U.S. patent application number 15/892861 was filed with the patent office on 2018-12-27 for methods and compositions for treating conditions of the eye.
The applicant listed for this patent is Massachusetts Eye and Ear Infirmary. Invention is credited to Evangelos S. Gragoudas, Joan W. Miller, Vasiliki Poulaki.
Application Number | 20180369380 15/892861 |
Document ID | / |
Family ID | 36927985 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180369380 |
Kind Code |
A1 |
Gragoudas; Evangelos S. ; et
al. |
December 27, 2018 |
Methods and Compositions for Treating Conditions of the Eye
Abstract
Provided are methods and compositions for treating ocular
conditions characterized by the presence of unwanted choroidal
neovasculature, for example, neovascular age-related macular
degeneration. The selectivity and sensitivity of, for example, a
photodynamic therapy (PDT)-based approach can be enhanced by
combining the PDT with an anti-FasL factor, for example, an
anti-FasL neutralizing antibody.
Inventors: |
Gragoudas; Evangelos S.;
(Cambridge, MA) ; Poulaki; Vasiliki; (Roslindale,
MA) ; Miller; Joan W.; (Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Eye and Ear Infirmary |
Boston |
MA |
US |
|
|
Family ID: |
36927985 |
Appl. No.: |
15/892861 |
Filed: |
February 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15155495 |
May 16, 2016 |
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15892861 |
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13948722 |
Jul 23, 2013 |
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15155495 |
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12862245 |
Aug 24, 2010 |
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13948722 |
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11359887 |
Feb 22, 2006 |
7803375 |
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12862245 |
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60655723 |
Feb 23, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2875 20130101;
A61K 39/39541 20130101; A61P 27/02 20180101; A61K 39/39541
20130101; A61K 31/353 20130101; A61K 39/3955 20130101; A61K 31/555
20130101; A61K 41/0071 20130101; A61K 2300/00 20130101; A61K
41/0076 20130101; A61K 38/1793 20130101; C07K 2317/76 20130101;
A61K 31/409 20130101; A61K 2039/505 20130101; A61K 31/655
20130101 |
International
Class: |
A61K 41/00 20060101
A61K041/00; A61K 39/395 20060101 A61K039/395; A61K 31/409 20060101
A61K031/409; C07K 16/28 20060101 C07K016/28; A61K 31/353 20060101
A61K031/353; A61K 38/17 20060101 A61K038/17; A61K 31/655 20060101
A61K031/655; A61K 31/555 20060101 A61K031/555 |
Claims
1. A method of treating unwanted choroidal neovasculature in a
mammal, the choroidal neovasculature comprising endothelial cells,
the method comprising the steps of: (a) administering to the mammal
an anti-FasL factor in an amount sufficient to permit an effective
amount to localize in the choroidal neovasculature; (b)
administering to the mammal an amount of photosensitizer sufficient
to permit an effective amount to localize in the choroidal
neovasculature; and (c) irradiating the choroidal neovasculature
with laser light such that the light is absorbed by the
photosensitizer so as to occlude the choroidal neovasculature
2. The method of claim 1, wherein the mammal is a primate.
3. The method of claim 2, wherein the primate is a human.
4. The method of claim 1, wherein the anti-FasL factor is
administered to the mammal prior to administration of the
photosensitizer.
5. The method of claim 1, wherein the photosensitizer is an amino
acid derivative, an azo dye, a xanthene derivative, a chlorin, a
tetrapyrrole derivative, or a phthalocyanine.
6. The method of claim 5, wherein the photosensitizer is lutetium
texaphyrin, a benzoporphyrin, a benzoporphyrin derivative, a
hematoporphyrin, or a hematoporphyrin derivative.
7. The method of claim 1, wherein the anti-FasL factor comprises an
anti-FasL antibody.
8. An improved method of treating unwanted choroidal neovasculature
in a mammal, the improvement comprising: administering to the
mammal an effective amount of an anti-FasL factor so as to mitigate
side effects associated with a method for treating unwanted
choroidal neovasculature.
9. The method of claim 8, wherein the anti-FasL factor comprises an
anti-FasL antibody.
10. The method of claim 8, wherein the side effects include
photoreceptor cell death.
11. The method of claim 10, wherein the anti-FasL factor reduces
apoptotic cell death of photoreceptor cells during the method of
treating unwanted choroidal neovasculature.
12. The method of claim 8, wherein the method of treating unwanted
choroidal neovasculature comprises photodynamic therapy using a
benzoporphyrin derivative photosensitizer.
13. The method of claim 8, wherein the method of treating unwanted
choroidal neovasculature comprises administering an effective
amount of an anti-VEGF aptamer.
14. The method of claim 8, wherein the method of treating unwanted
choroidal neovasculature comprises administering an effective
amount of an anti-VEGF antibody.
15. The method of claim 8, wherein the method of treating unwanted
choroidal neovasculature comprises administering an effective
amount of an anti-VEOF siRNA.
16. The method of claim 8, wherein the method of treating unwanted
choroidal neovasculature ameliorates the symptoms of age-related
macular degeneration.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of and priority
to U.S. Patent Application Ser. No. 60/655,723, filed Feb. 23,
2005, the entire disclosure of which is incorporated by reference
herein for all purposes.
FIELD OF THE INVENTION
[0002] The invention relates generally to methods and compositions
for treating ocular conditions and, more specifically, the
invention relates to photodynamic therapy-based methods and
compositions for treating ocular conditions characterized by
unwanted choroidal neovasculature.
BACKGROUND
[0003] Choroidal neovascularization can lead to hemorrhage and
fibrosis, with resulting visual loss in a number of conditions of
the eye, including, for example, age-related macular degeneration,
ocular histoplasmosis syndrome, pathologic myopia, angioid streaks,
idiopathic disorders, choroiditis, choroidal rupture, overlying
choroid nevi, and certain inflammatory diseases. One of the
disorders, namely, age-related macular degeneration (AMD), is the
leading cause of severe vision loss in people aged 65 and above
(Bressler et al. (1988) Surv. Ophthalmol. 32, 375-413, Guyer et al.
(1986) Arch. Ophthalmol. 104, 702-705, Hyman et al. (1983) Am. J.
Epidemiol. 188, 816-824, Klein & Klein (1982) Arch. Ophthalmol.
100, 571-573, Leibowitz et al. (1980) Surv. Ophthalmol. 24,
335-610). Although clinicopathologic descriptions have been made,
little is understood about the etiology and pathogenesis of the
disease.
[0004] Dry AMD is the more common form of the disease,
characterized by drusen, pigmentary and atrophic changes in the
macula, with slowly progressive loss of central vision. Wet or
neovascular AMD is characterized by subretinal hemorrhage, fibrosis
and fluid secondary to the formation of choroidal neovasculature
(CNV), and more rapid and pronounced loss of vision. While less
common than dry AMD, neovascular AMD accounts for 80% of the severe
vision loss due to AMD. Approximately 200,000 cases of neovascular
AMD are diagnosed yearly in the United States alone.
[0005] Currently, treatment of the dry form of age-related macular
degeneration includes administration of antioxidant vitamins and/or
zinc. Treatment of the wet form of age-related macular
degeneration, however, has proved to be more difficult. Currently,
two separate methods have been approved in the United States of
America for treating the wet form of age-related macular
degeneration. These include laser photocoagulation and photodynamic
therapy using a benzoporphyrin derivative photosensitizer. During
laser photocoagulation, thermal laser light is used to heat and
photocoagulate the neovasculature of the choroid. A problem
associated with this approach is that the laser light must pass
through the photoreceptor cells of the retina in order to
photocoagulate the blood vessels in the underlying choroid. As a
result, this treatment destroys the photoreceptor cells of the
retina creating blind spots with associated vision loss. During
photodynamic therapy, a benzoporphyrin derivative photosensitizer
is administered to the individual to be treated. Once the
photosensitizer accumulates in the choroidal neovasculature,
non-thermal light from a laser is applied to the region to be
treated, which activates the photosensitizer in that region. The
activated photosensitizer generates free radicals that damage the
vasculature in the vicinity of the photosensitizer (see, U.S. Pat.
Nos. 5,798,349 and 6,225,303). This approach is more selective than
laser photocoagulation and is less likely to result in blind spots.
Under certain circumstances, this treatment has been found to
restore vision in patients afflicted with the disorder (see, U.S.
Pat. Nos. 5,756,541 and 5,910,510).
[0006] During clinical studies, however, it has been found that
recurrence of leakage appears in at least a portion of the CNV by
one to three months post-treatment. Increasing photosensitizer or
light doses do not appear to prevent this recurrence, and can even
lead to undesired non-selective damage to retinal vessels (Miller
et al. (1999) Archives of Ophthalmology 117: 1161-1173). Another
avenue of investigation is to repeat the PDT procedure over
prolonged periods of time. The necessity for repeated PDT
treatments can nevertheless be expected to lead to cumulative
damage to the retinal pigment epithelium (RPE) and
choriocapillaris, which may lead to progressive treatment-related
vision loss. In addition, PDT can cause transient visual
disturbances, injection-site adverse effects, transient
photosensitivity reactions, infusion-related back pain, and vision
loss.
[0007] Therefore, there is still a need for improved methods for
treating AMD characterized by unwanted choroidal neovasculature
that increase the efficacy and selectivity of treatment, and which
reduce or delay a recurrence of the disorder.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to methods and
compositions for treating ocular conditions associated with
unwanted choroidal neovasculature. Such conditions include, for
example, neovascular AMD, ocular histoplasmosis syndrome,
pathologic myopia, angioid streaks, idiopathic disorders,
choroiditis, choroidal rupture, overlying choroid nevi, and certain
inflammatory diseases. The invention, for example, provides a more
effective PDT-based method for treating unwanted CNV that has one
or more of the following advantages: increased efficacy of
treatment; increased selectivity for CNV; and reduced or delayed
recurrence of the condition following PDT.
[0009] In one aspect, the invention provides a method of treating
unwanted CNV in a mammal, wherein the CNV comprises endothelial
cells, for example, capillary endothelial cells. The method
comprises the steps of: (a) administering to the mammal, for
example, a primate, preferably, a human, an anti-FasL factor in an
amount sufficient to permit an effective amount to localize in the
CNV; (b) administering to the mammal an amount of a photosensitizer
(PDT dye) sufficient to permit an effective amount to localize in
the CNV; and (c) irradiating the CNV with laser light such that the
light is absorbed by the photosensitizer so as to occlude the CNV.
During practice of this method, the anti-FasL factor can enhance
the activity of PDT. For example, the anti-FasL factor and the PDT
may act synergistically.
[0010] A variety of anti-FasL factors may be used in the invention.
Useful anti-FasL factors, include, for example, anti-FasL
neutralizing antibody (available, for example, from Pharmingen, San
Diego, Calif.); peptides and nucleic acids (for example, anti-FasL
aptamers) that bind FasL to prevent or reduce its binding to its
cognate receptor; certain antibodies and antigen binding fragments
thereof and peptides that bind preferentially to the Fas receptor;
antisense nucleotides and double stranded RNA for RNAi that
ultimately reduce or eliminate the production of either FasL or the
Fas receptor; soluble Fas; soluble FasL; decoy receptor-3 (DcR3)
analogues; matrix metalloproteinases (MMPs); vasoactive intestinal
peptide (VIP); pituitary adenylate cyclase-activating polypeptide
(PACAP); forskolin; combined use of benazepril and valsartan;
nonpeptidic corticotropin-releasing hormone receptor type 1
(CRH-R1)-specific antagonists; mimosine; peptides that produce a
defective Fas-FasL complex; platelet-activating factor (PAF); and
endothelin-1 (ET-1). These anti-FasL factors can act as direct or
indirect antagonists of FasL activity.
[0011] The term "antibody," as used herein, includes, for example,
a monoclonal antibody or an antigen binding fragment thereof (for
example, an Fv, Fab, Fab' or an (Fab').sub.2 molecule), a
polyclonal antibody or an antigen binding fragment thereof, or a
biosynthetic antibody binding site, for example, an sFv (U.S. Pat.
Nos. 5,091,513; 5,132,405; 5,258,498; and 5,482,858) that binds
specifically to a target ligand. As used herein, the terms binds
"specifically" or "preferentially" are understood to mean that the
targeting molecule, for example, the antibody, binds to the
complementary or target ligand with a binding affinity of at least
10.sup.5 M.sup.-1, and more preferably 10.sup.7 M.sup.-1.
[0012] The anti-FasL factor may, under certain circumstances, be
co-administered simultaneously with the photosensitizer.
Alternatively, the anti-FasL factor may be administered before or
after the photosensitizer. In a preferred embodiment, however, the
anti-FasL factor is administered to the mammal prior to
administration of the photosensitizer.
[0013] In another aspect, the invention provides an improved method
of treating unwanted choroidal neovasculature in a mammal. The
improvement includes administering to the mammal an effective
amount of an anti-FasL factor so as to relieve side effects
associated with a method for treating unwanted choroidal
neovasculature. The anti-FasL factor can include an anti-FasL
antibody, and the side effects can include photoreceptor cell
death. In certain instances, the anti-FasL factor reduces apoptotic
cell death of photoreceptor cells during the method of treating
unwanted choroidal neovasculature. The method of treating unwanted
choroidal neovasculature can include photodynamic therapy using a
benzoporphyrin derivative photosensitizer; can include
administering an effective amount of an anti-VEOF aptamer, an
effective amount of an anti-VEGF antibody, and/or an effective
amount of an anti-VEOF siRNA; and/or can include ameliorating the
symptoms of age-related macular degeneration.
[0014] In all the methods disclosed herein, it is contemplated that
any photosensitizer useful in PDT may be useful in the practice of
the invention. Useful photosensitizers include, for example, amino
acid derivatives, azo dyes, xanthene derivatives, chlorins,
tetrapyrrole derivatives, phthalocyanines, and assorted other
photosensitizers. Preferred photosensitizers, include, for example,
lutetium texaphyrin, benzoporphyrin and derivatives thereof, and
hematoporphyrin and derivatives thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The foregoing and other objects, features, and advantages of
the present invention, as well as the invention itself, may be more
fully understood from the following description of preferred
embodiments, when read together with the accompanying drawings, in
which:
[0016] FIG. 1 depicts a schematic drawing of the FasL apoptotic
pathway;
[0017] FIG. 2 provides evidence that anti-FasL treatment prevents
cleavage of bid after PDT;
[0018] FIG. 3 provides evidence that PDT increases the expression
of the Fas receptor in the rat retina;
[0019] FIG. 4 provides evidence that anti-FasL treatment prevents
activation of caspase 3 after PDT;
[0020] FIG. 5 provides evidence that anti-FasL treatment reduces
angiographic leakage after PDT;
[0021] FIG. 6 provides evidence that anti-FasL treatment reduces
angiographic leakage in laser-induced CNV;
[0022] FIG. 7 provides evidence that anti-FasL treatment reduces
PDT-induced caspase 8 activation in laser-induced CNV;
[0023] FIG. 8 provides evidence that anti-FasL treatment reduces
PDT-induced cytochrome c release in laser-induced CNV;
[0024] FIG. 9 provides evidence that anti-FasL treatment reduces
PDT-induced Bax upregulation in laser-induced CNV; and
[0025] FIG. 10 provides evidence that anti-FasL treatment reduces
PDT-induced Bcl-2 downregulation in laser-induced CNV.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The invention relates to an improved method for treating
ocular conditions characterized as having unwanted CNV. Such
conditions include, for example, neovascular AMD, ocular
histoplasmosis syndrome, pathologic myopia, angioid streaks,
idiopathic disorders, choroiditis, choroidal rupture, overlying
choroid nevi, and certain inflammatory diseases. The invention
provides one or more of the following advantages: increased
efficacy of treatment; increased selectivity for CNV; and reduced
or delayed recurrence of the condition following PDT.
[0027] The invention provides an improved method for treating
ocular disorders, for example, AMD, characterized by unwanted
choroidal neovasculature. The improved method comprises
administering to the mammal an effective amount of an anti-FasL
factor, for example, an anti-FasL antibody, for preserving
photoreceptor viability, i.e., reducing collateral retinal damage
during a treatment of unwanted choroidal neovasculature. For
example, the anti-FasL factor may be combined with an anti-VEGF
aptamer, for example the Macugen.RTM. aptamer (see the URL address
eyetk.com/science/science_vegf.asp), for treatment of AMD
(available from Eyetech Pharmaceuticals, Inc., NY, N.Y.).
Alternatively, the anti-FasL factor may be combined with a VEGF
specific RNAi for the treatment of AMD (see the URL address:
alnylam.com/therapeutic-programs/programs.asp) (available from
Alnylam Pharmaceuticals, Cambridge, Mass.). Similarly, the
anti-FasL factor may be combined with an anti-VEGF antibody or
antibody fragment for the treatment of AMD (see the URL address:
gene.com/gene/products/information/oncology/avastin/index.jsp)
(available from Genentech, Inc., San Francisco, Calif.).
[0028] In one aspect, the invention provides an improved PDT-based
method for treating unwanted target CNV. The method involves
administration of a photosensitizer to a mammal in need of such
treatment in an amount sufficient to permit an effective amount
(i.e., an amount sufficient to facilitate PDT) of the
photosensitizer to localize in the target CNV. After administration
of the photosensitizer, the CNV then is irradiated with laser light
under conditions such that the light is absorbed by the
photosensitizer. The photosensitizer, when activated by the light,
generates singlet oxygen and free radicals, for example, reactive
oxygen species, that result in damage to surrounding tissue. For
example, PDT-induced damage of endothelial cells results in
platelet adhesion and degranulation, leading to stasis and
aggregation of blood cells and vascular occlusion.
[0029] An increase in efficacy and/or selectivity of the PDT,
and/or reduction or delay of recurrence of the CNV, can be achieved
by administering an anti-FasL factor to the mammal prior to,
concurrent with, or after administration of the photosensitizer. It
is contemplated that a variety of photosensitizers useful in PDT
may be useful in the practice of the invention and include, for
example, amino acid derivatives, azo dyes, xanthene derivatives,
chlorins, tetrapyrrole derivatives, phthalocyanines, and assorted
other photosensitizers.
[0030] Amino acid derivatives include, for example,
5-aminolevulinic acid (Berg et al. (1997) Photochem. Photobiol. 65:
403-409; El-Far et al. (1985) Cell. Biochem. Function 3, 115-119).
Azo dyes, include, for example, Sudan I, Sudan II, Sudan III, Sudan
IV, Sudan Black, Disperse Orange, Disperse Red, Oil Red O, Trypan
Blue, Congo Red, .beta.-carotene (Mosky et al. (1984) Exp. Res.
155, 389-396). Xanthene derivatives, include, for example, rose
bengal.
[0031] Chlorins include, for example, lysyl chlorin p6 (Berg et al.
(1997) supra) and etiobenzochlorin (Berg et al. (1997) supra),
5,10,15,20-tetra (m-hydroxyphenyl) chlorin (M-THPC), N-aspartyl
chlorin e6 (Dougherty et al. (1998) J. Natl. Cancer Inst. 90:
889-905), and bacteriochlorin (Korbelik et al. (1992) J. Photochem.
Photobiol. 12: 107-119).
[0032] Tetrapyrrole derivatives include, for example, lutetium
texaphrin (Lu-Tex, PCI-0123) (Dougherty et al. (1998) supra, Young
et al. (1996) Photochem. Photobiol. 63: 892-897), benzoporphyrin
derivative (BPD) (U.S. Pat. Nos. 5,171,749, 5,214,036, 5,283,255,
and 5,798,349, Jori et al. (1990) Lasers Med. Sci. 5, 115-120),
benzoporphyrin derivative mono acid (BPD-MA) (U.S. Pat. Nos.
5,171,749, 5,214,036, 5,283,255, and 5,798,349, Berg et al. (1997)
supra, Dougherty et al. (1998) supra), hematoporphyrin (Hp) (Jori
et al. (1990) supra), hematoporphyrin derivatives (HpD) (Berg et
al. (1997) supra, West et al. (1990) In. J. Radiat. Biol. 58:
145-156), porfimer sodium or Photofrin (PHP) (Berg et al. (1997)
supra), Photofrin II (PII) (He et al. (1994) Photochem. Photobiol.
59: 468-473), protoporphyrin IX (PpIX) (Dougherty et al. (1998)
supra, He et al. (1994) supra), meso-tetra (4-carboxyphenyl)
porphine (TCPP) (Musser et al. (1982) Res. Commun. Chem. Pathol.
Pharmacol. 2, 251-259), meso-tetra (4-sulfonatophenyl) porphine
(TSPP) (Musser et al. (1982) supra), uroporphyrin I (UROP-I)
(El-Far et al. (1985) Cell. Biochem. Function 3, 115-119),
uroporphyrin III (UROP-III) (El-Far et al. (1985) supra), tin ethyl
etiopurpurin (SnET2), (Dougherty et al. (1998) supra 90: 889-905)
and 13,17-bis[1-carboxypropionyl]
carbamoylethyl-8-etheny-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetr-
anethyl 6 porphyrin sodium (ATX-S10(Na)) Mori et al. (2000) JPN. J.
CANCER RES. 91:753-759, Obana et al. (2000) Arch. Ophthalmol.
118:650-658, Obana et al. (1999) Lasers Surg. Med. 24:209-222).
[0033] Phthalocyanines include, for example, chloroaluminum
phthalocyanine (AlPcCl) (Rerko et al. (1992) Photochem. Photobiol.
55, 75-80), aluminum phthalocyanine with 2-4 sulfonate groups
(AlPcS2-4) (Berg et al. (1997) supra, Glassberg et al. (1991)
Lasers Surg. Med. 11, 432-439), chloro-aluminum sulfonated
phthalocyanine (CASPc) (Roberts et al. (1991) J. Natl. Cancer Inst.
83, 18-32), phthalocyanine (PC) (Jori et al. (1990) supra), silicon
phthalocyanine (Pc4) (He et al. (1998) Photochem. Photobiol. 67:
720-728, Jori et al. (1990) supra), magnesium phthalocyanine
(Mg2+-PC) (Jori et al. (1990) supra), and zinc phthalocyanine
(ZnPC) (Berg et al. (1997) supra). Other photosensitizers include,
for example, thionin, toluidine blue, neutral red and azure c.
[0034] However, useful photosensitizers, include, for example,
Lutetium Texaphyrin (Lu-Tex), a new generation photosensitizer
having favorable clinical properties including absorption at about
730 nm permitting deep tissue penetration and rapid clearance.
Lu-Tex is available from Alcon Laboratories, Fort Worth, Tex. Other
useful photosensitizers include benzoporphyrin and benzoporphyrin
derivatives, for example, BPD-MA and BPD-DA, available from QLT
Inc., Vancouver, Canada.
[0035] The photosensitizer preferably is formulated into a delivery
system that delivers high concentrations of the photosensitizer to
the CNV. Such formulations may include, for example, the
combination of a photosensitizer with a carrier that delivers
higher concentrations of the photosensitizer to CNV and/or coupling
the photosensitizer to a specific binding ligand that binds
preferentially to a specific cell surface component of the CNV.
[0036] In one embodiment, the photosensitizer can be combined with
a lipid based carrier. For example, liposomal formulations have
been found to be particularly effective at delivering the
photosensitizer, green porphyrin, and more particularly BPD-MA to
the low-density lipoprotein component of plasma, which in turn acts
as a carrier to deliver the photosensitizer more effectively to the
CNV. Increased numbers of LDL receptors have been shown to be
associated with CNV, and by increasing the partitioning of the
photosensitizer into the lipoprotein phase of the blood, it may be
delivered more efficiently to the CNV. Certain photosensitizers,
for example, green porphyrins, and in particular BPD-MA, interact
strongly with lipoproteins. LDL itself can be used as a carrier,
but LDL is more expensive and less practical than a liposomal
formulation. LDL, or preferably liposomes, are thus preferred
carriers for the green porphyrins since green porphyrins strongly
interact with lipoproteins and are easily packaged in liposomes.
Compositions of green porphyrins formulated as lipocomplexes,
including liposomes, are described, for example, in U.S. Pat. Nos.
5,214,036, 5,707,608 and 5,798,349. Liposomal formulations of green
porphyrin can be obtained from QLT Inc., Vancouver, Canada. It is
contemplated that certain other photosensitizers may likewise be
formulated with lipid carriers, for example, liposomes or LDL, to
deliver the photosensitizer to CNV.
[0037] Furthermore, the photosensitizer can be coupled or
conjugated to a targeting molecule that targets the photosensitizer
to CNV. For example, the photosensitizer may be coupled or
conjugated to a specific binding ligand that binds preferentially
to a cell surface component of the CNV, for example, neovascular
endothelial homing motif. It appears that a variety of cell surface
ligands are expressed at higher levels in new blood vessels
relative to other cells or tissues.
[0038] Endothelial cells in new blood vessels express several
proteins that are absent or barely detectable in established blood
vessels (Folkman (1995) Nature Medicine 1:27-31), and include
integrins (Brooks et al. (1994) Science 264: 569-571; Friedlander
et al. (1995) Science 270: 1500-1502) and receptors for certain
angiogenic factors like vascular endothelial growth factor (VEGF).
In vivo selection of phage peptide libraries have also identified
peptides expressed by the vasculature that are organ-specific,
implying that many tissues have vascular "addresses" (Pasqualini et
al. (1996) Nature 380: 364-366). It is contemplated that a suitable
targeting moiety can direct a photosensitizer to the CNV
endothelium thereby increasing the efficacy and lowering the
toxicity of PDT.
[0039] Several targeting molecules may be used to target
photosensitizers to the neovascular endothelium. For example,
.alpha.-v integrins, in particular .alpha.-v .beta. and .alpha.-v
.beta.5, appear to be expressed in ocular neovascular tissue, in
both clinical specimens and experimental models (Corjay et al.
(1997) Invest. Ophthalmol. Vis. Sci. 38, S965; Friedlander et al.
(1995) supra). Accordingly, molecules that preferentially bind
.alpha.-v integrins can be used to target the photosensitizer to
CNV. For example, cyclic peptide antagonists of these integrins
have been used to inhibit neovascularization in experimental models
(Friedlander et al. (1996) Proc. Natl. Acad. Sci. USA
93:9764-9769). A peptide motif having an amino acid sequence, in an
N- to C-terminal direction, ACDCRGDCFC (SEQ ID NO: 1)--also know as
RGD-4C--has been identified that selectively binds to human
.alpha.-v integrins and accumulates in tumor neovasculature more
effectively than other angiogenesis targeting peptides (Arap et al.
(1998) Nature 279:377-380; Ellerby et al. (1999) Nature Medicine 5:
1032-1038). Angiostatin may also be used as a targeting molecule
for the photosensitizer. Studies have shown, for example, that
angiostatin binds specifically to ATP synthase disposed on the
surface of human endothelial cells (Moser et al. (1999) Proc. Natl.
Acad. Sci. USA 96:2811-2816).
[0040] Clinical and experimental evidence strongly supports a role
for vascular endothelial growth factor (VEGF) in ocular
neovascularization, particularly ischemia-associated
neovascularization (Adamis et al. (1996) Arch. Ophthalmol.
114:66-71; Tolentino et al. (1996) Arch. Ophthalmol. 114:964-970;
Tolentino et al. (1996) Ophthalmology 103:1820-1828). Potential
targeting molecules include antibodies that bind specifically to
either VEGF or the VEGF receptor (VEOF-2R). Antibodies to the VEOF
receptor (VEGFR-2 also known as KDR) may also bind preferentially
to neovascular endothelium.
[0041] The targeting molecule may be synthesized using
methodologies known and used in the art. For example, proteins and
peptides may be synthesized using conventional synthetic peptide
chemistries or expressed as recombinant proteins or peptides in a
recombinant expression system (see, for example, "Molecular
Cloning" Sambrook et al. eds, Cold Spring Harbor Laboratories).
Similarly, antibodies may be prepared and purified using
conventional methodologies, for example, as described in "Practical
Immunology", Butt, W. R. ed., 1984 Marcel Deckker, New York and
"Antibodies, A Laboratory Approach" Harlow et al., eds. (1988),
Cold Spring Harbor Press. Once created, the targeting agent may be
coupled or conjugated to the photosensitizer using standard
coupling chemistries, using, for example, conventional cross
linking reagents, for example, heterobifunctional cross linking
reagents available, for example, from Pierce, Rockford, Ill.
[0042] Once formulated, the photosensitizer may be administered in
any of a wide variety of ways, for example, orally, parenterally,
or rectally. Parenteral administration, such as intravenous,
intramuscular, or subcutaneous, is preferred. Intravenous injection
is especially preferred. The dose of photosensitizer can vary
widely depending on the tissue to be treated; the physical delivery
system in which it is carried, such as in the form of liposomes; or
whether it is coupled to a target-specific ligand, such as an
antibody or an immunologically active fragment.
[0043] It should be noted that the various parameters used for
effective, selective photodynamic therapy in the invention are
interrelated. Therefore, the dose should also be adjusted with
respect to other parameters, for example, fluence, irradiance,
duration of the light used in PDT, and time interval between
administration of the dose and the therapeutic irradiation. All of
these parameters should be adjusted to produce significant damage
to CNV without significant damage to the surrounding tissue.
[0044] Typically, the dose of photosensitizer used is within the
range of from about 0.1 to about 20 mg/kg, preferably from about
0.15 to about 5.0 mg/kg, and even more preferably from about 0.25
to about 2.0 mg/kg. Furthermore, as the dosage of photosensitizer
is reduced, for example, from about 2 to about 1 mg/kg in the case
of green porphyrin or BPD-MA, the fluence required to close CNV may
increase, for example, from about 50 to about 100 Joules/cm.sup.2.
Similar trends may be observed with the other photosensitizers
discussed herein.
[0045] After the photosensitizer has been administered, the CNV is
irradiated at a wavelength typically around the maximum absorbance
of the photosensitizer, usually in the range from about 550 nm to
about 750 nm. A wavelength in this range is especially preferred
for enhanced penetration into bodily tissues. Preferred wavelengths
used for certain photosensitizers include, for example, about 690
nm for benzoporphyrin derivative mono acid, about 630 nm for
hematoporphyrin derivative, about 675 nm for chloro-aluminum
sulfonated phthalocyanine, about 660 nm for tin ethyl etiopurpurin,
about 730 nm for lutetium texaphyrin, about 670 nm for ATX-S10(NA),
about 665 nm for N-aspartyl chlorin e6, and about 650 nm for
5,10,15,20-tetra (m-hydroxyphenyl) chlorin.
[0046] As a result of being irradiated, the photosensitizer in its
triplet state is thought to interact with oxygen and other
compounds to form reactive intermediates, such as singlet oxygen
and reactive oxygen species, which can disrupt cellular structures.
Possible cellular targets include the cell membrane, mitochondria,
lysosomal membranes, and the nucleus. Evidence from tumor and
neovascular models indicates that occlusion of the vasculature is a
major mechanism of photodynamic therapy, which occurs by damage to
the endothelial cells, with subsequent platelet adhesion,
degranulation, and thrombus formation.
[0047] The fluence during the irradiating treatment can vary
widely, depending on the type of photosensitizer used, the type of
tissue, the depth of target tissue, and the amount of overlying
fluid or blood. Fluences preferably vary from about 10 to about 400
Joules/cm.sup.2 and more preferably vary from about 50 to about 200
Joules/cm.sup.2. The irradiance varies typically from about 50
mW/cm.sup.2 to about 1800 mW/cm.sup.2, more preferably from about
100 mW/cm.sup.2 to about 900 mW/cm.sup.2, and most preferably in
the range from about 150 mW/cm.sup.2 to about 600 mW/cm.sup.2. It
is contemplated that for many practical applications, the
irradiance will be within the range of about 300 mW/cm.sup.2 to
about 900 mW/cm.sup.2. However, the use of higher irradiances may
be selected as effective and having the advantage of shortening
treatment times.
[0048] The time of light irradiation after administration of the
photosensitizer may be important as one way of maximizing the
selectivity of the treatment, thus minimizing damage to structures
other than the target tissues. The optimum time following
photosensitizer administration until light treatment can vary
widely depending on the mode of administration, the form of
administration such as in the form of liposomes or as a complex
with LDL, and the type of target tissue. For example,
benzoporphyrin derivative typically becomes present within the
target neovasculature within one minute post administration and
persists for about fifty minutes, lutetium texaphyrin typically
becomes present within the target neovasculature within one minute
post administration and persists for about twenty minutes,
N-aspartyl chlorin e6 typically becomes present within the target
neovasculature within one minute post administration and persists
for about twenty minutes, and rose bengal typically becomes present
in the target vasculature within one minute post administration and
persists for about ten minutes.
[0049] Effective vascular closure generally occurs at times in the
range of about one minute to about three hours following
administration of the photosensitizer. However, as with green
porphyrins, it is undesirable to perform the PDT within the first
five minutes following administration to prevent undue damage to
retinal vessels still containing relatively high concentrations of
photosensitizer.
[0050] The efficacy of PDT may be monitored using conventional
methodologies, for example, via fundus photography or angiography.
Closure can usually be observed angiographically by
hypofluorescence in the treated areas in the early angiographic
frames. During the later angiographic frames, a corona of
hyperfluorescence may begin to appear which then fills the treated
area, possibly representing leakage from the adjacent
choriocapillaris through damaged retinal pigment epithelium in the
treated area. Large retinal vessels in the treated area typically
perfuse following photodynamic therapy.
[0051] Minimal retinal damage is generally found on histopathologic
correlation and is dependent on the fluence and the time interval
after irradiation that the photosensitizer is administered. It is
contemplated that the choice of appropriate photosensitizer,
dosage, mode of administration, formulation, timing post
administration prior to irradiation, and irradiation parameters may
be determined empirically.
[0052] It is contemplated that a variety of anti-FasL factors may
be combined with other treatments for treating unwanted CNV. The
anti-FasL factor can synergistically enhance the activity of the
treatment, for example, PDT. In addition, the anti-FasL factor can
be used to reduce or delay the recurrence of the condition. The
term "anti-FasL factor" is understood to mean any molecule, for
example, a protein, peptide, nucleic acid (ribonucleic acid (RNA)
or deoxyribonucleic acid (DNA)), peptidyl nucleic acid, organic
compound or inorganic compound, that decreases or eliminates the
activity of FasL in a mammal. An "effective amount" of an anti-FasL
factor is an amount of an anti-FasL factor sufficient to decrease
or eliminate the activity of FasL. For example, an effective amount
of an anti-FasL antibody is an amount sufficient to reduce or
eliminate the binding of FasL to its cognate receptor.
[0053] FIG. 1 depicts a representative drawing of the FasL
apoptotic pathway. An important mediator of apoptosis is the
signaling pathway triggered by the interaction of the receptor Fas
with its ligand (FasL). Fas (also known as Apo1/CD95) is a
transmembrane death receptor which, upon crosslinking with its
natural ligand, FasL, induces apoptosis in Fas receptor-bearing
cells. Fas is expressed in a wide variety of tissues, such as the
liver, ovary, lung and heart, as well as in myeloid and
lymphoblastoid cells. Its cytoplasmic domain contains a 68 amino
acid motif that is both necessary and sufficient for the induction
of apoptosis (death domain, DD). Upon cross-linking (by the Fas
Ligand or polyvalent antibodies), Fas recruits the adaptor FADD
(Fas-Associating protein with a Death Domain), also known as MORT1,
which, via its N-terminal DED then recruits the pro-enzyme form of
caspase-8. The aggregation of Fas, FADD and caspase-8, named the
death-inducing signaling complex (DISC), catalyses the proteolytic
autoactivation of caspase-8 (induced-proximity model). The
resulting subunits p10 and p18 are released into the cytoplasm,
where they form an enzymatically active complex that triggers the
downstream apoptotic caspase cascade.
[0054] In age-related macular degeneration, retinal pigment
epithelial cells inhibit choroidal vessel growth through Fas/FasL
mediated apoptosis of choroidal endothelial cells. In murine
carcinoma, FasL+T lymphocytes suppress tumor vessel growth.
Anti-FasL neutralizing antibody has been shown to have
anti-angiogenic properties in a murine model of oxygen-induced
retinopathy of prematurity. The anti-FasL neutralizing antibody has
also been shown to be safe and effective in reducing vascular
leakage and endothelial cell damage in a rat model of
streptozotocin-induced diabetes. In this model, anti-FasL
neutralizing antibody also has a neuroprotective effect by reducing
the amount of DNA fragmentation, and therefore apoptosis, in the
ganglion cell layer, the outer nuclear and RPE layer, and
endothelial cell layer of the retina. The amount of caspase
activation is also reduced. Administration of the anti-FasL
neutralizing antibody protects against apoptosis in numerous models
of inflammatory diseases and toxic insults. Additionally, mice
injected with an anti-CD95L antibody 30 minutes after induction of
stroke showed a decrease in both infarct volumes and mortality.
Thus, the anti-FasL antibody has anti-angiogenic and
anti-permeability properties. Moreover, administration of the
anti-FasL antibody protects against apoptosis in numerous models of
inflammatory diseases and toxic insults.
[0055] Studies utilizing an anti-FasL neutralizing antibody, which
was administered in a rat model of laser-induced CNV, reduced
angiographic leakage and neovascular formation. Furthermore, there
is a synergistic effect with PDT in treating CNV, and it reduces
significantly the recurrences of CNV. PDT treatment in the rat
model of laser-induced CNV induces apoptotic cell death selectively
in the endothelial and retinal pigment epithelial cells overlying
the treated CNV. This is associated with activation of mitochondria
and of executional caspases that ultimately lead to cell demise.
Administration of anti-FasL neutralizing antibody in combination
with PDT reduces the activation of the mitochondria, reduces the
activation of caspases, and, ultimately, decreases the apoptotic
death in the endothelial and RPE cell layer. Thus, without being
bound to theory, it appears that PDT-related retinal apoptosis
occurs through a Fas/FasL mechanism. As a result, the anti-FasL
neutralizing antibody can reduce the collateral damage to the
retina when given in combination with PDT, for example, by reducing
apoptosis of adjacent retinal cells. Furthermore, the combination
therapy increases the effectiveness of PDT, reduces the recurrence
of CNV, and protects the retina from PDT-induced apoptosis.
[0056] Numerous anti-FasL factors are well known and thoroughly
documented in the art. Examples of anti-FasL factors useful in the
practice of the invention, include, for example, an anti-FasL
neutralizing antibody (available, for example, from Pharmingen, San
Diego, Calif.); peptides and nucleic acids (for example, anti-FasL
aptamers) that bind FasL to prevent or reduce its binding to its
cognate receptor; certain antibodies and antigen binding fragments
thereof and peptides that bind preferentially to the Fas receptor,
antisense nucleotides (and PNAs) and double stranded RNA for RNAi
that ultimately reduce or eliminate the production of either FasL
or the Fas receptor; soluble Fas; soluble FasL; decoy receptor-3
(DcR3) analogues; matrix metalloproteinases (MMPs); vasoactive
intestinal peptide (VIP); pituitary adenylate cyclase-activating
polypeptide (PACAP); forskolin; combined use of benazepril and
valsartan; nonpeptidic corticotropin-releasing hormone receptor
type 1 (CRH-R1)-specific antagonists; mimosine; peptides that
produce a defective Fas-FasL complex; platelet-activating factor
(PAF); and endothelin-1 (ET-1). These anti-FasL factors can act as
direct or indirect antagonists of FasL activity.
[0057] The anti-FasL factor may be synthesized using methodologies
known and used in the art. For example, proteins and peptides may
be synthesized and purified using conventional synthetic peptide
chemistries and purification protocols, or expressed as recombinant
proteins or peptides in a recombinant expression system (see, for
example, "Molecular Cloning" Sambrook et al. eds, Cold Spring
Harbor Laboratories). Similarly, antibodies may be prepared and
purified using conventional methodologies, for example, as
described in "Practical Immunology", Butt, W. R. ed., 1984 Marcel
Deckker, New York and "Antibodies, A Laboratory Approach" Harlow et
al., eds. (1988), Cold Spring Harbor Press.
[0058] Antibodies (e.g., monoclonal or polyclonal antibodies)
having sufficiently high binding specificity for the marker or
target protein (for example, FasL or its receptor) can be used as
anti-FasL factors. As noted above, the term "antibody" is
understood to mean an intact antibody (for example, a monoclonal or
polyclonal antibody); an antigen binding fragment thereof, for
example, an Fv, Fab, Fab' or (Fab').sub.2 fragment; or a
biosynthetic antibody binding site, for example, an sFv, as
described in U.S. Pat. Nos. 5,091,513; 5,132,405; and 4,704,692. A
binding moiety, for example, an antibody, is understood to bind
specifically to the target, for example, FasL or its receptor, when
the binding moiety has a binding affinity for the target greater
than about 10.sup.5 M.sup.-1, more preferably greater than about
10.sup.7 M.sup.-1.
[0059] Antibodies against FasL or its receptor may be generated
using standard immunological procedures well known and described in
the art. See, for example, Practical Immunology, Butt, N. R., ed.,
Marcel Dekker, N Y, 1984. Briefly, isolated FasL or its receptor is
used to raise antibodies in a xenogeneic host, such as a mouse,
goat or other suitable mammal. The FasL or its receptor is combined
with a suitable adjuvant capable of enhancing antibody production
in the host, and injected into the host, for example, by
intraperitoneal administration. Any adjuvant suitable for
stimulating the host's immune response may be used. A commonly used
adjuvant is Freund's complete adjuvant (an emulsion comprising
killed and dried microbial cells). Where multiple antigen
injections are desired, the subsequent injections may comprise the
antigen in combination with an incomplete adjuvant (for example, a
cell-free emulsion).
[0060] Polyclonal antibodies may be isolated from the
antibody-producing host by extracting serum containing antibodies
to the protein of interest. Monoclonal antibodies may be produced
by isolating host cells that produce the desired antibody, fusing
these cells with myeloma cells using standard procedures known in
the immunology art, and screening for hybrid cells (hybridomas)
that react specifically with the target protein and have the
desired binding affinity.
[0061] Antibody binding domains also may be produced
biosynthetically and the amino acid sequence of the binding domain
manipulated to enhance binding affinity with a preferred epitope on
the target protein. Specific antibody methodologies are well
understood and described in the literature. A more detailed
description of their preparation can be found, for example, in
Practical Immunology, Butt, W. R., ed., Marcel Dekker, New York,
1984.
[0062] To the extent that the anti-FasL factor is a nucleic acid or
peptidyl nucleic acid, such compounds may be synthesized by any of
the known chemical oligonucleotide and peptidyl nucleic acid
synthesis methodologies known in the art (see, for example,
PCT/EP92/20702 and PCT/US94/013523) and used in antisense therapy.
Anti-sense oligonucleotide and peptidyl nucleic acid sequences,
usually 10 to 100 and more preferably 15 to 50 units in length, are
capable of hybridizing to a gene and/or mRNA transcript and,
therefore, may be used to inhibit transcription and/or translation
of a target protein.
[0063] Fas or FasL gene expression can be inhibited by using
nucleotide sequences complementary to a regulatory region of the
Fas or FasL gene (e.g., the Fas or FasL promoter and/or a enhancer)
to form triple helical structures that prevent transcription of the
Fas or FasL gene in target cells. See generally, Helene (1991)
ANTICANCER DRUG DES. 6(6): 569-84, Helene et al. (1992) ANN. NY
ACAD. SCI. 660: 27-36; and Maher (1992) BIOESSAYS 14(12): 807-15.
The antisense sequences may be modified at a base moiety, sugar
moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility of the molecule. For example, in the
case of nucleotide sequences, phosphodiester linkages may be
replaced by thioester linkages making the resulting molecules more
resistant to nuclease degradation. Alternatively, the deoxyribose
phosphate backbone of the nucleic acid molecules can be modified to
generate peptide nucleic acids (see Hyrup et al. (1996) BIOORG.
MED. CHEM. 4(1): 5-23). Peptidyl nucleic acids have been shown to
hybridize specifically to DNA and RNA under conditions of low ionic
strength. Furthermore, it is appreciated that the peptidyl nucleic
acid sequences, unlike regular nucleic acid sequences, are not
susceptible to nuclease degradation and, therefore, are likely to
have greater longevity in vivo. Furthermore, it has been found that
peptidyl nucleic acid sequences bind complementary single stranded
DNA and RNA strands more strongly than corresponding DNA sequences
(PCT/EP92/20702). Similarly, oligoribonucleotide sequences
generally are more susceptible to enzymatic attack by ribonucleases
than are deoxyribonucleotide sequences, such that
oligodeoxyribonucleotides are likely to have greater longevity than
oligoribonucleotides for in vivo use.
[0064] Additionally, RNAi can serve as an anti-FasL factor. To the
extent RNAi is used, double stranded RNA (dsRNA) having one strand
identical (or substantially identical) to the target mRNA (e.g. Fas
or FasL mRNA) sequence is introduced to a cell. The dsRNA is
cleaved into small interfering RNAs (siRNAs) in the cell, and the
siRNAs interact with the RNA induced silencing complex to degrade
the target mRNA, ultimately destroying production of a desired
protein (e.g., Fas or FasL). Alternatively, the siRNA can be
introduced directly.
[0065] Additionally, aptamers can be used as an anti-FasL factor
and may target Fas or FasL. Methods for identifying suitable
aptamers, for example, via systemic evolution of ligands by
exponential enrichment (SELEX), are known in the art and are
described, for example, in Ruckman et al. (1998) J. Biol. Chem.
273: 20556-20567 and Costantino et al. (1998) J. Pharm. Sci. 87:
1412-1420. Furthermore, to the extent that the anti-FasL factor is
an organic or inorganic compound, such compounds may be
synthesized, extracted and/or purified by standard procedures known
in the art.
[0066] The type and amount of anti-FasL factor to be administered
may depend upon the PDT and cell type to be treated. It is
contemplated, however, that optimal anti-FasL factors, modes of
administration and dosages may be determined empirically. The
anti-FasL factor 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.
[0067] Protein, peptide or nucleic acid based FasL factor
inhibitors can be administered at doses ranging, for example, from
about 0.001 to about 500 mg/kg, more preferably from about 0.01 to
about 250 mg/kg, and most preferably from about 0.1 to about 100
mg/kg. With regard to intravitreal administration, the anti-FasL
factor, for example, anti-FasL neutralizing antibody, typically is
administered periodically as boluses at dosages ranging from about
10 .mu.g to about 5 mg/eye and more preferably from about 100 .mu.g
to about 2 mg/eye.
[0068] The anti-FasL factor preferably is administered to the
mammal prior to PDT, although it may alternatively or additionally
be administered during and/or after PDT. Accordingly, it is
preferable to administer the anti-FasL factor prior to
administration of the photosensitizer. The anti-FasL factor, like
the photosensitizer, may be administered in any one of a wide
variety of ways, for example, orally, parenterally, or rectally.
However, parenteral administration, such as intravenous,
intramuscular, subcutaneous, subtenons, transcleral, and
intravitreal, is preferred. Administration may be provided as a
periodic bolus (for example, intravenously or intravitreally) or as
continuous infusion from an internal reservoir (for example, from a
bioerodable implant disposed at an intra- or extra-ocular location)
or from an external reservoir (for example, from an intravenous
bag). The anti-FasL factor 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 trans-scleral controlled release into the choroid (see,
PCT/US00/00207), or the anti-FasL factor may be administered
systemically. Additionally, the anti-FasL factor can be
administered as an ointment, encapsulated in microspheres or
liposomes, or placed in a device for longer release.
[0069] The present invention, therefore, includes the use of an
anti-FasL factor in the preparation of a medicament for treating,
preferably by a PDT-based method, an ocular condition, that
preferably is associated with choroidal neovasculature. The
anti-FasL factor may be provided in a kit which optionally may
comprise a package insert with instructions for how to treat such a
condition. A composition comprising both a photosensitizer and an
anti-FasL factor may be provided for use in the present invention.
The composition may comprise a pharmaceutically acceptable carrier
or excipient. Thus, the present invention includes a
pharmaceutically acceptable composition comprising a
photosensitizer and an anti-FasL factor, as well as the composition
for use in medicine. More preferably, however, the invention is for
use in combination therapy, whereby an anti-FasL factor and a
photosensitizer are administered separately. Preferably, the
anti-FasL factor is administered prior to administration of the
photosensitizer. Instructions for such administration may be
provided with the anti-FasL factor and/or with the photosensitizer.
If desired, the anti-FasL factor and photosensitizer may be
provided together in a kit, optionally including a package insert
with instructions for use. The anti-FasL factor and photosensitizer
preferably are provided in separate containers. For each
administration, the anti-FasL factor and/or photosensitizer may be
provided in unit-dosage or multiple-dosage form. Preferred dosages
of photosensitizer and anti-FasL factor, however, are as described
above.
[0070] In addition, the efficacy and selectivity of the PDT method
may be enhanced by combining the PDT with an apoptosis-modulating
factor. An apoptosis-modulating factor can be any factor, for
example, a protein (for example a growth factor or antibody),
peptide, nucleic acid (for example, an antisense oligonucleotide),
peptidyl nucleic acid (for example, an antisense molecule), organic
molecule or inorganic molecule, that induces or represses apoptosis
in a particular cell type. For example, it may be advantageous to
prime the apoptotic machinery of CNV endothelial cells with an
inducer of apoptosis prior to PDT so as to increase their
sensitivity to PDT. Endothelial cells primed in this manner are
contemplated to be more susceptible to PDT. This approach may also
reduce the light dose (fluence) required to achieve CNV closure and
thereby decrease the level of damage on surrounding cells such as
RPE. Alternatively, the cells outside the CNV may be primed with an
a repressor of apoptosis so as to decrease their sensitivity to
PDT. In this approach, the PDT at a particular fluence can become
more selective for CNV.
[0071] 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). At the
core of this process lies a conserved set of proenzymes, called
caspases, and two important members of this family are caspases 3
and 7 (Nicholson et al. (1997) TIBS 22:299-306). Monitoring their
activity can be used to assess on-going apoptosis.
[0072] It has been suggested that apoptosis is associated with the
generation of reactive oxygen species, and that the product of the
Bcl-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-2 belongs to a
growing family of apoptosis regulatory gene products, which may
either be death antagonists (Bcl-2, Bcl-xL . . . ) 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).
[0073] The apoptosis-inducing factor preferably is a protein or
peptide capable of inducing apoptosis in cells for example,
endothelial cells, disposed in the CNV. One apoptosis inducing
peptide comprises an amino sequence having, in an N- to C-terminal
direction, KLAKLAKKLAKLAK (SEQ ID NO: 2). This peptide reportedly
is non-toxic outside cells, but becomes toxic when internalized
into targeted cells by disrupting mitochondrial membranes (Ellerby
et al. (1999) supra). This sequence may be coupled, either by means
of a crosslinking agent or a peptide bond, to a targeting domain,
for example, the amino acid sequence known as RGD-4C (Ellerby et
al. (1999) supra) that reportedly can direct the apoptosis-inducing
peptide to endothelial cells. Other apoptosis-inducing factors
include, for example, constatin (Kamphaus et al. (2000) J. Biol.
Chem. 14: 1209-1215), tissue necrosis factor .alpha. (Lucas et al.
(1998) Blood 92: 4730-4741) including bioactive fragments and
analogs thereof, cycloheximide (O'Connor et al. (2000) Am. J.
Pathol. 156: 393-398), tunicamycin (Martinez et al. (2000) Adv.
Exp. Med. Biol. 476: 197-208), and adenosine (Harrington et al.
(2000) Am. J. Physiol. Lung Cell Mol. Physiol. 279: 733-742).
Furthermore, other apoptosis-inducing factors may include, for
example, anti-sense nucleic acid or peptidyl nucleic acid sequences
that reduce or turn off the expression of one or more of the death
antagonists, for example (Bcl-2, Bcl-xL). Antisense nucleotides
directed against Bcl-2 have been shown to reduce the expression of
Bcl-2 protein in certain lines together with increased
phototoxicity and susceptibility to apoptosis during PDT (Zhang et
al. (1999) Photochem. Photobiol. 69: 582-586). Furthermore, an
18mer phosphorothiate oligonucleotide complementary to the first
six codons of the Bcl-2 open reading frame, and known as 03139, is
being tested in humans as a treatment for non-Hodgkins'
lymphoma.
[0074] Apoptosis-repressing factors include, survivin, including
bioactive fragments and analogs thereof (Papapetropoulos et al.
(2000) J. Biol. Chem. 275: 9102-9105), CD39 (Goepfert et al. (2000)
Mol. Med. 6: 591-603), BDNF (Caffe et al. (2001) Invest.
Ophthalmol. Vis. Sci. 42: 275-82), FGF2 (Bryckaert et al. (1999)
Oncogene 18: 7584-7593), Caspase inhibitors (Ekert et al. (1999)
Cell Death Differ 6: 1081-1068) and pigment epithelium-derived
growth factor including bioactive fragments and analogs thereof.
Furthermore, other apoptosis-repressing factors may include, for
example, anti-sense nucleic acid or peptidyl nucleic acid sequences
that reduce or turn off the expression of one or more of the death
agonists, for example (Bax, Bak).
[0075] To the extent that the apoptosis-modulating factor is a
protein or peptide, nucleic acid, peptidyl nucleic acid, or organic
or inorganic compound, it may be synthesized and purified by one or
more the methodologies described relating to the synthesis of the
anti-FasL factor.
[0076] The type and amount of apoptosis-modulating factor to be
administered may depend upon the PDT and cell type to be treated.
It is contemplated, however, that optimal apoptosis-modulating
factors, modes of administration and dosages may be determined
empirically. The apoptosis modulating factor 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.
[0077] Protein, peptide or nucleic acid based apoptosis modulators
can be administered at doses ranging, for example, from about 0.001
to about 500 mg/kg, more preferably from about 0.01 to about 250
mg/kg, and most preferably from about 0.1 to about 100 mg/kg. For
example, nucleic acid-based apoptosis inducers, for example, G318,
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 modulators, for example, antibodies, may be administered
periodically as bolus dosages ranging from about 10 .mu.g to about
5 mg/eye and more preferably from about 100 .mu.g to about 2
mg/eye.
[0078] The apoptosis-modulating factor preferably is administered
to the mammal prior to PDT. Accordingly, it is preferable to
administer the apoptosis-modulating factor prior to administration
of the photosensitizer. The apoptosis-modulating factor, like the
photosensitizer and anti-FasL factor, may be administered in any
one of a wide variety of ways, for example, orally, parenterally,
or rectally. However, parenteral administration, such as
intravenous, intramuscular, subcutaneous, and intravitreal is
preferred. Administration may be provided as a periodic bolus (for
example, intravenously or intravitreally) or by continuous infusion
from an internal reservoir (for example, bioerodable implant
disposed at an intra- or extra-ocular location) or an external
reservoir (for example, and intravenous bag). The apoptosis
modulating factor 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
trans-scleral controlled release into the choroid (see,
PCT/US00/00207).
[0079] The foregoing methods and compositions of the invention are
useful in treating unwanted choroidal neovasculature and thereby
ameliorate the symptoms of ocular disorders including, for example,
AMD, ocular histoplasmosis syndrome, pathologic myopia, angioid
streaks, idiopathic disorders, choroiditis, choroidal rupture,
overlying choroid nevi, and inflammatory diseases, and it is
contemplated that the same methods and compositions may also be
useful in treating other forms of ocular neovasculature. More
specifically, the methods and compositions of the invention may
likewise be useful at treating and removing or reducing corneal
neovasculature, iris neovasculature, retinal neovasculature,
retinal angiomas and choroidal hemangiomas.
[0080] The invention is illustrated further by reference to the
following non-limiting example.
Example 1. Anti-FasL Factor Enhances PDT
[0081] PDT is an effective treatment for CNV but may require
multiple treatments to limit vision loss. Preclinical studies have
demonstrated damage to adjacent retinal structures which may
accumulate with multiple treatments. The neuroprotective properties
of an anti-FasL neutralizing antibody may offer neuroprotection and
improve the effectiveness of PDT. To investigate the efficacy of
PDT in combination with an anti-FasL neutralizing antibody in a
laser injury model of CNV in the rat, the following experiment was
undertaken.
Methods
Induction of Choroidal Neovascularization
[0082] Choroidal neovascular membranes were induced in Brown-Norway
rats using an Argon/dye laser. Briefly, Brown-Norway rats (Charles
River Laboratory, Wilmington, Mass.) were anesthetized via an
intramuscular injection of 50 mg/kg of ketamine hydrochloride and
10 mg/kg of xylazine. Pupils were dilated with a topical
application of 5% phenylephrine and 0.8% tropicamide. Six laser
spots were induced in each eye using an Argon/dye laser (532
Argon/dye laser, Coherent medical laser, Santa Clara, Calif.) by a
single investigator.
Administration of Anti-FasL Antibody In Vivo
[0083] These rats were continuously administered (via subcutaneous
pump) either anti-FasL neutralizing antibody or isotype-matched
control at a total dose of about 5 mg/kg. Briefly, to achieve
steady drug levels in the circulation of animals, the anti-FasL
antibody (anti-rat antibody MFL4, Armenian hamster IgG, Pharmingen,
San Diego, Calif.) or the isotype-matched control antibody
(Armenian hamster anti-TNP IgG, Pharmingen, San Diego, Calif.) was
administered by slow intraperitoneal release from osmotic pumps
(Alzet, Cupertino, Calif.) instead of repeated intraperitoneal
injections. Immediately following the induction of choroidal
neovascularization, osmotic pumps (Alzet, Cupertino, Calif.) were
implanted. Two hundred microliters of each antibody at a
concentration of 5 mg/ml were inserted in each osmotic pump, which
released 0.5 .mu.l/hr for 14 days.
Fluorescein Angiography
[0084] Fluorescein angiography was performed 14 days after the
initial laser treatment using a digital fundus camera system (Model
TRC 501 A, Topcon, Paramus, N.J.) and standard fluorescein filters
to determine if CNV induction was successful. CNV closure on
fluorescein angiograms was assessed 24 hours and seven days after
verterporfin PDT in a masked fashion using grading standards. Each
animal was given a bolus injection of 1 ml of 1% sodium fluorescein
(Akorn Inc, Decatur, Ill.) in saline intraperitoneally, and the
timer was started as soon as the fluorescein bolus was injected.
All angiograms were evaluated in masked fashion by two independent
retina specialists using grading standards and severity of CNV
(baseline FAs) and CNV closure (24 hour FAs).
Photodynamic Therapy in Rats
[0085] PDT with verteporfin was performed 14 days after CNV
induction. Briefly, rats were anesthetized, and verteporfin, at a
dose of 3 mg/m.sup.2, was injected into the tail veins of rats
immobilized in a stereotactic frame. The body surface area of each
rat was determined based on their weight according to a nomogram
developed by Gilpin. One eye of each animal was selected, avoiding
eyes that had large subretinal hemorrhages that the PDT spot could
not cover. Fifteen minutes after the injection, laser light at 689
nm was administered through the pupil with a diode laser (Coherent
medical laser, Santa Clara, Calif.) delivered through a slit lamp
adaptor (Laserlink, Coherent medical laser, Santa Clara, Calif.).
The laser spot size was set at 759 .mu.m on the plane of the
retina. The laser had a constant irradiance of 600 mW/cm.sup.2 and
a fluence of 25 J/cm.sup.2 which was delivered for 17 or 42 seconds
to achieve total energy doses of 10 J/cm.sup.2.
Western Blotting
[0086] The levels of protein expression of caspase 3 and caspase 8,
Bax, Bid, Bcl-2, and cytochrome c (Pharmacia) were evaluated by
Western blotting. Briefly, whole retinae were lysed for 30 minutes
on ice in lysis buffer (50 mM Tris-HCl, pH 8, with 120 mM NaCl and
1% NP-40) supplemented with the Complete-mini mixture of proteinase
inhibitors. The samples were cleared by micro-centrifugation
(14,000 rpm, 30 minutes, 4.degree. C.) and assessed for protein
concentration. Thirty micrograms of protein/sample were
electrophoresed in a 12% sodium dodecyl sulphate
(SDS)-polyacrylamide gel (SDS-PAGE) and electroblotted onto
nitrocellulose membranes. After a one hour incubation in blocking
solution (20% IgG-free normal horse serum in phosphate-buffered
saline (PBS)), the membranes were exposed overnight at 4.degree. C.
to the respective primary antibody. Following washing in PBS, the
respective secondary peroxidase-labeled antibody was applied at a
1:10,000 dilution for one hour at room temperature. The proteins
were visualized with the enhanced chemiluminescence technique
(Amersham Pharmacia Biotech, Piscataway, N.J.).
Enzyme Activity Assays for Caspase 3 and 8
[0087] The enzymatic activity of caspases 3 and 8 were detected in
retinal lysates with the Apo Alert kit (Clontech, Palo Alto,
Calif.).
Tunel Staining
[0088] Apoptotic cells were analyzed using the TUNEL technique.
Briefly, free 3'OH DNA termini were labeled using the TUNEL
procedure according to the manufacturer's recommendations
(Intergen, NY). TUNEL was performed with horseradish peroxidase
detection in sections from formalin-fixed, paraffin embedded
retinas. Whole eyes from rats with or without laser-induced CNV
treated with the anti-FasL antibody or the isotype-matched control
antibody were fixed in 4% paraformaldehyde overnight at 4.degree.
C. Then, TUNEL staining was performed.
Statistics
[0089] Differences in CNV induction between treatment groups were
evaluated using chi-square tests. Lesions that did not show
significant leakage were excluded from the statistical analysis.
Retinal levels of Bcl-2, Bax, Bcl-xL, and Bid were measured by
Western blotting, and cystolic and mitochondrial levels of
cytochrome c were measured by a modified ELISA method. Activation
of caspases-3 and -6 were measured with a modified ELISA method in
whole retinal lysates.
Results
Anti-FasL Antibody Treatment Reduces Angiographic Leakage in
Laser-Induced CNV
[0090] To assess whether the anti-FasL antibody treatment
influences the angiographic leakage in the laser-induced CNV model,
the percentage of lesions that were closed in rats that received
the anti-FasL antibody treatment was compared to the percentage
from those that received the control antibody treatment. The
percentage of angiographically "leaky" lesions (grade IIA+IIB)
among the anti-FasL antibody treated rats was 82% and was 97.4% in
the control antibody treated rats (P<0.0001). Whereas, the
percentage of the angiographically non-leaky and less leaky lesions
(grade 1+0) was 19% in rats that received the anti-FasL antibody
treatment and 2.6% in the control antibody treated rats (FIG. 6).
Thus, FIG. 6 provides evidence that anti-FasL antibody treatment
reduces angiographic leakage in laser-induced CNV.
Anti-FasL Antibody Treatment Reduces PDT-Induced Angiographic
Leakage in Laser-Induced CNV
[0091] To assess whether the anti-FasL treatment influences the
angiographic leakage after PDT in the laser induced CNV model, the
percentage of lesions that were closed in rats that received PDT
and the anti-FasL antibody treatment was compared to the percentage
from those that received the control treatment. Because the
anti-FasL antibody treatment reduced the angiographic leakage in a
statistically significant manner relative to the control antibody,
lesions on which to perform PDT were selected that had an equal
degree of angiographic leakage in the two antibody-treated
populations. The percentage of closed lesions among the anti-FasL
antibody and PDT treated rats was 100% and was 69% in the control
and PDT treated rats (FIG. 5). Thus, FIG. 5 provides evidence that
anti-FasL antibody treatment reduces angiographic leakage after
PDT. Because the anti-FasL antibody had a marginal effect on
non-leaky lesions as shown by the comparison between laser-induced
CNV animals receiving anti-FasL antibody treatment and control
antibody treatment (19% vs. 2.6%, FIG. 6), the fact that 100% of
the animals receiving combined anti-FasL antibody and PDT treatment
showed closed lesions in comparison to 69% of the animals receiving
combined control antibody and PDT treatment indicates that the
combination produces more than an additive effect and is
synergistic.
PDT Increases the Expression of the Fas Receptor in the Rat
Retina
[0092] It is understood that treatment with the photosensitizer
verteporfin increases the apoptotic cell death both in vivo and in
vitro. It was suggested that PDT acts in concert with the Fas
apoptotic signaling pathway, because a Fas-activating antibody can
potentiate the PDT-induced cell death of thymic cells in vivo. This
experiment indicated that verteporfin PDT increases the retinal
levels of the apoptotic death receptor, Fas, an indication that the
Fas/FasL pathway plays a causative role in the apoptotic retinal
cell death after PDT (FIG. 3). Thus, FIG. 3 provides evidence that
PDT increases the expression of the Fas receptor in the rat
retina.
Anti-FasL Antibody Treatment Reduces PDT-Induced Apoptosis in
Laser-Induced CNV
[0093] To investigate the role of Fas/FasL in verteporfin-PDT, the
occurrence of apoptotic cell death in the retinas of rats that had
received, concurrent with PDT, the anti-FasL antibody treatment or
the control antibody was studied. It was found that rats that had
received the control antibody treatment plus PDT showed apoptotic
cells in the RPE, photoreceptor and endothelial cell layer, whereas
the anti-FasL antibody treatment significantly reduced this
apoptotic death from PDT. Rats that received only the anti-FasL
antibody or control antibody showed a minimal amount of apoptotic
death.
Anti-FasL Antibody Treatment Prevents PDT-Induced Activation of
Caspases 3 and 8 in Laser-Induced CNV
[0094] Fas/FasL mediated apoptotic cell death involves the
activation of apical (receptor mediated) and executional caspases.
Rats treated with PDT and control antibody showed cleavage of the
proform of caspases 3 and 8 to their respective activated
fragments. The immunoblotting results were confirmed with caspase
activity assays. The anti-FasL antibody inhibited the PDT-induced
activation of caspases (FIGS. 4 and 7). Thus, FIG. 4 provides
evidence that anti-FasL antibody treatment prevents activation of
caspase 3 after PDT, and FIG. 7 provides evidence that anti-FasL
antibody treatment reduces PDT-induced caspase 8 activation in
laser-induced CNV.
Anti-FasL Antibody Treatment Reduces PDT-Induced Bax Upregulation
and Bcl-2 Downregulation in Laser-Induced CNV
[0095] Apoptosis and cell survival is the outcome of a delicate
balance between anti-apoptotic genes, such as Bcl-2, and
pro-apoptotic genes, such as Bax, which were shown to influence
Fas-mediated apoptosis in a variety of models. In the present
animal model, PDT-induced apoptosis is associated with
downregulation of Bcl-2 protein levels and upregulation of Bax
protein levels. Treatment with the anti-FasL antibody, but not the
control antibody, attenuates the PDT-induced downregulation of
Bcl-2 level and upregulation of Bax level (FIGS. 9 and 10). Thus,
FIG. 9 provides evidence that anti-FasL antibody treatment reduces
PDT-induced Bax upregulation in laser-induced CNV, and FIG. 10
provides evidence that anti-FasL antibody treatment reduces
PDT-induced Bcl-2 downregulation in laser-induced CNV.
Anti-FasL Antibody Treatment Prevents PDT-Induced Cleavage of Bid
in Laser-Induced CNV
[0096] In the present animal model, PDT-induced apoptosis is
associated with cleavage of Bid. Treatment with the anti-FasL
antibody, but not the control antibody, attenuates the PDT-induced
cleavage of Bid (FIG. 2). Thus, FIG. 2 provides evidence that
anti-FasL antibody treatment prevents cleavage of Bid after
PDT.
Anti-FasL Antibody Treatment Reduces PDT-Induced Cytochrome c
Release in Laser-Induced CNV
[0097] In the present animal model, PDT-induced apoptosis is
associated with release of mitochondrial cytochrome c into the
cytoplasm. Treatment with the anti-FasL antibody, but not the
control antibody, attenuates the PDT-induced release of
mitochondrial cytochrome c into the cytoplasm (FIG. 8). Thus, FIG.
8 provides evidence that anti-FasL antibody treatment reduces
PDT-induced cytochrome c release in laser-induced CNV.
Discussion
[0098] In the present study, the efficacy of PDT in combination
with a FasL neutralizing antibody was investigated. It was found
that continuous subcutaneous administration of the anti-FasL
antibody, but not the isotype matched control antibody, reduced the
angiographic leakage from CNV and increased the efficacy of
verteporfin PDT on CNV closure.
[0099] These findings suggest that apoptotic mechanisms may
participate in retinal cell loss during PDT. The involvement of the
Fas/FasL receptor/ligand pair and the Bcl-2 family members were
also investigated in this model. The Bcl-2 family includes several
anti-apoptotic members, such as Bcl-2 and Bcl-xL, whereas Bax and
the cleaved form of Bid promote apoptosis. The balance between pro-
and anti-apoptotic members of the Bcl-2 family regulates the fate
of mitochondrial cytochrome c. When the pro-apoptotic stimuli
predominate, cytochrome c moves from the mitochondria to the
cytoplasm, where, together with Apaf1, it activates pro-caspase-9.
The active form of caspase-9 then activates the executioner
caspases-3 and -6. PDT decreased the retinal levels of Bcl-2 and
Bcl-xL, increased the levels of Bax, and induced Bid cleavage. The
release of cytochrome c to the cytosol and activation of caspases-3
and -6 also were detected.
[0100] Treatment with the anti-FasL neutralizing antibody, but not
the isotype matched control antibody, reversed the above-mentioned
changes. This suggests a role for the Fas/FasL pathway in
triggering this apoptotic cascade. The Fas receptor can activate
caspase-9 and, subsequently, caspases-3 and -6, via two pathways.
The first pathway is activation of caspase-8, which can directly
cleave and activate caspase-9 (type I pathway). The second pathway
is, when the level of caspase-8 activation is insufficient to
cleave caspase-9 due to low endogenous pro-caspase-8 expression or
due to the presence of caspase-8 inhibitors such as FLIP,
amplification of the apoptotic signal of caspase-8 via the
mitochondria (type II pathway). Specifically, caspase-8 cleaves Bid
into its active, pro-apoptotic form. This then triggers cytochrome
c release from the mitochondria and the above-mentioned activation
of caspase-9 and the downstream executioner caspases (See FIG. 1).
The ability of a Fas/FasL neutralizing agent to inhibit the
downstream apoptotic signaling pathway in this study confirms its
role in triggering retinal cell apoptosis in PDT.
[0101] The ability of a Fas/FasL inhibitor to suppress retinal cell
apoptosis in PDT-treated retinae may also have therapeutic
implications. Concurrent treatment with a Fas/FasL neutralizing
agent, such as an anti-Fas or anti-FasL antibody, or possibly with
a caspase inhibitor, may limit the damage to adjacent normal
structures that occurs during PDT. This may allow more intensive
PDT treatments, thus potentially improving PDT results. Due to the
involvement of the Fas apoptotic pathway in several other disease
models, such as hepatitis, there is strong interest in drug
development of Fas/FasL inhibitors that would be effective and safe
for human use.
[0102] In conclusion, continuous subcutaneous administration of the
anti-FasL neutralizing antibody, and not the isotype matched
control antibody, reduced the angiographic leakage from CNV (97.4%
of the lesions in the animals treated with the control antibody
were stage IIA and IIB versus 82% in the anti-FasL antibody treated
group, P<0.001) and increased the efficacy of verteporfin PDT on
CNV closure (69% of the lesions in the animals treated with the
control antibody and PDT were angiographically not perfused versus
100% of lesions treated with anti-FasL antibody and PDT). PDT
decreased the retinal levels of Bcl-2 and Bcl-xL, increased the
levels of Bax, and induced cleavage of Bid and release of
cytochrome c into the cytosol. PDT also induced activation of
caspases-3 and -6 in the retina. Treatment with the anti-FasL
neutralizing antibody, but not the isotype-matched control,
reversed these changes.
[0103] Anti-FasL antibody administration decreased the angiographic
leakage, increased the efficacy of Verteporfin PDT for CNV closure
in a rat model, and reduced the collateral apoptotic damage induced
by PDT. This suggests that the combination of PDT with anti-FasL
neutralizing agents (i.e., anti-FasL factors) may limit damage to
normal structures and improve PDT results.
EQUIVALENTS
[0104] 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.
INCORPORATION BY REFERENCE
[0105] The entire disclosure of each of the patent documents and
scientific publications disclosed hereinabove is expressly
incorporated herein by reference for all purposes.
Sequence CWU 1
1
2110PRTArtificial SequenceSynthetic sequence 1Ala Cys Asp Cys Arg
Gly Asp Cys Phe Cys1 5 10214PRTArtificial SequenceSynthetic
sequence 2Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys1
5 10
* * * * *