AU721179B2 - Sulfated oligosaccharides having anticoagulant/antithrombotic activity - Google Patents

Description

WO 98/40081 PCT/AU98/00151 -1- SULFATED OLIGOSACCHARIDES HAVING ANTICOAGULANT/ANTITHROMBOTIC ACTIVITY FIELD OF THE INVENTION This invention relates to sulfated oligosaccharides, and in particular to the use of certain sulfated oligosaccharides as anticoagulant and/or antithrombotic agents.

BACKGROUND TO THE INVENTION Heparin, a sulfated glycosaminoglycan produced by mammalian mast cells, has been used extensively during the last 50 years as an anticoagulant for the prevention of post-operative thrombosis and the treatment of acute venous thrombosis. Heparin predominantly exerts its anticoagulant activity by accelerating the ability of the protease inhibitor antithrombin III to inactivate a number of key proteases in the coagulation cascade most notably factor Xa and thrombin, thrombin being the terminal enzyme in the cascade which converts soluble fibrinogen to insoluble fibrin. In fact, a specific pentasaccharide has been identified in heparin which binds with high affinity to antithrombin III and produces a conformational change in the inhibitor This pentasaccharide is sufficient to enhance inactivation of proteases, such as factor Xa, by antithrombin III. In contrast, heparin needs to crosslink antithrombin III with thrombin for enhanced thrombin inactivation to occur Despite its widespread clinical use as an anticoagulant, however, heparin suffers from a number of major disadvantages. First, it is structurally extremely diverse, varying in its molecular weight, monosaccharide sequence and sulfation pattern As a result of this diversity, less than 50% of heparin molecules in most preparations actually contain the antithrombin III binding pentasaccharide Thus, the quality control of different heparin batches is difficult. Second, heparin is an animal byproduct and consequently suffers from the risk of contamination with animal pathogens, a major concern for present day regulatory WO 98/40081 PCT/AU98/00151 -2agencies. Third, heparin can only be administered intravenously as, due to its high molecular weight, it exhibits poor bioavailability when injected subcutaneously Also, the high molecular weight of heparin (10-15kDa) precludes effective oral delivery of the drug. Fourth, in terms of clinical efficacy, heparin exhibits an extremely steep dose response curve. Thus, the coagulation time of patient's plasma must be continually monitored to ensure that drug overdose does not occur. Administration of appropriate heparin doses is further confounded by significant patient variability in heparin responsiveness. These difficulties lead to unacceptable bleeding being a complication of heparin therapy, particularly when the drug is being used as a long term treatment Finally, a significant number of patients develop heparin-induced thrombocytopenia (HIT) following prolonged heparin exposure a condition which precludes future heparin use in these individuals.

In an attempt to overcome some of the problems associated with heparin, a number of low molecular weight (LMW) (mol. wt. 4000-6500) heparin preparations have been developed and licenced These preparations have retained the anti-factor Xa and anti-thrombotic activities of native heparin but are less potent anticoagulants. As a result, the LMW heparins are less likely to induce bleeding complications in patients. Furthermore, due to their smaller size, they have improved bioavailability and can be administered subcutaneously.

An alternative approach has been to use the related glycosaminoglycan, dermatan sulfate, as an antithrombotic agent Interestingly dermatan sulfate, unlike heparin, catalyses thrombin inhibition by a second natural inhibitor of thrombin, heparin cofactor II However, despite the availability of LMW heparins and dermatan sulfate, there is still a considerable need for structurally well-defined anticoagulants and/or antithrombotics which are not animal derived, give reproducible patient responses, can be orally administered and are not prone to inducing thrombocytopenia.

Prior International Patent Application No. PCT/AU96/00238 discloses the preparation of a class of sulfated oligosaccharides based on polymers of monosaccharide units linked by 1-2, 1-3, 1-4 and/or 1-6 glycosidic bonds and consisting of from 3 to 8 WO 98/40081 PCT/AU98/00151 -3monosaccharide units, and the use of these sulfated oligosaccharides as potent inhibitors of human angiogenesis, tumour metastasis and inflammation.

In work leading to the present invention, it has been shown that these sulfated oligosaccharides may be used as anticoagulant/antithrombotic agents to overcome the many problems associated with glycosaminoglycan-derived anticoagulants/antithrombotics. In particular, it has been shown that the sulfated penta- or hexa-saccharides possess a well defined structure, a broad therapeutic window, a highly reproducible patient response, a probably reduced chance of inducing HIT-like syndromes and the potential for oral delivery.

Furthermore, these sulfated oligosaccharides do not act via antithrombin III but appear to inhibit coagulation by activating heparin cofactor II. Thus, the active sulfated oligosaccharides to some extent resemble the sulfated polysaccharide dermatan sulfate, rather than heparin, in their mode of action. Such oligosaccharides may be used for both prophylaxis and treatment of many thrombotic and cardiovascular diseases, the most notable of these being deep venous thrombosis, pulmonary embolism, thrombotic stroke, peripheral arterial thrombosis, unstable angina and myocardial infarction. Furthermore, effective oral delivery of the sulfated oligosaccharides makes these agents an alternative to warfarin, a widely used oral anticoagulant with severe side effects.

SUMMARY OF THE INVENTION In accordance with one aspect, the present invention provides a method for the anticoagulant and/or antithrombotic treatment of a human or other warm blooded animal patient in need of such treatment, which comprises administration to the patient of an effective amount of at least one sulfated oligosaccharide, wherein the oligosaccharide has the general formula I: Ri-(Rx)n-R 2 P:\OPER\JMS\P05562.PCT 28/4/00 -4wherein R 1 and R 2 and each Rx represents a hexose monosaccharide unit, all of which may be the same or different, adjacent monosaccharide units being linked by 1-2, 1-3, 1-4 and/or 1-6 glycosidic bonds; and n is an integer of from 1 to 6, preferably 3 or 4.

Preferably, the sulfated oligosaccharides have the general formula II: R-(R)n-R

(II)

wherein each R groupis the same and each represents a hexose monosaccharide unit, adjacent monosaccharide units being linked by 1-2, 1-3, 1-4 and/or 1-6 glycosidic bonds, and n is an integer of from 1 to 5, preferably 3 or 4.

As used herein, the term "anticoagulant and/or antithrombotic treatment" is intended to encompass both prophylactic and therapeutic treatment of a patient in need of such treatment.

DETAILED DESCRIPTION OF THE INVENTION The sulfated oligosaccharides which are used in accordance with this invention are based on polymers of monosaccharide units, which may be linked by 1-2, 1-3, 1-4 and/or 1-6 glycosidic bonds and which may consist of from 3 to 8 monosaccharide units. Preferably, the ;oligosaccharides consist of from 3 to 6 monosaccharide units (that is n is from 1 to more preferably from 5 to 6 monosaccharide units (n is from 3 to The polymers may comprise homopolymers containing only one type of monosaccharide unit, or heteropolymers containing two or more different types of monosaccharide units, although homopolymers are preferred..

The monosaccharide units which are linked together to form the oligosaccharides are referably hexoses, and may be either furanoses such as fructose or pyranoses such as glucose, S manV e, altrose, allose, talose, galactose, idose, or gulose. Particularly preferred hexoses are 7. glue s and mannose. The hexoses may be in either the D- or the L-configuration.

i^O j WO 98/40081 PCT/AU98/00151 Each monosaccharide unit may be a hexose, hexuronic, hexosamine or Nacetylhexosamine.

The oligosaccharides of general formulae I and II also include compounds wherein the monosaccharide units are derivatised, in particular where the units are phosphate, acetyl or other ester derivatives of monosaccharides.

In general, the sulfated oligosaccharides of this invention may be prepared by sulfation of oligosaccharides by methods known per se in the art to give their corresponding O-sulfated derivatives. Suitable sulfation methods are described in International Patent Application No.

PCT/AU96/00238, the contents of which are incorporated by reference. The oligosaccharides to be sulfated may be naturally occurring products including oligosaccharides prepared by enzymatic or chemical degradation of naturally occurring polysaccharides (such as amylose, dextran, cellulose, laminarin, pectin, chitin, chitosan, mannan, and a phosphomannan exopolysaccharide from the yeast Pichia holstii). Alternatively, the oligosaccharides may be prepared synthetically by the process disclosed in International Patent Application No.

PCT/AU96/00238.

As previously described, sulfated oligosaccharides falling within the scope of this invention have been shown to exhibit anticoagulant and/or antithrombotic activity, and accordingly in yet another aspect the present invention extends to the use of a sulfated oligosaccharide as described above as an anti-anticoagulant and/or antithrombotic agent in the treatment of a warm-blooded animal (including a human) patient.

The invention also extends to the use in the manufacture of a medicament for the anticoagulant and/or antithrombotic treatment of a human or other warm-blooded animal patient of at least one sulfated oligosaccharide as described above.

Furthermore, this invention also provides a pharmaceutical or veterinary composition for anticoagulant and/or antithrombotic treatment, which comprises at least one sulfated WO 98/40081 PCT/AU98/00151 -6oligosaccharide as described above, together with a pharmaceutically and veterinarily acceptable carrier or diluent therefor.

The active component is administered in therapeutically effective amounts. A therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.

The formulation of therapeutic compositions is well known to persons skilled in this field. Suitable pharmaceutically or veterinarily acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically and veterinarily active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the' active ingredient, use thereof in the pharmaceutical and veterinary compositions of the present invention is contemplated.

Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human or animal subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired WO 98/40081 PCT/AU98/00151 -7therapeutic effect in association with the required pharmaceutical or veterinary carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active ingredient for the particular treatment.

A variety of administration routes are available, although oral delivery is preferred because of the convenience to the patient. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects.

Such modes of administration include oral, rectal, topical, nasal, transdermal or parenteral (e.g.

subcutaneous, intramuscular and intravenous) routes. Formulations for oral administration include discrete units such as capsules, tablets, lozenges and the like. Other routes include intrathecal administration directly into spinal fluid, direct introduction such as by various catheter and balloon angioplasty devices well known to those of ordinary skill in the art, and intraparenchymal injection into targeted areas.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in polyethylene glycol and lactic acid.

WO 98/40081 PCT/AU98/00151 Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Other delivery systems can include sustained release delivery systems. Preferred sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules. Many types of sustained release delivery systems are available. These include, but are not limited to: erosional systems in which the active component is contained within a matrix, and diffusional systems in which the active component permeates at a controlled rate through a polymer. In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.

As previously described, in accordance with this invention the sulfated oligosaccharides may be used in treatment of many thrombotic and cardiovascular diseases, the most notable of these being deep venous thrombosis, pulmonary embolism, thrombotic stroke, peripheral arterial thrombosis, unstable angina and myocardial infarction.

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

Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

WO 98/40081 PCT/AU98/00151 -9- In the accompanying drawings: Figure 1 shows an analysis of the anticoagulant activity of a series of maltose based sulfated oligosaccharides of different chain length as measured by the activated partial thromboplastin time (APTT) test. Heparin (anticoagulant activity 120 U/mg) is included for comparison. Data obtained with pooled plasma from 90 donors, the coagulation time of the plasma being 30.8 seconds in the absence of inhibitors. Results representative of four separate experiments.

Figure 2 shows a comparison of the anticoagulant activity of different sulfated pentasaccharides as measured by the APTT test, with heparin being included for comparison.

Other details as in legend to Figure 1.

Figure 3 shows a comparison of the anti-thrombin III mediated anti-factor Xa activity of heparin, LMW heparin (Fragmin) and the sulfated oligosaccharide mannopentaose phosphate sulfate (PI-88). Factor Xa activity was determined with the chromogenic (Helena) anti-Xa assay run on a Roche COBAS Fara biochemistry analyser.

EXAMPLE 1 Preparation of Sulfated Oligosaccharides In the present Examples, two sources of oligosaccharides were used for subsequent sulfation. The first source represents oligosaccharides derived from naturally occurring polysaccharides. The usual preparation procedure involves partial depolymerisation of the polysaccharides by enzymatic or chemical means and size fractionation of the resultant oligosaccharides. Examples of polysaccharides from which oligosaccharides were generated are amylose, dextran, cellulose, laminaran, pectin, chitin, chitosan, mannan and a phosphomannan exopolysaccharide from the yeast Pichia holstii. The second source represents totally synthetic WO 98/40081 PCT/AU98/00151 hexose-containing oligosaccharides prepared by chemical polymerisation ofhexose monomers.

Prior International Patent Application No. PCT/AU96/00238 discloses a method for the manufacture of these totally synthetic oligosaccharides. The same patent application describes a procedure for the isolation of a mannopentaose phosphate of the structure P-6-Man-a-(1-3)- Man-a-(1-3)-Man-a-(1-3)-Man-a-(l-2)Man from the exopolysaccharide of the yeast Pichia holstii. The other naturally occurring oligosaccharides were purchased from Seikagaku, Tokyo, Japan. Finally, all of the oligosaccharides were sulfated by a procedure disclosed in Prior International Patent Application No. PCT/AU96/00238.

EXAMPLE 2 Anticoagulant Activity of the Sulfated Oligosaccharides Table 1 summarises the anticoagulant activity of 19 sulfated oligosaccharides, as measured by the activated partial thromboplastin time (APTT) test. Similar results were obtained with the thrombin time test. At the outset it is clear that 11 of the sulfated oligosaccharides tested exhibit considerable anticoagulant activity when compared with heparin and Fragmin (LMW heparin). Chain length, monosaccharide makeup and linkage, all appear to be critical factors. The influence of chain length is particularly evident with the maltose series where the di-, tri- and tetrasaccharides exhibit low activity whereas the maltopentaose, maltohexaose and maltoheptaose sulfates are quite effective anticoagulants (Table 1, Fig. Furthermore, a striking feature of the maltose series is that chain elongation by a single residue from the maltotetraose to maltopentaose results in a 19-fold increase in anticoagulant activity. The importance of monosaccharide composition is highlighted by chitosan hexamer sulfate, which is composed of pl-4 linked glucosamine, and exhibits negligible anticoagulant activity compared with p1-4 linked glucohexaose (cellohexaose, Table The glucose series of oligosaccharides emphasizes the importance of monosaccharide linkage on activity. Thus, the sulfated laminarin series (31-3 linked glucose) exhibits much lower anticoagulant activity than the sulfated a 1-4, al-6 and 31-4 linked WO 98/40081 PCT/AU98/00151 11 glucose containing oligosaccharides (Table There are additional, more subtle, effects of monosaccharide linkage on anticoagulant activity. Generally the penta- and hexasaccharide sulfates of either glucose or mannose linked al-2, al-3, al-4 and al-6 possess comparable anticoagulant activity (Table The one exception is isomaltopentaose sulfate, which is a substantially less effective anticoagulant than the maltopentaose and mannopentaose sulfates (Table 1, Fig. This suggests that anticoagulant activity is expressed by shorter chain al-4 than l 1-6 glucose polymers. Similarly, when glucose is 31-4 linked even shorter chain sulfated oligosaccharides are active, in this case the tetrasaccharide cellotetraose, being a reasonably potent anticoagulant (Table Finally, two mannopentaose phosphate sulfates (PI-88's) with slightly different degrees of sulfation, were essentially equally active suggesting that providing sulfation of the oligosaccharides is 50% anticoagulant activity is assured.

Based on the data presented in Table 1 and Figures 1 and 2 it is clear that many of the active sulfated oligosaccharides display comparable anticoagulant activities, the slopes of the dose response curves of the seven most active being from 4.5 to 5.9-fold less steep than that of heparin. Thus, these oligosaccharide-based anticoagulants possess a much wider therapeutic window than heparin, a desirable feature if one wishes to tightly control the anticoagulation state of a patient.

Another feature of the sulfated oligosaccharide-based anticoagulants is that their dose response curves are extremely linear, with the correlation coefficients of the linear regression lines being 0.99 in most cases (Table Such an extreme linearity in responsiveness is not seen with heparin. This reproducibility in responsiveness was confirmed when the anticoagulant activity of mannopentaose phosphate sulfate (PI-88) was tested on plasma from different donors. When plasma was anticoagulated to a similar extent by heparin and PI- 88, overall the PI-88 gave a more reproducible prolongation of coagulation time, ie, 88.3 11.9 sec compared with 77.5 20.8 sec for heparin (Table In fact, one donor failed to respond to heparin but responded, as expected, to PI-88 (Table 2).

WO 98/40081 PCT/AU98/00151 -12- The manner by which the sulfated oligosaccharides inhibit the coagulation cascade was then investigated. Initial studies showed that PI-88 did not enhance the ability of antithrombin II to inhibit thrombin. In fact, two different chromogenic assays revealed that PI- 88 is 700-7000 fold less active than heparin in enhancing anti-thrombin III mediated antithrombin activity (data not shown). When examined for anti-thrombin I mediated factor Xa inhibitory activity it was found that PI-88 was at least 100-1000-fold less active than heparin or LMW heparin (Fragmin) (Fig. These results imply that PI-88, and probably the other active sulfated oligosaccharides, do not exert their anticoagulant activity via antithrombin III.

However, subsequent studies showed that the sulfated oligosaccharides with anticoagulant activity are probably acting via heparin cofactor II, thus resembling dermatan sulfate in their anticoagulant properties. In a plasma free system in which purified heparin cofactor II was added to thrombin and a chromogenic thrombin substrate, it was found that PI-88 enhanced the ability of heparin cofactor II to inhibit thrombin as effectively as heparin and dermatan sulfate (data not shown).

Collectively, these data indicate that sulfated oligosaccharide, like PI-88, resemble dermatan sulfate and therefore may have considerable potential as anti-thrombotic agents.

Some additional biological properties of one of the sulfated oligosaccharides, PI-88, were defined. It was found to be a very weak inhibitor of collagen- and ADP-induced platelet aggregation (Table further highlighting the specificity of action of sulfated oligosaccharides on the coagulation system. Protamine is frequently used clinically to reverse the anticoagulant effects of heparin and Fragmin. It was found that protamine also can effectively inactivate the anticoagulant activity of PI-88 (Table indicating that protamine could be used as an effective in vivo antidote for sulfated oligosaccharides with anticoagulant properties. Finally, PI-88 was examined for its ability to act as an anticoagulant in a range of mammalian species. The compound was found to be an effective anticoagulant of human, marmoset, sheep, dog, rat and mouse plasma but was only weakly active in rabbit plasma (Table Thus, sulfated oligosaccharides potentially can be used as anticoagulants/antithrombotics not only in humans but in a wide range of mammalian species.

U,'

o TABLE 1 Comparison of the Anticoagulant. Activity of Different Sulfated Oligosaccharides as Measured by the Activated Partial Thromboplastin Time (APTT) Test Compound Monosaccharide Linkage Degree of Increase in Correlation Constituents Sulfationa Coagulation Coefficientc Time (sec)/j.Lgb Heparin Gic, IdoA, GlcNAc 131-4, al1- ND 29.69 0.977 LMW Heparin (Fragniin) Gic, IdoA, GlcNAc 131-4, al-4 ND 11.20 0.997 Maltose S04 Glc: al-4 6/8 0.31 0.995 Maltotriose S04 Glc al-4 9/11 0.25 0.892 Maltotetraose 504 Glc al-4 11/14 0.28 0.992 Maltopentaose S04 Gic al-4 9/17 5.25 0.996 Maltohexaose S04 GIc cxlA 16.6/20 5.85 0.999 Maltoheptaose S04 Gic a1-4 ND 5.9 Isomaltopentaose S04 Glc al-6 14/17 1.85 0.999 Isomaltohexaose S04 Glc al-6 12.8/20 6.56 0.999 Cellotetraose 504 Glc 131-4 ND 4.2 Cellopentaose 504 Glc; 01-4 ND Cellohexaose 504 Glc 131-4 ND 4.8 Laminaritetraose 504 Glc f31-3 ND 2.9 Laminarihexaose 504 Glc: 11-3 ND 0.9 Laniinariheptaose 504 Gic 131-3 ND 0.5 Chitosan hexamer S0 4 GlcN 131-4 ND 0.40.8 Mannopentaose S 0 4 d Man ccl-3, al-6 11/17 5.32 0.985 Mannopentaose phosphate S 0 4

(PI.

88 )e Man a1l-3, al-2 12.5/16 5.67095 Mannopentaose phosphate SQ 4

(PI-

88 )e Man al-3, al-2 14/16 5.04 0.995 Sucrose S04 Gic, Frc al-2 8/8 (100%) 0.39 0.994

C

Mtfintkes (Table 1) a Actual number of sulfate groups attached/theoretical maximum number of sulfate groups which can be coupled to each molecule.

Values in brackets represents sulfation. In cases where degree of sulfation not determined (ND) oligosaccharides sulfated under conditions which would result in maximum sulfation.

b Data expressed as increase in coagulation time (seconds) resulting from a 1g/ml increase in concentration of the test compound.

Values obtained from a linear regression analysis of the coagulation data.

c Correlation coefficient obtained following linear regression analysis of the coagulation data. When no value given insufficient data points for a correlation coefficient to be calculated.

d Totally synthetic mannopentaose.

e Mannopentaose phosphate isolated from the yeast Pichia holstii which contains mannose-6-phosphate at the reducing terminus.

ND not determined m:

C

o r /Au 9 8 U U 1 5 1 RECEIVED 1 4 MAY 199 P:\OPERUMS\PO5562.PCT 1415/98 TABLE 2 Assessment of Donor Variability in the Anticoagulant Activity of Heparin and Mannopentaose Phosphate S04 (PI-88) Normal Donor Coagulation Time (sec)a Control Heparin Mannopentaose 1Ag/ml) phosphate S04 *PU-88) (8.6tg/ml) 1 30.5 72.8 77.3 2 34.8 93.7 89.6 3 31.6 95.4 94.0 4 31.3 78.1 85.6 33.4 110.5 101.9 6 31.3 78.2 95.0 7 38.9 110.6 115.3 8 34.9 101.6 101.9 9 31.5 70.1 85.2 30.5 72.7 78.0 11 34.9 89.5 93.6 12 33.1 73.8 82.9 13 35.8 40.1 85.5 14 31.1 85.6 89.7 34.6 69.7 81.2 16 27.9 64.1 72.6 17 34.8 101.9 108.0 18 27.3 48.7 70.4 19 35.4 73.4 93.6 30.6 57.0 72.6 Mean SD 32.8 2.8 77.5 20.8 88.3 11.9 a Coagulation times estimated using the APTT test. Similar results were obtained in duplicate assays. Concentrations of heparin and mannopentaose phosphate SO 4

(PI-

88) were used which prolonged the coagulation time by approximately 2.5 fold.

Donor 13 highlighted in bold italics as this donor failed to respond to heparin but responded to mannopentaose phosphate SO 4 SUBSTITUTE SHEET (Rule 26) V~U' I I RECEIVItZ... 1 4 MAY 1998 P:\OPERMSO562. 145/98 -16- TABLE 3 Inhibition of Platelet Aggregation by Mannopentaose Phosphate S04 (P1-88) Relative Rate of Platelet Aggregation' PI-88 Concentration Collagen-induced ADP-induced Aggregation Aggregation 0 114±8 81±1 21 97±8 74±1 210 86±10 72±1 2100 79±0 56±2 a Human platelet aggregation induced by either collagen (9j tglml) or ADP (1014M) and relative rates of platelet aggregation estimated, in the presence or absence of P1-88, using a platelet aggregometer. Data SD representative of two platelet donors.

TABLE 4 Reversal of Anticoagulant Activity of Hleparin, Fragmin and P1-88 by Protamine Sulfate Anticoagulant Protamnine Sulfate Coagulation Concentration Time (seconds)' Nil 31.6 Nil 10 /g/n1 34.4 Heparin (2.1 jig/m1) -91.9 Heparin (2.1 A~glml) 10 Ag/ml 33.4 Fragmin (6.25 /Ag/ml) -109.0 Fragmin (6.25 ftg/ni1) 10 Jg/ml 38.0 PI-88 (8.6 j.g/m1) -79.7 P1-88 (8.6 Ag/ml) 10 jug/mn1 43.6 a Coagulation times estimated by the APTT test.

1-88 sulfated mannopentaose phosphate from Pichia holstii.

SUBSTITUTE SHEET (Rule 26) REC'EI/V U4 UAY 199 1 RECEIVD 1 4 MAY 1991 P:%OPER\JMS\P05562.PC 145/98 -17- TABLE 5 Anticoagulant Activity of Heparin, Fragmin and PI-88 with Plasma from Different Mammalian Species.

Relative Increase in Coagulation Time 2 Species Heparin Fragmin PI-88 PI-88 (0.83 14g/ml) (6.25 ig/ml) (2.24 jig/ml) (22.4 tig/ml) Human 1.31 3.02 1.42 4.65 Marmoset 1.26 2.71 1.40 2.86 Sheep 1.10 1.42 1.88 >4.98 Rabbit 1.25 1.85 1.02 1.20 Dog 1.01 1.32 1.24 2.42 Rat 1.02 1.90 1.54 >5.88 Mouse ND ND 1.13 3.03 a Coagulation times estimated by the APTT test. Data expressed as fold increase in coagulation time induced by the anticoagulant compared with untreated control.

PI-88 sulfated mannopentaose phosphate from Pichia holstii.

ND not determined.

SUBSTITUTE SHEET (Rule 26) FLT/AU 9 8 R EC E IVEXD /U U1 51 1 MAY 199E *P:\OPER\UMS\P05562.PCT 141/98 18 References 1. Bjork, 1. and Lindalil, U. (1982) Mol. Cell Biochem. 48, 161-182.

2. Bj6rk, Olson, S.T. and Shane, J.D. (1989). In "Heparin: Chemical and Biological Properties, Clinical Applications" (Lane, D.L. and Lindalil, eds) pp 229-255, Edward Arnold Ltd., London.

3. Olsen, S.T. and Bjork, 1. (1994). Regulation of thrombin activity by antithrombin and heparin. Sem. 7Tromb. Hem. 20, 373-409.

4. Lindahi, U. (1989). In "Heparin" (Lane, D.A. and Lindalil, eds) pp. 159- 189, CRC Press.

Hirsch, J. and Levine, M.N. (1992) Low-molecular-weight heparin. Blood 79, 1-17.

6. Beijering, ten Cate, H. and ten Cate, J.W. (1996). Ann. Hematol. 72, 177-183.

7. Agnelli, Cosmi, Di Filippo, P. et al. (1992). Thromb. Haemost. 67, 203-208.

8. Bourin, and Lindalhl, U. (1993). Biochem. J. 289, 313-330.

SUBSTITUTE SHEET (Rule 26)

Claims (12)

1. (Amended) A method for the anticoagulant and/or antithrombotic treatment of a human or other warm-blooded animal patient in need of such treatment, which comprises administration to the patient of an effective amount of at least one sulfated oligosaccharide, wherein the oligosaccharide has the general formula I: RI-(Rx)n-R 2 (I) wherein R 1 and R 2 and each R x represents a hexose monosaccharide unit, all of which may be the same or different, adjacent monosaccharide units being linked by 1-2, 1-3, 1-4 and/or 1-6 glycosidic bonds and n is an integer of from 1 to 6.

2. A method according to claim 1 wherein in the oligosaccharide of general formula I n is 3 or 4.

3. (Amended) A method according to claim 1 or claim 2, wherein in the oligosaccharide of general formula I, the monosaccharide units are selected from the group consisting of fructose, glucose, mannose, altrose, allose, talose, galactose, idose and gulose.

4. (Amended) A method according to claim 1, wherein the oligosaccharide has the general formula II: R-(R)n-R (II) AMENDED SHEET IPEA/AU P:\OPERUMS\PCTAU981.SPE- 12/1/99 PCT/AU98/00151 Received 13 January 1999 wherein each R group is the same and each represents a hexose monosaccharide unit, adjacent monosaccharide units being linked by 1-2, 1-3, 1-4 and/or 1-6 glycosidic bonds; and n is an integer of from 1 to A method according to claim 4, wherein in the oligosaccharide of general formula II, n is from 3 or 4.

6. (Amended A method according to claim 4 or claim 5, wherein in the oligosaccharide of general formula II, R represents a monosaccharide unit which is selected from the group consisting of glucose, mannose, altrose, allose, talose, galactose, idose and gulose.

7. A method according to claim 6, wherein in the oligosaccharide of general formula II, R represents glucose or mannose.

8. A method according to claim 1, wherein the oligosaccharide is prepared by enzymatic or chemical degradation of a naturally occurring polysaccharide.

9. (Amended) A method according to claim 8, wherein the oligosaccharide is an amylose-, dextran-, cellulose-, laminarin-, or mannan-oligosaccharide. A method according to claim 9, wherein the oligosaccharide is selected from maltopentaose, malothexaose, maltoheptaose, isomaltopentaose, isomaltohexaose, cellotetraose, cellopentaose, cellohexaose, laminaritetraose and mannopentaose.

11. A method according to claim 8, wherein the oligosaccharide is mannopentaose phosphate from the yeast Pichia holstii. AMENDED SHEET IPEA/AU P:\OPER\FMS\P05562.PCT 28/4/00 -21

12. A method according to any of claims 1 to 11, wherein the treatment comprises treatment of a thrombotic or cardiovascular disease.

13. A method according to claim 12, wherein the treatment comprises treatment of deep-vein thrombosis, pulmonary embolism, thrombotic stroke, peripheral arterial thrombosis, unstable angina or myocardial infarction.

14. Use of a sulfated oligosaccharide as defined in claim 1, as an anticoagulant and/or antithrombotic agent in the treatment of a warm-blooded animal (including a human) patient. Use in the manufacture of a medicament for the anticoagulant and/or antithrombotic treatment of a human or other warm-blooded animal patient of at least one sulfated oligosaccharide as defined in claim 1. Dated this 28 t day of April 2000. The Australian National University By its Patent Attorneys Davies Collison Cave 00o0f"f ftftt .t ft f