Note: Descriptions are shown in the official language in which they were submitted.
~ ~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
-- 2 --
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.
2 0 7
-- 3 --
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
,
`.
2 0 7
-- 6 --
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
-- 7
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~
-- 8 --
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 , . , :
2 0 7
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:
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~c~35207
-- 10 --
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
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Parameter Result of treatment in the ~ test
groups after treatment for 4 weeks
1 2 3
_______________________~___________________________ ___
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
- 14 -
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
- 15 -
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
- 16 -
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
-- 17 --
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
- 18 -
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
-- 19 --
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
- 20 -
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
-- 21 --
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
-- 22 --
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
- 23 -
~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
- 25 -
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
" : :~
.,"
:, ,
,.
. : . ~ .. ::
