U.S. patent application number 10/429428 was filed with the patent office on 2003-10-02 for methods and compositions for treating conditions of the eye.
Invention is credited to Gragoudas, Evangelos S., Miller, Joan W., Renno, Reem Z..
Application Number | 20030185834 10/429428 |
Document ID | / |
Family ID | 22665146 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030185834 |
Kind Code |
A1 |
Miller, Joan W. ; et
al. |
October 2, 2003 |
Methods and compositions for treating conditions of the eye
Abstract
Provided are methods and compositions for the photodynamic
therapy (PDT) of ocular conditions characterized by the presence of
unwanted choroidal neovasculature, for example, neovascular
age-related macular degeneration. The selectivity and sensitivity
of the PDT method can be enhanced by combining the PDT with an
anti-angiogenesis factor, for example, angiostatin or endostatin,
or with an apoptosis-modulating factor. Furthermore, the
selectivity and sensitivity of the PDT may be further enhanced by
coupling a targeting moiety to the photosensitizer so as to target
the photosensitizer to choroidal neovasculature.
Inventors: |
Miller, Joan W.;
(Winchester, MA) ; Gragoudas, Evangelos S.;
(Lexington, MA) ; Renno, Reem Z.; (Boston,
MA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
22665146 |
Appl. No.: |
10/429428 |
Filed: |
May 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10429428 |
May 5, 2003 |
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09780142 |
Feb 9, 2001 |
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60181641 |
Feb 10, 2000 |
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Current U.S.
Class: |
424/178.1 ;
514/185; 514/410; 604/20 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 47/64 20170801; A61K 38/484 20130101;
A61P 27/02 20180101; A61K 41/0057 20130101; A61K 45/06 20130101;
A61K 38/10 20130101; A61K 38/10 20130101; A61K 38/484 20130101;
A61P 27/06 20180101; A61K 47/6849 20170801; A61K 41/0071 20130101;
A61K 41/0076 20130101; A61N 5/062 20130101; A61P 43/00
20180101 |
Class at
Publication: |
424/178.1 ;
514/185; 514/410; 604/20 |
International
Class: |
A61K 039/395; A61N
001/30; A61K 031/555; A61K 031/409 |
Claims
What is claimed is:
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-angiogenesis 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,
wherein damage to the endothelial cells resulting from steps (a),
(b), and (c) is greater than that resulting only from steps (b) and
(c).
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-angiogenesis 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 derivatives, 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-angiogenesis factor is
angiostatin, endostatin, a peptide containing a RGD tripeptide
sequence and capable of binding the .alpha.vB3 integrin, a COX-2
inhibitor or pigment epithelium derived growth factor.
8. The method of claim 1, wherein occlusion of the choroidal
neovasculature resulting from steps (a), (b) and (c) is greater
than that resulting from steps (b) and (c) alone.
9. The method of claim 1, wherein the method more selectively
occludes choroidal neovasculature relative to the same treatment
lacking administration of the anti-angiogenesis factor.
10. 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 amount of photosensitizer to permit an effective amount to
localize in the neovasculature, the photosensitizer comprising a
targeting moiety that binds preferentially to endothelial cells of
the neovasculature; and (b) irradiating the neovasculature with a
laser light such that the light is absorbed by the photosensitizer
so as to occlude the choroidal neovasculature.
11. The method of claim 10, wherein the mammal is a primate.
12. The method of claim 11, wherein the mammal is a human.
13. The method of claim 10, wherein the targeting moiety is a
peptide.
14. The method of claim 13, wherein the peptide binds specifically
to an .alpha.-v .beta. integrin or is an antibody that binds
specifically to a vascular endothelial growth factor receptor.
15. The method of claim 14, wherein the integrin is .alpha.-v
.beta.3 integrin or .alpha.-v .beta.5 integrin.
16. The method of claim 14, wherein the antibody is a monoclonal
antibody or an antigen binding fragment thereof, a polyclonal
antibody or an antigen binding fragment thereof, or a biosynthetic
antibody binding site.
17. The method of claim 10, wherein the photosensitizer is an amino
acid derivatives, an azo dye, a xanthene derivative, a chlorin, a
tetrapyrrole derivative, and a phthalocyanine.
18. The method of claim 17, wherein the photosensitizer is lutetium
texaphryin, a benzoporphyrin, a benzoporphyrin derivative, a
hematoporphyrin or a hematoporphyrin derivative.
19. The method of claim 10, wherein the targeting moiety enhances
the specificity of the photosensitizer to choroidal neovasculature
relative to photosensitizer lacking the targeting moiety.
20. A method of treating unwanted choroidal neovasculature in a
mammal, the method comprising the steps of: (a) administering to
the mammal, an apoptosis-modulating factor in an amount sufficient
to permit an effective amount to localize in the choroidal
neovasculature or tissue surrounding the choroidal neovasculature;
(b) administering to the mammal an amount of photosensitizer
sufficient to permit an effective amount of 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,
wherein the level of cell damage to the choroidal neovasculature
relative to the tissue surrounding the choroidal neovasculature
resulting from steps (a), (b) and (c) is greater than that
resulting from steps (b) and (c) alone.
21. The method of claim 20, wherein the mammal is a primate.
22. The method of claim 21, wherein the mammal is a human.
23. The method of claim 20, wherein the factor is administered to
the primate before administration of the photosensitizer.
24. The method of claim 20, wherein the photosensitizer is an amino
acid derivatives, an azo dye, a xanthene derivative, a chlorin, a
tetrapyrrole derivative, or a phthalocyanine.
25. The method of claim 20, wherein the photosensitizer is lutetium
texaphyrin, a benzoporphyrin, a benzoporphyrin derivative, a
hematoporphyrin or a hematoporphyrin derivative.
26. The method of claim 20, wherein the apoptosis modulating factor
induces or represses apoptosis.
27. The method of claim 26, wherein the factor is a peptide.
28. The method of claim 27, wherein the peptide selectively binds
to neovasculature.
29. The method of claim 27, wherein the peptide induces apoptosis
in endothelial cells.
30. The method of claim 29, wherein the peptide comprises an amino
acid sequence comprising, in an N- to C-terminal direction,
KLAKLAKKLAKLAK (SEQ. ID. NO 1).
31. The method of claim 30, wherein the peptide further comprises
an RGD-4C peptide sequence.
32. The method of claim 1, wherein the method ameliorates the
symptoms of a disorder selected from the group consisting of
age-related macular degeneration, ocular histoplasmosis syndrome,
pathologic myopia, angioid streaks, idiopathic disorders,
choroiditis, choroidal rupture, overlying choroid nevi, and
inflammatory diseases.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Serial No. 60/181,641, filed Feb. 10, 2000, the
disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to photodynamic
therapy-based 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 there is no treatment for dry AMD. Until recently,
laser photocoagulation has been the only therapy available for
selected cases of neovascular AMD. Unfortunately, the majority of
patients with neovascular AMD do not meet the criteria for laser
photocoagulation therapy. Approximately 85% of patients with
neovascular AMD have poorly defined, occult, or relatively
extensive subfoveal choroidal neovascularization, none of which is
amenable to laser therapy. In addition, laser photocoagulation
relies on thermal damage to the CNV tissue, which damages the
overlying neurosensory retina with consequent loss of visual
function. Laser photocoagulation also is plagued by recurrences
that occur in approximately 50% of cases.
[0006] Photodynamic therapy (PDT) has shown promising results as a
new treatment for removing unwanted CNV and for treating
neovascular AMD (Miller et al. (1999) ARCHIVES OF OPHTHALMOLOGY
117: 1161-1173, Schmidt-Erfurth et al. (1999) ARCHIVES OF
OPHTHALMOLOGY 117:1177-1187, TAP Study Group (1999) ARCHIVES OF
OPHTHALMOLOGY 117: 1329-45, Husain et al. (1999) PHILADELPHIA:
MOSBY; 297-307). PDT involves the systemic administration of a
photosensitizer or PDT dye (photosensitizer) that accumulates in
proliferating tissues such as tumors and newly formed blood
vessels; followed by irradiation of the target tissue with
low-intensity, non-thermal light at a wavelength corresponding to
the absorption peak of the dye (Oleinick et al. (1998) RADIATION
RESEARCH: 150: S146-S156). Excitation of the dye leads to the
formation of singlet oxygen and free radicals-better known as
reactive oxygen species which cause photochemical damage to the
target tissue (Weishaupt et al. (1976) CANCER RES. 36:
2326-2329).
[0007] Studies using PDT for the treatment of CNV have demonstrated
that, with the proper treatment parameters of photosensitizer dose,
laser light dose, and timing of irradiation, relative selective
damage to experimental CNV can be achieved, sparing retinal
vessels, large choroidal vessels, and with minimal changes in the
neurosensory retina (Husain et al. (1996) ARCH OPHTALMOL. 114:
978-985, Husain et al. (1997) SEMINARS IN OPHTHALMOLOGY 12: 14-25,
Miller et al. (1995) ARCH OPHTHALMOL. 113: 810-818, Kramer et al.
(1996) OPHTHALMOLOGY 103(3): 427-438). Moreover, a PDT-based
procedure using a green porphyrin dye recently has been approved in
a variety of countries for use in the treatment of neovascular
AMD.
[0008] 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). Several
multicenter Phase 3 trials are underway to study repeated PDT
treatments, applied every three months. The interim data look
promising in terms of decreased rates of moderate vision loss (TAP
Study Group (1999) ARCHIVES OF OPHTHALMOLOGY 117: 1329-45). 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.
[0009] Therefore, there is still a need for improved PDT-based
methods that increase the efficacy and selectivity of treatment,
and which reduce or delay a recurrence of the disorder.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to PDT-based 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 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.
[0011] 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-angiogenesis
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 damage to
endothelial cells disposed within the choroidal neovasculature is
greater than the damage experienced by endothelial cells in a
similar treatment lacking administration of the anti-angiogenesis
factor. Furthermore, the anti-angiogenesis factor can potentiate
the cytotoxicity of PDT. For example, the anti-angiogenesis factor
and the PDT may act synergistically to selectively kill capillary
endothelial cells, while at the same time sparing retinal cells,
for example, retinal pigment epithelial cells and cells disposed in
the neurosensory retina, for example, photoreceptor cells and
Mueller cells.
[0012] The anti-angiogenesis factor can enhance the selectivity of
the PDT by, for example, occluding the CNV while at the same
sparing surrounding blood vessels, for example, normal choroidal
and retinal vasculature, and/or tissue, for example, the overlying
neurosensory retina. Accordingly, inclusion of the
anti-angiogenesis factor makes the PDT method more selective for
capillary endothelial cells. Furthermore, practice of the invention
can slow down or delay the recurrence of choroidal
neovasculature.
[0013] A variety of anti-angiogenesis factors may be used in the
invention. Useful anti-angiogenesis factors, include, for example:
angiostatin; endostatin; a peptide containing a RGD tripeptide
sequence and capable of binding the .alpha.v.beta. integrin; a
COX-2 selective inhibitor; halofuginone; anecotave acetate;
antibodies and other peptides that bind vascular endothelial growth
factor receptor; antibodies, other peptides, and nucleic acids that
bind vascular endothelial growth factor to prevent or reduce its
binding to its cognate receptor; tyrosine kinase inhibitors;
thrombospondin-1; anti-epidermal growth factor; hepatocyte growth
factor; thromboxane; and pigment endothelial-derived growth factor.
Preferred anti-angiogenic factors include angiostatin, endostatin
and pigment epithelium-derived growth factor.
[0014] The anti-angiogenesis factor may, under certain
circumstances, be co-administered simultaneously with the
photosensitizer. In a preferred embodiment, however, the
anti-angiogenesis factor is administered to the mammal prior to
administration of the photosensitizer.
[0015] In another aspect, the invention provides a method of
treating unwanted CNV in a mammal. The method comprises the steps
of: (a) administering to a mammal, for example, a primate,
preferably, a human, an amount of a photosensitizer to permit an
effective amount to localize in the CNV, the photosensitizer
comprising a targeting moiety that binds preferentially to cell
surface ligands disposed on endothelial cells, for example,
capillary endothelial cells, present in the CNV; and (b)
irradiating the CNV with laser light such that the light is
absorbed by the photosensitizer so as to occlude the CNV. The
targeting moieties bind preferentially to CNV and, therefore, can
increase the effective concentration of photosensitizer in the CNV
relative to surrounding cells and tissues. Accordingly, such a
method increases the selectivity of the PDT method for CNV while
sparing surrounding retinal and large choroidal blood vessels and
overlying neurosensory retina.
[0016] The targeting moiety can be any molecule, for example, a
protein, peptide, nucleic acid, peptidyl-nucleic acid, organic
molecule or inorganic molecule that has an affinity for endothelial
cells within CNV. However, targeting proteins and peptides are
preferred. For example, the targeting peptide can be a peptide that
targets .alpha.v.beta. integrin, for example, .alpha.v.beta. 3
integrin or .alpha.v.beta. 5 integrin. Alternatively, the targeting
peptide can be an antibody, for example, a monoclonal antibody or
an antigen binding fragment thereof, a polyclonal antibody or an
antigen binding fragment thereof, or a biosynthetic antibody
binding site that binds preferentially to a cell surface ligand
disposed at elevated concentrations or densities in CNV. By way of
example, the targeting moiety may be an antibody that binds
specifically to the vascular endothelial growth factor
receptor.
[0017] In another aspect, the invention provides a method of
treating unwanted CNV in a mammal. The method comprises the steps
of: (a) administering to the mammal, for example, a primate, and
more preferably, a human, an apoptosis-modulating factor in an
amount sufficient to permit an effective amount to localize in the
CNV or tissue surrounding the CNV; (b) administering to the mammal
an amount of photosensitizer sufficient to permit an effective
amount of 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. Cytotoxicity of the PDT can be enhanced
and/or made more specific for CNV relative to a similar treatment
lacking the apoptosis-modulating factor.
[0018] The apoptosis-modulating factor may be any molecule, for
example, a protein, peptide, nucleic acid, peptidyl-nucleic acid,
organic molecule or inorganic molecule, that enhances or stimulates
apoptosis in cells or tissues of the CNV or that represses
apoptosis in cells or tissues surrounding the CNV. In a preferred
embodiment, the apoptosis-modulating factor is a peptide capable of
inducing apoptosis in cells, for example, endothelial cells,
present in CNV. The peptide may comprise, for example, an amino
sequence comprising, in an N- to C-terminal direction,
KLAKLAKKLAKLAK (SEQ ID NO: 1) which is designed to be non-toxic
outside cells, but which is toxic when internalized into target
cells because it disrupts mitochondrial membranes. Furthermore,
this peptide may be targeted towards endothelial cells by inclusion
of a targeting amino acid sequence, for example, in an N- to
C-terminal direction, ACDCRGDCFC (SEQ ID NO: 2), also known as
RGD4C.
[0019] The apoptosis-modulating factor may be co-administered
simultaneously with the photosensitizer. However, in a preferred
embodiment, the apoptosis-modulating factor is administered to the
primate before administration of the photosensitizer and/or
irradiation.
[0020] In all the foregoing methods, it is contemplated that any
photosensitizer useful in PDT may be useful in the practice of the
invention. Preferred photosensitizers include, for example, amino
acid derivatives, azo dyes, xanthene derivatives, chlorins,
tetrapyrrole derivatives, phthalocyanines, and assorted other
photosensitizers. However, preferred photosensitizers, include, for
example, lutetium texaphyrin, benzoporphyrin and derivatives
thereof, and hematoporphyrin and derivatives thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] 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:
[0022] FIGS. 1A and 1B are bar charts showing the in vitro survival
of bovine retinal capillary endothelial (BRCE) cells (FIG. 1A) and
retinal pigment epithelial (RPE) cells (FIG. 1B) upon exposure to
Lutetium Texaphyrin (Lu-Tex)/PDT in the presence or absence of
angiostatin. Cells were plated and exposed to angiostatin 18 hours
before Lu-Tex/PDT. The surviving fraction was measured using a
1-week proliferation assay. Data represent the mean of triplicate
experiments.+-.SD;
[0023] FIGS. 2A-2C are graphs showing the kinetics of Caspase
3-like activation following Lu-Tex/PDT in BRCE (diamonds) and RPE
(squares). BRCE and RPE cells were exposed to Lu-Tex/PDT at
fluences of 10 J/cm.sup.2 (FIG. 2A), 20 J/cm.sup.2 (FIG. 2B) and 40
J/cm.sup.2 (FIG. 2C). At the indicated times thereafter, cells were
collected and lysed. Aliquots (50 .mu.g of protein) were incubated
with Ac-DEVD-AFC at 37.degree. C. for 30 min. The amount of
fluorochrome released was determined by comparison to a standard
curve in lysis buffer and the data represent the mean of three
independent experiments; and
[0024] FIG. 3 is a graph showing Caspase 3-like activity in BRCE
following Angiostatin/Lu-Tex/PDT versus Lu-Tex/PDT alone. BRCE were
exposed to angiostatin (500 ng/ml) alone (diamonds), Lu-Tex/PDT (20
J/cm.sup.2 (squares), 40 J/cm.sup.2 (crosses)) alone and
combination of angiostatin/Lu-Tex/PDT (triangles). At the indicated
times thereafter, cells were collected and lysed. Aliquots (50%g of
protein) were incubated with Ac-DEVD-AFC at 37.degree. C. for 30
min. The amount of fluorochrome released was determined by
comparison to a standard curve in lysis buffer and the data
represent the means of three independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention relates to an improved PDT-based 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.
[0026] The method of the invention relates to a PDT-based method of
treating unwanted target CNV. The method requires 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 aggregration of blood cells and vascular
occlusion.
[0027] An increase in efficacy and/or selectivity of the PDT,
and/or reduction or delay of recurrence of the CNV can be achieved
by (i) administering an anti-angiogenic factor to the mammal prior
to or concurrent with administration of the photosensitizer, (ii)
using a photosensitizer with a targeting molecule that targets the
photosensitizer to the CNV, (iii) administering an
apoptosis-modulating factor to the mammal prior to or concurrent
with administration of the photosensitizer, (iv) a combination of
any two of the foregoing, for example, a combination of the
anti-angiogenesis factor and the targeted photosensitizer, a
combination of the anti-angiogenesis factor and the apoptosis
modulating agent, or a combination of the targeted photosenitizer
and the apoptosis modulating agent, or (v) a combination of all
three of the foregoing.
[0028] 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 photo sensitizers.
[0029] 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 1, Sudan II, Sudan III, Sudan
IV, Sudan Black, Disperse Orange, Disperse Red, Oil Red O, Trypan
Blue, Congo Red, P-carotene (Mosky et al. (1984) EXP. RES. 155,
389-396). Xanthene derivatives, include, for example, rose
bengal.
[0030] 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).
[0031] 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-hy- droxyiminoethyliden
e-2,7,12,18-tetranethyl 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).
[0032] Phthalocyanines include, for example, chloroaluminum
phthalocyanine (AlPcCl) (Rerko et al. (1992) PHOTOCHEM. PHOTOBIOL.
55, 75-80), aluminum phthalocyanine with 2-4 sulfonate groups
(AlPcS.sub.24) (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
(Mg.sup.2+-PC) (Jori et al. (1990) supra), zinc phthalocyanine
ZnPC) (Berg et al. (1997) supra). Other photosensitizers include,
for example, thionin, toluidine blue, neutral red and azure c.
[0033] However, preferred photosensitizers, include, for example,
Lutetium Texaphyrin (Lu-Tex), a new generation photosensitizer
currently in clinical trial for CNV because of its favorable
clinical properties including absorption at about 730 nm permitting
deep tissue penetration and rapid clearance which is available from
Alcon Laboratories, Fort Worth, Tex. Other preferred
photosensitizers, include benzoporhyrin and benzoporphyrin
derivatives, for example, BPD-MA and BPD-DA, available from QLT
Phototherapeutics, Inc., Vancouver, Canada.
[0034] 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.
[0035] In one preferred 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 photosenstizer 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
considerably 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 Phototherapeutics, 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.
[0036] Furthermore, the photosensitizer can be coupled 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.
[0037] 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.
[0038] Several targeting molecules may be used to target
photosensitizers to the neovascular endothelium. For example,
.alpha.-v integrins, in particular .alpha.-v .beta.3 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 a-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: 2)--also know as
RGD-4C--has been identified that selectively binds to human a-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)
[0039] Another potential targeting molecule is an antibody for
vascular endothelial growth factor receptor (VEGF-2R). Clinical and
experimental evidence strongly supports a role for 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). Antibodies to
the VEGF receptor (VEGFR-2 also known as KDR) may also bind
preferentially to neovascular endothelium. As used herein, the term
"antibody" 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, and
more preferably 10.sup.7 M.sup.-1.
[0040] 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 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] It is contemplated that a variety of anti-angiogenic factors
may be combined with PDT to treat unwanted CNV. The
anti-angiogenesis factor can potentiate the cytotoxity of the PDT
thereby enhancing occlusion of the choroidal neovasculature. In
addition, the anti-angiogenesis factor can enhance the selectivity
of PDT, for example, by occluding the CNV while at the same sparing
the surrounding blood vessels, for example, the retinal and large
choroidal blood vessels and/or surrounding tissue, for example, the
retinal epithelium. Furthermore, the anti-angiogenesis factor can
be used to reduce or delay the recurrence of the condition.
[0052] The term "anti-angiogensis factor" is understood to mean any
molecule, for example, a protein, peptide, nucleic acid (ribose
nucleic acid (RNA) or deoxyribose nucleic acid (DNA)), peptidyl
nucleic acid, organic compound or inorganic compound, that reduces
or inhibits the formation of new blood vessels in a mammal. It is
contemplated that useful angiogenesis inhibitors, if not already
known, may be identified using a variety of assays well known and
used in the art. Such assays include, for example, the bovine
capillary endothelial cell proliferation assay, the chick
chorioallantoic membrane (CAM) assay or the mouse corneal assay.
However, the CAM assay is preferred (see, for example, O'Reilly et
al. (1994) CELL 79: 315-328 and O'Reilly et al. (1997) CELL 88:
277-285). Briefly, embryos with intact yolks are removed from
fertilized three day old white eggs and placed in a petri dish.
After incubation at 37.degree. C., 3% CO.sub.2 for three days, a
methylcellulose disk containing the putative angiogenesis inhibitor
is applied to the chorioallantoic membrane of an individual embryo.
After incubation for about 48 hours, the chorioallantoic membranes
are observed under a microscope for evidence of zones of
inhibition.
[0053] Numerous anti-angiogenesis factors are well known and
thoroughly documented in the art (see, for example,
PCT/US99/08335). Examples of anti-angiogenesis factors useful in
the practice of the invention, include, for example,
protein/peptide inhibitors of angiogenesis such as: angiostatin, a
proteolytic fragment of plasminogen (O'Reilly et al. (1994) CELL
79: 315-328, and U.S. Pat. Nos. 5,733,876; 5,837,682; and
5,885,795) including full length amino acid sequences of
angiostatin, bioactive fragments thereof, and analogs thereof;
endostatin, a proteolytic fragment of collagen XVIII (O'Reilly et
al. (1997) CELL 88: 277-285, Cirri et al. (1999) INT. BIOL. MARKER
14: 263-267, and U.S. Pat. No. 5,854,205) including full length
amino acid sequences of endostatin, bioactive fragments thereof,
and analogs thereof; peptides containing the RGD tripeptide
sequence and capable of binding the .alpha..sub.v.beta..sub.3
integrin (Brooks et al. (1994) CELL 79: 1157-1164, Brooks et al.
(1994) SCIENCE 264: 569-571); certain antibodies and antigen
binding fragments thereof and peptides that bind preferentially to
the .alpha..sub.v.beta..sub.3 integrin found on tumor vascular
epithelial cells (Brooks et al., supra, Friedlander et al. (1996)
PROC. NATL. ACAD. Sci. USA 93: 9764-9769); certain antibodies and
antigen binding fragments thereof and peptides that bind
preferentially to the epidermal growth factor receptor (Ciardello
et al. (1996) J. NATL. CANCER INST. 88: 1770-1776, Ciardello et al.
(2000) CLIN. CANCER RES. 6:3739-3747); antibodies, proteins,
peptides and/or nucleic acids that bind preferentially to and
neutralize vascular endothelial growth factor (Adamis et al. (1996)
ARCH OPTHALMOL 114:66-71), antibodies, proteins, and/or peptides
that bind preferentially to and neutralize vascular endothelial
growth factor receptor; anti-fibroblast growth factor,
anti-epidermal growth factor (Ciardiello et al. (2000) CLIN. CANCER
RES. 6: 3739-3747) including full length amino acid sequences,
bioactive fragments and analogs thereof, and pigment
epithelium-derived growth factor (Dawson (1999) SCIENCE 2035:
245-248) including full length amino acid sequences, bioactive
fragments and analogs thereof. Bioactive fragments refer to
portions of the intact protein that have at least 30%, more
preferably at least 70%, and most preferably at least 90% of the
biological activity of the intact proteins. Analogs refer to
species and allelic variants of the intact protein, or amino acid
replacements, insertions or deletions thereof that have at least
30%, more preferably at least 70%, and most preferably 90% of the
biological activity of the intact protein.
[0054] Other angiogenesis inhibitors include, for example: COX-2
selective inhibitors (Masferrer et al. (1998) PROC. AMER. Assoc.
CANCER RES. 39: 271; Ershov et al. (1999) J. NEUROSCI. RES. 15:
254-261; Masferrer et al. (2000) CURR. MED. CHEM. 7: 1163-1170);
tyrosine kinase inhibitors, for example, PD 173074 (Dimitroff et
al. (1999) INVEST. NEW DRUGS 17: 121-135), halofuginone
(Abramovitch et al. (1999) NEOPLASIA 1: 321-329; Elkin et al.
(1999) CANCER RES. 5: 1982-1988), AGM-1470 (Brem et al. (1993) J.
PED. SURGERY 28: 1253-1257), angiogenic steroids, for example,
hydrocortisone and anecortave acetate (Penn et al. (2000) INVEST.
OPHTHALMOL. VIS. SCI. 42: 283-290), thrombospondin-1 (Shafiee et
al. (2000) INVEST. OPHTHALMOL. VIS. SCI. 8: 2378-2388; Nor et al.
(2000) J. VASC. RES. 37: 09-218), UCN-01 (Kruger et al. (1998-1999)
INVASION METASTASIS 18: 209-218), CM101 (Sundell et al. (1997)
CLIN. CANCER RES. 3: 365-372); fumagillin and analogues such as
AGM-1470 (Ingber et al (1990) NATURE 348: 555-557), and other small
molecules such as thalidomide (D'Amato et al. (1994) PROC. NATL.
ACAD. SCI. USA 91: 4082-4085).
[0055] Several cytokines including bioactive fragments thereof and
analogs thereof have also been reported to have anti-angiogenic
activity and thus can be useful in the practice of the invention.
Examples include, for example, IL-12, which reportedly works
through an IFN-.gamma.-dependent mechanism (Voest et al. (1995) J.
NATL. CANC. INST. 87: 581-586); IFN-.alpha., which has been shown
to be anti-angiogenic alone or in combination with other inhibitors
(Brem et al. (1993) J. PEDIATR. SURG. 28: 1253-1257). Furthermore,
the interferons IFN-.alpha., IFN-.beta. and IFN-.gamma. reportedly
have immunological effects, as well as anti-angiogenic properties,
that are independent of their anti-viral activities. However,
preferred anti-angiogenic factors include endostatin and
angiostatin.
[0056] The anti-angiogenesis 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.
[0057] To the extent that the anti-angiogenesis 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. It is appreciated, however, that
oligoribonucleotide sequences generally are more susceptible to
enzymatic attack by ribonucleases than are deoxyribonucleotide
sequences. Hence, oligodeoxyribonucleotide's are preferred over
oligoribonucleotides for in vivo use. In the case of nucleotide
sequences, phosphodiester linkages may be replaced by thioester
linkages making the resulting molecules more resistant to nuclease
degradation. 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). Furthermore, to the extent that the
anti-angiogenesis factor is an organic or inorganic compound, such
compounds may be synthesized, extracted and/or purified by standard
procedures known in the art.
[0058] The type and amount of anti-angiogenesis factor to be
administered may depend upon the PDT and cell type to be treated.
It is contemplated, however, that optimal anti-angiogenesis
factors, modes of administration and dosages may be determined
empirically. The anti-angiogensis 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.
[0059] Protein, peptide or nucleic acid based angiogenesis
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. For example, antibodies that bind vascular epithelial growth
factor may be administered intravenously at doses ranging from
about 0.1 to about 5 mg/kg once every two to four weeks.
Endostatin, for example, may be administered intravenously on a
daily basis at dosages ranging from about 1 to about 50 mg/kg per
day. With regard to intravitreal administration, the
anti-angiogenesis factor, for example, antibodies that bind
vascular epithelial growth factor, typically is administered
periodically as boluses at dosages ranging from about 10 fig to
about 5 mg/eye and more preferably from about 100 .mu.g to about 2
mg/eye.
[0060] The anti-angiogenesis factor preferably is administered to
the mammal prior to PDT. Accordingly, it is preferable to
administer the anti-angiogenesis factor prior to administration of
the photosensitizer. The anti-angiogenesis 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, and intravitreal, is preferred. Administration may be
provided as a periodic bolus (for example, intravenously or
intavitreally) 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-angiogenesis 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).
[0061] The present invention, therefore, includes the use of an
anti-angiogenesis factor in the preparation of a medicament for
treating, preferably by a PDT-based method, an ocular condition,
that preferably is associated with choriodal neovasculature. The
anti-angiogenesis 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-angiogenesis 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-angiogenesis factor; as well as the
composition for use in medicine; More preferably, however, the
invention is for use in combination therapy, whereby an
anti-angiogenesis factor and a photosensitizer are administered
separately. Preferably the anti-angiogenesis factor is administered
prior to administration of the photosensitizer. Instructions for
such administration may be provided with the anti-angiogenesis
factor and/or with the photosensitizer. If desired, the
anti-angiogenesis factor and photosensitizer may be provided
together in a kit, optionally including a package insert with
instructions for use. The anti-angiogenesis factor and
photosensitizer preferably are provided in separate containers. For
each administration, the anti-angiogenesis factor and/or
photosensitizer may be provided in unit-dosage or multiple-dosage
form. Preferred dosages of photosensitizer and anti-angiogenic
factor, however, are as described above.
[0062] 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 decreasing 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.
[0063] 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.
[0064] It has been suggested that apoptosis is associated with the
generation of reactive oxygen species, and that the product of the
Bcl-.sub.2 gene protects cells against apoptosis by inhibiting the
generation or the action of the reactive oxygen species (Hockenbery
et al. (1993) CELL 75: 241-251, Kane et al. (1993) SCIENCE 262:
1274-1277, Veis et al. (1993) CELL 75: 229-240, Virgili et al.
(1998) FREE RADICALS BIOL. MED. 24: 93-101). BCI-.sub.2 belongs to
a growing family of apoptosis regulatory gene products, which may
either be death antagonists (Bcl-.sub.2, BCl-x.sub.L..) or death
agonists (Bax, Bak.) (Kroemer et al. (1997) NAT. MED. 3: 614-620).
Control of cell death appears to be regulated by these interactions
and by constitutive activities of the various family members
(Hockenbery et al. (1993) CELL 75: 241-251). Several apoptotic
pathways may coexist in mammalian cells that are preferentially
activated in a stimulus-, stage-, context-specific and cell-type
manner (Hakem et al. (1998) CELL 94: 339-352).
[0065] 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: 1). This peptide reportedly
is non-toxic outside cells, but become 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
exanple, 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 a (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), 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-.sub.2, Bcl-x.sub.L). Antisense nucleotides directed
against Bcl-.sub.2 have been shown to reduce the expression of
Bcl-.sub.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 18 mer
phosphorothiate oligonucleotide complementary to the first six
codons of the Bcl-.sub.2 open reading frame, and known as G3139, is
being tested in humans as a treatment for non-Hodgkins'
lymphoma.
[0066] 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).
[0067] To the extent that the apoptosis-modulating factor is a
protein or peptide, nucleic acid, peptidyl nucleic acid, organic or
inorganic compound, it may be synthesized and purified by one or
more the methodologies described relating to the synthesis of the
anti-angiogenesis factor.
[0068] 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.
[0069] 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 apopotosis 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 boluses a 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.
[0070] 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-angiogenesis 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).
[0071] Although the foregoing methods and compositions of the
invention may be useful in treated unwanted choroidal
neovasculature and thereby ameliorating 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, 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.
[0072] The invention is illustrated further by reference to the
following non-limiting examples.
EXAMPLE 1
Anti-Angiogenesis Factor Potentiates the Effect of PDT on
Endothelial Cells
[0073] Experiments were performed to determine whether the
cytotoxicity resulting from PDT can be potentiated by the addition
of an anti-angiogenesis factor. Cells of interest were treated by
PDT either alone or in combination with an anti-angiogenesis factor
and the effect on cytotoxicity of the PDT assessed via a cell
proliferation assay.
[0074] Bovine retinal capillary endothelial (BRCE) cells (from
Patricia A. D'Amore, Schepens Eye Research Institute, Boston,
Mass.) and Human retinal pigment epithelial (RPE) cells (from
Anthony P. Adamis, Massachusetts Eye & Ear Infirmary, Boston,
Mass.) were grown at 37.degree. C., 5% CO.sub.2 in Dulbecco's
modified Eagle's medium (DMEM; Sigma, St. Louis, Mo.), 5%
heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island,
N.Y.), supplemented with L-glutamine, penicillin, and streptomycin
(Gibco Grand Island, N.Y.). Lutetium Texaphyrin (Lu-Tex) was
obtained from Alcon Laboratories, Inc. (Fort Worth, Tex.) as a
stock solution of 2 mg/ml, stable in the dark at 4.degree. C., and
used in accordance with the manufacturer's guidelines.
[0075] Cell survival was measured using a cell proliferation assay.
Briefly, BRCE or RPE cells were plated at a density of 10.sup.5
cells in DMEM with 5% FBS and incubated at 37.degree. C. in 5%
CO.sub.2. After eighteen hours, and if desired, recombinant human
angiostatin (Calbiochem, La Jolla, Calif.) was added at a
concentration of 500 ng/ml. Eighteen hours later, the medium was
removed and replaced by 3 .mu.g/ml Lu-Tex in complete media. Thirty
minutes later, the cultures were exposed to timed irradiation using
an argon/dye Photocoagulator at 732 nm and laser delivery system
(model 920, Coherent Inc., Palo Alto, Calif.). Irradiance was
delivered at a rate of 10 mw/cm.sup.2 to give a total dose of 5 to
20 J/cm.sup.2, and irradiation time ranged from 7 to 28 minutes.
After irradiation, the medium was removed and replaced with
complete medium. Cultures were returned to the incubator for 7
days, after which cells were dispersed in trypsin, counted in a
masked fashion, and the surviving fraction determined. The results,
reported as the mean of triplicate.+-.SD, are summarized in Table
1. Cultures were photographed at various times following Lu-Tex/PDT
using a 16X-0.32 numeric aperture on a phase contrast inverted
microscope (Diaphot, Nikon, Melville, N.Y.).
1TABLE 1 Summary of Cellular Survival (%) as a Function of
Treatment* Angiostatin Lu-Tex/PDT Cell followed by Lu- followed by
Line Lu-Tex/PDT Angiostatin Tex/PDT Angiostatin BRCE 79.13 .+-.
4.05 87.39 .+-. 5.76 55.22 .+-. 3.65 77.61 .+-. 3.52 (5J/cm.sup.2)
53.17 .+-. 0.32 38.11 .+-. 2.50 67.16 .+-. 3.20 (10J/cm.sup.2)
33.34 .+-. 2.26 0.90 .+-. 0.32 32.97 .+-. 2.20 (20J/cm.sup.2) RPE
94.55 .+-. 1.60 99.09 .+-. 0.8 91.84 .+-. 7.97 (5J/cm.sup.2) 59.59
.+-. 3.56 56.84 .+-. 6.61 (10J/cm.sup.2) 53.47 .+-. 3.18 45.83 .+-.
5.51 (20J/cm.sup.2) *The interactive in vitro anti-endothelial
effect of combined treatment with angiostatin and Lu-Tex/PDT are
greater than additive when compared with the sum of expected
effects of each treatment alone. The potentiation of Lu-Tex/PDT
effect on BRCE was effective with preexposure to angiostatin only.
No effect of angiostatin was observed on RPE. Data are mean %
cellular survival .+-. SD.
[0076] In order to assess the effect of combining angiostatin to
Lu-Tex/PDT on BRCE cell survival, cells were pre-treated for 18
hours with 500 ng/ml angiostatin after which they were treated with
Lu-Tex/PDT at various fluences. Cellular survival was measured by
the 1-week cellular proliferation assay. When exposed to
angiostatin alone, the proliferation assay demonstrated a 12.61%
killing of BRCE cells at the angiostatin dose used (Table 1).
Pre-exposing BRCE cells to angiostatin did not appear to interfere
with the subsequent cellular uptake of Lu-Tex. More importantly,
the results showed a synergistic cytotoxic effect of angiostatin
and Lu-Tex/PDT on BRCE cells at all fluences used (5, 10 and 20
J/cm.sup.2), consistently exceeding the cytotoxicity resulting from
Lu-Tex/PDT alone, angiostatin alone or the arithmetic sum of their
respective toxicity's (Table 1, FIG. 1A). Controls consisted of
cells exposed to light only because no dark toxicity was observed
at the concentration of Lu-Tex used. Furthermore, it was observed
that angiostatin was not effective in potentiating the effect of
Lu-Tex/PDT if delivered after PDT.
[0077] In contrast to the results obtained with BRCE cells, no
cytotoxicity was observed when human RPE cells were treated with
human angiostatin, and no interactive killing was observed
following exposure to angiostatin and Lu-Tex/PDT (FIG. 1B, Table
1). When combined with angiostatin, Lu-Tex/PDT had a lethal dose
(LD.sub.100) of 20 J/cm.sup.2 for BRCE cells whereas Lu-Tex/PDT
alone required 40 J/cm.sup.2 to achieve the same effect on BRCE
cells. Previous studies showed that at fluences of 20 and 40
J/cm.sup.2, RPE cell survival is about 43% and 21%,
respectively.
[0078] The data show a specific anti-proliferative effect of
angiostatin on BRCE cells as demonstrated by the reduction in cell
number in a 1-week proliferation assay. In contrast, no effect of
angiostatin was observed on RPE cells. Accordingly, BRCE cells
appear to be another endothelial cell line, along with bovine
adrenal cortex-microvascular cells, bovine adrenal cortex capillary
cells, bovine aortic cells, human umbilical vein cells and human
dermal microvascular endothelium cells (Mauceri et al. (1998)
NATURE 394: 287-291, Lucas et al. (1998) BLOOD 92: 4730-41), that
is specifically targeted by angiostatin. In this study, BRCE cells
were used a representative capillary endothelial line of the
posterior segment to test the anti-angiogenic effect of
angiostatin. The finding that angiostatin induces apoptosis in BRCE
cells suggests that cell death might contribute to the overall
reduction of cell number. However, little is known concerning the
exact anti-angiogenic mechanism of angiostatin (Lucas et al. (1998)
BLOOD 92: 4730-4741).
[0079] In summary, the studies show that Lu-Tex/PDT and angiostatin
have combined cytotoxic effects on retinal capillary endothelial
cells, but not on pigment epithelial cells. However, when
angiostatin was administered after PDT, the combination did not
potentiate the effects of PDT. In the combination of angiostatin
before Lu-Tex/PDT, a fluence of 20 J/cm.sup.2 sufficed to achieve
nearly 100% mortality of BRCE. In the absence of angiostatin, a
light dose of 40 J/cm.sup.2 was required to achieve this level of
cytotoxicity. At the light dose of 20 J/cm.sup.2, RPE cell survival
post-PDT was improved by 20%. The results thus support the
potential of combining angiostatin with Lu-Tex/PDT to improve CNV
eradication and to decrease deleterious effects on the RPE.
EXAMPLE 2
Cellular Morphology Following PDT With Anti-Angiogenic Factor
[0080] Experiments were performed to establish how PDT effects the
cellular morphology of BRCE and RPE cells. The cells were treated
and exposed to PDT either alone or in combination with angiostatin
as described in Example 1. Although cells appeared severely damaged
immediately after PDT, subsequent recovery occurred in certain
circumstances. One week after PDT, some cells disappeared while
those that remained regained their spindle shape and their ability
to attach.
[0081] In BRCE cells that were first primed with angiostatin
followed by PDT, widespread and massive cell death was observed at
one week. Only remnants of cells and densely refractive bodies of
dying cells were observed floating in the medium. Particles were
recovered and placed in fresh complete media but none showed any
sign of reattachment or proliferation onto a new dish. The
combination of angiostatin and Lu-Tex/PDT, therefore, appears to be
lethal to BRCE under the conditions used.
[0082] Control BRCE cells and RPE cells which were treated with
angiostatin alone for 18 hours continued to proliferate and reached
confluence. No additive effect of angiostatin to Lu-Tex/PDT was
observed in RPE cells. RPE cells subjected to Lu-Tex/PDT alone or
with angiostatin appeared unchanged as evidenced by their
morphology.
EXAMPLE 3
Caspase 3-Like (DEVD-ase) Activation in BRCE and RPE Following
PDT
[0083] In order to investigate the role of apoptosis in Lu-Tex/PDT
mediated cell death in BRCE and RPE, the activation of Caspase
3-like (DEVD-ase) protease, a hallmark of apoptosis (Nicholson
(1997) TIBS 22: 299-306), was monitored. The kinetics of activation
were measured spectrofluorometrically by assaying the hydrolysis of
a substrate that can be cleaved only by the caspase 3-like protease
family members.
[0084] Various times after Lu-Tex/PDT, 10.sup.6 cells were
collected by centrifugation, and the washed cell pellet resuspended
in 500 .mu.l of ice-cold lysis buffer (pH 7.5) containing 10 mM
Tris, 130 mM NaCl, 1% Triton X-100, 10 mM NaF, 10 mM NaPi, 10 mM
NaPPi, 16 .mu.g/ml benzamidine, 10 .mu.g/ml phenanthroline, 10
.mu.g/ml aprotinin, 10 .mu.g/ml leupeptin, 10 .mu.g/ml pepstatin
and 4 mM 4(2-aminoethyl)-benzene-sulfanyl fluoride hydrochloride
(AEBSF). Cellular lysates were stored in aliquots at -84.degree. C.
for later protease activity assay or Western blot analysis. A
protein assay (Coomassie Plus protein assay (Pierce, Rockford,
Ill.) with a bovine serum albumin (BSA) standard was used to assay
protein concentration in cell extract.
[0085] In order to measure protease activity, aliquots containing
50 .mu.g of cellular protein were incubated with 14 .mu.M (final
concentration N-acetyl (Asp-Glu-Val-Asp (SEQ. ID NO:
3)--(7-amino-4-trifluoromethly coumarin) (Ac-DEVD-AFC); (Pharmingen
San Diego, Calif.) in 1 ml protease assay buffer pH 7.2 (20 mM
piperazine-N-N.sup.1-bis (2-ethanesulfuric acid) (PIPES), 100 mM
NaCl, 10 mM dithiothreitol (DTT), 1 mM EDTA, 0.1%(w/v)
3-[(3-Cholamidopropyl) dimethyl ammonio]-1-propane sulfonate
(CHAPS), and 10% sucrose) at 37.degree. C. for 1 hour. Fluorescence
was measured using a Perkin-Elmer MPF-44A spectrofluorometer
(.lambda..sub.excitation, 400 nm; .lambda..sub.emission 505 nm).
Cellular protein served as the blank. Results were compared with a
standard curve constructed with AFC (Sigma, St. Louis, Mo.) and are
shown in FIG. 2.
[0086] FIG. 2 illustrates the time course of Ac-DEVD-AFC cleavage
after Lu-Tex/PDT at three different light doses in BRCE and RPE.
FIGS. 2A, 2B and 2C represent data generated using light does of
10, 20 and 40 J/cm.sup.2, respectively. The results show a rapid
elevation of caspase 3-like activity immediately after
Lu-Tex/PDT--as early as 10 min post-Lu-Tex/PDT and peaking at 40
min--in both BRCE and RPE cells and at all doses used. The rate of
entry into apoptosis was time and dose-dependent in each cell line.
However, the amount of caspase 3-like activation was always
significantly higher in BRCE cells compared to RPE cells.
Furthermore, whereas at 10J/cm.sup.2 and 20J/cm.sup.2, the amount
of caspase 3-like activation increased by about 50% in BRCE cells
as compared to RPE cells; at 40J/cm.sup.2 (equivalent to the
LD.sub.100 for BRCE cells), the levels in BRCE were 5-fold those in
RPE cells.
[0087] In order to examine the effect of combining angiostatin and
Lu-Tex/PDT on DEVD-ase activation in BRCE cells, cells were treated
with angiostatin alone, Lu-Tex/PDT alone and
angiostatin/Lu-Tex/PDT, following which caspase 3-like activity was
assayed as described above. The results are summarized in FIG. 3.
Fluences of 20 and 40 J/cm.sup.2 were used, corresponding to
LD.sub.100 of combination angiostatin/Lu-Tex/PDT and Lu-Tex/PDT
alone respectively. Results demonstrated that the combination of
angiostatin/Lu-Tex/PDT induced a statistically significant increase
of caspase 3-like activity as compared to Lu-Tex/PDT alone using a
fluence of 20J/cm.sup.2 (FIG. 3). However, while both Lu-Tex/PDT
(40J/cm.sup.2) and the combination of angiostatin/Lu-Tex/PDT
(20J/cm.sup.2) resulted in 100% lethality of BRCE cells; Lu-Tex/PDT
(40J/cm.sup.2) resulted in increased levels of caspase 3-like
activity as compared to angiostatin/Lu-Tex/PDT (20J/cm.sup.2). As
in the case of BRCE cells treated with Lu-Tex/PDT alone, the rate
of entry into apoptosis of BRCE cells treated with combination of
angiostatin/Lu-Tex/PDT was time-dependent. Nevertheless, the time
courses differed significantly in that the induction of caspase
3-like activation occurred abruptly and more rapidly as a result of
angiostatin/Lu-Tex/PDT, peaking at 30 minutes and reaching minimum
levels at 90 minutes post-treatment.
EXAMPLE 4
Modulation of Bcl-2 Family Members in BRCE and RPE Cells after
Lu-Tex/PDT
[0088] In order to evaluate the expression of Bcl-2 family members
in BRCE and RPE cells after Lu-Tex/PDT, BRCE and RPE cells were
subjected to Lu-Tex/PDT-and the resultant cellular lysates
subjected to Western blot analysis for detection of the
anti-apoptotic Bcl-2, Bcl-XL markers, and the pro-apoptotic Bax and
Bak markers.
[0089] Cell lysates were produced as described in Example 3. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis of proteins was
performed with 12% SDS-polyacrylamide gels. All samples were boiled
in denaturing sample buffer, and equal amounts of proteins were
loaded per lane. Proteins were separated at room temperature under
reducing conditions at 120 V. Western blot transfer of separated
proteins was performed at room temperature, using polyvinylidene
fluoride membranes at 50 mA for 1 hr. To verify equal protein
loading, blots were stained with 0.1% ponceau red (Sigma) diluted
in 5% acetic acid. Afterwards, blots were blocked for 1 hr in Tris
buffered saline (TBS; 10 mM Tris-HCI, pH 7.5, and 150 mM NaCl)
containing 5% non-fat dried milk. Next, the membranes were probed
with an appropriate dilution (1:250 to 1: 1000) of primary antibody
in TBS containing 2.5% non-fat dried milk for one and a half hours.
Mouse polyclonal antibodies against Bcl-2, Bcl-x.sub.L, Bax and Bak
were purchased from Pharmingen. After incubation with primary
antibody, the blots were washed for 30 minutes with frequent
changes of TBS, blocked in 1% non-fat dried milk in TBS for 30
minutes, followed by incubation in a peroxidase-coupled secondary
antibody for 1 hour in TBS containing 1% non-fat dried milk. The
blots were washed for 1 hour with frequent changes of TBST (TBS,
0.1% Tween). Immunoblot analysis was performed using enhanced
chemiluminescence plus Western blotting detection reagents
(Amersham, Pharmacia Biotec Piscataway, N.J.) followed by exposure
to x-ray film (ML Eastman Kodak, Rochester, N.Y.).
[0090] Results showed a differential expression of members of Bcl-2
family members in BRCE and RPE cells. Specifically, Bcl-2 and Bax
were detected in BRCE cells whereas Bcl-x.sub.L and Bak were
detected in RPE cells (Table 2). After Lu-Tex/PDT at LD.sub.50,
downregulation of Bcl-2 and upregulation of Bax was observed in
BRCE cells resulting in an increase of the cellular ratio of Bax to
Bcl-2 protein. In RPE cells, there was an upregulation of both
Bcl-x.sub.L and Bak up to 4 hours post-PDT, after which,
Bcl-x.sub.L levels reached a plateau, and Bak level started to
decline. The upregulation of Bax in BRCE cells appeared to be
dose-dependent, however, the upregulation of its pro-apoptotic
counterpart Bak in RPE exhibited dose-dependence only until 20
J/cm.sup.2; after which it began to decline.
[0091] Lu-Tex/PDT induced caspase 3-like activation in both BRCE
and RPE cells in a dose- and time-dependent fashion, suggesting
that apoptosis is a mediator of Lu-Tex/PDT cytotoxicity in these
cell lines. Furthermore, the data indicate that Lu-Tex/PDT induced
apoptosis in BRCE cells through the modulation of Bcl-2 and Bax in
a dose- and time-dependent fashion, and in RPE cells through the
modulation of Bcl-x.sub.L and Bak. As a result, Lu-Tex/PDT may
cause different modes of death in each of the different cell
types.
2TABLE 2 Summary of Immunodetection of Bcl.sub.2 Family Members in
BRCE and RPE Cells Cell Line Bcl.sub.2 family member BRCE RPE
Bcl.sub.2 + - Bcl-x.sub.L - + Bax + - Bak - + Detectable (+) or
undetectable (-).
[0092] After incremental PDT doses, the pro-apoptotic Bak was
upregulated in RPE cells until 20 J/cm.sup.2 following which Bak
levels started declining despite an increase of PDT dose to 40
J/cm.sup.2. Without wishing to be bound by theory, it is possible
that a protective survival response is mounted in RPE cells at
these lethal doses to counteract the apoptotic trigger. Such a
hypothesis is further supported by the histologic evidence of RPE
cells recovery post-PDT in vivo (Kramer et al. (1996) OPHTHALMOLOGY
103(3): 427438, Husain et al. (1999) INVEST OPHTHALMOL VISL SCI.
40: 2322-31) and by reports from other investigators showing that
overexpression of anti-apoptotic Bcl-.sub.2 family members render
cells partially resistant to PDT (He et al. (1996) PHOTOCHEMISTRY
AND PHOTOBIOLOGY 64: 845-852) and inhibits the activation of
caspase-3 after PDT (Granville et al. (1998) FEBS 422:
151-154).
[0093] The data show that the combination of angiostatin to
Lu-Tex/PDT in BRCE cells resulted in an increase in DEVD-ase
activity compared with a same dose of Lu-Tex/PDT applied alone.
This suggests that the potentiating action of angiostatin on the
effect of Lu-Tex/PDT in BRCE cells proceeds through apoptosis.
However, the time course of caspase 3-like activity for
angiostatin/Lu-Tex/PDT differed from that of Lu-Tex/PDT alone in
that it proceeded faster without latency and peaked as soon as 20
minutes after Lu-Tex/PDT. The latter may be explained on the basis
that perhaps the apoptotic cascade was already primed by
pre-incubation with angiostatin first, and thus the application of
Lu-Tex/PDT benefited from an already lowered threshold of
activation to rapidly amplify the apoptotic response. However, this
does not exclude the possibility of the interplay of more than one
apoptotic pathway, especially since PDT is known to initiate
cytotoxicity through the generation of reactive oxygen species
(Weishaupt et al. (1976) supra) whereas angiostatin was recently
shown to act on human endothelial cells by binding to the
.alpha.-subunit of adenosine triphosphate synthase present on the
cell surface (Moser et al. (1999) PROC NATL ACAD SCI USA 96:
2811-2816). Furthermore, whereas angiostatin/Lu-Tex/PDT (20
J/cm.sup.2) resulted in a 100% lethality of BRCE cells as did
Lu-Tex/PDT (40 J/cm.sup.2) alone, the levels of DEVD-ase activation
were significantly higher in the former regimen. This supports the
theory that Lu-Tex/PDT and Angiostatin/Lu-Tex/PDT operate through
different apoptotic pathways in BRCE cells.
EXAMPLE 5
Targeted Delivery of Photosensitizer to the Choroidal
Neovasculature
[0094] It is contemplated that a photosensitizer can be directed to
the CNV endothelium by coupling the photosensitizer to a
neovascular endothelium binding moieties in order to increase the
efficacy and lower the toxicity of PDT. Several targeting molecules
may be used to target photosensitizers to the neovascular
endothelium. The .alpha.-v integrins, in particular .alpha.-v
.beta.-3 and .alpha.-v .beta.-5 integrins, appear to be expressed
in ocular neovascular tissue, in both clinical specimens and
experimental models (Corjay et al. (1997) supra; Friedlander et al.
(1995) supra). 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 ACDCRGDCFC (SEQ ID NO: 2)--also
called RGD-4C--was 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). Another potential targeting molecule is
an antibody for vascular endothelial growth factor receptor
(VEGF-2R). Clinical and experimental evidence strongly supports a
role for 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).
Antibody to the VEGF receptor (VEGFR-2 also known as KDR) can be
expected to bind preferentially to neovascular endothelium.
[0095] Experimental Design
[0096] The photosensitizer Verteporfin (QLT Phototherapeutics,
Inc., Vancouver BC) or Lutetium Texaphryin (Alcon Laboratories,
Fort Worth, Tex.) will be coupled to a targeting moiety, for
example, an RGD-4C peptide, or an anti-VEGF receptor antibody using
standard coupling chemistries. The spectral characteristics
(emission & excitation) of the resulting photosensitizer
complex can be measured in vitro. Subsequently, in vitro studies
can be carried out using BRCE and RPE cells, to assess cellular
uptake and phototoxicity following PDT. Experiments may address PDT
treatment parameters including optimal timing as well as drug and
light dosimetry for selective phototoxicity in vitro. Then, the
efficacy and selectivity of PDT using the bound photosensitizer in
vivo in the rat model of CNV can be tested. The results of PDT with
photosensitizer comprising the targeting molecule may then be
compared to the results of PDT with the same photosensitizer
lacking the targeting molecule.
[0097] CNV can be induced in animals using a krypton laser, and
documented by digital fundus fluorescein angiography. More
specifically, the laser injury model in the rat is a modification
of a similar model in the monkey (Dobi et al (1989) ARCH.
OPHTHALMOL. 107:264-269; Ryan (1982) ARCH. OPHTHALMOL.
100:1804-1809; Tobe et al. (1994) J. JPN. OPHTHALMOL. SOC.
98:837-845). Briefly, 5-6 high intensity krypton laser bums (100
.mu.m spot size, 0.1-second duration, 160 mW) can be placed in a
peripapillary fashion. CNV as evidenced by hyperfluorescence and
late leakage can be documented using digital fluorescein
angiography and is expected to develop in at least 60% of the
lesions within 2-3 weeks of laser injury.
[0098] PDT can then be performed over areas of CNV and normal
choroid and the effects assessed angiographically and
histologically. More specifically, PDT may be carried out using
tail vein injection of the photosensitizer either containing or
lacking a targeting molecule, followed by laser irradiation of the
treatment area. PDT may also be applied to areas of CNV in one eye
and to areas of normal choroid in the fellow eye. Photosensitizer
and laser parameters will be based on previous experiments using
Verteporfin and Lu-Tex in the monkey model, as well as some
preliminary dosimetry in the rat model.
[0099] The efficacy of PDT can be assessed as follows:
[0100] (a) Efficacy of CNV closure. Effective closure of CNV can be
assessed by the absence of leakage from CNV via fluorescein
angiography 24 hours after PDT. This methodology has been well
established in the laser injury in the monkey. Histopathology can
be carried out using light microscopy.
[0101] (b) Selectivity of Effect. Since CNV in this model develops
in an area of laser injury, it is difficult to assess the effects
of PDT on retina and choroid when areas of CNV are treated.
Therefore, to demonstrate the selectivity of PDT to CNV, PDT may
also be applied to areas of normal retina and choroid and a
published histopathologic grading scheme used to quantify damage to
RPE, photoreceptors, retinal and choroidal vessels (Kramer et al.
(1996) OPHTHALMOLOGY 103:427-438).
[0102] (c) Comparison of the Effects of PDT versus combined PDT
regimens. The effects of PDT may be compared between groups of CNV
animals treated with PDT using photosensitizer alone, and groups
receiving modified PDT (i.e. targeted photosensitizer). PDT may be
applied to the CNV and normal areas. First, it may be determined if
CNV closure occurs at the same light dose (fluence J/cm.sup.2)
using the modified PDT as with PDT alone. Then, at the identified
light dose, the effects of modified PDT, and PDT alone, on normal
choroid may be compared. As an example, using the targeted
photosensitizer, one may be able to achieve closure of CNV at a
lower fluence than with unbound photosensitizer, and at this
fluence one may find much less damage to the RPE in normal areas
treated with PDT using targeted photosensitizer.
EXAMPLE 6
Combined Effects of Targeted Pro-apoptotic Peptides and PDT For
Choroidal Neovascularization Treatment
[0103] Experiments have shown that PDT induces cell death in
endothelial cells by apoptosis and that its toxicity towards the
RPE also proceeds by programmed cell death. Different apoptotic
pathways appear to be triggered by PDT in BRCE and RPE cells. It is
contemplated that by specifically priming the apoptotic machinery
of neovascular capillary endothelial cells prior to PDT it may be
possible to increase their sensitivity to PDT This approach may
reduce the light dose (fluence) required to achieve CNV closure and
thereby decrease the effect on the surrounding cells such as RPE
cells.
[0104] Studies have shown the efficacy of targeted pro-apoptotic
peptides in anti-cancer activity in significantly reducing the
tumor size (Ellerby et al. (1999) supra). These targeted
pro-apoptotic conjugates were comprised of two functional domains:
an antimicrobial peptide (KLAKLAKKLAKLAK; SEQ ID NO: 1) with low
mammalian toxicity and an angiogenic homing peptide (RGD-4C). The
antibacterial peptide preferentially disrupts prokaryotic membranes
and eukaryotic mitochondrial membranes rather than eukaryotic
plasma membranes (Ellerby et al (1999) supra). Thus the chimeric
peptide, therefore, may have the means to enter the cytosol of
targeted cells, where it induces mitochondrial-dependent apoptosis.
Endothelial cells primed with these conjugates are expected to be
more susceptible to PDT.
[0105] Experimental Design
[0106] Peptides of interest will first be tested in vitro in BRCE
and RPE cells to ascertain specificity and efficacy. Then, the
pro-apoptotic peptide/PDT regimen may be assessed in vitro, and
then compared with PDT alone and peptide alone in both BRCE and RPE
cells. BRCE and RPE cells may be grown using standard tissue
culture techniques. The ApoAlert assay kit (Clonetech) may be used
to assay for caspase-3 like activity in cells post-treatment. This
colorimetric assay follows the chromophore p-nitroanilide (pNA)
arising from cleavage of the substrate DEVD-pNA. DEVD-pNA is a
known substrate for active caspase-3 and can be added to cellular
extracts prepared at different time points after treatment, and
samples can be analyzed to assess caspase-3 activity.
[0107] Thereafter, experiments may be carried out to test the
efficacy and selectivity of targeted pro-apoptotic peptide in vivo
in the rat model of CNV. Targeted pro-apoptotic peptides may be
injected intravenously 4 hours prior to PDT. PDT may be performed
over areas of CNV and in normal eyes, comparing the effect on CNV
closure of PDT alone with PDT after pro-apoptotic peptide, and
comparing the selectivity in normal choroid as described in Example
5.
[0108] Equivalents
[0109] 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.
[0110] Incorporation by Reference
[0111] Each of the patent documents and scientific publications
disclosed hereinabove is expressly incorporated herein by
reference.
Sequence CWU 1
1
3 1 14 PRT Artificial Sequence Synthetic sequence 1 Lys Leu Ala Lys
Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 10 2 10 PRT Artificial
Sequence Synthetic sequence 2 Ala Cys Asp Cys Arg Gly Asp Cys Phe
Cys 1 5 10 3 4 PRT Artificial Sequence Synthetic sequence 3 Asp Glu
Val Asp 1
* * * * *