EP0868191A1 - Potentialisation des proprietes d'inhibition du complement et de la coagulation d'un inhibiteur de c1 - Google Patents

Potentialisation des proprietes d'inhibition du complement et de la coagulation d'un inhibiteur de c1

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Publication number
EP0868191A1
EP0868191A1 EP96941227A EP96941227A EP0868191A1 EP 0868191 A1 EP0868191 A1 EP 0868191A1 EP 96941227 A EP96941227 A EP 96941227A EP 96941227 A EP96941227 A EP 96941227A EP 0868191 A1 EP0868191 A1 EP 0868191A1
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Prior art keywords
inhibitor
esterase inhibitor
inhibition
complement
dxs
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Cornelis Erik Hack
Walter Wuillemin
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Stichting Sanquin Bloedvoorziening
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Stichting Sanquin Bloedvoorziening
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • Txtle Potentiatxon of complement and coaqulatxon inhibitory properties of Cl-inhibitor
  • 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.
  • Inflammatory reactions occur in the course of numerous human and animal diseases and are mediated by an array of so-called inflammatory mediators.
  • 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.
  • 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-macroglobul ⁇ n and the serine protein ⁇ ase inhibitors (serpms) antithrombin III, «1-ant ⁇ trypsm, (.1-ant ⁇ chymotrypsm, ⁇ .2-ant ⁇ plasm ⁇ n, Cl-mhibitor, and others.
  • serpms serine protein ⁇ ase inhibitors
  • 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.
  • Activation of complement results in the generation of biologically active peptides, also known as the anaphyla- toxms.
  • vasopermeability 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.
  • vasoactive eicosanoids thromboxane A2 and peptidoleukotrienes
  • LTC4, LTD4 peptidoleukotrienes
  • LTC4 terminal complement com ⁇ plexes
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • MAC soluble membrane attack complexes
  • 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,
  • 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.
  • factor XII also known as Hageman factor
  • 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.
  • 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.
  • Cl-in ibitor the classical complement pathway
  • 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.
  • each polypeptide chain 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.
  • disulfide linked heavy and light chains the latter each containing one active site.
  • each active site of factor XIa is regulated by plasma protease inhibitors including a1-ant ⁇ trypsm, antithrombm III, Cl-mhibitor, and . ⁇ -antiplasmm, each a member of the superfamily of serine protease inhibitors (serpins).
  • serpins serine protease inhibitors
  • 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.
  • 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.
  • 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.
  • 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.
  • Cl-mhibitor is a major inhibitor of activated factor XI. Mei ers J.C.M. et al . , 1988, Biochemistry 2 :
  • 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 he ⁇ ditary 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.
  • 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.
  • Cl-mhibitor belongs to a superfamily of homologous proteins known as the serme-protemase inhibitors, also called serpms.
  • serme-protemase inhibitors also called serpms.
  • On sodium dodecylsulphate polyacrylamide gels Cl-inhibitor has an apparent molecular weight of approximately 105 kD. Its plasma concentration is about 270 mg/1.
  • 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. ,
  • Serpins have specificity for cer- tain proteinases and this specificity is in part determined by the amino acid sequence of the reactive centre.
  • serpins may be influenced by glycos- aminoglycans, a heterogeneous group of macromolecular sulphated glycocon ugates linked to a protein core.
  • glycos- aminoglycans a heterogeneous group of macromolecular sulphated glycocon ugates linked to a protein core.
  • sulphated polysaccharides may exert additional anticoagulant act_v_t ⁇ es 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).
  • LACI lipoprotem-associated coagu ⁇ lation inhibitor
  • 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 nex ⁇ n-1 (PN-1).
  • glycosammoglycans in particular heparin, may also potentiate the function of other serpins including Cl- mhibitor:
  • heparin 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.
  • Heparin might, therefore, be considered as a therapeutic complement inhibitor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Cl-inhibitor a major inhibitor of various complement, clotting, contact system and fibrinolytic proteases
  • a semisynthetic polyanionic compound the sulphated polysaccharide dextran sulphate
  • 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.
  • 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.
  • FIG. 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.
  • 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)
  • 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 (k 2 , 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
  • PBS phosphate buffered saline
  • 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/C4b ⁇ /C4c and C3b/C3b ⁇ /C3c, respectively).
  • 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.
  • 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").
  • 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.
  • 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.
  • the present mvention 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.
  • 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 ⁇ g/ml) from Kabi Vitrum, 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, M ⁇ lndal, 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 ⁇ g/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,
  • 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 .
  • Tw total volume of 200 ⁇ l.
  • the initial change in o absorbance at 405 nm ( ⁇ A) was measured at 37 C using a Titertek twinreader (Flow Laboratories, Irvine, UK).
  • 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 ⁇ 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 maximum activity by comparison with the _A/m ⁇ n 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) .
  • DXS dextran sulfate
  • HS heparan sulfate
  • DS dermatan sulfate
  • CSA/CSC chondroitin sulphate A/C # final concentration.
  • DXS 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.
  • complement activators such as aggregated IgG, cobra venom factor, E.coli bacteria or zymosan.
  • 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 byhack CE. et al., 1988, J Immunol Meth 108: 77;
  • 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-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.
  • heparin inhibited complement activation in serum by aggregated IgG similarly as DXS MW 5,000.
  • 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 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 centr
  • Cl-inhibitor is mixed with DXS (for example, 100 ⁇ g per Unit of Cl-inhibitor), incubated for one hour; and then adminis ⁇ tered by intravenous injection.
  • DXS for example, 100 ⁇ g per Unit of Cl-inhibitor

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Abstract

On utilise du sulfate de dextrane pour potentialiser de manière sélective un inhibiteur d'estérase C1 par rapport à l'inhibition du complément et de la coagulation, mais non par rapport à l'inhibition des systèmes à contact et à fibrinolyse. L'inhibiteur d'estérase C1 à potentialiser à l'aide du sulfate de dextrane peut être un inhibiteur d'estérase C1 endogène ou un inhibiteur d'estérase C1 exogène à administrer conjointement avec le sulfate de dextrane ou indépendamment de celui-ci. On décrit également l'utilisation du sulfate de dextrane, seul ou en association avec l'inhibiteur d'estérase C1, dans la prévention ou le traitement des affections inflammatoires telles que la septicémie et l'infarctus du myocarde.
EP96941227A 1995-12-18 1996-12-18 Potentialisation des proprietes d'inhibition du complement et de la coagulation d'un inhibiteur de c1 Withdrawn EP0868191A1 (fr)

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EP96941227A EP0868191A1 (fr) 1995-12-18 1996-12-18 Potentialisation des proprietes d'inhibition du complement et de la coagulation d'un inhibiteur de c1

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EP95203537 1995-12-18
EP95203537 1995-12-18
PCT/NL1996/000488 WO1997022347A1 (fr) 1995-12-18 1996-12-18 Potentialisation des proprietes d'inhibition du complement et de la coagulation d'un inhibiteur de c1
EP96941227A EP0868191A1 (fr) 1995-12-18 1996-12-18 Potentialisation des proprietes d'inhibition du complement et de la coagulation d'un inhibiteur de c1

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NZ539472A (en) * 2002-10-17 2008-05-30 Pharming Intellectual Pty Bv Protein modification
RS56285B1 (sr) 2013-03-15 2017-12-29 Shire Viropharma Inc C1-inh kompozicije za upotrebu u prevenciji i lečenju naslednog angioedema (hae)
US20150104445A1 (en) * 2013-10-10 2015-04-16 Viropharma Holdings Limited Methods of inhibiting the alternative pathway of complement immune system activation and compositions used therein
SE539575C2 (en) 2015-07-30 2017-10-17 Tx Medic Ab Dextran sulfate for use in treating, inhibiting or preventing cardiac fibrosis
JP2023521916A (ja) 2020-04-15 2023-05-25 ティーエックス メディック エービー コロナウィルス感染症の治療薬

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DE3634392A1 (de) * 1986-10-09 1988-04-14 Knoll Ag Verwendung polysulfatierter niedermolekularer dextransulfate
IL85145A (en) * 1987-01-23 1994-08-26 Univ Australian Anti-metastatic pharmacological or veterinary preparations containing modified herpin with reduced anticoagulant activity
EP0293826A3 (fr) * 1987-06-02 1989-05-10 Stichting REGA V.Z.W. Application thérapeutique et prophylactique des polysaccharides sulfatés contre le SIDA
WO1991005566A1 (fr) * 1989-10-16 1991-05-02 Invitron Corporation Anticoagulant a base de nexine de protease i/sulfate de dextran
PT98779B (pt) * 1990-08-27 1999-06-30 Monsanto Co Processo para a preparacao de uma composicao anticoagulante contendo inibidor sulfatados e metodos para a sua utilizacao
WO1992022320A1 (fr) * 1991-06-14 1992-12-23 Genentech, Inc. Variantes de l'inhibiteur c1 et traitement des reactions inflammatoires avec l'inhibiteur c1
DE4227762A1 (de) * 1992-08-24 1994-03-03 Behringwerke Ag Verwendung eines Kallikrein-Inhibitors zur Herstellung eines Arzneimittels zur Prophylaxe und Therapie bestimmter Krankheiten

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See references of WO9722347A1 *

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WO1997022347A1 (fr) 1997-06-26
JP2000507204A (ja) 2000-06-13

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