U.S. patent application number 10/792551 was filed with the patent office on 2004-09-02 for prevention of retinal injury and degeneration by specific factors.
Invention is credited to LaVail, Matthew, Steinberg, Roy H., Yancopoulos, George D..
Application Number | 20040171529 10/792551 |
Document ID | / |
Family ID | 32873061 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040171529 |
Kind Code |
A1 |
LaVail, Matthew ; et
al. |
September 2, 2004 |
Prevention of retinal injury and degeneration by specific
factors
Abstract
Photoreceptor injury or cell death (retinal degeneration) is
prevented by the introduction into the living mammalian eye of
specific, survival-promoting factors. These specific factors
prevent damage and degeneration of photoreceptors when introduced
into the living eye prior to, during or after exposure to the
damaging effects of light and delay photoreceptor damage caused by
inherited disease.
Inventors: |
LaVail, Matthew; (San
Francisco, CA) ; Steinberg, Roy H.; (San Francisco,
CA) ; Yancopoulos, George D.; (Yorktown Heights,
NY) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
32873061 |
Appl. No.: |
10/792551 |
Filed: |
March 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10792551 |
Mar 2, 2004 |
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08897390 |
Jul 21, 1997 |
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08897390 |
Jul 21, 1997 |
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08334859 |
Nov 4, 1994 |
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5667968 |
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08334859 |
Nov 4, 1994 |
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07836090 |
Feb 14, 1992 |
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07836090 |
Feb 14, 1992 |
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07691612 |
Apr 25, 1991 |
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5438121 |
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07691612 |
Apr 25, 1991 |
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07570657 |
Aug 20, 1990 |
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5229500 |
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07570657 |
Aug 20, 1990 |
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07400591 |
Aug 30, 1989 |
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5180820 |
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Current U.S.
Class: |
424/85.2 ;
514/20.8; 514/8.5; 514/9.1 |
Current CPC
Class: |
C07K 16/22 20130101;
F02B 2075/027 20130101; C07K 16/2863 20130101; G01N 2333/475
20130101; A61K 38/00 20130101; G01N 2333/4709 20130101; C07K 14/71
20130101; C07K 14/48 20130101; C07K 14/475 20130101 |
Class at
Publication: |
514/008 |
International
Class: |
A61K 038/18 |
Claims
We claim:
1. A method of reducing or preventing degeneration of retinal
neurons in a mammal caused by exposure to light or other
environmental trauma comprising administering to the mammal, prior
to, during or following such exposure, a therapeutically effective
dose of neurotrophic factor.
2. The method of claim 1 wherein said neurotrophic factor is brain
derived neurotrophic factor, ciliary neurotrophic factor,
neurotrophin-3 or a combination thereof.
3. The method of claim 2 wherein said retinal neurons are
photoreceptors.
4. The method of claim 3 wherein said administration is
intraocular.
5. The method of claim 4 wherein said administration is into the
vitreous or into the subretinal (interphotoreceptor) space.
6. The method of claim 3 wherein said administration is systemic
delivery.
7. The method of claim 6 wherein said neurotrophic factor has been
modified in such a way as to increase its ability to be transported
across the blood-retinal barrier.
8. The method of claim 7 wherein said modification comprises
increasing the lipophilicity of the factor.
9. The method of claim 7 wherein said modification comprises
glycosylation of the factor.
10. The method of claim 7 wherein said modification comprises
increasing the net positive charge on said factor.
11. The method of claim 6 wherein said systemic delivery is by an
oral route.
12. The method of claim 7 wherein said systemic delivery is by
subcutaneous, intravenous or intramuscular injection.
13. A method of preventing or reducing degeneration of retinal
neurons in a mammal caused by exposure to light or other
environmental trauma comprising administering to the mammal, prior
to, during or following said exposure, a therapeutically effective
dose of one or more factors selected from the group consisting of
acidic fibroblast growth factor (aFGF), bFGF plus heparin, aFGF
plus heparin, interleukin-1 beta (IL-1.beta.) and tumor necrosis
factor-alpha (TNF-.alpha.).
14. The method of claim 13 wherein said retinal neurons are
photoreceptors.
15. The method of claim 14 wherein said administration is
intraocular.
16. The method of claim 15 wherein said administration is into the
vitreous or into the subretinal (interphotoreceptor) space.
17. The method of claim 14 wherein said administration is delivered
systemically.
18. The method of claim 17 wherein said systemic delivery is by an
oral route.
19. The method of claim 18 wherein said systemic delivery is by
subcutaneous, intravenous or intramuscular injection.
20. A method of reducing or preventing degeneration of retinal
neurons in a mammal having a pathological condition wherein retinal
degeneration occurs, comprising administering to said mammal a
therapeutically effective dose of a neurotrophic factor.
21. The method of claim 20 wherein said pathological condition is
retinal detachment, age-related or other maculopathies, photic
retinopathies, surgery-induced retinopathies (either mechanically
or light-induced), toxic retinopathies, diabetic retinopathies,
retinopathy of prematurity, viral retinopathies such as CMV or HIV
retinopathy related to AIDS; uveitis; ischemic retinopathies due to
venous or arterial occlusion or other vascular disorder,
retinopathies due to trauma or penetrating lesions of the eye,
peripheral vitreoretinopathy or inherited retinal
degenerations.
22. The method of claim 21 wherein said neurotrophic factor is
brainderived neurotrophic factor, ciliary neurotrophic factor,
neurotrophin-3 or a combination thereof.
23. The method of claim 22 wherein said retinal neurons are
photoreceptors.
24. The method of claim 23 wherein said administration is
intraocular.
25. The method of claim 24 wherein said administration is into the
vitreous or into the subretinal (interphotoreceptor) space.
26. The method of claim 23 wherein said administration is by
systemic delivery.
27. The method of claim 26 wherein said systemic delivery is by an
oral route.
28. The method of claim 27 wherein said systemic delivery is by
subcutaneous, intravenous or intramuscular injection.
29. A method of reducing or preventing degeneration of retinal
neurons in a mammal having a pathological condition wherein retinal
degeneration occurs, comprising administering to said mammal a
therapeutically effective dose of one or more factors selected from
the group consisting of acidic fibroblast growth factor (aFGF),
bFGF plus heparin, aFGF plus heparin, IL-1.beta., TNF-.alpha. and
IGF-2.
30. The method of claim 29 wherein said retinal neurons are
photoreceptors.
31. The method of claim 30 wherein said administration is
intraocular.
32. The method of claim 31 wherein said administration is into the
vitreous or into the subretinal (interphotoreceptor) space.
33. The method of claim 30 wherein said administration is systemic
delivery.
34. The method of claim 33 wherein said systemic delivery is by an
oral route.
35. The method of claim 34 wherein said systemic delivery is by
subcutaneous, intravenous or intramuscular injection.
36. A method of assessing the survival-promoting ability of an
agent on retinal neurons or photoreceptors comprising (i) injecting
the agent intravitreally into an albino mammal eye, prior to,
during, or after exposure of the mammal to continuous light, (ii)
evaluating the injected eye for degeneration of retinal neurons or
photoreceptors as compared to a control eye exposed to the same
light in the absence of injection of the agent; wherein decreased
retinal degeneration as compared to the control eye correlates
positively with survival-promoting ability of the agent.
37. The method of claim 36 wherein said mammal is a rat.
38. The method of claim 36 wherein said control eye is in the same
mammal as the intravitreally injected eye.
Description
[0001] This application claims priority of U.S. patent application
Ser. No. 08/334,859 filed Nov. 4, 1994, which is a continuation of
U.S. patent application Ser. No. 07/836,090 filed Feb. 14, 1992,
which is a continuation-in-part of U.S. patent application Ser. No.
07/691,612 filed Apr. 25, 1991, which is a continuation-in-part of
U.S. patent application Ser. No. 07/570,657 filed Aug. 20, 1990 and
issued as U.S. Pat. No. 5,229,500, which is a continuation-in-part
of Ser. No. 07/400,591 filed on Aug. 30, 1989 and issued as U.S.
Pat. No. 5,180,820.
INTRODUCTION
[0002] The present invention relates to a method of preventing or
delaying retinal degeneration caused by exposure to light or other
environmental trauma, or by any pathological condition wherein
death or injury of retinal neurons or photoreceptors occurs. It is
based on the discovery that specific survival promoting factors,
when introduced into the living mammalian eye, prevent damage and
degeneration of photoreceptors caused by light and on the further
discovery that such factors can delay photoreceptor degeneration
associated with inherited diseases of the retina.
BACKGROUND OF THE INVENTION
[0003] Trophic factors play a major role in neuronal survival and
growth during development, in addition to the maintenance of
differentiated neurons. Such factors also appear to play a role in
the survival and regeneration of injured neurons in the central as
well as in the peripheral nervous system.
[0004] In mammals, a number of diseases of the retina involve
injury or degeneration of retina-associated neurons. Trophic
factors capable of rescuing these neurons may provide useful
therapies for the treatment of such diseases.
[0005] There is some evidence that the neurotrophic factor NGF
(nerve growth factor) enables axonal regrowth of retinal ganglion
cells in response to optic nerve section. (Carmigr al. Dev. Brain
Res. 6 (1983) 77-83). BDNF (brain derived neurotrophic factor)
purified from brain promotes the survival of retinal ganglion cells
in vitro. (Johnson, et al. J. Neuroscience 6 (1986): 3031-3038;
Thanos, et al. Eur. J. Neuroscience 1(1989): 19-26.) Other workers
have reported that retinal ganglion cells could be maintained by
extracts from the neonatal superior colliculus and that a factor
purified from such extracts promotes the survival and growth of
retinal ganglion cells in vivo. (Schultz, et al. J. Neurochemistry
55(1990): 832-303). Moreover, fibroblast growth factors promote the
survival of adult rat ganglion cells after application to
transected optic nerves (Sievers, et al., Neurosci. Let. 76
(1987):157-162).
[0006] In addition to the survival of retinal ganglion cells, there
is some evidence that certain cellular factors may promote the
survival and/or regeneration of photoreceptors. Photoreceptors
consist of rods and cones which are the photosensitive cells of the
retina. The rods contain rhodopsin, the rod photopigment, and the
cones contain 3 distinct photopigments, which respond to light and
ultimately trigger a neural discharge in the output cells of the
retina, the ganglion cells. Ultimately, this signal is registered
as a visual stimulus in the visual cortex.
[0007] The retinal pigment epithelial (RPE) cells produce, store
and transport a variety of factors that are responsible for the
normal function and survival of photoreceptors. RPE are
multifunctional cells that transport metabolites to the
photoreceptors from their blood supply, the chorio capillaris of
the eye. The RPE cells also function to recycle vitamin A as it
moves between the photoreceptors and the RPE during light and dark
adaptation. RPE cells also function as macrophages, phagocytizing
the rhythmically-shed tips of the outer segments of rods and cones.
Various ions, proteins and water move between the RPE cells and the
interphotoreceptor space, and these molecules ultimately effect the
metabolism and viability of the photoreceptors.
[0008] RCS (Royal College of Surgeons) rats, which have an
inherited retinal dystrophy due to mutant gene expression in the
RPE, with secondary photoreceptor cell death (Mullen & LaVail,
Science 192 (1976):799-801), provide a useful model system to study
the role of trophic factors on the retina. Using such rats, delay
of photoreceptor degeneration caused by the inherited defect was
obtained by the juxtaposition of normal RPE cells to the
photoreceptors before their degeneration both in experimental
chimeras (Mullen & LaVail, Science 192 (1976):799-801) and in
transplantation experiments (Li & Turner, Exp. Eye Res. 47:
911-917, 1988). In these experiments, the "rescue" extended beyond
the boundaries of the normal RPE cells.
[0009] These findings suggested the presence of a diffusable factor
produced by the RPE cells. It was subsequently determined that
subretinal or intravitreal injection of basic fibroblast growth
factor (bFGF) resulted in extensive photoreceptor rescue in RCS
rats (Faktorovich, et al., Nature 347 (1990):83-86). Basic FGF was
also shown to induce retinal regeneration from the RPE in chick
embryos (Park & Hollenberg, Dev. Biol. 134 (1989):
201-205).
[0010] Although the results obtained with in)ection of bFGF were
encouraging, therapeutic applications of bFGF could be very
limited. Given its mitogenic and angiogenic properties, harmful
side effects can be expected. As an example, intravitreal inje
(1990):83-86). Finally, bFGF is unable to remedy one particular
defect seen in RCS rats, which is the inability of the RPE to
phagoc, viosize degenerated neurons.
[0011] More limited rescue of photoreceptors in RCS rats has been
reported with the injection of phosphate buffered saline (PBS)
(Silverman & Hughes, Current Eye Res. 9 (1990): 183-191;
Faktorovich, et. al, Nature 347 (1990):83-86), as well as in
surgical controls. Such studies indicated a localized effect caused
by the possible release of protective factors from RPE or other
cells damaged during injection. In such instances, however, the
level of rescue differed quantitatively from that obtained using
bFGF, i.e. it was much more restricted to the area of the needle
track.
[0012] In the albino rat, normal illumination levels of light, if
continuous, can cause complete degeneration of photoreceptors.
Results obtained using such rats as a model to identify survival
enhancing factors appear to correlate well with data obtained using
RCS rats. Moreover, different factors can be compared and
complications can be assessed more quickly in the light damage
model than can be assessed by testing factors in models which are
based on the slowly evolving dystrophy of the RCS rat. Furthermore,
since the mechanism of cell death in light damage is better defined
than that in the RCS rats, the results in the light damage model
can be more readily applied to human diseases.
[0013] Using albino rats, it has been determined that a number of
agents, when administered systemically (intraperitoneally) can be
used to ameliorate retinal cell death or injury caused by exposure
to light. In general, exposure to light generates oxygen free
radicals and lipid peroxidation products. Accordingly, compounds
that act as antioxidants or as scavengers of oxygen free radicals
reduce photoreceptor degeneration. Agents such as ascorbate
(Organisciak et al, Investigative Ophthalmology & Visual
Science 26 (1985):158-1588), flunarizine (Edward, et al.,
Laboratory Science 109 (1991): 554562) and dimethylthiourea (Lam,
et al., Archives of Ophthalmology 108 (1990): 1751-1757) have been
used to ameliorate the damaging effects of constant light. There is
no evidence, however, that these compounds will act to ameliorate
other forms of photoreceptor degeneration and their administration
can generate potentially harmful side effects. Further, these
studies are limited because they utilize systemic delivery. Such
delivery often provides an inadequate means of assessing the
efficacy of a particular factor. It is difficult to assess the
amount of agent that actually reaches the retina. A large amount of
agent must be injected to attain a sufficient concentration at the
site of the retina. In addition, systemic toxic effects may result
from the injection of certain agents.
[0014] Other than the use of bFGF to delay inherited photoreceptor
degeneration in RCS rats, there is no demonstrated use of any
specific neurotrophic or other cellular factor to prevent injury or
death of mammalian photoreceptors. In copending U.S. application
Ser. No. 07/400,591 which is incorporated by reference herein, a
BDNF expressing clone was isolated from a retinal cDNA library.
Based on that discovery, as well as the expression for the first
time of purified BDNF using recombinant technology, a means was
provided for the use of a purified neurotrophic factor for the
treatment of diseases such as retinitis pigmentosa and other
retinal degenerations. As described in greater detail below, the
efficacy of BDNF, in addition to other neurotrophic and cellular
factors, has been demonstrated, providing the first pharmacological
means to treat most forms of inherited, age-related or
environmentally-induced retinal degenerations.
SUMMARY OF THE INVENTION
[0015] An object of the present invention is to provide a method of
preventing injury or death of retinal neurons.
[0016] Another object of the invention is to provide a method of
treating pathological diseases wherein degeneration of the retina
occurs.
[0017] Yet another object of the invention is to provide a method
of treating the living eye prior to or following exposure to light
or other environmental trauma thereby preventing degeneration of
retinal cells.
[0018] A further object of the present invention is to provide a
method of preventing photoreceptor injury and degeneration in the
living eye.
[0019] Another object of the invention is to provide a method of
protecting retinal neurons without the induction of side
effects.
[0020] Another object of the invention is to provide a method of
allowing injured photoreceptors to recover or regenerate.
[0021] Another object of the invention is to provide an in vivo
assay system for assessing the survival-promoting activity of
neurotrophic and other cellular hctors on retinal neurons and
photoreceptors.
[0022] These and other objects are achieved by treating the eye
with an effective amount of a neurotrophic factor such as
brain-derived neurotrophic factor (BDNF), ciliary neurotrophic
factor (CNTF), neurotrophin-3 (NT-3) or neurotrophin-4 (NT-4), or a
cellular factor such as acidic fibroblast growth factor (aFGF),
basic fibroblast growth factor (bFGF) plus heparin, aFGF plus
heparin, interleukin-1 beta (IL-1.beta.), tumor necrosis
factor-alpha (TNF-.alpha.) and insulin-like growth factor-2
(IGF-2). Similar effects, but to a lesser degree, may be achieved
using other neurotrophic or cellular factors that may, alone, or in
combination with other factors described herein, have
therapeutically beneficial effects. Such factors include nerve
growth factor (NGF), heparin, epidermal growth factor (EGF),
platelet derived growth factor (PDGF) and insulin-like growth
factor-1 (IGF-1).
DESCRIPTION OF THE FIGURES
[0023] FIG. 1 is a histogram illustrating the ONL thickness
obtained using the neurotrophic and cellular factors--CyL=cyclic
light; CL=constant light; PBS=phosphate buffered saline; bFGF=basic
fibroblast growth factor; aFGF=acidic fibroblast growth factor;
NGF=nerve growth factor; NT-3=neurotrophin-3; BDNF=brain derived
neurotrophic factor; CNTF=ciliary neurotrophic factor;
EGF=epidermal growth factor; PDGF=platelet derived growth factor;
IGF=insulin related growth factor; IL-6=interleukin-6, and
TNF=tumor necrosis factor.
[0024] FIG. 2 is a histogram illustrating the degree of
photoreceptor rescue obtained using the neurotrophic and cellular
factors. (Abbreviations: same as in FIG. 1).
[0025] FIG. 3A-3C is a composite of three light micrographs showing
FIG. 3A) control retina from a rat not exposed to light; FIG. 3B)
control retina from a rat exposed to light after PBS injection; and
FIG. 3C) BDNF-treated rat retina after exposure to light.
[0026] FIG. 4 is a histogram illustrating the degree of macrophage
incidence observed using the neurotrophic and cellular factors.
(Abbreviations: same as in FIG. 1).
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention provides for the utilization of
neurotrophic, as well as other cellular factors to delay, prevent
or rescue photoreceptors, as well as other retinal cells, including
neurons or supportive cells (e.g. Muller cells or RPE cells) from
injury and degeneration. Other retinal neurons include, but are not
limited to, retinal ganglion cells, displaced retinal ganglion
cells, amacrine cells, displaced amacrine cells, horizontal and
bipolar neurons.
[0028] As contemplated herein, neurotrophic or other cellular
factors are utilized to treat any condition which results in injury
or death of photoreceptors or other retinal cells. Examples of
conditions include: retinal detachment; age-related and other
maculopathies, photic retinopathies; surgery-induced retinopathies
(either mechanically or light-induced); toxic retinopathies
including those resulting from foreign bodies in the eye; diabetic
retinopathies; retinopathy of prematurity; viral retinopathies such
as CMV or HIV retinopathy related to AIDS; uveitis; ischemic
retinopathies due to venous or arterial occlusion or other vascular
disorders; retinopathies due to trauma or penetrating lesions of
the eye; peripheral vitreoretinopathy; and inherited retinal
degenerations.
[0029] The factors which are useful in practicing this invention
include one or more neurotrophic factor such as brain-derived
neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF),
neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) or functional
derivatives or analogs thereof, or one or more cellular factor such
as basic fibroblast growth factor (bFGF) plus heparin, acidic
fibroblast growth factor (aFGF), aFGF plus heparin, interleukin-1
beta (IL-1.beta.), tumor necrosis factor-alpha (TNF-.alpha.), and
insulin-like growth factor2 (IGF-2), or functional derivatives or
analogs thereof. Other factors that appears to be effective, but to
a lesser extent, include nerve growth factor (NGF), heparin,
epidermal growth factor (EGF), platelet derived growth factor
(PDGF) and insulin-like growth factor-1 (IGF-1). A functional
derivative of a factor is a compound which is an analog or an
active fragment of the compound or its analog. Combinations of the
neurotrophic factors and cellular factors may also be used to
achieve optimum results.
[0030] Each of the factors utilized may be obtained by methods
known by those skilled in the art. For example, they may be
purified from a natural source. Alternatively, they may be made by
recombinant means utilizing available sequence data. (See, for
example, for CNTF; Masiakowski, et al. J. Neurochemistry 57(1991):
1003-1012; NT-3; Maisonpierre, et al. Science 247(1990):
1446-1451).
[0031] Of particular suitability in practicing the subject
invention are the neurotrophic factors. As used herein,
neurotrophic factors are proteins responsible for the development
and maintenance of the nervous system. Widespread neuronal cell
death accompanies normal development of the central and peripheral
nervous systems, and apparently plays a crucial role in regulating
the number of neurons which project to a given target field (Berg,
D. K., 1982, Neuronal Development 297-331). Ablation and
transplantation studies have shown that neuronal cell death results
from the competition among neurons for limiting amounts of survival
factors ("neurotrophic factors"). The important neurotrophic
factors identified to date are NGF, BDNF, CNTF, NT-3 and NT-4.
[0032] In a preferred embodiment of the invention, BDNF is utilized
to treat any condition which results in injury or death of
photoreceptors or other retina-related cells. With the molecular
cloning of BDNF, as well as the resultant production and
purification of purified recombinant BDNF, as described in U.S.
Ser. No. 400,591, it became possible to determine the physiological
effects of BDNF on developing neurons, as well as to quantify the
levels of BDNF in tissues by immunoassay and to localize BDNF in
tissues using immunocytochemistry. Furthermore, a BDNF cDNA was
found in a retinal library and BDNF mRNA was found to be expressed
in adult retinas (Maisonpierre, et al. Neuron, 5 (1990): 501-509),
suggesting production of the protein in the retina and a possible
role for the factor in promoting retinal cell survival.
[0033] As described herein, treatment of the eye with BDNF results
in the increased survival of photoreceptors upon exposure to
environmental trauma such as light. Suprisingly, BDNF does not
cause the influx of macrophages observed when treating the retina
with bFGF. Furthermore, BDNF is not anticipated to have the side
effects of bFGF as it does not have similar angiogenic or mitogenic
properties.
[0034] In another preferred embodiment, ciliary neurotrophic factor
(CNTF) is used to prevent or delay photoreceptor degeneration.
CNTF, like BDNF, effectively protects photoreceptors without
macrophage influx and the mitogenic and angiogenic properties of
bFGF.
[0035] In still another embodiment, aFGF is used to prevent
photoreceptor degeneration. This factor, unlike bFGF, appears to
provide protection without the influx of macrophages observed when
bFGF is used.
[0036] In yet another embodiment, bFGF is used in conjunction with
a compound that suppresses the influx of macrophages observed using
bFGF alone. Heparin appears to be useful for this purpose.
Combinations of heparin and bFGF prevent photoreceptor injury
without macrophage influx, and heparin enhances the action of aFGF,
as well as bFGF (see FIG. 4).
[0037] In another embodiment, other factors such as IL-1.beta. and
TNF-.alpha. provide a substantial amount of retinal protection.
IL-.beta. however, has been observed to cause folding and rosette
formation and a somewhat greater incidence of macrophages than is
observed in control retinas or those protected with BDNF or CNTF.
Use of TNF-.alpha. may also be associated with a slightly greater
than normal incidence of macrophages.
[0038] In additional embodiments, the light damage model may be
used to evaluate the effect of various survival-promoting factors
on the retina. As shown herein, the intravitreal administration of
various factors into the eyes of albino rats enabled the rapid
assessment of both the ability of the factors to rescue
photoreceptors from degeneration and the side effects, such as
incidence of macrophages, associated with each factor. Although the
model described herein is the albino rat, the eyes of other albino
mammals, such as mice and rabbits, are also useful for this
purpose.
[0039] Although the light damage model has been used previously to
assess the effect of various agents such as antioxidents on the
retina, such studies have always been conducted using systemic
(intraperitoneal) administration. As described herein, the
intravitreal injection of potential survival promoting factors
represents a novel method of assessing factors, with several
advantages over systemic application. The amount of any specific
agent that reaches the retina can be more accurately determined,
since the eye is a round, relatively contained structure and the
agent is injected directly into it. Morover, the amount of agent
that need to be injected is miniscule compared to systemic
injections. For example, a single microliter in volume (about 1
microgram of agent) is used for intravitreal injection, as compared
to one to several milliliters (ten to several hundred milligrams of
agent) necessary for systemic injections. In addition, the
intravitreal route of administration avoids the potentially toxic
effect of some agents.
[0040] According to the present invention, the factors used herein
prevent the degeneration of retinal cells. It has been further
observed that when animals that have been exposed to damaging light
are returned to normal light, they will regenerate their inner and
outer segments. Thus, the factors of the present invention are able
not only to protect and prevent photoreceptors from degeneration,
but also to promote regeneration of retinal cells.
[0041] The factors of the present invention can be delivered to the
eye through a variety of routes. They may be delivered
intraocularly, by topical application to the eye or by intraocular
injection into, for example the vitreous or subretinal
(interphotoreceptor) space. Alternatively, they may be delivered
locally by insertion or injection into the tissue surrounding the
eye. They may be delivered systemically through an oral route or by
subcutaneous, intravenous or intramuscular injection.
Alternatively, they may be delivered by means of a catheter or by
means of an implant, wherein such an implant is made of a porous,
non-porous or gelatinous material, including membranes such as
silastic membranes or fibers, biodegradable polymers, or
proteinaceous material. The factors may be administered prior to
the onset of the condition, to prevent its occurrence, for example,
during surgery on the eye, or immediately after the onset of the
pathological condition or during the occurrence of an acute or
protracted condition.
[0042] The factors of the present invention may be modified to
enhance their ability to penetrate the blood-retinal barrier. Such
modifications may include increasing their lipophilicity by, for
example, glycosylation, or increasing their net charge by methods
known in the art.
[0043] The factors may be delivered alone or in combination, and
may be delivered along with a pharmaceutically acceptable vehicle.
Ideally, such a vehicle would enhance the stability and/or delivery
properties. The invention also provides for pharmaceutical
compositions containing the active factor or fragment or derivative
thereof, which can be administered using a suitable vehicle such as
liposomes, microparticles or microcapsules. In various embodiments
of the invention, it may be useful to use such compositions to
achieve sustained release of the active component.
[0044] The amount of factor which will be effective in the
treatment of a particular disorder or condition will depend on the
nature of the disorder or condition and can be determined by
standard clinical techniques.
EXAMPLE 1
[0045] Use of Neurotrophic and Cellular Factors to Prevent Light
Induced Photoreceptor Injury
[0046] Albino rats of either the F344 or Sprague-Dawley strain were
used at 2-5 months of age. The rats were maintained in a cyclic
light environment (12 hr on: 12 hr off at an in-cage illuminance of
less than 25 ft-c) for 9 or more days before being exposed to
constant light. The rats were exposed to 1 or 2 weeks of constant
light at an illuminance level of 115-200 ft-c (most rats received
125-170 ft-c) provided by two 40 watt General Electric "cool-white"
fluorescent bulbs with a white reflector that was suspended 60 cm
above the floor of the cage. During light exposure, rats were
maintained in transparent polycarbonate cages with stainless steel
wire-bar covers.
[0047] Two days before constant light exposure, rats anesthetized
with a ketamine-xylazine mixture were injected intravitreally with
1 .mu.l of the various factors dissolved in phosphate buffered
saline (PBS) at a concentration of 50-1000 ng/.mu.1. The injections
were made with the insertion of a 32 gauge needle through the
sclera, choroid and retina approximately midway between the ora
serrate and equator of the eye. The factor-injected animals were
compared to either uninjected littermates or to those that received
intravitreal injections of 1 .mu.l of PBS alone, as well as to
animals that were not exposed to constant light. Controls included
the injection of 1 .mu.l of PBS alone, or the insertion of a dry
needle with no injection. In all cases, the injections were made
into the superior hemisphere of the eye.
[0048] Immediately following constant light exposure, the rats were
killed by overdose of carbon dioxide followed immediately by
vascular perfusion of mixed aldehydes. The eyes were embedded in
epoxy resin for sectioning at 1 .mu.m thickness to provide sections
of the entire retina along the vertical meridian of the eye. The
degree of light-induced retinal degeneration was quantified by two
methods. The first was by measuring outer nuclear layer (ONL)
thickness, which is used as an index of photoreceptor cell loss. A
mean ONL thickness was obtained from a single section of each
animal with the aid of a Bioquant morphometry system. In each of
the superior and inferior hemispheres, ONL thickness was measured
in 9 sets of 3 measurements each (total of 27 measurements in each
hemisphere). Each set was centered on adjacent 440-.mu.m lengths of
retina (the diameter of the microscope field at 400.times.
magnification). The first set of measurements was taken at
approximately 440 .mu.m from the optic nerve head, and subsequent
sets were located more peripherally. Within each 440->m length
of retina, the 3 measurements were made at defined points separated
from one another by 75 .mu.m using an eyepiece micrometer. In this
way, the 54 measurements in the two hemispheres sampled
representative regions of almost the entire retinal section. The
results obtained with each of the factors tested are summarized in
FIG. 1.
[0049] The second method of assessing the degree of photoreceptor
rescue was by a 0-4+ pathologist's scale of rescue, 4+ being
maximal rescue and almost normal retinal integrity. The degree of
photoreceptor rescue in each section, as based on comparison to the
control eye in the same rat, was scored by four individuals. This
method has the advantage of considering not only the ONL thickness,
but also more subtle degenerative changes to the photoreceptor
inner and outer segments, as well as spatial degenerative gradients
within the eye. Data obtained from this method is summarized in
FIG. 2. The number of eyes examined for each factor was 10 or more,
except for insulin and laminin, which was 6 each.
[0050] Results and Discussion
[0051] The data obtained using the light damage model of
photoreceptor injury is presented in FIGS. 1, 2 and 3A-3C.
Neurotrophic factors BDNF and CNTF provided a high degree of
rescue. The factors bFGF, aFGF, bFGF plus heparin, aFGF plus
heparin, TNF-.alpha., IL-1.beta., NT-3 and IGF-2 also provided a
significant amount of rescue. Notably, all of the factors other
than bFGF enhanced survival without inducing a high incidence of
macrophages, as seen in FIG. 4 (IL-1.beta. and TNF-.alpha. were
associated with a slightly higher incidence of macrophages). Some
factors actually suppressed the incidence of macrophages as
compared to control retinas (retinas in the same animal that were
injected with PBS). Such factors included BDNF, aFGF, and bFGF plus
heparin.
[0052] Acidic fibroblast growth factor (aFGF), which had previously
been reported to be ineffective as compared to bFGF in the RCS rat,
was shown to provide significant protection of photoreceptors in
the light-damage model. In addition, the influx of macrophages
normally observed with injections of bFGF were not seen when bFGF
was used in combination with heparin, thus eliminating a side
effect that potentially would have obviated the use of bPGP.
[0053] Some degree of rescue, although to a lesser extent, was
observed with heparin, PDGF, NGF, EGF and IGF-1.
[0054] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
* * * * *