AU717123B2 - Glycosylation-mediated inhibition of factor X - Google Patents

Description

21I 12i 1

AUSTRALIA

Patents Act 1990 COR THERAPEUTICS,

INC.

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Glycosylation-mediated inhibition of factor X The following statement is a full description of this invention including the best method of performing it known to us:a -1- GLYCOSYLATION-MEDIATED INHIBITION OF FACTOR X Technical Field The invention relates to agents that inhibit the activation of Factor X, and thus are candidates for treatment or prevention of thrombosis. More specifically, the invention concerns agents that affect the normal glycosylation pattern of Factor X and thus diminish its conversion to its activated form. The activated form of Factor X, Factor Xa, is essential for the formation of thrombin, which is, in turn, essential for the formacion of blood clots, as well as an inducer of smooth muscle cell proliferation.

Backqround Ar: Thrombin is a multifunctional protease that reulaces several key biological processes. For example, Ehrombin is among the most potent of the known platelet activators. In addition, thrombin is essential for the cleavage of fibrinogen to fibrin to initiate clot 25 formation. These two elements are involved in normal hemostasis but in atherosclerotic arteries can initiate the formation of a thrombus, a major factor in pathogenesis of vasoocclusive conditions such as myocardial infarction, unstable angina, nonhemorrhagic 30 stroke and reocclusion of coronary arteries after angioplasty or thrombolytic therapy. Thrombin is also a potent inducer of smooth cell proliferation and may therefore be involved in a variety of proliferative S.responses such as restenosis after angioplasty and graftinduced atherosclerosis. In addition, thrombin is chemotactic for leukocytes and may therefore play a role in inflammation. (Hoover, et al. Cell (1978) 11:423; Etingin, et al., Cell (1990) 61:657.) These observations indicate that inhibition of thrombin formation or inhibition of thrombin itself may be effective in preventing or treating thrombosis, limiting restenosis and controlling inflammation.

The formation of thrombin is the result of the proteolytic cleavage of its precursor prothrombin at the Arg-Thr linkage at positions 271-272 and the Arg-Ile linkage at positions 320-321. This activation is catalyzed by the prothrombinase complex, which is assembled on the membrane surfaces of platelets, monocytes, and endothelial cells. The complex consists of Factor Xa (a serine protease), Factor Va (a cofactor), calcium ions and the acidic phospholipid surface.

Factor Xa is the activated form of its precursor, Factor X, which is secreted by the liver as a 58 kd precursor and is converted to the active form, Factor Xa, in both the extrinsic and intrinsic blood coagulation pathways. It is known that the circulating levels of Faccor X, and of the precursor of Factor Va, Factor

V,

are on the order of 10- 7 M. There has been no determination of the levels of the corresponding active 25 Factors Va and Xa.

The complete amino acid sequences of human Factor X and Factor Xa are known, and are as described by :Davie, in Hemostasis and Thrombosis, Second Edition, R.W. Coleman et al., eds. (1987) p. 250. Factor 30 X is a member of the calcium ion binding, gamma •carboxyglutamyl (Gla)-containing, vitamin K dependent, blood coagulation glycoprotein family, which also includes Factors VII and IX, prothrombin, protein C and S* protein S (Purie, et al., Cell (1988) 51:505).

f1 C- -3- The mature Factor X protein is preceded by a pre-pro leader sequence which is removed during intracellular processing and secretion. The mature Factor X precursor of Factor Xa is then cleaved to the two-chain form by deletion of the three amino acids RKR between the light chain C-terminus and activation peptide/heavy chain N-terminus. Finally, the two chain Factor X is converted to Factor Xa by deletion of the 52 amino acid "activation peptide" sequence generating a light chain of 139 residues and a heavy chain of 254 residues. These are linked through a single disulfide bond between position 128 of the light chain and position 108 of the heavy chain. .The light chain contains the Gla domain and an epidermal growth factor-like domain; the protease activity resides in the heavy chain and involves the histidine at position 42, the aspartic at position 88, and a serine at position 185.

Bovine Factor X has also been studied, and the amino acid sequence of bovine Factor X has been reported by Titani, Proc Natl Acad Sci USA (1975) 72:3082- 3086. The activation peptide of bovine Factor X is 51 amino acids long and contains almost equal masses of polypeptide and carbohydrate (Discipio, R.G. et al., Biochemistry (1977) 16:5253) 25 There are two known pathways for the activation of the two-chain Factor X in vivo. Activation must occur before the protease is incorporated into the prothrombinase complex (Steinberg, et al., in Hemostasis and Thrombosis, Coleman, et al. eds.

S 30 (1987) J.B. Lippencott, Philadelphia, PA, p. 112). In the intrinsic pathway, Factor X is cleaved to release the 52-amino acid activation peptide by the "tenase" complex which consists of Factor IXa, Factor VIIIa and calcium ions assembled on cell surfaces. In the extrinsic pathway, the cleavage is catalyzed by Factor VIIa which -4is bound to a tissue Factor on membranes. Factor X may also be converted to Factor Xa by in vitro cleavage using a protease such as that contained in Russel's viper venom. This protease is described by DiScipio, et al., Biochemistry (1977) £:5253.

Factor X variants exist that are differentially activated in the extrinsic versus intrinsic pathway (Fair, D.S. et al., J Clin Invest (1979) £48:884-894).

For example, Factor X "Vorarlberg" has 15% of the activity of normal Factor X with respect to the extrinsic pathway but 75% of normal Factor X with respect to the intrinsic pathway (Watzke, H.H. et al., J Biol Chem (1990). 2j5:11982-11989).

Clearly, the action of thrombin in platelet aggregation and clot formation could be prevented by suitable manipulation of factor X/Xa, in view of the essential role of factor Xa in the formation of thrombin from its precursor.

The activity of Factor Xa in effecting the conversion of prochrombin to thrombin is dependent on its inc -usion in the prothrombinase complex. Therefore, onr approach co this manipulation has been directed to preventing the participation of factor Xa in the thrombinase complex. The formation of the prothrombinase 25 complex (which is 278,000 fold faster in effecting the conversion of prothrombin to thrombin than Factor Xa in soluble form) has been studied (Nesheim, et al., SBio Chem (1979) 254:10952). These studies have utilized the active site-specific inhibitor, dansyl glutamyi 30 glycyl arginyl (DEGR) chloromethyl ketone, which covalently attaches a fluorescent reporter group into Factor Xa. Factor Xa treated with this inhibitor lacks protease activity, but is incorporated into the prothrombinase complex with an identical stoichiometry to that of Factor Xa and has a dissociation constant of 2.7 x 10-6 2.7 x 10-6 M (Nesheim, J Biol Chem (1981) 25:6537-6540; Skogen, et al., J Biol Chem (1984) 256:2306-2310; Krishnaswamy, et al., J Biol Chem (1988) 263:3823-3824; Husten, et al., J Biol Chem (1987) 262:12953-12961).

Other approaches to inhibition of Factor Xa include the use of lipoprotein-associated coagulation inhibitor (LACI), now called tissue factor pathway inhibitor (TFPI) (Girard, et al., Nature (1989) 3:518; Girard, et al., Science (1990) 248:1421), leech-derived antistatin (Dunwiddie, et al., J Biol Chem (1989) 264:16694), and tick-derived TAP (Waxman,

L.,

et al., Science (1990) 248:593). Alternatively, agents S which inhibit the vitamin K-dependent Gla conversion 1 enzyme, such as.coumarin, have been used. None of these approaches have proved satisfactory due to lack of specificity, the large dosage required, toxic side effects, and the long delay in effectiveness.

PCT publication US 91/06337 discloses an additional approach wherein the active site of Factor Xa is modified to prevent its enzymic accivity while retaiing the ability to form the prochrombinase complex.

The invention approach is directed to the 25 inhibition of the conversion of Factor X to its active form, Factor Xa. Specifically, this mode of inhibition is directed to manipulation of the glycosylation residues associated with Factor X.

Bovine, but not human, Factor X has been 30 subjected to detailed studies with respect to its 3 glycosylation patterns. The heavy chain of bovine Factor X contains N-linked glycosylation at residue 36 and O-linked glycosylation at residue 300 (Mizuochi, T. et al., J Biol Chm (1980) 255:3526-3531). While the S glycosylation pattern of human Factor X is not known, it has been noted that the activation peptide contains two potential N-linked glycosylation sites at positions 39 and 49 (Davie, in "Hemeostasis and Thrombosis", Second Edition, R.W. Coleman eds. (1987) p. 250). In addition, serine-linked sugar residues have been reported on the first epidermal growth factor-like domain of the related bovine factor IX (Hase, S. et al., J Biol Chem (1990) 265:1858-1861); analogously, the EGF-like domains on human Factor X light chain may contain O-linked sugars.

Accordingly, the invention offers an alternative approach to inhibit the conversion of Factor X to Factor Xa, thus preventing formation of an active prothrominase complex.

Disclosure of the Invention The invention provides effective therapeutic agents for the prevention and treatment of thrombus formation and other pathological processes in the vasculature induced by thrombin such as restenosis and nflammaion. This is highly significant as thrombus fcrraion is the leading cause of death in Western societies, and rescenosis is an expanding problem with increased use of angioplasty and other invasive 25 procedures. The therapeutic materials of the invention are capable of preventing or of diminishing the generation of human Factor Xa.

These pharmaceucicals are especially useful in acute settings to prevent thrombosis. This includes 30 preventing thrombus formation in the coronary arteries of o patients with rest angina, preventing rethrombosis after thrombolysis, and prevention of thrombosis during complicated angioplasties. These pharmaceuticals will also be useful in preventing smooth muscle cell proliferation following angioplasty or other vascular -7invasive procedures. The invention therapeutics offer considerable advantage over the now standard treatment which involves heparin (Hanson, et al., Proc Natl Acad S c i (1988) 8:3184). In one embodiment, the compounds of the invention are lectins that specifically bind the carbohydrate moieties associated with human Factor X, thus preventing its conversion to Factor Xa.

In another embodiment, the compounds of the invention are themselves capable of altering the glycosylation structure.

In one aspect, the invention is directed to a method to inhibit human Factor X activation, which method comprises contacting human Factor X with a specific carbohydrate-binding reagent capable of binding the terminal sialic acid-linked a(2-6) to a galactose or N-acetyl galactosamine residue. These target glycosyl residues are abbreviated herein SA(2-6)aGal and SA(2-6)GalNAc, respectively. The reagents are "SA/Gal/GalNAc" binding reagents. The carbohydratebinding reagent, when contacted with Factor X under suitable conditions of time, temperature and pH, is effective to prevent the conversion of human Factor X to hran Factor Xa by either the extrinsic or intrinsic pathway. Thus, in another aspect, the invention is 25 directed to a complex comprising said carbohydrate binding reagent and Factor X.

In another aspect, the invention is directed to factors which render Factor X unsusceptible to activation by cleavage of the carbohydrate moieties to which it is 30 derivatized, or by inhibiting the sialylation of this Factor.

Other aspects of the invention include pharmaceutical compositions of the therapeutically useful carbohydrate-binding or carbohydrate cleavage reagents and to methods to prevent or treat thrombosis or other -8pathological events initiated by thrombin using these compositions.

Brief Description of the Drawings Figures 1A and 1B show the results of glycosidase treatment of bovine Factor X and human Factor X, respectively, with respect to the coagulation activation of these factors.

Modes of Carrying Out the Invention The invention is based on the discovery that blockage of specific carbohydrate residues or cleavage of the carbohydrate moieties in human Factor X effectively inhibit its conversion to its activated form which is essential for the formation of the prothrombinase complex. Specifically, those reagents which are capable of binding to sialic acid residues at the non-reducing terminus of the glycosylation moieties, wherein the sialic acid residues linked a(2-6) to galactose or N-acetyl galactosamine residues (SA/Gal/GalNAc binding reagent) are capable of this inhibition. These reagents include various lectins, such as Sambucus nigra agglutinin (SNA) (elderberry bark lectin), and certain manrralian lectins, as well as other small molecules, such 25 as peptides which are known, or can be shown, to bind this target. In particular, peptides derived from Factor IXa/VIIIa or from tissue factor/VIIa are useful in this respect An additional important class of SA/Gal/GalNAc 30 binding reagents are antibodies specifically immunoreactive with this target. Such antibodies can conveniently be produced by rendering either of these disaccharide moieties immunogenic through coupling to S• immunogenicity-conferring carriers such as keyhole limpet hemocyanin, human serum albumin, bovine serum albumin, diphtheria toxoid, rotavirus VP6 carrier protein, and the like. Suitable techniques for coupling carbohydrates to immunogenic carriers of this type, for example Coupling the carbonyl moiety of the carbohydrate to an available amino group on the carrier followed by reduction, are well known in the art.

The disaccharide moiety, coupled itself or in the context of a larger carbohydrate to an immunogenicity-conferring carrier, is then administered in standard immunization protocols to suitable subjects to induce the production of antibodies. The effectiveness of the protocols can be monitored by standard irmmunoassays on the sera of the injected animals using the immunogen, or preferably the relevant disaccharide portion thereof, as antigen, specific binding partner. Such immunoassays, such as radioimnunoassay, enzyme-linked i.rmunoassays fluorescence-type immunoassays, and the like are well known in the art. When sufficient titers are obtained, the an-isera may be used directly after purification of the imunclob-!ins, or the antibody-producing cells suc i!!ias :he slen or a anioyrdcn suc a s h e s -en cells or as peripheral blood lymphocytes o-f the innunized animals can be used for the production of monoclonal for.s of these antibodies immunospecific for the disaccharide moiety. In general, techniques for immortalization of the antibody-producing cells in the form of, for example, hybridomas for the production of such antibodies, are also now standard procedure *o In addition to these SA/Gal/GalNAc binding 30 reagents, antibodies, including monoclonal antibodies, that bind generally to the carbohydrate moieties of the activation peptide of Factor x may inhibit Factor Xa formation.

35 In an alternative approach, prevention of the formation of the sialyl termination residues of the glycosylated Factor X may be an effective way of providing a Factor X that resists activation. In one approach, administration of inhibitors of a(2-6)sialyl transferase to the organism can achieve this end. Thus, the organism will produce Factor X which is devoid of the essential substitutent needed for its activation. In a variation of this approach, inhibitors of the transferases which catalyze the glycosylation of Factor X in general are also effective, since the internal saccharide chains must be provided as acceptors for the sialyl residue. The inhibitors are supplied to the organism in a manner effective to alter its repertoire of glycosylated Factor X. These inhibitors thus behave as preventive as well as prophylactic agents with regard to conditions characterized by an excess of Factor Xa or which are benefited by a diminution in the levels of Faccor Xa. Examples of such inhibitors include Amphomycin, Castanospermine, 2,3-Dehydro-2-deoxy-Nacenyl-neuraminic acid, 1-Deoxymannojirimycin hydrochloride, l-Deoxynojirlmycin, N-Methyl-ldeoxynojir mycin, Swainsonine and Ttunicamycin.

In still another approach, a soluble analog cf the SA/Gal/GalNAc sequence that is evidently required for Factor X-to-Factor Xa conversion may be used as a 25 competitor for the essential disaccharide residue. Such competitors are introduced so as to be present under those conditions wherein Factor X-to-Factor Xa conversion SS would otherwise be achieved. Thus, in vitro, the soluble analog is introduced along with the components of the 30 intrinsic or extrinsic pathway for conversion. In the context of a whole living organism, the soluble analog is administered to prevent this conversion in situ. The soluble analogs are low molecular weight molecules which have space and charge contours similar to those of the SA(2-6)cGal or SA(2-6)aGalNAc residues as they occur on -11the glycosylation side chains of Factor X. Among such soluble analogs are SA(2-6)aGal and SA(2-6)aGalNAc per se, as well as analogous di- and trisaccharides or alternative complex structures which exhibit these characteristics.

In still another alternative, Factor X-to- Factor Xa conversion can be inhibited by reagents that remove the glycosylation moieties from Factor X, thus rendering it unsusceptible to activation. Suitable such agents include neuraminidases and certain specific glycosidases that remove sialyl residues.

The preferred method for inhibiting Factor Xto-Factor Xa conversion untilizes binding reagents that are capable of forming complexes with the SA(2-6)aGal and SA(2-6)aGalNAc residues on the glycosylation side chains of Factor X. In general, reagents that are useful SA/Gal/GalNAc binding reagents for use in the method of the invencion can be identified using simple specific binding assays with the disaccharide moiety, or a molecule containing same, as the specific binding partner for t h e candidate compound to be tesced. Such assays are conduccec in a manner analogous to imm unoassays wherein, for example, in a typical protocol, the disaccharide moiety to be targeted is coupled to a solid support and 25 the coupled support treated with the candidate reagent.

The treated support is then washed to remove any unbound candidate. Candidate remaining bound to the support is detected by any suitable means such as by an additional moiety which specifically binds the candidate which is 30 itself labeled using a detectable label, for example a radiolabel or enzyme label. Thus, candidate SA/Gal/GalNAc binding reagents can be quickly screened for efficacy in such simple and straightforward protocols.

-12- In the invention method, the SAa(2-6)Gal and/or SAa(2-6)GalNAc binding agent, as identified by the above method, is contacted with the Factor X whose conversion is to be inhibited. The resultant of such contact is a complex which is resistant to conversion to the active form by either the extrinsic or intrinsic pathway. Said contacting is with an excess of said reagent, preferably a 2- to 10-fold molar excess and at temperature, pH, and salt conditions appropriate for the formation of the complex. Generally such contacting is at 40C to room temperature for 4 hours to overnight and in the presence of physiological saline and pH.

The SA/Gal/GalNAc binding compounds-of theinvention that are lectins may be prepared by isolation of native sources or, under some circumstances, may be prepared recombinantly. Alternatively, synthetic peptides containing the SA/Gal/GalNAc binding domain may be substituted for the full-length lectins. These pe tides may be synthesized using standard solid-phase or solution-phase peptide synthesis techniques as are known a: and indeed commercially available in the art.

The SA/Gal/GalNAc binding reagents of the invention and alternative methods set forth above that are capable of inhibiting the activation of Factor X are 25 useful in procedures complicated by thrombosis and in conditions whose pathogenesis involves thrombin generation. These conditions include those involving S•arterial thrombosis, such as unstable rest) angina and abrupt vessel closure during vascular interventions 30 including coronary and peripheral angioplasty and *o atherectomy, and during and after vascular bypass procedures (peripheral and coronary), reocclusion after thrombolytic therapy for myocardial infarction, thrombotic stroke (stroke in evolution), and thrombosis due to vasculitis (Kawasaki's disease). Also included -13are conditions involving venous thrombosis, such as deep venous thrombosis of the lower extremities, pulmonary embolism, renal vein, hepatic vein, inferior vena cava thrombosis, and cavernous sinus thrombosis. Other target conditions are those involving diffuse activation of the coagulation system, such as sepsis with disseminated intravascular coagulation, disseminated intravascular coagulation in other settings, thrombotic thrombocytopenic purpura, and rare conditions of unknown etiology (Lupus anticoagulant).

The SA/Gal/GalNAc binding agents of the invention and alternative methods for inhibition of Factor X activation are also useful as an.anticoagulant and anti-inflammatory for cardiopulmonary bypass, in harvesting organs, in preparation of blood products or samples and in transport and implantation of organs and associated treatment of the recipient. These reagents and methods are especially useful in a slow release form in indwelling intravascular devices catheters, 20 grafts, patches) Thrxobosis also plays a role in rescenosis following vascular interventions such as angioplasty, atherectomy, or endarterectomy by directly or indirectly causing smooth muscle cell proliferation, and the 25 reagents of the invention is also useful in treating this condition.

Adult respiratory distress syndrome (ARDS) is thought to be an "endotoxin" disease in which a prothrombotic endothelium is likely to exist, with inflammatory and proliferative components; the invention reagents and methods are also useful in treatment of

ARDS.

The therapeutic reagents of the invention which are lectins or their peptide-binding domains or which are antibodies or fragments thereof, or other peptide-based -14substances are formulated for administration using excipients conventional for administration of proteins or peptides, typically by injection, as set forth, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, latest edition, Easton, PA. For the antithrombosis effect, the lectins or other peptides or proteins are administered systemically, preferably by injection, and preferably by intravenous injection.

Dosage levels depend on a number of Factors, including the condition of the subject and the specific embodiment chosen. However, suitable dosage ranges are on the order of 1-50 mg per patient per continuous injected dose. For injection, the protein is dissolved or suspended in liquid medium, for example, Hank's solution, Ringer's solution, dextrose solution, and various buffers.

Additional excipients such as stabilizers can also be employed.

Besides injection, the lectins or other ~therapeutic agents of the invention can be administered 20 systemically, via suppository, oral administration, transmucosal administration, including intranasal sprays, and by slow release formulations. Additional formulation techniques include encapsulation formulations, such as liposomes.

25 In addition to utility as a therapeutic, the invention reagents can be used to raise polyclonal antisera or to produce cells which can be fused to immortalizing partners to obtain sources of monoclonal antibodies specific for these reagents. These antibodies are useful as diagnostic tools to monitor therapy with the invention reagents.

SThe ability of the SA/Gal/GalNAc binding reagents of the invention or other candidate substances to inhibit the conversion of Factor X to Factor Xa can be studied by determining the effect of the candidate inhibitor on the ability of Factor X subjected to putative activation conditions characteristic of the extrinsic or intrinsic pathway to generate an enzymic activity characteristic of Factor Xa. Thus, the presence of generated Factor Xa can be measured by kinetics of hydrolysis of chromozym X (N-methoxycarbonyl-D-norleucylglycyl-arginine-4-nitranilide acetate, Boehringer Mannheim) hydrolysis. (Wolf, et al., J Biol Chem (1991) 266:13726-13730.) 1 0 To generate the putative Factor Xa enzymes by the intrinsic pathway, Factor X is activated with 0.2 nM Factor IXa, 6.8 nM Factor VIIIa (from Cutter hemophilia concentrate), excess phospholipids in the mM range, and 5-10 mM calcium ions. The activation is conducted by incubating Factor X in physiological saline for about min at 37 0 C and detecting the Factor Xa formed by the cnromozym X hydrolysis described above. Activation of Factor VIII to VIIIa is conducted as described by Hill- Eubanks, J Biol Chem (1990) 17854-17858.

S 20 To determine the effect of the SA/Gal/GalNAc binding reagenz, the Factor X and the candidate binding reagent are first preincubated at low temperature for 4- 24 hours in buffered saline containing approximately 1 mM each of calcium, magnesium and manganese ions.

25 Similarly, candidate inhibiting enzymes which remove the glycosylation of Factor X are preincubated with this factor prior to addition of the components of the :intrinsic pathway. To test the inhibitory ability of the competitive soluble analogs of the SA/Gal/GalNAc residues, the soluble analog is added along with the components of the pathway. This assay method is, however, not appropriate for those agents that are inhibitors of glycosylation, as the effect of these inhibitors is dependent on Factor X formation in the living organism.

-16- For effect on the extrinsic pathway, the Factor X to Factor Xa conversion is conducted (as described for the intrinsic pathway activation) using 0.5-1 nM Factor VII, tissue factor (thromboplastin excess phospholipids and 5-10 mM calcium ions.

The foregoing procedures are illustrative of the manner in which candidate compounds which are SA/Gal/GalNAc binding reagents or which otherwise inhibit Factor Xa formation may be confirmed to show inhibition of Factor X activation. These assay procedures are used to determine the effectiveness of a glycosidase or other reagent capable of removing glycosylation units as well as the SA/Gal/GalNAc binding agents in the inhibition of the activation of Factor X. The assays set forth above are used as described, including pre-incubation with the candidate glycosidase or other glycosylation-removing enzymes or with the SA/Gal/GalNAc binding agent, and concomitant with soluble analogs of the SA/Gal/GalNAc sequence.

The following examples are intended to illustrate, but not limit the invention.

Example 1 25 Ability of Lectins of Various Bindina Secificities to Complex with Human and Bovine Factor X In a preliminary experiment, the ability of lectins with various binding specificities to complex with human and bovine Factor X and Xa was tested using the glycan differentiation kit (First Choice, Boehringer- Mannheim). In the procedure, conducted according to the manufacturer's instructions, the Factor X or Factor Xa was first immobilized on nitrocellulose or nylon membrane, and the specific binding of digoxygenin-labeled lectins from the kit was detected using antidigoxygenin -17antibody coupled to alkaline phosphatase. Table 1 summnarizes the results obtained using the various lectins of the kit.

Table 1 Galanthus nivalis agglutinin

(GNA)

Peanut agglutinin

(PNA)

Datu.ra s trarnioniun a utinin

(DSA)

S a--b u cu s ni'cr-a a lu in in

(S.NA)

Ma a ck a a-mu rens is 25 agglutinin

(MAA)

Specificity High mannose chains Bovine Human x Gal (,61-3)GalNAc, (w/o SA) Gal ()3-4)GalNAc; GlcNAc SAi~y2-6)Gal SA(a2-6)Ga1NAc SA(ty2-3)Ga.

S

9 9 1- Thus, DSA, SNA and MAA bind to human Factor X, and bovine Factor X is bound only by MAA. None of the activated forms bind to these lectins.

-18- Example 2 Inhibition of Factor X Activation in the Intrinsic Pathway Human or bovine Factor X (1 iM) and the candidate lectin (5 AM) were incubated overnight at in buffered saline containing i mM each of Ca+2, Mg 2 and Mn+2 ions. The incubation mixtures were activated with Factor IXa, VIIIa, phospholipids and calcium ions as described hereinabove. The Factor Xa liberated was assayed using the chromozym X substrate described above.

None of the lectins inhibited the activation of bovine Factor X; only SNA inhibited the activation of human Factor X. The extent of inhibition was 41%.

Exam Inhibition of Factor X Activation by the Extrinsic Pathway Bovine or human Factor X was incubated with the l ec:ins of Example 1 in the same manner as set forth in 20 Example 2 and then activated with Factor VIIa, tissue factor, phospholipids and calcium ions as described hereinabove. Generation of Factor Xa was determined as above. None of the lectins inhibited activation of bovine Factor X; again only SNA inhibited the activation 25 of human Factor X (67% inhibition).

Example 4 Dose Dependence of Human Factor X Activation by SNA Human Factor X (1 was incubated with concentrations of SNA ranging from 0-20 uM overnight at 4 0 C. Aliquots were assayed using intrinsic and extrinsic pathway activators as described in Examples 2 and 3 above. Bovine Factor X was used as a control, and showed only slight inhibition at the highest concentration of

SNA.

-19- In the intrinsic pathway activation using IXa/VIIIa, virtually no inhibition was observed up to 1 1 M SNA; at a concentration of 1.5 M, inhibition was increasing to about 47% at 20 AM.

In the extrinsic activation pathway, virtually no inhibition was obtained at 0.5 AM SNA; however, 1 AM SNA showed an inhibition of about 27%, which increased to an inhibition of 80% at 20 pM SNA.

Example Effect of Various Glycosidases Glycosidases of various specificities were used to treat human and bovine Factor X by treating Factor X with the glycosidase according to the manufacturer's instructions. In general, digestion is conducted at 37 0

C

overnight, although neuraminidase gave complete digestion after shorter time periods. The reaction mixtures were subjected to SDS/PAGE, followed by staining with Coomassie blue, and the shift in mobility of the treated 20 Factor X proteins, as compared to untreated Factor X, was determined. Neither bovine nor human Factor X showed a shift in mobility for the light chain for any of the glycosidases. Certain of the glycosidases tested, however, did result in alteration of mobility of the 25 heavy chain as set forth in Table 2 below.

The effects of deglycosylation on activity was tested in several ways. First, the deglycosylated material was tested in standard coagulation assays--the activated partial thromboplastin time assay (APTT) and the prothrombin time assay The assays were conducted using standard procedures but with Factor X-deficient human plasma and using an automatic coagulometer. The results of these assays are also shown in Table 2.

In addition, the glycosidase-treated proteins were activated using the. Factor IXa/VIIla complex or the Factor VIla/tissue factor complex and the activated Factor Xa detected using chromozym X. These results are shown in Figures 1A and 1B for bovine Factor X and human Factor X. respectively.

0 0* 0 0 9* 0* 0 00 00 0 0 9 000 0 S 00 0 0

U-I

00 (D 0Table 2 Control 0- glycosidase Endoglycosidase H Neurarninidase N-glucosidase F

AHLC

(kd) 6.2 0 4.5 7 Human X

APTT

100 61 49 12 148 100 108 60 8 37 Bovine X AH1 C (kd) 3.3 0 4.5 7 AP rr 100 116 88 100 106 106 2 102 Table 2 shows, in addition to the shifts in heavy chain mobility, the results of the APTT and PT coagulation tests as a percentage of the activity of the control. Only neuraminidase was dramatically effective in reducing coagulation activity; however, endoglycosidase H was partially effective with respect to human Factor X.

Figures 1A and 1B show the results for bovine Factor X and human Factor X, respectively, in activation by the extrinsic or intrinsic pathways and assayed by chromozym X. Consistent with the results in the coagulation activities in Table 2, only neuraminidase is effective in substantially reducing the activation by either pathway.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

*o

Claims (37)

1. A method to inhibit the conversion of human Factor X to human Factor Xa which method includes contacting human Factor X with a reagent capable of cleaving glycosylation moieties from said Factor X in an amount and for a time sufficient to cleave said glycosylation moieties.

2. The method of claim 1 wherein said glycosidase is a neuraminidase.

3. A method to inhibit the conversion of human Factor X to human Factor Xa which method includes contacting human Factor X with a reagent capable of binding to the glycosylation moieties of the activation peptide of Factor X in an amount and for a time sufficient to bind said peptide.

4. A method to inhibit the conversion of human Factor X to human Factor Xa, which method includes contacting human Factor X with at least one soluble analog of SA/Gal/GalNAc, said contacting being under conditions which would normally, in the absence of said soluble analog, effect the 20 conversion of Factor X to Factor Xa.

5. The method of claim 4 wherein said conditions are those of the extrinsic or intrinsic pathway. 25 6. The method of claim 4 wherein said contacting is effected by administering said soluble analog to a living organism.

7. A method to inhibit the conversion of human Factor X to human SFactor Xa in a living organism by administration of an effective amount of an 30 inhibitor of glycosylation.

8. The method of claim 7 wherein said inhibitor inhibits a sialyl transferase.

9. A method to identify an agent that inhibits the conversion of human Factor X to human Factor Xa, which method includes: 24 treating a disaccharide moiety which is SA(a2-6)Gal or SA(c2- 6)GalNAc with a candidate compound: assessing whether said compound binds or does not bind to said disaccharide, and identifying any compound that binds to said disaccharide and also has activity in inhibiting the conversion of human Factor X to human Factor Xa.

10. The method of claim 9 wherein said compound is a lectin.

11. The method of claim 10 wherein said substances is a mammalian lectin.

12. A complex comprising human Factor X and an agent identified by the method of anyone of claims 9-11.

13. A method to inhibit the conversion of human Factor X to human Factor Xa which method includes contacting human Factor X with a reagent capable of binding to the glycosylation moieties of the activation peptide of Factor X in an amount and for a time sufficient to bind said peptide.

14. The method of claim 13, wherein said reagent specifically binds to 25 sialic acid residues that are a2-6 linked to galactose or galactosamine 25 residues. The method of claim 14, wherein said reagent is a lectin. o

16. The method of claim 15, wherein said lectin is Sambucus nigra agglutinin.

17. The method of claim 15, wherein said lectin is a mammalian lectin.

18. The method of claim 14, wherein said reagent is an antibody.

19. The method of claim 18, wherein said antibody is a monoclonal antibody. The method of claim 14, wherein said reagent is a peptide derived from Factor IXa/VIIIa or from Tissue Factor/VIIa.

21. The method of any one of claims 13, 14 and 16, wherein said reagents are formulated for administration using conventional excipients.

22. A pharmaceutical composition useful in inhibiting the conversion of Factor X to Factor Xa, which composition includes, as active ingredient, a SA(a2-6)Gal or SA(a2-6) GalNAc binding agent in admixture with a pharmaceutically acceptable excipient; wherein said binding agent is not a plant-derived lectin.

23. The composition of claim 22, wherein said SA(a2-6)Gal or SA(a2-6) GalNAc binding agent is a mammalian lectin.

24. The composition of claim 22, wherein said SA(a2-6)Gal or SA(a2- 6)GalNAc binding agent is a mammalian lectin. S, 25. The method of claim 4, wherein said analog is a disaccharide or a Si trisaccharide.

26. The use of a reagent capable of cleaving glycosylation moieties from 25 Factor X in the preparation of a medicament for inhibiting the conversion of human Factor X to human Faxtor Xa.

27. The use of a reagent as in claim 26, wherein said glycosidase is a neuramidase. •28. The use of a reagent capable of binding to the glycosylation moieties from Factor X in the preparation of a medicament for inhibiting the conversion of human Factor X to human Factor Xa.

29. The use of at least one soluble analog of SA/Gal/GalNAc in the preparation of a medicament for inhibiting the conversion of human Factor X to human Factor Xa, wherein said inhibition occurs under conditions that would normally, in the absence of said analog, effect the conversion of Factor X to Factor Xa.

30. The use of at least one soluble analog of SA/Gal/GalNAc as in claim 29, wherein said conditions are those of the extrinsic or intrinsic pathway.

31. The use of at least one soluble analog of SA/Gal/GalNAc as in claim 29, wherein said medicament is prepared for administration to a living organism.

32. The use of an inhibitor of glycosylation in the preparation of a medicament for inhibiting the conversion of human Factor X to human Factor Xa.

33. The use of an inhibitor of glycosylation as in claim 32 wherein said inhibitor inhibits a sialyl transferase.

34. The use of an agent identified in any one of claims 9-11 in the preparation of a medicament for inhibiting the conversion of human Factor X Sto human Factor Xa, wherein said agent is capable of forming a complex with S: human Factor X. The use of a reagent capable of binding to the glycosylation moieties 25 of the activation peptide of Factor X in the preparation of a medicament for inhibiting the conversion of human Factor X to human Factor Xa.

36. The use of a reagent as in claim 35, wherein said reagent specifically "binds to sialic acid residues that are a2-6 linked to galactose or 30 galactosamine. o

37. The use of a reagent as in claim 36, wherein said reagent is a lectin.

38. The use of a lectin as in claim 37, wherein said lectin is Sambucus nigra agglutinin. 27

39. The use of a lectin as in claim 37, wherein said lectin is a mammalian lectin. The use of a reagent as in claim 36, wherein said reagent is an antibody.

41. The use of an antibody as in claim 40, wherein said antibody is a monoclonal antibody.

42. The use of a reagent as in claim 36, wherein said reagent is a peptide derived from Factor IXa/VIIIa or from Tissue Factor/VIIa.

43. The use of a reagent as in any one of claims 35, 36 or 38, wherein said reagent is formulated for administration using conventional excipients.

44. The use of at least one soluble analog of SA/Gal/GalNAc as in claim 29, wherein said analog is a disaccharide or a trisaccharide. Dated this eighth day of December 1999. COR THERAPEUTICS, INC. Patent Attorneys for the Applicant: F.B. RICE CO. 9 9 9 *9 99 9 9 9 *9*999