U.S. patent application number 09/949200 was filed with the patent office on 2002-10-31 for methods and compositions for producing a neurosalutary effect in a subject.
This patent application is currently assigned to Children's Medical Center Corporation. Invention is credited to Benowitz, Larry I..
Application Number | 20020160933 09/949200 |
Document ID | / |
Family ID | 24635087 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160933 |
Kind Code |
A1 |
Benowitz, Larry I. |
October 31, 2002 |
Methods and compositions for producing a neurosalutary effect in a
subject
Abstract
Methods and compositions for producing a neurosalutary effect in
a subject are provided. These methods generally involve
administering to a subject a therapeutically effective amount of a
compound that modulates the activity of N-kinase, or analog
thereof. Pharmaceutical and packaged formulations including the
compounds of the invention, e.g., compounds that modulate the
activity of N-kinase, are also provided.
Inventors: |
Benowitz, Larry I.; (Newton
Centre, MA) |
Correspondence
Address: |
NIXON PEABODY LLP
101 FEDERAL ST
BOSTON
MA
02110
US
|
Assignee: |
Children's Medical Center
Corporation
300 Longwood Avenue
Boston
MA
02115
|
Family ID: |
24635087 |
Appl. No.: |
09/949200 |
Filed: |
September 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09949200 |
Sep 7, 2001 |
|
|
|
09656915 |
Sep 7, 2000 |
|
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Current U.S.
Class: |
514/1 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
9/10 20180101; A61P 25/24 20180101; A61P 25/02 20180101; G01N
33/6896 20130101; A61P 43/00 20180101; A61P 25/16 20180101; A61P
25/18 20180101; A61P 25/00 20180101; A61P 25/28 20180101; A61P
25/08 20180101; G01N 2500/00 20130101; C12N 9/1205 20130101; A61P
25/14 20180101 |
Class at
Publication: |
514/1 |
International
Class: |
A61K 031/00 |
Claims
We claim:
1. A method comprising administering to a subject a therapeutically
effective amount of a compound that modulates the activity of
N-kinase, thereby producing a neurosalutary effect in said
subject.
2. The method of claim 1, wherein the neurosalutary effect is
produced in said subject by modulating neuronal survival.
3. The method of claim 1, wherein the neurosalutary effect is
produced in said subject by modulating neuronal regeneration.
4. The method of claim 1, wherein the neurosalutary effect is
produced in said subject by modulating neuronal axonal
outgrowth.
5. The method of claim 1, wherein the neurosalutary effect is
produced in said subject by modulating axonal outgrowth of central
nervous system neurons.
6. The method of claim 5, wherein the central nervous system
neurons are retinal ganglion cells.
7. The method of claim 1, wherein the compound that modulates the
activity of N-kinase is administered by introduction into a region
of neuronal injury.
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 region selected from the
group consisting of a cerebral ventricle, the lumbar area, and the
cisterna magna of the subject.
11. 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.
12. The method of claim 11, wherein the pharmaceutically acceptable
formulation is a dispersion system.
13. The method of claim 11, wherein the pharmaceutically acceptable
formulation comprises a lipid-based formulation.
14. The method of claim 13, wherein the pharmaceutically acceptable
formulation comprises a liposome formulation.
15. The method of claim 13, wherein the pharmaceutically acceptable
formulation comprises a multivesicular liposome formulation.
16. The method of claim 11, wherein the pharmaceutically acceptable
formulation comprises a polymeric matrix.
17. The method of claim 11, wherein the pharmaceutically acceptable
formulation is contained within a minipump.
18. The method of claim 11, 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.
19. 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.
20. The method of claim 1, wherein the subject is a mammal.
21. The method of claim 20, wherein the mammal is a human.
22. The method of claim 1, wherein said subject is suffering from a
neurological disorder.
23. The method of claim 22, wherein said neurological disorder is a
spinal cord injury.
24. The method of claim 23, wherein the spinal cord injury is
characterized by monoplegia, diplegia, paraplegia, hemiplegia and
quadriplegia.
25. The method of claim 22, wherein said neurological disorder is
epilepsy.
26. The method of claim 22, wherein said neurological disorder is
stroke.
27. The method of claim 22, wherein said neurological disorder is
Alzheimer's disease.
28. A method comprising administering a therapeutically effective
amount of a compound that modulates the activity of N-kinase to a
subject suffering from a neurological disorder, thereby treating
said subject suffering from a neurological disorder.
29. The method of claim 28, further comprising making a first
assessment of a nervous system function prior to administering the
compound that modulates the activity of N-kinase to the subject and
making a second assessment of the nervous system function after
administering the compound that modulates the activity of N-kinase
to the subject.
30. The method of claim 29, wherein the nervous system function is
a sensory function, cholinergic innervation, or a vestibulomotor
function.
31. A method for identifying a compound capable of producing a
neurosalutary effect in a subject, comprising contacting N-kinase,
or a biologically active fragment thereof, with a test compound and
determining the ability of the test compound to modulate the
activity of N-kinase, thereby identifying a compound capable of
producing a neurosalutary effect in a subject.
32. The method of claim 31, wherein the N-kinase is human
N-kinase.
33. The method of claim 32, wherein the human N-kinase is a
recombinantly produced N-kinase.
34. The method of claim 31, wherein the N-kinase is bovine
N-kinase.
35. The method of claim 34, wherein the bovine N-kinase is purified
from a bovine source.
36. The method of claim 31, further comprising determining the
ability of the test compound to modulate axonal outgrowth of a
central nervous system neuron.
37. The method of claim 31, wherein the test compound inhibits the
activity of N-kinase.
38. The method of claim 31, wherein the test compound stimulates
the activity of N-kinase.
39. The method of claim 31, 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.
40. A method for identifying a compound capable of producing a
neurosalutary effect in a subject, comprising contacting N-kinase
or a biologically active fragment thereof, 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
capable of producing a neurosalutary effect in a subject.
41. The method of claim 40, wherein the N-kinase substrate is a
histone HF-1 protein.
42. The method of claim 40, wherein the radioactive ATP is
[.gamma.-.sup.32P] ATP.
43. The method of claim 40, wherein the N-kinase is human
N-kinase.
44. The method of claim 43, wherein the human N-kinase is a
recombinantly produced N-kinase.
45. The method of claim 40, wherein the N-kinase is bovine
N-kinase.
46. The method of claim 45, wherein the bovine N-kinase is purified
from a bovine source.
47. The method of claim 40, further comprising determining the
ability of the test compound to modulate axonal outgrowth of a
central nervous system neuron.
48. A compound capable of producing a neurosalutary effect in a
subject identified by the method of claim 40.
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 in the presence of 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.
58. An isolated nucleic acid molecule that encodes the polypeptide
of SEQ ID NO:1.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. application Ser.
No. 09/656,915, filed on Sep. 7, 2000, the entire contents of which
are incorporated herein by this reference.
BACKGROUND OF THE INVENTION
[0002] Disorders of the peripheral and central nervous system are
widespread, and for many of these conditions effective therapeutic
interventions are lacking.
SUMMARY OF THE INVENTION
[0003] The present invention provides methods and compositions for
producing a neurosalutary effect in a subject, e.g., a subject
suffering from a neurological condition. Such an effect includes
promoting neuronal survival, axonal outgrowth, neuronal
regeneration or normalized neurological function in a subject. The
present 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.
The identification of N-kinase as a critical intracellular mediator
of axonal outgrowth, and the chemical characterization of its
structure, now provides for the ability to produce a neurosalutary
effect in a subject by modulating N-kinase activity. Furthermore,
this purification and characterization of N-kinase now allows for
its use in screening assays to identify additional compounds
capable of producing a neurosalutary effect in a subject.
[0004] Accordingly, in one aspect, the present invention provides a
method which includes administering to a subject a therapeutically
effective amount of a compound that modulates the activity of
N-kinase, thereby producing a neurosalutary effect in the
subject.
[0005] In one aspect, the compound that modulates the activity of
N-kinase is administered to a subject in accordance with the
present invention such that the compound is brought into contact
with neurons of the central nervous system of the subject. For
example, the compound may be administered into the cerebrospinal
fluid of the subject into the intrathecal space by introducing the
compound into a cerebral ventricle, the lumbar area, or the
cisterna magna. In such circumstances, the compound that modulates
the activity of N-kinase can be administered locally to cortical
neurons or retinal ganglion cells to produce a neurosalutary
effect.
[0006] In certain embodiments, the compound that modulates the
activity of N-kinase may be administered to a subject using a
pharmaceutically acceptable formulation. The pharmaceutically
acceptable formulation may allow for sustained delivery, providing
effective amounts of the compound that modulates the activity of
N-kinase to a subject for at least one week, or in other
embodiments, at least one month, after the pharmaceutically
acceptable formulation is initially administered to the subject.
Approaches for achieving sustained delivery of a compound of the
invention include the use of a slow release polymeric capsule, a
bioerodible matrix, or an infusion pump that disperses the
compounds of the invention. The infusion pump may be implanted
subcutaneously, intracranially, or in other locations as would be
medically desirable. In certain embodiments, the compounds of the
invention would be dispensed by the infusion pump via a catheter
either into the cerebrospinal fluid, or to a site where local
delivery was desired, such as a site of neuronal injury or a site
of neurodegenerative changes.
[0007] 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.
[0008] In yet another aspect, the invention features a method which
includes administering a therapeutically effective amount of a
compound that modulates the activity of N-kinase to a subject
suffering from a neurological disorder, thereby treating the
subject suffering from a neurological disorder. In one embodiment,
the method further includes making a first assessment of a nervous
system function, e.g., a sensory function, cholinergic innervation,
or a vestibulomotor function, prior to administering the compound
that modulates the activity of N-kinase to the subject and making a
second assessment of the nervous system function after
administering the compound to the subject.
[0009] In a further aspect, the invention features a method for
identifying a compound capable of producing a neurosalutary effect
in a subject by contacting N-kinase, or a biologically active
fragment thereof, with a test compound and determining the ability
of the test compound to modulate the activity of N-kinase, thereby
identifying a compound capable of producing a neurosalutary effect
in a subject. 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.
[0010] 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.
[0011] 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.
[0012] In another aspect, the invention features a method for
identifying a compound capable of producing a neurosalutary effect
in a subject. The method includes contacting N-kinase, or a
biologically active fragment thereof, 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
capable of producing a neurosalutary effect in a subject. 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.
[0013] In another aspect, the invention features a compound capable
of producing a neurosalutary effect in a subject identified by any
of the foregoing methods.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] In a further aspect, the invention features an isolated
nucleic acid molecule that encodes the polypeptide of SEQ ID
NO:1.
[0018] Pharmaceutical compositions, and packaged formulations,
comprising a composition of the invention (e.g., a compound that
modulates the activity of N-kinase) and a pharmaceutically
acceptable carrier are also provided by the invention.
[0019] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] 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.
[0021] 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
[0022] The present invention provides methods and compositions for
producing a neurosalutary effect in a subject, e.g., a subject
suffering from a neurological disorder. 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.
[0023] Accordingly, the present invention is directed to methods
which include administering to a subject a therapeutically
effective amount of a compound that modulates the activity of
N-kinase, thereby producing a neurosalutary effect in the
subject.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] As used herein, a "neurosalutary effect" means a response or
result favorable to the health or function of a neuron, of a part
of the nervous system, or of the nervous system generally. Examples
of such effects include improvements in the ability of a neuron or
portion of the nervous system to resist insult, to regenerate, to
maintain desirable function, to grow or to survive. The phrase
"producing a neurosalutary effect" includes producing or effecting
such a response or improvement in function or resilience within a
component of the nervous system. For example, examples of producing
a neurosalutary effect would include stimulating axonal outgrowth
after injury to a neuron; rendering a neuron resistant to
apoptosis; rendering a neuron resistant to a toxic compound such as
.beta.-amyloid, ammonia, or other neurotoxins; reversing
age-related neuronal atrophy or loss of function; or reversing
age-related loss of cholinergic innervation.
[0031] 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 neurological disorder.
[0032] 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).
[0033] 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.
[0034] The term "administering" to a subject includes dispensing,
delivering or applying an active compound in a pharmaceutical
formulation to a subject by any suitable route for delivery of the
active compound to the desired location in the subject, including
delivery by either the parenteral or oral route, intramuscular
injection, subcutaneous/intradermal injection, intravenous
injection, buccal administration, transdermal delivery and
administration by the rectal, colonic, vaginal, intranasal or
respiratory tract route.
[0035] 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.
[0036] As used herein, the term "subject" is intended to include
animals. In particular embodiments, the subject is a mammal, a
human or nonhuman primate, a dog, a cat, a horse, a cow or a
rodent.
[0037] As used herein, the term "effective amount" includes an
amount effective, at dosages and for periods of time necessary, to
achieve the desired result, such as sufficient to produce a
neurosalutary effect in a subject. An effective amount of an active
compound as defined herein may vary according to factors such as
the disease state, age, and weight of the subject, and the ability
of the active compound to elicit a desired response in the subject.
Dosage regimens may be adjusted to provide the optimum therapeutic
response. An effective amount is also one in which any toxic or
detrimental effects of the active compound are outweighed by the
therapeutically beneficial effects.
[0038] A therapeutically effective amount or dosage of an active
compound may range from about 0.001 to 30 mg/kg body weight, with
other ranges of the invention including about 0.01 to 25 mg/kg body
weight, about 0.1 to 20 mg/kg body weight, about 1 to 10 mg/kg, 2
to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, and 5 to 6 mg/kg body
weight. The skilled artisan will appreciate that certain factors
may influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of an active
compound can include a single treatment or a series of treatments.
In one example, a subject is treated with an active compound in the
range of between about 0.1 to 20 mg/kg body weight, one time per
week for between about 1 to 10 weeks, alternatively between 2 to 8
weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks.
It will also be appreciated that the effective dosage of an active
compound used for treatment may increase or decrease over the
course of a particular treatment.
[0039] "Neurological disorder" is intended to include a disease,
disorder, or condition which directly or indirectly affects the
normal functioning or anatomy of a subject's nervous system.
Elements of the nervous system subject to disorders which may be
effectively treated with the compounds and methods of the invention
include the central, peripheral, somatic, autonomic, sympathetic
and parasympathetic components of the nervous system, neurosensory
tissues within the eye, ear, nose, mouth or other organs, as well
as glial tissues associated with neuronal cells and structures.
Neurological disorders may be caused by an injury to a neuron, such
as a mechanical injury or an injury due to a toxic compound, by the
abnormal growth or development of a neuron, or by the misregulation
(such as downregulation or upregulation) of an activity of a
neuron. Neurological disorders can detrimentally affect nervous
system functions such as the sensory function (the ability to sense
changes within the body and the outside environment); the
integrative function (the ability to interpret the changes); and
the motor function (the ability to respond to the interpretation by
initiating an action such as a muscular contraction or glandular
secretion). Examples of neurological disorders include traumatic or
toxic injuries to peripheral or cranial nerves, spinal cord or to
the brain, cranial nerves, traumatic brain injury, stroke,
ischemia, cerebral aneurism, and spinal cord injury. Other
neurological disorders include cognitive and neurodegenerative
disorders such as Alzheimer's disease, dementias related to
Alzheimer's disease (such as Pick's disease), Parkinson's and other
Lewy diffuse body diseases, senile dementia, Huntington's disease,
Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic
lateral sclerosis, hereditary motor and sensory neuropathy
(Charcot-Marie-Tooth disease), diabetic neuropathy, progressive
supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease.
Autonomic function disorders include hypertension and sleep
disorders. Also to be treated with compounds and methods of the
invention are neuropsychiatric disorders such as depression,
schizophrenia, schizoaffective disorder, Korsakoff's psychosis,
mania, anxiety disorders, or phobic disorders, learning or memory
disorders (such as amnesia and age-related memory loss), attention
deficit disorder, autism, dysthymic disorder, major depressive
disorder, mania, obsessive-compulsive disorder, psychoactive
substance use disorders, anxiety, phobias, panic disorder, bipolar
affective disorder, psychogenic pain syndromes, and eating
disorders. Other examples of neurological disorders include
injuries to the nervous system due to an infectious disease (such
as meningitis, high fevers of various etiologies, HIV, syphilis, or
post-polio syndrome) and injuries to the nervous system due to
electricity (including contact with electricity or lightning, and
complications from electro-convulsive psychiatric therapy). The
developing brain is a target for neurotoxicity in the developing
central nervous system through many stages of pregnancy as well as
during infancy and early childhood, and the methods of the
invention may be utilized in preventing or treating neurological
deficits in embryos or fetuses in utero, in premature infants, or
in children with need of such treatment, including those with
neurological birth defects. Further neurological disorders include,
for example, those listed in Harrison's Principles of Internal
Medicine (Braunwald et al., McGraw-Hill, 2001) and in the American
Psychiatric Association's Diagnostic and Statistical Manual of
Mental Disorders DSM-IV (American Psychiatric Press, 2000) both
incorporated herein by reference in their entirety.
[0040] The term "stroke" is art recognized and is intended to
include sudden diminution or loss of consciousness, sensation, and
voluntary motion caused by rupture or obstruction (for example, by
a blood clot) of an artery of the brain.
[0041] "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, and cerebral hypoxia, which can, in turn,
lead to severe secondary events due to low cerebral blood flow.
[0042] In 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.
[0043] In yet another aspect, the invention features an isolated
nucleic acid molecule encoding an N-kinase polypeptide, e.g., the
N-kinase polypeptide of SEQ ID NO:1.
[0044] Various aspects of the invention are described in further
detail in the following subsections:
[0045] Methods for Identifying a Compound Capable of Producing a
Neurosalutary Effect in a Subject
[0046] In one aspect, the invention features a method for
identifying a compound capable of producing a neurosalutary effect
in a subject by contacting an N-kinase polypeptide, or a
biologically active fragment thereof, with a test compound and
determining the ability of the test compound to modulate the
activity of N-kinase, thereby identifying a compound capable of
producing a neurosalutary effect in a subject.
[0047] 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).
[0048] 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. U.S.A. 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.
[0049] 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.).
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] The ability of a test compound to produce a neurosalutary
effect in a subject may further be assessed using any of a variety
of known procedures and assays. For example, the ability of a test
compound to re-establish neural connectivity and/or function after
an injury, such as a CNS injury, may be determined histologically
(either by slicing neuronal tissue and looking at neuronal
branching, or by showing cytoplasmic transport of dyes). The
ability of compounds of the invention to re-establish neural
connectivity and/or function after an injury, such as a CNS injury,
may also be assessed by monitoring the ability of the test compound
to fully or partially restore the electroretinogram after damage to
the neural retina or optic nerve; or to fully or partially restore
a pupillary response to light in the damaged eye.
[0070] Other tests that may be used to determine the ability of an
N-kinase modulator to produce a neurosalutary effect in a subject
include standard tests of neurological function in human subjects
or in animal models of spinal injury (such as standard reflex
testing, urologic tests, urodynamic testing, tests for deep and
superficial pain appreciation, proprioceptive placing of the hind
limbs, ambulation, and evoked potential testing). In addition,
nerve impulse conduction can be measured in a subject, such as by
measuring conduct action potentials, as an indication of the
production of a neurosalutary effect.
[0071] Animal models suitable for use in the assays of the present
invention include the rat model of partial transection (described
in Weidner et al. (2001) Proc. Natl. Acad. Sci. USA 98:3513-3518).
This animal model tests how well a compound can enhance the
survival and sprouting of the intact remaining fragment of an
almost fully-transected cord. Accordingly, after administration of
the N-kinase modulator these animals may be evaluated for recovery
of a certain function, such as how well the rats may manipulate
food pellets with their forearms (to which the relevant cord had
been cut 97%).
[0072] Another animal model suitable for use in the assays of the
present invention includes the rat model of stroke (described in
Kawamata et al. (1997) Proc. Natl. Acad. Sci. USA
94(15):8179-8184). This paper describes in detail various tests
that may be used to assess sensorimotor function in the limbs as
well as vestibulomotor function after an injury. Administration to
these animals of an N-kinase modulator of the invention can be used
to assess whether a given compound, route of administration, or
dosage provides a neurosalutary effect, such as increasing the
level of function, or increasing the rate of regaining function or
the degree of retention of function in the test animals.
[0073] Standard neurological evaluations used to assess progress in
human patients after a stroke may also be used to evaluate the
ability of an N-kinase modulator to produce a neurosalutary effect
in a subject. Such standard neurological evaluations are routine in
the medical arts, and are described in, for example, "Guide to
Clinical Neurobiology" Edited by Mohr and Gautier (Churchill
Livingstone Inc. 1995).
[0074] For assessing function of the peripheral nervous system,
standard tests include electromyography, nerve conduction velocity
measurements, evoked potentials assessment and upper/lower
extremity somato-sensory evoked potentials. Electromyography tests
record the electrical activity in muscles, and is used to assess
the function of the nerves and muscles. The electrode is inserted
into a muscle to record its electrical activity. It records
activity during the insertion, while the muscle is at rest, and
while the muscle contracts. The nerve conduction velocity test
evaluates the health of the peripheral nerve by recording how fast
an electrical impulse travels through it. A peripheral nerve
transmits information between the spinal cord and the muscles. A
number of nervous system diseases may reduce the speed of this
impulse. Electrodes placed on the skin detect and record the
electrical signal after the impulse travels along the nerve. A
second stimulating electrode is sends a small electrical charge
along the nerve; the time between the stimulation and response will
be recorded to determine how quickly and thoroughly the impulse is
sent.
[0075] Standard tests for function of the cranial nerves, as known
to those skilled in the neurological medical art, include facial
nerve conduction studies; orbicularis oculi reflex studies (blink
reflex studies); trigeminal-facial nerve reflex evaluation as used
in focal facial nerve lesions, Bell's palsy, trigeminal neuralgia
and atypical facial pain; evoked potentials assessment; visual,
brainstem and auditory evoked potential measurements;
thermo-diagnostic small fiber testing; and computer-assisted
qualitative sensory testing.
[0076] Compounds Capable of Producing a Neurosalutary Effect in a
Subject
[0077] In another aspect, the invention features a compound capable
of producing a neurosalutary effect in a subject identified by any
of the foregoing methods.
[0078] In one embodiment, the compound capable of producing a
neurosalutary effect in a subject 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).
[0079] 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-carboxymethylaminomet- hyluracil, 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-isopenten- yladenine,
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).
[0080] 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.
[0081] 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).
[0082] 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. Nos. 4,987,071; and 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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 SurfZAP.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 2:725-734; 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/Technology 9:1373-1377;
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.
[0091] 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.
[0092] In one embodiment, the compound capable of producing a
neurosalutary effect in a subject 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.).
[0093] 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.
[0094] In still another embodiment, the compound capable of
producing a neurosalutary effect in a subject 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.
[0095] In still another embodiment, the compound capable of
producing a neurosalutary effect in a subject 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.
[0096] In still another embodiment, the compound capable of
producing a neurosalutary effect in a subject is an N-kinase
nucleic acid molecule or a portion thereof.
[0097] In a preferred embodiment, the compound that modulates the
activity of N-kinase is capable of entering a cell and acting
intracellularly. For example, membrane translocation domains may be
used to guide peptidic modulators of the N-kinase into a cell. Such
membrane translocation domains are known in the art and include,
but are not limited to, the third helix of the antennapedia
homeodomain protein and the HIV-1 protein Tat and are described in,
for example, Derossi et al., (1994) J. Biol. Chem. 269,
10444-10450; Lindgren et al., (2000) Trends Pharmacol. Sci. 21,
99-103; Ho et al., Cancer Research 61, 474-477 (2001); U.S. Pat.
Nos. 5,888,762; 6,015,787; 5,846,743; 5,747,641; 5,804,604; and
Published PCT applications WO 98/52614, WO 00/29427 and WO
99/29721. The entire contents of each of the foregoing references
are incorporated herein by reference.
[0098] Further art known techniques for facilitating the transfer
of the compound that modulates the activity of N-kinase (i.e., the
N-kinase modulator) into a cell include those described in, for
example, Lindgren M et al. (2000) Trends Pharmacol Sci.
21(3):99-103; Gariepy J. et al. (2001) Trends Biotechnol
19(1):21-28; Vyas S P et al. (2001) Crit Rev Ther Drug Carrier Syst
18(1):1-76; Morris M C et al. (2000) Curr Opin Biotechnol
11(5):461-466; and Derossi D et al. (1998) Trends Cell Biol.
8(2):84-87, the contents of all of which are incorporated herein by
reference.
[0099] Constructs expressing therapeutic genes, antisense
oligonucleotides and ribozymes can be delivered into cells by viral
vectors, as well as by non-viral delivery systems including those
described in, for example, Hope M J et al. (1998) Mol Membr Biol
15(1):1-14.
[0100] Pharmaceutically Acceptable Formulations
[0101] Pharmaceutical compositions and packaged formulations
comprising a compound that modulates the activity of N-kinase and a
pharmaceutically acceptable carrier are also provided by the
invention.
[0102] In a 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 may include the N-kinase modulator 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.
[0103] 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
an N-kinase modulator such that treatment of a targeted condition,
such as a neurological disorder, occurs. The terms also include
co-polymers and homopolymers such as synthetic or naturally
occurring. Linear polymers, branched polymers, and cross-linked
polymers are also meant to be included.
[0104] 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 the active compound 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. Polymers can be
prepared using methods known in the art.
[0105] The polymeric formulations can be formed by dispersion of
the active compound 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 the active compound, the types of
solvent used, and reaction times.
[0106] The active therapeutic compound 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.
[0107] 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,
multilamellar liposomes and unilamellar liposomes can all be used
so long as a sustained release rate of the encapsulated active
compound can be established. Methods of making controlled release
multivesicular liposome drug delivery systems are described in PCT
application Ser. Nos. US96/11642, US94/12957 and US94/04490, the
contents of which are incorporated herein by reference.
[0108] 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.
[0109] Examples of lipids useful in synthetic membrane vesicle
production include phosphatidylglycerols, phosphatidylcholines,
phosphatidylserines, phosphatidylethanolamines, sphingolipids,
cerebrosides, and gangliosides, with preferable embodiments
including egg phosphatidylcholine, dipalmitoylphosphatidylcholine,
distearoylphosphatidylcholine, dioleoylphosphatidylcholine,
dipalmitoylphosphatidylglycerol, and
dioleoylphosphatidylglycerol.
[0110] In preparing lipid-based vesicles containing an active
compound such variables as the efficiency of active compound
encapsulation, lability of the active compound, homogeneity and
size of the resulting population of vesicles, active
compound-to-lipid ratio, permeability, instability of the
preparation, and pharmaceutical acceptability of the formulation
should be considered.
[0111] Prior to introduction, the formulations can be sterilized,
by any of the umerous available techniques of the art, such as with
gamma radiation or electron beam sterilization.
[0112] Administration of the Pharmaceutically Acceptable
Formulation
[0113] The pharmaceutically acceptable formulations of the
invention are administered such that the active compound comes into
contact with a subject's nervous system to thereby produce a
neurosalutary effect. Both local and systemic administration of the
formulations are contemplated by the invention. Desirable features
of local administration include achieving effective local
concentrations of the active compound as well as avoiding adverse
side effects from systemic administration of the active compound.
In one embodiment, the active compound is administered by
introduction into the cerebrospinal fluid of the subject. In
certain aspects of the invention, the active compound is introduced
into a cerebral ventricle, the lumbar area, or the cisterna magna.
In another aspect, the active compound is introduced locally, such
as into the site of nerve or cord injury, into a site of pain or
neural degeneration, or intraocularly to contact neuroretinal
cells.
[0114] The pharmaceutically acceptable formulations can be
suspended in aqueous vehicles and introduced through conventional
hypodermic needles or using infusion pumps.
[0115] In one embodiment, the active compound 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 hours after
the injury has occurred, for example within 24, 12, or 6 hours from
the time of injury.
[0116] In another embodiment of the invention, the active compound
formulation is administered into a subject intrathecally. As used
herein, the term "intrathecal administration" is intended to
include delivering an active compound 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 an active compound to any of the above mentioned
sites can be achieved by direct injection of the active compound
formulation or by the use of infusion pumps. Implantable or
external pumps and catheter may be used.
[0117] For injection, the active compound 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 active compound 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 (such as using infusion pumps) of the active
compound formulation.
[0118] In one embodiment of the invention, the active compound
formulation is administered by lateral cerebroventricular injection
into the brain of a subject, preferably within 100 hours of when an
injury (resulting in a condition characterized by aberrant,
insufficient or inadequate axonal outgrowth of central nervous
system neurons) occurs (such as 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 (such as 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 active compound formulation is
administered by injection into the cisterna magna, or lumbar area
of a subject, preferably within 100 hours of when an injury occurs
(such as within 6, 12, or 24 hours of the time of the injury).
[0119] An additional means of administration to intracranial tissue
involves application of compounds of the invention to the olfactory
epithelium, with subsequent transmission to the olfactory bulb and
transport to more proximal portions of the brain. Such
administration can be by nebulized or aerosolized
prerparations.
[0120] In another embodiment of the invention, the active compound
formulation is administered to a subject at the site of injury,
preferably within 100 hours of when an injury occurs (such as
within 6, 12, or 24 hours of the time of the injury).
[0121] Duration and Levels of Administration
[0122] In a preferred embodiment of the method of the invention,
the active compound is administered to a subject for an extended
period of time to produce a neurosalutary effect, such as effect
modulation of axonal outgrowth. Sustained contact with the active
compound can be achieved by, for example, repeated administration
of the active compound 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
active compound provides sustained delivery, such as "slow release"
of the active compound to a subject. For example, the formulation
may deliver the active compound 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 active
compound for at least 30 days (either by repeated administration or
by use of a sustained delivery system, or both).
[0123] As used herein, the term "sustained delivery" is intended to
include continual delivery of the active compound 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 active compound can be demonstrated by, for
example, the continued therapeutic effect of the active compound
over time (such as sustained delivery of the active compound can be
demonstrated by continued production of a neurosalutary effect in a
subject). Alternatively, sustained delivery of the active compound
may be demonstrated by detecting the presence of the active
compound in vivo over time.
[0124] Preferred approaches for sustained delivery include use of a
polymeric capsule, a minipump to deliver the formulation, a
bioerodible implant, or implanted transgenic autologous cells (as
described in U.S. Pat. No. 6,214,622). Implantable infusion pump
systems (such as Infusaid; see such as 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.
[0125] 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 active compound and that
dosage ranges set forth herein are exemplary only and are not
intended to limit the scope or practice of the claimed
invention.
[0126] The invention, in another embodiment, provides a
pharmaceutical composition consisting essentially of an N-kinase
modulator 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
producing a neurosalutary effect in a subject having a neurological
disorder. Use of an N-kinase modulator derived factor in the
manufacture of a medicament for modulating the axonal outgrowth of
neurons is also encompassed by the invention.
[0127] In vitro Treatment of CNS Neurons
[0128] 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
dissagregation 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.
[0129] 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.
[0130] 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
[0131] Isolation and Characterization of the N-kinase
Polypeptide
[0132] 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.
[0133] 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.
[0134] 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).
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] Equivalents
[0140] 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
* * * * *