Comparison of Efficacy of Serum, EDTA, Tetracycline, Doxycycline, Minocycline, and NAC for Collagenase Inhibition (PDF)

Comparison of the efficacy of various concentrations and combinations of serum, ethylenediaminetetraacetic acid, tetracycline, doxycycline, minocycline, and N-acetylcysteine for inhibition of collagenase activity in an in vitro corneal degradation model Beth A. Kimmitt dvm...

Comparison of the efficacy of various concentrations and combinations of serum, ethylenediaminetetraacetic acid, tetracycline, doxycycline, minocycline, and N-acetylcysteine for inhibition of collagenase activity in an in vitro corneal degradation model Beth A. Kimmitt dvm OBJECTIVE To compare the efficacy of various concentrations and combinations of se- George E. Moore dvm, phd rum, EDTA, 3 tetracyclines, and N-acetylcysteine (NAC) for collagenase Jean Stiles dvm, ms inhibition in an in vitro corneal degradation model. SAMPLE Recieved August 3, 2017. Accepted September 7, 2017. Grossly normal corneas from recently euthanized dogs and horses and fresh serum from healthy dogs and horses. From the Department of Veterinary Clinical Scienc- PROCEDURES es (Kimmitt, Stiles) and Veterinary Administration Serum was pooled by species for in vitro use. For each species, sections of (Moore), College of Veterinary Medicine, Purdue Uni- cornea were dried, weighed, and incubated with clostridial collagenase (800 versity, West Lafayette, IN 47907. U/mL) in 5 mL of a 5mM calcium chloride–saline (0.9% NaCl) incubation solution and 500 µL of 1 of 19 treatments (homologous serum; 0.3%, 1.0%, Address correspondence to Dr. Stiles (stilesj@ or 2% EDTA; 0.1%, 0.5%, or 1.0% tetracycline, doxycycline, or minocycline; purdue.edu). 0.5%, 1.0%, or 5.0% NAC; serum with 0.5% tetracycline; serum with 1.0% EDTA; or 1.0% EDTA with 0.5% tetracycline). Positive and negative control specimens were incubated with 5 mL of incubation solution with and with- out collagenase, respectively. Each control and treatment was replicated 4 times for each species. Following incubation, corneal specimens were dried and reweighed. The percentage corneal degradation was calculated and compared among treatments within each species. RESULTS Treatments with tetracyclines at concentrations ≥ 0.5%, with EDTA at con- centrations ≥ 0.3%, and with NAC at concentrations ≥ 0.5% were more effective at preventing corneal degradation than serum in both species. The efficacy of each combination treatment was equal to or less than that of its components. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested EDTA, tetracyclines, and NAC may be beneficial for topi- cal treatment of keratomalacia, but in vivo studies are required. (Am J Vet Res 2018;79:555–561) D omesticated species commonly develop ulcer- ative keratitis, which can result in devastating consequences for the eye because ulceration can corneal degradation by those proteinases. However, when proteinase inhibitors are overwhelmed by pro- teinases released following corneal insult, keratoma- progress to corneal perforation. Loss of the corneal lacia develops and the corneal ulcer deepens.1,2 stroma following the initial corneal insult occurs ow- Many compounds have anticollagenase proper- ing to release of proteinases by etiologic agents (ie, ties and have been suggested for the treatment of bacteria or fungi), neutrophils, and corneal epithelial corneal ulcers to prevent corneal degradation and cells. The major proteinases that play a role in cor- keratomalacia. Some of those compounds are se- neal degradation are MMPs and serine proteinases. rum, plasma, EDTA, NAC, tetracyclines, and tetanus Anticollagenase enzymes (ie, proteinase inhibitors) antitoxin.3–6,a Results of a study3 in which the in vitro are present within the cornea and work to prevent effects of various concentrations of serum, NAC, and tetanus antitoxin on equine corneas were evaluated indicate that all 3 compounds provide corneas equal ABBREVIATIONS MMP Matrix metalloproteinase protection against collagenase digestion. In another NAC N-acetylcysteine in vitro study,4 serum, 0.2% EDTA, 0.1% doxycycline, TGF-β1 Transforming growth factor-β1 10% NAC, 0.2% ilomostat, and 0.1% and 0.5% α-1 pro- AJVR Vol 79 No. 5 May 2018 555 Unauthenticated | Downloaded 07/21/24 12:00 AM UTC teinase inhibitor all inhibited MMP activity in the tear corneal weight recorded. Collagenase derived from film of horses with ulcerative keratitis, with inhibi- Clostridium histolyticumb (800 U/mL) was added to tion of MMP activity greatest in samples treated with 5mM calcium chloride–saline (0.9% NaCl) solution to EDTA and ilomostat. prepare the incubation fluid. Saline solution was also Although the in vitro effects of single concen- used to create stock solutions of 2% EDTA,c 5% NAC,d trations of many anticollagenase compounds have 1% tetracycline,e doxycycline,f and minocycline.g been assessed, to our knowledge, various concentra- Those stock solutions were then diluted to create test tions of those compounds have not been compared solutions of 1% and 0.3% EDTA; 0.5% and 0.1% tetra- to determine the most effective concentration. Nor cycline, doxycycline, and minocycline; and 1% and have the effects of those compounds been evaluated 0.5% NAC. For each species, 4 replicates were created when used in combination. Determination of the ef- for each of the 5 stock solutions and 13 test solutions ficacy of combination treatment with anticollagenase as well as homologous serum and negative and posi- compounds is important because most patients with tive controls. For each replicate, a 5-mL aliquot of in- ulcerative keratitis are concurrently treated with mul- cubation fluid was added to a 10-mL tube followed tiple compounds. Therefore, the purpose of the study by 500 µL of homologous serum or one of the stock reported here was to compare the effect of various or test solutions and a homologous piece of cornea. anticollagenase compounds, alone and in combina- For each combination test solution, 500 µL of each tion, as well as at various concentrations, for the pre- individual compound was added to the 10-mL tube. vention of corneal degradation in an in vitro model. For each negative control replicate, 5 mL of 5mM cal- cium chloride in saline solution was added to a 10-mL Materials and Methods tube followed by a piece of cornea. For each positive control replicate, 5 mL of incubation fluid was added Corneal specimens to a 10-mL tube followed by a piece of cornea; no Corneas were obtained from recently euthanized inhibitory compounds were added to the positive dogs and horses at the Purdue University Veterinary control replicates. Replicate tubes were incubated on Teaching Hospital or local animal shelter. All animals a rocker at 40°C for 4 hours. Following incubation, were euthanized for reasons unrelated to the study. the remaining corneal specimens were retrieved by All corneas were examined by direct illumination pouring tube contents through a filter paper.h Each prior to harvest and determined to have no evidence retrieved specimen was placed in its original plastic of disease. Each cornea was removed and sectioned weigh boat and dried in an oven at 40°C for 3 hours. into 4 (canine) or 6 (equine) pieces of approximately The corneal specimens were weighed, and the post- equal size. Corneal pieces were labeled by species treatment corneal weight for each specimen was and date and individually stored at –80°C until use. recorded. For each specimen, the percentage of cor- Corneal pieces were evenly distributed among con- neal weight loss (degradation) was calculated as (pre- trol and treatment groups on the basis of storage time. treatment corneal weight – posttreatment corneal Serum samples weight)/pretreatment corneal weight X 100. Blood samples (each approx 6 mL) were obtained from 5 healthy dogs and 5 healthy horses (donor Statistical analysis animals). All dogs and some horses were owned by For each species, the mean ± SD percentage cor- Purdue University employees, whereas the remain- neal degradation was calculated for each of the 19 ing horses were teaching animals owned by Purdue treatments (5 stock solutions, 13 test solutions, and se- University. Consent was obtained from the owners rum) and negative and positive controls. A mixed lin- of all employee-owned animals prior to blood col- ear regression model was used to compare the mean lection. All live-animal procedures were approved percentage corneal degradation among treatments by the Purdue University Animal Care and Use Com- for each species. Each model contained a fixed effect mittee. All donor animals were manually restrained, for treatment and a random effect for cornea nested and blood was collected by jugular or cephalic veni- within donor animal. The Scheffe post hoc method puncture into serum separator tubes. Blood samples was used to control for type I error inflation owing were allowed to sit undisturbed at room temperature to multiple pairwise comparisons. Values of P < 0.05 (approx 22°C) for 1 hour after collection. Then, the were considered significant. Results were graphically serum from each sample was harvested and pooled reported as box-and-whisker plots owing to the small by species. The pooled samples were stored at room number (n = 4) of replicates for each treatment and to temperature and used within 2 hours after collection. demonstrate the distribution of results for each treat- ment relative to those for the negative and positive In vitro corneal degradation controls. The methodology used for in vitro corneal deg- radation was as previously described.6 Briefly, cor- Results neal pieces were individually placed in plastic weigh The mean ± SD percentage corneal degradation boats and dried in an oven at 40°C for 3 hours, af- was 91.0 ± 9.0% and 89.0 ± 9.6% for canine and equine ter which they were weighed and the pretreatment positive control samples, respectively, and 11.2 ± 7.4% 556 AJVR Vol 79 No. 5 May 2018 Unauthenticated | Downloaded 07/21/24 12:00 AM UTC  and 5.8 ± 4.6% for canine and equine negative control samples, respectively. For both canine and equine assays, the mean percentage corneal degradation was significantly lower (ie, treatment was more effective) than that for se- rum for treatments with tetracycline concentrations ≥ 0.5%, treatments with EDTA concentrations ≥ 0.3%, and treatments with NAC concentrations ≥ 0.5% (Figure 1). Additionally, for both species, the mean percentage corneal degradation did not differ significant- ly among the 3 concentrations (0.3%, 1.0%, and 2.0%) of EDTA (P = 0.99 for all 3 comparisons) or the 3 concentra- tions (0.5%, 1.0%, and 5.0%) of NAC (P = 0.99 for all comparisons of canine as- says and P ≥ 0.89 for all comparisons of equine assays). For canine assays, the mean per- centage corneal degradation was signif- icantly lower than that for the positive control for all treatments, except se- rum (P = 0.99), 0.1% minocycline (P = 0.99), and serum with 0.5% tetracycline (P = 0.20). The mean percentage cor- neal degradation for treatments with 0.1% tetracycline, 0.1% doxycycline, and 0.1% minocycline was significantly greater (ie, treatment was less effec- tive), compared with that for their re- spective 0.5% and 1.0% counterparts. However, the mean percentage corneal degradation did not differ significantly (P = 0.99 for all comparisons) between the 0.5% and 1% treatments for any of the 3 tetracyclines. For equine assays, the mean per- centage corneal degradation was signif- Figure 1—Box-and-whisker plots of the percentage corneal degradation (weight loss) for negative (NC) and positive (PC) controls and 19 anticollagenase treatments (serum icantly lower than that for the positive [S]; 0.1% [0.1% D], 0.5% [0.5% D], and 1.0% [1% D] doxycycline; 0.1% [0.1% M], 0.5% control for all treatments, except 0.1% [0.5% M], and 1.0% [1% M] minocycline; 0.1% [0.1% T], 0.5% [0.5% T], and 1.0% [1% doxycycline (P = 0.11) and 0.1% tetracy- T] tetracycline; 0.3% [0.3% E], 1.0% [1% E], and 2.0% [2% E] EDTA; 0.5% [0.5% N], cline (P = 0.064). The 0.1% tetracycline 1.0% [1% N], and 5.0% [5% N] NAC; serum with 1.0% EDTA [S + 1% E]; serum with and 0.1% doxycycline treatments were 0.5% tetracycline [S + 0.5% T]; and 1.0% EDTA with 0.5% tetracycline [1% E + 0.5% T]) following in vitro incubation with canine (A) and equine (B) corneal specimens. Each significantly (P < 0.001 for all compari- control and treatment was replicated 4 times for each species. For each treatment, sons) less effective than their 0.5% and replicates consisted of the incubation of a corneal specimen with 5 mL of incubation 1% counterparts. However, the mean fluid (Clostridium histolyticum–derived collagenase [800 U/mL] in 5mM calcium chloride percentage corneal degradation did in saline [0.9% NaCl] solution) and 500 µL of the assigned treatment (or 500 µL of each constituent for combined treatments). For the negative control replicates, corneal not differ significantly (P = 0.99 for all specimens were incubated with 5 mL of 5mM calcium chloride in saline solution. For the comparisons) between the 0.5% and positive control replicates, corneal specimens were incubated with 5 mL of incubation 1.0% treatments for either tetracycline fluid without any anticollagenase compounds (ie, treatment). For each plot, the lower or doxycycline or among the 3 minocy- and upper limits of the box represent the 25th and 75th percentiles, the horizontal line within the box represents the median, and the whiskers delimit the range. Within cline (0.1%, 0.5%, and 1.0%) treatments. each set of 3 concentrations for single-compound treatments, means for treatments The efficacy for each of the 3 com- denoted by different lowercase letters differ significantly (P < 0.05); the lack of lower- bination treatments was compared with case letters indicates the means did not differ significantly among the 3 concentrations. the efficacy for each of their respective *Mean differs significantly (P < 0.05) from that for the positive control. †Mean differs significantly (P < 0.05) from that for the serum treatment. ‡Mean differs significantly (P constituents. For the canine assays, the < 0.05) from that for the 0.5% tetracycline treatment. §Mean differs significantly (P < efficacy of the serum with 1.0% EDTA 0.05) from that for the 1.0% EDTA treatment. treatment was significantly (P < 0.001) AJVR Vol 79 No. 5 May 2018 557 Unauthenticated | Downloaded 07/21/24 12:00 AM UTC greater than that of the serum treatment but did not dif- In veterinary patients, serum is often used as fer significantly (P = 0.22) from that of the 1.0% EDTA the first line of defense against keratomalacia, and treatment. For the equine assays, the efficacy of the the in vitro effects of serum on corneal degrada- serum with 1% EDTA treatment was significantly (P < tion have been described for various veterinary 0.001) less than that of the 1.0% EDTA treatment but did species.6,a However, results of the present study not differ significantly (P = 0.22) from that of the serum indicated that other compounds are as efficacious treatment. For both canine and equine assays, the effi- as or more efficacious than serum for the preven- cacy of the serum with 0.5% tetracycline treatment was tion of corneal degradation. Although we do not significantly (P < 0.001 for both comparisons) less than discourage the use of serum in patients with kera- that of the 0.5% tetracycline treatment but did not differ tomalacia, we propose that it may be beneficial to significantly (P = 0.99 for both comparisons) from that consider other anticollagenase compounds for use of the serum treatment. Additionally, for both canine in conjunction with serum early in the treatment and equine assays, the efficacy of the 1.0% EDTA with process. In addition to its anticollagenase proper- 0.5% tetracycline treatment did not differ significantly ties, serum contains growth factors, such as epi- from that of the 1.0% EDTA (P = 0.99 for both compari- thelial growth factor, TGF-β1, and platelet-derived sons) or 0.5% tetracycline (P ≥ 0.69 for both compari- growth factor, that may be beneficial in the treat- sons) treatment. ment of keratomalacia and corneal ulceration.7 Epithelial growth factor promotes corneal epithe- Discussion lial migration and proliferation that improves and Results of the present in vitro study indicated accelerates wound healing.7 Expression of TGF-β1 that, for both canine and equine corneal specimens, in corneal epithelium increases during stromal re- the mean percentage degradation following treatment pair.7 Even though TGF-β1 decreases keratocyte with tetracyclines at concentrations ≥ 0.5%, EDTA at migration, it works synergistically with platelet- concentrations ≥ 0.3%, and NAC at concentrations ≥ derived growth factor to promote myofibroblast 0.5% was significantly less than that for the positive differentiation.8 control (corneal specimens incubated with collage- On the basis of the findings of the present study, nase but without an inhibitor), which suggested that tetracyclines, as a group, may be beneficial in reduc- those treatments might be efficacious for the preven- ing clinical keratomalacia. Tetracyclines chelate both tion of corneal degradation in dogs and horses with calcium (a MMP cofactor) and zinc (a stabilizing ion ulcerative keratitis. Additionally, the mean percentage of MMPs), which leads to MMP inhibition.4 In general, corneal degradation was significantly less than that tetracyclines chelate zinc to a greater extent than cal- of the positive control for equine specimens treated cium, and doxycycline is able to bind zinc more tight- with serum but not for canine specimens treated with ly than other members of the tetracycline group.9 For serum. Serum was also found to be less efficacious each of the 3 tetracyclines (tetracycline, doxycycline, than the treatments with tetracycline (tetracycline, and minocycline) evaluated in the present study, con- doxycycline, or minocycline) concentrations ≥ 0.5%, centrations ≥ 0.5% significantly decreased corneal treatments with EDTA concentrations ≥ 0.3%, and degradation (ie, inhibited collagenase) relative to that treatments with NAC concentrations ≥ 0.5%. In both of the positive controls for both canine and equine canine and equine assays, the 0.5% tetracycline and specimens. Results of another study4 indicate that 1% EDTA treatments when used alone were as effica- 0.1% doxycycline significantly inhibits proteases in cious as or more efficacious than when used in com- the tear film of horses, but that concentration of dox- bination with serum. In the present study, following ycycline was not effective in inhibiting collagenase incubation with serum, the mean ± SD percentage activity in the present study. However, the investiga- corneal degradation was 79 ± 15% for canine speci- tors of that study4 used gel zymography to measure mens and 57 ± 16% for equine specimens, which was protease activity rather than a corneal degradation similar to the mean ± SD percentage corneal degra- model as was used in the present study, and differ- dation for canine (74 ± 15%) and equine (38 ± 12%) ences between the experimental models may have specimens incubated with serum in another study6 contributed to the apparently conflicting results be- performed at our institution. However, in that study,6 tween the 2 studies. serum significantly decreased corneal degradation The ability of doxycycline to penetrate the ocular relative to the positive controls for both species, tear film following oral administration has also been whereas in the present study, serum decreased cor- investigated. In 1 study,10 peak tear film doxycycline neal degradation only in the equine assays. Although concentration ranged from 8.21 to 9.83 µg/mL (or results of that other study6 suggest that the anticolla- 0.0008% to 0.0010%) following oral administration genase activity is similar between serum and plasma, of the drug (20 mg/kg, q 24 h for 5 days) to horses, we chose to use serum in the present study so that which was quite low relative to the doxycycline con- we could compare our results with those of other in centrations (0.1%, 0.5%, and 1.0%) evaluated in the vitro studies3,4 in which the extent of corneal protec- present study. In another study,11 oral administration tion for various anticollagenase compounds, includ- of minocycline (4 mg/kg, q 12 h for 5 days) to ponies ing serum rather than plasma, was evaluated. resulted in a maximum tear film drug concentration of 558 AJVR Vol 79 No. 5 May 2018 Unauthenticated | Downloaded 07/21/24 12:00 AM UTC only 11.8 µg/mL (or 0.0012%), which was insufficient EDTA was evaluated at concentrations ranging from to inhibit MMP-2 and MMP-9 in vitro. In yet another 0.0001% to 1.0%, and the mean ± SD corneal dam- study,12 oral administration of doxycycline (10 mg/ age percentage was 5.03 ± 39.53% for the 0.0001% kg, q 12 h for 5 days) to dogs resulted in a maximum concentration and 57.86 ± 37.32% for the 1.0% con- tear film drug concentration of only 4.32 ng/mL (or centration. That finding suggests that the EDTA con- 0.0000432%), and varying the doxycycline dose from centrations evaluated in the present study have the 5 mg/kg to 10 mg/kg had no effect on the mean tear potential to induce damage to the corneal epithelium film drug concentration. Results of the present study in vivo, and further investigation into the potential suggested that the 0.1% doxycycline treatment was sig- corneal toxicity of EDTA in vivo is warranted. nificantly less effective at inhibiting collagenase, com- All 3 concentrations (0.5%, 1.0%, and 5.0%) of pared with the 0.5% and 1.0% doxycycline treatments. NAC evaluated in the present study significantly re- Because reported tear film drug concentrations follow- duced corneal damage relative to the positive con- ing oral administration of tetracyclines are much lower trols. N-acetylcysteine is a derivative of the amino acid than 0.1%, it is questionable whether oral administra- l-cysteine and inhibits collagenase irreversibly by re- tion of tetracyclines is clinically beneficial for patients ducing disulphide bonds and chelating calcium and with keratomalacia. Therefore, if a tetracycline is be- zinc. It also inhibits MMP-9, although the exact mecha- ing considered for a patient with keratomalacia solely nism by which it does so is unknown.17,18 In another for its anticollagenase effects, the topical route may be in vitro study3 of the anticollagenase activity of NAC, a the preferred route of administration. Currently, oxy- 2.0% concentration of NAC effectively decreased cor- tetracycline is the only tetracycline available in com- neal degradation but concentrations of 0.2% and 0.02% mercially available eye ointments. However, patients did not. Because the results of that study3 suggest that with keratomalacia often require frequent treatments, the concentration breakpoint for NAC efficacy against and solutions may be more beneficial than ointments corneal degradation is between 2% and 0.2%, we chose for those patients. Also, in horses with keratomalacia, to evaluate NAC concentrations of 0.5% and 1.0% in the subpalpebral lavage is facilitated by the use of anticol- present study, both of which resulted in significant de- lagenase solutions. creases in corneal degradation. The highest concentra- For patients with corneal defects, topical admin- tion of NAC chosen for evaluation in the present study istration of tetracyclines, particularly oxytetracy- was set at 5% because results of other studies3,19 indi- cline, promotes corneal epithelial wound healing.13 cate that NAC concentrations > 5% can be irritating to That effect is suspected, but not yet proven, to be the eyes. However, results of an in vivo study20 in which result of tetracycline-induced upregulation of TGF-β1 the effect of NAC concentrations of 3%, 10%, and 20% and other specific growth and transcription factors on corneal wound healing in dogs was investigated in- that promote corneal reepithelialization. Therefore, dicate that only the 3% solution significantly decreased tetracyclines may promote corneal healing in addi- wound healing time relative to that for control eyes tion to their anticollagenase activities. Tetracyclines that were treated with saline solution, although no ad- are also antimicrobial agents that can reduce the bac- verse effects were reported for eyes treated with NAC terial load in corneal ulcers, which decreases the col- at any of the 3 concentrations. lagenase load and aids in the prevention of further In both the canine and equine assays of the pres- corneal degradation. ent study, the mean percentage corneal degradation Ethylenediaminetetraacetic acid is a metal che- for the serum with 0.5% tetracycline (combined) lator and, similar to tetracyclines, chelates both cal- treatment did not differ from that for the serum only cium and zinc.14 It also decreases stimulation of neu- treatment but was significantly greater than that for trophil migration to corneal ulcers, which in turn the 0.5% tetracycline only treatment. Tetracycline decreases the release of proteinases.3 All 3 concentra- binds to proteins in a variable manner; therefore, it is tions (0.3%, 1.0%, and 2.0%) of EDTA evaluated in the possible that a portion of the tetracycline in the com- present study effectively decreased corneal degrada- bined treatment became bound to serum proteins, tion. The 0.3% EDTA treatment was chosen to mimic which decreased its anticollagenase activity relative an EDTA solution that can be easily produced by the to when it was used alone. Further research is nec- addition of 1.5 mL of sterile water to the contents of a essary to assess the use of multiple anticollagenase 3-mL EDTA blood collection tube, whereas the 1.0% agents simultaneously in a clinical setting. Specifical- and 2.0% EDTA treatments represented commonly ly, additional investigation is required to determine compounded EDTA solutions. It is important to note whether the traditionally recommended 5-minute that, although all 3 concentrations of EDTA effectively interval between administration of topical ophthal- prevented corneal degradation in vitro, EDTA can be mic medications is sufficient to prevent interaction potentially toxic to corneal epithelium in vivo. The between the medications. effect of EDTA on corneal tissue has been investigat- For the canine assays of the present study, the ed15 but only at concentrations (0.00001% to 0.01%) mean percentage corneal degradation for the serum appropriate for use as a preservative, which are much with 1.0% EDTA treatment was significantly lower lower than those required for anticollagenase activi- than that for the serum only treatment but did not dif- ty. However, in 1 study,16 corneal epithelial toxicity of fer significantly from that for the 1% EDTA only treat- AJVR Vol 79 No. 5 May 2018 559 Unauthenticated | Downloaded 07/21/24 12:00 AM UTC ment. Therefore, the serum with 1% EDTA treatment are necessary to further evaluate the efficacy of anti- did not appear to have a synergistic effect on the pre- collagenase compounds in patients with keratomala- vention of corneal degradation, but incubation with cia, results of the present study suggested that topical those 2 compounds in combination did not adversely administration of EDTA, NAC, and tetracyclines may affect the anticollagenase activity of EDTA, the more be useful for the prevention of corneal degradation. efficacious of the 2 compounds. Conversely, for the equine assays, the mean percentage corneal degrada- Acknowledgments tion for the serum with 1.0% EDTA treatment did not Supported by a grant from the Willis E. and Mary J. Armstrong differ from that for the serum only treatment and was Memorial Fund for Research in Anomalies of the Eye. significantly greater than that for the 1.0% EDTA only treatment. Finally, the 1.0% EDTA with 0.5% tetracy- Footnotes cline treatment did not have an additive or diminish- a. Ben-Shlomo G, Roecker H. Efficacy evaluation of canine se- ing effect on the mean percentage corneal degrada- rum and plasma against protease activity for treatment of keratomalacia (abstr). Vet Ophthalmol 2014;17:E36. tion, compared with the mean percentage corneal b. Collagenase from Clostridium histolyticum, type XI, Sigma- degradation for each of its constituent compounds. Aldrich Corp, St Louis, Mo. In the present study, corneal degradation was c. EDTA, Sigma-Aldrich Corp, St Louis, Mo. measured on the basis of estimated collagen loss in d. N-Acetyl-L-cysteine, Sigma-Aldrich Corp, St Louis, Mo. e. Tetracycline hydrochloride, Sigma-Aldrich Corp, St Louis, specimens following incubation with a commercially Mo. available collagenase. The C histolyticum–derived f. Doxycycline hyclate, Sigma-Aldrich Corp, St Louis, Mo. collagenase used in this study is composed of 7 dis- g. Minocycline hydrochloride, Sigma-Aldrich Corp, St Louis, tinct proteases that fall into the categories of class Mo. I (γ) and class II (ζ) collagenases.21 These are zinc- h. Whatman filter paper, Sigma-Aldrich Corp, St Louis, Mo. dependent enzymes that are functionally related to MMPs; therefore, it was not surprising that collage- References nase inhibition was variable among the EDTA, NAC, 1. Rehman AA, Ahsan H, Khan FH. α-2-macroglobulin: a physi- ological guardian. J Cell Physiol 2013;228:1665–1675. and tetracycline treatments evaluated. Although that 2. Borth W. α2-macroglobulin, a multifunctional binding pro- collagenase has been used to approximate keratoma- tein with targeting characteristics. FASEB J 1992;6:3345– lacia in other in vitro studies,3,4,6 the collagenolysis it 3353. induces may not completely mimic keratomalacia in 3. Haffner JC, Fecteau KA, Eiler E. Inhibition of collagenase breakdown of equine corneas by tetanus antitoxin, equine vivo because other sources of collagenases, including serum and acetylcysteine. Vet Ophthalmol 2003;6:67–72. exogenous pathogens and endogenous cells, may be 4. Ollivier FJ, Brooks DE, Kallberg ME, et al. Evaluation of vari- present in the diseased eyes of live patients. In serum, ous compounds to inhibit activity of matrix metalloprotein- the most abundant proteinase inhibitor is α-2 macro- ases in the tear film of horses with ulcerative keratitis. Am J globulin, which is nonspecific and inhibits MMPs as Vet Res 2003;64:1081–1087. 5. Burns FR, Stack MS, Gray RD, et al. Inhibition of purified col- well as other classes of proteinases such as serine lagenase from alkali-burned rabbit corneas. Invest Ophthal- proteinases, of which neutrophil elastase is the most mol Vis Sci 1989;30:1569–1575. abundant in vivo. The corneal degradation model 6. Conway ED, Stiles J, Townsend WM, et al. Comparison of the used in this study did not account for different types in vitro anti-collagenase efficacy of homologous serum and plasma on degradation of corneas of cats, dogs, and horses. of proteinases, continued release of proteinases, or Am J Vet Res 2016;77:627–633. repeated treatment with anticollagenase compounds. 7. Anitua E, Muruzabal F, Tayebba A, et al. Autologous serum Therefore, in vivo studies of the anticollagenase ef- and plasma rich in growth factors in ophthalmology: preclin- fects of the compounds evaluated in this study are ical and clinical studies. Acta Ophthalmol 2015;93:e605– warranted. e614. 8. Lopez-Garcia JS, Murube del Castillo J, Garcia Lozano I, et al. To our knowledge, the present study was the first Autologous serum and blood derivatives in ophthalmology. to compare the anticollagenase efficacy of multiple Arch Soc Esp Oftalmol 2012;87:376–377. compounds at various concentrations alone and in 9. Brion M, Lambs L, Berthon G. 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