U.S. patent application number 10/494004 was filed with the patent office on 2005-03-03 for non-neurotoxic plasminogen activating factors for treating stroke.
Invention is credited to Medcalf, Robert, Schleuning, Wolf-Dieter, Sohngen, Mariola, Sohngen, Wolfgang.
Application Number | 20050048027 10/494004 |
Document ID | / |
Family ID | 7704253 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050048027 |
Kind Code |
A1 |
Sohngen, Mariola ; et
al. |
March 3, 2005 |
Non-neurotoxic plasminogen activating factors for treating
stroke
Abstract
The invention concerns the use and the production of
non-neurotoxin plasminogen activating factors, derived for example
from the common vampire Desmodus rotundus (DSPA), for therapeutic
treatment of stroke in humans. The invention provides a novel
therapeutic base for treating stroke in humans.
Inventors: |
Sohngen, Mariola;
(Deutschland, DE) ; Sohngen, Wolfgang;
(Deutschland, DE) ; Schleuning, Wolf-Dieter;
(Deutschland, DE) ; Medcalf, Robert; (Blackburn,
AU) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Family ID: |
7704253 |
Appl. No.: |
10/494004 |
Filed: |
September 24, 2004 |
PCT Filed: |
October 31, 2002 |
PCT NO: |
PCT/EP02/12204 |
Current U.S.
Class: |
424/85.1 ;
514/12.2; 514/14.7; 514/14.8; 514/17.7; 514/2.4 |
Current CPC
Class: |
A61K 38/49 20130101;
A61P 7/02 20180101; C12Y 304/21069 20130101; A61P 9/00 20180101;
A61P 31/00 20180101; A61P 25/28 20180101; A61K 45/06 20130101; A61P
9/10 20180101; A61P 29/00 20180101; C12N 9/6459 20130101; A61P
25/00 20180101; A61P 43/00 20180101; A61K 31/7068 20130101; C12Y
304/21068 20130101 |
Class at
Publication: |
424/085.1 ;
514/012 |
International
Class: |
A61K 038/19 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 2, 2001 |
DE |
10153601.1 |
Dec 17, 2001 |
EP |
01130006.8 |
Claims
1. A method for treating stroke comprising administered to a
subject in need of such treatment, a therapeutically effective
amount of a plasminogen activating factor, wherein the the
plasminogen activating factor has an enhanced fibrin specificity
compared to wild-type t-PA.
2. The method according to claim 1, wherein the plasminogen
activating factor comprises at least a histidine or serine residue
forming together with an aspartic acid residue at least a part of a
zymogen triade.
3. The method according to claim 2, wherein the serine residue is
located at a position at least partly homologous to position 292 of
t-PA, the histidine residue is located at a position at least
partly homologous to position 305 of t-PA and the aspartic acid
residue is located at a position at least partly homologous to
position 447 of t-PA.
4. The factor according to claim 3, wherein the plasminogen
activating factor is selected from the group consisting of:
t-PA/R275E; t-PA/R275E, F305H; t-PA/R275E, F305H, A292S.
5. The method according to claim 1, wherein the plasminogen
activating factor contains a point mutation at Asp194 or of an
aspartic acid residue in a homologous position, which reduces
stability of the catalytically active conformation of the
plasminogen activating factor in the absence of fibrin.
6. The method according to claim 5, wherein Asp194 is substituted
by glutamatic acid or asparagine.
7. The method according to claim 6, wherein t-PA carries a
substitution at Asp194 to Glu194 or Asn194.
8. The method according to claim 1, wherein the plasminogen
activating factor comprises at least one mutation in its autolysis
loop, which reduces functional interactions between plasminogen and
plasminogen activating factor in the absence of fibrin.
9. The method according to claim 8, wherein at least one mutation
in the autolysis loop affects the amino acids at positions 420 to
423 of wild-type t-PA or at positions homologous thereof.
10. The method according to claim 9, wherein the mutation is
selected from the group consisting of: L420A, L420E, S421G, S421E,
P422A, P422G, P422E, F423A and F423E.
11. The method according to claim 1, wherein the plasminogen
activating factor is a zymogen comprising at least one point
mutation preventing catalysis by plasmin.
12. The method according to claim 11, wherein the point mutation is
located at position 15 or 275 of t-PA or at a position homologous
thereto.
13. The method according to claim 12, wherein glutamatic acid is at
position 15 or 275.
14. The method according to claim 1, wherein the plasminogen
activating factor is isolated from the saliva of the vampire bat
(DSPA).
15. The method according to claim 1, wherein said plasminogen
activating factor is administered to a human at least 3 hours after
onset of a stroke in said human.
16. The method according to claim 1, wherein said plasminogen
activating factor is administered to a human at least 6 hours after
onset of a stroke in said human.
17. The method according to claim 1, wherein said plasminogen
activity factor is administered to a human at least 9 hours after
onset of a stroke in said human.
18. The method according to claim 1, wherein onset of stroke in
said subject is temporally not exactly determined.
19. The method according to claim 1, wherein said plasminogen
activating factor does not exhibit neurotoxicity of wild-type
t-PA.
20. An isolated tissue plasminogen activating factor comprising an
autolysis loop comprising His420, Asn421, Ala422 and Cys423.
21. The isolated tissue plasminogen activating factor according to
claim 20, wherein said factor further comprises a point mutation at
position 194, which reduces stability of the catalytically active
conformation of the plasminogen activating factor in the absence of
fibrin.
22. An isolated tissue plasminogen activating factor according to
claim 21, wherein said point mutation Phe194.
23. An isolated tissue plasminogen activating factor according to
claim 20, wherein said factor comprises at least one point mutation
which prevents catalysis by plasmin.
24. An isolated tissue plasminogen activating factor according to
claim 23, wherein said point mutation is Glu275.
25. An isolated tissue plasminogen activating factor consisting of
an amino acid sequence as shown in Seq. ID No. 1.
26. An isolated urokinase comprising an autolysis loop comprising
Val420, Thr421, Asp422 and Ser423.
27. The isolated urokinase according to claim 26, wherein said
urokinase has a point mutation at position 194, which reduces
stability of the catalytic active conformation of the urokinase in
absence of fibrin.
28. The isolated urokinase according to claim 27, wherein said
point mutation is Glu194.
29. The isolated urokinasee according to claim 26, wherein said
urokinase has at least one point mutation which prevents catalysis
by plasmin.
30. The isolated urokinase according to claim 29, wherein said
point mutation is Ile275.
31. An isolated urokinase consisting of an amino acid sequence as
shown in Seq. ID No. 2.
32. A pharmaceutical composition comprising a plasminogen
activating factor according to claim 1 and at least one additional
pharmaceutically active component or a pharmaceutically acceptable
salt thereof.
33. The pharmaceutical composition according to claim 32, wherein
said component is a neuroprotective agent.
34. The pharmaceutical composition according to claim 33, wherein
said component is a glutamate receptor antagonist.
35. The pharmaceutical composition according to claim 34 wherein
said component is a competitive or non-competitive antagonist.
36. The pharmaceutical composition according to claim 33, wherein
said component is a thrombin inhibitor.
37. The pharmaceutical composition according to claim 33, wherein
said component is an anticoagulant agent.
38. The pharmaceutical composition according to claim 33, wherein
said component is an anti-inflammatory agent.
39. The pharmaceutical component according to claim 33, wherein
said composition is an antibiotic agent.
40. The pharmaceutical composition according to claim 33, wherein
said component is citicholine.
41. (Cancelled).
42. A method for the production of a non-neurotoxic plasminogen
activating factor comprising at least one of the following steps:
introducing a mutation in at least a part of a zymogen triade of a
plasminogen activating factor; introducing a substitution mutation
at Asp194 or a homologous aspartic acid residue to reduce
stabilization of the catalytic active conformation in the absence
of fibrin; introducing a substitution mutation in the hydrophobic
amino acid residues in the autolysis loop or in homologous regions
thereof; and introducing a mutation in a zymogen to prevent
catalysis of the zymogen by plasmin.
43. A drug produced by the process according to claim 42.
44. The pharmaceutical composition according to claim 36, wherein
said thrombin inhibitor is selected from the group consisting
thrombomodulin, thrombomodulin analogues, triabin, pallidipin and
solulin.
45. The pharmaceutical composition according to claim 37, wherein
said anticoagulant is selected from the group consisting hirudin,
heparin, acetylsalicylic acid and ancrod.
Description
[0001] The invention pertains to the therapeutic use of
non-neurotoxic plasminogen activators especially from the saliva of
Desmodus rotundus (DSPA) preferentially for the treatment of
stroke.
[0002] Different clinical pictures are summarized under the term
"stroke" which correlate in their clinical symptoms. According to
the respective pathogenesis a first differentiation between these
clinical pictures in so called ischaemic and haemorrhagic insults
is possible.
[0003] Ischaemic insults (ischaemia) are characterized in a
reduction or interruption of the blood circulation in the brain due
to a lack of arterial blood supply. Often this is caused by
thrombosis of an arteriosclerotic stenosed vessel or by arterio
arterial, respecitively, cardial embolisms.
[0004] Haemorrhagic insults are based inter alia on the perforation
of brain supplying arterias damaged by arterial hypertonia.
However, only approximately 20% of all cerebral insults are caused
by haemorrhagic insults. Thus, stroke due to thrombosis is much
more relevant.
[0005] In comparison to other tissue ischaemias the ischaemia of
the neuronal tissue is widely accompanied by necrosis of the
effected cells. The higher incidence of necrosis in neuronal tissue
can be explained with the new understanding of the phenomenon
"excitotoxicity" which is a complex cascade comprising a plurality
of reaction steps. The cascade is initiated by ischaemic neurons
affected by a lack of oxygen which then lose ATP instantaneously
and depolarize. This results in an increased postsynaptic release
of the neurotransmitter glutamate which activates membrane bound
glutamate receptors regulating cation channels. However, due to the
increased glutamate release glutamate receptors become over
activated.
[0006] Glutamate receptors regulate voltage dependent cation
channels which are opened by a binding of glutamate to the
receptor. This results in a Na.sup.+ and Ca.sup.2+ influx into the
cell massively disturbing the Ca.sup.2+ dependent cellular
metabolism. Especially the activation of the Ca.sup.2+ dependent
catabolic enzymes could give reason to the subsequent cell death
(Lee, Jin-Mo et al., "The changing landscape of ischaemic brain
injury mechanisms"; Dennis W. Zhol "Glutamate neurotoxicity and
diseases of the nervous system").
[0007] Although the mechanism of glutamate mediated neurotoxicity
is not yet entirely understood it is agreed upon that it
contributes in a large extent to the neuronal cell death following
cerebral ischaemia (Jin-Mo Lee, et al.).
[0008] Besides safeguarding vital functions and stabilizing
physiological parameter the re-opening of the closed vessel has
priority in the therapy of acute cerebral ischaemia. The re-opening
can be performed by different means. The mere mechanical
re-opening, as e.g. the PTCA after heart attack, so far has not yet
led to satisfying results. Only with a successful fibrinolysis an
acceptable improvement of the physical condition of patients can be
achieved. This can be accomplished by a local application using a
catheter (PROCAT, a study with prourokinase). However, despite
first positive results this method has not yet been officially
approved as a pharmaceutical treatment.
[0009] The naturally occurring fibrinolysis is based on the
proteolytic activity of the serine protease plasmin which
originates from its inactive precursor by catalysis (activation).
The natural activation of plasminogen is catalyzed by the
plasminogen activators u-PA (urokinase type plasminogen activator)
and t-PA (tissue plasminogen activator) occurring naturally in the
body. In contrast to u-PA, t-PA forms a so called activator complex
together with fibrin and plasminogen. Thus, the catalytic activity
of t-PA is fibrin dependent and is enhanced in its presence
approximately 550-fold. Besides fibrin also fibrinogen can
stimulate t-PA mediated catalysis of plasminogen to plasmin--even
though to a smaller extent. In the presence of fibrinogen the t-PA
activity is only increases 25-fold. Also the cleavage products of
fibrin (fibrin degradation products (FDP)) are stimulating
t-PA.
[0010] Early attempts of thrombolytic treatment of acute stroke go
back to the 1950s. First extensive clinical trials with
streptokinase, a fibrinolytic agent from betahaemolysing
streptococci, started only in 1995. Together with plasminogen
streptokinase forms a complex which catalyzes other plasminogen
molecules into plasmin.
[0011] The therapy with streptokinase has severe disadvantages
since it is a bacterial protease and therefore can provoke allergic
reactions in the body. Furthermore, due to a former streptococci
infection including a production of antibodies the patient may
exhibit a so called streptokinase resistance making the therapy
more difficult. Besides this, clinical trials in Europe
(Multicenter Acute Stroke Trial of Europe (MAST-E), Multicenter
Acute Stroke Trial of Italy (MAST-I)) and Australia (Australian
Streptokinase Trial (AST)) indicated an increased mortality risk
and a higher risk of intracerebral bleeding (intracerebral
haemorrhage, ICH) after treating patients with streptokinase. These
trials had to be terminated early.
[0012] Alternatively, urokinase--also a classical fibrinolytic
agent--can be applicated. In contrast to streptokinase it does not
exhibit antigenic characteristics since it is an enzyme naturally
occurring in various body tissues. It is an activator of
plasminogen and independent of a co-factor. Urokinase is produced
in kidney cell cultures.
[0013] Extensive experience on therapeutic thrombolysis is
available for the tissue type plasminogen activator--the so called
rt-PA--(see EP 0 093 619, U.S. Pat. No. 4,766,075), which is
produced in recombinant hamster cells. In the 90s several clinical
trials were performed world-wide using t-PA--with acute myocardial
infarction as the main indication--leading to partially
non-understood and contradictory results. In the so called European
Acute Stroke Trial (ECASS) patients were treated within a time
frame of 6 hours after the onset of the symptoms of a stroke
intravenously with rt-PA. After 90 days the mortality rate as well
as the Barthel-Index were examined as an Index for the disability
or the independent viability of patients. No significant
improvement of the viability was reported but an--even though not
significant--increase of mortality. Thus, it could be concluded, a
thrombolytic treatment with rt-PA of patients being individually
selected according to their respective case history immediately
after the beginning of the stroke could possibly be advantageous.
However, a general use of rt-PA within the time frame of 6 hours
after the onset of stroke was not recommended since an application
during this time increases the risk of intracerebal haemorrhage
(ICH) (C. Lewandowski C and Wiliam Barsan, 2001: Treatment of Acute
Stroke; in: Annals of Emergency Medicine 37:2; S. 202 ff.).
[0014] The thrombolytic treatment of stroke was also subject of a
clinical trial conducted by the National Institute of Neurologic
Disorder and Stroke (so called NINDS rtPA Stroke Trial) in the USA.
This trial concentrated on the effect of intravenous rt-PA
treatment within only three hours after the onset of the symptoms.
Patients were examined three months after the treatment. Due to the
observed positive effects of this treatment on the viability of
patients, rt-PA treatment within these limited time frame of three
hours was recommended although the authors found a higher risk for
ICH.
[0015] Two further studies (ECASS II Trial: Alteplase Thrombolysis
for Acute Noninterventional Therapy in Ischaemic Stroke (ATLANTIS))
examined whether the positive effects of rt-PA treatment within
three hours after the onset of stroke could be repeated even with a
treatment within six hours time. However, this question could not
be answered affirmatively since no improvement of the clinical
symptoms or any decrease in mortality was observed. The higher risk
for ICH remained.
[0016] Those partially contradictory results have led to a high
caution in the use of rt-PA. Already 1996 a publication of the
American Heart Association pointed out. the strong skepticism among
doctors with respect to thrombolytic treatment of stroke; whereas
there is no such skepticism with respect to fibrinolytica in the
therapy of myocardical infarct (van Gijn J, MD, FRCP,
1996--Circulation 1996, 93: 1616-1617).
[0017] A rational behind this scepticism was firstly given in a
summary of all stroke trials published 1997 (updated in March
2001). According to this review all thrombolytica treatments
(urokinase, streptokinase, rt-PA or recombinant urokinase) resulted
in a significant higher mortality within the first 10 days after
the stroke while the total number of either dead or disabled
patients was reduced when the thrombolytica where applied within
six hour after stroke onset. This effects were mainly due to ICH.
The broad use of thrombolytica for the treatment of stroke was
therefore not recommended.
[0018] Even before, such results gave reason to some other authors
mere sarcastic statement that stroke patients had the choice to
either die or to survive disabled (SCRIP 1997: 2265, 26).
[0019] Nevertheless, so far the therapy with rt-PA is the only
treatment of acute cerebral ischaemia approved by the Food and Drug
Administration (FDA) in the USA. However, it is restricted to an
application of rt-PA within three hours after the onset of
stroke.
[0020] The approval of rt-PA was reached in 1996. Before, in the
year 1995, first announcements about negative side effects of t-PA
became known, which provide. an explanatory basis for its dramatic
effects when applied in stroke treatment outside the three hour
time frame. Accordingly, microglia cells and neuronal cells of the
hippocampus produce t-PA which contributes to the glutamate
mediated excitotoxicity. This is concluded from a comparative study
on t-PA deficient and wild type mice when glutamate agonists were
injected in their hippocampus, respectively. The t-PA deficient
mice showed a significant higher resistance against external
(inthrathecal) applicated glutamate (Tsirka S E et al., Nature,
Vol. 377, 1995, "Excitoxin-induced neuronal degeneration and
seizure are mediated by tissue plasminogen activator"). These
results were confirmed in 1998 when Wang et al. could prove nearly
a double quantity of necrotic neuronal tissue in t-PA deficient
mice when t-PA was injected intravenously. This negative effect of
external t-PA on wild type mice was only approximately 33% (Wang et
al., 1998, Nature, "Tissue plasminogen activator (t-PA) increases
neuronal damage after focal cerebral ischaemia in wild type and
t-PA deficient mice".)
[0021] Further results on the stimulation of excitotoxicity by t-PA
were published by Nicole et al. in the beginning of 2001 (Nicole
O., Docagne F Ali C; Margaill I; Carmeliet P; MacKenzie E T, Vivien
D and Buisson A, 2001: The proteolytic activity of
tissue-plasminogen activator enhances NMDA receptor-mediated
signaling; in: Nat Med 7, 59-64). They could prove that t-PA being
released by depolarized cortical neurons could interact with the so
called NR1 sub-unit of the glutamate receptor of the NMDA type
leading to a cleavage of NR1. This increases the receptor's
activity resulting in a higher tissue damage after glutamate
agonist NMDA was applied. The NMDA agonist induced excitotoxicity.
Thus, t-PA exhibits a neurotoxic effect by activating the glutamate
receptor of the NMDA type.
[0022] According to a further explanatory concept the neurotoxicity
of t-PA results indirectly from the conversion of plasminogen in
plasmin. According to this model plasmin is the effector of
neurotoxicity (Chen Z L and Strickland S, 1997: Neuronal Death in
the hippocampus is promoted by plasmin-catalysed degradation of
laminin. Cell: 91, 917-925).
[0023] A summarizing outline of the time depending neurotoxic
effect of t-PA is given in FIG. 9. Therein also the increased
toxicity of the recombinant t-PA compared to endogenic t-PA becomes
evident. This is probably due to rt-PA being able to enter into
tissue in higher concentrations.
[0024] Despite its neurotoxic side effect and its increasing effect
on the mortality t-PA was approved by FDA. This can only be
explained by the lack of harmless and effective alternatives--thus
it is due to a very pragmatic cost benefit analysis. Therefore,
there is still a need for safe therapies. However, if they were
still based on thrombolytica--in case it is not possible to find
alternatives to thrombolysis--the problem of neurotoxicity has to
be considered (see for example Wang et al. a.a.O.; Lewandowski and
Barson 2001 a.a.O.).
[0025] Therefore, further examination of known thrombolytica
including DSPA (Desmodus rotundus Plasminogen Activator) in order
to develop new drugs for stroke was terminated although principally
all thrombolytica are potentially suitable. Especially in case of
DSPA its potential suitability for this medical indication was
pointed out earlier (Medan P; Tatlisumak T; Takano K; Carano R A D;
Hadley S J; Fisher M: Thrombolysis with recombinant Desmodus saliva
plasminogen activator (rDSPA) in a rat embolic stroke model; in:
Cerebrovasc Dis 1996:6; 175-194 (4.sup.th International Symposium
on Thrombolic Therapy in Acute Ischaemic Stroke). DSPA is a
plasminogen activator with a high homology (resemblance) to t-PA.
Therefore--and in addition to the disillusionment resulting from
the neurotoxic side effects of t-PA--there were no further
expectations, for DSPA being a suitable drug for stroke
treatment.
[0026] Instead, recent strategies aiming to improve known
thrombolytic treatments try to apply the thrombolytic substance no
longer intravenously but intraarterially via a catheter directly
close to the intravascular thrombus. First experience is available
with recombinant produced urokinase. Thus, possibly, the necessary
dose for thrombolysis and therewith negative side effects could be
reduced. However, this application requires a high technical
expenditure and is not available everywhere. Furthermore, the
patient has to be prepared in a time consuming action. Time,
however, is often limited. Thus, the preparation provides for an
additional risk.
[0027] Presently, new concepts are directed to anticoagulants such
as heparin, aspirin or ancrod, which is the active substance in the
poison of the malayan pit viper. Two further clinical trials
examining the effects of heparin (International Stroke Trial (IST)
and Trial of ORG 10172 in Acute Stroke Treatment (TOAST)) however,
do not indicate a significant improvement of mortality or a
prevention of stroke.
[0028] A further new treatment focuses neither on thrombus nor on
blood thinning or anti coagulation but attempts to increase the
vitality of cells damaged by the interruption of blood supply (WO
01/51613 A1 and WO 01/51614 A1). To achieve this antibiotics from
the group of quinons, aminoglycosides or chloramphenicol are
applied. For a similar reason it is further suggested to begin with
the application of citicholin directly after the onset of stroke.
In the body, citicholin is cleaved to cytidine and choline. The
cleavage products form part of the neuronal cell membrane and thus
support the regeneration of damaged tissue (U.S. Pat. No.
5,827,832).
[0029] Recent research on safe treatment is based on the new
finding that a part of the fatal consequences of stroke is caused
only indirectly by interrupted blood supply but directly to the
excito- or neurotoxicity including over activated glutamate
receptors. This effect is increased by t-PA (see above). A concept
to reduce excitotoxicity is therefore to apply so called
neuroprotectives. They can be used separately or in combination
with fibrinolytic agents in order to minimize neurotoxic effects.
They can lead to a reduced excitotoxicity either directly e.g. as a
glutamate receptor antagonist or indirectly by inhibiting voltage
dependent sodium or calcium channels (Jin-Mo Lee et al.
a.a.O.).
[0030] A competitive inhibition (antagonistic action) of the
glutamate receptor of NMDA type is possible e.g. with
2-amino-5-phosphonovalerate (APV) or 2-amino-5-phosphonoheptanoate
(APH). A non competitive inhibition can be achieved e.g. by
substances binding to the phencyclidine side of the channels. Such
substances can be phencyclidine, MK-801, dextrorphane or
cetamine.
[0031] So far, treatments with neuroprotectives have not shown the
expected success, possibly because neuroprotectives had to be
combined with thrombolytic agents in order to exhibit their
protective effects. This applies to other substances (see also FIG.
10).
[0032] Even a combination of t-PA and neuroprotective agents
results only in a limited damage. Nevertheless, the disadvantageous
neurotoxicity of the fibrinolytic agent as such is not avoided.
[0033] It is therefore an object of the present invention to
provide a new therapeutic concept for the treatment of stroke in
humans.
[0034] According to the invention the use of non-toxic plasminogen
activating factors is suggested as outlined in claim 1 for the
therapeutic treatment of stroke. Further advantageous uses are
subject of independent claims, respectively, as well as of further
dependent claims.
[0035] The central idea of the invention is the use of a
plasminogen activator in the treatment of stroke, of which the
mature enzyme exhibits an activity, which is selectively increased
by fibrin manifold, namely more than the 650-fold.
[0036] The use of the plasminogen activators according to the
invention is based on the following findings: Due to tissue damage
in the brain caused by stroke the blood brain barrier is damaged or
destroyed. Thus, fibrinogen circulating in the blood can enter into
the neuronal tissue of the brain. There, it activates t-PA
which--indirectly by activating the glutamate receptor or
plasminogen--results in further tissue damage. In order to avoid
this effect the invention suggests the use of a plasminogen
activator which is highly fibrin selective and--as an inversion of
the argument--has a reduced potential to be activated by
fibrinogen. Thus, this plasminogen activator is not--or compared to
t-PA at least substantially less--activated by fibrinogen entering
from the blood into neuronal tissue as a result of damaged blood
brain barrier, since t-PA's activator fibrin cannot enter the
neuronal tissue due to its size. The plasminogen activators
according to the invention therefore are non-neurotoxic.
[0037] According to a preferred embodiment of the invention,
non-toxic plasminogen activators are used, which comprise at least
one element of the so called cymogene triade. A comparable triade
is known from the catalytic center of serine proteases of the
chymotrypsine family consisting of three interacting amino acids
aspartate 194, histidine 40 and serine 32. However, this triade
does not exist in t-PA which belongs also to the family of
chymotrypsine like serine proteases. Nevertheless, it is known,
that the directed mutagenesis of native t-PA for the purpose of
introducing at least one of the above amino acids at a suitable
position results in a reduced activity of the pro-enzyme (single
chain t-PA) and to an increased activity of the mature enzyme
(double chain t-PA) in the presence of fibrin. Therefore, the
introduction of at least one amino acid of the triade--or of an
amino acid with the respective function in the triade--can increase
the cymogenity of t-PA (i.e. the ratio between the activity of the
mature enzyme an the activity of the pro-enzyme). As a result the
fibrin specificity is remarkably increased. This is due to
conformational interaction between the introduced amino acid
residue and/or amino acid residues of the wild type sequence.
[0038] It is known that the mutagenesis of the native t-PA with
substitution of Phe305 by His (F305H) and of Ala 292 by Ser (A292S)
leads to a 20-fold increase of the cymogenity, whereas the variant
F305H alone already leads to 5 times higher cymogentity (E L
Madison, Kobe A, Gething M-J; Sambrook J F, Goldsmith E J 1993:
Converting Tissue Plasminogen Activator to a Zymogen: A regulatory
Triad of Asp-His-Ser; Science: 262, 419-421). In the presence of
fibrin these t-PA mutants show an activity increase of 30,000 times
(F305H) and 130,000 times (F305H, A292S) respectively. In addition
these mutants comprise a substitution of Arg275 to R275E in order
to prevent cleavage by plasmin at the cleavage site Aug275-Ile276,
thereby converting the single chain t-PA to the double chain form.
The mutant site R275E alone leads to a 6,900 fold increase of the
fibrin specificity of t-PA (K Tachias, Madison E L 1995: Variants
of Tissue-type Plasminogen Activator Which Display Substantially
Enhanced Stimulation by Fibrin, in: Journal of Biological Chemistry
270, 31: 18319-18322).
[0039] The positions 305 and 292 of t-PA are homologous to the
positions His40 and Ser32 of the known triade of the chymotryptic
serine proteases. By the corresponding substitutions introducing
histidine or respectively serine, these amino acids can interact
with the aspartate477 of t-PA resulting in a functional triade in
the t-PA mutants (Madison et al., 1993).
[0040] These t-PA mutants can be used for the treatment of stroke
according to the invention because they show no or--compared to
wild type t-PA--a significantly reduced neurotoxicity due to their
increased fibrin specificity. For the purpose of disclosure of the
mentioned t-PA mutants F305H; F305H; A292S alone or in combination
with R275E we incorporate the publications of Madison et al.,
(1993) and Tachias and Madison (1995) hereby are fully incorporated
by reference.
[0041] The increase of fibrin specificity of plasminogen activators
can alternatively be achieved by a point mutation of Asp194 (or an
aspartate at a homologous position). Plasminogen activators belong
to the group of serine proteases of the chymotrypsin family and
therefore comprise the conserved amino acid Asp194, which is
responsible for the stability of the catalytic active conformation
of the mature proteases. It is known that Asp194 interacts with
His40 in the cymogenic form of serine proteases. After the cymogene
is activated by cleavage this specific interaction is interrupted
and the side chain of the Asp194 rotates about 170.degree. in order
to form a new salt bridge with Ile16. This salt bridge essentially
contributes to the stability of the oxyanione pocket of the
catalytic center of the mature serine proteases. It is also present
in t-PA.
[0042] The introduction of a point mutation replacing Asp194 prima
facie impedes the formation or respectively the stability of the
catalytic confirmation of serine proteases. Despite this the
mutated plasminogen activators show a significant increase of
activity in the presence of their co-factor fibrin--especially in
comparison to the mature wild type form--which can only be
explained in a way that the interaction with fibrin allows a
conformational change promoting catalytic activity (L Strandberg,
Madison E L, 1995: Variants of Tissue-type Plasminogen Activator
with Substantially Enhanced Response and Selectivity towards Fibrin
co-factors, in: Journal of Biological Chemistry 270, 40:
2344-2349).
[0043] In conclusion, the Asp194 mutants of the plasminogen
activators show a high increase of activity in presence of fibrin
which enables their use according to the invention.
[0044] In a preferred embodiment according to the invention, a
mutant t-PA is used, in which Asp194 is substituted by glutamate
(D194E) or respectively by asparagine (D194N). In these mutants the
activity of t-PA is reduced 1 to 2000 fold in the absence of
fibrin, whereas in the presence of fibrin, an increase of activity
by a factor of 498,000 to 1,050,000 can be achieved. These mutants
can further comprise a substitution of Arg15 to R15E, which
prevents the cleavage of the single chain t-PA at the peptide bond
Arg15-Ile16 by plasmin, leading to the double chain form of t-PA.
This mutation alone increases the activation of t-PA by fibrin by
the factor 12,000. For reasons of disclosure of the t-PA mutations
at positions 194 and 15, the publications of Strandberg and Madison
(1995) are fully incorporated by reference.
[0045] An increase of the fibrin dependency of plasminogen
activators can also be achieved by the introduction of point
mutations in the so called "autolysis loop". This element is known
from trypsine; it can also be found as a homologous part in serine
proteases and is especially characterized by three hydrophobic
amino acids (Leu, Pro and Phe). The autolysis loop in plasminogen
activators is responsible for the interaction with plasminogen.
Point mutations in this area can have the effect that the
protein-protein interaction between plasminogen and plasminogen
activators cannot be effectively formed any longer. These mutations
are only functionally relevant in the absence of fibrin. In the
presence of fibrin, they, in contrast, are responsible for an
increased activity of the plasminogen activators (K Song-Hua,
Tachias K, Lamba D, Bode W, Madison E L, 1997: Identification of a
Hydrophobic exocite on Tissue Type Plasminogen Activator That
Modulates Specificity for Plasminogen, in: Journal of Biological
Chemistry 272; 3, 1811-1816).
[0046] In a preferred embodiment t-PA is used showing point
mutations in the positions 420 to 423. If these residues are
substituted by directed mutagenesis this increases the fibrin
dependency of t-PA is increased by a factor up to 61,000 (K
Song-Hua et al.). Song-Hua et al. examined the point mutations
L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E.
These publications are fully incorporated by reference for
disclosure of the use according to the invention.
[0047] According to a further advantageous embodiment a modified
tissue plasminogen activator with an amino acid sequence according
to SEQ ID No. 1 (FIG. 13) is used. This modified t-PA differs from
the wild type t-PA by the exchange of the hydrophobic amino acids
in the position 420 to 423 in the autolysis loop as follows:
His420, Asp421, Ala422 and Cys423. This t-PA preferentially
contains a phenyl alanine at the position 194. Further the position
275 can be occupied by glutamate. Advantageously the position 194
is occupied by phenyl alanine.
[0048] Further, a modified urokinase can be used according to the
invention. The urokinase according to the invention can comprise
the amino acid sequence according to SEQ ID No. 2 (FIG. 14) in
which the hydrophobic amino acids of the autolysis loop are
substituted by Val420, Thr421, Asp422 and Ser423. Advantageously
the urokinase is carrying an Ile275 and a Glu194. This mutant
shows--in comparison to wild type urokinase--a 500-fold increased
fibrin specificity.
[0049] Both mutants--urokinase as well as t-PA--were analyzed in
semi quantitative tests and showed a increased fibrin specificity
in comparison to the wild type t-PA.
[0050] The plasminogen activator (DSPA) from the saliva of the
vampire bat (Desmodus rotundus) also shows a highly increased
activity in the presence of fibrin--in specific a 100,000-fold
increase. Thus it can be used preferentially according to the
invention. The term DSPA comprises four different proteases, which
fulfill an essential function for the vampire bat, namely an
increased duration of bleeding of the wounds of pray (Cartwright,
1974). These four proteases (DSPA.alpha.1, DSPA.alpha.2,
DSPA.beta., DSPA.gamma.) display a high similarity (homology) to
each other and to the human t-PA. They also show similar
physiological activities, leading to a common classification under
the generic term DSPA. DSPA is disclosed in the patents EP 0 352
119 A1 and of U.S. Pat. Nos. 6,008,019 and 5,830,849 which are
hereby fully incorporated by reference for purpose of
disclosure.
[0051] DSPA.alpha. so far is the best analyzed protease from this
group. It has an amino acid sequence with a homology greater than
72% in comparison to the known human t-PA amino acid sequence
(Krtzschmar et al, 1991). However, there are two essential
differences between t-PA and DSPA. Firstly all DSPA has full
protease activity as a single chain molecule, since it is--in
contrast to t-PA--not converted into a double chain form (Gardell
et al., 1989; Krtzschmar et al., 1991). Secondly, the catalytic
activity of DSPA is nearly absolutely dependent on fibrin (Gardell
et al., 1989; Bringmann et al., 1995; Toschie et al., 1998). For
example the activity of DSPA.alpha.1 is increased 100,000 fold in
the presence of fibrin whereas the t-PA activity is only increased
550 fold. In contrast, DSPA activity is considerably less strongly
induced by fibrinogen, since it only shows a 7 to 9 fold increase
(Bringmann et al., 1995). In conclusion, DSPA is considerably more
dependent of fibrin and much more fibrin specific as wild type t-PA
which is only activated 550-fold by fibrin.
[0052] Because of its fibrinolytic characteristics and the strong
similarity to t-PA, DSPA is an interesting candidate for the
development of a thrombolytic agent. Despite this, the therapeutic
use of DSPA as a thrombolytic agent was restricted to the treatment
of myocardinal infarction in the past, because--due to the
contribution of t-PA to the glutamate induced neurotoxicity--no
justified hopes existed, that a plasminogen activator which is
related to t-PA could reasonably be used for a treatment of acute
stroke.
[0053] Surprisingly it has been shown that DSPA has no neurotoxic
effects even though it shows a high resemblance (homology) to t-PA
and even though the physiological effects of the molecules are
comparable to a large extent. The above conclusion led to the idea
that DSPA after all may be successfully used as a thrombolytic
agent for the therapy of stroke without causing severe risks of
neuronal tissue damage. Especially interesting is the fact, that
DSPA can also be used later than 3 hours after the onset of stroke
symptoms.
[0054] A further teaching of the present invention that evolved
from the above findings is the option to modify or produce further
plasminogen activators in such a way that they reveal the essential
characteristics of DSPA, especially the lack of the neurotoxicity
of t-PA. The basis for this is the investigated relationship
between structure and biochemical effects, making it possible to
transform neurotoxic plasminogen activators into non-neurotoxic
plasminogen activators and thereby to produce non-neurotoxic
plasminogen activators on the basis of known or newly discovered
neurotoxic plasminogen activators.
[0055] The new teaching is based on in vivo comparative
examinations of the neurodegenerative effect of t-PA on one side
and of DSPA on the other side which are performed by using the so
called kainic acid model and a model for the examination of NMDA
induced lesion of the striatum.
[0056] The kainic acid model (also kainic acid injury model) is
based on the stimulation of the neurotoxic glutamate cascade by the
external application of kainic acid (KA) as an agonist of the
glutamate receptor of the kainic acid type (KA type) and of the
NMDA and AMPA glutamate receptors. Using a t-PA deficient mouse
stem as an experimental model it was possible to show that the
sensitivity of the laboratory animals against kainic acid only
reached the level of wild type mice after a supplementary
application of external t-PA. In contrast, an infusion of an
equimolar concentration of DSPA under the same experimental
conditions does not restore the sensitivity to kainic acid (KA). It
was concluded that the neurotoxic effect of t-PA was not induced by
DSPA. A summary of these results is shown in table 2.
1 TABLE 2 Hippocampal length intact (mm) Number Contralateral Ipsi-
Treatment of side lateral side Percentage group animals mean (SEM)
mean (SEM) remaining t-PA infusion 12 15.99 (0.208) 3.63 (0.458)
22.7* (1.85 uM) + KA DSPA infusion 11 16.07 (0.124) 13.8 (0.579)
85.87 (1.85 uM) + KA t-PA infusion 3 16.75 (0.381) 17.08 (0.363)
101.97 (1.85 uM) + PBS DSPA infusion 3 15.75 (0.629) 15.83 (0.363)
100.50 (1.85 uM) + PBS t-PA infusion 3 15.60 (0.702) 5.07 (1.09)
32.5 (0.185 uM) + KA DSPA infusion 3 16.06 (0.176) 13.80 (1.22)
85.93 (18.5 uM) + KA *P < 0.0001
[0057] Quantitative examinations based on this model revealed that
even a 10-fold increase of the DSPA concentration could not restore
the sensitivity of the t-PA deficient mice to the KA treatment
whereas already a 10-fold lower t-PA concentration led to KA
induced tissue damages. This leads to the conclusion that DSPA
possesses an at least 100 fold lower neurotoxic potential as t-PA
with respect to the stimulation of the neurodegeneration after KA
treatment (see also FIGS. 11 and 12).
[0058] In the second model of neurodegeneration, the possible
effects of t-PA as well as DSPA on the stimulation of the NMDA
dependent neurodegeneration were compared to wild type mice. For
this purpose, NMDA (as an agonist of the glutamate receptor of the
NMDA type) was injected in wild type mice alone or in combination
with either t-PA or DSPA. This model allows the comparison of the
effects of these proteases under conditions, which always lead to a
neurodegeneration and to an influx of plasma proteins due to the
breake down of the blood brain barrier (Chen et al., 1999).
[0059] While working on this model the injection of NMDA led to
reproducible lesions in the striatum of mice. The volume of lesions
was increased by a combined injection of t-PA and NMDA by at least
50%. The co-injection with DSPA.alpha.1 in contrast did not lead to
an increase or extension of the lesions caused by NMDA. Even in the
presence of plasma proteins which can freely diffuse in the region
of the lesion induced by NMDA, DSPA did not result in an increase
neurodegeneration (see also table 3).
2 TABLE 3 Treatment Number of Mean lesion group wild-type mice
volume (mm.sup.3) NMDA alone 8 1.85 (0.246) NMDA + t-PA 8 3.987
(0.293)* NMDA + DSPA 8 1.656 (0.094)** t-PA alone 3 0.20 (0.011)
DSPA alone 3 0.185 (0.016) **Not significant *P < 0.0001
[0060] These results show that fibrin-free DSPA--in contrast to
t-PA--behaves like an almost inert protease in the central nervous
system of a mammal and also of a human--and therefore does not
contribute to the neurotoxic effects caused by KA or NMDA. Despite
of the prejudice against the therapeutic use of t-PA like proteins
in stroke, this lacking neurotoxicity makes DSPA a suitable
thrombolytic agent for the treatment of acute stroke.
[0061] First results of the clinical trials show the
transferability of these results also for the treatment of stroke
in humans. It was found that significant improvements can be
achieved in patients after a successful perfusion (improvement by 8
points NIHSS or NIHSS score 0 to 1). Table 1 shows the data.
3TABLE 1 Base- NIHSS Patient line Post Tmt Day 7 Day 30 Day 90 sAEs
1001 12 7 4 4 * Re-Infarction 1002 8 9 2 0 0 1003 8 10 12 10 * 1004
8 4 2 0 0 1005 11 11 4 5 * 1006 9 7 1 * * 1007 14 6 * * * 2001 19
20 -- -- -- ICH, death 2002 15 21 -- -- -- ICH, death 3001 8 7 6 5
* 3002 15 16 9 8 * 3003 10 19 21 * -- Death day 39
[0062] The lacking neurotoxicity of DSPA and of the other
non-neurotoxic plasminogen activators (see above) offer the special
advantage in stroke treatment that the use of these plasminogen
activators--in contrast to the wild type t-PA--is not limited to a
short maximum period of only 3 hours after the onset of stroke. In
contrary, the treatment can be initiated later--for example after 6
hours or even later, since there is nearly no risk of stimulating
excitotoxic responses. First clinical trials with DSPA prove a safe
treatment of patients even in a time range of over 6 to 9 hours
after the onset of stroke symptoms.
[0063] This option of a timely unlimited treatment with
non-neurotoxic activators is of special importantance, since it
allows for the first time to treat patients with acute stroke
symptoms safely even when diagnosis is delayed or the onset of the
stroke cannot be determined with sufficient security. In the prior
art, this group of patients was excluded from thrombolytic therapy
with plasminogen activators due to unfavorable risk estimation.
Consequently, an essential contra-indication for the authorized use
of a thrombolytic agent for stroke is eliminated.
[0064] DSPA as well as further non-neurotoxic plasminogen
activators show no tissue damaging side effects. However, it can be
advantageous to apply them in combination with a neuroprotective
agent for the treatment of stroke in order to limit the tissue
damages induced by the glutamate occurring naturally in the human
body. Neuroprotective agents inhibiting the glutamate receptor
competitively or non-competitively can be used. Useful combinations
are e. g. with the known inhibitors of the glutamate receptors of
the NMDA type, the kainic acid type or the quisqualate type, as for
example APV, APH, phencyclidine, MK-801, dextrorphane or
cetamine.
[0065] Further a combination with cations can be advantageous since
cations, especially Zn-ions, block the cation channel regulated by
the glutamate receptor and can therefore reduce neurotoxic
effects.
[0066] In a further advantageous embodiment, non-neurotoxic
plasminogen activators can be combined with at least one further
therapeutic agent or with a pharmaceutically tolerable carrier. The
combination with a therapeutic agent which supports the reduction
of tissue damage by vitalizing the cells is especially
advantageous, since it contributes to the regeneration of already
damaged tissue or serves for the prevention of further stroke
incidents. Advantageous examples are combinations with antibiotics
as quinones, anticoagulants as heparin or hirudin as well as with
citicholine or acetylsalicylic acid.
[0067] A combination with at least one thrombin inhibitor can also
be advantageous. Preferentially, thrombomodulin and thrombomodulin
analogs like for example solulin, triabin or pallidipin can be
used. Further combinations with anti-inflammatory substances are
advantageous, since they influence the infiltration by
leucocytes.
[0068] Comparing Examinations of t-PA and DSPA Are Methods:
[0069] 1. Animals
[0070] Wild-type mice (c57/Black 6) and t-PA deficient mice
(t-PA-/-mice) (c57/Black 6) (Carmeliet et al., 1994) were supplied
by Dr. Peter Carmeliet, Leuven, Belgium.
[0071] 2. Protein Extraction from Brain Tissue
[0072] The assessment of proteolytic activity in brain tissue
following infusion of either t-PA or DSPA.alpha.1 was performed by
zymographic analysis (Granelli-Piperno and Reich, 1974). After an
infusion over a period of seven days into the hippocampus, mice
were anaesthetised, then transcardially perfused with PBS and the
brains removed. The hippocampus region was removed, transferred to
eppendorf tubes and incubated in an equal volume (w/v) (approx.
30-50 .mu.m) of 0.5% NP-40 lysis buffer containing no protease
inhibitors (0.5% NP-40, 10 mM Tris-HCl pH 7.4, 10 mM NaCL, 3 mM
MgCl2, 1 mM EDTA). The brain extracts were homogenized by means of
a hand-held glass homogeniser and left on ice for 30 minutes. The
samples were then centrifuged and the supematant was removed. The
amount of proteins present was determined (Bio-Rad-reagent).
[0073] 3. Zymographic Analysis of the Proteases
[0074] The proteolytic activity in the samples and the brain tissue
extracts was determined by zymographic analysis according to the
method of Granelli, Piperno and Reich (1974). The samples with
recombinant proteins (up to 100 nM) or the brain tissue extracts
(20 .mu.g) were subjected to a (10%) SDS-PAGE under non-reducing
conditions. The gels were removed from the plates, washed in 1%
triton X 100 for 2 hours and then overlaid onto an agarose gel
containing polymerized fibrinogen and plasminogen (Granelli,
Piperno and Reich, 1974). The gels were incubated at 37.degree. C.
in a humified chamber until proteolysed zones appeared.
[0075] 4. Intra-hippocampal Infusion of t-PA, DSPA and Subsequent
Injection of Kainic Acid
[0076] The kainic acid injury model was based on studies of Tsirka
et al. (1995). The animals were injected intraperitoneally (i. p.)
with atropine (4 mg/kg) and then anaesthetised with an i. p.
injection of sodium pentobarbitol (70 mg/kg). Afterwards mice were
placed in a stereotaxic frame and a micro-osmotic pump (Alzet model
1007D, Alzet Calif. USA) containing 100 .mu.l of either PBS or
recombinant human t-PA (0.12 mg/ml, 1.85 .mu.M) or DSPA.alpha.1
(1.85 .mu.M) was implanted subcutaneously between the shoulder
blades. The pumps were connected via sterile tubes to a brain
cannula and inserted through a burr opening made through the skull
at coordinates bregma -2.5 mm, midiolateral 0.5 mm and dorsoventral
1.6 mm in order to introduce the liquid near the midline. The
cannula was fixed at the desired position and the pumps were
allowed to infuse the respective solutions at a rate of 0.5 .mu.l
per hour for a total of 7 days.
[0077] Two days after infusion of the proteases the mice were
reanaesthetised and again placed in the stereotaxic frame.
Afterwards 1.5 nmol of kainic acid (KA) in 0.3 .mu.l, PBS was
injected unilaterally into the hippocampus. The coordinates were:
bregma -2.5 mm, medial-lateral 1.7 mm and dorsoventral 1.6 mm. The
excitotoxin (KA) was delivered for a duration of 30 seconds. After
the kainic acid treatment the injection needle remained at these
coordinates for further 2 minutes in order to prevent a reflux of
the liquid.
[0078] 5. Brain Processing Procedure
[0079] Five days after KA injection, the animals were anaesthetised
and transcardially perfused with 30 ml PBS followed by 70 ml of a
4% paraformaldehyd solution, post fixed in the same fixative
followed by incubation in 30% sucrose for further 24 hours. Coronal
sections (40 .mu.m) of the brain were then cut on a freezing
microtome and either counter-stained with thionin (BDH, Australia)
or processed for immunohistochemical examination as described
below.
[0080] 6. Quantification of Neuronal Loss within the
Hippocampus
[0081] The quantification of neuronal loss in the CA1-CA3
hippocampal subfields was performed as previously described (Tsirka
et al., 1995; Tsirka et al., 1996). Five consecutive parts of the
dorsal hippocampus from all treatment groups were prepared taking
care that the parts indeed comprised the place of the CA-injection
and lesion area. The hippocampal subfields (CA1-CA3) of these
sections were traced by means of camera lucida drawings of the
hippocampus. The entire lengths of the subfields was measured by
comparison to 1 mm standards traced under the same magnification.
The lengths of tissue with viable pyramidal neurons (having normal
morphology) and lengths of tissue devoid of neurons (no cells
present, no thionin staining) was determined. The lengths,
representing intact neurons and neuronal losses over each
hippocampal subfield were averaged across sections and the standard
deviations were determined.
[0082] 7. Intra-striatal NMDA Excitotoxic Lesions with or without
t-PA or DSPA
[0083] Wild type mice (c57/Black 6) were anaesthetised and placed
in a sterertaxic frame (see above). Mice then received an
unilateral injection of 50 nmol NMDA in the left stratum, injected
alone or in combination with either 46 .mu.M rt-PA or 46 .mu.M
DSPA.alpha.1. As controls t-PA and DSPA were also injected alone
(both at a concentration of 46 .mu.M). The injection coordinates
were: bregma -0.4 mm, midiolateral 2.0 mm and dorsoventral 2.5 mm.
The solutions (1 .mu.l total volume for all treatments) were
transferred over a period of 5 minutes at a rate of 0.2 .mu.l/min
and the needle was left in place for further 2 minutes after the
injection in order to minimize the reflux of fluid. After 24 hours
the mice were anaesthetised and perfused transcardially with 30 ml
PBS followed by 70 ml of a 4% paraformaldehyd solution, post fixed
in the same fixative for 24 hours with followed by incubation in
30% sucrose for further 24 hours. Brains were then cut (40 .mu.m)
on a freezing microtome and mounted onto gelatin coated glass
slides.
[0084] 8. Quantification of the Lesion Volume Following NMDA
Injection
[0085] The quantification of the striatal lesion volume was
performed using the method described by Callaway et al. (2000). Ten
consecutive coronal sections spanning the lesioned area were
prepared. The lesioned area was visualised using the Callaway
method and the lesion volume was quantified by the use of a micro
computer imaging device (MCID, Imaging Research Inc., Brock
University, Ontario, Canada).
[0086] 9. Immunohistochemistry
[0087] Immunohistochemistry was performed using standard
methodologies. Coronal sections were immersed in a solution of 3%
H.sub.2O.sub.2 and 10% methanol for 5 minutes followed by an
incubation in 5% normal goat serum for 60 minutes. The sections
were incubated over night either with an anti-GFAP antibody
(1:1,000; Dako, Carpinteria, Calif., USA) for the detection of
astrocytes, with an anti-MAC-1 antibody (1:1,000; Serotec, Raleigh,
N.C., USA) for the detection of microglia or with polyclonal
anti-DSPA antibodies (Schering A G, Berlin). After rinsing, the
sections were incubated with the appropriate biotinylated secondary
antibodies (Vector Laboratories, Burlingame, Calif., USA). This was
followed by a final incubation with avidin/biotin-complex (Vector
Laboratories, Burlingame, Calif., USA) for 60 minutes before
visualisation with 3,3'-diaminebebcidine/0.03% H.sub.2O.sub.2.
Sections were then mounted on gelatin coated slides, dried,
dehydrated and coverslipped with permount.
[0088] B. Results
[0089] 1. Infusion of t-PA or DSPA Disperses into the Hippocampus
of t-PA-/-Mice and Retains Proteolytic Activity
[0090] The initial experiments were designed to confirm that both
DSPA and t-PA retain their proteolytic activity for the 7 day
period of the infusion. To this end, aliquots of t-PA and DSPA (100
nmol) were incubated at 37.degree. C. and at 30.degree. C. for 7
days in a water bath. In order to determine the proteolytic
activity, 5 fold serial dilutions of the probes were subjected to
SPS-PAGE under non-reducing conditions and proteolytic activity was
assessed by zymographic analyses. An aliquot of t-PA and DSPA which
had been kept frozen for a period of 7 days was used as a control.
As can be seen in FIG. 1 there was only a minor loss of DSPA or
t-PA activity at an incubation with either 30.degree. C. or
37.degree. C. over this period of time.
[0091] 2. t-PA and DSPA Activity is Recovered in Hippocampal
Extracts Prepared from t-PA-/-Mice Following Infusion
[0092] First it had to be confirmed that the infused proteases were
present in the brain of the infused animals and also retained their
proteolytic activity while being in this compartment. To address
this point, t-PA-/- were infused for seven days with either t-PA or
DSPA (see above). Mice were then transcardially perfused with PBS
and the brains removed. The ipsilateral and contralateral
hippocampal regions were isolated as well as a region of the
cerebellum (taken as a negative control). Tissue samples (20 .mu.g)
were subjected to SDS-PAGE and zymographic analysis according to
the description in the methods section. As can be seen in FIG. 2,
both t-PA and DSPA activities were detected in the ipsilateral
region of the hippocampus, while some activity was also detected on
the contralateral side. This indicates that the infused proteases
not only retained their activity in the brain but had also diffused
within the hippocampal region. As a control, no activity could be
detected in the extract prepared from the cerebellum.
[0093] 3. Immunohistochemical Assessment of DSPA
[0094] To further confirm that DSPA had indeed diffused into the
hippocampal region, coronal brain sections of t-PA-/-mice were
analysed immunohistochemically after DSPA infusion. DSPA-antigen
was detected in the hippocampal region with the most prominent
staining in the area of the infusion site. This result confirms
that the infused DSPA is soluble and is indeed present in the
hippocampus.
[0095] 4. DSPA Infusion Does Not Restore Kainic-acid Mediated
Neurodegeneration in vivo
[0096] t-PA-/-mice are characteristically resistant to kainic acid
(KA) mediated neurodegeneration. However, intrahippocampal infusion
of rt-PA completely restores the sensitivity to KA-mediated injury.
To determine whether DSPA could be substituted for t-PA in this
model, t-PA-/-mice were infused intrahipocampically with either
t-PA or DSPA using a mini-osmotic pump. For both groups 12 mice
were tested. 2 days later the animals were injected with kainic
acid and left to recover. 5 days later the animals were killed and
the brains removed and prepared (see above). As controls,
t-PA-/-mice were also infused with PBS prior to KA treatment
(N=3).
[0097] Coronal brain sections were prepared and the neurons
detected by Nissl staining. As shown in FIG. 4, t-PA-/-mice infused
with PBS were resistant to subsequent challenge with KA. However,
infusion of recombinant t-PA restored sensitivity to KA treatment.
In contrast, infusion of the same concentration of DSPA into the
hippocampal region did not alter the sensitivity of the animals to
KA.
[0098] A quantitation of those results was based on data obtained
from 12 mice in each group. In 2 of the 12 mice infused with DSPA a
small extend of neurodegeneration was observed. The reason for that
in unclear and possibly not related to the presence of DSPA. The
combined data consider this minor effect that was observed in the
case of these 2 animals. All 12 mice treated with t-PA were
sensitive against the KA treatment. These results show that in case
of an infusion of t-PA or DSPA.alpha.1 in equimolar concentrations
only the administering of t-PA led to the restoration of
sensitivity to KA induced neurodegeneration.
[0099] 5. DSPA Infusion Does Not Result in Microglial
Activation
[0100] The restauration of the KA sensitivity of the t-PA-/-mice
caused by a t-PA infusion also results in a microglia activation
(Rogove et al., 1999). To assess the degree of microglial
activation following t-PA or DSPA infusion and subsequent KA
treatment, coronal sections of mice were subjected to an
immunohistochemical staining for activated microglia cells using
the Mac-1 antibody. The restauration of KA sensitivity following
t-PA infusion resulted in a clear increase in Mac-1 positive cells.
This was not observed in mice infused with DSPA. Hence, the
presence of DSPA does not result in the activation of microglia
cells following KA treatment.
[0101] 6. Titration of DSPA and t-PA in the Mice Hippocampus
Region.
[0102] The concentration of t-PA used for the infusion was based on
the concentration described by Tsirka et al. (1995) (100 .mu.l of
0.12 mg/ml [1.85 .mu.M]). The KA-injury experiments were repeated
using a 10-fold lower of t-PA (0.185 .mu.M) and a 10-fold higher
amount of DSPA (18.5 .mu.M). The lower t-PA concentration was still
able to restore the sensitivity to KA treatment (n=3). Of special
interest was the finding that the infusion of 10 fold increased
DSPA concentration only caused a little neuronal loss following KA
treatment. These data strongly point out that DSPA does not lead to
an increase of sensitivity to KA.
[0103] 7. Effect of t-PA and DSPA on NMDA-dependent
Neurodegeneration in Wild Type Mice
[0104] The effects of t-PA and DSPA were also examined in a model
of neurodegeneration in wild type mice. The injection of t-PA in
the striatum of these mice provably led to an increase of the
neurodegenerative effects caused by the glutamate analogue NMDA
(Nicole et al., 2001).
[0105] NMDA was injected into the striatal region of wild type mice
in the presence of t-PA or DSPA (each 46 .mu.M) with a total volume
of 1 .mu.l. After 24 hours the brains were removed and the size of
the lesions was quantified according to the Callaway method
(Callaway et al., 2000) (see above). As can be seen in FIG. 7,
injection of NMDA alone caused a reproducible lesion in all treated
mice (N=4). When t-PA and NMDA were applied together, the size of
the lesions was increased about 50% (P<0,01, n=4). In a clear
contrast the co-injection of NMDA and the same concentration of
DSPA did not lead to an increase in lesion size compared to NMDA
alone.
[0106] Injection of t-PA or DSPA alone did not lead to a detectable
neurodegeneration. The lacking effect of t-PA when being
administered alone is consistent with the results of Nicole et al.
(2001). These data show that the presence of DSPA does not increase
neurodegeneration even during a neurodegenerative event.
[0107] In order to confirm that the injection of DSPA had indeed
spread into the hippocampal region, immunohistochemistry was
performed on coronal sections by use of the DSPA antibody. The
examination showed that DSPA did indeed enter the striatal
region.
[0108] Kinetic Analysis of the Plasminogen Activation by Indirect
Chromogen Test
[0109] Indirect chromogen tests of the t-PA activity were performed
using the substrate Lys-plasminogen (American Diagnostica) and
spectrocyme PL (American Diagnostica) according to Madisan E. L.,
Goldsmith E. J., Gerard R. D., Gething M.-J., Sambrook J. F. (1989)
Nature 339 721-724; Madison E. L O., Goldsmith E. J., Gething M.
J., Sambrook J. F. and Bassel-Duby R. S. (1990) Proc. Natl. Acad.
Sci U.S.A 87, 3530-3533 as well as Madison E. L., Goldsmith E. J.,
Gething M. J., Sambrook J. F. and Gerard R. D. (1990) J. Biol. Chem
265, 21423-21426. Tests were performed both in the presence and
absence of the co-factor DESAFIB (American Diagnostica). DESAFIB is
a preparation of soluble fibrin monomeres gained by the cleavage of
highly pure human fibrinogen with the protease batroxobin.
Batroxobin cleaves the Arg.sup.16-Gly.sup.17-binding in the
A.alpha.-chain of fibrinogen and thereby releases fibrinopeptid A.
The resulting des-AA-fibrinogen representing fibrin I monomers is
soluble in the absence of the peptide Gly-Pro-Arg-Pro. The
concentration of Lys-plasminogen was varied from 0.0125 up to 0.2
.mu.M in the presence of DESAFIB and from 0.9 to 16 .mu.M in
absence of the co-factor.
[0110] Indirect Chromogen Tests in the Presence of Different
Stimuli.
[0111] Indirect chromogen standard tests were performed according
to the publications cited above. Probes of 100 .mu.l total volume
containing 0.25-1 ng enzyme, 0.2 .mu.M Lys-plasminogen and 0.62 mM
spectrocyme PL were used. The tests were performed either in the
presence of buffer, 25 .mu.g/ml DESAFIB, 100 .mu.g/ml cyanogen
bromide fragments of fibrinogen (American Diagnostica) or 100
.mu.g/ml of the stimulatory 13 amino acid peptide P368. The
analysis were performed in microtiter-plates and the optic density
was determined at a wave length of 405 nm every 30 seconds for 1
hour in a "Molecular Devices Thermomax". The reaction temperature
was 37.degree. C.
Sequence CWU 1
1
2 1 476 PRT Artificial Modified t-PA 1 Met Val Asn Thr Met Lys Thr
Lys Leu Leu Cys Val Leu Leu Leu Cys 1 5 10 15 Gly Ala Val Phe Ser
Leu Pro Arg Gln Glu Thr Tyr Arg Gln Leu Ala 20 25 30 Arg Gly Ser
Arg Ala Tyr Gly Val Ala Cys Lys Asp Glu Ile Thr Gln 35 40 45 Met
Thr Tyr Arg Arg Gln Glu Ser Trp Leu Arg Pro Glu Val Arg Ser 50 55
60 Lys Arg Val Glu His Cys Gln Cys Asp Arg Gly Gln Ala Arg Cys His
65 70 75 80 Thr Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn
Gly Gly 85 90 95 Thr Cys Gln Gln Ala Leu Tyr Phe Ser Asp Phe Val
Cys Gln Cys Pro 100 105 110 Glu Gly Phe Ala Gly Lys Cys Cys Glu Ile
Asp Thr Arg Ala Thr Cys 115 120 125 Tyr Glu Asp Gln Gly Ile Ser Tyr
Arg Gly Thr Trp Ser Thr Ala Glu 130 135 140 Ser Gly Ala Glu Cys Thr
Asn Trp Asn Ser Ser Ala Leu Ala Gln Lys 145 150 155 160 Pro Tyr Ser
Gly Arg Arg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn 165 170 175 His
Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr 180 185
190 Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala
195 200 205 Cys Ser Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro Gln
Phe His 210 215 220 Ser Thr Gly Gly Leu Phe Ala Asp Ile Ala Ser His
Pro Trp Gln Ala 225 230 235 240 Ala Ile Phe Ala Lys His Arg Arg Ser
Pro Gly Glu Arg Phe Leu Cys 245 250 255 Gly Gly Ile Leu Ile Ser Ser
Cys Trp Ile Leu Ser Ala Ala His Cys 260 265 270 Phe Gln Glu Arg Phe
Pro Pro His His Leu Thr Val Ile Leu Gly Arg 275 280 285 Thr Tyr Arg
Val Val Pro Gly Glu Glu Glu Gln Lys Phe Glu Val Glu 290 295 300 Lys
Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asp Asn Asp 305 310
315 320 Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser Ser Arg Cys Ala Gln
Glu 325 330 335 Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp
Leu Gln Leu 340 345 350 Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr
Gly Lys His Glu Ala 355 360 365 Leu Ser Pro Phe Tyr Ser Glu Arg Leu
Lys Glu Ala His Val Arg Leu 370 375 380 Tyr Pro Ser Ser Arg Cys Thr
Ser Gln His Leu Leu Asn Arg Thr Val 385 390 395 400 Thr Asp Asn Met
Leu Cys Ala Gly Asp Thr Arg Ser Gly Gly Pro Gln 405 410 415 Ala Asn
Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val 420 425 430
Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ile Ser Trp Gly 435
440 445 Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr Thr Lys Val
Thr 450 455 460 Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro 465
470 475 2 477 PRT Artificial Modified Urokinase 2 Met Val Asn Thr
Met Lys Thr Lys Leu Leu Cys Val Leu Leu Leu Cys 1 5 10 15 Gly Ala
Val Phe Ser Leu Pro Arg Gln Glu Thr Tyr Arg Gln Leu Ala 20 25 30
Arg Gly Ser Arg Ala Tyr Gly Val Ala Cys Lys Asp Glu Ile Thr Gln 35
40 45 Met Thr Tyr Arg Arg Gln Glu Ser Trp Leu Arg Pro Glu Val Arg
Ser 50 55 60 Lys Arg Val Glu His Cys Gln Cys Asp Arg Gly Ser Asn
Glu Leu His 65 70 75 80 Gln Val Pro Ser Asn Ser Cys Asp Glu Pro Arg
Cys Leu Asn Gly Gly 85 90 95 Thr Cys Val Ser Asn Lys Tyr Phe Ser
Ile His Trp Cys Asn Cys Pro 100 105 110 Lys Lys Phe Gly Gly Gln His
Cys Glu Ile Asp Lys Ser Lys Thr Cys 115 120 125 Tyr Glu Gly Asn Gly
His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr 130 135 140 Met Gly Arg
Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln 145 150 155 160
Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu Gly Lys 165
170 175 His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys
Tyr 180 185 190 Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met
Val His Asp 195 200 205 Cys Ala Asp Phe Gln Cys Gly Gln Lys Thr Leu
Arg Glu Pro Arg Phe 210 215 220 His Ser Thr Gly Gly Glu Phe Thr Thr
Ile Glu Asn Gln Pro Trp Phe 225 230 235 240 Ala Ala Ile Tyr Arg Arg
His Arg Gly Gly Ser Gly Val Thr Tyr Val 245 250 255 Cys Gly Gly Ser
Leu Met Ser Pro Cys Trp Val Ile Ser Ala Thr His 260 265 270 Cys Phe
Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly 275 280 285
Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys Phe Glu Val 290
295 300 Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp Thr His His
Asn 305 310 315 320 Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly
Arg Cys Ala Gln 325 330 335 Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu
Pro Ser Met Tyr Asn Asp 340 345 350 Pro Gln Phe Gly Thr Ser Cys Glu
Ile Thr Gly Phe Gly Lys Glu Asn 355 360 365 Ser Thr Asp Tyr Leu Tyr
Pro Glu Gln Leu Lys Met Thr Val Val Lys 370 375 380 Leu Ile Ser His
Arg Glu Cys Gln Gln Pro His Tyr Tyr Gly Ser Glu 385 390 395 400 Val
Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln Trp Lys Glu Ile 405 410
415 Tyr Pro Asn Val Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu
420 425 430 Val Cys Ser Leu Gln Gly Arg Met Thr Leu Thr Gly Ile Val
Ser Trp 435 440 445 Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val
Tyr Thr Arg Val 450 455 460 Ser His Phe Leu Pro Trp Ile Arg Ser His
Thr Lys Leu 465 470 475
* * * * *