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Figure 1.
Two applications of prostaglandin E2 (PGE2). The eyes received 2 transcorneal diffusions of PGE2 at 1- or 2-week intervals. AUC indicates area under the curve, a measurement of aqueous flare.

Two applications of prostaglandin E2 (PGE2). The eyes received 2 transcorneal diffusions of PGE2 at 1- or 2-week intervals. AUC indicates area under the curve, a measurement of aqueous flare.

Figure 2.
Changes in flare intensity after transcorneal diffusion of prostaglandin E2 (PGE2) with or without topical 2-time instillation of betamethasone phosphate. A, Double instillations 4 and 2 hours, and B, 1 and 0.5 hour before PGE2application. Transcorneal application of PGE2, 25 µg/mL or 7.09 × 10 −2mmol/L, occurred for 4 minutes (black arrow). White arrows indicate topical instillation of 0.1% betamethasone phosphate.

Changes in flare intensity after transcorneal diffusion of prostaglandin E2 (PGE2) with or without topical 2-time instillation of betamethasone phosphate. A, Double instillations 4 and 2 hours, and B, 1 and 0.5 hour before PGE2application. Transcorneal application of PGE2, 25 µg/mL or 7.09 × 10 −2mmol/L, occurred for 4 minutes (black arrow). White arrows indicate topical instillation of 0.1% betamethasone phosphate.

Table 1. 
Anti-inflammatory and Antiallergic Ophthalmic Solutions
Anti-inflammatory and Antiallergic Ophthalmic Solutions
Table 2. 
Effects of Double Instillation of Eyedrops on PGE2-Induced Aqueous Flare Elevation in Pigmented Rabbits*
Effects of Double Instillation of Eyedrops on PGE2-Induced Aqueous Flare Elevation in Pigmented Rabbits*
Table 3. 
Effects of Single Instillation of Eyedrops on PGE2-Induced Aqueous Flare Elevation in Pigmented Rabbits*
Effects of Single Instillation of Eyedrops on PGE2-Induced Aqueous Flare Elevation in Pigmented Rabbits*
1.
Hirata  HHiraki  SKaji  YTakeda  NFukuo  YTachinami  K The effects of transcorneal administration of prostaglandin E2 on rabbit eyes. Nippon Ganka Gakkai Zasshi. 1994;98927- 934
2.
Numata-Watanabe  KHirata  HHiraki  SHayasaka  S Decreased aqueous-flare reaction to repeated applications of prostaglandin E2 to the cornea in pigmented rabbits. Ophthalmic Res. 199628147- 152
3.
Watanabe  KHayasaka  SHiraki  S  et al.  Effects of topical clonidine on prostaglandin E2-induced aqueous flare elevation in pigmented rabbits. Ophthalmic Res. 2000;32210- 214Article
4.
Yanagisawa  SHayasaka  SZhang  XYHayasaka  YNagaki  YKitagawa  K Effect of topical betaxolol on acute rise of aqueous flare induced by prostaglandin E2 in pigmented rabbits. Jpn J Ophthalmol. 2001;45669- 671Article
5.
Hayasaka  YHayasaka  SZhang  XYNagaki  Y Effects of topical antiglaucoma eye drops on prostaglandin E2–induced aqueous flare elevation in pigmented rabbits. Invest Ophthalmol Vis Sci. 2002;431142- 1145
6.
Othenin-Girard  PTritten  JJPittet  NHerbort  CP Dexamethasone versus diclofenac sodium eyedrops to treat inflammation after cataract surgery. J Cataract Refract Surg. 1994;209- 12Article
7.
Miyake  KMasuda  KShirato  S  et al.  Comparison of diclofenac and fluorometholone in preventing cystoid macular edema after small incision cataract surgery: a multicentered prospective trial. Jpn J Ophthalmol. 2000;4458- 67Article
8.
Berdy  GJAbelson  MB Antihistamines and mast cell stabilizers in allergic ocular disease. Albert  DMJacobiec  FAeds.Principles and Practice of Ophthalmology. Philadelphia, Pa WB Saunders Co1994;1028- 1042
9.
Abelson  MBButrus  S Corticosteroids in ophthalmic practice. Albert  DMJacobiec  FAeds.Principles and Practice of Ophthalmology. Philadelphia, Pa WB Saunders Co1994;1013- 1022
10.
O'Banion  MKSadowski  HBWinn  VYoung  DA A serum- and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein. J Biol Chem. 1991;26623261- 23267
11.
Masferrer  JLReddy  STZweifel  BS  et al.  In vivo glucocorticoids regulate cyclooxygenase-2 but not cyclooxygenase-1 in peritoneal macrophages. J Pharmacol Exp Ther. 1994;2701340- 1344
12.
To  KAbelson  MBNeufeld  A Nonsteroidal anti-inflammatory drugs. Albert  DMJacobiec  FAeds.Principles and Practice of Ophthalmology. Philadelphia, Pa WB Saunders Co1994;1022- 1027
13.
Battistini  BBotting  RBakhle  YS COX-1 and COX-2: toward the development of more selective NSAIDs. Drug News Perspect. 1994;7501- 512
14.
Sawa  MTsurimaki  YTsuru  TShimizu  H New quantitative method to determine protein concentration and cell number in aqueous in vivo. Jpn J Ophthalmol. 1988;32132- 142
15.
Leibowitz  HMBerrospi  ARKupferman  ARestropo  GVGalvis  VAlvarez  JA Penetration of topically administered prednisolone acetate into the human aqueous humor. Am J Ophthalmol. 1977;83402- 406
16.
Noonan  WDSamples  JR Diclofenac sodium. J Toxicol Cutan Ocul Toxicol. 1993;12265- 272Article
17.
Sanders  DRKraff  M Steroidal and nonsteroidal anti-inflammatory agents. Arch Ophthalmol. 1984;1021453- 1456Article
18.
Flach  AJKraff  MCSanders  DRTanenbaum  L The quantitative effect of 0.5% ketorolac tromethamine solution and 0.1% dexamethasone sodium phosphate solution on postsurgical blood-aqueous barrier. Arch Ophthalmol. 1988;106480- 483Article
19.
Kraff  MCSanders  DRMcGuigan  LRaanan  MG Inhibition of blood-aqueous humor barrier breakdown with diclofenac. Arch Ophthalmol. 1990;108380- 383Article
20.
Tilden  MEBoney  RSGoldenberg  MMRosenbaum  JT The effects of topical S(+)–ibuprofen on interleukin-1 induced ocular inflammation in a rabbit model. J Ocul Pharmacol. 1990;6131- 135Article
21.
Miller  JDEakins  KEAtwal  M The release of PGE2-like activity into aqueous humor after paracentesis and its prevention by aspirin. Invest Ophthalmol. 1973;12939- 942
22.
Toris  CBCamras  CBYablonski  MEBrubaker  RF Effects of exogenous prostaglandins on aqueous humor dynamics and blood-aqueous barrier function. Surv Ophthalmol. 1997;41suppl 2S69- S75Article
Laboratory Sciences
July 2002

Effects of Topical Anti-inflammatory and Antiallergic Eyedrops on Prostaglandin E2–Induced Aqueous Flare Elevation in Pigmented Rabbits

Author Affiliations

From the Department of Ophthalmology, Toyama Medical and Pharmaceutical University, Toyama, Japan.

Arch Ophthalmol. 2002;120(7):950-953. doi:10.1001/archopht.120.7.950
Abstract

Objective  To evaluate the role of topical instillation of anti-inflammatory or antiallergic agents on experimental elevation of aqueous flare induced by prostaglandin E2 (PGE2) in pigmented rabbits.

Methods  Transcorneal diffusion of PGE2, 25 µg/mL (7.09 × 10 −2mmol/L), by means of a glass cylinder produced aqueous flare elevation. Anti-inflammatory or antiallergic agents were topically administered once or twice before PGE2 application. Aqueous flare was measured with a laser flare-cell meter. Results are given as mean ± SD.

Results  Double instillations of 0.1% betamethasone sodium phosphate and 0.1% fluorometholone acetate at 4 and 2 hours before PGE2 application inhibited 61% ± 11% and 46% ± 14%, respectively, of flare elevation. Double instillations of 0.1% diclofenac sodium and 0.1% pranoprofen at 4 and 2 hours before PGE2 application did not inhibit flare elevation. Double instillations of 0.1% betamethasone, 0.1% fluorometholone, 0.1% diclofenac, and 0.1% pranoprofen at 1 and 0.5 hour before PGE2 application inhibited 16% ± 10%, 16% ± 6%, 24% ± 9%, and 23% ± 10%, respectively, of flare elevation. Double instillations of 2% cromolyn sodium, 0.5% tranilast, 0.025% levocabastine hydrochloride, 0.1% pemirolast potassium, and 0.01% ibudilast at 1 and 0.5 hour before PGE2 application did not inhibit flare elevation. Single instillation of 0.1% betamethasone 6 hours before PGE2 application inhibited 88% of PGE2-induced aqueous flare elevation. Single instillation of 0.1% diclofenac 1 hour before PGE2 application inhibited 23% of PGE2-induced aqueous flare elevation.

Conclusions  Betamethasone needed several hours after topical instillation to inhibit flare elevation, but diclofenac needed 1 hour. Antiallergic agents did not affect disruption of the blood-aqueous barrier in rabbits.

Clinical Relevance  Corticosteroid eyedrops may need several hours from instillation to show action.

PREVIOUS STUDIES from our laboratory have shown that transcorneal diffusion of prostaglandin E2 (PGE2) by means of a glass cylinder induced aqueous flare elevation in pigmented rabbits, and that the elevation was reproducible when PGE2 was applied at an interval of more than 1 week.1,2 We also found that topical instillation of 0.25% clonidine hydrochloride, 0.5% betaxolol hydrochloride, 1.15% apraclonidine hydrochloride, 1.25% epinephrine bitartrate, or 0.04% dipivefrin hydrochloride inhibited flare elevation in rabbits.35

Elevation of aqueous flare after cataract surgery was inhibited by anti-inflammatory agents. Othenin-Girard et al6 reported that diclofenac sodium seemed to be as effective as dexamethasone sodium phosphate. Miyake et al7 reported that the amount of flare after cataract surgery was lower in a diclofenac group than that in a fluorometholone group. Mast cells contain many small granules of substances that mediate inflammation. Histamine produces the itching and redness.8 In the present study, therefore, we evaluated the effects of anti-inflammatory or antiallergic agents on PGE2-induced aqueous flare elevation in pigmented rabbits.

Corticosteroids bind to a specific receptor and enter into the cytoplasm and nucleus, which leads to the synthesis of specific proteins.9

Dexamethasone decreased a 4-kilobase messenger RNA encoding a cyclooxygenase-related protein.10 Glucocorticoids play a major role in the in vivo regulation of the cyclooxygenase 2 gene.11 Nonsteroidal anti-inflammatory drugs block prostaglandin biosynthesis by inhibitory effects on cyclooxygenases 1 and 2.12,13 Thus, the mechanisms of actions of corticosteroids and nonsteroidal anti-inflammatory drugs are different. Therefore, several time points for application of eyedrops were chosen in the present study.

MATERIALS AND METHODS
ANIMALS

We used pigmented male rabbits (Japanese mongrel) that weighed 2.5 to 3.5 kg each. The animals were housed and treated according to the Association for Research in Vision and Ophthalmology Resolution on Use of Animals in Research. The study was approved by the Institutional Animal Care and Utilization Committee, Toyama Medical and Pharmaceutical University, Toyama, Japan. One eye of each animal was used for the experiment. Four to 8 eyes were used in each group.

CHEMICAL SOLUTIONS

We used the following anti-inflammatory and antiallergic agents, which were purchased as ophthalmic solutions: betamethasone sodium phosphate (Shionogi Pharmaceutical Co Ltd, Osaka, Japan); fluorometholone acetate, levocabastine hydrocloride, and pemirolast potassium (Santen Inc, Napa, Calif); diclofenac sodium (Wakamoto Pharmaceutical Co, Ltd, Tokyo, Japan); pranoprofen (Senju Pharmaceutical Co, Ltd, Osaka); cromolyn sodium (cromoglicate sodium) (Fujisawa Pharmaceutical Co, Ltd, Osaka); tranilast (Kissei Pharmaceutical Co, Ltd, Nagano, Japan); and ibudilast (Senju Pharmaceutical Co, Ltd) (Table 1). The PGE2 (Funakoshi Chemicals, Tokyo) was dissolved in 100% ethanol and stored at −70°C. Prostaglandin E2solution was diluted in 5% ethanol with 0.9% sodium chloride (NaCl) just before use.

In 1 eye, 50 µL of eyedrops or placebo (0.9% NaCl) was topically instilled. Instillation took place once or twice before PGE2 application. The investigator who administered the eyedrops was masked to the contents.

TRANSCORNEAL DIFFUSION OF PGE2

For transcorneal diffusion, a glass cylinder (11 mm in diameter) was attached to the cornea, as described by Hirata et al.1 Next, 600 µL of PGE2 solution containing the study drug at a dose of 25 µg/mL, or 7.09 × 10 −2mmol/L, was delivered into the cylinder and pipetted out 4 minutes later. The cylinder was removed, and the corneal surface and conjunctival sac were rinsed with 20 mL of 0.9% NaCl. The eyes received 2 transcorneal applications of PGE2 at 1- or 2-week intervals (Figure 1). The eyes pretreated with anti-inflammatory or antiallergic agents or placebo (0.9% NaCl) were used initially for PGE2-induced flare elevation. After the interval, the same eyes received PGE2 application only.

AQUEOUS FLARE MEASUREMENT

Aqueous flare was measured with a laser flare-cell meter (FC 1000; Kowa Co, Ltd, Tokyo, Japan) according to the method described by Sawa et al.14 A laser flare-cell meter was used to measure intracameral protein levels. Five measurements were taken at each time point to obtain the mean value. The measurement was taken in the midportion of the anterior chamber, an area measuring 0.075 mm3. Aqueous flare elevation was expressed as the area under the curve (AUC) and inhibition was estimated by the following equation:

The investigator (X.-Y.Z.) who measured flare intensity was masked to the treatment.

STATISTICS

Statistical analysis was performed using the Scheffé multiple comparisons procedure. A probability (P) value of less than .05 was considered significant. Unless otherwise indicated, data are expressed as mean ± SD.

RESULTS

No remarkable changes in the systemic condition were noted after the transcorneal diffusion of PGE2 and the topical instillation of anti-inflammatory or antiallergic agents. Double instillation (1 and 0.5 hour before PGE2 application) of 0.1% betamethasone, 0.1% fluorometholone, 0.1% diclofenac, 0.1% pranoprofen, 2% cromolyn, 0.5% tranilast, 0.025% levocabastine, 0.1% pemirolast, and 0.01% ibudilast did not induce aqueous flare elevation.

After PGE2 was administered, aqueous flare increased, reached its maximum (450-470 photon counts/ms) at 60 to 90 minutes, and then gradually decreased and returned to baseline levels at 7 to 8 hours (Figure 2). When 0.1% betamethasone was topically instilled 4 and 2 hours before PGE2 application, aqueous flare elevated to 225 photon counts/ms at 60 minutes and then gradually decreased (Figure 2A). When 0.1% betamethasone was instilled 1 and 0.5 hour before PGE2 application, aqueous flare elevated to 365 photon counts/ms at 60 minutes and then gradually decreased (Figure 2B).

The effects of double instillation of anti-inflammatory and antiallergic eyedrops on aqueous flare elevation are shown in Table 2. Double instillations of 0.1% betamethasone and 0.1% fluorometholone at 4 and 2 hours before PGE2 application inhibited 61% ± 11% and 46% ± 14%, respectively, of the flare elevation.

Double instillations of 0.1% diclofenac and 0.1% pranoprofen at 4 and 2 hours before PGE2 application did not inhibit the flare elevation. Double instillations of 0.1% betamethasone, 0.1% fluorometholone, 0.1% diclofenac, and 0.1% pranoprofen at 1 and 0.5 hour before PGE2 application inhibited 16% ± 10%,16% ± 6%, 24% ± 9%, and 23% ± 10%, respectively, of the flare elevation. Double instillations of 2% cromolyn, 0.5% tranilast, 0.025% levocabastine, 0.1% pemirolast, and 0.01% ibudilast at 1 and 0.5 hour before PGE2 application did not inhibit flare elevation.

Betamethasone needed several hours after topical instillation to exhibit inhibition of flare elevation (Table 3). When instilled 1 hour before PGE2 application, a single drop of 0.1% diclofenac inhibited flare elevation more strongly (23% ± 10%) than did 0.1% betamethasone (12% ± 6%). When instilled 6 hours before PGE2 application, a single drop of 0.1% diclofenac did not inhibit flare elevation, but 0.1% betamethasone did (88% ± 10%).

COMMENT

In the present study, 0.1% betamethasone and 0.1% fluorometholone instilled 4 and 2 hours before PGE2 application showed stronger inhibition of flare elevation than those instilled 1 and 0.5 hour before PGE2application. Single instillations of 0.1% betamethasone 6 hours before PGE2 application inhibited 88% of aqueous flare elevation. The peak of prednisolone acetate concentration occurred 30 to 45 minutes after topical instillation in humans and rabbits.15 Corticosteroids might need several hours after administration to show action by inhibitory effects on expression of the messenger RNA–encoding cyclooxygenase-related protein.10 Diclofenac and pranoprofen instilled 1 and 0.5 hour before PGE2 application inhibited flare elevation. A single instillation of 0.1% diclofenac 1 hour before PGE2 application inhibited 23% of aqueous flare elevation. Diclofenac directly inhibits cyclooxygenase.16 The different times from administration to inhibition between betamethasone and diclofenac may be due to the dissimilar mechanisms of action of these agents. Several authors have reported that nonsteroidal anti-inflammatory drugs were more effective than corticosteroids in inhibiting blood-aqueous barrier breakdown after cataract surgery.7,1719 However, the action of diclofenac was weaker than that of betamethasone in the present study. A quantitative study of inhibitory effects on postsurgical inflammation by these agents is needed. Some authors compared topical instillations of corticosteroid and nonsteroidal anti-inflammatory drugs 1 hour before the production of experimental uveitis and then at hourly intervals for 6 hours.20 We believe that further studies looking at different dosing schedules could prove beneficial.

Antiallergic agents did not inhibit flare elevation. It is unlikely that these agents affected disruption of the blood-aqueous barrier in rabbits.

Another study found PGE2-like activity in the aqueous humor after paracentesis in rabbits.21 The PGE2 may be involved in traumatic iridocyclitis. The blood-aqueous barrier in rabbits has unique sensitivity to prostaglandins.22 Therefore, the findings in the present study are not always identical to those seen in humans. The exact mechanisms of inhibition by corticosteroids and nonsteroidal anti-inflammatory drugs in rabbits and humans should be studied further.

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Article Information

Submitted for publication June 29, 2001; final revision received January 14, 2002; accepted March 20, 2002.

Corresponding author and reprints: Yoriko Hayasaka, MD, Department of Ophthalmology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan (e-mail: ophthal@ms.toyama-mpu.ac.jp).

References
1.
Hirata  HHiraki  SKaji  YTakeda  NFukuo  YTachinami  K The effects of transcorneal administration of prostaglandin E2 on rabbit eyes. Nippon Ganka Gakkai Zasshi. 1994;98927- 934
2.
Numata-Watanabe  KHirata  HHiraki  SHayasaka  S Decreased aqueous-flare reaction to repeated applications of prostaglandin E2 to the cornea in pigmented rabbits. Ophthalmic Res. 199628147- 152
3.
Watanabe  KHayasaka  SHiraki  S  et al.  Effects of topical clonidine on prostaglandin E2-induced aqueous flare elevation in pigmented rabbits. Ophthalmic Res. 2000;32210- 214Article
4.
Yanagisawa  SHayasaka  SZhang  XYHayasaka  YNagaki  YKitagawa  K Effect of topical betaxolol on acute rise of aqueous flare induced by prostaglandin E2 in pigmented rabbits. Jpn J Ophthalmol. 2001;45669- 671Article
5.
Hayasaka  YHayasaka  SZhang  XYNagaki  Y Effects of topical antiglaucoma eye drops on prostaglandin E2–induced aqueous flare elevation in pigmented rabbits. Invest Ophthalmol Vis Sci. 2002;431142- 1145
6.
Othenin-Girard  PTritten  JJPittet  NHerbort  CP Dexamethasone versus diclofenac sodium eyedrops to treat inflammation after cataract surgery. J Cataract Refract Surg. 1994;209- 12Article
7.
Miyake  KMasuda  KShirato  S  et al.  Comparison of diclofenac and fluorometholone in preventing cystoid macular edema after small incision cataract surgery: a multicentered prospective trial. Jpn J Ophthalmol. 2000;4458- 67Article
8.
Berdy  GJAbelson  MB Antihistamines and mast cell stabilizers in allergic ocular disease. Albert  DMJacobiec  FAeds.Principles and Practice of Ophthalmology. Philadelphia, Pa WB Saunders Co1994;1028- 1042
9.
Abelson  MBButrus  S Corticosteroids in ophthalmic practice. Albert  DMJacobiec  FAeds.Principles and Practice of Ophthalmology. Philadelphia, Pa WB Saunders Co1994;1013- 1022
10.
O'Banion  MKSadowski  HBWinn  VYoung  DA A serum- and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein. J Biol Chem. 1991;26623261- 23267
11.
Masferrer  JLReddy  STZweifel  BS  et al.  In vivo glucocorticoids regulate cyclooxygenase-2 but not cyclooxygenase-1 in peritoneal macrophages. J Pharmacol Exp Ther. 1994;2701340- 1344
12.
To  KAbelson  MBNeufeld  A Nonsteroidal anti-inflammatory drugs. Albert  DMJacobiec  FAeds.Principles and Practice of Ophthalmology. Philadelphia, Pa WB Saunders Co1994;1022- 1027
13.
Battistini  BBotting  RBakhle  YS COX-1 and COX-2: toward the development of more selective NSAIDs. Drug News Perspect. 1994;7501- 512
14.
Sawa  MTsurimaki  YTsuru  TShimizu  H New quantitative method to determine protein concentration and cell number in aqueous in vivo. Jpn J Ophthalmol. 1988;32132- 142
15.
Leibowitz  HMBerrospi  ARKupferman  ARestropo  GVGalvis  VAlvarez  JA Penetration of topically administered prednisolone acetate into the human aqueous humor. Am J Ophthalmol. 1977;83402- 406
16.
Noonan  WDSamples  JR Diclofenac sodium. J Toxicol Cutan Ocul Toxicol. 1993;12265- 272Article
17.
Sanders  DRKraff  M Steroidal and nonsteroidal anti-inflammatory agents. Arch Ophthalmol. 1984;1021453- 1456Article
18.
Flach  AJKraff  MCSanders  DRTanenbaum  L The quantitative effect of 0.5% ketorolac tromethamine solution and 0.1% dexamethasone sodium phosphate solution on postsurgical blood-aqueous barrier. Arch Ophthalmol. 1988;106480- 483Article
19.
Kraff  MCSanders  DRMcGuigan  LRaanan  MG Inhibition of blood-aqueous humor barrier breakdown with diclofenac. Arch Ophthalmol. 1990;108380- 383Article
20.
Tilden  MEBoney  RSGoldenberg  MMRosenbaum  JT The effects of topical S(+)–ibuprofen on interleukin-1 induced ocular inflammation in a rabbit model. J Ocul Pharmacol. 1990;6131- 135Article
21.
Miller  JDEakins  KEAtwal  M The release of PGE2-like activity into aqueous humor after paracentesis and its prevention by aspirin. Invest Ophthalmol. 1973;12939- 942
22.
Toris  CBCamras  CBYablonski  MEBrubaker  RF Effects of exogenous prostaglandins on aqueous humor dynamics and blood-aqueous barrier function. Surv Ophthalmol. 1997;41suppl 2S69- S75Article
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