U.S. patent application number 12/355507 was filed with the patent office on 2009-05-14 for pharmaceutical preparations and medicines capable of generating, and/or containing, thrombin.
This patent application is currently assigned to BIO & BIO LICENSING SA. Invention is credited to Johann Eibl.
Application Number | 20090123453 12/355507 |
Document ID | / |
Family ID | 30773865 |
Filed Date | 2009-05-14 |
United States Patent
Application |
20090123453 |
Kind Code |
A1 |
Eibl; Johann |
May 14, 2009 |
Pharmaceutical Preparations And Medicines Capable Of Generating,
And/Or Containing, Thrombin
Abstract
The invention relates to a pharmaceutical active ingredient
preparation for producing a medicament that contains thrombin or
has a thrombin-generating capacity and compositions comprising
thereof. The inventive preparation contains: (A) prothrombin
obtained from plasma or by means of genetic engineering
(coagulation factor II), (B) coagulation factors V, VIII, IX, X
obtained from plasma or by means of genetic engineering, which can
be at least partially in the activated state, and coagulation
factor X1a obtained from plasma or by means of genetic engineering,
and (C) phospholipids which are safe from prions and contribute to
the clotting process, said phospholipids being optionally contained
in liposomes.
Inventors: |
Eibl; Johann; (Wien,
AT) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
30 ROCKEFELLER PLAZA, 44TH FLOOR
NEW YORK
NY
10112-4498
US
|
Assignee: |
BIO & BIO LICENSING SA
Luxembourg
LU
|
Family ID: |
30773865 |
Appl. No.: |
12/355507 |
Filed: |
January 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11041165 |
Jan 21, 2005 |
7494971 |
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12355507 |
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PCT/AT03/00208 |
Jul 23, 2003 |
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11041165 |
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Current U.S.
Class: |
424/94.64 |
Current CPC
Class: |
A61K 38/37 20130101;
A61K 47/24 20130101; A61K 38/4846 20130101; A61K 38/4833 20130101;
A61K 38/36 20130101; A61K 9/127 20130101; A61K 38/36 20130101; A61K
2300/00 20130101; A61K 38/37 20130101; A61K 2300/00 20130101; A61K
38/4833 20130101; A61K 2300/00 20130101; A61K 38/4846 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/94.64 |
International
Class: |
A61K 38/48 20060101
A61K038/48 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2002 |
AT |
A 1113/2002 |
Jul 21, 2003 |
AT |
PCT/AT03/00204 |
Claims
1-36. (canceled)
37. A method for the manufacture of a pharmaceutical preparation
containing thrombin characterized in that a pharmaceutical
substance capable of generating thrombin is mixed with Ca ions,
resulting in a thrombin-containing product which is subsequently
purified by chromatography.
38. A method according to claim 37 wherein the pharmaceutical
preparation is rendered virally safe by virus inactivation during
or after thrombin generation as well as virus partitioning after
thrombin generation is complete.
39-52. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] The present application is a continuation of International
Patent Application No. PCT/AT03/000208, filed Jul. 23, 2003,
published in German on Feb. 5, 2004 as International Patent
Publication No. WO04/011024, which claims priority to Austrian
Application No. A 1132/2002, filed Jul. 23, 2002 and International
Patent Application No. PCT/AT03/000204, filed Jul. 21, 2003,
published as WO2004011023, all of which are incorporated herein in
their entireties.
INTRODUCTION
[0002] The invention concerns a pharmaceutical preparation capable
of generating, and containing, thrombin, and medicines manufactured
thereof.
BACKGROUND OF THE INVENTION
[0003] When blood vessels are injured, blood escapes from the
vascular space and coagulates. The injured blood vessels are closed
by the coagulating blood and, in that manner, protect the organism
from massive blood loss. Blood coagulation is caused by the enzyme
thrombin, which is generated from its zymogen, prothrombin, and
causes the transformation of the protein fibrinogen, which is
present in blood plasma, into insoluble fibrin. The exiting blood
coagulates within minutes, with only one sixth of the fibrinogen
contained in plasma being transformed into fibrin. Thrombin
generation continues in the coagulated blood, which still contains
large amounts of fibrinogen, until all of the residual fibrinogen
in the coagulated blood has been transformed into fibrin. This
process of continued thrombin formation and coagulation proceeds
slowly over a prolonged period of time and may take several hours
until it is completed.
[0004] When fibrinogen is transformed into fibrin, thrombin causes
fibrinopeptides A and B to split or partially split from the two
ends of the fibrinogen molecule, whereby fibrin monomers are
formed, which remain soluble for some time. These aggregate
laterally to fibrils, forming a fibrin network in the further
course (Blomback). This fibrin network, which has formed in the
coagulated blood, adheres to the injured tissue or wound bed and,
in that manner, leads to hemostasis and wound closure.
[0005] Thrombin also causes the transformation of coagulation
factor XIII, the zymogen of coagulation factor XIIIa, into a
transglutaminase. The latter cross-links proteins such as fibrin,
fibrin monomers, fibrinogen, and other proteins occurring in blood
plasma by covalent bonds. The cross-linking of fibrin, in
particular, is of great importance for the stability of the nascent
fibrin network (Siebenlist et al.).
[0006] Thrombin also transforms the thrombin-activatable
fibrinolysis inhibitor (TAFI) into a carboxypeptidase (TAFIa),
which splits carboxyterminal lysine residues from fibrin and
thereby inhibits the formation of the
tissue-plasminogen-activator-plasminogen-fibrin-complex, which is
necessary for the transformation of plasminogen into plasmin
(Booth).
[0007] In vitro, the formation of thrombin in blood or blood plasma
and thus the onset of the coagulation process is rendered possible
by tissue extracts. Such extracts can best be obtained from brain
using aqueous media or organic solvents (Morawitz).
[0008] The coagulation-active material, which can be extracted
using a suitable buffer and which is termed thromboplastin,
consists of an apoprotein, the tissue factor, and
coagulation-active lipids. Small amounts of thromboplastin, when
added to blood or plasma, suffice to generate rapid
coagulation.
[0009] Using organic solvents, it is possible to separate the
coagulation-active lipids from apoprotein. The aqueous apoprotein
part is then termed partial thromboplastin, which, in the presence
of kaolin, glass powder, and other surface-active substances also
leads to rapid coagulation when blood or blood plasma are
added.
[0010] This has led to the concept of an activation cascade of
enzymes resulting in the formation of the enzyme thrombin, which
causes blood coagulation (Davie et al.; MacFarlane).
[0011] The coagulation process which is triggered by thromboplastin
is referred to as the extrinsic pathway of blood coagulation, in
contrast to the coagulation process which is triggered by partial
thromboplastin, the latter being termed the intrinsic pathway. The
two processes have in common that they transform coagulation factor
X into Xa, albeit by different routes. Accordingly, a
differentiation is made between the extrinsic and intrinsic tenase
pathways in the first part of the coagulation process, which leads
to activation of coagulation factor X. In another enzyme complex,
prothrombinase, coagulation factor Xa converts prothrombin into
thrombin. This process is referred to as the common pathway. The
enzyme system which generates coagulation factor Xa with the aid of
thromboplastin is termed the extrinsic tenase complex, in contrast
to the enzyme system in which partial thromboplastin plays a role,
i.e. the intrinsic tenase complex.
[0012] The prevailing view has it that tissue factor, jointly with
coagulation factor VIIa and thrombocytes trigger blood coagulation
after injuries (Rapaport et al.). Tissue factor occurs in almost
all tissues in very varying amounts along with coagulation-active
lipids, and the two substances, when in contact with blood, form
the extrinsic tenase complex, since small amounts of activated
coagulation factor VII are always present in blood (Drake et al.).
The extrinsic tenase complex transforms both, coagulation factors X
and IX into Xa and IXa, respectively, and IXa transforms X into Xa.
The activation of factor X and the resulting formation of thrombin,
however, come to a halt rapidly by the tissue factor pathway
inhibitor (TFPI). The temporarily formed extrinsic tenase complex
as well as coagulation factor XIa independently lead to the
activation of the intrinsic tenase complex, which, as far as the
activation of coagulation factor X is concerned, is 50-fold more
active than the extrinsic tenase complex. The intrinsic tenase
complex consists of activated coagulation factors IXa, VIIIa, and
coagulation-active phospholipids and is not inhibited by TFPI (von
dem Borne et al.). In the intrinsic pathway, the activity of the
enzyme coagulation factor IXa, which transforms coagulation factor
X in Xa, is increased 100,000 to 1,000,000-fold. This increase is
caused by co-factor VIIIa, which itself is not an enzyme, and
certain phospholipids at an optimum calcium ion concentration.
[0013] Since the intrinsic tenase complex transforms coagulation
factor X into Xa very rapidly, and the latter, jointly with
cofactor Va and coagulation-active phospholipids, activates
prothrombin, great amounts of thrombin are formed in a very short
time. (Mann et al.).
[0014] The individual coagulation factors and platelets--the
essential components of blood responsible for the process of
coagulation--are normally present in abundance. Only if one of
these components is reduced by 90% or more, an increased propensity
to bleeding can be noticed. Bleedings become life-threatening only
in deficiency states where a coagulation factor and/or platelets
drop to several percent of their normal values. The central
importance of tissue factor in triggering the coagulation process
and its dissemination in all organs is beyond doubt, however,
severe disturbances of blood coagulation occur in tissues with low
tissue factor content in cases where a factor of the intrinsic
tenase complex or of the prothrombinase complex is pathologically
reduced. A case in point are patients suffering from hemophilia A
or B. The blood of these patients still coagulates in most
instances, however, because the intrinsic tenase complex is
deficient or absent, thrombin formation in the coagulated blood is
insufficient and the clot dissolves rapidly, so that no
satisfactory hemostasis is achieved.
[0015] Thrombin, mostly of bovine origin, is used as a medicinal
product for non-parenteral administration to achieve hemostasis in
cases of superficial injuries. Its hemostyptic effect could be
improved decisively when administered jointly with medicinal
products containing fibrinogen (Grey; Young et al.). Because of
their species-specific use, fibrinogen and thrombin are obtained
primarily from allogenic source material today. By combining the
application of thrombin with fibrinogen-containing medicinal
products, one attempts to mimic and improve the physiological blood
coagulation and accompanying hemostasis. This can be achieved also
in patients with severe blood coagulation disturbances (Matras et
al.).
[0016] By combining the application of fibrinogen concentrates,
whose fibrinogen content amounts to 10- to 20-fold the fibrinogen
content of blood, and great amounts of thrombin (100-1000 U per
mL), it is possible to reduce the coagulation time in such a
fibrinogen-thrombin mixture to a matter of seconds and obtain a 10
to 100-fold reduction compared to the physiological bleeding time.
This has made it possible to practically achieve instantaneous
hemostasis when such fibrinogen-thrombin mixtures are applied in an
optimal manner, provided that no larger blood vessels, particularly
arterial vessels, were injured (Spangler).
[0017] The fibrinogen transformed into fibrin by thrombin adheres
to the wound bed as does coagulated blood, the transglutaminases
which were activated by the action of thrombin obviously causing
covalent bonds between the injured tissue and the fibrin formed.
This strong adherence of the formed fibrin to tissue can be used
also to glue non-bleeding tissue, since the formed fibrin does not
impair the healing of the glued tissue in most cases and is largely
degraded in a matter of days or weeks (Matras et al.).
[0018] When fibrinogen-thrombin mixtures are used to achieve
hemostasis, an as rapid as possible initiation of the coagulation
process is desirable. In contrast, a gradual onset of the
coagulation process is preferable in cases where parts of tissue
are glued and also for sealing purposes. This makes it possible for
the tissue parts to be adapted more appropriately, and similar
adaptations are necessary in sealing. Thus far, a slowing of the
coagulation process has been achieved by a reduction of the
thrombin concentration to approximately 1% of the amount of
thrombin used to achieve hemostasis. However, the use of both, high
and low concentrations of thrombin is accompanied by
disadvantages.
[0019] Highly viscous fibrinogen solutions containing between 5 and
10% fibrinogen can be brought to coagulate with thrombin amounts
ranging from 100 to 1000 units within seconds. This short
coagulation time is necessary to achieve hemostasis rapidly after
the application of such a mixture to a bleeding site and arrest the
bleeding. The disadvantage of such a procedure is that the
fibrinogen and thrombin solutions are poorly mixed, since the high
viscosity of the mixture does not allow satisfactory mixing in a
short time. What results is a non-homogenous coagulation with an
attendant impairment in biomechanical quality.
[0020] On the other hand, if fibrinogen-thrombin mixtures are used
not to achieve hemostasis but rather to glue parts of tissue, it is
necessary in most cases to slow down the coagulation of the
fibrinogen-thrombin mixture in order to be able to optimize the
adaptation of the parts to be glued or sealed before coagulation
occurs. At present, this is attempted by reducing the amount of
thrombin to between one tenth to one hundredth of the amount of
thrombin used to achieve hemostasis at the disadvantage that not
all of the fibrinogen present is converted into fibrin and not all
of the factor XIII is converted to factor XIIIa. This procedure is
also not suitable for obtaining an optimal fibrin clot, since the
low thrombin concentration is not sufficient to transform TAFI into
TAFIa.
[0021] A further problem is the use of bovine materials for the
manufacture of medicinal products containing thrombin. Since a risk
of transmission of prions by any bovine organs cannot be excluded
with certainty, bovine thrombin is hardly used anymore today.
[0022] Bovine thrombin has the further disadvantage that it is
antigenic for other species and can provoke allergies and
anaphylaxes. In addition, patients treated with bovine thrombins
have been observed to develop coagulation disorders, which is
attributable to the fact that bovine thrombin can cause the
formation of antibodies against coagulation factors which
cross-react with human coagulation factors, thus retarding the
coagulation process.
[0023] In manufacturing thrombin from prothrombin, thromboplastin
from animal source material, mostly bovine brain, has often been
used to activate thrombin, which increases the yield. Because of
the risk of transmission of prions, thromboplastin from bovine
source is rarely used anymore in the manufacture of thrombin, and a
poor thrombin yield is put up with instead.
SUMMARY OF THE INVENTION
[0024] The invention relates to a pharmaceutical active ingredient
preparation for producing a medicament that contains thrombin or
has a thrombin-generating capacity and compositions comprising
thereof. The inventive preparation contains: (A) prothrombin
obtained from plasma or by means of genetic engineering
(coagulation factor II), (B) coagulation factors V, VIII, IX, X
obtained from plasma or by means of genetic engineering, which can
be at least partially in the activated state, and coagulation
factor X1a obtained from plasma or by means of genetic engineering,
and (C) phospholipids which are safe from prions and contribute to
the clotting process, said phospholipids being optionally contained
in liposomes.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The subject invention aims inter alia at making available a
virally safe pharmaceutical preparation which is capable of
generating thrombin and
[0026] which, when formulated into a medicinal product capable of
generating thrombin and when mixed with a pharmaceutical
preparation containing fibrinogen, gives a complete and homogenous
mixture of the two components, without too rapid a coagulation
process preventing complete mixing. The aim, however, is not only
to render possible complete mixing, but also the adaptation of
tissue parts to be glued or sealed before coagulation begins.
Thrombin generation must continue also after the onset of
coagulation in order to convert all of the fibrinogen, factor XIII,
and TAFI into fibrin, factor XIIIa, and TAFIa, respectively, so
that the wound closure obtained by fibrin be durable, and
[0027] in which the prothrombin it contains is converted as
completely as possible into thrombin and the latter can be further
purified and used for the manufacture of a medicinal product
containing thrombin. This medicinal product can be used as such or
jointly with other medicinal products containing fibrinogen.
[0028] The medicinal products manufactured from the pharmaceutical
preparations capable of generating thrombin should contain the
allogenic material of the species to which they are intended to be
applied only. Likewise, only species-specific proteins should be
used in their manufacture. All source and auxiliary materials used
should exclude a risk of transmission of prions. The generation of
thrombin from prothrombin in vivo and in vitro should be as
complete as possible, since it is only the prothrombin present in
an already formed fibrinogen clot from which thrombin can be
generated.
[0029] The pharmaceutical preparation or substance according to the
invention is suitable for the manufacture of a medicinal product
containing, or capable of, generating thrombin and is characterized
in that it contains: [0030] (A) prothrombin obtained from plasma or
by recombinant technology (coagulation factor II), [0031] (B)
coagulation factors V, VIII, IX, X obtained from plasma or by
recombinant technology, which may be present at least partly in
activated form, and plasmatic or recombinant coagulation factor
XIa, and [0032] (C) prion-safe, coagulation-active phospholipids,
which may be incorporated in liposomes.
[0033] The pharmaceutical preparation according to the invention
contains preferably coagulation factor VII obtained from plasma or
by recombinant technology or a mixture of plasmatic or recombinant
coagulation factors VII and VIIa. In addition, it contains
preferably a recombinant tissue factor as such or in relipidated
form.
[0034] The pharmaceutical preparation according to the invention
may also consist of two or more plasma fractions and the
coagulation active phospholipids, the individual plasma fraction
containing one or more coagulation factors and/or activated
coagulation factors of components (A) and (B). The whole plasma
fraction mixture must, however, contain all coagulation factors
and/or activated coagulation factors contained in components (A)
and (B), tissue factor preferably being added.
[0035] The coagulation factors, in particular, are manufactured
exclusively from the blood plasma of a particular mammalian species
or the corresponding recombinant coagulation factors or activated
coagulation factors, including tissue factor.
[0036] In the pharmaceutical preparation according to the
invention, prothrombin, coagulation factors V, VIII, IX, X, XIa,
and coagulation factors VII and VIIa, if present, as well as tissue
factor are preferably rendered virally safe by virus inactivation
and/or virus partitioning.
[0037] The pharmaceutical preparation according to the invention
may be present in deep-frozen or freeze-dried form.
[0038] The invention also concerns a medicinal product capable of
generating thrombin which can be manufactured from a pharmaceutical
preparation according to the invention.
[0039] The medicinal product capable of generating thrombin
according to the invention may be present in deep-frozen or in
freeze-dried form.
[0040] A preferred embodiment of the medicinal product capable of
generating thrombin according to the invention is characterized in
that it contains thrombin in such amounts that it will contain no
more than 1 U of thrombin per mL after thawing or
reconstitution.
[0041] The invention also concerns a medicinal product containing
thrombin and which can be manufactured from a pharmaceutical
preparation capable of generating thrombin according to the
invention. It may be provided in deep-frozen or freeze-dried
state.
[0042] The invention also concerns a medicinal product mixture
which coagulates within 30 to 300 seconds by mixing a medicinal
product according to the invention with a medicinal product
containing fibrinogen prior to application.
[0043] The invention also concerns a medicinal product containing
thrombin which, if mixed with the medicinal product mixture
described above will coagulate within 3 to 10 seconds.
[0044] A preferred method for the manufacture of a pharmaceutical
preparation containing thrombin according to the invention is
characterized in that a pharmaceutical preparation capable of
generating thrombin according to the invention is mixed with
Ca-ions, which results in a product containing thrombin and which
is subsequently purified by chromatography.
[0045] A preferred embodiment of the process according to the
invention is that the pharmaceutical preparation is rendered
virally safe by subjecting it to virus inactivation during or after
thrombin generation and to virus removal after thrombin generation
is complete.
[0046] The invention also concerns a process for the manufacture of
a pro-cofactor-concentrate containing coagulation factors V and
VIII and which is characterized in that plasmatic cryoprecipitate
is dissolved and fibrinogen is separated from the solution, whereby
a solution is obtained which contains coagulation factors V and
VIII and which can be mixed with calcium chloride.
[0047] Another process according to the invention concerns the
manufacture of a prothrombin complex concentrate containing
coagulation factor XI in addition to coagulation factors II, VII,
VIIa, IX, X, and XI, and containing per 1.0 .mu.g prothrombin at
least 0.030 .mu.g coagulation factor XIa and which is characterized
in that plasmatic cryoprecipitate supernatant is adsorbed onto an
anion exchanger and a prothrombin complex containing coagulation
factor XIa is eluted after washing the adsorbate and storage in the
cold.
[0048] The invention is based on the finding that a virally safe
intrinsic tenase-prothrombinase-complex is responsible for a
next-to-complete, timewise controllable formation of
.alpha.-thrombin from virally safe prothrombin. The intrinsic
tenase-prothrombinase-complex is generated by activation of the
respective coagulation factors, which either are or have been
rendered virally safe, in the presence of optimal
Ca-ion-concentrations, with prion-safe, coagulation-active
phospholipids and the virally safe activator substance coagulation
factor XIa and/or the virally safe activator substances coagulation
factors VIIa and tissue factor.
[0049] Thus, the aims of the present invention are accomplished by
a pharmaceutical preparation, consisting of three components
(Components A, B, and C), for the manufacture of a virally safe
medicinal product capable of generating, or containing, thrombin,
the pharmaceutical preparation being characterized in that
[0050] Component A contains virally safe prothrombin obtained from
plasma or genetically engineered, as a pharmaceutical active
substance,
[0051] Component B contains virally safe, plasma derived or
genetically engineered coagulation factors V, VIII, IX, X, XIa as
pharmaceutical active substances and, in case the generation of
extrinsic tenase complex is desirable in addition to the generation
of prothrombinase and intrinsic tenase complexes, also contains
virally safe coagulation factors VII and/or VIIa as well as tissue
factor as pharmaceutical active substances, and
[0052] Component C contains fully synthetic, coagulation-active
phospholipids as pharmaceutical active substances having a
transformation point of the paracrystalline into the
liquid-crystalline form at beyond 15.degree. C. A mixture of the
pharmaceutical active substances, i.e. the coagulation factors
and/or activated coagulation factors contained in Components A and
B can also be obtained by mixing two or more plasma fractions
containing all necessary pharmaceutical active substances and to
which tissue factor has been added, if necessary.
[0053] After mixing of the three components and addition of an
optimum amount of CaCl.sub.2, thrombin is generated
temperature-dependent between 0.degree. and 40.degree. C., the rate
of thrombin generation increasing with rising temperatures.
[0054] Component A is manufactured by dissolution of freeze-dried,
virally safe prothrombin or thawing of a frozen solution of virally
safe prothrombin and preparation of a prothrombin stock solution
containing 10-100 units of prothrombin per mL and 0.1% sodium
citrate at pH 7.3.
[0055] Potency of the stock solution is assessed by determination
of the prothrombin units using coagulation factor-II-deficient
plasma, and establishing a calibration curve using a reference
normal plasma.
[0056] With the aid of ecarin, the maximum amount of
meizothrombin/thrombin is determined which can be generated from
one unit of prothrombin. The meizothrombin/thrombin determination
is carried out with chromogenic substrate S-2238. In addition, the
amount of thrombin in NIH units is determined which is generated
from one unit of prothrombin after the addition of thromboplastin.
A corresponding standard curve for the determination of thrombin is
generated using the international .alpha.-thrombin standard.
[0057] Component B is manufactured by mixing stock solutions of
virally safe coagulation factors V, VIII, IX, X, XIa as well as
coagulation factors VII, VIIa, and tissue factor. The
concentrations of the individual coagulation factors in their stock
solutions lie between 10 and 100 units per mL; the tissue factor
stock solution contains 25 .mu.g/mL. Instead of a stock solution of
coagulation factor V, a stock solution of coagulation factor Va may
be used. The content of an individual coagulation factor in a stock
solution is determined using the corresponding deficient plasmas,
and a standard curve with pooled normal plasma is established to
determine each individual coagulation factor.
[0058] In order to determine what amounts of the individual
coagulation factors are necessary to still obtain an optimum
thrombin generation, different mixtures of Components A and B are
prepared, containing one unit each of coagulation factors II, V,
VIII, IX, X, XIa, and II, XIa, VII, VIIa, and 2.5 .mu.g tissue
factor, respectively, per mL of sample. After addition of the
liposome emulsion and subsequent recalcification, thrombin
generation is determined at 26.degree. C. at different time
intervals. In the samples, prothrombin consumption and the
generation of .beta.- and .gamma.-thrombin are also determined.
Prothrombin is transferred into thrombin in the presence of 0.1%
PEG and the amounts of coagulation factors and tissue factor per mL
are determined which are necessary to still achieve an optimum
thrombin generation.
[0059] Mixtures of Components A and B may also be obtained from two
or more plasma fractions to the extent they contain as
pharmaceutical active substances all coagulation factors and
activated coagulation factors necessary for the generation of the
intrinsic tenase and prothrombinase complexes. The intrinsic tenase
pathway is activated either by coagulation factor XIa or via the
extrinsic tenase pathway, particularly when TFPI is absent. It is
also possible to activate the intrinsic tenase pathway by
coagulation factor XIa and via the extrinsic tenase pathway
simultaneously. In the presence of coagulation factor XIa, or by
its addition to, a mixture of coagulation factors which are
necessary for the generation of the intrinsic tenase complex, it is
also possible to determine whether or not at least 90% of the
prothrombin in the mixture have been transferred into thrombin, and
if that is not the case, what amounts of factor VIIa and tissue
factor are necessary to achieve a complete transfer of prothrombin
into thrombin. A surplus of coagulation-active phospholipids is
used to generate thrombin. The activation is carried out at
26.degree. C. or 37.degree. C. and at an optimum Ca-ion
concentration.
[0060] The coagulation factors used and/or the plasma fractions
containing the coagulation factors as well as the genetically
engineered tissue factor are rendered virally safe by virus
inactivation, e.g. the solvent/detergent method, and subsequent
virus removal by nanofiltration.
[0061] Component C is manufactured from mixtures of fully
synthetic, and therefore, prion-safe, choline- or
serinephospholipids or from
choline-serine-ethanolamin-phospholipids. Only phospholipids are
used which transfer from the paracrystalline gel state into the
liquid-crystalline state between 15.degree. and 40.degree. C. From
the phospholipid mixtures, emulsions can be produced which contain
liposomes having diameters ranging from 20 nm to 1000 nm and which
have the polar parts of the phospholipids in an outer membrane.
Using temperatures between 50.degree. and 100.degree. C., liposomes
can be produced having diameters of 20 nm-200 nm. Such emulsions
can be sterilized using bacterial-tight filters, sterile filled,
dried, and stored under nitrogen. The freeze-dried emulsions can be
reconstituted with water.
[0062] The coagulation factors used and the non-relipidated tissue
factor can be rendered virally safe by virus inactivation,
preferably the solvent/detergent method and subsequent removal of
the solvent/detergent. Quantifiable traces of the solvent used,
tri-n-butylphosphate, and of the detergent, Tween 80, remain in the
thrombin-containing solution. Virus removal is achieved preferably
by nanofiltration after prior clarifying filtration using a series
of filters with narrowing pores, decreasing from 5000 nm to 35 nm.
Preferably, after filtration through a 35 nm filter, also
nanofilters with pore sizes of 20 nm and 15 nm may be used.
[0063] Thrombin generation of this pharmaceutical preparation is
highly temperature dependent. Therefore, it is possible to inhibit
the formation of thrombin from prothrombin in mixtures of
Components A, B, and C, even at optimum Ca-ion concentration, if
low temperatures around freezing-point are used in processing. The
pharmaceutical preparation according to the invention can be
deep-frozen for storage or can be freeze-dried and stored between
0.degree. and 25.degree. C.
[0064] The pharmaceutical preparation capable of generating
thrombin can be formulated at low temperatures into a medicinal
product having a thrombin generation capacity of preferably 100 to
1000 units of thrombin per mL, sterile filtered, portioned, and
filled.
[0065] The pharmaceutical preparation capable of generating
thrombin is also used for the manufacture of thrombin-containing
medicinal products. Thrombin generation is preferably carried out
at temperatures ranging between 200 and 40.degree. C.
[0066] For the manufacture of thrombin from a pharmaceutical
preparation capable of generating thrombin containing one or more
components that have not been virus inactivated, virus inactivation
can be carried out during or after thrombin generation. The virus
inactivating substances can be removed in the course of the
subsequent purification of the generated thrombin. It is possible
to obtain a thrombin-containing pharmaceutical substance having a
thrombin content of 1000 to 10000 units per mL, which is subjected
to virus removal by clarifying filtrations and subsequent
nanofiltration. The virally safe substance can then be formulated,
sterile filtered, and processed into a medicinal product.
[0067] Medicinal products capable of generating, and/or containing,
thrombin can be used in combination with fibrinogen-containing
medicinal products to achieve hemostasis, to glue sutured tissue,
and seal body cavities and vessels against leakage of gasses and
body fluids.
[0068] By mixing a medicinal product capable of generating thrombin
with a fibrinogen-containing medicinal product and addition, if
any, of low amounts of thrombin, the coagulation time of such a
mixture can be adjusted to 30 to 300 seconds. In that manner,
complete mixing of the medicinal products in such a mixture is
possible, even if the viscosity is high. Also, after complete
mixing of the medicinal products, enough time remains for the
necessary adaptation of the tissue parts that need to be glued or
sealed. Once coagulation sets in--similarly to the physiological
coagulation process--more thrombin is generated in the partly
coagulated fibrinogen, so that after some time--up to several
hours--practically all prothrombin has converted into thrombin and
the latter transforms all of the fibrinogen into fibrin. Such a
continued thrombin generation renders possible not only the
homogenous generation of fibrin in the already coagulated
fibrinogen, but also the uniform activation of factor XIII and
subsequent onset of the cross-linking of the generated fibrin, as
well as the activation of TAFI and the subsequent splitting-off of
the end-terminal lysine residues of fibrin.
[0069] The application of fibrinogen-containing mixtures for
hemostasis requires a very short coagulation time. In order to
achieve this, and at the same time guarantee a homogenous formation
of the coagulated fibrinogen and the activation of zymogens such as
factor XIII and/or TAFI, fibrinogen-containing medicinal products
can be mixed completely with medicinal products capable of
generating thrombin, and such mixtures can be applied to the
bleeding site jointly with a thrombin solution having a high
thrombin content. Although the onsetting coagulation does not
immediately lead to a homogenous fibrin clot, the generation of
thrombin in the fibrin clot continues as the thrombin-generating
medicinal product contained in the clot continues to transform
prothrombin into thrombin, resulting in a homogenous coagulated
fibrin clot with homogenously distributed factor XIIIa and
homogenously distributed TAFIa. This makes it possible to achieve
an excellent fibrin structure and high resistance against
fibrinolytic influences even with rapidly coagulating mixtures of
thrombin-containing and fibrinogen-containing medicinal
products.
[0070] Another possibility to trigger spontaneous thrombin
generation in a mixture of coagulation factors capable of
generating thrombin which consists of prothrombin complex and
factor VIII is by addition of factors of the contact system. For
that purpose, coagulation factors XI and XII need to be added,
unless they are already contained in the mixture.
[0071] The activation of coagulation factor XI in vivo as yet is
not understood in all detail. What is known is that blood cells and
cells of the endothelium play an important role in this activation
process. In blood, factor XI is bound to high molecular kininogen
(K.sub.d10.sup.-8) and prothrombin (K.sub.d10.sup.-7) in
non-covalent form. These complexes may accumulate on activated
platelets, this leading to activation of coagulation factor XI if
thrombin and/or coagulation factor XIIa are present.
[0072] In vitro, coagulation factor XI can be activated by
coagulation factor XIIa on negatively charged surfaces, coagulation
factor XIIa being generated from coagulation factor XII by
kallikrein. The formation of kallikrein from prekallikrein present
in blood can occur by different mechanisms, such as by
prekallikrein activator or metal salts of ellagic acid.
[0073] According to the invention, thrombin can be generated
spontaneously in a mixture of coagulation factors capable of
generating thrombin and consisting of prothrombin complex and
coagulation factor VIII, by activation of the coagulation factor XI
contained therein, all components of the mixture having been virus
inactivated. Preferably, this activation is performed in the
presence of prion-safe, coagulation-active phospholipids with
virally safe kallikrein. Instead of kallikrein, also virally
inactivated prekallikrein with virally inactivated prekallikrein
activator or prekallikrein activators can be used, such as metal
salts of ellagic acid.
[0074] For the manufacture of thrombin-containing pharmaceutical
substances, virus inactivation can also be performed during or
after thrombin generation.
[0075] The following examples describe the invention in greater
detail.
EXAMPLES
Preparation of Component A
[0076] 100 mg of virally inactivated prothrombin are dissolved in 1
L of 0.1% sodium citrate solution at pH 7.5, and the prothrombin
content per mL is determined using prothrombin-deficient plasma.
After the addition of ecarin, the generated amounts of
meizothrombin and thrombin are determined by splitting of
chromogenic substrate S-2238. In addition, thrombin generation is
determined using a prothrombinase preparation. The generated
thrombin is determined with the aid of a fibrinogen solution as
thrombin time. With this fibrinogen solution, a standard curve was
previously established using the international standard for
.alpha.-thrombin. This prothrombin stock solution can be
deep-frozen at -20.degree. C. and stored for at least six
months.
[0077] Preparation of Component B from Individual Coagulation
Factors.
[0078] Virally safe, highly purified coagulation factor
concentrates manufactured from plasma or obtained by genetic
engineering are dissolved in 0.1% sodium citrate pH 7.5 and mixed
under stirring: 3.2 mg of factor V, 0.2 mg of factor VIII, 5.0 mg
of factor IX, 11 mg of factor X, and 0.5 mg of factor XIa. After
sampling, the solution is deep-frozen and stored at -20.degree. C.
or temperatures below that. An equivalent sample is used to
determine the amount of a 1M CaCl.sub.2-solution which is necessary
to adjust the Ca-ion-activity to 5 mM-8 mM.
[0079] Preparation of a Mixture of Components A and B.
[0080] The frozen solutions of Component A, prepared according to
Example 1, and Component B, prepared according to Example 2, are
carefully thawed. Care must be taken for the temperature not to
rise above 4.degree. C. From the solutions thus obtained, a mixture
of equal parts of Components A and B is prepared, the necessary
samples are drawn, and the mixture of the two Components is again
frozen and stored at -20.degree. C.
[0081] Preparation of a Mixture of Plasma Fractions Containing the
Coagulation Factors of Components A and B.
[0082] From 10 L of deep-frozen Source Plasma that has been
carefully thawed at a temperature not exceeding 4.degree. C., the
cryoprecipitate is separated from the cryo-supernatant by
centrifugation at 6000 g for 15 minutes.
[0083] Prothrombin complex concentrate containing coagulation
factor XIa:
[0084] The cryo-supernatant is mixed with 16 g of weak anion
exchanger DEAE-Sephadex A-50, the pH is adjusted to between 7.8 and
8.8 using 0.1 normal sodium hydroxide, and the mixture is stirred
at a temperature of between 30 and 6.degree. C. for one hour. The
mixture can be kept at this temperature until the ion exchanger is
separated by filtration and/or centrifugation, which must be
accomplished within no more than 24 hours. The liquid released from
the ion exchanger is stored for preparation of other plasma
fractions, and the separated ion exchanger is used for the
manufacture of prothrombin complex. The ion exchanger obtained in
the manner described can be stored at a temperature of between
4.degree. and 6.degree. C. for up to 100 hours. For further
processing, the separated ion exchanger is washed twice, each time
with 1 L of 0.5% saline, and the prothrombin-complex-containing
coagulation factor XIa is obtained by elution of the ion exchanger
with 1000 mL of a 0.3 NaCL solution at pH 7.5. After elution, the
ion exchanger is washed with 500 ml of a 0.3 M NaCl solution at pH
7.5 once more, and the rinse is added to the eluate. Using ecarin,
the amount of prothrombin present in the total eluate is
determined, and the total eluate is adjusted by dilution to the
desired prothrombin content, which may lie between 2 and 5
prothrombin units per mL. The total eluate, which is coagulation
factor-XIa-containing prothrombin complex concentrate, may be
frozen and stored at -20.degree. C. Using samples drawn before
freezing, the contents of coagulation factors and activated
coagulation factors are determined.
[0085] Preparation of a Pro-Cofactor Concentrate:
[0086] The cryoprecipitate sedimented from plasma by centrifugation
is dissolved in 750 mL of 0.3% citrate buffer pH 7.0 and mixed with
110 g glycine. After stirring at a temperature of between 0.degree.
and 2.degree. C. for one hour, the precipitated fibrinogen is
separated by centrifugation at 3000 g for 15 minutes, and the
supernatant, which contains coagulation factors V and VIII, is
frozen. In a sample of the supernatant which has been diluted with
water at a ratio of 1:10, the content of factor VIII is determined,
as well as the amount of CaCl.sub.2 which is necessary for
recalcification, in order to obtain an ion activity equivalent to
that of a 5 mM CaCl.sub.2 solution. The frozen solution can be
stored at -20.degree. C.
[0087] Preparation of a Mixture of
Coagulation-Factor-XIA-Containing Prothrombin Concentrate and
Pro-Cofactor Concentrate:
[0088] A recalcified sample of pro-cofactor concentrate prepared
according to Example 4 b. which has been diluted with distilled
water 1:10, is added to prothrombin concentrate prepared according
to Example 4 a. in amounts of 0.1, 0.2, 0.4, and 0.8 mL per mL of
prothrombin concentrate. To each of these mixtures, 0.1 mL of a
liposome emulsion prepared according to Example 5 is added. After
incubation at 26.degree. C. for eight hours, the amount of
pro-cofactor concentrate is determined which is necessary to
transfer 90% or more of the prothrombin present into thrombin.
[0089] For the preparation of the mixture from prothrombin complex
concentrate containing coagulation factor XIa, and pro-cofactor
concentrate which have been obtained from 10 L of deep-frozen
plasma and have been stored frozen, these concentrates are thawed
and mixed at the optimum ratio previously determined. The mixture
is frozen and stored at -20.degree. C.
[0090] Manufacture of Component C without Emulgator.
[0091] Fully synthetic, and therefore, virally and prion-safe
phospholipids are used. 200 mg
2Na-1,2-di-oleoyl-sn-glycero-3-phospho-L-serine, 400 mg
1,2,di-oleoyl-sn-glycero-3-phosphocholine, and 400 mg
di-oleoyl-ethanolamine-phospholipid are dissolved in 10 mL
chloroform, and the solution is evaporated in a 250-mL
round-bottomed flask by heating and continuous rotation until most
of the chloroform is removed. Residual chloroform is removed by a
nitrogen stream, and 10 mL of 0.1% citrate buffer solution pH 7.3
are added. The phospholipid film on the inner wall of the
round-bottomed flask is completely emulsified in the buffer
solution at 65.degree. C. During emulsification, the flask is
repeatedly shaken in a Vortex device until the lipid film has
completely disappeared. The emulsion as such or diluted 1:10 with
0.1% citrate buffer pH 7.0, is filtered through a filter having a
pore size of 0.45 .mu.m and is subsequently sterile filtered
through a 0.22 .mu.m filter. This liposome emulsion can be stored
sterile in a refrigerator and is shaken in a Vortex device for 1
minute prior to use. The emulsion is tested for sterility and
pyrogenicity according to the European Pharmacopoeia (Eur. Pharm.
4.sup.th Edition 2002, pages 123-126, 2.6.1. Sterility. Pages
131-132, 2.6.8. Pyrogens).
[0092] Preparation of Component C Using Sodium Cholate.
[0093] 250 mg 2Na-1,2-di-oleoyl-sn-glycero-3-phospho-L-serine, 750
mg 1,2,di-oleoyl-sn-glycero-3-phosphocholine and 500 mg sodium
cholate are dissolved in 10 mL of a 1+1 mixture of chloroform and
methyl alcohol, and the two solvents are evaporated under vacuum so
that a thin, uniform film settles on the inner wall of a
round-bottomed flask. The film is washed off with 10 mL 0.1%
citrate buffer pH 7.3, and the emulsion obtained in that manner is
processed and sterile filtered as described in Example 5.
[0094] Virally Safe Activator Substances for the Activation of the
Intrinsic Tenase Pathway.
[0095] Coagulation factor XIa alone or in combination with
coagulation factor XI, or coagulation factor VIIa, preferably with
tissue factor, are used as activator substances. Coagulation factor
XIa can also be used jointly with coagulation factor VIIa and
tissue factor. For use as pharmaceutical active substances, the
activated coagulation factors and tissue factor are virus
inactivated by a solvent/detergent method, and after removal of the
solvent/detergent, are subjected to virus partitioning by
nanofiltration.
[0096] The following pharmaceutical active substances, dissolved in
0.3% citrate buffer pH 7.3, are used as stock solutions of the
activator substances:
[0097] 30 .mu.g factor XIa/mL;
[0098] 30 .mu.g factor XIa and 50 ng factor XI/mL;
[0099] 30 .mu.g factor XIa and 20 ng factor VIIa/mL;
[0100] 30 .mu.g factor XIa and 20 ng factor VIIa and 10 ng tissue
factor/mL;
[0101] 10 .mu.g/mL tissue factor.
[0102] For the purpose of determining the best suitable activator
substance, 10 .mu.L each of a dilution series of the individual
activator substances are added to 1 mL each of the coagulation
factor mixtures prepared according to Examples 3 or 4 and are mixed
with a 10 .mu.L of a 1:100 dilution of a liposome emulsion prepared
according to Example 5. After recalcification, the mixtures are
kept at 260 or 37.degree. C. The amount of factor Xa generated is
determined using chromogenic substrate S-2251 at intervals of 10,
30, 60, and 120 min. The activator substance which generates the
greatest amount of factor Xa from the coagulation factor
concentrates produced according to Examples 3 and 4 after
recalcification, is used for the manufacture of a pharmaceutical
substance capable of generating thrombin.
[0103] Determination of the amounts of Component C necessary to
transform at least 90% of the prothrombin present in a mixture of
Components A and B at 26.degree. C. within eight hours or
37.degree. C. within two hours.
[0104] Coagulation factor mixtures according to Examples 3 or 4, to
which activator substances in amounts determined according to
Example 7 may have been added, are mixed with increasing amounts of
liposome emulsions. To 10 mL-samples of each coagulation factor
mixture, 0.1, 0.2, 0.4, or 0.8 mL of a 1:100 dilution of a liposome
emulsion produced according to Example 5 are added after
recalcification, the mixtures are incubated for eight hours at
26.degree. or two hours at 37.degree. C. The minimum amount of
liposome emulsion is determined which is necessary to transform at
least 90% of the prothrombin present into thrombin.
[0105] Preparation of a Virally Safe Pharmaceutical Substance
Capable of Generating Thrombin.
[0106] A virally safe coagulation factor mixture produced according
to Example 3 (Components A and B) is thawed, if necessary mixed
with an appropriate amount of a virally safe activator substance
selected as described in Example 7, and the mixture is stirred at
0.degree. to 2.degree. C. for 15 minutes. To the mixture, the
amount of liposome emulsion (Component C) determined in Example 5
is added. The mixture is adjusted to a concentration of 0.9% NaCl
using solid sodium chloride, subjected to clarifying filtration,
and sterile filtered through 0.22 .mu.m and/or 0.1 .mu.m filters.
The whole manipulation, including sterile filtration, is carried
out at a temperature between 0.degree. and 2.degree. C. The
pharmaceutical substance capable of generating thrombin obtained in
that manner is sampled, frozen, and stored at -20.degree. C. The
necessary tests for stability and pyrogenicity are carried out
according the European Pharmacopoeia. The capacity of generating
thrombin is determined in in-process controls and in the sterile
filtrate after recalcification at 37.degree. C. over two hours,
along with residual amounts of prothrombin which can be activated
by ecarin, in order to guarantee an at least 90% conversion of
prothrombin into thrombin.
[0107] Preparation of a Virally Safe Pharmaceutical Preparation
Containing Thrombin.
[0108] The frozen mixture produced from a factor-XIa-containing
prothrombin complex concentrate and a pro-cofactor concentrate
according to Example 4 is thawed, and, if desirable after Example
7, mixed with a selected, calculated amount of activator substance.
By addition of 10 mg Tween 80 and 0.3 mg tri-n-butylphosphate per g
protein of the coagulation factor mixture at 26.degree. C. after
recalcification and addition of the amount of liposome emulsion
determined according to Example 6, the major amount of the
prothrombin is converted into thrombin within eight hours, and
virus inactivation is carried out at the same time.
[0109] The thrombin-containing solution can be either frozen and
stored or be used immediately or after thawing. By the addition of
sodium citrate solution, the Ca-ion concentration is adjusted to 25
mM and the pH to 6.5. Under continued stirring at room temperature,
polyethylene glycol having a molecular weight of between 6000 and
8000 is added in an amount to achieve a 0.1% solution. A 20%
CM-sepharose suspension that has been washed with 0.025 M sodium
citrate pH 6.5 is then added under stirring until at least 95% of
the available thrombin has been adsorbed onto the CM-sepharose. The
required amount of CM-sepharose is determined previously by
estimating the lowest amount of the 20% emulsion which adsorbs at
least 95% of the available thrombin. The CM-sepharose is
centrifuged off between 3000 and 5000 g for 30 minutes, the
sediment is resuspended in 25 mM citrate buffer pH 6.5 and filled
into an appropriate column. The column is washed at pH 6.5 with 1%
citrate buffer containing also 0.1% PEG and eluted with a sodium
chloride gradient in the presence of 0.1% citrate and 0.1% PEG at
pH 6.5. The sodium chloride gradient is established between 10 and
200 mM. The fractions containing most of the thrombin are
collected, diafiltered, the concentration being thereby adjusted to
approximately 500 U thrombin per mL. Such a solution can be frozen,
stored after freeze-drying or further processed without delay.
[0110] After clarifying filtration by filters of different pore
sizes ranging from 5000 nm to 75 nm, the thrombin-containing
solution is filtered through a 35 nm-nanofilter and preferably
through further nanofilters having pore sizes of 20 nm and 15 nm.
The virally partitioned thrombin solution obtained in that manner
is concentrated to a content of more than 5000 U thrombin per mL
using a 30 KD-filter, frozen or freeze-dried, and stored.
[0111] Preparation of a Virally Safe Medicinal Product Capable of
Generating Thrombin.
[0112] An equivalent sample of the virally safe pharmaceutical
substance that has been produced according to Example 9 and has
been frozen or freeze-dried, is diluted with a 0.9% NaCl solution
in a manner to ensure it will generate 500.+-.50 U thrombin per mL
after recalcification at 37.degree. C. within two hours.
[0113] Such a diluted sample is mixed with an equal amount of a
5-10% fibrinogen-containing medicinal product which is planned to
form part of a fibrin sealant kit, and the coagulation time of this
mixture is determined after recalcification. If the coagulation
time at 37.degree. C. exceeds 150 seconds, the pharmaceutical
substance according to Example 7 is admixed with an amount of
thrombin produced as described in Example 8 appropriate to lower
the coagulation time to between 100 and 150 seconds.
[0114] The amounts of thrombin determined in that manner are added
to the substance obtained as described in Example 9 under stirring
at a temperature of between 0.degree. and 2.degree. C., and the
mixture is clarified by filtration and sterile filtered. The
resulting sterile bulk solution is portioned and filled into final
containers, preferably at the same temperature, and is
freeze-dried. The freeze-dried powder is reconstituted with a
sterile 5 mM calcium chloride solution.
[0115] Preparation of a Virally Safe Medicinal Product Containing
Thrombin.
[0116] A pharmaceutical substance according to Example 10 is thawed
or reconstituted and adjusted to a concentration of 0.9% NaCl using
sodium chloride. Isotonic saline is added to adjust the thrombin
concentration to between 500 and 5000 U per mL, as desired. PEG is
added in an amount to produce a 0.1% solution, and a 10% CaCl.sub.2
solution is added in an amount to obtain a 5 mM CaCl.sub.2
solution. This solution is sterile filtered, portioned and filled
into final containers under sterile conditions. It can be frozen or
freeze-dried for storage. Freeze-dried thrombin solution is
reconstituted with Water for Injections.
[0117] Use of a Medicinal Product Capable of Generating Thrombin in
Combination with a Medicinal Product Containing Thrombin.
[0118] 1 mL of fibrinogen-containing medicinal product containing
50-100 mg fibrinogen per mL is mixed with 1 mL of a medicinal
product capable of generating thrombin according to Example 11. The
mixture of the two medicinal products is stirred under sterile
conditions for approximately 20 seconds, and the homogenous mixture
obtained in that manner is applied to tissues to be glued or sealed
after they have been adapted to their desired positions. The glued
or sealed tissues are held in the adjusted positions until the
mixture of the two medicinal products has coagulated. If necessary,
they may be arrested in the fixed position for a longer period of
time, even for several hours.
[0119] Use of a Medicinal Product Containing Thrombin.
[0120] To 2.0 mL of a mixture of medicinal products according to
Example 13, 0.2 mL of a medicinal product containing thrombin
according to Example 12 at a concentration of 2000 u thrombin/mL
are added and mixed rapidly. To achieve hemostasis, this mixture is
applied to bleeding sites without delay, preferably after
accumulated blood has been removed by sucking, swabbing or blowing
it off.
[0121] Determination of coagulation factor XI and of coagulation
factor XII in ion-exchanger eluates according to Example 4 a which
are not stored after charging but eluted immediately after washing
of the charged ion exchanger.
[0122] Determination is Carried Out Using
Coagulation-Factor-Deficient Plasmas by American Diagnostica.
[0123] Determination of kallikrein and prekallikrein in eluates of
ion exchangers which are charged with prothrombin complexes and are
eluted immediately after washing or only after storage for up to
100 hours at refrigerator temperature.
[0124] Kallikrein activity is determined using chromogenic
substrate S-2403. A 4 mM chromogenic substrate solution is used,
which gives a final concentration of 0.4 mM. The prekallikrein
concentration is also determined with chromogenic substrate S-2403,
a 1:100 dilution of the eluate being mixed with an equal volume of
a 0.5% kaolin suspension and incubated at 37.degree. C. under
stirring for five minutes. Prior to the measured extinction, the
extinction which is obtained without activation of the kaolin
suspension is deducted, and the amounts or units prekallikrein are
determined using a standard curve.
[0125] Determination of Prekallikrein Activator.
[0126] Prekallikrein activator determination is performed in the
same manner as the determination of prekallikrein, except that a
concentration series of prekallikrein activators is used instead of
kaolin suspensions. If copper salts of ellagic acid are used, a
concentration series of between 10 ng and 10000 ng is used, which
generates the maximum amount of kallikrein from a prekallikrein
sample at 37.degree. C. within five minutes.
[0127] Preparation of plant and/or or synthetic phospholipid
emulsions from L-.alpha.-phosphatidylcholine,
L-a-phosphatidyl-L-serine, and L-a-phosphatidyl-ethanolamine, which
is the same as described in Example 5.
[0128] Determination of TAFIa.
[0129] TAFIa is determined using chromogenic TAFI activity kits by
American Diagnostica without the addition of activator (thrombin).
The content of TAFIa is calculated from a standard curve, which was
established using dilutions of plasma and subsequent activation
with thrombin and measurement of the TAFIa-activity with a
chromogenic substrate.
[0130] Determination of Coagulation Factor XIIIa.
[0131] Determination of activated factor XIII is carried out
according to European Pharmacopoeia Suppl. 4.5. 07/2003, p. 3687
without the use of activators of factor XIII (thrombin).
[0132] Ultrasound Treatment of Phospholipid Emulsions.
[0133] From the phospholipid mixtures, 1% suspensions are produced
by dilution with isotonic saline, and 2 mL are subjected to
ultrasonic irradiation. Ultrasonic irradiation is carried out with
Ultrasound Desintegrator Sonifire II W-250. The output is a maximum
of 200 watts at a frequency of 20 kHz. Konverter 102 C was used
with standard resonator 1/2'' and microtip 101-148-062. Ultrasonic
irradiation is carried out for between 10 and 100 seconds without
cooling of the irradiated emulsion at step 1 and at 10% pulsating
interval. The activity of the irradiated phospholipid emulsion is
determined by assessment of the partial thromboplastin time using a
reference plasma and a kaolin suspension. 100 .mu.L of plasma are
incubated with 50 .mu.L of a liposome emulsion and 50 .mu.L of a
0.5% kaolin suspension at 37.degree. C. for five minutes,
recalcified, and the coagulation time at 37.degree. C. is
determined. The required amount of liposome emulsion is determined
which causes the shortest coagulation time. The activities of the
liposome emulsions described in Examples 5 and 6, and of the
phospholipid emulsions according to Example 18 can be determined by
this method.
[0134] Determination of the Appropriate Ca-Ion Concentration for
the Desired Adjustment of the Coagulation Time of a Pharmaceutical
Substance Capable of Generating Thrombin.
[0135] Pharmaceutical preparations generating thrombin are
produced, and a geometric dilution series of the pharmaceutical
preparation to be tested is mixed with Ca-ions, the highest Ca-ion
concentration being 1 mM. The dilution series ranges from 125 .mu.M
to 1000 .mu.M. Similarly, a Ca-ion concentration series is prepared
ranging from 10 mM to 640 mM, and the coagulation time is
determined. The required Ca-ion concentration is determined using a
standard curve.
[0136] In a chessboard sample, the optimum mixture of a
phospholipid mixture with kaolin is determined which, under optimum
ultrasonic irradiation, shows the highest activity in APTT with
plasma. The test is carried out using 100 .mu.L of the irradiated
mixture, which has been incubated with 100 .mu.L of plasma at
37.degree. C. for 5 minutes, and which is recalcified by addition
of 100 .mu.L of a CaCl.sub.2-solution. To determine the optimum
irradiation intensity, ultrasonic irradiation is carried out as
described in Example 21, an 80% pulsating interval being used in
steps 1, 2, and 3, and the samples being irradiated for 10, 30,
100, or 300 seconds.
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