Note: Descriptions are shown in the official language in which they were submitted.
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THE USE OF A PROTEIN TYROSINE KINASE PATHWAY INHIBITOR
IN THE TREATMENT OF OCULAR DISORDERS
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for the
prophylactic and therapeutic treatment of age-related
macular degeneration, as well as methods for the
prophylactic and therapeutic treatment of degeneration of
the retina, degeneration of the choroid, and thickening
of Bruch's membrane.
BACKGROUND OF THE INVENTION
As the average human life span is continually
lengthened by improvements in medical technology, there
is an increasing need to address medical issues
associated with age. The aging process results in
physical and chemical changes in the eye, which lead to
loss of visual acuity, decreased contrast sensitivity
and, ultimately, complete vision loss. Blindness is
perhaps the leading debilitating condition afflicting the
elderly population. Age-related macular degeneration is
the leading cause of blindness in patients over 65 years
of age. In fact, vision loss attributed to age-related
macular degeneration was found in approximately 10% of
the United States population over 65 years of age
(Gerster et al. Age Ageing 20: 60-69 (1991)). As the
elderly population of the world increases, the incidence
of age-related macular degeneration is expected to
increase dramatically, reaching a predicted 7.5 million
cases in the United States alone by the year 2030 (Hyman
et al. Am J Epidemiol 118: 213-227 (1983)).
Age-related macular degeneration is a progressive,
degenerative disorder of the eye resulting in loss of
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visual acuity. Symptoms of age-related macular
degeneration include blurred vision, decreased ability to
read, especially in dim light, trouble with dark
adaptation and, in relatively few cases, abrupt vision
loss. Complications associated with advanced age-related
macular degeneration are divided into two categories,
atrophic and exudative. Atrophic complications are
associated with retinal pigment epithelial cell loss
resulting in atrophy of the retinal pigment epithelium
(RPE). Exudative complications, which appear in
approximately 1096 of age-related macular degeneration
cases, include disciform scars (i.e., scarring involving
fibrous elements) and neovascularization. Ultimately,
blindness from age-related macular degeneration stems
from degeneration of the RPE and the subsequent death of
photoreceptors.
Risk factors associated with age-related macular
degeneration include age, heredity, systemic disease,
environmental factors, such as smoking and light exposure
(see e.g., Chesapeake Bay Waterman Study, Taylor et al.,
Arch. Ophthalmol. 110: 99-104 (1992)), and nutritional
deficiency. Hyperopia and iris color also have been
linked to the disease. Age-related macular degeneration
has been correlated to light-colored irises, perhaps due
to chronic exposure to damaging light which is normally
absorbed by dark-colored irises. The disease also
appears more often in women than men.
The best documented mechanism for the development of
age-related macular degeneration involves molecular
degradation in the RPE. Incomplete digestion of abnormal
molecules, most likely altered post-synthetically,
results in the formation of pockets of waste in RPE cells
which eventually interfere with the normal metabolism of
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the cell. Consequently, aberrant excretions from RPE
cells aggregate within Bruch ' s membrane as basal laminar
deposits, drusen and debris. It is believed that such
deposits invoke neovascularization and/or the death of
RPE cells (Young, Survey of Ophthalmology 3/(5): 291-306
(1987)).
There is currently no known prophylactic or
therapeutic treatment for age-related macular
degeneration (AMD). While not a cure for AND, laser
photocoagulation is used to treat choroidal
neovascularization associated with age-related macular
degeneration. Such treatment has been proven to reduce
the risk of severe vision loss. Laser photocoagulation
also has been used to treat drusen. However, laser
treatment may cause permanent blind spots corresponding
to the treated areas, leading to a decrease in visual
acuity. Laser treatment may also cause persistent or
recurrent hemorrhage and increase the risk of sensory
retinal detachment. Many patients eventually experience
severe vision loss in spite of treatment. Likewise,
there currently is no method available for the
prophylactic or therapeutic treatment of physical and
chemical changes in the eye associated with the aging
process, in general.
Given the prevalence of age-related macular
degeneration, there remains a need for an effective
prophylactic and therapeutic treatment of age-related
macular degeneration. Accordingly, it is a principal
object of the present invention to provide a method of
prophylactically and therapeutically treating age-related
macular degeneration, including treatment of the atrophic
and exudative complications associated with the disease.
A need also exists in the art for an effective
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,
prophylactic and therapeutic treatment for deterioration
of the eye, such as degeneration of the retina,
degeneration of the choroid and thickening of Bruch's
membrane. The present invention provides such methods of
treatment. These and other objects of the present
invention, as well as additional inventive features, will
become apparent from the detailed description provided
herein.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed to a method for
the prophylactic and therapeutic treatment of age-related
macular degeneration, including treatment of the atrophic
and exudative complications associated with the disease.
The present invention is also directed to a method for
the prophylactic and therapeutic treatment of an animal
for degeneration of the retina. A method of
prophylactically or therapeutically treating an animal
for degeneration of the choroid is also provided, as is a
method of prophylactically or therapeutically treating an
animal for thickening of Bruch's membrane. The methods
involve the administration of an inhibitor of the protein
tyrosine kinase pathway. Preferably, the inhibitor of
the protein tyrosine kinase pathway is a compound of
formula:
x z
Y
1.1 1
o
1101
w
V
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wherein V, W and X are selected from the group consisting
of hydro, hydroxyl, alkoxy, halo, an ester, an ether, a
carboxylic acid group, a pharmaceutically acceptable salt
5 of a carboxylic acid group, and -SR, in which R is
hydrogen or an alkyl group, and Y is selected from the
group consisting of oxygen, sulfur, C(OH), and C=0, and Z
is selected from the group consisting of hydro and
C(0)0R1, wherein R1 is an alkyl. Preferably, the alkoxy
is a C1-C6 alkoxy. Preferably, the halo is fluorine,
chlorine or bromine. Preferably, the ester is a C1-C6
ester. Preferably, the ether is a C1-C6 ether. Preferred
pharmaceutically acceptable salts of the carboxylic acid
group include sodium and potassium salts. Preferably,
the alkyl groups are C1-C6 alkyl groups. Desirably, the
protein tyrosine kinase pathway inhibitor is genistein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated on the discovery
that an inhibitor of the protein tyrosine kinase pathway,
specifically genistein, is effective in treating
prophylactically and therapeutically age-related macular
degeneration, including the exudative and atrophic
complications associated with age-related macular
degeneration, and ocular disorders, such as degeneration
of the retina, degeneration of the choroid and thickening
of Bruch's membrane. Accordingly, the present invention
provides a method for the prophylactic and therapeutic
treatment of age-related macular degeneration. By
"prophylactic" is meant the protection, in whole or in
part, against age-related macular degeneration, in
particular choroidal neovascularization and retinal
pigment epithelium atrophy. By "therapeutic" is meant
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the amelioration of age-related macular degeneration,
itself, and the protection, in whole or in part, against
further age-related macular degeneration, in particular
choroidal neovascularization and retinal pigment
epithelium atrophy. Preferably, age-related macular
degeneration is that which results from age, hyperopia,
systemic disease, such as cardiovascular disease or
hypertension, smoking, light exposure or nutritional
deficiency.
The method comprises the administration of an
inhibitor of the protein tyrosine kinase pathway in an
amount sufficient to treat the macula for age-related
macular degeneration prophylactically or therapeutically.
Any inhibitor of the protein tyrosine kinase (PTK)
pathway can be used in the method of the present
invention as long as it is safe and efficacious.
The present invention additionally provides a method
for the prophylactic and therapeutic treatment of both
atrophic and exudative complications associated with age-
related macular degeneration. Atrophic complications
include pigmentary disturbance of the retinal pigment
epithelium, hard, soft and/or confluent drusen, basal
laminar deposits, a thickening of Bruch's membrane, and
RPE atrophy. Drusen are yellow or white excresences
between the basement membrane of the RPE and Bruch's
membrane which are often precursors for
neovascularization. Exudative complications include
choroidal neovascularization, RPE detachment, RPE tears,
disciform scarring, vitreous hemorrhage and subretinal
hemorrhage. Prophylactic and therapeutic treatment of
exudative complications can be effected by prevention or
inhibition of disruption of the integrity of the basement
membrane.
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The method comprises the administration of an
inhibitor of the PTK pathway in an amount sufficient to
treat an exudative or atrophic complication of age-
related macular degeneration prophylactically or
therapeutically. Any safe and efficacious PTK inhibitor
can be used.
The present invention further provides a method for
the prophylactic and therapeutic treatment of an animal
for degeneration of the retina, in particular that which
is due to aging. As used herein, degeneration of the
retina, in particular that which is due to aging,
includes, for example, pigment epithelial abnormalities
and ganglion cell loss. Degeneration of the retina also
includes loss of photoreceptor cells, which can result in
decreased contrast sensitivity and vision loss. By
"prophylactic" is meant the protection, in whole or in
part, against further degeneration of the retina. By
"therapeutic" is meant the amelioration of degeneration
of the retina, itself, and the protection, in whole or in
part, against further degeneration of the retina.
The method comprises the administration of an
inhibitor of the PTK pathway in an amount sufficient to
treat degeneration of the retina, in particular that
which is due to aging, prophylactically or
therapeutically. As indicated above, any PTK pathway
inhibitor can be used in this method as long as it is
safe and efficacious.
In addition, the present invention provides a method
for the prophylactic and therapeutic treatment of an
animal for degeneration of the choroid, in particular
that which is due to aging. Degeneration of the choroid,
in particular that which is due to aging, includes, for
example, flattening of the choroid. Degeneration of the
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choroid due to aging also includes atrophy of
choriocapillaries. By "prophylactic" is meant the
protection, in whole or in part, against degeneration of
the choroid. By "therapeutic" is meant the amelioration
of degeneration of the choroid, itself, and the
protection, in whole or in part, against further
degeneration of the choroid.
The method comprises the administration of an
inhibitor of the PTK pathway in an amount sufficient to
treat degeneration of the choroid, in particular that
which is due to aging, prophylactically or
therapeutically. Any safe and efficacious PTK pathway
inhibitor can be used.
Ocular disorders further include thickening of
Bruch's membrane, which is located between the retina and
the choroid, in particular thickening of Bruch's membrane
as a result of aging. Thickening of Bruch's membrane can
interfere with vision by separating photoreceptor cells
from the vascular-rich choroid and causing abnormalities
in the retina. Accordingly, the present invention
provides a method for the prophylactic and therapeutic
treatment of an animal for thickening of Bruch's
membrane. By "prophylactic" is meant the protection, in
whole or in part, against thickening of Bruch's membrane.
By "therapeutic" is meant the amelioration of thickening
of Bruch's membrane, itself, and the protection, in whole
or in part, against further thickening of Bruch's
membrane.
The method comprises the administration of an
inhibitor of the PTK pathway in an amount sufficient to
treat Bruch's membrane for thickening prophylactically or
therapeutically. Any safe and efficacious inhibitor of
the PTK pathway can be used.
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Herein, "PTK inhibitor" will be used to refer to
such compounds and is intended to encompass any and all
compounds that inhibit the PTK pathway, irrespective of
at which point in. the pathway the compound effects
inhibition. PTK inhibitors are known in the art; others
can be identified in accordance with assays known in the
art. One of ordinary skill in the art will appreciate
that, while complete inhibition of the PTK pathway by the
PTK inhibitor is preferred, partial inhibition of the PTK
pathway can be sufficient to achieve a prophylactic or
therapeutic effect in the context of the present
invention.
Preferably, the PTK inhibitor is genistein (5,7-
dihydroxy-3-(4-hydroxypheny1)-4H-1-benzopyran-4-one) or a
pharmaceutically acceptable, PTK pathway-inhibiting
analogue or prodrug thereof or a pharmaceutically
acceptable salt of any of the foregoing. Accordingly,
the PTK inhibitor can be a compound of the following
formula:
11111
0
11111
wherein V, W and X are selected from the group consisting
of hydro, hydroxyl, an alkoxy, halo, an ester, an ether,
a carboxylic acid group, a pharmaceutically acceptable
salt of a carboxylic acid group, and -SR, in which R is
hydrogen or an alkyl group, and Y is selected from the
group consisting of oxygen, sulfur, C(OH), and 0=0, and Z
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is selected from the group consisting of hydro and
C(0)0R1, wherein R1 is an alkyl. Preferably, the alkoxy
is a C1-C6 alkoxy. Preferably, the halo is fluorine,
chlorine or bromine. Preferably, the ester is a C1-C6
5 ester. Preferably, the ether is a C1-C6 ether. Preferred
pharmaceutically acceptable salts of the carboxylic acid
group include sodium and potassium salts. Preferably,
the alkyl groups are C1-C6 alkyl groups. Desirably, the
PTK pathway inhibitor is genistein.
10 The prodrug can be any pharmaceutically acceptable
prodrug of genistein, a PTK pathway-inhibiting analogue
of genistein, or a pharmaceutically acceptable salt of
either of the foregoing. One of ordinary skill in the
art will appreciate, however, that the prodrug used must
be one that can be converted to an active PTK inhibitor
in or around the macula, retina, choroid or Bruch's
membrane. A preferred prodrug is a prodrug that
increases the lipid solubility of genistein, a PTK
pathway-inhibiting analogue of genistein, or a
pharmaceutically acceptable salt of either of the
foregoing. A preferred prodrug is one in which one or
more of V, W and X are independently derivatized with an
ester, such as pivalic acid.
Compounds of the above formula are widely available
commercially. For example, genistein is available from
LC Laboratories (Woburn, MA). Those compounds that are
not commercially available can be readily prepared using
organic synthesis methods known in the art.
Whether or not a particular analogue, prodrug or
pharmaceutically acceptable salt of a compound in
accordance with the present invention can treat macular
degeneration, degeneration of the retina, degeneration of
the choroid, or thickening of Bruch's membrane, any one
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of which can be due to aging, prophylactically or
therapeutically can be determined by its effect in the
mouse model used in Examples 2 and 4-6. Alternatively,
analogues, prodrugs and pharmaceutically acceptable salts
of inhibitors of the PTK pathway can be tested by in
vitro assays such as, for example, the method set forth
in Example 1.
In addition, color Doppler imaging can be used to
evaluate the action of a drug in ocular pathology (Valli
et al., Ophthalmologica 209(13): 115-121 (1995)). Color
Doppler imaging is a recent advance in ultrasonography,
allowing simultaneous two-dimensional imaging of
structures and the evaluation of blood flow.
Accordingly, atrophic complications, such as retinal
pigment epithelium atrophy, and exudative complications,
such as choroidal neovascularization, can be analyzed
using such technology. Similarly, complications
associated with degeneration of the retina, such as
photoreceptor loss and ganglion cell loss, as well as
complications associated with degeneration of the
choroid, such as atrophy of the choriocapillaries and
flattening of the choroid, can be analyzed using color
Doppler imaging.
A PTK inhibitor can be bound to a suitable matrix,
such as a polymeric matrix, if desired, for use in the
present inventive method. Any of a wide range of
polymers can be used in the context of the present
invention provided that, if the polymer-bound compound is
to be used in vivo, the polymer is biologically
acceptable (see, e.g., U.S. Patent Nos. 5,384,333 and
5,164,188).
An advantage of genistein is that it is very safe
and efficacious. For example, when genistein was orally
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administered to Zucker diabetic fatty rats, genistein was
found to be nontoxic to the retina at dosages ranging
from 75 mg/kg/day to 300 mg/kg/day over a period of six
months as measured by electroretinography. In addition,
oral administration of genistein was found to have no
effect on food intake and body weight for male and female
rats. Also, no effect of orally administered genistein
was found with respect to the weight of the ovaries and
the uterus in female rats.
The PTK inhibitor, which is preferably genistein, a
PTK pathway-inhibiting analogue of genistein, a PTK
pathway-inhibiting prodrug of genistein, or a
pharmaceutically acceptable salt of any of the foregoing,
can be administered in accordance with the present
inventive methods by any suitable route. Suitable routes
of administration include systemic, such as orally or by
injection, topical, intraocular, periocular (e.g.,
subTenon's), subconjunctival, subretinal, suprachoroidal
and retrobulbar. The manner in which the PTK inhibitor
is administered is dependent, in part, upon whether the
treatment of age-related macular degeneration,
degeneration of the retina, degeneration of the choroid,
or thickening of Bruch's membrane is prophylactic or
therapeutic. For instance, the manner in which the PTK
inhibitor is administered for therapeutic treatment of
age-related macular degeneration is dependent, in part,
upon the cause of the disease.
For example, given that the appearance of drusen and
RPE hyperpigmentation is often the first indication of
age-related macular degeneration, the PTK inhibitor can
be administered prophylactically as soon as drusen and
RPE hyperpigmentation are detected. For the prophylactic
treatment of age-related macular degeneration, the PTK
=
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inhibitor is preferably administered systemically, e.g.,
orally or by injection. For the therapeutic treatment of
age-related macular degeneration, i.e., treatment of
atrophic and/or exudative complications, the PTK
inhibitor can be administered systemically, e.g., orally
or by injection, intraocularly, topically,
subconjunctivally or periocularly (e.g., subTenon's), for
example.
The PTK inhibitor is preferably administered as soon
as possible after it has been determined that an animal,
such as a mammal, specifically a human, is at risk for
age-related macular degeneration (prophylactic treatment)
or has begun to develop age-related macular degeneration
(therapeutic treatment). Treatment will depend, in part,
upon the particular PTK inhibitor used, the amount of the
PTK inhibitor administered, the route of administration,
and the cause and extent, if any, of macular degeneration
realized. Likewise, the PTK inhibitor is preferably
administered as soon as possible after it has been
determined that an animal, specifically a human, is at
risk for retinal or choroidal degeneration or thickening
of Bruch's membrane (prophylactic treatment) or has begun
to develop degeneration of the retina or choroid or
thickening of Bruch's membrane (therapeutic treatment).
One skilled in the art will appreciate that suitable
methods of administering a PTK inhibitor, which is useful
in the present inventive method, are available. Although
more than one route can be used to administer a
particular PTK inhibitor, a particular route can provide
a more immediate and more effective reaction than another
route. Accordingly, the described routes of
administration are merely exemplary and are in no way
limiting.
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The dose administered to an animal, particularly a
human, in accordance with the present invention should be
sufficient to effect the desired response in the animal
over a reasonable time frame. One skilled in the art
will recognize that dosage will depend upon a variety of
factors, including the strength of the particular PTK
inhibitor employed, the age, species, condition or
disease state, and body weight of the animal, as well as
the amount of the macula, retina or choroid about to be
affected or actually affected by degeneration or the
amount of Bruch's membrane about to be affected or
actually affected by thickening. The size of the dose
also will be determined by the route, timing and
frequency of administration as well as the existence,
nature, and extent of any adverse side effects that might
accompany the administration of a particular PTK
inhibitor and the desired physiological effect. It will
be appreciated by one of ordinary skill in the art that
various conditions or disease states, in particular,
chronic conditions or disease states, may require
prolonged treatment involving multiple administrations.
Suitable doses and dosage regimens can be determined
by conventional range-finding techniques known to those
of ordinary skill in the art. Generally, treatment is
initiated with smaller dosages, which are less than the
optimum dose of the compound. Thereafter, the dosage is
increased by small increments until the optimum effect
under the circumstances is reached. The present
inventive method will typically involve the
administration of from about 1 mg/kg/day to about 100
mg/kg/day, preferably from about 15 mg/kg/day to about 50
mg/kg/day, if administered systemically. Intraocular
administration typically will involve the administration
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of from about 0.1 mg total to about 5 mg total,
preferably from about 0.5 mg total to about 1 mg total.
A preferred concentration for topical administration is
100 M.
5
Compositions for use in the present inventive method
preferably comprise a pharmaceutically acceptable carrier
and an amount of a PTK inhibitor sufficient to treat age-
related macular degeneration and/or an atrophic or
exudative complication thereof prophylactically or
10 therapeutically. Alternatively, compositions for use in
the present inventive method of treating degeneration of
the retina, such as that which is due to aging,
degeneration of the choroid, such as that which is due to
aging, or thickening of Bruch's membrane, such as that
15 which is due to aging, preferably comprise a
pharmaceutically acceptable carrier and an amount of a
PTK inhibitor sufficient to treat degeneration of the
retina, degeneration of the choroid or thickening of
Bruch's membrane, respectively, prophylactically or
therapeutically. The carrier can be any of those
conventionally used and is limited only by chemico-
physical considerations, such as solubility and lack of
reactivity with the compound, and by the route of
administration. It will be appreciated by one of
ordinary skill in the art that, in addition to the
following described pharmaceutical compositions, the PTK
inhibitor can be formulated as polymeric compositions,
inclusion complexes, such as cyclodextrin inclusion
complexes, liposomes, microspheres, microcapsules and the
like (see, e.g., U.S. Patent Nos. 4,997,652, 5,185,152
and 5,718,922).
The PTK inhibitor can be formulated as a
pharmaceutically acceptable acid addition salt. Examples
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of pharmaceutically acceptable acid addition salts for
use in the pharmaceutical composition include those
derived from mineral acids, such as hydrochloric,
hydrobromic, phosphoric, metaphosphoric, nitric and
sulfuric acids, and organic acids, such as tartaric,
acetic, citric, malic, lactic, fumaric, benzoic,
glycolic, gluconic, succinic, and arylsulphonic, for
example p-toluenesulphonic, acids.
The pharmaceutically acceptable excipients described
herein, for example, vehicles, adjuvants, carriers or
diluents, are well-known to those who are skilled in the
art and are readily available to the public. It is
preferred that the pharmaceutically acceptable carrier be
one which is chemically inert to the PTK inhibitor and
one which has no detrimental side effects or toxicity
under the conditions of use.
The choice of excipient will be determined in part
by the particular PTK inhibitor, as well as by the
particular method used to administer the composition.
Accordingly, there is a wide variety of suitable
formulations of the pharmaceutical composition of the
present invention. The following formulations are merely
exemplary and are in no way limiting.
Injectable formulations are among those that are
preferred in accordance with the present inventive
method. The requirements for pharmaceutically effective
carriers for injectable compositions are well-known to
those of ordinary skill in the art (see Pharmaceutics and
Pharmacy Practice, J.B. Lippincott Co., Philadelphia, PA,
Banker and Chalmers, eds., pages 238-250 (1982), and ASHP
Handbook on Injectable Druqs, Toissel, 4th ed., pages 622-
630 (1986)). It is preferred that such injectable
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compositions be administered intramuscularly,
intravenously, or intraperitoneally.
Topical formulations are well-known to those of
skill in the art. Such formulations are suitable in the
context of the present invention for application to the
skin. The use of patches, corneal shields (see, e.g.,
U.S. Patent No. 5,185,152), and ophthalmic solutions
(see, e.g., U.S. Patent No. 5,710,182) and ointments,
e.g., eye drops, is also within the skill in the art.
Formulations suitable for oral administration can
consist of (a) liquid solutions, such as an effective
amount of the compound dissolved in diluents, such as
water, saline, or orange juice; (b) capsules, sachets,
tablets, lozenges, and troches, each containing a
predetermined amount of the active ingredient, as solids
or granules; (c) powders; (d) suspensions in an
appropriate liquid; and (e) suitable emulsions. Liquid
formulations may include diluents, such as water and
alcohols, for example, ethanol, benzyl alcohol, and the
polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant,
suspending agent, or emulsifying agent. Capsule forms
can be of the ordinary hard- or soft-shelled gelatin type
containing, for example, surfactants, lubricants, and
inert fillers, such as lactose, sucrose, calcium
phosphate, and corn starch. Tablet forms can include one
or more of lactose, sucrose, mannitol, corn starch,
potato starch, alginic acid, microcrystalline cellulose,
acacia, gelatin, guar gum, colloidal silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, calcium
stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents,
disintegrating agents, moistening agents, preservatives,
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flavoring agents, and pharmacologically compatible
excipients. Lozenge forms can comprise the active
ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active
ingredient in an inert base, such as gelatin and
glycerin, or sucrose and acacia, emulsions, gels, and the
like containing, in addition to the active ingredient,
such excipients as are known in the art.
Formulations suitable for parenteral administration
include aqueous and non-aqueous, isotonic sterile
injection solutions, which can contain anti-oxidants,
buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and
preservatives. The inhibitor can be administered in a
physiologically acceptable diluent in a pharmaceutical
carrier, such as a sterile liquid or mixture of liquids,
including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol,
isopropanol, or hexadecyl alcohol, glycols, such as
propylene glycol or polyethylene glycol,
dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-
1,3-dioxolane-4-methanol, ethers, such as
poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty
acid ester or glyceride, or an acetylated fatty acid
glyceride, with or without the addition of a
pharmaceutically acceptable surfactant, such as a soap or
a detergent, suspending agent, such as pectin, Carbomers
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
* trade mark
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Oils, which can be used in parenteral formulations
include petroleum, animal, vegetable, or synthetic oils.
Specific examples of oils include peanut, soybean,
sesame, cottonseed, corn, olive, petrolatum, and mineral.
Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and
isostearic acid. Ethyl oleate and isopropyl myristate
are examples of suitable fatty acid esters.
Suitable soaps for use in parenteral formulations
include fatty alkali metals, ammonium, and
triethanolamine salts, and suitable detergents include
(a) cationic detergents such as, for example, dimethyl
dialkyl ammonium halides, and alkyl pyridinium halides,
(b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and
monoglyceride sulfates, and sulfosuccinates, (c) nonionic
detergents such as, for example, fatty amine oxides,
fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric
detergents such as, for example, alkyl-p-
aminopropionates, and 2-alkyl-imidazoline quaternary
ammonium salts, and (e) mixtures thereof.
The parenteral formulations will typically contain
from about 0.5 to about 25% by weight of the active
ingredient in solution. Preservatives and buffers may be
used. In order to minimize or eliminate irritation at
the site of injection, such compositions may contain one
or more nonionic surfactants having a hydrophile-
lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations will
typically range from about 5 to about 15% by weight.
Suitable surfactants include polyethylene sorbitan fatty
acid esters, such as sorbitan monooleate and the high
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molecular weight adducts of ethylene oxide with a
hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral formulations
can be presented in unit-dose or multi-dose sealed
5 containers, such as ampules and vials, and can be stored
in a freeze-dried (lyophilized) condition requiring only
the addition of the sterile liquid excipient, for
example, water, for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions can be
10 prepared from sterile powders, granules, and tablets of
the kind previously described.
Such compositions can be formulated as intraocular
formulations, sustained-release formulations or devices
(see, e.g., U.S. Patent No. 5,378,475). For example,
15 gelantin, chondroitin sulfate, a polyphosphoester, such
as bis-2-hydroxyethyl-terephthalate (BHET), or a
polylactic-glycolic acid (in various proportions) can be
used to formulate sustained-release formulations.
Implants (see, e.g., U.S. Patent Nos. 5,443,505,
20 4,853,224 and 4,997,652), devices (see, e.g., U.S. Patent
Nos. 5,554,187, 4,863,457, 5,098,443 and 5,725,493), such
as an implantable device, e.g., a mechanical reservoir,
an intraocular device or an extraocular device with an
intraocular conduit (e.g., 100 - 1 mm in diameter), or
an implant or a device comprised of a polymeric
composition as described above, can be used.
The present inventive method also can involve the
co-administration of other pharmaceutically active
compounds. By "co-administration" is meant
administration before, concurrently with, e.g., in
combination with the PTK inhibitor in the same
formulation or in separate formulations, or after
administration of a PTK inhibitor as described above.
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For example, corticosteroids, e.g., prednisone,
methylprednisolone, dexamethasone, or triamcinalone
acetinide, or noncorticosteroid anti-inflammatory
compounds, such as ibuprofen or flubiproben, can be co-
administered. Similarly, vitamins and minerals, e.g.,
zinc, anti-oxidants, e.g., carotenoids (such as a
xanthophyll carotenoid like zeaxanthin or lutein), and
micronutrients can be co-administered. In addition,
other types of inhibitors of the protein tyrosine kinase
pathway, which include natural protein tyrosine kinase
inhibitors like quercetin, lavendustin A, erbstatin and
herbimycin A, and synthetic protein tyrosine kinase
inhibitors like tyrphostins (e.g., AG490, AG17, AG213
(RG50864), AG18, AG82, AG494, AG825, AG879, AG1112,
AG1296, AG1478, AG126, RG13022, RG14620 and AG555),
dihydroxy- and dimethoxybenzylidene malononitrile,
analogs of lavendustin A (e.g., AG814 and AG957),
quinazolines (e.g., AG1478), 4,5-dianilinophthalimides,
and thiazolidinediones, can be co-administered with
genistein or an analogue, prodrug or pharmaceutically
acceptable salt thereof (see Levitzki et al., Science
267: 1782-1788 (1995); and Cunningham et al., Anti-Cancer
Drug Design 7: 365-384 (1992)). In this regard,
potentially useful derivatives of genistein include those
set forth in Mazurek et al., U.S. Patent No. 5,637,703.
Neutralizing proteins to growth factors, such as a
monoclonal antibody that is specific for a given growth
factor, e.g., VEGF (for an example, see Aiello et al.,
PNAS USA 92: 10457-10461 (1995)), or phosphotyrosine
(Dhar et al., Mol. Pharmacol. 37: 519-525 (1990)), can be
co-administered. Other various compounds that can be co-
administered include inhibitors of protein kinase C (see,
e.g., U.S. Patent Nos. 5,719,175 and 5,710,145), cytokine
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modulators, an endothelial cell-specific inhibitor of
proliferation, e.g., thrombospondins, an endothelial
cell-specific inhibitory growth factor, e.g., TNFa, an
anti-proliferative peptide, e.g., SPARC and prolferin-
like peptides, a glutamate receptor antagonist,
aminoguanidine, an angiotensin-converting enzyme
inhibitor, e.g., angiotensin II, calcium channel
blockers, y-tectorigenin, ST638, somatostatin analogues,
e.g., SMS 201-995, monosialoganglioside GM1, ticlopidine,
neurotrophic growth factors, methy1-2,5-
dihydroxycinnamate, an angiogenesis inhibitor, e.g.,
recombinant EPO, a sulphonylurea oral hypoglycemic agent,
e.g., gliclazide (non-insulin-dependent diabetes), ST638
(Asahi et al., FEBS Letter 309: 10-14 (1992)),
thalidomide, nicardipine hydrochloride, aspirin,
piceatannol, staurosporine, adriamycin, epiderstatin,
(+)-aeroplysinin-1, phenazocine, halomethyl ketones,
anti-lipidemic agents, e.g., etofibrate, chlorpromazine
and spinghosines, aldose reductase inhibitors, such as
tolrestat, SPR-210, sorbinil or oxygen, and retinoic acid
and analogues thereof (Burke et al., Drugs of the Future
17(2): 119-131 (1992); and Tomlinson et al., Pharmac.
Ther. 54: 151-194 (1992)). Selenoindoles (2-thioindoles)
and related disulfide selenides, such as those described
in Dobrusin et al., U.S. Patent No. 5,464,961, are useful
PTK inhibitors. Those patients that exhibit systemic
fluid retention, such as that due to cardiovascular or
renal disease and severe systemic hypertension, can be
additionally treated with diuresis, dialysis, cardiac
drugs and antihypertensive agents.
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EXAMPLES
The following examples further illustrate the
present invention but, of course, should not be construed
as in any way limiting its scope.
EXAMPLE 1
This example demonstrates that genistein is
effective in inhibiting disruption of the integrity of
basement membrane and subsequent invasion of basement
membrane by retinal endothelial cells.
Retinal capillary endothelial cells were cultured on
Matrigel*(Collaborative Research, Bedford, MA) in the
presence of bFGF (1-10 ng/ml) and in the presence or
absence of genistein (0.2 mg/100 ml). Matrigelkis
derived from basement membrane-producing tumor tissue and
is comprised of type IV collagen and laminin as is the
retinal basement membrane, differing only in the relative
proportions of the components.
Retinal capillary endothelial cells cultured in the
presence of bFGF alone invaded Matrigel*. The presence of
genistein reduced retinal capillary endothelial cell
invasion into Matriger'by .8 mm, i.e., genistein
inhibited invasion by approximately 32s.
This example illustrates the ability of genistein to
inhibit disruption of the integrity of and the subsequent
retinal capillary endothelial cell invasion of a basement
membrane.
EXAMPLE 2
This example demonstrates the ability of genistein
to inhibit photoreceptor loss as associated with age-
related macular degeneration and degeneration of the
retina associated with aging.
* trade mark
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Two strains of mice, senile accelerated mice strain
9 (SAM9) and resistant mice strain 4 (R4) (both strains
were gifts from Kyoto University, Japan and are currently
maintained at Johns Hopkins University), were exposed to
a light source in order to determine the effect of
genistein on light-induced damage of the macula. SAM9
mice are prone to accelerated age-associated changes in
the skin and eye. R4 mice are identical to SAM9, except
that R4 mice are resistant to physical changes associated
with accelerated aging.
A total of about 20-25 mice of each strain were
divided into control and treatment groups. The treatment
groups received genistein (150 mg/kg food) by oral lavage
once daily up to about 4 weeks. Mice were sacrificed at
1, 2 and 3 weeks following light exposure. Eyes were
enucleated and cut serially for histological study.
Specifically, the photoreceptor nuclei were counted to
determine the degree of photoreceptor loss over specific
areas of the posterior and peripheral retina.
At week 1, an average of six layers of photoreceptor
nuclei were observed in both the treated and control
groups for both strains of mice. There was no
significant difference in the amount of light-induced
damage between the control and treatment groups. At week
2, genistein had significantly reduced the amount of
light-induced damage in the treatment groups of both
strains. An average of two to three layers of
photoreceptor nuclei were observed in the SAM9 mice and
the R4 mice treated with genistein. Approximately 0-1
layer of photoreceptor nuclei was observed in control
groups. At week 3, an average of 0-1 layers of
photoreceptor nuclei was observed in both treated and
untreated animals of both strains.
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An additional strain of senile accelerated mice,
strain 8 (SAM8), along with resistant strain 1 (R1), were
exposed to a light source in order to determine the
effect of genistein on light-induced photoreceptor cell
5 loss. All subjects were exposed to constant, white
fluorescent light of 800-lux, after dark adaptation for
24 hours. Each of the SAM8 and R1 strains of mice was
divided into two groups: the treated group, which
received genistein (50 mg/kg body weight/day) orally by
10 the lavage method, and the control group, which received
no treatment. After one week of light exposure, the
thickness and cell count in the outer nuclear layer (ONL)
was greater in the genistein treated groups of both SA48
and R1, than in the control groups. After two weeks of
15 light exposure, the ONL thickness was greater in the
genistein treated R1 than in the corresponding control
group. After three weeks of light exposure, the ONL
thickness was greater in the genistein treated R1 than in
the corresponding control group. There was no difference
20 by treatment in the SAM8 group after two and three weeks
of light exposure.
The example illustrates the ability of genistein to
inhibit photoreceptor loss, such as that associated with
degeneration of the retina, such as that which is due to
25 aging, and age-related macular degeneration.
EXAMPLE 3
This example demonstrates the ability of genistein
to inhibit the progression of age-related macular
degeneration after disruption of Bruch's membrane.
Bruch's membrane in three cynomolgus monkeys was
disrupted using a laser beam and subsequent choroidal
neovascularization was evaluated. Genistein was
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administered to two monkeys via a single intraocular
injection of 100 mg before laser-induced disruption of
Bruch's membrane. Thirteen of seventeen lesions
developed neovascularization. One monkey was
administered genistein orally (300 mg/kg body weight), in
addition to a single intraocular injection (100 mg),
every other day starting two days prior to laser
treatment, for a total of three doses. Of eighteen
lesions resulting from laser treatment, only two
developed choroidal neovascularization.
These results indicate that genistein, when given
prior to disruption of Bruch's membrane, can inhibit or
prevent secondary development of neovascularization,
thereby inhibiting the progression of age-related macular
degeneration.
EXAMPLE 4
This example describes a method of determining the
usefulness of a PTK pathway inhibitor as described herein
to inhibit or ameliorate degeneration of the retina, such
as that which is due to aging.
Two strains of mice, a senile accelerated prone
strain (SAMP) and a resistant strain (SAMR) are obtained
from Takeda Chemical Industries Ltd., Osaka, Japan. As
discussed above, SAMP mice are prone to accelerated age-
associated changes in the eye, while SAMR mice are
resistant to physical changes associated with accelerated
aging.
Subjects of each strain are divided into control and
treatment groups. All mice are bred under specific
pathogen-free conditions with a high efficiency
particulate device with a temperature- and a light-
controlled regimen (68-72 F and a light/dark cycle with
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lights on at 7:00 a.m. to 9:00 p.m.) and free access to
food (PMI Nutrition International, Inc, 1401, St. Louis,
MO) ad libitum and water from an automated filtered
system. The treatment groups receive genistein (50
mg/kg/day) by oral lavage once daily or, alternatively,
genistein is incorporated into food (150 mg/kg food).
For analysis, eyes are enucleated and, optionally,
cut serially for histological study. For light or
electron microscopy, eye cups are fixed for 2 hours in 2%
osmium tetroxide in phosphate buffer, alcohol dehydrated
and embedded in epoxy resin. Two micron thick sections
are stained with toluidine blue and are used for light
microscopy. For electron microscopy, thin sections are
stained with lead citrate and uranyl acetate and examined
in a JOEL JEM-100 CX2 electron microscope (Hitachi, Tokyo,
Japan). Ganglion cell loss and retinal pigment epithelium
changes in control and treated subjects are then examined.
Specifically, if desired, the ganglion cell nuclei are
counted to determine the degree of ganglion cell loss.
Example 5
This example describes a method of determining the
usefulness of a PTK pathway inhibitor as described herein
to inhibit or ameliorate degeneration of the choroid,
such as that which is due to aging.
Two strains of mice, a senile accelerated prone
strain (SAMP) and a resistant strain (SAMR), obtained
from Takeda Chemical Industries Ltd., Osaka, Japan, are
employed to analyze the effect of genistein on
degeneration of the choroid. The animals are kept as
described in Example 4. Changes in the choriocapillaries,
including atrophy of the choriocapillaries, are examined
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via light and electron microscopy, as described in Example
4.
Choroidal vascular casts are also useful in analyzing
the extent of choriocapillary atrophy. Subjects are
anaesthetized with an intramuscular injection of 50 mg/kg
body weight of Ketamine + Xylazine. The left ventricle is
perfused with 50 ml of heparinized lactated Ringer's
solution. The mice are then sacrificed prior to injecting
mercox solution (Ladd Research Industries, Burlington, VT)
through the left ventricle. The eyes are enucleated and
the anterior segment is separated by micro-dissection.
Corrosion casts of the posterior segment are made in 0.1
mol KOH. After complete bleaching of the tissues, the
retinal vessels are separated from the choroidal
vasculature by careful micro-dissection under water.
Choroidal vascular casts are mounted on aluminum tubs and
sputter-coated with gold palladium prior to scanning
electron microscopy (JOEL JSM-840A scanning electron
microscope, Hitachi, Tokyo, Japan). Quantitative analysis
of the choroidal vascular bed is performed by recording
random areas in the posterior pole of each eye at 400 X
magnification. Microplan II image analysis program
(Laboratory Computer Systems Inc., MA) is used for
measuring the area of choroidal vascular bed in each 400x
photograph by tracing the area of choriocapillaries. The
resultant values are tabulated and analyzed by paired
student's t test. P values less than 0.05 are typically
regarded as significant.
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29
Example 6
This example describes a method of determining the
usefulness of a PTK pathway inhibitor as described herein
to inhibit or ameliorate thickening of Bruch's membrane,
which is often associated with aging.
Two strains of mice, a senile accelerated prone
strain (SAMP) and a resistant strain (SAMR), are obtained
from Takeda Chemical Industries Ltd., Osaka, Japan, and
employed to analyze the effect of genistein on thickening
of Bruch's membrane. The animals are kept as described
in Example 4. Changes in Bruch's membrane, including
thickening of Bruch's membrane, are examined via light and
electron microscopy, as described in Example 4.
20
