U.S. patent application number 10/460550 was filed with the patent office on 2005-09-08 for neuroprotective synergy of erythropoietin and insulin-like growth factors.
Invention is credited to Digicaylioglu, Murat, Lipton, Stuart A..
Application Number | 20050197284 10/460550 |
Document ID | / |
Family ID | 29739984 |
Filed Date | 2005-09-08 |
United States Patent
Application |
20050197284 |
Kind Code |
A9 |
Digicaylioglu, Murat ; et
al. |
September 8, 2005 |
Neuroprotective synergy of erythropoietin and insulin-like growth
factors
Abstract
The present invention provides a method of providing acute
neuroprotection by inducing the erythropoietin (EPO) signaling
pathway in neuronal cells close to or subsequent to the time of
excitatory insult; and inducing an insulin-like growth factor (IGF)
signaling pathway in the neuronal cells close to or subsequent to
the time of excitatory insult, thereby producing a synergistic
acute neuroprotective effect in the neuronal cells. The invention
also provides a method of preventing or reducing the severity of a
neurologic condition in a subject by administering to the subject
EPO or an active fragment or analog thereof at a dose of at most
2000 U/kg; and administering to the subject an IGF or an active
fragment or analog thereof, thereby providing neuroprotection and
preventing or reducing the severity of the neurologic condition.
Such a method can be used to prevent or reduce the severity of, for
example, Alzheimer's disease, Parkinson's disease, Huntington's
disease, epilepsy, amyotrophic lateral sclerosis, multiple
sclerosis, a movement disorder, HIV-associated dementia,
HIV-associated neuropathy, neuropathic pain, migraine, glaucoma,
drug addiction, drug withdrawal, drug dependency, depression or
anxiety.
Inventors: |
Digicaylioglu, Murat; (San
Diego, CA) ; Lipton, Stuart A.; (Rancho Santa Fe,
CA) |
Correspondence
Address: |
Cathryn Campbell
McDERMOTT, WILL & EMERY
Suite 700
4370 La Jolla Village Drive
San Diego
CA
92122
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0092444 A1 |
May 13, 2004 |
|
|
Family ID: |
29739984 |
Appl. No.: |
10/460550 |
Filed: |
June 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60388058 |
Jun 11, 2002 |
|
|
|
60458145 |
Mar 26, 2003 |
|
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Current U.S.
Class: |
514/7.7 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
21/02 20180101; A61K 38/30 20130101; A61P 25/36 20180101; A61P
25/00 20180101; A61P 25/06 20180101; A61P 25/16 20180101; A61P
27/06 20180101; A61P 25/22 20180101; A61P 31/18 20180101; A61P
25/04 20180101; A61P 25/14 20180101; A61P 25/28 20180101; A61P
25/30 20180101; A61K 38/30 20130101; A61P 43/00 20180101; A61P
25/24 20180101; A61K 2300/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/30; A61K
038/18 |
Goverment Interests
[0002] This application was made with government support under P01
HD29587, R01 NS43242 and NS43242 awarded by the National Institute
of Health. The government has certain rights in the invention.
Claims
We claim:
1. A method of providing acute neuroprotection, comprising: (a)
contacting neuronal cells with erythropoietin (EPO) or an active
fragment or analog thereof close to or subsequent to the time of
excitatory insult; and (b) contacting said neuronal cells with an
insulin-like growth factor (IGF) or an active fragment or analog
thereof close to or subsequent to the time of excitatory insult,
thereby producing a synergistic acute neuroprotective effect in
said neuronal cells.
2. The method of claim 1, wherein step (a) comprises contacting
said neuronal cells with EPO or an active fragment thereof.
3. The method of claim 2, wherein said EPO is human EPO or an
active fragment thereof.
4. The method of claim 3, comprising contacting said neuronal cells
with human EPO.
5. The method of claim 1, wherein step (a) comprises contacting
said neuronal cells with an EPO analog.
6. The method of claim 5, wherein said EPO analog is selected from
the group consisting of
3 GGTYSCHFGPLTWVCKPQGG; (SEQ ID NO: 7) GGDYHCRMGPLTWVCKPLGG; (SEQ
ID NO: 8) GGVYACRMCPITWVCSPLGG; (SEQ ID NO: 9)
VGNYMCHFGPITWVCRPGGG; (SEQ ID NO: 10) GGLYLCRFGPVTWDCGYKGG; and
(SEQ ID NO: 11) GGCRIGPITWVCGG. (SEQ ID NO: 12)
7. The method of claim 5, wherein said EPO analog is a
peptidomimetic.
8. The method of claim 5, wherein said EPO analog is a small
molecule.
9. The method of claim 1, wherein said EPO or active fragment or
analog thereof has at least 10-fold higher affinity for the EPO
receptor than native human EPO.
10. The method of claim 1, wherein said EPO or active fragment or
analog thereof is oligomeric.
11. The method of claim 10, wherein said oligomeric EPO or active
fragment or analog thereof is dimeric.
12. The method of claim 11, wherein said dimeric EPO or active
fragment or analog thereof comprises dimeric GGTYSCHFGPLTWVCKPQGG
(EMP1) (SEQ ID NO: 7).
13. The method of claim 1, wherein said EPO or active fragment or
analog thereof has a half-life greater than native human EPO.
14. The method of claim 1, wherein said EPO or active fragment or
analog thereof is hyper-glycosylated compared to native human
EPO.
15. The method of claim 14, wherein step (a) comprises contacting
said neuronal cells with Darbepoietin.
16. The method of claim 1, further comprising contacting said
neuronal cells with soluble EPO receptor.
17. The method of claim 1, wherein step (b) comprises contacting
said neuronal cells with an IGF or an active fragment thereof.
18. The method of claim 17, wherein said IGF or active fragment
thereof is IGF-I or an active fragment thereof.
19. The method of claim 18, wherein said IGF-I is human IGF-I or an
active fragment thereof.
20. The method of claim 19, comprising contacting said neuronal
cells with human IGF-I.
21. The method of claim 1, wherein step (b) comprises contacting
said neuronal cells with an IGF analog.
22. The method of claim 21, wherein said IGF analog is a
peptidomimetic.
23. The method of claim 21, wherein said IGF analog is a small
molecule.
24. The method of claim 21, wherein said IGF analog is an IGF-I
analog.
25. The method of claim 1, wherein said IGF or active fragment or
analog thereof has at least 10-fold higher affinity for the IGF-I
receptor than native human IGF-I.
26. The method of claim 1, wherein said IGF or active fragment or
analog thereof has an altered affinity for an IGF-binding protein
(IBP).
27. The method of claim 1, wherein said IGF or active fragment or
analog thereof has a half-life greater than native human IGF.
28. The method of claim 1, wherein step (a) and step (b) are in
vitro.
29. The method of claim 1, wherein step (a) and step (b) are in
vivo.
30. A method of preventing or reducing the severity of an acute
neurologic condition in a subject, comprising: (a) administering to
said subject EPO or an active fragment or analog thereof close to
or subsequent to the time of acute injury; and (b) administering to
said subject an IGF or an active fragment or analog thereof close
to or subsequent to the time of acute injury, thereby providing a
synergistic acute neuroprotective effect and preventing or reducing
the severity of the acute neurologic condition.
31. The method of claim 30, wherein said acute neurologic condition
is stroke.
32. The method of claim 30, wherein said acute neurologic condition
is head or spinal cord trauma.
33. The method of claim 30, wherein said acute neurologic condition
is seizure.
34. The method of claim 30, wherein step (a) comprises
administering EPO or an active fragment thereof.
35. The method of claim 34, wherein said EPO is human EPO or an
active fragment thereof.
36. The method of claim 35, comprising administering human EPO.
37. The method of claim 30, wherein step (a) comprises
administering an EPO analog.
38. The method of claim 37, wherein said EPO analog is selected
from the group consisting of
4 GGTYSCHFGPLTWVCKPQGG; (SEQ ID NO: 7) GGDYHCRMGPLTWVCKPLGG; (SEQ
ID NO: 8) GGVYACRMGPITWVCSPLGG; (SEQ ID NO: 9)
VGNYMCHFGPITWVCRPGGG; (SEQ ID NO: 10) GGLYLCRFGPVTWDCGYKGG; and
(SEQ ID NO: 11) GGCRIGPITWVCGG. (SEQ ID NO: 12)
39. The method of claim 37, wherein said EPO analog is a
peptidomimetic.
40. The method of claim 37, wherein said EPO analog is a small
molecule.
41. The method of claim 30, wherein said EPO or active fragment or
analog thereof has at least 10-fold higher affinity for the EPO
receptor than native human EPO.
42. The method of claim 30, wherein said EPO or active fragment or
analog thereof is oligomeric.
43. The method of claim 42, wherein said oligomeric EPO or active
fragment or analog thereof is dimeric.
44. The method of claim 43, wherein said dimeric EPO or active
fragment or analog thereof comprises dimeric GGTYSCHFGPLTWVCKPQGG
(SEQ ID NO: 7).
45. The method of claim 30, wherein said EPO or active fragment or
analog thereof has a half-life greater than native human EPO.
46. The method of claim 30, wherein said EPO or active fragment or
analog thereof is hyper-glycosylated compared to native human
EPO.
47. The method of claim 30, wherein step (a) comprises
administering Darbepoietin.
48. The method of claim 30, further comprising administering
soluble EPO receptor.
49. The method of claim 30, wherein step (b) comprises
administering an IGF or an active fragment thereof.
50. The method of claim 49, wherein said IGF is IGF-I or an active
fragment thereof.
51. The method of claim 50, wherein said IGF-I is human IGF-I or an
active fragment thereof.
52. The method of claim 50, wherein said IGF-I is human IGF-I.
53. The method of claim 30, wherein step (b) comprises
administering an IGF analog.
54. The method of claim 53, wherein said IGF analog is a
peptidomimetic.
55. The method of claim 53, wherein said ICF analog is a small
molecule.
56. The method of claim 53, wherein said IGF analog is an IGF-I
analog.
57. The method of claim 30, wherein said IGF or active fragment or
analog thereof has at least 10-fold higher affinity for the IGF-I
receptor than native human IGF-I.
58. The method of claim 30, wherein said IGF or active fragment or
analog thereof has an altered affinity for an IGF-binding protein
(IBP).
59. The method of claim 30, wherein said IGF or active fragment or
analog thereof has a half-life greater than native human IGF.
60. A method of preventing or reducing the severity of a neurologic
condition in a subject, comprising: (a) administering to said
subject EPO or an active fragment or analog thereof at a dose of at
most 2000 U/kg; and (b) administering to said subject an IGF or an
active fragment or analog thereof, thereby providing
neuroprotection and preventing or reducing the severity of the
neurologic condition.
61. The method of claim 60, wherein said neurologic condition is
selected from the group consisting of Alzheimer's disease,
Parkinson's disease, Huntington's disease, epilepsy, amyotrophic
lateral sclerosis, multiple sclerosis, a movement disorder,
HIV-associated dementia, HIV-associated neuropathy, neuropathic
pain, migraine, glaucoma, drug addiction, drug withdrawal, drug
dependency, depression and anxiety.
62. The method of claim 60, wherein step (a) comprises
administering EPO or an active fragment thereof.
63. The method of claim 62, wherein said EPO is human EPO or an
active fragment thereof.
64. The method of claim 63, comprising administering human EPO.
65. The method of claim 60, wherein step (a) comprises
administering an EPO analog.
66. The method of claim 65, wherein said EPO analog is selected
from the group consisting of
5 GGTYSCHFGPLTWVCKPQGG; (SEQ ID NO: 7) GGDYHCRMGPLTWVCKPLGG; (SEQ
ID NO: 8) GGVYACRMGPITWVCSPLGG; (SEQ ID NO: 9)
VGNYMCHFGPITWVCRPGGG; (SEQ ID NO: 10) GGLYLCRFGPVTWDCGYKGG; and
(SEQ ID NO: 11) GGCRIGPITWVCGG. (SEQ ID NO: 12)
67. The method of claim 65, wherein said EPO analog is a
peptidomimetic.
68. The method of claim 65, wherein said EPO analog is a small
molecule.
69. The method of claim 60, wherein said EPO or active fragment or
analog thereof has at least 10-fold higher affinity for the EPO
receptor than native human EPO.
70. The method of claim 60, wherein said EPO or active fragment or
analog thereof is oligomeric.
71. The method of claim 70, wherein said oligomeric EPO or active
fragment or analog thereof is dimeric.
72. The method of claim 71, wherein said dimeric EPO or active
fragment or analog thereof comprises dimeric GGTYSCHFGPLTWVCKPQGG
(SEQ ID NO: 7).
73. The method of claim 60, wherein said EPO or active fragment or
analog thereof has a half-life greater than native human EPO.
74. The method of claim 60, wherein said EPO or active fragment or
analog thereof is hyper-glycosylated compared to native human
EPO.
75. The method of claim 74, wherein step (a) comprises
administering Darbepoietin.
76. The method of claim 60, further comprising administering
soluble EPO, receptor.
77. The method of claim 60, wherein step (b) comprises
administering an IGF or an active fragment thereof.
78. The method of claim 77, wherein said IGF is IGF-I or an active
fragment thereof.
79. The method of claim 78, wherein said IGF-I is human IGF-I or an
active fragment thereof.
80. The method of claim 79, wherein said IGF-I is human IGF-I.
81. The method of claim 60, wherein step (b) comprises
administering an IGF analog.
82. The method of claim 81, wherein said IGF analog is a
peptidomimetic.
83. The method of claim 81, wherein said IGF analog is a small
molecule.
84. The method of claim 81, wherein said IGF analog is an IGF-I
analog.
85. The method of claim 60, wherein said IGF or active fragment or
analog thereof has at least 10-fold higher affinity for the IGF-I
receptor than native human IGF-I.
86. The method of claim 60, wherein said IGF or active fragment or
analog thereof has an altered affinity for an IGF-binding protein
(IBP).
87. The method of claim 60, wherein said IGF or active fragment or
analog thereof has a half-life greater than native human IGF.
88. A method of preventing or reducing the severity of a cerebral
neurologic condition in a subject, comprising: (a) transnasally
administering to said subject EPO or an active fragment or analog
thereof at a dose of at most 2000 U/kg; and (b) transnasally
administering to said subject an IGF or an active fragment or
analog thereof, thereby providing acute neuroprotection and
preventing or reducing the severity of the cerebral neurologic
condition.
89. A method of differentiating neuronal precursor cells to produce
an enriched neuronal cell population, comprising (a) contacting the
neuronal precursor cells with EPO or an active fragment or analog
thereof; and (b) contacting the neuronal precursor cells with IGF
or an active fragment or analog thereof, thereby differentiating
the neuronal precursor cells to produce an enriched cell population
containing at least 70% neurons.
90. The method of claim 89, which produces an enriched cell
population containing at least 80% neurons.
91. The method of claim 89, which produces an enriched cell
population containing at least 90% neurons.
92. The method of claim 89, further comprising transplanting said
cells into a subject.
93. The method of claim 92, wherein said cells are transplanted
into a tissue or organ selected from the group consisting of brain,
eye and spinal cord.
94. A method of preventing or reducing the severity of a neurologic
condition in a subject, comprising intranasally administering to
said subject EPO or an active fragment or analog thereof, thereby
preventing or reducing the severity of the neurologic
condition.
95. The method of claim 94, wherein said EPO or an active fragment
or analog thereof is administered at a dose of at most 2000
U/kg.
96. The method of claim 94, comprising administering EPO or an
active fragment thereof.
97. The method of claim 96, wherein said EPO is human EPO or an
active fragment thereof.
98. The method of claim 97, comprising administering human EPO.
99. The method of claim 94, comprising administering an EPO
analog.
100. The method of claim 99, wherein said EPO analog is selected
from the group consisting of
6 GGTYSCHFGPLTWVCKPQGG; (SEQ ID NO: 7) GGDYHCRMGPLTWVCKPLGG; (SEQ
ID NO: 8) GGVYACRMGPITWVCSPLGG; (SEQ ID NO: 9)
VGNYMCHFGPITWVCRPGGG; (SEQ ID NO: 10) GGLYLCRFGPVTWDCGYKGG; and
(SEQ ID NO: 11) GGCRIGPITWVCGG. (SEQ ID NO: 12)
101. The method of claim 99, wherein said EPO analog is a
peptidomimetic.
102. The method of claim 99, wherein said EPO analog is a small
molecule.
103. The method of claim 94, wherein said EPO or active fragment or
analog thereof has at least 10-fold higher affinity for the EPO
receptor than native human EPO.
104. The method of claim 94, wherein said EPO or active fragment or
analog thereof is oligomeric.
105. The method of claim 104, wherein said oligomeric EPO or active
fragment or analog thereof is dimeric.
106. The method of claim 105, wherein said dimeric EPO or active
fragment or analog thereof comprises dimeric GGTYSCHFGPLTWVCKPQGG
(SEQ ID NO: 7).
107. The method of claim 94, wherein said EPO or active fragment or
analog thereof has a half-life greater than native human EPO.
108. The method of claim 94, wherein said EPO or active fragment or
analog thereof is hyper-glycosylated compared to native human
EPO.
109. The method of claim 94, comprising administering
Darbepoietin.
110. The method of claim 94, further comprising administering
soluble EPO receptor.
111. The method of claim 94, wherein said neurologic condition is
an acute neurologic condition.
112. The method of claim 94, wherein said neurologic condition is a
chronic neurologic condition.
113. The method of claim 94, wherein said neurologic condition is
selected from the group consisting of stroke, head or spinal cord
trauma, Alzheimer's disease, Parkinson's disease, Huntington's
disease, epilepsy, amyotrophic lateral sclerosis, multiple
sclerosis, a movement disorder, dementia, HIV-associated dementia,
fronto-temporal dementia, HIV-associated neuropathy, chronic pain,
neuropathic pain, migraine, glaucoma, drug addiction, drug
withdrawal, drug dependency, depression and anxiety.
Description
[0001] This application claims benefit of the filing date of U.S.
Provisional Application No. 60/388,058, filed Jun. 11, 2002, and of
U.S. Provisional Application No. 60/458,145, filed Mar. 26, 2003,
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates to the prevention and treatment of
acute and chronic neurodegenerative conditions, and to
erythropoietin and insulin-like growth factors and analogs of these
factors.
BACKGROUND INFORMATION
[0004] For a variety of serious neurodegenerative diseases, there
exist no effective therapies or cures. For example, Parkinson's
disease is a progressive and ultimately fatal neurodegenerative
disorder characterized by loss of the pigmented dopaminergic
neurons of the substantia nigra. The symptoms of Parkinson's
disease often can be managed initially by administration of L-DOPA,
the immediate precursor of dopamine. However, reduced efficacy of
L-DOPA treatment typically occurs over time. Programmed cell death
(apoptosis) has been implicated in this neurodegenerative
disorder.
[0005] In Alzheimer's disease, the most common neurodegenerative
disease and most frequent cause of dementia, progressive failure of
memory and degeneration of temporal and parietal association cortex
result in speech impairment and loss of coordination, and, in some
cases, emotional disturbance. Alzheimer's disease generally
progresses over many years, with patients gradually becoming
immobile, emaciated and susceptible to pneumonia.
[0006] Neuroprotective therapy has been sought for a variety of
acute and chronic neurological conditions, including stroke,
Parkinson's disease, Alzheimer's disease, Huntington's disease,
amyotrophic lateral sclerosis, multiple sclerosis, epilepsy and
pain. Present therapies are relatively ineffective or are
accompanied by unwanted side effects. In particular, erythropoietin
(EPO) can be neuroprotective when administered in high doses;
however, such doses also promote the formation of new red blood
cells, consequently causing side effects such as "sludging" of the
blood and leading to increased risk of stroke. Thus, there is a
need for novel methods of using erythropoietin to achieve
neuroprotection, that do not rely on excessively high doses of the
factor. The present invention satisfies this need and provides
related advantages as well.
SUMMARY OF THE INVENTION
[0007] The present invention provides a method of providing acute
neuroprotection by inducing the erythropoietin (EPO) pathway in
neuronal cells close to or subsequent to the time of excitatory
insult; and inducing an insulin-like growth factor (IGF) pathway in
the neuronal cells close to or subsequent to the time of excitatory
insult, thereby producing a synergistic acute neuroprotective
effect in the neuronal cells.
[0008] The present invention further provides a method of providing
acute neuroprotection by contacting neuronal cells with EPO or an
active fragment or analog thereof close to or subsequent to the
time of excitatory insult; and contacting the neuronal cells with
an IGF or an active fragment or analog thereof close to or
subsequent to the time of excitatory insult, thereby producing a
synergistic acute neuroprotective effect in the neuronal cells.
[0009] Many forms of erythropoietin, as well as active fragments
and analogs thereof, can be useful in the methods of the invention.
In one embodiment, neuronal cells are contacted with EPO or an
active fragment thereof, for example, with human EPO or an active
fragment thereof. In another embodiment, neuronal cells are
contacted with an EPO analog, which can be, without limitation, a
peptide, peptidomimetic, small molecule or nucleic acid EPO analog.
In further embodiments, the invention is practiced with an EPO
analog that includes the amino acid sequence GGTYSCHFGPLTWVCKPQGG
(SEQ ID NO: 7); GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 8);
GGVYACRMGPITWVCSPLGG (SEQ ID NO: 9); VGNYMCHFGPITWVCRPGGG (SEQ ID
NO: 10); GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 11); or GGCRIGPITWVCGG
(SEQ ID NO: 12).
[0010] In another embodiment, the invention is practiced with EPO,
or an active fragment or analog thereof, which has at least 10-fold
higher affinity for the EPO receptor than native human EPO. In
another embodiment, the invention is practiced with EPO or an
active fragment or analog thereof which is oligomeric, for example,
dimeric. As an example, such a dimeric form of EPO can be a dimer
in which each monomer contains the amino acid sequence
GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7).
[0011] In a further embodiment, the invention is practiced with EPO
or an active fragment or analog thereof that has a half-life
greater than the half-life of native human EPO. In an additional
embodiment, the invention is practiced with EPO or an active
fragment or analog thereof that is hyper-glycosylated-compared to
native human EPO. In yet a further embodiment, the invention is
practiced by contacting neuronal cells with Darbepoietin. In any
embodiment of the invention, soluble EPO receptor can be optionally
included, for example, to prolong the half-life of EPO or an active
fragment or analog thereof.
[0012] A variety of forms of IGF and active fragments and analogs
thereof also are useful in the invention. In one embodiment, the
invention is practiced by contacting neuronal cells with an IGF or
an active fragment thereof, for example, IGF-I or an active
fragment thereof. In an additional embodiment, the invention is
practiced by contacting neuronal cells with human IGF-I or an
active fragment thereof. In further embodiments, the invention is
practiced with an IGF analog such as a peptide, peptidomimetic,
small molecule or nucleic acid IGF analog including, without
limitation, peptide, peptidomimetic, small molecule and nucleic
acid IGF-I analogs.
[0013] In another embodiment, the invention is practiced with an
IGF or active fragment or analog thereof which has at least 10-fold
higher affinity for the IGF-I receptor than native human IGF-I. In
a further embodiment, the invention is practiced with an IGF or
active fragment or analog thereof which has an altered affinity for
an IGF-binding protein (IBP). In yet a further embodiment, the
invention is practiced with an IGF or active fragment or analog
thereof which has a half-life greater than the half-life of native
human IGF-I. In the methods of the invention, the two contacting
steps can be performed in vitro or in vivo and further can be
performed simultaneously or in any order.
[0014] Also provided herein is a method of preventing or reducing
the severity of an acute neurologic condition in a subject by
administering to the subject EPO or an active fragment or analog
thereof close to or subsequent to the time of acute injury; and
administering to the subject an IGF or an active fragment or analog
thereof close to or subsequent to the time of acute injury, thereby
providing a synergistic acute neuroprotective effect and preventing
or reducing the severity of the acute neurologic condition. Such an
acute neurologic condition can be, without limitation, stroke, head
or spinal cord trauma, or seizure.
[0015] A method of the invention for preventing or reducing the
severity of an acute neurologic condition can be practiced, for
example, with EPO or an active fragment thereof, such as human EPO
or an active fragment thereof. A method of the invention also can
be practiced with an EPO analog, which can be, without limitation,
a peptide, peptidomimetic, small molecule or nucleic acid analog.
In particular embodiments, the invention is practiced with an EPO
analog containing one of the following amino acid sequences:
GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7); GGDYHCRMGPLTWVCKPLGG (SEQ ID
NO: 8); GGVYACRMGPITWVCSPLGG (SEQ ID NO: 9); VGNYMCHFGPITWVCRPGGG
(SEQ ID NO: 10); GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 11); or
GGCRIGPITWVCGG (SEQ ID NO: 12).
[0016] In one embodiment, the invention is practiced with EPO or an
active fragment or analog thereof which has at least 10-fold higher
affinity for the EPO receptor than native human EPO. In another
embodiment, the invention is practiced with EPO or an active
fragment or analog thereof which is oligomeric, for example,
dimeric. As an example, a method of the invention can be practiced
with a dimeric form of EPO in which each monomer contains the amino
acid sequence GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7).
[0017] In a further embodiment, the invention is practiced with EPO
or an active fragment or analog thereof which has a half-life
greater than the half-life of native human EPO. Such a form of EPO
can be hyper-glycosylated as compared to native human EPO and
further can be, for example, Darbepoietin. The methods of the
invention also optionally include the step of administering soluble
EPO receptor to the subject.
[0018] A variety of forms of IGF and active fragments and analogs
thereof are useful in the invention. In one embodiment, the
invention is practiced by administering IGF or an active fragment
thereof, for example, IGF-I or an active fragment thereof. In an
additional embodiment, the invention is practiced by administering
human IGF-I or an active fragment thereof. The invention also can
be practiced by administering an IGF analog such as a peptide,
peptidomimetic, small molecule or nucleic acid analog including,
but not limited to, a variety of IGF-I analogs.
[0019] In one embodiment, a method of the invention for preventing
or reducing the severity of an acute neurological condition is
practiced with an IGF or active fragment or analog thereof which
has at least 10-fold higher affinity for the IGF-I receptor than
native human IGF-I. In another embodiment, the invention is
practiced with an IGF or active fragment or analog thereof which
has an altered affinity for an IGF-binding protein. In a further
embodiment, the invention is practiced with an IGF or active
fragment or analog thereof which has a half-life greater than the
half-life of native human IGF. In the methods of the invention, EPO
and IGF, or active fragments or analogs thereof, can be
administered simultaneously or in any order and in the same or
different pharmaceutical compositions.
[0020] Also provided by the invention is a method of preventing or
reducing the severity of a neurologic condition in a subject by
administering to the subject EPO or an active fragment or analog
thereof at a dose of at most 2000 U/kg; and administering to the
subject an IGF or an active fragment or analog thereof, thereby
providing neuroprotection and preventing or reducing the severity
of the neurologic condition. The EPO and IGF, or active fragments
or analogs thereof, can be administered to the subject
simultaneously or in any order and in the same or different
pharmaceutical compositions. A variety of acute and chronic
neurologic conditions can be treated according to a method of the
invention including, but not limited to, Alzheimer's disease;
Parkinson's disease; Huntington's disease; epilepsy; amyotrophic
lateral sclerosis; multiple sclerosis; movement disorders;
HIV-associated dementia; HIV-associated neuropathy; retinal
degeneration including macular degeneration and light-induced
retinal degeneration such as photoreceptor degeneration;
neuropathic pain; migraine; glaucoma; drug addiction; drug
withdrawal; drug dependency; and depression or anxiety.
[0021] Various forms of EPO and active fragments and analogs
thereof are useful for preventing or reducing the severity of a
neurologic condition according to a method of the invention. As an
example, the invention can be practiced with EPO or an active
fragment thereof such as human EPO or an active fragment thereof. A
method of the invention also can be practiced with an EPO analog,
which can be, without limitation, a peptide, peptidomimetic, small
molecule or nucleic acid analog. In particular embodiments, the
invention is practiced with an EPO analog containing one of the
following amino acid sequences: GGTYSCHFGPLTWVCKPQGG (SEQ ID NO:
7); GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 8); GGVYACRMGPITWVCSPLGG (SEQ
ID NO: 9); VGNYMCHFGPITWVCRPGGG (SEQ ID NO: 10);
GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 11); or GGCRIGPITWVCGG (SEQ ID NO:
12).
[0022] In one embodiment, a method of the invention for preventing
or reducing the severity of a neurologic condition is practiced
with EPO or an active fragment or analog thereof which has at least
10-fold higher affinity for the EPO receptor than native human EPO.
In another embodiment, the invention is practiced with EPO or an
active fragment or analog thereof which is oligomeric, for example,
dimeric. The invention can be practiced, for example, with a
dimeric form of EPO in which each monomer contains the amino acid
sequence GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7). In a further
embodiment, the invention is practiced with EPO or an active
fragment or analog thereof which has a half-life greater than the
half-life of native human EPO. Such a form of EPO can be
hyper-glycosylated as compared to native human EPO and further can
be, for example, Darbepoietin. The methods of the invention further
optionally include the step of administering soluble EPO receptor
to the subject.
[0023] A variety of forms of IGF and active fragments and analogs
thereof also are useful in the invention. In one embodiment, the
invention is practiced by administering an IGF or an active
fragment thereof, for example, IGF-I or an active fragment thereof.
In an additional embodiment, the invention is practiced by
administering human IGF-I or an active fragment thereof. In a
further embodiment, the invention is practiced by administering an
IGF analog such as a peptide, peptidomimetic, small molecule or
nucleic acid analog including, but not limited to, a variety of
IGF-I analogs.
[0024] In one embodiment, a method of the invention for preventing
or reducing the severity of a neurological condition is practiced
with an IGF or active fragment or analog thereof which has at least
10-fold higher affinity for the IGF-I receptor than native human
IGF-I. In another embodiment, the invention is practiced with an
IGF or active fragment or analog thereof which has an altered
affinity for an IGF-binding protein. In a further embodiment, the
invention is practiced with an IGF or active fragment or analog
thereof which has a half-life greater than the half-life of native
human IGF.
[0025] The present invention also provides a method of preventing
or reducing the severity of a cerebral neurologic condition in a
subject by transnasally administering to the subject EPO or an
active fragment or analog thereof at a dose of at most 2000 U/kg;
and transnasally administering to the subject an IGF or an active
fragment or analog thereof, thereby providing acute neuroprotection
and preventing or reducing the severity of the neurologic
condition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows the nucleic acid and corresponding amino acid
sequence of human erythropoietin (EPO). (A) Nucleotide sequence
(SEQ ID NO: 1) of human EPO (Genbank accession X02157, version
X02157.1). The coding sequence is shown as nucleotides 182 to 763
of SEQ ID NO: 1, with nucleotides 263 to 763 of SEQ ID NO: 1
encoding mature EPO. (B) The corresponding amino acid sequence (SEQ
ID NO: 2) of human EPO. Residues 1 to 27 make up the signal
sequence, with residues 28 to 193 constituting mature human EPO.
(C) The amino acid sequence (SEQ ID NO: 3) of mature human EPO.
[0027] FIG. 2 shows the nucleic acid and corresponding amino acid
sequence of human insulin-like growth factor-I (IGF-I). (A) The
nucleotide sequence (SEQ ID NO: 4) human IGF-I (Genbank accession
X00173, version X00173.1). The coding sequence is shown as
nucleotides 12 to 473 of SEQ ID NO: 4. (B) The corresponding amino
acid sequence (SEQ ID NO: 5) of human IGF-I. Residues 1 to 21 make
up the signal sequence; residues 22 to 48 constitute a propeptide;
residues 49 to 118 constitute mature IGF-I; and residues 119 to 153
make up the carboxy-terminal propeptide (domain E). (C) Mature
human IGF-I (SEQ ID NO: 6) is shown schematically.
[0028] FIG. 3 shows that simultaneous co-administration of EPO and
IGF-I ameliorates neuronal apoptosis induced by the excitotoxin
N-methyl-D-aspartate (NMDA) acting at the NMDA receptor, a
glutamate receptor in the brain. Overstimulation of this receptor
mediates, at least in part, a wide range of acute and chronic
neurologic disorders by permitting excessive Ca.sup.2+ influx and
subsequent free radical formation (nitric oxide and reactive oxygen
species). Cultured rat cerebrocortical cells were incubated with
NMDA (200 .mu.M) for 20 minutes, a paradigm known to induce
apoptotic neuronal cell death (Bonfoco et al., PNAS 92:7162-7166
(1995); Budd et al., PNAS 97:6161-6166 (2000)). EPO (10 U/ml),
IGF-I (100 ng/ml), or 10 U/ml EPO plus 100 ng/ml IGF-I were applied
three hours prior to or concurrent with NMDA exposure. Apoptotic
neurons are represented by the percentage of MAP2-positive cells
co-labeled by TUNEL 16 hours after NMDA exposure. In this and
subsequent figures, results are mean.+-.S.E. (n=3-5). p<0.05 by
ANOVA versus NMDA (*) or versus simultaneous addition of NMDA and
EPO or IGF-I (.dagger.).
[0029] FIG. 4 shows that inhibition of PI3-kinase abrogates the
anti-apoptotic effects of combined EPO and IGF-I treatment.
Cerebrocortical cultures were exposed to 10 .mu.M LY294002 (gray
bars) for 30 minutes prior to incubation with EPO, IGF-I or EPO and
IGF-I for three hours, at which time cells were subject to NMDA
exposure. Neuronal apoptosis was assessed 16 hours after NMDA
exposure by determining the percentage of MAP2 positive cells that
were also TUNEL positive. *, p<0.05 or .dagger., p<0.01 by
ANOVA versus same treatment plus LY294002.
[0030] FIG. 5 shows that EPO and IGF-I each signal through
PI3-kinase. (A) Treatment with EPO induces association of the p85
subunit of PI3-kinase with the EPO-R. Cerebrocortical cells were
stimulated with 5 or 10 U/ml EPO for 30 minutes. EPO-R was
immunoprecipitated from total cell lysates and separated on a
SDS-polyacrylamide gel. The blot was then probed with anti-EPO-R
antibody, stripped and reblotted with anti-p85 antibody. (B)
Cerebrocortical cells were stimulated with EPO (5 U/ml, upper
panel) or IGF-I (100 ng/ml, lower panel) for the indicated amount
of time. Whole cell lysates were run on SDS-polyacrylamide gels;
blots were probed with anti-phospho-p85 antibody, stripped, and
then reprobed with anti-p85 antibody.
[0031] FIG. 6 shows that combined treatment with EPO and IGF-I
induces Akt phosphorylation. Mixed neuronal/glial rat
cerebrocortical cultures were treated for 20 minutes with 10 U/ml
EPO, 100 ng/ml IGF-I, or both EPO and IGF-I, with or without
simultaneous application of 200 .mu.M NMDA. NIH3T3 cells stimulated
with 100 .mu.g/ml platelet-derived growth factor (PDGF) for 10
minutes served as a positive control. After three hours, cells were
lysed, and whole-cell lysates subjected to immunoblot analysis with
anti-phospho-Akt antibody; the blot was stripped and reprobed with
an anti-Akt antibody.
[0032] FIG. 7 shows that Akt contributes to neuroprotection
mediated by combined treatment with EPO and IGF-I. Cerebrocortical
cultures were exposed to an adenoviral vector encoding a wild-type
(wt) or dominant-negative (dn) form of Akt for four hours. Other
cultures were coinfected with wt and dn Akt in a molar ratio of 1:1
or 2:1 (wt-Akt:dn-Akt). Thirty-six hours later, cells were
incubated with 10 U/ml EPO, 100 ng/ml IGF-I, or both EPO/IGF-I,
with or without simultaneous application of 200 .mu.M NMDA. After
an additional 16 hours, cells staining positive for TUNEL (to
identify apoptotic cells) and MAP2 (to specifically identify
neurons) were scored as apoptotic neurons. *, p<0.05 by ANOVA
versus EPO/IGF-I plus NMDA; t, p<0.01 versus EPO/IGF-I plus NMDA
with or without wt-Akt.
[0033] FIG. 8 shows that combined treatment with EPO and IGF-I
promotes long-term neuronal survival downstream of caspase
activation. (A) Survival of neurons in mixed neuronal-glial
cultures after exposure to 200 .mu.M NMDA for 20 minutes (black
bars). EPO/IGF-I treatment (10 U/ml and 100 ng/ml, respectively) at
the time of NMDA exposure promoted neuronal survival for up to 48
hours after the insult (white bars; *, p<0.05). (B)
Cerebrocortical cultures were exposed to NMDA or EPO/IGF-I, or
concurrently exposed to EPO/IGF-I and NMDA. Culture lysates were
subjected to immunoprecipitation with an antibody specifically
reactive with the active form of caspase-3. The immunoprecipitates
were separated by electrophoresis and transferred to nitrocellulose
membranes. EPO/IGF-I (with or without NMDA exposure) led to an
increase in the amount of XIAP associated with active caspase-3.
(C) Densitometry of XIAP, a protein inhibitor of apoptosis,
revealed a 2.5 to 3-fold increase in the amount of XIAP bound to
active caspase-3. (D) Caspase-3-like activity is reduced by
EPO/IGF-I. Rat cerebrocortical cells were infected with an
adenoviral vector encoding dominant-negative Akt (dn-Akt) or
control vector. After 36 hours, cultures were incubated with EPO
(10 U/ml), IGF-I (100 ng/ml) or EPO/IGF-I. Cells were exposed to
200 .mu.M NMDA three hours after or simultaneously with EPO/IGF-I
incubation. Caspase activity in cell lysates was assessed 16 hours
after NMDA exposure and is shown as relative DEVDase activity
expressed as percent increase over control levels. (E)
Neuron-enriched cultures were exposed to NMDA insult (200 .mu.M for
20 minutes) with or without EPO/IGF-I, and neuronal survival
assessed at the indicated time points. Even in the absence of
non-neuronal cells, EPO/IGF-I was equally effective in promoting
long-term neuronal survival after exposure to NMDA.
[0034] FIG. 9 shows that combined treatment with EPO and IGF-I can
be effective in promoting neuronal survival when applied several
hours following NMDA exposure. Cells were exposed to NMDA as
described above and treated with 10 U/ml EPO and 100 ng/ml IGF-I
three hours or one hour prior to NMDA exposure, at the time of NMDA
exposure, or one hour, two hours, three hours or five hours
subsequent to NMDA exposure. The percentage of apoptotic neurons
was determined as described above.
[0035] FIG. 10 shows that EPO and IGF-I together are more effective
at reducing neuronal apoptosis than when used individually. Cells
were exposed to 200 .mu.M NMDA as described above and
simultaneously treated with EPO alone, IGF-I alone, or EPO in
combination with IGF-I at varying concentrations. Incubation 1: 0.5
U/ml EPO and 1 ng/ml IGF-l, alone or in combination. Incubation 2:
1.0 U/ml EPO and 10 ng/ml IGF-l, alone or in combination.
Incubation 3: 2.0 U/ml EPO and 20 ng/ml IGF-l, alone or in
combination. Incubation 4: 5.0 U/ml EPO and 50 ng/ml IGF-1, alone
or in combination. Incubation 5: 10 U/ml EPO and 20 ng/ml IGF-1,
alone or in combination. Incubation 6: 20 U/ml EPO and 400 ng/ml
IGF-1, alone or in combination. The percentage of apoptotic neurons
was determined 18 hours following NMDA exposure as described above.
Incubations 3, 4 and 5 produced results with the EPO/IGF
combination which were significantly different (p<0.02) from
treatment with each cytokine alone.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention is directed to the surprising
discovery that erythropoietin and insulin-like growth factor (IGF)
together mediate more rapid neuroprotection than either factor
mediates alone, indicating that combined EPO and IGF treatment can
be useful in providing neuroprotection in acute neurological
conditions such as stroke, trauma and seizure. The invention also
is directed to the surprising discovery that, together, EPO and
IGF-I synergize to reduce apoptosis in neurons, providing enhanced
neuroprotection at reduced concentrations, indicating that reduced
concentrations of EPO and IGF-I can be useful in treating acute and
chronic neurological disorders such as, without limitation,
Alzheimer's disease, amyotrophic lateral sclerosis (ALS),
Huntington's disease, glaucoma, HIV-associated dementia, multiple
sclerosis; Parkinson's disease, and neuropathic pain.
[0037] As disclosed herein in Example I, the neuroprotective
effects of concurrent EPO and IGF-I administration were compared to
treatment with EPO or IGF-I individually in rat primary
cerebrocortical cultures exposed to NMDA, an excitatory insult.
Neuronal apoptosis was quantified by double labeling for TUNEL
reactivity, which is indicative of apoptosis, and a neuron-specific
marker, microtubule associated protein-2 (MAP2) 16 hours after NMDA
insult. As shown in FIG. 3, a brief 20 minute exposure to NMDA
produced an apoptotic appearance and TUNEL reactivity in 76+7% of
MAP2-labeled neurons. Preincubation for three hours with either EPO
(10 U/ml) or IGF-I (100 ng/ml) alone significantly attenuated
neuronal apoptosis (p<0.05); however, treatment of neurons with
EPO or IGF-I individually at the time of NMDA insult did not
significantly reduce cell death (see FIG. 3). In contrast,
application of EPO together with IGF-I at the time of NMDA exposure
revealed a synergistic effect between the two factors, which
together reduced neuronal apoptosis from about 76% to about 35%. As
further shown in FIG. 9, combined EPO/IGF-I treatment dramatically
reduced apoptosis when applied concurrently with NMDA exposure or
when applied one hour, two hours or three hours following NMDA
exposure. These results demonstrate that IGF-I and EPO synergize to
mediate more rapid neuroprotection than either factor mediates in
the absence of the other.
[0038] As further disclosed herein in Example IIA, rat
cerebrocortical neurons were preincubated for three hours with EPO,
IGF-I, or EPO in combination with IGF-I (EPO/IGF-I) in the presence
or absence of 10 .mu.M LY294002, a specific PI3-kinase inhibitor.
The results shown in FIG. 4 demonstrate that LY294002 abolished the
neuroprotective effect of EPO and IGF-I either alone or in
combination (p<0.05) but did not itself cause neuronal apoptosis
in cerebrocortical cultures or increase the amount of apoptosis
induced by NMDA, indicating that PI3-kinase activity is required
for EPO- and IGF-I-mediated neuroprotection. Furthermore,
individual treatment with either EPO or IGF-I induced
phosphorylation of the p85 regulatory subunit of PI3-kinase in a
time-dependent manner; IGF-I incubation resulted in maximal
phosphorylation of the p85 subunit after 10 minutes and EPO-induced
phosphorylation was observed after 20 to 30 minutes (Example IIB;
FIG. 5A). Given that phosphorylation of the p85 regulatory subunit
is known to activate PI3-kinase by leading to release of the
catalytic subunit, these results indicate that both EPO and IGF-I
activate PI3-kinase.
[0039] As further disclosed herein in Example III, EPO and IGF-I
treatment of neuronal cells can cooperatively activate Akt kinase,
which is a kinase activated downstream of P13-kinase-mediated
production of 3' phospholipids. The Akt kinase is phosphorylated at
two critical sites, serine-473 and threonine-308. As shown in FIG.
6A, a three hour incubation with EPO or IGF-I alone resulted in
moderate Akt activation, as evidenced by increased
phospho-serine-473 Akt, while co-incubation with maximally
effective concentrations of EPO and IGF-I together resulted in a
much larger increase in phospho-serine-473 Akt. Furthermore,
cerebrocortical cultures expressing dominant negative Akt (dn-Akt)
and incubated with EPO/IGF-I displayed significantly higher levels
of NMDA-induced neuronal apoptosis in comparison with uninfected
cultures or cultures infected with wild type Akt (wt-Akt;
p<0.01). In sum, these results indicate that Akt serine-473
phosphorylation is synergistically induced by the combination of
EPO and IGF-I and that Akt phosphorylation and activation play a
role in the neuroprotection mediated by combined EPO/IGF-I
treatment.
[0040] The results disclosed in Example VA show that treatment with
EPO in combination with IGF-I prevents NMDA-induced neurotoxicity
in cerebrocortical cultures in the presence of the active form of
caspase-3. In particular, this result shows that prolonged survival
of neurons following NMDA exposure and EPO/IGF-I treatment
indicated that EPO/IGF-I neuroprotection occurs, at least in part,
downstream of initial caspase-3 activation, which is typically
associated with neuronal apoptosis within 16 hours of NMDA insult.
Furthermore, as shown in FIG. 8A, combined EPO/IGF-I treatment
resulted in long-term neuronal survival.
[0041] As disclosed herein in Example VC, cultures were incubated
with EPO, IGF-I and NMDA and, after immunoprecipitation of the
active form of caspase-3, immunoprecipitates were probed for the
presence of XIAP, an anti-apoptotic protein that functions to
inhibit caspase-3 activity. FIG. 8B shows that active caspase-3 was
associated with XIAP in cultured neurons and that treatment with
EPO and IGF-I increased the relative amount of XIAP associated with
active caspase-3, consistent with negative regulation of the active
form of caspase-3 by association with XIAP. Furthermore, the
proteolytic activity of caspase-3 was modulated by combined
EPO/IGF-I treatment. As shown in FIG. 8D, NMDA exposure resulted in
increased caspase-3 proteolytic activity as indicated by enhanced
DEVD substrate cleavage, while simultaneous application or 3 hour
preincubation with EPO/IGF-I diminished the NMDA-induced increase
in caspase-3-like activity. This reduction in caspase-3 activity
was partially inhibited by infection with a dominant negative Akt
in cultures preincubated with EPO and IGF-I, demonstrating that Akt
can play a role in regulating the proteolytic activity of neuronal
caspase-3 (see FIG. 8D).
[0042] Furthermore, as shown in FIG. 8E, prolonged survival
afforded by combined EPO and IGF-I treatment did not require
non-neuronal cells present in the mixed cerebrocortical cultures.
Taken together with the results described above, this experiment
demonstrates that combined EPO and IGF-I treatment promotes
neuronal survival downstream of caspase-3 activation by a signal
transduction pathway intrinsic to neurons.
[0043] Based on these discoveries, the present invention provides a
method of providing acute neuroprotection by inducing the
erythropoietin (EPO) pathway in neuronal cells close to or
subsequent to the time of excitatory insult; and inducing an
insulin-like growth factor (IGF) pathway in the neuronal cells
close to or subsequent to the time of excitatory insult, thereby
producing a synergistic acute neuroprotective effect in the
neuronal cells.
[0044] The present invention further provides a method of providing
acute neuroprotection by contacting neuronal cells with EPO or an
active fragment or analog thereof close to or subsequent to the
time of excitatory insult; and contacting the neuronal cells with
an IGF or an active fragment or analog thereof close to or
subsequent to the time of excitatory insult, thereby producing a
synergistic acute neuroprotective effect in the neuronal cells.
[0045] Many forms of erythropoietin, as well as active fragments
and analogs thereof, can be useful in the methods of the invention.
In one embodiment, neuronal cells are contacted with EPO or an
active fragment thereof, for example, with human EPO or an active
fragment thereof. In another embodiment, neuronal cells are
contacted with an EPO analog, which can be, without limitation, a
peptide, peptidomimetic, small molecule or nucleic acid EPO analog.
In further embodiments, the invention is practiced with an EPO
analog that includes the amino acid sequence GGTYSCHFGPLTWVCKPQGG
(SEQ ID NO: 7); GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 8);
GGVYACRMGPITWVCSPLGG (SEQ ID NO: 9); VGNYMCHFGPITWVCRPGGG (SEQ ID
NO: 10); GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 11); or GGCRIGPITWVCGG
(SEQ ID NO: 12).
[0046] In another embodiment, the invention is practiced with EPO,
or an active fragment or analog thereof, which has at least 10-fold
higher affinity for the EPO receptor than native human EPO. In
another embodiment, the invention is practiced with EPO or an
active fragment or analog thereof which is oligomeric, for example,
dimeric. As an example, such a dimeric form of EPO can be a dimer
in which each monomer contains the amino acid sequence
GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7).
[0047] In a further embodiment, the invention is practiced with EPO
or an active fragment or analog thereof that has a half-life
greater than the half-life of native human EPO. In an additional
embodiment, the invention is practiced with EPO or an active
fragment or analog thereof that is hyper-glycosylated compared to
native human EPO. In yet a further embodiment, the invention is
practiced by contacting neuronal cells with Darbepoietin. In any
embodiment of the invention, soluble EPO receptor optionally can be
included, for example, to increase the half-life of EPO or an
active fragment or analog thereof.
[0048] A variety of forms of IGF and active fragments and analogs
thereof also are useful in the invention. In one embodiment, the
invention is practiced by contacting neuronal cells with an IGF or
an active fragment thereof, for example, IGF-I or an active
fragment thereof. In an additional embodiment, the invention is
practiced by contacting neuronal cells with human IGF-I or an
active fragment thereof. In further embodiments, the invention is
practiced with an IGF analog such as a peptide, peptidomimetic,
small molecule or nucleic acid IGF analog including, without
limitation, peptide, peptidomimetic, small molecule and nucleic
acid IGF-I analogs.
[0049] In another embodiment, the invention is practiced with an
IGF or active fragment or analog thereof which has at least 10-fold
higher affinity for the IGF-I receptor than native human IGF-I. In
a further embodiment, the invention is practiced with an IGF or
active fragment or analog thereof which has an altered affinity for
an IGF-binding protein (IBP). In yet a further embodiment, the
invention is practiced with an IGF or active fragment or analog
thereof which has a half-life greater than the half-life of native
human IGF-I. In the methods of the invention, the two contacting
steps can be performed in vitro or in vivo and further can be
performed simultaneously or in any order.
[0050] As used herein, the term "neuronal cell" means a nerve cell
and is characterized, in part, by containing one or more markers of
neuronal differentiation. Such a marker can be, for example,
neurofilament, NeuN or MAP2. A neuronal cell further generally is
characterized as containing neuronal-like processes.
[0051] The methods of the invention produce a synergistic acute
neuroprotective effect in neuronal cells. As used herein, the term
"acute neuroprotective effect" means a rapid effect that functions
to reduce neuronal cell death or deterioration. An acute
neuroprotective effect generally occurs within several minutes to
about several hours. Thus, medicaments that produce an "acute
neuroprotective effect" need not be pre-incubated with the neuronal
cells prior to the time of excitatory insult, such as stroke,
trauma or seizure, etc. An acute neuroprotective effect can rapidly
function to reduce neuronal apoptosis.
[0052] The extent of apoptotic cell death can be determined by a
variety of assays well known in the art. Such methods include light
microscopy for determining the presence of one or more
morphological characteristics of apoptosis, such as condensed or
rounded morphology, shrinking and blebbing of the cytoplasm,
preservation of the structure of cellular organelles including
mitochondria, and condensation and margination of chromatin. The
percentage of apoptotic cells also can be determined by assaying
apoptotic activity using terminal deoxytransferase-mediated (TdT)
dUTP biotin nick end-labeling (TUNEL) in conjunction with condensed
cell morphology (Gavriel et al., J. Cell Biol. 119:493 (1992);
Gorczyca et al., Int. J. Oncol. 1:639 (1992); Studzinski (Ed.),
Cell Growth and Apoptosis, Oxford: Oxford University Press (1995)).
ApopTag.TM. (ONCOR, Inc., Gaithersburg, Md.) is a commercially
available kit for identification of apoptotic cells using
digoxygenin labeling. In addition, apoptotic cells can be
identified by detecting characteristic nucleosomal DNA fragments
using agarose gel electrophoresis (Studzinski, supra, 1995; Gong et
al., Anal. Biochem. 218:314 (1994)) or using DNA filter elution
methodology to detect apoptosis-associated DNA fragmentation
(Bertrand et al., Drug Devel. 34:138 (1995)). One skilled in the
art understands that these, or other assays for apoptosis, can be
performed using methodologies routine in the art.
[0053] The term "synergistic," as used herein in reference to an
acute neuroprotective effect means an acute neuroprotective effect
achieved by the combination of a particular dose of EPO, or active
fragment or analog thereof, and a particular dose of IGF, or active
fragment or analog thereof, that is significantly greater than the
additive acute neuroprotective effect ensuing from individual
treatment with the same doses of EPO and IGF, or active fragments
or analogs thereof. In particular embodiments, the synergistic
acute neuroprotective effect reduces neuronal cell death by at
least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
[0054] In the methods of the invention for providing acute
neuroprotection, neuronal cells are contacted with EPO or an active
fragment or analog thereof and IGF or an active fragment or analog
thereof close to or subsequent to the time of excitatory insult. As
used herein, the term "close to or subsequent to the time of
excitatory insult" means that the treatment or contact occurs at
any time after the stroke, trauma, seizure, poisoning or other
excitatory insult or that the treatment or contact occurs at most
an hour prior to the time of excitatory insult. Generally, the
neuronal cells are contacted with EPO and IGF from about the time
of insult to about 48 hours later and can be contacted, for
example, between 30 minutes and 8 hours following the insult. In
one embodiment, the EPO and IGF contacting steps occur within 30
minutes following excitatory insult. In another embodiment, the EPO
and IGF contacting steps occur within the first hour following
excitatory insult. In further embodiments, the EPO and IGF
contacting steps occur within the first two hours following the
excitatory insult, within the first three hours following the
excitatory insult, within the first 12 hours following the
excitatory insult, or within the first 24 hours following
excitatory insult.
[0055] Erythropoietin (EPO) is the principal growth factor that
induces proliferation and differentiation of erythroid progenitor
cells and is a member of the cytokine family that includes
interleukins 2 through 7, G-CSF, GM-CSF, TPO, growth hormone and
leptin (Koury and Bondurant, Transfusion 30:673-674 (1992)).
[0056] Binding of EPO to its receptor triggers signal transduction
by ligand-mediated receptor dimerization on the cell surface. Point
mutations that introduce cysteine residues into the membrane
proximal part of the extracellular domain of the EPO receptor, and
which result in disulfide-linked receptor dimers on the cell
surface, are constitutively active. Such receptors lead to cell
proliferation of EPO-dependent cell lines and other biological
effects of EPO in the absence of the hormone (Yoshimura et al.,
Nature 348:647-649 (1990); Watowich et al., Proc. Natl. Acad. Sci.,
USA 89:2140-2144 (1992); and Watowich et al., Mol. Cell. Biol.
14:3539-3549 (1994)). Expression of these constitutive EPO
receptors in mice results in erythroleukemia through unregulated
activation of the signaling pathway (Longmore and Lodish, Cell
67:1089-1102 (1991); Longmore et al., Mol. Cell. Biol. 14:2266-2277
(1994)). EPO receptor activation has been shown to follow a
sequential dimerization mechanism, with binding to a high affinity
site 1 on EPO preceding binding of the second receptor to a lower
affinity site 2 (Matthews et al., Proc. Natl. Acad. Sci., USA 93:
9471-9476 (1996)).
[0057] As used herein, the term "erythropoietin" is synonymous with
"EPO" and means a polypeptide that has substantially the amino acid
sequence of naturally occurring human EPO or a homolog thereof.
EPOs useful in the invention include human and other primate EPOs,
mammalian EPOs such as bovine, porcine, murine and rat homologs and
other vertebrate homologs such as Danio rerio homologs. Thus, the
term EPO encompasses species homologs, alternatively spliced forms,
isotype and glycosylation variants and precursors of the mature
human EPO sequence (SEQ ID NO: 3) shown in FIG. 1. An EPO generally
has an amino acid sequence with at least about 80% amino acid
identity to the sequence of naturally occurring, mature human EPO
(SEQ ID NO: 3) and can have, for example, 90% or 95% or more amino
acid identity with SEQ ID NO: 3.
[0058] A variety of forms of erythropoietin with varying
glycosylation patterns are available commercially, including but
not limited to, EPOGEN (Amgen; Thousand Oaks, Calif.); EPOGIN
(Chugai Pharmaceuticals; Tokyo, Japan); EPOMAX (Elanex; Bothell,
Wash.); EPREX (Janssen-Cilag; Beerse, Belgium); NEORECORMON and
RECORMON (Roche; Basel, Switzerland) and PROCRIT (Ortho Biotech;
Raritan, N.J.); various forms of EPO also are available generically
as EPOETIN ALFA, EPOETIN BETA and EPOETIN OMEGA. Thus, it is
understood that an EPO useful in the invention can be obtained
commercially or by a variety of well known methods, including,
without limitation, purification from a natural source, recombinant
expression, or peptide or chemical synthesis
[0059] A method of the invention can be practiced, if desired, with
an "EPO analog." As used herein, the term "EPO analog" means a
molecule that induces or enhances the expression, activity or
intracellular signaling of the erythropoietin receptor and that, in
combination with an insulin-like growth factor, produces a
synergistic acute neuroprotective effect in neurons. Such an analog
can be, without limitation, a protein, peptide, peptidomimetic,
small molecule, ribozyme, nucleic acid molecule, oligonucleotide,
oligosaccharide, cell, phage or virus, or a combination thereof. As
described further below, EPO analogs useful in the invention
encompass, yet are not limited to, erythropoietin mimetic peptides
(EMPs); cyclic molecules such as cyclic peptides or
peptidomimetics; dimeric and oligomeric EPO analogs; analogs with
increased plasma half-life; anti-EPO receptor antibodies; small
molecule drugs that induce EPO receptor dimerization;
hyper-glycosylated forms of EPO; EPO-encoding nucleic acid
molecules; and constitutive forms of the EPO receptor. It is
understood that the term EPO analog encompasses active fragments of
EPO, which are described hereinabove.
[0060] An EPO analog can be an erythropoietin mimetic peptide (EMP)
containing at least one copy of the amino acid sequence
YXCXXGPXTWXCXP, where X is any amino acid (Wrighton et al., Science
273:458-463 (1996)). In one embodiment, the EPO analog contains two
or more copies of YXCXXGPXTWXCXP (Wrighton et al., Nature Biotechn.
15:1261-1265 (1997). In another embodiment, the EPO analog contains
the sequence YXCXXGPXTWXCXP, where X is any amino acid and the
cyclic portion is indicated by underlining. Erythropoietin mimetic
peptides are known in the art and include EMP1 (SEQ ID NO: 7), EMP2
(SEQ ID NO: 8), EMP3 (SEQ ID NO: 9), EMP4 (SEQ ID NO: 10), EMP5
(SEQ ID NO: 11) and AF11154 (SEQ ID NO: 12) as shown in Table 1.
Additional cyclic molecules including cyclic peptides and
peptidomimetics and disulfide-bonded peptides and peptidomimetics
also can be EPO analogs useful in the invention.
1TABLE 1 Analog Sequence IC.sub.50 (.mu.m) SEQ ID NO: EMP1
GGTYSCHFGPLTWVCKPQGG 0.2 7 EMP2 GGDYHCRMGPLTWVCKPLGG 0.2 8 EMP3
GGVYACRMGPITWVCSPLGG 0.3 9 EMP4 VGNYMCHFGPITWVCRPGGG 0.5 10 EMP5
GGLYLCRFGPVTWDCGYKGG 1.0 11 AF11154 GGCRIGPITWVCGG 10.0 12
[0061] Recombinant human EPO forms a dimer upon extensive heating,
based on intermolecular disulfide bond formation involving
cysteines-7 and -161 as described, for example, in Derby et al.,
supra, 1996. Thus, oligomeric forms of erythropoietin, as well as
oligomeric EPO fragments and analogs thereof, can be useful in the
invention. See, in general, DePaolis et al., J. Pharm. Sci.
84:1280-1284 (1995), and Derby et al., Int. J. Peptide Protein Res.
47:201-208 (1996).
[0062] Oligomeric forms of EPO or active fragments-or analogs
thereof useful in the invention include dimers and trimers as well
as higher multimeric forms. An oligomeric form of EPO can include
two or more, three or more, four or more, five or more, six or
more, seven or more, eight or more, nine or more, ten or more,
fifteen or more, twenty or more, 50 or more, 100 or more, 200 or
more, 500 or more, or 1000 or more copies of EPO or an active
fragment or analog thereof. As examples, chemical cross-linking,
synthetic peptide chemistry, phage display and conjugation of
biotin-tagged EPO with streptavidin can be useful in generating
oligomeric EPO analogs. Dimeric and trimeric EPO analogs can be
formed using heterobifunctional crosslinking reagents, for example,
by chemically modifying a first pool of erythropoietin monomers to
contain free sulfhydryl residues and mixing this pool with a second
pool containing maleimido groups; the oligomeric EPO subsequently
can be purified, for example, by size exclusion HPLC as described,
in Sytkowski et al., supra, 1998.
[0063] Native human erythropoietin has a relatively short plasma
half-life of about 4 to 13 hours, while EPO analogs with a larger
molecular size can have a reduced rate of clearance and, therefore,
increased plasma survival and in vivo biological activity. Thus,
higher molecular weight EPO analogs including oligomeric forms of
EPO, or active fragments or analogs thereof, can exhibit an
increased plasma half-life as compared to the half-life of native
monomeric human EPO (Sytkowski et al., Proc. Natl. Acad. Sci. USA
95:1184-1188 (1998)). An oligomeric form of EPO can have, for
example, a half-life of at least 15, 18, 21, 24, 48, 72 or 96
hours. One skilled in the art recognizes that, if desired, soluble
EPO receptor can be included to increase the half-life of native
erythropoietin, or an active fragment or analog thereof, and,
therefore, therapeutic value.
[0064] As discussed above, binding of EPO to the EPO receptor
results in receptor dimerization, as is common for growth factor
and cytokine receptors. Dimerization of the EPO receptor can be
sufficient to induce a biological-response characteristic of EPO.
Thus, an EPO analog useful in the invention can promote
dimerization of the EPO receptor. In one embodiment, the invention
is practiced with an EPO analog which is a multivalent antibody,
such as a bivalent monoclonal antibody, that binds the
extracellular domain of the erythropoietin receptor and promotes
receptor dimerization. Such bivalent anti-EPO receptor antibodies
have been shown to mimic EPO activity as described, for example, in
Schneider et al., Blood 89:473-482 (1997), and Elliot et al., J.
Biol. Chem. 271: 24691-24697 (1996).
[0065] As used herein, the term "antibody" includes polyclonal and
monoclonal antibodies, as well as polypeptide fragments of
antibodies that retain binding activity for an EPO receptor of at
least about 1.times.10.sup.-5 M. One skilled in the art understands
that anti-EPO receptor antibody fragments, such as Fab,
F(ab').sub.2 and Fv fragments, can retain binding activity for EPO
receptor and, thus, are included within the definition of the term
antibody as used herein. The term antibody also encompasses
non-naturally occurring antibodies and fragments containing, at a
minimum, one V.sub.H and one V.sub.L domain, such as chimeric
antibodies, humanized antibodies and single chain antibodies that
specifically bind EPO receptor. Such non-naturally occurring
antibodies can be constructed using solid phase peptide synthesis,
produced recombinantly or obtained, for example, by screening
combinatorial libraries consisting of variable heavy chains and
variable light chains as described by Borrebaeck (Ed.), Antibody
Engineerinq (Second edition) New York: Oxford University Press
(1995).
[0066] A variety of bivalent antibodies are useful in the
invention, including, without limitation, naturally occurring
monoclonal and polyclonal antibodies such as monoclonal antibody
MoAb34 (Schneider et al., supra, 1997); F(ab').sub.2 fragments; and
"miniantibodies," which are functional analogs of bivalent whole
antibodies that assemble in E. coli. A miniantibody includes two
scFv fragments linked to a dimerization domain via a hinge region,
such as the murine IgG3 long upper hinge. Useful dimerization
domains include anti-parallel amphipathic helices, arranged as a
helix-turn-helix bundle (dHLX; see, for example, Borrebaeck, supra,
19.95). One skilled in the art can screen for bivalent antibody EPO
analogs using routine assays. A primary screen can be an ELISA
utilizing, for example, immobilized EPO receptor extracellular
domain (EPObp), and a secondary screen for EPO agonist activity can
be, for example, a thymidine uptake proliferation assay using a
cell line stably expressing EPO receptor (Schneider et al., supra,
1997).
[0067] Anti-EPO receptor antibodies can be prepared, for example,
using as an immunogen an EPO receptor fusion protein or a synthetic
peptide encoding a portion of the EPO receptor extracellular
domain. One skilled in the art understands that purified EPO
receptor or an extracellular domain thereof, including peptide
portions such as synthetic peptides, can be produced recombinantly
and used as immunogens. Furthermore, non-immunogenic fragments or
synthetic peptides of an EPO receptor can be made immunogenic by
coupling the hapten to a carrier molecule such as bovine serum
albumin (BSA) or keyhole limpet hemocyanin (KLH) by well known
methods as described, for example, by Harlow and Lane, Antibodies:
A Laboratory Manual (Cold Spring Harbor Laboratory Press,
1988).
[0068] An EPO analog also can be a small molecule drug that can
induce EPO receptor dimerization and mimic one or more biological
activities of naturally occurring erythropoietin. A small molecule
EPO analog can contain, for example, eight copies of
N-3-[2-(4-biphenyl)-6-chloro-5-meth- yl]indoyl-acetyl-L-lysine
methyl ester, for example, attached to a
polyamidoamino-octa-4-hydroxymethylbenzamide support via a chemical
linker and can be prepared as described in Qureshi et al., Proc.
Natl. Acad. Sci., USA 96:12156-12161 (1999). Additional small
molecule EPO analogs can be routinely identified, for example, by
screening compound libraries for molecules able to induce
dimerization of soluble EPO receptor. Convenient assays for
dimerization include assaying for retention of labeled recombinant
EPO-binding protein (rEBP), which is the extracellular domain of
the EPO receptor, to unlabeled rEMP immobilized on a plate in the
presence of test compound (see Qureshi et al., supra, 1999).
[0069] Native erythropoietin is heavily glycosylated, and EPO
prepared from Chinese hamster ovary (CHO) cells has three N-linked
and one O-linked glycosylation sites with the average carbohydrate
content being about 40%. In native EPO, carbohydrate plays an
important role in stability, biosynthesis, apical secretion and
biological activity. In particular, glycosylation appears to
increase both conformational stability and solubility of EPO,
although conformation is not affected. Thus, an EPO analog also can
be a form of EPO that is hyper-glycosylated compared to native
human EPO. Such analogs are known in the art and include, without
limitation, Darbepoietin.
[0070] An EPO analog also can be a nucleic acid molecule encoding
erythropoietin or an active fragment or analog thereof. An
exemplary nucleic acid analog of human EPO is provided herein as
SEQ ID NO: 1 (see FIG. 1). The skilled person understands that a
nucleic acid molecule encoding an active fragment of EPO or a
peptide analog of EPO such as one of those described hereinabove
also can be an EPO analog useful in the methods of the
invention.
[0071] As used herein, the term nucleic acid molecule means any
polymer of two or more nucleotides, which are linked by a covalent
bond such as a phosphodiester bond, a thioester bond, or any of
various other bonds known in the art as useful and effective for
linking nucleotides. A nucleic acid molecule can be linear,
circular or supercoiled, and can be single stranded or double
stranded. A nucleic acid molecule can be, for example, DNA or RNA,
or a DNA/RNA hybrid.
[0072] A nucleic acid EPO analog, including a sense or antisense
nucleic acid molecule or oligonucleotide, also can contain one or
more nucleotide analogs or phosphothioate bonds, which protect
against degradation by nucleases. A ribonucleotide containing a
2-methyl group, instead of the normal hydroxyl group, bonded to the
2'-carbon atom of ribose residues, is an example of a non-naturally
occurring RNA molecule that is resistant to enzymatic and chemical
degradation. Other examples of non-naturally occurring organic
molecules include RNA containing 2'-aminopyrimidines, such RNA
being 1000.times. more stable in human serum as compared to
naturally occurring RNA (see Lin et al., Nucl. Acids Res.
22:5229-5234 (1994); and Jellinek et al., Biochemistry
34:11363-11372 (1995)).
[0073] Additional nucleotide analogs also are well known in the art
and can be useful, for example, in an EPO analog. For example, RNA
molecules containing 2'-O-methylpurine substitutions on the ribose
residues and short phosphorothioate caps at the 3'- and 5'-ends
exhibit enhanced resistance to nucleases (Green et al., Chem. Biol.
2:683-695 (1995)). Similarly, RNA containing
2'-amino-2'-deoxypyrimidines or 2'-fluoro-2'-deoxypyrimidines is
less susceptible to nuclease activity (Pagratis et al., Nature
Biotechnol. 15:68-73 (1997)). Furthermore, L-RNA, which is a
stereoisomer of naturally occurring D-RNA, is resistant to nuclease
activity (Nolte et al., Nature Biotechnol. 14:1116-1119 (1996));
Klobmann et al., Nature Biotechnol. 14:1112-1115 (1996)). Such RNA
molecules and methods of producing them are well known and routine
in the art (see Eaton and Piekern, Ann. Rev. Biochem. 64:837-863
(1995)). DNA molecules containing phosphorothioate linked
oligodeoxynucleotides are nuclease-resistant and can be useful EPO
analogs (Reed et al., Cancer Res. 50:6565-6570 (1990)).
Phosphorothioate-3' hydroxypropylamine modification of the
phosphodiester bond also reduces the susceptibility of a DNA
molecule to nuclease degradation (see Tam et al., Nucl. Acids Res.
22:977-986 (1994)). Furthermore, thymidine can be replaced with
5-(1-pentynyl)-2'-deoxoridine (Latham et al., Nucl. Acids Res.
22:2817-2822 (1994)).
[0074] Viral vectors can be particularly useful for introducing a
nucleic acid analog into a neuronal cell or neuronal precursor cell
in a method of the invention. Such vectors include, for example,
retroviral vectors, lentiviral vectors, adenoviral vectors,
adeno-associated vectors (AAV) and herpesvirus vectors (see, for
example, Kaplitt and Loewy, Viral Vectors: Gene Therapy and
Neuroscience Applications Academic Press, San Diego, Calif. (1995);
Chang, Somatic Gene Therapy CRC Press, Boca Raton, Fla. (1995)).
Lentiviral, retroviral and adeno-associated vectors can be useful,
for example, for permanent expression, and adenovirus and
herpesvirus can be used to achieve transient expression lasting for
several months to about one year. It is understood that both
permanent and transient expression can be useful in the methods of
the invention.
[0075] A variety of techniques are known in the art for introducing
a nucleic acid molecule into a neuronal cell or neuronal precursor
cell. Such methods include microinjection, electroporation,
lipofection, calcium-phosphate mediated transfection,
DEAE-Dextran-mediated transfection, polybrene- or
polylysine-mediated transfection, and conjugation to an antibody,
gramacidin S, artificial viral envelope or other intracellular
carrier such as TAT. See Cibelli et al., Nat. Biotech. 16:642-646
(1998); Lamb and Gearhart, Cur. Opin. Gen. Dev. 5:342-348 (1995);
Choi (U.S. Pat. No. 6,069,010); and Current Protocols in Molecular
Bioloqy, John Wiley and Sons, pp 9.16.4-9.16.11 (2000).
[0076] The biological effects of erythropoietin also can be
mimicked by a constitutively active EPO receptor, which is another
EPO analog that can produce acute neuroprotective benefits
according to a method of the invention. As used herein, the term
"constitutive EPO receptor" is synonymous with "constitutively
active EPO receptor" and means a polypeptide having structural
similarity to the native human erythropoietin receptor and which
has one or more hormone-independent biological activities of the
native EPO receptor. Such a constitutive EPO receptor can have a
modified dimerization interface such that the receptor dimerizes in
the absence of ligand. In one embodiment, the constitutive EPO
receptor is a variant of the native human EPO receptor which has an
arginine to cysteine mutation at position 129 and which forms
disulfide-linked homodimers in the absence of EPO. In other
embodiments, a constitutive EPO receptor is a variant of a native
EPO receptor containing one or more non-naturally occurring
cysteines within the dimerization interface (see, for example,
Watowich et al., supra, 1992; Watowich et al., supra, 1994; and
Longmore et al., supra, 1994).
[0077] An EPO analog also can be a molecule that induces or
enhances the intracellular signal transduction cascade of the EPO
receptor. Signal is initiated following binding of EPO ligand and
results in dimerization of receptor. JAK2 is autophosphorylated and
subsequently phosphorylates the EPO receptor and STAT proteins,
which then are free to translocate to the nucleus and activate
transcription. In addition, cross talk between the JAK2 and NF-KB
signaling pathways, as well as Akt and bcl-X.sub.L upregulation,
can lead to neuroprotection (Digicaylioglu and Lipton, Nature
412:641-647 (2001)). Hematopoietic cell phosphatase (HCP), also
known as SHP1 or PTP1C, binds to phosphorylated EPO receptor and
dephosphorylates JAK2, thereby acting as a negative regulator of
the EPO receptor intracellular pathway. Thus, an EPO analog also
can be an inhibitor of HCP such as an HCP antisense molecule. HCP
inhibitors are known in the art and further can be identified by
routine methods (Barbone et al., Nephrol. Dial. Transplant.
14[Suppl. 2]:80-84 (1999)).
[0078] Naturally occurring human IGF-I, also known as somatomedin
C, is a hormone of 70 amino acids. IGF-I is a basic peptide (pI
8.4) with about 43% amino acid homology to proinsulin. Naturally
occurring IGF-II is a relatively neutral peptide (pI 6.4) with 60%
amino acid homology to native IGF-I.
[0079] The sequence of mature human IGF-I (SEQ ID NO: 6) is known
in the art and is shown in FIG. 2 (Rotwein et al., J. Biol. Chem.
261:4828-4832 (1986); and Jansen et al., Nature 306:609-611
(1983)). The 70 amino acids that make up mature human IGF-I have
been divided into four principle domains. The first 29 residues of
IGF-I bear a strong resemblance to the B chain of insulin and are
therefore denoted the "B domain." Similarly, IGF-I residues 42-62
are homologous to the insulin A chain and are consequently denoted
the "A domain." Intervening between the B and A domains (residues
30-41) is the "C domain;" the carboxy-terminal 7 amino acids
(residues 63-70) are known as the "D domain."
[0080] NMR solution structure of the core of human IGF-I shows
striking similarity to insulin (Cooke et al., Biochem. 30:5484-5491
(1991)), and IGF-I is known to bind the insulin receptor, although
with lower affinity than to the IGF type I receptor. Consistent
with this model, the IGF-I C and D domains may be "flaps," which
flank the insulin-conserved receptor binding cleft, and which
contribute to specific binding to the type 1 receptor. Mutagenesis
experiments have revealed that residues in the carboxy-terminal
extended region of the B domain and residues in the C domain
proximal to the B domain are involved in receptor binding, and that
tyrosines 24 and 31 are involved in specific receptor
interactions.
[0081] Unlike most other growth factors, the IGFs are present in
high concentrations in the circulation; however, only a relatively
small fraction is available in a "free" or unbound form. Specific
binding proteins of high molecular weight and with a high binding
capacity for IGF-I and IGF-II act as carrier proteins and modulate
IGF functions (Holly et al., Endocrin. 118:7-18 (1988)). Most IGFs
in blood circulate as part of a non-covalently associated ternary
complex made up of IGF-I or IGF-II, IGFBP-3 and a protein known as
the "acid-labile subunit" (ALS).
[0082] As used herein, the term "insulin-like growth factor" is
synonymous with "IGF" and means a polypeptide that has
substantially the amino acid sequence of naturally occurring human
IGF-I or naturally occurring human IGF-II or a homolog of one of
these proteins. Insulin-like growth factors useful in the invention
include human and other primate IGFs, mammalian IGFs such as
bovine, porcine, murine and rat homologs, and other vertebrate
homologs such as chicken and Danio rerio homologs. Thus, the term
IGF encompasses species homologs, alternatively spliced forms,
isotype variants and precursors of the mature human IGF-I sequence
shown in FIG. 2, and species homologs, alternatively spliced forms,
isotype variants and precursors of mature human IGF-II. An IGF-I
generally has an amino acid sequence with at least about 80% amino
acid identity to the sequence of naturally occurring mature human
IGF-I (SEQ ID NO: 6) and can have, for example, 90% or 95% or more
amino acid identity with SEQ ID NO: 6. Similarly, an IGF-II
generally has an amino acid sequence with at least about 80% amino
acid identity to the sequence of naturally occurring mature human
IGF-II and can have 90% or 95% or more amino acid identity with
mature human IGF-II. It is understood that an IGF useful in the
invention can be obtained by a variety of well known methods,
including, without limitation, purification from a natural source,
recombinant expression, and peptide or chemical synthesis. As an
example, IGF-I can be prepared in bacteria as a fusion peptide,
followed by subsequent cleavage of the fusion product as described,
for example, in U.S. Pat. No. 5,708,134.
[0083] An active fragment of an IGF also can be useful in the
invention. An active fragment has an amino acid sequence
corresponding to a portion of full-length IGF-I or IGF-II and
retains the ability to synergize with EPO to produce an acute
neuroprotective effect. Active fragments of IGF-I and IGF-II useful
in the invention include fragments with similar activity or with
improved activity or stability relative to the naturally occurring
full-length IGF. As a non-limiting example, such an active fragment
can lack the first 1 to 5 amino terminal residues of IGF-I.
Destripeptide IGF-I, which lacks the native N-terminal residues Gly
Pro Glu, stimulates protein and DNA synthesis at concentrations
between 4 and 50-fold lower than the levels required for
full-length IGF-I. The elimination of 1 to 5 amino acids from the
N-terminus of bovine or porcine IGF-I also results in enhanced
potency. These and other active fragments of IGF-I find use in the
methods of the invention.
[0084] A method of the invention can be practiced, if desired, with
an "IGF analog." As used herein, the term "IGF analog" means a
molecule that induces or enhances expression, activity or
intracellular signaling of the type 1 insulin-like growth factor
receptor and that, in combination with EPO, produces a synergistic
acute neuroprotective effect in neurons. Such an IGF analog can be,
without limitation, a protein, peptide, peptidomimetic, small
molecule, ribozyme, nucleic acid molecule, oligonucleotide,
oligosaccharide, cell, phage or virus, or a combination thereof. It
is understood that the term IGF analog encompasses active fragments
of IGF-I and IGF-II.
[0085] IGF analogs include molecules with improved characteristics
relative to native human IGF-I or IGF-II, for example, that
facilitate commercial production of the analog; that have improved
potency, size, stability or solubility; or that provide a more
desirable pharmaceutical formulation. As non-limiting examples,
more active IGF analogs can be produced, for example, by a
modification affecting the interaction of the IGF with its
receptor, an IGF-binding protein (IBP) or heparin.
[0086] An IGF analog can be a molecule retaining IGF activity but
having reduced affinity for one or more serum components, as
described, for example, in Applebaum et al. (U.S. Pat. No.
4,876,242). Such an analog can have equal potency relative to IGF-I
at the type I IGF receptor and can display, for example, a 5- to
10-fold increase in activity relative to human IGF-I. As an
example, an IGF analog can be a 71 amino acid analog of human IGF-I
containing the first 17 residues of the B chain of human insulin in
place of the first 16 residues of human IGF-I or another analog
incorporating a portion of insulin in place of the native IGF
sequence (see, also, Cascieri and Bayne in LeRoith and Raizada,
Current Directions in Insulin-like Growth Factor Research Plenum
Press: New York 1994). An IGF analog also can be a peptide or
peptidomimetic analog with a non-naturally occurring amino-terminal
sequence. Ballard et al. (U.S. Pat. Nos. 5,470,828 and 5,164,370)
describe IGF-I and IGF-II analogs where there is a substitution of
Glu3 in IGF-I, or a substitution of Glu5 and Glu6 in IGF-II. The
one or more glutamic acid residues can be substituted, without
limitation, with Ala, Asn, Phe, Ile, Met, Val, Ser, Pro, Thr, Tyr
or Cys. If desired, at least one of the surrounding Gly, Pro or Thr
residues also can be absent from the IGF analog.
[0087] While naturally occurring IGFs are single chain molecules,
an IGF analog can have two or more chains. Two-chain IGF analogs
include disulfide-bonded heterodimers composed of a first chain
containing the B and C domains, and a second chain containing the A
domain. Such a two-chain IGF analog can be an IGF-I analog in which
the C domain contains a deletion of the first 8, 10 or 12 residues
and can have increased IGF-I activity as compared to native human
IGF-I (see, for example, U.S. Pat. No. 5,622,932).
[0088] An IGF pathway also can be induced using a molecule which
increases the active concentration of an IGF, for example, by
inhibiting the interaction of IGF-I or IGF-II with one of their
binding proteins but not with the type 1 IGF receptor; such an IGF
analog can be used alone or in combination with IGF-I or IGF-II, or
an active fragment or analog thereof. Such IGF analogs, which can
be a small antagonist mimetics of an IGF binding protein acting as
indirect agonists, include peptides and peptidomimetics such as
those shown in Table 2. These molecules are well known in the art
as described, for example, in U.S. Pat. No. 6,251,865 and Lowman et
al., Biochemistry 37:8870-8878 (1998).
2 TABLE 2 Peptide sequence SEQ ID NO: ASEEVCWPVAEWYLCNMWGR 13
ASEEVCWPVAEWYLC 14 GPETCWPVAEWYLCN 15 EEVCWPVAEWYLCN 16
EVCWPVAEWYLCN 17 CWPVAEWYLCN 18 CPAGPLQWLCEKYFG 19
SEVGCRAGPLQWLCEKYFG 20
[0089] Additional IGF analogs useful in the invention include but
are not limited to LR3IGF-I, which contains an Arg for Glu
substitution at position 3 and a 13 residue amino-terminal
extension (Francis et al., J. Mol. Endocrinol. 8:213-223 (1992)) as
well as forms containing [Leu24] or [Leu24][Arg31] (Bayne et al.,
J. Biol. Chem. 265:15648-15652 (1990); Seigel et al., Molecular
Vision 6:157-163 (2000)), and the Val.sub.59 IGF-I analog that
simplifies production through application of cyanogen bromide (Ueda
et al., U.S. Pat. No. 4,745,179).
[0090] An IGF analog also can be a nucleic acid molecule that, for
example, encodes IGF-I or IGF-II or an active fragment or analog
thereof. An exemplary nucleic acid analog of human IGF-I is
provided herein as SEQ ID NO: 4. The skilled person understands
that a nucleic acid molecule encoding an active fragment of IGF-I
or IGF-II, or a peptide analog thereof, such as those described
hereinabove also can be a nucleic acid analog of IGF-I or IGF-II
useful in the methods of the invention.
[0091] Additional IGF analogs can be identified, if desired, by
routine methods. As an example, a kinase receptor activation assay
(KIRA) can be used to measure activation of the human type I IGF-I
receptor and thereby identify an IGF analog as described in Lowman
et al., Biochemistry 37:8870-8878 (1998). Briefly, human MCF-7
cells (ATCC-HTB 22), which express IGF and insulin receptors, are
grown overnight in 96 well plates with 50/50 F12/DMEM medium
(Gibco) at 37.degree. C. in 5% C02. Supernatants are decanted, and
stimulation media (50/50 F12/DMEM with 25 mM HEPES and 2.0% BSA)
containing either test compound or recombinant human IGF-I
standards are added. After stimulation at 37.degree. C. for 15
minutes, supernatants are decanted, and the cells lysed. Lysates
are transferred to an immunosorbant plate coated with polyclonal
anti-IGF-I receptor (Santa Cruz Biotechnology) and blocked with
BSA. After incubation for two hours at room temperature, unbound
receptor is removed by washing, and bound receptor detected with
biotinylated antibody 4G10 (anti-phosphotyrosine), followed by
development with HRP-conjugated dextran-streptavidin and
tetramethylbenzidine substrate solution. The product absorbance is
read at 450 nm with a reference at 650 nm.
[0092] An EPO or IGF analog useful in the invention also can be a
bifunctional molecule such as a biofunctional peptide having the
activity of EPO as well as an IGF. It is understood that such a
chimeric EPO/IGF peptide, which has the activity of both EPO and an
IGF, can be used in the methods of the invention in the absence of
additional EPO or IGF or an active fragment or analog thereof or,
if desired, in combination with one or both of EPO or IGF or active
fragments or analogs thereof. Thus, while in many cases, the
methods of the invention are practiced by contacting neuronal cells
or administering a combination of two factors (EPO and an IGF), the
methods of the invention also can be practiced by contacting
neuronal cells with or by administering to a subject a single
bifunctional analog in place of individual EPO and IGF proteins or
active fragments or analogs thereof. In one embodiment, the
invention is practiced by contacting neuronal cells with an EPO/IGF
bifunctional analog which is cleaved after uptake into the neuronal
cells to produce individual EPO and IGF analogs. In another
embodiment, the invention is practiced by administering to a
subject an EPO/IGF bifunctional analog, which is cleaved subsequent
to administration to produce individual EPO and IGF analogs.
[0093] Peptide analogs can be synthesized by well known
methodology, for example, utilizing an Applied Biosystems 430A
Peptide Synthesizer (Foster City, Calif.). Boc amino acid resins
and other reagents can be obtained from Applied Biosystems and
other commercial sources. Sequential Boc chemistry, using double
couple protocols and acetic anhydride capping can be applied to the
desired Boc-amino acid-4-(oxymethyl)phenylacetamidometh- yl [PAM]
resin. Asparagine, histidine, glutamine, arginine,
.alpha.-p-hydroxyphenyl) acetic acids, .beta.-p-hydroxyphenyl
proprionic acids, p-hydroxybenzoic acids, p-hydroxycinnamic acids
and p-hydroxyphenoxy acetic acids can be coupled using preformed
hydroxy benzotrizole esters. Other residues can be conveniently
coupled using preformed symmetrical anhydrides with
dicyclocarbodiamide (DCC). Recombinant hormones and active
fragments and analogs thereof also can be prepared using
recombinant methods in prokaryotic host cells or in yeast or other
eukaryotic host cells as described, for example, in U.S. Pat. No.
5,104,796 and U.S. Pat. No. 5,084,384 (see, also, U.S. Pat. No.
5,622,932). It is understood that recombinant forms of EPO or IGF
can be prepared as fusion proteins and can contain additional
heterologous sequences such as signal sequences.
[0094] The present invention also provides a method of preventing
or reducing the severity of an acute neurologic condition in a
subject by administering to the subject EPO or an active fragment
or analog thereof close to or subsequent to the time of acute
injury; and administering to the subject an IGF or an active
fragment or analog thereof close to or subsequent to the time of
acute injury, thereby providing a synergistic acute neuroprotective
effect and preventing or reducing the severity of the acute
neurologic condition. Such an acute neurologic condition can be,
without limitation, stroke, head or spinal cord trauma, or
seizure.
[0095] A method of the invention for preventing or reducing the
severity of an acute neurologic condition can be practiced, for
example, with EPO or an active fragment thereof, such as human EPO
or an active fragment thereof. A method of the invention also can
be practiced with an EPO analog, which can be, without limitation,
a peptide, peptidomimetic, small molecule or nucleic acid analog.
In particular embodiments, the invention is practiced with an EPO
analog containing one of the following amino acid sequences:
GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7); GGDYHCRMGPLTWVCKPLGG (SEQ ID
NO: 8); GGVYACRMGPITWVCSPLGG (SEQ ID NO: 9); VGNYMCHFGPITWVCRPGGG
(SEQ ID NO: 10); GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 11); or
GGCRIGPITWVCGG (SEQ ID NO: 12).
[0096] In one embodiment, the invention is practiced with EPO or an
active fragment or analog thereof which has at least 10-fold higher
affinity for the EPO receptor than native human EPO. In another
embodiment, the invention is practiced with EPO or an active
fragment or analog thereof which is oligomeric, for example,
dimeric. As an example, a method of the invention can be practiced
with a dimeric form of EPO in which each monomer contains the amino
acid sequence GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7). In a further
embodiment, the invention is practiced with EPO or an active
fragment or analog thereof which has a half-life greater than the
half-life of native human EPO. Such a form of EPO can be
hyper-glycosylated as compared to native human EPO and further can
be, for example, Darbepoietin. The methods of the invention also
optionally include the step of administering soluble EPO receptor
to the subject.
[0097] A variety of forms of IGF and active fragments and analogs
thereof are useful in the invention. In one embodiment, the
invention is practiced by administering IGF or an active fragment
thereof, for example, IGF-I or an active fragment thereof. In an
additional embodiment, the invention is practiced by administering
human IGF-I or an active fragment thereof. The invent-ion also can
be practiced by administering an IGF analog such as a peptide,
peptidomimetic, small molecule or nucleic acid analog including,
but not limited to, a variety of IGF-I analogs.
[0098] In one embodiment, a method of the invention for preventing
or reducing the severity of an acute neurological condition is
practiced with an IGF or active fragment or analog thereof which
has at least 10-fold higher affinity for the IGF-I receptor than
native human IGF-I. In another embodiment, the invention is
practiced with an IGF or active fragment or analog thereof which
has an altered affinity for an IGF-binding protein. In a further
embodiment, the invention is practiced with an IGF or active
fragment or analog thereof which has a half-life greater than the
half-life of native human IGF. In the methods of the invention, EPO
and IGF, or active fragments or analogs thereof, can be
administered simultaneously or in any order and in the same or
different pharmaceutical compositions.
[0099] As used herein, the term "subject" means any animal
containing neurons, for example, a mammal such as a mouse, rat,
dog, primate or human. A subject typically suffers from an acute or
chronic neurologic condition or is at high risk of developing a
neurologic condition.
[0100] As used herein, the term "acute neurological condition"
means any neurological disorder or disease having a short and
relatively severe course. As non-limiting examples, an "acute
neurologic condition" can be cerebral ischemia associated with
stroke; hypoxia; anoxia; poisoning by carbon monoxide, manganese or
cyanide; hypoglycemia; perinatal asphyxia; near death drowning;
mechanical trauma to the nervous system such as trauma to the head
or spinal cord; epileptic seizure; cardiac arrest; or cerebral
asphyxia associated, for example, with coronary bipass surgery.
Acute neurological conditions generally are distinguished from
chronic neurological conditions, in which the neurological
condition is of a relatively long duration, for example, several
months or years.
[0101] Also provided by the invention is a method of preventing or
reducing the severity of a neurologic condition in a subject by
administering to the subject EPO or an active fragment or analog
thereof at a dose of at most 2000 U/kg; and administering to the
subject an IGF or an active fragment or analog thereof, thereby
providing neuroprotection and preventing or reducing the severity
of the neurologic condition. The EPO and IGF, or active fragments
or analogs thereof, can be administered to the subject
simultaneously or in any order and in the same or different
pharmaceutical compositions using any of a variety of routes of
administration including, without limitation, oral, intravenous,
intraperitoneal, subcutaneous, intracerebroventricular,
intrathecal, transnasal, intravitreal and transcleral
administration. A variety of acute and chronic neurologic
conditions can be treated according to a method of the invention
including, but not limited to, Alzheimer's disease, Parkinson's
disease, Huntington's disease, amyotrophic lateral sclerosis,
multiple sclerosis, HIV-associated dementia, glaucoma, epilepsy,
light-induced retinal degeneration such as photoreceptor
degeneration, macular degeneration, and pain.
[0102] The present invention also provides a method of preventing
or reducing the severity of a cerebral neurologic condition in a
subject by transnasally administering to the subject EPO or an
active fragment or analog thereof at a dose of at most 2000 U/kg;
and transnasally administering to the subject an IGF or an active
fragment or analog thereof, thereby providing acute neuroprotection
and preventing or reducing the severity of the neurologic
condition.
[0103] Various forms of EPO and active fragments and analogs
thereof are useful for preventing or reducing the severity of a
neurologic condition according to a method of the invention. As an
example, the invention can be practiced with EPO or an active
fragment thereof such as human EPO or an active fragment thereof. A
method of the invention also can be practiced with an EPO analog,
which can be, without limitation, a peptide, peptidomimetic, small
molecule or nucleic acid analog. In particular embodiments, the
invention is practiced with an EPO analog containing one of the
following amino acid sequences: GGTYSCHFGPLTWVCKPQGG (SEQ ID NO:
7); GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 8); GGVYACRMGPITWVCSPLGG (SEQ
ID NO: 9); VGNYMCHFGPITWVCRPGGG (SEQ ID NO: 10);
GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 11); or GGCRIGPITWVCGG (SEQ ID NO:
12).
[0104] In one embodiment, a method of the invention for preventing
or reducing the severity of a neurologic condition is practiced
with EPO or an active fragment or analog thereof which has at least
10-fold higher affinity for the EPO receptor than native human EPO.
In another embodiment, the invention is practiced with EPO or an
active fragment or analog thereof which is oligomeric, for example,
dimeric. The invention can be practiced, for example, with a
dimeric form of EPO in which each monomer contains the amino acid
sequence GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7). In a further
embodiment, the invention is practiced with EPO or an active
fragment or analog thereof which has a half-life greater than the
half-life of native human EPO. Such a form of EPO can be
hyper-glycosylated as compared to native human EPO and further can
be, for example, Darbepoietin. The methods of the invention further
optionally include the step of administering soluble EPO receptor
to the subject.
[0105] A variety of forms of IGF and active fragments and analogs
thereof also are useful in the invention. In one embodiment, the
invention is practiced by administering an IGF or an active
fragment thereof, for example, IGF-I or an active fragment thereof.
In an additional embodiment, the invention is practiced by
administering human IGF-I or an active fragment thereof. In a
further embodiment, the invention is practiced by administering an
IGF analog such as a peptide, peptidomimetic, small molecule or
nucleic acid analog including, but not limited to, a variety of
IGF-I analogs.
[0106] In one embodiment, a method of the invention for preventing
or reducing the severity of a neurological condition is practiced
with an IGF or active fragment or analog thereof which has at least
10-fold higher affinity for the IGF-I receptor than native human
IGF-I. In another embodiment, the invention is practiced with an
IGF or active fragment or analog thereof which has an altered
affinity for an IGF-binding protein. In a further embodiment, the
invention is practiced with an IGF or active fragment or analog
thereof which has a half-life greater than the half-life of native
human IGF.
[0107] The term "neurological condition" as used herein,
encompasses all acute and chronic neurological conditions. Thus,
neurological conditions encompass, without limitation,
hypoxia-ischemia (stroke); head or spinal cord injury; epilepsy;
neurodegenerative disorders such as Parkinson's disease,
Huntington's disease, Alzheimer's disease, amyotrophic lateral
sclerosis or multiple sclerosis; optic neuropathies such as
glaucoma., light-induced retinal degeneration such as photoreceptor
degeneration, and macular degeneration; disorders of photoreceptor
degeneration such as retinitis pigmentosa; metabolic, mitochondrial
and infectious brain abnormalities such as encephalitis; and
neuropathic pain (Lipton and Rosenberg, New Enql. J. Med. 330: 613
(1994)). Chronic neurological conditions encompass
neurodegenerative diseases such as Alzheimer's disease,
Huntington's disease; disorders of photoreceptor degeneration such
as retinitis pigmentosa and light-induced retinal degeneration;
macular degeneration of the retina; forms of dementia including
fronto-temporal dementia and HIV-associated dementia (acquired
immunodeficiency syndrome dementia complex); neuropathic pain
syndromes such as causalgia or painful peripheral neuropathies and
other chronic pain syndromes; olivopontocerebellar atrophy;
Parkinson's disease; Parkinsonism; amyotrophic lateral sclerosis;
mitochondrial abnormalities and other biochemical disorders such as
MELAS syndrome, MERRF, Leber's disease, Wernicke's encephalopathy,
Rett syndrome, homocysteinuria, hyperhomocysteinemia,
hyperprolinemia, nonketotic hyperglycinemia, hydroxybutyric
aminoaciduria, sulfite oxidase deficiency, combined systems
disease, lead encephalopathy; hepatic encephalopathy, Tourette's
syndrome; drug addiction, tolerance, dependency; and depression or
anxiety syndromes.
[0108] The methods of the invention for preventing or reducing the
severity of a neurologic condition in a subject are based, in part,
on the discovery that, when administered in conjunction with an IGF
such as IGF-I, EPO can be neuroprotective at much lower doses than
previously observed. Thus, the methods of the invention are
practiced by administering a dose of at most 2000 U/kg EPO, or
active fragment or analog thereof. In particular embodiments, the
invention is practiced by administering a dose of at most 1500
U/kg, 1000 U/kg, 750 U/kg, 500 U/kg, 250 U/kg, 100 U/kg, 90 U/kg,
80 U/kg, 70 U/kg, 60 U/kg, 50 U/kg, 40 U/kg, 30 U/kg, 20 U/kg, 10
U/kg, 5 U/kg, 2.5 U/kg or 1 U/kg EPO or active fragment or analog
thereof. In further embodiments, the invention is practiced by
administering at most 2000 U/kg/day, 1500 U/kg/day, 1000 U/kg/day,
750 U/kg/day, 500 U/kg/day, 250 U/kg/day, 100 U/kg/day, 90
U/kg/day, 80 U/kg/day, 70 U/kg/day, 60 U/kg/day, 50 U/kg/day, 40
U/kg/day, 30 U/kg/day, 20 U/kg/day, 10 U/kg/day, 5 U/kg/day, 2.5
U/kg/day or 1 U/kg/day EPO or active fragment or analog thereof. In
still further embodiments, a method of the invention for preventing
or reducing the severity of a neurologic condition in a subject is
practiced by administering EPO, or active fragment or analog
thereof, in the range of 0.5 U/kg/day to 200 U/kg/day, 0.5 U/kg/day
to 100 U/kg/day, 0.5 U/kg/day to 50 U/kg/day, 0.5 U/kg/day to 25
U/kg/day, 0.5 U/kg/day to 15 U/kg/day, 0.5 U/kg/day to 10 U/kg/day,
0.5 U/kg/day to 5 U/kg/day, 1 U/kg/day to 200 U/kg/day, 1 U/kg/day
to 100 U/kg/day, 1 U/kg/day to 50 U/kg/day, 1 U/kg/day to 25
U/kg/day, 1 U/kg/day to 15 U/kg/day, 1 U/kg/day to 10 U/kg/day, 1
U/kg/day to 5 U/kg/day, 2 U/kg/day to 200 U/kg/day, 2 U/kg/day to
100 U/kg/day, 2 U/kg/day to 50 U/kg/day, 2 U/kg/day to 25 U/kg/day,
2 U/kg/day to 15 U/kg/day, 2 U/kg/day to 10 U/kg/day, 2 U/kg/day to
5 U/kg/day, 3 U/kg/day to 200 U/kg/day, 3 U/kg/day to 100 U/kg/day,
3 U/kg/day to 50 U/kg/day, 3 U/kg/day to 25 U/kg/day, 3 U/kg/day to
15 U/kg/day, 3 U/kg/day to 10 U/kg/day, 3 U/kg/day to 5 U/kg/day, 5
U/kg/day to 200 U/kg/day, 5 U/kg/day to 100 U/kg/day, 5 U/kg/day to
50 U/kg/day, 5 U/kg/day to 25 U/kg/day, 5 U/kg/day to 15 U/kg/day,
or 5 U/kg/day to 10 U/kg/day. In still further embodiments, a
method of the invention for preventing or reducing the severity of
a neurologic condition in a subject is practiced by administering
EPO, or active fragment or analog thereof, at 3 U/kg/day, 5
U/kg/day, 10 U/kg/day, 15 U/kg/day, 20 U/kg/day or 25 U/kg/day. A
unit of EPO, as defined by the World Health Organization and as
used herein, is the equivalent of 1.2 international units of EPO
activity.
[0109] The methods of the invention additionally involve
administering an IGF or active fragment or analog thereof to the
subject. Such an IGF or active fragment or analog thereof generally
is administered to a subject at from 0.5 ng/kg/day to 500
ng/kg/day. In particular embodiments, a method of the invention is
practiced by administering an IGF such as IGF-I, IGF-II or an
active fragment of analog of one of these factors, in a range of 1
ng/kg/day to 500 ng/kg/day, 1 ng/kg/day to 250 ng/kg/day, 1
ng/kg/day to 100 ng/kg/day, 1 ng/kg/day to 50 ng/kg/day, 1
ng/kg/day to 20 ng/kg/day, 1 ng/kg/day to 10 ng/kg/day, 5 ng/kg/day
to 500 ng/kg/day, 5 ng/kg/day to 250 ng/kg/day, 5 ng/kg/day to 100
ng/kg/day, 5 ng/kg/day to 50 ng/kg/day, 5 ng/kg/day to 20
ng/kg/day, 5 ng/kg/day to 10 ng/kg/day, 10 ng/kg/day to 500
ng/kg/day, 10 ng/kg/day to 250 ng/kg/day, 10 ng/kg/day to 100
ng/kg/day, 10 ng/kg/day to 50 ng/kg/day, 10 ng/kg/day to 20
ng/kg/day, 20 ng/kg/day to 500 ng/kg/day, 20 ng/kg/day to 250
ng/kg/day, 20 ng/kg/day to 100 ng/kg/day; 20 ng/kg/day to 50
ng/kg/day, 30 ng/kg/day to 500 ng/kg/day, 30 ng/kg/day to 250
ng/kg/day, 30 ng/kg/day to 100 ng/kg/day, 30 ng/kg/day to 50
ng/kg/day, 50 ng/kg/day to 500 ng/kg/day, 50 ng/kg/day to 250
ng/kg/day or 50 ng/kg/day to 100 ng/kg/day. In other embodiments, a
method of the invention is practiced by administering 5 ng/kg/day,
10 ng/kg/day, 20 ng/kg/day, 25 ng/kg/day, 30 ng/kg/day or 50
ng/kg/day of an IGF or active fragment or analog thereof.
[0110] In further embodiments, a method of the invention is
practiced by administering EPO, or an active fragment or analog
thereof, in a range of 1 U/kg/day to 100 U/kg/day, 1 to 50
U/kg/day, 1 U/kg/day to 15 U/kg/day, 2 U/kg/day to 100 U/kg/day, 2
U/kg/day to 50 U/kg/day, 2 U/kg/day to 15 U/kg/day, 3 U/kg/day to
100 U/kg/day, 3 U/kg/day to 50 U/kg/day, or 3 U/kg/day to 15
U/kg/day in combination with an IGF, or active fragment or analog
thereof, administered in a range of 1 ng/kg/day to 500 ng/kg/day, 5
ng/kg/day to 200 ng/kg/day or 10 ng/kg/day to 100 ng/kg/day.
[0111] EPO, or an active fragment or analog thereof, and an IGF or
an active fragment or analog thereof, generally are administered to
a subject in a pharmaceutical composition. It is understood that
the EPO and IGF can be administered in the same or separate
pharmaceutical compositions and further can be administered
simultaneously or in any order, and by the same or different routes
of administration. A pharmaceutical composition useful in the
invention includes EPO or an active fragment or analog thereof, or
an IGF or an active fragment or analog thereof, or both, each in a
concentration range of, for example, approximately 0.0001% to
approximately 0.1% weight by volume but not to exceed 2000 U/kg EPO
or active fragment or analog thereof. A pharmaceutical composition
useful in the methods of the invention further can include an
excipient well known in the art for preparing pharmaceutical
compositions including compositions suitable for intranasal
administration. Pharmaceutical compositions useful in the invention
further encompass, without limitation, those containing carrier
proteins such as albumin. As non-limiting examples, a
pharmaceutical composition of the invention can include about 0.1%
to 0.4% of a carrier protein such as albumin.
[0112] A pharmaceutical composition includes a pharmaceutically
acceptable carrier, which is any carrier that has substantially no
long term or permanent detrimental effect when administered.
Examples of pharmaceutically acceptable carriers include, without
limitation, water, such as distilled or deionized water; saline;
and other aqueous media. It is understood that the active
ingredients can be soluble or can be delivered as a suspension in a
suitable carrier.
[0113] A preservative or tonicity adjustor can be included, if
desired, in a pharmaceutical composition useful in the invention.
Useful preservatives include, without limitation, benzalkonium
chloride, chlorobutanol, thimerosal, phenylmercuric acetate, and
phenylmercuric nitrate. Tonicity adjustors useful in the invention
include salts such as sodium chloride, potassium chloride, mannitol
or glycerin and other pharmaceutically acceptable tonicity
adjustor.
[0114] Various buffers and means for adjusting pH can be-used to
prepare a pharmaceutical composition useful in the invention,
provided that the resulting preparation is pharmaceutically
acceptable. Such buffers include, without limitation, acetate
buffers, citrate buffers, phosphate buffers and borate buffers. It
is understood that acids or bases can be used to adjust the pH of
the composition as needed. Pharmaceutically acceptable antioxidants
useful in the invention include, yet are not limited to, sodium
metabisulfite, sodium thiosulfate, acetylcysteine, butylated
hydroxyanisole and butylated hydroxytoluene.
[0115] A variety of routes of administration can be useful in the
invention depending, in part, on the size and characteristics of
the polypeptide or analog to be administered and the history, risk
factors and symptoms of the subject to be treated. Routes of
administration suitable for the methods of the invention include
both systemic and local administration. In specific embodiments,
EPO or an active fragment or analog thereof, is administered
through oral, intravenous, subcutaneous, intraperitoneal,
intravitreal, transcleral, intranasal, intrathecal or epidural
administration, or via intracerebro ventricular injection or a
shunt surgically inserted into the cerebro ventricle. In further
embodiments, IGF-I or an active fragment or analog thereof, is
administered orally, intravenously, intranasally, intrathecally,
epidurally, or via cerebro ventricular injection or a shunt
surgically inserted into the cerebro ventricle.
[0116] Routes of administration useful in the methods of the
invention encompass, without limitation, oral delivery; intravenous
injection; intramuscular injection; subcutaneous injection;
intraperitoneal injection; transdermal diffusion and
electrophoresis; topical eye drops and ointments; periocular and
intraocular injection including subconjunctival injection; extended
release delivery devices including locally implanted extended
release devices including a bioerodible or reservoir-based
implants. It is understood that an implant useful in the invention
generally releases the implanted pharmaceutical composition over an
extended period of time.
[0117] Intranasal administration can be useful in the methods of
the invention for preventing or reducing the severity of a
neurological condition that affects the brain. Such conditions
include yet are not limited to cerebral ischemia; stroke; hypoxia
or anoxia; mechanical trauma to the head; HIV-associated dementia
(AIDs dementia complex); Alzheimer's disease or Parkinson's
disease; and drug addiction, tolerance and dependency, as well as
other neurological disorders that affect the brain described
hereinabove or known in the art. In one embodiment, intranasal
administration is used to deliver a peptide or peptidomimetic
analog of EPO. In another embodiment, intranasal administration is
used to deliver a peptide or peptidomimetic analog of IGF-I. In a
further embodiment, intranasal administration is used to deliver a
peptide or peptidomimetic analog of EPO and a peptide or
peptidomimetic analog of IGF-I. In further embodiments, the peptide
or peptidomimetic analogs of EPO administered intranasally have a
length of up to 50 residues, 40 residues, 30 residues, 25 residues,
20 residues, 15 residues, 12 residues or 10 residues. In yet
further embodiments, the peptide or peptidomimetic analogs of IGF
administered intranasally have a length of up to 50 residues, 40
residues, 30 residues, 25 residues, 20 residues, 15 residues, 12
residues or 10 residues.
[0118] Intranasal administration of EPO or IGF-I, or a peptide or
peptidomimetic of one of these factors, can bypass the blood-brain
barrier and thereby deliver the therapeutic agent to the brain. As
an example, Liu et al., J. Neur. Sci. 187:91-97 (2001), demonstrate
that recombinant human IGF-I can protect against focal cerebral
ischemic damage when administered intranasally. Intranasal
administration can be accomplished by routine methods, for example,
using a Rhinule (Ferring; Germany) to blow a liquid substance
containing the desired active ingredients into each nostril
(Pietrowsky et al., Biol. Psych. 39:332-240 (1996)). The Rhinule is
a small, flexible tube with a tip on one end that allows one to
deliver a defined volume of 0.2 ml of a liquid substance into a
nostril. Additional means of intranasal administration, including
the use of nose drops (Liu et al., supra, 2001), also are
encompassed by the methods of the invention. Nasal formulations of
EPO, IGF-I or both, can be prepared by routine methods. As an
example, a nasal formulation of IGF-I can contain 0.1-10% IGF-1 and
0.05 to 2.0% by weight carboxyvinyl polymer.
[0119] Any of the methods of the invention can include the
additional step of expressing in the neuronal cells, or in neuronal
precursor cells as described further below, one or more nucleic
acid molecules encoding gene products that are therapeutically
useful. As an example, for treatment of Parkinson's disease, a
neuronal cell or neuronal precursor cell can express, for example,
a nucleic acid molecule encoding the catecholamine enzyme tyrosine
hydroxylase, thereby increasing dopamine-62 -hydroxylase activity
upon intracerebral grafting (Jiao et al., Nature 362:450 (1993);
see, also, Dhawan et al., Science 254: 1509 (1991); and Barr and
Leiden, Science 254:1507 (1991)). Similarly, for treatment of
Alzheimer's disease, a neuronal cell or neuronal precursor cell can
express a nucleic acid molecule encoding nerve growth factor,
thereby promoting cell survival of the cholinergic neurons that are
typically lost in Alzheimer's disease (Rosenberg et al., Science
242:1575-1578 (1988)). In a similar manner, a neuronal cell or
neuronal precursor cell can be engineered to express encephalin for
treatment of neuropathic disorders involving intractable pain. One
skilled in the art recognizes that these and other combinations are
encompassed by the methods of the invention.
[0120] A neuronal cell or neuronal precursor cell further can be
engineered to express one or more anti-apoptotic gene products
including, without limitation, members of the Bcl-2 family such as
Bcl-2 and Bcl-XL and members of the inhibitor of apoptosis (IAP)
family such as c-IAP-1, c-IAP-2, XIAP or NIAP. ((Anderson, Trends
Pharm. Sci. 18:51 (1997); Gross and et al., Genes Dev. 13:1899-1911
(1999); and Deveraux and Reed, Genes Dev. 13:239-252 (1999)).
[0121] In any of the methods of the invention, the neuronal cells
or neuronal precursor cells optionally can be treated to promote
cell survival. In one embodiment, mature neuronal cells are treated
with a p38 inihibitor. In another embodiment, neuronal cells or
neuronal precursor cells are treated to inhibit caspase activity. A
variety of caspase inhibitors are useful in the invention
including, for example, nucleic acids, polypeptides, peptides,
peptidomimetics and non-peptide inhibitors such as small molecule
drugs known in the art. As used herein, the term "caspase
inhibitor" means any molecule that binds to and inhibits the
activity of one or more caspases. Caspase inhibitors useful in the
methods of the invention generally are cell permeable and have
inhibitory activity in vivo and include viral and cellular gene
products as well as synthetic inhibitors such as synthetic small
molecules (Ekert et al., Cell Death and Differentiation 6:1081-1086
(1999)).
[0122] A caspase inhibitor can be a general (non-selective) caspase
inhibitor or can be a selective caspase inhibitor. Selective
inhibitors are those inhibitors which do not inhibit non-caspase
cysteine proteases or serine proteases. Non-selective caspase
inhibitors, which also inhibit one or more non-caspase protease
inhibitors, include, for example, the cysteine protease inhibitor
iodoacetamide. A caspase inhibitor also can be selective for one or
more specific caspases. A caspase inhibitor can selectively inhibit
caspase-3 or caspase-7 or a combination thereof and can be
combined, for example, with any form of EPO or IGF or an active
fragment or analog thereof disclosed herein or known in the art.
Caspase inhibitors selective for caspases-3 and -7 include
non-peptide inhibitors such as isatin sulfonamides (see, for
example, Lee et al., J. Biol. Chem. 275:16007-16014 (2000)). A
selective caspase inhibitor also can be selective for caspase-3,
caspase-6, caspase-7 or caspase-8, or any combination thereof, and
can be combined, for example, with any form of EPO and IGF.
[0123] A caspase inhibitor can be, for example, the cytokine
response modifier A (CrmA) polypeptide, or an encoding nucleic acid
molecule, which inhibits caspases -1 and -8; or the p35 baculovirus
protein, or an encoding nucleic acid molecule, which inhibits
caspases-1, -3, -6, -7, -8 and -10 but does not inhibit non-caspase
cysteine proteases or serine proteases (Clem et al., Science
254:1388-1390 (1991)). A caspase inhibitor also can be an inhibitor
of apoptosis protein (IAP) or an encoding nucleic acid molecule.
IAPs useful as caspase inhibitors in a method of the invention
include XIAP and Survivin.
[0124] A caspase inhibitor also can be a synthetic caspase
inhibitor such as a pseudosubstrate which acts as a reversible or
irreversible competitive inhibitor of one or more caspases. Active
site mimetic peptide ketones are useful, for example, as selective
caspase inhibitors. Such caspase inhibitors include, for example,
benzylcarbonyl (z)-VAD-fluoromethylketone (fmk),
z-VAD-fmk/chloromethylketone (CMK), z-DEVD-fmk/cmk; and z-D-cmk.
Additional caspase inhibitors include the halomethyl ketone-linked
peptide YVAD, Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO,
t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO (Ekert et
al., supra, 1999). The skilled person understands that these and
other caspase inhibitors known in the art can be useful in the
methods of the invention. See, for example, Nicholson, Nature
407:810-816 (2000); WO 00/55114; and Garcia-Calvo et al., J. Biol.
Chem. 273:32608-32613 (1998)).
[0125] As disclosed herein, EPO or an active fragment or analog
thereof can cross the blood-brain barrier following intranasal
administration, whereby unwanted systemic effects such as increased
hematocrit can be avoided. In particular, intranasally applied
radiolabeled EPO migrated into olfactory tissue and was observed in
the rostral migratory stream (RMS). Furthermore, autoradiography
demonstrated that radiolabeled erythropoietin accumulated in brain
tissue when applied intranasally. These results demonstrate that
intranasal administration can be used to deliver erythropoietin and
active fragments and analogs thereof to the brain, for example, via
the rostral migratory stream. These results further indicate that
intranasal administration of EPO, or active fragments or analogs
thereof, can be useful for treating neurologic conditions such as
stroke or neurodegenerative conditions while avoiding side effects
associated with chronic systemic erythropoietin administration.
[0126] Thus, the present invention provides a method of preventing
or reducing the severity of a neurologic condition in a subject by
intranasally administering to the subject EPO or an active fragment
or analog thereof, thereby preventing or reducing the severity of
the neurologic condition. In one embodiment, the EPO or an active
fragment or analog thereof is administered at a dose of at most
2000 U/kg.
[0127] A variety of forms of erythropoietin, as well as active
fragments and analogs thereof, can be administered intranasally in
the methods of the invention. In one embodiment, a method of the
invention is practiced by intranasal administration of EPO or an
active fragment thereof, for example, by intranasal administration
of human EPO or an active fragment thereof. In another embodiment,
a method of the invention is practiced by intranasal administration
of an EPO analog, which can be, without limitation, a peptide,
peptidomimetic, small molecule or nucleic acid EPO analog. In
further embodiments, a method of the invention is practiced by
intranasal administration of an EPO analog that includes the amino
acid sequence GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7);
GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 8); GGVYACRMGPITWVCSPLGG (SEQ ID
NO: 9); VGNYMCHFGPITWVCRPGGG (SEQ ID NO: 10); GGLYLCRFGPVTWDCGYKGG
(SEQ ID NO: 11); or GGCRIGPITWVCGG (SEQ ID NO: 12).
[0128] In another embodiment, a method of the invention is
practiced by intranasal administration of EPO, or an active
fragment or analog thereof, which has at least 10-fold higher
affinity for the EPO receptor than native human EPO. In another
embodiment, a method of the invention is practiced by intranasal
administration of EPO or active fragment or analog thereof which is
oligomeric, for example, dimeric. As an example, such a dimeric
form of EPO can be a dimer in which each monomer contains the amino
acid sequence GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 7). In further
embodiments, the invention is practiced with EPO or an active
fragment or analog thereof that has a half-life greater than the
half-life of native human EPO, or with EPO or an active fragment or
analog thereof that is hyper-glycosylated compared to native human
EPO, for example, Darbepoietin. In any embodiment of the invention
involving intranasal administration of EPO or an active fragment or
analog thereof, soluble EPO receptor can be optionally included,
for example, to prolong the half-life of EPO or the active fragment
or analog thereof.
[0129] It is understood that any of a variety of acute and chronic
neurologic conditions can be treated by intranasal administration
of EPO, or an active fragment or analog thereof, in a method of the
invention. Such neurologic conditions include, for example, stroke
and neurodegenerative disorders. The methods of the invention which
rely on intranasal administration of EPO or an active fragment or
analog thereof can be useful for preventing or reducing the
severity of neurologic conditions such as, without limitation,
stroke, head or spinal cord trauma, Alzheimer's disease,
Parkinson's disease, Huntington's disease, epilepsy, amyotrophic
lateral sclerosis, multiple sclerosis, a movement disorder,
dementia, HIV-associated dementia, fronto-temporal dementia,
HIV-associated neuropathy, chronic pain, neuropathic pain,
migraine, glaucoma, drug addiction, drug withdrawal, drug
dependency, depression and anxiety. One skilled in the art
understands that these and other acute and chronic neurologic
conditions can be treated by intranasal administration of EPO or an
active fragment or analog thereof according to a method of the
invention.
[0130] Intranasal administration can be accomplished by any method
wherein the EPO or an active fragment or analog thereof is
introduced via the nasal cavity, with or without enhancers or
mechanical devices such as a Rhinule (Pietrowsky et al., supra,
1996). One skilled in the art understands that nose drops can be
useful for intranasal administration, as can other nasal
formulations including polymers and formulations as described in
Shimoda et al., Biol. Pharm. Bull. 18:734-739 (1995).
[0131] The present invention also provides a method of
differentiating neuronal precursor cells to produce an enriched
neuronal cell population by contacting the neuronal precursor cells
with EPO or an active fragment or analog thereof; and contacting
the neuronal precursor cells with IGF-I or an active fragment or
analog thereof, thereby differentiating the neuronal precursor
cells to produce an enriched cell population containing at least
70% neurons. In particular embodiments, a method of the invention
produces an enriched cell population containing at least 80%
neurons, at least 90% neurons or at least 95% neurons. If desired,
the neuronal precursor cells further can be contacted with a
differentiating agent, for example, retinoic acid. In particular
embodiments, the invention is practiced by contacting the neuronal
precursor cells with a concentration of at most 2000 U/ml, 1500
U/ml, 1000 U/ml, 750 U/ml, 500 U/ml, 250 U/ml, 100 U/ml or 50 U/ml
EPO or active fragment or analog thereof.
[0132] Neuronal precursor cells useful in the methods of the
invention can be, for example, human stem cells; embryonic stem
cells such as human embryonic stem cells; or hematopoietic neuronal
precursor cells such as human hematopoietic stem cells. Neuronal
precursor cells useful in the methods of the invention also can be,
without limitation, selected CD133-positive (AC133-positive);
CD133-positive/CD34-positive; CD133-positive/CD34-negative;
CD133-positive/CD34-negative/CD45-negative;
CD34-negative/CD38-negative/Lin-negative; or
CD34-positive/CD38-negative/- Lin-negative/Thy-1-negative human
neuronal precursor cells.
[0133] A method of the invention for differentiating neuronal
precursor cells can further include the step of introducing into
the neuronal precursor cells a nucleic acid molecule encoding a
MEF2 polypeptide or an active fragment thereof. Such a MEF2
polypeptide can be, for example, human MEF2C, or an active fragment
thereof, and further can be, if desired, a constitutively active
MEF2 polypeptide such as a MEF2/VP16 fusion protein or a
constitutively active MEF2 polypeptide containing one or more
serine/threonine to aspartic acid/glutamic acid substitutions in
the MEF2 transactivation domain. A method of the invention also can
include, if desired, the step of inhibiting caspase activity;
treating with a protective factor such as minocycline or another
tetracycline derivative; or expressing a Bcl-X.sub.L family member
in the neuronal precursor cells, for example, to prolong survival
during subsequent transplantation.
[0134] As used herein, the term "MEF2 polypeptide" means a
polypeptide that has MEF2 DNA binding activity in addition to
activity as a transcriptional activator and includes polypeptides
having substantially the amino acid sequence of MEF2A, MEF2B, MEF2C
or MEF2D. Thus, a MEF2 polypeptide can have, for example,
substantially the amino acid sequence of human MEF2A; human MEF2B;
human MEF2C; or human MEF2D. A MEF2 polypeptide includes a MADS
domain, a MEF2 domain and a transcriptional activation domain. It
is understood that, while the MADS domain and MEF2 domains of a
MEF2 polypeptide will be similar in structure to the MADS domain
and MEF2 domain of a naturally occurring MEF2 polypeptide such as
human MEF2C, the transcriptional activation domain of a MEF2
polypeptide may be structurally unrelated and can be, for example,
a synthetic transcriptional activation or a heterologous
transcriptional activation domain derived, for example, from VP16
or GAL4. One skilled in the art appreciates that a fragment of a
MEF2 polypeptide that retains MEF2 DNA binding activity and
transcriptional activity also can be useful in the methods of the
invention.
[0135] The term MEF2 polypeptide encompasses a polypeptide having
the sequence of a naturally occurring human MEF2A polypeptide
(GenBank accession NM 005587), naturally occurring human MEF2B
polypeptide (GenBank accession NM 005919), naturally occurring
human MEF2C polypeptide (GenBank accession L08895) or naturally
occurring human MEF2D polypeptide (GenBank accession NM 005920) and
is intended to include related polypeptides having substantial
amino acid sequence similarity to one of these polypeptides. Such
related polypeptides typically exhibit greater sequence similarity
to hMEF2A, hMEF2B, hMEF2C or hMEF2D than to other MADS box proteins
such as serum response factor (SRF) and include species homologs
such as primate, mouse, rat and D. rerio homologs, alternatively
spliced forms, and isotype variants of human MEF2A, MEF2B, MEF2C
and MEF2D.
[0136] Induction of the MEF2 pathway also can be achieved using a
MEF2 activator, which is a small molecule that results in increased
expression or activity of a MEF2 polypeptide or which is a mimetic
of MEF2 function. A MEF2 activator can result in increased
expression or activity of one or more MEF2 polypeptides, for
example, may result in increased expression or activity of MEF2C
without effecting expression or activity of MEF2A, MEF2B or MEF2D.
Such a MEF2 activator can be an organic chemical, drug, nucleic
acid molecule, peptide, peptidomimetic, polypeptide or other
naturally or non-naturally occurring organic molecule, and can be,
for example, a MEF2 mimetic. Exemplary MEF2 activators are
transcription factors that upregulate MEF2 expression, molecules
that compete for binding to a MEF2 inhibitor such as Cabin1,
histone deacetylase inhibitors including, but not limited to,
VX-563 (Vertex Pharmaceuticals; Cambridge, Mass.), and kinases that
activate MEF2 polypeptides such as p38.alpha.. It is understood
that a MEF2 activator can be useful in any of the methods of the
invention disclosed herein.
[0137] A variety of differentiating agents optionally are useful in
the methods of the invention including, for example, retinoic acid.
Other differentiating agents useful in a method of the invention
include, without limitation, neurotrophic factor 3, epidermal
growth factor, IGF-I, platelet-derived growth factor and other
agents that increase cAMP.
[0138] A method of the invention for differentiating neuronal cells
optionally includes the step of transplanting into a subject cells
treated to induce the EPO and IGF pathways. In a method of the
invention, cells can be transplanted, for example, into the brain,
eye (retina) or spinal cord after neuronal injury or damage. Thus,
cells treated to induce the EPO and IGF pathways can be
transplanted into a subject having or at risk of, for example,
stroke or a neurodegenerative disease such as Alzheimer's disease;
Huntington's disease; amyotrophic lateral sclerosis; Parkinson's
disease; epilepsy; brain or spinal cord trauma; multiple sclerosis;
optic neuropathy such as glaucoma, macular degeneration, or
light-induced retinal degeneration such as photoreceptor
degeneration; infection of the central nervous system; multiple
system atrophy affecting the brain; or another acute or chronic
neurodegenerative condition. Upon transplantation, the cells begin
to differentiate or continue differentiating to produce a cell
population containing protected neuronal cells.
[0139] Cells can be transplanted into a subject, for example, into
the eye, brain or spinal cord using well known methods for
transplanting or "grafting" neurons as described, for example, in
McDonald et al., Nat. Med. 5:1410-1412 (1999), and summarized in
Dunnett et al., Brit. Med. Bulletin 53:757-776 (1997). Methods for
preventing or ameliorating rejection, for example, using
cyclosporine A treatment, also are known in the art.
[0140] Those skilled in the art understand that the steps of
inducing the EPO pathway, inducing an IGF pathway, and optionally
contacting the neuronal precursor cells with a differentiating
agent can be performed in any order or simultaneously. It further
is understood that a neuronal precursor population in which the EPO
and IGF pathways have been induced can be transplanted into a
subject prior to, during or after differentiation of the neuronal
precursor cells into neuronal cells. In one embodiment, cells are
transplanted prior to or during differentiation. Where cells are
transplanted prior to differentiation, the neuronal environment can
drive the cells into the desired neuronal cell type due to the
presence of the appropriate environmental cues. In view of the
above, it is clear that differentiation can occur in vitro or in
vivo, or can occur partially in vitro and partially in vivo.
[0141] As used herein in reference to a neuronal cell, the term
"protected" means a cell that is more resistant to injury,
apoptosis or cell death than a cell in which EPO and IGF pathways
have not been induced, or in which these pathways have been induced
to a lesser extent. Thus, a population containing protected
neuronal cells can exhibit reduced apoptosis as compared to a
population that does not contain "protected" neuronal cells. Assays
for determining the extent of apoptosis are known in the art, as
described hereinabove.
[0142] It is understood that a method of the invention can be used
to produce a population that contains protected neuronal cells and
in which a large proportion of the cell population is neuronal. For
example, a method of the invention can be used to produce a cell
population containing, for example, at least 50% neuronal cells. In
other embodiments, the population produced includes at least 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more neuronal cells. The
proportion of neuronal cells can be determined by assaying for one
or more characteristic neuronal markers such as the presence of
NeuN, neurofilament or MAP2.
[0143] The methods of the invention for differentiating neuronal
precursor cells can include, if desired, contacting the neuronal
precursor cells with a differentiating agent. As used herein, the
term "differentiating agent" means a naturally occurring or
synthetic cytokine, growth factor or other compound that causes or
enhances a neuronal precursor cell to have one or more
characteristics of a neuronal cell. A differentiating agent useful
in the invention can be, for example, a retinoic acid such as
all-trans retinoic acid; neurotrophic factor 3 (NT3); epidermal
growth factor (EGF); EPO; IGF-I; platelet derived growth factor
(PDGF), or a combination of two or more of these factors. For
example, EGF, IGF-I and PDGF can be used together as a
differentiating agent. Basic fibroblast growth factor (bFGF) or
another factor that enhances proliferation of precursor cells
optionally can be used prior to treating with a differentiating
agent such as EGF, IGF-I and PDGF. One skilled in the art
understands that, if desired, one or more factors such as
brain-derived neurotrophic factor (BDNF) also can be added to
promote neuronal cell survival. A neuronal precursor cell also can
be engineered to express one or more factors that promote
differentiation including, for example, MEF2, neuroD, neuroD2,
neuroD3, neurogenin1, neurogenin2, neurogenin3, MATH1 or MATH2
(Lee, Curr. Opin. Neurobiol. 7:13-20 (1997)). Such a factor can be
expressed instead of or in addition to application of an extrinsic
differentiating agent.
[0144] As used herein, the term "neuronal precursor cell" means any
cell that is not a neuron but which is capable of differentiating
into a neuron under the appropriate conditions. Neuronal precursor
cells can be multipotent or unipotent and can be stem cells,
precursor cells, primary cells or established cells. Neuronal
precursor cells such as stem cells generally can be distinguished
from neurons in that they lack neuronal markers such as the nuclear
protein NeuN, neurofilament and microtubule-associated protein 2
(MAP2) as well as the neuronal-like processes characteristic of
mature neurons. In one embodiment, the neuronal precursor cells are
primary cells, which is a well known term in the art for cells
which are derived directly from an organism and which have limited
growth capacity in culture.
[0145] A neuronal precursor cell useful in the invention can be
multipotent or unipotent. As used herein in reference to a neuronal
precursor cell, the term "multipotent" is synonymous with
"pluripotent" and means a neuronal precursor cell capable of
differentiating into two or more distinct lineages, including the
neuronal lineage. Multipotent neuronal precursor cells such as stem
cells, which are generally nestin-positive cells, are distinguished
from unipotent precursor cells, which are generally Hu-positive
cells. Expression of nestin and Hu can be determined, for example,
by well-established immunocytochemistry methods. A multipotent
neuronal precursor cell is capable of differentiating into at least
three or more, four or more, or five or more distinct lineages,
including the neuronal lineage.
[0146] As used herein, the term "stem cell" means a pluripotent
cell type which can differentiate under the appropriate conditions
to give rise to all cellular lineages. Thus, a stem cell
differentiates to neuronal cells, hematopoietic cells, muscle
cells, adipose cells, germ cells and all other cellular lineages. A
stem cell can be an embryonic stem cell. Where the term
"hematopoietic stem cell" is used, it is understood that this term
refers to cells that are committed to the hematopoietic lineage but
which can differentiate to all cells of the hematopoietic
lineage.
[0147] As used herein, the term "embryonic stem cell" is synonymous
with "ES cell", and means a pluripotent cell type derived from an
embryo which can differentiate to give rise to all cellular
lineages. Examples of cell markers that indicate a human embryonic
stem cell include the Oct-4 transcription factor, alkaline
phosphatase, SSEA-4, TRA 1-60, and the GCTM-2 epitope. Examples of
cell markers that indicate a differentiated neuronal cell including
neurofilament proteins, .beta.-tubulin, Map2a+b, synaptophysin,
glutamic acid decarboxylase, TuJ1, SNAP 25, transcription factor
Brn-3, and GABA.sub.A .alpha.2 receptor subunit as described in
Reubinoff et al., Nat. Biotech. 18:399-404 (2000); Ghosh and
Greenberg, Neuron 15:89-103 (1995); Bain et al., Devel. Biol.
168:342-357 (1995); and Williams et al., Neuron 18:553-562
(1997).
[0148] Embryonic stem cells useful in the methods of the invention
can be obtained from a variety of sources. Embryonic stem cells can
be obtained, for example, from mice, cows, primates and humans by
methods well known in the art. As an example, murine embryonic
cells can be isolated from a mouse as described in Forrester et
al., Proc. Natl. Acad. Sci. USA 88:7514-7517 (1991), or Bain et
al., Devel. Biol. 168:342-357 (1995). Briefly, two-stage cell
embryos are isolated from fertilized female mice about 45 hours
after injection with human chorionic gonadotropin. The two
blastomeres are fused by electrical impulse and cultured in M16
medium until the four cell stage is reached. The ES cells are grown
on gelatin coated tissue culture flasks in DMEM (Dulbeco's modified
Eagle's medium) containing high glucose and 1 mM glutamine (BRL)
supplemented with 10% fetal bovine serum, 10% newborn calf serum,
nucleosides, 1000 units/ml leukemia inhibitory factor, and 0.1 mM
2-mercaptoethanol.
[0149] Embryonic stem cells also can be isolated from primates as
described in Thomson (U.S. Pat. No. 5,843,780). Briefly,
blastocysts are removed from fertilized female monkeys 6-8 days
after onset of ovulation, treated with pronase (Sigma) to remove
the zona pellucida, rabbit anti-rhesus monkey spleen cell antiserum
and guinea pig complement (Gibco BRL), and washed in DMEM. The
inner cell mass (ICM) is removed from the lysed blastocyst with a
pipette and plated on mouse gamma irradiated embryonic fibroblasts.
After 7 to 21 days, the ICM derived masses are removed with a
micropipette, treated with 0.05% trypsin-EDTA (Gibco BRL) and 1%
chicken serum, and replated on embryonic feeder cells. Colonies
demonstrating ES morphology, characterized by compact colonies with
a high nucleus to cytoplasm ratio and prominent nucleoli, are
subcultured. The ES cells are divided, for example, by
trypsinization or exposure to Dulbecco's phosphate buffered saline
containing 2 mM EDTA every 1-2 weeks when cultures become
dense.
[0150] Embryonic stem-like cells also can be isolated from cows as
described in Cibelli et al., Nat. Biotech. 16:642-646 (1998).
Briefly, oocytes are removed from freshly slaughtered cows and
placed in maturation medium M199 (Gibco), 10% fetal calf serum
(FCS), 5 ug/ml bovine leutinizing hormone (Nobl) and 10 ug/ml
pen-strep (Sigma) for 22 hours at 38.5.degree. C. Oocytes are
subsequently fertilized in vitro and cultured on mouse embryonic
fibroblast feeder layers and CR2 with 6 mg/ml BSA until they reach
the blastocyst stage. ES cells are isolated from the blastocyst by
mechanical removal of the zona pellucida and trophoblast with a 22
gauge needle and placed under mouse embryonic fibroblast feeder
layers for one week. A small colony of the resulting cell mass is
removed and cultured on top of a gamma irradiated mouse embryonic
fibroblast feeder layer as cultures become dense.
[0151] Embryonic stem cells also can be isolated from human
blastocysts as described in Reubinoff et al., supra, 2000. Briefly,
fertilized oocytes are cultured to the blastocyst stage and the
zona pellucida digested by pronase (Sigma). The inner cell mass is
removed by immunosurgery with anti-human serum antibody (Sigma) and
exposure to Guinea pig complement (BRL), and cultured on a
mitomycin C mitotically inactivated mouse embryonic feeder cell
layer in DMEM (BRL) supplemented with 20% fetal bovine serum
(Hyclone), 0.1 mM 2-mercaptoethanol, 1% non essential amino acids,
2 mM glutamine, 50 units/ml penicillin and 50 ug/ml streptomycin
(BRL) and 2,000 units/ml recombinant leukemia inhibitory factor.
Cell mass clumps are removed with a micropipette and replated on
fresh feeder layer every six to eight days.
[0152] Human stem cells can be obtained, for example, from cord
blood, which is highly enriched in primitive cells and contains a
CD133-positive/CD34-positive population. These cells can be
efficiently isolated by methods well known in the art, for example,
the Miltinyl MACS system. If desired, the
CD133-positive/CD34-positive population can be expanded by
culturing in vitro with Flt3L+TPO to produce as much as an 160-fold
expansion in long-term culture potential and a 2.times.10.sup.6
fold expansion in the number of neuronal precursor cells.
[0153] Human neuronal precursor cells useful in the invention
include human embryonic stem cells, human hematopoietic stem cells
and other neuronal precursor cells isolated from adult human blood
or from cord blood of newborn infants. A neuronal precursor cell
population can be enriched, for example, in CD133
(AC133)-positive/CD34-positive or CD133-positive/CD34-negative
neuronal precursor cells. Such enriched neuronal precursor cells
can be isolated, for example, with magnetic-activated cell sorting,
fluorescence-activated cell sorting (FACS), or related methods well
known in the art as described further below. It further is
understood that in vitro expansion of neuronal precursor cells such
as human stem cells can be performed, if desired, in the presence
of one or more of the following factors: SCF, IL-3, IL-6, flt3L,
LIF, IL-II, TGF-.beta., TPO, and bFGF, which are commercially
available, for example, from Biosource (Camarillo, Calif.), R &
D Systems (Minneapolis, Minn.) and Chemicon (Temecula, Calif.).
Various protocols for expansion and useful concentrations of
particular factors are well known in the art.
[0154] Human neuronal precursor cells can be obtained, for example,
from peripheral blood. Donors can be treated with recombinant human
G-CSF (rhG-CSF), such as Neupogen (Amgen; Thousand Oaks, Calif.),
or recombinant human GM-CSF (rhGM-CSF), such as Leukine (Immunex;
Seattle, Wash.), or both. The human neuronal precursor cells can be
primitive cells characterized as CD34+, Thy-/dim, CD38-, which can
be obtained, if desired, from G-CSF or GM-CSF treated to donors to
increase long-term-culture potential. Apheresis can be used to
collect white blood cells, for example, four to five days following
treatment with G-CSF, GM-CSF or a combination of G-CSF and GM-CSF,
generally yielding 4.times.10.sup.6 CD34-positive cells per
kilogram of body weight.
[0155] A Ceprate SC immunoaffinity column commercially available
from Cellpro (Bothell, Wash.) can be used to isolate a
CD133-positive neuronal precursor cell population. The desired cell
population binds the column matrix via a biotin conjugated antibody
linked to the column matrix and is released by mechanical shaking.
Ceprate SC immunoaffinity can be used to yield about 50%
CD34-positive cells with about 16-99% purity. CD133-positive human
neuronal precursor cells also can be isolated, for example, using
an Isolex 300 magnetic cell separator (Baxter Healthcare
Corporation; Deerfield, Ill.), which relies on mouse monoclonal
IgG1 antibodies and magnetic beads coated with anti-mouse IgG1
antibody. Release of the precursor cells by peptidase treatment
yields about 50% CD34-positive cells with 33-100% purity.
[0156] Additional art-accepted procedures for isolation of human
stem and other neuronal precursor cells include the magnetic
activated cell sorting system (MACS) commercially available from
Miltenyi Biotech (Auburn, Calif.) and fluorescence-activated cell
sorting (FACS). In the MACS sorting system, small magnetic beads
coated with secondary antibody are bound to the primary
antibody-treated cells and retained on a ferromagnetic matrix
column by a strong magnet. Cells are released by removal of the
magnet to give greater than 50% recovery and greater than 90%
purity of the desired cells. Fluorescence-activated cell sorting
(FACS) is a well known method whereby cells are selected by
attachment of fluorescent-conjugated antibodies to give greater
than 90% purity of the recovered stem or other neuronal precursor
cells. If desired, isolated stem or other neuronal precursor cells
can be assayed for the ability to repopulate bone marrow of a
sublethally irradiated nonobese diabetic/severe combined
immunodeficient (NOD-SCID) mouse using methods well known in the
art, as described, for example, in Miyoshi et al., Science 283:
682-686 (1999).
[0157] Human CD34-negative bone marrow cells such as
CD133-positive/CD34-negative cells or
CD133-positive/CD34-negative/Lin-ne- gative cells can be useful in
the methods of the invention. Such cells can be, for example,
CD34-negative/Lin-negative cells, which can have characteristics of
stromal cells and are capable of repopulating the bone marrow of
NOD/SCID mice following sublethal irradiation. Methods of preparing
neuronal precursor cell populations enriched for particular markers
are well known in the art. As an example, CD133-positive/CD34-pos-
itive hematopoietic stem and other neuronal precursor cells can be
prepared as set forth in Yin et al., Blood 90:5002-5012 (1997);
CD133-positive/CD34-negative/CD45-negative neuronal precursor cells
cells can be prepared as described, for example, in Uchida et al.,
Proc. Natl. Acad. Sci., USA 97:14720-14725 (2000); and
CD34-negative/CD38-negative/Li- n-negative human hematopoietic stem
cells and CD34-positive/CD38-negative/- Lin-negative/Thy-1-negative
hematopoietic stem cells can be prepared, for example, as described
in Bhatia et al., Nature Medicine 4:1038-1045 (1998).
[0158] In the methods of the invention, neuronal precursor cells
such as embryonic stem cells can be contacted with a
differentiating agent to induce differentiation of the cells along
the neuronal pathway. Methods for differentiating embryonic stem
cells by growth of the cells to high density are described in
Reubinoff et al., supra, 2000. Methods differentiating expanded CNS
cells by initial growth in the presence of a mitogen such as basic
fibroblast growth factor (bFGF) followed by removal of bFGF are
described in Johe et al., Genes Develop. 10:3129-3140 (1996).
Induction of neurogenesis by addition of growth factors can be
achieved with platelet derived growth factor (PDGF) such as
PDGF-AA, PDGF-AB or PDGF-BB administered in the absence of bFGF as
described in Johe et al., supra, 1996. Induction of neuronal
differentiation also can be achieved in vitro by removal of
fibroblast growth factor-2 and subsequent addition of insulin like
growth factor-1, heparin or neurotrophin-3 as described in Brooker
et al., J. Nerosci. Res. 59:332-341 (2000) and Ghosh and Greenberg,
Neuron 15:89-103 (1995); addition of platelet-derived growth factor
as described in Williams et al., Neuron 18:553-562 (1997); addition
of insulin-like growth factor-1 alone or in combination with brain
derived neurotrophic factor as described in Arsenijevic and Weiss,
J Neurosci. 18:2118-2128 (1998); and exposure to retinoic acid as
described in Bain et al., Devel. Biol. 168:342-357 (1995).
[0159] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Amelioration Of NMDA-Induced Neurotoxicity by EPO in Combination
with IGF-I does not Require Preincubation Before Neurotoxic
Insult
[0160] This example demonstrates that simultaneous application of
EPO and IGF-I at the time of neurotoxic insult effectively reduces
apoptosis of mature neurons in rat primary cerebrocortical cultures
exposed to NMDA.
[0161] The neuroprotective effects of concurrent EPO and IGF-I
administration were compared to individual treatment with EPO or
IGF-I in rat primary cerebrocortical cultures. Incubation of the
cultures with 200 .mu.M NMDA for 20 minutes induced death only in
neurons in the mixed neuronal/glial cultures as reported previously
(Bonfoco et al., supra, 1995).
[0162] Neuronal apoptosis was quantified by double labeling for
TUNEL reactivity, which in conjunction with condensed morphology is
indicative of apoptosis, and the neuron-specific protein
microtubule associated protein-2 (MAP2) 16 hours after NMDA insult.
As shown in FIG. 3, a brief 20 minute exposure to NMDA produced an
apoptotic appearance and TUNEL reactivity in 76+7% of MAP2-labeled
neurons. Preincubation for three hours with either EPO (10 U/ml) or
IGF-I (100 ng/ml) alone significantly attenuated neuronal apoptosis
(p<0.05). The EPO and IGF-I doses assayed represent maximally
effective concentrations of these factors as previously determined
from dose-response curves (Heck et al., Biol. Chem. 274:9828-9835
(1999); and Digicaylioglu and Lipton, Nature 412:641-647 (2001)).
Control incubations with IGF-I or EPO in the absence of NMDA
exposure did not affect neuronal viability.
[0163] As expected, treatment of neurons with EPO or IGF-I
individually at the time of NMDA insult did not significantly
reduce cell death (see FIG. 3). In contrast, application of EPO
together with IGF-I at the time of NMDA exposure was about as
effective in preventing apoptosis as a three hour preincubation
with both factors either alone or together. In particular, modest,
5-10%, reductions in neuronal apoptosis were seen with either EPO
or IGF-I alone, while about 50% neuronal apoptosis was suppressed
with combined EPO and IGF-I treatment at the time of NMDA insult.
These results demonstrate that signal transduction pathways
activated by IGF-I and EPO converge to promote more rapid
neuroprotection than either factor mediates in the absence of the
other. These results further indicate that IGF-I and EPO synergize
to mediate acute neuroprotection.
[0164] For these experiments, mixed neuronal/glial cerebrocortical
cultures were prepared as described previously (Bonfoco et al.,
supra, 1995); Lei et al., Neuron 8:1087-1099 (1992); and Lipton et
al., Nature 364:626-632 (1993)). In brief, cortical tissue from
embryonic day 16 Sprague-Dawley rats was dissociated in 0.5%
trypsin and plated on poly-L-lysine coated glass coverslips at a
density of 10.sup.5 cells per 35 mm culture dish in
serum-containing medium. Prior to experimental use, cerebrocortical
neurons were kept at 37.degree. C. and 5% CO.sub.2 for at least 17
days to permit full expression of NMDA receptors (Lei et al.,
supra, 1992; and Lipton et al., supra, 1993).
[0165] EPO and IGF-I incubations were performed essentially as
follows. Human recombinant EPO purchased from Amgen (Epoietin alfa,
2000 IU/ml) was diluted in cell culture medium to a final
concentration of 5-10 U/ml. Insulin-like growth factor-I (IGF-I)
(Calbiochem; La Jolla, Calif.) was diluted in cell culture medium
to a final concentration of 50-100 ng/ml.
[0166] NMDA incubations were performed as follows. Cerebrocortical
cultures were exposed to 200 .mu.M NMDA in nominally Mg.sup.2+-free
Earle's Balanced Salt Solution (EBSS), containing 1.8 mM CaCl.sub.2
and 5 .mu.M glycine for 20 minutes. After NMDA exposure, cultures
were washed with EBSS and then placed in conditioned tissue culture
medium filtered with a 0.2 .mu.M Acrodisk filter (Perkin-Elmer;
Wellesley, Mass.) to remove activated microglia. Where indicated,
EPO, IGF-I or both EPO and IGF-I were added to the conditioned
media.
[0167] Detection of apoptotic neurons was performed essentially as
follows. After experimental incubation, cerebrocortical cultures
were fixed in PBS (150 mM NaCl, 1.7 mM monobasic sodium phosphate,
9.1 mM dibasic sodium phosphate) with 4% paraformaldehyde, and
permeabilized in PBS containing 1% Tween-20. Apoptotic cells were
identified by condensed morphology in conjunction with TUNEL using
the Apoptosis Detection System (Promega; Madison, Wis.). Cultures
were labeled according to the manufacturer's protocol using
FITC-labeled 12-dUTP to visualize DNA strand breaks.
[0168] In order to specifically evaluate neuronal apoptosis in
cerebrocortical cultures, neurons were double-labeled for TUNEL
reactivity and MAP2 16 hours after NMDA insult. MAP-2 staining was
performed essentially as described in Budd et al., Proc. Natl.
Acad. Sci., USA 97:6161-6166 (2000). Apoptotic neurons were
identified using a Zeiss inverted Axiovert microscope equipped with
camera and software for deconvolution (Intelligent Imaging
Innovations; Denver, Colo.).
EXAMPLE II
PI3-Kinase is Required for Neuroprotection by EPO And IGF-I
[0169] This example demonstrates that the PI3 kinase can play a
role in mediating the neuroprotective effects of EPO and IGF-I.
[0170] PI3-kinase is involved in IGF-I and EPO signaling (Mayeux et
al., supra, 1993; Kermer et al., supra, 2000; and Damen et al., J.
Biol. Chem. 270:23402-23408 (1995)). In order to elucidate the role
of PI3-kinase in the neuroprotective effects of EPO and IGF-I, rat
cerebrocortical neurons were preincubated for three hours with EPO,
IGF-I, or EPO in combination with IGF-I (EPO/IGF-I) in the presence
or absence of 10 .mu.M LY294002, a specific PI3-kinase inhibitor.
As shown in FIG. 4, neuronal apoptosis resulting from NMDA exposure
(200 .mu.M NMDA and 5 .mu.M glycine for 20 minutes) decreased in
cells preincubated with EPO, IGF-I or EPO/IGF-I. As further shown
in FIG. 4, LY294002 abolished the neuroprotective effect of EPO and
IGF-I either alone or in combination (p<0.05) but did not itself
cause neuronal apoptosis in cerebrocortical cultures or increase
the amount of apoptosis induced by NMDA. These results indicate
that PI3-kinase activity is required for EPO- and IGF-I-mediated
neuroprotection.
[0171] For PI3-kinase inhibition, cultures were prepared and
incubated with NMDA alone, or with EPO or IGF-I individually, or
EPO in combination with IGF-I, as described above. PI3-kinase
activity was inhibited pharmacologically with 10 .mu.M LY294002
(Calbiochem; San Diego, Calif.) dissolved in dimethylsulfoxide
(DMSO, Sigma) and added to cultures 30 minutes prior to the
addition of the indicated growth factor or factors. Neuronal
apoptosis was assessed 16 hours after NMDA application by
determining the percentage of MAP2-positive cells that were also
TUNEL positive, as described above.
[0172] PI3-kinase is activated by phosphorylation of a p85
regulatory subunit, which leads to release of the catalytic subunit
(Pleiman et al., Science 263:1609-1612 (1994)). To determine if EPO
or IGF-I induces phosphorylation of the p85 subunit of PI3-kinase,
rat cerebrocortical cultures were treated with EPO or IGF-I for 10,
20 or 30 minutes, and protein lysates from these cultures analyzed
by immunoblotting for the presence of phospho-p85. As shown in FIG.
5B (upper and lower panels), both factors induced p85
phosphorylation in a time-dependent manner. However, IGF-I
incubation resulted in maximal phosphorylation of p85 subunit of
PI3-kinase after 10 minutes, whereas EPO-induced phosphorylation
was observed after 20 to 30 minutes. Equal protein loading was
confirmed by analyzing the amounts of total p85 with a second
antibody. These results indicate that EPO and IGF-I induce
phosphorylation of the p85 regulatory subunit of PI3 kinase, with
IGF-I activation occurring more rapidly.
[0173] In the murine interleukin-3-dependent cell line DA-3,
liganded EPO receptor (EPO-R) has been shown to directly associate
with PI3-kinase (Damen et al., supra, 1995; He et al., Blood
82:1609-1612 (1993)). To determine if binding of EPO to the EPO
receptor promotes a PI3-kinase/EPO-R interaction in primary
neurons, EPO-R complex was immunoprecipitated from cerebrocortical
culture lysates that were either untreated or treated with EPO for
5 or 10 minutes. Blots of the immunoprecipitated proteins were
probed with antibody recognizing the p85 subunit of PI3-kinase
(FIG. 5A, top), and equal protein loading confirmed by probing with
anti-EPO-R antibody (FIG. 5A, bottom). As shown in the FIG. 5B,
brief incubation with EPO (5-10 minutes) promoted an association
between EPO-R and the p85 subunit of PI3-kinase. However, in the
absence of EPO, only very low levels of the p85/EPO-R complex were
detected. These results demonstrate that ligand binding to neuronal
EPO-R promotes the association of PI3-kinase with this receptor and
indicate that this association can result in phosphorylation and
activation of PI3-kinase (Scheid and Woodgett, Nat. Rev. Mol. Cell.
Biol. 2:760-768 (2001)).
[0174] Preparation of total cell extracts for immunoblotting or
immunoprecipitation was performed essentially as follows. Cultures
were washed briefly in cold PBS. After addition of ice cold lysis
buffer (50 mM Tris-Cl, 150 mM NaCl, 1.1 mM PMSF, 10 .mu.g/ml
Aprotinin, 1% Triton X-100, pH 8.0, 2 mM Ortho-Vanadate, 0.1%
deoxycholate), cells were scraped off the culture dishes. Lysates
were transferred to a microcentrifuge tube, vortexed for 15 seconds
at 4.degree. C., and cleared by centrifugation for 20 minutes at
14,000.times.g at 40.degree. C. Protein concentrations were
determined using a BCAProtein assay kit (Pierce; Rockford,
Ill.).
[0175] Immunoblotting was performed as follows. Total protein (30
mg) was resolved on a 10% NuPage Bis-Tris SDS gel (Invitrogen;
Carlsbad, Calif.) with MOPS electrode buffer under reducing
conditions and electroblotted onto a nitrocellulose membrane
(Amersham; Piscataway, N.J.) for three hours. Nonspecific binding
was blocked by incubation with 5% non-fat dry milk in Tris-buffered
saline (pH 7.5) containing 0.1% Tween-20 for one hour at room
temperature. After blocking, the blots were incubated overnight at
4.degree. C. with the appropriate primary antibody diluted in
blocking buffer. Anti-EPO-R was used at 1:200 dilution (R&D;
Minneapolis, Minn.), and anti-p85 was used at 1:400 dilution
(Upstate; Waltham, Mass.). After the primary antibody incubations,
membranes were washed in Tris buffered saline with 0.1% Tween-20
for 10 minutes with three changes and incubated with the secondary
antibody conjugated with horseradish peroxidase (Vector Labs,
1:400) for one hour at room temperature and washed again in Tris
buffered saline with 0.1% Tween-20. Blots were developed with an
enhanced chemiluminescence kit obtained from Amersham and exposed
to X-ray film.
[0176] Immunoprecipitations were performed using precleared
whole-cell lysates, which were incubated with 2 .mu.g of anti-EPO-R
antibody (Cell Signaling Technologies; Beverly, Mass.) for one hour
at room temperature, followed by addition of protein A/G-Sepharose
beads (Santa Cruz Biotechnology; Santa Cruz, Calif.).
Immunoprecipitates were run on SDS gels as described above and
probed with the appropriate antibody. For loading controls, blots
were stripped and reprobed with the indicated primary antibody and
appropriate secondary antibody.
EXAMPLE III
EPO And IGF-I Cooperate in Activating Akt
[0177] This example demonstrates that the Akt kinase can be
cooperatively activated by EPO and IGF-I in neuronal cells.
[0178] Akt-kinase is activated downstream of PI3-kinase-mediated
production of 3' phospholipids. In response to production of
phosphotidylinositol-3,4,5-trisphosphate, Akt is phosphorylated at
two critical sites: serine-473 and threonine-308 (Russell et al.,
Nuerobiol. 36:455-467 (1998); Scheid and Woodgett, supra,
2001).
[0179] To assess possible activation of the Akt kinase,
cerebrocortical-cultures were exposed to EPO or IGF-I for three
hours and immunoblotted as described above using anti-phospho Akt
(anti-pAkt) and anti-Akt antibodies from Cell Signaling
Technologies at 1:2000 dilution. A three hour incubation with EPO
or IGF-I resulted in moderate Akt activation, as evidenced by
increased phospho-serine-473 Akt detected by western blotting (FIG.
6A). Co-incubation with EPO and IGF-I resulted in a much larger
increase in phospho-serine-473 Akt. Furthermore, a 20-minute
exposure to NMDA reduced constitutive levels of Akt phosphorylation
but did not inhibit the Akt phosphorylation mediated by EPO in
combination with IGF-I. Total Akt, measured by reprobing with an
anti-Akt antibody, was unchanged either by the addition of
EPO/IGF-I or NMDA. Taken together, these results indicate that
phosphorylation of Akt on serine-473 is synergistically induced by
the combination of EPO and IGF-I.
[0180] As described above, rat cerebrocortical cultures contain a
mixture of neuronal and glial cells. Double immunofluorescence
labeling with anti-phosphorylated Akt (serine-473) and
neuron-specific MAP2 antibodies demonstrated that Akt activation in
response to EPO/IGF-I treatment occurred predominantly in neurons.
In cerebrocortical cultures treated with EPO in combination with
IGF-I and exposed to NMDA, there was an increase in phospho-Akt
labeling similar to that observed in cultures treated with
EPO/IGF-I alone. These results demonstrate that EPO and IGF-I
synergize to produce activated Akt in neuronal cells.
[0181] Double immunofluorescence staining for phosphorylated Akt
and the neuron-specific marker MAP2 was performed as follows.
Cerebrocortical cells were fixed in ice-cold PBS containing 4%
paraformaldehyde for 10 minutes at >4.degree. C., rinsed 3 times
in PBS, and permeabilized in PBS containing 1% Tween-20 for 10
minutes at room temperature. Phosphorylated Akt was detected by
incubation with specific polyclonal antibodies (Cell Signaling
Technologies) diluted 1:1000, followed by detection with
FITC-conjugated secondary antibody at 1:125 dilution (Sigma; St.
Louis, Mo.). Neurons were identified by using a primary antibody
against MAP2 (Sigma) and a secondary antibody conjugated to
Texas-Red (Vector Labs; Irvine, Calif.).
EXAMPLE IV
The Akt Kinase can Play a Role in Mediating the Neuroprotective
Effects of EPO and IGF-I
[0182] This example demonstrates that inhibition of Akt activity
with a dominant negative form of Akt reduces the neuroprotective
effects of EPO and IGF-I.
[0183] The role of Akt in the EPO/IGF-I signaling pathway was
analyzed using a dominant negative Akt construct (dn-Akt) in which
a critical phosphorylation site has been mutated (Fujio and Walsh,
J. Biol. Chem. 274:16349-16354 (1999)). Cerebrocortical cultures
were infected with an adenoviral vector encoding dn-Akt (Fujio and
Walsh, supra, 1999) and assayed for the effects of NMDA alone or in
cultures also treated with EPO in combination with IGF-I. Parallel
cerebrocortical cultures were infected with an adenoviral vector
encoding wild-type Akt (wt-Akt) as a control. As shown in FIG. 7,
infection for 36 hours with either dominant negative or wild type
Akt had no significant effect on neuronal viability. Overexpression
of wt-Akt also had no significant effect on NMDA-induced apoptosis
or EPO/IGF-I-mediated neuroprotection of cultures exposed to NMDA.
Furthermore, overexpression of dn-Akt did not affect neuronal
apoptosis-consequent to NMDA exposure. However, cerebrocortical
cultures expressing dn-Akt and incubated with EPO/IGF-I displayed
significantly higher levels of NMDA-induced neuronal apoptosis in
comparison with uninfected cultures or cultures infected with
wt-Akt (p<0.01). Overexpression of wt-Akt abrogated the
pro-apoptotic effects of dn-Akt expression when co-infected in a
molar excess ratio of 2:1 (wt-Akt:dn-Akt), indicating that the
increased neuronal apoptosis seen in dn-Akt infected cells resulted
from specific expression of dn-Akt rather than a non-specific
effect of protein overexpression. In sum, these results indicate
that Akt phosphorylation and activation play a role in the
neuroprotection mediated by the combination of EPO and IGF-I.
[0184] Cultures were prepared and treated with NMDA, EPO, IGF-I, or
EPO in combination with IGF-I, where indicated, as described above,
and the percentage of apoptotic neurons was determined by TUNEL in
combination with morphology and MAP2-reactivity as described above.
An adenoviral vector expressing hemagglutinin (HA)-tagged,
nonphosphorylatable, dominant-negative mutant of Akt (Fujio and
Walsh, supra, 1999) was obtained from Dr. K. Walsh. Infections were
performed with a multiplicity of infection (MOI) of 10 and using
HA-tagged wild type Akt as a control. After a four hour exposure to
the adenovirus, primary neurons were incubated in filtered
preconditioned medium for 24-36 hours before being used in
experiments. Expression of the viral Akt constructs was confirmed
by immunofluorescence labeling with monoclonal anti-HA antibody
(1:100, Roche; Basel, Switzerland).
EXAMPLE V
EPO/IGF-I Treatment does not Prevent NMDA-Induced Caspase-3
Activation
[0185] This example demonstrates that treatment with EPO in
combination with IGF-I prevents NMDA-induced neurotoxicity in the
presence of the active form of caspase-3, indicating that
EPO/IGF-I-mediated neuroprotection occurs downstream of
caspase-3.
[0186] In cerebrocortical cultures briefly exposed to an
excitotoxic dose of NMDA, mitochondrial cytochrome c is released,
followed by activation of the intrinsic caspase pathway (Budd et
al., Proc. Natl. Acad. Sci. USA 97:6161-6166 (2000); Yuan and
Yankner, Nature 407:802-809 (2000)). Constitutively active Akt
prevents activation of caspase-9 and caspase-3 downstream of
cytochrome c release (Zhou et al., supra, 2000), indicating that
Akt is an important factor that regulates the intrinsic caspase
cascade (see, also, Kermer et al., supra, 2000).
[0187] The effect of EPO/IGF-I incubation on caspase activation was
analyzed in cerebrocortical cells following NMDA insult.
Cerebrocortical cultures were exposed to NMDA with or without
co-administration of EPO in combination with IGF-I, and the
cultures fixed 6, 12, 16, or 48 hours following NMDA exposure.
Caspase-3 activation was assessed by immunodetection using an
antibody recognizing only the active form of caspase-3. The results
showed that many MAP2 labeled neurons expressed the active form of
caspase-3 in their condensed nuclei as early as 16 hours after NMDA
exposure, and EPO/IGF-I treatment did not prevent initial
activation of caspase-3 by NMDA. However, EPO-IGF-I treated neurons
showed reduced immunoreactivity for active caspase-3 and did not
lose MAP2 labeling in their processes. Prolonged survival of the
neurons following treatment indicates that neuroprotection mediated
by the combination of EPO and IGF-I occurs, at least in part,
downstream of initial caspase-3 activation, which is typically
associated with neuronal apoptosis within 16 hours of NMDA insult
(Tenneti et al., supra, 1998, Budd et al., supra, 2000).
[0188] For these experiments, neuron-rich cerebrocortical cultures
were continuously exposed to EPO and IGF-I (10 U/ml and 100 ng/ml,
respectively) from the time of NMDA exposure (200 .mu.M for 20
minutes) until they were fixed. Immunofluorescence assays were
performed as described above, with neurons expressing active
caspase-3 identified by double labeling with monoclonal anti-MAP2
and a polyclonal antibody specific for the cleaved form of
caspase-3 (Cell Signaling Technologies) using a 1:100 dilution.
[0189] In order to determine if prolonged neuroprotective effects
are mediated by combined EPO and IGF-I treatment, the number of
surviving cerebrocortical neurons was determined at various
intervals after exposure to NMDA. The total number of viable MAP2
positive cells was assayed to avoid the effects of secondary
necrosis seen at longer survival times (Bonfoco et al., supra,
1995). As shown in FIG. 8A, NMDA exposure alone resulted in a
dramatic decrease in the number of surviving neurons. Combined
application of EPO and IGF-I prevented neuronal cell death at 6,
12, 16 and 48 hours after NMDA exposure. These results demonstrate
that EPO/IGF-I supports long-term neuronal survival after NMDA
exposure to a greater extent than observed with either factor
alone.
[0190] Primary cultures were prepared and incubated with NMDA with
or without EPO/IGF-I as described above. Neuronal survival was
assayed as the total number of MAP2 positive cells to avoid the
confounding effects of secondary necrosis, which can occur at later
time points and obfuscate the number of apoptotic cells (Bonfoco et
al., supra, 1995).
[0191] In mixed cerebrocortical cultures, EPO is known to induce
expression of XIAP and c-IAP, two related factors capable of
inhibiting caspase-3 proteolytic activity (Digicaylioglu and
Lipton, supra, 2001; and Holcik and Korneluk, Nat. Rev. Mol. Cell.
Biol. 2:550-556 (2001)). Here, cerebrocortical cultures were
analyzed for the effect of combined EPO and IGF-I treatment on
association of XIAP and active caspase-3. Cultures were incubated
with EPO, IGF-I and NMDA and, after immunoprecipitation of the
active form of caspase-3, immunoprecipitates were probed for the
presence of XIAP using western blotting. As shown in FIG. 8B,
active caspase-3 was associated with XIAP in cultured neurons, and
treatment with EPO and IGF-I increased the relative amount of XIAP
associated with active caspase-3. In particular, in cultures
treated with EPO and IGF-I for 16 hours, densitometric analysis
revealed a 2.5 to 3-fold increase in the amount of XIAP bound to
active caspase-3 (see FIG. 8C), although NMDA exposure alone also
produced a modest increase in the amount of XIAP associated with
active caspase-3. The presence of an active caspase-3/XIAP complex
in neurons indicates that, following activation of caspase-3 by
proteolytic cleavage, the active form can be negatively regulated
by association with XIAP.
[0192] To study the effect of combined EPO/IGF-I treatment on
proteolytic activity of caspase-3, protein lysates were prepared
from cerebrocortical cultures 16 hours after NMDA exposure, and
caspase-3 activity was assayed using the fluorescent caspase-3
substrate, DEVD-7-amino-4-trifluoromethyl- -coumarin (DEVD-AFC).
FIG. 8D shows that, despite the presence of basal levels of XIAP in
the lysates, NMDA exposure resulted in increased DEVD cleavage.
Simultaneous application or 3 hour preincubation with EPO in
combination with IGF-I diminished the NMDA-induced increase in
caspase-3-like activity by increased XIAP expression. Furthermore,
the reduction in caspase-3 activity was partially inhibited by
infection with dn-Akt in cultures preincubated with EPO and IGF-I
(FIG. 8D). These results indicate that Akt can play a role in
regulating the proteolytic activity of neuronal caspase-3.
[0193] To perform these experiments, cerebrocortical cultures were
exposed to NMDA, or EPO and IGF-I, or concurrently exposed to NMDA
and EPO/IGF-I as described above. Immunoprecipitation of culture
lysates was performed using precleared whole-cell lysates,
incubated with a 1:100 dilution of antibody specifically
recognizing the cleaved form of caspase-3 (Cell Signaling
Technologies) for one hour at room temperature, followed by
addition of protein A/G-Sepharose beads and immunoprecipitation as
described above. The immunoprecipitates were separated by
electrophoresis and transferred to a nitrocellulose membrane, which
was blotted with a 1:200 dilution of anti-XIAP antibody (Trevigen;
Gaithersburg, Md.).
[0194] Caspase-3 (DEVD) cleavage assays were performed as described
previously. Briefly, cerebrocortical cultures were lysed in cold
buffer containing 10% sucrose, 0.1% CHAPS, 100 mM HEPES (pH 7.5),
and 10 mM dithiothreitol (DTT). Cytoplasmic protein extracts (200
.mu.g) were incubated for 30 minutes at 37.degree. C. with 80 .mu.M
caspase-3 peptide substrate DEVD-AFC (Enzyme Systems Products;
Livermore, Calif.). Free fluorescent AFC released by caspase-3
activity was measured on a FluoroMax2 fluorometer at 400 nm
excitation and 505 nm emission.
[0195] To determine if the prolonged survival afforded by combined
EPO and IGF-I treatment was mediated by non-neuronal cells in the
mixed cerebrocortical cultures, primary cultures were grown under
conditions that inhibit growth of non-neuronal cells. As shown in
FIG. 8E, the relatively pure neuronal cultures also were protected
from NMDA-induced apoptosis by the combination of EPO and IGF-I
treatment. These results indicate that non-neuronal cells are not
required for the neuroprotective benefits of combined treatment
with EPO and IGF-I. Taken together, these results demonstrate that
combined EPO and IGF-I treatment promotes neuronal survival
downstream of caspase-3 activation by a signal transduction pathway
intrinsic to neurons.
[0196] Relatively pure neuronal cultures were prepared from rat
cortices in a similar manner as preparation of mixed neuronal/glial
cultures but with the following modifications. Culture medium was
replaced on the second day after plating with Neurobasal medium
containing B27 supplements (Life Technologies; Carlsbad, Calif.).
The cultures were then maintained for an additional 15 to 16 days;
the cultures were composed of greater than 95% neurons (Johnson et
al., J. Neurosci. 19:2996-3006 (1999)).
[0197] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0198] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
Sequence CWU 1
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ctg tcg gaa gct gtc ctg cgg ggc cag gcc ctg ttg gtc aac tct tcc 517
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105 110 cag ccg tgg gag ccc ctg cag ctg cat gtg gat aaa gcc gtc agt
ggc 565 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser
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873 gcctgtccca tggacactcc agtgccagca atgacatctc aggggccaga
ggaactgtcc 933 agagagcaac tctgagatct aaggatgtca cagggccaac
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1233 ggaaccatga agacaggatg ggggctggcc tctggctctc atggggtcca
agttttgtgt 1293 attcttcaac ctcattgaca agaactgaaa ccaccaaaaa
aaaaaaaaa 1342 2 193 PRT Homo sapiens 2 Met Gly Val His Glu Cys Pro
Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu
Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp
Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala
Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55
60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg
65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu
Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu
Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val
Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu
Arg Ala Leu Arg Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp
Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp
Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg
Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185
190 Arg 3 166 PRT Homo sapiens 3 Ala 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 Leu 65
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 Arg 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 Ala 145 150 155 160 Cys Arg Thr
Gly Asp Arg 165 4 725 DNA Homo sapiens CDS (12)...(473) 4
cttcagaagc a atg gga aaa atc agc agt ctt cca acc caa tta ttt aag 50
Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys 1 5 10 tgc tgc
ttt tgt gat ttc ttg aag gtg aag atg cac acc atg tcc tcc 98 Cys Cys
Phe Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser 15 20 25
tcg cat ctc ttc tac ctg gcg ctg tgc ctg ctc acc ttc acc agc tct 146
Ser His Leu Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser 30
35 40 45 gcc acg gct gga ccg gag acg ctc tgc ggg gct gag ctg gtg
gat gct 194 Ala Thr Ala Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val
Asp Ala 50 55 60 ctt cag ttc gtg tgt gga gac agg ggc ttt tat ttc
aac aag ccc aca 242 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe
Asn Lys Pro Thr 65 70 75 ggg tat ggc tcc agc agt cgg agg gcg cct
cag aca ggt atc gtg gat 290 Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro
Gln Thr Gly Ile Val Asp 80 85 90 gag tgc tgc ttc cgg agc tgt gat
cta agg agg ctg gag atg tat tgc 338 Glu Cys Cys Phe Arg Ser Cys Asp
Leu Arg Arg Leu Glu Met Tyr Cys 95 100 105 gca ccc ctc aag cct gcc
aag tca gct cgc tct gtc cgt gcc cag cgc 386 Ala Pro Leu Lys Pro Ala
Lys Ser Ala Arg Ser Val Arg Ala Gln Arg 110 115 120 125 cac acc gac
atg ccc aag acc cag aag gaa gta cat ttg aag aac gca 434 His Thr Asp
Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala 130 135 140 agt
aga ggg agt gca gga aac aag aac tac agg atg tag gaagaccctc 483 Ser
Arg Gly Ser Ala Gly Asn Lys Asn Tyr Arg Met * 145 150 ctgaggagtg
aagagtgaca tgccaccgca ggatcctttg ctctgcacga gttacctgtt 543
aaactttgga acacctacca aaaaataagt ttgataacat ttaaaagatg ggcgtttccc
603 ccaatgaaat acacaagtaa acattccaac attgtcttta ggagtgattt
gcaccttgca 663 aaaatggtcc tggagttggt agattgctgt tgatctttta
tcaataatgt tctatagaaa 723 ag 725 5 153 PRT Homo sapiens 5 Met Gly
Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe 1 5 10 15
Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20
25 30 Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr
Ala 35 40 45 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala
Leu Gln Phe 50 55 60 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys
Pro Thr Gly Tyr Gly 65 70 75 80 Ser Ser Ser Arg Arg Ala Pro Gln Thr
Gly Ile Val Asp Glu Cys Cys 85 90 95 Phe Arg Ser Cys Asp Leu Arg
Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110 Lys Pro Ala Lys Ser
Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125 Met Pro Lys
Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 130 135 140 Ser
Ala Gly Asn Lys Asn Tyr Arg Met 145 150 6 70 PRT Homo sapiens 6 Gly
Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 1 5 10
15 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly
20 25 30 Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu
Cys Cys 35 40 45 Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr
Cys Ala Pro Leu 50 55 60 Lys Pro Ala Lys Ser Ala 65 70 7 20 PRT
Artificial Sequence synthetic construct 7 Gly Gly Thr Tyr Ser Cys
His Phe Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 15 Pro Gln Gly Gly
20 8 20 PRT Artificial Sequence synthetic construct 8 Gly Gly Asp
Tyr His Cys Arg Met Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 15 Pro
Leu Gly Gly 20 9 20 PRT Artificial Sequence synthetic construct 9
Gly Gly Val Tyr Ala Cys Arg Met Gly Pro Ile Thr Trp Val Cys Ser 1 5
10 15 Pro Leu Gly Gly 20 10 20 PRT Artificial Sequence synthetic
construct 10 Val Gly Asn Tyr Met Cys His Phe Gly Pro Ile Thr Trp
Val Cys Arg 1 5 10 15 Pro Gly Gly Gly 20 11 20 PRT Artificial
Sequence synthetic construct 11 Gly Gly Leu Tyr Leu Cys Arg Phe Gly
Pro Val Thr Trp Asp Cys Gly 1 5 10 15 Tyr Lys Gly Gly 20 12 14 PRT
Artificial Sequence synthetic construct 12 Gly Gly Cys Arg Ile Gly
Pro Ile Thr Trp Val Cys Gly Gly 1 5 10 13 20 PRT Artificial
Sequence synthetic construct 13 Ala Ser Glu Glu Val Cys Trp Pro Val
Ala Glu Trp Tyr Leu Cys Asn 1 5 10 15 Met Trp Gly Arg 20 14 15 PRT
Artificial Sequence synthetic construct 14 Ala Ser Glu Glu Val Cys
Trp Pro Val Ala Glu Trp Tyr Leu Cys 1 5 10 15 15 15 PRT Artificial
Sequence synthetic construct 15 Gly Pro Glu Thr Cys Trp Pro Val Ala
Glu Trp Tyr Leu Cys Asn 1 5 10 15 16 14 PRT Artificial Sequence
synthetic construct 16 Glu Glu Val Cys Trp Pro Val Ala Glu Trp Tyr
Leu Cys Asn 1 5 10 17 13 PRT Artificial Sequence synthetic
construct 17 Glu Val Cys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn 1
5 10 18 11 PRT Artificial Sequence synthetic construct 18 Cys Trp
Pro Val Ala Glu Trp Tyr Leu Cys Asn 1 5 10 19 15 PRT Artificial
Sequence synthetic construct 19 Cys Arg Ala Gly Pro Leu Gln Trp Leu
Cys Glu Lys Tyr Phe Gly 1 5 10 15 20 19 PRT Artificial Sequence
synthetic construct 20 Ser Glu Val Gly Cys Arg Ala Gly Pro Leu Gln
Trp Leu Cys Glu Lys 1 5 10 15 Tyr Phe Gly
* * * * *