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1 From the Department of Pharmacology, Tokyo Research Laboratories, Kowa Company, Ltd., Tokyo, Japan; and the 2 Department of Ophthalmology, University of Tokyo, School of Medicine, Japan.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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1,ß-blocker
with a nitric oxide donative action, on
N-methyl-D-aspartate (NMDA)–induced retinal
damage in rats and to determine whether topically instilled nipradilol
penetrates the ipsilateral posterior retina–choroid at
pharmacologically active concentrations in rabbits. METHODS. To determine effects on NMDA-induced damage, drugs were injected alone or with NMDA into the vitreous of one eye, and cell loss in the ganglion cell layer (GCL) and thinning of the retinal neural cell layers were histologically evaluated. To evaluate posterior penetration, first, [14C]-nipradilol was instilled, and its tissue concentration was measured. Second, nipradilol or timolol was instilled, and their effects on intravitreal injection of endothelin-1–induced retinal artery contraction were compared, to evaluate whether a pharmacologically active level of nipradilol penetrates the inner limiting layer by topical application.
RESULTS. Intravitreous injection of NMDA reduced cell numbers in the GCL and the thickness of the inner plexiform layer (IPL) to 50.4% ± 2.6% and 47.8% ± 4.9% (n = 8) of control, respectively. Nipradilol alone had no effect. Coadministration of nipradilol with NMDA reduced cell numbers in the GCL and IPL thickness to 67.8% ± 2.2% and 74.4% ± 5.2% of control, respectively (P < 0.05–0.01). Sodium nitroprusside, but not timolol or bunazosin, also significantly prevented the NMDA-induced reduction of cell numbers in the GCL and IPL thickness. Radioactivity of nipradilol was found in the ipsilateral posterior retina–choroid at 318.6 ± 42.9 ng/g (n = 4), which was significantly higher than in the contralateral control (107.4 ± 21.8 ng/g). Topical application of nipradilol, but not timolol, significantly suppressed the endothelin-1–induced contraction of the retinal artery (83.95% ± 8.15% and 35.24% ± 5.62% of baseline vessel diameter for nipradilol and timolol, respectively).
CONCLUSIONS. Nipradilol suppressed the NMDA-induced retinal damage in rats for which nitric oxide released from nipradilol may be responsible. Posterior penetration studies suggested that an effective concentration of nipradilol reached the posterior retina after topical application.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1-receptor blocking
properties1
2
3
with a nitric oxide (NO) donative
action.4
Topical administration of 0.25% nipradilol
lowered intraocular pressure (IOP) both in rabbits and humans by
reducing aqueous production and by increasing uveoscleral
outflow.5
6
The ocular hypotensive effect was equipotent
to that of 0.5% timolol in glaucoma patients with less systemic
ß-blocking effects,7
and nipradilol has therefore been
registered as an antiglaucoma ophthalmic solution in Japan. Further,
unilateral topical application of nipradilol significantly increased
the optic nerve head (ONH) blood velocity on the ipsilateral treated
side in rabbits, possibly through the NO donative and/or
1-blocking action of nipradilol, which
penetrated locally.5 Evidence has been accumulating that NO plays a crucial role in neural degeneration, including the loss of retinal ganglion cells.8 9 10 11 12 Vorwerk et al.10 have shown that the NO released from neuronal NO synthase (nNOS) is a prerequisite for the full expression of excitotoxicity in the retina by use of nNOS-deficient mice. Additionally, Neufeld et al.13 reported that the inhibition of inducible NOS (iNOS) prevented ganglion cell loss in an elevated IOP model. These results suggest that NO released from several isoforms of NOS participates in various degenerated states and tissues. On the contrary, NO plays important roles in physiologic maintenance such as basal tone in the retinal circulation and visual transduction,8 and reports have suggested that it has neuroprotective effects.14 15 16 17 18
To investigate these, we first investigated the neuroprotective effects
of nipradilol on N-methyl-D-aspartate
(NMDA)–induced retinal damage in comparison with those of the
nonselective ß-blocker timolol, an
1-selective blocker bunazosin,19
and a NO-donor sodium nitroprusside (SNP) in rats. Second, we studied
by two methods in rabbits whether topically instilled nipradilol could
penetrate the posterior retina and choroid. The first method was direct
measurement of topically applied
[14C]-nipradilol in retina–choroid, and the
second was a look at whether topical nipradilol reaches the retinal
inner limiting layer at a pharmacologically active level by use of the
inhibitory action of nipradilol against endothelin (ET)-1–induced
retinal artery contraction as an in vivo parameter. The results of the
second study may also be applicable to other topical drugs.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Effects on NMDA-Induced Retinal Damage
Male Sprague–Dawley rats (7 weeks old; Japan Laboratory
Animals, Tokyo) were anesthetized by intraperitoneal injection of
sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago,
IL). The methods for determining the volume of intravitreal injection,
doses, and histologic evaluations were essentially the same as
described in previous reports.20
21
22
23
Briefly, body
temperature was kept at 37°C with a heating pad (KN-474-S; Natsume,
Tokyo, Japan) throughout the experiment. A 33-gauge needle was inserted
into the midvitreous of one eye chosen at random under a stereoscopic
microscope with care to avoid lens injury. The other eye received the
vehicle solution as a control. A single 5-µl injection of the drug
into one eye was completed over 1 minute. The following drugs were
administered with or without 4 x
10-2 M NMDA (Sigma, St.
Louis, MO): 2 x 10-3
M
(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine
hydrogen maleate (MK-801; Nacalai Tesque, Kyoto, Japan),
10-10 to
10-6 M nipradilol
hydrochloride, 10-8 to
10-6 M timolol maleate
(Sigma), 10-8 to
10-6 M bunazosin
hydrochloride, and 10-8 to
10-6 M SNP (Sigma).
Nipradilol (purity, more than 99.5% by high–performance liquid
chromatography [HPLC]) and bunazosin (purity, more than 99.5% by
HPLC) were synthesized in our laboratory. Nipradilol was dissolved in
equimolar amounts of hydrogen chloride to obtain a
10-2-M solution and
diluted by 0.1 M phosphate buffer (pH 7.0). Other drugs were dissolved
in 0.1 M phosphate buffer (pH 7.0).
Seven days after the injection, both eyes were enucleated, and three sequential meridian sections (3 µm thick) were made through the optic disc. Sections were stained with hematoxylin and eosin, and the number of cells in the ganglion cell layer (GCL), and the thickness of the inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) were measured as previously reported.20 21 22 Morphologically distinguishable glial cells and vascular endothelial cells were excluded from the cell count, as described by Lam et al.24 Data from three sequential sections were averaged for each eye. The values for the treated eye of each animal were normalized to those for the contralateral vehicle-treated control and were shown as a percentage. All histologic measurements were performed by an investigator masked to the treatment.
Posterior Penetration of Topically Instilled Nipradilol
[3-14C]-nipradilol (Code CFQ11032,
radiochemical purity 98%, specific radioactivity 1.55 MBq (43
µCi)/mg) was obtained from Amersham Pharmacia Biotech
(Buckinghamshire, UK). Four male Japanese White (JW) rabbits (10 weeks
old, Japan Laboratory Animals) were used. Rabbits were placed in a
restraining cage and [14C]-nipradilol (1%, 100
µl, 1.5 MBq [41 µCi]/dose) was instilled twice daily (10
AM and 6 PM) for 7 days into the lower cul-de-sac of the right eye.
Thirty minutes after the final instillation, rabbits were bled from the
marginal ear artery and deeply anesthetized with pentobarbital. To
minimize contamination of the intraocular tissues, enucleation and
dissection of the eye were performed by the following procedure. A cut
was made along the edge of the orbit, and both eyelids were clamped by
surgical clip. Grasping the clamped eyelids enabled enucleation of the
globe together with the eyelids and surrounding tissues, as a pouch.
This pouch was immersed immediately in a mixture of hexane and solid
carbon dioxide for 2 minutes and then stored at -15°C. The next day,
posterior surrounding connective tissues were removed, and the frozen
globe was cut circle-wise at a radius of 5 mm with the optic nerve for
its center for division into anterior and posterior cups. Vitreous and
then retina–choroid were exfoliated from the vitreous side of the
posterior cup. The lens, iris, aqueous humor, and cornea were isolated
from the vitreous side of the anterior cup. Isolation of the tissue was
performed under semifrozen conditions. All tissues were dissolved, and
radioactivity was measured by a liquid scintillation counter (Tri-Carb
Liquid Scintillation Analyzer 2700TR; Packard Instruments, Meriden,
CT).
Thirteen male JW rabbits (10 weeks old; Japan Laboratory Animals) were used in the intravitreal endothelin-1 (ET-1 human; Sigma) injection study. To serve as a control for the later experiments, physiological saline (50 µl) was instilled twice daily (10 AM and 6 PM) for 7 days into the lower cul-de-sac of both eyes. Thirty minutes after the final instillation, 20 µl of 5 x 10-8 M ET-1 was injected into the vitreous of both eyes. Care was taken to inject accurately between the lens and posterior fundus. Photographs of the ocular fundus were obtained with a fundus camera (RC-XV3; Kowa, Tokyo, Japan) at 5 minutes before and 15, 30, 45, and 60 minutes after the ET-1 injection and were captured in an image filing system (VK-2; Kowa). The diameter of the two major retinal arteries just at the rim of the ONH and the diameter of the ONH were measured. The average diameter of the arteries was normalized by the ONH diameter and was expressed as a percentage of the diameter 5 minutes before the ET-1 injection. IOP was measured 5 minutes before the final instillation of nipradilol or timolol and 5 minutes before the ET-1 injection. Nipradilol (0.25%, 50 µl) or timolol (0.5%, 50 µl) was instilled in one randomly selected eye for 7 days according to the schedule described earlier. Vehicle or saline was instilled in the other eye. Fundus photographs were obtained 5 minutes before and 60 minutes after the ET-1 injection at the base of the control experiment. All procedures were performed by an investigator masked to the treatment.
All data are presented as the mean ± SEM. Data were analyzed using the paired t-test, Wilcoxon signed-rank test, or Dunnett or Bonferroni multiple comparison test, as appropriate. P < 0.05 was considered statistically significant.
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Results |
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Discussion |
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NMDA-induced cell loss in the GCL and reduction of IPL thickness were
significantly suppressed by the coadministration of nipradilol.
Assuming that the volume of the rat vitreous is approximately 50
µl,37
the effective concentration at the retinal surface
would be one tenth of that obtained when nipradilol diffused uniformly,
or somewhat higher. Because the negative logarithm of dissociation
constant for competitive antagonist (pA2) values of
nipradilol were 8.9 for nonselective ß-blocking and 6.5 for selective
1-blocking actions,
respectively,1
2
3
it is conceivable that the effective
concentration of nipradilol elicits both receptor blocking actions at
the retina. We also examined the effects of a nonselective ß-blocker,
timolol,38
and a selective
1-blocker, bunazosin,19
on the
NMDA-induced retinal damage. But neither blocker had any effect at
concentrations sufficient to cause ß- or
1-receptor blockade.
In this study, we found a protective effect of nipradilol, as well as of SNP, on NMDA-induced damage. Nipradilol was not histologically toxic to normal retina up to 10-4 M (data not shown). The underlying mechanism by which nipradilol or the related NO donor SNP protects neural cells against NMDA-induced retinal damage is not clear. There is evidence that NMDA-induced retinal ganglion cell damage can be modulated by the ß1-selective blocker, betaxolol, the mechanism for which is reported at least as a Ca2+ channel-blocking action.39 40 This does not apply to nipradilol, however, because nipradilol does not have Ca2+ channel-blocking activity. One explanation for the protective effect is that it causes vasodilation. A previous report4 and Table 2 in this article suggest that nipradilol has vasodilating activity in the retinal artery. Moreover, the result obtained with SNP, which also has vasodilating activity, suggests that vasodilating agents can have neuroprotective effects. Another possibility is that the NMDA receptor has a redox modulatory site that consists of thiol groups, where disulfide bonds form on the surface of the receptor, and redox agents modulate the receptor activity through changes at this site.14 41 42 It is reported that reducing agents upregulate receptor activity and oxidizing agents downregulate it by forming disulfide bonds. NO acting as an oxidant may cause S-nitrosylation of NMDA-receptor thiol to downregulate the receptor activity.14 41 42 Thus, it may be possible that nipradilol and SNP downregulate the NMDA receptor activity and produce the neuroprotective effect through NO-donating action, but this mechanism of action should be studied in an in vitro system without blood supply.
The current results indicate that nipradilol has neuroprotective properties in NMDA-induced retinal damage, but it was not known whether topically applied nipradilol could reach the retina at a pharmacologically active concentration. To investigate this issue, the ocular penetration of topically instilled nipradilol was studied in rabbits.
It is difficult to enucleate an eyeball free of contamination when enucleation scissors are inserted through the conjunctiva, which has a very high concentration of radioactivity. To minimize such contamination, we enucleated the eye through the orbital skin, bisected the frozen globe into anterior and posterior cups, and isolated the tissue from the vitreous side on which the radioactivity was thought to be the lowest. Additionally, it was confirmed that no radioactivity was present in the hexane that was used to freeze the eyeballs. Each tissue in the treated eye had a higher concentration of nipradilol than that in the nontreated contralateral control, where nipradilol concentrations were approximately the same as or lower than those in blood. Nipradilol concentrations measured in the cornea, aqueous humor or lens were comparable to those found in the previous study for nipradilol43 and for betaxolol.40 The nipradilol concentration in the posterior retina–choroid was 0.98 and 0.33 µM in the treated eye and nontreated contralateral control, respectively. The former value was significantly higher than the latter. The difference was not considered attributable to contamination during the dissecting procedure, because the concentration in the posterior vitreous isolated first was much lower, and the concentration in the retina–choroid in the control eye and was similar to that in the blood.
Rather, we believe this difference indicates that topically applied nipradilol reaches the posterior retina–choroid at an effective concentration, not through the vitreous but through an as yet unidentified route, at least in the rabbit eye. One possibility may be the periocular route. Sponsel et al.44 reported that Tenon capsules accumulate betaxolol or timolol at much higher concentrations than can be obtained intraocularly after long-term topical therapy and suggested that periocular accumulation provides more immediate access to the posterior segment and proximal ocular vasculature for topical drugs. Additionally, Geroski and Edelbauser45 documented that the sclera is quite permeable to a wide range (molecular weight: 285–70,000) of solutes, and the permeability constant for transscleral solutes was inversely related to solute molecular weight. The molecular weight of nipradilol is 326.35, and there is therefore a possibility that nipradilol reaches the retina–choroid through the periocular transscleral route.
In this study, we used 1% nipradilol (100 µl) to evaluate the tissue concentration of drug. The clinical dose and volume of nipradilol are 0.25% and 50 µl, respectively. The minimal estimated concentration of nipradilol in the posterior retina–choroid after application of a 0.25% solution and 50-µl drop would be 0.12 and 0.04 µM in the treated eye and contralateral control, respectively, and the concentration in blood, 0.03 µM. At these concentrations, nipradilol is considered to show pharmacologic activity,1 2 3 4 including neuroprotective action as seen in the current rat experiment. But there is a possibility that most of the drug is bound to tissue(s) and not pharmacologically active, and it was difficult to determine the concentration in the retina in this way because of difficulty in isolating the retinal layer from the choroid in a frozen state.
To determine whether pharmacologically active nipradilol could reach the retinal layer after topical application, we designed the ET-1 intravitreal injection study. Nipradilol exhibits NO-related vasodilating activity by activating the soluble guanylate cyclase and increasing the production of cyclic guanosine monophosphate in the vascular cells.4 We focused on the retinal artery that is present between the vitreous and the retinal inner limiting layer and examined the inhibitory effect of topical nipradilol on ET-1–induced retinal artery contraction. Topical nipradilol significantly suppressed the ET-1–induced contraction of the retinal artery.
The vascular contraction in the vehicle-treated side in the nipradilol group tended to be less than that in the saline- or timolol-treated group. This may be because of the effect of systemically absorbed nipradilol. According to the result of the first rabbit experiment, the nipradilol concentration in blood in the second rabbit experiment would be 0.03 µM or higher. The effective vasodilating concentration of nipradilol was 0.01 µM and higher.4 The suppressive effect against ET-1–induced contraction, however, was significantly stronger in the nipradilol-treated than the vehicle-treated eye. On the contrary, timolol had an equipotent ocular hypotensive effect with nipradilol but no effect on ET-1–induced contraction of the retinal artery, which suggests that the effect of nipradilol was not mediated by the ocular hypotensive effect.
These results suggest that topically applied nipradilol reached the ipsilateral retina at a pharmacologically active concentration in the normal rabbit eye, not through the systemic circulation, but through local penetration. A route through the vitreous seems unlikely, because the concentration in the vitreous was very low. The active nipradilol concentration in the ipsilateral retina is not known, but it should be higher than the lowest vasodilating concentration (0.01 µM).4 This concentration range is not far from the estimated vitreous concentration of nipradilol that showed neuroprotective effect against NMDA-induced retinal damage.
The results obtained from the posterior penetration experiment for nipradilol may be also applicable to other topically applied drugs with a similar molecular weight and lipophilicity. For example, betaxolol has a molecular weight similar to that of nipradilol but is much more lipophilic.46 It is possible that topically instilled betaxolol would reach the ipsilateral retina at a higher concentration than presently observed for nipradilol in normal rabbits.40 The results of the posterior penetration studies should not be directly extrapolated to the human eye, but the possibility that a topically applied drug can reach the posterior parts of the human eye at a pharmacologic concentration is not to be excluded.
In summary, nipradilol had a dose-dependent and significant protective
effect on NMDA-induced retinal damage in the rat eye. SNP, but not the
ß- and 1-blockers, showed a comparable
effect on the NMDA-induced retinal damage. Moreover, it was indicated
that nipradilol could reach the posterior retina after topical
application at pharmacologically active concentrations by local
penetration in the normal rabbit eye. These properties may be
advantageous in an antiglaucoma eye drop.
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Acknowledgements |
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Footnotes |
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Commercial relationships policy: E (KM, TK, MY); N (MA).
Corresponding author: Ken Mizuno, Department of Pharmacology, Tokyo Research Laboratories, Kowa Co., Ltd., 2-17-43 Noguchicho, Higashimurayama, Tokyo 189-0022, Japan. kmizuno{at}kowa.co.jp
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-blocking and nitroglycerin-like activities, on aqueous humor dynamics and fundus circulation Invest Ophthalmol Vis Sci 39,736-743 blocking and nitroglycerin-like activities, on intraocular pressure and aqueous dynamics in humans Br J Ophthalmol 84,293-299
-nitro-L-arginine methyl ester protects retinal neurons against N-methyl-D-aspartate-induced neurotoxicity in vivo Eur J Pharmacol 328,45-49[Medline][Order article via Infotrieve]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 4 x
10-2 M of NMDA (
) and 2 x
10-3 M of MK-801 (
) on cell number in GCL
and thickness of IPL, INL, and ONL. Data are the mean ± SEM;
**P < 0.01 versus vehicle (Dunnett multiple
comparison test); n = 8.
