WO1992017506A1

WO1992017506A1 - MODULATION OF tPA ACTIVITY BY HEPARIN FRAGMENTS

Info

Publication number: WO1992017506A1
Application number: PCT/US1992/002472
Authority: WO
Inventors: H. Edward Conrad; John C. Klock; Jay M. Edelberg; Salvatore V. Pizzo
Original assignee: Glycomed, Inc.
Priority date: 1991-03-29
Filing date: 1992-02-27
Publication date: 1992-10-15
Also published as: AU1680692A

Abstract

Disaccharides or oligosaccharides which include disaccharide subunits containing at least two negatively charged residues and fragment mixtures formed by partial or complete depolymerization of heparin by nitrous acid are capable of enhancing the ability of endogenous or administered tPA to dissolve blood clots. Disaccharide units which include, in particular, at least two sulfate residues, lack anticoagulant activity but are effective in enhancing the fibrin-specific clotting dissolution activity of tPA.

Description

MODULATION OF tPA ACTIVITY BY HEPARIN FRAGMENTS

Technical Field

The invention relates to therapeutics useful in controlling blood clots. More particularly, it concerns the use of short oligosaccharides to enhance tPA activity in blood clot dissolution, both when the tPA is administered and when endogenous tPA is relied upon.

Background Art The use of tPA to dissolve blood clots in therapeutic, prophylactic and preventive methods to control cardiovascular disorders is by now reasonably well established. Tissue plasminogen activator (tPA) is an endogenous compound which plays this important physiological role in normal subjects. However, in individuals suffering from cardiovascular disease or having the propensity therefor, endogenous tPA is insufficient to control blood clot formation and to dissolve any clots which may already have formed. tPA converts plasminogen to plas in, which represents a necessary step in the cascade of events leading to fibrin dissolution. The activity of tPA is, in fact, catalyzed by the presence of fibrin so that the tPA acts uniquely at the site of the blood clot to effect dissolution. Other proteolytic enzymes which have been used for the same purpose, but which have activity independently of the presence of fibrin, include urokinase and streptokinase. The perceived disadvantage of these clot-dissolving drugs is that they are activated systemically, and their activity is thus quickly dissipated. tPA, on the other hand, is converted from its more stable to its activated, less stable, form only in the presence of the clot.

It is known to administer anticoagulants, heparin in particular, in conjunction with tPA or urokinase or streptokinase treatment. The rationale is that the anticoagulant prevents the formation of additional clots. Furthermore, it has been demonstrated in in vitro assays that heparin stimulates the activity of tPA up to 25-fold (Andrade-Gordon, P., et al..

Biochemistry (1986) 2j5:4033-4040; Pacques, E.P. , et al. , Thromb Res (1986) .59:799-805; Fears, R. , Bioche J (1988) 249:77-81; Edelberg, J.M. , et al., Biochemistry (1990) 2J3:5906-5911) . Thus, the coadministration of heparin along with the clot-dissolving protease is thought both to stimulate the initial dissolution of the clot and prevent the formation of further fibrin clots. The use of fractionated heparin to provide this therapy has been suggested by Strickland (PCT application O90/04975) . In addition, it is known that tPA is bound to heparan sulfate on clearance cells; thus, administration of heparin, or its fragments, may prevent the rapid clearance of the drug by competition with this heparan sulfate for binding. Thus, administration of the competitor may enhance the concentration of endogenous tPA and may also prolong the half-life of tPA administered.

The use of complete heparin, or even low molecular weight forms thereof, suffers from the disadvantage of inability separately to control the anticoagulant activity and the tPA-stimulating activity of the protocol. Also, the competition for tPA binding may not be very efficient.

By providing a defined composition of oligosaccharide subunits related to those present in hepa in but lacking anticoagulant activity, a more controlled regimen of tPA activity stimulation or clearance reduction can be obtained. Suitable nonanticoagulant heparin fragments and disaccharides or oligosaccharides related thereto are provided by the invention.

Disclosure of the Invention

The invention provides tPA activity-stimulating saccharides which can be used to enhance the activity of either endogenous or administered tPA. The saccharides are either mixtures obtainable by complete nitrous acid digestion of heparin or are synthetic or purified individual saccharides, containing at least two sulfates and/or phosphates in a disaccharide unit, which lack anticoagulant activity and which, nevertheless, bind tPA and/or other fibrinolytic proteases or alter the rate constant with respect to conversion of plasminogen to plasmin. Accordingly, in one aspect, the invention is directed to a pharmaceutical composition formulated for systemic, preferably oral, administration which comprises an amount of a size-fractionated fragment mixture obtainable by partial or complete heparin degradation by nitrous acid in an amount effective to maintain elevated levels of tPA activity in a subject to which the composition is administered. In another aspect, the invention is directed to such compositions wherein the active ingredient is a disaccharide having at least two sulfate and/or phosphate moieties or a 3-12 unit oligosaccharide containing a disaccharide subunit of this type. The invention is also directed to methods to enhance the dissolution of blood clots by administering the compositions of the invention and to saccharide/tPA complexes which are useful pharmaceuticals. The invention methods may also involve the administration of tPA and/or other fibrinolytic agents.

In other aspects, the invention is directed to a process to prepare a sulfated and/or phosphorylated saccharide having the ability to stimulate the fibrin-dependent clot-dissolving activity of tPA. The disaccharides or disaccharide subunit-containing oligosaccharide pathway of the invention may be produced synthetically using a chemical synthesis reaction as disclosed herein or may be obtained by digesting heparin and carrying out separation procedures based on size and charge as disclosed herein.

In order to produce the polysaccharide compounds of the invention synthetically it is first necessary to synthesize an iduronic acid or glucuronic acid reaction synthon. Next, a glycosamine reaction synthon is produced. The glycuronic acid synthon and glycosamine synthon are reacted to produce a disaccharide reaction synthon. The disaccharide units can be reacted to form oligosaccharides containing 4, 6, 8 or any multiple thereof of saccharide units and/or can be reacted with either an iduronic or glycosamine reaction synthon to provide polysaccharides containing any odd number of saccharide units. These oligosaccharides can also be digested in nitrous acid to yield size-fractionated fragment mixtures included in the invention.

In order to obtain the fragment mixtures and components thereof by digestion, heparin can be obtained from a natural source and subjected to digestion with nitrous acid under conditions which favor complete digestion. Following the digestion the mixture is separated according to size and those portions corresponding to a molecular weight characteristic of di-, tetra-, hexa- and octasaccharides are segregated away and recovered. The recovered portions may then be separated according to charge in order to obtain the more highly charged portions. These portions will contain disaccharide units which contain at least two sulfate residues.

Brief Description of the Drawings

Figure 1 shows the elution pattern of a heparin digest from a P10 column. Figure 2 shows the effect of various heparin fractions on tPA activity.

Figure 3 shows the effect of lipoprotein A on the fibrinolytic reaction.

Modes of Carrying Out the Invention

The invention involves systemic administration of a small heparin-related saccharide either alone or in conjunction with tPA administration. When the saccharide is administered alone, it is believed to enhance the activity of endogenous tPA and/or other endogenous fibrinolytic proteins. The saccharide fragment is related to degradation products obtainable when heparin is degraded to completion using nitrous acid.

Preparation and Nature of the Invention Saccharides

If the saccharide is to be prepared from natural sources, heparin is preferably used as the starting material. To place the compositions discussed below in context, it may be noted that heparin and heparan sulfate are members of the GAG family which are classified by the nature of the hexosamine/aldouronic acid repeating units. For example, in chondroitin sulfates, the aldouronic acid is primarily D-glucuronic acid, and the hexosamine is N-acetylated 2-amino-2-deoxy- D-galactose, more commonly known as N-acetyl galactosamine and abbreviated as GalNAc.

In dermatan sulfate (chondroitin sulfate B) the aldouronic acid is mostly L-iduronic acid and the hexosamine is GalNAc. In keratan sulfate, the aldouronic acid is replaced by D-galactose, and the hexosamine is mostly N-acetylated 2-amino-2-deoxy-D-glucose, more commonly known as N-acetyl glucosamine and abbreviated as GlcNAc. In the compositions of interest herein, heparan sulfate and heparin, the hexosamine is mostly N- acetylated or N-sulfated glucosamine (GlcNH₂) , and the aldouronic acid is mostly L-iduronic in heparin and mostly D-glucuronic acid in heparan sulfate. Heparan sulfate is commonly considered to have a higher proportion of glucuronic acid than heparin.

Problems of heterogeneity in preparations of heparan sulfate or heparin isolated from tissues make sharp distinctions difficult, since the oligosaccharides are related by the biosynthesis pathway, as explained below. Conventional heparin (used as an anticoagulant) has a molecular weight of 5-25 kd and is extracted as a mixture of various chain lengths by conventional procedures. These procedures involve autolysis and extraction of suitable tissues, such as beef or porcine lung, intestine or liver, and removal of other GAGs and nonpolysaccharide components.

The molecular weight of the chains in the extract is significantly lower than the 60-100 kDa known to exist in the polysaccharide chains of the heparin proteoglycan synthesized in the tissue. The GAG moiety is synthesized bound to a peptide matrix at a serine residue through a tetrasaccharide linkage region of the sequence D-GlcA-D-Gal-D-Gal-D-Xyl → protein, which is then elongated at the D-GlcA residue with alternate additions of GlcNAc and GlcA.

The polysaccharide side chains are modified by a series of enzymes which sequentially deacetylate the N- acetyl glucosamine and replace the acetyl group with sulfate, epimerize the hydroxyl at C5 of the D-glucuronic acid residue (to convert it to L-iduronic acid and the GAG chain from the heparan type to a heparin type) , sulfate the 0-2 of the resulting L-iduronic acid and the 0-6 of the glucosamine residue, either at the heparan or heparin stage. This further sulfation is associated with the active site for antithrombin (anticlotting) activity. Other chemically possible sulfation sites are on the 0-2 of D-glucuronic acid; however, these are found as minor constituents.

In the representations of oligomers produced synthetically and those derived from GAG, the following abbreviations are used: D-glucuronic acid = GlcA; L- iduronic acid = IdoA; D-glucosamine = GlcNH₂; N-acetyl- D-glucosamine = GlcNAc; N-acetyl-D-galactosamine = GalNAc; D-glucosamine N-sulfate = GlcNS; 2,5- anhydromannose = AMan; 2,5-anhydromannitol = AManH; D- xylose = Xyl; glycosa inoglycan = GAG; acetyl = Ac. The location of the O-linked sulfate residues is indicated by "S" and the number of the position of sulfation where the sulfate residue is linked to oxygen on the sugar residue. In the synthetic saccharides of the invention, or in modified hydrolysis products, one or more sulfates may be replaced by phosphate; in these embodiments the phosphate is represented by "P". In these designations, also, the alpha and beta anomeric linkages are as those conventionally found in heparin, and the indicated D or L configurations, as conventionally found, pertains. The locations of the sulfates are shown below the abbreviation for the sugar to which they apply; thus, for example,

IdoA-GlcNS 2S 6S

refers to L-iduronic acid and D-glucosamine N-sulfate with sulfates connected respectively at the 2 and 6 positions of the sugar residues;

IdoA-GlcNS 2P 6P

refers to the corresponding diphosphate.

By "heparin/heparan sulfate" or "heparin" is meant a preparation obtained from tissues in a manner conventional for the preparation of heparin as an anti¬ coagulant or otherwise synthesized and corresponding to that obtained from tissue. This preparation may include residues of D-glucuronic acid (GlcA) , as characteristic of heparan sulfate as well as L-iduronic acid (IdoA) as characteristic of heparin. However, although both GlcA and IdoA are present in both, they are present in different proportional amounts. The conversion of D-glucuronic acid to L-iduronic acid is a result of epimerization at the 5-carbon of GlcA in a heparan-type intermediate. This sequence of steps involved in such epimerization and conversion is understood in the art. To the extent that full conversion has not been made, heparan sulfate characteristics remain in the prepara¬ tion. Because the precise nature of the polymeric chains in the preparations of heparin is not generally determined, and varies from preparation to preparation, the term "heparin/heparan sulfate" or "heparin" is intended to cover the range of mixtures encountered.

The "heparin/heparan sulfate" preparation can be obtained from a variety of mammalian tissues, including, if desired, human tissue. Generally, porcine or bovine sources are used, and vascularized tissue is preferred. A preferred source of heparin/heparan sulfate starting material is porcine intestinal mucosa, and preparations labeled "heparin" prepared from this tissue source are commercially available. In general, the heparin/heparan sulfate starting material is prepared from the selected tissue source by allowing the tissue to undergo autolysis and extracting the tissue with alkali, followed by coagulation of the protein, and then precipitation of the heparin-protein complex from the supernatant by acidification. The complex is recovered by reprecipitation with a polar nonaqueous solvent, such as ethanol or acetone or their mixtures, and the fats are removed by extraction with an organic solvent such as ethanol and proteins by treatment with a proteolytic enzyme, such as trypsin. Suitable procedures for the preparation of the heparin starting material are found, for example, in Charles, A.F., et al., Biochem J (1936) 3J):1927-1933, and modifications of this basic procedure are also known, such as those disclosed by Coyne, E. , in Chemistry and Biology of Heparin. Elsevier Publishers, North Holland, New York, Lunblad, R.L., et al., eds. (1981) . The starting material is then depolymerized with nitrous acid under conditions which effect partial or complete depolymerization. There is an extensive body of art concerning depolymerization of heparin/heparan sulfate chains and separation of products by size. Particularly relevant is the report of Guo, Y., et al., Anal Biochem (1988) 168:54-62, which discloses the results of structure determination after the 2,5- anhydromannose at the reducing terminus is reduced to the corresponding 2,5-anhydromannitol. It is known that nitrous acid cleaves specifically at the linkage between -lo¬

an N-sulfated or N-unsubstituted glucosamine residue and the uronic acid coupled to the l-position of the glucosamine. Glucosamine residues which are^' acetylated on the nitrogen provide 1,4-linkages which are resistant to nitrous acid cleavage. It is also known that the conditions of nitrous acid cleavage effect the deamination and recyclization of the sugar at the reducing terminus to obtain a 2,5-D-anhydromannose at this position. In general, the nitrous acid is generated in situ from sodium nitrite by adjusting the pH to acidic pH, preferably around pH 1.5. The total nitrous acid in such a reaction mixture may be controlled by controlling the amount of sodium nitrite added, and may be in amounts either less than or greater than required for complete cleavage of all of the susceptible bonds in the heparin present in the reaction mixture. In either case, the reaction is continued to completion, as judged by cessation of nitrogen evolution.

Thus, in typical situations, the nitrous acid is prepared in situ by solution of sodium nitrite in cold acidic solution at a concentration of about 50 mM, and the reagent is used to treat the heparin at a concentration of about 100 mg/ml, at a pH of about 1.2 to about 1.8, preferably about 1.5. The reaction is conducted at room temperature. The mixture can be neutralized by addition of a suitable reagent at the desired stage of digestion; however, with respect to the present invention, the reaction is carried to completion. Other depolymerization methods can also be used as long as they produce active components, i.e., components which (1) are di-, tetra- or hexasaccharides; (2) are sulfated so that the disaccharide or a disaccharide subunit contains at least two sulfates; (3) have substantial ability to stimulate the fibrin-dependent clot-dissolving activity of tPA or other proteins; and (4) have insignificant or no anticlotting activity.

The depolymerized saccharides can then be size-separated by a variety of means. In a particularly effective method, the mixture is chromatographed in a Biogel P10 system in which two columns are connected in tandem. A system containing two 5-cm x 128-cm columns can accommodate as much as 160 ml of the polymerization mixture. Fractions eluting from the columns are analyzed for saccharides using any convenient method; a particularly convenient method is the carbazol procedure for quantification of uronic acid content. In the Biogel P10 system, the peaks elute in reverse order of size, largest fragments first. The identified, size-fractionated fractions from the size-separation protocol can then be analyzed for their activity with respect to tPA clot dissolution enhancement and can also be separated into their individual components. Anion-exchange chromatography is conveniently used to separate molecules with differing charges. Thus, the more highly sulfated forms of the saccharides in the mixture can conveniently be separated from the less highly charged members. Further separation by high pressure liquid chromatography (HPLC) permits the isolation of individual compounds. In particular, HPLC separations are most conveniently performed on C18 columns run in the reversed phase ion-pairing mode or on polyethylene imine columns developed with salt gradients.

The mixture of disaccharides has been found to contain, as its major component, IdoA-AMan; minor

2S 6S components are: GlcA-AMan and GlcA-AMan.

2S 6S 3S,6S

These subunits also occur either at the reducing terminus, or internally, in the higher molecular weight saccharide oligo ers which are contained in the tetra-, hexa- and octasaccharide fractions. If desired, the sulfates may be replaced by phosphate moieties using conventional technology. Defined di-, tetra- and higher molecular weight oligosaccharides can also be prepared synthetically. The synthetic approach generally involves preparation of an iduronic acid or glucuronic acid synthon and reaction therewith by a glycosamine reaction synthon. The resulting disaccharide unit can be directly treated under the conditions of nitrous acid depolymerization to effect the conversion of the glucosamine residue to the anhydromannose. The dimer, however, prior to such conversion, is also useful in the invention. In addition, the anhydromannose residue can further be reduced to convert the exocyclic aldehyde to an alcohol; these reduced compounds are also included within the invention. They can also be prepared by reduction of the depolymerization fragment mixtures. The disaccharides can also be extended to form oligosaccharides containing any even number of saccharide units; this conversion can be followed by treatment to convert the reducing glycosamine to an anhydromannose or a reduced form thereof, if desired. In addition, using a water-soluble carbodiimide and sodium borohydride, any of the uronic acid carboxyl residues can also be reduced to an alcohol. These reduced forms of uronic acid saccha¬ rides are also included in the compounds of the invention. In addition, the even-numbered saccharide may be reacted with either a uronic acid or glycosamine reaction synthon to provide polysaccharides containing any odd number of saccharide units. In the case of those saccharides having glycosamine residues at the reducing terminus, the above-mentioned conversions can also be employed.

In all of the foregoing synthetic routes, the sulfate or phosphate moieties derivatized to the saccharide units may be added or exchanged to the finished oligomer, or may be introduced on the starting monomeric materials.

As was the case with regard to the depolymerization fragments, the synthetic saccharides must contain at least one disaccharide subunit which contains at least two sulfates. It is believed that the disulfated disaccharide subunit is responsible for binding to the kringle region of tPA. Although the larger saccharides appear to affect rate constant but not the Michaelis constant for the reaction catalyzed by tPA, the disaccharide affects both. This indicates that there must be an association of these saccharides with tPA.

Thus, the invention is directed to saccharides which lack anticoagulant activity and which contain 2-12 sugar units. Within the saccharide must be a disaccharide uronic acid-glycosamine (or derivative thereof) subunit with at least two sulfate residues. The uronic acid residue may either be in its reduced or nonreduced form. Anticoagulant activity is assured to be absent in saccharides of less than 5 units, since this appears to be the minimum structure required; with regard to saccharides of greater length, assay for anticoagulation activity, for example by assessing ability to bind antithrombin III, is employed. Preferred saccharide compounds of the invention include those wherein at least one uronic acid-glycosamine-derived unit contains at least two sulfates and is selected from the group consisting of:

IdoA-AMan; GlcA-AMan; GlcA-AMan; IdoA-GlcNH- . 2S 6S 2S 6S 3S 6S 2S 2 , 6-dlS Also preferred are oligosaccharides where at least one subunit is of the formula

Ido-GlcNAc-IdoA-GlcNAc 2S 6S 2S 6S or

IdoA-GlcNAc-IdoA-AMan, 2S 6S 2S 6S including, for example,

IdoA-GlcNAc-IdoA-GlcNAc-IdoA-GlcNAc-IdoA-AMan(2 ,5) ; 2S 6S 2S 6S 2S 6S 2S 6S

IdoA-GlcNAc-IdoA-GlcNAc-IdoA-GlcNAc-IdoA-AMan(2,5) ; 2S 6S 2S 6S 2S 6S 6S

IdoA-GlcNAc-IdoA-GlcNAc-IdoA-GlcNAc-IdoA-AMan(2,5); 2S 6S 2S 6S 2S 2S 6S

IdoA-GlcNAc-IdoA-GlcNAc-IdoA-GlcNAc-IdoA-AMan(2,5) ; 2S 6S 2S 6S 6S 2S 6S

IdoA-GlcNAc-IdoA-GlcNAc-IdoA-GlcNAc-IdoA-AMan(2 ,5) ; 2S 6S 2S 2S 6S 2S 6S IdoA-GlcNAc-IdoA-GlcNAc-IdoA-GlcNAc-IdoA-AMan(2,5); and 2S 6S 6S 2S 6S 2S 6S

IdoA-GlcNAc-IdoA-GlcNAc-IdoA-GlcNAc-IdoA-AMan(2,5), 2S 2S 6S 2S 6S 2S 6S and their counterparts wherein one or more sulfate is replaced by phosphate.

Assays Systems

The ability of the saccharides of the invention to enhance tPA activity is assessed as follows. Various concentrations of plasminogen (40-200 nM) are incubated with tPA (35 IU/ml) in the presence of the plasmin substrate D-Val-L-Leu-L-Lys-p-nitroanilide (VLK-pNA, 300 μM) in a buffer containing 50 mM Tris-HCl, 0.05% gelatin, and 0.01% Tween-80, pH 7.4. The reaction was monitored by change in absorbance due to liberation of P-nitroaniline from the plasmin substrate. The assay is described in detail in Edelberg, J.M. , et al., Biochemistry (1990) 23:5906-5911. The effects of increasing concentrations of various heparin fragments or synthetic saccharides to be tested on the reaction rate, as measured by absorbance, are determined. Steady state kinetics are determined by initial rate measurements as described by Urano, T., et al., Biochemistry (1978) 27:6522-6528, and by Hoylaerts, M. , et al., J Biol Chem (1982) 257:2912-2919.

Briefly, the initial rate of plasminogen activation was determined using the equation V- = b(l+K_E/S )/ek , where b is the initial rate of substrate hydrolysis derived from a plot of instantaneous rate of substrate cleavage versus time; K_E is the apparent Michaelis constant of VLK-pNA hydrolysis by plasmin (0.3 mM) ; k is the catalytic rate constant for hydrolysis of VLK-pNA by plasmin 2 (2.3 x 10³ M/mol plasmin s) ; and e is the molar extinction coefficient of the hydrolyzed substrate at 405 n (8.8 x 10 3 M—1 cm—1) .

The kinetic constants for plasminogen activation were determined by a double reciprocal plot of initial rates versus plasminogen concentrations in the absence of any test saccharides. The activity with respect to tPA binding can also be conveniently assessed in a competitive assay using radiolabeled heparin in competition with the candidate fragment or compound. As one metabolic role of the invention saccharides is to bind endogenous tPA to prevent its clearance, this assay also gives a measure of physiological activity.

Although the examples below show the influence of the saccharides on the tPA-catalyzed conversion of plasminogen to plasmin in the presence of fibrinogen fragments and lipoprotein A, this effect may not be important in vivo if the approximately 100-fold activation of tPA by fibrin dominates all of the activation and inhibition reactions of other materials. Of course, this is adjustable by supplying varying amounts of the saccharides. In any case, an additional effect is to enhance the apparent activity by preventing scavenging of the endogenous (or administered) tPA by heparan sulfate on the surface of the endothelial cells. The inhibition of this binding by the saccharide materials of the invention would prevent this rapid clearance of tPA.

The absence of anticoagulant activity can also be verified using standard methods, such as antithro - bin-III binding. Typical methods for assessing anticoagulant activity are set forth, for example in Kazmir, F.J., in "Chemistry and Biology of Heparin," Lundblad, R.L. , et al. , eds. (1981), Elsevier/North Holland, New York, pages 615-623; Fareed, J. , in "Perspectives in Homeostasis," Fareed, J. , et al., eds. (1981) Pergamon Press, New York, pages 310-347.

As set forth above, assessment of this null property is not necessary in oligosaccharides of less than 5 saccharide units.

Administration Protocols

The invention compounds are administered systemically, either along with a clot-dissolving protease or as a means to enhance the activity of endogenous tPA or other fibrinolytic protease. In either case, administration may be by any known systemic route, including injection intravenously, subcutaneously, intraperitoneally, or introduction using slow drug-release systems which are applied to the exterior to blood vessels, such as those described by Urquhart in U.S. patent 3,797,485. When tPA is coadministered. administration by injection is preferred. However, if the compounds of the invention are to be administered to enhance the endogenous protease, while injection can be used, oral means of administration are preferred. The short-chain oligosaccharides or disaccharides of the invention can readily be absorbed through the digestive tract into the bloodstream.

Formulations suitable for these modes of administration are known in the art, and a suitable compendium of formulations is found in Remington' s

Pharmaceutical Sciences. Mack Publishing Company, Easton, Pennsylvania, latest edition. Suitable formulations for injection include liquid formulations in buffers, physiological saline, or carriers such as Hank's Solution or Ringer's Solution, or the saccharides may be prepared in solid form and taken up in liquid suspension or solution for administration.

For oral administration, the invention saccharides are conveniently provided as syrups, tablets, capsules, powders, or can be added to foods or beverages to enhance acceptability and patient compliance. Thus, the disaccharides or oligosaccharides can be included in juices, soft drinks, or milk-based confections such as milk shakes and eggnog, or can be added in recipes for solid foods such as pastries and pastas. In addition, the oligosaccharides can be used to coat cereals or in frostings. In general, the saccharide compositions of the invention can be included in food and beverage compositions in a manner analogous to ordinary sugar. The invention compositions may be administered as pure compounds or as mixtures of two or more of these active ingredients. The compositions may include the saccharide enhancers of fibrinolytic activity as the sole active ingredient or may also include the fibrinolytic protease, preferably tPA. The subjects to whom the saccharides of the invention will be administered include those who are suffering from heart attack or other cardiovascular disturbance or at risk for these conditions. More particularly, the compositions of the invention may be used in the treatment of deep vein thrombosis, whether familial or nonfamilial, including when said thrombosis is surgically related. Also susceptible to treatment by the compositions of the invention are Atrophe blanche or livido vasculitis, systemic vasculitis, myocardial infarction, stroke and pulmonary emboli (which may, themselves, be a result of deep vein thrombosis) .

Further, the invention compositions are useful in treating, therapeutically or prophylactically, patients with high levels of fibrinolytic inhibitors, including lipoprotein A. Persons assessed with high levels of lipoprotein A are advantageously administered those embodiments of the invention compositions which are the disaccharide or tetrasaccharides as the activity of tPA is not inhibited in the presence of lipoprotein A when these embodiments are used as the stimulant. In patients of these high titers, use of heparin itself or of the invention compositions of higher molecular weight is less preferred. Typical dosages are designed to result in concentrations of the saccharides of the invention in the bloodstream of approximately 20-100 μg/ml. Accordingly, typical dosage ranges are on the order of 60-300 g. If administered intravenously, suitable dosages are in the range of 0.1-10 mg/kg/hour on a constant basis over a period of about 5-30 days. In particular, preferred dosage is about 0.3 mg/kg/hour or about 35 mg/hour or 840 mg/day for a 70 kg adult.

As the conditions for tPA therapy are well recognized in the art, one of ordinary skill would readily recognize the appropriate subject for administration of the saccharides of the invention, either alone or with the accompaniment of tPA administration.

Preparation of Antibodies

Antibodies immunoreactive with the saccharide compounds of the invention are useful in monitoring therapy and in immunoassays in general. These antibodies are prepared using conventional immunization protocols involving repeated administration of the saccharides to mammalian subjects such as rabbits, mice, rats, sheep, and the like. Antibody titers in the serum can be monitored using conventional immunoassays using the saccharides of the invention as antigen.

As saccharides are notably nonimmunogenic, the administration of the saccharides should be in a form wherein they are conjugated to an antigenically neutral carrier to provide immunogenicity. Suitable carriers include those conventional in the art, such as keyhole limpet hemacyanin (KLH) and various serum albumins. In addition, the VP6 protein of rotavirus has been shown to be a particularly effective carrier. Coupling to carrier is conducted by conventional means, including reductive amination, or usage of suitable linkers such as those available from Pierce Chemical Co. (Rockford, IL) .

The invention further includes the saccharides conjugated to the carriers employed in these immunization protocols, as well as the resulting antibodies. Polyclonal antisera may be satisfactory for many utilities; preparation of monoclonal forms of these antibodies can also be effected using the standard Kohler-Milstein procedure with improvements, as is well known in the art. The antibodies of the invention are thus useful in immunoassays, not only of the type described below involving competition between labeled composition and the analyte saccharide in the sample, but also for direct immunoassay of the saccharide. Alternate protocols involving direct assays are also of wide variety and well known. Typically, the analyte bound to antibody is detected by means of an additional reactive partner which bears a label or other means of detection. Thus, in typical sandwich assays, for example, the binding of the antibodies of the invention to analyte can be detected by further reaction with a labeled preparation of these same antibodies or by labeled antibody immunoreactive with this preparation by virtue of species differences.

Additional Conjugates

The compositions of the invention may also be labeled using typical methods such as radiolabeling, fluorescent labeling, chromophores or enzymes, and used in a competitive assay for the amount of the saccharides of the invention in a biological sample. Suitable protocols for competitive assays of analytes in biological samples are well known in the art, and generally involve treatment of the sample, in admixture with the labeled competitor, with a specific binding partner which is reactive with the analyte such as, typically, an immunoglobulin or fragment thereof. The antibodies prepared according to the invention are useful for this purpose. The binding of analyte and competitor to the antibody can be measured by removing the bound complex and assaying either the complex or the supernatant for the label. The separation can be made more facile by preliminary conjugation of the specific binding partner to a solid support. Such techniques are well known in the art, and the protocols available for such competitive assays are too numerous and too well known to be set forth in detail here.

The labeled fragments can be utilized to detect the location of tPA in vivo, and, as tPA preferentially binds to blood clots, to localize blood clots, as well. For use in the application, the conjugates are administered to the subject, preferably by injection, and the label detected by suitable radioscintographic means. Appropriate labels for such detection include techneceum-99, iodine-131, and indium.

The saccharides of the invention conjugated to solid supports are also useful for the purification of specifically binding moieties, including tPA, as well as for purification protocols for antibodies prepared as set forth above. Similarly, antibodies of the invention conjugated to solid support are useful for the purification of the invention saccharides.

EXAMPLES The following examples illustrate the invention and are not intended to limit its scope. Unless indicated otherwise, parts are parts by weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

Example 1 Preparation of Heparin Fragments Heparin (10 g) and 345 g sodium nitrite were dissolved in 80 ml water at pH 6. The pH of the solution was adjusted to 1.5 with 6N HC1 and maintained by dropwise addition of either 6N HCl or 2 M sodium carbonate. The reaction was maintained until completion was reached, as shown by the cessation of nitrogen evolution. In general, the time required was approximately 6 minutes. The pH was then adjusted to 8.5 with 2 M sodium carbonate and the mixture was centrifuged for 10 minutes at 8000 rpm in a Sorvall GSA rotor to remove a fine white precipitate which forms during the rise in pH. The supernatant is decanted, degassed under vacuum, and concentrated by lyophilization or rotary evaporation to a concentration of 300 mg heparin fragment/ml. The preparation process is scalable.

The concentrated mixture was then chromatographed on a Biogel P10 system containing two columns in tandem, each approximately 5 cm x 128 cm and containing a total of 5 1 of Biogel P10. The columns were packed and run in 0.5 M ammonium carbonate at a flow rate of 0.7 ml/min. Fractions of 18 ml each were analyzed for oligosaccharides using the carbazol procedure of Bitter, T. , et al., Anal Biochem (1962)

4_.:330-334. The saccharides elute sequentially by size, largest first; the last peak contains mostly disaccharides.

The disaccharide fraction was further fractionated by gel filtration on a Biogel P10 column (1 cm x 60 cm) to obtain unsulfated, onosulfated and disulfated disaccharides.

Example 2 tPA Activity Enhancement by the Fragment Mixtures

The fractions eluting from the Biogel P10 column were assayed for the ability to enhance tPA activity in the assay for conversion of plasminogen to plasmin described hereinabove with and without the addition of fragments prepared by CNBr digestion of fibrinogen to simulate fibrin stimulation.

When assayed without the presence of fibrinogen fragments, Lineweaver-Burke plots obtained by varying the concentration of plasminogen permitted the calculation of the effect of the saccharides on 1^ and k .. These results are shown in Table 1 below.

(Determination for the di- and decasaccharides were performed under two sets of conditions) The higher chain length saccharides, while not affecting the Michaelis constant, are capable of raising the reaction rate constant on the order of 3-4-fold. This ability appears to taper off gradually with oligomers of 10 residues or more. These oligomers are less preferred anyway, as their anticoagulant activities become significant and need to be verified by assay. The disaccharide, on the other hand, while putatively less effective with respect to the rate constant, lowers the Michaelis constant. Thus, the overall rate of conversion of plasminogen to plasmin is enhanced on the order of 6- to 25-fold at physiological (blood) concentrations of plasminogen.

The effect of various fragment mixtures in comparison with heparin itself in the absence and presence of fibrinogen fragments and absence and presence of lipoprotein A is shown in Figure 2. As the desired effect of the saccharide is to enhance the performance of tPA in the presence of clots, the effect in "the presence of fibrinogen fragments is extremely important. The first conclusion that can be drawn from the data shown in Figure 2 is that the disaccharide and octasaccharide fragments are at least slightly more potent than any other saccharides tested in this respect.

In more detail it is seen that the initial rate is 0.01 as determined above when no saccharide is added. Not shown in the figure are the results that this initial rate is the same (0.01) when lipoprotein A alone is added to the reaction mixture, and that the initial rate is increased to 0.04 when fibrinogen fragments alone are added. This is consistent with the known ability of fibrin to stimulate the tPA-catalyzed conversion of plasminogen to plasmin.

While heparin provides dramatic stimulation of initial rate (0.11 in the absence of fibrinogen fragments) , it appears that the dramatic nature of this stimulation is lost when fibrinogen fragments are present. Furthermore, the presence of lipoprotein A (in the absence of fibrinogen, as tested here) diminishes the ability of heparin to stimulate the conversion, as shown. The initial rate is now only 0.03.

On the other hand, the saccharide fragment mixtures, while much less capable of stimulating tPA, as compared to heparin in the absence of fibrinogen fragments (0.03-0.04, as initial rates), are comparable or more effective in this stimulation in the presence of fibrinogen fragments. This is significant as it more closely approximates physiological conditions. Furthermore, lipoprotein A appears to make little difference when employed in the presence of the small fragments. As lipoprotein A has no effect in the absence of heparin (initial rate 0.01, comparable to the control) , heparin must mediate the inhibition by lipoprotein A. This effect is clearly not present in the case of the di- and tetrasaccharides. Presumably the smaller saccharides do not bind both tPA and lipoprotein A to the same saccharide backbone as may be the case for high molecular weight heparin.

The effect of lipoprotein A (LPA) dependent on the size of the heparin fragment is shown more clearly in Figure 3. Figure 3A shows the V^ values obtained in the plasminogen → plasmin assay described above in the presence of 50 μg/ml of each heparin fragment alone (shaded) or with 50 μg/ml of heparin fragment plus 50 nM LPA (striped) . (Fibrinogen fragments are not present.) This figure clearly shows that lipoprotein A has no discernible affect on the initial reaction velocity when the disaccharide fragment is used; there is an almost negligible effect when the tetrasaccharide is used. On the other hand, with increasing chain length, the effect of lipoprotein A increases dramatically. This is shown in another form in Figure 3B, as a percent of possible activity.

As the physiological situation requires the efficacy of tPA in the presence of fibrin (fibrinogen fragments) , the advantages of the saccharides of the invention over the use of heparin per se are readily seen.

Example 3 In Vivo Models

Guinea pigs, hamsters, or rabbits can be used. Radiolabeled fibrin, which accumulates in the lungs, is first injected, and tPA is administered. The appearance of labeled fibrin fragments in the blood is then measured, with and without the administration of the candidate saccharide. This jLn vivo model is useful in assessing the effects of the saccharide additives in in vivo contexts.

Claims

1. A pharmaceutical composition formulated for systemic administration which comprises an amount of a disaccharide or a 3-12 unit oligosaccharide which contains a disaccharide subunit, which disaccharide or subunit contains at least two negatively charged moieties independently selected from sulfate and phosphate, in an amount effective to maintain elevated levels of tPA activity in a subject to which the composition is administered.

2. The composition of claim 1 wherein said disaccharide or 3-12 unit oligosaccharide is obtainable as a size-fractionated heparin fragment mixture obtained by complete or partial degradation of heparin by nitrous acid.

3. The composition of claim 1 wherein the disaccharide or disaccharide subunit is a conjugate of a reduced or nonreduced uronic acid selected from L- iduronic acid and D-glucuronic acid with a 2,5-D-anhydro- mannose or glucosamine, and/or wherein said moieties are sulfate, and/or wherein the disaccharide or disaccharide subunit is conjugated either α-1,4, β-1,4, or β-1,3, and/or wherein the disaccharide has a structure selected from the group consisting of

IdoA-AMan ; GlcA-AMan ; IdoA-GlcNH- 2S 6S 2S 6S 2S 2,6-dlS and GlcA-AMan

3S,6S

4. The pharmaceutical composition of claims 1-3 which further contains tissue plasminogen activator (tPA) or a fibrinolytic fragment thereof.

5. Use of the composition of claims 1-4 for the manufacture of a medicament for the conduct of a method to enhance the dissolution of blood clots in a subject.

6. An oligosaccharide/tPA complex which comprises tPA or a fibrinolytically active fragment thereof associated with a disaccharide or a 3-12 unit oligosaccharide which contains a disaccharide subunit, which disaccharide or disaccharide subunit contains at least two negatively charged moieties selected independently from sulfate and phosphate.

7. The complex of claim 6 wherein said disaccharide or subunit is obtainable as a size- fractionated heparin fragment mixture obtained from complete or partial degradation of heparin by nitrous acid.

8. The complex of claim 6 wherein the disaccharide or disaccharide subunit is a conjugate of a reduced or nonreduced uronic acid selected from L- iduronic acid and D-glucuronic acid with a 2,5-D-anhydro- mannose or glucosamine, and/or wherein said moieties are sulfate, and/or wherein the disaccharide or disaccharide subunit is conjugated either α-1,4, β-1,4, or β-1,3, and/or wherein the disaccharide has a structure selected from the group consisting of IdoA-AMan ; GlcA-AMan ; IdoA-GlcNH₂ ; 2S 6S 2S 6S 2S 2,6-dιS and GlcA-AMan

3S,6S

9. An antibody composition specifically immunoreactive with the disaccharide or oligosaccharide of claims 1-3.

10

10. A method to detect the presence, absence or amount of the size-fractionated disaccharide or a 3-12 unit oligosaccharide which contains a disaccharide subunit, which disaccharide or subunit contains at least

₁₅ two negatively charged moieties independently selected from sulfate and phosphate which enhances activity in converting plasminogen to plasmin, which method comprises contacting a sample suspected of containing said disaccharide or oligosaccharide with the antibodies of

₂₀ claim 9 under conditions wherein said antibodies will form a complex with said disaccharide or oligosaccharide; and detecting the presence, absence or amount of said complex.

25

11. A conjugate comprising the saccharide or oligosaccharide of claims 1-3 coupled to an affinity support.

_₀

12. A method to purify antibodies immunoreactive with the disaccharide or a 3-12 unit oligosaccharide which contains a disaccharide subunit, which disaccharide or subunit contains at least two negatively charged moieties independently selected from

_₅ sulfate and phosphate which enhances activity in converting plasminogen to plasmin, which method comprises contacting a sample containing said antibodies with the conjugate of claim 11 under conditions wherein said antibodies are adsorbed; and eluting the adsorbed antibodies from the conjugate.

13. A conjugate which comprises the disaccharide or oligosaccharide of claims 1-3 coupled with an immunogenicity-conferring carrier.

14. A method to prepare antibodies immunoreactive with the disaccharide or a 3-12 unit oligosaccharide which contains a disaccharide subunit, which disaccharide or subunit contains at least two negatively charged moieties independently selected from sulfate and phosphate which enhances activity in converting plasminogen to plasmin, which method comprises administering to a mammalian subject an amount of the carrier of claim 13 effective to raise antibody titers in said mammal; and recovering said antibodies.

15. A conjugate comprising the disaccharide or oligosaccharide of claims 1-3 coupled with a label.

16. A method to localize blood clots in a mammalian subject, which method comprising administering to said subject the conjugate of claim 15, allowing time for the conjugate to localize at said blood clots, and detecting the presence of the label.