WO2013016181A1

WO2013016181A1 - Compositions and methods for anti-coagulation

Info

Publication number: WO2013016181A1
Application number: PCT/US2012/047577
Authority: WO
Inventors: Andreas Greinacher; Krystin KRAUEL
Original assignee: Paringenix, Inc.
Priority date: 2011-07-22
Filing date: 2012-07-20
Publication date: 2013-01-31

Abstract

The present application provides pharmaceutical compositions and unit dosage forms comprising ODSH and an anticoagulant heparin in an ODSH:anticoagulant heparin (ODSH:H) weight ratio ranging from about 1:1 to about 4:1. Also provided herein are methods of anticoagulating, comprising administering ODSH and an anticoagulant heparin to subjects in need of such treatment.

Description

COMPOSITIONS AND METHODS FOR ANTI-COAGULATION

1. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of provisional application no. 61/510,985, filed July 22, 2011, the contents of all of which are incorporated herein in their entireties by reference thereto.

2. BACKGROUND

[0002] Thrombosis, or the formation of a blood clot inside a vessel, can arise in a wide variety of circumstances and jeopardize a patient's life. Heparins, such as unfractionated heparin or low molecular weight heparin, is administered to patients at risk for thrombosis as well as patients suffering from thrombosis. However, heparin can also trigger a serious auto-immune condition called heparin-induced thrombocytopenia (HIT) in which a subject receiving heparin experiences a substantial drop in platelet count, usually accompanied by major arterial, venous, or microvascular thrombosis.

[0003] As a very effective and low cost anti-coagulant, heparin remains widely used and is an essential drug according to the World Health Organization. However, there is a need for ways of anti-coagulating patients using heparin that do not also carry the attendant risk of triggering HIT. There remains a significant need for anti-coagulant treatments using heparins that carry a reduced risk of HIT.

[0004] U.S. Patent no. 7,468,358 describes the use of non-anticoagulant O-desulfated heparin, including 2-0, 3-0 desulfated heparin (ODSH), in the treatment or amelioration of HIT. See also, Rao et al, 2010, "Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands," Am. J. Physiol. Cell Physiol 299:97-110. U.S. Patent no.

7,875,596 discloses dermatan sulfates and O-desulfated heparins useful in treating and preventing heparinoid-induced autoimmune responses, in particular HIT. But while O- desulfated heparins are useful in treating and preventing HIT, they do not retain the anticoagulant activity of other heparins. WO 2006/023397 discloses the combined use of 5 to 50 parts non-anticoagulant heparin, including 2-0 desulfated heparin, to 0 to 1 part unmodified fully anticoagulant heparin to treat inflammation, but provides no guidance on HIT or on anticoagulation. 3. SUMMARY

[0005] Applicants have discovered that substantially non-anticoagulant 2-0, 3-0 desulfated heparin (ODSH) combined with anticoagulant heparin in weight ratios ranging from about 2: 1 to about 4: 1 provides anti-coagulation effectiveness equivalent to that of heparin alone, while inhibiting molecular interactions that can give rise to heparin-induced thrombocytopenia (HIT). In light of this discovery, it is now appreciated that a composition comprising both ODSH and heparin in defined weight ratios is useful to anti- coagulate patients and that such a composition is further useful in anti-coagulating subjects at risk for HIT. Provided herein are compositions and unit dosage forms comprising ODSH and heparin, as well as methods of anticoagulating subjects in need of such treatment.

[0006] In one aspect, the present disclosure provides pharmaceutical compositions comprising ODSH and an anticoagulant heparin, such as unfractionated heparin (UFH) or low molecular weight heparin (LMWH), in an ODSH:anticoagulant heparin (ODSH:H) weight ratio ranging from about 1 : 1 to about 4: 1.

[0007] The pharmaceutical compositions may be suitable for parenteral administration, such as intravenous or subcutaneous administration. For intravenous administration, pharmaceutical compositions can be formulated for administration as a bolus or as a continuous infusion. Pharmaceutical compositions can also be formulated for subcutaneous administration.

[0008] The pharmaceutical compositions containing ODSH and anticoagulant heparin in an ODSH:anticoagulant heparin (ODSH:H) weight ratio ranging from about 1:1 to about 4:1 contain anticoagulant heparin in amounts and at concentrations suited to the chosen route of administration. The amount and concentration of anticoagulant heparin in the compositions and unit dosage forms described herein can be expressed either in units of activity, such as International Units (IU) or U.S. Pharmacopeia Units (U), or weight amounts. In some embodiments, the pharmaceutical compositions comprise from about 2 U/mL to about 20,000 U/mL UFH. In some embodiments, the pharmaceutical compositions comprise from about 60 mg/mL to about 160 mg/mL LMWH. In some embodiments, the pharmaceutical compositions comprise from about 10,000 IU/mL to about 25,000 IU/mL LMWH. In some embodiments, the pharmaceutical compositions comprise from about 1 mg/mL ODSH to about 1000 mg/mL ODSH.

[0009] In another aspect, the present disclosure provides unit dosage forms of the pharmaceutical composition containing from 1 to 1000 mL of the pharmaceutical composition as a sterile, non-pyrogenic fluid. When used for intravenous bolus administration or subcutaneous administration, unit dosage forms can range from 0.1 to 1 mL. When used for intravenous infusion, unit dosage forms can range up to 1000 mL.

[0010] In another aspect, the present disclosure provides methods of anticoagulation, comprising administering ODSH and an anticoagulant heparin to a subject in need of anticoagulation, in an ODSH:anticoagulant heparin weight ratio ranging from about 1 : 1 to about 4:1.

[0011] The ODSH and heparin can be administered concurrently, either as a single composition comprising both ODSH and heparin or as two separate compositions.

Whether administered as a single composition or as two separate compositions, ODSH and anticoagulant heparin can each be administered intravenously or subcutaneously. ODSH and anticoagulant heparin can be administered via the same or via different routes.

[0012] The methods of the present disclosure are useful to treat any condition in which anticoagulant treatment is called for, including any disease or condition that may be treated with UFH or LMWH. By way of example, the present methods may be used in the treatment and/or prevention of any of the following: venous thrombosis (including deep vein thrombosis), peripheral arterial embolism, ischemic complications of unstable angina or myocardial infarction, acute myocardial infarction, clotting in arterial and cardiac or cardiovascular surgery (including percutaneous cardiac intervention, or PCI), pulmonary embolism, acute and chronic consumptive coagulopathies, and atrial fibrillation with embolization. Furthermore, the present methods may be used for any indication where use of heparin is desirable, but contraindicated due to a risk of HIT.

[0013] Suitable subjects for the present methods include, but are not limited to, subjects at risk for any of the preceding diseases or conditions, subjects undergoing surgical procedures, e.g., abdominothoracic surgery, joint (e.g., hip or knee) replacement surgery, subjects with severely restricted mobility during acute illness, hospitalized subjects, subjects on dialysis, and subjects at risk for HIT, including patients with antibodies to platelet factor 4 ("PF4")/heparin complex, patients with a prior incidence of HIT, patients previously treated with heparin.

4. BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 provides a graph showing the percent of PF4 bound to platelets at increasing concentrations of UFH or ODSH;

[0015] FIG. 2 provides a graph showing the percent of PF4/UFH complex bound to resting or activated platelets at increasing concentrations of ODSH;

[0016] FIG. 3 provides a graph showing the percent of PF4/UFH complexes bound to platelets at increasing concentrations of UFH or ODSH;

[0017] FIG. 4 provides a graph showing the percent of PF4/UFH complexes bound to platelets incubated with 0.52 μg/mL UFH and increasing amounts of ODSH;

[0018] FIG. 5 provides a graph showing the percent of anti-PF4/UFH antibody bound to PF4/UFH complexes at increasing concentrations of ODSH or UFH;

[0019] FIG. 6 provides a graph showing the number of donor sera showing platelet activation in response to 0.2 IU/mL of UFH or LMWH and increasing concentrations of ODSH (open and filled squares, respectively) or 0.2 IU/mL of LMWH and increasing concentrations of UFH (filled triangles); and

[0020] FIG. 7 provides a graph showing the number of donor sera showing platelet activation in sera from patients exposed to increasing concentrations of ODSH and UFH.

5. DETAILED DESCRIPTION

5.1. Overview

[0021] Thrombosis, or clotting, can occur in a variety of settings. Patients can experience or be at risk of thrombosis not only by virtue of having a disease or condition affecting clotting but also as a result of surgical intervention. The prophylactic use of heparin is standard practice in a variety of surgical settings. While heparin is a low cost and highly effective anticoagulant, it can induce an auto-immune condition called heparin-induced thrombocytopenia (HIT). Heparin complexes with platelet-binding platelet factor-4 ("PF4") when present in approximately equimolar amounts. The complex is antigenic and can lead to the development of antibodies. In subjects with antibodies to the PF4/heparin complex, administration of heparin can trigger a chain reaction in which PF4 forms a complex with heparin, which is bound by anti-PF4/heparin antibodies. The

antibody/antigen complex then activates platelets, causing the formation of microparticles which can develop into clots and cause a precipitous drop in platelet count. As shown in the Examples below, ODSH interferes with (a) PF4/UFH complex formation, (b) PF4 binding to platelets, and (c) platelet activation by PF4/UFH antibodies. As such ODSH has the ability to disrupt the interactions that lead to HIT.

[0022] Applicants have newly discovered that, while ODSH disrupts heparin's interactions with PF4, it does not interfere with heparin's anticoagulant activity when combined with heparin in defined weight ratios and used in amounts in which it is shown to prevent or reduce the interactions that lead to HIT. See Examples below.

Consequently, ODSH and anticoagulant heparin are useful in treating subjects in need of anticoagulation, including, but not limited to, subjects who could be at risk for HIT.

Provided herein, as described below in further detail, are compositions and unit dosage forms of ODSH and heparin, as well as methods of treatment comprising administering ODSH and heparin.

5.2. ODSH and anticoagulant heparin

[0023] ODSH for use in the compositions, unit dosage forms and methods described herein can be synthesized by cold alkaline hydrolysis of USP porcine intestinal heparin, which removes the 2-0 and 3-0 sulfates, leaving N- and 6-0 sulfates and carboxylates intact. Fryer, A. et al, 1997, J. Pharmacol. Exp. Therap. 282: 208-219. Using this method, ODSH can be produced with an average molecular mass of 11.7 ± 0.3 kDa, and low affinity for anti-thrombin III (¾ = 339 μΜ or 4 mg/ml vs. 1.56 μΜ or 22 μg/ml for heparin), consistent with the observed low level of anticoagulant activity. Accordingly, ODSH is substantially non-anticoagulant. Methods for the preparation of 2-0, 3-0 desulfated non-anticoagulant heparin may also be found, for example, in U.S. Patent nos. 5,668,118, 5,912,237, and 6,489,311, and WO 2009/015183, the contents of which are incorporated in their entirety herein, and in U.S. Patent nos. 5,296,471, 5,969,100, and 5,808,021.

[0024] Anticoagulant heparins for use in the compositions, unit dosage forms and methods described herein includes unfractionated heparin (UFH) and low molecular weight heparin (LMWH), which are readily available. UFH can be heparin sodium or heparin calcium, typically from porcine intestines, or heparin sodium from bovine lung. Commonly used low molecular weight heparins include, but are not limited to, enoxaparin, dalteparin, tinzaparin, reviparin, and fondaparinux, a synthetic pentasaccharide. UFH or LMWH, compliant with the requirements established by the U.S. and European Pharmacopeia, can be used in the compositions, dosage forms, and methods described below.

5.3. Pharmaceutical compositions

[0025] In a first aspect, the present disclosure provides pharmaceutical compositions comprising ODSH and an anticoagulant heparin, in an ODSHranticoagulant heparin (ODSH:H) weight ratio ranging from about 1 : 1 to about 4: 1. In some embodiments, the weight ratio ranges from about 1 : 1 to about 3:1 , from about 2: 1 to about 4: 1, from about 2: 1 to about 3: 1 , from about 3:1 to about 4:1, or a range including nonintegral ratios (e.g., from about 1.5: 1 to about 3.5: 1). In certain embodiments, the weight ratio is about 1 : 1, about 2: 1, about 3: 1, about 4: 1, including nonintegral ratios between about 1 : 1 and about 4: 1.

[0026] The pharmaceutical compositions can be formulated for administration by a variety of routes, typically for parenteral administration, e.g., via intravenous or subcutaneous injection. Pharmaceutical compositions can be formulated in volumes and concentrations suitable for intravenous bolus injection, intravenous continuous infusion, or subcutaneous administration, to provide the desired dose of ODSH and anticoagulant heparin, examples of which are provided below.

[0027] The pharmaceutical compositions can be in the form of a sterile, non-pyrogenic, fluid composition. In some embodiments, the pharmaceutical composition can comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or diluents. The specific carriers, excipients, preservative, and/or diluents used will depend on the desired mode of administration. The compositions may further include one or more buffering agents, sugars or salts (e.g., sodium chloride). In some embodiments, the pharmaceutical compositions are preservative-free.

[0028] The amount and concentration of anticoagulant heparin in the compositions and unit dosage forms described herein can be expressed either in units of activity, such as International Units (IU) or U.S. Pharmacopeia Units (U), or weight amounts. For UFH, units of anti-Factor Ila activity per mg of material can be determined according to the U.S. Pharmacopeia monograph on heparin sodium for injection. See, e.g., Pharmacopeial Forum, Vol. 35(5), Sept.-Oct. 2009, noting that the potency of heparin sodium, calculated on a dried basis, is no less than 180 USP Heparin Units in each mg. For LMWH, units of anti-Factor Xa activity per mg can be determined relative to the W.H.O. First International Low Molecular Weight Heparin Reference Standard.

[0029] In some embodiments, the pharmaceutical compositions comprise from about 2 U/mL to about 20,000 U/mL UFH. In some embodiments, the pharmaceutical compositions comprise UFH suitable for subcutaneous administration of doses ranging from about 5,000 U to about 30,000 U. In some embodiments, the pharmaceutical compositions comprise UFH suitable for intravenous administration of doses ranging from about 1,000 U to about 30,000 U. In some embodiments, the pharmaceutical compositions comprise UFH suitable for intravenous infusion of doses ranging from about 50 U/ 24 hours to about 50,000 U/ 24 hours.

[0030] In some embodiments, the pharmaceutical compositions comprise LMWH. In some embodiments, the pharmaceutical compositions comprise from about 60 mg/mL to about 160 mg/mL LMWH. In some embodiments, the pharmaceutical compositions comprise LMWH suitable for subcutaneous administration of doses ranging from about 0.5 mg/kg to about 2 mg/kg. In some embodiments, the pharmaceutical compositions comprise LMWH suitable for subcutaneous administration of doses ranging from about 25 mg to about 70 mg. In some embodiments, the pharmaceutical compositions comprise LMWH suitable for intravenous administration of doses ranging from about 25 mg to about 40 mg.

[0031] In some embodiments, the pharmaceutical compositions comprise from about 10,000 lU/mL to about 25,000 IU/mL LMWH. In some embodiments, the pharmaceutical compositions comprise LMWH suitable for subcutaneous administration of doses ranging from about 2,500 IU to about 5,000 IU. In some embodiments, the pharmaceutical compositions comprise LMWH suitable for subcutaneous administration of doses ranging from about 120 IU/kg to about 200 IU/kg.

[0032] The amount of ODSH in the pharmaceutical compositions described herein can range from about 1 to about 4 times the weight amount of anticoagulant heparin (H) in the composition. In various embodiments, the ODSH:H ratio can be about 1 : 1, about 2: 1, about 3:1, or about 4:1, including nonintegral ratios between about 1 :1 and about 4:1. In one embodiment, the pharmaceutical composition comprises ODSH in a sterile vial at a concentration of 50 mg/mL.

[0033] In some embodiments, the pharmaceutical compositions comprise ODSH and anticoagulant heparin in a weight ratio that provides an anti-coagulating effect with reduced platelet binding.

[0034] As shown in Example 3 below, combination of ODSH with anticoagulant heparin can inhibit the formation of PF4/ anticoagulant heparin complexes and significantly reduce binding of such complexes to platelets. Thus, in some embodiments, the pharmaceutical compositions comprise ODSH and anticoagulant heparin in a weight ratio that provides an anticoagulating effect with reduced platelet binding. Platelet binding can be measured using the assay set forth in Example 3 below.

5.4. Unit dosage forms

[0035] Pharmaceutical compositions can be conveniently presented in unit dosage forms. Unit dosage forms contain ODSH and anticoagulant heparin in amounts and volumes suitable for administration in the doses described herein, with a weight amount of ODSH ranging from about 1 to about 4 times the weight amount of anticoagulant heparin.

[0036] Unit dosage forms can contain UFH in an amount ranging from about 1,000 U, up to about 5,000 U, up to about 10,000 U, up to about 20,000 U, up to about 25,000 U, up to about 50,000U, or up to about 75,000 U. In some embodiments, unit dosage forms contain LMWH in amounts ranging from 1000 IU to about 18,000 IU (or from about 10 mg to about 116 mg). In some embodiments, unit dosage forms contain LMWH in an amount of 95,000 IU. In some embodiments, unit dosage forms contain ODSH in an amount ranging from 10 mg to 2000 mg. In some embodiments, unit dosage forms contain a single dose. In some embodiments, unit dosage forms contain multiple doses. Typically, unit dosage forms can range in volume from 1 mL to 1000 mL, for example, 0.1 mL, 0.2 mL, 0.3 mL, 0.5 mL, 1 mL, 5 mL, 10 mL, 30 mL, 100 mL, 200 mL, 500 mL, or 1000 mL.

[0037] Unit dosage forms have containers appropriate for the volumes and intended route of administration, including for example ampules, vials, preloaded syringes, infusion bags, cartridge units with Luer lock connectors, single or multidose containers.

-g- 5.5. Methods of treatment

[0038] In another aspect, the present disclosure provides methods of anticoagulation. Generally, the method comprises administering ODSH and an anticoagulant effective amount of anticoagulant heparin (H), in an ODSH:H weight ratio of about 1: 1 to about 4: 1, to a subject in need of anti-coagulation.

[0039] The methods of the present disclosure are useful to treat any condition in which anticoagulant treatment is called for, including any disease or condition that may be treated with UFH or LMWH. By way of example, the present methods may be used in the treatment and/or prevention of one or more of the following: venous thrombosis (including deep vein thrombosis), arterial thrombosis (including stroke, myocardial infarction or "heart attack", and acute leg ischemia), peripheral arterial embolism, clotting in arterial and cardiac surgery, pulmonary embolism, acute and chronic consumptive coagulopathies, and atrial fibrillation with embolization. Furthermore, the present methods may be used for any indication where use of heparin is contraindicated due to a risk of HIT.

[0040] Suitable subjects for the present methods include, but are not limited to, subjects at risk for, or suffering from, any of the preceding diseases or conditions, subjects undergoing major abdominothoracic surgery, subjects undergoing joint (e.g., hip or knee) replacement surgery, subjects with severely restricted mobility during acute illness, and subjects at risk for HIT, including patients with antibodies to PF4/heparin complex, patients with a prior incidence of HIT, patients previously treated with heparin. The subject treated may be any animal, for example, a mammal, particularly a human.

[0041] ODSH and heparin are administered for a time and in an amount sufficient to provide anticoagulation. Anticoagulation dosing regimens and amounts for heparin are well known. See, e.g., product labeling information for "Heparin Sodium Injection" at www.drugs.com/pro/heparin.html; product label for dalteparin (FRAGMIN®); product label information for enoxaparin (LOVENOX®). Anticoagulation can be determined using standard assays, such as, but not limited to, assays measuring activated partial thromboplastin time (aPTT) or anti-Factor Xa. When using aPTT, an anticoagulating effect is observed when aPTT is 1.5 to 2.5 times the upper limit of a normal reading. See Hirsch et al, 2008, Chest 133: 141S-159S. In some embodiments, ODSH and

anticoagulant heparin (H) are administered in amounts and weight ratios that reduce PF4/H complex formation and platelet binding. Reduced platelet binding can be measured by assays known to those skilled in the art. One such an assay is provided in Example 3 below.

[0042] In various embodiments, ODSH and anticoagulant heparin are administered one or more times over a period of 2 weeks to indefinitely, a period of 2 weeks to 6 months, a period of 3 months to 5 years, a period of 6 months to 1 or 2 years, or the like. ODSH and anticoagulant heparin administration can be repeated, for example, once daily, twice daily, every two days, three days, five days, one week, two weeks, or one month. The repeated administration can be at the same dose or at different doses. In some embodiments, an initial dose is administered at a higher dose amount and/or via a different route, e.g., subjects treated by subcutaneous injection may receive an initial intravenous dose, or subjects treated by intravenous infusion may receive an initial bolus injection.

[0043] Administration of anticoagulant heparin, such as UFH or LM WH, can be carried out according to standard regimens, known to those skilled in the art.

[0044] In some embodiments, UFH is administered in amounts ranging from about 5,000 U to about 20,000 U, typically with an initial intravenous injection of 5,000 U to 10,000 U. In some embodiments, UFH is administered as an intravenous injection, at a dose of 5,000 U to 10,000 U, every 4 to 6 hours. In some embodiments, UFH is administered as an infusion, at a dose of 20,000 U/24 hours to 40,000 U/24 hours. In some embodiments, UFH is administered as a subcutaneous injection, at a dose of 8,000 to 10,000 U every 8 hours or 15,000- 20,000 U every 12 hours. Exemplary doses and amounts are based on a subject weighing about 68 kg and can be adjusted up or down depending on the actual subject's weight.

[0045] In some embodiments, LMWH is administered at doses of about 120 IU/kg every 12 hours, or about 150 to about 200 IU/kg once daily. In some embodiments, LMWH is administered at a dose of about 2,500 IU to about 5,000 IU once daily. In some embodiments, LMWH is administered at a dose of about 25 mg to about 40 mg, once daily. In some embodiments, LMWH is administered at a dose ranging from about 0.75 mg/kg up to 2 mg/kg once daily.

[0046] In the methods disclosed herein, ODSH is administered according to the same dosing regimen (i.e. route and schedule) used for heparin and in a dose amount ranging from 1 to 4 times the weight amount of the heparin administered. In some embodiments, ODSH is administered at a dose or amount per kilogram of patient body weight from about 0.5 mg/kg to about 10 mg/kg for subcutaneous doses, or about 0.5 mg/kg to about 25 mg/kg for bolus doses, and from about 0.05 mg/kg/hr to about 5 mg/kg/hr for infusions.

[0047] The ODSH and anticoagulant heparin can be administered concurrently, either as a single composition comprising both ODSH and anticoagulant heparin or as two separate compositions. Whether administered as a single composition or as two separate compositions, ODSH and anticoagulant heparin can be administered intravenously or subcutaneously.

6. EXAMPLES

Example 1 : Effect of ODSH on binding of platelet factor-4 (PF4) to

platelets

[0048] This experiment demonstrates the effects of 2-0, 3-0 desulfated heparin (used interchangeably with ODSH throughout Examples) on the binding of platelet factor-4 (PF4) to platelets.

1.1 Materials & methods

[0049] Materials. A solution of 50 mg/mL ODSH (in 0.9% saline), and UFH (Braun, Melsungen, Germany), 150 IU/mg, were used as starting materials in each of the experiments described in the following examples.

[0050] Preparation of platelets. Platelet-rich plasma (PRP) was obtained from hirudinized (10 μg/mL, lepirudin [Refludan], Pharmion, Hamburg, Germany) whole blood of healthy volunteers by centrifugation (120 g, 20 min, 30°C). Gel-filtered platelets (GFPs) were obtained by adding PRP onto a Sepharose CL-2B liquid chromatography column (30 mL; Sigma-Aldrich, Taufkirchen, Germany) preequilibrated with buffer (137 mM NaCl, 2.7 mM KC1, 2 mM MgCl₂ x 6 H₂0, 2 raM CaCl₂ x 2 H₂0, 12 mM NaHC0₃, 0.4 mM NaH₂P0₄, 0.4% BSA, 0.1% glucose, pH 7.2).

[0051] Assay for PF4 binding to platelets. GFPs (40,000/μί) were incubated (30 min, 37°C) with 25 μg/mL PF4 (ChromaTec, Greifswald,Germany) in the presence of increasing concentrations (0.07, 0.13, 0.26, 0.52, 1.04, 2.08, 4.17, 8.33, 16.67, 33.33 g/mL) of unfractionated heparin (UFH, 150 IU/mg; Braun, Melsungen, Germany), 2-0, 3-0 desulfated heparin, or buffer. Then GFPs were fixed (1% paraformaldehyde, Merck, Darmstadt, Germany; 20 min, 4°C), washed twice with buffer (600 g, 7 min, 4°C), incubated with rabbit anti-human PF4 (Dianova, Marl, Germany) FITC-labelled with FluoReporter FITC Protein Labelling Kit (Molecular Probes, Eugene, OR), and mouse anti-human CD42a-PE (BD Biosciences, San Jose, CA), or isotype controls (30 min, 4°C), washed, and analysed by flow cytometry (Cytomics FC 500, Beckman Coulter; STADT, Germany). Antibody binding was quantified by geometric mean fluorescent intensity (MFI).

1.2 Results

[0052] Gel-filtered platelets (GFPs) were incubated with 25 μg/mL PF4 in the presence of increasing concentrations of UFH or 2-0, 3-0 desulfated heparin. PF4 binding was detected with a FITC-labelled anti-human PF4 antibody using flow cytometry and antibody binding was quantified by geometric mean fluorescent intensity (MFI). PF4 binding to platelets without any anticoagulants was arbitrarily set at 100% (baseline). Data are mean ± SD of 4 independent experiments. UFH showed the typical effect on PF4 binding to platelets. At low concentrations, UFH enhanced PF4 binding to platelets up to four fold over baseline, reaching maximal enhancement of binding when UFH is added at 0.52 μg/mL (P=0.0009). FIG. 1. At concentrations above 0.52 μg/mL, the effect on PF4 tapered off until, at concentrations at or above 8.33 μg/mL, UFH reduced PF4 binding to platelets to below baseline levels (P=0.0068). FIG. 1.

[0053] PF4 binding to platelets in the presence of 2-0, 3-0 desulfated heparin was only weakly enhanced at low ODSH concentrations as compared to the effect seen with UFH, showing less than two-fold increase over baseline at most. Maximal enhancement of PF4 binding in the presence of 2-0, 3-0 desulfated heparin was observed at a concentration of 0.52 μg/mL (P=0.0024) and was significantly lower than binding observed with UFH (P=0.0005). FIG. 1. At concentrations of 2.08 \iglmL and greater, ODSH was significantly more effective in preventing PF4 from binding to platelets than UFH at the same concentrations (P=0.0039 for 2.08 μg/mL ODSH versus UFH). FIG. 1. Example 2: ODSH and activated versus resting platelets

[0054] This experiment demonstrates that higher concentrations of ODSH are necessary to inhibit PF4 binding to activated platelets as compared to resting platelets.

2.1 Materials & methods

[0055] In a first series of experiments, gel-filtered platelets (GFPs) (40,000/μί) were preloaded with PF4/UFH complexes (25 μg/mL PF4 and 0.52 μg/mL UFH, 30 min, 37°C), allowing binding of PF4/UFH complexes to the platelets at experimentally- determined optimal concentrations. Then, platelets with bound PF4/heparin complexes were washed (137 mM NaCl, 2.7 raM KC1, 12 mM NaHC0₃, 0.4 raM NaH₂P0₄, 0.4% BSA, 0.1% glucose, 2.5 U/mL apyrase, 0.1 ug mL hirudin, pH 6.3; 600 g, 7 min, 37°C) to remove unbound PF4 and UFH, and subsequently incubated with UFH, ODSH (per concentrations described above in Example 1), or buffer, fixed, and processed as described above, before flow cytometric analysis. The amount of PF4 bound after preincubation of platelets with PF4/UFH complexes (formed at optimal ratios) was defined as 100% PF4 binding (baseline).

[0056] In a second series of experiments, the effect of incubating GFPs with PF4, UFH, and ODSH or buffer was tested. GFPs were preincubated with convulxin (100 ng/mL; provided by Dr. Clemetson, Bern, Switzerland) and a monoclonal GPIIb/IIIa-specific antibody, abciximab (4 μg/mL; ReoPro, Centocor, Leiden, Netherlands), followed by concurrent incubation with PF4 (25 μg/mL), UFH (0.52 μg/mL), and ODSH (per concentrations described above) or buffer. GFPs were then fixed and processed as described above for flow cytometric analysis. Abciximab was used to block platelet aggregation and convulxin was used to activate platelets (Polgar et al, 1997, J. Biol. Chem. 272: 13576-13583).

2.2 Results

[0057] The ability of ODSH to reduce or prevent PF4/UFH complex binding to platelets and/or complex formation was examined in resting and activated platelets. PF4 binding to platelets in the presence of UFH alone was defined as 100% binding (baseline). In resting platelets, ODSH reduced PF4 binding below baseline levels when added at concentrations of 0.26 μg/mL or higher, with values reaching statistical significance at 2.08 μg/mL (P=0.0474), even when platelets were incubated concurrently with ODSH and UFH. See FIG. 2, filled symbols. When platelets were activated before incubation with PF4, UFH, and ODSH (open symbols, FIG. 2), higher ODSH concentrations were needed to inhibit PF4 binding to platelets: 4.17 μg/mL (P=0.0002), 8.33 μg/mL (P=0.0006), 16.67 μg/mL (P=0.0001) and 33.33 μg/mL (P=0.0048) for activated platelets compared to resting platelets.

Example 3: ODSH inhibits formation of PF4/heparin complexes

[0058] This experiment demonstrates that ODSH inhibits formation of PF4/heparin complexes.

3.1 Materials & methods

[0059] Assay for PF4/heparin complex binding to platelets. GFPs (40,000/μΙ-.) were preloaded with PF4/heparin complexes (25 μg/mL PF4 and 0.52 μg/mL UFH, 30 min, 37°C), allowing binding of PF4/heparin complexes at experimentally-determined optimal concentrations. Then, platelets with bound PF4/heparin complexes were washed (137 mM NaCl, 2.7 mM KC1, 12 mM NaHC0₃, 0.4 mM NaH₂P0₄, 0.4% BSA, 0.1% glucose, 2.5 U/mL apyrase, 0.1 μg/mL hirudin, pH 6.3; 600 g, 7 min, 37°C) to remove unbound PF4 and heparin, incubated with UFH, or 2-0, 3-0 desulfated heparin (at concentrations described in Example 1) or buffer, fixed, and processed as described in Example 1, before flow cytometric analysis. The amount of PF4 bound after preincubation of platelets with PF4/heparin complexes (formed at optimal ratios) was defined as 100% PF4 binding. In a second series of experiments, GFPs were incubated concurrently with PF4 (25 μg/mL) and UFH (0.52 μg/mL), with or without 2-0, 3-0 desulfated heparin (at the concentrations described in Example 1) before fixation.

3.2 Results

[0060] Binding of PF4/heparin complexes to the platelet surface is necessary to trigger PF4/heparin antibody induced platelet activation. GFPs preincubated with PF4/heparin complexes and then washed, were incubated with increasing concentrations of UFH or 2- O, 3-0 desulfated heparin. PF4 binding was detected with a FITC-labelled anti-human PF4 antibody using flow cytometry and antibody binding was quantified by geometric mean fluorescent intensity (MFI). Preincubation of platelets with PF4/heparin complexes was defined as 100% binding. Data are mean ± SD of 4 independent experiments. ODSH and UFH equally displaced PF4/heparin complexes from the platelet surface. 2-0, 3-0 desulfated heparin was not only effective in displacing PF4 but also displaced PF4 heparin complexes from the platelet surface at concentrations of 1.04 μg/mL (P=0.0496) and greater (FIG. 3).

[0061] A test was performed to confirm that 2-0, 3-0 desulfated heparin can prevent PF4 heparin complex formation and binding to platelets in combination with UFH. GFPs were incubated with 25 μg/mL PF4 in the presence of 0.52 μg/mL UFH and increasing concentrations of ODSH concurrently. PF4 binding was detected with a FITC -labelled anti-human PF4 antibody using flow cytometry and antibody binding was quantified by geometric mean fluorescent intensity (MFI). PF4 binding in the presence of UFH alone (at optimal ratios) was defined as 100% binding. Data are mean ± SD of at least 3 independent experiments. ODSH reduced PF4/heparin complex binding even if incubated concurrently with UFH. 2-0, 3-0 desulfated heparin, incubated concurrently with UFH, significantly reduced PF4/heparin complex binding starting at concentrations of 2.08 μg/mL (P=0.0474) (FIG. 4).

[0062] ODSH's influence on PF4/heparin antibody binding to PF4/heparin complexes coated to a solid phase was shown using a PF4/heparin ELISA. The signal of PF4/heparin antibody binding without any ODSH or anticoagulant was set 100%. Data are mean ± SD of 6 independent experiments. 2-0, 3-0 desulfated heparin reduced antibody binding starting at concentrations of 4.17 g/mL (P=0.0124) with the same efficacy as UFH (FIG. 5). These results confirm that 2-0, 3-0 desulfated heparin disrupts PF4/heparin complexes.

Example 4: ODSH inhibits platelet activation by antibodies to the

PF4/heparin complex

[0063] This experiment demonstrates that ODSH inhibits platelet activation triggered by antisera against PF4/heparin complexes.

4.1 Materials & methods

[0064] PF4/heparin antibody assays. In an enzyme-linked immunosorbent assay (ELISA) (Juhl et al, 2006, Eur. J. Haematol. 76(5):420-426) that detects anti-PF4/heparin IgG antibodies, UFH or 2-0, 3-0 desulfated heparin was added at the concentrations described in Example 1 to the patient sera containing anti-PF4/heparin IgG and then incubated on a PF4/heparin-coated microtiter plate. The plate was then washed and incubated with peroxidase-conjugated goat anti-human IgG, and washed again.

Tetramethylbenzidine was added, and the reaction was stopped with 1 M H2SO4. Sera from 16 patients with sero logically-confirmed HIT were tested.

[0065] The heparin-induced platelet activation (HIPA) test was performed as described (Warkentin and Greinacher, 2007, Laboratory Testing for Heparin-induced

Thrombocytopenia, pp. 227-238, in HEPARIN-I DUCE THROMBOCYTOPENIA, Warkentin and Greinacher, eds., New York, NY, Informa Healthcare) using a low molecular weight heparin (LMWH), reviparin, at 0.2 IU/mL (which induces maximal platelet activation in the presence of anti-PF4/heparin antibodies), or increasing concentrations of 2-0, 3-0 desulfated heparin (as described in Example 1) to test for any cross-reactivity.

[0066] To assess whether ODSH inhibits platelet activation by PF4/heparin antibodies, 0.2 IU/mL LMWH or UFH combined with increasing concentrations of 2-0, 3-0 desulfated heparin was added to donor sera (concentrations described in Example 1). The number of donor sera showing platelet activation at 45 minutes was determined. High concentrations of UFH (100 IU/mL) were used to show inhibition in all functional experiments.

4.2 Results

[0067] 2-0, 3-0 desulfated heparin did not induce platelet activation in the presence of PF4/heparin antibodies at any concentrations tested (n=3 sera). Furthermore, ODSH inhibited platelet activation by PF4/heparin antibodies in the presence of 0.2 IU/mL LMWH. 8.33 μg/mL ODSH inhibited platelet activation by 0.2 IU/mL LMWH in half the sera tested (FIG. 6). Starting at concentrations of 16.67 μg/mL ODSH and 8.33 μg/mL UFH, platelet activation was inhibited in 4 out of 5 donor platelets by a donor serum containing anti-PF4/heparin IgG (FIG. 7).

Example 5: Anticoagulation by unfractionated heparin in the

presence of ODSH

[0068] This experiment demonstrates that ODSH in combination with unfractionated heparin provides substantially the same anticoagulation effect, as measured by activated partial thromboplastin time (aPTT) and anti-factor Xa activity (anti-Xa), as unfractionated heparin alone.

5.1 Materials & methods

[0069] Activated partial thromboplastin time (aPTT) and anti-factor Xa activity (anti-Xa; UFH standard curve) were analyzed by standard methods (BCS XP; Siemens Healthcare, Marburg, Germany) in pooled plasma (n=25 healthy blood donors) spiked with UFH (2 μg/mL ~ 0.3 IU/mL) alone, 2-0, 3-0 desulfated heparin (1.04, 2.08, 4.17, 8.33, 16.67 μg/mL·) alone, or combined UFH (2 μg/mL) and 2-0, 3-0 desulfated heparin (1.04, 2.08, 4.17, 8.33, 16.67 μg/ L). Saline 0.9% was added as buffer control to measure the baseline activity of the pool plasma. Statistical analysis was preformed by t-test of paired samples. P values less than 0.5 were considered statistically significant.

5.2 Results

[0070] ODSH had only minimal effects on the aPTT and anti-Xa activity in pooled plasma containing UFH at a fixed concentration of 2 g/mL (-0.3 IU/mL). ODSH was added in increasing concentrations shown in Example 1 to be effective in displacing PF4/heparin complexes from platelets. Up to 8 μg/mL 2-0, 3-0 desulfated heparin had only minimal effects on the aPTT and anti-Xa activity of 2 \iglvnL UFH (Table 1).

[0071] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0072] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).

Claims

WHAT IS CLAIMED IS:

1. A sterile pharmaceutical composition comprising 2-0, 3-0 desulfated heparin (ODSH) and unfractionated heparin (UFH) in an ODSH:UFH weight ratio of about 1 : 1 to about 4:1.

2. The pharmaceutical composition of claim 1, which is suitable for intravenous administration to a patient in need of anticoagulation.

3. The pharmaceutical composition of claim 1 comprising about 1 mg/mL to about 1000 mg/mL ODSH.

4. The pharmaceutical composition according to any one of claims 1 to 3 comprising from about 2 U/mL to about 20,000 U/mL UFH.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the ODSH:UFH weight ratio ranges from about 2: 1 to about 4: 1.

6. A sterile pharmaceutical composition comprising ODSH and UFH in a weight ratio of ODSH:UFH which has an anticoagulating effect with reduced platelet binding as determined in the assay according to Example 3, wherein the pharmaceutical composition is suitable for intravenous administration.

7. The pharmaceutical composition of claim 6 comprising about 1 mg/mL to about 1000 mg/mL ODSH.

8. The pharmaceutical composition of claim 6 comprising about 2 U/mL to about 20,000 U/mL UFH.

9. The pharmaceutical composition according to claim 6, 7, or 8, wherein the anticoagulating effect is determined in vitro.

10. A sterile pharmaceutical composition comprising 2-0, 3-0 desulfated heparin (ODSH) and low molecular weight heparin (LMWH) in an ODSH:LMWH weight ratio of about 1 : 1 to about 4:1.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is suitable for subcutaneous administration to a patient in need of anticoagulation.

12. The pharmaceutical composition of claim 10 or 11 comprising about 1 mg/mL to about 1000 mg/mL ODSH.

13. The pharmaceutical composition of any one of claims 10 to 12 comprising about 10,000 IU/mL to about 25,000 IU/mL LMWH.

14. The pharmaceutical composition according to any one of claims 10 to 13, wherein the ODSH:LMWH weight ratio ranges from about 2: 1 to about 4: 1.

15. A sterile pharmaceutical composition comprising ODSH and LMWH in a weight ratio of ODSH:LMWH which has an anticoagulating effect with reduced platelet binding as determined in the assay according to Example 3, wherein the pharmaceutical composition is suitable for subcutaneous administration.

16. The pharmaceutical composition of claim 15 comprising about 1 mg/mL to about 1000 mg/ml ODSH.

17. The pharmaceutical composition of claim 15 comprising about 10,000 IU/mL to about 25,000 IU/ml LMWH.

18. The pharmaceutical composition according to claim 15, 16, or 17, wherein the anticoagulating effect is determined in vitro.

19. The pharmaceutical composition according to any one of the preceding claims, further comprising one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

20. A unit dosage form for sterile, intravenous administration to a patient in need of anticoagulation, containing about 1 to about 1000 ml of a composition comprising ODSH and UFH in an ODSH:UFH weight ratio of about 1: 1 to about 4:1.

21. The unit dosage form of claim 20, comprising about 1,000 U to about 50,000 U

UFH.

22. The unit dosage form of claim 20 comprising about 10 mg to about 2000 mg

ODSH.

23. The unit dosage form according to any one of claims 20 to 22, wherein the ODSH:UFH weight ratio ranges from about 1:1 to about 2:1.

24. The unit dosage form according to any one of claims 20 to 22, wherein the ODSH:UFH weight ratio ranges from about 2:1 to about 4:1.

25. A unit dosage form for sterile, subcutaneous administration to a patient in need of anticoagulation, containing about 1 to about 30 ml of a composition comprising ODSH and LMWH in an ODSH:LMWH weight ratio of about 1 :1 to about 4: 1.

26. The unit dosage form of claim 25, comprising about 1,000 IU to about 95,000 IU LMWH.

27. The unit dosage form of claim 25 comprising about 10 mg to about 2000 mg

ODSH.

28. The unit dosage form according to any one of claims 25 to 27, wherein the ODSH:LMWH weight ratio ranges from about 1 :1 to about 2:1, about 2:1 to about 3:1, or about 3: 1 to about 4: 1.

29. A method of anticoagulating a subject comprising administering to a subject in need of anticoagulation an amount of a pharmaceutical composition according to any one of claims 1 to 19 which is effective to anti-coagulate.

30. The method of claim 29, wherein the pharmaceutical composition is administered intravenously.

31. The method of claim 30, wherein the pharmaceutical composition is administered as a bolus.

32. The method of claim 30, wherein the pharmaceutical composition is administered as an infusion.

33. The method of claim 29, wherein the pharmaceutical composition is administered subcutaneously.

34. A method of anticoagulating a subject comprising concurrently administering ODSH and UFH in an ODSH:UFH weight ratio of about 1 : 1 to about 1 :4 and a UFH amount effective to anticoagulate, to a subject in need of anticoagulation.

35. The method of claim 34, wherein the ODSH and UFH are each administered intravenously.

36. The method of claim 35, wherein the pharmaceutical composition is administered as a bolus.

37. The method of claim 35, wherein the pharmaceutical composition is administered as an infusion.

38. A method of anticoagulating a subject comprising concurrently administering ODSH and LMWH in an ODSH:LMWH weight ratio of about 1 :1 to about 1:4 and an LMWH amount effective to anticoagulate, to a subject in need of anticoagulation.

39. The method of claim 38, wherein LMWH is administered subcutaneously.

40. The method of claim 38, wherein the ODSH and LMWH are each

administered subcutaneously.

41. The method of any one of claims 29 to 40, wherein the subject is at risk for, or suffering from, venous thrombosis (including deep vein thrombosis), arterial thrombosis (including stroke, myocardial infarction or "heart attack", and acute leg ischemia), peripheral arterial embolism, clotting in arterial and cardiac surgery, pulmonary embolism, acute or chronic consumptive coagulopathies, or atrial fibrillation with embolization.

42. The method of any one of claims 29 to 40, wherein the subject is undergoing abdominothoracic surgery or joint replacement surgery, has severely restricted mobility during acute illness, or is at risk for HIT.

43. The method of claim 29, wherein the pharmaceutical composition is packaged in a unit dosage form according to any one of claims 20 to 28.