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sheets. The cells retain their epithelial morphology nea and iris-ciliary body of the rabbit. Invest Ophthalmol Vis and synthesize cAMP in response to /3-adrenergic but Sci 17:1069, 1978. 4. Klyce SD, Palkama KA, Harkonen M, Marshall WS, Huht- not serotonergic stimulation. aniity S, Mann KP, and Neufeld AH: Neural serotonin stim- Key words: epithelium, cornea, rabbit, cell culture, /3-ad- ulates chloride transport in the rabbit corneal epithelium. In- renergic response, growth factors, cholera toxin, cAMP, vest Ophthalmol Vis Sci 23:181, 1982. serotonergic response 5. Neufeld AH, Ledgard SE, Jumblatt MM, and Klyce SD: Se- rotonin-stimulated cyclic AMP synthesis in the rabbit corneal Acknowledgments: The authors thank Sally Ledgard for epithelium. Invest Ophthalmol Vis Sci 23:193, 1982. technical assistance and Yasuo Ishii for electron micros- 6. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein copy. measurement with the Folin phenol reagent. J Biol Chem 193:265, 1951. From the Eye Research Institute of Retina Foundation and 7. Gipson IK and Grill SM: A technique for obtaining sheets of Department of Ophthalmology, Harvard Medical School, Boston, intact rabbit corneal epithelium. Invest Ophthalmol Vis Sci Massachusetts. Supported in part by USPHS grants EY-02360 and 23:269, 1982. EY-02367. Submitted for publication: July 27, 1982. Reprint re- 8. Barnes D and Sato G: Methods for growth of cultured cells quests: Marcia M. Jumblatt, Eye Research Institute of Retina in serum-free medium. Anal Biochem 102:255, 1980. Foundation, 20 Staniford Street, Boston, MA 02114. 9. Taylor-Papadimitriou J, Purkis P, and Fentiman IS: Cholera toxin and analogues of cyclic AMP stimulate the growth of References cultured human mammary epithelial cells. J Cell Physiol 102:317, 1980. 1. Sundar-Raj CV, Freeman IL, and Brown SI: Selective growth 10. Green H: Cyclic AMP in relation to proliferation of the epi- of rabbit corneal epithelial cells in culture and basement mem- dermal cell: a new view. Cell 15:801, 1978. brane collagen synthesis. Invest Ophthalmol Vis Sci 19:1222, 11. Jumblatt MM and Neufeld AH: Characterization of cyclic 1980. AMP mediated wound closure of the rabbit corneal epithe- 2. Sun TT and Green H: Cultured epithelial cells of cornea, con- lium. Curr Eye Res 1:189, 1981. junctiva and skin: Absence of marked intrinsic divergence of 12. Jumblatt MM, Fogle JA, and Neufeld AH: Cholera toxin stim- their differentiated states. Nature 269:489, 1977. ulates adenosine 3',5'-monophosphate synthesis and epithelial 3. Neufeld AH, Zawistowski KA, Page ED, and Bromberg BB: wound closure in the rabbit cornea. Invest Ophthalmol Vis Influences on the density of/8-adrenergic receptors in the cor- Sci 19:1321, 1980.

A Comparison of the Ocular Anti-inflammatory Activity of Steroidal and Nonsteroidal Compounds in the Rat

P. Dharrocherjee, R. N. Williams, and K. E. Eakins

The anti-inflammatory activities of steroidal and nonste- factorily for comparative evaluation of anti-inflammatory roidal compounds have been evaluated in the rat model of agents. Invest Ophthalmol Vis Sci 24:1143-1146, 1983 ocular inflammation induced by subcutaneous injection of lipopolysaccharides. Dexamethasone sodium phosphate, BW755C, flurbiprofen, indomethacin, and benoxaprofen The anti-inflammatory drugs such as aspirin and were administered orally or topically for 24 or 48 hrs. Oral indomethacin, inhibit the enzyme, cyclo-oxygenase, administration of dexamethasone, BW755C, and flurbipro- which converts arachidonic acid into prostaglandins.' fen inhibited iris-vasodilatation and leukocyte accumulation Unlike the aspirin-like drugs, the anti-inflammatory in the anterior chamber in a dose-dependent manner. In- corticosteroids reduce the formation of both cyclo- domethacin and benoxaprofen were active only at high oxygenase and lipoxygenase products of arachidonate doses. Topical administration of these compounds inhibited metabolism by preventing the release of the precursor the inflammatory responses in a similar manner. The in- acid, arachidonic acid.2'3 This additional inhibition hibitory effect on leukocyte accumulation by these com- of leukotriene formation might be responsible for the pounds was greater than their effect on vasodilatation. wider profile of anti-inflammatory activity exhibited BW755C, a phenyl pyrazoline derivative, which is an in- hibitor of both the cyclooxygenase and lipoxygenase path- by the corticosteroids. ways of arachidonic acid metabolism was the most active Although effective clinically as anti-inflammatory nonsteroidal compound and had an anti-inflammatory pro- agents, corticosteroids have a large number of un- file similar to dexamethasone. The results of this study also, desirable side-effects when used either systemically or indicate that the model of rat ocular inflammation induced topically. A compound having the ability of a corti- by subcutaneous injection of endotoxin can be used satis- costeroid to prevent the formation of the products of

0146-0404/83/0800/1143/$ 1.00 © Association for Research in Vision and Ophthalmology

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JL ment of the treatments. Dilatation of iris blood vessels 100 JL 1 JL was assessed, and the severity was scored (0 for nor- mal, 1 as mild, 2 moderate, and 3 as^severe). At the JL JL end of the experimental period, animals were killed with sodium pentobarbitone, the anterior chamber Ji Ji 50 punctured, and the aqueous humor collected in a

"T 1 1 ft disposable graduated capillary tube and mixed with

• 0.1 ml heparinized saline. Leukocytes were counted in a single blind manner in a Neubauer Chamber. To 1 1 compare the activity of various compounds and to 1 T analyse the data statistically, each value of the va- 100 T i 1 sodilation score and leukocyte count was converted to the percent of the mean of the control. The mean Jl Jl of these values were then compared statistically with Ti Jl Jl Jl JL that of the control using unpaired t-test for groups 50 Jl J, consisting of unequal number of observations. J- Preparation of drug solutions. Dexamethasone So-

q q q dium phosphate and BW755C were dissolved in sa- in q q

.25 o in .03 8 .125 o o in o o* o o in CM in in in o CM line immediately prior to administration. Indometh- _ o 6 o *- rsi Dexamethasone BW755C Flurbiprolen Indomethacin Benoxaprofen acin, flurbiprofen and benoxaprofen were dissolved mg/kg mg/kg mg/kg mg/kg mg/kg in saline with the aid of equimolar concentration of Fig. 1. The inhibitory activity of the anti-inflammatory agents sodium carbonate yielding clear solutions with pH administered orally three times in 24 hrs on the iris-vasodilatation and leukocyte accumulation in the aqueous humor. Each column between 7.0 and 7.5. expressed as the percent of control values is the mean of 16-20 Oxyphenbutazone being insoluble in water was so- eyes. The figures within the columns are the doses and the bars lubilised in polysorbate mono-oleate and then diluted represent ±SEM. *P < 0.05. with phosphate buffer, pH 7.5 to a final concentration of 5% polysorbate mono-oleate. both pathways of arachidonic acid metabolism but Results. The anti-inflammatory effects of the test free of their undesirable side-effects, would be valu- compounds following oral administration on the re- able as a topical ocular anti-inflammatory drug. The sponses of the eye to endotoxin are summarized in nonsteroidal anti-inflammatory compound BW755C Figure 1. Dexamethasone effectively inhibited both (3-amino-1 -(3 trifluromethylphenyl)-2-pyrazoline) the vasodilation and leukocyte accumulation in a may be representative of such a class of drugs, since dose-dependent manner. Both the phenyl pyrazoline it is equi-active in inhibiting cyclo-oxygenase and li- derivative, BW755C, and flurbiprofen exhibited sim- poxygenase activity, and possesses a wider profile of ilar anti-inflammatory activity but were both less po- anti-inflammatory activity than the aspirin-like drugs.4 tent than dexamethasone. Benoxaprofen and indo- In the present study, we have compared the anti- methacin at 20 mg/kg and 10 mg/kg respectively re- inflammatory activity of the aspirin-like drugs, flur- duced leukocyte accumulation by about 40% without biprofen, indomethacin and benoxaprofen, with a significant modification of the initial vasodilation. corticosteroid, dexamethazone, and BW755C, fol- This inhibition was maximal for both compounds lowing both oral and topical administration using the since higher concentrations did not result in any fur- newly described rat model of ocular inflammation.56 ther reduction in leukocyte accumulation. Materials and Methods. Ocular inflammation in The effects of topical administration of these com- the rat was induced by injecting Shigella endotoxin pounds for a period of 24 hrs are seen in Figure 2. into the foot pad as described by Bhattacherjee, Wil- With the exception of dexamethasone, none of the liams, and Eakins.6 Immediately after the injection, compounds significantly altered either the vasodila- groups of 8-10 rats were treated with the anti-inflam- tion or the leukocyte accumulation at this period of matory compounds either orally three times a day for treatment. Dexamethasone on the other hand was 24 hrs or topically three times a day either for 24 or highly effective on both parameters of inflammation 48 hrs. A constant volume of 10 n\ and 500 n\ was following 24 hrs of treatment. used respectively for topical and oral administration. However, the anti-inflammatory activity of some At each dose level, a control group treated with ve- but not all, of these compounds became apparent hicle alone was always included. when the period of treatment was extended to 48 hrs Animals were then examined under a slit lamp 24 (Fig. 3). For example, both BW755C and flurbipro- and 48 hrs after endotoxin injection and commence- fen, which were without a significant inhibitory action

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on the vascular and leukocyte responses in the first 24 hrs of treatment, were significantly more effective LT Xi^x at the end of the 48 hrs. BW755 was found to inhibit both the vascular and the cellular component of the inflammatory response whereas flurbiprofen, even at X 48 hrs, was without effect on the vascular response, X except at one dose. Interestingly, flurbiprofen pro- duced a biphasic dose-response curve on leukocyte accumulation, low doses (up to 2.5 i*g) inhibiting and higher doses (5 and 10 ng) potentiating the appear- ance of cells in the anterior chamber. The maximal I 1 inhibition of the cellular response achieved with flur- I biprofen was only 50%. Jjx X ^ 1

Neither indomethacin nor benoxaprofen exhibited a? significant anti-inflammatory activity after treatment S 50 o -x for 48 hours. Oxyphenbutazone, which was only tested for 48 hrs, was effective in reducing leukocyte accumulation at relatively large doses (25 ^g/eye). It

should be noted, however, that the oxyphenbutazone Dexamethasone BW755C Flurbiprofen Indomethacin Benoxaprolen data are not strictly comparable with those of the ug/eye ug/eye ug/eye ug/eye ug/eye other compounds since the vehicle used to solubilise Fig. 2. The effect of anti-inflammatory agent administered top- the former was different from that used for the other ically three times a day for one day on the responses to endotoxin. The values expressed as the percent of controls are the mean compounds (saline only) and may have enhanced the ± SEM of 16-20 eyes. *P < 0.05. bioavailability of oxyphenbutazone. Discussion. The results of the present study show that this model of ocular inflammation is suitable for ocular responses of the rat to endotoxin in a well- the evaluation of anti-inflammatory activity of both defined, dose-dependent manner following both oral steroids and nonsteroidal compounds administered and topical administration. either orally or topically. Compounds like dexameth- The nonsteroidal compounds, administered orally, asone, with proven clinical efficacy, suppressed the had variable effects on the inflammatory responses,

100 JL T I T T T T 75 IT T T T T T

50 - r

25 Fig. 3. The inhibition of responses to endotoxin by anti-inflammatory agents administered topically three times a day for 2 days. The values expressed as the per- 100 T cent of controls are the mean I JL ± SEM of 16-20 eyes. *P I T J 75 1 < 0.05. T f 50 S•LL T- j. 25

o o o 10 10.0 5.0

Dexamethasone BW755C Flurbiprofen Indomethacin Oxyphenbutazone Benoxaprofen ug/eye pg/eye vg/eye ug/eye ug/eye ug/eye

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the effect being more pronounced on leukocyte ac- Acknowledgments. The authors wish to acknowledge the cumulation than on vasodilation. Of these com- excellent technical assistance of Mr. B. R. Hammond and pounds, flurbiprofen and BW755C significantly in- Miss Tracey A. Dopson and to thank Ms. Lorraine Joyce hibited both the leukocyte and vascular responses. for the secretarial help. On the other hand inhibition of leukocyte accumu- From The Wellcome Research Laboratories, Langley Court, lation but not of vasodilation was seen only at high Beckenham, Kent, England. Submitted for publication: October doses of indomethacin and benoxaprofen. There is 14, 1982. Reprint requests: P. Bhattacherjee, PhD, The Wellcome general agreement in the literature that following oral Research Laboratories, Langley Court, Beckenham, Kent BR3 administration, drugs such as indomethacin and the 3BS, England. salicylates will inhibit cell migration at high doses.4'7'8 Flurbiprofen and benoxaprofen also inhibit cell mi- References gration at doses which are well above those required 1. Vane JR: Inhibition of prostaglandin synthesis as a mechanism to inhibit prostaglandin biosynthesis.4 of action for aspirin-like drugs. Nature (New Biol.) 231:232, 1971. It is not clear why BW755C, flurbiprofen, and oxy- 2. Gryglewski RJ, Panczenko B, Korbut R, Grodzinska L, and phenbutazone were without effect after 24 hrs; pos- Ocetkiewicz A: Corticosteroids inhibit prostaglandin release sibly their rate of absorption following topical ad- from perfused lungs of sensitized guinea pig. Prostaglandins ministration is slow. The reversal of the dose response 10:343, 1975. curve for flurbiprofen following topical administra- 3. Hong SL and Levine L: Inhibition or arachidonic acid release from cells as the biochemical action of anti-inflammatory cor- tion of either 5 or 10 Mg/eye would seem paradoxical ticosteroids. Proc Natl Acad Sci USA 73:1730, 1976. but might be explained if the higher doses of flurbi- 4. Higgs GA, Eakins KE, Mugridge KG, Moncada S, and Vane profen were irritating to the ocular tissues. JR: The effects of nonsteroid anti-inflammatory drugs on leu- Indomethacin was without effect on either param- kocyte migration in carrageenin-induced inflammation. Eur eter of inflammation after 48 hrs. It is of interest that J Pharmacol 66:81, 1980. 5. Rosenbaum JT, McDevitt HO, Guss RB, and Egbert PR: En- topical administration of indomethacin was found to dotoxin-induced uveitis in rats as a model for human disease. enhance significantly the number of polymorpho- Nature 286:611, 1980. nuclear leukocytes in the aqueous humour of the rab- 6. Bhattacherjee P, Williams RN, and Eakins KE: An evaluation bit with immunogenic uveitis while betamethasone of ocular inflammation following the injection of bacterial en- and dexamethasone, inhibited white cell entry into dotoxin into the rat foot pad. Invest Ophthalmol Vis Sci 9 24:196, 1983. the anterior chamber. No sign of enhanced leukocyte 7. Phelps P and McCarty DJ Jr: Suppressive effects of indo- accumulation was found in the present study follow- methacin on crystal-induced inflammation in canine joints ing indomethacin treatment. These differences might and neutrophilic motility in vitro. J Pharmacol Exp Ther result from the different species involved, however, 158:546, 1967. oral administration of low doses of indomethacin 8. Blackham A and Owen RT: Prostaglandin synthetase inhibi- tors and leucocytic emigration. J Pharm Pharmacol 27:201, have been found to enhance the accumulation of leu- 1975. kocytes in inflammatory exudates, at doses which 9. Kulkarni PS, Bhattacherjee P, Eakins KE, and Srinivasan BD: significantly reduce prostaglandin production, prior Anti-inflammatory effects of betamethasone phosphate, dexa- to inhibiting cell migration at higher doses.4 methasone phosphate and indomethacin on rabbit ocular in- It is now well established that prostaglandins and flammation induced by bovine serum albumin. Curr Eye Res 1:43, 1981. the lipoxygenase products of arachidonic acid are in- 10. Miller JD, Eakins KE, and Atwal M: Release of PGE2-like volved in the ocular responses to trauma and im- activity into aqueous humor after paracentesis and its preven- munogenic or non-immunogenic stimuli.10"14 The tion by aspirin. Invest Ophthalmol 12:939, 1973. mediators involved in endotoxin-induced ocular in- 11. Eakins KE, Whitelocke RAF, Perkins ES, Bennett A, and flammation have yet to be identified. However, the Unger WG: Release of prostaglandins in ocular inflammation in the rabbit. Nature, (New Biol) 239:248, 1972. fact that BW755C, which inhibits both the cyclo-ox- 12. Bhattacherjee P: Release of prostaglandin-like substances by ygenase and lipoxygenase pathways of arachidonic Shigella endotoxin and its inhibition by non-steroidal anti-in- acid metabolism, reduced vasodilation and leukocyte flammatory compounds. Br J Pharmacol 54:489, 1975. accumulation, suggests that both prostaglandins and 13. Yamauchi H, Iso T, Iwao J, and Iwata H: The role of pros- hydroxy products are involved in this model of ocular taglandins in experimental ocular inflammations. Agents Ac- tions 9:280, 1979. inflammation. 14. Bhattacherjee P, Hammond B, Salmon JA, Stepney R, and Eakins KE: Chemotactic response to some arachidonic acid Key words: rat, ocular, inflammation, evaluation, anti-in- lipoxygenase products in the rabbit eye. Eur J Pharmacol flammatory agents 73:21, 1981.

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