U.S. patent application number 12/244655 was filed with the patent office on 2009-02-05 for method of inhibiting choroidal neovascuarization.
This patent application is currently assigned to University of Pennsylvania. Invention is credited to Alan M. Laties, Zhijun Luo, Rong Wen.
Application Number | 20090036479 12/244655 |
Document ID | / |
Family ID | 32030800 |
Filed Date | 2009-02-05 |
United States Patent
Application |
20090036479 |
Kind Code |
A1 |
Wen; Rong ; et al. |
February 5, 2009 |
METHOD OF INHIBITING CHOROIDAL NEOVASCUARIZATION
Abstract
The present invention relates to compositions and methods for
inhibiting unwanted angiogenesis, particularly those of ocular
tissues. The treatment, inhibition, and/or prevention of choroidal
neovasculature (CNV) is provided, along with an animal model for
CNV and imaging techniques that permit the screening of potential
agents as anti-angiogenesis and anti-CNV agents.
Inventors: |
Wen; Rong; (Bala Cynwyd,
PA) ; Luo; Zhijun; (Chestnut Hill, MA) ;
Laties; Alan M.; (Philadelphia, PA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
University of Pennsylvania
Philadelphia
PA
|
Family ID: |
32030800 |
Appl. No.: |
12/244655 |
Filed: |
October 2, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10665203 |
Sep 18, 2003 |
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12244655 |
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60412088 |
Sep 18, 2002 |
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Current U.S.
Class: |
514/291 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
17/02 20180101; A61K 31/445 20130101; A61P 27/02 20180101; A61P
27/00 20180101; A61P 33/02 20180101; A61K 31/436 20130101; A61P
43/00 20180101; Y10S 514/912 20130101; A61P 35/00 20180101; A61P
27/10 20180101; A61P 7/02 20180101 |
Class at
Publication: |
514/291 |
International
Class: |
A61K 31/436 20060101
A61K031/436; A61P 27/00 20060101 A61P027/00 |
Goverment Interests
GOVERNMENT INTERESTS
[0002] This invention was supported in part by Grant Nos. Y12727
from the U.S. National Institutes of Health. The Government may
have certain rights in this invention.
Claims
1. A method to treat a disease affecting the choroid or retina of
an eye in a patient having diabetic retinopathy or age related
macular degeneration, the method comprising administering to the
eye a composition comprising between 0.25% (w/w) to 2.5% (w/w) of
rapamycin in a pharmaceutically acceptable topical formulation for
a duration to achieve an amount of rapamycin in the choroid or
retina sufficient to treat the disease.
2. The method of claim 1, wherein the patient has age related
macular degeneration.
3. The method of claim 1, wherein the patient has diabetic
retinopathy.
4. The method of claim 1, wherein the composition comprises 0.25%
(w/w) of rapamycin.
5. The method of claim 4, wherein the patient has age related
macular degeneration.
6. The method of claim 4, wherein the patient has diabetic
retinopathy.
7. The method of claim 1, wherein the composition comprises 2.5%
(w/w) of rapamycin.
8. The method of claim 7, wherein the patient has age related
macular degeneration.
9. The method of claim 7, wherein the patient has diabetic
retinopathy.
10. A method to treat a disease affecting the choroid or retina of
an eye in a patient having diabetic retinopathy or age related
macular degeneration, the method comprising administering to the
eye a composition comprising between 0.1% (w/w) to 2.5% (w/w) of
rapamycin in a pharmaceutically acceptable topical formulation for
a duration to achieve an amount of rapamycin in the choroid or
retina sufficient to treat the disease.
11. The method of claim 10, wherein the patient has age related
macular degeneration.
12. The method of claim 10, wherein the patient has diabetic
retinopathy.
13. The method of claim 10, wherein the composition comprises 0.1%
(w/w) of rapamycin.
14. The method of claim 13, wherein the patient has age related
macular degeneration.
15. The method of claim 13, wherein the patient has diabetic
retinopathy.
16. The method of claim 10, wherein the composition comprises 1%
(w/w) of rapamycin.
17. The method of claim 16, wherein the patient has age related
macular degeneration.
18. The method of claim 16, wherein the patient has diabetic
retinopathy.
19. A method to treat a disease affecting the choroid or retina of
an eye in a patient having diabetic retinopathy or age related
macular degeneration, the method comprising administering to the
eye a composition comprising between 0.1% (w/w) to 5% (w/w) of
rapamycin in a pharmaceutically acceptable topical formulation for
a duration to achieve an amount of rapamycin in the choroid or
retina sufficient to treat the disease.
20. The method of claim 19, wherein the patient has age related
macular degeneration.
21. The method of claim 19, wherein the patient has diabetic
retinopathy.
22. The method of claim 19, wherein the composition comprises 2% of
rapamycin.
23. The method of claim 22, wherein the patient has age related
macular degeneration.
24. The method of claim 22, wherein the patient has diabetic
retinopathy.
25. A method to treat a disease affecting at least one of the
choroid, retina, or uvea of an eye in a patient having diabetic
retinopathy, age related macular degeneration, or retinitis
pigmentosa, the method comprising administering to the eye a
composition comprising between 0.1% (w/w) to 5% (w/w) of rapamycin
in a pharmaceutically acceptable topical formulation for a duration
to achieve an amount of rapamycin in the choroid, retina, or uvea
sufficient to treat the disease.
26. The method of claim 9, wherein the composition comprises 2% of
rapamycin.
27. A method to treat a disease affecting the choroid or retina of
an eye in a patient having diabetic retinopathy or age related
macular degeneration, the method comprising administering to the
eye a composition comprising between 0.1% (w/w) to 5% (w/w) of a
therapeutic agent selected from the group consisting of
immunophilin binding compounds in a pharmaceutically acceptable
topical formulation for a duration to achieve an amount of the
therapeutic agent in the choroid or retina sufficient to treat the
disease.
28. The method of claim 27, wherein the patient has age related
macular degeneration.
29. The method of claim 27, wherein the patient has diabetic
retinopathy.
30. The method of claim 27, wherein the therapeutic agent is
selected from the group consisting of rapamycin, tacrolimus,
everolimus, pimecrolimus, SDZ-RAD, CCl-779, AP23841, ABT-578, and
analogs and derivatives thereof.
31. The method of claim 30, wherein the patient has age related
macular degeneration.
32. The method of claim 30, wherein the patient has diabetic
retinopathy.
33. The method of claim 30, wherein the therapeutic agent is
selected from the group consisting of rapamycin and tacrolimus.
34. The method of claim 33, wherein the patient has age related
macular degeneration.
35. The method of claim 33, wherein the patient has diabetic
retinopathy.
36. The method of any of claims 27, 30 or 33, wherein the
composition comprises 2% of the therapeutic agent.
37. The method of claim 36, wherein the patient has age related
macular degeneration.
38. The method of claim 36, wherein the patient has diabetic
retinopathy.
39. The method of claim 33, wherein the therapeutic agent is
tacrolimus and the composition comprises 1% of the therapeutic
agent.
40. The method of claim 39, wherein the patient has age related
macular degeneration.
41. The method of claim 39, wherein the patient has diabetic
retinopathy.
42. A method to treat a disease affecting at least one of the
choroid, retina, or uvea of an eye in a patient having diabetic
retinopathy, age related macular degeneration, or retinitis
pigmentosa, the method comprising administering to the eye a
composition comprising between 0.1% (w/w) to 5% (w/w) of a
therapeutic agent selected from the group consisting of
immunophilin binding compounds in a pharmaceutically acceptable
topical formulation for a duration to achieve an amount of
therapeutic agent in the choroid, retina, or uvea sufficient to
treat the disease.
43. The method of claim 42, wherein the therapeutic agent is
selected from the group consisting of rapamycin, tacrolimus,
everolimus, pimecrolimus, SDZ-RAD, CCl-779, AP23841, ABT-578, and
analogs and derivatives thereof.
44. The method of claim 43, wherein the therapeutic agent is
selected from the group consisting of rapamycin and tacrolimus.
45. The method of any of claims 42, 43 or 44, wherein the
composition comprises 2% of the therapeutic agent.
46. The method of claim 44, wherein the therapeutic agent is
tacrolimus and the composition comprises 1% of the therapeutic
agent.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/665,203, filed Sep. 18, 2003, which claims the benefit of
U.S. Provisional Application No. 60/412,088, filed Sep. 18, 2002,
both of which are incorporated herein in their entirety.
TECHNICAL FIELD
[0003] The present invention relates to compositions and methods
for inhibiting unwanted angiogenesis, including that of ocular
tissues. In particular, compositions and methods for the treatment
of choroidal neovasculature (CNV) in ocular diseases are provided.
While the invention is exemplified with respect to rapamycin
(sirolimus) and tacrolimus, the invention provides for the use of
the family of "limus" compounds to inhibit unwanted
angiogenesis.
BACKGROUND OF THE INVENTION
[0004] The retina of the eye contains the cones and rods that
detect colors. In the center of the retina is the macula lutea,
which is about 1/3 to 1/2 cm in diameter. The macula provides
detailed vision, particularly in the center (the fovea), because
the cones are higher in density. Blood vessels, ganglion cells,
inner nuclear layer and cells, and the plexiform layers are all
displaced to one side (rather than resting above the cones),
thereby allowing light a more direct path to the cones.
[0005] Under the retina are the choroid, comprising a collection of
blood vessels embedded within a fibrous tissue, and the deeply
pigmented epithelium, which overlays the choroid layer. The
choroidal blood vessels provide nutrition to the retina
(particularly its visual cells).
[0006] There are a variety of retinal disorders, whose current
treatment is not optimal. The retina may tear, form holes and
separate from the underlying choroid.
[0007] Age-related macular degeneration (AMD) is the major cause of
severe visual loss in the United States for individuals over the
age of 55. AMD occurs in either an atrophic or (less commonly) an
exudative form. In exudative AMD, blood vessels grow from the
choriocapillaris through defects in Bruch's membrane, and in some
cases the underlying retinal pigment epithelium (choroidal
neovascularization or angiogenesis). Organization of serous or
hemorrhagic exudates escaping from these vessels results in
fibrovascular scarring of the macular region with attendant
degeneration of the neuroretina, detachment and tears of the
retinal pigment epithelium, vitreous hemorrhage and permanent loss
of central vision. This process is responsible for more than 80% of
cases of significant visual loss in patients with AMD.
[0008] Several studies have recently described the use of laser
photocoagulation in the treatment of initial or recurrent
neovascular lesions associated with AMD (Macular Photocoagulation
Study Groups (1991) in Arch. Ophthal. 109:1220; Arch. Ophthal.
109:1232; Arch Ophthal. 109:1242). Unfortunately, AMD patients with
subfoveal lesions subjected to laser treatment experienced a rather
precipitous reduction in visual acuity (mean 3 lines) at 3 months
follow-up. Moreover, at two years post-treatment treated eyes had
only marginally better visual acuity than their untreated
counterparts (means of 20/320 and 20/400, respectively). Another
drawback of the procedure is that vision after surgery is
immediately worse.
[0009] Choroidal neovascularization (CNV) has proven recalcitrant
to treatment in most cases. Laser treatment can ablate CNV and help
to preserve vision in selected cases not involving the center of
the retina, but this is limited to only about 10% of the cases.
There is no other treatment available to correct CNV.
Unfortunately, even with successful laser photocoagulation, the
neovascularization recurs in about 50-70% of eyes (50% over 3 years
and >60% at 5 years). (Macular Photocoagulation Study Group,
Arch. Opthalmol. 204:694-701 (1986)). In addition, many patients
who develop CNV are not good candidates for laser therapy because
the CNV is too large for laser treatment, or the location cannot be
determined so that the physician cannot accurately aim the laser.
Thus, until the present invention, there has been a long-felt need
for methods that will prevent or significantly inhibit choroidal
neovascularization.
[0010] In addition to AMD, choroidal neovascularization is caused
by such retinal disorders as: presumed ocular histoplasmosis
syndrome, myopic degeneration, angioid streaks and ocular trauma.
Angiogenic damage associated with retinal and intravitreal
neovascularization occurs in a wide range of disorders including
diabetic retinopathy, venous occlusions, sickle cell retinopathy,
retinopathy of prematurity, retinal detachment, ocular ischemia and
trauma.
[0011] Citation of the above documents is not intended as an
admission that any of the foregoing is pertinent prior art. All
statements as to the date or representation as to the contents of
these documents is based on the information available to the
applicant and does not constitute any admission as to the
correctness of the dates or contents of these documents.
DISCLOSURE OF THE INVENTION
[0012] The present invention provides compositions and methods that
are effective in inhibiting unwanted angiogenesis, specifically
choroidal neovascularization (CNV) that is associated with ocular
diseases such as age related macular degeneration (AMD) and
histoplasmosis syndrome. Compositions of the invention for
inhibiting angiogenesis comprise active agents in the "limus"
family of compounds, which bind to members of the immunophilin
family of cellular proteins, including cyclophilins and
FK506-binding proteins (FKBPs), to inhibit angiogenesis in
choroidal membranes. Non-limiting members of the "limus" family of
compounds include sirolimus (rapamycin) and its water soluble
analog SDZ-RAD, tacrolimus, everolimus, pimecrolimus, CCI-779
(Wyeth), AP23841 (Ariad), and ABT-578 (Abbott Laboratories) as well
as analogs and derivatives thereof.
[0013] A therapeutic amount of the active agents of the invention
may be administered to a patient by a variety of different routes
and can be given in dosages that are safe and provide angiogenic
inhibition at internal sites. The present invention thus provides
methods of treating mammalian diseases characterized by undesired
and uncontrolled angiogenesis by administering a composition
comprising one or more active agents of the invention. In a
particular embodiment of the invention, methods to inhibit or treat
choroidal neovascularization (CNV) of the eye are provided.
[0014] Thus, the present invention is especially useful for
treating certain ocular neovascular diseases such as macular
degeneration, including age-related macular degeneration (AMD). The
invention is particularly useful in the treatment or inhibition of
the wet form of AMD wherein blood vessels grow from their normal
location in the choroids into an undesirable position under the
retina. Leakage and bleeding from these new blood vessels results
in vision loss and possibly blindness. The invention also provides
methods for inhibiting the transition from the dry form of AMD
(wherein the retinal pigment epithelium or RPE degenerates and
leads to photoreceptor cell death and the formation of yellow
deposits called drusen under the retina) to the wet form of AMD.
The invention thus also provides methods for the treatment of the
dry form of AMD.
[0015] Compounds which are contemplated for use in the present
invention are administered to the patient to halt the progression
of the disease and permit reductions in, or regression of, the
neovascularization. Other diseases that can be treated using the
present invention include, but are not limited to, diabetic
retinopathy, neovascular glaucoma and retrolental fibroplasia.
[0016] Accordingly, and in a first aspect, the invention provides
compounds, compositions, kits and methods to inhibit unwanted
angiogenesis as well as neovascularization in the retina of a human
or animal. In a second aspect, the invention provides a treatment
for diseases mediated by angiogenesis or choroidal
neovascularization in a subject. In a further aspect, the invention
provides methods for preventing, inhibiting or treating the wet
form of AMD, including inhibiting the loss of vision associated
therewith.
[0017] Another aspect of the invention is the use of the above
described methods in combination with other methods known for the
treatment of angiogenesis, neovascularization, and the wet form of
AMD as well as reducing the loss of visual acuity associated
therewith. Moreover, the invention provides methods for the
visualization of blood vessels by use of lipophilic dyes in a body
as well as an animal model for choroidal neovascularization that
can be applied as an assay for rapid identification of additional
anti-angiogenic, anti-neovascularization, and anti-AMD
compounds.
[0018] Advantages and novel features of the invention will be set
forth in part in the description, examples and figures which
follow, all of which are intended to be for illustrative purposes
only, and not intended in any way to limit the invention, and in
part will become apparent to those skilled in the art on
examination of the following, or may be learned by practice of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a three dimensional reconstruction of retinal
blood vessels. Scale bar: 100 .mu.m.
[0020] FIG. 2 shows CNV in tissue from a Matrigel injected eye.
Scale bar: 100 .mu.m.
[0021] FIG. 3 shows inhibition of CNV formation by rapamycin. Scale
bar: 100 .mu.m.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF INVENTION
[0022] The invention provides methods for the treatment of
disorders involving angiogenesis and neovascularization, including
ophthalmic disorders, and in particular retinal disorders involving
macular degeneration, choroidal neovascularization, and the like in
the retina or between the retina and its underlying choroidal
tissue, or involving choroidal tissue, as described above. The
methods comprise a novel use for the immune suppressor, rapamycin,
which is also known as macrocyclic lactone sirolimus (commercially
available as Rapamune.RTM., Wyeth-Ayerst) (See, Physician's Desk
Reference, 55.sup.th edition). According to the Merck Index,
12.sup.th edition, Rapamune is also known as RAPA, RPM, sirolimus,
AY22989, and NSC-226080.
[0023] Although Sirolimus is known as an immunosuppressant, it has
been reported as an anti-angiogenic compound in the context of
primary and metastatic tumors (Guba et al., Nature Medicine
18(2):128-135 (February 2002) and Guba et al., Chir. Forum Exp.
Klin. Forsch. Band 30, pages 37-39 (2001)). In those studies, there
is no discussion related to other types of neovascularization, such
as choroidal neovascularization. Instead, there is a discussion of
the involvement of vascular endothelial growth factor (VEGF) and
the serum levels thereof. VEGF is a factor implicated in numerous
indications without certainty that therapies directed thereto are
efficacious in treating said indications. For example, VEGF has
been suggested as involved in the formation of pathogenic new
vessels in AMD, although VEGF activity has never been tested in an
animal model with regard to AMD. Therefore, the ability to treat
AMD by targeting VEGF activity remains experimental.
[0024] In addition to the use of sirolimus, the invention provides
for the use of other immunophilin binding compounds as well as
rapamycin derivatives and analogs to treat angiogenesis and
neovascularization. Non-limiting examples of such compounds include
SDZ-RAD, tacrolimus, everolimus, pimecrolimus, CCl-779 (Wyeth),
AP23841 (Ariad), and ABT-578 (Abbott Laboratories) as well as those
described in U.S. Pat. Nos. 5,527,907; 6,376,517; and 6,329,386.
Additional derivatives include those disclosed in published U.S.
Patent application 2002/0123505. All of these documents are hereby
incorporated by reference as if fully set forth.
[0025] The invention also provides for the use of the above agents
in combination with other agents and therapies for the treatment of
angiogenesis or neovascularization, particularly CNV. Non-limiting
examples of such additional agents and therapies include
pyrrolidine, dithiocarbamate (NF.kappa.B inhibitor); squalamine;
TPN 470 analogue and funagillin; PKC (protein kinase C) inhibitors;
Tie-1 and Tie-2 kinase inhibitors; inhibitors of VEGF receptor
kinase; proteosome inhibitors such as Velcade.TM. (bortezornib, for
injection; ranibuzurnab (Lucentis.TM.) and other antibodies
directed to the same target; pegaptanib (Macugen.TM.); vitronectin
receptor antagonists, such as cyclic peptide antagonists of
vitronectin receptor-type integrins; .alpha.-v/.beta.-3 integrin
antagonists; .alpha.-v/.beta.-1 integrin antagonists;
thiazolidinediones such as rosiglitazone or troglitazone;
interferon, including .gamma.-interferon or interferon targeted to
CNV by use of dextran and metal coordination; pigment epithelium
derived factor (PEDF); endostatin; angiostatin; anecortave acetate;
acetonide; triarncinolone; tetrathiomolybdate; RNA silencing or RNA
interference (RNAi) of angiogenic factors, including ribozymes that
target VEGF expression; Accutane.TM. (13-cis retinoic acid); ACE
inhibitors such as quinopril or perindozril; inhibitors of mTOR
(mammalian target of rapamycin); 3-aminothalidomide;
pentoxifylline; 2-methoxyestradiol; colchicines; AMG-1470;
cyclooxygenase inhibitors such as nepafenac, rofecoxib, and
diclofenac; t-RNA synthase modulator; metalloprotease 13 inhibitor;
acetylcholinesterase inhibitor; potassium channel blockers;
endorepellin; purine analog of 6-thioguanine; cyclic peroxide
ANO-2; (recombinant) arginine deiminase;
epigallocatechin-3-gallate; cerivastatin; analogues of suramin;
VEGF trap molecules; Visudyne.TM. and other photosensitizers with
photodynarnic therapy (PDT); and laser photocoagulation.
[0026] "Macular degeneration" is characterized by the excessive
buildup of fibrovascular deposits in or beneath the macula and
retina and the atrophy and/or dislodgement of the retinal pigment
epithelium (RPE). The administration of rapamycin appears to limit
excessive angiogenesis, such as choroidal neovascularization in
age-related macular degeneration (AMD), which may occur without
such treatment. As used herein, the term "angiogenesis" means the
generation of new blood vessels ("neovascularization") into a
tissue or organ. An "angiogenesis-mediated disease or condition" of
the eye or retina is one in which new blood vessels are generated
in a pathogenic manner in the eye or retina, resulting in loss of
vision or other problem, e.g., choroidal neovascularization
associated with AMD.
[0027] The methods of the invention include a preferred embodiment
using rapamycin and or other active agents in vitro or in vivo.
When administered in vitro, the method is used, for example, to
screen for, or assay the effects of, additional candidate active
agents for the activity of controlling or reducing
neovascularization or angiogenesis in retinal or choroidal tissue
or cells. This may be used as a helpful assay for additional
anti-angiogenesis or CNV agents. When administered in vivo the
method is used, for example, to treat a patient having a
predisposition to develop the choroidal neovascularization
typically seen in AMD, or to prevent or inhibit choroidal
neovascularization in such a patient, or to reduce choroidal
neovascularization in an AMD patient. Prevent, inhibit and reduce
are given their ordinary meanings with regard to the effect of the
active agents of the invention on choroidal neovascularization. A
patient having a predisposition or in need of prevention may be
identified by the skilled practitioner by established methods and
criteria in the field. The skilled practitioner may also readily
diagnose individuals as in need of inhibition or treatment based
upon established criteria in the field for identifying unwanted
angiogenesis and/or neovascularization.
[0028] An effective amount of the drug is that amount which
provides the therapeutic effect sought, e.g., an therapeutically
effective dose of rapamycin or drug equivalent would be the amount
which reduces choroidal neovascularization in an AMD patient, or
which inhibits or completely prevents choroidal neovascularization
in a patient predisposed to AMD, or who, even without
predisposition, shows early signs of AMD. Thus, the therapeutically
effective dose may not be the same in every patient treated with
rapamycin. An effective amount also refers to the amount of drug
which inhibits angiogenesis or neovascularization in a model or
assay therefor, such as that disclosed by the present
invention.
[0029] "Patient" preferably refers to a subject who has, or who may
develop, choroidal neovascularization associated with exudative AMD
unless treated by the preferred methods of the present invention.
Such a patient is preferably a mammal, more preferably a human,
although the present methods are also application to model
experimental animals and veterinary animal subjects.
[0030] An active agent of the invention, such as rapamycin, is
preferably administered orally, intravenously, topically,
intraocularly, intramuscularly, locally or in an ocular device.
More preferably the mode of administration is selected from the
following: intraocular injection, subretinal injection, subscleral
injection, intrachoroidal injection, subconjunctival injection,
topical administration, oral administration and parenteral
administration. Most preferably the active agent is directly
administered to the retinal area by subretinal injection, although
less invasive modes of administration may be developed that are
equally as effective. Formulations for timed release or delayed
release over time are also provided by the present invention.
[0031] The dosage of the active agent will depend on the condition
being treated, the particular agent, and other clinical factors
such as weight and condition of the human or animal and the route
of administration of the agent. It is to be understood that the
present invention has application for both human and veterinary
use. For administration to humans, an effective dosage is one that
inhibits choroidal neovascularization. In the case of rapamycin, an
inhibiting amount that can be employed ranges generally between
about 0.1 to 300 mg/kg/day, preferably between approximately 0.5
and 50 mg/kg/day, and most preferably between approximately 1 to 10
mg/kg/day. Dosages of various agents of the invention for treating
various conditions can be refined by the use of clinical trials on
the present invention. Additionally, dose ranges for the practice
of the invention include those disclosed in U.S. Pat. Nos.
6,376,517 and 5,387,589, which are hereby incorporated by reference
as if fully set forth.
[0032] An active agent of the invention, such as rapamycin, may be
subjected to conventional pharmaceutical operations, such as
sterilization and/or may contain conventional adjuvants, such as
preservatives, stabilizers, wetting agents, emulsifiers, buffers
etc. The agents may also be formulated with pharmaceutically
acceptable excipients for clinical use to produce a pharmaceutical
composition. Formulations of the present invention suitable for
oral administration may be presented as discrete units such as
capsules, cachets or tablets each containing a predetermined amount
of the active ingredient; as a powder or granules; as a solution or
a suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil emulsion and as a
bolus, etc. Stated differently, the active agents of the invention
may be used to prepare a medicament for the treatment of any of the
conditions described herein.
[0033] For administration, an active agent such as rapamycin may be
combined with one or more adjuvants appropriate for the indicated
route of administration. The active agent may be admixed with
lactose, sucrose, starch powder, cellulose esters of alkanoic
acids, stearic acid, talc, magnesium stearate, magnesium oxide,
sodium and calcium salts of phosphoric and sulphuric acids, acacia,
gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl
alcohol, and tableted or encapsulated for conventional
administration. Alternatively, the compounds of this invention may
be dissolved in polyethylene glycol, propylene glycol,
carboxymethyl cellulose colloidal solutions, ethanol, corn oil,
peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or
various buffers. Other adjuvants and modes of administration are
well known in the pharmaceutical art and may be used in the
practice of the invention. The carrier or diluent may include time
delay material, such as glyceryl monostearate or glyceryl
distearate alone or with a wax, or other materials well known in
the art.
[0034] The formulations of the invention include those suitable for
oral, ophthalmic, (including intravitreal or intracarneral) nasal,
topical (including buccal and sublingual), or parenteral (including
subcutaneous, intramuscular, intravenous, intradermal,
intratracheal, and epidural) administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by conventional pharmaceutical techniques. Such techniques include
the step of bringing into association the active ingredient and the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
associate the active ingredient with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0035] Further provided in another aspect of the invention is a
pharmaceutical composition comprising: a therapeutically effective
amount of an active agent of the invention, such as rapamycin, and
a pharmaceutically acceptable carrier suitable for administration
to the eye or eye tissue.
[0036] In addition, a kit is provided, comprising at least one vial
comprising a therapeutically effective amount of an active agent of
the invention, such as rapamycin, and a second vial comprising a
pharmaceutically acceptable carrier suitable for administration to
the eye or eye tissue. Other kits of the invention comprise
components such as the active agent of the invention for use in the
practice of the methods disclosed herein, wherein containers, each
with one or more of the various reagents (typically in concentrated
form) utilized in the methods, including, for example, buffers and
other reagents as necessary, are also included. A label or
indicator describing, or a set of instructions for use of, kit
components in a method of the present invention, will also be
typically included, where the instructions may be associated with a
package insert and/or the packaging of the kit or the components
thereof.
[0037] Diseases associated with retinal/choroidal
neovascularization that can be treated according to the present
invention include, but are not limited to, diabetic retinopathy,
macular degeneration, retinopathy of prematurity, infections
causing a retinitis or choroiditis, presumed ocular histoplasmosis,
myopic degeneration, angioid streaks, ocular trauma, and AMD. Other
non-limiting examples of diseases and unwanted conditions that may
be treated with the present invention include, but are not limited
to, pseudoxanthoma elasticum, vein occlusion, artery occlusion,
carotid obstructive disease, Sickle Cell anemia, Eales disease,
myopia, chronic retinal detachment, hyperviscosity syndromes,
toxoplasmosis, trauma and post-laser complications. Other diseases
include, but are not limited to, diseases associated with rubeosis
(neovasculariation of the angle) and diseases caused by the
abnormal proliferation of fibrovascular or fibrous tissue,
including all forms of proliferative vitreoretinopathy, whether or
not associated with diabetes.
[0038] In addition to the compositions and methods for the
treatment of unwanted conditions, the invention also provides
methods for the visualization of blood vessels in a body. Such
methods may also be viewed as methods of detectably labeling blood
vessels in a body for subsequent visualization. Conventional ways
to process tissue samples for blood vessel visualization are both
labor intensive and time consuming. To improve the efficiency in
tissue preparation, a technique called Vessel Painting is provided
by the present invention. The basic concept of Vessel Painting is
to selectively stain the inner lining of blood vessels with
fluorescent dye. Blood vessels are lined with endothelial cell
membrane, a lipid bilayer membrane that can be stained directly
with a lipophilic dye. The key to this technique is a specially
formulated solution, the Vessel Paint, which contains a lipophilic
dye. Non-limiting examples of such dyes are available from
Molecular Probes, and they include DiI, DiO, DiO, DiD, DiA, and
DiR, which are long-chain dialkylcarbocyanines and
dialkylaminostyryl dyes used as neuronal tracers. By intracardiac
perfusion of an animal with Vessel Paint followed by a wash,
optionally in a fixative solution such as, but not limited to 4%
paraformaldehyde solution, blood vessels are stained
instantaneously. Tissues can be viewed by fluorescence microscopy
immediately after perfusion. The staining is remarkably bright with
very low background so that high contrast images can be obtained
using objective lenses of different magnification powers.
[0039] The invention also provides for an animal model of ocular
neovascularization. The model is discussed in detail below, but
generally it is based upon the injection of material into the
subretinal space of an animal's eye. Any suitable non-human animal
model for ocular disease may be used, and the injected material may
range from Matrigel.TM., an extract of extracellular matrix (ECM)
proteins from the murine EHS (Engelbreth-Holm-Swarm) tumor that is
widely used as reconstituted basement membrane in cell culture
experiments, to a simple solution of rat tail collagen I, bovine
collagen I, and human collagen I (such as that available from BD
Biosciences). See Gautreau, A. et. al., PNAS, 96: 7300. (1999);
Abir R. et al. Hum Reprod, 14:299.(1999); and Abir R. et al. Fertil
Steril, 75:141. (2001). Other sources of collagen may be used,
including those produced by using lyophilized collagen (such as rat
tail collagen from Roche) dissolved in 0.1.times.DME pH 4.0 (DME
power from Life technologies without NaHCO.sub.3, make a 10.times.
solution but with the pH color indicator, and use HCl to adjust pH
to 4.0, then dilute this solution with water to make the 0.1.times.
DME solution) and a 10% acetic acid solution.
[0040] Without being bound by theory, and offered to improve the
understanding of the invention, the injection of a collagen (or
protein) solution is believed to be sufficient to mimic the
abnormal deposits that occur in AMD after injection into the
subretinal space of rats to induce new blood vessels invasion. Such
animal models may be advantageously used to screen candidate active
agents of the invention for activity against angiogenesis,
neovascularization (such as CNV), and AMD. Non-limiting examples of
such methods include those comprising the administration (by any
method disclosed herein) of a candidate agent to said animal and
determining the effect (increase, decrease, or no change) on
angiogenesis or neovascularization in said animal.
[0041] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all and only experiments performed. Efforts
have been made to ensure accuracy with respect to numbers used
(e.g. amounts, temperature, etc.) but some experimental errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric.
EXAMPLE 1
Matrigel.TM. Based Animal Model
[0042] Within this application, unless otherwise stated, the
techniques utilized may be found in any of several well-known
references, such as: Molecular Cloning: A Laboratory Manual
(Sambrook et al., Cold Spring Harbor Laboratory Press (1989);
"Guide to Protein Purification" in Methods in Enzymology (M. P.
Deutshcer, ed., (1990) Academic Press, Inc.); Culture of Animal
Cells: A Manual of Basic Technique, 2nd Ed., Liss, Inc., New York,
N.Y., (1987).
[0043] In search of effective treatments for CNV, a simple animal
model was created by injecting 2-3 .mu.l Matrigel.TM. to the
subretinal space of adult Sprague-Dawley rats using a 33-gauge
needle connected to a 10 .mu.l Hamilton microsyringe. A week or
more later the animals were sacrificed by CO.sub.2 inhalation and
perfused with Vessel Paint, a new visualization technique recently
developed in the inventors' lab that enables screening agents,
including chemical compounds and proteins, for their potential in
inhibiting CNV (Wen, ARVO Abstract, March 2002). Vessel Painting
comprises the use of a solution containing DiI, followed by 4%
paraformaldehyde solution, and is discussed further below.
[0044] The anterior portion of the eye, including the cornea and
lens, was removed, and the eyecup was imbedded in 5% agarose.
Serial thick sections (100 .mu.m) of the eye were cut on a
vibratome and mounted on glass slides. Eye sections were examined
by fluorescence microscopy for neovascularization in the
Matrigel.TM. deposit. Serial optical sections were obtained using
confocal microscopy. Three-dimensional reconstruction of newly
developed blood vessels was achieved using Auto Visualiz-3D
(Autouant Imaging, Inc.). Protein leakage of the new vessels was
detected by assessing color change in the Matrigel.TM. deposit
after intravenous injection of Evan's Blue dye.
[0045] Neovascularization was observed as early as 7 days after
Matrigel.TM. injection, and extensive CNV was evident 10 days after
Matrigel.TM. injection in all of the eyes injected. New blood
vessels, originated from choriocapillaris exclusively, invaded the
Matrigel.TM. deposit and formed extensive networks 14 days after
Matrigel.TM. injection. Three-dimensional reconstruction clearly
showed that the new vessels originated from the choroid. The
Matrigel.TM. deposit became light blue in color after Evan's blue
injection, as compared with pale white surrounding tissue,
indicating the lack of barrier. Discform scar was observed 30 days
after Matrigel.TM. injection.
[0046] Thus, the subretinal Matrigel.TM. deposits induce CNV in the
subretinal space, mimicking the pathology seen in exudative, or wet
form, AMD, and thereby providing an improved animal model for
researching the pathology of CNV and for testing potential
therapies.
EXAMPLE 2
Inhibition of CNV
[0047] In the initial characterization of the model, it was
suspected that there was a possible involvement of an inflammatory
reaction to Matrigel.TM. in the generation of neovascularization.
As a result, two known immunosuppressants were tested, cyclosporin
and rapamycin.
[0048] Oral administration of cyclosporin (15 mg/kg/d, given 4 day
before Matrigel.TM. injection thorough 10 days after injection) had
no effect on CNV. In marked contrast, however, oral rapamycin
(Rapamune.RTM., 1.5 mg/kg/d, given 4 days before Matrigel.TM.
injection thorough 10 day after injection) resulted in complete
inhibition of CNV development in the 16 eyes tested. Rapamycin is
commercially available as oral solution, marketed as Rapamune Oral
Solution by Wyeth-Ayerst. Thus, the anti-CNV properties of
rapamycin by local administration were further examined.
[0049] Since rapamycin is not soluble in water, it was either
dissolved in DMSO (tested in 8 eyes) or suspended in PBS (tested in
6 eyes), then mixed with Matrigel.TM.. The mixed
Matrigel.TM.-containing rapamycin was injected to the subretinal
space. At a dose of 25 .mu.g/.mu.l rapamycin (or 30
.mu.g/injection, since each injection used 1.2 .mu.l of
Matrigel.TM.), there was again a complete inhibition of CNV in
rapamycin-treated eyes.
[0050] In further experiments, and using the methods described
above, the amount of rapamycin was reduced to 2.5 .mu.g/l. At this
amount (the word "concentration" is not being used since rapamycin
is not soluble in water), the crystals of rapamycin were clearly
visible, even after 10 days. In each case, there was no detectable
neovascularization in treated eyes. Concerns with insolubility, if
they exist, may be addressed by the use of soluble active agents of
the invention, such as, but not limited to, SDZ-RAD.
[0051] Rapamycin thus has the potential to inhibit or prevent CNV
in human patients. In addition, local administration of rapamycin
is evidently a practical approach to CNV treatment, which is
particularly advantageous given the potential disadvantages effects
of systemic administration.
EXAMPLE 3
Vessel Painting
[0052] The retina from a normal 3 months old Sprague-Dawley rat,
sacrificed by CO.sub.2 overdose was perfused with Vessel Paint
(DiI, 0.1 mg/ml), followed by 4% paraformaldehyde. The retina was
dissected and postfixed in the same fixative for 1 hr, rinsed in
phosphate buffered saline (PBS), and flat-mounted on glass slides.
Microphotographs were taken on a Nikon E800 microscope.
[0053] Microphotographs of the flat-mount retina showed that
vessels stain brightly with a low background. Endothelial cell
nuclei were also stained and easily identifiable. The spatial
structure of the vasculature was well preserved and the deep
retinal capillaries were also visible.
[0054] The vascular network is better appreciated by images to show
its three-dimensionality. Excellent 3-D images of vasculature can
be obtained using the corrosion-casting technique and scanning
electron microscopy (SEM), see Konerding M A (1991) Scanning
electron microscopy of corrosion casting in medicine. Scanning
Microsc. 5:851-865. However, corrosion casting is technically
challenging and time-consuming, as is SEM. Alternatively, 3-D
images can be reconstructed from a stack of serial optical sections
by confocal microscopy. Bright staining and a high signal-to-noise
ratio by Vessel Painting make it possible to obtain serial optical
sections of samples as thick as 100-150 .mu.m by laser scanning
confocal microscopy without significant deterioration of signal
from the bottom of the sample even using objective lenses of low
magnification power. High quality of 3-D images at different
viewing angles can be reconstructed from a stack of 2-D digital
images using commercially available software. FIG. 1 shows 3-D
reconstructed images of a flat-mounted retina, processed as
described above. A stack of 78 optical sections along the Z-axis
(z-step=1 .mu.m) was taken by confocal microscopy using a 20.times.
objective lens on a Bio-Rad MRC-1024 confocal microscope. Three-D
images were reconstructed to show the retinal vasculature at an
angle of 0.degree. (FIG. 1A) or 180.degree. (FIG. 1B). Both depict
an artery on the retinal surface with connections to the deep
capillaries.
EXAMPLE 4
[0055] CNV in Matrigel.TM. Injected Area
[0056] Matrigel.TM. is an extract of extracellular matrix (ECM)
proteins from the murine EHS (Engelbreth-Holm-Swarm) tumor and is
widely used as reconstituted basement membrane in cell culture
experiments. It is also used to assess angiogenic or antiangiogenic
agents in an in vivo assay, the Matrigel.TM. plug assay (Passaniti
A, Taylor R M, Pili R, et al. (1992) A simple, quantitative method
for assessing angiogenesis and antiangiogenic agents using
reconstituted basement membrane, heparin, and fibroblast growth
factor. Lab Invest. 67:519-528). Pathological studies indicate an
association between CNV and abnormal deposits of extracellular
matrix (ECM) in the location between the retinal pigment epithelium
(RPE) and Bruch's membrane. In order to mimic the abnormal deposits
that occur in AMD, Matrigel.TM. was injected into the subretinal
space of rats. New blood vessels invade Matrigel.TM. deposits
shortly after Matrigel.TM. injection.
[0057] In the following, Matrigel.TM. (1.2 .mu.l) was introduced to
the subretinal space by injection into eyes of Sprague-Dawley rats.
At a given time after injection, the animal was sacrificed and
perfused with Vessel Paint, followed by 4% paraformaldehyde
solution. The anterior portion of the eye was removed and the
eyecup was then embedded in 5% agarose. Serial cross sections (100
.mu.m thick) were cut on a vibratome.
[0058] New blood vessels originating from the choriocapillaris were
detectable as early as 4 days after Matrigel.TM. injection and
became well developed by 10 days after injection. A DIC image of a
cross section of an eye, from a 2 month old animal 10 days after
Matrigel.TM. injection, is shown in FIG. 2A. The DIC image was
superimposed with an optical section to show CNV along with
choroidal and retinal vasculature (FIG. 2A). New blood vessels
penetrate Bruch's membrane at a single site (yellow arrowhead) and
then ramify in the Matrigel.TM. layer between the RPE and the
retina. A stack of 42 optical sections were taken from this sample
and a 3-D image was reconstructed (FIG. 2B). The 3-D image clearly
shows that the new blood vessels originated from the
choriocapillaris through a single penetration site (yellow
arrowhead).
[0059] The permeability of newly formed blood vessels was assessed
by Evans Blue assay. Evans Blue (60 mg/kg in PBS) was injected
intravenously to a Sprague-Dawley rat whose eyes had been injected
with Matrigel.TM. 10 days before. A choroid-retina preparation was
dissected, flat-mounted and viewed by fluorescence microscopy.
Evans Blue staining was only seen in the Matrigel.TM. injected
area, indicating the leaky nature of the new vessels.
EXAMPLE 5
Inhibition of CNV by Rapamycin
[0060] In initial screens of potential anti angiogenic agents using
the Matrigel.TM. model, rapamycin demonstrated a remarkable ability
to inhibit new blood vessel formation. Rapamycin, clinically used
as immunosuppressant (Kahan B D (2001) Sirolimus: a comprehensive
review. Expert Opin. Pharmacother. 2:1903-1917), binds to a FKBP
(FK506 binding protein) to form an FKBP-rapamycin complex, which in
turn inhibits the function of mTOR (mammalian target of rapamycin),
a central controller of cell growth (Schmelzle T, Hall M N (2000)
TOR, a central controller of cell growth. Cell 103:253-262).
Rapamycin inhibits endothelial cell proliferation (Vinals F,
Chambard J C, Pouyssegur J (1999) p70 S6 kinase-mediated protein
synthesis is a critical step for vascular endothelial cell
proliferation. J. Biol. Chem. 274:26776-26782) and the response to
VEGF (Yu Y, Sato J D (1999) MAP kinases, phosphatidylinositol
3-kinase, and p70 S6 kinase mediate the mitogenic response of human
endothelial cells to vascular endothelial growth factor. J. Cell
Physiol. 178:235-246) and bFGF (basic fibroblast growth factor) and
PDGF (platelet-derived growth factor). See Cao et al. (1995)
Effects of rapamycin on growth factor-stimulated vascular smooth
muscle cell DNA synthesis. Inhibition of basic fibroblast growth
factor and platelet-derived growth factor action and antagonism of
rapamycin by FK506. Transplantation 59(3):390-5 and Ruygrok et al.
(2003) Rapamycin and cardiovascular medicine. Intern. Med. J. 33
(3):103-9. It has been shown to block tumor angiogenesis (Guba M,
von Breitenbuch P, Steinbauer M, et al. (2002) Rapamycin inhibits
primary and metastatic tumor growth by antiangiogenesis:
involvement of vascular endothelial growth factor. Nat. Med.
8:128-135).
[0061] Adult Sprague-Dawley rats (n=22) were injected with
Matrigel.TM. to the subretinal space. Animals were fed with
rapamycin once a day at a dose of 3 mg/kg started 4 days before
Matrigel.TM. injection. Eyes were collected at 10 days (n=18) or 20
days (n=4) after Matrigel.TM. injection and processed as described
in Example 4. Tissue sections were examined by fluorescence
microscopy. None of the eyes collected at 10 days contained any new
blood vessels invading the subretinal space in rapamycin treated
animals. In the 20 days group, some newly formed blood vessels
started to invade the Matrigel.TM. area. The amount of CNV in this
group was semiquantified as (+). In comparison, CNV in control
animals (10 days) were graded as (+++.about.++++), as shown in FIG.
2.
[0062] In another group of animals, rapamycin was mixed with
Matrigel.TM. (suspension, 1 .mu.g/.mu.l, n=6; 10 .mu.g/.mu.l, n=11)
and co-injected to the subretinal space with Matrigel.TM. (in-gel
delivery). Eyes were collected 10 days after injection and
processed as described in Example 4. No CNV was found in any of the
eyes treated with rapamycin by in-gel delivery. FIG. 3 shows a DIC
image of a section from an eye, injected with Matrigel and
rapamycin mixture at a high dose (10 .mu.g/.mu.l suspension) 10
days before tissue collection, to show rapamnycin particles in the
subretinal space. Rapamycin particles in Matrigel.TM. are clearly
seen in the DIC image, which is superimposed on a confocal image of
Vessel Paint staining to show the choroidal and retinal blood
vessels. No CNV was found in any eyes injected with rapamycin.
[0063] In all experiments, rapamycin had no discernable effect on
the normal vasculature of the eye. Cyclosporin, also an
immunosuppressant, failed to inhibit CNV formation either when
administrated orally (100 mg/kg/d, n=3) or in-gel (25 .mu.g/.mu.l,
n=3) by the same experimental paradigm.
EXAMPLE 5
[0064] CNV index in FK506 tacrolimus treated eyes FK506, which is
not water soluble, was mixed with Matrigel.TM. (as a suspension) at
10 .mu.g/.mu.l, and 1.2 .mu.l were injected into the subretinal
space as described above. Eyes were collected 10 days after
injection and blood vessels were stained with Vessel Paint. Eyes
were embedded in 5% agarose and serial sections were cut (100 .mu.m
thick) on a vibratome. CNV was examined by fluorescence microscopy
and CNV index of each eye was calculated. Results are shown
below.
TABLE-US-00001 FK506 Mean = 12.67 (n = 6) SEM = 2.76 Control Mean =
32.00 (n = 10) SEM = 6.41 Student's t-test P = 0.042 Therefore,
FK506 inhibited CNV by 60%.
[0065] All references cited herein, including patents, patent
applications, and publications, are hereby incorporated by
reference in their entireties, whether previously specifically
incorporated or not.
[0066] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0067] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
* * * * *