Georgoulas, Stylianos D.; (2010) Novel methods for the modulation of wound healing after glaucoma filtration surgery. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Scarring is known to be the leading cause of trabeculectomy failure. Anti-metabolic drugs (5-FU and MMC) are currently used in an attempt to inhibit scarring after glaucoma filtration surgery (GFS), but they are only effective in a limited number of patients in the long term and can cause severe side effects. There is a clinical need for better therapies to ensure that the healing process can be better mediated after GFS. Serum Amyloid P (SAP) was examined to determine if it has potential to inhibit scarring after GFS. It has been shown that non-activated, bone-marrow derived peripheral monocyte precursor CD 14+ fibrocytes circulating in the blood participate in scarring and that myofibroblasts may not originate from tissue fibroblasts, but from this bone-marrow- derived precursor. SAP is a protein that belongs in the pentraxin family of proteins and is a potent modulator of monocyte differentiation in vitro and in vivo. It has been suggested that SAP may also have broad anti-fibrotic activity in many tissues, organs and species. It was found that administration of SAP by injection to the bleb significantly prolonged bleb survival as well as maintained normal lOP (with no post-surgical hypotony) in a clinically validated in vivo rabbit model of GFS. There was decreased local collagen deposition and cell number in the treated eyes. These results suggest that the circulating monocytes transformed into fibroblasts in the bleb area after GFS may participate in the scarring process after GFS and that SAP may have potential for development as a therapeutic agent to prevent fibrosis following GFS. Another class of potentially less toxic but relevant molecules that could serve to inhibit fibrosis are matrix metalloproteinase (MMP) inhibitors. Our group had previously found that multiple injections of a MMP inhibitor called ilomastat could inhibit fibrosis in vitro and in vivo. As with both SAP and with the currently used cytotoxic agents, the pharmacokinetics of multiple subconjunctival injections to control fibrosis is not optimal. Such injections are uncomfortable to the patient and carry some risk of infection. Upon injection into the bleb, there is rapid clearance of the medicine leading to dose dumping, resulting in the need to use high doses in the hope of administering enough of the medicine to provide clinical benefit. The strategy to down regulate an undesirable mediator using siRNA to treat disease has recently been shown to have potential. Since the activated fibrotic cells in the bleb may have a longer local residence time than that of an injected drug, it was thought it may be possible to use siRNA to stop the production of MMPs. In vitro experiments confirmed that while collagen contraction was inhibited using siRNA, unfortunately it was not possible to decrease bleb failure in vivo. Noting that ilomastat was used as an excellent positive control during the experiments with SAP and siRNA, it was felt that if a prolonged therapeutic concentration of ilomastat could be maintained in the bleb, then multiple injections could be avoided. Furthermore, a prolonged therapeutic concentration of ilomastat might result in better overall healing. Since the subconjunctiva is open during GFS surgery, it was decided to formulate a new single application prolonged release ilomastat tissue tablet that could be left in the bleb at the time of surgery. Since it is known that the first 30 days post surgery is a very critical period for the scarring process after GFS, it was hypothesised that an ilomastat tablet designed to last 30 days in the bleb would serve to inhibit fibrosis. Since ilomastat is poorly soluble (0.038 mg/ml), it was hypothesised that a tablet comprised entirely of ilomastat without excipients would be dissolved at a low rate since the volume and flow in the bleb are at non-sink conditions for such a poorly soluble molecule. Release studies of the tablet in vitro showed that the excipient free ilomastat tissue tablets achieved a constant release of active ilomastat in a concentration significantly higher than the therapeutic dose for at least 30 days. HPLC analysis confirmed that ilomastat did not degrade and remained active after sterilization and after incubation in an aqueous environment for 30 days at 37°C. Ilomastat released from the tablets significantly inhibited contraction of HTF collagen I gels in vitro. In vivo, the survival of the blebs treated with the ilomastat tablet was found to be significantly superior to the sterile water group (negative control) and with the MMC group (positive control). An inert tablet comprised of only ethyl cellulose, a common excipient, did not result in bleb survival beyond the negative control. Histological analysis showed that the ilomastat treatment group had significantly less subconjunctival scarring compared to the negative and the positive controls. No ilomastat was detectable in serum or in the ocular tissues. This work in this thesis has served to increase our understanding about the potential benefit of prolonging the local pharmacokinetics of a medicine in the bleb. This may mediate the healing process after GFS and leads the way to a possible improved treatment for scarring after glaucoma surgery and other situations when scarring is a problem.

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