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Figure 1.
Chemical structures of ethacrynic acid and ticrynafen. Although the righthand portions of the molecules are identical, ethacrynic acid has substantial sulfhydryl reactivity, whereas ticrynafen does not.

Chemical structures of ethacrynic acid and ticrynafen. Although the righthand portions of the molecules are identical, ethacrynic acid has substantial sulfhydryl reactivity, whereas ticrynafen does not.

Figure 2.
Intraocular pressure (IOP) in 6 cynomolgus monkeys under ketamine hydrochloride anesthesia during twice-daily topical application of ticrynafen in one eye and vehicle in the opposite eye. Data are mean±SEM IOP (A) in ticrynafen-treated and control eyes, or IOP difference (B) between eyes. BL indicates baseline. Time 0 hours on both days is at 9 AM. Time 0 hours on day 5 is 18 hours after the afternoon treatment on day 4 and immediately precedes the morning treatment on day 5. Asterisk indicates significantly different from ipsilateral pretreatment BL by the 2-tailed paired t test (P<.05).

Intraocular pressure (IOP) in 6 cynomolgus monkeys under ketamine hydrochloride anesthesia during twice-daily topical application of ticrynafen in one eye and vehicle in the opposite eye. Data are mean±SEM IOP (A) in ticrynafen-treated and control eyes, or IOP difference (B) between eyes. BL indicates baseline. Time 0 hours on both days is at 9 AM. Time 0 hours on day 5 is 18 hours after the afternoon treatment on day 4 and immediately precedes the morning treatment on day 5. Asterisk indicates significantly different from ipsilateral pretreatment BL by the 2-tailed paired t test (P<.05).

Figure 3.
Intraocular pressure (IOP) in 4 monkey eyes with laser-induced glaucoma at baseline (BL) before (A) and after topical application of vehicle (B) or 2% ticrynafen ointment (C-G). All data are mean±SEM. H, Vehicle-treated minus ipsilateral BL IOP at the corresponding time point. I through M, Ticrynafen-treated minus ipsilateral vehicle-treated IOP at the corresponding time point. Time 0 (always 9:30 AM) on vehicle and all ticrynafen days immediately precedes topical treatment; on treatment days 2 through 5, time is 18 hours after the afternoon ticrynafen treatment on the previous day. Significantly different from 0.0 by the 2-tailed paired t test: asterisk indicates P<.10; dagger, P<.02; double dagger, P<.05.

Intraocular pressure (IOP) in 4 monkey eyes with laser-induced glaucoma at baseline (BL) before (A) and after topical application of vehicle (B) or 2% ticrynafen ointment (C-G). All data are mean±SEM. H, Vehicle-treated minus ipsilateral BL IOP at the corresponding time point. I through M, Ticrynafen-treated minus ipsilateral vehicle-treated IOP at the corresponding time point. Time 0 (always 9:30 AM) on vehicle and all ticrynafen days immediately precedes topical treatment; on treatment days 2 through 5, time is 18 hours after the afternoon ticrynafen treatment on the previous day. Significantly different from 0.0 by the 2-tailed paired t test: asterisk indicates P<.10; dagger, P<.02; double dagger, P<.05.

Figure 4.
Intraocular pressure (IOP) in 4 untreated normotensive cynomolgus monkey eyes at baseline (BL) before (A) and during topical application of vehicle (B) or 2% ticrynafen ointment (C-G) in the opposite laser-induced glaucomatous eye (Figure 3). All data are mean±SEM. Time 0 (always 9:30 AM) on vehicle and on all ticrynafen days immediately precedes topical ticrynafen in the opposite glaucomatous eye; on treatment days 2 through 5, time 0 is 18 hours after the previous afternoon contralateral ticrynafen treatment.

Intraocular pressure (IOP) in 4 untreated normotensive cynomolgus monkey eyes at baseline (BL) before (A) and during topical application of vehicle (B) or 2% ticrynafen ointment (C-G) in the opposite laser-induced glaucomatous eye (Figure 3). All data are mean±SEM. Time 0 (always 9:30 AM) on vehicle and on all ticrynafen days immediately precedes topical ticrynafen in the opposite glaucomatous eye; on treatment days 2 through 5, time 0 is 18 hours after the previous afternoon contralateral ticrynafen treatment.

Total Outflow Facility After Intracameral or Topical Ticrynafen or Vehicle in Cynomolgus Monkeys*
Total Outflow Facility After Intracameral or Topical Ticrynafen or Vehicle in Cynomolgus Monkeys*
1.
Epstein  DLFreddo  TFBassett-Chu  SChung  MKarageuzian  L Influence of ethacrynic acid on outflow facility in the monkey and calf eye. Invest Ophthalmol Vis Sci. 1987;282067- 2075
2.
Croft  MAHubbard  WCKaufman  PL Effect of ethacrynic acid on aqueous outflow dynamics in monkeys. Invest Ophthalmol Vis Sci. 1994;351167- 1175
3.
Epstein  DHooshmand  LBEpstein  MPM Thiol adducts of ethacrynic acid increase outflow facility in enucleated calf eyes. Curr Eye Res. 1992;11253- 258Article
4.
Liang  LLEpstein  DLDe Kater  AWSchahstafaei  AErickson-Lamy  KA Ethacrynic acid increases facility of outflow in the human eye in vitro. Arch Ophthalmol. 1992;110106- 109Article
5.
Croft  MAKaufman  PL Effect of daily topical ethacrynic acid on aqueous humor dynamics in monkeys. Curr Eye Res. 1995;14777- 781Article
6.
Wang  RPodos  SMSerle  JBLee  PNeufeld  AHDeschenes  R Effects of topical ethacrynic acid ointment vs timolol on intraocular pressure in glaucomatous monkey eyes. Arch Ophthalmol. 1994;112390- 394Article
7.
Tingey  DPOzment  RRSchroeder  AEpstein  DL The effect of intracameral ethacrynic acid on the intraocular pressure of living monkeys. Am J Ophthalmol. 1992;113706- 711
8.
Melamed  SKostas-Neumann  RBarak  AEpstein  DL The effect of intracamerally injected ethacrynic acid on intraocular pressure in patients with glaucoma. Am J Ophthalmol. 1992;113508- 512
9.
Koechel  DABudd  GCBretz  NS Acute effects of alkylating agents on canine renal function and ultrastructure: high-dose ethacrynic acid vs dihydroethacrynic acid and ticrynafen. J Pharmacol Exp Ther. 1984;228799- 809
10.
Weiner  IMMudge  GH Diuretics and other agents employed in the mobilization of edema fluid. Gilman  AGGoodman  LSRall  TWMurod  Feds.Goodman and Gilman's the Pharmacological Basis of Therapeutics 7th ed. New York, NY Macmillan Publishing Co Inc1985;903- 904
11.
Tingey  DPSchroeder  AEpstein  MPMEpstein  DL Effects of topical ethacrynic acid adducts on intraocular pressure in rabbits and monkeys. Arch Ophthalmol. 1992;110699- 702Article
12.
Epstein  DLRoberts  BCSkinner  LL Nonsulfhydryl-reactive phenoxyacetic acids increase aqueous humor outflow facility. Invest Ophthalmol Vis Sci. 1997;381526- 1534
13.
Bárány  EH Simultaneous measurements of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest Ophthalmol. 1964;3135- 143
14.
Kaufman  PLBárány  EH Loss of acute pilocarpine effect on outflow facility following surgical disinsertion and retrodisplacement of the ciliary muscle from the scleral spur in the cynomolgus monkey. Invest Ophthalmol. 1976;15793- 807
15.
Bill  AHellsing  K Production and drainage of aqueous humor in the cynomolgus monkey (Macaca irus). Invest Ophthalmol. 1965;4920- 926
16.
Kaufman  PLDavis  GE "Minified" Goldmann applanating prism for tonometry in monkeys and humans. Arch Ophthalmol. 1980;98542- 546Article
17.
Erickson  KAGonnering  RSKaufman  PLDortzbach  RK The cynomolgus monkey as a model for orbital research, III: effects on ocular physiology of lateral orbitotomy and isolation of the ciliary ganglion. Curr Eye Res. 1984;3557- 564Article
18.
Erickson-Lamy  KAKaufman  PLMcDermott  MLFrance  NK Comparative anesthetic effects on aqueous humor dynamics in the cynomolgus monkey. Arch Ophthalmol. 1984;1021815- 1820Article
19.
Kaufman  PLBárány  EH Cytochalasin B reversibly increases outflow facility in the eye of the cynomolgus monkey. Invest Ophthalmol Vis Sci. 1977;1647- 53
20.
Kaufman  PLTrue-Gabelt  BAErickson-Lamy  KA Time-dependence of perfusion outflow facility in the cynomolgus monkey. Curr Eye Res. 1988;7721- 726Article
21.
Kaufman  PL Pressure-dependent outflow. Ritch  RKrupin  THeds.The Glaucomas Vol 12nd ed. St Louis, Mo Mosby–Year Book1996;307- 335
22.
Gabelt  BTRobinson  JCHubbard  WC  et al.  Apraclonidine and brimonidine effects on anterior ocular and cardiovascular physiology in normal and sympathectomized monkeys. Exp Eye Res. 1994;59633- 644Article
23.
Lee  PYPodos  SMHoward-Williams  JRSeverin  CHRose  ADSiegel  MJ Pharmacological testing in the laser-induced monkey glaucoma model. Curr Eye Res. 1985;4775- 781Article
24.
Kaufman  PLBárány  EH Residual pilocarpine effects on outflow facility after ciliary muscle disinsertion in the cynomolgus monkey. Invest Ophthalmol. 1976;15558- 561
25.
Schroeder  AErickson  K Cholinergic agonists do not increase trabecular outflow facility in the human eye [abstract]. Invest Ophthalmol Vis Sci. 1994;35(suppl)2054
26.
Schroeder  AErickson  K Low dose cholinergic agonists increase trabecular outflow facility in the human eye in vitro [abstract]. Invest Ophthalmol Vis Sci. 1995;36(suppl)S722
27.
Zhang  XWang  NSchroeder  AErickson  K Expression of adenylate cyclase subtypes II and IV in the human outflow pathway [abstract]. Invest Ophthalmol Vis Sci. 1998;39(suppl)S799
28.
Lee  PYPodos  SMSerle  JBCamras  CBSeverin  CH Intraocular pressure effects of multiple doses of drugs applied to glaucomatous monkey eyes. Arch Ophthalmol. 1987;105249- 252Article
29.
Kaufman  PL Epinephrine, norepinephrine, and isoproterenol dose-outflow facility response relationships in cynomolgus monkey eyes with and without ciliary muscle retrodisplacement. Acta Ophthalmol (Copenh). 1986;64356- 363Article
30.
Crawford  KSGange  SJGabelt  BT  et al.  Indomethacin and epinephrine effects on outflow facility and cyclic adenosine monophosphate formation in monkeys. Invest Ophthalmol Vis Sci. 1996;371348- 1359
31.
Robinson  JCKaufman  PL Effects and interactions of epinephrine, norepinephrine, timolol and betaxolol on outflow facility in the cynomolgus monkey. Am J Ophthalmol. 1990;109189- 194
32.
Bill  A Uveoscleral drainage of aqueous humor: physiology and pharmacology. Bito  LZStjernschantz  Jeds.The Ocular Effects of Prostaglandins and Other Eicosanoids New York, NY Alan R. Liss Inc1989;417- 427
33.
Bárány  EH Topical epinephrine effects on true outflow resistance and pseudofacility in vervet monkeys studied by a new anterior chamber perfusion technique. Invest Ophthalmol. 1968;788- 104
34.
Freddo  TFPatterson  MMScott  DREpstein  DL Influence of mercurial sulfhydryl agents on aqueous outflow pathways in enucleated eyes. Invest Ophthalmol Vis Sci. 1984;25278- 285
35.
Erickson-Lamy  KASchroeder  AEpstein  DL Ethacrynic acid induces reversible shape and cytoskeletal changes in cultured cells. Invest Ophthalmol Vis Sci. 1992;332631- 2640
36.
Gabelt  BTKaufman  PL Pharmacologic enhancement of aqueous humor outflow. Van  Buskirk EMed.100 Years of Progress in Glaucoma Philadelphia, Pa Lippincott-Raven1997;257- 269
37.
Peterson  JATian  BKiland  JA  et al.  Latrunculin (LAT)-A & staurosporine, but not swinholide (SWIN)-A, increase outflow facility in the monkey [abstract]. Invest Ophthalmol Vis Sci. 1996;37(suppl)S825
38.
Tian  BKaufman  PLVolberg  TGabelt  BTGeiger  B H-7 disrupts the actin cytoskeleton and increases outflow facility. Arch Ophthalmol. 1998;116633- 643Article
39.
Mobley  PLHedberg  KBonin  LChen  BGriffith  OH Decreased phosphorylation of four 20-kDa proteins precedes staurosporine-induced disruption of the actin/myosin cytoskeleton in rat astrocytes. Exp Cell Res. 1994;21455- 66Article
40.
O'Donnell  MEBrandt  JDCurry  FE Na-K-Cl cotransport regulates intracellular volume and monolayer permeability of trabecular meshwork cells. Am J Physiol. 1995;268C1067- C1074
41.
Palfrey  JCLeung  S Inhibition of Na-K-2Cl cotransport and bumetanide binding by ethacrynic acid, its analogs, and adducts. Am J Physiol. 1993;264C1270- C1277
42.
Epstein  DLRoberts  BCSkinner  LL Non-sulfhydryl reactive phenoxyacetic acids increase outflow facility and disrupt the trabecular and endothelial cell cytoskeleton [abstract]. Invest Ophthalmol Vis Sci. 1996;37(suppl)S894
43.
Epstein  DLDeKater  AWErickson-Lamy  KFay  FSSchroeder  AHooshmand  L The search for a sulfhydryl drug for glaucoma: from chemistry to cytoskeleton. Lütjen  Drecoll Eed.Basic Aspects of Glaucoma Research III Stuttgart, Germany Schattauer1993;345- 353
Citations 0
Laboratory Sciences
November 1998

Effect of Ticrynafen on Aqueous Humor Dynamics in Monkeys

Author Affiliations

From the Departments of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison (Ms Croft and Dr Kaufman), Mt Sinai School of Medicine, New York, NY (Drs Wang and Podos), and Washington University Medical School, St Louis, Mo (Dr Neufeld).

Arch Ophthalmol. 1998;116(11):1481-1488. doi:10.1001/archopht.116.11.1481
Abstract

Objective  To determine the effect of ticrynafen, a non–sulfhydryl-reactive compound similar to ethacrynic acid, on outflow facility in normotensive monkey eyes and on intraocular pressure (IOP) in monkey eyes with laser-induced glaucoma.

Methods  In normotensive eyes, facility (perfusion) was measured shortly before and after bolus or exchange intracameral infusion of ticrynafen or vehicle in opposite eyes, and 3.5 to 4.5 hours after 5 days of twice-daily 2% ticrynafen or vehicle ointment. In glaucomatous eyes, baseline and vehicle diurnal IOP curves were established, 2% ticrynafen ointment was given twice daily for 5 days, and IOP was measured immediately before and 0.5 to 6 hours after each morning treatment.

Results  In normotensive eyes, exchange 2-mL infusion of 0.2-, 1-, or 4-mmol/L ticrynafen increased facility by 33%±6% (mean±SEM), 73%±18%, and 60%±11%, respectively. Day 5 posttreatment facility was higher in the ticrynafen group than in controls by 28%±9%. In glaucomatous eyes, maximum IOP decline, from approximately 35 mm Hg, was 7.5±2.0 mm Hg on day 4 and 9.8±2.4 mm Hg on day 5 of twice-daily ticrynafen treatment.

Conclusion  The facility-increasing, IOP-lowering action of ticrynafen, ethacrynic acid, and derivatives may not depend entirely on sulfhydryl reactivity.

Clinical Relevance  Whether such drugs as ethacrynic acid and ticrynafen prove valuable for glaucoma therapy, at the least they are useful probes to study aqueous outflow mechanisms.

ETHACRYNIC ACID increases outflow facility in living monkey eyes,1,2 enucleated calf eyes,1,3 and organ-cultured, perfused human anterior ocular segments4 and decreases intraocular pressure (IOP) in living normotensive and glaucomatous monkey57 and glaucomatous human8 eyes.

Ethacrynic acid is an alkylating agent, giving it sulfhydryl reactivity.9,10 This property may be responsible for both its facility-increasing and toxic corneal effects.5,11 Ticrynafen is a nonalkylating compound,9 structurally similar to ethacrynic acid (Figure 1) but putatively devoid of sulfhydryl reactivity.12 We have determined the effect of ticrynafen on IOP, outflow facility, and anterior segment biomicroscopic appearance in cynomolgus monkey eyes with normal IOP and with laser-induced glaucoma.

MATERIALS AND METHODS
CYNOMOLGUS MONKEYS

Thirty-nine juvenile and adult cynomolgus monkeys (Macaca fascicularis) of both sexes were studied; 35 were bilaterally ocular normotensive with no biomicroscopically visible anterior chamber cells or flare or other ocular abnormalities, while 4 had stable unilateral ocular hypertension induced by repeated argon laser photocoagulation of the trabecular meshwork. All experiments were conducted in accordance with National Institutes of Health (Bethesda, Md) and institutional guidelines, and with the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research.

ANESTHESIA

For applanation tonometry and administration of ticrynafen ointment, intramuscular ketamine hydrochloride, 10 to 12 mg/kg, supplemented at approximately 45-minute intervals by 5 mg/kg, was used, supplemented in glaucomatous monkeys by topical 0.5% proparacaine hydrochloride. For anterior chamber perfusion, intramuscular ketamine hydrochloride, 10 mg/kg, was followed by intramuscular pentobarbital sodium, 35 mg/kg.

OUTFLOW FACILITY MEASUREMENT AND INTRACAMERAL DRUG DELIVERY

Total outflow facility was measured by 2-level constant-pressure (approximately 3 and 12 mm Hg above spontaneous IOP) perfusion of the anterior chamber (AC) with Bárány mock aqueous humor.13,14 Two variations of the basic method were used. With a 1-needle technique (bolus intracameral infusion of drug and no drug in the reservoir), a second cannulation is avoided; however, the AC contents are not completely mixed or replaced, and drug concentration declines with a half-life of approximately 30 minutes (varying somewhat with the facility of the individual eye). The AC and reservoir exchange15 with drug solution requires 2 needles rather than 1, but allows rapid administration, more complete mixing, and closer approximation of a specific intracameral drug concentration during the posttreatment facility measurements, since the perfusand contains the desired drug concentration.

IOP MEASUREMENT

The IOP was determined with a minified Goldmann applanation tonometer16 in monkeys with normotensive eyes, and with a calibrated pneumatic applanation tonometer (Pneumatonometer model 30, Digilab Inc, Cambridge, Mass) in animals with laser-induced glaucoma.

TICRYNAFEN

Ticrynafen powder and 2% ticrynafen ointment (2% ticrynafen in mineral oil petrolatum base) were obtained (Telor Ophthalmic Pharmaceuticals, Woburn, Mass). The same 2% ticrynafen ointment preparation was used for both the bilaterally normotensive and the unilaterally glaucomatous monkeys. The manufacturer also prepared ethacrynic acid ointment with the use of a similar mineral oil petrolatum base for other studies cited herein.5,6 We chose the concentration of ticrynafen based on an IOP-effective concentration of ethacrynic acid.6

MONKEYS WITH BILATERALLY NORMOTENSIVE EYES
Protocol 1
Experimental Design

After baseline slitlamp examination, facility was measured simultaneously in both eyes of 11 monkeys for approximately 45 minutes, immediately before and beginning 1 hour after a 10-µL bolus intracameral infusion of 2.5-mmol/L (group A, n=6) or 25-mmol/L (group B, n=6) ticrynafen in one eye and vehicle alone in the other (1 monkey was used in both protocols). These doses achieved initial concentrations of 0.25-mmol/L (group A) and 2.5-mmol/L (group B) ticrynafen in the 100-µL cynomolgus anterior chamber.17,18 Ticrynafen or vehicle was injected into the inflow tubing of the perfusion apparatus (via a T-piece) of opposite eyes and allowed to wash into the anterior chambers for 5 minutes. The eyes were then exposed to cold air for 3 minutes to enhance convection mixing.19 The perfusion apparatus was closed to inflow during the interval between baseline and postdrug facility measurements.

Drug Preparation

For 6 animals in group A, the phosphate buffer was adjusted to pH 6.8 with 1.0N hydrochloric acid and filtered through a 0.2-µm acetate filter. Then, 41 mg of ticrynafen was dissolved in 40 mL of phosphate buffer by ultrasonication and further diluted with buffer to 50.0 mL to achieve a final ticrynafen concentration of 2.5 mmol/L. For 1 monkey in group A and all 5 monkeys in group B, 82.8 mg of ticrynafen and 60.6 mg of TRIS (base) were dissolved in 8.0 mL of purified water, the pH adjusted to 7.0 with 1N hydrochloric acid, and then further diluted with purified water to 10 mL. The solution was then filtered through a 0.2-µm nylon or acetate filter. The vehicle solution was prepared identically, except that 20.0 mg of sodium chloride was substituted for the higher ticrynafen concentration. Solutions were made immediately before the experiment.

Protocol 2
Experimental Design

Total outflow facility was measured in both eyes for approximately 45 minutes, immediately before and beginning 0.5 hour after a 10-minute AC exchange with 2 mL of 0.04-, 0.2-, 1-, and 4-mmol/L ticrynafen in one eye and vehicle alone in the other in 4 groups of 4, 5, 7, and 9 monkeys, respectively. The system was closed to inflow during the interval between baseline and postdrug facility measurements. During postexchange facility measurements, the reservoirs contained the respective ticrynafen or vehicle solution.

Drug Preparation

TRIS, 242 mg, was dissolved in 7 mL of purified water; ticrynafen, 331 mg, was added and vortexed until dissolved; and water was added to 10 mL. A 1.0-mL aliquot of ticrynafen solution was added to 20 mL of Bárány solution and adjusted to pH 7.4 with 50-µL drops of 1.0N hydrochloric acid while being mixed, and Bárány solution was added to 25 mL. Vehicle solution was prepared identically except for omission of ticrynafen and substitution of 5.0N for 1.0N hydrochloric acid. Both solutions were filtered through a 0.2-µm nylon filter. These solutions are stable for 1 month, but were prepared 1 week before the experiment.

Protocol 3

A 1-cm strip of ointment containing 2% ticrynafen was administered to the cul de sac of one eye and placebo ointment to the opposite eye, randomly selected, of 6 ketamine-anesthetized monkeys with bilaterally normotensive eyes (3 from one of the previous protocols, 5 with 1 previous AC perfusion) twice daily for 5 consecutive days at approximately 9 AM and 3:30 PM. Excess ointment was wiped away 15 minutes after application. On day 1, baseline IOP was measured immediately before treatment. On day 5, IOP was measured immediately before and 1 and 3 hours after morning treatment. Clinical biomicroscopy was performed by a trained ophthalmologist immediately before baseline and final IOP measurements on days 1 and 5. Total outflow facility was determined 3.5 to 4.5 hours after morning treatment on day 5.

All monkeys had biomicroscopically clear ACs bilaterally at the time of the study. One monkey appeared in 4 groups, and 4 others appeared in 2 groups, but no monkey appeared twice in the same group.

MONKEYS WITH UNILATERAL LASER-INDUCED GLAUCOMA

The IOP was measured at approximately 9:30 AM and repeated 0.5, 1, 2, 3, 4, 5, and 6 hours later to establish a baseline diurnal curve in both eyes of 4 monkeys with unilateral glaucoma under ketamine anesthesia. The following day, IOP was measured in both eyes starting at approximately 9:30 AM, immediately before and then 0.5, 1, 2, 3, 4, 5, and 6 hours after administration of a 1-cm strip of placebo ointment to the glaucomatous eye only. Beginning 2 days later, 2% ticrynafen ointment was administered twice daily to the glaucomatous eye, at 9:30 AM and 3:30 PM, for 5 days. On each day, IOP was measured in both eyes immediately before the morning 2% ticrynafen dose in the glaucomatous eye and 0.5, 1, 2, 3, 4, 5, and 6 hours thereafter. Immediately after the last IOP measurement, the second dose of 2% ticrynafen was given. Slitlamp examination was performed immediately before the first IOP measurement of the day, and just before IOP measurement every 2 hours thereafter. The contralateral normotensive eyes were untreated throughout the experiment.

DATA ANALYSIS

Data are presented as mean±SEM values. Differences between or ratios of ticrynafen-treated and contralateral vehicle-treated control eyes were tested against 0 or 1, respectively, by the 2-tailed paired t test. The Bonferroni t test was used for the analysis of the multiple-dose study in glaucomatous monkey eyes. The correlation between treated vs control IOP differences and treated/control facility response was examined by least-squares linear regression, with and without adjusting for baseline IOP.

RESULTS
MONKEYS WITH BILATERALLY NORMOTENSIVE EYES

Pretreatment baseline facilities in the ticrynafen and contralateral control eyes were similar in all intracameral drug protocols.

Protocol 1

One hour after unilateral bolus intracameral infusion of 10 µL of 2.5-mmol/L ticrynafen (group A, Table 1), the mean postdrug-baseline facility ratio averaged 1.15±0.05 (n=6) in the ticrynafen-infused eye and 1.27±0.09 in the vehicle-injected eye. These 15% and 27% increases were both statistically significant (P=.03), were of the magnitude expected for perfusion-induced resistance washout in this system,20 and did not differ significantly (treated-control postdrug facility ratio, 1.03±0.10; treated-control, postdrug-baseline facility ratio, 0.92±0.06; neither differing significantly from 1.0). Thus, there was no apparent ticrynafen-induced facility change. Similar results were seen with the 10-fold higher dose (10 µL of 25-mmol/L ticrynafen; group B, Table 1); the treated-control, postdrug-baseline facility ratio averaged 1.12±0.11 (n=6).

Protocol 2

Facility did not increase after 0.04-mmol/L ticrynafen exchange (Table 1). However, after AC exchange with 0.2-, 1-, and 4-mmol/L ticrynafen, facility relative to predrug baseline and adjusted for control eye washout increased significantly, by 33%±6% (0.2 mmol/L), 73%±18% (1 mmol/L), and 48%±11% or 60%±11% (4 mmol/L) (Table 1). In the group treated with 4-mmol/L ticrynafen, there were 2 animals in which the postexchange facilities in the control eyes nearly doubled compared with baseline, so that the facility increase in the control eyes averaged 30%±13% for 9 monkeys (Table 1). When these 2 animals were excluded, the perfusion-induced facility increase in the 7 remaining control eyes was a more typical 14%±8% and the facility increase in the contralateral ticrynafen-treated eyes remained at approximately 80%, so that the ticrynafen-vehicle ratios for postdrug facility and postdrug-predrug facility increased to 1.48 and 1.60, respectively (Table 1).

Protocol 3

Average pretreatment IOP was 13.8 mm Hg in both eyes (Figure 2). The IOP in the control eyes 18 and 19 hours after the afternoon day 4 treatment (ie, immediately before and 1 hour after the day 5 morning treatment) was essentially identical to day 1 pretreatment baseline, but decreased by approximately 3 mm Hg at 3 hours on treatment day 5. The IOP in the ticrynafen-treated eyes averaged 1 to 1.5 mm Hg less than in the control eyes at the day 3 pretreatment and the 1- and 3-hour posttreatment measurements, but these differences were not statistically significant. On day 5, 3-hour posttreatment IOP was significantly lower than day 1 pretreatment baseline in both ticrynafen-treated (−3.3±0.8 mm Hg; n=6; P=.004) and control (−3.0±0.9 mm Hg; P=.02) eyes. Slitlamp examination disclosed only mild superficial punctate keratopathy associated with repeated tonometry, with no differences in frequency or severity between ticrynafen- and vehicle-treated eyes.

After 5 days of twice-daily unilateral topical administration of 2% ticrynafen ointment, facility in the ticrynafen-treated eyes averaged 28%±9% higher than in the contralateral vehicle-treated controls (Table 1; n=6; P=.03). There was no significant correlation between treated vs vehicle eye IOP differences and treated-vehicle eye facility response, with or without adjustment for baseline IOP differences.

MONKEYS WITH UNILATERAL LASER-INDUCED GLAUCOMA

Six-hour baseline diurnal IOP (day −4) averaged between 34.0±3.1 and 37.5±4.0 mm Hg (Figure 3, A). The IOP immediately before and for 6 hours after vehicle treatment the next day (day −3) averaged between 34.0±2.6 and 37.3±3.3 mm Hg (Figure 3, B). Since there was no apparent effect of the vehicle (Figure 1 3, H), the vehicle-treated IOP diurnal curve was used for comparison with ticrynafen treatment at the same time of the day in the same eye. The onset of IOP reduction after ticrynafen treatment did not occur until after the seventh dose beginning on day 4. The maximum IOP decline on day 4 averaged 7.5±2.0 mm Hg (P<.05; n=4) at hour 4, and on day 5 averaged 9.8±2.4 mm Hg (P<.05; n=4) at hour 2. The IOP in the untreated contralateral normotensive eyes displayed normal diurnal fluctuation during the course of the experiment, averaging between 15.8±1.2 and 18.5±0.7 mm Hg (Figure 4).

Slitlamp examination showed no abnormalities in any vehicle-treated eye or in 3 of 4 ticrynafen-treated eyes. Corneal epithelial edema was noted in 1 ticrynafen-treated eye 2 hours after dosing on days 1 and 4; the edema lessened by 4 hours and disappeared by 6 hours after dosing. However, a confluent epithelial defect occurred in the same eye on day 5 of treatment.

COMMENT

In normotensive cynomolgus monkeys, ticrynafen produced an increase in outflow facility when given by AC and reservoir exchange, but not at similar doses given by intracameral bolus injection. Anterior chamber and reservoir exchange, as performed here, allows more rapid administration of drug, more complete mixing of the AC, and better maintenance of the drug concentration during posttreatment facility measurements (as the perfusate in the reservoir contains the desired drug concentration). These pharmacodynamic differences may explain ticrynafen's greater efficacy when given by AC-reservoir exchange.

In normotensive cynomolgus monkeys, ticrynafen administered by AC exchange infusion (with the drug concentration maintained during postdrug facility measurements) and ethacrynic acid given by bolus intracameral infusion (with initial drug concentration declines during postdrug measurements) (M.A.C., P.L.K., unpublished data, 1997) both increase facility dose-dependently. Ticrynafen, 1 mmol/L, appears maximal and increased facility by a washout-corrected 73%±18% (n=7) relative to baseline; ethacrynic acid, 0.25 mmol/L, which is also maximal (M.A.C., P.L.K., unpublished data, 1997), produced a nearly identical baseline- and washout-corrected 71%±15% (n=6) facility increase.2

Topical 2% ticrynafen ointment, administered twice daily for 5 days, produced a 28%±9% facility increase and no ocular abnormalities, while 1.5% ethacrynic acid ointment, administered once daily for 5 days, produced a 40%±15% increase in facility and ocular abnormalities in all eyes (12 of 12 eyes).5 In glaucomatous monkey eyes, topical 2% ticrynafen ointment, administered twice-daily for 5 days, produced an IOP drop similar in magnitude and with fewer corneal abnormalities than once-daily 2.5% or 1.5% ethacrynic acid ointment in a similar base of mineral oil and petrolatum (ticrynafen: 9.8 mm Hg, corneal abnormalities in 1 of 4 eyes; 2.5% ethacrynic acid: 8.5 mm Hg, corneal abnormalities in 3 of 4 eyes; 1.5% ethacrynic acid: 6.5 mm Hg, corneal abnormalities in 2 of 4 eyes6). In monkeys with bilaterally normotensive eyes, IOP after unilateral 2% ticrynafen ointment, administered twice daily for 5 days, averaged 1 to 1.5 mm Hg less in the ticrynafen-treated eyes than in the control eyes at the day 3 pretreatment and the 1- and 3-hour posttreatment measurements, but these differences were not statistically significant. Ethacrynic acid, 1.5% ointment, given once daily for 5 days, significantly lowered IOP by 2.8, 1.7, and 3.7 mm Hg before and 1 and 3 hours, respectively, after treatment on day 5 compared with contralateral control eyes.5

The small IOP reduction induced by topical ticrynafen in normotensive monkeys is not surprising given the low baseline IOP. When IOP is low, even a substantial effect on inflow or outflow may have little effect on IOP; this is evident from the Goldmann equation.21 In our experiments, IOP of the control eye on day 5, 3 hours after the morning treatment (immediately before facility measurements) was 10.3 mm Hg. Assuming an episcleral venous pressure of 10 mm Hg, and episcleral venous pressure, aqueous formation, and uveoscleral outflow to be the same in both eyes, a 28% higher trabecular facility in the ticrynafen-treated eyes would predict an IOP of 10.2 mm Hg by the Goldmann equation, ie, essentially the same as in the control eyes. In fact, IOP of the ticrynafen-treated eyes on day 5, 3 hours after the morning treatment (immediately before facility measurements) averaged 9.3 mm Hg. As an even more striking example, assume an aqueous formation rate of 1.5 µL/min, IOP of 13 mm Hg, trabecular facility of 0.33 µL · min−1 · mm Hg−1, episcleral venous pressure of 10 mm Hg, and uveoscleral outflow of 0.5 µL/min. By the Goldmann equation, even a 52% facility increase to 0.50 µL · min−1 · mm Hg−1 would only yield a 1–mm Hg decrease in IOP.

Monkeys with normotensive eyes under ketamine anesthesia and receiving no other drug exhibit a time-dependent decrease in IOP during 6 to 8 hours, perhaps related to ketamine itself, to depth of anesthesia, or to the normal diurnal rhythm for IOP.22 This may partly or completely account for the small but significant contralateral IOP reduction in our ticrynafen-treated animals, rather than a contralateral effect of ticrynafen itself. As we did not measure baseline facility, we cannot say whether facility increased in the control eye. The contralateral (nonglaucomatous) eye in the unilaterally glaucomatous monkeys demonstrated only the normal diurnal IOP decline, with IOP returning to the morning baseline each day, ie, no contralateral effect was evident.

The glaucomatous monkeys had received laser treatment in the midtrabecular meshwork of all 4 quadrants, and we did not measure their perfusion or tonographic outflow facility after they received ticrynafen. However, pilocarpine23 does produce a substantial IOP decrease and an outflow facility increase in this model, indicating that the meshwork can still respond functionally to mechanical distortion24 or a direct drug effect.2527 Epinephrine also lowers IOP in the glaucomatous monkey model,23,28 but it is not known whether this effect is caused by enhanced trabecular or uveoscleral outflow.2933 Some trabecular meshwork between the burns may have been unaffected by the laser treatment or subsequent scarring and remained responsive to the drug, or even damaged trabecular meshwork could be affected functionally by ticrynafen.

Ethacrynic acid produced a small but significant transient IOP rise after the day 5 treatment compared with day 5 baseline values in cynomolgus monkeys with normotensive eyes.5 A similar trend was seen with ticrynafen in our normotensive monkeys, but the magnitude was even smaller and not statistically significant. Some intracameral doses of some sulfhydryl-reactive compounds may produce an acute facility decrease in enucleated calf and primate eyes,34 perhaps because of trabecular cell swelling. No postticrynafen IOP rise was seen on any day in the glaucomatous monkey eyes. In 4 other glaucomatous monkeys, IOP was measured hourly for 6 hours on the third day before, and then daily for 5 days during, once-daily treatment with normal saline. The IOP tended to decline over 5 days, but the change from baseline was not statistically significant at any time point (R.F.W., unpublished data).

Four- or 5-fold excess concentrations of cysteine block the ethacrynic acid–induced facility increase in enucleated calf eyes1 and living cynomolgus monkeys (M.A.C., P.L.K., unpublished data, 1997) and the ethacrynic acid–induced shape change in cultured human trabecular meshwork cells.35 This suggests that the ethacrynic acid effects might result from sulfhydryl reactivity. However, ticrynafen, which is structurally similar to ethacrynic acid but putatively lacks sulfhydryl reactivity,9,12 significantly lowers IOP in glaucomatous and increases outflow facility in normotensive monkey eyes. In addition, a recently published study reported that, in excised bovine eyes, 0.125-mmol/L ticrynafen increases facility by 102% relative to baseline, significantly more than the 50% washout-induced facility increase in control eyes, and that, in contrast to ethacrynic acid, the effect is maintained in the presence of a 5-fold excess concentration of cysteine.12 Cytoskeletal drugs such as cytochalasins,36 latrunculins,37 and certain protein kinase inhibitors such as H-738 and staurosporine37,39 may exert their facility-increasing effect by altering the shape and adhesion of endothelial cells in the meshwork and along the inner canal wall. Ethacrynic acid and ticrynafen both induced shape changes in cultured calf pulmonary artery endothelial cells and human trabecular meshwork cells,12,35 but ethacrynic acid had a greater effect at comparable doses. This might explain why, in our present study, a 4-fold higher concentration of ticrynafen delivered via AC exchange ([ticrynafen]AC maintained during facility measurements) was required to produce a facility increase similar to that with ethacrynic acid given via bolus injection.2 Nonetheless, given that ticrynafen and ethacrynic acid are of reasonably comparable potency and efficacy, and that the drugs likely lower IOP and increase outflow facility by similar mechanisms (eg, cell shape change; ethacrynic acid,35 ticrynafen12), these data suggest that the facility-increasing, IOP-lowering action of ethacrynic acid and derivatives may not depend entirely, if at all, on sulfhydryl reactivity.

Despite evidence of changes in cell shape,12,35 volume regulation,40 and adhesion consequent to drug effects on cytoskeletal proteins such as actin and tubulin12,35 or sodium-potassium-chloride ion cotransport,40,41 the cellular biophysical mechanism by which ethacrynic acid and related compounds such as ticrynafen lower IOP and increase outflow facility remains elusive. Whatever the mechanism, it is intriguing to note that ticrynafen increases facility and lowers IOP, apparently in conjunction with a change in cell shape, as does ethacrynic acid, but apparently without microtubule disruption,12,42 and does so in the absence of sulfhydryl reactivity. Whether drugs such as ethacrynic acid and ticrynafen prove valuable for glaucoma therapy, they are useful probes for studying aqueous outflow mechanisms.43

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Article Information

Accepted for publication May 13, 1998.

This study was supported by grants from the National Eye Institute, Bethesda, Md (EY02698 to the University of Wisconsin, Madison, and EY01867 to Mount Sinai School of Medicine, New York, NY); unrestricted grants from Research to Prevent Blindness Inc, New York, NY (to University of Wisconsin and Mount Sinai School of Medicine); and Telor Ophthalmic Pharmaceuticals Inc, Woburn, Mass (to University of Wisconsin and Mount Sinai School of Medicine).

Francis X. Smith, PhD (Bio-Concepts Laboratories Inc, Salem, NH) advised on preparation of ticrynafen solutions. Ms Croft and Drs Wang, Podos, and Kaufman had no personal proprietary interest or received any personal payment or considerations relative to ticrynafen or Telor Ophthalmic Pharmaceuticals Inc. On projects unrelated to ticrynafen, Dr Kaufman has served as a paid consultant to Telor, and Drs Kaufman and Podos have served as paid consultants for other pharmaceutical companies. Telor contributed to a nonprofit charitable organization for which Dr Kaufman serves as an unpaid officer and member of the Board of Directors. Dr Neufeld formerly was an employee and equity holder of Telor.

Reprints: Paul L. Kaufman, MD, Department of Ophthalmology and Visual Sciences, University of Wisconsin, Clinical Sciences Center, 600 Highland Ave, Madison, WI 53792-3220 (e-mail: kaufman@macc.wisc.edu).

References
1.
Epstein  DLFreddo  TFBassett-Chu  SChung  MKarageuzian  L Influence of ethacrynic acid on outflow facility in the monkey and calf eye. Invest Ophthalmol Vis Sci. 1987;282067- 2075
2.
Croft  MAHubbard  WCKaufman  PL Effect of ethacrynic acid on aqueous outflow dynamics in monkeys. Invest Ophthalmol Vis Sci. 1994;351167- 1175
3.
Epstein  DHooshmand  LBEpstein  MPM Thiol adducts of ethacrynic acid increase outflow facility in enucleated calf eyes. Curr Eye Res. 1992;11253- 258Article
4.
Liang  LLEpstein  DLDe Kater  AWSchahstafaei  AErickson-Lamy  KA Ethacrynic acid increases facility of outflow in the human eye in vitro. Arch Ophthalmol. 1992;110106- 109Article
5.
Croft  MAKaufman  PL Effect of daily topical ethacrynic acid on aqueous humor dynamics in monkeys. Curr Eye Res. 1995;14777- 781Article
6.
Wang  RPodos  SMSerle  JBLee  PNeufeld  AHDeschenes  R Effects of topical ethacrynic acid ointment vs timolol on intraocular pressure in glaucomatous monkey eyes. Arch Ophthalmol. 1994;112390- 394Article
7.
Tingey  DPOzment  RRSchroeder  AEpstein  DL The effect of intracameral ethacrynic acid on the intraocular pressure of living monkeys. Am J Ophthalmol. 1992;113706- 711
8.
Melamed  SKostas-Neumann  RBarak  AEpstein  DL The effect of intracamerally injected ethacrynic acid on intraocular pressure in patients with glaucoma. Am J Ophthalmol. 1992;113508- 512
9.
Koechel  DABudd  GCBretz  NS Acute effects of alkylating agents on canine renal function and ultrastructure: high-dose ethacrynic acid vs dihydroethacrynic acid and ticrynafen. J Pharmacol Exp Ther. 1984;228799- 809
10.
Weiner  IMMudge  GH Diuretics and other agents employed in the mobilization of edema fluid. Gilman  AGGoodman  LSRall  TWMurod  Feds.Goodman and Gilman's the Pharmacological Basis of Therapeutics 7th ed. New York, NY Macmillan Publishing Co Inc1985;903- 904
11.
Tingey  DPSchroeder  AEpstein  MPMEpstein  DL Effects of topical ethacrynic acid adducts on intraocular pressure in rabbits and monkeys. Arch Ophthalmol. 1992;110699- 702Article
12.
Epstein  DLRoberts  BCSkinner  LL Nonsulfhydryl-reactive phenoxyacetic acids increase aqueous humor outflow facility. Invest Ophthalmol Vis Sci. 1997;381526- 1534
13.
Bárány  EH Simultaneous measurements of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest Ophthalmol. 1964;3135- 143
14.
Kaufman  PLBárány  EH Loss of acute pilocarpine effect on outflow facility following surgical disinsertion and retrodisplacement of the ciliary muscle from the scleral spur in the cynomolgus monkey. Invest Ophthalmol. 1976;15793- 807
15.
Bill  AHellsing  K Production and drainage of aqueous humor in the cynomolgus monkey (Macaca irus). Invest Ophthalmol. 1965;4920- 926
16.
Kaufman  PLDavis  GE "Minified" Goldmann applanating prism for tonometry in monkeys and humans. Arch Ophthalmol. 1980;98542- 546Article
17.
Erickson  KAGonnering  RSKaufman  PLDortzbach  RK The cynomolgus monkey as a model for orbital research, III: effects on ocular physiology of lateral orbitotomy and isolation of the ciliary ganglion. Curr Eye Res. 1984;3557- 564Article
18.
Erickson-Lamy  KAKaufman  PLMcDermott  MLFrance  NK Comparative anesthetic effects on aqueous humor dynamics in the cynomolgus monkey. Arch Ophthalmol. 1984;1021815- 1820Article
19.
Kaufman  PLBárány  EH Cytochalasin B reversibly increases outflow facility in the eye of the cynomolgus monkey. Invest Ophthalmol Vis Sci. 1977;1647- 53
20.
Kaufman  PLTrue-Gabelt  BAErickson-Lamy  KA Time-dependence of perfusion outflow facility in the cynomolgus monkey. Curr Eye Res. 1988;7721- 726Article
21.
Kaufman  PL Pressure-dependent outflow. Ritch  RKrupin  THeds.The Glaucomas Vol 12nd ed. St Louis, Mo Mosby–Year Book1996;307- 335
22.
Gabelt  BTRobinson  JCHubbard  WC  et al.  Apraclonidine and brimonidine effects on anterior ocular and cardiovascular physiology in normal and sympathectomized monkeys. Exp Eye Res. 1994;59633- 644Article
23.
Lee  PYPodos  SMHoward-Williams  JRSeverin  CHRose  ADSiegel  MJ Pharmacological testing in the laser-induced monkey glaucoma model. Curr Eye Res. 1985;4775- 781Article
24.
Kaufman  PLBárány  EH Residual pilocarpine effects on outflow facility after ciliary muscle disinsertion in the cynomolgus monkey. Invest Ophthalmol. 1976;15558- 561
25.
Schroeder  AErickson  K Cholinergic agonists do not increase trabecular outflow facility in the human eye [abstract]. Invest Ophthalmol Vis Sci. 1994;35(suppl)2054
26.
Schroeder  AErickson  K Low dose cholinergic agonists increase trabecular outflow facility in the human eye in vitro [abstract]. Invest Ophthalmol Vis Sci. 1995;36(suppl)S722
27.
Zhang  XWang  NSchroeder  AErickson  K Expression of adenylate cyclase subtypes II and IV in the human outflow pathway [abstract]. Invest Ophthalmol Vis Sci. 1998;39(suppl)S799
28.
Lee  PYPodos  SMSerle  JBCamras  CBSeverin  CH Intraocular pressure effects of multiple doses of drugs applied to glaucomatous monkey eyes. Arch Ophthalmol. 1987;105249- 252Article
29.
Kaufman  PL Epinephrine, norepinephrine, and isoproterenol dose-outflow facility response relationships in cynomolgus monkey eyes with and without ciliary muscle retrodisplacement. Acta Ophthalmol (Copenh). 1986;64356- 363Article
30.
Crawford  KSGange  SJGabelt  BT  et al.  Indomethacin and epinephrine effects on outflow facility and cyclic adenosine monophosphate formation in monkeys. Invest Ophthalmol Vis Sci. 1996;371348- 1359
31.
Robinson  JCKaufman  PL Effects and interactions of epinephrine, norepinephrine, timolol and betaxolol on outflow facility in the cynomolgus monkey. Am J Ophthalmol. 1990;109189- 194
32.
Bill  A Uveoscleral drainage of aqueous humor: physiology and pharmacology. Bito  LZStjernschantz  Jeds.The Ocular Effects of Prostaglandins and Other Eicosanoids New York, NY Alan R. Liss Inc1989;417- 427
33.
Bárány  EH Topical epinephrine effects on true outflow resistance and pseudofacility in vervet monkeys studied by a new anterior chamber perfusion technique. Invest Ophthalmol. 1968;788- 104
34.
Freddo  TFPatterson  MMScott  DREpstein  DL Influence of mercurial sulfhydryl agents on aqueous outflow pathways in enucleated eyes. Invest Ophthalmol Vis Sci. 1984;25278- 285
35.
Erickson-Lamy  KASchroeder  AEpstein  DL Ethacrynic acid induces reversible shape and cytoskeletal changes in cultured cells. Invest Ophthalmol Vis Sci. 1992;332631- 2640
36.
Gabelt  BTKaufman  PL Pharmacologic enhancement of aqueous humor outflow. Van  Buskirk EMed.100 Years of Progress in Glaucoma Philadelphia, Pa Lippincott-Raven1997;257- 269
37.
Peterson  JATian  BKiland  JA  et al.  Latrunculin (LAT)-A & staurosporine, but not swinholide (SWIN)-A, increase outflow facility in the monkey [abstract]. Invest Ophthalmol Vis Sci. 1996;37(suppl)S825
38.
Tian  BKaufman  PLVolberg  TGabelt  BTGeiger  B H-7 disrupts the actin cytoskeleton and increases outflow facility. Arch Ophthalmol. 1998;116633- 643Article
39.
Mobley  PLHedberg  KBonin  LChen  BGriffith  OH Decreased phosphorylation of four 20-kDa proteins precedes staurosporine-induced disruption of the actin/myosin cytoskeleton in rat astrocytes. Exp Cell Res. 1994;21455- 66Article
40.
O'Donnell  MEBrandt  JDCurry  FE Na-K-Cl cotransport regulates intracellular volume and monolayer permeability of trabecular meshwork cells. Am J Physiol. 1995;268C1067- C1074
41.
Palfrey  JCLeung  S Inhibition of Na-K-2Cl cotransport and bumetanide binding by ethacrynic acid, its analogs, and adducts. Am J Physiol. 1993;264C1270- C1277
42.
Epstein  DLRoberts  BCSkinner  LL Non-sulfhydryl reactive phenoxyacetic acids increase outflow facility and disrupt the trabecular and endothelial cell cytoskeleton [abstract]. Invest Ophthalmol Vis Sci. 1996;37(suppl)S894
43.
Epstein  DLDeKater  AWErickson-Lamy  KFay  FSSchroeder  AHooshmand  L The search for a sulfhydryl drug for glaucoma: from chemistry to cytoskeleton. Lütjen  Drecoll Eed.Basic Aspects of Glaucoma Research III Stuttgart, Germany Schattauer1993;345- 353
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