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

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.^3-5

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.

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.

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.

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 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.

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.

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%).

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.^7,17-19 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.

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).