WO1990006123A1

WO1990006123A1 - Therapeutic use of calcium entry blockers in retinal or optic nerve dysfunction

Info

Publication number: WO1990006123A1
Application number: PCT/US1989/005487
Authority: WO
Inventors: Craig E. Crosson; David E. Potter
Original assignee: Houston Biotechnology Incorporated
Priority date: 1988-12-05
Filing date: 1989-12-05
Publication date: 1990-06-14
Also published as: WO1990006118A1; AU4807790A; AU4754790A; CA2004617A1; CA2004616A1

Abstract

Ischemia or edema of the retina or optic nerve results in retinal dysfunction. This retinal dysfunction can be associated with the activation of calcium channels and/or excitatory amino acid receptors. The prophylactic or therapeutic administration of compounds to block these processes can ameliorate or prevent retinal dysfunction. These compounds include the classes of calcium channel antagonists. Therapeutic treatment with compounds include dihydropyridines and diphenylipiperazines as calcium channel antagonists. Such compounds also exhibit a prophylactic effect to ischemia and edema of the retina or optic nerve. Rat models are provided for screening compounds capable of ameliorating retinal dysfunction. Particularly, calcium entry blockers and excitatory amino acid antagonists can be screened and the host eyes examined for the effect of the drugs. The model animal is a rat having a salt-inducible retinal dysfunction and/or a retinal dysfunction as a result of vascular occlusion resulting in ischemia.

Description

-1-

TITLE: THERAPEUTIC USE OF CALCIUM ENTRY BLOCKERS

IN RETINAL OR OPTIC NERVE DYSFUNCTION

Technical Field

The subject invention is drawn to the use of calcium channel antagonists and excitatory amino acid antagonists in the treatment of retinal and optic nerve dysfunction and in vivo bioassays for screening such compounds. Background of the Invention

Retinal vascular disease and ischemia are associated with malfunction of neuroendocrine regulation and autoregulation of the choroidal and retinal circulations, respectively. It has been postulated that excessive elevation of intracellular calcium (calcium overload) in retinal blood vessels and neurons may be involved in the pathogenesis of retinal vasculopathy, ischemia and ultimately, retinal damage. Some specific pathologic events triggered by excess intracellular calcium ions include: generation of free radicals, activation of proteases, endonucleases and lipases, and interference with energy production in mitochondria.

Blood flow to the retina is supplied by two separate vascular systems: the retinal vessels supplying the inner retinal layers and choroidal vessels supplying the outer retinal layers. In primates, approximately 35% of the total retinal blood flow is derived from the retinal vessels, while 65% is from the choroidal vessels. Although the choroidal blood flow is of greater magnitude, retinal ischemia is usually associated with a reduction of flow in the inner retinal vessels. This greater propensity for ischemia in the inner retina may result from several factors: (1) the high rate of choroidal blood flow over that required to meet the metabolic needs of the outer retina; (2) the large diameter capillaries i the choroid are less likely to be occluded by emboli; (3) the lack of anastomoses in the retinal vessels; and (4) the larger percentage of oxygen extracted from the retina arterioles/capillaries (35%) as compared to the choroidal circulation (3-4%). To maintain an adequate supply of nutrients to the inner retina under various systemic and ocular conditions, blood flow through normal retinal vessels is highly autoregulated by metabolic (oxygen and carbon dioxide), myogenic and possibly local hormonal (paracrine and autocrine) factors.

A number of systemic and ocular disorders have been associated with ischemic conditions o£ the retina or opti nerve. Ocular manifestations of systemic disorders include: diabetes, atherosclerosis, hyperlipidemia, and hypertension. Specific ocular disorders include: retinitis of AIDs, macular degeneration, anterior ischemi optic neuropathy, ocular.-, hypertension, glaucoma, retinopathy of prematurity, retinal vessel occlusion, diabetic retinopathy and hypertensive retinopathy. In addition, edemic conditions of the retina or optic nerve are evidenced in diabetes, hypertension and cystoid macular edema. Newer evidence also suggests that excessive influx of calcium ions into vascular and neuronal tissue is a primary contributor to the pathogenesis of ischemic injury and_. the. development of vasculopathy and neuropathy.

It is therefore of substantial interest to identify compounds which may be used in the therapeutic treatment of or prophylactic treatment against vasculopathies and neuropathies associated with the eye.

Further, it is of great interest to develop a reproducible and sensitive bioassay which is a good -3-

predictor of the utility of a compound as a therapeutic for various ischemic retinopathies. Desirable characteristics of such a bioassay are the use of relatively small animals with ocular vasculature and neural retina similar to that of humans, particularly rodentiae, which provides for constitutive retinal dysfunction or the ability to reproducibly induce such dysfunction, ease of access to the major arteries supplying the retina, ease of identifying the existence of the dysfunction and the effect of addition of a candidate compound on occurrence of such dysfunction or the effect on progression of such dysfunction. Relevant Literature

The publications cited herein are incorporated by reference as if each publication were specifically and individually indicated to be incorporated by reference.

Choi (1985) Neuroscience Letters 58:293-297, described the calcium dependence of glutamate neurotoxicity in cortical cell culture. Meldrum (1985) Clinical Science 63:113-122, describes potential therapeutic applications of antagonists of excitatory amino acid neurotransmitters. Sinclair et al., (1982) J. American Academy of Ophthalmo1o y 8_9:748-750, describe retinal vascular autoregulation in diabetes mellitus. Rhie et al., (1982) Diabetes .31:1056-1060, describe retinal vascular reactivity to norepinephrine and angiotensin II in normals and diabetics. Fleckenstein et al., (1985) Am. J. Cardiol. 56:3H-14H, describe the experimental basis of long-term therapy of arterial hypertension with calcium antagonists. Fleckenstein et al., (1987) Ibid. 59:177B-187B, describe future directions in the use of calcium antagonists in the treatment of cardiovascular disease. Godfraind (1987) Ibid. 5_9:11B-23B, provides a classification of calcium antagonists. Fleckenstein et al., (1987) TIPS 8:496-501, describe investigation of the role of calcium in the pathogenesis of experimental arteriosclerosis. Katz and Leach (1987) J___ Clin. Pharmacol. 22:825-834, describe a therapeutic application of 1,4-dihydropyridine calcium channel blockers. Gelmers et al., (1988) N^ Engl. J. Med 318:203-207, describe an investigation of nimodipine in acute ischemic stroke. Cook and Hof (1988) Br. J.

Pharmacol. )3_:121-131, describe the cardiovascular effect of apa in and BRL 34915 in rats and rabbits. Nihard (1982) Anqiology 32:37-45, describes the effect of calcium-entry-blockers on arterioles, capillaries and venules of the retina. Corbiere, French Patent No. 2,585,574 describes the use of ocular pharmaceuticals containing (nitrophenyl)dihydropyridinedicarboxylates. Triggle and Janis (1987) Ann. Rev. Pharmacol. Toxicol. 22:347-369, describe structure-function relationships fo calcium channel ligands, particularly 1,4-dihydropyridines.

Articles concerned with rat models for chronic or acute retinal dysfunction include von Sallmann and Grimes (1974) Investigative Ophthalmology 13^1010-1015; Frank e al., (1986) Science 231:376-378 and Stefansson et al., (1988) Invest. Ophthalmol. Vis. Sci. 29:1050-1055. SUMMARY OF THE INVENTION

Azaheterocycle calcium entry blockers are useful in the treatment of subjects, such as mammals, including man suffering from ischemia or edema of the retina or optic nerve. Such calcium entry blockers may be grouped as calcium channel antagonists and excitatory amino acid receptor antagonists. Associated with retinal dysfunctio are techniques for assessing neural retinal function. I addition, such compounds exhibit prophylactic effects in preventing such conditions. Methods are further provide for screening compounds associated with regulation of calcium channels by employing in vivo bioassays using rat with inducible retinal dysfunction. DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Compounds associated, either directly or indirectly, with the modulation of calcium entry exhibit a therapeuti or prophylactic effect to subjects suffering from ischemi or edema of the retina or optic nerve. Such conditions are evidenced in the systemic and ocular ischemic and edemic disorders cited above. These compounds may be divided into two categories. The first are the calcium channel antagonists, which may be further divided into dihydropyridines and diphenylpiperazines. The second category are excitatory amino acid antagonists, which include NMDA, quisqualate and kainate receptor antagonists.

Among dihydropyridines of interest are nifedipine, having the structural formula:

H₃

nimodipine, having the structural formula:

nisoldipine, having structural formula:

nitrendipine, having structural formula:

1,l-Dimethyl-2-[N-(3,3-diphenylpropyl₎-N-methyl- amino]ethyl methyl l,4-dihydro-2,6-dimethyl-4- ⁽3-nitrophenyl⁾-3,5-pyridinedicarboxylate hydrochloride, having the structural formula:

HC1

Among diphenylpiperazmes of interest are cinnarizine and flunarizine, having structural formula:

and

In addition, the calcium entry blockers of this invention may include such calcium channel antagonists as phenylalkylamines, such as verapamil and adipamil, and benzothiazepines, such as diltiazem.

Excitatory amino acid receptor antagonists include MK-801, 2-APV and CNQX, having the structural formula:

and

respectively.

Further, the pharmaceutically acceptable salts of any of the above-designated compounds may be employed as the calcium entry blocker in accordance with the invention. Combinations of the aforementioned compounds may likewise be used.

Calcium entry blockers of this invention may be administered orally, parenterally or topically. In acute situations, parenteral and/or topical administration is preferred in order to more rapidly introduce the calcium entry blocker to the target site. For chronic therapy, oral administration is normally preferred since it is more easily administered. The compounds for use in this invention are administered in their pure form or in admixture with a pharmaceutically acceptable carrier such as an organic or inorganic solid or liquid excipient (depending on the desired administration). The pharmaceutical preparations may thus be administered as a solid, semi-solid, lyophilized powder, liquid dosage form, tablets, pills, capsules, powders, solutions, suspensions, emulsions, creams, lotions, ointments, or granules, as well as injectable solutions. The nature of the composition in the pharmaceutical carrier or diluent will, of course, depend upon the intended route of administration.

When the pharmaceutical composition is in the form of a solution or suspension, examples of appropriate pharmaceutical carriers or diluents (depending on the intended route of administration) include for aqueous systems, water; for non-aqueous systems, ethanol, glycerin, propylene glycol, corn oil, olive oil, syrup, cottonseed oil, peanut oil, sesame oil, parafins and mixtures thereof with water; and for solid systems, lactose, kaolin, mannitol, sucrose, gelatin and agar.

In addition to conventional pharmaceutical carriers or excipients, the pharmaceutical compositions may include other medicinal agents, pharmaceutical agents, adjuvants, stabilizers, anti-oxidents, preservatives, lubricants, suspending agents, and viscosity modifiers, etc.

The dosage level of the calcium entry blocker within this invention is dependent upon the conditions of the disease to be treated, the administration route employed, the subject and the pharmacokinetic and pharmacodynamic characteristics of the active ingredient. The dosage of the active ingredient is generally within the range from about 0.1 to about 100 mg/kg administered orally, parenterally or topically.

When administered either parenterally or topically, the physiological pH is generally in the range of about pH 6.5 to 8.

Methods are further described for screening compounds capable of reversing retinal malfunction the effect of retinal dysfunction, where an in vivo bioassay is employed involving rats with inducible retinal dysfunction. Specific compounds for treating retinal dysfunction are provided associated with modulation of calcium channel activity and/or the activation of excitatory amino acid receptors. Particularly, calcium channel antagonists or other compounds having equivalent effect (excitatory amino acid antagonists) can be used in the treatment of retinal vasculopathy. One methodology involves the use of Dahl salt-sensitive (SS) rats which are available from Harlan Sprague-Dawley. The rats will generally be in the age group of three to twenty weeks, usually in the age group of four to twelve weeks. When placed on a high salt diet, the animals rapidly develop (2-4 weeks) a systemic hypertension. Other rats which may be used are normal Sprague Dawley (albino) rats, Long Evans pigmented rats or spontaneously hypertensive (SHR) (albino) rats.

All of these rats may be employed as models by creation of acute retinal ischemia in their eyes. The ischemia may be created by reversibly occluding the short posterior ciliary arteries and the central retinal artery. Electroretmograms are recorded prior to, during and after occlusion. The occlusion is reversed after a brief period, usually one minute to three hours, preferably five minutes to two hours and reperfusion occurs. During reperfusion ERGs are taken to provide an index of retinal function, followed by a histologic examination to determine changes in normal retinal structure. Ophthalmoscopic examination of the eyes is also performed to document the absence of retinal blood flow and gross ischemic damage. Drug efficacy is related to the ability of the candidate composition to reduce or prevent pathologic changes noted in ERG and histologic examinations.

For histological examination, the eyes may be fixed by cardiac perfusion with a fixative, such as a combination of paraformaldehyde and glutaraldehyde in an appropriate buffer. After removal of the eyes, the globe may be opened at the ora serrata and fixation continued for four to twenty-four hours. Segments of the central and peripheral retinal are then dissected free, the tissue washed and then post fixed in an appropriate fixative, e.g., osmium tetroxide. Following dehydration, the sample may be sectioned in accordance with conventional techniques for light and electron microscopy. Changes in thickness on the retinal layer or number of cell bodies per unit area in the inner and outer nuclear layers may then be observed and reported. In addition, the retinas may be reported as "normal", if all layers are intact with no abnormalities; "mild degeneration", if thinning of the inner and outer segment or visible reduction in cell bodies of the inner and oute nuclear layers has occurred; and "severe degeneration", i extensive loss of any individual or multiple layers of th retina has occurred.

To evaluate retinal function, an electroretinogram (ERG) may be employed. Functional assessment of the inne and outer layers of the neural retina and the non-neural retina (RPE) is made by means of full field ERGs. The wave forms of the ERG result from the electrophysiological processes involved in visual transduction in the retina. Reduction in these waves provides a direct measurement of retinal function. The initial negative deflection, terme the "a-wave", originates in the photoreceptors. The subsequent b-wave is produced by the Muller and bipolar cells from the inner retina. The much slower positive c-wave arises from the RPE but is generally reduced or absent in adult albino rats. Whereas the photoreceptors and RPE are nourished by the choroidal circulation, the Muller and bipolar cells are nourished primarily by the retinal vessels. An initial indication as to the site of retinal ischemia may be related to selective reductions in the individual wave forms.

Base-line ERGs may be obtained prior to induction of retinal ischemia. Thereafter, ERGs are determined at convenient intervals, e.g. hourly, daily or weekly. These subsequent ERGs are then normalized to preischemic values and are expressed as the percent of control (i.e. baseline) values. Prior to dark adaptation, the rat host receives an ophthalmoscopic examination to ensure the absence of cataracts or other gross abnormalities. Since rats are primarily a rod-dominated (98%) animal, ERGs are performed under dark-adapted conditions (12-14 hours). Rats are anesthetized and placed on a heating pad to maintain normal body temperature. To record ERGs, small agar-Ag/AgCl electrodes are placed on the cornea and tongue. A reference ground electrode is placed under the scalp. ERG signals may be amplified by an appropriate differential amplifier and recorded. Light stimulation is provided by an appropriate photostimulator in conjunction with a series of neutral density filters.

Single flash (10 μsec duration) of white light is used to generate individual ERGs. The amplitude of the b-waves is measured from base line to peak in the absence of an a-wave or from the trough of the a-wave to the peak of the b-wave. a-Waves are measured form the base line to the peak of the a-wave. The time interval from the onset of the flash to the peak of the a- and b-waves is used for measurements of latency.

Group data are compared by means of a two-way analysis of variance. Comparisons involving two means employ Students t-test for non-paired data. Differences between groups (control vs. drug-treated) are regarded as significant if P-values are 0.05.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL The methodology involves the creation of acute retinal ischemia in the eyes of normal Sprague-Dawley (albino) or Long-Evans (pigmented) rats, which are available from Harlan Sprague-Dawley. Adult rats were used, ranging in weight from 175 g to 250 g. These rats were housed under normal conditions and fed standard rat chow. Rats were anesthetized with 50 mg/kg sodium pentobarbital mtraperitoneally (i.p.) and the iris of the eye dilated with one drop of 10% atropine solution. Total retinal ischemia in these animals was created by reversibly occluding the short posterior ciliary arteries and the central retinal artery. The duration of the occulsions varied from five to 120 minutes. Prior to the occlusion, baseline ERGs were recorded and used as an -13-

index of normal retinal function. Complete retinal occlusion was determined by the absence of ERG. At the end of the occlusion period, the retina was allowed to reperfuse, and changes in normal retinal structure and function determined by histological observations and ERGs During the reperfusion period, ERGs were evaluated at one to two minute intervals for the first 30 minutes and thereafter at ten minute intervals through 120 minutes. Additional, ERG evaluations in selected animals were made at 24 hours. Drug efficacy was based on the ability of a compound to minimize or prevent the pathologic changes in retinal structure and/or function induced by acute retina ischemia (e.g. the appearance of necrotic cells within th retina or a significant reduction or loss of normal wave forms in the ERG. )

Example 1 illustrates an in vivo bioassay which can be employed for determining the efficacy of compounds in the treatment of retinal dysfunction.

Examples 2-3, conducted in accordance with the procedure of Example 1, demonstrate that pretreatment wit Ca channel antagonists can protect retinal function (as measured by ERG recovery) from ischemic injury. Values are means ± standard errors and have been normalized (0-100%) to preocclusion control values. At each time point tested, significant improvement in b-wave recovery when compared to control-treated animals is exhibited.

Example 4 is drawn to the use of an excitatory amino acid antagonist. Example 1 The subject invention provides for retinal degeneration models as evidenced by both structural and functional changes. Associated with the retinal dysfunction and/or degeneration is a dramatic reduction in retinal perfusion. These rats are therefore good models for screening compounds having activities as calcium channel antagonists or excitatory amino acid antagonists a their use in preventing or ameliorating retinal degenerati Four different periods of retinal ischemia in Long-Evans and Sprague-Dawley rats were examined. In normal Sprague-Dawley rats occlusions of five minutes resulted in the rapid return to control level of both a- and b-waves of the ERG, while occlusions of two hours result in the irreversible loss of retinal function, as measured by the ERG. Occlusion for periods between five minutes to two hours in both Long-Evans and Sprague-Dawley rats resulted in a partial but permanent loss of retinal function, that was amenable by drug therapy.

Reperfusion following 30 minutes of total retinal ischemia resulted in rapid recovery of the a-wave in one to two minutes. The recovery of the b-wave was considerably different. The b-wave was first observed between 16 and 22 minutes. From this point the b-wave slowly recovered over the next 60 to 120 minutes, but remained significantly reduced from the control levels. By 120 minutes, the b-wave has recovered to approximately 30% of control values. By 24 hours the mean b-wave was still only 40% of control values. For shorter periods of occlusion (e.g. 15 minutes), the a-wave again rapidly recovered in one to two minutes. The initial appearance of the b-wave also occurred at 16 to 22 minutes of reperfusion, but the magnitude of the a-wave recovery at 90 minutes and 24 hours was 61% and 100% of control levels (as compared to 26% and 40%, respectively, for the 30 minute occlusion). These data indicate that total retinal ischemia for 30 minutes results in the partial loss of retinal function. This loss appears to be permanent, as the b-wave recovery was only 40% of control values after 24 hours of reperfusion. The rapid return of the a-wave and gradual return of the b-wave indicates that the primary site of acute retina ischemic injury is the inner retinal layer. -15-

Example 2

Long-Evans rats were treated i.p. with control (10% TWEEN 80) or nifedipine 30 minutes prior to the occlusion of retinal vessels.

Table I shows the effect of nifedipine i.p. on b-wav recovery following 30 minutes of total retinal ischemia (*P<0.05).

TABLE I

The ability of the 3.3 mg/kg dose to provide apparently better protection of retinal function than the 10 and 33 mg/kg dose likely reflects cardiovascular side effects of nifedipine, as significantly greater reductions in heart rate and blood pressure were observed in these animals. Hence, the resulting dose-related reduction in cardiac output and peripheral vasodilation likely reduces retinal perfusion in the ischemic eye and reduces functional recovery (e.g. ERG's) of the retina.

Example 3

Long-Evans rats were treated mtraperitoneally with either 10% TWEEN 80 as a control or 1,1-Dimethyl-2-[N-(3,3-diphenylpropyl)- N-methyl-amino]ethyl methyl 1,4-dihydro-2,6-dimethyl-

4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate hydrochloride 30 minutes prior to the occlusion of retinal vessels. Statistical comparisons were made and the results tabulated at each time point. (*P<0.05).

Unlike Ca channels, which are located in both retinal neurons and vessels, excitatory amino acid receptor are located only in the retina. Hence, the in vitro chick retina assay, an assay independent of retina blood flow, was used to evaluate these excitatory amino acid receptor antagonists. Chick retinas were isolated from a day 14 embryo. Isolated retinas were then incubated for 40 or 60 minutes in a control Ringer's solution (5 mM glucose under an atmosphere of 95% air, 5 C0₂ ) or in a test Ringer's solution (0 mM glucose under a atmosphere of 95% N₂, 5% C0₂) . In selected experiments, the NMDA antagonist, MK 801 (10~⁶ to 10~⁴M), was added t retinas incubated in the test Ringer's solution. At the end of the incubation period retinas were fixed in 4% paraformaldehyde, dehydrated in ethanol and embedded in paraffin. Thick (4μm) cross-section of the retina were then cut, stained with haematoxylin and eosin, and evaluated by light microscopy to determine the degree of retinal degeneration.

Control retinas (i.e. incubate in Ringer's with glucose under 95% air) showed no damage or alteration in retinal structure following incubation up to 60 minutes. Retinas incubated in the test Ringer's solution showed signs of cellular degeneration in the ganglionic and inne plexiform layers and edema in the inner nuclear, outer plexiform and inner plexiform layers. The administratio of 10~⁶ M to 10~⁴ M MK 801 to retinas incubated in test Ringer's caused a dose related improvement in these -17-

structural integrity of the retina, with all layers present in the MK 801-treated retinas, when compared to nontreated retinas. In addition, the edema noted in retinas incubated in the test Ringer's was reduced by the administration of MK 801.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

-18-CLAIMS

1. A method of treating a subject suffering from ischemia or edema of the retina or optic nerve which comprises administering to said subject a therapeutically effective amount of a calcium entry blocker.

2. The method of claim 1, wherein said compound is azaheterocyclic.

3. The method of claim 2, wherein said compound is selected from the group consisting of calcium channel antagonists and excitatory amino acid antagonists.

4. The method of claim 1, wherein said compound is administered topically, parenterally or orally.

5. The method of claim 3, wherein said compound is a calcium channel antagonist.

6. The method of claim 5, wherein said calcium channel antagonist is a dihydropyridine.

7. The method of claim 6, wherein said dihydropyridine is selected from the group consisting of nifedipine, nimodipine, nisoldipine, nitrendipine and 1,l-dimethyl-2-[N-(3,3-diphenylpropyl)-N-methyl-amino] ethyl methyl l,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)- 3,5-pyridinedicarboxylate hydrochloride.

8. The method of claim 7, wherein said compound is nifedipine.

9. The method of claim 7 , wherein said compound is 1,l-dimethyl-2-[N-(3,3-diphenylpropyl)-N-methyl-amino] ethyl methyl l,4-dihydro-2,6-dimethyl-4-(3-nitropheryl)- 3,5-pyridinedicarboxylate hydrochloride. -19-

10. The method of claim 5, wherein said calcium channel antagonist is a diphenylpiperazine.

11. The method of claim 10, wherein said diphenylpiperazine is selected from the group consisting of cinnarizine and flunarizine.

12. The method of claim 5, wherein said calcium channel antagonist is a phenylalkylamine.

13. The method of claim 12, wherein said phenylalkylamine is selected from the group consisting of verapamil and adipamil.

14. The method of claim 5, wherein said calcium channel antagonist is a benzothiazepine.

15. The method of claim 14, wherein said benzothiazepine is diltiazem.

16. The method of claim 3, wherein said compound is an excitatory amino acid antagonist.

17. The method of claim 16, wherein said excitatory amino acid antagonist is selected from the group consisting of MK-801, 2-APV and CNQX.

18. A method of preventing ischemia or edema of the retina or optic nerve which comprises administering to a subject a prophylactically effective amount of a calcium entry blocker.

19. The method of claim 18, wherein said calcium entry blocker is a compound selected from the group consisting of calcium channel antagonists and excitatory amino acid antagonists.

20. The method of claim 18, wherein said compound is administered topically, parenterally or orally.

21. A method for evaluating a physiologically active compound for the treatment of retinal dysfunction resulting from cellular calcium overload, said method comprising: administering to a rat suffering from retinal ischemia, a calcium channel modulating amount of a drug, which is a calcium channel antagonist or excitatory amino acid antagonist, wherein said retinal ischemia is a result of occlusion of at least one of the short posterior ciliary arteries and the central retinal artery; and evaluating at least one of an ERG, histopathology of the retina of said rat or ophthalmoscopic examination of the retina of said rat, as an indication of the effect of said drug.

22. The method according to claim 21, wherein said rat is an adult Sprague-Dawley (albino) rat, Long-Evans pigmented rat, hypertensive Dahl salt-sensitive (albino) rat or spontaneously hypertensive (SHR) (albino) rat.

23. The method according to claim 22, wherein said drug is an azaheterocycle calcium channel antagonist.

24. The method according to claim 22, wherein said drug is an excitatory amino acid antagonist.

25. The method according to claim 21, wherein the a-, and b-waves are determined in said ERG.

26. The method according to claim 21, wherein said occlusion is for 5 to 120 minutes.

27. The method according to claim 22, wherein both the posterior ciliary arteries and/or the central retinal artery are occluded.