U.S. patent application number 15/387208 was filed with the patent office on 2017-07-13 for methods and compositions for preserving the viability of photoreceptor cells.
The applicant listed for this patent is Massachusetts Eye and Ear Infirmary. Invention is credited to Toru Nakazawa.
Application Number | 20170196806 15/387208 |
Document ID | / |
Family ID | 35149240 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170196806 |
Kind Code |
A1 |
Nakazawa; Toru |
July 13, 2017 |
METHODS AND COMPOSITIONS FOR PRESERVING THE VIABILITY OF
PHOTORECEPTOR CELLS
Abstract
Provided are methods and compositions for maintaining the
viability of photoreceptor cells following retinal detachment. The
viability of photoreceptor cells can be preserved by administering
a neuroprotective agent, for example, a substance capable of
suppressing endogenous MCP-1, a MCP-1 antagonist, a substance
capable of suppressing endogenous TNF-alpha, a TNF-alpha
antagonist, a substance capable of suppressing endogenous IL-1
beta, an IL-1 beta antagonist, a substance capable of inducing
endogenous bFGF, exogenous bFGF, a bFGF mimetic, and combinations
thereof, to a mammal having an eye with retinal detachment. The
neuroprotective agent maintains the viability of the photoreceptor
cells until such time that the retina becomes reattached to the
underlying retinal pigment epithelium and choroid. The treatment
minimizes the loss of vision, which otherwise may occur as a result
of retinal detachment.
Inventors: |
Nakazawa; Toru; (Sendai
Miyagi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Eye and Ear Infirmary |
Boston |
MA |
US |
|
|
Family ID: |
35149240 |
Appl. No.: |
15/387208 |
Filed: |
December 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11587022 |
Aug 7, 2007 |
9549895 |
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PCT/US05/13710 |
Apr 22, 2005 |
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15387208 |
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60564717 |
Apr 23, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0051 20130101;
A61K 38/2006 20130101; A61P 27/02 20180101; A61K 2039/505 20130101;
A61K 38/06 20130101; A61K 38/19 20130101; C07K 16/241 20130101;
A61K 9/0048 20130101; A61K 38/1793 20130101; A61K 45/06 20130101;
A61K 38/1825 20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 38/20 20060101 A61K038/20; A61K 38/18 20060101
A61K038/18; A61K 38/19 20060101 A61K038/19 |
Claims
1. A method of preserving the viability of photoreceptor cells
disposed within a retina of a mammalian eye following retinal
detachment, the method comprising: administering to a mammal having
an eye in which a region of the retina has been detached an amount
of a neuroprotective agent selected from the group consisting of a
substance capable of suppressing endogenous MCP-1, an MCP-1
antagonist, and combinations thereof sufficient to preserve the
viability of photoreceptor cells disposed within the region of the
detached retina.
2. The method of claim 1, wherein the neuroprotective agent is
administered to the mammal prior to reattachment of the region of
detached retina.
3. The method of claim 1, wherein the neuroprotective agent is
administered to the mammal after reattachment of the region of
detached retina.
4. The method of claim 1, wherein the neuroprotective agent is
administered locally or systemically.
5-6. (canceled)
7. The method of claim 4, wherein at least one neuroprotective
agent is administered by intraocular, intravitreal, or transcleral
administration.
8. The method of claim 1, wherein the neuroprotective agent reduces
the number of photoreceptor cells in the region that die following
retinal detachment.
9. The method of claim 1, wherein the retinal detachment occurs as
a result of a retinal tear, retinoblastoma, melanoma, diabetic
retinopathy, uveitis, choroidal neovascularization, retinal
ischemia, pathologic myopia, or trauma.
10-18. (canceled)
19. A method of preserving the viability of photoreceptor cells
disposed within a retina of a mammalian eye following retinal
detachment, the method comprising: administering to a mammal having
an eye in which a region of the retina has been detached an amount
of a neuroprotective agent selected from the group consisting of a
substance capable of suppressing endogenous IL-1 beta, an IL-1 beta
antagonist, and combinations thereof sufficient to preserve the
viability of photoreceptor cells disposed within the region of the
detached retina.
20. The method of claim 19, wherein the neuroprotective agent is
administered to the mammal prior to reattachment of the region of
detached retina.
21. The method of claim 19 or 20, wherein the neuroprotective agent
is administered to the mammal after reattachment of the region of
detached retina.
22. The method of claim 19, wherein the neuroprotective agent is
administered locally or systemically.
23-24. (canceled)
25. The method of claim 22 or 21, wherein at least one
neuroprotective agent is administered by intraocular, intravitreal,
or transcleral administration.
26. The method of claim 19, wherein the neuroprotective agent
reduces the number of photoreceptor cells in the region that die
following retinal detachment.
27. The method of claim 19, wherein the retinal detachment occurs
as a result of a retinal tear, retinoblastoma, melanoma, diabetic
retinopathy, uveitis, choroidal neovascularization, retinal
ischemia, pathologic myopia, or trauma.
28. A method of preserving the viability of photoreceptor cells
disposed within a retina of a mammalian eye following retinal
detachment, the method comprising: administering to a mammal having
an eye in which a region of the retina has been detached an amount
of a neuroprotective agent selected from the group consisting of a
substance capable of inducing endogenous bFGF, a bFGF mimetic, and
combinations thereof sufficient to preserve the viability of
photoreceptor cells disposed within the region of the detached
retina.
29. The method of claim 28, wherein the neuroprotective agent is
administered to the mammal prior to reattachment of the region of
detached retina or after reattachment of the region of detached
retina.
30. (canceled)
31. The method of claim 28, wherein the neuroprotective agent is
administered locally or systemically.
32-33. (canceled)
34. The method of claim 31, wherein at least one neuroprotective
agent is administered by intraocular, intravitreal, or transcleral
administration.
35. The method of claim 28, wherein the neuroprotective agent
reduces the number of photoreceptor cells in the region that die
following retinal detachment.
36. The method of claim 28, wherein the retinal detachment occurs
as a result of a retinal tear, retinoblastoma, melanoma, diabetic
retinopathy, uveitis, choroidal neovascularization, retinal
ischemia, pathologic myopia, or trauma.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority to and the benefit
of U.S. Ser. No. 60/564,717, filed on Apr. 23, 2004, the entirety
of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to methods and compositions
for preserving the viability of photoreceptor cells following
retinal detachment, and more particularly the invention relates to
compositions including, for example, a neuroprotective agent, and
their use in maintaining the viability of photoreceptor cells
following retinal detachment.
BACKGROUND
[0003] The retina is a delicate neural tissue lining the back of
the eye that converts light stimuli into electric signals for
processing by the brain. Within the eye, the retina is disposed
upon underlying retinal pigment epithelium and choroid, which
provide the retina with a supply of blood and nutrients. A common
and potentially blinding condition known as retinal detachment
occurs when the retina becomes disassociated from its underlying
retinal pigment epithelium and/or choroid with the accumulation of
fluid in the intervening space. The loss of visual function appears
to be more pronounced when the retinal detachments involve the
central macula.
[0004] Unless treated, retinal detachments often result in
irreversible visual dysfunction, which can range from partial to
complete blindness. The visual dysfunction is believed to result
from the death of photoreceptor cells, which can occur during the
period when the retina is detached from its underlying blood and
nutrient supply. Reattachment of the retina to the back surface of
the eye typically is accomplished surgically, and despite the good
anatomical results of these surgeries (i.e., reattachment of the
retina) patients often are still left with permanent visual
dysfunction.
[0005] There is still a need for new methods and compositions for
maintaining the viability of photoreceptor cells following retinal
detachment and for preserving vision when the retina ultimately
becomes reattached.
SUMMARY
[0006] Monocyte chemoattractant protein-1 (MCP-1), tumor necrosis
factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and/or
basic fibroblast growth factor (bFGF) mRNA and/or protein
expression levels are increased in the retina following retinal
detachment. Modulating the activity of these targets provides a
neuroprotective effect in the retina. Thus, modulating MCP-1,
TNF-alpha, IL-1 beta, and/or bFGF can maintain the viability of
photoreceptor cells following retinal detachment and preserve
vision when the retina is reattached.
[0007] In one aspect, the invention provides a method of preserving
the viability of photoreceptor cells disposed within a retina of a
mammalian eye following retinal detachment. The method includes
administering to a mammal having an eye in which a region of the
retina has been detached an amount of a neuroprotective agent
selected from a substance capable of suppressing endogenous MCP-1,
a MCP-1 antagonist, a substance capable of suppressing endogenous
TNF-alpha, a TNF-alpha antagonist, a substance capable of
suppressing endogenous IL-1 beta, an IL-1 beta antagonist, a
substance capable of inducing endogenous bFGF, exogenous bFGF, a
bFGF mimetic, and combinations thereof sufficient to preserve the
viability of photoreceptor cells disposed within the region of the
detached retina. Suppressing endogenous cytokines such as MCP-1,
TNF-alpha or IL-1 beta includes, but is not limited to, suppressing
or otherwise interfering with expression of the gene encoding the
cytokine, suppressing or otherwise interfering with the
transcription of the gene into mRNA, and/or suppressing or
otherwise interfering with the translation of the mRNA from the
cytokine gene into a functional protein.
[0008] This aspect can have any of the following features. The
neuroprotective agent can be administered to the mammal prior to
reattachment of the region of detached retina. The neuroprotective
agent can be administered to the mammal after reattachment of the
region of detached retina. The neuroprotective agent can be
administered locally or systemically. A plurality of
neuroprotective agents can be administered to the mammal. At least
one neuroprotective agent can be administered by intraocular,
intravitreal, or transcleral administration. The neuroprotective
agent can reduce the number of photoreceptor cells in the region
that die following retinal detachment. The retinal detachment
occurs as a result of a retinal tear, retinoblastoma, melanoma,
diabetic retinopathy, uveitis, choroidal neovascularization,
retinal ischemia, pathologic myopia, or trauma.
[0009] In another aspect, the invention provides a method of
preserving the viability of photoreceptor cells in a mammalian eye
following retinal detachment. More particularly, the invention
provides a method of preserving the viability of photoreceptor
cells disposed within a region of a retina that has become detached
from its underlying retinal pigment epithelium and/or choroid. The
method comprises administering to a mammal in need of such
treatment an amount of a neuroprotective agent sufficient to
preserve the viability of photoreceptor cells, for example, rods
and/or cones, disposed within the region of the detached retina.
Administration of the neuroprotective agent minimizes the loss of
visual function resulting from the retinal detachment.
[0010] The neuroprotective agent reduces the number of
photoreceptor cells in the region of the retina that, without
treatment, would die following retinal detachment. It is understood
that photoreceptor cells in the retina may die via a variety of
cell death pathways, for example, via apoptotic and necrotic cell
death pathways. It has been found, however, that upon retinal
detachment, the photoreceptor cells undergo apoptotic cell death in
the detached portion of the retina. Furthermore, one or more
caspases, for example, caspase 3, caspase 7 and caspase 9,
participate in the cascade of events leading to apoptotic cell
death. Accordingly, neuroprotective agents useful in the practice
of the invention can include, for example, an apoptosis inhibitor,
for example, a caspase inhibitor, for example, one or more of, a
caspase 3 inhibitor, a caspase 7 inhibitor, and a caspase 9
inhibitor.
[0011] Because photoreceptors die as a result of retinal
detachment, administration of neuroprotective agents minimize or
reduce the loss of photoreceptor cell viability until such time the
retina becomes reattached to the choroid and an adequate blood and
nutrient supply is once again restored. The neuroprotective agent
minimizes the level of photoreceptor cell death, and maintains
photoreceptor cell viability prior to reattachment of the detached
region of the retina. Under certain circumstances, however, it may
be beneficial to administer the neuroprotective agent for a period
of time after a retinal detachment has been detected and/or the
retina surgically reattached. This period of time may vary and can
include, for example, a period of a week, two weeks, three weeks, a
month, three months, six months, nine months, a year, and two
years, after surgical reattachment.
[0012] The neuroprotective agent, for example, can be administered,
either alone or in combination with a pharmaceutically acceptable
carrier or excipient, by one or more routes. For example, the
neuroprotective agent may be administered systemically, for
example, via oral or parenteral routes, for example, via
intravascular, intramuscular or subcutaneous routes. Alternatively,
the neuroprotective agent may be administered locally, for example,
via intraocular, intravitreal, intraorbital, or transcleral routes.
Furthermore, it is contemplated that a plurality of neuroprotective
agents, for example, a substance capable of suppressing endogenous
MCP-1, a MCP-1 antagonist, a substance capable of suppressing
endogenous TNF-alpha, a TNF-alpha antagonist, a substance capable
of suppressing endogenous IL-1 beta, an IL-1 beta antagonist, a
substance capable of inducing endogenous bFGF, exogenous bFGF, a
bFGF mimetic, one or more caspase inhibitors, and combinations
thereof, may be administered to the mammal to maintain viability of
the photoreceptor cells disposed within the detached portion of the
retina.
[0013] It is contemplated that the practice of the invention will
be helpful in maintaining the viability of photoreceptor cells in
retinal detachments irrespective of how the retinal detachments
were caused. For example, it is contemplated that the practice of
the method of the invention will be helpful in minimizing visual
dysfunction resulting from retinal detachments caused by one or
more of the following: a retinal tear, retinoblastoma, melanoma,
diabetic retinopathy, uveitis, choroidal neovascularization,
retinal ischemia, pathologic myopia, and trauma.
[0014] The foregoing aspects and embodiments of the invention may
be more fully understood by reference to the following figures,
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The objects and features of the invention may be more fully
understood by reference to the drawings described below in
which:
[0016] FIG. 1 depicts a bar chart showing the ratio of cleaved
caspase 3 to pro-caspase 3 in densitometry units in detached
retinas (hatched bars) and attached retinas (solid bars) at one,
three and five days post retinal detachment;
[0017] FIG. 2 depicts a bar chart showing the ratio of cleaved
caspase 9 to pro-caspase 9 in densitometry units in detached
retinas (hatched bars) and attached retinas (solid bars) at one,
three and five days post retinal detachment;
[0018] FIG. 3 depicts a bar chart showing the level of caspase 7 in
densitometry units in detached retinas (hatched bars) and attached
retinas (solid bars) at one, three and five days post retinal
detachment;
[0019] FIG. 4 depicts a bar chart showing the ratio of cleaved
poly-ADP ribose-polymerase (PARP) to pro-PARP in densitometry units
in detached retinas (hatched bars) and attached retinas (solid
bars) at one, three and five days post retinal detachment;
[0020] FIG. 5 depicts a bar chart showing the retinal mRNA
expression of various types of mRNA in detached retina versus
non-detached retina;
[0021] FIG. 6 depicts a bar chart showing retinal mRNA expressional
changes for nineteen genes in detached retina versus non-detached
retina at 72 hours post detachment;
[0022] FIG. 7 depicts a bar chart showing retinal mRNA expressional
changes for nineteen genes in detached retina of the right eye, or
oculus dexter (OD), versus non-detached retina of the left eye, or
oculus sinister (OS), at 72 hours post detachment;
[0023] FIG. 8 depicts a bar chart showing mRNA expressional changes
in the retinal pigment layer (RPE), the outer nuclear layer (ONL),
the inner nuclear layer (INL) and the ganglion cell layer (GCL) of
the retina, for each of four genes (bFGF, TNF-alpha, IL-1 beta, and
MCP-1), in detached retina versus non-detached retina at 72 hours
post detachment;
[0024] FIGS. 9A-E depict four bar charts and a line graph showing
mRNA expressional changes at 1 hour (FIG. 9A), 3 hours (FIG. 9B), 6
hours (FIG. 9C), and 24 hours (FIG. 9D) post retinal detachment,
and a time course (FIG. 9E) showing these changes for certain
genes;
[0025] FIG. 10A depicts a plot showing cytokine levels, as
determined by ELISA, for TNF-alpha, IL-1 beta, and MCP-1 in
detached retinas from right eyes (RD+) and undetached retinas from
left eyes (RD-), at 6 and 72 hours post retinal detachment;
[0026] FIG. 10B depicts an alternative view of the data depicted in
FIG. 10A, with the y-axis showing the ratio of the average ELISA
results for TNF-alpha, IL-1 beta, and MCP-1 in detached retinas
from right eyes (OD) versus undetached retinas from left eyes (OS),
at 6 and 72 hours post retinal detachment;
[0027] FIG. 11A depicts a bar chart showing quantitative results of
TdT-dUTP Terminal Nick End-Labeling (TUNEL) staining of detached
retina 24 hours after both retinal detachment and subretinal
administration of TNF-alpha, IL-1 beta, MCP-1, or control;
[0028] FIG. 11B depicts an alternative view of the data depicted in
FIG. 11A, with the y-axis showing TUNEL-positive cells per square
millimeter of detached retina 24 hours after both retinal
detachment and subretinal administration of TNF-alpha, IL-1 beta,
MCP-1, or control;
[0029] FIG. 12 depicts a bar chart showing quantitative results of
TUNEL staining of cells in the ONL of rats with a detached retina
72 hours after retinal detachment and subsequent treatment with
etanercept;
[0030] FIG. 13 depicts a bar chart showing quantitative results of
TUNEL staining of cells in the ONL of rats with a detached retina
72 hours after retinal detachment and subsequent treatment with
either normal goat serum (NGS) or goat anti-TNF-alpha antibody
(TNFa);
[0031] FIG. 14 depicts a bar chart showing quantitative results of
TUNEL staining of cells in the ONL of detached retina of either
knockout mice lacking the MCP-1 gene (CCL2) or wild-type control
mice, 72 hours after retinal detachment;
[0032] FIG. 15 depicts a bar chart showing quantitative results of
TUNEL staining of cells in the ONL of detached retina of either
knockout mice deficient in TNF-alpha (TNF KO), knockout mice
deficient in TNF Receptors 1A and 1B (TNFR KO), or wild-type
control mice 72 hours after retinal detachment; and
[0033] FIG. 16 depicts a bar chart showing retinal mRNA
expressional levels in detached retina from rats treated with an
intravitreal injection of 5.0 .mu.g pigment epithelium-derived
factor (PEDF) versus detached retina from rats treated with an
intravitreal injection of PBS control.
DETAILED DESCRIPTION
[0034] During retinal detachment, the entire retina or a portion of
the retina becomes dissociated from the underlying retinal pigment
epithelium and choroid. As a result, the sensitive photoreceptor
cells disposed in the detached portion of the retina become
deprived of their normal supply of blood and nutrients. If
untreated, the retina or more particularly the sensitive
photoreceptor cells disposed within the retina die causing partial
or even complete blindness. Accordingly, there is an ongoing need
for methods and compositions that preserve the viability of
photoreceptor cells following retinal detachment. If photoreceptor
cell death can be minimized during retinal detachment, the affected
photoreceptors likely will survive once the retina is reattached to
the underlying retinal pigment epithelium and choroid, and the
photoreceptors regain their normal blood and nutrient supply.
[0035] Retinal detachment can occur for a variety of reasons. The
most common reason for retinal detachment involves retinal tears.
Retinal detachments, however, can also occur because of, for
example, retinoblastomas and other ocular tumors (for example,
angiomas, melanomas, and lymphomas), diabetic retinopathy, retinal
vascular diseases, uveitis, retinal ischemia and trauma.
Furthermore, retinal detachments can occur as a result of formation
of choroidal neovascularizations secondary to, for example, the
neovascular form of age-related macular degeneration, pathologic
myopia, and ocular histoplasmosis syndrome. It is understood that
the clinical pathologies of retinal detachments are different from
those of degenerative retinal disorders, for example, retinitis
pigmentosa and age-related macular degeneration. However, the
neuroprotective agents discussed herein may be useful in treating
retinal detachments that occur secondary to an underlying
degenerative retinal disorder. Accordingly, it is contemplated that
the methods and compositions of the invention may be useful in
minimizing or otherwise reducing photoreceptor cell death following
retinal detachment, irrespective of the cause of the
detachment.
[0036] The invention provides a method of preserving the viability
of photoreceptor cells in a mammalian, for example, a primate, for
example, a human, eye following retinal detachment. More
particularly, the invention provides a method of preserving the
viability of photoreceptor cells disposed within a region of a
retina, which has become detached from its underlying retinal
pigment epithelium and/or choroid. The method may be particularly
helpful in preventing vision loss when the region of detachment
includes at least a portion of the macula. The method comprises
administering to a mammal in need of such treatment an amount of a
neuroprotective agent sufficient to preserve the viability of
photoreceptor cells disposed within the region of the detached
retina.
[0037] As used herein, the term "neuroprotective agent" means any
agent that, when administered to a mammal, either alone or in
combination with other agents, minimizes or eliminates
photoreceptor cell death (including both necrotic and apoptotic
cell death) in a region of the retina that has become detached from
the underlying retinal pigment epithelium and/or choroid. It is
contemplated that useful neuroprotective agents include, for
example, a substance capable of suppressing endogenous MCP-1, a
MCP-1 antagonist, a substance capable of suppressing endogenous
TNF-alpha, a TNF-alpha antagonist, a substance capable of
suppressing endogenous IL-1 beta, an IL-1 beta antagonist, a
substance capable of inducing endogenous bFGF, exogenous bFGF, a
bFGF mimetic, combinations thereof, and apoptosis inhibitors, for
example, caspase inhibitors, and certain neurotrophic factors that
prevent the onset or progression of apoptosis. More specifically,
useful neuroprotective agents may include, for example, a protein
(for example, a growth factor, antibody or an antigen binding
fragment thereof), a peptide (for example, an amino acid sequence
less than about 25 amino acids in length, and optionally an amino
acid sequence less that about 15 amino acids in length), a nucleic
acid (for example, a deoxyribonucleic acid, ribonucleic acid, an
antisense oligonucleotide, or an aptamer), a peptidyl nucleic acid
(for example, an antisense peptidyl nucleic acid), an organic
molecule or an inorganic molecule, which upon administration
minimizes photoreceptor cell death following retinal detachment.
Additionally, interfering RNA (RNAi) techniques can be used.
Neuroprotective agents alternatively or additionally may protect
against gliosis.
[0038] It is understood that photoreceptor cell death during
retinal detachments may occur as a result of either necrotic or
apoptotic (also known as programmed cell death) pathways. Both of
these pathways are discussed in detail in, for example, Kerr et al.
(1972) BR. J. CANCER 26: 239-257, Wyllie et al. (1980) INT. REV.
CYTOLOGY 68: 251-306; Walker et al. (1988) METH. ACHIE. EXP.
PATHOL. 13: 18-54 and Oppenheim (1991) ANN. REV. NEUROSCI. 14:
453-501. Apoptosis involves the orderly breakdown and packaging of
cellular components and their subsequent removal by surrounding
structures (Afford & Randhawa (2000) J. CLIN. PATHOL.
53:55-63). In general, apoptosis, also referred to as an apoptotic
pathway, does not result in the activation of an inflammatory
response. This is in contrast to necrotic cell death, which is
characterized by the random breakdown of cells in the setting of an
inflammatory response. Typically, during necrosis, also known as a
necrotic pathway, a catastrophic event, for example, trauma,
inflammation, ischemia or infection, typically causes uncontrolled
death of a large group of cells. There are a variety of assays
available for determining whether cell death is occurring via a
necrotic pathway or an apoptotic pathway (see, for example, Cook et
al. (1995) INVEST. OPHTHALMOL. VIS. SCI. 36:990-996).
[0039] Apoptosis involves the activation of a genetically
determined cell suicide program that results in a morphologically
distinct form of cell death characterized by cell shrinkage,
nuclear condensation, DNA fragmentation, membrane reorganization
and blebbing (Kerr et al. (1972) BR. J. CANCER 26: 239-257). Assays
for detecting the presence of apoptotic pathways include measuring
morphologic and biochemical stigmata associated with cellular
breakdown and packaging, such as pyknotic nuclei, apoptotic bodies
(vesicles containing degraded cell components) and
internucleosomally cleaved DNA. This last feature is specifically
detected by binding and labeling the exposed 3'--OH groups of the
cleaved DNA with the enzyme terminal deoxynucleotidyl transferase
in the staining procedure often referred to as the TdT-dUTP
Terminal Nick End-Labeling (TUNEL) staining procedure. It is
believed that, at the core of this process lies a conserved set of
serine proteases, called caspases, which are activated specifically
in apoptotic cells.
[0040] There are approximately fourteen known caspases, and the
activation of these proteins results in the proteolytic digestion
of the cell and its contents. Each of the members of the caspase
family possess an active-site cysteine and cleave substrates at
Asp-Xxx bonds (i.e., after the aspartic acid residue). In general,
a caspase's substrate specificity typically is determined by the
four residues amino-terminal to the cleavage site. Caspases have
been subdivided into subfamilies based on their substrate
specificity, extent of sequence identity and structural
similarities, and include, for example, caspase 1, caspase 2,
caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8,
caspase 9, caspase 10, caspase 11, caspase 12, caspase 13 and
caspase 14. Monitoring their activity can be used to assess the
level of on-going apoptosis.
[0041] Furthermore, it has been suggested that apoptosis is
associated with the generation of reactive oxygen species, and that
the product of the Bcl-.sub.2 gene protects cells against apoptosis
by inhibiting the generation or the action of the reactive oxygen
species (Hockenbery et al. (1993) CELL 75: 241-251, Kane et al.
(1993) SCIENCE 262: 1274-1277, Veis et al. (1993) CELL 75: 229-240,
Virgili et al. (1998) FREE RADICALS BIOL. MED. 24: 93-101).
Bcl-.sub.2 belongs to a growing family of apoptosis regulatory gene
products, which may either be death antagonists (Bcl-.sub.2,
Bcl-x.sub.L) or death agonists (Bax, Bak) (Kroemer et al. (1997)
NAT. MED. 3: 614-620). Control of cell death appears to be
regulated by these interactions and by constitutive activities of
the various family members (Hockenbery et al. (1993) CELL 75:
241-251). Several apoptotic pathways may coexist in mammalian cells
that are preferentially activated in a stimulus-, stage-,
context-specific and cell-type manner (Hakem et al. (1998) CELL 94:
339-352). However, it is contemplated that agents that upregulate
the level of the Bcl-2 gene expression or slow down the rate of
breakdown of the Bcl-.sub.2 gene product may be useful in the
practice of the invention.
[0042] Useful apoptosis inhibitors include, for example, (i)
proteins, for example, growth factors, cytokines, antibodies and
antigen binding fragments thereof (for example, Fab, Fab', and Fv
fragments), genetically engineered biosynthetic antibody binding
sites, also known in the art as BABS or sFv's, and (ii) peptides,
for example, synthetic peptides and derivatives thereof, which may
be administered systemically or locally to the mammal. Other useful
apoptosis inhibitors include, for example, deoxyribonucleic acids
(for example, antisense oligonucleotides), ribonucleic acids (for
example, antisense oligonucleotides and aptamers) and peptidyl
nucleic acids, which once administered reduce or eliminate
expression of certain genes, for example, caspase genes as in the
case of anti-sense molecules, or can bind to and reduce or
eliminate the activity of a target protein or receptor as in the
case of aptamers. Additionally, RNAi techniques can be used. Other
useful apoptosis inhibitors include small organic or inorganic
molecules that reduce or eliminate apoptotic activity when
administered to the mammal.
[0043] One set of apoptosis inhibitors useful in the practice of
the invention include caspase inhibitors. Caspase inhibitors
include molecules that inhibit or otherwise reduce the catalytic
activity of a target caspase molecule (for example, a classical
competitive or non-competitive inhibitor of catalytic activity) as
well as molecules that prevent the onset or initiation of a caspase
mediated apoptotic pathway.
[0044] With regard to the inhibitors of catalytic function, it is
contemplated that useful caspase inhibitors include, on the one
hand, broad spectrum inhibitors that reduce or eliminate the
activity of a plurality of caspases or, on the other hand, specific
caspase inhibitors that reduce or eliminate the activity of a
single caspase. In general, caspase inhibitors act by binding the
active site of a particular caspase enzyme and forming either a
reversible or an irreversible linkage to target caspase molecule.
Caspase inhibitors may include inhibitors of one or more of caspase
1, caspase 2, caspase 3, caspase 4, caspase 6, caspase 7, caspase
8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, and
caspase 14.
[0045] Useful caspase inhibitors include commercially available
synthetic caspase inhibitors. Synthetic caspase inhibitors
typically include a peptide recognition sequence attached to a
functional group such as an aldehyde, chloromethylketone,
fluoromethylketone, or fluoroacyloxymethylketone. Typically,
synthetic caspase inhibitors with an aldehyde fu group reversibly
bind to their target caspases, whereas the caspase inhibitors with
the otl functional groups tend to bind irreversibly to their
targets. Useful caspase inhibitors, wl-modeled with
Michaelis-Menten kinetics, preferably have a dissociation constant
of the enzyme-inhibitor complex (K.sub.i) lower than 100 .mu.M,
preferably lower than 50 .mu.M, more preferably lower than 1 .mu.M.
The peptide recognition sequence corresponding to that found in
endogenous substrates determines the specificity of a particular
caspase. For example, peptides with the Ac-Tyr-Val-Ala-Asp-aldehyde
sequence are potent inhibitors of caspases 1 and 4 (K.sub.i=10 nM),
and are weak inhibitors of caspases 3 and 7 (K.sub.i.gtoreq.50
.mu.M). Removal of the tyrosine residue, however, results in a
potent but less specific inhibitor. For example,
2-Val-Ala-Asp-fluoromethylketone inhibits caspases 1 and 4 as well
as caspases 3 and 7.
[0046] Exemplary synthetic caspase 1 inhibitors, include, for
example, Ac-N-Me-Tyr-Val-Ala-Asp-aldehyde,
Ac-Trp-Glu-His-Asp-aldehyde, Ac-Tyr-N-Me-Val-Ala-N-Me-Asp-aldehyde,
Ac-Tyr-Val-Ala-Asp-Aldehyde, Ac-Tyr-Val-Ala-Asp-chloromethylketone,
Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone,
Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetol,
Ac-Tyr-Val-Lys-Asp-aldehyde,
Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone,
biotinyl-Tyr-Val-Ala-Asp-chloromethylketone,
Boc-Asp(OBzl)-chloromethylketone,
ethoxycarbonyl-Ala-Tyr-Val-Ala-Asp-aldehyde (pseudo acid),
Z-Asp-2,6-dichlorobenzoyloxymethylketone,
Z-Asp(OlBu)-bromomethylketone,
Z-Tyr-Val-Ala-Asp-chloromethylketone,
Z-Tyr-Val-Ala-DL-Asp-fluoromethlyketone,
Z-Val-Ala-DL-Asp-fluoromethylketone, and
Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone, all of which can be
obtained from Bachem Bioscience Inc., PA. Other exemplary caspase 1
inhibitors include, for example, Z-Val-Ala-Asp-fiuoromethylketone,
biotin-X-Val-Ala-Asp-fluoromethylketone, Ac-Val-Ala-Asp-aldehyde,
Boc-Asp-fluoromethylketone,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Pro-Tyr-Val-Al-
a-Asp-aldehyde (SEQ ID NO: 1),
biotin-Tyr-Val-Ala-Asp-fluoroacyloxymethylketone,
Ac-Tyr-Val-Ala-Asp-acyloxymethylketone, Z-Asp-CH2-DCB,
Z-Tyr-Val-Ala-Asp-fluoromethylketone, all of which can be obtained
from Calbiochem, Calif.
[0047] Exemplary synthetic caspase 2 inhibitors, include, for
example, Ac-Val-Asp-Val-Ala-Asp-aldehyde, which can be obtained
from Bachem Bioscience Inc., PA, and
Z-Val-Asp-Val-Ala-Asp-fluoromethylketone, which can be obtained
from Calbiochem, Calif.
[0048] Exemplary synthetic caspase 3 precursor protease inhibitors
include, for example, Ac-Glu-Ser-Met-Asp-aldehyde (pseudo acid) and
Ac-Ile-Glu-Thr-Asp-aldehyde (pseudo acid) which can be obtained
from Bachem Bioscience Inc., PA. Exemplary synthetic caspase 3
inhibitors include, for example, Ac-Asp-Glu-Val-Asp-aldehyde,
Ac-Asp-Met-Gin-Asp-aldehyde, biotinyl-Asp-Glu-Val-Asp-aldehyde,
Z-Asp-Glu-Val-Asp-chloromethylketone,
Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone, and
Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone which can be obtained from
Bachem Bioscience Inc., PA. Other exemplary caspase 3 inhibitors
include, for example,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Gl-
u-Val-Asp-aldehyde (SEQ ID NO: 2),
Z-Asp-Glu-Val-Asp-fluoromethylketone,
biotin-X-Asp-Glu-Val-Asp-fluoromethylketone,
Ac-Asp-Glu-Val-Asp-chloromethylketone, which can be obtained from
Calbiochem, Calif.
[0049] Exemplary synthetic caspase 4 inhibitors include, for
example, Ac-Leu-Glu-Val-Asp-aldehyde and
Z-Tyr-Val-Ala-DL-Asp-fluoromethylketone, which can be obtained from
Bachem Bioscience Inc., PA, and
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Gl-
u-Val-Pro-aldehyde (SEQ ID NO: 3), which can be obtained from
Calbiochem, Calif.
[0050] Exemplary synthetic caspase 5 inhibitors include, for
example, Z-Trp-His-Glu-Asp-fluoromethylketone, which can be
obtained from Calbiochem, Calif., and Ac-Trp-Glu-His-Asp-aldehyde
and Z-Trp-Glu(O-Me)-His-Asp(O-Me) fluoromethylketone, which can be
obtained from Sigma Aldrich, Germany.
[0051] Exemplary synthetic caspase 6 inhibitors include, for
example, Ac-Val-Glu-Ile-Asp-aldehyde,
Z-Val-Glu-Ile-Asp-fluoromethylketone, and
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gl-
u-Ile-Asp-aldehyde (SEQ ID NO: 4), which can be obtained from
Calbiochem, Calif.
[0052] Exemplary synthetic caspase 7 inhibitors include, for
example, Z-Asp(OMe)-Gln-Met-Asp(OMe) fluoromethylketone,
Ac-Asp-Glu-Val-Asp-aldehyde,
Biotin-Asp-Glu-Val-Asp-fluoromethylketone,
Z-Asp-Glu-Val-Asp-fluoromethylketone,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Gl-
u-Val-Asp-aldehyde (SEQ ID NO: 2), which can be obtained from Sigma
Aldrich, Germany.
[0053] Exemplary synthetic caspase 8 inhibitors include, for
example, Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Ile-Glu-Pro-Asp-aldehyde,
Ac-Ile-Glu-Thr-Asp-aldehyde, Ac-Trp-Glu-His-Asp-aldehyde and
Boc-Ala-Glu-Val-Asp-aldehyde which can be obtained from Bachem
Bioscience Inc., PA. Other exemplary caspase 8 inhibitors include,
for example,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Gl-
u-Thr-Asp-aldehyde (SEQ ID NO: 5) and
Z-Ile-Glu-Thr-Asp-fluoromethylketone, which can be obtained from
Calbiochem, Calif.
[0054] Exemplary synthetic caspase 9 inhibitors, include, for
example, Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Leu-Glu-His-Asp-aldehyde,
and Ac-Leu-Glu-His-Asp-chloromethylketone which can be obtained
from Bachem Bioscience Inc., PA. Other exemplary caspase 9
inhibitors include, for example,
Z-Leu-Glu-His-Asp-fluoromethylketone and
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Gl-
u-His-Asp-aldehyde (SEQ ID NO: 6), which can be obtained from
Calbiochem, Calif.
[0055] Furthermore, it is contemplated that caspase specific
antibodies (for example, monoclonal or polyclonal antibodies, or
antigen binding fragments thereof), for example, an antibody that
specifically binds to and reduces the activity of, or inactivates a
particular caspase may be useful in the practice of the invention.
For example, an anti-caspase 3 antibody, an anti-caspase 7
antibody, or an anti-caspase 9 antibody may be useful in the
practice of the invention. Additionally, it is contemplated that an
anti-caspase aptamer that specifically binds and reduces the
activity of, or inactivates a particular caspase, for example, an
anti-caspase 3 aptamer, an anti-caspase 7 aptamer, or an
anti-caspase 9 aptamer may be useful in the practice of the
invention.
[0056] Alternatively, endogenous caspase inhibitors can be used to
reduce, or inhibit caspase activity. For example, one useful class
of endogenous caspase inhibitor includes proteins known as
inhibitors of apoptosis proteins (IAPs) (Deveraux et al. (1998)
EMBO JOURNAL 17(8): 2215-2223) including bioactive fragments and
analogs thereof. One exemplary IAP includes X-linked inhibitor of
apoptosis protein (XIAP), which has been shown to be a direct and
selective inhibitor of caspase-3, caspase-7 and caspase-9. Another
exemplary IAP includes survivin (see, U.S. Pat. No. 6,245,523;
Papapetropoulos et al. (2000) J. BIOL. CHEM. 275: 9102-9105),
including bioactive fragments and analogs thereof. Survivin has
been reported to inhibit caspase-3 and caspase-7 activity. It is
also contemplated that molecules that act through IAPs will also be
useful, for example, VEGF has anti-apoptotic activity by acting
through survivin.
[0057] In addition, it is contemplated that useful neuroprotective
agents may include one or more neurotrophic factors, which may
serve as effective apoptosis inhibitors (Lewis et al. (1999)
INVEST. OPHTHALMOL. VIS. SCI. 40: 1530-44; LaVail et al. (1998)
INVEST. OPHTHALMOL. VIS. SCI. 39: 592-602). Exemplary neurotrophic
factors include, for example, Brain Derived Growth Factor (Caffe et
al. (2001) INVEST OPHTHALMOL. VIS. SCI. 42: 275-82) including
bioactive fragments and analogs thereof; Fibroblast Growth Factor
(Bryckaert et al. (1999) ONCOGENE 18: 7584-7593) including
bioactive fragments and analogs thereof; PEDF including bioactive
fragments and analogs thereof; and Insulin-like Growth Factors, for
example, IGF-I and IGF-II (Rukenstein et al. (1991) J NEUROSCI.
11:2552-2563) including bioactive fragments and analogs thereof;
and cytokine-associated neurotrophic factors.
[0058] Bioactive fragments refer to portions of an intact template
protein that have at least 30%, more preferably at least 70%, and
most preferably at least 90% of the biological activity of the
intact proteins. Analogs refer to species and allelic variants of
the intact protein, or amino acid replacements, insertions or
deletions thereof that have at least 30%, more preferably at least
70%, and most preferably 90% of the biological activity of the
intact protein.
[0059] With reference to the foregoing proteins, the term "analogs"
includes variant sequences that are at least 80% similar or 70%
identical, more preferably at least 90% similar or 80% identical,
and most preferably 95% similar or 90% identical to at least a
portion of one of the exemplary proteins described herein, for
example, Brain Derived Growth Factor. To determine whether a
candidate protein has the requisite percentage similarity or
identity to a reference polypeptide, the candidate amino acid
sequence and the reference amino acid sequence are first aligned
using the dynamic programming algorithm described in Smith and
Waterman (1981) J. MOL BIOL. 147:195-197, in combination with the
BLOSUM62 substitution matrix described in FIG. 2 of Henikoff and
Henikoff (1992), PROC. NAT. ACAD. SCI. USA 89:10915-10919. An
appropriate value for the gap insertion penalty is -12, and an
appropriate value for the gap extension penalty is -4. Computer
programs performing alignments using the algorithm of
Smith-Waterman and the BLOSUM62 matrix, such as the GCG program
suite (Oxford Molecular Group, Oxford, England), are commercially
available and widely used by those skilled in the art. Once the
alignment between the candidate and reference sequence is made, a
percent similarity score may be calculated. The individual amino
acids of each sequence are compared sequentially according to their
similarity to each other. If the value in the BLOSUM62 matrix
corresponding to the two aligned amino acids is zero or a negative
number, the pairwise similarity score is zero; otherwise the
pairwise similarity score is 1.0. The raw similarity score is the
sum of the pairwise similarity scores of the aligned amino acids.
The raw score is then normalized by dividing it by the number of
amino acids in the smaller of the candidate or reference sequences.
The normalized raw score is the percent similarity.
[0060] Alternatively, to calculate a percent identity, the aligned
amino acids of each sequence are again compared sequentially. If
the amino acids are non-identical, the pairwise identity score is
zero; otherwise the pairwise identity score is 1.0. The raw
identity score is the sum of the identical aligned amino acids. The
raw score is then normalized by dividing it by the number of amino
acids in the smaller of the candidate or reference sequences. The
normalized raw score is the percent identity. Insertions and
deletions are ignored for the purposes of calculating percent
similarity and identity. Accordingly, gap penalties are not used in
this calculation, although they are used in the initial
alignment.
[0061] Furthermore, by way of example, cAMP elevating agents may
also serve as effective apoptosis inhibitors. Exemplary cAMP
elevating agents include, for example,
8-(4-chlorophenylthio)-adenosine-3':5'-cyclic-monophosphate
(CPT-cAMP) (Koike (1992) PROG. NEURO-PSYCHOPHARMACOL. BIOL.
PSYCHIAT. 16: 95-106), forskolin, isobutyl methylxanthine, cholera
toxin (Martin et al. (1992) J. NEUROBIOL. 23:1205-1220), and
8-bromo-cAMP, N.sup.6, O.sup.2'-dibutyryl-cAMP and
N.sup.6,O.sup.2'dioctanoyl-cAMP (Rydel and Greene (1988) PROC. NAT.
ACAD. SCI. USA 85: 1257-1261).
[0062] Furthermore, other exemplary apoptosis inhibitors can
include, for example, glutamate inhibitors, for example, NMDA
receptor inhibitors (Bamford et al. (2000) EXP. CELL RES. 256:
1-11) such as eliprodil (Kapin et al. (1999) INVEST. OPHTHALMOL.
VIS. SCI 40,1177-82) and MK-801 (Solberg et al. INVEST. OPHTHALMOL.
VIS. SCI (1997) 38,1380-1389) and
n-acetylated-.alpha..pi..pi..eta..alpha.-linked-acidic dipeptidase
inhibitors, such as, 2-(phosphonomethyl) pentanedioic acid (2-PMPA)
(Harada et al. NEUR. LETT. (2000) 292,134-36); steroids, for
example, hydrocortisone and dexamethasone (see, U.S. Pat. No.
5,840,719; Wenzel et al. (2001) INVEST. OPHTHALMOL. VIS. SCI. 42:
1653-9); nitric oxide synthase inhibitors (Donovan et al. (2001) J.
BIOL. CHEM. 276: 23000-8); serine protease inhibitors, for example,
3,4-dichloroisocoumarin and N-tosyl-lysine chloromethyl ketone
(see, U.S. Pat. No. 6,180,402); cysteine protease inhibitors, for
example, N-ethylmaleimide and iodoacetamide, or an interleukin-1
.beta.-converting enzyme inhibitor, for example, Z-Asp-2,
6-dichlorobenzoyloxymethylketone (see, U.S. Pat. No. 6,180,402);
and anti-sense nucleic acid or peptidyl nucleic acid sequences that
lower of prevent the expression of one or more of the death
agonists, for example, the products of the Bax, and Bak genes.
[0063] In addition, or in the alternative, it may be useful to
inhibit expression or activity of members of the caspase cascade
that are upstream or downstream of caspase 3, caspase 7 and caspase
9. For example, it may be useful to inhibit PARP, which is a
component of the apoptosis cascade downstream of caspase 7. An
exemplary PARP inhibitor includes 3-aminobenzamide (Weise et al.
(2001) CELL DEATH DIFFER. 8:801-807). Other examples include
inhibitors of the expression or activity of Apoptosis Activating
Factor-1 (Apaf-1) and/or cytochrome C. Apaf-1 and cytochrome C bind
the activated form of caspase 9 to produce a complex, which is
known to propagate the apoptosis cascade. Thus, any protein (for
example, antibody), nucleic acid (for example, aptamer), peptidyl
nucleic acid (for example, antisense molecule) or other molecule
that inhibits or interferes with the binding of caspase 9 to
Apaf-1/cytochrome C can serve to inhibit apoptosis.
[0064] Under certain circumstances, it may be advantageous to also
administer to the individual undergoing treatment with the
neuroprotective agent an anti-permeability agent and/or an
inflammatory agent so as to minimize photoreceptor cell death. An
anti-permeability agent is a molecule that reduces the permeability
of normal blood vessels. Examples of such molecules include
molecules that prevent or reduce the expression of genes encoding,
for example, Vascular Endothelial Growth Factor (VEGF) or an
Intercellular Adhesion Molecule (ICAM) (for example, ICAM-1, ICAM-2
or ICAM-3). Exemplary molecules include antisense oligonucleotides
and antisense peptidyl nucleic acids that hybridize in vivo to a
nucleic acid encoding a VEGF gene, an ICAM gene, or a regulatory
element associated therewith. Other suitable molecules bind to
and/or reduce the activity of, for example, the VEGF and ICAM
molecules (for example, anti-VEGF and anti-ICAM antibodies and
antigen binding fragments thereof, and anti-VEGF or anti-ICAM
aptamers). Other suitable molecules bind to and prevent ligand
binding and/or activation of a cognate receptor, for example, the
VEGF receptor or the ICAM receptor. Such molecules may be
administered to the individual in an amount sufficient to reduce
the permeability of blood vessels in the eye. An anti-inflammatory
agent is a molecule that prevents or reduces an inflammatory
response in the eye and in some instances can be considered a
neuroprotective agent. Exemplary anti-inflammatory agents include
steroids, for example, hydrocortisone, dexamethasone sodium
phosphate, and methylpredisolone. Such molecules may be
administered to the individual in an amount sufficient to reduce or
eliminate an inflammatory response in the eye.
[0065] It is contemplated that the foregoing and other
neuroprotective agents now known or hereafter discovered may be
assayed for efficacy in minimizing photoreceptor cell death
following retinal detachment using a variety of model systems.
Basic techniques for inducing retinal detachment in various animal
models are known in the art (see, for example, Anderson et al.
(1983) INVEST. OPHTHALMOL. VIS. SCI. 24: 906-926; Cook et al.
(1995) INVEST. OPHTHALMOL. VIS. SCI. 36: 990-996; Marc et al.
(1998) OPHTHALMOL. VIS. SCI. 39: 1694-1702; Mervin et al. (1999)
AM. J. OPHTHALMOL. 128: 155-164; Lewis et al. (1999) AM. J.
OPHTHALMOL. 128: 165-172). Once a suitable animal model has been
created (see, Example 1 below) an established or putative
neuroprotective agent can be administered to an eye at different
dosages. The ability of the neuroprotective agent and dosage
required to maintain cell viability may be assayed by one or more
of (i) tissue histology, (ii) TUNEL staining, which quantifies the
number of TUNEL-positive cells per section, (iii) electron
microscopy, (iv) immunoelectron microscopy to detect the level of,
for example, apoptosis inducing factor (AIF) in the samples, and
(v) immunochemical analyses, for example, via Western blotting, to
detect the level of certain caspases in a sample.
[0066] The TUNEL technique is particularly useful in observing the
level of apoptosis in photoreceptor cells. By observing the number
of TUNEL-positive cells in a sample, it is possible to determine
whether a particular neuroprotective agent is effective at
minimizing or reducing the level of apoptosis, or eliminating
apoptosis in a sample. For example, the potency of the
neuroprotective agent will have an inverse relationship to the
number of TUNEL-positive cells per sample. By comparing the
efficacy of a variety of potential neuroprotective agents using
these methods, it is possible to identify neuroprotective factors
most useful in the practice of the invention.
[0067] The neuroprotective agent may be administered to the mammal
from the time the retinal detachment is detected to the time the
retina is repaired, for example, via surgical reattachment. It is
understood, however, that under certain circumstances, it may be
advantageous to administer the neuroprotective agent to the mammal
even after the retina has been surgically repaired. For example,
even after the surgical reattachment of a detached retina in
patients with rhegmatogenous retinal detachments, persistent
subretinal fluid may exist under the fovea as detected by ocular
coherence tomography long after the surgery has been performed
(see, Hagimura et al. (2002) AM. J. OPHTHALMOL. 133:516-520). As a
result, even after surgical repair the retina may still not be
completely reattached to the underlying retinal pigment epithelium
and choroid. Furthermore, when retinal detachments occur secondary
to another disorder, for example, the neovascular form of
age-related macular degeneration and ocular melanomas, it may be
beneficial to administer the neuroprotective agent to the
individual while the underlying disorder is being treated so as to
minimize loss of photoreceptor cell viability. Accordingly, in such
cases, it may be advantageous to administer the neuroprotective
agent to the mammal for one week, two weeks, three weeks, one
month, three months, six months, nine months, one year, two years
or more (i) after retinal detachment has been identified, and/or
(ii) after surgical reattachment of the retina has occurred, and/or
(iii) after detection of an underlying degenerative disorder, so as
to minimize photoreceptor cell death.
[0068] Once the appropriate neuroprotective agents have been
identified, they may be administered to the mammal of interest in
any one of a wide variety of ways. It is contemplated that a
neuroprotective agent, for example, a caspase inhibitor, can be
administered either alone or in combination with another
neuroprotective agent, for example, a neurotrophic agent. It is
contemplated that the efficacy of the treatment may be enhanced by
administering two, three, four or more different neuroprotective
agents either together or one after the other. Although the best
means of administering a particular neuroprotective agent or
combination of neuroprotective agents may be determined
empirically, it is contemplated that neuroprotective agents may be
administered locally or systemically.
[0069] Systemic modes of administration include both oral and
parenteral routes. Parenteral routes include, for example,
intravenous, intrarterial, intramuscular, intradermal,
subcutaneous, intranasal and intraperitoneal routes. It is
contemplated that the neuroprotective agents administered
systemically may be modified or formulated to target the
neuroprotective agent to the eye. Local modes of administration
include, for example, intraocular, intraorbital, subconjuctival,
intravitreal, subretinal or transcleral routes. It is noted,
however, that local routes of administration are preferred over
systemic routes because significantly smaller amounts of the
neuroprotective agent can exert an effect when administered locally
(for example, intravitreally) versus when administered systemically
(for example, intravenously). Furthermore, the local modes of
administration can reduce or eliminate the incidence of potentially
toxic side effects that may occur when therapeutically effective
amounts of neuroprotective agent (i.e., an amount of a
neuroprotective agent sufficient to reduce, minimize or eliminate
the death of photoreceptor cells following retinal detachment) are
administered systemically.
[0070] Administration may be provided as a periodic bolus (for
example, intravenously or intravitreally) or as continuous infusion
from an internal reservoir (for example, from an implant disposed
at an intra- or extra-ocular location (see, U.S. Pat. Nos.
5,443,505 and 5,766,242)) or from an external reservoir (for
example, from an intravenous bag). The neuroprotective agent may be
administered locally, for example, by continuous release from a
sustained release drug delivery device immobilized to an inner wall
of the eye or via targeted transscleral controlled release into the
choroid (see, for example, PCT/US00/00207, PCT/US02/14279, Ambati
et al. (2000) INVEST. OPHTHALMOL. VIS. SCI. 41:1181-1185, and
Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI. 41:1186-1191). A
variety of devices suitable for administering a neuroprotective
agent locally to the inside of the eye are known in the art. See,
for example, U.S. Pat. Nos. 6,251,090, 6,299,895, 6,416,777,
6,413,540, and 6,375,972, and PCT/US00/28187.
[0071] The neuroprotective agent also may be administered in a
pharmaceutically acceptable carrier or vehicle so that
administration does not otherwise adversely affect the recipient's
electrolyte and/or volume balance. The carrier may comprise, for
example, physiologic saline or other buffer system.
[0072] In addition, it is contemplated that the neuroprotective
agent may be formulated so as to permit release of the
neuroprotective agent over a prolonged period of time. A release
system can include a matrix of a biodegradable material or a
material which releases the incorporated neuroprotective agent by
diffusion. The neuroprotective agent can be homogeneously or
heterogeneously distributed within the release system. A variety of
release systems may be useful in the practice of the invention,
however, the choice of the appropriate system will depend upon rate
of release required by a particular drug regime. Both
non-degradable and degradable release systems can be used. Suitable
release systems include polymers and polymeric matrices,
non-polymeric matrices, or inorganic and organic excipients and
diluents such as, but not limited to, calcium carbonate and sugar
(for example, trehalose). Release systems may be natural or
synthetic. However, synthetic release systems are preferred because
generally they are more reliable, more reproducible and produce
more defined release profiles. The release system material can be
selected so that neuroprotective agents having different molecular
weights are released by diffusion through or degradation of the
material.
[0073] Representative synthetic, biodegradable polymers include,
for example: polyamides such as poly(amino acids) and
poly(peptides); polyesters such as poly(lactic acid), poly(glycolic
acid), poly(lactic-co-glycolic acid), and poly(caprolactone);
poly(anhydrides); polyorthoesters; polycarbonates; and chemical
derivatives thereof (substitutions, additions of chemical groups,
for example, alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art),
copolymers and mixtures thereof. Representative synthetic,
non-degradable polymers include, for example: polyethers such as
poly(ethylene oxide), poly(ethylene glycol), and
poly(tetramethylene oxide); vinyl polymers-polyacrylates and
polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl
methacrylate, acrylic and methacrylic acids, and others such as
polyvinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl
acetate); poly(urethanes); cellulose and its derivatives such as
alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various
cellulose acetates; polysiloxanes; and any chemical derivatives
thereof (substitutions, additions of chemical groups, for example,
alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art),
copolymers and mixtures thereof.
[0074] One of the primary vehicles currently being developed for
the delivery of ocular pharmacological agents is the
poly(lactide-co-glycolide) microsphere for intraocular injection.
The microspheres are composed of a polymer of lactic acid and
glycolic acid, which are structured to form hollow spheres. These
spheres can be approximately 15-30 .mu.m in diameter and can be
loaded with a variety of compounds varying in size from simple
molecules to high molecular weight proteins such as antibodies. The
biocompatibility of these microspheres is well established (see,
Sintzel et al. (1996) EUR. J. PHARM. BIOPHARM. 42: 358-372), and
microspheres have been used to deliver a wide variety of
pharmacological agents in numerous biological systems. After
injection, poly(lactide-co-glycolide) microspheres are hydrolyzed
by the surrounding tissues, which cause the release of the contents
of the microspheres (Zhu et al. (2000) NAT. BIOTECH. 18: 52-57). As
will be appreciated, the in vivo half-life of a microsphere can be
adjusted depending on the specific needs of the system.
[0075] The type and amount of neuroprotective agent administered
may depend upon various factors including, for example, the age,
weight, gender, and health of the individual to be treated, as well
as the type and/or severity of the retinal detachment to be
treated. As with the modes of administration, it is contemplated,
that the optimal neuroprotective agents and dosages of those
neuroprotective agents may be determined empirically. The
neuroprotective agent preferably is administered in an amount and
for a time sufficient to permit the survival of at least 25%, more
preferably at least 50%, and most preferably at least 75%, of the
photoreceptor cells in the detached region of the retina.
[0076] By way of example, protein-, peptide- or nucleic acid-based
neuroprotective agents can be administered at doses ranging, for
example, from about 0.001 to about 500 mg/kg, optionally from about
0.01 to about 250 mg/kg, and optionally from about 0.1 to about 100
mg/kg. Nucleic acid-based neuroprotective agents may be
administered at doses ranging from about 1 to about 20 mg/kg daily.
Furthermore, antibodies that are neuroprotective agents may be
administered intravenously at doses ranging from about 0.1 to about
5 mg/kg once every two to four weeks. With regard to intravitreal
administration, the neuroprotective agents, for example,
antibodies, may be administered periodically as boluses in dosages
ranging from about 10 .mu.g to about 5 mg/eye, and optionally from
about 100 .mu.g to about 2 mg/eye. With regard to transcleral
administration, the neuroprotective agents may be administered
periodically as boluses in dosages ranging from about 0.1 .mu.g to
about 1 mg/eye, and optionally from about 0.5 .mu.g to about 0.5
mg/eye.
[0077] The present invention, therefore, includes the use of a
neuroprotective agent, for example, a substance capable of
suppressing endogenous MCP-1, a MCP-1 antagonist, a substance
capable of suppressing endogenous TNF-alpha, a TNF-alpha
antagonist, a substance capable of suppressing endogenous IL-1
beta, an IL-1 beta antagonist, a substance capable of inducing
endogenous bFGF, exogenous bFGF, a bFGF mimetic, a caspase
inhibitor, and combinations thereof, in the preparation of a
medicament for treating an ocular condition associated with a
retinal detachment, for example, a loss of vision as a result of
photoreceptor cell death in the region of retinal detachment. A
composition comprising one or more neuroprotective agents, one
agent optionally being a caspase inhibitor, may be provided for use
in the present invention. The neuroprotective agent or agents may
be provided in a kit which optionally may comprise a package insert
with instructions for how to treat the patient with the retinal
detachment. For each administration, the neuroprotective agent may
be provided in unit-dosage or multiple-dosage form. Preferred
dosages of the neuroprotective agents, however, are as described
above.
[0078] It has also been observed, as more fully described in
Examples 3 and 4 below, that mRNA levels of bFGF, TNF-alpha, and
IL-1 beta, and protein levels of bFGF, TNF-alpha, and IL-1 beta
increase in retinas in response to detachment of the retina from
the underlying choroidal tissue. bFGF is a cytokine that has
anti-inflammatory activity, while TNF-alpha and IL-1 beta are
cytokines with pro-inflammatory activity. Accordingly, to the
extent the viability of photoreceptor cells disposed within a
retina (as well as other cells disposed within the retina) is to be
preserved, steps may be taken to exploit these natural biological
responses by either enhancing the anti-inflammatory substance or
suppressing the pro-inflammatory substance.
[0079] For example, insofar as bFGF has anti-inflammatory activity,
it is possible to enhance the level of bFGF by either further
inducing its production or exogenously adding it, in order to
provide further anti-inflammatory activity. Similarly, it is
possible to add exogenous molecules that mimic the activity of bFGF
(a bFGF mimetic). Any of these routes may preserve the viability of
photoreceptor cells disposed within a retina. Examples of the
neuroprotective agents that can enhance the level of bFGF,
supplement the level of bFGF, or mimic the activity of bFGF,
include proteins or peptides that are inducers of the bFGF gene,
exogenous bFGF itself (whether isolated from a natural source or
manufactured using recombinant DNA techniques), peptides from the
active portion of the full size bFGF protein, and small molecules.
In some instances, one or more of the following may be useful to
modulate bFGF: growth hormone (increases bFGF-mRNA), TGF-beta 1
(upregulates bFGF-mRNA expression and bFGF levels), cell-associated
heparin-like molecules and heparan sulfate proteoglycans (controls
bioavailability of bFGF to ocular cells), prostaglandin E2
(stimulates bFGF-mRNA expression), prolactin (stimulates bFGF-mRNA
expression), nicotine (regulates bFGF production and increases
bFGF-mRNA), CS23 (acid-stable mutein of bFGF which up-regulates
bFGF-mRNA expression), forskolin and PMA (increases bFGF-mRNA
expression), acidosis (enhances bFGF-mRNA expression as well as
bFGF secretion), NYAG (Naoyi'an granule, may enhance bFGF
expression and suppress TNF), huangpi when paired with TGF-beta 1
(may increase bFGF), lansoprazole (increases bFGF levels),
Silicone-containing gel dressing (Silastic (SGS) and ClearSite)
(increases levels of bFGF), angiotensin II (increases bFGF
expression), Luteinizing hormone (increases bFGF expression), and
finasteride (decreases bFGF levels--to the extent bFGF decrease is
desired). It should be understood that any of the dosage
strategies, drug formulations, or administration schedules
described above are applicable to all of these neuroprotective
agents.
[0080] Conversely, insofar as TNF-alpha and IL-1 beta have
pro-inflammatory activity, one may decrease the level of one or
both of these cytokines or otherwise antagonize one or both of
their activities, in order to reduce the pro-inflammatory activity.
Examples of the neuroprotective agents that can suppress the level
of TNF-alpha or antagonize the activity of TNF-alpha, include, for
example, etanercept (Example 5, below), estrogen (decreases TNF
gene expression), inhibition of p38 MAPK (decreases TNF-alpha
expression), chronic garlic administration (decreases TNF-alpha
expression), eicosapentaenoic acid (decreases TNF-alpha
expression), and TBP2 and TBP3 (decrease TNF-alpha activity).
Neuroprotective agents can also include growth factors, cytokines,
antibodies, for example TNF-alpha blocking antibody as described in
Example 6, below, and Di62-anti-TNF-alpha monoclonal antibody
(decreases. TNF-alpha activity), and antigen binding fragments
thereof (for example, Fab, Fab', and Fv fragments), genetically
engineered biosynthetic antibody binding sites, also known in the
art as BABS or sFv's, and peptides, for example, synthetic peptides
and derivatives thereof, which may be administered to systemically
or locally to the mammal. Other useful neuroprotective agents
include, for example, deoxyribonucleic acids (for example,
antisense oligonucleotides), ribonucleic acids (for example,
antisense oligonucleotides, aptamers, and interfering RNA) and
peptidyl nucleic acids, which once administered reduce or eliminate
expression of certain genes or can bind to and reduce or eliminate
the activity of a target protein or receptor as in the case of
aptamers. Other useful neuroprotective agents include small organic
or inorganic molecules that reduce or eliminate activity when
administered to the mammal. Any of these routes may preserve the
viability of photoreceptor cells disposed within a retina. It
should be understood that any of the dosage strategies, drug
formulations, or administration schedules described above are
applicable to all of these neuroprotective agents.
[0081] Examples of the neuroprotective agents that can suppress the
level of IL-1 beta or antagonize the activity of IL-1 beta, include
pseudo-ICE and ICEBERG (block IL-1 beta secretion),
polymorphonuclear cell (PMN) inhibitors (decrease IL-1),
glucocorticoids (decrease IL-1 beta expression), cyclosporine
combined with a steroid (decreases IL-1 expression),
15-deoxy-Delta(12,14)-PGJ2 (PGJ2, decreases IL-1 beta and TNF-alpha
expression and increases IL-1rn expression), PPARgamma ligands
(decrease IL-1 secretion), cPKC and nPKC (decrease IL-1 beta and
TNF-alpha production), IL-1rn (IL-1 antagonist), PMA (increases
IL-1rn expression), IL-10 (increases IL-1rn), retinoic acid
(upregulates IL-1 beta--to the extent an increase in IL-1 is
desired), phorbol esters (increase IL-1 beta expression, --to the
extent an increase in IL-1 is desired), IFNs (increase or decrease
IL-1/IL-1rn), and lipopolysaccharide (increases IL-1rn expression
and increases IL-1). Neuroprotective agents also include growth
factors, cytokines, antibodies and antigen binding fragments
thereof (for example, Fab, Fab', and Fv fragments), genetically
engineered biosynthetic antibody binding sites, also known in the
art as BABS or sFv's, and (ii) peptides, for example, synthetic
peptides and derivatives thereof, which may be administered to
systemically or locally to the mammal. Other useful neuroprotective
agents include, for example, deoxyribonucleic acids (for example,
antisense oligonucleotides), ribonucleic acids (for example,
antisense oligonucleotides, aptamers, and interfering RNA) and
peptidyl nucleic acids, which once administered reduce or eliminate
expression of certain genes or can bind to and reduce or eliminate
the activity of a target protein or receptor as in the case of
aptamers. Other useful neuroprotective agents include small organic
or inorganic molecules that reduce or eliminate activity when
administered to the mammal. Any of these routes may preserve the
viability of photoreceptor cells disposed within a retina. It
should be understood that any of the dosage strategies, drug
formulations, or administration schedules described above are
applicable to all of these neuroprotective agents.
[0082] One or more of these cytokines can be modulated to preserve
the viability of photoreceptor cells disposed within a retina (as
well as other cells disposed within the retina). It should be
understood that any of the dosage strategies, drug formulations, or
administration schedules described above are applicable to all of
these neuroprotective agents. Additionally, while treatments
involving these cytokine responses in detached retinas above are
described with relation to their anti- or pro-inflammatory activity
and potential for inducing cell death, to the extent another
mechanism is involved (for example, the ability of these cytokines
to affect a undesirable proliferative change in photoreceptor cells
or apoptosis), similar strategies can be used to choose
neuroprotective agents that modulate the cytokines.
[0083] In addition, as shown in Examples 3 and 4, MCP-1 mRNA and
MCP-1 protein are increased in detached retinas as compared to
non-detached retinas. The increase in this factor can induce
migration of microglia and macrophages to the detachment area for
phagocytosis of the debris produced by apoptotic photoreceptors.
These monocytes also may be related to secretion of TNF-alpha, and
further destruction of photoreceptors. Immunohistochemistry data
indicates that MCP-1 (and bFGF) was increased in Muller cells three
days after retinal detachment, and microglia migrate toward the
Muller cells, which increase the MCP-1 protein. Insofar as
microglia and macrophages may be secondarily toxic to
photoreceptors, suppression of the increase of MCP-1 following
retinal detachment may be beneficial.
[0084] Examples of the neuroprotective agents that can suppress the
level of MCP-1 or antagonize the activity of MCP-1, include, for
example, ADR7 and ADR22 (MCP-1 antagonists), renin-angiotensin
system (RAS) inhibitors (decreases MCP-1 expression), naked plasmid
encoding 7ND (MCP-1 antagonist), dilazep (inhibits MCP-1 mRNA
expression), fenofibric acid (inhibits MCP-1 mRNA expression),
cetirizine (decreases MCP-1 levels), tenidap (decreases MCP-1
expression), dexamethasone (decreases MCP-1 mRNA expression),
IFN-gamma (inhibits lipopolysaccharide-inducible MCP-1), blockers
of PTK and PKC which are needed for MCP-1 gene expression in human
monocytes, triple helix-forming oligonucleotides (TFO's) (selective
inhibitor of MCP-1 gene expression), LY294002 (inhibits MCP-1
expression), olmesartan (inhibits MCP-1 and TNF expression),
suppressors of NF-kappaB (inhibits MCP-1 expression), wogonin
(inhibits MCP-1 expression), and Platelet-activating factor (PAF)
(stimulates MCP-1 expression--to the extent an increase in IL-1 is
desired). Neuroprotective agents can also include growth factors,
cytokines, antibodies and antigen binding fragments thereof (for
example, Fab, Fab', and Fv fragments), genetically engineered
biosynthetic antibody binding sites, also known in the art as BABS
or sFv's, and (ii) peptides, for example, synthetic peptides and
derivatives thereof, which may be administered to systemically or
locally to the mammal. Other useful neuroprotective agents include,
for example, deoxyribonucleic acids (for example, antisense
oligonucleotides), ribonucleic acids (for example, antisense
oligonucleotides, aptamers, and interfering RNA) and peptidyl
nucleic acids, which once administered reduce or eliminate
expression of certain genes or can bind to and reduce or eliminate
the activity of a target protein or receptor as in the case of
aptamers. Other useful neuroprotective agents include small organic
or inorganic molecules that reduce or eliminate activity when
administered to the mammal. Any of these routes may preserve the
viability of photoreceptor cells disposed within a retina. These
neuroprotective agents can be used in combination with the
neuroprotective agents described above, for example with the
neuroprotective agents related to the cytokines described above. It
should be understood that any of the dosage strategies, drug
formulations, or administration schedules described above are
applicable to all of these neuroprotective agents.
[0085] Throughout the description, where compositions are described
as having, including, or comprising specific components, or where
processes are described as having, including, or comprising
specific process steps, it is contemplated that compositions of the
present invention also consist essentially of, or consist of, the
recited components, and that the processes of the present invention
also consist essentially of, or consist of, the recited processing
steps. Further, it should be understood that the order of steps or
order for performing certain actions are immaterial so long as the
invention remains operable. Moreover, two or more steps or actions
may be conducted simultaneously.
[0086] In light of the foregoing description, the specific
non-limiting examples presented below are for illustrative purposes
and not intended to limit the scope of the invention in any
way.
EXAMPLES
Example 1: Detection of Caspase Activity Following Retinal
Detachment
[0087] This example demonstrates that certain caspases,
particularly caspases 3, 7 and 9, are activated in photoreceptor
cells following retinal detachment.
[0088] Experimental retinal detachments were created using
modifications of previously published protocols (Cook et al. (1995)
INVEST. OPHTHALMOL. VIS. SCI. 36(6):990-6; Hisatomi et al. (2001)
AM. J. PATH. 158(4):1271-8). Briefly, rats were anesthetized using
a 50:50 mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml).
Pupils were dilated using a topically applied mixture of
phenylephrine (5.0%) and tropicamide (0.8%). A 20 gauge
micro-vitreoretinal blade was used to create a sclerotomy
approximately 2 mm posterior to the limbus. Care was taken not to
damage the lens during the sclerotomy procedure. A Glaser
subretinal injector (20 gauge shaft with a 32 gauge tip,
Becton-Dickinson, Franklin Lakes, N.J.) connected to a syringe
filled with 10 mg/ml of Healon.RTM. sodium hyaluronate (Pharmacia
and Upjohn Company, Kalamazoo, Mich.) then was introduced into the
vitreous cavity. The tip of the subretinal injector was used to
create a retinotomy in the peripheral retina, and then the sodium
hyaluronate was slowly injected into the subretinal space to
elevate the retina from the underlying retinal pigment epithelium.
Retinal detachments were created only in the left eye (OS) of each
animal, with the right eye (OD) serving as the control. In each
experimental eye, approximately one half of the retina was
detached, allowing the attached portion to serve as a further
control.
[0089] Following creation of the experimental retinal detachment,
intraocular pressures were measured before and immediately after
retinal detachment with a Tono-pen. No differences in intraocular
pressures were noted. The retinal break created by the subretinal
injector was confined only to the site of the injection.
[0090] Light microscopic analysis of the detached retinas showed an
increase in morphologic stigmata of apoptosis as a function of time
after detachment. Eyes then were enucleated one, three, five and
seven days after creation of the retinal detachment. For light
microscopic analysis, the cornea and lens were removed and the
remaining eyecup placed in a fixative containing 2.5%
glutaraldehyde and 2% formaldehyde in 0.1M cacodylate buffer (pH
7.4) and stored at 4.degree. C. overnight. Tissue samples then were
post-fixed in 2% osmium tetroxide, dehydrated in graded ethanol,
and embedded in epoxy resin. One-micron sections were stained with
0.5% toluidine blue in 0.1% borate buffer and examined with a Zeiss
photomicroscope (Axiophot, Oberkochen, Germany).
[0091] At one day after creation of the detachment, pyknosis in the
ONL was confined to the area of the peripheral retinotomy site
through which the subretinal injector was introduced. By three
days, however, pyknotic nuclei were seen in the whole ONL of the
retina in the area of the detachment. Extrusion of pyknotic nuclei
from the ONL into the subretinal space was observed. The remaining
layers of the retina appeared morphologically normal. No
inflammatory cells were seen, and there was no apparent disruption
of the retinal vasculature. Similar changes were seen in sections
from retinas detached for up to one week. No pyknotic nuclei were
seen in the area of the attached retina or in the fellow,
non-detached eye. The amount of ONL pyknosis was similar between
detachments of three-day or one week duration.
[0092] Disruption of the photoreceptor outer segments was a
prominent feature in the detached retinas. Outer segments of the
control eyes and the attached portions of the experimental eyes had
an orderly, parallel arrangement. Detachments produced
artifactually during tissue processing in these eyes did not alter
the photoreceptor morphology. In contrast, the photoreceptor outer
segments of detached retinas were severely disorganized and lost
their normal structural organization. Additionally, outer segments
in attached areas had similar lengths, whereas the outer segments
in detached areas showed variable lengths.
[0093] Internucleosomal DNA cleavage in photoreceptor cells was
detected via TUNEL staining. For TUNEL staining, the cornea and
lens were not removed after enucleation, but rather the whole eye
was fixated overnight at 4.degree. C. in a phosphate buffered
saline solution of 4% paraformaldehyde solution (pH 7.4). Then, a
section was removed from the superior aspect of the globe and the
remaining eyecup embedded in paraffin and sectioned at a thickness
of 6 .mu.m. TUNEL staining was performed on these sections using
the TdT-Fragel DNA Fragmentation Detection Kit (Oncogene Sciences,
Boston, Mass.) in accordance with the manufacturer's instructions.
Reaction signals were amplified using a preformed avidin:
biotinylated-enzyme complex (ABC-kit, Vector Laboratories,
Burlingame, Calif.). Internucleosomally cleaved DNA fragments were
stained with diaminobenzidine (DAB) (staining indicates
TUNEL-positive cells) and sections were then counterstained with
methylene green.
[0094] TUNEL-positive cells were detected at all time points tested
(one, three, five and seven days post-detachment). TUNEL-positive
staining was confined only to the photoreceptor cell layer. Two
eyes with retinal detachments that persisted for two months were
monitored. The TUNEL assay at two months did not reveal any
staining indicating the presence of internucleosomally cleaved DNA.
The prolonged detachment was associated with a marked reduction in
the thickness of and number of cell bodies contained in the ONL as
compared to the non-detached retina.
[0095] Antibodies specific for caspases 3, 7, 9 and PARP were used
in Western blots to probe total retinal protein extracts at various
times after creation of the retinal detachment. For Western blot
analysis, retinas from both experimental and control eyes were
manually separated from the underlying retinal pigment
epithelium/choroid at days one, three and five after creation of
the retinal detachment. In eyes with retinal detachments, the
experimentally detached portion of the retina was separated from
the attached portion of the retina and analyzed separately. Retinas
were homogenized and lysed with buffer containing 1 mM ethylene
diaminetetraacetic acid/ethylene glycol-bis
(2-aminoethylethel-N,N,N',N'-tetraacetic acid/dithiothreitol, 10 mM
HEPES pH 7.6, 0.5% (octylphenoxy)polyethoxyethanol (IGEPAL), 42 mM
potassium chloride, 5 mM magnesium chloride, 1 mM
phenylmethanesulfonyl fluoride and 1 tablet of protease inhibitors
per 10 ml buffer (Complete Mini, Roche Diagnostics GmbH, Mannheim,
Germany). Samples were incubated for 15 minutes on ice, and then
centrifuged at 21,000 rpm at 4.degree. C. for 30 minutes. The
protein concentration of the supernatant was determined using the
Bio-Rad D.sub.C Protein Assay reagents (Bio-Rad Laboratories,
Hercules, Calif.).
[0096] Proteins were separated via sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (7.5% and 15% Tris-HCL
Ready-Gels, Bio-Rad Laboratories), in which 30 .mu.g of total
retinal protein were applied in each lane. The fractionated
proteins were transferred to a PVDF membrane (Immobilon-P,
Millipore, Bedford, Mass.). The resulting membrane was blocked with
5% non-fat dry milk in 0.1% TBST IGEPAL. The blocked membranes then
were incubated with antibodies against caspase 7 (1:1,000; Cell
Signaling Technology, Beverly, Mass.), caspase 9 (1:1,000; Medical
& Biological Laboratories, Naka-ku Nagoya, Japan),
cleaved-caspase 3 (1:1,000; Cell Signaling Technology, Beverly,
Mass.), caspase 3 (1:2000; Santa Cruz, Santa Cruz, Calif.) or PARP
(1:1000; Cell Signaling Technologies, Beverly, Mass.) overnight at
4.degree. C. Bands were detected using the ECL-Plus reagent
(Amersham, Pharmacia, Piscataway, N.J.). Membranes were exposed to
HyperFilm (Amersham) and densitometry was preformed using
ImageQuant 1.2 software (Molecular Dynamics, Inc., Sunnyvale,
Calif.). For each eye tested, densitometry levels were normalized
by calculating the ratio of the cleaved-form to the pro-form of the
protein of interest. Pro-caspase 7 levels were normalized to the
densitometry readings from a non-specific band detected by the
secondary IgG. Five eyes were used for each time point, except for
the PARP levels for day 5 after detachment for which only four eyes
were used. All statistical comparisons were performed using a
paired t-test.
[0097] The cleaved, or active form of caspase 3 was elevated in the
detached retinas as compared to the attached retinas. The level of
cleaved-caspase 3 increased as a function of time after detachment,
with a peak at approximately three days (see, FIG. 1). No
cleaved-caspase 3 was detected in the control eye or in the
attached portion of the retina in the experimental eye.
[0098] The ratio of the active to inactive form of caspase 9 also
increased as a function of time after creation of the experimental
retinal detachment (see, FIG. 2). The peak level of cleaved-caspase
9 was seen at three to five days after creation of the detachment.
The caspase 7 antibody was able only to detect the pro-form of the
protein. There was, however, a significant difference in the amount
of the pro-form detected in the protein extract from the detached
retinas as compared to the attached retinas (see, FIG. 3). Western
blotting with antibodies against PARP (a component of the apoptosis
cascade downstream of caspase 7) detected an increase in the level
of cleaved-PARP that was maximal at five days after detachment
(see, FIG. 4). P-values for the comparisons between detached and
attached retinas are shown in FIGS. 1-4.
[0099] The results demonstrate that caspase 3, caspase 7 and
caspase 9 are all activated in photoreceptor cells following
retinal attachment.
Example 2: Preservation of Photoreceptor Viability Following
Retinal Detachment
[0100] The type of experiment provided herein may show that the
viability of photoreceptor cells in a detached region of a retina
can be maintained by administering a caspase inhibitor to an
affected eye.
[0101] Retinal detachments are surgically induced in Brown-Norway
rats as discussed in Example 1. The caspase inhibitor,
Z-Val-Ala-Asp-fluoromethylketone is dissolved in dimethyl sulfoxide
(DMSO) to give the final concentrations of 0.2 mM, 2 mM, and 20 mM.
After creating the retinotomy with the subretinal injector, a small
amount of Healon.RTM. sodium hyaluronate is injected in the
subretinal space so as to elevate the retina. After retinal
elevation, a Hamilton syringe with a 33 gauge needle is introduced
through the retinotomy site, and 25 .mu.l of inhibitor is injected
into the region of detachment. About 25 minutes later, Healon.RTM.
sodium hyaluronate is injected, via the same retinotomy site, to
maintain the retinal detachment. Healon.RTM. sodium hyaluronate is
injected until resistance is detected.
[0102] Only the right eyes of rats are used in evaluating the role
of the Z-VAD-FMK inhibitor. The left eyes serve as the controls.
Five animals are used for each concentration of inhibitor (namely,
no inhibitor, 0.2 mM inhibitor, 2 mM inhibitor, and 20 mM
inhibitor). For the no inhibitor control, 25 .mu.l of DMSO is
injected into the region of detachment followed by Healon.RTM.
sodium hyaluronate.
[0103] After 72 hours, the eyes are enucleated and the rats
euthanized. The enucleated eyes are paraffin embedded as described
in Example 1. Then, 6 .mu.m sections from the posterior segments
are analyzed by TUNEL staining as described in Example 1. It is
contemplated that there will be fewer photoreceptor cells in the
region of retinal detachment that stain TUNEL-positive in eyes
treated with the caspase inhibitor relative to eyes that have not
been treated with the caspase inhibitor.
Example 3: Detection of mRNA Levels of bFGF, TNF-Alpha, IL-1 Beta,
and MCP-1
[0104] This example demonstrates that mRNA levels of bFGF,
TNF-alpha, IL-1 beta, and MCP-1 increase in retinal samples
following retinal detachment.
[0105] Two groups of eyes from six adult male Brown Norway rats
(300-450 g, Charles River, Boston, Mass.) were examined. One group
of eyes was the control group having non-detached retinas, while
the other group of eyes was the experimental group having detached
retinas (each rat had one control and one experimental eye). Rats
were anesthetized with a 50:50 mixture of ketamine (100 mg/mL) and
xylazine (20 mg/mL) (both from Phoenix Pharmaceutical, St. Joseph,
Mo.). Pupils were dilated with a topically applied mixture of
phenylephrine (5.0%) and tropicamide (0.8%). A sclerotomy was
created approximately 2 mm posterior to the limbus with a 20-gauge
needle. Care was taken not to damage the lens during creation of
the sclerotomy. A Glaser subretinal injector (20-gauge shaft with a
32-gauge tip, BD Biosciences, Franklin Lakes, N.J.) connected to a
syringe filled with 10 mg/mL Healon.RTM. sodium hyaluronate
(Pharmacia and Upjohn Co., Kalamazoo, Mich.) was then introduced
into the vitreous cavity. A retinotomy was created in the
peripheral retina with the tip of the subretinal injector, and the
sodium hyaluronate was slowly injected into the subretinal space.
Thus, one half of the retina was detached. Retinal detachments were
created only in the right eye of each animal, with the left eye
serving as the control. Three days after retinal detachment, the
rats were sacrificed with an overdose of sodium pentobarbital and
the eyes were enucleated. The neural retina was dissected in a cold
pool of PBS and frozen immediately with powdered dry ice.
[0106] Total RNA was extracted with a Micro-to-Midi.TM. Total RNA
Purification System (Invitrogen, Carlsbad, Calif.) according to the
manufacture's instructions. Each retina was homogenized manually
with 600 .mu.ul of RNA lysis solution, and the same volume of 70%
ethanol was added. The mixture was applied to an RNA spin cartridge
and centrifuged at 12,000.times.g for 15 seconds at 25.degree. C.
The cartridge was washed with wash buffer I and twice with wash
buffer II. Total RNA was eluted with 30 .mu.l of RNase-free water.
To decrease the contamination of genomic DNA, DNase I (Invitrogen)
was added followed by a 15 minute incubation at room temperature.
The concentration of total RNA was measured with a UV spectrometer
(MUTO, JAPAN). Three micrograms of total RNA was used in a reverse
transcription reaction using the SuperScript III First-Strand
Synthesis System for RT-PCR (Invitrogen). First-strand cDNA was
amplified using an ABI7700 real-time PCR system with a SYBR.RTM.
green PCR core kit (Applied Biosystems) and PCR primer sets as
listed in Table 1, in which the forward primers (F1) appear in the
5' to 3' direction from left to right and the reverse primers (R1)
appear in the 3' to 5' direction from left to right.
[0107] PCR amplification was performed for 40 cycles with two step
PCR methods. Denaturation was at 96.degree. C. for 30 seconds and
annealing and extension were at 60.degree. C. for 90 seconds. The
quality of PCR products was evaluated by agarose gel
electrophoresis and staining with ethidium bromide. For relative
comparison of each gene, the Ct value of real-time PCR data was
analyzed with the 2(-Delta Delta C(T)) method (Livak et al. (2001)
METHODS. 25: 402-8). To standardize the amount of sample cDNA added
to each reaction, the Ct value of each target gene was subtracted
by the Ct value of endogenous control (18rRNA).
[0108] The data from the experimental and control groups were
analyzed using a T-test and StatView 4.11J software for the
Macintosh computer (Abacus concepts Inc., Berkeley, Calif.). The
significance level was set at P<0.05 (*). All values are
expressed as the mean.+-.standard deviation (SD). The bar chart
shown in FIG. 6 indicates the expressional change of each mRNA
examined by real-time PCR of the six rats 3 days following retinal
detachment. The Y axis presents the relative value of the
experimental right eye versus the untreated left control eye. bFGF,
TNF-alpha, IL-1 beta, and MCP-1 were significantly increased 3 days
after retinal detachment.
[0109] Additionally, for each type of mRNA of interest (bFGF, PEDF,
TNF-alpha, IL-1 beta, HGF, CNTF, TGF beta, and IGF 1), the amount
of mRNA from each of the six eyes of a group was averaged. Then,
the average amount of a type of mRNA from the detached group was
compared with the amount of a type of mRNA from the non-detached
group. The results are shown in FIG. 5. As can be seen, the level
of bFGF is approximately 11 times greater in the detached group
than in the non-detached group; the level of TNF-alpha is
approximately 8 times greater in the detached group than in the
non-detached group; and the level of IL-1 beta is approximately 3
to 4 times greater in the detached group than in the non-detached
group.
TABLE-US-00001 TABLE 1 Primer Set for real-time PCR. 18rRNA
F1-CAGTGAAACTGCGAATGGCTCATT (SEQ ID NO: 7)
R1-CCCGTCGGCATGTATTAGCTCTAGA (SEQ ID NO: 8) bFGF
F1-TCTTCCTGCGCATCCATCCAGA (SEQ ID NO: 9) R1-CAGTGCCACATACCAACTGGAG
(SEQ ID NO: 10) BDNF F1-CTTGGACAGAGCCAGCGGATTTGT (SEQ ID NO: 11)
R1-CCGTGGACGTTTGCTTCTTTCATG (SEQ ID NO: 12) NT-3
F1-TCTGCCACGATCTTACAGGTGAACA (SEQ ID NO: 13)
R1-CGCCTGGATCAACTTGATAATGAGG (SEQ ID NO: 14) NT-4
F1-TACCCTGGCAAGAGAGACGAGGAA (SEQ ID NO: 15)
R1-CCACCGTGCATGGTTTATGATACG (SEQ ID NO: 16) GDNF
F1-TGCCCTTCGCGCTGACCAGTGACA (SEQ ID NO: 17)
R1-TTCGAGGAAGTGCCGCCGCTTGTT (SEQ ID NO: 18) IGF-1
F1-TTCAGTTCGTGTGTGGACCAAGG (SEQ ID NO: 19)
R1-GCTTCAGCGGAGCACAGTACATCT (SEQ ID NO: 20) HGF
F1-AGATGAGTGTGCCAACAGGTGCAT (SEQ ID NO: 21)
R1-AGGTCAAATTCATGGCCAAACCC (SEQ ID NO: 22) PDGFA
F1-CACTGTTAAGCATGTGCCGGAGAA (SEQ ID NO: 23)
R1-CCAGATCAAGAAGTTGGCCGATGT (SEQ ID NO: 24) PDGFB
F1-CTTGAACATGACCCGAGCACATTCT (SEQ ID NO: 25)
R1-ATCGATGAGGTTCCGCGAGATCT (SEQ ID NO: 26) TNF-alpha
F1-CCCAGACCCTCACACTCAGATCAT (SEQ ID NO: 27)
R1-GCAGCCTTGTCCCTTGAAGAGAA (SEQ ID NO: 28) IL-1 beta
F1-TCAGGAAGGCAGTGTCACTCATTG (SEQ ID NO: 29)
R1-ACACACTAGCAGGTCGTCATCATC (SEQ ID NO: 30) TGEbeta2
F1-AATGGCTCTCCTTCGACGTGACA (SEQ ID NO: 31)
R1-CCTCCAGCTCTTGGCTCTTATTTGG (SEQ ID NO: 32) CNTF
F1-GCCGTTCTATCTGGCTAGCAAGGA (SEQ ID NO: 33)
R1-GCCTCAGTCATCTCACTCCAACGA (SEQ ID NO: 34) MCP-1
F1-ATGCAGGTCTCTGTCACGCTTCTG (SEQ ID NO: 35)
R1-GACACCTGCTGCTGGTGATTCTCTT (SEQ ID NO: 36) VEGF
F1-TCTTCCAGGAGTACCCCGATGAGA (SEQ ID NO: 37)
R1-GGTTTGATCCGCATGATCTGCAT (SEQ ID NO: 38) Angiopoietin-1
F1-GCCCAGATACAACAGAATGCGGTT (SEQ ID NO: 39)
R1-CTCCAGCAGTTGGATTTCAAGACG (SEQ ID NO: 40) Angiopoietin-2
F1-CTCGGAAACTGACTGATGTGGAAGC (SEQ ID NO: 41)
R1-TGTCCTCCATGTCCAGCACTTTCTT (SEQ ID NO: 42) CTGF
F1-ACCCAACTATGATGCGAGCCAACT (SEQ ID NO: 43)
R1-AATTTTAGGCGTCCGGATGCACT (SEQ ID NO: 44) PEDF
F2-GCTGTTTCCAACTTCGGCTACGAT (SEQ ID NO: 45)
R2-AGAGAGCCCGGTGAATGACAGACT (SEQ ID NO: 46)
Example 4: Characterization of Cytokine Response in Experimental
Retinal Detachment
[0110] This example characterizes the molecular and cellular
responses that occur after retinal detachment by quantifying growth
factors, cytokines, and chemokines in a rat model of experimental
retinal detachment.
[0111] Initial experiments characterized cytokine, chemokine, and
growth factor responses to retinal detachment by determining
changes in gene expression in the whole neural retina following
retinal detachment using 19 different PCR primer sets. An
identified subset of cytokines and growth factors was further
investigated to determine the cellular origin and time course of
gene expression by combining laser capture microdissection (LCM)
with quantitative real-time PCR (QPCR), and by
immunohistochemistry. LCM allows capture of specific cells in a
histological section using laser irradiation (see, for example,
Sgroi et al. (1999) CANCER RES. 59: 5656-61). Real-time PCR detects
small changes in gene expression using PCR amplification and
quantification of mRNA levels with the 2(-Delta Delta C(T)) method
(Livak et al. (2001) METHODS. 25: 402-8). In order to pinpoint
cellular sources of cytokine production, LCM and QPCR techniques
were combined to isolate cells from various retinal layers and
quantify the mRNA in those layers. Data from these techniques
demonstrates a relationship between retinal detachment, cytokine
activation and photoreceptor cell death. Additionally, for certain
cytokines and growth factors, protein levels after retinal
detachment were examined.
[0112] Methods
[0113] Retinal detachment was induced by subretinal injection of
sodium hyaluronate. Retinal tissues were collected at various time
points (1, 3, 6, 24, 72 hours) after inducing retinal detachment.
Neural retina was homogenized, and mRNA expression was quantified
by QPCR. To identify the cellular sources of expressed genes,
samples from various retinal layers were obtained using LCM.
Immunohistochemistry and Enzyme Linked-Immuno-Sorbent Assay (ELISA)
were performed to show expressional changes of proteins. TUNEL
staining was used in order to assess photoreceptor death induced by
retinal detachment with or without subretinal administration of
cytokines.
[0114] Retinal Detachment Procedure
[0115] Fifty-five adult male Brown Norway rats (200-300 g, Charles
River, Boston, Mass.) were used in this study. Rats were
anesthetized with a 1:1 mixture of ketamine (100 mg/mL) and
xylazine (20 mg/mL; both from Phoenix Pharmaceutical, St. Joseph,
Mo.). Pupils were dilated with a topically applied mixture of
phenylephrine (5.0%) and tropicamide (0.8%; Massachusetts Eye and
Ear Infirmary internal formulary preparation). A sclerotomy was
created approximately 2 mm posterior to the limbus with a 22-gauge
needle. Care was taken not to damage the lens during creation of
the sclerotomy. A Glaser subretinal injector (20-gauge shaft with a
32-gauge tip, BD Biosciences, San Jose, Calif.) connected to a
syringe filled with 10 mg/mL Healon.RTM. sodium hyaluronate
(Pharmacia and Upjohn Co., Kalamazoo, Mich.) was then introduced
into the vitreous cavity. A retinotomy was created in the
peripheral retina with the tip of the subretinal injector, and the
sodium hyaluronate was slowly injected into the subretinal space,
causing detachment of one half of the retina.
[0116] Retinal detachments were created only in the right eye of
each animal, with the left eye serving as a control. At specified
days after retinal detachment, rats were sacrificed with an
overdose of sodium pentobarbital, and the eyes were enucleated.
Each neural retina was immersed and dissected in cooled PBS and was
frozen immediately with dry ice. Samples were kept at -80.degree.
C. until used in further experiments.
[0117] RNA Extraction and RT-PGR Examination
[0118] Total RNA was extracted with a Micro-to-Midi.TM. Total RNA
Purification System (Invitrogen Corp., Carlsbad, Calif.) according
to the manufacturer's instructions. Each retina was homogenized
manually with 600 .mu.l of RNA lysis solution and added to an
equivalent volume of 70% ethanol. The mixture was applied to an RNA
spin cartridge and centrifuged at 12,000.times.g for 15 seconds at
25.degree. C. The cartridge was washed once with wash buffer I and
twice with wash buffer II. Total RNA was eluted using 30 .mu.l of
RNase-free water. To prevent contamination of genomic DNA, DNase I
(Invitrogen Corp., Carlsbad, Calif.) was added, followed by a 15
minute incubation at room temperature. Total RNA concentration was
measured using a UV spectrophotometer (UV-1201, Shimadzu corp.,
Kyoto, JAPAN). Three micrograms of total RNA was used in a reverse
transcription reaction using the SuperScript.TM. III First-Strand
Synthesis System for RT-PCR (Invitrogen, Carlsbad, Calif.).
First-strand cDNA was amplified using an ABI7700 real-time PCR
system with a SYBR.RTM. green PCR core kit (Applied Biosystems,
Foster City, Calif.) and the PCR primer sets listed in Table 1.
[0119] PCR amplification was performed for 40 cycles with two-step
PCR methods. Denaturation was at 96.degree. C. for 30 seconds and
annealing and extension were at 60.degree. C. for 90 seconds. The
quality of the PCR products was evaluated by agarose gel
electrophoresis and staining with ethidium bromide. For relative
comparison of each gene, the Ct value of real-time PCR data was
analyzed with the 2(-Delta Delta C(T)) method (Livak et al. (2001)
METHODS. 25: 402-8). To normalize the amount of sample cDNA added
to each reaction, the Ct value of each target gene was subtracted
by the Ct value of endogenous control (18rRNA).
[0120] Laser Capture Microdissection (LCM)
[0121] Three days after retinal detachment, eyes were enucleated
and embedded in Tissue-Tek.degree. OCT.TM. compound (Sakura
Finetechnical Co., Ltd., Tokyo, Japan). Transverse cryosections
measuring 12 microns and including the optic nerve were made with a
cryostat (Micron, Germany) and mounted on Superfrost.RTM. Plus
slides (Fisherbrand, Pittsburgh, Pa.). Before LCM, sections were
dehydrated using 75% ethanol, DEPC water twice, 75% ethanol, 95%
ethanol, and 100% ethanol for one minute each and Xylen for 5
minutes.
[0122] Tissue separation with LCM was achieved using a laser at 70
mW laser power for 0.75 seconds with a spot size of 7.5 .mu.m for
the GCL and for the RPE. These settings were changed to 90 mW for
1.2 seconds with a 15 .mu.m spot size for the INL and for the ONL.
LCM was performed on each nuclear layer (GCL, INL, ONL, RPE) from
16 sections in the area of the detached retina and from the
corresponding area of the undetached, left eye. Samples collected
from cell layers of the left, intact eye served as controls. After
collection of aimed cells with the LCM caps (Arcturus, Mountain
View, Calif.), total RNA was extracted with a PicoPure RNA
Isolation Kit (Arcturus, cat# KIT0202) according to the
manufacturer's instructions with the recommended optional DNase
treatment (Qiagen, catalog#79254, Valencia, Calif.). Total RNA was
eluted with 30 .mu.l of Elution Buffer. Subsequently, 24 .mu.l of
the solution containing total RNA was used for QPCR.
[0123] ELISA of TNF-Alpha, IL-1 Beta, and MCP-1
[0124] Samples of neural retina were collected at 6 and 72 hours
after retinal detachment. For each retina, protein was extracted
with 200 .mu.l of RIPA lysis buffer (50 mM Tris [pH 8.0], 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl) containing one
tablet of protease inhibitor cocktail (Complete, Roche Diagnostics,
Alameda, Calif.) and was sonicated at 10 watts using a Branson
Sonifier.RTM. 250 (Branson Ultrasonics Corp., Danbury, Conn.) for 2
seconds. A 30 minute incubation on ice followed. The supernatant
was collected after centrifugation at 14,000.times.g (Micromax RF,
Thermo IEC, Needham Heights, Mass.) for 30 minutes at 4.degree. C.,
and the total protein concentration was measured with a DC protein
assay kit (Bio-Rad, Hercules, Calif.). One hundred micrograms of
total protein were used for ELISA (Biosource, Camarillo, Calif.),
and ELISA was performed according to the protocol that was provided
with the kit. The absorbance at 450 nm wavelength was measured
using a 96-well plate-reading spectrophotometer (Spectramax 190,
Molecular Devices, Sunnyvale Calif.).
[0125] Immunohistochemisny
[0126] Isolated retinas were fixed in 4% paraformaldehyde (PFA) at
4.degree. C. overnight and then cryoprotected with PBS (0.1 M
phosphate buffer [pH, 7.4], 0.15 M NaCl) containing 20% sucrose.
Retinal specimens were frozen in Tissue-Tek.RTM. OCT.TM. compound,
and 10 .mu.m sections were prepared with a cryostat to include the
optic nerve. Sections were mounted onto Superfrost.RTM. slides,
placed in blocking buffer (PBS containing 10% goat serum, 0.5%
gelatin, 3% BSA, and 0.2% Tween20), and incubated with rabbit
anti-rat polyclonal bFGF (Santa Cruz Biotech. Inc., Santa Cruz,
Calif., 1:200), rabbit anti-rat polyclonal TNF-alpha (1:200),
rabbit anti-rat polyclonal IL-1 beta (Pierce Biotechnology, Inc.,
Rockford, Ill., 1:200) or rabbit anti-rat polyclonal MCP-1
(Peprotec, Rocky Hill, N.J., 1:200). For double staining, mouse
monoclonal antibody against glial fibrillary acidic protein (GFAP,
Sigma-Aldrich, 1:400), as a marker of astrocytes, or mouse
monoclonal antibody against glutamine synthetase (BD Biosciences,
San Jose, Calif., 1:200), as a marker of Muller cells, were used.
The same procedure was used for the negative control but without
the primary antibody. Sections were then incubated with
fluorescence-conjugated secondary antibody, either goat anti-mouse
immunoglobulin G (IgG) conjugated to Alexa Fluor 488 (green color)
or anti-rabbit IgG conjugated to Alexa Fluor 546 (red color)
(Molecular Probes, Eugene, Oreg.). Sections were mounted with
Vectashield mounting media with propidium iodide (Vector
Laboratories, Burlingame, Calif.). Photomicrographs of retinal
sections were taken 2 mm from the center of the optic nerve head
using fluorescent microscopy (DMRXA camera, Leica, Germany) and
OpenLab software, version 2.2.5 (Improvision Inc., Lexington,
Mass.).
[0127] TUNEL Staining
[0128] Subretinal administration of select cytokines was performed
following the creation of retinal detachment. Twenty four hours
after retinal detachment and subretinal administration of 5 .mu.l
of PBS, rat recombinant TNF-alpha (0.1 .mu.g/.mu.1), rat
recombinant IL-1 beta (0.1 .mu.g/.mu.l), or rat recombinant MCP-1
(0.1 .mu.g/.mu.l), the eyes were harvested, fixed overnight with 4%
PFA, and cryoprotected with 20% sucrose. TUNEL staining was
performed using the ApopTag.RTM. Fluorescein In Situ Apoptosis
detection kit (S7110, Chemicon International, Inc., Temecula,
Calif.). The center of the retinal detachment lesion was
photographed. TUNEL-positive cells were counted in a masked
fashion, and standard error was determined.
[0129] Statistics
[0130] Data from the experimental and control groups were analyzed
with an unpaired T-test using StatView 4.11J software for a
Macintosh computer (Abacus concepts Inc., Berkeley, Calif.). The
significance level was set to P<0.05 (*). Except where otherwise
noted, values were expressed as mean.+-.standard deviation
(SD).
[0131] Results
[0132] Significant increases in bFGF, TNF-alpha, IL-1 beta, and
MCP-1 mRNA were observed in neural retina 72 hours after retinal
detachment. LCM revealed increased expression of mRNA for bFGF and
MCP-1 in all retinal layers, with bFGF especially evident in the
ONL and MCP-1 evident in the INL. TNF-alpha mRNA was significantly
increased in ONL and INL. IL-1 beta mRNA was significantly
increased in GCL.
[0133] Time course experiments showed that bFGF mRNA was increased
after 24 hours and that MCP-1 mRNA was detectable after 1 hour.
However, the curve of time dependent increase for MCP-1 mRNA was
similar to that of bFGF mRNA. TNF-alpha and IL-1 beta mRNA were
increased within 1 hour following retinal detachment; however,
TNF-alpha mRNA showed a second increase after 6 hours. ELISA
analysis revealed that TNF-alpha, IL-1 beta, and MCP-1 proteins
were increased significantly at 6 hours after retinal detachment.
Immunohistochemistry indicated bFGF and TNF-alpha protein
expression in the whole retina, while IL-1 beta protein was
specifically expressed in the astrocytes and MCP-1 protein was
expressed in the Muller cells. Subretinal administration of
exogenous MCP-1 protein increased TUNEL-positive cells in ONL 24
hours after retinal detachment.
[0134] Gene-Expression Following Retinal Detachment
[0135] Retinal detachment was induced in the rats, and the
expression of 19 different genes was examined by QPCR at 72 hours
following detachment. The nineteen genes that were examined
included bFGF; brain-derived neurotrophic factor (BDNF);
neurotrophin-3,4 (NT-3,4); glial cell line-derived neurotrophic
factor (GDNF); insulin growth factor-1 (IGF-1); hepatocyte growth
factor (HGF); platelet-derived growth factor A,B (PDGFA,B);
TNF-alpha; IL-1 beta; transforming growth factor beta 2
(TGF-beta2); ciliary neurotrophic factor (CNTF); MCP-1; vascular
endothelial growth factor (VEGF); angiopoietin-1,2 (Angio-1,2);
connective tissue growth factor (CTGF): and PEDF.
[0136] FIG. 7, which displays certain data also shown in FIG. 6,
indicates the expression pattern of these nineteen genes and shows
the average fold increase in expression of each gene's mRNA in
detached retinas of right eyes (OD) as compared to the expression
of the same gene's mRNA in undetached retinas of left eyes (OS),
for six rats. The genes are shown on the x-axis and the average
fold increase is shown on the y-axis. The average fold increase in
mRNA expression of bFGF (11.6.+-.6.0, p<0.0001), TNF-alpha
(5.7.+-.3.3, p=0.0015), IL-1 beta (3.8.+-.2.5, p=0.0003), and MCP-1
(149.3.+-.53.3, p<0.0001) was significant at 72 hours following
retinal detachment. Other cytokines and growth factors showed no
significant change in the average fold increase in expression in
the detached retinas in right eyes (OD) versus the non-detached
retinas in left eyes (OS).
[0137] LCM Analysis
[0138] QPCR analysis was performed on samples collected from
various retinal layers using LCM. The RPE contains retinal pigment
epithelial cells; the ONL primarily contains photoreceptors; the
INL is composed of multiple cell types including amacrine cells,
bipolar cells, horizontal cells, and Miller cells, whose cellular
processes span the retina; and the GCL primarily contains retinal
ganglion cells (RGC), displaced amacrine cells, and, to some
extent, astrocytes and endothelial cells.
[0139] FIG. 8 shows results of QPCR analysis on LCM-collected
samples of three to five rats. The average fold increase in mRNA
expression of bFGF was significantly increased in all layers. The
average fold increase in mRNA expression of TNF-alpha was
significantly decreased in the RPE (0.2.+-.0.1, p=0.0185), and
increased in the ONL (15.6.+-.12.8, p=0.0211) and in the INL
(54.4.+-.20.9, p=0.0008), but not significantly changed in the GCL.
The average fold increase in mRNA expression of IL-1 beta was
significantly increased only in the GCL (9.4.+-.3.5, p=0.0467), but
not in the other layers. The average fold increase in mRNA
expression of MCP-1 was increased significantly in ONL
(18.6.+-.8.0, p=0.0493), INL (187.0.+-.67.1, p=0.0104), and GCL
(4.0.+-.1.2, p=0.0164), but unchanged in RPE. These data suggest
that the distribution of genes induced after retinal detachment is
specific to various retinal layers. The highest level of gene
induction was, for bFGF, in the ONL, for TNF-alpha and MCP-1 in the
INL, and for IL-1 beta in the GCL.
[0140] Time Course Evaluation Following Retinal Detachment
[0141] Three days following retinal detachment, significant average
fold increases in mRNA expression were detected for bFGF,
TNF-alpha, IL-1 beta, and MCP-1, as shown in FIG. 7. To see earlier
time points for these responses, retinal tissues were harvested at
1 h, 3 h, 6 h, and 24 h after induction of detachment. FIGS. 9A-D
show the time course evaluation of the average fold increase in
mRNA expression in total retina with detachment in right eyes (OD)
as compared to total retina without detachment in left eyes (OS) at
1 hour (FIG. 9A), 3 hours (FIG. 9B), 6 hours (FIG. 9C), and 24
hours (FIG. 9D).
[0142] As shown in FIG. 9A, at one hour after retinal detachment
the average fold increases in mRNA expression of TNF-alpha, IL-1
beta, and MCP-1 were significant. However, MCP-1 mRNA levels were
not as elevated at 1 hour compared to 72 hours following retinal
detachment (as shown in FIG. 7). The average fold increase of mRNA
expression of bFGF was not changed at this time point.
[0143] As shown in FIG. 9B, at three hours after retinal detachment
the average fold increases of mRNA expression of IL-1 beta and
MCP-1 were significant, but the fold increase of IL-1 beta was
lower than after 1 hour. The average fold increases of mRNA
expression of TNF-alpha and bFGF were not changed
significantly.
[0144] As shown in FIG. 9C, at six hours after retinal detachment
the average fold increase of mRNA expression of TNF-alpha was at
its peak level, but the average fold increases of mRNA expression
of bFGF, IL-1 beta, and MCP-1 were not significantly changed from
their 3 hour levels.
[0145] As shown in FIG. 9D, at twenty-four hours after retinal
detachment the average fold increase of mRNA expression of bFGF
increased significantly. The average fold increases in mRNA
expression of TNF-alpha, IL-1 beta, and MCP-1 remained
significant.
[0146] These data indicate that gene expressional changes for
TNF-alpha, IL-1 beta, and MCP-1 can be detected as early as one
hour following retinal detachment, and that increased expression of
bFGF becomes significant by 24 hours after retinal detachment. As
FIG. 9E shows, the peak of IL-1 beta induction was 1 hour after
retinal detachment. After 3 hours IL-1 beta dropped and then
remained constant. TNF-alpha showed relatively heightened levels at
1 hour, dropped at 3 hours, and peaked at 6 hours (FIGS. 9A-C). As
shown in FIG. 9E, the curve of MCP-1 induction was similar to that
of bFGF, however MCP-1 increased significantly within 1 hour. These
data suggest that inducible factors, MCP-1 and bFGF, overlap 24
hours later and that there may be a functional redundancy between
these genes.
[0147] ELISA of TNF-Alpha, IL-1 Beta, and MCP-1
[0148] To investigate changes in cytokine expression at the protein
level, ELISA was performed on neural retina at 6 and 72 hours after
retinal detachment. Total protein levels in detached retinas versus
undetached retinas did not vary significantly, as shown in Table 2.
However, the ratios of TNF-alpha, IL-1 beta, and MCP-1 proteins in
the right eye (OD, with detached retina) as compared to the left
eye (OS, with non-detached retina) were significantly increased at
six hours. Seventy-two hours after retinal detachment, the
significant increase in MCP-1 expression was sustained. FIGS. 10A
and 10B show the quantitative results of TNF-alpha, IL-1 beta, and
MCP-1 protein expression as assessed by ELISA. In FIG. 10A, the
y-axis represents measured cytokine levels (mean.+-.SD) in eyes
with detached retinas (RD+) and in eyes with undetached retinas
(RD-) with statistical significance determined using the
Mann-Whitney U test. FIG. 10B shows and alternate view of the same
data, in which the y-axis shows the ratio of the average ELISA
result from detached retinal samples (OD) versus control retinal
samples (OS) at 6 and 72 hours after detachment for 4 rats (n=4,
mean.+-.SD). At six hours after retinal detachment in the untreated
control left eye, the average concentration of TNF-alpha was 1.24
pg/.mu.g total protein (n=4), IL-1 beta was 1.12 pg/.mu.g total
protein, and MCP-1 was 0.87 pg/.mu.g total protein, while in the
right eye with retinal detachment, TNF-alpha was 1.57 pg/.mu.g
total protein, IL-1 beta was 1.41 pg/.mu.g total protein, and MCP-1
was 1.35 pg/.mu.g total protein. These data indicate that 6 hours
after retinal detachment the relative protein levels of TNF-alpha,
IL-1 beta, and MCP-1 were significantly increased.
TABLE-US-00002 TABLE 2 Total protein per retina of detached retina
and control. RD RD (-) RD6 h (.mu.g/retina) 921.5 .+-. 22.2 929.0
.+-. 46.9 N.S RD72 h(.mu.g/retina) 960.3 .+-. 83.5 950.3 .+-. 38.4
N.S
[0149] Immunohistochemistry of bFGF, TNF-Alpha, IL-1 Beta and MCP-1
after Retinal Detachment
[0150] The distribution of bFGF, TNF-alpha, IL-1 beta, and MCP-1
proteins was analyzed at 6 and 72 hours after retinal detachment by
immunohistochemistry using polyclonal antibodies against these
proteins. In untreated retinal sections, immunoreactivity of bFGF
and TNF-alpha was weakly detectable in the entire retina;
immunoreactivity of IL-1 beta was distributed in the vitreal
surfaces of the GCL; and MCP-1 was slightly detectable in the cell
bodies in the GCL and INL.
[0151] Six hours following retinal detachment, immunoreactivity of
bFGF was unchanged. But after 72 hours, the immunoreactivity of
bFGF was increased in the ONL, INL, and GCL, and especially in the
vitreal surfaces of the GCL and in the middle row of the INL
(Miller cells). Monocytes localized on the outer segment of
photoreceptors, which appeared at 72 hours after retinal
detachment, also showed immunoreactivity for bFGF.
[0152] TNF-alpha was increased in the ONL and INL at 6 hours after
retinal detachment, but, by 72 hours, the intensity of the signal
was decreased. The infiltrating monocytes showed TNF-alpha
immunoreactivity at 72 hours after retinal detachment.
[0153] IL-1 beta immunoreactivity was significantly increased in
the GCL 6 hours and 72 hours after retinal detachment. However, the
signal after 6 hours was stronger than at 72 hours, and monocytes
in the subretinal space were not stained. Double labeling with
antibody against GFAP, an astrocyte marker, demonstrated that the
IL-1 beta immunoreactivity co-localized with astrocytes.
[0154] Immunoreactivity for MCP-1 was significantly increased in
the INL, with the appearance of spindle-shaped cells that are
indicative of Muller cells, by 6 hours. Greater staining was
observed at 72 hours. The subretinal monocytes also expressed the
MCP-1 protein. The expression of MCP-1 in the INL was co-localized
with glutamine synthetase, a Muller cell marker.
[0155] These data suggested that bFGF and TNF-alpha were increased
in the whole retina, while IL-1 beta and MCP-1 were specifically
increased in astrocyte or Muller cells, respectively, in the neural
retina.
[0156] TUNEL Staining 24 Hours after Retinal Detachment with
Subretinal Administration of Cytokines
[0157] To investigate effects of cytokines on photoreceptor cell
death induced by retinal detachment, TUNEL staining was performed
24 hours after retinal detachment with subretinal administration of
PBS, TNF-alpha (0.1 .mu.g/.mu.1), IL-1 beta (0.1 .mu.g/.mu.l), or
MCP-1 (0.1 .mu.g/.mu.l). FIG. 11A shows the average cell
TUNEL-positive cells counted per field, with error bars
representing standard error. In the control condition with PBS,
TUNEL-positive cells were detected in the ONL 24 hours after
retinal detachment (15.3.+-.5.4 cells/field, n=7). The number of
TUNEL-positive cells did not change significantly with subretinal
administration of TNF-alpha and IL-1 beta. However, subretinal
administration of MCP-1 increased the number of TUNEL-positive
cells in the ONL 24 hours after retinal detachment (67.7.+-.46.5
cells/field, p=0.0138, n=7). FIG. 11B shows an alternate view of
the data shown in FIG. 11A, with the y-axis showing the average
cell TUNEL-positive cells counted per square millimeter, with error
bars representing standard error. These data suggest that MCP-1
enhances photoreceptor cell death induced by retinal
detachment.
[0158] Discussion
[0159] Experiments described in this example characterized the
expressional gene changes of growth factors, cytokines, and
chemokines in an animal model of retinal detachment. These
experiments showed that mRNAs of TNF-alpha, IL-1 beta, and MCP-1
were detected very early after retinal detachment, and bFGF began
to increase at 24 hours. By 72 hours following retinal detachment,
bFGF, TNF-alpha, IL-1 beta, and MCP-1 were significantly increased.
Examination of the distribution of these genes after retinal
detachment using QPCR with samples collected by LCM showed that
mRNA of bFGF was most increased in the photoreceptor layer,
although the induction was also detected significantly in all other
nuclear layers (RPE, INL, GCL). TNF-alpha was increased in the ONL
and the INL, and decreased in the RPE. IL-1 beta was specifically
increased in the GCL, and MCP-1 was increased in the ONL and the
INL.
[0160] LCM sampling of cells from the various layers of neural
retina correlated with the distribution of bFGF, TNF-alpha, IL-1
beta, and MCP-1 detected by immunohistochemistry. Astrocytes,
Muller cells, and subretinal monocytes produced bFGF, TNF-alpha,
IL-1 beta, and/or MCP-1 in the retina. Specifically, the
immunoreactivity of bFGF and TNF-alpha was increased over the
entire neural retina, while IL-1 beta and MCP-1 were increased in
the astrocytes or Muller cells, respectively. Subretinal
administration of MCP-1 with retinal detachment significantly
enhanced the number of TUNEL-positive cells in the ONL 24 hours
after retinal detachment, demonstrating that MCP-1 enhances
photoreceptor retinal detachment-induced cell death.
[0161] The other cytokines and growth factors assessed in the
experiments described in this example showed no significant change
in the average fold increase in expression of mRNA in the detached
retinas in the right eyes (OD) versus the non-detached retinas in
the left eyes (OS). Regulation of gene expression of bFGF,
TNF-alpha, IL-1 beta, and MCP-1 offers therapeutic avenues to treat
retinal detachment and prevent photoreceptor loss, retinal gliosis,
and proliferative changes, all of which cause significant vision
loss in patients.
Example 5: Neuroprotective Effect of TNF-Alpha Suppression
Following Retinal Detachment
[0162] This example confirms that administration of agents that
suppress TNF-alpha, in this case goat TNF-alpha blocking antibody
and etanercept, protects against cell death following retinal
detachment.
[0163] Retinal detachments were experimentally induced in the right
eye of Adult Male Norway Brown rats, using the retinal detachment
procedure described in Example 4. Retinal detachments were created
only in the right eye of each animal, and the left eye served as a
control. Following retinal detachment, various treatments were
administered subretinally using a Hamilton syringe equipped with a
32-gauge needle. The tip of needle was introduced through a
sclerotomy and retinal hole into the subretinal space, and 5 .mu.l
of solution, either normal goat serum (0.1 mg/mL), goat
anti-TNF-alpha antibody (0.1 mg/mL), or etanercept (2 mg/mL), was
injected over 3 minutes. Seventy-two hours after retinal
detachment, the eyes were subjected to TUNEL analysis.
[0164] FIGS. 12 and 13 show TUNEL-positive responses in the ONL at
72 hours following retinal detachment. As shown in FIG. 12, animals
treated with etanercept following retinal detachment had a
significantly decreased TUNEL-positive response in the ONL at 72
hours as compared to controls. Similarly, FIG. 13 shows that
animals treated with goat anti-TNF-alpha antibody (TNFa) following
retinal detachment had a significantly decreased TUNEL-positive
response in the ONL at 72 hours following retinal detachment as
compared to animals treated with normal goat serum (NGS) or
controls left untreated (RD72 hours). These data indicate that
administration of agents that suppress TNF-alpha, such as goat
TNF-alpha blocking antibody or etanercept, protects against
photoreceptor cell death following retinal detachment.
Example 6: TNF-Alpha, TNF Receptor, and MCP-1 Deficient Animals
Show Less Apoptotic Cell Death Following Retinal Detachment
[0165] This example shows that TNF-alpha deficient mice (TNF-/-),
TNF Receptors 1A and 1B double deficient mice (TNFR-/-), and MCP-1
deficient mice exhibit less apoptotic cell death following retinal
detachment as compared to wild-type mice.
[0166] Knock-out mice deficient in TNF-alpha, TNF Receptors 1A and
1B, or MCP-1 were anesthetized by intraperitoneal injection of a
ketamine (62.5 mg/kg) and xylazine (12.5 mg/kg) mixture. For each
animal, after dilation of the animal's pupil with 1% cyclopentolate
and 2.5% phenylephrine hydrochloride, a scleral puncture was made
at the supemasal equator using a glass micropipette. One microliter
of vitreous fluid was removed to reduce ocular pressure. Then, a
glass micropipette was introduced into the subretinal space, and
one microliter of Healon.RTM. GV sodium hyaluronate (Pharmacia
& Upjohn, Uppsala, Sweden) was injected into the subretinal
space. Mice receiving scleral punctures served as a control. At 72
hours after retinal detachment, the eyes were subjected to TUNEL
analysis.
[0167] As shown in FIG. 14, mice deficient in MCP-1 (CCL2) showed
less apoptosis than wild-type (wild) mice at 72 hours following
retinal detachment. Additionally, as shown in FIG. 15, mice
deficient in TNF-alpha (TNF KO) and mice deficient in TNF Receptors
1A and 1B (TNFR KO) showed less apoptosis than wild-type (Wild)
mice at 72 hours following retinal detachment. These data further
link TNF-alpha, TNF Receptors 1A and 1B, and MCP-1 with apoptosis
of photoreceptors and validate them as appropriate treatment
targets.
Example 7: Neuroprotective Effect of MCP-1 Suppression Following
Retinal Detachment
[0168] This example contemplates that administration of agents that
suppress MCP-1 protects against cell death following retinal
detachment.
[0169] Retinal detachments are experimentally induced in the right
eye of Adult Male Norway Brown rats, using the retinal detachment
procedure described in Example 4. Retinal detachments are created
only in the right eye of each animal, and the left eye serves as a
control. Following retinal detachment, various treatments are
administered subretinally using a Hamilton syringe equipped with a
32-gauge needle. The tip of needle is introduced through a
sclerotomy and retinal hole into the subretinal space, and 5 .mu.l
of solution containing a suitable concentration of a MCP-1
suppressing agent is injected over 3 minutes.
[0170] It is contemplated that treatment with an MCP-1 suppressing
agent, including any of those listed herein, following retinal
detachment will show decreased TUNEL-positive responses in detached
retinas at 72 hours as compared to controls. This indicates that
administration of agents that suppress MCP-1 protects against
photoreceptor cell death following retinal detachment.
Example 8: Neuroprotective Effect of IL-1 beta Suppression
Following Retinal Detachment
[0171] This example contemplates that administration of agents that
suppress IL-1 beta protects against cell death following retinal
detachment.
[0172] Retinal detachments are experimentally induced in the right
eye of Adult Male Norway Brown rats, using the retinal detachment
procedure described in Example 4. Retinal detachments are created
only in the right eye of each animal, and the left eye serves as a
control. Following retinal detachment, various treatments are
administered subretinally using a Hamilton syringe equipped with a
32-gauge needle. The tip of needle is introduced through a
sclerotomy and retinal hole into the subretinal space, and 5 .mu.l
of solution containing a suitable concentration of an IL-1 beta
suppressing agent is injected over 3 minutes.
[0173] It is contemplated that treatment with an IL-1 beta
suppressing agent, including any of those listed herein, following
retinal detachment will show decreased TUNEL-positive responses in
detached retinas at 72 hours as compared to controls. This
indicates that administration of agents that suppress IL-1 beta
protects against photoreceptor cell death following retinal
detachment.
Example 9: Neuroprotective Effect of bFGF Induction Following
Retinal Detachment
[0174] This example contemplates that administration of agents that
induce bFGF protect against cell death following retinal
detachment.
[0175] Retinal detachments are experimentally induced in the right
eye of Adult Male Norway Brown rats, using the retinal detachment
procedure described in Example 4. Retinal detachments are created
only in the right eye of each animal, and the left eye serves as a
control. Following retinal detachment, various treatments are
administered subretinally using a Hamilton syringe equipped with a
32-gauge needle. The tip of needle is introduced through a
sclerotomy and retinal hole into the subretinal space, and 5 .mu.l
of solution containing a suitable concentration of a bFGF inducing
agent is injected over 3 minutes.
[0176] It is contemplated that treatment with an bFGF inducing
agent, including any of those listed herein, following retinal
detachment will show decreased TUNEL-positive responses in detached
retinas at 72 hours as compared to controls. This indicates that
administration of agents that induce bFGF protects against
photoreceptor cell death following retinal detachment.
Example 10: Intravitreal Administration of PEDF
[0177] This example confirms that an intravitreal administration of
PEDF has no significant effect on the mRNA expression of bFGF,
TNF-alpha, or IL-1 beta, and fails to significantly protect against
photoreceptor cell death following retinal detachment.
[0178] In these experiments, retinal detachments were created in
Brown Norway rats by injecting 10% hyaluronic acid into the
subretinal space using a transvitreous approach. Treatment with
PEDF (BioProducts MD, Middletown, Md.) or control vehicle was
administered immediately after detachment. In an initial set of
experiments, the treatment groups included: intravitreal
administration of 2.5 .mu.g PEDF (n=6); intravitreal administration
of 5.0 .mu.g PEDF (n=9); subretinal administration of 2.5 .mu.g
PEDF (n=6); subretinal administration of 5.0 .mu.g PEDF (n=9);
intravitreal administration of control vehicle (n=3); and
subretinal administration of control vehicle (n=3). In a second set
of experiments, the treatment groups included: intravitreal
administration of 5.0 .mu.g PEDF (n=15); subretinal administration
of 5.0 .mu.g PEDF (n=15); intravitreal administration of control
vehicle (n=9); and subretinal administration of control vehicle
(n=9).
[0179] For both sets of experiments, all eyes were enucleated 72
hours after retinal detachment and embedded in paraffin. Four
micron sections through the area of detachment, including the optic
nerve, were obtained. Light microscopy was performed at 3.times.
and at 32.times. magnification using hematoxylin-eosin staining.
TUNEL staining was performed using a commercial kit (Oncogene, San
Diego, Calif.) and viewed at 20.times. magnification using green
fluorescein staining for TUNEL and blue DAPI nuclear staining.
TUNEL-positive cells were counted per millimeter of tissue by a
masked observer. The mean number of TUNEL-positive cells per
millimeter of tissue was determined by averaging the number of
TUNEL-positive cells per mm of tissue in three sections of each
eye. Additionally, a realtime polymerase chain reaction (PCR) was
performed for a variety of cytokines, including bFGF, TNF-alpha,
and IL-1 beta, on extracted retina from normal eyes (n=6),
untreated eyes with retinal detachment (n=6), PEDF-treated eyes
with retinal detachment (n=6) and PBS-treated eyes with retinal
detachment (n=6).
[0180] Light microscopic analysis of detached retinas showed the
presence of pyknotic nuclei in the outer nuclear layer, disruption
of the normal organization of the photoreceptor outer segments, and
loss of photoreceptor nuclei. In the initial set of experiments,
TUNEL-staining of detached retina from rats treated with a
subretinal injection of control vehicle showed multiple
TUNEL-positive cells in the ONL, whereas TUNEL-staining of detached
retina from rats treated with a subretinal injection of 5.0 .mu.g
PEDF showed fewer TUNEL-positive cells in the ONL. Quantitatively,
as shown in Table 3, the initial set of experiments showed that the
mean number of TUNEL-positive cells per millimeter of tissue was
greater in control eyes with detached retinas having a subretinal
sham injection (mean SD, 63.8.+-.11.9) versus eyes with detached
retinas that were subretinally injected with 2.5 .mu.s PEDF
(17.6.+-.15.5) or 5.0 .mu.s PEDF (30.4.+-.18.1). These differences
were statistically significant by a two-tailed t-test (p=0.007 and
p=0.016, respectively). There was no statistically significant
difference in the number of TUNEL-positive cells found in the
intravitreally-injected control eyes versus the
intravitreally-injected PEDF eyes.
TABLE-US-00003 TABLE 3 Number of TUNEL-Positive Cells in Initial
Set of Experiments. Treatment Mean SD P Intravitreal Control 23.3
11.0 Intravitreal PEDF 2.5 .mu.g 16.9 14.3 0.100 Intravitreal PEDF
5.0 .mu.g 28.4 15.7 0.190 Subretinal Control 63.8 11.9 Subretinal
PEDF 2.5 .mu.g 17.7 15.5 0.007 Subretinal PEDF 5.0 .mu.g 30.4 18.1
0.016
[0181] However, as shown in Table 4, the second set of experiments
conducted with larger numbers of sample and control eyes showed no
statistical difference between control eyes with detached retinas
having a subretinal sham injection (mean.+-.SD, 38.6.+-.32.2) and
eyes with detached retinas that were subretinally injected with 5.0
.mu.s PEDF (35.5.+-.42.1). As with the initial set of experiments,
the second set of experiments showed no statistically significant
difference in the number of TUNEL-positive cells found in the
intravitreally-injected control eyes versus intravitreally-injected
PEDF eyes.
TABLE-US-00004 TABLE 4 Number of TUNEL-Positive Cells in Second Set
of Experiments. Treatment Mean SD P Intravitreal Control 30.2 22.0
Intravitreal PEDF 5.0 .mu.g 30.3 28.5 0.122 Subretinal Control 38.6
32.2 Subretinal PEDF 5.0 .mu.g 35.5 42.1 0.847
[0182] A realtime PCR was performed for a variety of cytokines,
including bFGF, PEDF, TNF-alpha, IL-1 beta, HGF, CNTF, TGF beta,
and IGF 1. The amount of each mRNA from each of six eyes treated
with a 5.0 .mu.g intravitreal injection of PEDF just after
detachment was averaged. That average was compared with the average
amount of mRNA from each of six eyes treated with the PBS control
vehicle just after detachment. The results for the intravitreal
injection are shown in FIG. 16. As can be seen, intravitreal
administration of PEDF does not significantly alter the mRNA levels
of bFGF, TNF-alpha, IL-1 beta, or any of the cytokines
measured.
[0183] Overall, the experiments described in this example show that
in a larger sample set, administration of PEDF, either subretinally
or intravitreally, has no significant effect on photoreceptor cell
death following retinal detachment as measured using TUNEL.
INCORPORATION BY REFERENCE
[0184] The entire content of each patent and non-patent document
disclosed herein is expressly incorporated herein by reference for
all purposes.
EQUIVALENTS
[0185] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
Sequence CWU 1
1
46119PRTArtificial SequenceCaspase 1 inhibitor 1Xaa Ala Val Ala Leu
Leu Pro Ala Val Leu Leu Ala Leu Leu Pro Tyr1 5 10 15Val Ala
Xaa220PRTArtificial SequenceCaspase 3 inhibitor 2Xaa Ala Val Ala
Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Asp Glu Val
Xaa 20320PRTArtificial SequenceCaspase 4 inhibitor 3Xaa Ala Val Ala
Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Leu Glu Val
Xaa 20420PRTArtificial SequenceCaspase 6 inhibitor 4Xaa Ala Val Ala
Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Val Glu Ile
Xaa 20520PRTArtificial SequenceCaspase 8 inhibitor 5Xaa Ala Val Ala
Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Ile Glu Thr
Xaa 20620PRTArtificial SequenceCaspase 9 inhibitor 6Xaa Ala Val Ala
Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Leu Glu His
Xaa 20724DNAArtificial SequencePrimer nucleotide 7cagtgaaact
gcgaatggct catt 24825DNAArtificial SequencePrimer nucleotide
8agatctcgat tatgtacggc tgccc 25922DNAArtificial SequencePrimer
Nucleotide 9tcttcctgcg catccatcca ga 221022DNAArtificial
SequencePrimer Nucleotide 10gaggtcaacc atacaccgtg ac
221124DNAArtificial SequencePrimer Nucleotide 11cttggacaga
gccagcggat ttgt 241224DNAArtificial SequencePrimer Nucleotide
12gtactttctt cgtttgcagg tgcc 241325DNAArtificial SequencePrimer
Nucleotide 13tctgccacga tcttacaggt gaaca 251425DNAArtificial
SequencePrimer Nucleotide 14ggagtaatag ttcaactagg tccgc
251524DNAArtificial SequencePrimer Nucleotide 15taccctggca
agagagacga ggaa 241624DNAArtificial SequencePrimer Nucleotide
16gcatagtatt tggtacgtgc cacc 241724DNAArtificial SequencePrimer
Nucleotide 17tgcccttcgc gctgaccagt gaca 241824DNAArtificial
SequencePrimer Nucleotide 18ttgttcgccg ccgtgaagga gctt
241923DNAArtificial SequencePrimer Nucleotide 19ttcagttcgt
gtgtggacca agg 232024DNAArtificial SequencePrimer Nucleotide
20tctacatgac acgaggcgac ttcg 242124DNAArtificial SequencePrimer
Nucleotide 21agatgagtgt gccaacaggt gcat 242223DNAArtificial
SequencePrimer Nucleotide 22cccaaaccgg tacttaaact gga
232324DNAArtificial SequencePrimer Nucleotide 23cactgttaag
catgtgccgg agaa 242424DNAArtificial SequencePrimer Nucleotide
24tgtagccggt tgaagaacta gacc 242525DNAArtificial SequencePrimer
Nucleotide 25cttgaacatg acccgagcac attct 252623DNAArtificial
SequencePrimer Nucleotide 26tctagagcgc cttggagtag cta
232724DNAArtificial SequencePrimer Nucleotide 27cccagaccct
cacactcaga tcat 242823DNAArtificial SequencePrimer Nucleotide
28aagagaagtt ccctgttccg acg 232924DNAArtificial SequencePrimer
Nucleotide 29tcaggaaggc agtgtcactc attg 243024DNAArtificial
SequencePrimer Nucleotide 30ctactactgc tggacgatca caca
243123DNAArtificial SequencePrimer Nucleotide 31aatggctctc
cttcgacgtg aca 233225DNAArtificial SequencePrimer Nucleotide
32ggtttattct cggttctcga cctcc 253324DNAArtificial SequencePrimer
Nucleotide 33gccgttctat ctggctagca agga 243424DNAArtificial
SequencePrimer Nucleotide 34agcaacctca ctctactgac tccg
243524DNAArtificial SequencePrimer Nucleotide 35atgcaggtct
ctgtcacgct tctg 243625DNAArtificial SequencePrimer Nucleotide
36ttctcttagt ggtcgtcgtc cacag 253724DNAArtificial SequencePrimer
Nucleotide 37tcttccagga gtaccccgat gaga 243823DNAArtificial
SequencePrimer Nucleotide 38tacgtctagt acgcctagtt tgg
233924DNAArtificial SequencePrimer Nucleotide 39gcccagatac
aacagaatgc ggtt 244024DNAArtificial SequencePrimer Nucleotide
40gcagaacttt aggttgacga cctc 244125DNAArtificial SequencePrimer
Nucleotide 41ctcggaaact gactgatgtg gaagc 254225DNAArtificial
SequencePrimer Nucleotide 42ttctttcacg acctgtacct cctgt
254324DNAArtificial SequencePrimer Nucleotide 43acccaactat
gatgcgagcc aact 244423DNAArtificial SequencePrimer Nucleotide
44tcacgtaggc ctgcggattt taa 234524DNAArtificial SequencePrimer
Nucleotide 45gctgtttcca acttcggcta cgat 244624DNAArtificial
SequencePrimer Nucleotide 46tcagacagta agtggcccga gaga 24
* * * * *