PROSTAGLANDINS, NONSTEROIDAL ANTI-INFLAMMATORY AGENTS and EYE DISEASE* by Steven M

Home , Arachidonic acid, Clonixin, Fenamic acid, Oxyphenbutazone, Pirprofen, Prostaglandin antagonist

PROSTAGLANDINS, NONSTEROIDAL ANTI-INFLAMMATORY AGENTS AND EYE DISEASE* BY Steven M. Podos, MD

DURING THE LAST DECADE AN EVER-INCREASING NUMBER OF INVESTIGATIONS HAVE been carried out on the prostaglandins and their inhibitors. It has become clear that the prostaglandins are an important group of compounds. One function ofthese compounds is the mediation ofcertain aspects ofthe inflammatory response. Moreover, a host of nonsteroidal anti-inflammatory agents that inhibit the action of the prostaglandins have been elucidated. It is the purpose of this report to review the interrelationships of the prostaglandins, their inhibitors, and their role in disorders of the ocular system and to present the results ofstudies designed to answer some ofthe questions about their actions and inhibition with respect to the eye.

PROSTAGLANDINS AND THE EYE Mechanical irritation of the iris produces miosis, increased vascular permeability, and elevation of intraocular pressure in rabbits.' Rabbit iris contains a substance which contracts smooth muscles and mimics the effects of stroking the iris. This substance is an unsaturated hydroxy fatty acid which Ambache named irin.2 After paracentesis, irin appears in secondary aqueous humor.3 Acidic lipids extracted from human vesicular glands, which von Euler called prostaglandins, contract smooth muscle.4 The prostaglandins are 20 carbon, unsaturated fatty acids.§ Arachidonic acid (5, 8, 11, 14- eicosatetraenoic acid) and dihomo-y-linolenic acid (8, 11, 14-eicosatri- enoic acid) are the precursors of prostaglandin E2 (PGE2) and prostaglandin E, (PGE,), respectively. Other prostaglandins are reported

*From the Departments of Ophthalmology, Mount Sinai School of Medicine of The City University of New York, New York, and Washington University School of Medicine, St. Louis, Missouri. Supported in part by research grants EY-00004, EY-01333, and EY-01661 from the National Eye Institute, Bethesda, Maryland. TR. AM. OPHTH. Soc., vol. LXXIV, 1976 638 Podos such as the A, F, and B series. The activity of the prostaglandin synthesis system varies among different tissues of the body. The prostaglandins exist in virtually ever.y organ system and their actions are diverse.6 They are involved in inflammation, pain, fever, smooth muscle contraction, gastric secretion, lipid and carbohydrate metabolism, cardiovascular responses, renal physiology, and blood coagulation, for example. Suggested therapeutic implications of this family of compounds include their use as bronchial dilators, gastric antisecretogogues, abor- tifacients, and antihypertensive agents. Different members of the family may have different actions on the same system. The iris of rabbits and sheep contains PGE2 and prostaglandin Fa (PGF2,) which are felt to be the vasoactive agents of irin.7 Moreover, the iris of the pig eye, in vitro, is able to convert fatty acid precursor to prostaglandin.8 Thus, the eye contains these compounds and the machinery to manufacture them. The effects of prostaglandins on the eye have been reviewed.9'10 Prostaglandins, administered topically, systemically, or intracamerally, elevate intraocular pressure in the rabbit, cat, and monkey. 11-17 Very small doses of prostaglandin produce this effect. With topical application of0.55,g or greater of PGE 1, a significant rise in intraocular pressure occurs with a maximum elevation within the first 30 minutes. 16 This is followed by a gradual decrease. Two hours after 50 ,.ug of PGE1 the intraocular pressure still remains above baseline values. The PGEs induce the greatest pressure response while PGF2a, PGA, and PGF1. are respectively less effective. 12 Differences between species of animals are noted. The mechanism of the intraocular pressure response appears to be increased aqueous humor production. No decrease of outflow facility occurs after intracameral injections of PGE1 in cannulated eyes of anes- thetized rabbits. 14 In awake, non-cannulated rabbits, tonography reveals an increase in total facility of outflow after topical PGE1.16 The intraocular pressure response is less in the monkey after equivalent doses of PGE but a rise of outflow facility also occurs. 17 PGE1 increases trans- epithelial short circuit current in isolated ciliary body preparations,18 implying increased active transport. A breakdown of the blood aqueous barrier and increased pseudofacility caused by PGE may provide other possible explanations for its pressure effects. After topical application of PGE, extravasation of thorotrast particles occurs in the stroma of the ciliary and iris processes.19 After injection of PGE1 into the vitreous humor there is alteration of the tight junctions of the non-pigmented epithelium allowing for leakage of protein into the anterior chamber in Prostaglandins 639 rabbits.20 In addition, anterior segment fluorescein angiography shows increased vascular permeability in rabbit eyes.21 A marked increase of aqueous-humor protein is a concomitant feature in cat, monkey, and rabbit eyes after treatment with PGE1 or PGE2. 12-14,16 The protein level in rabbit aqueous humor is ten to fifteen times as high one hour after the topical application of 5 ug of PGE1 as compared to aqueous humor from untreated or control eyes. 16 The reason for the increased aqueous humor production is not entirely resolved. Other related features of the effect of prostaglandins on the eye are of interest. Most investigators report concomitant miosis. 11-13 A consensual response in the fellow eye after treating one eye with intracameral prostaglandin is reported. 12 The secondary messenger cyclic adenosine-3',5' -monophosphate (cyclic AMP, c-AMP) is involved in the actions of prostaglandins.22'23'24 Adenyl cyclase, the enzyme which generates cyclic AMP, and c-AMP phosphodiesterase, which converts cyclic AMP to the metabolically inactive adenosine-5'-monophosphate, exist in ciliary processes and iris tissue. 25,26 Cyclic AMP is present in aqueous humor, iris, ciliary body, and other ocular tissues.27 Adrenergic agents increase the amount of anterior but not posterior chamber aqueous humor c-AMP,28'29 and c-AMP increases outflow facility.30 Ciliary process adenyl cyclase is responsive to PGE,25 as is the cyclic AMP of rabbit iris and ciliary body in vitro. 27 The following studies were carried out to delineate the effect oftopically applied PGE2 on aqueous humor cyclic AMP in rabbits in vivo.

MATERIALS AND METHODS 3H-c-AMP (38.4 Ci/mMole) was obtained from New England Nuclear, c-AMP from Sigma, and scintillation fluid (3a40) from Research Products International Corporation. c-AMP-dependent protein kinase was purified from rabbit skeletal muscle by the method of Wastila and associates.31 The protein kinase inhibitor was purified through the trichloracetic acid precipitation step from rabbit skeletal muscle by the method of Walsh and co-workers.32 Charcoal (3 gm, Nutural A from Sigma) was suspended in 250 ml of 50 mM potassium phosphate buffer, pH 4.0, and stirred for 30 minutes at 4°C. Bovine serum albumin (0.5 gm, Sigma) and dextran (0.3 gm, mol. wt. 8200, Sigma) were dissolved in 250 ml of the same buffer and stirred for at least 30 minutes. The dextran and bovine serum albumin solution was then added to the charcoal suspension and was allowed to stir overnight at 4°C. This was made up fresh each week and was stirred for 30 minutes before each use. 640 Podos Cyclic AMP was measured by a modification of the protein-binding method of Gilman.33 All procedures were carried out in an ice bath except where indicated. All components of the reaction mixture except the protein kinase were made up or diluted in 50 mM sodium acetate buffer, pH 4.0. Protein kinase was diluted in 10 mM potassium phosphate, pH 7.0. The concentration of the components of the reaction mixture was adjusted so that there was enough 3H-c-AMP to give 25,000 counts per minute, enough protein kinase to bind between 12 and 20% of the c-AMP, and a maximal effective concentration ofprotein kinase inhibitor. Volumes normally used were 110 ul sodium acetate buffer, 20 ,1u 3EH-c- AMP, 20 ul protein kinase inhibitor, and 50 ul binding protein. Test tubes containing the air dried aqueous humor samples were stirred vigorously with buffer on a vortex mixer to insure all c-AMP was in solution. The reaction was initiated by the addition of protein kinase. After two hours the reaction was stopped by the addition of 1 ml charcoal and the test tube was quickly stirred on a vortex mixer and centrifuged for five minutes at 1300 X g. Centrifugation was done at room temperature. The aqueous phase was decanted off the charcoal and counted in 10 ml scintillation fluid. A standard curve of0.25 to 16 p moles c-AMP was run with each experi- ment and duplicates were run at each concentration. Each milligram of PGE2 was dissolved in 0.1 ml of 95% ethanol and 0.9 ml of 0.02% sodium carbonate aqueous solution. Baseline intraocular pressure was measured in awake, wrapped animals under topical 0.5% proparacaine anesthesia with the Mackay-Marg tonometer. PGE2, 5 ,ug in 5 ,ul, was applied to one eye and 5 ,ul of diluent to the other eye. Other rabbits received just the diluent. Thirty minutes later, intraocular pressure was remeasured and 0.1 ml of anterior chamber aqueous humor was removed with a tuberculin syringe. Duplicate samples of 100 ,1 aqueous humor were immediately pipetted into 1.5 ml absolute ethanol in 12 X 75 mm polystyrene test tubes. These were placed in boiling water until the ethanol boiled and were then cooled in an ice bath. They were then dried under a stream ofair and the c-AMP assay was run in the same test tube.

RESULTS At 30 minutes after instillation ofPGE2, the cyclic AMP in aqueous humor was significantly higher in the treated eyes than in the fellow eyes (Table I). This was associated with a significant elevation of intraocular pressure in the PGE2-treated eyes. Ten eyes of rabbits treated just with diluent Prostaglandins 641

TABLE I: EFFECT OF TOPICAL PGE2 ON AQUEOUS HUMOR CYCLIC AMP IN 15 RABBITS Mean c-AMP + SE Mean AIOP + SE (nM/L) (mm Hg) PGE2 (5 AO 53.3 3.3* + 10.7 ± 1.4* Felloweye 34.1 ±2.7 -0.3 ±0.6 *Significant difference between eyes treated with PGE2 and eyes treated with diluent at 30 minutes after treatment, paired t-test, p < 0.001. had a mean c-AMP level of 25.7 + 1.1 (SE) nM/L in their anterior chamber aqueous humor. The higher levels in the fellow eyes may re- flect a consensual response. The results of cyclic AMP in anterior chamber aqueous humor of untreated animals agree well with those previously reported.27 It is not surprising but reassuring that PGE2, in vivo, produces an increase of aqueous humor c-AMP. Further studies are needed to explain why the epinephrine-induced elevation of ocular cyclic AMP is associated with a fall in intraocular pressure while the PGE-induced aqueous humor cyclic AMP elevation is associated with a rise of intraocular pressure.

INHIBITION OF PROSTAGLANDINS The direct actions of prostaglandins may be inhibited by a variety of compounds such as polyphloretin phosphate (PPP). The smooth muscle contraction in isolated preparations induced by PGE2 and PGFv is inhibited by PPP. In the rabbit, close-arterial infusion or subconjunctival injection ofPPP antagonizes the intraocular pressure elevation due to PGE. 15 Topical instillation of PPP only limited the PGF2a- induced pressure rise. A low molecular weight fraction of PPP contains the major prostaglandin-blocking activity. Subconjunctival PPP also antagonizes the increased permeability of the iris due to topical PGE as documented by fluorescein iris angiography.21 Intraocular PPP fails to modify the effects of prostaglandin in the monkey eye. 14 Some prostaglandin congeners selectively antagonize smooth muscle actions of the prostaglandins,34 but no information is reported with respect to ocular studies along these lines. In our unpublished experiments, one of these compounds, 7-oxa-13 prostynoic acid administered topically in doses of 500 ,g, does not ablate the intraocular pressure response of rabbit eyes to 5 ,ug of topical PGE1. Another agent capable of blocking prostaglandin-induced intraocular pressure elevation in rabbits is imidazole.B5 If the elevation of intraocular pressure induced by prostaglandins is related to the generation of increased levels of intraocular cyclic 64%2 Podos AMP,25'26'27 agents which increase the activity of phosphodiesterase, its inactivating enzyme, should antagonize the prostaglandin effect by lowering the cyclic AMP concentration. Although a variety ofdrugs, such as the methylxanthines, are known to inhibit phosphodiesterase,36 very few cyclic AMP phosphodiesterase stimulators are described. Imidazole is capable of increasing the activity of cyclic AMP phosphodiesterase in vitro.37 The specificity ofthis effect is demonstrated by other studies.36 Our in vivo studies demonstrate the capability of imidazole to block the prostaglandin-induced intraocular pressure elevation in awake rabbits. 38 However, imidazole also inhibits the elevation of intraocular pressure produced by topical nitrogen mustard, an action that is not prostaglandin-mediated. Thus imidazole is not a specific prostaglandin inhibitor but does have promising anti-inflammatory properties. 9 On the other hand, the endogenous synthesis and release of prostaglandins may be inhibited and thus their effects prevented. The aspirin- like compounds are such inhibitors. Aspirin and indomethacin inhibit the generation of PGE2 and PGF2, from arachidonic acid added to homogenates of guinea pig lung.40 The release of E prostaglandins from rabbit spleen is enhanced by the addition of arachidonic acid and abolished by aspirin and indomethacin.41 Aspirin, in vivo and in vitro, inhibits the production of prostaglandin in human platelets. " Vane suggests that a mechanism of action of aspirin-like drugs, which are potent anti-inflammatory, analgesic and antipyretic agents, appears to be via inhibition of prostaglandin synthesis.40 Intermediary substances may be involved.43 Many families of compounds have such inhibitory action including the indomethacin analogs, arylacetic acids, fenamates, salicy- lates, pyrazolones, and others."'45'46'47 Very similar rank order of poten- cies are described for these drugs in the in vitro cell-free synthetase assay and the in vivo standard carrageenin edema rat paw assay.'"45 The corticosteroids are very potent in vivo anti-inflammatory drugs. Yet, confficting results are reported with respect to their inhibition of prostaglandin biosynthesis. Some investigators find little effect,44 while others report inhibition of PGE2 synthesis from arachidonic acid in skin homogenates." Possibly, corticosteroids inhibit the release rather than the synthesis of prostaglandins.49 A differential sensitivity of the prostaglandin synthetase system exists in different tissues. For instance, iris enzyme in vitro is 400 times less sensitive to indomethacin than spleen cell-free prostaglandin synthetase preparations. 0 Prostaglandin synthetase in the conjunctiva appears to be somewhat more sensitive than that of iris. In vitro studies of rabbit eyes show that aspirin and dexamethasone have little or no ef- Prostaglandins 643 fect on uveal PG synthetase whereas indomethacin, phenylbutazone, and especially indoxole are quite active in this regard.51 In vivo, in the eye, one experimental model is capable of specifically delineating the effects of inhibitors of prostaglandin synthesis, although other prostaglandin-mediated ocular insults are responsive to such therapy. Arachidonic acid, applied topically to rabbit and monkey eyes, elevates intraocular pressure with a peak response at about 30 minutes.5253 Arachidonic acid also increases anterior chamber protein and facility of outflow measured tonographically. These effects of arachidonic acid are similar to those of PGE2 applied topically. Pretreatment with indomethacin, 10 mg/kg or greater intraperitoneally, or aspirin as a 600 mg suppository, prevents the elevation of intraocular pressure, aqueous humor protein, and PGE induced by topical 2% arachidonic acid. Indomethacin, 2 mg/kg intraperitoneally, reduces the arachidonic acid effect on intraocular pressure by about 50% in rabbits. Topically applied indomethacin also is effective.53 The effect of indomethacin appears to be on the synthesis or release of prostaglandin from arachidonic acid in the eye as indomethacin has no effect on the rise of intraocular pressure seen after instilling 5 gg of PGE1- or PGE2. These data suggest that the presence, action, and inhibition of ocular prostaglandin synthesis can be demonstrated in vivo. Further studies show that indomethacin blocks the arachidonic acid-associated elevation of aqueous humor PGE itself.54 Either exogenous or endogenous precursor could be sub- strate. Conversion of arachidonic acid PGE2 may be occurring in the eye or its adnexa.50 A study was carried out of the effect of topical and intraperitoneal indomethacin on the elevation of intraocular pressure induced by arachidonic acid injected into the vitreous in order to clarify this point.

MATERIALS AND METHODS Awake restrained rabbits were given indomethacin, 10 mg/kg, in aqueous suspension by intraperitoneal injection. Other uninjected rabbits were used as controls. Baseline intraocular pressure was measured one hour later by Mackay-Marg tonometry. Arachidonic acid, 1 mg in .02 ml of peanut oil, was injected into the vitreous humor of one eye and .02 ml diluent into the vitreous humor of the other eye of rabbits pretreated with indomethacin or no drug. Equal numbers of right and left eyes were treated with arachidonic acid. Intraocular pressure measurements were repeated at 30 minutes, 1 hour, 2 hours, and 4 hours after the injection of arachidonic acid. Similar experiments were carried out topically pretreating one eye of rabbits with 2 drops of 0.5% indo- 644 Podos methacin suspension in water. Thirty minutes later intraocular pressure was determined, both eyes received an injection of arachidonic acid into the vitreous humor, and pressure was monitored.

RESULTS Topical and systemic administration of indomethacin effectively reduced the intraocular pressure elevation induced by injection of arachidonic acid (Table II and III). This implies that there is intraocular production or release of prostaglandin from arachidonic acid and that the indomethacin had penetrated the eye. The in vivo system using topical arachidonic acid is applicable to the screening of potential ocular anti-inflammatory agents. 5355 Subconjunc- tival injections of arachidonic acid or topical administration of aqueous solutions of sodium arachidonate may also be employed.56 The questions now arise as to which synthetase inhibitors are most effective in the eye and which clinical conditions are most amenable to such therapy. PROSTAGLANDIN-MEDIATED OCULAR CONDITIONS As the prostaglandins are physiologic constituents of uveal tissue that are capable of elevating intraocular pressure, a relationship to open-angle glaucoma can be envisioned. In one paper it is stated that none out of nine specimens of aqueous humor from patients with cataract contained greater than 2 ng/ml of PGE1-like activity as compared to six out of eight aqueous humor samples from patients with open-angle glaucoma.57 The fact that only the patients with glaucoma were being treated with irritating drops casts doubt on the significance ofthis study. No significant difference is seen between aqueous humor from glaucomatous and non- glaucomatous eyes with regard to levels of PGE, as determined by radioimmunoassay, in another small series of patients in which the speci-

TABLE II: EFFECT OF INTRAPERITONEAL INDOMETHACIN ON THE INTRAOCULAR PRESSURE RESPONSE TO INTRAVITREAL ARACHIDONIC ACID IN RABBITS Mean Pressure SD Number of Intraocular ± (mm Hg) Treatment Animals 0 min 30 min Indomethacin (10 mg/kg) 8 Arachidonicacid (1 mg) 14.1 ± 2.0 16.6 ± 4.8 Peanut oil 14.1 ± 2.0 16.0 ± 2.0 No Indomethacin 8 Arachidonic acid (1 mg) 16.1 ± 1.7 21.5 ± 4.2* Peanut oil 16.8 ± 1.8 15.6 ± 2.0 *Significant mean difference between eyes of individual rabbits receiving arachidonic acid into one vitreous and peanut oil into other vitreous, paired t-test, p < 0.01. Prostaglandins 64%5

TABLE III: EFFECT OF TOPICAL INDOMETHACIN ON THE INTRAOCULAR PRESSURE RESPONSE TO INTRAVITREAL ARACHIDONIC ACID IN 8 RABBITS Mean Intraocular Pressure ± SD (mm Hg) Treatment 0 min 30 min Arachidonic acid (1 mg OU) Diluent 13.9 ± 1.6 25.8 ± 5.1* Indomethacin 0.5% 14.4 ± 1.4 17.8 ± 2.8 *Significant mean difference between eyes of individual rabbits receiving indomethacin 0.5% to one eye and diluent to other followed 30 minutes ltery by arachidonic acid 1 mg into the vitreous of both eyes, paired t-test, p < 0.001. mens were obtained both at surgery by outpatient paracentesis.56 It is most unlikely that prostaglandins relate to the etiology of primary open- angle glaucoma. On the other hand paracentesis and uveitis are associated with elevated levels of aqueous humor prostaglandin. Following paracentesis, the site of disruption of the blood-aqueous barrier occurs within the ciliary processes.59 Aspirin (600 mg PR) prevents the disruption of the blood- aqueous barrier in the rabbit eye. 0 Protein concentrations in secondary aqueous humor after paracentesis, in vivo, are much reduced in aspirin-pretreated rabbits. Paracentesis-produced PGE2-like activity is diminished by pretreatment of rabbits with aspirin (200 mg/kg).61 In rhesus monkeys, however, pretreatment with systemic aspirin or indomethacin does not inhibit the rise of anterior chamber protein after paracentesis or intraocular pressure after subconjunctival arachidonic acid in one study.62 Yet, pretreatment with aspirin does decrease the effusion of protein into the anterior chamber of humans after paracentesis. 3 The inconsistency of results in two primate species is dis- turbing and needs further study. It is attractive to examine the relationships between prostaglandins and uveitis. Many systemic inflammatory responses may be mediated by the prostaglandins. 64 Bovine serum albumin injected into the vitreous of rabbit eyes creates an immunogenic uveitis. Eakins reports that PGE1-like activity equivalent to 45-150 ng/ml is found in the aqueous humor of such treated eyes as compared to less than 2 ng/ml in control aqueous.65 After paracentesis alone, the predominant PGE appears to be E2, 4-16 ng/ml.61 These differences are attributed to the migration of white blood cells into the eye in the former case and the prostaglandin release from these cells as opposed to from uveal tissue. Analysis of human aqueous humor for prostaglandin-like activity reveals that samples from untreated patients with uveitis contain substantial 6466 Podos amounts (20-56 ng/ml assayed as PGE2) compared to specimens from eyes with cataracts or uveitis controlled with corticosteroids.66 Elevated levels of PGE also are reported to occur in the aqueous humor of human eyes with glaucomatocyclitic crisis and Behvet's disease.67 In all of these studies the activity is measured on smooth muscle strips, sometimes specifically confirmed by chromatography. Quantitative analysis of the prostaglandins is a difficult process. Nevertheless, it can be concluded that prostaglandins are involved in inflammation in the eye as they are in inflammation elsewhere in the bQdy. A transport system exists for moving prostaglandins out of the eye,68 and experimentally induced uveitis in rabbits inhibits the in vitro active accumulation of prostaglandins by the isolated ciliary body.69'70 In addition, the ocular tissues have little prostaglandin dehydrogenase activity,7 the enzyme responsible for their degradation. These factors may be responsible, in part, for the PGE accumulation and contribution to the pathogenesis of chronic uveitis.69 Other organic anions are transported out of the eye. 72 Probenecid blocks the secretion of some of these compounds,72 permitting anterior chamber accumulation. If this be the case, probenecid may enhance the in vivo ocular response to prostaglandins.

MATERIALS AND METHODS Awake, restrained rabbits were pretreated with probenecid, 250 mg/kg intraperitoneally. Other rabbits were not so pretreated. Intraocular pressure was measured with a Mackay-Marg tonometer. One hour later, intraocular pressure was remeasured. Either PGE2 (5 ,ug in 5 ,1) or arachidonic acid (2% in peanut oil) was instilled in all eyes. Repeat measurements ofintraocular pressure were carried out at 30 minutes, 1 hour, and 2 hours.

RESULTS No differences in the pressure response to either PGE2 or arachidonic acid occurred comparing the animals pretreated with probenecid to those animals not pretreated with probenecid (Table IV). Thus, probenecid may not inhibit the transport system for prostaglandins in vivo or the prostaglandin effect on pressure may be maximal. Probenecid is not a useful adjunctive agent for enhancing the in vivo ocular effect of prostaglandins in order to compare the effects of various prostaglandin inhibitors. In other experiments, mechanical stimulation of the rabbit eye produces elevations of intraocular pressure and PG-activity that are significantly lower in indomethacin pretreated animals.73 Formaldehyde or Prostaglandins 647

TABLE IV: EFFECT OF INTRAPERITONEAL PROBENECID ON THE INTRAOCULAR PRESSURE RESPONSE TO TOPICAL PGE, OR ARACHIDONIC ACID IN RABBITS Mean Intraocular Pressure + SD (mm Hg) No. 0 min 30 min PGE2 (5 tg) OU Probenecid (250 mg/kg) 10 15.0 ± 1.1 31.0 ± 4.6 Control 10 15.9 ± 1.7 31.2 ± 2.7 Arachidonic acid (2%) OU Probenecid (250 mg/kg) 10 14.0 ± 0.9 21.1 ± 1.6 Control 10 13.6 ± 1.8 23.8 ± 4.0 trigeminal nerve stimulation induce a rise in pressure that is not amelio- rated by indomethacin and no significant PG activity. It is of interest that polyphloretin phosphate has a high molecular weight fraction that has almost no prostaglandin blocking activity but is a potent antagonist of hyaluronidase and formaldehyde. 15'74 PPP also limits the rise of intraocular pressure and aqueous humor protein induced by topical nitrogen mustard.75 Yet, aspirin has little effect on the response ofthe eye to nitrogen mustard. 6 It appears that this irritative stimulus is mediated neuro- logically and not by prostaglandin.76 Sensory denervation with retrobulbar alcohol prevents the protein effect of nitrogen mustard. The disruption of the blood-aqueous barrier after paracentesis, however, is not blocked by retrobulbar anesthesia. Argon laser radiation to rabbit irides also breaks down the blood-aqueous barrier and aspirin reduces this response.60 E prostaglandins found in the aqueous humor during peak laser response are reduced in indomethacin-pretreated animals. 77 Pretreatment of humans and rabbits with aspirin or indomethacin prevents the rebound rise of intraocular pressure after ocular compression.78 Preoperative compression may lead to an elevated ocular tension at the time of opening the globe and the therapeutic role of aspirin in this situation needs further study. Indomethacin also prevents the sustained elevation of pressure in rabbit eyes after the injection of alpha-chymotrypsin into the posterior chamber.79 The secondary rise of intraocular pressure after alkali burns in rabbits appears to be prostaglandin- mediated.10 Prostaglandin-like activity is elevated in aqueous humor after application of 20 ,u of 2N sodium hydroxide. Pretreatment with aspirin or indomethacin abolished this increase ofintraocular pressure and aqueous prostaglandin.81 In addition, the hyperemia of the iris and conjunctiva produced after superior cervical ganglionectomy in the rabbit can be blocked by indomethacin.82 This interaction of the synthesis of prostaglandins and adrenergic responses suggests that epinephrine- 648 Podos induced hyperemia also may be amenable to therapy with synthetase inhibitors. Thus, a large number of ocular inflammations appear to be mediated by prostaglandins and many clinical conditions might be aided by treatment with the appropriate inhibitor of prostaglandin synthesis. Further- more, the use of such drugs would obviate the toxic side effects of the corticosteroids. One is hampered by the paucity of knowledge about which clinical entities are so mediated and the experimental knowledge about which of the many PG synthesis inhibitors are most effective, especially when administered topically. The first step is to look at the nonophthalmologic literature in order to identify potential compounds of this nature. Next one should review the ophthalmologic literature in order to assess their relative potency on in vitro eye tissue synthetase, which may be very different from other tissues. However, most im- portantly, their relative in vivo ocular potency and toxicity must be dis- covered. This may differ from in vitro assessments for many reasons, including variations in the chemical state of the drug, ocular penetration, and the size of particle in suspension.

INHIBITORS OF PROSTAGLANDIN SYNTHESIS What compounds are known presently to be inhibitors of prostaglandin synthesis in any system? In vitro, in nonocular tissue many agents show such activity. 4445,46,83,84 Great variability exists between various tissues. 50 A partial listing by families of compounds would include indole acetic acids: indomethacin, fluoroindomethacin; arylacetic acids: ibuprofen, naproxen, metiazinic acid; anthranilic acids: meclofenamic acid, flufenamic acid, mefenamic acid, niflumic acid; pyrazolones: phenylbutazone, oxyphenbutazone, aminopyrine; and miscellaneous: indoxole, thiabendazole, aspirin, paracetamol, and clofibrate.4"45 In subcellular fractions, the ID50 in order of decreasing potency generally is meclofenamic acid > niflumic acid or indomethacin > mefenamic acid > flufenamic acid > naproxen > phenylbutazone > aspirin or ibuprofen. 83 The ID50 doses of these compounds against prostaglandin synthetase correlate well with the ID50 as anti-inflammatory agents,4"45 except for the fenamates.45 A general order ofpotency in vivo appears to be indomethacin > ibuprofen > metiazinic acid > fenamates > phenylbutazone > oxyphenbutazone > aspirin > aminopyrine in the rat paw test.45 In another in vitro study, the activities are indomethacin > naproxen > aspirin,85 and this rank correlated well with antipyretic, anti-inflammatory, and analgesic action. In addition, certain psychotropic drugs also inhibit prostaglandin synthesis. 86 Prostaglandins 649 The anti-inflammatory and analgesic properties of aspirin, indomethacin, and phenylbutazone are well documented. Further studies on nonophthalmologic in vivo nonsteroidal anti-inflammatory activity are well reviewed for certain new compounds.84 Fenoprofen is effective in delaying the erythema of human skin after ultraviolet irradiation,87 reducing the pain and stiffness of rheumatoid arthritis,"8 and suppressing fever.89 Flurbiprofen is a potent nonsteroidal anti-inflammatory agent. It is more potent than indomethacin and aspirin in certain types of rat inflammation.'° In a controlled study of 35 patients with ankylosing spondylitis, flurbiprofen is reported to be almost as effective as phenylbutazone when given orally.91 Ibuprofen is another agent in this group effective in the rheumatic disorders.92 The fenamates demonstrate antipyretic and anti-inflammatory activity,45 such as that of flufenamic acid in leprosy93 or rheumatoid arthritis. 94 Clonixin is a new nonsteroidal anti-inflammatory drug95 with a nicotinic acid structure which reduces carrageenin pellet edema more potently than aspirin but less so than indomethacin. 9 Naproxen inhibits rat paw edema as well as indomethacin and more so than aspirin or phenylbutazone. 97 Another agent worth exploring is centchroman.98 Very few such new drugs have been investigated in the eye. In vitro, the effect of inhibitors on prostaglandin synthesis is different for the en- zymes of spleen, uvea, and conjunctiva.51 Naproxen, phenylbutazone, and indomethacin are equally potent on anterior uvea, the latter being more potent on conjunctiva. Aspirin and dexamethasone have little activity. Indoxole is more active than any of the above drugs. In vivo, using the arachidonic acid model, our preliminary trials with topically administered drugs,5355 indicate that aspirin and dexamethasone have little effect, whereas 10% oxyphenbutazone ointment, fenoprofen 2% solution and indomethacin 1% suspension are approximately equiactive. Parenthetically, A'-tetrahydrocannabinol, given intravenously, also inhibits the arachidonic acid-induced elevation of intraocular pressure and aqueous humor protein.99 In other studies, sodium arachidonate produces greater effects on intraocular pressure and aqueous humor protein than does arachidonic acid. " Pretreatment with topically applied nonsteroidal anti-inflammatory agents reduces these effects. Indomethacin is more potent than either indoxole or pirprofen in vivo. The following in vivo study was designed to compare the topical effects of a large number of inhibitors of prostaglandin synthesis on the intraocular pressure elevation produced by arachidonic acid in rabbits. MATERIALS AND METHODS Awake rabbits were restrained in canvas wraps. Intraocular pressure 650 Podos was measured with a Mackay-Marg tonometer after topical 0.5% proparacaine anesthesia. Baseline measurements were made. One eye was treated with 2 drops of a solution or suspension of each drug made up just before use. The other eye of each animal received diluent. Equal numbers of right and left eyes were used. Thirty minutes later intraocular pressure was determined and 2 drops of 5% arachidonic acid made up freshly in peanut oil were applied to the cornea of both eyes. Intraocular pressure measurements were repeated at 30 minutes, 1, 2, and 4 hours thereafter. The drugs to be tested were made up in concentrations of 0.01%, 0.1%, 1.0% and 2.0% , and each concentration was tested topically in at least five eyes. The following preparations were tested: solutions of naproxen sodium, fenoprofen sodium, sodium salt of flufenamic acid and sodium salt of meclofenamic acid in distilled water; suspensions of niflumic acid, indoxole, centchroman, clonixin, indomethacin, and oxyphenbutazone in distilled water; solution of flurbiprofen and suspensions of mefenamic acid, flufenamic acid and meclofenamic acid in distilled water and 1 N KOH buffered with saturated KH2PO4 to a pH of 7.4 to 7.8. In addition, the testing was repeated with mefenamic acid, flufenamic acid, meclofenamic acid, indoxole, and centchroman adding two drops of polysorbate 80 (Tween 80) to the preparations.-5 The data were analyzed for each drug, time, and concentration by tabulating the mean difference between control and treated eye ± SE and the mean change in the treated eye divided by the mean change in the control eye. The latter number subtracted from one, times one hundred, indicated percent of inhibition. This was plotted against log concentration and the approximate concentration of drug to produce 50% inhibition of the response to arachidonic acid was calculated.

RESULTS As the maximum elevation of intraocular pressure produced by arachidonic acid occurred at 30 minutes, the comparison of the inhibiting effects of the various nonsteroidal anti-inflammatory agents was best demonstrated at this time (Table V). Dose-response relationships were found by both methods of calculation (see Methods). The optimum effective concentration was different for different agents. Some of the drugs appeared to be more effective inhibitors than others. Indomethacin, flurbiprofen, clonixin, and meclofenamic acid at the 1% and 2% concentrations produced at least 65% inhibition of the arachidonic acid effect. At the 0.1% concentration only flurbiprofen was this active. Prostaglandins 651 I I Q 0em V~~~~~t t 0ot - C'w CrI t

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U "~Z U '20 4.0 rA ~ ~ ~ ~~~ 652 Podos When polysorbate 80 was added to the drugs in suspension in order to provide better dispersion, an appreciably enhanced effect was seen only with indoxole. Indoxole treated in this manner almost completely blocked the arachidonic acid effect at the 1% concentration and was more effective than flurbiprofen at the 0.1% concentration (Table VI). Although the dose-response curves were not linear, the approximate concentration of drug to half-inhibit the arachidonic acid-induced ocular hypertension could be calculated. This provided another means of rank- ing the drugs. Indoxole-polysorbate, flurbiprofen, meclofenamic acid, indomethacin, and clonixin were the most effective, in descending order of potency (Table VII). In this comparative study of the topical administration of 14 nonsteroidal anti-inflammatory agents, an aqueous solution of flurbiprofen and aqueous suspensions of indoxole with polysorbate, indomethacin, meclofenamic acid, and clonixin demonstrate the greatest activity. Indoxole requires the addition of polysorbate 80, an emulsifying and dispersing compound, to be highly effective in blocking the arachidonic acid-induced elevation of intraocular pressure. Many factors must be considered before extrapolating these results to the treatment ofclinical disease. The arachidonic acid system is a good model ofprostaglandin-mediated inflammation with a numerical endpoint of inhibition. It is obvious that the role of prostaglandins is not elucidated for most human clinical entities. Other mediators of inflammation exist, many of which appear susceptible to the effects of corticosteroids. The corticosteroids are weak inhibitors of prostaglandin synthesis. However, the agents tested may be less specific in their actions

TABLE VI: INHIBITION OF INTRAOCULAR PRESSURE RESPONSE TO ARACHIDONIC ACID BY VARIOUS TOPICAL NONSTEROIDAL ANTI-INFLAMMATORY SUSPENSIONS WITH POLYSORBATE-80 Mean /IOP + SE, Control - Drug at 30 Min. (Mean AIOPd4,,JMean AIOP,.fti), 30-0 Min. Arachidonic Acid 5% OU No. 2% 1% 0.1% 0.01% Mefenamic acid 6 3.7 + 1.0 4.0 + 1.3 2.2 ± 1.5 1.0 ± 1.4 (.54) (.55) (.78) (.89) Flufenamic acid 3 3.0 ± 1.7 2.3 ± 2.1 0.3 ± 2.1 0.3 ± 1.5 (.72) (.74) (.96) (.97) Meclofenamic acid 6 5.0 ± 1.0* 4.0 ± 1.0* 3.7 ± 1.5 0.7 ± 1.5 (.42) (.29) (.45) (.91) Indoxole 6 - 8.0 ± 1.4* 6.5 ± 1.3* 3.0 ± 1.6 (.03) (.26) (.67) Centchroman 3 3.3 ± 2.9 2.7 ± 1.5 2.0 ± 1.0 -0.7 ± 1.5 (.60) (.77) (.80) (1.09) *Significant difference between the eyes treated with drug and those treated with diluent, paired t-test, p <0.01. Prostaglandins 653

TABLE VII: DRUGS RANKED BY APPROXIMATE CONCENTRATION TO PRODUCE 50% INHIBITION OF RESPONSE TO ARACHIDONIC ACID Drug Concentration (%) Indoxole + Polysorbate 80 0.03 Flurbiprofen 0.06 Meclofenamic acid 0.09 Indomethacin 0.2 Clonixin 0.4 Fenoprofen 1.0 Mefenamic acid 1.1 Flurenamic acid 1.8 Niflumic acid 1.8 Indoxole 1.8 Meclofenamic acid, sodium salt 2.0 Centchroman >2.0 Oxyphenbutazone >2.0 Flufyenamic acid, sodium salt >2.0 Naproxen >2.0 and should be tested in other ocular inflammations. In addition, the effect of arachidonic acid on intraocular pressure shows some variability. Comparing the effect of potential inhibitors by calculating the ratio of pressure rise in the treated and control eyes takes this variability into account. The preparation of drug certainly plays a role in its effect. Permea- bility is an important issue. The comparative penetrability through the cornea of the agents tested is unknown. Some are solutions and others are suspensions. Flurbiprofen is the only highly effective solu- ble drug in the present study. Dispersion may affect permeability and the dose-response characteristics. At high concentrations the differences between 1% and 2% may be small in drugs that disperse poorly. Niflumic acid, oxyphenbutazone, and indoxole do not disperse well. Indoxole is much more effective in a dispersed suspension. Additives that alter pH, dispersion, and solubility may be important in finding the best topical nonsteroidal anti-inflammatory compounds. Noncorticosteroid anti-inflammatory agents are effective in a variety of human and animal ocular conditions. 100'101 Hydrocortisone 2%% and indomethacin 1%, or these drugs given systemically, reduce intraocular inflammation created in rabbits by intracameral bovine serum. 102 Indo- methacin 0.1% is the minimally active dose in this model.'03 Indo- methacin 1% does not alter wound healing or the course ofcorneal herpes infection. 103 In other studies, indomethacin 0.5% is as effective as dexamethasone 0.1% in rabbit uveitis,104 and indomethacin 0.25% inhibits xenograft reactions.'05 Water suspensions of indomethacin 1% enter the rabbit eye.106 Inflammation enhances its penetration. Oral 654 Podos oxyphenbutazone is effective in human ocular inflammatory disease. 107'108 Aqueous suspensions of this drug are anti-inflammatory in rabbit uveitis in concentrations down to 0.5%.109 Oxyphenbutazone, as a 10% ointment, enters the human eye, does not elevate human intraocular pressure, and is as effective as prednisolone 2.5% in postoperative inflammation.110 It also is effective in the treatment of superficial eye injuries111 and episcleritis.112 It does not hasten galactose-induced cataracts in rats.113 Fenoprofen 1% is effective in rabbit uveitis. 114 The obvious next steps will involve therapeutic trials of the best agents found in the arachidonic acid studies in models of animal disease and ultimately human disease states.

SUMMARY AND CONCLUSIONS The prostaglandins produce elevation of intraocular pressure and breakdown ofthe blood-aqueous barrier. They act via the secondary messenger system, cyclic AMP. Although the pathogenesis of many forms of ocular inflammation, both external and internal, is unclear, it is evident that some forms of ocular inflammation are prostaglandin-mediated, at least in part. Others may be totally mediated by prostaglandin synthesis. At present the corticosteroids are the mainstay of therapy of these conditions. However, the corticosteroids are poor inhibitors of prostaglandin synthesis and have many deleterious side effects such as induction of ocular hypertension, cataract, and infection. The search for new agents that will obviate these side effects and be more specific for the disease process is crucial. The discovery that the mode of action of many nonsteroidal anti-inflammatory agents is via inhibition of prostaglandin synthesis places a premium on elucidating which of these agents is most effective and least toxic in the eye and by which route of administration. The arachidonic acid screening model is ideal for initially choosing which agent has the greatest potential clinically. Arachidonic acid, a PGE2 precursor, when given topically also elevates intraocular pressure and aqueous humor protein, and these effects are blocked by the nonsteroidal anti-inflammatory drugs. This occurs if the arachidonic acid is injected into the vitreous humor, too, providing evidence that this in vivo model involves intraocular mechanisms. Utilizing the arachidonic acid system, a comparative study of nonsteroidal inhibitors of prostaglandin synthesis shows that the most effective of 14 agents were flurbiprofen solution and suspensions of polysorbate-dispersed indoxole, meclofenamic acid, indomethacin, and clonixin. Animal uveitis is not an ideal model for the human condition. Nevertheless, proving the superior efficacy of a Prostaglandins 655 screened drug in this system will identify those drugs to be tested in the human disease states. Only after the very few best drugs of this nature are identified should the ultimate steps of human testing be initiated.

ACKNOWLEDGMENTS Centchroman was provided by the Centra Drug Research Institute, Lucknow, India; oxyphenbutazone by Geigy Pharmaceuticals, Summit, New Jersey; fenoprofen sodium by Lilly Laboratories for Clinical Re- search, Indianapolis, Indiana; indomethacin by Merck, Sharp and Dohme, West Point, Pennsylvania; mefenamic acid, flufenamic acid and sodium salt, and meclofenamic acid and sodium salt by Parke-Davis and Com- pany, Ann Arbor, Michigan; clonixin by Schering Corporation, Bloomfield, New Jersey; niflumic acid by The Squibb Institute for Medi- cal Research, Princeton, New Jersey; naproxen sodium by Syntex Re- search, Palo Alto, California, and indoxole and flurbiprofen by the Upjohn Company, Kalamazoo, Michigan. Segments ofthe older literature review for this paper were freely adapted from a paper "Prostaglandins and the Eye" by Steven M. Podos and Bernard Becker presented at thfe 1973 American Academy of Ophthalmology and Otolaryngology meeting and published in Symposium on Ocular Therapy, volume 7, edited by I. H. Leopold, C. V. Mosby Co., 1974. Dr Kenneth Eakins suggested the use of polysorbate 80.

REFERENCES 1. Duke-Elder PM, Duke-Elder WS: The vascular responses of the eye. Proc Roy Soc Biol 109:19, 1931. 2. Ambache N: Properties of irin, a physiological constituent of the rabbit's iris. J Physiol 135:114, 1957. 3. Ambache N, Kavanagh L, Whiting J: Effect of mechanical stimulation on rabbits' eyes: Release of active substance in anterior chamber perfusates. J Physiol 176:378, 1965. 4. von Euler US: On the specific vasodilating and plain muscle stimulating substance from accessory genital glands in man and certain animals (prostaglandin and vesiglandin). J Physiol 88:213, 1937. 5. Samuelsson B: Biosynthesis of prostaglandins. Fed Proc 31:1442, 1972. 6. Higgins CB, Braunwald E: The prostaglandins: Biochemical, physiologic and clinical considerations. Am J Med 53:92, 1972. 7. Ambache N, Brummer HC: A simple chemical procedure for distinguishing E from F prostaglandins, with application to tissue extracts. BrJ Pharm Chemother 33:162, 1968. 8. Van Dorp DA, Jouvenaz GH, Struijk CB: The biosynthesis of prostaglandin in pig eye iris. Biochim Biophys Acta 137:396, 1967. 9. Neufeld AH, Sears ML: Prostaglandin and eye. Prostaglandins 4:157, 1973. 10. Perkins ES: Prostaglandins and the eye. Adv Ophthalmol 29:2, 1975. 11. Waitzman MB, King CD: Prostaglandin influences on intraocular pressure and pupil size. Am J Physiol 212:329, 1967. 656 Podos 12. Beitch BR, Eakins KE: The effects of prostaglandins on the intraocular pressure of the rabbit. Br J Pharmacol 37:158, 1969. 13. Eakins KE: Increased intraocular pressure produced by prostaglandins El and E2 in the cat eye. Exp Eye Res 10:87, 1970. 14. Kelly GM, Starr MS: Effects of prostaglandins and a prostaglandin antagonist on intraocular pressure and protein in the monkey eye. Can J Ophthalmol 6:205, 1971. 15. Bethel RA, Eakins KE: The mechanism of the antagonism of experimentally induced ocular hypertension by polyphloretin phosphate. Exp Eye Res 13:83, 1971. 16. Kass MA, Podos SM, Moses RA, Becker B: Prostaglandin El and aqueous humor dynamics. Invest Ophtalmol 11:1022, 1972. 17. Casey WJ: Prostaglandin E2 and aqueous humor dynamics in the rhesus monkey eye. Prostaglandins 8:327, 1974. 18. Cole DF, Nagasubramanian S: The effect of natural and synthetic vasopressins and other substances on active transport in the ciliary epithelium of the rabbit. Exp Eye Res 13:45, 1972. 19. Tamura T: Effects of prostaglandins El and E2 on the ciliary body of albino rabbits: An electron microscopic study. Jap J Ophthalmol 18:135, 1974. 20. Meyers RL, Shabo AL, Maxwell DS: Effect of prostaglandin on the blood aqueous barrier in the rabbit ciliary process. Prostaglandins 9:167, 1975. 21. Whitelocke RAF, Ealdns KE: Vascular changes in the anterior uvea of the rabbit produced by prostaglandins. Arch Ophthalmol 89:495, 1973. 22. Hinman JW: Prostaglandins. Ann Rev Biochem 41:170, 1972. 23. Waitzman MB: Possible new concepts relating prostaglandins to various ocular func- tions. Survey Ophthalmol 14:301, 1970. 24. Sears ML, Neufeld AH, Jampol LM: Editorial: Prostaglandin. Invest Ophthalmol 12:161, 1973. 25. Waitzman MB, Woods WD: Some characteristics of an adenyl cyclase preparation from rabbit ciliary process tissue. Exp Eye Res 12:99, 1971. 26. ShantaTR, Woods WD, Waitzman MB, Boume GM: Histochemical method for locali- zation of cyclic 3',5'-nucleotide phosphodiesterase. Histochemie 7:177, 1966. 27. Neufeld AH, Sears ML: Cyclic-AMP in ocular tissues of the rabbit, monkey, and human. Invest Ophthalmol 13:475, 1974. 28. Neufeld AH, Jampol LM, Sears ML: Cyclic AMP in the aqueous humor: The effects of adrenergic agents. Exp Eye Res 14:242, 1972. 29. Neufeld AH, Chavis RM, Sears ML: Cyclic AMP in the aqueous humor: The effects of repeated topical epinephrine administration and sympathetic denervation. Exp Eye Res 16:265, 1973. 30. Neufeld AH, Dueker DK, Vegge T, Sears ML: Adenosine 3',5'-monophosphate increases the outflow of aqueous humorfrom the rabbit eye. Invest Ophthalmol 14:40, 1975. 31. Wastila WB, Stull JT, Mayer SE, Walsh DA: Measurement of cyclic 3',5'-adenosine monophosphate by the activation of skeletal muscle protein kinase. i Biol Chem 246:1996, 1971. 32. Walsh DA, Ashby CD, Gonzolez C, Calkins D, Fischer EH, Krebs EG: Purification and characterization of a protein inhibitor of adenosine 3',5'-monophosphate-dependent proteinkinase. J Biol Chem 246:1977, 1971. 33. Gilman AG: A protein binding assay for adenosine 3',5'-cyclic monophosphate. Proc Natl Acad Sci USA 67:305, 1970. 34. Fried J, Santhanakrishnan TS, Himizu J, Lin CH, Ford SH, Rubin B, Grigas EO: Prostaglandin antagonist: Synthesis and smooth muscle activity. Nature 223:208, 1969. 35. Zink HA, Podos SM, Becker B: Inhibition by imidazole of the increase in intraocular pressure induced by topical prostaglandin E. Nature [New Biol] 245:21, 1973. 36. Robison GA, ButcherRW, Sutherland EW: Cyclic-AMP. Academic Press, New York, 1971, p 84. Prostaglandins 657 37. Butcher RW, Sutherland EW: Adenosine 3',5'-phosphate in biological materials. J Biol Chem 237:1244, 1962. 38. Zink HA, Podos SM, Becker B: Modification by imidazoles of ocular inflammatory and pressure responses. Invest Ophthalmol 14:280, 1975. 39. Puig-Parellada P, Garcia-Gasulla G, Puig-Muset P: Pharmacological properties of imidazole. I. Analgesic and anti-inflammatory actions. Pharmacology 10:161, 1973. 40. Vane JR: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin- like drugs. Nature [New Bioll 231:232, 1971. 41. Ferreira SH, Moncada S, Vane JR: Indomethacin and aspirin abolish prostaglandin release from spleen. Nature [New Biol] 231:237, 1971. 42. Smith JB, Willis AL: Aspirin selectively inhibits prostaglandin production in human platelets. Nature [New Biol] 231:235, 1971. 43. Collier HOJ: Prostaglandins and aspirin. Nature 231:235, 1971. 44. Flower R, Gryglewski R, Herbaczynska-Cedro K, Vane JR: Effects ofanti-inflammatory drugs on prostaglandin biosynthesis, Nature [New Biol] 238:104, 1972. 45. Ham EA, Cirillo VJ, Zanetti M, Shen TY, Kuehl FA Jr: Studies on the mode of action of nonsteroidal anti-inflammatory agents. In Prostaglandins in Cellular Biol- ogy, Ramwell PW, Pharriss BB (eds), New York, Plenum Press, 1972, p 345. 46. Vane JR: Prostaglandins and aspirin-like drugs. Proc 5th Int Cong Pharmacol vol 5, Basel, Karger, 1973, p 352. 47. Flower RJ: Drugs which inhibit prostaglandin biosynthesis. Pharmacol Rev 26:33, 1974. 48. Greaves MW, McDonald-Gibson W: Inhibition of prostaglandin biosynthesis by corticosteroids. Br Med J 2:83, 1972. 49. Lewis GP, Piper PJ: Inhibition of release of prostaglandins as an explanation of some of the actions of anti-inflammatory corticosteroids. Nature 254:308, 1975. 50. Bhattacherjee P, Eakins KE: Inhibition of the prostaglandin synthetase systems in ocular tissues by indomethacin. Br J Pharmacol 50:227, 1974. 51. Bhattacherjee P, Eakins KE: A comparison of the inhibitory activity of compounds on ocular prostaglandin biosynthesis. Invest Ophthalmol 13:967, 1974. 52. Podos SM, Becker B, Kass MA: Indomethacin blocks arachidonic acid-induced elevation of intraocular pressure. Prostaglandins 3:7, 1973. 53. Podos SM, Becker B, Kass MA: Prostaglandin synthesis, inhibition and intraocular pressure. Invest Ophthalmol 12:426, 1973. 54. Jaffe BM, Podos SM, Becker B: Indomethacin blocks arachidonic acid-associated elevation of aqueous humor prostaglandin E1. Invest Ophthalmol 12:621, 1973. 55. Podos SM, Becker B: Fenoprofen and ocular prostaglandin production. Invest Ophthalmol 13:235, 1974. 56. Bhattacherjee P, Eakins KE: Inhibition of the ocular effects of sodium arachidonate by anti-inflammatory compounds. Prostaglandins 9:175, 1975. 57. Wyllie AM, Wyllie JH: Prostaglandins and glaucoma. Br Med J 3:615, 1971. 58. Podos SM, Jaffe BM, Becker B: Prostaglandins and glaucoma. Br MedJ 4:232, 1972. 59. Unger WG, Cole DF, Hammond B: Disruption ofthe blood-aqueous barrier following paracentesis in the rabbit. Exp Eye Res 20:255, 1975. 60. Neufeld AH, Jampol LM, Sears ML: Aspirin prevents the disruption of the blood- aqueous barrier in the rabbit eye. Nature 238:158, 1972. 61. Miller JD, Eakins KE, Atwal M: The release of prostaglandins Erlike activity into aqueous humor after paracentesis and its prevention by aspirin. Invest Ophthalmol 12:939, 1973. 62. Kass MA, Neufeld AH, Sears ML: Aspirin and indomethacin do not prevent the response of the monkey eye to trauma. Invest Ophthalmol 14:604, 1975. 63. Zimmerman TJ, Gravenstein N, Sugar A, Kaufman HE: Aspirin stabilization of the blood-aqueous barrier in the human eye. Am J Ophthalmol 79:817, 1975. 64. Willis AL, Davison P, Ramwell PW, Brocklehurst WE, Smith B: Release and actions of prostaglandins in inflammation and fever: Inhibition by anti-inflammatory and anti- 658 Podos pyretic drugs. In Prostaglandins in Cellular Biology. Ramwell PW, Pharriss BB (eds), New York, Plenum Press, 1972, p 227. 65. Eakins KE, Whitelocke RAF, Perkins ES, Bennett A, Unger WG: Release of prostaglandins in ocular inflammation in the rabbit. Nature [New Bioll 239:248, 1972. 66. Eakins KE, Whitelocke RAF, Bennett A, Martenet AC: Prostaglandin-like activity in ocular inflammation. Br Med J 3:452, 1972. 67. Masuda K, Izawa Y, Mishima S: Prostaglandins and uveitis: A preliminary report. Jap J Ophthalmol 17:166, 1973. 68. Bito LZ, Salvador EV: Intraocular fluid dynamics. III. The site and mechanism of prostaglandin transfer across the blood intraocular fluid barriers. Exp Eye Res 14: 233, 1972. 69. Bito LZ: Inhibition of uveal prostaglandin transport in experimental uveitis. In Prostaglandins and Cyclic AMP, Kahn RH, Lands WEM (eds), New York, Academic Press, 1973, p 213. 70. Bito LZ: The effects of experimental uveitis on anterior uveal prostaglandin transport and aqueous humor composition. Invest Ophthalmol 13:959, 1974. 71. Eakins KE, Atwal M, Bhattacherjee P: Inactivation of prostaglandin El by ocular tissues in vitro. Exp Eye Res 19:141, 1974. 72. Forbes M, Becker B: The transport of organic anions by the rabbit eyes. II. In vivo transport of iodopyracet (Diodrast). Am J Ophthalmol 50:867, (Part II), 1960. 73. Cole DF, Unger WG: Prostaglandins al mediators for the responses of the eye to trauma. Exp Eye Res 17:357, 1973. 74. Cole DF: Formaldehyde-induced ocular hypertension: The effects of polyphloretin phosphate and (+) catechin. Exp Eye Res 19:533, 1974. 75. Cole DF: Prevention of experimental ocular hypertension with polyphloretin phosphate. Br J Ophthalmol 45:482, 1961. 76. Jampol LM, Neufeld AH, Sears ML; Pathways for the response of the eye to injury. Invest Ophthalmol 14:184, 1975. 77. Unger WG, Perkins ES, Bass MS: The response ofthe rabbit eye to laser irradiation of the iris. Exp Eye Res 19:367, 1974. 78. Obstbaum SA, Podos SM: Ocular compression and noncorticosteroidal anti-inflammatory agents. Am J Ophthalmol 79:1008, 1975. 79. Sears D, Sears M: Blood-aqueous barrier and alpha chymotrypsin glaucoma in rabbits. Am J Ophthalmol 77:378, 1974. 80. Paterson CA: Intraocular pressure changes after alkali bums. Arch Ophthalmol 91:211, 1974. 81. Paterson CA, Pfister RR: Prostaglandin-like activity in the aqueous humor following alkali burns. Invest Ophthalmol 14:177, 1975. 82. Neufeld AH, Chavis RM, Sears ML: Degeneration release of norepinephrine causes transient ocular hyperemia mediated by prostaglandins. Invest Ophthalmol 12:167, 1973. 83. Flower RJ: Drugs which inhibit prostaglandin biosynthesis. Pharmacol Rev 26:33, 1974. 84. Willikens RF, O'Brien WM: New nonsteroidal anti-inflammatory drugs. Bull Rheum Dis 24:770, 1974. 85. Tomlinson RV, Ringold HJ, Qureshi MC, Forchielli E: Relationship between inhibition of prostaglandin synthesis and drug efficacy: Support for the current theory on mode of action of aspirin-like drugs. Biochem Biophys Res Comm 46:552, 1972. 86. Lee RE: The influence of psychotropic drugs on prostaglandin synthesis. Prosta- glandins 5:63, 1974. 87. Gruber CM, Ridolfo AS, Nickander R, Mikulaschek WM: Delay of erythema of human skin by anti-inflammatory drugs after ultraviolet irradiation. Clin Pharmacol Ther 13:109, 1972. 88. Huskisson EC, Wojtulewski JA, Berry H, et al: Treatment of rheumatoid arthritis with fenoprofen: Comparison with aspirin. Br Med J 1:176, 1974. Prostaglandins 659 89. Gruber CM: Antipyresis with fenoprofen. J Clin Pharmacol 14:215, 1974. 90. Glenn EM, Rohloff N, Bowman BJ, Lyster SC: The pharmacology of 2-(2-fluoro-4- biphenyl) propionic acid (flurbiprofen), a potent nonsteroidal anti-inflammatory drug. Agents Actions 3/4:210k, 1973. 91. Calin A, Grahame R: Double-blind cross-over trial of flurbiprofen and phenylbutazone in ankylosing spondylitis. Br Med J 2:496, 1974. 92. Davies EF, Avery GS: Ibuprofen: A i'eview of pharmacological properties and therapeutic efficacy in rheumatic disorders. Drugs 2:416, 1971. 93. Goodwin CS: Antipyretic and anti-inflammatory action of flufenamic acid in acute reaction of lepromatous leprosy. Lancet 2:854, 1968. 94. Rajan KT, Hill AGS, Barr A, et al: Flufenamic acid in rheumatoid arthritis. Ann Rheum Dts 26:43, 1967. 95. Katchen B, Buxbaum S, Mayer J, Ning J: Metabolsim and pharmacokinetics of a new nonsteroid anti-inflammatory agent, 2-(3-chloro-o-toluidino) nicotinic acid (clonixin) in rats, dogs and monkeys. J Pharmacol Exp Ther 181:453, 1973. 96. Watnick AS, Taber RI, Tabachnick IA: Anti-iinflammatory and analgesic properties of clonixin. Arch Int Pharmacodyn Ther 190:78, 1971. 97. Roszkowski AP, Rooks WH, Tomolonis AJ, Miller LM: Anti-inflammatory and analgetic properties of d-2-(6'-methoxy-2'-naphthyl)-propionic acid (naproxen). J Pharmacol Exp Ther 179:114, 1971. 98. Dhawan BN, Srimal RC: Anti-inflammatory and some other pharmacological effects of 3,4-trans-2,2-dimethyl-3-phenyl-4-[p(18-pyrrolidinoethoxy)-phenyll-7-methoxy- chroman (Centchroman). BrJ Pharmacol 49:64, 1973. 99. Green K, Podos SM: Antagonism of arachidonic acid-induced ocular effects by A'-tetrahydrocannabinol. Invest Ophthalmol 13:422, 1974. 100. Leopold IH: Advances in ocular therapy: Noncorticosteroid anti-inflammatory agents. AmJ Ophthalmol 78:759, 1974. 101. Sears ML: Use of aspirin and aspirin-like drugs in ophthalmology. Symposium on Ocular Therapy, vol 7, Leopold IH (ed), St Louis, CV Mosby Co, 1974, p 104. 102. Hanna C, Keatts HC: Indomethacin in ocular inflammation in rabbits. Arch Ophthal- mol 77:554, 1967. 103. Hanna C: Nonsteroid anti-inflammatory agents: Past and present. In Symposium on Ocular Anti-inflammatory Compounds, Kaufman HE (ed), Springfield, Charles C Thomas, 1970, p 139. 104. Baldwin HA, Borgmann AR: An improved immunogenic uveitis test in rabbits for evaluation of anti-inflammatory compounds. Proc Soc Exp Biol Med 133:1326, 1970. 105. Lorenzetti DWC, Ellison EM, Kaufman HE: Quantitative steroid effect on graft reaction. Arch Ophthalmol 79:64, 1968. 106. Hanna C, Sharp JD: Ocular absorption of indomethacin by the rabbit. Arch Ophthal- mol 88:196, 1972. 107. Werner LEJ: Hydroxy-phenylbutazone (Tanderil) in the treatment of ocular disease. BrJ Ophthalmol 44:755, 1960. 108. Watson PG, Lobascher DJ, Sabiston DW, et al: Double-blind trial of the treatment of episcleritis-scleritis with oxyphenbutazone or prednisolone. BrJ Ophthalmol 50: 463,1966. 109. Thomas AH, Hanna C: Oxyphenbutazone in ocular inflammation. Arch Opthalmol 89:340, 1973. 110. Wilhelmi E, et al: Experimental and clinical investigation of a nonhormonal anti- inflammatory eye ointment. Ophthalmol Res 5:253, 1973. 111. Valk LEM: A new possibility to treat superficial eye injuries with an ointment, pre- senting the advantages, but not the disadvantages of local application of corticosteroids: A double-blind trial on the effect of oxyphenbutazone 10% eye ointment (Tanderil) as a therapy in superficial eye injuries. Ophthalmologica 167:422, 1973. 660 Podos 112. Warson PG, McKay DAR, Clemett RS, Wilkinson P: Treatment of episcleritis: A double-blind trial comparing betamethasone 0.1 percent, oxyphenbutazone 10 percent, and placebo eye ointments. Br J Ophthalmol 57:866, 1973. 113. Koch HR, Dumling H, Hockwin 0, Rast F: Investigations on the influences of oxyphenbutazone on formation of galactose-induced cataracts in rats. Ophthalmol 2:60, 1971. 114. Harris LS, Kahanowicz Y: Ocular anti-inflammatory effect of fenoprofen. Arch Ophthalmol 93:501, 1975.