NZ618786B2 - Resorption enhancers as additives to improve the oral formulation of non-anticoagulant sulfated polysaccharides - Google Patents

Abstract

The disclosure relates to a method of enhancing blood coagulation in a subject, comprising orally administering to the subject a procoagulant amount of a non-anticoagulant sulphated polysaccharide (NASP) in combination with a gastrointestinal epithelial barrier permeation enhancer comprising chitosan and bromelain in a manner sufficient to enhance blood coagulation in the subject. The NASP may be a naturally occurring NASP selected from the group consisting of N-acetyl-heparin (NAH), N-acetyl-de-O-sulfated-heparin (NA-de-o-SH), de-N-sulfated-heparin (De-NSH), de-N-sulfated-acetylated-he-parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS) or fucoidans. The NASP may also be a synthetic NASP selected from the group consisting of sulfated oligomers, sulfated pentoses, sulfated hexoses or sulfated cyclodextrins. The gastrointestinal epithelial barrier permeation enhancer may be a tight junction modulator selected from the group consisting of proteases, bile acids, polysaccharides, fatty acids and salts thereof. The disclosure also relates to a composition comprising the NASP and gastrointestinal epithelial barrier permeation enhancer. n and bromelain in a manner sufficient to enhance blood coagulation in the subject. The NASP may be a naturally occurring NASP selected from the group consisting of N-acetyl-heparin (NAH), N-acetyl-de-O-sulfated-heparin (NA-de-o-SH), de-N-sulfated-heparin (De-NSH), de-N-sulfated-acetylated-he-parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS) or fucoidans. The NASP may also be a synthetic NASP selected from the group consisting of sulfated oligomers, sulfated pentoses, sulfated hexoses or sulfated cyclodextrins. The gastrointestinal epithelial barrier permeation enhancer may be a tight junction modulator selected from the group consisting of proteases, bile acids, polysaccharides, fatty acids and salts thereof. The disclosure also relates to a composition comprising the NASP and gastrointestinal epithelial barrier permeation enhancer.

Description

PCT/U52012/047248 RESORPTION ENHANCERS AS ADDITIVES TO IMPROVE THE ORAL FORMULATION OF NON-ANTICOAGULANT SULFATED POLYSACCHARIDES CROSS-REFERENCE To RELATED APPLICATION Pursuant to 35 U .S.C. §119(e), this application claims priority to United States Provisional Application Serial No. 61/509,514 filed on July 19, 2011, the disclosure of which is herein incorporated by reference.

UCTION Bleeding is one of the most serious and significant. manifestations of disease, and may occur from a local site or be systemic. Localized bleeding may be associated with lesions and may be further complicated by a defective haemostatic mechanism. Blood clotting is inadequate in bleeding disorders, which may be caused by ital coagulation disorders, ed coagulation disorders, or hemorrhagic conditions induced by trauma. Congenital or acquired deficiencies of any of the coagulation factors may be associated with a hemorrhagic tendency.

Some congenital coagulation disorders include hemophilia, a recessive X-linked disorder involving a ncy of coagulation factor VIII (hemophilia A) or factor IX (hemophilia B) and von Willebrands disease, a rare bleeding disorder involving a severe ency of von Willebrands factor. ed coagulation disorders may arise in individuals without a previous history of bleeding as a result of a disease process. For e, acquired coagulation ers may be caused by inhibitors or autoimmunity against blood coagulation factors, such as factor VIII, von Willebrand factor, factors IX, V, XI, XII and XIII; or by hemostatic ers such as caused by liver disease, which may be associated with decreased synthesis of coagulation factors.

SUMMARY s of the invention include methods for enhancing blood coagulation in a subject.

In practicing methods according to certain embodiments, an amount of a non-anticoagulant sulfated polysaccharide (NASP) in combination with a gastrointestinal epithelial r permeation enhancer is orally administered to a subject in a manner sufficient to e blood coagulation in the subject. Compositions and kits for practicing methods of the invention are also described.

In embodiments of the invention, an amount of a NASP in ation with a intestinal epithelial r permeation enhancer is orally administered to a subject in a manner sufficient to e blood coagulation. In certain ments, the gastrointestinal epithelial barrier permeation enhancer is a tight junction modulator. For example, tight junction modulators provided by the invention may include, but are not limited to proteases, bile acids, WO 12954 polysaccharides, fatty acids and salts f and any combination thereof. For example, the tight junction modulator may be a bile acid, such as for instance deoxycholate. In other instances, the tight junction modulator may be a protease, such as bromelain or an tic ent of bromelain. In yet other instances, the tight junction modulator may be a polysaccharide, such as chitosan. In still other instances, the tight junction modulator may be a fatty acid or a fatty acid salt, such as sodium e. In certain embodiments, gastrointestinal epithelial barrier permeation enhancers of the invention are a combination of tight on modulators. For example, in certain instances, a combination of bromelain and chitosan are orally administered with a NASP to a subject in a manner sufficient to enhance blood coagulation in the subject.

In certain embodiments, the present invention provides a method for ing blood coagulation by orally administering a composition having an amount of a NASP in ation with a gastrointestinal epithelial barrier tion enhancer to a subject, where the NASP is a natural NASP. In some instances, the l NASP is N-acetyl-heparin (NAH), N-acetyl-de—O- sulfated’heparin (NA-de-o—SH), de-N-sulfated-heparin (De-NSH), de-N—sulfated-acetylated—he- parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived oiigosaccharides (HDO), or pentosan polysulfate (PPS). In certain instances, the natural NASP is a fucoidan. For example, in these embodiments, the an may be Fucoidan GFS 5508005, Undaria pinnatifida, depyrogenated; Fucoidan GFS 5508004, Undaria pinnatifida; Fucoidan GFS 3, Undaria pinnatz‘fida; Fucoidan 5307002, Friars vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucus vesiculosus, hydrolyzed sample of 2 of lower MW, max. MW peak 22.5 kD; Fucoidan 5308004, Fucus vesiculoxus; Fucoidan 5308005, Fucus vesiculasus; Fucoidan L/FVF1091, Fucus vesiculosus; Fucoidan 96A, Fucus vesiculosus; Fucoidan VGZOIO96B, Fucus vesiculosus; Fucoidan VG57, Undaria pinnatifida, high charge (high sulphation, deacetylated); Fucoidan VGSO, Ascophyllum nodosum, max. MW peak 149.7 kD; and combinations thereof.

In other embodiments, the present invention provides a method for ing blood coagulation by orally administering a composition having an amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer to a subject, where the NASP is a synthetic NASP. For example, the synthetic NASP may be a sulfated er, such as a sulfated oligosaccharide or a sulfated aliphatic. In certain instances, synthetic NASPs are sulfated pentoses, sulfated hexoses or sulfated cyclodextrins. For e, synthetic NASPs may include, but are not limited to sulfated maltopentoses, sulfated beta-cyclodexlrins, sulfated 6-Carboxyicodextrin and derivatives thereof.

In certain embodiments, methods of invention further include orally administering a blood coagulation factor to the subject in conjunction with the composition containing a PCTIUS20121047248 procoagulant amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer. In these instances, the blood coagulation factor may include but are not limited to factor Xa, factor IXa, factor XIa, factor XIIa, VIIIa, lekrein, and high-molecular weight kininogen, tissue factor, factor VIIa, factor Va, factor Xa, factor II, factor V, factor VII, U! factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, von rands factor, and combinations thereof. For example, in some embodiments, methods of the ion include orally stering to a subject an amount of a NASP in combination with a gastrointestinal epithelial r permeation enhancer and factor VIII. In another embodiment, methods include orally administering to a subject an amount of a NASP in combination with a gastrointestinal epithelial r permeation enhancer and factor IX.

In other embodiments, the present invention provides a method of inhibiting TFPI activity in a subject. For example, methods may further include inhibiting TFPI activity in a subject by orally administering an amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer in a manner sufficient to inhibit TFPI ty in the subject. In n instances, a procoagulant amount of a NASP and a gastrointenstinal epithelial barrier permeation enhancer are combined with a biological sample (e.g., plasma) that includes TFPI and ing the TFPI activity of the biological sample. In other instances, methods include combining a procoagulant amount of a NASP and a gastrointestinal epithelial barrier permeation enhancer with a biological sample, adding TFPI to the composition and measuring 2O TFPI acitivty of the biological .

In certain embodiments, compositions of interest demonstrated ed permeation, for example, when determined by resorption studies in CaCo-Z cell models as compared to compositions in the absence of gastrointestinal epithelial permeation enhancers.

ASPECTS OF THE INVENTION 1. A method of enhancing blood coagulation in asubject, the method comprising: orally administering to the t a procoagulant amount of a non-anticoagulant sulfated polysaccharide (NASP) in combination with a gastrointestinal epithelial barrier permeation enhancer in a manner sufficient to enhance blood coagulation in the subject. 3O 2. The method according to Claim 1, wherein the amount of NASP administered to the subject ranges from 0.01 mg/kg to about 100 trig/kg. 3. The method according to Claim 1, wherein the NASP is a lly occurring or synthetic NASP. 4. The method according to Claim 3, wherein the NASP is a naturally occurring NASP. 5. The method according to Claim 4, n the naturally occurring NASP is ed from the group consisting of N-acetyl-heparin (NAH), N—acetyl-de-O-sulfated-heparin (NA-de-o— PCT/U52012/047248 SH), de-N-sulfated-heparin H), ulfated-acetylated-he- parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran e (DXS), heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS) and fucoidans, and ations thereof. 6. The method according to Claim 5, wherein the naturally occurring NASP is a fucoidan. 7. The method according to Claim 3, wherein the NASP is a synthetic NASP. 8. The method according to Claim 7, wherein the synthetic NASP is a sulfated oligomer. 9. The method according to Claim 1, wherein the gastrointestinal epithelial barrier permeation enhancer is a tight junction modulator. 10. The method according to Claim 9, n the tight junction modulator is selected from the group consisting of proteases, bile acids, polysaccharides, fatty acids and salts f, and combinations thereof. 11. The method according to Claim 10, wherein the tight junction moldulator ses a protease. 12. The method according to Claim 11, n the tight junction modulator is bromelain or an tic component thereof. 13. The method according to Claim 10, wherein the tight on modulator is a bile acid. 14. The method according to Claim 13, wherein the bile acid is deoxycholate.

. The method according to Claim 10, wherein the tight junction modulator is a polysaccharide. 16. The method according to Claim 15, wherein the polysaccharide is chitosan. 17. The method according to Claim 10, wherein the tight junction modulator is a fatty acid or salt thereof. 18. The method according to Claim 17, n the tight junction modulator is sodium caprate. 19. The method according to Claim 1, wherein the gastrointestinal epithelial barrier permeation enhancer comprises a chitosan and a bromelain.

. The method according to Claim 19, wherein the gastrointestinal epithelial barrier permeation enhancer comprises about 0.3% to about 3% chitosan and about 0.05 mg/mL to about 3O 0.5 mg/mL bromelain. 21. The method according to Claim 20, wherein the gastrointestinal epithelial barrier permeation enhancer ses about 3% chitosan and about 0.5 mg/mL bromelain. 22. The method according to Claim 1, wherein the method further comprising administering to the subject one or more factors selected from the group consisting of factor XI, factor XII, likrein, high molecular weight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factor XIII, factor 11, von Willebrands factor, tissue factor, factor VIIa, factor Va, and factor Xa, factor IXa, factor XIa, factor XIIa, and VIIla.

PCTIUS2012/047248 23. The method according to Claim 1, n the subject has a bleeding er selected from the group consisting of a chronic or acute bleeding disorder, a congenital coagulation disorder caused by a blood factor deficiency, and an acquired coagulation disorder. 24. The method Claim 23, wherein the bleeding er is a blood factor deficiency of one or more factors selected from the group consisting of factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, and von Willebrand factor; a fibrinogen disorder; a ombin disorder; or a platelet dysfunction.

. The method according to Claim 1, wherein the subject is in need of enhanced blood coagulation e of prior administration of an anticoagulant. 26. The method according to Claim 25, wherein the anticoagulant is selected from the group consisting of heparin, a coumarin derivative, tissue factor pathway inhibitor (TFPI), antithrombin III, lupus anticoagulant, nematode anticoagulant peptide (NAPCZ), active-site blocked factor VIIa (factor VHai), factor IXa inhibitors, factor Xa tor, factor Va inhibitor, factor VIIIa inhibitor, thrombin inhibitor, and an antibody that binds a clotting factor. 27. The method according to Claim 26, wherein the anticoagulant is an antibody that binds a clotting factor selected from the group consisting of Factor V, Factor VII, Factor VIH, Factor 1X, Factor X, Factor XIII, Factor II, Factor XI, Factor XH, von rands factor, prekallikrein, and high molecular weight kininogen . 28. The method according to Claim 1, wherein the subject is in need of enhanced blood coagulation because of a al or other invasive procedure. 29. The method according to Claim 1, wherein the method is a method of inhibiting TFPI activity in the subject.

. An oral dosage composition comprising: (a) a pro-coagulant amount, of a non—anticoagulant ed polysaccharide (NASP); (b) a gastrointestinal epithelial barrier permeation enhancer; and (c) an oral dosage delivery vehicle; wherein the oral dosage composition is in unit dosage form. 3]. The oral dosage composition ing to Claim 30, wherein the amount of NASP 3O present in the composition provides a dose in the range of 0.01 mg/kg to about 100 mg/kg. 32. The oral dosage composition according to Claim 30, wherein the NASP is a naturally ing or synthetic NASP. 33. The oral dosage composition according to Claim 32, wherein the NASP is a naturally occurring NASP. 34. The oral dosage composition according to Claim 33, wherein the naturally occurring NASP is selected from the group consisting of N—acetyl-heparin (NAH), N-acetyl-de-O-sulfated- n -o-SH), de-N-sulfated-heparin (De-NSH), de-N-sulfated-acetylated-he- parin PCT/US20121’047248 (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically ed alginic acid , chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin- derived oligosaccharides (HDO), pentosan polysulfate (PPS) and fucoidans, and combinations thereof.

. The oral dosage composition according to Claim 34, wherein the naturally occurring NASP is a fucoidan. 36. The oral dosage composition according to Claim 32, wherein the NASP is a tic NASP. 37. The oral dosage composition according to Claim 36, wherein the tic NASP is a sulfated oligomer. 38. The oral dosage composition according to Claim 30, wherein the gastrointestinal epithelial barrier permeation enhancer is a tight junction modulator. 39. The oral dosage composition according to Claim 38, wherein the tight junction tor is selected from the group consisting of ses, bile acids, polysaccharides, fatty acids and salts f, and combinations thereof. 40. The oral dosage composition according to Claim 39, wherein the tight junction moldulator comprises a protease. 41. The oral dosage composition according to Claim 40, wherein the tight on modulator is bromelain or an enzymatic component f. 42. The oral dosage composition according to Claim 39, n the tight junction modulator is a bile acid. 43. The oral dosage composition according to Claim 42, wherein the bile acid is holate. 44. The oral dosage composition according to Claim 39, wherein the tight junction modulator is a polysaccharide. 45. The oral dosage composition according to Claim 44, wherein the polysaccharide is chitosan. 46. The oral dosage composition according to Claim 39, wherein the tightjunction tor is a fatty acid or salt thereof. 47. The oral dosage composition according to Claim 46, wherein the tight junction modulator is sodium e. 48. The oral dosage composition according to Claim 30, wherein the gastrointestinal epithelial barrier permeation enhancer comprises a chitosan and a bromelain. 49. The oral dosage composition according to Claim 48, wherein the gastrointestinal epithelial barrier permeation enhancer comprises about 0.3% to about 3% chitosan and about 0.05 mg/mL to about 0.5 mg/mL bromelain. 50. The oral dosage composition, according to Claim 49, wherein the gastrointestinal epithelial barrier permeation enhancer ses about 3% chitosan and about 0.5 mg/mL bromelain. 51. The oral dosage composition according to Claim 30, r comprising one or more factors selected from the group consisting of factor XI, factor XII, prekallikrein, high molecular weight kininogen , factor V, factor VII, factor VIII, factor IX, factor X, factor XIII, factor 11, von Willebrands factor, tissue factor, factor Vila, factor Va, and factor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa. 52. The oral dosage composition according to Claim 30, wherein the composition is a liquid. 53. The oral dosage composition according to Claim 30, wherein the composition is a solid. 54. Use of a procoagulant amount of a non-anticoagulant sulfated polysaccharide (NASP) in combination with a gastrointestinal epithelial r permeation enhancer for the manufacture of a medicament for enhancing blood coagulation in a subject.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the experimental setup for CaC02 bioavailability screening to determine the % resorption of fucoidans.

Figure 2 shows an example of the amount ofNASP in ation with a gastrointestinal epithelial barrier permeation enhancer resorbed in CaC02 bioavailability ing for fucoidan Fucus vesiculosus L/FVF—109l.

Figure 3 shows the condition of the cell layer in the CaCo~2 bioavailability screening for fucoidan Fucus vesiculosus L/FVFle9l in ation with the gastrointestinal epithelial barrier permeation er bromelain as measured by transepithelial electrical resistance. s 4a-b show an example of resorption data acquired in the CaCo~2 bioavailability screening for fucoidan BAXS 13 in combination with gastrointestinal epithelial barrier permeation enhancers.

Figures 5a-c show an example of resorption data acquired in the CaCo-Z bioavailability screening for fucoidan F.v. L/FVF-1091 in combination with intestinal epithelial barrier permeation enhancers.

Figures 6a-b show an e of resorption data acquired in the CaCo~2 bioavailability screening for fucoidan U.p. 5508005 in combination with gastrointestinal epithelial barrier tion enhancers.

Figures 7a-b show an example of resorption data acquired in the CaCo-2 bioavailability screening for fucoidan F .v.F DSlOOl 108B in ation with gastrointestinal epithelial barrier permeation ers.

Figures 8a—b show an example of resorption data acquired in the CaCo-2 bioavailability screening for fucoidan FVF SKl lOl44B in combination with gastrointestinal epithelial barrier permeation enhancers. -7A- PCTfUS2012/047248 Figure 9 show an example of resorption data acquired in the CaCo-Z bioavailability screening for synthetic NASP sulfated B-cyclodextrin in combination with gastrointestinal epithelial barrier tion enhancers.

Figure 10 show an example of resorption data acquired in the CaCo—Z bioavailability screening for synthetic NASP sulfated maltopentaose in combination with gastrointestinal epithelial barrier tion enhancers.

Figures llaub show show an example of resorption data acquired in the CaCo-Z bioavailability screening for fucoidans F.v. L/FVF—1091 and F.v.F DSlOOl 108B in combination with gastrointestinal lial barrier permeation enhancers at g concentrations.

Figures 12a—b shows the condition of the cell layer in the CaCo-2 bioavailability screening for synthetic NASP sulfated B-cyclodextrin in the absence and in combination with the gastrointestinal epithelial barrier tion enhancer bromelain as measured by transepithelial electrical resistance.

Figures l3a—c shows the condition of the cell layer in the CaCo-2 bioavailability screening for synthetic NASP sulfated maltopentaose in the absence and in combination with the gastrointestinal epithelial barrier permeation enhancers deoxycholate and chitosan as measured by transepithelial electrical resistance.

Figures 14a-b show an example of tion data acquired in the CaCo-Z bioavailability screening for fucoidan B-cyclodextrin in combination with gastrointestinal epithelial barrier permeation ers.

Figures lSa-b show an example of tion data acquired in the CaCo~2 bioavailability screening for fucoidan sulfated maltopentaose in combination with gastrointestinal epithelial barrier permeation enhancers.

RELEVANT DEFINITIONS In describing the present invention, the ing terms will be employed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appended , the ar forms ' a , an” and “the” include plural referents unless the content clearly dictates otherwise. 3O Thus, for example, reference to a “ NASP” may e a mixture of two or more NASPs, as desired An “NASP” as used herein refers to sulfated polysaccharides (SP) that exhibit non- anticoagulant and anticoagulant activity in any of the various clotting assays described herein.

NASPs may be natural sulfated ccharides, such as those extracted from a biological source or tic sulfated polysaccharides, where the sulfated polysaccharide is partially or wholly ed by synthetic methods (e.g., chemical synthesis). One measure of activity is to compare PCT/U820121047248 the clotting time demonstrated by a NASP with the anticoagulant activity displayed by heparin.

For example, NASPs of interest exhibit anticoagulant activity in the dilute prothrombin time (dPT) or activated partial mromboplastin time (aPTT) clotting assay that is no more than one- third, such as less than one-tenth, the molar anticoagulant activity of unfractionated heparin (MW range 8,000 to 30,000; mean 18,000 daltons). As such, NASPs of st demonstrate a 2-fold or more lower anticoagulant activity as compared to heparin, such as a 5-fold or more lower anticoagulant ty as compared to heparin, such as a 10-fold or more lower agulant activate as compared to heparin, such as a 25—fold or more lower anticoagulant activity as compared to heparin, such as a 50-fold or more lower anticoagulant activity as compared to heparin, including a lOO-fold or more lower anticoagulant activity as compared to heparin, by ing methods and compositions as ed herein.

NASPs of interest may range in molecular weight from 10 daltons to 1,000,000 daltons, such as for example, from 100 daltons to 900,000 daltons, such as from 500 daltons to 500,000 s, such as from 1000 s to 250,000 daltons, including 5000 daltons to 150,000 daltons. NASPs may range in average molecular weight from about 10 daltons to about 500,000 daltons, such as from about 100 daltons to about 0 daltons, such as from 1000 daltons to 250,000 daltons, including 1000 daltons to 150,000 daltons.

In some instances, NASPs of st may include, but are not limited to yl- heparin (NAH), N-acetyl-de-O-sulfated-heparin -o-SH), de-N-sulfated-heparin (De- 2O NSH), de-N-sulfated-acetylated-he- parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived accharides (HDO), pentosan polysulfate (PPS), sulfated maltopentoses, sulfated beta-cyclodextrins, sulfated 6- Carboxyicodextrin and derivatives thereof. In certain instances, the NASP is a fucoidan. For example, the an may be Fucoidan 5307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucus losus, yzed sample of 5307002 of lower MW, max. MW peak 22.5 kD; Fucoidan V657, Undaria pinnan‘fida, high charge (high sulphation, deacetylated); Fucoidan GFS (5508005), Undaria pinnatifida, genated; Fucoidan GFS (L/FVF—0109l), Fucus vesiculosus, depyrogenated, max. MW peak 125 kD; Fucoidan GFS (L/FVF-01092), 3O Fucus vesiculosus, depyrogenated, max. MW peak 260 kD; Fucoidan GFS (L/FVF-01093), Fucus vesiculosus, hydrolyzed depyrogenated, max. MW peak 36 kD; Maritech® Ecklonia radiala t; Maritech® Ecklorzz’a maxima extract; Maritech® Macrocystis pyrifera extract; Maritech® Immune trial Fucoidan Blend; and combinations thereof.

NASPs in combination with a gastrointestinal epithelial barrier permeation enhancer may be used in the methods of the invention for improving hemostasis, in treating bleeding disorders, such as those associated with deficiencies of coagulation s or for reversing the effects of anticoagulants, in particular when enhanced resorption by the gastrointestinal system is necessary PCT/U52012/047248 or desired. The ability of NASPs to promote clotting and reduce ng may be ined using various in vitro clotting assays (e.g., TFPI-dPT, thrombin generation and thromboelastography (TEG) assays) and in vivo bleeding models (ag. tail snip, transverse cut, whole blood clotting time, or cuticle bleeding time determination in hemophilic mice or dogs).

U! See, e.g., PDR Staff. Physicians' Desk Reference. 2004, Anderson et a1. (1976) Thromb. Res. 9:575-580; Nordfang et al. (1991) Thromb Haemost. 66:464-467; Welsch et a1. (1991) Thrombosis Research -222; Broze et al. (2001) Thromb Haemost 851747-748; Scallan et a1. (2003) Blood. 102:2031-2037; Pijnappels et al. (1986) Thromb. Haemost. 55:70-73; and Giles et al.(1982) Blood 601727-730, and the examples herein.

A “procoagulant” is used herein in its conventional sense to refer to any factor or reagent capable of initiating or accelerating clot formation. A procoagulant of the invention includes but is not limited to any activator of the sic or extrinsic coagulation pathways, such as a clotting factor selected from the group consisting of factor Xa, factor IXa, factor Xla, factor X1121, and VIlIa, prekallelqein, high-molecular weight gen, tissue factor, factor Vita, and factor Va, as well as other reagents that promote clotting e kallikrein, APTT initiator (i. e., a t containing a phospholipid and a contact activator), Russel‘s viper venom (RVV time), and thromboplastin (for dPl‘). In some embodiments, t activators may be employed as procoagulant reagents. For example, t tors may include micronized silica particles, ellagic acid, sulfatides, kaolin or the like. Procoagulants may be from a crude natural t, a blood or plasma sample, isolated and substantially purified, synthetic, or recombinant.

Procoagulants may include naturally occurring clotting factors or fragments, variants or covalently modified derivatives thereof that retain biological activity (116., promote clotting).

The term “polysaccharide,” as used herein, refers to a polymer containing two or more covalently linked saccharide es. Saccharide residues may be linked for e by glycosidic, ester, amide, or oxime linking moieties. The average molecular weight of polysaccharides may vary widely, such as for example ranging from 100 to 1,000,000 daltons and more, such as 100 to 500,000 daltons and more, such as 1000 to 250,000 s and more, such as 1000 to 100,000 s and more, such as 10,000 to 50,000 daltons and more.

Polysaccharides may be straight chained (i.e., linear) or branched or may contain discrete regions 3O of linear and branched portions. Polysaccharides may also be fragments of polysaccharides generated by degradation (e.g., hydrolysis) of larger polysaccharides. Degradation can be achieved by any convenient protocol including treatment of polysaccharides with acid, base, heat, oxidants or enzymes to yield fragmented polysaccharides. Polysaccharides may be chemically altered and may be modified, including but not limited to, sulfation, polysulfation, esterification, and methylation. lar weight, as discussed herein, can be sed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to lar weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using for e, gel tion chromatography or other liquid chromatography techniques.

The term “derived from” is used herein to fy the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant methodologies.

The terms “variant,39 nanalog” and “mutein” refer to biologically active derivatives of a reference le, that retain desired activity, such as clotting activity in the treatment of a bleeding disorder. The terms “variant” and “analog” in reference to a polypeptide (e.g., clotting ) refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or ons, ve to the native molecule, so long as the modifications do not destroy biological activity and which are “substantially homologous” to the reference molecule as defined below. The amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of 50% or more, such as 60% or more, such as 70% or more, such as 80% or more, such as 90% or more, such as 95% or more, including 99% or more when the two sequences are aligned. In some instances, analogs will include the same number of amino acids but will include substitutions. The term “mutein” further es ptides having one or more amino acid-like molecules including but not limited to compounds contain only amino and/or imino molecules, polypeptides containing one or more s of an amino acid (including, for example, synthetic turally occuring amino acids, etc.), ptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non—naturally ing (e. g., tic), cyclized, branched molecules and the like. The term also includes molecules comprising one or more N-substituted glycine residues (a “peptoid”) and other tic amino acids or peptides. (See, e.g., US. Patent Nos. ,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chem Biol. (2000) 7:463—473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367—9371 for descriptions of peptoids). In embodiments of the invetion, analogs and muteins have at least the same clotting activity as the native molecule. 3O As discussed above, analogs may include substitutions that are conservative, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. cally, amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine, glutamine, ne, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are in some instances classified as aromatic amino acids. For example, an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a PCT/U82012/047248 , or a r conservative ement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non—conservative amino acid substitutions, or any U! integer between 5-25, so long as the desired function of the molecule remains .

By “derivative” is meant any suitable modification of the reference molecule of interest or of an analog thereof, such as sulfation, acetylation, glycosylation, phosphorylation, polymer ation (such as with polyethylene glycol), or other addition of n moieties, so long as the desired biological activity (6.3., clotting activity, inhibition of TFPI activity) of the reference molecule is retained. For example, polysaccharides may be derivatized with one or more organic or inorganic groups. Examples include but are not d to polysaccharides substituted in at least one hydroxyl group with another moiety (e. g., a sulfate, carboxyl, phosphate, amino, nitrile, halo, silyl, amido, acyl, aliphatic, aromatic, or a saccharide , or where a ring oxygen has been replaced by , nitrogen, a methylene group, etc. Polysaccharides may be chemically altered, for example, to improve procoagulant function. Such modifications may include, but are not limited to, sulfation, polysulfation, esterification, and methylation.

By “fragment” is meant a molecule containing a part of the intact full-length sequence and ure. In some instances, a fragment of a polysaccharide may be generated by degradation (e.g., hydrolysis) of a larger polysaccharide. Active fragments of a polysaccharides of the invention may include about 2-20 saccharide units of the full-length polysaccharide, such as about 5-10 saccharide units of the full—length molecule, and including any integer between 2 saccharide units and the full-length molecule, so long as the fragment retains biological activity, such as for example, ng activity or the ability to inhibit TFPI activity. A fragment of a polypeptide can include a C-terminal deletion, an N-terminal deletion, or an internal deletion of the native polypeptide. Active fragments of a particular protein may include, in some embodiments, about 5—10 contiguous amino acid es of the full-length molecule or more, such as about 1525 contiguous amino acid residues of'the full-length molecule or more, such as about 20-50 contiguous amino acid residues of the full—length molecule or more, and including any r between 5 amino acids and the full—length sequence, so long as the fragment in question retains biological activity, such as for example, clotting activity.

By “substantially purified” is meant the isolation of a substance (e.g., non-anticoagulant sulfated polysaccharide) such that the substance includes the ty of the sample in which it s. For example, a sample that is ntially purified contains 50% or more of the substance of interest, such as 60% or more of the substance of interest, such as 75% or more of 3S the substance of interest, such as 90% or more of the substance of interest, such as 95% or more of the substance of interest, including 99% or more of the substance of interest. Any convenient protocol may be ed for ing ccharides, polynucleotides, and polypeptides of PCT/U820121047248 interest and include, but are not limited to ultrafiltration, selective precipitation, llization, ion-exchange chromatography, affinity chromatography and ntation according to density.

By “isolated” is meant, when, referring to a ccharide or polypeptide, that the indicated molecule is separate and discrete from the whole sm with which the molecule is found in nature or is present in the substantial e of other biological macro-molecules of the same type.

By ogy” is meant the percent identity between two polypeptide moieties. As referred to herein, two polypeptide sequences are antially homologous” to each other when the sequences exhibit about 50% or more sequence identity, such as 60% or more sequence identity, such as 75% or more sequence identity, such as 85% or more sequence identity, such as 90% or more sequence identity, such as 95% or more sequence identity, including 99% or more sequence identity. In some embodiments, substantially homologous polypeptides include sequences having complete identity to a specified sequence.

By ity” is meant an exact t to subunit correspondence of two polymeric sequences. For example, an identical polypeptide is one that has an exact amino acid-to-amino acid pondence to another ptide or an identical polynucleotide is one that has an exact nucleotide-to-nucleotide correspondence to another polynucleotide. Percent identity can be determined by a direct ison of the sequence information between two molecules (the 2O reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the ces, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100.

Any convenient protocol may be employed to determine percent identity between two polymeric sequences, such as for example, ALIGN, Dayhoff, MO. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 35353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 22482—489, 1981 for e analysis.

By “subject” is meant any member of the subphylum chordata, including, without limitation, humans and other primates, including non—human es such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age.

Thus, both adult and newborn individuals are of st.

The term "patient," is used in its conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a NASP 0f the invention, and includes both humans and non-human animals.

By “biological sample” is meant a sample of tissue or fluid ed from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, inal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.

By “therapeutically effective dose or amount” is meant an amount that, when administered as described herein, brings about the d therapeutic response, such as for example, reduced bleeding or shorter clotting times.

By “bleeding disorder” is meant any disorder associated with excessive bleeding, such as a congenital coagulation er, an acquired coagulation disorder, administration of an anticoagulant, or a trauma induced hemorrhagic ion. As discussed below, bleeding disorders may include, but are not limited to, hemophilia A, hemophilia B, von Willebrand disease, thic ocytopenia, a deficiency of one or more t factors, such as Factor XI, Factor XII, prekallikrein, and high lar weight kininogen (HMWK), a deficiency of one or more factors ated with clinically significant bleeding, such as Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia), and von rands factor, a vitamin K deficiency, a disorder of fibrinogen, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia, an alphaZ-antiplasmin deficiency, and excessive bleeding such as caused by liver disease, renal disease, thrombocytopenia, et dysfunction, hematomas, internal hemorrhage, hemarthroses, surgery, trauma, hypothermia, menstruation, and pregnancy.

DETAILED DESCRIPTION Aspects of the invention include methods for enhancing blood coagulation in a subject.

In cing methods according to n embodiments, an amount of a non—anticoagulant sulfated polysaccharide (NASP) in combination with a‘ gastrointestinal epithelial barrier permeation enhancer is orally administered to a subject in a manner sufficient to enhance blood coagulation in the subject. Compositions and kits for practicing methods of the invention are also described.

Before the invention is described in r detail, it is to be understood that the invention is not d to particular embodiments described herein as such embodiments may vary. It is also to be understood that the terminology used herein is for the purpose of describing U.) U1 particular ments only, and the terminology is not intended to be limiting. The scope of the invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ry skill in the art to which this ion belongs. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, n the upper and lower limit of that range and any other stated or ening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Certain ranges are presented herein with numerical values being preceded by the term " about. " The term "about“ is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number, which, in the context in which it is presented, provides the substantial equivalent of the specifically d . All publications, patents, and patent applications cited in this specification are incorporated herein by reference to the same extent as if each dual publication, , or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated herein by reference to disclose and be the subject matter in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the invention described herein is not entitled to antedate such publication by virtue of prior ion. Further, the dates of publication provided might be different from the actual publication dates, which may need to be independently med.

It is noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely, n «only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation. As will be nt to those of skill in the art upon reading this disclosure, each of the individual embodiments described and rated herein has discrete components and features which may be readily separated from or combined with the features of any of the other l embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the invention, representative illustrative methods and materials are now described.

In r describing the subject invention, s for ing blood coagulation in a subject by orally administering a NASP in combination with a gastrointestinal epithelial barrier tion er are described first in greater detail. Next, compositions and kits for practicing methods of the t invention are also bed.

METHODS FOR ENHANCING BLOOD COAGULATION IN A SUBJECT As summarized above, aspects of the invention include methods for enhancing blood coagulation by orally administering a composition having an amount of a NASP in ation with a gastrointestinal epithelial permeation enhancer to a subject. The term “enhancing blood coagulation” is used in its conventional sense to refer to accelerating the initiation (i.e., reducing the amount time for coagulation to begin) of blood coagulation as well as the overall rate of blood coagulation of the subject (i.e., ng the amount of time for blood coagulation to be complete). In some embodiments, methods of the invention accelerate the initiation of blood coagulation. For example, methods of the invention may reduce the amount of time required for the blood to begin coagulating by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 90% or more, such as 95% or more, as compared to a suitable control. In other embodiments, methods of the invention increase the rate of blood coagulation. For example, methods of the invention may increase the rate of blood coagulation by 2% or more, such as by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 100% or more, such as by 200% or more, including by 500% or more, as compared to a suitable control.

In embodiments of the present disclosure, a procoagulant amount of a NASP is orally administered in combination with a gastrointestinal epithelial barrier permeation enhancer. ing on the physiology of the subject, the phrase “gastrointestinal epithelial” as used , refers to the epithelial tissue of the digestive tract, such as the stomach and intestinal tract (e.g,, duodenum, jejunum, ileum), and may further include other structures which participate in the gastrointestinal functions of the body including the lower part of the esophagus, the rectum and the anus. By gastrointestinal permeation enhancer is meant a compound that, when orally administered, increases the amount of NASP that is resorbed by the gastrointestinal .

Furthermore, gastrointestinal permeation enhancers may also accelerate the initiation (i.e., reducing the amount time for resorption to begin) of NASP resorption through the 3O gastrointestinal epithelium as well as accelerate the l rate of ort of the NASP across the gastrointestinal epithelium of the t (i.e., reducing the amount of time for NASP resorption by the gastrointestinal system to be complete).

In some embodiments, gastrointestinal epithelial barrier permeation enhancers increase the amount of NASP resorbed by the intestinal system. For e, gastrointestinal epithelial barrier permeation enhancers may increase the amount of NASP resorbed by the gastrointestinal system by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or ~16- PCT/U82012/047248 more, such as by 75% or more, such as by 90% or more, such as 95% or more, as compared to a le control. In other embodiments, gastrointestinal epithelial barrier permeation ers accelerate the initiation of NASP resorption through the gastrointestinal epithelium. For example, gastrointenstinal lial barrier permeation ers of the invention may reduce the amount of time required to initiate resorption of the NASP by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 90% or more, such as 95% or more, as compared to a suitable control. In yet other embodiments, gastrointestinal epithelial r permeation enhancers of the invention increase the rate of resorption of the NASP by the intestinal system. For example, gastrointestinal epithelial barrier permeation enhancers may increase the rate of NASP resorption by 2% or more, such as by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 100% or more, such as by 200% or more, including by 500% or more, as compared to a suitable control. In some instances, gastrointestinal epithelial tion enhancers of the invention may increase the tion of NASPs as determined by Caco-2 cell models, as described in greater detail below. For example, gastrointestinal epithelial barrier permeation enhancers of the invention may increase the resorption as determined by Caco-Z cell models by 2% or more, such as by 5% or more, such as by 10% or more, such as by % or more, such as by 50% or more, such as by 75% or more, such as by 100% or more, such as by 200% or more, including by 500% or more, as compared to a suitable control.

In embodiments of the invention, gastrointestinal epithelial barrier permeation enhancers may vary, depending on the ular blood coagulation disorder, the physiology of the subject and the desired enhancement of resorption by the gastrointestinal . In some embodiments, gastrointestinal epithelial barrier tion enhancers are tight junction modulators. The term “tight junction” is employed in it conventional sense to refer to the closely associated cellular areas where membranes of adjacent cells are joined together. As such, in certain embodiments, methods of the invention include orally administering a composition having a procoagulant amount of a NASP in combination with a compound which modulates the permeation of the NASP through the tight junctions of the gastrointestinal epithelium. By “modulates” is meant modifying or increasing the permeation of the NASP through the tight junctions of the gasnointestinal epithelium. As such, tight 3O junction modulators modify or increase the resorption of NASPs by the gastrointestinal . In embodiments of the ion, tight on modulators may include, but are not limited to enzymes, bile acids, ccharides, fatty acids and salts thereof and any combination thereof.

In some instances, tight junction tors are polysaccharides. For example, the polysaccharide tight junction modulator may be chitosan. Chitosan, as used herein refers to the linear copolymer of 2-acetarnidedeoxy-l3-D-glucopyranose and o-[3-D~glucopyranose produced by the N-deactylation of chitin. Polysaccharide tight junction modulators may also include tives of chitosan such as N-alkyl chitosan, acylated chitosan, thiolated chitosan, PCT/U320121047248 phosphorylated chitosan, chitosan cyclodextrin, N-(aminoalkyl) chitosan, succinyl chitosan and octanoyl chitosan, among others.

In other instances, tightjunction modulators are bile acids. The term “bile acid” is used in its conventional sense to refer to the steroidal acids and salts thereof commonly found in the bile of ’Jl mammals. Suitable bile acids may include, but are not limited to, cholic acid (cholate), deoxycholic acid (deoxycholate), chenodeoxycholic acid (chenodeoxycholate), ursodeoxycholic acid (ursodeoxycholate), glycocholic acid (glycocholate), taurocholic acid (taurocholate) and holic acid cholate), among others.

In other instances, tight junction modulators are s. For example, the enzyme tight junction modulators may be a protease, such as bromelain or an enzymatic fragment of bromelain.

Bromelain, as used herein refers to the group of enzymes commonly derived from the fruit, stem and leaves of Ananas s and may also include elements such as cysteine proteases, amylase, acid phosphatase, peroxidases and cellulases.

In yet other instances, tight junction modulators are fatty acids and fatty acid salts f.

Fatty acid tight junction modulators of the invention may vary, and may include any one or a combination of medium chain fatty acids, such as for e C8 (caprylate), C10 (caprate) and C12 (laurate) fatty acids and fatty acid salts f. In certain instances, for example, the fatty acid tight junction modulator is sodium caprate.

The concentration of gastrointestinal epithelial barrier permeation enhancer that is stered in combination with the NASP may vary depending on the effects as desired.

Depending on the gastrointestinal epithelial barrier permeation enhancer, the concentration may be 0.01% or more of the total mass of the composition, such as 0.1% or more, such as 1% or more, such as 2% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more and including 50% or more of the total mass of the composition. In other embodiments, the concentration of the gastrointestinal epithelial barrier permeation er that is administered in combination with the NASP is 0.01 mg/mL or more, such as 0.05 mg/mL or more, such as 0.1 mg/mL or more, such as 1 mg/mL or more and including 5 mg/mL or more. In yet other ments, the concentration of the gastrointestinal epithelial barrier permeation enhancer that is administered in combination with the NASP is 0.1 mM or more, such as 0.5 mM or more, such as 1 mM or more, such as 5 mM or more, such as 10 mM or more, such as 25 mM or more and including 50 mM or moreln certain ments, two or more gastrointestinal epithelial barrier permeation enhancers are employed concurrently. For example, two or more tight junction modulators may be employed in combination with a NASP, such as three or more tight junction modulators, including four or more tight on modulators. Any combination of tight junction modulators may be employed, such as for example, a polysaccharide and a protease, a fatty acid and polysaccharide, a ccharide and a bile acid, a polysaccharide, a fatty acid and a bile acid, two different polysaccharides or two -18— PCT/U82012/047248 different bile acids, among other combinations. Where more than one gastrointestinal epithelial barrier permeation enhancer is employed, the mass tage of each gastrointestinal epithelial barrier permeation enhancer may vary, ranging from 1% or more of the total mass of the composition, such as 2% or more, such as 5% or more, such as 10% or more, such as 25% or U] more and including 50% or more of the total mass of the composition. For example, where two gastrointestinal lial barrier permeation enhancers are employed, the mass ratio of the first gastrointestinal epithelial barrier permeation er and the second intestinal epithelial barrier permeation enhancer may vary, g between 1:1 and 1:25; 1:25 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of the first gastrointestinal epithelial barrier permeation enhancer to the second gastrointestinal epithelial barrier permeation enhancer may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio of the second gastrointestinal epithelial barrier permeation er to the first gastrointestinal epithelial r permeation enhancer ranges between 1:1 and 1:25; 1:2.5 and and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 121000, or a range thereof. For example, the mass ratio of the second gastrointestinal epithelial barrier permeation enhancer to the first gastrointestinal epithelial barrier permeation enhancer may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In certain instances, the gastrointestinal epithetial barrier permeation enhancer includes a chitosan and a bromelain.

Where a chitosan and a bromelain are employed, the mass ratio of the chitosan and the bromelain may vary, ranging between 1:] and 122.5; 1:25 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200;1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For e, the mass ratio of the chitosan to the bromelain may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio of the ain to the chitosan ranges between 1:1 and 112.5; 1:25 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 3O 1:1000, or a range thereof. For example, the mass ratio of the bromelain to the chitosan range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000.

Where a combination of chitosan and ain are ed, in certain embodiments, the concentration of an may vary, ranging from about 0.1% to about 5%, such as about 0.15% to about 4.5%, such as 0.2% to about 4%, such as about 0.25% to about 3.5%, such as 0.3% to about 3%, such as 0.5% to about 2.5%, including about 0.5% to 1.5%. Likewise, where a combination of chitosan and bromelain are employed, in certain embodiments, the W0 2013/012954 concentration of bromelain may vary, ranging from about 0.01 mg/mL to about 1.0 mg/mL, such as about 0.2 mg/mL to about 0.9 mg/mL, such as 0.25 mg/mL to about 0.75 mg/mL, such as about 0.3 mg/mL to about 0.6 mg/mL, including about 0.4 mg/mL to about 0.5 mg/mL. As such, in these embodiments, methods include administering a NASP in combination with both chitosan and bromelain. For example, the NASP may be a natural or synthetic NASP, such as those bed above, including N-acetyl-heparin (NAH), N—acetyl-de-O-su1fated-heparin (NA-de—o— SH), de-N—sulfatedheparin (De—NSH), de-N-sulfated-acetylated~he— parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically ed pectin (CSP), dextran sulfate (DXS), heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS), ed maltopentoses, sulfated beta- cyclodextrins, sulfated 6-Carboxyicodextrin and tives thereof. For instance, the NASP may be a fucoidan, such as an 5307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucus vesiculosus, hydrolyzed sample of 5307002 of lower MW, max. MW peak 22.5 kD; Fucoidan VG57, Undaria pinnatifida, high charge (high sulphation, deacetylated); Fucoidan GFS (5508005), Undaria pinnatifida, depyrogenated; Fucoidan GFS (L/FVF~01091), Fucus vesiculosus, depyrogenated, max. MW peak 125 kD; Fucoidan GFS -01092), Fucus vesiculosus, genated, max. MW peak 260 kD; Fucoidan GFS (L/FVF-01093), Fucus losus, hydrolyzed depyrogenated, max. MW peak 36 kD; Maritech® ia radiata t; Maritech® Ecklom'a maxima extract; Maritech® Macrocysti: pyriflra extract; Maritech® Immune trial Fucoidan Blend; and combinations thereof. As noted above, in certain instances the concentration of chitosan ranges from about 0.1% to about 5%, such as about 3% and the concentration of bromelain ranges from 0.1 mg/mL to about 1 mg/mL, such as about 0.5 mg/mL.

Where two or more gastrointestinal epithetial barrier permeation enhancers are employed, in some embodiments, the combination is a synergistically effective combination of gastrointestinal epithetial barrier permeation enhancers. The term “synergistically effective” is meant that the combination of gastrointestinal epithetial barrier permeation enhancers produces an effect (i.e., es intestinal epithelial barrier permeation) which is greater than would be achieved by the sum of the individual gastrointestinal epithetial barrier permeation enhancers. For example, the combination of more than one intestinal epithelial barrier permeation enhancer produces an effect that is 2-fold or greater than would be achieved by the sum of the individual gastrointestinal epithelial r permeation enhancers, such as 3—fold or greater, such as 4-fold or greater, such as 5-fold or greater, such as 10-fold or greater and including d or greater than would achieved with the sum the individual gastrointestinal epithelial barrier permeation ers. As such, where two gastrointestinal epithelial barrier permeation enhancers are ed, synergistically effective combinations of the present invention produce an effect which is 2-fold or greater, such as 5-fold or greater, such as 10-fold or greater and including 25 ~fold or greater than would be achieved by the sum of the two individual gastrointestinal epithelial barrier permeation enhancers. Likewise, where three gastrointestinal epithelial barrier permeation enhancers are combined, synergistically ive combinations of the present invention produce an effect which is 2—fold or greater, such as 5-fold or greater, such as IO-fold or greater and including 25-fold or greater than would be achieved by the sum of the three individual gastrointestinal epithelial barrier permeation enhancersln certain embodiments, synergistically effective combinations of the present invention include a ation of chitosan and ain. In these embodiments, the combination of chitosan and bromelain has a greater effect on enhancing permeation through the gastrointestinal epithelial r than is achieved by the sum of chitosan and bromelain individually. For example, in some ces, the combination of chitosan and bromelain enhances permeation through the gastrointestinal epithelial barrier by 2—fold or r, such as 5-fold or greater, such as 10-folder or greater and ing 25 -fold or greater than is achieved by the sum of chitosan and bromelain individually. In certain instances, synergistically effective combinations include a combination of bromelain having a concentration ranging from 0.l mg/mL to about 0.5 mg/mL, such as 0.15 mg/mL to about 0.4 mg/mL, including 0.25 mg/mL and an having a concentration ranging from about 1% to about 5% w/v, such as 1.5% to about 4.5% w/v, such as 2% to about 4% w/v and including about 3% w/v. In certain embodiments, istically effective combinations of bromelain and an include a ation of 0.5mg/mL ain and 3% wiv chitosan. In other embodiments, istically effective combinations of bromelain and chitosan include 0.25 mg/mL bromelain and 1.5% w/v chitosan. In yet other embodiments, synergistically effective combinations of bromelain and chitosan include 0.12 mg/mL bromelain and 0.75% w/v chitosan.

In embodiments of the ion, methods for enhancing blood coagulation by orally administering a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer to a subject are provided. By “subject” is meant the person or organism ing the blood coagulation enhancement. As such, subjects of the ion may include but are not limited to humans and other primates, such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory s including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.

In some embodiments, the t methods may be employed to treat bleeding disorders, such as a chronic or acute bleeding disorder, a congenital coagulation disorder caused by a blood factor deficiency, an acquired coagulation disorder and administration of an anticoagulant. For example, bleeding disorders may include, but are not limited to hemophilia A, hemophilia B, von Willebrand disease, idiopathic thrombocy‘topenia, a deficiency of one or more contact factors, PCT/U52012/047248 such as Factor XI, Factor XII, prekallikrein, and high molecular weight lcininogen (HMWK), a deficiency of one or more factors associated with clinically significant bleeding, such as Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia), and von Willebrands factor, a vitamin K deficiency, a disorder of fibrinogen, including U! afibrinogenemia, hypofibrinogenemia, and rinogenemia, an alphaz-antiplasmin deficiency, and excessive bleeding such as caused by liver disease, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hroses, surgery, , hypothermia, menstruation, and pregnancy.

In other embodiments, the subject methods may be employed to e blood coagulation in order to reverse the effects of an anticoagulant in a subject. For example, the t may have been treated with an anticoagulant including, but not limited to, heparin, a coumarin derivative, such as warfarin or dicumarol, TFPI, AT III, lupus anticoagulant, nematode anticoagulant peptide (NAPcZ), active-site blocked factor VIIa (factor VIIai), factor IXa inhibitors, factor Xa inhibitors, including fondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906), tors of s Va and VIIIa, including activated protein C (APC) and e thrombomodulin, thrombin tors, including hirudin, bivalirudin, argatroban, and ximelagatran. In certain embodiments, the anticoagulant in the subject may be an antibody that binds a clotting factor, including but not limited to, an antibody that binds to Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II, Factor XI, Factor XII, von Willebrands factor, prekallilqein, or high molecular weight gen (HMWK).

Aspects of the invention e orally administering to a subject a composition having an procoagulant amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer to enhance blood coagulation. As described above, NASPs of the invention may be l NASPs or synthetic NASPs. In some embodiments, NASPs are natural NASPs.

By “natural” is meant that the NASP is found or derived from a naturally occurring source, such as from an animal or plant source and encompass a broad range of subclasses including heparins, glycosarninoglycans, fucoidans, carrageenans, pentosan polysulfates, dermatan sulfates and dextran sulfates. In some embodiments, natural NASPS may be extracted from a biological 3O source. By “biological source” is meant a lly-occurring organism or part of an sm.

For example, NASPs of interest may be extracted from plants, animals, fungi or bacteria. In ular, NASPs of interest may be extracted from edible seaweeds, brown algae, echinoderms (e.g., sea urchins, sea ers) and the like. Any convenient protocol can be employed for extracting the NASP from the biological source. For ce, the NASP can be extracted from the biological source by acid-base extraction, enzymatic degradation, selective precipitation, filtration, among other procedures. Methods for extracting and isolating NASPs from biological sources such as edible seaweeds and brown algae are described in detail in co-pending US.

W0 2013/012954 PCT/U82012/047248 Patent Application Serial No. ,712, filed February 25, 2010, the disclosure of which is herein incorporated by reference, in its entirety.

In certain ments, natural NASPs of the invention include, but are not d to N-acetyl-heparin (NAH), yl—de—O-sulfated-heparin (NA-de-o-Sl—l), ulfated-heparin (De-NSH), de—N-sulfated-acetylated~he- parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS) and ations thereof. In some instances, the NASP may be a low lar weight fragment of a naturally occurring NASP. In other instances natural NASPs may also include biochemical or chemical derivatives of naturally occurring NASPs. In certain instances, natural NASPs are fucoidans. As described in greater detail below, fucoidans are naturally-occurring complex sulfated polysaccharides compounds which may be extracted from certain edible ds, brown algae and derms (e.g, sea s, sea ers). As used herein the term, “fucoidan” refers to a diverse group of moieties ted from a biological source of low e polymers rather than a single chemical entity. In certain instances, fucoidans of the invention include, but are not limited to Fucoidan GFS 5508005, Undaria pinnatifida, depyrogenated; Fucoidan GFS 5508004, Undaria pinnatilida; Fucoidan GFS 5508003, Undaria pinnatifida; Fucoidan 5307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucus vesiculosus, hydrolyzed sample of 5307002 of lower MW, max. MW 2O peak 22.5 kD; Fucoidan 5308004, Fucus vesiculosus; Fucoidan 5308005, Fucus vesiculosus; Fucoidan L/FVF1091, Fucus vesiculosus; Fucoidan VC1201096A, Fucus vesiculosus; Fucoidan 96B, Fucus vesiculosus; Fucoidan VG57, Undaria pinnatifida, high charge (high sulphation, deacetylated); Fucoidan VGSO, Ascophyllum nodosum, max. MW peak 149.7 kD; and combinations thereof.

Examples of suitable NASPs are described in greater detail in United States Patent Application Serial No. 11/140,504, filed on May 27, 2005, now United States Patent No. 7,767,654, and United States Patent Application Serial No. 13/006,396, filed on Januaryl3, 2011, the disclosures of which is herein orated by reference in their entirety.

In other embodiments, NASPs are synthetic NASPs. By “synthetic” is meant that the sulfated polysaccharide is partially or wholly produced by man-made methods (cg, chemical synthesis). For e, the synthetic NASP may be a sulfated oligomer, such as a sulfated oligosaccharide or a sulfated tic. In certain instances, synthetic NASPs are ed es, sulfated hexoses or sulfated cyclodextrins. For example, synthetic NASPs may include, but are not limited to sulfated maltopentoses, sulfated beta-cyclodextrins, sulfated 6- Carboxyicodextrin and derivatives thereof.

Examples of other suitable synthetic NASPs are described in greater detail in United States Provisional Patent Application Serial No. 61/592,554, filed on January 30, 2012 and PCTI’U320121047248 United States Provisional Patent Application Serial No. 61/592,549, filed on y30, 2012, the disclosures of which is herein incorporated by reference in their entirety.

Depending on the desired s and potency of the NASPS, one or more NASPS may employed together. For example, two or more NASPs may be employed together, such as three or more NASPs and including four or more NASPs. Where more than one NASP is employed, all of the NASPs may be natural NASPs, all of the NASPs may be synthetic NASPS or any combination thereof. Where more than one NASP is employed, the mass percentage of each NASP in the composition may vary, ranging from 1% or more of the total mass of the composition, such as 2% or more, such as 5% or more, such as 10% or more, such as 25% or more and including as 50% or more of the total mass of the composition.

In embodiments of the ion, the mass ratio of the NASP and the gastrointestinal epithelial barrier permeation er may vary, ranging between 1:1 and 1:2.5; 122.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and mm; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of the NASP to the gastrointestinal epithelial barrier permeation enhancer may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio of the gastrointestinal epithelial barrier permeation enhancer to the NASP ranges between 1:1 and 1:25; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of the gastrointestinal epithelial barrier permeation enhancer to the NASP may range between 1:1 and :5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000.

The NASP and the gastrointestinal epithelial barrier permeation enhancer may be stered to the subject in any order. In some instances, the NASP is orally administered prior to orally administering the gastrointestinal epithelial barrier permeation enhancer. In other instances, the NASP is orally administered after orally administering the gastrointestinal epithelial r permeation enhancer. In yet other instances, the NASP is orally administered in conjunction with orally administering the gastrointestinal epithelial barrier permeation enhancer.

If both the NASP and the gastrointestinal epithelial barrier permeation enhancer are provided at the same time, each can be provided in the same or in a different composition. Where the NASP and the gastrointestinal epithelial barrier tion enhancer are administered at the same time, the NASP may be mixed with the gastrointestinal epithelial r tion enhancer the blood ation factor before stering the composition to the subject. Any convenient mixing protocol may be used, such as by dry shaking, on or suspension mixing, industrial mixing protocols and the like. Thus, NASPs and gastrointestinal epithelial barrier permeation enhancers can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is ed administration to a subject such that the therapeutic effect of the WO 12954 combination of the NASP and gastrointestinal epithelial barrier permeation enhancer is caused in the subject oing therapy. Similarly, one or more NASPs and one or more gastrointestinal epithelial barrier permeation ers can be orally administered in at least one therapeutic dose.

U] Any suitable ation of NASP and gastrointenstinal epithelial barrier tion enhancer may be administered. In n embodiments, s include administering a natural NASP (as described above) with one or more of intenstinal epithelial barrier permeation enhancers. In these ments, methods may include administering a combination of a natural NASP with one or more of sodium caprate, deoxycholate, bromelain and an. In certain embodiments, methods include administering a natural NASP with sodium caprate. For instance, one or more of Undaria pinnatifida U.p.5508005 and Fucus vesiculosus Ev. L/FVF 1091 may be administered with sodium caprate. In other embodiment, methods include administering a natural NASP with deoxycholate. For instance, one or more of Undaria pinnatifida U.p.5508005, Fucus losus Ev. L/FVF 1091, Fucus vesiculosus Ev. D81001108, Fucus vesiculosus Ev. SK110144B may be stered with deoxycholate. In yet other embodiments, methods include administering a natural NASP with bromelain. For instance, one or more of Undarz‘a pinnatifida U.p.5508005, Fucus vesiculosus Ev. L/FVF 1091, Fucus vesiculosus Ev.

DSlOOl 108, Fucus vesiculosus Ev. SK110144B may be administered with bromelain. In yet other embodiments, methods include stering a natural NASP with chitosan. For instance, one or more of Undaria pinnatifida U .p.5508005 Fucus vesiculosus Ev. L/FVF 1091, Fucus vesiculosus Ev. DSlOOl 108, Fucus vesiculosus F.v. SK110144B may be administered in combination with chitosan. In yet other embodiments, methods include administering a l NASP with a combination of bromelain and chitosan( as described above). For instance, one or more of Undaria pinnatifida U.p.5508005, Fucus lasus Ev. L/FVF 1091, Fucus vesiculosus Ev. DSIOOI 108, Fucus vesiculosus Frv. SKl 10144B may be administered with a combination of bromelain and chitosan.

In certain embodiments, methods e administering a synthetic NASP (as described above) with one or more of gastrointenstinal epithelial r permeation enhancers. In these embodiments, methods may include administering a combination of a synthetic NASP widl one or more of sodium caprate, deoxycholate, bromelain and chitosan. In certain embodiments, methods include administering a synthetic NASP with sodium caprate. For instance, one or more of a sulfated B-cyclodextrin and a sulfated maltopentose may be administered with sodium caprate. In other embodiment, methods include administering a tic NASP with deoxycholate. For instance, one or more of a 24 kD sulfated 6-carboxyicodextrin, a 14 kD 6- carboxyicodextrin, a sulfated B—cyclodextrin and a sulfated maltopentose may be administered with deoxycholate. In yet other embodiments, methods include administering a synthetic NASP PCT/U82012/047248 with bromelain. For instance, one or more of a 24 kD sulfated 6-carboxyicodextrin, a 14 kD 6- carboxyicodextrin, a sulfated B-cyclodextrin and a sulfated maltopentose may be administered with bromelain. In yet other ments, methods include administering a synthetic NASP with an. For instance, one or more of a 24 kD sulfated 6-carboxyicodextrin, a 14 kD 6— carboxyicodextrin, a sulfated B-cyclodextrin and a sulfated maltopentose may be administered in combination with chitosan. In yet other embodiments, methods include administering a tic NASP with a combination of bromelain and chitosan( as described above). For instance, one or more of a 24 kD sulfated 6-carboxyicodextrin, a 14 kD, 6-carboxyicodextrin, a sulfated B- cyclodextrin and a sulfated maltopentose may be administered with a combination of bromelain and chitosan.

In certain embodiments, aspects of the invention include enhancing blood coagulation in a subject by orally administering to the subject, a composition that contains a procoagulant amount of a NASP and a intestinal lial r permeation er in ation with a blood coagulation factor. For example, the subject may be orally administered a procoagulant amount of a composition containing a NASP and a gastrointestinal epithelial barrier tion enhancer in combination with one or more blood coagulation factors which include, but are not limited to factor XI, factor XII, prekallikrein, high molecular weight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factor XIII, factor II, factor VIIa, and von Willebrands factor, factor Xa, factor [Xa, factor Xla, factor XIla, and VIIIa, prekallekrein, and high-molecular weight kininogen, tissue factor, factor VIIa, factor Va, and factor Xa.

Where a composition that contains a NASP and a gastrointestinal epithelial barrier tion enhancer is orally administered with a blood coagulation factor to the subject, the mass ratio of the composition that contains the NASP and gastrointestinal epithelial barrier Ix) U! permeation enhancer to the blood coagulation factor may vary, ranging between 1:1 and 122.5; 122.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1150;1250 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range f. For example, the mass ratio of the composition that contains the NASP and intestinal epithelial barrier permeation enhancer to the blood ation factor may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio of the blood coagulation factor to the composition that contains the NASP and gastrointestinal epithelial barrier permeation enhancer ranges between 1:1 and 1:25; 122.5 and 1:5; 1:5 and 1:10;1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and , or a range thereof. For example, the mass ratio of the blood coagulation factor to the ition that contains the PCT/U82012i047248 NASP and gastrointestinal epithelial barrier permeation enhancer may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000.

The blood coagulation factor and the composition that contains the NASP and gastrointestinal epithelial barrier permeation enhancer may be administered to the t in any order. In some instances, the composition that contains the NASP and gastrointestinal epithelial barrier permeation enhancer is orally administered prior to administering the blood coagulation factor In other instances, the composition that contains the NASP and gastrointestinal epithelial r permeation enhancer is orally administered in ction with administering the blood coagulation factor. In yet other ces, the ition that contains the NASP and gastrointestinal epithelial barrier permeation enhancer is orally administered after administering the blood coagulation factor. Where the composition that contains the NASP and gastrointestinal epithelial barrier permeation enhancer is orally administered in conjunction with the blood coagulation , the composition that contains the NASP and gastrointestinal epithelial barrier permeation enhancer may be mixed with the blood coagulation factor before orally administering the composition to the subject. Any convenient mixing protocol may be used, such as a by dry shaking, solution or suspension mixing, industrial mixing ols and the like.

Aspects of the invention include methods and compositions for treating bleeding disorders by orally administering a gulant amount of a NASP in combination with gastrointestinal epithelial r permeation enhancer. NASPs in ation with a gastrointestinal epithelial barrier permeation enhancer as disclosed herein can be administered alone (i.e., as single agents), or in combination with other hemostatic agents. As desired, a procoagulant amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer may be employed in the treatment of a subject that has been diagnosed as having a bleeding disorder, including congenital coagulation disorders, acquired coagulation ers, administration of an anticoagulant, and trauma d hemorrhagic conditions.

In some ces, a subject may be diagnosed as having a blood clotting disorders that includes, but is not d to hemophilia A, hemophilia 13, von Willebrand disease, idiopathic thrombocytopenia, a ency of one or more contact factors, such as Factor XI, Factor XII, prekallikrein, and high molecular weight kininogen (HMWK), a deficiency of one or more factors associated with clinically significant bleeding, such as Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia), and von Willebrands factor, a vitamin K deficiency, a disorder of ogen, including afibrinogenemia, rinogenemia, and inogenemia, an alphaz-antiplasmin deficiency, and excessive bleeding such as caused by liver disease, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hemarthroses, surgery, trauma, ermia, uation, and pregnancy, In other instances, a subject may be diagnosed as having a blood clotting disorder that includes a congenital coagulation disorder or an acquired coagulation disorder caused by a blood PCT/U52012/047248 factor deficiency. For example, the blood factor deficiency may be caused by deficiencies of one or more factors, including but not limited to, factor V, factor VII, factor VIII, factor IX, factor XI, factor XII, factor XIII, and von Willebrand factor.

In yet other instances, a subject may be diagnosed as having a blood clotting disorder resulting from the administration of an anticoagulant to the subject. For example, the anticoagulant may include but is not d to, heparin, a coumarin derivative, such as warfarin or dicumarol, tissue factor pathway inhibitor , antithrombin III, lupus anticoagulant, nematode anticoagulant peptide (NAPc2), active-site blocked factor VIIa (factor VIIai), factor lXa inhibitors, factor Xa inhibitors, including fondaparinux, idraparinux, DX—9065a, and razaxaban 6), inhibitors of factors Va and Villa, ing activated protein C (APC) and soluble thrombomodulin, in inhibitors, including hirudin, bivalirudin, argatroban, and ximelagatran. In certain embodiments, the anticoagulant may be an antibody that binds a ng factor, including but not limited to, an dy that binds to Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II, Factor XI, Factor XII, von Willebrands factor, prekallikrein, orhigh molecular weight kininogen (HMWK).

In yet other instances, methods of the invention include a method of inhibiting TFPI activity in a subject. For example, methods may further include orally administering to a subject, an amount of a NASP in combination with a intestinal lial barrier permeation enhancer in a manner sufficient to inhibit TFPI activity in the t. In certain instances, a procoagulant amount of a NASP in combination with a gastrointenstinal epithelial barrier permeation enhancer is combined with a biological sample (e.g., blood plasma) that includes TFPI and measuring the TFPI ty of the biological sample. In other instances, methods e combining a procoagulant amount of a NASP and a gastrointestinal epithelial r permeation er with a biological sample, adding TFPI to the composition and measuring the TFPI acitivty of the biological sample. In certain instances, the biological sample is a plasma , such as for e, normal blood plasma or Factor VIII-inhibited blood .

In practicing methods of the invention, protocols for enhancing blood coagulation in a subject may vary, such as for example by age, weight, severity of the blood clotting disorder, the general health of the subject, as well as the ular composition and concentration of the 3O NASPs and gastrointestinal epithelial barrier permeation enhancers being administered. In embodiments of the invention, the concentration of NASPs achieved in a subject following oral administration and resorption by the gastrointestinal system may vary, in some instances, ranging from 0.01 nM to 500 nM. NASPs of interest are procoagulant at their optimal concentration. By “optimal concentration” is meant the concentration in which NASPS exhibit the highest amount of procoagulant activity. Since many of the NASPs also demonstrated anticoagulant activity at much higher concentrations than the optimal tration, NASPs of the invention show non- anticoagulant behavior in the range of its optimal concentration. As such, depending on the -28— potency of the NASP as well as the desired effect, the optimal concentration of NASPS provided by methods of the invention may range, from 0.01nM to 500 nM, such as 0.1 nM to 250 nM, such as 0.1 nM to 100 nM , such as 0.1 nM to 75 nM, such as 0.1 nM to 50 nM, such as 0.1 nM to 25 nM, such as 0.1 nM to 10 nM, and including 0.1 nM to 1 nM. Optimal concentrations and activity level as determined by calibrated automated thrombography (CAT) assay of NASPs of interest are described in greater detail in United States Patent Application Serial No. 11/140,504, filed on May 27, 2005, now United States Patent No. 7,767,654, and United States Patent ation Serial No. 13/006,396, filed on yl3, 2011, the disclosures of which is herein incorporated by reference in their entirety. se, the concentration of gastrointestinal epithelial barrier permeation enhancers achieved in a subject following oral administration and resorption by the gastrointestinal system may vary, in some instances, ranging from 0.01 nM to 500 nM. For example, depending on the inherent absorptivity of the NASP as well as the desired effect, the concentration of intestinal epithelial barrier permeation enhancers provided by methods of the invention may range, from 0.0lnM to 500 nM, such as 0.1 nM to 250 nM, such 0.1 nM to 100 nM , such as 0.1 nM to 75 nM, such as 0.1 nM to 5011M, such as 01 nM to 25 nM, such as 0.1 nM to 10 nM, and ing 0.1 nM to 1 nM.

Therefore, the oral dosage of compositions containing NASPs in combination with gastrointestinal epithelial barrier permeation enhancers of interest may vary, ranging from about 0.01 mg/kg to 500 mg/kg per day, such as from 0.01 mg/kg to 400 mg/kg per day, such as 0.01 mg/kg to 200 mg/kg per day, such as 0.1 mg/kg to 100 mg/kg per day, such as 0.01 mg/kg to 10 mg/kg per day, such as 0.01 mg/kg to 2 mg/kg per day, including 0.02 mg/kg to 2 mg/kg per day.

In other embodiments, the oral dosage may range from 0.01 to 100 mg/kg four times per day (QID), such as 0.01 to 50 mg/kg QID, such as 0.01 mg/kg to 10 mg/kg QID, such as 0.01 mg/kg to 2 tug/kg QlD, such as 0.01 to 0.2 trig/kg QID. In other embodiments, the oral dosage range from 0.01 mg/kg to 50 mg/kg three times per day (TID), such as 0.01 mg/kg to 10 mg/kg TED, such as 0.01 mg/kg to 2 mg/kg TlD, and including as 0.01 mg/kg to 0.2 mg/kg TID. In yet other ments, the oral dosage may range from 001 mg/kg tolOO mg/kg two times per day (BID), such as 0.01 mykg to 10 mg/kg BID, such as 0.01 mg/kg to 2 mg/kg BID, including 0.01 mg/kg to 0.2 mg/kg BID. The amount of compound administered will depend on the potency and concentration of the specific NASP, the magnitude or procoagulant effect desired, the inherent tivity of the NASP, as well as the desired enhancement of gastrointestinal resorption.

As discussed above, compositions ning a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer as provided by s of the invention may be orally administered in ation with other NASPs, gastrointestinal epithelial barrier tion enhancers or other therapeutic agents, such as hemostatic agents, blood factors, or other tions according to a dosing schedule relying on the judgment of the clinician and needs of the subject. As such, dosing schedules may include, but is not limited to administration five times per day, four times per day, three times per day, twice per day, once per day, three times per week, twice per week, once per week, twice per month, once per month, and any combination thereof.

’Jl In some embodiments, the bleeding disorder may be a chronic condition (e.g., a congenital or acquired coagulation factor ncy) requiring the subject methods and compositions in multiple doses over an extended period. Alternatively, methods and compositions of the invention may be administered to treat an acute condition (e.g., bleeding caused by surgery or trauma, or factor inhibitor/autoimmune episodes in subjects receiving coagulation replacement therapy) in single or multiple doses for a relatively short period, for example one to two weeks.

In practicing embodiments of the invention, one or more therapeutically effective cycles of treatment will be administered to a subject. By peutically effective cycle of ent” is meant a cycle of ent that when administered, brings about the d therapeutic response with respect to treatment, For example, one or more therapeutically effective cycles of treatment may increase the rate of blood clotting as determined by blood clotting assays (e.g., CAT, aPI‘T, described in detail below) by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 20% or more, such as 30% or more, such as 40% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, including increasing the rate of blood clot formation by 99% or more. In other instances, one or more therapeutically effective cycles of treatment may increase the rate of blood clot formation by 1.5-fold or more, such as 2-fold or more, such as 5-fold or more, such as lO-fold or more, such as 50-fold or more, including increasing the rate of blood clot formation by lOO-fold or more. In some embodiments, subjects treated by s of the invention exhibit a positive therapeutic response. By “positive therapeutic response" is meant that the subject exhibits an improvement in one or more symptoms of a bleeding disorder. For example, a subject exhibiting a positive therapeutic response to methods provided by the invention may include but is not d to responses such as shortened blood clotting times, reduced ng, reduced need for factor ement therapy or a combination thereof. In n embodiments, more than one 3O therapeutically effective cycle of treatment is administered.

As reviewed above, in cing methods according to certain embodiments, a ition having a procoagulant amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer is administered to a subject to enhance blood coagulation in the subject. Any convenient mode of administration may be employed so long as the ition is ed through the gastrointestinal epithelium. As such, modes of administration may include oral stration or by stric tube (e.g., feeding tube or NG- tube). As discussed in greater detail below, pharmaceutical compositions of the invention may PCT/U52012/047248 be in the form of a liquid solution or suspension, syrup, tablet, capsule, powder, gel, or any combination thereof. Where a ition having a procoagulant amount of a NASP and gastrointestinal epithelial barrier permeation enhancer is orally administered in combination with a blood coagulation factor, as discussed in detail above, the mode of administration for the NASP and gastrointestinal epithelial barrier permeation enhancer component may be the same or different than for the blood ation factor. For example, in some instances, the composition having a procoagulant amount of a NASP and gastrointestinal epithelial barrier permeation enhancer may be administered orally, whereas the blood coagulation factor may be locally d (e.g., as a cream). In other instances, both the composition having a procoagulant amount of a NASP gastrointestinal epithelial barrier permeation er and the blood coagulation factor are administered orally.

In certain embodiments, methods of the invention provide for orally administering composition having a procoagulant amount Of a NASP and gastrointestinal epithelial barrier permeation enhancer prophylactically, such as for example before planned surgery. The composition may be adtninistered prophylactically as desired, such as one hour or more prior to a d procedure, such as 10 hours prior to a planned procedure, such as 24 hours prior to a planned ure, and including one week prior to a planned procedure. In some instances, the composition administered prior to or during a planned procedure may be a sustained-release formulation (e.g., sustained release caplets or s), as bed in greater detail below.

In certain embodiments, compositions of the invention can be orally administered prior to, concurrent with, or subsequent to other agents for treating related or unrelated conditions. If provided at the same time as other agents, compositions of the invention can be provided in the same or in a different ition. Thus, NASPs and gastrointestinal epithelial barrier permeation enhancers of interest and other agents can be presented in an oral dosage form to the individual by way of concurrent therapy. For example, concurrent therapy may be ed by administering itions of the invention and a pharmaceutical composition having at least one other agent, such as a hemostatic agent or ation factor (e. g. FVHI or FIX), which in combination comprise a eutically effective dose, according to a particular oral dosing regimen. Similarly, one or more NASPs in combination with one or more gastrointestinal epithelial barrier tion enhancers and eutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), so long as the therapeutic effect Of the combination of these substances is caused in the subject oing therapy.

COMPOSITIONS PCT/U820121047248 Aspects of the invention also include oral dosage compositions for enhancing blood coagulation in a subject. In embodiments of the invention, compositions include a procoagulant amount of a NASP in ation with a gastrointestinal epithelial barrier permeation enhancer.

Compositions also include a combination of a procoagulant amount of a NASP with a U! gastrointestinal epithelial barrier permeation enhancer and a blood coagulation factor. As described in detail above, gastrointestinal lial barrier permeation enhancers include compounds that when orally administered, increase the amount of NASP that is resorbed by the gastrointestinal system. Furthermore, intestinal permeation enhancers may also accelerate the initiation (i.e., reducing the amount time for tion to begin) of NASP resorption through the gastrointestinal epithelium as well as accelerate the overall rate of transport. of the NASP across the gastrointestinal epithelium of the subject (i.e., reducing the amount of time for NASP resorption by the gastrointestinal system to be complete).

As noted above, gastrointestinal lial barrier permeation enhancers may increase the amount of NASP ed by the gastrointestinal system. For example, gastrointestinal epithelial barrier permeation enhancers may increase the amount of NASP resorbed by the gastrointestinal system by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 90% or more, such as 95% or more, as compared to a suitable control. In other embodiments, gastrointestinal epithelial barrier permeation enhancers in oral compositions of the invention accelerate the initiation of NASP resorption through the gastrointestinal epithelium. For example, gastrointenstinal epithelial barrier permeation enhancers of the invention may reduce the amount of time required to initiate resorption of the NASP by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 90% or more, such as 95% or more, as compared to a suitable control. In yet other embodiments, gastrointestinal epithelial barrier tion enhancers in oral compositions of the invention increase the rate of resorption of the NASP. For e, gastrointestinal epithelial barrier tion enhancers may se the rate of NASP resorption by 2% or more, such as by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 100% or more, such as by 200% or more, including by 500% or more, as compared to a suitable. control. 3O In n instances, gastrointestinal epithelial tion ers in oral compositions of the invention may se the tion of NASPs as determined by Caco-2 cell models. For e, gastrointestinal epithelial barrier permeation enhancers of the invention may increase the resoprtion as determined by Caco-Z cell models by 2% or more, such as by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 100% or more, such as by 200% or more, including by 500% or more, as compared to a suitable control. 2012/047248 As sed in detail above, oral dosage compositions of the invention include one or more NASPs in combination with one or more gastrointestinal epithelial barrier permeation enhancers.

Gastrointestinal lial barrier permeation enhancers in the compositions of interest may vary. In some embodiments, gastrointestinal epithelial barrier permeation enhancers are tight on modulators. For example, tight on tors in oral dosage compositions of the invention may include, but are not limited to enzymes, bile acids, polysaccharides, fatty acids and salts f and any combination thereof.

In some instances, tight junction modulators are polysaccharides. For example, the polysaccharide tight junction tor, in certain instances may be chitosan. an, as discussed above, refers to the linear copolymer of 2-acetamidedeoxy-B-D‘glucopyranose and 2-amino-l3~D- glucopyranose made by N-deactylation of chitin. ccharide tight junction modulators may also include, derivatives of chitosan such as N—alkyl an, acylated Chitosan, thiolated Chitosan, phosphorylated Chitosan, chitosan cyclodextrin, N-(aminoalkyl) Chitosan, succinyl chitosan and octanoyl chitosan, among others.

In other instances, tight junction modulators are bile acids. Suitable bile acid tight junction modulators may include but are not limited to cholic acid (cholate), holic acid (deoxycholate), chenodeoxycholic acid deoxycholate), ursodeoxycholic acid (ursodeoxycholate), glycocholic acid (glycocholate), taurocholic acid (taurocholate) and lithocholic acid (lithocholate), among others.

In other instances, tight junction modulators are enzymes, For example, in certain itions of the invention, the enzyme tight junction tors is a protease, such as bromelain.

In yet other instances, tight junction modulators are fatty acids and fatty acid salts thereof.

Fatty acid tight junction modulators in compositions of the invention may vary, and may include any one or a combination of medium chain fatty acids, such as for example C8 (caprylate), C10 (caprate) and C12 (laurate) fatty acids and fatty acid salts thereof. In certain instances, for example, the fatty acid tight junction modulator is sodium caprate.

In certain embodiments, oral dosage compositions of the invention include two or more gastrointestinal epithelial barrier permeation enhancers. For example, itions may include two or more tight junction modulators, such as three or more tight junction modulators, including four or more tight junction modulators. Compositions may include any combination of tight junction modulators, such as for example, a polysaccharide and a protease, a fatty acid and polysaccharide, a polysaccharide and a bile acid, a polysaccharide, a fatty acid and a bile acid, two different ccharides or two different bile acids, among other combinations. Where compositions include more than one gastrointestinal epithelial barrier permeation enhancer, the mass tage of each gastrointestinal epithelial barrier permeation enhancer may vary, ranging from 1% or more of the total mass of the composition, such as 2% or more, such as 5% or more, such as 10% or more, such as 25% or more and including 50% or more of the total mass of the composition. 2012/047248 In certain instances, compositions of the invention include chitosan and bromelain.

Where the composition includes a combination of chitosan and bromelain, the mass ratio of chitosan and ain may vary, ranging between 1:1 and 1:25; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150:11150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of chitosan to bromelain may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio of bromelain to chitosan ranges between 1:1 and 1:25; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100;1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of bromelain to chitosan may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000.

Where compositions of the invention include a combination of chitosan and ain, the concentration of chitosan may also vary, ranging from about 0.1% to about 5%, such as about 0.15% to about 4.5%, such as 0.2% to about 4%, such as about 0.25% to about 3.5%, such as 0.3% to about 3%, such as 0.5% to about 25%, including about 0.5% to 1.5%. se, where a combination of chitosan and bromelain are employed, in certain ments, the concentration of bromelain may also vary, ranging from about 0.01 mg/mL to about 1.0 mg/mL, such as about 0.2 mg/mL to about 0.9 mg/mL, such as 0.25 mg/mL to about 0.75 mg/mL, such as about 0.3 mg/mL to about 0.6 mg/mL, ing about 0.4 mg/mL to about 0.5 mg/mL. In certain instances, the concentration of chitosan is about 3% and the concentration of bromelain is about 0.5 mg/mL.

As described above, NASPs in oral dosage compositions of the invention are ed polysaccharides that demonstrate procoagulant activity. The non-anticoagulant properties of NASPs may be determined using clotting assays, ing calibrated automated thrombography (CAT) in Factor VIII and/or Factor 1X deficient plasma, dilute prothrombin time (dPT) or activated l thromboplastin time (aPIT) clotting assays. One measure of noncoagulant activity is to compare the NASP in question with the known anticoagulant heparin. For example, NASPS may exhibit one—third or less, such as one~tenth or less of the anticoagulant activity (measured by statistically significant increase in clotting time) of unfractionated heparin (MW range 8,000 to 30,000; mean 18,000 Daltons). Thus, a NASP can demonstrate at least a two-fold lower anticoagulant activity as compared to n, such as a two- to five-fold or lower anticoagulant activity as compared to heparin, and including a two- to 10-fold or lower anticoagulant activity as compared to heparin, using any of the various clotting assays detailed herein.

In some embodiments, oral dosage compositions of the invention e a natural NASP in ation with a gastrointestinal epithelial barrier permeation enhancer. As PCT/U82012/047248 discussed above, natural NASPs may be NASPs found or derived from a lly occurring source, such as from an animal or plant source and may encompass a broad range of subclasses including derivatives of heparins, glycosaminoglycans, fucoidans, carrageenans, pentosan polysulfates, dermatan sulfates and dextran es. In some embodiments, natural NASPs of the invention are extracted from a biological source. By “biological source” is meant a naturally- ing organism or part of an organism. For example, NASPs of st may be extracted from plants, animals, fungi or bacteria. In particular, NASPs of interest may be extracted from edible seaweeds, brown algae, echinoderms (e.g., sea urchins, sea cucumbers) and the like. For instance, the NASP can be extracted from the biological source by acid»base extraction, enzymatic degradation, selective precipitation, filtration, among other procedures. l NASPs such as those extracted from biological sources, ing but not limited to edible seaweeds and brown algae are described in detail in co-pending U.S. Patent Application Serial No. 12/449,712, filed February 25, 2010, the sure of which is herein incorporated by reference, in its entirety.

In certain embodiments, natural NASPs of the invention include, but are not limited to N-acetyl-heparin (NAH), N-acetyl~de-O-sulfated—heparin (NA-de-o-SH), de-N—sulfated—heparin (De~NSH), ulfated-acetylated-he- parin (De-NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated c acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived oligosaccharides (HDO), pentosan lfate (FPS) and combinations thereof. In some ces, the NASP may be a low lar weight fragment of a naturally occurring NASP. In other instances, natural NASPs may also include biochemical or chemical derivatives of naturally occurring NASPS. In certain ces, l NASPs are fucoidans. As described above, fucoidans are naturally—occurring complex sulfated polysaccharide compounds which may be extracted from certain edible seaweeds, brown algae and echinoderms (e.g., sea urchins, sea cucumbers). Examples of suitable NASPs are also described in greater detail in United States Patent Application Serial No. 11/140,504, filed on May 27, 2005, now United States Patent No. 7,767,654 and in United States Patent Application Serial No. 13/006,396 filed on January 13, 2011, the disclosures of which are herein incorporated by reference in their entirety.

NASPs of st may range in average molecular weight from about 10 daltons to about 500,000 daltons, such as from about 100 daltons to about 0 daltons, such as from 1000 daltons to 250,000 daltons, including 1000 daltons to 150,000 daltons. Molecular weights of NASPs can be ined by any convenient protocol, such as for example, gel permeation chromatography or high-performance size-exclusion chromatography (HPSEC), ary electrophoresis, PAGE (polyacrylamide gel electrophoresis), agarose gel electrophoresis, among others.

In some embodiments, NASPs of interest may be geneous mixtures of sulfated polysaccharides having varying molecular weights. For example, in some instances, 5% or more of PCT/U52012/047248 the NASP composition has a molecular weight that ranges from 10 to 30,000 daltons, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including 95% or more of the NASP composition has a molecular weight that ranges from 10 to ,000 daltons. In other embodiments, 5% or more of the NASP composition has a molecular weight LII that ranges from 30,000 daltons to 75,000 daltons, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, ing 95% or more of the NASP composition has a molecular weight that ranges from 30,000 to 75,000 daltons. In yet other embodiments, 5% or more of the NASP composition has a molecular weight that are greater than 75,000 daltons, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, ing 95% or more of the NASP composition has a molecular weight that is greater than 75,000 daltons.

In certain embodiments, low molecular weight NASPS may be employed for enhancing blood ation as provided by s and compositions of the invention. By “low molecular weight NASP” is meant a NASP having a weight average molecular weight that ranges from about to 30,000 daltons, such as for example 100 to 30,000 daltons, such as 500 to 25,000 daltons, such as 1000 to 15,000 daltons and including 5000 to 10,000 daltons. Examples of low molecular weight NASPs may include, but are not limited to naturally occurring or synthetic NASPS having a molecular weight g from 10 to 30,000 daltons, such as from 5000 to 10,000 daltons, fragments of larger molecular weight NASPs produced by acid or enzyme hydrolysis of the larger molecular 2O weight NASP, or may be isolated fractions having molecular weights ranging from 10 to 30,000 daltons,such as 5000 to 10,000 daltons from a fractionated NASP sample.

In certain embodiments, itions of the present invention include a gastrointenstinal epithelial barrier permeation enhancer (e.g., sodium caprate, deoxycholate, bromelain or an) and a low molecular weight natural NASP. For instance, compositions of interest may e a Fucus vesiculosus fucoidan having a molecular weight of 20,000 daltons or less in combination with a gastrointenstinal epithelial barrier permeation enhancer. For e, compositions of interest may include one or more of Fucus vesiculosus Ev. DSlOOl 108, Fucus vesiculosus F.v. SK110144B in combination with one or more of deoxycholate, bromelain and chitosan. In r example, compositions of interest may include one or more of FUCHS vesiculosus Ev. D8100] 108, Furns vesiculosus F.v. SKl 10144B in combination with a mixture of chitosan and bromelain. In certain other embodiments, itions of the present ion include a gastrointestinal epithelial barrier permeation er (e.g., sodium caprate, deoxycholate, bromelain or chitosan) and low lar weight synthetic NASP. For instance, certain compositions may include one or more of a sulfated 6- carboxyicodextrin, a ed B-cyclodextrin and a sulfated maltopentose having a molecular weight of 25,000 daltons or less in combination with a gastrointestinal epithelial r permeation enhancer. For example, compositions of interest may include one or more of a 24,000 dalton sulfated WO 12954 6-carboxyicodextrin, a 14,000 dalton sulfated 6-carboxyicodextrin, a sulfated B-cyclodextrin and a sulfated maltopentose in combination with one or more of deoxycholate, bromelain and chitosan. In another example, itions of interest may include one or more of a 24,000 dalton sulfated 6— carboxyicodextrin, a 14,000 dalton sulfated 6-carboxyicodextrin, a sulfated B-cyclodextrin and a sulfated maltopentose in combination with a mixture of chitosan and bromelain.

In some embodiments, NASPs are ted from a biological source and may be fractionated to isolate low molecular weight NASPs (i.e., fractions containing NASPs having molecular weight ranging from lO-30,000 daltons). Any convenient protocol may be used to fractionate NASPs of interest, including but not limited to size exclusion chromotagraphy, gel tion chromotagraphy, capillary electrophoresis, among others.

In certain instances, low molecular weight NASPs obtained by fractionating a NASP sample may be employed for ing blood coagulation as provided by the methods and compositions of the invention. For example, NASPs extracted from a biological source may be fractionated to isolate NASPs having molecular weights that range from 10 to 30,000 daltons, such as 10 to 5000 daltons, such as 5000 to 10,000 daltons, such as 10,000 to 15,000 daltons, and including 15,000 to 30,000 daltons. In certain embodiments, one or more of these fractions may be orally administered in combination with a gastrointestinal epithelial barrier permeation enhancer for enhancing blood coagulation in a subject, such as by the methods described above.

In certain embodiments, different molecular weight ons may be prepared by acid- hydrolysis or radical depolymerization of high molecular weight NASP. The molecular weight ranges of the resulting products may be adjusted based upon the stringency of the hydrolysis or depolymerization conditions employed. ons may then be further purified using ion exchange chromatography. For instance, to obtain middle and low molecular weight fractions of NASP, high molecular weight NASP may be hydrolyzed using an acid such as HCl (or any other suitable acid) at concentrations ranging from 0.02 to 1.5 M and at temperatures g from 25°C to 80°C.

Hydrolysis on times will typically range from 15 minutes to several hours. The resulting hydrolyzed on mixture is then neutralized by on of base (e.g., sodium hydroxide). Salts are subsequently removed, for example, by electrodialysis, and the hydrolysis products are analyzed to ine weight average molecular weight, saccharide content, and sulfur content, using conventional analytical techniques for carbohydrate is. Alternatively, enzymatic s may be employed to degrade NASPs using, e.g., glycosidases. NASPs for use in the invention may be heterogeneous or homogeneous, depending upon the degree of separation employed.

In certain ments, oral dosage compositions of the ion include a blood coagulation factor in ation with a gastrointestinal lial barrier permeation enhancer and a fucoidan, such as for example, Fucoidan 5307002, Fucus vesiculosus, max. MW peak 126.7 kD; Fucoidan VG49, Fucu: vesiculosus, hydrolyzed sample of 5307002 of lower MW, max. MW peak PCT/U320121047248 22.5 kD; Fucoidan VG57, Undaria pinnarifida, high charge (high sulfation, deacetylated); Fucoidan GFS (5508005), Undarz‘a pinnatifida, depyrogenated; Fucoidan GFS (L/FVF-01091), Fucus vesiculosus, depyrogenated, max. MW peak 125 kD; Fucoidan GFS (L/FVF—O1092), Fucus vesiculosus, depyrogenated, max. MW peak 260 kD; Fucoidan GFS (L/FVF-01093), Fucus UI vesiculosus, yzed depyrogenated, max. MW peak 36 kD; Maritech® ia a extract; Maritech® Ecklom‘a maxima extract; Maritech® ystis pyrifera extract; Maritech® Immune trial Fucoidan Blend; and any combinations thereof, In other embodiments, oral dosage compositions of the invention may include a synthetic NASP in combination with a gastrointestinal epithelial barrier permeation enhancer. Synthetic NASPs are ed polysaccharides which are partially or wholly produced by man—made methods (e.g., chemical synthesis). For example, the synthetic NASP may be a sulfated oligomer, such as a sulfated oligosaccharide or a sulfated aliphatic. In certain ces, tic NASPs are ed pentoses, sulfated hexoses or sulfated cyclodextrins. For example, synthetic NASPs may include, but are not limited to sulfated maltopentoses, sulfated betacyclodextrins , sulfated 6-Carboxyicodextrin and derivatives f.

Oral dosage compositions of the ion may include one or more NASPs, as desired.

For example, two or more NASPS may be combined, such as three or more NASPs and including four or more NASPs. Where more than one NASP is combined together, all of the NASPs may be natural NASPs, all of the NASPS may be synthetic NASPS or any combination f.

Where oral compositions e more than one NASP, the mass percentage of each NASP in the composition may vary, g from 1% or more of the total mass of the composition, such as 2% or more, such as 5% or more, such as 10% or more, such as 25% or more and including as 50% or more of the total mass of the composition.

In embodiments of the invention, the mass ratio of the one or more NASPs and the one or more gastrointestinal epithelial barrier permeation enhancers in the oral dosage compositions may vary, ranging between 1:1 and 1:25; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of the NASP to the gastrointestinal epithelial barrier permeation enhancer may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio of the gastrointestinal epithelial barrier permeation enhancer to the NASP ranges between 1:1 and 1:25; 1:2.5 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of the gastrointestinal epithelial barrier permeation enhancer to the NASP may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. -38— In addition, oral dosage compositions of the invention may also include one or more blood coagulation factors. For example, compositions may include an amount of one or more NASPS and one or more gastrointestinal epithelial barrier permeation enhancers in combination with one or more blood coagulation factors. Blood coagulation s of interestinclude, but are LII not limited to factor XI, factor XII, prekallikrein, high molecular weight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factor XIII, factor II, factor VIIa, and von Willebrands , factor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen, tissue factor, factor VIIa, factor Va, and factor Xa.

The amount (i.e., mass) of each of the NASP, the gastrointestinal epithelial barrier '10 permeation enhancer and blood coagulation factor in oral dosage compositions of interest may vary, ranging from 0.001 mg to 1000 mg, such as 001 mg to 500 mg , such as 0.1 mg to 250 mg, such as 0.5 mg to 100 mg, such as 1 mg to 50 mg, including 1 mg to 10 mg. As such, in the subject compositions, the mass ratio of the NASP and gastrointestinal epithelial barrier permeation enhancer to blood coagulation factor may vary, and in some instances may range between 1:1 and 1:25; 1:25 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and :50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of the NASP and gastrointestinal epithelial barrier permeation enhancer to blood coagulation factor may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 1:1000. In some embodiments, the mass ratio of the blood coagulation factor to the NASP and gastrointestinal epitheliaI barrier permeation enhancer ranges n 1:1 and 1:25; 1:25 and 1:5; 1:5 and 1:10; 1:10 and 1:25; 1:25 and 1:50; 1:50 and 1:100; 1:100 and 1:150; 1:150 and 1:200; 1:200 and 1:250; 1:250 and 1:500; 1:500 and 1:1000, or a range thereof. For example, the mass ratio of the blood coagulation factor to the composition that contains a NASP and gastrointestinal epithelial barrier permeation enhancer may range between 1:1 and 1:10; 1:5 and 1:25; 1:10 and 1:50; 1:25 and 1:100; 1:50 and 1:500; or 1:100 and 111000.

Oral dosage compositions may be homogeneous, containing only a single type of NASP and single type of gastrointestinal lial permeation barrier enhancer. In other ments, itions of interest are heterogenous mixtures of two or more NASPs or two or more gastrointestinal lial permeation barrier enhancers. For example, heterogenous mixtures may 3O n two or more NASPs and two or more gastrointestinal epithelial permeation barrier ers.

In other instances, heterogeneous es may contain one NASP and two or more gastrointestinal epithelial permeation barrier enhancers. In yet other instances, heterogeneous mixutres may contain two or more NASPs and one gastrointestinal epithelial permeation barrier enhancer, In certain embodiments, oraI dosage compositions of the invention may further include one or more pharmaceuticaliy acceptable excipients or oral dosage delivery vehicle as part of a pharmaceutical ition. Excipients may include, but are not d to, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, water, alcohols, polyols, PCT/U820121047248 glycerine, vegetable oils, phospholipids, buffers, acids, bases, and any ations thereof. A carbohydrate such as a sugar, 21 derivatized sugar such as an alditol, aldonic acid, an fied sugar, and/or a sugar polymer may also be employed. Some carbohydrate excipients of st include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D- U! mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raftinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, l, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. Inorganic salts may include, but are not limited to citric acid, sodium chloride, potassium chloride, sodium e, potassium nitrate, sodium ate monobasic, sodium phosphate dibasic, and any combinations thereof.

In certain embodiments, oral dosage compositions of the invention may also include an antimicrobial agent for preventing or deterring microbial growth, such as for example konium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and any combinations thereof.

One or more antioxidants may also be employed. Antioxidants, which can reduce or prevent oxidation and thus deterioration of the composition, may include, for example, ascorbyl ate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium dehyde sulfoxylate, sodium metabisulfite, and any combinations thereof.

One or more surfactants may also be included in compositions of the invention. For example, suitable surfactants may include, but are not limited to polysorbates, such as "Tween " and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as olipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other s.

Acids or bases may also be present in oral dosage compositions of the invention. For example, acids may include but are not limited to hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and any combinations thereof. Examples bases include, but are not limited to sodium hydroxide, sodium acetate, ammonium hydroxide, potassium ide, ammonium acetate, potassium acetate, sodium phosphate, ium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium te, and any combinations f.

The amount of any individual excipient in the oral dosage composition will vary ing on the nature and function of the excipient, oral dosage delivery vehicle and particular needs of the composition. Typically, the l amount of any individual excipient is PCT/U32012/047248 determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant e effects. Generally, however, the excipient(s) will be present in the oral dosage composition in an amount of about 1% to about 99% by weight, such as from about 5% to about 98% by weight, such as from about 15 to about 95% by weight of the excipient, including less than 30% by weight. Pharmaceutical excipients along with other excipients that may be employed in compositions of interest are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk nce", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of ceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000, the sure of which is herein incorporated by reference.

As described above, compositions of the ion may be stered by convenient mode of administration so long as the ition is resorbed through the gastrointestinal epithelium (e.g., orally or by nasogastric tube). As such, the formulation may vary. For example, compositions of the invention may be powders or lates that can be reconstituted with a solvent prior to use, dry insoluble compositions for combination with vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration.

Diluents for reconstituting solid compositions may include, but are not limited to bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, ’s solution, saline, sterile water, deionized water, and any combinations thereof. In some embodiments, pharmaceutical compositions of the invention may be in the form of a liquid solution or suspension, syrup, tablet, capsule, powder, gel, or any combination thereof for ingestion or ation by a nasogastric tube. For e, oral dosage compositions of the invention be pre-loaded into a tablet, a e, caplet device, or the like, depending upon the intended use.

In certain embodiments, the compositions are in unit dosage form, such that an amount of the composition is ready in a single oral dose, in a premeasured or pre-packaged form.

UTILITY The t methods and compositions find use in any situation where there is a desire to e blood coagulation in a subject, a desire to enhance resorption of NASPs through the gastrointestinal system and the subject is responsive to treatment with a NASP and a gastrointestinal epithelial barrier permeation enhancer. In certain ments, the subject methods and compositions may be employed to treat bleeding disorders, such as a chronic or acute bleeding disorder, a congenital coagulation disorder caused by a blood factor deficiency, an acquired coagulation disorder and stration of an agulant. For example, bleeding PCTIU52012/047248 disorders may include, but are not limited to hemophilia A, hemophilia B, von Willebrand disease, idiopathic thrombocytopenia, a deficiency of one or more contact factors, such as Factor XI, Factor XII, prekallikrein, and high lar weight kininogen (HMWK), a deficiency of one or more factors associated with clinically significant bleeding, such as Factor V, Factor VII, U! Factor VIII, Factor IX, Factor X, Factor XIII, Factor II rothrombinemia), and von Willebrands factor, a vitamin K deficiency, a er of fibrinogen, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia, an alphaz—antiplasmin deficiency, and excessive bleeding such as caused by liver e, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hemarthroses, y, trauma, hypothermia, menstruation, and ncy.

The subject methods and compositions also find use in enhancing blood coagulation to treat a congenital coagulation er or an acquired coagulation disorder caused by a blood factor deficiency. The blood factor deficiency may be caused by deficiencies of one or more factors, including but not limited to, factor V, factor VII, factor VIII, factor IX, factor XI, factor XII, factor XIII, and von Willebrand .

The subject methods and compositions also find use in enhancing blood coagulation in order to improve hemostasis in treating bleeding disorders, such as those associated with deficiencies of coagulation factors or for reversing the effects of anticoagulants in a subject. For e, enhancing blood coagulation by methods and compositions of the invention may be employed to treat bleeding ers such as congenital coagulation disorders, acquired coagulation disorders, and hemorrhagic conditions induced by trauma. Examples of bleeding disorders that may be treated with NASPs and gastrointestinal epithelial barrier permeation enhancers include, but are not limited to, hemophilia A, hemophilia B, von rand disease, thic thrombocytopenia, a deficiency of one or more contact factors, such as Factor XI, Factor XII, prekallikrein, and high molecular weight kininogen (HMWK), a deficiency of one or more factors associated with clinically significant bleeding, such as Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia), and von Willebrands factor, a vitamin K ency, a er of fibrinogen, including nogenemia, hypofibrinogenemia, and dysfibrinogenenna, an alphayantiplasmin deficiency, and excessive 3O bleeding such as caused by liver disease, renal disease, thrombocytopenia, et dysfunction, hematomas, internal hemorrhage, hemarthroses, surgery, trauma, hypothermia, menstruation, and pregnancy. In certain embodiments, methods and compositions of the ion are used to treat congenital coagulation ers including hemophilia 1A, hemophilia B, and von Willebrands disease. In other embodiments, NASPs are used to treat acquired coagulation disorders, including deficiencies of factor VIII, von Willebrand factor, factor IX, factor V, factor XI, factor X11 and factor XIII, particularly disorders caused by inhibitors or munity against blood PCT/U82012/047248 coagulation factors, or haemostatic disorders caused by a disease or ion that results in reduced synthesis of coagulation factors.

In some ments, the bleeding disorder may be a c ion (e.g., a congenital or acquired coagulation factor deficiency) requiring the subject methods and compositions in multiple doses over an extended . Alternatively, methods and compositions of the ion may be orally administered to treat an acute condition (e. g., bleeding caused by surgery or trauma, or factor inhibitor/autoimmune episodes in ts receiving coagulation ement therapy) in single or multiple doses for a relatively short period, for example one to two weeks.

IO The subject methods and compositions also find use in enhancing blood coagulation in a subject undergoing a surgical or invasive procedure.

The subject methods and compositions also find use in enhancing blood coagulation in order to e the s of an anticoagulant in a subject, the method comprising stering a eutically effective amount of a composition comprising a procoagulant amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer to the subject. In certain embodiments, the subject may have been treated with an anticoagulant including, but not limited to, heparin, a coumarin derivative, such as warfarin or dicumarol, TF'PI, AT III, lupus anticoagulant, de anticoagulant peptide (NAPCZ), active-site blocked factor VIIa (factor Vflai), factor IXa inhibitors, factor Xa inhibitors, including fondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906), inhibitors of factors Va and VIIIa, including activated protein C (APC) and soluble thrombomodulin, thrombin inhibitors, including hirudin, bivalirudin, argatroban, and ximelagatran. In certain embodiments, the anticoagulant in the subject may be an antibody that binds a clotting factor, including but not limited to, an antibody that binds to Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II, Factor XI, Factor XII, von Willebrands factor, prekallikrein, or high molecular weight kininogen (HMWK).

In another aspect, the invention provides a method for treating a subject undergoing a surgical or ve procedure wherein ed blood clotting would be desirable, comprising orally administering a therapeutically effective amount of a composition comprising a 3O procoagulant amount of a NASP in combination with a gastrointestinal epithelial barrier permeation enhancer as detailed herein to the subject. In certain embodiments, the NASP and gastrointestinal epithelial barrier permeation er can be coadministered with one or more different NASPs and one or more different gastrointestinal epithelial r permeation enhancers, and/or in combination with one or more other therapeutic agents to the subject oing a surgical or invasive procedure. For example, the subject may be administered a therapeutically effective amount of one or more s selected from the group consisting of factor XI, factor XII, prekallikrein, high molecular weight kininogen (HMWK), factor V, factor PCT/U82012l047248 VII, factor VIII, factor IX, factor X, factor XIII, factor II, factor VIIa, and von Willebrands factor. Treatment may further comprise administering a procoagulant, such as an activator of the intrinsic coagulation pathway, including factor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen; or an activator of the extrinsic coagulation UI pathway, including tissue factor, factor VIIa, factor Va1 and factor Xa. Therapeutic agents used to treat a subject undergoing a surgical or invasive procedure can be administered in the same or different compositions and concurrently, , or after administration of the NASP and the gastrointestinal epithelial barrier permeation enhancer.

As disclosed above, hemostatic agents, blood factors, and medications may also be employed. For e, the subject may be administered one or more blood ation s such as factor XI, factor XII, prekallikrein, high molecular weight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factor XIII, factor II, factor VIIa, von Willebrands factor, factor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa, prekallekrein, and high~ molecular weight kininogen, tissue factor, factor VIIa, factor Va, and factor Xa. 1(rrs Also provided are kits for use in practicing the subject methods, where the kits may include one or more of the above compositions, e.g., an NASP composition, a gastrointestinal epithelial r permeation enhancer composition and/or blood coagulation factor, as bed above. The kit may further include other components, e.g., administration devices, fluid sources, etc., which may find use in practicing the subject methods. Various ents may be packaged as desired, e.g., together or tely.

In addition to above mentioned ents, the subject kits may further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally ed on a suitable recording medium. For example, the instructions may be printed, such as on paper or plastic, etc. As such, the instructions may be present in the kits as a e , in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable 3O computer readable storage medium, e.g. CD~ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote , e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the ctions can be aded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

WO 12954 PCT/'US2012/047248 EXPERIMENTAL The ing es are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used (e. g., amounts, temperatures, etc), but some mental error and deviation should, of course, be d for.

Bioavailability and Resorgtion Studies of NASPs in combination with Gastrointestinal Epithelial Barrier Permeation Enhancers The bioavailability of NASPs in combination with gastrointestinal epithelial r permeation enhancers of st were studied using the CaCo-2 cell model screening. This method utilizes a human colon carcinoma cell line that expresses a wide range of transporter proteins on its cell membranes. Cell layers are grown on a membrane surface that separates two compartments (24-well plate). An example of the experimental setup for these experiments is illustrated in Figure 1. Selected NASP and gastrointestinal epithelial barrier permeation enhancer samples were dissolved in RPMI cell medium at a concentration of 1 mg/mL and applied onto the cells in the apical compartment. Cells were incubated at 37 °C in 5 % C02.

Medium samples were removed from the basolateral and apical tment at different time points. The condition of the cell layer was monitored by measurement of the transepithelial electrical resistance . Samples were analyzed by thrombin generation assay (CAT).

NASP concentration was calculated based on activity from CAT assay. Experimental details of thrombin generation assays and other blood coagulation assays are described in United States Patent ation Serial No. 11/140,504, filed on May 27, 2005, now United States Patent No. 7,767,654, and United States Patent Application Serial No. 13/006,396, filed on January13, 201 1, the disclosures of which is herein incorporated by reference.

All NASP and gastrointestinal epithelial barrier permeation enhancer samples were d in such a way that the sample concentration was in the range of increasing procoagulant activity. Based on the initial load concentration values, apical and basolateral concentrations were determined at 2 hour increments (e.g., 2 hours, 4 hours, 6 hours, 8 hours, including 24 hours). Based on the determined basolateral concentrations, the t resorption was determined for each combination of compounds (i.e., NASP and gastrointestinal lial barrier permeation ers).

An example of the resorption s described herein is illustrated in Tables 1-3 which summarize the apical and basolateral concentrations of the NASP, Fucoidan F.v. 1091 in combination with the gastrointestinal epithelial barrier permeation enhancer, Bronielain (0.5 mg/mL) in the Caco-2 cell model. Table 1 illustrates that the starting apical concentration of RV.

PCTx’U$2012/047248 91 in combination with Bromelain is about 840 ug/mL. After about 8 hours, the apical concentration of RV. L/FVF 1091 is reduced by about 25% to an average of about 627 ug/mL.

Table 1: Sample Start (mg/mL) 8 hours (mg/mL) Ev. LflWF 1091 —Set 1 670 F.v. L/FVF1091— Set 2 840 F.v. L/FVF1091— Set 3 Table 2 illustrates the basolateral concentrations of RV. L/FVF 1091 in combination with ain at various time points. Figure 2 shows the basolateral concentrations of RV. L/FVF 1091 in the presence of ain (0.5 nag/ml.) in the Caco-2 system. Figure 3 shows Lhe condition of the cell layer as measured by the corresponding transepithelial electrical resistance curves of each trial.

Table 2: Sample Concentration (us/mm Ev. L/FVF 1091 —Set 1 5.3 14.5 292 Ev. L/FVF1091— Set 2 05 2.1 6.5 F.v.L/FVF1091—- Set 3 Theoretical Maximum Table 3 illustrates the percent (%) resorption of RV. L/FVF 1091 in the presence of Bromelain at various time .

Table 3: Percent Resorption “T753135 4 hours 6 hours 8 hours _' Dilution Factor I 0.74 F.v. L/FVF 1091 —Set 1 20.7 F.v. L/FVF1091— Set 2 4.6 Ev. L/FVF 1091 — Set 3 Based on Tables 2 and 3, the average resorption is 19 mg/mL of RV. L/FVF 1091 in combination with Bromelain at 8 hours and trate that the basolateral concentration of RV.

UFVF 1091 increases with time in the presence of the gastrointestinal epithelial r permeation enhancer, ain.

Table 4 is a summary of percent resorption ofnatural NASPs in the Caco-2 cell model in the presence of several gastrointestinal epithelial permeation enhancers at various concentrations. As shown in Table 4, all of the natural NASPs demonstrated increased resorption in the presence of the gastronintestinal epithelial barrier tion ers of interest.

Table 4: Average Percent U.p.5508005 F.v. L/FVF F.v. F.v.

Resorption at 8 hours 1091 DS1001108 SK110144B Molecular Weight 380 kD 143 kD 18 kD 12 kl) No enhancer 0.1 0.7 0.4 4.0 Sodium Caprate . n/a 12 mM Deoxycholate - 0.06% . 10.7 Deoxycholate - 0.08% 4.7 10.9 10.4 22.6 Bromelain - 0.5 mg/mL 6.2 8.6 28.9 Chitosan - 3% 2.5 2.5 1.6 9.0 Bromelain/Chitosan 19.6 36.9 _1 28.8 55.0 0.5 mg/mL:3% __‘- BromelainiChitosan n/a 3.1 6.7 n/a 0.05 mg/mL:3% Bromelain/Chitosan n/a 4.4 7.3 n/a 0.25 mg/mL:1.5% Bromelain/Chitosan n/a 2.6 8.7 n/a 0.5 L:O.3 % Table 5 is a y of percent resorption of synthetic NASPs in the Caco~2 cell model in the presence of several gastrointestinal epithelial permeation enhancers at various concentrations. As shown in Table 5, all of the synthetic NASPs demonstrated increased resorption in the presence of the gastronintestinal epithelial barrier permeation enhancers of interest.

Table 5: 2012/047248 Average Percent Sulfated 6— Sulfated 6- ed {3- Sulfated Resorptiou at 8 hours Carboxyicodextrin Carboxyicodextrin extrin Maltopentose Molecular Weight 24kD 14kD No enhancer Sodium Caprate 12 mM Deoxycholate - 0.06% Deoxycholate - 0.08% Bromelain ~ 0.5 mg/mL Chitosan - 3% Bromelain/Chitosan 0.5 3% Figures 4-8 illustrate resorption of some natural NASPs of interest in Caco-2 cell models as determined by TGA and liquid chromatography/mass spectrometry in combination with different gastrointestinal epithelial barrier permeation enhancers. Figures 9 and 10 illustrate resorption of some synthetic NASPS of interest in Caco-Z cell models as determined by TGA and liquid chromatography/mass spectrometry in combination with different gastrointestinal epithelial barrier permeation enhancers.

Figures 4a—b illustrate the NASP, BAXS 13 resorption in Caco—Z cell models in the absence of any gastrointestinal epithelial barrier permeation enhancer as well as in the ce of bromelain, chitosan and in the presence of a combination of bromelain and chitosan. As rated in Figures 4a—b, basolateral concentrations of BAX513 are increased in the presence of the gastrointestinal epithelial barrier tion enhancer, indicating that resorption of BAXS 13 increases when administered in combination with a gastrointestinal epithelial barrier permeation enhancer.

Figures Sa-c rate the NASP, Fucoidan F.v. L/FVF 1091 resorption in Caco-2 cell models in the absence of any gastrointestinal epithelial barrier permeation enhancer as well as in the presence of several ent gastrointestinal epithelial r permeation enhancers sodium caprate, deoxycholate, bromelain, chitosan and in the presence of a combination of bromelain and chitosan. s Sa—c demonstrates that the basolateral concentrations of Fucoidan Fiv.

L/FVF 1091 increased in the presence of the gastrointestinal epithelial barrier permeation enhancer, indicating that resorption of an F.v. L/FVF 1091 increases when administered in combination with a gastrointestinal epithelial barrier permeation er. In particular, the tion of Fucoidan F.v. L/FVF 1091 increases substantially when administered in the WO 12954 PCT/U82012/047248 ce of a combination of chitosan and bromelain.

Figures 6a—b show the NASP, an U.p. 5 resorption in Caco-2 cell models in the absence of any gastrointestinal epithelial barrier permeation enhancer as well as in the presence of gastrointestinal epithelial r permeation enhancers sodium caprate, deoxycholate, bromelain, chitosan and in the presence of a combination of ain and chitosan. Figures 6a—b demonstrates that the basolateral concentrations of Fucoidan U.p. 5508005 increased in the presence of a gastrointestinal epithelial barrier permeation enhancer, indicating that resorption of Fucoidan U.p. 5508005 increases when administered in combination with a gastrointestinal epithelial barrier tion enhancer. In particular, the resorption of Fucoidan Up. 5508005 increased substantially in the presence of sodium caprate, bromelain and in the ce of a combination of chitosan and bromelain.

Figures 7a-b depict the NASP, Fucoidan FvF DSIOOllOSB resorption in Caco-2 cell models in the absence of any gastrointestinal epithelial barrier permeation enhancer as well as in the ce of gastrointestinal lial barrier permeation enhancers deoxycholate (at different concentrations), bromelain, chitosan and in the presence of a combination of bromelain and chitosan. Figures 7a-b illustrates that the basolateral concentrations of Fucoidan FvF DSlOOl 108B increased in the presence of a gastrointestinal epithelial barrier permeation enhancer, indicating that resorption of an FVF DS 1001 108B increases when administered in combination with a gastrointestinal epithelial barrier permeation enhancer. In particular, the resorption of an FVF DSlOOl 108B increases substantially when administered in the presence of a combination of chitosan and bromelain.

Figures 8a-b show the NASP, Fucoidan FVF SKI lOl44B resorption in Caco-2 cell models in the absence of any gastrointestinal epithelial barrier permeation er as well as in the presence of gastrointestinal epithelial barrier permeation enhancers deoxycholate (at different concentrations), bromelain, chitosan and in the presence of a combination of bromelain and chitosan. Figures 8a-b demonstrates that the teral concentrations of Fucoidan FVF SKIlOl44B increased in the presence of a gastrointestinal epithelial barrier permeation enhancer, indicating that resorption of Fucoidan FvF SKI 101448 increases when administered in combination with a gastrointestinal lial barrier tion enhancer. In ular, the 3O resorption of Fucoidan FVF 8K110144B increased substantially in the presence of 0.08% deoxycholate, bromelain, and in the presence of a combination of an and bromelain.

Figures 9 and 14 show the synthetic NASP, sulfated B-cyclodextrin resorption in Caco-Z cell models in the absence of any gastrointestinal epithelial barrier permeation enhancer as well as in the presence of gastrointestinal epithelial barrier permeation enhancers deoxycholate, bromelain and sodium caprate. Figure 9 demonstrates that the basolateral concentrations of sulfated B-cyclodexttin increased in the presence of a intestinal epithelial ban‘ier S2012/047248 permeation enhancer, indicating that tion of sulfated B-cyclodextrin ses when administered in combination with a gastrointestinal epithelial barrier permeation enhancer. In particular, the resorption of sulfated B—cyclodextrin increased substantially in the presence of all three of holate, bromelain, and sodium caprate at 8 hours.

Figures 10 and 15 show the tic NASP, sulfated maltopentaose resorption in Caco- 2 cell models in the absence of any gastrointestinal epithelial barrier permeation enhancer as well as in the ce of gastrointestinal epithelial barrier permeation ers deoxycholate, bromelain, chitosan and sodium caprate. Figure 10 trates that the basolateral concentrations of sulfated maltopentaose increased in the presence of a gastrointestinal epithelial barrier permeation enhancer, indicating that resorption of sulfated maltopentaose increases when administered in combination with a gastrointestinal epithelial barrier permeation enhancer. In particular, the resorption of sulfated maltopentaose increased substantially in the presence of deoxycholate and bromelain at 8 hours. Sulfated maltopentaose also showed strong increased resorption in the presence of chitosan and sodium caprate.

As demonstrated in Figures 4-10, the basolateral concentrations of both l and synthetic NASPs are increased in the presence of each of the gastrointestinal epithelial barrier permeation enhancers, indicating that resorption of the NASP increases when administered in combination with a gastrointestinal lial barrier tion enhancer as compared to resorption in the absence of gastrointestinal epithelial barrier permeation enhancer.

Figures lla-b illustrates the effect of different concentrations of chitosan and bromelain when a combination of an and ain is employed in combination with NASPs Fucoidan F.v. L/FVF 1091 (Figure 11a) and Fucoidan FVF DSlOOl 1088 (Figure 11b). In particular, combinations of chitosan and brornelain include 0.3% chitosan and 0.5 mg/ml bromelain; 1.5% chitosan and 0.25 mg/mL bromelain; 3% chitosan and 0.05 mg/mL bromalein; and 3% an and 0.5 mg/mL bromelain. As illustrated in Figures lOa-b, a combination of 3% chitosan and 0.5 mg/mL bromelain demonstrated the strongest tion of the NASP.

Table 6 is a summary of percent resorption of natural and synthetic NASPs in the Caco—2 cell model. As shown in Table 4, all of the NASPs demonstrated increased resorption in the presence of the gastronintestinal epithelial barrier tion enhancers of interest.

Table 6 Avg. BAX513 F.v.L/FVF Up. 5508005 ‘F.V.D81001108B Sulfated tion at 1091 Maltopentaose 8 hours cyclodextrin 180w— 380w PCT/U82012/047248 No enhancer 0.3 0.6 Sodium Caprate (12 mM) Deoxycholate 67.4 (0.06%) Deoxycholate (0.08%) Bromelain 48.6 (0.5 mg/mL) Chitosan (3%) Bromelajn (0.5 mg/mL) and Chitosan (3%) Figures lZa-b and 13a-c shows the ion of the cell layer as measured by transepithelial electrical resistance in the ce and absence of a gastrointestinal epithelial barrier permeation enhancers when tested with sulfated B-cyclodextrin and sulfated (ll maltopentose, respectively. As depicted in Figures lZa—b, in the ce of sulfated B- cyclodextrin, gastrointestinal epithelial r permeation enhancer bromelain reduce the transepithelial electrical resistance values substantially (<300 ohms/cmz). The transepithelial electrical resistance values remained low after on of fresh medium. Treatment with the gastrointestinal epithelial barrier permeation er alone however, resulted in the recovery of the transepithelial electrical resistance. Likewise, as depicted in s l3a-c, in the ce of sulfated maltopentose, gastrointestinal epithelial barrier permeation enhancers bromelain, deoxycholate and chitosan reduce the transepithelial electrical resistance values substantially. r, in contrast to studies as ed in Figures lZa-b for sulfated B—cyclodextrin, the transepithelial electrical resistance increased after removal of the gastrointestinal epithelial barrier permeation enhancer and the maltopentaose.

PCT/U52012/047248 Although the foregoing ion has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this ion that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention, It will be appreciated that those skilled in the art will be able to devise various arrangements which, gh not itly described or shown herein, embody the principles of the invention and are included within its spirit and scope. rmore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the ors to furthering the art, and are to be construed as being without tion to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any ts developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims (15)

What is claimed is:

1. Use of a procoagulant amount of a non—anticoagulant sulfated polysaccharide (NASP) selected from the group consisting of N—acetyl-heparin (NAH), N-acetyl—de-O-sulfated— n (NA—de—o-SH), de-N—sulfated—hepan'n (De-NSH), de—N—sulfated—acetylated-heparin (De—NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin—derived oligosaccharides (HDO), pentosan polysulfate (PPS), fucoidans, sulfated maltopentoses, sulfated beta-cyclodextrins, sulfated 6~Carboxyicodextrin in combination with chitosan and ain in the manufacture of a medicament for ing blood coagulation in a subject.

2. The use according to Claim 1, wherein the amount of NASP formulated for administration to the subject ranges from 0.01 mg/kg to about 100 mg/kg.

3. The use according to any of Claims 1—2, n the NASP is a naturally occurring synthetic NASP.

4. The use according to any of Claims 1-3, wherein the NASP is a naturally occurring NASP ed from the group ting of N—acetyl-heparin (NAH), N—acetyl-de-O- ed-heparin (NA—de-o—SH), de—N—sulfated—heparin (De-NSH), de-N—sulfated—acetylated— he- parin (De-NSAH), ate~oxidized heparin (POH), chemically sulfated rin (CSL), chemically sulfated alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS) and fucoidans, and combinations thereof.

5. The use according to any of Claims 1—3, wherein the NASP is a synthetic NASP selected from the group consisting of sulfated oligomers, sulfated pentoses, sulfated hexoses or sulfated extrins.

6. The use according to any of Claims 1—5, wherein the medicament further comprises one or more factors ed from the group consisting of factor XI, factor XII, prekallikrein, high molecular weight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factor XIII, factor II, von Willebrands factor, tissue factor, factor Vlla, factor Va, and factor Xa, factor IXa, factor Xla, factor Xlla, and VIIIa.

7. The use according to any of Claims 1—6, wherein the subject: (a) has a bleeding disorder selected from the group ting of a chronic or acute bleeding disorder, a ital coagulation disorder caused by a blood factor deficiency, and an acquired coagulation disorder; or (b) is in need of enhanced blood coagulation because of prior administration of an anticoagulant; or (c) is in need of enhanced blood coagulation e of a surgical or other invasive procedure.

8. The use according to any of Claims 1-7, wherein the ment inhibits TFPI activity in the subject.

9. The use according to any one of claims 1-8, wherein the ratio of chitosan to bromelain is sufficient to enhance tion 2-fold or greater than would be achieved by the sum of an or bromelain individually.

10. The use according to any one of claims 1-9, n the amount of chitosan formulated for administration to the subject is about 0.73% to about 3% and the amount of bromelain formulated for administration to the subject is about 0.05 mg/mL to about 0.5 mg/mL.

11. An oral dosage composition comprising: (a) a pro—coagulant amount of a non—anticoagulant sulfated polysaccharide (NASP) selected from the group consisting of N-acetyl~heparin (NAH), N—acetyl—de—O- sulfated—heparin (NA-de-o-SH), de-N~sulfated—heparin (De—NSH), de—N—sulfated-acetylated— heparin (De—NSAH), periodate-oxidized heparin (POH), chemically sulfated laminarin (CSL), chemically ed alginic acid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS), heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS), ans, sulfated maltopentoses, sulfated beta-cyclodextrins, sulfated 6-Carboxyicodextrin; (b) chitosan and bromelain; and (c) an oral dosage delivery vehicle; wherein the oral dosage composition is in unit dosage form.

12. The oral dosage composition according to claim 11, wherein the ratio of chitosan to bromelain is sufficient to enhance permeation 2—fold or r than would be achieved by the sum of chitosan or bromelain individually.

13. The oral dosage composition according to claim 11, wherein the amount of an formulated for administration to the subject is about 0.3% to about 3% and the amount of bromelain formulated for administration to the t is about 0.05 mg/rnL to about 0.5 mg/mL.

14. The use of a procoagulant amount of a non—anticoagulant sulfated ccharide (NASP) in combination with chitosan and bromelain according to any one of claims 1 to 10, substantially as described herein.

15. An oral dosage composition according to any one of claims 11 to 13, substantially described herein. PCTIU