Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

2. Anti-inflammatory agents

Conjunctival inflammation and uveitis after trabeculectomy hasten the scarring response. The grading system used in our long-term trial showed a good correlation between inflammation and the long-term outcome, and is an example of how phenotypic classification may later be used to predict who received therapeutic interventions (www.blebs.net) (Figure 15.6). Topical steroids are part of the routine post-operative

management, and there is good evidence for their effectiveness (Kent et al., 1998). Systemic steroids do not seem to have an additional beneficial effect (Azuara-Blanco et al., 1999). Topical NSAIDs may be effective (Kent et al., 1998), but their use is still controversial. Newer agents affecting aspects of the inflammation pathway include cyclosporine and cyclooxygenase-2 inhibitors, although the effect of intraoperative or post-operative application of cyclosporine

in an animal model was not encouraging (Lattanzio et al., 2005). Another novel approach to the modulation of inflammatory cytokines is the development of dendrimers, hyperbranched nanomolecules that can be chemically synthesized to have precise structural characteristics. In an experimental model of GFS, water-soluble conjugates of D( )-glucosamine and D( )-glucosamine 6-sulfate with immuno-modulatory and anti-angiogenic properties used together increased the long-term success from 30% to 80% (Shaunak et al., 2004). Actions include the blockage of the toll-like receptors. This is far more effective than that seen with conventional steroids. This may be because of the synergistic action of the molecule which may be an increasingly important principle for therapeutic enhancement (Figures 15.7, 15.8 and 15.9). Human amniotic membrane appears to have anti-angio- genic, anti-inflammatory and antifibrotic characteristics. Based on that, the effects of the use of amniotic membrane were

tested both in animal models (Barton et al., 2001; Demir et al., 2002), and in humans (Fujishima et al., 1998; Bruno et al., 2006). The subconjunctival placement of amniotic membrane after experimental filtration surgery increased bleb survival as result of reduced fibrosis.

3. Anticlotting agents

Fibrinolytic agents such as tissue plasminogen activator and urokinase have been used to lyse blood clots after surgery (WuDunn, 1997). In the short term, fibrinolytic agents may lower intraocular pressure, but there is a risk of further extraand intraocular haemorrhage. The breakdown molecules may have a longer-term stimulatory effect on wound healing (Gray et al., 1993).

4. Growth factor modulators

Flowing through the bleb in the aqueous are a large number growth factors or

MWt 13 600

35 Angstroms

TLR-4 MD-2

Macrophages and

dendritic cells

cytokines (Chang et al., 2000). One growth factor, transforming growth factor beta (TGF-β), stimulates more activity than the other growth factors found in the aqueous (Khaw et al., 1994). TGF-β may even neutralize the effect of MMC in vivo (Khaw

et al., 1994). Various agents may affect the activity of growth factors including TGF-β. Tranilast ((N-(3 , 4 -dimethoxycinnamoyl) anthranilic acid) inhibits TGF-βactivity, and has antiscarring potential when used in GFS. Genistein and Suramin also suppress

TGF-β activity. Suramin reduces postoperative scarring in an experimental model of GFS (Mietz et al., 1998), and an early pilot study has been encouraging (Mietz and Krieglstein, 2001). Interferon-alpha, an antifibrotic cytokine, reduces scarring activity of fibroblasts, although a clinical trial did not show it to be significantly better than current antimetabolites (Gillies et al., 1999).

When we had established the important role of TGF-β relative to other growth factors in the eye (Khaw et al., 1994) we used different methods to target TGF-β activity, including antisense oligonucleotides (Cordeiro et al., 2003) and a human monoclonal antibody specific to the active form of human TGF-β2, the predominant isoform in the aqueous (Lerdelimumab, Trabio™, Cambridge Antibody Technology, Cambridge, UK). One of the theoretical advantages of the antibody is that, unlike antimetabolites, it only acts if there is TGF- β2 in the wound minimizing the chance of hypotony (Figure 15.10). In an animal model of aggressive conjunctival scarring, it significantly improved GFS outcome (Cordeiro et al., 1999). Compared to the effects of MMC treatment, it appeared much less destructive to local tissue. A pilot clinical study of this antibody in GFS showed no significant side effects or inflammatory reaction. There was a promising intraocular pressure reduction of 10.4 mmHg in the antibody treated group at 1 year, compared with 4.6 mmHg in the placebo group, and fewer interventions in the antibody treated group (Siriwardena et al., 2002), without the very cystic blebs seen with antimetabolites (Figure 15.11). Two larger randomized controlled trials have not shown a significant effect. It may be that the dose based on an earlier study was not adequate, and subsequent experiments from our lab have shown a significantly enhanced effect with a prolonged dosing regimen (Mead et al., 2003), and the data also suggested an enhanced effect when the antibody is combined with intraoperative 5-FU.

CAT-152 human TGF- 2 antibody:

"autoregulating"

FIGURE 15.10 Schematic view of antibody in the presence of high and low concentrations of TGF-β2 in the conjunctiva

FIGURE 15.11 Diffuse non-cystic bleb in the human eye after glaucoma surgery, with TGF-β antibody

Other inhitors of TGF-β have been used, including a natural inhibitor of this factor, decorin, which is a small proteoglycan. In a rabbit model of glaucoma filtration surgery the outcome of pre-operative and post-operative application of decorin was encouraging, as delayed increase of IOP and decreased fibrosis were observed after the surgery (Grisanti et al., 2005). SiRNA against TGF-β receptor II mRNA in vitro decreased the production of the TGF-β receptor II, as well as the production of fibronectin and the migration of human corneal fibroblasts. In vivo the same SiRNA molecule reduced inflammation and the deposition of ECM in an in vivo scarring animal model equivalent to the subconjunctival scarring after

the glaucoma filtration surgery (Nakamura et al., 2004). Additionally, SiRNA molecules against TGF-β 2 encapsulated in poly(D, L-lactide-co-glycolide) microspheres and applied after trabeculectomy with one injection in an experimental model led to 100% survival of the bleb for more than a month (Gomes dos Santos et al., 2006).

Future treatment approaches might target intracellular signaling events downstream of the TGF-β receptor. Within the cell, the main TGF-β signaling pathway runs through proteins that activate gene transcription: the Smad proteins. Of particular relevance is Smad3, which is essential for TGF-β induced production of extracellular matrix proteins (Chen et al., 1999; Massague, 1999). The use of Smad3 as a therapeutic target in immediate post-operative applications might prove beneficial (Leask and Abraham, 2004). Smad 7, which interferes with Smad2/3 activation, is another potential therapeutic target. Gene transfer of the Smad7 gene has been shown in animal models to have a protective effect against the development of lung, liver, and renal fibrosis (Schiller et al., 2004). Inhibiting the p38 intracellular pathway, where Smad proteins are not involved, with specific MAPK p38 inhibitors, resulted in an in vitro study in inhibition of TGF-β stimulated differentiation of human Tenon’s fibroblasts into myofibroblasts (Meyer-Ter-Vehn et al., 2006).

5. Antiproliferative agents

At present, 5-FU and MMC are the main antifibrotic agents used to modulate healing after trabeculectomy. β-Irradiation, which we have shown to have similar effects on growth arrest of fibroblasts (Khaw et al., 1991), significantly improved the success of GFS in a large African trial we conducted recently (Kirwan et al., 2006), and may also be useful in pediatric GFS (Akimoto et al., 1998). 5-FU and MMC have profound and long-term effects on cell proliferation in vitro and in vivo, and have revolutionized the long-term prospects of GFS (1989; Chen,

1983; Cheung et al., 1997; Shigeeda et al., 2006). 5-FU injections improve the success (no reoperation and IOP 21mmHg) of GFS from 50 to 73% at 1 year after surgery, and from 26 to 49% at 5 years (1996). Complications of 5-FU injections are mainly post-operative corneal epithelial toxicity and late-onset bleb leaks. Intraoperative 5-FU based on laboratory studies (Khaw et al., 1992, 1993) has been shown to significantly improve the success of patients undergoing first time GFS for at least 80 months without a significant increase in sight threatening complications (Khaw et al., 2004).

Because of its higher biological potency and long-lasting effects on fibroblasts, intraoperative MMC is favored in eyes at high risk of bleb failure, and carries a high success rate of 88.7% at 3 years after surgery (Cheung et al., 1997). Intraoperative MMC and post-operative subconjunctival injections of 5-FU are often combined to achieve maximum effects. However, in patients with low to moderate risk of failure, MMC does not offer advantages over intraoperative 5-FU (Singh et al., 2000) with appropriate follow-up care. In addition, serious complications are more common in MMC treated eyes, underlying the importance of carefully tailoring the choice of adjunct to the patient’s needs (Membrey et al., 2000). However, newer application techniques, such as the broad surface application of antifibrotic agents to promote diffuse rather than focal filtration, dramatically reduce the risk of complications (Cordeiro et al., 1997).

Photodynamic therapy with a diffuse blue light coupled with a photosensitizing agent to kill fibroblasts may also be another way to control surface area of treatment and modulate healing (Grisanti et al., 2000). Pilot clinical trials have shown promising results (Diestelhorst and Grisanti, 2002; Jordan et al., 2003). Other approaches to control proliferation include overexpressing genes which inhibit proliferation, such as p21 WAF-1/CIP-1 introduced via an adenovirus system (Perkins et al., 2002), antagonizing integrins and their receptors

(Paikal et al., 2000), or altering intracellular gene transcription (Akimoto et al., 1998).

6. Modulators of cell movement, and extra cellular matrix synthesis and contraction

Photographic analysis of blebs reveals that tissue contraction is a critical component of filtration surgery failure. We have a much more detailed understanding of the processes that occur during tissue contraction, and have recently been able to image cells and matrix simultaneously during the process of contraction (Figure 15.12). The matrix metalloproteinases (MMPs) are enzymes that degrade the extracellular matrix. Cell-mediated collagen contraction can be inhibited using MMP inhibitors (Daniels et al., 2003). In an experimental model of glaucoma surgery, the use of an MMP inhibitor leads to a dramatic reduction of scarring, with retention of normal tissue morphology. This action is equivalent to MMC, but without the deleterious side effects (Wong et al., 2003, 2005).

A number of agents can affect the cytoskeleton of the cell and hence inhibit migration. Taxol and etoposide (microtubulestabilizing agents) have been used in models of filtration surgery, and prolong bleb survival (Jampel and Moon, 1998). β-Aminopropionitrile and D-penicillamine interfere with molecular cross-linking of

x

FIGURE 15.12 Simultaneous cell and matrix imaging in conjunctival tissue

collagen, and there is experimental and clinical evidence that they may work in filtration surgery (Jampel et al., 1998). MMP inhibition also surprisingly results in a reduction of collagen synthesis in vitro (Daniels et al., 2003), which may help to explain the dramatic reduction in scar tissue formation in vivo (Wong et al., 2003).

F. Pterygium

Surgery is indicated to treat pterygia when the visual axis is threatened, when eye movements are restricted, or when the patient feels that the cosmetic appearance is unacceptable. Surgical excision down to bare sclera is straightforward, but local recurrences are common (30–89%) and tend to be more aggressive than the original lesion. A wide range of antifibrotic agents have been used to prevent pterygium recurrence, including β-irradiation, 5-FU, MMC, thiotepa, and daunorubicin. MMC in particular has proven to be very effective, but there have been reports of sight-threatening complications after its use, including secondary glaucoma, corneal edema and perforation, corectopia, iritis, cataract formation, scleral calcification and incapacitating photophobia and pain (Rubinfeld et al., 1992). Recent studies avoided complications by using single intraoperative applications of MMC at weaker concentrations, while maintaining high success rates (Young et al., 2004). Intraoperative MMC, together with AMT transplantation, had a good therapeutic effect after excision of primary, recurrent and pseudopterygia and no recurrences were observed up to 14 months following surgery (Tseng et al., 2005; Nakamura et al., 2006a). Daunorubicin and β-irradiation, though not in widespread use, seem to be similarly effective (Dadeya et al., 2002; Jurgenliemk-Schulz et al., 2004).

G. Ocular Cicatricial Pemphigoid (OCP)

The treatment of acute inflammatory episodes in OCP is based on the use of

systemic and topical anti-inflammatory and immunosuppressive agents. However, subconjunctival injections of 0.4 mg/ml MMC successfully prevented recurrences of synechiae and induced conjunctival quiescence in two small case series (Secchi and Tognon, 1996; Donnenfeld et al., 1999). Furthermore, intraoperative application of MMC in severe cicatricial ocular pemphigoid resulted in reduction of conjunctival inflammation and facilitated amniotic membrane that was transplanted to recreate a deep fornix after symblepharon lysis. Good visual acuity and corneal transparency results were described (Tseng et al., 2005; Nakamura et al., 2006b). TGF-β might play an important role in the development of fibrosis in OCP (Razzaque et al., 2003), but to date no trials specifically targeting this cytokine have been published.

H. Cataract Surgery

Cataract is the commonest cause of blindness and severe visual impairment in the world, with 135 million people affected. It can be cured by surgery and intraocular lens (IOL) implantation, but posterior capsular opacification (PCO) leads to renewed visual loss in up to 50% of cases over a 5 year period. This is a very significant problem in the developing world. Lens epithelial cells (LECs), the only cell lineage present in the capsular bag, mediate this type of fibrotic response. Following cataract surgery, residual equatorial LECs proliferate and migrate to form peripheral Elschnig pearls and Soemmering’s cataract. In addition, the cuboidal anterior capsular LECs transdifferentiate into myofibroblastlike cells on the inner surface of the lens capsule, and together with transformed equatorial cells lead to the clinical picture of fibrotic PCO (Apple et al., 2000) (Figure 15.13). Apart from cataract surgery in developing countries the great interest in PCO lies in the fact that controlling PCO may enable the development of a fully accommodative IOL. Many advances have been

FIGURE 15.13 Fibrotic posterior capsule opacification. Control of capsular fibrosis will be needed to make a fully accommodating lens viable

made in reducing the incidence of PCO, mainly through improvements in surgical techniques and IOL technology. Pharmacological interventions have so far shown promise in in vitro and in vivo studies, but translation to human use is complicated by safety concerns about collateral damage to intraocular tissues.

1. Surgical technique

Adequate removal of cortical matter reduces PCO, as does in-the-bag fixation and good centration of the IOL, as they enhance the barrier effect of the optic part of the lens implant. An intact continuous curvilinear capsulorhexis (CCC) slightly smaller than the diameter of the IOL optic is regarded as a vital step in the prevention of PCO, halving the relative area of PCO and minimizing capsular wrinkling (Hollick et al., 1999).

(a) IOL technology – Convexity of the posterior surface of the IOL optic and angulation of the haptics create a tight contact between the IOL optic and the posterior capsule. Together with the “shrink wrapping” of the capsule around the IOL, the resulting “no space, no cell” situation prevents LEC proliferation. In addition, the square edge of the optic of some lenses

induces a sharp bend in the posterior capsule and acts as a barrier to LEC migration onto the visual axis (Nishi and Nishi, 1999). The importance of biocompatibility of IOL materials in the prevention of PCO is still controversial. In IOLs with square edge design, different IOL materials have similar PCO scores (Nishi et al., 2004).

(b)Sealed capsular irrigation/physical destruction of LECs – Sealed capsular irrigation is a promising new technique to prevent PCO (Maloof et al., 2003). A special device designed by Maloof is used to form a seal around the opening of the CCC. In theory, any pharmacological agent deemed effective at preventing PCO can be irrigated into the bag without collateral damage to other ocular tissues. Currently clinical trials are under way, using distilled deionized water, based on an idea by Crowston to osmotically disrupt the residual LECs in the sealed capsular bag. Other ways to physically destruct residual LECs include cryotherapy to the capsular equator, which reduces central PCO by 20%, but is associated with collateral damage to tissues and increased inflammation. The use of bipolar diathermy to the capsular equator leads to permanent eradication of PCO in the rabbit, but this procedure has not been reported in humans.

(c)Photodynamic therapy – Photodynamic therapy (PDT) involves the sensitization of cells in a target tissue using a photosensitizing agent followed by light irradiation to selectively destroy them. Rose bengal mixed with sodium hyalorunate, injected into the capsular bag after lens extraction and irradiated for 2 minutes, effectively reduces PCO (Koh et al., 2002).

2. Anti-inflammatory agents

Steroids and non-steroidal antiinflammatory drugs (NSAIDs) are in routine clinical use to control the post-oper- ative inflammation after cataract surgery. Increased inflammation probably contributes to the higher incidence of PCO

observed in patients with uveitis. However, in a small prospective randomized double masked trial comparing dexamethasone 0.1%, diclofenac 0.1%, and saline 0.9% drops for 3 weeks post-operatively, no improvement in PCO scores and Nd:YAG rate were observed at 2 years (Zaczek et al., 2004). It is interesting that the Nd:YAG rate was actually lower and statistically different in the placebo group compared to the dexamethasone and the diclofenac group in the 4 year follow-up, although Nd:YAG rate cannot be considered as an accurate marker of the development of after-cata- ract POC (Laurell and Zetterstrom, 2002). A recent study has shown that dexamethasone promotes the survival and accumulation of abnormal cells across the lens capsule, and both dexamethasone and diclofenac increase the concentration of collagen I; these pathological changes enhance the possibility and subsequently the severity of PCO (Symonds et al., 2006).

In another study, the comparison of postoperative topical application of diclofenac 0.1% or dexamethasone 0.1% after cataract surgery revealed slightly reduced levels of PCO at the diclofenac group compared to the dexamethasone treated eyes, but the difference was not statistically significant (Barequet et al., 2002). Additionally, previous in vitro experiments have shown the inhibitory effect of diclofenac in human lens epithelial cell proliferation in a dose dependent manner (Cortina et al., 1997). Other in vivo studies have also compared diclofenac therapy with other therapeutic approaches, like betamethasone phosphate (Tsuchiya et al., 2003) and ketorolac trimethamine (Flach and Dolan, 2000), no significant differences were found as regards the prevention of PCO. It has been proposed that only a single intraoperative sub-Tenon’s capsule injection of 40mg triamcinolone acetonate has comparable results with 1% prednisone eye drop treatment (Paganelli et al., 2004). Higher intraocular doses of dexamethasone and diclofenac, achieved by injection during hydrodissection and subsequent capsular

irrigation, reduced, but did not prevent, PCO in a rabbit model (Inan et al., 2001). The evaluation of heparin coating of IOLs is so far inconclusive. Cyclosporin A, which inhibits LEC proliferation in vitro, has not been investigated in vivo.

3. Antiproliferative agents

Mitomycin C (MMC), applied as a subconjunctival adjunct during combined trabeculectomy and cataract surgery, reduces PCO requiring an Nd:YAG capsulotomy (Shin et al., 1998). All other published data about MMC in cataract surgery have been acquired in animal models. In the rabbit, MMC delivered by hydrodissection and capsule irrigation at 0.5 mg/ml or 0.04 mg/ ml for 3 minutes significantly reduces PCO, with a greater inhibitory effect on cell proliferation than on fibrosis (Inan et al., 2001). Mixing of MMC sodium hyaluronate has similar effects. Rabbit experiments with 5-FU have shown conflicting results. Daunorubicin, daunomycin, ethylenediaminotetraacetic acid (EDTA), salmosin, colchicine, retinoic acid and saporin have all shown some promise in in vivo studies. Their main potential problem is intraocular toxicity which is well recognized and dose dependent. However, assessment of inflammation and corneal endothelial cell loss in the rabbit is difficult, as there often is an intense fibrinous response, and as rabbit endothelium is able to regenerate. Currently no clinical use of these drugs to prevent PCO is practiced. Octreotide, a synthetic analogue of somatostatin, has been shown to inhibit in vitro the proliferation of human epithelial lens cells, but further studies have to be done (Baldysiak-Figiel et al., 2005) (Figure 15.14).

Two clinical studies have investigated specific antibodies against LECs, coupled with Ricin A (Clark et al., 1998; Meacock et al., 2000). Internalization by the target cell causes cell death by inhibiting protein synthesis. The results showed PCO reduction, but increased levels of early post-operative

FIGURE 15.14 Posterior capsular opacification in an experimental model

uveitis requiring increased steroid administration. This has inhibited further development. Adenoviral transfer of the herpes simplex virus thymidine kinase gene (HSV-tk) into proliferating LECs, followed by induction of cell death of transduced cells by gancyclovir treatment, successfully reduces PCO in vivo and in vitro (Malecaze et al., 1999). However, this is associated with intense fibrinous reaction, corneal edema and endothelial cell loss in rabbits.

4. Modulators of matrix remodeling and contraction

MMP inhibition using the broad spectrum inhibitor Ilomastat (GM6001) reduces both the migration of LECs and capsular wrinkling in an in vitro capsular bag culture model (Wong et al., 2004). Minoxidil, which inhibits lysyl hydroxylase, resulting in reduced collagen crosslinking, also reduces LEC proliferation and migration.

5. Growth factor modulators

In an in vitro human lens capsule culture model, a monoclonal antibody to human TGF-β2 effectively inhibited capsular wrinkling and myofibroblast transformation, but did not affect cell proliferation (Wormstone

et al., 2002). α νβ 6 integrin has been supported as the main activator of TGF-β1 in the human lens capsule after cataract extraction, and could become a new therapeutic target for PCO prevention (Sponer et al., 2005).

After injury or cataract extraction, lens epithelial cells are transformed to myofibroblasts (epithelial–mesenchymal transition (EMT)) that express αSMA, after which the after-cataract opacity occurs. TGF-β2 that exists in high levels in aqueous humor activates Smad3/4 signaling in lens epithelial cells and it has been found that this signaling is important for EMT. Adenoviral transfer of Smad7 which inhibits phosphorylation and subsequently activation of Smad 3 has been shown to prevent EMT and capsular fibrosis in an animal model (Saika et al., 2004a). Additionally, it is proposed that development of small molecule inhibitors of Smad3 could have a beneficial effect in the prevention of fibrosis (Roberts et al., 2006).

Bone morphogenic protein-7 (BMP-7), a member of the TGF-β superfamily, has been discovered to reduce EMT in renal epithelial cells by antagonizing TGF-β (referred to in Saika et al., 2006). Also decreased expression of BMP-7 occurs in experimental chronic pyelonephritis which is characterized by severe fibrotic changes (Biyikli et al., 2005). BMP-7 has been supported to slow down fibrosis by inducing the expression of inhibitors of differentiation 2 and 3 (Id2 and Id3). Adenoviral transfer of BMP-7, Id2 or Id3 has been applied in the lens epithelium of an animal model with successful results, as the EMT was suppressed in all cases indicating a potential use in the prevention of capsular fibrosis (Saika et al., 2006).

I. Age-Related Macular Degeneration

(AMD)

AMD is the leading cause of visual impairment and blindness in the elderly in the developed world, and is estimated to affect up to 25 million people. Neovascular

FIGURE 15.15 Wet age-related -macular degeneration

– the scarring component is a critical component of the permanent visual loss

AMD is characterized by choroidal neovascularization (CNV) that invades the subretinal space leading to photoreceptor damage and loss of central vision, the vessels ultimately being replaced by a fibrovascular scar in the long term. The scarring response is a central component of the damage that results in permanent visual loss and modulation of this process may fundamentally alter long-term outcome (Figure 15.15).

1. Anti-angiogenesis therapy

AMD is managed with a variety of treatments including transpupillary thermotherapy (Algvere et al., 2003) and photodynamic therapy (PDT), while radiotherapy has demonstrated only a modest treatment benefit (Marcus et al., 2004). PDT showed statistically significant visual benefit at 1–2 years of follow-up in eyes with AMD-related subfoveal predominantly classic CNV (1999). PDT in combination with verteporfin (Visudyne) safely reduces the risk of moderate and severe vision loss in patients with subfoveal occult-only CNV at 2 years (Bressler 2001, 2002; Azab et al., 2005). More recently, a pilot study of PDT and intravitreal triamcinolone acetonide demonstrated improved visual acuity with reduced treatment frequency; however, elevated IOP seems to be the most frequent early side effect of treatment (Spaide et al., 2003).