U.S. patent application number 16/141724 was filed with the patent office on 2019-01-17 for tissue protective peptides and uses thereof.
This patent application is currently assigned to Araim Pharmaceuticals, Inc.. The applicant listed for this patent is Araim Pharmaceuticals, Inc.. Invention is credited to Michael Brines, Anthony Cerami, Thomas Coleman.
Application Number | 20190016769 16/141724 |
Document ID | / |
Family ID | 37728020 |
Filed Date | 2019-01-17 |
View All Diagrams
United States Patent
Application |
20190016769 |
Kind Code |
A1 |
Cerami; Anthony ; et
al. |
January 17, 2019 |
TISSUE PROTECTIVE PEPTIDES AND USES THEREOF
Abstract
The present invention is directed to novel tissue protective
peptides. The tissue protective peptides of the invention may bind
to a tissue protective receptor complex. In particular, the present
invention is drawn to tissue protective peptides derived from or
sharing consensus sequences with portions of cytokine receptor
ligands, including Erythropoietin (EPO), that are not involved in
the binding of the ligand to the receptor complex, e.g., to the EPO
receptor homodimer. Accordingly, the tissue protective peptides of
the invention are derived from the amino acid sequences of regions
of cytokine receptor ligands that are generally located on or
within the region of the ligand protein that is opposite of the
receptor complex, i.e., are generally derived from amino acid
sequences of regions of the ligand protein that face away from the
receptor complex while the ligand is bound to the receptor. The
invention is further directed to the consensus sequences for use in
engineering a synthetic tissue protective peptide. These tissue
protective peptides also include fragments, chimeras, as well as
peptides designed to mimic the spatial localization of key amino
acid residues within the tissue protective receptor ligands, e.g.,
EPO. The invention further encompasses methods for treating or
preventing a disease or disorder using tissue protective peptides
of the current invention. The invention also encompasses methods
for enhancing excitable tissue function using tissue protective
peptides of the current invention.
Inventors: |
Cerami; Anthony; (La Jolla,
CA) ; Brines; Michael; (Woodbridge, CT) ;
Coleman; Thomas; (Mount Kisco, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Araim Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Araim Pharmaceuticals, Inc.
Tarrytown
NY
|
Family ID: |
37728020 |
Appl. No.: |
16/141724 |
Filed: |
September 25, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15147848 |
May 5, 2016 |
10100096 |
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16141724 |
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14248030 |
Apr 8, 2014 |
9340598 |
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15147848 |
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13278131 |
Oct 20, 2011 |
8716245 |
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14248030 |
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11997898 |
Oct 29, 2008 |
8071554 |
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PCT/US2006/031061 |
Aug 7, 2006 |
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13278131 |
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60831737 |
Jul 18, 2006 |
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60706276 |
Aug 8, 2005 |
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60705741 |
Aug 5, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 15/00 20180101;
Y02A 50/411 20180101; A61P 37/02 20180101; A61P 1/00 20180101; A61P
7/00 20180101; A61P 17/02 20180101; C07K 14/505 20130101; A61P
39/00 20180101; A61P 29/00 20180101; A61P 43/00 20180101; A61K
38/00 20130101; A61P 5/00 20180101; A61P 3/00 20180101; A61P 9/00
20180101; A61P 9/14 20180101; A61P 13/12 20180101; A61P 19/08
20180101; A61P 17/00 20180101; C07K 14/435 20130101; A61P 27/02
20180101; C07K 14/71 20130101; A61P 25/02 20180101; A61P 1/04
20180101; A61P 19/00 20180101; A61P 33/06 20180101; A61P 9/04
20180101; A61P 25/00 20180101; A61P 7/04 20180101; A61P 25/28
20180101; A61P 11/00 20180101; Y02A 50/30 20180101; A61P 13/00
20180101; A61P 15/08 20180101; A61P 3/10 20180101; A61P 9/10
20180101; C07K 14/575 20130101; C07K 2319/00 20130101 |
International
Class: |
C07K 14/505 20060101
C07K014/505 |
Claims
1. An isolated polypeptide consisting of a sequence of no more than
30 amino acids and comprising the amino acid motif: (a)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.2, wherein n is 0-5; (b)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-L.sub.1, wherein n is 0-5; or (c)
L.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.1, wherein n is 0-5; and
wherein H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1
and N.sub.2 are negatively charged amino acids, X is any amino
acid, and L.sub.1 is a polar amino acid; and wherein said
polypeptide has cellular protective activity in a responsive cell,
tissue or organ.
2. An isolated polypeptide consisting of a sequence of no more than
30 amino acids and comprising the amino acid motif: (a)
H.sub.1-N.sub.1-(L).sub.n-P.sub.1-H.sub.2, wherein n is 0-1; or (b)
H.sub.1-P.sub.1-(L).sub.n-N.sub.1-H.sub.2, wherein n is 0-1; and
wherein H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1 is
a negatively charged amino acid, L.sub.1 is a polar amino acid, and
P.sub.1 is a positively charged amino acid; and wherein said
polypeptide has cellular protective activity in a responsive cell,
tissue or organ.
3. An isolated polypeptide comprising: (i) an amino acid sequence
having less than 90% sequence identity with any portion of SEQ ID
NO:1 having the same number of amino acid residues as said
polypeptide; and (ii) the amino acid motif: (a)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.2, wherein n is 0-5; (b)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-L.sub.1, wherein n is 0-5; or (c)
L.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.1, wherein n is 0-5; and
wherein H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1
and N.sub.2 are negatively charged amino acids, X is any amino
acid, and L.sub.1 is a polar amino acid; and wherein said
polypeptide has cellular protective activity in a responsive cell,
tissue or organ.
4. An isolated polypeptide comprising: (i) an amino acid sequence
having less than 90% sequence identity with any portion of SEQ ID
NO:1 having the same number of amino acid residues as said
polypeptide; and (ii) the amino acid motif: (a)
H.sub.1-N.sub.1-(L).sub.n-P.sub.1-H.sub.2, wherein n is 0-1; or (b)
H.sub.1-P.sub.1-(L).sub.n-N.sub.1-H.sub.2, wherein n is 0-1; and
wherein H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1 is
a negatively charged amino acid, L.sub.1 is a polar amino acid, and
P.sub.1 is a positively charged amino acid; and wherein said
polypeptide has cellular protective activity in a responsive cell,
tissue or organ.
5. The isolated polypeptide of claim 1 or 3, wherein the distance
between N.sub.1 and N.sub.2 as a result of the tertiary structure
of said polypeptide is between about 3 .ANG. to about 5 .ANG..
6. The isolated polypeptide of claim 5, wherein said distance is
between about 4 .ANG. to about 5 .ANG..
7. The isolated polypeptide of claim 6, wherein said distance is
between about 4.4 .ANG. to about 4.8 .ANG..
8. The isolated polypeptide of claim 2 or 4, wherein the distance
between N.sub.1 and P.sub.2 as a result of the tertiary structure
of said polypeptide is between about 3 .ANG. to about 5 .ANG..
9. The isolated polypeptide of claim 8, wherein said distance is
between about 4 .ANG. to about 5 .ANG..
10. The isolated polypeptide of claim 9, wherein said distance is
between about 4.4 .ANG. to about 4.8 .ANG..
11. An isolated polypeptide consisting of a sequence of no more
than 30 amino acids and comprising the amino acid motif: (a)
H.sub.1N.sub.1N.sub.2H.sub.2; (b) H.sub.1N.sub.1N.sub.2L; or (c)
that is formed as a result of the of the tertiary structure of said
polypeptide such that the distance between the carbonyl carbons of
N.sub.1 and N.sub.2 is about 3 .ANG. to about 5 .ANG., wherein
H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1 and
N.sub.2 are negatively charged amino acids, and L.sub.1 is a polar
amino acid; and wherein said polypeptide has cellular protective
activity in a responsive cell, tissue or organ.
12. An isolated polypeptide consisting of a sequence of no more
than 30 amino acids and comprising the amino acid motif: (a)
H.sub.1N.sub.1(L).sub.nPiH.sub.2, wherein n is 0-1; or (b)
H.sub.1P.sub.1(L).sub.nN.sub.1H.sub.2, wherein n is 0-1; that is
formed as a result of the of the tertiary structure of said
polypeptide such that the distance between the carbonyl carbons of
N.sub.1 and P.sub.2 is about 3 .ANG. to about 5 .ANG., wherein
H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1 is a
negatively charged amino acid, L.sub.1 is a polar amino acid, and
P.sub.1 is a positively charged amino acid; and wherein said
polypeptide has cellular protective activity in a responsive cell,
tissue or organ.
13. An isolated polypeptide comprising: (i) an amino acid sequence
having less than 90% sequence identity with any portion of SEQ ID
NO:1 having the same number of amino acid residues as said
polypeptide; and (ii) the amino acid motif: (a)
H.sub.1N.sub.1N.sub.2H.sub.2; (b) H.sub.1N.sub.1N.sub.2L.sub.1; or
(c) that is formed as a result of the of the tertiary structure of
said polypeptide such that the distance between the carbonyl
carbons of N.sub.1 and N.sub.2 is about 3 .ANG. to about 5 .ANG.,
wherein H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1
and N.sub.2 are negatively charged amino acids, and L.sub.1 is a
polar amino acid; and wherein said polypeptide has cellular
protective activity in a responsive cell, tissue or organ.
14. An isolated polypeptide comprising: (i) an amino acid sequence
having less than 90% sequence identity with any portion of SEQ ID
NO:1 having the same number of amino acid residues as said
polypeptide; and (ii) the amino acid motif: (a)
H.sub.1N.sub.1(L).sub.nPiH.sub.2, wherein n is 0-1; or (b)
H.sub.1P.sub.1(L).sub.nN.sub.1H.sub.2, wherein n is 0-1; that is
formed as a result of the of the tertiary structure of said
polypeptide such that the distance between the carbonyl carbons of
N.sub.1 and P.sub.2 is between about 3 .ANG. and about 5 .ANG.,
wherein H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1 is
a negatively charged amino acid, L.sub.1 is a polar amino acid, and
P.sub.1 is a positively charged amino acid; and wherein said
polypeptide has cellular protective activity in a responsive cell,
tissue or organ.
15. The isolated polypeptide of any one of claims 1, 3, and 5-7,
wherein said amino acid motif is (a) and wherein H.sub.1 and
H.sub.2 are the same hydrophobic amino acid.
16. The isolated polypeptide of claim 11 or 13, wherein said amino
acid motif is (a) and wherein H.sub.1 and H.sub.2 are the same
hydrophobic amino acid.
17. The isolated polypeptide of any one of claims 1, 3, and 5-7,
wherein said amino acid motif is (a) and wherein H.sub.1 and
H.sub.2 are different hydrophobic amino acids.
18. The isolated polypeptide of claim 11 or 13, wherein said amino
acid motif is (a) and wherein H.sub.1 and H.sub.2 are different
hydrophobic amino acids.
19. The isolated polypeptide of any one of claims 2, 4, and 8-10,
wherein said amino acid motif is (a) or (b) and wherein H.sub.1 and
H.sub.2 are the same hydrophobic amino acid.
20. The isolated polypeptide of claim 12 or 14, wherein said amino
acid motif is (a) or (b) and wherein H.sub.1 and H.sub.2 are the
same hydrophobic amino acid.
21. The isolated polypeptide of any one of claims 2, 4, and 8-10,
wherein said amino acid motif is (a) or (b) and wherein H.sub.1 and
H.sub.2 are different hydrophobic amino acids.
22. The isolated polypeptide of claim 12 or 14, wherein said amino
acid motif is (a) or (b) and wherein H.sub.1 and H.sub.2 are
different hydrophobic amino acids.
23. The isolated polypeptide of any one of claims 1, 3, and 5-7,
wherein said amino acid motif is (a), (b), or (c) and wherein
N.sub.1 and N.sub.2 are the same negatively charged amino acid.
24. The isolated polypeptide of claim 11 or 13, wherein said amino
acid motif is (a), (b), or (c) and wherein N.sub.1 and N.sub.2 are
the same negatively charged amino acid.
25. The isolated polypeptide of any one of claims 1, 3, and 5-7,
wherein said amino acid motif is (a), (b), or (c) and wherein
N.sub.1 and N.sub.2 are different negatively charged amino
acids.
26. The isolated polypeptide of claim 11 or 13, wherein said amino
acid motif is (a), (b), or (c) and wherein N.sub.1 and N.sub.2 are
different negatively charged amino acids.
27. The isolated polypeptide of any one of claims 1-26, wherein
said peptide is derived from a type 1 cytokine.
28. The isolated peptide of claim 27, wherein the type 1 cytokine
is granulocyte-macrophage colony stimulating factor, interleukin-3,
Thrombopoietin, Ciliary Neurotrophic Factor or Leukemia Inhibitory
Factor.
29. The isolated polypeptide of any one of claims 1-28, wherein
said polypeptide further comprises at least one other of the
following amino acids motifs: (a)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.2, wherein n is 0-5; (b)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-L.sub.1, wherein n is 0-5; (c)
L.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.1, wherein n is 0-5 (d)
H.sub.1-N.sub.1-(L).sub.n-P.sub.1-H.sub.2, wherein n is 0-1; (e)
H.sub.1-P.sub.1-(L).sub.n-N.sub.1-H.sub.2, wherein n is 0-1 (f)
H.sub.1N.sub.1N.sub.2H.sub.2; (g) H.sub.1N.sub.1N.sub.2L.sub.1; (h)
L.sub.1N.sub.1N.sub.2H.sub.1 (i) H.sub.1N.sub.1(L).sub.nPiH.sub.2,
wherein n is 0-1; or (j) H.sub.1P.sub.1(L).sub.nN.sub.1H.sub.2,
wherein n is 0-1; wherein motif (f), (g), or (h) is formed as a
result of the of the tertiary structure of said polypeptide such
that the distance between the carbonyl carbons of N.sub.1 and
N.sub.2 is about 3 .ANG. to about 5 .ANG.; wherein motif (i) or (j)
is formed as a result of the of the tertiary structure of said
polypeptide such that the distance between the carbonyl carbons of
N.sub.1 and P.sub.2 is about 3 .ANG. to about 5 .ANG.; and wherein
H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1 and
N.sub.2 are negatively charged amino acids, X is any amino acid,
L.sub.1 is a polar amino acid, and P.sub.1 is a positively charged
amino acid.
30. The isolated polypeptide of claim 29, wherein at least two of
said amino acid motifs are different.
31. The isolated polypeptide of any one of claims 1-29, wherein
said polypeptide is chimeric peptide further comprising an
amphipathic peptide helix.
32. The isolated polypeptide of claim 31, wherein said amphipathic
peptide helix comprises the amino acid sequence ALSILVLLQAGS (SEQ
ID NO:48); VALLPCPPCRA (SEQ ID NO:49); NAIIKNAYKKG (SEQ ID NO:50);
GSWQRSLQDTE (SEQ ID NO:51); GGSAARPAPP (SEQ ID NO:52); NALAENDTPYY
(SEQ ID NO:53); GALAEAYPSKP (SEQ ID NO:54); GCSSQHWSYGL (SEQ ID
NO:55); VMIVMLAICFL (SEQ ID NO:56); LRRYINMLTRP (SEQ ID NO:28); or
LALSILVLYQA (SEQ ID NO:57).
33. The isolated polypeptide of any one of claims 1-30, wherein
said polypeptide does not increase hemoglobin in the recipient.
34. The isolated polypeptide of any one of claims 1-31, wherein the
cellular protective activity is protecting, maintaining, enhancing
or restoring the function and/or viability of said cell, tissue or
organ.
35. The isolated polypeptide of any one of claims 1-32, wherein
said polypeptide has cellular protective activity in neuronal,
bone, eye, adipose, connective, hair, teeth, mucosal, pancreas,
endocrine, ear, epithelial, skin, muscle, heart, lung, liver,
kidney, intestine, adrenal, capillary, endothelial, testes, ovary,
or endometrial cells or tissues.
36. The isolated polypeptide of any one of claims 1-32 wherein said
polypeptide has cellular protective activity in a stem cell.
37. The isolated polypeptide of claim any one of claims 1-32,
wherein said polypeptide has cellular protective activity in
excitable tissue.
38. The isolated peptide of claim 35, wherein said excitable tissue
is central nervous system tissue, peripheral nervous system tissue,
cardiac tissue or retinal tissue.
39. The isolated peptide of any one of claims 1-36, wherein said
peptide is capable of traversing an endothelial cell barrier.
40. The isolated peptide of claim 37, wherein the endothelial cell
barrier comprises the blood-brain barrier, the blood-eye barrier,
the blood-testes barrier, blood-ovary barrier, blood-nerve or the
blood-spinal cord barrier.
41. A pharmaceutical composition comprising the isolated
polypeptide of any of claims 1-38 and a pharmaceutically acceptable
carrier.
42. The pharmaceutical composition of claim 39, wherein said
composition is formulated for oral, intranasal, ocular,
inhalational, transdermal, rectal, sublingual or parenteral
administration.
43. The pharmaceutical composition of claim 39, wherein said
composition is formulated as a perfusate solution.
44. A method for protecting, maintaining or enhancing the viability
of a responsive cell, tissue or organ isolated from a mammalian
body comprising exposing said cell, tissue or organ to a
pharmaceutical composition comprising exposing said cell, tissue or
organ to the pharmaceutical composition of claim 39, 40, or 41.
45. Use of an isolated peptide of any one of claims 1-38 for the
preparation of a pharmaceutical composition for the protection
against and/or prevention of a tissue injury, for the restoration
of, or for the rejuvenation of tissue and/or tissue function in a
subject in need thereof.
46. Use of an isolated peptide of any one of claims 1-38 for the
preparation of a pharmaceutical composition for the prevention,
therapeutic treatment or prophylactic treatment in a subject in
need thereof of a cardiovascular disease, cardiopulmonary disease,
respiratory disease, kidney disease, disease of the urinary system,
disease of the reproductive system, done disease, skin disease,
gastrointestinal disease, endocrine abnormality, metabolic
abnormality, cognitive dysfunction, or a disease or disorders of
the central or peripheral nervous system.
47. The use of claim 43 or 44 wherein the subject is a mammal.
48. The use of claim 45 wherein the mammal is a human.
49. A method for facilitating the transcytosis of a molecule across
an endothelial cell barrier in a subject in need thereof comprising
administration to said subject a composition comprising said
molecule in association with an isolated peptide of any one of
claims 1-38.
50. The method of claim 47, wherein said association is a labile
covalent bond, a stable covalent bond, or a non-covalent
association with a binding site for said molecule.
51. An isolated nucleic acid comprising the nucleotide sequence
encoding the isolated peptide of any one of claims 1-38.
52. A vector comprising the nucleic acid of claim 49.
53. The vector of claim 50 which is an expression vector.
54. A host cell containing the expression vector of claim 51.
55. A method of recombinantly producing the isolated peptide of any
one of claims 1-38, said method comprising i) culturing in a medium
the host cell of claim 52, under conditions suitable for the
expression of said peptide, and ii) recovery and isolation of said
peptide from said medium.
56. The isolated peptide of claim 2, wherein said peptide comprises
the amino acid sequence peptide A (APPRLICDSRVLERYLLEAKEAE, SEQ ID
NO:32), peptide C (NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29), peptide D
(QQAVEVWQGLALLSEAVLRGQALLV, SEQ ID NO:30), peptide E
(GCAEHCSLNENITVPDTKVN, SEQ ID NO:31), peptide F (RYLLUNITTGC, SEQ
ID NO:33), peptide G (QEQLERALNSS, SEQ ID NO:40), peptide I
(CSLNENIQEQLERALNSS, SEQ ID NO:43), peptide J
(QEQLERALNSSLRRYINMLTRTR, SEQ ID NO:41), peptide K (WEHVNAIQEARRLL,
SEQ ID NO:35), or peptide L (KIRSDLTALTESYVKH, SEQ ID NO:37).
57. The isolated peptide of any one of claims 1-38, wherein said at
least one cellular protective activity is neuroprotection, which
activity is evaluated in vitro by a method comprising: (a)
contacting a test culture of primary hippocampal neurons with
N-methyl-D-aspartate and said peptide; and (b) determining the cell
viability at 48 hours post said contact, such that if the cell
viability determined in step (b) is greater than that of a control
culture in the absence of said peptide, the peptide possesses
cellular protective activity.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 15/147,848, filed May 5, 2016, which is a continuation of U.S.
application Ser. No. 14/248,030, filed Apr. 8, 2014, now issued
U.S. Pat. No. 9,340,598, issued May 17, 2016, which is a
continuation of U.S. application Ser. No. 13/278,131, filed Oct.
20, 2011, now issued U.S. Pat. No. 8,716,245, issued May 6, 2014,
which is a continuation of U.S. application Ser. No. 11/997,898,
now issued U.S. Pat. No. 8,071,554, issued Dec. 6, 2011, which is a
U.S. National Stage Application under 35 U.S.C. .sctn. 371 of
International Patent Application No. PCT/US2006/031061, filed Aug.
7, 2006, which claims the benefit of priority to U.S. Provisional
Patent Application No. 60/831,737, filed Jul. 18, 2006, U.S.
Provisional Patent Application No. 60/706,276, filed Aug. 8, 2005,
and U.S. Provisional Patent Application No. 60/705,741, filed Aug.
5, 2005, the entire contents of which are each incorporated herein
by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 20, 2018, is named 12110-031-999_SEQ_LISTING.txt and is
23,030 bytes in size.
1. INTRODUCTION
[0003] The present invention is directed to novel tissue protective
peptides. The tissue protective peptides of the invention may bind
to a tissue protective receptor complex. In particular, the present
invention is drawn to tissue protective peptides derived from or
sharing consensus sequences with portions of cytokine receptor
ligands, including Erythropoietin (EPO), that are not involved in
the binding of the ligand to the receptor complex, e.g., to the EPO
receptor homodimer. Accordingly, the tissue protective peptides of
the invention are derived from the amino acid sequences of regions
of cytokine receptor ligands that are generally located on or
within the region of the ligand protein that is opposite of the
receptor complex, i.e., are generally derived from amino acid
sequences of regions of the ligand protein that face away from the
receptor complex while the ligand is bound to the receptor. The
invention is further directed to the consensus sequences for use in
engineering a synthetic tissue protective peptide. These tissue
protective peptides also include fragments, chimeras, as well as
peptides designed to mimic the spatial localization of key amino
acid residues within the tissue protective receptor ligands, e.g.,
EPO.
[0004] The invention also encompasses methods for treating,
preventing or ameliorating a disease or disorder and or treating,
restoring or ameliorating a tissue injury using tissue protective
peptides of the current invention. The invention also encompasses
methods for enhancing excitable tissue function using tissue
protective peptides of the current invention.
2. BACKGROUND OF THE INVENTION
[0005] Erythropoietin ("EPO") is a glycoprotein hormone commonly
associated with the maintenance of hematocrit and, more recently,
tissue protection. Mature human EPO protein comprises 165 amino
acids and has a molecular weight of 34 kDa, with glycosyl residues
contributing about 40% of the weight of the molecule. The EPO
molecule comprises four helices that interact via their hydrophobic
domains to form a predominantly globular structure within an
aqueous environment (Cheetham et al., 1998, Nat. Struct. Biol.
5:861-866, which is hereby incorporated by reference in its
entirety). The invention derives from the discovery that certain
amino acids facing the aqueous environment (i.e., away from the
hydrophobic, globular central core) mediate tissue protection.
Peptides can be derived or designed from an understanding of the
tissue protective regions that have been identified by the
Applicants.
[0006] As noted above, EPO is pluripotent. In its hormonal role,
EPO regulates hematocrit through its role in the maturation of
erythroid progenitor cells into erythrocytes. EPO acts as an
anti-apoptotic agent during the maturation process of erythroid
progenitor cells, permitting progenitor cells to mature into
erythrocytes. Decreased levels of tissue oxygen (hypoxia) trigger
an increased production of erythropoietin by the kidney, which
results in increased erythropoiesis. Given that the kidney normally
produces the majority of the serum erythropoietin, the loss of
kidney function, such as in chronic renal failure, results in
decreased production of EPO and often anemia. Similarly, anemia may
result from other chronic conditions, such as cancer, or treatments
associated with these illnesses, such as chemotherapy, which
directly suppress the production of EPO. Commercially available
recombinant erythropoietin has been available under the trademarks
of PROCRIT, available from Ortho Biotech Inc., Raritan, N.J., and
EPOGEN, available from Amgen, Inc., Thousand Oaks, Calif. and has
been used to treat anemia resulting from end stage renal disease,
therapy with AZT (zidovudine) in HIV-infected patients, oncology
patients, and chemotherapy. Currently a hyperglycosylated
erythropoietin, ARANESP.TM. (Amgen, Thousand Oaks, Calif.), is
available for the treatment of anemia. Additionally, these
compounds have been used to increase the hematocrits of patients
undergoing surgery to reduce the need for allogenic blood
transfusions.
[0007] Recently, several lines of evidence have suggested that EPO
also functions locally in a paracrine-autocrine manner to minimize
tissue damage. For example, EPO improves an hypoxic cellular
microenvironment and decreases programmed cell death caused by
metabolic stress. Both of these activities are moderated, in part,
through EPO's interaction with a specific cell surface receptor
comprised, in part, by the erythropoietin receptor ("EPOR")
protein. EPOR is an approximately 66 kDa protein and is a member of
the Type-1 cytokine receptor family. This family comprises
receptors that are grouped together based on the shared homology of
their extracellular domains and includes receptors for interleukin
IL-2, IL3, IL4, IL5, IL6, IL7, IL9, IL11, granulocyte
macrophage-colony stimulating factor (GM-CSF), granulocyte colony
stimulating factor (G-CSF), leukemia inhibiting factor (LIF),
ciliary neurotrophic factor (CNTF), thrombopoietin, growth hormone
and prolactin. The conserved extracellular domain of these
receptors has a length of approximately 200 amino acids, comprises
four positionally conserved cysteine residues in the amino-terminal
region (Cys 294, Cys 283, Cys 248, and Cys 238, which appear to be
critical to the maintenance and the structural integrity of the
receptors (Murray, 1996, Harpers Biochemistry 24.sup.th ed. pp.
524-526, Appilion & Lange, Ltd.; Caravella et al., 1996,
Protein: Struct. Funct. Gen. 24:394-401, each of which is hereby
incorporated by reference in its entirety)), and a
Trp-Ser-X-Trp-Ser (SEQ ID NO:58) motif located proximal to the
transmembrane domain.
[0008] In connection with erythropoiesis, EPOR functions in a
manner similar to other receptors within the Type-1 cytokine
receptor family. First, the receptor ligand, e.g., EPO, binds to a
preformed dimer of EPOR, (EPOR).sub.2. It has been determined that
EPO interacts with the extracellular domain of the classic
(EPOR).sub.2 homodimer receptor via two distinct regions on the
ligand surface: a high affinity receptor binding site (site 1) and
a low affinity receptor binding site (site 2). The amino acid
sequences of EPO associated with site 1 are TKVNFY, SEQ ID NO:2,
corresponding to amino acids 44-49 of SEQ ID NO:1, and SNFLRG, SEQ
ID NO:3, corresponding to amino acids 146-151 of SEQ ID NO:1; the
sequences associated with site 2 are VLERY, SEQ ID NO:4,
corresponding to amino acids 11-15 of SEQ ID NO:1, and SGLRS, SEQ
ID NO:5, corresponding to amino acids 100-104 of SEQ ID NO:1
(Cheetham et al., 1998, Nature Structural Biology 5:861-866, hereby
incorporated by reference in its entirety). EPOR homodimer
activation leads to tyrosine phosphorylation of signaling proteins
that are associated with EPOR, e.g., Jak2 tyrosine kinases, that
may in turn activate several different pathways including, for
example, the phosphatidylinositol (PI) 3-kinase pathway, the
Ras/MAP kinase pathway, and/or the STAT pathway. These pathways
trigger the anti-apoptotic functions necessary for erythropoiesis
that are mediated by erythropoietin (Kirito et al., 2002, Blood
99:102-110; Livnah et al., 1999, Science 283:987-990; Naranda et
al., 2002, Endocrinology 143:2293-2302; Remy et al., 1999, Science
283:990-993; and Yoshimura et al., 1996, The Oncologist 1:337-339,
each of which is hereby incorporated by reference in its
entirety).
[0009] Recently, Applicants have discovered that the tissue
protective properties of EPO are mediated by a receptor that
comprises not only EPOR but also another receptor protein, the beta
common receptor (".beta..sub.c"). The EPOR/.beta..sub.c receptor
is, in contrast to the homodimer (EPOR).sub.2, a heterocomplex (see
infra) and is known to play a role in the protection of excitable
tissues. See, e.g., WO 2004/096148 and PCT no. PCT/US01/49479,
filed Dec. 28, 2001, U.S. patent application Ser. No. 09/753,132,
filed Dec. 29, 2000, and Ser. No. 10/188,905, filed Jul. 3, 2002,
each of which is hereby incorporated by reference in its entirety.
Although Applicants had established that the .beta.c receptor is
central to the tissue protective pathways in these excitable
tissues, the structure of the activating ligands for the receptors
was still unknown.
3. SUMMARY
[0010] The present invention is drawn to isolated polypeptides that
have at least one cellular protective activity in a responsive
cell, tissue, or organ, which polypeptides contain amino acid
motifs comprising the consensus sequence (a)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.2, wherein n is 0, 1, 2, 3,
4 or 5; (b) H.sub.1-N.sub.1-(X).sub.n-N.sub.2-L.sub.1, wherein n is
0, 1, 2, 3, 4 or 5; (c) L.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.1,
wherein n is 0, 1, 2, 3, 4 or 5; (d)
H.sub.1-N.sub.1-(L).sub.n-P.sub.1-H.sub.2, wherein n is 0 or 1; or
(e) H.sub.1-P.sub.1-(L).sub.n-N.sub.1-H.sub.2, wherein n is 0 or 1,
and wherein H.sub.1 and H.sub.2 are hydrophobic amino acids,
N.sub.1 and N.sub.2 are negatively charged amino acids, X is any
amino acid, L.sub.1 is a polar amino acid, and P.sub.1 is a
positively charged amino acid. In certain embodiments, the peptides
of the invention also lack erythoropoietic activity, e.g., do not
increase hemoglobin or hematocrit in a recipient. In further
embodiments, the isolated polypeptides of the invention consist of
no more than 10, no more than 15, no more than 20, or no more than
30 amino acids. In other embodiments, the isolated peptide has less
than 90%, less than 85%, less than 80%, less than 75%, less than
70%, less than 65%, less than 60%, less than 55%, less than 50%,
less than 45%, less than 40%, less than 35%, less than 30%, or less
than 20 percent sequence identity with any portion of the amino
acid sequence of mature human erythropoietin ("EPO") set forth in
SEQ ID NO:1, wherein said portion of EPO contains the same number
of amino acid residues as said peptide.
[0011] In certain embodiments of the invention described
hereinabove, wherein the isolated polypeptide comprises the
structural motif (a) H.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.2,
wherein is 0, 1, 2, 3, 4 or 5 (embodied by sequence identifiers
6-11, respectively, discussed infra); (b)
H.sub.1-N.sub.1-(L).sub.n-P.sub.1-H.sub.2, wherein n is 0 or 1
(embodied by sequence identifiers 24-25, respectively, discussed
infra); or (e) H.sub.1-P.sub.1-(L).sub.n-N.sub.1-H.sub.2, wherein n
is 0 or 1 (embodied by sequence identifiers 26-27, respectively,
discussed infra), H.sub.1 and H.sub.2 may be the same hydrophobic
amino acid. In other embodiments of the invention described
hereinabove, wherein the isolated polypeptide comprises the
structural motifs (a) H.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.2,
wherein n is 0, 1, 2, 3, 4 or 5; (d)
H.sub.1-N.sub.1-(L).sub.n-P.sub.1-H.sub.2, wherein n is 0 or 1; or
(e) H.sub.1-P.sub.1-(L).sub.n-N.sub.1-H.sub.2, wherein n is 0 or 1,
H.sub.1 and H.sub.2 may be different hydrophobic amino acids. In
other embodiments, the invention provides for an isolated
polypeptide comprising the amino acid motif (a)
H.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.2, wherein n is 0, 1, 2, 3,
4 or 5; (b) H.sub.1-N.sub.1-(X).sub.n-N.sub.2-L.sub.1, wherein n is
0, 1, 2, 3, 4 or 5; (c) L.sub.1-N.sub.1-(X).sub.n-N.sub.2-H.sub.1,
wherein n is 0, 1, 2, 3, 4 or 5, and wherein N.sub.1 and N.sub.2
may the same or may be different negatively charged amino
acids.
[0012] The invention provides for isolated polypeptides comprising
the amino acid motifs described hereinabove, wherein said motifs
are formed by consecutive amino acids within the amino-acid
sequence of said polypeptide. In specific examples in accordance
with this embodiment, the invention provides for an isolated
polypeptide comprising the amino acid motif
H.sub.1-N.sub.1-N.sub.2-H.sub.2 (SEQ ID NO:6),
H.sub.1-N.sub.1-X-N.sub.2-H.sub.2 (SEQ ID NO:7),
H.sub.1-N.sub.1-X-X-N.sub.2-H.sub.2 (SEQ ID NO:8),
H.sub.1-N.sub.1-X-X-X-N.sub.2-H.sub.2 (SEQ ID NO:9),
H.sub.1-N.sub.1-X-X-X-X-N.sub.2-H.sub.2 (SEQ ID NO:10),
H.sub.1-N.sub.1-X-X-X-X-X-N.sub.2-H.sub.2 (SEQ ID NO:11),
H.sub.1-N.sub.1-N.sub.2-L.sub.1 (SEQ ID NO:12),
H.sub.1-N.sub.1-X-N.sub.2-L.sub.1 (SEQ ID NO:13),
H.sub.1-N.sub.1-X-X-N.sub.2-L.sub.1 (SEQ ID NO:14),
H.sub.1-N.sub.1-X-X-X-N.sub.2-L.sub.1 (SEQ ID NO:15),
H.sub.1-N.sub.1-X-X-X-X-N.sub.2-L.sub.1 (SEQ ID NO:16),
H.sub.1-N.sub.1-X-X-X-X-X-N.sub.2-L.sub.1 (SEQ ID NO:17),
L.sub.1-N.sub.1-N.sub.2-H.sub.2 (SEQ ID NO:18),
L.sub.1-N.sub.1-X-N.sub.2-H.sub.2 (SEQ ID NO:19),
L.sub.1-N.sub.1-X-X-N.sub.2-H.sub.2 (SEQ ID NO:20),
L.sub.1-N.sub.1-X-X-X-N.sub.2-H.sub.2 (SEQ ID NO:21),
L.sub.1-N.sub.1-X-X-X-X-N.sub.2-H.sub.2 (SEQ ID NO:22),
L.sub.1-N.sub.1-X-X-X-X-X-N.sub.2-H.sub.2 (SEQ ID NO:23),
H.sub.1-N.sub.1-P.sub.1-H.sub.2 (SEQ ID NO:24),
H.sub.1-N.sub.1-L.sub.1-P.sub.1-H.sub.2 (SEQ ID NO:25),
H.sub.1-P.sub.1-N.sub.1-H.sub.2 (SEQ ID NO:26), or
H.sub.1-P.sub.1-L.sub.1-N.sub.1-H.sub.2 (SEQ ID NO:27), wherein
H.sub.1 and H.sub.2 are hydrophobic amino acids, N.sub.1 and
N.sub.2 are negatively charged amino acids, X is any amino acid,
L.sub.1 is a polar amino acid, and P.sub.1 is a positively charged
amino acid. In certain aspects consistent with this embodiment,
wherein the isolated polypeptide comprises a motif having the amino
acid residues H.sub.1 and H.sub.2, H.sub.1 and H.sub.2 may the same
or may be different hydrophobic amino acids. In other aspects
consistent with this embodiment, wherein the isolated polypeptide
comprises a motif having the amino acid residues N.sub.1 and
N.sub.2, N.sub.1 and N.sub.2 may the same or may be different
negatively charged amino acids.
[0013] In other embodiments, the invention provides isolated
polypeptides wherein the amino acid motif is formed due to the
spatial organization of amino acids within the tertiary structure
of a polypeptide, i.e., the amino acids forming the motif are
spatially adjacent to one another in the three dimensional
structure, i.e. tertiary structure, of the polypeptide but may be
separated by 1 or more amino acids within the primary amino acid
sequence of the polypeptide chain. In a specific example in
accordance with this embodiment, the amino acid motif comprising
amino acid residues H.sub.1, N.sub.1, N.sub.2, and H.sub.2
analogous to SEQ ID NO:6, discussed supra, may form as a result of
the tertiary structure adopted by, i.e., protein folding of
peptides comprising, e.g., SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10 or SEQ ID NO:11, wherein the amino acid residues
between N.sub.1 and N.sub.2, e.g. (X).sub.n, fold such that N.sub.1
and N.sub.2 become linearly adjacent. Accordingly, the invention
encompasses isolated peptides comprising the amino acid motif
H.sub.1N.sub.1N.sub.2H.sub.2; H.sub.1N.sub.1N.sub.2L.sub.1;
L.sub.1N.sub.1N.sub.2H.sub.1; H.sub.1N.sub.1(L).sub.nPiH.sub.2,
wherein n is 0 or 1; or H.sub.1P.sub.1(L).sub.nN.sub.1H.sub.2,
wherein n is 0 or 1, which motifs are formed as a result of the
tertiary structure of said polypeptide. In related embodiments,
wherein the amino acid motif comprises N.sub.1 and N.sub.2, the
tertiary structures form such that the distance between the
carbonyl carbons of N.sub.1 and N.sub.2 is about 3 .ANG. to about 5
.ANG., preferably about 4 .ANG. to about 5 .ANG., and more
preferably about 4.4 .ANG. to about 4.8 .ANG.. In other
embodiments, wherein the amino acid motif comprises N.sub.1 and
N.sub.2, the tertiary structures form such that the distance
between N.sub.1 and N.sub.2 are confined spatially such that the
charge separation, e.g., the charged side chains, of the two is
between about 6.5 .ANG. to about 9 .ANG.. In a related embodiment,
N.sub.1 and N.sub.2 are thus spatially confined as a result of
being in an amino acid sequence that forms all or a portion of an
alpha helix, and may be separated by 1, 2, or more than 2 amino
acids in the sequence of said amino acids forming said helix. In
other related embodiments, wherein the amino acid motif comprises
N.sub.1 and P.sub.1, the tertiary structures form such that the
distance between the carbonyl carbons of N.sub.1 and P.sub.1 is
about 3 .ANG. to about 5 .ANG., preferably about 4 .ANG. to about 5
.ANG., and more preferably about 4.4 .ANG. to about 4.8 .ANG.. In
other embodiments, wherein the amino acid motif comprises N.sub.1
and P.sub.1, the tertiary structures form such that the distance
between N.sub.1 and P.sub.1 are confined spatially such that the
charge separation, e.g., the charged side chains, of the two is
between about 6.5 .ANG. to about 9 .ANG.. In a related embodiment,
N.sub.1 and P.sub.1 are spatially confined as a result of being in
an amino acid sequence that forms all or a portion of an alpha
helix, and may be separated by 1, 2, or more than 2 amino acids in
the sequence of said amino acids forming said helix. In certain
embodiments, the amino acids forming the motif within the tertiary
structure of said polypeptide are separated from each other by an
equal number of intervening amino acid residues in the linear amino
acid sequence of said polypeptide. In yet other embodiments, the
amino acids forming the motif within the tertiary structure of said
polypeptide are separated from each other by a different number of
intervening amino acid residues in the linear amino acid sequence
of said polypeptide. In certain embodiments, the isolated
polypeptide of the inventions forms a regular tertiary structure,
e.g., .alpha.-helix or .beta.-pleated sheet, such that the surface
of said structure presents the amino acids comprising said motif,
and thus the motif itself, to the interface of the protein
structure and the aqueous environment, i.e., presents the motif on
the surface of folded the polypeptide. In preferred embodiments,
the tertiary structures of the polypeptides of the invention form
in an aqueous environment at physiological conditions, e.g., PBS
(13 mM NaH.sub.2PO.sub.4, 137 mM NaCl, pH 7.4) at 37.degree. C.
[0014] In specific embodiments, the invention provides for isolated
polypeptides comprising the amino acid motifis described herein
above, e.g., peptide A (APPRLICDSRVLERYLLEAKEAE, SEQ ID NO:32),
peptide C (NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29), peptide D
(QQAVEVWQGLALLSEAVLRGQALLV, SEQ ID NO:30), peptide E
(GCAEHCSLNENITVPDTKVN, SEQ ID NO:31), peptide F (RYLLUNITTGC, SEQ
ID NO:33), peptide G (QEQLERALNSS, SEQ ID NO:40), peptide I
(CSLNENIQEQLERALNSS, SEQ ID NO:43), peptide J
(QEQLERALNSSLRRYINMLTRTR, SEQ ID NO:41), peptide K (WEHVNAIQEARRLL,
SEQ ID NO:35), or peptide L (KIRSDLTALTESYVKH, SEQ ID NO:37).
[0015] In certain embodiments, the invention provides isolated
polypeptides comprising 1 or more, 2 or more, 3 or more, 4 or more,
5 or more, 6 or more, or more than 6 amino acid motifs described
herein. In specific aspects of the invention in accordance with
this embodiment, wherein the isolated polypeptide comprises at
least two of the amino acid motifs described herein above, said at
least two motifs may be the same motif or they may be different
motifs.
[0016] In certain aspects, the invention provides for isolated
polypeptides lacking an erythropoietic activity, e.g., increasing
hemoglobin in a recipient. Preferably, the isolated polypeptides
lack other activities including, but not limited to, vasoactive
action (e.g., vasoconstriction), hyperactivating platelets,
pro-coagulant activities and stimulating proliferation and/or
production of thrombocytes and/or erythropoietic-dependent cells
(see, Coleman et al., 2006, PNAS 103:5965-5970, hereby incorporated
by reference in its entirety). In other aspects, the invention
provides isolated polypeptides that comprise at least one cellular
protective activity. Such cellular protective activity includes,
but is not limited to, protecting, maintaining, enhancing or
restoring the function or viability of a responsive mammalian cell,
tissue, or organ. Accordingly, in one aspect, the present invention
is directed to the use of an isolated polypeptide described herein
for the preparation of pharmaceutical compositions for protecting,
maintaining, enhancing, or restoring the function or viability of
responsive mammalian cells and their associated cells, tissues, and
organs. In related embodiments, the compositions are for
administration to a subject in need thereof. In preferred
embodiments, said subject is a mammal and, preferably, a human.
[0017] In other aspects, the present invention is directed to the
use of an isolated polypeptide described herein for the preparation
of a pharmaceutical composition for the protection against and/or
prevention of a responsive tissue injury, for the restoration of,
or for the rejuvenation of responsive tissue and/or responsive
tissue function in a subject in need thereof. In one particular
aspect, the responsive mammalian cells and their associated cells,
tissues, or organs are distal to the vasculature by virtue of a
tight endothelial cell barrier. In another particular aspect, the
cells, tissues, organs or other bodily parts are isolated from a
mammalian body, such as those intended for transplant. By way of
non-limiting examples, a responsive cell or tissue may be neuronal,
eye (e.g., retinal), adipose, connective, hair, teeth, mucosal,
pancreas, endocrine, ear, epithelial, skin, muscle, heart, lung,
liver, kidney, intestine, adrenal (e.g., adrenal cortex, adrenal
medulla), capillary, endothelial, testes, ovary, bone, skin, or
endometrial cells or tissue. Further, non-limiting examples of
responsive cells include photoreceptor (rods and cones), ganglion,
bipolar, horizontal, amacrine, Muller, Purkinje, myocardium, pace
maker, sinoatrial node, sinus node, junction tissue,
atrioventricular node, bundle of His, hepatocytes, stellate,
Kupffer, mesangial, renal epithelial, tubular interstitial, goblet,
intestinal gland (crypts), enteral endocrine, glomerulosa,
fasciculate, reticularis, chromaffin, pericyte, Leydig, Sertoli,
sperm, Graffian follicle, primordial follicle, islets of
Langerhans, .alpha.-cells, .beta.-cells, .gamma.-cells, F-cells,
osteoprogenitor, osteoclasts, osteoblasts, endometrial stroma,
endometrial, stem and endothelial cells. These examples of
responsive cells are merely illustrative. In one aspect, the
responsive cell or its associated cells, tissues, or organs are
excitable cells, tissues, or organs, or predominantly comprise
excitable cells or tissues. In certain aspects of the invention,
the excitable tissue is central nervous system tissue, peripheral
nervous system tissue, cardiac tissue or retinal tissue. In another
aspect, the responsive cell or its associated cells, tissues, or
organs are not excitable cells, tissues, or organs, nor do they
predominantly comprise excitable cells or tissues.
[0018] The erythropoietic and/or cellular protective activity of
the isolated polypeptide of the invention in responsive cells may
be evaluated and/or determined by any method described herein and
or known in the art. In certain embodiments, the erythropoietic
and/or cellular protective activity is determined in an in vitro
assay. In other embodiments, the erythropoietic and/or cellular
protective activity is determined in an in vivo assay. In a related
embodiment, wherein the cellular protective activity is
neuroprotection, the invention provides for a method of evaluating
said activity in vitro by (a) contacting a test culture of primary
hippocampal neurons with N-methyl-D-aspartate and said peptide; and
(b) determining the cell viability at 48 hours post said contact,
such that if the cell viability determined in step (b) is greater
than that of a control culture in the absence of said peptide, the
peptide possesses cellular protective activity.
[0019] In a particular embodiment, the mammalian cell, tissue, or
organ for which an aforementioned isolated peptide is used are
those that have expended or will expend a period of time under at
least one condition adverse to the viability of the cell, tissue,
or organ. In accordance with this embodiment, the isolated peptides
of the invention provide protection against and/or prevention of a
tissue injury resulting from such conditions, provide for the
restoration of, or provide for the rejuvenation of tissue and/or
tissue function in a subject in need thereof before, during or
after such conditions arise. Such conditions include traumatic in
situ hypoxia or metabolic dysfunction, surgically-induced in situ
hypoxia or metabolic dysfunction, or in situ toxin exposure, the
latter may be associated with chemotherapy or radiation therapy. In
other embodiments, the isolated peptides of the invention provide
protection against and/or prevention of a tissue injury resulting
from a disease or disorder, provide for the restoration of, or
provide for the rejuvenation of tissue and/or tissue function in a
subject in need thereof before, during or after such conditions
arise. In related embodiments said injury is caused by a seizure
disorder, multiple sclerosis, stroke, hypotension, cardiac arrest,
ischemia, myocardial infarction, inflammation, age-related loss of
cognitive function, radiation damage, cerebral palsy,
neurodegenerative disease, Alzheimer's disease, Parkinson's
disease, mitochondrial disease, AIDS dementia, memory loss,
amyotrophic lateral sclerosis, alcoholism, mood disorder, anxiety
disorder, attention deficit disorder, autism, Creutzfeld-Jakob
disease, brain or spinal cord trauma or ischemia, heart-lung
bypass, chronic heart failure, macular degeneration, diabetic
neuropathy, diabetic retinopathy, hepatitis, pancreatitis,
glaucoma, retinal ischemia, retinal trauma, cardiovascular disease,
cardiopulmonary disease, respiratory disease, kidney disease,
disease of the urinary system, disease of the reproductive system,
bone disease, skin disease, connective tissue disease,
gastrointestinal disease, endocrine abnormality, metabolic
abnormality, or a disease or disorder of the central or peripheral
nervous system. In still other embodiments, the adverse conditions
are a result of cardiopulmonary bypass (heart-lung machine), as is
used for certain surgical procedures. In still other embodiments,
said injury is cognitive dysfunction. In a particular embodiment,
the mammalian cell, tissue, or organ for which an aforementioned
isolated peptide is used express the .beta..sub.c receptor.
[0020] In certain embodiments, the invention is also directed to
pharmaceutical compositions comprising the aforementioned isolated
polypeptides for administration to a subject in need thereof. In
specific aspects in accordance with this embodiment, the
pharmaceutical composition of the invention further comprises a
pharmaceutically acceptable carrier. Such pharmaceutical
compositions may be formulated for oral, intranasal, ocular,
inhalational, transdermal, rectal, sublingual, vaginal, or
parenteral administration, or in the form of a perfusate solution
for maintaining the viability of cells, tissues, or organs ex vivo.
In related embodiments of the invention the subject is a mammalian
animal, preferably a human.
[0021] In other aspects, the invention provides a method for
facilitating the transcytosis of a molecule across an endothelial
cell barrier in a subject in need thereof comprising administration
to said subject a composition comprising said molecule in
association with an isolated peptide of the invention described
hereinabove. In a related embodiment, association is a labile
covalent bond, a stable covalent bond, or a non-covalent
association with a binding site for said molecule.
[0022] According to another aspect of the invention, the isolated
peptide of the invention, as described herein above, is capable of
traversing an endothelial cell barrier. In a related embodiment,
the endothelial cell barrier comprises the blood-brain barrier, the
blood-eye barrier, the blood-testis barrier, the blood-ovary
barrier, blood-placenta, blood-heart, blood-kidney, blood-nerve, or
blood-spinal cord barrier.
[0023] According to one aspect of the invention, there is provided
an isolated nucleic acid molecule that comprises a nucleotide
sequence which encodes a polypeptide comprising the isolated
polypeptide as described herein above.
[0024] In another embodiment of the invention, there is provided an
isolated nucleic acid molecule that comprises a nucleotide sequence
(i.e., a cDNA, a nucleotide sequence interrupted by introns, or
uninterrupted by introns), which encodes a polypeptide comprising
or consisting of the isolated polypeptide of the invention as
described herein above. In one embodiment, the nucleotide sequence,
encoding the isolated polypeptide of the invention, is synthesized
using preferred codons that facilitate optimal expression in a
particular host cell. Such preferred codons can be optimal for
expression in cells of a species of plant, bacterium, yeast,
mammal, fungus, or insect.
[0025] The invention also provides for a vector comprising the
nucleic acid molecule. The invention also provides for an
expression vector comprising the nucleic acid molecule and at least
one regulatory region operably linked to the nucleic acid molecule.
In another embodiment, the invention provides for a cell comprising
the expression vector. In yet another embodiment, there is provided
a genetically-engineered cell which comprises the nucleic acid
molecule.
[0026] In another embodiment, the invention provides for a method
of recombinantly producing the isolated peptide of the invention,
described herein above, comprising culturing in a medium a host
cell containing a nucleic acid molecule comprising a nucleotide
sequence encoding a polypeptide of the invention, under conditions
suitable for the expression of said peptide, and recovering and/or
isolating the expressed polypeptide from said medium.
3.1 Terminology
[0027] As used herein, the terms "about" or "approximately" when
used in conjunction with a number refer to any number within 1, 5,
or 10% of the referenced number.
[0028] The term "administered in conjunction with" in the context
of the methods of the invention means administering a compound
prior to, at the same time as, and/or subsequent to the onset of a
disease, disorder, or condition.
[0029] The term "amino acid" or any reference to a specific amino
acid is meant to include naturally occurring proteogenic amino
acids as well as non-naturally occurring amino acids such as amino
acid analogs. Those skilled in the art would know that this
definition includes, unless otherwise specifically noted, includes
naturally occurring protogenic (L)-amino acids, their optical
(D)-isomers, chemically modified amino acids, including amino acid
analogs such as penicillamine (3-mercapto-D-valine), naturally
occurring non-proteogenic amino acids such as norleucine and
chemically synthesized proteins that have properties known in the
art to be characteristic of an amino acid. As used herein, amino
acids will be represented wither by their three letter acronym or
one letter symbol as follows: alanine=Ala or A, arginine=Arg or R,
asparagine=Asn or N, aspartic acid=Asp or D, cysteine=Cys or C,
glutamic acid=Glu or E, glutamine=Gln or Q, glycine=Gly or G,
histidine=His or H, isoleucine=Ile or I, leucine=Leu or L,
lysine=Lys or K, methionine=Met or M, phenylalanine=Phe or F,
proline=Pro or P, serine=Ser or S, threonine=Thr or T,
tryptophan=Trp or W, tyrosine=Tyr or Y, and valine=Val or V.
Additionally, the term "amino acid equivalent" refers to compounds
that depart from the structure of the naturally occurring amino
acids, but which have substantially the structure of an amino acid,
such that they can be substituted within a peptide, which retains
its biological activity despite the substitution. Thus, for
example, amino acid equivalents can include amino acids having side
chain modifications or substitutions, and also include related
organic acids, amides or the like. The term "amino acid" is
intended to include amino acid equivalents. The term "residues"
refers both to amino acids and amino acid equivalents. Amino acids
may also be classified into the following groups as is commonly
known in the art: (1) hydrophobic amino acids: His, Trp, Tyr, Phe,
Met, Leu, Ile, Val, Ala; (2) neutral hydrophilic amino acids: Cys,
Ser, Thr; (3) polar amino acids: Ser, Thr, Asn, Gln; (4)
acidic/negatively charged amino acids: Asp, Glu; (5) charged amino
acids: Asp, Glu, Arg, Lys, His; (6) positively charged amino acids:
Arg, Lys, His; and (7) basic amino acids: His, Lys, Arg.
[0030] As used herein, "excitable tissue" means tissue that
contains excitable cells. Excitable cells are cells that respond
actively to an electric stimulus and have an electrical charge
differential across their cellular membranes. Excitable cells are
generally capable of undergoing an action potential. Such cells
typically express channels, such as voltage-gated, ligand-gated,
and stretch channels, which allow flow of ions (potassium, sodium,
calcium, chloride, etc.) across the membrane. Excitable tissue
includes neuronal tissue, muscle tissue, and glandular tissue.
Excitable tissue includes, but is not limited to, neuronal tissues
such as tissue of the peripheral nervous system (ear and retina)
and central nervous system (brain and spinal cord); cardiovascular
tissue such as the cells of the heart and associated nerves; and
glandular tissue such as the pancreas where T-type calcium channels
along with cell-to-cell gap junctions participate in secretion of
insulin. An exemplary list of excitable tissue includes organs and
tissues that include nerves, skeletal muscle, smooth muscle,
cardiac muscle, uterus, central nervous system, spinal cord, brain,
retina, olfactory system, auditory system, etc.
[0031] The term "host cell" as used herein refers to the particular
subject cell transfected with a nucleic acid molecule and the
progeny or potential progeny of such a cell. Progeny of such a cell
may not be identical to the parent cell transfected with the
nucleic acid molecule due to mutations or environmental influences
that may occur in succeeding generations or integration of the
nucleic acid molecule into the host cell genome.
[0032] An "isolated" or "purified" polypeptide is substantially
free of cellular material or other contaminating proteins from the
cell or tissue source from which the protein or polypeptide is
derived, or substantially free of chemical precursors or other
chemicals when chemically synthesized. The language "substantially
free of cellular material" includes preparations of a polypeptide
in which the polypeptide is separated from cellular components of
the cells from which it is isolated or recombinantly produced.
Thus, a polypeptide that is substantially free of cellular material
includes preparations of polypeptides having less than about 30%,
20%, 10%, or 5% (by dry weight) of heterologous protein (also
referred to herein as a "contaminating protein"). When the
polypeptide is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, 10%, or 5% of the volume of the
protein preparation. When the polypeptide is produced by chemical
synthesis, it is preferably substantially free of chemical
precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the protein. Accordingly such preparations of the polypeptide
have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical
precursors or compounds other than the antibody of interest. In a
preferred embodiment, polypeptides of the invention are isolated or
purified.
[0033] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
In a specific embodiment, a nucleic acid molecule(s) encoding a
polypeptide of the invention is isolated or purified.
[0034] As used herein in reference to a structure within a
polypeptide, the term "motif" refers either to a set of consecutive
amino acids within the amino acid sequence of the polypeptide chain
and/or to a set of linearly adjacent amino acids within the
tertiary structure of said polypeptide. Because the motif may be
formed all or in part as a result of protein folding, amino acids
that are adjacent in the described motif may be separated by 0, 1
or more, 5 or more, 10 or more, 15 or more or 20 or more amino
acids within the linear amino acid sequence of the polypeptide.
[0035] As used herein, the terms "peptide," "polypeptide" and
"protein" are used interchangeably and in their broadest sense to
refer to constrained (that is, having some element of structure as,
for example, the presence of amino acids which initiate a .beta.
turn or .beta. pleated sheet, or for example, cyclized by the
presence of disulfide bonded Cys residues) or unconstrained (e.g.,
linear) amino acid sequences. In certain embodiments, the peptide
of the invention consists of less than 30 amino acids. However,
upon reading the instant disclosure, the skilled artisan will
recognize that it is not the length of a particular peptide but its
ability to bind a tissue protective receptor complex and/or compete
with the binding of a peptide described herein that distinguishes
the peptide of the invention. The terms "peptide," "polypeptide,"
and "protein" also refer to compounds containing amino acid
equivalents or other non-amino acid groups, while still retaining
the desired functional activity of a peptide. Peptide equivalents
can differ from conventional peptides by the replacement of one or
more amino acids with related organic acids (such as PABA), amino
acids or the like or the substitution or modification of side
chains or functional groups.
[0036] The term "preventing a disease, disorder, or condition"
means delaying the onset, hindering the progress, hindering the
appearance, protection against, inhibiting or eliminating the
emergence, or reducing the incidence, of such disease, disorder, or
condition. Use of the term "prevention" is not meant to imply that
all patients in a patient population administered a preventative
therapy will never develop the disease, disorder, or condition
targeted for prevention, but rather that the patient population
will exhibit a reduction in the incidence of the disease, disorder,
or condition. For example, many flu vaccines are not 100% effective
at preventing flu in those administered the vaccine. One skilled in
the art can readily identify patients and situations for whom
preventative therapy would be beneficial, such as, but not limited
to, individuals about to engage in activities that may lead to
trauma and injury (e.g., soldiers engaging in military operations,
race car drivers, etc.), patients for whom surgery is planned,
patients at risk for inherited diseases, disorders, or conditions,
patients at risk for diseases, disorders, or conditions
precipitated by environmental factors, or portions of the
population at risk for particular diseases, disorders, or
conditions such as the elderly, infants, or those with weakened
immune systems, or those patients with genetic or other risk
factors for a disease, disorder, or condition.
[0037] As used herein, the terms "subject" and "patient" are used
interchangeably. As used herein, the terms "subject" and "subjects"
refer to an animal, preferably a mammal including a non-primate
(e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a
non-primate (e.g., a monkey or a human), and more preferably a
human.
[0038] As used herein, the term "tissue protective activity" or
"tissue protection" refers to the effect of inhibiting or delaying
damage or death of a cell, tissue, or organ. Unless otherwise
noted, the "delay" in damage or death of a cell, tissue or organ is
evaluated relative to a control condition in the absence of a
peptide of the invention. The tissue protective activity is useful
in various conditions, diseases, and cellular, organ, and/or tissue
damage, for example, those described in section 5.3. Tissue
protective activity is specific to tissue, cells, and/or organs
expressing a tissue protective receptor complex (i.e., a responsive
tissue cell, and. or organ, respectively), such as, but not limited
to, the tissues of the central nervous system. In specific
embodiments, the responsive cells are not erythrocyte progenitor
cells.
[0039] The term "tissue protective receptor complex" as used herein
means a complex comprising at least one erythropoietin receptor
subunit and at least one beta common receptor subunit. The tissue
protective receptor complex may contain multiple erythropoietin
receptor subunits and/or beta common receptor subunits, as well as
other types of receptors or proteins. See WO 2004/096148, which is
hereby incorporated by reference herein in its entirety.
[0040] To determine the percent identity of two amino acid
sequences, the sequences are aligned for optimal comparison
purposes. The amino acid residues at corresponding amino acid
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue as the corresponding
position in the second sequence, then the molecules are identical
at that position. The percent identity between the two sequences is
a function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions X 100%). In one embodiment, the
two sequences are the same length. In an alternate embodiment, the
sequences are of different length and, accordingly, the percent
identity refers to a comparison of the shorter sequence to a
portion of the longer sequence, wherein said portion is the same
length as said shorter sequence.
4. BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 depicts the results of an in vivo sciatic nerve
injury model to compare the efficacy of peptide J (SEQ ID NO:41) to
the tissue protective molecule carbamylated EPO (CEPO), wherein
peptide J, SEQ ID NO:41, is a chimeric peptide consisting of the
external facing amino acids of helix B of EPO (i.e., peptide G, SEQ
ID NO:40) combined with an amphipathic helix from pancreatic
polypeptide (LRRYINMLTRP, SEQ ID NO:28)
[0042] FIG. 2 depicts the tissue protective effects of peptides of
the invention as tested in an in vivo sciatic nerve injury model.
In the assay, the right sciatic nerve of rats (n=6 per group) was
injured and the animal immediately dosed with PBS, or PBS
containing equal molar concentrations of carbamylated EPO, EPO
peptide A (SEQ ID NO:32, corresponding to amino acids 1-23 of SEQ
ID NO:1), peptide D (SEQ ID NO:30, corresponding to amino acids
58-82 of SEQ ID NO:1), or peptide G (SEQ ID NO:40). Peptide G (SEQ
ID NO:40) is based on those amino acids within Helix B of EPO that
face outward from the globular center of the EPO molecule into the
hydrophilic environment, i.e., present on the surface of the
polypeptide. Additionally, a 20-mer constructed from a region of
pigment epithelium-derived factor known to be tissue protective via
another receptor was included as a negative control. The recovery
from injury over the next 4 days demonstrates that peptide G, SEQ
ID NO:40, and peptide D, SEQ ID NO:30, exhibit a tissue protective
effect in this in vivo model assay that is equivalent to or better
than carbamylated EPO (CEPO).
[0043] FIG. 3 depicts the erythropoietic effects of peptide D, SEQ
ID NO:30, and CEPO, known to lack erythropoietic activity, as
tested in a UT-7 assay for erythropoietic activity. The results of
this in vitro assay demonstrate that neither peptide D, SEQ ID
NO:30, nor CEPO exhibit erythropoietic activity at doses up to
10,000 pM.
[0044] FIG. 4 depicts the results of an in vivo assay to determine
whether peptide F (SEQ ID NO:33, corresponding to amino acids 14-29
of SEQ ID NO:1) and peptide G (SEQ ID NO:40) are erythropoietic or
elicit neutralizing antibodies against EPO. The results demonstrate
that neither protein increased hemoglobin levels in the rats when
administered at 0.8 m/kg, 3 days/week sub-cutaneously (s.c.) over
the course of 130 days. In addition, neither peptide elicits an
antibody response, in contrast to the administration of EPO.
[0045] FIG. 5 depicts the results of in vitro studies that
demonstrate that peptide D, SEQ ID NO:30, protects motor neurons
against kainate induced death.
[0046] FIG. 6 shows that peptide D, SEQ ID NO:30, at doses of 0.1
ng/ml and 1 ng/ml protects P-19 cells against apoptosis associated
with serum deprivation.
[0047] FIGS. 7 A-B depict the results of a middle cerebral artery
occlusion assay in rats. FIG. 7A depicts a graph demonstrating that
peptide D (SEQ ID NO:30, corresponding to amino acids 58-82 of SEQ
ID NO:1) at a single dose of 4.4 ug/kg is able to reduce the volume
of the infarct in the brain as robustly as four doses of 4.4 ug/kg
administered 2 hours apart. FIG. 7B depicts the results of a foot
fault assay to determine the behavioral deficit caused by the
middle cerebral artery occlusion. FIG. 7B shows that rats
demonstrated behavioral improvements when administered peptide D,
SEQ ID NO:30, at both a single dose schedule (1.times.4.4 ug/kg)
and a multiple dose schedule (4.times.4.4 ug/kg).
[0048] FIGS. 8 A-B depict the results of an in vivo assay of a
diabetic neuropathy assay. Diabetes is induced in rats using
streptozotocin. After verification of induced diabetes, the rats
were treated with peptide D, SEQ ID NO:30, or PBS five times a week
at a dose of 4 ug/kg-bw i.p. for a period of two weeks. Both the
nerve conduction velocity and the hot plate latency of the rats
were observed. FIG. 8A demonstrates that the rats treated with
peptide D, SEQ ID NO:30 exhibited improved conduction velocities in
comparison to the untreated rats. FIG. 8B demonstrates that
hotplate latency for the treated rats was reduced relative to the
untreated rats, further demonstrating the improvement in conduction
velocity.
[0049] FIGS. 9 A-B depict the results of treatment of cisplatin
induced neuropathy and nephropathy with EPO Helix B chimera. FIG.
9A demonstrates that the animals treated with peptide G (SEQ ID
NO:40, a Helix B chimera) exhibited improved results when tested in
a hotplate latency assay. FIG. 9B demonstrates that the urine
production, a measure of kidney function, was maintained as normal
in the peptide G (SEQ ID NO:40) treated animals.
[0050] FIG. 10 depicts the effects of peptide D (SEQ ID NO:30) on
retinal leakage associated with diabetic retinopathy. The figure
demonstrates that peptide D (SEQ ID NO:30) was able to
substantially reduce retinal leakage in the treated animals.
[0051] FIG. 11 depicts the results of peptide F (SEQ ID NO:33) or
peptide G (SEQ ID NO:40) on a model of kidney ischemia-reperfusion.
The figure demonstrates that both peptides reduced the injury score
resulting from an ischemia-reperfusion injury of 60 minutes when
assessed after 72 hours
[0052] FIG. 12 illustrates that the administration of peptide F
(SEQ ID NO:33) protects mice from experimental cerebral
malaria.
[0053] FIG. 13 Clinical Score in murine EAE model treated with
Peptide E, SEQ ID NO:31. FIG. 13 depicts the clinical course of
neurological function in mice with experimental autoimmune
encephalomyelitis. 4.4 .mu.g/kg Peptide E was administered i.p.
daily. Administration of peptide E significantly improved
neurological function relative to control. Clinical staging; 1,
flaccid tail; 2, ataxia and/or hind-limb paresis, or slow righting
reflex; 3, paralysis of hind limb and/or paresis of forelimbs; 4,
paresis of forelimb; 5, moribund or death.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Tissue Protective Peptides
[0054] The erythropoietic activity of erythropoietin ("EPO") has
been well characterized in the art (see, e.g., Cheetham et al.,
1998, Nat. Struct. Biol. 5; 861-866, herein incorporated by
reference in its entirety). EPO initiates erythropoiesis by binding
to the extracellular portion of a preformed erythropoietin receptor
(EPOR) homodimer (i.e., (EPOR).sub.2) in a manner that bridges
between specific locations on the individual EPOR subunits. When
EPO binds to the (EPOR).sub.2, large portions of the globular
ligand are remote from the binding regions and face outward, away
from the complex of EPO and (EPOR).sub.2 into the aqueous medium.
The Applicants have determined that tissue protection, as distinct
from erythropoiesis, is mediated through a receptor other than
(EPOR).sub.2, which consists of an EPOR monomer in conjunction with
another receptor, CD131 (also known as the .beta.-common receptor
subunit (.beta..sub.c)). EPOR and .beta..sub.c interact to form the
receptor heterodimer, EPOR-13c. Whether other proteins are involved
in this interaction is currently unknown. The instant invention
discloses tissue protective peptides derived from the three
dimensional structure of EPO, and in particular, from the portions
of EPO facing away from the EPOR binding sites, i.e., not
interacting with, the classical, erythropoietic EPOR (EPOR).sub.2
homodimer. Not wishing to be bound by any particular theory, the
Applicants believe that this portion of the EPO molecule interacts
with the tissue protective receptor and thereby mediates tissue
protection.
[0055] The three dimensional structure of EPO is accepted as
described by Cheetham et al., 1998, Nat. Struct. Biol. 5; 861-866,
hereby incorporated by reference in its entirety, and as set forth
in SEQ ID NO:1 (also available as data deposited in the Protein
Data Bank of the National Center for Biotechnology Information as
entry "1BUY"). The portions of the EPO molecule that face away from
the membrane-proximal portion of the EPOR homodimer when bound to
said receptor (i.e., away from the cell membrane when the
(EPOR).sub.2 homodimer is expressed on the surface of a cell)
consist of the following secondary structures: loop AB
(corresponding to amino acids 29-55 of SEQ ID NO:1), helix B
(corresponding to amino acids 56-82 of SEQ ID NO:1), loop BC
(corresponding to amino acids 83-92 of SEQ ID NO:1) and loop CD
(corresponding to amino acids 112-138 of SEQ ID NO:1). In one
embodiment of the invention, the tissue protective peptides consist
of the amino acid sequences corresponding to these distinct
structures of the EPO molecule.
[0056] Not wishing to be bound to any particular theory, the
Applicants believe that the Tissue Protective Receptor is
preformed, i.e. that the EPOR and .beta..sub.c protein subunits are
functionally associated prior to their interaction with EPO. EPO is
a member of the type I cytokine superfamily. Members of type 1
cytokine superfamily branch are characterized by four helices which
interact hydrophobically to form a globular protein whose exterior
surface interfaces with the aqueous medium and is termed
"externally-facing". Unexpectedly, the Applicants have determined
that more than one peptide derived from the externally-facing
portion of the EPO molecule is tissue-protective. A further
surprising discovery is that peptides derived from portions of the
EPO molecule that are buried within the EPO:(EPOR).sub.2 complex
and peptides that may also contain portions of erythropoiesis
binding sites 1 or 2 are also be highly potent in tissue
protection. To account for these discoveries, Applicants propose
that successful activation of the tissue protective receptor is due
to an appropriate, spatially compact charge configuration within
the peptide ligand. Further, this compact charge configuration is
embodied by two distinct structural motifs: (1) two negatively
charged amino acids adjacent to each other, and flanked by
hydrophobic amino acids; or (2) a positive and a negative (i.e.,
basic and acidic) amino acid immediately adjacent to one another,
and flanked by single hydrophobic or polar amino acid residues. The
proximity of these charges may occur via the linear structure
imposed by peptide bonding, i.e., the structure may be formed by
consecutive amino acids in a polypeptide chain, or alternatively,
proximity can also occur via a spatial relationship between
different parts of the EPO molecule (or other related type 1
cytokine molecules) imparted by the protein's tertiary structure,
i.e., three dimensional structure. Not wishing to be bound to any
specific theory, Applicants believe that, in general, this
requirement dictates that a tissue protective peptide will have a
distinct tertiary structure (e.g., helices or pleated sheets) that
provides for the required spatial location of the pair of charged
amino acids (i.e., the two negatively charges amino acids and/or
the positive and negative amino acid). A simple exception is a
linear peptide wherein the amino acid pair is immediately adjacent
to each other, with the required rigidity imparted by the peptide
backbone. Accordingly, the structural motif (1), is encompassed by
a linear sequence of amino acid residues, e.g.,
H.sub.1-N.sub.1-N.sub.1-H.sub.2 (SEQ ID NO:6), or by a linear
sequence of amino acid residues wherein N.sub.1 and N.sub.2 are
separated by 1, 2, 3, 4, 5, 6, or more intervening residues, e.g.,
H.sub.1-N.sub.1-X-X-X-X-X-N.sub.1-H.sub.2 (SEQ ID NO:11).
[0057] For tissue protection, the pair of charged amino acids must
be spatially oriented such that the carbonyl carbons are about 3
angstroms (.ANG.) to about 5 .ANG. apart, preferably, about 4 .ANG.
to about 5 .ANG. apart, and more preferably about 4.4 .ANG. to
about 4.8 .ANG. apart. This can be accomplished in a number of
ways, for example, by adjacent charged amino acids in a simple
linear peptide (see, e.g., Example 2 and peptide G, SEQ ID NO:40,
Table 1) or for peptides that can form an alpha helix, charged
amino acids separated by an intervening amino acid residue (see,
e.g., Example 2 and peptide F, SEQ ID NO:33, Table 1). It is to be
noted that tertiary structure (e.g., an alpha helix in amphipathic
peptides) can also be imparted when the peptide is within a
specific microenvironment, such as at the extracellular-cell
surface membrane interface (see, Segrest, 1990, Proteins 8:103-117,
hereby incorporated by reference in its entirety).
[0058] Further, tissue protective activity is predicted for
peptides that contain pairs of charged amino acids such that the
charged side-chains (either positive and negative or two negatives)
be confined spatially to within about 6.5 .ANG. to about 9 .ANG. of
each other. This can be provided for in an alpha helix by the
charged pair being separated by one or two amino acids, which will
provide for the charges to be more or less on the same side of the
helix with the required about 6.5 .ANG. to about 9 .ANG.
separation. A non-limiting example of such a peptide is found in
peptide F (see, Example 2, SEQ ID NO:33, Table 1). One skilled in
the art can devise a tertiary structure for the peptide that is
generally required to obtain the appropriate three dimensional
location of the charged amino acids, as well as the design of small
molecules to mimic the charge separation within the peptide.
[0059] The spatial distances between the carbamyl carbons of any to
amino acids or between the side chains of any two amino acids can
be deduced by any method known in the art or described herein. For
example, where the three-dimensional structure of the protein is
known, the charge separation of two side chains or the spatial
distance between two carbamyl carbons within a portion of interest
of said protein can be calculated based on the published, or
otherwise art-accepted, three-dimensional coordinates of the amino
acid residues in said portion of interest. Where the
three-dimensional structure of the protein and, therefore, the
portion of interest is unknown, or wherein a fully synthetic
peptide is constructed based on the teachings herein, whose three
dimensional structure is unknown, the charge separation of two side
chains or the spatial distance between two carbamyl carbons within
said peptide can be estimated using the three-dimensional structure
predicted by protein modeling software as is known in the art.
Non-limiting examples of such software are MOE.TM. by Chemical
Computing Group (Quebec, Canada) and Modeler by Accelrys (San
Diego, Calif.). Similarly such predictive software, available from
the above-noted companies as well, is also known in the art for the
design of small molecules as and, accordingly, one of ordinary
skill in the art, based upon the teachings herein, would be able to
make small molecules that emulate the disclosed structural
motifs.
[0060] Non-naturally occurring or chimeric peptides can be designed
that mimic the critical spatial proximities described herein above
via a linear sequence of amino acids. The present invention is,
therefore, directed to novel tissue protective peptides, including
those that exhibit these structural motifs that trigger tissue
protection.
[0061] The present invention also relates to the use of tissue
protective fragments of other type 1 cytokines, including, but not
limited to, granulocyte-macrophage colony stimulating factor
(GM-CSF), interleukin-3 (IL-3), Thrombopoietin (TPO), Ciliary
Neurotrophic Factor (CNTF) and Leukemia Inhibitory Factor (LIF),
that are structurally homologous with the above noted
externally-presenting amino acid sequences of EPO and/or contain
the structural motifs described above.
[0062] Further, the tissue protective peptides may be chimeric
compounds based upon structural motifs described above combining
non-adjacent structural elements and surface presenting amino acids
solely. In particular, the applicants have determined that the
addition of an amphipathic peptide helix to the above noted
sequences increases the potency of the peptide.
[0063] Additionally, the tissue protective peptides of the present
invention include fusion peptides resulting from the combination of
two or more of the above noted peptides, or with a related or
unrelated macromolecule for specific transport, such as native EPO,
insulin or leptin.
5.1.1 Fragments
A. EPO-Derived Peptide Fragments
[0064] The present invention relates to novel tissue protective
peptides that in one embodiment are comprised of fragments of the
amino acid sequences of EPO, derived from the three dimensional
structure of the EPO protein, and in particular, were derived from
those regions of EPO facing away from the ligand binding sites
and/or the internal portion of the EPOR homodimer. These fragments
are derived from the following EPO structures: (1) loop AB and
N-terminal portion of helix B (NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29,
corresponding to amino acids 38-57 of SEQ ID NO:1); (2)C-terminal
portion of helix B (QQAVEVWQGLALLSEAVLRGQALLV, SEQ ID NO:30,
corresponding to amino acids 58-82 of SEQ ID NO:1), and (3) a
portion of the A-B loop consisting of a small cysteine loop and a
.beta.-pleated sheet (GCAEHCSLNENITVPDTKVN, SEQ ID NO:31,
corresponding to amino acids 28-47 of SEQ ID NO:1). These peptide
fragments are all demonstrated in Example 2 (see FIG. 1 and Table
1) to exhibit tissue protective properties.
[0065] Unexpectedly, some peptides derived from other regions of
the EPO molecule that are buried and other peptides that include
portions of the binding sites to (EPOR).sub.2 are also tissue
protective. For example, a peptide consisting of the N-terminal
portion of Helix A (APPRLICDSRVLERYLLEAKEAE, SEQ ID NO:32,
corresponding to amino acids 1-23 of SEQ ID NO:1) that contains a
portion of EPOR binding Site 2 (underlined) is tissue protective
(see Example 2 and Table 1). However, the presence of Site 2 amino
acids does not account for the tissue protective activity, as a
peptide consisting of amino acids 14-19 of SEQ ID NO:1
(RYLLEAKEAENITTGC, SEQ ID NO:33) and lacking amino acids 11-13 of
SEQ ID NO:1 (i.e., VLE; the site 2 amino acids that are required
for binding of EPO to the EPOR dimer, (EPOR).sub.2, is also tissue
protective (see, Example 2 and Table 1, also Elliott et al., 1997,
Blood 89:493, hereby incorporated by reference in its entirety).
Applicants have previously shown that mutations within the
erythropoiesis binding sites that abolish erythropoiesis do not
modify the tissue protective properties of EPO (Leist et al.
Science (2004) 305:239, hereby incorporated by reference in its
entirety).
[0066] One of ordinary skill in the art will recognize that
fragments of varying lengths can form a tissue protective peptide,
although the fragment is preferably less than 30 amino acids in
length. Further, judicious selection of other molecules for
inclusion, e.g., D-amino acids or polyethylene glycol, will also
constitute a tissue protective peptides, but with enhanced
biological half-lives.
[0067] A. Structural Motifs
[0068] Specifically, the following structural motifs have been
identified that trigger the Tissue Protective Receptor complex:
[0069] (a) A Negative Charge Configuration ("Structural Motif
A").
[0070] In this structural motif, the peptide possesses two
negatively charged amino acids, which can be separated by up to 5
amino acids, flanked by hydrophobic amino acids. Structurally this
can be represented as:
[0071] (a1) HNNH;
[0072] (a2) HNXNH;
[0073] (a3) HNXXNH;
[0074] (a4) HNXXXNH;
[0075] (a5) HNXXXXNH; or
[0076] (a6) HNXXXXXNH,
where H represents hydrophobic amino acids (e.g., the moderately
hydrophobic amino acids: glycine, proline, cysteine, tyrosine, and
tryptophan, and preferably the highly hydrophobic amino acids:
alanine, valine, isoleucine, methionine, leucine, phenylalanine), N
represents a negatively charged amino acid such as glutamate or
aspartate, and X represents any amino acid, although preferably a
hydrophilic one. In certain embodiments, the flanking hydrophobic
amino acids are the same. In other embodiments, the flanking amino
acids are different.
[0077] A variation of this structural motif involves a peptide
where one of the flanking hydrophobic amino acids has been replaced
with a polar amino acid such as serine, threonine, asparagine, or
glutamine.
[0078] As an alternative to peptide linkages establishing the
mutual proximity of the two negative charges in a linear sequence,
the necessary charge proximity may also be accomplished by a three
dimensional structure as discussed herein above, (Section 5.1). For
example, the negatively charged amino acids may be spatially
immediately adjacent on the external surface of a helix, but will
be separated by additional amino acids in the linear peptide
sequence. For example, in helix A of EPO (corresponding to amino
acids 10-28 of SEQ ID NO:1), E18 and E21 are adjacent on the three
dimensional structure, but have two intervening amino acids between
them in the linear peptide sequence. As an additional example, in
helix B (peptide D, SEQ ID NO:30; corresponding to amino acids
58-82 of SEQ ID NO:1) E62 and E72 are separated by two amino acids
(Q65 and L69) on the surface of the helix, but have 9 amino acids
between them within the linear peptide. Peptides constructed from
helix A or helix B are tissue protective (See Example 2 and Table
1, infra). In contrast, peptide B (NITTGCAEHCSLNE, SEQ ID NO:34) a
peptide with dual negative charges (underlined) at the appropriate
distance but lacking a flanking hydrophobic amino acid, is not
tissue protective (See Example 2 and Table 1, infra).
[0079] (b) Negative/Positive Amino Acid Configuration ("Structural
Motif B").
[0080] In this structural motif, the peptide has a positive amino
acid next to a negative amino acid and both charged amino acids are
flanked by single hydrophobic amino acids. Structurally this can be
represented as:
[0081] (b1) HNPH; or
[0082] (b2) HPNH,
where P represents positively charged amino acids such as arginine,
lysine or histidine and N represents the negatively charged amino
acids glutamate or aspartate. As with the first motif, the mutual
proximity of the two opposite charges may be accomplished by three
dimensional structure. For example, a positive and a negatively
charged amino acid may be spatially adjacent on the surface of a
helix, but will be separated by one or more amino acids in the
linear peptide sequence. For example, in helix B (corresponding to
amino acids 58-82 of SEQ ID NO:1) E72 and R76 are immediately
adjacent to each other on the external surface of the helix and a
peptide constructed from this helix is tissue protective (see
Example 2 and Table 1).
[0083] In a variation of this particular motif, the negative and
positive amino acids can be separated by a polar amino acid,
e.g.,
[0084] (b3) HNLPH;
[0085] (b4) HPLNH,
wherein L represents a polar amino acids such as serine, threonine,
asparagine, or Glutamine. An example of this motif is peptide E
(GCAEHCSLNENITVPDTKVN, SEQ ID NO:34), which is tissue protective
(see Example 2 and Table 1).
[0086] Given that the core of the above structural motif is four
amino acids in length, a peptide of this core structural motif may
trigger the Tissue Protective Receptor. In certain embodiments the
polypeptides of the invention comprise 1 structural motif. In
alternate embodiments, the polypeptides of the invention comprise
more than 1, more than 2, more than 3 or more than 4 of the
structural motifs. In certain embodiments, wherein the polypeptide
comprises at least two structural motifs, the motifs are the same.
In alternate embodiments, wherein the polypeptide comprises at
least two structural motifs, the motifs are different. Preferably,
the multiple peptides of the present invention that one skilled in
the art can generate are less than 30 amino acids in length.
[0087] One of ordinary skill in the art will recognize that it is
the above noted structural motifs, as opposed to the actual amino
acid sequence of EPO that is important to the current invention.
Thus one of ordinary skill in the art would recognize that the
isolated peptide may have less than 90%, less than 85%, less than
80%, less than 75%, less than 70%, less than 65%, less than 60%,
less than 55%, less than 50%, less than 45%, less than 40%, less
than 35%, less than 30%, or less than 20 percent sequence identity
with any portion of the amino acid sequence of mature human
erythropoietin ("EPO") set forth in SEQ ID NO:1, wherein said
portion of EPO contains the same number of amino acid residues as
said peptide.
[0088] Additionally, U.S. Pat. No. 5,700,909 to O'Brien et al.,
hereby incorporated by reference in its entirety) discloses a 17
amino acid peptide sequence of EPO (SEQ ID NO:11 of O'Brien) which
induces biological activity in NS20Y, SK-N-MC, and PC12 cells
including sprouting, differentiation, neuroprotection, and
prevention of neuronal cell death. SEQ ID NO:11 of O'Brien
(designed epopeptide AB), although prophetically disclosed to have
erythropoietic activity, in fact lacks such erythropoietic activity
and was subsequently found to lack in vivo activity. When
epopeptide AB was injected into the muscle of mice, the frequency
of motor end plate sprouting in the adjacent muscles increased in a
manner similar to that induced by ciliary neurotrophic factor.
These data are interpreted within the concept that neuronal (but
not hematological) cells respond to a peptide sequence within EPO
and that EPO may have separate domains for neurotrophic and
hematotrophic activity (Campana et al., Int. J. Mol. Med. (1998)
1(1):235-241; J. S. O'Brien in U.S. Pat. No. 5,700,909, issued Dec.
23, 1997; J. S. O'Brien in U.S. Pat. No. 5,571,787, issued Nov. 5,
1996; J. S. O'Brien in U.S. Pat. No. 5,714,459, issued Feb. 3,
1998; and J. S. O'Brien and Y. Kashimoto in U.S. Pat. No.
5,696,080, issued Dec. 9, 1997). However, O'Brien did not
appreciate the current structural motifs based upon the proximity
of charged amino acids in the tertiary structure of the
peptide.
[0089] C. Type 1 Cytokine Fragments.
[0090] Given the spatially compact charge configuration able to
activate the tissue protective receptor, Applicants have discovered
that certain fragments of type-1 cytokines are expected to cross
react with the tissue protective receptor. This cytokine family
includes, but is not limited to, interleukin (IL)-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, granulocyte macrophage-colony
stimulating factor (GM-CSF), leptin, granulocyte colony stimulating
factor (G-CSF), leukemia inhibiting factor (LIF), ciliary
neurotrophic factor (CNTF), thrombopoietin (TPO), growth hormone,
macrophage colony stimulating factor (M-CSF), erythropoietin (EPO)
and prolactin.
[0091] Consideration of the secondary structure of EPO provides
guidance for the preparation of a candidate tissue protective
peptide via the spatial arrangement of amino acids derived from
homologous amino acids located within homologous secondary
structures within other type-1 cytokine receptor ligands: e.g.,
GM-CSF and IL-3 (Kannan, 2000, Neuroimmunomod. 8:132-141, hereby
incorporated by reference in its entirety), among others, have been
shown to possess potent neurotrophic and neuroprotective
activities, due in large part, the Applicants believe, by
stimulating a tissue protective receptor. For example, considering
helix B of these type I cytokines: Homologous amino acids in
thrombopoietin (TPO; Protein Data Bank (PDB) accession 1V7M)
comprise D62, G65, T68, L69, E72, A76 and Q80, where these amino
acids are spatially adjacent to one another in a linear
arrangement; homologous amino acids in leukemia inhibitory factor
(LIF; PDB accession 1EMR) comprise E61, R64, Y68, S72, N75, and
D79; homologous amino acids in ciliary neurotrophic factor (CNTF;
PDB accession 1CNT) comprise E71, E75. These all are examples of
Motif A described above (section 5.1.1), wherein the underlined
amino acids are negatively charged.
[0092] Examples of peptides derived from the Type-1 cytokines that
exemplify the structural Motif B described herein above (section
5.1.1) include, but are not limited to, GM-CSF helix A fragment,
WEHVNAIQEARRLL (SEQ ID NO:35); TPO helix A fragment, LSKLLRDSHVLH
(SEQ ID NO:36); TPO helix B fragment: E56, K59; CNTF helix A
fragment, KIRSDLTALTESYVKH (SEQ ID NO:37); CNTF helix B fragment:
R89, E92. LIF helix B fragment, GTEKAKLVELYRIVVYL (SEQ ID NO:38);
and interleukin 3 (IL-3) helix A fragment SIMIDEIIHHLKRPPNPL (SEQ
ID NO:39).
[0093] These aforementioned amino acids are merely exemplary from
some members of the cytokine superfamily that signal through Type 1
cytokine receptors, and homologous regions on other members of the
cytokine superfamily will be readily identified by the skilled
artisan.
5.1.2 Chimeras
[0094] "Chimeric" tissue protective peptides--linear amino acid
sequences that incorporate non-linear structural elements of the
externally-facing amino acids of EPO molecule exhibit the
above-noted structural motifs--are also contemplated by the current
invention. Chimeric tissue protective peptides of the current
invention may consist of combining structural elements of separate
amino acid sequences into a single peptide. In other words, a
chimeric tissue protective peptide may be comprised of amino acid
sequences derived from non-linear but adjacent structural elements
such as a fragment derived from amino acid sequences 110-115,
133-136, and 160-165, of SEQ ID NO:1 which would allow structural
elements of the C terminal portion of helix C and N-terminal
portion of loop C-D, the .beta.-pleated sheet in loop C-D, and the
C-terminal portion of EPO to be contained in a single peptide.
Additionally, chimeric tissue protective peptides may be used to
select out the important features of a particular structure, for
example the externally-facing amino acids of a particular tertiary
structure. Thus, a chimeric tissue protective peptide may consist
of a fragment comprised of helix B amino acids 58, 62, 65, 69, 72,
76, 79, 80, 83, 84, and 85 (e.g., peptide G, QEQLERALNSS, SEQ ID
NO:40) or, in other words, all of the exterior-presenting amino
acids of helix B of EPO. This peptide is shown to be tissue
protective in Example 2, infra (see Table 1).
[0095] Furthermore, the potency of the current tissue protective
peptides may be increased by attaching an amphipathic peptide
helix. Amphipathic peptide helices are well known in the art, e.g.
from peptides that signal through the Class B G-protein coupled
receptors (e.g., Segrest et al., 1990, Proteins 8:103, hereby
incorporated by reference in its entirety), serving to localize the
peptide ligand to the cell membrane. Examples of such helices
include, but are not limited to, the highly hydrophobic regions
from: calcitonin (ALSILVLLQAGS, SEQ ID NO:48); corticotropin
releasing hormone (VALLPCPPCRA, SEQ ID NO:49); beta endorphin
(NAIIKNAYKKG, SEQ ID NO:50); glucagon (GSWQRSLQDTE, SEQ ID NO:51);
secretin (GGSAARPAPP, SEQ ID NO:52); vasointestinal polypeptide
(NALAENDTPYY, SEQ ID NO:53); neuropeptide Y (GALAEAYPSKP, SEQ ID
NO:54); gonadotropin releasing hormone (GCSSQHWSYGL, SEQ ID NO:55);
parathyroid hormone (VMIVMLAICFL, SEQ ID NO:56); pancreatic
polypeptide (LRRYINMLTRP, SEQ ID NO:28); and calcitonin gene
related peptide (LALSILVLYQA, SEQ ID NO:57) (disclosed in Grace et
al., 2004, PNAS 101:12836, hereby incorporated by reference in its
entirety). For example, a chimeric peptide made from a peptide with
the surface charge motif of helix B of EPO (QEQLERALNSS, SEQ ID
NO:40) joined at the carboxy terminus to the ampipathic helix of
pancreatic polypeptide (LRRYINMLTRP, SEQ ID NO:28) for a chimeric
peptide. Further modifications may be made to the carboxy terminus
of the amphipathic helix without affecting its tissue protective
properties. Thus, a further example, replacing the terminal proline
of the above chimeric peptide with the sequence TR
(QEQLERALNSSLRRYINMLTRTR, SEQ ID NO:41) generates a molecule with
potent tissue protective activity as demonstrated in the sciatic
nerve assay (see, FIG. 1).
[0096] Additionally, instead of the above-noted helices, other
tertiary structures can be attached to the tissue protective
peptides. For example, the helix B exterior-presenting amino acids
can be linked to the beta pleated sheet (CSLNENI, SEQ ID NO:42)
found within the AB loop of EPO to form a chimeric peptide having
the sequence CSLNENIQEQLERALNSS (SEQ ID NO:43), which is tissue
protective (see Example 2 and Table 1). Additionally, the
presenting amino acids of the terminal portion of helix C (ALGKA,
SEQ ID NO:44, corresponding to amino acids 111,112,113,116, and 118
of SEQ ID NO:1) may be combined with all or part of loop CD-partial
(LGAQKEAISPPDAASAAPLRTI, SEQ ID NO:45, corresponding to amino acids
112-133 of SEQ ID NO:1). Preferably, a linking arm will be present
between the fused peptides to provide for flexibility so that the
joined peptides can assume the proper structural orientation to
bind with the tissue protective receptor complex. Such fusion
peptides may have a synergistic effect, obtaining a greater tissue
protective effect jointly as opposed to individually possibly
through enhanced binding with the tissue protective receptor
complex or increased biological half life.
[0097] One of ordinary skill in the art will recognize the benefit
of combining various desired structural elements in to a single
peptide for maximizing the tissue protective effects of such
compounds. Such chimeras may comprise amino acids peptides, and
non-amino acid elements, such as linkers or bridging atoms or
moieties.
5.1.3 Fusion Peptides
[0098] The present invention further contemplates that two or more
of the above noted tissue protective peptides, fragment derived or
chimera, may be linked to a related or unrelated protein such as
erythropoietin, albumin, etc.
5.1.4 Manufacture of Tissue Protective Peptides
[0099] Tissue protective peptides of the current invention may be
made using recombinant or synthetic techniques well known in the
art. In particular, solid phase protein synthesis is well suited to
the relatively short length of the tissue protective peptides and
may provide greater yields with more consistent results.
Additionally, the solid phase protein synthesis may provide
additional flexibility regarding the manufacture of the tissue
protective peptides. For example, desired chemical modifications
may be incorporated into the tissue protective peptide at the
synthesis stage: homocitrulline could be used in the synthesis of
the peptide as opposed to lysine, thereby obviating the need to
carbamylate the peptide following synthesis.
[0100] Synthesis
[0101] In solid-phase synthesis of a peptide an amino acid with
both .alpha.-amino group and side chain protection is immobilized
on a resin. See e.g. Nilsson, B., Soellner, M., and Raines, R.
Chemical Synthesis of Proteins, Annu. Rev. Biomol. Struct. 2005.
34:91-118; Meldal M. 1997. Properties of solid supports. Methods
Enzymol. 289:83-104 and Songster M F, Barany G. 1997. Handles for
solid-phase peptide synthesis. Methods Enzymol. 289:126-74.
Typically, two types of a-amino-protecting groups are used: an
acid-sensitive tert-butoxycarbonyl (Boc) group or a base-sensitive
9-fluorenylmethyloxycarbonyl (Fmoc) group. Wellings D A, Atherton
E. 1997. Standard Fmoc protocols. Methods Enzymol. 289:44-67. After
the quick and complete removal of these a-amino-protecting groups
another protected amino acid with an activated carboxyl group can
then be coupled to the unprotected resin-bound amine. By using an
excess of activated soluble amino acid, the coupling reactions are
forced to completion. The cycle of deprotection and coupling is
repeated to complete the sequence. With side chain deprotection and
cleavage, the resin yields the desired peptide. Guy C A, Fields G
B. 1997. Trifluoroacetic acid cleavage and deprotection of
resin-bound peptides following synthesis by Fmoc chemistry. Methods
Enzymol. 289:67-83, and Stewart J M. 1997. Cleavage methods
following Boc-based solid-phase peptide synthesis. Methods Enzymol.
289:29-44. Additional methods for performing solid phase protein
synthesis are disclosed in Bang, D. & Kent, S. 2004. A One-Pot
Total Synthesis of Crambin. Angew. Chem. Int. Ed. 43:2534-2538;
Bang, D., Chopra, N., & Kent, S. 2004. Total Chemical Synthesis
of Crambin. J. Am. Chem. Soc. 126:1377-1383; Dawson, P. et al.
1994. Synthesis of Proteins by Native Chemical Ligation. Science.
266:776-779; Kochendoerfer et al. 2003. Design and Chemical
Synthesis of a Homogenous Polymer-Modified Erythropoiesis Protein.
Science. 299: 884-887. (Each reference recited in this paragraph is
hereby incorporated by reference in its entirety.)
[0102] If necessary, smaller peptides derived from solid phase
peptide synthesis may be combined through peptide ligations such as
native chemical ligation. In this process, the thiolate of an
N-terminal cysteine residue of one peptide attacks the C-terminal
thioester of a second peptide to affect transthioesterification. An
amide linkage forms after rapid S.fwdarw.N acyl transfer. See
Dawson, P. et al. 1994. Synthesis of Proteins by Native Chemical
Ligation. Science. 266:776-779, which is hereby incorporated by
reference in its entirety.
[0103] Further, one of ordinary skill in the art would recognize,
that the tissue protective peptides of the current invention may
encompass peptidomimetics, peptides including both naturally
occurring and non-naturally occurring amino acids, such as
peptoids. Peptoids are oligomers of N-substituted glycines,
glycoholic acid, thiopronine, sarcosine, and thiorphan. These
structures tend to have a general structure of
(--(C.dbd.O)--CH.sub.2--NR--).sub.n with the R group acting as the
side chain. Such peptoids can be synthesized using solid phase
synthesis in accordance with the protocols of Simon et al.,
Peptoids: A molecular approach to drug discovery, Proc. Natl. Acad.
Sci USA, 89:9367-9371 (1992) and L.sub.1 et al., Photolithographic
Synthesis of Peptoids, J. AM. CHEM. SOC. 2004, 126, 4088-4089, each
of which is hereby incorporated by reference in its entirety.
Additionally, the current invention contemplates the use of
peptidemimetics or peptide mimetics, non-peptide drugs with
properties analogous to those of the template peptide. (Fauchere,
J. (1986) Adv. Drug Res. 15:29; Veber and Friedinger (1985) TINS p.
32; and Evans et al. (1987) J. Med. Chem 30:1229, which are
incorporated by reference). Synthesis of various types of
peptidomimetics has been reviewed for example in: Methods of
Organic Chemistry (Houben-Weyl), Synthesis of Peptides and
Peptidomimetics--Workbench Edition Volume E22c (Editor-in-Chief
Goodman M.) 2004 (George Thieme Verlag Stuttgart, New York, hereby
incorporated by reference in its entirety).
Recombinant Techniques
[0104] A variety of host-expression vector systems may be utilized
to produce the tissue protective peptides of the invention. Such
host-expression systems represent vehicles by which the tissue
protective peptide of interest may be produced and subsequently
purified, but also represent cells that may, when transformed or
transfected with the appropriate nucleotide coding sequences,
exhibit the modified erythropoietin gene product in situ. These
include but are not limited to, bacteria, insect, plant, mammalian,
including human host systems, such as, but not limited to, insect
cell systems infected with recombinant virus expression vectors
(e.g., baculovirus) containing the tissue protective peptide coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing
erythropoietin-related molecule coding sequences; or mammalian cell
systems, including human cell systems, e.g., HT1080, COS, CHO, BHK,
293, 3T3, harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells, e.g.,
metallothionein promoter, or from mammalian viruses, e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter.
[0105] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications and processing of protein products may be important
for the function of the protein. As known to those of ordinary
skill in the art, different host cells have specific mechanisms for
the post-translational processing and modification of proteins and
gene products. Appropriate cell lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells that possess
the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
may be used. Such mammalian host cells, including human host cells,
include but are not limited to HT1080, CHO, VERO, BHK, HeLa, COS,
MDCK, 293, 3T3, and WI38.
[0106] For long-term, high-yield production of recombinant
peptides, stable expression is preferred. For example, cell lines
that stably express the recombinant tissue protective
cytokine-related molecule gene product may be engineered. Rather
than using expression vectors that contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements, e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, and
the like, and a selectable marker. Following the introduction of
the foreign DNA, engineered cells may be allowed to grow for 1-2
days in an enriched media, and then are switched to a selective
media. The selectable marker in the recombinant plasmid confers
resistance to the selection and allows cells to stably integrate
the plasmid into their chromosomes and grow to form foci that in
turn can be cloned and expanded into cell lines. This method may
advantageously be used to engineer cell lines that express the
tissue-protective product. Such engineered cell lines may be
particularly useful in screening and evaluation of compounds that
affect the endogenous activity of the EPO-related molecule gene
product.
[0107] Further Modifications
[0108] Additional modifications can be made to the tissue
protective peptides. For example, the peptide may be synthesized
with one or more (D)-amino acids. The choice of including an (L)-
or (D)-amino acid into a peptide of the present invention depends,
in part, upon the desired characteristics of the peptide. For
example, the incorporation of one or more (D)-amino acids can
confer increasing stability on the peptide in vitro or in vivo. The
incorporation of one or more (D)-amino acids can also increase or
decrease the binding activity of the peptide as determined, for
example, using the bioassays described herein, or other methods
well known in the art.
[0109] Replacement of all or part of a sequence of (L)-amino acids
by the respective sequence of entatiomeric (D)-amino acids renders
an optically isomeric structure in the respective part of the
polypeptide chain. Inversion of the sequence of all or part of a
sequence of (L)-amino acids renders retro-analogues of the peptide.
Combination of the enantiomeric (L to D, or D to L) replacement and
inversion of the sequence renders retro-inverso-analogues of the
peptide. It is known to those skilled in the art that enantiomeric
peptides, their retro-analogues, and their retro-inverso-analogues
maintain significant topological relationship to the parent
peptide, and especially high degree of resemblance is often
obtained for the parent and its retro-inverso-analogues. This
relationship and resemblance can be reflected in biochemical
properties of the peptides, especially high degrees of binding of
the respective peptides and analogs to a receptor protein. The
synthesis of the properties of retro-inverso anologues of peptides
have been discussed for example in Methods of Organic Chemistry
(Houben-Weyl), Synthesis of Peptides and Peptidomimetics--Workbench
Edition Volume E22c (Editor-in-chief Goodman M.) 2004 (George
Thieme Verlag Stuttgart, New York), and in references cited
therein, all of which are hereby incorporated by reference herein
in their entireties.
[0110] Amino acid "modification" refers to the alteration of a
naturally occurring amino acid to produce a non-naturally occurring
amino acid. Derivatives of the peptides of the present invention
with non-naturally occurring amino acids can be created by chemical
synthesis or by site specific incorporation of unnatural amino
acids into polypeptides during biosynthesis, as described in
Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C.
Griffith, Peter G. Schultz, 1989 Science, 244:182-188, hereby
incorporated by reference herein in its entirety.
[0111] Peptide mimetics that are structurally similar to
therapeutically useful peptides may be used to produce an
equivalent therapeutic or prophylactic effect. Generally,
peptidomimetics are structurally similar to a paradigm polypeptide
(i.e., a polypeptide that has a biochemical property or
pharmacological activity), but have one or more peptide linkages
optionally replaced by a linkage selected from the group consisting
of: --CH.sub.2--NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-(cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--,
and --CH.sub.2SO--, by methods known in the art and further
described in the following references: Spatola, A. F. in "Chemistry
and Biochemistry of Amino Acids, Peptides, and Proteins," B.
Weinstein, eds., Marcel Dekker, New York, p 267 (1983); Spatola, A.
F., Vega Data (March 1983), Vol. 1. Issue 3, "Peptide Backbone
Modifications" (general review); Morely, J. S., Trends Pharma Sci
(1980) pp. 463-468 (general review); Hudson, D. et al., (1979) Int
J Pept Prot Re 14: 177-185 (--CH.sub.2--NH--,
--CH.sub.2--CH.sub.2--); Spatola, A. F. et al., (1986) Life Sci
38:1243-1249 (--CH.sub.2--S); Hann, M. M., (1982) J Chem Soc Perkin
Trans I 307-314 (--CH.dbd.CH--, cis and trans); Almquist, R. G. et
al., (1980) J Med Chem 23: 1392 (--COCH.sub.2--); Jennings-White, C
et al., (1982) Tetrahedron Lett 23:2533 (--COCH.sub.2--); Szelke, M
et al., European Appln. EP 45665 (1982) CA: 97: 39405 (1982)
(--CH(OH)CH.sub.2--); Holladay, M. W. et al., (1983) Tetrahedron
Lett 24:4401-4404 (--C(OH)CH.sub.2--); and Hruby, V. J., (1982)
Life Sci 31:189-199 (--CH.sub.2--S--); each of which is
incorporated herein by reference.
[0112] In another embodiment, a particularly preferred non-peptide
linkage is --CH.sub.2NH--. Such peptide mimetics may have
significant advantages over polypeptide embodiments, including, for
example: more economical production, greater chemical stability,
enhanced pharmacological properties (half-life, absorption,
potency, efficacy, etc.), altered specificity (e.g., a
broad-spectrum of biological activities), reduced antigenicity, and
others.
[0113] A variety of designs for peptide mimetics are possible. For
example, cyclic peptides, in which the necessary conformation is
stabilized by non-peptides, are specifically contemplated, U.S.
Pat. No. 5,192,746 to Lobl, et al., U.S. Pat. No. 5,576,423 to
Aversa, et al., U.S. Pat. No. 5,051,448 to Shashoua, and U.S. Pat.
No. 5,559,103 to Gaeta, et al., all hereby incorporated by
reference, describe multiple methods for creating such compounds.
Synthesis of nonpeptide compounds that mimic peptide sequences is
also known in the art. Eldred et al., J. Med. Chem. 37:3882 (1994),
hereby incorporated by reference herein in its entirety) describe
non-peptide antagonists that mimic the peptide sequence. Likewise,
Ku et al., J. Med. Chem 38:9 (1995) (hereby incorporated by
reference herein in its entirety) further elucidates the synthesis
of a series of such compounds.
[0114] Further modifications following synthesis may be
implemented. For example, the tissue protective peptides may be
further chemically modified, i.e. carbamylated, acetylated,
succinylated, etc., in accordance with U.S. patent application Ser.
No. 10/188,905, which published as 20030072737-A1 on Apr. 17, 2003
and discloses chemically modified EPO, and in accordance with U.S.
patent application Ser. No. 10/612,665, filed Jul. 1, 2003, and
U.S. patent application Ser. No. 09/753,132, filed Dec. 29, 2000,
which are incorporated by reference herein in their entirety.
[0115] Additionally, the tissue protective peptides may consist of
recombinant tissue protective peptides--muteins. The disclosed
mutations may include substitutions, deletions, including internal
deletions, additions, including additions yielding fusion proteins,
or conservative substitutions of amino acid residues within and/or
adjacent to the amino acid sequence, but that result in a "silent"
change, and non-conservative amino acid changes and larger
insertions and deletions, as previously disclosed in PCT/US03/20964
entitled Recombinant Tissue Protective Cytokines and Encoding
Nucleic Acids Thereof for Protection, Restoration, and Enhancement
of Responsive Cells, Tissues, and Organs (which is incorporated by
reference herein in its entirety)
[0116] Either conservative or non-conservative amino acid
substitutions can be made at one or more amino acid residues. Both
conservative and non-conservative substitutions can be made.
Conservative replacements are those that take place within a family
of amino acids that are related in their side chains. Genetically
encoded amino acids can be divided into four families: (1)
acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine;
(3) nonpolar (hydrophobic)=cysteine, alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan,
glycine, tyrosine; and (4) uncharged polar=asparagine, glutamine,
serine, threonine. Non-polar may be subdivided into: strongly
hydrophobic=alanine, valine, leucine, isoleucine, methionine,
phenylalanine and moderately hydrophobic=glycine, proline,
cysteine, tyrosine, tryptophan. In alternative fashion, the amino
acid repertoire can be grouped as (1) acidic=aspartate, glutamate;
(2) basic=lysine, arginine, histidine, (3) aliphatic=glycine,
alanine, valine, leucine, isoleucine, serine, threonine, with
serine and threonine optionally be grouped separately as
aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine,
tryptophan; (5) amide=asparagine, glutamine; and (6)
sulfur-containing=cysteine and methionine. (See, for example,
Biochemistry, 4th ed., Ed. by L. Stryer, WH Freeman and Co., 1995,
which is incorporated by reference herein in its entirety).
[0117] Alternatively, mutations can be introduced randomly along
all or part of the coding sequence of a tissue protective peptide,
such as by saturation mutagenesis, and the resultant mutants can be
screened for biological activity to identify mutants that retain
activity. Following mutagenesis, the encoded peptide can be
expressed recombinantly and the activity of the recombinant tissue
protective peptide can be determined.
[0118] In another embodiment, the tissue protective peptide may be
further modified through the additions of polymers (such as
polyethylene glycol), sugars, or additional proteins (such as a
fusion construct) in an effort to extend the half-life of the
tissue protective peptide or enhance the peptide's tissue
protective effects. Examples of such modifications are disclosed
within WO/04022577 A3 and WO/05025606 A1, which are incorporated
herein by reference.
5.2 Assays for Testing Tissue Protective Peptides
5.2.1 Biological Screens or Assays
[0119] Tissue protective peptides in accordance with the present
invention may be tested for tissue protective activity, e.g.,
protecting cells, tissues or organs. Protective activities may be
further tested using in vitro and in vivo assays. In vitro tests
that are indicative of tissue protective activity include, for
example, cell proliferation assays, cell differentiation assays, or
detecting the presence of proteins or nucleic acids upregulated by
tissue protective receptor complex, e.g. tissue protective cytokine
receptor complex, activity, e.g., nucleolin, neuroglobin,
cytoglobin, or frataxin. Neuroglobin, for example, may be involved
in facilitating the transport or the short-term storage of oxygen.
Therefore, oxygen transport or storage assays may be used as an
assay to identify or screen for compounds which modulate tissue
protective activity.
[0120] Neuroglobin is expressed in cells and tissues of the central
nervous system in response to hypoxia or ischemia and may provide
protection from injury (Sun et al. 2001, PNAS 98:15306-15311;
Schmid et al., 2003, J. Biol. Chem. 276:1932-1935, each of which is
incorporated by reference herein in its entirety). Cytoglobin may
play a similar role in protection, but is expressed in a variety of
tissues at varying levels (Pesce et al., 2002, EMBO 3:1146-1151,
which is incorporated by reference herein in its entirety). In one
embodiment of the invention, the levels of an upregulated protein
in a cell may be measured before and after contacting the tissue
protective peptide to a cell. In certain embodiments, the presence
of an upregulated protein associated with tissue protective
activity in a cell, may be used to confirm the tissue protective
activities of a peptide.
[0121] Nucleolin may protect cells from damage. It plays numerous
roles in cells including modulation of transcription processes,
sequence specific RNA-binding protein, cytokinesis, nucleogensis,
signal transduction, apoptosis induced by T-cells, chromatin
remodelling, or replication. It can also function as a cell surface
receptor DNA/RNA helicase, DNA-dependent ATPase, protein shuttle,
transcription factor component, or transcriptional repressor
(Srivastava and Pollard, 1999, FASEB J., 13:1911-1922; and Ginisty
et al., 1999, J. Cell Sci., 112:761-772, each of which is
incorporated by reference herein in its entirety).
[0122] Frataxin is a protein involved with mitochondrial iron
metabolism and has previously been shown to be strongly
up-regulated by EPO both in vivo and in vitro (Sturm et al. (2005)
Eur J Clin Invest 35: 711, which is incorporated by reference
herein in its entirety)
[0123] Expression of an upregulated protein may be detected by
detecting mRNA levels corresponding to the protein in a cell. The
mRNA can be hybridized to a probe that specifically binds a nucleic
acid encoding the upregulated protein. Hybridization may consist
of, for example, Northern blot, Southern blot, array hybridization,
affinity chromatography, or in situ hybridization.
[0124] Tissue protective activity of the polypeptide of the
invention can also be detected using in vitro neuroprotection
assays. For example, primary neuronal cultures may be prepared from
new born rat hippocampi by trypsinization, and cultured as by any
method known in the art and/or described herein e.g. in MEM-II
growth medium (Invitrogen), 20 mM D-glucose, 2 mM L-glutamine, 10%
Nu-serum (bovine; Becton Dickinson, Franklin Lakes, N.J.), 2% B27
supplement (Invitrogen), 26.2 mM NaHCO.sub.3, 100 U/ml penicillin,
and 1 mg/ml streptavidin (see, e.g., Leist et al., 2004, Science
305:239-242, hereby incorporated by reference in its entirety). One
day after seeding, 1 .mu.M cytosinearabino-furanoside is added.
Thirteen day old cultures are then preincubated with increasing
doses of EPO or CEPO (3-3000 pM) for 24 h. On day 14, the medium is
removed and the cultures challenged with 300 .mu.M NMDA in PBS at
RT. After 5 min, pre-conditioned medium is returned to the cultures
which are then returned to the incubator for 24 h. The cells are
fixed in paraformaldehyde, stained by Hoechst 33342 (Molecular
Probes, Eugene, Oreg.) and condensed apoptotic nuclei may be
counted. NGF (50 ng/ml) and MK801 (1 .mu.M) are included as
positive controls.
[0125] Animal model systems can be used to demonstrate the tissue
protective activity of a compound or to demonstrate the safety and
efficacy of the compounds identified by the screening methods of
the invention described above. The compounds identified in the
assays can then be tested for biological activity using animal
models for a type of tissue damage, disease, condition, or syndrome
of interest. These include animals engineered to contain the tissue
protective receptor complex coupled to a functional readout system,
such as a transgenic mouse.
[0126] Animal models that can be used to test the efficacy of the
cell or tissue protective activity of an identified compound
include, for example, protection against the onset of acute
experimental allergic encephalomyelitis (EAE; see, Example 12) in
Lewis rats, restoration or protection from diminished cognitive
function in mice after receiving brain trauma, cerebral ischemia
("stroke"; Example 5) or seizures stimulated by excitotoxins
(Brines et al., 2000, PNAS, 97:10295-10672, which is incorporated
by reference herein in its entirety), protection from induced
retinal ischemia (Rosenbaum et al., 1997, Vis. Res. 37:3443-51
which is incorporated by reference herein in its entirety),
protection from injury to the sciatic nerve (see, Example 2), and
protection from ischemia-reperfusion injury to the heart (in vitro
cardiomyocyte studies and in vivo ischemia-reperfusion injury, see,
e.g., Calvillo et al., 2003, PNAS 100:4802-4806 and Fiordaliso et
al., 2005, PNAS 102:2046-2051, each of which is hereby incorporated
by reference in its entirety). Such assays are described in further
detail in Grasso et al. (2004) Med Sci Monit 10: BR1-3 or PCT
publication no. WO02/053580, each of which is incorporated by
reference herein in its entirety. The in vivo methods described
therein are directed towards administration of EPO, however, tissue
protective proteins administered in place of EPO have been
identified to also exhibit similar biologic activity, e.g., Leist
et al. (2004) Science 305: 239-242, which is incorporated by
reference herein in its entirety. Peptides may be substituted for
testing as well. Other assays for determining tissue protective
activity of a peptide are well known to those of skill in the
art.
5.2.2 Cell Binding Assays
[0127] Alternatively, cell binding assays can be for evaluation of
the polypeptides of the invention. For example, the tissue
protective peptide of interest can be bound to a biological marker
such as a fluorescent or radiolabled marker for ease of detection
and tested for binding to transfected BaF3 cells expressing EPOR
and/or .beta..sub.c receptor. In a 96 well plate, eight 1:2 serial
dilutions of the tissue protective peptide of interest in growth
medium (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 2
mM L-glutamine) are plated, such that the final volume in each well
is about 100 .mu.l. The BaF3 parental line and BaF3 cells
transfected with EPOR and/or .beta..sub.c receptor can be washed
three times in growth media (see above), pellets resuspended in
growth medium, and cells counted and diluted in growth media to
5,000 cells/100 .mu.l. 100 .mu.l of diluted cells are then added to
each peptide dilution. The assay plate is then incubated in a
37.degree. C. incubator for three to four days. The plate/cells are
then washed and the plate is read on a fluorescent plate reader or
by other suitable method to detect the level of biomarker
associated with the biological activity of the tissue protective
peptide of interest.
[0128] Similarly, a competitive assay can be utilized to determine
if a tissue protective peptide is tissue protective. In the
competitive assay, a compound known to be tissue protective
including, but not limited to, tissue protective cytokines such as
those disclosed in U.S. patent application Ser. Nos. 10/188,905 and
10/185,841 (each of which is incorporated by reference herein in
its entirety), can be attached to a suitable bio marker.
[0129] In a 96 well plate eight 1:2 serial dilutions of a known
tissue protective compound/biomarker in suitable growth medium, and
the same dilution series of the known tissue protective
compound/biomarker and an excess of the tissue protective peptide
of interest are plated. The final volume of each dilution should be
about 100 .mu.l. Once again, the BaF3 cells are seeded into the
plates as disclosed supra and allowed to incubate. After an
appropriate amount of time, the cells are washed and the plate is
read on a fluorescent plate reader or by any other suitable method
known in the art to detect the biomarker. If the readout of the
plates and/or wells containing the known tissue protective
compound/biomarker and tissue protective peptide of interest is
less than the readout of the plates containing only the known
tissue protective compound/biomarker then the tissue protective
peptide of interest is tissue protective.
5.2.3 Cytokine and Cell Proliferation/Differentiation Activity
[0130] Many protein factors discovered to date, including all known
cytokines, have exhibited activity in one or more factor-dependent
cell proliferation assays, and hence these assays serve as a
convenient confirmation of cytokine activity. The activity of a
tissue protective peptide can be evidenced by any one of a number
of routine factor dependent cell proliferation assays for cell
lines including, without limitation, 32D, DA2, DA1G, T10, B9,
B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2,
CTLL2, TF-1, Mole and CMK. These cells are cultured in the presence
or absence of a tissue protective peptide, and cell proliferation
is detected by, for example, measuring incorporation of tritiated
thymidine or by colorimetric assay based on the metabolic breakdown
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) (Mosman, 1983, J. Immunol. Meth. 65:55-63, which is
incorporated by reference herein in its entirety).
5.2.4 Other Assays
[0131] If a tissue protective peptide exhibits a tissue protective
activity, one of ordinary skill in the art would recognize that it
would be beneficial to verify the result using one of the
neuroprotective and tissue protective assays known to those skilled
in the art, such as, but not limited to, P-19 and PC-12 cell
assays. Additionally, various in vivo models such as animal models
related to spinal cord injury, ischemic stroke, peripheral nerve
damage, heart, eyes, kidneys, etc. would be helpful in further
characterizing the tissue protective peptide. Suitable in vitro and
in vivo assays are disclosed in U.S. patent application Ser. Nos.
10/188,905 and 10/185,841, each of which is incorporated by
reference herein in its entirety.
5.3 Therapeutic Use
[0132] One of ordinary skill in the art would recognize that the
tissue protective peptides of the current invention are useful as
therapeutics for treatment or prevention of various diseases,
disorders, and conditions. One skilled in the art would also
recognize that such peptides can be used to achieve modulation of a
tissue protective receptor complex, e.g., tissue protective
cytokine complex. Both in vitro and in vivo techniques that can be
used for assessing the therapeutic indications of, for example, the
compounds identified by the inventive assays disclosed above are
disclosed in PCT Application No. PCT/US01/49479, U.S. patent
application Ser. Nos. 10/188,905 and 10/185,841, incorporated
herein by reference.
[0133] The aforementioned tissue protective peptides of the
invention may be useful generally for the prevention, therapeutic
treatment, or prophylactic treatment of human diseases or disorders
of the central nervous system or peripheral nervous system which
have primarily neurological or psychiatric symptoms, ophthalmic
diseases, cardiovascular diseases, cardiopulmonary diseases,
respiratory diseases, kidney, urinary and reproductive diseases,
bone diseases, skin diseases, connective tissue diseases,
gastrointestinal diseases and endocrine and metabolic
abnormalities. Examples of use include, but are not limited to,
protection against and repair of injury resulting from trauma and
resulting inflammation to the brain (ischemic stroke, blunt trauma,
subarachnoid hemorrhage), spinal cord (ischemia, blunt force
trauma), peripheral nerves (sciatic nerve injury, diabetic
neuropathy, carpal tunnel syndrome), retinal (macular edema,
diabetic retinopathy, glaucoma), and heart (myocardial infarct,
chronic heart failure). In particular, such diseases, disorders,
and conditions include hypoxic conditions, which adversely affect
responsive tissues, such as excitable tissues in the central
nervous system tissue, peripheral nervous system tissue, or cardiac
tissue or retinal tissue such as, for example, brain, heart, or
retina/eye. Therefore, the tissue protective peptides of the
invention can be used to treat or prevent damage to responsive
tissue resulting from hypoxic conditions in a variety of conditions
and circumstances. Non-limiting examples of such conditions and
circumstances are provided in the table herein below.
[0134] The tissue protective polypeptides are also of interest in
the modulation of stem cell activity. It has been established that
cytokines exhibiting tissue protective activity, e.g. EPO, are able
to mobilize stem cells, stimulating the migration to regions of
injury and aiding the repair process, e.g. in a regenerative role.
For example, in experimental stroke, EPO mediates the migration of
neuroblasts into a region of ischemic injury to regenerate neurons
during the period of recovery (Tsai et al, J. Neurosci (2006)
26:1269-74, incorporated herein by reference in its entirety). As
another example, EPO and CEPO mobilize endothelial progenitor cells
from the bone marrow into the circulation. These cells then home to
distance regions and are involved in the formation of new blood
vessels (for effect of EPO, see, Bahlmann et al, 2003, Kidney Int.
64:1648-1652, incorporated by reference herein in its entirety).
While not wishing to be bound to any particular theory, the
isolated polypeptides disclosed herein are believed to have a
similar effect on the migration of stem cells.
[0135] In the example of the protection of neuronal tissue
pathologies treatable and preventable using tissue protective
peptides of the invention, such pathologies include those which
result from reduced oxygenation of neuronal tissues. Any condition
which reduces the availability of oxygen to neuronal tissue,
resulting in stress, damage, and finally, neuronal cell death, can
be treated using tissue protective peptides of the present
invention. Generally referred to as hypoxia and/or ischemia, these
conditions arise from or include, but are not limited to, stroke,
vascular occlusion, prenatal or postnatal oxygen deprivation,
suffocation, choking, near drowning, carbon monoxide poisoning,
smoke inhalation, trauma, including surgery and radiotherapy,
asphyxia, epilepsy, hypoglycemia, chronic obstructive pulmonary
disease, emphysema, adult respiratory distress syndrome,
hypotensive shock, septic shock, anaphylactic shock, insulin shock,
sickle cell crisis, cardiac arrest, dysrhythmia, nitrogen narcosis,
and neurological deficits caused by heart-lung bypass
procedures.
[0136] In one embodiment, for example, the tissue protective
peptides of the present invention identified using the inventive
assay could be administered alone or as part of a composition to
prevent injury or tissue damage resulting from risk of injury or
tissue damage prior to, during, or subsequent to a surgical
procedure or a medical procedure. For example, surgical procedures
may include tumor resection or aneurysm repair and medical
procedures may include labor or delivery. Other pathologies caused
by or resulting from hypoglycemia which are treatable using tissue
protective peptides of the present invention include insulin
overdose, also referred to as iatrogenic hyperinsulinemia,
insulinoma, growth hormone deficiency, hypocortisolism, drug
overdose, and certain tumors.
[0137] Other pathologies resulting from excitable neuronal tissue
damage include seizure disorders, such as epilepsy, convulsions, or
chronic seizure disorders. Other treatable conditions and diseases
include, but are not limited to, diseases such as stroke, multiple
sclerosis, hypotension, cardiac arrest, Alzheimer's disease,
Parkinson's disease, cerebral palsy, brain or spinal cord trauma,
AIDS dementia, age-related loss of cognitive function, memory loss,
amyotrophic lateral sclerosis, seizure disorders, alcoholism,
retinal ischemia, optic nerve damage resulting from glaucoma, and
neuronal loss. The specific tissue protective peptides of the
present invention may be used to treat or prevent inflammation
resulting from disease conditions or various traumas, such as
physically or chemically induced inflammation. The tissue
protective peptides are also contemplated for the treatment and
prevention of inflammatory conditions in one or more organs or
tissues including, but not limited to, the brain, spinal cord,
connective tissue, heart, lung, kidney and urinary tract, pancreas,
eyes and prostate. Non-limiting examples of such trauma include
tendonitis, angitis, chronic bronchitis, pancreatitis,
osteomyelitis, rheumatoid arthritis, glomerulonephritis, optic
neuritis, temporal arteritis, encephalitis, meningitis, transverse
myelitis, dermatomyositis, polymyositis, necrotizing fascilitis,
hepatitis, and necrotizing enterocolitis. Further the tissue
protective cytokines may used to treat or prevent inflammation
resulting from ischemic and non-ischemic conditions including, but
not limited to, allergies, rheumatic diseases, sports related
injuries, infections including viral, fungal, and bacterial. The
inflammation may be acute or chronic. Further applications in the
field of inflammation are noted within PCT/US2004/031789 filed Sep.
29, 2004 and published as WO 2005/032467, hereby incorporated by
reference in its entirety.
[0138] The specific tissue protective peptides of the present
invention may be used to treat central nervous and peripheral
nervous system diseases resulting from demyelination or impairment
of the mylin sheath. These diseases are defined as mainly involving
inflammatory myelin sheath lesions of unknown origin, with the
exception of myelination deficiency diseases, such as
leukodystrophy, and diseases due to obvious causes. Multiple
sclerosis (MS) is a typical disease among demyelinating diseases,
and pathologically, it is characterized by changes, mainly,
inflammatory demyelination, and gliosis. Since its etiology is
unknown, its diagnosis is made based on its clinical features,
i.e., spatial multiplicity and multiplicity over time of central
nervous system lesions. Furthermore, acute disseminated
encephalomyelitis (ADEM), inflammatory diffuse sclerosis, acute and
subacute necrotizing hemorrhagic encephalomyelitis, and transverse
myelitis are included in demyelinating diseases. Also, peripheral
nervous tissues rely upon Schwann's cells to maintain the myelin
sheath, if these cells are impaired, peripheral demyelinating
disease is caused.
[0139] The tissue protective peptides of the present invention may
be used to treat or prevent conditions of, and damage to the heart
including any chronic or acute pathological event involving the
heart and/or associated tissue (e.g., the pericardium, aorta and
other associated blood vessels), including ischemia-reperfusion
injury; congestive heart failure; cardiac arrest; myocardial
infarction; atherosclerosis, mitral valve leakage, atrial flutter,
cardiotoxicity caused by compounds such as drugs (e.g.,
doxorubicin, herceptin, thioridazine and cisapride); cardiac damage
due to parasitic infection (bacteria, fungi, rickettsiae, and
viruses, e.g., syphilis, chronic Trypanosoma cruzi infection);
fulminant cardiac amyloidosis; heart surgery; heart
transplantation; angioplasty, laparoscopic surgery, traumatic
cardiac injury (e.g., penetrating or blunt cardiac injury, and
aortic valve rupture), surgical repair of a thoracic aortic
aneurysm; a suprarenal aortic aneurysm; cardiogenic shock due to
myocardial infarction or cardiac failure; neurogenic shock and
anaphylaxis. The tissue protective peptides of the current
invention may also be used to treat those individuals at risk for
heart disease such as cardiac failure (i.e., where the heart is not
able to pump blood at a rate required by the metabolizing tissues,
or when the heart can do so only with an elevated filling
pressure). Such at risk patients would include patients having or
being at risk of having cardiac infarction, coronary artery
disease, myocarditis, chemotherapy, cardiomyopathy, hypertension,
valvular heart diseases (most often mitral insufficiency and aortic
stenosis) and toxin-induced cardiomyopathy (e.g. ethanol, cocaine,
etc.) and the like.
[0140] The tissue protective peptides of the present invention may
be used to treat or prevent conditions of, and damage to, the eyes,
e.g., retinal tissue. Such disorders include, but are not limited
to retinal ischemia, macular degeneration, retinal detachment,
retinitis pigmentosa, arteriosclerotic retinopathy, hypertensive
retinopathy, retinal artery blockage, retinal vein blockage,
hypotension, and diabetic retinopathy.
[0141] In another embodiment, the tissue protective peptides of the
present invention and principles of the invention may be used to
prevent or treat injury resulting from radiation damage to
responsive tissue. A further utility of the tissue protective
peptides of the present invention is in the treatment of poisoning,
such as neurotoxin poisoning (e.g., domoic acid shellfish
poisoning), toxins (ethanol, cocaine, etc.), as the result of
chemotherapeutic agents of radiation exposure; neurolathyrism; Guam
disease; amyotrophic lateral sclerosis; and Parkinson's
disease.
[0142] As mentioned above, the present invention is also directed
to tissue protective peptides of the present invention for use in
enhancing tissue function in responsive cells, tissues and organs
in a mammal by peripheral administration of a tissue protective
cytokine as described above. Various diseases and conditions are
amenable to treatment using this method. For example this method is
useful for enhancing function in excitable tissues resulting in an
increase in cognitive function even in the absence of any condition
or disease. Further, the tissue protective cytokines are useful for
improving the quality of wound healing, reducing the time required
to heal, improving the quality of the healed tissues and reducing
the incidence of adhesions resulting from the wound. See
PCT/US2004/031789 filed Sep. 29, 2004 and published as WO
2005/032467, hereby incorporated by reference in its entirety.
These uses of the present invention are describe in further detail
below and include enhancement of learning and training in both
human and non-human mammals.
[0143] Conditions and diseases treatable or preventable using
tissue protective peptides of the present invention directed to the
central nervous system include but are not limited to mood
disorders, anxiety disorders, depression, autism, attention deficit
hyperactivity disorder, and cognitive dysfunction. These conditions
benefit from enhancement of neuronal function. Other disorders
treatable in accordance with the teachings of the present invention
include sleep disruption, for example, sleep apnea and
travel-related disorders; subarachnoid and aneurismal bleeds,
hypotensive shock, concussive injury, septic shock, anaphylactic
shock, and sequelae of various encephalitides and meningitides, for
example, connective tissue disease-related cerebritides such as
lupus. Other uses include prevention of or protection from
poisoning by neurotoxins, such as domoic acid shellfish poisoning,
neurolathyrism, and Guam disease, amyotrophic lateral sclerosis,
Parkinson's disease; postoperative treatment for embolic or
ischemic injury; whole brain irradiation; sickle cell crisis; and
eclampsia.
[0144] A further group of conditions treatable or preventable using
tissue protective peptides of the present invention include
mitochondrial dysfunction, of either a hereditary or acquired
nature, which are the cause of a variety of neurological diseases
typified by neuronal injury and death. For example, Leigh disease
(subacute necrotizing encephalopathy) is characterized by
progressive visual loss and encephalopathy, due to neuronal drop
out, and myopathy. In these cases, defective mitochondrial
metabolism fails to supply enough high energy substrates to fuel
the metabolism of excitable cells. A tissue protective peptide
optimizes failing function in a variety of mitochondrial diseases.
As mentioned above, hypoxic conditions adversely affect excitable
tissues. The excitable tissues include, but are not limited to,
central nervous system tissue, peripheral nervous system tissue,
and heart tissue. In addition to the conditions described above,
the tissue protective peptides of the present invention are useful
in the treatment of inhalation poisoning such as carbon monoxide
and smoke inhalation, severe asthma, adult respiratory distress
syndrome, and choking and near drowning. Further conditions which
create hypoxic conditions or by other means induce responsive
tissue, such as excitable tissue damage include hypoglycemia that
may occur in inappropriate dosing of insulin, or with
insulin-producing neoplasms (insulinoma).
[0145] Various neuropsychologic disorders which are described to
originate from excitable tissue damage are treatable using tissue
protective peptides of the present invention. Chronic disorders in
which neuronal damage is involved and for which treatment or
preventable by the present invention is provided include disorders
relating to the central nervous system and/or peripheral nervous
system including age-related loss of cognitive function and senile
dementia, chronic seizure disorders, Alzheimer's disease,
Parkinson's disease, dementia, memory loss, amyotrophic lateral
sclerosis, multiple sclerosis, tuberous sclerosis, Wilson's
Disease, cerebral and progressive supranuclear palsy, Guam disease,
Lewy body dementia, prion diseases, such as spongiform
encephalopathies, e.g., Creutzfeldt-Jakob disease, Huntington's
disease, myotonic dystrophy, Freidrich's ataxia and other ataxias,
as well as Gilles de la Tourette's syndrome, seizure disorders such
as epilepsy and chronic seizure disorder, stroke, brain or spinal
cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia,
glaucoma, autonomic function disorders such as hypertension and
sleep disorders, and neuropsychiatric disorders that include, but
are not limited to schizophrenia, schizoaffective disorder,
attention deficit disorder, dysthymic disorder, major depressive
disorder, mania, obsessive-compulsive disorder, psychoactive
substance use disorders, anxiety, panic disorder, as well as
unipolar and bipolar affective disorders. Additional
neuropsychiatric and neurodegenerative disorders include, for
example, those listed in the American Psychiatric Association's
Diagnostic and Statistical Manual of Mental Disorders (DSM), the
most current version of which in incorporated herein by reference
in its entirety.
[0146] A further group of conditions treatable or preventable using
tissue protective peptides of the present invention include kidney
diseases such as renal failure, acute and chronic. Blood supply to
the kidneys can be cut off due to several causes including shock
from infections invading the bloodstream (septicemia), internal or
external hemorrhaging, loss of fluid from the body as a result of
severe diarrhea or burns, reactions to transfusions, cardiac arrest
or arythmias, surgical trauma and kidney transplantations. The
reduced flow of blood to the kidneys resulting from the above
conditions may reduced blood flow to dangerously low levels for a
time period great enough to cause the development of acute renal
failure. The depressed blood flow also results in necrosis, or
tissue death, in the kidney, damaging the renal tubular cells.
Renal failure may also result from diseases (interstitial and
diabetic) nephrotic syndromes, infections, injury (CPB-induced),
toxins (contrast-induced, chemotherapy-induced, cyclosporine),
autoimmune inflammation (e.g. Lupus, erythrotosis, etc.) The tissue
protective peptides of the current invention assist in the repair
or prevention of this damage helping to ameliorate acute renal
failure.
[0147] The following table lists additional exemplary, non-limiting
indications as to the various conditions and diseases amenable to
treatment by the aforementioned tissue protective peptides.
TABLE-US-00001 Cell, tissue or Dysfunction or organ pathology
Condition or disease Type Heart Ischemia Coronary artery disease
Acute, chronic Stable, unstable Myocardial infarction Dressler's
syndrome Angina Congenital heart disease Valvular Cardiomyopathy
Prinzmetal angina Cardiac rupture Aneurysmatic Septal perforation
Angiitis Arrhythmia Tachy-, bradyarrhythmia Stable, unstable
Supraventricular, Hypersensitive carotid sinus ventricular node
Conduction abnormalities Congestive heart failure Left, right,
bi-ventricular, Cardiomyopathies, such as systolic, diastolic
idiopathic familial, infective, metabolic, storage disease,
deficiencies, connective tissue disorder, infiltration and
granulomas, neurovascular Myocarditis Autoimmune, infective,
idiopathic Cor pulmonale Radiation injury Blunt and penetrating
trauma Toxins Cocaine toxicity, adriamycin Vascular Hypertension
Primary, secondary Decompression sickness Fibromuscular hyperplasia
Aneurysm Dissecting, ruptured, enlarging Lungs Obstructive Asthma
Chronic bronchitis, Emphysema and airway obstruction Ischemic lung
disease Pulmonary embolism, Pulmonary thrombosis, Fat embolism
Environmental lung diseases Ischemic lung disease Pulmonary
embolism Pulmonary thrombosis Interstitial lung disease Idiopathic
pulmonary fibrosis Congenital Cystic fibrosis Cor pulmonale Trauma
Pneumonia and Infectious, parasitic, pneumonitides toxic,
traumatic, burn, aspiration Sarcoidosis Pancreas Endocrine Diabetes
mellitus, type I Beta cell failure, dysfunction and II Diabetic
neuropathy Other endocrine cell failure of the pancreas Exocrine
Exocrine pancreas failure pancreatitis Bone Osteopenia Primary
Hypogonadism Secondary immobilisation Postmenopausal Age-related
Hyperparathyroidism Hyperthyroidism Calcium, magnesium, phosphorus
and/or vitamin D deficiency Osteomyelitis Avascular necrosis Trauma
Paget's disease Skin Alopecia Areata Primary Totalis Secondary Male
pattern baldness Vitiligo Localized Primary Generalized secondary
Ulceration Diabetic Pressure sores, pressure ulcers, Decubitis bed
sores Peripheral vascular disease Surgical wounds, lacerations Burn
injuries Autoimmune Lupus erythematosus, disorders Sjogren's
syndrome, Rheumatoid arthritis, Glomerulonephritis, Angiitis
Langerhan's histiocytosis Eye Optic neuritis Blunt and penetrating
injuries, Infections, Sarcoid, Sickle C disease, Retinal
detachment, Temporal arteritis Retinal ischemia, Macular
degeneration, Retinitis pigmentosa, Arteriosclerotic retinopathy,
Hypertensive retinopathy, Retinal artery blockage, Retinal vein
blockage, Hypotension, Diabetic retinopathy, glaucoma and Macular
edema Embryonic and Asphyxia fetal disorders Ischemia CNS Chronic
fatigue syndrome, acute and chronic hypoosmolar and hyperosmolar
syndromes, AIDS Dementia, Electrocution Cerebral malaria
Encephalitis Rabies, Herpes, Meningitis Subdural hematoma Nicotine
addiction Drug abuse and Cocaine, heroin, crack, withdrawal
marijuana, LSD, PCP, poly-drug abuse, ecstasy, opioids, sedative
hypnotics, amphetamines, caffeine Obsessive-compulsive disorders
Spinal stenosis, Transverse myelitis, Guillian Barre, Trauma, Nerve
root compression, Tumoral compression, Heat stroke ENT Tinnitus
Meuniere's syndrome Hearing loss Traumatic injury, barotraumas
Kidney Renal failure Acute, chronic Vascular/ischemic, interstitial
disease, diabetic kidney disease, nephrotic syndromes, infections,
injury, contrast-induced, chemotherapy-induced, cyclosporine,
CPB-induced, or preventive Radiation injury Henoch Schonlein
purpura Striated muscle Autoimmune disorders Myasthenia gravis
Dermatomyositis Polymyositis Myopathies Inherited metabolic,
endocrine and toxic Heat stroke Crush injury Rhabdomyolysis
Mitochondrial disease Infection Necrotizing fasciitis Sexual
Central and peripheral Impotence secondary to dysfunction (e.g.
erectile dysfunction) medication, (diabetes) Liver Hepatitis Viral,
bacterial, parasitic Ischemic disease Cirrhosis, fatty liver
Infiltrative/metabolic diseases Gastrointestinal Ischemic bowel
disease Inflammatory bowel disease Necrotizing enterocolitis Organ
Treatment of donor and transplantation recipient Reproductive
Infertility Vascular tract Autoimmune Uterine abnormalities
Implantation disorders Endocrine Glandular hyper- and hypofunction
General Shock Septic, hemodynamic Parasitemia Malaria,
trypanosomiasis, Leshmaniasis
[0148] As mentioned above, these diseases, disorders or conditions
are merely illustrative of the range of benefits provided by the
tissue protective peptides of the present invention. Accordingly,
this invention generally provides preventative, therapeutic, or
prophylactic treatment of the consequences of mechanical trauma or
of human diseases. Prevention or therapeutic or prophylactic
treatment for diseases, disorders or conditions of the CNS and/or
peripheral nervous system are contemplated. Prevention or
therapeutic or prophylactic treatment for diseases, disorders or
conditions which have a psychiatric component is provided.
Prevention or therapeutic or prophylactic treatment for diseases,
disorders or conditions including but not limited to those having
an ophthalmic, cardiovascular, cardiopulmonary, respiratory,
kidney, urinary, reproductive, gastrointestinal, endocrine, or
metabolic component is provided.
[0149] In one embodiment, such a pharmaceutical composition
comprising a tissue protective peptide can be administered
systemically to protect or enhance the target cells, tissue or
organ. Such administration may be parenterally, via inhalation, or
transmucosally, e.g., orally, nasally, rectally, intravaginally,
sublingually, ocularly, submucosally or transdermally. Preferably,
administration is parenteral, e.g., via intravenous or
intraperitoneal injection, and also including, but is not limited
to, intra-arterial, intramuscular, intradermal and subcutaneous
administration.
[0150] For other routes of administration, such as by use of a
perfusate, injection into an organ, or other local administration,
a pharmaceutical composition will be provided which results in
similar levels of a tissue protective peptide as described above. A
level of about 15 pM -30 nM is preferred.
[0151] The pharmaceutical compositions of the invention may
comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable carrier. In a specific embodiment, the
term "pharmaceutically acceptable" means approved by a regulatory
agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized foreign pharmacopeia for
use in animals, and more particularly in humans. The term "carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is administered. Such pharmaceutical carriers can be
sterile liquids, such as saline solutions in water and oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. A saline solution is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene glycol, water, ethanol and the
like. The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, capsules, powders, sustained-release formulations
and the like. The composition can be formulated as a suppository,
with traditional binders and carriers such as triglycerides. The
compounds of the invention can be formulated as neutral or salt
forms. Pharmaceutically acceptable salts include those formed with
free amino groups such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with free carboxyl groups such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin, hereby
incorporated by reference herein in its entirety. Such compositions
will contain a therapeutically effective amount of the compound,
preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
patient. The formulation should suit the mode of
administration.
[0152] Formulations for increasing transmucosal adsorption of
peptides such as long acting tissue protective peptides are also
contemplated by the current invention. Pharmaceutical compositions
adapted for oral administration may be provided as capsules or
tablets; as powders or granules; as solutions, syrups or
suspensions (in aqueous or non-aqueous liquids); as edible foams or
whips; or as emulsions. Tablets or hard gelatine capsules may
comprise lactose, starch or derivatives thereof, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate,
stearic acid or salts thereof. Soft gelatine capsules may comprise
vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
Solutions and syrups may comprise water, polyols and sugars.
[0153] An active agent intended for oral administration may be
coated with or admixed with a material that delays disintegration
and/or absorption of the active agent in the gastrointestinal tract
(e.g., glyceryl monostearate or glyceryl distearate may be used).
Thus, the sustained release of an active agent may be achieved over
many hours and, if necessary, the active agent can be protected
from being degraded within the stomach. Pharmaceutical compositions
for oral administration may be formulated to facilitate release of
an active agent at a particular gastrointestinal location due to
specific pH or enzymatic conditions.
[0154] Pharmaceutical compositions adapted for transdermal
administration may be provided as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. Pharmaceutical compositions adapted for
topical administration may be provided as ointments, creams,
suspensions, lotions, powders, solutions, pastes, gels, sprays,
aerosols or oils. For topical administration to the skin, mouth,
eye or other external tissues a topical ointment or cream is
preferably used. When formulated in an ointment, the active
ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water base or a
water-in-oil base. Pharmaceutical compositions adapted for topical
administration to the eye include eye drops. In these compositions,
the active ingredient can be dissolved or suspended in a suitable
carrier, e.g., in an aqueous solvent. Pharmaceutical compositions
adapted for topical administration in the mouth include lozenges,
pastilles and mouthwashes.
[0155] Pharmaceutical compositions adapted for nasal and pulmonary
administration may comprise solid carriers such as powders
(preferably having a particle size in the range of 20 to 500
microns). Powders can be administered in the manner in which snuff
is taken, i.e., by rapid inhalation through the nose from a
container of powder held close to the nose. Alternatively,
compositions adopted for nasal administration may comprise liquid
carriers, e.g., nasal sprays or nasal drops. Alternatively,
inhalation of compounds directly into the lungs may be accomplished
by inhalation deeply or installation through a mouthpiece into the
oropharynx. These compositions may comprise aqueous or oil
solutions of the active ingredient. Compositions for administration
by inhalation may be supplied in specially adapted devices
including, but not limited to, pressurized aerosols, nebulizers or
insufflators, which can be constructed so as to provide
predetermined dosages of the active ingredient. In a preferred
embodiment, pharmaceutical compositions of the invention are
administered into the nasal cavity directly or into the lungs via
the nasal cavity or oropharynx.
[0156] Pharmaceutical compositions adapted for rectal
administration may be provided as suppositories or enemas.
Pharmaceutical compositions adapted for vaginal administration may
be provided as pessaries, tampons, creams, gels, pastes, foams or
spray formulations.
[0157] Pharmaceutical compositions adapted for parenteral
administration include aqueous and non-aqueous sterile injectable
solutions or suspensions, which may contain antioxidants, buffers,
bacteriostats and solutes that render the compositions
substantially isotonic with the blood of an intended recipient.
Other components that may be present in such compositions include
water, alcohols, polyols, glycerine and vegetable oils, for
example. Compositions adapted for parenteral administration may be
presented in unit-dose or multi-dose containers, for example sealed
ampules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of a sterile
liquid carrier, e.g., sterile saline solution for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets. In one embodiment, an autoinjector comprising an
injectable solution of a tissue protective peptide may be provided
for emergency use by ambulances, emergency rooms, and battlefield
situations, and even for self-administration in a domestic setting,
particularly where the possibility of traumatic amputation may
occur, such as by imprudent use of a lawn mower. The likelihood
that cells and tissues in a severed foot or toe will survive after
reattachment may be increased by administering a tissue protective
peptide to multiple sites in the severed part as soon as
practicable, even before the arrival of medical personnel on site,
or arrival of the afflicted individual with severed toe in tow at
the emergency room.
[0158] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or
water-free concentrate in a hermetically-sealed container such as
an ampule or sachette indicating the quantity of active agent.
Where the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampule of sterile saline can be provided so that the
ingredients may be mixed prior to administration.
[0159] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0160] A perfusate composition may be provided for use in
transplanted organ baths, for in situ perfusion, or for
administration to the vasculature of an organ donor prior to organ
harvesting. Such pharmaceutical compositions may comprise levels of
tissue protective peptides, or a form of tissue protective peptides
not suitable for acute or chronic, local or systemic administration
to an individual, but will serve the functions intended herein in a
cadaver, organ bath, organ perfusate, or in situ perfusate prior to
removing or reducing the levels of the tissue protective peptide
contained therein before exposing or returning the treated organ or
tissue to regular circulation.
[0161] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0162] In another embodiment, for example, a tissue protective
peptide can be delivered in a controlled-release system. For
example, the peptide may be administered using intravenous
infusion, an implantable osmotic pump, a transdermal patch,
liposomes, or other modes of administration. In one embodiment, a
pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref.
Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek
et al., 1989, N. Engl. J. Med. 321:574, each of which is
incorporated by reference herein in its entirety). In another
embodiment, the compound can be delivered in a vesicle, in
particular a liposome (see Langer, Science 249:1527-1533 (1990);
Treat et al., in Liposomes in the Therapy of Infectious Disease and
Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.
353-365 (1989); WO 91/04014; U.S. Pat. No. 4,704,355;
Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Press: Boca Raton, Fla., 1974; Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley:
New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev.
Macromol. Chem. 23:61, 1953; see also Levy et al., 1985, Science
228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al.,
1989, J. Neurosurg. 71:105, (each of which is incorporated by
reference herein in its entirety).
[0163] In yet another embodiment, a controlled release system can
be placed in proximity of the therapeutic target, i.e., the target
cells, tissue or organ, thus requiring only a fraction of the
systemic dose (see, e.g., Goodson, pp. 115-138 in Medical
Applications of Controlled Release, vol. 2, supra, 1984, which is
incorporated by reference herein in its entirety). Other controlled
release systems are discussed in the review by Langer (1990,
Science 249:1527-1533, which is incorporated by reference herein in
its entirety).
[0164] In another embodiment, tissue protective peptide, as
properly formulated, can be administered by nasal, oral, rectal,
vaginal, ocular, transdermal, parenteral or sublingual
administration.
[0165] In a specific embodiment, it may be desirable to administer
a tissue protective peptide of the invention locally to the area in
need of treatment; this may be achieved by, for example, and not by
way of limitation, local infusion during surgery, topical
application, e.g., in conjunction with a wound dressing after
surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as silastic membranes, or fibers. A non-limiting example of
such an embodiment would be a coronary stent coated with a tissue
protective peptide of the present invention.
[0166] Selection of the preferred effective dose will be readily
determinable by a skilled artisan based upon considering several
factors, which will be known to one of ordinary skill in the art.
Such factors include the particular form of tissue protective
peptide, and its pharmacokinetic parameters such as
bioavailability, metabolism, half-life, etc., which will have been
established during the usual development procedures typically
employed in obtaining regulatory approval for a pharmaceutical
compound. Further factors in considering the dose include the
condition or disease to be treated or the benefit to be achieved in
a normal individual, the body mass of the patient, the route of
administration, whether administration is acute or chronic,
concomitant medications, and other factors well known to affect the
efficacy of administered pharmaceutical agents. Thus the precise
dosage should be decided according to the judgment of the
practitioner and each patient's circumstances, e.g., depending upon
the condition and the immune status of the individual patient, and
according to standard clinical techniques.
[0167] In another aspect of the present invention, a pharmaceutical
composition according to the present invention may include a tissue
protective peptide in a formulation with at least one small
molecule that exhibits tissue protective functionality. Suitable
small molecules include, but are not limited to, steroids (e.g.,
lazaroids and glucocorticoids), antioxidants (e.g., coenzyme
Q.sub.10, alpha lipoic acid, and NADH), anticatabolic enzymes
(e.g., glutathione peroxidase, superoxide dimutase, catalase,
synthetic catalytic scavengers, as well as mimetics), indole
derivatives (e.g., indoleamines, carbazoles, and carbolines),
nitric acid neutralizing agents, adenosine/adenosine agonists,
phytochemicals (flavanoids), herbal extracts (ginko biloba and
turmeric), vitamins (vitamins A, E, and C), oxidase electron
acceptor inhibitors (e.g., xanthine oxidase electron inhibitors),
minerals (e.g., copper, zinc, and magnesium), non-steriodal
anti-inflammatory drugs (e.g., aspirin, naproxen, and ibuprofen),
and combinations thereof. Additionally agents including, but not
limited to, anti-inflammatory agents (e.g., corticosteroids,
prednisone and hydrocortisone), glucocorticoids, steroids,
non-steriodal anti-inflammatory drugs (e.g., aspirin, ibuprofen,
diclofenac, and COX-2 inhibitors), beta-agonists, anticholinergic
agents and methyl xanthines), immunomodulatory agents (e.g., small
organic molecules, T cell receptor modulators, cytokine receptor
modulators, T-cell depleting agents, cytokine antagonists, monokine
antagonists, lymphocyte inhibitors, or anti-cancer agents), gold
injections, sulphasalazine, penicillamine, anti-angiogenic agents
(e.g., angiostatin), TNF-.alpha. antagonists (e.g., anti-TNF.alpha.
antibodies), and endostatin), dapsone, psoralens (e.g., methoxalen
and trioxsalen), anti-malarial agents (e.g., hydroxychloroquine),
anti-viral agents, and antibiotics (e.g., erythromycin and
penicillin) may be used in conjunction with the current
pharmaceutical compositions.
[0168] In another aspect of the invention, a perfusate or perfusion
solution is provided for perfusion and storage of organs for
transplant, the perfusion solution includes an amount of a tissue
protective peptide effective to protect responsive cells and
associated cells, tissues or organs. Transplant includes but is not
limited to allotransplantation, where an organ (including cells,
tissue or other bodily part) is harvested from one donor and
transplanted into a different recipient, both being of the same
species; autotransplantation, where the organ is taken from one
part of a body and replaced at another, including bench surgical
procedures, in which an organ may be removed, and while ex vivo,
resected, repaired, or otherwise manipulated, such as for tumor
removal, and then returned to the original location or
xenotransplantation, where tissues or organs or transplanted
between species. In one embodiment, the perfusion solution is the
University of Wisconsin (UW) solution (U.S. Pat. No. 4,798,824,
hereby incorporated by reference herein in its entirety) which
contains from about 1 to about 25 U/ml (10 ng=1 U) of tissue
protective peptide, 5% hydroxyethyl starch (having a molecular
weight of from about 200,000 to about 300,000 and substantially
free of ethylene glycol, ethylene chlorohydrin, sodium chloride and
acetone); 25 mM KH.sub.2PO.sub.4; 3 mM glutathione; 5 mM adenosine;
10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium gluconate; 1.5 mM
CaCl.sub.2; 105 mM sodium gluconate; 200,000 units penicillin; 40
units insulin; 16 mg dexamethasone; 12 mg Phenol Red; and has a pH
of 7.4-7.5 and an osmolality of about 320 mOsm/l. The solution is
used to maintain cadaveric kidneys and pancreases prior to
transplant. Using the solution, preservation can be extended beyond
the 30-hour limit recommended for cadaveric kidney preservation.
This particular perfusate is merely illustrative of a number of
such solutions that can be adapted for the present use by inclusion
of an effective amount of a tissue protective peptide. In a further
embodiment, the perfusate solution contains from about 1 to about
500 ng/ml of a tissue protective peptide, or from about 40 to about
320 ng/ml tissue protective peptide. As mentioned above, any form
of tissue protective peptide can be used in this aspect of the
invention.
[0169] While the preferred recipient of a tissue protective peptide
for the purposes herein throughout is a human, the methods herein
apply equally to other mammals, particularly domesticated animals,
livestock, companion, and zoo animals. However, the invention is
not so limiting and the benefits can be applied to any mammal.
[0170] In further aspects of the ex-vivo invention, any tissue
protective peptide such as but not limited to the ones described
above may be employed.
[0171] In another aspect of the invention, methods and compositions
for enhancing the viability of cells, tissues or organs which are
not isolated from the vasculature by an endothelial cell barrier
are provided by exposing the cells, tissue or organs directly to a
pharmaceutical composition comprising a tissue protective peptide,
or administering or contacting a pharmaceutical composition
containing a tissue protective peptide to the vasculature of the
tissue or organ. Enhanced activity of responsive cells in the
treated tissue or organ is responsible for the positive effects
exerted.
[0172] Similar to other tissue protective compounds based on
erythropoietin, it is possible that the tissue protective peptides
of the present invention may be transported from the luminal
surface to the basement membrane surface of endothelial cells of
the capillaries of organs with endothelial cell tight junctions,
including, for example, the brain, retina, and testis. Thus,
responsive cells across the barrier may be susceptible targets for
the beneficial effects of tissue protective peptides, and others
cell types or tissues or organs that contain and depend in whole or
in part on responsive cells therein may be targets for the methods
of the invention. While not wishing to be bound by any particular
theory, after transcytosis of the tissue protective peptide may
interact with an tissue-protective receptor on a responsive cell,
for example, neuronal, eye (e.g., retinal), adipose, connective,
hair, tooth, mucosal, pancreatic, endocrine, aural, epithelial,
skin, muscle, heart, lung, liver, kidney, small intestine, adrenal
(e.g. adrenal cortex, adrenal medulla), capillary, endothelial,
testes, ovary, or endometrial cell, and receptor binding can
initiate a signal transduction cascade resulting in the activation
of a gene expression program within the responsive cell or tissue,
resulting in the protection of the cell or tissue, or organ, from
damage, such as by toxins, chemotherapeutic agents, radiation
therapy, hypoxia, etc. In another embodiment, the tissue protective
peptide can be cross-linked to a compound that can cross the
barrier, such as carbamylated erythropoietin, to be transported
across the barrier in accordance with the teaching of PCT
Application No. PCT/US01/49479, U.S. patent application Ser. Nos.
10/188,905 and 10/185,841, incorporated herein by reference. Thus,
methods for protecting responsive cell-containing tissue from
injury or hypoxic stress, and enhancing the function of such tissue
are described in detail herein below.
[0173] In the practice of one embodiment of the invention, a
mammalian patient is undergoing systemic chemotherapy for cancer
treatment, including radiation therapy, which commonly has adverse
effects such as nerve, lung, heart, ovarian or testicular damage.
Administration of a pharmaceutical composition comprising a tissue
protective peptide as described above is performed prior to and
during chemotherapy and/or radiation therapy, to protect various
tissues and organs from damage by the chemotherapeutic agent, such
as to protect the testes. Treatment may be continued until
circulating levels of the chemotherapeutic agent have fallen below
a level of potential danger to the mammalian body.
[0174] In the practice of another embodiment of the invention,
various organs are planned to be harvested from a victim of an
automobile accident for transplant into a number of recipients,
some of which required transport for an extended distance and
period of time. Prior to organ harvesting, the donor is infused
with a pharmaceutical composition comprising tissue protective
peptides as described herein. Harvested organs for shipment are
perfused with a perfusate containing tissue protective peptides as
described herein, and stored in a bath comprising tissue protective
peptides. Certain organs are continuously perfused with a pulsatile
perfusion device, utilizing a perfusate containing tissue
protective peptides in accordance with the present invention.
Minimal deterioration of organ function occurs during the transport
and upon implant and reperfusion of the organs in situ.
[0175] In another embodiment of the present invention, a
participant in a hazardous activity, one could take a dose of a
pharmaceutical composition containing a tissue protective peptide
sufficient to either prevent (i.e. delaying the onset of,
inhibiting, or stopping), protect against, or mitigate the damage
resulting from an injury to a responsive cell, tissue, or organ. In
particular, this method of treatment may have application in
various professions susceptible to injury such as, but not limited
to, professional athletes (divers, race car drivers, football
players, etc.), military personnel (soldiers, paratroopers),
emergency personnel (police, fire, EMS, and disaster relief
personnel), stuntmen, and construction workers. Additionally, the
prophylactic use of tissue protective peptides is contemplated in
such recreational endeavors including, but not limited to, rock
climbing, rappelling, sky diving, racing, bicycling, football,
rugby, baseball, and diving that pose a risk of injury.
[0176] In another embodiment of the invention, a surgical procedure
to repair a heart valve requires temporary cardioplegia and
arterial occlusion. Prior to surgery, the patient is infused with a
tissue protective peptide. Such treatment prevents hypoxic ischemic
cellular damage, particularly after reperfusion. Additionally, the
pharmaceutical compositions of the present invention may be used
prophylactically to prepare an individual for surgery in an effort
to limit the trauma associated with the surgical procedure or aide
in the recovery of the individual from the surgical procedure.
Although the present method of treatment using pharmaceutical
compositions containing tissue protective peptides provides a
prophylactic use for surgical procedures, it may be particularly
useful in procedures that induce temporary ischemic events
including, but not limited to, bypass procedures (coronary bypass),
angioplasty procedures, amputations, and transplantations, as well
as, those performed directly upon responsive cells, tissues, or
organs such as brain and spinal cord surgery, and open heart
procedures. Such procedures may involve the use of cardiopulmonary
(heart lung) bypass.
[0177] In another embodiment of the invention, in any surgical
procedure, such as in cardiopulmonary bypass surgery, a tissue
protective peptide of the invention can be used. In one embodiment,
administration of a pharmaceutical composition comprising tissue
protective peptides as described above is performed prior to,
during, and/or following the bypass procedure, to protect the
function of brain, heart, and other organs.
[0178] In the foregoing examples in which a tissue protective
peptide of the invention is used for ex-vivo applications, or for
in vivo applications to treat responsive cells such as neuronal
tissue, retinal tissue, heart, lung, liver, kidney, small
intestine, adrenal cortex, adrenal medulla, capillary endothelial,
testes, ovary, or endometrial cells or tissue, the invention
provides a pharmaceutical composition in dosage unit form adapted
for protection or enhancement of responsive cells, tissues or
organs distal to the vasculature which comprises an amount within
the range from about 0.01 pg to 7.5 mg, 0.5 pg to 6.5 mg, 1 pg to 5
mg, 500 pg to 5 mg, 1 ng to 5 mg, 500 ng to 5 mg, 1 .mu.g to 5 mg,
500 .mu.g to 5 mg, or 1 mg to 5 mg of a tissue protective peptide,
and a pharmaceutically acceptable carrier. In a preferred
embodiment, the amount of tissue protective peptide is within the
range from about 0.5 pg to 1 mg. In a preferred embodiment, the
formulation contains tissue protective peptides that are
non-erythropoietic.
[0179] In a further aspect of the invention, administration of
tissue protective peptides may be used to restore cognitive
function in mammals having undergone brain trauma. After a delay of
either 5 days or 30 days, administration of tissue protective
peptides should be able to restore function as compared to
placebo-treated mammals, indicating the ability of the tissue
protective peptide to regenerate or restore brain activity. Thus,
the invention is also directed to the use of tissue protective
peptides for the preparation of a pharmaceutical composition for
the treatment of brain trauma and other cognitive dysfunctions,
including treatment well after the injury (e.g. three days, five
days, a week, a month, or longer). The invention is also directed
to a method for the treatment of cognitive dysfunction following
injury by administering an effective amount of tissue protective
peptides. Any tissue protective peptide as described herein may be
used for this aspect of the invention.
[0180] Furthermore, this restorative aspect of the invention is
directed to the use of any tissue protective peptides herein for
the preparation of a pharmaceutical composition for the restoration
of cellular, tissue or organ dysfunction, wherein treatment is
initiated after, and well after, the initial insult responsible for
the dysfunction. Moreover, treatment using tissue protective
peptides of the invention can span the course of the disease or
condition during the acute phase as well as a chronic phase.
[0181] A tissue protective peptide of the invention may be
administered systemically at a dosage between about 1 ng and about
100 .mu.g/kg body weight, preferably about 5-50 .mu.g/kg-body
weight, most preferably about 10-30 .mu.g/kg-body weight, per
administration. This effective dose should be sufficient to achieve
serum levels of tissue protective peptides greater than about 80,
120, or 160 ng/ml of serum after administration. Such serum levels
may be achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours
post-administration. Such dosages may be repeated as necessary. For
example, administration may be repeated daily, as long as
clinically necessary, or after an appropriate interval, e.g., every
1 to 12 weeks, preferably, every 1 to 3 weeks. In one embodiment,
the effective amount of tissue protective peptide and a
pharmaceutically acceptable carrier may be packaged in a single
dose vial or other container. In another embodiment, the tissue
protective peptides, which are capable of exerting the activities
described herein but not causing an increase in hemoglobin
concentration or hematocrit, are used. Such tissue protective
peptides are preferred in instances wherein the methods of the
present invention are intended to be provided chronically.
5.4 Transcytosis
[0182] Carrier Molecule and Tissue Protective Peptide.
[0183] The present invention is further directed to a method for
facilitating the transport of a Tissue Protective Peptide across an
endothelial cell barrier in a mammal by administering a composition
which comprises the tissue protective peptide in association with a
carrier peptide, a peptide capable of crossing an endothelial cell
barrier, such as erythropoietin, as described hereinabove. Tight
junctions between endothelial cells in certain organs in the body
create a barrier to the entry of certain molecules. For treatment
of various conditions within the barriered organ, means for
facilitating passage of the tissue protective peptide may be
desired.
[0184] Tissue Protective Peptide as Carrier Molecule.
[0185] Tissue protective peptides of the invention may be useful as
carriers for delivering other molecules across the blood-brain and
other similar barriers which they can travel across. A composition
comprising a molecule desirous of crossing the barrier with a
tissue protective peptide is prepared and peripheral administration
of the composition results in the transcytosis of the composition
across the barrier. The association between the molecule to be
transported across the barrier and the tissue protective peptide
may be a labile covalent bond, in which case the molecule is
released from association with the tissue protective peptide after
crossing the barrier. If the desired pharmacological activity of
the molecule is maintained or unaffected by association with tissue
protective peptides, such a complex can be administered.
[0186] The skilled artisan will be aware of various means for
associating molecules with tissue protective peptides of the
invention and the other agents described above, by covalent,
non-covalent, and other means. Furthermore, evaluation of the
efficacy of the composition can be readily determined in an
experimental system. Association of molecules with tissue
protective peptides may be achieved by any number of means,
including labile, covalent binding, cross-linking, etc.
Biotin/avidin interactions may be employed; for example, a
biotinylated tissue protective peptides of the invention may be
complexed with a labile conjugate of avidin and a molecule
desirably transported. As mentioned above, a hybrid molecule may be
prepared by recombinant or synthetic means, for example, a fusion
or chimeric polypeptide which includes both the domain of the
molecule with desired pharmacological activity and the domain
responsible for the peptides tissue-protective receptor activity
modulation. Protease cleavage sites may be included in the
molecule.
[0187] A molecule may be conjugated to a tissue protective peptide
of the invention through a polyfunctional molecule, i.e., a
polyfunctional crosslinker. As used herein, the term
"polyfunctional molecule" encompasses molecules having one
functional group that can react more than one time in succession,
such as formaldehyde, as well as molecules with more than one
reactive group. As used herein, the term "reactive group" refers to
a functional group on the crosslinker that reacts with a functional
group on a molecule (e.g., peptide, protein, carbohydrate, nucleic
acid, particularly a hormone, antibiotic, or anti-cancer agent to
be delivered across an endothelial cell barrier) so as to form a
covalent bond between the cross-linker and that molecule. The term
"functional group" retains its standard meaning in organic
chemistry. The polyfunctional molecules that can be used are
preferably biocompatible linkers, i.e., they are noncarcinogenic,
nontoxic, and substantially non-immunogenic in vivo. Polyfunctional
cross-linkers such as those known in the art and described herein
can be readily tested in animal models to determine their
biocompatibility. The polyfunctional molecule is preferably
bifunctional. As used herein, the term "bifunctional molecule"
refers to a molecule with two reactive groups. The bifunctional
molecule may be heterobifunctional or homobifunctional. A
heterobifunctional cross-linker allows for vectorial conjugation.
It is particularly preferred for the polyfunctional molecule to be
sufficiently soluble in water for the cross-linking reactions to
occur in aqueous solutions such as in aqueous solutions buffered at
pH 6 to 8, and for the resulting conjugate to remain water soluble
for more effective bio-distribution. Typically, the polyfunctional
molecule covalently bonds with an amino or a sulfhydryl functional
group. However, polyfunctional molecules reactive with other
functional groups, such as carboxylic acids or hydroxyl groups, are
contemplated in the present invention.
[0188] The homobifunctional molecules have at least two reactive
functional groups, which are the same. The reactive functional
groups on a homobifunctional molecule include, for example,
aldehyde groups and active ester groups. Homobifunctional molecules
having aldehyde groups include, for example, glutaraldehyde and
subaraldehyde. The use of glutaraldehyde as a cross-linking agent
was disclosed by Poznansky et al., Science 223, 1304-1306 (1984).
Homobifunctional molecules having at least two active ester units
include esters of dicarboxylic acids and N-hydroxysuccinimide. Some
examples of such N-succinimidyl esters include disuccinimidyl
suberate and dithio-bis-(succinimidyl propionate), and their
soluble bis-sulfonic acid and bis-sulfonate salts such as their
sodium and potassium salts. These homobifunctional reagents are
available from Pierce, Rockford, Ill.
[0189] The heterobifunctional molecules have at least two different
reactive groups. The reactive groups react with different
functional groups, e.g., present on the peptide and the molecule.
These two different functional groups that react with the reactive
group on the heterobifunctional cross-linker are usually an amino
group, e.g., the epsilon amino group of lysine; a sulfhydryl group,
e.g., the thiol group of cysteine; a carboxylic acid, e.g., the
carboxylate on aspartic acid; or a hydroxyl group, e.g., the
hydroxyl group on serine.
[0190] Of course, certain of the various tissue protective peptides
of the invention, may not have suitable reactive groups available
for use with certain cross-linking agent; however, one of skill in
the art will be amply aware of the choice of cross-linking agents
based on the available groups for cross-linking in tissue
protective peptides of the invention.
[0191] When a reactive group of a heterobifunctional molecule forms
a covalent bond with an amino group, the covalent bond will usually
be an amido or imido bond. The reactive group that forms a covalent
bond with an amino group may, for example, be an activated
carboxylate group, a halocarbonyl group, or an ester group. The
preferred halocarbonyl group is a chlorocarbonyl group. The ester
groups are preferably reactive ester groups such as, for example,
an N-hydroxy-succinimide ester group.
[0192] The other functional group typically is either a thiol
group, a group capable of being converted into a thiol group, or a
group that forms a covalent bond with a thiol group. The covalent
bond will usually be a thioether bond or a disulfide. The reactive
group that forms a covalent bond with a thiol group may, for
example, be a double bond that reacts with thiol groups or an
activated disulfide. A reactive group containing a double bond
capable of reacting with a thiol group is the maleimido group,
although others, such as acrylonitrile, are also possible. A
reactive disulfide group may, for example, be a 2-pyridyldithio
group or a 5, 5'-dithio-bis-(2-nitrobenzoic acid) group. Some
examples of heterobifunctional reagents containing reactive
disulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)
propionate (Carlsson, et al., 1978, Biochem J., 173:723-737),
sodium S-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate,
and
4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.
N-succinimidyl 3-(2-pyridyldithio) propionate is preferred. Some
examples of heterobifunctional reagents comprising reactive groups
having a double bond that reacts with a thiol group include
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and
succinimidyl m-maleimidobenzoate.
[0193] Other heterobifunctional molecules include succinimidyl
3-(maleimido) propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)
butyrate, sulfosuccinimidyl
4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,
maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate
salt of succinimidyl m-maleimidobenzoate is preferred. Many of the
above-mentioned heterobifunctional reagents and their sulfonate
salts are available from Pierce Chemical Co., Rockford, Ill.
USA.
[0194] The need for the above-described conjugated to be reversible
or labile may be readily determined by the skilled artisan. A
conjugate may be tested in vitro for desirable pharmacological
activity. If the conjugate retains both properties (the properties
of the conjugated molecule and the properties of the tissue
protective peptide), its suitability may then be tested in vivo. If
the conjugated molecule requires separation from the tissue
protective peptide for activity, a labile bond or reversible
association with long acting erythropoietin or the long acting
tissue protective cytokine will be preferable. The lability
characteristics may also be tested using standard in vitro
procedures before in vivo testing.
[0195] Additional information regarding how to make and use these
as well as other polyfunctional reagents may be obtained from the
following publications or others available in the art: [0196]
Carlsson, J. et al., 1978, Biochem. J. 173:723-737; [0197] Cumber,
J. A. et al., 1985, Methods in Enzymology 112:207-224; [0198] Jue,
R. et al., 1978, Biochem 17:5399-5405; [0199] Sun, T. T. et al.,
1974, Biochem. 13:2334-2340; [0200] Blattler, W. A. et al., 1985,
Biochem. 24:1517-152; [0201] Liu, F. T. et al., 1979, Biochem.
18:690-697; [0202] Youle, R. J. and Neville, D. M. Jr., 1980, Proc.
Natl. Acad. Sci. U.S.A. 77:5483-5486; [0203] Lerner, R. A. et al.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:3403-3407; [0204] Jung, S.
M. and Moroi, M., 1983, Biochem. Biophys. Acta 761:162; [0205]
Caulfield, M. P. et al., 1984, Biochem. 81:7772-7776; [0206]
Staros, J. V., 1982, Biochem. 21:3950-3955; [0207] Yoshitake, S. et
al., 1979, Eur. J. Biochem. 101:395-399; [0208] Yoshitake, S. et
al., 1982, J. Biochem. 92:1413-1424; [0209] Pilch, P. F. and Czech,
M. P., 1979, J. Biol. Chem. 254:3375-3381; [0210] Novick, D. et
al., 1987, J. Biol. Chem. 262:8483-8487; [0211] Lomant, A. J. and
Fairbanks, G., 1976, J. Mol. Biol. 104:243-261; [0212] Hamada, H.
and Tsuruo, T., 1987, Anal. Biochem. 160:483-488; or [0213]
Hashida, S. et al., 1984, J. Applied Biochem. 6:56-63, each of
which is hereby incorporated by reference in its entirety.
[0214] Additionally, methods of cross-linking are reviewed by Means
and Feeney, 1990, Bioconjugate Chem. 1:2-12, hereby incorporated by
reference in its entirety.
[0215] Barriers which are crossed by the above-described methods
and compositions of the present invention include but are not
limited to the blood-brain barrier, the blood-eye barrier, the
blood-testes barrier, the blood-ovary barrier, blood-nerve barrier,
blood-spinal cord barrier, and blood-placenta barrier.
[0216] Candidate molecules for transport across an endothelial cell
barrier include, for example, hormones, such as growth hormone,
neurotrophic factors, antibiotics, antivirals, or antifungals such
as those normally excluded from the brain and other barriered
organs, peptide radiopharmaceuticals, antisense drugs, antibodies
and antivirals against biologically-active agents, pharmaceuticals,
and anti-cancer agents. Non-limiting examples of such molecules
include hormones such as growth hormone, nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), ciliary neurotrophic
factor (CNTF), basic fibroblast growth factor (bFGF), transforming
growth factor .beta.1 (TGF.beta.1), transforming growth factor
.beta.2 (TGF.beta.2), transforming growth factor .beta.3
(TGF.beta.3), interleukin 1, interleukin 2, interleukin 3, and
interleukin 6, AZT, antibodies against tumor necrosis factor, and
immunosuppressive agents such as cyclosporin. Additionally, dyes or
markers may be attached to the tissue protective peptides of the
present invention in order to visualize cells, tissues, or organs
within the brain and other barriered organs for diagnostic
purposes. As an example, a marker used to visualize plaque within
the brain could be attached to a tissue protective peptide in order
to determine the progression of Alzheimer's disease within a
patient.
[0217] The present invention is also directed to a composition
comprising a molecule to be transported via transcytosis across an
endothelial cell tight junction barrier and a tissue protective
peptide as described above. The invention is further directed to
the use of a conjugate between a molecule and a tissue protective
peptide cytokine as described above for the preparation of a
pharmaceutical composition for the delivery of the molecule across
a barrier as described above.
[0218] Various animal models and in-vitro tests of neuroprotection
and transcytosis are provided in PCT/US01/49479 (hereby
incorporated by reference herein in its entirety) to demonstrate
the effectiveness of the tissue protective peptides of the
invention. For transcytosis, model proteins conjugated to the long
acting erythropoietins of the invention are evaluated for transport
into the brain following parenteral administration. These tests in
in-vitro models and animal models are predictive of the efficacy of
the present compounds in other mammalian species including
humans.
[0219] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed, however, as limiting
the broad scope of the invention.
6. EXAMPLES
Example 1: Method of Peptide Synthesis
[0220] A. Synthesis of Peptide A (SEQ ID NO:32, corresponding to
EPO amino acid sequence 38-57) and Peptide B (SEQ ID NO:34,
corresponding to EPO amino acid sequence 58-82).
[0221] Peptide A, SEQ ID NO:32, and Peptide B, SEQ ID NO:34,
fragments of EPO (see Table 1), were synthesized using "in situ
neutralization" Boc Chemistry stepwise solid-phase peptide
synthesis, as described in Band, D., Chopra, N and Kent, S., "Total
Synthesis of Crambin," J. AM. CHEM. SOC. 2004, 126, 1377-1383
(incorporated by reference herein in its entirety). Briefly, two
fragments corresponding to EPO amino acid sequence 38-57 (peptide
C, NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29) and EPO amino acid sequence
58-82 (peptide D, QQAVEVWQGLALLSEAVLRGQALLV, SEQ ID NO:30) were
synthesized on --OCH.sub.2-Pam-resins (free .sup..alpha.carboxyl
peptides) or on HSCH.sub.2CH.sub.2CO-Leu-OCH.sub.2-Pam-Resin
(.sup..alpha.thioester peptides). During synthesis the side chains
of various amino acids were protected as follows: Arg(Tos),
Asn(Xan), Asp(OcHex), Cys(4-CH.sub.3Bzl) or Cys(ACM), Glu(OcHex),
Lys(2-Cl--Z), Ser(Bzl), Thr(Bzl), Tyr(Br--Z). After the peptide
chain was assembled, the peptides were deprotected and
simultaneously cleaved from the resin support by treatment with
anhydrous HF containing p-cresol (90:10, v/v) for 1 hr at 0.degree.
C. After evaporation of the HF under reduced pressure, crude
products were precipitated and triturated with chilled diethyl
ether, and the peptides were dissolved in 50% aqueous acetonitrile
containing 0.1% TFA and purified by the preparative HPLC system.
Peptide compositions were confirmed using LC-MS.
Example 2. Validation of Peptide-Mediated Tissue Protection
[0222] The tissue protective peptides were tested for any tissue
protective activity using a Sciatic Nerve Assay. Sprague-Dawley
rats (250-300 grams) (six per group, including control) were
anesthetized using isoflurane (Baxter NPC 10019-773-60) and a Table
Top Laboratory Anesthesia System (flowmeter set to 2-3
liters/minute @ 55 psi) for at least 3 minutes. The rat was then
placed on a homeothermic blanket to ensure that the core
temperature of the rat was maintained at 35-37.degree. C. during
the operation. Core temperature was monitored via a rectal probe.
The right sciatic nerve of the anesthetized rat was exposed at mid
thigh through a quadriceps muscle dissection; a 2 cm incision with
a 15 blade scalpel was made through the skin parallel and over the
quadriceps muscle and the quadriceps muscle was cut to expose the
sciatic nerve using a pair of dissecting scissors. The sciatic
nerve was then freed from the surrounding membranes. A 2-0 braided
silk thread (Ethicon, 685-G) was passed under the nerve and the
ends of the suture passed through a guide which was maintained
perpendicular to the nerve. The end of the suture was then tied to
a non-elastic cord which was then draped around the pulley system
(a NYL pulley bearing MTD 1/4''B (PO Number 04174-01) with
stabilizer) and a 100 gram weight attached to the non-elastic cord
was slowly released. The weight was allowed to hang for 1 minute
before the silk suture was cut to release the weight.
[0223] A 289 pmol/kg dose of carbamylated erythropoietin, a 289
pmol/kg dose of one peptide from the series A-J (see Table 1), or
PBS was then injected into the caudal vein using a 1/2 cc insulin
syringe. A 20mer fragment (corresponding to amino acids 102-121)
from pigment epithelium-derived growth factor (PEDF) which does not
follow the teaching above was used as control.
[0224] The muscle and surgical incision were then closed and 5 ml
of Lactated Ringers solution was injected subcutaneously into the
rat. The core temperature of the rat was maintained at
35-37.degree. C. using a heat blanket during recovery.
[0225] Over the next four days the rear toe splaying of the rats
was determined by placing the rat in an acrylic tube with a
diameter of 30 cm on the scanning surface of a digital scanner.
After waiting 5 minutes in order to permit acclimation, a scan was
taken of the rat's back feet that clearly displayed all 5 toes.
Three acceptable scans of each rat were taken. From the scans, the
Toe Spread (the distance between the ball of the first toe and the
ball of the fifth toe) and the Intermediate Toe Spread (the
distance between the ball of the second toe and the ball of the
fourth toe) were measured. The static sciatic index was then
computed in accordance with S. Erbayraktar et al., 2003, Proc Natl
Acad Sci USA 100, 6741-6746 (hereby incorporated by reference in
its entirety) and statistical analysis performed.
[0226] All peptides except B (SEQ ID NO:34), H (SEQ ID NO:47) and
the PEDF derivative were equally protective, providing a static
sciatic index ("SSI") of about -0.57 versus a SSI of about -67 to
about -68 for PBS/PEDF fragment (FIG. 2). FIG. 2 also shows that
the efficacy of the positive peptides was at least equivalent, if
not improved, over that of the carbamylated erythropoietin.
[0227] Table 1 also presents the approximate distance between
carbonyl carbons for the tested peptides. Distances were calculated
using the three-dimensional coordinates provided by Cheetham et
al., 1998, Nat. Struct. Biol. 5:861-866, hereby incorporated by
reference. The peptides which tested positive for tissue protective
activity each had a carbonyl carbon to carbonyl carbon
distance/separation of between about 3 .ANG. to about 5 .ANG..
TABLE-US-00002 TABLE 1 Tissue protective efficacy of representative
peptides using an in vivo bioassay (sciatic nerve injury model).
Appprox Distance Between carbonyl Dose Sciatic EPO Carbons [nmoles/
nerve Peptide Class peptide sequence Structure (Angstroms) kg-bw]
assay A) EPO fragment A 1-23 APPRLICDSRVLERYLLEAKEAE 4.6 29, 290, +
(SEQ ID NO: 32) 1450 APPRLICDSRVLERYLLEAKEAE 4.4 (SEQ ID NO: 32) B
24-37 NITTGCAEHCSLNE (SEQ ID NO: 34) 2.8 290 - C 38-57
NITVPDTKVNFYAWKRMEVG (SEQ ID NO: 29) 4.6 290 + D 58-82
QQAVEVWQGLALLSEAVLRGQALLV 4.8 29, 290, + (SEQ ID NO: 30) 1450 E
28-47 GCAEHCSLNENITVPDTKVN (SEQ ID NO: 31) 4.4 290 + F 14-29
RYLLEAKEAENITTGC (SEQ ID NO: 33) 3.6 290 + B) Helix face G 58, 62,
65, QEQLERALNSS (SEQ ID NO: 40) 3.6 290 + 69, 72, 76 79, 80, 83,
84, 85 H 71, 72, 75, SELRGQ (SEQ ID NO: 47) 7.2 290 - 76, 77 C)
chimera I Peptide G + CSLNENIQEQLERALNSS (SEQ ID NO: 43) 290 +
.beta.-pleated sheet (33- 39) J Peptide G + QEQLERALNSSLRRYINMLTRTR
290 + pancreatic (SEQ ID NO: 41) polypeptide helix D) Type 1
cytokine motif K GM-CSF WEHVNAIQEARRLL (SEQ ID NO: 35) 3.6 290 +
helix A WEHVNAIQEARRLL (SEQ ID NO: 35) 4.6 (13-26) WEHVNAIQEARRLL
(SEQ ID NO: 35) 4.6 L CNTF helix KIRSDLTALTESYVKH (SEQ ID NO: 37)
4.7 290 + A (26-41)
Example 3. Tissue Protective Peptides are Non Erythropoietic
A. In Vitro Assessment:
[0228] UT-7epo, a human erythropoietin-dependent leukemia cell
line, was used for the determination of the erythropoietic potency
of the peptides. UT-7epo cells (Deutsche Sammlung von
Mikroorganismen and Zellkulturen (DSMZ), Cat. No. ACC 363) were
grown in a complete RPMI-1640 medium with 10% FBS and 5 ng/ml
erythropoietin. The proliferation/survival (=viability increase)
response of the cells exposed to erythropoietin is mediated by the
classical erythrocyte-type erythropoietin receptor and is a
quantitative measure of the capacity of erythropoietin-variants to
stimulate the classical erythropoietin receptor.
[0229] UT-7epo cells were transferred to fresh complete RPMI 1640
medium containing 10% donor calf serum, 4 mM L-glutamine, and
supplemented with 5 ng/ml of recombinant human erythropoietin. The
cells were maintained in 75 cm.sup.2 flasks with 20 ml of
medium/flask in a humidified incubator with 5% CO.sub.2 at
37.degree. C. for 48 h. On day two of the assay, i.e., at 48 h, the
cells were transferred from the flask into a 50-ml conical tube and
centrifuged at 1,000 rpm for 5 minutes at room temperature. The
supernatant was discarded and the cells washed two times with 10 ml
of starvation media (3% donor calf serum, 4 mM L-glutamine). The
cells were then re-suspended in starvation media, using up and down
pipette action to obtain a single cell suspension. The re-suspended
cells were diluted with starvation media to a obtain a density of
4.times.10.sup.5 cells/ml, and plated at a total culture volume of
10 ml per 25 cm.sup.2 flask. Following a 4 h incubation, the cells
were again transferred to a 50-ml conical tube. Control cells were
maintained throughout with 5 ng/ml of rhu-erythropoietin.
[0230] Cells were diluted to 200,000 cells/ml in starvation medium,
plated at 100 .mu.l/well in a 96 well plate and exposed to varying
concentrations of erythropoietin, carbamylated erythropoietin, and
Peptide D, SEQ ID NO:30. A series of 10 fold dilutions in RPMI 1640
medium containing 3% serum was used to generate concentrations of
test compounds from 0.2 pM to 20 nM. Following a further for 48 h
incubation, a solution of 15 ml WST-1 Cell Proliferation Reagent
(Roche) was added to each well, and incubated for 1 hour at
37.degree. C. in CO.sub.2. After mixing for 1 minute, the plate was
read in a plate reader (absorption at 450 nm, subtracted from
background absorption at 650 nm).
[0231] Peptide D exhibited no erythropoietic activity at doses as
high as 10,000 pM (FIG. 3). Preferably, the peptide will have no
erythropoietic activity for a dose lower than 1 .mu.g/ml, and more
preferably for a dose lower than 10 .mu.g/ml.
B. In Vivo Assessment:
[0232] To evaluate the erythropoietic activity of tissue protective
peptide F (SEQ ID NO:33) or peptide G (SEQ ID NO:40, as discussed
supra a peptide constructed of the presenting residues of Helix B),
the peptides were administered 0.8 m/kg subcutaneously three time
per week to male Sprague Dawley rats. The dosage schedule
corresponded to the equivalent dose (on a molar basis) of EPO
previously determined to be elicit maximum erythropoiesis.
Hemoglobin concentration was determined periodically by use of an
automated analyzer (Keska Corporation).
[0233] Neither Peptide B nor Peptide C showed any increase in
hematocrit over the course of the study (FIG. 4; the response to an
equimolar dosage of EPO is presented for comparison). The decrease
in hemoglobin noted for EPO after 3 weeks is due to the production
of anti-EPO neutralizing antibodies which cause pure red cell
aplasia. In contrast, no neutralizing antibody response was
observed for either peptide G or peptide F.
Example 4. Peptide is Tissue Protective in In Vitro Assays
[0234] Peptides can be readily assessed for tissue protection using
any number of in vitro assays. For example, protection from
excitoxicity can be determined using kainite-induced death of mouse
motoneurons. Spinal cords were obtained from 15-day old
Sprague-Dawley rat embryos as previously described (Siren et al.,
2001, PNAS 98:4044, hereby incorporated by reference in its
entirety). The ventral horn was trypsinized and centrifuged through
a 4% BSA cushion for 10 min at 300.times.g. Cells (representing
mixed neuron-glia culture) were seeded at a density of 2,000
cells/cm.sup.2 into 24-mm well plates precoated with poly-DL
ornithine and laminin. Motoneurons were further purified by
immunopanning and the cells were seeded at low density (20,000
cells/cm.sup.2) onto 24-mm well plates precoated with
poly-DL-ornithine and laminin, and containing complete culture
medium [Neurobasal/B27 (2%); 0.5 mM L-glutamine; 2% horse serum; 25
mM 2 mercaptoethanol; 25 mM glutamate; 1% penicillin and
streptomycin; 1 ng/ml BDNF]. The medium (without glutamate) was
re-added to cultures on days 4 and 6.
[0235] Cell death was induced on day 6 in culture by incubation for
48 h with kainic acid (5 mM for mixed neuron-glia cultures; 50 mM
for purified cultures). Peptide D (5 ng/mL) or vehicle was added to
the cultures 72 h before induction of cell death, and treatment
continued for 48 h. The medium was then discarded and the cells
fixed with 4% (vol/vol) paraformaldehyde in PBS for 40 min,
permeabilized with 0.2% Triton X-100, blocked with 10% (vol/vol)
FCS in PBS, incubated with antibodies against non-phosphorylated
neurofilaments (SMI-32; 1:9,000) overnight, and visualized by using
the avidin-biotin method with diaminobenzidine. Viability of
motoneurons was assessed morphologically by counting SMI-32
positive cells across four sides of the cover slip and staining for
apoptotic bodies was done by using H33258.
[0236] Peptide D (SEQ ID NO:30, corresponding to amino acids 58-82
of SEQ ID NO:1) completely protected motoneurons from injury caused
by kainate (FIG. 5).
[0237] Alternatively, tissue protection afforded by peptides can be
determined using an assay employing mouse P19 cells, which are
neuronal-like and die via apoptosis upon withdrawal of serum.
Tissue protection of peptide D (SEQ ID NO:30) was compared to that
of EPO using P19 clone P19S1801A1 as previously published (Siren et
al., 2001, PNAS 98:4044, nereby incorporated by reference in its
entirety). Cells were maintained undifferentiated in DMEM
supplemented with 2 mM Lglutamine; 100 units/ml penicillin G; 100
mg/ml streptomycin sulfate (GIBCO); 10% (vol/vol) FBS (HyClone),
containing 1.2 g/liter NaHCO.sub.3 and 10 mM Hepes buffer,
hereafter referred to as complete medium. Serum-free medium
contained the same components as above with the deletion of serum
and the addition of 5 mg/ml of insulin; 100 mg/ml of transferrin;
20 nM progesterone; 100 mM putrescine; 30 nM Na2SeO3 (from Sigma).
For the experiments, 50% confluent cells were pretreated overnight
with EPO or vehicle, dissociated with trypsin, washed in serum-free
medium, and plated in 25-cm.sup.2 tissue culture flasks at a final
density of 104 cells/cm.sup.2 in serum-free medium alone or with
added EPO. Cell viability and was determined by trypan blue
exclusion and a hemacytometer.
[0238] Peptide C, SEQ ID NO:29 (corresponding to amino acids 38-57
of SEQ ID NO:1) was at least 10 time more potent on a weight basis
than EPO in preventing apoptosis of p19 cells (FIG. 6).
Example 5. Middle Cerebral Artery Occlusion Model
[0239] Male Crl:CD(SD)BR rats weighing 250-280 g were obtained from
Charles River, Calco, Italy. Surgery was performed in accordance
with the teachings of Brines et al., 2000, PNAS USA 97:10526-10531
(hereby incorporated by reference in its entirety. Briefly, the
rats were anesthetized with chloral hydrate (400 mg/kg-bw, i.p.),
the carotid arteries were visualized, and the right carotid was
occluded by two sutures and severed. A burr hole adjacent and
rostral to the right orbit allowed visualization of the middle
cerebral artery ("MCA"), which was cauterized distal to the rhinal
artery. To produce a penumbra (border zone) surrounding this fixed
MCA lesion, the contralateral carotid artery was occluded for 1
hour by using traction provided by a fine forceps and then
re-opened.
[0240] Sprague Dawley rats (8 per group) were subjected to the
above noted MCAO protocol. The rats were administered PBS,
carbamylated erythropoietin (44 ug/kg), or peptide D (aa 58-82; 4.4
ug/kg) upon release of the occlusion. Additionally, peptide D (aa
58-82; 4.4 ug/kg) was administered in four doses at 2 hour
intervals following the occlusion to a separate group. For
assessment of injury, rats were subjected to behavioral testing or
the volume of the lesion was determined by tetrazolium staining of
brain sections performed 24 hours post surgery in accordance with
the previously noted protocol.
[0241] FIG. 7A presents a graph demonstrating the volume of lesions
resulting from the MCAO protocol. Treatment with peptide D (SEQ ID
NO:30), either as a single dose or by multiple doses, reduced the
lesion volume resulting from the MCAO surgery by about two thirds:
statistically equivalent to the tissue protective effects of
carbamylated erythropoietin.
[0242] (b) Therapeutic Window of Tissue Protective Cytokines
[0243] The MCAO protocol as outlined above was repeated for the
instant example. Following the occlusion procedure, PBS,
carbamylated erythropoietin (44 ug/kg, i.v.), or peptide D (SEQ ID
NO:30) (4.4 ug/kg) were administered to the rats immediately after
recirculation was established in the carotid (i.e., one hour from
the onset of ischemia). In addition, peptide D (SEQ ID NO:30) was
administered in four doses(each 4.4 ug/kg-bw) at 2 hours intervals
following the occlusion. (8 rats per group).
[0244] (c) Behavioral Testing.
[0245] A separate group of rats was also tested in a foot fault
behavioral protocol. Rats were tested on an elevated stainless
steel grid floor 30 cm.times.30 cm with grid size of 30 mm
according to the protocol of Markgraf et al., 1992, Brain Research
575:238-246 (hereby incorporated by reference in its entirety).
When placed on the grid, rat would attempt to move around and
occasionally place a foot, rather than on the grid, through a grid
opening ("foot fault"). The number of foot faults was measured for
a 1 minute period.
[0246] The rats treated with peptide D (SEQ ID NO:30) following
reperfusion suffered from fewer foot faults than those treated with
PBS (FIG. 7B). No significant additional benefit was observed
following the administration of multiple doses of peptide D (SEQ ID
NO:30). Although the mean number of foot faults was less in the
group receiving multiple doses of peptide, the difference observed
was not significantly different from the group receiving a single
dose.
Example 6. Diabetic Neuropathy
[0247] Diabetes was induced in male Sprague Dawley rats (Charles
River, Calco, IT) using streptozocin administered at a single dose
of 60 mg/kg ip in fasting rats as previously described (Bianchi et
al., 2004, Proc Natl Acad Sci USA 101, 823-828, hereby incorporated
by reference in its entirety). Diabetes was confirmed by increased
serum glucose levels to greater than 300 mg per deciliter (mg %)
(normal levels are <100 mg %). Diabetic animals were then
treated with peptide D (SEQ ID NO:30; 4 .mu.g/kg) or vehicle 5
times a week intraperitoneally. Two weeks after induction of the
diabetic state, nerve conduction velocity was determined using the
caudal nerve.
[0248] As shown in FIG. 8A, the diabetic animals exhibited a
reduction in caudal nerve conduction velocity from about 22 m/s
(normal) to about 19 m/s. Administration of peptide D (SEQ ID
NO:30) was associated with an increase in conduction velocity to
about 23 m/s.
[0249] Additionally, the thermal nociceptive threshold was
quantified by measurement of the time to paw withdrawal in a "hot
plate" test. Withdrawal latency was defined as the time between
placement on the hot plate and the time of withdrawal and licking
of hind paw. Each animal was tested twice separated by a 30 min
rest interval. Hind paw thermal thresh-hold was measured 4 weeks
after induction of diabetes. Peptide D (SEQ ID NO:30) reduced the
latency time spent on the hot plate by the diabetic animal (FIG.
8B).
Example 7: Protection of Sciatic Nerve and Kidney from
Cisplatinum-Induced Injury
[0250] Cisplatinum (CDDT) was administered intraperitoneally to
male Sprague-Dawley rats at 2 mg/kg twice weekly for 5 weeks as
described in Bianchi et al., 2006, Clin Cancer Res 12: 2607-2612,
hereby incorporated by reference in its entirety. Animals were
separated into groups of 6 each. During the 5 week CDDT
administration, animals also received either peptide G, (SEQ ID
NO:40) at 0.4 .mu.g/kg-bw or PBS i.p. three times per week. A
control group received PBS instead of CDDT. Hot plate latency was
determined as described in Example 6 above.
[0251] Animals that received CDDT and only PBS exhibited an
increase in latency compared to controls: i.e., CDDT was associated
with impaired thermal sensitivity. In contrast, animals that
received the peptide exhibited normal hot plate latency (FIG.
9A).
[0252] Treatment with peptide also prevented CDDT-induced
polyuria(FIG. 9B) Specifically, animals that had received PBS
exhibited a significant increase in daily urine production from
about 30 mL/day to about 47 mL/day. In contrast, animals having
received the tissue-protective peptide did not significantly differ
from controls animals that received PBS instead of CDDT.
Example 8: Protection from Diabetes-Induced Retinal Vascular
Leak
[0253] Beneficial effects of tissue-protective peptides on
hyperglycemia-induced retinal vascular leakiness can be determined
using a rat model of diabetic retinopathy. In this model, Evans
blue is used to determine leakage from the blood vessel into
tissues as described by Xu et al., 2001, Invest. Ophthal. Vis. Sci
42: 789-794 (hereby incorporated by reference in its entirety).
Evans blue is tightly bound to albumin and is therefore retained
within the circulation unless leakiness of the vessel wall occurs,
such as caused by uncontrolled diabetes mellitus.
[0254] In this model, fasting male Sprague-Dawley rats receive a
single dose of streptozotocin (60 mgkg ip). Two days later,
following verification of the development of diabetes mellitus
(fasting serum glucose greater than 300 mg %, animals were divided
into groups of 6 animals each as well as a control group that did
not receive streptozotocin. The two diabetic groups were
administered either peptide D (SEQ ID NO:30) at 4 .mu.gkg
intraperitoneally 5 days a week or PBS on the same schedule. After
three weeks of uncontrolled diabetes, animals were anesthetized and
administered Evans Blue dye (30 mg/kg) intravenously, which was
allowed to circulate for 2 hours. Using transcardiac puncture, the
animals were then perfused with PBS until the effluent was clear
followed by 4% paraformaldehyde. The eyes were then removed and the
retinas carefully dissected from the globe. The retinal content of
Evans Blue was determined by incubating the retinas in formamide at
80.degree. C. for 18 hours. The supernatant was then removed and
saved for analysis and the retinas completely dried and weighed.
Concentration of Evans Blue in the supernatant was determined by a
spectrophotometer and a standard curve of Evans Blue dissolved in
formamide established.
[0255] As seen in FIG. 10, animals that received administration of
peptide D (SEQ ID NO:30) experienced no increase in Evans Blue dye
within the retina, compared to control. In contrast, diabetic
animals that received only PBS exhibited an increase in retinal
Evans blue content, indicating the vascular leakage had
occurred.
Example 9: Protection from Acute Renal Failure
[0256] Tissue-protective peptides are also effective in preventing
injury to the kidneys in the setting of ischemia. Adult male Wistar
rats were anesthetized and an abdominal incision made to visualize
both renal arteries. Using an atraumatic vascular clamp, both
arteries were compressed for 60 minutes, completely arresting renal
blood flow. The clamps were then removed to restore circulation and
peptide F (SEQ ID NO:33) or peptide G (SEQ ID NO:40) was
administered at 290 pmol/kg-bw intravenously. An additional group
undergoing ischemia received only PBS intravenously.
[0257] Seventy two hours following reperfusion, the animals were
anesthetized and underwent perfusion-fixation using
paraformaldehyde. Fixed animals were sectioned sagittally into
halves and further fixed by immersion in 10% formaldehyde at room
temperature for one day. Histological evaluation of the kidneys was
performed according to the protocol of Sharples et al., 2005, J
Amer Soc Nephrol: 15: 2115 (hereby incorporated by reference in its
entirety). Briefly, after dehydration using graded ethanol, pieces
of kidney were embedded in parafin, cut into 5 micrometer sections
and mounted on glass slides. Sections on slides were deparaffinized
with xylene, counterstained with hematoxylin and eosin, and
examined under a light microscope. One hundred fields were examined
for each kidney, and a score from 0 to 3 was given for each tubular
profile: 0, normal histology; 1, tubular cell swelling, brush
border loss, and nuclear condensation with up to one third nuclear
loss; 2, as for score 1 but greater than one third and less than
two thirds tubular profiles showing nuclear loss; and 3, greater
than two thirds tubular profile showing nuclear loss. The
histologic score for each kidney was calculated by addition of all
scores, with a maximum score of 300.
[0258] Administration of either peptide F (SEQ ID NO:33) or peptide
G (SEQ ID NO:40) was associated with a significant reduction in
injury score (p<0.05) compared to the controls.
Example 11. Efficacy of Tissue Protective Peptides in Cerebral
Malaria
[0259] A rodent model of cerebral malaria was developed according
to Kaiser et al., 2006, J. Infect. Dis. 193:987-995 (hereby
incorporated by reference in its entirety. Female CBA/J mice 7
weeks old were separated into groups of 20 animals. Each group was
infected with Plasmodium berghei Anka (PbA) administered
intraperitoneally as a dose of 10.sup.6 PbA infected erythrocytes.
Mice received either PBS or peptide F (SEQ ID NO:33) on days 4, 5,
and 6 as intraperitoneal injection at a dose of 2.6 .mu.g/kg.
Clinical status and blood smear data were gathered during the
follow-up (end point D30). Cumulative long-term survival was
calculated according to the Kaplan-Meier method and groups were
compared with the log rank test. Survival time was the dependant
variable. A p-value of <0.05 was considered significant.
[0260] As shown in FIG. 12, all mice in the control group (saline)
died by day 8. In contrast, mice that received peptide F (SEQ ID
NO:33) exhibited prolonged survival, significantly different from
the control group (p<0.005), using a log-rank test.
Example 12: Efficacy of Tissue Protective Peptides in a Murine EAE
Model
[0261] Experimental autoimmune encephalomyelitis ("AEA") was
induced in C57BL/6 female mice (6-8 weeks of age) according to
Savino et al., 2006, J Neuroimmunol 172:27-37, hereby incorporated
by reference in its entirety. EAE was induced by subcutaneous
immunization in the flanks with a total of 200 .mu.g of MOG35-55
(Multiple Peptide Systems, San Diego, Calif., USA) in incomplete
Freund's adjuvant (Sigma, St. Louis, Mo., USA) supplemented with 8
mg/ml of Mycobacterium tuberculosis (strain H37RA; Difco, Detroit,
Mich., USA). Animals were housed in specific pathogen-free
conditions, allowing access to food and water ad libitum. Mice
received 500 ng of pertussin toxin (Sigma) i.v. at the time of
immunization and 48 h later. Weight and clinical score were
recorded daily (0=healthy, 1=flaccid tail, 2=ataxia, and/or
hind-limbs paresis, or slow righting reflex, 28 C. 3=paralysis of
hind limb and/or paresis of forelimbs, 4=paraparesis of fore limb,
5=moribund or death). Food pellets and the drinking water were
placed on Petri plates on the floor of the cage to enable sick mice
to eat and drink. Peptide E (SEQ ID NO:31) was administered daily
subcutaneously at a dose of 4.4 micrograms/kg-bw, starting on day 4
after immunization.
[0262] Administration of peptide E (SEQ ID NO:31) significantly
reduced both the time course and severity of the clinical
presentation of AEA in the treated animals (p<0.01) (FIG.
13).
[0263] The invention is not to be limited in scope by the specific
embodiments described which are intended as single illustrations of
individual aspects of the invention, and functionally equivalent
methods and components are within the scope of the invention.
Indeed various modifications of the invention, in addition to those
shown and described herein will become apparent to those skilled in
the art from the foregoing description and accompanying drawings.
Such modifications are intended to fall within the scope of the
appended claims.
[0264] All references cited herein are incorporated by reference
herein in their entireties for all purposes.
Sequence CWU 1
1
581165PRTArtificial SequenceErythropoietin peptide 1Ala Pro Pro Arg
Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu
Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30
Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35
40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val
Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly
Gln Ala Leu65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu
Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu
Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile
Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn
Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala145 150 155 160
Cys Arg Thr Gly Asp 165 26PRTArtificial SequenceEPO amino Acids
44-49 associated with a high affinity receptor binding site 1 2Thr
Lys Val Asn Phe Tyr1 5 36PRTArtificial SequenceEPO amino acids
146-151 associated with a high affinity receptor binding site 1
3Ser Asn Phe Leu Arg Gly1 5 45PRTArtificial SequenceEPO amino acids
11-15 interacting with a low affinity receptor binding Site 2 4Val
Leu Glu Arg Tyr1 5 55PRTArtificial SequenceEPO amino acids 100-104
interacting with a low affinity receptor binding site 2 5Ser Gly
Leu Arg Ser1 5 64PRTArtificial SequenceIsolated polypeptides
comprising amino acid structural motif AVARIANT1,4Xaa = any
hydrophobic amino acid (same or different)VARIANT2,3Xaa = any
negatively charged amino acid (same or different) 6Xaa Xaa Xaa Xaa1
75PRTArtificial SequenceVariant of isolated EPO polypeptides
comprising amino acid structural motif AVARIANT1,5Xaa = Any
hydrophobic amino acid (same or different)VARIANT2, 4Xaa = Any
negatively charged amino acid (same or different)VARIANT3Xaa = Any
amino acid 7Xaa Xaa Xaa Xaa Xaa1 5 86PRTArtificial SequenceVariant
of isolated EPO polypeptides comprising amino acid structural motif
AVARIANT1, 6Xaa = Any hydrophobic amino acid (same or
different)VARIANT2,5Xaa = Any negatively charged animo acid (same
or different)VARIANT3,4Xaa = Any amino acid (same or different)
8Xaa Xaa Xaa Xaa Xaa Xaa1 5 97PRTArtificial SequenceVariant of
isolated EPO polypeptides comprising amino acid structural motif
AVARIANT1, 7Xaa = Any hydrophobic amino acid (same or
different)VARIANT2,6Xaa = Any negatively charged amino acid (same
or different)VARIANT3,4,5Xaa = Any amino acid (same or different)
9Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 108PRTArtificial SequenceVariant of
isolated EPO polypeptides comprising amino acid structural motif
AVARIANT1,8Xaa = Any hydrophobic amino acid (same or
different)VARIANT2, 7Xaa = Any negatively charged amino acid (same
or different)VARIANT3,4,5,6Xaa = Any amino acid (same or different)
10Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 119PRTArtificial
SequenceVariant of isolated EPO polypeptides comprising amino acid
structural motif AVARIANT1, 9Xaa = Any hydrophobic amino acid (same
or different)VARIANT2, 8Xaa = Any negatively charged amino acid
(same or different)VARIANT3,4,5,6,7Xaa = Any amino acid (same or
different) 11Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
124PRTArtificial SequenceVariant of isolated EPO polypeptides
comprising amino acid structural motif AVARIANT1Xaa = Any
hydrophobic amino acidVARIANT2, 3Xaa = Any negatively charged amino
acid (same or different)VARIANT4Xaa = Any polar amino acid 12Xaa
Xaa Xaa Xaa1 135PRTArtificial Sequenceariant of isolated EPO
polypeptides comprising amino acid structural motif AVARIANT1Xaa =
Any hydrophobic amino acidVARIANT2,4Xaa = Any negatively charged
amino acidVARIANT3Xaa = Any amino acidVARIANT5Xaa = Any polar amino
acid 13Xaa Xaa Xaa Xaa Xaa1 5 146PRTArtificial SequenceVariant of
isolated EPO polypeptides comprising amino acid structural motif
AVARIANT1Xaa = Any hydrophobic amino acidVARIANT2,5Xaa = Any
negatively charged amino acid (same or different)VARIANT3,4Xaa =
Any amino acid (same or different)VARIANT6Xaa = Any polar amino
acid 14Xaa Xaa Xaa Xaa Xaa Xaa1 5 157PRTArtificial SequenceVariant
of isolated EPO polypeptides comprising amino acid structural motif
AVARIANT1Xaa = Any hydrophobic amino acidVARIANT2, 6Xaa = Any
negatively charged amino acid (same or different)VARIANT3,4,5Xaa =
Any amino acid (same or different)VARIANT7Xaa = Any polar amino
acid 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 168PRTArtificial
SequenceVariant of isolated EPO polypeptides comprising amino acid
structural motif AVARIANT1Xaa = Any hydrophobic amino acidVARIANT2,
7Xaa = Any negatively charged amino acid (same or
different)VARIANT3,4,5,6Xaa = Any amino acid (same or
different)VARIANT8Xaa = Any polar amino acid 16Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 179PRTArtificial SequenceVariant of isolated EPO
polypeptides comprising amino acid structural motif AVARIANT1Xaa =
Any hydrophobic amino acidVARIANT2, 8Xaa = Any negatively charged
amino acid (same or different)VARIANT3,4,5,6,7Xaa = Any amino acid
(same or different)VARIANT9Xaa = Any polar amino acid 17Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 184PRTArtificial SequenceVariant of
isolated EPO polypeptides comprising amino acid structural motif
AVARIANT1Xaa = Any polar amino acidVARIANT2,3Xaa = Any negatively
charged amino acid (same or different)VARIANT4Xaa = Any hydrophobic
amino acid 18Xaa Xaa Xaa Xaa1 195PRTArtificial SequenceVariant of
isolated EPO polypeptides comprising amino acid structural motif
AVARIANT1Xaa = Any polar amino acidVARIANT2,4Xaa = Any negatively
charged amino acid (same or different)VARIANT3Xaa = Any amino
acidVARIANT5Xaa = Any hydrophobic amino acid 19Xaa Xaa Xaa Xaa Xaa1
5 206PRTArtificial SequenceVariant of isolated EPO polypeptides
comprising amino acid structural motif AVARIANT1Xaa = Any polar
amino acidVARIANT2, 5Xaa = Any negatively charged amino acid (same
or different)VARIANT3,4Xaa = Any amino acid (same or
different)VARIANT6Xaa = Any hydrophobic amino acid 20Xaa Xaa Xaa
Xaa Xaa Xaa1 5 217PRTArtificial SequenceVariant of isolated EPO
polypeptides comprising amino acid structural motif AVARIANT1Xaa =
Any polar amino acidVARIANT2,6Xaa = Any negatively charged amino
acid (same or different)VARIANT3,4,5Xaa = Any amino acid (same or
different)VARIANT7Xaa = Any hydrophobic amino acid 21Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5 228PRTArtificial SequenceVariant of isolated EPO
polypeptides comprising amino acid structural motif AVARIANT1Xaa =
Any polar amino acidVARIANT2,7Xaa = Any negatively charged amino
acid (same or different)VARIANT3,4,5,6Xaa = Any amino acid (same or
different)VARIANT8Xaa = Any hydrophobic amino acid 22Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 239PRTArtificial SequenceVariant of isolated
EPO polypeptides comprising amino acid structural motif
AVARIANT1Xaa = Any polar amino acidVARIANT2,8Xaa = Any negatively
charged amino acid (same or different)VARIANT3,4,5,6,7Xaa = Any
amino acid (same or different)VARIANT9Xaa = Any hydrophobic amino
acid 23Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 244PRTArtificial
SequenceVariant of isolated EPO polypeptides comprising amino acid
structural motif AVARIANT1,4Xaa = Any hydrophobic amino acid (same
or different)VARIANT2Xaa = Any negatively charged amino
acidVARIANT3Xaa = Any positively charged amino acid 24Xaa Xaa Xaa
Xaa1 255PRTArtificial SequenceVariant of isolated EPO polypeptides
comprising amino acid structural motif AVARIANT1,5Xaa = Any
hydrophobic amino acidVARIANT2Xaa = Any negatively charged amino
acidVARIANT3Xaa = Any polar amino acidVARIANT4Xaa = Any positively
charged amino acid 25Xaa Xaa Xaa Xaa Xaa1 5 264PRTArtificial
SequenceVariant of isolated EPO polypeptides comprising amino acid
structural motif AVARIANT1,4Xaa = Any hydrophobic amino
acidVARIANT2Xaa = Any positively charged amino acidVARIANT3Xaa =
Any negatively charged amino acid 26Xaa Xaa Xaa Xaa1
275PRTArtificial SequenceVariant of isolated EPO polypeptides
comprising amino acid structural motif AVARIANT1,5Xaa = Any
hydrophobic amino acidVARIANT2Xaa = Any positively charged amino
acidVARIANT3Xaa = Any polar amino acidVARIANT4Xaa = Any negatively
charged amino acid 27Xaa Xaa Xaa Xaa Xaa1 5 2811PRTArtificial
SequenceAmphipathic helix from pancreatic polypeptide 28Leu Arg Arg
Tyr Ile Asn Met Leu Thr Arg Pro 1 5 10 2920PRTArtificial
SequencePeptide C - loop AB and N-terminal portion of helix B
corresponding to EPO amino acids 38-57 29Asn Ile Thr Val Pro Asp
Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 1 5 10 15 Met Glu Val Gly
20 3025PRTArtificial SequencePeptide D - the C-terminal portion of
helix B corresponding to EPO amino acids 58-82 30Gln Gln Ala Val
Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala 1 5 10 15 Val Leu
Arg Gly Gln Ala Leu Leu Val 20 25 3120PRTArtificial SequencePeptide
E - a portion of the A-B loop consisting of a small cysteine loop
and a Beta-pleated sheet corresponding to EPO amino acids 28-47
31Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp 1
5 10 15 Thr Lys Val Asn 20 3223PRTArtificial SequencePeptide A -
consists of the N-terminal portion of Helix A corresponding to EPO
amino acids 1-23 32Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu
Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu 20
3316PRTArtificial SequencePeptide F - consists of EPO amino acids
14-19 33Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly
Cys 1 5 10 15 3414PRTArtificial SequencePeptide B - consists of EPO
amino acids 24-37 34Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu
Asn Glu 1 5 10 3514PRTArtificial SequenceGM-CSF helix A fragment
35Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu 1 5 10
3612PRTArtificial SequenceTPO helix A fragment 36Leu Ser Lys Leu
Leu Arg Asp Ser His Val Leu His 1 5 10 3716PRTArtificial
SequenceCNTF helix A fragment 37Lys Ile Arg Ser Asp Leu Thr Ala Leu
Thr Glu Ser Tyr Val Lys His 1 5 10 15 3817PRTArtificial SequenceLIF
helix B fragment 38Gly Thr Glu Lys Ala Lys Leu Val Glu Leu Tyr Arg
Ile Val Val Tyr 1 5 10 15 Leu3918PRTArtificial SequenceInterleukin
3 (IL-3) helix A fragment 39Ser Ile Met Ile Asp Glu Ile Ile His His
Leu Lys Arg Pro Pro Asn 1 5 10 15 Pro Leu4011PRTArtificial
SequencePeptide G - All exterior-presenting amino acids of helix B
of EPO 40Gln Glu Gln Leu Glu Arg Ala Leu Asn Ser Ser 1 5 10
4123PRTArtificial SequencePeptide J - A chimera peptide of Peptide
G linked to pancreatic polypeptide helix 41Gln Glu Gln Leu Glu Arg
Ala Leu Asn Ser Ser Leu Arg Arg Tyr Ile 1 5 10 15 Asn Met Leu Thr
Arg Thr Arg 20 427PRTArtificial SequenceBeta-pleated sheet found
within AB loop of EPO 42Cys Ser Leu Asn Glu Asn Ile1 5
4318PRTArtificial SequencePeptide I - A chimera peptide linking the
helix B exterior-presenting amino acids to the beta pleated sheet
found within the AB loop of the EPO 43Cys Ser Leu Asn Glu Asn Ile
Gln Glu Gln Leu Glu Arg Ala Leu Asn 1 5 10 15 Ser
Ser445PRTArtificial SequenceTerminal portion of helix C
corresponding to amino acids 111,112,113,116, and 118 of EPO 44Ala
Leu Gly Lys Ala1 5 4522PRTArtificial SequenceLoop CD-partial
corresponding to amino acids 112-133 of EPO 45Leu Gly Ala Gln Lys
Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala 1 5 10 15 Ala Pro Leu
Arg Thr Ile 20 464PRTArtificial Sequenceamino acid fragment of
Peptide F 46Glu Ala Lys Glu1 476PRTArtificial SequencePeptide H -
consists of EPO amino acids 61, 72, 75, 76, 77 47Ser Glu Leu Arg
Gly Gln1 5 4812PRTArtificial SequenceCalcitonin 48Ala Leu Ser Ile
Leu Val Leu Leu Gln Ala Gly Ser 1 5 10 4911PRTArtificial
SequenceCorticotrophin Releasing Hormone 49Val Ala Leu Leu Pro Cys
Pro Pro Cys Arg Ala 1 5 10 5011PRTArtificial SequenceBeta Endorphin
50Asn Ala Ile Ile Lys Asn Ala Tyr Lys Lys Gly 1 5 10
5111PRTArtificial SequenceGlucagon 51Gly Ser Trp Gln Arg Ser Leu
Gln Asp Thr Glu 1 5 10 5210PRTArtificial SequenceSecretin 52Gly Gly
Ser Ala Ala Arg Pro Ala Pro Pro 1 5 10 5311PRTArtificial
SequenceVIP 53Asn Ala Leu Ala Glu Asn Asp Thr Pro Tyr Tyr 1 5 10
5411PRTArtificial SequenceNP-Y 54Gly Ala Leu Ala Glu Ala Tyr Pro
Ser Lys Pro 1 5 10 5511PRTArtificial SequenceGNRH 55Gly Cys Ser Ser
Gln His Trp Ser Tyr Gly Leu 1 5 10 5611PRTArtificial
SequenceParathyroid Hormone 56Val Met Ile Val Met Leu Ala Ile Cys
Phe Leu 1 5 10 5711PRTArtificial SequenceCGRP 57Leu Ala Leu Ser Ile
Leu Val Leu Tyr Gln Ala 1 5 10 585PRTArtificial SequenceA motif
located proximal to the transmembrane domain of the Erythropoietin
Receptor ("EPOR") proteinVARIANT3Xaa = any amino acid 58Trp Ser Xaa
Trp Ser1 5
* * * * *