Note: Descriptions are shown in the official language in which they were submitted.
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AMELIORATION OF CATARACTS, MACULAR DEGENERATION
AND OTHER OPHTIi.~LMIC DTSEASES
CROSS-REFERENCE TO RELATED APPLrCATIONS
Continuation-in-part of United States Application No. 101440,583, filed May
19,
2003, also claiming benefit of United States Provisional Application No.
60/523,803, filed
November 20, 2003, the entire contents of each of which are incorporated by
reference
herein.
FIELD OF THE INVENTION
The present invention is directed to compositions that ameliorate the
development of
cataracts in the eye of a patient and to methods for effecting such
amelioration. In preferred
embodiments of the invention, cataract development or growth is essentially
halted. The
present invention is also directed to the treatment of macular degeneration in
the eye and to
certain other uses. In accordance with preferred embodiments, the compositions
of this
invention are capable of administration to patients without the need for
injections and can be
formulated into eye drops for such administration. Methods for treatment of
cataracts and
macular degeneration are also provided, as are methods for the preparation of
the novel
compounds and compositions useful in the practice of the invention.
BACKGROUND OF THE INVENTION
Various patents and other publications are referenced herein. The contents of
each of
these patents and publications are incorporated by reference herein, in their
entireties. The
entire contents of commonly-owned co-pending U.S. Application No. 10/440,583,
filed May
19, 2003, are incorporated by reference herein.
Aging-related cataract results from gradual opacification of the crystalline
lens of the
eye. This disease is presently treated by surgical removal and replacement of
the affected
lens. It is believed that once begun, cataract development proceeds via one or
more common
pathways that culminate in damage to Iens fibers. This condition progresses
slowly and
occurs predominantly in the elderly. Alternatively, cataract may form because
of surgical,
radiation or drug treatment of a patient, e.g. after surgery of an eye to
repair retinal damage
(vitrectomy) or to reduce elevated intraocular pressure; x-irradiation of a
tumor; or steroid
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drug treatment. A significant retardation of the rate of cataract development
in such patients
may eliminate the need for many surgical cataract extractions. This reduction
would provide
tremendous benefits both to individual patients and to the public health
system.
A less serious but more pervasive condition of the ocular lens is presbyopia.
The lens
is enveloped by a tough collagen capsule that is thought to impart elasticity
to the lens,
enabling it to focus at different distances through ciliary muscle-controlled
changes of
curvature. As the lens ages, it increases in volume and hence progressively
loses its
elasticity, which diminishes an individual's ability to focus on near objects.
This condition is
known as presbyopia, and occurs in a large percentage of the aging population.
In addition to cataract and presbyopia, the eye can experience numerous
diseases and
other deleterious conditions that affect its ability to function normally.
lVlany such conditions
can be found in the interior and most particularly at the rear of the eye,
where lies the optic
nerve and the retina, seven layers of alternating cells and processes that
convert a light signal
into a neural signal. Diseases and degenerative conditions of the optic nerve
and retina are
the leading causes of blindness throughout the world.
A significant degenerative condition of the retina is macular degeneration,
also
referred to as age-related macular degeneration (AlVID). AMD is the most
common cause of
vision loss in the United States in those 50 or older, and its prevalence
increases with age.
AMD is classified as either wet (neovascular) or dry (non-neovascular). The
dry form of the
disease is most common. It occurs when the central retina has become
distorted, pigmented,
or most commonly, thinned. The wet form of the disease is responsible for most
severe loss
of vision. The wet form of macular degeneration is usually associated with
aging, but other
diseases that can cause wet macular degeneration include severe myopia and
some intraocular
infections like histoplasmosis, which may be exacerbated in individuals with
AI~S. A
variety of elements may contribute to macular degeneration, including genetic
makeup, age,
nutrition, smoking and exposure to sunlight.
Retinopathy associated with diabetes is a leading cause of blindness in type 1
diabetes, and is also common in type 2 diabetes. The degree of retinopathy
depends on the
duration of the diabetes, and generally begins to occur ten or more years
after onset of
diabetes. Diabetic retinopathy may be classified as (1) non-proliferative or
background
retinopathy, characterized by increased capillary permeability, edema,
hemorrhage,
microaneurysms, and exudates, or 2) proliferative retinopathy, characterized
by
neovascularization extending from the retina to the vitreous, scarring,
fibrous tissue
formation, and potential for retinal detachment. Diabetic retinopathy is
believed to be
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caused, at least in part, by the development of glycosylated proteins due to
high blood
glucose. Glycosylated proteins generate free radicals, resulting in oxidative
tissue damage
and depletion of cellular reactive oxygen species (ROS) scavengers, such as
glutathione.
Several other less common, but nonetheless debilitating retinopathies include
choroidal neovascular membrane (CNVM), cystoid macular edema (CME, also
referred to as
macular edema or macular swelling), epi-retinal membrane (ERM) (macular
pucker) and
macular hole. In CNVM, abnormal blood vessels stemming from the choroid grow
up
through the retinal layers. The fragile new vessels break easily, causing
blood and fluid to
pool within the layers of the retina. In CME, which can occur as a result of
disease, injury or
surgery, fluid collects within the layers of the macula, causing blurred,
distorted central
vision. ERM (macular pucker) is a cellophane-like membrane that forms over the
macula,
affecting the central vision by causing blur and distortion. As it progresses,
the traction of the
membrane on the macula may cause swelling. ERM is seen most often in people
over 75
years of age. Its etiology is unknown, but may be associated with diabetic
retinopathy,
posterior vitreous detachment, retinal detachment or trauma, among other
conditions.
Another disease of the interior of the eye is uveitis, or inflammation of the
uveal tract.
The uveal tract (uvea) is composed of the iris, ciliary body, and choroid. The
uvea is the
intermediate of the three coats of the eyeball, sandwiched between the sclera
and the retina in
its posterior (choroid) portion. Uveitis may be caused by trauma, infection or
surgery, and
can affect any age group. Uveitis is classified anatomically as anterior,
intermediate,
posterior, or diffuse. Anterior uveitis affects the anterior portion of the
eye, including the iris.
Intermediate uveitis, also called peripheral uveitis, is centered in the area
immediately behind
the iris and lens in the region of the ciliary body. Posterior uveitis may
also constitute a form
of retinitis, or it may affect the choroids or the optic nerve. Diffuse
uveitis involves all parts
of the eye.
Glaucoma is made up of a collection of eye diseases that cause vision loss by
damage
to the optic nerve. Elevated intraocular pressure (IOP) due to inadequate
ocular drainage is a
primary cause of glaucoma. Glaucoma can develop as the eye ages, or it can
occur as the
result of an eye injury, inflammation, tumor or in advanced cases of cataract
or diabetes. It
can also be caused by certain drugs such as steroids. Further, glaucoma can
develop in the
absence of elevated IOP. This farm of glaucoma has been associated with
inheritance (i.e.,
family history of normal-tension glaucoma) Japanese ancestry, as well as
systemic heart
disease, such as irregular heartbeat.
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The eye produces about one teaspoon of aqueous humor daily. Normally, this
fluid
escapes from the eye through a spongy mesh of connective tissue called the
trabecular
meshwork at the same rate at which it is produced. Free radicals and other
reactive oxygen
species (ROS) cause gradual damage to the trabecular meshwork over a period of
time. As a
result, the trabecular meshwork becomes partially blocked, outflow facility
decreases and the
IOP builds up as more aqueous humor is formed. Though the IOP does not rise
high enough
to cause any noticeable symptoms initially, when pressure remains elevated or
continues to
rise, fibers in the optic nerve are compressed and destroyed, leading to a
gradual loss of
vision over a period of years. Izzotti et al. provide convincing evidence
linking oxidative
DNA damage in a small but critical tissue structure in the outflow system to
glaucoma
Izzotti A, Sacca SC, Cartiglia C, De Flora S. Oxidative deoxyribonucleic
damage in the eyes
of glaucoma patients. Am J Med. 2003; 114:638--646). They observed a more than
threefold
increase in the amount of 8-oxo-deoxyguanosine (8-OH-dG) in the trabecular
meshwork
tissue of glaucoma patients. The increased oxidative DNA damage correlated
further with
clinical parameters, such as intraocular pressure indexes and visual field
loss.
The primary features of the optic neuropathy in glaucoma include
characteristic
changes in the optic nerve head, a decrease in number of surviving retinal
ganglion cells, and
loss of vision. It has been proposed that a cascade of events links
degeneration of the optic
nerve head with the slow death of retinal ganglion cells observed in the
disease, and that this
cascade of events can be slowed or prevented through the use of
neuroprotective agents
(Osborne et al., 2003, Eur. J. Ophthalmol. 13 Su 3 : S19-S26), of which
antioxidants and
free radical scavengers are an important class (Hartwick, 2001, Optometry and
Vision
Science 78: 85-94).
The eye's outermost layer, the cornea, controls and focuses the entry of light
into the
eye. The cornea must remain transparent to refract light properly. The cornea
also helps to
shield the rest of the eye from germs, dust, and other harmful matter, and,
significantly, it
serves as a filter to screen out some of the most damaging ultraviolet (UV)
wavelengths in
sunlight. Without this protection, the lens and the retina would be highly
susceptible to injury
from UV radiation.
The cornea and surrounding conjunctiva are also subject to a variety of
deleterious
conditions that can impair vision. These include inflammatory responses, such
as those
resulting from allergic reaction, infection or trauma, and a variety of
dystrophies (conditions
in which one or more parts of the cornea lose their normal clarity due to a
buildup of cloudy
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material), such as Fuchs' dystrophy, keratoconus, lattice dystrophy, and map-
dot-fingerprint
dystrophy, to name a few, as well as other disorders (e.g., dry eye syndrome).
Ocular surface and lacrimal gland inflammation has been identified in dry eye
that
plays a role in the pathogenesis of ocular surface epithelial disease, termed
keratoconjunctivitis sicca. Both oxidative tissue damage and polymorphonuclear
leukocytes
indicating an oxidative potential occur in the tear film of patients suffering
from dry eyes.
These reactions lead to severe damage of the involved tissue. Free radicals
and inflammation
may be involved in the pathogenesis or in the self-propagation of the disease.
(Augustin, A.J.
et al., "Oxidative reactions in the tear fluid of patients suffering from dry
eyes" Graefe's
Arch. Clin.l Exp.l Ophthalmol. 1995, 11:694-698).
Blepharitis is an inflammation of the eyelids. Blepharoconjunctivitis is an
inflammation of the eyelids and the conjunctiva of the eye. Both conditions
are associated
with the condition known as ocular rosacea, though other causes can be
present. Blepharitis
is an abnormal condition wherein the tears produced contain an excess of
lipids (the oily
ingredient in natural tears) and, in some cases, contain an irritating oil as
well. As explained
hereinafter, this oil ingredient serves to prevent evaporation of the aqueous
layer that wets the
corneal epithelium of the eye and helps spread the aqueous layer over the
normally aqueous-
resistant cornea during a blink. If excess oil is present, the lipid layer
will tend to adhere to
the cornea itself. If the eye is unable to clear this oil from the surface of
the cornea, a "dry"
area occurs on the cornea since the aqueous layer is unable to hydrate this
area.
Rosacea is a disease of the skin (acne rosacea) and eyes (ocular rosacea) of
unknown
etiology and a variety of manifestations. The clinical and pathological
features of the eye
disease are nonspecific, and the disease is widely underdiagnosed by
ophthalmologists.
Retinal phototoxicity is induced by exposure of the eye to retinal
illumination from an
operating microscope positioned for temporal approach eye surgery or from
lasers used by
the military. These light sources have the potential for light-induced injury
to the fovea
(M.A. Pavilack and R.D. Brod "Site of Potential Operating Microscope Light-
induced
Phototoxicity on the Human Retina during Temporal Approach Bye Surgery"
Ophthalmol.
2001, 108(2):381-385; H.F. McDonald and M.J. Harris "Operating microscope-
induced
retinal phototoxicity during pats plane vitrectomy" Arch. Ophthalmol. 1988
106:521-523;
Harris M.D. et al. "Laser eye injuries in military occupations" Aviat. Space
Environ. Med.
2003, 74(9):947-952). Damage may also occur upon treatment of ablated surface
of corneas
after excimer laser phototherapy (Seiji Hayashi et al. "Oxygen free radical
damage in the
cornea after excimer laser therapy" Br. J. Ophthalmol. 1997, 81:141-144).
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Certain corneal disorders are not correctable and may be remedied only by
corneal
transplant, while others may be corrected by phototherapeutic keratectomy
(PTK), i.e.,
eximer laser surgery, the process of which is also known to cause an
inflammatory response,
causing corneal hazing or areas of corneal opacification.
The skin around the eyes is also subject to disease and disorders. In
particular,
rosacea of the eyelids and blepharitis are disorders which can be severe.
Ocular rosacea is a
common and potentially blinding eye disorder with an uncertain etiology (Stone
D.U. and J.
Chodosh, 2004 Curr. Opin. Ophthalmol. 15(6):499-502). Blepharitis of the eyes
may be
Staphylococcal blepharitis, seborrheic blepharitis, mixed forms of these, or
the most severe
form, ulcerative blepharitis.
Oxidative stress has been implicated in the development or acceleration of
numerous
ocular diseases or disorders, including AMD and the various retinopathies
described above
(see, e.g., Ambati et al., 2003, Survey of Ophthalmology 48: 257-293; Berra et
al., 2002,
Arch. Gerontol. Geriatrics 34: 371-377), as well as uveitis (e.g., ~amir et
al., 1999, Free Rad.
Biol. Med. 27: 7-15), cataract (e.g., M. Lou, 2003, Prog. Retinal & Eye Res.
22; 657-682),
glaucoma (e.g., Babizhayev & Bunin, 2002, Curr. Op. Ophthalmol. 13: 61-67),
corneal and
conjuctival inflammations, various corneal dystrophies, post-surgical or UV-
associated
corneal damage (e.g., Cejkova et al., 2001, Histol. Histopathol. 16: 523-533;
Kasetsuwan et
al., 1999, Arch. Ophthalmol. 117: 649-652), and presbyopia (Moffat et al.,
1999, Exp. Eye
Res. 69: 663-669). For this reason, agents with anti-oxidative properties have
been
investigated as potential therapeutic agents for the treatment of such
disorders. Many
investigations have focused on the biochemical pathways that generate reducing
power in
cells, for example, glutathione synthesis and cycling. Enzymes, such as
superoxide
dismutase, that reduce activated oxygen species have also been studied to
determine whether
they diminish cellular oxidative stress. Compounds for inhibiting lipid
oxidation in cell
membranes by direct radical scavenging have also been considered to be
promising
therapeutic interventions.
Nitroxides are stable free radicals that are reducible to their corresponding
hydroxylamines. These compounds are of interest because of their radical
scavenging
properties, mimicking the activity of superoxide dismutase and exerting an
anti-inflammatory
effect in various animal models of oxidative damage and inflammation. Due to
their
comparative lack of toxicity, hydroxylamines are preferable to nitroxides as
therapeutic
agents.
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It has been known to provide certain hydroxylamine compositions for the
prevention
or retardation of cataracts. U.S. Patent 6,001,853, in the name of Zigler, et
al., the content of
which is incorporated herein by reference, reflects work performed at the
National Institutes
of Health of the United States. Zigler et al. identified a class of
hydroxylamines which, when
administered to the eye of a test animal, ameliorated cataract genesis or
development. Such
administration was necessarily via injection for physico-chemical reasons.
While Zigler et al.
disclosed that it would be clinically convenient to deliver tempol-H by liquid
eye drops, no
working example was reported, Zigler's hydroxylamines being actually
administered by
subconjunctival injections. Zigler's materials were also accompanied by the co-
administratian of a reducing agent, either via injection, systemically or
otherwise. It is
believed that subsequent work at the iVational Institutes of Health was
directed to the
identification of effective hydroxylamines that could be administered
topically, however
those efforts were not successful,
Accordingly, it has been the object of intense research activity to identify
compounds
and compositions containing them that can ameliorate cataract formation and
development in
the eyes of patients, without the need for unpleasant, inconvenient and
potentially dangerous
intraocular injections. In particular, a long-felt need has existed, which has
not been fulfilled,
far such compounds and compositions which can be administered via topical
application,
especially via eye drops.
SI~JMMAItY OF THE INVENTION
The present invention provides compositions for the treatment of cataracts in
the eyes
of patients either who are developing cataracts or who are known or suspected
of being at risk
far formation of cataracts. Compositions are else provided for the treatment
of macular
degeneration, various retinopathies, glaucoma, uveitis, certain disorders of
the cornea, eye lid
or conjuctiva, and presbyopia in the eyes of patients who exhibit or are at
risk of developing
such diseases or degenerative conditions. In accordance with preferred
embodiments, such
compositions are formulated in topical liquid form, especially as eye drops.
Periodic
application of the compositions of this invention retards or halts development
of cataracts or
macular degeneration in treated eyes. The invention provides compositions,
which need not
be applied via injection or other uncomfortable or inconvenient routes.
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In accordance with preferred embodiments, the present invention provides
compositions comprising an ophthalmologically acceptable carrier or diluent
and a compound
having the formula:
R5
R1
R;
OH
In such compounds, R1 and RZ are, independently, H or C1 to C3 alkyl and R3
and R4
are, independently CI to C3 alkyl. It is also possible, in accordance with
certain
embodiments, that Rl and R~, taken together, or R3 and R4, taken together, or
both form a
cycloalkyl moiety. In the compounds of the invention, RS is H, OH, or Cl to C6
alkyl while
R6 is Cl to C6 alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl. R~ is
C1 to C6 alkyl,
alkenyl, alkynyl, or substituted alkyl or alkenyl or Cl-C6 cycloalkyl or
heterocyclic. It is also
possible for R~ and R~, or R5, R6 and R~, taken together, to form a carbocycle
or heterocycle
having from 3 to 7 atoms in the ring. The term '°ophthalmic," as used
herein, means to have
usefulness in the treatment of the eye and its diseases.
In the compounds used in the compositions of the invention, the substituted
alkyl or
alkenyl species can be substituted with at least one hydroxy, alkoxy,
alkylthio, alkylamino,
dialkylamino, aryloxy, arylamino, benzyloxy, benzylamino or heterocyclic or
YCO-Z where
Y is O, N, or S and Z is alkyl, cycloalkyl or heterocyclic or aryl
substituent. In accordance
with some embodiments, the heterocycle is a 5, 6, or 7 membered ring with at
least one
oxygen, sulfur, or nitrogen atom in the ring. In one preferred composition, R6
and R~, taken
together are cyclopropyl, while in others, R6 and R~, taken together are
tetrahydrofuranyl and
R5, R~ and R~ taleen together are furanyl.
For certain preferred compounds, each of Rl through R4 is Cl to C3 alkyl, most
especially ethyl or methyl, most especially, methyl. For some preferred
embodiments, the
compounds of the invention R~ is Cl to C6 alkyl substituted with at least one
C1 to C6 alkoxy
or benzyloxy group.
_g_
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In other preferred compounds, each of Rr through R4 is methyl, RS is H or
methyl, R6
is methyl substituted with benzyloxy or Cl to C6 alkoxy and R~ is methyl or
where R6 and R~
form a cyclopropyl group. In others, each of Rl through R4 is methyl, RS is
methyl, RG is
ethoxy methyl and R~ is methyl. In still others, each of Rl through R4 is
methyl, R5 is methyl,
R6 is benzyloxy methyl and R~ is methyl, while compounds where each of Rl
through R4 is
methyl, R5 is methyl, R~ is hydroxymethyl and R~ is methyl also find utility.
Also preferred for some embodiments, are compounds wherein each of R1 through
R4
is methyl and R5, R6, and R~ form a furanyl group or where RS is H and R6 and
R~ form a
tetrahydrofuranyl group. A further embodiment provides compounds where Rl
through R4
are all methyl, RS is H, and RG and R~ form a cyelopropyl ring.
It is preferred that the compositions of the invention tie formulated into an
aqueous
medium, which may be delivered in topical liquid form to the eye, via eye
drops for example.
Accordingly, pH and other characteristics of compositions of the invention are
ophthalmologically acceptable for topical application to the eye of a patient.
For some
embodiments, the compound is in the form of a salt, preferably a hydrochloride
or similar
salt.
The compositions of the invention may contain more than one compound of the
invention. Furthermore, the compositions may contain another compound known in
the art
far use in the treatment of a particular indication in combination with the
compounds) of the
invention. In some embodiments, the compounds of the invention are
administered
simultaneously. rn other embodiments, the compounds of the invention are
administered
sequentially. Likewise, other compounds known in the art for use in the
treatment of the
diseases and disorders described herein may be administered with the
compounds) of the
invention either simultaneously or sequentially. The invention also provides
methods of
treatment of diseases and disorders using such combination therapy.
Since the compounds of the invention contain oxidizable hydroxylamine
moieties,
which are most effective in the chemically reduced state, in certain
embodiments the
compositions preferably further comprise an anti-oxidant agent, especially a
sulfhydryl
compound. Exemplary compounds include mercaptopropionyl glycine, N-
acetylcysteine, (3-
mercaptoethylamine, glutathione and similar species, although other anti-
oxidant agents
suitable for ocular administration, e.g. ascorbic acid and its salts or
sulfite or sodium
metabisulfite may also be employed. The amount of hydroxylamines may range
from about
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0.1% weight by volume to about 20.0%; weight by volume and preferred is about
0.25%-
weight by volume to about 10.0% weight by volume.
The invention can also be seen to provide ophthalmologic compositions
comprising
an ophthalmologically acceptable carrier or diluent together with a compound
having an N-
hydroxypiperidine portion bound to a solubility modifying portion. In this
way, the active
moiety, hydroxylamine, can be delivered to the lens of an eye in need of
treatment in a
"stealth" form, that is, in the form of a chemical compound that can have the
hydroxylamine
portion cleaved from the balance of the molecule. The compound is broken down
in the eye
to give rise to the active hydroxylamine species for effective treatment of
cataracts, macular
degeneration or any of the other eye disorders referred to herein. The
compound thus
provided has a solubility in water at 25°C of at least about 0.2% by
weight and a water- n-
octanol partition coefficient at 25°C of at least about 3. In
accordance with preferred
embodiments, the water solubility is greater than about 0.5% by weight,
preferably greater
than about 2.0% and the partition coefficient is greater than about 5,
preferably greater than
about 10.
Accordingly, it is desired that the compounds used be such that, upon
administration
topically to the eye, they penetrate the cornea and are converted to the
desired
hydroxylamine, preferably, an N-hydroxypiperidine. It is preferred that this
conversion
occurs through enzymatic cleavage of the compound. In one preferred
embodiment, the
hydroxylamine portion comprises -1,4-dihydroxy-2,2,6,6-tetramethylpiperidine.
The invention includes methods for ameliorating - either slowing or arresting
entirely
- the development of a cataract in the lens of a patient. Likewise, the
invention includes
methods for ameliorating the development of, or otherwise treating,
presbyopia, macular
degeneration, various retinopathies, glaucoma, uveitis, corneal disorders
(particularly those
associated with trauma, inflammation (both of which can be caused by eximer
laser surgery),
aging, UV exposure and other oxidative-related disorders), disorders of the
conjunctiva
(conjunctivitis), dry eye syndrome, blepharitis and rosacea of the eye. The
invention also
provides methods for protecting retinal pigment epithelium against
photooxidative damage.
In one embodiment, the methods comprise administering to the eye an
ophthalmologic
composition comprising an ophthalmologically acceptable carrier or diluent in
the form of
eye drops containing a compound having one or more of the foregoing compounds
as an
active ingredient therein. It is preferred that the administration takes place
a plurality of time
and, in certain preferred embodiments, chronic, periodic administration is
performed.
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In another aspect of the invention, ophthalmic compositions of the invention
are used
as a prophylactic treatment to prevent or delay development of certain age-
related ocular
conditions, including cataracts, presbyopia, corneal degeneration and
dystrophy, glaucoma,
macular degeneration and photooxidative retinal damage. In preferred
embodiments the
compositions are formulated as eyedrops or eye washes. They are administered
to the eye
prior to, or at the initial stages of, development of age-related conditions
of the eye, or to
prevent progression of later stage disease.
The invention also provides methods for identifying pharmaceuticals that can
be
delivered to the lens of a patient in the form of eye drops. These methods
comprise selecting
a compound having a water solubility at 25°C of at least about 0.1% by
weight and a water/n-
octanol partition coefficient of at least about 5 at 25°C, which
compound is enzymatically
cleavable under conditions obtaining in the eye of a patient to give rise to a
proximate drug
for treatment of a condition of the eye, preferably the lens. Preferably, the
active
pharmaceutical species is a hydroxylamine, especially one having an N-
hydroxypiperidine
nucleus.
BRIEF DESCRxPTION OF TkIE DRA~'6'I~TGS
Figure 1 depicts aqueous humor levels of tempol-H (1,4-dihydroxy- 2,2,6,6-
tetramethylpiperidine) in rabbit eyes treated topically with Compound 1 of the
invention or
with tempol-H.
Figure 2 depicts levels of tempol-H in various tissues of rabbit eyes
following topical
administration of Compound 1, at selected time points post-treatment.
Figure 3 shows that various concentrations of tempol-H protect blue light-
illuminated
A2E-laden RPE cells.
Figure 4 shows the effect of 1 mM tempol-H in protection of blue
light~illuminated
A2E-laden RPE cells.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention provides compounds and compositions that can be
administered
topically to the eyes of patients who exhibit, or who are developing or are at
risk of
developing cataracts, presbyopia, uveitis, macular degeneration or other
retinopathies,
including but not limited to diabetic retinopathy, choroidal neovascular
membrane (CNVM),
cystoid macular edema (CME), epi-retinal membrane (ERM) (macular pucker) and
macular
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hole, as well as disorders of the cornea (and surrounding eye lids and
conjuctiva), including
but not limited to inflammation, trauma, irradiation damage, corneal
neovascularization and
various dystrophies such as Fuch's dystrophy, keratoconus, lattice dystrophy
and map-dot-
fingerprint dystrophy, as well as dry eye syndrome, blepharitis and rosacea of
the eye. While
such compounds may be seen to include as a chemical fragment, hydroxylamine
species
previously known to be effective in retarding cataract development, the
achievement of
compounds that can be topically applied is a very significant advance in the
therapeutic arts.
Indeed, the National Institutes of Health, assignee of the Zigler patent;
tried, but failed to
identify compounds that could be efficacious in therapies for cataracts or
macular
degeneration through topical application. In this context, it is noted that
the Zigler patent
recites administration of certain compositions such as tempol-H via injection
and recognizes
the desirability of topical administration via eye drops, however, this
proposed route of
administration was not found to be available in practice. Accordingly, the
present invention
should be viewed as "pioneering" and as having satisfied a long - felt, but
unserved need in
the art.
The inverxtors have demonstrated that the compounds of the present invention
are
absorbed into and across the cornea and sclera, through the uvea and into the
lens and interior
of the eye. Enzymatic processes within these tissues cleave the N-
hydroxypiperidine portion
of the compound from the acid to which it was esterified. The N-
hydroxypiperidine moiety,
once liberated, then performs the same functions with the same efficacy as
demonstrated by
Zigler. Additionally, a further advantage of the compounds of the invention is
that, even in
their esterified form, they have been found to possess free radical-scavenging
and antioxidant
activities as seen in tempol-H and other non-esterified hydroxylamine
compounds.
The compounds of the invention have not been known heretofore for
administration to
the eye. They have certainly not been known for use in the treatment of
cataract, presbyopia,
corneal disorders, macular degeneration, retinopathies, glaucoma or uveitis.
U.S. Patent
5,981,548, in the name of Paolini, et al., the content of which is
incorporated herein by
reference, depicts certain N-hydroxylpiperidine esters and their use as
antioxidants in a
number of contexts. However, Paolini does not disclose ophthalmologic
formulations or
topical treatment of the eyes of patients. Paolini does disclose, however,
useful syntheses for
certain molecules of this type.
Gupta et al. in U.S. Patent 4,404,302, the content of which, disclose the use
of certain
N-hydroxylamines as light stabilizers in plastics formulations. Mitchell et
al. in U.S. Patent
5,462,946, the content of which is incorporated herein by reference, discloses
certain
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nitroxides deriving from substituted oxazolidines for protection of organisms
from oxidative
stress. U.S. Patent 3,936,456, the content of which is incorporated herein by
reference, in the
name of Ramey et al., provides substituted piperazine dione oxyls and
hydroxides for the
stabilization of polymers. U.S. Patent 4,691,015, to Behrens et al., the
content of which is
incorporated herein by reference, describes hydroxylamines derived from
hindered amines
and the use of certain of them for the stabilization of polyolefins.
In one aspect, the present invention provides compositions comprising a
pharmaceutically carrier or diluent and a compound having the formula:
R5
R1
R-
OH
where R1 and R2 are, independently, H or C1 to C3 alkyl;
R3 and R~. are, independently C1 to C3 alkyl; and
where Rl and RZ, taken together, or R3 and R4, taken together, or both may be
cycloalkyl;
RS is H, QH, or Cl to C6 alkyl;
R6 is C1 to C~ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl;
R~ is Cl to C6 alkyl, alkenyl, alkynyl, substituted alkyl, aIkenyl,
cycloalkyl, or
heterocycle
or where RG and R~, or R5, R6 and R~, taken together, form a carbocycle or
heterocycle
having from 3 to 7 atoms in the ring. These compounds may also be used with
ophthalmically acceptable carriers for use in ophthalmic compositions.
In another aspect, the present invention provides an ophthalmically acceptable
carrier
or diluent and a compound having an N-hydroxy piperidine portion bound to a
solubility
modifying portion, the compound having a solubility in water at 25°C of
at least about 0.25%
by weight and a water - n-octonal partition coefficient at 25°C of at
least about 5. The
composition may have the N-hydroxy piperidine portion cleavable from the
compound under
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conditions found in the eye. It is foreseeable that this portion is cleaved
under conditions in
the lens of the eye. The N-hydroxy piperidine portion may be cleaved
enzymatically. The
compositions may also exist wherein the N-hydroxy piperidine portion is 1-oxyl-
4-hydroxy-
2,2,6,6-tetramethylpiperidyl.
The term Cl to Cn alkyl, alkenyl, or alkynyl, in the sense of this invention,
means a
hydrocarbyl group having from 1 to n carbon atoms in it. The term thus
comprehends
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-
butyl, and the various
isomeric forms of pentyl, hexyl, and the like. Likewise, the term includes
ethenyl, ethynyl,
propenyl, propynyl, and similar branched and unbranched unsaturated
hydrocarbon groups of
up to n carbon atoms. As the context may admit, such groups may be
functionalized such as
with one or more hydroxy, alkoxy, alkylthio, alkyIamino, dialkylamino,
aryloxy, arylamino,
benzyloxy, benzylamino, heterocycle, or YCO-Z, where Y is O, N, or S and Z is
alkyl,
cycloalkyl, heterocycle, or aryl substituent.
The term carbocycle defines cyclic structures or rings, wherein all atoms
forming the
ring are carbon. Exemplary of these are cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl,
cycloheptyl, etc. Cyclopropyl is one preferred species. Heterocycle defines a
cyclic structure
where at least one atom of the ring is not carbon. Examples of this broad
class include furan,
dihydrofuran, tetxahydrofuran, pyran, oxazole, oxazoline, oxazolidine,
imidazole and others,
especially those with an oxygen atom in the ring. Five, six and seven membered
rings with at
least one oxygen or nitrogen atom in the ring are preferred heterocycles.
Furanyl and
tetrahydrofuranyl species are among those preferred.
It is preferred for certain embodiments that each of Rl through R4 be lower
alkyl that
is C1 to C3 alkyl. Preferably, all these groups are methyl for convenience in
synthesis and
due to the known efficacy of moieties having such substitution at these
positions. However,
other substituents may be used as well.
In certain embodiments, compounds are employed where R6 is Cl to CG alkyl
substituted with at least one C1 to CG alkoxy or benzyloxy group. Preferred
among these are
compounds having ethoxy or benzyloxy substituents. Among preferred compounds
are those
where each of Rl through R4 is methyl, RS is H or methyl, RG is methyl
substituted with
benzyloxy or C1 to C~ alkoxy, and R~ is methyl or where R6 and R~ form a
cyclopropyl group
as well as the compound in which each of Rl through R4 is methyl, RS is
methyl, R6 is ethoxy
or benzyloxy methyl, and R~ is methyl. An additional preferred compound is one
in which
each of Rl through R4 is methyl, R5 is methyl, R6 is hydroxymethyl, and R~ is
methyl.
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Other useful compounds are those wherein each of Rl through R4 is methyl, and
R5,
R6, and R~ form a furanyl group, or in which R6 and R~ form a
tetrahydrofuranyl group. The
compound where Rl through R4 is methyl, RS is H and, R~ and R~ form a
cyclopropyl ring is
a further preferred species are as those set forth in the examples below.
The compounds of the invention are formulated into compositions for
application to
the eye of patients in need of therapy. Thus, such compositions are adapted
for
pharmaceutical use as an eye drop or in contact lenses, inserts or the like,
as described in
greater detail below. Accordingly, formulation of compound into sterile water
containing any
desired diluents, salts, pH modifying materials and the like as are known to
persons skilled in
the pharmaceutical formulations art may be performed in order to achieve a
solution
compatible with administration to the eye. It may be that eye drops, inserts,
contact lenses,
gels and other topical liquid forms may require somewhat different
formulations. All such
formulations consistent with direct administration to the eye are comprehended
hereby.
The compositions of the invention may also have antioxidants in ranges that
vary
depending on the kind of antioxidant used. The usage also depends on the
amount of
axltioxidant needed to allow at least 2 years shelf-life for the
pharmaceutical composition.
One or more antioxidants may be included in the formulation. Certain commonly
used
antioxidants have maximum levels allowed by regulatory authorities. As such,
the amount of
ar~tioxidant(s) to be administered should be enough to be effective while not
causing any
untoward effect. Such doses may be adjusted by a physician as needed, within
the maximum
levels set by regulatory authorities, and is well within the purview of the
skilled artisan to
determine the proper and effective dose. Reasonable ranges are about 0.01% to
about 0.15%
weight by volume of EDTA, about 0.01 % to about 2.0% weight volume of sodium
sulfite,
and about 0.01% to about 2.0% weight by volume of sodium metabisulfite. One
skilled in
the art may use a concentration of about 0.1 % weight by volume for each of
the above. N-
Acetylcysteine may be present in a range of about 0.1 % to about 5.0% weight
by volume,
with about 0.1% to about 10% of hydroxylamine concentration being preferred.
Ascorbic
acid or salt may also be present in a range of about 0.1% to about 5.0% weight
by volume
with about 0.1% to about 10% weight by volume of hydroxylamine concentration
preferred.
Other sulfhydryls, if included, may be the same range as for N-acetylcysteine.
Other
exemplary compounds include mercaptopropior~yl glycine, N-acetyl cysteine, (3-
mercaptoethylamine, glutathione and similar species, although other anti-
oxidant agents
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suitable for ocular administration, e.g. ascorbic acid and its salts or
sulfite or sodium
metabisulfite may also be employed.
A buffering agent may be used to maintain the pH of eye drop formulations in
the
range of about 4.0 to about 8.0; this is necessary to prevent corneal
irritation. Because the
compounds of this invention are esters, the pH is preferably maintained at
about 3.5 to about
6.0, preferably about 4.0 to about 5.5, in order to prevent hydrolysis of the
ester bond and to
ensure at least a 2-year shelf life, for the product. This pH also ensures
that most of the
hydroxylamine is in its protonated form for highest aqueous solubility. The
buffer may be
any weak acid and its conjugate base with a pKa of about 4.0 to about 5.5;
e.g. acetic
acid/sodium acetate; citric acid/sodium citrate. The pKa of the hydroxylamines
is about 6Ø
For direct intravitreal or intraocular injection, formulations should be at pH
7.2 to 7.5,
preferably at pH 7.3-7.4.
The compounds of the present invention may also include tonicity agents
suitable for
administration to the eye. Among those suitable is sodium chloride to make
formulations of
the present invention approximately isotonic with 0.9% saline solution.
In certain embodiments, the compounds of the invention are formulated with
viscosity
enhancing agents. Exemplary agents are hydroxyethylcellulose,
hydroxypropylcellulose,
methylcellulose, and polyvinylpyrrolidone. The viscosity agents may exists in
the
compounds up to about 2.0% weight by volume. It may be preferred that the
agents are
present in a range from about 0.2% to about 0.5% weight by volume. A preferred
range for
polyvinylpyrrolidone may be from about 0.2% to about 2.0%weight by volume. One
skilled
in the art may prefer any range established as acceptable by the Food and Drug
Administration.
The compounds of the invention may have co-solvents added if needed. Suitable
cosolvents may include glycerin, polyethylene glycol (PEG), polysorbate,
propylene glycol,
mannitol and polyvinyl alcohol. The presence of the co-solvents may exist in a
range of
about 0.2% to about 4.0% weight by volume. It may be preferred that mannitol
may be
formulated in the compounds of the invention in a range of about 0.5% to about
4.0% weight
by volume. It may also be preferred that polyvinyl alcohol may be formulated
in the
compounds of the invention in a range of about 0.1 % to about 4.0% weight by
volume. One
skilled in the art may prefer ranges established as acceptable by the Food and
Drug
Administration.
Preservatives may be used in the invention within particular ranges. Among
those
preferred are up to 0.013% weight by volume of benzallconium chloride, up to
0.013% weight
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by volume of benzethonium chloride, up to 0.5% weight by volume of
chlorobutanol, up to
0.004% weight by volume or phenylmercuric acetate or nitrate, up to 0.01%
weight by
volume of thimerosal, and from about 0.01% to about 0.2% weight by volume of
methyl or
propylparabens.
Formulations for injection are preferably designed for single-use
administration and
do not contain preservatives. Injectable solutions should have isotonicity
equivalent to 0.9%
sodium chloride solution (osmolality of 290-300 mOsmoles). This may be
attained by
addition of sodium chloride or other co-solvents as listed above, or
excipients such as
buffering agents and antioxidants, as listed above. Injectable formulations
are sterilized and,
in one embodiment, supplied in single-use vials or ampules. In another
embodiment,
injectable products may be supplied as sterile, freeze-dried solids for
reconstitution and
subsequent injection.
The compositions of the invention may contain more than one compound of the
invention. Thus the compositions of the invention contain at least one
compound of the
invention. In some embodiments, the compounds of the invention are
administered
simultaneously. In other embodiments, the compounds of the invention are
administered
sequentially. The methods of the invention include combination therapy.
In some embodiments of the invention, the compounds) of the invention are
administered with another compound known in the art that is useful for
treating a disease or
disorder that is the target of the compounds of the invention. Thus the
composition of the
invention may further contain at least one other compound known in the art for
treating the
disease or disorder to be treated. The other compounds) known in the art may
be
administered simultaneously with the compounds) of the invention, or may be
administered
sequentially. Similarly, the methods of the invention include using such
combination
therapy.
The tissues, including the lens, of the anterior chamber of the eye are bathed
by the
aqueous humor. This fluid is in a highly reducing redox state because it
contains antioxidant
compounds and enzymes. The lens is also a highly reducing environment, which
maintains
the hydroxlamine compounds in the preferred reduced form. Therefore, it may be
advantageous to include a reducing agent in the eye drop formulation, or to
dose separately
with a reducing agent to maintain the hydroxylamine in its reduced form.
Preferred reducing agents may be N-acetylcysteine, ascorbic acid or a salt
form, and
sodium sulfite or metabisulfite, with ascorbic acid and/or N-acetylcysteine or
glutathione
being particularly suitable for injectable solutions. A combination of N-
acetylcysteine and
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sodium ascorbate may be used in various formulations. A metal chelator
antioxidant, such as
EDTA (ethylenediaminetetraacetic acid) or possibly DTPA
(diethylenetriaminepentaacetic
acid) may also be added to keep the hydroxylamine in the reduced form in the
eye drop
formulation.
It is noteworthy that the compounds of the invention exert their anti-
caractogenic and
other therapeutic effects in two ways. First, the ester compounds are
hydrolyzed iv situ to
form hydroxylamine components that exert therapeutic activity. Second, the
esterified
compounds themselves possess anti-caractogenic and antioxidant activity,
thereby supporting
the therapeutic efficacy of pharmaceutical preparations comprising the
compounds.
In connection with the first basis for activity of the compounds of the
invention, i.e.,
cleavage to liberate hydroxyIamine components, numerous esterases are known to
be present
in ocular tissues, especially the cornea. The specific esterase(s) that
cleaves the esters of the
present series need not be identified in order to practice the invention. The
cleavage of the
esters occurs rapidly and essentially completely on administering the
compounds to the eyes
of rabbits. This is shown by the presence of tempol-H in the aqueous humor at
all times (30,
60, 90 and 120 minutes) examined after topical dosing. In contrast, the esters
are stable in
aqueous solutions; e.g. solution of Ester 4 at 40°C, in acetate buffer
at pH 4.6, is stable for 3
months.
The present invention has optimal use in ameliorating the development of a
cataract in
the eye of a patient. Another optimal use includes the treatment of macular
degeneration in
the retina of a patient. Yet other optimal uses include treatment, reduction
or prevention of
the development of diabetic retinopathy and various other retinopathies as
described herein,
as well as the treatment of uveitis and glaucoma. Still another optimal use is
the treatment of
corneal disorders, particularly those associated with oxidative stress, such
as inflammation or
txauma (which can be, but are not necessarily, associated with surgery) and
various
dystrophies. The compounds and compositions of the invention may also be used
to reduce,
prevent or ameliorate photooxidative damage to retinal pigment epithelium, and
for
amelioration of irntation and inflammation during laser surgery of the eye,
including
trabeculectomy treatment for glaucoma and keratectomy for corneal reshaping.
The
compounds and compositions may also be used to treat diseases and disorders of
the
conjunctiva and eyelids. Further, the compounds and compositions of the
invention may be
used to treat alopecia and damage to rectal tissue following radiation
therapy.
Many of the disorders and conditions described herein, particularly cataract,
presbyopia and macular degeneration, are progressive conditions of the aging
process.
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Accordingly, the compositions of the invention may be used to advantage as a
prophylactic
treatment to prevent or delay development of these age-related ocular
conditions. In
preferred embodiments the compositions are formulated as eyedrops or eye
washes. They are
administered to the eye prior to, or at the initial stages of, development of
age-related
conditions of the eye.
Compositions comprising the compounds of the invention may be delivered to the
eye
of a. patient in one ar more of several delivery modes known in the art. In a
preferred
embodiment, the compositions are topically delivered to the eye in eye drops
or washes. In
another embodiment, the compositions are delivered in a topical ophthalmic
ointment, which
is particularly useful for treating conditions of the cornea, conjuctiva or
surrounding skin,
such as dry-eye and bIepharitis. In another emi~odiment, the compositions may
be delivered
to various locations within the eye via periodic subconjunctival or
intraocular injection, or by
infusion in an irrigating solution such as BSS~ or BSS PLUS~ (Alcon USA, Fort
Worth,
T~) or by using pre-formulated solutions of the hydroxylamines in compositions
such as
BSS~ or BSS PLUS. In one embodiment, the use of the compounds of the invention
in
vitrectomy may be effective in reducing or preventing the development of
vitrectomy-
associated cataracts.
Alternatively, the compasitions may be applied in other ophthalmologic dosage
forms
known to those skilled in the art, such as pre-formed or in situ-formed gels
or liposomes, for
example as disclosed in U.S. Patent 5,718,922 to Herrero-Vanrell. A direct
injection of drugs
into the vitreous body used for treating diseases has been used, in which
microspheres or
liposomes were used to release drugs slowly (Moritera, T. et al. "Microspheres
of
biodegradable polymers as a drug-delivery system in the vitreous" Invest.
Ophtlzalfrzol. Vis.
Sci. 1991 32(6):1785-90).
In another embodiment, the composition may be delivered to or through the lens
of an
eye in need of treatment via a contact lens (e.g. Lidofilcon B, Bausch & Lomb
CW79 or
DELTACON (Deltafilcon A) or other object temporarily resident upon the surface
of the eye.
For example, U.S. Pat. No. 6,410,045 describes a contact lens-type drug
delivery device
comprising a polymeric hydrogel contact lens containing drug substance in a
concentration of
between 0.05% and 0.25% by weight absorbed in said contact lens which is
capable of being
delivered into the ocular fluid.
In other embodiments, supports such as a collagen corneal shield (e.g. BIO-COR
dissolvable corneal shields, Summit Technology, Watertown, Mass.) can be
employed. The
compositions can also be administered by infusion into the eyeball, either
through a cannula
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from an osmotic pump (ALZET~, Alza Corp., Palo Alto, Calif.) or by
implantation of timed-
release capsules (OCCUSENT~) or biodegradable disks (OCULEX~, OCUSERT~) which
contain the compositions. These routes of administration have the advantage of
providing a
continuous supply of the composition to the eye.
Many types of drug delivery systems are known in the art and can be used for
delivery
of compositions of the present invention. Non-limiting examples have been set
forth above,
and more are listed below.
A preferred method to treat dry eye symptoms utilizes aqueous based solutions
or
gels, which may be formulated to contain one or more compounds of the present
invention.
The "active" ingredients in these artificial tear formulations are common
water soluble or
dispersable polymers such as hydroxyethylcellulose,
hydroxypropylmethylcellulose,
methylcellulose, carboxymethylcellulose, polyvinyl alcohol, polyvinyl
pyrrolidone,
polyethylene glycol, carborners and poloxamers.
U.S. Patent 6,429,194 describes aqueous ophthalmic preparations for
instillation into
the eye, or in which to pre soak or store an object to be inserted into the
eye, such as a contact
lens, an ointment, or a solid device to be inserted into the conjunctiva) sac.
The ophthalmic
preparation includes a mucin component, similar to that found at the normal
human ocular
surface. U.S. patent No. 6,281,192 also describes the ophthalmic applications
of mucin.
An ophthalmic solution must provide an effective and long lasting treatment
for
symptoms of dry eye. One approach to achieving these aims is to provide a
solution with
tailored rheological properties, that is, a high viscosity solution that
yields or flows under
stress. Examples of this approach are disclosed in U.S. Pat. Nos. 5,075,104
and 5,209,927,
where the rheological properties of the ophthalmic solutions are attained
through the use of
carbomer polymers. These carbomer polymers have been found to be bio-adhesive
as
described in U.S. Pat. Nos. 5,225,196; 5,188,828; 4,983,392 and 4,615,697. It
is believed
that the bio-adhesive properties of the carbomer contributes to longer
retention times in the
eye. In fact, U.S. Pat. Nos. 5,075,104 and 5,209,927, teach that the carbomer
polymers appear
to function by maintaining or restoring the normal hydration equilibrium of
the epithelial
cells, protecting the cornea in a manner similar to that believed to be
provided by the mucin
component of normal tears.
U.S. patent No. 4,883,658 describes a synergistic combination of an aqueous
solution
of a partially hydrolyzed polyvinyl acetate) and a fully hydrolyzed polyvinyl
acetate), i.e.
polyvinyl alcohol), exhibiting a low surface tension at the water-air
interface, while forming
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a completely wettable absorbed Layer over hydrophobic solids. The combination
suitable for
use as an aqueous vehicle for topically used ophthalmic drugs or nutrients.
Emulsioxi based formulations for treating dry eye symptoms have emerged
recently.
One approach, as disclosed in U,S. Patent Nos. 5,578,586; 5,371,108;
5,294,607; 5,278,151;
4,914-,088, utilize methods and compositions for reducing evaporation of the
aqueous layer
from the surface of the eye. The method comprises applying an admixture of a
charged
phospholipid and a non-polar oil over the eye, preferably in the form of a
finely divided oil-
in-water emulsion. Another approach is described in U.S. Pat. Nos. 4,818,537
and 4,804,539,
where liposome compositions in the form of emulsions are reported to enhance
retention on
ocular surfaces and thereby alleviate the symptoms of dry eye.
Several other types of delivery systems are available that are particularly
suitable for
delivering pharmaceutical compositions to the interior or posterior of the
eye. For instance,
U,S. Patent 6,154,671 to Parel et al. discloses a device for transferring a
medicament into the
eyeball by iontophoresis. The device utilizes a reservoir for holding the
active agent, which
contains at least one active surface electrode facing the eye tissue lying at
the periphery of the
cornea. The reservoir also has a return electrode in contact with the
patient's partly closed
eyelids. U.S. Patent 5,869,079 to along et al. discloses combinations of
hydrophilic and
hydrophobic entities in a biodegradable sustained release ocular implant. In
addition, U.S.
Patent 6,375,972 to Guo et al., U.S. Patent 5,902,598 to Chen et al., U.S.
Patent 6,331,313 to
along et al., U.S, Patent 5,707,643 to Ogura et al., U.S. Patent 5,466,233 to
Weiner et al. and
U,S. Patent 6,25Z,09Q to Avery et aI. each describes intraocuIar implant
devices and systems
that may be used to deliver pharmaceutical compositions comprising compounds
of the
present invention.
U.S. Pat. No. 4,014,335 describes an ocular drug delivery device placed in the
cul-de-
sac between the sclera and lower eyelid for administering the drug and acting
as a reservoir.
The device comprises a three-layered laminate of polymeric materials holding
the drug in a
central reservoir region of the laminate. The drug diffuses from the reservoir
through at least
one of the polymeric layers of the laminate.
Solid devices, in the form of ocular inserts, have been utilized for longer
term
symptomatic relief of dry eye. These devices are placed in the eye and slowly
dissolve or
erode to provide a thickened tear film. Examples of this technology are given
in U.S. Pat.
Nos. 5,518,732; 4,343,787, and 4,287,175.
The compounds of this invention can have uses in fields broader than
ophthalmology.
These areas may include, for example, protection of hair follicles and rectum
from radiation
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damage during radiation therapy for cancer. Other forms of administration of
the
compositions of the present invention, wherein the delivery to the eye is not
called for, may
include oral tablets, liquids and sprays; intravenous, subcutaneous and
intraperitoneal
injections; application to the skin as a patch or ointment; enemas,
suppositories, or aerosols.
For effective treatment of cataract, presbyopia, macular degeneration,
glaucoma or
any of the other retinopathies, corneal disorders or eye conditions described
herein, one
skilled in the art may recommend a dosage schedule and dosage amount adequate
for the
subject being treated. The dosing may occur less frequently if the
compositions are
formulated in sustained delivery vehicles, or are delivered Via implant. For
topical delivery,
it may be preferred that dosing occur one to four times daily for as long as
needed. The
dosage amount may be one or two drops per dose. The dosage schedule may also
vary
depending on the active drug concentration, which may depend on the
hydroxylamine used
and on the needs of the patient. It may be preferred that the active amount be
from about
0.1% to about 10,0% weight by volume in the formulation. In some embodiments,
it is
preferable that the active drug concentration be 0.25% to about 10.0% weight
by volume.
The concentration of the hydroxylamine component will preferably be in the
range of about
0.1 p.M to about 10 mM in the tissues and fluids. In some embodiments, the
range is from 1
~m to 5 mM, in other embodiments the range is about 10 p.M to 2.5 mM. In other
embodiments, the range is about 50 ~M to 1 mM. Most preferably the range of
hydroxylamine concentration will be from 1 to 100 ~,M. The concentration of
the reducing
agent will be from 1 ~M to 5 mM in the tissues or fluids, preferably in the
range of 10 ~,M to
2 rnM. The concentrations of the components of the composition are adjusted
appropriately
to the route of administration, by typical pharmacokinetic and dilution
calculations, to
achieve such local concentrations. Alternatively, penetration of cornea and
absorption into
other tissues in the interior of the eye is demonstrated using radiolabeled
hydroxylamine.
An ophthalmologist or one similarly skilled in the art will have a variety of
means to
monitor the effectiveness of the dosage scheme and adjust dosages accordingly.
For
example, effectiveness in treating cataract may be determined by the
ophthalmologist by
observing the degree of opacity of the lens at intervals by slit-lamp
examination, or other
means known in the art. Effectiveness in the treatment of macular degeneration
or other
retinopathies may be determined by improvement of visual acuity and evaluation
for
abnormalities and grading of stereoscopic color fundus photographs. (Age-
Related Eye
Disease Study Research Group, NEI, NIH, AREDS Report No. 8, 2001, Arch.
Ophthalmol.
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119: 1427-1436). Effectiveness in the treatment of uveitis may be determined
by
improvement in visual acuity and vitreous haze and control of inflammation
(Foster et al.,
2003, Arch. Ophthalmol. 121: 437-40). Following such evaluation, the
ophthalmologist may
adjust the freguency and/or concentration of the dose, if needed.
Another aspect of the invention features a method of identifying a
pharmaceutical for
delivery to the eye of a patient in the form of eye drops, which comprises
selecting a
compound having a water solubility at 25°C of at least about 0.25% by
weight and a water -
n-octonal partition coefficient of at least about 5 at 25°C, which
compound is enzymatically
cleavable under conditions obtained in the lens of the eye of a patient to
give rise to a
hydroxylamine, preferably an N-.hydroxy piperidine. Preferably, the selected
compound is an
ester of the hydroxylamine.
It may be preferred that at least 0.1 % solubility is needed for an eye drop,
even for a
suspension formulation. Completely water-insoluble compounds may not be
effective.
Esters that are soluble in water (>0.1% weight by volume) are preferred.
Esters with less
than 0.1 % solubility may be used in the form of suspensions or ointments or
other
formulations. Solubility is determined by mixing 100mg of test compound with 1
ml of
water, at room temperature and adding additional 1 ml quantities of water,
with mixing, until
ester dissolves completely.
Corneal penetration is shown by measuring a substantial concentration (e.g.
>5p,M) of
the effective hydxoxylamine and/or ester in the aqueous humor after
administering a solution
of the compound ifz vivo to the eyes of rabbits. This is determined by
electron spin
resonance (ESR), high performance liquid chromatography (HPLC) or gas
chromatography
(GC) assay of the rabbit aqueous humor. Irc vitro corneal penetration methods
may also be
used prior to the ih vivo testing method particularly for screening compounds.
Penetration of
compounds to the interior or posterior of the eye is likewise shown by
measuring the
concentration of the compound in the vitreous humor, uvea or xetina after
administering a
solution of the compound to the eyes of rabbits.
Esters are selected for these tests based on their calculated or measured
octanol/water
partition coefficient (P). Hydrophilic compounds such as tempol-H cannot
penetrate the
lipophilic epithelial layer of the cornea. Partition coefficients of tempol-H
and esters that
penetrate are as follows:
P (Calculated)*
Tempol-H 0.8 (measured, 0.5)
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Ester 4 16.4.
Ester 8 8.2
Ester 14 6.3
* Clog P version 4.0, Biobyte Corporation
Enzymatic conversion is essentially complete at greater than 90% hydrolysis of
the
ester in vivo to the alcohol and acid after administering the compound to the
eye of rabbits.
The conversion may be determined by HPLC ox GC assay of a selected eye tissue
(e.g.,
aqueous humor). Alternatively, the enzymatic conversion may be determined by
incubating
the compound in plasma or eye tissue homogenate and assaying samples
periodically by
HPLC or GC to monitor the rate of breakdown. Esters with a half life of less
than about 1 or
2 hours are preferred candidates. This method may be the preferred screening
procedure
before in vivo testing.
Esters should have less than about 10% hydrolysis at 40°C, after 3
months, in aqueous
solution at pH 4.0-5Ø This extrapolates to a shelf life of the ester in
solution of at least 18
months at room temperature, which may be preferred for an eye drop product.
EXAMPLES
The presexit invention is illustrated in certain embodiments by reference to
the
following examples. The examples are for purposes of illustration only and are
not intended
to be limiting in any way.
Example 1: Detenninatiozz of Ester Compound Stability In Aqueous Solution.
lVXethod: A 0.1-0.5% solution of the ester compound was prepared in buffer (pH
4.5-5.0)
containing DTPA or EDTA. The solution was filled into amber glass vials, which
were
sealed and placed in a controlled temperature container maintained at
40°C. Sample vials
were removed periodically and stored at 0-5°C until analyzed by HPLC,
GC, or GC/MS
analytical methods, and found to be stable after 3 months under these
conditions.
To be useful as an anti-cataract drug the agent must penetrate into the lens.
This may
be included in the method for selecting an anti-cataract compound. A
description of method
for tempol-H follows:
Example 2: Drug Perzetration of Organ CultuYed Rat Lenses
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In contrast to drugs tested previously as anti-cataract agents, tempol-H and
tempol
have a remarkable ability to penetrate lens tissue from the surrounding fluid.
The
experiments described in this section determined the time course, active
compound
concentrations and compound distribution in the lens, after incubation with
rat lenses under
the organ culture conditions.
Method: Rat lenses were cultured as follows: Rat lenses were obtained from
Sprague-
Dawley rats. The lenses were incubated in 24-well cluster dishes in modified
TC-199
medium and were placed in a 37°C incubator with a 95% air/5% C02
atmosphere. The
lenses were incubated in 2 ml of culture medium, which was adjusted to 300
milliosmoles
(mOsm). Lenses were incubated, for 1 to 24 hours, in the culture medium with
4.0 mM
tempol-H, or with 4~.0 mM of the oxidized form, tempol. At the appropriate
time, the lenses
were removed from the medium, blotted dry, homogenized and were analyzed for
active
compound by electron spin resonance method ($SR). In one experiment, lenses
were
incubated for 4 hours and dissected into epithelial, cortical and nuclear
sections before
analysis.
Results: Concentrations (mM, in lens water) of tempol-H reached 0.4 mM, 0.8 mM
and 1.0 mM, respectively, after 1, 2 and 4 hours incubation of active
compound. Levels of
tempol-H found, after incubation of lenses with the oxidized form tempol,
reach 0.6 mM, 1.5
mM and 2.8 mlV.l respectively. Zn the latter case, only a trace (S°Io
or less) of the oxidized
form tempol, was found in the lens; it was almost completely converted to the
reduced form
tempol-H.
Distribution of tempol-H between the lens epithelium, cortex and nucleus was
fairly
even, after a 4-hour incubation period with tempol-H. Levels of tempol-H
reached 1.5 mM,
0.8 mM and 1.0 mM, respectively, in the epithelium, cortex and nucleus. Levels
of tempol-
H/tempol in lenses incubated with the oxidized form, tempol, were 1.2 mM, 2.9
mM and 2.0
mM, respectively. In the latter case, all compounds in the nucleus were in the
reduced form
with only about S°lo in the epithelium in the oxidized form.
Conclusion: Both the reduced and oxidized forms of the active agent readily
penetrated into the cultured rat lens from the bath medium and distributed to
the epithelium,
cortex and nucleus. Incubation of lenses with the oxidized form tempol,
results in high
concentrations of reduced compound tempoI-H throughout the lens.
Example 3: 1-oxyl-4-(3'-ethoxy-2',2'-dimethyl)propanecarbonyloxy-2,2,6,6-
tetramethylpiperidine
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O
I
O~
1,1'-carbonyldiimidazole was added in small portion (1.27 g, 7.84 mmol) to a
stirred
solution of 3-ethoxy-2,2-dimethylpropionic acid (750 mg, 7.13 mmol; prepared
according to
the procedure described in J. Org. Chem., 38, 2349, 1975, the content of which
was
incorporated herein by reference) in dry DMF (10 mL). A vigorous gas evolution
was
observed. This solution was heated at 100°C for 1 h. To this mixture
was then added tempol
(900 mg, 5.23 mmol) and 1,8-diazabicyclo [5,4,0]undec-7-ene (DBU) (800 mg,
5.26 mmol)
and continue heating for 12 h. The reaction mixture was concentrated under
reduced
pressure. The residue was dissolved in ethyl acetate (I00 mL) and was washed
successively
with 1N HCl, saturated NaHC03 and brine, was dried over anhydrous sodium
sulfate and was
concentrated in vacuo to give red colored solid (1.48g). This was purified by
column
chromatography on silica gel using cyclohexane: ethyl acetate (8:1) as eluent
to give a red
colored crystalline solid (1.22 g, 70.0 %).
IR (KBr, cm-1): 1360 (N-O~), 1725 (ester)
Example 4: 1-hydroxy-4-(3'-ethoxy-2',2'-dimethyl)propanecarbonyloxy-2,2,6,6-
tetramethylpiperidine hydrochloride
O
O O~
-N \
OH HCI
The nitroxide of Example 2 (1.02 mg, 3.34 mmol) was added to a solution of
saturated hydrogen chloride in ethanol (2D mL). The red color disappears
quickly and the
resulting yellow colored solution was boiled to give a clear colorless
solution. The solution
was concentrated iyz vacuo, was dissolved in 100 mL ethyl acetate and was
washed with
saturated NaHC03 to obtain the hydroxylamine free-base. The ethyl acetate
layer was
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separated and concentrated to give a red colored oiI which was mostly
nitroxide, by TLC.
This oil was purified by column chromatography on silica gel using
cyclohexane:ethyl
acetate (4:1) as eluent to give a red colored crystalline solid (700 mg). The
solid was
dissolved in a solution of saturated hydrogen chloride in ethanol (20 mL), was
concentrated
ifz vacuo, and was recrystallized from ethyl acetate:diisopropylether (2:1, 50
mL) to give
white crystalline solid (320 mg). m.p.140-142°C (dec.).
1H-NMR (270 MHz, D20) ppm: 1.48 (6H, s); 1.57 (3H, t); 1.63 (12H, s); 1.82
(2H, s);
2.02 (2H, t); 2.40 (2H, d), 3.88 (2H, q); 5.44 (1H, m)
IR (KBr, cm-1): 3487 (OH), 1726 (ester)
Mass Spec. (EI, m/z) 301 (M+)
Example Sa: 1-oxyl-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine
O
O
~N \
I
O~
A suspension of sodium hydride (60% ire oil, 1.0 g, 25mmol) in dry THF (50 mL)
was
stirred at room temperature for 5 min and to this mixture was added tempol
(4.0 g, 23 mmol).
The mixture was stirred for 1 h, cyclopropanecarbonyl chloride (2.4g, 23 mmol)
was added
dxopwise over 5 min and then it was refluxed for 1 h. The reaction mixture was
concentrated
under reduced pressure. The residue was taken up in pentane (100 mL) and the
supernatant
was separated and concentrated under reduced pressure to give red solid. This
solid was
purified by column chromatography on silica gel using cyclohexane:ethyl
acetate (3:1) as
eluent to give a red colored crystalline solid (1.4 g, 5.8 mmol, 25.3 %).
IR (I~Br, cm-1): 1361 (N-O~), 1720 (ester)
Example 5b: Alternative method - 1-oxyl-4-cyclopropanecarbonyloxy-2,2,6,6-
tetramethylpiperidine
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O
O
~N \
O~
1,1'-Carbonyldiimidazole (1.78 g, 11 mmol) was added in small portions to a
stirred
solution of cyclopropanecarboxylic acid (860 mg, 10 mmol) in dry DMF (10 mL).
A
vigorous gas evolution was observed. This solution was heated at 40°C
for 1 h. To this
mixture was then added tempol (1.72 g, 10 mmol) and 1,8-
diazabicyclo[5,4,0]under-7-
ene(DBU) (1.52 g, 10 mmol) and it was heated at 40°C for anotherl2 h.
The reaction
mixture was concentrated under reduced pressuxe. The residue was dissolved in
ethyl acetate
(100 mL) and was washed successively with 1N HCI, saturated NaHCO3 and brine.
The
ethyl acetate Layer was separated, dried over anhydrous sodium sulfate and
concentrated ifa
uacuo to give red colored solid. This solid was purified by column
chromatography on silica
gel using cyclohexane:ethyl acetate (8:1) as eluent to give a red colored
crystalline solid
(720mg, 30.0 %).
IR (KBr, c m 1): 1360 ( N-O~), 1720 (ester)
Example 5c: Alternative method - 1-oxyl-4-cyclopropanecarbonyloxy-
2,2,6,6-tetramethylpiperidine [DCC/DMAP esterification method
O
O
~N \
O.
To a stirred solution of tempol (1.72 g, 0.01 mmole), cyclopropanecarboxylic
acid
(0.946g, .011 mmole), and DMAP (0.12, .001 mmole) in dichloromethane (25 ml)
was added
DCC (2.27 g, 0.11 mmole) and the mixture was stirred overnight at room
temperature. The
mixture was filtered over celite and the solution was evaporated under reduced
pressure. The
product was isolated by silica gel column chromatography using first hexane
and then 10%
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ethyl acetate in hexane. Yield: 2.26 g (94.2). IR and NMR were consistent with
the assigned
structure.
Example 6: 1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine
hydrochloride (Compound 1)
O
O
~N \
OH HCI
The nitroxide of Example 5a (2.2g, 9.15 mmol) was added to a solution of
saturated
hydrogen chloride in ethanol (20 mL). The red color disappeared quickly and
the resulting
yellow colored solution was boiled to give clear colorless solution. The
solution was
concentrated in vacuo, dissolved in 100 mL ethyl acetate and was washed with
saturated
NaHC03 to obtain the hydroxylamine free-base. The ethyl acetate layer was
separated,
acidified with ethereal HCI, and concentrated to give white solid, which was
recrystallized
from ethanol (10 mL) as a white crystalline solid 1.15g (4.13 mmol, 45.1 %).
rn.p. 224-228°C
(dec.).
1H-NMR (270 MHz, D20) pprn: 0.97 (4~-I,d); 1.43 (1H, m); 1.44 (6H, s), 1.46
(6H,s);
1.90 (2H,t); 2.28 (2H,t); 5.2(lH,m)
IR (I~Br, cm-1): 3478 (OH), 1720 (ester)
Mass Spec. (EI, m/z) 240 (M+)
Example 7: 1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine
hydrochloride (Alternate method)
OH HCI
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The nitroxide of Example 5a (700 mg, 2.91 mmol) was added to a solution of
saturated hydrogen chloride in ethanol (20 mL). The red color disappeared
quickly and the
resulting yellow colored solution was boiled to give a clear colorless
solution. The solution
was concentrated ifi vacuo, dissolved in 100 mL ethyl acetate and concentrated
to half
volume to give a white crystalline solid, 627 mg (2.25 mmol, 77.5 %.). m.p.224-
227°C (dec.).
1H-NMR (270 MHz, D20) ppm: 0.97 (4H,d); 1.43 (1H, m); 1.44 (6H, s), 1.46
(6H,s);
1.90 (2H,t); 2.28 (2H,t); 5.2(lH,m)
IR (KBr, cm-1): 3476 (OH), 1720 (ester)
Mass Spec. (EI, m/z) 240 (M+)
Example S: 1-oxyl-4-(3'-benzyIoxy-2',2'-dimethyl)propanecarbonyloxy-2,2,6,6-
tetramethylpiperidine
O
0 0
N'
i
O~
To a stirred solution of 3-benzyloxy-2,2-.dimethylpropionic acid (1.04 g, 5
mmol),
(prepared by a method similar to that described in J. Org. Chem., 38,
2349,1975), in dry
DMF (5 mL), was added 1,1'-carbonyldiimidazole in small porkions. A vigorous
gas
evolution was observed. This solution was heated at 50°C for 30 min. To
this mixture was
then added tempol (900 mg, 5.23 mmol) and 1,8-diazabicyclo[5,4,0]undec-7-
ene(DBU) (800
mg, 5.26 mmol). The mixture was heated at 50°C for 3 days (monitored by
TLC) and then it
was concentrated under reduced pressure. The residue was dissolved in ethyl
acetate (100
mL), washed successively with 1N HCI, saturated NaHC03 and brine, and dried
over
anhydrous sodium sulfate. The dried solution was concentrated in vacuo to give
red colored
solid (1.48g). This solid was purified by columxi chromatography on silica gel
using
cyclohexane:ethyl acetate (3:1) as eluent to give a red colored crystalline
solid (1.02 g, 2.8
mmol, 56.2alo).
1R (KBr, cm-1): 1359 (N-O~), 1732 (ester)
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Example 9: 1-hydroxy-4-(3'-benzyloxy 2',2'-dimethyl)propanecarbonyloxy-
2,2,6,6-tetramethylpiperidine hydrochloride
O
O
\ /
HCI
The nitroxide of Example 8 (1.02 mg, 3.34 xnmol) was added to a solution of
saturated hydrogen chloride in ethanol (20 mL). The red color disappears
quickly and the
resulting yellow colored solution was boiled to give clear colorless solution.
The solution
was concentrated in vacuo and the residue dissolved in ethyl acetate (20 mL).
Hexane (20
mL) was added and product began to oil out; the mixture was then allowed to
stand for 12 h.
An oily residue was obtained by decantation of the solvent and it was treated
with was
isopropyl ether and warmed. Upon cooling the mixture, a waxy solid was
obtained and
recrystallized from ethyl acetate to give white crystalline solid (0.6g, 1.5
mmol, 45%).
1H-NMR (270 MHz, I?20) ppm: 1.26 (6H, s), 1.51 (6H, s); 1.65 (6H, s); 2.01
(2H, t);
2.44 (2H, d), 5.40 (1H, m); 3.46 (2H, s), 4.55 (2I3, S), 7.31 (5H, s)
IR (KBr, c;m-1): 3480 (OH), 1712 (ester), 710 (aromatic)
Mass Spec. (EI, m/z) 262 (M+)
Example 10: 1-hydroxy-4-(3'-hydroxy-2',2'-dimethyl)propanecarbonyloxy-2,2,6,6-
tetramethylpiperidine hydrochloride
O
O ~~ -OH
-N \
I
OH HCI
Pd/C (5 %, 100 mg) was added to a solution of 1-oxyl-4-(3'-benzyloxy-2',2'-
dimethyl)propanecarbonyloxy-2,2,6,6-tetramethylpiperidine (l.Og, 3.83 mmol) in
ethanol,
and the mixture was hydrogenated in a Paar hydrogenation apparatus at 45 psi
for 12 h. The
reaction mixture was filtered through celite and concentrated in vacuo to give
a clear
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colorless oil, which was purified by column chromatography on silica gel using
cyclohexane:ethyl acetate (3:1) as eluent to give a colorless oil. The oil was
dissolved in a
solution of saturated hydrogen chloride in ethanol (20 mL) and concentrated in
vacuo.
Product crystallized upon standing, and was recrystallized from ethanol (123
rng, 0.4 mmol,
10.4 %). m.p.210-215°C (dec.).
1H-NMR of the free base (270 MHz, CDC13) ppm: 1.14 {6H, s), 1.44 (6H, s); 1.57
(6H, s); 1.70 {2H, m); 2.8 (1H, s, br), 3.65 (2H, s) 5.16 (1H, m)
IR (KBr, cm-1): 3480 (OH), 1712 (ester), 710 (aromatic)
Mass Spec. (EI, m/z) 262 (M+)
Example 1I: I-oxyl-4-(I-methyl-cyclopropane)carbonyloxy-2,2,6,6-
tetramethylpiperidine
O
O
~N
O~
A suspension of sodium hydride (60% in oil 2.2g), in dry THF (80 mL) was
stirred at
room temperature for 5 min and then tempol (3.4g, 17.44 mmol) was added. The
mixture
was stirred for 30 min, 1-methyl-cyclopropanecarbonyl chloride (2.2g, 18.71
mmol) was
added drop wise over 5 min and then it was refluxed for 12 h. The reaction
mixture was
concentrated under reduced pressure and the residue crystallized immediately.
The product
was purified by column chromatography on silica geI using cyclohexane:ethyl
acetate (3:I) as
eluent to give a red colored crystalline solid (2.0 g, 7.86 mmol, 45.1 %).
IR (I~Br, cm-1): 1314 (N-O~), 1722 (ester)
Example 12: 1-hydroxy,4-(1-methyl-cyclopropane)carbonyloxy-2,2,6,6-
tetramethylpiperidine hydrochloride
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O
O
~N \
OH HCI
The nitroxide of Example 11 (700 mg, 2.91 mmol) was added to a solution of
saturated hydrogen chloride in ethanol (10 rnL). The red color disappears
quickly and the
resulting yellow colored solution was boiled to give a clear colorless
solution. The solution
was concentrated ifa vacuo to give white crystalline solid, which was
filtered, washed with
ethyl acetate and dried in vacuo (0.700 mg, 2.4mmol, 82.7%) m.p. 215°C -
220°C (dec.).
1H-NMR (270 MHz, D20) ppm: 0.80 (2H, d); 1.19 (2H, m); 1.21 (2H,s); 1.44 (15H,
s); 2.03 (4H, m); 5.10 (1H, m)
lViass Spec. (EI, m/z) 254 (M+)
Examgle l3: 1-oxyl-4-(2-furan)carbonyloxy-2,2,6,6-tetramethylpiperidine
O
O /
O
N'
I
O~
A stirred mixture of sodium methoxide (25% sodium methoxide in methanol, 200
mg)
in benzene (I00 mL) was heated to reflux and the benzene was gradually
distilled off to half
volume to obtain a fine suspension of solid sodium methoxide. To this mixture
was added
tempol (1.76 g, 10 mmol), methyl 2-furoate (1.26 g, 10 mmole) and benzene (50
mL).
Distillation of benzene was continued for 8 h to remove formed methanol. The
volume of
benzene in the flask was maintained by adding more benzene. The benzene layer
was
washed with 1 N HCI, then with water, dried over anhydrous sodium sulfate and
evaporated
to dryness to give a red solid (1.72g), which was recrystallized from hexane
to give 1.45g of
product. It was further purified by column chromatography on silica gel using
cyclohexane:ethyl acetate (3:1) as eluent to give a red colored crystalline
solid (1.02 g, 3.82
mmol, 32.8 %).
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IR (.KBr, cm-I): 1364 (i~-O~), 2716 (ester), 706 (aromatic)
Example 14: 1-hydroxy-4-(2'-furan)carbonyloxy-2,2,6,6-tetramethylpiperidine
hydrochloride
O
O
O
N
I
OH HCI
The nitroxide of Example 13 (300 mg, l.I3mmo1) was added to a solution of
saturated hydrogen chloride in ethanol (10 mL). The red color disappeared
quickly and the
resulting yellow colored solution was boiled to give clear colorless solution.
The solution
was kept at room temperature for I h and a white crystalline solid separated.
It was filtered,
washed with ethyl acetate and dried ih vacuo to afford the hydroxylamine (220
mg,
0.72mmo1, 64.5°Io, m.p. 209.4°C 210.4°C).
1H-NMR (270 MHz, D20) ppm: 1.49 (6H, s); 1.62 (6H, s); 2.03 (2H, t); 2.42 (2H,
d),
5.49 (1H, m); 6.63 (1H, q); 6.64 (1H, d), 7.34 (1H, d), 7.74 (1H, s)
Mass Spec. (EI, m/z) 266 (M+)
Example 15: 1-oxyl-4-(3'-tetrahydrofuran)carbonyloxy-2,2,6,6-
tetramethylpiperidine
O
O
N "~
I
O~
To a stirred solution of 3-tetrahydrofuancarboxylic acid ( 1.5 g, 13 mmol) in
dry DMF
(20 mL) was added 1,1'-carbonyldiimadazole (2.3 g, 14.18 mmol) in small
portions. A
vigorous gas evolution was observed. This solution was heated at 70°C
for 1 h. To this
mixture was then added tempol (2.23 g, 12.97 mmol) and 1,$-
diazabicyclo(5,4,0]undec-7-
ene(DBU) (2.0 g, 13.14 mmol) and heating was continued for 12 h. The reaction
mixture
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was poured into 250 mL water and extracted with ether (2 x 100 mL). The
ethereal layers
were combined and washed successively with 1N HCI, saturated NaHC03 and brine,
dried
over anhydrous sodium sulfate and concentrated ifZ vacuo to give red colored
solid (2.05 g),
that recrystallized from ethyl acetate:Hexane (1:2) to obtain pure red
crystalline solid
nitroxide (1.45 g, 5.36 mmol, 37.8°l0).
IR (I~Br, cm-1): 1360 (N-O~), 1725 (ester)
Example 16: 1-hydroxy-4-(3'-tetrahydrofuran)carbonyloxy-2,2,6,6-
tetramethylpiperidine hydrochloride
O
O
O
N
OM HCI
The nitroxide of Example 15 (300 mg, 1.11 mmol) was added to a solution of
saturated hydrogen chloride in ethanol (10 mL). The red color disappeared
quickly and the
resulting yellow colored solution was boiled to give a clear colorless
solution. The solution
was kept at room temperature for 1 h and a white crystalline solid separated.
The solid was
filtered, washed with ethanol and dried in vacuo to afford product (146 mg,
0.48 mmol,
42.86°Io, m.p. 221.0°C-223.2°C).
1H-NMR (270 MHz, DMSO-d6) ppm: 0.84 (2H, m); 0.90 (2H, m); 1.35 (6H, s); 1.46
(6H, s); 1,65 (1H, m); 2.13 (2H, t); 2.44 (2H, d), 5.14 (1H, m)
Example 17: Absorption of Representative Compounds across the Coxneas of
Animals
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R~
Rabbit corneal esterases
IH
OH
Tempol-H
Compound 1 Rl=
Compound 2 Rl=
O
OH
Compound 3 R1=
Groups of six New Zealand White rabbits were used in the study to evaluate the
absorption of tempol-H and comlaound 1. The test compounds Were prepared in
sterile saline
solutions at a concentration of 3.S% weigl?t by volume. The animals were held
in restraining
boxes during instillation of eye drops, 50pL in each eye, using a
micropipette. After dosing,
the eye was gently held closed for 60 seconds. The rabbits were dosed twice
daily for 4
consecutive days. On the fifth d~.y, rabbits were dosed once and then
euthanized at 30
minutes post dosE (2 rabbits), 60 minutes post-dose (2 rabbits) and at 120
minutes post-dose
(2 rabbits). Immediately after euthanization, aqueous humor was collected from
each rabbit.
The aqueous concentration of tempol-H in each sample was measured using the
electron spin
resonance (ESR) method.
Aqueous humor levels of tempol-H after dosing with tempol-H, were below
detectable limits of the assay at all time points (see Figure 1). Aqueous
humor
concentrations of tempol-H after dosing with compound 1 were maximal at 30
minutes post-
dose but were still present at 2 hours post-dose. (see Figure 1 and Table 1).
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Table I: Aqueous Humor Concentrations: Absorption Studies in Rabbits
Dose: 50~L of 3.5% solution, single dose (N=4 eyes / timepoint)
Concentration
of Tempol-H
~M
30 minutes 60 minutes 120 minutes
51.0 20.0 1.5
30.0 30.0 1.2
Compound 1
18.0 5.0 7.0
30.0 3.0 8.0
Mean 32.3 14.8 4.4
p g/ml (5.5) (2.5) (0.75)
Example 18: Identifccation of Metabolites of Compound 1 ifz Rabbit Eye
Aqueous humor samples, from the in vivo rabbit study described in Example 16
were
identified by GCIMS for the presence of compound 1 and its metabolites, tempol-
H and
carboxylic acid (R1COOH), formed by hydrolysis of compound 1 by ocular
esterases,. Both
the metabolites were observed but not Compound 1. This confirmed that Compound
1 was
completely converted to its metabolites.
A sample of aqueous humor was freeze dried in a lOmL amber colored glass vial
containing a tiny magnetic bar. To this was added 1mL of methylene chloride
and the
solution was stirred for two minutes and allowed to stand for five minutes. A
3~L aliquot of
the methylene chloride layer was injected into the GC column. The
cyclopropanecarboxylic
acid was detected by a mass spectrometer detector at 13.02 (retention time)
with m/z=85 (GC
model 5989B and MS model 5890 series II (both made by HP)). Agilent DB-5
column 25m
length, 0.2 mm diameter was used. Carrier gas He at 22cm/sec. Inlet
temperature was 250°C,
detector 280°C. For every injection, the temperature was held at
35°C for 5 minutes, then
was increased to 240°C at 10°C/min, and was held at 240°C
for 3 minutes. Splitless injection
was used.
Example 19: Tolerance of compound 1 in vivo in Rabbit Eyes
Eyedrops containing 3.5 °Io compound 1 were administered six times, at
1-hour
intervals, to each eye of two conscious rabbits. The drug was well tolerated
and no adverse
findings were noted in this preliminary study.
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Example 20: OeularBioavailability i~t Rabbit
The ocular bioavailability of compounds 2 and 3 was evaluated in New Zealand
White rabbits. Each compound was dissolved in lOmM phosphate buffer, pH 7.0 to
a
concentration of 125mM. This concentration was equal to ~ 3.5% for compounds 2
and 3.
Fifty ~1 was instilled onto the cornea of both eyes of each rabbit 6 times at
1-hour intervals.
Two rabbits were used for each compound. One rabbit treated with each compound
was
euthanized 30 minutes after the last dose and the second was euthanized 90
minutes after the
final dose.
After death, the eyes of each rabbit were immediately enucleated and a blood
sample
was collected from the orbit. Aqueous humor was collected from each eye with a
syringe and
then the lens was dissected from the eye. The capsule/epithelium was carefully
separated
from the fiber mass and both parts were frozen on dry ice, the
capsule/epithelium in 100,1 of
mM DTPA (diethylenetriaminepentaacetic acid) solution and the fiber mass in a
sealed vial
without added liquid. Likewise, the aqueous and blood samples were quick
frozen. The rest
of each eye including the cornea, retina, sclera and vitreous were frozen for
possible future
dissection and analysis. All samples were transported to the lab on dry ice
and were stored at
-75°C until processed.
The aqueous concentration of tempol-H in each sample was measured using the
electron spin resonance (ESR) method. Analysis of the aqueous humor reveals
that both
compounds penetrated the cornea and entered the aqueous chamber. The highest
concentrations fox both compounds was present in the 30-minute sample with
tlae 90-minute
samples being significantly reduced in concentration. Small amounts (2-3~,M of
each
compound) were also detected in the blood.
Example 21: Aqueous FXumor Cofzcerttratiotzs of Co~rtpoufzels 2 arZel 3; ift
Rabbits
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Table II: Dose of Compound 2 and 3: 50~tL of 125mM solution, at hourly
intervals x
6 (N=2 eyes / timepoint)
Concentration
of Tempol-H
pM
30 minutes 90 minutes Blood
31.4 11.6 2.3
Compound 3
22.2 9.0 2.5
Mean 26.8 10.8 2.4
pg/ml (4.6) ( 1.9) (0.4)
52.4 6.0 3.6
Compound 2
35.2 5.7 0.6
Mean 43.8 5.9 2.1
~ g/ml (7.5) ( 1.0) (0.4)
Example 22 Aqueous Solubility Data
Table III: Solubility of Compound of Example 6 was determined at room
temperature in various systems.
Conditions Solubility Solubility
mg/ml % w/v
Water 74.9 7.5
0.9% Sodium chloride 40.5 4.1
O.O1M Acotate buffer at 68.6 6.9
pH 4.8
O.OlM Citrate buffer at 71.1 7.1
pH 4.8
Water + 1% w/v glycerin 62.2 6.2
Water + 1% w/v propylene 63.8 6.4
glycol
Similarly, the solubility compounds of Examples 10 and 16 in water were
determined
to be >3.5% w/v (>35 mg/ml) in water whereas the compound of Example 12 is
soluble at
approximately 0.1% w/v in watex.
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Table IV: Partition Coefficient of Ester Compounds
OR
N
i
OH
Exam les R Calculated PC
23 H 0.8
7.2
CH3
24
0
25 ~ 16.2
0
50. I
26
0
27 ~OH~ 53.7
0
28 ~ 125.9
0
0 91.2
29
0 6.3
0
114.8
31 ~0.,.,~''
34.7
32 ~~''OH~
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Exam les R Calculated PC
199.5
33 ~-"~ '~H~
4.3
34 "CHI
~~H 8.1
35
144.5
36 ~0
~~H~ 10
37
~.~H~ 51.3
38 H "CHI
575.4
39
p 34.7
40
69.4
41 ~H
67.6
42
39.8
43 H
t
CHI
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Table V: Melting Points of Ester Compounds
R~
Examples R1 R2 M.P. (°C)
44 O 97.2-98.2
44 OH (as HCl salt) ~ 224-228°C
(dec.).
45 OH (as HCl salt) .~ 224-227°C
(dec.).
46 O ~' ~ 103.9-105.2
O
47 OH (as HCl salt) 209.4-220.1
r~
O
48 O N 150-152.3
49 OH (as HCl salt) N 250.6-253.2
50 O 64.8-66.1
\ ~ OCOCH3
51 OH (as HCl salt) 229.0-230.9
\ ~ OCOCH3
52 O OH 107-109.3
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Examples R1 R2 M.P. (°C)
53 OH (as HCl salt) \ / OH 220.0-223.0
54 O 111.1-112.3
OCH3
55 OH (as HCl salt) 228.0-231.2
OCH3
56 O OCH3 121.2-122.9
OCH3
OCH3
57 OH (as HCl salt) ~~~3 241.8-244.6
OCH3
OCH3
58 O HO 145.2-146.4
59 OH (as HCl salt) ~~ 237.8-269.1
60 O O 132-133.0
61 OH (as HCl salt) O 267.9-270
62 O 68.3-69.9
63 OH (as HCl salt) 264.8-266.3
Spectral Data for the Ester Cornpouhds
1H-NMR (270 MHz, DMSO-d~) ppm: spectral data that was common to all 4-
substtuted-1-hydroxy-2,2,6,6-tetramethylpiperidine hydrochloride portion 1.35
(6H, s); 1.46
(6H, s); 2.13 (2H, t); 2.44 (2H, d), 5.14 (1H, m)
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Examples IR CKBr) cm-1 iH-NMR (270 MHz, DMSO-d6) ppm:
Carbonyls) for
the ester moiety
57 1716 3.75 (S, 9H); 6.95 (s, 2H)
61 1738 2.79 (t,2H); 3.31 (t,2H); 7.45
1687 (m,2H); 7.55
(m,lH);7.93 (d, 2H)
53 1682 6.87 (d,2H); 7.83 (d,2H), 10.3
(br, s, 1H)
51 1718 3.9 (s,3H), 7.18 (d,2H), 8.07
1755 (d,2H)
59 1723 6.54, 7.85 (dd, J=16.0 Hz); 6.84
(m, 1H); 7.24
(m,lH);7.54 (d, 1H); 7.86 (d,lH);
10/2 (br, s,
1H)
61 1718 1.96 (m, 2H); 2.30 (m, 4H); 2.78
(m, 2H)
49 1688 2.88 (S, 6H); 6.65 (d, 2H); 7.73
(d, 2H)
53 1682 3.70 (s, 3H); 7.20 (d,2H); 7.72
(d, 2H)
The following Tables VI to XII describe methods used in the synthesis of
additional
examples of the ester compounds of the invention. The appropriate carboxylic
acid listed in
the Tables is converted to the ester nitroxide by the DCC/DMAP esterification
method of
Example 5c. The ester nitroxide is converted to the corresponding 1-
hydroxypiperidine by
the methods described in Examples 6 and 7.
3-acyloxy-2.2-dimethylpropionic acids were prepared by the method described in
U.S. Patent 4,851,436, the content of which is incorporated herein by
reference, for the
synthesis of 3-acetoxy-2,2-dimethylpropionic acid.
Table VI: Substitute cyclopropanecarboxylic acid with following compounds in
DCC/DMAP esterification method
Examples Starting material Chemical name
(structure)
3-Acetoxy-2,2-dimethylpropionic
~OCH3 acid
H 0
G
2 I'
0
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Examples Starting material Chemical name
(structure)
0
Ha2C~ 3-Pivaloloxy-2,2-dimethylpropionic
acid
0
H a~C~'a 3-Cyclopropanecarbonyloxy-2,2-
a dimethylpropionic acid
H a2C~''a
lox
l-c
ro
anecarbon
)-
3-(1-Meth
clo
y
p
y
y
p
y
a
2,2-dimethylpropionic acid
3-(2-Methyl-cyclopropanecarbonyloxy)-
H a~C"a ~'' 2,2-dimethylpropionic acid
a
3-(2,2-Dimethyl-
H a2G~a cyclopropanecarbonyloxy)-2,2-
dimethylpropionic acid
a 3-(3-Tetrahydrofurancarbonyloxy)-2,2-
~'a id
h
l
i
di
i
Ha~ met
y
prop
on
c ac
a
64 3-( 1-Methyl-3-
a
H a~C~ tetrahydrofurancarbonyloxy)-2,2-
dimethylpropionic acid
Table VII
3-alkoxy-2.2-dimethylpropionic acids and 3-alkoxyalkyl-2.2-dimethylpropionic
acids
were prepared by the method described in J. Org. Chem. 38, 2349 (1975).
Substitute cyclopropanecarboxylic acid with following compounds in the
DCC/DMAP esterification method (Example 5c):
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Starting material Chemical name
(structure)
3-Methoxy-2,2-dimethylpropionic
' acid
o'~..
HOiC
3-propoxy-2.2-dimethylpropionic
H0~0'1" acid
3-isopropoxy-2.2-dimethylpropionic
0 acid
'
H02C
3-Cyclopropylmethoxy-2,2-
dimet
hylpropiomc acid
3-(2-Methoxy-ethoxy)-2,2-
~~
"'~~ dimeth 1 ro ionic acid
~''a'~ yp P
3-Ethoxymethoxy-2,2-dimethylpropionic
0
'
HG~rC acid
Table VIII
3-N-substituted-2.2-dimethylpropionic acids are prepared by the method
described in
U.S. Patent No. 5,475,013 to Talley et al., the content of which is
incorporated herein by
reference. Substitute cyclopropanecarboxylic acid with the following compounds
in the
DCC/DMAP esterification method (Example Sc):
Starting material Chemical name
(structure)
3-Amino-2,2-dimethylpropionic
H O~C'~''N H~ acid
HQ~~ H~, 3-Dimethylamino-2,2-dimethylpropionic
acid
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Starting material Chemical name
(structure)
2,2-Dimethyl-3-piperidin-1-yl-propionic
H acid
~
HO~C
"'
0 2,2-Dimethyl-3-(4-oxo-piperidin-1-yl)-
H propionic acid
H 0~0
2,2-Dimethyl-3-thiomorpholin-4-yl-
H0 propionic acid
~~-'H
2
H~ 2,2-Dimethyl-3-(4-methyl-piperazin-1-yl)-
'
l propionic acid
~h~
H O~C
3-Imidazol-1-yl-2,2-dimethyl-propionic
~
H acid
H 0~,~
Table IX
3-S-substitted-2.2-dimethylpropionic acids are prepared by the method
described in
U.S. Patent 5,475,013. Substitute cyclopropanecarboxylic acid with following
compounds in
the DCC/DMAP esterification method (Example 5c):
Starting material Chemical name
(structure)
2,2-Dimethyl-3-methylsulfanylpropionic
~~
H 02 C acid
~'
3-Methanesulfinyl-2,2-dimethylpropionic
~'~0' acid
H 0
C '~
2
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2,2-Dimethyl-3-phenylsulfanylpropionic
HQ2~~S '"~ acid
~,..
0 .- 3-Benzenesulfonyl-2,2-dimethylpropionic
Hp~O~~ ~ % acid
0
Table X
3-Substitted-2.2-dimethylpropionic acids are prepared by the method described
in
U.S. Patent 5,475,013. Substitute cyclopropanecarboxylic acid with following
compounds in
the DCC/DMAP esterification method (Example 5c):
Starting material Chemical name
(structure)
,x..- I 2,2-Dimethyl-3-phenylpropionic acid
H O~C
.~'~~ 2,2-Dimethyl-3-pyridin-4-yl-propionic acid
I
HOG
Table XI
Various NSA)D (nonsteroidal anti-inflammatory drugs containing carboxylic acid
group) are commercially available. Substitute cyclopropanecarboxylic acid with
following
compounds in the DCC/DMAP esterification method:
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Starting material Chemical name
(structure)
Ketorolac or 5-Benzoyl-2,3-dihydro-1H-
r' ~~ ~aZH pyrrolizine-1-carboxylic acid
0
Flurbibrofen or 2-(2-Fluoro-biphenyl-4-
F
yl)propionic acid
r~' ~Q~t-~
Ibuprofen or 2-(4-Isobutyl-
~, phenyl)propionic acid
CQzH
Naproxen or 2-(5-Methoxy-naphthalen-2-
'~ - ~~~H yl)propionic acid
-0 ~ ~ H3
Aspirin
0
H02C
Table XII
Various carboxylic acids are commercially available. Substitute
cyclopropaneearboxylic acid with the following compounds in DCC/DMAP
esterification
method:
Starting material Chemical name
(structure)
~ p2H Cyclopent-3-enecarboxylic acid
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Starting material Chemical name
(structure)
H ~~0 But-3-enoic acid
Tetrahydro-furan-2-carboxylic
~CO~H acid
Tetrahydro-thiophene-2-carboxylic
~CO~H acid
Gi~2H Tetrahydro-thiophene-3-carboxylic
S acid
GO~H ~-Oxo-thiazolidine-4-carboxylic
acid
SNH
~ ~
0
OQ~H 2-Oxo-oxazolidine-4-carboxylic
acid
NH
0
0 ~~H 2-Oxo-imidazolidine-4-carboxylic
acid
NN f~H
~~
0
O~~H 2-Oxo-[1,3]dioxolane-4-carboxylic
acid
~0 ~0
'~'
0
C a~H 1-Methyl-pyrrolidine-3-carboxylic
acid
H
i
CH3
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Starting material Chemical name
(structure)
1-methyl-pyrrolidine-2-carboxylic
--CO~H acid
N
i
GH3
Tetrahydro-pyran-4-carboxylic
acid
~.-GQ~H
Tetrahydro-thiopyran-4-carboxylic
acid
S~~--- G O~H
1-Methyl-piperidine-4-carboxylic
Ha'~ W~-CO~H acid
3-hydroxy-2-methylpropionic
~OH acid
HOG
3-amino-2-methylpropionic acid
,,NHS
H 0~2G
3-mercapto-2-methylpropionic
~SH acid
H ~~C
3-methoxy-2-methylpropionate
(synthesis:
H~ U.S. Patent No. 4,617,154,
00.,~ the content of
~ which is incorporated herein
by reference)
Example 65: Plzarmacokinetics, Ocular Tissue Distributiofz, and Urinary
Excretiofz
of Total Radioactivity Following Single Topical Dose Administration of
Compound 1 in
Rabbits. The pharmacokinetics, ocular tissue distribution and urinary
excretion of total
radioactivity following a single topical dose administration of the tritiated
hydrochloride of
Compound 1 in rabbits was evaluated. Eighteen male New Zealand White (Harlan)
rabbits,
age 3-6 months, weight 2.1 to 3.0 kg were utilized.
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Experiynental Design: The experimental design was a randomized, single-
treatment
study in one group of rabbits administered a single topical application of
[3H]Compound 1-
HCl into both eyes. The group consisted of 6 subgroups of 3 animals. At six
specified times
(0.5, 1, 2, 4, 8 and 24 hours) post-dose, three animals per subgroup were
euthanized and
terminal samples collected. Animals designated to the 24 hour post-dose
subgroup were
placed in metabolism cages and urine was collected from 0-12 and I2-24 hours
post-dose.
Terminal blood was collected by cardiac puncture technique. From each eye, the
following
ocular tissues were harvested: aqueous humor; vitreous humor; lens; and
cornea. Total
radioactivity was determined in all samples.
The design and dosages are shown in the table below.
Group#/Sex Dose VolumeDose Dose Concn
Level
Per Eye (mg/kg) (~.Ci/k (mg/ml) (mCi/ml)
(ml) )
1 18/M 0.04 -0.8 ~34 to 30 1.275
to 1.2 49
Animals received a single topical ocular dose in each eye, right (OD) followed
by left
(OS). While slightly pulling away the lower eyelid from the globe, the dose
was accurately
pipetted onto the cornea and allowed to collect in the lower conjunctival sac.
The eyelids
were gently held shut for I minute after drug instillation and then carefully
released.
Data analysis and statistical evaluation: Counts per minute for all assayed
samples
were converted to disintegrations per minute (DPM) using a Beckman LS6000
counter.
Following appropriate background subtraction, DPM values were converted to
concentrations
(DPM per ml or g). Average DPM per ml or g values were calculated. Mean
concentration
values of 3H radioactivity were calculated and converted to equivalents per ml
or g based on
the measured specific activity and nominal concentration of the administered
test article
formulation as determined by oxidation. Concentrations of total radioactivity
in blood and
plasma, amount (% of dose) and concentration in ocular tissue and urine
samples were
determined. Elimination kinetics including blood, plasma, and tissue half
Lives were
calculated. Urinary excretion data were also tabulated. Statistical evaluation
consisted of
descriptive statistical analysis. Pharmacokinetics of radioactivity were
evaluated by model
independent analyses pending suitability of sample assay results. Non-
truncated numerical
values were used in the calculations.
Representative results: A single drop (40u1 ) of a 3~/o solution of Compound 1
yielded the following peals tissue concentrations (nanogram equivalents/gram),
at 30 minutes
post-dose, in cornea, aqueous, lens, vitreous, blood and plasma, respectively:
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Cornea Adueous Lens Vitreous Blood Plasma
13090 5930 310 I50 240 360
As shown in Figure 2, radioactivity was still measurable in all tissues at 24
hours
post-dose. It was previously established that dosing rabbits with a 3%
solution of Compound
1, 4 times daily, for one week caused no toxicity.
Example 66: Effect of Compound 1 or Tempol-H Treatment on Photocyaernical
Retinal Injury in a Rabbit Model.
The ability of light to produce retinal injury well below the levels capable
of
producing thermal damage has been termed photochemical retinal injury. The
mechanism of
action for photochemical retinal injury is believed to be the light induced
production of free
radicals. The ability of commonly used light sources in clinical ophthalmology
to produce
photochemical retinal injury is well recognized. The operating microscopes
used in
ophthalmic surgery have been shown to have the capability of producing retinal
photochemical injury. This observation has been utilized to produce a model in
the rabbit
eye to test the ability of various agents to block photochemical retinal
injury. It has been
determined that opthalmoscopically visible retinal lesions are detectable
after as little as 2.5
minutes of exposure to an operating microscope. The protocol set forth below
is utilized to
determine whether Compound 1 or tempol-H given via intravitreal injection has
the ability to
block the development of an ophthalmoscopically visible photochemical retinal
lesion
produced by a operating microscope.
Materials and Methods: One week prior to treatment, a baseline Fundus
photograph
and FA is performed on each animal. One day prior to being exposed to the
light from a
operating microscope, one eye of each animal receives an intravitreal
injection of 0.1 cc of
BSS~ as a control and the fellow eye receives 0.1 cc of Compound 1 or Tempo-H
in a '
BSS~ vehicle. The animals are anesthetized with a IM injection of a
60°70-40% mixture of
ketamine hydrochloride 100mglml and Xylazine ZOmg/ml. A speculum is inserted
into the
eye receiving the injection, and the injection is performed using a 30g needle
passed through
the sclera just behind the limbus and angled to clear the lens, placing the
tip in the mid
vitreous cavity. Following injection, the intraocular pressure is monitored
using a hand-held
Applanation tonometer. If necessary, a paracentesis is performed to return
intraocular
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pressure to normal levels, prior to returning the animals to their cages.
Alternatively,
Compound 1 or tempol-H may be administered to the rabbit eye by multiple
instillations of
topical eye drops prior to the procedure.
Forty eyes in 20 pigmented rabbits are exposed to the light from a Zeiss model
OpMi 1 operating microscope for various periods of time up to one hour. The
rabbits are
anesthetized with a IM injection of a 60%-40% mixture of ketamine
hydrochloride 100mg/ml
and Xylazine 20mg/ml. The rabbits are then positioned on a table; a lid
speculum is inserted
in the eye to be exposed. The pupil is dilated with tropicamide HCL 1%, and
the cornea
irrigated with BSS~. The light from the microscope is positioned 20 cm from
the animal in
a manner so that the light is centered and parallel to the eye. The light
filaments are centered
in sharp focus on the cornea. Forty-eight hours after exposure, the animals
are examined and
a repeat Fundus photograph and FA performed. The animals are killed using
standard
techniques and selected eyes are harvested and stored in chilled glutaldehyde
solution, for
additional analyses.
Example 67: Evaluation of Compound 1 or Tempol-H Incorporation into Retinal
Tissue of Rabbits Following Intravitreous Injection
To assess the efficacy of Compound 1 or tempol-H at preventing AMD and other
retinal disorders, it must be determined that the drug is incorporated into
retinal tissue. The
protocol set forth in this example utilizes scintillation technique to
establish quantity and
duration of Compound 1 or tempol-H incorporation into retinal tissue following
intravitreous
injection in the rabbit. The New Zealand White Rabbit is well characterized in
experiments
involving intravitreous injection as the large eye of the rabbit provides a
suitable template for
treatment administration (Hosseini et al., 2003, Lasers Surg. Med. ~32: 265-
270).
Furthermore, the New Zealand White Rabbit has been used extensively in
experiments
assessing drug uptalce into retinal tissue via scintillation technique (Ahmed
et al., 1987, J.
Pharm. Sci. 76: 583-586).
Materials and Methods: Twenty-four New Zealand White Rabbits, all male,
weighing between 2.5 and 3 kilograms, are used in this protocol. The rabbits
are randomized
into one of two treatment arms. Initially, rabbits are given baseline ocular
exams to ensure
that all eyes are free of irritation and infection. Following randomization
and baseline exams,
rabbits are anesthetized by subcutaneous injection of 100 mg/ml ketamine / 20
mg/ml
xylazine. One drop of commercially available 0.5% proparacaine hydrochloride
is applied to
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both eyes. Rabbits are then treated bilaterally by intravitreous injection in
accordance with
the masked randomization scheme shown below.
Masked Treatment Arms:
1. Bilateral intravitreous injection with labeled Compound 1 or tempol-H.
(N=12)
2. Bilateral intravitreous injection with placebo (vehicle). (N=12)
On Day 1, 7, 14 and 28, respectively, six rabbits, three from each treatment
arm
respectively, are sacrificed with a lethal dose of sodium pentobarbital.
Immediately
following sacrifice, bilateral enucleation is performed and the retinal tissue
is removed for
scintillation analysis (retinal tissue may be frozen following enucleation to
prevent fluid
loss). Retinal tissue is ground into a slurry and dissolved in an alkali or
quaternary
ammonium compound. Scintillation readings are conducted to quantify the amount
of
Compound 1 or tempol-H present in the retinal tissue.
For statistical analysis, data from all rabbits that are dosed with test
articles is
considered evaluable. Primary and secondary efficacy variables are for
statistical
significance. Two-sided nonparametric statistical tests are used, and p < 0.05
is considered
statistically significant.
Example 68: EfJ"-zcacy of Tempol-H in Protection Against Photooxidative
Processes
in the Retinal Pigmefzt Epithelium (RPE)
Background: Although the etiology of atrophic age-related macular degeneration
(AMD) is not fully understood, it is generally accepted that AMD begins with
the death of
retinal pigment epithelial cells, the degeneration of photoreceptor cells, and
resultant loss of
vision, occurring thereafter. There is a growing body of evidence linking the
lipofuscin that
accumulates in RPE with the death of these cells. For instance, not only are
lipofuscin levels
highest in RPE cells underlying the macula, the monitoring RPE lipofuscin by
detection of
fundus autofluorescence has also shown that areas of RPE atrophy develop at
sites of
previously increased fluorescence. Studies concerned with examining
associations between
RPE lipofuscin and RPE cell death have shown that a major constituent of RPE
lipofuscin,
the bisretinoid fluorophore A2E, can perturb cellular membranes and can
mediate blue light
damage to RPE. The photoexcitation of A2E leads to the generation of singlet
oxygen and
the addition of the latter to carbon-carbon double bonds along the retinoid-
derived side arms
of A2E such that epoxides are formed. In this way, A2E is converted into a
mixture of
compounds (A2E-epoxides) bearing epoxides of varying numbers. These highly
reactive
epoxides have been shown to damage the cell. Ultimately, the photochemical
events
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CA 02546053 2006-05-15
WO 2005/055926 PCT/US2004/039716
provoked by the irradiation of A2E in RPE cells initiates cell death by way of
a pathway that
involves the participation of cysteine-dependent proteases (caspases) to
cleave cellular
substrates and that is modulated by the mitochondria) protein bcl-2.
The ability of tempo)-H to protect against A2E-mediated blue light damage was
examined.
Materials ahd Methods: ARPE-19 cells that have accumulated A2E in culture were
exposed to 430 +/- 20 nm light delivered from a halogen source. This
wavelength of light is
relevant since (i) the excitation maximum of A2E is approximately 430 nm, and
(ii) light of
this wavelength reaches the RPE ire vivo while light of wavelengths longer
than 400 nm are
not absorbed by the cornea and lens. Cell death in blue light-illuminated A2E-
laden RPE was
compared with and without pre-treatment (24 hours) with tempo)-H. The loss of
cell viability
was quantified by:
1. A calorimetric microtiter assay based on ability of healthy metabolically
active
cells to cleave the yellow tetrazolium salt MTT to purple formazan crystals.
Cell viability
was assessed 24 hours after blue light illumination. Triplicate wells were
assayed in each of
2 experiments.
2. Two-color fluorescence assay in which the nuclei of all dead cells appear
red due to
staining by a membrane-impermeant dye (Dead Red nuclei acid stain, Molecular
Probes)
with the nuclei of all cells stained blue with DAPI. Cell viability was
assessed 8 hours after
blue light illumination. Replicates were assayed by counting cells within 5
microscopic
fields within the area of illumination in each well and 3 wells were assayed
per experiment.
Data are based on of 3 experiments.
Results: As shown in Fig. 3 and Fig. 4, tempo)-H affords a dose-dependent
protection against the death of A2E-laden RPE in this model of macular
degeneration. This
represents what would be expected from Compound, I as well as tempo)-H is the
active
metabolite of Compound I.
While the present invention has been particularly shown and described with
reference
to the presently preferred embodiments, it is understood that the invention is
not limited to
the embodiments specifically disclosed and exemplified herein. Numerous
changes and
modifications may be made to the preferred embodiment of the invention, and
such changes
and modifications may be made without departing from the scope and spirit of
the invention
as set forth in the appended claims.
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