U.S. patent application number 11/969346 was filed with the patent office on 2008-05-01 for use of beta-adrenoceptor antagonists for the manufacture of a medicament for the treatment of disorders of the outer retina.
Invention is credited to Robert J. Collier, Jr., Louis JR. Desantis, Michael A. Kapin.
Application Number | 20080103211 11/969346 |
Document ID | / |
Family ID | 22609644 |
Filed Date | 2008-05-01 |
United States Patent
Application |
20080103211 |
Kind Code |
A1 |
Collier, Jr.; Robert J. ; et
al. |
May 1, 2008 |
USE OF BETA-ADRENOCEPTOR ANTAGONISTS FOR THE MANUFACTURE OF A
MEDICAMENT FOR THE TREATMENT OF DISORDERS OF THE OUTER RETINA
Abstract
Compositions and methods for treating disorders of the outer
retina with .beta.-adrenoceptor antagonists are disclosed.
Inventors: |
Collier, Jr.; Robert J.;
(Arlington, TX) ; Desantis; Louis JR.; (Fort
Worth, TX) ; Kapin; Michael A.; (Arlington,
TX) |
Correspondence
Address: |
ALCON
IP LEGAL, TB4-8
6201 SOUTH FREEWAY
FORT WORTH
TX
76134
US
|
Family ID: |
22609644 |
Appl. No.: |
11/969346 |
Filed: |
January 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11415824 |
May 2, 2006 |
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11969346 |
Jan 4, 2008 |
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10130408 |
May 15, 2002 |
7081482 |
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PCT/US00/32575 |
Nov 29, 2000 |
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11415824 |
May 2, 2006 |
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60167993 |
Nov 30, 1999 |
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Current U.S.
Class: |
514/651 |
Current CPC
Class: |
A61K 31/343 20130101;
A61K 31/165 20130101; Y10S 514/912 20130101; A61K 31/404 20130101;
A61K 31/5377 20130101; A61K 31/222 20130101; A61K 31/34 20130101;
A61P 43/00 20180101; A61K 31/4704 20130101; A61K 31/138 20130101;
A61P 27/02 20180101 |
Class at
Publication: |
514/651 |
International
Class: |
A61K 31/138 20060101
A61K031/138; A61P 27/02 20060101 A61P027/02 |
Claims
1-6. (canceled)
7. An ophthalmic composition for treating disorders of the outer
retina, said composition comprising from 0.001% to 5% w/v of a
.beta.-adrenoceptor antagonist in a pharmaceutical acceptable
carrier.
8. The composition of claim 7, wherein the concentration of the
.beta.-adrenoceptor antagonist is from 0.01% to 2% w/v.
9. The composition of claim 8, wherein the concentration of the
.beta.-adrenoceptor antagonist is from 0.25% to 0.75% w/v.
10. The composition of claim 7, further comprising poly(styrene
divinylbenzene) Sulfonic Acid.
11. The composition of claim 10, wherein the .beta.-adrenoceptor
antagonist is levobetaxolol.
Description
[0001] This application claims continuation from U.S. Ser. No.
10/130,408 filed May 15, 2002: which is a 371 application of
PCT/US00/32575 filed Nov. 29, 2000; which claims benefit of U.S.
Ser. No. 60/167,993 filed Nov. 30, 1999.
[0002] This invention is directed to the use of .beta.-adrenoceptor
antagonists, such as, betaxolol, for treating disorders of the
outer retina.
BACKGROUND OF THE INVENTION
[0003] To date, more than 100 genes have been mapped or cloned that
may be associated with retinal degeneration. The pathogenesis of
retinal degenerative diseases such as age-related macular
degeneration (ARMD) and retinitis pigmentosa (RP) is multifaceted
and can be triggered by environmental factors in those who are
genetically predisposed. One such environmental factor, light
exposure, has been identified as a contributing factor to the
progression of retinal degenerative disorders such as ARMD (Young,
Survey of Ophthalmology, 1988, Vol. 32: 252-269). Photo-oxidative
stress leading to light damage to retinal cells has been shown to
be a useful model for studying retinal degenerative diseases for
the following reasons: damage is primarily to the photoreceptors
and retinal pigment epithelium (RPE) of the outer retina (Noell, et
al., Investigative Ophthalmology & Visual Science, 1966, Vol.
5: 450-472; Bressler, et al., Survey of Ophthalmology, 1988, Vol.
32: 375-413; Curcio, et al., Investigative Ophthalmology &
Visual Science, 1996, Vol. 37: 1236-1249); they share a common
mechanism of cell death, apoptosis (Ge-Zhi, et al., Transactions of
the American Ophthalmology Society, 1996, Vol. 94: 411-430; Abler,
et al., Research Communications in Molecular Pathology and
Pharmacology, 1996, Vol. 92: 177-189); light has been implicated as
an environmental risk factor for progression of ARMD and RP
(Taylor, et al., Archives of Ophthalmology, 1992, Vol. 110: 99-104;
Naash, et al., Investigative Ophthalmology & Visual Science,
1996, Vol. 37: 775-782); and therapeutic interventions which
inhibit photo-oxidative injury have also been shown to be effective
in animal models of heredodegenerative retinal disease (LaVail, et
al., Proceedings of the National Academy of Science, 1992, Vol. 89:
11249-11253; Fakforovich, et al., Nature, 1990, Vol. 347:
83-86).
[0004] A number of different classes of compounds have been
reported to minimize retinal photic injury in various animal
models, including: antioxidants, such as, ascorbate (Organisciak,
et al., Investigative Ophthalmology & Visual Science, 1985,
Vol. 26: 1580-1588), dimethylthiourea (Organisciak, et al.,
Investigative Ophthalmology & Visual Science, 1992, Vol. 33:
1599-1609; Lam, et al., Archives of Ophthalmology, 1990, Vol. 108:
1751-1757), .alpha.-tocopherol (Kozaki, et al., Nippon Ganka Gakkai
Zasshi, 1994, Vol. 98: 948-954), and .beta.-carotene (Rapp, et al.,
Current Eye Research, 1996, Vol. 15: 219-223); calcium antagonists,
such as, flunarizine, (Li, et al., Experimental Eye Research, 1993,
Vol. 56: 71-78; Edward, et al., Archives of Ophthalmology, 1992,
Vol. 109: 554-622); growth factors, such as, basic-fibroblast
growth factor (bFGF), brain-derived nerve factor (BDNF), ciliary
neurotrophic factor (CNTF), and interleukin-1-.beta. (LaVail, et
al., Proceedings of the National Academy of Science, 1992, Vol. 89:
11249-11253); glucocorticoids, such as, methylprednisolone (Lam, et
al., Graefes Archives of Clinical & Experimental Ophthalmology,
1993, Vol. 231: 729-736), dexamethasone (Fu, J., et al.,
Experimental Eye Research, 1992, Vol. 54: 583-594);
NMDA-antagonists, such as, eliprodil and MK-801 (Collier, et al.,
Investigative Ophthalmology & Visual Science, 1999, Vol. 40,
pg. S159) and iron chelators, such as, desferrioxamine (Li, et al.,
Current Eye Research, 1991, Vol. 2: 133-144).
[0005] Ophthalmic .beta.-adrenergic antagonists, also referred to
as .beta.-adrenoceptor antagonists or .beta.-blockers are well
documented IOP-lowering agents for therapy of glaucoma. Currently,
several ophthalmic .beta.-blockers are approved for use worldwide.
The majority of these are nonselective .beta.-blockers; betaxolol
is a cardioselective .beta.-blocker marketed as Betoptic.RTM. or
Betoptic.RTM.S (Alcon Laboratories, Inc., Fort Worth, Tex.).
[0006] As a potential treatment for glaucoma and other inner retina
pathologies, Osborne, et al. (Brain Research, 1997, Vol. 751:
113-123) have shown that betaxolol is neuroprotective in a rat
ischemia/reperfusion injury model. Ischemia/reperfusion results in
a reduction of the electroretinogram (ERG) b-wave amplitude, a
measure of inner retina function, not photoreceptor or RPE
function. This ERG b-wave deficit was protected by treatment with
betaxolol. Consistent with the inner retinal protection was
preservation of choline acetyltransferase and calretinin
immunoreactivity in the inner plexiform layer and cell bodies in
the ganglion cell layer and inner nuclear layer by treatment with
betaxolol. In vitro studies by Osborne, et al. have also shown that
betaxolol can prevent the kainate induced elevation of
intracellular calcium in chick retinal cells, partially inhibited
changes in GABA immunoreactivity in the rabbit inner retina
following glucose-oxygen deprivation, and partially prevented the
glutamate-induced release of lactate dehydrogenase in cortical
cultures. P-adrenoceptor antagonists have also been shown to relax
KCl-induced contraction of porcine ciliary artery (Hester, et al.,
Survey of Ophthalmology, Vol. 38: S125-S134, 1994). Moreover,
certain .beta.-blockers have been shown to produce vasorelaxation
unrelated to their .beta.-adrenergic blocking action (Yu, et al.,
Vascular Risk Factors and Neuroprotection in Glaucoma, pp. 123-134,
(Drance, S. ed.) Update, 1996; Hoste, et al., Current Eye Research,
Vol. 13: 483-487, 1994; and Bessho, et al., Japanese Journal of
Pharmacology, Vol. 55: 351-358, 1991.) There is experimental
evidence that this is due to the ability of certain .beta.-blockers
to act as calcium channel blockers and to reduce the entry of
calcium ion into vascular smooth muscle cells where it participates
in the contraction response and reduces the diameter of the lumen
of the blood vessel and decreases blood flow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows the prevention of photic retinopathy by the
systemic administration of the selective .beta.-blockers, betaxolol
and its isomers.
[0008] FIG. 2 shows the prevention of photic retinopathy by the
systemic administration of the non-selective .beta.-blocker,
timolol.
[0009] FIG. 3 compares the protection of the retina from photic
retinopathy by betaxolol and levobetaxolol following topical ocular
administration.
[0010] FIG. 4 shows preservation of retinal function in P23H mutant
rhodopsin transgenic rats.
[0011] FIG. 5 shows upregulation of endogenous retinal neurotrophic
factor mRNA levels following a single administration of
levobetaxolol compared to other agents.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to .beta.-adrenoceptor
antagonists which have been discovered to be useful in treating
disorders of the outer retina, particularly: ARMD; RP and other
forms of heredodegenerative retinal disease; retinal detachment and
tears; macular pucker; ischemia affecting the outer retina; damage
associated with laser therapy (grid, focal, and panretinal)
including photodynamic therapy (PDT); trauma; surgical (retinal
translocation, subretinal surgery, or vitrectomy) or light induced
iatrogenic retinopathy; and preservation of retinal transplants. As
used herein, the outer retina includes the RPE, photoreceptors,
Muller cells (to the extent that their processes extend into the
outer retina), and the outer plexiform layer. The compounds are
formulated for systemic or local ocular delivery.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0013] Neurotrophic factors can be potent neuroprotective agents,
but as peptides, are difficult to deliver to the retina or central
nervous system. We have demonstrated that betaxolol upregulates
CNTF and bFGF mRNA retinal expression and this can prevent
light-induced apoptotic cell death to the outer retina. We have
found that treatment with betaxolol can completely prevent
photo-oxidative induced retinopathy and significantly reduce loss
of retinal function. The safety advantages of the compound make it
particularly desirable for both acute and chronic therapies. Such
an agent would have utility in the treatment of various outer
retinal degenerative diseases.
[0014] In our light damage paradigms, antioxidants were either
ineffective (alpha-tocopherol) or marginally effective at high
doses (ascorbate, vitamin E analogs). Similarly, some calcium
antagonists (flunarizine, nicardipine) were moderately effective
while others (nifedipine, nimodipine, verapamil) had no effect in
preventing light-induced functional or morphological changes.
However, it has been discovered that .beta.-adrenoceptor
antagonists are effective in these light damage paradigms and
therefore are useful for treating disorders of the outer
retina.
[0015] Disorders of the outer retina encompass acute and chronic
environmentally induced (trauma, ischemia, photo-oxidative stress)
degenerative conditions of the photoreceptors and RPE cells in
normal or genetically predisposed individuals. This would include,
but not be limited to, ARMD, RP and other forms of
heredodegenerative retinal disease, retinal detachment, tears,
macular pucker, ischemia affecting the outer retina, damage
associated with laser therapy (grid, focal and panretinal)
including photodynamic therapy (PDT), thermal or cryotherapy,
trauma, surgical (retinal translocation, subretinal surgery or
vitrectomy) or light induced iatrogenic retinopathy and
preservation of retinal transplants.
[0016] The invention contemplates the use of any
.beta.-adrenoceptor antagonist, including their isomers and
pharmaceutically acceptable salts, for treating disorders of the
outer retina. Preferred .beta.-adrenoceptor antagonists also
exhibit neurotrophic activity and may have calcium antagonist
activity.
[0017] Representative .beta.-adrenoceptor antagonists useful
according to the present invention include, but are not limited to:
betaxolol (R or S or racemic), timolol, carteolol, levobunolol,
metipranolol, befunolol, propranolol, metoprolol, atenolol,
pendolol, and pinbutolol.
[0018] The preferred .beta.-adrenoceptor antagonist is betaxolol,
and/or its R or S isomer. The S-isomer is also referred to as
levobetaxolol.
[0019] In general, for degenerative diseases, the .beta.-blockers
of this invention are administered orally with daily dosage of
these compounds ranging between 0.001 and 500 milligrams. The
preferred total daily dose ranges between 1 and 100 milligrams.
Non-oral administration, such as, intravitreal, topical ocular,
transdermal patch, subdermal, parenteral, intraocular,
subconjunctival, or retrobulbar injection, iontophoresis or slow
release biodegradable polymers or liposomes may require an
adjustment of the total daily dose necessary to provide a
therapeutically effective amount of the compound. The
.beta.-blockers can also be delivered in ocular irrigating
solutions used during surgery, see, for example, U.S. Pat. No.
4,443,432. This patent is herein incorporated by reference.
Concentrations should range from 0.001 .mu.M to 100 .mu.M,
preferably 0.01 .mu.M to 5 .mu.M.
[0020] The .beta.-blockers can be incorporated into various types
of ophthalmic formulations for topical delivery to the eye. They
may be combined with ophthalmologically acceptable preservatives,
surfactants, viscosity enhancers, gelling agents, penetration
enhancers, buffers, sodium chloride, and water to form aqueous,
sterile ophthalmic suspensions or solutions or preformed gels or
gels formed in situ. Ophthalmic solution formulations may be
prepared by dissolving the compound in a physiologically acceptable
isotonic aqueous buffer. Further, the ophthalmic solution may
include an ophthalmologically acceptable surfactant to assist in
dissolving the compound. The ophthalmic solutions may contain a
viscosity enhancer, such as, hydroxymethylcellulose,
hydroxyethylcellulose, hydroxypropylmethylcellulose,
methylcellulose, polyvinyl-pyrrolidone, or the like, to improve the
retention of the formulation in the conjunctival sac. In order to
prepare sterile ophthalmic ointment formulations, the active
ingredient is combined with a preservative in an appropriate
vehicle, such as, mineral oil, liquid lanolin, or white petrolatum.
Sterile ophthalmic gel formulations may be prepared by suspending
the active ingredient in a hydrophilic base prepared from the
combination of, for example, carbopol-940, or the like, according
to the published formulations for analogous ophthalmic
preparations; preservatives and tonicity agents can be
incorporated.
[0021] If dosed topically, the .beta.-blockers are preferably
formulated as topical ophthalmic suspensions or solutions, with a
pH of about 4 to 8. The .beta.-blockers will normally be contained
in these formulations in an amount 0.001% to 5% by weight, but
preferably in an amount of 0.01% to 2% by weight. Thus, for topical
presentation, 1 to 2 drops of these formulations would be delivered
to the surface of the eye 1 to 4 times per day according to the
discretion of a skilled clinician.
[0022] The preferred .beta.-blocker, betaxolol (or its R or S
isomer), is orally bioavailable, demonstrates a low incidence of
adverse effects upon administration, and effectively crosses the
blood-brain barrier indicating that effective concentrations are
expected in the target tissue, the retina. Betaxolol is described
in U.S. Pat. Nos. 4,252,984 and 4,311,708, the contents of which
are incorporated herein by reference.
[0023] .beta.-adrenoceptor antagonists were evaluated in our
photo-oxidative induced retinopathy paradigm, a model of retinal
degenerative diseases that may have utility for identifying agents
for treatment of RP and ARMD. Unexpectedly betaxolol and its
enantiomers, demonstrated marked potency and efficacy as a
neuroprotective agent. Both photoreceptor and RPE cells were
completely protected from light-induced functional changes and
morphologic lesions. Timolol was also neuroprotective, but was
significantly less potent. Additional evaluation of levobetaxolol
in a transgenic rat model that has a rhodopsin mutation, which is
similar to a defect observed in some human patients with
heredodegenerative disease, provided significant protection of
retinal function.
EXAMPLE 1
Prevention of Photo-oxidative Induced Retinopathy by Betaxolol and
its Enantiomers
[0024] Photic retinopathy results from excessive excitation of the
RPE and neuroretina by absorption of visible or near ultraviolet
radiation. Lesion severity is dependent upon wavelength,
irradiance, exposure duration, species, ocular pigmentation, and
age. Damage may result from peroxidation of cellular membranes,
inactivation of mitochondrial enzymes such as cytochrome oxidase,
and/or increased intracellular calcium. Cellular damage resulting
from photo-oxidative stress leads to cell death by apoptosis,
(Shahinfar, et al., 1991, Current Eye Research, Vol. 10: 47-59;
Abler, et al., 1994, Investigative Ophthalmology & Visual
Science, Vol. 35(Suppl): 1517). Oxidative stress induced apoptosis
has been implicated as a cause of many ocular pathologies,
including, iatrogenic retinopathy, macular degeneration, RP and
other forms of heredodegenerative disease, ischemic retinopathy,
retinal tears, retinal detachment, glaucoma and retinal
neovascularization (Chang, et al., 1995, Archives of Ophthalmology,
Vol. 113: 880-886; Portera-Cailliau, et al., 1994, Proceedings of
National Academy of Science (U.S.A.), Vol. 91: 974-978; Buchi, E.
R., 1992, Experimental Eye Research, Vol. 55: 605-613; Quigley, et
al., 1995, Investigative Ophthalmology & Visual Science, Vol.
36: 774-786). Photic induced retinal damage has been observed in
mice (Zigman, et al., 1975, Investigative Ophthalmology &
Visual Science, Vol. 14: 710-713), rats (Noell, et al., 1966,
Investigative Ophthalmology and Visual Science, Vol. 5: 450-473;
Kuwabara, et al., 1968, Archives of ganzfeld. ERGs to a series of
light flashes increasing in intensity were digitized to analyze
temporal characteristics of the waveform and response voltage-log
intensity relationship.
Results:
[0025] Experiment 1: Comparison of betaxolol with its R and S
isomer: Vehicle Dosed Rats. Blue-light exposure for 6 hours
resulted in a significant diminution of the ERG response amplitude
(ANOVA, p<0.001) compared to controls when measured after a
5-day recovery period (FIG. 1). Maximum a-wave and b-wave
amplitudes were reduced approximately 66% in vehicle-dosed rats
compared to controls. In addition, threshold responses were lower
and evoked at brighter flash intensities.
[0026] Betaxolol (racemic). Systemic (IP) dosing with betaxolol
(racemic) provided dose-dependent protection of outer and inner
retina function against this light-induced retinal degeneration in
rats after a 5-day recovery period (FIG. 1). Maximum a-wave
response amplitudes in betaxolol dosed rats with 20 and 40 mg/kg
were 1.9 and 2.1 fold higher, respectively, than vehicle dosed
rats.
[0027] Levobetaxolol (S-isomer). Systemic administration of
levobetaxolol provided dose-dependent protection of outer retina
function when the ERGs were measured 5 days after induction of this
severe photo-oxidative induced retinopathy. Systemic dosing with 20
mg/kg and 40 mg/kg levobetaxolol afforded significant protection of
retinal function to this oxidative insult (FIG. 1). ERG amplitudes
in rats dosed with 20 mg/kg were 69% of normal and twice the
amplitude of vehicle-dosed rats. Complete protection of the retinal
response to a flash of light was measured after a 5-day recovery
period in rats dosed with levobetaxolol (40 mg/kg). This protection
persisted after a 4-week recovery period.
[0028] Betaxolol (R-isomer). Partial but significant protection of
outer and inner retina function against light-induced retinal
degeneration was measured in rats dosed with 20 and 40 mg/kg (FIG.
1). ERGs were approximately 64% of normal in rats dosed (20 or 40
mg/kg) with the R-isomer of betaxolol. This protection persisted
after a 4-week recovery period.
Experiment 2: Prevention of Photic Retinopathy by Timolol
[0029] Five days after blue-light exposure, outer retinal function
in vehicle dosed rats was reduced by 54% and inner retina function
was reduced 52% (FIG. 2). Systemic administration (IP) of timolol
at 10, 20, and 40 mg/kg afforded no significant protection of
retinal function to this photo-oxidative insult (FIG. 2). ERGs
recorded from rats dosed with 80 mg/kg were significantly better
than responses measured in vehicle dosed rats.
Conclusion
[0030] Systemic administration of the .beta.-adrenoceptor
antagonists, betaxolol and its enantiomers, provided dose-dependent
neuroprotection of outer and inner retina function when measured
5-days or 4-weeks after induction of a severe photo-oxidative
induced retinopathy. Significant retinal protection was measured in
rats dosed with these .beta.-adrenoceptor antagonists at 20 and 40
mg/kg. This photic-induced retinopathy was prevented in rats dosed
with levobetaxolol. Timolol, a non-selective .beta.-blocker, was
also effective in reducing the severity of oxidative damage to the
retina as a result of this light exposure.
EXAMPLE 2
[0031] Prevention of Photo-oxidative Induced Retinopathy by Topical
Ocular dosing with Levobetaxolol
[0032] The purpose of this experiment was to determine the degree
of retinal protection that could be measured in rats following
topical ocular dosing. Levobetaxolol (0.5%), (racemic) betaxolol
(0.5%), and vehicle were evaluated in the photic retinopathy model.
Induction of photochemical lesions and evaluation of retinal
function with the ERG were performed as described in the
photo-oxidative induced retinopathy paradigm used in Example 1.
Subjects and Dosing
[0033] Male Sprague Dawley rats were randomly assigned to either a
vehicle dosed group (N=10), (racemic) betaxolol (0.5%) dosed group
(N=10) or levobetaxolol (0.5%) dosed group (N=10). Rats were dosed
topical ocular (b.i.d.) with two drops per eye. Rats were pre-dosed
for 17 days prior to light exposure and dosed an additional two
days after the light exposure. Control rats (N=4) were housed in
their home cage under normal cyclic light exposure.
Results
[0034] Blue-light exposure to vehicle dosed rats resulted in a
significant reduction in retinal function (ANOVA, p<0.004), as
measured by the electroretinogram (ERG), when measured five days
after light exposure (FIG. 3). Maximum a-wave response amplitudes
were reduced by 58% and inner retina function was reduced 56%.
[0035] Topical ocular dosing with levobetaxolol (b.i.d.) provided
significant protection when compared to vehicle dosed rats (FIG.
3). Further, levobetaxolol completely Ophthalmology, Vol. 79:
69-78; LaVail, M. M., 1976, Investigative Ophthalmology &
Visual Science, Vol. 15: 64-70), rabbit (Lawwill, T., 1973,
Investigative Ophthalmology & Visual Science, Vol. 12: 45-51),
and squirrel (Collier, et al., 1989; In LaVail et al., Inherited
and Environmentally Induced Retinal Degenerations. Alan R. Liss,
Inc., New York; Collier, et al., 1989, Investigative Ophthalmology
& Visual Science, Vol. 30: 631-637), non-human primates (Tso,
M. O. M., 1973, Investigative Ophthalmology & Visual Science,
Vol. 12: 17-34; Ham, et al., 1980, Vision Research, Vol. 20:
1105-1111; Sperling, et al., 1980, Vision Research, Vol. 20:
1117-1125; Sykes, et al., 1981, Investigative Ophthalmology &
Visual Science, Vol. 20: 425-434; Lawwill, T., 1982, Transactions
of the American Ophthalmology Society, Vol. 80: 517-577), and man
(Marshall, et al., 1975, British Journal of Ophthalmology, Vol. 59:
610-630; Green, et al., 1991, American Journal of Ophthalmology,
Vol. 112: 520-27). In man, chronic exposure to environmental
radiation has also been implicated as a risk factor for ARMD
(Young, R. W., 1988, Survey of Ophthalmology, Vol. 32: 252-269;
Taylor, et al., 1992, Archives of Ophthalmology, Vol. 110: 99-104;
Cruickshank, et al., 1993, Archives of Ophthalmology, Vol. 111:
514-518).
Systemic Dosing
[0036] The purpose of Experiment 1 was to determine if selective
.beta.-adrenoceptor antagonists, in particular betaxolol (racemic),
levobetaxolol (S-isomer), and betaxolol (R-isomer) are
neuroprotective and can rescue retinal cells from a photo-oxidative
induced retinopathy. The purpose of Experiment 2 was to determine
the dose-dependent efficacy of timolol, a potent non-selective
.beta..sub.1- and .beta..sub.2-blocker, in this photo-oxidative
stress model. Male Sprague Dawley rats were randomly assigned to
drug or vehicle experimental groups. Rats received three
intraperitoneal (IP) injections of either vehicle or drug at 48,
24, and 0 hours prior to a 6-hour light exposure to spectrally
filtered blue light (.about.220 fc). Control rats were housed in
their home cage under normal cyclic light exposure. Control rats
were not dosed with either vehicle or drug. The ERG is a
non-invasive clinical measurement of the electrical response of the
eye to a flash of light. The a-wave and b-wave are two components
of the ERG that are diagnostic of retinal function. The a-wave
reflects outer retina function and is generated by interactions
between photoreceptor and RPE while the b-wave reflects inner
retina function, particularly on-bipolar cells. Although the inner
retina is not significantly damaged by this light exposure, the
b-wave is depressed due to the lack of photoreceptor input. Changes
in the a-wave amplitude or latency are diagnostic of outer retina
pathology. The ERG was recorded after a five day recovery period
from dark-adapted anesthetized rats (ketamine-HCl, 75 mg/Kg;
xylazine, 6 mg/Kg). The eye's electrical response to a flash of
light was elicited by viewing a ameliorated this photic induced
retinopathy as no significant difference in retinal function was
detected between control and levobetaxolol dosed rats.
[0037] No significant protection was measured in betaxolol
(racemic) dosed rats. In betaxolol dosed rats, ERG response
amplitudes were higher but not significantly different than
responses measured from vehicle dosed rats.
EXAMPLE 3
Preservation of Visual Function in Transgenic Rats by
Levobetaxolol
[0038] The P23H rhodopsin mutated transgenic rat has a specific
rhodopsin mutation that has been identified in subsets of patients
with RP. This degeneration is characterized by a slow degeneration
of retinal photoreceptors and marked reduction in the
electroretinogram. As in light damage, photoreceptor loss is
primarily through an apoptotic process.
Methods:
Subjects and Dosing
[0039] At the time of weaning, rats are randomly assigned to either
a drug or vehicle group. Rats were dosed (oral gavage) with vehicle
or levobetaxolol (40 mg/kg,) every other day. This dose was
evaluated based on its ability to completely ameliorate a photic
induced retinopathy. ERGs were recorded as described in Example
1.
Results
[0040] Oral dosing with levobetaxolol (40 mg/kg) every other day
significantly attenuated the loss of retinal function measured in
3- and 6-month old P23H mutant rhodopsin transgenic rats compared
to vehicle dosed rats (FIG. 4). Outer retinal function in 6-month
old rats was 32% better than responses measured in vehicle dosed
rats.
EXAMPLE 4
Upregulation of Retinal Endogenous Neurotrophic Factors by
Betaxolol
[0041] LaVail and others (Faktorovich, et al, Nature, Vol. 347:
83-86, 1990; LeVail, et al., Proceedings of the National Academy of
Science, 1992, Vol. 89: 11249-11253), have shown that intravitreal
injection of a number of growth factors can prevent light damage to
the retina. These neurotrophic factors are large peptides and don't
easily cross the blood-retinal barrier. In terms of a therapeutic
strategy for treatment of chronic degenerative retinal disease,
repeated intravitreal injections potentially present complications,
including hemorrhage, retinal detachment, and inflammation. An
alternative strategy is the use of adenovirus-mediated gene
transfer (bFGF in the RCS rat, Cayouette, et al, Journal of
Neuroscience, Vol. 18(22): 9282-93, 1998, and CNTF in the rd mouse,
Cayouette, et al., Human Gene Therapy, Vol. 8(4): 423-30, 1997),
which has had limited success in preventing photoreceptor loss due
to loss of expression over time and non-homogeneous infection of
cells. We have shown that placement of genetically engineered cells
into the vitreous that secrete CNTF are also effective in
preventing an oxidative induced retinopathy. A recent strategy has
been to identify pharmacologic agents that upregulate endogenous
growth factors. Wen et al, (WO 98/10758, 19 Mar. 1998), have shown
that .alpha..sub.2-adrenoceptor agonists can upregulate bFGF and
prevent photic injury. To determine if a .beta.-adrenergic
antagonist can induce endogenous production of neurotrophic
factors, levobetaxolol was evaluated.
Evaluation of Levobetaxolol:
[0042] Male albino Sprague Dawley rats were given a single IP
injection of either an .alpha..sub.2-adrenoceptor agonist
(brimonidine) (20 mg/kg), a P-adrenergic antagonist (levobetaxolol)
(20 mg/kg), or vehicle and maintained in the dark for 12 hours
prior to harvesting of retinal tissue. Dark-adapted normal control
rats were also evaluated. Endogenous retinal growth factor mRNA
upregulation was determined by Northern blot analysis. Retinas were
flash frozen in liquid nitrogen and stored until isolation of total
RNA. RNA samples were run on a 1.2% agarose gel, transferred to
nylon membranes, prehybridized, hybridized with labeled cDNA probes
for 16 hours, washed, and exposed to X-ray film. The blots were
then stripped and reprobed with an oligo specific for the 18S RNA.
The bands specific for bFGF, CNTF and 18S RNA were scanned in a gel
image scanner and analyzed.
Results
[0043] No difference was observed in the bFGF/18S or CNTF/18S ratio
between vehicle dosed and control rats (FIGS. 5).
[0044] A single dose of brimonidine (20 mg/kg) resulted in a 14
fold increase in bFGF mRNA expression (FIG. 5). However, CNTF mRNA
expression was not upregulated in these rats.
[0045] Similarly, levobetaxolol, a .beta.-adrenergic antagonist,
induced a 13-fold increase in bFGF mRNA expression in rats
receiving a single IP injection (20 mg/kg) (FIG. 5). In addition to
upregulating bFGF in these rodent retinas, endogenous CNTF mRNA
expression was upregulated by a factor of 2.3 compared to
background expression. Treatment with recombinant-CNTF has been
shown to be efficacious in prevention of photic retinopathy and
retinal heredodegenerative change.
Conclusion
[0046] We unexpectedly found that levobetaxolol was a potent
inducer of endogenous bFGF mRNA. Unlike .alpha.-adrenoceptor
agonists, levobetaxolol also resulted in a marked elevation of CNTF
mRNA expression. Further, we have demonstrated that dosing with
levobetaxolol, betaxolol (racemic) or its R-isomer provided
significant protection to the retina when stressed with a severe
photo-oxidative insult. The upregulation of CNTF mRNA is
particularly important in treatment of retinopathy. The efficacy of
CNTF or its analogue in preventing outer retinal degeneration has
been demonstrated in the rat and mouse phototoxicity model, RCS
dystrophic rat, Rdy cat suffering a rod-cone dystrophy, retinal
degeneration canine model, transgenic rat (P23H and Q344ter),
transgenic mouse (Q344ter), rd mouse and rds mouse. On the other
hand, bFGF has only demonstrated efficacy in the rat and mouse
phototoxicity model and RCS dystrophic rat.
[0047] Based on these novel findings we conclude that
.beta.-adrenoceptor antagonists, in particular levobetaxolol and
betaxolol, are neuroprotective in transgenic rat and
photo-oxidative stress models (FIGS. 1, 2, 3, and 4) and would be
effective in the treatment of various ophthalmic degenerative
diseases of the outer retina. Neuroprotection may be afforded by
upregulation of endogenous neurotrophic factors, including, CNTF
and bFGF (FIG. 5).
EXAMPLE 5
Levobetaxolol Hydrochloride Formulations
[0048] TABLE-US-00001 Concentration 0.25% 0.5% 0.75% Ingredient
Percent w/v Percent w/v Percent w/v Levobetaxolol hydrochloride
0.28.sup.a 0.56.sup.b 0.84.sup.c Poly(styrene 0.375 0.75 1.125
divinylbenzene) Sulfonic Acid Carbomer 974 P 0.35 0.35 0.35
Mannitol 4.5 4.0 3.67 Boric Acid 0.3 0.3 0.3 Disodium Edetate 0.01
0.01 0.01 Benzalkonium Chloride 0.01 + 5% excess.sup.d 0.01 + 5%
excess.sup.d 0.01 + 5% excess.sup.d N-Lauroylsarcosine 0.03 0.03
0.03 Tromethamine pH adjust to 6.5 pH adjust to 6.5 pH adjust to
6.5 Hydrochloric Acid 6.5 .+-. 0.2 6.5 .+-. 0.2 6.5 .+-. 0.2 (if
needed) Purified Water qs 100% qs 100% qs 100% .sup.aEquivalent to
0.25% betaxolol free base .sup.bEquivalent to 0.5% betaxolol free
base .sup.cEquivalent to 0.75% betaxolol free base .sup.dThe 5%
excess is added as an overage
[0049] TABLE-US-00002 Betoptic .RTM. S Ophthalmic Betaxolol
Ophthalmic Ingredient Suspension, 0.25% Suspension Racemic
Betaxolol 0.28 + 5% xs 0.28 Poly(styrene divinylbenzene 0.25 0.25
Sulfonic Acid) Carbomer 974P 0.2 0.45 Mannitol 4.5 4.5 Boric Acid
-- 0.4 Edetate Disodium 0.01 0.01 Benzalkonium Chloride 0.01 + 10%
excess 0.01 + 5% excess N-Lauroylsarcosine -- 0.03 Tromethamine
and, if needed, Adjust pH 7.6 .+-. 0.2 Adjust pH 7.0 .+-. 0.2
Hydrochloric Acid Purified Water qs 100 qs 100
* * * * *