U.S. patent application number 11/640811 was filed with the patent office on 2007-10-04 for methods and compositions for modulating axonal outgrowth of central nervous system neurons.
This patent application is currently assigned to Children's Medical Centre Corporation. Invention is credited to Larry I. Benowitz.
Application Number | 20070231376 11/640811 |
Document ID | / |
Family ID | 24635087 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070231376 |
Kind Code |
A1 |
Benowitz; Larry I. |
October 4, 2007 |
Methods and compositions for modulating axonal outgrowth of central
nervous system neurons
Abstract
Methods and compositions for modulating the axonal outgrowth of
central nervous system neurons are provided. Methods for
stimulating the axonal outgrowth of central nervous system neurons
following an injury (e.g., stroke, Traumatic Brain Injury, cerebral
aneurism, spinal cord injury and the like) and methods for
inhibiting the axonal outgrowth of central nervous system neurons
in conditions such as epilepsy, e.g., posttraumatic epilepsy, and
neuropathic pain syndrome, are also provided. These methods
generally involve contacting the central nervous system neurons
with a compound that modulates the activity of N-kinase, or analog
thereof. The methods and compositions are particularly useful for
modulating the axonal outgrowth of mammalian central nervous system
neurons, such as mammalian cortical neurons or retinal ganglion
cells. Pharmaceutical and packaged formulations that include the
compounds of the invention that modulate the activity of N-kinase
are also provided.
Inventors: |
Benowitz; Larry I.; (Newton
Centre, MA) |
Correspondence
Address: |
David S. Resnick;NIXON PEABODY LLP
100 Summer Street
Boston
MA
02110
US
|
Assignee: |
Children's Medical Centre
Corporation
Boston
MA
|
Family ID: |
24635087 |
Appl. No.: |
11/640811 |
Filed: |
December 18, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09656915 |
Sep 7, 2000 |
7172871 |
|
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11640811 |
Dec 18, 2006 |
|
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Current U.S.
Class: |
424/450 ;
424/486; 435/15; 435/375; 514/1; 530/300; 530/324; 530/326;
530/387.9 |
Current CPC
Class: |
A61P 25/24 20180101;
A61P 25/18 20180101; A61P 25/28 20180101; G01N 33/6896 20130101;
A61P 9/12 20180101; A61P 25/16 20180101; C12N 9/1205 20130101; A61P
25/02 20180101; A61P 9/10 20180101; A61P 25/08 20180101; G01N
2500/00 20130101; A61P 25/14 20180101; A61P 25/00 20180101; A61P
43/00 20180101 |
Class at
Publication: |
424/450 ;
424/486; 435/015; 435/375; 514/001; 530/300; 530/324; 530/326;
530/387.9 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/00 20060101 A61K031/00; C07K 14/00 20060101
C07K014/00; C07K 16/00 20060101 C07K016/00; C07K 7/00 20060101
C07K007/00; C12N 5/00 20060101 C12N005/00; C12Q 1/48 20060101
C12Q001/48 |
Claims
1. A method for treating a subject suffering or prone to suffering
from a condition characterized by aberrant axonal outgrowth of
central nervous system neurons, comprising administering to said
subject a compound that modulates the activity of N-kinase, thereby
treating the subject suffering or prone to suffering from a
condition characterized by aberrant axonal outgrowth of central
nervous system neurons.
2. The method of claim 1, wherein the condition characterized by
aberrant axonal outgrowth of central nervous system neurons is
spinal cord injury.
3. The method of claim 2, wherein the spinal cord injury is
selected from the group consisting of monoplegia, diplegia,
paraplegia, hemiplegia and quadriplegia.
4. The method of claim 1, wherein the condition characterized by
aberrant axonal outgrowth of central nervous system neurons is
epilepsy.
5. The method of claim 4, wherein the epilepsy is posttraumatic
epilepsy.
6. The method of claim 1, wherein the condition characterized by
aberrant axonal outgrowth of central nervous system neurons is
neuropathic pain syndrome.
7. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is administered by introduction into the
central nervous system of the subject.
8. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is introduced into the cerebrospinal fluid of
the subject.
9. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is introduced to the subject
intrathecally.
10. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is introduced into a cerebral ventricle of the
subject.
11. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is introduced into the lumbar area of the
subject.
12. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is introduced into the cisterna magna of the
subject.
13. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is administered to the subject in a
pharmaceutically acceptable formulation.
14. The method of claim 13, wherein the pharmaceutically acceptable
formulation is a dispersion system.
15. The method of claim 13, wherein the pharmaceutically acceptable
formulation comprises a lipid-based formulation.
16. The method of claim 15, wherein the pharmaceutically acceptable
formulation comprises a liposome formulation.
17. The method of claim 16 wherein the pharmaceutically acceptable
formulation comprises a multivesicular liposome formulation.
18. The method of claim 13, wherein the pharmaceutically acceptable
formulation comprises a polymeric matrix.
19. The method of claim 13, wherein the pharmaceutically acceptable
formulation is contained within a minipump.
20. The method of claim 13, wherein the pharmaceutically acceptable
formulation provides sustained delivery of the compound that
modulates the activity of N-kinase, to a subject for at least one
week after the pharmaceutically acceptable formulation is
administered to the subject.
21. The method of claim 13, wherein the pharmaceutically acceptable
formulation provides sustained delivery of the compound that
modulates the activity of N-kinase, to a subject for at least one
month after the pharmaceutically acceptable formulation is
administered to the subject.
22. The method of claim 1, wherein the subject is a mammal.
23. The method of claim 22, wherein the mammal is a human.
24. The method of claim 1, wherein the central nervous system
neurons are retinal ganglion cells.
25. A method for modulating axonal outgrowth of a central nervous
system neuron, comprising contacting the central nervous system
neuron with a compound that modulates the activity of N-kinase,
thereby modulating axonal outgrowth of the central nervous system
neuron.
26. The method of claim 25, wherein the outgrowth is
stimulated.
27. The method of claim 25, wherein the outgrowth is inhibited.
28. The method of claim 25, wherein said central nervous system
neurons are mammalian.
29. A method for modulating the axonal outgrowth of a central
nervous system neuron in a subject, comprising administering to
said subject a compound that modulates the activity of N-kinase,
such that axonal outgrowth in the subject is modulated.
30. A method for identifying a compound that modulates axonal
outgrowth of a central nervous system neuron, comprising contacting
N-kinase with a test compound and determining the ability of the
test compound to modulate the activity of N-kinase, thereby
identifying a compound that modulates axonal outgrowth of a central
nervous system neuron.
31. The method of claim 30, wherein the N-kinase is human
N-kinase.
32. The method of claim 31, wherein the human N-kinase is a
recombinantly produced N-kinase.
33. The method of claim 30, wherein the N-kinase is bovine
N-kinase.
34. The method of claim 33, wherein the bovine N-kinase is purified
from a bovine source.
35. The method of claim 30, further comprising determining the
ability of the test compound to modulate axonal outgrowth of a
central nervous system neuron.
36. The method of claim 30, wherein the test compound inhibits the
activity.
37. The method of claim 30, wherein the test compound stimulates
the activity.
38. The method of claim 30, wherein the ability of the test
compound to modulate the activity of N-kinase is determined by
assessing the ability of the test compound to modulate N-kinase
dependent phosphorylation of a substrate.
39. A method for identifying a compound that modulates axonal
outgrowth of a central nervous system neuron, comprising contacting
N-kinase with a test compound, an N-kinase substrate, radioactive
ATP, and Mn.sup.+2; and determining the ability of the test
compound to modulate N-kinase dependent phosphorylation of the
substrate, thereby identifying a compound that modulates axonal
outgrowth of a central nervous system neuron.
40. The method of claim 39, wherein the N-kinase substrate is a
histone HF-1 protein.
41. The method of claim 39, wherein the radioactive ATP is
[.gamma.-.sup.32P] ATP.
42. The method of claim 39, wherein the N-kinase is human
N-kinase.
43. The method of claim 42, wherein the human N-kinase is a
recombinantly produced N-kinase.
44. The method of claim 39, wherein the N-kinase is bovine
N-kinase.
45. The method of claim 44, wherein the bovine N-kinase is purified
from a bovine source.
46. The method of claim 39, further comprising determining the
ability of the test compound to modulate axonal outgrowth of a
central nervous system neuron.
47. A compound that modulates axonal outgrowth of a central nervous
system neuron identified by the method of claim 30.
48. A compound that modulates axonal outgrowth of a central nervous
system neuron identified by the method of claim 39.
49. An isolated N-kinase polypeptide of the type that: (a) is
present in neonatal brain tissue; (b) is inhibited in the presence
of 6-thioguanine; (c) is activated by Mn.sup.+2 but not by
Mg.sup.+2 or Ca.sup.+2; (d) has a molecular weight of approximately
49 kDa; and (e) is eluted from a Cibacron Blue column at a NaCl
concentration of 1.5-1.75 M.
50. An antibody which is specifically reactive with an epitope of
the N-kinase polypeptide of claim 49.
51. The antibody of claim 50, wherein the antibody is an
intracellular antibody.
52. The antibody of claim 50, wherein the epitope comprises an ATP
binding domain.
53. A fragment of the N-kinase polypeptide of claim 49, wherein the
fragment comprises at least 15 contiguous amino acids.
54. The fragment of claim 53, wherein the fragment comprises at
least 30 contiguous amino acids.
55. The fragment of claim 53, wherein the fragment comprises at
least 50 contiguous amino acids.
56. The fragment of claim 53, wherein the fragment comprises at
least 100 contiguous amino acids.
57. A fragment of the N-kinase polypeptide of claim 49, wherein the
fragment is able to elicit an immune response.
Description
BACKGROUND OF THE INVENTION
[0001] Past early childhood, injury to the central nervous system
(CNS) results in functional impairments that are largely
irreversible. Within the brain or spinal cord, damage resulting
from stroke, trauma, or other causes can result in life-long losses
in cognitive, sensory and motor functions, and even maintenance of
vital functions. Nerve cells that are lost are not replaced, and
those that are spared are generally unable to re-grow severed
connections, although a limited amount of local synaptic
reorganization can occur close to the site of injury. Functions
that are lost are currently untreatable.
[0002] Regenerative failure in the CNS has been attributed to a
number of factors, which include the presence of inhibitory
molecules on the surface of glial cells that suppress axonal
growth; absence of appropriate substrate molecules such as laminin
to foster growth and an absence of the appropriate trophic factors
needed to activate programs of gene expression required for cell
survival and differentiation.
[0003] By contrast, within the peripheral nervous system (PNS),
injured nerve fibers can re-grow over long distances, with eventual
excellent recovery of function. Within the past 15 years,
neuroscientists have come to realize that this is not a consequence
of intrinsic differences between the nerve cells of the peripheral
and central nervous system; remarkably, neurons of the CNS will
extend their axons over great distances if given the opportunity to
grow through a grafted segment of PNS (e.g., sciatic nerve).
Therefore, neurons of the CNS retain a capacity to grow if given
the right signals from the extracellular environment. Factors which
contribute to the differing growth potentials of the CNS and PNS
include certain growth-inhibiting molecules on the surface of the
oligodendrocytes that surround nerve fibers in the CNS, but which
are less abundant in the comparable cell population of the PNS
(Schwann cells); molecules of the basal lamina and other surfaces
that foster growth in the PNS but which are absent in the CNS
(e.g., laminin); and trophic factors, soluble polypeptides which
activate programs of gene expression that underlie cell survival
and differentiation. Although such trophic factors are regarded as
essential for maintaining the viability and differentiation of
nerve cells, the particular ones that are responsible for inducing
axonal regeneration in the CNS remain uncertain.
[0004] Moreover, the intracellular molecule(s) that mediates axonal
outgrowth of normal neuronal cells (e.g., upon stimulation with
extracellular factors or upstream secondary messengers) has not
been elucidated. One report has described the partial isolation of
a kinase, referred to as "protein kinase N", from rat
pheochromocytoma PC12 cells that is activated by NGF treatment of
the PC12 cells and sensitive to purine regulation (C. Volonte, et
al., (1989) J. Cell Biol. 109, 2395-403). However, as PC12 cells
are a rat phaeochromocytoma cell line from the adrenal medulla,
with many different properties than normal CNS neurons, these cells
present a limited model for the processes by which growth of normal
CNS neurons is stimulated and the results obtained in PC12 cells
may not be predictive of molecules involved in normal CNS neuron
growth. Furthermore, this protein kinase N was only partially
purified and remains to be molecularly characterized.
[0005] In view of the lack of understanding of the molecules
involved in mediating axonal outgrowth, effective treatments for
CNS injuries have not been developed. Accordingly, elucidation and
molecular characterization of such molecules is still necessary,
and methods and compositions for modulating the outgrowth of normal
CNS neurons by modulating the activity of such molecules are still
needed.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods and compositions for
modulating the axonal outgrowth of central nervous system neurons,
in particular normal mammalian central nervous system neurons. The
invention is based, at least in part, on the isolation of a highly
purified form of the N-kinase polypeptide from normal mammalian
neuronal tissue, molecular characterization of its chemical
structure (including amino acid sequence), demonstration of its
sensitivity to purine regulation and the discovery that this kinase
plays an active role in the axonal outgrowth of CNS neurons,
including mammalian CNS neurons, such as retinal ganglion neurons.
Identification of N-kinase as a critical intracellular mediator of
axonal outgrowth, and chemical characterization of its structure,
now provides for the ability to modulate axonal outgrowth by
modulating N-kinase activity. Furthermore, this purification and
characterization of N-kinase now allows for its use in screening
assays to identify additional modulators of axonal outgrowth.
[0007] Accordingly, in one aspect, the present invention is
directed to a method for treating a subject (e.g., a mammal, such
as a human) suffering or prone to suffering from a condition
characterized by aberrant, e.g., inadequate or insufficient, axonal
outgrowth of central nervous system neurons (e.g., stroke, CNS
trauma, or a neurodegenerate disease), by administering to the
subject a compound that modulates the activity of N-kinase, thereby
treating the subject suffering or prone to suffering from a
condition characterized by aberrant axonal outgrowth of central
nervous system neurons.
[0008] The methods of the invention for modulating, e.g.,
stimulating, the axonal outgrowth of central nervous system neurons
can be used following damage or other injury to the CNS neurons
(e.g., stroke, Traumatic Brain Injury, cerebral aneurism, spinal
cord injury and the like). The methods of the invention for
modulating, e.g., inhibiting, the axonal outgrowth of CNS neurons
can be used in neuroproliferative disorders where aberrant axonal
outgrowth may occur, such as epilepsy (e.g., post-traumatic
epilepsy) and neuropathic pain syndrome.
[0009] In one aspect, the compound that modulates the activity of
N-kinase is administered to a subject in accordance with the
present invention by introduction into the central nervous system
of the subject, for example into the cerebrospinal fluid of the
subject. In certain aspects of the invention, the compound that
modulates the activity of N-kinase is introduced intrathecally, for
example into a cerebral ventricle, the lumbar area, or the cisterna
magna. In a preferred embodiment, the method of the invention
modulates outgrowth of damaged cortical neurons. In yet another
preferred embodiment, the method of the invention modulates
outgrowth of damaged retinal ganglion cells. In such circumstances,
the compound that modulates the activity of N-kinase can be
administered locally to cortical neurons or retinal ganglion cells
to modulate axonal outgrowth.
[0010] In yet another aspect of the invention, the compound that
modulates the activity of N-kinase is administered in a
pharmaceutically acceptable formulation. The pharmaceutically
acceptable formulation can be a dispersion system, for example a
lipid-based formulation, a liposome formulation, or a
multivesicular liposome formulation. The pharmaceutically
acceptable formulation can also comprise a polymeric matrix,
selected, for example, from synthetic polymers such as polyesters
(PLA, PLGA), polyethylene glycol, poloxomers, polyanhydrides, and
pluronics or selected from naturally derived polymers, such as
albumin, alginate, cellulose derivatives, collagen, fibrin,
gelatin, and polysaccharides.
[0011] In a preferred embodiment, the pharmaceutically acceptable
formulation provides sustained delivery, e.g., "slow release" of
the compound that modulates the activity of N-kinase to a subject
for at least one week, more preferably at least one month, after
the pharmaceutically acceptable formulation is administered to the
subject. Preferred approaches for achieving sustained delivery of a
formulation of the invention include the use of a slow release
polymeric capsules or an infusion pump that includes the
formulation.
[0012] In one embodiment of the invention, the compound that
modulates the activity of N-kinase is a small molecule, the
N-kinase polypeptide or fragment thereof, an anti-N-kinase
antibody, an antisense N-kinase nucleic acid molecule, a ribozyme,
or the N-kinase gene or fragment thereof.
[0013] In another aspect, the invention features a method for
modulating, e.g., stimulating or inhibiting, axonal outgrowth of a
central nervous system neuron (such as a mammalian central nervous
system neuron) by contacting the central nervous system neuron with
a compound that modulates the activity of N-kinase, thereby
modulating axonal outgrowth of the central nervous system
neuron.
[0014] In yet another aspect, the invention features a method for
modulating the axonal outgrowth of a central nervous system neuron
in a subject, by administering to the subject a compound that
modulates the activity of N-kinase, such that axonal outgrowth in
the subject is modulated.
[0015] In a further aspect, the invention features a method for
identifying a compound that modulates axonal outgrowth of a central
nervous system neuron by contacting N-kinase with a test compound
and determining the ability of the test compound to modulate the
activity of N-kinase, thereby identifying a compound that modulates
axonal outgrowth of a central nervous system neuron. In a preferred
embodiment, the ability of the test compound to modulate the
activity of N-kinase is determined by assessing the ability of the
test compound to modulate N-kinase dependent phosphorylation of a
substrate, e.g., a histone HF-1 protein.
[0016] In one embodiment, the N-kinase used in the methods of the
invention is a human N-kinase, such as a recombinantly produced
human N-kinase. In another embodiment, the N-kinase used in the
methods of the invention is a bovine N-kinase, such as an N-kinase
which is purified from a bovine source, e.g., neonatal bovine brain
tissue.
[0017] In another embodiment, the screening method of the invention
further includes determining the ability of the test compound to
modulate axonal outgrowth of a central nervous system neuron.
[0018] In another aspect, the invention features a method for
identifying a compound that modulates axonal outgrowth of a central
nervous system neuron, comprising contacting N-kinase with a test
compound, an N-kinase substrate (e.g., a histone HF-1 protein),
radioactive ATP (e.g., [.gamma.-.sup.32P] ATP), and Mn.sup.+2; and
determining the ability of the test compound to modulate N-kinase
dependent phosphorylation of the substrate, thereby identifying a
compound that modulates axonal outgrowth of a central nervous
system neuron. In a preferred embodiment, the method of the
invention further includes determining the ability of the test
compound to modulate axonal outgrowth of a central nervous system
neuron.
[0019] In another aspect, the invention features a compound that
modulates axonal outgrowth of a central nervous system neuron
identified by any of the foregoing methods.
[0020] In yet another aspect, the invention features an isolated
N-kinase polypeptide of the type that: (a) is present in neonatal
brain tissue (e.g., neonatal human, bovine, rat, or mouse brain
tissue); (b) is inhibited by 6-thioguanine; (c) is activated by
Mn.sup.+2 but not by Mg.sup.+2 or Ca.sup.+2; (d) has a molecular
weight of approximately 49 kDa; and (e) is eluted from a Cibacron
Blue column at a NaCl concentration of 1.5-1.75 M.
[0021] In a further aspect, the invention features an antibody,
e.g., an intracellular antibody, which is specifically reactive
with an epitope of the N-kinase polypeptide. In a preferred
embodiment, the antibody is reactive with an epitope which includes
the ATP binding domain of the N-kinase.
[0022] In another aspect, the invention features a fragment of the
N-kinase polypeptide, for example, a fragment that includes at
least 15, 20, 25, 30, 40, 50, 100, 150, or 200 contiguous amino
acids of the N-kinase polypeptide. In a preferred embodiment, the
fragment of the N-kinase polypeptide is able to elicit an immune
response.
[0023] Pharmaceutical compositions, and packaged formulations,
comprising a composition of the invention (e.g., compound that
modulates the activity of N-kinase) and a pharmaceutically
acceptable carrier are also provided by the invention.
[0024] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIGS. 1A-C are SDS-PAGE gels depicting the purification of
the N kinase polypeptide. The N-kinase band at each stage of the
purification is indicated by an asterisk. FIG. 1A depicts a
prominent 49 kDa band which binds strongly to a Cibacron Blue
column and requires 1.5-1.75 M NaCl to be eluted. FIG. 1B depicts
the protein fractions obtained from the separation on a C4
hydrophobic-interaction column. Fractions 24-26 contain the
N-kinase polypeptide. FIG. 1C depicts the final stage of
purification which was accomplished by SDS-PAGE. The band indicated
by the asterisk coincided in its migration position with N-kinase
activity, as visualized in a parallel gel assayed with the in-gel
kinase method.
[0026] FIG. 2 depicts the amino acid (SEQ ID NO:1) sequence of the
human N-kinase. Direct matches between the purified protein and the
published sequence are shown in blue. K65 (bold type) lies in the
ATP-binding region of the kinase domain.
DETAILED DESCRIPTION
[0027] The present invention provides methods and compositions for
modulating the axonal outgrowth of central nervous system neurons,
in particular mammalian central nervous system neurons. The
invention is based, at least in part, on the isolation of a highly
purified form of the N-kinase polypeptide from a mammalian source
and the discovery that this kinase plays an active role in the
axonal outgrowth of CNS neurons, including mammalian CNS neurons,
such as retinal ganglion neurons, or cortical pyramidal cells.
[0028] Accordingly, the present invention is directed to a method
for treating a subject (e.g., a mammal, such as a human) suffering
or prone to suffering from a condition characterized by aberrant
axonal outgrowth of central nervous system neurons, (e.g., a
condition characterized by a failure of injured central nervous
system neurons to regrow their connections) by administering to the
subject a compound that modulates the activity of N-kinase, thereby
treating the subject suffering or prone to suffering from a
condition characterized by aberrant axonal outgrowth of central
nervous system neurons.
[0029] The methods of the invention for modulating, e.g.,
stimulating, the axonal outgrowth of central nervous system neurons
can be used following damage or other injury to the CNS neurons
(e.g., stroke, Traumatic Brain Injury, cerebral aneurism, spinal
cord injury and the like). The methods of the invention for
modulating, e.g., inhibiting, the axonal outgrowth of CNS neurons
can be used in neuroproliferative disorders where aberrant axonal
outgrowth may occur, such as epilepsy (e.g., post-traumatic
epilepsy) and neuropathic pain syndrome.
[0030] As used herein, the term "N-kinase" includes all forms of
N-kinase including but not limited to human N-kinase, bovine
N-kinase, murine N-kinase, rat N-kinase, and porcine N-kinase. The
amino acid and nucleotide sequences of the human N-kinase are
described in Zhou T-H. et al. (2000) J. Biol. Chem.
275(4):2513-2519 and in GenBank Accession Number AF083420, the
contents of which are incorporated herein by reference. The amino
acid sequence of the human N-kinase is shown in FIG. 2 and in SEQ
ID NO:1. In a preferred embodiment, the term "N-kinase" includes
the isoform of N-kinase that is inhibited by 6-thioguanine, that is
activated by Mn.sup.+2 but not by Mg.sup.+2 or Ca.sup.+2, and that
has a molecular weight of approximately 49 kDa.
[0031] As used herein, the language "a compound that modulates the
activity of N-kinase" includes any compound which has the ability
to modulate, e.g., stimulate or inhibit, the activity of N-kinase
as determined by, for example, the assays described herein. Such
compounds are able to modulate one or more of the following: (a)
the ability of N-kinase to phosphorylate a substrate, e.g., a
histone HF-1 protein; (b) the ability of N-kinase to interact with,
e.g., bind to, a non-N-kinase molecule, such as a downstream
molecule in the axonal outgrowth signaling pathway; (c) the ability
of N-kinase to bind ATP or Mn.sup.+2; or (d) the ability of
N-kinase to modulate the axonal outgrowth of central nervous system
neurons.
[0032] In a preferred embodiment, the compound that modulates the
activity of N-kinase acts downstream of AF-1 or other growth
factors in the axonal outgrowth signaling pathway. The ability of
the compound to act downstream of AF-1 or other growth factors in
the axonal outgrowth signaling pathway may be determined using one
of the assays described herein. For example, once a compound has
been determined to be capable of stimulating the activity of
N-kinase (e.g., stimulating the N-kinase dependent phosphorylation
of a substrate), N-kinase may be contacted both with this compound
and with 6-thioguanine. The inability of 6-thioguanine to inhibit
the stimulatory effect of the compound would indicate that the
compound is acting downstream of 6-thioguanine in the axonal
outgrowth signaling pathway, whereas the ability of 6-thioguanine
to inhibit the stimulatory effect of the compound would indicate
that the compound is acting upstream (or at the same point) in the
signaling pathway. Alternatively, once a compound has been
determined to be capable of inhibiting the activity of N-kinase
(e.g., inhibiting the N-kinase dependent phosphorylation of a
substrate), N-kinase may be contacted both with this compound and
with inosine. The inability of inosine to counteract the inhibitory
effect of the compound would indicate that the compound is acting
downstream of inosine in the axonal outgrowth signaling pathway,
whereas the ability of inosine to counteract the inhibitory effect
of the compound would indicate that the compound is acting upstream
(or at the same point) in the signaling pathway.
[0033] In certain embodiments of the invention, the compound that
modulates the activity of N-kinase can be any compound with the
proviso that it is not a purine base (e.g., guanine, inosine,
adenosine, and xanthine), such as a purine base linked to sugars,
such as ribose, deoxyribose, and analogs and derivatives thereof.
In certain other embodiments of the invention, the compound that
modulates the activity of N-kinase can be any compound with the
proviso that it is not a purine base analog or derivative
thereof.
[0034] Examples of compounds that modulate the activity of N-kinase
include small molecules, the N-kinase polypeptide or fragments
thereof, an anti-N-kinase antibody, an antisense N-kinase nucleic
acid molecule, a ribozyme, or the N-kinase gene or fragments
thereof.
[0035] As used herein, the term "small molecule" includes, but is
not limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e.,.
including heteroorganic and organometallic compounds) having a
molecular weight less than about 10,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 5,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 1,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 500 grams per
mole, and salts, esters, and other pharmaceutically acceptable
forms of such compounds.
[0036] As used herein, the language "modulating the axonal
outgrowth of central nervous system neurons" is intended to include
the capacity to stimulate or inhibit axonal outgrowth of central
nervous system neurons to various levels, e.g., to levels which
allow for the treatment of a targeted CNS condition.
[0037] As used herein, the term "outgrowth" (i.e., axonal
outgrowth) refers to the process by which axons grow out of a CNS
neuron. The outgrowth can result in a totally new axon or the
repair of a partially damaged axon. Outgrowth is typically
evidenced by extension of an axonal process of at least 5 cell
diameters in length. Moreover, axonal outgrowth can be evidenced by
GAP-43 expression (which can be detected by, for example,
immunostaining).
[0038] As used herein, the term "CNS neurons" is intended to
include the neurons of the brain and the spinal cord which are
unresponsive to nerve growth factor (NGF). The term is not intended
to include support or protection cells such as astrocytes,
oligodentrocytes, microglia, ependyma and the like, nor is it
intended to include peripheral nervous system (e.g., somatic,
autonomic, sympathetic or parasympathetic nervous system) neurons.
Preferred CNS neurons are mammalian neurons, more preferably human
neurons.
[0039] As used herein, the language "contacting" is intended to
include both in vivo or in vitro methods of bringing a compound
that modulates the activity of N-kinase into proximity with a CNS
neuron, such that the compound that modulates the activity of
N-kinase can modulate the outgrowth of axonal processes from the
CNS neuron.
[0040] As used herein, the term "subject" is intended to include
animals susceptible to conditions characterized by aberrant, e.g.,
insufficient, axonal outgrowth of central nervous system neurons,
preferably mammals, most preferably humans. In a preferred
embodiment, the subject is a primate. In an even more preferred
embodiment, the primate is a human. Other examples of subjects
include dogs, cats, goats, and cows.
[0041] As used herein, the term "condition characterized by
aberrant axonal outgrowth of central nervous system neurons" is
intended to include a disease, disorder, or condition which is
caused or characterized by an aberrant, e.g., increased,
insufficient, inadequate, or decreased, axonal outgrowth of central
nervous system neurons. Such conditions directly or indirectly
affect the normal functioning of the central nervous system (CNS).
A condition characterized by aberrant, e.g., insufficient, axonal
outgrowth of central nervous system neurons includes, but is not
limited to, an injury to the optic nerve, e.g., affecting retinal
ganglion cells; traumatic brain injury; stroke; cerebral aneurism;
spinal cord injury, including monoplegia, diplegia, paraplegia,
hemiplegia and quadriplegia; neuroproliferative disorders, e.g.,
Alzheimer's disease, dementias related to Alzheimer's disease (such
as Pick's disease), Parkinson's and other Lewy diffuse body
diseases, multiple sclerosis, amyotrophic lateral sclerosis,
progressive supranuclear palsy, epilepsy, Jakob-Creutzfieldt
disease, or AIDS related dementias; epilepsy, e.g., posttraumatic
brain injury; or neuropathic pain syndrome.
[0042] As used herein, the term "stroke" is art recognized and is
intended to include sudden diminution or loss of consciousness,
sensation, and voluntary motion caused by rapture or obstruction
(e.g., by a blood clot) of an artery of the brain.
[0043] As used herein, the term "Traumatic Brain Injury" is art
recognized and is intended to include the condition in which, a
traumatic blow to the head causes damage to the brain or connecting
spinal cord, often without penetrating the skull. Usually, the
initial trauma can result in expanding hematoma, subarachnoid
hemorrhage, cerebral edema, raised intracranial pressure (ICP), and
cerebral hypoxia, which can, in turn, lead to severe secondary
events due to low cerebral blood flow (CBF).
[0044] In another aspect, the invention features a method for
modulating, e.g., stimulating or inhibiting, axonal outgrowth of a
central nervous system neuron (such as a mammalian central nervous
system neuron) by contacting the central nervous system neuron with
a compound that modulates the activity of N-kinase, thereby
modulating axonal outgrowth of the central nervous system
neuron.
[0045] In yet another aspect, the invention features a method for
modulating the axonal outgrowth of a central nervous system neuron
in a subject, by administering to the subject a compound that
modulates the activity of N-kinase, such that axonal outgrowth in
the subject is modulated.
[0046] In yet another aspect, the invention features an isolated
N-kinase polypeptide of the type that: (a) is present in neonatal
brain tissue (e.g., neonatal human, bovine, rat, or mouse brain
tissue); (b) is inhibited by 6-thioguanine; (c) is activated by
Mn.sup.+2 but not by Mg.sup.+2 or Ca.sup.+2; (d) has a molecular
weight of approximately 49 kDa; and (e) is eluted from a Cibacron
Blue column at a NaCl concentration of 1.5-1.75 M. As used herein,
an "isolated" N-kinase polypeptide is substantially free (i.e.,
greater than 95% free) of cellular material or other contaminating
proteins from the cell or tissue source from which the N-kinase
protein is derived, or substantially free from chemical precursors
or other chemicals when chemically synthesized. The language
"substantially free of cellular material" includes preparations of
N-kinase in which the protein is separated from cellular components
of the cells from which it is isolated or recombinantly produced.
In one embodiment, the language "substantially free of cellular
material" includes preparations of N-kinase protein having less
than about 20% (by dry weight) of non-N-kinase protein (i.e.,
contaminating protein), more preferably less than about 10% of
non-N-kinase protein, still more preferably less than about 5% of
non-N-kinase protein, and most preferably less than about 3%
non-N-kinase protein. When the N-kinase protein or biologically
active portion thereof is recombinantly produced, it is also
preferably substantially free of culture medium, i.e., culture
medium represents less than about 20%, more preferably less than
about 10%, and most preferably less than about 5% of the volume of
the protein preparation.
[0047] Various aspects of the invention are described in further
detail in the following subsections:
Method for Identifying a Compound that Modulates Axonal Outgrowth
of a Central Nervous System Neuron
[0048] In one aspect, the invention features a method for
identifying a compound that modulates axonal outgrowth of a central
nervous system neuron by contacting N-kinase with a test compound
and determining the ability of the test compound to modulate the
activity of N-kinase, thereby identifying a compound that modulates
axonal outgrowth of a central nervous system neuron.
[0049] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[0050] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233.
[0051] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.).
[0052] In one embodiment, an assay is a cell-based assay in which a
cell which expresses an N-kinase protein or biologically active
portion thereof is contacted with a test compound and the ability
of the test compound to modulate N-kinase activity is determined.
Determining the ability of the test compound to modulate N-kinase
activity can be accomplished by monitoring, for example, the
production of one or more specific metabolites in a cell which
expresses N-kinase (see, e.g., Saada et al. (2000) Biochem Biophys.
Res. Commun. 269: 382-386). The cell, for example, can be of
mammalian origin, e.g., a neuronal cell.
[0053] Determining the ability of the test compound to modulate
N-kinase activity can further be accomplished by, for example,
determining the ability of N-kinase to phosphorylate a substrate.
The ability of N-kinase to phosphorylate a substrate (e.g., a
histone HF-1 protein) can be determined by, for example, an in
vitro kinase assay. Briefly, the N-kinase can be incubated with the
substrate and radioactive ATP, e.g., [.gamma.-.sup.32P] ATP, in a
buffer containing MnCl.sub.2, e.g., 5 mM MnCl.sub.2. Following the
incubation, the substrate can be immunoprecipitated or precipitated
with TCA or collected on a filter (if no other kinases are present)
and separated by SDS-polyacrylamide gel electrophoresis under
reducing conditions, transferred to a membrane, e.g., a PVDF
membrane, and autoradiographed. The appearance of detectable bands
on the autoradiograph indicates that the substrate has been
phosphorylated. Alternatively, the in-gel assays described in
Example 1 may be used to determine the ability of N-kinase to
phosphorylate a substrate. Phosphoaminoacid analysis of the
phosphorylated substrate can also be performed in order to
determine which residues on the protein are phosphorylated.
Briefly, the radiophosphorylated protein band can be excised from
the SDS gel and subjected to partial acid hydrolysis. The products
can then be separated by one-dimensional electrophoresis and
analyzed on, for example, a phosphoimager and compared to
ninhydrin-stained phosphoaminoacid standards.
[0054] The ability of the test compound to modulate N-kinase
binding to a non-N-kinase molecule, such as a downstream molecule
in the axonal outgrowth signaling pathway, can also be determined.
Determining the ability of the test compound to modulate N-kinase
binding to a non-N-kinase molecule can be accomplished, for
example, by coupling the non-N-kinase molecule with a radioisotope
or enzymatic label such that binding of the non-N-kinase molecule
to N-kinase can be determined by detecting the labeled non-N-kinase
molecule in a complex.
[0055] It is also within the scope of this invention to determine
the ability of a test compound to interact with, e.g., bind to,
N-kinase or biologically active portions thereof. Preferred
biologically active portions of the N-kinase proteins to be used in
assays of the present invention include fragments which participate
in interactions with non-N-kinase molecules, e.g., fragments with
high surface probability scores. Determining the ability of the
test compound to bind N-kinase can be accomplished, for example, by
coupling the compound with a radioisotope or enzymatic label such
that binding of the compound to N-kinase can be determined by
detecting the labeled N-kinase compound in a complex. For example,
test compounds can be labeled with .sup.125I, .sup.35S, .sup.14C,
or .sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemmission or by scintillation
counting. Alternatively, test compounds can be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to
product.
[0056] Determining the ability of a test compound to interact with
N-kinase may also be accomplished without the labeling of any of
the interactants. For example, a microphysiometer can be used to
detect the interaction of a compound with N-kinase without the
labeling of either the compound or the N-kinase. McConnell, H. M.
et al. (1992) Science 257:1906-1912. As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between a compound and N-kinase.
[0057] Determining the ability of the test compound to bind to
N-kinase or a biologically active portion thereof, can also be
accomplished using a technology such as real-time Biomolecular
Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)
Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.
Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for
studying biospecific interactions in real time, without labeling
any of the interactants (e.g., BIAcore). Changes in the optical
phenomenon of surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
[0058] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of N-kinase can be
accomplished by determining the ability of the N-kinase protein to
further modulate the activity of a downstream effector of an
N-kinase target molecule. For example, the activity of the effector
molecule on an appropriate target can be determined or the binding
of the effector to an appropriate target can be determined as
previously described.
[0059] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize any of the
reactants, e.g., N-kinase or a non-N-kinase molecule, to facilitate
separation of complexed from uncomplexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to N-kinase, or interaction of N-kinase
with a non-N-kinase molecule in the presence and absence of a test
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/N-kinase fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed non-N-kinase molecule or N-kinase, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of N-kinase binding or activity
determined using standard techniques.
[0060] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either N-kinase or a non-N-kinase molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated
N-kinase or non-N-kinase molecule can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with N-kinase
or a non-N-kinase molecule but which do not interfere with the
binding of N-kinase to its target non-N-kinase molecule can be
derivatized to the wells of the plate, and unbound non-N-kinase
molecule or N-kinase trapped in the wells by antibody conjugation.
Methods for detecting such complexes, in addition to those
described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with
N-kinase or a non-N-kinase molecule, as well as enzyme-linked
assays which rely on detecting an enzymatic activity associated
with N-kinase or a non-N-kinase target molecule.
[0061] In another embodiment, modulators of N-kinase expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of N-kinase mRNA or protein in the cell
is determined. The level of expression of N-kinase mRNA or protein
in the presence of the candidate compound is compared to the level
of expression of N-kinase mRNA or protein in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of N-kinase expression based on this comparison. For
example, when expression of N-kinase mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of N-kinase mRNA or protein expression.
Alternatively, when expression of N-kinase mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of N-kinase mRNA or protein expression. The level
of N-kinase mRNA or protein expression in the cells can be
determined by methods described herein for detecting N-kinase mRNA
or protein.
[0062] In yet another aspect of the invention, the N-kinase
proteins can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with N-kinase
("N-kinase-binding proteins" or "N-kinase-bp") and are involved in
N-kinase activity. Such N-kinase-binding proteins are also likely
to be involved in the propagation of signals by the N-kinase
proteins or N-kinase targets as, for example, downstream elements
of an N-kinase-mediated signaling pathway. Alternatively, such
N-kinase-binding proteins are likely to be N-kinase inhibitors.
[0063] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for an N-kinase
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming an N-kinase-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the N-kinase protein.
[0064] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating compound can be identified using a cell-based or a cell
free assay, and the ability of the compound to modulate the
activity of an N-kinase protein can be confirmed in vivo, e.g., in
an animal such as an animal model for a condition characterized by
aberrant, e.g., insufficient axonal outgrowth of central nervous
system neurons. Examples of such animal models are described in,
for example, Benowitz et al. (1999) PNAS 96(23): 13486-90. Epilepsy
animal models are also known in the art.
[0065] This invention further pertains to novel compounds
identified by the above-described screening assays. Accordingly, it
is within the scope of this invention to further use a compound
identified as described herein in an appropriate animal model. For
example, a compound identified as described herein (e.g., an
N-kinase modulating compound, an antisense N-kinase nucleic acid
molecule, an N-kinase-specific antibody, or an N-kinase-binding
partner) can be used in an animal model to determine the efficacy,
toxicity, or side effects of treatment with such a compound.
Alternatively, a compound identified as described herein can be
used in an animal model to determine the mechanism of action of
such a compound. Furthermore, this invention pertains to uses of
novel compounds identified by the above-described screening assays
for treatments as described herein.
[0066] In one embodiment, the N-kinase used in the methods of the
invention is a human N-kinase, such as a recombinantly produced
N-kinase. N-kinase, e.g., human N-kinase, may be introduced into a
recombinant expression vector using standard techniques and
expressed in prokaryotic or eukaryotic cells. For example,
N-kinase, e.g., human N-kinase, can be expressed in bacterial cells
such as E. coli, insect cells (using baculovirus expression
vectors) yeast cells or mammalian cells. Suitable host cells are
discussed further in Goeddel, Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. (1990).
Alternatively, a recombinant expression vector containing N-kinase,
e.g., human N-kinase, can be transcribed and translated in vitro,
for example using T7 promoter regulatory sequences and T7
polymerase.
[0067] In another embodiment, the N-kinase used in the methods of
the invention is a bovine N-kinase, such as an N-kinase which is
purified from a bovine source, e.g., neonatal bovine brain tissue,
as described herein in, for instance, Example 1.
[0068] In another embodiment, the method of the invention further
includes determining the ability of the test compound to modulate
axonal outgrowth of a central nervous system neuron. Determining
the ability of the test compound to modulate axonal outgrowth of a
central nervous system neuron can be accomplished by, for example,
using dissociated cultures of purified rat retinal ganglion cells.
Dissociated cultures of purified rat retinal ganglion cells can,
for example, be prepared by immunopanning as described in Barres et
al., Neuron, 1: 791-803,1988, the contents of which are
incorporated herein by reference. In brief, retinas from
Sparague-Dawley rats can be dissociated using papain activated with
cysteine. Macrophages are removed by incubation with an anti-rat
macrophage antibody (Accurate) followed by immunopanning with an
anti-rabbit IgG antibody. Ganglion cells are isolated by
immunopanning with an anti-Thy-1 antibody, then dislodged with
trypsin for use in low-density cultures. Rat retinal ganglion cells
are maintained at 37.degree. C in a CO.sub.2 incubator using the
same medium described above except for the presence of 30 mM
bicarbonate.
[0069] Samples are plated in quadruplicate in randomized positions
of a 24-well plate, contacted with the test compound, and the code
is concealed to ensure that growth is evaluated in a blinded
fashion. Each experiment may contain 4 wells of a negative control
(media plus supplements only) and 4 wells of a positive control
(e.g., a standardized AF-1 sample of known activity). Growth and
survival are assessed after 6 days for all ganglion cells in 25
consecutive fields of each well using phase contrast microscopy at
400.times. magnification (c. 150 ganglion cells counted per well).
Extension of a process 5 cell diameters in length is used as the
criterion for growth, since it clearly distinguishes stimulated
cells from negative controls (Schwalb et al., 1995). After the
completion of counting, the code is broken, the data tabulated, and
means and standard errors are calculated for the 4 replicate wells
of each sample using Cricket Graph (CA Associates, Islandia, N.Y.).
Data are normalized by subtracting the growth in the negative
controls (usually 4-5%) and dividing by the net growth in the
positive controls.
[0070] Goldfish retinal ganglion cells (Benowitz et al. (1998) J.
Biol. Chem. 273(45):29626-34) as well as mixtures of rat and
goldfish ganglion cells may also be used.
Compounds that Modulate Axonal Outgrowth of a Central Nervous
System Neuron
[0071] In another aspect, the invention features a compound that
modulates axonal outgrowth of a central nervous system neuron
identified by any of the foregoing methods.
[0072] In one embodiment, the compound that modulates axonal
outgrowth of a central nervous system neuron is an antisense
N-kinase nucleic acid molecule. An "antisense" nucleic acid
comprises a nucleotide sequence which is complementary to a "sense"
nucleic acid encoding a protein, e.g., complementary to the coding
strand of a double-stranded cDNA molecule or complementary to an
mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen
bond to a sense nucleic acid. The antisense nucleic acid can be
complementary to the entire N-kinase coding strand, or to only a
portion thereof. In one embodiment, an antisense nucleic acid
molecule is antisense to a "coding region" of the coding strand of
a nucleotide sequence encoding an N-kinase. The term "coding
region" refers to the region of the nucleotide sequence comprising
codons which are translated into amino acid residues. In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding N-kinase. The term "noncoding region" refers to 5' and 3'
sequences which flank the coding region that are not translated
into amino acids (i.e., also referred to as 5' and 3' untranslated
regions).
[0073] Given the coding strand sequence encoding N-kinase,
antisense nucleic acids of the invention can be designed according
to the rules of Watson and Crick base pairing. The antisense
nucleic acid molecule can be complementary to the entire coding
region of N-kinase mRNA, but more preferably is an oligonucleotide
which is antisense to only a portion of the coding or noncoding
region of N-kinase mRNA. For example, the antisense oligonucleotide
can be complementary to the region surrounding the translation
start site of N-kinase mRNA. An antisense oligonucleotide can be,
for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50
nucleotides in length. An antisense nucleic acid of the invention
can be constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0074] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding an N-kinase protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention include direct injection at a tissue site, e.g., in
the brain. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface. e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[0075] In yet another embodiment, the antisense N-kinase nucleic
acid molecule may be an .alpha.-anomeric nucleic acid molecule. An
.alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0076] In still another embodiment, the compound that modulates
axonal outgrowth of a central nervous system neuron is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity
which are capable of cleaving a single-stranded nucleic acid, such
as an mRNA, to which they have a complementary region. Thus,
ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and
Gerlach (1988) Nature 334:585-591)) can be used to catalytically
cleave N-kinase mRNA transcripts to thereby inhibit translation of
N-kinase mRNA. A ribozyme having specificity for an
N-kinase-encoding nucleic acid can be designed based upon the
nucleotide sequence of an N-kinase cDNA. For example, a derivative
of a Tetrahymena L-19 IVS RNA can be constructed in which the
nucleotide sequence of the active site is complementary to the
nucleotide sequence to be cleaved in an N-kinase-encoding mRNA.
See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.
U.S. Pat. No. 5,116,742. Alternatively, N-kinase mRNA can be used
to select a catalytic RNA having a specific ribonuclease activity
from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.
W. (1993) Science 261:1411-1418.
[0077] Alternatively, N-kinase gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the N-kinase (e.g., the N-kinase promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the N-kinase gene in target cells. See generally,
Helene, C. (1991) Anticancer Drug Des. 6(6): 569-84; Helene, C. et
al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)
Bioassays 14(12):807-15.
[0078] In still another embodiment, the compound that modulates
axonal outgrowth of a central nervous system neuron is an
anti-N-kinase antibody. A full-length N-kinase protein or,
alternatively, antigenic peptide fragments of N-kinase may be used
as immunogens to generate anti-N-kinase antibodies. The antigenic
peptide of N-kinase comprises at least 8 amino acid residues of the
amino acid sequence shown in SEQ ID NO:1 and encompasses an epitope
of N-kinase such that an antibody raised against the peptide forms
a specific immune complex with the N-kinase protein. Preferably,
the antigenic peptide comprises at least 10 amino acid residues,
more preferably at least 15 amino acid residues, even more
preferably at least 20 amino acid residues, and most preferably at
least 30 amino acid residues.
[0079] Preferred epitopes encompassed by the antigenic peptide are
regions of N-kinase that are located on the surface of the protein,
e.g., hydrophilic regions, as well as regions with high
antigenicity.
[0080] An N-kinase immunogen typically is used to prepare
antibodies by immunizing a suitable subject, (e.g., rabbit, goat,
mouse or other mammal) with the immunogen. An appropriate
immunogenic preparation can contain, for example, recombinantly
expressed N-kinase protein or a chemically synthesized N-kinase
polypeptide. The preparation can further include an adjuvant, such
as Freund's complete or incomplete adjuvant, or similar
immunostimulatory agent. Immunization of a suitable subject with an
immunogenic N-kinase preparation induces a polyclonal anti-N-kinase
antibody response.
[0081] The term "antibody" as used herein includes immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
which specifically binds (immunoreacts with) an antigen, such as an
N-kinase. Examples of immunologically active portions of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin. The invention provides polyclonal and monoclonal
antibodies that bind N-kinase molecules. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of N-kinase. A monoclonal antibody composition
thus typically displays a single binding affinity for a particular
N-kinase protein with which it immunoreacts.
[0082] Polyclonal anti-N-kinase antibodies can be prepared as
described above by immunizing a suitable subject with an N-kinase
immunogen. The anti-N-kinase antibody titer in the immunized
subject can be monitored over time by standard techniques, such as
with an enzyme linked immunosorbent assay (ELISA) using immobilized
N-kinase. If desired, the antibody molecules directed against
N-kinase can be isolated from the mammal (e.g., from the blood) and
further purified by well known techniques, such as protein A
chromatography to obtain the IgG fraction. At an appropriate time
after immunization, e.g., when the anti-N-kinase antibody titers
are highest, antibody-producing cells can be obtained from the
subject and used to prepare monoclonal antibodies by standard
techniques, such as the hybridoma technique originally described by
Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et
al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol.
Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological
Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A.
Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.
(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell
line (typically a myeloma) is fused to lymphocytes (typically
splenocytes) from a mammal immunized with an N-kinase immunogen as
described above, and the culture supernatants of the resulting
hybridoma cells are screened to identify a hybridoma producing a
monoclonal antibody that binds N-kinase.
[0083] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-N-kinase monoclonal antibody (see,
e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited
supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind N-kinase, e.g., using a
standard ELISA assay.
[0084] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-N-kinase antibody can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with N-kinase to thereby isolate immunoglobulin library members
that bind N-kinase. Kits for generating and screening phage display
libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT International Publication No. WO
92/18619; Dower et al. PCT International Publication No. WO
91/17271; Winter et al. PCT International Publication WO 92/20791;
Markland et al. PCT International Publication No. WO 92/15679;
Breitling et al. PCT International Publication WO 93/01288;
McCafferty et al. PCT International Publication No. WO 92/01047;
Garrard et al. PCT International Publication No. WO 92/09690;
Ladner et al. PCT International Publication No. WO 90/02809; Fuchs
et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins
et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991)
Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377;
Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al.
(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et
al. Nature (1990) 348:552-554.
[0085] Additionally, recombinant anti-N-kinase antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in Robinson et al. International Application No.
PCT/US86/02269; Akira, et al. European Patent Application 184,187;
Taniguchi, M., European Patent Application 171,496; Morrison et al.
European Patent Application 173,494; Neuberger et al. PCT
International Publication No. WO 86/01533; Cabilly et al. U.S. Pat.
No. 4,816,567; Cabilly et al. European Patent Application 125,023;
Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc.
Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.
139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA
84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et
al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl.
Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science
229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S.
Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;
Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988)
J. Immunol. 141:4053-4060.
[0086] In one embodiment, the compound that modulates axonal
outgrowth of a central nervous system neuron is an intracellular
antibody specific for N-kinase. The use of intracellular antibodies
to inhibit protein function in a cell is known in the art (see
e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca,
S. et al. (1990) EMBO J. 9:101-108; Werge, T. M. et al. (1990) FEBS
Letters 274:193-198; Carlson, J. R. (1993) Proc. Natl. Acad. Sci.
USA 90:7427-7428; Marasco, W. A. et al. (1993) Proc. Natl. Acad.
Sci. USA 90:7889-7893; Biocca, S. et al. (1994) Bio/Technology
12:396-399; Chen, S-Y. et al. (1994) Human Gene Therapy 5: 595-601;
Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA 91:5075-5079;
Chen, S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5932-5936;
Beerli, R. R. et al. (1994) J. Biol. Chem. 269:23931-23936; Beerli,
R. R. et al. (1994) Biochem. Biophys. Res. Commun. 204:666-672;
Mhashilkar, A. M. et al. (1995) EMBO J. 14:1542-1551; Richardson,
J. H. et al. (1995) Proc. Natl. Acad. Sci. USA 92:3137-3141; PCT
Publication No. WO 94/02610 by Marasco et al.; and PCT Publication
No. WO 95/03832 by Duan et al.).
[0087] To inhibit protein activity using an intracellular antibody,
a recombinant expression vector is prepared which encodes the
antibody chains in a form such that, upon introduction of the
vector into a cell, the antibody chains are expressed as a
functional antibody in an intracellular compartment of the
cell.
[0088] In still another embodiment, the compound that modulates
axonal outgrowth of a central nervous system neuron is a small
molecule. As used herein, the term "small molecule" includes, but
is not limited to, peptides, peptidomimetics, amino acids, amino
acid analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0089] In still another embodiment, the compound that modulates
axonal outgrowth of a central nervous system neuron is an N-kinase
polypeptide or portion thereof, e.g., a fragment that includes at
least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200
contiguous amino acids of the N-kinase polypeptide. For example,
the compound could comprise the constitutively active catalytic
domain of the N-kinase.
[0090] In still another embodiment, the compound that modulates
axonal outgrowth of a central nervous system neuron is an N-kinase
gene or portion thereof.
Pharmaceutically Acceptable Formulations
[0091] Pharmaceutical compositions, and packaged formulations,
comprising a composition of the invention (e.g., compound that
modulates the activity of N-kinase) and a pharmaceutically
acceptable carrier are also provided by the invention. In the
method of the invention, the compound that modulates the activity
of N-kinase can be administered in a pharmaceutically acceptable
formulation. Such pharmaceutically acceptable formulation typically
includes the compound that modulates the activity of N-kinase as
well as a pharmaceutically acceptable carrier(s) and/or
excipient(s). As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and anti fungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
For example, the carrier can be suitable for injection into the
cerebrospinal fluid. Excipients include pharmaceutically acceptable
stabilizers and disintegrants. The present invention pertains to
any pharmaceutically acceptable formulations, including synthetic
or natural polymers in the form of macromolecular complexes,
nanocapsules, microspheres, or beads, and lipid-based formulations
including oil-in-water emulsions, micelles, mixed micelles,
synthetic membrane vesicles, and resealed erythrocytes.
[0092] In one embodiment, the pharmaceutically acceptable
formulations comprise a polymeric matrix. The terms "polymer" or
"polymeric" are art-recognized and include a structural framework
comprised of repeating monomer units which is capable of delivering
a compound that modulates the activity of N-kinase such that
treatment of a targeted condition, e.g., a CNS injury, occurs. The
terms also include co-polymers and homopolymers e.g., synthetic or
naturally occurring. Linear polymers, branched polymers, and
cross-linked polymers are also meant to be included.
[0093] For example, polymeric materials suitable for forming the
pharmaceutically acceptable formulation employed in the present
invention, include naturally derived polymers such as albumin,
alginate, cellulose derivatives, collagen, fibrin, gelatin, and
polysaccharides, as well as synthetic polymers such as polyesters
(PLA, PLGA), polyethylene glycol, poloxomers, polyanhydrides, and
pluronics. These polymers are biocompatible with the nervous
system, including the central nervous system, they are
biodegradable within the central nervous system without producing
any toxic byproducts of degradation, and they possess the ability
to modify the manner and duration of compound that modulates the
activity of N-kinase release by manipulating the polymer's kinetic
characteristics. As used herein, the term "biodegradable" means
that the polymer will degrade over time by the action of enzymes,
by hydrolytic action and/or by other similar mechanisms in the body
of the subject. As used herein, the term "biocompatible" means that
the polymer is compatible with a living tissue or a living organism
by not being toxic or injurious and by not causing an immunological
rejection.
[0094] Polymers can be prepared using methods known in the art
(Sandler, S. R.; Karo, W. Polymer Syntheses; Harcourt Brace:
Boston, 1994; Shalaby, W.; Ikada, Y.; Langer, R.; Williams, J.
Polymers of Biological and Biomedical Significance (ACS Symposium
Series 540; American Chemical Society: Washington, D.C., 1994).
Polymers can be designed to be flexible; the distance between the
bioactive side-chains and the length of a linker between the
polymer backbone and the group can be controlled. Other suitable
polymers and methods for their preparation are described in U.S.
Pat. Nos. 5,455,044 and 5,576,018, the contents of which are
incorporated herein by reference.
[0095] The polymeric formulations can be formed by dispersion of
the compound that modulates the activity of N-kinase within
liquefied polymer, as described in U.S. Pat. No. 4,883,666, the
teachings of which are incorporated herein by reference or by such
methods as bulk polymerization, interfacial polymerization,
solution polymerization and ring polymerization as described in
Odian G., Principles of Polymerization and ring opening
polymerization, 2nd ed., John Wiley & Sons, New York, 1981, the
contents of which are incorporated herein by reference. The
properties and characteristics of the formulations are controlled
by varying such parameters as the reaction temperature,
concentrations of polymer and compound that modulates the activity
of N-kinase, types of solvent used, and reaction times.
[0096] The compound that modulates the activity of N-kinase can be
encapsulated in one or more pharmaceutically acceptable polymers,
to form a microcapsule, microsphere, or microparticle, terms used
herein interchangeably. Microcapsules, microspheres, and
microparticles are conventionally free-flowing powders consisting
of spherical particles of 2 millimeters or less in diameter,
usually 500 microns or less in diameter. Particles less than 1
micron are conventionally referred to as nanocapsules,
nanoparticles or nanospheres. For the most part, the difference
between a microcapsule and a nanocapsule, a microsphere and a
nanosphere, or microparticle and nanoparticle is size; generally
there is little, if any, difference between the internal structure
of the two. In one aspect of the present invention, the mean
average diameter is less than about 45 .mu.m, preferably less than
20 .mu.m, and more preferably between about 0.1 and 10 .mu.m.
[0097] In another embodiment, the pharmaceutically acceptable
formulations comprise lipid-based formulations. Any of the known
lipid-based drug delivery systems can be used in the practice of
the invention. For instance, multivesicular liposomes (MVL),
multilamellar liposomes (also known as multilamellar vesicles or
"MLV"), unilamellar liposomes, including small unilamellar
liposomes (also known as unilamellar vesicles or "SUV") and large
unilamellar liposomes (also known as large unilamellar vesicles or
"LUV"), can all be used so long as a sustained release rate of the
encapsulated compound that modulates the activity of N-kinase or
analogue thereof can be established. In one embodiment, the
lipid-based formulation can be a multivesicular liposome system.
Methods of making controlled release multivesicular liposome drug
delivery systems is described in PCT Application Serial Nos.
US96/11642, US94/12957 and US94/04490, the contents of which are
incorporated herein by reference.
[0098] The composition of the synthetic membrane vesicle is usually
a combination of phospholipids, usually in combination with
steroids, especially cholesterol. Other phospholipids or other
lipids may also be used.
[0099] Examples of lipids useful in synthetic membrane vesicle
production include phosphatidylglycerols, phosphatidylcholines,
phosphatidylserines, phosphatidylethanolamines, sphingolipids,
cerebrosides, and gangliosides. Preferably phospholipids including
egg phosphatidylcholine, dipalmitoylphosphatidylcholine,
distearoylphosphatidylcholine, dioleoylphosphatidylcholine,
dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol
are used.
[0100] In preparing lipid-based vesicles containing a compound that
modulates the activity of N-kinase or analogue thereof, such
variables as the efficiency of compound that modulates the activity
of N-kinase encapsulation, lability of the compound that modulates
the activity of N-kinase, homogeneity and size of the resulting
population of vesicles, compound that modulates the activity of
N-kinase-to-lipid ratio, permeability, instability of the
preparation, and pharmaceutical acceptability of the formulation
should be considered (see Szoka, et al., Annual Reviews of
Biophysics and Bioengineering, 9:467, 1980; Deamer, et al., in
Liposomes, Marcel Dekker, New York, 1983, 27; and Hope, et al.,
Chem. Phys. Lipids, 40:89, 1986, the contents of which are
incorporated herein by reference).
Administration of the Pharmaceutically Acceptable Formulation
[0101] The pharmaceutically acceptable formulations of the
invention are administered such that the compound that modulates
the activity of N-kinase, or analogue thereof, comes into contact
with central nervous system neurons to thereby modulate the axonal
outgrowth thereof. Both local and systemic administration of the
formulations are contemplated by the invention, although local
administration may be preferable to achieve effective local
concentrations of the compound that modulates the activity of
N-kinase, or analogue, as well as to avoid possible side effects
from systemic administration of the agent. In one embodiment, the
compound that modulates the activity of N-kinase is administered by
introduction into the central nervous system of the subject, e.g.,
into the cerebrospinal fluid of the subject. In certain aspects of
the invention, the compound that modulates the activity of N-kinase
is introduced intrathecally, e.g., into a cerebral ventricle, the
lumbar area, or the cisterna magna. In another aspect, the compound
that modulates the activity of N-kinase is introduced
intraocularly, to thereby contact retinal ganglion cells.
[0102] The pharmaceutically acceptable formulations can easily be
suspended in aqueous vehicles and introduced through conventional
hypodermic needles or using infusion pumps. Prior to introduction,
the formulations can be sterilized with, preferably, gamma
radiation or electron beam sterilization, described in U.S. Pat.
No. 436,742 the contents of which are incorporated herein by
reference.
[0103] In one embodiment, the compound that modulates the activity
of N-kinase formulation described herein is administered to the
subject in the period from the time of, for example, an injury to
the CNS up to about 100 or 200 hours after the injury has occurred,
for example, within 48, 36, 24, 12, or 6 hours from the time of
injury. In another embodiment, the compound that modulates the
activity of N-kinase is administered to a subject suffering from a
chronic injury to the CNS. Accordingly, the compound is
administered to the subject over the subject's life time.
[0104] In another embodiment of the invention, the compound that
modulates the activity of N-kinase formulation is administered into
a subject intrathecally. As used herein, the term "intrathecal
administration" is intended to include delivering a compound that
modulates the activity of N-kinase formulation directly into the
cerebrospinal fluid of a subject, by techniques including lateral
cerebroventricular injection through a burrhole or cisternal or
lumbar puncture or the like (described in Lazorthes et al. Advances
in Drug Delivery Systems and Applications in Neurosurgery, 143-192
and Omaya et al., Cancer Drug Delivery, 1: 169-179, the contents of
which are incorporated herein by reference). The term "lumbar
region" is intended to include the area between the third and
fourth lumbar (lower back) vertebrae. The term "cisterna magna" is
intended to include the area where the skull ends and the spinal
cord begins at the back of the head. The term "cerebral ventricle"
is intended to include the cavities in the brain that are
continuous with the central canal of the spinal cord.
Administration of a compound that modulates the activity of
N-kinase to any of the above mentioned sites can be achieved by
direct injection of the compound that modulates the activity of
N-kinase formulation or by the use of infusion pumps. For
injection, the compound that modulates the activity of N-kinase
formulation of the invention can be formulated in liquid solutions,
preferably in physiologically compatible buffers such as Hank's
solution or Ringer's solution. In addition, the compound that
modulates the activity of N-kinase formulation may be formulated in
solid form and re-dissolved or suspended immediately prior to use.
Lyophilized forms are also included. The injection can be, for
example, in the form of a bolus injection or continuous infusion
(e.g., using infusion pumps) of the compound that modulates the
activity of N-kinase formulation.
[0105] In one embodiment of the invention, the compound that
modulates the activity of N-kinase formulation is administered by
lateral cerebro ventricular injection into the brain of a subject,
preferably within 100 hours of when an injury (resulting in a
condition characterized by aberrant axonal outgrowth of central
nervous system neurons) occurs (e.g., within 6, 12, or 24 hours of
the time of the injury). The injection can be made, for example,
through a burr hole made in the subject's skull. In another
embodiment, the formulation is administered through a surgically
inserted shunt into the cerebral ventricle of a subject, preferably
within 100 hours of when an injury occurs (e.g., within 6, 12 or 24
hours of the time of the injury). For example, the injection can be
made into the lateral ventricles, which are larger, even though
injection into the third and fourth smaller ventricles can also be
made. In yet another embodiment, the compound that modulates the
activity of N-kinase formulation is administered by injection into
the cisterna magna, or lumbar area of a subject, preferably within
100 hours of when an injury occurs (e.g., within 6, 12, or 24 hours
of the time of the injury).
[0106] In another embodiment of the invention, the compound that
modulates the activity of N-kinase formulation is administered to a
subject at the site of injury, preferably within 100 hours of when
an injury occurs (e.g., within 6, 12, or 24 hours of the time of
the injury).
Duration and Levels of Administration
[0107] In a preferred embodiment of the method of the invention,
the compound that modulates the activity of N-kinase, or analog
thereof, is contacted with CNS neurons for an extended period of
time to effect modulation of axonal outgrowth. Sustained contact
with the compound that modulates the activity of N-kinase, or
analog, can be achieved by, for example, repeated administration of
the compound that modulates the activity of N-kinase or analog over
a period of time, such as one week, several weeks, one month or
longer. More preferably, the pharmaceutically acceptable
formulation used to administer the compound that modulates the
activity of N-kinase, or analog, provides sustained delivery, e.g.,
"slow release" of the compound that modulates the activity of
N-kinase, or analog, to a subject. For example, the formulation may
deliver the compound that modulates the activity of N-kinase, or
analog, for at least one, two, three, or four weeks after the
pharmaceutically acceptable formulation is administered to the
subject. Preferably, a subject to be treated in accordance with the
present invention is treated with the compound that modulates the
activity of N-kinase, or analog, for at least 30 days (either by
repeated administration or by use of a sustained delivery system,
or both).
[0108] As used herein, the term "sustained delivery" is intended to
include continual delivery of a compound that modulates the
activity of N-kinase or analogue thereof in vivo over a period of
time following administration, preferably at least several days, a
week, several weeks, one month or longer. Sustained delivery of the
compound that modulates the activity of N-kinase or analogue
thereof can be demonstrated by, for example, the continued
therapeutic effect of the compound that modulates the activity of
N-kinase or analogue thereof over time (e.g., sustained delivery of
the compound that modulates the activity of N-kinase or analogue
thereof can be demonstrated by continued outgrowth or by continued
inhibition of outgrowth of CNS neurons over time). Alternatively,
sustained delivery of the compound that modulates the activity of
N-kinase or analogue thereof may be demonstrated by detecting the
presence of the compound that modulates the activity of N-kinase or
analogue thereof in vivo over time.
[0109] Preferred approaches for sustained delivery include use of a
polymeric capsule or a minipump to deliver the formulation.
Polymeric capsules can be prepared as described herein. Implantable
infusion pump systems (e.g., Infusaid; see e.g., Zierski, J. et al.
(1988) Acta Neurochem. Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am.
Osteopath. Assoc. 94:487-493) and osmotic pumps (sold by Alza
Corporation) are available in the art. Another mode of
administration is via an implantable, externally programmable
infusion pump. Suitable infusion pump systems and reservoir systems
are also described in U.S. Pat. No. 5, 368,562 by Blomquist and
U.S. Pat. No. 4,731,058 by Doan, developed by Pharmacia Deltec
Inc.
[0110] The pharmaceutical formulation, used in the method of the
invention, contains a therapeutically effective amount of the
compound that modulates the activity of N-kinase or analogue
thereof. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired result. A therapeutically effective amount of the
compound that modulates the activity of N-kinase or analogue
thereof may vary according to factors such as the disease state,
age, and weight of the subject, and the ability of the compound
that modulates the activity of N-kinase or analogue thereof (alone
or in combination with one or more other agents) to elicit a
desired response in the subject. Dosage regimens may be adjusted to
provide the optimum therapeutic response. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the compound that modulates the activity of N-kinase or
analogue thereof are outweighed by the therapeutically beneficial
effects. A non-limiting dosage range is about 5 .mu.M-1000 .mu.M,
although the particular optimal dosage will vary depending upon,
among other factors, the particular compound that modulates the
activity of N-kinase, or analogue thereof, used.
[0111] It is to be noted that dosage values may vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compound that modulates the
activity of N-kinase or analogue thereof and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed invention.
[0112] The invention, in another embodiment, provides a
pharmaceutical composition consisting essentially of a compound
that modulates the activity of N-kinase and a pharmaceutically
acceptable carrier, as well as methods of use thereof to modulate
axonal outgrowth by contacting CNS neurons with the composition. By
the term "consisting essentially of" is meant that the
pharmaceutical composition does not contain any other modulators of
neuronal growth such as, for example, nerve growth factor (NGF). In
one embodiment, the pharmaceutical composition of the invention can
be provided as a packaged formulation. The packaged formulation may
include a pharmaceutical composition of the invention in a
container and printed instructions for administration of the
composition for treating a subject having a disorder associated
with an injury of central nervous system neurons, e.g., an injury
to retinal ganglion cells, a spinal cord injury or a traumatic
brain injury. Use of the compound that modulates the activity of
N-kinases, and analogues thereof, of the invention in the
manufacture of a medicament for modulating the outgrowth of CNS
neurons (e.g., mammalian CNS neurons) is also encompassed by the
invention.
In Vitro Treatment of CNS Neurons
[0113] CNS neurons can further be contacted with a compound that
modulates the activity of N-kinase, in vitro to modulate axonal
outgrowth in vitro. Accordingly, CNS neuron cells can be isolated
from a subject and grown in vitro, using techniques well known in
the art, and then treated in accordance with the present invention
to modulate axonal outgrowth. Briefly, a CNS neuron cell culture
can be obtained by allowing neuron cells to migrate out of
fragments of neural tissue adhering to a suitable substrate (e.g.,
a culture dish) or by disaggregating the tissue, e.g., mechanically
or enzymatically, to produce a suspension of CNS neuron cells. For
example, the enzymes trypsin, collagenase, elastase, hyaluronidase,
DNase, pronase, dispase, or various combinations thereof can be
used. Trypsin and pronase give the most complete disaggregation but
may damage the cells. Collagenase and dispase give a less complete
disaggregation but are less harmful. Methods for isolating tissue
(e.g., neural tissue) and the disaggregation of tissue to obtain
cells (e.g., CNS neuron cells) are described in Freshney R. I.,
Culture of Animal Cells, A Manual of Basic Technique, Third
Edition, 1994, the contents of which are incorporated herein by
reference.
[0114] Such cells can be subsequently contacted with a compound
that modulates the activity of N-kinase in amounts and for a
duration of time as described above. Once modulation of axonal
outgrowth has been achieved in the CNS neuron cells, these cells
can be re-administered to the subject, e.g., by implantation. It is
preferred that the cells are not allowed to differentiate
extensively in vitro, as cells that integrate most successfully in
a subject are primitive cells.
[0115] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, patents and published patent
applications cited throughout this application, as well as the
Figures and the Sequence Listing are hereby incorporated by
reference.
EXAMPLES
Example I
Isolation and Characterization of the N-kinase Polypeptide
[0116] Neocortical gray matter from bovine brain was homogenized in
buffer containing protease and phosphatase inhibitors, and
particulate material was centrifuged down. The soluble fraction
from approximately 1 kg of tissue was used as the starting material
for isolation of the kinase.
[0117] During the purification process, kinase activity was
monitored using an in-gel kinase method. In this method, a histone
HF-1 substrate protein is polymerized into a 10% polyacrylamide gel
before the samples to be analyzed are electrophoresed in the gel.
Following completion of the electrophoresis, proteins are partially
renatured with guanidium isothiocyanate and incubated in the
presence [.sup.32P]-ATP plus Mn.sup.2+, with or without 6-TG
present. (Activation by Mn.sup.2+, but not by Mg.sup.2+ or
Ca.sup.2+, is a distinctive property of N-kinase). The purification
of the kinase was monitored by looking for a 6-TG-inhibitable
radioactive band corresponding to the site where the kinase
phosphorylates the HF-1 substrate in the gel.
[0118] In the first step of the purification process the starting
material was subjected to cation-exchange chromatography (using a
Fast-S column, Pharmacia). A 6-TG inhibitable kinase activity bound
strongly to this column and eluted with 0.3 M NaCl. The
cation-exchange column fraction containing N-kinase was
subsequently separated on a Cibacron Blue column (Pharmacia), which
allows for the separation of adenine nucleotide-binding proteins. A
6-TG inhibitable, 47-50 kDa polypeptide bound strongly and required
a NaCl concentration of 1.5 M NaCl for elution (see FIG. 1A).
[0119] The eluted fraction containing the N-kinase polypeptide was,
then, subjected to reversed-phase chromatography with a C4
hydrophobic interaction column (Pharmacia). Briefly, the Cibacron
Blue column fraction containing the N-kinase was applied to the C4
column and eluted with a gradient of increasing
acetonitrile-isopropanol concentration. Evaluation of the kinase
activity of the column fractions by in-gel kinase assays showed
that the 6-TG-inhibitable, HF-1-phosphorylating activity eluted in
fractions 24-26 (FIG. 1B). To achieve a higher level of
purification, these fractions were pooled, re-applied to the same
column, and the separation was repeated with a gradient of
increasing acetonitrile-isopropanol concentration. The N-kinase
polypeptide, again, eluted at fractions 24-26.
[0120] The column fractions containing the highest concentrations
of N-kinase were lyophilized and applied to a 10% polyacrylamide
SDS gel. A small portion of the sample was run on a parallel gel to
carry out in-gel kinase assays. A band at 49 kDa was clearly
visible after staining the gel with Coomassie blue; this coincided
in its migration position with the 6-TG-inhibitable kinase
activity. This band was cut out and it was verified that it
contained HF-1-phosphorylating activity.
[0121] The gel band containing the N-kinase polypeptide was then
subjected to partial proteolytic digestion, the proteolytic
fragments were analyzed by mass spectroscopy and the masses of the
fragments compared to those of various known peptides using the
process described in, for example, Eng J. K. et al. (1994) J. Am.
Soc. Mass. Spectrom. 5:976-989; Chittum H. S. et al. (1998)
Biochemistry 37:10866-870; and LeRoy G. et al. (1998) Science
282:1900-04, the contents of which are incorporated herein by
reference.
[0122] This analysis revealed that N-kinase is an isoform of MST-3,
i.e., either MST-3 itself, MST-3b, or an as yet undefined isoform.
These proteins are members of the STE family of serine-threonine
kinases that are found throughout the animal kingdom. STE family
members are generally components of modular signaling cassettes
that are involved in various aspects of cellular differentiation.
FIG. 2 depicts the amino acid sequence of the N-kinase. Direct
matches between the purified protein and the published sequence are
shown in blue. K65 (bold type) lies in the ATP-binding region of
the kinase domain.
Equivalents
[0123] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
1 1 272 PRT Homo sapiens 1 Met Asp Ser Arg Ala Trp Gly Ala Asn Lys
Arg Arg Ala Thr His Gly 1 5 10 15 Gly Ser Thr Asn Lys Ala Asp Thr
Lys Lys Gly Lys Gly Ser Gly Val 20 25 30 Lys Gly Asp Asn Arg Thr
Lys Val Val Ala Lys Asp Ala Asp Asp Thr 35 40 45 Val Ser Cys Asp
Ser Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Lys Asp 50 55 60 Thr Lys
Trp Met Tyr Gly Gly Gly Ser Ala Asp Gly Asp Thr Ala Thr 65 70 75 80
Arg Lys Gly Asp Tyr His Ser Lys Lys His Arg Asp Lys Ala Ala Asn 85
90 95 Val Ser His Gly Val Lys Ala Asp Gly Val Ala Gly Thr Asp Thr
Lys 100 105 110 Arg Asn Thr Val Gly Thr Trp Met Ala Val Lys Ser Ala
Tyr Asp Ser 115 120 125 Lys Ala Asp Trp Ser Gly Thr Ala Ala Arg Gly
His Ser His Met Lys 130 135 140 Val Lys Asn Asn Thr Gly Asn Tyr Ser
Lys Lys Val Ala Cys Asn Lys 145 150 155 160 Ser Arg Thr Ala Lys Lys
His Lys Arg Asn Ala Lys Lys Thr Ser Tyr 165 170 175 Thr Asp Arg Tyr
Lys Arg Trp Lys Ala Ser His Asp Asp Ser Ser Ser 180 185 190 Asp Ser
Asp Ala Thr Asp Gly Ala Ser Gly Gly Ser Asp Ser Gly Asp 195 200 205
Trp Thr Arg Lys Asp Lys Asn Asn Gly Ala Ser Asp Asp Arg Asn Lys 210
215 220 Met Lys Asp Lys Arg Ser Cys Ser Thr Ser Ala Lys Lys Ser Ala
Cys 225 230 235 240 Gly Gly Asn Gly Ser Arg Gly Ala Tyr Ala Ala Cys
Gly Ser Asp Thr 245 250 255 Met Val Ala Val Arg Arg Tyr Ser Ser Gly
Gly Gly Thr Ser Ser His 260 265 270
* * * * *