 200 to about 40,000 are
usually selected for the purposes of the present invention. In
certain embodiments, PEGs having molecular weights from 200 to
2,000 daltons or from 200 to 500 daltons are used. Different forms
of PEG may also be used, depending on the initiator used for the
polymerization process, e.g., a common initiator is a
monofunctional methyl ether PEG, or methoxypoly(ethylene glycol),
abbreviated mPEG. Other suitable initiators are known in the art
and are suitable for use in the present invention.
[0217] Lower-molecular-weight PEGs are also available as pure
oligomers, referred to as monodisperse, uniform, or discrete. These
are used in certain embodiments of the present invention.
[0218] PEGs are also available with different geometries: branched
PEGs have three to ten PEG chains emanating from a central core
group; star PEGs have 10 to 100 PEG chains emanating from a central
core group; and comb PEGs have multiple PEG chains normally grafted
onto a polymer backbone. PEGs can also be linear. The numbers that
are often included in the names of PEGs indicate their average
molecular weights (e.g. a PEG with n=9 would have an average
molecular weight of approximately 400 daltons, and would be labeled
PEG 400.
[0219] As used herein, "PEGylation" is the act of coupling (e.g.,
covalently) a PEG structure to the hepcidin analogue of the
invention, which is in certain embodiments referred to as a
"PEGylated hepcidin analogue". In certain embodiments, the PEG of
the PEGylated side chain is a PEG with a molecular weight from
about 200 to about 40,000. In certain embodiments, the PEG portion
of the conjugated half-life extension moiety is PEG3, PEG4, PEG5,
PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In particular embodiments,
it is PEG11. In certain embodiments, the PEG of a PEGylated spacer
is PEG3 or PEG8. In some embodiments, a spacer is PEGylated. In
certain embodiments, the PEG of a PEGylated spacer is PEG3, PEG4,
PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain
embodiments, the PEG of a PEGylated spacer is PEG3 or PEG8.
[0220] In some embodiments, the present invention includes a
hepcidin analogue peptide (or a dimer thereof) conjugated with a
PEG that is attached covalently, e.g., through an amide, a thiol,
via click chemistry, or via any other suitable means known in the
art. In particular embodiments PEG is attached through an amide
bond and, as such, certain PEG derivatives used will be
appropriately functionalized. For example, in certain embodiments,
PEG11, which is
O-(2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol, has both
an amine and carboxylic acid for attachment to a peptide of the
present invention. In certain embodiments, PEG25 contains a diacid
and 25 glycol moieties.
[0221] 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. 6:
332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57
and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73:
721-729. The polymeric moiety may be straight-chain or branched. In
some embodiments, it has 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.
[0222] In some embodiments, a hepcidin analogue 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.
[0223] In some embodiments, the polymeric moiety may be coupled (by
covalent linkage) to an amino, carboxyl or thiol group of an amino
acid side chain. Certain 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.
[0224] The skilled worker will be well aware of suitable techniques
which can be used to perform the coupling reaction. For example, 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.
[0225] As used herein, disulfide bond oxidation can occur within a
single step or is a two-step process. As used herein, for a single
oxidation step, the trityl protecting group is often employed
during assembly, allowing deprotection during cleavage, followed by
solution oxidation. When a second disulfide bond is required, one
has the option of native or selective oxidation. For selective
oxidation requiring orthogonal protecting groups, Acm and Trityl is
used as the protecting groups for cysteine. Cleavage results in the
removal of one protecting pair of cysteine allowing oxidation of
this pair. The second oxidative deprotection step of the cysteine
protected Acm group is then performed. For native oxidation, the
trityl protecting group is used for all cysteines, allowing for
natural folding of the peptide.
[0226] A skilled worker will be well aware of suitable techniques
which can be used to perform the oxidation step.
[0227] In particular embodiments, a hepcidin analogue of the
present invention comprises a half-life extension moiety, which may
be selected from but is not limited to the following: Ahx-Palm,
PEG2-Palm, PEG11-Palm, isoGlu-Palm, dapa-Palm, isoGlu-Lauric acid,
isoGlu-Mysteric acid, and isoGlu-Isovaleric acid.
[0228] In particular embodiments, a hepcidin analogue comprises a
half-life extension moiety having the structure shown below,
wherein n=0 to 24 or n=14 to 24:
##STR00028##
[0229] In certain embodiments, a hepcidin analogue of the present
invention comprises a conjugated half-life extension moiety shown
in Table 6.
TABLE-US-00017 TABLE 6 Illustrative Half-Life Extension Moieties #
Conjugates C1 ##STR00029## C12 (Lauric acid) C2 ##STR00030## C14
(Mysteric acid) C3 ##STR00031## C16 (Palm or Palmitic acid) C4 C5
C6 C7 C8 C9 C10 C11 ##STR00032## Biotin C12
[0230] In certain embodiments, a half-life extension moiety is
conjugated directly to a hepcidin analogue, while in other
embodiments, a half-life extension moiety is conjugated to a
hepcidin analogue peptide via a linker moiety, e.g., any of those
depicted in Tables 5 or 7.
TABLE-US-00018 TABLE 7 Illustrative Linker Moieties # Linker Moiety
L1 ##STR00033## IsoGlu L2 ##STR00034## Dapa L3 ##STR00035## Ahx L4
Lipdic based linkers: ##STR00036## n = 1 to 24 L5 ##STR00037## PEG1
L6 ##STR00038## PEG2 L7 ##STR00039## PEG11 (40 atoms) L8
##STR00040## n = 1 to 25 PEG based linkers L9 ##STR00041## OEG L10
##STR00042## IsoGlu-Ahx L11 ##STR00043## IsoGlu-OEG-OEG L12
##STR00044## IsoGlu-PEG5 L13 ##STR00045## IsoGlu-PEGn L14
##STR00046## .beta.Ala-PEG2 L15 ##STR00047## .beta.Ala-PEG11 (40
atoms)
[0231] With reference to linker structures shown in Table 7,
reference to n=1 to 24 or n=1 to 25, or the like, (e.g., in L4, L8
or L13) indicates that n may be any integer within the recited
range. For example, for L4 shown in Table 7, n could be 1, 2, 3,
etc., wherein when n=5, L4 has the structure shown in L3 (Ahx).
[0232] In particular embodiments, a hepcidin analogue of the
present invention comprises any of the linker moieties shown in
Table 7 and any of the half-life extension moieties shown in Table
6, including any of the following combinations shown in Table
8.
TABLE-US-00019 TABLE 8 Illustrative Combinations of Linkers and
Half- Life Extension Moieties in Hepcidin Analogues Half-Life
Linker Extension Moiety L1 C1 L2 C1 L3 C1 L4 C1 L5 C1 L6 C1 L7 C1
L8 C1 L9 C1 L10 C1 L11 C1 L12 C1 L13 C1 L14 C1 L15 C1 L1 C2 L2 C2
L3 C2 L4 C2 L5 C2 L6 C2 L7 C2 L8 C2 L9 C2 L10 C2 L11 C2 L12 C2 L13
C2 L14 C2 L15 C2 L1 C3 L2 C3 L3 C3 L4 C3 L5 C3 L6 C3 L7 C3 L8 C3 L9
C3 L10 C3 L11 C3 L12 C3 L13 C3 L14 C3 L15 C3 L1 C4 L2 C4 L3 C4 L4
C4 L5 C4 L6 C4 L7 C4 L8 C4 L9 C4 L10 C4 L11 C4 L12 C4 L13 C4 L14 C4
L15 C4 L1 C5 L2 C5 L3 C5 L4 C5 L5 C5 L6 C5 L7 C5 L8 C5 L9 C5 L10 C5
L11 C5 L12 C5 L13 C5 L14 C5 L15 C5 L1 C6 L2 C6 L3 C6 L4 C6 L5 C6 L6
C6 L7 C6 L8 C6 L9 C6 L10 C6 L11 C6 L12 C6 L13 C6 L14 C6 L15 C6 L1
C7 L2 C7 L3 C7 L4 C7 L5 C7 L6 C7 L7 C7 L8 C7 L9 C7 L10 C7 L11 C7
L12 C7 L13 C7 L14 C7 L15 C7 L1 C8 L2 C8 L3 C8 L4 C8 L5 C8 L6 C8 L7
C8 L8 C8 L9 C8 L10 C8 L11 C8 L12 C8 L13 C8 L14 C8 L15 C8 L1 C9 L2
C9 L3 C9 L4 C9 L5 C9 L6 C9 L7 C9 L8 C9 L9 C9 L10 C9 L11 C9 L12 C9
L13 C9 L14 C9 L15 C9 L1 C10 L2 C10 L3 C10 L4 C10 L5 C10 L6 C10 L7
C10 L8 C10 L9 C10 L10 C10 L11 C10 L12 C10 L13 C10 L14 C10 L15 C10
L1 C11 L2 C11 L3 C11 L4 C11 L5 C11 L6 C11 L7 C11 L8 C11 L9 C11 L10
C11 L11 C11 L12 C11 L13 C11 L14 C11 L15 C11 L1 C12 L2 C12 L3 C12 L4
C12 L5 C12 L6 C12 L7 C12 L8 C12 L9 C12 L10 C12 L11 C12 L12 C12 L13
C12 L14 C12 L15 C12
[0233] In certain embodiments, a hepcidin analogue comprises two or
more linkers. In particular embodiments, the two or more linkers
are concatamerized, i.e., bound to each other.
[0234] In related embodiments, the present invention includes
polynucleotides that encode a polypeptide having a peptide sequence
present in any of the hepcidin analogues described herein.
[0235] In addition, the present invention includes vectors, e.g.,
expression vectors, comprising a polynucleotide of the present
invention.
[0236] Methods of Treatment
[0237] In some embodiments, the present invention provides methods
for treating a subject afflicted with a disease or disorder
associated with dysregulated hepcidin signaling, wherein the method
comprises administering to the subject a hepcidin analogue of the
present invention. In some embodiments, the hepcidin analogue that
is administered to the subject is present in a composition (e.g., a
pharmaceutical composition). In one embodiment, a method is
provided for treating a subject afflicted with a disease or
disorder characterized by increased activity or expression of
ferroportin, wherein the method comprises administering to the
individual a hepcidin analogue or composition of the present
invention in an amount sufficient to (partially or fully) bind to
and agonize ferroportin in the subject. In one embodiment, a method
is provided for treating a subject afflicted with a disease or
disorder characterized by dysregulated iron metabolism, wherein the
method comprises administering to the subject a hepcidin analogue
or composition of the present invention.
[0238] In some embodiments, methods of the present invention
comprise providing a hepcidin analogue or a composition of the
present invention to a subject in need thereof. In particular
embodiments, the subject in need thereof has been diagnosed with or
has been determined to be at risk of developing a disease or
disorder characterized by dysregulated iron levels (e.g., diseases
or disorders of iron metabolism; diseases or disorders related to
iron overload; and diseases or disorders related to abnormal
hepcidin activity or expression). In particular embodiments, the
subject is a mammal (e.g., a human).
[0239] In certain embodiments, the disease or disorder is a disease
of iron metabolism, such as, e.g., an iron overload disease, iron
deficiency disorder, disorder of iron biodistribution, or another
disorder of iron metabolism and other disorder potentially related
to iron metabolism, etc. In particular embodiments, the disease of
iron metabolism is hemochromatosis, HFE mutation hemochromatosis,
ferroportin mutation hemochromatosis, transferrin receptor 2
mutation hemochromatosis, hemojuvelin mutation hemochromatosis,
hepcidin mutation hemochromatosis, juvenile hemochromatosis,
neonatal hemochromatosis, hepcidin deficiency, transfusional iron
overload, thalassemia, thalassemia intermedia, alpha thalassemia,
beta thalassemia, sideroblastic anemia, porphyria, porphyria
cutanea tarda, African iron overload, hyperferritinemia,
ceruloplasmin deficiency, atransferrinemia, congenital
dyserythropoietic anemia, anemia of chronic disease, anemia of
inflammation, anemia of infection, hypochromic microcytic anemia,
iron-deficiency anemia, iron-refractory iron deficiency anemia,
anemia of chronic kidney disease, transfusion-dependent anemia,
hemolytic anemia, erythropoietin resistance, iron deficiency of
obesity, other anemias, benign or malignant tumors that overproduce
hepcidin or induce its overproduction, conditions with hepcidin
excess, Friedreich ataxia, gracile syndrome, Hallervorden-Spatz
disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular
carcinoma, cancer (e.g., liver cancer), hepatitis, cirrhosis of
liver, pica, chronic renal failure, insulin resistance, diabetes,
atherosclerosis, neurodegenerative disorders, dementia, multiple
sclerosis, Parkinson's disease, Huntington's disease, or
Alzheimer's disease.
[0240] In certain embodiments, the disease or disorder is related
to iron overload diseases such as iron hemochromatosis, HFE
mutation hemochromatosis, ferroportin mutation hemochromatosis,
transferrin receptor 2 mutation hemochromatosis, hemojuvelin
mutation hemochromatosis, hepcidin mutation hemochromatosis,
juvenile hemochromatosis, neonatal hemochromatosis, hepcidin
deficiency, transfusional iron overload, thalassemia, thalassemia
intermedia, alpha thalassemia.
[0241] In certain embodiments, the disease or disorder is one that
is not typically identified as being iron related. For example,
hepcidin is highly expressed in the murine pancreas suggesting that
diabetes (Type I or Type II), insulin resistance, glucose
intolerance and other disorders may be ameliorated by treating
underlying iron metabolism disorders. See Ilyin, G. et al. (2003)
FEBS Lett. 542 22-26, which is herein incorporated by reference. As
such, peptides of the invention may be used to treat these diseases
and conditions. Those skilled in the art are readily able to
determine whether a given disease can be treated with a peptide
according to the present invention using methods known in the art,
including the assays of WO 2004092405, which is herein incorporated
by reference, and assays which monitor hepcidin, hemojuvelin, or
iron levels and expression, which are known in the art such as
those described in U.S. Pat. No. 7,534,764, which is herein
incorporated by reference.
[0242] In certain embodiments, the disease or disorder is
postmenopausal osteoporosis.
[0243] In certain embodiments of the present invention, the
diseases of iron metabolism are iron overload diseases, which
include hereditary hemochromatosis, iron-loading anemias, alcoholic
liver diseases, heart disease and/or failure, cardiomyopathy, and
chronic hepatitis C.
[0244] In particular embodiments, any of these diseases, disorders,
or indications are caused by or associated with a deficiency of
hepcidin or iron overload.
[0245] In some embodiments, methods of the present invention
comprise providing a hepcidin analogue of the present invention
(i.e., a first therapeutic agent) to a subject in need thereof in
combination with a second therapeutic agent. In certain
embodiments, the second therapeutic agent is provided to the
subject before and/or simultaneously with and/or after the
pharmaceutical composition is administered to the subject. In
particular embodiments, the second therapeutic agent is iron
chelator. In certain embodiments, the second therapeutic agent is
selected from the iron chelators Deferoxamine and Deferasirox
(Exjade.TM.) In another embodiment, the method comprises
administering to the subject a third therapeutic agent.
[0246] The present invention provides compositions (for example
pharmaceutical compositions) comprising one or more hepcidin
analogues of the present invention and a pharmaceutically
acceptable carrier, excipient or diluent. A pharmaceutically
acceptable carrier, diluent or excipient refers to a non-toxic
solid, semi-solid or liquid filler, diluent, encapsulating material
or formulation auxiliary of any type. Prevention of the action of
microorganisms may be ensured by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents such as sugars, sodium
chloride, and the like.
[0247] The term "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. Gennaro (Ed.),
Mark Publishing Company, Easton, Pa., USA, 1985. For example,
sterile saline and phosphate-buffered saline at slightly acidic or
physiological pH may be used. 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, diethanolamine, histidine, arginine, lysine
or acetate (e.g. as sodium acetate), or mixtures thereof. The term
further encompasses any carrier agents listed in the US
Pharmacopeia for use in animals, including humans.
[0248] In certain embodiments, the compositions comprise two or
more hepcidin analogues disclosed herein. In certain embodiments,
the combination is selected from one of the following: (i) any two
or more of the hepcidin analogue peptide monomers shown therein;
(ii) any two or more of the hepcidin analogue peptide dimers
disclosed herein; (iii) any one or more of the hepcidin analogue
peptide monomers disclosed herein, and any one or more of the
hepcidin analogue peptide dimers disclosed herein.
[0249] It is to be understood that the inclusion of a hepcidin
analogue of the invention (i.e., one or more hepcidin analogue
peptide monomers of the invention or one or more hepcidin analogue
peptide dimers of the present invention) in a pharmaceutical
composition also encompasses inclusion of a pharmaceutically
acceptable salt or solvate of a hepcidin analogue of the invention.
In particular embodiments, the pharmaceutical compositions further
comprise one or more pharmaceutically acceptable carrier,
excipient, or vehicle.
[0250] In certain embodiments, the invention provides a
pharmaceutical composition comprising a hepcidin analogue, or a
pharmaceutically acceptable salt or solvate thereof, for treating a
variety of conditions, diseases, or disorders as disclosed herein
or elsewhere (see, e.g., Methods of Treatment, herein). In
particular embodiments, the invention provides a pharmaceutical
composition comprising a hepcidin analogue peptide monomer, or a
pharmaceutically acceptable salt or solvate thereof, for treating a
variety of conditions, diseases, or disorders as disclosed herein
elsewhere (see, e.g., Methods of Treatment, herein). In particular
embodiments, the invention provides a pharmaceutical composition
comprising a hepcidin analogue peptide dimer, or a pharmaceutically
acceptable salt or solvate thereof, for treating a variety of
conditions, diseases, or disorders as disclosed herein.
[0251] The hepcidin analogues of the present invention may be
formulated as pharmaceutical compositions which are suited for
administration with or without storage, and which typically
comprise a therapeutically effective amount of at least one
hepcidin analogue of the invention, together with a
pharmaceutically acceptable carrier, excipient or vehicle.
[0252] In some embodiments, the hepcidin analogue pharmaceutical
compositions of the invention are in unit dosage form. In such
forms, 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, e.g., any one of the routes and means of
administration disclosed herein.
[0253] In particular embodiments, the hepcidin analogue, or the
pharmaceutical composition comprising a hepcidin analogue, is
suspended in a sustained-release matrix. A sustained-release
matrix, as used herein, is a matrix made of materials, usually
polymers, which are degradable by enzymatic or acid-base hydrolysis
or by dissolution. Once inserted into the body, the matrix is acted
upon by enzymes and body fluids. A sustained-release matrix
desirably is chosen from biocompatible materials such as liposomes,
polylactides (polylactic acid), polyglycolide (polymer of glycolic
acid), polylactide co-glycolide (copolymers of lactic acid and
glycolic acid) polyanhydrides, poly(ortho)esters, polypeptides,
hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids,
fatty acids, phospholipids, polysaccharides, nucleic acids,
polyamino acids, amino acids such as phenylalanine, tyrosine,
isoleucine, polynucleotides, polyvinyl propylene,
polyvinylpyrrolidone and silicone. One embodiment of a
biodegradable matrix is a matrix of one of either polylactide,
polyglycolide, or polylactide co-glycolide (co-polymers of lactic
acid and glycolic acid).
[0254] In certain embodiments, the compositions are administered
parenterally, subcutaneously or orally. In particular embodiments,
the compositions are administered orally, intracisternally,
intravaginally, intraperitoneally, intrarectally, topically (as by
powders, ointments, drops, suppository, or transdermal patch,
including delivery intravitreally, intranasally, and via
inhalation) or buccally. The term "parenteral" as used herein
refers to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous,
intradermal and intra-articular injection and infusion.
Accordingly, in certain embodiments, the compositions are
formulated for delivery by any of these routes of
administration.
[0255] In certain embodiments, pharmaceutical compositions for
parenteral injection comprise pharmaceutically acceptable sterile
aqueous or nonaqueous solutions, dispersions, suspensions or
emulsions, or sterile powders, for reconstitution into sterile
injectable solutions or dispersions just prior to use. Examples of
suitable aqueous and nonaqueous carriers, diluents, solvents or
vehicles include water, ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like),
carboxymethylcellulose and suitable mixtures thereof,
beta-cyclodextrin, vegetable oils (such as olive oil), and
injectable organic esters such as ethyl oleate. Proper fluidity may
be maintained, for example, by the use of coating materials such as
lecithin, by the maintenance of the required particle size in the
case of dispersions, and by the use of surfactants. These
compositions may also contain adjuvants such as preservative,
wetting agents, emulsifying agents, and dispersing agents.
Prolonged absorption of an injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption,
such as aluminum monostearate and gelatin.
[0256] Injectable depot forms include those made by forming
microencapsule matrices of the hepcidin analogue in one or more
biodegradable polymers such as polylactide-polyglycolide,
poly(orthoesters), poly(anhydrides), and (poly)glycols, such as
PEG. Depending upon the ratio of peptide to polymer and the nature
of the particular polymer employed, the rate of release of the
hepcidin analogue can be controlled. Depot injectable formulations
are also prepared by entrapping the hepcidin analogue in liposomes
or microemulsions compatible with body tissues.
[0257] The injectable formulations may be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium just prior to use.
[0258] Hepcidin analogues of the present invention may also be
administered in liposomes or other lipid-based carriers. As is
known in the art, liposomes are generally derived from
phospholipids or other lipid substances. Liposomes are formed by
mono- or multi-lamellar hydrated liquid crystals that are dispersed
in an aqueous medium. Any non-toxic, physiologically acceptable and
metabolizable lipid capable of forming liposomes can be used. The
present compositions in liposome form can contain, in addition to a
hepcidin analogue of the present invention, stabilizers,
preservatives, excipients, and the like. In certain embodiments,
the lipids comprise phospholipids, including the phosphatidyl
cholines (lecithins) and serines, both natural and synthetic.
Methods to form liposomes are known in the art.
[0259] Pharmaceutical compositions to be used in the invention
suitable for parenteral administration may comprise sterile aqueous
solutions and/or suspensions of the peptide inhibitors made
isotonic with the blood of the recipient, generally using sodium
chloride, glycerin, glucose, mannitol, sorbitol, and the like.
[0260] In some aspects, the invention provides a pharmaceutical
composition for oral delivery. Compositions and hepcidin analogues
of the instant invention may be prepared for oral administration
according to any of the methods, techniques, and/or delivery
vehicles described herein. Further, one having skill in the art
will appreciate that the hepcidin analogues of the instant
invention may be modified or integrated into a system or delivery
vehicle that is not disclosed herein, yet is well known in the art
and compatible for use in oral delivery of peptides.
[0261] In certain embodiments, formulations for oral administration
may comprise 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 enzymatic inhibitors
(e.g. pancreatic trypsin inhibitors, diisopropylfluorophosphate
(DFF) or trasylol) to inhibit enzymatic degradation. In certain
embodiments, the hepcidin analogue 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. 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.
[0262] In particular embodiments, oral dosage forms or unit doses
compatible for use with the hepcidin analogues of the present
invention may include a mixture of hepcidin analogue and nondrug
components or excipients, as well as other non-reusable materials
that may be considered either as an ingredient or packaging. Oral
compositions may include at least one of a liquid, a solid, and a
semi-solid dosage forms. In some embodiments, an oral dosage form
is provided comprising an effective amount of hepcidin analogue,
wherein the dosage form comprises at least one of a pill, a tablet,
a capsule, a gel, a paste, a drink, a syrup, ointment, and
suppository. In some instances, an oral dosage form is provided
that is designed and configured to achieve delayed release of the
hepcidin analogue in the subject's small intestine and/or
colon.
[0263] In one embodiment, an oral pharmaceutical composition
comprising a hepcidin analogue of the present invention comprises
an enteric coating that is designed to delay release of the
hepcidin analogue in the small intestine. In at least some
embodiments, a pharmaceutical composition is provided which
comprises a hepcidin analogue of the present invention and a
protease inhibitor, such as aprotinin, in a delayed release
pharmaceutical formulation. In some instances, pharmaceutical
compositions of the instant invention comprise an enteric coat that
is soluble in gastric juice at a pH of about 5.0 or higher. In at
least one embodiment, a pharmaceutical composition is provided
comprising an enteric coating comprising a polymer having
dissociable carboxylic groups, such as derivatives of cellulose,
including hydroxypropylmethyl cellulose phthalate, cellulose
acetate phthalate and cellulose acetate trimellitate and similar
derivatives of cellulose and other carbohydrate polymers.
[0264] In one embodiment, a pharmaceutical composition comprising a
hepcidin analogue of the present invention is provided in an
enteric coating, the enteric coating being designed to protect and
release the pharmaceutical composition in a controlled manner
within the subject's lower gastrointestinal system, and to avoid
systemic side effects. In addition to enteric coatings, the
hepcidin analogues of the instant invention may be encapsulated,
coated, engaged or otherwise associated within any compatible oral
drug delivery system or component. For example, in some embodiments
a hepcidin analogue of the present invention is provided in a lipid
carrier system comprising at least one of polymeric hydrogels,
nanoparticles, microspheres, micelles, and other lipid systems.
[0265] To overcome peptide degradation in the small intestine, some
embodiments of the present invention comprise a hydrogel polymer
carrier system in which a hepcidin analogue of the present
invention is contained, whereby the hydrogel polymer protects the
hepcidin analogue from proteolysis in the small intestine and/or
colon. The hepcidin analogues of the present invention may further
be formulated for compatible use with a carrier system that is
designed to increase the dissolution kinetics and enhance
intestinal absorption of the peptide. These methods include the use
of liposomes, micelles and nanoparticles to increase GI tract
permeation of peptides.
[0266] Various bioresponsive systems may also be combined with one
or more hepcidin analogue of the present invention to provide a
pharmaceutical agent for oral delivery. In some embodiments, a
hepcidin analogue of the instant invention is used in combination
with a bioresponsive system, such as hydrogels and mucoadhesive
polymers with hydrogen bonding groups (e.g., PEG, poly(methacrylic)
acid [PMAA], cellulose, Eudragit.RTM., chitosan and alginate) to
provide a therapeutic agent for oral administration. Other
embodiments include a method for optimizing or prolonging drug
residence time for a hepcidin analogue disclosed herein, wherein
the surface of the hepcidin analogue surface is modified to
comprise mucoadhesive properties through hydrogen bonds, polymers
with linked mucins or/and hydrophobic interactions. These modified
peptide molecules may demonstrate increase drug residence time
within the subject, in accordance with a desired feature of the
invention. Moreover, targeted mucoadhesive systems may specifically
bind to receptors at the enterocytes and M-cell surfaces, thereby
further increasing the uptake of particles containing the hepcidin
analogue.
[0267] Other embodiments comprise a method for oral delivery of a
hepcidin analogue of the present invention, wherein the hepcidin
analogue is provided to a subject in combination with permeation
enhancers that promote the transport of the peptides across the
intestinal mucosa by increasing paracellular or transcellular
permeation. For example, in one embodiment, a permeation enhancer
is combined with a hepcidin analogue, wherein the permeation
enhancer comprises at least one of a long-chain fatty acid, a bile
salt, an amphiphilic surfactant, and a chelating agent. In one
embodiment, a permeation enhancer comprising sodium
N-[hydroxybenzoyl)amino] caprylate is used to form a weak
noncovalent association with the hepcidin analogue of the instant
invention, wherein the permeation enhancer favors membrane
transport and further dissociation once reaching the blood
circulation. In another embodiment, a hepcidin analogue of the
present invention is conjugated to oligoarginine, thereby
increasing cellular penetration of the peptide into various cell
types. Further, in at least one embodiment a noncovalent bond is
provided between a peptide inhibitor of the present invention and a
permeation enhancer selected from the group consisting of a
cyclodextrin (CD) and a dendrimers, wherein the permeation enhancer
reduces peptide aggregation and increasing stability and solubility
for the hepcidin analogue molecule.
[0268] Other embodiments of the invention provide a method for
treating a subject with a hepcidin analogue of the present
invention having an increased half-life. In one aspect, the present
invention provides a hepcidin analogue having a half-life of at
least several hours to one day in vitro or in vivo (e.g., when
administered to a human subject) sufficient for daily (q.d.) or
twice daily (b.i.d.) dosing of a therapeutically effective amount.
In another embodiment, the hepcidin analogue has a half-life of
three days or longer sufficient for weekly (q.w.) dosing of a
therapeutically effective amount. Further, in another embodiment,
the hepcidin analogue has a half-life of eight days or longer
sufficient for bi-weekly (b.i.w.) or monthly dosing of a
therapeutically effective amount. In another embodiment, the
hepcidin analogue is derivatized or modified such that is has a
longer half-life as compared to the underivatized or unmodified
hepcidin analogue. In another embodiment, the hepcidin analogue
contains one or more chemical modifications to increase serum
half-life.
[0269] When used in at least one of the treatments or delivery
systems described herein, a hepcidin analogue of the present
invention may be employed in pure form or, where such forms exist,
in pharmaceutically acceptable salt form.
[0270] The total daily usage of the hepcidin analogues and
compositions of the present invention can be decided by the
attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular
subject will depend upon a variety of factors including: a) the
disorder being treated and the severity of the disorder; b)
activity of the specific compound employed; c) the specific
composition employed, the age, body weight, general health, sex and
diet of the patient; d) the time of administration, route of
administration, and rate of excretion of the specific hepcidin
analogue employed; e) the duration of the treatment; 0 drugs used
in combination or coincidental with the specific hepcidin analogue
employed, and like factors well known in the medical arts.
[0271] In particular embodiments, the total daily dose of the
hepcidin analogues of the invention to be administered to a human
or other mammal host in single or divided doses may be in amounts,
for example, from 0.0001 to 300 mg/kg body weight daily or 1 to 300
mg/kg body weight daily. In certain embodiments, a dosage of a
hepcidin analogue of the present invention is 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. In particular
embodiments, a total dosage is about 1 mg, about 2 mg, about 3 mg,
about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9
mg, or about 10 mg about once or twice weekly, e.g., for a human
patient. In particular embodiments, the total dosage is in the
range of about 1 mg to about 5 mg, or about 1 mg to about 3 mg, or
about 2 mg to about 3 mg per human patient, e.g., about once
weekly.
[0272] In various embodiments, a hepcidin analogue 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
pharmaceutical composition selected by the skilled practitioner for
the particular subject. 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.
[0273] Such regular hepcidin analogue 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). In some embodiments, 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).
In other embodiments, 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. So, for example, a regular daily dosing
regimen with a peptide, a peptide analogue, or a dimer 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. A variety of other drug holiday regimens are envisioned to
be useful for administering the hepcidin analogues of the
invention.
[0274] Thus, the hepcidin analogues may be delivered via an
administration regime which comprises two or more administration
phases separated by respective drug holiday phases.
[0275] During each administration phase, the hepcidin analogue 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. Alternatively, the
administration pattern may comprise administration of a plurality
of doses of the hepcidin analogue to the recipient subject, wherein
said doses are spaced by dosing intervals.
[0276] 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.
[0277] 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 hepcidin
analogue of the present invention.
[0278] 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.
[0279] Where an administration pattern comprises a plurality of
doses, the duration of the following drug holiday phase is longer
than the dosing interval used in that administration pattern. Where
the dosing interval is irregular, the duration of the drug holiday
phase may be greater than the mean interval between doses over the
course of the administration phase. Alternatively the duration of
the drug holiday may be longer than the longest interval between
consecutive doses during the administration phase.
[0280] 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.
[0281] Within these constraints, 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.
[0282] 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.
[0283] 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 a hepcidin analogue 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.
[0284] In some embodiments, the present invention provides
compositions and medicaments comprising at least one hepcidin
analogue as disclosed herein. In some embodiments, the present
invention provides a method of manufacturing medicaments comprising
at least one hepcidin analogue as disclosed herein for the
treatment of diseases of iron metabolism, such as iron overload
diseases. In some embodiments, the present invention provides a
method of manufacturing medicaments comprising at least one
hepcidin analogue as disclosed herein for the treatment of diabetes
(Type I or Type II), insulin resistance, or glucose intolerance.
Also provided are methods of treating a disease of iron metabolism
in a subject, such as a mammalian subject, and preferably a human
subject, comprising administering at least one hepcidin analogue,
or composition as disclosed herein to the subject. In some
embodiments, the hepcidin analogue or the composition is
administered in a therapeutically effective amount. Also provided
are methods of treating diabetes (Type I or Type II), insulin
resistance, or glucose intolerance in a subject, such as a
mammalian subject, and preferably a human subject, comprising
administering at least one hepcidin analogue or composition as
disclosed herein to the subject. In some embodiments, the hepcidin
analogue or composition is administered in a therapeutically
effective amount.
[0285] In some embodiments, the invention provides a process for
manufacturing a hepcidin analogue or a hepcidin analogue
composition (e.g., a pharmaceutical composition), as disclosed
herein.
[0286] In some embodiments, the invention provides a device
comprising at least one hepcidin analogue of the present invention,
or pharmaceutically acceptable salt or solvate thereof for delivery
of the hepcidin analogue to a subject.
[0287] In some embodiments, the present invention provides methods
of binding a ferroportin or inducing ferroportin internalization
and degradation which comprises contacting the ferroportin with at
least one hepcidin analogue, or hepcidin analogue composition as
disclosed herein.
[0288] In some embodiments, the present invention provides kits
comprising at least one hepcidin analogue, or hepcidin analogue
composition (e.g., pharmaceutical composition) as disclosed herein
packaged together with a reagent, a device, instructional material,
or a combination thereof.
[0289] In some embodiments, the present invention provides a method
of administering a hepcidin analogue or hepcidin analogue
composition (e.g., pharmaceutical composition) of the present
invention to a subject via implant or osmotic pump, by cartridge or
micro pump, or by other means appreciated by the skilled artisan,
as well-known in the art.
[0290] In some embodiments, the present invention provides
complexes which comprise at least one hepcidin analogue as
disclosed herein bound to a ferroportin, preferably a human
ferroportin, or an antibody, such as an antibody which specifically
binds a hepcidin analogue as disclosed herein, Hep25, or a
combination thereof.
[0291] In some embodiments, the hepcidin analogue of the present
invention has a measurement (e.g., an EC50) of less than 500 nM
within the Fpn internalization assay. As a skilled person will
realize, the function of the hepcidin analogue is dependent on the
tertiary structure of the hepcidin analogue and the binding surface
presented. It is therefore possible to make minor changes to the
sequence encoding the hepcidin analogue that do not affect the fold
or are not on the binding surface and maintain function. In other
embodiments, the present invention provides a hepcidin analogue
having 85% or higher (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 99.5%) identity or homology to an amino acid
sequence of any hepcidin analogue described herein that exhibits an
activity (e.g., hepcidin activity), or lessens a symptom of a
disease or indication for which hepcidin is involved.
[0292] In other embodiments, the present invention provides a
hepcidin analogue having 85% or higher (e.g., 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity or homology
to an amino acid sequence of any hepcidin analogue presented
herein, or a peptide according to any one of the formulae or
hepcidin analogues described herein.
[0293] In some embodiments, a hepcidin analogue of the present
invention may comprise functional fragments or variants thereof
that have at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid
substitutions compared to one or more of the specific peptide
analogue sequences recited herein.
[0294] In addition to the methods described in the Examples herein,
the hepcidin analogues of the present invention may be produced
using methods known in the art including chemical synthesis,
biosynthesis or in vitro synthesis using recombinant DNA methods,
and solid phase synthesis. See e.g. Kelly & Winkler (1990)
Genetic Engineering Principles and Methods, vol. 12, J. K. Setlow
ed., Plenum Press, NY, pp. 1-19; Merrifield (1964) J Amer Chem Soc
85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart &
Young (1984) Solid Phase Peptide Synthesis, 2ed. Pierce, Rockford,
Ill., which are herein incorporated by reference. The hepcidin
analogues of the present invention may be purified using protein
purification techniques known in the art such as reverse phase
high-performance liquid chromatography (HPLC), ion-exchange or
immunoaffinity chromatography, filtration or size exclusion, or
electrophoresis. See Olsnes, S. and A. Pihl (1973) Biochem.
12(16):3121-3126; and Scopes (1982) Protein Purification,
Springer-Verlag, NY, which are herein incorporated by reference.
Alternatively, the hepcidin analogues of the present invention may
be made by recombinant DNA techniques known in the art. Thus,
polynucleotides that encode the polypeptides of the present
invention are contemplated herein. In certain preferred
embodiments, the polynucleotides are isolated. As used herein
"isolated polynucleotides" refers to polynucleotides that are in an
environment different from that in which the polynucleotide
naturally occurs.
EXAMPLES
[0295] The following examples demonstrate certain specific
embodiments of the present invention. The following examples were
carried out using standard techniques that are well known and
routine to those of skill in the art, except where otherwise
described in detail. It is to be understood that these examples are
for illustrative purposes only and do not purport to be wholly
definitive as to conditions or scope of the invention. As such,
they should not be construed in any way as limiting the scope of
the present invention.
Abbreviations
[0296] DCM: dichloromethane
DMF: N,N-dimethylformamide
NMP: N-methylpyrolidone
[0297] HBTU: O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate HATU:
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
DCC: Dicyclohexylcarbodiimide
NHS: N-hydoxysuccinimide
[0298] DIPEA: diisopropylethylamine EtOH: ethanol Et2O: diethyl
ether Hy: hydrogen TFA: trifluoroacetic acid TIS:
triisopropylsilane ACN: acetonitrile HPLC: high performance liquid
chromatography ESI-MS: electron spray ionization mass spectrometry
PBS: phosphate-buffered saline Boc: t-butoxycarbonyl
Fmoc: Fluorenylmethyloxycarbonyl
[0299] Acm: acetamidomethyl IVA: Isovaleric acid (or Isovaleryl) K(
): In the peptide sequences provided herein, wherein a compound or
chemical group is presented in parentheses directly after a Lysine
residue, it is to be understood that the compound or chemical group
in the parentheses is a side chain conjugated to the Lysine
residue. So, e.g., but not to be limited in any way, K-[(PEG8)]-
indicates that a PEG8 moiety is conjugated to a side chain of this
Lysine. Palm: Indicates conjugation of a palmitic acid
(palmitoyl).
[0300] As used herein "C( )" refers to a cysteine residue involved
in a particular disulfide bridge. For example, in Hepcidin, there
are four disulfide bridges: the first between the two C(1)
residues; the second between the two C(2) residues; the third
between the two C(3) residues; and the fourth between the two C(4)
residues. Accordingly, in some embodiments, the sequence for
Hepcidin is written as follows:
TABLE-US-00020 (SEQ ID NO: 39)
Hy-DTHFPIC(1)IFC(2)C(3)GC(2)C(4)HRSKC(3)GMC(4)C(1) KT-OH;
and the sequence for other peptides may also optionally be written
in the same manner.
Example 1
Synthesis of Peptide Analogues
[0301] Unless otherwise specified, reagents and solvents employed
in the following were available commercially in standard laboratory
reagent or analytical grade, and were used without further
purification.
[0302] Procedure for Solid-Phase Synthesis of Peptides
[0303] Peptide analogues of the invention were chemically
synthesized using optimized 9-fluorenylmethoxy carbonyl (Fmoc)
solid phase peptide synthesis protocols. For C-terminal amides,
rink-amide resin was used, although wang and trityl resins were
also used to produce C-terminal acids. The side chain protecting
groups were as follows: Glu, Thr and Tyr: 0-tButyl; Trp and Lys:
t-Boc (t-butyloxycarbonyl); Arg:
N-gamma-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; His,
Gln, Asn, Cys: Trityl. For selective disulfide bridge formation,
Acm (acetamidomethyl) was also used as a Cys protecting group. For
coupling, a four to ten-fold excess of a solution containing Fmoc
amino acid, HBTU and DIPEA (1:1:1.1) in DMF was added to swelled
resin [HBTU: 0-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate; DIPEA: diisopropylethylamine; DMF:
dimethylformamide]. HATU
(O-(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluronium
hexafluorophosphate) was used instead of HBTU to improve coupling
efficiency in difficult regions. Fmoc protecting group removal was
achieved by treatment with a DMF, piperidine (2:1) solution.
[0304] Procedure for Cleavage of Peptides Off Resin
[0305] Side chain deprotection and cleavage of the peptide
analogues of the invention (e.g., Compound No. 2) was achieved by
stirring dry resin in a solution containing trifluoroacetic acid,
water, ethanedithiol and tri-isopropylsilane (90:5:2.5:2.5) for 2
to 4 hours. Following TFA removal, peptide was precipitated using
ice-cold diethyl ether. The solution was centrifuged and the ether
was decanted, followed by a second diethyl ether wash. The peptide
was dissolved in an acetonitrile, water solution (1:1) containing
0.1% TFA (trifluoroacetic acid) and the resulting solution was
filtered. The linear peptide quality was assessed using
electrospray ionization mass spectrometry (ESI-MS).
[0306] Procedure for Purification of Peptides
[0307] Purification of the peptides of the invention (e.g.,
Compound No. 2) was achieved using reverse-phase high performance
liquid chromatography (RP-HPLC). Analysis was performed using a C18
column (3 .mu.m, 50.times.2 mm) with a flow rate of 1 mL/min.
Purification of the linear peptides was achieved using preparative
RP-HPLC with a C18 column (5 .mu.m, 250.times.21.2 mm) with a flow
rate of 20 mL/min. Separation was achieved using linear gradients
of buffer B in A (Buffer A: Aqueous 0.05% TFA; Buffer B: 0.043%
TFA, 90% acetonitrile in water).
[0308] Procedure for Oxidation of Peptides
[0309] Method a (Single Disulfide Oxidation).
[0310] Oxidation of the unprotected peptides of the invention was
achieved by adding drop-wise iodine in MeOH (1 mg per 1 mL) to the
peptide in a solution (ACN: H.sub.2O, 7: 3, 0.5% TFA). After
stirring for 2 min, ascorbic acid portion wise was added until the
solution was clear and the sample was immediately loaded onto the
HPLC for purification.
[0311] Method B (Selective Oxidation of Two Disulfides).
[0312] When more than one disulfide was present, selective
oxidation was often performed. Oxidation of the free cysteines was
achieved at pH 7.6 NH.sub.4CO.sub.3 solution at 1 mg/10 mL of
peptide. After 24 h stirring and prior to purification the solution
was acidified to pH 3 with TFA followed by lyophilization. The
resulting single oxidized peptides (with ACM protected cysteines)
were then oxidized/selective deprotection using iodine solution.
The peptide (1 mg per 2 mL) was dissolved in MeOH/H.sub.2O, 80:20
iodine dissolved in the reaction solvent was added to the reaction
(final concentration: 5 mg/mL) at room temperature. The solution
was stirred for 7 minutes before ascorbic acid was added portion
wise until the solution is clear. The solution was then loaded
directly onto the HPLC.
[0313] Method C (Native Oxidation).
[0314] When more than one disulfide was present and when not
performing selective oxidations, native oxidation was performed.
Native oxidation was achieved with 100 mM NH4CO3 (pH7.4) solution
in the presence of oxidized and reduced glutathione
(peptide/GSH/GSSG, 1:100:10 molar ratio) of (peptide: GSSG: GSH,
1:10, 100). After 24 h stirring and prior to RP-HPLC purification
the solution was acidified to pH 3 with TFA followed by
lyophilization.
[0315] Procedure of Cysteine Oxidation to Produce Dimers.
[0316] Oxidation of the unprotected peptides of the invention was
achieved by adding drop-wise iodine in MeOH (1 mg per 1 mL) to the
peptide in a solution (ACN: H2O, 7: 3, 0.5% TFA). After stirring
for 2 min, ascorbic acid portion wise was added until the solution
was clear and the sample was immediately loaded onto the HPLC for
purification.
[0317] Procedure for Dimerization.
[0318] Glyoxylic acid (DIG), IDA, or Fmoc-.beta.-Ala-IDA was
pre-activated as the N-hydoxysuccinimide ester by treating 1
equivalent (abbreviated "eq") of the acid with 2.2 eq of both
N-hydoxysuccinimide (NHS) and dicyclohexyl carbodiimide (DCC) in
NMP (N-methyl pyrolidone) at a 0.1 M final concentration. For the
PEG13 and PEG25 linkers, these chemical entities were purchased
pre-formed as the activated succinimide ester. The activated ester
.about.0.4 eq was added slowly to the peptide in NMP (1 mg/mL)
portionwise. The solution was left stirring for 10 min before 2-3
additional aliquots of the linker .about.0.05 eq were slowly added.
The solution was left stirring for a further 3 h before the solvent
was removed under vacuo and the residue was purified by reverse
phase HPLC. An additional step of stirring the peptide in 20%
piperidine in DMF (2.times.10 min) before an additional reverse
phase HPLC purification was performed.
[0319] One of skill in the art will appreciate that standard
methods of peptide synthesis may be used to generate the compounds
of the invention.
[0320] Linker Activation and Dimerization
[0321] Peptide monomer subunits were linked to form hepcidin
analogue peptide dimers as described below.
[0322] Small Scale DIG Linker Activation Procedure:
[0323] 5 mL of NMP was added to a glass vial containing IDA diacid
(304.2 mg, 1 mmol), N-hydroxysuccinimide (NHS, 253.2 mg, 2.2 eq.
2.2 mmol) and a stirring bar. The mixture was stirred at room
temperature to completely dissolve the solid starting materials. N,
N'-Dicyclohexylcarbodiimide (DCC, 453.9 mg, 2.2 eq., 2.2 mmol) was
then added to the mixture. Precipitation appeared within 10 min and
the reaction mixture was further stirred at room temperature
overnight. The reaction mixture was then filtered to remove the
precipitated dicyclohexylurea (DCU). The activated linker was kept
in a closed vial prior to use for dimerization. The nominal
concentration of the activated linker was approximately 0.20 M.
[0324] For dimerization using PEG linkers, there was no
pre-activation step involved. Commercially available pre-activated
bi-functional PEG linkers were used.
[0325] Dimerization Procedure:
[0326] 2 mL of anhydrous DMF was added to a vial containing peptide
monomer (0.1 mmol). The pH of the peptide was the adjusted to 8-9
with DIEA. Activated linker (IDA or PEG13, PEG 25) (0.48 eq
relative to monomer, 0.048 mmol) was then added to the monomer
solution. The reaction mixture was stirred at room temperature for
one hour. Completion of the dimerization reaction was monitored
using analytical HPLC. The time for completion of dimerization
reaction varied depending upon the linker. After completion of
reaction, the peptide was precipitated in cold ether and
centrifuged. The supernatant ether layer was discarded. The
precipitation step was repeated twice. The crude dimer was then
purified using reverse phase HPLC (Luna C18 support, 10 u, 100 A,
Mobile phase A: water containing 0.1% TFA, mobile phase B:
Acetonitrile (ACN) containing 0.1% TFA, gradient of 15% B and
change to 45% B over 60 min, flow rate 15 ml/min). Fractions
containing pure product were then freeze-dried on a
lyophilizer.
[0327] Conjugation of Half-Life Extension Moieties
[0328] Conjugation of peptides were performed on resin. Lys(ivDde)
was used as the key amino acid. After assembly of the peptide on
resin, selective deprotection of the ivDde group occurred using
3.times.5 min 2% hydrazine in DMF for 5 min. Activation and
acylation of the linker using HBTU, DIEA 1-2 equivalents for 3 h,
and Fmoc removal followed by a second acylation with the lipidic
acid gave the conjugated peptide.
Example 2
Activity of Peptide Analogues
[0329] Peptide analogues were tested in vitro for induction of
internalization of the human ferroportin protein. Following
internalization, the peptides are degraded. The assay measures a
decrease in fluorescence of the receptor.
[0330] The cDNA encoding the human ferroportin (SLC40 A1) was
cloned from a cDNA clone from Origene (NM_014585). The DNA encoding
the ferroportin was amplified by PCR using primers also encoding
terminal restriction sites for subcloning, but without the
termination codon. The ferroportin receptor was subcloned into a
mammalian GFP expression vector containing a neomycin (G418)
resistance marker in such that the reading frame of the ferroportin
was fused in frame with the GFP protein. The fidelity of the DNA
encoding the protein was confirmed by DNA sequencing. HEK293 cells
were used for transfection of the ferroportin-GFP receptor
expression plasmid. The cells were grown according to standard
protocol in growth medium and transfected with the plasmids using
Lipofectamine (manufacturer's protocol, Invitrogen). The cells
stably expressing ferroportin-GFP were selected using G418 in the
growth medium (in that only cells that have taken up and
incorporated the cDNA expression plasmid survive) and sorted
several times on a Cytomation MoFlo.TM. cell sorter to obtain the
GFP-positive cells (488 nm/530 nm). The cells were propagated and
frozen in aliquots.
[0331] To determine activity of the hepcidin analogues (compounds)
on the human ferroportin, the cells were incubated in 96 well
plates in standard media, without phenol red. Compound was added to
desired final concentration for at least 18 hours in the incubator.
Following incubation, the remaining GFP-fluorescence was determined
either by whole cell GFP fluorescence (Envision plate reader,
485/535 filter pair), or by Beckman Coulter Quanta.TM. flow
cytometer (express as Geometric mean of fluorescence intensity at
485 nm/525 nm). Compound was added to desired final concentration
for at least 18 hours but no more than 24 hours in the
incubator.
[0332] In certain experiments, reference compounds included native
Hepcidin, Mini-Hepcidin, and R1-Mini-Hepcidin, which is an analog
of mini-hepcidin. The "RI" in RI-Mini-Hepcidin refers to Retro
Inverse. A retro inverse peptide is a peptide with a reversed
sequence in all D amino acids. An example is that
Hy-Glu-Thr-His-NH2 becomes Hy-DHis-DThr-DGlu-NH2. The EC.sub.50 of
these reference compounds for ferroportin degradation was
determined according to the activity assay described above. These
peptides served as control standards.
TABLE-US-00021 TABLE 9 Reference compounds Potency EC50 Name
Sequence (nM) Hepcidin
Hy-DTHFPIC(1)IFC(2)C(3)GC(2)C(4)HRSKC(3)GMC(4)C(1) 34 KT-OH (SEQ ID
NO: 39) Mini- Hy-DTHFPICIF-NH.sub.2 (SEQ ID NO: 40) 712 Hepcidin
1-9 RI-Mini Hy-DPhe-DIle-DCys-DIle-DPro-DPhe-DHis-DThr-DAsp- >10
.mu.M Hepcidin NH.sub.2 (SEQ ID NO: 41)
[0333] The potency EC.sub.50 values (nM) determined for various
peptide analogues of the present invention are provided in Tables 3
and 4. These values were determined as described herein.
Example 3
In Vivo Validation of Peptide Analogues
[0334] Hepcidin analogues of the present invention were tested for
in vivo activity, to determine their ability to decrease free Fe2+
in serum.
[0335] A hepcidin analogue (Compound 1) or vehicle control were
administered to mice (n=3/group) at 1000 nmol/kg either
intravenously or subcutaneously. Serum samples were taken from
groups of mice administered with the hepcidin analog at 30 min, 1
h, 2 h, 4 h, 10 h, 24 h, 30 h, 36 h, and 48 h post-administration.
Iron content in plasma/serum was measured using a colorimetric
assay on the Cobas c 111 according to instructions from the
manufacturer of the assay (assay: IRON2: ACN 661). The data
obtained from the cobas Iron2 analysis is presented in FIG. 1A
(intravenous administration) and FIG. 1B (subcutaneous
administration) as mean values+/-SEM.
[0336] In another experiment, various hepcidin analogues or vehicle
control were administered to mice (n=3/group) at 1000 nmol/kg
subcutaneously. Serum samples were taken from groups of mice
administered with vehicle or hepcidin analog at 30 h and 36 h
post-administration. Iron content in plasma/serum was measured
using a colorimetric assay on the Cobas c 111 according to
instructions from the manufacturer of the assay (assay: IRON2: ACN
661). The data obtained from the cobas Iron2 analysis is presented
in FIG. 2 as mean values+/-SEM.
[0337] These studies demonstrate that hepcidin analogues of the
present invention reduce serum iron levels for at least 30 hours,
thus demonstrating their increased serum stability.
[0338] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0339] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Sequence CWU 1
1
41110PRTArtificial SequenceHepcidin analogue peptide
formulaMOD_RES(1)..(1)Xaa is Asp, isoGlu or IdaMOD_RES(4)..(4)Xaa
is Phe, Phe(4-F), Phe(4-CN), 4-BIP, Phe(4-OCH3), Tyr,
Phe(2,3-(OCH3)2), Phe(2,3-Cl2), or DpaMOD_RES(6)..(6)Xaa is Cys or
PenMOD_RES(7)..(7)Xaa is any amino acidmisc_feature(8)..(8)Xaa can
be any naturally occurring amino acidMOD_RES(10)..(10)Xaa is Lys,
Glu or absent 1Xaa Thr His Xaa Pro Xaa Xaa Xaa Phe Xaa 1 5 10
27PRTArtificial SequenceHepcidin analogue peptide
formulaMOD_RES(1)..(5)Xaa is any amino acidMOD_RES(6)..(6)Xaa is
Cys or PenMOD_RES(7)..(7)Xaa is Lys or absent 2Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 310PRTArtificial SequenceHepcidin analogue peptide
formulaMOD_RES(4)..(4)Phe or DpaMOD_RES(6)..(6)Cys or
PenMOD_RES(7)..(7)Ile or LysMOD_RES(8)..(8)Lys or
ArgMOD_RES(10)..(10)Lys, Glu or absent 3Asp Thr His Xaa Pro Xaa Xaa
Xaa Phe Xaa 1 5 10 47PRTArtificial SequenceHepcidin analogue
peptide formulaMOD_RES(5)..(5)Gly or SarMOD_RES(6)..(6)Cys or
PenMOD_RES(7)..(7)Lys or absent 4Pro Arg Ser Lys Xaa Xaa Xaa 1 5
517PRTArtificial SequenceSynthesized hepcidin analogue 5Asp Thr His
Phe Pro Cys Ile Lys Phe Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys
617PRTArtificial SequenceSynthesized hepcidin analogue 6Asp Thr His
Phe Pro Cys Ile Lys Phe Glu Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys
717PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(8)..(8)IsoGlu-Palm conjugated half-life extension
moiety 7Asp Thr His Phe Pro Cys Ile Lys Phe Glu Pro Arg Ser Lys Gly
Cys 1 5 10 15 Lys 817PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IsoGlu-Palm conjugated half-life extension
moiety 8Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg Ser Lys Gly
Cys 1 5 10 15 Lys 917PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(6)..(6)PenMOD_RES(10)..(10)IsoGlu-Palm conjugated
half-life extension moietyMOD_RES(16)..(16)Pen 9Asp Thr His Phe Pro
Xaa Ile Lys Phe Lys Pro Arg Ser Lys Gly Xaa 1 5 10 15 Lys
1017PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(6)..(6)PenMOD_RES(10)..(10)IsoGlu-Palm conjugated
half-life extension moiety 10Asp Thr His Phe Pro Xaa Ile Lys Phe
Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys 1117PRTArtificial
SequenceSynthesized hepcidin analogueMOD_RES(10)..(10)IsoGlu-Palm
conjugated half-life extension moietyMOD_RES(16)..(16)Pen 11Asp Thr
His Phe Pro Cys Ile Lys Phe Lys Pro Arg Ser Lys Gly Xaa 1 5 10 15
Lys 1217PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IsoGlu-Palm conjugated half-life extension
moietyMOD_RES(15)..(15)Sar (sarcosine0 12Asp Thr His Phe Pro Cys
Ile Lys Phe Lys Pro Arg Ser Lys Xaa Cys 1 5 10 15 Lys
1317PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(4)..(4)beta, beta
diphenylalanineMOD_RES(10)..(10)IsoGlu-Palm conjugated half-life
extension moiety 13Asp Thr His Xaa Pro Cys Ile Lys Phe Lys Pro Arg
Ser Lys Gly Cys 1 5 10 15 Lys 1417PRTArtificial SequenceSynthesized
hepcidin analogueMOD_RES(4)..(4)beta, beta
diphenylalanineMOD_RES(10)..(10)IsoGlu-Palm conjugated half-life
extension moietyMOD_RES(15)..(15)Sar (sarcosine) 14Asp Thr His Xaa
Pro Cys Ile Lys Phe Lys Pro Arg Ser Lys Xaa Cys 1 5 10 15 Lys
1516PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IsoGlu-Palm conjugated half-life extension
moiety 15Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg Ser Lys
Gly Cys 1 5 10 15 1616PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IsoGlu-Palm conjugated half-life extension
moietyMOD_RES(15)..(15)Sar (sarcosine0 16Asp Thr His Phe Pro Cys
Ile Lys Phe Lys Pro Arg Ser Lys Xaa Cys 1 5 10 15 1717PRTArtificial
SequenceSynthesized hepcidin analogueMOD_RES(10)..(10)IsoGlu-Palm
conjugated half-life extension moiety 17Asp Thr His Phe Pro Cys Lys
Lys Phe Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys 1817PRTArtificial
SequenceSynthesized hepcidin analogueMOD_RES(10)..(10)IsoGlu-Lauric
acid conjugated half-life extension moiety 18Asp Thr His Phe Pro
Cys Ile Lys Phe Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys
1917PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IsoGlu-Myristic acid conjugated half-life
extension moiety 19Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg
Ser Lys Gly Cys 1 5 10 15 Lys 2017PRTArtificial SequenceSynthesized
hepcidin analogueMOD_RES(10)..(10)IsoGlu-Bioitin conjugated
half-life extension moiety 20Asp Thr His Phe Pro Cys Ile Lys Phe
Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys 2117PRTArtificial
SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IsoGlu-Isovaleric acid conjugated
half-life extension moiety 21Asp Thr His Phe Pro Cys Ile Lys Phe
Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys 2217PRTArtificial
SequenceSynthesized hepcidin analogueMOD_RES(10)..(10)PEG2-Palm
conjugated half-life extension moiety 22Asp Thr His Phe Pro Cys Ile
Lys Phe Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys 2317PRTArtificial
SequenceSynthesized hepcidin analogueMOD_RES(10)..(10)PEG11-Palm
conjugated half-life extension moiety 23Asp Thr His Phe Pro Cys Ile
Lys Phe Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys 2417PRTArtificial
SequenceSynthesized hepcidin analogueMOD_RES(10)..(10)PEG11-Lauric
acid conjugated half-life extension moiety 24Asp Thr His Phe Pro
Cys Ile Lys Phe Lys Pro Arg Ser Lys Gly Cys 1 5 10 15 Lys
2517PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)PEG11-Myristic acid conjugated half-life
extension moiety 25Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg
Ser Lys Gly Cys 1 5 10 15 Lys 2617PRTArtificial SequenceSynthesized
hepcidin analogueMOD_RES(10)..(10)Palm conjugated half-life
extension moiety 26Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg
Ser Lys Gly Cys 1 5 10 15 Lys 2717PRTArtificial SequenceSynthesized
hepcidin analogueMOD_RES(10)..(10)PEG8 conjugated half-life
extension moiety 27Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg
Ser Lys Gly Cys 1 5 10 15 Lys 2817PRTArtificial SequenceSynthesized
hepcidin analogueMOD_RES(10)..(10)Ahx-Palm conjugated half-life
extension moiety 28Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg
Ser Lys Gly Cys 1 5 10 15 Lys 2917PRTArtificial SequenceSynthesized
hepcidin analogueMOD_RES(8)..(8)Ahx-Palm conjugated half-life
extension moietyMOD_RES(15)..(15)Sar (sarcosine) 29Asp Thr His Phe
Pro Cys Ile Lys Phe Glu Pro Arg Ser Lys Xaa Cys 1 5 10 15 Lys
3017PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)Ahx-Palm conjugated half-life extension
moietyMOD_RES(15)..(15)Sar (sarcosine) 30Asp Thr His Phe Pro Cys
Ile Lys Phe Lys Pro Arg Ser Lys Xaa Cys 1 5 10 15 Lys
3117PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)Ac conjugated half-life extension moiety
31Asp Thr His Phe Pro Cys Ile Lys Phe Lys Pro Arg Ser Lys Gly Cys 1
5 10 15 Lys 3217PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(8)..(8)PEG11-Palm conjugated half-life extension
moietyMOD_RES(15)..(15)Sar (sarcosine) 32Asp Thr His Phe Pro Cys
Ile Lys Phe Glu Pro Arg Ser Lys Xaa Cys 1 5 10 15 Lys
3317PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)PEG11-Palm conjugated half-life extension
moietyMOD_RES(15)..(15)Sar (sarcosine) 33Asp Thr His Phe Pro Cys
Ile Lys Phe Lys Pro Arg Ser Lys Xaa Cys 1 5 10 15 Lys
3417PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(8)..(8)isoGlu-Ahx-Palm conjugated half-life
extension moietyMOD_RES(15)..(15)Sar (sarcosine) 34Asp Thr His Phe
Pro Cys Ile Lys Phe Glu Pro Arg Ser Lys Xaa Cys 1 5 10 15 Lys
3517PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(8)..(8)OEG-OEG-isoGlu-(C18-diacid) conjugated
half-life extension moietyMOD_RES(15)..(15)Sar (sarcosine) 35Asp
Thr His Phe Pro Cys Ile Lys Phe Glu Pro Arg Ser Lys Xaa Cys 1 5 10
15 Lys 3610PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IsoGlu-Octanoic acid conjugated half-life
extension moiety 36Asp Thr His Phe Pro Cys Ile Arg Phe Lys 1 5 10
3711PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IDA (Iminodiacetic
acid)MOD_RES(11)..(11)beta alanine with Peg2-Palm conjugated
half-life extension moiety 37Asp Thr His Phe Pro Cys Ile Lys Phe
Xaa Xaa 1 5 10 3811PRTArtificial SequenceSynthesized hepcidin
analogueMOD_RES(10)..(10)IDA (Iminodiacetic
acid)MOD_RES(11)..(11)beta alanine with Peg11-Palm conjugated
half-life extension moiety 38Asp Thr His Phe Pro Cys Ile Lys Phe
Xaa Xaa 1 5 10 3925PRTHomo sapiens 39Asp Thr His Phe Pro Ile Cys
Ile Phe Cys Cys Gly Cys Cys His Arg 1 5 10 15 Ser Lys Cys Gly Met
Cys Cys Lys Thr 20 25 409PRTHomo sapiens 40Asp Thr His Phe Pro Ile
Cys Ile Phe 1 5 419PRTArtificial SequenceSynthesized retro inverse
peptide with D amino acidsMOD_RES(1)..(9)All amino acids are D
amino acids 41Phe Ile Cys Ile Pro Phe His Thr Asp 1 5
* * * * *