topical fibronectin therapy in persistent corneal ulceration

Australian and New Zealand Journal of Ophthalrnolonv 1987: 15: 257-262

TOPICAL FIBRONECTIN THERAPY IN PERSISTENT CORNEAL ULCERATION

PETER MCCLUSKEY FRACO

DENlS WAKEFIELD MD

Laboratory of Ocular Immunology, School of Pathology. University of New South Wales, Ophthalmology Department, St Vincent 5 Hospital, Sydney

Laboratory of Ocular Immunology, School of Pathology, University of New South Wales, Department of Immunology. Prince of Wales Hospital, Uveitis Research Clinic, Sydney €ye Hospital. Sydney

LUCY YORK BSC Laboratory of Ocular immunology. School of Pathology. University of New South Wales

Abstract fifteen patients with 20 episodes of perssstent corneal ulceration, resistant to conventional therapy, were treated with topical autologous fibronectin Thirteen corneal ulcers (eight patients) developed following penetrating keratoplasty, three patients had mucous membrane pemphigold, two patients had herpetic keratltis and one each had Slogfen's syndrome and a trophic corneal ulcer A standard protocol for fibronectin administration was followed This therapy healed 16 of the 20 ulcers after a mean duration of treatment of 2 3 months Corneal ulceration associated with mucous membrane pemphigord failed to respond to fibronectin Corneal ulcers whch recurred after cessation of hbronectm responded to rein troduction of this therapy Topical fibronectin is an effective therapy for refractory corneal ulceration and is free of malor side effects

Key words Fibronectin, persistent corneal ulceration

Corneal ulceration is one of the commonest pathological processes affecting the ocular surface. The vast majority of ulcers heal rapidly without complication. In compromised corneas, in some corneas after surgery, and in patients suffering from certain systemic illnesses involving the cornea, corneal healing is greatly diminished or absent. Previously established regimens for treating such persistent ulcers, including pressure patching, bandage contact lenses, debridement and corneal surgery, have a variable success rate and no one regimen is universally successful. Persistent epithelial defects are an uncommon, but important, management problem in corneal

disease. Patients with such ulcers are prone to secondary infection, corneal melting and corneal scarring, and post-keratoplasty defects may lead to graft failure. Although current treatment regimens are able to heal many of these defects, the response is slow and unpredictable, and may be incomplete. There is a group of patients with persistent corneal ulceration refractory to conventional therapy.

Autologous topical fibronectin may heal resistant ulcers and significantly shorten the healing time of slowly responding ulcers. Nishida et a1 initially reported the use of autologous topical fibronectin in two patients with long-standing

Reprint requests: Dr P. McCluskey, School of Pathology, University of New South Wales, PO Box I , Kensington, New South Wales 2033, Australia. This study was presented in part at the 18th Annual Scientific Meeting of the Royal Australian College of Ophthalmology in September 1986.

TOPICAL FIBRONECTIN THERAPY IN PERSISTENT CORNEAL ULCERATION 257

trophic ulcers, which responded to topical autologous fibronectin.' A subsequent study reported highly successful results in 20 patients with persistent ulcers following herpes simplex keratitis.2 Colvin et a1 reported successful results with topical fibronectin in five of six patients with herpetic keratitis, neurotrophic keratitis and keratitis associated with rheumatoid a r t h r i t i ~ . ~

Previous studies of the use of topical fibronectin in the treatment of persistent corneal ulceration have involved either small numbers of patients or a limited spectrum of disease. The follow-up has been short and there has been no comment on the efficacy of this form of treatment in corneal ulcers that recur after cessation of fibronectin therapy. This paper extends the clinical use of topical fibronectin by reporting its successful use in a number of types of persistent corneal ulceration including a large number of patients with persistent corneal ulceration following penetrating keratoplasty.

PATIENTS AND METHODS Patients Patients were referred for treatment because of the refractory nature of their corneal disease. For the purposes of this study arbitrary criteria were established for entry to the trial. Patients were eligible for treatment if corneal ulceration had been present for a minimum of four weeks despite the use of established treatment regimens. There was no restriction on the aetiology of the corneal ulceration. Patients with recurrence of corneal ulceration following fibronectin therapy were retreated immediately.

In all patients infection was excluded clinically and by appropriate microbiological investigation. In each patient, a detailed history was taken and a physical examination was performed, as well as appropriate investigations to exclude associated systemic disease. The tear film was carefully evaluated clinically, by rose bengal staining and by Schirmer's testing. Prior to treatment, patients were receiving a variety of topical and systemic medications. Topical medications consisted of corticosteroids, antibiotics and cycloplegics in varying combinations. Patients had also received trials of pressure patching or

therapeutic bandage soft contact lenses, where appropriate. All patients were receiving frequent proprietary topical ocular lubricants. Patient 4 was receiving oral prednisolone (100-10 mg per day) and methotrexate (2 mg per day) as immunosuppression for mucous membrane pemphigoid. Patient 14 was taking oral prednisolone (50-0 mg per day) for mucous membrane pemphigoid. Patient 15 was taking 5 mg daily of prednisolone orally.

The aetiology of the ulceration was determined clinically and confirmed by appropriate microbiological investigation when necessary, or by satisfying published criteria for mucous membrane pemphigoid4 and Sjogren's ~ y n d r o m e . ~

For this study, healing was defined as total reepithelialisation of the defect as determined clinically by slit lamp biomicroscopy and fluores- cein stainng. Duration of ulceration was defined as the time from which ulceration was first detected until presentation to the authors. Duration of follow-up was defined as the time from presentation to the authors until the present, and duration of treatment was defined as the time from the commencement of fibronectin therapy until healing occurred.

Patients were treated using a standard protocol of fibronectin administration. Each patient was reviewed weekly for the first month of treatment and then fortnightly until healing occurred. All current medication was continued unless investigations performed at the initial assessment indicated that a change in therapy was necessary. Topical fibronectin was administered four times daily until healing of the defect occurred. The patients completed their current bottle of fibronectin drops and therapy was then suspended. Other medications were continued as necessary.

Production of Autologous Fibronectin Plasma provides a convenient source of fibronectin. Drops were prepared at four-weekly intervals from 10 ml of fresh autologous plasma. A modification of the method of Nishida et al' was used to extract autologous fibronectin from fresh plasma using gelatin-coupled Sepharose 4B

258 AUSTRALIAN AND NEW ZEALAND JOURNAL OF OPHTHALMOLOGY

TABLE 1 Clinical Features of Patients Treated with Topical Fibronectin

Length of Result follow-up

of ulcer ulcer (months) f;:::;:; (months) Aetiology Duration of Duration Of Patient NO. Sex Ulcer

(years)

I 2 3 4 5

6

7 8 9

10 1 1

12 13 14 15

69, M 72, F 62, F 63, M 54, M

63, M

80, F 69, M 57, F 17, M 44, M

45, M 73, M 64, F 62, F

1 2 3 4 5 6 7 8 9

10 I 1 12 13 14 15 16 17 18 19 20

HSV PPK PPK MMP PPK Re1 Re1 Re1

PPK Re1

PPK HSV

Trophic PPK PPK Re1

PPK MMP MMP ss

8 2

18 24 15

1 0.5 0.5

6 1 8 3

1.5 3 2

0.5 7

12 3 I

1 2 8 3 1 2 1 1 1 1 7 1 3 3 1 1 4 3 2 1

Healed Healed Healed Failed Healed Healed Healed Healed Healed Healed Failed Healed Healed Healed Healed Healed Healed Failed Failed Healed

13 12 7 6

16

8

8 8 7 5 9

9 6 4 3

HSV = herpes simplex virus. PPK = post-penetrating keratoplasty . MMP = mucous membrane pemphigoid. SS = Sjogren's syndrome. Re1 = Relapse.

(Pharmacia) affinity chromatography. Urea was used as the eluent and was removed by gel filtration, using a Sepharose (3-25 (Pharmacia) desalting column. Fibronectin was sterilised by filtration through a 0.22 micron filter. Fibronectin drops were prepared by a 1 : l dilution with 0.5% chloramphenicol (Chloroptic, Allergan). The drops were prepared in a laminar flow hood and dispensed in sterile dropper bottles. All fibronectin extracted from plasma was dispensed back to the patient. SDS- polyacrylamide gel electrophoresis after reduction with mercaptoethanol was performed on the fibronectin drops and revealed a single 220K band. Using an ELISA to determine fibronectin concentration, a mean concentration of 98.9k63.0 pg/ml was detected. Fibronectin concentrations ranged from 50.0 to 296 pg/ml.

All equipment and materials including the columns were disposable and were discarded after a single use. Fibronectin drops were prepared so that patients received only fibronectin from their own plasma, thus eliminating the problems of transmission of microorganisms such as human immuno-

deficiency virus (HIV) and hepatitis B virus. All batches of drops were routinely cultured for microorganisms prior to use.

RESULTS There were 15 patients who sustained 20 episodes of corneal ulceration. Patients consisted of nine males (mean age of 61.9 k 11.2 years) and five females (mean age of 66.2k7.6 years). The clinical features of the patients are described in Table 1. The aetiologies of the ulcers and the outcome of therapy are detailed in Table 2. Healing occurred in 16 ulcers (80.0%) of 11 patients (73.3%). The mean pretreatment ulcer duration was 5.8k6.5 months, and the mean duration of treatment was 2.35 k 2.0 months. The mean duration of follow-up was 8.1 k 3 . 3 months.

Three patients developed recurrent epithelial defects after fibronectin treatment. Patient 5 was referred with an epithelial defect present for 15 months after his fifth penetrating keratoplasty. Originally this patient had a penetrating keratoplasty performed as an emergency for corneal perforation secondary to necrotising

TOPICAL FIBRONECTIN THERAPY IN PERSISTENT CORNEAL ULCERATION 259

TABLE 2 Aetiology of Ulcer and Outcome of Fibronectin Treatment

Aetiology Number of Number of Success of ulcer patients ulcers rate

Post- penetrating keratoplast y 8 13 92.3%

Herpes simplex virus

keratitis 2 2 100% Mucous

membrane emphigoid 3 3 0 070

Trophic 1 I 100% Sjogren’s syndrome I I 100%

herpetic ketatitis. The postoperative course was complex and necessitated two regrafts for a combination of recurrent herpetic keratitis and rejection. A further penetrating keratoplasty was required for graft rejection and at this time lens extraction was also performed. After operation severe secondary glaucoma developed; this was uncontrolled by medical therapy and required cyclocryotherapy to reduce the intraocular pressure to less than 25 mmHg on maximum tolerated medical treatment. Subsequent to this, the graft decompensated and the fifth keratoplasty was performed. The intraocular pressure was not well controlled and the epithelial defect recurred when the intraocular pressure became elevated. The defect persisted despite reduction of the intraocular pressure to less than 25 mmHg by the use of oral acetazola- mide (1000-500 mg per day) and further cyclocryotherapy. The patient was referred for fibronectin treatment at this time. Fibronectin enabled reepithelialisation to occur and the corneal graft remained clear. Recurrences of corneal ulceration were associated with fluctua- tions of intraocular pressure and all have healed promptly with reintroduction of fibronectin therapy. After the most recent episode of ulceration had healed, a Molten0 valve was placed in the eye and a stable intraocular pressure of less than 20 mmHg has been present for the last three months of follow-up. There have been no further episodes of ulceration and the corneal graft remains clear.

Patient 6 underwent uneventful penetrating keratoplasty for pseudophakic bullous kerato-

pathy. After operation an epithelial defect remained for six months. This healed with one month of fibronectin treatment. Three months later a severe episode of rejection resulted in graft failure. A repeat keratoplasty was performed. Epithelialisation of the graft was not complete four weeks after operation. With four weeks of fibronectin treatment epithelialisation had occurred. There has been no recurrence after three months of follow-up.

Patient 11 had a 15 year history of recurrent ulcerative keratitis. A precise diagnosis had not been established and it was considered that the patient had a variant of Terrien’s marginal corneal dystrophy. He has had more than 20 corneal procedures to each eye over this period. His left eye was phthisical. An anterior segment reconstruction using a freehand tectonic corneoscleral graft had been performed on his right eye 18 months previously. This was followed by an optical penetrating keratoplasty six months later. Over the subsequent period he had suffered recurrent episodes of corneal ulceration at the junction of the grafts. The aetiology of the defects clinically appeared non- inflammatory in origin and was presumed to be related to exposure and tear dysfunction rather than to active Terrien’s syndrome. Despite inten- sive use of ocular lubricants and a lateral tarsorrhaphy, healing of the ulcers was poor and tectonic lamellar corneal grafts were required to prevent perforation from progressive thinning. Topical fibronectin was added to the therapy of the two most recent episodes with rapid healing of the defects and no stromal thinning.

All patients with mucous membrane pemphigold failed to respond to treatment and their disease progressed despite fibronectin therapy. These three patients had advanced bilateral disease with severe conjunctival cicatrisation and ocular dryness. Subsequent to the unsuccessful use of fibronectin, they required major anterior segment surgery for disease control. Patient 4 was blind in the right eye and had undergone three penetrating keratoplasties to his left eye. The most recent graft had decompensated and contained a large area of corneal ulceration. Fibronectin therapy had no effect in this patient

260 AUSTRALIAN A N D NEW ZEALAND JOURNAL OF OPHTHALMOLOGY

and he subsequently underwent a kerato- prosthesis procedure to his remaining eye. Patients 13 and 14 subsequently required penetrating keratoplasty to the eyes which had previously failed to respond to fibronectin therapy. Again both patients had extremely dry eyes with opacified ulcerated corneas from advanced mucous membrane pemphigoid.

The other patient failure occurred in an 80-year-old patient who was referred with a n eight-month history of a total epithelial defect following penetrating keratoplasty. There was no change in the corneal defect with 28 weeks of topical fibronectin treatment. The patient then underwent uneventful repeat keratoplasty with no recurrence of a n epithelial defect.

DISCUSSION This study reports for the first time the successful use of topical fibronectin in post-keratoplasty persistent epithelial defects, in trophic ulceration associated with Sjogren’s syndrome, and in patients with recurrent episodes of ulceration. Twelve of 13 patients with persistent ulcers following keratoplasty were successfully cured with topical fibronectin therapy without complication. The potential therapeutic usefulness of fibronectin in ophthalmology has thus been broadened by this demonstration of its effective- ness in healing such non-responsive epithelial defects a f t e r pene t r a t ing ke ra top la s ty . Fibronectin greatly reduced the need for regrafting in the group of patients reported here and allowed long-term healing of ulcers resistant to other treatment.

Persistent epithelial defects are a n uncommon but significant complication of corneal graft surgery. They retard visual rehabilitation after surgery and may result in secondary infection, graft opacification and graft rejection. Some defects respond to conventional treatment regimens; however, there remains a group of ulcers which d o not heal. These patients require regrafting. In this study autologous topical fibronectin was able to rapidly heal resistant defects in all but two patients. One of the two patients responded slowly over eight months but

the defect healed and the corneal epithelium has remained intact.

Relapses were uncommon, but were responsive to further courses of treatment. Two of the three patients with relapses (Patients 5 and 1 l), represent unusual and difficult management problems, while the other patient failed to epithelialise a f t e r a fu r the r pene t ra t ing keratoplasty. Fibronectin rapidly healed the defects in each patient.

Topical autologous fibronectin has been used in several uncontrolled trials for the treatment of post-herpetic and trophic corneal ulceration and has been shown to be useful adjunctive therapy in such patients. In our study we have obtained similar results, albeit with small patient numbers, by using fibronectin therapy less often per day and by manufacturing the drops far less frequently, thus maximising the efficiency of this therapy.

The uniform lack of response in patients with mucous membrane pemphigoid was disap- pointing, but not unexpected, in view of the advanced nature of their disease. This lack of benefit shows that a severely compromised cornea, particularly in the presence of extreme dryness, remains a difficult management problem and can not be salvaged by attempting to restore the epithelium alone.

In-vitro evidence supports the concept that fibronectin is an essential cofactor for the main- tenance of normal cellular integrity. Fibronectin is a high molecular weight glycoprotein (440K daltons) present on the surface of cells, in connective tissue matrices, in extracellular fluids and found in significant concentrations in plasma. It is thought to be involved in a wide range of cellular activities related to intercellular adhesion and the attachment of cells t o matrix. Known properties of fibronectin include agglutination, attachment , spreading, increased mobility and resistance to detachment of cellsa6 Fibronectin is also a n essential growth factor.’ In-vitro studies of wound healing, using rabbit cornea, have demonstrated that fibronectin appears at the edge of the healing ulcer and is present in high levels beneath the migrating epithelial cell^.^,^

TOPICAL FIBRONECTIN THEKAPY I N PEKSISTENT CORNEAL UI.CEKA-TION 26 1

Thus far, fibronectin has been reserved for use in patients with corneal disease resistant to conventional therapy. Fibronectin will be a useful addition to the management of other more common types of epithelial disease. By promoting the rapid re-epithelialisation of the cornea following surgery, infection, trauma, chemical injury and recurrent erosion, fibro- nection may significantly reduce the morbidity from corneal disease.

ACKNOWLEDGEMENTS We wish to thank Drs I. B. Jack, L. Robinson, R. F. Taylor, D. Treloar and A. Wechsler for allowing their patients to be included in this study. References 1. Nishida T, Ohashi Y, Awata T, Manabe R. Fibronectin.

A new therapy for corneal trophic ulcer. Arch Ophthalmol 1983: 101: 1046-1048.

2. Nishida T, Nakagawa S , Manage R. Clinical evaluation of fibronectin eyedrops on epithelial disorders after herpetic keratitis. Ophthalmology 1985; 92: 213-216.

3. Colvin R, Phan T-M, Boruchoff S , Zagachin L, Foster C. Fibronectin therapy of persistent corneal epithelial defects. ARVO Abstracts 1985. Supplement to Invest Ophthalmol Vis Sci 1985; 26: 92.

4. Moutsopoulos H, Webber B, Vlagopoulos T. Differences in the clinical manifestations of sicca syndrome in the presence and absence of rheumatoid arthritis. Am J Med 1979; 66: 733-738.

5. Bairstow B. Cicatricial pemphigoid. Arch Dermatol 1971; 104: 454-459.

6. Yamada K , Olden K. Fibronectins - adhesive glycoproteins of cell surface and blood. Nature 1978; 275: 179-184.

7. Bitterman P, Rennard S, Adelberg S, Crystal R. Role of fibronectin as growth factor for fibroblasts. Cell Biol 1983;

8. Ohashi Y, Nakagawa S, Nishida T, Suda T, Watanabe K, Manabe R. Appearance of fibronectin in rabbit cornea after thermal burn. Jpn J Ophthalmol 1983; 27: 547-545.

9. Nishida T, Nakagawa S , Nishibayashi C, Tanaka H, Manabe R. Fibronectin enhancement of corneal epithelial wound healing of rabbits in vivo. Arch Ophthalmol 1984;

97: 1925-1932.

102: 455-456.

262 AUSTRALIAN AND NEW ZEALAND JOURNAL OF OPHTHALMOLOGY

topical fibronectin therapy in persistent corneal ulceration

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