Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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- 20465~8
DESCRIPTION
The human tissue plasminogen activator (t-PA)
possesses a great therapeutic importance in the
dissolving of blood coagula, e.g. in the case of
5 heart infarcts. t-PA acts on the blood coagulation
cascade by the activation of plasminogen to plasmin.
Plasmin in turn dissolves fibrin, the main component
of the protein matrix of coagulated blood.
Natural t-PA is composed of several functional
10 domains F, G, Kl, K2 and P. The domain P contains the
proteolytically active centre which brings about the
cleavage of plasminogen to plasmin. The gene-
technological preparation of t-PA or of different t-PA
muteins, in which some of the domains F, G, Kl and K2
15 are deleted, in eukaryotic and prokaryotic cells is
already known. In contradistinction to natural t-PA,
t-PA derivatives are thereby synthesised from pro-
karyotes in non-glycosylated form.
Proteins with t-PA activity dissolve in only
20 small concentration in the buffers usually employed
for the solubilisation of proteins, such as e.g.
50 mmole/l. Na citrate, 50 mmole/l. phosphate or
physiological NaCl. However, for the use as thera-
peutic active material, proteins with t-PA activity
25 should be present with a comparatively high enzymatic
activity of at least 0 . 25 MU/ml ., preferably of
0. 25 MU/ml . to 10 MU/ml .
~ 2046598
From EP-A 0 217 379, it is known to increase
the solubility of t-PA by neutral or slightly alkaline
arginine formulations. A disadvantage of this process
is, however, the low stability of highly concentrated
5 t-PA under neutral or slightly alkaline conditions.
U.S. Patent 4,777,043 discloses a pharmaceutical
composition with human t-PA and a pharmaceutically
compatible arginium ion-containing buffer with a
chloride ion concentration of up to 0. 3 mole/l .
EP-A 0 156 169, EP-A 0 303 351 and EP-A 0 297 294
disclose further possibilities of solubilising
proteins with t-PA activity in buffers by means of
amino acids, their salts, derivatives and homologues.
Furthermore, t-PA can be stabilised by addition of
gelatine according to EP-A 0 123 304, by addition of
albumin according to EP-A 0 122 940 or by addition of
a polysulphuric acid ester of a saccharide or of a
sulphated sugar according to EP-A 0 198 321. PCT/US88
04402 discloses a process for the increasing of the
t-PA solubility, wherein one uses an aqueous medium
with a basic amino acid, especially arginine, in a
concentration of 0. 02 to 0 . 2 mole/l ., together with a
citric acid group in a concentration of 0. 02 to 0. 08
mole/l. at a pH value of 5 to 8.
l~owever, these various cbmpositions are not
generally suitable for all proteins with t-PA propert-
ies. In particular, it was ascertained that non-
20~6598
glycosylated t-PA, non-glycosylated t-PA muteins and
glycosylated t-PA possess solubility properties
dif f ering greatly f rom one another
Consequently, it was the task of the invention
5 to develop pharmaceutical preparations which contain
glycosylated and non-glycosylated t-PA or t-PA muteins
with an activity of at least 0 . 25 MU/ml ., whereby the
t-PA is to be stable over a comparatively long period
of time.
In accordance with the invention there is
provided a pharmaceutical preparation of a protein
with t-PA activity of at least 0 25 MU/ml, preferably
at least 0 4 MU/ml ., and having a pH value of 4 . 5 to
9, which composition contains a substance of the group
15 citric acid, ascorbic acid, 2-oxoglutaric acid,
fumaric acid, Tris and EDTA in a concentration of at
least 0 . 2 mole/l.
Preferred is a concentration of the above
mentioned substances of 0 . 2 to 1 mole/1, especially
20 preferably of 0 . 3 to 1 mole/l .
Suitable for a composition according to the
invention is a pH value between 4 . 5 and 9, a pH value
of 6 to 7 . 5 is especially preferred.
By protein with t-PA activity according to the
25 present invention are to be understood non-modified t-
PA from prokaryotic and eukaryotic organisms, as well
as all t-PA muteins. Examples for t-PA muteins are
described e . g . by Harris ( Protein Engineering,
( 1987 ), 449-458 ) .
B
.
2046598
The composition according to the invention
preferably contains native glycosylated t-PA, e.g.
from CH0 cells. If the preparation according to the
invention contains a native glycosylated t-PA, then
5 its enzymatic activity is preferably to amount to at
least 1.4 MU/ml.
The uni~ U is a unit of the activity for t-PA
according to the definitlon of the WH0, National
Institute for Biological Standards and Control.
Further preferred is a non-glycosylated t-PA
from prokaryotes (t-PA pro), which is obtainable
according to DE-35 37 708. By t-PA pro, one under-
stands a t-PA which begins with the amino acids
-3 (Gly) to ~1 (Ser) and ends at 527 (Pro) (nomen-
15 clature according to Harris, Protein Engineering,
Volume 1 (1987) 449-458). If a preparation according
to the invention contains t-PA pro, then its enzymatic
activity should amount to at least 0. 25 MU/ml .
Further preferred is also a non-glycosylated t-PA
20 mutein with the domains Kl, K2 and P, which is
designated KlK2P pro (preparation according to DE
39 233 . 391.1 ) . KlK2P pro begins at amino acid 85-92
and ends at 527 (Pro). If a preparation according to
the invention contains KlK2P pro, then its enzymatic
25 activity is to amount to preferably at least 0.4 MU/ml.
Further preferred is also a non-glycosylated t-PA
mutein with the domains K2 and P, which is designated
~ 204~598
as K2P pro (preparation according to EP-A 0 382 174).
K2P pro begins at amino acid 174-179 and ends with
527 (Pro). K2P pro, as also KlK2P pro, can contain
partly or wholly the amino acids -3 (Gly) to +5 (Ile)
5 according to Harris, supra. If the preparation
according to the invention contains K2P pro, then its
enzymatic activity is preferably to amount to at least
1.4 MU/ml. However, all other t-PA variants from
prokaryotes or eukaryotes are also suitable.
In the following is set out a series of especially
preferred preparations according to the present
inve~tion .
One formulation contains 300 mmole/l. citric
acid/NaOH, pH 6. A urther formulation contains
15 300 mmole/l. ascorbic acid, pH 6. Furthermore, a
further formulation contains 300 mmole/l. 2-oxo-
glutaric acid, pH 6. Flrthermore, a further formul-
- ation contains 300 mmole/l. EDTA, pH 6. Furthermore,
a further formulation contains 300 mmole/l. fumaric
20 acid/NaOH, pH 6. Furthermore, a further formulation
contains 1 mole/l. Tris/HCl, pH 7.2.
Finally, a subject of the invention is also a
medicament based on a protein with t-PA activity as
active material in solution or as lyophilisate with
25 the given active materials and possibly also further
pharmaceutically compatible additive, adjuvant, carrier
and filling materials.
(o
2046598
The pharmaceutical preparations according to the
inve~tion are preferably used as injection or infusion
solutions. This can take place in that a solution
already ready for injection is made available which
5 possesses the composition according to the invention.
However, it is also possible to make available
pharmaceutical preparations in the form of lyophilisates.
These are then reconstituted with per se known agents
or solutions suitable for injection purposes. As
10 injection medium, water is preferably used which con-
tains the additives usual in the case of injection
solutions, such as stabilising agents, solubilising
agents, buffers and isotonic additives, for example a
physiological NaCl concentration. Such additives are,
15 for erample, mannitol, tartrate or citrate buffers,
ethanol, complex iormers, such as e.g. ethylenediamine-
tetraacetic acid and its non-toxic salts, as well as
high molecular polymers, such as liquid polyethylene
oxide, for viscosity regulation. Liquid carrier
20 materials for injection solutions must be sterile and
are preferably filled into ampouLes.
Finally, the present invention also comprises
the use of glycosylated proteins with t-PA activity
for the prepal~ation of pharmaceutical preparations
25 according to the invention.
The following Examples are to explain further
the concrete carrying out of the invention.
2046598
Examp 1 e
Influence of various substances on the solubility of
a non-glycosylated t-PA mutein with the domain
compos ition K2P .
In this Example, the solubility of K2P pro
(preparation according to EP-A 0 382 174) in various
buffer solutions is described. As is to be gathered
from Table 1, KrP pro dissolves in the buffer solutions
set out with a distinctly higher activity than 1.4
MU/ml.
Carrying out
170 ml. purified K2P pro (dissolved in 0.5 mole/l.
arginine/phorphoric acid pH 7.2) are concentrated by
ultra-filtration over an Amicon YM 10 membrane. In
each case, 1 ml. of the concentrate is dialysed against
the buffers set out in Table 1. After centrifuging of
the samples, the enzymatic activity is measured in the
supernatant .
The enzymatic activity is given as volume
activity in MU/ml. and as total activity in MU.
The measurement of the t-PA activity can thereby
be determined in the usual way by cleavage of a
chromogenic substrate (H. Lill, ZGIMAL, 42, (1987),
478-486). The unit U is one unit of the activity for
t-PA according to the definition of the WH0, National
Institute for Biological Standards and ~ontrol.
Table l 2046598
buf f er activity
MU/ml. MU
0 . 3 mole/l . citric acid/NaOH, pH 6 2 . 23 2 . 05
0.3 mole/l. ascorbic acid/NaOH, pH 6 1.68 1.95
1 mole/l. Tris/HCl, pH 7.2 8.76 5.8
0.3 mole/l. EDTA/NaOH, pH 6 1.95 1.95
0.3 mole/l. 2-oxo~glutaric acid, pH 6 2.64 3.17
0.3 mole/l. fumaric acid/NaOH, pH 6 1.85 4.08
10Example 2
Solubility of KlK2P pro
Purified KlK2P pro (dissolved in 0.5 mole/l.
arginine/H3PO4) is concentrated by ultrafiltration over
a YM 10 membrane (Amicon). In each case, 0.5 ml. of
15 the concentrate (activity: 3.5 MU/ml. ) is dialysed
against the buffer seC out in Table 2. After centri-
fuging of the samples, the enzymatic activity is
measured in the clear supernatant.
The enzymatic activity is given as volume unit
20 in MU/ml. and a total ac~ivity in MU.
The measurement of the t-PA activity can thereby
be determined in the usual way by cleavage of a
chromogenic ~substrate (H. Lill, ZGIMAL, 42 (1987),
478-486). The unit U is a unit of activity according
to the definition of the WHO, National Institute for
Biologlcal Standards and Control.
Table 2 2~46598
buffer activity
MU/ml. MU
1 mole/l. Tris/HCl, pH 7.2 1.65 1.07
5Examp 1 e 3
Solubility of tPA pro
Purified tPA pro (dissolved in 0.5 mole/l.
arginine/H3P04) is concentrated by ultrafiltration over
a YM 10 membrane (Amicon). In each case, 1 ml. of the
10 concentrate (activity: 2.4 MU/ml.) is dialysed against
the buffers set out in Table 3. After centrifuging of
the samples, the enzymatic activity ia measured in the
clear supernatant.
The enzymatic activity is given as volume unit
15 in MU/ml. and as total activity in MU.
The measurement of the tPA activity can thereby
be determined in the usual way by cleavage of a chromo-
genic substrate (H. Lill, ZGIMAL, 42 (1987), 478-486).
The unit U is a unit of activity according to the
20 definition of the WHO, National Institute for
Biological Standards and Control.
Table 3
buffer activity
MU/ml. MU
0.3 M citric acid/NaOI~, pl~ 6 0.29 0.16
l M Tris/HCl, pH 7.2 2.15 1.07
1~
Example 4 204659~
Solubility of CHO-tPA
Purifled CHO-tPA (dissolved in 0. 5 mole/l .
arginine/H3P04) is concentrated by ultrafiltration
5 over a YM 10 membrane (Amicon). In each case, 1 ml. of
the concentrate (activity: 6. 6 MU/ml . ) is dialysed
against the buffers set out in Table 4. After centri-
fuging of the samples, the enzymatic activity is
measured in the clear supernatant.
The enzymatic activity is given as volume units
in MU/ml. and as total activity in MU.
The measurement of the tPA activity can thereby
be determined in the usual way by cleavage of a chromo-
genic substrate (H. Lill, ZGIMAL, 42 (1987) 478-486).
15 The unit U is a unit of activity according to the
definition of the WHO, National Institute for
Biological Standards.
Table 4
buf f er - a c tivity
MU/ml. MU
1 M Tris/HCl, pH 7.2 6.16 6.49
0 . 3M citric acid/NaOH, pH 6 4.44 4.44
