Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL COMPOSITION COMPRISING FACTOR VII POLYPEPTIDES AND FACTOR XI
POLYPEPTIDES
FIELD OF THIS INVENTION
The present invention relates to a pharmaceutical composition comprising
factor VII or
a factor VII-related polypeptide and factor XI or a factor XI-related
polypeptide. The invention
also relates to the use of a combination of a factor VII or a factor VII-
related polypeptide and a
factor XI or a factor XI-related polypeptide for the manufacture of a
medicament for treatment
of subjects suffering from bleeding episodes, or prevention hereof. The
invention also relates to
a method for treatment of bleeding episodes in subjects and to a method for
enhancing clot
formation in a subject. The present invention also relates to kits comprising
these compounds.
BACKGROUND OF THE INVENTION
Haemostasis is initiated by the formation of a complex between tissue factor
(TF) being
exposed to the circulating blood following an injury to the vessel wall, and
FVlla which is present
in the circulation in an amount corresponding to about 1 % of the total FVII
protein mass. This
complex is anchored to the TF-bearing cell and activates FX into FXa and FIX
into FIXa on the cell
surface. FXa activates prothrombin to thrombin, which activates FVIII, FV, FXI
and FXIII. Further-
more, the limited amount of thrombin formed in this initial step of
haemostasis also activates
the platelets. Following the action of thrombin on the platelets these change
shape and expose
charged phospholipids on their surface. This activated platelet surface forms
the template for
the further FX activation and the full thrombin generation. The further FX
activation on the acti-
vated platelet surface occurs via a FIXa-FVllla complex formed on the surface
of the activated
platelet, and FXa then converts prothrombin into thrombin while still on the
surface. Thrombin
then converts fibrinogen into fibrin which is insoluble and whieh stabilizes
the initial platelet
plug. This process is compartmentalized, i.e., localised to the site of TF
expression or exposure,
thereby minimizing the risk of a systemic activation of the coagulation
system. The insoluble fi-
brin forming the plug is furthermore stabilised by FXIII-catalysed cross-
linking of the fibrin fibres.
FVlla exists in plasma mainly as a single-chain zymogen, which is cleaved by
FXa into its
two-chain, activated form, FVlla. Recombinant activated factor Vlla (rFVlla)
has been developed
as a pro-haemostatic agent. The administration of rFVlla offers a rapid and
highly effective pro-
haemostatic response in haemophilic subjects with bleedings who cannot be
treated with
coagulation factor products due to antibody formation. Also bleeding subjects
with a factor VII
deficiency or subjects having a normal coagulation system but experiencing
excessive bleeding
can be treated successfully with FVlla. In these studies, no unfavourable side
effects of rFVlla (in
particular the occurrence of thromboembolism) has been encountered.
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Extra exogenously administered FVlla increases the formation of thrombin on
the acti-
vated platelet surface. This occurs in haemophiliac subjects lacking FIX or
FVIII and therefore
missing the most potent pathway for full thrombin formation. Also in the
presence of a lowered
number of platelets or platelets with a defect function, extra FVlla increases
the thrombin for-
mation.
Commercial preparations of recombinant human FVlla are sold as NovoSeven~
(Novo
Nordisk AIS,Denmark). Novoseven~ is indicated for treatment of bleeding
episodes in haemo-
philia A and B patients. Novoseven~ is the only recombinant FVlla available on
the market for
effective and reliable treatment of bleeding episodes.
FXI is a component of the intrinsic pathway of coagulation. A deficiency of
FXI is asso-
ciated with a mild to moderate bleeding disorder especially from tissues with
a high local fibri-
nolytic activity. In contrast, it is believed that high levels of FXI are a
risk factor for venous
thrombosis. FXI is the zymogen of a trypsin-like serine protease that is
activated by FXlla, throm
bin and FXIa. Activated FXI (FXIa) participates in the activation of FIX,
which in turn (in combina
tion with FVIII) further activates FX and thus gives rise to generation of
thrombin.
It is well known that subjects who bleed excessively in association with
surgery or major
trauma and need blood transfusions develop more complications than those who
do not experi-
ence any bleeding. However, also moderate bleedings requiring the
administration of human
blood or blood products (platelets, leukocytes, plasma-derived concentrates
for the treatment of
coagulation defects, etc.) may lead to complications associated with the risk
of transferring hu-
man viruses (hepatitis, HIV, parvovirus, and other, by now unknown viruses).
Extensive bleedings
requiring massive blood transfusions may lead to the development of multiple
organ failure in-
cluding impaired lung and kidney function. Once a subject has developed these
serious complica-
tions a cascade of events involving a number of cytokines and inflammatory
reactions is started
making any treatment extremely difficult and unfortunately often unsuccessful.
Therefore a ma-
jor goal in surgery as well as in the treatment of major tissue damage is to
avoid or minimise the
bleeding. To avoid or minimise such bleeding it is of importance to ensure the
formation of sta-
ble and solid haemostatic plugs that are not easily dissolved by fibrinolytic
enzymes. Further-
more, it is of importance to ensure quick and effective formation of such
plugs or clots.
Today, subjects experiencing bleeding episodes, including trauma victims and
subjects
bleeding in association with surgery, are often treated with several
injections or infusions of
FVlla since the short half-life of FVlla (2.5 hours) may require more than one
administration to
maintain a certain level of haemostatic ability. A faster arrest of bleedings
would be an impor-
tant benefit to such subjects. So would a reduction in the number of
administrations needed to
stop bleeding and maintain haemostasis.
Japanese patent application No. 59-116213A concerns an aerosol composition for
use as
a tissue glue containing a blood coagulant as an active component. The blood
coagulant may be
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3
selected from blood coagulation factors I, II, III, IV, V, VII, VIII, IX, X,
XI, XII, and XIII, prekallikrein,
high polymer kininogen and thrombin. A combination of F XIII and thrombin is
preferred.
European Patent No. 225.160 (Novo Nordisk) concerns compositions of FVlla and
methods
for the treatment of bleeding disorders not caused by clotting factor defects
or clotting factor
inhibitors.
European Patent No. 82.182 (Baxter Travenol Lab.) concerns a composition of
factor Vlla
for use in counteracting deficiencies of blood clotting factors or the effects
of inhibitors to blood
clotting factors in a subject.
International Patent Publication No. WO 93/06855 (Novo Nordisk) concerns the
topical
application of FVlla.
US Patent No. 5,252,217 concerns a process for preparing a human factor XI
concentrate
intended for therapeutic use.
There is still a need in the art for improved treatment of subjects
experiencing bleeding
episodes, including subjects where the bleeding episodes are due to surgery,
trauma, or other
forms of tissue damage; induced coagulophathy, including coagulopathy in multi-
transfused sub-
jects; congenital or acquired coagulation or bleeding disorders, including
diminished liver func-
tion ("liver disease"); defective platelet function or decreased platelet
number; lacking or ab-
normal essential clotting "compounds" (e.g., platelets or von Willebrand
factor protein); in-
creased fibrinolysis; anticoagulant therapy or thrombolytic therapy; or stem
cell transplantation.
There remains a need in the art for an improved, reliable and widely
applicable method
of enhancing coagulation, enhancing or ensuring formation of stable
haemostatic plugs, or en-
hancing convenience for the treated subject, or achieving full haemostasis in
subjects, in particu-
lar in subjects having an impaired thrombin generation. There is also a need
for methods
wherein the amount of FVlla needed for achieving full haemostasis is lowered
and methods
wherein the time to bleeding arrest is shortened.
SUMMARY OF THE INVENTION
One object of the present invention is to provide compositions, which can
effectively be
used in the treatment or prophylaxis of bleeding episodes and coagulation
disorders.
A second object of the present invention is to provide compositions in single-
unit
dosage form, which can effectively be used in the treatment or prophylaxis of
bleeding episodes
or as a procoagulant. Another object of the present invention is to provide
compositions,
methods of treatment or kits exhibiting a synergistic effect.
A further object of the present invention is to provide compositions, methods
of
treatment or kits exhibiting no substantial side effects, such as a high level
of systemic activation
of the coagulation system.
Other objects of the present invention will become apparent upon reading the
present
description.
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In a first aspect the invention provides a pharmaceutical composition
comprising factor
VII or a factor VII-related polypeptide, and factor XI or a factor XI-related
polypeptide.
In a second aspect, the invention provides a kit of parts containing a
treatment for
bleeding episodes comprising
a) An effective amount of a preparation of a factor VII or factor VII-related
polypeptide
and a pharmaceutically acceptable carrier in a first unit dosage form;
b) An effective amount of a preparation of a factor XI or factor XI-related
polypeptide
and a pharmaceutically acceptable carrier in a second unit dosage form; and
c) Container means for containing said first and second dosage forms.
In different embodiments thereof, the kit further contains an effective amount
of a
TFPI-inhibitor and/or a factor VIII; the TFPI-inhibitor or the factor VIII (or
the combination of the
two) may be present in separate unit dosage forms or may be present in one of
the unit dosage
forms containing either factor VII or factor VII-related polypeptide, or the
factor XI or factor XI-
related polypeptide.
In a third aspect, the invention provides the use of a factor VII or factor
VII-related
polypeptide in combination with a factor XI or a factor XI-related polypeptide
for the
manufacture of a medicament for treating bleeding episodes in a subject. In a
further aspect, the
invention provides the use of a composition as described in any one of claims
1 to18, for the
manufacture of a medicament for treating bleeding episodes in a subject.
In different embodiments thereof, the medicaments are for reducing clotting
time, pro-
longing the clot lysis time, and increasing clot strength.
In another embodiment, the medicament is formulated for intravenous
administration,
preferably injection or infusion, in particular injection.
In one embodiment, the medicament is formulated in single-unit dosage form; in
another it is formulated in the form of a first unit dosage form comprising a
preparation of a
factor VII or factor VII-related polypeptide and a second unit dosage form
comprising a prepara-
tion of a factor XI or factor XI-related polypeptide.
In different embodiments, the medicaments are for treatment of subjects
experiencing
bleeding episodes due to surgery, trauma, or other forms of tissue damage;
coagulophathy, in-
cluding coagulopathy in multi-transfused subjects; congenital or acquired
coagulation or bleed-
ing disorders, including decreased liver function ("liver disease"); defective
platelet function or
decreased platelet number; lacking or abnormal essential clotting "compounds"
(e.g., platelets
or von Willebrand factor protein); increased fibrinolysis; anticoagulant
therapy or thrombolytic
therapy; stem cell transplantation. In one series of embodiments, the
bleedings occur in organs
such as the brain, inner ear region, eyes, liver, lung, tumour tissue,
gastrointestinal tract; in an-
other series of embodiments, it is diffuse bleeding, such as in haemorrhagic
gastritis and profuse
uterine bleeding. In another series of embodiments, the bleeding episodes are
bleeding in con-
nection with surgery or trauma in subjects having acute haemarthroses
(bleedings in joints),
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chronic haemophilic arthropathy, haematomas, (e.g., muscular, retroperitoneal,
sublingual and
retropharyngeal), bleedings in other tissue, haematuria (bleeding from the
renal tract), cerebral
haemorrhage, surgery (e.g., hepatectomy), dental extraction, and
gastrointestinal bleedings
(e.g., UGI bleeds). In one embodiment, the medicament is for treating bleeding
episodes due to
5 trauma, or surgery, or lowered count or activity of platelets, in a subject.
In a further aspect, the invention provides a method for treating bleeding
episodes in a
subject, the method comprising administering to a subject in need thereof a
first amount of a
preparation of a factor VII or factor VII-related polypeptide and a second
amount of a prepara-
tion of a factor XI or factor XI-related polypeptide, wherein the first and
second amount to-
gether are effective to treat bleedings.
In a further aspect, the invention provides a method for reducing clotting
time in a sub-
ject, the method comprising administering to a subject in need thereof a first
amount of a
preparation of a factor VII or factor VII-related polypeptide and a second
amount of a prepara-
tion of a factor XI or factor XI-related polypeptide wherein the first and
second .amount together
are effective to reduce clotting time.
In a further aspect, the invention provides a method to enhance haemostasis in
a sub-
ject, the method comprising administering to a subject in need thereof a first
amount of a
preparation of a factor VII or factor VII-related polypeptide and a second
amount of a prepara-
tion of a factor XI or factor XI-related polypeptide wherein the first and
second amount together
are effective to enhance haemostasis.
In a further aspect, the invention provides a method for prolonging the clot
lysis time in
a subject, the method comprising administering to a subject in need thereof a
first amount of a
preparation of a factor VII or factor VII-related polypeptide and a second
amount of a prepara-
tion of a factor XI or factor XI-related polypeptide wherein the first and
second amount together
are effective to prolong the clot lysis time.
In a further aspect, the invention provides a method for increasing clot
strength in a
subject, the method comprising administering to a subject in need thereof a
first amount of a
preparation of a factor VII or factor VII-related polypeptide and a second
amount of a prepara-
tion of a factor XI or factor XI-related polypeptide wherein the first and
second amount together
are effective to increase clot strength.
In one series of embodiments of the methods, the factor VII or factor VII-
related
polypeptide and the factor XI or factor XI-related polypeptide are
administered in single-unit
dosage form.
In another series of embodiments the factor VII or factor VII-related
polypeptide and
the factor XI or factor XI-related polypeptide are administered in the form of
a first-unit dosage
form comprising a preparation of a factor VII or factor VII-related
polypeptide and a second-unit
dosage form comprising a preparation of a factor XI or factor XI-related
polypeptide. In a series
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of embodiments thereof, the first-unit dosage form and the second-unit dosage
form are admi-
nistered with a time separation of no more than 15 minutes.
In a further aspect, the invention provides a kit containing a treatment for
bleeding
episodes comprising
d) An effective amount of a factor VII or factor VII-related polypeptide and
an effective
amount of a factor XI or factor XI-related polypeptide and a pharmaceutically
acceptable carrier in a one-unit dosage form; and
e) Container means for containing said one-unit dosage form.
In one series of embodiments of the invention, the factor VII or factor VII-
related
polypeptide is a factor VII-related polypeptide. In one series of embodiments
of the invention
the factor VII-related polypeptide is a factor VII amino acid sequence
variant. In one embodiment
the ratio between the activity of the factor VII-related polypeptide and the
activity of native
human factor Vlla (wild-type FVlla) is at least about 1.25 when tested in the
"In Vitro Hydrolysis
Assay" as described in the present description.
In one series of embodiments of the invention the factor VII or factor VII-
related
polypeptide is factor VII. In one embodiment said factor VII is human factor
VII. In one
embodiment the factor VII is bovine, porcine, canine, equine, murine or salmon
factor VII. In
another embodiment the factor VII is recombinantly made. In another embodiment
the factor VII
is derived from plasma. In a preferred embodiment the factor VII is
recombinant human factor
VII. In one series of embodiments of the invention the factor VII or factor
VII-related polypeptide
is in its activated form. In one preferred embodiment of the invention the
factor VII is
recombinant human factor Vlla.
In one series of embodiments the factor XI or factor XI-related polypeptide is
a factor
XI-related polypeptide. In one embodiment the factor XI-related polypeptide is
a factor XI amino
acid sequence variant. In one embodiment the ratio between the activity of
said factor XI-related
polypeptide and the activity of native human plasma factor XI (wild-type FXI)
is at least about
1.25 when tested in the "FXI chromogenic assay" as described in the present
description. In one
embodiment the factor XI or factor XI-related polypeptide is a factor XI
polypeptide. In one em-
bodiment the factor XI is human factor XI. In one embodiment the factor XI is
bovine, porcine,
canine, equine, murine or salmon factor XI. In a preferred embodiment the
factor XI is recombi-
nantly made. In another embodiment the factor XI is derived from plasma.. In
another embodi-
ment the factor XI is platelet-derived factor XI. In a preferred embodiment
the factor XI is re-
combinant human plasma factor XI. In one series of embodiments of the
invention the factor XI
or factor XI-related polypeptide is in its activated form. In one embodiment
the factor XI-related
polypeptide is a fragment of factor XI. In one embodiment the factor XI-
related polypeptide is a
hybrid factor XI polypeptide, e.g., a porcine/human hybrid. In one embodiment,
the factor XI is
human plasma activated factor XI (FXIa).
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In one embodiment the factor VII or factor VII-related polypeptide and the
factor XI or
factor-XI related polypeptide are present in a ratio by mass of between about
100:1 and about
1:100 (w/w factor Vll:factor XI).
In one embodiment, the factor VII-related polypeptides are amino acid sequence
variants having no more than 20 amino acids replaced, deleted or inserted
compared to wild-
type factor VII (i.e., a polypeptide having the amino acid sequence disclosed
in U.S. Patent No.
4,784,950), In another embodiment, the factor Vll variants have no more than
15 amino acids
replaced, deleted or inserted; in other embodiments, the factor VII variants
have no more than
amino acids, such as 8, 6, 5, or 3 amino acids, replaced, deleted or inserted
compared to wild-
10 type factor VII. In one embodiment, the factor VII variants are selected
from the list of L305V-
FVlla, L305V/M306D/D309S-FVlla, L3051-FVlla, L305T-FVlla, F374P-FVlla,
V158T/M298Q-FVlla,
V158D/E296V/M298Q-FVlla, K337A-FVlla, M298Q-FVlla, V158D/M298Q-FVlla,
L305V/K337A-FVlla,
V158D/E296V/M298Q/L305V-FVlla, V158D/E296V/M298Q/K337A-FVlla,
V158D/E296V/M298Q/L305V/K337A-FVlla, K157A-FVII, E296V-FVII, E296V/M298Q-FVII,
V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII
In a further embodiment, the factor VII-related polypeptides have increased
tissue
factor-independent activity compared to native human coagulation factor Vlla.
In another
embodiment, the increased activity is not accompanied by changes in the
substrate specificity. In
another embodiment of the invention, the binding of the factor VII-related
polypeptides to
tissue factor are not impaired and the factor VII-related polypeptides have at
least the activity of
wild-type factor Vlla when bound to tissue factor.
In a preferred embodiment, the factor VII or factor VII-related polypeptide
and the
factor XI or factor XI-related polypeptide are recombinant human factor Vlla
and recombinant
human plasma factor XI or recombinant human factor Vlla and recombinant human
plasma
factor Xla.
In one embodiment, the clotting time is reduced in mammalian blood. In another
embodiment the haemostasis is enhanced in mammalian blood. In another
embodiment the clot
lysis time is prolonged in mammalian blood. In another embodiment the clot
strength is
increased in mammalian blood. In one embodiment, the mammalian blood is human
blood. In
another embodiment, the mammalian blood is normal human blood; in one
embodiment, the
blood is blood from a subject having an impaired thrombin generation. In one
embodiment, the
blood is blood from a subject having a deficiency of one or more coagulation
factors; in another
embodiment, the blood is blood from a subject having inhibitors against one or
more
coagulation factors; in one embodiment, the blood is from a subject having a
lowered
concentration of fibrinogen; in one embodiment, the blood is factor XI-
deficient human blood.
In one series of embodiments, the blood is plasma.
In one embodiment, the factor VII or factor VII-related polypeptide and the
factor XI or
factor XI-related polypeptide prolong the in vitro clot lysis time in normal
human plasma. In
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another embodiment, the factor VII or factor VII-related polypeptide and the
factor XI or factor
XI-related polypeptide increase the maximal clot strength clot lysis time in
normal human plasma
in vitro. In another embodiment, the factor VII or factor VII-related
polypeptide and the factor XI
or factor XI-related polypeptide shorten the clotting time in normal human
plasma in vitro.
In one embodiment of the invention, the factor VII or factor VII-related
polypeptide and
the factor XI or factor XI-related polypeptide are the sole haemostatic agents
contained in the
composition. In another embodiment, the factor VII or factor VII-related
polypeptide and the
factor XI or factor XI-related polypeptide are the sole active haemostatic
agents contained in the
composition. In another embodiment, the factor VII or factor VII-related
polypeptide and the
factor XI or factor XI-related polypeptide are the sole coagulation factors
administered to the
subject. In one embodiment of the invention, the factor VII or factor VII-
related polypeptide and
the factor XI or factor XI-related polypeptide are the sole active agents
administered to the pa-
tient. In one embodiment, the composition is substantially free of
prothrombin; in another em-
bodiment, the composition is substantially free of FX; in another embodiment,
the composition
is substantially free of FXa.
In another embodiment, the pharmaceutical composition is formulated for
intravenous
administration, preferably injection or infusion, in particular injection. In
one embodiment, the
composition contains at least one pharmaceutical acceptable excipients or
carrier.
In one embodiment of the invention, the composition is in single-unit dosage
form
wherein the single-unit dosage form contains both coagulation factors. In one
embodiment of
the invention, the composition is in the form of a kit-of-parts comprising a
preparation of a
factor Vll or factor VII-related polypeptide as a first-unit dosage form and a
preparation of a
factor XI or factor XI-related polypeptide as a second-unit dosage form, and
comprising
container means for containing said first and second unit dosage forms. In one
embodiment the
composition or kit, as applicable, further contains directions for the
administration of the
composition or separate components, respectively.
In one embodiment of the invention, the factor VII or factor VII-related
polypeptide
and the factor XI or factor XI-related polypeptide are administered in single-
dosage form. In one
embodiment of the invention, the factor VII or factor VII-related polypeptide
and the factor XI or
factor XI-related polypeptide are administered in the form of a first unit
dosage form comprising
a preparation of a factor VII or factor VII-related polypeptide and a second
unit dosage form
comprising a preparation of a factor XI or factor XI-related polypeptide.
In one embodiment of the invention, the factor VII or factor VII-related
polypeptide
and the factor XI or factor XI-related polypeptide are administered
simultaneously. In another
embodiment, the factor VII or factor VII-related polypeptide and the factor XI
or factor XI-
related polypeptide are administered sequentially. In one embodiment, the
factor VII or factor
VII-related polypeptide and the factor XI or factor XI-related polypeptide are
administered with
a time separation of no more than 15 minutes, preferably 10, more preferred 5,
more preferred
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2 minutes. In one embodiment, the factor VII or factor VII-related polypeptide
and the factor XI
or factor XI-related polypeptide are administered with a time separation of up
to 2 hours,
preferably from 1 to 2 hours, more preferred up to 1 hour, more preferred from
30 minutes to 1
hour, more preferred up to 30 minutes, more preferred from 15 to 30 minutes.
In one embodiment, the effective amount of the factor VII or factor VII-
related
polypeptide is an amount from about 0.05 mg/day to about 500 mg/day (70-kg
subject). In one
embodiment, the effective amount of a preparation of a factor XI or factor XI-
related
polypeptide is from about 0.01 mglday to about 500 mg/day (70-kg subject).
In one embodiment the factor VII or factor VII-related polypeptide and factor
XI or
factor XI-related polypeptide are present in a ratio by mass of between about
100:1 and about
1:100 (w/w factor Vll:factor XI)
In one embodiment of the present invention, the pharmaceutical composition is
in
single-dosage form and consists essentially of a preparation of a factor VII
or factor VII-related
polypeptide and a preparation of a factor XI or factor XI-related polypeptide,
and one or more
of the components selected from the list of pharmaceutical acceptable
excipients or carriers,
stabilizers, detergents, neutral salts, antioxidants, preservatives, and
protease inhibitors.
In a further embodiment, the subject is a human; in another embodiment, the
subject
has an impaired thrombin generation; in one embodiment, the subject has a
lowered plasma
concentration of fibrinogen (e.g., a multi-transfused subject). ; in one
embodiment, the subject
has.a lowered plasma concentration of factor VIII.
In another aspect, the invention concerns a method to enhance haemostasis in a
subject
suffering from a factor VII responsive syndrome compared to when the subject
is treated with
factor VII as the only coagulation protein, the method comprising
administering to the subject in
need thereof a first amount of a preparation of a factor VII or factor VII-
related polypeptide and
a second amount of a preparation of a factor XI or factor XI-related
polypeptide, wherein the
first and second amounts together are effective to enhance haemostasis.
In another aspect, the invention concerns a method to enhance formation of
thrombin in
a subject, the method comprising administering to the subject in need thereof
a first amount of
a preparation of a factor VII or factor VII-related polypeptide and a second
amount of a prepara-
tion of a factor XI or factor XI-related polypeptide, wherein the first and
second amounts to-
gether are effective to enhance formation of thrombin.
In another aspect, the invention concerns a method to enhance formation of
thrombin in
a subject suffering from a factor VII responsive syndrome compared to when the
subject is trea-
ted with factor VII as the only coagulation protein, the method comprising
administering to the
subject in need thereof a first amount of a preparation of a factor VII or
factor VII-related poly-
peptide and a second amount of a preparation of a factor XI or factor XI-
related polypeptide,
wherein the first and second amounts together are effective to enhance
formation of thrombin.
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In another aspect, the invention concerns a method for reducing the number of
admini-
strations of coagulation factor protein needed to accomplish haemostasis in a
subject suffering
from a factor Vll responsive syndrome compared to the number of
administrations needed when
factor VII is administered to the subject as the only coagulation factor
protein, the method
5 comprising administering to a subject in need thereof a first amount of a
preparation of a factor
VII or factor VII-related polypeptide and a second amount of a preparation of
a factor XI or fac-
tor XI-related polypeptide, wherein the first and second amounts together are
effective to redu-
ce the number of administrations of coagulation factor protein.
In another aspect, the invention concerns a method of treating bleedings in a
subject
10 suffering from a factor Vll responsive syndrome, the method comprising
administering to the
subject in need thereof a first amount of a preparation of a factor VII or
factor VII-related poly-
peptide and a second amount of a preparation of a factor XI or factor XI-
related polypeptide,
wherein the first and second amounts together are effective in treating
bleedings.
In one embodiment, the factor VII is human recombinant factor Vlla (rFVlla).
In another
embodiment, the rFVlla is NovoSeven~ (Novo Nordisk A/S, Bagsvaerd, Denmark).
In one embodiment, the pharmaceutical composition is formulated for
intravenous ad-
ministration. In one embodiment, the composition further comprises an
inhibitor of the fibrino-
lytic system, including, without limitation, aprotinin, s -aminocaproic acid
or tranexamic acid. In
one embodiment, the composition further contains a TFPI inhibitor and/or
FVIII.
In one embodiment, the composition further contains a factor VIII. In one
embodiment,
the factor VIII is an activated factor VIII (factor Vllla). In a further
embodiment the factor VIII is a
recombinant factor Vllla. In a further embodiment the factor VIII is
recombinant human factor
Vllla.
In another aspect, the invention relates to the use of a factor Vlla in
combination with a
factor XI for the manufacture of a medicament for enhancing fibrin clot
formation in mammal-
ian plasma.
In another aspect, the invention relates to a method of enhancing fibrin clot
formation
in a subject, which method comprises administering to a subject in need
thereof a first amount
of a preparation of a factor VII or factor VII-related polypeptide and a
second amount of a
preparation of a factor XI or factor XI-related polypeptide, wherein the first
and second amounts
together are effective in treating bleedings.
In one embodiment of the present invention, the pharmaceutical composition
(when in
single-preparation form) consists essentially of a factor Vlla and a factor
XI, and, optionally, a
6 pharmaceutical acceptable excipient or carrier, and, optionally, a
stabiliser, and, optionally, a
detergent, and, optionally, a neutral salt, and, optionally, an antioxidant,
and, optionally, a
preservative, and, optionally, a protease inhibitor.
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In another embodiment of the present invention, the pharmaceutical composition
(when in single-preparation form) consists essentially of a factor Vlla and a
factor XI, and,
optionally, a pharmaceutical acceptable excipient or carrier, and, optionally,
a stabiliser, and,
optionally, a detergent, and, optionally, a neutral salt, and, optionally, an
antioxidant, and,
optionally, a preservative, and, optionally, a protease inhibitor, and a TFPI-
inhibitor.
In another embodiment of the present invention, the pharmaceutical composition
(when in single-preparation form) consists essentially of a factor Vlla and a
factor XI, and,
optionally, a pharmaceutical acceptable excipient or carrier, and, optionally,
a stabiliser, and,
optionally, a detergent, and, optionally, a neutral salt, and, optionally, an
antioxidant, and,
optionally, a preservative, and, optionally, a protease inhibitor, and a
factor VIII, and, optionally,
a TFPI-inhibitor.
In another embodiment, the pharmaceutical composition (when in form of a kit)
consists of a first unit dosage form consisting essentially of a factor Vlla
and, optionally, a
pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser,
and, optionally, a
detergent, and, optionally, a neutral salt, and, optionally, an antioxidant,
and, optionally, a
preservative, and, optionally, a protease inhibitor; and a second unit dosage
form consisting
essentially of a factor XI, and, optionally, a pharmaceutical acceptable
excipient or carrier, and,
optionally, a stabiliser, and, optionally, a detergent, and, optionally, a
neutral salt, and,
optionally, an antioxidant, and, optionally, a preservative, and, optionally,
a protease inhibitor.
In another embodiment, the pharmaceutical composition (when in form of a kit)
consists of a first unit dosage form consisting essentially of a factor Vlla
and, optionally, a
pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser,
and, optionally, a
detergent, and, optionally, a neutral salt and, optionally, an antioxidant,
and, optionally, a
preservative, and, optionally, a protease inhibitor; and a second unit dosage
form consisting
?5 essentially of a factor XI, and, optionally, a pharmaceutical acceptable
excipient or carrier, and,
optionally, a stabiliser, and, optionally, a detergent, and, optionally, a
neutral salt, and,
optionally, an antioxidant, and, optionally, a preservative, and, optionally,
a protease inhibitor;
wherein either the first unit dosage form or the second unit dosage form or
both dosage forms
further contain a factor VIII and/or a TFPI-inhibitor.
.0
LIST OF FIGURES
Figure 1: Addition of FVlla results in a dose-dependent prolongation of the
clot lysis
time. This effect was optimal at 10 nM FVlla.
Figure 2: In the presence of 10 nM FVlla, addition of FXI resulted in a
further
5 prolongation of the clot lysis time. The effect was dose-dependent and
optimal at 30 nM FXI.
Figure 3: Thromboelastography (roTEG) measurements were utilized to analyze
the
effect of FVlla and FXI on the Maximal Clot Firmness (MCF), as well as the
clots resistance to t-PA
mediated lysis.
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Figure 4: These results demonstrate that FVlla and FXI when added to plasma in
a
synergistic fashion shorten the clotting time in NHP.
DETAILED DESCRIPTION OF THIS INVENTION
Subjects, who bleed excessively in association with surgery or major trauma
thus nee-
ding blood transfusions, develop more complications than those who do not
experience any
bleeding. However, also moderate bleedings may lead to complications if they
require the admi-
nistration of human blood or blood products (platelets, leukocytes, plasma-
derived concentrates
for the treatment of coagulation defects, etc.) because this is associated
with the risk of transfer-
ring human viruses (e.g., hepatitis, HIV, parvovirus, or other, by now unknown
viruses) as well as
non-viral pathogens. Extensive bleedings requiring massive blood transfusions
may lead to the
development of multiple organ failure including impaired lung and kidney
function. Once a
subject has developed these serious complications a cascade of events
involving a number of cy-
tokines and inflammatory reactions is started making any treatment extremely
difficult and un-
fortunately often unsuccessful. A patient experiencing a major loss of blood
becomes clinically
unstable. Such patients are in risk of experiencing atrial fibrillation, which
may lead to a fatal
stop of cardiac activity; impaired renal function; or fluid extravasations in
lungs (so-called "wet
lungs" or ARDS). Therefore, a major goal in surgery as well as in the
treatment of major tissue
damage is to avoid or minimise the bleeding. To avoid or minimize such
unwanted bleedings it
is important to ensure formation of stable and solid haemostatic plugs that
are not readily dis-
solved by fibrinolytic enzymes. Furthermore, it is of importance to ensure
quick and effective
formation of such plugs or clots.
Subjects with thrombocytopenia (lowered count or activity of platelets) also
have an
impaired thrombin generation as well as a defective stabilization of the
fibrin plugs resulting in
haemostatic plugs prone to premature dissolution. Furthermore, subjects
subjected to major
trauma or organ damage and who, as a consequence, have obtained frequent blood
transfusions
often have lowered platelet counts as well as lowered levels of fibrinogen,
factor VIII, and other
coagulation proteins. These subjects experience an impaired (or lowered)
thrombin generation.
These subjects, therefore, have a defective, or less efficient, haemostasis
leading to the
formation of fibrin plugs that are easily and prematurely dissolved by
proteolytic enzymes, such
enzymes in addition being extensively released in situations characterized by
extensive trauma
and organ damage.
Bleedings in tissues may also lead to the formation of haematomas. The sizes
of (in par-
titular intercranial and spinal) haematomas are closely correlated to the
extent of loss of neuro-
logical function, rehabilitation difficulties, and/or the severity and degree
of permanent impair-
ments of neurological function following rehabilitation. The most severe
consequences of hae-
matomas are seen when they are located in the brain where they may even lead
to the death of
the patient.
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Thus, major objectives in treatment of bleedings are to obtain haemostasis in
a mini-
mum of time, thus keeping the blood loss at a minimum.
The present invention thus provides beneficial compositions, uses and methods
of
treatment for treatment of bleeding episodes in subjects in need of such
treatment. The compo-
sitions, uses and methods may be associated with beneficial effects such as
less blood loss before
haemostasis is obtained, less blood needed during surgery, blood pressure kept
at an acceptable
level until haemostasis is obtained, faster stabilisation of blood pressure,
shorter recovery time
for the treated patient, shorter rehabilitation time for the treated patient,
diminished formation
of haematomas or formation of smaller haematomas, including haematomas in the
brain, faster
arrest of bleedings, reduction in the number of administrations needed to stop
bleeding and
maintain haemostasis.
The administration of a preparation of a factor VII or factor VII-related
polypeptide,
e.g., factor Vlla, in combination with a preparation of a factor XI or factor
XI-related polypeptide
provides a shortened clotting time, a firmer clot and an increased resistance
to fibrinolysis com-
pared to the clotting time, clot firmness and resistance when either factor
Vlla or factor XI is ad-
ministered alone.
The administration of a preparation of a factor VII or factor VII-related
polypeptide,
e.g., factor Vlla, in combination with a preparation of a factor XI or factor
XI-related polypeptide
also provides for a reduced time to obtain bleeding arrest and a reduced
number of administra-
tions to maintain haemostasis compared to the situation when either factor
Vlla or factor XI is
administered alone. The present invention provides a beneficial effect of
simultaneous or se-
quential dosing of a preparation of a factor XI or factor X-related
polypeptide and a preparation
of a factor VII or factor VII-related polypeptide. The pharmaceutical
composition according to
the present invention may be in the form of a single composition or it may be
in the form of a
multi-component kit (kit-of-parts). The composition according to the present
invention is useful
as a therapeutic and prophylactic procoagulant in mammals, including primates
such as humans.
The present invention further provides a method for treating (including
prophylactically treating
or preventing) bleeding episodes in a subject, including a human being.
Whenever, a first or second or third, etc., unit dose is mentioned throughout
this
specification this does not indicate the preferred order of administration,
but is merely done for
convenience purposes.
A combination of a preparation of a factor VII or faetor VII-related
polypeptide and a
preparation of a factor XI or factor XI-related polypeptide is an advantageous
product ensuring
short clotting times, rapid formation of haemostatic .plugs, and formation of
stable haemostatic
plugs. It has been found by the present inventor that a combination of a
factor VII or factor VII-
related polypeptide and a factor XI or a factor XI-related polypeptide is an
advantageous
product ensuring the formation of solid, stable and quickly formed haemostatic
plugs.
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The present inventors have shown that a combination of a factor Vlla and a
factor XI
can reduce the clotting time of normal human plasma more effectively than
either factor Vlla or
factor XI alone. It has also been shown that a combination of a factor Vlla
and a factor XI can
increase the firmness of the clot more effectively than either factor Vlla or
factor XI alone. By
combining a factor Vlla at a concentration where no further increase in clot
firmness was
observed with a factor XI, also at a concentration where no further increase
in clot firmness was
observed, it was unexpectedly shown that a further increase in clot firmness
was obtained. It has
also been shown that combination of a factor Vlla and a factor XI can prolong
the in vitro clot
lysis time in normal human plasma more effectively than either factor Vlla or
factor XI alone. It
has also been shown that combination of a factor Vlla and a factor XI can
prolong the half-clot
lysis time in normal human plasma more effectively than either factor Vlla or
factor XI alone. It
has also been shown that combination of a factor Vlla and a factor XI can
protect the clot from
fibrinolysis, in particular tPA-mediated fibrinolysis, in normal human plasma
more effectively
than either factor Vlla or factor XI alone.
Thus, by enhancing coagulation a more effective treatment of bleeding in
subjects can
be obtained.
Without wishing to be bound by theory, it is believed that the full thrombin
generation
is necessary for a solid, stabile haemostatic plug to be formed, and thereby
for the maintenance
of haemostasis. The fibrin structure of such a plug is dependent on both the
amount of thrombin
formed and the rate of the initial thrombin generation. In the presence of an
impaired thrombin
generation a porous fibrin plug, which is highly permeable, is being formed.
The fibrinolytic en-
zymes normally present on the fibrin surface easily dissolve such a fibrin
plug. The formation of a
stable fibrin plug is also dependent on the presence of factor Xllla, which is
being activated by
thrombin and therefore also dependent on the full thrombin generation.
Furthermore, the re-
Gently described thrombin activatable fibrinolytic inhibitor, TAFI, requires
rather high thrombin
amounts for its activation. In the presence of a not fully adequate thrombin
formation the TAFI
may therefore not be activated resulting in the formation of a haemostatic
plug, which is easier
than normally dissolved by the normal fibrinolytic activity. In situations
with lowered number of
platelets, thrombocytopenia, a faster thrombin generation is initiated by the
administration of
exogenous extra faetor Vlla. However, the total thrombin generation is not
normalised by factor
Vlla even in high concentrations.
In subjects with lowered plasma concentrations of fibrinogen (multi-transfused
subjects
as a consequence of multiple trauma or extensive surgery) full thrombin
activation does not oc-
cur. A more effective haemostasis is then obtained by the administration of a
combination of a
factor VII and a factor XI.
Subjects with thrombocytopenia have an impaired thrombin generation as well as
a de-
fective stabilization of the fibrin plugs resulting in haemostatic plugs prone
to premature disso-
lution. Furthermore, subjects subjected to major trauma or organ damage and
who, as a conse-
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quence, have obtained frequent blood transfusions often have lowered platelet
counts as well as
lowered levels of fibrinogen, factor VIII, and other coagulation proteins.
These subjects experi-
ence an impaired (or lowered) thrombin generation. In addition, their lowered
fibrinogen level
interfere negatively with the activation of factor XIII. These subjects,
therefore, have a defective,
5 or less efficient, haemostasis leading to the formation of fibrin plugs
which are easily and pre-
maturely dissolved by proteolytic enzymes, such enzymes in addition being
extensively released
in situations characterized by extensive trauma and organ damage.
In order to facilitate the formation of fully stabilized plugs with full
capacity to main-
tain haemostasis in a subject, a composition according to the invention is
administered. This
10 composition is especially beneficial in subjects with a lowered number of
platelets and in subjects
with lowered plasma levels of fibrinogen and/or other coagulation proteins.
In the presence of a factor XI it is believed that lower concentrations of
factor Vlla may
be sufficient to ensure a sufficient haemostasis.
15 Factor VII Polypeptides:
In practicing the present invention, any factor VII polypeptide may be used
that is
effective in preventing or treating bleeding. This includes factor VII
polypeptides derived from
blood or plasma, or produced by recombinant means.
The present invention encompasses factor VII polypeptides, such as, e.g.,
those having
the amino acid sequence disclosed in U.S. Patent No. 4,784,950 (wild-type
human factor VII). In
some embodiments, the factor VII polypeptide is human factor Vlla, as
disclosed, e.g., in U.S.
Patent No. 4,784,950 (wild-type factor VII). In one series of embodiments,
factor Vll polypeptides
include polypeptides that exhibit at least about 10%, preferably at least
about 30%, more
preferably at least about 50%, and most preferably at least about 70%, of the
specific biological
activity of human factor Vlla. In one series of embodiments, factor Vll
polypeptides include
polypeptides that exhibit at least about 90%, preferably at least about 100%,
preferably at least
about 120%, more preferably at least about 140%, and most preferably at least
about 160%, of
the specific biological activity of human factor Vlla. In one series of
embodiments, factor VII
polypeptides include polypeptides that exhibit at least about 70 %, preferably
at least about 80
%, more preferably at least about 90 %, and most preferable at least about 95
%, of identity
with the sequence of wild-type factor VII as disclosed in U.S. Patent No.
4,784,950.
As used herein, "factor VII polypeptide" encompasses, without limitation,
factor VII, as
well as factor VII-related polypeptides. The term "factor VII" is intended to
encompass, without
limitation, polypeptides having the amino acid sequence 1-406 of wild-type
human factor VII (as
disclosed in U.S. Patent No. 4,784,950), as well as wild-type factor VII
derived from other species,
such as, e.g., bovine, porcine, canine, murine, and salmon factor VII, said
factor VII derived from
blood or plasma, or produced by recombinant means. It further encompasses
natural allelic
variations of factor VII that may exist and occur from one individual to
another. Also, degree and
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16
location of glycosylation or other post-translation modifications may vary
depending on the
chosen host cells and the nature of the host cellular environment. The term
"factor VII" is also
intended to encompass factor VII polypeptides in their uncleaved (zymogen)
form, as well as
those that have been proteolytically processed to yield their respective
bioactive forms, which
may be designated factor Vlla. Typically, factor VII is cleaved between
residues 152 and 153 to
yield factor Vlla.
"Factor VII-related polypeptides" include, without limitation, factor VII
polypeptides
that have either been chemically modified relative to human factor VII and/or
contain one or
more amino acid sequence alterations relative to human factor Vll (i.e.,
factor VII variants),
and/or contain truncated amino acid sequences relative to human factor VII
(i.e., factor VII
fragments). Such factor VII-related polypeptides may exhibit different
properties relative to
human factor VII, including stability, phospholipid binding, altered specific
activity, and the like.
The term "factor VII-related polypeptides" are intended to encompass such
polypeptides in their
uncleaved (zymogen) form, as well as those that have been proteolytically
processed to yield
their respective bioactive forms, which may be designated "factor Vlla-related
polypeptides" or
"activated factor VII-related polypeptides"
As used herein, "factor VII-related polypeptides" encompasses, without
limitation,
polypeptides exhibiting substantially the same or improved biological activity
relative to wild-
type human factor VII, as well as polypeptides in which the factor Vlla
biological activity has
been substantially modified or reduced relative to the activity of wild-type
human factor Vlla.
These polypeptides include, without limitation, factor VII or factor Vlla that
has been chemically
modified and factor VII variants into which specific amino acid sequence
alterations have been
introduced that modify or disrupt the bioactivity of the polypeptide.
It further encompasses polypeptides with a slightly modified amino acid
sequence, for
instance, polypeptides having a modified N-terminal end including N-terminal
amino acid
deletions or additions, and/or polypeptides that have been chemically modified
relative to
human factor Vlla.
Factor VII-related polypeptides, including variants of factor VII, whether
exhibiting
substantially the same or better bioactivity than wild-type factor VII, or,
alternatively, exhibiting
substantially modified or reduced bioactivity relative to wild-type factor
VII, include, without
limitation, polypeptides having an amino acid sequence that differs from the
sequence of wild-
type factor VII by insertion, deletion, or substitution of one or more amino
acids.
Factor VII-related polypeptides, including variants, encompass those that
exhibit at least
about 10%, at least about 20%, at least about 25%, at least about 30%, at
least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least about 75%,
at least about
80%, at least about 90%, at least about 100%, at least about 110%, at least
about 120%, or at
least about 130%, of the specific activity of wild-type factor Vlla that has
been produced in the
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same cell type, when tested in one or more of a clotting assay, proteolysis
assay, or TF binding
assay as described above.
Factor VII-related polypeptides, including variants, having substantially the
same or
improved biological activity relative to wild-type factor Vlla encompass those
that exhibit at least
about 25%, preferably at least about 50%, more preferably at least about 75%,
more preferably
at least about 100%, more preferably at least about 110%, more preferably at
least about 120%,
and most preferably at least about 130% of the specific activity of wild-type
factor Vlla that has
been produced in the same cell type, when tested in one or more of a clotting
assay, proteolysis
assay, or TF binding assay as described above.
Factor VII-related polypeptides, including variants, having substantially
reduced
biological activity relative to wild-type faetor Vlla are those that exhibit
less than about 25%,
preferably less than about 10%, more preferably less than about 5% and most
preferably less
than about 1 % of the specific activity of wild-type factor Vlla that has been
produced in the
same cell type when tested in one or more of a clotting assay, proteolysis
assay, or TF binding
assay as described above. factor VII variants having a substantially modified
biological activity
relative to wild-type factor VII include, without limitation, factor VII
variants that exhibit TF-
independent factor X proteolytic activity and those that bind TF but do not
cleave factor X.
In some embodiments the factor VII polypeptides are factor VII-related
polypeptides, in
particular variants, wherein the ratio between the activity of said factor VII
polypeptide and the
activity of native human factor Vlla (wild-type FVlla) is at least about 1.25
when tested in the "In
Vitro Hydrolysis Assay" (see "Assays", below); in other embodiments, the ratio
is at least about
2.0; in further embodiments, the ratio is at least about 4Ø In some
embodiments of the
invention, the factor VII polypeptides are factor VII-related polypeptides, in
particular variants,
wherein the ratio between the activity of said factor VII polypeptide and the
activity of native
human factor Vlla (wild-type FVlla) is at least about 1.25 when tested in the
"In Vitro Proteolysis
Assay" (see "Assays", below); in other embodiments, the ratio is at least
about 2.0; in further
embodiments, the ratio is at least about 4.0; in further embodiments, the
ratio is at least about
8Ø
In some embodiments, the factor VII polypeptide is human factor VII, as
disclosed, e.g.,
in U.S. Patent No. 4,784,950 (wild-type factor VII). In some embodiments, the
factor VII
polypeptide is human factor Vlla. In one series of embodiments, the factor VII
polypeptides are
factor VII-related polypeptides that exhibits at least about 10%, preferably
at least about 30%,
more preferably at least about 50%, and most preferably at least about 70%, of
the specific
biological activity of human factor Vlla. In some embodiments, the factor VII
polypeptides have
an amino acid sequence that differs from the sequence of wild-type factor VII
by insertion,
deletion, or substitution of one or more amino acids.
Non-limiting examples of factor VII variants having substantially the same or
better
biological activity compared to wild-type factor Vlla include, but are not
limited to, those
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18
described in Danish Patent Applications Nos. PA 2000 00734 and PA 2000 01360
(corresponding
to WO 01/83725), and PA 2000 01361 (corresponding to WO 02/22776). Non-
limiting examples of
factor VII variants having substantially the same or improved biological
activity as wild-type
factor VII include 552A-FVII, S60A-FVII (lino et al., Arch. Biochem. Biophys.
352: 182-192, 1998);
L305V-FVII, L305V/M306D1D309S-FVII, L3051-FVII, L305T-FVII, F374P-FVII,
V158T/M298Q-FVII,
V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-
FVII,
V158D1E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII,
V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII,
V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII; FVlla variants exhibiting
increased
proteolytic stability as disclosed in U.S. Patent No. 5,580,560; factor Vlla
that has been
proteolytically cleaved between residues 290 and 291 or between residues 315
and 316
(Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); and oxidized forms of
factor Vlla
(Kornfelt et al., Arch. Biochem. Biophys. 363:43-54, 1999). Non-limiting
examples of factor VII
variants having substantially reduced or modified biological activity relative
to wild-type factor
VII. include R152E-FVlla (Wildgoose et al., Biochem 29:3413-3420, 1990), 5344A-
FVlla (Kazama et
al.; J. Biol. Chem. 270:66-72, 1995), FFR-FVlla (Hoist et al., Eur. J. Vasc.
Endovasc. Surg. 15:515-520,
1998), and factor Vlla lacking the Gla domain, (Nicolaisen et al., FEBS Letts.
317:245-249, 1993).
Non-limiting examples of chemically modified factor VII polypeptides and
sequence variants are
described, e.g., in U.S. Patent No. 5,997,864.
The biological activity of factor Vlla in blood clotting derives from its
ability to (i) bind
to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of factor IX
or factor X to produce
activated factor IX or X (factor IXa or Xa, respectively).
For purposes of the invention, biological activity of factor VII polypeptides
("factor VII
biological activity") may be quantified by measuring the ability of a
preparation to promote
blood clotting using factor VII-deficient plasma and thromboplastin, as
described, e.g., in U.S.
Patent No. 5,997,864. In this assay, biological activity is expressed as the
reduction in clotting
time relative to a control sample and is converted to "factor VII units" by
comparison with a
pooled human serum standard containing 1 unit/ml factor VII activity.
Alternatively, factor Vlla
biological activity may be quantified by
(i) Measuring the ability of factor Vlla or a factor Vlla -related polypeptide
to produce
activated factor X (factor Xa) in a system comprising TF embedded in a lipid
membrane and factor X. (Persson et al., J. Biol. Chem. 272:19919-19924, 1997);
(ii) Measuring factor X hydrolysis in an aqueous system ("In Vitro Proteolysis
Assay", see
below);
'S (iii) Measuring the physical binding of factor Vlla or a factor Vlla -
related polypeptide to TF
using an instrument based on surface plasmon resonance (Persson, FEBS Letts.
413:359-363, 1997); and
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(iv) Measuring hydrolysis of a synthetic substrate by factor Vlla and/or a
factor Vlla -related
polypeptide ("In Vitro Hydrolysis Assay", see below); and
(v) Measuring generation of thrombin in a TF-independent in vitro system.
The term "factor VII biological activity" or "factor VII activity" is intended
to include the
ability to generate thrombin; the term also includes the ability to generate
thrombin on the surface
of activated platelets in the absence of tissue factor.
A factor Vlla preparation that may be used according to the invention is,
without limita-
tion, NovoSeven~ (Novo Nordisk A/S, Bagsvaerd, Denmark).
Factor XI polypeptides:
In practicing the present invention, any factor XI polypeptide may be used
that is
effective in preventing or treating bleeding. This includes factor XI
polypeptides derived from
blood or plasma, or produced by recombinant means. Furthermore, platelets may
contain a
structurally different form of FXI (possible due to alternative splicing of
the FXI gene). Platelet
factor XI is described in Lipscomb, M.S. & Walsh, P.N. (1979), Journal of
Clinical Investigation, 63,
1006-1014.
As used herein, "factor XI polypeptide" encompasses, without limitation,
factor XI, as
well as factor XI-related polypeptides. The term "factor XI" is intended to
encompass, without
limitation, polypeptides having the amino acid sequence as described in Sun,
Y. & Gailani, D.
(1996), J. Biol. Chem. 271: 29023-29028 (wild-type human factor XI, plasma),
as well as wild-type
factor XI derived from other species, such as, e.g., bovine, porcine, canine,
murine, and salmon
factor XI. In some embodiments, the factor XI polypeptide is wild-type human
factor XI, as
disclosed, e.g., in Sun, Y. & Gailani, D. (1996), J. Biol. Chem. 271: 29023-
29028.
It further encompasses natural allelic variations of factor XI that may exist
and occur
from one individual to another. Also, degree and location of glycosylation or
other post-
translation modifications may vary depending on the chosen host cells and the
nature of the
host cellular environment. The term "factor XI" is also intended to encompass
factor XI
polypeptides in their uncleaved (zymogen) form, as well as those that have
been proteolytically
processed to yield their respective bioactive forms, which may be designated
factor Xla.
"Factor XI-related polypeptides" include, without limitation, factor XI
polypeptides that
have either been chemically modified relative to human factor XI and/or
contain one or more
amino acid sequence alterations relative to human factor XI (i.e., factor XI
variants), and/or
contain truncated amino acid sequences relative to human factor XI (i.e.,
factor XI fragments).
Such factor XI-related polypeptides may exhibit different properties relative
to human factor XI,
including stability, phospholipid binding, altered specific activity, and the
like.
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The term "factor XI-related polypeptides" are intended to encompass such
polypeptides in their
uncleaved (zymogen) form, as well as those that have been proteolytically
processed to yield
their respective bioactive forms, which may be designated "factor Xla-related
polypeptides" or
"activated factor XI-related polypeptides".
5 As used herein, "factor XI-related polypeptides" encompasses, without
limitation,
polypeptides exhibiting substantially the same or improved biological activity
relative to wild-
type human factor XI, as well as polypeptides, in which the factor XI
biological activity has been
substantially modified or reduced relative to the activity of wild-type human
factor XI. These
polypeptides include, without limitation, factor XI or factor Xla that has
been chemically
10 modified and factor XI variants into which specific amino acid sequence
alterations have been
introduced that modify or disrupt the bioactivity of the polypeptide.
It further encompasses polypeptides with a slightly modified amino acid
sequence, for
instance, polypeptides having a modified N-terminal end including N-terminal
amino acid
deletions or additions, and/or polypeptides that have been chemically modified
relative to
15 human factor XI.
Factor XI-related polypeptides, including variants of factor XI, whether
exhibiting
substantially the same or better bioactivity than wild-type factor XI, or,
alternatively, exhibiting
substantially modified or reduced bioactivity relative to wild-type factor XI,
include, without
limitation, polypeptides having an amino acid sequence that differs from the
sequence of wild-
20 type factor XI by insertion, deletion, or substitution of one or more amino
acids.
Factor XI-related polypeptides, including variants, encompass those that
exhibit at least
about 10%, at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least about 90%,
at least about
100%, at least about 110%, at least about 120%, and at least about 130%, of
the specific activity
of wild-type factor XI that has been produced in the same cell type, when
tested in the factor XI
activity assay as described in the present specification.
Factor XI-related polypeptides, including variants, having substantially the
same or
improved biological activity relative to wild-type factor XI encompass those
that exhibit at least
about 25%, preferably at least about 50%, more preferably at least about 75%,
more preferably
at least about 100%, more preferably at least about 110%, more preferably at
least about 120%,
and most preferably at least about 130% of the specific biological activity of
wild-type human
factor XI that has been produced in the same cell type when tested in one or
more of the specific
factor XI activity assay as described. For purposes of the invention, factor
XI biological activity
may be quantified as described later in the present description ("assay
part").
Factor XI-related polypeptides, including variants, having substantially
reduced
biological activity relative to wild-type factor XI are those that exhibit
less than about 25%,
preferably less than about 10%, more preferably less than about 5% and most
preferably less
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21
than about 1 % of the specific activity of wild-type factor XI that has been
produced in the same
cell type when tested in one or more of the specific factor XI activity assays
as described above.
Non-limiting examples of factor XI polypeptides include plasma-derived human
factor XI
as described, e.g., in Gailani & Broze (1993), Blood Coagul. Fibrinolysis,
4:15-20, or Kerbiriou &
Griffin (1979), J. Biol. Chem., 254:12020-12207, or,
In some embodiments the factor XI are factor XI-related polypeptides wherein
the ratio
between the activity of said factor XI polypeptide and the activity of native
human factor XI
(wild-type factor XI) is at least about 1.25 when tested in the " FXI
chromogenic assay" (see be-
low); in other embodiments, the ratio is at least about 2.0; in further
embodiments, the ratio is
at least about 4Ø
Factor XI-related polypeptides also include fragments of factor XI or factor
XI-related
polypeptides retaining their characteristic haemostasis-related activity. The
haemostasis-related
activity of a factor XI polypeptide may, for example, be measured using the
factor XI-aetivity as-
say described in the present specification.
In preferred embodiments, the factor XI is human plasma factor XI or activated
human
plasma factor Xla. In one embodiment, the FXI is platelet factor XI. In
another embodiment, the
FXI is recombinantly made.
Definitions
In the present context the three-letter or one-letter indications of the amino
acids have
been used in their conventional meaning as indicated in table 1. Unless
indicated explicitly, the
amino acids mentioned herein are L-amino acids. It is to be understood, that
the first letter in,
for example, K337 represent the amino acid naturally present at the indicated
position wild-type
factor VII, and that, for example, [K337A]-FVlla designates the FVII-variant
wherein the amino
acid represented by the one-letter code K naturally present in the indicated
position is replaced
by the amino acid represented by the one-letter code A.
Table 1: Abbreviations for amino acids:
Amino acid Tree-letter code One-letter code
Glycine Gly G
Proline Pro P
Alanine Ala A
Valine Val V
Leucine Leu L
Isoleucine Ile I
Meth ionine Met M
Cysteine Cys C
Phenylalanine Phe F
Tyrosine Tyr Y
Tryptophan Trp W
Histidine His H
Lysine Lys K
Arginine Arg R
Glutamine Gln Q
Asparagine Asn N
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22
Glutamic Acid Glu E
Aspartic Acid Asp D
The term "factor Vlla" or "FVlla" may be used interchangeably. The term factor
Vlla in-
cludes zymogen factor VII (single-chain factor VII). The term "factor XI" or
"FXI" may be used
interchangeably. The term "factor VIII" or "FVIII" may be used
interchangeably. The term "factor
VIII" or "FVIII" included activated factor VIII (FVllla), variants and
truncated forms retaining the
characteristic FVIII-related haemostatic activity; the term includes
recombinantly made FVIII and
plasma-derived FVIII. Human FVIII and human recombinant FVIII are preferred
In this context, "subjects with an impaired thrombin generation" means
subjects who
cannot generate a full thrombin burst on the activated platelet surface and
ineludes subjects having
a generation of thrombin less that the thrombin-generation in subjects having
a fully functioning,
normal haemostatic system, including a normal amount and function of
coagulation factors,
platelets and fibrinogen (e.g., as in pooled, normal human plasma), and
includes, without
limitations, subjects lacking factor VIII; subjects with a lowered number of
platelets or platelets with
a defective function (e.g., thrombocytopenia or thrombasthenia Glanzmann or
subjects with
excessive bleeds); subjects having lowered levels of prothrombin, FX or FVII;
subjects having a
lowered level of several coagulation factors (e.g., due to exessive bleeding
as a consequence of
trauma or extensive surgery); and subjects with lowered plasma concentrations
of fibrinogen (e.g.,
multitransfused subjects).
By "level of thrombin generation" or "normal thrombin generation" is meant the
level of
the patient's level of thrombin generation compared to the level in healthy
subjects. The level is
designated as a percentage of the normal level. The terms may, where
appropriate, be used in-
terchangeably.
The term "enhancement of the haemostatic system" means an enhancement of the
ability
to generate thrombin. The term "enhancing haemostasis" is intended to
encompass the situations
when the measured thrombin generation for a test sample containing a
preparation of a factor VII
or factor VII-related polypeptide and a preparation of a factor XI or factor
XI-related polypeptide is
prolonged relative to the individual thrombin generation of a control sample
containing only the
factor VII or factor VII-related polypeptide or the factor XI or factor XI-
related polypeptide,
respectively, when tested in the same thrombin generation assay. The thrombin
generation may be
assayed as described in the thrombin generation assay of the present
description (see "assay part").
"Sole" agents or factors as used herein refers to situations in which the
factor VII or fac-
for VII-related polypeptide and the factor XI or factor XI-related
polypeptide, taken together,
are the only haemostatic agents, or active haemostatic agents, or coagulation
factors contained
in the pharmaceutical composition or kit, or are the only haemostatic
agents,or active haemo-
static agents, or coagulation factors administered to the patient in the
course of a particular
treatment, such as, e.g., in the course of a particular bleeding episode. It
will be understood that
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23
these situations encompass those in which other haemostatic agents or
coagulation factors, as
applicable, are not present in either sufficient quantity or activity so as to
significantly influence
one or more coagulation parameters.
Clot lysis time, clot strength, fibrin clot formation, and Blotting time are
clinical parameters
used for assaying the status of patient's haemostatic system. Blood samples
are drawn from the
patient at suitable intervals and one or more of the parameters are assayed by
means of, e.g.,
thromboelastograpy as described by, e.g., Meh et aL,Blood Coagulation &
Fibrinolysis 2001;12:627-
637; Vig et al., Hematology, Vol. 6 (3) pp. 205-213 (2001); Vig et al., Blood
coagulation & fibrinolysis,
Vol. 12 (7) pp. 555-561 (2001) Oct; Glidden et al., Clinical and applied
thrombosis/hemostasis, Vol. 6
(4) pp. 226-233 (2000) Oct; McKenzie et al., Cardiology, Vol. 92 (4) pp. 240-
247 (1999) Apr; or Davis
et al., Journal of the American Society of Nephrology, Vol. 6 (4) pp. 1250-
1255 (1995).
The term "prolonging clot lysis time" is intended to encompass the situations
when the
measured clot lysis time for a test sample containing a preparation of a
factor VII or factor VII-
related polypeptide and a preparation of a factor XI or factor XI-related
polypeptide is prolonged
relative to the individual clot lysis time of a control sample containing only
the factor VII or factor
VII-related polypeptide or the factor XI or factor XI-related polypeptide,
respectively, when tested in
the same clot lysis assay. The clot lysis time may be assayed as described
above.
The term "increasing clot strength" is intended to encompass the situations
when the
measured clot strength, e.g., mechanical strength, for a test sample
containing a preparation of a
factor VII or factor VII-related polypeptide and a preparation of a factor XI
or factor XI-related
polypeptide is increased relative to the individual clot lysis time of a
control sample containing only
the factor VII or factor VII-related polypeptide or the factor XI or factor XI-
related polypeptide,
respectively, when tested in the same clot strength assay. The clot strength
may be assayed as
described, e.g. in Carr et al, 1991. (Carr ME, Zekert SL. Measurement of
platelet-mediated force
development during plasma clot formation. AM J MED SCI 1991; 302: 13-8), or as
described above by
means of thromboelastography.
The term "enhancing fibrin clot formation" is intended to encompass the
situations when
the measured rate for or degree of fibrin clot formation for a test sample
containing a preparation
of a factor VII or factor VII-related polypeptide and a preparation of a
preparation of a factor XI or
factor XI-related polypeptide is increased relative to the individual rate for
or degree of fibrin clot
formation of a control sample containing only the factor VII or factor VII-
related polypeptide or the
factor XI or factor XI-related polypeptide, respectively, when tested in the
same clotting assay. The
fibrin clot formation may be assayed as described above.
The term "shortening clotting time" is intended to encompass the situations
when the
measured time for clot formation (clotting time) for a test sample containing
a preparation of a
factor VII or factor VII-related polypeptide and a preparation of a
preparation of a factor XI or
factor XI-related polypeptide is increased relative to the individual clotting
time of a control sample
containing only the factor VII or factor VII-related polypeptide or the factor
XI or factor XI-related
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24
polypeptide respectively, when tested in the same clotting assay. The clotting
time may be assayed
by means of standard PT og aPTT assays, which are known to the general skilled
person.
The term "lowered count or activity of platelets" refers to the number of
platelets (throm-
bocytes) present in the subject's plasma and to the biological, coagulation-
related activity of such
platelets. Lowered counts may be due, e.g., to increased platelet destruction,
decreased platelet
production, and pooling of a larger than normal fraction of platelets in the
spleen. Thrombocyto-
penia, for example, is defined as a platelet count less than 150,000 platelets
per microliter; the up-
per limit of the normal platelet count is generally considered to be between
350,000 and 450,000
platelets per microliter. Platelet count may be measured by automated platelet
counters; this is a
well known method to the skilled worker. Syndromes due to lowered platelet
count include, with-
out limitation, thrombocytopenia, coagulophathy. "Activity" includes, without
limitation, aggrega-
tion, adhesion, and coagulant activity of the platelets. Decreased activity
may be due, e.g., to glyco-
protein abnormalities, abnormal membrane-cytoskeleton interaction,
abnormalities of platelet
granules, abnormalities of platelet coagulant activity, abnormalities of
signal transduction and se-
cretion. Platelet activity, including aggregation, adhesion, and coagulant
activity, are measured by
standard methods known to the skilled worker, see e.g.,Platelets. A Practical
Approach, Ed. S.P.
Watson & K.S. Authi: Clinical Aspects of Platelet Disorders (K.J. Clemetson)
15:299-318, 1996, Oxford
University Press; Williams Hematology, Sixth Edition, Eds. Beutler, Lichtman,
Coller, Kipps & Selig-
sohn, 2001, McGraw-Hill. Syndromes due to lowered platelet activity includes,
without limitaion,
Glanzmann thrombathenis, Bernard-Soulier syndrome, anticoagulant treatment and
thrombolytic
treatment. "Lowered" refers to the count or activity of a sample of the test
plasma compared to
the count or activity in a sample of normal pooled plasma when measured in the
same assay
As used herein the term "bleeding disorder" reflects any defect, congenital,
acquired or in
duced, of cellular or molecular origin that is manifested in bleeding
episodes. Examples of bleeding
disorders include, but are not limited to, clotting factor deficiencies (e.g.
deficiency of coagula
tion factors VIII, IX, XI or VII), clotting factor inhibitors, defective
platelet function (e.g.,
Glanzmann thombasthenia and Bernard-Soulier syndrome), thrombocytopenia, von
Willebrand's
disease, and coagulophathy such as that caused by a dilution of coagulation
proteins, increased
fibrinolysis and lowered number of platelets due to bleedings and/or
transfusions (e.g., in multi
transfused subjects having been subjected to surgery or trauma).
Bleeding refers to extravasation of blood from any component of the
circulatory system.
The term "bleeding episodes" is meant to include unwanted, uncontrolled and
often excessive
bleeding in connection with surgery, trauma, or other forms of tissue damage,
as well as un-
wanted bleedings in subjects having bleeding disorders. Bleeding episodes may
occur in subjects
having a basically normal coagulation system but experiencing a (temporary)
coagulophathy, as
well as in subjects having congenital or acquired coagulation or bleeding
disorders. In subjects
having a defective platelet function, the bleedings may be likened to
bleedings caused by hae-
mophilia because the haemostatic system, as in haemophilia, lacks or has
abnormal essential
CA 02452677 2003-12-31
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clotting "compounds" (e.g., platelets or von Willebrand factor protein). In
subjects who experi-
ence extensive tissue damage, for example in association with surgery or vast
trauma, the normal
a haemostatic mechanism may be overwhelmed by the demand of immediate
haemostasis and
they may develop excessive bleeding in spite of a basically (pre-trauma or pre-
surgery) normal
5 haemostatic mechanism. Such subjects, who further often are multi
transfused, develop a (tem-
porary) coagulopathy as a result of the bleeding and/or transfusions (i.e., a
dilution of coagula-
tion proteins, increased fibrinolysis and lowered number of platelets due to
the bleeding and/or
transfusions). Bleedings may also occur in organs such as the brain, inner ear
region and eyes;
these are areas with limited possibilities for surgical haemostasis and thus
problems with achiev-
10 ing satisfactory haemostasis. Similar problems may arise in the process of
taking biopsies from
various organs (liver, lung, tumour tissue, gastrointestinal tract) as well as
in laparoscopic surgery
and radical retropubic prostatectomy. Common for all these situations is the
difficulty to provide
haemostasis by surgical techniques (sutures, clips, etc.) which also is the
case when bleeding is
diffuse (e.g., haemorrhagic gastritis and profuse uterine bleeding). Bleedings
may also occur in
15 subjects on anticoagulant therapy in whom a defective haemostasis has been
induced by the
therapy given; these bleedings are often acute and profuse. Anticoagulant
therapy is often given
to prevent thromboembolic disease. Such therapy may include heparin, other
forms of pro-
teoglycans, warfarin or other forms of vitamin K-antagonists as well as
aspirin and other platelet
aggregation inhibitors, such as, e.g., antibodies or other inhibitors of GP
Ilb/Illa activity. The
20 bleeding may also be due to so-called thrombolytic therapy which comprises
combined treat-
mentwith an antiplatelet agent (e.g., acetylsalicylic acid), an anticoagulant
(e.g., heparin), and a
fibrinolytic agent (e.g., tissue plasminogen activator, tPA). Bleeding
episodes are also meant to
inelude, without limitation, uncontrolled and excessive bleeding in connection
with surgery or
trauma in subjects having acute haemarthroses (bleedings in joints), chronic
haemophilic ar-
25 thropathy, haematomas, (e.g., muscular, retroperitoneal, sublingual and
retropharyngeal),
bleedings in other tissue, haematuria (bleeding from the renal tract),
cerebral haemorrhage,
surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings
(e.g., UGI bleeds).
The bleeding episodes may be associated with inhibitors against factor VIII;
haemophilia A;
haemophilia A with inhibitors; haemophilia B; deficiency of factor VII;
deficiency of factor XI;
thrombocytopenia; deficiency of von Willebrand factor (von Willebrand's
disease); severe tissue
damage; severe trauma; surgery; laparoscopic surgery; haemorrhagic gastritis;
taking biopsies;
anticoagulant therapy; upper gastroentestinal bleedings (UGI); or stem cell
transplantation. The
bleeding episodes may be profuse uterine bleeding; occurring in organs with a
limited possibility for
mechanical haemostasis; occurring in the brain; occurring in the inner ear
region; or occurring in the
eyes. The terms "bleeding episodes" and "bleedings" may, where appropriate, be
used inter-
changeably.
In this context, the term "treatment" is meant to include both prevention of
an expec-
ted bleeding, such as, for example, in surgery, and regulation of an already
occurring bleeding,
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26
such as, for example, in trauma, with the purpose of inhibiting or minimising
the bleeding. The
above-referenced "expected bleeding" may be a bleeding expected to occur in a
particular tissue
or organ, or it may be an unspecified bleeding. Prophylactic administration of
a preparation of a
factor VII or factor VII-related polypeptide and a preparation of a factor XI
or factor XI-related
polypeptide is thus included in the term "treatment".
The term "subject" as used herein is intended to mean any animal, in
particular mammals,
such as humans, and may, where appropriate, be used interchangeably with the
term "patient".
The faetor VII or factor VII-related polypeptides and factor XI or factor XI-
related poly-
peptides as defined in the present specification may be administered
simultaneously or sequen-
tially. The factors may be supplied in single-dosage form wherein the single-
dosage form con-
tains both coagulation factors, or in the form of a kit-of-parts comprising a
preparation of a fac
tor VII or factor VII-related polypeptide as a first unit dosage form and a
preparation of a factor
XI or factor XI-related polypeptide as a second unit dosage form. Whenever a
first or second or
third, etc., unit dose is mentioned throughout this specification this does
not indicate the prefer
red order of administration, but is merely done for convenience purposes
By "simultaneous" dosing of a preparation of a factor VII or factor VII-
related polypep-
tide and a preparation of a factor XI or factor XI-related polypeptide is
meant administration of
the coagulation factor proteins in single-dosage form, or administration of a
first coagulation
factor protein followed by administration of a second coagulation faetor
protein with a time se-
paratiori of no more than 15 minutes, preferably 10, more preferred 5, more
preferred 2 minu-
tes. Either factor may be administered first.
By "sequential" dosing is meant administration of a first coagulation factor
protein fol-
lowed by administration of a second coagulation factor protein with a time
separation of up to
2 hours, preferably from 1 to 2 hours, more preferred up to 1 hour, more
preferred from 30 mi
nutes to 1 hour, more preferred up to 30 minutes, more preferred from 15 to 30
minutes.
Either of the two unit dosage form, or coagulation factor proteins, may be
administered first.
Preferably, both products are injected through the same intravenous access.
By "level of factor XI" or "factor XI level" is meant the level of the
patient's clotting
factor XI activity compared to the level in healthy subjects. The level is
designated as a percenta-
ge of the normal level. The terms may, where appropriate, be used
interchangeably.
By "reduced level of factor XI" or "reduced factor XI level" is meant a
decrease in the
presence or activity of factor XI in the blood stream compared to the mean
factor XI level in a
population of subjects having no coagulation factor XI deficiency or
inhibitors to coagulation
factor XI. The level of circulating factor XI can be measured by either a
coagulant or an immuno-
logic assay. factor XI procoagulant activity is determined by the ability of
the patient's plasma to
correct the clotting time of factor XI-deficient plasma (e.g., an APTT assay,
see below; see also
"assay part" of the present description).
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One unit of factor XI has been defined as the amount of factor XI present in
one milli-
litre of normal (pooled) human plasma (corresponding to a factor XI level of
100 %).
One unit of factor VII is defined as the amount of factor VII present in 1 ml
of normal
plasma, corresponding to about 0.5 pg protein. After activation 50 units
correspond to about 1
Pg protein.
By "deficiency" is meant a decrease in the presence or activity of, e.g.,
factor XI in
plasma compared to that of normal healthy individuals. The term may, where
appropriate, be
used interchangeably with "reduced factor XI level".
By "APTT" or "aPTT" is meant the activated partial thromboplastin time
(described by,
e.g., Proctor RR, Rapaport SI: The partial thromboplastin time with kaolin; a
simple screening test
for first-stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212,
1961).
By "factor XI-responsive syndrome" is meant a syndrome where exogenous factor
XI
administered to the subject in need thereof may prevent, cure or ameliorate
any symptoms, con-
ditions or diseases, expected or present, caused by the syndrome. Included
are, without limita-
tion, syndromes caused by a reduced level of factor XI, e.g., bleeding
disorders caused by inhibi-
tors to factor XI. A factor XI-responsive syndrome may also be treated with a
composition accor-
ding to the present invention.
By "factor VII-responsive syndrome" is meant a syndrome where exogenous factor
VI1,
preferably factor Vlla, administered to the subject in need thereof may
prevent, cure'or amelio-
rate any symptoms, conditions or diseases, expected or present, caused by the
syndrome. Inclu-
ded are, without limitation, syndromes caused by a reduced level of clotting
factors VIII, IX, XL or
VII, clotting factor inhibitors, defective platelet function (e.g., Glanzmann
thombasthenia and
Bernard-Soulier syndrome), thrombocytopenia, von Willebrand's disease, and
coagulophathy
such as that caused by a dilution of coagulation proteins, increased
fibrinolysis and lowered
number of platelets due to bleedings and/or transfusions (e.g., in multi
transfused subjects hav-
ing been subjected to surgery or trauma).
"Half-life" refers to the time required for the plasma concentration of a
factor VII or
factor VII-related polypeptide or a factor XI or factor XI-related polypeptide
to decrease from a
particular value to half of that value.
By "primary haemostasis" is meant the initial generation of thrombin by FXa
and
TF:factor Vlla, the subsequent activation of platelets and formation of the
initial loose plug of
activated, adhered platelets which has not yet been stabilized by fibrin and,
finally, by cross-
linked fibrin. If not stabilized by the fibrin formed during the second step
of the haemostatic
process (maintained haemostasis), the plug is easily dissolved by the
fibrinolytic system.
By "secondary haemostasis" or "maintained haemostasis" is meant the secondary,
full,
and major, burst or generation of thrombin taking place on the surface of
activated platelets
and catalysed by factor Vllla and factor Vllla, the subsequent formation of
fibrin and the stabili-
zation of the initial platelet plug. Stabilization of the plug by fibrin leads
to full haemostasis.
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28
By "full haemostasis" is meant the formation of a stable and solid fibrin clot
or plug at
the site of injury which effectively stops the bleeding and which is not
readily dissolved by the
fibrinolytic system. In this context, the term haemostasis will be used to
represent full haemosta-
sis as described above.
The total amount of protein in a preparation may be measured by generally
known
methods, e.g, by measuring optical density. Amounts of factor XI coagulation
or factor VII prote-
in ("antigen") may be measured by generally known methods such as standard
Elisa immuno
assays. In general terms, such assay is conducted by contacting, e.g., a
solution of the factor XI
protein- containing preparation with an anti-FXl antibody immobilised onto the
elisa plate, sub-
sequently contacting the immobilised antibody-factor XI complex with a second
anti FXI antibo-
dy carrying a marker, the amounts of which, in a third step, are measured. The
amounts of each
coagulation factor may be measured in a similar way using appropriate
antibodies. The total
amount of coagulation factor protein present in a preparation is determined by
adding the
amounts of the individual coagulation factor proteins. In one embodiment, the
preparation
comprises isolated coagulation factor. In another embodiment the preparation
is free of coagu-
lation factor II and coagulation factor Ila (prothrombin and thrombin) and/or
factor X or Xa.
As used herein, the term "isolated" refers to coagulation factors, e.g.,
factor XI or fac-
for XI-related polypeptides that have been separated from the cell in which
they were synthesi-
zed or the medium in whieh they are found in nature (e.g., plasma or blood).
Separation of po-
lypeptides from their cell of origin may be achieved by any method known in
the art, including,
without limitation, removal of cell culture medium containing the desired
product from an
adherent cell culture; centrifugation or filtration to remove non-adherent
cells; and the like. Se-
paration of polypeptides from the medium in which they naturally occur may be
achieved by any
method known in the art, including, without limitation, affinity
chromatography, such as, e.g.,
on an anti-factor Vll or anti-factor XI antibody column, respectively;
hydrophobic interaction ,
chromatography; ion-exchange chromatography; size exclusion chromatography;
electrophoretic
procedures (e.g., preparative isoelectric focusing (IEF)), differential
solubility (e.g., ammonium
sulfate precipitation), or extraction and the like.
The term "TFPI inhibitor" means compounds inhibiting the anti-coagulative
activity of
TFPI (tissue factor pathway inhibitor). The term includes compounds such as
those disclosed in
European Patent No. 558 529, WO 96/28153 and US 5,622,988. "TFPI" and "EPI"
(extrinsic path-
way inhibitor) may be used interchangeably.
Within the present invention an "effective amount" of a factor VII polypeptide
and a
factor XI polypeptide is defined as the amount of a factor VII polypeptide,
e.g., FVlla, and a fac-
for XI polypeptide that together suffices to prevent or reduee bleeding or
blood loss, so as to
cure, alleviate or partially arrest the disease and its complications.
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29
The term "activity of factor Vlla" or "factor Vlla-activity" includes the
ability to generate
thrombin; the term also includes the ability to generate thrombin on the
surface of activated plate-
lets in the absence of tissue factor.
The composition according to the invention may further comprise a TFPI-
inhibitor. The
composition according to the invention may further comprise a factor VIII.
Such a composition
should preferably be administered to subjects who do not have inhibitors to
factor VIII.
Abbreviations
TF tissue factor
FVII factor VII in its single-chain, unactivated form
FVlla factor VII in its activated form
rFVlla recombinant factor Vll in its activated form
FXI factor XI in its zymogenic, unactivated form
FXIa factor XI in its activated form
rFXI recombinant FXI
rFXla recombinant FXIa
FVIII ' factor VIII in its zymogenic, unactivated form
rFVlll ~ recombinant FVIII
FVllla factor VIII in its activated form
rFVllla recombinant FVllla
TFPI tissue factor pathway inhibitor
Preparation of compounds:
Human purified factor Vlla suitable for use in the present invention is
preferably made by
DNA recombinant technology, e.g. as described by Hagen et al.,
Proc.NatLAcad.Sci. USA 83: 2412-
2416, 1986, or as described in European Patent No. 200.421 (ZymoGenetics,
Inc.).
Factor VII may also be produced by the methods described by Broze and Majerus,
J.BioLChem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J.Clin.lnvest. 71:
1836-1841, 1983. These
methods yield factor VII without detectable amounts of other blood coagulation
factors. An even
further purified factor VII preparation may be obtained by including an
additional gel filtration as
the final purification step. factor VII is then converted into activated
factor Vlla by known means,
e.g. by several different plasma proteins, such as factor Xlla, IX a or Xa.
Alternatively, as described
by Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565),
factor VII may be activated
by passing it through an ion-exchange chromatography column, such as Mono Q~
(Pharmacia fine
Chemicals) or the like.
Factor VII -related polypeptides may produced by modification of wild-type
factor VII or by
recombinant technology. factor VII -related polypeptides with altered amino
acid sequence when
compared to wild-type factor VII may be produced by modifying the nucleic acid
sequence encoding
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wild-type factor VII either by altering the amino acid codons or by removal of
some of the amino
acid codons in the nucleic acid encoding the natural factor VII by known
means, e.g. by site-specific
mutagenesis.
It will be apparent to those skilled in the art that substitutions can be made
outside the
5 regions critical to the function of the factor Vlla or factor XI-molecule
and still result in an active
polypeptide. Amino acid residues essential to the activity of the factor VII
or factor VII-related
polypeptide or factor XI or factor XI-related polypeptide, and therefore
preferably not subject to
substitution, may be identified according to procedures known in the art, such
as site-directed
mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells,
1989, Science
10 244: 1081-1085). In the latter technique, mutations are introduced at every
positively charged
residue in the molecule, and the resultant mutant molecules are tested for
coagulant, respec-
tively cross-linking activity to identify amino acid residues that are
critical to the activity of the
molecule. Sites of substrate-enzyme interaction can also be determined by
analysis of the three-
dimensional structure as determined by such techniques as nuclear magnetic
resonance analysis,
15 crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992,
Science 255: 306-312;
Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et
al., 1992, FEBS Letters
309: 59-64).
The introduction of a mutation into the nucleic acid sequence to exchange one
nucleo-
tide for another nucleotide may be accomplished by site-directed mutagenesis
using any of the
20 methods known in the art. Particularly useful is the procedure that
utilizes a super coiled, dou-
ble stranded DNA vector with an insert of interest and two synthetic primers
containing the de-
sired mutation. The oligonucleotide primers, each complementary to opposite
strands of the
vector, extend during temperature cycling by means of Pfu DNA polymerase. On
incorporation
of the primers, a mutated plasmid containing staggered nicks is generated.
Following tempera-
25 ture cyeling, the product is treated with Dpnl, which is specific for
methylated and hemi-
methylated DNA to digest the parental DNA template and to select for mutation-
containing syn-
thesized DNA. Other procedures known in the art for creating, identifying and
isolating variants
may also be used, such as, for example, gene shuffling or phage display
techniques.
Separation of polypeptides from their cell of origin may be achieved by any
method
30 known in the art, including, without limitation, removal of cell culture
medium containing the
desired product from an adherent cell culture; centrifugation or filtration to
remove non
adherent cells; and the like.
Optionally, factor VII or factor VII-related polypeptides may be further
purified. Purifi-
cation may be achieved using any method known in the art, including, without
limitation, affin-
ity chromatography, such as, e.g., on an anti-factor Vll antibody column (see,
e.g., Wakabayashi
et al., J. Biol. Chem. 261:11097, 1986; and Thim et al., Biochem. 27:7785,
1988); hydrophobic in-
teraction chromatography; ion-exchange chromatography; size exclusion
chromatography; elec-
trophoretic procedures (e.g., preparative isoelectric focusing (IEF),
differential solubility (e.g.,
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31
ammonium sulfate precipitation), or extraction and the like. See, generally,
Scopes, Protein Puri-
fication, Springer-Verlag, New York, 1982; and Protein Purification, J.C.
Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989. Following purification, the
preparation preferably con-
tains less than about 10% by weight, more preferably less than about 5% and
most preferably
less than about 1 %, of non-factor VII or factor VII-related polypeptides
derived from the host
cell.
Factor VII or factor VII-related polypeptides may be activated by proteolytic
cleavage,
using factor Xlla or other proteases having trypsin-like specificity, such as,
e.g., factor IXa, kallik-
rein, factor Xa, and thrombin. See, e.g., Osterud et al., Biochem. 11:2853
(1972); Thomas, U.S.
Patent No. 4,456,591; and Hedner et al., J. Clin. Invest. 71:1836 (1983).
Alternatively, factor VII or
factor VII-related polypeptides may be activated by passing it through an ion-
exchange chroma-
tography column, such as Mono Q~ (Pharmacia) or the like. The resulting
activated factor Vll or
factor VII-related polypeptide may then be formulated and administered as
described below.
Factor XI for use within the present invention may be prepared from plasma
according
to known methods, such as those disclosed by Koide et al. (Biochemistry 16:
2279-2286, 1977)
and Bouma et al. (J.BioI.Chem. 252: 6432-6437, 1977), incorporated herein by
reference. It is pre-
ferred, however, to use recombinant factor XI so as to avoid to the use of
blood- or tissue-
derived products that carry a risk of disease transmission. Methods for
preparing recombinant
factor XI are known in the art. See, for example, Kemball-Cook et al. (Gene
139(2): 275-279,
1994), Fujikawa et al. (Biochemistry 25: 2417-2424, 1986), Meijers et al.
(Blood 79(6): 1435-1440,
1992), which are incorporated herein by reference in their entirety.
Factor XI -related polypeptides may produced by modification of wild-type
factor XI or by
recombinant technology. factor XI -related polypeptides with altered amino
acid sequence when
compared to wild-type factor XI may be produced by modifying the nucleic acid
sequence encoding
wild-type factor XI either by altering the amino acid codons or by removal of
some of the amino
acid codons in the nucleic acid encoding the natural factor XI by known means,
e.g. by site-specific
mutagenesis, as described in more detail above. Separation of polypeptides
from their cell of ori-
gin may be achieved by any method known in the art, including, without
limitation, removal of
cell culture medium containing the desired product from an adherent cell
culture; centrifugation
or filtration to remove non-adherent cells; and the like. Optionally, factor
XI or factor XI-related
polypeptides may be further purified. Purification may be achieved using any
method known in
the art, including, without limitation, affinity chromatography, such as,
e.g., on an anti-factor XI
antibody column; hydrophobic interaction chromatography; ion-exchange
chromatography; size
exclusion chromatography; electrophoretic procedures (e.g., preparative
isoelectric focusing
(IEF), differential solubility (e.g., ammonium sulfate precipitation), or
extraction and the like, as
described in more detail above. Following purification, the preparation
preferably contains less
than about 10% by weight, more preferably less than about 5% and most
preferably less than
about 1 %, of non-factor XI or factor XI-related polypeptides derived from the
host cell. The re-
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32
suiting activated factor XI or factor XI-related polypeptide may then be
formulated and adminis-
tered as described below.
As will be appreciated by those skilled in the art, it is preferred to use
factor XI and fac-
tor Vlla proteins syngeneic with the subject in order to reduce the risk of
inducing an immune
response. Preparation and characterization of non-human factor XI has been
disclosed by , for
example, Gailani (Blood 90(3): 1055-1064, 1997). The present invention also
encompasses the use
of such factor XI and factor Vlla proteins within veterinary procedures.
Pharmaceutical Compositions and Methods of Use
The preparations of the present invention may be used to treat any factor VII
responsi-
ve syndrome, such as, e.g., bleeding disorders, including, without limitation,
syndromes caused
by a reduced level of clotting factors VIII, IX, XI or VII, clotting factor
inhibitors, defective platelet
function (e.g., Glanzmann thombasthenia and Bernard-Soulier syndrome),
thrombocytopenia,
von Willebrand's disease, and coagulophathy such as that caused by a dilution
of coagulation
proteins, increased fibrinolysis and lowered number of platelets due to
bleedings andlor transfu-
sions (e.g., in multi transfused subjects having been subjected to surgery or
trauma).
Pharmaceutical compositions comprising a preparation of a factor VII or factor
VII-related
polypeptide and a preparation of a factor XI or factor XI-related polypeptide
according to the
present invention are primarily intended for parenteral administration for
prophylactic and/or
therapeutic treatment. Preferably, the pharmaceutical compositions are.
administered
parenterally, i.e., intravenously, subcutaneously, or intramuscularly;
intravenously being most
preferred. They may also be administered by continuous or pulsatile infusion.
Pharmaceutical compositions or formulations according to the invention
comprise a factor
VII or a factor VII-related polypeptide, and factor XI or a factor XI-related
polypeptide, either
formulated in a single-unit dosage form or in the form of a kit-of parts,
preferably dissolved in, a
pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent.
Briefly,
pharmaceutical compositions suitable for use according to the present
invention is made by mixing
a factor Vlla, or a factor XI, or a factor Vlla in combination with a factor
XI, preferably in purified
form, with suitable adjuvants and a suitable carrier or diluent. A variety of
aqueous carriers may be
used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like.
The preparations of the
invention can also be formulated using non-aqueous carriers, such as, e.g., in
the form of a gel or as
liposome preparations for delivery or targeting to the sites of injury.
Liposome preparations are
generally described in, e.g., U.S. Patents Nos. 4,837,028, 4,501,728, and
4,975,282. The compositions
may be sterilised by conventional, well-known sterilisation techniques. The
resulting aqueous
solutions may be packaged for use or filtered under aseptic conditions and
lyophilised, the
lyophilised preparation being combined with a sterile aqueous solution prior
to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances
or
adjuvants, including, without limitation, pH adjusting and buffering agents
and/or tonicity
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33
adjusting agents, such as, for example, sodium acetate, sodium lactate, sodium
chloride,
potassium chloride, calcium chloride, etc.
Formulations may further include one or more diluents, emulsifiers,
preservatives,
buffers, excipients, etc. and may be provided in such forms as liquids,
powders, emulsions,
controlled release, etc. One skilled in this art may formulate the
compositions of the invention
an appropriate manner, and in accordance with accepted practices, such as
those disclosed in
Remin tq on's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,
Easton, PA, 1990.
Thus, a typical pharmaceutical composition for intravenous infusion could be
made up to contain
250 ml of sterile Ringer's solution and 10 mg of the preparation.
The compositions containing the preparations of the present invention can be
administered for prophylactic and/or therapeutic treatments. In therapeutic
applications,
compositions are administered to a subject already suffering from a disease,
as described above,
in an amount sufficient to cure, alleviate or partially arrest the clinical
manifestations of the
disease and its complications. An amount adequate to accomplish this is
defined as
"therapeutically effective amount". Effective amounts for each purpose will
depend on the
severity of the disease or injury as well as the weight and general state of
the subject. It will be
understood that determining an appropriate dosage may be achieved using
routine
experimentation, by constructing a matrix of values and testing different
points in the matrix.
Local delivery of the preparations of the present invention, such as, for
example, topical
application, may be carried out, e.g., by means of a spray, perfusion, double
balloon catheters,
stent, incorporated into vascular grafts or stents, hydrogels used to coat
balloon catheters, or
other well established methods. In any event, the pharmaceutical compositions
should provide a
quantity of the preparation sufficient to effectively treat the condition.
The concentration of factor VII or factor VII-related polypeptide, factor XI
or factor XI-
related polypeptide, or factor VII or factor VII-related polypeptide in
combination with factor XI
or factor XI-related polypeptide in these formulations can vary widely, i.e.,
from less than about
0.5% by weight, usually at or at least about 1 % by weight to as much as 15 or
20% by weight
and will be selected primarily by fluid volumes, viscosities, etc., in
accordance with the particular
mode of administration selected. Administration by injection or infusion, in
particular injection,
is preferred. Thus, the factor VII or factor VII-related polypeptide and the
factor XI or factor XI-
related polypeptide are prepared in a form suitable for intravenous
administration, such as a
preparation that is either a dissolved lyophilized powder or a liquid
formulation containing both
the factor VII or factor VII-related polypeptide and the factor XI or factor
XI-related polypeptide
in one dosage form, or a dissolved lyophilized powder or a liquid formulation
containing the
factor VII or factor VII-related polypeptide in one dosage form and dissolved
lyophilized powder
or a liquid formulation containing the factor XI or factor XI-related
polypeptide in another
dosage form.
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34
It is to be understood that the amount of factor VII or factor VII-related
polypeptide
and the amount of factor XI or factor XI-related polypeptide together comprise
an aggregate
effective amount for treating the bleeding episode.
It must be kept in mind that the materials of the present invention may
generally be
employed in serious disease or injury states, that is, life threatening or
potentially life
threatening situations. In such cases, in view of the minimization of
extraneous substances and
general lack of immunogenicity of factor Vlla and factor XI in humans, it is
possible and may be
felt desirable by the treating physician to administer a substantial excess of
these compositions.
In prophylactic applications, compositions containing a preparation of a
factor VII or
factor VII-related polypeptide and a preparation of a factor XI or factor XI-
related polypeptide
are administered to a subject susceptible to or otherwise at risk of a disease
state or injury to
enhance the subject's own coagulative capability. Such an amount is defined to
be a
"prophylactically effective dose." It is to be understood that the amount of
factor VII or factor
VII-related polypeptide and the amount of factor XI or factor XI-related
polypeptide together
comprise an aggregate effective amount for preventing a bleeding episode.
Single or multiple administrations of the compositions can be carried out with
dose
levels and patterns being selected by the treating physician. The compositions
may be
administered one or more times per day or week. An effective amount of such a
pharmaceutical
composition is the amount that provides a clinically significant effect
against bleeding episodes.
Such amounts will depend, in part, on the particular condition to be treated,
age, weight, and
general health of the subject, and other factors evident to those skilled in
the art.
The composition of the invention is generally administered in a single dose
before the
expected bleeding or at the start of the bleeding. It may however also be
given repeatedly (in
multiple doses) preferably with intervals of 2-4-6-12 hour, depending on the
dose given and the
condition of the subject.
For treatment in connection with deliberate interventions, the factor VII or
factor VII-
related polypeptide and the factor XI or factor XI-related polypeptide will
typically be administered
within about 24 hours prior to performing the intervention, and for as much as
7 days or more
thereafter. Administration as a coagulant can be by a variety of routes as
described herein.
The composition may be in the form of a single preparation (single-dosage
form)
comprising both a preparation of a preparation of a factor VII or factor VII-
related polypeptide and
a preparation of a preparation of a factor XI or factor XI-related polypeptide
in suitable
concentrations. The composition may also be in the form of a kit-of-parts
consisting of a first unit
dosage form comprising a preparation of a preparation of a factor VII or
factor VII-related
polypeptide and a second unit dosage form comprising a preparation of a
preparation of a factor XI
or factor XI-related polypeptide. In this case, the factor VII or factor VII-
related polypeptide and the
factor XI or factor XI-related polypeptide should be administered one after
the other, preferably
within about 15 minutes of each other, for example within 10 minutes of each
other or, preferably,
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within 5 minutes or, more preferred, within 2 minutes of each other. Either of
the two unit dosage
forms can be administered first.
The kit includes at least two separate pharmaceutical compositions. The kit
includes
container means for containing the separate compositions such as a divided
bottle or a divided foil
5 packet. Typically the kit includes directions for the administration of the
separate components. The
kit form is particularly advantageous when the separate components are
preferably administered in
different dosage forms, are administered at different dosage intervals, or
when titration of the
individual components of the combination is desired by the prescribing
physician.
The amount of factor VII or factor VII-related polypeptide and the amount of
factor XI or
10 factor XI-related polypeptide administered according to the present
invention may vary from a ratio
of between about 1:100 to about 100:1 (w/w). The ratio of factor VII to factor
XI may thus be, e.g.,
about1:100,or1:90,or1:80,or1:70or1:60,or1:50,or1:40,or1:30,or1:20,or1:10,or1:5,
or1:2,
or 1:1, or 2:1, or 5:1, or 10:1, or 20:1, or 30.1, or 40:1, or 50:1, or 60:1,
or 70:1, or 80:1, or 90:1, or
100:1; or between about 1:90 to about 1:1, or between about 1:80 to about 1:2,
or between about
15 1:70 to about 1:5, or between about 1:60 to about 1:10, or between about
1:50 to about 1:25, or
between about 1:40 to about 1:30, or between about 90:1 to about 1:1, or
between about 80:1 to
about 2:1, or between about 70:1 to about 5:1, or between about 60:1 to about
10:1, or between
about 50:1 to about 25:1, or between about 40:1 to about 30:1.
The dose of the factor VII or factor VII-related polypeptide ranges from what
20 corresponds to about 0.05 mg to about 500 mg/day of wild-type factor VII,
e.g., from about 1 mg
to about 200 mg/day, or, e.g., from about 5 mg to about 175 mg/day for a 70-kg
subject as
loading and maintenance doses, depending on the weight of the subject, the
condition and the
severity of the condition.
The dose of the factor XI or factor XI-related polypeptide ranges from what
corre-
25 sponds to about 0.05 mg to about 500 mg/day of wild-type factor XI, e.g.,
from about 1 mg to
about 200 mg/day, or, e.g., from about 1 mg to about 175 mg/day for a 70-kg
subject as loading
and maintenance doses, depending on the weight of the subject, the condition
and the severity
of the condition.
The combination of a factor Vlla and a factor XI shows a synergistic effect in
an in vitro
30 clot firmness- and fibrinolysis time-assay. Moreover, the combination of a
factor Vlla and a factor
XI shows a synergistic effect in forming stable fibrin clots, increasing the
half-clot lysis time,
increasing clot strength and increasing resistance to fibrinolysis.
The composition may be in the form of a single preparation comprising both a
factor Vlla
and a factor XI in suitable concentrations. The composition may also be in the
form of a kit
35 consisting of a first unit dosage form comprising a factor Vlla and a
second unit dosage form
comprising a factor XI and, optionally, one or more further unit dosage forms
comprising a factor
VIII and/or an TFPI inhibitor. In this case, the factor Vlla and the factor XI
should be administered
sequentially, preferably within about 1-2 hours of each other, for example
within 30 minutes of
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36
each other or, preferably, within 10 minutes or, more preferred, within 5
minutes of each other.
Either of the two unit dosage forms can be administered first.
Since the present invention relates to the prevention or treatment of bleeding
episodes or
for coagulative treatment by treatment with a combination of active
ingredients that may be
administered separately, the invention also relates to combining separate
pharmaceutical
compositions in kit form. The kit includes at least two separate
pharmaceutical compositions. The kit
includes container means for containing the separate compositions such as a
divided bottle or a
divided foil packet. Typically the kit includes directions for the
administration of the separate
components. The kit form is particularly advantageous when the separate
components are
preferably administered in different dosage forms, are administered at
different dosage intervals,
or when titration of the individual components of the combination is desired
by the prescribing
physician
Assays:
Test for factor Vlla activity:
A suitable assay for testing for factor Vlla activity and thereby selecting
suitable factor
Vlla variants can be performed as a simple preliminary in vitro test:
In Vitro Hydrolysis Assay
Native (wild-type) factor Vlla and factor Vlla variant (both hereafter
referred to as "fac-
tor Vlla") may be assayed for specific activities. They may also be assayed in
parallel to directly
compare their specific activities. The assay is carried out in a microtiter
plate (MaxiSorp, Nunc,
Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288,
Chromogenix, Swe-
den), final concentration 1 mM, is added to factor Vlla (final concentration
100 nM) in 50 mM
Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCh and 1 mg/ml bovine serum
albumin. The ab-
sorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader
(Molecular De-
vices, USA). The absorbance developed during a 20-minute incubation, after
subtraction of the
absorbance in a blank well containing no enzyme, is used to calculate the
ratio between the ac-
tivities of variant and wild-type factor Vlla:
Ratio = (A4os nm factor Vlla variant)/(A4os nm factor Vlla wild-type).
Based thereon, factor Vlla variants with an activity comparable to or higher
than native
factor Vlla may be identified, such as, for example, variants where the ratio
between the activity
of the variant and the activity of native factor VII (wild-type FVII) is
around, versus above 1Ø
The activity of factor Vlla or factor Vlla variants may also be measured using
a physio-
logical substrate such as factor X, suitably at a concentration of 100-1000
nM, where the factor
Xa generated is measured after the addition of a suitable chromogenic
substrate (eg. S-2765). In
addition, the activity assay may be run at physiological temperature.
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37
In Vitro Proteolysis Assax
Native (wild-type) factor Vlla and factor Vlla variant (both hereafter
referred to as "fac-
for Vlla") are assayed in parallel to directly compare their specific
activities. The assay is carried
out in a microtiter plate (MaxiSorp, Nunc, Denmark). factor Vlla (10 nM) and
factor X (0.8 mi-
croM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCl2 and
1 mg/ml bo-
vine serum albumin, are incubated for 15 min. factor X cleavage is then
stopped by the addition
of 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1
mg/ml bovine se-
rum albumin. The amount of factor Xa generated is measured by addition of the
chromogenic
substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final
concentration 0.5
mM. The absorbance at 405 nm is measured continuously in a SpectraMaxTM 340
plate reader
(Molecular Devices, USA). The absorbance developed during 10 minutes, after
subtraction of the
absorbance in a blank well containing no FVlla, is used to calculate the ratio
between the prote-
olytic activities of variant and wild-type factor Vlla:
Ratio = (A405 nm factor Vlla variant)/(A405 nm factor Vlla wild-type).
Based thereon, factor Vlla variants with an activity comparable to or higher
than native
factor Vlla may be identified, such as, for example, variants where the ratio
between the activity
of the variant and the activity of native factor VII (wild-type FVII) is
around, versus above 1Ø
Thrombin generation assay:
The ability of factor VII or factor VII-related polypeptides or factor XI or
factor XI-related
polypeptides (e.g., variants) to generate thrombin can be measured in an assay
comprising all
relevant coagulation factors and inhibitors at physiological concentrations
and activated plate-
lets (as described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99,
542-547 which is hereby
incorporated as reference).
Test for factor XI activity:
A suitable assay for testing for factor XI amidolytic activity and thereby
selecting
suitable factor XI variants can be performed as a simple in vitro test using a
chromogenic
substrate as described, for example, in Gailani et al. (Blood 97(10): 3117-
3122, 2001) ("the FXI
chromogenic assay'.
Factor XI biological activity may also be performed as a simple in vitro test
measuring
the activation of factor IX to IXa as described for example, in Gailani et al.
(Blood 97(10): 3117-
3122, 2001 ).
Test for factor VIII activity:
Suitable assays for testing for factor VIII activity, and thereby providing
means for
selecting suitable factor VIII variants for use in the present invention, can
be performed as simple
in vitro tests as described, for example, in Kirkwood TBL, Rizza CR, Snape TJ,
Rhymes IL, Austen
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38
DEG. Identification of sources of interlaboratory variation in factor VIII
assay. B J Haematol 1981;
37; 559-68.; or Kessels et al., British Journal of Haematology, Vol. 76
(Suppl.1) pp. 16 (1990)).
factor VIII activity may also be measured by a two-step chromogenic assay
based on the
amidolytic activity of generated FXa (Wagenvoord et al, 1989, Haemostasis,
19(4):196-X04).
factor VIII biological activity may also be quantified by measuring the
ability of a
preparation to correct the clotting time of factor VIII-deficient plasma,
e.g., as described in
Nilsson et al., 1959.(Nilsson IM, Blombaeck M, Thilen A, von Francken L,
Carriers of haemophilia
A - A laboratory study, Acta Med Scan 1959; 165:357). In this assay,
biological activity is expressed
as units/ml plasma (1 unit corresponds to the amount of FVIII present in
normal pooled plasma.
Aspects of the invention:
Aspect 1: A pharmaceutical composition comprising a factor VII or a factor VII-
related
polypeptide, and a factor XI or factor XI-related polypeptide.
Embodiment 2: The composition as in aspect 1, wherein said factor VII or
factor VII'-
related polypeptide is a factor VII-related polypeptide.
Embodiment 3: The composition as in aspect 1, wherein the factor Vlla is human
factor
Vlla
Embodiment 4: The composition as in aspect 1 or aspect 3, wherein the factor
Vlla and
the factor XI is recombinant human factor Vlla and recombinant human factor
XI.
Embodiment 5: The composition as in any one of aspects 1-4, wherein the factor
XI is
platelet factor XI.
Embodiment 6: The composition as in any one of aspects 1-5, wherein the factor
XI is
activated factor XI.
?5 Embodiment 7: The composition as in any one of aspects 1-6, wherein the
composition
further contains a TFPI inhibitor.
Embodiment 8: The composition as in any one of aspects 1-7, wherein the
composition
further contains a factor VIII.
Aspect 2: A kit containing a treatment for bleeding episodes comprising
a) an effective amount of a factor Vlla and a pharmaceutically acceptable
carrier in a first
unit dosage form;
b) an effective amount of a factor XI and a pharmaceutically acceptable
carrier in a second
unit dosage form; and
c) container means for containing said first and second dosage forms.
Embodiment 10: The composition as in aspect 2, comprising
a) an effective amount of a factor Vlla and a pharmaceutically acceptable
carrier in a first
unit dosage form;
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b) an effective amount of a factor XI and a pharmaceutically acceptable
carrier in a second
unit dosage form;
c) an effective amount of a TFPI inhibitor and a pharmaceutically acceptable
carrier in a
third unit dosage form; and
d) container means for containing said first, second and third dosage forms.
Aspect 3: A kit containing a treatment for bleeding episodes comprising
a) an effective amount of a factor Vlla and a TFPI inhibitor and a
pharmaceutically
acceptable carrier in a first unit dosage form;
b) an effeetive amount of a factor XI and a pharmaceutically acceptable
carrier in a second
unit dosage form; and
c) container means for containing said first and second dosage forms.
Aspect 4: A kit containing a treatment for bleeding episodes comprising
a) an effective amount of a factor Vlla and a pharmaceutically acceptable
carrier in a first
unit dosage form;
b) an effective amount of a factor XI and a TFPI inhibitor and a
pharmaceutically acceptable
carrier in a second unit dosage form; and
c) container means for containing said first and second dosage forms.
Embodiment 9: A kit as in any one of embodiments 2-4 further containing a
factor VIII,
either formulated in a separate unit dosage form, or contained within a unit
dosage
form also containing one or more of the compounds selected from the list of a
factor
Vlla, a factor XI or a TFPI inhibitor.
Aspect 6: Use of a factor Vlla in combination with a factor XI for the
manufacture of a
medicament for treating bleeding episodes in a subject.
Aspect 7: Use of a factor Vlla in combination with a factor XI for the
manufacture of a
medicament for reducing clotting time in a subject.
Aspect 8: Use of a factor Vlla in combination with a factor XI for the
manufacture of a
medicament for prolonging the clot lysis time in normal mammalian plasma.
Aspect 9: Use of a factor Vlla in combination with a factor XI for the
manufacture of a
medicament for increasing clot strength in normal mammalian plasma.
Aspect 10: Use of a factor Vlla in combination with a factor XI for the
manufacture of a
medicament for enhancing fibrin clot formation in normal human plasma.
Aspect 11: A method of enhancing fibrin clot formation in a subject, which
method
comprises administering to a subject an effective amount of a factor Vlla in
combination with an
effective amount of a factor XI.
Aspect 12: A method for treating bleeding episodes in a subject comprising
administer-
ing to a subject an effective amount of a factor Vlla in combination with an
effective amount of
a factor XI.
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Embodiment 10: Method as in aspect 19 or 20, wherein the factor Vlla and the
factor XI
are administered in one-dosage form.
Embodiment 11: Method as in aspect 19 or 20, wherein the factor Vlla and the
factor XI
5
is administered sequentially.
The present invention is further illustrated by the following examples, which,
however,
are not to be construed as limiting the scope of protection. The features
disclosed in the fore-
going description and in the following examples may, both separately and in
any combination
thereof, be material for realizing the invention in diverse forms thereof.
EXAMPLES
Example 1
Imarovina Haemostatic Clot Stability by Combining Coagulation factors Vlla and
XI
METHODS:
Clot lysis assay: Normal human plasma diluted 10-fold with buffer (20 mM
HEPES, 150 mM NaCI,
5 mM CaCI, pH 7.4) containing Innovin (bade Behring, 2000-fold dilution),
rFVlla (Novo Nordisk
A/S, Bagsvaerd, Denmark; various concentrations) and t-PA (American
Diagnostics, 8 nM) was
added to 96-well ELISA plates and turbidity at 650 nm was measured over time
at room
temperature. Where indicated, purified human FXI (Haematologic Technologies,
various
concentrations) was included.
Rotatonal thromboelastography (roTEG): Measurements was conducted on citrated
normal
human plasma added 5 nM t-PA and the effect of addition of 1 nM FVlla alone or
in
combination with 30 nM FXI (Haematologic Technologies) was analyzed. Clotting
was initiated
by addition of Innovin (final concentration 2000-fold diluted, Dade Behring #
526945) and
calcium (final concentration 15 mM) in a 20 mM HEPES, 150 mM NaCI, pH 7.4
buffer.
RESULTS:
Clot lysis assay: Addition of FVlla results in a dose-dependent prolongation
of the clot lysis time
(Fig. 1). This effect was optimal at 10 nM FVlla. In the presenee of 10 nM
FVlla, addition of FXI
resulted in a further prolongation of the clot lysis time (Fig. 2). The effect
was dose-dependent
and optimal at 30 nM FXI.
Thromboelastography: roTEG measurements were utilized to analyze the effect of
FVlla and FXI
on the Maximal Clot Firmness (MCF), as well as the clots resistance to t-PA
mediated lysis. Prior to
addition of FVlla/FXI, the MCF was 25 mm and the time required for half clot
lysis was 12.3
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minutes (Fig. 3). Addition of FXI (30 nM) did not alter MCF but prolonged the
half-clot lysis time
to 16.1 min (Fig. 3). Similarly, addition of FVlla (1 nM) resulted in clot
protection from t-PA-
mediated fibrinolysis (half-clot lysis time; 16.7 min) without any effect on
MCF (Fig. 3). However,
addition of FVlla (1 nM) together with FXI (30 nM) increases the MCF (29 mm),
as well as the
half-clot lysis time (24.2 min, Fig. 3).
CONCLUSION:
These results demonstrate that FVlla and FXI addition to plasma in a
synergistic fashion improve
clot mechanical strength and resistance to t-PA mediated fibrinolysis.
Example 2
Shortening the clotting time in normal human plasma by Combining Coagulation
factors Vlla
and XI
METHODS:
Clot assay: Aliquots (55 NI) of rFVlla (1 Ng/ml) alone, FXI (25 nM) alone, or
rFVlla and FXI in 50
mM Pipes, 100 mM NaCI, 2 mM EDTA, 1 % BSA, pH 7.2, were incubated for 5 min
with a 55 NI ali-
quot containing 100 NM PC/PS vesicles and 50 mM CaCl2 in the same buffer. A 55
NI aliquot of
normal human plasma (NHP) was then added and clotting followed for 400 seconds
in an ACL
clotting machine using the standard APTT program.
RESULTS:
Clot assay: Prior to addition of rFVlla or FXI, NHP did not clot within the
400 seconds monitoring
time. Following addition of FXI (25 nM) the clotting time was still longer
than 400 s. Addition of
FVlla (1 Ng/ml) reduced the clot time to 159.4 ~ 1.4 seconds (Fig 4). Addition
of both FVlla (1
Ng/ml) and FXI (25 nM) reduced the clot time to 95.0 ~ 1.4 seconds (Fig 4)
CONCLUSION:
These results demonstrate that FVlla and FXI addition to plasma in a
synergistic fashion shorten
the clotting time in NHP.
