The present invention provides bioconjugates comprising a sulfated polysaccharide such as alginate sulfate and hyaluronan sulfate and at least one bioactive polypeptide capable of binding a sulfate group of said sulfated polysaccharide. The bioactive polypeptide can be a heparin-binding polypeptide and/or a positively-charged polypeptide. Also, provided are delivery systems and methods for sustained release of said bioactive polypeptide(s) using said bioconjugates.

Description of the Invention

SUMMARY OF THE INVENTION

It has now been found according to the present invention that a bioconjugate comprising a sulfated polysaccharide, such as alginate sulfate and hyaluronan sulfate, and at least one bioactive peptide capable of binding a sulfate group of said sulfated polysaccharide, can direct the sustained release of said at least one bioactive peptide from said bioconjugate.

Thus, the present invention relates to a bioconjugate comprising a sulfated polysaccharide and at least one bioactive polypeptide capable of binding a sulfate group of said sulfated polysaccharide.

The present invention further relates to a pharmaceutical composition comprising a bioconjugate of the invention, in particular, as a delivery system for sustained release of bioactive polypeptide(s).

The present invention relates also to pharmaceutical compositions comprising sulfated polysaccharides and a pharmaceutically acceptable carrier, for treatment or inhibition of a disease or disorder caused by, or associated with, the activity of at least one bioactive polypeptide capable of binding a sulfate group of said sulfated polysaccharide.

Also provided is a method for treatment of a patient suffering from a disease or disorder caused by, or associated with, the activity of at least one bioactive polypeptide capable of binding a sulfate group of a sulfated polysaccharide, which comprises administering to said patient an effective amount of sulfated alginate, sulfated hyaluronan, or both.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to a bioconjugate comprising a sulfated polysaccharide and at least one bioactive polypeptide capable of binding a sulfate group of said sulfated polysaccharide. These bioconjugates are useful when administered to a mammal, for sustained release of said bioactive peptide(s) from said bioconjugate.

The at least one bioactive polypeptide may be a positively charged polypeptide, a heparin-binding polypeptide, or both.

The term "bioactive polypeptide" as used herein refers to a polypeptide exhibiting a variety of pharmacological activities in vivo, and include but are not limited to, growth factors, cytokines, chemokines, angiogenic factors, immunomodulators, hormones, and the like.

In the present application, the terms "polypeptide" and "proteins" are used interchangeably.

The term "positively charged polypeptides" refers to a polypeptide/protein that has a positive net charge at physiological pH of about pH=7.5. Examples of positively charged proteins include, but are not limited to, insulin, glatiramer acetate, antithrombin III, interferon .gamma. (also known as heparin-binding protein), IGF, somatostatin, erythropoietin, luteinizing hormone-releasing hormone (LH-RH) and interleukins such as IL-2 and IL-6.

The term "heparin-binding protein or polypeptide" refers to proteins that have clusters of positively-charged basic amino acids and form ion pairs with specially defined negatively-charged sulfo or carboxyl groups on the heparin chain (See Capila and Linhardt, 2002). Examples of heparin-binding proteins include, but are not limited to, thrombopoietin (TPO); proteases/esterases such as antithrombin III (AT III), serine protease inhibitor (SLP1), C1 esterase inhibitor (C1 INH) and Vaccinia virus complement control protein (VCP); growth factors such as a fibroblast growth factor (FGF, aFGF or bFGF), a FGF receptor, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), transforming growth factor .beta.1 (TGF-.beta.1), a platelet-derived growth factor (PDGF, PDGF-AA and PDGF-BB), and epidermal growth factor (EGF); chemokines such as platelet factor 4 (PF-4, now called CXC chemokine ligand 4 or CXCL4), stromal cell-derived factor-1 (SDF-1), IL-6, IL-8, RANTES (Regulated on Activation, Normal T Expressed and Secreted), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1 (MIP-1), lymphotactin, and fractalkine; lipid or membrane-binding proteins such as an annexin, apolipoprotein E (ApoE); pathogen proteins such as human immunodeficiency virus type-1 (HIV-1) coat proteins e.g. HIV-1 gp120, cyclophilin A (CypA), Tat protein, viral coat glycoprotein gC, gB or gD of herpes simplex virus (HSV), an envelope protein of Dengue virus, circumsporozoite (CS) protein of Plasmodium falciparum, bacterial surface adhesion protein OpaA; and adhesion proteins such as 1- and P-selectin, heparin-binding growth-associated molecule (HB-GAM), thrombospondin type I repeat (TSR), and amyloid P (AP).

In preferred embodiments of the present invention, the at last one heparin-binding polypeptide is selected from the group consisting of PDGF-BB, PDGF-AA, bFGF, aFGF, VEGF, TGF.beta.1, IL-6, TPO, SDF-1, HGF, EGF and IGF.

In a preferred embodiment of the invention, the at least one bioactive polypeptide is an angiogenic factor or a growth factor exhibiting angiogenic activity selected from the group consisting of TGF-.beta.1, VEGF, bFGF, aFGF, PDGF-BB, IGF, and a combination thereof.

In a more preferred embodiment of the invention, said at least one angiogenic factor consists of bFGF.

In yet another more preferred embodiment of the invention, the bioconjugate comprises a combination of VEGF, PDGF-BB and TGF-.beta.1.

In accordance with the present invention, the sulfated polysaccharides forming the bioconjugate may be composed of different recurring monosaccharide units, may be of different lengths, and may have different types of bonds linking said units. The sulfated polysaccharides may be linear as sulfated cellulose branched as sulfated glycogen, and may vary in length, for example, it may be as small as a sulfated trisaccharide. The sulfated polysaccharide may be a homopolysaccharide including, but not limited to, starch, glycogen, cellulose or chitin or a heteropolysaccharide including, but not limited to, alginic acid (alginate) salts and hyaluronic acid.

In a preferred embodiment of the invention, the polysaccharide comprises uronic acid residues such D-glucoronic, D-galacturonic, D-mannuronic, L-iduronic, and L-guluronic acids. Example of polysaccharides comprising uronic acid include, but are not limited to, alginic acid salts, preferably sodium alginate, pectin, gums and mucilages from plant sources; and glycosaminoglycans (GAGs) from animal sources including hyaluronic acid (hyaluronan). The sulfated polysaecharides comprising uronic acid can be chemically sulfated or may be naturally sulfated polysaccharides.

In one preferred embodiment of the present invention, the sulfated polysaccharide in the bioconjugate is alginate sulfate. In another embodiment the sulfated polysaccharide is hyaluronan sulfate.

Alginic acid is a linear polysaccharide obtained from brown algae and seaweed and consist of .beta.-1,4-linked glucuronic and mannuronic acid units.

Hyaluronic acid is composed of repeating dimmeric units of glucuronic acid and N-acetyl glucosamine and forms the core complex proteoglycans aggregates found in the extracellular matrix.

In a more preferred embodiment the bioconjugate is selected from the group consisting of aFGF-alginate sulfate, bFGF-alginate sulfate, PDGF-BB-alginate sulfate, PDGF-AA-alginate sulfate, VEGF-alginate sulfate, TGF.beta.1-alginate sulfate, IL-6-alginate sulfate, TPO-alginate sulfate, SDF-1-alginate sulfate, HGF-alginate sulfate, EGF-alginate sulfate, IGF-alginate sulfate, bFGF-hyaluronan sulfate and VEGF-hyaluronan sulfate.

The present invention is based on results obtained with the sulfated polysaccharides alginate sulfate and sulfated hyaluronan. We show herein that alginate and hyaluronic are sulfated and converted into reactive polymers capable of specifically interacting with at least one positively-charged polypeptide and/or heparin-binding polypeptide, forming a bioconjugate capable of sustaining the release of said at least one polypeptide. By sulfating the polysaccharides, we endowed them with properties which allowed binding and controlled release of important signal proteins such as various cytokines and growth factors. Alginate sulfate and hyaluronan sulfate were both found to mimic the biological specificities of heparan sulfate and heparin when forming the bioconjugates.

We prepared sulfated alginate and hyaluronan with different sulfation degrees and showed, by SPR technology, the interaction of the alginate sulfate and hyaluronan sulfate with various bioactive polypeptides. We determined that various positively-charged proteins and heparin-binding proteins bound specifically to the sulfated alginates and the sulfated hyaluronans with particular affinity-binding constants. Said proteins bound alginate sulfate and hyaluronan sulfate with high affinity and some of them exhibited superior binding to alginate sulfate and hyaluronan sulfate than to heparin (see, for example, bFGF, SDF-1, TGF.beta.1, and PDGF-BB binding in Table 3 (see Original Patent) hereinafter). We found that the pattern and kinetics of release of positively-charged proteins and heparin-binding proteins from these bioconjugates are dependent on the relative affinity of said proteins to the sulfated polysaccharide.

A bioconjugate according to the present invention can be injected to any part of the human body and serve as a delivery system for said bioactive polypeptide(s), for example, we show herein that administration of a bioconjugate comprising sulfated alginate and bFGF or a mixture of the three angiogenic factors, VEGF, TGF-b1 and PDGF-BB to animals, promoted sustained release of the factors and lead to superior vascularization and more mature blood vessels than the same factors supplied with non-modified alginate. The experiment with the three angiogenic factors demonstrate that the angiogenic factors work in a complementary and coordinated manner to form mature and high density blood vessels.

Thus in one aspect, the invention provides a pharmaceutical composition comprising a bioconjugate according to the invention and a pharmaceutically acceptable carrier.

In a preferred embodiment, the invention provides a pharmaceutical composition as a delivery system for sustained release of at least one bioactive polypeptide, comprising a bioconjugate composed of said at least one bioactive polypeptide and a sulfated polysaccharide, wherein said bioactive polypeptide is capable of binding a sulfate group of said sulfated polysaccharide.

For its use as a delivery system for the sustained release of the bioactive polypeptide(s), the bioconjugate of the invention may be injected or implanted in a mammal, optionally in association with or provided in a supporting matrix, used as scaffold for cell transplantation and tissue engineering. In the examples below, we show the successful sustained release of bioactive peptides from the bioconjugate of the invention present in capsules or in scaffolds formed by alginate.

Thus, in a preferred embodiment of the invention, the pharmaceutical composition further comprises a supporting matrix.

The matrix may serve as support or as a carrier for the bioconjugate and may be made up of particles or porous materials. The matrix material may be flexible and amenable to be fixed in place preventing its migration to an unintended location. The polymer matrix materials can be either natural or synthetic and include, but are not limited to, synthetic polymers such as polyethylene glycol (polyethylene oxide), poly(vinyl alcohol), polylactic acid, polyglycolic acid, and polyhydroxybutyrate, or natural polymers like collagen, fibrin, and gelatin, or polysaccharides like chitosan and alginate.

The matrix material is preferably biodegradable. Thus, physical removal of the matrix material from recipient's tissue following drug delivery is not necessary and there is no concern about effects of the residual matrix in the long term. Of advantage is the use of a matrix material which does not provoke a significant inflammatory or proliferative tissue response or which does not alter or interfere with the recipient's natural defense systems and healing processes.

The matrix may be in any form appropriate to the mode of delivery, for example, hydrogel, beads, microspheres (microbeads), hydrogel microcapsules, sponges, scaffolds, foams, colloidal dispersions, suspensions, and the like. Thus, a sustained release dosage form based on bioconjugates of sulfated polysaccharides and bioactive peptides may be fashioned as liquids, meshes, sponges, fibers and hydrogels.

In certain embodiments of the invention, the supporting matrix is selected from the group consisting of a polysaccharide, a protein, an extracellular matrix component a synthetic polymer and a mixture thereof.

In one preferred embodiment of the invention, the supporting matrix consists of polysaccharide, preferably of alginate hydrogel or hyaluronan hydrogel.

The term "pharmaceutically acceptable carrier" refers to a vehicle which delivers the active components to the intended target and which will not cause harm to humans or other recipient organisms. As used herein, "pharmaceutical" will be understood to encompass both human and veterinary pharmaceuticals. Useful carriers include, for example, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil and polymers composed of chemical substances like polyglycolic acid or polyhydroxybutyrate or natural polymers like collagen, fibrin or polysaccharides like chitosan and alginate. The carrier may be in any form appropriate to the mode of delivery, for example, solutions, colloidal dispersions, emulsions (oil-in-water or water-in-oil), suspensions, creams, lotions, gels, foams, mousses, sprays and the like. Methodology and components for formulation of pharmaceutical compositions are well known and can be found, for example, in Remington's Pharmaceutical Sciences, Eighteenth Edition, A. R. Gennaro, Ed., Mack Publishing Co. Easton Pa., 1990.

In one embodiment of the invention the carrier consists of an aqueous buffer.

In another embodiment of the invention, the carrier consists of a polysaccharide, and is preferably alginate hydrogel or hyaluronic acid.

The composition can be administered to a patient in need thereof in a variety of ways. The routes of administration include but are not limited to intraliver, intradermal, transdermal (e.g. in slow release formulations), intramuscular, intraperitoneal, intravenous, intracoronary, subcutaneous, oral, epidural, topical, and intranasal routes. Any other therapeutically efficacious route of administration can be used.

In a further aspect, the invention provides a method for the sustained released administration of at least one bioactive polypeptide which is capable of binding a sulfate group of a sulfated polysaccharide to a patient in need of treatment with said polypeptide, wherein the method comprises administering to said patient an effective amount of a bioconjugate of the invention comprising a sulfated polysaccharide and at least one bioactive polypeptide capable of binding a sulfate group of said sulfated polysaccharide.

Cochran et al. (J. Med. Chem. 2003, 46, 4601-4608) studied the binding interactions of the anticancer agent PI-88 and derivatives of PI-88 with the angiogenic factors FGF-1, FGF-2 and VEGF. PI-88 is a mixture of highly sulfated, monophosphorylated mannose oligosaccharide ranging in size from di- to hexasaccharide. The derivatives of the PI-88 which were studied had defined carbohydrate chain length (2 to 5 saccharide units) and were lacking a phosphate group. The results obtained in this binding study indicated that the two dominant components of the PI-88 mixture, namely, the penta- and tetrasaccharide components, have increased affinity for the angiogenic factors and therefore are responsible for the bulk of the antiangiogenic activity of PI-88. The binding studies demonstrated that PI-88 had greater affinity for FGF-1 and VEGF than heparin, heparan sulfate or polyanionic drugs such as sucrose octasulfate. The binding was highly dependent on the degree of sulfation and the chain length tested.

Alginate and hyaluronan are polysaccharides and not short oligosaccharide as PI-88, and are of different composition than PI-88. It was thus unexpected that their sulfated form would exhibit high affinity for angiogenic factors and in general for heparin-binding polypeptides and/or positively-charged bioactive polypeptides. Moreover, we found that alginate sulfate and hyaluronan bound bFGF, SDF-1, TGF.beta.1, and PDGF-BB with higher affinity than heparin (Tables 3 and 6 (see Original Patent)).

In view of the high affinity of alginate sulfate and hyaluronan sulfate to the bioactive polypeptides capable of binding sulfated polysaccharides, said sulfated polysaccharides themselves may be exploited for the elimination of said bioactive polypeptides in diseases or disorders caused by or associated with the activity of said bioactive polypeptides, for example, alginate sulfate or sulfated hyaluronan can be used for the treatment of: cancer which is known to be associated with growth factors and angiogenic factors; inflammatory diseases such as rheumatoid arthritis and bowel inflammatory diseases (e.g. Crohn's disease) associated with IL-6 activity; proliferative diabetic retinopathy associated with VEGF activity; myelodysplastic syndrome with myelofibrosis associated with TGFb and TPO activity; diabetic peripheral neuropathy associated with IFG activity; pulmonary arterial hypertension associated with PDGF-BB activity; and arteriosclerosis associated with PDGF-AA activity.

Thus, in another aspect, the invention provides a pharmaceutical composition comprising a sulfated polysaccharide selected from the group consisting of sulfated alginate, sulfated hyaluronan, and both, and a pharmaceutically acceptable carrier, for treatment or inhibition of a disease or disorder caused by, or associated with, the activity of at least one bioactive polypeptide capable of binding a sulfate group of said sulfated polysaccharide, preferably in diseases or disorders caused by, or associated with, the activity of a bioactive polypeptide selected from the group consisting of platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factor .beta. (TGF.beta.), acidic fibroblast growth factor (aFGF), interleukin-6 (IL-6), thrombopoietin (TPO), stromal cell derived factor-1 (SDF-1), hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulin growth factor (IGF), platelet-derived growth factor AA (PDGF-AA), and a combination thereof.

In a preferred embodiment, the invention provides a pharmaceutical composition comprising a sulfated alginate and/or sulfated hyaluronan and a pharmaceutically acceptable carrier for the treatment of cancer.

In another aspect, the invention relates to a method for treatment of a patient suffering from a disease or disorder caused by, or associated with, the activity of at least one bioactive polypeptide capable of binding a sulfate group of a sulfated polysaccharide, which comprises administering to said patient an effective amount of a sulfated alginate, sulfated hyaluronan, or both.

In a preferred embodiment, the invention relates to a method for the treatment of a patient suffering from cancer.
 

Claim 1 of 23 Claims

1. A bioconjugate comprising a sulfated polysaccharide selected from the group consisting of alginate sulfate and hyaluronan sulfate and at least one bioactive polypeptide selected from the group consisting of a positively-charged polypeptide, a heparin-binding polypeptide or both, wherein the bioactive polypeptide non-covalently associates with a sulfate group of the sulfated polysaccharide, thereby allowing sustained release of the bioactive polypeptide from the bioconjugate.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.