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Evaluation of various compounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ulcerative keratitis

Franck J. OllivierDepartment of Large Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.
Department of Small Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.

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Dennis E. BrooksDepartment of Large Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.
Department of Small Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.

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Maria E. KallbergDepartment of Large Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.
Department of Small Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.

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Andras M. KomaromyDepartment of Large Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.
Department of Small Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.

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Mary E. LassalineDepartment of Large Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.
Department of Small Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.

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Stacy E. AndrewGeorgia Veterinary Specialists, 455 Abernathy Rd NE, Atlanta, GA 30328.

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Kirk N. GelattDepartment of Large Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.
Department of Small Animal Clinical Sciences, College of Medicine, University of Florida, Gainesville, FL 32610.

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Gary R. StevensCollege of Veterinary Medicine, Departments of Statistics, College of Medicine, University of Florida, Gainesville, FL 32610.

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Timothy D. BlalockCollege of Veterinary Medicine, Departments of Obstetrics/Gynecology, College of Medicine, University of Florida, Gainesville, FL 32610.

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Gysbert-Botho van SettenSt Eriks Eyes Clinic, Karolinska Institute, Stockholm, Sweden.

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Gregory S. SchultzCollege of Veterinary Medicine, Departments of Obstetrics/Gynecology, College of Medicine, University of Florida, Gainesville, FL 32610.

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Abstract

Objective—To examine in vitro effects of various antiproteolytic compounds on activity of matrix metalloproteinase (MMP)-2 and -9 in the tear film of horses with active corneal ulcers.

Sample Population—Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis.

Procedure—Horses were sedated, and tear samples were collected from the lower fornix of 34 ulcerated eyes by use of capillary tubes. The protease inhibitors 0.2% EDTA, 0.1% doxycycline, 10% N-acetylcysteine (NAC), 0.1% solution of a modified dipeptide that contains hydroxamic acid (ie, ilomostat), 0.1% α1-proteinase inhibitor (PI), 0.5% α1-PI, and 100% fresh equine serum (ES) were used to treat pooled samples. Amount of latent and active MMP-2 and -9 was measured by optical density scanning of gelatin zymograms of treated and untreated tear samples.

Results—Pooled tear samples obtained from ulcerated eyes contained the latent and active forms of MMP-2 and -9. Compared with MMP activity in untreated samples, total MMP activity (sum of all bands detected) observed on the gelatin zymogram gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% α1-PI, 93.6% by 0.5% α1-PI, and 90.0% by ES.

Conclusions and Clinical Relevance—We documented that EDTA, doxycycline, NAC, ilomostat, α1- PI, and ES inhibited MMP activity in vitro. Because these compounds use different mechanisms to inhibit various families of proteases in the tear film of horses, a combination of these protease inhibitors may be beneficial for treatment of corneal ulcers in horses. (Am J Vet Res 2003;64:1081–1087)

Abstract

Objective—To examine in vitro effects of various antiproteolytic compounds on activity of matrix metalloproteinase (MMP)-2 and -9 in the tear film of horses with active corneal ulcers.

Sample Population—Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis.

Procedure—Horses were sedated, and tear samples were collected from the lower fornix of 34 ulcerated eyes by use of capillary tubes. The protease inhibitors 0.2% EDTA, 0.1% doxycycline, 10% N-acetylcysteine (NAC), 0.1% solution of a modified dipeptide that contains hydroxamic acid (ie, ilomostat), 0.1% α1-proteinase inhibitor (PI), 0.5% α1-PI, and 100% fresh equine serum (ES) were used to treat pooled samples. Amount of latent and active MMP-2 and -9 was measured by optical density scanning of gelatin zymograms of treated and untreated tear samples.

Results—Pooled tear samples obtained from ulcerated eyes contained the latent and active forms of MMP-2 and -9. Compared with MMP activity in untreated samples, total MMP activity (sum of all bands detected) observed on the gelatin zymogram gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% α1-PI, 93.6% by 0.5% α1-PI, and 90.0% by ES.

Conclusions and Clinical Relevance—We documented that EDTA, doxycycline, NAC, ilomostat, α1- PI, and ES inhibited MMP activity in vitro. Because these compounds use different mechanisms to inhibit various families of proteases in the tear film of horses, a combination of these protease inhibitors may be beneficial for treatment of corneal ulcers in horses. (Am J Vet Res 2003;64:1081–1087)