U.S. patent application number 10/145224 was filed with the patent office on 2002-09-12 for methods for modulating the axonal outgrowth of central nervous system neurons.
Invention is credited to Benowitz, Larry I..
Application Number | 20020128223 10/145224 |
Document ID | / |
Family ID | 25446158 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020128223 |
Kind Code |
A1 |
Benowitz, Larry I. |
September 12, 2002 |
Methods for modulating the axonal outgrowth of central nervous
system neurons
Abstract
Methods for modulating the axonal outgrowth of central nervous
system neurons are provided. Methods for stimulating the axonal
outgrowth of central nervous system neurons following an injury
(e.g., stroke, Traumatic Brain Injury, cerebral aneurism, spinal
cord injury and the like) and methods for inhibiting the axonal
outgrowth of central nervous system neurons are also provided.
Finally, a packed formulation comprising a pharmaceutical
composition comprising an inosine nucleoside and a pharmaceutically
acceptable carrier packed with instructions for use of the
pharmaceutical composition for treatment of a central nervous
system disorder is provided.
Inventors: |
Benowitz, Larry I.; (Newton
Centre, MA) |
Correspondence
Address: |
NIXON PEABODY LLP
101 FEDERAL ST
BOSTON
MA
02110
US
|
Family ID: |
25446158 |
Appl. No.: |
10/145224 |
Filed: |
May 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10145224 |
May 14, 2002 |
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08921902 |
Sep 2, 1997 |
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Current U.S.
Class: |
514/45 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/52 20130101; A61K 31/7076 20130101; A61P 25/00 20180101;
A61K 31/70 20130101; A61K 31/708 20130101; A61P 25/08 20180101 |
Class at
Publication: |
514/45 |
International
Class: |
A61K 031/708 |
Goverment Interests
[0001] Work described herein was supported, at least in part, under
grant R01EY05690 awarded by the National Eye Institute. The U.S.
government therefore may have certain rights in this invention.
Claims
We claim:
1. A method for modulating axonal outgrowth of central nervous
system neurons, the method comprising contacting the central
nervous system neurons with a pharmaceutical composition consisting
essentially of an effective amount of a purine nucleoside, or
analog thereof, such that axonal outgrowth is modulated.
2. The method of claim 1, wherein the outgrowth is stimulated.
3. The method of claim 2, wherein the purine nucleoside is
inosine.
4. The method of claim 2, wherein the purine nucleoside is
guanosine.
5. The method of claim 1, wherein the outgrowth is inhibited.
6. The method of claim 5, wherein the purine nucleoside is
6-thioguanine.
7. The method of claim 1, wherein said central nervous system
neurons are mammalian.
8. A method for stimulating the axonal outgrowth of central nervous
system neurons following an injury, comprising administering to a
subject a purine nucleoside, or analog thereof, such that axonal
outgrowth is stimulated.
9. The method of claim 8, wherein the injury is due to a stroke
episode.
10. The method of claim 8, wherein the injury is due to a Traumatic
Brain Injury (TBI) episode.
11. The method of claim 8, wherein the injury is due to a cerebral
aneurism.
12. The method of claim 8, wherein the injury is a spinal cord
injury.
13. The method of claim 12, wherein the spinal cord injury is
selected from the group consisting of monoplegia, diplegia,
paraplegia, hemiplegia and quadriplegia.
14. The method of claim 8, wherein the purine nucleoside or analog
thereof is administered by introduction into the central nervous
system of the subject.
15. The method of claim 14, wherein the purine nucleoside or analog
thereof is introduced into the cerebrospinal fluid of the
subject.
16. The method of claim 15, wherein the purine nucleoside or analog
thereof is introduced intrathecally.
17. The method of claim 15, wherein the purine nucleoside or analog
thereof is introduced into a cerebral ventricle.
18. The method of claim 15, wherein the purine nucleoside or analog
thereof is introduced into the lumbar area.
19. The method of claim 15, wherein the purine nucleoside or analog
thereof is introduced into the cisterna magna.
20. The method of claim 8, wherein the purine nucleoside is
inosine.
21. The method of claim 8, wherein the purine nucleoside is
guanosine.
22. The method of claim 8, wherein the subject is a mammal.
23. The method of claim 22, wherein the mammal is a human.
24. The method of claim 8, wherein the purine nucleoside or analog
thereof is administered in a pharmaceutically acceptable
formulation.
25. The method of claim 24, wherein the pharmaceutically acceptable
formulation is a dispersion system.
26. The method of claim 25, wherein the pharmaceutically acceptable
formulation comprises a lipid-based formulation.
27. The method of claim 26, wherein the pharmaceutically acceptable
formulation comprises a liposome formulation.
28. The method of claim 27, wherein the pharmaceutically acceptable
formulation comprises a multivesicular liposome formulation.
29. The method of claim 25, wherein the pharmaceutically acceptable
formulation comprises a polymeric matrix.
30. The method of claim 29, wherein the polymeric matrix is
selected from the group consisting of naturally derived polymers,
such as albumin, alginate, cellulose derivatives, collagen, fibrin,
gelatin, and polysaccharides.
31. The method of claim 29, wherein the polymeric matrix is
selected from the group consisting of synthetic polymers such as
polyesters (PLA, PLGA), polyethylene glycol, poloxomers,
polyanhydrides, and pluronics.
32. The method of claim 29, wherein the polymeric matrix is in the
form of microspheres.
33. The method of claim 24, wherein the pharmaceutically acceptable
formulation provides sustained delivery of the purine nucleoside to
a subject for at least one week after the pharmaceutically
acceptable formulation is administered to the subject.
34. The method of claim 24, wherein the pharmaceutically acceptable
formulation provides sustained delivery of the purine nucleoside to
a subject for at least two weeks after the pharmaceutically
acceptable formulation is administered to the subject.
35. The method of claim 24, wherein the pharmaceutically acceptable
formulation provides sustained delivery of the purine nucleoside to
a subject for at least three weeks after the pharmaceutically
acceptable formulation is administered to the subject.
36. The method of claim 24, wherein the pharmaceutically acceptable
formulation provides sustained delivery of the purine nucleoside to
a subject for at least four weeks after the pharmaceutically
acceptable formulation is administered to the subject.
37. The method of claim 20, wherein the inosine nucleoside is
administered at a concentration of 5-10 .mu.M.
38. The method of claim 20, wherein the inosine nucleoside is
administered at a concentration of 10-25 .mu.M.
39. The method of claim 20, wherein the inosine nucleoside is
administered at a concentration of 25-50 .mu.M.
40. The method of claim 21, wherein the guanosine nucleoside is
administered at a concentration of 25-50 .mu.M.
41. The method of claim 21, wherein the guanosine nucleoside is
administered at a concentration of 50-100 .mu.M.
42. The method of claim 21, wherein the guanosine nucleoside is
administered at a concentration of 100-150 .mu.M.
43. The method of claim 8, wherein the central nervous system
neurons are retinal ganglion cells.
44. A pa nervous system disorder.
Description
BACKGROUND OF THE INVENTION
[0002] Past early childhood, injury to the central nervous system
(CNS) results in functional impairments that are largely
irreversible. Within the brain or spinal cord, damage resulting
from stroke, trauma, or other causes can result in life-long losses
in cognitive, sensory and motor functions, and even maintenance of
vital functions. Nerve cells that are lost are not replaced, and
those that are spared are generally unable to regrow severed
connections, although a limited amount of local synaptic
reorganization can occur close to the site of injury. Functions
that are lost are currently untreatable.
[0003] Regenerative failure in the CNS has been attributed to a
number of factors, which include the presence of inhibitory
molecules on the surface of glial cells that suppress axonal
growth; absence of appropriate substrate molecules such as laminin
to foster growth and an absence of the appropriate trophic factors
needed to activate programs of gene expression required for cell
survival and differentiation.
[0004] By contrast, within the peripheral nervous system (PNS),
injured nerve fibers can regrow over long distances, with eventual
excellent recovery of function. Within the past 15 years,
neuroscientists have come to realize that this is not a consequence
of intrinsic differences between the nerve cells of the peripheral
and central nervous system; remarkably, neurons of the CNS will
extend their axons over great distances if given the opportunity to
grow through a grafted segment of PNS (e.g., sciatic nerve).
Therefore, neurons of the CNS retain a capacity to grow if given
the right signals from the extracellular environment. Factors which
contribute to the differing growth potentials of the CNS and PNS
include partially characterized, growth-inhibiting molecules on the
surface of the oligodendrocytes that surround nerve fibers in the
CNS, but which are less abundant in the comparable cell population
of the PNS (Schwann cells); molecules of the basal lamina and other
surfaces that foster growth in the PNS but which are absent in the
CNS (e.g., laminin); and trophic factors, soluble polypeptides
which activate programs of gene expression that underlie cell
survival and differentiation. Although such trophic factors are
regarded as essential for maintaining the viability and
differentiation of nerve cells, the particular ones that are
responsible for inducing axonal regeneration in the CNS remain
uncertain. As a result, to date, effective treatments for CNS
injuries have not been developed.
[0005] Accordingly, methods and compositions for modulating the
outgrowth of CNS neurons are still needed.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods and compositions for
modulating the axonal outgrowth of central nervous system neurons.
The invention is based, at least in part, on the discovery that
purine nucleosides and analogs thereof are capable of modulating
(i.e. either stimulating or inhibiting) axonal outgrowth of CNS
neurons. Accordingly, the method of the invention involves
contacting central nervous system neurons with a purine nucleoside
or analog thereof. In one aspect, the invention provides methods
for stimulating outgrowth, preferably using inosine or guanosine
nucleosides or analogs thereof. In another aspect, the invention
provides methods for inhibiting outgrowth, preferably using a
6-thioguanine nucleoside. In a preferred embodiment, the methods of
the invention modulate axonal outgrowth of retinal ganglion
cells.
[0007] The invention also provides methods for stimulating the
outgrowth of central nervous system neurons following damage or
other injury to the CNS neurons (e.g., stroke, Traumatic Brain
Injury, cerebral aneurism, spinal cord injury and the like). These
methods involve administering to a subject a purine nucleoside
(e.g., inosine or guanosine), or analog thereof, such that axonal
outgrowth is stimulated. In one aspect, the purine nucleoside or
analog thereof is administered by introduction into the central
nervous system of the subject, for example into the cerebrospinal
fluid of the subject. In certain aspects of the invention, the
purine nucleoside or analog thereof is introduced intrathecally,
for example into a cerebral ventricle, the lumbar area, or the
cisterna magna. In a preferred embodiment, the stimulatory method
of the invention promotes outgrowth of damaged retinal ganglion
cells. The purine nucleoside or analog thereof can be administered
locally to retinal ganglion cells to stimulate axonal
outgrowth.
[0008] In another embodiment, the invention provides methods for
inhibiting outgrowth of CNS neurons in which a purine nucleoside
(e.g., 6-thioguanine) is administered to a subject. The inhibitory
methods of the invention can be used to inhibit axonal outgrowth
in, for example, neuroproliferative disorders or neuropathic pain
syndromes.
[0009] In yet another aspect of the invention, the purine
nucleoside or analog thereof is administered in a pharmaceutically
acceptable formulation. The pharmaceutically acceptable formulation
can be a dispersion system, for example a lipid-based formulation,
a liposome formulation, or a multivesicular liposome formulation.
The pharmaceutically acceptable formulation can also comprise a
polymeric matrix, selected, for example, from synthetic polymers
such as polyesters (PLA, PLGA), polyethylene glycol, poloxomers,
polyanhydrides, and pluronics or selected from naturally derived
polymers, such as albumin, alginate, cellulose derivatives,
collagen, fibrin, gelatin, and polysaccharides.
[0010] In a further aspect of the invention, the pharmaceutically
acceptable formulation provides sustained delivery, e.g., "slow
release" of the purine nucleoside to a subject for at least one,
two, three, or four weeks after the pharmaceutically acceptable
formulation is administered to the subject. Sustained delivery of a
formulation of the invention may be provided by use of, for
example, slow release capsules or an infusion pump.
[0011] The invention, finally, provides a pharmaceutical
composition comprising a purine nucleoside or analog thereof and a
pharmaceutically acceptable carrier.
[0012] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-D are graphs showing the quantitation of purinergic
effects on axonal outgrowth.
[0014] FIG. 1A is a graph depicting axonal growth in response to
the nucleosides adenosine (A), guanosine (G), cytidine (C), uridine
(U), and thymidine (T) at a concentration of 1, 10, and 100 .mu.M
as indicated. Data are normalized by subtracting the level of
growth in the negative controls and then dividing by the net growth
in positive controls treated with 20-30% AF-1.
[0015] FIG. 1B is a graph depicting dose-response curves for
adenosine and guanosine. EC.sub.50 values estimated from these data
are 10-15 .mu.M for adenosine and 20-30 .mu.M for guanosine.
[0016] FIG. 1C is a graph depicting the effects of adenosine
nucleotides.
[0017] FIG. 1D is a graph depicting the effects of
membrane-permeable analogs of cyclic AMP (dBCAMP, dibutyryl cyclic
AMP; Sp-8-Br-cAMPS, 8-bromoadenosine-3',5' cyclic
monophosphorothioate) or cyclic GMP (8-Br cGMP, 8-bromo cyclic GMP;
8-pcpt-cGMP, 8-(4-chlorophenylthio) guanosine-3',5'-cyclic
monophosphate). Data represent means+standard errors of the mean
(SEM; not shown if<0.02) and are pooled from 2-4 independent
experiments. p values are based upon 2-tailed t-tests, comparing
growth to that of the negative controls. *p<0.05; **p<0.01;
***p<0.001.
[0018] FIG. 2 is a graph showing that adenosine does not stimulate
growth via extracellular receptors. Outgrowth stimulated by AF-1
(a-b), 100 .mu.M adenosine (Ado) (c-d), or 100 .mu.M guanosine
(Guo) (e-f), is unaffected by the addition of 20 pM 8-PST, an
inhibitor of A1 and A2 adenosine receptors (compare growth in a, c,
and e with b, d, and f). The nonhydrolyzable adenosine analog,
2-chloroadenosine (2-CA, 100 .mu.M) diminishes growth below
baseline levels (g) (p<0.001 in 3 experiments).
[0019] FIG. 3 is a graph showing that adenosine must be hydrolyzed
to stimulate outgrowth. Top: A graph depicting the effects of
deoxycoformycin (DCF) and exogenous adenosine deaminase (ADA) on
outgrowth induced by AF-1 (a-c), adenosine (d-f), and guanosine (g,
h). Bottom: A graph depicting the effects of deoxycoformycin (DCF)
and exogenous adenosine deaminase (ADA) on survival induced by AF-1
(a-c), adenosine (d-f), and guanosine (g,h). Whereas augmenting
adenosine hydrolysis with exogenous ADA leaves the activity of
adenosine unaltered (f), blocking endogenous ADA activity with DCF
causes adenosine to suppress growth (e, top) and survival (e,
bottom). ***p<0.001.
[0020] FIG. 4 is a graph depicting a dose-response curve for
inosine. At concentrations above 50 .mu.M, inosine stimulates about
60% the maximal level of growth achieved with AF-1. The EC.sub.50
for inosine is estimated to be 10-15 .mu.M. Hypoxanthine was
inactive, while 5' IMP appears to have less than {fraction (1/10 )}
the activity of inosine. Outgrowth stimulated by all concentrations
of inosine 10 .mu.M is significantly above background
(p<0.001).
[0021] FIG. 5 is a graph depicting that inosine and guanosine
stimulate growth through an intracellular mechanism. At 20 .mu.M,
NBTI, an inhibitor or purine transport, has no effect on the
activity of AF-1, but blocks c. 90% of the activity of inosine (50
.mu.M) or guanosine (100 .mu.M). *** differences in growth with and
without drugs are significant at p<0.001. Data is pooled from 4
independent experiments.
[0022] FIG. 6A is a graph showing that AF-1 contains no apparent
inosine activity. On a G-10 Sephadex column. AF-1 elutes with a
peak of 7 minutes, with no activity detected at the time of peak
inosine elution (i.e. 9-10 min).
[0023] FIG. 6B is a graph showing that the effects of inosine and
guanosine are independent of cell density. Data from multiple
independent experiments, each indicated by a single point, were
analyzed for the effect of plating density on cell outgrowth. In
all cases, the concentration of inosine or guanosine was maintained
at 100 .mu.M. The regression lines were calculated by
least-squares-fit (Cricket Graph) and are shown below the
symbols.
[0024] FIGS. 7A-D are graphs showing that the effects of AF-1 are
inhibited by 6-thioguanine but restored by inosine.
[0025] FIG. 7A shows that at 10 .mu.M, the purine analog 6-TG
suppressed growth induced by AF-1 below baseline (lane 2 vs. 1:
p<0.001) and reduced the growth induced by 25 .mu.M inosine
(Ino-25) by about 50% (lane 4 vs. 3); Growth induced by higher
concentrations of inosine or guanosine (Guo-100: lanes 8 vs. 7)
were unaffected. Inosine at 100 .mu.M restored all of the growth
induced by AF-1 in the presence of 10 .mu.M 6-TG (lane 10), which
is significantly higher than the growth induced by 100 .mu.M
inosine, either alone or with 10 .mu.M 6-TG (p<0.01).
[0026] FIG. 7B is a graph showing that the concentration of 6-TG
used here had no effect on cell survival.
[0027] FIG. 7C is a graph showing that AF-1 and inosine have
partially additive effects. Outgrowth was assessed for AF-1 and
inosine, each at 0, EC.sub.50, or saturating concentrations. While
the effects of half-maximal concentrations of each were additive
(lane 5), growth reached a plateau level in the presence of higher
concentrations of each (lanes 6, 8, 9).
[0028] FIG. 7D shows further studies on the effects of
6-thioguanine. Outgrowth stimulated by AF-1 was completely blocked
by 6-TG (10 .mu.M) and was not restored in the presence of NBTI (N,
20 .mu.M) and/or dipyridamole (D, 10 .mu.M), purine transport
blockers inhibitors that suppress the activity of inosine.
Inhibitory effects of 6-TG were not mimicked by two reducing
agents, a-tocopherol (a-toc, 30 .mu.M) or glutathione a-methyl
ester (MEG, 100 .mu.M).
[0029] FIG. 8 is a graph depicting the effects of purines on rat
retinal ganglion cells (quantitative studies). CNTF stimulated
growth is inhibited by 6-TG (10 .mu.M) but is fully restored by the
addition of 25 .mu.M inosine. Significance of differences from
control: *p=0.03; ***p<0.001. Results are pooled from 3
independent studies.
DETAILED DESCRIPTION
[0030] The present invention provides methods for modulating the
axonal outgrowth of central nervous system neurons. The invention
is based, at least in part, on the discovery that purine
nucleosides (e.g., inosine and guanosine) and analogs thereof
induce stimulation of axonal outgrowth from both goldfish as well
as mammalian retinal ganglion cells (see Examples I and XI,
respectively). As shown in Example II. purine nucleosides are more
active than their nucleotide counterparts, and they exert their
effect through an intracellular pathway (see Example VI). The
invention further is based, at least in part, on the discovery that
adenosine nucleosides and analogs thereof induce inhibition of
axonal outgrowth from retinal ganglion cells (see Example X).
[0031] Accordingly, the methods of the invention for modulating
axonal outgrowth of CNS neurons generally involve contacting the
central nervous system neurons with a purine nucleoside or analog
thereof such that axonal outgrowth is modulated.
[0032] 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 targeted CNS injuries.
[0033] As used herein, the term "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.
[0034] 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.
[0035] As used herein, the language "contacting" is intended to
include both in vivo or in vitro methods of bringing a purine
nucleoside or analog thereof into proximity with a CNS neuron, such
that the purine nucleoside or analog thereof can modulate the
outgrowth of axonal processes from said CNS neuron.
[0036] As used herein, the language "purine nucleoside" is art
recognized and is intended to include any purine base linked to a
sugar, or an analog thereof. For example, purine nucleosides
include guanine, inosine or adenine and analogs include
6-thioguanine (6-TG) and the like.
[0037] In one embodiment, the outgrowth of CNS neurons is
stimulated, preferably using inosine or guanosine nucleosides or
analogs thereof. In another embodiment, the outgrowth of CNS
neurons is inhibited, preferably using a 6-TG nucleoside.
[0038] The invention also provides methods for stimulating the
outgrowth of central nervous system neurons following an injury.
The method involves administering to a subject a purine nucleoside
(e.g., inosine or guanosine) or analog thereof.
[0039] As used herein, the term "subject" is intended to include
animals susceptible to CNS injuries, preferably mammals, most
preferably humans. In a preferred embodiment, the subject is a
primate. In an even more preferred embodiment, the primate is a
human. Other examples of subjects include dogs, cats, goats, and
cows.
[0040] As used herein, the term "injury" is intended to include a
damage which directly or indirectly affects the normal functioning
of the CNS. For example, the injury can be damage to retinal
ganglion cells; a traumatic brain injury; a stroke related injury;
a cerebral aneurism related injury; a spinal cord injury, including
monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia; a
neuroproliferative disorder or neuropathic pain syndrome.
[0041] As used herein, the term "stroke" is art recognized and is
intended to include sudden diminution or loss of consciousness,
sensation, and voluntary motion caused by rapture or obstruction
(e.g. by a blood clot) of an artery of the brain.
[0042] As used herein, the term "Traumatic Brain Injury" is art
recognized and is intended to include the condition in which, a
traumatic blow to the head causes damage to the brain, often
without penetrating the skull. Usually, the initial trauma can
result in expanding hematoma, subarachnoid hemorrhage, cerebral
edema, raised intracranial pressure (ICP), and cerebral hypoxia,
which can, in turn, lead to severe secondary events due to low
cerebral blood flow (CBF).
Pharmaceutically Acceptable Formulations
[0043] In the method of the invention, the purine nucleoside or
analog thereof can be administered in a pharmaceutically acceptable
formulation. The present invention pertains to any pharmaceutically
acceptable formulations, such as 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.
[0044] In one embodiment, the pharmaceutically acceptable
formulations comprise a polymeric matrix.
[0045] The terms "polymer" or "polymeric" are art-recognized and
include a structural framework comprised of repeating monomer units
which is capable of delivering a purine nucleoside or analog
thereof such that treatment of a targeted condition, e.g., a CNS
injury, occurs. The terms also include co-polymers and homopolymers
e.g., synthetic or naturally occurring. Linear polymers, branched
polymers, and cross-linked polymers are also meant to be
included.
[0046] 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 purine nucleoside 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.
[0047] Polymers can be prepared using methods known in the art
(Sandler, S. R.; Karo, W. Polymer Syntheses; Harcourt Brace:
Boston, 1994; Shalaby, W.; Ikada, Y.; Langer, R.; Williams, J.
Polymers of Biological and Biomedical Significance (ACS Symposium
Series 540; American Chemical Society: Washington, D.C., 1994).
Polymers can be designed to be flexible; the distance between the
bioactive side-chains and the length of a linker between the
polymer backbone and the group can be controlled. Other suitable
polymers and methods for their preparation are described in U.S.
Pat. Nos. 5,455,044 and 5,576,018, the contents of which are
incorporated herein by reference.
[0048] The polymeric formulations are preferably formed by
dispersion of the purine nucleoside 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 purine
nucleoside, types of solvent used, and reaction times.
[0049] In addition to the purine nucleoside and the
pharmaceutically acceptable polymer, the pharmaceutically
acceptable formulation used in the method of the invention can
comprise additional pharmaceutically acceptable carriers and/or
excipients. 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.
[0050] The purine nucleoside or analog thereof 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.
[0051] In another embodiment, the pharmaceutically acceptable
formulations comprise lipid-based formulations. Any of the known
lipid-based drug delivery systems can be used in the practice of
the invention. For instance, multivesicular liposomes (MVL),
multilamellar liposomes (also known as multilamellar vesicles or
"MLV"), unilamellar liposomes, including small unilamellar
liposomes (also known as unilamellar vesicles or "SUV") and large
unilamellar liposomes (also known as large unilamellar vesicles or
"LUV"), can all be used so long as a sustained release rate of the
encapsulated purine nucleoside or analogue thereof can be
established. In one embodiment, the lipid-based formulation can be
a multivesicular liposome system. Methods of making controlled
release multivesicular liposome drug delivery systems is described
in PCT Application Serial Nos. US96/11642, US94/12957 and
US94/04490. the contents of which are incorporated herein by
reference.
[0052] 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.
[0053] Examples of lipids useful in synthetic membrane vesicle
production include phosphatidylglycerols, phosphatidylcholines,
phosphatidylserines, phosphatidylethanolamines, sphingolipids,
cerebrosides, and gangliosides. Preferably phospholipids including
egg phosphatidylcholine, dipalmitoylphosphatidylcholine,
distearoylphosphatidylcholine, dioleoylphosphatidyicholine,
dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol
are used.
[0054] In preparing lipid-based vesicles containing a purine
nucleoside or analogue thereof, such variables as the efficiency of
purine nucleoside encapsulation, lability of the purine nucleoside,
homogeneity and size of the resulting population of vesicles,
purine nucleoside-to-lipid ratio, permeability, instability of the
preparation, and pharmaceutical acceptability of the formulation
should be considered (see Szoka, et al., Annual Reviews of
Biophysics and Bioengineering, 9:467, 1980; Deamer, et al., in
Liposomes, Marcel Dekker, New York, 1983, 27; and Hope, et al.,
Chem. Phys. Lipids, 40:89, 1986, the contents of which are
incorporated herein by reference).
Administration of the Pharmaceutically Acceptable Formulation
[0055] In one embodiment, the purine nucleoside or analog thereof
is administered by introduction into the central nervous system of
the subject, e.g., into the cerebrospinal fluid of the subject. In
certain aspects of the invention, the purine nucleoside or analog
thereof is introduced intrathecally, e.g., into a cerebral
ventricle, the lumbar area, or the cisterna magna. In another
aspect, the purine nucleoside or analog thereof is introduced
intraocullarly, to thereby contact retinal ganglion cells.
[0056] The pharmaceutically acceptable formulations can easily be
suspended in aqueous vehicles and introduced through conventional
hypodermic needles or using infusion pumps. Prior to introduction,
the formulations can be sterilized with, preferably, gamma
radiation or electron beam sterilization, described in U.S. Pat.
No. 436,742 the contents of which are incorporated herein by
reference.
[0057] In one embodiment, the purine nucleoside formulation
described herein is administered to the subject in the period from
the time of injury to 100 hours, for example within 24, 12 or 6
hours after the injury has occurred.
[0058] In another embodiment of the invention, the purine
nucleoside formulation is administered into a subject
intrathecally. As used herein, the term "intrathecal
administration" is intended to include delivering a purine
nucleoside formulation directly into the cerebrospinal fluid of a
subject, by techniques including lateral cerebroventricular
injection through a burrhole or cisternal or lumbar puncture or the
like (described in Lazorthes et al. Advances in Drug Delivery
Systems and Applications in Neurosurgery, 143-192 and Omaya et al.,
Cancer Drug Delivery, 1: 169-179, the contents of which are
incorporated herein by reference). The term "lumbar region" is
intended to include the area between the third and fourth lumbar
(lower back) vertebrae. The term "cisterna magna" is intended to
include the area where the skull ends and the spinal cord begins at
the back of the head. The term "cerebral ventricle" is intended to
include the cavities in the brain that are continuous with the
central canal of the spinal cord. Administration of a purine
nucleoside to any of the above mentioned sites can be achieved by
direct injection of the purine nucleoside formulation or by the use
of infusion pumps. For injection, the purine nucleoside 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 purine nucleoside formulation
may be formulated in solid form and re-dissolved or suspended
immediately prior to use. Lyophilized forms are also included. The
injection can be, for example, in the form of a bolus injection or
continuous infusion (e.g., using infusion pumps) of the purine
nucleoside formulation.
[0059] In one embodiment of the invention, said purine nucleoside
formulation is administered by lateral cerebro ventricular
injection into the brain of a subject in the inclusive period from
the time of the injury to 100 hours thereafter. The injection can
be made, for example, through a burr hole made in the subject's
skull. In another embodiment, said encapsulated therapeutic agent
is administered through a surgically inserted shunt into the
cerebral ventricle of a subject in the inclusive period from the
time of the injury to 100 hours thereafter. 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.
[0060] In yet another embodiment, said purine nucleoside
formulation is administered by injection into the cisterna magna,
or lumbar area of a subject in the inclusive period from the time
of the injury to 100 hours thereafter.
Duration and Levels of Administration
[0061] In another embodiment of the method of the invention, the
pharmaceutically acceptable formulation provides sustained
delivery, e.g., "slow release" of the purine nucleoside to a
subject for at least one, two, three, or four weeks after the
pharmaceutically acceptable formulation is administered to the
subject.
[0062] As used herein, the term "sustained delivery" is intended to
include continual delivery of a purine nucleoside or analogue
thereof in vivo over a period of time following administration,
preferably at least several days, a week or several weeks.
Sustained delivery of the purine nucleoside or analogue thereof can
be demonstrated by, for example, the continued therapeutic effect
of the purine nucleoside or analogue thereof over time (e.g.,
sustained delivery of the purine nucleoside or analogue thereof can
be demonstrated by continued outgrowth or by continued inhibition
of outgrowth of CNS neurons over time). Alternatively, sustained
delivery of the purine nucleoside or analogue thereof may be
demonstrated by detecting the presence of the purine nucleoside or
analogue thereof in vivo over time.
[0063] In one embodiment, the pharmaceutically acceptable
formulation provides sustained delivery of the purine nucleoside or
analogue thereof to a subject for less than 30 days after the
purine nucleoside or analogue thereof is administered to the
subject. For example, the pharmaceutically acceptable formulation,
e.g., "slow release" formulation, can provide sustained delivery of
the purine nucleoside or analogue thereof to a subject for one,
two, three or four weeks after the purine nucleoside or analogue
thereof is administered to the subject. Alternatively, the
pharmaceutically acceptable formulation may provide sustained
delivery of the purine nucleoside or analogue thereof to a subject
for more than 30 days after the purine nucleoside or analogue
thereof is administered to the subject.
[0064] The pharmaceutical formulation, used in the method of the
invention, contains a therapeutically effective amount of the
purine nucleoside or analogue thereof. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired result. A therapeutically
effective amount of the purine nucleoside or analogue thereof may
vary according to factors such as the disease state, age, and
weight of the subject, and the ability of the purine nucleoside or
analogue thereof (alone or in combination with one or more other
agents) to elicit a desired response in the subject. Dosage
regimens may be adjusted to provide the optimum therapeutic
response. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the purine nucleoside or
analogue thereof are outweighed by the therapeutically beneficial
effects. A non-limiting range for a therapeutically effective
concentration of inosine is 5 .mu.M to 1 mM. A non-limiting range
for a therapeutically effective concentration of guanosine is at
least 25 .mu.M to 1 mM. In a particularly preferred embodiment, the
therapeutically effective concentration of the inosine nucleoside
is 10-25 .mu.M, or 25-50 .mu.M. In a particularly preferred
embodiment, the therapeutically effective concentration of the
guanosine nucleoside is 25-50 .mu.M, 50-100 .mu.M, or 100-150
.mu.M. Adenosine can be used to inhibit neurite outgrowth at
relatively high doses, e.g., higher than 5 mM, (so that its
conversion to inosine is inhibited). At such concentrations,
however, adenosine may become toxic. Adenosine analogs, e.g.,
6-thioguanine are, therefore, preferable for administration in
mammalian subjects to inhibit axonal growth. It is to be noted that
dosage values mav 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
purine nucleoside or analogue thereof and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed invention.
[0065] The invention, in another embodiment, provides a
pharmaceutical composition consisting essentially of a purine
nucleoside or analog thereof and a pharmaceutically acceptable
carrier and methods of use thereof to modulate axonal outgrowth by
contacting CNS neurons with the composition. By the term
"consisting essentially of" is meant that the pharmaceutical
composition does not contain any other modulators of neuronal
growth such as, for example, nerve growth factor (NGF). In one
embodiment, the pharmaceutical composition of the invention can be
provided as a packaged formulation. The packaged formulation may
include a pharmaceutical composition of the invention in a
container and printed instructions for administration of the
composition for treating a subject having a disorder associated
with an injury of central nervous system neurons, e.g., an injury
to retinal ganglion cells, a spinal cord injury or a traumatic
brain injury.
In Vitro Treatment of CNS Neurons
[0066] CNS neurons can further be contacted with a therapeutically
effective amount of a purine nucleoside or analog thereof, in
vitro. Accordingly, CNS neuron cells can be isolated from a subject
and grown in vitro, using techniques well known in the art.
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.
[0067] Such cells can be subsequently contacted with a purine
nucleoside or analog thereof at levels 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.
[0068] 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 are hereby
incorporated by reference.
EXAMPLES
[0069] In the following examples, the following methodologies were
used:
[0070] Sample Preparation
[0071] Axogenesis factor-1 was obtained essentially as described in
Schwalb et al., 1995, and Schwalb et al., Neuroscience, 72:
901-910,1996, the contents of which are incorporated herein by
reference). Optic nerves were dissected, cut into fragments 1 mm in
length, and incubated in a ratio of 6 nerves in 3 ml of either L-15
media (Gibco BRL) or phosphate-buffered saline (Gibco BRL). After
3-4 hours, nerve fragments were removed by filtering through a 0.22
.mu.m pore low protein-binding filter (Gelman). A low molecular
weight fraction of the conditioned media was prepared by
ultrafiltration, first with a molecular weight cut-off of 3 kDa
(Amicon Centriprep-3), then with a cut-off of 1 kDa (Filtron). The
filtrate was used as a positive control at 20-30% final
concentration. Adenosine, adenosine 5' monophosphate, adenosine
deaminase, adenosine diphosphate, adenosine triphosphate, 8-bromo
3',5'-cyclic guanosine monophosphate, 3',5' cyclic adenosine
monophosphate, 5' cyclic guanosine monophosphate, cytidine,
guanosine, hypoxanthine, inosine, 5'-inosine monophosphate,
a-tocopherol, 6-thioguanine, thymidine, uridine, and xanthine were
all obtained from Sigma Chemical Co., St. Louis, Mo.,
8-p-sulphophenyl-theophylline, dibutyryl cyclic adenosine
monophosphate and 2-deoxycoformycin were from Calbiochem,
2-chioroadenosine, erythro-9-(2-hydroxy-3nonyl) adenine and IB-MECA
from Research Biochemicals, Inc. (Natick, Mass.), and
4(nitrobenzyl-6-thioinosine) from Aldrich Chemicals, Inc. The
membrane-permeable, nonhydrolyzable analogs of cAMP and cGMP,
8bromoadenosine-3',5' cyclic monophosphorothioate and
8-(4-chlorophenylthio) guanosine-3',5'-cyclic monophosphate were
from Biolog.
Dissociated Retinal Cultures
[0072] Goldfish (Comet Variety, Mt. Parnell Fisheries, Mt. Parnell
Pa.), 6-10 cm in length, were dark-adapted and their retinas
dissected. Retinas were incubated with papain (20 .mu.g/ml),
activated with cysteine (2.8 mM) for 30 minutes at room
temperature, then dissociated by gentle trituration. Repeated
cycles of trituration and sedimentation yielded cultures nearly
homogeneous in ganglion cells, which are readily identified by
their oval shape, phase-bright appearance, size (diameter 15
.mu.m), and extension of only 1 or 2 neurites of uniform caliber;
these criteria have been verified by retrograde labeling (see
Schwartz & Agranoff, Brain Res. 206: 331-343,1981 and Schwalb
et al., J. Neuroscience 15: 5514-5625, 1995, the contents of which
are incorporated herein by reference). Low density cultures were
achieved by plating c. 5.times.10.sup.3 cells/well into poly
L-lysine coated, 24-well culture dishes (Costar, Cambridge, Mass.).
Cells were maintained at 21.degree. C. in serum free, defined media
containing insulin, selenium, transferrin, bovine serum albumin,
catalase, superoxide dismutase, hormones, and vitamins in Eagle's
L-15 media as described in Schwalb et al., 1995, the contents of
which are incorporated herein by reference). Dissociated cultures
of purified rat retinal ganglion cells were 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 postnatal day 8 Sparague-Dawley
rats were dissociated using papain activated with cysteine.
Macrophages were removed by incubation with an anti-rat macrophage
antibody (Accurate) followed by immunopanning with an anti-rabbit
IgG antibody. Ganglion cells were isolated by immunopanning with an
anti-Thy-1 antibody, then dislodged with trypsin for use in
low-density cultures. Rat retinal ganglion cells were 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.
Experimental Design
[0073] In a typical experiment, samples were plated in
quadruplicate in randomized positions of a 24-well plate and the
code was concealed to ensure that growth was evaluated in a blinded
fashion. Each experiment contained 4 wells of a negative control
(media plus supplements only) and 4 wells of a positive control (a
standardized AF-1 sample of known activity). Growth and survival
were 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 was the criterion for
growth. since it clearly distinguishes stimulated cells from
negative controls (see Schwalb et al., 1995). After the completion
of counting, the code was broken, the data tabulated, and means and
standard errors were calculated for the 4 replicate wells of each
sample using Cricket Graph (CA Associates, Islandia, N.Y.). Data
were normalized by subtracting the growth in the negative controls
(usually 4-5%) and dividing by the net growth in the positive
controls. In the most favorable experiments, more than 50% of
retinal ganglion cells (RGCs) exposed to AF-1 extended axons 5 cell
diameters in length after 6 days. Group comparisons were based upon
pairvise, 2-tailed Student's t-tests. Several independent
experiments were performed for most samples, as noted in the figure
legends. In some cases, cell viability was assessed with the dye
5,6-carboxyfluorescein diacetate. Cell survival is reported as the
number of viable RGCs per high-powered field.
Example I
Purine Induced Stimulation of Axonal Outgrowth From Goldfish
Retinal Ganglion Cells
[0074] The low molecular weight growth factor AF-1, secreted by
optic nerve glia, induced dramatic outgrowth from goldfish retinal
ganglion cells. Little outgrowth occurred in the control condition
using defined media alone. These two limits were the basis for
normalizing results for other factors. When nucleosides were tested
at concentrations between 1-100 .mu.M, adenosine and guanosine
stimulated almost as much outgrowth from goldfish retinal ganglion
cells as AF-1. Pyrimidine bases had no activity over this
concentration range. A more complete dose-response curve for the
purines shows that adenosine is the more active of the two, with an
EC.sub.50 of 10-15 .mu.M (see FIG. 1B). At concentrations of 50-100
.mu.M, adenosine induced a maximal response equal to 60% the level
induced by AF-1, but at higher concentrations, outgrowth decreased.
Guanosine had a higher EC.sub.50 than adenosine (25 .mu.M, see FIG.
1B), and at concentrations of 100 .mu.M, it stimulated the same
maximal level of activity as adenosine, with no obvious decrease in
activity at higher concentrations.
Example II
Purine Nucleotides are Less Active than Nucleosides
[0075] Extracellularly, adenosine could be stimulating either
P.sub.1 receptors, which are optimally responsive to adenosine per
se, or P.sub.2 receptors, which respond maximally to ATP or other
nucleotides. AMP and ADP showed a marginally significant level of
activity at 100 .mu.M (p 0.05), as did ATP at 10 .mu.M (but not at
100 .mu.M). Since the activity of the purine nucleotides is
considerably lower than that of the purines themselves, it is
unlikely that P2 receptors are involved. Plausibly, the purines
could function intracellularly as precursors for cyclic nucleotides
that might serve as second messengers in axogenesis. The biological
activity of membrane-perneable analogs of cAMP and cGMP was,
therefore examined. Neither dibutyryl cAMP (dBcAMP) nor 8-Br cGMP
showed any activity between 1-100 .mu.M (see FIG. 1D). More
recently developed nonhydrolyzable, membrane-permeable analogs of
cAMP (8-bromoadenosine-3',5' cyclic monophosphorothioate:
Sp-8-Br-cAMPS) and cGMP (8-(4-chlorophenylthio)
guanosine-3',5'-cyclic monophosphate: 8-pcpt-cGMP)) were also found
to be inactive when tested at concentrations up to 1 mM (see FIG.
1D).
Example III
The Positive Effects of Adenosine are not Mediated Through
Extracellular Adenosine Receptors
[0076] 8-p-(sulfophenyl theophylline) (8-PST), described in Collis
et al., Brit. J. Pharmacol. 92:69-75, 1987, the contents of which
are incorporated herein by reference, is an inhibitor of the two
most common adenosine receptors (A1 and A2). At 20 .mu.M, a dosage
that almost completely blocks receptor-mediated effects of
adenosine in rats, 8-PST had no effect on outgrowth stimulated by
adenosine, guanosine, or AF-1 (see FIG. 2). Further evidence that
the positive effects of adenosine are not mediated through
extracellular adenosine receptors comes from studies using the
non-hydrolyzable analog 2-chloroadenosine (2CA), which is an
agonist at the A1, A2 and A3 receptors. At concentrations of 10 and
100 .mu.M, 2-CA caused a small but significant decrease in growth
below the baseline in 3 out of 3 independent experiments (see FIG.
2).
Example IV
Adenosine Must be Hydrolyzed to Inosine to Stimulate Growth
[0077] To investigate whether the activity of adenosine is due to
the formation of an active metabolite, the activity of ADA was
inhibited using either deoxycoformycin (DCF) or
erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). In the presence of 10
.mu.M DCF, 100 .mu.M adenosine not only failed to stimulate growth,
but caused it to decline below baseline levels (FIG. 3, lanes e vs.
d). Cell survival also decreased when adenosine hydrolysis was
blocked. In the presence of 10 .mu.M DCF, 10 .mu.M adenosine caused
survival to decrease by 20% (not shown), and 100 .mu.M adenosine
caused survival to decline by 57% (FIG. 3, bottom, lane e). The
effects of DCF on outgrowth and survival were specifically related
to the presence of nonhydrolyzed adenosine, since they did not
occur when DCF was used alone with AF- 1, or with guanosine (FIG.
3, lanes b and h). Like DCF, 10 .mu.M EHNA rendered adenosine (100
.mu.M) ineffective in stimulating outgrowth and caused cell
survival to decline by about 30% (data not shown). EHNA also
exhibited nonspecific effects, however, reducing growth stimulated
by either guanosine or AF-1 by about 50%, though not altering cell
survival. Further evidence that the positive effects of adenosine
require its hydrolysis comes from experiments in which exogenous
ADA was added. At 0.4 U/ml. the enzyme did not diminish axon
outgrowth stimulated by 100 .mu.M adenosine or affect cell survival
(see FIG. 3, lane f).
Example V
Inosine is the Active Metabolite
[0078] Inosine, the primary product of adenosine dearnidation,
proved to be a potent activator of axon outgrowth. As shown in FIG.
4, the EC.sub.50 for inosine was 10-15 .mu.M, and a maximal
response, equal to about 60% the level achieved with AF-1, was
attained at concentrations above 25 .mu.M. While the EC.sub.50 and
maximum response induced by inosine were similar to those of
adenosine, one notable difference was that at higher
concentrations, inosine did not cause growth to decline. unlike the
case for adenosine. Further hydrolysis of inosine yields
hypoxanthine, which showed no activity at all (see FIG. 4). Inosine
5' monophosphate (5' IMP) was inactive at 10 .mu.M, and at 100
.mu.M it showed less activity than inosine at 10 .mu.M (see FIG.
4).
Example VI
Purines Stimulate Growth Through an Intracellular Pathway
[0079] Two inhibitors of the purine transporter, nitrobenzylthio
inosine (NBTI) and dipyridamole, were used to investigate whether
inosine and guanosine needed to enter neurons to stimulate
outgrowth. At 20 .mu.M, NBTI blocked about 90% of the growth
induced by either inosine or guanosine (see FIG. 5; 86% loss of
activity for 50 .mu.M inosine, p<0.001; 93% loss of activity for
100 .mu.M guanosine, p<0.01). Dipyridamole (10 .mu.M) also
diminished the growth induced by inosine (114% decrease; p<0.01;
not shown; guanosine not tested). In contrast, AF-1 showed little
inhibition by NBTI (10% decline, n.s.) and slightly more with
dipyridamole (25% decline, n.s., not shown). The NBTI-related loss
in activity for the purines was far greater than for AF-1
(p<0.001).
Example VII
AF-1 Activity is not Due to Inosine
[0080] To address whether AF-1 preparations might still contain
purines that could account for some of their biological activity,
native AF-1 and inosine were chromatographed on a size-exclusion
column with Sephadex G-10 (Pharmacia Biotech, Uppsala, Sweden), 1
cm in diameter and 10 cm in length. Samples were loaded in a volume
of 0.5 ml and collected in 1 ml fractions. The column buffer was
either 20% methanol in distilled water or 0.14 M NaCl. Fractions
were bioassayed at 30% concentration. As shown in FIG. 6A, the peak
of inosine activity was at 9-10 minutes, whereas for AF-1 it
occurred at 7 minutes.
[0081] Example VIII.
Axonal Outgrowth is the Effect of Inosine and Guanosine and not the
Effect of a Secondary Factor
[0082] The cultures used here contained 70-90% ganglion cells, with
the remainder representing other neural and non-neuronal elements
of the retina (see Schwartz & Agranoff, 1982 and Schwalb et al,
1995, the contents of which are incorporated herein by reference).
This heterogeneity raised the possibility that inosine or guanosine
could act first upon another cell population, which secretes a
secondary factor that stimulates retinal ganglion cells to grow. In
this case, the effect of the purines would be expected to vary with
cell density, since the concentration of any secondary factor would
increase proportionately with increasing density. To examine this,
axonal outgrowth was investigated in response to a fixed
concentration of inosine or guanosine over a 3-4-fold range of cell
densities. The regression lines for both the inosine and the
guanosine data demonstrate that growth is not a function of cell
density (see FIG. 6B), arguing against the presence of a
concentration-dependent secondary factor.
Example IX
Induction of Phosphoprotein GAP-43 Expression by Purines
[0083] One hallmark of optic nerve regeneration in vivo is the
enhanced expression of the membrane phosphoprotein GAP-43. To
investigate whether this upregulation is induced by purines,
immunohistochemistry was carried out using a polyclonal rabbit
antiserum against recombinant goldfish GAP-43. Recombinant
zebrafish GAP-43 was made by transforming E. coli with a cDNA
isolated by Dr. Eva Reinhard, University of Basel, Switzerland (see
Reinhard et al., Development, 120: 1757-1775, 1994, the contents of
which are incorporated herein by reference) subcloned into the
prokaryotic expression vector pTrcHisB (Invitrogen). The protein
produced was purified by Ni.sup.2+-NTA-affinity chromatography and
used to immunize rabbits. The specificity of the resulting antibody
was demonstrated in western blots, where the antibody recognized a
unique 48 kDa band that is enriched in retinal ganglion cells
undergoing regeneration or in synaptosomal plasma membranes from
goldfish brain.
[0084] AF-1, inosine, and guanosine all caused a large increase in
GAP-43 levels relative to L-15 treated controls. A
semi-quantitative analysis was carried out by assigning a level for
GAP-43 immunoreactivity of 0 (none), 1 (moderate) or 2 (intense),
and correlating the staining intensity with the length of a cell's
axon for 150-200 cells treated with L-15, inosine, or AF-1. Inosine
produced a 5.5-fold increase in the number of intensely stained
cells over L-15 whereas AF-1 produced a 8-fold increase. In all 3
cases, the intensity of GAP-43 immunostaining correlated strongly
with axonal length.
Example X
Blockade of Axonal Outgrowth with 6-thioguanine (6-TG)
[0085] In goldfish RGCs 6-TG at 10 .mu.M blocked all growth
stimulated by AF-1 (see FIG. 7A, lane 2), but had no effect on cell
survival (see FIG. 7B). The same concentration of 6-TG reduced
outgrowth stimulated by 25 .mu.M inosine by only 50% (see FIG. 7A,
lanes 3 and 4), and had no effect on growth stimulated by either
100 .mu.M inosine or 100 .mu.M guanosine (see FIG. 7A, lanes 5-8).
At 100 .mu.M, inosine fully restored the growth induced by AF-1 in
the presence of 10 .mu.M 6-TG back to its original level, which was
significantly higher than the level of growth induced by inosine
alone (see FIG. 7A, lanes 10 vs. 6). Therefore, inosine and 6-TG
appear to be acting competitively at a level of intracellular
signaling that is also utilized by AF-1 to stimulate outgrowth.
Further evidence that inosine may activate the same pathway that is
utilized by AF-1 signaling came from the observation that when the
two were combined at their EC.sub.50 levels, they showed additive
effects, whereas at saturating concentrations, growth saturates at
the level stimulated by high AF-1 levels alone (see FIG. 7C, lane
9). Since 6-TG has a free thiol, it could be acting as a reducing
agent rather than as a purine analog. However, two other reducing
agents, a -tocopherol at 30 .mu.M or glutathione a-methyl ester
(MEG) at 100 .mu.M had no effect on outgrowth stimulated by AF-1
(see FIG. 7D). Another possibility is that inosine might block the
inhibitory effect of 6-TG on outgrowth by interfering with its
transport into cells. However, the two transport inhibitors that
blocked the activity of inosine, NBTI and dipyridamole, failed to
prevent 6-TG from blocking outgrowth stimulated by AF-1 (see FIG.
7D).
Example XI
Mammalian Retinal Ganglion Cells Extend Axons in Response to
Inosine
[0086] Retinal ganglion cells were isolated from 8 day old rats by
immunopanning as described in Barres et al., Neuron, 1:
791-803,1988, the contents of which are incorporated herein by
reference, and grown in defined media. Inosine at 25 or 50 .mu.M
stimulated a 50% increase in the number of cells extending axons 5
cell diameters in length (see FIG. 8). Ciliary neurotrophic factor
(CNTF) induced a much larger increase in outgrowth (see FIG. 8) and
enhanced cell survival. At 10 .mu.M, 6-TG blocked CNTF-induced
outgrowth. The addition of inosine at 50 .mu.M restored
CNTF-induced outgrowth nearly to its original level (see FIG.
8).
Equivalents
[0087] 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.
* * * * *