To determine whether inhibition of integrin α5β1 altered the outcome of glaucoma surgery in vivo, CLT-28643 was assessed for efficacy in preservation of the bleb in the mouse model. Daily clinical examinations revealed that none of the animals showed any pain-distress. No regimen-related differences in pre- and posttreatment body weights during follow-up were detected (data not shown).
Bleb area and survival were analyzed every 2 days after surgery as a measure of filtration surgery outcome. Analysis of the bleb area and survival showed that all vehicle-treated eyes failed by day 17 (
Fig. 3). All the other regimens significantly improved bleb area and bleb survival compared with vehicle (
P < 0.05). The dose regime of repeated injections of 2 μg of CLT-28643 was significantly superior to MMC with respect to bleb area (
P < 0.001,
Fig. 3A) and both these groups showed 100% bleb survival for the duration of the study (
Fig. 3B). The effect of a single SCJ injection of 2-μg CLT-28643 immediately after surgery on bleb area and survival was similar to that of MMC (
P = 0.28, and
P = 0.32, respectively). Administration of 10-μg drops of CLT-28643, 3× daily, also increased bleb area versus vehicle and was comparable to MMC. By Day 28, 75% of the blebs in the drops treated group failed versus MMC's 100% survival (
P = 0.003). Repeated injections of a low-dose (1 μg) CLT-28643 was found to be less effective compared with MMC in bleb characteristics and resulted in a significantly reduced bleb area as compared with the antimitotic agent (
P < 0.001) and increased bleb failure (75% vs. 100%) on day 28 (
P = 0.006).
Figure 4 shows representative photographs of the blebs after treatment on different postoperative days. Repeated injections of CLT-28643 were clearly associated with a large bleb compared with the flat and scarred blebs observed in the respective vehicle control group. Importantly, grading corneal opacity indicated that 2-minute administration of 0.02% MMC significantly increased corneal toxicity as compared with vehicle (
P < 0.001), whereas treatment with CLT-28643 was similar to vehicle (
P > 0.05;
Table 2). While all groups showed some transient corneal opacification postoperatively, 8/40 eyes (20%) of MMC-treated eyes had scores of greater than or equal to 1.0 compared with 3/200 eyes in other treatment groups.
Thus, our data revealed a dose-dependent effect in the efficacy of the integrin inhibitor CLT-28643. While a 2-μg single injection of CLT-28643 improved bleb characteristics in a similar way as 10-μg topical administration and MMC, repeated injections of 2 μg induced an even better response in surgical outcome, and were statistically superior to MMC in terms of bleb area. Importantly, MMC increased corneal opacification, to a greater extent compared with the CLT-28643- or vehicle-treated groups.