DEVICE FOR DELIVERY OF ANTIFIBROTIC AGENTS & METHOD (Docket No. 9602a)

RELATED PATENT APPLICATIONS & INCORPORATION BY REFERENCE

This application, is a PCT application which claims the benefit under 35 USC 119(e) of U. S. Provisional Patent Application No. 60/808,446, entitled "DEVICE FOR DELIVERY OF ANTIFIBROTIC AGENTS & METHOD," filed May 25, 2006. This related application is incorporated herein by reference and made a part of this application. If any conflict arises between the disclosure of the invention in this PCT application and that in the related provisional application, the disclosure in this PCT application shall govern. Moreover, any and all U. S. patents, U. S. patent applications, and other documents, hard copy or electronic, cited or referred to in this application are incorporated herein by reference and made a part of this application.

DEFINITIONS

The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. The words "consisting/' "consists of," and other forms thereof, are intended to be equivalent in meaning and be closed ended in that an item or items following any one of these words is meant to be an exhaustive listing of such item or items and limited to only the listed item or items.

BACKGROUND

Subconjunctival fibrosis is a major complication associated with eye surgery. While several surgical procedures and medical implants are available to treat a number of diseases and disorders of the eye, resultant tissue fibrosis often leads to unsatisfactory post-operative outcomes. Glaucoma is a multifactorial optic neuropathy in which there is a characteristic acquired loss of retinal ganglion cells and atrophy of the optic nerve.

Major risk factors for glaucoma include elevated intraocular pressure, positive family history, African heritage, and older age. Reduction and control of the intraocular pressure remains the main stay of treatment in the management of glaucoma. The increase in the intraocular pressure is thought mainly due to outflow resistance. Elevated intraocular pressure can be reduced pharmacologically, following surgical filtration procedures (GFS) or by the use of glaucoma drainage devices (GDD). The success rate of these operations is about 70-80% at one year and 40-50% at five years. Excessive postoperative fibrosis at the wound site significantly reduces surgical success following glaucoma surgery. Pharmacological attempts to prevent fibrosis following glaucoma surgery have thus far proven unsatisfactory. Topical steroids have been the mainstay for suppressing the inflammatory /fibrous reaction that follows the various glaucoma filtering surgeries, but with variable results. The use of antifibrotic medications or agents such as, for example, 5-fluorouracil (5-FU) and mitomycin-C (MMC), help prevent the postsurgical fibrosis and improve the surgical outcomes following trabeculectomy operation (85-90% at one year compared to 70% without these agents). However, the current technique of delivering the MMC or 5-FU in the form of soaking a small wedge of sponge in a given concentration of the drug and applying to the operation site for variable time periods before washing the drug from the surgical site leads to inconsistent results. The inflammatory /fibrous reaction that follows an operation implanting a glaucoma drainage device differs from that of trabeculectomy in that the inflammatory reaction is ongoing probably due to the biomaterial of the glaucoma drainage device. This leads to bleb encapsulation and elevated intraocular pressures in both the short term, that is, in the hypertensive phase and in the long term leading to elevated intraocular pressure and failure of the operation. The failure rate for the glaucoma drainage devices has been reported at 10% per post-operative year, thus reaching 50% failure rate in 5 years. Antifibrotic agents have also been used during glaucoma drainage device implant operations, but with variable and questionable results. Topical application of such medications at the time of surgery appears to be less effective 7-9 mm from the limbus for such implant operations compared to the effects seen at the limbus during a trabeculectomy operation. It is therefore possible that one-time application of these agents may not be sufficient to decrease the chances of long-term fibrous reaction that occurs following an implant operation.

In the recent past, non-penetrating filtering procedures like the viscocanulostomy and deep sclerectomy with or without a collagen implant are gaining popularity as a means of controlling the intraocular pressure, at the same time avoiding the immediate postoperative complications of a penetrating filtering procedure like the trabeculectomy such as hypotony, flat anterior chamber, choroidal effusions etc. The success rate of these operations, however, is less than 50% at 12 months because of scar tissue formation. The inflammatory /fibrous reaction that follows implant operations defers from that of the trabeculectomy operation in that the inflammatory reaction is on going, probably due to the biomaterial that makes up the implanted glaucoma drainage device and the micro-motion exhibited by an end-plate with ocular movements and the presence of the aqueous medium, in the subconjunctival space and a lot of other factors that are not well understood at the present time. In glaucoma filtration non-penetrating surgery (NPS), excessive postoperative scarring at the wound site significantly reduces surgical success. Pharmacological attempts to prevent fibrosis following glaucoma surgery have thus far proven unsatisfactory. Topical steroids have been the mainstay for suppressing the inflammatory /fibrous reaction that follows the various glaucoma filtering surgeries, but with variable results. The use of antifibrotic agents such as, for example, 5-FU and MMC help prevent the postsurgical fibrosis and improve the surgical outcomes. However, the current technique of delivering 5-FU and MMC in the form of soaking a small wedge of sponge in a given concentration of the medication and applying to the operation site for a variable time period before washing the medication from the surgical site leads to inconsistent results. Therefore, there is a need for more specific treatments directed against the inflammatory /fibrous reaction both in terms of the ability to deliver the medication in a dose dependent and predictable fashion. Desirably such treatment may also be able to deliver new antifibrotic agents having less associated problems than the current ones in use.

SUMMARY

My ophthalmological device and method of inhibiting inflammatory cell proliferation have one or more of the features depicted in the illustrative embodiments discussed in the section entitled "DETAILED DESCRIPTION OF SOME ILLUSTRATIVE EMBODIMENTS."

My ophthalmological device and method provide sustained, slow release over a prolonged period of an antifϊbrotic agent that decreases fibrosis formations either around an implanted ocular device or within and around an area of surgical intervention. My ophthalmological device includes an implant member carrying the antifibrotic agent that is inserted into a wound in an eye produced by surgery. The member may comprise a porous or non-porous material and it may or may not be bio-degradable. The slow release of the antifibrotic agent from the member retards the fibrous reaction and thus enhances the success rate of the operation. The claims that follow define my invention; however, without limiting the scope of my invention as expressed by these claims,, in general terms, some, but not necessarily all, of the features of my device and method are: One, the antifibrotic agent is released slowly over a prolonged period when the implant member is inserted into a wound in an eye. The prolonged period may be in excess of 1 week and may be released at a rate of substantially from 0.03 to 0.09 milligrams per hour. Two, the surface of the implant member may be coated with the antifibrotic agent, and this antifibrotic agent may be selected from the group consisting of Mitomycin-C, 5-Flurouracil, Rapamycin, a transforming growth factor (TGF) antibody, a form of corticosteroid, an immunesuppresive agent, and heparin. The transforming growth factor (TGF) antibody may be selected from the group consisting of TGF-B 2 monoclonal antibody, Interlukin 1 or 6 antibody, and a cytokine antibody. The form of corticosteroid may be dexamethasone, and the immunesuppresive agent may be selected from the group consisting of cyclosporin and FK57. Three, the implant member may comprise a porous polymeric material, which may have a width substantially from 4 millimeters (mm) to 15 mm, a length substantially from 4 mm to 15 mm, and a thickness substantially from 0.25 mm to 0.50 mm. This porous polymeric material may be a P-HEMA matrix and it may be bio-degradable. For example, the bio-degradable material may be selected from the group consisting of collagen, chitosan, polysaccharides, proteins, polylactides, a polylactide and glycolic acid copolymer, polyanhydrides, and polyesters. In one embodiment, my ophthalmological device may comprise a distribution plate including the porous polymeric member. The distribution plate may have a tube extending from it having a free end adapted to be inserted into the intraocular chamber of the eye.

In another embodiment, my ophthalmological device may comprise a pressure responsive valve including a tube extending from the valve and having a free end adapted to be inserted into the intraocular chamber of the eye. The aqueous medium in the intraocular chamber flows through the tube and over or through the implant member to wash the antifibrotic agent from the member and into the wound. Alternately, my ophthalmological device may be a sheet or membrane that is inserted into the wound created during eye surgery. My method delivers the antifibrotic agent into a wound produced by surgical intervention in the eye. For example, an implant member carrying the antifibrotic agent may be inserted into the subconjunctival space, and the agent is released over a prolonged period in a slow and sustained fashion to reduce postoperative fibrosis. The prolonged period may be in excess of one (1) week, for example, from about 1 to about 3 weeks. The antifibrotic agent may be released at a rate of substantially from 0.03 to 0.09 milligrams per hour. My ophthalmological device may be used where the free end of the tube is inserted into an intraocular chamber of the eye during surgery. The implant member may be inserted locally in a limbal pocket of the eye to inhibit limbal scar tissue from obstructing the eye's deep scleral pocket or canulostomy opening in deep sclerectomy/ viscocanulostomy eye operations. The eye surgery may a trabeculectomy. These features are not listed in any rank order nor is this list intended to be exhaustive.

DESCJRIPTION OF THE DRAWING

Some embodiments of my device and method will now be discussed in detail in connection with the accompanying drawing, which is for illustrative purposes only. This drawing includes the following figures (Figs.), with like numerals indicating like parts:

Fig. 1 depicts the synthesis of P-HEMA hydrogel with incorporated MMC using a redox polymerization method. Fig. 2 shows representative photographs of cell culture dishes used for P- HEMA toxicity tests after fixation and toluidine blue staining. Fig. 3 shows photomicrographs of COS-I cells cultured for 7 days in the presence or absence of hydrogel polymers.

Fig. 4 shows the sensitivity of COS-I and early passage human conjunctival fibroblasts to P-HEMA hydrogels. Fig. 5 shows the sensitivity of early passage human conjunctival fibroblasts to salt-free P-HEMA hydrogels. Fig. 6 shows photomicrographs of human conjunctival fibroblasts cultured for 7 days in the presence or absence of salt-free hydrogel polymers. Fig. 7 is a perspective view of one embodiment of my ophthalmological device. Fig. 8 is a perspective view of another embodiment of my ophthalmological device similar to that depicted in Fig. 7. Fig. 9 is a modified Baerveldt implant adapted to use with my method. Fig. 10 is a modified Ahmed valve implant adapted to use with my method. Fig. 11 is a perspective view of another embodiment of my ophthalmological device. Fig. 11 A is a cross-sectional view taken along line HA-HA of Fig. 11. Fig. 12 is a cross-sectional view showing my ophthalmological device surgically implanted into an eye. Fig. 13 is a cross-sectional view showing my device illustrated in Fig. 11 surgically implanted into a closed wound. Fig. 13A is an end view taken along line 13A-13A of Fig. 13.

DETAILED DESCRIPTION OF SOME ILLUSTRATIVE EMBODIMENTS

General

My ophthalmological device and method inhibits inflammatory cell proliferation to prevent fibrous tissue formation and prevent growth of fibrous tissue after eye surgery by placing in the vicinity of a wound created during surgery a member carrying an antifibrotic agent released into the wound slowly over a prolonged period. The aqueous liquid in contact with the member washes from the member or dissolves the antifibrotic agent. The member may be a component of an implanted ophthalmological device such as, for example, the Baerveldt distribution plate disclosed in U. S. Patent No. 5,476,445 or the Ahmed valve disclosed in U. S. Patent No. 5,071,408. It may comprise a porous material with the antifibrotic agent retained in its pores. Or, it

may be or non-porous polymeric material having a surface coated with the antifibrotic agent. The polymeric material may be a bio-degradable material may be naturally occurring, for example, polymers such as collagen, chitosan, polysaccharides (starch sugar, and cellulose), and proteins, or synthetic, for example, polylactides (PLA) and its copolymer with glycolic acid (PLGA), polyanhydrides, and polyesters. My method may be used in eye surgery such as, for example, a trabeculectomy or implanting drainage device such as, for example, the Baerveldt distribution plate or the Ahmed valve. Inhibiting fibrous tissue formation enhances the success of either implanting drainage devices or in non-penetrating surgery. The member may be inserted locally in a limbal pocket of the eye to inhibit limbal scar tissue from obstructing the eye's deep scleral pocket or canulostomy opening in deep sclerectomy/ viscocanulostomy eye operations.

Experiments

The following experiments were conducted to test delivering antifibrotic agents to wounds resulting from eye surgery: 1. To create a devices or membrane that is loaded with an antifibrotic agent that is released slowly over a 1-3 week period and test its efficacy in the lab using cultured fibroblasts in a Petri dish model. 2. To test a simple plate-tube model of a device having a portion coated with my tomycin C in a rabbit model and study the histology of the bleb. 3. To test a NPS membrane in a rabbit model.

The following antifibrotic agents were tested; Mitomycin C, 5-Flurouracil, Rapamycin, transforming growth factor (TGF) antibodies (specifically, TGF-B 2 monoclonal antibody, Interlukin 1 or 6 antibody or other cytokine antibodies), a form of corticosteroid such as, for example, dexamethasone, immunesuppresive agents such as, for example, cyclosporin or FK57, and heparin. Delivery methods varied: (1) local delivery of such antifibrotic agents from the surface an end-plate of a glaucoma drainage device; (2) delivery from the surface of a thin biomaterial membrane (both degradable and non-degradable) inserted at the time of non-penetrating surgery (NPS membrane); (3) delivery involving a co- mixture with polymers (both degradable and non-degradable) to hold the

antifibrotic agent to the glaucoma drainage device or to the membrane to be used at the time of the non-penetrating surgery; (4) delivery by entrapping the antifibrotic agent in the material comprising the glaucoma drainage device or the NPS membrane modified to contain microspores or channels; (5) delivery by including covalent binding of the antifibrotic agent to the glaucoma drainage device or the NPS membrane via solution chemistry techniques such as, for example, via the Carmeda process or dry chemistry techniques, for example, vapor deposition methods such as rf-plasma polymerization; and (6) combinations of these delivery methods.

Experiment 1

INHIBITION OF CELL PROLIFERATION BY MITOMYCIN C INCORPORATED INTO P-HEMA HYDROGELS

In this Experiment 1 a device including a P-HEMA matrix was tested that releases low concentrations of mitomycin- C over a 3-week period using an in vitro model.

MATERIALS AND METHODS

Materials: Neutral buffered formalin, toluidine blue, hematoxylin and eosin solution, sodium dodecyl sulfate, mitomycin C (Streptomyces caespitosus), reagents for tissue culture (culture medium, trypsin, antibiotics) and reagents for hydrogel synthesis (2-hydroxy ethyl methacrylate, N, N'-methylene-bisacrylamide, N,N,N',N'- tetramethylethylenediamine, and ammonium persulfate) were purchased from Sigma/ Aldrich (St. Louis, MO). Tissue culture dishes (60 mm diameter) were from Corning-Costar (Corning, NY). COS-I cells were obtained from the American Type Culture Collection (Manassas, VA). Early passage human conjunctival fibroblasts were established in 1994 from a biopsy sample of a 43 year old white male. The primary cultures were frozen in liquid nitrogen at 2 ^nd passage. This cell strain does not have an infinite life span and early passage cultures {4^-6^ passage) were used for the present experiments. Both the COS-I cell line and the fibroblast strain were maintained in DMEM containing 100 u/ml penicillin G, 0.25 μg/ml amphotericin B, 100 u/ml streptomycin and 10% fetal bovine serum (Hydone Laboratories, Logan, UT).

1 Hydrogel synthesis; In a typical P-HEMA hydrogel synthesis, 0.0508 g of N,

2 N'-methylene-bisacrylamide (MBA) was dissolved in 4 ml distilled water containing

3 400 μL HHN'/N'-tetramethylethylenediamine (TEMED). This was followed by the

4 addition of 4 ml of 2-hydroxyethyl methacrylate (HEMA solution, 99%) and

5 thorough mixing. A 1 ml aliquot of ammonium persulfate (AMP) in water (0.5 mol

6 % with respect to HEMA monomer) was subsequently added. Following mixing of

7 all components, the solution was cast between two sealed glass slides and allowed to

8 react at least 12 hours at room temperature to form the hydrogel. The product was a

9 polymer sheet approximately 2 mm thick. The reaction followed typical redox

10 pathways. For all syntheses, the crosslinker MBA was included at a 1:100 mol ratio

11 of crosslinker to monomer.

^■ j o * * * * * * * * * * * * * * * * *

13 Mitomycin C (MMC) was loaded into the hydrogels by two different

14 procedures. In Procedure 1, the drug was directly mixed in with the hydrogel

15 precursors before polymerization/ crosslinking. After hydrogel formation, the solid

16 polymer hydrogel was cut into circular discs of 8 mm diameter using a trephine. The

17 disks were sterilized by UV-irradiation for a period of 2 hours, and then used in cell

18 culture studies. In Procedure 2, the polymer was synthesized first and cut into

19 circular disks of 8 mm diameter. Unreacted, low molecular weight species were

20 removed from the polymer was by repeated washing with a 50/50 (v/v) solution of

21 distilled water and ethanol. MMC at the required concentrations was dissolved in

22 ethanol and then incubated with the disks in standard 10 ml vials. The solvent was

23 then slowly evaporated during which time MMC diffuses into the swollen polymer

24 as a consequence of the concentration gradient. When all the bulk solvent had

25 evaporated, the disks containing MMC were gently air-dried and then sterilized by

26 UV irradiation. The use of ethanol as a solvent in drug loading procedures also

27 helped sterilize the polymer matrix. 28

29 Measurement of polymer cytotoxicity in vitro: A single 8 mm disk of sterile

30 polymer was affixed to the center of each 60 mm tissue culture dish with clear RTV

31 silicone cement. The silicone cement was sterile as it came from the tube and care

32 was taken during this procedure to maintain the sterility of both the polymer and 3 the tissue culture dish. The cement was allowed to cure for 1-2 hours in a sterile

34 environment, until no odor of acetic acid could be detected. COS-I cells or

35 conjunctival fibroblasts (1.5 x 10 ^s cells/60 mm culture dish) were added to the

polymer-containing dishes and the cells were maintained at 37° C in a humidified 5% CO _z -95% air atmosphere; culture medium was replenished on day 3 and 5 after the cells were plated. After 7 days of culture, cell accumulation in each culture dish was assessed by a modification of the method of Leavesley (Leavesley DI, Ferguson GD, Wayner EA, Cheresh DA: Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen, J.Cell Biol. (1992) 117, 1101-1107). The culture medium was removed and the cell layer was gently washed 3 times with 5 ml of phosphate-buffered saline. The polymer piece was removed from each dish, cells were fixed for 30 minutes in 5 ml of neutral buffered formalin, then stained for one hour with 5 ml of 1% toluidine blue in neutral buffered formalin. The dye solution was removed, the cell layer was washed 4 times with 5 ml of distilled water, and the dishes were allowed to air-dry overnight at room temperature. Dye bound to the fixed cells was solubilized by the addition of 2 ml of 2% aqueous sodium dodecyl sulfate, followed by incubation for 15 minutes. The amount of dye in each dish, -which was proportional to the number of cells, was measured as the absorbance at 650 nm on a Shimadzu UV-Visible spectrophotometer (Model UV-1601, Shimadzu Scientific Instruments Inc., Houston, TX). In some cases, the solutions had to be diluted with 2% aqueous sodium dodecyl sulfate to bring the absorbance within the linear range of the spectrophotometer. For some experiments, the dishes were photographed before the dye was solubilized. In other experiments, replicate dishes were fixed in neutral buffered formalin and then stained with hematoxylin/ eosin to monitor cell morphology.

Statistical Analyses: All cytotoxicity tests were performed in quadruplicate and values reported are mean ± SD. Where appropriate, the Student's t-test provided in the Microsoft Excel™ software was employed to determine statistical significance. The dose-response curve for MMC toxicity was fit using the exponential model and software contained in SlideWritePlus for Windows™ ¹ , ver. 6.10.

RESULTS The incorporation of an antifibrotic agent into a slow release polymer could have significant advantages in slowing the fibrosis that is so often the cause of bleb failure after glaucoma surgery. A first step in the development of such polymers is the demonstration that 1) the polymer matrix itself has little or no cytotoxicity and 2)

the antiproliferative agent is released from the polymer in an active form. The hydrogels used in the present study were synthesized by a standard redox mechanism as depicted in Fig. 1. The inclusion of the crosslinker, MBA at a concentration of 1 mol % provided hydrogel sheets that were firm enough to be easily manipulated in subsequent experiments.

Cy toxicity of polymers prepared by Procedure 1: COS-I cells (a readily available, immortalized cell line ⁹ ) were used in preliminary experiments to establish the parameters of the study (number of cells in the initial inoculum, days in culture, number of medium changes, method of quantifying cell number). Once these parameters had been determined, the study was repeated with early passage cultures human conjunctival fibroblasts, the cell type against which these slow release polymers will ultimately be targeted. An inoculum of 1.5 x 10 ⁵ cells per 60 mm culture dish produced a confluent monolayer after 7 days in culture. Two medium changes were incorporated into the protocol; these medium changes insured that nutrients did not become limiting during the experimental period and also periodically removed cytotoxins released from the polymers, in a manner analogous to the natural "flushing" of a wound site with interstitial fluids. The method of Leavesley was used in a slightly modified form to assess cell number in these experiments. Cells were cultured in the presence of test agent for 7 days, then the medium was removed and the cell layer was lightly fixed. Toluidine blue, a basic dye that binds primarily to basophilic structures, including nuclei, ribosomes, and glycosaminoglycans, was subsequently added. After extensive washing to remove unbound dye, the culture dishes were dried overnight. Fig. 2 shows a representative sampling of fixed and stained culture dishes after a test of polymers prepared by Procedure 1. Accordingly, culture dishes (60 mm) with an affixed 8 mm disk of polymer prepared by Procedure 1 were inoculated with COS-I cells as described above. The cultures were fixed and stained 7 days after inoculation to provide Panels A through G, Fig. 2: Panel A, no additions; Panel B, silicone cement alone; Panel C, P-HEMA hydrogel with no incorporated MMC; Panel D, P-HEMA hydrogel with 0.031 mg/ g incorporated MMC; Panel E, P- HEMA hydrogel with 0.156 mg/g incorporated MMC; Panel F, P-HEMA hydrogel with 0.311 mg/g incorporated MMC; Panel G, P-HEMA hydrogel with 0.420 mg/g incorporated MMC

Panel A shows the stained, confluent monolayer produced by COS-I cells after 7 days in culture in the absence of a polymer sample. Panel B shows a culture dish to which the silicone cement but no polymer has been added. Panel C shows a dish cultured for 7 days with a polymer disk tihat contained no added MMC, and Panels D through F show dishes which has been cultured with polymer disks containing 0.031, 0.156, 0.311, and 0.420 mg/g incorporated MMC, respectively. While the silicone cement used to affix the polymer disks had no discernible effect on cell proliferation (compare Panels A and B) it was clear from the examination of the stained dishes that even the polymer that contained no incorporated MMC was inhibiting cell proliferation. Evidence of cytotoxicity was also observable at the cellular level, as shown by the photomicrographs in Fig. 3 including Panels A through F: Panel A, cells cultured in the absence of polymer; Panel B, cells cultured in the presence of P-HEMA hydrogel prepared by Procedure 1, with no incorporated MMC; Panels C-F, cells cultured in the presence of P-HEMA hydrogels prepared by Procedure 1, with incorporated MMC at the concentrations (mg/gram of gel) of 0.031, 0.156, 0.311, and 0.42, respectively. All photomicrographs were prepared at an identical magnification (approximately 30Ox). COS-I cells cultured in the absence of polymer (Panel A, Fig. 3) formed a confluent monolayer of uniformly shaped cells, as did cells cultured in dishes containing silicone cement alone (data not shown). Cells cultured in the presence of polymer, however, showed obvious signs of distress, including irregular shaped and giant cells and the presence of vacuoles (Panels B-F, Fig. 3). Note the morphological changes in those cells cultured with a polymer sample that lacked incorporated MMC (Panel B, Fig. 3). The obvious toxicity of the polymer matrix made it difficult to distinguish the effects of the polymer from those of the incorporated MMC, especially at the lower MMC concentrations (see Panels C and D, Fig. 3). The effect of the polymers on cell proliferation was quantified by solubilizing the toluidine blue in the stained dishes and measuring the absorbance in a spectrophotometer. A single 8 mm disk of polymer prepared by Procedure 1 was affixed to each culture dish with silicone cement and cells were fixed and stained 7 days after inoculation, as described above. After solubilization of the cell-bound dye, the color (quantified as absorbance at 650 run) was a measure of the number of cells in each dish. "Control" indicates data from cells inoculated into culture dishes with no additions; "Cement", indicates data from cells inoculated into dishes containing

silicone cement, but no polymer. Panel A, Fig. 3, COS-I cells; Panel B, Fig. 3, human conjunctival fibroblasts. Data is presented as mean + SD (n=4); All test groups were compared to the hydrogel sample with no incorporated MMC using Student's t-test, *, p<0.005. Fig. 4, graph A, shows the absorbance values obtained with COS-I cells cultured for a 7-day experimental period. As expected from the visual inspection of the stained dishes, cells cultured in the presence of silicone cement achieved an absorbance equivalent to that in dishes with no additions. The presence of polymer that contained no MMC in the culture dish reduced the number of cells after 7 days in culture to 48% of that observed in control cultures. In fact, the toxicity of the polymer matrix was so high that it obliterated any dose-response relationship when increasing amounts of MMC were incorporated into the hydrogel. Only those dishes containing polymer with the highest MMC concentration (0.420 mg/g polymer) had a significantly lower number of cells than did the dishes containing the polymer with no MMC. The toxicity of the polymer matrix was even more pronounced when early passage conjunctival fibroblasts were used in these experiments, as shown in Fig. 4, graph B. The effect of the polymer matrix on cell growth was so profound that incorporation of MMC into the polymer had no additional cytotoxic effect.

Cytotoxicity of polymers prepared by Procedure 2: The low molecular weight components present in the polymer matrix (unreacted monomer, TEMED, APS) responsible for the cytotoxicity of the polymers prepared by Procedure 1 were hypothesized. A method for removing these components from the polymer matrix before MMC was incorporated was developed. In preliminary experiments with COS-I cells, polymer matrix prepared by Procedure 2 had no detectable cytotoxicity in the absence of MMC (data not- shown). The human conjunctival fibroblasts were more sensitive than COS-I cells to these low molecular components. As illustrated in Fig. 5, the sensitivity to polymers prepared by Procedure 2 using this cell line were quantified A single 8 mm disk of polymer was affixed to each culture dish with silicone cement and cells were fixed and stained 7 days after inoculation, as described in Methods. After solubilization of the cell-bound dye, the color (measured as absorbance at 650 run) was a measure of the number of cells in each dish. "Control" indicates data from cells inoculated into culture dishes with no additions. Data is

presented as mean ± SD (n=4); All test groups were compared to the hydrogel sample with no incorporated MMC using Student's t-test, *, p<0.016. A salt-free polymer matrix with no incorporated MMC has no significant effect on the proliferation of the fibroblasts. Incorporated MMC inhibited cell proliferation in a dose-dependent fashion. The dose response to MMC could be fit to an exponential (see Fig. 5 insert); the r ² value for the fit was 0.95 and the concentration of MMC that inhibited 50% of cell proliferation (IC ₅₀ ) was calculated to be 0.15 mg/g dried gel. The morphology of fibroblasts cultured in the presence or absence of the polymer samples was also assessed, as shown in Fig. 6. In Fig. 6, Panel A shows cells cultured in the absence of polymer; Panel B shows cell cultured in the presence of P- HEMA hydrogen prepared by Procedure 2, with no incorporated MMC; Panels C-F shows cells cultured in the presence of P-HEMA hydrogels with incorporated MMC at the concentrations (mg/gram of gel) of 0.04, 0.12, 0.36, and 2.0, respectively. All photomicrographs were prepared at an identical magnification (approximately300x). As expected from the proliferation experiments, culture for 7 days in the presence of polymer with no incorporated MMC had little or no effect on the morphology of the cells (compare Panels A and B in Fig. 6). As increasing quantities of MMC were added to the polymer samples the cells, the morphology became more and more atypical, as shown in Panels C-F of Fig. 6.

DISCUSSION Sub-conjunctival fibrosis is the main cause of glaucoma surgery failure. The use of antifibrotic agents such as MMC has increased the success rate of the trabeculectomy operation. However, the use of MMC has little or no effect on the success of the glaucoma drainage devices. The reason for this phenomenon is not well understood. It may be that the conjunctival fibroblasts are less sensitive to one time application of MMC. It could be that the presence of a biomaterial leads to ongoing inflammation and attraction of fibroblasts. The current experiments are an attempt to create a device that can potentially alter the bleb following glaucoma drainage device surgery, so that lower intraocular pressures comparable to trabeculectomy operations can be achieved. It is of interest that contaminants (low molecular weight molecules) of polymer itself can exhibit toxicity as is demonstrated in the first part of the experiment. The cytotoxic effect of the polymer disappeared after the contaminants

were removed. It has been shown that physical deformity in the form of a dent in the smooth surface of drainage devices can attract fibroblasts potentially contributing to bleb failure. Experiment 2 A white albino rabbit was used for the experiment sedated with IM ketamine 35mg/kg and xylazine 5mg/kg per vivarium protocol. The area of the eye was shaved to prevent hair from contaminating the field. The field was prepped with betadine, and draped in a sterile fashion. Topical anesthesia with proparicane 0.5% was used to prevent any further discomfort. After this is done, the supero-temporal sub-conjunctival space was exposed opening the conjunctiva with wescott scissors. Once the tenon's tissue was separated a glaucoma drainage device end-plate measuring 1.5mmxl.5mm was inserted into the space and secured to the underlying sclera with two interrupted sutures. The tube attached to the plate was then inserted into the anterior chamber through a 23 gauge needle tract. The conjunctiva was closed with a running chromic suture. The animal was treated with topical tobradex ointment twice a day to prevent infection for 1 week. At one week after the surgery, clinical examination has revealed a large avascular cystic bleb on the surface of the end plate. The animal was euthanized per vivarium protocol on day 8. The eye was enucleated taking care not to disturb the end plate or the bleb. The eye was injected with 10% formalin and preserved in a formalin jar. It was sent for histological examination by an independent ocular pathologist well versed with rabbit histology.

Fathology The rabbit eye was in fixative and measured 17X17X16 mm. A "plastic" exoplanted reservoir was present superiorly measuring approximately 12mm in largest dimension. This was removed before opening the globe. The eye was opened through the plane of the exoplant device. The eye was phakic and the retina attached. Microscopic sections show edematous conjunctival tissue superiorly (bleb) with minimal round cell infiltration. The inner surface of the space formed between the superior conjunctiva and episclera was lined by fibinrous material containing few cells. There were no areas of granulomatous inflammation or necrosis noted. There was no vasculitis. There was no pathologic change noted inside the eye.

DEVICES

Fig. 7 schematically depicts one embodiment of my ophthalmological device identified by the numeral 10. The ophthalmological device 10 may be used to treat glaucoma and includes a distribution plate 12 carrying an antifibrotic agent and a tube 14 attached to the plate 12 near an inner end 14b so fluid exiting this inner end flows onto the plate. The plate 12 may be solid or porous, and its dimensions may be, for example, substantially 8 mm x 8 mm x 0.5 mm. If the plate is solid, the antifibrotic agent is applied as a coating to the exterior surface of the plate. If the plate is porous, the antifibrotic agent is absorbed in the pores of the plate material. Both the plate 12 and the tube 14 may be made of silicone. The hydrogel prepared as discussed above, or another material including an antifibrotic agent, is applied to the plate 12 in a sufficient quantity so the antifibrotic agent is released slowly over a prolonged period, for example, 1-3 weeks, when inserted into a wound in an eye produced by surgery. When implanted in a patient's eye to treat glaucoma in accordance with conventional surgical procedures, the aqueous humor from the eye' intraocular anterior chamber AC flows into the open end 14a of the tube 14 and over the plate 12. As illustrated in Fig. 12, the plate 12 is sutured to the bare sclera d under the conjunctival /Tenons' pocket approximately 7-10 mm from the limbus with the help of non-absorbable sutures such as prolene or nylon. Then the open end 14a of the tube 14 is inserted into the anterior chamber AC through a needle tract. The scleral portion of the tube is then covered with scleral or pericardial patch graft £ to prevent future tube conjunctival erosion, and secured to the surrounding sclera e. Then, the conjunctiva is secured to the limbus. The aqueous humor exits the anterior chamber AC through the inner end 14b flowing across the plate 12 and into the subconjunctival space d and is absorbed by surface blood vessels. Another embodiment of my ophthalmological device identified by the numeral 20, also used to treat glaucoma, is schematically depicted in Fig. 8. The ophthalmological device 20 includes a pair of silicone plates 22 and 24 having a sheet of matrix material 26, for example, the P-HEMA-Drug matrix carrying the antifibrotic agent, is sandwitched between the pair of plates. A silicone tube 28 has its inner open end 28b in contact with the matrix material 26 and its outer open end 28a is adapted to be placed into the eye as discussed above so the eye's aqueous humor may flow into the sheet of matrix material 26. The plate 22 has a plurality of

hole 30 therein. The dimensions of the plates 22 and 24 may be, for example, 8 mm x 8 mm x 0.5 mm. The dimensions of the sheet of matrix material 26 may be, for example, 8 irtrn x 8 mm x 0.5 mm. The ophthalmological device 20 is implanted in an eye to treat glaucoma in essentially the same manner as discussed above in connection with the device 10. Fig. 9 schematically depicts yet another embodiment of my ophthalmological device identified by the numeral 30. This device 30 is a conventional Baerveldt implant device modified by inserting a sheet of material 32 carrying the antifibrotic agent onto the surface of a distribution plate 34 of the Baerveldt implant device. A tube 36 attached to the distribution plate 34 provides a passageway for the aqueous humor flowing from the eye to wash the antifibrotic agent from the sheet of material 32 when the device 30 is implanted in an eye essentially in the same manner as discussed above. Fig. 10 schematically depicts still another embodiment of my ophthalmological device identified by the numeral 40. This device 40 includes an Ahmed valve 42 and a distribution plate 44 similar to the device 20 including a pair of silicone plates 22 and 24 having a sheet of matrix material 26 carrying the antifibrotic agent sandwitched between the pair of plates. A tube 43 has one open end 43b in communication with an inlet 42a of the valve the valve 42 that opens at an outlet end 42b in response to a differential in pressure across the valve. The outlet end 42b is in contact with the sheet of matrix material 26, so, when the ophthalmological device 40 is implanted as discussed above, the aqueous humor from the eye flows first through the tube 43 and out the open outlet end 42b, washing the antifibrotic agent from the sheet of matrix material 26. Figs. 11 and HA schematically depicts still another embodiment of my ophthalmological device identified by the numeral 50. This ophthalmological device 50 is made of a biodegradable material in the form of a sheet or plate 52 folded, cut, or otherwise formed into a suitable shape as required for placement in a wound created during eye surgery. As shown in Fig. HA, the sheet or plate 52 may have a substantially U-shaped configuration, which is a convenient shape for use in several eye surgical procedures. The sheet or plate 52 may have dimensions of 15 mm x 15 mm x 0.25 mm. This sheet or plate 52 is impregnated or coated with the antifibrotic agent in a sufficient amount to release slowly over a 1-3 week period after surgery. After this release period, the sheet or plate 52 disintegrates without any toxic reaction or other side effects. As illustrated in Figs. 13 and 13A, non-penetrating

ϊ surgery may use the sheet or plate 52. Fluid from the wound created by the surgery

2 contacts the sheet or plate 52 to slowly release the antifibrotic agent.

3

4 SCOPE OP THE INVENTION

5

6 The above presents a description of the best mode contemplated of carrying

7 out my invention, and of the manner and process of making and using it, in such

8 full, dear, concise, and exact terms as to enable any person skilled in the art to which

9 it pertains to make and use my invention. My invention is, however, susceptible to LO modifications and alternate constructions from the illustrative embodiments .1 discussed above which are fully equivalent. Consequently, it is not the intention to .2 limit my invention to the particular embodiments disclosed. On the contrary, my - 3 intention is to cover all modifications and alternate constructions coming within the .4 spirit and scope of my invention as generally expressed by the following claims, .5 which particularly point out and distinctly claim the subject matter of my invention:

6.