PROTECTION OF INTRAOCULAR STRUCTURES

Abstract: This invention relates to the protection of intraocular structures against disintegration caused by irritative stimuli. The protection is carried out by applying proteinase inhibitor into the eye.

Description: PROTECTION OF INTRAOCULAR STRUCTURES FIELD OF THE INVENTION This invention relates to protection of intraocular structures against disintegration caused by irritative stimuli, e.g. mechanical or chemical irritation. Especially the invention relates to the protection of the integrity of the endothelial cells of the cornea. The invention can be used especially in connection with intraocular surgical operations. BACKGROUND OF THE INVENTION Signs of ocular irritation such as miosis, breakdown of the blood aqueous barrier with increased protein in the aqueous humour, uveal and pericorneal vasodilatation, and elevation of the intraocular pressure are often seen in response to various irritative stimuli, e.g. mechanical irritation (surgery) or chemically induced trauma of the eye. The term ocular irritative response (OIR) comprises all these symptoms. Intraocular surgery of the anterior segment of the eye is known to be accompanied by loss of endothelial cells. This may in part be due to surgical manipulations, use of irrigation solutions or direct toxic effects drugs. Postoperative intraocular inflammation as well as chronic anterior segment disease such as heterochromic cyclitis may cause further damage to endothelial cells especially when leucoytes are present in the anterior chamber. Human corneal endothelial cells show in contrast to other species only a very limited regenerative capacity. In primates endothelial wound healing is achieved mainly not by cell division but by enlargement of neighbouring endothelial cells covering the defect by migration and expansion of their surface resulting in a permanent decrease of endothelial cell density and by this in an impairment of functional capacity. In humans any endothelial cell loss is a serious complication since the endothelial cell S UBSTITUTESHEET layer plays a key role in the maintance of corneal stromal dehydration and transparency. The endothelial cell damage can be partially reduced by the use of gentle operation techniques, often in combination with viscoelastic substances such as sodium hyaluronate. Also the preoperative use of inhibitors of prostaglandin -synthetase has been suggested. However, it seems that endothelial cell loss as side effect of anterior segment surgery cannot be avoided with current surgical techniques (Draeger, J. , Winter R. : Developments in intracapsular cataract extraction. In: Cataract surgery (Steele, A.D. , Drews, R.C., Eds.) Butterworth International Mediacal Reviews, Ophthalmology 2, Butterworth & Co (Publishers), London, UK, 1984, pp 72-85) . STATEMENT OF THE INVENTION It has been now discovered that mechanical irritation (surgical trauma) of ocular structures in the anterior segment of the eye and induction of ocular irritative response is associated with activation of the intraocular fibrinolytic system leading to occurrence-.of proteolytic activity in the anterior chamber fluid. We have discovered that this proteolytic activity which was at least in part due to plasmin, could be inhibited by proteinase inhibitor. The general idea of the invention resides a method for inhibition of proteolytic"activity in intraocular fluids by applying a therapeutically effective amount of proteinase inhibitor, e.g. plasmin inhibitor such as aprotinin, in the form of a physiologically acceptable preparation. The invention and some of its preferred embodiments are precisely defined in the appending claims. DETAILED DESCRIPTION OF THE INVENTION When protein as inhibitor is applied into the eye under OIR, the fibrinolysis caused by proteinases SUBSTITUTE SHEET present, and in this way also the disintegration of cells is prevented. In connection with surgical operations the application might be pre-, per-, or postoperative depending on the intention to prevent or to therapy occurence of proteolytic activity in intraocular fluids. The way of drug application may be topical into the conjuctival sac as drop; intracameral, intravitreal or subretinal injections or intravenous infusions. Experiments The occurence of proteolytic activity in ocular fluids after either surgical trauma (mechanical irritation of intraocular structure of the anterior segment of the eye) or chemical induction of sensory axon reflex with capsaicin (neurogenic inflammation) and the inhibition of the proteolytic activity by the use of proteinase inhibitor aprotinin is described hereinafter. Materials and procedure All experiments were performed in accordance to The Guiding Principles in The Care and Use of Animals (DHEW Publication, NIH 80-23) and the Declaration of Helsinki. Anterior chamber fluid of ten patients with healthy eyes with collected prior to cataract surgery immediately after the primary incision into the anterior chamber with a 1 ml syringe and a Sautter needle. Seven adult New Zealand albino rabbits (3,0-4,2 kg b.w.) were used for the experiments. General anesthesia was accomplished by intravenous injection of ketamin-xylazin dilution (Ketalar® (Parke-Davis, S.A., Barcelona, Spain) 10 mg/kg b.w. Rompun® vet. (Bayer, Leverkusen, FRG) 13 mg/kg b.w.). Topical anesthetics (Oftan-Obucain®, Star, Tampere, Finland) were administered twice before sugical manipulation, subconjunctival or intraocular application of capsaicin or the control substance. Before use, SUBSTITUTESHEET capsaicin (8-methyl-N-vanillyl-6-nomenamide) (Sigma, St. Louis, USA) (250 mg) was solved in 2,5 ml ethanol, 2,5 ml tween-80 and diluted in 0,9 % NaCl to a final concentration of 1 %. Experimental procedure OIR was provoked mechanically by rubbing with the aspiration needle on the surface of the iris of the right eye of five rabbits. Anterior chamber fluid (ACF) specimens were collected from these eyes before, 15 minutes after and 1 hour after irritation during paracenthesis with a sharp needle attached to a 1 ml syringe. To provoke OIR after subconjunctival application of chemical irritants, 0,5-1,0 ml 1 % capsaicin solution was injected subconjunctivally to the right eye of four rabbits. The left eye received 0,5-1,0 ml of a control solution (2,5 ml ethanol, 2,5 ml tween-80 and 120 ml 0,9 % NaCl) which was administered subconjunctivally. Before the application of the substances ACF was collected from both eyes by the use of 1 ml syringes and a sharp needle. 60 minutes after application ACF was collected again from both eyes. For the provokation of OIR after intracameral injection of capsaicin, ACF was collected before and 30 minutes after injection of 50-150 microl capsaicin (1 %) into the anterior chamber of five rabbits using a sharp needle. To the control eyes only paracenthesis and aspiration of ACF was performed in the same time intervals without injection of any substance. All specimens collected were transferred into Eppendorf-tubes, immediately frozen on dry ice and stored at -70 °C. Determination of proteolytic activity A modification of the radial caseinolysis (Saksela, O. : Radial caseinolysis in agaraose: a simple method for SUBSTITUTESHEET detection of plasminogen activator in presence of inhibitory substances and serum, Anal Biochem 111, 1981, 276-282) was used. For this assay agarose (Agarose C, Pharmacia, Sweden) was prepared to a concentration of 1,2 % (w/v) in 0,1 M Tris-HCl buffer, pH 8,0, containing 0,03 % (w/v) NaN3. Fat-free milk powder (Valio, Finland) served as the source of casein. The casein mixture was made in 0,1 M Tris-HCl buffer, pH 8,0, and stored at -20 °C as a 15 % (w/v) stock solution. The casein was added to the melted agarose at 50-56 °C and the mixture cast on a glass plate. For the assay of PA, purified plasminogen (Plasminogen, Kabi Diagnostica, Stockholm, Sweden, > 20 CU/mg protein) was added to the agarose at 50-56 °C. The final concentration of plasminogen in the agarose was 4 microg/ml. Sample wells were punched into the gel and the specimens of ACF were added (8 microl) . The agarose plate was incubated in a humidified chamber at 37 °C. The development of caseinolysis was recorded at 24 hour intervalls. The total incubation time was 48 hours. Reactions with specific peptides revealed that the caseinolytic activity observed was due to plasmin. Human plasmin (Kabi Diagnostica, Stockholm, Sweden: > 15 CU/mg) was used as a standard for plasmin-like activity. Its activity was checked against a WHO-standard (Human plasmin, National Institute for Biological Standards and Control, London, GB) . Human urokinase (MW 54 000; Calbiochem, USA) was used as a standard for demonstrating PA activity. Aprotinin (Antagosan®, respond Aprotinin 100 ky, Behring, Germany) was used in inhibitor studies to inhibit the proteolytic activity of anterior chamber fluid. Parallel analysis of plasmin activity in ACF specimens was performed by the use of a rapid plasmin-specific assay. SUBSTITUTESHEET Results Human anterior chamber fluid of healthy eyes, void of any sign of inflammation, was shown to contain no proteolytic activity. Mechanical irritation of the iris surface caused a significant elevation of plasmin in the ACF of all five rabbits after 15 to 60 minutes (Table 1) . The presence of plasmin was associated with a sligth bleeding from iris vessels in one eye and exudation of fibrin into the anterior chamber in another eye. As shown by the latter experiments, the bare, carefully performed puncture of the AC with aspiration of ACF in volumes of about 100 microl did not lead to detectable increase of proteolytic activity in the ACF. Table 1: ACTIVITIES OF PLASMIN IN THE ANTERIOR CHAMBER FLUID BEFORE , 15 AND 60 MINUTES AFTER MECHANICAL IRRITATION OF THE IRIS TIME INTERVAL AFTER- MECHANICAL IRRITATION RABBIT BEFORE 15 MINUTES 60 MINUTES Subconjuctival injection of capsaicin led to miosis of the treated eyes within the next 5 to 15 minutes whereas the placebo treated eyes retained normal iris diameter. In the samples of ACF collected before the subconjunctival injection no plasmin activity could be detected (Table 2) . One hour after injection 80 % of the ACF samples from capsaicin treated eyes showed notable activity of plasmin (Table 2) . In the control group the SUBSTITUTESHEET ACF of only one eye contained plasmin activity. In this eye the plasmin activity was lower than in the treated eyes and associated with a bleeding into the anterior chamber due to accidental contact of the aspiration needle with the iris during the first aspiration. Table 2: ACTIVITIES OF PLASMIN IN THE ANTERIOR CHAMBER FLUID BEFORE AND 60 MINUTES AFTER SUBCONJUNCTIVAL INJECTION OF CAPSAICIN Intracameral injection of capsaicin led in the treated eyes to immediate miosis and enlargment of the iris vessels. Also in this experiment no plasmin activity was present in the ACF samples collected before treatment. 30 minutes after the intracameral injection of capsaicin plasmin was present in 80 % of the treated eyes (Table 3) . It is notable that the eye that did not show any elevation of plasmin activity was one of those treated two days earlier with subconjunctival injection of capsaicin. However, the other two eyes, also treated two days earlier with subconjunctival capsaicin injection showed a marked elevation of plasmin activity in ACF in response to intracameral injection of capsaicin (Table 3) . SUBSTITUTESHEET Table 3: ACTIVITIES OF PLASMIN IN THE ANTERIOR-CHAMBER FLUID BEFORE AND 30 MINUTES AFTER INTRACAMERAL INJECTION OF CAPSAICIN Proteolytic activity of ACF specimens was shown to be due to plasmin and to be successfully inhibited in vitro by the use of aprotinin. Discussion of results It was found that proteolytic activity occured in the anterior chamber fluid after mechanical stimulation and chemical induction of OIR. This proteolytic activity was shown to be at least in part due to enzymes of the fibrinolytic system, e.g. plasmin, and could be inhibited by proteinase inhibitor, such as aprotinin. The importance of the fibrinolytic system in formation and resolution of inflammatory reactions in the anterior segment of the eye has been proposed (Pandolfi, M. , Nilsson, I.M. , Martinsson, G. : Coagulation and fibrinolytic components in primary and plasmoid aqueous humour of rabbit, Acta Ophthalmol (Copenh) 42, 1964, 820-825). Besides" this early report, data on the presence of plasmin in the aqueous humour of the eye are scarce (Cejkova, J. , Lojda, Z., Salonen, E.-M., Vaheri, A.: Histochemical SUBSTITUTESHEET study of alkali-burned rabbit anterior segment in which severe lesions were prevented by aprotinin treatment, Histochemistry 92, 1989, 441-448). In the latter study plasmin was demonstrated in the rabbit aqueous humour after severe alkaline burn. However, no levels were given. The model with severe alkaline burn is also likely to be associated with secondary infection and inflammation of the whole anterior segment as well as with total loss of corneal endothelial cells in the area beneath the stromal damage (Chung, J.-H., Fagerholm, P. : Endothelial healing in rabbit corneal alkali wounds, Acta Ophthalmol (Copenh) 65, 1987, 648-656) . The damage of intraocular structures caused by the penetration of alkaline substances into the anterior segment can neither be predicted nor clearly quantified. In the present study primary ACF did not contain plasmin activity. This is in accordance to earlier results (Pandolfi, M. , Kwaan, H.C.: Fibrinolysis in the anterior segment of the eye, Arch ophthalmol 77, 1967, 99-104) . The most important result of our study is that OIR produced either by mechanical irritation of the iris (similar to surgical manipulations) or by chemical stimulation of axon reflexes in the anterior segment of the eye was followed by the occurrence of free plasmin in the ACF. Notably the stimulation methods used did not cause any permanent tissue damage. Occurence of plasmin activity in ACF as part of OIR speaks for the development of a acute failure of the inhibitory regulation of the plasmin-plasminogen activator system in the aqueous humor leading to a tilt of the equilibrance towards fibrinolysis. This, in turn, might be due to the initial increase of PA from damaged or affected intraocular tissues such as the iris which is known to contain t-PA activity (Pandolfi and Kwaan, ibid) . In the present study not only the induction of OIR by mechanical manipulation of the iris, but even SUBSTITUTESHEET minor trauma caused by slight contact of the aspiration needle with the iris and associated with consecutive bleeding (one eye) led the occurrence of plasmin in the ACF. Basically the same mechanism is expected to lead to even higher elevation of plasmin activity after the traumatising procedure used in intraocular surgery. The present study shows that the activation of the fibrinolytic system is also essential part of the normal response of the eye to surgical manipulations or induction of OIR. It is even possible that there is a link between the intraocular fibrinolytic system and the intraocular (patho-)physiology of neuropeptides and prostaglandins. Treatment with intraocular proteinase inhibitors such as aprotinin, similar to the preoperative application of corticosteroids or inhibitors of prostaglandin-synthesis used in today's practice, might be beneticial in reducing the occurrence and the effects of OIR. It might furthermore enhance the wound healing of structures localized in the anterior segment of the eye after acute trauma (operations) , corrosion injuries, or acute inflammation such as serious intraocular infections (endophthalmitis) . Peroperative administration of proteinase inhibitor may even have protective effects on the adherence of the corneal endothelium to the underlying stroma. Similarly the use of fibrin as a temporary sealant to plug retinal holes (Nasaduke, I., Peyman, G.A. : The use of autogenous fibrin sealant to plug retinal holes in experimental detachments, Ann ophthalmol 18(11), 1986, 324-327) or the treatment of lens capsule perforations with fibrinogen (Buschmann, W. : Microsurgical treatment of lens capsule perforations - part II: Clinical applications and results, Ophthalmic Surg 18(4), 1987, 276-282) migth offer possible indication for the intraocular use of proteinase inhibitor in order to prevent the temporary undesired fibrinolysis. In some SUBSTITUTESHEET cases the combination of aprotinin with thrombin might offer additional therapeutic possibilities. Thrombin has been shown to catalyse the conversation of fibrinogen to fibrin and appears to be nontoxic to the corneal endothelium (Mannis, M.J. , Sweet, E. , Landers, M.B., 3rd; Lewis, R.A. : Uses of thrombin in ocular surgery. Effect on the corneal endothelium, Arch Ophthalmol 106, 1988, 251-253). Intraocular inhibition of plasmin would also diminish the cleavage of fibronectin molecules inthe anterior segment. Because fragments of fibronectin have been shown to be chemotactic for leucoytes the inhibition of fibronectin fragmentation might also lead to diminished invasion of leucocytes into the anterior chamber. Activated leucocytes, in turn, especially polymorphonuclear leucocytes (PMNLs) display proteolytic enzymes such as collagenase and plasmin (Prause, J.U. : Cellular and biochemical mechanisms involved in the degradation and healing of the cornea, Acta Ophthalmol (Copenh) Suppl. 168, 1984, 9-10, Kao, W.W.-Y., Ebert, J. , Kao, C.W.-C, Covington, H. , Cintron, C: Development of monoclonal antibodies recognizing collagenase from rabbit PMN; the presence of this enzyme in ulcerating corneas Curr Eye Res 5, 1986, 801-815) . For corneal epithelial wound healing the beneficiency of topical aprotinin application has been shown (Salonen, E.-M., Tervo, T. , Torma, E. , Tarkkanen, A., Vaheri, A.: Plasmin in tear fluid of patients with corneal disease: basis for new therapy, Acta Ophthalmol (Copenh) 64, 1987, 3-12, Tervo, T. , Salonen, E.-M., Vaheri, A., Immonen, I., van Setten, G.-B., Himberg, J.J., Tarkkanen, A.: Elevation of tear fluid plasmin in corneal disease, Acta Ophthalmol (Copenh) 66, 1988, 393-399, Tervo, T. , van Setten, G.-B.: Aprotinin for inhibition of plasmin on the ocular surface: principles and clinical observations. In: Advances in applied biotechnology series Voll: Healing Processes in the SUBSTITUTE SHEET Cornea (Eds. R.W. Beuerman, C.E. Crosson, H.E. Kaufman) , Portfolio Publishing Company, The Wodlands, TX, USA, 1989, pp 151-163) . For indications which would profite from enchancement of localized or general intraocular fibrinolysis the intraocular use of proteinase inhibition in combination with PAs would offer a possibility to make the intraocular use of PAs safer and better adjustable, provided that proper diagnostic methods are available for continuous control. Recent reports on the intraocular use of t-PA have shown promising results in lysis of postoperative fibrin formations or in therapy of obstructed filtration channels after glaucoma surgery (Williams, G.A. , Lambrou, F.H. , Jaffe, G.A. , Snyder, R.W. , Green, G.D., Denevyi, R.G., Abrams, G.W. : Treatment of postvitrectomy fibrin ormation with intraocular tissue plasminogen activator, Arch Ophthalmol 106(8), 1988, 1055-1058, Ortiz, J.R., Walker, S.D., McManus, P.E., Martinez, L.A. , Brown, R.H. , Jaffe, G.J. : Filtering bleb thrombolysis with tissue plasminogen activator, Am J Ophthalmol 106, 1988, 624-625) . The intraocular use of proteinase inhibitor gives new perspectives for the therapy of acute and possibly also chronic changes of the fibrinolytic system in intraocular fluids of the eye. The initation of the therapy with proteinase inhibitor (both alone or in combination with other substances) might be as well pre-, per- or post-operative, the way of administration topical to the external surface of the eye, directly into the anterior or posterior part of the eye or intravenously. Temporary intravenous application of proteinase inhibitor might be as efficient in intraocular inhibition of proteolysis as it has already been shown for the therapy of acute pancreatitis. Although the present invention has been described in conjunction with the preferred embodiments it is to be SUBSTITUTESHEET understood that modifications and variations may be resorted to without departing from the spirit and the scope of the invention as those skilled in the art will readily understand. Such modifications and variations are considered to be within the purview and the scope of the invention and the appended claims. SUBSTITUTE SHEET

Claims: CLAIMS 1. A method for the protection of intraocular structures against irritative stimuli in a patient, comprising applying proteinase inhibitor into the eye of the patient. 2. A method as claimed in claim 1 for the protection of the intraocular structures against irritative structures caused by intraocular surgery. 3. A method as claimed in claim 2 wherein during the surgical operation the eye is rinsed with liquid containing proteinase inhibitor. 4. A method as claimed in any of claims 1 - 3 for the protection of the endothelial cell structure of cornea. 5. A method as claimed in any of claims l to 4 wherein the proteinase inhibitor is plasmin inhibitor. 6. A method as claimed in claim 5 wherein the plasmin inhibitor is aprotinin. 7. A method for carrying out intraocular surgical operations in a patient, in which method proteinase inhibitor is administrated into the patient's eye. 8. A composition to be used for the protection of intraocular structures in a patient against irritative stimuli, which composition comprises proteinase inhibitor. 9. A composition to be used as a rinsing liquid for the eye during intraocular surgical operations, which composition comprises proteinase inhibitor. SUBSTITUTESHEET

Inventors: TERVO TIMO, VAN SETTEN GYSBERT-BOSTHO, TERVO, TIMO, VAN SETTEN, GYSBERT-BOSTHO
Applicants: HUHTAMAEKI OY
Application year: 1990
Main classification: A61K37
IPC codes: A61K38