U.S. patent application number 14/812553 was filed with the patent office on 2016-05-26 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 Joan Miller, David N. Zacks.
Application Number | 20160144024 14/812553 |
Document ID | / |
Family ID | 27613340 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160144024 |
Kind Code |
A1 |
Zacks; David N. ; et
al. |
May 26, 2016 |
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
an apoptosis inhibitor to a mammal having an eye with retinal
detachment. The apoptosis inhibitor 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: |
Zacks; David N.; (Ann Arbor,
MI) ; Miller; Joan; (Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Eye and Ear Infirmary |
Boston |
MA |
US |
|
|
Family ID: |
27613340 |
Appl. No.: |
14/812553 |
Filed: |
July 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12862234 |
Aug 24, 2010 |
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14812553 |
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10892787 |
Jul 16, 2004 |
7811832 |
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12862234 |
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PCT/US03/01648 |
Jan 17, 2003 |
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10892787 |
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60349918 |
Jan 18, 2002 |
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Current U.S.
Class: |
424/172.1 ;
514/20.8; 514/44A; 514/44R |
Current CPC
Class: |
A61K 38/16 20130101;
A61K 38/02 20130101; A61K 38/06 20130101; A61K 39/3955 20130101;
A61K 38/55 20130101; A01N 1/0226 20130101; A61P 27/02 20180101;
A61K 31/7088 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7088 20060101 A61K031/7088; A61K 38/16
20060101 A61K038/16; A61K 38/02 20060101 A61K038/02 |
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's
retina, the mammal having an eye in which a region of the retina
has been detached, an amount of an apoptosis inhibitor sufficient
to preserve the viability of photoreceptor cells disposed within
the region of the detached retina, wherein the apoptosis inhibitor
comprises an agent that inactivates, reduces or inhibits at least
one of FAS-ligand or FAS-receptor.
2. The method of claim 1, wherein the apoptosis inhibitor is
administered to the mammal prior to reattachment of the region of
detached retina.
3. The method of claim 1, wherein the apoptosis inhibitor is
administered to the mammal after reattachment of the region of
detached retina.
4. (canceled)
5. The method of claim 1, wherein a plurality of apoptosis
inhibitors are administered to the mammal.
6. The method of claim 1, wherein at least one apoptosis inhibitor
is administered by intraocular, intravitreal, or transcleral
administration.
7. The method of claim 1, wherein the apoptosis inhibitor reduces
the number of photoreceptor cells in the region that die following
retinal detachment relative to the number of photoreceptor cells
that die in the absence of the apoptosis inhibitor.
8-14. (canceled)
15. 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.
16. A method of preserving the viability of photoreceptor cells
disposed within a retina of a mammalian eye following retinal
detachment from the retinal pigment epithelium (RPE) or choroid,
the method comprising: administering to a mammal's retina, the
mammal having an eye in which a region of the retina has been
detached, an amount of an apoptosis inhibitor sufficient to
preserve the viability of photoreceptor cells disposed within the
region of the detached retina.
17-29. (canceled)
30. The method of claim 1, wherein the apoptosis inhibitor is
selected from the group consisting of a protein, a peptide, a
nucleic acid, a peptidyl nucleic acid, a small organic molecule,
and a small inorganic molecule.
31. The method of claim 30, wherein the protein is selected from
the group consisting of a cytokine, an antibody or an antigen
binding fragment thereof, and a genetically engineered biosynthetic
antibody binding site(s).
32. The method of claim 30, wherein the peptide comprises an amino
acid sequence of less than about 25 amino acids in length.
33. The method of claim 30, wherein the peptide comprises an amino
acid sequence of less than about 15 amino acids in length.
34. The method of claim 30, wherein the peptide comprises a
synthetic peptide or a derivative thereof.
35. The method of claim 30, wherein the nucleic acid comprises a
deoxyribose nucleic acid, a ribose nucleic acid, an antisense
oligonucleotide, and an aptamer.
36. The method of claim 35, wherein the deoxyribose nucleic acid
comprises an antisense oligonucleotide and/or an aptamer.
37. The method of claim 1, wherein the retinal detachment occurs
secondary to an underlying degenerative retinal disorder.
38. The method of claim 5, wherein the apoptosis inhibitors are
administered concurrently or sequentially.
39. The method of claim 16, wherein the detachment of the retina
from the RPE is caused by apoptosis of the RPE or cell death of the
RPE.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US03/01648, filed Jan. 17, 2003, which claims
the benefit of U.S. Provisional Application No. 60/349,918, filed
Jan. 18, 2002, the entire disclosures of which are incorporated by
reference herein.
FIELD OF THE INVENTION
[0002] The invention relates generally to compositions and their
use for preserving the viability of photoreceptor cells following
retinal detachment, and more particularly the invention relates to
compositions comprising an apoptosis inhibitor 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] 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
predominantly undergo apoptotic cell death in the detached portion
of the retina. In addition, it has been found that, among other
things, one or more caspases, for example, caspase 3, caspase 7,
caspase 8, and caspase 9, participate in the cascade of events
leading to apoptotic cell death.
[0007] In one 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 an apoptosis inhibitor 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 apoptosis inhibitor minimizes the loss of visual function
resulting from the retinal detachment. The apoptosis inhibitor
reduces the number of photoreceptor cells in the region of the
retina that, without treatment, would die following retinal
detachment.
[0008] Useful apoptosis inhibitors include agents capable of
modulating, for example, the receptor mediated pathway and/or the
intrinsic pathway. Useful apoptosis inhibitors include agents
capable of modulating the activity of a caspase selected from the
group consisting of caspase 3, caspase 7, caspase 8, and caspase 9.
Furthermore, it is contemplated that, under certain circumstances,
it can be advantageous to administer along with the apoptosis
inhibitor, another neuroprotective agent, for example, another
apoptosis inhibitor or a neurotrophic factor.
[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 caspase inhibitor, for example, a caspase
3 inhibitor, a caspase 7 inhibitor, a caspase 8 inhibitor or a
caspase 9 inhibitor, or a combination of two or more of such
caspase inhibitors, sufficient to preserve the viability of
photoreceptor cells disposed within the region of the detached
retina.
[0010] Because photoreceptors die as a result of retinal
detachment, administration of the apoptosis inhibitor minimizes or
reduces 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 apoptosis inhibitor
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 apoptosis inhibitor for a period of
time after a retinal detachment has been detected and/or the retina
surgically reattached. This period of time may vary depending on
the circumstances 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.
[0011] The apoptosis inhibitor, for example, a caspase inhibitor,
can be administered, either alone or in combination with a
pharmaceutically acceptable carrier or excipient, by one or more
routes. For example, the apoptosis inhibitor may be administered
systemically, for example, via oral or parenteral routes, for
example, via intravascular, intramuscular or subcutaneous routes.
Alternatively, the apoptosis inhibitor may be administered locally,
for example, via intraocular, intravitreal, intraorbital,
subretinal, or transcleral routes. Furthermore, it is contemplated
that the apoptosis inhibitor, for example, a caspase inhibitor, may
be administered with another type of neuroprotective agent, for
example, a neurotrophic factor, to maintain viability of the
photoreceptor cells disposed within the detached portion of the
retina. The apoptosis inhibitor and the neuroprotective agent may
be co-administered either simultaneously or one after the other,
for example, the apoptosis inhibitor is administered after the
neuroprotective agent or the neuroprotective agent is administered
after the apoptosis inhibitor.
[0012] 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.
[0013] In another aspect, the invention provides an improved method
of reattaching a detached retina in a mammal, for example, a human.
The improvement comprises administering, either locally to the eye
or systemically, an apoptosis inhibitor in an amount sufficient to
preserve the viability of photoreceptor cells in the eye. The
apoptosis inhibitor can be a caspase inhibitor, for example, a
caspase 3 inhibitor, a caspase 7 inhibitor, a caspase 8 inhibitor,
or a caspase 9 inhibitor. The method may also comprise
co-administering the apoptosis inhibitor with a neuroprotective
agent, for example, a neurotrophic factor.
[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 is a schematic representation of the intrinsic and
the FAS-mediated apoptosis pathways, however, for clarity some of
the intermediates in each pathway are not shown and the
abbreviations include Cyto C--cytochrome C, Apaf-1--apoptosis
activating factor 1, Casp 3--caspase 3, Casp 7--caspase 7, Casp
8--caspase 8, Casp9--caspase 9, tBID--truncated BID, and
PARP--poly-ADP ribose-polymerase;
[0017] FIG. 2 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;
[0018] FIG. 3 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;
[0019] FIG. 4 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;
[0020] FIG. 5 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
[0021] FIG. 6 depicts a bar chart showing the kinetics of
FAS-receptor/FAS-ligand complex formation as a function of time
after retinal detachment (the units on the ordinate axis correspond
to normalized densitometry readings of immunoprecipitated
complexes);
[0022] FIGS. 7a and 7b depict bar charts showing the kinetics of
intrinsic pathway activation as measured by caspase 9 activity
levels as a function of time after retinal detachment (FIG. 7a) and
by caspase 9/cytochrome C complex formation as a function of time
after retinal detachment (FIG. 7b);
[0023] FIG. 8 depicts a bar chart showing the inhibition of caspase
9 activity 24 hours after retinal detachment, as measured in vitro,
following injection of either DMSO solvent alone or DMSO solvent
containing the caspase 9 inhibitor zLEHD.fmk at the time of
detachment; and
[0024] FIG. 9 depicts a bar chart showing the inhibition of caspase
9 activity 24 hours after retinal detachment, as measured in vitro,
following injection of anti-FAS-receptor (anti-FAS) neutralizing
antibody, or anti-FAS-ligand (anti-FASL) neutralizing antibody at
the time of detachment.
DETAILED DESCRIPTION
[0025] 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.
[0026] 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 neovascularization 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
apoptosis inhibitors 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.
[0027] 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).
[0028] 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.
[0029] In general, during retinal detachment as shown in FIG. 1,
apoptosis is activated by one of two main pathways, the
receptor-mediated pathway (Walczak & Krammer (2000) EXP. CELL
RES. 256: 58-66) and the intrinsic (mitochondrial) pathway
(Loeffler & Kraemer (2000) EXP. CELL RES. 256: 19-26). The
receptor mediated pathway is understood to involve the components
of the FAS/FAS-ligand system; the prototypical receptor-mediated
apoptosis pathway. Both FAS and FAS-ligand are surface membrane
proteins that belong to the tumor necrosis factor-.alpha.
superfamily of proteins (Love (2003) PROG. NEURO. BIOL. PSYCH. 27:
267-82). As shown in FIG. 1, cleaved caspase 8 can either directly
activate caspase 3 or directly activate BID, a member of the Bcl-2
family of proteins, which in turn then feeds into the intrinsic
pathway by stimulating the release of mitochondrial cytochrome
C.
[0030] In addition to the receptor-mediated pathway, apoptosis can
also become activated via an intrinsic pathway. It is understood
that the intrinsic pathway does not involve a surface receptor, but
rather results from the modification of intracellular pools of
proteins. Such modulators include BID (activated by the
FAS-mediated pathway) as well as other members of the Bcl-2 family.
Environmental or intracellular stressors result in post-translation
modification of these proteins, which then exert their effect on
the mitochondria to release cytochrome C. It is understood that the
released cytochrome C then binds with apoptosis activating factor-1
and caspase 9 to form a complex known as the apoptosome, which in
turn activates more downstream apoptosis reactions. In particular,
the apoptosome, can induce the conversion of pro-caspase 9 into
active cleaved caspase 9, which itself then induces the conversion
of pro-caspase 3 into active cleaved caspase 3. Activated caspase 3
(either activated by the FAS-mediated pathway or the intrinsic
pathway) then initiates apoptosis optionally via intermediates
caspase 7 and PARP.
[0031] 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 an
apoptosis inhibitor sufficient to preserve the viability of
photoreceptor cells disposed within the region of the detached
retina. The apoptosis inhibitor is capable of modulating, for
example, decreasing, the activity of one or more of caspase 3,
caspase 7, caspase 8 and caspase 9, and/or preventing or reducing
the activation of one or more of caspase 3, caspase 7, caspase 8,
and caspase 9.
[0032] As used herein, the term "apoptosis inhibitor" is understood
to mean any agent other than a naturally occurring neurotrophic
factor that, when administered to a mammal, reduces apoptotic cell
death in photoreceptor cells. It is understood that the apoptosis
inhibitor excludes certain naturally occurring neurotrophic
factors, including brain-derived neurotrophic factor, glial cell
line-derived neurotrophic factor, neurotrophin, insulin-like growth
factor, ciliary neurotrophic factor, fibroblast growth factor
(acidic and basic), transforming growth factor .alpha., and
transforming growth factor is understood that certain useful
apoptosis inhibitors act by reducing or eliminating the activity of
one or more members of the intrinsic apoptotic pathway and/or the
FAS-mediated apoptotic pathway. For example, it is understood that
an agent that inactivates or reduces the activity of the FAS-ligand
and/or the FAS-receptor is considered to be an apoptosis inhibitor.
Furthermore, it is understood that an agent that either directly or
indirectly affects the activity of a particular caspase, for
example, caspase 3, caspase 7, caspase 8, and caspase 9, is
considered to be an apoptosis inhibitor.
[0033] 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.
[0034] 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-10
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.
[0035] Although photoreceptors may undergo either apoptotic cell
death or necrotic cell death following retinal detachment it is
believed that the primary mechanism of cell death is via apoptosis.
Accordingly, apoptosis inhibitors preferably are used in the
practice of the invention.
[0036] Useful apoptosis inhibitors include, for example, proteins,
for example, 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. Other useful apoptosis inhibitors include,
for example, peptides, for example, an amino acid sequence less
than about 25 amino acids in length, and optionally an amino acid
sequence less than 15 amino acids in length. Peptides useful in the
invention comprise, for example, synthetic peptides and derivatives
thereof. Other useful apoptosis inhibitors include, for example,
deoxyribose nucleic acids (for example, antisense oligonucleotides
and aptamers), ribose nucleic 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. Other useful
apoptosis inhibitors include small organic or inorganic molecules
that reduce or eliminate apoptotic activity when administered to
the mammal.
[0037] 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.
[0038] 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.
[0039] Useful caspase inhibitors include commercially available
synthetic (i.e., non naturally occurring) caspase inhibitors. The
synthetic caspase inhibitors may comprise less that 25, optionally
less than 15, and optionally less than 10 amino acids or amino acid
derivatives. 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 functional group reversibly bind to their target
caspases, whereas the caspase inhibitors with the other functional
groups tend to bind irreversibly to their targets. Useful caspase
inhibitors, when 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.
[0040] 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-fluoromethylketone,
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, CA.
[0041] 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, CA.
[0042] 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),
biotin-X-Asp-Glu-Val-Asp-fluoromethylketone,
Ac-Asp-Glu-Val-Asp-chloromethylketone, which can be obtained from
Calbiochem, CA. Another exemplary caspase 3 inhibitor includes, the
caspase 3 inhibitor
N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyketone
(z-Asp-Glu-Val-Asp-fnk), which can be obtained from Enzyme Systems
Products, CA.
[0043] 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, CA.
[0044] Exemplary synthetic caspase 5 inhibitors include, for
example, Z-Trp-His-Glu-Asp-fluoromethylketone, which can be
obtained from Calbiochem, CA, 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.
[0045] 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, CA.
[0046] 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.
[0047] 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-Va-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, CA.
[0048] 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, CA.
[0049] 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, an anti-caspase 8 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,
an anti-caspase 8 aptamer, or an anti-caspase 9 aptamer may be
useful in the practice of the invention.
[0050] Alternatively, certain endogenous caspase inhibitors other
than naturally occurring neurotrophic factors 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 J. 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.
[0051] 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).
[0052] 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. SC 40, 1177-82) and MK-801 (Solberg et al. INVEST. OPHTHALMOL.
VIS. SCI (1997) 38, 1380-1389) and
n-acetylated-.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; 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.
[0053] 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 the apoptosome 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.
[0054] It is contemplated that the foregoing and other apoptosis
inhibitors 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 apoptosis inhibitor can be
administered to an eye at different dosages. The ability of the
apoptosis inhibitor 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.
[0055] 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 apoptosis inhibitor is effective at minimizing
or reducing the level of apoptosis, or eliminating apoptosis in a
sample. For example, the potency of the apoptosis inhibitor will
have an inverse relationship to the number of TUNEL positive cells
per sample. By comparing the efficacy of a variety of potential
apoptosis inhibitors using these methods, it is possible to
identify apoptosis inhibitors most useful in the practice of the
invention.
[0056] In addition, the apoptosis inhibitor may be co-administered
with a neuroprotective agent. 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 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, 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 deoxyribose nucleic acid,
ribose nucleic 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.
[0057] It is contemplated that useful neuroprotective agents may
include one or more neurotrophic factors. 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; Ciliary Neurotrophic
Factor 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. 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.
[0060] Under certain circumstances, it may be advantageous to also
administer to the individual undergoing treatment with the
apoptosis inhibitor an anti-permeability agent and/or an
anti-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. Exemplary anti-inflammatory agents include
steroids, for example, hydrocortisone, dexamethasone sodium
phosphate, methylpredisolone, and triamcinolone acetonide. Such
molecules may be administered to the individual in an amount
sufficient to reduce or eliminate an inflammatory response in the
eye.
[0061] As a result, the invention provides an improved method for
treating a retinal detachment. The method involves administering an
apoptosis inhibitor before and/or during and/or after surgical
reattachment of the detached retina. The apoptosis inhibitor 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
apoptosis inhibitor 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 apoptosis inhibitor 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.
[0062] Once the appropriate apoptosis inhibitors 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 an
apoptosis inhibitor, for example, a caspase inhibitor, can be
administered either alone or in combination with a 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 agents either together or one after
the other. Although the best means of administering a particular
apoptosis inhibitor or combination of an apoptosis inhibitor with
another neuroprotective agent may be determined empirically, it is
contemplated that the active molecules may be administered locally
or systemically.
[0063] 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 apoptosis inhibitors administered
systemically may be modified or formulated to target the apoptosis
inhibitor 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 apoptosis inhibitor 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 an apoptosis
inhibitor (i.e., an amount of an apoptosis inhibitor sufficient to
reduce, minimize or eliminate the death of photoreceptor cells
following retinal detachment) are administered systemically.
[0064] 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 apoptosis inhibitor 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 an apoptosis
inhibitor 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.
[0065] The apoptosis inhibitor 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.
[0066] In addition, it is contemplated that the apoptosis inhibitor
may be formulated so as to permit release of the apoptosis
inhibitor over a prolonged period of time. A release system can
include a matrix of a biodegradable material or a material which
releases the incorporated apoptosis inhibitor by diffusion. The
apoptosis inhibitor 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
apoptosis inhibitor having different molecular weights are released
by diffusion through or degradation of the material.
[0067] 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
poly(vinyl 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.
[0068] 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.
[0069] The type and amount of apoptosis inhibitor 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 apoptosis inhibitors and dosages of those
apoptosis inhibitors may be determined empirically. The apoptosis
inhibitor 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.
[0070] By way of example, protein-, peptide- or nucleic acid-based
apoptosis inhibitors 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 apoptosis inhibitors may be administered
at doses ranging from about 1 to about 20 mg/kg daily. Furthermore,
antibodies 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 apoptosis inhibitors,
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 g to about 2 mg/eye. With regard to
transcleral administration, the apoptosis inhibitors, 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.
[0071] The present invention, therefore, includes the use of a
apoptosis inhibitor, for example, a caspase inhibitor, 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 apoptosis
inhibitors, one agent optionally being a caspase inhibitor, may be
provided for use in the present invention. The apoptosis inhibitor
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 apoptosis
inhibitor may be provided in unit-dosage or multiple-dosage form.
Preferred dosages of the apoptosis inhibitors, however, are as
described above.
[0072] 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.
[0073] In light of the foregoing description, the specific
non-limiting examples presented below are for illustrative purposes
and are not intended to limit the scope of the invention in any
way.
Examples
Example 1
Detection of Caspase Activity Following Retinal Detachment
[0074] This example demonstrates that certain caspases,
particularly caspases 3, 7 and 9, are activated in photoreceptor
cells following retinal detachment. The example also demonstrates
that photoreceptor death is mediated by the intrinsic apoptotic
pathway following retinal detachment.
[0075] 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 sodium hyaluronate (Healon.RTM., 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 Healon
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.
[0076] 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.
[0077] 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).
[0078] At one day after creation of the detachment, pyknosis in the
outer nuclear layer 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 outer nuclear layer of the retina in the area of the
detachment. Extrusion of pyknotic nuclei from the outer nuclear
layer into the subretinal space were 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 outer nuclear layer pyknosis was similar between
detachments of three-day or one week duration.
[0079] 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.
[0080] 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.
[0081] 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 outer
nuclear layer as compared to the non-detached retina.
[0082] 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 min. The protein
concentration of the supernatant was determined using the Bio-Rad
D.sub.C Protein Assay reagents (Bio-Rad Laboratories, Hercules,
Calif.). 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.
[0083] 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. 2). No
cleaved-caspase 3 was detected in the control eye or in the
attached portion of the retina in the experimental eye.
[0084] 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. 3). 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. 4). 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. 5). P-values for the comparisons between detached and
attached retinas are shown in FIGS. 2-5.
[0085] The results demonstrate that caspase 3, caspase 7 and
caspase 9 are all activated in photoreceptor cells following
retinal attachment.
Example 2
Activation of FAS-Mediated Apoptotic Pathway in the Retina
Following Retinal Detachment
[0086] The purpose of this example was to determine whether only
the intrinsic pathway becomes activated during retinal detachment,
or whether the receptor-mediated pathway also contributes to
photoreceptor death.
[0087] Experimental retinal detachments were created in
Brown-Norway rats by injecting 10% hyaluronic acid into the
subretinal space. Retinal tissue was harvested at 2, 4, 8, 24, 72
and 168 hours after creation of the detachment. Immunoprecipitation
was performed to assess for FAS-receptor/FAS-ligand complex
formation, and activation of caspase 8 and BID was assessed by
Western blot analysis. Caspase 9 activity assay and
immunoprecipitation of the caspase 9/cytochrome C complex was
performed at these same time points. The results demonstrate that
the FAS-mediated apoptotic pathway is activated during retinal
detachment, and that FAS pathway activation precedes that of
intrinsic pathway.
[0088] 2.1. Animal Model
[0089] The experiments described in Examples 2-4 were performed in
accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research and the guidelines established by
the University Committee on Use and Care of Animals of the
University of Michigan. Retinal detachments were created in adult
male Brown-Norway rats (300-400 gm) essentially as described in
Example 1 but with minor modifications.
[0090] Briefly, rats were anesthetized with a 50:50 mix of ketamine
(100 mg/ml) and xylazine (20 mg/ml), and pupils were dilated with
topical phenylephrine (2.5%) and tropicamide (1%). A sclerotomy was
created approximately 2 mm posterior to the limbus with a 20-guage
microvitreoretinal blade (Walcott Scientific, Marmora, N.J.), with
special caution to not damage the lens. A Glaser subretinal
injector (32-gauge tip; BD Ophthalmic Systems, Sarasota, Fla.)
connected to a syringe filled with 10 mg/ml sodium hyaluronate
(Healon.RTM.; Pharmacia and Upjohn Co., Kalamazoo, Mich.) was
introduced through the sclerotomy into the vitreous cavity. The tip
of the subretinal injector was introduced into the subretinal space
through a peripheral retinotomy, and the sodium hyaluronate was
slowly injected. The neurosensory retina was thus detached from the
underlying retinal pigment epithelium. In all experiments,
approximately one-third to one-half of the retina was detached.
Detachments were made in the left eye, with the right eye serving
as the control. For control eyes, a sham surgery was performed in
which all components of the procedure were performed except
introduction of the subretinal injector and creation of the
detachment. In experimental eyes, only the detached portion of the
retina was harvested for analysis.
[0091] 2.2. Western Blot Analysis
[0092] Retinas from experimental and control eyes were dissected
from the RPE-choroid at 3 and 7 days after retinal detachment.
Retinas were homogenized and lysed with buffer containing 10 mM
HEPES (pH 7.6), 0.5% IGEPAL, 42 mM KCL, 1 mM PMSF, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, 5 mM MgCL.sub.2, and 1 tablet of protease
inhibitors per 10 mL buffer (Complete Mini; Roche Diagnostics GmbH,
Mannheim, Germany). The homogenates were incubated on ice and
centrifuged at 22,000 g at 4.degree. C. for 60 minutes. The protein
concentration of the supernatant was determined using the Dc
Protein Assay kit (Bio-Rad Laboratories; Hercules Calif.). The
protein samples were loaded and run on SDS-Polyacrylamide gels
(4-20% Tris-HCL ready gels, Bio-Rad Laboratories). After
electrophoretic separation the proteins were transferred onto
polyvinylidene fluoride (PVDF) membranes (Immobilon-P). Protein
bands were visualized with Ponceau S staining and the lanes
assessed for equal loading by densitometry on a non-specific band
present across all lanes. Membranes were then placed in 5% nonfat
powdered milk in TBS (150 mM NaCl, 50 mM Tris; pH 7.6) and
incubated overnight at 4.degree. C. on a shaker. Membranes were
then incubated with the primary antibody in 2.5% powdered milk in
TBS for overnight at 4.degree. C. Membranes were washed extensively
with TBS-T (0.1% Tween 20), and then incubated with horseradish
peroxidase labeled secondary antibody (1:3000, Santa Cruz
Biotechnology) for 1 hour at room temperature. Bands were
visualized with ECL-Plus (Amersham, Piscataway, N.J.) according to
the manufacturer's instructions. Antibodies against the following
proteins were used: caspase-8 (1:800 dilution, Santa Cruz
Biotechnology, Santa Cruz, Calif.), caspase-9 (1:2000 dilution,
MBL, Nakaku, Japan), cytochrome C (1:1000 dilution, BD Biosciences,
San Jose, Calif.), BID (1:1000 dilution, Santa Cruz Biotechnology),
FAS (1:1000 dilution, Santa Cruz Biotechnology), and FAS-ligand
(1:2000 dilution, MBL).
[0093] 2.3. Immunoprecipitation
[0094] Retinal samples were isolated as described in Section 2.2.
For each condition tested, 20 .mu.g of protein extract was placed
in 100 .mu.l of immunoprecipitation buffer-A (IP-A)+PMSF (20 mM
Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride) and 100 .mu.l of IP-B buffer (100 mM Tris pH 7.5, 100 mM
NaCl, 0.4% Triton X-100). Samples were first incubated overnight
with an anti-FAS antibody (0.2 .mu.g anti-FAS rabbit polyclonal IgG
(Santa Cruz, sc-716)) at 4.degree. C. with gentle rocking, then
incubated for 2 hours in 35 .mu.l of 50% suspension of protein G
sepharose beads at 4.degree. C. with gentle rocking. Beads were
prewashed 4 times with 1 ml of cold IP-C buffer (50 mM tris pH 7.5,
100 mM NaCl, 0.2% Triton X-100), then pelleted at 2200 rpm for 6
minutes. Resuspended beads with attached proteins were diluted with
Laemmli Dye loading buffer and heated at 95.degree. C. for 10
minutes prior to running on a 4-20% SDS-PAGE ready gel (Bio-Rad).
Western blot analysis was performed as described above using a
monoclonal antibody against FAS-Ligand (MBL, D057-3).
Immunoprecipitation of the caspase 9/cytochrome C complex was
performed using a similar protocol, except the antibodies used were
anti-caspase 9 (rabbit polyclonal IgG (Santa Cruz, sc-7885)) and a
monoclonal antibody against cytochrome C (MBL, BV-3026-3).
Densitometry of Western blot bands was performed using a Kodak
440CF Image Station (Kodak Company, Rochester, N.Y.). For each time
point, the densitometry reading of the detached retina was
normalized against the densitometry reading of attached retina at
the same time point.
[0095] 2.4. Caspase 9 Activity Assay
[0096] Caspase 9 activity was measured using a colorimetric
tetrapeptide LEHD-pNA cleavage assay kit according to the
manufacturer's instructions (BioVision, Mountain View, Calif.). In
this assay, 100 .mu.g of total retinal protein from either attached
or detached retinas were incubated with substrate (LEHD-pNA, 200
.mu.M final concentration) at 37.degree. C. for 60 min. Absorbance
was measured at 405 nm in a microplate reader (SpectraMAX 190,
Molecular Devices). As a negative control, retinal protein was
incubated with assay buffer lacking any tetrapeptide. A second
negative control was used in which assay buffer alone was incubated
with the tetrapeptide. As a positive control, purified caspase 9
was incubated with the tetrapeptide alone. For each time point, the
caspase 9 activity in the detached retina was normalized against
the caspase 9 activity in attached retina at the same time
point.
[0097] 2.5. Results
[0098] The initial experiment determined whether or not the FAS
pathway becomes activated upon retinal detachment.
Immunoprecipitation studies demonstrated that the
receptor/FAS-ligand complex is formed upon retinal detachment (data
not shown). Activation of caspase 8 and BID was demonstrated on
Western blot analysis by formation of their cleaved forms, as would
be expected by the formation of functional FAS-receptor/FAS-ligand
complex. The peak of FAS-receptor/FAS-ligand complex formation
occurred 8 hours after retinal detachment (see, FIG. 6). This
preceded the peak of caspase 9 activity, which occurred 24 hours
after creation of the detachment (see, FIG. 7a), which corresponded
to the peak of caspase 9/cytochrome C complex formation (see, FIG.
7b). Normalizing the densitometry readings for any decrease in
outer nuclear layer thickness that might result from the retinal
detachment did not significantly alter the relative values
shown.
[0099] These experiments demonstrate that retinal detachment up
regulates and activates the FAS/FAS-ligand pathway. This up
regulation occurs at the transcription level, as demonstrated by
the increased levels of messenger RNA (data not shown). These
components are not just present at increased levels of pro-form,
but become activated by the detachment as evidenced by their
cleavage into enzymatically active states. The data also shows that
FAS activation precedes that of the intrinsic pathway, when taken
in conjunction with the ability to decrease the latter's activity
by inhibition of the former suggests a direct linkage of activation
between the two.
Example 3
Modulation of Caspase 9 and FAS Receptor Activity Following Retinal
Detachment
[0100] This Example demonstrates that it is possible to module the
activity of caspase 9 in vivo following retinal detachment. Direct
inhibition of the intrinsic pathway was performed using the caspase
9 inhibitor z-Leu-Glu-His-Asp-fluoromethylketone (zLEHD.fmk).
Indirect inhibition (via inhibition of FAS complex formation) was
performed using neutralizing antibodies against either the
FAS-receptor or FAS-ligand. Injection of zLEHD.fmk into the
subretinal space of a detached retina resulted in decreased caspase
9 activity, as did injection of anti-FAS-receptor antibody into
either the subretinal space or intravitreally.
[0101] In these experiments the retina was detached with sodium
hyaluronate according to the protocol described in Example 2,
followed immediately by the injection of 5 .mu.l of inhibitor. In
one experiment, the direct inhibitor of caspase 9--zLEHD.fmk was
tested. Five microliters of the zLEHD.fmk (2 mM solution in DMSO)
(BioVision) was injected into the subretinal space of the detached
retina using a Hamilton Syringe (Hamilton Corp, Reno, Nev.). Five
microliters of DMSO was injected into the subretinal space of the
detached retinas as a control for the solvent in which the
zLEHD.fmk was dissolved. In another experiment, the neutralizing
antibody against the FAS-receptor (5 .mu.g in phosphate buffered
saline) (clone ZB4, Upstate, Lake Placid, N.Y.) or FAS-ligand (5
.mu.g in phosphate buffered saline) (clone NOK-1, BD-Biosciences)
was injected either into the subretinal space or the vitreous
cavity.
[0102] In all inhibition experiments, the retinas were harvested at
24 hours after detachment, as this was the peak of caspase 9
activity seen after detachment (as shown in Example 2). The caspase
9 activity in the detached retina was normalized against the
caspase 9 activity in attached retina at the same time point.
[0103] Caspase 9 activity levels were used as a measurement of
intrinsic pathway activation. The activity levels were tested 24
hours after the retinal detachment was created and inhibitor
applied, as this was the time of peak caspase 9 activity (FIG. 7a).
Injection of the caspase 9 inhibitor zLEHD.fmk into the subretinal
space of a detached retina significantly reduced caspase 9 activity
to approximately 50% of the control level (p=0.05) (FIG. 8).
[0104] Injection of neutralizing antibodies against either the
FAS-receptor or the FAS-ligand into the subretinal space of the
detached retina also resulted in the reduction of caspase 9
activity by approximately 50% (p=0.05) (FIG. 9). The effect of
intravitreal injection of these antibodies was less than that seen
with a subretinal injection, and did not reach statistical
significance. Intravitreally injected anti-FAS-receptor antibody
reduced caspase 9 activity by only about 30% (p=0.13). Intravitreal
injection of anti-FAS-ligand antibody resulted in only a 10%
reduction of caspase 9 activity (p=0.54).
Example 4
Preservation of Photoreceptor Viability Following Retinal
Detachment
[0105] This example demonstrates that administration of an
apoptosis inhibitor can preserve photoreceptor cells following
retinal detachment. The administration of a caspase 9 inhibitor
reduced the number of apoptotic cells following retinal
detachment.
[0106] Briefly, the retinal detachments were created in the left
eyes of three Brown Norway rats, as described in Example 2, section
2.1. The detachment was located on the temporal portion of the
retina, and comprised approximately one third of the total retinal
area.
[0107] A first rat received the retinal detachment only. A second
rat received the retinal detachment and a caspase 9 inhibitor in
DMSO. Briefly, immediately after the retina was detached, 5 .mu.l
of the zLEHD.fmk (2 mM solution in DMSO) (BioVision) was injected
into the subretinal space of the detached retina using a Hamilton
Syringe (Hamilton Corp, Reno, Nev.). A third rat received the
retinal detachment and DMSO (solvent control). Briefly, 5 .mu.l of
DMSO was injected into the subretinal space of the detached retinas
as a control for the solvent in which the zLEHD.fmk was
dissolved.
[0108] The rats were allowed to recover from the surgery and were
returned to their cages, as per standard animal care protocols.
Seventy-two hours (3 days) after creation of the detachments, the
eyes were enucleated and immersion-fixed in 4% paraformaldehyde
solution for 24 hours. The fixed eyes were then embedded in
paraffin and sectioned for histologic analysis. TUNEL staining was
performed on the sections using a commercially-available kit
(TdT-Fragel DNA Fragmentation Detection Kit: Oncogene, Boston,
Mass.) according to the manufacturer's instructions.
[0109] The number of TUNEL-positive cells/100 cells in the outer
nuclear layer were counted for 3 high power fields per section for
2 separate slides. The results are summarized in Table 1.
TABLE-US-00001 TABLE 1 Sample % TUNEL positive cells Attached
retina (right eye) 1.3% TUNEL positive Detached retina (left eye)
21.6% TUNEL positive Detached retina (left eye) 33.3% TUNEL
positive plus DMSO only Detached retina (left eye) 4.6% TUNEL
positive plus caspase 9 inhibitor
[0110] The results in Table 1 demonstrate that in the eyes with the
detached retinas, the administration of the caspase 9 inhibitor
significantly reduced the percentage of apoptotic cells and,
therefore, preserved photoreceptor viability following retinal
detachment.
INCORPORATION BY REFERENCE
[0111] The entire disclosure of each of the patent and non-patent
documents disclosed herein is expressly incorporated herein by
reference for all purposes.
EQUIVALENTS
[0112] 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 SequenceDescription of Artificial Sequence
Synthetic caspase 1 inhibitor peptide 1Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Pro Tyr 1 5 10 15 Val Ala Asp
220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic caspase 3 inhibitor peptide 2Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Asp Glu Val Asp 20
320PRTArtificial SequenceDescription of Artificial Sequence
Synthetic caspase 4 inhibitor peptide 3Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Leu Glu Val Pro 20
420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic caspase 6 inhibitor peptide 4Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Val Glu Ile Asp 20
520PRTArtificial SequenceDescription of Artificial Sequence
Synthetic caspase 8 inhibitor peptide 5Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Ile Glu Thr Asp 20
620PRTArtificial SequenceDescription of Artificial Sequence
Synthetic caspase 9 inhibitor peptide 6Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Leu Glu His Asp 20
74PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Leu Glu His Asp 1 84PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Tyr
Val Ala Asp 1 94PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 9Tyr Val Ala Asp 1 104PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Trp
Glu His Asp 1 114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 11Tyr Val Ala Asp 1 124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Tyr
Val Ala Asp 1 134PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 13Tyr Val Ala Asp 1 144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Tyr
Val Ala Asp 1 154PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 15Tyr Val Lys Asp 1 164PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Tyr
Val Lys Asp 1 174PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 17Tyr Val Ala Asp 1 185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Ala
Tyr Val Ala Asp 1 5 194PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Tyr Val Ala Asp 1
204PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Tyr Val Ala Asp 1 214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Tyr
Val Ala Asp 1 224PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Tyr Val Ala Asp 1 235PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Val
Asp Val Ala Asp 1 5 245PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 24Val Asp Val Ala Asp 1 5
254PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Glu Ser Met Asp 1 264PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Ile
Glu Thr Asp 1 274PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 27Asp Glu Val Asp 1 284PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Asp
Met Gln Asp 1 294PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Asp Glu Val Asp 1 304PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Asp
Glu Val Asp 1 315PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 31Xaa Asp Glu Val Asp 1 5
324PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Asp Glu Val Asp 1 334PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Asp
Glu Val Asp 1 344PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 34Leu Glu Val Asp 1 354PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Trp
His Glu Asp 1 364PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 36Trp Glu His Asp 1 374PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Val
Glu Ile Asp 1 384PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 38Val Glu Ile Asp 1 394PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Asp
Gln Met Asp 1 404PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 40Asp Glu Val Asp 1 414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Asp
Glu Val Asp 1 424PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 42Ile Glu Pro Asp 1 434PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Ala
Glu Val Asp 1 444PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 44Ile Glu Thr Asp 1 454PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Leu
Glu His Asp 1 464PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 46Leu Glu His Asp 1
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