EP0868191A1

EP0868191A1 - Potentiation of complement and coagulation inhibitory properties of c1-inhibitor.

Info

Publication number: EP0868191A1
Application number: EP96941227A
Authority: EP
Inventors: Cornelis Erik Hack; Walter Wuillemin
Original assignee: Stichting Sanquin Bloedvoorziening
Current assignee: Stichting Sanquin Bloedvoorziening
Priority date: 1995-12-18
Filing date: 1996-12-18
Publication date: 1998-10-07
Also published as: AU1042597A; JP2000507204A; WO1997022347A1

Abstract

Dextran sulphate is used to potentiate C1-esterase inhibitor selectively with respect to inhibition of complement and coagulation, but not with respect to inhibition of the contact and fibrinolytic systems. The C1-esterase inhibitor to be potentiated by the dextran sulphate may be endogenous C1-esterase inhibitor, or exogenous C1-esterase inhibitor which is to be administered together with or separate from the dextran sulphate. Use of the dextran sulphate, alone or together with C1-esterase inhibitor, in prophylactic or therapeutic treatment of inflammatory conditions, such as sepsis and myocardial infarction.

Description

Txtle: Potentiatxon of complement and coaqulatxon inhibitory properties of Cl-inhibitor

Field of the Invention

This invention is m the fields of immunology and biochemistry and describes a method to modify the inhibitory spectrum of Cl-mhibitor, 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 Cl-inhibitor can be potentiated up to over 100-fold, without affecting its inhibitory properties towards fibrinolytic or contact system proteases. Th s potentiation is achieved by incubating Cl-inhxbitor with the synthetic sulfated polysaccharide dextran sulphate. Pharmaceutical compositions containing potentiated Cl-mhibitor have considerable applications, for example as antl-mflammatory 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 animal diseases and are mediated by an array of so-called inflammatory mediators. Gallm Jl, 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; histamm and serotonin; neuropeptides ; reactive oxygen species; and nitric oxide and related compounds. Also the manor plasma cascade systems, which include the coagulation, fibrinolytic, contact and complement 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-macroglobulιn and the serine protein¬ ase inhibitors (serpms) antithrombin III, «1-antιtrypsm, (.1-antιchymotrypsm, <.2-antιplasmιn, Cl-mhibitor, 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: Gallin Jl, Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, Raven Press Ltd, p.33. Activation of complement results in the generation of biologically active peptides, also known as the anaphyla- toxms. These anaphylatoxms, m particular C3a and C5a, are chemotactic for neutrophils and able to aggregate, activate and degranulate these cells. Vogt W. , 1986, Complement 3: 177; Goldstein IM, 1992, In: Gallin Jl, Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, Raven Press Ltd, p.63; Hugli TE, 1984, Springer Semin Immunopathol 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, LTD4 and LTE4 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 H.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 by the in vivo administration 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. As πust mentioned, complement can be activated via two different pathways, the classical and the alternative pathway. The latter will not be discussed here since Cl- mhibitor 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 complex of 5 proteins, one Clq, two Cir and two Cis proteins. The 'q protein of the Cl complex binds to an activator, for mple immune complexes, which leads lo activation of both and both Cis subcomponents. Schumaker VN et al. , 1987, ev Immunol 5: 21; Cooper N.R., 1985, Adv Immunol _3_7 : 151. During activation Cir and Cis are converted from smqle peptide-cham inactive proteins into two-chain active serine proteinases. The activated Cl complex then activates the complement factors C4 and C2 , which together form the bi- molecular C4b,2a complex. Polley MJ et al. , 1968, J Exp Med 128: 533; Kerr MA, 1980, Biochem J 1_89: 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 anaphylatoxm 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 Jl, 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 proteins can inhibit activation of the classical pathway of complement, notably, Cl-mhibitor, C4- binding protein and the seπne-proteinase factor I. Muller- Eberhard H.J., 1992, In: Gallin Jl, Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, Raven Press, p.33; Schumaker VN et al., 1987, Ann Rev Immunol 5: 21; Cooper NR, 1985, Adv

Immunol 37: 151. Of these, Cl-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 kallikrem-kinin system. Colman R.W. , 1984, J Clin Invest _7_3: 1249; Kaplan A.P. et al., 1987, Blood 2_0: 1; Kozin F. et dl. , 1992, In: Gallin Jl , Goldstein IM,

Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, Raven Press, p.103. 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 smgle-cham inactive into a two-chain active serine proteinase. Tans G. 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 kallikrem.

Kallikrem in turn may activate bound but not yet activated factor XII (reciprocal activation). Factor Xlla may activate factor XI, which in turn can activate factor IX to start activation of coagulation. Cochrane CG. et al., 1982, Adv Immunol 3J: 290; Colman R.W. , 1984, J Clin Invest 73: 1249; Kaplan A.P. et al., 1987, Blood 70: 1; Kozm F. et al., 1992, In: Gallin Jl, Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, Raven Press, p.103. Activation of the contact system is controlled by the same protein that also inhibits the classical complement pathway, Cl-in ibitor, and which will be discussed below. During activation of the contact system several biologically active fragments are formed such as bradykinin, kallikrem 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 7 : 1249; Kozm F. et al., 1992, In: Gallin Jl , 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 23: 1249; Kaplan A.P. et al. , 1987,

Blood 20: 1; Kozm r. et al . , 1992, In: Gallin Jl , Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, 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 deficiency of factor XII may have an increased risk for thromboembolic disease. This, together with its tissue type-plasminogen-like structure (Tans 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 m agreement herewith. Levi M. et al., 1991, J Cl n Invest 8: 1155. Factor XI is often considered as a member of the contact system since n vitro it can be activated by factor XII. Kurachi K. et al. , 1977, 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, J Biol Chem 252: 6432; Van der Graaf F. et al., 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 a1-antιtrypsm, antithrombm III, Cl-mhibitor, and .^-antiplasmm, 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: 1615; Damus P.S. et al. , 1973, Nature TΛ6 : 355; Forbes CD. et al. , 1970, J Lab Clm Med 16: 809; Saito H. et al., 1979, Proc Natl Acad Sci USA 16: 2013. Initial studies suggested l-antitrypsm to be the main inhibitor of factor XIa in plasma. Scott CF. et al . , 1982, J Cl n Invest 69: 844. However, studies with enzyme-linked lmmunosorbent assays to quantitate complexes between factor XIa and its inhibitors in plasma demonstrated Cl-inhibitor to be a major inhibitor of factor XIa. Wuillemm W.A. et al. , 1995, Blood 85: 1517.

The rn_. 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 Chem 266: 7353; Gailani D. et al., 1991, 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 3_0: 10363; Broze Jr. G.J., 1992, Seminars Hematol 2_9 : 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 hepaπn-mediated potentiation of antithrombm III. Cl-mhibitor

Cl-mhibitor, also known as Cl-esterase inhibitor, refers to a protein that is present in blood and is the mam inhibitor of the classical pathway of complement and of the contact system. Cl-inhibitor can inhibit the activated form of the first component of complement and activated factor XII, and it is also a major inhibitor of kallikrem. Schapira M. et al. , 1985, Complement 2: 111; Davis A.E., 1988, Ann Rev Immunol 6: 595; Sim R.B. et al., 1979, FEBS Lett 9_7: 111; De Agostim A. et al. , 1984, J Clin Invest JJ : 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 I . et al. , 1983, J Cl n Invest 71: 149; Harpel P.C et al., 1975, T Clin Invest 55: 593. Thus, Cl-mhibitor regulates the activity of two plasma cascade systems, i.e. the complement and contact systems, that during activation generate biologically active peptides. Cl-inhibitor is, therefore, an important regulator of inflammatory reactions. In addition, Cl-mhibitor is a major inhibitor of activated factor XI. Mei ers J.C.M. et al . , 1988, Biochemistry 2 :

959; Wuillemin W.A. et al. , 1995, Blood 85: 1517. Conside¬ ring the possible function of factor XI as discussed above, Cl-inhibitor should therefore also be considered as a coa¬ gulation inhibitor. Also tissue-type plasminogen activator and plasm 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 5_5: 149; Booth N.A. et al. , 1987, Blood 6_9: 1600. Cl-mhibitor should therefore also be considered as a (weak) fibrinolytic inhibitor.

Cl-inhibitor has been purified from plasma at large scale and used for clinical application, particularly m the treatment of heπditary angioedema, a disease caused by a genetic deficiency of C1-inhibitor. Furthermore, adimnis- tration of Cl-inhibitor has been claimed to have beneficial effects other diseases as well, such as systemic inflam¬ matory responses in mammals [Fong S., 1992, WO 92/22320 (Genentech Inc)], and of complications of severe burns, pancreatitis, bone marrow transplantation, cytokine therapy and the use of extracorporeal circuits [Eisele B. et al., 1994, DE-A-4227762 (Behringwerke AG) ] . The present invention relates to these therapeutical applications of Cl-mhibitor in that it provides a novel method to enhance the inhibitory activity of Cl -inhibitor, and hence reduces the amount of Cl-inhibitor needed for these therapies.

Full-length genomic and cDNA coding for Cl-mhibitor has been cloned. Bock S.C et al . , 1986, Biochemistry 25 : 4292; Carter P.E. et al. , 1988, Eur J Biochem 173: 163. Functional recombinant Cl-inhibitor protem has been expressed COS cells and found to be similar to the plasma protem. Eldennq E. et al., 1988, J Biol Chem 263: 11776. Several variants of recombinant Cl-inhibitor with ammo acid mutations at the Pl 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. Eldermg E. et al. , 1988, J Biol Chem 263: 11776; Eldenng E. et al., 1993, J Biol Chem 167: 7013; Eldermg E. et al., 1993, J Cl n Invest £1 : 1035; Patent Cetus Corp,

US617920; Davis A.E. et al., 1992, Nature Genetics 1: 354; Eldermg E. et al. , 1995, J Biol Chem 270: 2579; Verpy et al., 1995, J Cl n Invest £5: 350.

Cl-mhibitor belongs to a superfamily of homologous proteins known as the serme-protemase inhibitors, also called serpms. Travis J. et al., 1983, Ann Rev Biochem 5_2 : 655; Carrel R.W. et al. , 1985, Trends Bioch Sci 1_0: 20. On sodium dodecylsulphate polyacrylamide gels Cl-inhibitor has an apparent molecular weight of approximately 105 kD. Its plasma concentration is about 270 mg/1. Schapira M et al., 1985, Complement 2: 111; Nui ens JH et al. , 1989, J Clin Invest 84: 443. Cl-mhibitor is an acute phase protein whose levels may increase up to 2-fold during uncomplicated infections and other inflammatory conditions. Kalter ES et al., 1985, J Infect Dis 151: 1019. The increased synthesis of Cl-inhibitor in inflammatory conditions is most probably meant to protect the organism against the deleterious effects of (intravascular) activation of the complement and contact systems during acute phase reactions . In patients with rheumatoid arthritis the synthetic rate of Cl-inhibitor may increase up to 2.5 times the normal rate. Woo et al. ,

1985, Clin Exp Immunol 6J : 1. Metabolic studies with radiolabeled Cl-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 oϊ about 20 hours. Woo et al., 1985, Clin Exp Immunol 61: 1; Quastel M. et al., 1983, J Clin Invest 11: 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 Biochem 5_2 : 655. Like other serpins Cl-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 8_1 : 56. Serpins have specificity for cer- tain proteinases and this specificity is in part determined by the amino acid sequence of the reactive centre.

The activity of serpins may be influenced by glycos- aminoglycans, a heterogeneous group of macromolecular sulphated glycocon ugates linked to a protein core. Kjellen L. et al., 1991, Annu Rev Biochem 6.0: 443; Poole A.R. , 1986, J Biochem 236 : 1; Bourin M.-C et al . , 1993, Biochemical J 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, Immunology Today 14: 500. Glycosaminoglycans have been claimed to have anti-metastatic and/or anti-inflammatory activities based on their properties to inhibit endoglyco- sidases, particularly heparinase. Parish CR. et al., 1988, WO 88/05301 (Australia University). This effect of glycos¬ ammoglycans is unrelated to the present invention.

Its enhancing effects on the function of antithrombm underlie the therapeutical use of heparin. Furthermore, sulphated polysaccharides may exert additional anticoagulant act_v_tιes the presence of lipoprotem-associated coagu¬ lation inhibitor (LACI), which effect has been patented for therapeutic application. Tze-Che Wun, 1992, EP-A-0473564 (Monsanto Company). The semisynthetic sulphated polysaccha- ride dextran sulphate has less enhancing effects on anti¬ thrombm III then heparin, although it may potentiate other inhibitors of coagulation such as protease nexιn-1 (PN-1). Scott R.W., 1991, WO 91/05566 ( invitron Corp.). These effects of sulphated polysaccharides on clotting inhibitors are unrelated to the present mvention, which is dealing with the interaction of dextran sulphate and Cl-inhibitor. The hepaπn-antithrombm III interaction is probably the best studied example of glycosam oglycan-enhanced function of a serpin. However, a number of studies have also shown that glycosammoglycans, in particular heparin, may also potentiate the function of other serpins including Cl- mhibitor: In kinetic assays with purified proteins heparin has been shown to potentiate the inhibition of Cis by Cl- mhibitor 15- to 35-fold, whereas the inhibition of activated Cl or Cir is less enhanced. Rent R. et al., 1976, Clin Exp Immunol 23: 264; Sim R.B. et al., 1980, Biochim Biophys Acta 612: 433; Caughman G.B. et al., 1982, Mol Immunol 1_9: 287; Nilsson T. et al. , 1983, Eur J Biochem 129: 663; Lennick M. et al. , 1986, Biochemistry 2_.5: 3890; Hortm G.L. et al., 1991, Immunol Invest 20: 75. This enhanced interaction of Cis occurs at the expense of an increased proteolytic inactivation of Cl-mhibitor. Weiss V. et al. , 1983, Hoppe-Seyler ' s Z Physiol Chem 3_64 : 295. In addition to these effects on Cl-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 Cl-esterase and on the formation of the classical C3- convertase. Raepple E. et al., 1976, Immunochemistry 13: 251; Loos M. et al., 1976, Immunochemistry 13: 257; Strunk R. et al., 1976, Clin Immunol Immunopathol 6: 248. Heparin might, therefore, be considered as a therapeutic complement inhibitor. However, the complement-inhibiting effects 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 15: 701. However, this does not obviate another disadvantage of the use of heparin (or any other glycosaminoglycan) , i.e., that it has to be purified from animal sources.

The mechanism by which glycosammoglycans potentiate Cl-inhibitor towards inhibition of its target proteases Cis and factor XIa is not known. However, in analogy to what is known for heparin-accelerated inhibition of thrombin by antithrombm III, several mechanisms are postulated: (I)

Glycosammoglycans may induce a conformational change in the inhibitor, rendering it more active; (II) Glycosammoglycans may work as a template on which inhibitor and target protease may assemble; (III) Glycosammoglycans 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., 1994, J Biol Chem 269: 15957. Which one of these mechanism(s) applies to the observed glycosaminoglycan- induced enhancement of Cl-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. , 1988, EP-A-0293826 (Stichting Rega V.Z.W.). In addition, dextran sulphate may be useful for the treatment of arteriosclerosis. Herr D., 1988, EP-A-0276370 (Knoll AG) . These effects are unrelated to the present invention. Furthermore, high molecular weight species of dextran 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 Cl-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 Cl-inhibitor selectively potentiated up to over 100-fold regarding its complement and clotting inhibitory properties. Therefore, the present invention contemplates a pharmaceutical composition containing Cl-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 Cl-mhibitor 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 invention.

Brief Description of the Drawings

Figure 1. Influence of glycosammoglycans or DXS on the amidolytic activity of factor XIa. The a idolytic activity of factor XIa was determined as the initial change in absorbance at 405 nm at 37 C using the chromogenic substrate S-2366 at a final concentration of 0.4 mmol/l n a buffer containing 0.1 mol/1 Tris-HCl, pH 7.4, 0.14 mol/1 NaCl, and 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/1 factor XIa, in the absence of any glycosammoglycans, remaining after addition of varying amounts of different glycosammoglycans (ng/ml, final concentrations). figure 2. Kinetics of the inactivation of factor XIa by Cl-inhibitor in the absence of glycosammoglycans or DXS. Factor XIa (final concentration 6 nmol/1) was incubated at 37°C with different concentrations of Cl-inhibitor in 0.1 mol/1 Tris-HCl, pH 7.4, 0.14 mol/1 NaCl, 0.1 . Tw. At various times, aliquots were removed and assayed for residual amidolytic activity of factor XIa. (Panel A)

Inactivation of factor XIa was assessed in the presence of Cl-inhibitor at 0 (solid circles), 0.32 (open circles), 0.64 (solid squares), 0.96 (open squares) or 1.28 (plus signs) umol/1. The natural logarithm of residual factor XIa amidolytic activity was plotted against time. (Panel B) The pseudo first-order rate constants (k, mm^-1) were calculated from the slopes of the plots shown in panel A and plotted as a function of the Cl-inhibitor concentration. The slope of the line represents the second order rate constant (k₂, min^{^}- , M-¹ ) .

Figure 3. Kinetics of the inactivation of factor XIa by Cl-inhibitor in the presence of glycosammoglycans or DXS. Factor XIa (final concentrations 3 to 8 nmol/1) was incubated at 37°C with Cl-inhibitor (final concentration 0.32 umol/1) m 0.1 mol/1 Tris-HCl, pH 7.4, 0.14 mol/1 NaCl, 0.1 o 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 Cl-mhibitor 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 glycosammoglycans or DXS. Figure 4. Pseudo first-order rate constants of factor XIa inhibition by Cl-mhibitor in the presence of glycos¬ ammoglycans or DXS. The pseudo first-order rate constants were determined as described in legend to Figure 2, in the presence of varying concentrations of Cl-inhibitor and in the presence of DXS MW 500,000, DXS MW 5,000, heparin, heparan sulfate or dermatan sulfate, or in the absence of glycosammoglycans or DXS. The slope of the lines represents the second order rate constants (k₂ , mm^-1, M^_i). Figure 5. Inhibition of Cis by Cl-mhibitor in the presence of various glycosammoglycans or DXS. Cis at a final concentration of 3 nmol/1 was incubated with Cl- mhibitor (final concentration 15 nmol/1) and various glycosammoglycans (each tested at 10 ng/ml) in phosphate buffered saline (PBS)-0.05 ° T ., 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 Cis by Cl-mhibitor. Condi- tions used are the same as described in Figure 5.

Figure 7. Dose-response of the enhancing effect of DXS MW 5,000 on the inhibition of Cis by Cl-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 CaCl (final concentration). After 15 mm at 37°C a clot had formed, which was removed by centrifugation for 10 mm at 2,000 x g at 4°C One vol of recalcified plasma was then incubated with one vol veronal buffered saline containing aggregated human IgG at a o concentration of 5 mg/ml for 20 min at 37 C Complement activation durmg this incubation was then measured by assessing the generation of Cls-Cl-inhibitor complexes, C4 and C3 activation products (C4b/C4bι/C4c and C3b/C3bι/C3c, respectively). Aggregated IgG was prepared as described. Hack CE. et al. , 1981, J Immunol 127 : 1450. Cls- Cl-inhibitor complexes and C3 and C4 activation products were measured as previously described. Nuijens J.H. et al . , 1989, J Clin Invest M : 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, lOOo 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 5,000 was used.

Figure 10. Inhibi ion 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 11. 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 Cl-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 glycosammoglycans on the inhibition of Cis have been des- cribed previously (see section "Background of invention"). However, these glycosammoglycans are obtained from animal sources and to a varying extent also potentiate antithrombm 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 unacceptable risk of bleeding. The advantages of the present invention are: a) DXS has stronger enhancing effects on the inhibition of factor XIa and Cis than any glycosammoglycan, as is illustrated below; b) only the larger forms of DXS may have some stimulating effects on antithrombm 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 quantities, whereas glycosammoglycans such as heparin are purified from animals.

To more clearly define the present mvention, it will be described in three sections. The first section describes the effects of DXS on the inhibition of target proteases factor XIa, factor Xlla, kallikrem and Cis by Cl- hibitor in purified systems. Results obtained with glycosammo¬ 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-heparm, glycos¬ ammoglycans sometimes used as complement inhibitors, are also given for comparison. The third section describes the application of DXS in therapeutical compositions containing Cl-inhibitor.

The effects of DXS on the inhibition of target proteases by Cl-inhibitor m purified systems

In this section the effects of DXS on the inhibition of target proteases factor XIa, factor Xlla, kallikrem and Cis by Cl-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-mhibitor, and the effects of DXS on these rate constants. The determination of rate constants for the inhibition of factor XIa by Cl- mhibitor will be shown in detail, whereas that of the constants describing the inhibition of kallikrem, factor Xlla or Cis will be described more briefly. The effects of various glycosammoglycans on the rate constants is also shown to illustrate that DXS is more potent in enhancing Cl- hibitor than any glycosaminogJyean. Finally, in case of factor XIa the effects ol DXS or glycosammoglycans on the inhibition by antithrombm III, u 2. -a.ntlplasmin and αl- antitrypsm are also shown as these inhibitors significantly contribute to the inhibition of factor XIa plasma.

Dextran sulfate (MW 500,000, sulfur content 17°_) was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden; dextran sulfate (MW 5,000), heparan sulfate ( rom bovme intestinal mucosa) and soybean-trypsin inhibitor (SBTI, type I-S) from Sigma Chemical Co., St.Louis, MO; unfractionated heparin (1 U/ml corresponding to 7 μg/ml) from Kabi Vitrum, Stockholm, Sweden; dermatan sulfate (chondroitin sulfate B) . Hexadimethrme bromide (Polybrene) was from Janssen Chimica, Beerse, Belgium; Tween-20 (Tw) from J.T. Baker Chemical, Phillipsburg, N . The chromogenic substrates Glu-Pro-Arg-p- nitroanilide (S-2366; factor XIa substrate) and H-D-Pro-Phe- Arg-p-nitroanilide (S-2302; factor Xlla and kallikrem substrate) were from Chromogenix, Mάlndal, Sweden; H-D-Val- Ser-Arg-p-nitroanilide (S-2314; Cis substrate) from Kabi Diagnostica (Stockholm, Sweden) .

Purified human factor XIa was obtained from Kordia Laboratory Supplies, Leiden, The Netherlands, and was stored at -70°C in 0.1 mol/1 Tris-HCl , pH 7.4, 0.14 mol/1 NaCl, 0.1° (wt/vol) Tw. This preparation was made by incubating factor XI with factor Xlla, after which factor Xlla was removed by absorption onto a corn trypsin inhibitor column. Factor XIa preparation migrated as a smgle band at 160 kD on non-reducing, and as two bands at 50 and 30 kD, 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 and blocks its catalytic activity (Dors D.M. et al . , 1992, Thromb Hae ost 67 : 644) was added to the factor XIa preparation (80 μg/ml final concentration) to block traces of contaminating factor Xlla. Factor XIa concentrations were expressed as the molar concentration of the 80 kD subunits. Purified human (.-factor Xlla was obtained from Kordia Laboratory Supplies, Leiden, The Netherlands. Kallikrein, |ϊ-factor Xlla and Cis were purified as described (Nuijens J.H. et al., 1987, Thromb Haemost 5j3: 778; Nuijens J.H. et al., 1987, Immunology 61: 387). Purified Cl-inhibitor preparations were obtained from Behringwerke AG (Marburg, Germany) and from the department of Development of plasma products from our institute (CLB), <.1-antitrypsin, u2- antiplasmin and antithrombm III were from Calbiochem (La Jolla, CA).

Amidolytic activity of factor XIa was determined in wells of microtiterplates (Greiner GmbH, Frickenhausen, Germany) by using the chromogenic substrate S-2366 at a final concentration of 0.4 mmol/l in a buffer containing 0.1 mol/1 Tris-HCl, pH 7.4, 0.14 mol/1 NaCl, and 0.1 .

(wt/vol) Tw (total volume of 200 μl). The initial change in o absorbance at 405 nm (όA) was measured at 37 C using a Titertek twinreader (Flow Laboratories, Irvine, UK).

Glycosammoglycans 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/I Tris-HCl, pH 7.4, 0.14 mol/1 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/1) to the reaction mixtures, 10 μl aliquots were removed at various times and residual amidolytic activity of factor XIa was assessed by diluting in 190 μl buffer and substrate as described above. The observed _A/min, which was constant during the time of measurement, was converted to percentage of maximum activity by comparison with the _A/mιn 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 Cl-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 XIa amidolytic activity was plotted against time (Fig. 2A) .

Under these conditions, the equation ln(E/E₀)=-k x t, where E₀ is the initial concentration of factor XIa, and E the concentration of remaining 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^-1. The inhibition of factor XIa by Cl-inhibitor in the presence of various amounts of dex- tran sulfate, heparin, heparan sulfate or dermatan sulfate also 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 500,000 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 kι = 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 activity against time demonstrating that the reaction was 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- glycans potentiated the inhibition of factor XIa by Cl- inhibitor, the semisynthetic compound DXS was the compound that best potentiated Cl-inhibitor regarding inhibition of factor XIa, i.e. up to over 100-fold. DXS also potentiated inhibition of factor XIa by antithrombm III (ATIII), but this effect was not greater than 5-fold, and it was also much weaker than that of heparin on AT III (Table I). The inhibition of factor XIa by _*ι2-antiplasmin (a2AP) or <ιl- antitrypsin (alAT) was hardly enhanced by DXS.

Table I. Second-order rate constants for the inactivation of factor XIa by Cl-inhibitor (Cllnh), ul-antitrypsin (alAT), (.2-antiplasmin (a2AP), and antithrombm III (ATIII) in the presence of various glycosaminoglycans (GAG) or DXS

( 10³M-¹s-¹) no GAG DXS¹ DXS² Hep* HS^* DS

Cllnh 1.8 210 160 85 42 6 ulAT 0.1 0.02 nd 0.06 0.08 0.06 u2AP 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 μg/ml, final concentration]; ² DXS MW 5,000 [10 μg/ml]; * Hep, heparin [50 U/ml]; HS, heparan sulfate [1 mg/ml]; DS, dermatan sulfate [1 mg/ml]; nd = not determined.

In an analogous way as described above, the effects of glycosaminoglycans and DXS on the inhibition of factor Xlla or kallikrein by Cl-inhibitor were investigated. No poten¬ tiation of Cl-inhibitor was observed in these experiments (Table II) .

Table II. Second-order rate constants for the inactivation of (/-factor Xlla, (.-factor Xlla and kallikrein by Cl- inhibitor in the presence of various glycosaminoglycans (GAG) or DXS

1 (10³M"¹s-¹ ) no GAG DXS¹ DXS² HS* D ..S..*

α-FXIIa: 8.0 3.1 7.2 6.8 10.3

I'.-FXIIa: 9.8 5.4 1.7 8.2 11.9 kallikrein 25.5 22.1 19.4 24.5 26.0

¹ DXS MW 500,000 [125 μg/ml, final concentration]; ² DXS MW 5,000 [125 μg/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 Xlla or kallikrein by Cl-inhibitor. The inhibition of Cis was analyzed using second order conditions . It appeared that the various glycosaminoglycans also potentiated the inhibition of Cis by Cl-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 Cis (3 nM) by Cl-Inh (15 nM) . Fig. 6 shows that this effect of DXS is optimal at DXS concentration of 10-20 μg/ml. Similar results were obtained with DXS MW 5,000 (see Fig. 3). Compiling these data yields second order rate constants of inhibition given in Table III. Table III. Second-order rate constants for the inactivation of Cis by Cl-inhibitor in the presence of various glycos¬ aminoglycans (GAG)

blank — 0.453

DXS 500,000 100 μg/ml 58.75

DXS 5,000 100 μg/ml 34.05 heparin 50 U/ml 26.24

N-ac-heparin 1 mg/ml 4.856

HS 1 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 Cis or factor XIa by Cl-inhibitor can be potentiated by incubating Cl-inhibitor with DXS, whereas inhibition of the contact system is not affected.

The effects of DXS on complement activation in plasma

Examples 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 Cl-inhibitor in serum may be tested by adding DXS to fresh o human serum, followed by incubation at 37 C of the mixture with complement activators such as aggregated IgG, cobra venom factor, E.coli bacteria or zymosan. After this 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 CE. et al., 1988, J Immunol Meth 108: 77; Hack CE. 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 μg/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-Cl-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). Effects 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 1 U of purified Cl-inhibitor were directly compared with the effects of increasing Cl-inhibi¬ tor concentrations by assessing the effects of DXS-treated Cl-inhibitor with those of a dose-response curve in a CH50 determination. To this, 1 U of Cl-inhibitor preincubated with DXS, or various concentrations of Cl-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 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 with DXS.

Table IV. Comparison of the effect of 1 U of Cl-inhibitor potentiated with DXS with those of untreated Cl-inhibitor on the hemolytic activity of recalcified plasma as determined by CH50 assay

plasma plus CH50 titer

(Units/ml)

buffer 44

DXS 5,000 (100 μg/ml) 32 DXS 500,000 (100 μg/ml) 25

Cl-Inh (1 U)/DXS 5,000 (100 μg/ml) 29

Cl-Inh (1 U)/DXS 500,000 (100 μg/ml) 26

Cl-Inh (1 U)/DXS 5,000 (10 μg/ml) 38

Cl-Inh (1 U)/DXS 500,000 (10 μg/ml) 33 Cl-Inh (5 U) 44

Cl-Inh (15 U) 43

Cl-Inh (45 U) 37

Cl-Inh (135 U) 27

* DXS, dextran sulfate; Cl-Inh, Cl-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 containing Cl-inhibitor

In the preferred embodiment of the invention, the therapeutic composition contains plasma-derived Cl-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 freeze-dried preparation in the final container. Brummelhuis H.G.J. et al., 1983, Vox Sang. 45: 205, Tersmette et al. , 1986, Vox Sang. 51: 239. Cl-inhibitor is prepared from human plasma, depleted of vitamin K-dependent coagulation factors, 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 Chemicals, Uppsala, Sweden) at a concentration of 2 g/kg, for 60 minutes at 8-10°C; 3) the DEAE-Sephadex is collected and washed with 150 mM sodium chloride, pH 7.0, and eluted with 10 mM trisodium citrate, 2 M sodium chloride, pH 7.0; 4) ammonium sulphate is added to the eluate to yield a final concentration of 50%, v/v; 5) after centrifugation at 13,000 rpm, ammonium sulphate is added to the supernatant to yield a final concentration of 65%, v/v; 6) the precipitate is collected by centrifugation and dissolved in 10 mM trisodium citrate, pH 7.0; 7) a diafiltration is performed to remove the ammonium sulphate and to concentrate the solution to a protein concentration of 40-50 mg/ml; 8) after the addition of Hepatitis B immunoglobulin (0.4 IU/ml), the solution is filtered through a 0.22 μm 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,

Cl-inhibitor is mixed with DXS (for example, 100 μg per Unit of Cl-inhibitor), incubated for one hour; and then adminis¬ tered by intravenous injection.

Claims

1. A pharmaceutical composition comprising a dextran sulphate species which selectively potentiates Cl-esterase inhibitor with respect to inhibition of complement and coagulation but not with respect to inhibition of the contact and fibrinolytic systems, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein said dextran sulphate species is low molecular weight dextran sulphate.

3. The pharmaceutical composition of claim 2, wherein said dextran sulphate species is dextran sulphate having a molecular weight of about 5,000.

4. The pharmaceutical composition of claim 1, wherein said dextran sulphate species is dextran sulphate having a molecular weight of about 500,000.

5. The pharmaceutical composition of claim 1, further comprising Cl-esterase inhibitor.

6. The pharmaceutical composition of claim 5, wherein said Cl-esterase inhibitor is selected from the group consisting of Cl-esterase inhibitor purified from plasma, Cl-esterase inhibitor purified from biological material other than plasma, recombinant Cl-esterase inhibitor, and a mutant of recombinant Cl-esterase inhibitor.

7. The pharmaceutical composition of claim 5, wherein said Cl-esterase inhibitor is selected from the group consisting of Cl-esterase inhibitor purified from human plasma, Cl-esterase inhibitor purified from biological human material other than plasma, human recombinant Cl-esterase inhibitor, and a mutant of human recombinant Cl-esterase inhibitor.

8. The pharmaceutical composition of claim 5, wherein said Cl-esterase inhibitor and said dextran sulphate species are chemically linked to eachother.

9. The pharmaceutical composition of claim 1 or claim 5, comprising said dextran sulphate species in an amount which is effective to selectively potentiate Cl- esterase inhibitor with respect to inhibition of complement and coagulation but not with respect to inhibition of the contact and fibrinolytic systems.

10. The pharmaceutical composition of claim 1 or claim 5, for use as an anti-inflammatory composition.

11. The pharmaceutical composition of claim 1 or claim 5, for the prophylactic or therapeutic treatment of sepsis or myocardial infarction.

12. A method of a prophylactic or therapeutic treatment of a mammal, which method comprises administration to said mammal of an effective amount of a dextran sulphate species which potentiates Cl-esterase inhibitor selectively with respect to inhibition of complement and coagulation, but not with respect to inhibition of the contact and fibrinolytic systems.

13. The method of claim 12, further comprising administration to said mammal of a physiologically effective amount of Cl-esterase inhibitor.

14. The method of claim 13, wherein said dextran sulphate species and said Cl-esterase inhibitor are adminis¬ tered in the form of a physical mixture, or chemically linked to eachother, or in separate compositions.

15. A dextran sulphate species for use in a method of a prophylactic or therapeutic treatment of a mammal to potentiate Cl-esterase inhibitor selectively with respect to inhibition of complement and coagulation, but not with res- pect to inhibition of the contact and fibrinolytic systems.

16. Use of a dextran sulphate species for preparing a pharmaceutical composition for specifically potentiating Cl- esterase inhibitor with respect to inhibition of complement and coagulation, but not with respect to inhibition of the contact and fibrinolytic systems.