U.S. patent application number 09/922350 was filed with the patent office on 2002-02-28 for use of texaphyrin in ocular diagnosis and therapy.
This patent application is currently assigned to Pharmacyclics, Inc.. Invention is credited to Blumenkranz, Mark S., Miller, Richard A., Woodburn, Kathryn W., Young, Stuart W..
Application Number | 20020025298 09/922350 |
Document ID | / |
Family ID | 46256498 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025298 |
Kind Code |
A1 |
Blumenkranz, Mark S. ; et
al. |
February 28, 2002 |
Use of texaphyrin in ocular diagnosis and therapy
Abstract
The use of texaphyrins for ocular diagnosis and therapy is
provided, especially use of photosensitive texaphyrins for
photodynamic therapy of conditions of the eye characterized by
abnormal vasculature, such as macular degeneration, or pterygium,
for example. The photosensitive texaphyrin may be a free-base
texaphyrin or may be metallated with a diamagnetic metal.
Preferably, the photosensitive texaphyrin is metallated with
lutetium. Due to the dual wavelength absorption of texaphyrins,
i.e., at 400-500 nm and at 700-800 nm, especially about 732 nm, as
compared to porphyrins, texaphyrins are more effective and
versatile for use in humans as compared to porphyrins. Texaphyrins
serve as effective contrast agents in ocular angiography.
Inventors: |
Blumenkranz, Mark S.;
(Portola Valley, CA) ; Woodburn, Kathryn W.;
(Sunnyvale, CA) ; Miller, Richard A.; (Portola
Valley, CA) ; Young, Stuart W.; (Portola Valley,
CA) |
Correspondence
Address: |
Vinit G. Kathardekar
Pharmacyclics Inc.
995 E. Arques Avenue
Sunnyvale
CA
94085-4521
US
|
Assignee: |
Pharmacyclics, Inc.
|
Family ID: |
46256498 |
Appl. No.: |
09/922350 |
Filed: |
August 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09922350 |
Aug 2, 2001 |
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09329720 |
Jun 10, 1999 |
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6270749 |
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09329720 |
Jun 10, 1999 |
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PCT/US97/22661 |
Dec 11, 1997 |
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60093058 |
Dec 11, 1996 |
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Current U.S.
Class: |
424/9.6 ;
514/185; 514/410; 604/20 |
Current CPC
Class: |
A61K 49/0021 20130101;
A61K 41/0076 20130101; Y10S 514/912 20130101 |
Class at
Publication: |
424/9.6 ;
514/185; 514/410; 604/20 |
International
Class: |
A61K 049/00; A61K
031/555; A61K 031/407 |
Claims
1. A method for treating an ocular condition characterized by
abnormal vasculature of a subject; the method of comprising:
administering a photosensitive texaphyrin to the subject; and
photoirradiating the vasculature.
2. The method of claim 1 wherein the photosensitive texaphyrin is
fluorescent and the treating is further in combination with
observing the ocular condition of the subject by observing
fluorescence of the texaphyrin.
3. The method of claim 1 wherein the ocular condition is selected
from the group consisting of macular degeneration, glaucoma, disc
neovascularization, retinal neovascularization, pannus, pterygium,
retinal neovasculature, choroidal neovasculature, ocular
histoplasmosis syndrome, myopia, ocular inflammatory disease,
central serous retinopathy, subretinal neovascular membrane, and
neovasculature induced by neoplasm.
4. The method of claim 1 wherein the ocular condition of the
subject is macular degeneration.
5. The method of claim 1 where the photosensitive texaphyrin has
structure I: 3or structure II: 4wherein M is a diamagnetic metal
cation; R.sub.1-R.sub.4, R.sub.7 and R.sub.8 are independently
hydrogen, halide, hydroxyl, alkyl, alkenyl, alkynyl, aryl,
haloalkyl, nitro, formyl, acyl, hydroxyalkyl, alkoxy,
hydroxyalkoxy, hydroxyalkenyl, hydroxyalkynyl, saccharide, carboxy,
carboxyalkyl, carboxyamide, carboxyamidealkyl, amino, aminoalkyl, a
site-directing molecule, a catalytic group, or a couple that is
coupled to a site-directing molecule or to a catalytic group;
R.sub.6 and R.sub.9 are independently selected from the groups of
R.sub.1-R.sub.4, R.sub.7 and R.sub.8, with the proviso that the
halide is other than iodide and the haloalkyl is other than
iodoalkyl; R.sub.5 and R.sub.10-R.sub.12 are independently
hydrogen, alkyl, alkenyl, alkynyl, aryl, hydroxyalkyl, alkoxy,
hydroxyalkoxy, hydroxyalkenyl, hydroxyalkynyl, carboxyalkyl,
carboxyamide, carboxyamidealkyl, amino, aminoalkyl, or a couple
that is coupled to a saccharide, to a site-directing molecule, or
to a catalytic group; R.sub.13 is alkyl, alkenyl, oxyalkyl, or
hydroxyalkyl having up to about 3 carbon atoms and having
rotational flexibility around a first-bound carbon atom; and n is
an integer value less than or equal to 5.
6. The method of claim 1 wherein the photosensitive texaphyrin is
selected from the group consisting of texaphyrins A1-A108 of Tables
A and B.
7. The method of claim 1 wherein the photosensitive texaphyrin is
bound to a diamagnetic metal cation and the diamagnetic metal
cation is Lu(III), La(III), In(III), Zn(II) or Cd(II).
8. The method of claim 1 wherein the photosensitive texaphyrin is
bound to a diamagnetic metal cation and the diamagnetic metal
cation is Lu(III).
9. The method of claim 1 wherein the photoirradiating is with light
having a wavelength range of about 700 to 800 nanometers.
10. The method of claim 1 wherein the photoirradiating is with
light having a wavelength range of about 725-740 nanometers.
11. The method of claim 1 further comprising: administering a
paramagnetic texaphyrin to the subject, and imaging the subject by
magnetic resonance imaging.
12. The method of claim 1 wherein the photosensitive texaphyrin is
LuT2BET.
13. A method for observing vasculature in an eye of a subject, the
method comprising: administering a detectable texaphyrin to the
subject; and observing the vasculature of the eye.
14. The method of claim 13 where the detectable texaphyrin is
fluorescent and the observing is by fluorescence.
15. The method of claim 14 where the fluorescent texaphyrin is in a
metal complex with Y(III), Lu(III), or Gd(III).
16. The method of claim 14 where excitation light to induce
fluorescence has a wavelength of about 400-500 nm.
17. The method of claim 14 where excitation light to induce
fluorescence has a wavelength of about 700-800 nm.
18. The method of claim 13 where the detectable texaphyrin is
complexed with a paramagnetic cation and the observing is by
magnetic resonance imaging.
19. The method of claim 13 where the observing is by x-ray
imaging.
20. A method for photodynamic therapy of macular degeneration of a
subject comprising: administering a photosensitive texaphyrin to
the subject; and photoirradiating the macula.
21. The method of claim 20 where the photosensitive texaphyrin has
structure I: 5or structure II: 6wherein M is a diamagnetic metal
cation; R.sub.1-R.sub.4, R.sub.7 and R.sub.8 are independently
hydrogen, halide, hydroxyl, alkyl, alkenyl, alkynyl, aryl,
haloalkyl, nitro, formyl, acyl, hydroxyalkyl, alkoxy,
hydroxyalkoxy, hydroxyalkenyl, hydroxyalkeynl, saccharide, carboxy,
carboxyalky, carboxyamide, carboxyamiodealkyl, amino, aminoalkyl, a
site-directing molecule, a catalytic group, or a couple that is
coupled to a site-directing molecule or to a catalytic group;
R.sub.6 and R.sub.9 are independently selected from the groups of
R.sub.1-R.sub.4, R.sub.7 and R.sub.8, with the proviso that the
halide is other than iodide and the haloalkyl is other than
iodoalkyl; R.sub.5 and R.sub.10-R.sub.12 are independently
hydrogen, alkyl, alkenyl, alkynyl, aryl, hydroxyalkyl, alkoxy,
hydroxyalkoxy, hydroxyalkenyl, hydroxyalkynyl, carboxyalkyl,
carboxyamide, carboxyamidealkyl, amino, aminoalkyl, or a couple
that is coupled to a saccharide, to a site-directing molecule, or
to a catalytic group; R.sub.13 is alkyl, alkenyl, oxyalkyl, or
hydroxyalkyl having up to about 3 carbon atoms and having
rotational flexibility around a first-bound carbon atom; and n is
an integer value less than or equal to 5.
22. The method of claim 21 wherein the photosensitive texaphyrin is
selected from the group consisting of texaphyrins A1-A108 of Tables
A and B.
23. The method of claim 21 wherein the photosensitive texaphyrin is
bound to a diamagnetic metal cation and the diamagnetic metal
cation is Lu(III), La(III), In(III), Zn(II) or Cd(II).
24. The method of claim 21 wherein the photosensitive texaphyrin is
bound to a diamagnetic metal cation and the diamagnetic metal
cation is Lu(III).
25. A method for observing and treating an ocular condition
characterized by abnormal vasculature of a subject using a single
agent, the method comprising: administering a photosensitive
fluorescent texaphyrin to the subject; observing the ocular
condition of the subject by fluorescence of the texaphyrin; and
photoirradiating the vasculature.
26. The method of claim 5 wherein the photosensitive texaphyrin has
structure I.
27. The method of claim 26 wherein: R.sub.1-R.sub.4, R.sub.7 and
R.sub.8 are independently alkyl, hydroxyalkyl, or hydroxyalkoxy;
and R.sub.5, R.sub.6 and R.sub.9-R.sub.12 are hydrogen.
28. The method of claim 27 wherein R.sub.1 is hydroxyalkyl; and
R.sub.2, R.sub.3 and R.sub.4 are alkyl.
29. The method of claim 1 wherein the photosensitive texaphyrin is
administered by rapid infusion as a bolus.
30. The method of claim 29 comprising administering 0.01 to 5
.mu.mol/kg of the photosensitive texaphyrin in a sterile aqueous or
aqueous propylene glycol solution.
31. The method of claim 30 wherein the photosensitive texaphyrin is
LuT2BET.
32. The method of claim 21 wherein the photosensitive texaphyrin
has structure I.
33. The method of claim 32 wherein: R.sub.1-R.sub.4, R.sub.7 and
R.sub.8 are independently alkyl, hydroxyalkyl, or hydroxyalkoxy;
and R.sub.5, R.sub.6 and R.sub.9-R.sub.12 are hydrogen.
34. The method of claim 33 wherein R.sub.1 is hydroxyalkyl; and
R.sub.2, R.sub.3 and R.sub.4 are alkyl.
35. The method of claim 21 wherein the photosensitive texaphyrin is
administered by rapid infusion as a bolus.
36. The method of claim 35 comprising administering 0.01 to 5
.mu.mol/kg of the photosensitive texaphyrin in a sterile aqueous or
aqueous propylene glycol solution.
37. The method of claim 36 wherein the photosensitive texaphyrin is
LuT2BET.
Description
[0001] This application is a continuation application of copending
international application PCT/US97/22661 filed Dec. 11, 1997, which
claims priority to USSN 60/093,058, filed Dec. 11, 1996. The
applications are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] The retina is a thin layer of light-sensitive tissue that
lines the inside wall of the back of the eye. When light enters the
eye, it is focused by the cornea and the lens onto the retina. The
retina then transforms the light images into electrical impulses
that are sent to the brain through the optic nerve.
[0003] The macula is a very small area of the retina responsible
for central vision and color vision. The macula allows us to read,
drive, and perform detailed work. Surrounding the macula is the
peripheral retina which is responsible for side vision and night
vision. Macular degeneration is damage or breakdown of the macula,
underlying tissue, or adjacent tissue. Macular degeneration is the
leading cause of decreased visual acuity and impairment of reading
and fine "close-up" vision. Age-related macular degeneration (ARMD)
is the most common cause of legal blindness in the elderly.
[0004] The most common form of macular degeneration is called "dry"
or involutional macular degeneration and results from the thinning
of vascular and other structural or nutritional tissues underlying
the retina in the macular region. A more severe form is termed
"wet" or exudative macular degeneration. In this form, blood
vessels in the choroidal layer (a layer underneath the retina and
providing nourishment to the retina) break through a thin
protective layer between the two tissues. These blood vessels may
grow abnormally directly beneath the retina in a rapid uncontrolled
fashion, resulting in oozing, bleeding, or eventually scar tissue
formation in the macula which leads to severe loss of central
vision. This process is termed choroidal neovascularization
(CNV).
[0005] CNV is a condition that has a poor prognosis; effective
treatment using thermal laser photocoagulation relies upon lesion
detection and resultant mapping of the borders. Angiography is used
to detect leakage from the offending vessels but often CNV is
larger than indicated by conventional angiograms since the vessels
are large, have an ill-defined bed, protrude below into the retina
and can associate with pigmented epithelium.
[0006] Neovascularization results in visual loss in other eye
diseases including neovascular glaucoma, ocular histoplasmosis
syndrome, myopia, diabetes, pterygium, and infectious and
inflammatory diseases. In histoplasmosis syndrome, a series of
events occur in the choroidal layer of the inside lining of the
back of the eye resulting in localized inflammation of the choroid
and consequent scarring with loss of function of the involved
retina and production of a blind spot (scotoma). In some cases, the
choroid layer is provoked to produce new blood vessels that are
much more fragile than normal blood vessels. They have a tendency
to bleed with additional scarring, and loss of function of the
overlying retina. Diabetic retinopathy involves retinal rather than
choroidal blood vessels resulting in hemorrhages, vascular
irregularities, and whitish exudates. Retinal neovascularization
may occur in the most severe forms.
[0007] Current diagnosis of ocular disorders often includes use of
a dye such as fluorescein or indocyanine green in an angiogram.
Fluorescein is a low molecular weight molecule that rapidly leaks
from vessels, however, due to its low absorption and emission
wavelengths and due to autofluorescence, only superficial
morphology can be imaged; subretinal hemorrhage blocks the
detection of the underlying chorioretinal vasculature. In contrast,
ICG, having an absorption band at 805 nm and fluorescence at 835
nm, is able to image deeper choroidal vessels better than
fluorescein due to spectral differences and also due to its longer
retainment in the diseased vasculature, possibly in association
with albumin. In this procedure, the dye is injected into the blood
stream through a vein in the arm. Special filters are placed in the
light path, and in front of the film, to permit the fluorescent dye
to be seen as it passes through the vessels in the retina. Pictures
of the vascular anatomy are taken of the retina and macula as the
dye passes through the blood vessels of the back of the eye.
Vascular occlusions or leakage of dye generally indicates abnormal
vasculature, however, a problem with fluorescein is that of
leakage. Optical coherence tomography is another technique that
uses noncontact imaging and provides high-depth resolution in
cross-sectional tomographs of the retina.
[0008] Current treatment of neovascularization relies on ablation
of blood vessels using thermal laser photocoagulation. However,
such treatment is nonselective, requires thermal destruction of the
tissue, and is accompanied by full-thickness retinal damage, as
well as damage to medium and large choroidal vessels. Further, the
patient is left with an atrophic scar and visual scotoma. Moreover,
recurrences are common, and the prognosis for the patient's
condition is poor.
[0009] Developing strategies have sought more selective closure of
the blood vessels to preserve the overlying neurosensory retina.
One such strategy is photodynamic therapy (PDT), a treatment
technique that uses a photosensitizing dye and non-damaging light
corresponding to the sensitizer's absorption profile to produce
cytotoxic materials, such as singlet oxygen, from benign precursors
when irradiated in the presence of oxygen. Other reactive species
such as superoxide, hydroperoxyl, or hydroxyl radicals may be
involved in the consequent irreversible damage to biological
components. At the doses used, neither the light nor the drug has
any independent activity against the target.
[0010] The effectiveness of PDT is predicated on three additional
factors: i) The photosensitive dyes used in PDT preferably have the
ability to localize at the treatment site as opposed to surrounding
tissue. ii) The high reactivity and short lifetime of activated
oxygen means that it has a very short range (.about.0.1 m) and is
unlikely to escape from the region in which it is produced;
cytotoxicity is therefore restricted to the precise region of
photoactivated drug. iii) Developments in light delivery, such as
lasers, light emitting diodes, and fiber optics, allow a beam of
intense, non-damaging, light to be delivered accurately to many
parts of the body. For a review of photodynamic therapy, see U.S.
Pat. No. 5,252,720 (incorporated by reference herein).
[0011] Photodynamic therapy of conditions in the eye characterized
by neovascularization has been attempted using conventional
porphyrin derivatives such as hematoporphyrin derivative
(dihematoporphyrin ether), PHOTOFRIN.RTM. porfimer sodium, and tin
ethyl etiopurpurin. Problems have been encountered in this context
due to interference from eye pigments, as described in U.S. Pat.
No. 5,576,013 to Williams, et al. for example. In addition,
phthalocyanine and benzoporphyrin derivatives have been used in
photodynamic treatment. PCT publication WO 95/24930 and Miller et
al., (Archives of Ophthalmology, June, 1995) relate to treatment of
eye conditions characterized by unwanted neovasculature comprising
administering a green porphyrin to the neovasculature and
irradiating the neovasculature with light having a wavelength of
550-695 nm. U.S. Pat. Nos. 5,166,197 and 5,484,778 relate to
phthalocyanine derivatives reportedly useful for macular
degeneration. Asrani and Zeimer (British Journal of Ophthalmology,
1995, 79:766-770) relate to photoocclusion of ocular vessels using
a phthalocyanine encapsulated in heat-sensitive liposomes. Levy
(Semin. Oncol. 1994, 21/6, suppl. 15 (4-10)) relates to
photodynamic therapy with porfimer sodium (PHOTOFRIN.RTM.,
requiring light of 630 nm and causing cutaneous photosensitivity
that may last for up to 6 weeks), and benzoporphyrin derivative
(BPD verteporfin, causing cutaneous photosensitivity of a few
days). Lin et al. (IOVS 34:1303 Abstract 2953, 1993) relate to the
photodynamic occlusion of choroidal vessels using benzoporphyrin
derivative BPD-MA. Baumal et al. (Invest. Ophthalmol. Vis. Sci.
37/3:S122 (abstract) 1996) relates to PDT of experimental choriodal
neovascularization with tin ethyl etiopurpurin (SnET2) and 665 nm
irradiation. BPD and SnET2 are insoluble in aqueous solutions and
require hydrophobic vehicles for administration. Further
limitations of prior art photosensitizers include inadequate light
penetration through hemorraghic and pigmented tissue, systemic
cutaneous photosensitivity, lack of selectivity, normal tissue
damage, and reopening of diseased vessels. Hydrophobic vehicle
solubilizers require clearance time prior to irradiation. Bolus
injection and instantaneous illumination cannot be performed.
[0012] Texaphyrins are aromatic pentadentate macrocyclic "expanded
porphyrins" useful as MRI contrast agents, as radiosensitizers, as
chemosensitizers, and in photodynamic therapy. Texaphyrin is
considered as being an aromatic benzannulene containing both
18.pi.- and 22.pi.-electron delocalization pathways. Texaphyrin
molecules absorb strongly in the tissue-transparent 700-900 nm
range, and they exhibit inherent selective uptake or
biolocalization in certain tissues, particularly regions such as,
for example, liver, atheroma or tumor tissue. Texaphyrins have
exhibited significant tumor selectivity as detected by magnetic
resonance imaging and fluorescence detection. Texaphyrins and
water-soluble texaphyrins, method of preparation and various uses
have been described in U.S. Pat. Nos. 4,935,498; 5,162,509;
5,252,720; 5,256,399; 5,272,142; 5,292,414; 5,369,101; 5,432,171;
5,439,570; 5,451,576; 5,457,183; 5,475,104; 5,504,205; 5,525,325;
5,559,207; 5,565,552; 5,567,687; 5,569,759; 5,580,543; 5,583,220;
5,587,371; 5,587,463; 5,591,422; 5,594,136; 5,595,726; 5,599,923;
5,599,928; 5,601,802; 5,607,924; and 5,622,946; PCT publications WO
90/10633; 94/29316; 95/10307; 95/21845; 96/09315; 96/38461;
96/40253; 97/26915; and 97/35617; PCT application PCT/US97/09501;
allowed U.S. patent applications 08/458,347; 08/484,551; and
08/591,318; and pending U.S. patent applications 08/763,451;
08/903,099; and 08/914,272; each patent, publication, and
application is incorporated herein by reference.
[0013] The present invention provides texaphyrins for ocular
diagnosis and therapy. The use of texaphyrins circumvents problems
seen in prior art methods, in part, because of the dual wavelengths
for absorption of light, solubility in aqueous solutions, rapid
clearance, use of a bolus injection, and the provision of a single
agent for visualization and treatment.
SUMMARY OF THE INVENTION
[0014] The present invention provides use of a texaphyrin in the
preparation of a pharmaceutical composition for use in ocular
diagnosis and therapy, in particular, therapy involving
photodynamic therapy of conditions of the eye characterized by
abnormal vasculature. Accordingly, an aspect of the invention is
directed to use of a texaphyrin for carrying out angiography of the
eye, i.e., observing vasculature of an eye of a subject. The method
of use comprises the steps of administering a detectable texaphyrin
to the subject and observing the vasculature state of the eye. When
the detectable texaphyrin is fluorescent, observing may be by
fluorescence; when the detectable texaphyrin is complexed with a
paramagnetic cation, the observing may be by magnetic resonance
imaging. Further, imaging of a detectable texaphyrin may be carried
out using x-rays, Raman Scattering, magnetometry (bioluminescence)
or optical coherence tomography. In a further aspect of the
invention, use of a texaphyrin for treating an ocular condition of
a subject characterized by abnormal vasculature is provided. The
method of use comprises the steps of administering a photosensitive
texaphyrin to the subject; and photoirradiating the vasculature.
The method of use may further comprise the step of observing the
ocular condition of the subject by imaging the texaphyrin as stated
herein.
[0015] Use of a texaphyrin for photodynamic therapy of macular
degeneration of a subject, the method of use comprising the steps
of administering a photosensitive texaphyrin to the subject and
photoirradiating the macula is another aspect of the invention.
[0016] Use of a texaphyrin for observing and treating an ocular
condition of a subject characterized by abnormal vasculature using
a single agent is also an aspect of the invention. The method of
use comprises the steps of administering a photosensitive
fluorescent texaphyrin to the subject, observing the ocular
condition of the subject by fluorescence of the texaphyrin, and
photoirradiating the vasculature.
[0017] For angiography, texaphyrins may be activated by 400-500 nm
light (the Soret band) or 700-800 nm light (the Q band) and,
therefore, provide considerable versatility for use in humans (see
FIG. 1). For phototherapy, texaphyrins may be irradiated at 400-500
nm and at longer wavelengths of light where ocular tissues are
relatively transparent, especially where light can penetrate blood
and vascular tissue, i.e., 700-800 nm, especially at about 732 nm.
Texaphyrins are particularly effective as visualizing agents in
angiography of ocular blood vessels due to their localization in
areas of abnormal permeability or damage. Texaphyrins are
particularly effective in PDT in that the wavelength of light used
with texaphyrin is readily transmitted through blood and other
endogenous pigments to effect photodynamically-mediated destruction
of pigmented and pigment-related tissue as described in U.S.Ser.
No. 08/914,272, incorporated by reference herein. PDT requires
higher levels of light than for imaging.
[0018] The aqueous solubility of texaphyrins is an advantage in the
ocular methods of use provided herein, providing for rapid infusion
as a bolus as compared to BPD, mTBPC, or SnET2 which require
solubilizing vehicles such as lipid environments, for example; and
further obviating the need for a lipophilic carrier, use of
liposomes, or use of a pump. The primary excitation (470 nm) and
emission peaks (750 nm) for texaphyrin are very widely spaced with
a secondary excitation peak available in the infrared range (732
nm) if necessary. Texaphyrins can be administered in a bolus
injection, allowing for a sufficiently large amount of drug to be
present in the blood and for fast-turnaround between dosing and
treatment. Texaphyrins are cleared quickly from the body; no
toxicity to the eye has been observed in the use of texaphyrins in
the present invention. A further advantage to rapid clearing is
that larger vessels clear faster, allowing for imaging and
treatment of a neovascular complex. Diagnostic imaging and therapy
can be performed with one agent by using texaphyrin as both an
angiographic and a PDT agent, thus enabling both accurate
determination of dye localization prior to treatment and immediate
confirmation of photodynamic closure following treatment.
[0019] Following long-standing patent law convention, the terms "a"
and "an" mean "one or more" when used in this application,
including the claims.
1 ABBREVIATIONS ARMD Age related macular degeneration BPD
Benzoporphyrin derivative IN Inferonasal IT Inferotemporal CNV (M)
Choroidal neovascularization (membrane) FWHM full width half
maximum HDL high-density lipoproteins ICG indocynanine green LDL
Low density lipoprotein Lu(III)T2BET lutetium texaphyrin, T2BET
mTHPC Tetra(m-hydroxyphenyl)chlorin NZW New Zealand White OD Right
eye OS Left eye PDT Photodynamic therapy SN Superonasal SnET2 Tin
etiopurpurin ST Superotemporal TGF-b Transforming growth factor-b
Txp Texaphyrin VEGF Vascular endothelial growth factor
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. Absorption spectrum (solid line) and fluorescence
profile (broken line) of LuT2BET.
[0021] FIG. 2. LuT2BET fluorescent angiogram of the eye of an NZW
rabbit in which a lesion (laser coagulation) in the inferior nasal
quadrant had been produced a few weeks prior to the angiogram. The
image was obtained immediately after injection of 4 .mu.mol
LuT2BET/kg injection and reveals the retinal vessels emanating from
the optic disc, which is the dark area, at 9:00 o'clock to the left
of the center of the eye. The vessels crossing horizontally
represent the branches of the ophthalmic artery and vessels running
vertically represent the arteries and veins in the choroid. An
acute lesion (occlusion) has also been placed in one of the main
arteries emanating from the optic disc (seen better in the late
phase angiogram, FIG. 3). The round hypovascular area at 4:00 and
5:00 o'clock at the edge of the visualized portion of the retina is
the area where the previous thermal ablation occurred.
[0022] FIG. 3. Late phase LuT2BET fluorescent angiogram of the
animal detailed in FIG. 2 revealing the occluded vessel in the
upper left hand corner. Just off center the chronic lesion is
easily visualized. Neovascular vessels are identified around an
avascular scar.
[0023] FIG. 4. Early stage fluorescein angiogram revealing leaking
of the dye out of the acute lesion.
[0024] FIG. 5. Late stage fluorescein angiogram of the lower right
hand quadrant of the animal in FIG. 4.
[0025] FIG. 6. Late stage ICG fluorescent angiogram of the lower
right hand quadrant. The texaphyrin fluorescent angiogram of this
same lesion is viewed in FIG. 3. The ICG image is amorphic and does
not detail the neovascular vessels that are distinctly noted in
FIG. 3.
[0026] FIG. 7. Fluorescein angiogram of a rabbit's eye that had a
lesion acutely induced in the medullary ray. The fluorescein is
leaking from the laser-induced lesion.
[0027] FIG. 8. Fluorescein angiogram of the rabbit in FIG. 7 after
receiving PDT using LuT2BET. The image depicts closure of the
vessels as evidenced by fluorescein staining and retention in the
PDT targeted lesion.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The present invention provides for the use of texaphyrins in
the preparation of a pharmaceutical composition for use in ocular
diagnosis and therapy; especially diagnostic angiograms, and
photodynamic therapy of conditions of the eye characterized by
abnormal vasculature. "Abnormal vasculature", as used herein, means
undesirable vasculature; neovasculature; irregular, occluded,
weeping, or inflamed ocular vessels or ocular tissues; inflammatory
ocular membranes; or abnormal conditions having to do with
channeling of fluids in the ocular area, especially blood vessels.
Conditions having abnormal vasculature include conditions such as
macular degeneration, glaucoma, disc or retinal neovascularization
in diabetic retinopathy, pannus which is abnormal superficial
vascularization of the cornea or conjunctiva, pterygium which is
thickening of the bulbar conjunctiva on the cornea, conditions
having retinal or choroidal neovasculature, ocular histoplasmosis
syndrome, myopia, ocular inflammatory diseases, central serous
retinopathy, subretinal neovascular membrane, or neovasculature
induced by neoplasm, such as melanoma or retinal blastoma, for
example. Treatment of abnormal vasculature related to pigmented and
nonpigmented ocular cancers, including melanoma is also
contemplated in the use of the present invention. Ocular melanoma
includes uveal melanoma, and melanoma of the skin of the eyelids,
the conjunctiva, the orbit of the eye, or retinal melanoma.
[0029] "Observing the vasculature", as used herein, means carrying
out an imaging procedure and collecting information from an
angiogram, x-ray, magnetic resonance image, or optical coherence
tomogram, for example, to interpret the condition of the eye. The
condition of the eye may be normal, or may include vascular leakage
or occlusions, for example. As used herein, "eye" or "ocular"
includes the eye, underlying and adjacent tissue, and related
tissues near and around the eye that have an influence on the
functioning of the eye.
[0030] The texaphyrin or texaphyrin metal complex for use in ocular
diagnosis or photodynamic therapy may have structure I: 1
[0031] or may have structure II: 2
[0032] M is a divalent metal cation selected from the group
consisting of Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II),
Hg(II), Fe(II), Sm(II), and UO.sub.2(II); or a trivalent metal
cation selected from the group consisting of Mn(III), Co(III),
Ni(III), Fe(III), Ho(III), Ce(III), Y(III), In(III), Pr(III),
Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Er(III),
Tm(III), Yb(III), Lu(III), La(III), and U(III).
[0033] R.sub.1-R.sub.4, R.sub.7 and R.sub.8 are independently
hydrogen, halide, hydroxyl, alkyl, alkenyl, alkynyl, aryl,
haloalkyl, nitro, formyl, acyl, hydroxyalkyl, alkoxy,
hydroxyalkoxy, hydroxyalkenyl, hydroxyalkynyl, saccharide, carboxy,
carboxyalkyl, carboxyamide, carboxyamidealkyl, amino, aminoalkyl, a
site-directing molecule, a catalytic group, or a couple that is
coupled to a site-directing molecule or to a catalytic group.
[0034] R.sub.6 and R.sub.9 are independently selected from the
groups of R.sub.1-R.sub.4, R.sub.7 and R.sub.8, with the proviso
that the halide is other than iodide and the haloalkyl is other
than iodoalkyl.
[0035] R.sub.5 and R.sub.10-R.sub.12 are independently hydrogen,
alkyl, alkenyl, alkynyl, aryl, hydroxyalkyl, alkoxy, hydroxyalkoxy,
hydroxyalkenyl, hydroxyalkynyl, carboxyalkyl, carboxyamide,
carboxyamidealkyl, amino, aminoalkyl, or a couple that is coupled
to a saccharide, to a site-directing molecule, or to a catalytic
group; and n is an integer value less than or equal to 5.
[0036] R.sub.13 is alkyl, alkenyl, oxyalkyl, or hydroxyalkyl having
up to about 3 carbon atoms and having rotational flexibility around
a first-bound carbon atom. Rotational flexibility allows the rest
of the group to be positioned outside the plane of the texaphyrin.
Thus, for example, a preferred alkenyl is
CH.sub.2--CH.dbd.CH.sub.2. The pyrrole nitrogen substituent is most
preferably a methyl group.
[0037] Fluorescent texaphyrins are used for angiography. The term
"fluorescent", as used herein, means that upon photoirradiation by
light associated with the absorption profile of texaphyrin, light
is emitted at a longer wavelength by the irradiated texaphyrin. All
texaphyrins are fluorescent, albeit to varying degrees, and
texaphyrins complexed with Y(III), Lu(III), Gd(III), Dy(III),
Eu(III), or Mn(III) are preferred as fluorescent texaphyrins, for
example. Lutetium texaphyrin, in particular, possesses a strong,
broad fluorescence emission profile in the near-infrared centered
at 750 nm (FWHM=60 nm) that is not obstructed by endogneous
chromophores, thereby exhibiting significant advantages over
conventional fluorescein angiography. Further, lutetium texaphyrin
exhibits rapid plasma clearance in humans (T1/2.alpha. of 0.25
hours), thereby minimizing cutaneous phototoxicity compared to
other photosensitizers.
[0038] In addition to fluorescent detection, texaphyrins may be
imaged by x-radiation, Raman scattering, magnetometry, or by
optical coherence tomography; further, texaphyrins complexed with a
paramagnetic metal cation may be used for magnetic resonance
imaging. Preferred paramagnetic metal cations include Mn(II),
Mn(III), Fe(III), or trivalent lanthanide metals other than
La(III), Lu(III), and Pm(III). More preferably, the paramagnetic
metal is Mn(II), Mn(III), Dy(III), or Gd(III); most preferably,
Gd(III). Any of various types of magnetic resonance imaging can be
employed in the practice of the invention, including, for example,
nuclear magnetic resonance (NMR), NMR spectroscopy, and electronic
spin resonance (ESR). The preferred imaging technique is NMR.
[0039] Photosensitive texaphyrins are used for photodynamic
therapy. A photosensitive texaphyrin may be a free-base texaphyrin
or may be metallated with a diamagnetic metal. The term
"photosensitive", as used herein, means that upon photoirradiation
by light associated with the absorption profile of texaphyrin,
texaphyrin effects the generation of oxygen products that are
cytotoxic. Cytotoxic oxygen products may be singlet oxygen,
hydroxyl radicals, superoxide, hydroperoxyl radicals, or the like.
For generating singlet oxygen, the preferred metal is a diamagnetic
metal. A preferred diamagnetic metal is Lu(III), La(III), In(III),
Zn(II), or Cd(II) and a most preferred diamagnetic metal is
Lu(III).
[0040] In the above-described structure I, "n" will typically be an
integer value less than or equal to 5. In the context of the basic
macrocycle with a divalent or trivalent metal cation, n is 1 or 2;
however, one skilled in the art in light of the present disclosure
would realize that the value of n would be altered due to charges
present on substituents R.sub.1-R.sub.12 and charges present on the
covalently bound site-directing molecule. It is understood by those
skilled in the art that the complexes described in the present
invention have one or more additional ligands providing charge
neutralization and/or coordinative saturation to the metal ion.
Such ligands include chloride, nitrate, acetate, and hydroxide,
among others.
[0041] Representative examples of alkanes useful as alkyl group
substituents of the present invention include methane, ethane,
straight-chain, branched or cyclic isomers of propane, butane,
pentane, hexane, heptane, octane, nonane and decane, with methane,
ethane and propane being preferred. Alkyl groups having up to about
thirty, or up to about fifty carbon atoms are contemplated in the
present invention. Representative examples of substituted alkyls
include alkyls substituted by two or more functional groups as
described herein.
[0042] Representative examples of alkenes useful as alkenyl group
substituents include ethene, straight-chain, branched or cyclic
isomers of propene, butene, pentene, hexene, heptene, octene,
nonene and decene, with ethene and propene being preferred. Alkenyl
groups having up to about thirty or fifty carbon atoms, and up to
about five double bonds, or more preferably, up to about three
double bonds are contemplated in the present invention.
[0043] Representative examples of alkynes useful as alkynyl group
substituents include ethyne, straight-chain, branched or cyclic
isomers of propyne, butyne, pentyne, hexyne, heptyne, octyne,
nonyne and decyne, with ethyne and propyne being preferred. Alkynyl
groups having up to about thirty, or up to about fifty carbon
atoms, and having up to about five or up to about three triple
bonds are contemplated in the present invention.
[0044] The aryl may be a compound whose molecules have the ring
structure characteristic of benzene, naphthalene, phenanthrene,
anthracene, and the like, i.e., either the 6-carbon ring of benzene
or the condensed 6-carbon rings of the other aromatic derivatives.
For example, an aryl group may be phenyl or naphthyl, unsubstituted
or substituted with a nitro, carboxy, sulfonic acid, hydroxy,
oxyalkyl or halide substituent. In this case, the substituent on
the phenyl or naphthyl may be added in a synthetic step after the
condensation step which forms the macrocycle.
[0045] Among the halide substituents, chloride, bromide, fluoride
and iodide are contemplated in the practice of this invention with
the exception of iodide for R.sub.6 and R.sub.9. R.sub.6 and
R.sub.9 may have chloride, bromide or fluoride substituents.
Representative examples of haloalkyls used in this invention
include halides of methane, ethane, propane, butane, pentane,
hexane, heptane, octane, nonane and decane, with halides,
preferably chlorides or bromides, of methane, ethane and propane
being preferred.
[0046] "Hydroxyalkyl" means alcohols of alkyl groups. Preferred are
hydroxyalkyl groups having one to twenty, more preferably one to
ten, hydroxyls. "Hydroxyalkyl" is meant to include glycols and
polyglycols; diols of alkyls, with diols of C.sub.1-10 alkyls being
preferred, and diols of C.sub.1-3 alkyls being more preferred; and
polyethylene glycol, polypropylene glycol and polybutylene glycol
as well as polyalkylene glycols containing combinations of
ethylene, propylene and butylene.
[0047] Representative examples of oxyalkyls include the alkyl
groups as herein described having ether linkages. "Oxyalkyl" is
meant to include polyethers with one or more functional groups. The
number of repeating oxyalkyls within a substituent may be up to
200, preferably is from 1-20, and more preferably, is 1-10, and
most preferably is 1-5. A preferred oxyalkyl is
O(CH.sub.2CH.sub.2O).sub.xCH.sub.3 where x=1-100, preferably 1-10,
and more preferably, 1-5.
[0048] Oxyhydroxyalkyl means alkyl groups having ether or ester
linkages, hydroxyl groups, substituted hydroxyl groups, carboxyl
groups, substituted carboxyl groups or the like.
[0049] Representative examples of thioalkyls include thiols of
ethane, thiols of straight-chain, branched or cyclic isomers of
propane, butane, pentane, hexane, heptane, octane, nonane and
decane, with thiols of ethane (ethanethiol, C.sub.2H.sub.5SH) or
propane (propanethiol, C.sub.3H.sub.7SH) being preferred.
Sulfate-substituted alkyls include alkyls as described above
substituted by one or more sulfate groups, a representative example
of which is diethyl sulfate ((C.sub.2H.sub.5).sub.2SO.sub.4).
[0050] Representative examples of phosphates include phosphate or
polyphosphate groups. Representative examples of
phosphate-substituted alkyls include alkyls as described above
substituted by one or more phosphate or polyphosphate groups.
[0051] Representative examples of phosphonate-substituted alkyls
include alkyls as described above substituted by one or more
phosphonate groups.
[0052] Representative examples of carboxy groups include carboxylic
acids of the alkyls described above as well as aryl carboxylic
acids such as benzoic acid. Representative examples of
carboxyamides include primary carboxyamides (CONH.sub.2), secondary
(CONHR) and tertiary (CONR'R") carboxyamides where each of R' and
R" is a functional group as described herein.
[0053] Representative examples of useful amines include a primary,
secondary or tertiary amine of an alkyl as described
hereinabove.
[0054] "Carboxyamidealkyl" means alkyl groups with secondary or
tertiary amide linkages or the like. "Carboxyalkyl" means alkyl
groups having hydroxyl groups, carboxyl or amide substituted
ethers, ester linkages, tertiary amide linkages removed from the
ether or the like.
[0055] The term "saccharide" includes oxidized, reduced or
substituted saccharide; hexoses such as D-glucose, D-mannose or
D-galactose; pentoses such as D-ribose or D-arabinose; ketoses such
as D-ribulose or D-fructose; disaccharides such as sucrose,
lactose, or maltose; derivatives such as acetals, amines, and
phosphorylated sugars; oligosaccharides, as well as open chain
forms of various sugars, and the like. Examples of
amine-derivatized sugars are galactosamine, glucosamine, sialic
acid and D-glucamine derivatives such as
1-amino-1-deoxysorbitol.
[0056] In an embodiment of the present invention, texaphyrins are
further coupled to site-directing molecules to form conjugates for
targeted in vivo delivery. "Site-directing" means having
specificity for targeted sites. "Specificity for targeted sites"
means that upon contacting the texaphyrin-conjugate with the
targeted site, for example, under physiological conditions of ionic
strength, temperature, pH and the like, specific binding will
occur. The interaction may occur due to specific electrostatic,
hydrophobic, entropic or other interaction of certain residues of
the conjugate with specific residues of the target to form a stable
complex under conditions effective to promote the interaction. A
site-directing molecule may have binding specificity for
localization to a treatment site. Exemplary site-directing
molecules contemplated in the present invention include, but are
not limited to: lipoproteins including low density lipoprotein;
cholesterol; polyamides including peptides having affinity for an
ocular receptor; proteins such as antibodies or an immunologically
active fragment thereof; oligonucleotides complementary to an
ocular DNA or RNA; histamine; hormone mimics such as morphine; a
catalytic group; and further macrocycles such as sapphyrins and
rubyrins. Ocular-specific site-directing molecules may include
molecules directed at vascular endothelial growth factor (VEGF), or
the class of integrins, both of which are important in ocular
angiogenesis. Further site-directing molecules may include
b-endorphin, or TGF-b, for example.
[0057] The term "catalytic group" means a chemical functional group
that assists catalysis by acting as a general acid, Brnsted acid,
general base, Brnsted base, nucleophile, or any other means by
which the activation barrier to reaction is lowered or the ground
state energy of the substrate is increased. Exemplary catalytic
groups contemplated include, but are not limited to, imidazole;
guanidine; substituted saccharides such as D-glucosamine,
D-mannosamine, D-galactosamine, D-glucamine and the like; amino
acids such as L-histidine and L-arginine; derivatives of amino
acids such as histamine; polymers of amino acids such as
poly-L-lysine, (LysAla).sub.n, (LysLeuAla).sub.n where n is from
1-30 or preferably 1-10 or more preferably 2-7 and the like;
derivatives thereof; and texaphyrin metal complexes. The term
"appended to the texaphyrin complex-site directing molecule
conjugate" means that the catalytic groups are attached either
directly to the texaphyrin metal complex or to the texaphyrin
complex via a linker or couple of variable length, or are attached
to the ligand portion of a texaphyrin complex-ligand conjugate
either with or without a linker or couple of variable length.
[0058] A preferred site-directing molecule for coupling to
texaphyrin is low density lipoprotein (LDL). Human LDL is a
physiologic serum protein metabolized by cells via uptake by high
affinity receptors. Neovascularization has been shown to have
increased numbers of LDL receptors; and by increasing the
partitioning of the texaphyrin into the lipoprotein phase of the
blood, LDL is expected to more efficiently deliver texaphyrin to
the target tissue. A texaphyrin-LDL conjugate is selective for
neovascularization since leakage of the conjugate is expected to
occur only in neovasculature due to the large size of the
conjugate. LDL can be isolated and purified according to the
procedure of Hauel et al., (J. Clin. Invest., 34:1345, 1995).
[0059] A couple may be described as a linker, i.e., the covalent
product formed by reaction of a reactive group designed to attach
covalently another molecule at a distance from the texaphyrin
macrocycle. Exemplary linkers or couples are amides, amine,
disulfide, thioether, ether, ester, or phosphate covalent
bonds.
[0060] In most preferred embodiments, conjugates and appended
groups are covalently bonded to the texaphyrin via a carbon-carbon,
carbon-nitrogen, carbon-sulfur, or a carbon-oxygen bond, more
preferably a carbon-oxygen or a carbon-nitrogen bond.
[0061] Preferred functionalizations are: when R.sub.6 and R.sub.9
are other than hydrogen, then R.sub.5 and R.sub.10 are hydrogen or
methyl; and when R.sub.5 and R.sub.10 are other than hydrogen, then
R.sub.6 and R.sub.9 are hydrogen, hydroxyl, or halide other than
iodide. Other preferred functionalizations are where R.sub.6 and
R.sub.9 are hydrogen, then R.sub.5, R.sub.10, R.sub.11 and R.sub.12
are lower alkyl or lower hydroxyalkyl. The lower alkyl is
preferably methyl or ethyl, more preferably methyl. The lower
hydroxyalkyl is preferably of 1 to 6 carbons and 1 to 4 hydroxy
groups, more preferably 3-hydroxypropyl. In a presently preferred
embodiment, R.sub.1 is (CH.sub.2).sub.2CH.sub.2OH, R.sub.2 and
R.sub.3 are CH.sub.2CH.sub.3, R.sub.4 is CH.sub.3, R.sub.7 and
R.sub.8 are O(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.2OCH.sub.3,
R.sub.5, R.sub.6, and R.sub.9-R.sub.12 are H, and M is Lu. This
texaphyrin is named herein as LuT2BET.
[0062] In other presently preferred texaphyrin compounds,
R.sub.1-R.sub.12 are as in Tables A and B for texaphyrins A1-A108,
and M is as defined hereinabove. However, while the above-described
texaphyrins are presently preferred compounds for use in the
present invention, the invention is not limited thereto and any
photosensitive texaphyrin may be useful for PDT, and any
fluorescent texaphyrin may be useful for angiography.
2TABLE A Representative Substituents for Texaphyrin Macrocycles
A1-A108 of the Present Invention. Substituents for R.sub.1-R.sub.6
are provided in TABLE A and for R.sub.7-R.sub.12 in TABLE B. TXP
R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 R.sub.6 A1
CH.sub.2(CH.sub.2).sub.2OH CH.sub.2CH.sub.3 CH.sub.2CH.sub.3
CH.sub.3 H H A2 " " " " " " A3 " " " " " " A4 " " " " " " A5 " " "
" " " A6 " " " " " " A7 " " " " " " A8 " " " " " " A9 " " " " " "
A10 " " " " " " A11 " " " " " " A12 " COOH COCH " " " A13
CH.sub.2(CH.sub.2).sub.2OH COOCH.sub.2 COOCH.sub.2 CH.sub.3 H H
CH.sub.3 CH.sub.3 A14 CH.sub.2CH.sub.2CON CH.sub.2CH.sub.3
CH.sub.2CH.sub.3 " " " (CH.sub.2CH.sub.2OH).sub.- 2 A15
CH.sub.2CH.sub.2ON " " " " " (CH.sub.3)CH.sub.2-
(CHOH).sub.4CH.sub.2 OH A16 CH.sub.2CH.sub.3 " " " " " A17
CH.sub.2(CH.sub.2).sub.2OH " " " " " A18 " " " " " " A19 " " " " "
" A20 CH.sub.2CH.sub.3 CH.sub.3 CH.sub.2CH.sub.2 " " " COOH A21 " "
CH.sub.2CH.sub.2 " " " CO-VEGF A22 CH.sub.2(CH.sub.2).sub.2OH
CH.sub.2CH.sub.3 CH.sub.2CH.sub.3 " " " A23 " " " " " " A24 " " " "
" " A25 " " " " " " A26 " " " " " " A27 " COOH COOH " " " A28 "
COOCH.sub.2 COOCH.sub.2 " " " CH.sub.3 CH.sub.3 A29
CH.sub.2CH.sub.2CO- CH.sub.2CH.sub.3 CH.sub.2CH.sub.3 CH.sub.3 H H
VEGF A30 CH.sub.2CH.sub.2O- " " " " " VEGF A31
CH.sub.2(CH.sub.2).sub.2OH " CH.sub.2CH.sub.2 " " " CO-VEGF A32 " "
" " " " A33 CH.sub.2CH.sub.3 CH.sub.3 CH.sub.2CH.sub.2 " " " COOH
A34 " " CH.sub.2CH.sub.2 " " " CO-VEGF A35 CH.sub.2CH.sub.3
CH.sub.2CH.sub.3 CH.sub.2CH.sub.3 " " " A36 " " " " " " A37 " " " "
" " A38 " " " " " " A39 CH.sub.2(CH.sub.2).sub.2OH CH.sub.2CH.sub.3
CH.sub.2CH.sub.3 CH.sub.3 H COOH A40 " " " " " COOH A41 " " " " "
CON- HCH- (CH.sub.2 OH).sub.2 A42 " " " " " " A43 " " " " " H A44 "
" " " " OCH.sub.3 A45 " " " " " " A46 " " " " " " A47 " " " " " "
A48 " " " " " " A49 " " " " " " A50 " " " " " CH.sub.3 A51 " " " "
" " A52 " " " " " " A53 " " " " " " A54 " " " " CH.sub.3 H A55 " "
" " " " A56 " " " " " " A57 CH.sub.2(CH.sub.2).sub.2OH
CH.sub.2CH.sub.3 CH.sub.2CH.sub.3 CH.sub.3 CH.sub.3 H A58 " " " " "
" A59 " " " " " " A60 " " " " " " A61 " " " " " " A62 " " " " " "
A63 " " " " " OH A64 " " " " " F A65 " " " " CH.sub.2 H
(CH.sub.2).sub.6 OH A66 " " " " H Br A67 " " " " " NO.sub.2 A68 " "
" " " COOH A69 " " " " " CH.sub.3 A70 " " " " C.sub.6H.sub.5 H A71
" COOH COOH " CH.sub.2 " CH.sub.3 A72 " COOCH.sub.2 COOCH.sub.2 "
CH.sub.3 " CH.sub.3 CH.sub.3 A73 CH.sub.2CH.sub.2CON
CH.sub.2CH.sub.3 CH.sub.2CH.sub.3 " " " (CH.sub.2CH.sub.2OH).sub.2
A74 CH.sub.2CH.sub.2ON " " " " " (CH.sub.3)CH.sub.2(CH-
OH).sub.4CH.sub.2OH A75 CH.sub.2CH.sub.3 " " " CH.sub.2 "
(CH.sub.2).sub.6 OH A76 CH.sub.2(CH.sub.2).sub.2OH CH.sub.2CH.sub.3
CH.sub.2CH.sub.3 CH.sub.3 CH.sub.3 or H CH.sub.2 CH.sub.3 A77 " " "
" " " A78 " " " " " " A79 " " " " " " A80 " " " " " " A81 " " " " "
" A82 " " " " " " A83 " " " " " " A84 " " " " " " A85 " " " " H "
A86 " " " " " " A87 " " " " CH.sub.3 or " CH.sub.2 CH.sub.3 A88 " "
" " " " A89 " " " " H H A90 " " " " " " A91 " " " " " " A92 " " " "
" " A93 " COOH COOH " " " A94 " COOCH2 COOCH2 " " " CH3 CH3 A95
CH.sub.2(CH.sub.2).sub.2OH CH2CH3 CH2CH2- " " " COVEGF A96
CH.sub.2CH.sub.3 CH.sub.3 CH2CH2- " " " COOH A97 " " CH2CH2- " " "
COVEGF A98 CH.sub.2CH.sub.3 " " " " " A99 " " " " " " A100 " " " "
" " A101 " " " " " " A102 " " " " " " A103 " " " " " " A104 " " " "
" " A105 CH.sub.2(CH.sub.2).sub.2OH " " " " " A106 " " " " " " A107
" " " " " " A108 " " " " " "
[0063]
3TABLE B Representative Substituents for Texaphyrin Macrocycles
A1-A108 of the Present Invention. Substituents for R.sub.1-R.sub.6
are provided in TABLE A and for R.sub.7-R.sub.12 in TABLE B. TXP
R.sub.7 R.sub.8 R.sub.9 R.sub.10 R.sub.11 R.sub.12 A1
O(CH.sub.2).sub.3OH O(CH.sub.2).sub.3 H H H H OH A2
O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2 " " " " CH.sub.3
CH.sub.2O).sub.3 CH.sub.3 A3 O(CH.sub.2).sub.nCON- " " " " "
linker-VEGF, n=1-10 A4 O(CH.sub.2).sub.nCON- H " " " " linker-VEGF,
n=1-10 A5 OCH.sub.2CO- " " " " " VEGF A6 O(CH.sub.2CH.sub.2O).sub.3
" " " " " CH.sub.3 A7 OCH.sub.2CON- O(CH.sub.2 " " " " linker-VEGF
CH.sub.2 O).sub.3 CH.sub.3 A8 OCH.sub.2CO- " " " " " VEGF A9
O(CH.sub.2CH.sub.2 " " " " " O).sub.100CH.sub.3 A10 OCH.sub.2H " "
" " CON(CH.sub.2 CH.sub.2OH).sub.2 A11 CH.sub.2CON(CH.sub.3) " " "
" " CH.sub.2(CHOH).sub.4 CH.sub.2OH A12 " " " " " " A13
CH.sub.2CON(CH.sub.3) H H H H H CH.sub.2(CHOH).sub.4 CH.sub.2OH A14
" " " " " " A15 OCH.sub.3 OCH.sub.3 " " " " A16 OCH.sub.2CO.sub.2-
H " " " " VEGF A17 O(CH.sub.2).sub.n " " " " " COOH, n=1-10 A18
(CH.sub.2).sub.n " " " " " -CON-linker -VEGF, n=1-10 A19
YCOCH.sub.2- " " " " " linker-VEGF, Y=NH,O A20
O(CH.sub.2).sub.2CH.sub.2 O(CH.sub.2).sub.2 " " " " OH CH.sub.2OH
A21 " " " " " " A22 OCH.sub.2COOH O(CH.sub.2 " " " " CH.sub.2
O).sub.3CH.sub.3 A23 O(CH.sub.2).sub.nCO- H " " " " VEGF, n=1-10
A24 O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2 " " " " CH.sub.3
CH.sub.2O).sub.n- linker- VEGF, n=1-10 A25 OCH.sub.3 OCH.sub.2CO- "
" " " VEGF A26 " CH.sub.2CO- " " " " VEGF A27 " " " " " " A28
OCH.sub.3 CH.sub.2CO- H H H H VEGF A29 " OCH.sub.3 " " " " A30 " "
" " " " A31 H O(CH.sub.2).sub.n " " " " COOH, n=1-10 A32 "
(CH.sub.2).sub.n- " " " " CON- linker- VEGF, n=1-10 A33 OCH.sub.3
O(CH.sub.2 " " " " CH.sub.2 O).sub.3-CH.sub.3 A34 " " " " " " A35 H
O(CH.sub.2).sub.n " " " " CO- VEGF, n=1-10 A36 OCH.sub.3 " " " " "
A37 O(CH.sub.2CH.sub.2O).sub.3 " " " " " CH.sub.3 A38 " O(CH.sub.2
" " " " CH.sub.2 O).sub.n- VEGF, n=1-10 A39 O(CH.sub.2).sub.3OH
O(CH.sub.2).sub.3 O(CH.sub.2).sub.3 H H H OH OH A40
O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2 COOH " " " CH.sub.2 CH.sub.3
O).sub.3CH.sub.3 A41 O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2CH.sub.2
(CH.sub.2).sub.3 " " " CH.sub.3 OH O).sub.3CH.sub.3 A42 " "
O(CH.sub.2CH.sub.2 " " " O).sub.3CH.sub.3 A43 " O(CH.sub.2).sub.3 "
" " " COOH A44 H OCH.sub.2 OCH.sub.3 " " " COOH A45 " OCH.sub.2 " "
" " COOH A46 " O(CH.sub.2 " " " " CH.sub.2O).sub.3 CH.sub.3 A47
O(CH.sub.2CH.sub.2O).sub.3 " " " " " CH.sub.3 A48 " OCH.sub.2CO- "
" " " VEGF A49 " OCH.sub.2 " " " " COOH A50 " O(CH.sub.2 O(CH.sub.2
" " " CH.sub.2O).sub.3 CH.sub.2O).sub.3 CH.sub.3 CH.sub.3 A51 "
OCH.sub.2 " " " " COOH A52 O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2
OCH.sub.3 " " " CH.sub.3 CH.sub.2O).sub.100 CH.sub.3 A53 H
OCH.sub.2CO- " " " " VEGF A54 O(CH.sub.2)OH O(CH.sub.2).sub.3 H
CH.sub.3 " " OH A55 H O(CH.sub.2 " " " " CH.sub.2O).sub.3 CH.sub.3
A56 O(CH.sub.2CH.sub.2O).sub.3 " " " " " CH.sub.3 A57 H
OCH.sub.2CO- H CH.sub.3 " " VEGF A58 " OCH.sub.2CO- " " " " VEGF
A59 " OCH.sub.2 " " " " CON (CH.sub.2CH.sub.2 OH).sub.2 A60
O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2 " " " " CH.sub.3
CH.sub.2O).sub.100 CH.sub.3 A61 " OCH.sub.2CO- " " " " VEGF A62 H
CH.sub.2CON " " " " (CH.sub.3)CH.sub.2 (CHOH).sub.4 CH.sub.2OH A63
O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2 OH " " " CH.sub.3
CH.sub.2O).sub.3 CH.sub.3 A64 " " F " " " A65 " " H CH.sub.2 " "
(CH.sub.2).sub.6 OH A66 " " Br H " " A67 " " NO.sub.2 " " " A68 " "
COOH " " " A69 " " CH.sub.3 " " " A70 " " H C.sub.6H.sub.5 " " A71
" " " CH.sub.2 " " CH.sub.3 A72 " " " CH.sub.3 " " A73 " " " " " "
A74 OCH.sub.3 OCH.sub.3 " " " " A75 H OCH.sub.2CO- " CH.sub.2 " "
VEGF (CH.sub.2).sub.6 OH A76 O(CH.sub.2).sub.3OH
O(CH.sub.2).sub.3OH H CH.sub.3 or CH.sub.3 or CH.sub.3 CH.sub.2
CH.sub.2 or CH.sub.3 CH.sub.3 CH.sub.2 CH.sub.3 A77
O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2CH.sub.2 " " " " CH.sub.3
O).sub.3CH.sub.3 A78 O(CH.sub.2).sub.3OH O(CH.sub.2CH.sub.2 " " " "
O).sub.3CH.sub.3 A79 H O(CH.sub.2).sub.n " " " " CO-VEGF, n=1,2,3
A80 H O(CH.sub.2).sub.n " " " " CO-VEGF, n=1,2,3 A81 H
O(CH.sub.2).sub.3 " " " " OH A82 O(CH.sub.2).sub.3OH
O(CH.sub.2).sub.n " " " " CO-VEGF, n=1,2,3 A83
O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2).sub.n " " " " CH.sub.3
CO-VEGF, n=1,2,3 A84 " O(CH.sub.2).sub.n " " " " CO-VEGF, n=1,2,3
A85 " O(CH.sub.2 " " " " CH.sub.2O).sub.3 CH.sub.3 A86 " " " "
CH.sub.2 CH.sub.2 (CH.sub.2).sub.2 (C- OH H.sub.2).sub.2 OH A87 " "
" CH.sub.3 or " " CH.sub.2 CH.sub.3 A88 " O(CH.sub.2 " " " "
CH.sub.2O).sub.3 CH.sub.3 A89 O(CH.sub.2CH.sub.2O)hd 3 O(CH.sub.2 "
" " " CH.sub.2-CH.sub.2- CH.sub.2O).sub.120 CH.sub.3- VEGF A90 H
VEGF " " " " A91 OCH.sub.2CO- OCH.sub.2CO- " " " " VEGF VEGF A92
CH.sub.2CO-VEGF CH.sub.2CO- " " " " VEGF A93 " " " " " " A94 " " "
" " " A95 H YCOCH.sub.2- " " " " linker- VEGF Y=NH,O A96
O(CH.sub.2CH.sub.2O).sub.3 O(CH.sub.2 " " " " CH.sub.3
CH.sub.2O).sub.5- VEGF A97 " " " " " " A98 H O(CH.sub.2).sub.3 " "
" " CO-VEGF A99 " " " " " " A100 OCH.sub.3 " " " " " A101
O(CH.sub.2CH.sub.2O).sub.3 " " " " " CH.sub.3 A102 "
O(CH.sub.2CH.sub.2 " " " " O).sub.n-VEGF, n=1-10 A103 "
O(CH.sub.2CH.sub.2 " " " " O).sub.n-VEGF, n=6 A104 "
O(CH.sub.2CH.sub.2 " " " " O).sub.n-VEGF, n=3 A105 " " " " " " A106
OCH.sub.3 O(CH.sub.2CH.sub.2 " " " " O).sub.n-VEGF, n=1-10 A107 H "
" " " " A108 O(CH.sub.2CH.sub.2O).sub.x " " " " "
CH.sub.3,x=1-10
[0064] Importantly, texaphyrins may be synthesized using certain
substituents to effect a lipid-water distribution coefficient that
is optimal for use in the eye, i.e., sufficiently water soluble for
uptake into vascular tissues and for ease of handling. "Water
soluble" means soluble in aqueous fluids to about 1 mM or better.
U.S. Patents, PCT publications, and pending applications to
texaphyrins, methods of making and uses thereof have been listed
herein and incorporated by reference herein. Sapphyrin compounds
are disclosed in U.S. Pat. Nos. 5,041,078; 5,159,065; 5,120,411;
5,302,714; and 5,457,195; each patent is incorporated by reference
herein.
[0065] One skilled in the art of organic synthesis in light of the
present disclosure and the disclosures in the patents, applications
and publications incorporated by reference herein could extend and
refine the referenced basic synthetic chemistry to produce
texaphyrins having various substituents. For example,
polyether-linked polyhydroxylated groups, saccharide substitutions
in which the saccharide is appended via an acetal-like glycosidic
linkage, an oligosaccharide or a polysaccharide may be similarly
linked to a texaphyrin. A doubly carboxylated texaphyrin in which
the carboxyl groups are linked to the texaphyrin core via aryl
ethers or functionalized alkyl substituents could be converted to
various esterified products wherein the ester linkages serve to
append further hydroxyl-containing substituents. Polyhydroxylated
texaphyrin derivatives may be synthesized via the use of secondary
amide linkages. Saccharide moieties may be appended via amide
bonds. Polyhydroxylated texaphyrin derivatives containing branched
polyhydroxyl (polyol) subunits may be appended to the texaphyrin
core via aryl ethers or ester linkages.
[0066] Treatment of carboxylated texaphyrins with thionyl chloride
or p-nitrophenol acetate would generate activated acyl species
suitable for attachment to monoclonal antibodies or other
biomolecules of interest. Standard in situ coupling methods (e.g.,
1,1'-carbonyldiimidazole) could be used to effect the
conjugation.
[0067] For the above-described uses, texaphyrins are provided as
pharmaceutical preparations. A pharmaceutical preparation of a
texaphyrin may be administered alone or in combination with
pharmaceutically acceptable carriers, in either single bolus or
multiple doses. Preferable pharmaceutical carriers include sterile
aqueous solutions. The pharmaceutical compositions formed by
combining a texaphyrin of the present invention and the
pharmaceutically acceptable carriers are then easily administered
in a variety of dosage forms. Administration may be intravenous,
intramuscular, subcutaneous, ophthalmic, retrobulbar, juxtabulbar,
or topical, with intravenous, topical, ophthalmic, or retrobulbar
administration being preferred, and intravenous being more
preferred.
[0068] Solutions of the texaphyrin in aqueous propylene glycol or
in sterile aqueous solution may be employed. Such aqueous solutions
should be suitably buffered if necessary and the liquid diluent
first rendered isotonic with sufficient saline or glucose. In this
connection, sterile aqueous media which can be employed will be
known to those of skill in the art in light of the present
disclosure. Topical creams, emulsions, solutions, and the like are
contemplated for applications to surface areas of the ocular
area.
[0069] Pharmaceutical forms include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. Preferably, the
form is sterile, fluid to the extent that easy use with a syringe
exists, stable under the conditions of manufacture and storage, and
is preserved against the contaminating action of microorganisms,
such as bacteria and fungi. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars such as mannitol or dextrose or sodium chloride. A
more preferable isotonic agent is a mannitol solution of about 2-8%
concentration, and, most preferably, of about 5% concentration.
Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0070] Sterile solutions are prepared by incorporating the
texaphyrin in the required amount in the appropriate solvent with
various of the other ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the various sterilized active ingredients
into a sterile vehicle which contains the basic dispersion medium
and the required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum-drying and freeze-drying techniques which yield a powder of
the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0071] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
permeation enhancers, antibacterial and antifungal agents, isotonic
and absorption delaying agents and the like. The use of such media
and agents for pharmaceutically active substances is well known in
the art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated. Supplementary active ingredients can
also be incorporated into the compositions.
[0072] The texaphyrin to be used in the angiographic or
photodynamic methods of the invention will be administered in a
pharmaceutically effective amount. By "pharmaceutically effective"
is meant a dose that will provide an image for angiography or, upon
exposure to light of the appropriate wavelength, provide
substantial treatment of abnormal vasculature. The specific dose
will vary depending on the particular texaphyrin chosen, the dosing
regimen to be followed, photoirradiation exposure, timing of
administration, the tissue or disease to be treated, the
sensitivity of the detector, the filters used for visualization,
and the physical delivery system in which it is carried. Such dose
can be determined without undue experimentation by methods known in
the art or as described herein.
[0073] The parameters used for effective angiography and effective
treatment in the invention are interrelated. Therefore, the dose is
adjusted with respect to other parameters, for example, fluence,
irradiance, duration of the light used in photodynamic therapy, and
the time interval between administration of the dose and the
therapeutic irradiation. Such parameters should be adjusted to
produce significant damage to abnormal vascular tissue without
significant damage to the surrounding tissue or, on the other hand,
to enable the observation of blood vessels in the eye without
significant damage to the surrounding tissue. Typically, the dose
of texaphyrin used is within the range of from about 0.001
.mu.mol/kg to about 15 .mu.mol/kg/treatment, and preferably from
about 0.01-5 .mu.mol/kg/treatment. The lower dosage limit is
dependent upon the detection system used and, as methods of
detection improve, may be decreased. Further, as the texaphyrin
dose is reduced, the fluence required to treat neovascular tissue
may change.
[0074] After the photosensitizing texaphyrin has been administered,
the tissue being treated in the eye is irradiated at a wavelength
similar to the absorbance of the texaphyrin, usually either about
400-500 nm or about 700-800 nm. In the present photodynamic therapy
methods, the light source may be a laser, a light-emitting diode,
or filtered light from, for example, a xenon lamp; the light may
have a wavelength range of about 400-900 nm, preferably about
400-500 nm or 700-800 nm, more preferably about 450-500 nm or about
710-760 nm, or most preferably about 450-500 nm or about 725-740
nm; and the light may be administered topically, endoscopically, or
interstitially (via, e.g., a fiber optic probe). Preferably, the
light is administered using a slit-lamp delivery system. A
wavelength in this range is especially preferred since blood and
retinal pigment epithelium (RPE) are relatively transparent at
longer wavelengths and, therefore, treatment results in less tissue
damage and better light penetration. The fluence and irradiance
during the irradiating treatment can vary depending on type of
tissue, depth of target tissue, and the amount of overlying fluid
or blood.
[0075] As a result of being irradiated, the texaphyrin 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, melanosome, endoplasmic reticulum, the Golgi apparatus,
and the nucleus. Occlusion of the vasculature is thought to be a
major mechanism of photodynamic therapy which occurs by damage to
endothelial cells with subsequent platelet adhesion, degranulation,
and thrombus formation.
[0076] The optimum length of time following texaphyrin
administration until light treatment can vary depending on the mode
of administration, the form of administration, and the type of
target tissue. For example, a time interval of seconds to about 5 h
should be appropriate for vasculature in the eye. The time of light
irradiation after administration of the texaphyrin 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 human, it is believed that the texaphyrin begins to reach the
retinal and choroidal vasculature within seconds following
administration. Typically, the texaphyrin persists for a period of
minutes to hours, depending on the texaphyrin, the formulation, the
dose, the infusion rate, as well as the type of vessel and vessel
size.
[0077] In addition, texaphyrin can be used to observe the condition
of blood vessels as a single agent, or in concert with other dyes
such as fluorescein or indocyanine green to follow the progress of
destruction of abnormal vascular tissue. In an angiographic system,
a sufficient amount of texaphyrin is administered to produce an
observable fluorescent emission when excited by light, preferably
light having a wavelength range of about 400-900 nm, preferably
about 400-500 nm or 700-800 nm, more preferably about 450-500 nm or
about 710-760 nm, or most preferably about 450-500 nm or about
725-740 nm. Images are recorded by illuminating the eye with light
in the excitation wavelength range and detecting the amount of
fluorescent light emitted at the emission wavelength of about
700-800 nm, preferably about 730-760 nm. One such device, which
both emits and receives light in the 430-760 nm range, is the
TOPCON.TM. 50VT camera in the Ophthalmic Imaging System (Ophthalmic
Imaging System Inc., 221 Lathrop Way, Suite 1, Sacramento Calif.).
Generally, visualization is with a laser scanning microscope, and
the activating wavelength for fluorescence detection is that of the
laser. Imaging using wavelengths of light in the near-infrared
range eliminates autofluorescence and enables efficient light
penetration through tissue, particularly hemorrhagic and pigmented
tissue.
[0078] A camera is used to collect the emitted fluorescent light,
digitize the data, and store it for later depiction on a video
screen, as a hard paper copy, or in connection with some other
imaging system. While a film-recording device may be used when
additional dyes such as fluorescein are being used in combination
with the texaphyrin, a CCD camera (charge-coupled device) is
preferable as being able to capture emissions at higher
wavelengths. As a result, one can obtain more sophisticated
information regarding the pattern and extent of vascular structures
in different ocular tissue layers, giving the ability to detect the
"leakiness" that is characteristic of new or inflamed blood
vessels. Further, it is preferable to use a camera that is capable
of providing the excitation light, appropriately filtered to
deliver only light of the desired excitation wavelength range, and
then to capture the emitted, fluorescent light with a receiving
device, appropriately filtered to receive only light in the desired
emission wavelength range.
[0079] The following example is included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLE
Texaphyrins for Angiography and for Photodynamic Therapy of
Vascular Lesions of the Eye
[0080] The present example provides results from studies using
Lu(III)T2BET, cited herein, for fluorescent digital angiography of
the eye and for photodynamic therapy of vascular lesions of the
eye. LuT2BET has broad absorption bands at 470 and 732 nm
(.epsilon.=32,000 M.sup.-1 cm.sup.-1 at 732 nm), is highly
fluorescent, and exhibits an intense emission profile centered at
750 nm as shown in FIG. 1. The wavelength range from 700-800 nm is
diagnostically important since autofluorescence from endogneous
chromophores (<700 nm) does not hinder the emission signal.
[0081] Fluorescence angiography comparing LuT2BET, fluorescein, and
ICG was performed on normal NZW rabbits. Normal rabbits (n=4)
received an intravenous injection of LuT2BET from 1-10 .mu.mol/kg
after intramuscular sedation and pupillary dilatation. Extensive
fundoscopy was performed immediately and up to 2 hours post
texaphyrin, fluorescein, or ICG administration. Fluorescein and ICG
angiography, in addition to H&E histology, revealed no evidence
of normal tissue damage, demonstrating that lutetium texaphyrin
fluorescence angiography appears safe. In further studies, vascular
lesions including neovascularization was induced in albino rabbits
(NZW) using photocoagulation. PDT using LuT2BET was carried out on
the induced lesions. The results demonstrate that vascular
occlusions, areas of decreased perfusion, and areas of
neovascularization can be identified, showing that texaphyrin is an
effective agent for fluorescent angiography. The results also
demonstrate closure of induced vascular lesions using PDT with
texaphyrin, showing that texaphyrin is an effective agent for PDT
of abnormal vasculature of the eye. Therefore, selective
accumulation of Lu(III)T2BET in areas of neovascularization should
facilitate diagnosis and photodynamic therapy of age-related
macular degeneration and other disorders related to abnormal
vasculature.
[0082] Dyes. Lutetium texaphyrin, Lu(III)T2BET, was dissolved in 5%
mannitol at a concentration of 2 mM. Indocyanine-green (ICG,
Cardio-Gree.sup.n.RTM., Becton Dickinson and Company, Cockeysville,
Md.) and fluorescein (Alcon Laboratories, Fort Worth, Tex.)
fluorescent angiograms were performed at concentrations of 0.31
mg/kg and 7.5 mg/kg, respectively. All administrations were
performed via the ear vein.
[0083] Animals. Studies were designed and performed in accordance
with the Association for Research in Vision and Ophthalmology
(ARVO) resolution on the use of animals in research. Male New
Zealand White rabbits, weighing 3 to 4 kg, were obtained from
R&R Rabbitry (Stanwood, Wash.). Rabbits received regular Purina
Laboratory Rabbit Chow and water ad libidum for the entire duration
of the study. Each rabbit studied was anesthetized with a Ketamine
(8.4 mg/kg)/Rompun (1.2 mg/kg) cocktail administered via the ear
vein. Pupils were dilated with tropicamide (MYDRIACYL.RTM.1%, Alcon
Laboratories, Fort Worth, Tex.) and 0.5% proparacaine
(ALCAINE.RTM., Alcon Laboratories, Fort Worth, Tex.), 10%
phenylephrine (AK-DILATETM, Akom, Inc., Abita Springs, La.) was
used for topical anesthesia. No measures were taken to protect the
animals from ambient light.
[0084] Photography. Fundus photography and fluorescence angiograms
were performed with the TOPCON.TM.50VT camera coupled to the
Ophthalmic Imaging System (Ophthalmic Imaging System Inc.,
Sacramento, Calif.). Lu-Tex imaging was performed with conventional
excitation and interference filters. ICG was excited at 790-805 nm
and fluorescence detected at 835 nm. Fluorescein protocol included
excitation at 465-490 nm, with emission at 520-530 nm. A xenon lamp
was used to activate the dyes.
[0085] Induction of Neovascularization. Photocoagulation bums were
introduced in the right macular retina of seven rabbits with an
argon dye laser (Coherent Inc., Palo Alto, Calif.). On average,
five lesions were induced with 577 nm light using a spot size of 50
.mu.m using enough power (typically 450 mW) to cause bleeding into
the retina. The eyes were photographed immediately after
photocoagulation. Fluorescein and ICG angiograms were performed
11-15 days after photocoagulation. Neovascular lesions exhibited
hyperfluorescence immediately after injection followed by leakage.
Two animals had a grid of ten lesions induced in the medullary ray
(200 .mu.m spot size for 0.2 seconds using a set power of 300 mW).
Four control animals, having no induced lesions, were given
fluorescence angiograms with the respective dyes.
[0086] PDT Protocol. Animals received Lu(III)T2BET, 30 minutes post
injection laser light at 488 nm, spot size of 1000 .mu.m, was
directed to each lesion via a slitlamp system. Fluorescein
angiography was performed throughout the procedure, from prior to
until after PDT.
[0087] Plasma Analysis. Rabbits were injected via the ear vein with
1 .mu.mol Lu(III)T2BET/kg and blood was collected at 15 min, 30
min, 3 hr, and 5 hr post administration. EDTA was used as the
anticoagulant. Plasma samples (25 .mu.l) were mixed with 10 mM
Triton X100 (3 ml) and analyzed by fluorescence. Fluorescence
spectra were acquired with a SLM 48000S instrument, excitation was
between 450-480 nm and emission monitored from 700-800 nm. In vivo
binding of Lu(III)T2BET to rabbit plasma proteins and lipoproteins
was assessed using KBr density-gradient ultracentrifugation to
fractionate the protein/lipoprotein mixture, followed by
fluorescence spectroscopy to determine the relative distribution of
Lu(III)T2BET among the low-density lipoproteins (LDL), high-density
lipoproteins (HDL), and heavy proteins.
[0088] Histologic Evaluation. Eyes were enucleated under deep
anesthesia and fixed in 10% buffered formalin. The rabbits were
then sacrificed with an overdose of pentobarbital (100 mg/kg).
Tissues were embedded in paraffin and sequential sections, taken
every 5 mm to the center of the eye, were stained with hematoxylin
and eosin (H&E) for light microscopic evaluation.
[0089] FIG. 2 provides a LuT2BET angiogram of the right eye of an
NZW rabbit immediately following a 4 .mu.mol/kg injection of
lutetium texaphyrin. Approximately two weeks prior to the study,
the rabbit had a laser-induced lesion placed in the inferior
temporal quadrant of the eye (darkened area at approx. 4:00) and
just prior to the angiogram, a retinal vessel in the medullary ray
had been occluded temporal to the disc by a focal application of
577 nm light from a dye-pumped argon laser. The excitation was 500
nm (50 nm bandwidth) and the filter used for the fluorescence image
was 750 nm (10 nm bandwidth).
[0090] The late LuT2BET angiogram is displayed in FIG. 3. An
occluded retinal vessel with staining of the vessel wall is seen in
the upper left of the angiogram and neovascularization around a
relatively avascular area is seen in the lower right of the
angiogram (the inferior temporal lesion noted in FIG. 2).
[0091] Early and late fluorescein angiograms are shown in FIG. 4
and FIG. 5, respectively. The damaged and induced neovascular
lesions exhibited hyperfluorescence immediately after fluorescein
injection followed by extensive leakage (FIG. 5).
[0092] FIG. 6 depicts the ICG angiogram of the laser-induced
neovascular lesion (inferior temporal lesion, same region as in
FIG. 3). The ICG has contrasted an amorphic region, the vessels
imaged with LuT2BET can not be delineated.
[0093] FIG. 7 and FIG. 8 demonstrate fluorescein angiograms of a
rabbit's eye having an induced lesion before and after receiving
PDT with LuT2BET.
[0094] The concentration of LuT2BET in rabbit plasma after an
intravenous injection of 1 .mu.mol/kg was found to be 2.68 .mu.g/ml
at 15 min, 1.79 .mu.g/ml at 30 min, 0.18 .mu.g/ml at 3 hr, and 0.12
.mu.g/ml at 5 h. The relative in vivo binding of LuT2BET to plasma
protein components was assessed using density gradient
ultracentrifugation. More sensitizer was associated with the HDL
fraction (49%) compared to 40.5% for the heavy proteins, and 10.5%
to the LDL portion.
[0095] The angiograms for ICG, fluorescein, and Lu(III)T2BET
demonstrated different patterns of fluorescence. The retinal and
choroidal vasculature of the rabbit were well delineated by LuT2BET
angiography. Experimentally-induced retinal and choroidal vascular
lesions were enhanced by LuT2BET and demonstrated subtly different
patterns of staining than either fluorescein or ICG, consistent
with the different wavelength employed and probable differential
binding characteristics of LuT2BET. Fluorescein, a small molecule
that is not bound to plasma elements, was diffusely present
throughout the lesion with some central sparing in the
ischemic/scarred zone. Indocyanine green, binding mainly to
albumin, had a different fluorescence pattern than Lu(III)T2BET.
Lu(III)T2BET, predominantly bound to lipoproteins and to some
extent albumin, retained more angiographic information of the
remaining vascular anatomy. These three agents reveal different
features of ophthalmic disease. Histologic analysis revealed no
evidence of retinal toxicity following photography.
[0096] FIG. 7 and FIG. 8 demonstrate fluorescein angiograms of a
rabbit's eye having an induced lesion before and after receiving
PDT with LuT2BET. Thirty minutes after injection with LuT2BET,
laser light at 488 nm, spot size of 1000 .mu.m, was directed to the
lesion via a slitlamp lens system. Prior to PDT, fluorescence
fluorescein angiography revealed some hyperfluorescence at early
phases, which leaked over time (shown in FIG. 7). After PDT, the
fluorescein angiogram revealed hyperfluorescence in the vessel
(FIG. 8), indicative of reduced blood flow and therefore of vessel
closure. There was limited fluorescence leakage outside of the
PDT-treated vessel (compare FIG. 7 with FIG. 8).
[0097] These studies confirm that Lu(III)T2BET is capable of
serving as a contrast agent for angiography of the eye, especially
for visualizing the retinal and choroidal vascular systems, and as
a photosensitive agent for photodynamic therapy of vascular lesions
of the eye. Lu(III)T2BET demonstrated filling of retinal and
choroidal vasculature, accumulation and fluorescence in areas of
abnormal vasculature such as laser-induced choroidal
neovascularization of the choroid and occlusive vasculopathy of the
retina.
[0098] Closure of induced lesions was observed after PDT with
LuT2BET. No damage to surrounding vessels or tissues was observed.
Further, no damage was observed in control normal choroid or
retinal tissue. The PDT treatment therefore demonstrated
selectivity, an advantage over techniques, such as laser
photocoagulation, for example.
[0099] Due to differences in solubility, partition coefficient,
molecular weight, protein binding, and other variables,
Lu(III)T2BET angiography results in different information than that
obtained with either of the two most commonly used contrast agents,
Na-fluorescein and indocyanine green. Additionally, Lu(III)T2BET
has relatively more efficient energy-coupling capabilities than
either agent. Lu(III)T2BET may be sequentially used advantageously
for both angiography and PDT following a single or multiple
administration.
[0100] The uses and methods disclosed and claimed herein can be
executed without undue experimentation in light of the present
disclosure. While the uses and methods of this invention have been
described in terms of preferred embodiments, it will be apparent to
those of skill in the art that variations may be applied to the
uses and methods and in the steps or in the sequence of steps of
the methods described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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