U.S. patent application number 12/719152 was filed with the patent office on 2010-07-01 for compounds with 5-ht1a activity useful for treating disorders of the outer retina.
This patent application is currently assigned to ALCON, INC.. Invention is credited to Robert J. Collier, JR., Thomas R. Dean, Mark R. Hellberg, Michael A. Kapin.
Application Number | 20100168121 12/719152 |
Document ID | / |
Family ID | 29270198 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100168121 |
Kind Code |
A1 |
Collier, JR.; Robert J. ; et
al. |
July 1, 2010 |
COMPOUNDS WITH 5-HT1A ACTIVITY USEFUL FOR TREATING DISORDERS OF THE
OUTER RETINA
Abstract
Compositions and methods for treating disorders of the outer
retina with compounds with 5-HT.sub.1A agonist activity are
disclosed.
Inventors: |
Collier, JR.; Robert J.;
(Arlington, TX) ; Kapin; Michael A.; (Arlington,
TX) ; Hellberg; Mark R.; (Arlington, TX) ;
Dean; Thomas R.; (Weatherford, TX) |
Correspondence
Address: |
ALCON
IP LEGAL, TB4-8, 6201 SOUTH FREEWAY
FORT WORTH
TX
76134
US
|
Assignee: |
ALCON, INC.
Hunenberg
CH
|
Family ID: |
29270198 |
Appl. No.: |
12/719152 |
Filed: |
March 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11187474 |
Jul 22, 2005 |
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12719152 |
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10221070 |
Sep 9, 2002 |
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PCT/US2001/005700 |
Feb 23, 2001 |
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11187474 |
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60190279 |
Mar 17, 2000 |
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Current U.S.
Class: |
514/252.15 ;
514/252.14; 514/252.18; 514/252.19; 514/254.04; 514/254.06;
514/254.11; 514/277; 514/278; 514/337; 514/373; 514/657 |
Current CPC
Class: |
A61K 31/496 20130101;
Y10S 514/912 20130101; A61P 27/02 20180101; A61K 31/519 20130101;
A61K 31/506 20130101 |
Class at
Publication: |
514/252.15 ;
514/252.14; 514/254.04; 514/252.19; 514/373; 514/277; 514/254.11;
514/252.18; 514/278; 514/254.06; 514/337; 514/657 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 31/496 20060101 A61K031/496; A61K 31/428 20060101
A61K031/428; A61K 31/4418 20060101 A61K031/4418; A61K 31/438
20060101 A61K031/438; A61K 31/4433 20060101 A61K031/4433; A61K
31/135 20060101 A61K031/135; A61P 27/02 20060101 A61P027/02 |
Claims
1. A method for treating disorders of the outer retina which
comprises administering a pharmaceutically effective amount of a
compound with 5-HT.sub.1A agonist activity.
2. The method of claim 1, wherein the compound is selected from the
group consisting of: tandospirone, urapidil, ziprasidone, repinotan
hydrochloride, xaliproden hydrochloride (SR-57746A), buspirone,
flesinoxan, EMD-68843, DU-127090, gepirone, alnespirone, PNU-95666,
AP-521, flibanserin, MKC-242, lesopitron, sarizotan hydrochloride,
E-5842, SUN-N4057, Org-13011, Org-12966 and 8-OH-DPAT.
3. The method of claim 1 wherein the disorder is selected from the
group consisting of: AMD; RP and other forms of heredodegenerative
retinal disease; retinal detachment and tears; macular pucker;
ischemia affecting the outer retina; diabetic retinopathy; 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.
4. The method of claim 3 wherein the disorder is AMD.
5. The method of claim 3 wherein the compound is selected from the
group consisting of: tandospirone, urapidil, ziprasidone, repinotan
hydrochloride, xaliproden hydrochloride (SR-57746A), buspirone,
flesinoxan, EMD-68843, DU-127090, gepirone, alnespirone, PNU-95666,
AP-521, flibanserin, MKC-242, lesopitron, sarizotan hydrochloride,
E-5842, SUN-N4057, Org-13011, Org-12966 and 8-OH-DPAT.
6. The method of claim 5 wherein the disorder is selected from the
group consisting of AMD, RP, and diabetic retinopathy.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/187,474 filed Jul. 22, 2005, which claims
benefit to patent application Ser. No. 10/221,070 filed Sep. 9,
2002, which is a 371 of PCT/US01/05700 filed Feb. 23, 2001, which
claims benefit of U.S. Ser. No. 60/190,279 filed Mar. 17, 2000.
[0002] The present invention is directed to compounds with
5-HT.sub.1A agonist activity useful for treating disorders of the
outer retina resulting from acute or chronic degenerative
conditions or diseases of the eye.
BACKGROUND OF THE INVENTION
[0003] Age-related macular degeneration (AMD) is the leading cause
of blindness in the elderly, with an incidence of about 20% in
adults 65 years of age increasing to 37% in individuals 75 years or
older. Non-exudative AMD is characterized by drusen accumulation
and atrophy of rod and cone photoreceptors in the outer retina,
retinal pigment epithelium (RPE), Bruch's membrane and
choriocapillaris; while exudative AMD leads to choroidal
neovascularization (Green and Enger, Ophthalmol, 100:1519-35, 1993;
Green et al., Ophthalmol, 92:615-27, 1985; Green and Key, Trans Am
Ophthalmol Soc, 75:180-254, 1977; Bressler et al., Retina,
14:130-42, 1994; Schneider et al., Retina, 18:242-50, 1998; Green
and Kuchle (1997). In: Yannuzzi, L. A., Flower, R. W., Slakter, J.
S. (Eds.) Indocyanine green angiography. St. Louis: Mosby, p.
151-6). Retinitis pigmentosa (RP) represents a group of hereditary
dystrophies characterized by rod degeneration with secondary
atrophy of cone photoreceptors and underlying pigment epithelium.
(Pruett, Trans Am Ophthalmol Soc, 81:693-735, 1983; Heckenlively,
Trans Am Ophthalmol Soc, 85:438-470, 1987; Pagon, Sur Ophthalmol,
33:137-177, 1988; Berson, Invest Ophthalmol Vis Sci, 34:1659-1676,
1993; Nickells and Zack, Ophthalmic Genet, 17:145-65, 1996). The
pathogenesis of retinal degenerative diseases such as AMD and RP is
multifaceted and can be triggered by environmental factors in
normal individuals or in those who are genetically predisposed. To
date more than 100 genes have been mapped or cloned that may be
associated with various outer retinal degenerations.
[0004] Light exposure is an environmental factor that has been
identified as a contributing factor to the progression of retinal
degenerative disorders such as AMD (Young, Sur Ophthal, 32:252-269,
1988; Taylor, et al., Arch Ophthal, 110:99-104, 1992; Cruickshank,
et al., Arch Ophthal, 111:514-518, 1993). 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, the same
cells that are affected in heredodegenerative diseases (Noell et
al., Invest Ophthal Vis Sci, 5, 450-472, 1966; Bressler et al., Sur
Ophthal, 32, 375-413, 1988; Curcio et al., Invest Ophthal Vis Sci,
37, 1236-1249, 1996); apoptosis is the cell death mechanism by
which photoreceptor and RPE cells are lost in AMD and RP, as well
as following a photo-oxidative induced cell injury (Ge-Zhi et al.,
Trans AM Ophthal Soc, 94, 411-430, 1996; Abler et al., Res Commun
Mol Pathol Pharmacol, 92, 177-189, 1996; Nickells and Zack,
Ophthalmic Genet, 17:145-65, 1996); light has been implicated as an
environmental risk factor for progression of AMD and RP (Taylor et
al., Arch Ophthalmol, 110, 99-104, 1992; Naash et al., Invest
Ophthal Vis Sci, 37, 775-782, 1996); 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., Proc Nat Acad Sci, 89, 11249-11253, 1992;
Fakforovich et al., Nature, 347, 83-86, 1990; Frasson et al., Nat.
Med. 5, 1183-1187, 1990).
[0005] A number of different compound classes have been identified
in various animal models that minimize retinal photo-oxidative
injury. They include: antioxidants such as ascorbate (Organisciak
et al., Invest Ophthal Vis Sci, 26:1589-1598, 1985),
dimethylthiourea (Organisciak et al., Invest Ophthal Vis Sci,
33:1599-1609, 1992; Lam et al., Arch Ophthal, 108:1751-1752, 1990),
.alpha.-tocopherol (Kozaki et al., Nippon Ganka Gakkai Zasshi,
98:948-954, 1994) and .beta.-carotene (Rapp et al., Cur Eye Res,
15:219-232, 1995); calcium antagonists such as flunarizine (Li et
al., Exp Eye Res, 56: 71-78, 1993; Edward et al., Arch Ophthal,
109, 554-622, 1992; Collier et al., Invest Ophthal Vis Sci,
36:S516); growth factors such as basic-fibroblast growth factor,
brain derived nerve factor, ciliary neurotrophic factor, and
interleukin-1-.beta. (LaVail et al., Proc Nat Acad Sci, 89,
11249-11253, 1992); glucocorticoids such as methylprednisolone (Lam
et al., Graefes Arch Clin Exp Ophthal, 231, 729-736, 1993) and
dexamethasone (Fu et al., Exp Eye Res, 54, 583-594, 1992); iron
chelators such as desferrioxamine (Li et al., Cur Eye Res, 2,
133-144, 1991); NMDA-antagonists such as eliprodil and MK-801
(Collier et al., Invest Ophthal Vis Sci, 40:S159, 1999).
[0006] Serotonergic 5-HT.sub.1A agonists (i.e., buspirone,
ziprasidone, urapidil) have either been registered or launched for
the treatment of anxiety, hypertension, schidzophrenia, psychosis
or depression-bipolar disorders. In addition, 5-HT.sub.1A agonists
have been shown to be neuroprotective in various animal models and
are being evaluated in the clinic to treat cerebral ischemia, head
trauma, Alzheimer's Disease, Multiple Sclerosis and amytrophic
lateral sclerosis. The 5-HT.sub.1A agonists, 8-OH-DPAT
(8-hydroxy-2-(di-n-propylamino)tetralin) and ipsapirone, were shown
to prevent NMDA-induced excitotoxic neuronal damage in the rat
magnocellular nucleus basalis (Oosterink et al., Eur J Pharmacol,
358:147-52, 1998), dosing with Bay-x-3702 significantly reduced
ischemic damage in a rat acute subdermal hematoma model (Alessandri
et al., Brain Res, 845:232-5, 1999), while 8-OH-DPAT, Bay-x-3702,
urapidil, gepirone and CM 57493 significantly reduced cortical
infarct volume in the rat (Bielenberg and Burkhardt, Stroke,
21(Suppl): IV161-3; Semkova et al., Eur J Pharmacol, 359:251-60,
1998; Peruche et al., J Neural Transm--Park Dis Dement Sect,
8:73-83, 1994) and mouse (Prehn et al., Eur J Pharmacol,
203:213-22, 1991; Prehn et al., Brain Res, 630:10-20, 1993) after
occlusion of the middle cerebral artery. In addition, treatment of
rats with SR 57746A, a potent 5-HT.sub.1A agonist, has been shown
to be neuroprotective following 4-vessel transient global ischemia,
vincristine sulphate induced septohippocampal lesions,
acrylamide-induced peripheral neuropathy, and sciatic nerve crush
(Fournier et al., Neurosci, 55:629-41, 1993) and has been shown to
delay the progression of motor neuron degeneration in pmn mice
(Fournier et al., Br J Pharmacol, 124:811-7, 1998).
[0007] This class of compounds has been disclosed for the treatment
of glaucoma (lowering and controlling IOP), see e.g., WO 98/18458
(DeSantis, et al) and EP 0771563A2 (Mano, et al.). Osborne, et al.
(Ophthalmologica, Vol. 210:308-314, 1996) teach that
8-hydroxydipropylaminotetralin (8-OH-DPAT) (a 5-HT.sub.1A agonist)
reduces IOP in rabbits. Wang, et al. (Current Eye Research, Vol.
16(8):769-775, August 1997, and IVOS, Vol. 39(4), 5488, March,
1998) disclose that 5-methylurapidil, an .alpha..sub.1A antagonist
and 5-HT.sub.1A agonist lowers IOP in the monkey, but due to its
.alpha..sub.1A receptor activity. Also, 5-HT.sub.1A antagonists are
disclosed as being useful for the treatment of glaucoma (elevated
IOP) (e.g. WO 92/0338, McLees). Furthermore, DeSai, et al. (WO
97/35579) and Macor, et al. (U.S. Pat. No. 5,578,612) disclose the
use of 5-HT.sub.1 and 5-HT.sub.1-like agonists for the treatment of
glaucoma (elevated IOP). These anti-migraine compounds are
5-HT.sub.1B,D.E,F agonists, e.g., sumatriptan and naratriptan and
related compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A and 1B show the preservation of the ERG a- and
b-wave function in rats dosed systemically with 8-OH-DPAT and
exposed to a severe photo-oxidative insult.
[0009] FIG. 2 shows protection of retinal morphology
(photoreceptors and RPE) in rats dosed systemically with 8-OH-DPAT
and exposed to a severe photo-oxidative insult.
[0010] FIG. 3 shows protection of retinal DNA, a measure of retinal
cell number (A), and complete protection of retinal morphology
(photoreceptors) in rats dosed systemically with buspirone and
exposed to a severe photo-oxidative insult.
[0011] FIGS. 4A and 4B show the preservation of the ERG a- and
b-wave function in rats dosed systemically with SR-57746A and
exposed to a severe photo-oxidative insult.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to 5-HT.sub.1A agonists
which have been discovered to be useful in treating disorders of
the outer retina, particularly: AMD; RP and other forms of
heredodegenerative retinal disease; retinal detachment and tears;
macular pucker; ischemia affecting the outer retina; diabetic
retinopathy; 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.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0013] Serotonergic 5-HT.sub.1A agonists have been shown to be
potent neuroprotective agents following varying insults to the
central nervous system. Unexpectedly, we have demonstrated that
8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), buspirone and
SR-57746A exhibit potent neuroprotective activity in the retina and
prevent light-induced apoptotic cell death to photoreceptors and
RPE cells. We have found that treatment with buspirone can
completely prevent photo-oxidative induced retinopathy and
significantly reduce loss of retinal DNA and ONL thinning. The
safety advantages of some of these compounds make them 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 5-HT.sub.1A agonists are
100-fold more potent in these light damage paradigms and therefore
are useful for treating disorders of the outer retina.
[0015] The invention contemplates the use of any pharmaceutically
acceptable 5-HT.sub.1A agonist, including pharmaceutically
acceptable salts, for treating disorders of the outer retina
(Compounds). Pharmaceutically acceptable means the Compounds can be
safely used for the treatment of diseases of the outer retina. 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.
[0016] 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 limited to, AMD, RP and other forms of heredodegenerative
retinal disease, retinal detachment, tears, macular pucker,
ischemia affecting the outer retina, diabetic retinopathy, 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.
[0017] Compounds of the present invention have potent affinity for
5-HT.sub.1A receptors with IC.sub.50 values that range up to about
500 nM (preferably less than 100 nM). These Compounds are also
either full or partial agonists with IC.sub.50 values ranging up to
about 1 .mu.M (preferably less than 500 nM). Representative
5-HT.sub.1A agonists useful according to the present invention
include, but are not limited to: tandospirone, urapidil,
ziprasidone, repinotan hydrochloride, xaliproden hydrochloride
(SR-57746A), buspirone, flesinoxan, EMD-68843, DU-127090, gepirone,
alnespirone, PNU-95666, AP-521, flibanserin, MKC-242, lesopitron,
sarizotan hydrochloride, Org-13011, Org-12966, E-5842, SUN-N4057,
and 8-OH-DPAT.
[0018] Receptor binding and agonist activity according to this
invention can be determined using the following methods.
Method 1
5-HT.sub.1A Receptor Binding Assay
[0019] 5-HT.sub.1A binding studies were performed with human cloned
receptors expressed in Chinese hamster ovary (CHO) cells using
(.sup.3H)8-OH DPAT as the ligand. Membranes from Chinese hamster
ovary cells (CHO) expressing cloned 5-HT.sub.1A receptors
(manufactured for NEN by Biosignal, Inc., Montreal, Canada) were
homogenized in approximately 40 volumes of 50 mM Tris pH 7.4 for 5
sec. Drug dilutions were made using a Beckman Biomek 2000 robot
(Beckman Instruments, Fullerton, Calif.). Incubations were
conducted with membrane prep, test compounds, and 0.25 nM
[.sup.3H]8-OH-DPAT (NEN, Boston, Mass.) in the same buffer at
27.degree. C. for 1 h. Assays were terminated by rapid vacuum
filtration over Whatman GF/B glass fiber filters pre-soaked in 0.3%
polyethyleneimine. Bound radioactivity was measured using liquid
scintillation spectrometry. Data were analyzed using non-linear
curve fitting programs (Sharif et al., J Pharmac Pharmacol, 51:
685-694, 1999).
[0020] Ligand binding studies can also be run using membrane
preparations from calf and rat brain (local source) and human
cortex membranes. Specific brain regions were dissected out,
homogenized in 10 volumes of 0.32 M sucrose and centrifuged for 10
min at 700.times.g. The resulting supernatant was centrifuged at
43,500.times.g for 10 min and the pellet re-suspended in 50 mM
Tris-HCl (pH 7.7, 25.degree. C.) using a 10 sec polytron treatment.
Aliquots were stored at -140.degree. C. To remove endogenous
serotonin, the preps were incubated at 37.degree. C. for 10 min
prior to the experiment. Assay incubations were terminated by rapid
filtration over Whatman GF/C filters using a Brandel cell
harvester. K.sub.i values were calculated using the Cheng-Prusoff
equation (De Vry et al., J Pharm Exper Ther, 284:1082-1094,
1998.)
Method 2
5-HT.sub.1A Functional Assays
[0021] The function of Compounds of the present invention can be
determined using a variety of methods to assess the functional
activity of 5-HT.sub.1A agonists. One such assay is performed using
hippocampal slices from male Sprague-Dawley rats, measuring the
inhibition of forskolin-stimated adenylate cyclase (J Med Chem,
42:36, 1999; J Neurochem, 56:1114, 1991; J Pharm Exper Ther,
284:1082, 1998). Rat hippocampal membranes were homogenized in 25
volumes of 0.3 M sucrose containing 1 mM EGTA, 5 mM EDTA, 5 mM
dithiothreitol, and 20 mM Tris-HCl, pH 7.4 at 25.degree. C. The
homogenate was centrifuged for 10 m in at 1,000.times.g. The
supernatant subsequently was centrifuged at 39,000.times.g for 10
min. The resulting pellet was re-suspended in homogenization buffer
at a protein concentration of approximately 1 mg/ml and aliquots
were stored at -140.degree. C. Prior to use, the membranes were
rehomogenized in a Potter-Elvehjem homogenizer. Fifty .mu.l of the
membrane suspension (50 .mu.g protein) were added to an incubation
buffer containing 100 mM NaCl, 2 mM magnesium acetate, 0.2 mM ATP,
1 mM cAMP, 0.01 mM GTP, 0.01 mM forskolin, 80 mM Tris-HCl, 5 mM
creatine phosphate, 0.8 U/.mu.l creatine phosphokinase, 0.1 mM
IBMX, 1-2 .mu.Ci .alpha.-[.sup.32P]ATP. Incubations with test
compounds (10 min at 30.degree. C.) were initiated by the addition
of the membrane solution to the incubation mixture (prewarmed 5 min
at 30.degree. C.). [.sup.32P]cAMP was measured according to the
method of Salomon (Adv Cyclic Nucleotide Res, 10:35-55, 1979).
Protein was measured using the Bradford (Anal Biochem, 72:248-254,
1976) assay.
[0022] Functional activity can also be determined in recombinant
human receptors according to the method of Schoeffter et al.
(Neuropharm, 36:429-437, 1997). HeLa cells transfected with
recombinant human 5-HT.sub.1A receptors were grown to confluence in
24-well plates. The cells were rinsed with 1 ml of Hepes-buffered
saline (in mM) NaCl 130, KCl 5.4, CaCl.sub.2 1.8, MgSO.sub.4 0.8,
NaH.sub.2PO.sub.4 0.9, glucose 25, Hepes 20, pH 7.4, and phenol red
5 mg/l. The cells were labelled with 6 .mu.Ci/ml of
[.sup.3H]adenine (23 Ci/mmol, Amersham, Rahn AG, Zurich,
Switzerland) in 0.5 ml of saline at 37.degree. C. for 2 hr. The
plates were subsequently rinsed twice with 1 ml of buffered saline
containing 1 mM isobutylmethylxanthine. The cells were incubated
for 15 min in 1 ml of this solution (37.degree. C.) in the presence
or absence of 10 .mu.M forskolin and the test compound. The buffer
was then removed and 1 ml of 5% trichloroacetic acid (TCA)
containing 0.1 mM cAMP and 0.1 mM ATP was added to extract the
samples. After 30 min at 4.degree. C., the TCA extracts were
subjected to chromatographic separation on Dowex AG 50W-X4 and
alumina columns (Salomon, Meth Enzymol, 195: 22-28, 1991). Cyclic
AMP production was calculated as the ratio
[.sup.3H]cAMP/([.sup.3H]cAMP+[.sup.3H]ATP).
[0023] The above procedures described in Methods 1 and 2 were used
to generate the following data.
TABLE-US-00001 TABLE 1 5-HT.sub.1A Receptor Binding and Functional
Assay Data. Receptor Binding Compound (IC.sub.50 nM, SEM) cAMP
Inhibition (EC.sub.50) (R,S) 8-OH-DPAT 1.5 nM 4.7 nM (R) 8-OH-DPAT
0.5 nM 2.6 nM SR-57746A 2.5 nM 3.7 nM
[0024] In general, for degenerative diseases, the 5-HT.sub.1A
agonists of this invention are s administered orally with daily
dosage of these compounds ranging between about 0.001 and about 500
milligrams. The preferred total daily dose ranges between about 1
and about 100 milligrams. Non-oral administration, such as,
intravitreal, topical ocular, transdermal patch, subdermal,
parenteral, intraocular, subconjunctival, or retrobulbar or
subtenon's injection, trans scleral (including 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 5-HT.sub.1A
agonists can also be delivered in ocular irrigating solutions.
Concentrations should range from about 0.001 .mu.M to about 100
.mu.M, preferably about 0.01 .mu.M to about 5 .mu.M.
[0025] The 5-HT.sub.1A agonists can be incorporated into various
types of ophthalmic formulations for delivery to the eye (e.g.,
topically, intracamerally, or via an implant). 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.
[0026] If dosed topically, the 5-HT.sub.1A agonists are preferably
formulated as topical ophthalmic suspensions or solutions, with a
pH of about 4 to 8. The 5-HT.sub.1A agonists 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.
[0027] The following topical ophthalmic formulations are useful
according to the present invention administered 1-4 times per day
according to the discretion of a skilled clinician.
Example 1
TABLE-US-00002 [0028] Ingredients Amount (wt %) Buspirone 0.01-2%
Hydroxypropyl methylcellulose 0.5% Dibasic sodium phosphate
(anhydrous) 0.2% Sodium chloride 0.5% Disodium EDTA (Edetate
disodium) 0.01% Polysorbate 80 0.05% Benzalkonium chloride 0.01%
Sodium hydroxide/Hydrochloric acid For adjusting pH to 7.3-7.4
Purified water q.s. to 100%
Example 2
TABLE-US-00003 [0029] Ingredients Amount (wt %) Buspirone 0.01-2%
Methyl cellulose 4.0% Dibasic sodium phosphate (anhydrous) 0.2%
Sodium chloride 0.5% Disodium EDTA (Edetate disodium) 0.01%
Polysorbate 80 0.05% Benzalkonium chloride 0.01% Sodium
hydroxide/Hydrochloric acid For adjusting pH to 7.3-7.4 Purified
water q.s. to 100%
Example 3
TABLE-US-00004 [0030] Ingredients Amount (wt %) Compound 0.01-2%
Guar gum 0.4-6.0% Dibasic sodium phosphate (anhydrous) 0.2% Sodium
chloride 0.5% Disodium EDTA (Edetate disodium) 0.01% Polysorbate 80
0.05% Benzalkonium chloride 0.01% Sodium hydroxide/Hydrochloric
acid For adjusting pH to 7.3-7.4 Purified water q.s. to 100%
Example 4
TABLE-US-00005 [0031] Ingredients Amount (wt %) Xaliproden
hydrochloride 0.01-2% White petrolatum and mineral oil and lanolin
Ointment consistency Dibasic sodium phosphate (anhydrous) 0.2%
Sodium chloride 0.5% Disodium EDTA (Edetate disodium) 0.01%
Polysorbate 80 0.05% Benzalkonium chloride 0.01% Sodium
hydroxide/Hydrochloric acid For adjusting pH to 7.3-7.4
Example 5
TABLE-US-00006 [0032] 10 mM IV Solution w/v % Buspirone 0.384%
L-Tartaric acid 2.31% Sodium hydroxide pH 3.8 Hydrochloric acid pH
3.8 Purified water q.s. 100%
Example 6
TABLE-US-00007 [0033] 5 mg Capsules mg/capsule Ingredient (Total
Wt. 22a mg) Buspirone Hydrochloride 5 Lactose, anhydrous 55.7
Strach, Sodium carboxy-methyl 8 Cellulose, microcrystalline 30
Colloidal silicon dioxide .5 Magnesium sterage .8
Method 3
Neuroprotective Effects in the Rat Photo-Oxidative Induced
Retinopathy Model
[0034] The retinal protective effect of these 5-HT.sub.1A agonists
were evaluated in our photo-oxidative induced retinopathy
paradigm.
[0035] Induction of Photochemical Lesion. Photochemical lesions
were induced in dark adapted rats (24 hour) by exposure to (220 fc)
blue light (half-amplitude bandpass=435-475 nm) for 6 hours.
Animals were allowed to recover for 5 days in darkness prior to
electrodiagnostic evaluation of retinal function. Rats were single
housed in clear polycarbonate cages during this light exposure.
[0036] Electrodiagnostic Evaluation. The electroretinogram (ERG)
was recorded from anesthetized rats after a 24-hour dark-adaptation
period. Rats were anesthetized by IP injection with Ketamine-HCl
(75 mg/Kg) and Xylazine (6 mg/Kg). Flash ERGs recorded from a
platinum-iridium wire loop electrode positioned on the cornea were
elicited by viewing a ganzfeld. Electrical responses to a series of
light flashes increasing in intensity were digitized to analyze
temporal characteristics of the waveform and response voltage-log
intensity (VlogI) relationship. Changes in the ERG a-wave are
associated with photoreceptor and retinal pigment epithelium damage
while damage to the inner retina is reflected in changes in the ERG
b-wave.
[0037] Assessment of Retinal Morphology. Ocular tissues were
obtained from control and drug or vehicle dosed rats and fixed by
immersion into a mixture of 2% paraformaldehyde and 2%
glutaraldehyde. Fixed eyeballs were dehydrated in an ascending
ethanol series, embedded in JB-4 plastic resin, and 1 to 1.5-micron
thick sections were analyzed using a quantitative computer image
analysis system attached to the microscope. Retinal layer thickness
(retinal pigment epithelium, RPE; outer nuclear layer thickness,
ONL; inner nuclear layer thickness, INL; and length of
photoreceptor inner and outer segments, IS+OS) was measured.
[0038] Assessment of DNA Chances. Albino rats were euthanized by
CO.sub.2 inhalation and individual retinas were frozen in separate
tubes. Each retina had been sonicated in 0.8 ml (2.0 M NaCl, 50 mM
NaPO.sub.4, pH 7.4, 2 mM EDTA) to yield a uniform homogenate, and
stored frozen. Aliquots (0.1 ml) of each sample were diluted
10-fold with 2.0 M NaCl, 50 mM NaPO.sub.4. pH 7.4, 2 mM EDTA
containing 1.1 .mu.g/ml bisbenzimidazole (Hoechst 33258). A
standard curve was constructed using calf thymus DNA from 0 to 25
.mu.g/ml in the same buffer. Triplicate 0.2 ml aliquots of each
retina sample and standard were pipetted into a 96 well plate for
fluorescence measurements in the Cytofluor II. The excitation
wavelength was 360 nm, and the emission wavelength was 460 nm.
[0039] Subjects and Dosing. Male Sprague Dawley rats were randomly
assigned to drug and vehicle experimental groups. Control rats were
housed in their home cage under normal cyclic light exposure. All
rats were dosed 48, 24 and 0 hours prior to a 6-hour blue-light
exposure. Dosing was as follows:
[0040] 1.) 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin): Rats
receiving either vehicle (N=10) or 8-OH-DPAT (0.5 mg/kg [N=5] or
1.0 mg/kg [N=10]) were given three subcutaneous (SC) injections
prior to light exposure. Five rats were used as controls. Retinal
protection was assessed by analyzing the ERG response and measuring
changes in retinal morphology.
[0041] 2.) Buspirone: For DNA quantitation, six rats per treatment
group were dosed IP with vehicle or buspirone (0.5 and 1 mg/kg)
prior to light exposure. Retinas from seven normal rats were used
as controls. To evaluate changes in retinal morphology, rats were
dosed (IP) with either vehicle (N=8) or buspirone (1.0 mg/kg
[N=9]). Six rats were used as controls. Retinal protection was
assessed by quantitating changes in retinal DNA and measuring
changes in retinal morphology.
[0042] 3.) SR-57746A: Rats were dosed (IP) with vehicle (N=15) or
SR-57746A (0.5 mg/kg [N=5] or 1 mg/kg [N=15]). Eleven rats were
used as controls. The ERG was analyzed after a 5-day recovery
period to assess retinal protection.
[0043] 8-OH-DPAT Evaluation Results. Blue-light exposure for 6
hours resulted in a significant diminution of the ERG response
amplitude (ANOVA, p<0.001; Bonferroni t-test, p<0.05)
compared to normals when measured after a 5-day recovery period
(FIGS. 1A and B). Blue-light exposure resulted in a 75% reduction
in the maximum a- and b-wave amplitudes in vehicle dosed rats
compared to controls. In addition, threshold responses were lower
and evoked at brighter flash intensities.
[0044] Rats dosed with 8-OH-DPAT showed dose-dependent protection
of outer and inner retina function against this photo-oxidative
induced retinopathy (FIGS. 1A and B). Maximum a- and b-wave
response amplitudes in 8-OH-DPAT (0.5 mg/kg) dosed rats were not
different than vehicle dosed rats and were approximately 27% of
control amplitudes. However, maximum a- and b-wave response
amplitudes from 8-OH-DPAT (1.0 mg/kg) dosed rats were approximately
53% and 61% of normal, respectively, and significantly higher than
responses measured in vehicle dosed rats (FIGS. 1A and 1B).
[0045] Consistent with these ERG changes, morphometric analysis of
these retinas after a 3-week recovery period demonstrated a
significant (ANOVA, p<0.01) loss of photoreceptor cells,
shortening of photoreceptor inner+outer segment length, and
flattening of the RPE in vehicle dosed animals. No significant
changes in the thickness of the INL were detected. ONL thickness
was reduced 73%, inner+outer segment length was reduced 82%, and
RPE thickness was reduced 59% compared to controls (FIG. 2).
Lesions observed in rats dosed with 8-OH-DPAT (0.5 mg/kg) were not
significantly different than lesions measured in vehicle dosed
rats. While ERGs were reduced approximately 63%, the ONL thickness
was reduced by 53%, photoreceptor segment length was reduced 60%,
and the RPE thickness was reduced 34%. However, photic lesions
observed in rats dosed with 8-OH-DPAT (1.0 mg/kg) were
significantly different from vehicle dosed rats. While ERG response
amplitudes were greater than 50% of normal, the ONL thickness was
2.4 fold thicker, photoreceptor segment length was 2.9 times
longer, and RPE thickness was 1.9 times thicker compared to vehicle
dosed rats.
[0046] Buspirone Evaluation Results. As seen in FIG. 3A, vehicle
dosed retinal DNA levels were significantly reduced (ANOVA,
p=0.017) about 30% from control levels. No significant differences
were measured between groups dosed with vehicle or 0.1 mg/kg
buspirone. Retinal protection was measured in rats dosed with
buspirone (1 mg/kg). Retinal DNA levels were significantly higher
than measured in vehicle dosed rats, but not significantly
different than controls.
[0047] Blue-light exposure for 6 hours resulted in a significant
reduction in photoreceptor number (ANOVA, p<0.05). Morphometric
analysis of these retinas after a 4-week recovery period
demonstrated a 54% thinning of the outer nuclear layer in vehicle
dosed rats compared to controls (FIG. 3B). However, no significant
difference in ONL thickness was measured between normal and
buspirone treated rats. In rats dosed with buspirone (1 mg/kg) the
ONL thickness was 28.3.mu. compared to 30.4.mu. in normal rats.
[0048] SR-57746A Evaluation Results. Significant protection of
retinal function was measured in light-exposed rats dosed with
SR-57746A (0.5 and 1.0 mg/kg). Maximum a- and b-wave response
amplitudes were reduced by 50% in vehicle dosed rats compared to
controls (FIGS. 4A and B). Maximum responses were 82% of controls
in rats dosed with SR-57746A (0.5 mg/kg) and 70% of normal in rats
dosed with 1 mg/kg.
[0049] Conclusion. These 5-HT.sub.1A agonists (8-OH-DPAT,
buspirone, and SR-57746A) demonstrated good potency and efficacy in
this oxidative model of retinal degenerative disease. Functional
and structural protection were achieved in rats dosed on three
consecutive days with a dose as low as 1 mg/kg.
* * * * *