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1From the Wound Healing and Glaucoma Research Units, Institute of Ophthalmology and Moorfields Eye Hospital, London, United Kingdom; and 2Cambridge Antibody Technology, Cambridge, United Kingdom.
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Abstract |
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METHODS. In a randomized, controlled, masked-observer study, after modified glaucoma surgery, 48 rabbits were randomly allocated to receive a postoperative course of seven subconjunctival injections of CAT-152 (1 mg/mL), 5-FU (50 mg/mL), or no treatment. Bleb characteristics, the presence of subconjunctival drainage, and local reaction to treatment were assessed. Animals were killed on days 10, 21, and 30. Immunohistochemistry, histologic staining and electron microscopy were performed to demonstrate the mechanism of CAT-152–mediated effects on the extracellular matrix.
RESULTS. CAT-152 significantly improved surgical outcome (log rank test, P < 0.001) and reduced subconjunctival collagen deposition (P < 0.01) compared with 5-FU and control. Median bleb survival was increased in the CAT-152 group (23.5 days) compared with the 5-FU (20 days) and control (16 days) treatment groups. CAT-152 treatment improved bleb morphology (P < 0.05) and was well tolerated. 5-FU prolonged the duration of corneal epitheliopathy (P < 0.01).
CONCLUSIONS. Postoperative administration of CAT-152 significantly improved surgical outcome, reduced subconjunctival scarring, and minimized the risk of corneal side effects compared with the anti-scarring agent 5-FU. These findings suggest that CAT-152 may offer therapeutic benefit as a postoperative agent to prevent subconjunctival scarring after glaucoma filtration surgery.
Of all the growth factors involved in the wound-healing cascade, TGF-ß has been shown to be one of the most potent stimulators of scarring in the eye and is involved in the pathogenesis of cataract, proliferative vitreoretinopathy, and conjunctival scarring.25 26 27 28 TGF-ß2, the most predominant of the three mammalian isoforms in the eye, is the most potent growth factor in the aqueous at stimulating conjunctival fibroblast function. Elevated levels of this isoform are found in the aqueous of glaucomatous eyes compared with normal eyes.29 30 31 32 33 34 These findings suggest that neutralizing the effects of TGF-ß2 may reduce conjunctival scarring after glaucoma filtration surgery.
CAT-152 (lerdelimumab) is a novel human monoclonal antibody that was isolated and developed in vitro by the technique of antibody phage display. It displays high affinity and specificity for the active form of TGF-ß2 and has been designed for therapeutic use. We have demonstrated in vitro that CAT-152 inhibits TGF-ß2–induced human Tenon’s fibroblast migration and proliferation. Furthermore, in our in vivo model of aggressive conjunctival scarring we have shown that subconjunctival administration of CAT-152 at the time of glaucoma surgery and in the immediate postoperative period successfully improves surgical outcome, reduces subconjunctival fibrosis, and is safe and well tolerated.26 In the first human trial of CAT-152 in patients undergoing trabeculectomy, good tolerance and safety were reported with a treatment regimen of both intra- and postoperative injection.35 A multicenter phase II study is under way.
However to date, we have made no assessment of isolated postoperative application of CAT-152. Using the same model of glaucoma filtration surgery, the present study was designed to determine whether postoperative application alone of CAT-152 can improve bleb survival and to compare the effectiveness of CAT-152 with the currently used postoperative antiscarring agent 5-FU.
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Postoperative Subconjunctival Administration of CAT-152 after Rabbit Filtration Surgery
The animals were randomly allocated to receive a postoperative course of seven subconjunctival injections (100 µL) of CAT-152 (1 mg/mL), 5-FU (50 mg/mL), or no treatment. The subconjunctival injections were given on days 2, 3, 4, 7, 9, 11, and 14 after surgery (day 0) under topical anesthesia (proxymetacaine hydrochloride 0.5% eye drops, 1 drop per eye), using a 30-gauge needle (Myjector 100u; Terumo, Tokyo, Japan). CAT-152 was injected 5 mm behind the limbus at the nasal margin of the superior rectus muscle. 5-FU was administered 180° from the site of surgery. The rationale for the injection site selection was in keeping with the method used in the original studies of subconjunctival 5-FU in glaucoma filtration surgery and widespread clinical practice.5 6 Injections were given by an independent clinician, because the different injection sites for each treatment precluded masking.
Clinical Evaluation of Postoperative CAT-152 Effects in Filtration Surgery
Baseline observations were performed before glaucoma filtration surgery. Measurement of intraocular pressure in both eyes was made with a handheld tonometer (Tonopen; Mentor, Norwell, MA) after topical instillation of 0.5% proxymetacaine HCl eye drops, 1 drop per eye, with a mean reading of three recordings being documented per time point. The conjunctival appearance and the drainage area were observed. All animals were examined by a masked observer at set times after surgery. Assessment of both eyes (contralateral untreated eye used as control) was made daily from days 0 to 4 and thereafter at regular periods, at least twice weekly, until death. Bleb characteristics, including length, width, and height, were measured with calipers, and intraocular pressure was recorded. Previous experience with this surgical model has identified a small area of avascularity (<3 mm) that appears in the nasal conjunctiva. This is a transient clinical observation and appears to develop independently of any treatment given. The incidence of avascularity was noted (normal vascularization, 0; avascular region, 1). The drainage bleb vascularity characteristics were assessed independently from this area and were graded by dividing the conjunctival areas into quadrants (superior, nasal, and temporal) and scoring the appearance (0, avascular; +1, normal vascularity; +2, hyperemic; and +3, very hyperemic). Slit lamp examination was performed to identify both anterior chamber activity (0, quiet; 1, cells; 2, fibrin; and 3, hypopyon) and anterior chamber depth, which was recorded as deep (+2), shallow (+1), or flat (0).
An assessment of the duration of corneal epitheliopathy was made after topical installation of lignocaine fluorescein into the left eye and was graded according to the area of the cornea affected (0, nil; 1, <25%; 2, < 50%; 3, < 75%; 4, < 90%; 5, up to 100%).
Data Evaluation
Bleb survival was taken as the primary efficacy end point in the analysis of CAT-152 in rabbit filtration surgery. Bleb failure was defined as the appearance of a flat, vascularized, and scarred bleb in the presence of a deep anterior chamber. Kaplan-Meier and log rank statistics were used to compare treatment groups in bleb and intraocular pressure failure (defined as the return of the intraocular pressure in the surgical eye to baseline level). Bleb area and height, anterior chamber depth and activity, and conjunctival vascularity per quadrant were all analyzed with a repeated-measures procedure and the generalized linear model (SPSS; SPSS Inc., Chicago, IL). This allowed comparison of treatment groups over the whole study period, using between-subject tests. Duration corneal epitheliopathy and avascularity were analyzed using the Kruskal-Wallis test and the Dunn test. Finally, intraocular pressure was analyzed with the multivariate analysis of variance with Bonferroni’s modification, to compare differences between treatments and the effects of time and treatment. The level of significance applied to the statistical analysis was P < 0.05.
The experiment was performed in two phases. In the first phase, 24 rabbits underwent the experimental protocol and were killed on day 30. Observational analysis of the effect of CAT-152 on glaucoma surgery and histologic analysis of the ocular tissue were performed on these animals. In the second phase, the experiment was repeated using an additional 24 rabbits which were killed on days 10 (n = 12) and 21 (n = 12) to provide histologic information at these earlier time points.
Analysis of Rabbit Tissues
Histology
On days 10, 21, and 30 animals were killed with a lethal intravenous injection of phenobarbitone, and the tissues were processed for histology. Both eyes were enucleated. The upper lid was left intact, attached to the globe to preserve the architecture of the superior fornix and conjunctival tissues around the drainage site. All the eyes were fixed in 10% buffered formal saline for 24 hours, stored in 70% alcohol and fixed in paraffin wax. Sequential 5-µm sections of the operative wound site were prepared, and histologic staining was performed to demonstrate cellularity and extracellular matrix deposition including: hematoxylin and eosin (for total cellularity); picrosirius red and Gamori’s trichrome (for scar formation, collagen density, and orientation), oxidation aldehyde fuchsin (elastic fibers), 
-smooth muscle actin immunohistochemistry (myofibroblast phenotype identification), and proliferating cell nuclear antigen (PCNA) immunohistochemistry (recent cell division). These parameters were graded by two masked observers, using a modified scoring system (based on that described by Shah et al.36 ) which incorporated visual reference standards for each grade on a scale of 0 to 4: 0, same as control eye; 1, 1%–25% of control; 2, 26%–50% of control; 3, 51%–75% of control; 4, more than 75% of control. All surgical eyes were compared with contralateral, nonsurgical eyes. Mean scores of histologic parameters for each treatment group per time point were calculated, and this semiquantitative data were analyzed by analysis of variance (SPSS).
Electron Microscopy
We also compared the electron micrograph characteristics of two rabbit eyes treated with postoperative CAT-152 and two untreated eyes. The surgical technique and postoperative injection procedure used in the main study were repeated. Animals were killed on day 10, and the operative wound site was processed for both scanning and transmission electron microscopy. The day 10 time point was selected to provide maximum information on the comparative bleb appearances between treated and untreated animals before the onset of bleb failure.
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Effects on Filtration Surgery
CAT-152 significantly improved glaucoma filtration surgery outcome in this animal model of aggressive postsurgical scarring. CAT-152 significantly prolonged bleb survival compared with the 5-FU group and the untreated control group, as shown in the Kaplan-Meier survival curve in Figure 1A (log rank test; P = 0.0009). The rate of bleb failure and percentage survival in each treatment group is shown in Table 1 . All the blebs in the control and 5-FU groups had failed by day 22; however, 62.5% of the CAT-152 treatment group had functioning blebs. By day 30 all but one of the operations had failed; the only animal with a functioning bleb had received CAT-152. The median (range) survival rates were 23.5 (20–30), 20 (16–22), and 16 (14–21) days in the CAT-152, 5-FU, and control groups, respectively.
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Local reaction to treatment was assessed by the degree of anterior chamber inflammation and conjunctival vascularity. No significant difference was found between treatment groups for either of these indicators of the inflammatory response (vascularity, superior P = 0.402, temporal P = 0.434, nasal P = 0.668; anterior chamber inflammation P = 0.430).
The depth of the anterior chamber was assessed as an indirect indicator of drainage. On day 1 after surgery the anterior chamber was flat in most of the animals. Over the next 7 days the anterior chamber gradually deepened. No significant difference was found between the treatment groups in the time taken for the anterior chamber to deepen (P = 0.302).
Histologic Effects
CAT-152 treatment reduced scarring at a microscopic level. The greatest histologic difference between treatment groups was seen on day 10 (Fig. 3) . At this time point, total scar formation, as judged by the staining characteristics of picrosirius red, was significantly reduced by CAT-152 treatment (P = 0.01, Figs. 3D 3E 3F , Fig. 5 ). In addition, CAT-152 significantly reduced the population of cells expressing -smooth muscle actin, indicating less fibroblast differentiation into the myofibroblast phenotype (P = 0.01; Figs. 4 5 ). No other significant differences were found between treatment groups in total cellularity, elastic fiber deposition, or proliferating cell nuclear antigen (PCNA) at day 10. By day 30 subconjunctival scarring at the wound site consisted characteristically of densely packed collagen and fibroblasts. In contrast, the only surviving CAT-152–treated bleb showed much looser architecture with visible conjunctival bleb formation (Fig. 6) .
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Discussion |
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The anti-metabolites 5-FU and MMC are currently the backbone of anti-scarring treatments. The intraoperative regimen of mitomycin C has gained favor due to the convenience of a single treatment and the delivery of lower intraocular pressure in certain eyes.37 However, even short exposure to MMC results in local irreversible tissue destruction.12 20 The advantage of CAT-152 over MMC and 5-FU lies in its more physiological method of action, potentially providing long-term titratable intraocular pressure control, while maintaining normal tissue architecture in the absence of side effects.
Rabbit models exhibit an exaggerated healing response compared with human tissue. Demonstration of efficacy in such models is therefore likely to be reproduced in the clinical setting. The surgical model of glaucoma surgery used in this study localizes scarring to the level of the conjunctiva. This is achieved by maintaining a permanent fistula to drain aqueous into the subconjunctival wound site. Experience has shown that intraocular pressure is not a reliable indicator of filtration in this model of glaucoma surgery. Furthermore, in this study no significant differences were detected in IOP between groups. This can be explained by the fact that basal preoperative intraocular pressure in this model of glaucoma filtration surgery is within the normal range (this is a model of subconjunctival scarring, not of glaucoma). Given this, bleb failure rather than intraocular pressure has always been defined as the primary outcome variable representing failure of surgery in this model.
We found that isolated postoperative 5-FU had the same bleb survival end point as the control in this experiment. This model exhibits an extremely aggressive scarring response that may explain this finding. However, conversely, all the treatments that have improved bleb survival in this model have worked in a subsequent clinical setting.12 26 Only a limited number of studies have looked at efficacy of 5-FU as an isolated postoperative agent in animal experiments. Doyle et al.38 compared the effect of five postoperative injections of 5-FU with single intraoperative 5-FU, and combined intra- and postoperative 5-FU in rabbit filtration surgery. Control animals received intraoperative distilled water only. No significant difference was shown in bleb survival between postoperative 5-FU treatment and control in this study. However, what can be seen in the survival curve from Doyle et al. and our data is that postoperative injections of 5-FU appear to shift the survival curve to the right of the control at the early time points, without affecting the final end point.
The landmark Fluorouracil Filtering Surgery Study6 represents the only definitive report in which postoperative injections of 5-FU were shown to improve surgical outcome. In this study, 21 subconjunctival injections were administered: two injections per day on days 1 to 7 after surgery and one injection per day on days 7 to 14 after surgery. If this number of injections had been used, we may well have shown efficacy of postoperative 5-FU in this model. When designing the protocol we chose a postoperative regimen that more closely reflected current clinical subconjunctival 5-FU use.
Anterior chamber depth was included in the observations as an indirect indicator of the drainage of fluid through the tube into the subconjunctival space. The rabbit anterior segment is very crowded with a very large lens and small anterior chamber. Therefore, in practice, this measurement was fairly difficult to grade. In most of the animals, the anterior chamber was flat on day 1 and gradually deepened over the next 7 days. No significant difference was found between the treatments in the time taken for the anterior chamber to deepen. Overall, this observation may not be as true a representation of anterior fluid dynamics as had been originally anticipated.
Ideally all subconjunctival injections would have been given at the same site. In all the preclinical and clinical studies of CAT-152 in glaucoma filtration surgery, the drug has been administered by subconjunctival injection in the superior nasal quadrant adjacent to the drainage bleb. The same injection site was therefore selected in this study. Clinically, the site of subconjunctival 5-FU administration varies, depending on clinical preference. Some clinicians favor injections adjacent to the bleb and some 90° and others 180° from the operation site. The main concern with injecting 5-FU adjacent to the bleb relates to the possibility of intraocular penetration, because the pH of 5-FU is 9. We decided to base our study protocol on the pioneering studies of 5-FU in glaucoma surgery performed by the Fluorouracil Filtering Surgery Study group. In this study 5-FU was injected 180° from the site of surgery.5 Given the different injection sites for the CAT-152 and 5-FU groups a universal injection site for a control vehicle was not possible and therefore the no-treatment control group was introduced.
Aqueous flow bathes the wound and provides a unique and changeable environment that influences postoperative healing. Of all the growth factors in the aqueous TGF-ß is the most potent stimulator of human Tenon’s fibroblast activity.29 Latent TGF-ß2 is produced by tissues within the eye (ciliary body and trabecular meshwork) before activation by plasmin and thrombospondin released from blood components.32 39 40 41 Aqueous humor in glaucomatous eyes contains increased level of TGF-ß2.33 After glaucoma surgery, elevated levels of activated TGF-ß2 at the wound site are therefore likely to be related to aqueous concentration, the flow of aqueous, and breakdown of the blood aqueous barrier. In addition TGF-ß also displays the ability to autoinduce its own production thereby initiating a perpetuating cascade of activation.42 43 In a mouse model of conjunctival scarring, peak levels of TGF- ß2 have been shown at the wound site at day 7.25 Without treatment, the rabbit model fails by day 14. We propose that isolated postoperative administration of CAT-152, between days 2 and 14 in this model, can still neutralize subconjunctival TGF-ß2 levels at the wound site below a threshold required to mediate downstream effects on the extracellular matrix.
Histologic analysis of the rabbit tissues showed that CAT-152 significantly reduced subconjunctival collagen deposition compared with both the 5-FU and control groups. CAT-152 also significantly reduced the population of cells expressing -smooth muscle actin, indicating an inhibition of fibroblast differentiation into the myofibroblast phenotype. Myofibroblasts are specialized fibroblasts that play an important role in wound healing. They are present transiently during tissue repair and are thought to generate the contractile force that is integral to normal wound closure. Excessive or abnormal contraction of granulation tissue leads to pathologic scarring. Fibroblast differentiation into the myofibroblast phenotype, characterized by the expression and assembly of -smooth muscle actin into stress fibers, is modulated by cytokines.44 TGF-ß has been shown to be a direct inducer of the myofibroblast phenotype and is capable of upregulating -smooth muscle actin, both in vivo and in vitro.45 46 We have shown in vitro that CAT-152 significantly inhibits TGF-ß2–stimulated collagen contraction.26 These observations suggest that the beneficial effects of CAT-152 in glaucoma surgery may be mediated by a reduction in TGF-ß2–induced collagen production and contraction.
Repeated injections of subconjunctival 5-FU in clinical practice are known to cause corneal epitheliopathy. Similar findings were associated with the use of 5-FU in this study. The more serious complications of avascular bleb formation, bleb-related infection and chronic hypotony reflect the cytotoxic mechanism of action of antiproliferative agents. CAT-152 treatment provides a more physiological alternative. We present evidence that postoperative TGF-ß2 inhibition with this novel monoclonal antibody can prevent failure of experimental glaucoma surgery by inhibition of subconjunctival scarring. This represents a potentially useful development in the prevention of late surgical failure and may provide us with a safer therapy to maintain maximal IOP control in the longer term.
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Footnotes |
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Submitted for publication September 24, 2002; revised February 4, 2003; accepted March 11, 2003.
Disclosure: A.L. Mead, Cambridge Antibody Technology (F); T.T.L. Wong, None; M.F. Cordeiro, None; I.K. Anderson, Cambridge Antibody Technology (E); P.T. Khaw, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Anna L. Mead, Wound Healing Unit, Department of Pathology, Institute of Ophthalmology and Moorfields Eye Hospital, 11-43 Bath Street, London EC1V 9EL, UK; a.mead{at}ucl.ac.uk.
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