EP0691976A1

EP0691976A1 - Anticoagulant compounds

Info

Publication number: EP0691976A1
Application number: EP94909884A
Authority: EP
Inventors: Robert Kisilvesky; Walter A. Szarek; Alan R. Giles; Michael E. Nesheim
Original assignee: Queens University at Kingston
Current assignee: Queens University at Kingston
Priority date: 1993-03-29
Filing date: 1994-03-29
Publication date: 1996-01-17
Also published as: CA2159649A1; WO1994022885A1

Abstract

A series of sulfated disaccharides having blocked anomeric centers have been shown to exhibit blood anticoagulant properties specific to inhibition of factor IXa, and can be used in vivo to inhibit clotting, extracorporeally, or in vitro to coat surfaces which come into contact with blood to inhibit the formation of clots thereon.

Description

ANTICOAGULANT COMPOUNDS FIELD OF INVENTION

This invention relates to novel pharmaceutical compositions for use as blood anticoagulants. More particularly this invention relates to pharmaceutical compositions which inhibit Factor IXa in the blood coagulation cascade, and to compositions which may be used to render material surfaces anticoagulative, such as prosthetic implant surfaces and artificial devices and blood carrying devices such as tubing, prosthetic heart valves, and extracorporeal devices such as renal dialysis machines. BACKGROUND OF INVENTION

Heparin and polysaccharide derivatives thereof have long been used for selective anticoagulation activity both in vivo and in vitro. Heparin is a sulfate-containing polysaccharide which can be extracted from bovine and porcine lung and intestinal mucosa. Heparin is not, however, a pure compound but is a mixture of polysaccharides with a continuous distribution of molecular weights in the range 1,500 to 30,000 daltons. The activity thereof is somewhat variable, depending on the source and molecular weight. This can cause problems, such as risk of bleeding complications, because different patients react very differently to a given dosage. Heparin is not orally active and must be given parenterally. While several synthetic oligo- and polysaccharides based on the structure of heparin have been described in the literature there remains a need for a simple, relatively low molecular weight, synthetic saccharide, which may be orally active, for use as an anticoagulant pharmaceutical and as a coating for prostheses, tubing, glass and similar surfaces which come into contact with blood so as to reduce thrombotic problems. Vejay Nair, in U.S. patent 4,021,544, describes a series of sulfated maltoses and oligosaccharides of the maltose series having a formula:

1 where M is hydrogen or a salt of an alkali metal, alkaline earth metal, ammonium, tri(loweralkyl)amine (C₁-C₆), piperidine, pyrazine, alkanolamine (C_rC₆) and cycloalkanolamine ( - ); and n is 2-10, which are used to inhibit the complement system of warm blooded animals. "Complements" refer to a complex group of proteins in blood and other body fluids that, working together with antibodies or other factors, play an important role as mediators of immune, allergic, immunochemical and/or immunopathological reactions. Complement inhibitors can be used therapeutically for such non-immunologic diseases as paroxysmal nocturnal haemoglobinuria and hereditary angio-neurotic edema. While activation of the complement system may also accelerate blood clotting, there is no evidence that inhibition thereof would have any anticoagulant activity and, indeed, the opposite is probably the case. In this series of compounds, however, the anomeric hydroxyl group is not blocked but may be sulfated. It has now been found that sulfated and sulfonated oligosaccharides preferably having blocked anomeric centers, surprisingly exhibit anticoagulant properties specific to inhibition of Factor IXa. OBJECT OF INVENTION

It is, therefore, one object of the present invention to provide novel compounds having anticoagulant activity both in vivo and in vitro.

Another object of this invention is to provide a composition and method for producing antithrombotic surfaces for implanted prostheses, extracorporeal devices, laboratory equipment and the like.

2 BRIEF STATEMENT OF INVENTION

By one aspect of this invention there is provided a disaccharide containing at least one sulfur based anion, which exhibits antithrombotic properties specific to inhibition of Factor IXa.

By another aspect of this invention there is provided an anticoagulant preparation comprising a pharmaceutically effective amount of a disaccharide containing at least one sulfur based anion, which exhibits antithrombotic properties specific to inhibition of Factor IXa, and a pharmaceutically acceptable carrier therefor. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a sketch illustrating the blood coagulation cascade;

Figure 2 is a sketch similar to Figure 1 but showing the blood coagulation cascade in a simplified form;

Figure 3 is a graph showing APTT prolongation at various concentrations of test compounds in plasma.

Figure 4 is a graph illustrating Factor IXa clotting time with increasing concentrations of selected inhibitors;

Figure 5 is a graph illustrating Factor X activation versus concentration of inhibitor #3;

Figure 6 is a graph illustrating Factor X intrinsic fluorescence versus concentration of inhibitor #3;

Figure 7 is a graph illustrating Factor IXa fluorescence versus concentration of inhibitor #3;

Figure 8 is a graph illustrating Factor X activation and intrinsic fluorescence versus concentration of inhibitor #3; and Figure 9 is a graph illustrating Factor X activation and Factor Ixa intrinsic fluorescence versus concentration of inhibitor #3. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Sulfated compounds of the present invention fall into two groups namely non- reducing disaccharides having a general formula:

where R¹ is H or (CH₂OSO^" ₃ M⁺)n, n is an integer from 1-6,

R₂ is OSO^" ₃ M⁺ and M is selected from NH₄, trialkylammonium, cyclic ammonium, alkali metals and alkaline earth metals, and R is

and reducing disaccharides having a blocked anomeric center of the type Illustrative, but not limiting, examples of compounds within these general formulae include:

(a) Sucrose octasulfate sodium salt:

0

OSO-₃Na ^" H

(b) Trehalose octasulfate sodium salt:

and (c) Methyl ø-D-lactoside heptasulfate sodium salt:

Example 1

Preparation of sucrose octasulfate ammonium salt.

2-Picoline was fractionally distilled to remove the water azeotrope and the collected "dry" picoline was dried over CaH₂ and re-distilled. The solvent was stored in a sealed flask until used. A dry 1-L three-necked flask, fitted with a sealed mechanical stirrer, a 50-mL vented dropping funnel, and a stopper, was charged with dry 2-picoline (92 mL). The flask was placed in an oil bath held at 45°C and brisk stirring was begun. The dropping funnel was charged with chlorosulfonic acid (16 mL, 240.7 mmol) and the top of the funnel was fitted with a calcium chloride drying tube. The chlorosulfonic acid was carefully dripped into the 2-picoline over 1.5 h. The bath temperature was raised to 60°C, and powdered sucrose (10.0 g, 29.2 mmol) was added, rinsing the sides of the joint and the flask clean with a small amount of dry 2-picoline from a dry pipette. Stirring was continued for 1-1.5 hours more, to afford a viscous purple solution.

The mixture was cooled to room temperature over -1 h, and then the flask was immersed in an ice-water bath. Stirring became difficult, but was continued as 1/2 concentrated aqueous ammonium hydroxide solution (70 mL) was added. At this point, a drop of the clear purple solution placed on moist Ph paper produced the green color of Ph ~8. Ethanol (95% v/v, 250 Ml) was added portionwise to the stirred solution, to produce a tan solid precipitate. The suspension was stirred vigorously for 30 min. The precipitate was collected by filtration, washed sequentially with 95% ethanol (2 x 50 Ml), acetone (50 Ml), and ether (50 Ml). The product was left in a vacuum desiccator overnight, to remove traces of 2-picoline. Crude recovery was 35.43 g.

The tan powder was dissolved in water (60 Ml). The solution was decolorized and filtered; the filtrate was added to a separatory funnel, and the Ph was adjusted to 8.5 with concentrated aqueous ammonium hydroxide. A 500-M1 Erlenmeyer flask was fitted with a mechanical stirrer, and ethanol (300 Ml) was added. The product solution was slowly dripped into the vigorously stirred ethanol over 2 h. A white precipitate was formed, which was collected by filtration, washed with absolute ethanol, and then with acetone. The product was dried in vacuo. yield 27.5 g (84%); mp 155-158°C (dec).

Example 2 Preparation of sucrose octasulfate sodium salt (Lazaridis method)

Sucrose octasulfate ammonium salt (10 g) prepared in Example 1 was dissolved in water (50 Ml), and the Ph was raised to 9.0 with 20% w/v aqueous sodium hydroxide. Ethanol (95%) was added to the solution with vigorous mechanical stirring, until an oily precipitate was formed. Scratching of the precipitate eventually caused solidification, and the solid was collected by filtration. This was dissolved in a minimum amount of water, the solution was filtered, and the Ph of the filtrate was adjusted to 9 with 20% sodium hydroxide. Alcohol was added, with vigorous stirring, to form a fine powder. When addition was complete, the suspension was stirred for several hours more. The product was collected by filtration, washed sequentially with absolute alcohol, acetone, and ether, and dried in vacuo overnight over sodium hydroxide. Yield of white powder, 8.96 g (87%); mp 159-166°C (dec); MW 1158. Example 3 Preparation of trehalose octasulfate sodium salt

Using the apparatus described in Example 1, chlorosulfonic acid (17.9 Ml, 269 mmol) was added dropwise to 2-picoline (90 Ml) at 45°C, over 1.25 h. The temperature was raised to 60°C, and powdered and dried , α-trehalose monohydrate (11.00 g, 29.1 mmol) was added. Stirring was continued for 1.5 h. The mixture was then cooled on an ice-water bath. Water (150 Ml) was added cautiously, followed by barium hydroxide octahydrate (48.14 g, 152.6 mmol). The mixture was stirred for 1 h. The suspension was poured into centrifuge flasks, and centrifuged at -2000 φm for 15 min. The supernatant

8 was decanted, the solids were re-suspended in a small amount of water, and centrifuged again. The supernatant was added to the first supernatant, and the deep red-puφle liquid was vigorously stirred mechanically while 95% ethanol (400 Ml) was slowly added dropwise. This procedure caused an oily material to separate; stirring overnight caused this oil to solidify. The solid was collected by filtration, and washed with 95% ethanol, and then with ether. The powdery material was allowed to stand in vacuo to remove traces of picoline. The crude barium salt weighed 34.15 g.

This barium salt was dissolved in water (100 Ml), and the solution was decolorized by the addition of Norit® charcoal (5 g). The solution was filtered through Celite^®, and the filtrate was diluted to a volume of 750 Ml. This solution was passed down a 2 cm x 90 cm column containing Amberlite^® IR-120(Na⁺). The column was flushed with water (500 Ml) and the combined eluate was concentrated to a volume of 300 mL on the rotary evaporator, maintaining a bath temperature of <40°C. This solution was freeze- dried to provide a fluffy white powder, 20.0 g (59%); MW 1164. Example 4 Preparation of methyl g-D-lactoside heptasulfate ammonium salt

Following a procedure similar to that used in Example 1, 2-picoline • S0₃ complex was prepared at 45°C from chlorosulfonic acid (15.4 mL, 232 mmol) and 2-picoline (990 mL). The temperature was raised to 60°C, and methyl 3-D lactoside (10.00 g, 28.06 mmol) was added. The mixture was stirred at 60°C for lh before being cooled. The solution was chilled in an ice-water bath, as half concentrated ammonium hydroxide solution (75 mL) was added. Ethanol (95%, 500 mL) was added with vigorous stirring. This procedure caused separation of an oily material, which was allowed to settle. The red supernatant was decanted, and more ethanol was added to the residue, which was ground up thoroughly. This procedure induced solidification, and the ethanolic suspension of the product was stirred vigorously overnight. The crude ammonium salt (35.1 g) was collected by filtration.

The salt was dissolved in water (60 Ml) and the solution was decolorized by addition of Norit® charcoal (1 g). The solution was filtered through Celite^®, and the filtrate was vigorously stirred as 95% ethanol (-200 Ml) was added. This procedure caused separation of an oily white material. The supernatant was decanted, and the oily material was triturated with ethanol and ground into a fine powder. The powder was dried in vacuo over sodium hydroxide pellets for 2 days, yield 28.11 g (97%). Example 5 Preparation of methyl g-D-lactoside heptasulfate sodium salt

Methyl 0-D-lactoside heptasulfate ammonium salt (12 g, 11.6 mmol), as prepared by Example 4, was dissolved in water (60 Ml), and the solution was filtered. The Ph of the filtrate was adjusted to 8 with 20% w/v sodium hydroxide solution. While the aqueous solution was being vigorously stirred, 95% ethanol (150 Ml) was added. This procedure caused separation of an oily material. The supernatant was decanted, the oily residue was dissolved in water (100 Ml) and the solution was freeze-dried. The resulting powder was dissolved again in water (100 Ml) and the solution was freeze-dried. The powdery product was dried 24 h in vacuo at 60°C. Yield 10.41 g (84%); mp 167-174°C (dec).

10

TE SHEET Example 6

Screening tests for anticoagulation effects

(a) In Vitro Testing

The compositions produced according to Example 5, Example 3 and Example 2, and sucrose (MW360) as a control, were designated compounds 1, 2, 3 and 4, respectively for blind screening tests of their anticoagulation properties. Each compound was added to human plasma at increasing concentrations and the Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Clotting Time (TCT) were measured using standard protocols (Figure 3). Compound 4 showed no anticoagulant activity but in the case of compounds 1, 2 and 3 the TCT and PT remained the same as the control, while the APTT showed a dose related response by prolongation of APTT.

(b) In Vivo Studies

In vivo recovery and survival of anticoagulant activity This study was designed to measure the prolongation of the APTT (activated partial thromboplastin time) as well as the platelet and white blood cell (WBC) counts in an animal model. A 4.5 kg normal male New Zealand white rabbit was selected for this puφose. 50 mg, in 5 ml PBS, of sucrose octasulfate sodium salt (compound 3) was infused into the right ear vein of an unaesthetized male NZW rabbit over a 40 second period. In a second study, a male NZW rabbit was anesthetized and a stomach tube placed so that the test solution, sucrose octasulfate at 200 mg/kg body weight, could be administered in a 200 mg/ml water. The infusions were well tolerated. Blood samples were drawn from the left ear vein via a 21 gauge butterfly needle immediately pre- infusion and then at 1, 5, 10, 20, 46, 70, 123, 182 and 240 minutes post infusion (injected

11 samples) and at 10 minute intervals over 2 hours in the case of the orally administered dose. The blood samples were collected into 1/lOth volume of buffered citrate and also into pediatric EDTA tubes for cell counts. The citrated samples were centrifuged to obtain platelet-poor plasma and frozen. Complete blood counts were performed on a

Baker System 9000 automated haemotology instrument. Cell counts were expressed as percent of the pre-infusion value. APTT tests were performed manually in glass tubes as follows: 100 μl plasma was put into a tube 37°C, 100 μl APTT reagent (Organon

Teknika) was added and incubated for 3 minutes at 37°C. 100 μl 25 mM CaCl₂ was then added and the time to clotting was then measured with a stopwatch. A standard line was constructed by adding known amounts of test material to citrated normal rabbit plasma. The prolongation of the APTT (in seconds) was plotted against the concentration of anticoagulant and the prolongation of each of the test samples was read as apparent activity from this line.

The predicted 100% recovery for the amount infused (4.3 x 10^"5 moles infused)

40 ml/kg x 4.5kg was 239 μM. As can be seen, the measured activity in the 1 minute sample was 130μm which is an actual recovery of about 55%, and a half life of approximately 5 minutes.

Immediately post infusion the platelet count was low, but recovered within 5 minutes. There was no apparent change in the WBC count.

12 The results are tabulated below:

Table 1

Time APTT Sec pits x 10⁹/L WBC x 10⁹/L [cmpd #3]

(manual) Prolongation pre 25" 27" 0 326 (100%) 7.6 (100%) 0

1' 34" 37" 9 125 (38%) 8.2 (108%) 130/ιM

5' 30" 32" 5" 264 (81%) 7.5 (98%) 75μM

10' 28" 28" 3 310 (95%) 7.4 (97%) 35μM

20' 24" 23" -1 288 (88%) 7.2 (95%)

46' 28" 27" 3 300 (92%) 6.7 (88%)

70' 26" 25" 1 291 (89%) 6.5 (86%)

123' 27" 26" 2 352 (108%) 7.6 (100%)

182' 27" 28" 2"

240' 27" 29" 2"

Table 2

Oral Dosing Experiments

Sucrose Octasulfate 200 mg/kg body weight

Results mean (x) of 2 studies x APTT PROLONG- x PLATELET CT

TIME (SEC) ATION (SEC) x 10⁹/L

Pre 7 min 30.5 298

Pre 6 min 28.5 0

ORAL DOSE GIVEN AT ZERO TIME

Post 15 min 34.0 5.5 ^' 233

Post 30 min 33.5 5.0 249

Post 45 min 34.5 6.0 248

Post 60 min 35.0 6.5 263

Post 75 min 34.0 5.5 265

Post 90 min 32.0* 3.5 400*

* N = 1 (1 specimen clotted)

13 Conclusion

A 5-6 second prolongation of APTT observed 15 min after oral ingestion that was

sustained for 90 minutes.

It will be appreciated by reference to Figure 2 that the APTT is simply a measure of blood coagulation in the overall blood coagulation cascade and no inferences as to where the inhibitor is active can be drawn. The PT, however, is a measure of the activation of Factor X to Factor Xa (prothrombinase) and activation of prothrombin to thrombin via the extrinsic pathway, and the TCT is a measure of the conversion of fibrinogen to fibrin by thrombin alone. It can be deduced that as there is no increase in PT or TCT, which measures the extrinsic pathway and the conversion of fibrinogen, respectively, the anticoagulant activity of the test compounds is confined to the inhibition of one or more components of the intrinsic pathway, i.e defined by the activation of Factor XII to Xlla, and/or XI to XIa and/or IX to IXa, and subsequent action of IXa.

These tests do not, however, establish precisely where in the intrinsic pathway that the anticoagulation effect occurs. It was, therefore, necessary to conduct a further series of tests to determine what was being affected in the intrinsic pathway. The further tests, described below in Examples 7, 8, and 9, involved (a) plasma systems using activated blood Factors XIa, Xa and XIa and (b) a purified components approach. Example 7 Activated Factor clotting times

Respective aliquots of human blood plasma were mixed, in vitro, with 15 μg ml of purified bovine Factor XIa (Enzyme Research Labs Inc.), or 300 ng/ml of purified human Factor IXa (Enzyme Research Labs Inc.), or 9 ng/ml purified human Factor Xa

14

SUBSTITUTE SHEET (Queen's University, Kingston). All concentrations refer to the final concentrations achieved in the assay. Each aliquot was then divided into four smaller aliquots and 0, 40, 90 and 180 μM of each of compounds 1-4 (as defined in Example 6) added and the clotting time was determined. No anticoagulant effect was observed with respect to Factor XIa or Factor Xa, but a marked effect was noted with respect to compounds 1-3 in plasma to which Factor IXa had been added. This effect is illustrated in Figure 4. Example 8 Prothrombinase activity using PAPA

This assay is described by Nesheim et al. in Biochemistry 18:996-1003 (1979) and was designed to measure prothrombin activating potential of prothrombinase (see Figure 1) contained within highly purified components. Activation of prothrombin was monitored with a freshly prepared fluorescent thrombin inhibitor DAP A. Reaction mixtures consisted of purified bovine prothrombin (1.4 μM) in tris-buffered saline (TBS) pH 7.4, consisting of 2 mM CaCl₂ and 3 μM DAPA at ambient temperature. Purified bovine factor Va was added to 5 nM final, phospholipid vesicles (PCPS = 75% phosphatidylcholine + 25% phosphatidylserine) added to 10 μM final and the anticoagulant compounds 1-4 to be tested were added to a final concentration of 100 μM. The reaction was started with the addition of purified bovine factor Xa to a final concentration of 5 nM.

No appreciable inhibition of prothrombinase (as defined by Figure 1) was seen with any of these compounds.

15 Example 9

Tenase activity measured chromogenically

This assay was performed to determine Factor X activating potential of an activating enzyme complex comprising highly purified components. Reaction mixtures consisted of 0.5 μM purified human factor X in HEPES buffered saline (HBS) pH 7.4, containing 5 mM CaCl₂ , 10 μM PCPS vesicles and 0.2 mM chromogenic substrate S- 2222. To 0.7 ml of this mixture at ambient temperature was added purified recombinant F.VIII(rF.VIII) to a final concentration of 2.5 nM followed by thrombin (1 nM final to activate the rF.VIII). The anticoagulant test compounds were added in a 2 μl volume and the reaction initiated with the addition of purified human F.IXa (1.4 nM final). The cleavage of S-2222 by F.Xa was monitored at 405 nM and the slopes plotted vs the concentration of inhibitor.

Half maximal inhibition was obtained at a concentration of 2 μM with compound 3 as shown in Figure 5. No inhibition was seen with compound 4 (sucrose). Example 10 Intrinsic fluorescence measurements

Binding of the anticoagulant compounds to and/or induction of conformational changes in the individual components of the tenase complex (see Figure 1) was analyzed by changes in intrinsic fluorescence. Samples at 22°C were excited at a wavelength of 280 nm at a slit width of 1 nm and emission was measured at 340 nm. The buffer used was HEPES-buffered saline (HBS) pH 7.4 containing 5 mM CaCl₂ and 0.01% Tween®- 80 and was filtered through a 0.2 μ filter. Purified proteins were tested as follows: recombinant F.VIII at a concentration of 55 nM, purified F.X at a concentration of 600 nM and purified F.IXa/3 at a concentration of 200 nM. Concentrated solutions of the

16 test compounds were prepared in a solution containing the protein to be tested. Aliquots of these solutions were added to a cuvette containing a solution of the test protein at the same concentration. After each addition, intrinsic fluorescence of the protein was measured. The inclusion of the protein in the concentrated solution eliminated the need to correct for dilution of the protein during the titration and thereby allowed highly accurate measurements of small relative changes in fluorescence. Titrations of anticoagulants covered the concentration range of 0.1 to 30 μM. The change in intrinsic fluorescence induced by the addition of anticoagulant was expressed as a ratio relative to initial fluorescence intensity obtained in the absence of the compound.

Anticoagulant compound 3 caused no change with rF.VIII, a decrease in intrinsic fluorescence with F.X (Figure 6) and an increase with FIXa (Figure 7). The change in F.X did not saturate with increasing concentrations of compound 3 and the concentration of compound 3 required to produce half-maximal change in intrinsic fluorescence was much higher than the concentration of compound 3 that produced half-maximal anticoagulant activity in the tenase assay (Figure 8). The increases in intrinsic fluorescence induced by compound 3 on F.IXa/3 were saturable, with the concentration to produce half-maximal response virtually identical to that for the anticoagulant activity (Figure 9). No changes were induced by compound 4. Compounds 1 and 2 were subsequently assayed and found to have very similar values to those obtained for 3. Oral administration

Thus far the invention has been described primarily with reference to therapeutic compositions for administration to a patient parenterally. The pharmaceutical composition may be administered parenterally in the form of a sterile aqueous solution

17 or isotonic saline. However, oral administration, in a pharmaceutically acceptable carrier such as lactose or calcium carbonate, is also possible, as described in Example 6.

It will be appreciated by those skilled in the art that the demonstrated anticoagulant activity of the compounds of this invention as assessed by APTT suggests that they will have antithrombotic activity as this test is used traditionally to monitor proven anticoagulant activity of therapeutics such as heparin. The range of increase of APTT associated with the therapeutic benefit of heparin has been established and the dose of the compounds required to achieve an equivalent increase in APTT has also been established (see Figure 3). Therefore, assuming that the effect on APTT by the claimed compounds, compared with that of heparin, translates into an equivalent antithrombotic activity, the dosage of the compounds of this invention can be readily calculated on a weight per kilogram of body weight basis designed to elevate the APTT into the therapeutic range, given that for heparin an APTT of 55-70 seconds can be achieved at a dosage rate of 0.2 - 0.3 units heparin/ml plasma.

The invention does, however, also contemplate the treatment of surfaces which come into contact with blood in vivo, extracoφoreally or in vitro so as to render them anticoagulative. It will be appreciated that in recent years silicones and silicone rubbers, in addition to many other materials, have been used to manufacture many medical devices, such as implants and equipment to transport or transfer blood, i.e. tubing, in which blood comes into contact with the silicone surface. Silicones are inert to human tissue and the body is able to tolerate silicone materials without undue adverse effects. However, blood tends to coagulate when in contact with silicone surfaces and this has been seen as a serious shortcoming to the extended use of such materials. Heparin coated surfaces have been suggested in the past to overcome this shortcoming, but there

18

SUB is considerable difficulty in bonding heparin to a silicone surface. One of the few effective methods involves exposing the heparin-coated surface to ionizing radiation. Another method, described in U.S. patent 3,508,959, involves sulfonating a partially cured silicone rubber by coating the rubber with an organosilane or organosiloxane and curing the rubber so as to sulfonate the surface thereof. In accordance with the present invention silicone and other surfaces, such as metals, glass and plastics materials with which blood comes into contact, may be activated by reacting the surface with a chemical linking agent, to which a selected compound of the present invention is then chemically attached.

19

Claims

CLAIMS:

1. A disaccharide containing at least one sulphur based anion, which exhibits antithrombotic properties specific to inhibition of Factor IXa.

2. A disaccharide as claimed in claim 1 wherein said sulphur based anion is selected from the group consisting of sulfate and sulfonate.

3. A disaccharide as claimed in claim 2, having blocked anomeric centers.

4. A disaccharide as claimed in claim 3, having a general formula

where R¹ is H, or (CH₂OSO-₃ M⁺)n, n is an integer from 1-6, R₂, R₃ and R₄ are the same or different and selected from OH, NH,SO-₃M⁺, and SO-₃M where M is selected from ammonium, cyclic ammonium, alkali metals and alkaline earth metals,

20

SUBSTITUTE SHEET The sulfated disaccharide of claim 3 having a general formula:

where R¹ is H or (CH₂OSO-₃M⁺)n or (CH₂SO-₃M⁺)n n is an integer from 1-6,

R₂, R₃ and R₄ are the same or different and selected from OH, NH, SO³- M⁺, OS0₃-M⁺ and HS0₃-M⁺

M is selected from NH₄, trialkylammonium, cyclic ammonium, alkali metals and alkaline earth metals; and R is:

H ,

21 6. The sulfated disaccharide of claim 4 selected from the group consisting o sύc'rδse octasulfate sodium salt and trehalose octasulfate sodium salt.

7. The sulfated disaccharide of claim 5, comprising methyl 3-D-lactoside

heptasulfate sodium salt.

8. An anticoagulant preparation comprising a pharmaceutically effective amount of a disaccharide containing at least one sulphur based anion, which exhibits antithrombotic properties specific to inhibition of Factor IXa, and a pharmaceutically acceptable carrier therefor.

9. An anticoagulant preparation as claimed in claim 8 wherein said sulphur based anion is selected from the group consisting of sulfate and sulfonate.

10. An anticoagulant preparation as claimed in claim 9 having at least one blocked anomeric center.

11. An anticoagulant preparation as claimed in claim 10 wherein all anomeric centers are blocked.

12. An anticoagulant preparation as claimed in claim 9 wherein said disaccharide has a formula:

where R_r is.H or (CH^OSO^M^n or (CH₂SO-₃M)n n is an integer from 1-6,

R₂, R₃ and R₄ are the same or different and selected from OH, NH,SO-₃M⁺OSO-₃M⁺ and where

22 M is selected from NH₄ trialkylammonium, cyclic ammonium, alkali metals

and alkaline earth metals; and R is

13. An anticoagulant preparation as claimed in claim 8 wherein said sulfated disaccharide has a formula:

where R_x is H, (CH₂OS0₃M⁺)n or (CH₂HS0₃M⁺)n,

R_ls R₂, R₃ and R₄ are the same or different and selected from OH, NH, SO-₃M⁺ and SO-₃M⁺ and where n is an integer from 1-6,

23 M is selected from NH₄ trialkylammonium, cyclic ammonium, alkali metals and

alkaline earth metals; and R is

H , R

14. An anticoagulant preparation as claimed in claim 13 wherein said disaccharide is selected from the group consisting of sucrose octasulfate sodium salt, and methyl _/3-D-lactoside heptasulfate sodium salt.

15. An article having an antithrombotic surface, comprising a substrate having a coating comprising a disaccharide containing a sulphur based anion which exhibits antithrombotic properties specific to inhibition of Factor IXa.

16. An article as claimed in claim 15 wherein said coating comprises a sulfated disaccharide having a formula:

H , R

24 where R_j is H, (CH₂HSO-₃M or (CH₂OS0₃M and n is an integer from 1-6;

R₂, R₃ and R₄ are the same or different and selected from OH, NH, SO- M⁺

OSO-₃M⁺ and SO-₃M⁺ where M is selected from NH₄, trial yl ammomum, cyclic ammonium, alkali metals and alkaline earth metals and

25

SUBSTITUTE SHEET l An article as claimed in claim 15 wherein said coating comprises a sulfated disaccharide having a formula:

18. An article as claimed in claim 15 wherein said sulfate disaccharide is selected

from the group consisting of sucrose octasulfate sodium salt, and methyl β-D- lactoside heptasulfate sodium salt.

26

SUBSTITUTE SHEET