Sequential Therapy with Saratin, Bevacizumab and Ilomastat to Prolong Bleb Function following Glaucoma Filtration Surgery in a Rabbit Model

<div><p>To determine if sequential treatment with Bevacizumab (Avastin), a monoclonal, VEGF antibody that blocks angiogenesis; Saratin, a 12 kD polypeptide with anti-inflammatory and anti-thrombotic properties; and Ilomastat, a matrix metalloproteinase (MMP) inhibitor, prolongs bleb life following glaucoma filtration surgery (GFS) in a rabbit model. Thirty-two New Zealand White rabbits (eight rabbits per group) underwent GFS in the left eye. Group 1 received a perioperative injection of both Saratin and Bevacizumab, and later, subconjuctival injections of Ilomastat on days 8 and 15. Group 2 received only Saratin perioperatively, and also received Ilomastat injections on days 8 and 15. Group 3, the negative control, received a single perioperative injection of Balanced Saline Solution (BSS) along with post-operative BSS injections on days 8 and 15. Group 4, the positive control, received topical treatment with Mitomycin-C (MMC) at the time of surgery with no further treatment. Blebs were evaluated by an observer masked to treatment every third day. Histology was obtained on two eyes in each group on post-op day twelve as well as all eyes following bleb failure. Eyes in group 1 had a mean bleb survival time of 29 ± 2.7 days, whereas those in group 2 that received the experimental treatment without Bevacizumab had a mean survival time of 25.5 ± 2.7 days. An ANOVA test showed that the Saratin/Ilomastat/Bevacizumab group demonstrated a significant prolongation of bleb survival compared to the BSS control—mean survival time of 19.7 ±2.7 days—(p = 0.0252) and was not significantly different from the MMC positive control group (p = 0.4238)—mean survival time of 32.5 ± 3.3. From tissue histology at day 12, the four different groups showed marked differences in the cellularity and capsule fibrosis. The MMC eyes showed minimal cellularity, were avascular and had minimal fibrous tissue. BSS group showed high cellularity, moderate to high fibrosis, and thicker and more defined capsules than either of the treatment groups and the positive control. Both the Saratin/Ilomastat/Bevacizumab and Saratin/Ilomastat only eyes showed moderate cellularity with minimal fibrosis, with less cellularity and fibrosis present in the triple treatment group. Sequential therapy with multiple agents, including Bevacizumab, prolonged bleb function following GFS in the rabbit model and were significantly better than the negative BSS control. The experimental group did not show the same surface tissue histological thinning and side effects associated with MMC treatment.</p></div

Masson’s Trichrome stained representative sections near implants site taken from POD 12, Masson’s Trichrome (magnification 10x, black scale bars = 100 μm).

<p>(A) Saratin/Ilomostat; (B) Saratin/Bevacizumab/Ilomostat; (C) BSS; (D) MMC (all representative, come from areas of roughly the same location in relation to tube placement)</p

Kaplan-Meier bleb survival plot of eyes in each treatment group.

<p>For each rabbit, bleb failure was declared after the bleb appeared flat in two consecutive masked clinical examinations. The first of the two dates was recorded as the endpoint.</p

Harris Hematoxylin and Eosin stained representative sections near implants site taken on Post-Operative Day 12 (magnification 10x, black scale bars = 100 μm).

<p>(A) Saratin/Ilomostat; (B) Saratin/Bevacizumab/Ilomostat; (C) BSS; (D) MMC (all representative, come from areas of roughly the same location in relation to tube placement).</p

Box and Whisker bleb survival plot of eyes treated with MMC, BSS, Saratin/Bevacizumab/Ilomastat, or Saratin/Ilomastat.

<p>For each rabbit, bleb failure was declared after the bleb appeared flat in two consecutive masked clinical examinations. The first of the two dates was recorded as the endpoint.</p

Representative sections of the implant site taken on POD 12, Masson’s Trichrome 20x; Left: Topical+2 injections of saratin; <i>asterisk</i>, mild collagen density and moderate fibroblast proliferation Middle: Balanced saline solution; Right: Mitomycin-C; <i>asterisk</i>, acellular fibrosis and necrosis.

<p>Representative sections of the implant site taken on POD 12, Masson’s Trichrome 20x; Left: Topical+2 injections of saratin; <i>asterisk</i>, mild collagen density and moderate fibroblast proliferation Middle: Balanced saline solution; Right: Mitomycin-C; <i>asterisk</i>, acellular fibrosis and necrosis.</p

Box-and-whisker plot of bleb survival for each treatment group.

<p>First and third quartiles are delineated by boxes. Whiskers represent maxima and minima.</p

Representative sections of the bleb conjunctiva taken on POD 12, H&E 250x; (A) Topical saratin; (B) Topical+2 injections of saratin; (C) Balanced saline solution; (D) Mitomycin-C.

<p>Representative sections of the bleb conjunctiva taken on POD 12, H&E 250x; (A) Topical saratin; (B) Topical+2 injections of saratin; (C) Balanced saline solution; (D) Mitomycin-C.</p

Bleb survival of LBCF and FBCF rabbits using 22 and 26-gauge cannulas.

<p>Bleb survival of LBCF and FBCF rabbits using 22 and 26-gauge cannulas.</p

Experimental groups.

<p>Experimental groups.</p