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but only delayed excystation of A. polyphaga organ- 5. Hirst LW, Green WR, Merz W, Kaufman C, Visvesvara GS, isms. The reason for the apparent greater effectiveness Jensen A, and Howard M: Management of Acanthamoeba ker- of this system over the other two chemical disinfection atitis. A case report and review of the literature. Ophthalmology 91:1105, 1984. systems is not clear. It is also unclear whether different 6. Samples JR, Binder PS, Luibel FJ, Font RL, Visvervara GS, and strains of Acanthamoeba species would be variably Peter CR: Acanthamoeba keratitis possibly acquired from a hot susceptible to these disinfection systems. However, re- tub. Arch Ophthalmol 102:707, 1984. sults of this study indicate that heat disinfection was 7. Sculley RE, Mark EJ, and McNeely BU: Case records of the more effective overall in killing Acanthamoeba tro- Massachusetts General Hospital. N Engl J Med 312:634, 1985. 8. Blackman HJ, Rao NA, Lemp MA, and Visvesvara GS: Acanth- phozoites and cysts as compared to cold disinfection amoeba keratitis successfully treated with penetrating kerato- systems. plasty: suggested immunogenic mechanisms of action. Cornea Key words: Acanthamoeba, keratitis, soft contact lens, dis- 3:125, 1984. infection 9. Cohen EJ, Buchanan HW, Laughrea PA, Adams CP, Galentine PG, Visvesvara GS, Folberg R, Arentsen JJ, and Laibson PR: From the National Eye Institute,* Bethesda, Maryland, and De- Diagnosis and management of Acanthamoeba keratitis. Am J partments of Ophthalmology! and Microbiology,^ Cleveland Clinic Ophthalmol 100:389, 1985. Foundation, Cleveland, Ohio, and the Centers for Disease Control,§ 10. Moore MB, McCulley JP, Luckenbach M, Gelender H, Newton Atlanta, Georgia. Submitted for publication: August 14, 1985. Reprint C, McDonald MB, and Visvervara GS: Acanthamoeba keratitis requests: David Meisler, MD, Department of Ophthalmology, associated with soft contact lenses. Am J Ophthalmol 100:396, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 1985. 44106. 11. Krachmer JH and Purcell JJ Jr: Bacterial corneal ulcers in cos- metic soft contact lens wearers. Arch Ophthalmol 96:57, 1978. References 12. Yamamoto GK, Pavan-Langston D, Stowe GC, and Albert DM: 1. Visvesvara GS: Free-living pathogenic amoebae. In Manual of Fungal invasion of a therapeutic soft contact lens and cornea. Clinical Microbiology, 3rd ed, Lennette EH, Balows A, Hausler Ann Ophthalmol 11:1731, 1979. WJ, and Truant JP, editors, Washington, DC, American Society 13. Wilson LA, Schlitzer RL, and Ahearn DG: Pseudomonas corneal for Microbiology, 1980, pp. 704-708. ulcers associated with soft contact lens wear. Am J Ophthalmol 2. Culbertson CG: Pathogenic Acanthamoeba (Hartmannella). Am 92:546, 1981. JClinPathol 35:195, 1961. 14. Myrowitz E, Pearlman P, and Goldberg HK: A case of Pseu- 3. Page FC: Re-definition of the genus Acanthamoeba with descrip- domonas keratitis in a soft contact lens wearer using contami- tions of three species. J Protozool 14(4):709, 1967. nated chemical disinfection solution. Contact Intraocul Lens Med 4. Nagington J and Richards JE: Chemotherapeutic compounds J7(4):337, 1981. and Acanthamoebae from eye infections. J Clin Pathol 29:648, 15. Freedman H and Sugar J: Pseudomonas keratitis following cos- 1976. metic soft lens wear. Contact Lens Journal 10:21, 1976.

Bioavailability and Corneal Anti-Inflammatory Effect of Topical Suprofen

Howard M. Leibowirz,* William J. Ryan,* Allan Kupferman,*f and Louis DeSanrisJ

The bioavailability in rabbit cornea and aqueous humor of an centrations of suprofen when administered according to this ophthalmic formulation of suprofen, a nonsteroidal anti-in- regimen. Invest Ophthalmol Vis Sci 27:628-631, 1986 flammatory drug, was evaluated following topical adminis- tration of a single dose to the eye. The drug penetrated rapidly into the uninflamed cornea with intact epithelium; highest Locally administered corticosteroids effectively sup- levels occurred during the first 30 to 45 min after instillation press corneal inflammation, but their use carries the and decreased thereafter. The bioavailability of suprofen in risk of several ocular complications, including cataract, cornea and aqueous humor following administration of a 1.0% glaucoma, and the enhancement of actively replicating concentration was twice that produced by a 0.5% concentration herpes simplex virus. This has prompted the search for of the drug. Topical application of multiple doses of suprofen other effective but potentially less toxic compounds. failed to suppress polymorphonuclear leukocyte invasion of We have studied suprofen, a nonsteroidal anti-inflam- the cornea if treatment was started after the induction of in- matory agent, and report here our data on its bioavail- flammation. Suprofen therapy initiated prior to the induction of corneal inflammation and maintained into the post-inflam- ability and anti-inflammatory effectiveness in the cor- mation period did produce a significant (P < 0.01) decrease nea following topical administration to the rabbit eye. in the numbers of PMNs that invaded the inflamed cornea. Materials and Methods. Bioavailability studies: So- There was no significant difference (P > 0.05) in the corneal dium thiamylal was administered intravenously to New anti-inflammatory effect achieved by the 0.5% and 1.0% con- Zealand albino rabbits (1.8-2.4 kg), producing light

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anesthesia for approximately 10 min and allowing op- Table 1. Mean concentration of suprofen following timal control of the administration of radiolabeled topical application to the normal rabbit cornea* drug. A single 0.05-ml dose of 3H-suprofen 0.5% (17.6 Cornea (fig/gm) Aqueous humor (iig/ml) AtCi/mg) or 1.0% (16.0 /iCi/mg) was placed on the cen- Time tral corneal surface (epithelium intact) of both non- (min) 0.5% 1.0% 0.5% 1.0% inflamed eyes using a microsyringe. The 0.05-ml dose was contained within the conjunctival sac while the 5 17 (±3) 25 (±5) 0.076 (±0.007) 0.192 (±0.031) 15 11 (±3) 18 (±3) 0.120 (±0.015) 0.719 (±0.020) lids were manually blinked three times and taped 30 14(±l)t 22 (±4)f 0.514 (±0.014) 1.342 (±0.271) closed. At fixed times thereafter animals were killed by 45 10 (±2) 16 (±4) 0.673 (±0.018)* 0.863 (±0.250) intracardiac pentobarbital sodium; three saline-soaked, 60 7(±l)t 6 (±2)f 0.508 (±0.047) 1.570 (±0.346)§ 120 4(±1) 10(±l) 0.354 (±0.044) 0.699 (±0.146)§ cotton-tipped applicators, followed by a dry, cotton- 240 4(±D 6(±1) 0.279 (±0.023) 0.430 (±0.038) tipped applicator, were gently rolled over the corneal 360 2(±1) 6(±D 0.151 (±0.019) 0.238 (±0.030)

surface to remove radiolabeled surface contaminants. * Table entries are the arithmetic mean (±SEM) of data derived from six This procedure did not disrupt the epithelium, as de- uninflamed eyes with intact epithelium following topical application of a single 0.05-ml dose. Only one eye of an animal was used to obtain data for a given termined by biomicroscopic observation and fluores- time period. cein staining. t 60-minute value is significantly less (P < 0.01) than the 30-min value in the same column. An aqueous humor sample was aspirated from the % Peak value—significantly greater (P < 0.05) than all other values in the anterior chamber with a 27-gauge needle attached to same column. a tuberculin syringe, and an 8-mm full thickness central § The 120-min value is significantly less (P < 0.05) than 60-min value. corneal specimen was obtained by trephination. The corneal samples were weighed and dissolved in 1.5 ml of 0.5N quaternary ammonium hydroxide in toluene PMN invasion of the inflamed cornea was examined (Soluene 100, Packard Instruments; Downers Grove, both when treatment was initiated after induction of IL) at 37 °C over a 24-hr period. Each sample was acid- inflammation and when treatment was initiated prior ified with 0.2 ml concentrated HC1 to eliminate che- to inflammation and continued after induction of the miluminescence produced by the Soluene. Thereafter inflammatory event. Each regimen is outlined in Table the corneal and aqueous samples were handled iden- 4. A control group treated with suprofen vehicle (a tically. Each was diluted with 15 ml of scintillation proprietary buffered ophthalmic vehicle containing counting solution (Ultrafluor, National Diagnostics; preservative and xanthine derivatives) was run with Somerville, NJ) and counted for a minimum of 10 each experimental trial. One hour after completion of min in a Packard 460C scintillation spectrometer. The each treatment protocol a 10-mm full thickness corneal corrected counts per minute were then converted and button was removed by trephination, and the tissue samples were solubilized with a commercially available expressed as micrograms of suprofen per gram of cor- solubilizing agent (Soluene 100, Packard), as described neal tissue or per milliliter of aqueous humor. The final above. The soluble samples were counted for a mini- figure reported for each designated time period is based mum of 10 min, quantitatively measuring the amount on data from six eyes collected in a minimum of two of radioactivity in the cornea. The specific calculations individual experimental trials. The geometric mean was used to determine the data have been reported.1 Sta- plotted as a function of time, a smooth curve was con- tistical evaluation of the data was performed primarily structed, and the area under the curve was determined with analysis of variance using the Scheffe test. This by use of the trapezoid rule. The area thus obtained investigation conformed to the ARVO Resolution on was used to determine the ability of suprofen, with The Use of Animals in Research. time, to concentrate in the cornea and aqueous humor. Half life and the "elimination constant" of topically Results. Bioavailability studies: Measurable quan- applied suprofen in rabbit cornea and aqueous humor tities of suprofen were detected in both cornea and also were calculated. Anti-inflammatory effectiveness of suprofen: Each animal was anesthetized with intravenous thiamylal Table 2. Concentration with time of suprofen in the sodium, and a bilateral inflammatory response was rabbit cornea and aqueous humor* produced by intracorneal inoculation of 0.03 ml lab- Cornea Aqueous humor oratory grade clove oil. Prior to induction of inflam- Drug (ng-min/gm) (ng-min/ml) mation all animals received three intravenous inocu- Suprofen 0.5% 1571 119 lations of 0.05 fid/kg of tritiated thymidine (6.7 Ci/ Suprofen 1.0% 3732 255 mmole) at 24-hr intervals. The intracorneal injection * Table entries are derived from data obtained from uninflamed eyes with of clove oil was given concomitantly with the third intact epithelium following topical application of a single 0.05-ml dose, as thymidine injection. The ability of suprofen to suppress reported in Table 1.

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Table 3. Half life and "elimination constant" of topically applied suprofen in rabbit cornea and aqueous humor

Cornea Aqueous humor

Half life Elimination constant Half life Elimination constant (Tm) (K*1 X 10~3) (fC1 X IO~3)

Suprofen 0.5% 127 min 3.5 min"1 165 min 4.2 min"1 Suprofen 1.0% 200 min 5.5 min"1 148 min 4.7 min"1

aqueous humor (Table 1). Highest corneal levels oc- during the two subsequent days did result in a signif- curred during the first 30-45 min after instillation icant decrease (P < 0.01) in the numbers of radiolabeled without a definite peak. Thereafter corneal suprofen polymorphonuclear leukocytes that invaded the cornea levels decreased; 60-min values were significantly less in comparison to simultaneously run, vehicle treated (P< 0.01) than 30-min values after instillation of both controls. There was no significant difference (P> 0.05) concentrations of suprofen. Suprofen 0.5% produced between the anti-inflammatory effect achieved by the a definite peak in aqueous humor concentration 45 0.5% and 1.0% concentrations of the drug. Moreover, min after application, whereas suprofen 1.0% yielded a less aggressive pretreatment regimen (ie, administra- a prolonged peak during the first 60 min after instil- tion of suprofen only 1 day prior to induction of in- lation with no significant difference (P > 0.05) between flammation and elimination of its use on the day in- each of the values measured during this interval. flammation was induced) was ineffective. Specific val- Thereafter the aqueous humor suprofen level decreased ues are presented in Table 4. significantly (P < 0.05). Discussion. The accumulation of polymorphonu- The area under the time-concentration curve, an clear leukocytes (PMNs) is a prominent event in acute expression of the total quantity of drug in cornea and inflammation of the cornea.2 A number of substances aqueous humor, is presented in Table 2. The data show present in the inflammatory locus exert PMN che- that 1.0% suprofen produces twice the drug bioavail- motactic activity; these include denatured and degraded ability in both ocular locations than does the 0.5% proteins, fibrin degradation products, leukotriene B4, 3 concentration. Mean values for the half life (Ti/2) and complement fragments, and bacterial factors. Pre- the "elimination constant" (Kel) of topically admin- sumably the primary role of the PMN in the inflamed istered suprofen are presented in Table 3. cornea is to phagocytize such material as tissue debris, Anti-inflammatory effectiveness of suprofen: The foreign particles, immune complexes, and microor- anti-inflammatory effectiveness in the cornea of su- ganisms. Unfortunately, in this process lysosomal profen was directly dependent on whether treatment neutral proteases, oxygen-derived free radicals, and was started before or after the induction of inflam- metabolites of arachidonic acid are liberated into the mation. Treatment started either immediately after in- extracellular environment. These factors exert a pro- duction of inflammation or 24 hr later was ineffective. inflammatory effect4 that on balance may be detri- In contrast, treatment initiated 48 hr prior to clove oil mental to the optical integrity of the cornea. injection and continued on the day of injection and Suprofen, currently under clinical investigation as a

Table 4. Mean decrease in corneal inflammatory activity*

Day If Day2f Day if Day4f DaySf % Decrease IV thymidine IV thymidine IV thymidine intracorneal clove oil

no treatment no treatment suprofen vehicle suprofen vehicle suprofen vehicle 12.8 (±1.4) suprofen vehicle suprofen vehicle suprofen vehicle suprofen vehicle suprofen vehicle 9.6 (±4.1) no treatment no treatment no treatment suprofen 0.5% suprofen 0.5% 14.9 (±4.7) no treatment no treatment suprofen 0.5% suprofen 0.5% suprofen 0.5% 13.5 (±5.1) no treatment no treatment suprofen 1.0% suprofen 1.0% suprofen 1.0% 16.9 (±4.7) suprofen 0.5% suprofen 0.5% suprofen 0.5% suprofen 0.5% suprofen 0.5% 32.4 (±5.1)§ suprofen 1.0% suprofen 1.0% suprofen 1.0% suprofen 1.0% suprofen 1.0% 38.9 (±2.5)§ no treatment suprofen 0.5% no treatment suprofen 0.5% suprofen 0.5% 18.1 (±4.9)

* Table entries are the arithmetic mean ± standard error of the mean of t All treatment regimens = 0.5 ml qlh X 8. data derived from the study of 12 eyes (six animals). Values are expressed as $ All treatment regimens = 0.5 ml qlh X 6. percent difference from the mean of 12 simultaneously run, untreated control § Values are significantly different (P < 0.01) from vehicle controls but are eyes (six animals). not significantly different (P > 0.05) from each other.

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topical ophthalmic drug, reaches measurable levels in Key words: suprofen, nonsteroidal anti-inflammatory agent, the cornea following a single topical instillation. More- corneal inflammation, bioavailability, polymorphonuclear over, it does so in the noninflamed eye with intact cor- leukocyte neal epithelium, a circumstance usually associated with From the Departments of Ophthalmology* and Pharmacology]", comparatively low drug bioavailability. Higher corneal Boston University School of Medicine, Boston, Massachusetts, and levels of suprofen can be anticipated in the inflamed the Research and Development Division, Alcon Laboratories, Inc.4 eye, a situation in which there is a partial breakdown Fort Worth, Texas. Supported in part by Public Health Service Grant in the epithelial lipophilic barrier to drug penetration, EY-00544 from the National Eye Institute, by grants from Research to Prevent Blindness, Inc., the Massachusetts Lions Eye Research and in the absence of an intact epithelial layer. None- Fund, Inc., and Alcon Laboratories, Inc. Submitted for publication: theless, suprofen did not produce a significant (P July 8, 1985. Reprint requests: H. M. Leibowitz, MD, Boston Uni- > 0.05) reduction in neutrophils invading the inflamed versity School of Medicine, 80 E. Concord St., Boston, MA 02118. cornea unless therapy was initiated prior to the induc- tion of inflammation. Although it is known that most References nonsteroidal anti-inflammatory drugs inhibit the pro- duction of prostaglandins and thromboxanes,5 it must 1. Leibowitz HM and Kupferman A: Anti-inflammatory effective- ness in the cornea of topically administered prednisolone. Invest be emphasized that there is no unifying hypothesis that Ophthalmol 13:757, 1974. explains adequately how and why NSAIDs act to sup- 2. Aronson SB and Moore TE Jr: Corticosteroid therapy in central press inflammation. Even the mode of action of aspirin, stromal keratitis. Am J Ophthalmol 67:873, 1969. the oldest member of the group, is obscure. 3. O'Flaherty JT and Ward PA: Chemotactic factors and the neu- trophil. Semin Hematol 16:163, 1979. The effect of NSAIDs on leukocyte migration is 4. Abramson S and Weissman G: The release of inflammatory me- controversial. Several investigators studying indo- diators from neutrophils. Ricerca in Clinica E in Laboratorie methacin have reported a suppressive effect on PMN 11:91, 1981. accumulation,67 while others have found that this drug 5. Vane JR: Inhibition of prostaglandin synthesis as a mechanism causes an increase in PMNs.8'9 There appears to be no of action for aspirin-like drugs. Nature (New Biol) 231:232, 1971. satisfactory explanation for these contradictory results. 6. Higgs GA, Eakins ICE, Mugridge KG, Moncada S, and Vane JR: The effects of non-steroid anti-inflammatory drugs on leu- Similarly, the mechanism of action for the effects of kocyte migration in carrageenin-induced inflammation. Eur J suprofen in the inflamed cornea is not known. Pharmacol 66:81, 1980. The present findings suggest that optimal use of su- 7. Salmon JA, Simmons PM, and Moncada S: The effect of profen may be in situations in which the ophthalmol- BW755C and other anti-inflammatory drugs on eicosanoid con- ogist induces the inflammation and so controls its on- centrations and leukocyte accumulation in experimentally-in- duced acute inflammation. J Pharmacol 35:808, 1983. set. In contrast to corticosteroids, suprofen does not 8. Higgs CA, Eakins KE, Moncada S, and Vane JR: Arachidonic 10 inhibit the healing of stromal wounds. Thus, this drug acid metabolism in inflammation and the mode of action of may be especially useful for treatment of milder forms anti-inflammatory drugs. Agents Actions (SuppI) 6:167, 1979. of inflammation associated with ocular surgery and 9. Lundberg C and Gerdin B: The inflammatory reaction in an laser therapy, particularly if it proves to be free from open model of open wounds in the rat. The effect of arachidonic acid metabolites. Eur J Pharmacol 97:229, 1984. other toxic side effects that accompany corticosteroid 10. Lee BP, Kupferman A, and Leibowitz HM: Effect of suprofen therapy. on corneal wound healing. Arch Ophthalmol 103:95, 1985.

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