U.S. patent application number 13/793116 was filed with the patent office on 2013-08-08 for mini-hepcidin peptides and methods of using thereof.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFONIRA. The applicant listed for this patent is The Regents of the University of Califonira. Invention is credited to Tomas Ganz, Elizabeta Nemeth, Gloria Preza, Piotr Ruchala.
Application Number | 20130203662 13/793116 |
Document ID | / |
Family ID | 42233878 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130203662 |
Kind Code |
A1 |
Ganz; Tomas ; et
al. |
August 8, 2013 |
Mini-Hepcidin Peptides and Methods of Using Thereof
Abstract
Disclosed herein are peptides which exhibit hepcidin activity
and methods of making and using thereof.
Inventors: |
Ganz; Tomas; (Los Angeles,
CA) ; Nemeth; Elizabeta; (Sherman Oaks, CA) ;
Preza; Gloria; (Montebello, CA) ; Ruchala; Piotr;
(Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of Califonira; |
Oakland |
CA |
US |
|
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFONIRA
Oakland
CA
|
Family ID: |
42233878 |
Appl. No.: |
13/793116 |
Filed: |
March 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13131792 |
May 31, 2011 |
8435941 |
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PCT/US09/66711 |
Dec 4, 2009 |
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13793116 |
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61120277 |
Dec 5, 2008 |
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Current U.S.
Class: |
514/5.4 ;
530/317; 530/328; 530/350; 530/387.3 |
Current CPC
Class: |
A61P 3/00 20180101; C07K
14/575 20130101; A61K 38/00 20130101; C07K 7/06 20130101 |
Class at
Publication: |
514/5.4 ;
530/328; 530/317; 530/350; 530/387.3 |
International
Class: |
C07K 7/06 20060101
C07K007/06 |
Goverment Interests
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with Government support of Grant
Nos. DK 075378 and DK 065029, awarded by the National Institutes of
Health. The Government has certain rights in this invention.
Claims
1. An isolated peptide having the following structural formula
A1-A2-A3-A4-A5-A6-A7-A8-A9-A10 wherein A1 is Asp, Glu,
pyroglutamate, Gln, Asn, or an unnatural amino acid commonly used
as a substitute thereof; A2 is Thr, Ser, Val, Ala, or an unnatural
amino acid commonly used as a substitute thereof; A3 is His, Asn,
Arg, or an unnatural amino acid commonly used as a substitute
thereof; A4 is Phe, Leu, Ile, Trp, Tyr, or an unnatural amino acid
commonly used as a substitute thereof which includes
cyclohexylalanine; A5 is Pro, Ser, or an unnatural amino acid
commonly used as a substitute thereof; A6 is Ile, Leu, Val, or an
unnatural amino acid commonly used as a substitute thereof; A7 is
Cys, Ser, Ala, or an unnatural amino acid commonly used as a
substitute thereof which includes S-tertiary butyl-cysteine; A8 is
Ile, Leu, Thr, Val, Arg, or an unnatural amino acid commonly used
as a substitute thereof; A9 is Phe, Leu, Ile, Tyr, or an unnatural
amino acid commonly used as a substitute thereof which includes
cyclohexylalanine; and A10 is Cys, Ser, Ala, or an unnatural amino
acid commonly used as a substitute thereof; wherein the
carboxy-terminal amino acid is in amide or carboxy-form; wherein at
least one sulfhydryl amino acid is present as one of the amino
acids in the sequence; and wherein A1, A2, A3, A1 to A2, A1 to A3,
A10, A9 to A10, A8 to A10, or a combination thereof are optionally
absent.
2. The peptide of claim 1, wherein A1 is D-Asp, D-Glu,
D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida, or N-MeAsp; A2 is D-Thr,
D-Ser, D-Val, Tle, Inp, Chg, bhThr, or N-MeThr; A3 is D-His, D-Asn,
DArg, Dpa, (D)Dpa, or 2-aminoindan; A4 is D-Phe, D-Leu, D-Ile,
D-Trp, Phg, bhPhe, Dpa, Bip, 1Nal, bhDpa, Amc, PheF5, hPhe, Igl, or
cyclohexylalanine; A5 is D-Pro, D-Ser, Oic, bhPro, trans-4-PhPro,
cis-4-PhPro, cis-5-PhPro, Idc; A6 is D-Ile, D-Leu, Phg, Chg, Amc,
bhIle, Ach, and MeIle; A7 is D-Cys, D-Ser, D-Ala, Cys(S-tBut),
homoC, Pen, (D)Pen, Dap(AcBr), and Inp; A8 is D-Ile, D-Leu, D-Thr,
D-Val, D-Arg, Chg, Dpa, bhIle, Ach, or MeIle; A9 is D-Phe, D-Leu,
D-Ile, PheF5, N-MePhe, benzylamide, bhPhe, Dpa, Bip, 1Nal, bhDpa,
cyclohexylalanine; or A10 is D-Cys, D-Ser, D-Ala; or a combination
thereof.
3. The peptide of claim 1, wherein A1 is Ala, D-Ala, Cys, D-Cys,
Phe, D-Phe, Asp or D-Asp linked to Cys or D-Cys, Phe or D-Phe
linked to a PEG molecule linked to chenodeoxycholate,
ursodeoxycholate, or palmitoyl, or Dpa or (D)Dpa linked to
palmitoyl; A2 is Ala, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, or D-Gly;
A3 is Ala, D-Ala, Cys, D-Cys, Dpa, Asp or D-Asp linked to Dpa or
(D)Dpa; A4 is Ala, D-Ala, Pro, or D-Pro; A5 is Ala, D-Ala, Pro,
D-Pro, Arg, D-Arg; A6 is Ala, D-Ala, Phe, D-Phe, Arg, D-Arg, Cys,
D-Cys; A7 is His, or D-His; A8 is Cys, or D-Cys; or A9 is Phe or
D-Phe linked to RA, Asp, D-Asp, Asp or D-Asp linked to RB, bhPhe
linked to RC, or cysteamide, wherein RA is
--CONH.sub.2--CH.sub.2--CH.sub.2--S, -D-Pro linked to Pro-Lys or
Pro-Arg, -bhPro linked to Pro linked to Pro-Lys or Pro-Arg, -D-Pro
linked to bhPro-Lys or bhPro-Arg, wherein RB is
-PEG11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, -(PEG11)-(GPHyp)10, and wherein
RC is -D-Pro linked to Pro-Lys or Pro-Arg, -D-Pro linked to
bhPro-Lys or bhPro-Arg; or a combination thereof.
4. The peptide of claim 1, wherein A1 is Asp; A2 is Thr; A3 is His;
A4 is Phe; A5 is Pro; A6 is Ile; A7 is Ala; A8 is Ile; A9 is Phe;
and A10 is Cys in amide form; wherein A1 or A1 to A2 are optionally
absent.
5. The peptide of claim 1, wherein A1 is Asp, A2 is Thr, A3 is His,
A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol
amino acid, A8 is Ile, A9 is Phe in amide form, and A10 is
absent.
6. The peptide of claim 1, wherein A1 and A2 are absent, A3 is His,
A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol
amino acid, A8 is Ile in amide form, and A9 and A10 are absent.
7. The peptide of claim 1, wherein A1 and A2 are absent, A3 is His,
A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol
amino acid in amide form, and A8 to A10 are absent.
8. The peptide of claim 1, wherein the peptide is a cyclic
peptide.
9. The peptide of according to claim 1, wherein the sequence is
retroinverted such that A1 is the C-terminus and A10 is the
N-terminus.
10. The peptide according to claim 1, wherein the peptide has an
addition at the N-terminus, C-terminus, or both.
11. The peptide according to claim 1, wherein the peptide is
selected from the group consisting of: Hep3-8, Hep3-9, Hep1-8,
Hep1-9, Hep1-10 C7A, Hep9F4A, Hep9C7-SStBut, (D)C, homoC, Pen,
(D)Pen, Cyc-1, Pr10, Pr11, Pr12, riHep7.DELTA.DT, Pr23, Pr24, Pr25,
Pr27, Pr28, F4bhPhe, F4Dpa, F4Bip, F4 1Nal, F4bhDpa, F9bhPhe,
F9Dpa, F9Bip, F91Nal, F9bhDpa, Pr39, Pr40, Pr41, Pr42, Pr43, Pr44,
Pr45, Pr46, Pr13, Pr14, Pr15, Pr16, Pr17, Pr18, Pr19, Pr20, Pr21,
Pr22, Pr-1, Pr-2, Pr-3, and Pr-4.
12. The peptide according to claim 1, wherein the peptide exhibits
hepcidin activity.
13. The peptide according to claim 1, wherein the peptide binds
ferroportin.
14. A composition which comprises at least one peptide according to
claim 1.
15. A method of binding a ferroportin or inducing ferroportin
internalization and degradation which comprises contacting the
ferroportin with at least one peptide according to any one of claim
1 or the composition according to claim 14.
16. A method of treating a disease of iron metabolism in a subject
which comprises administering at least one peptide according to
claim 1 or the composition according to claim 14 to the
subject.
17. The method of claim 16, wherein the disease of iron metabolism
is an iron overload disease.
18. A kit comprising at least one peptide according to claim 1 or
the composition according to claim 14 packaged together with a
reagent, a device, instructional material, or a combination
thereof.
19. A complex comprising at least one peptide according to claim 1
bound to a ferroportin or an antibody.
20. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/120,277, filed 5 Dec. 2008, which is herein
incorporated by reference in its entirety. FPIC
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention generally relates to peptides which
exhibit hepcidin activity.
[0005] 2. Description of the Related Art
[0006] Hepcidin, 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, and 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. (2009) 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.
[0007] Abnormal hepcidin activity is associated with iron overload
diseases which include hereditary hemochromatosis and iron-loading
anemias. Hereditary hemochromatosis (HH) is a genetic iron overload
disease that is mainly caused by hepcidin deficiency, or very
rarely 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 (hepatic cirrhosis,
hepatocellular carcinoma), diabetes, and heart failure. Currently,
the only treatment for HH is regular phlebotomy, which is effective
but very burdensome for the patients.
[0008] 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 untransfused
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.
SUMMARY OF THE INVENTION
[0009] The present invention generally relates to peptides which
exhibit hepcidin activity and methods of using thereof.
[0010] The present invention provides peptides, which may be
isolated and/or purified, comprising, consisting essentially or
consisting of the following structural formula
A1-A2-A3-A4-A5-A6-A7-A8-A9-A10
wherein [0011] A1 is Asp, Glu, pyroglutamate, Gln, Asn, or an
unnatural amino acid commonly used as a substitute thereof; [0012]
A2 is Thr, Ser, Val, Ala or an unnatural amino acid commonly used
as a substitute thereof; [0013] A3 is His, Asn, Arg, or an
unnatural amino acid commonly used as a substitute thereof; [0014]
A4 is Phe, Leu, Ile, Trp, Tyr or an unnatural amino acid commonly
used as a substitute thereof which includes cyclohexylalanine;
[0015] A5 is Pro, Ser, or an unnatural amino acid commonly used as
a substitute thereof; [0016] A6 is Ile, Leu, Val, or an unnatural
amino acid commonly used as a substitute thereof; [0017] A7 is Cys,
Ser, Ala, or an unnatural amino acid commonly used as a substitute
thereof which includes S-tertiary butyl-cysteine; [0018] A8 is Ile,
Leu, Thr, Val, Arg, or an unnatural amino acid commonly used as a
substitute thereof; [0019] A9 is Phe, Leu, Ile, Tyr or an unnatural
amino acid commonly used as a substitute thereof which includes
cyclohexylalanine; and [0020] A10 is Cys, Ser, Ala, or an unnatural
amino acid commonly used as a substitute thereof;
[0021] wherein the carboxy-terminal amino acid is in amide or
carboxy-form;
[0022] wherein at least one sulfhydryl amino acid is present as one
of the amino acids in the sequence; and
[0023] wherein A1, A2, A3, A1 to A2, A1 to A3, A10, A9 to A10, A8
to A10, or a combination thereof are optionally absent,
[0024] with the proviso that the peptide does not consist of amino
acid residues 1 to 6 of Hep25.
[0025] In some embodiments, the peptides of the present invention
are not Hep4-7, Hep3-7, Hep1-7, Hep9C7-tBut, Hep9-C7A, Hep9-7CS,
(D)Pen, Cyc-2, Cyc-3, Cyc-4, or Pr26.
[0026] In some embodiments, the peptides of the present invention
contain only one amino acid residue having a thiol capable of
forming a disulfide bond.
[0027] In some embodiments, the peptides of the present invention
contain only two amino acid residues which each have a thiol
capable of forming a disulfide bond.
[0028] In some embodiments, [0029] A1 is D-Asp, D-Glu,
D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida, or N-MeAsp; [0030] A2 is
D-Thr, D-Ser, D-Val, Tie, Inp, Chg, bhThr, or N-MeThr; [0031] A3 is
D-His, D-Asn, DArg, Dpa, (D)Dpa, or 2-aminoindan; [0032] A4 is
D-Phe, D-Leu, D-Ile, D-Trp, Phg, bhPhe, Dpa, Bip, 1Nal, bhDpa, Amc,
PheF5, hPhe, Igl, or cyclohexylalanine; [0033] A5 is D-Pro, D-Ser,
Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, Idc; [0034] A6
is D-Ile, D-Leu, Phg, Chg, Amc, bhIle, Ach, and MeIle; [0035] A7 is
D-Cys, D-Ser, D-Ala, Cys(S-tBut), homoC, Pen, (D)Pen, Dap(AcBr),
and Inp; [0036] A8 is D-Ile, D-Leu, D-Thr, D-Val, D-Arg, Chg, Dpa,
bhIle, Ach, or MeIle; [0037] A9 is D-Phe, D-Leu, D-Ile, PheF5,
N-MePhe, benzylamide, bhPhe, Dpa, Bip, 1Nal, bhDpa,
cyclohexylalanine; or [0038] A10 is D-Cys, D-Ser, D-Ala; or a
combination thereof.
[0039] In some embodiments, [0040] A1 is Ala, D-Ala, Cys, D-Cys,
Phe, D-Phe, Asp or D-Asp linked to Cys or D-Cys, Phe or D-Phe
linked to a PEG molecule linked to chenodeoxycholate,
ursodeoxycholate, or palmitoyl, or Dpa or (D)Dpa linked to
palmitoyl; [0041] A2 is Ala, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, or
D-Gly; [0042] A3 is Ala, D-Ala, Cys, D-Cys, Dpa, Asp or D-Asp
linked to Dpa or (D)Dpa; [0043] A4 is Ala, D-Ala, Pro, or D-Pro;
[0044] A5 is Ala, D-Ala, Pro, D-Pro, Arg, D-Arg; [0045] A6 is Ala,
D-Ala, Phe, D-Phe, Arg, D-Arg, Cys, D-Cys; [0046] A7 is His, or
D-His; [0047] A8 is Cys, or D-Cys; or [0048] A9 is Phe or D-Phe
linked to RA, Asp, D-Asp, Asp or D-Asp linked to RB, bhPhe linked
to RC, or cysteamide, wherein RA is
--CONH.sub.2--CH.sub.2--CH.sub.2--S, -D-Pro linked to Pro-Lys or
Pro-Arg, -bhPro linked to Pro linked to Pro-Lys or Pro-Arg, -D-Pro
linked to bhPro-Lys or bhPro-Arg, wherein RB is
-PEG11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, -(PEG11)-(GPHyp)10, and wherein
RC is -D-Pro linked to Pro-Lys or Pro-Arg, -D-Pro linked to
bhPro-Lys or bhPro-Arg; or a combination thereof.
[0049] In some embodiments, A1 is Asp; A2 is Thr; A3 is His; A4 is
Phe; A5 is Pro; A6 is Ile; A7 is Ala; A8 is Ile; A9 is Phe; and A10
is Cys in amide form; wherein A1 or A1 to A2 are optionally
absent.
[0050] In some embodiments, A1 is Asp, A2 is Thr, A3 is His, A4 is
Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol amino
acid, A8 is Ile, A9 is Phe in amide form, and A10 is absent.
[0051] In some embodiments, A1 and A2 are absent, A3 is His, A4 is
Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol amino
acid, A8 is Ile in amide form, and A9 and A10 are absent.
[0052] In some embodiments, A1 and A2 are absent, A3 is His, A4 is
Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol amino
acid in amide form, and A8 to A10 are absent.
[0053] In some embodiments, the peptides are cyclic peptides.
[0054] In some embodiments, the peptides are retroinverted such
that A1 is the amidated C-terminus and A10 is the N-terminus, and
all amino acids are D-amino acids instead of the natural L-amino
acids.
[0055] In some embodiments, the peptides have an addition at the
N-terminus, C-terminus, or both.
[0056] In some embodiments, the peptides are selected from the
group consisting of: Hep3-8, Hep3-9, Hep1-8, Hep1-9, Hep1-10 C7A,
Hep9F4A, Hep9C7-SStBut, (D)C, homoC, Pen, (D)Pen, Cyc-1, Pr10,
Pr11, Pr12, riHep7.DELTA.DT, Pr23, Pr24, Pr25, Pr27, Pr28, F4bhPhe,
F4Dpa, F4Bip, F4 1Nal, F4bhDpa, F9bhPhe, F9Dpa, F9Bip, F91Nal,
F9bhDpa, Pr39, Pr40, Pr41, Pr42, Pr43, Pr44, Pr45, Pr46, Pr13,
Pr14, Pr15, Pr16, Pr17, Pr18, Pr19, Pr20, Pr21, Pr22, Pr-1, Pr-2,
Pr-3, and Pr-4.
[0057] In some embodiments, the peptides exhibit hepcidin activity.
In some embodiments, the peptides bind ferroportin, preferably
human ferroportin.
[0058] In some embodiments, the present invention provides
compositions and medicaments which comprise at least one peptide as
disclosed herein. In some embodiments, the present invention
provides method of manufacturing medicaments for the treatment of
diseases of iron metabolism, such as iron overload diseases, which
comprise at least one peptide as disclosed herein. Also provided
are methods of treating a diseases of iron metabolism in a subject,
such as a mammalian subject, preferably a human subject, which
comprises administering at least one peptide or composition as
disclosed herein to the subject. In some embodiments, the peptide
is administered in a therapeutically effective amount.
[0059] 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 peptide or composition as disclosed herein.
[0060] In some embodiments, the present invention provides kits
comprising at least one peptide or composition as disclosed herein
packaged together with a reagent, a device, instructional material,
or a combination thereof.
[0061] In some embodiments, the present invention provides
complexes which comprise at least one peptide as disclosed herein
bound to a ferroportin, preferably a human ferroportin, or an
antibody, such as an antibody which specifically binds a peptide as
disclosed herein, Hep25, or a combination thereof.
[0062] Both the foregoing general description and the following
detailed description are exemplary and explanatory only and are
intended to provide further explanation of the invention as
claimed. The accompanying drawings are included to provide a
further understanding of the invention and are incorporated in and
constitute part of this specification, illustrate several
embodiments of the invention, and together with the description
serve to explain the principles of the invention.
DESCRIPTION OF THE DRAWINGS
[0063] This invention is further understood by reference to the
drawings wherein:
[0064] FIG. 1 is a graph showing the relative hepcidin activity of
alanine substitutions in Hep25.
[0065] FIG. 2A is a graph showing the relative hepcidin activities
of F4 substitutions in Hep25.
[0066] FIG. 2B is a graph showing the relative hepcidin activities
of F9 substitutions in Hep25.
[0067] FIG. 3A is a graph showing the hepcidin activities of Hep1-9
and Hep1-10 C7A relative to Hep25 (A).
[0068] FIG. 3B is a graph showing the hepcidin activities of Hep1-7
and Hep1-8 relative to Hep1-9 or Hep25.
[0069] FIG. 3C is a graph showing the hepcidin activities of
Hep4-7, Hep3-7, Hep3-8 and Hep3-9 relative to Hep1-9.
[0070] FIG. 4 is a graph showing the hepcidin activities of C7
modified peptides relative to Hep25.
[0071] FIG. 5 is a graph showing in vivo effect (as measured by
serum iron levels in mice) of mini-hepcidins Hep1-9, Pr6 and Pr12
compared to Hep25 or control (PBS).The peptides were injected
intraperitoneally, 50 .mu.g peptide per mouse
[0072] FIG. 6 is a graph showing in vivo effect (as measured by
serum iron levels in mice) of mini-hepcidin Pr27 injected
intraperitoneally (20 and 200 nmoles). The amount of injected Hep25
was 20 nmoles.
[0073] FIG. 7 is a graph showing in vivo effect (as measured by
serum iron levels in mice) of mini-hepcidin riHep7.DELTA.DT
injected intraperitoneally (20 and 200 nmoles). The amount of
injected Hep25 was 20 nmoles.
[0074] FIG. 8 is a graph showing in vivo effect (as measured by
serum iron levels in mice) of mini-hepcidins Pr27 and Pr28 which
were first mixed with liposomes and injected intraperitoneally (20
nmoles). The amount of injected Hep25 was 20 nmoles.
[0075] FIG. 9 is a graph showing in vivo effect (as measured by
serum iron levels in mice) of mini-hepcidin Pr27 after oral
administration by gavage (200 nmoles).
DETAILED DESCRIPTION OF THE INVENTION
[0076] The present invention provides peptides which are useful in
the study and treatment of diseases of iron metabolism.
[0077] 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,
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.
[0078] In some cases the diseases and disorders included in the
definition of "disease of iron metabolism" 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, these diseases are
encompassed under the broad definition. Those skilled in the art
are readily able to determine whether a given disease is a "disease
or iron metabolism" 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.
[0079] In preferred 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.
[0080] As used herein, the terms "protein", "polypeptide" and
"peptide" are used interchangeably to refer to two or more amino
acids linked together. Except for the abbreviations for the
uncommon or unnatural amino acids set forth in Table 2 below, the
three-letter and one-letter abbreviations, as used in the art, are
used herein to represent amino acid residues. Except when preceded
with "D-", the amino acid is an L-amino acid. Groups or strings of
amino acid abbreviations are used to represent peptides. Except
when specifically indicated, peptides are indicated with the
N-terminus on the left and the sequence is written from the
N-terminus to the C-terminus.
[0081] The peptides of the present invention may be made 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 peptides 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 peptides 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
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.
[0082] In some embodiments, the peptides of the present invention
are substantially purified. As used herein, a "substantially
purified" compound refers to a compound that is removed from its
natural environment and is at least about 60% free, preferably
about 75% free, and most preferably about 90% free from other
macromolecular components with which the compound is naturally
associated.
[0083] As used herein, an "isolated" compound refers to a compound
which is isolated from its native environment. For example, an
isolated peptide is a one which does not have its native amino
acids, which correspond to the full length polypeptide, flanking
the N-terminus, C-terminus, or both. For example, isolated Hep1-9
refers to an isolated peptide comprising amino acid residues 1-9 of
Hep25 which may have non-native amino acids at its N-terminus,
C-terminus, or both, but does not have a cysteine amino acid
residue following its 9.sup.th amino acid residue at the
C-terminus. As set forth herein, references to amino acid positions
correspond to the amino acid residues of Hep25. For example,
reference to amino acid position 9, corresponds to the 9.sup.th
amino acid residue of Hep25.
[0084] The peptides of the present invention bind ferroportin,
preferably human ferroportin. Preferred peptides 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.
[0085] The peptides of the present invention that mimic the
hepcidin activity of Hep25, the bioactive human 25-amino acid form,
are herein referred to as "mini-hepcidins". As used herein, a
compound having "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.
[0086] In some embodiments, the peptides 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 vitro activity may be 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.
peptide) that elicits 50% of the maximal loss of fluorescence
generated by the reference Hep25 preparation. EC.sub.50 of Hep25
preparations in this assay range from 5 to 15 nM and preferred
mini-hepcidins have EC.sub.50 values in in vitro activity assays of
about 1,000 nM or less.
[0087] Other methods known in the art for calculating the hepcidin
activity and in vitro activity of peptides according to the present
invention may be used. For example, the in vitro activity of
compounds may be 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, the in vitro activity of compounds
may be 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.
Design of Mini-Hepcidins
[0088] Previous studies indicate that the N-terminal segment of
Hep25 is important for its hepcidin activity and is likely to form
the contact interface with ferroportin. However, the importance of
each N-terminal amino acid to hepcidin activity was unknown.
Therefore, alanine-scanning mutagenesis was performed on residues
1-6 of Hep25 to determine the contribution of each N-terminal amino
acid to hepcidin activity. As shown in FIG. 1, the T2A substitution
did not substantially impact hepcidin activity. Phenylalanine
substitutions (F4A or F9A) caused the largest decrease, more than
about 70%, in hepcidin activity. The remaining alanine
substitutions had detectable decreases in hepcidin activity which
were not as significant as the F4A or F9A substitutions.
[0089] To determine whether the highly conserved and apparently
structurally important F4 phenylalanine is important for hepcidin
activity, the F4 amino acid of Hep25 was systematically substituted
with other amino acids. As shown in FIG. 2A, making the side-chain
more polar (F4Y) led to substantial loss of hepcidin activity as
did the substitution with D-phenylalanine (f) or charged amino
acids (D, K and Y). However, hepcidin activity was maintained when
the F4 residue was substituted with nonaromatic cyclohexylalanine,
thereby indicating that a bulky hydrophobic residue is sufficient
for activity.
[0090] To determine whether the highly conserved and apparently
structurally important F9 phenylalanine is important for hepcidin
activity, the F9 amino acid of Hep25 was substituted with other
amino acids. As shown in FIG. 2B, hepcidin activity not only
decreased when F9 was substituted with alanine, but also when it
was substituted with nonaromatic cyclohexylalanine, thereby
indicating that an aromatic residue may be important for
activity.
[0091] Mutational studies indicate that C326, the cysteine residue
at position 326 of human ferroportin, is the critical residue
involved in binding hepcidin. Thus, various N-terminal fragments of
Hep25 containing a thiol, i.e. Hep4-7, Hep3-7, Hep3-8, Hep3-9,
Hep1-7, Hep1-8, Hep1-9, and Hep 1-10 C7A, were chemically
synthesized, refolded and their activities relative to Hep25 were
assayed using flow-cytometric quantitation of the ferroportin-GFP
degradation, iron efflux estimation based on measurements of
cellular ferritin, and radioisotopic iron efflux studies. The
sequences and EC.sub.50's of these N-terminal fragments are shown
in Table 1.
[0092] Remarkably and unexpectedly, as shown in FIG. 3, Hep1-9 and
Hep 1-10 C7A were found to be quite active in the flow-cytometry
assay of ferroportin-GFP internalization. On a mass basis, Hep1-9
and Hep1-10 C7A were only about 4-times less potent and on a molar
basis, about 10-times less potent than Hep25. Thus, Hep1-9 and
Hep1-10 C7A were used as the basis to construct other peptides
having hepcidin activity.
[0093] To determine the importance of the cysteine thiol on the
hepcidin activity of Hep1-9, the C7 residue of Hep1-9 was
substituted with amino acids that have a similar shape but cannot
form disulfide bonds to give Hep9-C7S (serine substitution) and
Hep9C7-tBut (t-butyl-blocked cysteine) or with a cysteine modified
by disulfide coupled tertiary butyl, which can participate in
disulfide exchange with HS-t-butyl as the leaving group, to give
Hep9C7-SStBut. As shown in FIG. 4, amino acid substitutions that
ablated the potential for disulfide formation or exchange caused a
complete loss of hepcidin activity, thereby indicating that
disulfide formation is required for activity. Other C7 amino acid
substitutions and their resulting hepcidin activities are shown in
Table 1.
[0094] Other peptides based on Hep1-9 and Hep1-10 C7A were
constructed to be disulfide cyclized, have unnatural amino acid
substitutions, be retroinverted, have modified F4 and F9 residues,
or have a positive charge. The C-terminal amino acid was the
amidated form. The modifications and the resulting hepcidin
activities are shown in Table 1.
[0095] As shown in Table 1, with the exception of Pr40 and Pr41,
mini-hepcidins which exhibit EC.sub.50's of about 1000 nM or less
contain at least 6 contiguous amino acid residues which correspond
to residues 3-8 of Hep25 (see Hep3-8). Thus, in some embodiments,
preferred mini-hepcidins have at least 6 contiguous amino acid
residues that correspond to 6 contiguous amino acid residues of
Hep1-9, preferably residues 3-8. The amino acid residues may be
unnatural or uncommon amino acids, L- or D-amino acid residues,
modified residues, or a combination thereof.
[0096] In some embodiments, the mini-hepcidins of the present
invention have at least one amino acid substitution, a
modification, or an addition. Examples of amino acid substitutions
include substituting an L-amino acid residue for its corresponding
D-amino acid residue, substituting a Cys for homoC, Pen, (D)Pen,
Inp, or the like, substituting Phe for bhPhe, Dpa, bhDpa, Bip,
1Nal, and the like. The names and the structures of the
substituting residues are exemplified in Table 2. Other suitable
substitutions are exemplified in Table 1. Examples of a
modification include modifying one or more amino acid residues such
that the peptide forms a cyclic structure, retroinversion, and
modifying a residue to be capable of forming a disulfide bond.
Examples of an addition include adding at least one amino acid
residue or at least one compound to either the N-terminus, the
C-terminus, or both such as that exemplified in Table 1.
[0097] As shown in Table 1, a majority of the mini-hepcidins which
exhibit EC.sub.50's of about 100 nM or less contain at least one
Dpa or bhDPA amino acid substitution. Thus, in some embodiments,
the mini-hepcidins of the present invention have at least one Dpa
or bhDPA amino acid substitution.
[0098] In view of the alanine substitution data of FIG. 1, in some
embodiments, the mini-hepcidins of the present invention may have
an Ala at amino acid positions other than amino acid position 4 and
9 as long as there is an available thiol for forming a disulfide
bond at amino acid position 7. See Hep9F4A and Hep9C-SStBut in
Table 1.
[0099] In view of the position 4 amino acid substitution data of
FIG. 2 and Table 1, the mini-hepcidins of the present invention may
have an amino acid substitution at position 4 which does not result
in a substantial change of its charge or polarity as compared to
that of Hep25, Hep1-9 or Hep 1-10 C7A. Preferred amino acid
substitutions at position 4 of Hep1-9 or Hep1-10 C7A include Phe,
D-Phe, bhPhe, Dpa, bhDpa, Bip, 1Nal, or the like.
[0100] The mini-hepcidins according to the present invention have
the following structural formula
A1-A2-A3-A4-A5-A6-A7-A8-A9-A10
wherein [0101] A1 is Asp, Glu, pyroglutamate, Gln, Asn, or an
unnatural amino acid commonly used as a substitute thereof; [0102]
A2 is Thr, Ser, Val, Ala, or an unnatural amino acid commonly used
as a substitute thereof; [0103] A3 is His, Asn, Arg, or an
unnatural amino acid commonly used as a substitute thereof; [0104]
A4 is Phe, Leu, Ile, Trp, Tyr, or an unnatural amino acid commonly
used as a substitute thereof which includes cyclohexylalanine;
[0105] A5 is Pro, Ser, or an unnatural amino acid commonly used as
a substitute thereof; [0106] A6 is Ile, Leu, Val, or an unnatural
amino acid commonly used as a substitute thereof; [0107] A7 is Cys,
Ser, Ala, or an unnatural amino acid commonly used as a substitute
thereof which includes S-tertiary butyl-cysteine; [0108] A8 is Ile,
Leu, Thr, Val, Arg, or an unnatural amino acid commonly used as a
substitute thereof; [0109] A9 is Phe, Leu, Ile, Tyr, or an
unnatural amino acid commonly used as a substitute thereof which
includes cyclohexylalanine; and [0110] A10 is Cys, Ser, Ala, or an
unnatural amino acid commonly used as a substitute thereof;
[0111] wherein the carboxy-terminal amino acid is in amide or
carboxy-form;
[0112] wherein a Cys or another sulfhydryl amino acid is present as
one of the amino acids in the sequence; and
[0113] wherein A1, A2, A3, A1 to A2, A1 to A3, A10, A9 to A10, A8
to A10, or a combination thereof are optionally absent.
[0114] In some embodiments, A1 is Asp; A2 is Thr; A3 is His; A4 is
Phe; A5 is Pro; A6 is Ile; A7 is Ala; A8 is Ile; A9 is Phe; and A10
is Cys in amide form; wherein A1 or A1 to A2 are optionally
absent.
[0115] In some embodiments, A1 is Asp, A2 is Thr, A3 is His, A4 is
Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol amino
acid, A8 is Ile, A9 is Phe in amide form, and A10 is absent.
[0116] In some embodiments, A1 and A2 are absent, A3 is His, A4 is
Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol amino
acid, A8 is Ile in amide form, and A9 and A10 are absent.
[0117] In some embodiments, A1 and A2 are absent, A3 is His, A4 is
Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol amino
acid in amide form, and A8 to A10 are absent.
[0118] In some embodiments, the unnatural amino acid of A1, A2, A3,
A4, A5, A6, A7, A8, A9, A10, or a combination thereof is the
corresponding D-amino acid. For example, for A1, the unnatural
amino acid may be D-Asp, D-Glu, D-Gln, D-Asn, or the like.
[0119] In some embodiments, the unnatural amino acid for: [0120] A1
is D-Asp, D-Glu, D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida, or
N-MeAsp; [0121] A2 is D-Thr, D-Ser, D-Val, Tle, Inp, Chg, bhThr, or
N-MeThr; [0122] A3 is D-His, D-Asn, DArg, Dpa, (D)Dpa, or
2-aminoindan; [0123] A4 is D-Phe, D-Leu, D-Ile, D-Trp, Phg, bhPhe,
Dpa, Bip, 1Nal, bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine;
[0124] A5 is D-Pro, D-Ser, Oic, bhPro, trans-4-PhPro, cis-4-PhPro,
cis-5-PhPro, Idc; [0125] A6 is D-Ile, D-Leu, Phg, Chg, Amc, bhIle,
Ach, and MeIle; [0126] A7 is D-Cys, D-Ser, D-Ala, Cys(S-tBut),
homoC, Pen, (D)Pen, Dap(AcBr), and Inp; [0127] A8 is D-Ile, D-Leu,
D-Thr, D-Val, D-Arg, Chg, Dpa, bhIle, Ach, or MeIle; [0128] A9 is
D-Phe, D-Leu, D-Ile, PheF5, N-MePhe, benzylamide, bhPhe, Dpa, Bip,
1Nal, bhDpa, cyclohexylalanine; and [0129] A10 is D-Cys, D-Ser,
D-Ala.
[0130] In some embodiments, the amino acid substitution (and
addition, if indicated) for: [0131] A1 is Ala, D-Ala, Cys, D-Cys,
Phe, D-Phe, Asp or D-Asp linked to Cys or D-Cys, Phe or D-Phe
linked to a PEG molecule linked to chenodeoxycholate,
ursodeoxycholate, or palmitoyl, or Dpa or (D)Dpa linked to
palmitoyl; [0132] A2 is Ala, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, or
D-Gly; [0133] A3 is Ala, D-Ala, Cys, D-Cys, Dpa, Asp or D-Asp
linked to Dpa or (D)Dpa; [0134] A4 is Ala, D-Ala, Pro, or D-Pro;
[0135] A5 is Ala, D-Ala, Pro, D-Pro, Arg, D-Arg; [0136] A6 is Ala,
D-Ala, Phe, D-Phe, Arg, D-Arg, Cys, D-Cys; [0137] A7 is His, or
D-His; [0138] A8 is Cys, or D-Cys; and [0139] A9 is Phe or D-Phe
linked to RA, Asp, D-Asp, Asp or D-Asp linked to RB, bhPhe linked
to RC, or cysteamide, wherein RA is
--CONH.sub.2--CH.sub.2--CH.sub.2--S, -D-Pro linked to Pro-Lys or
Pro-Arg, -bhPro linked to Pro linked to Pro-Lys or Pro-Arg, -D-Pro
linked to bhPro-Lys or bhPro-Arg, wherein RB is
-PEG11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, -(PEG11)-(GPHyp)10, and wherein
RC is -D-Pro linked to Pro-Lys or Pro-Arg, -D-Pro linked to
bhPro-Lys or bhPro-Arg.
[0140] In some embodiments, the mini-hepcidin is a 10-mer sequence
wherein A7 is Ala and A10 is Cys.
[0141] In some embodiments, the mini-hepcidin forms a cyclic
structure by a disulfide bond.
[0142] In some embodiments, the mini-hepcidin is a retroinverted
peptide such that A1 is the C-terminus and A10 is the N-terminus
and the amino acid residues are D-amino acids. In some embodiments,
the retroinverted peptide has at least one addition at the
N-terminus, C-terminus, or both. In some embodiments, the
retroinverted peptide contains at least one L-amino acid.
[0143] In some embodiments, the mini-hepcidin has an amino acid
substitution at position 4, position 9, or both. In some
embodiments, the amino acid substituent is Phg, Phe, D-Phe, bhPhe,
Dpa, Bip, 1Nal, Dpa, bhDpa, Amc, or cysteamide.
[0144] In some embodiments, the mini-hepcidin has an amino acid
substitution at position 7. In some embodiments, the amino acid
substituent is Cys(S-tBut), Ala, D-Ala, Ser, D-Ser, homoC, Pen,
(D)Pen, His, D-His, or Inp.
[0145] Examples of some preferred mini-hepcidins according to the
present invention are provided in Table 1.
TABLE-US-00001 TABLE 1 EC.sub.50 Name 1 2 3 4 5 6 7 8 9 10 (nM)
Hep25 10 DTHFPICIFCCGCCHRSKCGMCCKT (SEQ ID NO: 1) Hep10wt D T H F P
I C I F C (SEQ ID NO: 2) Length Hep4 (Hep4-7) -- -- -- F P I C --
-- -- >10,000 (SEQ ID NO: 3) Hep5 (Hep3-7) -- -- H F P I C -- --
-- >10,000 (SEQ ID NO: 4) Hep6 (Hep3-8) -- -- H F P I C I -- --
1000 (SEQ ID NO: 5) Hep7.DELTA.DT (Hep3-9) -- -- H F P I C I F --
700 (SEQ ID NO: 6) Hep7 (Hep1-7) D T H F P I C -- -- -- >10,000
(SEQ ID NO: 7) Hep8 (Hep1-8) D T H F P I C I -- -- 2000 (SEQ ID NO:
8) Hep9 (Hep1-9) D T H F P I C I F -- 76 (SEQ ID NO: 9) Hep10
(Hep1-10 D T H F P I A I F C 100 C7A) (SEQ ID NO: 10) Thiol
Modified Hep9F4A D T H A P I C I F -- >3000 (SEQ ID NO: 11)
Hep9C7-SStBut D T H A P I CS-S-tBut I F -- 700 Hep9C7-tBut D T H A
P I C-tBut I F -- >10,000 Hep9-C7A D T H F P I A I F --
>10,000 (SEQ ID NO: 12) Hep9-C7S D T H F P I S I F -- >10,000
(SEQ ID NO: 13) (D)C D T H F P I C I F -- 1000 homoC D T H F P I
homoC I F -- 900 Pen D T H F P I Pen I F -- 700 (D)Pen D T H F P I
(D)Pen I F -- 3000 Dap(AcBr) D T H F P I Dap(AcBr) I F -- >10000
Disulfide Cyclized Cyc-1 C-D T H F P I C I F -- 300 (SEQ ID NO: 14)
Cyc-4 D T H F P I C I F-R1 -- >10000 Cyc-2 -- C H F P I C I F --
>10000 (SEQ ID NO: 15) Cyc-3 -- -- H F P I C I F-R1 -- >10000
Unnatural AA's Pr10 D Tle H Phg Oic Chg C Chg F -- >3000 Pr11 D
Tle H P Oic Chg C Chg F -- >3000 Retroinverted Pr12 F I C I P F
H T D -- 900* riHep7.DELTA.DT F I C I P F H -- -- -- 150* Modified
Retroinverted Pr23 R2-F I C I P F H T D -- 100 Pr24 R3-F I C I P F
H T D -- 1000* Pr25 F I C I P F H T D-R6 -- 600 Pr26 F I C I P F H
T D-R7 -- >10,000 Pr27 R4-F I C I P F H T D -- 20* Pr28 R5-F I C
I P F H T D -- 3000 Modified F4 and F9 F4bhPhe D T H bhPhe P I C I
F -- 700 F4Dpa D T H Dpa P I C I F -- 30 F4Bip D T H Bip P I C I F
-- 150 F4 1Nal D T H 1Nal P I C I F -- 110 F4bhDpa D T H bhDpa P I
C I F -- 80 F9bhPhe D T H F P I C I bhPhe -- 150 F9Dpa D T H F P I
C I Dpa -- 70 F9Bip D T H F P I C I Bip -- 150 F91Nal D T H F P I C
I 1Nal -- 200 F9bhDpa D T H F P I C I bhDpa -- 100 Pr39 D T H Dpa P
I C I Dpa -- 35 Pr40 D -- Dpa -- P I C I F -- 70 Pr41 D -- Dpa -- P
I C I Dpa -- 300 Pr42 D T H Dpa P R C R Dpa -- 30 Pr43 D T H Dpa P
R C R Dpa -- 200 Pr44 D T H Dpa Oic I C I F -- 30 Pr45 D T H Dpa
Oic I C I Dpa -- 150 Pr46 D T H Dpa P C C C Dpa -- 80 Positive
Charge Pr13 D T H F P I C I F-R8 -- 100 Pr14 D T H F P I C I F-R9
-- 90 Pr15 D T H F P I C I F-R10 -- 150 Pr16 D T H F P I C I F-R11
-- 50 Pr17 D T H F P I C I F-R12 -- 300 Pr18 D T H F P I C I F-R13
-- 1000 Pr19 D T H F P I C I bhPhe-R8 -- 700 Pr20 D T H F P I C I
bhPhe-R9 -- 200 Pr21 D T H F P I C I bhPhe-R12 -- 500 Pr22 D T H F
P I C I bhPhe-R13 -- 600 Pr-1 C Inp (D)Dpa Amc R Amc Inp Dpa
Cysteamide** -- 1500 Pr-2 C P (D)Dpa Amc R Amc Inp Dpa Cysteamide**
-- 2000 Pr-3 C P (D)Dpa Amc R Amc Inp Dpa Cysteamide** -- 1000 Pr-4
C G (D)Dpa Amc R Amc Inp Dpa Cysteamide** -- 2000 R1 =
--CONH.sub.2--CH.sub.2--CH.sub.2--S R2 = Chenodeoxycholate-(PEG11)-
R3 = Ursodeoxycholate-(PEG11)- R4 = Palmitoy1-(PEG11)- R5 =
2(PalmitoyI)-Dap(PEG11)-, wherein "Dap" = diaminopropionic acid R6
= -PEG11-GYIPEAPRDGQAYVRKDGEWVLLSTFL R7 = -(PEG11)-(GPHyp)10,
"GPHyp" = Gly-Pro-hydroxyproline R8 = -PPK R9 = -PPR R10 = -bhProPK
R11 = -bhProPR R12 = -PbhProK R13 = -PbhProR Underlined residues =
D amino acids "--" indicates a covalent bond, e.g. point of
attachment to the given peptide Double underlined = residues
connected by a disulfide link to form a cyclized structure *active
in vivo **oxidized The PEG compound may be PEG11, i.e.
O-(2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol
TABLE-US-00002 TABLE 2 Uncommon or Unnatural Amino Acids
##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036##
[0146] In some embodiments, one or more peptides as described
herein, are provided in the form of a composition which comprises a
carrier suitable for its intended purpose. The compositions may
also include one or more additional ingredients suitable for its
intended purpose. For example, for assays, the compositions may
comprise liposomes, niclosamide, SL220 solubilization agent (NOF,
Japan), cremophor EL (Sigma), ethanol, and DMSO. For treatment of
an iron overload disease, the compositions may comprise different
absorption enhancers and protease inhibitors, solid microparticles
or nanoparticles for peptide encapsulation (such as chitosan and
hydrogels), macromolecular conjugation, lipidization and other
chemical modification.
[0147] The present invention also provides kits comprising one or
more peptides and/or compositions of the present invention packaged
together with reagents, devices, instructional material, or a
combination thereof. For example, the kits may include reagents
used for conducting assays, drugs and compositions for diagnosing,
treating, or monitoring disorders of iron metabolism, devices for
obtaining samples to be assayed, devices for mixing reagents and
conducting assays, and the like.
[0148] As the peptides of the present invention exhibit hepcidin
activity, i.e. act as agonists of ferroportin degradation, they may
be used to treat iron overload diseases. For example, one or more
peptides (preferably at least one mini-hepcidin) according to the
present invention may be administered to a subject to ameliorate
the symptoms and/or pathology associated with iron overload in
iron-loading anemias (especially .beta.-thalassemias) where
phlebotomy is contraindicated and iron chelators are the mainstay
of treatment but are often poorly tolerated. One or more peptides,
preferably at least one mini-hepcidin, according to the present
invention may be used to treat hereditary hemochromatosis,
especially in subjects who do not tolerate maintenance phlebotomy.
One or more peptides, preferably at least one mini-hepcidin,
according to the present invention may be used to treat acute iron
toxicity.
[0149] Thus, one or more peptides of the present invention may be
administered to a subject, preferably a mammal such as a human. In
some embodiments, the peptides are administered in a form of a
pharmaceutical composition. In some embodiments, the peptides are
administered in a therapeutically effective amount. As used herein,
a "therapeutically effective amount" is an amount which ameliorates
the symptoms and/or pathology of a given disease of iron metabolism
as compared to a control such as a placebo.
[0150] A therapeutically effective amount may be readily determined
by standard methods known in the art. The dosages to be
administered can be determined by one of ordinary skill in the art
depending on the clinical severity of the disease, the age and
weight of the subject, or the exposure of the subject to iron.
Preferred effective amounts of the compounds of the invention
ranges from about 0.01 to about 10 mg/kg body weight, preferably
about 0.1 to about 3 mg/kg body weight, and more preferably about
0.5 to about 2 mg/kg body weight for parenteral formulations.
Preferred effective amounts for oral administration would be up to
about 10-fold higher. Moreover, treatment of a subject with a
peptide or composition of the present invention can include a
single treatment or, preferably, can include a series of
treatments. It will be appreciated that the actual dosages will
vary according to the particular peptide or composition, the
particular formulation, the mode of administration, and the
particular site, host, and disease being treated. It will also be
appreciated that the effective dosage used for treatment may
increase or decrease over the course of a particular treatment.
Optimal dosages for a given set of conditions may be ascertained by
those skilled in the art using conventional dosage-determination
tests in view of the experimental data for a given peptide or
composition. Changes in dosage may result and become apparent by
standard diagnostic assays known in the art. In some conditions
chronic administration may be required.
[0151] The pharmaceutical compositions of the invention may be
prepared in a unit-dosage form appropriate for the desired mode of
administration. The compositions of the present invention may be
administered for therapy by any suitable route including oral,
rectal, nasal, topical (including buccal and sublingual), vaginal
and parenteral (including subcutaneous, intramuscular, intravenous
and intradermal). It will be appreciated that the preferred route
will vary with the condition and age of the recipient, the nature
of the condition to be treated, and the chosen peptide and
composition.
[0152] Pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of at least one peptide
as disclosed herein, and an inert, pharmaceutically acceptable
carrier or diluent. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration and known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is
contemplated.
[0153] Supplementary active compounds can also be incorporated into
the compositions. Supplementary active compounds include
niclosamide, liposomes, SL220 solubilization agent (NOF, Japan),
cremophor EL (Sigma), ethanol, and DMSO.
[0154] Toxicity and therapeutic efficacy of the peptides and
compositions of the present invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Peptides which exhibit
large therapeutic indices are preferred. While peptides that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such peptides to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0155] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of peptides of the present invention lies
preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon the dosage form employed and
the route of administration utilized. For any peptide used in the
method of the invention, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
Peptide Synthesis
[0156] Hep25 was synthesized at the UCLA Peptide Synthesis Core
Facility using solid phase 9-fluorenylmethyloxycarbonyl (fmoc)
chemistry. Specifically, the peptides were synthesized on an ABI
431A peptide synthesizer (PE Biosystems, Applied Biosystems, Foster
City, Calif.) using fmoc amino acids, Wang resin (AnaSpec, San
Jose, Calif.), and double coupling for all residues. After
cleavage, 30 mg crude peptides was reduced with 1000-fold molar
excess of dithiothreitol (DTT) in 0.5 M Tris buffer (pH 8.2), 6 M
guanidine hydrochloride, and 20 mM EDTA at 52.degree. C. for 2
hours. Fresh DTT (500-molar excess) was added and incubated for an
additional hour at 52.degree. C. The reduced peptides were purified
on the 10-g C18 SEP-PAK cartridges (Waters, Milford, Mass.)
equilibrated in 0.1% TFA and eluted with 50% acetonitrile. The
eluates were lyophilized and resuspended in 0.1% acetic acid. The
reduced peptides were further purified by reversed-phase
high-performance liquid chromatography (RP-HPLC) on VYDAC C18
column (218TP510; Waters) equilibrated with 0.1% trifluoroacetic
acid and eluted with an acetonitrile gradient. The eluates were
lyophilized, dissolved in 0.1% acetic acid, 20% DMSO, to the
approximate concentration of 0.1 mg/ml (pH 8), and air oxidized by
stirring for 18 hours at room temperature. The refolded peptides
were also purified sequentially on the 10-g C18 SEP-PAK cartridge
and on the RP-HPLC VYDAC C18 column using an acetonitrile gradient.
The eluates were lyophilized and resuspended in 0.016% HCl. The
conformation of refolded synthetic hepcidin derivatives was
verified by electrophoresis in 12.5% acid-urea polyacrylamide gel
electrophoresis (PAGE), and peptide masses were determined by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS; UCLA Mass Spectrometry Facility, Los
Angeles, Calif).
[0157] The other peptides set forth in Table 1 were synthesized by
the solid phase method using either Symphony.RTM. automated peptide
synthesizer (Protein Technologies Inc., Tucson, Ariz.) or CEM
Liberty automatic microwave peptide synthesizer (CEM Corporation
Inc., Matthews, N.C.), applying 9-fluorenylmethyloxycarbonyl (Fmoc)
chemistry (Fields & Noble (1990) Int J Pept Protein Res
35:161-214) and commercially available amino acid derivatives and
reagents (EMD Biosciences, San Diego, Calif. and Chem-Impex
International, Inc., Wood Dale, Ill.). Peptides were cleaved from
resin using modified reagent K (TFA 94% (v/v); phenol, 2% (w/v);
water, 2% (v/v); TIS, 2% (v/v); 2 hours) and precipitated by
addition of ice-cold diethyl ether. Subsequently, peptides were
purified by preparative reverse-phase high performance liquid
chromatography (RP-HPLC) to >95% homogeneity and their purity
evaluated by matrix-assisted laser desorption ionization
spectrometry (MALDI-MS, UCLA Mass Spectrometry Facility, Los
Angeles, Calif.) as well as analytical RP-HPLC employing Varian
ProStar 210 HPLC system equipped with ProStar 325 Dual Wavelength
UV-Vis detector with the wavelengths set at 220 nm and 280 nm
(Varian Inc., Palo Alto, Calif.). Mobile phases consisted of
solvent A, 0.1% TFA in water, and solvent B, 0.1% TFA in
acetonitrile. Analyses of peptides were performed with a
reversed-phase C18 column (Vydac 218TP54, 4.6.times.250 mm, Grace,
Deerfield, Ill.) applying linear gradient of solvent B from 0 to
100% over 100 min (flow rate: 1 ml/min).
[0158] Other methods known in the art may be used to synthesize or
obtain the peptides according to the present invention. All
peptides were synthesized as carboxyamides (--CONH.sub.2) which
creates a charge-neutral end more similar to a peptide bond than
the negatively charged --COOH end. Nevertheless, peptides having
the negatively charged --COOH end are contemplated herein.
Activity Assays
[0159] FLOW CYTOMETRY. The activity of peptides of the present
invention was measured by flow cytometry as previously described.
See Nemeth et al. (2006) Blood 107:328-333, which is herein
incorporated by reference. ECR293/Fpn-GFP, a cell line stably
transfected with a ponasterone-inducible ferroportin construct
tagged at the C-terminus with green fluorescent protein was used.
See Nemeth et al. (2004) Science 306:2090-2093, which is herein
incorporated by reference. Briefly, the cells were plated on
poly-D-lysine coated plates in the presence of 20 .mu.M FAC, with
or without 10 .mu.M ponasterone. After 24 hours, ponasterone was
washed off, and cells were treated with peptides for 24 hours.
Cells were then trypsinized and resuspended at 1.times.10.sup.6
cells/ml, and the intensity of green fluorescence was analyzed by
flow cytometry. Flow cytometry was performed on FACSCAN
(fluorescence activated cell scanner) Analytic Flow Cytometer
(Becton Dickinson, San Jose, Calif.) with CELLQUEST version 3.3
software (Becton Dickinson). Cells not induced with ponasterone to
express Fpn-GFP were used to establish a gate to exclude background
fluorescence. Cells induced with ponasterone, but not treated with
any peptides, were used as the positive control. Each peptide was
tested over the range of concentrations (0, 0.01, 0.03, 0.1, 0.3,
1, 3 and 10 .mu.M). Each peptide treatment was repeated
independently 3 to 6 times. For each concentration of peptide, the
results were expressed as a fraction of the maximal activity
(F.sub.Hep25) of Hep25 (in the dose range 0.01-10 .mu.M), according
to the formula
1--((F.sub.x-F.sub.Hep25)/(F.sub.untreated-F.sub.Hep25)), where F
was the mean of the gated green fluorescence and x was the peptide.
The IEC.sub.50 concentrations are set forth in the Table 1.
[0160] FERRITIN ASSAY. Cells treated with peptides having hepcidin
activity will retain iron and contain higher amounts of ferritin.
Thus, following ferritin assay may be used to identify
mini-hepcidins according to the present invention. Briefly,
HEK293-Fpn cells are incubated with 20 .mu.M FAC with or without 10
.mu.M ponasterone. After 24 hours, ponasterone is washed off, and
hepcidin derivatives are added for 24 hours in the presence of 20
.mu.M FAC. Cellular protein is extracted with 150 mM NaCl, 10 mM
EDTA, 10 mM Tris (pH 7.4), 1% Triton X-100, and a protease
inhibitor cocktail (Sigma-Aldrich, St Louis, Mo.). Ferritin levels
are determined by an enzyme-linked immunosorbent assay (ELISA)
assay (Ramco Laboratories, Stafford, Tex., or Biotech Diagnostic,
Laguna Niguel, Calif.) according to the manufacturer's instructions
and are normalized for the total protein concentration in each
sample, as determined by the bicinchoninic acid (BCA) assay
(Pierce, Rockford, Ill.).
[0161] IN VIVO ASSAYS. Serum iron assay. The decrease in serum iron
after peptide administration is the principal measure of hepcidin
activity. Thus, as provided herein, the hepcidin activity of
selected peptides of the present invention were assayed in vivo by
measuring serum iron in test subjects. Briefly, C57/B16J mice were
maintained on NIH31 rodent diet (333 parts per million (ppm) iron;
Harlan Teklad, Indianapolis, Ind.). Two weeks before the
experiment, the mice were switched to a diet containing about 2-4
ppm iron (Harlan Teklad, Indianapolis, Ind.) in order to suppress
endogenous hepcidin. Peptide stocks were diluted to desired
concentrations in sterile phosphate buffered saline (PBS) or other
diluents as described next. Mice were subjected to the following
treatments: (a) Injected intraperitoneally either with 100 .mu.l
PBS (control) or with 50 .mu.g peptide in 100 .mu.l PBS; (b)
Injected with 100 .mu.l of peptide (or PBS) mixed with 500 .mu.g
empty liposomes COATSOME EL series (NOF, Tokyo, Japan) (prepared as
per manufacturer's recommendation); (c) Injected with 100 .mu.l
peptides (or PBS) solubilized with SL220 solubilization agent (NOF,
Tokyo, Japan); (d) Gavaged with 250 .mu.l of peptide (or PBS) in
1.times. solvent (Cremophor EL (Sigma)/ethanol/PBS;
(12.5:12.5:75)). Mice were sacrificed 4 hours later, blood was
collected by cardiac puncture, and serum was separated using
MICROTAINER tubes (Becton Dickinson, Franklin Lakes, N.J.). Serum
iron was determined by using a colorimetric assay (Diagnostic
Chemicals, Oxford, Conn.), which was modified for the microplate
format so that 10 .mu.l serum was used per measurement. The results
were expressed as the percentage of decrease in serum iron when
compared with the average value of serum iron levels in PBS-treated
mice.
[0162] As shown in FIG. 5, intraperitoneal (i.p.) administration of
50 .mu.g Pr12 per mouse in PBS caused a significant decrease in
serum iron after 4 hours, when compared to i.p. administration of
PBS. The serum iron decrease was similar to that caused by i.p.
injection of 50 .mu.g of Hep25. Injection (i.p.) of Hep9 did not
result in a serum iron decrease. Pr12 is a retroinverted form of
Hep9, and is resistant to proteolysis because of the retroinverted
structure. The experiment indicates that increased proteolytic
resistance improves the activity of mini-hepcidins.
[0163] As shown in FIG. 6, i.p. administration of 200 nmoles of
riHep7.DELTA.DT in PBS resulted in serum iron concentrations
significantly lower than those achieved after injection of PBS, and
also lower than i.p. injection of 20 nmoles of Hep25.
Administration of 20 nmoles of riHep7.DELTA.DT slightly but not
significantly reduced serum iron concentrations. The experiment
indicates that after i.p. injection peptides as small as 7 amino
acids are able to display activity comparable to Hep25.
[0164] As shown in FIG. 7, i.p. administration of 20 nmoles Pr27 in
PBS caused a serum iron decrease comparable to that caused by i.p.
administration of 20 nmoles Hep25. This indicated that
mini-hepcidin can achieve similar potency to Hep25 in vivo. Higher
concentration of Pr27 (200 nmoles) caused even greater decrease in
serum iron concentrations.
[0165] As shown in FIG. 8, i.p. administration of 20 nmoles Pr27 in
liposomal solution also caused a serum iron decrease similar to
that caused by i.p. administration of 20 nmoles Hep25.
Administration of liposomal solution by itself did not affect serum
iron levels. The liposomal solution was prepared by mixing 100
.mu.l of PBS with 500 .mu.g empty liposomes COATSOME EL series
(NOF, Tokyo, Japan) (prepared as per manufacturer's
recommendation). Mini-hepcidin Pr28 dissolved in liposomal
solution, however, showed lesser ability to decrease serum iron
than Pr27. The experiment indicates that suspension of peptides in
liposomes does not affect their activity. Thus, liposomes may be
useful for oral administration of peptides according to the present
invention.
[0166] As shown in FIG. 9, oral administration of Pr27 200 nmoles
by gavage in a cremophore EL solution caused a decrease in serum
iron in mice as compared to oral administration of PBS in the same
formulation. Cremophor EL increases solubility of chemicals, and is
frequently used excipient or additive in drugs. Cremophor EL
solution was prepared by mixing Cremophor EL (Sigma), ethanol and
PBS in a ratio 12.5:12.5:75. 250 .mu.l of the solution was
administered by gavage to mice.
[0167] Thus, the present invention may be used to decrease serum
iron in subjects. A preferred mini-hepcidin according to the
present invention is a retroinverted peptide which comprises a PEG
molecule, such as PEG11, linked to its N-terminal amino acid. In
some embodiments, the PEG molecule is linked to palmitoyl group or
diaminopropionic acid linked to one or more palmitoyl groups.
[0168] In addition to assaying the effect on serum iron content,
other in vivo assays known in the art may be conducted to identify
mini-hepcidins according to the present invention and/or determine
the therapeutically effective amount of a given peptide or
mini-hepcidin according to the present invention. Examples of such
assays include the following:
[0169] Tissue iron assay. In addition to or instead of the serum
iron assay above, tissue iron distribution can be determined by
enhanced Perl's stain of liver and spleen sections obtained from
the treated mice. Briefly, the tissue sections are fixed in 4%
paraformaldehyde/PBS, incubated in Perl's solution (1:1, 2% HCl and
2% potassium ferrocyanide) and diaminobenzidine in 0.015% hydrogen
peroxide. Tissue non-heme iron may be quantitated using the
micromethod of Rebouche et al. See Rebouche et al., J Biochem
Biophys Methods. 2004 Mar. 31; 58(3):239-51.; Pak et al. Blood.
2006 Dec. 1; 108(12):3730-5. 100 mg pieces of liver and spleen are
homogenized and acid is added to release non-heme bound iron which
is detected by colorimetric reaction using ferrozine and compared
to controls. Treatment with mini-hepcidins would be expected to
cause redistribution of iron from other tissues to the spleen. Over
weeks to months, the administration of mini-hepcidins would be
expected to decrease tissue iron content in all tissues because of
diminished dietary iron absorption.
[0170] Hematology assays. Hematology assays may be used to identify
mini-hepcidins according to the present invention and/or determine
the therapeutically effective amount of a given peptide or
mini-hepcidin according to the present invention. Briefly, blood
from treated subjects is collected into heparin-containing tubes.
Hemoglobin, RBC, MCV, EPO, white cell parameters, reticulocyte
counts, and reticulocyte Hgb content are determined using methods
known in the art and compared to controls. Treatment with
mini-hepcidins would be expected to cause a decrease in MCV and
diminish the Hgb content of reticulocytes. Administration of
mini-hepcidins in excessive amounts would be expected to decrease
Hgb.
[0171] IRON EXPORT ASSAYS. Iron (.sup.55Fe) export assays known in
the art using primary hepatocytes and macrophages may be used to
identify mini-hepcidins according to the present invention and/or
determine the therapeutically effective amount of a given peptide
or mini-hepcidin according to the present invention. Peptides
having hepcidin activity will diminish or decrease the release of
.sup.55Fe from cells. Briefly, cells are incubated with
.sup.55Fe-NTA or .sup.55Fe-Tf for 36 hours. After washing off
unincorporated .sup.55Fe, cells are treated with a given peptide or
a control. In case of ferroportin mutants, the transfection is
performed prior to addition of .sup.55Fe and expression allowed to
proceed during the 36 hour iron-loading period. Aliquots of the
media are collected after 1, 4, 8, 24, 36, 48 and 72 hours and
radioactivity is determined by a scintillation counter.
Cell-associated radioactivity can be measured by centrifuging cells
through silicone oil to lower the non-specific binding of
radiolabeled iron to cells using methods known in the art.
[0172] To determine whether a given peptide modifies the
internalization and degradation of endogenous ferroportin, the
protein levels and cellular distribution of ferroportin in
hepatocytes and macrophages treated with the peptide may be assayed
using Western blotting, immunohistochemistry and ferroportin
antibodies known in the art.
[0173] To the extent necessary to understand or complete the
disclosure of the present invention, all publications, patents, and
patent applications mentioned herein are expressly incorporated by
reference therein to the same extent as though each were
individually so incorporated.
[0174] Having thus described exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
within disclosures are exemplary only and that various other
alternatives, adaptations, and modifications may be made within the
scope of the present invention. Accordingly, the present invention
is not limited to the specific embodiments as illustrated herein,
but is only limited by the following claims.
Sequence CWU 1
1
15125PRTHomo sapiens 1Asp 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 210PRTHomo sapiens 2Asp Thr His Phe Pro Ile Cys Ile Phe Cys 1
5 10 34PRTHomo sapiens 3Phe Pro Ile Cys 1 45PRTHomo sapiens 4His
Phe Pro Ile Cys 1 5 56PRTHomo sapiens 5His Phe Pro Ile Cys Ile 1 5
67PRTHomo sapiens 6His Phe Pro Ile Cys Ile Phe 1 5 77PRTHomo
sapiens 7Asp Thr His Phe Pro Ile Cys 1 5 88PRTHomo sapiens 8Asp Thr
His Phe Pro Ile Cys Ile 1 5 99PRTHomo sapiens 9Asp Thr His Phe Pro
Ile Cys Ile Phe 1 5 1010PRTHomo sapiens 10Asp Thr His Phe Pro Ile
Ala Ile Phe Cys 1 5 10 119PRTHomo sapiens 11Asp Thr His Ala Pro Ile
Cys Ile Phe 1 5 129PRTHomo sapiens 12Asp Thr His Phe Pro Ile Ala
Ile Phe 1 5 139PRTHomo sapiens 13Asp Thr His Phe Pro Ile Ser Ile
Phe 1 5 1410PRTHomo sapiensDISULFID(1)..(8) 14Cys Asp Thr His Phe
Pro Ile Cys Ile Phe 1 5 10 158PRTHomo sapiensDISULFID(1)..(6) 15Cys
His Phe Pro Ile Cys Ile Phe 1 5
* * * * *