Note: Descriptions are shown in the official language in which they were submitted.
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Title: Potentiation of complement and coagulation inhibitory
properties of C1-inhibitor
Field of the InventiQn
This invention is in the fields of immunology and
biochemistry and describes a method to modify the inhibitory
spectrum of C1-inhibitor, a major plasma inhibitor of
multiple proteases of the complement, contact, fibrinolytic
and coagulation plasma cascade systems. More specifically,
it is demonstrated that inhibition of complement and
clotting proteases by C1-inhibitor can ~e potentiated up to
over 100-fold, without affecting its inhibitory properties
towards fibrinolytic or contact system proteases. This
potentiation is achieved by incubating C1-inhibitor with the
synthetic sulfated polysaccharide dextran sulphate.
Pharmaceutical compositions containing potentiated
C1-inhibitor have considerable applications, for example as
anti-inflammatory agent for the prophylactic or therapeutic
treatment of sepsis or myocardial infarction.
Background of the Invention
Inflammatory reactions occur in the course of numerous
human and Anim~l diseases and are mediated by an array of
so-called inflammatory mediators. Gallin JI, Goldstein IM,
Snyderman R (eds): Inflammation: Basic Principles and
Clinical Correlates, New York, Raven Press Ltd, 1992.
Inflammatory mediators include activated monocytes, macro-
phages, neutrophils, eosinophils, basophils, mast cells,
platelets and endothelial cells; cytokines; prostaglandins;
leukotrienes; platelet activating factor; histamin and
serotonin; neuropeptides; reactive oxygen species; and
nitric oxide and related compounds.
Also the major plasma cascade systems, which include
the coagulation, fibrinolytic, contact and complement
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systems, contribute to inflammatory reactions since during
activation of these systems fragments are generated, which
have potent biological effects and are therefore considered
to be inflammatory mediators. The plasma cascade systems
each consist of a series of plasma proteins, most of which
are synthesized by the liver and circulate in blood as
inactive precursors, also called factors. Activation of the
first factor of a system comprises conversion by limited
proteolysis of the inactive, often single-chain precursor
into a cleaved often two-chain active protein. This
activated first factor subsequently activates, again by
limited proteolysis, a number of inactive second factors,
which in turn each activate a number of third factors and so
on. This reaction pattern resembles a cascade. Excessive
activation of the plasma cascade systems is regulated by the
presence of a series of inhibitors including the multi-
specific inhibitor ~2-macroglobulin and the serine protein-
ase inhibitors (serpins) antithrombin III, (~l-antitrypsin,
~ antichymotrypsin, .L2-antiplasmin, C1-inhibitor, and
others.
The complement system
The complement system constitutes one of the plasma
cascade systems. Its physiological role is to defend the
body against invading micro-organisms and to remove necrotic
tissue and cellular debris.
The complement system can be activated via two path-
ways, a classical and an alternative pathway, which both can
trigger activation of a common terminal pathway. Cooper
N.R., 1985, Adv Immunol 37: 151; Muller-Eberhard H.J. et
al., 1980, Adv Immunol 29: 1; Muller-Eberhard H.J., 1992,
In: &allin JI, Goldstein I~, Snyderman R (eds):
In~lammation: Basic Principles and Clinical Correlates, New
York, Raven Press Ltd, p.33.
~ctivation of complement results in the generation of
biologically active peptides, also known as the anaphyla-
toxins. These anaphylatoxins, in particular C3a and C5a, are
chemotactic for neutrophils and able to aggregate, activate
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and degranulate these cells. vogt W., 1986, Complement 3:
177; Goldstein IM, 19~2, In: Gallin JI, Goldstein IM,
Snyderman R ~eds): Inflammation: Basic Principles and
Clinical ~orrelates, New York, Raven Press Ltd~ p.63; Hugli
TE, 1984, Springer Semin Tmmll nopathol 7: 193. Furthermore,
they may enhance vasopermeability, stimulate adhesion of
neutrophils to endothelium, activate platelets and endothe-
lial cells, and induce degranulation of mast cells and the
production of vasoactive eicosanoids, thromboxane A2 and
peptidoleukotrienes such as LTC4, ~TD4 and ~TE4 by mono-
nuclear cells. Also the so-called terminal complement com-
plexes (TCC), formed upon activation of the common pathway,
have important biological effects including the capability
to lyse target cells and, at sublytic concentrations, to
induce cells to release mediators, such as cytokines,
proteinases and eicosanoids. Muller-Eberhard ~.J., 1986, Ann
Rev Immunol 4: 503; Hansch GM, 1992, Immunopharmacol 24:
107. Finally, complement activation products may induce the
expression of tissue factor by cells and thereby initiate
and enhance coagulation. Osterud B et al., 1984, Haemostasis
14: 386; Hamilton KK et al., 1990, J Biol Chem 265: 3809.
Thus, complement activation products have a number of
biological effects, which may induce or enhance inflammatory
reactions.
Activation of complement is considered to play an
important role in the pathogenesis of a number of inflamma-
tory disorders, including sepsis and septic shock; toxicity
induced ~y the in yivo a~mi ni ~tration of cytokines or
monoclonal antibodies ~mAbs); immune complex diseases such
as rheumatoid arthritis, systemic lupus erythematosus and
vasculitis; multiple trauma; ischaemia-reperfusion injuries;
myocardial infarction; and so on. The pathogenetic role of
complement activation in these conditions is likely related
in some way or another to the aforementioned biological
effects of its activation products. Inhibition of complement
activation may, therefore, add to the treatment of these
conditions.
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As just mentioned, complement can be acti~ated via two
different pathways, the classical and the alternative
pathway. The latter will not be discussed here since C1-
inhibitor is not known to have an effect on this pathway.
Classical pathway activation starts with activation of the
first component, which consists of a macromolecular comple~
~f 5 proteins, one Clq, two Clr and two Cls proteins. The
'q protein of the Cl complex binds to an activator, for
~mple immune complexes, which leads to activation of=both
and both Cls subcomponents. Schumaker VN et al., 1987,
ev Immunol 5: 21, Cooper N.R., 1985, Adv Immunol 37
151. During activation Clr and Cls are converted from single
peptide-chain inactive proteins into two-chain active serine
proteinases. The activated Cl complex then activates the
complement factors C4 and C2, which together ~orm the bi-
molecular C4b,2a complex. Polley MJ et al., 1968, J Exp Med
128: 533; Kerr MA, 1980, Biochem J 189: 173. This complex
then activates C3, the third component of complement, by
cleaving it into the smaller fragment C3a and the larger
C3b. The C4b,2a complex is hence called a C3-convertase.
Cleavage of C5 by a C5-convertase, which is generated
by fixation of an additional C3b molecule to a C3-
convertase, yields the anaphylatoxin C5a and nascent C5b,
which latter together with C6 forms the bimolecular C5b,C6
complex, which in turn binds C7. The C5b,C6,C7 complex
either inserts into a membrane or interacts with S protein.
Interaction with S protein finally yields soluble membrane
attack complexes (MAC). C5b,C6,C7 inserted into a membrane
forms a receptor for C8. Subsequently, the tetramolecular
C5b-8 complex will bind and polymerize C9, yielding fully
assembled membrane-inserted MAC complexes, each consisting
of the C5b-8 complex and one or more C9 molecules. Muller-
Eberhard H.J., 1992, In: Gallin JI, Goldstein IM, Snyderman
R (eds): Inflammation: BasiC Principles and Clinical
Correlates, New York, Raven Press, p.33; Muller-Eberhard HJ,
1986, Annu Rev Immunol 4: 503.
Several plasma pro~eins can inhibit activation o~ the
classical pathway of complement, notably, C1-inhibitor, C4-
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binding protein and the serine-proteinase factor I. Muller-
Eberhard H.J., 1992, In: Gallin JI, Goldstein IM, Snyderman
(eds): Inflammation: Basic Principles and Clinical
Correlates, New York, Raven Press, p.33; Schumaker VN et
al., 1987, Ann Rev Imrnunol 5: 21; Cooper NR, 1985, Adv
Immunol 37: 151. Of=these, C1-inhibitor will be described in
more detail below.
The contact system
The contact system consists of a set of proteins, which
circulate in blood as inactive precursor proteins. The
system is also known as the contact system of coagulation or
the kallikrein-kinin system. Colman R.W., 1984, J Clin
Invest 73: 1249; Kaplan A.P. et al., 1987, Blood 70: 1;
Kozin F. et al., 1992, In: Gallin JI, Goldstein IM,
Snyderman R ~eds): Inflammation: Basic Principles and
Clinical Correlates, New York, Raven Press, p.l03. The
contact system constitutes one of the major plasma cascade
systems, and is often regarded as one of the two pathways of
clotting, the so-called extrinsic pathway of coagulation
being the other.
Activation of the contact system starts with the
binding of factor XII, also known as Hageman factor, to an
activator. SubsequentLy, bound factor XII may become
activated, during which process it is converted from a
single-chain inactive into a two-chain active serine
proteinase. Tans &. et al., 1987, Sem Thromb Hemost 13: 1.
Activated factor XII then activates prekallikrein, that via
its cofactor high molecular weight kininogen is bound to the
activator, into the active serine proteinase kallikrein.
Rallikrein in turn may activate bound hut not yet activated
factor XII ~reciprocal activation). Factor XIIa may activate
factor XI, which in turn can activate factor IX to start
activation of coagulation. Cochrane C.G. et al., 1982, Adv
Immunol 33: 290; Colman R.W., 1984, J Clin Invest 73: 1249;
Kaplan A.P. et al., 1987, Blood 70: 1; Kozin F. et al.,
1992, In: Gallin JI, Goldstein IM, Snyderman R (eds):
Inflammation: Basic Principles and Clinical Correlates, New
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York, Raven Press, p.103. Activation of the contact system
is controlled by the same protein that also inhibits the
classical complement pathway, C1-inhibitor, and which will
be discussed below. During activation of the contact system
several biologically active fragments are formed such as
bradykinin, kallikrein and activated factor XII. These
fragments may enhance activation and degranulation of
neutrophils, increase vasopermeability and decrease vascular
tonus. Colman R.W., 1984, J Clin Invest 73: 1249; Kozin F.
et al., 1992, In: Gallin JI, Goldstein IM, Snyderman R
(eds): Inflammation: Basic Principles and Clinical
Correlates, New York, Raven Press, p.103.
It is generally accepted that the contact system
becomes activated in inflammatory conditions. Colman R.W.,
1984, J Clin Invest 73: 1249; Kaplan A.P. et al., 1987,
Blood 70: 1; Kozin F. et al., 1992, In: Gallin JI, Goldstein
IM, Snyderman R ~eds): In~lammation: Basic Principles and
Clinical Correlates, New Yor~, Raven Press, p.103. However,
its precise role in inflammation as well as that under
physiological conditions is not well understood. Persons
with a genetic deficienc~ of factor XII may have an
increased risk for thromboembolic disease. This, together
with its tissue type-plasminogen-like structure lTans G. et
al., 1987, Sem Thromb Hemost 13: 1), suggests that factor
XII participates in fibrinolysis. In vivo observations on
the contribution of factor XII to plasminogen activation in
homo- and heterozygous factor XII deficient individuals are
in agreement herewith. Levi M. et al., 1991, J Clin Invest
88: 1155.
Factor XI is often considered as a member of the
contact system since in vitro it can be activated by factor
XII. Kurachi K. et al., lg77, Biochemistry 16: 5831. It is a
dimeric glycoprotein consisting of two identical polypeptide
chains held together by a disulfide bond. Upon activation,
each polypeptide chain can be cleaved at an internal peptide
bond giving rise to disulfide linked heavy and light chains,
the latter each containing one active site. Bouma B.N. et
al., 1977, ~ Biol Chem 252: 6432; Van der Graaf F. et al.,
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1983, J Biol Chem 258: 9669; Fujikawa K. et al., 1986,
Biochemistry 25: 2417. The activity of each active site of
factor XIa is regulated by plasma protease inhibitors
including ~ antitrypsin, antithrombin III, C1-inhibitor,
and ~2-antiplasmin, each a member of the superfamily of
serine protease inhibitors (serpins). Soons H. et al., 1987,
Biochemistry 26: 4624-4629. Heck L.W. et al., 1974, J Exp
Med 140: 1~15; Damus P.S. et al., 1973, Nature 246: 355;
Forbes C.D. et al., 1970, J Lab Clin Med 76: 809; Saito H.
et al., 1979, Proc Natl Acad Sci US~ 76: 2013. Initial
studies suggested ~I1-antitrypsin to be the main inhibitor of
factor XIa in plasma. Scott C.F. et al., 1982, J Clin Invest
69: 844. However, studies with enzyme-linked immunosorbent
assays to quantitate complexes between factor XIa and its
inhibitors in plasma demonstrated Cl-inhibitor to be a major
inhibitor of factor XIa. Wui~lemin W.A. et al., 1995, Blood
85: 1517.
T~e in vivo role of factor XI may be unrelated to
contact activation: recent studies have suggested that
activation of factor XI may occur independently from factor
XII via thrombin and contribute to activation of factor IX.
Naito K. et al., 1991, J Biol ~hem 266: 7353; Gailani D. et
al., 19gl, Science 253: 909. In this view factor XI acts to
enhance thrombin generation, initially induced by the
extrinsic pathway. Davie E.W. et al., 1991, Biochemistry 30:
10363; Broze Jr. G.J., 1992, Seminars Hematol 29: 159. This
supposed role of factor XI in the coagulation system is
consistent with clinical data that the only deficiency of a
contact system protein, which results in a (mild~ bleeding
disorder, is that of factor XI. This, together with the lack
of evidence that in vivo the contact system participates in
the process of coagulation, raises serious doubts on whether
factor XI should be considered as a contact system protein.
Anyway, regardless the precise role of factor XI in the
clotting mechanism, inhibition of factor XIa will attenuate
coagulation, without the risk of a severe bleeding tendency
as for example is induced by heparin-mediated potentiation
of antithrombin III.
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Cl-inhibitor
Cl-inhibitor, also known as Cl-esterase inhibitor,
refers to a protein that is present in blood and is the main
inhibitor of the classical pathway of complement and of the
contact system. C1-inhibitor can inhibit the activated ~orm
of the first component of complement and activated factor
XII, and it is also a major inhibitor of kallikrein.
Schapira M. et al., 1985, Complement 2: 111; Davis A.E.,
1988, Ann Rev Immunol 6: 595; Sim R.B. et al., 1979, FEBS
Ilett 97: 111; De Agostini A. et al., 1984, J Clin Invest 73:
1542; Pixley R.A. et al., 1985, J Biol Chem 260: 1723;
Schapira M. et al., 1982, J Clin Invest 69: 462; Van der
Graaf F. et al., 1983, J Clin Invest 71: 149; Harpel P.C. et
al., 1975, J Clin Invest 55: 593. Thus, Cl-inhibitor
regulates the activity of two plasma cascade systems, i.e.
the complement and contact systems, that during activation
generate biologically active peptides. C1-inhibitor is,
therefore, an important regulator of inflammatory reactions.
In addition, C1-inhibitor is a major inhibitor of activated
factor XI. Meijers J.C.M. et al., 1988, Biochemis~ry 27:
959; Wuillemin W.A. et al., 1995, Blood 85: 1517. Conside-
ring the possible function of factor XI as discu~sed above,
C1-inhibitor should therefore also be considered as a coa-
gulation inhibitor. ~lso tissue-type plasminogen activator
and plasmin are inhibited to some extent by Cl-inhibitor,
although this inhibitor is not the major inhibitor of these
proteinases. Harpel P.C. et al., 1975, J Clin Invest 55
149; Booth N.A. et al., 1987, Blood 69: 160Q. Cl-inhi~itor
should therefore also be considered as a (weak) fibrinolytic
inhibitor.
C1-inhibitor has been purified from plasma at large
scale and used for clinical application, particularly in the
treatment of heriditary angioedema, a disease caused by a
genetic deficiency of C1-inhibitor. Furthermore, a~mini~-
tration of Cl-inhibitor has been claimed to have beneficial
effects in other diseases as well, such as systemic inflam-
matory responses in m~mm~ls r~ong S., 1992, WO 92/22320
~GenentecA Inc)], and of complica~ions of severe burns,
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pancreatitis, bone marrow transplantation, cytokine therapy
and the use of extracorporeal circuits LEisele B. et al.,
1994, DE-A-4227762 (Behringwerke AG)]. The present invention
relates to these therapeutical applications of Cl-inhibitor
- 5 in that it provides a novel method to enhance the inhibitory
activity of C1-inhibitor, and hence reduces the amount of
C1-inhibitor needed for these therapies.
Full-length genomic and cDNA coding for C1-inhibitor
has been cloned. Bock S.C~ et al., 1986, Biochemistry 25:
~292; Carter P.E. et al., 1988, Eur J Biochem 173: 163.
Functional recombinant C1-inhibitor protein has been
expressed in COS cells and found to be similar to the plasma
protein. Eldering E. et al., 1988, J Biol Chem 263: 11776.
Several variants of recombinant C1-inhibitor with amino acid
mutations a~ the P1 and the P3 and/or P5 position of the
reactive centre as well as variants isolated from patients
with hereditary angioedema have been expressed in the same
system. Eldering E. et al., 1988, J Biol Chem 263: 11776;
Eldering E. et al., 1993, J Biol Chem 267: 7013; Eldering E.
et al., 19g3, J Clin Invest 91: 103S; Patent cetus Corp,
US617920; Davis A.E. et al., 1992, Nature Genetics 1:~354;
~ldering E. et al., 1995r J Biol Chem ~70: 2579; Verpy et
al., 1995, J Clin Invest 95: 350.
C1-inhibitor belongs to a superfamily of homologous
proteins known as the serine-proteinase inhibitors, also
called serpins. Travis J. et al., 1983, Ann Rev Biochem 52:
655; Carrel R.W. et al., 1985, Trends Bioch Sci lQ: 20. on
sodium dodecylsulphate polyacrylamide gels C1-inhibitor has
an apparent molecular weight of approximately 105 kD. Its
plasma concentration is about 270 mg/l. Schapira M et al.,
1985, Complement 2 111; Nuijens JH et al., 1989, J Clin
~nvest 34: 443. Cl-inhibitor is an acute phase protein whose
levels may increase up to 2-fold during uncomplicated
infections and other inflammatory conditions. Kalter BS et
- 35 al~ t 1985, J Infect Dis 151: 1019. The increased synthesis
of C1-inhibitor in inflammatory conditions is most probably
meant to protect the organism against the deleterious
e~fects of (intravascular) activation of the complement and
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contact systems during acute phase reactions. In patients
with rheumatoid arthritis the synthetic rate of C1-inhibitor
may increase up to 2.5 ~imes the normal rate. Woo et al.,
1985, Clin Exp Immunol 61: 1. Metabolic studies with
radiolabeled C1-inhibitor in normal volunteers have yielded
a fractional catabolic rate (FCR) of 2.5~ of the plasma pool
per hour and an apparent plasma half-life time of clearance
of about 20 hours. Woo et al., 1985, Clin Exp Immunol 61- 1;
Quastel M. et al., 1983, J Clin Invest 71: 1041. __
The serpins share a similar mechanism of inhibition,
which is characterized by forming stable bi-molecular
complexes with the proteinase to be inhibited. In these
complexes the active site of the proteinase is bound to the
so-called reactive centre of the serpin and hence rendered
inactive. Travis J. et al., 1983, Ann Rev ~iochem 52: 655.
Like other serpins C1-inhibitor inhibits proteinases by
forming stable complexes with these proteinases, which are
rapidly cleared from the circulation. De Smet B.J.G.L. et
al., 1993, Blood 81: 56. Ser~ins have specificity for cer-
tain proteinases and this specificity is in part determined
by the amino acid sequence o~ the reactive centre.
The activity of serpins may be influenced by glycos-
aminoglycans, a heterogeneous group o~ macromolecular
sulphated glycoconjugates linked to a protein core. Kjellen
L. et al., 1991, Annu Rev Biochem 60: 443; Poole ~.R., 1986,
J Biochem 236: 1; ~ourin M.-C. et al., 1993, Biochemical
289: 313. This group includes the physiological compounds
heparin, heparan sulfate and dermatan sulfate. Poole A.R.,
1986, J Biochem 236: 1. For example, heparan sulfate and
heparin-like molecules are endothelial cell-associated
glycosaminoglycan in the vascular bed. Ausprunk D.H. et al.,
1981, Am J Pathol 103: 353; Marcum J.A. et al., 1985,
Biochem Biophys Res Comm 126: 365; Ihrcke N.S. et al., 1993,
Immunolo~y Today 14: 500. Glycosaminoglycans have been
claimed to ha~e anti-metastatic and/or anti-inflammatory
activities based on their properties to inhibit endoglyco-
sidases, particularly heparinase. Parish C.R. et al., 1988,
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11
WO 88/05301 (Australia University). This effect of glycos-
aminoglycans is unrelated to the present invention.
Its enhancing effects on the function of antithrombin
underlie the therapeutical use of heparin. Furthermore,
sulphated polysaccharides may exert additional anticoagulant
activities in the presence of lipoprotein-associated coagu-
lation inhibitor (LACI ), which effect has been patented for
therapeutic application. Tze-Chein Wun, 1992, ~P-A-0473564
(Monsanto Company). The semisynthetic sulphated polysaccha-
ride dextran sulphate has less enhancing effects on anti-
thrombin III then heparin, although it may potentiate other
inhibitors of coagulation such as protease nexin-l (PN-l).
Scott R.W., l991r WO 91/05566 (Invitron Corp.). These
effects of sulphated polysaccharides on clotting inhibitors
are unrelated to the present invention, which is dealing
with the interaction of dextran sulphate and Cl-inhibitor.
The heparin-antithrombin III interaction is probably the
best studied example of glycosaminoglycan-enhanced function
of a serpin. However, a number of studies have also shown
that glycosaminoglycans, in particular heparin, may also
potentiate the function of other serpins including C1-
inhibitor: In kinetic assays with purified proteins heparin
has been shown to potentiate the inhibition of Cls by C1-
inhibitor 15- to 35-fold, whereas the inhibition of
2~ activated C1 or Clr is less enhanced. Rent R. et al., 197G,
Clin Exp Immunol 23: 264; Sim R.B. et al., i980, Biochim
Biophys Acta 612: 433; Caughman G.B. et al., 1982, Mol
Immunol 12: 287; Nilsson T. et al., 1983, Eur J Biochem 129:
663; ~ennick M. et al., 1986, Biochemistry 25: 3890; Hortin
G.L. et al., 1991, Immunol Invest 20: 75. This enhanced
interaction o~ Cls occurs at the expense of an increased
proteolytic inactivation of Cl-inhibitor. Weiss V. et al.,
1983, Hoppe-Seyler's Z Physiol Chem 364: 295. In addition to
these effects on C1-inhibitor heparin has multiple other
effects on the complement system such as inhibiting effects
on the binding of Clq to an activator, on the activity of
C1-esterase and on the formation of the classical C3-
convertase. Raepple E. et al., 1976, Immunochemistry 13:
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251; Loos M. et al., 1976, Immunochemistry 13: 257, Strunk
R. et al., 1976, Clin Immunol Immunopathol 6: 248. ~eparin
might, therefore/ be considered as a therapeutic complement
inhibitor. However, the complement-inhibiting eff~cts of
heparin are observed at concentrations at least one order
higher than those required for anticoagulant effects, and
using such doses in vivo carries the unacceptable risk of
bleeding. To reduce its anticoagulant properties a N-
desulfated, N-acetylated form of heparin has been developed,
which preparation has been shown to possess significant
complement inhibitory properties. Weiler J.M. et al., 1992,
J Immunol 148: 3210; Friedrichs G.S. et al., 1994, Circ Res
75: 701. However, this does not obviate another disadvantage
o~ the use of heparin (or any other glycosaminoglycan),
i.e., that it has to be purified ~rom ~nim~l sources.
The mechanism by which glycosaminoglycans potentiate
Cl-inhibitor towards inhibition of its target proteases Cls
and factor XIa is not known. ~owever, in analogy to what is
known ~or heparin-accelerated inhibition of thrombin by
antithrombin III, several mechanisms are postulated: (I)
Glycosaminoglycans may induce a conformational change in the
inhibitor, rendering it more active; (II) Glycosaminoglycans
may work as a template on which inhibitor and target
protease may assemble; ( III) Glycosaminoglycans may
neutralize positive charges either on the inhibitor or on
the protease or both, thereby allowing a more easy inter-
action. Evans D.L. et al., 1992, Biochemistry 31: 12629;
Bode W. et al., 1994, Fibrinolysis 8: 161; Potempa J. et
al., 1~94, J Biol Chem 269: 15957. Which one of these
mech~ni~m(s~ applies to the observed glycosaminoglycan-
induced enhancement of C1-inhibitor function remains to_be
shown in further studies.
In the present invention the synthetic sulphated
polysaccharide dextran sulphate is used to enhance the
inhibitory activity of Cl-inhibitor. Dextran sulphate and
related compounds may be effective inhibitors of human
immune deficiency virus type 1. De Clercq E.D.A. et al.,
lg88, EP-A-0293826 (Stichting Rega V.Z.W.). In addition,
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dextran sulphate may be useful for the treatment of
arteriosclerosis. Herr D., 1988, EP-A-0276370 (Knoll ~G).
These effects are unrelated to the present invention.
Furthermore, high molecular weight species of dextran
5 sulphate, but not low molecular weight species, are able to
enhance auto-activation of factor XII of the contact system.
Samuel M. et al., 1992, J Biol Chem 267:~ 19691.
Summary of the Invention -
It has now been found that inhibitory properties of
C1-inhibitor, a major inhibitor of various complement,
clotting, contact system and fibrinolytic proteases, can be
modified by incubation with a semisynthetic polyanionic
compound, the sulphated polysaccharide dextran sulphate,
yielding a C1-inhibitor selectively potentiated up to over
100-~old regarding its complement and clotting inhibitory
properties. There~ore, the present invention contemplates a
pharmaceutical composition containing C1-inhibitor with
selectively enhanced function, that can be used
prophylactically or therapeutically to inhibit activation of
complement and/or coagulation in vivo. The pharmaceutical
composition comprises C1-inhibitor and dextran sulphate
species. Exemplary compositions may contain Cl~inhibitor
derived from human plasma or any other biological source, or
recombinant Cl-esterase inhibitor, or mutants derived
therefrom. Exemplary compositions may also contain dextran
sulphate of varying molecular weight, or any other synthetic
polyanionic compound with comparable effects.
The invention will be more fully understood after a
consideration of the following description of the inven~ion.
Brief ~escription of the Drawings
Figure 1. Influence of glycosaminoglycans or DXS on the
amidolytic activity of factor XIa. The amidolytic activity
of factor XIa was determined as the initial change in
a~sorbance at 405 nm at 37 C using the chromogenic substrate
S-2366 at a final concentration of 0.4 mmol/l in a buffer
cont~in;ng 0.1 mol/l Tris-HCl, pH 7.4, 0.14 mol/l NaCl, and
-
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W O 97/22347 14 PCT/NL96/00488
0.1 ~ (wt/vol) Tw. The effect of different amounts of DXS MW
500,000 (solid circles), DXS MW 5,000 (triangles)/ heparin
(open circles), heparan sulfate (solid squares), or dermatan
sulfate (open squares) was tested. Results are expressed as
the percentage of the activity of 1 nmol/l factor XIa, in
the absence of any glycosaminoglycans, rem~ining after
addition of varying amounts of different glycosaminoglycans
(I~g/ml, final concentrations~.
Figure 2. Kinetics of the inactivation of factor=XIa by
Cl-inhibitor in the absence of glycosaminoglycans or DXS.
Factor XIa (final concentration 6 nmol/l) was incubated at
37~C with different concentrations of C1-inhibitor in
0.1 mol/l Tris-HCl, pH 7.4, 0.14 mol/l NaCl, 0.1 % Tw. At
various times, aliquots were removed and assayed for
residual amidolytic activity of factor XIa. (Panel A)
Inactivation of ~actor XIa was assessed in the presence of
C1-inhibitor at 0 (solid circles), 0.32 (open circles), 0.64
(solid squares), 0.96 (open squares) or 1.28 (plus signs)
~mol/l. The natural lo~arithm o~ residual factor XIa
amidolytic activity was plotted against time. (Panel B) The
pseudo first-order rate constants (k, min-1) were calculated
from the slopes of the plots shown in panel A and plo~ted as
a function of the C1-inhibitor concentration. The slope of
the line represents the second order rate constant (k2,
min-l, M-1)
Figure 3. Kinetics of the inactivation of factor XIa by
Cl-inhibitor in the presence of glycosaminoglycans or DXS.
Factor XIa (final concentrations 3 to 8 nmol/l~ was
incubated at 37~C with C1-inhibitor (final concentration
0.32 ~molfl) in 0.1 mol/l Tris-~Cl, pH 7.4, 0.14 mol/l NaCl,
O.1 ~ Tw, and the pseudo first-order rate constants were
determined as described in legend to Figure 2. Results are
expressed as the potentiation factor of the inhibition of
factor XIa by C1-inhibitor in the presence of varying
amounts of DXS MW 500,000, DXS MW 5,000, heparin, heparan
sulfate or dermatan sulfate, compared with the inhibition
rate in the absence of glycosaminoglycans or DXS.
CA 02239787 1998-06-17
W O 97/Z2347 PCT/NL96/00488
Figure 4. Pseudo first-order rate constan~s of factor
XIa inhibition by C1-inhibitor in the presence of glycos-
aminoglycans or DXS. The pseudo first-order rate constants
were determined as described in legend to Figure 2, in the
presence of varying concentrations of C1-inhibitor and in
the presence of D~S MW 500,000, DXS MW 5,000, heparin,
heparan sulfate or derma~an sulfate, or in the absence of
glycosaminoglycans or DXS. The s~ope of the lines represents
the second order rate constants tk2, min-l, M-l).
Figure 5. Inhibition of Cls by C1-inhibitor in the
presence of various glycosaminoglycans or DXS. Cls at a
final concentration of 3 nmol/l was incubated with C1-
inhibitor ~final concentration 15 nmol/l) and various
glycosaminoglycans (each tested at 10 ~g/ml) in phosphate
buffered saline ~PBS)-0.0~ % Tw, containing the chromogenic
substrate S2314 at a final concentration of 0.8 mmol/l at
37 C. The change in absorbance at 405 nm in time is shown.
Figure 6. Dose-response of the enhancing effect of DXS
MW 500,000 on the inhibition of Cls by C1-inhibitor. Condi-
tions used are the same as described in Figure 5.
Figure 7. Dose-response of the enhancing effect of DXS
MW ~,000 on the inhibition of Cls by C1-inhibitor. Condi-
tions used are the same as described in Figure 5.
Figure 8. Inhibition by DXS MW 500,000 of complement
activation in recalcified plasma by aggregated human IgG.
Citrated (10 mmol/l, final concentration) blood was recalci-
fied by adding 10 mM CaCl2 (final concentration). After
15 min at 37~C a clot had formed, which was removed by
centrifugation for 10 min at 2,000 x g at 4~C. One vol of
recalcified plasma was then incubated with one vol veronal
buffered saline cont~ining aggregated human IgG at a
concentration of 5 mg/ml for 20 min at 37 C. Complement
activation during this incubation was then measured by
assessing the generation of Cls-Cl-inhibitor complexes, C4
and C3 activation products (C4b/C4bi/C4c and C3b/C3bi/C3c,
respectively). Aggregated IgG was prepared as described.
Hack C.E. et al., 1981, J Immunol 127: 1450. Cls-
C1-inhibitor complexes and C3 and C4 activation products
,
CA 02239787 l998-06-l7
W O 97/22347 PCT/NL96/00488 16
were measured as previously described. Nuijens J.H. et al.,
1989, J Clin Invest 84: 443; Wolbink G.J. et al., 1993, J
Immunol Meth 163: 67. Results (mean and standard deviation
of 3 experiments) are shown as % inhibition, 0% being the
generation of activation products in the absence of DXS,
100% being the generation of complement activation products
in the absence of aggregated IgG and DXS.
Figure 9. Inhibition by DXS MW 5,000 of complement
activation in recalcified plasma by aggregated human IgG.
The experiment was performed similarly as the one described
in Figure 8, except that DXS MW 5,0~0 was used.
Figure 10. Inhibition by heparin of complement
activation in recalcified plasma by aggregated human IgG.
The experiment was performed similarly as that described in
Figure 8, except that heparin was used.
Figure ll. Inhibition by N-acetyl-heparin of complement
activation in recalcified plasma by aggregated human IgG.
The experiment was performed similarly as the one described
in Figure 8, except that N-acetyl-heparin was used.
Detailed Description of the Invention
Several patents/patents applications and scientific
articles are referred to below that discuss various aspects
of the materials and methods used to realize the invention.
It is intended that all of the references be entirely
incorporated by reference.
The kernel of the present invention is the realization
that Cl-inhibitor, a major inhibitor of complement,
clotting, contact system and fibrinolytic proteases in
plasma can be modified regarding its inhibitory spectrum by
the semisynthetic compound dextran sulphate (DXS): the
inhibitory properties of C1-inhibitor towards complement and
coagulation systems are potentiated up to over 100-fold,
whereas those towards contact and fibrinolytic systems are
not affected. Virtually every method to modify the inhibi-
tory function of Cl-inhibitor by DXS is intended to come
into the scope of this invention. Potentiating effects of
glycosaminoglycans on the inhibition of Cls have been des-
CA 02239787 l998-06-l7
WO 97/22347 17 PCT/NL96/00488
cribed previously (see section Background of invention ).
However, these glycosaminoglycans are obtained from ~nim~l
sources and to a varying extent also potentiate antithrombin
III and heparin cofactor II. Low doses of these glycosamino-
glycans are used in a clinical setting to treat thrombo-
embolic diseases. To obtain inhibition of complement in
patients, doses of heparin of at least one order higher are
needed, which have the unacceptab~e risk of bleeding. The
advantages of the present invention are: a) DXS has stronger
enhancing effects on the inhibition of factor XIa and Cls
than any glycosaminoglycan, as is illustrated ~elow; b) only
the lar~er forms of DXS may have some stimulating effects on
antithrombin III and treatment with the low MW forms of this
compound, therefore, does not have the risk of bleeding
tendency; and c) DXS is a semisynthetic compound that can be
produced in large ~uantities, whereas glycosaminoglycans
such as heparin are puri~ied from ~nim~l S .
To more clearly define the present invention, it will
be described in three sections. The first section describes
the effects of DXS on the inhibition of target proteases
factor XIa, factor XIIa, kallikrein and Cls by C1-inhibitor
in purified systems. Results obtained with glycosamino-
glycans are also given for comparison. The second section
describes the effects of DXS on complement activation in
plasma. The effects of heparin and N-acetyl-heparin, glycos-
aminoglycans sometimes used as complement inhibitors, are
also given for comparison. The third section describes the
application of DXS in therapeutical compositions containing
C1-inhibitor.
The effects of DXS on the inhibition of tarqet proteases by
C1-inhibitor in purified systems
~ In this section the effects of DXS on the inhibition of
target proteases factor XIa, factor XIIa, kallikrein and Cls
by C1-inhibitor are presented. The type of experiments shown
is the determination of pseudo-first order and second order
rate constants, which constants describe the kinetics of the
inhibition of target proteases by Cl-inhibitor, and the
CA 02239787 1998-06-17
W O 97/22347 18 PCT/NL96/00488
effects of DXS on these rate constants. The determination of
rate constants for the inhibition of factor XIa by C1-
inhibitor will be shown in detail, whereas that of the
constants describing the inhibition of kallikrein, factor
XIIa or Cls will be described more briefly. The effects of
various glycosaminoglycans on the rate constants is also
shown to illustrate that DXS is more potent in enhancing C1-
inhibitor than any glycosaminoglycan. Finally, in case of
factor XIa the effects of DXS or glycosaminoglycans on the
inhibition by antithrombin III, ~2-antiplasmin and ~
antitrypsin are also shown as these inhibitors significantly
contribute to the inhibition of factor XIa in plasma.
Dextran sulfate (MW 500,000, sulfur content 17%) was
obtained from Pharmacia Fine ~hemicals, Uppsala, Sweden;
dextran sulfate (MW 5,000), heparan sulfate ~from bovine
intestinal mucosa) and soybean-trypsin inhibitor (SBTI, type
I-S) from Sigma Chemical Co., St.Louis, MO; unfractionated
heparin (l U/ml corresponding to 7 ~g/ml) from Kabi Vitrum,
Stockholm, Sweden; dermatan sulfate (chondroitin sulfate B).
Hexadimethrine bromide (Polybrene) was from Janssen Chimica,
Beerse, Belgium; Tween-20 (Tw) from J.T. Baker Chemical,
Phillipsburg, NJ. The chromogenic substrates Glu-Pro-Arg-p-
nitroanilide (S-2366; factor XIa substrate~=and H-D-Pro-Phe-
Arg-p-nitroanilide ~S-2302; factor XIIa and kallikrein
substrate) were from Chromogenix, Molndal, Sweden; ~-D-Val-
Ser-Arg-p-nitroanilide (S-2314; Cls substrate) ~rom Kabi
Diagnostica (Stockholm, Sweden).
Purified human factor X~a was obtained from Kordia
Laboratory Supplies, Leiden, The Netherlands, and was stored
at -70 C in 0.1 mol/l Tris-HCl, p~ 7.4, 0.14 mol/l NaCl,
0.1% (wt/vol) Tw. This preparation was made by incubating
factor XI with factor XIIa, after which factor XIIa was
removed by absorption onto a corn trypsin inhibitor column.
Factor XIa preparation migrated as a single band at 160 kD
on non-reducing, and as two bands at 50 and 30 k~,
respectively, on reducing SDS/10-15~ ~wt/vol)-polyacrylamide
gel electrophoresis. Monoclonal antibody (mAb) OT-2, which
is directed against the light chain of activated factor XII
CA 02239787 1998-06-17
W O 97/22347 ~9 PCT/NL96/00488
and blocks its catalytic activity (Dors D.M. et al., 1992,
Thromb Haemost 67: 644) was added to the factor XIa
preparation (80 ,ug/ml final concentration) to block traces of
contaminating factor XIIa. Factor XIa concentrations were
5 expressed as the molar concentration of the 80 kD subunits.
Purified human ~ factor XIIa was obtained from Kordia
Laboratory Supplies, Leiden, The Netherlands. Kallikrein,
~-factor XI~a and Cls were purified as described (Nuijens
J.H. et al., 1987, Thromb Haemost 58: 778; Nuijens J.H. et
al., 1987, Immunoloqy 61: 387). Purified C1-inhibitor
preparations were obtained ~rom Behringwerke AG (Marburg,
Germany) and from the department of Development o~ plasma
products from our institute (C~B), .~1-antitrypsin, ~,~2-
antiplasmin and antithrombin III were from Calbiochem (La
Jolla, CA).
Amidolytic activity of factor XIa was determined in
wells of microtiterplates (Greiner GmbH r Frickenhausen,
Germany) by using the chromogenic substrate S-2366 at a
final concentration of 0.4 mmol/l in a buffer contA;ning
0.1 mol/l Tris-HCl, pH 7.4, 0.14 mol/l NaCl, and 0.1 %
(wt/vol) Tw (total volume of 200 ,ul). The initial change in
absorbance at 405 nm (~iA) was measured at 37 C using a
Titertek twinreader (Flow Laboratories, Irvine, UK).
Glycosaminoglycans and DXS may affect directly the
amidolytic activity of kallikrein. Tankersley D.L. et al.,
1983, Blood 62: 448. Heparin, heparan sulfate or dermatan
sulfate had no measurable effect on the amidolytic activity
of factor XIa, whereas DXS MW 500,000, but not DXS MW 5,000
dose-dependently inhibited this activity up to 50% (Fig. 1).
In further experiments, results obtained with DXS were
corrected for this effect.
Factor XIa and inhibitors were incubated in the
presence or absence of glycosaminoglycans or DXS in 0.5 ml
polypropylene tubes at 37~C with 0.1 mol/l Tris-HCl, pH 7.4,
0.14 mol/l NaCl, 0.1 % (wt/vol) Tw as a buffer. Before
incubation the various components of the mixtures were
prewarmed at 37~C for 5 min. After addition of prewarmed
factor XIa (final concentrations 3 to 8 nmol/l) to the
CA 02239787 1998-06-17
W O 97/22347 PCT/NL96/00488
reaction mixtures, 10 ~l aliquots were removed at various
times and residual amidolytic activity of factor XIa was
assessed by diluting in 190 ~l buffer and substrate as
described above. The observed ~A/min, which was constant
during the time of measurement, was converted to percentage
of m~i m-lm activity by comparison with the ~A/min of the
sample containing factor XIa and glycosaminoglycan but no
protease inhibitor. The kinetics of the inhibition were
studied under pseudo first-order conditions with the
inhibitors in 13 to 210-fold molar excess over factor XIa.
Inactivation of factor XIa by C1-inhibitor indeed appeared
to follow first-order kinetics under pseudo first-order
conditions, as was concluded from the straight lines
obtained when the natural logarithm of residual factor ~Ia
amidolytic activity was plotted against time (Fig. 2A).
Under these conditions, the equation ln(E/EO)=-k x t, where
Eo is the initial concentration of factor XIa, and E the
concentration of re~ining factor XIa at time t, describes
the inhibitory kinetics (Soons H., 1987, Biochemistry 26:
4624). According to this equation, the values of the
apparent first-order rate constants, k, were calculated from
the slopes of these lines and were found to be directly
proportional to the Cl-inhibitor concentrations (Fig. 2B).
Therefore, inhibition was found to be second-order, in
agreement with previous studies. Soons H., 1987,
Biochemistry 26: 4624. The rate constant describing the
reaction was calculated by linear regression analysis and
found to be 1.8 x 103 M-1 s-l The inhibition of factor XIa
by C1-inhibitor in the presence of various amounts of dex-
tran sulfate, heparin, heparan sulfate or dermatan sulfatealso appeared to be first-order under pseudo first-order
conditions. However, the rate constants increased with
increasing amounts of the glycosaminoglycans (Fig. 3). DXS
MW 500,OOO and DXS MW 5,000 appeared to be more potent in
enhancing the inhibition of factor XIa by Cl-inhibitor than
any of the physiological glycosaminoglycans tested (Fig. 4).
Similar experiments were performed with the other inhibitors
of factor XIa except that the equation
CA 02239787 1998-06-17
W O 97/22347 21 PCT/NL~61'~C188
kl= k2 x ~Cl-inhibitor] was used to calculate second order
rate constants. Again, straight lines were obtained in a
semilogarithmic plot of the residual factor XIa amidolytic
A activity against time demonstrating that the reaction was
5 first-order. The values of the apparent second-order rate
constants were calculated and are given in Table I. Each
rate constant was determined at least twice, the variation
between the different determinations was 9.6 + 0.5 % (mean +
standard error of mean). Thus, though various glycosamino-
10 glycans potentiated the inhibition of factor XIa by C1-
inhibitor, the semisynthetic compound DXS was the compound
that best potentiated C1-inhibitor regarding inhibition of
factor XIa, i.e. up to over 100-fold. DXS also potentiated
inhibition of factor XIa by antithrombin III (ATIII), but
15 this effect was not greater than 5-foldr and it was also
much weaker than that of heparin on AT III (Table I). The
inhibition of factor XIa by (l2-antiplasmin ~a2AP) or ~1-
antitrypsin (alAT) was hardly enhanced by DXS.
20 Table I. Second-order rate constants for the inactivation of
factor XIa by C1-inhibitor (ClInh), ~1-antitrypsin (alAT),
x2-antiplasmin (a2AP), and antithrombin III (ATIII) in the
presence of various glycosaminoglycans (GAG) or DXS
(103M-lS-l~
no GAG DXSl DXS2 Hep HS DS
____________________________________________________________
ClInh 1.8 210 160 85 42 6
(xlAT 0.1 0.02 nd 0.06 0.08 0.06
~2AP 0.43 0.19 nd 0.51 0.57 0.65
ATIII 0.32 1.54 nd 4.4 1.27 1.24
1 DXS MW 500,000 [10 ~g/ml, final concentration]; 2 DXS MW
5,000 [10 ~g/ml~; Hep, heparin [50 u/ml]; HS, heparan~ 35 sulfate [1 mg/mll; DS, dermatan sulfate [1 mg/ml~; nd = not
det~rmined.
In an analogous way as described above, the effects of
glycosaminoglycans and DXS on the inhibition of factor XIIa
CA 02239787 l998-06-l7
W O 97/22347 22 PCT/NL96/00488
or kallikrein by C1-inhibitor were investigated. No poten-
tiation of C1-inhibitor was observed in these experiments
(Table II).
Table II. Second-order rate constants for the inactivation
of .~-factor XIIa, I,-factor XIIa and kallikrein by C1-
inhibitor in the presence of various glycosaminoglycans
(GAG) or DXS
( 103M-lS-l )
no GAG DXS1 DXS 2 HS DS
_______________________________________________________
<~-FXIIa: 8.0 3.1 7.2 6.8 10.3
I;-FXIIa: 9.8 5.4 1.7 8.2 11.9
kallikrein 2~.5 22.1 19.4 24.5 26.0
1 DXS MW 500,0Q0 [125 ~g/ml, final concentration]; 2 DXS MW
5,000 L 125 ~g/ml]; HS, heparan sulfate [1 mg/ml]; DS,
dermatan sulfate [1 mg/ml].
Thus, in spite of their enhancing effects on the
inhibition of factor XIa by Cl-inhibitor, DXS and glycos-
aminoglycans hardly had an effect, if any, on the inhibition
of factor XIIa or kallikrein by Cl-inhibitor.
The inhibition of Cls was analyzed using second order
conditions. It appeared that the various glycosaminoglycans
also potentiated the inhibition of Cls by C1-inhibitor. In
Fig. 5 it is shown that DXS MW 500,000 best potentiates the
inhibitory activity of Cl-inhibitor (15 nM) on the amido-
lytic activity of Cls (3 nM~ by Cl-Inh ~15 nM~. Fig. 6 shows
that this effect of DXS is optimal at DXS concentration of
10-20 ~g~ml. Similar results were obtained with DXS MM 5,000
( see Fig. 3). Compiling these data yields second order rate
constants of inhibition given in Table III.
CA 02239787 1998-06-17
W O 97/22347 PCTANL96/00488 23
Table I~I. Second-order rate constants for the inactivation
of Cls by C1-inhibitor in the presence of various glycos-
aminoglycans (GAG)
GAG concentration# ( 1O5M-1S-1 )
___________________________________________________
blank -- 0.453
DXS 500,000 100 ~g/ml 58.75
DXS 5,000 100 ~g/ml 34.05
heparin 50 U/ml 26.24
N-ac-heparin 1 mg/ml 4.856
HS l mg/ml 8.755
DS 1 mg/ml 13.43
CSA 1 mg/ml 2.509
CSC 1 mg/ml 3.606
DXS, dextran sulfate; HS, heparan sulfate; DS, dermatan
sulfate; CSA/CSC, chondroitin sulphate A/C. # final
concentration.
Thus, the experiments shown in this section indicate
that the inhibition of Cls or factor XIa by C1-inhi~itor can
be potentiated by incubating C1-inhibitor with DXS, whereas
inhibition of the contact system is not affected.
The effects of DXS on complement activation in plasma
~ xamples presented in this and the following section
are meant to further illustrate the invention, and are not
to be considered as limiting the scope of the invention. For
example, variation in the source, type, or method of
producing DXS species; different assays; different labels
and/or signals; test supports of different materials and
- configurations may be employed without departing from the
scope of the present invention.
The effects of DXS on the inhibition of complement by
C1-inhibitor in serum may be tested by adding DXS to fresh
human serum, followed by incubation at 37 C of the mixture
with complement activators such as aggregated IgG, cobra
venom factorr E.coli bacteria or zymosan. After this
CA 02239787 1998-06-17
W O 97/22347 PCTANL96/00488 24
incubation EDTA is added to prevent further activation and
the mixture is tested for the presence of complement
activation products such as C3a, C4a, C5a, C3b/bi/c,
C4b/bi/c or C5b-C9. Assays for these complement activation
products are well known in the art and can be obtained
commercially. The preferred assays are those described by
Hack C.E. et al., 1988, J Immunol Meth 108: 77; Hack C.E. et
al., 1990, J Immunol 144: 4249; Nuijens J.H. et al., 1989, J
Clin Invest 84: 443; and Wolbink G.J. et al., 1993, J
Immunol Meth 163: 67.
As is shown in Fig. 8 and Fig. 9 DXS MW 500,000 as well
as DXS MW 5,000 both significantly inhibited complement
activation in serum by aggregated IgG: Both DXS species at a
concentration of about 100-200 ~g/ml nearly completely
inhibited the generation of activated C4 and C3 in serum by
the classical pathway activator aggregated IgG. In addition,
DXS MW 500,000, but not DXS MW 5,000, also inhibited the
generation of Cls-C1-inhibitor complexes, probably
reflecting a direct effect of DXS MW 500,000 on the binding
of Clq to aggregated IgG. The effects of heparin and N-
acetyl-heparin were explored in similar experiments. As is
shown in Fig. 10 heparin inhibited complement activation in
serum by aggregated IgG similarly as DXS MW 5,000. In con-
trast, N-acetyl-heparin appeared to be a weaker complement
inhibitor than heparin or DXS (Fig. 10). ~ffects of this
heparin-species with reduced anticoagulant properties on the
generation of activated C3 were hardly observed, whereas
inhibition of C4 activation was not complete unless
concentrations of 1 mg/ml were tested.
The effects of DXS on l u of purified C1-inhibitor were
directly compared with the effects of increasing C1-inhibi-
tor concentrations by assessing the effects of DXS-treated
C1-inhibitor with those of a dose-response curve in a CH50
determination. To this, 1 U of C1-inhibitor preincubated
with DXS, or various concentrations of C1-inhibitor without
DXS, were added to recalcified plasma, and the CH50 titer of
the mixtures were determined. The results, shown in Table
IV, indicate that the decrease of CH50 titer upon addition
CA 02239787 1998-06-17
W O 97~2347 25 PCT~NL96/00488
of high doses of Cl-inhibitor, i.e., up to 135 U, was only
moderate, i.e. from 44 to 27 U/ml. A similar effect was
observed with 1 U of Cl-inhibitor potentiated wi~h ~XS.
Table IV. Comparison of the effect of 1 U of C1-inhibitor
potentiated with DXS with those of untreated C1-inhibitor on
the hemolytic activity of recalci~ied plasma as determined
by CH50 assay
10 plasma plus* C~50 titer
(Units/ml)
_______________ ____________________________________________
buffer 44
DXS 5,000 (100 ~g/ml) 32
15 DXS 500,000 (100 ~g/ml) 25
Cl-Inh (1 U)/DXS 5,000 (100 ~g/ml) 29
Cl-Inh (1 U)/DXS 500,000 (100 ~g/ml) 26
C1-Inh (1 U)/DXS 5,000 (10 ~g/ml) 38
C1-Inh (1 U)/DXS 500,000 ~10 ~g/ml) 33
20 C1-Inh (5 U) 44
C1-Inh (15 U) 43
Cl-Inh (45 U) 37
C1-Inh (135 U) 27
* DXS, dextran sulfate; C1-Inh, C1-inhibitor
Thus, the experiments described in this section indi-
cate that DXS is able to potentiate Cl-inhibitor in serum
and to reduce the generation of complement activation
products.
Application of DXS in therapeutical compositions cont~i n; ng
- C1-inhibitor
In ~he preferred embodiment of the invention, the
therapeutic composition contains plasma-derived C1-inhibitor
as the active ingredient, for example as prepared according
to Voogelaar E.F. et al., 1974, Vox Sang. 26: 118. The virus
safety of this preparation is guaranteed by the addition of
hepatitis B-immunoglobulin and a heat treatment of the
-
CA 02239787 l998-06-l7
W O 97/22347 PCT/NL~G/00188 26
freeze-dried preparation in the final container. Brllmm~lhuis
H.G.J. et al., 1983, Vox Sang. 45: 205r Tersmette et al.,
1986r Vox Sang. 51: 239. C1-inhibitor is prepared from human
plasmar depleted of vitamin K-dependent coagulation factorsr
according to a procedure which involves the following
purification steps: 1) the starting plasma is 1 to 10
diluted with sterile destilled water; 2) the diluted plasma
is incubated with DEAE-Sephadex A50 (Pharmacia Fine
Chemicalsr Uppsalar Sweden) at a concentration of 2 g/kgr
for 60 minutes at 8-10~C; 3) the D~AE-Sephadex is collected
and washed with 150 mM sodium chloride, pH 7.0, and eluted
with 10 mM trisodium citrater 2 M sodium chlorider pH 7.0;
4) ammonium sulphate is added to the eluate to yield a final
concentration of 50%r v/v; 5) a~ter centrifugation at 13rO00
rpmr ammonium sulphate is added to the supernatant to yield
a final concentration of 65%r v/v; 6) the precipitate is
collected by centrifugation and dissolved in 10 mM trisodium
citrater pH 7.0; 7) a diafiltration is performed to le~l~ve
the ammonium sulphate and to concentrate the solution to a
protein concentration of 40-50 mg/ml; 8) after the addition
of Hepatitis ~ immunoglobulin (0.4 IU/ml)r the solution is
filtered through a 0.22 ~m filter, dispensed in vials and
freeze-dried; 9) the freeze-dried product is heat-treated
for 72 hours at 60~C.
In the preferred embodiment of the invention,
C1-inhibitor is mixed with DXS (for example, 100 ~g per Unit
of C1-inhibitor), incubated for one hour; and then ~min;~-
tered by intravenous injection.
