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


METHODS
Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis.


METHODS
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% alpha1-proteinase inhibitor (PI), 0.5% alpha1-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% alpha1-PI, 93.6% by 0.5% alpha1-PI, and 90.0% by ES.


CONCLUSIONS
We documented that EDTA, doxycycline, NAC, ilomostat, alpha1PI, 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.
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D003320:Corneal Ulcer / Q000201:enzymology / Q000662:veterinary*; D004318:Doxycycline / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004586:Electrophoresis / Q000662:veterinary; D006734:Horse Diseases / Q000201:enzymology*; D006736:Horses; D006877:Hydroxamic Acids / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors / Q000494:pharmacology*; D013666:Tears / Q000201:enzymology*; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D006877:Hydroxamic Acids; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D000515:alpha 1-Antitrypsin; D004492:Edetic Acid; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D004318:Doxycycline; D000111:Acetylcysteine