Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL COMPOSITION COMPRISING FACTOR Vll POLYPEPTlDES AND FACTOR V
POLYPEPTIDES
FIELD OF THIS INVENTION
The present invention relates to a pharmaceutical composition comprising
factor VII or
a fiactor VII-related polypeptide and factor V or a factor V-related
polypeptide. The invention
also relates to the use of a combination of factor VII or a factor VII-related
polypeptide, and a
factor V or a factor V-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 fihe 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 FVlll, 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 which 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-
finking 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. 1n 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 A/S,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.
Factor V is a large glycoprotein synthesized as a single chain molecule and
circulates in
blood as an inactive cofactor at a concentration of 30 nM. Approximately 25 %
of Factor V in
blood is present in the a granules of platelets, whereas the rest is present
in plasma. In the co-
agulation cascade Factor V acts as a cofactor for the serine protease
activated factor X. Before it
can act effectively as a cofactor, Factor V must be activated by limited
proteolysis. Both factor Ila
and Xa are able to do so. The first Factor V cDNA sequence was published by
(Cane et al. (PNAS
83:6800, 1986) but the complete genomic sequence is still not known. Factor V-
deficiency is a
rare recessive inherited disorder associated with a bleeding tendency due to
loss-of-function mu-
tations in the Factor V gene (Guasch et al., Thromb. Haemost. 77:252, 1997).
Some discussion re-
mains whether complete Factor V-deficiency is compatible with life. Published
studies elaim rela-
tively mild bleeding tendencies in affected individuals. At the same time, the
Factor V knockout
mouse display a severe phenotype and approximately one-half do not make it
pasfi mid-
gestation, whereas the remaining embryos come to term normally but die within
hours after
birth from excessive hemorrhage. In some cases, Factor V-deficiency co-
inherits with factor VIII-
deficiency (Seligsohn et al., NEJM 307:1191, 1982). The most common genetic
defect in Factor V
is the so-called factor Vieiden mutation, which is associated with an
increased thrombotic risk due
to activated protein c resistance. This genetic abnormality is prevalent in
approximately 3% of
Caucasians, and numerous studies have shown factor Vieiden to be the most
common genetic risk
factor for thrombosis.
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
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3
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.
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.
Cripe et al. (Biochemistry 31:3777, 1992), Jenny et al. (PNAS 84: 4846, 1987),
Kane et al.
(Biochemistry 26: 6508, 1987), and Kane & Davie (PNAS 83: 6800, 1986) concern
the structure,
amino acid sequence, and DNA encoding Factor V.
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 time to bleeding arrest is shortened.
SUMMARY OF THE INVENTION
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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.
In a first aspect the invention provides a pharmaceutical composition
comprising factor
VII or a factor VII-related polypeptide, and factor V or a factor V-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 factor VII or a factor VII-related
polypeptide
and a pharmaceutically acceptable carrier in a first unit dosage form;
b) An effective amount of a preparation of factor V or a factor V-related
polypeptide
and a pharmaceutically acceptable carrier in a second unit dosage form; and
c) Container means for containing said first- and second-unit dosage forms.
In a third aspect, the invention provides the use of factor VII or a factor
VII-related
polypeptide in combination with a factor V or a factor V-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 to19, for the
manufacture of a medicament for treating bleeding episodes in a subject.
In different embodiments thereof, the medicaments are for reducing time needed
to
obtain full haemostasis, reducing time needed to maintain haemostasis,
reducing clotting time,
prolonging the clot lysis time, and increasing clot strength.
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
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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),
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
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 factor VII or a factor VII-related polypeptide, and a second
amount of a prepara-
tion of factor V or a factor V-related polypeptide, wherein the first and
second amount together
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 factor VII or a factor VII-related polypeptide, and a second
amount of a prepara-
tion of factor V or a factor V-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 factor VII or a factor VII-related polypeptide, and a second
amount of a prepara-
tion of factor V or a factor V-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 factor VII or a factor VII-related polypeptide, and a second
amount of a prepara-
tion of factor V or a factor V-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 factor VII or a factor VII-related polypeptide, and a second
amount of a prepara-
tion of factor V or a factor V-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 V or factor V-related polypeptide are administered
in single-unit
dosage form.
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In another series of embodiments the factor VII or factor VII-related
polypeptide and
the factor V or factor V-related polypeptide are administered in the form of a
first-unit dosage
form comprising a preparation of factor VII or a factor VII-related
polypeptide and a second-unit
dosage form comprising a preparation of factor V or a factor V-related
polypeptide. In a series of
embodiments thereof, the first-unit dosage form and the second-unit dosage
form are adminis-
tered 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 factor VII or a factor VII-related polypeptide, and
an effective
amount of factor V or a factor V-related polypeptide, and a pharmaceutically
acceptable carrier in a single-unit dosage form; and
e) Container means for containing said single-unit dosage form.
In one series of embodiments of the invention, the factor Vll or factor Vll-
related
polypeptide is a factor VIl-related polypeptide. In one series of embodiments
of the invention
the factor Vll-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 VI1. !n one embodiment said factor Vll is human factor
VII. !n 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 V or factor V-related polypeptide is a
factor V-
related polypeptide. In one embodiment the factor V-related polypeptide is a
factor V amino
acid-sequence variant. In one embodiment the ratio between the activity of
said factor V-related
polypeptide and the activity of native human plasma factor V (wild-type factor
V) is at least
about 1.25 when tested in the "factor V assay" as described in the present
description. In one
embodiment the factor V or factor V-related polypeptide is a factor V
polypeptide. In one em-
bodiment the factor V is human factor V. In one embodiment the factor V is
bovine, porcine, ca-
nine, equine, murine, rat or salmon factor V. In a preferred embodiment the
factor V is recombi-
nantly made. In another embodiment the factor V is derived from plasma. In
another embodi-
ment the factor V is derived from platelets. In a preferred embodiment the
factor V is recombi-
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7
pant human plasma factor V. In another embodiment the factor V is human
activated factor V
(FVa). In one embodiment the factor V-related polypeptide is a fragment of
factor V. In one em-
bodiment the factor V-related polypeptide is a hybrid factor V polypeptide,
e.g., a por-
cine/human hybrid.
In one embodiment the factor VII or factor VII-related polypeptide and the
factor V or
factor V-related polypeptide are present in a ratio by mass of between about
100:1 and about
1:100 (w/w factor Vll:factor V).
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 VII variants have no more than
15 amino acids
replaced, deleted or inserted; in other embodiments, the factor VII variants
have no more than
10 amino acids, such as 8, 6, 5, or 3 amino acids, replaced, deleted or
inserted compared to wild-
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 V or factor V-related polypeptide are recombinant human factor Vlla and
recombinant
human factor V. In another preferred embodiment, the factor VII or factor VII-
related
polypeptide and the factor V or factor V-related polypeptide are recombinant
human factor Vlla
and recombinant human factor Va.
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
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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 V-
deficient human blood. In
one series of embodiments, the blood is plasma.
In one embodiment of the invention, the factor VII or factor Vll-related
polypeptide and
the factor V or factor V-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 V or factor V-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 V or factor V-related polypeptide are the sole coagulation factors
administered to the sub-
ject. In one embodiment of the invention, the factor VII or factor VII-related
polypeptide and the
factor V or factor V-related polypeptide are the sole active agents
administered to the patient.
In one embodiment, the composition is substantially free of thrombin or
prothrombin; in an-
other embodiment, the composition is substantially free of 1=X; 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 factor
VII or a factor VII-related polypeptide as a first-unit dosage form and a
preparation of factor V or
a factor V-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 V or factor V-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 V or
factor V-related polypeptide are administered in the form of a first-unit
dosage form comprising
a preparation of factor Vll or a factor VII-related polypeptide and a second-
unit dosage form
comprising a preparation of factor V or a factor V-related pofypeptide.
In one embodiment of the invention, the factor VII or factor VII-related
polypeptide
and the factor V or factor V-related polypeptide are administered
simultaneously. In another
embodiment, the factor VII or factor VII-related polypeptide and the factor V
or factor V-related
polypeptide are administered sequentially. In one embodiment, the factor VII
or factor VII-
related polypeptide and the factor V or factor V-related polypeptide are
administered with a
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9
time separation of no more than 15 minutes, preferably 10, more preferred 5,
more preferred 2
minutes. In one embodiment, the factor VII or factor VII-related polypeptide
and the factor V or
factor V-related polypeptide are administered with a time separation of up to
2 hours,
preferably from 1 to z 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 factor V or a factor V-
related polypeptide
is from about 0.01 mg/day to about 500 mg/day (70-kg subject).
In one embodiment the factor Vll or factor VII-related polypeptide and factor
V or
factor V-related polypeptide are present in a ratio by mass of between about
100:1 and about
1:100 (w/w factor Vll:factor V)
In one embodiment of the present invention, the pharmaceutical composition is
in
single-unit dosage form and eonsists essentially of a preparation of factor
VII or a factor VII-
related polypeptide, and a preparation of facfior V or a factor V-related
polypeptide, and one or
more of the components selected from the list of pharmaceutical acceptable
carriers, stabilizers,
detergents, neutral salts, antioxidants, preservatives, and protease
inhibitors.
In another embodiment of the present invention, the pharmaceutical composition
is in
the form of a kit-of-parts with the first-unit dosage form consisting
essentially of a preparation
of factor VII or a factor VII-related polypeptide, and one or more of the
components selected
from the list of pharmaceutical acceptable carriers, stabilizers, detergents,
neutral salts,
antioxidants, preservatives, and protease inhibitors; and with the second-unit
dosage form
consisting essentially of a preparation of factor V or a factor V-related
polypeptide and one or
more of the components selected from the list of pharmaceutical acceptable
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 or FIX.
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 Vll as the only coagulation protein, the method comprising
administering to the subject in
need thereof a first amount of a preparation of factor VII or a factor VII-
related polypeptide, and
a second amount of a preparation of factor V or a factor V-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
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a preparation of factor VII or a factor VII-related polypeptide and a second
amount of a prepara-
tion of factor V or a factor V-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
5 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 factor VII or a
faetor VII-related
polypeptide and a second amount of a preparation of factor V or a factor V-
related polypeptide,
wherein the first and second amounts together are effective to enhance
formation of thrombin.
10 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 VII 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 com-
prising administering to a subject in need thereof a first amount of a
preparation of factor VII or
a factor VII-related polypeptide and a second amount of a preparation of
factor V or a factor V-
related polypeptide, wherein the first and second amounts together are
effective to reduce the
number of administrations of coagulation factor protein.
In another aspect, the invention concerns a method of treating bleedings in a
subject
suffering from a factor VII responsive syndrome, the method comprising
administering to the
subject in need thereof a first amount of a preparation of factor VII or a
factor VII-related poly-
peptide and a second amount of a preparation of factor V or a factor V-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/5, Bagsvaerd, Denmark).
In another aspect, the invention relates to the use of factor VII or a factor
VII-related
polypeptide in combination with a factor V for the manufacture of a medicament
for enhancing
fibrin clot formation in mammalian 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 factor VII or a factor VII-related polypeptide and a
second amount of a
preparation of factor V or a factor V-related polypeptide, wherein the first
and second amounts
together are effective in treating bleedings.
LIST OF FIGURES
Figure 1: Addition of FVlla results in a significant shortening of the
clotting time,
further shortened by the addition of FV. Addition of FV alone did not reduce
the clotting time.
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DETAILED DESCRIPTION OF THIS INVENTION
Subjects, who bleed excessively in association with surgery or major trauma
thus need-
ing 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 ad-
s ministration of human blood or blood products (platelets, leukocytes, plasma-
derived concen-
trates for the treatment of coagulation defects, etc.) because this is
associated with the risk of
transferring 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
cytokines and inflammatory reactions is started making any treatment extremely
difficult and
unfortunately 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-
ticular 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.
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.
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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 factor VII or a factor VII-related
polypeptide,
e.g., factor Vlla, in combination with a preparation of factor V or a factor V-
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 V is ad-
ministered alone.
The administration of a preparation of factor VII or a factor VII-related
polypeptide,
e.g., factor Vlla, in combination with a preparation of factor V or a factor V-
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 V is
administered alone. The present invention provides a beneficial effect of
simultaneous or se-
quential dosing of a preparation of factor V or a factor V-related polypeptide
and a preparation
of factor VII or a factor VIl-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 factor VII or a factor VII-related
polypeptide and a
preparation of factor V or a factor V-related polypeptide is an advantageous
product ensuring
short clotting times, rapid formation of haemostatic plugs, and formation of
stable haemostatic
plugs. ft has been found by the present inventor that a combination of factor
VII or a factor VII-
related polypeptide and a factor V or a factor V-related polypeptide is an
advantageous product
ensuring the formation of solid, stable and quickly formed haemostatic plugs.
The present inventors have shown that a combination of factor Vlla and factor
V can
increase the firmness of the clot more effectively than either factor Vlla or
factor V alone. It has
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also been shown that combination of factor VII or a factor VII-related
polypeptide and a factor V
can prolong the in vitro clot lysis time in normal human plasma more
effectively than either
factor Vlla or factor V alone. It has also been shown that combination of
factor VII or a factor VII-
related polypeptide and a factor V can prolong the half-clot lysis time in
normal human plasma
more effectively than either factor Vlla or factor V alone. It has also been
shown that
combination of factor VII or a factor VII-related polypeptide and a factor V
can protect the clot
from fibrinolysis, in particular tPA-mediated fibrinolysis, in normal human
plasma more
effectively than either factor Vlla or factor V 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-
cently 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 factor 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
factor VII or a factor VII-related polypeptide, and a factor V.
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-
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 levels
interfere negatively with the activation of factor Xlll. These subjects,
therefore, have a defective,
or less efficient, haemostasis leading to the formation of fibrin plugs which
are easily and pre-
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14
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
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.
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 VII 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 VII
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 VIl-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
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
"faetor 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.
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"Factor VII-related polypeptides" include, without limitation, factor VII
polypeptides
that have either been chemically modified relative to human factor VII andlor
contain one or
more amino acid sequence alterations relative to human factor VII (i.e.,
factor VII variants),
and/or contain truncated amino acid sequences relative to human factor VII
(i.e., factor VII
5 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
10 "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.
15 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
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%,
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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 factor 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 substifiution 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
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 Vll variants having substantially the same or improved biological
activity as wild-type
factor VII include S52A-FVII, S60A-FVII (lino et al., Arch. Biochem. Biophys.
352: 182-192, 1998);
L305V-FVII, L305V/M306D/D309S-FVII, L3051-FVII, L305T-FVII, F374P-FVII,
V158T/M298Q-FVII,
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V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-
FVII,
V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII,
V158D/E296V1M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII,
V158D1E296V-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), S344A-
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 ("!n Vitro Proteolysis
Assay", see
below);
(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
(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.
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The term "factor VII biological activity" or "factor Vll 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 V polypeptides:
The present invention encompasses Factor V polypeptides, such as, e.g., those
having
the amino acid sequence disclosed in Cripe et al. (Biochemistry 31:3777,
1992), Jenny et al. (PNAS
84: 4846, 1987), Kane et al. (Biochemistry 26: 6508, 1987), and Kane & Davie
(PNAS 83: 6800, 1986)
(wild-type human Factor V).
In practicing the present invention, any factor V polypeptide may be used that
is
effective in preventing or treating bleeding. This includes factor V
polypeptides derived from
blood or plasma, or produced by recombinant means.
As used herein, "factor V polypeptide" encompasses, without limitation, factor
V, as
well as factor V-related polypeptides. The term "factor V" is intended to
encompass, without
limitation, polypeptides having the amino acid sequence as described in Cripe
et al. (Biochemistry
31:3777, 1992), Jenny et al. (PNAS 84: 4846, 1987), Kane et al. (Biochemistry
26: 6508, 1987), and
Kane & Davie (PNAS 83: 6800, 1986) (wild-type human factor V), as well as wild-
type factor V
derived from other species, such as, e.g., bovine, porcine, canine, murine,
rat and salmon factor
V. It further encompasses natural allelic variations of factor V 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 V" is also intended to encompass factor V
polypeptides in their
zymogen form, as well as those that have been processed to yield their
respective bioactive
forms.
"Factor V-related polypeptides" include, without limitation, factor V
polypeptides that
have either been chemically modified relative to human factor V and/or contain
one or more
amino acid sequence alterations relative to human factor V (i.e., factor V
variants), and/or
contain truncated amino acid sequences relative to human factor V (i.e.,
factor V fragments).
Such factor V-related polypeptides may exhibit different properties relative
to human factor V,
including stability, phospholipid binding, altered specific activity, and the
like.
The term "factor V-related polypeptides" are intended to encompass such
polypeptides
in their zymogen form, as well as those that have been processed to yield
their respective
bioactive forms.
As used herein, "factor V-related polypeptides" encompasses, without
limitation,
polypeptides exhibiting substantially the same or improved biological activity
relative to wild-
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19
type human factor V, as well as polypeptides, in which the factor V biological
activity has been
substantially modified or reduced relative to the activity of wild-type human
factor V. These
polypeptides include, without limitation, factor V that has been chemically
modified and factor
V 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 V.
Factor V-related polypeptides, including variants of factor V, whether
exhibiting
substantially the same or better bioactivity than wild-type factor V, or,
alfiernatively, exhibiting
substantially modified or reduced bioactivity relative to wild-type factor V,
include, without
limitation, polypeptides having an amino acid sequence that differs from the
sequence of wild-
type factor V by insertion, deletion, or substitution of one or more amino
acids.
Factor V-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 V that has been produced in the same cell type, when
tested in the factor V
activity assay as described in the present specification.
Factor V-related polypeptides, including variants, having substantially the
same or
improved biological activity relative to wild-type factor V 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 V that has been produced in the same cell type when tested in one or
more of the specific
factor V activity assay as described. For purposes of the invention, Factor V
biological activity may
be quantified by measuring the ability of a preparation to clot plasma, as
described, e.g., in
Thorelli et al., Thromb Haemost 80: 92, 1998. In this assay, biological
activity is expressed as the
reduction in clotting time relative to a control sample.
Factor V-related polypeptides, including variants, having substantially
reduced
biological activity relative to wild-type factor V 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 V that has been
produced in the same
cell type when tested in one or more of the specific factor V activity assays
as described above.
Non-limiting examples of Factor V equivalents include plasma-derived human
Factor V as
described, e.g., in Dahlback, J.Clin.lnvest. 66:583-591, 1980; ICane &
Majerus, J.BioLChem.
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256:1002-1007, 1981; or Katzmann efi al., P.N.A.S., USA 78:162-166;
recombinant human Factor V
as described, e.g., in Kane & Davie, P.N.A.S., USA 83:6800-6804, 1986 or Jenny
et al., P.N.A.S., USA
84:4846-4850, 1987; platelet-derived Factor V as described, e.g., in Tracy et
al., Blood 60:59-63,
1982 or in Tracy et al., J.BioLChem. 260:2119-2124, 1985, bovine Factor V as
described, e.g. in Ne-
5 sheim et al., J.BioLChem. 254:508-517,1979a or Esmon, J.BioI.Chem. 254:964-
973, 1979.
In some embodiments the Factor V are Factor V equivalents wherein the ratio
between
the activity of said Factor V polypeptide and the activity of native human
Factor V (wild-type Fac-
tor V) is at least about 1.25 when tested in the "factor V assay" (Thorelli et
al, see above); in
other embodiments, the ratio is at least about 2.0; in further embodiments,
the ratio is at least
10 about 4Ø
Factor V-related polypeptides also include fragments of factor V or factor V-
related poly-
peptides retaining their characteristic haemostasis-related activity. The
haemostasis-related activ-
ity of a factor V polypeptide may, for example, be measured using the factor V-
activity assay de-
scribed in the present specification.
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 One-letter
code 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
PhenylalaninePhe F
Tyrosine Tyr Y
Tryptophan Trp W
Nistidine His N
Lysine Lys K
Arginine Arg R
Glutamine Gln Q
Asparagine Asn N
Glutamic AcidGlu E
Aspartic AcidAsp D
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The term "factor Vlla" or "FVlla" may be used interchangeably.
In this context, "subjects with an impaired thrombin generation" means
subjects who
cannot generate a full thrombin burst on the activated platelet surface and
includes subjects having
a generafiion 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 having 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 factor VII or
a factor VII-related polypeptide and a preparation of factor V or a factor V-
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 V or factor V-
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 Vll or fac-
for VII-related polypeptide and the factor V or factor V-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 haemostatic
agents, or coagulation factors administered to the patient in the course of a
particular treat-
ment, such as, e.g., in the course of a particular bleeding episode. It will
be understood that
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 clotting time are
clinical parameters
used for assaying the status of patient's haemostatic system. Blood samples
are drawn from the
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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; McICenzie 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 factor
VII or a factor VII-
related polypeptide and a preparation of factor V or a factor V-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 V or factor V-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
factor VII or a factor VII-related polypeptide and a preparation of factor V
or a factor V-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 V or factor V-
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 factor VII or a factor VII-related polypeptide and a preparation of a
preparation of factor V or a
factor V-related polypeptide is increased relative to the individual rate for
or degree of fibrin clot
formation of a confirol sample containing only the factor VII or factor VII-
related polypeptide or the
factor V or factor V-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
factor Vll or a factor VII-related polypeptide and a preparation of a
preparation of factor V or a
factor V-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
V or factor V-related
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
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23
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)
15299-318, 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
Lion 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 sysfiem.
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
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
haemostatic mechanism may be overwhelmed by the demand of immediate
haemostasis and
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24
they may develop excessive bleeding in spite of a basically (pre-trauma or pre-
surgery) normal
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 andlor
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-
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
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
bleeding may also be due to so-called thrombolytic therapy which comprises
combined treat-
ment with 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
include, without limitation, uncontrolled and excessive bleeding in connection
with surgery or
trauma in subjects having aeute haemarthroses (bleedings in joints), chronic
haemophilic ar-
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 V;
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 ex-
petted bleeding, such as, for example, in surgery, and regulation of an
already occurring bleed-
ing, 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
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tissue or organ, or it may be an unspecified bleeding. Prophylactic
administration of a prepara-
tion of factor VII or a factor VII-related polypeptide and a preparation of
factor V or a factor V-
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 present invention also encompasses the use of factor VII or FVII-related
polypeptides, and
tPA inhibitors within veterinary procedures.
The factor VII or factor VII-related polypeptides and factor V or factor V-
related poly-
peptides as defined in the present specification may be administered
simultaneously or sequen-
10 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 factor
VII or a factor VII-related polypeptide as a first unit dosage form and a
preparation of factor V or
a factor V-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 pre-
15 (erred order of administration, but is merely done for convenience purposes
By "simultaneous" dosing of a preparation of factor VII or a factor VII-
related polypep-
tide and a preparation of factor V or a factor V-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 factor
protein with a time
20 separation of no more than 15 minutes, preferably 10, more preferred 5,
more preferred 2 min-
utes. 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
25 minutes 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 V" or "factor V level" is meant the level of the patient's
factor V ac-
tivity 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 interchangeably.
By "reduced level of factor V" or "reduced factor V level" is meant a decrease
in the
presence or activity of factor V in the blood stream compared to the mean
factor V level in a
population of subjects having no factor V deficiency or inhibitors to factor
V. The level of circu-
lating factor V can be measured by either a coagulant or an immunologic assay.
Factor V activity
is determined by the ability of the patient's plasma to correct the clotting
time of factor V-
deficient plasma (e.g., an APTT assay, see below; see also "assay part" of the
present descrip-
tion).
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One unit of factor V has been defined as the amount of factor V present in one
millili-
tre of normal (pooled) human plasma (corresponding to a factor V 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 ~,g protein. After activation 50 units
correspond to about 1
~g protein.
By "deficiency" is meant a decrease in the presence or activity of, e.g.,
factor V in
plasma compared to that of normal healthy individuals. The term may, where
appropriate, be
used interchangeably with "reduced factor V level".
By "APTT" or "aPTT" is meant the activated partial thromboplastin time
(described by,
e.g., Proctor RR, Rapaport 51: 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 V-responsive syndrome" is meant a syndrome where exogenous factor V
ad-
ministered to the subject in need thereof may prevent, cure or ameliorate any
symptoms, condi-
tions or diseases, expected or present, caused by the syndrome. Included are,
without limitation,
syndromes caused by a reduced level of factor V, e.g., bleeding disorders
caused by inhibitors to
factor V. A factor V-responsive syndrome may also be treated with a
composition according to
the present invention.
By "factor VII-responsive syndrome" is meant a syndrome where exogenous factor
VII,
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. In-
cluded are, 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 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 factor
VII or a
factor Vil-related polypeptide, or factor V or a factor V-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
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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.
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 V- or factor Vll
protein("anti-
gen") 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 V protein-
containing preparation with an anti-thromobomodulin antibody immobilised onto
the elisa
plate, subsequently contacting the immobilised antibody-factor V complex with
a second anti-
factor V antibody 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 anti-
bodies. 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
essentially free of coagulation 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 V or factor
V-related polypeptides that have been separated from the cell in which they
were synthesized or
the medium in which they are found in nature (e.g., plasma or blood).
Separation of polypep-
tides from their cell of origin may be achieved by any method known in the
art, including, with-
out 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. Separation 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 VII or anti-factor V antibody column, respectively; hydrophobic
interaction chroma-
tography; ion-exchange chromatography; size exclusion chromatography;
electrophoretic proce-
dures (e.g., preparative isoelectric focusing (IEF)), differential solubility
(e.g., ammonium sulfate
precipitation), or extraction and the like.
Within the present invention an "effective amount" of factor VII or a factor
VII-related
polypeptide, and factor V or a factor V-related polypeptide is defined as the
amount of factor VII
or a factor VII-related polypeptide, e.g., FVlla, and factor V or a factor V-
related polypeptide,
that together suffices to prevent or reduce bleeding or blood loss, so as to
cure, alleviate or par-
tially arrest the disease and its complications.
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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.
Abbreyiations
TF tissue factor
FVII factor VII in its single-chain, unactivated form
FVlla factor VII in its activated form
rFVlla recombinant factor VII in its activated form
TAFI TAFI in its zymogenic, unactivated form
Factor V Factor V in its zymogenic, unactivated form
Factor Va Factor V in its activated form
rFactor V recombinant Factor V
rFactor Va recombinant Factor Va
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 Vla, 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
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
regions critical to the function of the factor Vlla or factor V-molecule and
still result in an active
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29
polypeptide. Amino acid residues essential to the activity of the factor VII
or factor VII-related
polypeptide or factor V or factor V-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
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,
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
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-
ture cycling, 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
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 VII 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.,
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
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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 Vla or other proteases having trypsin-like specificity, such as,
e.g., factor IXa, kallik-
5 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 VII or
factor VII-related polypeptide may then be formulated and administered as
described below.
10 Factor V for use within the present invention may be isolated from plasma
according to
known methods, such as those disclosed by ICatzmann et al., Proc. Natl. Acad.
Sci. USA 78: 162,
1981. It is preferred, however, to use recombinant Factor V so as to avoid to
the use of blood- or
tissue-derived products that carry a risk of disease transmission. Methods for
preparing recom-
binant Factor V are known in the art; see, for example, ICane & Davie, Proc.
Natl. Acad. Sci. U.S.A.
15 83: 6800, 1986. Activated Factor V is described in, for example, Esmon,
J.BioLChem. 254: 964-973,
1979; Nesheim, J.BioI.Chem. 254:1326-1334, 1979; Suzuki et al., J.BioLChem.
257:6556-6564,1982;
Nesheim et al, J.BioLChem. 259:3187-3196, 1984a; and Jenny et al., P.N.A.S.,
USA 84:4846-
4850,1987. Factor V variants may be produced by means of site-directed
mutagenesis as de-
scribed above.
20 Factor V -related polypeptides may produced by modification of wild-type
factor V or by
recombinant technology. factor V -related polypeptides with altered amino acid
sequence when
compared to wild-type factor V may be produced by modifying the nucleic acid
sequence encoding
wild-type factor V 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 V by known means, e.g.
by site-specific
25 mutagenesis, as described in more detail above. Separation of polypeptides
from their Bell 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 V
or factor V-related
polypeptides may be further purified. Purification may be achieved using any
method known in
30 the art, including, without limitation, affinity chromatography, such as,
e.g., on an anti-factor V
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 V or factor V-related polypeptides derived from the
host cell. The result-
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31
ing activated factor V or factor V-related polypeptide may then be formulated
and administered
as described below.
As will be appreciated by those skilled in the art, it is preferred to use
Factor V polypep-
tides and Factor Vli polypeptides syngeneic with the subject in order to
reduce the risk of induc-
ing an immune response. Preparation and characterization of non-human Factor V
has been dis-
closed by, for example, Katzmann et al., Proc. Natl. Acad. Sci. USA, 78: 162,
1981. The present
invention also encompasses the use of such Factor V 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
respon-
sive 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 and/or transfu-
sions (e.g., in multi transfused subjects having been subjected to surgery or
trauma).
Pharmaceutical compositions comprising a preparation of factor VII or a factor
VII-related
polypeptide and a preparation of factor V or a factor V-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 V or a factor V-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
factor VII or a factor VII-related polypeptide, or a factor V, or factor VII
or a factor VII-related
polypeptide in combination with a factor V, 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
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32
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
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
Remington'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 Vll or factor VII-related polypeptide, factor V or
factor V-
related polypeptide, or factor VII or factor VII-related polypeptide in
combination with factor V
or factor V-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 V or factor V-
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 V or factor V-
related polypeptide
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33
in one dosage form, or a dissolved lyophilized powder or a liquid formulation
containing the
factor Vll or factor VII-related polypeptide in one dosage form and dissolved
lyophilized powder
or a liquid formulation containing the factor V or factor V-related
polypeptide in another
dosage form.
It is to be understood that the amount of factor VII or factor VII-related
polypeptide
and the amount of factor V or factor V-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 V 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 factor
VII or a
factor VII-related polypeptide and a preparation of factor V or a factor V-
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 V or factor V-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 V or factor V-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 factor Vll or a factor VII-
related polypeptide and
a preparation of a preparation of factor V or a factor V-related polypeptide
in suitable
concentrations. The composition may also be in the form of a kit-of-parts
consisting of a first unit
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34
dosage form comprising a preparation of a preparation of factor VII or a
factor VII-related
polypeptide and a second unit dosage form comprising a preparation of a
preparation of factor V
or a factor V-related polypeptide. In this case, the factor VII or factor VII-
related polypeptide and
the factor V or factor V-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,
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
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 V or
factor V-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
V 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
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 Vll or factor VII-related polypeptide ranges from what
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 V or factor V-related polypeptide ranges from what
corresponds
to about 0.05 mg to about 500 mg/day of wild-type factor V, 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 main-
tenance doses, depending on the weight of the subject, the condition and the
severity of the
condition.
The combination of factor VII or a factor VII-related polypeptide and Factor V
or a
factor V-related polypeptide shows a synergistic effect in an in vitro
clotting time assay.
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The composition may be in the form of a single preparation comprising both
factor VII or a
factor VII-related polypeptide and Factor V or a factor V-related polypeptide
in suitable
concentrations. The composition may also be in the form of a kit consisting of
a first unit dosage
form comprising factor VII or a factor VII-related polypeptide, and a second
unit dosage form
5 comprising factor V or a factor V-related polypeptide. In this case, the
factor VII or factor VII-related
polypeptide and the factor V or factor V-related polypeptide should be
administered sequentially,
preferably within about 1-2 hours of each other, for example within 30 minutes
of 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.
10 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
ineludes container means for containing the separate compositions such as a
divided bottle or a
15 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 Assa~r
Native (wild-type) factor Vlla and factor Vlla variant (both hereafter
referred to as "fac-
for 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 CaCla 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).
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36
Based thereon, factor Vlla variants with an acfiivity 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.
In Vitro Proteolysis Assa~r
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, ZO 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 acfiivities of variant and wild-type factor Vlla:
Ratio = (A405 nm factor Vlla variant)l(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 V or
factor V-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 V activity:
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37
Factor V polypeptides useful in accordance with the present invention may be
selected
by suitable assays that can be performed as simple preliminary in vitro tests,
such as, e.g., as the
clotting assay described by Thorelli et al., Thromb Haemost 80: 92, 1998 (the
"factor V assay")
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
METHODS:
Clot assay: Aliquots (55 NI) of rFVlla (1 Ng/ml) in 50 mM Pipes, 100 mM NaCI,
2 mM EDTA, 1
BSA, pH 7.2, were incubated for 5 min with an 55 NI aliquot containing 100 NM
PC/PS vesicles and
50 mM CaCl2 in the same buffer. A 55 NI aliquot of either normal human plasma
(NHP) or factor
VIII-deficient plasma (FVIII-DP) was then added and clotting followed for 400
seconds in an ACL
clotting machine using the standard APTT program. Where indicated, Factor V
(30 nM) was
included.
RESULTS:
Clot assay: Prior to addition of Factor Vlla or Factor V, NHP as well as FVIII-
DP did not clot within
the 400 seconds monitoring time. Addition of Factor Vlla (1 Ng/ml) reduced the
clot time to 184.0
~ 1.9 seconds in NHP and 126.6 ~ 3.1 seconds in FVIII-DP (Fig 1). Addition of
both Factor Vlla (1
Ng/ml) and Factor V (30 nM) reduced the clot time to 116.2 ~ 0.8 and 109.8 ~
1.41 seconds in NHP
and FVIII-DP, respectively (Fig 1).
CONCLUSION:
These results demonstrate that Factor Vlla and Factor V addition to plasma in
a synergistic
fashion shorten the clotting time of normal and FVIII-DP.
