U.S. patent application number 14/013957 was filed with the patent office on 2014-02-20 for methods for treating bleeding disorders.
The applicant listed for this patent is Baxter Healthcare S.A., Baxter International Inc.. Invention is credited to Michael Dockal, Friedrich Scheiflinger, Peter Turecek.
Application Number | 20140050717 14/013957 |
Document ID | / |
Family ID | 41542352 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140050717 |
Kind Code |
A1 |
Dockal; Michael ; et
al. |
February 20, 2014 |
Methods for Treating Bleeding Disorders
Abstract
A method of factor X1-dependent blood coagulation enhancement in
a subject in need of enhanced blood coagulation comprising
administering a therapeutically effective amount of a composition
comprising a non-anticoagulant sulfated polysaccharide (NASP) to
the subject. A method of factor X1-dependent blood coagulation
enhancement in a subject in need of enhanced blood coagulation
comprising: (i) selecting a subject that is not deficient for
factor X1; and (ii) administering a therapeutically effective
amount of a composition comprising a non-anticoagulant sulfated
polysaccharide (NASP) to the subject, wherein the NASP enhances
blood coagulation in a factor X1-dependent manner. A method of
identifying a non-anticoagulant sulfated polysaccharide (NASP)
which is capable of enhancing blood coagulation in dependence on
FXI, the method comprising: a) combining a blood or plasma sample
comprising activation competent FXI with a composition comprising a
sulfated polysaccharide and measuring the clotting or thrombin
generation parameters of the blood or plasma sample; b) combining a
corresponding blood or plasma sample deficient in activation
competent FXI with a composition comprising the sulfated
polysaccharide and measuring the clotting or thrombin generation
parameters of the blood or plasma sample; and c) comparing the
clotting or thrombin generation parameters of the blood or plasma
samples as determined in steps (a) and (b) with each other, wherein
a decrease in the clotting time of the blood sample or an increase
in peak thrombin or decrease in peak time of the plasma sample
comprising activation competent FXI compared to the clotting time
of the blood sample or peak thrombin or peak time of the plasma
sample deficient in activation competent FXI is indicative of a
NASP which is capable of enhancing blood coagulation in dependence
on FXI.
Inventors: |
Dockal; Michael; (Vienna,
AT) ; Scheiflinger; Friedrich; (Vienna, AT) ;
Turecek; Peter; (Klosterneuburg, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxter Healthcare S.A.
Baxter International Inc. |
Glattpark (Opfikon)
Deerfield |
IL |
CH
US |
|
|
Family ID: |
41542352 |
Appl. No.: |
14/013957 |
Filed: |
August 29, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13002501 |
Jan 3, 2011 |
8546096 |
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PCT/EP2009/006082 |
Aug 21, 2009 |
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14013957 |
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61189734 |
Aug 22, 2008 |
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Current U.S.
Class: |
424/94.64 ;
514/54; 514/56 |
Current CPC
Class: |
A61K 38/37 20130101;
A61K 38/4846 20130101; A61K 38/37 20130101; A61K 38/36 20130101;
G01N 33/86 20130101; A61K 31/727 20130101; A61K 31/737 20130101;
A61K 38/4853 20130101; A61P 7/04 20180101; A61K 38/366 20130101;
A61K 31/737 20130101; A61K 38/4853 20130101; A61K 38/4846 20130101;
A61K 45/06 20130101; A61K 38/36 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/727
20130101 |
Class at
Publication: |
424/94.64 ;
514/54; 514/56 |
International
Class: |
A61K 31/737 20060101
A61K031/737; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of factor XI-dependent blood coagulation enhancement in
a subject in need of enhanced blood coagulation comprising
administering a therapeutically effective amount of a composition
comprising a non-anticoagulant sulfated polysaccharide (NASP) to
the subject, wherein the NASP enhances blood coagulation in a
factor XI-dependent manner.
2. The method of claim 1, wherein the NASP is selected from the
group consisting of pentosan polysulfate (PPS), fucoidan,
N-acetyl-heparin (NAH), N-acetyl-de-O-sulfated-heparin
(NA-de-o-8H), de-N-sulfated-heparin (De-NSH),
de-N-sulfatecl-acetylated-heparin (De-NSAH), periodate-oxidized
heparin (POH), chemically sulfated laminarin (CSL), chemically
sulfated alginic acid (CSAA), chemically sulfated pectin (CSP),
dextran sulfate (DXS) and heparin-derived oligosaccharides
(HDO).
3. The method of claim 2, wherein the NASP is PPS.
4. The method of claim 2, wherein the NASP is fucoidan.
5. The method of claim 2, wherein the NASP enhances the activation
of factor XI.
6. The method of claim 1, wherein the NASP is administered at a
dosage of about 0.01 mg/kg to about 200 mg/kg.
7. The method of claim 1, wherein the subject has a bleeding
disorder 25 selected from the group consisting of a congenital
coagulation disorder caused by a blood factor deficiency, a chronic
or acute bleeding disorder, and an acquired Coagulation
disorder.
8. The method of claim 7, wherein the blood factor deficiency is of
one or more factors selected from the group consisting of factor V,
factor VII, factor VIII, factor IX, factor X, factor XI, factor
XII, factor XIII, and von Willebrand factor.
9. The method of claim 1, wherein the cause of the need for
enhanced blood coagulation is prior administration of an
anticoagulant or surgery or other invasive procedure.
10. The method of claim 1, further comprising administering an
agent selected from the group consisting of a procoagulant, an
activator of the intrinsic coagulation pathway, an activator of the
extrinsic coagulation pathway, and a second NASP.
11. The method of claim 10, wherein the activator of the intrinsic
coagulation pathway is selected from the group consisting of factor
Xa, factor IXa, factor XIa, factor XIIa, and kallikrein.
12. The method of claim 11, wherein the activator of the extrinsic
coagulation pathway is selected from the group consisting of tissue
factor, factor VIIa, thrombin, and factor Xa.
13. The method of claim 1, further comprising administering one or
more factors selected from the group consisting of factor XI,
factor XII, prekallikrein, high molecular weight kininogen (HMWK),
factor V, factor Va, factor VII, factor VIII, factor VIIIa, factor
IX, factor X, factor XIII, factor II, factor VIIa, and von
Willebrand factor.
14. The method of claim 9, wherein the anticoagulant is selected
from the group consisting of heparin, a coumarin derivative, such
as warfarin or dicumarol, tissue factor pathway inhibitor (TFPI),
antithrombin III, lupus anticoagulant, nematode anticoagulant
peptide (NAPc2), factor VIIa inhibitors, active-site blocked factor
VIIa (factor VIIai), factor IXa inhibitors, active-site blocked
factor IXa (factor IXai), factor Xa inhibitors, including
fondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906),
active-site blocked factor Xa (factor Xai), inhibitors of factors
Va and VIIIa, including activated protein C (APC) and soluble
thrombomodulin, thrombin inhibitors, including hirudin,
bivalirudin, argatroban, and ximelagatran, and an antibody or
antibody fragment that binds a clotting factor.
15. The method of claim 14, wherein the anticoagulant is an
antibody orantibody fragment that binds a clotting factor selected
from the group consisting of Factor V, Factor VII, Factor VIII,
Factor IX, Factor X, Factor XIII, Factor II, Factor XI, Factor XII,
von Willebrand factor, prekallikrein, and high molecular weight
kininogen (HMWK).
16. The method of claim 1 wherein the subject is deficient in
factor XI, the method further comprising administering factor
XI.
17. The method of claim 1 wherein the subject is deficient in
factor VIII, the method further comprising administering factor
VIII or a procoagulant bypassing agent.
18. The method of claim 17 wherein the patient has inhibitor
antibodies against factor VIII.
19. The method of claim 1 wherein the subject is deficient in
factor IX, the method further comprising administering factor
IX.
20. The method of claim 19 wherein the patient has inhibitor
antibodies against factor IX.
21. The method of claim 1 wherein the NASP is administered via a
non-intravenous route.
22. A method of factor Xi-dependent blood coagulation enhancement
in a subject in need of enhanced blood coagulation comprising: (i)
selecting a subject that is not deficient for factor XI; and (ii)
administering a therapeutically effective amount of a composition
comprising a non-anticoagulant sulfated polysaccharide (NASP) to
the subject, wherein the NASP enhances blood coagulation in a
factor Xi-dependent manner.
23. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods for treating bleeding
disorders, particularly congenital coagulation disorders caused by
a blood factor deficiency, chronic or acute bleeding disorders, or
acquired coagulation disorders.
BACKGROUND OF THE INVENTION
[0002] Bleeding disorders, and particularly congenital or acquired
deficiencies in coagulation factors, are typically treated by
factor replacement. Congenital coagulation disorders include
hemophilia, a recessive X-linked disorder involving a deficiency of
coagulation factor VIII (hemophilia A) or factor IX (hemophilia B),
and von Willebrand's disease, a rare bleeding disorder involving a
severe deficiency of von Willebrand factor. Hemophilia C is a
milder form of hemophilia caused by a deficiency in factor XI. It
is usually asymptomatic, but factor replacement therapy may be
required during surgery. Acquired coagulation disorders may arise
in individuals without a previous history of bleeding as a result
of a disease process. For example, acquired coagulation disorders
may be caused by inhibitors or autoimmunity against blood
coagulation factors, such as factor VIII, von Willebrand factor,
factors IX, V, XI, XII and XIII; or by hemostatic disorders such as
caused by liver disease, which may be associated with decreased
synthesis of coagulation factors. As many as 20% of patients
receiving chronic factor replacement therapy may generate
neutralizing antibodies to replacement factors. Protein
therapeutics are produced by recombinant technology or are prepared
from plasma and can only be administered intravenously, which is
inconvenient. Conventional therapy for hemophilia A and factor VIII
inhibitor patients is accomplished by therapeutics like recombinant
factor VIII or procoagulant bypassing agents, for example FEIBA or
recombinant factor VIIa. Although effective, development of
inhibitory antibodies which render the therapy ineffective is a
common occurrence. FVIIa and FEIBA as therapeutics for the
treatment of FVIII inhibitor patients have quite short half lives
and so require frequent intravenous administration.
[0003] Naito and Fujikawa (1991) J Biol Chem 266: 7353-7358 and
Gailani and Broze Jr (1993) Blood 82: 813-819 both disclose that
negatively charged surfaces such as dextran sulfate, sulfatide or,
heparin can facilitate the activation of Factor XI by thrombin or
Factor XIa in vitro. However, such materials would not have been
considered suitable for therapy of blood coagulation disorders.
Typical dextran sulfate and heparin compounds have anticoagulant
effects in vivo. Furthermore, these agents would activate contact
activation factors (Factor XII, high molecular weight kininogen or
prekallikrein) in vivo, which could be dangerous. Localized contact
activation on platelets was suggested to be of physiologic
relevance (Smith S A and Morrissey J H, Thromb Haemost. 2008 Jul.
26. [Epub ahead of print]). Systemic contact activation might lead
to a systemic increase in the level of bradykinin which is
generated by the cleavage of HMWK by kallikrein-like enzymes.
Unregulated bradykinin release might increase vascular
permeability, vascular leakage and possibly edema formation. Such a
clinical phenotype is known from the disease hereditary angioedema
which is characterised by a functional deficiency in the FXIIa
inhibitor C1-Inhibitor.
[0004] There is a need for non-protein therapeutics for treating
bleeding disorders, which are safe, convenient and effective.
[0005] The listing or discussion of a prior-published document in
this specification should not be taken as an acknowledgement that
the document is part of the state of the art or is common general
knowledge.
SUMMARY OF THE INVENTION
[0006] A first aspect of the invention provides a method of factor
XI-dependent blood coagulation enhancement in a subject in need of
enhanced blood coagulation comprising administering a
therapeutically effective amount of a composition comprising a
non-anticoagulant sulfated polysaccharide (NASP) to the subject,
wherein the NASP enhances blood coagulation in a factor
XI-dependent manner.
[0007] A second aspect of the invention provides a method of factor
XI-dependent blood coagulation enhancement in a subject in need of
enhanced blood coagulation comprising: [0008] (i) selecting a
subject that is not deficient for factor XI; and [0009] (ii)
administering a therapeutically effective amount of a composition
comprising a non-anticoagulant sulfated polysaccharide (NASP) to
the subject, wherein the NASP enhances blood coagulation in a
factor XI-dependent manner.
[0010] A third aspect of the invention provides a method of
identifying a non-anticoagulant sulfated polysaccharide (NASP)
which is capable of enhancing blood coagulation in dependence on
FXI, the method comprising: [0011] a) combining a blood or plasma
sample comprising activation competent FXI with a composition
comprising a sulfated polysaccharide and measuring the clotting or
thrombin generation parameters of the blood or plasma sample;
[0012] b) combining a corresponding blood or plasma sample
deficient in activation competent FXI with a composition comprising
the sulfated polysaccharide and measuring the clotting or thrombin
generation parameters of the blood or plasma sample; and [0013] c)
comparing the clotting or thrombin generation parameters of the
blood or plasma samples as determined in steps (a) and (b) with
each other, wherein a decrease in the clotting time of the blood
sample or an increase in peak thrombin or decrease in peak time of
the plasma sample comprising activation competent FXI compared to
the clotting time of the blood sample or peak thrombin or peak time
of the plasma sample deficient in activation competent FXI is
indicative of a NASP which is capable of enhancing blood
coagulation in dependence on FXI.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0014] According to a first aspect of the invention, blood
coagulation is enhanced in a factor XI-dependent manner.
[0015] Coagulation factor XI is a member of the intrinsic (contact
activation) pathway. Human factor XI is located on chromosome 4,
187.42-187.45 Mb. The accession number in the Swissprot database is
P03951. Although synthesized as a single polypeptide chain, FXI
circulates as a homo-dimer. Each chain has a relative molecular
mass of approximately 80000 g/mol. Typical plasma concentrations of
factor XI are 5 mg/l, corresponding to a plasma concentration (of
factor XI dimers) of approximately 30 nM. In its activated form,
factor XIa activates factor IX by selectively cleaving arg-ala and
arg-val peptide bonds.
[0016] Enhancement of blood coagulation by a chemical agent can be
determined experimentally using techniques that are known in the
art. In vitro tests are preferred. Suitable techniques include
rotation thromboelastography with whole blood preparations as
described in Example 1, and calibrated automated thrombography with
plasma preparations as described in Example 2. Typically, normal
blood or plasma may be used in such experiments. By "normal" is
meant that the blood is from a person or pooled from several
persons not having a coagulation disorder. In rotation
thromboelastography, enhancement of blood coagulation can be
inferred from a reduction in the clotting time (CT) and/or clot
formation time (CFT) in the presence of an agent compared with the
same parameter in the absence of the agent in normal blood. The CT
or CFT may be reduced by at least 5%, at least 10%, preferably at
least 50%. In calibrated automated thrombography, enhancement of
blood coagulation can be inferred from a reduction in peak time
and/or an increase in peak thrombin in the presence of an agent
than in the absence of the agent in normal plasma. Thrombin
generation time or peak time is the time interval from the start of
thrombin generation, to the time of the thrombin peak maximum. In
the assay described in Example 2, the start of thrombin generation
is the addition of the fluorogenic substrate-calcium mix to the
other components in the assay. Thrombin peak maximum, also referred
to as Peak IIa or Peak time is the maximal thrombin concentration
generated during the assay. Peak time may be reduced by at least 1
min, at least 2 minutes, preferably at least 5 minutes, more
preferably at least 10 minutes. Peak thrombin may be increased by
at least 5%, at least 10%, preferably at least 20%, more
preferably, at least 50%, 100%, 200% or 300%. The skilled person
will appreciate that different concentrations of any given agent
may need to be tested in order to identify an effect on blood
coagulation in the above assays. Typically, concentrations to test
are 0.1-500 .mu.g/mL, and generally from 1 to 50 .mu.g/mL.
[0017] The ability of NASPs to promote clotting and reduce bleeding
may also be readily determined using other in vitro clotting assays
(e.g., dPT and aPTT assays) or in vivo bleeding models (e.g. tail
snip, transverse cut, whole blood clotting time, or cuticle
bleeding time determination in hemophilic mice or dogs). See, for
example, PDR Staff. Physicians' Desk Reference. 2004, Anderson et
al. (1976) Thromb. Res. 9:575-580; Nordfang et al. (1991) Thromb.
Haemost. 66:464-467; Welsch et al. (1991) Thrombosis Research
64:213-222; Broze et al. (2001) Thromb Haemost 85:747-748; Scallan
et al. (2003) Blood. 102:2031-2037; Pijnappels et al. (1986)
Thromb. Haemost. 55:70-73; and Giles et al. (1982) Blood
60:727-730.
[0018] When an agent that enhances blood coagulation is identified,
its dependency on FXI can be determined by techniques such as
rotation thromboelastography and calibrated automated
thrombography, as described above. The assay is performed in normal
blood or plasma and also in blood or plasma lacking
activation-competent FXI. When the enhancement of coagulation
parameters is greater in the presence than in the absence of
activation-competent FXI, the mechanism of action of the agent on
coagulation is dependent on FXI. This is so even if there is a
FXI-independent component to the activity. By "activation-competent
FXI" is meant FXI that is capable of being activated to FXIa.
"Activation-competent FXI" may also be referred to as coagulation
competent FXI or FXI:c. It may be determined by an aPTT based
activity assay, such as the assay described in Ingram G I, Knights
S F, Arocha-Pi{umlaut over (n)}ango C L, Shepperd J P,
Perez-Requejo J L, Mills D K. Simple screening tests for the
diagnosis of isolated clotting factor defects. With special
reference to `contact factor` defects. J Clin Pathol. 1975 July;
28(7):524-30. Blood or plasma from a person genetically deficient
in FXI, i.e. a person having hemophilia C, lacks
activation-competent FXI, or has a lower concentration of
activation-competent FXI than blood or plasma from a healthy
person. A healthy person has on average 100 IU/dL of FXI:c in their
plasma. Severe FXI deficiency is defined as a plasma FXI activity
of less than 20 IU/dL, and partial FXI deficiency as 20-70 IU/dL
(Gomez and Bolton-Maggs (2008) Hemophilia e-publication ahead of
print: doi:10.1111/j.1365-2516.2008.01667.x). Deficiencies in FXI
may also arise as a consequence of the development of inhibitors,
particularly antibody inhibitors, of FXI (Salomon O et al (2006)
Sem Hematology 43, S10-12; Bern M M et al (2005) Haemophilia, 11,
20-25.) Normal blood or plasma, which contains activation-competent
FXI, can be made deficient in activation-competent FXI by
incubation with an inhibitor of FXI activation. Typically, an
antibody is used, such as a polyclonal antibody or plasma
containing a polyclonal antibody. A suitable affinity purified
polyclonal antibody is "GAFXI-AP" from Enzyme Research Laboratories
(South Bend Ind., USA).
[0019] According to the first aspect of the invention, the
composition is administered to a subject in need of enhanced blood
coagulation. A need for enhanced blood coagulation may arise due to
any bleeding disorder.
[0020] By "subject" is included any member of the subphylum
chordata, including, without limitation, humans and other primates,
including non-human primates such as chimpanzees and other apes and
monkey species; farm animals such as cattle, sheep, pigs, goats and
horses; domestic mammals such as dogs and cats; laboratory animals
including rodents such as mice, rats and guinea pigs; birds,
including domestic, wild and game birds such as chickens, turkeys
and other gallinaceous birds, ducks, geese, and the like. The term
does not denote a particular age. Thus, both adult and newborn
individuals are intended to be covered. The invention described
herein is intended for use in any of the above vertebrate species.
The term "patient" refers to a living organism suffering from or
prone to a condition that can be prevented or treated by
administration of a NASP of the invention, and includes both humans
and animals.
[0021] At least one therapeutically effective cycle of treatment
with a NASP will be administered to a subject. By "therapeutically
effective cycle of treatment" is intended a cycle of treatment that
when administered, brings about a positive therapeutic response
with respect to treatment of an individual for a bleeding disorder.
Of particular interest is a cycle of treatment with a NASP that
improves hemostasis. A "positive therapeutic response" is one in
which the individual undergoing treatment according to the
invention exhibits an improvement in one or more symptoms of a
bleeding disorder, including such improvements as shortened blood
clotting times and reduced bleeding and/or reduced need for factor
replacement therapy.
[0022] The composition comprising the NASP is typically, although
not necessarily, administered orally, via injection
(subcutaneously, intravenously or intramuscularly), by infusion, or
locally. The pharmaceutical preparation can be in the form of a
liquid solution or suspension immediately prior to administration,
but may also take another form such as a syrup, cream, ointment,
tablet, capsule, powder, gel, matrix, suppository, or the like.
Additional modes of administration are also contemplated, such as
pulmonary, rectal, transdermal, transmucosal, intrathecal,
pericardial, intra-arterial, intracerebral, intraocular,
intraperitoneal, and so forth. The respective pharmaceutical
compositions comprising NASPs and other agents may be administered
using the same or different routes of administration in accordance
with any medically acceptable method known in the art.
[0023] In a particular embodiment, a composition comprising a NASP
is used for localized delivery of a NASP, for example for the
treatment of bleeding as a result of a lesion, injury, or surgery.
The compositions are also suitable for local treatment. For
example, a NASP may be administered by injection at the site of
bleeding or in the form of a solid, liquid, or ointment, preferably
via an adhesive tape or a wound cover. Suppositories, capsules, in
particular gastric-juice-resistant capsules, drops or sprays may
also be used. The particular preparation and appropriate method of
administration are chosen to target the site of bleeding.
[0024] The compositions comprising a NASP may be administered
prophylactically, for example before planned surgery. Such
prophylactic uses will be of particular value for subjects with
known pre-existing blood coagulation disorders. In another
embodiment of the invention, the pharmaceutical composition
comprising a NASP is in a sustained-release formulation, or a
formulation that is administered using a sustained-release device.
Such devices are well known in the art, and include, for example,
transdermal patches, and miniature implantable pumps that can
provide for drug delivery over time in a continuous, steady-state
fashion at a variety of doses to achieve a sustained-release effect
with a non-sustained-release pharmaceutical composition.
[0025] In one aspect, NASPs may be used in the methods of the
invention for improving hemostasis in treating bleeding disorders,
particularly those associated with deficiencies of coagulation
factors or for reversing the effects of anticoagulants in a
subject. NASPs may be administered to a subject to treat bleeding
disorders, including congenital coagulation disorders, acquired
coagulation disorders, and hemorrhagic conditions induced by
trauma. Examples of bleeding disorders that may be treated with
NASPs include, but are not limited to, hemophilia A, hemophilia B,
von Willebrand's disease, idiopathic thrombocytopenia, a deficiency
of one or more contact factors, such as Factor XI, Factor XII,
prekallikrein, and high molecular weight kininogen (HMWK), a
deficiency of one or more factors associated with clinically
significant bleeding, such as Factor V, Factor VII, Factor VIII,
Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia),
and von Willebrand's factor, a vitamin K deficiency, a disorder of
fibrinogen, including afibrinogenemia, hypofibrinogenemia, and
dysfibrinogenemia, an alpha.sub.2-antiplasmin deficiency, and
excessive bleeding such as caused by liver disease, renal disease,
thrombocytopenia, platelet dysfunction, hematomas, internal
hemorrhage, hemarthroses, surgery, trauma, hypothermia,
menstruation, and pregnancy. In certain embodiments, NASPs are used
to treat congenital coagulation disorders including hemophilia A,
hemophilia B, and von Willebrand's disease. In other embodiments,
NASPs are used to treat acquired coagulation disorders, including
deficiencies of factor VIII, von Willebrand factor, factor IX,
factor V, factor XI, factor XII and factor XIII, particularly
disorders caused by inhibitors or autoimmunity against blood
coagulation factors, or haemostatic disorders caused by a disease
or condition that results in reduced synthesis of coagulation
factors.
[0026] The needs of the patient will depend on the particular
bleeding disorder being treated. For example, a NASP may be
administered to treat a chronic condition (e.g., a congenital or
acquired coagulation factor deficiency) in multiple doses over an
extended period. Alternatively, a NASP may be administered to treat
an acute condition (e.g., bleeding caused by surgery or trauma, or
factor inhibitor/autoimmune episodes in subjects receiving
coagulation replacement therapy) in single or multiple doses for a
relatively short period, for example one to two weeks. In addition,
NASP therapy may be used in combination with other hemostatic
agents, blood factors, and medications. For example, the subject
may be administered a therapeutically effective amount of one or
more factors selected from the group consisting of factor XI,
factor XII, prekallikrein, high molecular weight kininogen (HMWK),
factor V, factor Va, factor VII, factor VIII, factor VIIIa, factor
IX, factor X, factor XIII, factor II, factor VIIa, and von
Willebrand's factor. Treatment may further comprise administering a
procoagulant, such as an activator of the intrinsic coagulation
pathway, including factor Xa, factor IXa, factor XIa, factor XIIa,
and kallikrein; or an activator of the extrinsic coagulation
pathway, including tissue factor, factor VIIa, thrombin, and factor
Xa. In addition, transfusion of blood products may be necessary to
replace blood loss in subjects experiencing excessive bleeding, and
in cases of injury, surgical repair may be appropriate to stop
bleeding. Depending on the bleeding disorder, it may not be
appropriate to administer prekallikrein, high molecular weight
kininogen (HMWK) and/or FXII. Typically, where the NASP is
administered in combination with a clotting factor, the dose and/or
frequency of administration is reduced compared to the dose and/or
frequency that would be appropriate if the clotting factor was to
be administered without the NASP. Suitably, the dose of clotting
factor is at least 1%, and up to 5, 10, 25, 50, 75% or 100% of the
appropriate dose that would be used if the clotting factor were
administered without the NASP.
[0027] According to the first aspect of the invention, the
composition administered to the subject comprises a
non-anticoagulant sulfated polysaccharide (NASP). "NASP" as used
herein refers to a sulfated polysaccharide that exhibits
anticoagulant activity in a dilute prothrombin time (dPT) or
activated partial thromboplastin time (aPTT) clotting assay that is
no more than one-third, and preferably less than one-tenth, the
molar anticoagulant (statistically significant increase in clotting
time) activity of unfractionated heparin (MW range 8,000 to 30,000;
mean 18,000 Daltons). NASPs may be purified and/or modified from
natural sources (e.g. brown algae, tree bark, animal tissue) or may
be synthesized de novo and may range in molecular weight from 100
Daltons to 1,000,000 Daltons. NASPs may be used in the methods of
the invention for improving hemostasis in treating bleeding
disorders, particularly those associated with deficiencies of
coagulation factors or for reversing the effects of anticoagulants.
NASPs are "non-anticoagulant," in that they do not significantly
increase clotting times over the range of concentrations studied.
Such compounds can be used in the methods of the present invention
provided that any anticoagulant activity that they may exhibit only
appears at concentrations significantly above the concentration at
which they exhibit procoagulant activity. The ratio of the
concentration at which undesired anticoagulant properties occur to
the concentration at which desired procoagulant activities occur is
referred to as the therapeutic index for the NASP in question. The
therapeutic index for NASPs of the present invention may be 5, 10,
30, 100, 300, 1000 or more.
[0028] As a class, sulfated polysaccharides are characterized by a
plethora of biological activities with often favorable tolerability
profiles in animals and humans. These polyanionic molecules are
often derived from plant and animal tissues and encompass a broad
range of subclasses including heparins, glycosaminoglycans,
fucoidans, carrageenans, pentosan polysulfates, and dermatan or
dextran sulfates (Toida et al. (2003) Trends in Glycoscience and
Glycotechnology 15:29-46). Lower molecular weight, less
heterogeneous, and chemically synthesized sulfated polysaccharides
have been reported and have reached various stages of drug
development (Sinay (1999) Nature 398:377-378; Orgueira et al.
(2003) Chemistry 9:140-169; Williams et al. (1998) Gen. Pharmacol.
30:337-341).
[0029] Sulfated polysaccharides with potential NASP activity
include, but are not limited to, glycosaminoglycans (GAGs),
heparin-like molecules including N-acetyl heparin (Sigma-Aldrich,
St. Louis, Mo.) and N-desulfated heparin (Sigma-Aldrich),
sulfatoids, polysulfated oligosaccharides (Karst et al. (2003)
Curr. Med. Chem. 10:1993-2031; Kuszmann et al. (2004) Pharmazie.
59:344-348), chondroitin sulfates (Sigma-Aldrich), dermatan sulfate
(Celsus Laboratories Cincinnati, Ohio), fucoidan (Sigma-Aldrich),
pentosan polysulfate (PPS) (Ortho-McNeil Pharmaceuticals, Raritan,
N.J.), fucopyranon sulfates (Katzman et al. (1973) J. Biol. Chem.
248:50-55), and novel sulfatoids such as GM1474 (Williams et al.
(1998) General Pharmacology 30:337) and SR 80258A (Burg et al.
(1997) Laboratory Investigation 76:505), and novel heparinoids, and
their analogs. NASPs may be purified and/or modified from natural
sources (e.g. brown algae, tree bark, animal tissue) or may be
synthesized de novo and may range in molecular weight from 100
Daltons to 1,000,000 Daltons. Additional compounds with potential
NASP activity include periodate-oxidized heparin (POH) (Neoparin,
Inc., San Leandro, Calif.), chemically sulfated laminarin (CSL)
(Sigma-Aldrich), chemically sulfated alginic acid (CSAA)
(Sigma-Aldrich), chemically sulfated pectin (CSP) (Sigma-Aldrich),
dextran sulfate (DXS) (Sigma-Aldrich), heparin-derived
oligosaccharides (HDO) (Neoparin, Inc., San Leandro, Calif.).
[0030] In principle, any free hydroxyl group on a monosaccharide
component of a glycoconjugate can be modified by sulfation to
produce a sulfated glycoconjugate for potential use as a NASP in
the practice of the invention. For example, such sulfated
glycoconjugates may include without limitation sulfated
mucopolysaccharides (D-glucosamine and D-glucuronic acid residues),
curdlan (carboxymethyl ether, hydrogen sulfate, carboxymethylated
curdlan) (Sigma-Aldrich), sulfated schizophyllan (Itoh et al.
(1990) Int. J. Immunopharmacol. 12:225-223; Hirata et al. (1994)
Pharm. Bull. 17:739-741), sulfated glycosaminoglycans, sulfated
polysaccharide-peptidoglycan complex, sulfated alkyl
malto-oligosaccharide (Katsuraya et al. (1994) Carbohydr Res.
260:51-61), amylopectin sulfate, N-acetyl-heparin (NAH)
(Sigma-Aldrich), N-acetyl-de-O-sulfated-heparin (NA-de-o-SH)
(Sigma-Aldrich), de-N-sulfated-heparin (De-NSH) (Sigma-Aldrich),
and De-N-sulfated-acetylated-heparin (De-NSAH) (Sigma-Aldrich).
[0031] The term "polysaccharide", as used herein, refers to a
polymer comprising a plurality (i.e., two or more) of covalently
linked saccharide residues. Linkages may be natural or unnatural.
Natural linkages include, for example, glycosidic bonds, while
unnatural linkages may include, for example, ester, amide, or oxime
linking moieties. Polysaccharides may have any of a wide range of
average molecular weight (MW) values, but generally are of at least
about 100 Daltons. For example, the polysaccharides can have
molecular weights of at least about 500, 1000, 2000, 4000, 6000,
8000, 10,000, 20,000, 30,000, 50,000, 100,000, 500,000 Daltons or
even higher. Polysaccharides may have straight chain or branched
structures. Polysaccharides may include fragments of
polysaccharides generated by degradation (e.g., hydrolysis) of
larger polysaccharides. Degradation can be achieved by any of a
variety of means known to those skilled in the art including
treatment of polysaccharides with acid, base, heat, or enzymes to
yield degraded polysaccharides. Polysaccharides may be chemically
altered and may have modifications, including but not limited to,
sulfation, polysulfation, esterification, and methylation.
[0032] A NASP may be a derivative or fragment of a
polysaccharide.
[0033] By "derivative" is intended any suitable modification of the
reference molecule of interest or of an analog thereof, such as
sulfation, acetylation, glycosylation, phosphorylation, polymer
conjugation (such as with polyethylene glycol), or other addition
of foreign moieties, so long as the desired biological activity
(e.g., clotting activity) of the reference molecule is retained.
For example, polysaccharides may be derivatized with one or more
organic or inorganic groups. Examples include polysaccharides
substituted in at least one hydroxyl group with another moiety
(e.g., a sulfate, carboxyl, phosphate, amino, nitrile, halo, silyl,
amido, acyl, aliphatic, aromatic, or a saccharide group), or where
a ring oxygen has been replaced by sulfur, nitrogen, a methylene
group, etc. Polysaccharides may be chemically altered, for example,
to improve procoagulant function. Such modifications may include,
but are not limited to, sulfation, polysulfation, esterification,
and methylation. Methods for making analogs and derivatives are
generally available in the art.
[0034] By "fragment" is intended a molecule consisting of only a
part of the intact full-length sequence and structure. A fragment
of a polysaccharide may be generated by degradation (e.g.,
hydrolysis) of a larger polysaccharide. Active fragments of a
polysaccharide will generally include at least about 2-20
saccharide units of the full-length polysaccharide, preferably at
least about 5-10 saccharide units of the full-length molecule, or
any integer between 2 saccharide units and the full-length
molecule, provided that the fragment in question retains biological
activity, such as clotting activity.
[0035] Preferably, the NASP is not an activator of the contact
pathway. By this, we mean it does not contribute to activation of
Factor XII. Preferably, the NASP does not activate HMWK or
prekallikrein.
[0036] Preferably, the NASP is selected from the group consisting
of pentosan polysulfate (PPS), fucoidan, N-acetyl-heparin (NAH),
N-acetyl-de-O-sulfated-heparin (NA-de-o-SH), de-N-sulfated-heparin
(De-NSH), de-N-sulfated-acetylated-heparin (De-NSAH),
periodate-oxidized heparin (POH), chemically sulfated laminarin
(CSL), chemically sulfated alginic acid (CSAA), chemically sulfated
pectin (CSP), dextran sulfate (DXS) and heparin-derived
oligosaccharides (HDO).
[0037] More preferably, the NASP is PPS or fucoidan. Fucoidan is a
polysaccharide composed largely of sulfated esters of fucose, with
a variable degree of branching. Linkages may be predominantly
.alpha.(1.fwdarw.2) or .alpha.(1.fwdarw.3). .alpha.(1.fwdarw.4)
linkages may also be present. The fucose esters are predominantly
sulfated at position 4 and/or 2 and/or 3. Monosulfated fucoses
dominate, although desulfated fucose may also be present. In
addition to sulfated fucose esters, fucoidan may also contain
non-sulfated fucose, D-xylose, D-galactose, uronic acid, glucoronic
acid or combinations of more than one of these. F-fucoidan is
>95% composed of sulfated esters of fucose, whereas U-fucoidan
is approximately 20% glucuronic acid.
[0038] Preferably, the NASP enhances the activation of factor XI.
In this embodiment, the first aspect of the invention provides a
method of enhancing the activation of factor XI in a subject in
need of enhanced blood coagulation. By "enhancing the activation of
factor XI" we mean that factor XI is activated more quickly and or
to a greater extent in the presence than the absence of an
effective concentration of the NASP. Without wishing to be bound by
theory, NASPs may activate factor XI directly, indirectly, or by a
combination of direct and indirect means. Methods such as rotation
thromboelastography with whole blood preparations and calibrated
automated thrombography with plasma preparations, or other methods
as described above which are useful to determined enhancement of
blood coagulation, and factor XI-dependency of such enhancement,
may be used to identify activation of factor XI. Typically, factor
XI-dependent enhancement of blood coagulation is established for
the NASP as described above. Then, blood or plasma deficient in
activation-competent factor XI is supplemented with activated
factor XI. If the NASP fails to enhance blood coagulation in the
supplemented blood or plasma, compared to supplemented blood or
plasma lacking the NASP, yet exhibits a factor XI-dependent
enhancement of blood coagulation, it can be inferred that the NASP
acts by enhancing the activation of factor XI. Factor XIa may be
used at a concentration of about 20 to 200 pM, suitably 60 pM.
[0039] Preferably according to the method of the first aspect, the
NASP is administered at a dosage of about 0.005 mg/kg to about 200
mg/kg, typically from about 0.01 mg/kg to about 200 mg/kg.
Generally, a therapeutically effective amount will range from about
0.01 mg/kg to 200 mg/kg of a NASP daily, more preferably from about
0.01 mg/kg to 20 mg/kg daily, even more preferably from about 0.02
mg/kg to 2 mg/kg daily. Preferably, such doses are in the range of
0.01-50 mg/kg four times a day (QID), 0.01-10 mg/kg QID, 0.01-2
mg/kg QID, 0.01-0.2 mg/kg QID, 0.01-50 mg/kg three times a day
(TID), 0.01-10 mg/kg TID, 0.01-2 mg/kg TID, 0.01-0.2 mg/kg TID,
0.01-100 mg/kg twice daily (BID), 0.01-10 mg/kg BID, 0.01-2 mg/kg
BID, or 0.01-0.2 mg/kg BID. The amount of compound administered
will depend on the potency of the specific NASP and the magnitude
or procoagulant effect desired and the route of administration. The
specific dosing schedule will be known by those of ordinary skill
in the art or can be determined experimentally using routine
methods. Suitable daily or twice daily doses are 0.005 mg/kg to 0.5
mg/kg by intravenous administration, 0.02 to 2 mg/kg by
subcutaneous administration, or 1 to 100 mg/kg by per oral
administration. Exemplary dosing schedules include, without
limitation, administration five times a day, four times a day,
three times a day, twice daily, once daily, three times weekly,
twice weekly, once weekly, twice monthly, once monthly, and any
combination thereof. Preferred compositions are those requiring
dosing no more than once a day.
[0040] Suitably, the subject has a bleeding disorder selected from
the group consisting of a congenital coagulation disorder caused by
a blood factor deficiency, a chronic or acute bleeding disorder,
and an acquired coagulation disorder. Typically, the blood factor
deficiency is of one or more factors selected from the group
consisting of factor V, factor VII, factor VIII, factor IX, factor
X, factor XI, factor XII, factor XIII, and von Willebrand
factor.
[0041] Alternatively, the cause of the need for enhanced blood
coagulation is prior administration of an anticoagulant or surgery
or other invasive procedure. Where there has been prior
administration of an anticoagulant, the method is for reversing the
effects of the anticoagulant in the subject.
[0042] The method of the first aspect of the invention may further
comprise administering an agent selected from the group consisting
of a procoagulant, an activator of the intrinsic coagulation
pathway, an activator of the extrinsic coagulation pathway, and a
second NASP. A NASP (again, preferably provided as part of a
pharmaceutical preparation) can be administered alone or in
combination with other NASPs or therapeutic agents, such as
hemostatic agents, blood factors, or other medications used to
treat a particular condition or disease according to a variety of
dosing schedules depending on the judgment of the clinician, needs
of the patient, and so forth.
[0043] A NASP can be administered prior to, concurrent with, or
subsequent to other agents. If provided at the same time as other
agents, the NASP can be provided in the same or in a different
composition. Thus, NASPs and other agents can be presented to the
individual by way of concurrent therapy. By "concurrent therapy" is
intended administration to a subject such that the therapeutic
effect of the combination of the substances is caused in the
subject undergoing therapy. For example, concurrent therapy may be
achieved by administering a dose of a pharmaceutical composition
comprising a NASP and a dose of a pharmaceutical composition
comprising at least one other agent, such as a hemostatic agent or
coagulation factor (e.g. FVIII or FIX), which in combination
comprise a therapeutically effective dose, according to a
particular dosing regimen. Similarly, one or more NASPs and
therapeutic agents can be administered in at least one therapeutic
dose. Administration of the separate pharmaceutical compositions
can be performed simultaneously or at different times (i.e.,
sequentially, in either order, on the same day, or on different
days), so long as the therapeutic effect of the combination of
these substances is caused in the subject undergoing therapy.
[0044] A "procoagulant" as used herein refers to any factor or
reagent capable of initiating or accelerating clot formation. A
procoagulant includes any activator of the intrinsic or extrinsic
coagulation pathways, such as a clotting factor selected from the
group consisting of factor Xa, factor IXa, factor XIa, factor XIIa,
kallikrein, tissue factor, factor VIIa, and thrombin. Other
reagents that promote clotting include prekallikrein, APTT
initiator (i.e., a reagent containing a phospholipid and a contact
activator), Russell's viper venom (RVV time), and thromboplastin
(for dPT). Contact activators that can be used in the methods of
the invention as procoagulant reagents include micronized silica
particles, ellagic acid, sulfatides, kaolin or the like known to
those of skill in the art. Procoagulants may be from a crude
natural extract, a blood or plasma sample, isolated and
substantially purified, synthetic, or recombinant. Procoagulants
may include naturally occurring clotting factors or fragments,
variants, analogs or muteins thereof that retain biological
activity (i.e., promote clotting). Optimal concentrations of the
procoagulant can be determined by those of skill in the art.
Depending on the bleeding disorder, it may not be appropriate to
administer contact activators, such as prekallikrein, kallikrein,
high molecular weight kininogen (HMWK) and/or FXII.
[0045] The terms "variant", "analog" and "mutein" refer to
biologically active derivatives of the reference molecule that
retain desired activity, such as clotting activity, in the
treatment of a bleeding disorder described herein. In general, the
terms "variant" and "analog" in reference to a polypeptide (e.g.,
clotting factor) refer to compounds having a native polypeptide
sequence and structure with one or more amino acid additions,
substitutions (generally conservative in nature) and/or deletions,
relative to the native molecule, so long as the modifications do
not destroy biological activity and which are "substantially
homologous" to the reference molecule as defined below. In general,
the amino acid sequences of such analogs will have a high degree of
sequence homology to the reference sequence, e.g., amino acid
sequence homology of more than 50%, generally more than 60%-70%,
even more particularly 80%-85% or more, such as at least 90%-95% or
more, when the two sequences are aligned. Often, the analogs will
include the same number of amino acids but will include
substitutions, as explained herein. The term "mutein" further
includes polypeptides having one or more amino acid-like molecules
including but not limited to compounds comprising only amino and/or
imino molecules, polypeptides containing one or more analogs of an
amino acid (including, for example, unnatural amino acids, etc.),
polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring (e.g., synthetic), cyclized, branched
molecules and the like. The term also includes molecules comprising
one or more N-substituted glycine residues (a "peptoid") and other
synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos.
5,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chem. Biol.
(2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA
(1992) 89:9367-9371 for descriptions of peptoids). Preferably, the
analog or mutein has at least the same clotting activity as the
native molecule. Methods for making polypeptide analogs and muteins
are known in the art and are described further below.
[0046] As explained above, analogs generally include substitutions
that are conservative in nature, i.e., those substitutions that
take place within a family of amino acids that are related in their
side chains. Specifically, amino acids are generally divided into
four families: (1) acidic--aspartate and glutamate; (2)
basic--lysine, arginine, histidine; (3) non-polar--alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar--glycine, asparagine,
glutamine, cysteine, serine threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino
acids. For example, it is reasonably predictable that an isolated
replacement of leucine with isoleucine or valine, an aspartate with
a glutamate, a threonine with a serine, or a similar conservative
replacement of an amino acid with a structurally related amino
acid, will not have a major effect on the biological activity. For
example, the polypeptide of interest may include up to about 5-10
conservative or non-conservative amino acid substitutions, or even
up to about 15-25 conservative or non-conservative amino acid
substitutions, or any integer between 5-25, so long as the desired
function of the molecule remains intact. One of skill in the art
may readily determine regions of the molecule of interest that can
tolerate change by reference to Hopp/Woods and Kyte-Doolittle
plots, well known in the art.
[0047] By "fragment" is intended a molecule consisting of only a
part of the intact full-length sequence and structure. A fragment
of a polypeptide can include a C-terminal deletion, an N-terminal
deletion, and/or an internal deletion of the native polypeptide.
Active fragments of a particular protein will generally include at
least about 5-10 contiguous amino acid residues of the full-length
molecule, preferably at least about 15-25 contiguous amino acid
residues of the full-length molecule, and most preferably at least
about 20-50 or more contiguous amino acid residues of the
full-length molecule, or any integer between 5 amino acids and the
full-length sequence, provided that the fragment in question
retains biological activity, such as clotting activity, as defined
herein.
[0048] "Homology" refers to the percent identity between two
polynucleotide or two polypeptide moieties. Two nucleic acid, or
two polypeptide sequences are "substantially homologous" to each
other when the sequences exhibit at least about 50%, preferably at
least about 75%, more preferably at least about 80%-85%, preferably
at least about 90%, and most preferably at least about 95%-98%
sequence identity over a defined length of the molecules. As used
herein, substantially homologous also refers to sequences showing
complete identity to the specified sequence.
[0049] In general, "identity" refers to an exact
nucleotide-to-nucleotide or amino acid-to-amino acid correspondence
of two polynucleotides or polypeptide sequences, respectively.
Percent identity can be determined by a direct comparison of the
sequence information between two molecules (the reference sequence
and a sequence with unknown % identity to the reference sequence)
by aligning the sequences, counting the exact number of matches
between the two aligned sequences, dividing by the length of the
reference sequence, and multiplying the result by 100. Readily
available computer programs can be used to aid in the analysis,
such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and
Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National
Biomedical Research Foundation, Washington, D.C., which adapts the
local homology algorithm of Smith and Waterman Advances in Appl.
Math. 2:482-489, 1981 for peptide analysis. Programs for
determining nucleotide sequence identity are available in the
Wisconsin Sequence Analysis Package, Version 8 (available from
Genetics Computer Group, Madison, Wis.) for example, the BESTFIT,
FASTA and GAP programs, which also rely on the Smith and Waterman
algorithm. These programs are readily utilized with the default
parameters recommended by the manufacturer and described in the
Wisconsin Sequence Analysis Package referred to above. For example,
percent identity of a particular nucleotide sequence to a reference
sequence can be determined using the homology algorithm of Smith
and Waterman with a default scoring table and a gap penalty of six
nucleotide positions.
[0050] Another method of establishing percent identity in the
context of the present invention is to use the MPSRCH package of
programs copyrighted by the University of Edinburgh, developed by
John F. Collins and Shane S. Sturrok, and distributed by
IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of
packages the Smith-Waterman algorithm can be employed where default
parameters are used for the scoring table (for example, gap open
penalty of 12, gap extension penalty of one, and a gap of six).
From the data generated the "Match" value reflects "sequence
identity." Other suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art, for example, another alignment program is BLAST, used with
default parameters. For example, BLASTN and BLASTP can be used
using the following default parameters: genetic code=standard;
filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;
Descriptions=50 sequences; sort by .dbd.HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+Swiss protein+Spupdate+PIR. Details of these programs
are readily available.
[0051] "Recombinant" as used herein to describe a nucleic acid
molecule means a polynucleotide of genomic, cDNA, viral,
semisynthetic, or synthetic origin which, by virtue of its origin
or manipulation is not associated with all or a portion of the
polynucleotide with which it is associated in nature. The term
"recombinant" as used with respect to a protein or polypeptide
means a polypeptide produced by expression of a recombinant
polynucleotide. In general, the gene of interest is cloned and then
expressed in transformed organisms. The host organism expresses the
foreign gene to produce the protein under expression
conditions.
[0052] Preferably, the activator of the intrinsic coagulation
pathway is factor Xa, factor IXa or factor XIa. In certain
circumstances it may also be factor XIIa or kallikrein. Preferably,
the activator of the extrinsic coagulation pathway is tissue
factor, factor VIIa, thrombin, and factor Xa.
[0053] The method of the first aspect of the invention may further
comprise administering one or more factors selected from the group
consisting of factor XI, factor XII, prekallikrein, high molecular
weight kininogen (HMWK), factor V, factor Va, factor VII, factor
VIII, factor VIIIa, factor IX, factor X, factor XIII, factor II,
factor VIIa, and von Willebrand factor.
[0054] Factor XI may be provided as fresh frozen plasma (FFP) or as
a factor XI concentrate. Suitable factor XI concentrates are
Hemoleven.RTM. (Laboratoire francais du Fractionnement et des
Biotechnologies, Les Ulis, France) and factor XI concentrate (Bio
Products Laboratory, Elstree, Hertfordshire, United Kingdom).
Recombinant Factor XI is also envisaged. FXII, prekallikrein, HMWK
or Factor V may be provided as fresh frozen plasma (FFP). Factor
VII may be provided as a concentrate, suitably Factor VII
concentrate from Baxter BioScience or Bio Products Laboratory.
FVIII Immunate.RTM. and Advate.RTM. FVIII are both recombinant
FVIII products available from Baxter BioScience (Vienna, Austria).
Bebulin VH.RTM. factor IX complex is available from Baxter
BioScience (Vienna, Austria). Factor X may be provided as fresh
frozen plasma or as a component in a prothrombin complex
concentrate. Factor XIII may be provided as fresh frozen plasma, or
as a FXIII concentrate, such as Fibrogammin.RTM. P (Centeon Pharma
GmbH, Marburg, Germany). Factor II may be provided as fresh frozen
plasma or as component in a prothrombin complex concentrate.
NovoSeven.RTM. recombinant activated FVII is available from Novo
Nordisk A/S (Denmark). Von Willebrand factor (vWF) is available as
Humate-P.RTM. (CSL BEHRING, King of Prussia, Pa.). Recombinant vWF
can be obtained as in Schlokat, et al. (1995), "Large Scale
Production of Recombinant von Willebrand Factor", Thrombosis and
Haemostasis 78, 1160 or U.S. Pat. No. 6,114,146 (Baxter AG). FEIBA
VH Immuno from Baxter BioScience (Vienna, Austria) is a
freeze-dried sterile human plasma fraction with Factor VIII
inhibitor bypassing activity. In vitro, FEIBA VH Immuno shortens
the activated partial thromboplastin time (APTT) of plasma
containing Factor VIII inhibitor. It contains Factors II, IX, and
X, mainly non-activated, and Factor VII mainly in the activated
form. The product contains approximately equal units of Factor VIII
inhibitor bypassing activity and Prothrombin Complex Factors.
Prothrombin complex concentrates (PCCs) may be used, for example to
increase factor X levels. PCC contains factors II, VII, IX, and X
and protein C. Infusion of fresh frozen plasma may be used to
provide coagulation factors which are deficient in the subject.
[0055] As noted above, where a clotting factor is administered with
a NASP, the dose of the clotting factor may be reduced compared to
the dose that would be suitable in the absence of the NASP.
Typically, rFVIII is administered at about 10 to 60 U/kg in
hemophilia A patients. When rFVIII is administered in combination
with a NASP, a dose of at least 0.1 or 0.6 U/kg, and up to 1, 2, 5,
7.5, 10, 12, 30, 45 or 60 U/kg may be suitable, for example a dose
of 0.1 to 0.6, 1 to 6, 2 to 12, 5 to 30, 7.5 to 45, or 10 to 60
U/kg. Typically, FEIBA is administered at about 50-100 U/kg in
hemophilia A inhibitor patients. When FEIBA is administered in
combination with a NASP, a dose of at least 0.5 or 1 U/kg, and up
2.5, 5, 10, 12.5, 25, 37.5, 50, 75 or 100 U/kg may be suitable, for
example a dose of 0.5 to 1, 2.5 to 5, 5 to 10, 12.5 to 25, 25 to
50, 37.5 to 75 or 50 to 100 U/kg. Similarly, rFVIIa is typically
administered at about 90 .mu.g/kg in hemophilia A inhibitor
patients. When rFVIIa is administered in combination with a NASP, a
dose of at least 0.9 .mu.g/kg and up to 4.5, 9, 22.5, 45, 67.5 or
90 .mu.g/kg may be suitable. A typical dose of Factor XI in Factor
XI replacement therapy, such as in treatment of hemophilia C, is 30
U/kg or less, and is usually provided in the form of a Factor XI
concentrate. When Factor XI is administered in combination with a
NASP, a dose of up to 0.3, 1.5, 3, 7.5, 15, 22.5 or 30 U/kg may be
suitable.
[0056] Preferably, when the method is for reversing the effects of
the anticoagulant in the subject, the subject has been treated with
an anticoagulant including, but not limited to, heparin, a coumarin
derivative, such as warfarin or dicumarol, TFPI, AT III, lupus
anticoagulant, nematode anticoagulant peptide (NAPc2), factor VIIa
inhibitors, active-site blocked factor VIIa (factor VIIai),
active-site blocked FIXa (factor IXai), factor IXa inhibitors, a
factor Xa inhibitor, including fondaparinux, idraparinux, DX-9065a,
and razaxaban (DPC906), active-site blocked FXa (factor Xai), an
inhibitor of factor Va or VIIIa, including activated protein C
(APC) and soluble thrombomodulin, a thrombin inhibitor, including
hirudin, bivalirudin, argatroban, or ximelagatran. In certain
embodiments, the anticoagulant in the subject may be an antibody or
antibody fragment that binds a clotting factor, including but not
limited to, an antibody or antibody fragment that binds to Factor
V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII,
Factor II, Factor XI, Factor XII, von Willebrand factor,
prekallikrein, or high molecular weight kininogen (HMWK). As an
alternative to an antibody or antibody fragment, the anticoagulant
may be a small drug-like molecule, peptide or aptamer which binds
to a coagulation protein and thereby inhibits its activation or its
interaction with another coagulation protein or cell surface.
[0057] Suitably, in the method of the first aspect of the
invention, the subject is deficient in factor XI, and the method
further comprises administering factor XI. By "deficient in factor
XI" is intended a subject having no more than 70% of the plasma
factor XI:c of a healthy vertebrate of the same species as the
subject. Where the subject is a human, they may have a partial
deficiency, defined as a plasma factor XI:c activity of 20-70
IU/dL, or a severe deficiency, defined as a plasma factor XI:c
activity of less than 20 IU/dL. Factor XI deficiency in humans is
referred to as hemophilia C. About 20-50% of individuals with
partial deficiency have excessive bleeding, but identifying these
persons in advance is difficult. Most individuals with severe
deficiency do not spontaneously bleed, but they are at risk of
bleeding after surgery. The conventional therapy for hemophilia C
is administration of fresh frozen plasma, factor XI concentrate or
antifibrinolytic agents like tranexamic acid and
.epsilon.-aminocaproic acid. Factor XI of recombinant origin is
also envisaged. Although the coagulation enhancing effect of a NASP
according to the present invention is dependent on factor XI, it is
believed that the small quantities of factor XI present in subjects
having a factor XI deficiency may be sufficient for administration
of a NASP to be effective. However, administration of a NASP and
factor XI will increase the effectiveness of the NASP in enhancing
blood coagulation in factor XI deficient subjects.
[0058] Suitably, in the method of the first aspect of the
invention, the subject is deficient in factor VIII, and the method
further comprises administering factor VIII or a procoagulant
bypassing agent. Suitable factor VIII products are FVIII
Immunate.RTM. and Advate.RTM. FVIII (Baxter BioScience, Vienna,
Austria). A suitable bypassing agent is FEIBA VH Immuno (Baxter
BioScience, Vienna, Austria). The inventors have found that the
coagulation enhancing effect of NASPs is additive with the effect
of exogenous FVIII in FVIII deficient plasma. Thus NASPs may be
used as an adjunct therapy in treatment or prophylaxis of
hemophilia A. In this embodiment of the invention, the patient may
have inhibitor antibodies against factor VIII. Typically, inhibitor
patients are treated with a bypassing agent, such as FEIBA. Such
inhibitor patients may have either a high titer response of greater
than 5 BU or a low titer response of between 0.5 and 5 BU. For
clinical purposes, the magnitude of the antibody response can be
quantified through the performance of a functional inhibitor assay
from which the Bethesda unit (BU) inhibitor titer can be obtained.
The International Society of Thrombosis and Haemostasis (ISTH)
definition of a high titer response is 5 BUs and its definition of
a low titer response is between 0.5 and 5 BUs. The magnitude of the
antibody response to FVIII can be quantified using a functional
inhibitor assay, such as that described in Kasper C K et al (1975)
Proceedings: A more uniform measurement of factor VIII inhibitors.
Thromb. Diath. Haemorrh. 34(2):612.
[0059] Suitably, in the method of the first aspect of the
invention, the subject is deficient in factor IX, and the method
further comprises administering factor IX. A suitable factor IX is
Bebulin VH.RTM. factor IX complex (Baxter BioScience, Vienna,
Austria). In this embodiment of the invention, the patient may have
inhibitor antibodies against factor IX. FIX inhibitors could be
quantified by an aPTT assay as described by Kasper (supra).
Suitably, factor IX and/or FEIBA are also administered to the
factor IX deficient subject.
[0060] Preferably, according to the method of the first aspect of
the invention, a NASP is administered via a non-intravenous
route.
[0061] A NASP composition for use in the method of the first aspect
of the invention may further comprise one or more pharmaceutically
acceptable excipients to provide a pharmaceutical composition.
Suitable excipients are described in "Remington: The Science &
Practice of Pharmacy", 19th ed., Williams & Williams, (1995),
the "Physician's Desk Reference", 52nd ed., Medical Economics,
Montvale, N.J. (1998), and Kibbe, A. H., Handbook of Pharmaceutical
Excipients, 3rd Edition, American Pharmaceutical Association,
Washington, D.C., 2000. Exemplary excipients include, without
limitation, carbohydrates, inorganic salts, antimicrobial agents,
antioxidants, surfactants, buffers, acids, bases, and combinations
thereof. Excipients suitable for injectable compositions include
water, alcohols, polyols, glycerine, vegetable oils, phospholipids,
and surfactants. A carbohydrate such as a sugar, a derivatized
sugar such as an alditol, aldonic acid, an esterified sugar, and/or
a sugar polymer may be present as an excipient. Specific
carbohydrate excipients include, for example: monosaccharides, such
as fructose, maltose, galactose, glucose, D-mannose, sorbose, and
the like; disaccharides, such as lactose, sucrose, trehalose,
cellobiose, and the like; polysaccharides, such as raffinose,
melezitose, maltodextrins, dextrans, starches, and the like; and
alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,
sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
The excipient can also include an inorganic salt or buffer such as
citric acid, sodium chloride, potassium chloride, sodium sulfate,
potassium nitrate, sodium phosphate monobasic, sodium phosphate
dibasic, and combinations thereof.
[0062] The amount of the NASP (e.g., when contained in a drug
delivery system) in the composition will vary depending on a number
of factors, but will optimally be a therapeutically effective dose
when the composition is in a unit dosage form or container (e.g., a
vial). A therapeutically effective dose can be determined
experimentally by repeated administration of increasing amounts of
the composition in order to determine which amount produces a
clinically desired endpoint.
[0063] The NASP compositions herein may optionally include one or
more additional agents, such as hemostatic agents, blood factors,
or other medications used to treat a subject for a condition or
disease. Particularly preferred are compounded preparations
including one or more blood factors such as factor XI, factor V,
factor VII, factor VIII, factor IX, factor X, factor XIII, factor
II, factor VIIa, and von Willebrand factor. Preparations may also
include prekallikrein, high molecular weight kininogen (HMWK)
and/or factor XII. NASP compositions may also include other
procoagulants, such as an activator of the intrinsic coagulation
pathway, including but not limited to, factor Xa, factor IXa,
factor XIa, factor XIIa, and kallikrein; or an activator of the
extrinsic coagulation pathway, including but not limited to, tissue
factor, factor VIIa, thrombin, and factor Xa. NASP compositions may
include naturally occurring, synthetic, or recombinant clotting
factors or fragments, variants or covalently modified derivatives
thereof that retain biological activity (i.e., promote clotting).
Alternatively, such agents can be contained in a separate
composition from the NASP and co-administered concurrently, before,
or after the NASP composition of the invention.
[0064] A second aspect of the invention provides a method of factor
XI-dependent blood coagulation enhancement in a subject in need of
enhanced blood coagulation comprising: [0065] (i) selecting a
subject that is not deficient for factor XI; and [0066] (ii)
administering a therapeutically effective amount of a composition
comprising a non-anticoagulant sulfated polysaccharide (NASP) to
the subject, wherein the NASP enhances blood coagulation in a
factor XI-dependent manner.
[0067] Typically, the factor XI status of the subject is determined
in order to identify whether they are suitable for treatment
according to this aspect of the invention. Deficiency in FXI:c may
be determined by an aPTT based activity assay, such as in Ingram G
I et al (supra). ELISAs to detect FXI antigen may also be used,
and/or genetic analyses to identify a mutation in the FXI gene. If
the subject is deficient in factor XI, it may be appropriate to
treat them according to the method of the first aspect of the
invention, such as by administering factor XI and a NASP. If the
subject is not deficient in factor XI, they may suitably be treated
according to the method of the second aspect of the invention.
[0068] In this aspect of the invention, the NASP enhances blood
coagulation in a factor XI-dependent manner. Factor XI-dependent
enhancement of blood coagulation may be determined as described in
relation to the first aspect of the invention.
[0069] As described in the Examples, factor XI-dependent
enhancement of blood coagulation by a NASP is more readily detected
under conditions in which the tissue factor concentration is low.
In a subject, tissue factor concentration is likely to be low at
sites which bleed spontaneously, or in response to mild trauma, for
example muscles or joints. Hemophilia A or B patients may bleed at
these sites. Hemophilia A patients may also be subject to
spontaneous bleeding in the brain or digestive tract. As the factor
XI-dependent effect of a NASP in enhancing blood coagulation is
likely to be important in the treatment of such bleeds, it is
preferred to select a subject which is not deficient in factor XI.
Preferably, the subject has at least 70 IU/dL and typically about
100 IU/dL of FXI:c in their plasma.
[0070] The method of the second aspect of the invention may also be
useful where the subject is in need of enhanced blood coagulation
for other reasons, for example to reverse the effect of
administered anti-coagulants.
[0071] According to a third aspect of the invention is provided a
method of identifying a non-anticoagulant sulfated polysaccharide
(NASP) which is capable of enhancing blood coagulation in
dependence on FXI.
[0072] In steps (a) and (b), blood or plasma comprising or
deficient in activation competent factor XI is combined with a
sulfated polysaccharide and the clotting or thrombin generation
parameters of the blood or plasma samples are measured. Techniques
and blood or plasma preparations as described in relation to the
first aspect of the invention are suitable for this purpose.
[0073] The blood or plasma sample deficient in activation competent
FXI is a "corresponding" sample to the blood or plasma sample
comprising activation competent FXI. By "corresponding" is meant
that the samples are similar other than with respect to the
presence of activation competent FXI. Typically they are from the
same species, and preferably have similar levels of other
coagulation factors and molecules that influence coagulation.
Suitably, the samples are obtained from the same subject, and one
is treated to make it deficient in activation competent FXI.
Alternatively, the sample deficient in FXI may be obtained from a
genetically FXI deficient subject, or pooled material from two or
more such subjects. The sample comprising activation competent FXI
may be obtained from a normal subject, or pooled material from two
or more such subjects.
[0074] Step (c) of the method of the third aspect comprises
comparing the clotting or thrombin generation parameters of the
blood or plasma samples as determined in steps (a) and (b), wherein
a decrease in the clotting time of the blood sample or an increase
in peak thrombin or decrease in peak time of the plasma sample
comprising activation competent FXI compared to the clotting time
of the blood sample or peak thrombin or peak time of the plasma
sample deficient in activation competent FXI is indicative of a
NASP which is capable of enhancing blood coagulation in dependence
on FXI.
[0075] A NASP identified as being capable of enhancing blood
coagulation in dependence on factor XI may be used in a method
according to the first or second aspects of the invention.
[0076] It is typical to include tissue factor in an assay to
measure the clotting or thrombin generation properties of a blood
or plasma sample. However, in the method of the third aspect of the
invention, it may be necessary to inhibit or reduce clotting or
thrombin generation driven by the extrinsic pathway, in order to
detect a factor XI-dependent NASP-mediated enhancement of blood
coagulation. It has been found that the factor XI-dependent
component of NASP-mediated enhancement of blood coagulation in
normal human blood or plasma is more readily detected where the
tissue factor concentration is low. Suitably, the tissue factor
concentration in a plasma assay may be less than 40 pM, less than
20 pM, 5 pM, 1 pM, 0.5 pM, less than 0.2 pM or approximately 0 pM.
Suitably, the tissue factor concentration in a blood assay may be
less than 1 pM, less than 500 fM, less than 100 fM, less than 50,
20 or 10 fM. It may also be necessary to inhibit or reduce the
first step of the intrinsic pathway, that of activation of FXII, in
order to identify a factor XI-dependent NASP-mediated enhancement
of blood coagulation. Factor XII deficient blood or plasma could be
used. Alternatively, an inhibitor of factor XII may be included in
the assay, such as corn trypsin inhibitor (CTI). A concentration of
40 .mu.g/mL CTI may be effective. Other features of suitable
assays, and components that may be included, are known to the
person of ordinary skill in the art, and are also illustrated in
the Examples.
[0077] The present invention will be further illustrated in the
following examples, without any limitation thereto.
Example 1
Fucoidan Improves Clot Formation in Whole Blood
[0078] Fucoidan improves clotting parameters in FVIII inhibited
blood, and so may be useful in the treatment of hemophilia A.
Materials
[0079] Blood samples from a healthy individual were drawn into
citrated Venojecte tubes (Terumo Europe, Leuven, Belgium (127
mmol/L)) mixing one part of citrate with nine parts of blood by a
21-G butterfly needle. The first tube aspirated was discarded. A
proportion of these blood samples were incubated with high titer
heat inactivated anti-human FVIII antiserum raised in goat (3876
BU/ml; Baxter BioScience, Vienna, Austria) resulting in 150 BU/mL.
Test samples were prepared by dissolving quantities of sulfated
polysaccharide in Hepes buffered saline and adding human serum
albumin (Sigma-Aldrich Corporation, St. Louis, Mo., USA) to a
concentration of 5 mg/mL. A control sample was prepared in which no
sulfated polysaccharide was included. The sulfated polysaccharide
was Undaria pinnatifida fucoidan of .about.127 Da (Kraeber GmbH
& Co; Ellerbek, Germany).
Method
[0080] Continuous visco-elastic assessment of human whole blood
clot formation and firmness was performed by rotation
thromboelastography with whole blood preparations in the presence
or absence of sulfated polysaccharides. Briefly, blood is added
into a disposable cuvette in a heated cuvette holder. A disposable
pin (sensor) is fixed on the tip of a rotating axis. The axis is
guided by a high precision ball bearing system and rotates back and
forth. The axis is connected with a spring for the measurement of
elasticity. The exact position of the axis is detected by the
reflection of light on a small mirror on the axis. The loss of
elasticity when the sample clots leads to a change in the rotation
of the axis. The data obtained are analysed on a computer and
visualized in a thromboelastogram. The thromboelastogram shows
elasticity (mm) versus time (s). An elasticity of close to zero is
observed before clot formation begins. Mirror image traces above
and below the zero line indicate the effect of clot formation on
the rotation of the axis.
[0081] Recordings were made using a ROTEG thromboelastography
coagulation analyser (Pentapharm, Munich, Germany) at 37.degree. C.
Before starting each experiment, the citrated whole blood was mixed
with corn trypsin inhibitor (CTI) (Hematologic Technologies, Inc.,
Essex Junction, Vt., USA) providing a final concentration of 52
.mu.g/mL for specific inhibition of FXIIa, in order to inhibit
FXIIa-mediated contact activation. The analytical set-up was as
follows: To 20 .mu.L of test sample or control, 300 .mu.L of
pre-warmed (37.degree. C.) CTI treated citrated whole blood was
added, followed by 20 .mu.L of a 1:15 dilution of TF PRP reagent
containing recombinant human tissue factor (rTF, 3 .mu.M) (TS40,
Thrombinoscope BV, Maastricht, The Netherlands). Coagulation was
initiated by the addition of 20 .mu.L 200 mM CaCl.sub.2
(star-TEM.RTM., Pentapharm, Munich, Germany) and recordings were
allowed to proceed for at least 120 min. The final concentration of
rTF in the assay was 11 fM.
[0082] The thromboelastographic parameters of clotting time (CT),
clot formation time (CFT) and maximum clot firmness (MCF) were
recorded in accordance with the manufacturer's instructions. CT is
defined as the time from the start of measurement to the start of
clot formation. CFT is defined as the time from the start of clot
formation until an amplitude of 20 mm is reached. MCF is the
maximum difference in amplitude between the two traces during the
assay. The first derivative of the data of the thromboelastogram
are plotted to obtain a graph of velocity (mm/s) against time (s).
From this graph, the maximum velocity (maxV) is determined. The
time at which the maximum velocity is obtained (maxV-t) is also
determined.
Results
[0083] The effect of fucoidan from Undaria pinnatifida on
thromboelastographic parameters was tested at two concentrations in
FVIII-inhibited blood. Two controls were performed in which no
fucoidan was present. One used FVIII-inhibited blood and the other
used normal blood. Results are shown in Table 1 below. The
FVIII-inhibited blood had a characteristically long clotting time
and clot formation time. The clotting time and clot formation time
were both shorter in the FVIII-inhibited blood containing fucoidan,
with the fucoidan exerting a concentration dependent effect on both
parameters. Fucoidan also reduced CT and CFT in normal blood.
TABLE-US-00001 TABLE 1 Clotting parameters Fucoidan/blood CT (s)
CFT (s) MCF (mm) Control - FVIII-inhibited blood 2447 881 55 U.p.
10 nM - FVIII inhibited blood 1163 419 55 U.p. 100 nM - FVIII
inhibited blood 956 330 50 Control - Normal blood 869 274 45 U.p.
10 nM - Normal blood 767 225 46 U.p. 100 nM - Normal blood 382 105
54
Example 2
Calibrated Automated Thrombography (CAT) to Study Thrombin
Generation
[0084] The procoagulant activity of sulfated polysaccharides was
examined in several plasmas from patients with congenital
coagulation factor deficiencies, in order to study the mechanism of
action. This example describes the basic method which is used in
the later examples.
Materials
[0085] Plasmas from patients with congenital coagulation factor
deficiencies were obtained from George King, Bio-Medical Inc.
Kansas USA. According to the supplier, the residual coagulation
factor activity for each of the plasmas was lower than 1% except
for prothrombin deficient plasma which was 4%. As a model for
antibody mediated FVIII deficiency fresh frozen pooled normal
plasma (George King, Bio-Medical Inc., Kansas, USA) was incubated
with high titer heat inactivated anti-human FVIII plasma raised in
goat (4490 BU/ml; Baxter BioScience, Vienna, Austria) giving rise
to 50 BU/mL. In some experiments FXI activity of pooled normal
plasma or FVIII deficient plasma was blocked by an anti human FXI
antibody (GAFXI-AP, Enzyme Research Laboratories, South Bend, Ill.,
USA) at a final concentration of 100 nM. If not indicated
otherwise, the plasmas were mixed with corn trypsin inhibitor (CTI)
(Hematologic Technologies, Inc., Essex Junction, Vt., USA),
providing a final concentration of 40 .mu.g/mL, for specific
inhibition of factor XIIa.
[0086] Test samples were prepared by dissolving quantities of
sulfated polysaccharide in Hepes buffered saline and adding human
serum albumin (Sigma-Aldrich Corporation, St. Louis, Mo., USA) to a
concentration of 5 mg/mL. The sulfated polysaccharides and their
sources are indicated in Table 2 below.
TABLE-US-00002 TABLE 2 Sulfated polysaccharide MW (kDa) Source
Pentosan polysulfate 5.9 CF Pharma Ltd. (Budapest, sodium (PPS)
Hungary) Fucoidan LMW, 7.5 Kraeber GmbH & Co (Ellerbek,
Ascophyllum nodosum Germany) Fucoidan, Fucus ~115.5 F6531;
Sigma-Aldrich Chemie vesiculosus GmbH (Taufkirchen, Germany)
Fucoidan, Undaria ~127 Kraeber GmbH & Co (Ellerbek, pinnatifida
Germany) Fucoidan HMW, ~600 Kraeber GmbH & Co (Ellerbek,
Ascophyllum nodosum Germany) Fucoidan, Laminaria >1000 Kraeber
GmbH & Co (Ellerbek, japonica Germany)
[0087] Reference samples were prepared from reference proteins
FVIII Imrnunate.RTM. reference standard (Baxter BioScience, Vienna,
Austria); Factor eight inhibitor by-passing activity (FEIBA)
reference standard (Baxter BioScience, Vienna, Austria);
NovoSeven.RTM. recombinant activated FVII (Novo Nordisk A/S,
Denmark) and purified human plasma FIX (Enzyme Research
Laboratories, South Bend, Ill., USA). A proprietary thrombin
calibrator compound was obtained from Thrombinoscope BV,
Maastricht, The Netherlands.
Method
[0088] The influence of each sulfated polysaccharide on thrombin
generation was measured in duplicate via calibrated automated
thrombography in a Fluoroskan Ascent.RTM. reader (Thermo
Labsystems, Helsinki, Finland; filters 390 nm excitation and 460 nm
emission) following the slow cleavage of the fluorogenic substrate
Z-Gly-Gly-Arg-AMC (Hemker H C. Pathophysiol Haemost Thromb 2003;
33: 4 15). To each well of a 96 well micro-plate (Immulon 2HB,
clear U-bottom; Thermo Electron) 80 .mu.L of pre-warmed (37.degree.
C.) plasma was added. For triggering thrombin generation by tissue
factor, 10 .mu.L of PPP reagent containing a certain amount of
recombinant human tissue factor (rTF) and phospholipid vesicles
composed of phosphatidylserine, phosphatidylcholine and
phosphatidylethanolamine (48 .mu.M) (Thrombinoscope BV, Maastricht,
The Netherlands) was added. Alternatively, a mix of rTF
(Innovin.RTM., Siemens Healthcare Diagnostics Inc., Tarrytown,
N.Y., USA) and a phospholipid emulsion composed of
phosphatidylcholine, phosphatidylserine and sphingomyelin
(Phospholipid-TGT, Rossix, Molndal, Sweden) was used. If thrombin
generation was triggered by factor XIa, a mix of human factor XIa
(0.72 nM) (Enzyme research Laboratories, South Bend, Ind., USA) and
Phospholipid-TGT (48 .mu.M) was added. If thrombin generation
without the addition of any trigger was studied, just 10 .mu.L
Phospholipid-TOT (48 .mu.M) diluted in Hepes buffered saline was
included. Just prior to putting the plate into the pre-warmed
(37.degree. C.) reader, 10 .mu.L of test or reference sample or
calibrator compound was added. Thrombin generation was started by
dispensing 20 .mu.L of FluCa reagent (Thrombinoscope BV,
Maastricht, The Netherlands) containing fluorogenic substrate and
Hepes buffered CaCl.sub.2 (100 mM) into each well and fluorescence
intensity was recorded at 37.degree. C.
[0089] The parameters of the resulting thrombin generation curves
were calculated using the Thrombinoscope.TM. software
(Thrombinoscope BV, Maastricht, The Netherlands) and thrombin
calibrator to correct for inner fitter and substrate consumption
effects (Hemker H C. Pathophysiol Haemost Thromb 2003; 33: 4 15).
With the thrombin calibrator as a reference, the molar
concentration of thrombin in the test wells was calculated by the
software. The thrombin amounts at the peak of each thrombin
generation curve (peak thrombin, nM) were plotted against the peak
thrombin obtained from standard concentrations of a reference
protein (FVIII Immunate.RTM. reference standard, FEIBA reference
standard) and fitted by a non-linear algorithm. Based on this
calibration, FVIII Immunate.RTM., FEIBA and FIX equivalent
activities were calculated. Other parameters recorded were lag time
(time interval between starting measurement and start of thrombin
generation), peak time (time interval between starting measurement
and peak thrombin) and endogenous thrombin potential (area under
curve of thrombin concentration versus time).
Example 3
Tissue Factor and FVIII Dependency of Thrombin Generation
[0090] Tissue factor and FVIII dependency of thrombin generation
were assessed using the CAT assay. Pooled normal plasma and FVIII
inhibited plasma were tested in the presence of tissue factor at 1,
5 or 20 pM. As expected, at each concentration of tissue factor,
the peak thrombin was reduced in the FVIII inhibited plasma
compared to normal plasma, and the peak time was increased. The
most pronounced difference between the thrombin generation
parameters of the two plasmas was observed at the lowest tissue
factor concentration. Results are shown in Table 3 below.
TABLE-US-00003 TABLE 3 Plasma Tissue Ratio Normal/ Factor (pM)
Normal FVIII inhibited FVIII inhibited Peak thrombin (nM) 1 94.4
29.9 3.16 5 276.5 173.3 1.60 20 398.1 346.5 1.15 Peak time (min) 1
11.89 17.92 0.66 5 5.24 6.15 0.85 20 3.43 3.48 0.99
[0091] As the deficiency of thrombin generation in FVIII deficient
plasma was most pronounced at low tissue factor concentrations as
indicated by the highest ratio (normal:FVIII inhibited) of Peak
thrombin, and lowest ratio (normal:FVIII inhibited) of Peak time,
later experiments designed to identify the effect of sulfated
polysaccharides on thrombin generation were generally performed at
a low tissue factor concentration.
Example 4
Hemophilia Therapeutics Improve Thrombin Generation in FVIII
Inhibited or Deficient Plasma
[0092] To provide a reference with which to compare the efficacy of
sulfated polysaccharides, hemophilia therapeutics were tested in
the CAT assay at a range of concentrations in FVIII inhibited or
hemophiliac plasma. A control using normal plasma was run for
comparison. FVIII Immunate.RTM. was tested at 0, 25, 100, 250, 500
and 1000 mU/ml in hemophilia A plasma. FEIBA was tested at 0, 10,
40, 100, 250 and 500 mU/ml in FVIII inhibited plasma. rFVIIa
Novoseven.RTM. was tested at 0, 0.04, 0.2, 1, 5 and 25 nM. For each
hemophilia therapeutic, peak thrombin increased and peak time
decreased at increasing concentrations of therapeutic agent. The
highest concentrations of FVIII Immunate.RTM. and FEIBA tested gave
rise to thrombin generation parameters that were comparable to that
of normal plasma. At the highest concentration of rFVIIa
Novoseven.RTM. tested, peak time was comparable to that of normal
plasma, and peak thrombin was about 60% of the level obtained with
normal plasma.
Example 5
Sulfated Polysaccharides are Most Effective at Improving Thrombin
Generation at Intermediate Concentrations
[0093] Sulfated polysaccharides were tested at a range of
concentrations in hemophilia A plasma. The concentration of tissue
factor was 1 pM. At concentrations of up to 100 nM, fucoidan from
Fucus vesiculosus improved thrombin generation parameters (i.e.
increased peak thrombin and decreased peak time) in a
concentration-dependent manner. At higher fucoidan concentrations
of 250, 500, 1000, 1500 and 2000 nM, thrombin generation parameters
deteriorated in a concentration-dependent manner. A similar pattern
was observed for each of the sulfated polysaccharides tested, with
an optimal effect on thrombin generation at an intermediate
concentration, and sub-optimal effects at lower and higher
concentrations. The optimal effect was achieved at a comparable
.mu.g/ml concentration of each sulfated polysaccharide, although
the nM concentrations varied over two orders of magnitude. The
FVIII equivalent activity of the concentration of each of the six
sulfated polysaccharides tested that had the most beneficial effect
on peak thrombin was estimated. Results are shown in Table 4
below.
TABLE-US-00004 TABLE 4 Sulfated Conc Conc FVIII EA polysaccharide
(nM) (.mu.g/ml) (mU/ml) PPS 1000 5.9 733 Fuc An LMW 1000 7.5 937
Fuc Fv 100 11.6 874 Fuc Up 100 12.7 869 Fuc An HMW 25 15.0 794 Fuc
Lj 10 10.0 826
[0094] Further data are given in Table 5 below, indicating the
"therapeutic window" for each sulfated polysaccharide. The
"therapeutic window" is the concentration range at which the
sulfated polysaccharide provides for a peak thrombin in severe
hemophilia A plasma (FVIII activity below 1% of normal plasma)
which is at least the peak thrombin provided by the addition of 10
mU/mL (1% of normal) of factor VIII (Immunate.RTM.) to severe
hemophilia A plasma. Also shown is the FVIII equivalent activity of
the optimal concentration of sulfated polysaccharide and, in
brackets, the FVIII equivalent activity of the polysaccharide
concentration at either end of the therapeutic window. The results
indicate that each sulfated polysaccharide has a procoagulant
effect across a broad concentration range.
TABLE-US-00005 TABLE 5 Sulfated Therapeutic window polysaccharide
MW (kD) nM .mu.g/mL FVIII EA PPS 5.9 50-20000* 0.3-118* 733 (10-46)
A.n. LMW 7.5 25-20000* 0.2-150* 937 (16-577) F.v. 115 5-2000*
0.6-230* 874 (17-334) U.p. 127 5-2000* 0.6-254* 869 (28-101) A.n.
HMW 600 2.5-500 1.5-300 794 (67-129) L.j. >1000 1-250 1-250 826
(42-170) *indicates that the upper limit given for the therapeutic
window was the highest concentration of sulfated polysaccharide
tested.
[0095] Similar experiments were performed with the sulfated
polysaccharides in FVIII inhibited plasma. In each case, FEIBA
equivalent activities were estimated rather than FVIII equivalent
activities. Results are presented in the following two tables, and
are broadly consistent with the results obtained using hemophilia A
plasma.
TABLE-US-00006 TABLE 6 Sulfated Conc Conc FEIBA EA polysaccharide
(nM) (.mu.g/ml) (mU/ml) PPS 1000 5.9 587 Fuc An LMW 1000 7.5 773
Fuc Fv 100 11.6 625 Fuc Up 100 12.7 1047 Fuc An HMW 25 15.0 1226
Fuc Lj 10 10.0 1090
TABLE-US-00007 TABLE 7 Sulfated Therapeutic window FEIBA EA
polysaccharide MW (kD) nM .mu.g/mL mU/mL PPS 5.9 50-10000 0.3-59
587 (25-189) A.n. LMW 7.5 50-20000* 0.4-150 773 (18-386) F.v. 115
10-2000* 1.2-230 625 (23-230) U.p. 127 10-2000* 1.3-254 1047
(65-85) A.n. HMW 600 2.5-500 1.5-300 1226 (54-150) L.j. >1000
2.5-250 2.5-250 1090 (288-175) *indicates that the upper limit
given for the therapeutic window was the highest concentration of
sulfated polysaccharide tested.
[0096] A comparison of the FEIBA equivalent activity in FVIII
inhibited plasma and the FVIII equivalent activity in hemophilia A
plasma for the optimum concentration of each sulfated
polysaccharide is shown in Table 8 below.
TABLE-US-00008 TABLE 8 Optimum Sulfated Concentration FEIBA EA
FVIII EA polysaccharide (nM) (.mu.g/ml) (mU/ml) (mU/ml) PPS 1000
5.9 587 733 A.n. LMW 1000 7.5 773 937 F.v. 100 11.6 625 874 U.p.
100 12.7 1047 869 A.n. HMW 25 15.0 1226 794 L.j. 10 10.0 1090
826
Example 6
Sulfated Polysaccharides Act Additively with Hemophilia
Therapeutics in Promoting Thrombin Generation
[0097] Experiments were performed to examine the effect of fucoidan
on peak thrombin in hemophilia A or FVIII inhibited plasma in the
presence of increasing concentrations of hemophilia therapeutics.
The CAT assay was used to determine peak thrombin with a tissue
factor concentration of 1 pM.
[0098] A range of concentrations of FVIII Immunate.RTM. were tested
in hemophilia A plasma, namely 0, 0.1, 1, 10, 100 and 1000 mU/mL.
For each concentration, fucoidan from Undaria pinnatifida was added
at a concentration of 100 nM and a corresponding control was
performed in the absence of fucoidan. The ratio of peak thrombin in
the presence and absence of fucoidan was calculated for each
concentration of Immunate.RTM.. Similar experiments were performed
using FEIBA at 0, 10, 40, 100, 250 or 500 mU/mL as the hemophilia
therapeutic and FVIII inhibited plasma, and using rFVIIa
NovoSeven.RTM. at 0, 0.04, 0.2, 1, 5 and 25 nM as the hemophilia
therapeutic and FVIII inhibited plasma. Results are shown in Table
9 below.
TABLE-US-00009 TABLE 9 Therapeutic/ Parameter Concentration of
therapeutic plasma measured 0 0.1 1 10 100 1000 FVIII mU/ml/ Peak
thrombin 102.04 105.85 105.59 110.07 134.86 208.25 Hem A plasma
(+fucoidan) Peak thrombin 36.75 36.14 36.37 38.23 50.56 102.31
(-fucoidan) Ratio 2.8 2.9 2.9 2.9 2.7 2.0 0 10 40 100 250 500 FEIBA
mU/ml/ Peak thrombin 120.77 124.47 130.16 152.29 198.27 258.35
FVIII inhibited (+fucoidan) Peak thrombin 27.58 30.27 36.96 47.04
72.12 102.71 (-fucoidan) Ratio 4.4 4.1 3.5 3.2 2.7 2.5 0 0.04 0.2 1
5 25 rFVIIa nM/ Peak thrombin 119.59 127.08 145.22 179.39 208.54
212.37 FVIII inhibited (+fucoidan) Peak thrombin 28.05 34.43 43.63
53.87 62.04 65.19 (-fucoidan) Ratio 4.3 3.7 3.3 3.3 3.4 3.3
[0099] The results show that increasing the quantity of hemophilia
therapeutic results in an increase in peak thrombin. The
enhancement of peak thrombin caused by the addition of fucoidan was
slightly greater at lower than higher quantities of FVIII or FEIBA
tested, and roughly comparable at all quantities of rFVIIa tested.
Thus, fucoidan appears to act additively with hemophilia
therapeutics, particularly when the concentration of hemophilia
therapeutic is not high enough to promote a physiological amount of
thrombin generation. (In this assay, normal plasma produces a peak
thrombin of about 100 nM.) Thus, fucoidan may be useful as an
adjunct therapy in hemophilia treatment.
Example 7
Tissue Factor Dependency of the Fucoidan Effect
[0100] Tissue factor dependency of the effect of fucoidan on
thrombin generation parameters was tested by the CAT assay. Peak
time and peak thrombin were determined for four different plasma
and fucoidan combinations at 0, 0.2, 0.5, 1, 5 and 20 pM tissue
factor. The plasmas were pooled normal plasma and FVIII inhibited
plasma. Each was tested in the presence or absence of 100 nM
fucoidan from Undaria pinnatifida. Results are shown in Table 10
below.
TABLE-US-00010 TABLE 10 Fucoidan Tissue factor (pM) Plasma (100 nM)
Parameter 0 0.2 0.5 1.0 5.0 20.0 Normal - Peak time (min) 44.43
24.20 19.02 15.84 9.15 4.31 Peak thrombin 22.67 53.36 71.91 86.60
186.1 301.2 (nM) Normal + Peak time (min) 15.84 13.84 12.16 10.66
6.31 3.97 Peak thrombin 291.7 199.2 198.0 208.7 263.8 309.8 (nM)
FVIII - Peak time (min) 120.0 53.67 43.67 35.67 15.17 5.33
inhibited Peak thrombin 0.26 0.73 4.47 9.48 65.32 206.6 (nM) FVIII
+ Peak time (min) 55.00 33.67 28.83 24.33 9.83 4.67 inhibited Peak
thrombin 1.50 12.26 31.71 51.28 134.0 259.7 (nM)
[0101] The results show that the effect of fucoidan in reducing
peak time and increasing peak thrombin in normal plasma is most
pronounced at the lowest concentrations of tissue factor and
particularly when no tissue factor is added. When the concentration
of tissue factor is high, thrombin is generated almost exclusively
through the extrinsic pathway. Under those conditions, sulfated
polysaccharides do not increase thrombin generation. In FVIII
inhibited plasma, there is also a trend for fucoidan to have a more
pronounced effect on peak time and peak thrombin at low tissue
factor concentrations. However, to achieve a physiologically
relevant thrombin generation in this assay, some tissue factor is
required. Any effect of fucoidan in the total absence of tissue
factor may not be meaningful. Even at the highest concentration of
tissue factor tested, fucoidan still increased peak thrombin.
[0102] As indicated above, fucoidan from Undaria pinnatifida is
capable of stimulating thrombin generation at low concentrations of
tissue factor and even in the absence of tissue factor (in normal
plasma). Other sulfated polysaccharides were tested for their
effect on thrombin generation in the absence of tissue factor in
the CAT assay. Each compound was tested at the optimal
concentration as determined in Example 5, with the exception of
A.n. HMW which was tested at 10 nM. (Peak thrombin is only slightly
lower where A.n. F-IMW is used as 10 nM, compared to 25 nM.)
Results are shown in Table 11 below.
TABLE-US-00011 TABLE 11 Peak thrombin (nM) Peak time (min) Normal
Normal Compound plasma FVIII inhibited plasma FVIII inhibited
Control 22.67 0.26 44.43 >120.00 PPS 26.22 0.24 31.56 68.33 A.
n. LMW 53.25 0.30 26.04 52.83 F. v. 46.15 0.22 28.72 58.83 U. p.
291.74 1.50 15.84 55.00 A. n. HMW 120.56 0.20 21.02 69.83 Fuc Lj
61.10 0.37 26.88 75.33
[0103] The results show that each of the compounds tested are
capable of increasing Peak thrombin and reducing Peak time in
normal plasma. In the total absence of tissue factor, the compounds
did not enhance Peak thrombin or reduce Peak time in FVIII
inhibited plasma. This can be explained by the fact that the
extrinsic pathway is inactive in the absence of tissue factor, and
the intrinsic pathway is inactive in the absence of FVIII.
Example 8
Fucoidan Acts Independently of FXII to Promote Thrombin
Generation
[0104] Thrombin generation parameters were tested in FXII deficient
plasma in the presence or absence of 100 nM fucoidan from Undaria
pinnatifida, at a tissue factor concentration of 1 pM. Under these
conditions, residual FXII activity was lower than 1% of normal, but
corn trypsin inhibitor was still included at 40 .mu.g/mL as a
precaution. Fucoidan was found to increase peak thrombin and
decrease peak time, as in previous experiments. FXII is the
starting point of the intrinsic (contact activation) pathway. The
fact that fucoidan improves thrombin generation parameters in FXII
deficient plasma indicates that it does not act on FXII.
Example 9
Coagulation Factor Dependency of the Effect of Sulfated
Polysaccharides
[0105] To examine the mechanism of action of sulfated
polysaccharides further, CAT assays were performed in further
coagulation factor deficient plasmas. No tissue factor was added in
order to minimise the contribution of the extrinsic pathway to
thrombin generation. The following fucoidans were tested:
Ascophyllum nodosum, high MW, 10 nM; Fucus vesiculosus, 100 nM;
Undaria pinnatifida, 100 nM; Ascophyllum nodosum, low MW, 1000
nM.
[0106] When prothrombin deficient plasma was tested, there was
essentially no peak thrombin in the control lacking fucoidan. In
the presence of each of the fucoidans, there were small peaks,
which may be explained by the fact that the prothrombin deficient
plasma retained about 4% of the prothrombin activity of normal
plasma. When FX deficient plasma was used, no thrombin peaks were
observed in the absence or presence of any of the fucoidans. This
shows that FX, which is essential for both intrinsic and extrinsic
pathways, as it is part of the common pathway, is required for
sulfated polysaccharides to promote thrombin generation. Similarly,
in FV deficient plasma, no thrombin peaks were observed. FV is part
of the prothrombin activating complex of the common pathway, and is
necessary for sulfated polysaccharides to promote thrombin
generation. In FVII deficient plasma, all fucoidans were capable of
generating a thrombin peak, but there was no peak in the absence of
fucoidan. FVII is the starting point of the extrinsic pathway, and
is not necessary for sulfated polysaccharides to promote thrombin
generation. In FIX deficient plasma, a small thrombin peak was
observed in the presence of Undaria pinnatifida fucoidan, but not
in the other samples. FIX is activated in the intrinsic pathway,
and appears to be necessary for sulfated polysaccharides to promote
substantial thrombin generation. In FVIII deficient plasma, a very
small thrombin peak was observed in the presence of Undaria
pinnatifida fucoidan, but not in the other samples. FVIII is
activated in the intrinsic pathway, and appears to be necessary for
sulfated polysaccharides to promote substantial thrombin
generation. In FXI deficient plasma, no thrombin peaks were
observed in the presence or absence of NASPs. FXI is activated in
the intrinsic pathway, and appears to be necessary for sulfated
polysaccharides to promote thrombin generation. In FXII deficient
plasma, a small thrombin peak was observed in the absence of
fucoidan. Each of the fucoidans caused a substantial increase in
peak thrombin and a reduction in peak time. Thus FXII, which is
required for the intrinsic pathway, is not necessary for sulfated
polysaccharides to promote thrombin generation. Results are
summarised in Table 12 below.
TABLE-US-00012 TABLE 12 Necessary for mechanism of Coagulation
factor Role sulfated polysaccharides? prothrombin common pathway
yes FX common pathway yes FV common pathway yes FVII extrinsic
pathway no FIX intrinsic pathway yes FVIII intrinsic pathway yes
FXI intrinsic pathway yes FXII intrinsic pathway no
[0107] The coagulation factors of the intrinsic pathway are
necessary for sulfated polysaccharide enhancement of thrombin
generation, with the exception of FXII, the first coagulation
factor of that pathway. The order in which the coagulation factors
act in the intrinsic pathway is FXII, followed by FXI, then FIX and
FVIII in combination. Finally FX and FV act in combination in the
common pathway. The first coagulation factor of this pathway that
is required for the sulfated polysaccharides to enhance thrombin
generation is FXI. The data therefore suggest that sulfated
polysaccharides act on the intrinsic pathway by enhancing the
activation of FXI.
[0108] The fact that sulfated polysaccharides enhance thrombin
generation in the absence of FVII and tissue factor implies that
their mechanism of action is independent of the extrinsic pathway
and is fully driven through the intrinsic pathway.
Example 10
A FXI-Dependent Mechanism of Fucoidan Activity Contributes to
Thrombin Generation when Tissue Factor Concentration is Low
[0109] The FXI dependency of the effect of sulfated polysaccharides
was studied in pooled normal plasma and FXI deficient plasma by CAT
assay at different concentrations of tissue factor. The sulfated
polysaccharide tested was the Undaria pinnatifida fucoidan at 100
nM. As observed in previous experiments, the stimulatory effect of
the fucoidan was greater at lower concentrations of tissue factor
in normal plasma. As in the previous experiment, fucoidan did not
have a stimulatory effect in FXI deficient plasma in the absence of
added tissue factor. However, a stimulatory effect was observed at
1, 5 and 20 pM tissue factor in FXI deficient plasma. Results are
shown in Table 13 below.
TABLE-US-00013 TABLE 13 Peak thrombin (nM) Plasma/ Tissue factor
(pM) fucoidan 0 0.2 0.5 1 5 20 Normal 22.67 53.36 71.91 86.60
186.06 301.24 plasma - fucoidan Normal 291.74 199.20 198.00 208.74
263.81 309.80 plasma + fucoidan FXI deficient 0.00 Not Not 37.24
172.17 358.87 plasma - tested tested fucoidan FXI deficient 2.41
Not Not 102.73 279.60 392.51 plasma + tested tested fucoidan
[0110] At increasing concentrations of tissue factor, the
contribution of the extrinsic pathway to thrombin generation
increases. The stimulatory effect of fucoidan in the presence of
tissue factor in FXI deficient plasma may be mediated by the
extrinsic pathway. By comparing the stimulatory effect of fucoidan
at 0 or 1 pM tissue factor between normal plasma and FXI deficient
plasma, it can be seen that at these low tissue factor
concentrations, fucoidan has a greater stimulatory effect in normal
than FXI deficient plasma. It follows that a FXI dependent
mechanism of fucoidan activity contributes to thrombin generation
when tissue factor concentration is low.
[0111] A further experiment was conducted but instead of using FXI
deficient plasma, FXI was inhibited in pooled normal plasma by
pre-incubation with anti-FXI antibody. The antibody was polyclonal
goat anti-human FXI affinity purified "GAFXI-AP" from Enzyme
Research Laboratories (South Bend, Ill., USA). It was used at a
concentration of 150 nM to fully inhibit FXI. As a control, the
same pooled normal plasma was used untreated. Otherwise, the
experiment was performed in the same way as the preceding
experiment. Results are shown in Table 14 below.
TABLE-US-00014 TABLE 14 Peak thrombin (nM) Plasma/ Tissue factor
(pM) fucoidan 0 0.2 0.5 1 5 20 Normal 13.56 30.22 39.28 57.77
150.39 301.64 plasma - fucoidan Normal 287.07 187.86 191.16 199.59
254.10 303.78 plasma + fucoidan FXI-inhibited 0.56 5.89 16.16 33.17
133.06 303.02 plasma - fucoidan FXI-inhibited 6.15 27.06 57.80
99.53 241.15 310.10 plasma + fucoidan
[0112] The results confirm the conclusion that at low tissue factor
concentrations, fucoidan stimulates thrombin generation by a
FXI-dependent mechanism.
Example 11
Fucoidans Stimulate Thrombin Generation in FXI Deficient Plasma
Supplemented with FXI
[0113] An experiment was performed to examine the effect of
supplementing FXI deficient plasma with exogenous FXI. Sulfated
polysaccharide-stimulated thrombin generation was measured by CAT
assay. No tissue factor was used in this experiment. The sulfated
polysaccharide tested was the Undaria pinnatifida fucoidan at 100
nM. FXI deficient patient plasma was obtained form George King
(Bio-Medical Inc., Kansas, US). It was supplemented with purified
human factor XI (Enzyme Research Laboratories, South Bend, Ind.,
USA) to a concentration of 0, 0.3, 3 or 30 nM exogenous factor XI.
30 nM factor XI is the concentration found in normal human plasma.
Thrombin peak time and peak thrombin were tested in the presence or
absence of fucoidan. Results are shown in Table 15 below.
TABLE-US-00015 TABLE 15 Factor XI (nM) Compound Parameter 0 0.3 3
30 +fucoidan Peak time (min) 38.8 38.8 28.0 21.8 Peak thrombin (nM)
2.8 14.3 56.9 134.8 -fucoidan Peak time (min) >120 >120
>120 53.7 Peak thrombin (nM) 0.2 0.2 0.3 25.2
[0114] Results show that fucoidan stimulates thrombin generation in
a manner that is dependent on Factor XI concentration.
[0115] A further experiment was performed comparing the effects of
four different sulfated polysaccharides on thrombin generation in
the presence or absence of 30 nM Factor XI in Factor XI deficient
plasma. The following fucoidans were tested: Ascophyllum nodosum,
high MW, 10 nM; Fucus vesiculosus, 100 nM; Undaria pinnatifida, 100
nM; Ascophyllum nodosum, low MW, 1000 nM. Results are shown in
Table 16 below.
TABLE-US-00016 TABLE 16 +30 nM Factor XI No added Factor XI Peak
Peak Peak Peak Compound time (min) thrombin (nM) time (min)
thrombin (nM) A.n. LMW 28.8 67.9 46.5 2.9 F. v. 29.8 70.8 44.5 2.4
U. p. 26.3 116.6 42.5 2.5 A. n. HMW 26.0 86.8 48.3 2.3 None 51.7
36.0 >120 0.2
[0116] Results show that all fucoidans stimulated thrombin
generation in FXI deficient plasma supplemented with Factor XI. In
the absence of added Factor XI, no thrombin peaks were generated by
fucoidan. These results verify the Factor XI-dependency of the
stimulation of thrombin generation by fucoidans.
Example 12
Fucoidans Act by Activating FXI
[0117] The FXI-dependent mechanism of fucoidan stimulation of
thrombin generation was studied in a CAT assay in which activated
FXI (FXIa) was added to FXI deficient plasma. No tissue factor was
added. The following fucoidans were tested: Ascophyllum nodosum,
high MW, 10 nM; Fucus vesiculosus, 100 nM; Undaria pinnatifida, 100
nM; Ascophyllum nodosum, low MW, 1000 nM.
[0118] A thrombin peak was observed in FXI deficient plasma to
which 60 pM human plasma FXIa (Enzyme Research Laboratories, South
Bend, Ill., USA) was added. However, the addition of fucoidans to
the FXI deficient plasma+FXIa did not increase peak thrombin or
decrease peak time. From this experiment, it appears that the
fucoidans normally act to activate or enhance the activation of FXI
to FXIa. When FXIa is provided, the fucoidans had no further
stimulatory effect.
Example 13
Fucoidans Stimulate Thrombin Generation in Extrinsically
Compromised Plasma
[0119] The effect of fucoidan stimulation of thrombin generation
was studied in FVII deficient plasma at a range of concentrations
of tissue factor. As FVII is the first coagulation factor in the
extrinsic pathway, FVII deficient plasma is extrinsically
compromised. In the absence of fucoidan, there was only a small
thrombin peak which had a large peak time at high tissue factor
concentration (20 pM). The thrombin peak may have been caused by
residual FVII. Tissue factor concentrations lower than 5 .mu.M gave
no thrombin generation. When 100 nM Undaria pinnatifida fucoidan
was included, a large thrombin peak was obtained. Increasing the
concentration of tissue factor had little effect on peak thrombin,
and reduced peak time only slightly. Results are shown in Table 17
below.
TABLE-US-00017 TABLE 17 Peak Peak Peak Peak Peak Peak Peak Peak
thrombin time thrombin time thrombin time thrombin time (nM) (min)
(nM) (min) (nM) (min) (nM) (min) Tissue factor (pM) 0 1 5 20
+fucoidan 303.1 15.8 305.9 14.0 313.4 12.5 285.3 11.7 -fucoidan 0
>120 0 >120 0 >120 27.6 51.0
[0120] The results show that when the extrinsic pathway is
prevented from acting by the absence of FVII, tissue factor has
little effect on the stimulation of thrombin generation by
fucoidan. In a further experiment, fucoidan stimulation of thrombin
generation was studied in FVII deficient plasma or FVIII inhibited
FVII deficient plasma at a range of tissue factor concentrations.
Thrombin peaks stimulated by 100 nM Undaria pinnatifida fucoidan in
the FVIII inhibited FVII deficient plasma were small and delayed,
even in the presence of high tissue factor concentrations, compared
to the peaks stimulated in the FVII deficient plasma. Results are
shown in Table 18 below.
TABLE-US-00018 TABLE 18 Tissue factor (pM) 0 1 5 20 Peak Peak Peak
Peak Peak Peak Peak Peak thrombin time thrombin time thrombin time
thrombin time Plasma (nM) (min) (nM) (min) (nM) (min) (nM) (min)
FVII 303.1 15.8 305.9 14.0 313.4 12.5 285.3 11.7 deficient FVIII 0
>120 2.6 38.3 15.4 32.2 62.7 26.8 inhibited FVII deficient
[0121] The results show that in extrinsically compromised plasma,
fucoidan acts to stimulate thrombin generation via the intrinsic
pathway, even at high tissue factor concentrations.
Example 14
Sulfated Polysaccharides May be Useful in Place of Coagulation
Factor Therapy in Hemophilia
[0122] Patients with less than 1% normal FVIII are considered to
have severe hemophilia, with 1-5% moderately severe hemophilia, and
with more than 5% but less than 40% mild hemophilia. An experiment
was performed to evaluate fucoidan stimulated thrombin generation
at low concentrations of FVIII that reflect the levels of FVIII
present in plasma of patients with hemophilia A. No tissue factor
was added. Fucoidans from Undaria pinnatifida (100 nM) and
Ascopliyllum nodosum, high MW (10 nM) were tested. FVIII was added
to FVIII deficient plasma at a range of concentrations to provide
FVIII at 0, 0.2, 0.5, 1, 2 or 10% of the FVIII present in normal
plasma. These figures do not take into account any residual FVIII
present in the FVIII deficient plasma. Results are shown in Table
19 below. In a control experiment, the plasma was pre-incubated
with anti-FXI antibody. No thrombin peaks were observed in the
presence of either fucoidan at any of the concentrations of FVIII
tested (not shown).
TABLE-US-00019 TABLE 19 % FVIII compared to normal plasma 0 0.2 0.5
1 2 10 fucoidan Peak thrombin (nM) U. pinnatifida 5.6 35.0 54.1
65.9 74.7 93.3 A. nodosum 0 2.4 6.4 10.2 19.3 35.8 -- 0 0 0 0 0 0
fucoidan Peak time (min) U. pinnatifida 50.2 42.7 38.3 34.2 33.0
28.3 A. nodosum >120 56.9 51.4 46.5 42.3 34.2 -- >120 >120
>120 >120 >120 >120
[0123] Results show that even at low concentrations of FViil,
sulfated polysaccharides stimulate thrombin generation. The absence
of any thrombin peaks when FXI is inhibited show that a FXI
dependent mechanism is responsible for this activity. Thus,
sulfated polysaccharides may be useful in treating hemophiliacs via
a FXI-dependent mechanism.
Example 15
Sulfated Polysaccharides May be Useful in Place of Coagulation
Factor Therapy in Hemophilia B
[0124] Sulfated polysaccharides were tested at a range of
concentrations in hemophilia B plasma in the CAT assay. The
concentration of tissue factor was 1 pM. At concentrations of up to
100 nM, fucoidan from Fucus vesiculosus improved thrombin
generation parameters (i.e. increased peak thrombin and decreased
peak time) in a concentration-dependent manner. At higher fucoidan
concentrations of 250, 800 and 2000 nM, thrombin generation
parameters deteriorated in a concentration-dependent manner. A
similar pattern was observed for each of the sulfated
polysaccharides tested, with an optimal effect on thrombin
generation at an intermediate concentration, and sub-optimal
effects at lower and higher concentrations. The optimal effect was
achieved at a comparable .mu.g/ml concentration of each sulfated
polysaccharide, although the nM concentrations varied over two
orders of magnitude. The FIX equivalent activity of the
concentration of each of the six sulfated polysaccharides tested
that had the most beneficial effect on peak thrombin was estimated.
Results are shown in Table 20 below.
TABLE-US-00020 TABLE 20 Sulfated polysaccharide Conc (nM) Conc
(.mu.g/ml) FIX EA (mU/ml) PPS 1000 5.9 32 Fuc An LMW 1000 7.5 58
Fuc Fv 100 11.6 42 Fuc Up 100 12.7 41 Fuc An HMW 25 15.0 80 Fuc Lj
10 10.0 76
[0125] Further data are given in Table 21 below, indicating the
"therapeutic window" for each sulfated polysaccharide. The
"therapeutic window" is the concentration range at which the
sulfated polysaccharide provides for a peak thrombin in severe
hemophilia B plasma (FIX activity below 1% of normal plasma) which
is at least the peak thrombin provided by the addition of 10 mU/mL
(1%) of factor IX to severe hemophilia B plasma. Also shown is the
FIX equivalent activity of the optimal concentration of sulfated
polysaccharide and, in brackets, the FIX equivalent activity of the
polysaccharide concentration at either end of the therapeutic
window. The results indicate that each sulfated polysaccharide has
a procoagulant effect across a broad concentration range.
TABLE-US-00021 TABLE 21 Sulfated Therapeutic window FIX EA
polysaccharide MW (kD) nM .mu.g/mL (mU/ml) PPS 5.9 250-6667* 1.5-39
32 (27-12) A.n. LMW 7.5 100-20000* 0.8-150 58 (13-28) F.v. 115
20-2000* 2.3-232 42 (26-12) U.p. 127 8-2000* 1.0-254 41 (11-19)
A.n. HMW 600 5-333 3-200 80 (28-22) L.j. >1000 2.5-100 2.5-100
76 (22-23) *indicates that the upper limit given for the
therapeutic window was the highest concentration of sulfated
polysaccharide tested.
Example 16
Treatment of a Patient Dependent on Factor XI Status
[0126] A patient may consult a physician prior to elective surgery.
As the surgery carries the risk of bleeding, the physician may plan
to administer a sulfated polysaccharide before or shortly after
surgery, in the event that the patient suffers undue bleeding
following surgery. The physician will wish to check whether the
patient is suitable for such therapy, and will therefore check the
patient's records and/or perform testing to determine whether the
patient has hemophilia C. Certain patients may be at particular
risk of hemophilia C, for example patients having a family history
of the condition.
[0127] If the patient has a normal level of plasma factor XI:c
activity (greater than 70 IU/dL), the patient can be administered a
sulfated polysaccharide either before surgery, or following surgery
in the event that they suffer bleeding.
[0128] If the patient has a partial deficiency (plasma factor XI:c
activity of 20-70 IU/dL), or a severe deficiency (plasma factor
XI:c activity of less than 20 IU/dL), the physician may decide to
administer the sulfated polysaccharide before or after surgery in
combination with factor XI concentrate or fresh frozen plasma.
* * * * *