Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: TEERAPEUTIC USE OF CALCIUM BNTRY BLOCX~RS , ' `~
IN RETINAL OR OPTIC NERVE DYSFUNCTION
INVENTORS: CRAIG E. CROSSON and DAVID E. POTTER ;
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 t`i~
and in vivo bioassays for screening such compounds.
Backqround of the Invention '~ s
Retinal vascular disease and ischemia are associated .
with malfunction of neuroendocrine regulation and
autoregulation of the choroidaI and retinal circulations,
10 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
rctinal 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
: ~,-': `.
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flow in the inner retinal vessels. This greater
propensity for ischemia in the inner retina may result
from several factors: (l) the high rate of choroidal
blood flow over that required to meet the metabolic needs
of the outer retina; (2) the large diameter capillaries in
the choroid are less likely to be occluded by emboli; (3) ,- ~
the lack of anastomoses in the retinal vessels; and (4)^? i
the larger percentage of oxygen extracted from the retinal
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
lS (paracrine and autocrine) factors. -~
A number of systemic and ocular disorders have been
associated with ischemic conditions of the retina or optic
nerve. Ocular manifestations of systemic disorders ~
include: diabetes, atherosclerosis, hyperlipidemia, and -- ;
20 hypertension. Specific ocular disorders include: - i`
retinitis of AIDs, macular degeneration, anterior ischemic
optic neuropathy, ocular hypertension, glaucoma,
retinopathy of prematurity, retinal vessel occlusion,
diabetic retinopathy and hypertensive retinopathy. In
25 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 i
of or prophylactic treatment against vasculopathies and
35 neuropathies associated with the eye. `
Further, it is of great interest to develop a ; - ~-
reproducible and sensitive bioassay which is a good ;- ~
.. .....
2~04~616
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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
10 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 68:113-122, describes potential
therapeutic applications of antagonists of excitatory ~ ~ y;`
amino acid neurotransmitters. Sinclair et al., (1982) J ; ,~ ?
American AcademY of ODhthalmologY 89:748-750, 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
30 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.
59:11B-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
' ~ , :-:
2~0~616
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.:'':
: ;
Leach (1987) J. Clin. Pharmacol. 27:825-834, describe a -
therapeutic application of 1,4-dihydropyridine calcium
channel blockers. Gelmers et al., (1988) N. Enql. J. Med.
318:203-207, describe an investigation of nimodipine in
acute ischemic stroke. Cook and Hof (1988) Br. J.
Pharmacol. 93:121-131, describa the cardiovascular effects
of apamin and BRL 34915 in rats and rabbits. Nihard ~ ;
(1982) Anqioloqy 33:37-45, 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.
27:347-369, describe structure-function relationships for
15 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 O~hthalmology 13:1010-1015; Frank et ;
al., (1986) Science 231:376-378 and Stefansson et al.,
(1988) Invest. O~hthalmol. Vis. Sci. 29:1050-1055.
S ~ ARY OF 1~ INVENTION
Azaheterocycle calcium entry blockers are useful in - :
the treatment of subjects, such as mammals, including man, ~ ;
25 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 dysfunction ;;
are techniques for assessing neural retinal function. In
30 addition, such compounds exhibit prophylactic effects in ~ ;~
preventing such conditions. Methods are further provided
for screening compounds associated with regulation of
calcium channels by employing in vivo bioassays using rats
with inducible retinal dysfunction.
35 DESCRIPTION OF THE SPECIFIC E~ ODIMENTS ~ ~`f5
Compounds associated, either directly or indirectly, -;
with the modulation of calcium entry exhibit a therapeutic
.
2(~0~616
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or prophylactic effect to subjects suffering from ischemia
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~
~4~
COOC ~ COOC~
~C~C~
nimodipine, having the structural formula: ~
~0
~&OOC ~ cooc~ ~c~~ Oct~
i r t~
nisoldipine, having structural formula~
NO~
~C--~ COOC ~ COOC~, .: . . ` - ;,
~C' ~,C~c~' " ,' ~
.: ~, ,.. ". . ~:,
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nitrendipine, having structural formula~
.' ' '~" ~', ~" ''.',
N
~C~OOC ~ COOC~ ; and :~
'' ' "' .,:',~" ~ i'
''''''.'.''..,'"~
,,"." ''`'~". ,''~ ,.' . '','' '
'; " i ' ''..'.'`""~',:'' '
,,,.,,, ~, ,.'',,,~
"' ' " ''.'; ~ i. '~ '"',.'
1,1-Dimethyl-2-[N-(3,3-diphenylpropyl)-N-methyl- ~ :
amino]ethyl methyl 1,4-dihydro-2,6-dimethyl-4-
5 (3-nitrophenyl)-3,5-pyridinedicarboxylate hydrochloride, : .~
having the structural formula: . ~.
~ 2
H3 ~ ~3
~3 ~ CB
~ )046~6 ~ :
-7-
Among diphenylpiperazines of interest are cinnarizine and
flunarizine, having structural formula~
C,H, C=C
H C,H,
and
F~CH--1~1 CH,~ C/ :
H C,H, :
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:
~00~6~
-8-
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 i; `
, "~
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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,
5 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
10 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 ~-
15 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, ~;
20 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
25 capable of reversing retinal malfunction the effect of
retinal dysfunction, where an in vlvo bioassay is employed
involving rats with inducible retinal dysfunction.
Specific compounds for treating retinal dysfunction are
provided associated with modulation of calcium channel ~ ;y"
30 activity and/or the activation of excitatory amino acid `~ ~ `
receptors. Particularly, calcium channel antagonists or
other compounds having equivalent effect (excitatory amino i
acid antagonists) can be used in the treatment of retinal
vasculopathy.
35One 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
2(~046~6 - ~ :
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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 : ~,
5 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.
Electroretinograms 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 i -
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 i;
for four to twenty-four hours. Segments of the central `
30 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 i,'~,
nuclear layers may then be observed and reported. In
2~0~6~6
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 segments
or visible reduction in cell bodies of the inner and outer ~ ~
5 nuclear layers has occurred; and "severe degeneration", if ~ ~-
extensive loss of any individual or multiple layers of the
retina has occurred.
To evaluate retinal function, an electroretinogram
(ERG) may be employed. Functional assess~ment of the inner
10 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, termed
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. ~ereas 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 a normalities. 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. -
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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 ~sec 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
15 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
25 retinal ischemia in the eyes of normal Sprague-Dawley s.
(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
30 chow~ Rats were anesthetized with 50 mg/kg sodium ,
pentobarbital intraperitoneally (i.p.) and the iris of the -ci
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 ;` -
:~:
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2~0461~ ~
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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
10 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 retinal
ischemia (e.g. the appearance of necrotic cells within the
retina or a significant reduction or loss of normal wave
15 forms in the ERG.) `
Example 1 illustrates an ln 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 with
Ca+~ channel antagonists can protect retinal function (as
measured by ERG recovery) from ischemic injury. Values `
are means ~ standard errors and have been normalized ,~,~r
(0-100%) to preocclusion control values. At each time ~ i j , h'"
25 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. ` ~;
Exam~le 1 ~7
The subject invention provides for retinal
degeneration models as evidenced by both structural and i:
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 entry
blockers or excitatory amino acid antagonists and their
use in proventing or ameliorating retinal degeneration.
Z(~0~Çi16
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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 fun~tion, 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 :
10 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% o control values after
24 hours of reperfusion. The rapid return of the a-wave i ~
and gradual return of the b-wave indicates that the ~ ~ -
primary site of acute retina ischemic injury is the inner `~
retinal layer.
: ~ ' '.`':,'
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2~046~6
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Exam~le 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-wave
recovery following 30 minutes of total retinal ischemia
(*P<0.05). ~ -:
TABLE I
TIME FROM
10REPERFUSIONCONTROL 1 mg/kg3.3 mg/kg 10 mg/kg 33 mg/kg
(min) (N=7) (n=4) (n=5) (n=6) (n=5)
5 + 2 13 + 3*20 + 6* 13 + 2* 15 + 5* i. .. -
19 + 4 47 + 2* 60 + 9* 39 + 5* 41 + 6* : . ~ i~
28 + 6 66 + 2*81 + 12* 58 + 9* 51 + 6* ""''' ,'~''''t ' `'l.~'.'
15 120 32 + 6 72 + 2*84 + 13* 65 + 11* 54 + 6* ::
180 34 + 5 70 _ 5*81 + 9* 67 + 9* 53 + 6* :-~
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
20 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 intraperitoneally with
either 10% TWEEN 80 as a control or
1,1-Dimethyl-2-[N-(3,3-diphenylpropyl)-
30 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).
: : ;
X(~0~-~616 ~ : ~
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TABLE IV
TIME FROM
REPERFUSION CONTROL 0.33 mg/kg - -~
(min) (n=7) (n-5)
7.0 i 2 13 i 5* -~
19 i 3 36 i 6*
31 i 2 52 i 5*
120 35 i 2 60 i 3*
180 36 i 6 64 i 4*
10 Exam~le 4 . :~;-:
Unlike Ca channels, which are located in both ;, `~
retinal neurons and vessels, excitatory amino acid
receptor are located only in the retina. Hence, the 1n -~
vitro chick retina assay, an assay independent of retinal -i~ ;
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%
20 CO2) or in a test Ringer's solution (0 mM glucose under an -
atmosphere of 95% N2, 5% CO2). In selected experiments,
the NMDA antagonist, MK 801 (10 5 to 10 4M), was added to
retinas incubated in the test Ringer's solution. At the
end of the incubation period retinas were fixed in 4%
25 paraformaldehyde, dehydrated in ethanol and embedded in ` `. ,
paraffin. Thick (4~m) 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 ceilular degeneration in the ganglionic and inner
plexiform layers and edema in the inner nuclear, outer
plexiform and inner plexiform layers. The administration
of 10 6 M to 10 4 M MK 801 to retinas incubated in test
Ringer's caused a dose related improvement in these
:~' , '.'' '., `'
;~Q0461~
-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
5 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.
:. ....,,,.~:. :"
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