WO1999007353A1

WO1999007353A1 - USE OF α1A-ADRENERGIC RECEPTOR ANTAGONISTS IN GLAUCOMA THERAPY

Info

Publication number: WO1999007353A1
Application number: PCT/US1998/015652
Authority: WO
Inventors: Steven M. Podos; Thomas W. Mittag
Original assignee: The Mount Sinai School Of Medicine Of The City University Of New York
Priority date: 1997-08-05
Filing date: 1998-07-29
Publication date: 1999-02-18
Also published as: AU8667898A

Abstract

The present invention relates to the identification and use of agents which act as selective antagonists at the α1A-adrenergic receptor to decrease intraocular pressure.

Description

USE OF αl A-ADRENERGIC RECEPTOR ANTAGONISTS IN GLAUCOMA THERAPY

SPECIFICATION

INTRODUCTION The present invention relates to the use of agents which act as specific antagonists of the αl A adrenergic receptor to decrease intraocular pressure in subjects in need of such treatment, for example, in patients suffering from glaucoma, and for methods of identifying compounds which may be useful in decreasing intraocular pressure.

BACKGROUND OF INVENTION

Glaucoma is a major eye disease which can cause progressive loss of vision leading to blindness. The majority of human glaucomas are associated with increased intraocular pressure ("IOP"). High IOP is considered the major risk factor for glaucomatous visual impairment resulting from death of retinal ganglion cells, loss of the nerve fiber layer in the retina, and the destruction of the axons in the optic nerve. Current treatments are aimed at reducing IOP.

Several classes of drugs acting by three different mechanisms are used as topically administered ocular therapies to lower IOP. Drugs which act primarily by decreasing the formation of aqueous humor ("AH") within the eye include beta- adrenergic blockers (e.g., timolol), topical carbonic anhydrase inhibitors (e.g., dorzolamide) and alpha-2-adrenergic receptor agonists (e.g., clonidine derivatives). A drug which lowers IOP by increasing the outflow of AH via the uveoscleral route is represented by the prostaglandin derivative latanoprost. The third mechanism for lowering IOP is to decrease the resistance to AH outflow in the trabecular meshwork outflow channels. Pilocarpine and epinephrine act primarily by this mechanism. These older agents have been gradually replaced over recent years by alternative drugs acting by other mechanisms which have greater efficacy and fewer ocular side effects. It is generally considered that the ideal ocular hypotensive agent for treating glaucoma would act by a trabecular mechanism, at the site of the primary pathology in most open angle glaucomas.

SUMMARY OF THE INVENTION The present invention relates to the use of adrenergic antagonist compounds with specificity for the αl A adrenergic receptor subtype for decreasing IOP. It is based, at least in part, on the discovery that selective αl A adrenergic receptor antagonists such as 5-methylurapidil ("5-MU") and WB 4101 hydrochloride increase outflow facility of aqueous humor and decrease IOP in a primate model of human glaucoma. It is further based on the discovery that the activity of the drug 5- methylurapidil ("5-MU") derives from its ability to specifically act at the αl A adrenergic receptor, and that the 5-hydroxytryptamine 1A ("5HT_1A") agonist activity of 5-MU is not responsible for the decrease in IOP.

Previous studies identified the subclass of αl -antagonists as potential ocular hypotensives. However the efficacy of this subclass of drugs was found to be variable, the mechanism of action in the primate eye uncertain, and the occurrence of side-effects unpredictable. At the present time, at least three subtypes of αl -receptors (using the current NC-IUPHAR classification) have been identified by cloning and binding studies, namely α 1 A, IB and 1C or ID, although only two (1A and IB) have been identified in pharmacological response studies (see Table A; Minneman and Esbenshade, 1994, Annu. Rev. Pharmacol. 34:117-133; Gross et al, 1988, European J. Pharmacol. 151:333-335). Many αl-blockers have low subtype selectivity acting at more than one of these receptors. It is likely that the variability of responses and side effects observed with αl -antagonists such as prazosin, thymoxamine or corynanthine is due to their lack of specificity for the different subtypes of αl -adrenergic receptors. According to the present invention, agents which act as antagonists with specificity for the αlA-adrenergic receptor, and particularly agents which have a greater than 20-fold greater affinity for binding at the αlA receptor relative to the α IB and/or αlC receptors, including but not limited to 5-MU and WB-4101 and compounds which compete with 5-MU or WB 4101 for binding at the αl A receptor with the required specificity, may be used as ocular hypotensive agents.

DESCRIPTION OF THE FIGURES FIGURE 1. Effects of a single dose of 1 % (open circles) or 2% (closed circles) 5-mefhylurapidil on intraocular pressure (IOP) in 8 normal monkeys. Points represent mean change in IOP from baseline values and the limits ± SEM change. Asterisks indicate statistically significant reductions in IOP compared with baseline values (two-tailed paired t test, p < 0.05).

FIGURE 2. Effects of a single dose of 1% (open circles) or 2% (closed circles) 5-mefhylurapidil on pupillary diameter (PD) in 8 normal monkeys. Points represent mean change in PD from baseline values and the limits ± SEM change. Asterisks indicate statistcally significant reductions in PD compared with baseline values (two-tailed paired t test, p < 0.05).

FIGURE 3. Effects of a single dose of 1% (open circles) or 2% (closed circles) 5-methylurapidil on intraocular pressure (IOP) in 8 unilaterally glaucomatous monkeys. Points represent mean change in IOP from baseline values and the limits ± SEM change. Asterisk indicates statistically significant reduction in IOP compared with the baseline day values (two-tailed paired t test, p < 0.05).

FIGURE 4. Effects of twice-daily administration of 2% 5- methylurapidil to eyes with glaucoma of 7 unilaterally glaucomatous monkeys for 5 days. Points represent mean values of IOP on vehicle-treated day (open circles), day 1 (closed circles), day 3 (open squares), and day 5 (closed squares) of treatment and bars indicate ± SEM. Asterisks indicate statistically significant reductions in intraocular pressure (IOP) compared with vehicle-treated values (two-tailed paired t test, p < 0.05).

FIGURE 5. Effects on IOP of the contralateral non-glaucomatoous eyes of twice-daily administration of 2% 5-MU for 5 successive days to the eyes with glaucoma in 7 unilateral glaucomatous monkeys. Points represent means of IOP on vehicle-treated day (open circles), day 1 (closed circles) and day 5 (closed squares) of treatment and bars indicate ± SEM. Asterisks indicate statistically significant reductions in intraocular pressure (IOP) compared with vehicle-treated values (two- tailed Bonferroni t-test, ρ<0.05). DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for decreasing intraocular pressure in a subject in need of such treatment (including, but not limited to, a subject suffering from glaucoma), comprising administering, to the subject, an effective amount of a selective αlA adrenergic receptor antagonist. Preferably, the selective antagonist has an affinity for binding to the αl A adrenergic receptor which is at least 20 times greater than the affinity for the antagonist to bind to the αlB or the αlC adrenergic receptors, using measurements as described in (Minneman and Esbenshade, 1994, Annu. Rev. Pharmacol. Toxicol. 34: 1 17-133 or Gross et al., 1988, Eur. J. Pharmacol. 151:333-335). Examples of selective antagonists include 5- methylurapidil, having the following chemical structure (I), and WB-4101 hydrochloride, having the following chemical structure (II):

(II) WB-4101 hydrochloride as well as agents which compete with either 5-methylurapidil or WB-4101 hydrochloride (and structurally related compounds) for binding with the αl A adrenergic receptor and/or are selective in binding to that receptor.

The present invention also provides for a method for the identification of a compound which may be useful in decreasing intraocular pressure in a subject in need of such treatment, comprising (i) determining whether the compound acts as an antagonist at the αl adrenergic receptor and (ii) measuring the affinity of the compound for binding to the αl A adrenergic receptor, the α IB adrenergic receptor, and the αlC adrenergic receptor, wherein the ability of the compound to act as an antagonist at the αl adrenergic receptor and to selectively bind to the αl A receptor has a positive correlation with the ability of the compound to be effective in decreasing intraocular pressure, αl adrenergic receptor antagonist action is defined herein as the ability of a compound to inhibit effects mediated by the αl adrenergic receptor, such effects including, but not limited to, a hypertensive effect (wherein antagonism results in a hypotensive effect), a positive inotropic effect, cellular Ca²⁺ mobilization via opening of channels or release of intracellular stores, and phosphoinositide hydrolysis (see Takanashi et al., 1991, Naunyn Schmiedeberg' s Arch. Pharmacol. 343:669 and Valenta et al., 1990, Br. J. Pharmacol. 99:713). In a non-limiting example, selective binding to αl A adrenergic receptors may be measured as follows. The cellular effect of activation of αl receptors is mobilization of Ca²⁺ via Ca²⁺ channels or release from intracellular stores. When sub-types of cloned α receptors are selectively expressed in a cell line, the Ca²⁺ mobilization can be determined for each subtype by Ca²⁺-sensitive fluorescent dyes. In whole tissues or animals, Ca²⁺ mobilization by αl A or αlB receptors may be measured by contraction of smooth muscle or another physiological response, such as the inotropic response. In these cases selective antagonism of αlA receptors by a drug may be determined, for example, by comparison with a known selective agent such as chloroethyl clonidine ("CEC"), an irreversible blocker. In binding studies αlA selectivity can be determined by competition against prazosin (which non-selectively binds to all l receptor subtypes) and comparing with CEC, WB-4101 and 5-MU, the last two of which are known to be αl A receptor selective. The preferred method is to use cell lines which express the specific subtypes as a preliminary selection method, and then to determine the specificity of a test antagonist by measuring its IC50 in blocking norepinephrine-induced Ca²⁺ mobilization.

Such a method may be used to screen a plurality of test compounds in order to identify a compound useful in decreasing intraocular pressure. As discussed above, such selective binding is preferably manifested as an at least 20-fold greater affinity for the αl A adrenergic receptor relative to the αlB or αlC/D adrenergic receptors. In other embodiments, such a compound competes with 5-MU or WB-4101 (but is distinct from 5-MU or WB-4101) for selectively binding to the αl A adrenergic receptor.

Prior to the present invention, the effectiveness or side effects of αl adrenergic receptor antagonists were unpredictable because such agents are not sufficiently selective for binding to the αl A adrenergic receptor and it had not been discovered that it is the αl A receptor mediated action which is responsible for decreasing intraocular pressure. The present invention therefore provides a method for identifying compounds which would more likely be clinically useful. The present invention further provides for a method of decreasing intraocular pressure in a subject in need of such treatment comprising administering, to the subject, an effective amount of a compound identified by the foregoing method. Further, it has been discovered that the administration of such an antagonist may become more effective with repeated dosing, as tachyphylaxis was not observed with repeat 5-MU administration; rather, the effectiveness was observed to increase. The present invention is illustrated by the following nonlimiting working examples, which demonstrate that (i) the selective αlA adrenergic receptor antagonists 5-MU and WB-4101 decrease IOP and increase outflow facility of AH in normal and glaucomatous monkey eyes; (ii) the activity of 5-MU in decreasing IOP does not derive from its 5HT_1A agonist activity; and (iii) 5-MU selectively acts as an antagonist at the l A-adrenergic receptor.

EXAMPLE: 5-MU LOWERS INTRAOCULR PRESSURE Materials And Methods

Eight normal and 8 unilaterally glaucomatous female cynomolgus monkeys, each weighing 3 -5kg, were involved in this study. For all experiments the monkeys were anesthetized with intramuscular ketamine hydrochloride (l-5mg/kg), and one drop of 0.5% proparacaine hydrochloride was topically administered to the eye 5 minutes before measurements. Glaucoma had been previously induced by repeated argon laser photocoagulation (65-120 spots; power, 1.1-1.5W; size, 50μm; duration, 0.5 sec) or diode photocoagulation (50-120 spots; power, 1.2W; size, 75μm; duration, 0.5 sec) of the mid-trabecular meshwork for 360°. Eight female albino rabbits, each weighing 2-3kg, were used to measure uveoscleral outflow.

On each day of the study IOP was measured with a calibrated pneumotonometer (model classic, Mentor, Norwell, MA) at 0 hr (prior to drug administration), 0.5 hour and then hourly until 6 hours after drug administration. The pupil diameter (PD) was measured with a millimeter ruler under normal room light at the same time and same intervals as IOP measurements in normal monkeys. Slit-lamp examinations were performed at 0 hour (pre-treatment) and 1, 3 and 5 hours (post- treatment) for detection of corneal changes and aqueous humor flare or cells, using maximum magnification in a dark room.

5-Methylurapidil (Research Biochemicals, Inc., MA) was freshly prepared as a 1% or 2% (w/v) solution by dissolving in 0.1 M hydrochloric acid followed by adjusting the pH to 7.0 with NaOH. Single-dose testing was performed with 1% and 2% concentrations in eyes of both normal and glaucomatous monkeys (n = 8). In normal monkeys, 50μl (25μl x 2, five minutes apart) of 5-MU was randomly applied topically to one eye and an equal volume of vehicle (saline) was applied to the contralateral (no-drug) eye. The 0 hour (pre-drug) measurement was taken as the baseline value against which drug-treated eye and contralateral eye IOP or PD measurements were compared. In glaucomatous monkeys, 50μl of normal saline was topically applied to the glaucomatous eye at 9:30 a.m. on the first day and 50μl of 5- MU to the glaucomatous eye at 9:30 a.m. on the second day. Diurnal IOP curves on the saline-treated day, considered as the baseline, were compared with drug-treated diurnal IOP curves. A multiple-dose study was carried out in 7 monkeys with unilateral glaucoma following one baseline (untreated) and one vehicle-treated day (50μl of saline applied twice a day). Fiftyμl of 2% 5-MU was topically applied to the glaucomatous eye twice daily (at 9:30 a.m. and 3:30 p.m.) for 5 consecutive days. Diurnal IOP measurements were performed daily. For statistical analysis, IOP on each of the five drug treatment days was compared to that on the vehicle-treated day. The 2% dose of 5-MU was used to evaluate the mechanism by which the drug reduced IOP in 8 normal monkeys. Outflow facility was measured using a electronic indentation tonograph (Alcon EDT-103) prior to drug administration and was repeated 1 hour after unilateral application of 2 X 25μl of 2% 5-MU. Animals were anesthetized as described above and placed in the supine position for outflow facility measurements. Aqueous humor flow measurements were performed using a scanning computerized fluorophotometer and a software analysis package (Coherent Fluorotron, Coherent, Palo Alto, CA) . For this procedure the animals were anesthetized as described above and seated in specially designed chairs. Fluorescein was iontophoresed into the central corneas of both eyes (with an electrode of 10% fluorescein in 2% agar gel) for 7 minutes at 4:00 p.m. on the day prior to aqueous flow measurements. Baseline aqueous humor flow rates were taken at 1 hour intervals for 4 hours beginning at 9:30 a.m. The following day, flow measurements were repeated, as done on the baseline day, beginning 1 hour after drug application. The washout period between each test on the same animal was at least one week.

Uveoscleral outflow was determined with a perfusion technique using fluorescein isothiocyanate-dextran beginning 1 hour after 50μl of topical 2% 5-MU in 8 rabbits. Naive albino rabbits weighing 2-3 kg were used. The rabbits were restrained in cloth wrappers for all measurements. The care and use of animals conformed to the Declaration of Helsinki and The Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 86-23). Baseline IOPs were measured at 9:00 a.m. with a calibrated pneumatonometer following topical application of one drop of proparacaine hydrochloride 0.5%. Fifty μl of 2% 5-MU were then applied topically to one eye and an equal volume of normal saline to the contralateral control eye. Similar experiments were also performed in untreated rabbits as additional controls. IOP measurements were repeated 1 hour after instillation of 5-MU. Five to 10 minutes following the IOP measurements the outflow ("Fu") was measured as described in Bill, 1966, Exp. Eye Res. 5:45-54; Suguro et al., 1985, Invest. Ohpthamol. Vis. Sci. 26:810-813; Goh et al., 1989, Graefe's Arch. Clin.Exp. Ophthalmol. 227:476-481; and Gabelt and Kaufman, 1989, Exp. Eye Res. 49:389-402 (with modifications). The rabbits were anesthetized with intravenous urethane 40% (Sigma Chemical CO., St. Louis, MO, initial dose 0.8g/kg body weight, additional doses given when required). When the rabbit was anesthetized the head was then held securely in place with a head holder. One drop of proparacaine 0.5% was applied to the cornea and two 23-gauge needles were inserted through the peripheral cornea into the anterior chamber in each eye. Needle A was attached to polyethylene tubing and a 5 ml infusion syringe filled with 10^"4 M fluorescein isothiocyanate-dextran (FITC- dextran) (71,200 MW, Sigma Chemical CO., St. Louis MO) in phosphate-buffered saline (PBS), pH 7.4. Needle B was attached to polyethylene tubing leading to a 5 ml withdrawl syringe or a buret through a two-way stopcock. The syringes were controlled by an infusion/withdrawal pump (Model 944, Harvard Apparatus, South Natick, MA). After the needles were inserted into the anterior chamber, 1ml of 10^"4 M FITC-dextran was used to flush the anterior chamber at a rate of 0.5 ml/min for 2 min using both of syringes. The syringe from needle B was then disconnected and the buret was connected to the anterior chamber using the stopcock. The FITC-dextran solution was infused into the anterior chamber using syringe A until the solution in the buret was 20 cm above the anterior chamber to establish an IOP of 15 mmHg. The syringe B was then re-connected and the buret was disconnected from the anterior chamber. Then the eye was continuously perfused with the FITC-dextran solution from syringe A to syringe B at the rate of 10 ul/min for 30 minutes. The anterior chamber was then washed thoroughly with 2ml of PBS at a rate of 0.5 ml/min for 4 minutes. It was assumed that fluorescein tracer labeled fluid in the conventional outflow paths had been washed out. The animal was killed by an overdose of intravenous urethane, the needles withdrawn, and the eye enucleated. The cornea was then removed and the eye was washed thoroughly with PBS. After the lens was discarded, the eye was dissected into uvea, fluid of posterior segment plus vitreous, and sclera. The sclera was minced with scissors. All samples were homogenized in 5-7 ml of PBS and centrifuged for 30 min. After centrifugation , the volume of each sample was measured and the supernatant was measured for the concentration of the FITC-dextran by using a fluorophotometer (Coherent Fluorotron Master, Palo Alto,CA). Forty-two different concentration of FITC-dextran were measured to construct a standard curve by computer.

Uveoscleral outflow (Fu) was considered to be the volume (Vu) of labeled anterior chamber fluid required to have deposited the amount of tracer recovered from the ocular tissues, divided by the duration (T) of the labeled infusion. For each tissue and fluid compartment, the quantity of tracer in tissue or fluid (ng) was determined and Fu calculated as follows: Quantity of tracer in tissue or fluid (ng)

Quantity of tracer in tissue or fluid (ng) Vu =

(concentration of tracer during perfusion (ng/ul)

T= Time of anterior chamber perfusion

ΣVu Fu =

The two-tailed paired t-test was used for all statistical analyses. All experimental studies complied with the ARVO Resolution on the Use of Animals in Research and were approved by the Mt. Sinai School of Medicine Institutional Animal Care and Utilization Committee.

Results

The unilateral topical application of 5-MU to 8 normal monkey eyes significantly (p <0 .05) reduced IOP bilaterally for 2 hours (1% dose) and for 4 hours (2% dose) after drug administration, as compared with baseline pre-drug values (FIGURE 1). The maximum reduction in IOP occurred 1 hour after drug administration, and was 2.8 ± 0.7mmHg (1%) and 4.4 ± 0.5mmHg (2%) in drug- treated eyes, and 2.3 ± O.δmmHg (1%) and 3.0 ± 0.7mmHg (2%) in contralateral no- drug eyes (mean ± SEM). Both 1% and 2% doses significantly (p < 0.05) reduced pupil size for 6 hours in treated eyes, and for 2 hours (1%) and for 3 hours (2%) in contralateral eyes (FIGURE 2). One of the 8 eyes treated with the 1% dose developed mild corneal edema and corneal punctate erosion; 2 of the 8 eyes treated with the 2% dose developed mild corneal edema and conjunctival mucous discharge.

In 8 monkeys with unilateral glaucoma, single-dose administration of 5-MU significantly (p < 0.05) reduced IOP for 1 hour and 5 hours following 1% and 2% concentrations, respectively. The maximum reduction in IOP was 6.5 ± l.OmmHg (1%) and 7.5 ± 0.8mmHg (2%) at 1 hour after drug administration

(FIGURE 3). Mucous discharge of the conjunctiva was observed in 1 of the 8 eyes treated with the 2% dose. The corneas of all animals remained clear.

The ocular hypotensive effect was observed to increase with successive dosing in a 5-day twice-daily administration of 2% 5-MU study (FIGURES 4 and 5). The reduction in IOP became more pronounced at each interval from day 1 to day 5. The maximum reduction in IOP (the differences in IOP between drug - treated and vehicle - treated values) at 1 hour after application was 6.9 ± 1.3mmHg on day 1 and 15.1 ± 2.9mmHg on day 5, (p < 0.05). (Significant differences in IOP were not observed when comparing the baseline and vehicle-treated days.) Mild corneal edema was observed in one of seven drug-treated eyes on day 4, but was not noted in any of the eyes on the day 5 examination. Mucous discharge was observed throughout the experiment in two animals during the 5 -day treatment. After instillation of the drug, these two animals squeezed their lids shut for about 30 minutes. At the peak of IOP change, one hour after administration of 2% 5-MU to one eye of the normal monkeys, outflow facility was significantly increased by 51% (p < 0.01) in the treated eyes, and by 16%) (p > 0.10, not significant) in the contralateral fellow eyes compared with baseline values. IOP was significantly (p < 0.05) reduced at 1 hour in both eyes when measured tonographically. After application of 2% 5-MU to the normal monkey, aqueous humor flow rates were increased by 11% (p < 0.01) over a period of 4 hours in the treated eyes, and by 3% (p > 0.10) in the contralateral fellow eyes compared with baseline values (Table B). TABLE B EFFECTS OF 2% 5-METHYLURAPIDIL ON AQUEOUS HUMOR DYNAMICS IN 8 NORMAL MONKEYSf

t Values are mean ± SEM. Intraocular pressure was measured with an electronic tonography apparatus.

* Significantly different as compared with baseline measurement (two-tailed paired t test, p < 0.05).

In 8 rabbits, uveoscleral outflow was unchanged (p > 0.30) at 1 hour after administration of 2% 5-MU compared with baseline values. Intraocular pressure was significantly (p < 0.05) reduced at 1 hour in the treated eyes (Table C). Systolic and diastolic blood pressures were significantly reduced (p<0.005) by up to 12± 2 and 11 ± 2 mm Hg respectively for the first 2 hours after bilateral topical administration of 2% 5-MU (Table D). TABLE C EFFECT OF 2% 5-METHYLURAPIDIL ON

UVEOSCLERAL OUTFLOW IN 8 TREATED RABBITS

Values are mean ± SEM. Intraocular pressure was measured with a calibrated pneumotonometer.

Significantly different as compared with baseline measurement (two-tailed paired t test, p < 0.05).

TABLE D EFFECT OF BILATERAL TOPICAL ADMINISTRATION OF 2% 5-METHYLURAPIDIL ON SYSTEMIC BLOOD PRESURE IN 6 NORMAL MONKEYS BLOOD PRESSURE ( mmHg)

t Values are mean ± SEM. Blood pressure was measured with a sphingomanometer for newborns.

Significantly different as compared with baseline measurement (two- tailed paired t-test, p<0.005). Discussion

Some α,-adrenergic antagonist drugs produce miosis and a reduction of intraocular pressure in animals and humans. Thymoxamine, a selective α_r adrenergic antagonist, produces miosis without lowering IOP in normal human eyes (Wand and Grant, 1976, Invest. Ohpthalmol. 15:400-403; Wand and Grant, 1980, Surg. Ophthalmol. 25:75-84). However, prazosin, which is also a selective α_r adrenergic antagonist, has little pupillary effect, but can significantly lower IOP in animals (Rowland and Potter, 1980, Eur.J. Pharmacol. 64:361-363; Smith et al., 1989, Arch. Ophthalmol. 79:1933-1936). Corynanthine, another α,-adrenergic antagonist, reduces IOP but tachyphylaxis develops in humans (Serle et al., 1985, Ophthalmol. 92:977-980) and animals (Serle et al., 1984, Arch. Ophthalmol. 102:1385-1388). The differences in response among various α, -blocking drugs could be due to differences in numbers and/or activities of α, -receptor subtypes at different sites or alternative signal transduction systems linked to the subtypes of α,-adrenoceptors present in different tissues.

Three α,-adrenergic receptor subtypes (α_1A, α_1B, α_{lc D} ) have now been cloned and pharmacologically characterized (Minneman and Esbenshade, 1994, Annu. Rev. Pharmacol. Toxicol. 34:117-133; Bylund, 1992, FASEB J. 6:832-839; Harrison et al., 1991, Trends Pharmacol. Sci. 12:62-67). Each subtype is a product of a separate gene and may have a unique drug specificity and tissue distribution. 5-MU is one of the currently available drugs which has substantially different affinities for the pharmacologically defined α_1A and α_1B-subtypes. It is 50-100 fold selective for antagonism of the α_1A over the α_1B subtype (Minneman and Esbenshade, 1994, Annu. Rev. Pharmacol. Toxicol. 34: 117-133). However, 5-MU is also a potent agonist at the 5-HT_1A receptor (Hoyer et al., 1984, Pharmacol. Rev. 46: 157-203). A population of 5- HT_1A receptors is present in the iris-ciliary body of the rabbit (Chidlow et al., 1995, Invest. Ophthalmol. Vis. Sci. 36:2238-2245) and human (Hoyer et al., 1984, Pharmacol. Rev. 46:157-203). The effect of selective 5-HT_IA agonists on IOP has not been reported yet, although a number of studies have shown the effects of serotonin, which is non-selective, and some of its analogues on IOP in animals and humans. However, ketanserin, a 5-HT_2A antagonist, lowers IOP in normotensive and ocular hypertensive eyes of rabbits (Krootila et al., 1987, J. Ocular Pharmacol. 3:279-290; Conway and Lewis, 1989,Invest. Ophthalmol. Vis. Sci. 30:24) and humans (Costagliola et al., 1990, Cardiovasc. Drug. Ther. 4:95-97; Costagliola et al., 1991, Exp. Eye Res. 52:507-510; Costagliola et al., 1993, Br. J. Ophthalmol. 77:344-348) either by increasing outflow facility or suppressing aqueous humor formation.

Ketanserinol, which is a metabolite of ketanserin, is reported to have a greater ocular hypotensive effect than does ketanserin in normotensive rabbits (Espino and Musson, 1993, InvestOphthalmol. Vis. Sci. 34:1111).

Prior to the completion of these studies, it was believed that since 5- MU is a dual-action drug, its effect on aqueous humor dynamics could result from a combination of antagonism at α,-receptors (α_1A-subtype) and agonism at 5-HT receptors (5-HT_1A subytpe). Some β - blockers used as ophthalmic drugs, such as carteolol, are dual - action drugs because they are β₂ - receptor antagonists, but have sympathomimetic activity at other types of adrenergic receptors. This study on the effects of 5-MU on IOP and aqueous humor dynamics shows that 5-MU does reduce IOP bilaterally following unilateral administration in monkey eyes. The reduction in IOP appears to be dose-dependent. The magnitude and the duration of IOP reduction is greater in the treated eyes than in contralateral fellow eyes, and greater in glaucomatous than in the normal monkey eyes. The bilateral reduction in IOP may be caused by systemic transfer of drug, or mediation through the central nervous system, or through a non-adrenergic mechanism. The drug produces miosis probably by inhibiting α, -receptor-mediated contraction of the dilator muscle of the iris. The multiple-dose study in monkeys demonstrates an increase of the ocular hypotensive effect with repeated dosing, which differs from findings with another α,-antagonist drug, corynanthine, where tachyphylaxis develops (Serle et al, 1985, Ophthalmol. 92:977-980). By day 5 the ocular hypotensive effects were substantial with 5-MU. We noted that a few irritative effects occured following single-dose and multiple-dose applications in some monkeys. These include mucous discharge, mild corneal edema and corneal punctate epithelial erosions, but not anterior chamber inflammation. Improvement of the formulation may reduce these side effects. The mechanism of action of α, -adrenergic antagonists on aqueous humor dynamics are not fully understood. Thymoxamine has little effect on IOP and does not substantially increase outflow facility (Wand and Grant, 1976, Invest. Ohpthalmol. 15:400-403; Wand and Grant, 1980, Surg. Ophthalmol. 25:75-84) or aqueous humor flow (Lee et al., 1981, Invest. Ophthalmol. 21:805-811). Prazosin reduces IOP by reducing aqueous humor formation in animal eyes when administered topically (Krupin et al., 1980, Arch. Ophthalmol. 98:1639-1642). Corynanthine reduces IOP without altering outflow facility or the rate of aqueous humor flow (Serle et al., 1984, Arch. Ophthalmol. 102:1385-1388). The dual action of 5-MU which combines antagonist activity at the α, _A-adrenergic receptor subtype and agonist activity at the 5-HT_1A receptor subtype had effects on aqueous humor dynamics that are quite different from previously studied α, - receptor blockers. Topical application of 5-MU to monkey eyes reduced IOP by increasing outflow facility up to 51% in the treated eye with a small, but significant increase in aqueous humor flow rate. Considering the high expense of terminal experiments in monkeys, we have investigated the effect of 2% 5-MU on uveoscleral outflow only in rabbits. The result shows that 5-MU does not change uveoscleral outflow. However drug effects on uveoscleral outflow may be species-dependent, and could be different in the monkey eye. The increase in outflow facility may relate to effects of 5-MU on trabecular tissue and to α-adrenoreceptor and 5-HT receptor systems in trabecular tissue or associated ciliary muscle. Vascular mechanisms may also be involved in the ocular hypotensive responses to 5-MU. Oral administration of the α, -receptor blocker prazosin reduces blood pressure. The effect of this drug on aqueous humor production may be mediated in part by a decrease in systemic blood pressure

(Rowland and Potter, 1980, Eur. J. Pharmacol. 64:361-363). 5-MU also decreases blood pressure and heart rate by activation of a CNS 5-HT_1A-receptor system (Dreteler et al, 1990, Eur. J. Pharmacol. 180:339-349; Dreteler et al., 1981, J. Cardiovasc. Pharmacol. 17:488-493). This drug may thus affect the tone of arterial or venous vessels that could influence ocular blood flow, aqueous formation and outflow facility. In summary, 5-methylurapidil, a newly recognized dual-action drug which is an α,_A-adrenergic antagonist and a 5-HT,_A agonist, reduced IOP and pupil size in normal and glaucomatous monkey eyes with few adverse effects. The most significant findings in this study is that drugs with the above receptor profile of activity can cause a reduction in IOP in primates by increasing outflow facility and that multiple dosing does not result in tachyplylaxis but causes a cumulative increase in response.

EXAMPLE: 5-MU DOES NOT ACT VIA 5HT_1Λ AGONIST ACTION A series of experiments were performed in monkey eyes using the 5HT_1A antagonist, pMPPI hydrochloride (4-iodo-N-[2-[4-(methoxyphenyl)-l- piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride; Kung et al., 1995, J. Pharmacol. Exp. Ther. 272:429-437; Kung et al., 1994, Synapse 18:459-466; Kung et al., 1994, Life Sci. 5_5: 1459- 1462), having the following chemical structure:

p-MPPI The first experiment, in which 50 μl of 0.2 percent p-MPPI alone was given topically to one eye, showed no IOP effect in either eye over a 6 hour period. In a second experiment, p-MPPI pre-treatment was given to one eye, vehicle to the fellow eye, and one hour later both eyes were treated topically with 2% 5-MU. The results are shown in Table E. TABLE E. EFFECT OF TOPICAL OCULAR PRE TREATMENT WITH

THE 5HTι_A.RECEPTOR ANTAGONIST 0.15% p-MPPI (GIVEN 1 HOUR PRIOR TO 5-MU) ON THE IOP RESPONSES TO TOPICAL 2% 5-MU GIVEN AT 0 TIME IN 6 NORMAL MONKEY EYES

Intraocular Pressure (mean mmHg ± SEM) at time (hrs) pre- and post- treatment

* p-values when compared to pretreatment IOP of 18.8 ± 0.71, 16.8 ± 0.48 and

17.0 ± 0.45, respectively. ** p-values for comparison of IOP in vehicle and p-MPPI pre-treated eyes.

In a third set of experiments, a 50 μl dose of 0.15%> p-MPPI was applied to one eye of six normal monkeys. IOP and pupil size were measured before treatment and then hourly for six hours after dosing. After a two week washout period, 50 μl of 0.2%) p-MPPI was randomly applied to one eye of six normal monkeys, and the same volume of saline was applied to the fellow control eye. One hour after the p-MPPI or saline dosing, 50 μl of 2% 5-MU was applied to both eyes of all six monkeys. IOP was measured prior to the p-MPPI pre-treatment, prior to the 5- MU dosing, and then hourly up to six hours after 5-MU treatment.

The results of these experiments were as follows. Unilateral topical application of 0.15% p-MPPI did not alter the IOP or pupil size, as compared with either contralateral control eyes or baseline values (p>0.80). The IOP was significantly (p<0.05) reduced for 4 hours both in the 5-MU treated, p-MPPI pre- treated eyes and in eyes treated only with 5-MU. The reduction in IOP between these two 5-MU-treated groups was not significantly different (p>0.80). We conclude from these studies that p-MPPI has no effect on the duration or magnitude of the response to 5-MU. This indicates that the receptor mechanism by which 5-MU lowers IOP is associated with the specific αl A adrenergic receptor antagonism of the drug as opposed to any 5-HT_1A serotonin antagonist effect.

EXAMPLE: WB 4101 DECREASES IOP Fifty microliters of a 1% solution of WB 4101 hydrochloride (was applied to one eye of six normal monkeys and vehicle was applied to the fellow eyes. Then, 1.5 hours after dosing, the IOP and outflow facility was measured in the drug- treated and fellow eyes of each monkey. The results are shown in Table F.

TABLE F. EFFECT OF WB 4101 ON IOP AND OUTFLOW FACILITY

* significantly different as compared with the baseline value (p<0.02) and the fellow eyes (p<0.03), two-tailed paired t-test.

+ significantly different as compared with the baseline value (p<0.01), two- tailed paired t-test.

The present invention therefore provides for the use of effective concentrations of WB4101 and/or a salt thereof, including, but not limited to, WB4101 hydrochloride, in methods of decreasing intraocular, in particular in the treatment of glaucoma.

Various publications are cited herein, which are hereby incorporated by reference in their entireties.

Claims

1. A method for the identification of a compound which may be useful in decreasing intraocular pressure in a subject in need of such treatment, comprising (i) determining whether the compound acts as an antagonist at the ╬▒l adrenergic receptor and (ii) measuring the affinity of the compound for binding to the ╬▒lA adrenergic receptor, the ╬▒lB adrenergic receptor, and the ╬▒lC adrenergic receptor, wherein the ability of the compound to act as an antagonist at the ╬▒l adrenergic receptor and to selectively bind to the ╬▒l A receptor has a positive correlation with the ability of the compound to be effective in decreasing intraocular pressure.

2. The method of claim 1, wherein an affinity for binding of the compound to the ╬▒l A adrenergic receptor which is at least 20-fold greater than the affinity of the compound for the ╬▒lB adrenergic receptor or the ╬▒lC adrenergic receptor has a positive correlation with the ability of the compound to be effective in decreasing intraocular pressure.

3. A method of screening a plurality of test compounds in order to identify one or more compound which may be used to decrease intraocular pressure in a subject, comprising (i) determining, for each test compound, whether the test compound acts as an antagonist at the ╬▒l adrenergic receptor and (ii) measuring the affinity of the test compound for binding to the ╬▒l A adrenergic receptor, the ╬▒lB adrenergic receptor, and the ╬▒lC adrenergic receptor, wherein the ability of the test compound to act as an antagonist at the ╬▒l adrenergic receptor and to selectively bind to the ╬▒l A receptor has a positive correlation with the ability of the test compound to be effective in decreasing intraocular pressure.

4. The method of claim 3, wherein an affinity for binding of the compound to the ╬▒l A adrenergic receptor which is at least 20-fold greater than the affinity of the compound for the ╬▒lB adrenergic receptor or the ╬▒lC adrenergic receptor has a positive correlation with the ability of the compound to be effective in decreasing intraocular pressure.

5. A method of decreasing intraocular pressure in a subject in need of such treatment, comprising administering, to the subject, an effective amount of a compound which is structurally related to WB-4101, which is a selective antagonist of the ╬▒lA adrenergic receptor, and which competes with WB-4101 for binding to the ╬▒lA adrenergic receptor.

6. The method of claim 5 which is WB-4101.

7. The method of claim 5 which is used to treat a subject suffering from glaucoma.

8. The method of claim 6 which is used to treat a subject suffering from glaucoma.