the effects of topical s(+)-ibuprofen on interleukin-1 induced ocular inflammation in a rabbit model

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JOURNAL OF OCULAR PHARMACOLOGY Volume 6, Number 2, 1990 Mary Ann Liebert. Inc.. Publishers The Effects of Topical S(+)-Ibuprofen on Interleukin-1 Induced Ocular Inflammation in a Rabbit Model MORRIS E. TILDEN,1 RICHARD S. BONEY,1 MARVIN M. GOLDENBERG,2 and JAMES T. ROSENBAUM1 'Departments of Ophthalmology, Medicine and Cell Biology, Oregon Health Sciences University, Portland, OR 2Ophthalmic Pharmaceutical Discovery, American Cyanamid/Storz, Pearl River, NY ABSTRACT Topical non-steroidal anti-inflammatory drugs may serve as an alternative or adjunct to topical corticosteroid therapy for iritis. We have tested the efficacy of topically given S(+)-ibuprofen in a rabbit model of uveitis secondary to the intravitreal injection of human recombinant interleukin 1-alpha. Topically administered S(+)-ibuprofen was found to inhibit increased vascular permeability associated with this model. These results with topical S(+)-ibuprofen compare favorably to the results seen with topical prednisolone and are significantly superior to the results seen with topical flurbiprofen. Topical S(+)-ibuprofen did not significantly reduce the cellular infiltration associated with interleukin-1 induced inflammation. These findings suggest a potential role for topical S(+)-ibuprofen in the treatment of ocular inflammation and, in this animal model, it appears to be superior to an alternative non-steroidal compound, but further studies are indicated to assess its activity in alternate models of ocular inflammation. INTRODUCTION The mainstay of current therapy for idiopathic iritis is topical corticosteroids. While frequently effective, topical corticosteroids such as prednisolone acetate are associated with cataractogenesis. Corticosteroids elevate intra-ocular pressure in a subset of patients and may interfere with recovery from an infectious process (1). Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of inflammation such as arthritis. For ocular inflammation, very few published studies have compared the efficacy of topical non- steroidal anti-inflammatory drugs to corticosteroids or other NSAIDs (2, 3). Ibuprofen (R,S 2-(4 isobutylphenyl)propionic acid) is an optically active 2-aryl-propionic acid derivative with potent non-steroidal anti-inflammatory activity. Clinically, oral ibuprofen is used as a racemate although, in vitro, the s(+)-enantiomer is 100 times more active an inhibitor of the prostaglandin synthetase system than is the R(-)-enantiomer (4). However, this large difference in potency between the two isomers is not observed in vivo (4). A pharmacokinetic study in healthy volunteers showed that peak-plasma concentrations are higher and reached faster following the oral administration of the S(+)-enantiomer as compared to the R(-)-enantiomer or to the racemate (5). A limited clinical study in four rheumatoid arthritis patients shows the S(+)-enantiomer to be somewhat more active than the racemate form (5). S(+)-ibuprofen has greater water solubility than the racemate ibuprofen and, therefore, may have better corneal penetrability. Previous unpublished studies (Marvin Goldenberg, American Cyanamid) show that the topical S(+)-enantiomer of ibuprofen is transported through the cornea within twenty minutes. The aim of 131

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Page 1: The Effects of Topical S(+)-Ibuprofen on Interleukin-1 Induced Ocular Inflammation in a Rabbit Model

JOURNAL OF OCULAR PHARMACOLOGYVolume 6, Number 2, 1990Mary Ann Liebert. Inc.. Publishers

The Effects of Topical S(+)-Ibuprofen onInterleukin-1 Induced Ocular Inflammation

in a Rabbit ModelMORRIS E. TILDEN,1 RICHARD S. BONEY,1 MARVIN M. GOLDENBERG,2

and JAMES T. ROSENBAUM1'Departments of Ophthalmology, Medicine and Cell Biology, Oregon Health Sciences University, Portland, OR

2Ophthalmic Pharmaceutical Discovery, American Cyanamid/Storz, Pearl River, NY

ABSTRACT

Topical non-steroidal anti-inflammatory drugs may serve as an alternative or adjunct to topicalcorticosteroid therapy for iritis. We have tested the efficacy of topically given S(+)-ibuprofen in a rabbitmodel of uveitis secondary to the intravitreal injection of human recombinant interleukin 1-alpha. Topicallyadministered S(+)-ibuprofen was found to inhibit increased vascular permeability associated with this model.These results with topical S(+)-ibuprofen compare favorably to the results seen with topical prednisolone andare significantly superior to the results seen with topical flurbiprofen. Topical S(+)-ibuprofen did notsignificantly reduce the cellular infiltration associated with interleukin-1 induced inflammation. Thesefindings suggest a potential role for topical S(+)-ibuprofen in the treatment of ocular inflammation and, inthis animal model, it appears to be superior to an alternative non-steroidal compound, but further studies areindicated to assess its activity in alternate models of ocular inflammation.

INTRODUCTION

The mainstay of current therapy for idiopathic iritis is topical corticosteroids. While frequently effective,topical corticosteroids such as prednisolone acetate are associated with cataractogenesis. Corticosteroidselevate intra-ocular pressure in a subset of patients and may interfere with recovery from an infectious process(1). Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of inflammation suchas arthritis. For ocular inflammation, very few published studies have compared the efficacy of topical non-steroidal anti-inflammatory drugs to corticosteroids or other NSAIDs (2, 3).

Ibuprofen (R,S 2-(4 isobutylphenyl)propionic acid) is an optically active 2-aryl-propionic acid derivativewith potent non-steroidal anti-inflammatory activity. Clinically, oral ibuprofen is used as a racematealthough, in vitro, the s(+)-enantiomer is 100 times more active an inhibitor of the prostaglandin synthetasesystem than is the R(-)-enantiomer (4). However, this large difference in potency between the two isomersis not observed in vivo (4). A pharmacokinetic study in healthy volunteers showed that peak-plasmaconcentrations are higher and reached faster following the oral administration of the S(+)-enantiomer as

compared to the R(-)-enantiomer or to the racemate (5). A limited clinical study in four rheumatoid arthritispatients shows the S(+)-enantiomer to be somewhat more active than the racemate form (5).

S(+)-ibuprofen has greater water solubility than the racemate ibuprofen and, therefore, may have bettercorneal penetrability. Previous unpublished studies (Marvin Goldenberg, American Cyanamid) show that thetopical S(+)-enantiomer of ibuprofen is transported through the cornea within twenty minutes. The aim of

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Page 2: The Effects of Topical S(+)-Ibuprofen on Interleukin-1 Induced Ocular Inflammation in a Rabbit Model

the present study is to compare the efficacy of the topically applied S(+)-enantiomer of ibuprofen withcurrently available topical therapies. To induce inflammation, we used an intravitreal injection of humanrecombinant Interleukin-1 (IL-1) in rabbits. This produces an acute inflammatory response which serves asan excellent model for uveitis (2,6). We demonstrate that topically applied S(+)-ibuprofen appears to besuperior to flurbiprofen in this animal model.

MATERIALS AND METHODS

Animals

New Zealand white rabbits of both sexes, weighing between 2.0 and 2.4 Kg were purchased commerciallyand housed in animal care facilities of the Oregon Health Sciences University, Portland. Animals were fedstandard laboratory chow. The animals were handled in accordance with standards set by the AmericanAssociation for Accreditation of Laboratory Animal Care.

Interleukin-1 Administration

The intravitreal injection of recombinant human interleukin-1 produces an acute inflammatory responsewhich is characterized by anterior chamber cellular infiltrates, iris vessel dilatation, and protein extravasation(6). These changes become evident within 6 hours after injection and reach a maximum at 24 hours (6).

Recombinant human interleukin-1-alpha (rIL-1) was obtained from Hoffmann-LaRoche Inc.(Nutley, N.J.)and contained less than 20 ng of endotoxin per 106 units of rIL-1 (Limulus assay performed by the supplier).The activity of the rIL-1 was determined by thymocyte proliferation assay, with 1 unit equivalent to anamount inducing 50% of maximal stimulation (7).

Fifty microliter injections of rIL-1 were made into the central vitreous using a 28 gauge needle. Carewas taken to avoid the lens and retina by using direct visualization during the injection. Prior to injectionthe rabbits received topical proparacaine 0.5% for anesthesia. The rIL-1 was diluted to desired concentrationin pyrogen free saline containing 0.025% human serum albumin as a carrier protein.

Treatments

Four groups of five rabbits were injected with 250 units of recombinant human interleukin-1 alpha.Rabbits received 50 microliters topical S(+)-ibuprofen 0.9% (American Cyanamid-Pearl River, NY),flurbiprofen sodium 0.03% (Allergan, Puerto Rico), prednisolone acetate 1% (Allergan, Puerto Rico), or

phosphate buffer carrier, pH 7.0 (American Cyanamid, Pearl River, NY) one hour prior to the administrationof intravitreal rIL-1 and then at hourly intervals until the conclusion of the experiment. In addition, tworabbits received intravitreal rIL-1 alone and did not receive any topical medications and three rabbits wereadministered topical S(+)-ibuprofen hourly but were not injected with rIL-1.

Aqueous Humor Protein Determinations

Rabbits were sacrificed 6 hours after rIL-1 injection by barbiturate overdose. Vascular permeability andcellular infiltration were determined by measurement of accumulated protein and leukocytes in the aqueoushumor aspirated at time of death. Direct quantitation of aqueous humor protein levels was performed bybinding of brilliant blue (Biorad, Richmond CA) as described by Bradford (8). Cell counts were performedin a hemocytometer. Results of the cell counts were expressed as number of cells per microliter. Whenadequate sample was obtained, Wright-stained slides were prepared by cytocentrifugation for differential cellcounts.

Statistical Analysis

The results are presented as a mean +_ standard error. Statistical comparisons between groups were

performed using Student's t test. A p value less than 0.05 was considered significant.

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RESULTS

Interleukin-1 Induced Ocular Vascular Permeability (Figure 1)

Three rabbits were tested with topical S(+)-ibuprofen every hour for seven doses to evaluate theinflammatory effects of topical S(+)-ibuprofen alone. Protein levels in aqueous humor showed a mean levelof only 0.6 +_ 0.3 mg/ml (N=3), which is indistinguishable from normal aqueous humor. In contrast, sixhours after intra-vitreal injection of 250 units of IL-1, without any topical therapy, there was an average risein aqueous humor protein levels to 35.3 mg/ml (N=2). A comparable increase in aqueous humor proteinwas found in rabbits which received IL-1 followed by hourly administration of the topical carrier (34.48 +.10.09 mg/ml, N=5). Topical flurbiprofen had no apparent effect on IL-1 induced aqueous humor proteinlevels with a mean level of 34.08 ±_ 3.06 mg/ml (N=5). Topical therapy with prednisolone or S(+)-ibuprofen resulted in an attenuation of this increase in protein levels to 17.52 +_ 7.66 mg/ml (N=5) and18.89 +. 10.40 mg/ml (N=5), respectively, with p< 0.01 relative to either carrier or flurbiprofen treatedrabbits in both cases.

Protein (mg/ml)

The Effect of Topical S(+)-Ibuprofen on Aqueous Humor Protein LevelsInduced by Intravitreal Interleukin-1

Aqueous humor protein levels were measured 6 hours after intravitrealinterleukin-1 injection and hourly treatment with topical carrier,

1% prednisolone acetate, 0.9% S(+)-ibuprofen, or 0.03% flurbiprofen.

Interleukin-1 Induced Anterior Chamber Cellular Infiltration (Figure 2)

The intravitreal injection of 250 units of IL-1 usually induces an accumulation of anterior chamberleukocytes within six hours. In all samples tested, drug treated or non-treated rabbits, between 96% and100% of leukocytes present were polymorphonuclear leukocytes. For rabbits treated with topical S(+)-ibuprofen hourly for seven doses, 0.83 +_ 1.44 leukocytes/ul (N=3) were present in the aqueous humor.In contrast, in those rabbits receiving IL-1 alone, without topical therapy, six hours after intravitreal injectionthere were an average of 120 leukocytes/ul (N=2) present in the aqueous humor. In those rabbits treatedwith topical carrier before and after IL-1 injection, a comparable leukocyte count of 164 +_ 87.6 (N=5) was

present. Topical prednisolone reduced the anterior chamber accumulation of cells induced by IL-1 injectionto 56 ± 35.5 leukocytes/ul (N=5) (p< 0.01 relative to flurbiprofen treated rabbits and p< 0.025 relativeto carrier rabbits), whereas topical flurbiprofen actually demonstrated an increase in mean cell counts with288 +_ 97.2 leukocytes/ul (N=3) present. Topical S(+)-ibuprofen had little effect on the reduction ofaqueous humor cell counts, with 126 _+ 91.3 leukocytes/ul (N=5) present, but appeared to be superior toflurbiprofen in that it did not increase the cell count (p < 0.01). The flurbiprofen group includes five rabbits,but two outliers were present, one with 2.5 leukocytes/ul and one with 1175 leukocytes/ul. N=3 representsdata calculated with the outliers excluded. Outliers were also excluded from statistical comparisons.Inclusion of the outliers would increase the mean cell count in flurbiprofen treated rabbits to 408 ±451leukocytes/ul, further increasing the gap between flurbiprofen and other agents.

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Leukocytes/ul300 -i 288

WM ControlBIL-1(HUB Carrier

FlurbiprofenPrednisolone

üü Ibuprofen

FIGURE 2The Effect of Topical S(+)-Ibuprofen on Aqueous Humor Cell Counts

Induced by Intravitreal Interleukin-1Aqueous humor cells were quantitated 6 hours after intravitreal

interleukin-1 injection and hourly treatment with topical carrier,1% prednisolone acetate, 0.9% S(+)-ibuprofen, or 0.03% flurbiprofen.

DISCUSSION

Topical non-steroidal anti-inflammatory drugs have been approved for the treatment of miosis associatedwith ophthalmic surgery (9). They have also been tested for the treatment of postoperative cystoid macularedema following cataract extraction (10, 11), the treatment of chronic cystoid macular edema (11), and forthe treatment of ocular inflammation (2, 3).

Topical S(+)-ibuprofen was well tolerated in this study since the protein and cell counts subsequent to

S(+)-ibuprofen alone are comparable to those seen in normal rabbits. Our data indicate that S(+)-ibuprofensignificantly reduced disruption of the blood-aqueous barrier induced by intravitreal IL-1. The results withtopical S(+)-ibuprofen are comparable to results with topical prednisolone with regard to reducing disruptionof the blood-aqueous barrier and statistically significantly superior to the results seen with flurbiprofen.Although the cell counts are not significantly reduced by topical S(+)-ibuprofen, the data suggest that S(+)-ibuprofen is superior to flurbiprofen with regard to its effects on cellular inflammation associated with localIL-1 injection (p < 0.01). It has been reported that S(+)-ibuprofen causes inhibition of neutrophil chemotaxisand endothelial cell adherence of neutrophils in vitro (13). Flurbiprofen treated rabbits had higher aqueoushumor cell counts than carrier-treated controls, although this difference did not reach statistical significancedue to variability in the cellular response subsequent to IL-1. Although not statistically significant(p < 0.1), prednisolone may be superior to S(+)-ibuprofen in reducing the aqueous humor cell counts in thismodel. The anti-inflammatory actions of non-steroidal anti-inflammatory drugs are mediated by a differentmechanism than that of corticosteroids, which require the secondary induction of lipocortins (14). Due tothe different mechanisms of these two drug classes, conceivably initiating pretreatment with steroids earliermay result in an even greater reduction of aqueous humor protein levels and cell counts.

In a previous study (2) we had reported that topical flurbiprofen had no ability to alter IL-1 inducedcellular infiltration but it was effective in reducing the disruption of the blood-aqueous barrier secondary toIL-1. In the present study we used a slightly greater amount of IL-1 (250 vs 200 units) and noted greateraqueous humor protein levels in controls (35.3 vs 14.9 mg/ml). The failure to demonstrate efficacy offlurbiprofen in the present study may relate to the greater activity of the inflammation or a type II statisticalerror relating to the size of the treatment group. Although our previous study had noted a benefit fromflurbiprofen, the present study indicates that S(+)-ibuprofen appears to be more potent in this model.

Since topical prednisolone therapy may be associated with significant toxicity including cataractogenesisand glaucoma, efforts should be made to find alternative modes of anti-inflammatory therapy. Topical S(+)-ibuprofen appears to be superior to flurbiprofen, a presently marketed alternative non-steroidal anti-inflammatory medication. Further studies are indicated to determine if S(+)-ibuprofen may have additiveeffects with corticosteroids and if its beneficial effects can be demonstrated in additional models of ocularinflammation.

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ACKNOWLEDGEMENTS

Supported in part by American Cyanamid/Storz and grants EY06477 & EY06484 from the NationalInstitute of Health. Dr. Goldengerg is an employee of American Cyanamid/Storz. S(+)-ibuprofen was

provided by American Cyanamid/Storz.REFERENCES

1. O'Day, D.M. and Jones, B.R. Herpes Simplex Keratitis. In Clinical Ophthalmology, Duane, T.D., Ed.Harper and Row, Medical Dept., Hagerstown, 1989, V.4, Chapt. 19, p. 10-11

2. Rubin, R.M. and Rosenbaum, J.T. A platelet-activating factor antagonist inhibits Interleukin-1 inducedinflammation. Biochem. Biophvs. Res. Com.. 154:429-436, 19882.

3. Hotchkiss, ML., Robin, A.L., Pollack, I.P., Quigley, H.A. Nonsteroidal anti-inflammatory agents afterargon laser trabeculoplasty; a trial with flurbiprofen and indomethacin. Ophthalmol,. 91:969-974, 1984

4. Adams, S.S., Bresloff, P. and Mason, C.G. Pharmacological differences between the optical isomers ofibuprofen: evidence for metabolic inversion of the (-) isomer. J. Pharm. Pharmac. 28:256-257, 1976

5. Geissinger, G., Stock, K.P., Bach, G.L., Loew, D. and Brune, K. Pharmacological differences betweenR(-) and S(+)-ibuprofen. Agents and Actions. 27:455-457, 1989

6. Rosenbaum, J.T., Samples, J.R., Hefeneider, S.H. and Howes, E.L. Jr. Ocular inflammatory effectsof intravitreal Interleukin-1. Arch. Ophthalmol.. 105:1117-1120, 1987

7. Gery, I., Gershon, R.K. and Waksman, B.H. Potentiation of the T-lymphocyte response to mitogens: I.The responding Cell. J. Exp. Med.. 136:128-142, 1972

8. Bradford, M.M. Rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding. Anal. Biochem.. 72:248-254, 1976

9. Vander, J.F., Greven, CM., Macguire, J.I., Marino, R.J., Shakin, E.P., and Lucier, A.C. Flurbiprofensodium to prevent intraoperative myosis during vitreoretinal surgery.Am. J. Ophthalmol.. 108:288-291, 1989

10. Flach, A.J., Lavell, C.J., Olander, K.W., Ritzloff, J.A., and Sorenson, L.W. The effect of ketorolactromethamine solution 0.5% in reducing post-operative inflammation after cataract extraction andintraocular lens implantation. Ophthalmol.. 95:1279-1284, 1988

11. Jampol, L.M. Pharmacologie therapy of aphakic and pseudoaphakic cystoid macular edema.Ophthalmol.. 92:807-810, 1985

12. Flach, A.J., Dolan, B.J., Irvine, A.R. Effectiveness of ketorolac tromethamine 5% ophthalmic solutionfor chronic aphakic and pseudoaphakic cystoid macular edema. Am. J. Ophthalmol.. 103:479-486, 1987

13. Neilson, V.G. and Webster, R.O. Inhibition of human polymorphonuclear leukocyte functions byibuprofen. rmmunopharmacology. 13:61-71, 1987

14. Wallner, B.P., Mattaliano, R.J., Hession, C, Cate, R.L., Tizard, R., Sinclair, L.K., Foeller, C,Chow, E.P., Browning, J.L., Ramachandran, K.L. and Pepinsky, R.B. Cloning and expression of humanlipocortin, aphospholipase A2inhibitor with potential anti-inflammatory activity. Nature. 320:77-81, 1986

Date Received: March 5, 1990Accepted for Publication: April 24, 1990

Reprint Requests: James T„ Rosenbaum, M.D.Oregon Health Sciences UniversityMail Code L329A3181 Sam Jackson Park RoadPortland OR 97201USA

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