CA2095207A1 - Use of inhibitors of plasminogen activators for the treatment of inflammations and wounds - Google Patents

Abstract

BEHRINGWERKE AKTIENGESELLSCHAFT HOE 92/B 009 - Ma 949 Dr. Bc/Wr Abstract of the disclosure:

The use of inhibitors of plasminogen activators for the treatment of inflammations and wounds The invention relates to inhibitors of plasminogen activators, such as urokinase, uPA or tPA, for intra- and post-operative therapy, for the treatment of injuries and for the treatment and prophylaxis of inflammatory dis-orders. Particularly suitable inhibitors are PAI-1 and PAI-2.

Description

~ ~3 9 ~ 2 ~ 7 BEHRINGWERKE AKTIENGESELLSCHAFT KOE 92/B 009 - Ma 9~9 Dr. Bc/Wr The use of inhibitors of plasminogen activators for the treatment of inflammat.ions and wounds Regulation of plasmin activity is impaired in various disorders. Plasmin is formed from plasminogen by plas-minogen activators (PA) such as, for example, uPA (urin-ary PA) or tissue plasminogen activator (tPA). The PAs are inhibited by plasminogen activator inhibitors (PAI).
At present, two different types of physiolo~ically relevant PAIs are known: plasminogen activator inhibitor type 1 (PAI-l) and plasminogen activator inhibitor type 2 (PAI-2). Both PAIs are inhibitors of the serpin type.

In general, in cases of inflammations of the eyes, of the ears or of the skin, for example wh~n there is damage to the cornea, there is an increase in the plasmin activity in the lacrimal fluid. Plasmin is in turn thought to be responsible for the breakdown of fibron~ctin in th~
extracellular matrix, for a promotion of angiogenesis and for an activation of procollagenase to active collagen-ase, which results in the breakdown of collagen molecules (Barlati et al. Exp. Eye Res. 51, 1-9 (1990~). Lower molecular weight substances such as 2-aminocaproic acid or tranexamic acid (trans-4-aminoethyl-cyclohexan~-carboxylic acid) have already been employed ~or the I treatment of various pathological states in which increased plasmin activities play a part. Thus, or example, tranexamic acid has proved ~o be suitable for the treatment of osteoarthritis. It was likewi e possible to reduce the excessive fibrinolytic activity in ulcer-; ative colitis and related disorders with the aid of tranexamic acid. The abovementioned lower molecular weight plasmin inhibitors are, however, disadvantageous because of their toxic side effects.

:, :
.,:

. . ~ . ~ .

.~ . -.................... . .
, ~ :: ., :

() 7 Aprotinin represents another available plasmin inhibitor.
It has proven conditionally suitable for a tr0atment of corneal ulcer.

It has now been found, suxprisiLngly, that inflammations, especially of the eyes, of the ears and/or of the skin but also, for example, osteoarthritis or ulcerative colitis can advantageously be treated with inhibitors of plasminogen activators.

The invention therefore relates to the use of inhibitors of plasminogen activators for the preparation of a pharmaceutical for the therapy and/or prophylaxis of inflammations of the eyes, of the ears, of the skin, of os~eoarthritis and/or of ulcerative colitis.

Particularly inhibited are urokinase (uPA) and/or tPA, especially uPA and/or tPA. Suitable inhibitors are, in particular, PAI-1 and/or PAI-2 and the mutants or vari-ants thereof, especially PAI-2 J in particular fragments of PAI 2, for example deletions at the N-terminus, especially of the N-terminal amino acids l-B2 or 1-84 of matur~ PAI-2. The inhibitors can be used either alone or in combination with other medicam~nts, for example antibiotics, and both topical and systemic administration of them is possible. For osteoarthritis, the active substances are preferably ac~inistered intraarticularly or intraoperatively ~open or arthroscopic). In general, an effective dose for topical administration i5 1-300,000 urokinase inhibiting units and preferably 500-50,000 urokinase inhibiting units, and for i.v. - or for solubilized i.m. ac~inistration it is 50-750,000 urokinase inhibiting units per kg of body weight.

The inhibitors, especially PAI-2, can be stabilized by addition of physiologically tolerated stabilizers such as albumin, polygeline, gelatin hydrolysate, glycine, cysteine, glucose, lactose, maltose and/or -~ucrose.

Indications for the inhibitors are generally the topical therapy and/or prophylaxis of inflammatory eye disorders such as corneal ulcer, uveitis, conjunctivitis, for in~ra- and post-operative treatment, for protecting the structure of the eye, especially with an inflammatory course, or of inflammatory ear disorders such as, for example, inflammation of the middle ear, inflammation of the eardrum~ rupture of the eardrum with an inflammatory course, but also of skin disorders with inflammatory symptoms such as erythema, especially when there is infiltration of tissue by inflammatory cells such as macrophages, monocytes and granulocytes, acantholysis, vesiculation such as, for example, associated with pemphigus, ec~ema, contact dermatitis and atopic dermatitis, burns with an inflammatory course, vascul-itis, promotion of wound healinq, therapy of open wounds and dermal ulcers. Other indications are the treatment of epithelial lesions, osteoarthritis, ulcerative colitis, Crohn's disease, IBD (inflammatory bowel disease), pancreatitis, acne, treatment of epithelial lesions, corneal lesions, scarring associated with eye injuries, scleritis, non-healing coxneal erosions, xerosis, keratosis. Furthermore, the inhibitors are suitable, alone or in combination with other active substances, preferably as irrigation solutions for the intra- and post-operative treatment and~or for the treatment of open wounds of the said inflammations. Another advantageous mode of use is the treatment of contact lenses before wearing with a solution of the said inhibitors alone or in combination with other active substances to inhibit neovascularization, for the therapy of eye injuries and of eye inflammations and/or for the treatment of corneal infiltrates of the eyes and of corneal inflammations of the eyes of contact lens wearers.

Some preferred uses of inhibitors of plasminogen activ-ators such as, for example, PAI-2 are described in detail hereinafter:

.

, , ., ~ .

~,0~3~7 . ~

1. The use for treatment after injury to the cornea of ~he eye by laser keratectomy, but also by kerato-tomy. This laser keratectomy method can be used to correct the refractive i.ndex in short- and long-sighted people, to correct astigmatism, to correct corneal defects, to remove scars, for keratoplasty, for smoothing the cornea when there are manifes-tations of desiccation, for fresh in~uries to the cornea, for infections of the eye, for example by viruses (for example herpes), baoteria, fungi or parasites, for calcific deposits below the epi~
thelial layer (a manifes~ation of aging), for adhesions of the conjunctiva with the cornea (pterygia).

2. The use for inflammations of the eye and opacities of the eye, for example after laser treatment, normal ophthalmic surgery (for example vitrectomy, cataract surgery, extracapsular cataract extraction, lens operations, lens replacement, len~ implanta-2Q tion, keratoplasty, corneal transplantations), for conjunctivitis, keratoconjunctivitis, kerato-conjunctivitis sicca, iritis, iridocyclitis, kera-titis, Grave's ophthalmopathy, Mooren's ulcer~
vasculitis, uveitis, for allergic manifestations in the eye, infections, metabolic disorders, inflam-matory diseases and autoimmune diseases ~for example I systemic lupus erythematosus~ Wegener~s granulo-matosis, rheumatoid arthritis, sarcoidosis, poly-arthritis, pemphigus, pemphigoid, erythema multi-forme, Sjogren's syndrome, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, keratitis, scleritis.

3. The u~;e to prevent ~carring on the eye after surgics~l intervention or after injury.

4. The use for increasing the rate of wound healing and optimal regeneration tfor example smooth boundary between old stroma ~nd newly Eormed epithelium), for normalization and stabilization of the metabolic functions of the epithelium ~nd of the stroma after injury or sllrgical intervention.

5. The use to inhibit neovAsculari~ation associated with retinopathy, especially in diabetics; ~or detached retina, retinal vessel injury, for inflam-mation of the retina and uvea, for corneal trans-plantations.

6. The use in tr~nsplantations on the eye, especially for corneal transplantations. PAI~2 can in this case, owing to a recluction in the attraction of PMN, reduce the sensitization, prevent neovascularization without simultaneously having an adverse effect on wound healing. This diminishes the risk of rejection.

7. ~he use for edemas in the region of the eye, for example macula edema, submacula ede~lar edema after photocoagulation, corneal edema, con~unctival edema, retinal edema, edema in the vicinity of the eye.

8. The use for infiltrations in the region of the eye, espPcially of the cornea, of the chamber of the eye, of the conjuctiva and of the sclera.

9. The use for therapy of corneal ulcer.

PAI-2 can be prepared as follows~ for example: ~s des-cribed in EP 0,238,275, PAI-2 cDNA was isolated, cloned, ligated into vectors and transformed or transfected into suitable host cells by processes known to the person skilled in the art. The cells were fermented and dis-rupted by known processes. Suitable Eor purifying rPAI-2 , `.

from, for example, E.coli lysates is a combination of various chromatography methods (for example ion exchange chromatography on Q-Sepharose, hydrophobic chromatography on phenyl-Sepharose, gel fi:Ltration on Sephacryl or Fractogel). A process suitable for purifying rPAI-~ from yeast lysates comprises basic purification (cell removal, cell disruption by ball mill, cross-flow filtration, cross-flow concentration) and subsequent final purification (chromatographic processes as described, for example, in WO 91/02057 or by i.on exchange chromatography and immunoaffinity chromatography). The specific activity is generally 150,000 U/mg or more based on the inhibition of human urokinase.

The preparation of PAI-l can be prepared in an analogous way, for example as disclosed in WO 90/13648.

The advantages of the inhibitors of plasminogen activ-ators are in particular a monotherapy, which is generally non-toxic and free of side effects, of the said inflamma-tions with simultaneous inhibition of neovascularization.
Since the inhibition of the plasminogen activators allows considerably lower administration concentrations than in the case of plasmin inhibitors, simultaneous administration or combination with other groups of acti~e substances such as, for example, antibiotics is particul-arly advantageous. Furthermore, the amount of inhibitorsof plasminogen activators which can be administered is distinctly less than in the case of plasmin inhibitors and, moreover, the risk of an allergic reaction is generally reduced. An advantageously long duration of action compared with plasmin inhibitors has al~o been observed.

' . ' :, ,, ,.. ~

:. :

2~9~207 Example l:

Use of PAI-2 for the therapy of corneal ulcer In the rabbit model of corneal ulcer described by Cejkova et al. 1975 (Histochemistry 45: 71-75), PAI-2 was admin-istered in various concentrations (5-100 ~g/ml) locally into the eye (25 ~g/ml three times a day; 100 ~g/ml twice a day). The controls receiv~d the same volumes (1 ml) of the solvent (physiological sa]ine, pH 7.2) and were administered twice a day. The reference substance was aprotinin (5,000 IU/ml in the first two weeks, twice a day; the dose was reduced to 2,500 TU/ml in the following two weeks).

After the treatment with 0.75 M sodium hydroxide with the aid of a plastic tube of internal diameter 11 mm there is a massive influx of inflammatory cells: these secrete proteolytic activity and simultaneously induce keratino-cytes to synthesize a different proteoglycan spectrum on the corneal epithelium. This results in disturbances in the order structure of the cornea, which in turn leads to loss of transparency.

The following parameters were measured: perforation of the cornea, ulcer formation, neovascularization, plasmin activity in the lacrimal fluid, inflammatory features such as influx of inflammatory cells, transparency of the eye, visibility of the iris (Table 1). The following advantages compared with the placebo group emerge for the PAI-2 groups: it was possible to reduce the plasmin activity to a minimum. In order to chieve the s~me degree of reduction of plasmin activity with aprotinin, administration must be more frequent and more often. With PAI-2 the perforation and the ulcer formation were completely prevented. It was also possible with aprotinin to achieve these effects. Surprisinglyr with PAI-2 there was a drastic and lasting reduction, specifically to the . . ~ . ~ . . ., ; ~ , ;

. . . .:

~3~20~

normal extent found in healthy tis~ueV in the nul~er of inflammatory cells flowing in as a consequence of the experimental stimulus.

The following beneficial effects were, surprisingly, S additionally achievable: PAI-2 brought about the inhib-ition, which is absolutely necessary for complete healing, of neovascularization so that it was possible to restore the transparency of the eye, and the iris became visible again. Although on treatment with aprotinin ~he ulceration and the perforation of the cornea is pre-vented, the transparency remains lost. The bPneficial effects achieved even with relatively low PAI-2 dosages were not achievable with higher aprotinin dosages either.

Inhibition of plasminogen activatoxs by PAI-2 addition-ally displays even further advantages compared with the inhibition of plasmin, for example by aprotinin: treat-ment exclusively with inhibitors of plasminogen activators is sufficient for successful treatment, which represents an advantage compared with conventional therapy. The extremely high potency of the active prin-cipal makes it possible to use very low inhibitor concentrations so that a combination therapy, for example with antibiotics such as chloramphenicol, i5 also readily possible too.

In another experiment, the treatment o the corneal ulcer was started only after 10 days, not immediately after the induction, for example by exposure to alkali. At this late time, th~ plasmin activity in the lacrimal fluid has already risen considerably to values of 2-2~5 ~g/ml.

Starting aprotinin treatment (5,000 IU/ml; local adminis-tration twice a day) at the same time (10 days after the induction) results in the concentration of active plasmin detectable in the lacrimal fluid returning to normal values. This effect is, however, maintained for only . ~

:, . l , . , :

g about two hours, and then the plasmin levels rise again.

Starting PAI-2 treatment (fox example 100 ~g/ml); local administration) at this time re~ults in the acti~e plasmin concentration fal].ing to normal values (0.4-0.6 ~g/ml) again. The effect of a single PAI-2 administration surpxisingly persists for a long time.
Thus, for example, the plasmin concentration in the lacrimal fluid 48 hours after a single PAI-2 administration is still 1 ~g/ml. In this connection, all three PAI-2 concentrations t~sted (5, 25, 100 ~g/ml) were suitable for reducing the ulcerative process. The best therapy results were achieved with a PAI-2 concentration of 25 ~g/ml.

This surprisingly long-lasting efect is observed only on inhibition of plasminogen activators by PAI-2 but not on inhibition of plasmin by aprotinin.

Whereas local administra~ion of steroids, for example of dexamethasone-sodium phosphate, during treatment of the eye is associated with side effects such as, for Pxample, change in the swelling of the vi~reous body and in the intraocular pressure, treatment of the eye with PAI-2 shows no side effects in the normal eye. Thus, the histology such as, for example, the pattern of the lamellar structure of the anteriox stroma, as well as biochemical findings such as, for example, the hydrogen-ation of the cornea, enzymatic activities (lactate dehydrogenase, acid phosphatase, acid glycosidase, alkaline phosphatase, succinate dehydrogenase) remain unchanged. It was additionally possible to show that, besides the successful inhibition of inflammation, there was no inhibition of reepithelization.

Measurement of the plasmin activity in the lacrLmal fluid:

.

~c~35207 Small paper disks (Whatman filter paper; 5 mm diameter) were impregnated in a substrate solution of D-Val-Leu-Lys-trifluoromethylaminocoumarin (FCA, from Enzymes Systems Products, Livermore, CA, US~) and dried. The substrate solution was pxepared as follows: 1 mg of substrate was dissolved in 4 drops of N,N-dimethyl-formamide, and 1 ml of 0.1 M tris-HCl pH 7.2 was added.
Plasmin solutions with 0.2-0.5 ~g/ml were prepared by dissolving plasmin (Sigma) in 0.1 M tris-HCl buffer of pH 7.2. A 20 ~l drop of each concentration was placed on the paper disks pretreated with substrate solution, and they were then incubated in a humidity chamber at 37C
(in a thermostat) together with the paper disks impregnated with lacrimal fluid. To determine the plasmin concentration in the lacrimal fluid, the paper disks were placed on th~ cornea, and the lacrimal fluid was absorbed for 5 seconds. During the incubation of the paper disks at 37C they were examined under W light at intervals of 2 hours. The intensity of the yellow fluorescence, emitted under W light, of the lacrimal fluid samples was compared with the samples of known plasmin concentration (calibration plot from paper disks of known plasmin concentration), and the plasmin activity of the samples was thus determined.

Table 1:
Treatmen~ of alkali-induced corneal ulcPr Test groups:

Group 1: Placebo group (physiological saline;
administered twice a day) 30 Group 2: Aprotinin group (5,000 IU/ml administered twice a day) Group 3: rPAI-2 (lO0 ~g~ml; administered twice a day) Group 4: rPAI-2 ( 25 ~g/ml; administered three times a day) . ~

2 ~9~2~7 Parameter Result of treatment in the ~ test groups after treatment for 4 weeks _______________________~___________________________ ___ 5 Transparency Iris visible - - - ~
Inflammation ~ weak - -Increased plasmin activity + - - -Neovascularization ~ + weak 10 Ulcer formation + +
Perforation +

~xample 2:

Use of PAI-2 for regeneration of the corneal epithelium The effect of PAI-2 on the regeneration of corneal epithelium was investigated in a rabbit model.

Description of the model:

The investigations were carried out with adult chinchilla rabbits (body weight 2.5-3.0 kg). The animals were anesthetized by i.v. administration of thiopental (25 mg/ml/kg of body weight). The eyes were then immobilized and the cornea was mechanically injured: the entire cornea was carefully deepitheli~ed twice (at an I interval o one week) using a Graefe knife. Subsequently PAI-2 (in PBS buffer, pH 7.2) was administered in various concentrations (5-50 ~g/ml) locally into the eye (3 times a day). The control animals received the same volumes (0~5 ml) of PBS buffer (pH 7.2) or aprotinin (60 ~g/ml3 or flurbiprofen (0.1% solution) in PBS buffer, pH 7.2.
The animals were sacrificed at various times (after 1, 4, 30 7, 14, 21, 28 days) after production of the injury. The corneal structure was examined histologically and histo-chemically. The plasmin activity in the lachrymal fluid ,, .

- ~2 ~ 5207 was determined and correlated with the histological data.
Twice a week, the optical appearance of the eyes was photographically recorded. The plasmin activity was determined as described in Example 1. After sacrifice of the animals, the eyes were immediately enucleated, the cornea or the complete anterior segment of the eye was excised.

Some corneae were subjected to a McGovern silver impreg-nation technique (1955). After the epithelium had been stripped off, the specimens were placed in gelatin. All the other methods were carried out with the complete anterior segments of the eye. One part was quenched in light petroleum spirit which was cooled with a mixture of acetone and dry iceO In a cryostat, sections (12 ~m thick) were performed parallel or perpendicular to the surface of the eye. The sections were then transferred to slides or semipermeable membranes which had not been precooled and were used for investigations for glucos-aminoglycans, aminopeptidase M (APM), dipeptidylpeptidase IV (DPP IV), gamma-glutamyltransferase (GGT), alkaline phosphatase (AlP), Na+/K+-dependent adenosine triphos-phatase (ATPase) and dehydrogenases (succinate dehydro-genase SDH, lactate dehydrogenase LDH).
Before detection of APM, DPP IV, GGT and AlP, the sec-tions were fixed for 2 minutes with a cold mixture of chloroform and acetone (l:l). AlP was detected by an azo coupling method using naphthol-AS-MX-phosphate (Calbiochem, La Jolla; Ca, USAj and fast blue B salt (Michrome, Gurr, Poole, UX). APM was detected using Ala-4-methoxy-~-naphthylamine~Ala-MNA (Bachem, Bubendorf, Switzerland) and fast blue B salt (FBB, Michrome, Gurr, UK), DPP IY using Gly-Pro-MNA (Bachem; Switzerland) and fast blue B salt (Michrome, Gurrl UK), GGT using gamma-Glu-MNA (Bachem, Switzerland), Gly-Gly (Lachema, Brno, Czechoslovakia) and fast blue B by the method of Lojda et al. (1979~. Sections on semipermeable membranes were used for the detection of acid phosphatase (AcP) - by means of .
.. ..

3~07 ~ 13 -an azo coupling reaction with naphthol-AS-BI-phosphate (Calbiochem) and hexazonium p-rosaniline, which was prepaxed from acridine-free rosaniline (Merck, Darmstadt, Germany) - and of ~-glucuronidase (p-glu) and N-acetyl-~-D~glucosaminidase (Ac-Glu) - substrates: naphthol-AS-BI-~-glucuronide and naphthol-AS-BI-N-ace-tyl-~-D-glucos-amir~ide (both from Calbiochemj; the coupling reagent hexazonium p-rosaniline was used for both. Acid ~-galac-tosidase (~-Gal) was detected by means of the indigogenic method of Lojda (Cejkova and Lojda, 1975; Lojda et al., 1979) using 4-Cl-5-Br-3-indolyl ~-D-galactoside (Cyclo Chemicals, Los Angeles, CA, USA).
ATPase was detected by the method of Cejkova and Lojda (1978) using ATP-Tris and Ba2+ salt. Dehydrogenases were detected in unfixed cryostat sections by the method of Lojda et al. (1979) using nitro BT (Lachema, Brno, Czechoslovakia) as electron acceptor. To detect glucos-aminoglycans, the sections were fixed in cold ethanol (5 minutes) and stained with a 1% strength alcian blue solution which contained various concentrations of MgCl2 (Cejkova et al., 1973), but also with a 1% strength solution of alcian blue in 3% strength acetic acid, pH
2.5. The remaining part of the anterior segment of the eye was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, and then sectioned in a cryostat.
12 ~m-thick section~ were transferred to albumin-coated slides, thawed and dried. Fixed sections were used to detect DPP IV (with Gly-Arg-MNA, Bachem, Swit2erland, and nitrosalicylic aldehydP, NSA, Merck) and DPP II (with Lys-Pro-MNA and fast blue B) (method of Lojda, 1985 and Cejkova and Lojda 1986). Some of the fixed corneae were examined for morphological alteration (using hematoxylin-eosin staining).

Buffer control:
In the untreated eye, the plasmin activity increases from 0.2-0.4 ~g/ml within 2 hours ater the injury. A plasmin activity of l.0-2.0 ~g/ml is reached after one day and .

352~7 remains at this level up to day 7, then falls to 0.2-0.4 ~g/ml by day 14 and reaches a level below 0.2 ~g/ml after 21 days (see Tab. 2).
Epithelial regeneration starts from the limbus, and the S epithelium contains various enzyme activities (DPP IV, acid glycosidases and lysosomal proteases (see Tab. 3)).
Within 4 days after the deepithelization, a massive influx of inflammatory cells (especially polymorpho-nuclear neutrophilic granulocytes, PMN) into the stroma is observed. Corneal neovascularization takes place in the periphery. Some keratinocytes become necrotic. These are replaced by keratinocytes from the vicinity. The reepithelization is complete after 14 21 days. Adverse consequences which should be mentioned are angiogenesis and loss of transparency ~the transparency was not completely restored). The corneal opacity which occurred was irreversible.

Treatment with flurbiprofen:
The plasmin activity in the lacrimal fluid increased in a similar way to the buffer control group (see Tab. 2).
In contrast to the buffer control, the influx of inflam matory cells into the stroma was less, and neovasculariz-ation was impeded but not blocked. In some eyes no reepithelization took place, and in some cases it was even inhibited. The corneal epithelium showed only very low activities of ATPase and GGT. The data are compiled in Tab. 3.
I

Treatment with aprotinin or PAI-20 Throughout the course of the experiment the plasmin activities were significantly reduced. The maximum levels were 1.0 ~g/ml in the aprotinin group and only 0.4 ~g/ml in the PAI-2 groups (see Tab. 2). The activities of DPP
IV, acid glycosidases, lysosomal hydrolases are only relatively weak in both treatment groups, and those of ATPase and of GGT are normal. On treatment with aprotinin, only relatively few inflammatory cells migrate .~
, ~ ~
: . .' ~0~3~ 2 07 into the stroma, and almost none are now detectable in the PAI-2 groups~ Transparency was restored in both treatment groups. Wound healing was not complete in the aprotinin group until 14 days had elapsed, while wound healing was already complete again in the PAI-2 groups on day 10 a~ter deepithelization. ~leovascularization of the cornea could not be pre~ented by aprotinin treatment, whereas treatment with PAI-2 (in all concentrations used) completely blocked angiogenesis.

The advantages of trPatment with PAI-2 or inhibitors of plasminogen activators thus comprise an increase in the rate of reepithelization, a complete blockade of neo-vascularization, a complete and rapid (more rapid than with aprotinin or another substance) restoration of transparency. PAI-2 suppresses inflammation of the cornea and of the stroma very well. Thus, for example, scarcely any PMN are detected as having migrated into the region of inflammation. The enzymatic activities of the corneal epithelium normalize again very rapidly during treatment with PAI-2. An additional advantage is regarded as being the ~act that the PAI-2 concentrations used were ~ar lower than those of aprotinin. PAI-2 was still completely active even at concentrations of 10 ~g/ml. Associated with this is the advantage of PAI-2 that PAI-2 can possibly also be administered simultaneously with other active substances.

2~9~2~7 Tab. 2 PL~S~IN ACTIVITY IN THE TE~ FL~D (~g~ml) Repeated de-epithelialization Time ~h,d) ~uffer r-P~I-2 Aprotinin Flurbi-pro~en .

2 h 0.2-0.4 0.2-0.4 < 0.20.2-0.4 1 d 1.0-2.0 0.2-0.4 0.2-0.41.0-2.0 4 d 1.0-2.0 0.2-0.4 0.4-1.01.0-2.0 7 d 1.0-2.0 0.2-0.4 0.2-9.41.0-2.0 14 d 0.2-0.4 < 0.2 < 0.20.2-0.4 21 d < 0.2 < 0.2 .

;: , ~ .,- ' :

~V~2~7 Tab. 3 Repeated co~e~ de ~pithelis~io of the ra~bit ~ye 'rest g~oups Parameter Bu~Plur~iprofen Apro~in rPA~
con~ol l 10 mg/ml ] ~o ~g/ml ~ [ 10 ~Lg/ml 3 .. . __ ~

1. Plas~un ac~vity slig~t no in tear fluid ~ease Increase Inc~ease ~c~ease Enymatic ac~ es in coTneal epi~elium - DP~ IV I low slight slight - AQd ~Iycosidases I low sli~t slight - Lysosomal hydrolases ~ low slight slight - Na+ - K+ -ATPase low - GGI - low ~ ~

3. PhIN in strom~ Numerous 10w low AN~yt 4. ReepithelisationNosmal Enabled Accelera~d after tl~ di~) Pro10~ 14d~y3) (1Odays) wound healin~

5. Vascu~arisationP~onounced~ ~d ) Pronounc~d Blocked 6. Transparency ~ve~iblePar~y Restored R~tored of co~nea Cloody C~ V ~9~ I Y) .

' :

~0~35~7 Example 3:
PAI-2 for the treatment ~f eye injuries and for intra-and post-operative treatment The experiments were carried out and analyzed as described in Example 2. ~owever~ in this case, the epithelium was not stripped off, but the cornea was injured by a scalpel incision which penetrated into the stroma to a depth of 60-80% of the thickness of the stroma and covered the entire surface of the cornea (5 mm from the limbus). After an incision into the cornea, the plasmin activity in the lacrimal fluid increases x~la-tively rapidly (within 2 hours) and it remains at rela-tively high levels for several days (see Table 4).
Inflammatory cells migrate into the area of the wound after only one day. It is then possible also to detect increased activities of lysosomal proteases and of glycosidases in the direct vicinity thereof. Epithelial cells migrate in to the incision wound from day 1 to day 7. The incision wound is refilled with epithelial cells after 10 days. Adhesion of the cells which have migrated in to the underlying stroma is, however, very poor. The actual wound closure therefore tends to take place slowly and haltingly. In the wound healing the arrangement of the collagen fibrils was irregular.
Glycosaminoglycans were not detected at the site of the wound. The activities of glycosidases, especially of ~-galactosidases, were increased in the same region. It was evident from the macroscopic appearance that the transparency of the cornea was in some cases temporarily lost in the region of the incision wound ~ the maximum was between day 4 and day 7 during the wound-healing phase. The cornea healed with a non-transparent scar tissue. The changes in the corneal transparency were irreversible. The data are compiled in Tab. 5~

::

~ 0 9 ) 2 0 7 Treatment with PAI-2:
Only a minimal plasmin activity was detectable in the lacrimal fluid of the animals treated with PAI-2 throughout the treatment time. The maximum activity reached was 0.4 ~g/ml. In contrast to the buffer control group, inflammation was complPtely stopped in the animals treated with PAI-2. Thus, for example, on day 1 no inflammatory cells were detectable in the immediate vicinity of the stroma wound on the eyes managed with PAI-2. Activation of keratinocytes took place relatively early. The epithelial cells wh:ich migrated in had only relatively low activities of lysosomal hydrolases, whereas the activities of GGT, ATPase and SD~ were normalized again very early. After only 4 ~ays the incision wound was completely refilled with epithelial cells. These adhered very well to the underlying stroma.
The originally gaping incision wound which had been produced was completely leveled oPf again as soon as day 10. The lower-lying layers of the stroma healed with involvement (metabolic and cell-division activity) by the neighboring keratinocytes. Wound healing was complete after 14 days. The newly formed keratinocytes showed normal metabolic activity (for example concerning GGT), nor was there any evidence of inflammation. Overall, treatment with PAI-2 made it possible for wound healing to take place at a consid~rably greater rate and with a considerable improvement in terms of quality. The corneal wound healing resulted in completely transparent tissue, and scar formation was en~irely absent. The findings which are important for the patient wPre achievable with both PAI-2 concentrations (10 mg/ml and 20 ~g/ml).

The PAI-2 data are compiled in Table 5.

Treatment with aprotinin:
Compared with the buffer control group, the plasmin concentration in the lacrimal fluid was reduced (see Tab.
3). The incision wound heals at the normal rate ~lower ,.

.

2~2~7 than with PAI-2). The activity of lysosomal hy~rolases and glycosidases was reduced, comparable with the effect of PAI-2, in the epithelial cells which migrated in. The adhesion of the newly formed epithelium to the stroma was not as strong as in animals treated with PAI-2. The incision wound healed more slowly but resulted in trans-parent tissue. The histochemistry of the site of the wound was very similar to that in animals treated with PAI-2.

Buffer control group.
In the animals in the buffer control group, the plasmin activity in the lacrimal fluid was increased for a longer time (see Tab. ~). At the same time, a marked influx of inflammatory cells (predominantly PMN) into the cornea i5 observed. The lytic potential of the epithelium, composed of, for example, lysosomal hydrolases, was increased, while the activity of GGT and ATPase was reduced. Wound closure itself was very slow (about 10 days), adhesion of the newly formed epithelium to the underlying stroma was very poor, white scars were formed, and transparency was partly, and irreversibly, lost.

The advantages of treatment with PAI-~ or with inhibitors o~ the plasmin/plasminogen activator system are accord-ingly that only in this way is there rapid and completely satisfactory - completely transparent, no scars, good adhesion, no neovascularization, normal metabolic activ-ity of the cornea ~ restoration of the cornea, also on a histological basis.

20!J~207 Tab. 4 PhA~YIN ACTIVITY IN T~E T~AR ~L~ID ~g/ml) Corneal deep incision wound ~ime (h,d) 9uffsr r-PAI-2 Aprotini~
_ 2 h 0.2-0.4 0.2-0.4 c 0.2 1 d0.4~1.0 0.2-0.4 0.2 0.4 4 d 1.0-2.0 0.2-0.4 0.4-1.0 7 d 1.0-2.0 0.2-0.4 0.4-1.0 14 d 0.4-1.0 0.2-0.4 0.2-0.4 21 d 0.2-0.4 < 0.2 < 0.2 28 d < 0.2 '~ '`: ' .,. ~ . .

' ' '` .` ' .' . ',~', "'` . ,' ' ~ .. '' '' ', , ' ~. . . ' ' . ~

^' .'. ' ' . ~,'' ,, " , ' . i. ` .' . .

~09~ 2~7 Tab. 5 Co~e~ deep illci~ion woundL
o~ the ra~bit; eye . . . _... ,...... . . . _ . __ r~t ~oups pa~ eter Buffer control Apro~in rPAI-2 [~O~Lg/ml] 1 l0sr20~Lg _ _ . ._ _ _ _ _ Tea~ id - Plasmin ac~vity high increase Reduced nearly no increase ~uqltra~on p~'s Numerow R~e Abs~t Epi~elium - Lysosomal HydroIasesIn~eased Low ~ow - GGT Decreased Nonnal Nomal (early - ATP~se De~seased No~nal Nom~ y Wound he~
- Wound c~osureSlowly Nonnal accele~ated ~lOda~) (4days) - Epithelial adhesion Bad Acceptable Very good to s~roma - Scar fo~ma~on ~
- TranspasencyPar~ally ]Dst T~an~a~ent Transpasent ~rever~ible - ;;; .

,: ~ <. - : .

"
' i ' ' :' ,' - ~ ' ~ :

2 0~f3 ~2 ~7 ~xample ~:

Use of PAI-2 for treatment after laser suxgery Photorefractive laser keratectomy was performed on rabbits by the method described by Lohmann et al. 1991 (193 nm axcimer laser). Subsequently, PAI-2 was adminis-tered locally into the eye at a concentration of 20 ~g/ml (two to three times a day). The control group received the same volume of the solvent (20 mM glycine pH 7.2;
150 mM NaCl, 0.3% gelatin hydrolysate). The re~erence group received either aprotinin (5,000 IU/ml; twice a day; 2 weeks) or 0.1% prednisolone acetate (twice a day, 1 week; once a day, 11 weeks) or 0.1~ flurbiprofen (see prednisolone acetate). The parameters measured were the development of opacity, plasmin concentration in the lacrimal fluid and the histological findings.

In the control group the plasmin concentration rose within a short time from about 1 ~gtml to levels of Up to ~g/ml, but normalized again after one week. The closure area between the newly formed epithelium and the remaining stroma was not smooth. In addition, newly formed matrix material, for example collagen, was detect-able. A few Yacuoles were observed in the stroma. Without treatment, there is a tendency to regress to short-sightedness. As a conse~uence of the formation of faulty structures and of de novo syntheses, opacity of the eye was to be observed. The result observed on treatment with prednisolone acetate is in principle the same, merely the de novo synthesis of collagen being somewhat reduced, whereas on treatment with 1urbiprofen a significant de novo synthesis of collagen is to be observed, beside the opacity. On treatment with aprotinin, the boundary between old stroma and the newly formed epithelium is smooth. Nevertheless, opacity of the cornea related to reflection of light is observed. No vacuoles were observed, and the basal cells were of normal size. New - . . ~

: ,;

~.

2~35~07 - ~4 -collagen was synthesized, however. Treatment with PAI-2 results in an absolutely clear, transparenk eye (see Table 6). No opacity is found even after more than three months - this normally appears no later than after three to five weeks. The epithelium is perfectly regenerated:
the boundary between old stroma and newly formed epithe-lium is smooth. There are no deposits, no vacuoles, no de novo synthesis of collagen and no hyperplasia of the epithelium. No regression to shortsightedness was observed. Even if all the reference substances are combined together the result of treatment is no better than that in the group treated with PAI-2. The disadvan-tages of this combination therapy and of therapy with reference substances compared with treatment with PAI-2 are:
- Topical administration of steroids for three months or longer entails an unwanted and high risk (depen-ding on the degree of ablation) of the formation of secondary glaucomas.
- Aprotinin has proven incompatible with prednisolone - Hypersensitivity reactions to aprotinin are known - BSE problems, degree of purity, foreign-protein nature are against the use of aprotinin.

.

~9~207 TAB. 6 THERAPY AFTER LASER KERATECTOMY:
Duration Opacity Epithe- De novo of treat- lial collagen ment surface synthesis (months) (-> light reflection) Predni- 3 + irregular solone 10 Flurbi- 1 -3 ~ irregular ++
profen Aprotinin 0.5-1 + regular ++

P.prot./Pred.
Flurbi-15 profen 3 + regular _ PAI-2 1 - regular 1) but de novo synthesis of an as yet unknown substance " : :~
.,"
:, , ,.
. : . ~ .. ::

Claims (22)

1. The use of inhibitors of plasminogen activators for the preparation of a pharmaceutical for the therapy and/or prophylaxis of inflammations of the eyes, of the ears, of the skin, of the bones, of the joints, of the intestine or other internal organs and/or for intra- and/or post-operative treatment and/or for the treatment of open wounds.

2. A pharmaceutical as claimed in claim 1, wherein the plasminogen activators are urokinase and/or tPA.

3. A pharmaceutical as claimed in claim 1, wherein the inhibitors are PAI-1 and/or PAI-2, preferably fragments of PAI-2.

4. A pharmaceutical as claimed in claim 1 for the treatment of corneal ulcer, uveitis, conjunctivitis, inflammations of the middle ear, inflammations of the eardrum, rupture of the eardrum with inflam-matory course, erythema of the skin, acantholysis, pemphigus, eczema, contact dermatitis, atopic dermatitis, burns with inflammatory course, vas-culitis, dermal ulcers, osteoarthritis, IBD (Inflam-matory Bowel Disease), pancreatitis, acne, treatment of epithelial lesions, treatment of corneal lesions, for the treatment of scarring associated with eye injuries, for the treatment of scleritis, non-healing corneal erosions, xerosis and/or keratosis.

5. A pharmaceutical as claimed in claim 1 for the intra- and post-operative treatment and/or the treatment of open wounds of the said inflammations.

6. A pharmaceutical as claimed in claim 1 for the inhibition of neovascularization, for the treatment of corneal infiltrates of the eyes or of corneal inflammations of the eyes of contact lens wearers.

7. A pharmaceutical as claimed in claim 1, wherein the operative treatment is a laser keratectomy and/or the open wounds or inflammations are a consequence of laser keratectomy.

8. A pharmaceutical as claimed in claim 1 for the treatment of inflammations of the eye and opacities of the eye, for example after laser treatment, normal ophthalmic surgery (for example vitrectomy, cataract surgery, extracapsular cataract extraction, lens operations, lens replacement, lens implanta-tion, keratoplasty, corneal transplantations), and as a consequence of the following disorders: con-junctivitis, keratoconjunctivitis, kerato-conjunctivitis sicca, iritis, iridocyclitis, kera-titis, Grave's ophthalmopathy, Mooren's ulcer, vasculitis, uveitis, for allergic manifestations in the eye, infections, metabolic disorders, inflam-matory diseases and autoimmune diseases (for example systemic lupus erythematosus, Wegener's granulo-matosis, rheumatoid arthritis, sarcoidosis, poly-arthritis, pemphigus, pemphigoid, erythema multi-forme, Sj?gren's syndrome, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, keratitis, scleritis.

9. A pharmaceutical as claimed in claim 1, to prevent scarring on the eye after surgical intervention or after injury.

10. A pharmaceutical as claimed in claim 1, to inhibit neovascularization associated with retinopathy, especially in diabetics; for detached retina, retinal vessel injury or inflammation of the retina and uvea, or for corneal transplantations.

11. A pharmaceutical as claimed in claim 1 for use in transplantations on the eye, especially for corneal transplantations.

12. A pharmaceutical as claimed in claim 1 for the treatment of edemas in the region of the eye, for example macula edema, submacula edema, edema after photocoagulation, corneal edema, conjunctival edema, retinal edema, edema in the vicinity of the eye.

13. A pharmaceutical as claimed in claim 1 for the treatment of infiltrations in the region of the eye, especially of the cornea, of the chamber of the eye, of the conjunctiva and of the sclera.

14. A pharmaceutical as claimed in claim 1 for increas-ing the rate of wound healing, especially in ophthalmology.

15. A pharmaceutical as claimed in claim 1 for local injection, such as, for example, injection into subconjunctiva, vitreous body, chamber of the eye or sclera.

16. A pharmaceutical as claimed in claim 1 for adminis-tration by microinjection.

17. A pharmaceutical as claimed in claim 1 for use as irrigation solution.

18. The use of inhibitors as claimed in claim 1 in combination with a biodegradable vehicle system.

19. The use of inhibitors as claimed in claim 1 in combination with a pharmaceutically acceptable carrier system such as, for example, cellulose derivatives, polymeric matrices or contact lenses.

20. The use of inhibitors as claimed in claim 1 in combination with stabilizers.

21. The use of inhibitors as claimed in claim 1 in combination with other active substances.

22. The use of inhibitors as claimed in claim 1, wherein one or more antibiotics are used as other active substances.