SK41593A3 - Using of inhibitors of plasminogene activators for the production of medicament for a therapy of ignitions and wounds - Google Patents

Abstract

The invention relates to inhibitors of plasminogen activators such as urokinase, uPA or tPA for intra- and postoperative therapy, for the treatment of injuries and for the treatment and prophylaxis of inflammatory disorders. Suitable inhibitors are, in particular, PAI-1 and PAI-2.

Description

Technical field

The invention relates to the use of plasminogen activator inhibitors for the manufacture of medicaments for the therapy and / or prophylaxis of inflammatory diseases.

BACKGROUND OF THE INVENTION

In various diseases, regulation of plasma activity is impaired. Plasmins are generated from β-azminogen by plasminogen activators (PA), such as urinary β-azminogen activator (uPA) or tissue β-azminogen activator (tPA). Plasminogen activators (PAs) are inhibited by plasmin activator inhibitors (PAIs).

Two different physiologically relevant types of PAI are currently known: Plasminogen Activator Inhibitor Type 1 (PAI-1) and Plasminogen Activator Inhibitor Type 2 (P AI-2). Both PAIs are a new type of pump.

In case of inflammation of eyes, ears or skin, eg. in case of corneal damage, plasmin activity in the tear fluid is generally increased. Plasma n is responsible for the breakdown of fibronectin in the extracellular matrix, for stimulating anginogenesis, and for activating procollagenase to more active collagenase, resulting in the degradation of collagen molecules (Barlati et al., Exp.Eye Res.51,

1-9 (1990)). Previously, low molecular weight substances such as α1α-aminocaproic acid, or trans-4-aminoethylcyclohexanecarboxylic acid (tranexamic acid) have been used to treat various pathological conditions in which plasmin activity is increased. Thus, for example, tranexamic acid has been shown to be useful in the treatment of osteoarthritis. By using tranexamic acid, excessive fibrin activity in corneal ulcer and similar diseases could be reduced. However, a disadvantage of the aforementioned low molecular weight plasmin inhibitors is their toxic side effects.

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Another possible reptile is new aprotinin, conditionally suitable (corneal at 1 c e r).

The inhibitor has been shown to treat corneal ulcer

SUMMARY OF THE INVENTION

A surprising finding was that inflammations, especially eyes, ears and skin, but also e.g. osteoarthritis, or colitis ulcerosa, can be successfully treated with inhibitors of β 1 azminogen activators.

Accordingly, it is an object of the invention to use β 1 azminogen activator inhibitors for the manufacture of a medicament for the therapy and / or prophylaxis of inflammation of the eyes, ears, skin, osteoarthritis and / or colitis in cerebrospinal fluid.

In particular, urokinase (uPA) and / or tPA, in particular uPA and / or tPA, was inhibited. Suitable inhibitors are, in particular, PAI-1 and / or PAI-2 and mutants or variants thereof, in particular PAI-2, in particular fragments of PAI-2, for example deletion at the N-terminus of particularly N-terminal amino acids 1-82 and 1-84, respectively. at PAI-2. Inhibitors can be used either alone or in combination with other drugs, for example anti-biotic agents, where they can be used both locally and for systemic treatment. In osteoarthritis, the active compounds were administered primarily intra-articularly or intraoperatively (often arthroscopically). Generally, an effective dose for topical use lies between 1-300,000 urokinase inhibiting units, preferably between 500-50,000 urokinase inhibiting units, for i.v. application, or for i.m. application using a solvent between 50-750,000 urokinase inhibiting units per kg body weight i.

Inhibitors, in particular PAI-2, can be stabilized by the addition of physiologically acceptable stabilizers such as albumin, polygelin, gelatin hydrolyzate, glycine, cysteine, glucose, lactose, maltose and / or sucrose.

Fields of application of inhibitors are generally topical therapies and / or prophylaxis of inflammatory ocular diseases such as corneal ulcer, uveitis, for surgical or post-operative treatment, for the protection of ocular structures;

especially in the case of inflammatory courses, in inflammatory ear diseases such as e.g. middle ear inflammation, eardrum inflammation, rupture of the eardrum with an inflammatory course, but also in skin diseases accompanied by inflammations such as rose, especially in tissue infiltration by inflammatory cells such as macrophages, monocytes and granulocytes, acantholysis, such as blisters. pemphigus, eczema and contact atopic skin inflammations, inflammatory burns, vasculitis, to promote wound healing, open wound therapy and skin ulcers. Other areas of application are treatment of epithelial lesions, osteoarthritis, colitis ulcerosa, Cron's disease, IBD (inflammatory bowel disease), pancreatitis, acne, treatment of epithelial damage, corneal damage, scar formation in eye injuries, scleritis, non-treatment ulcer erosions , xerosis and keratosis. Furthermore, the inhibitors are suitable either alone or in combination with other active ingredients, in particular as wash solutions for intra and post-operative treatment and / or for the treatment of said open wound inflammation. Another preferred use is the treatment of contact lenses prior to use with a solution of said inhibitor, either alone or in combination with other active agents for inhibiting neovascular disease, for the treatment of eye injury and inflammation and / or for treating corneal infiltrate or corneal inflammation induced by contact wear. lenses.

The following is a description of several preferred uses of plasminogen activator inhibitors such as P AI-2:

Use in treatment after corneal injury with 1 aserkeratek tome, but also with keratotomy. These aserkeratectomy methods 1 can be used in refractive index correction for nearsightedness and farsightedness, for correction of astigmatic grain, for correction of corneal defects, for scar removal, for keratope 1, for corneal smoothing during drying, for fresh corneal injury, in eye infections such as viruses (such as herpes virus), bacteria, fungi or parasites, lime deposits under the epithelium (a phenomenon associated with

Aging), with the corneal conjunctiva (pituitary).

Use in eye inflammation and eye opacity, for example after laser surgery, normal eye surgery (e.g. vitrectomy, cataractchi rurgi i, extracapsular cataractextraction, lens surgery, lens replacement, lens implantation, keratop 1 asti i, corneal transplantation) in conjunctivitis vitju , keratoconjugate vitis sicca, iritis, ir idocyclitis, keratitis, cataract, Mooren ulcer, vasculitis uveitis, eye allergies, infections, metabolic disorders, inflammatory diseases, and auto-immune diseases (e.g. systemic Lupus erythovirus, Worm, rheumatoid arthritis, sarcoidosis, after 1yarthri tis, pemphigus, pemphigoid, erythema multiforme, Sjoegren's syndrome, intestinal inflammatory diseases, sclerosis mu 11 ip 1 ex, Myasthenia gravis, keratitis, scleritis.

Use to prevent scar formation in the eyes after surgery or injury.

Use for accelerated wound healing and optimal regeneration (for example, smooth contact areas between old tissue and newly formed epithelium), to normalize and stabilize epithelial function in metabolism and tissue after injury or after surgery.

Use for inhibiting neovascularization in retinopathy, particularly in diabetic patients, in retinal wash, retinal vessel breakdown, retinal inflammation and uvea, in corneal transplants.

Use in eye transplants, especially corneal transplants. Herein, PAI-2 can reduce sensitivity by reducing the affinity of PMN, preventing neovascularization without, at the same time, adversely affecting wound healing. This reduces the body's negative reactions.

Use in eye swelling such as macula and submacula swelling, photocoagulation swelling, corneal swelling, conjunctival swelling, retinal swelling, and swelling around the eye.

Use in eye infiltration such as cornea, eye chamber, conjunctiva and sclera.

Use in Corneal ulcer therapy.

PAI-2 can e.g. according to the method disclosed in EP-O-238-275, PAI-2 - cDNA was isolated in a conventional manner, cloned, transferred to vectors and transformed in suitable host cells. The cells were regenerated and disintegrated in a known manner. For purification of rPAI-2 from Escherichia coli lysates, e.g. suitable combinations of various chromatographic methods (eg ion exchange chromatography on Q-Sepharose, hydrophobic chromatography and on Phenylsepharose, gel filtration through Sephacryl or Fractogel. For purification of rPAI-2 from yeast lysates, a procedure consisting of a coarse purification (cell separation, desinte ball-mill cells, countercurrent filtration and countercurrent concentration) Subsequent purification to a higher degree (by chromatographic methods such as disclosed in WO-91/02057, or by ion exchange chromatography and immunoaffinity chromatography) Specific activity is generally 1 50 000 units / mg or more, related to the inhibition of human interest and name.

PAI-1 can be prepared in an analogous manner, for example according to WO 90/13648.

In particular, the benefits of inflammations without inhibitors of activators of β 1 azminogens in the generally non-toxic monotherapy of side effects, while at the same time relying on the above mentioned neovascularization. Since the plasma inhibitor concentrations of the novel activators used are substantially lower than the plasma concentrations of the novel inhibitors, co-administration with other active substances is possible, of which a combination with antibiotics is particularly advantageous. Furthermore, since the useful amount of plasma inhibitors of the novel activators is clearly lower than that of the plasma of the novel inhibitors, the risk of allergic reactions is generally reduced. The advantage is an i d 1 hour effect compared to new reptiles.

Example 1

Use of PAI-2 for Corneal ulcer therapy

In the rabbit model described by Cejkova et al. (H stochemistry 45: 71-75) for Corneal ulcer, PAI-2 was applied at 5-100 µg / ml) to (25 µg / ml eye of various concentrations)

3 times a day, 100 µg / ml twice a day). The control group received equal volumes (1 mL) of solvent (saline, pH 7.2) applied twice daily. The reference substance was aprotinin (5000 International Units / ml in the first two weeks, twice daily; in the next two weeks the dose was reduced to 2500 International Units / ml).

Treatment with 0.75 M sodium hydroxide using a plastic tube with an internal diameter of 11 mm induced a massive increase in inflammatory cells. others exhibited proteolytic activity and at the same time induced keratinocytes synthesizing a different spectrum of proteoglycans on the corneal epithelium. As a result, the proper structure of the cornea is disrupted, further leading to a loss of transparency.

The following parameters were measured: perforation ulcer formation, neovascularization, plasma new activity fluid, signs of inflammation such as eye transparency, visibility with the control group, the following advantages were observed in the PAI-2 group: plasmin activity could be reduced to a minimum.

current of inflammatory cornea, in tear cells, iris (Tab1). Compared to

More frequent application is required to reduce plasma activity of the same by aprotinin equally. For PAI-2, perforation and ulceration were completely prevented. These effects can also be achieved by aprotinin. In PAI-2, there was a surprisingly high and sustained reduction in the incidence of inflammatory cells that occurred after experimental intervention to the level of healthy tissue.

The subsequent positive effects, surprisingly due to PAI-2, which prevented neovascularization, led to complete recovery. Thus, the transparency of the eye and the iris visibility could be restored. Although aprotinin treatment does not cause ulceration and perforation of the cornea, it does not restore transparency. Positive results already achieved with a relatively low dose of PAI-2 were not achieved even with higher aprotinin doses.

Inhibition of plasminogen activators by PAI-2 has, unlike inhibition of plasmin, e.g. aprotinin, yet other advantages. Exclusive treatment with plasminogen activator inhibitors is sufficient for a successful treatment, which is an advantage over previously used therapy. The extremely high effect of the active ingredient used permits the application of the inhibitor at very low concentrations, which also allows for combination therapy, for example with antibiotics such as chloramphenicol.

In another experiment, treatment of the corneal ulcer was not performed directly after induction, for example with caustic, but started only after 10 days. At this later time, the reptile activity in the tear fluid has increased to 2 - 2.5 zum / ml. If aprotinin treatment (5000 International Units / ml, topical administration twice daily) is initiated at this time (10 days after induction), the detectable concentration of active plasmin decreases to normal. However, this effect lasts only about two hours after which the plasmin value rises again.

When PAI-2 treatment is initiated at this point in time (e.g., 100 µg / ml, topical application), the active plasmin concentration decreases to normal values (0.4 - 0.6 zug / ml). Surprisingly, the effect of a single application of PAI-2 lasts for a very long time. The value of plasmin concentration in the tear fluid is, for example, after 48 hours. after a single application

PAI-2 still only 1 zug / ml. At all three concentrations of PAI-2 (5.25,100 zug / ml) tested, the ulcerative process could be suppressed. The best therapeutic results were achieved using a PAI-2 concentration of 25 µg / ml.

This surprisingly long-lasting effect was only observed with inhibitors of plasminogen activators PAI-2, but not with healing by plasmas with novaprotinin.

While topical application of steroids, such as dexamethasone sodium phosphate, is associated with side effects in the treatment of the eye, such as changes in ocular sphere volume and ocular pressure, treatment of the eye with PAI-2 in the normal eye has no side effects. Histology, e.g. a sample of the lamellar structure of the anterior tissue, but also biochemical findings such as e.g. corneal hydration, enzymatic activities (lactate dehydrogenase, acid phosphatase, acid glycosidase, alkaline phosphatase, succinate dehydrogenase) remain unchanged. In addition, it has been shown that, in addition to successful inflammation, it did not inhibit repercussion.

Measurement of plasmin activity in tear fluid:

The small wheels of filter paper (W hat man filter paper with a diameter of 5 mm) were soaked in the substrate solution D-va 1 - 1 eu-1-s-1 rifluoromethylamine amine (FCA, Enzyme System Products, Livermore, CA, USA) and dried. The substrate solution was prepared as follows: 1 mg of the substrate was dissolved in 4 drops of Ν, Ν-dimethylformamide and 1 ml of 0.1 M β-HCl pH 7.2 was added. Plasmin solutions at a concentration of 0.2 0.5 µg / ml were prepared by dissolving plasmin (Sigma) in 0.1 M 1 mM HCl-buffer pH 7.2. 20 _of each concentration of 1 was dropped on a paper circles that were pre-impregnated with a solution of a substrate. they were then incubated in a humid chamber at 37 ° C (in a thermostate), together with paper wheels impregnated with tear liquid. To determine the concentration of plasmin in the lacrimal fluid, the paper wheel was placed on the cornea and the lacrimal fluid was soaked for 5 seconds. During the incubation of the paper wheels at 37 ° C, the paper wheels were inspected at 2 hour intervals in UV light. The intensity of the yellow tear fluid fluorescence induced by UV irradiation was compared with the fluorescence of a known plasmin concentration (a calibration curve obtained from paper wheels at a known concentration of plasmin) to determine the plasmin activity of the samples.

Table 1

Caustic-induced corneal ulcer treatment

Tested groups

Group 1

Group 2

Group 3

Group 4

Parameter Control group (saline; twice daily) aprotinin group (5000 International Units / ml; twice daily) rPAI-2 (100 zug / ml, twice daily) rPAI-2 (25 zug / ml, three times) daily)

Treatment result of 4 test groups after 4 weeks

3 4

Transparencia - - - + Visibility of the iris - - - + inflammation + weak - - Increased plasma activity nov + - - - Neovasculature 1 + + weak - Ulcer formation + + - -

perforation

1

Example 2

Use of PAI-2 to regenerate corneal epithelium

The effect of PAI-2 on corneal epithelial regeneration was studied in a rabbit model

Mode description 1 u

Experiments were carried out on chinchilla adult rabbits (2.5-3.0 kg body weight. Anesthesia was also administered with thiopental (25 mg / kg body weight). The eyes were then fixed and the cornea mechanically damaged: the whole cornea was cautiously twice deepite1 izova1a (within one week) using a Graef knife, then PAI-2 (in PBS buffer, pH 7.2) was applied topically to the eye three times daily at various concentrations (5-50 zug / ml). volume (0.5 mL) of PBS buffer solution (pH 7.2) or aprotinin (60 µg / ml) or flurbiprofen (0.1% solution) in PBS buffer solution, pH 7.2.

Animals were killed at various times after injury (1, 4, 7, 14, 21, 28 days). The structure of the cornea was examined histologically and biochemically.

Plasmin activity was determined in the tear fluid and correlated with histological data. An optical image of the condition of the eyes was photographed twice a week.

Plasmin activity was determined as described in Example 1. After the animals were sacrificed, the eyes were immediately removed and the cornea or whole anterior eye segment was excised.

Several corneas were subjected to silver impregnation according to McGovern (1955). After contraction of the epithelium, the preparations were placed in gelatin. All other methods were performed with whole anterior segments. A portion was immersed in light gasoline, which was cooled with a mixture of acetone and solid carbon dioxide. Sections (12 µm thick) were made in the cryostate parallel or perpendicular to the eye surface. The sections were then transferred in advance

2 uncooled preparation carrier or semipermeable membrane, and assayed for g 1 ukosamycin, am inopeptidase M (APM), dipeptide 1 peptidase IV (DPP IV), gamma -glutamyltransferase (GGT), alkaline phosphatase (A1P), aden, Na * -K * '(ATPase) and dehydrogenase (succinate dehydrogenase SDH, 1 actate dehydrogenase LDH).

Prior to APM, DPP IV, GGT, and A1P detection, sections were fixed for two minutes in cold chloroform / acetone (1: 1). AlP detection was performed by the azocopy method using naphthol-AS-MX-phosphate (Calbiochem, La Jolla: Ca, USA) and Fast Blue-B salt (Michrome, Gurr, Polle, UK). AMP was detected with A1a-4-methoxy-beta-naphtha-1-amino-a1a-MNA (Bachem, Bubendorf, CH) and Fast Blue-B salt (FBB, Michrome Gurr, UK), DPP IV with gly-pro-MNA (Bachem , CH) and Fast Blue-B salts (Michrome Gurr, UK), GGT using gamma-glu-MNA (Bachem, CH), gly (Lachema, Brno, CSFR) and Fast Blue-B, according to Lojda et al. (1979). Sections on semipermeable membranes were used to detect acid phosphatase (AcP) by an azocope of fasting with naphthol-AS-BI-phosphate (Calbiochem) and hexazonium p-rosin, prepared from rosaline pure acridine. (Merck, Darmstadt, Germany) and from beta-g 1 ukuronidase (beta-glu) and from N-acetyl-beta-D-glucosaminidase (Ac-Glu), Substrates: Naphthol-AS-BI-beta-glucuronide and naphtho 1-AS -BI-acetyl-beta-D-glycosamine (both from Ca1 biochem). Both substrates were subjected to a coupling reaction with hexzonium-β-rosaniline. Acid beta-galactosidase was detected using the Lojda indigomethod (Cejkova, Lojda, 1975, Lojda et al., 1979) using 4-C1-5-Br-3-indolyl beta-D. -ga 1 activity (Cyc 10 Chemica 1, Los Angeles, CA, USA).

ATPase was detected according to Cejkova and Lojda (1978) using ATP-Tris and barium salt.

Dehydrogenases were detected using nitro BT (Lachema, Brno, ČSFR) as electron acceptor in non-fixed cryostat sections according to Lojda et al. (1979).

For evidence of g 1 ukosaminoglycans, sections were fixed for 5 min. in cold ethanol and stained with 1% alcian blue solution containing different concentrations of magnesium chloride (Cejkova et al., 1,973), but also with 1% alcian blue solution

3 in 3% acetic acid, pH 2.5.

The remaining portion of the anterior eye segments was fixed in 4% paraformaldehyde in 0.1M phosphate buffered pH

7.2 and then cut in a cryostate. 12 µm thick sections were transferred to a carrier coated with anbumin, thawed and dried. Fixed sections were used to detect DPP IV (with Gly-arg-MNA, Bachem, CH, and nitrosylic acid aldehyde, NSA, Merck) and DPP II (with lys-pro-MNA and Fast Blue-B ( Lojda, 1985, Cejkova and Lojda, 1986) Morphologic changes were examined for ingestion of hematoxa1 by inesosin in some fixed corneas.

Control - buffer solution

In the untreated eye, plasmin activity rises to 0.2-0.4 zug / ml within two hours after injury. After one day, the plasmin activity reaches 1.0 - 2.0 zug / ml and remains at that value until day 7, and then decreases to day 0.2 to 0.4 xug / ml by day 14, reaching 21 less than 0.2 zug / m 1 (Table 2).

Epithelium regenerates and contains various enzymatic activities (DPP IV, acid glycosidases and lysosomal proteases (Table 3). Massive growth of inflammatory cells (especially polymorphic neutrophil granulocytes, PMN) observed in supporting connective tissue occurs within 4 days after deepitelization. Some keratinocytes do not necrotize and are replaced by local keratinocytes After 14 to 21 days, the relapse is complete and the negative consequences include angiogenesis and loss of transparency (transparency and not completely recovering again).

The resulting corneal opacity was also reverse.

Flurbiprofen treatment:

Plasmin activity in a single liquid is increasing similar to that of the buffer solution control group (Table 2).

Unlike the buffer solution control, fewer inflammatory cells flowed into the supporting connective tissue, neovascularization attenuated but did not stop. In a few cases, relapse 1 did not occur, often even inhibiting. Corneal epithelium showed only very low activity of APlase and GGT. The results are shown in Table 3.

Aprotinin or P AI-2 treatment:

Throughout the course of the experiment, the activity of plasmin decreased significantly. The maximum values in the versus new group reached ľ0.0 µg / ml, in the PAI-2 group only 0.4 µg / ml (Table 2). The activities of DPP IV, acid glycosidases and 1-isosomal hydrolase are relatively low in both groups and ATPase and GGT activities are normal.

When treated with aprotinin, only a relatively small amount of inflammatory cells enter the supporting connective tissue, almost none were found in the PAI-2 group.

In both groups, transparency was restored again.

In the aprotinin group, the wound recovered only after 14 days, while in the PAI-2 group, the wound was re-occluded as early as 10 days after treatment. The use of aprotinin cannot prevent neovascularization of the cornea, while the use of PAI-2 (at all concentrations used) completely blocked the diagnosis.

Thus, the advantages of treatment with PAI-2, optionally with inhibitors of β-amino-activator activators, are the acceleration of re-epithelialization, complete cessation of neovascularization, complete and rapid (faster than aprotinin or other agents) restoration of transparency. PAI-2 suppresses corneal inflammation and supporting connective tissue very well. For example, almost no PMNs were found in the inflammation region. Corneal epithelial enzymatic activities normalized very rapidly using PAI-2. As a further advantage, it is appreciated that the concentrations of PAI-2 used are much less than that of aprotinin.

PAI-2 is still fully active even at a concentration of even 10 µg / ml. This is associated with the advantage of PAI that:

5 can be applied concurrently with other active substances.

Table 2

Plasmin activity in tear fluid (zug / ml)

Repeated deepitelization

Time (H, d) buffer solution R-PAI-2 A p r o t i n n flurbiprofen 2 h 0.2-0.4 0.2-0.4 <0.2 0.2-0.4 2 d 1, 0-2.0 0.2-0.4 0, 2-0-4 1, 0-2.0 4 d 1.0-2.0 0.2-0.4 0.2-0.4 1, 0-2.0 7 d 1.0-2.0 0.2-0.4 0.2-0.4 1.0-2.0 14 d 0.2-0.4 <0.2 <0.2 0.2-0.4 21 d <0.2 <0.2

6

Table 3

Repeated deepite1 and rabbit eyes

Tested groups

parameter

Buffer solution

F 1 urb i profen

A rtin t rinPI-2

| (k o n t r o 1 ä) 1 [10 g / m 1] [60 zug / ml] [10 mg / ml] 1. r 1 Activity p 1 and z m n u | weak no in 1 with liquid I increase increase increase increase 2. 1 Enzymatic activities in the epithelium [cornea | - DPP IV - acid.gly- | + low weak weak k o z d i z | + low weak with 1 abá - lysosome. | hydrolase + low weak weak - To * -K * - | - low + + ATPase | - GGT | | - low + + 3. 1 PMN in tissue- | nive | numerous low low nearly no 4. 1 Reep i te1 i zá- normal p r e díí - impossible zrých1 e- cia po za- (14 days) a woman (<1 4 days) on the only morning in | (10 days) tissue

7

Table 3, continued

5. Vasku 1 a- | r i zác i a | 1 | a clear i nh i bo- (no blocked a clear b 1 forged 1 6. Transparen- | i nver z. partially restored restored corneal cervix 1 cloudy cloudy (Close) (Easy)

Example 3

Use of PAI-2 to treat eye injury and post-operative treatment

The experiments were performed and evaluated similar to those described in Example 2. However, here the epithelium was not selected, but the cornea was disrupted by incision at 1 and 1 u into the supporting connective tissue to a depth of 60-80% of the thickness of the supporting connective tissue. 5 mm from the edge). After corneal incision, plasmin activity in the tear fluid increases (over 2 hours) and remains at relatively high levels for several days (Table 4).

Inflammatory cells migrate to the site of injury as early as 1 day. Increased activity of 1-isosomal proteases and glycosidases can also be demonstrated in the immediate vicinity.

From day one to day seven, epithelial cells migrate into the incision wound. After 10 days, the incision is refilled with epithelial cells. However, the adhesion of the growing cells to the underlying connective tissue is very poor. This heals the wound slowly and laboriously. The arrangement of co-agene fibrils is irregular in this healing.

G 1 y goats inog1 yko1 y were not found at the wound site. The activities of glycosidases, in particular beta-galactosidases, were increased in the same region.

From a macroscopic perspective, the corneal transparency in the area of the incision is temporarily lost; the maximum loss is between 4 and 7 days of the healing phase.

The cornea was cured to form a non-transparent scar tissue. Changes in corneal transparency were irreversible. The data are summarized in Table 5.

Treatment with PAI-2;

During the entire treatment period, minimal plasmin activity was detected in the tear fluid in PAI-2 treated animals. The maximum activity achieved was about 0.4 µg / ml.

In contrast to the control group (buffered solution), inflammation in PAI-2-treated animals stopped completely. Thus, for example, no inflammatory cells in PAI-2-treated eyes in the immediate vicinity of the supporting connective tissue injury were detected on the first day.

Keratinocytes activated relatively quickly. The epithelial cells that migrated migrated had only relatively low activities of 1 isosomal hydrolases, whereas GGT, ATPase and SDH activities again normalized rapidly.

After only 4 days, the incision was completely filled with epithelial cells. These cells very well at Tn 1 as well as the underlying fibrous ligament underlying them. Already after 10 days the originally wounding wound was quite flat. Adjacent keratinocytes (metabolic activity and cell division activity) were involved in the cure of the underlying layers of supporting connective tissue. After 14 days, wound healing was complete. ^Novo formed keratinocytes displayed normal metabolic activities (e.g., the activity of GGT). There was also no inflammation in any case.

Treatment with PAI-2 allows, in summary, a substantial acceleration of wound healing and a substantial improvement in its quality. When the wound is healing on the cornea, a completely transparent tissue is formed and no scarring occurs. These are important for patients

Effects can be obtained at both concentrations (10 µg / ml and 20 µg / ml). The results obtained with PAI-2 are summarized in Table 5.

Ošetrenieaprotinínom:

Compared to the control group (buffer solution), the concentration of plasmin in the tear fluid decreased (cf. Table 3).

The wound heals at a normal rate (slower than P AI-2).

Compared to the effect of PAI-2, the activity of 1-isosomal hydrolase decreased in the doped epithelial cells.

The adhesion of the newly formed epithelium to the supporting connective tissue was not as strong as in animals treated with PAI-2. Cutting wounds heal more slowly, but transparent tissue has formed. Histochemically, the site of injury was very similar to that of PAI-2 animals.

Control group (with buffer solution):

Plasma activity in the lacrimal fluid was increased for a longer period in the control group (buffered) animals (Table 4). At the same time, a strong incidence of inflammatory cells (predominantly PMN) was observed in the cornea.

The lytic potential of the epithelium consisting of, for example, lysosomal hydrolase, increased, while the activity of GGT and ATPase was reduced. The wound closure itself was very slow (approximately 10 days) and the adhesion of the newly formed epithelium to the underlying fibrous tissue below was very poor. A white scar and transparency was formed and was partially- irreversibly lost.

The advantages of treatment with PAI-2 and / or inhibitors of the system of plasminogen include the fact that this route results in a faster and more satisfactory restoration of the cornea in a transparent manner, without scars, with good adhesion, without neovascularization and with normal corneal metabolic activity, even on a histological basis.

Table 4

Plasmin activity in tear fluid (/ µg / ml)

Deep cut in cornea

Time buffer R-PAI-2 and a (h, d) solution

2 h 0.2-0.4 0.2-0.4 <0.2 2 d 0,4-1,0 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

Table 5

Deep cut in the rabbit eye

Tested groups parameter buffer aprotinin R-PAI / 2 inspection [60 / gg / ml] [10 or 20 zug / m 1] tear drip on i - activity high reduced almost uneven plasmin increase infiltration -PMN numerous rare a mistake Epithelium i um - 1 yzozomá1. increased low low hydro Iase -GGT lowered normal normal (comming soon) -ATPase lowered normal normal (comming soon) Wound healing -uzatvore- porn 1 é standard 1 no accelerated not wounds (10 days) (4 d n i) - a d h é z i a epithelium i a of 1 á acceptors very of a t e r n good -Creation scars + - - -transpa-

Table 5. continued rencia | Partial scars to irreves. tissue lost transparent transpar e n t a n

Example 4

Use of PAI-2 for treatment after laser surgery

Photoreactive laser keratectomy (193 nm excimer laser) was performed on rabbits as described by Lohman et al. (1991). PAI-2 was then applied topically to the eyes (2 to 3 times daily) at a concentration of 20 µg / ml. The control group received an equal volume of solvent (20 mM glycine pH 7.2, 150 mM N and Cl, 0.3% gelatin hydrolyzate). The reference group contained either aprotinin (5000 International Units / ml, 2 times a day, 2 weeks), or 0.1% before it with 1-acetate (2 times a day, 1 week; once a day, 11 weeks), or 0, 1% flurbiprofen (preferably 1 onacetate).

Turbidity formation, plasmin concentration in the lacrimal fluid and histological findings were used as measurement parameters.

In the control group, plasmin concentration increased from about 1 µg / ml in a short time to values up to 50 µg / ml. However, after a week it normalized. The newly formed area between the epithelium and the surviving connective tissue was not smooth. In addition, it was possible to prove a newly formed matrix material, for example co-agen.

Occasionally, vacuoles were observed in the supporting connective tissue. Untreated, there is a tendency to regression to myopia.

Eye opacity was observed as a result of the formation of defective structures and neosynthesis. In principle, the same result was observed with anterior solonacetate treatment, but only a new synthesis of the wheel 23 gene was somewhat reduced, while in flurbiprofen treatment a new collagen synthesis was observed alongside the haze.

In aprotinin treatment, the boundary area between the old supporting connective tissue and the newly formed epithelium is smooth. However, corneal opacification due to light reflection was observed. No vacuoles were observed and also the size of the underlying cells was normal. However, a new collagen was synthesized.

Treatment with PAI-2 resulted in an absolutely clean, transparent eye (see Table 6). No turbidity was observed even after three months, although it develops normally after three to five weeks. Epithelium perfectly regenerated:

The boundary area between the old supporting connective tissue and the newly formed epithelium is smooth. No scarring, no vacuoles, no new collagen synthesis and no epithelial hyperplasia were observed.

No regression to myopia was observed.

Even when the combination of all reference substances is applied together, the treatment results are no better than. treatment results of the PAI-2 treated group.

The disadvantages of this combination therapy or reference therapy compared to PAI-2 treatment are as follows:

- three months and longer topical application of steroids presents an undesirable and high risk (depending on the degree of damage) of secondary glaucoma formation

- aprotinin cannot be combined with prednisolone

- allergic reactions to aprotinin are known

- BSE, the degree of purity and the nature of the foreign protein also oppose the use of aprotinin.

4

Table 6

Laser keratectomy therapy

Time Turbidity treatment (months) surface epithelium New synthesis 1 gene => r e f 1 e x i a world 1 o) prednisolone 3 + irregularly Delno + F 1urb i profen 1-3 + irregularly Delno + + aprotinin 0,5-1 + regular + + Aprot./Pred. flurbiprofen 3 + regular - 1> PAI-2 1 regular -

) Synthesis of a new yet unknown substance

Claims (22)

PATENT CLAIM Use of plasminogen activator inhibitors for the manufacture of / δZczt / tV for the treatment and / or prophylaxis of inflammation of the eyes, ears, skin, bones, joints, intestines or other internal organs, and / or for surgical and / or postoperative treatment, and / or on the *. treatment of open wounds. Use according to claim 1, characterized in that the β-aminogen activators are urokinase and / or tPA. Use according to claim 1, characterized in that the inhibitors are PAI-1 and / or PAI-2, preferably fragments of PAI-2. The use of the Plasminogen Activator Inhibitors according to claim 1 for the treatment of corneal inflammation, uveitis. t 'i v tids, inflammation of the middle ear, inflammation of the eardrum, will break! inflammatory eardrum, redness of the skin, acanthilosis pemphigus, eczema, tactile and atopic skin inflammations, inflammatory burns, vasculitis, skin ulcers, bone joint inflammations, intestinal inflammations, i. IBD, pank4 reatitis, acne, as well as for the treatment of epithelium, for the treatment of corneal injury, for the treatment of scars in eye injuries, for scleritis, non-healing corneal erosions, xerosis and / or keratosis. Use of plasminogen activator inhibitors according to claim 1, for operative and / or postoperative treatment and / or for the treatment of open wounds or for the treatment of the aforementioned inflammations. Use of plasminogen activator inhibitors according to claim 1, for neovascularization, for the treatment of ocular corneal infiltrates and / or corneal inflammations in persons using contact lenses. Use according to claim 1, characterized in that the surgical treatment is laser-keratectomy and / or open wounds or inflammations are the result of laser-keratectomy. The use of the Plasminogen Activator Inhibitors according to claim 1 for the treatment of ocular inflammations and cataracts, for example after laser treatment, after normal eye surgery. surgery, such as vitrectomy, surgical surgery, extracapsular cataract surgery, ophthalmic lens surgery, ophthalmic lens replacement, ophthalmic lens implantation, keratoplasty, corneal transplantation, as well as the consequences of other diseases such as conjunctivitis, keratoconjunctivitis , keratoconjunctivitis sicca, Irit d dy, iridocyclitidy, keratitis, cataract, More _n: ovho ulcer, vasculitis, uveitis, ocular allergic phenomena, infections, metabolic disorders, inflammatory diseases and diseases associated with autoimmunity, such as systemic lupus erythematosus, Wegener's syndrome, rheumatoid arthritis, sarcoidosis, polyarthritis, pemphigus, pemphigoid, erythma multiforme, S y egren's syndrome, inflammatory skin diseases, inflammatory bowel diseases, sclerosis mu 11 ip 1 ex, myasthenia da, keratitis. C Use of plasminogen activator inhibitors according to claim 1 for preventing scar formation in the eye after surgery. ¹ procedure or injury. Use of the Plasminogen Activator Inhibitors according to claim 1 for inhibiting neovascularization in retinopathy, particularly in diabetics, retinal detachment, retinal vessel injury or retinal inflammation, and uvea or corneal transplantation. Use of plasminogen activator inhibitors according to claim 1 in ocular transplantation, in particular in corneal transplantation. Use of the Plasminogen Activator Inhibitors according to claim 1 for the treatment of edema around the eye, for example, macular edema, submacula edema, photocoagulation edema, corneal edema, conjunctival edema, retinal edema, eye edema. Use of plasminogen activator inhibitors for the treatment of infiltrates in the eye area, particularly of the cornea, ocular ventricle, conjunctivae and whites. · ' Use of plasminogen activator inhibitors according to claim 1 for accelerating the treatment of wounds, particularly in the ophthalmological field. C Use of a plasminogen activator inhibitor according to claim 1 for topical injection such as injection into a subconject, vitreous humor, ocular ventricle or whites. Use of plasminogen activator inhibitors according to claim 1 for microinjection. Use of plasminogen activator inhibitors according to claim 1 as irrigation solution. . Use of plasminogen activator inhibitors according to claim 1 in combination with a biodegradable carrier system; i! ί Use of plasminogen activator inhibitors according to claim 1 in combination with a pharmaceutically acceptable carrier system such as cellulose derivatives, polymer matrices or contact lenses. Use of the plasminogen activator inhibitors according to claim 1 in combination with stabilizers. i í s 21. Use of plasminogen activator inhibitors according to i I This claim 1 in combination with other active ingredients. 22. Use of the inhibitors according to claim 1, characterized in that at least one antibiotic is used from the other active substances.