U.S. patent application number 10/418965 was filed with the patent office on 2004-02-19 for use of green porphyrins to treat neovasculature in the eye.
Invention is credited to Gragoudas, Evangelos S., Miller, Joan W..
Application Number | 20040034007 10/418965 |
Document ID | / |
Family ID | 46251298 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040034007 |
Kind Code |
A1 |
Miller, Joan W. ; et
al. |
February 19, 2004 |
Use of green porphyrins to treat neovasculature in the eye
Abstract
Photodynamic therapy of conditions of the eye characterized by
unwanted neovasculature, such as age-related macular degeneration,
is effective using green porphyrins as photoactive agents,
preferably as liposomal compositions.
Inventors: |
Miller, Joan W.;
(Winchester, MA) ; Gragoudas, Evangelos S.;
(Lexington, MA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
46251298 |
Appl. No.: |
10/418965 |
Filed: |
April 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10418965 |
Apr 18, 2003 |
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09824155 |
Apr 2, 2001 |
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6610679 |
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09824155 |
Apr 2, 2001 |
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09347382 |
Jul 6, 1999 |
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6225303 |
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09347382 |
Jul 6, 1999 |
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08942475 |
Oct 2, 1997 |
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08942475 |
Oct 2, 1997 |
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08390591 |
Feb 17, 1995 |
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5798349 |
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08390591 |
Feb 17, 1995 |
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08209473 |
Mar 14, 1994 |
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5707986 |
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Current U.S.
Class: |
514/185 ;
424/450; 514/410 |
Current CPC
Class: |
A61K 9/1275 20130101;
A61F 9/008 20130101; A61K 9/0019 20130101; A61K 31/555 20130101;
A61K 31/409 20130101; Y10S 514/912 20130101; A61K 41/0071 20130101;
A61K 49/0036 20130101; A61K 49/0084 20130101 |
Class at
Publication: |
514/185 ;
514/410; 424/450 |
International
Class: |
A61K 031/555; A61K
031/409; A61K 009/127 |
Claims
1. A method to treat conditions of the eye characterized by
unwanted neovasculature, which method comprises: administering to a
subject in need of such treatment an amount of liposomal
formulation of green porphyrin sufficient to permit an effective
amount to localize in said neovasculature; permitting sufficient
time to elapse to allow an effective amount of said green porphyrin
to localize in said neovasculature; and irradiating neovasculature
with light absorbed by the green porphyrin.
2. The method of claim 1 wherein the neovasculature is choroidal
neovasculature.
3. The method of claim 1 wherein the green porphyrin is of the
formula shown in FIGS. 1-3 or 1-4.
4. The method of claim 1 wherein said green porphyrin is of a
formula shown in FIG. 1 or a mixture thereof wherein each of
R.sup.1 and R.sup.2 is independently selected from the group
consisting of carbalkoxyl (2-6C), alkyl (1-6C), arylsulfonyl
(6-10C), cyano and --CONR.sup.5CO wherein R.sup.1 is aryl (6-10C)
or alkyl (1-6C); each R.sup.3 is independently carboxyl,
carboxyalkyl (2-6C) or a salt, amide, ester or acyl hydrazone
thereof, or is alkyl (1-6C); R.sup.4 is CH.dbd.CH.sub.2 or
--CH(OR.sup.4.)CH.sub.3 wherein R.sup.4. is H, or alkyl (1-6C)
optionally substituted with a hydrophilic substituent.
5. The method of claim 4 wherein said green porphyrin is of the
formula shown in FIGS. 1-3 or 1-4 or a mixture thereof and wherein
each of R.sup.1 and R.sup.2 is independently carbalkoxyl (2-6C);
one R.sup.3 is carboxyalkyl (2-6C) and the other R.sup.3 is the
ester of a carboxyalkyl (2-6C) substituent; and R.sup.4 is
CH.dbd.CH.sub.2 or --CH(OH)CH.sub.3.
6. The method of claim 5 wherein said green porphyrin is of the
formula shown in FIGS. 1-3 and wherein R.sup.1 and R.sup.2 are
methoxycarbonyl; one R.sup.3 is --CH.sub.2CH.sub.2COOCH.sub.3 and
the other R.sup.3 is CH.sub.2CH.sub.2COOH; and R.sup.4 is
CH.dbd.CH.sub.2.
7. A method to treat unwanted choroidal neovasculature ,which
method comprises administering to a subject in need of such in need
of such treatment an amount of green porphyrin sufficient to permit
an effective amount to localize in said choroidal neovasculature;
permitting sufficient time to elapse to allow an effective amount
of said green porphyrin to localize in said choroidal
neovasculature; and irradiating said choroidal neovasculature with
light absorbed by the green porphyrin.
8. The method of claim 7 wherein said green porphyrin is complexed
with low-density lipoprotein.
9. The method of claim 7 wherein said green porphyrin is contained
in a liposomal preparation.
10. The method of claim 7 wherein the green porphyrin is of the
formula shown in FIGS. 1-3 or 1-4.
11. The method of claim 7 wherein said green porphyrin is of a
formula shown in FIG. 1 or a mixture thereof wherein each of
R.sup.1 and R.sup.2 is independently selected from the group
consisting of carbalkoxyl (2-6C), alkyl (1-6C), arylsulfonyl
(6-10C), cyano and --CONR.sup.5CO wherein R.sup.5 is aryl (6-10C)
or alkyl (1-6C); each R.sup.3 is independently carboxyl,
carboxyalkyl (2-6C) or a salt, amide, ester or acyl hydrazone
thereof, or is alkyl (1-6C); R.sup.4 is CH.dbd.CH.sub.2 or
--CH(OR.sup.4.)CH.sub.3 wherein R.sup.4. is H or alkyl (1-6C)
optionally substituted with a hydrophilic substituent.
12. The method of claim 11 wherein said green porphyrin is of the
formula shown in FIGS. 1-3 or 1-4 or a mixture thereof and wherein
each of R.sup.1 and R.sup.2 is independently carbalkoxyl (2-6C);
one R.sup.3 is carboxyalkyl (2-6C) and the other R.sup.3 is the
ester of a carboxyalkyl (2-6C) substituent; and R.sup.4 is
CH.dbd.CH.sub.2 or --CH(OH)CH.sub.3.
13. The method of claim 12 wherein said green porphyrin is of the
formula shown in FIGS. 1-3 and wherein R.sup.1 and R.sup.2 are
methoxycarbonyl; one R.sup.3 is --CH.sub.2CH.sub.2COOCH.sub.3 and
the other R.sup.3 is CH.sub.2CH.sub.2COOH; and R.sup.4 is
CH.dbd.CH.sub.2.
14. A method to treat age-related macular degeneration (AMD) which
method comprises administering to a subject in need of such
treatment an amount of green porphyrin sufficient to permit an
effective amount to localize in the choroid; permitting sufficient
time to elapse to allow an effective amount of said green porphyrin
to localize in said choroid; and irradiating said choroid with
light absorbed by the green porphyrin.
15. The method of claim 14 wherein said green porphyrin is
complexed with low-density lipoprotein.
16. The method of claim 14 wherein said green porphyrin is
contained in a liposomal preparation.
17. The method of claim 14 wherein the green porphyrin is of the
formula shown in FIGS. 1-3 or 1-4.
18. The method of claim 14 wherein said green porphyrin is of a
formula shown in FIG. 1 or a mixture thereof wherein each of
R.sup.1 and R.sup.2 is independently selected from the group
consisting of carbalkoxyl (2-6C), alkyl (1-6C), arylsulfonyl
(6-10C), cyano and --CONR.sup.5CO wherein R.sup.5 is aryl (6-10C)
or alkyl (1-6C); each R.sup.3 is independently carboxyl,
carboxyalkyl (2-6C) or a salt, amide, ester or acyl hydrazone
thereof, or is alkyl (1-6C); R.sup.4 is CH.dbd.CH.sub.2 or
--CH(OR.sup.4.)CH.sub.3 wherein R.sup.4. is H, or alkyl (1-6C)
optionally substituted with a hydrophilic substituent.
19. The method of claim 18 wherein said green porphyrin is of the
formula shown in FIGS. 1-3 or 1-4 or a mixture thereof and wherein
each of R.sup.1 and R.sup.2 is independently carbalkoxyl (2-6C);
one R.sup.3 is carboxyalkyl (2-6C) and the other R.sup.3 is the
ester of a carboxyalkyl (2-6C) substituent; and R.sup.4 is
CH.dbd.CH.sub.2 or --CH(OH)CH.sub.3.
20. The method of claim 19 wherein said green porphyrin is of the
formula shown in FIGS. 1-3 and wherein R.sup.1 and R.sup.2 are
methoxycarbonyl; one R.sup.3 is --CH.sub.2CH.sub.2COOCH.sub.3 and
the other R.sup.3 is CH.sub.2CH.sub.2COOH; and R.sup.4 is
CH.dbd.CH.sub.2.
Description
[0001] This is a continuation-in-part of U.S. Ser. No. 08/209,473
filed Mar. 14, 1994, the contents of which are incorporated herein
by reference.
TECHNICAL FIELD
[0002] The invention is in the field of photodynamic therapy,
specifically related to ocular conditions. More particularly, the
invention concerns the use of green porphyrins in photodynamic
therapeutic treatment of conditions characterized by unwanted
neovasculature in the eye.
BACKGROUND ART
[0003] Choroidal neovascularization leads to hemorrhage and
fibrosis, with resultant visual loss in a number of eye diseases,
including macular degeneration, ocular histoplasmosis syndrome,
myopia, and inflammatory diseases. Age-related macular degeneration
(AMD) is the leading cause of new blindness in the elderly, and
choroidal neovascularization is responsible for 80% of the severe
visual loss in patients with this diseases. Although the natural
history of the disease is eventual quiescence and regression of the
neovascularization process, this usually occurs at the cost of
sub-retinal fibrosis and vision loss.
[0004] Current treatment of AMD relies on occlusion of the blood
vessels using laser photocoagulation. However, such treatment
requires thermal destruction of the neovascular tissue, and is
accompanied by full-thickness retinal damage, as well as damage to
medium and large choroidal vessels. Further, the subject is left
with an atrophic scar and visual scotoma. Moreover, recurrences are
common, and visual prognosis is poor.
[0005] Developing strategies have sought more selective closure of
the blood vessels to preserve the overlying neurosensory retina.
One such strategy is photodynamic therapy, which relies on low
intensity light exposure of photosensitized tissues to produce
photochemical effects. Photosensitizing dyes are preferentially
retained in tumors and neovascular tissue, which allows for
selective treatment of the pathologic tissue. As a result of the
invention, PDT may be used to cause vascular occlusion in tumors by
damaging endothelial cells, as well as a direct cytotoxic effect on
tumor cells.
[0006] Photodynamic therapy of conditions in the eye characterized
by neovascularization has been attempted over the past several
decades using the conventional porphyrin derivatives such as
hematoporphyrin derivative and Photofrin porfimer sodium. Problems
have been encountered in this context due to interference from eye
pigments. In addition, phthalocyanine has been used in photodynamic
treatment.
[0007] A newer photosensitizer, a member of the group designated
"green porphyrins", is in the class of compounds called
benzoporphyrin derivatives (BPD). This photosensitizer has also
been tested to some extent in connection with ocular conditions.
For example, Schmidt, U. et al. described experiments using BPD
coupled with low density lipoprotein (LDL) for the treatment of
Greene melanoma (a nonpigmented tumor) implanted into rabbit eyes
and achieved necrosis in this context (IOVS (1992) 33:1253 Abstract
2802) . This abstract also describes the success of LDL-BPD in
achieving thrombosis in a corneal neovascularization model. The
corneal tissue is distinct from that of the retina and choroid.
[0008] The present applicants have described treating choroidal
neovascularization using LDL-BPD in several abstracts published
Mar. 15, 1993. These abstracts include those by Schmidt-Erfurth, U.
et al. (abstract 2956); by Haimovici, R. et al. (abstract 2955);
and by Walsh, A. W. et al. (abstract 2954). In addition, Lin, S. C.
et al. described photodynamic closure of choroidal vessels using
liposomal BPD in (abstract 2953). All of the foregoing are
published in IOVS (1993) 34:1303. An additional abstract of the
present applicants describing LDL-BPD to inhibit choroidal
neovasculature is by Moulton, R. S. et al. (abstract 2294), IOVS
(1993) 34:1169.
[0009] The green porphyrins offer advantages in their selectivity
for neovasculature. The present applicants have further determined
that coupling of the green porphyrins to a carrier such as LDL or
as contained in a liposomal formulation provides an advantageous
delivery method for the drug to the desired ocular location.
DISCLOSURE OF THE INVENTION
[0010] The invention is directed to diagnosis and treatment of
certain conditions of the eye using photodynamic methods and
employing green porphyrins as the photoactive compounds. The green
porphyrins of the invention are described in U.S. Pat. Nos.
4,883,790; 4,920,143; 5,095,030; and 5,171,749, the entire contents
of which are incorporated herein by reference. These materials
offer advantages of selectivity and effectiveness when employed in
protocols directed to the destruction of unwanted ocular
neovasculature, especially in the choroid.
[0011] Accordingly, in one aspect, the invention is directed to a
method to treat conditions of the eye characterized by unwanted
neovasculature, which method comprises administering to a subject
in need of such treatment an amount of a liposomal formulation of
green porphyrin that will localize in said neovasculature; and
irradiating the neovasculature with light absorbed by the green
porphyrin.
[0012] In another aspect, the invention is directed to a method to
treat conditions of the choroid characterized by unwanted
neovascularization, such as AMD, which method comprises
administering to a subject in need of such treatment an amount of a
green porphyrin that will localize in the neovascularized choroid;
and irradiating the choroid with light absorbed by the green
porphyrin.
[0013] In still another aspect, the invention is directed to a
method to treat age-related macular degeneration (AMD) which method
comprises administering to a subject in need of such treatment an
amount of green porphyrin that will localize in the choroid and
irradiating the choroid with light absorbed by the green
porphyrin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows preferred forms of the green porphyrins useful
in the methods of the invention.
MODES OF CARRYING OUT THE INVENTION
[0015] In general, the green porphyrin is of a formula shown in
FIG. 1 or a mixture thereof.
[0016] Referring to FIG. 1, in preferred embodiments each of
R.sup.1 and R.sup.2 is independently selected from the group
consisting of carbalkoxyl (2-6C), alkyl (1-6C), arylsulfonyl
(6-10C), cyano and --CONR.sup.5CO wherein R.sup.5 is aryl (6-10C)
or alkyl (1-6C); each R.sup.3 is independently carboxyl,
carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone
thereof or is alkyl (1-6C); R.sup.4 is CH.dbd.CH.sub.2 or
--CH(OR.sup.4.)CH.sub.3 wherein R.sup.4. is H, or alkyl (1-6C)
optionally substituted with a hydrophilic substituent. Especially
preferred also are green porphyrins of the formula shown in FIGS.
1-3 or 1-4 or mixtures thereof.
[0017] More preferred are embodiments are those wherein the green
porphyrin is of the formula shown in FIGS. 1-3 or 1-4 or. a mixture
thereof and wherein each of R.sup.1 and R.sup.2 is independently
carbalkoxyl (2-6C); one R.sup.3 is carboxyalkyl (2-6C) and the
other R.sup.3 is an ester of a carboxyalkyl (2-6C) substituent; and
R.sup.4 is --CH.dbd.CH.sub.2 or --CH(OH) CH.sub.3.
[0018] Still more preferred are embodiments wherein green porphyrin
is of the formula shown in FIGS. 1-3 and wherein R.sup.1 and
R.sup.2 are methoxycarbonyl; one R.sup.3 is
--CH.sub.2CH.sub.2COOCH.sub.3 and the other R.sup.3 is
CH.sub.2CH.sub.2COOH; and R.sup.4 is CH.dbd.CH.sub.2; i.e.,
BPD-MA.
[0019] The green porphyrin is formulated into a delivery system
that delivers high concentrations to the target tissue. Such
formulations may include coupling to a specific binding ligand
which may bind to a specific surface component of the
neovasculature or by formulation with a carrier that delivers
higher concentrations to the target tissue.
[0020] In one preferred embodiment, the green porphyrin is prepared
as a liposomal formulation. Liposomal formulations are believed to
deliver the green porphyrin selectively to the low-density
lipoprotein component of plasma which, in turn acts as a carrier to
deliver the active ingredient more effectively to the
neovasculature. Increased numbers of LDL receptors have been shown
to be associated with neovascularization, and by increasing the
partitioning of the green porphyrin into the lipoprotein phase of
the blood, it appears to be delivered more efficiently to the
neovasculature. Green porphyrins, and in particular BPD-MA,
strongly interact with such 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
involving lipocomplexes, including liposomes, are described in U.S.
Pat. No. 5,214,036 and in U.S. Ser. No. 07/832,542 filed Feb. 5,
1992, the disclosures of both of these being incorporated herein by
reference. Liposomal BPD can also be obtained from Quadra Logic
Technologies, Inc., Vancouver, British Columbia.
[0021] When injected intravenously, BPD-MA is cleared from the
bloodstream with a half-life of about 10-30 minutes, with the
highest tissue levels being reached in about three hours after
administration by injection and declining rapidly in the first 24
hours. BPD-MA is cleared primarily via bile and feces (60%), with
only 4% being cleared via the kidneys and urine. Thus, skin
photosensitivity occurs with BPD-MA only transiently, with minimal
reactivity after 24 hours in in vivo models.
[0022] The green porphyrin can 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.
[0023] The dose of green porphyrin 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.
[0024] 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 photodynamic therapy, and time
interval between administration of the dose and the therapeutic
irradiation. All of these parameters should be adjusted to produce
significant damage to neovascular tissue without significant damage
to the surrounding tissue. Typically, the dose of green porphyrin
used is within the range of from about 0.1 to about 20 mg/kg,
preferably from about 0.15-2.0 mg/kg, and even more preferably from
about 0.25 to about 0.75 mg/kg.
[0025] Specifically, as the green porphyrin dose is reduced from
about 2 to about 1 mg/kg, the fluence required to close choroidal
neovascular tissue tends to increase, for example, from about 50 to
about 100 Joules/cm.sup.2.
[0026] After the photosensitizing green porphyrin has been
administered, the neovascular tissue or tumor being treated in the
eye is irradiated at the wavelength of maximum absorbance of the
green porphyrin, usually between about 550 and 695 nm. A wavelength
in this range is especially preferred for enhanced penetration into
bodily tissues.
[0027] As a result of being irradiated, the green porphyrin in its
triplet state is thought to interact with oxygen and other
compounds to form reactive intermediates, such as singlet oxygen,
which can cause disruption of 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
endothelial cells, with subsequent platelet adhesion,
degranulation, and thrombus formation.
[0028] The fluence during the irradiating treatment can vary
widely, depending on type of tissue, depth of target tissue, and
the amount of overlying fluid or blood, but preferably varies from
about 50-200 Joules/cm.sup.2.
[0029] The irradiance typically varies from about 150-900
mW/cm.sup.2, with the range between about 150-600 mW/cm.sup.2 being
preferred. However, the use of higher irradiances may be selected
as effective and having the advantage of shortening treatment
times.
[0030] The optimum time following green porphyrin 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.
As a specific example, an exposure type of 1-20 minutes is often
appropriate for retinal neovascular tissue, about 120 minutes for
choroidal neovascular tissue, and up to about three hours for
tumors. Thus, effective vascular closure generally occurs at times
in the range of about one minute to about three hours following
administration of the green porphyrin.
[0031] The time of light irradiation after administration of the
green porphyrin may be important as one way of maximizing the
selectivity of the treatment, thus minimizing damage to structures
other than the target tissues. For a primate, it is believed that
the green porphyrin begins to reach the retinal vasculature by
about 7-15 seconds following administration. Typically, the green
porphyrin persists for a period of about 5-15 minutes, depending on
the dose given. Treatment within the first five minutes following
administration of the green porphyrin should generally be avoided
to prevent undue damage to retinal vessels still containing
relatively high concentrations of the green porphyrin.
[0032] Clinical examination and fundus photography typically reveal
no color change immediately following photodynamic therapy,
although a mild retinal whitening occurs in some cases after about
24 hours. Closure of choroidal neovascularization, however, is
preferably confirmed histologically by the observation of damage to
endothelial cells. Vacuolated cytoplasm and abnormal nuclei can
become apparent as early as 1-2 hours following photodynamic
therapy, with disruption of neovascular tissue typically becoming
more apparent by about 24 hours after light treatment. Associated
damage to the retinal pigment epithelium (RPE), pyknotic nuclei in
the outer nuclear layer, and loss of photoreceptors may also be
observed. However, the inner retina usually appears relatively
undamaged, as shown by control studies using photodynamic therapy
with BPD-MA on a normal retina and choroid showing no damage to
large choroidal and retinal vessels.
[0033] Closure can usually be observed angiographically by about 40
seconds to a minute in the early frames by hypofluorescence in the
reated areas. During the later angiographic frames, a corona of
hyperfluorescence begins to appear and 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 perfuse following
photodynamic therapy, but tend to demonstrate late staining.
[0034] Minimal retinal damage is generally found on histopathologic
correlation and is dependent on the fluence and the time interval
after irradiation that the green porphyrin is administered.
Histopathologic examination usually reveals vessel remnants in the
area of choroidal neovascular tissue, but the retinal vessels
typically appear normal. Further, there is no indication of
systemic toxicity, and cutaneous photosensitization does not appear
to develop.
[0035] As a result of the invention, photodynamic therapy can be
used more selectively, relying on the low intensity light exposure
of green porphyrins that have become localized within vascular
tissue. Complications, such as hemorrhage, are not noted with the
invention method. Thus, photodynamic therapy with a green porphyrin
appears to have broad application to clinical ophthalmology in
treating such diseases as age-related macular degeneration,
neovascular glaucoma, and persistent disc neovascularization in
diabetic retinopathy.
[0036] The following examples are to illustrate but not to limit
the invention.
EXAMPLE 1
Control of Experimental Choroidal Neovascularization Using PDT with
BPD-MA/LDL at Low Irradiance
[0037] Cynomolgus monkeys weighing 3-4 kg were anesthetized with an
intramuscular injection of ketamine hydrochloride (20 mg/kg),
diazepam (1 mg/kg), and atropine (0.125 mg/kg), with a supplement
of 5-6 mg/kg of ketamine hydrochloride as needed. For topical
anesthesia, proparacaine (0.5%) was used. The pupils were dilated
with 2.5% phenylephrine and 0.8% tropicamide.
[0038] Choroidal neovascularization was produced in the eyes of the
monkeys using a modification of the Ryan model, in which burns are
placed in the macula, causing breaks in Bruch's membrane, with a
Coherent Argon Dye Laser #920, Coherent Medical Laser, Palo Alto,
Cailf. (Ohkuma, H. et al. Arch. Ophthalmol. (1983) 101 : 1102-1110;
Ryan, S. J. Arch Ophthalmol (1982) 100:1804-1809). Initially, a
power of 300-700 mW for 0.1 seconds was used to form spots of about
100 .mu.m, but improved rates of neovascularization were obtained
with 50.mu. spots formed using a power of about 300-450 mW for 0.1
second.
[0039] The resulting choroidal neovascularizations were observed by
(1) fundus photography (using a Canon Fundus CF-60Z camera, Lake
Success, Long Island, N.Y.); (2) by fluorescein angiography (for
example, by using about 0.1 ml/kg body weight of 10% sodium
fluorescein via saphenous vein injection); and (3) histologic
examination by light and electron microscopy.
[0040] Immediately before use, BPD-MA was dissolved in dimethyl
sulfoxide (Aldrich Chemical Co., Inc., Milwaukee, Wis.) at a
concentration of about 4 mg/ml. Dulbeccos phosphate buffered salt
solution (Meditech, Washington, D.C.) was then added to the stock
to achieve a final BPD concentration of 0.8 mg/ml. Human
low-density-lipoprotein (LDL) prepared from fresh frozen plasma was
added at a ratio of 1:2.5 mg BPD-MA:LDL. The green porphyrin dye
and dye solutions were protected from light at all times. After
mixing, the dye preparation was incubated at 37.degree. for 30
minutes prior to intravenous injection. The monkeys were then
injected intravenously via a leg vein with 1-2 mg/kg of the BPD-MA
complexed with LDL over a five-minute period, followed by a flush
of 3-5 cc of normal saline.
[0041] Following this intravenous injection, the eyes of the
monkeys were irradiated with 692 nm of light from an argon/dye
laser (Coherent 920 Coherent Medical Laser, Palo Alto, Calif.),
using a Coherent LDS-20 slit lamp. The standard fiber was coupled
to larger 400 .mu.m silica optical fiber (Coherent Medical Laser,
Pal Alto, Calif.) to allow larger treatment spots as desired.
Seventeen (17) areas of choroidal neovascularization were treated
using a 1250 .mu.m spot.. Treatment spot sizes were confirmed at
the treatment plane using a Dial caliper micrometer. Some areas of
choroidal neovascularization were treated with several adjacent
treatment spots to treat the whole area of choroidal
neovascularization. One large choroidal neovascular membrane was
treated with photodynamic therapy to the nasal half only.
[0042] The photodynamic irradiation treatments were carried out
with a plano fundus contact lens (OGFA, Ocular Instruments, Inc.,
Bellvue, Mass.). Power was verified at the cornea by a power meter
(Coherent Fieldmaster, Coherent, Auborn, Calif.). The fluence at
each treatment spot was 50, 75, 100 or 150 Joules/cm.sup.2.
Initially, the irradiance was set at 150 mW/cm.sup.2 to avoid any
thermal effect but, as the experiment proceeded, the irradiance was
increased to 300 mW/cm.sup.2 or 600 mW/cm.sup.2 to reduce the
treatment duration time. The time interval between injection of the
green porphyrin dye and the treatment irradiating step ranged from
about 1 to about 81 minutes.
[0043] A number of different combinations of parameter values were
studied and are summarized below in Table 1:
1TABLE 1 IRRADIANCE AT 150 mW/cm.sup.2 Number Dye Duration of Time
after of CNV dose Fluence Treatment Injection Closure by Treated
(mg/kg) (J/cm.sup.2) (mins) (mins) Angiography 2 2 50 5.6 18, 38
2/2 1 2 75 8.3 81 1/1 1 2 100 11.2 22 1/1 2 1 50 5.6 5, 30 0/2 3 1
100 11.2 1, 2 and 5 3/3 4 1 150 16.6 14-43 3/4
[0044] "Dye only" controls, which were exposed to dye but not to
laser light, were examined in the areas of normal retina/choroid.
Areas of choroidal neovascularization were examined
angiographically and histologically. "Light only" controls were not
performed, since the irradiances used for photodynamic therapy were
well below the levels used for clinical laser photocoagulation. (In
a related experiment, a minimally detectable lesion using
"light-only" required an irradiance of 37 W/cm.sup.2, about 100
times the light levels used for photodynamic therapy.)
[0045] Following photodynamic therapy, the monkeys were returned to
an animal care facility. No attempt was made to occlude the
animals' eyes, but the room in which they were housed was darkened
overnight.
[0046] The condition of the choroidal neovasculature was followed
by fundus photography, fluorescein angiography, and histologic
examination. In particular, the eyes of the monkeys were examined
by fluorescein angiography acutely and at 24 hours after the
photodynamic therapy was given. In some cases, follow-up by
fluorescein angiography was performed at 48 hours and at one week,
until the eyes were harvested and the animals killed at the
following time points: acutely, at 24 hours, 48 hours, and 8 days
following photodynamic therapy. Animals were sacrificed with an
intravenous injection of 25 mg/mg Nembutal.
[0047] To perform the histologic examination, all eyes were
enucleated under deep anesthesia and fixed overnight in modified
Karnovsky's fixative, and then transferred to 0.1M phosphate
buffer, pH 7.2 at 4.degree. C. Both light microscopy and electron
microscopy were used for these studies. For light microscopy,
tissue samples were dehydrated, embedded in epon and serially
sectioned at one micron. The sections were stained with tolnizin
blue and examined with an Olympus photomicroscope. For electron
microscopy, tissue samples were post-fixed in 2% osmium tetroxide
and dehydrated in ethanol. Sections were stained with uranyl
acetate in methanol, stained with Sato's lead stain, and examined
with a Philips #CM 10 transmission electron microscope.
[0048] Using the low irradiance level of 150 mW/cm.sup.2 to
minimize any thermal component of the treatment, green porphyrin
doses of 1-2 mg/kg of BPD-MA/LDL, and fluences of 50-150
Joules/cm.sup.2, choroidal neovascularization was effectively
closed. Using the higher 2mg/kg dose effectively closed choroidal
neovascularizations at even the lowest 50 Joules/cm.sup.2 fluence.
When the green porphyrin dose was decreased to the decrease the
damage to surrounding tissues to 1 mg/kg of BPD-MA/LDL, the fluence
required to effectively close choroidal neovascular tissue
increased to 100 Joules/cm.sup.2. At 100 and 150 Joules/cm.sup.2,
the treated choroidal neovascular tissue was angiographically
closed, as shown by hypofluorescence in the area of treatment.
[0049] Prior to photodynamic therapy, the areas of choroidal
neovascularization exhibited a gray sub-retinal elevation that
leaked profusely on fluorescein angiography. There was no apparent
color change in the treated areas either during or immediately
after photodynamic treatment. However, 24 hours after the
irradiating step, there was mild retinal whitening in the treated
areas.
[0050] Further fluorescein angiography showed hypofluorescence in
the treated areas, with no apparent filling of the associated
neovascular tissues. Retinal vessels within the treated areas were
perfused, but stained later. A hyperfluorescent rim at the border
of the treated area was apparent in the later frames of the
angiograph, and the rim then progressed to fill the treated area.
Although mild staining of retinal vessels was noted
angiographically, no complications, such as serous retinal
detachment or hemorrhage, were noted.
[0051] On histopathologic examination of the 2 mg/kg dose samples,
there was marked disruption of the treated choroidal neovascular
tissue with disrupted endothelial cells. The choriocapillaris was
also occluded. Although large choroidal vessels were unaffected,
extravasated red blood cells were noted in the choroid. Retinal
pigment epithelium (RPE) damage was noted as well with vacuolated
cells, with the outer nuclear layer demonstrating pyknotic nuclei
and disrupted architecture. No histologic abnormality of the
retinal vessels was seen.
[0052] Histopathologic examination of the 1 mg/kg dose samples
showed damage to endothelial cells in the choroidal neovascular
tissue, with abnormal nuclei and disrupted cytoplasm in the
endothelial cells. The lumens of the vessels in the choroidal
neovascular tissue were occluded by fibrin acutely and were closed
by 24 hours after treatment. Closure of the choriocapillaris was
also noted. At 24 hours, the retinal pigment epithelium (RPE)
appeared abnormal with vacuolated cytoplasm. Pyknotic nuclei in the
inner and outer layer indicated damage secondary to the laser
injury used to induce the neovascularization in this model. Retinal
vessels appeared to be undamaged.
[0053] Choroidal neovascular tissue that was treated and followed
for eight days showed persistent closure, as shown by
hypofluorescence in the early frames of the angiogram.
Histologically, the treated areas demonstrated degraded vessel
lumens empty of debris. The choriocapillaris was sparse but patent
in the treated area. In contrast, areas of choroidal
neovascularization not treated by photodynamic therapy demonstrated
branching capillaries between Bruch's membrane and the outer
retina.
[0054] No adverse effects of photodynamic therapy with the green
porphyrin were noted. There was no associated serous retinal
detachment, retinal or sub-retinal hemorrhage, or post-treatment
inflammation. Further, no adverse systemic effects of the dye
administration were noted. However, the low irradiance forced
treatment times to be long--about 16.6 minutes to yield 150
Joules/cm.sup.2.
EXAMPLE 2
Control of Experimental Choroidal Neovascularization Using PDT with
BPD-MA/LDL at Higher Irradiances
[0055] To make clinical treatments shorter, additional experiments
were performed using higher irradiance values. Experience with
higher irradiance indicated that no thermal damage would take place
with irradiances as high as 1800 mW/cm.sup.2. Moulton et al.,
"Response of Retinal and Choroidal Vessels to Photodynamic Therapy
Using Benzoporphyrin Derivative Monoacid", IOVS 34, 1169 (1993),
Abstract 2294-58. Therefore, irradiances of 300 mW/cm.sup.2 and 600
mW/cm.sup.2 were also used to treat choroidal neovascular tissue in
accordance with the procedures described in Example 1. The results
showed that shortened treatment times effectively closed the
choroidal neovascular tissue, as indicated below in Table 2.
2TABLE 2 IRRADIANCE OVER 150 mW/cm.sup.2 Duration Closure Number
Dye of Time after by of CNV dose Fluence Irradiance Treatment
Injection Angio- Treated (mg/kg) (J/cm.sup.2) (mW/cm.sup.2) (mins)
(mins) graphy 2 1 150 300 8.3 5, 53 2/2 2 1 150 600 4.7 22, 69
2/2
[0056] Occlusion of the choroidal neovascular tissue and subjacent
choriocapillaris was observed, as well as damage to the retinal
pigment epithelium and outer retina.
EXAMPLE 3
Control of Experimental Choroidal Neovascularization Using PDT with
BPD-MA Liposomes
[0057] The following experiment of photodynamic therapy using a
liposomal preparation of BPD-MA was conducted to determine the
optimal time interval after intravenous injection as a bolus of the
BPD-MA over about 20 seconds, followed by a 3-5 cc saline flush, to
begin the irradiating step. Choroidal neovascularization in
cynomolgus monkeys was treated to demonstrate efficacy of the
photodynamic therapy. Normal choroid tissue was treated to assess
relative damage to adjacent tissues.
[0058] The monkeys were initially injected with a green porphyrin
dose of 1 mg/kg. At predetermined time intervals following this
injection, the eyes of the monkeys were irradiated with an
irradiance of 600 mW/cm.sup.2, and a fluence of 50 J/cm.sup.2. The
irradiating light was from an argon/dye laser (Coherent 920
Coherent Medical Laser, Palo Alto, Calif.) equipped with a 200
micron fiber adapted through a LaserLink (Coherent Medical Laser)
and a split lamp delivery system (Coherent). Other than these
differences, the eye membranes were treated in the same manner as
described in Example 1. All areas of treated choroidal
neovasculature for all time points after the liposomal BPD-MA
injection showed whitening of the retina and early hypofluorescense
on fluorescein angiography when measured one week after treatment.
On histology, there was evidence of partial closure of choroidal
neovasculature at the early time points, no effect at mid-time
points, and more effective closure at late irradiation time points,
e.g., at 80 and 100 minutes.
[0059] The normal choroid treated with the same parameters showed
whitening of the retina, early hypofluorescence at all time points,
and histologic evidence of choriocapillaris (c-c) accompanied by
damage to the choroid and retina, particularly at early time
points.
EXAMPLE 4
Using PDT with BPD-MA Liposomes at Lower Green Porphyrin Doses
[0060] Using the general procedure of Example 1, additional
experiments were performed using the intravenous injection of
liposomal BPD-MA at doses of 0.25, 0.5 and 1 mg/kg. Photodynamic
therapy was performed with an irradiance of 600 mW/cm.sup.2, a
fluence of 150 J/cm.sup.2, and a treatment duration of four
minutes, nine seconds.
[0061] The effects of treatment were assessed by fundus photography
and fluorescein angiography, and then confirmed by light and
electron microscopy. Photodynamic therapy of normal choroid tissue
demonstrated the effect on adjacent structures, such as the retina,
while the treatment of choroid neovascular tissue demonstrated
efficacy.
[0062] Table 3 below describes the lesions produced on normal
choroids by administration of 0.5 mg/kg BPD-MA at time points
ranging from 5 to 60 minutes:
3TABLE 3 0.5 mg/kg, NORMAL CHOROID Time after injection min
Fluorescein Angiography Histology 5 Hypofluorescence c-c and large
choroidal vessel closure; outer and inner retina damage. 20
Hypofluorescence; cc closure; damage retinal vessels - to outer
retina normal 40 Mild early cc open (not center hypofluorescence of
lesion); outer retina damage 60 Early cc closed; outer
hypofluorescence; retina damage; less than the 20- inner retina
fairly minute lesion good. described above
[0063] When 0.5 mg/kg BPD-MA was also used to treat choroidal
neovasculature under the same conditions, marked hypofluorescence,
corresponding to closure of choroid neovasculature was exhibited in
areas irradiated at times of 5, 20 and 40 minutes after injection.
When 50 minutes after injection were allowed to elapse before
photodynamic irradiation was begun, there was less hypofluorescence
and presumably less effective closure.
[0064] The study was then repeated with the green porphyrin dose
decreased to 0.25 mg/kg. Table 4 below describes the lesions
produced on normal choroids by treatments with 0.25 mg/kg, 600
mW/cm.sup.2, and 150 J/cm.sup.2 at time points ranging from 5 to 60
minutes:
4TABLE 4 0.25 mg/kg, NORMAL CHOROID Time after injection min
Fluorescein Angiography Histology 10 Early c-c closure;
hypofluorescence choroidal vessel - normal; RPE damaged; retinal
vessels - normal; mild damage to outer retina 20 Early Same as
10-minute hypofluorescence lesion above 40 Faint early Patchy cc
closure; hypofluorescence; less damage to RPE late staining and
outer retina 60 Not demonstrated No effect on cc; mild
vacuolization of RPE
[0065] When the above study was repeated using the same green
porphyrin dose of 0.25 mg/kg and irradiance of 600 mW/cm.sup.2, but
with a reduced fluence of 100 J/cm.sup.2, the same angiographic and
histologic pattern was exhibited as described above. However, cc
was open in the 40-minute lesion.
[0066] In the last portion of these experiments, a green porphyrin
dose of 0.25 mg/kg was used to treat experimental choroidal
neovascularization with an irradiance of 600 mW/cm.sup.2 and a
fluence of 150 J/cm.sup.2 at elapsed time points ranging from 5 to
100 minutes. This combination of conditions caused effective cc
closure with only minimal damage to the outer retina. The results
are shown in Table 5 below:
5TABLE 5 0.25 mg/kg, PDT over CNV Time after injection (min)
Fluorescein Angiography Histology 5 Early Partially closed
hypofluorescence CNV; c-c closed; damage to inner retina 20 Early
CNV - open vessel, hypofluorescence; fibrin and clots; less than
the 5- inner retina looks minute lesion fine 30 Some Minimal effect
on hypofluorescense CNV next to CNV 40 Hypofluorescence; Minimal
effect on questionable change CNV compared to previous reaction 60
Hypofluorescence Minimal effect on CNV 80 Hypofluorescence Partial
closure of CNV; retina over CNV looks intact 100 Hypofluorescence
CNV partially closed
[0067] Thus, fluorescein angiography and histopathology in the
above series of experiments demonstrated early hypofluorescence at
early time points. Further, the histopathology study showed partial
CNV closure at all time points after injection using 80 and 100
minutes as the post-injection interval before the irradiating
treatment.
[0068] In summary, acceptable destruction of choroidal neovascular
tissue at all tested doses of BPD-MA was shown by fluorescein
angiography and histology. However, the lower doses appeared to
increase selectivity, as assessed by treatment of a normal choroid.
Effective choriocapillaris closure in normal choroids with minimal
retinal damage was produced by irradiating at a time about 10
minutes, 20 seconds after injection of the green porphyrin at a
dose of 0.25 mg/kg. By adjusting the dose, the time of irradiation
after green porphyrin injection, and fluence, one can improve even
further the selectivity of the green porphyrin. However, the
liposomal preparation of BPD-MA was clearly demonstrated to be a
potent photosensitizer.
EXAMPLE 5
Additional Data Using Liposomal BPD
[0069] Using the techniques of Examples 1-4, a total of 61 areas of
experimental CNV in 9 monkeys were treated with PDT using BPD-MA.
Effective CNV closure was demonstrated by fluorescein angiography
at all tested dye doses: 1, 0.5, 0.375, and 0.25 mg/kg. The lower
the dose, the shorter the time interval after dye injection in
which laser irradiation produced CNV closure.
[0070] The fundus appearance was unchanged immediately after
treatment, and only slight deep retinal whitening corresponding to
the laser irradiation spot appears 24 hours later. CNV closure was
determined angiographically at 24 hours by early hypofluorescence
corresponding to the treated area. As the angiogram progressed most
lesions demonstrated staining starting at the periphery of the
lesion.
[0071] Table 6 summarizes the effect of PDT on CNV, using different
dye doses and variable treatment times after dye injection. PDT
using a dye dose of 1 mg/kg was performed over 7 membranes in 1
monkey. Laser irradiation was performed at each of the following
times after dye injection: 5, 20, 40, 60, 80, 100 and 120 minutes.
CNV closure was induced in all lesions when irradiation was
performed 5-100 minutes after dye injection.
6TABLE 6 Angiographic Closure of CNV Time (min) of Dye Dose No.
Lesions Rx after dye mg/kg CNV injection CNV closure 1 7 5-100 6/7
>100 0/1 0.5 11 <60 7/8 60-100 0/3 0.375 29 <50 16/18
50-100 3/11 0.25 14 <20 2/2 20-40 2/4 40-100 0/8
[0072] PDT using dye dose of 0.5 mg/kg was performed on 11
membranes in 2 monkeys, with laser irradiation at 10, 20, 30, 40,
50, 60, 80, 100 minutes after dye injection. PDT effect was
assessed 24 hours after treatment. CNV closure was induced in 7/11
membranes, that were irradiated at 10, 20, 30, 40 and 50 minutes
after dye injection. Only 1/2 membranes irradiated at 50 minutes
after dye injection showed angiographic closure. The treatments
performed 60 minutes and more after dye injection showed no
angiographic closure of the membranes.
[0073] 29 areas of CNV in 5 monkeys were treated with PDT using
BPD-MA at dose of 0.375 mg/kg. All treated CNV membranes were
assessed angiographically at 24 hours. As indicated in Table 6, 7/8
CNV irradiated within 50 minutes after injection demonstrated
angiographic closure. Only 3/11 membranes irradiated more than 50
minutes after dye injection demonstrated angiographic closure.
[0074] A dye dose of 0.25 mg/kg was found to be the threshold dose
for PDT using a light dose of 150 J/cm.sup.2 and 600 mW/cm.sup.2.
CNV closure was demonstrated in 2/2 membranes that were irradiated
within 20 minutes after dye injection. Only 2/4 CNV irradiated
20-40 minutes after dye injection showed closure. No effect was
demonstrated in the CNV that were irradiated more than 40 minutes
after dye injection.
[0075] Histologic confirmation of CNV closure was evident at all
tested dye doses: 1, 0.5, 0.375, and 0.25 mg/kg.
[0076] On light microscopy the closed CNV showed vessels packed
with red blood cells (RBCs), occasional extravasated RBCs and
pockets of fibrin within the tissue as well as in the subretinal
space. Most of the stromal cells appeared undamaged.
[0077] On electron microscopy the closed vessels appeared packed
with RBCs and platelets. The endothelial cells were missing or
severely damaged. Extravasated RBCs and occasional white blood
cells (WBCS) were found near the vessel remnants. At 0.25 mg/kg the
vessels were packed with RBCs but the endothelial cells seemed to
be surviving the treatment.
[0078] Treatment Selectivity
[0079] Treatment selectivity was investigated by performing PDT in
normal retina/choroid using, the same dye doses and time points of
laser irradiation after dye injection. In most cases the closure of
the choriocapillaris in normal choroid followed a similar time
course as the closure of CNV. When PDT was performed using dye
doses of 0.5, 0.375, 0.25 mg/kg, the retinal structure was well
preserved. In none of the cases were retinal detachment or
hemorrhage observed. reducing the dye dose resulted in more
selective closure of the choriocapillaris with minimal damage to
the adjacent tissues. RPE cells were typically damaged at all dye
doses.
[0080] The assessment of the damage to the retina and choroid was
graded according to the histologic findings for the retina/choroid
at different levels, as follows:
7 Grade 1: RPE only or RPE + slight photoreceptor changes +
occasional pyknosis in the ONL; with or without choriocapillaris
(c-c) closure; Grade 2: Choriocapillaris closure + RPE +
photoreceptors + 10-20% pyknosis in the ONL; Grade 3: C-c closure +
RPE + photoreceptors + ONL pyknosis >50%; Grade 4: C-c closure +
RPE + photoreceptors + ONL pyknosis >50%; Grade 5: C-c closure +
RPE + photoreceptors + ONL pyknosis >50% + choroidal vessel
damage or retinal vessel or inner retinal damage;
[0081] A total of 38 PDT spots were placed in normal
retina/choroid. The treatment parameters and the degree of effect
are summarized in Table 7.
8TABLE 7 PDT effect on normal retina/choroid Time (min) No. of
lesions per of Rx after histologic grading Dye Dose dye injection
No. Lesions 1 2 3 4 5 1 mg/kg <60* 2 2 60-100* 3 3 0.5 mg/kg
<20 1 1 20-60 3 3 0.375 mg/kg <20 3 1 1 1 20-50 9 2 4 2 1
50-100 11 1 5 5 0.25 mg/kg <20 1 1 20-40 1 1 40-60 2 2 *The
lesions irradiated at 40 and 120 minutes were not identified
histologically.
[0082] PDT using a dye dose of 1 mg/kg led to damage of both inner
and outer retina. The early treatments (5 and 20 minutes after dye
injection) demonstrated grade 5 effect with damage to the inner
retina, and lesions induced 60 minutes and more after dye injection
showed a grade 4 effect.
[0083] At 0.5 mg/kg, only the lesion irradiated 5 minutes after dye
injection demonstrated damage to the inner retina (grade 5).
Lesions irradiated at 20 minutes and later did not affect the inner
retina, but showed pyknosis in the outer nuclear layer (ONL),
vacuolization and disorganization of the photoreceptors' inner and
outer segments, and damage to the RPE (grade 4).
[0084] At 0.375 mg/kg, 2/3 lesions irradiated 10 minutes after dye
injection showed some congestion of the small retinal vessels, but
the inner-nuclear layer (INL) was preserved. Lesions applied 20
minutes and later after dye injection showed some pyknosis in the
ONL, some vacuolization and disorientation of the photoreceptors'
inner and outer segments, and damage to the RPE. Most lesions
demonstrated damage of 1 or 2 or 3, with some lesions demonstrated
grade 4 damage.
[0085] 0.25 mg/kg was found to be a threshold dose for induction of
choriocapillaris closure. This was achieved with almost no effect
on the overlying retina. There was mild damage to some RPE cells,
minimal swelling of photoreceptors, and a few pyknotic nuclei in
the ONL.
[0086] "Dye only" control areas of normal retina/choroid showed no
effect by fluorescein angiography or histologic examination.
* * * * *