Note: Descriptions are shown in the official language in which they were submitted.
~ 2141~42
Thrombocyte-stabilizing Factor IX-fragments, their preparation and
use and drugs containing these.
The invention refers to fragments of the coagulation factor IX
which contain intact G~A- and EGF-like domain as a substance for
stabilizing platelets in vivo.
Furthermore the invention refers to the production of these
substances and drugs containing these substances.
Haemostasis is a complex process which includes the participation
of several plasma proteins, blood cells and vessel components.
Its physiological function is to protect the organism against
blood loss, while performing blood coagulation and at the same
time avoiding escalation of the coagulation process. Thereby
hemostasis maintains the bloodflow in the whole organism.
Disorder in haemostasis might lead to intravascular occlusions
(thrombosis).
Prophylaxis and treatment of thrombosis diseases, like heart
attack, venous thrombosis, DIC and stroke are very complicated,
since inhibition o~ blood coagulation also causes risks o~ live-
threatening bleeding complications.
Well known are three classes of antithrombotic substances.
These are thrombin inhibitors, vitamin-K-antagonists and
inhibitors of platelet-aggregation.
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In clinical praxis, heparin catalysing the complex-formation of
coagulation proteases with AT III is the most used anticoagulant.
The anticoagulant action of heparin mainly consists of thrombin
inhibition.
Using heparin causes risks of bleeding complications, since
complete thrombin inhibition also inhibits extrinsic coagulation.
Therefore heparin dosage has to be adjusted exactly by means of in
vitro coagulation tests. The use of heparin is a difficult balance
between an effective antithrombotic action without risking a
bleeding disorder.
1-5 ~ of heparin treated patients show a decrease in platelet
count.
The most potent natural inhibitor of thrombin is hirudin, a leech
protein. Hirudin forms a stoechiometric complex with thrombin. Up
to now no clinically tested antidot exists, therefore bleeding
complications can not be excluded.
Further anticoagulants are the vitamin-K-antagonists, which like
Warfarin prevent the gamma-carboxylation in order to decrease
plasma level of active vitamin-K-depending coagulation factors II,
VII, IX and X. Full anticoagulatory effect occur with a delay of
36-72 hours after injection of antagonists. Thus they are not
suited for the treatment of acute thrombosis. Use of these
substances might cause bleeding complications equally to those
seen using heparin.
Platelet aggegation inhibitors, for example acetyl salicylic acid
belong to the`third class of antithrombotic substances applied
2141642
today.
They are not able to inhibit activation of plasmatic coagulation
factors and their action is predominantly limited to the treatment
or phrophylaxis of arterial thrombosis.
These examples show that treatment of thrombosis using available
drugs has some decisive disadvantages. Mainly the not well
controlled inhibition of the extrinsic coagulation which protects
against blood loss and the fact that the drugs used today
interfere with the coagulation cascade at its end and may result
in uncontrolled bleeding complications are disadvantages of these
drugs.
In general it is desirable for the antithrombotic therapy to
interfere at an early stage with the coagulation system in order
to prevent irreversible processes (e.g. platelet activation),
because otherwise the therapeutic treatment has only a limited
effect in preventing possible damage.
The aim o~ the invention was on the one hand to inhibit the
plasmatic coagulation and at the same time prevent a decrease in
platelet count, while the function of the protecting extrinsic
coagulation remains intact.
According to the invention the problem was solved by the
preparation of proteolytical fragments of factor IX, containing an
intact gamma-carboxyglutamate-domain (Gla) and an epidermal growth
facter-like domain (EGF) and having a molecular weight of about
12.000 to 50.000 Dalton.
Surprisingly it was found, that the factor IX fragments according
21416 12
23251-67
to the invention are able to inhibit the factor IXa induced
decrease of the thromobcyte count in vivo. The factor IX has
an imported role in hemostasis because the "extrinsic" and the
"intrinsic" pathway are crossing here (Mann, R. G., Jenny, R. J.
et al., Annu. Rev. Biochem, 57 (1988), 915-956). Factor IX is
a vitamin-K-dependent plasma glycoprotein with 415 amino acids.
It contains an amino terminal domain with gamma-carboxy-
glutamic acid residues and two domains~ containing sequences
which resemble these from epidermal growth factor (EGF)
(Thompson, A. R., Blood, 67r (1986), 565-572).
According to one aspect of the present invention,
there is provided drug with antithrombotic efficacy on the
basis of proteolytic fragments of the human blood coagulation
factor IX, where the fragments a.) are containing the Gla-
domain and at least one EGF-like domain, b.) are showing no
factor IX coagulation activity, c.) are not chemically modified
and d.) have a molecular weight of about 12~000 to 15,000 D.
According to a further aspect of the present
invention,there is provided method for the preparation of a
drug as defined above where the factor IX is bound on a carrier
which binds the factor IX on the amino terminal end, the factor
IX is cleaved proteolytically, the fragments are eluted from
the carrier, purified and if necessary lyophilized.
According to another aspect of the present invention,
there is provided method for the preparation of a drug as
defined above where the factor IX is cleaved proteolytically
in solution after the addition of protecting substances and
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23251-67
the fragments are separated, purified and if necessary
lyophilized.
According to a still further aspect of the present
invention, there is provided an antithrombotic compound
comprising a proteolytic fragment of human blood coagulation
factor IX wherein said fragment a.) contains a Gla-domain and
at least one EGF-like domain, b.) shows no factor IX
coagulation activity, c.) is not chemically modified and d.)
has a molecular weight of about 12,000 to 50,000 D.
According to another aspect of the present invention,
there is provided a use of a drug as defined above in the
treatment of a thrombotic disease.
The results of the previous experiments about the
efficacy and the inhibition of factor IX or factor IXa in
difficultthrombosis models are based on the competitive
inhibition of the binding to the factor IXa receptors of
endothelialcells. In these described coagulation processes
the thrombocyte-mediated coagulation played only a minor role.
In EP 0 263 529 it was demonstrated that peptides of
the EGF-like domain of factor IX or a sequential part o~ it
with a molecular weight of 500 to 1,000 Dalton can inhibit the
generation of a thrombus in Wessler's stasis-thrombosis-model.
The basis for this was the inhibition of the interaction of
the injected factor IXa with endothelial cells in an isolated
part of a vessel by the EGF-like sequence containing peptide.
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. ~ .
~ 23251-67
In Wessler's stasis animal model the generation of a
thrombus formed under total stasis of the blood stream in an
isolated part of the vessel is the decisive characteristic.
In this model the
21~16~2
. ~
thrombocyte-mediated coagulation of the physiological blood stream
is of minor importance.
It is unknown, if the EGF-like peptides have any influence on the
physiological thrombocyte-mediated coagulation with the
simultanous decrease of the thrombocyte count.
To get a signiicant inhibition of thrombus generation very high
concentrations of peptides were neccessary. The mass relation
peptide to factor IXa was about 1660 to 1, which means a molecular
relation of peptide to factor IXa of 65.000 to 1.
Disadvantages of the use of such small peptides are also their
short half-life time of a few minutes.
Astermark and Stenflo, J, Biol. Chem., 260, 2438-2443 ex~mined
different domains of bovine favctor IX, their effects on blood
coagulation in vitro and their interaction with factor IX-
receptors on bovine endothelial cells in vitro. An effficacy of
the isolated factor IX-domains was not described. With native
factor IX it was not possible to inhibit thrombosis (Benedict,
Ryan, et al. J. Clin.Invest., 88, (1991), 1760-1765).
With chemically inactivated factor IXa it was possible to inhibit
the formation of fibrin clots in tumor tissue of mice in which
tumor necrosis factor was injected. With the zymogen factor IX no
difference to the control was detectable (Tijburg, Ryan et al., J.
Biol. Chem. 266, (1991), 12067-12074).
In other e~mln~tions it was shown that endothelial cells and
thrombocytes in the presence of factor VIII and factor X have high
-
214164Z
. ~ .
affinity binding sites for factor IXa, but not for factor IX,
which are important for the further course of the coagulation
(Stern, Nawroth, J. Biol. Chem. 260, 6717-6722; Hoffmann, Monroe,
Thromb. and Hemost., 68, (1993), 74-78 and Ahmad, Rawala-Sheik, J.
Biol. Chem., 264, (1989), 20012-20016).
Because of this knowledge about the characteristics of factor IX
obtained until now, it could in no case be expected that the use
of factor IX fragments according to the invention makes the
stabilization of thrombocytes possible.
Surprisingly it was found that factor IX fragments according to
the invention lead to a stabilization of thrombocytes, although
normally a massive participation of thrombocytes takes place under
physiological conditions during intravascular fibrin formation
which leads to a consumption of thrombocytes.
Preparation of factor IX fragments
For the preparation of the factor IX fragments according to the
invention purified factor IX-fractions from plasma as well as
other, e.g. by genetic engeneering prepared factor IX containing
solutions can be used as source material.
According to the invention the factor IX is treated with proteases
under conditions where the Gla- and at least one EGF-like domain
stay largely intact and where fragments of factor IX are formed
with a molecular weight of about 12.000 to 50.000 D. This can be
achieved e.g. with proteases having the same or a similar
specificity like e.g. Russel's viper venom and where the factor IX
2141642
fragments should have no residual factor IX activity and shouldn't
be chemically modified.
If proteases with a broad specificity are used like e.g.
chymotrypsin or trypsin the proteolysis is performed under
conditions where the amino terminal end of factor IX is protected.
Preferrably the factor IX is bound reversible, that means non
covalent, with its amino terminal end onto a solid support and
treated in the immobilized form with the proteases. After that the
antithrombotic efficaciuos fragments of factor IX are eluted from
the support.
As supports, anion exchangers or affinity supports with affinity
to factor IX on its amino terminal end, can be used. Especially
preferable the factor IX is bound onto polymers carrying alpha-
hydroxy-amino-propyl-groups, e.g. Fractogel Amino-TSK.
Another possibility for the preparation of the factor IX fragments
according to the invention is-the proteolytical cleavage of factor
IX in solution with proteases like e.g. chymotrypsin or trypsin,
preferably immobilized on a carrier, where the amino terminal end
of factor IX is protected with low molecular substances binding to
the Gla- and EGF-like domain. Preferably low molecular amines and
especially preferable 1-amino-2-propanol are used as such
protecting substances. Also possible is the use of other
protecting substances like e.g. protamine.
The proteolytic factor IX fragments may be treated with~mild
reducing agents, e.g. dithiothreitol under gentle conditions. Thus
it is principally possible to isolate inhibitory active factor IX
21416g2
fragments which are not covalently modified and have no factor IX
activity.
It is known, that with the conventional treatment of the factor IX
with alpha-chymotrypsin, fragments are generated which have no
intact Gla- or EGF-like domains or where the yield of fragments is
only very poor ~Wildgoose et al., Biochem and Biophys. Res. Comm.,
152 (1988), 1207-1212). Controlled proteolytic cleavage of factor
IX was a problem because a mixture of different fragments was
always generated.
Using the method according to the invention it was for the first
time possible to prepare efficacious factor IX fragments in a
technically reproducible manner and with high yield in a pure
form.
The advantage of the preparation methods according to the
invention are among others:
1. The avoidance of highly toxic protease inhibitors (e.g.
diisopropyl-
fluorophosphate) for the inactivation of the proteases used
for the cleavage.
2. The avoidance of a chemical modification of the factor IX
fragments.
3. A gentle proteolytical cleavage of the factor IX because o~
the fixation
of the amino terminal end onto a matrix, preferrable a matrix
21~1642
.
containing 2-hydroxy-amino-propyl groups.
4. A gentle proteolytical cleavage of the factor IX because of
the use of
low molecular amines for protection of the amino terminal
domains.
5. A chromatographic separation of the antithrombotic efficacious
factor IX fragments from ineffective fragments.
Example 1:
Preparation of factor IX-fragments from native factor IX, which is
bound during the proteolytic cleavage with alpha-chymotrypsin onto
Fractogel Amino TSK.
A 50 ml column with Fractogel Amino-TSK (Merck, Darmstadt), a
copolymerisate of glycidylmethacrylat, pentaerythroldimethacrylate
and polyvinylalcohol with alpha-hydroxyl-amino-propyl-groups was
equilibrated with a tris-HCL-buffer (0,05 M Tris-HCl, 0,05 M NaCl,
pH 7,3). 16 mg of factor IX with a specific activity of 110 U/mg
dissolved in Tris-HCl-buffer were applied onto the column.
Subsequently the column was washed with the same buffer to which
10 mM calcium chloride was added.
For the fragmentation the bound Factor IX was incubated with
chymotrypsin, dissolved in the calcium-containing tris-HCl-buffer.
The concentration of the protease in the buffer was 0,025 mg/ml.
21416q2
The column was washed with the same buffer with a flow rate of 100
ml/h. After one column-volume eluate, a protein fraction was
eluted containing chymotrypsin and factor IX fragments which have
no affinity to the Fractogel Amino-TSK-column under the chosen
conditions. The fraction containing protease was rejected.
The column is washed with 2.5 column-volumes of the tris-HCl-
chymotrypsin-buffer without protease and calcium until no protein
was eluted from it.
In a further step the matrix was washed with tris-HCl-buffer (0.05
M tris-HCL, 0.15 M NaCl, pH 7.3) until no protein was eluted.
For the elution of the efficacious factor IX fragments the NaCl
concentration of the tris-HCl-buffer was increased to 0.5 M. The
desired fraction was eluted with a volume of 70 ml. The factor IX
fragments were dialyzed against 75 mM NaCl and subsequently
lyophilized.
The ~ragments had no more ~actor IX activity and inhibited the
factor X activation in the presence of endothelial cells as well
as the coagulation of factor IXa containing plasma under
participation of phospholipids in vitro. On the other hand they
didn't inhibit the coagulation of a plasma sample to which
thrombin was added.
In SDS-gel-electrophoresis of the fragment-fraction under non-
reducing conditlons two bands with a molecular weight of 47.000
and 34.000 Dalton were detectable, which could be assigned to the
amino terminal end of factor IX.
The yield of efficacious factor IX fragments in relation to the
applied factor IX protein was 60 ~.
12
~ 2141642
Example 2:
Preparation of factor IX-fragments from native factor IX, which is
bound during the proteolytic cleavage with trypsin on Fractogel
amino TSK.
10 mg factor IX were treated in the same way as in example 1, with
the difference that trypsin instead of chymotrypsin was used in a
concentration of 0.05 mg/ml.
In SDS-gel electrophoresis under reducing conditions a main band
of approximately 33.000 Dalton was detectable which could be
assigned to the amino terminal end of factor IX.
The yield o eicacious ~actor IX ~ragments in relation to the
applied ~actor IX protein was 57 ~.
The factor IX fragments had no more factor IX activity. In vitro
they inhibited the factor X activation in the presence of
endothelial cells as well as the coagulation of factor IXa
containing plasma under participation of phospholipids.
The coagulation of a plasma sample to which thrombin was added was
not inhibited.
Example 3:
~ 21~1fi42
Preparation of factor IX-fragments in the presence of 1-amino-2-
propanol
To 15 mg factor IX with a specific activity of 110 U/mg dissolved
in 60 ml tris-HCl-buffer (0,05 M Tris-HCl, 0,15 M NaCl, pH 7,5) 10
mM 1-amino-2-propanol were added. This solution was applied to a
15 ml column with alpha-chymotrypsin-Sepharose with a flow rate of
200 ml/h. Every 15 minutes the remaining factor IX activity in the
solution was determined with the one stage assay. When no
r~m~- n 1 ng factor IX activity was detectable in the solution the
column was washed with buffer until no more protein was eluted
from the column.
The solution with the factor IX fragments was dialyzed against
tris-HCl (0,05 M Tris-HCl, 0,2 M NaCl, pH 7).
A purification of the desired factor IX fragments was achieved by
chromatography with a carrier with 2-hydroxyl-amino groups
(Fractogel Amino-TSK). A 50 ml column with Fractogel Amino-TSK was
equilibrated with tris-HCl-buffer (0,05 M Tris-HCl, 0,1 M NaCl, pH
7). The solution with the factor IX fragments was diluted with two
parts of aqua dest and applied to the column with a flow rate of
300 ml/h. The antithrombotic effective factor IX fragments were
bound onto the column whereas the uneffective fragments and other
impurities were eluted.
After a washing step with tris-HCl-buffer (0,05 M Tris-HCl, 0,15 M
NaCl, pH 7) the antithrombotic effective fragments were eluted
with tris-HCl-buffer (0,05 M Tris-HCl, 0,5 M NaCl, pH 7) in a
volume of 40 - 60 ml. They have the same characteristics as in
14
- = =
2i91642
. ~
example 1. Their molecular weight was around 50.000 Dalton which
could be assigned to the amino terminal end of factor IX.
After a repeated dialysis against 0,05 M Tris-HCl, 0,075 M NaCl,
pH 7 the factor IX fragments were lyophilized.
Example 4:
Preparation of factor IX-fragments from activated factor IX, which
was bound to Fractogel Amino-TSK during the proteolysis.
Russel viper venom (RW, Sigma, Heidelberg) was immobilized on
CNBr-activated Sepharose 4 B according to the instruction of the
manufacturer (Pharmacia, Sweden). 1 ml gel contained 1.5 mg RW. A
factor IX solution with a specific activity of 110 U/mg was
applied onto the column until no further increase of activity was
detectable. The solution was dialyzed against tris-HCl-buffer
(0.05 M tris-HCl, 0.05 M NaCl, pH 7,3).
A 30 ml column with Fractogel Amino-TSK was equilibrated with
tris-HCl-buffer. The activated factor IX-solution was applied onto
the column and the column was washed with the same buffer to which
10 mM calcium chloride was added.
To the buffer with calcium chloride, alpha-chymotrypsin was added
in a concentration of 0.015 mg/ml and this solution was pumped in
circulation over the column with a flow rate of 75 ml/h. After one
column volume eluate, a fraction was obtained containing alpha-
chymotrypsin and fragments of factor IXa, without affinity to
214164~
Amino-TSK.
After washing with 3 column volumes of the chymotrypsin-containing
buffer the column was washed with tris-HCl-buffer without protease
until no more protein was eluted.
The matrix was washed with 0.05 M tris-HCl, 0.15 M NaCl, pH 7
until no more protein was eluted and subsequently eluted with 0.05
M tris-HCl, 0.5 M NaCl, pH 7.
The factor IX fragments were dialyzed against 0.075 M NaCl and
lyophilized.
The yield of eficacious factor IX fragments in relation to the
applied factor IX protein was 60 ~. ~
In SDS-gel electrophoresis under non-reducing conditions one main
band with a molecular weight of 31.000 Dalton was detectable,
which could be assigned to the amino terminal end of factor IX.
The factor IX fragments had no more factor IX activity. In vitro
they inhibited the factor X activation in the presence of
endothelial cells as well as the coagulation of factor IXa
containing plasma under participation of phospholipids.
On the other hand they didn't inhibit the coagulation of a plasma
sample to which thrombin was added.
Surprisingly an lnhibition of the factor IXa induced drastic
decrease of thrombocytes in vivo could be achieved by the factor
IX fragments according to the invention. Factor IXa is a strong
thrombogen which induces intravascular thrombosis in rabbits.
~n 1 m~ 1 experiments with rabbits
16
2141642
To 5 anesthetised rabbits 0.12 mg/kg body weight FIXa was injected
with a cannula in the ear vein.
To another 5 animals a dose of 1.8 mg/kg body weight factor IX
fragments from example 1 were injected 5 to 10 minutes before the
application of factor IXa.
5 control animals recieved 0.2 ml/kg body weight 0.9 ~ NaCl-
solution instead o~ Factor IXa.
During the whole period of the experiment of 60 minutes the
arterial blood pressure, the breething frequency and the standard
EKG were registered and in fixed intervalls arterial blood was
drawn in citrate tubes. In the drawn blood the thrombocyte count
and in the plasma of some animals the APTT and thrombin time was
determined.
Except the preparation of the rabbits for the experiment the
vessel system of the animals wasn't impaired. The physiological
blood flow in the vessels was ensured.
In the control group a slight decrease of the thrombocyte count
was detectable. This is an indication that even the preparation of
the animals and the narcosis has an influence on the stability of
the thrombocytes.
In the animals recieving the factor IXa compared to the NaCl-
control group a drastic, significant drop of the thrombocyte count
was detectable. The animals showd symptoms of a disseminated
intravasale coagulation (DIC). A heavy decrease of blood pressure
17
~. 21416g2
as well as strong deviations in the breething frequency and EKG
occured.
A distinct indication for an activation of the coagulation was the
coagulation of some of the blood samples in the citrate tubes and
the shortening of the APTT respectively the increase of the
thrombin time, which indicated a consumption of fibrinogen and the
formation of fibrin clots in the vessels.
The rabbits which recieved the factor IX fragments according to
the invention 5 - 10 minutes before the factor IXa dose showed
none of the above described incompatibility reactions. The
thrombocyte count remained surprisingly in the range of the
control gro'up. In no case a coagulation in the test tubes was
observed. The APTT and the thrombin time remained relatively
constant. This means that the coagulation activation was
prevented, without affecting the protecting extravascular
coagulation.
The single dose of 2 mg/kg body weight factor IX fragments showed
no signs of incompatibility reactions and kept the thrombocyte
count above the level of the control animals. The consequences of
the preparation of the ~n;m~l s and the narcosis on the thrombocyte
count were avoided by the factor IX fragments.
With the factor IX fragments according to the invention it is
possible to avoid a massive thrombocyte-mediated coagulation
activation in vivo and to stabilize at the same time the
thrombocytes. The protecting extravascular coagulation is not
affected by this treatment.
