Compositions and methods for modulation of vascular structure and/or function   

The present invention relates to compositions comprising semi-crystalline poly-β-1→4-N-acetylglucosamine (p-GlcNac) polysaccharide polymers and methods utilizing such polymers for stimulating, in a p-GlcNac concentration-dependent manner, transient, localized stimulation of endothelin-1 release, vasoconstriction, and/or reduction in blood flow out of a breached vessel. These effects, individually and/or collectively, contribute or lead to cessation of bleeding. More specifically, the methods of the present invention comprise topical administration of compositions and materials comprising semi-crystalline polymers of N-acetylglucosamine that are free of proteins and substantially free of single amino acids and other organic and inorganic contaminants, and whose constituent monosaccharide sugars are attached in a β-1→4 conformation.

Vascular homeostasis depends, in part, upon the regulated secretion of biochemical modulators by endothelial cells. Under normal physiological conditions, endothelial cells synthesize and secrete nitric oxide, prostacyclin, PG12, adenosine, hyperpolarizing factor, tissue factor pathway inhibitor, and scuplasminogen activator. Endothelial cells also activate antithrombin III and protein C, which, collectively, mediate vascular dilation, inhibit platelet adhesion, platelet activation, thrombin formation and fibrin deposition. Nitric oxide, in particular, plays a critical role in vascular homeostasis (Pearson, J. D. (2000) Lupus 9 (3): 183-88; Becker et al., (2000) Z Kardiol 89 (3): 160-7; Schinin-Kerth, V. B. (1999) Transfus Clin Biol 4 (6): 355-63).

Production of nitric oxide and prostacyclin, which are powerful vasodilators and inhibitors of platelet aggregation and activation, underlies the antithrombotic activity of the endothelium (Yang et al. (1994) Circulation 89 (5): 2666-72). Nitric oxide is synthesized at a constitutive, basal level from arginine by nitric oxide synthase, and this synthesis is stimulated by the vaso-active agents acetylcholine and bradykinin. It has been shown that inhibition of nitric oxide synthase by the arginine analogues monomethyl-L-arginine (L-NMMA) and nitro-L-arginine methyl ester (L-NAME) reduces nitric oxide levels and leads not only to vasoconstriction, as measured by intravascular ultrasound imaging, but also to an increase in platelet aggregation (Yao et al. (1992) Circulation 86 (4): 1302-9; Emerson et al. (1999) Thromb Haemost 81 (6): 961-66).

Perturbation of the endothelium as the result of atherosclerosis, diabetes, postischemic reperfusion, inflammation or hypertension for example, leads to a prothrombotic state in which the endothelium elaborates a further set of biochemical modulators including TNF-α, IL-8, von Willebrand factor, platelet activating factor, tissue plasminogen activator, and type 1 plasminogen activator inhibitor. (Pearson, J. D. (2000) Lupus 2 (3): 183-88; Becker et al. (2000) Z Kardiol 12 (3): 160-7; Schinin-Kerth, V. B. (1999) Transfus Clin Biol 6 (6): 355-63). In addition, the vascular endothelium synthesizes and elaborates the endothelins, which are the most potent vasoconstrictor peptides known.

The endothelins are a family of 21-amino acid peptides, i.e., endothelin-1, endothelin-2, and endothelin-3, originally characterized by their potent vasoconstricting and angiogenic properties (see, e.g., Luscher et al. (1995), Agents Actions Suppl. (Switzerland) 45: 237-253; Yanagisawa et al. (1988) Nature 332: 411-415). The three isopeptides of the endothelin family, endothelin-1, endothelin-2, and endothelin-3, possess highly conserved amino acid sequences that are encoded by three separate genes (see, e.g., Inoue et al. (1989) Proc Natl Acad Sci USA 86: 2863-67; Saida et al. (1989) J Biol Chem 264: 14613-16). Although the endothelins are synthesized in several tissues including smooth muscle cells, endothelin-1 is exclusively synthesized by the vascular endothelium (Rosendorff, C. (1997) Cardiovasc Drugs 10 (6): 795-802). The endothelins are synthesized as preproendothelins of two hundred and three amino acids. The endothelin signal sequence is cleaved and the protein is then further proteolytically processed to yield the mature, biologically active 21 amino acid form (see, e.g., Kashiwabara et al. (1989) FEBS Lett 24: 337-40). Endothelin synthesis is regulated via autocrine mechanisms including endothelin and non-endothelin converting enzymes as well as by chymases (Baton et al. (1999) Curr Opin Nephrol Hypertens 8 (5): 549-56). Elaboration of endothelin-1 from the endothelium is stimulated by angiotensin II, vasopressin, endotoxin, and cyclosporin inter alia (see e.g. Brooks et al. (1991) Eur J Pharm 194: 115-17) and is inhibited by nitric oxide.

Endothelin activity is mediated via binding with preferential affinities to two distinct G protein-coupled receptors, ETA and ETB, in an autocrine/paracrine manner (see, e.g., Hocher et al. (1997) Eur. J. Clin. Chem. Clin. Biochem. 35 (3): 175-189; Shichiri et al. (1991) J. Cardiovascular Pharmacol. 17: S76-S78). ETA receptors are found on vascular smooth muscle linked to vasoconstriction and have been associated with cardiovascular, renal, and central nervous system diseases. ETB receptors are more complex and display antagonistic actions. ETB receptors in the endothelium have the dual roles of clearance and vasodilation, while ETB receptors on smooth muscle cells also mediate vasoconstriction (Dupuis, J. (2000) Can J Cardiol 16 (1): 903-10). The ETB receptors on the endothelium are linked to the release of nitric oxide and prostacycline (Rosendorff, C. (1997) Cardiovasc Drugs 14 (6): 795-802). There are a variety of agonists and antagonists of endothelin receptors (Webb et al. (1997) Medicinal Research Reviews 17 (1): 17-67), which have been used to study the mechanism of action of the endothelins. Because endothelin is known to have powerful vasoconstrictive activity, endothelin antagonists in particular (also termed “endothelin receptor antagonists” in the art) have been studied with regard to their possible role in treating human disease, most notably, cardiovascular diseases such as hypertension, congestive heart failure, atherosclerosis, restenosis, and myocardial infarction (Mateo et al. (1997) Pharmacological Res. 36 (5): 339-351).

Moreover, endothelin-1 has been shown to be involved in the normal functioning of the menstrual cycle. Menstruation represents a remarkable example of tissue repair and replacement, involving the regulated remodeling and regeneration of a new layer of endometrial tissue lining the uterus. This repair and remodeling process is remarkable in that it is accomplished without scarring, a phenomenon generally not seen in other organs of the body. Defects in that repair process are believed to be the basis of excessive or abnormal endometrial bleeding in women with documented menorrhagia as well as in women carrying subcutaneous levonorgestrel implants (NORPLANT) for contraceptive purposes. In both of these groups of patients, only very low levels of endometrial endothelin-1 have been detected as compared with control populations. Moreover, it has been indicated that endothelin-1 not only may play a role in effecting cessation of menstrual bleeding but endothelin-1 may also have a mitogenic activity required for regenerating and remodeling of endometrial tissue after menstruation (see Salamonsen et al. 1999, Ballière\'s Clinical Obstetrics and Gynaecology (2): 161-79; Goldie 1999, Clinical and Experimental Pharmacology and Physiology 26: 145-48; Salamonsen et al. 1999, Clin. Exp. Phamaol. Physiol. 26 (2): 154-57).

In summary, vascular homeostasis reflects a dynamic balance between two physiological states mediated by the vascular endothelium. The first, which has been termed antithrombotic, is characterized inter alia by the production of nitric oxide, vasodilation, inhibition of platelet attachment and activation, and by repression of endothelin-1 synthesis. The second or prothrombotic physiological state is characterized inter alia by the production of endothelin-1, vasoconstriction, platelet activation, and hemostasis (Warner (1999), Clinical and Experimental Physiology 26: 347-52; Pearson, (2000), Lupus 2(3): 183-88).

In light of the physiological importance of vascular homeostasis, there is a need for methods and compositions that are capable of modulating one or more aspects of the above processes. More specifically, there is a need for compositions and methods for the modulation of endothelin release, vasoconstriction, and blood flow out of a breached vessel and which would therefore be useful for effecting cessation of bleeding. That is, although such compositions and methods would act in a manner that is not dependent upon physical barrier formation, coagulation, or blood clot formation, such compositions and methods would nevertheless contribute, inter alia, to the achievement of hemostasis. Accordingly, such methods and compositions would be expected to have therapeutic applications for the treatment of diseases or conditions arising as a consequence of the perturbation of vascular homeostasis. Moreover, in view of the systemic effects resulting, e.g., from administration to patients of endothelin-1 antagonists as described supra, there is an even greater need for compositions and methods that produce localized and transient physiological responses, including, but not limited to, stimulation of endothelin-1 release, in such patients.

SUMMARY

The present invention relates to methods and compositions for the treatment or amelioration of vascular disorders including bleeding disorders. More specifically, the invention relates to compositions comprising semi-crystalline poly-β-1→4-N-acetylglucosamine (p-GlcNac) polysaccharide polymers, and use of such polymers in methods to effect transient localized, modulation of vascular structure and/or function by, e.g., stimulation of endothelin-1 release, vasoconstriction, and/or reduction in blood flow out of a breached vessel, thereby contributing to or effecting cessation of bleeding.

The present invention is based in part on the Applicants\' discovery that topical application of semi-crystalline poly-β-1→4-N-acetylglucosamine (p-GlcNac) polysaccharide polymers to a vascular surface induces not only contraction of that vessel, thereby decreasing the lumen of that vessel, but also induction of a transient, localized stimulation of endothelin-1 release in those tissues contiguous with the applied compositions and materials disclosed herein.

The present invention relates, in one aspect, to a method for achieving transient, localized, modulation of vascular structure and/or function in a patient, comprising topical administration of a material comprising semi-crystalline poly-β-1→4 N-acetylglucosamine polymers, which are free of protein, substantially free of other organic contaminants, and substantially free of inorganic contaminants. Administration of these materials induces transient, localized physiological responses including, but not limited to, stimulation of endothelin-1 release, vasoconstriction, and reduction in blood flow out of a breached vessel.

In one embodiment of the present invention, endothelin-1 is released from vascular endothelial cells. In other aspects of this embodiment, endothelin-1 release is stimulated from other endothelial tissues or from platelets.

In one embodiment, the poly-β-1→4 N-acetylglucosamine polymer comprises about 50 to about 4,000 N-acetylglucosamine monosaccharides covalently attached in a β-1→4 conformation, and has a molecular weight of about 10,000 daltons to about 800,000 daltons.

In another embodiment, the poly-β-1→4 N-acetylglucosamine polymer comprises about 50 to about 10,000 N-acetylglucosamine monosaccharides covalently attached in a (β-1→4 conformation, and has a molecular weight of about 10,000 daltons to about 2 million daltons. In yet another embodiment, the poly-β-1→4 N-acetylglucosamine polymer comprises about 50 to about 50,000 N-acetylglucosamine monosaccharides covalently attached in a β-1→4 conformation, and has a molecular weight of about 10,000 daltons to about 10 million daltons. In another embodiment, the poly-β-1→4 N-acetylglucosamine polymer comprises about 50 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a β1→4 conformation, and has a molecular weight of about 10,000 daltons to about 30 million daltons.

In preferred embodiments of the invention, the disclosed method is used for the treatment of a mammalian patient, and in more preferred embodiments, for the treatment of a human in need of such treatment. More specifically, modulation of vascular structure and/or function is used to effect cessation of bleeding, particularly in a patient afflicted with a coagulopathy. Such a disorder can be the result of a genetic defect, such as hemophilia, or a medical treatment, including for example, administration of systemic anticoagulants, e.g. coumadin, to a dialysis patient, cardiac patient, or other patient with an increased risk of vessel blockage. Similarly, the present method is used to effect a temporary, localized, reduction in blood flow out of a breached vessel during surgical repair of an aneurysm or excision of a tumor or polyp, particularly in a patient having a coagulopahtic condition, thereby minimizing blood loss during such a procedure. In other embodiments, the method of the present invention is used for the treatment of bleeding ulcers or varices, particularly esophageal varices. While not wishing to be bound by a particular theory or mechanism, it is believed that such cessation of bleeding by the methods disclosed herein occurs in a coagulation-independent manner.

In other embodiments of the method of the invention, the p-GlcNac-containing material is topically administered to the skin of the patient or to the surface of another organ, or the material may be applied directly to a vascular structure to be modulated, which may be a capillary, vein, or artery.

In yet another embodiment of the method of the invention, where the vascular structure is a breached blood vessel, topical application of the p-GlcNac-containing materials of the invention is used to achieve cessation of bleeding.

In a further embodiment of the invention, the extent of the transient, localized modulation of vascular structure and/or function is substantially proportional to the amount of semi-crystalline poly-β-1→4 N-acetylglucosamine applied.

The invention is also directed toward a biodegradable material comprising semi-crystalline poly-β-1→4 N-acetylglucosamine polymers which are free of protein, substantially free of other organic contaminants, and are substantially free of inorganic contaminants. In one embodiment, the semi-crystalline poly-β-1→4 N-acetylglucosamine polymers comprise about 50 to about 4,000 N-acetylglucosamine monosaccharides covalently attached in a β-1→4 conformation and have a molecular weight of about 10,000 daltons to about 800,000 daltons. In another embodiment, the semi-crystalline poly-β-1→4 N-acetylglucosamine polymer comprises about 50 to about 10,000 N-acetylglucosamine monosaccharides covalently attached in a β-1→4 conformation, and has a molecular weight of about 10,000 daltons to about 2 million daltons. In yet another embodiment, the poly-β-1→4 N-acetylglucosamine polymer comprises about 50 to about 50,000 N-acetylglucosamine monosaccharides covalently attached in a β-1→4 conformation, and has a molecular weight of about 10,000 daltons to about 10 million daltons. In another embodiment, the poly-β-1→4 N-acetylglucosamine polymer comprises about 50 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a β-1→4 conformation, and has a molecular weight of about 10,000 daltons to about 30 million daltons.

In another embodiment, the biodegradable material comprising semi-crystalline poly-β-1→4 N-acetylglucosamine polymers is a non-barrier-forming material.

In still another embodiment, the semi-crystalline poly-β-1→4 N-acetylglucosamine polymer comprises at least one N-acetylglucosamine monosaccharide that is deacetylated. In other aspects of this embodiment the poly-β-1→4 N-acetylglucosamine polymer may comprise about 10%, 20%, 30%, 40%, 50% or 60% deacetylated residues, provided the partially-deacetylated poly-β-1→4 N-acetylglucosamine polymer retains its semi-crystalline structure as demonstrated by sharp, discrete peaks when the polymer is analyzed by IR absorption spectroscopy, as described in Example 6, below.

FIG. 1. Chemical structure of 100% p-GlcNAc. “n” refers to an integer ranging up to about 150,000.

FIG. 2. Carbohydrate analysis of p-GlcNAc, Gas Chromatography-Mass Spectroscopy data. Solid squares represent p-GlcNAc purified using the acid treatment/neutralization variation of the Chemical/Biological method, as described in Section 5.3.2, below.

FIG. 3A. Circular dichroism spectra of solid membranes of pure p-GlcNAc.

FIG. 3B. Circular dichroism spectra of solid membranes of Deacetylated p-GlcNAc. The disappearance of the 211 nm minimum and 195 nm maximum observed in pure p-GlcNAc (FIG. 3A) indicates complete deacetylation under the conditions used, as described in Section 5.4 below.

FIG. 4A. Infra-red spectra analyses of thin membranes of pure diatom p-GlcNAc prepared by the mechanical force purification method, top, and the chemical/biological purification method, bottom.

FIG. 4B. Infra-red spectra analyses of two preparations of commercial “chitin” cast into membranes according to the methods detailed in Section 5.5, below.

FIG. 4C. Infra-red spectra analyses of pure p-GlcNAc which was modified by heat denaturation (top) and by chemical deacetylation (bottom), according to the methods detailed in Section 5.4, below.

FIG. 4D. Infra-red spectrum analysis of a p-GlcNAc membrane derived from the diatom Thalassiosira fluviatilis, using the chemical/biological purification method, as detailed in Section 5.3.2, below.

FIG. 4E. Infra-red spectrum analysis of a p-GlcNAc membrane prepared by the mechanical force purification method, as described in Section 53.1, below, following autoclaving.

FIG. 5A. NMR analysis of p-GlcNAc purified using the chemical/biological purification method as described in Section 5.3.2, below. Chart depicting peak amplitudes, areas, and ratios relative to reference controls. Ratio of total areas of peaks.

FIG. 5B. NMR analysis of p-GlcNAc purified using the chemical/biological purification method as described in Section 5.3.2. The graph depicts the ratios of total areas of peaks.

FIGS. 6A-B. Transmission electron micrographs (TEM) of a p-GlcNAc membrane prepared by the mechanical force purification method as described in Section 5.3.1, below. Magnification: (FIG. 6A), 4190×; (FIG. 6B), 16,250×.

FIGS. 7A-B. Transmission electron micro graphs (TEM) of a p-GlcNAc membrane by HF treatment as described in the discussion of the chemical/biological purification method in Section 5.3.2, below. Magnification: (FIG. 7A), 5270×; (FIG. 78) 8150×.

FIGS. 8A-B. Transmission electron micrographs (TEM) of a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the chemical/biological purification method, as described in Section 5.3.2, below. Magnification: (FIG. 8A), 5270×; (FIG. 8B), 16,700×.

FIG. 9A. Scanning electron micrograph depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the chemical/biological purification method as described in Section 5.3.2, below. Magnification: 200×.

FIG. 9B. Scanning electron micrograph depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the chemical/biological purification method as described in Section 5.3.2, below. Magnification: 1000×.

FIG. 9C. Scanning electron micrograph depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the chemical/biological purification method as described in Section 5.3.2, below. Magnification: 5000×.

FIG. 9D. Scanning electron micrograph depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the chemical/biological purification method as described in Section 5.3.2, below. Magnification: 10,000×.

FIG. 9E. Scanning electron micrograph depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the chemical/biological purification method as described in Section 5.3.2, below. Magnification: 20,000×.

FIGS. 10A-B. Scanning electron micrographs of a pure p-GlcNAc membrane made from material which was initially produced using the cell dissolution/neutralization purification method described in Section 5.3, below, dissolved in dimethylacetamide/lithium chloride, and reprecipitated in H2O into a mat, as described below in Section 5.5. Magnification: (FIG. 10A), 1000×, (FIG. 10B), 10,000×.

FIGS. 11A-B. Scanning electron micrographs of a deacetylated p-GlcNAc mat. Magnification: (FIG. 11A), 1000×, (FIG. 11B), 10,000×.

FIGS. 12A-B. Photographs of diatoms. Note the p-GlcNAc fibers extending from the diatom cell bodies.

FIG. 13. Diagram depicting some of the possible p-GlcNAc and deacetylated derivatives of the p-GlcNAc starting material. (Adapted from S. Hirano, “Production and Application of Chitin and Chitosan in Japan”, in “Chitin and Chitosan,” 1989, Skjak-Braek, Anthonsen, and Sanford, eds. Elsevier Science Publishing Co., pp. 37-43.)

FIG. 14. Transformed NMR data curves, used to obtain areas for each carbon atom and to then calculate the CH3 (area) to C-atom (area) ratios.

FIG. 15. Typical p-GlcNAc C13—NMR spectrum. The individual peaks represent the contribution to the spectrum of each unique carbon atom in the molecule.

FIG. 16. Transformed NMR spectrum data representing values calculated for CH3 (area) to C-atom (area) ratios. Top: Graphic depiction of data; bottom: numerical depiction of data.

FIGS. 17A-G. Three-dimensional p-GlcNAc matrices produced in various solvents.

Specifically, the p-GlcNAc matrices were produced in distilled water (FIG. 17A, FIG. 17D), 10% methanol in distilled water (FIG. 17B), 25% methanol in distilled water (FIG. 17C), 10% ethanol in distilled water (FIG. 17E), 25% ethanol in distilled water (FIG. 17F) and 40% ethanol in distilled water (FIG. 17G). Magnification: 200×. A scale marking of 200 microns is indicated on each of these figures.

FIG. 18. A typical standard curve obtained using the procedure described, below, in Section 18.1. A standard curve such as this one was used in the lysozyme-chitinase assay also described, below, in Section 18.1.

FIG. 19. p-GlcNAc lysozyme digestion data. The graph presented here depicts the accumulation of N-acetylglucosamine over time, as p-GlcNAc membranes are digested with lysozyme. The graph compares the degradation rate of fully acetylated p-GlcNAc to partially (50%) deacetylated p-GlcNAc, and demonstrates that the degradation rate for the partially deacetylated p-GlcNAc was substantially higher than that of the fully acetylated p-GlcNAc material.

FIG. 20. p-GlcNAc lysozyme digestion data. The graph presented here depicts the accumulation of N-acetylglucosamine over time, as p-GlcNAc membranes are digested with lysozyme. The graph compares the degradation rate of two partially deacetylated p-GlcNAc membranes (specifically a 25% and a 50% deacetylated p-GlcNAc membrane). The data demonstrate that the degradation rate increases as the percent of deacetylation increases, with the degradation rate for the 50% deacetylated p-GlcNAc membrane being substantially higher than that of the 25% deacetylated p-GlcNAc membrane.

FIGS. 21A-21E. p-GlcNAc in vivo biodegradability data. FIGS. 21A-21C depict rats which have had prototype 1 (fully acetylated p-GlcNAc) membrane abdominally implanted, as described, below, in Section 18.1. FIG. 21A shows a rat at day 0 of the implantation; FIG. 21B shows a rat at day 14 post-implantation; FIG. 21C shows a rat at day 21 post-implantation. FIGS. 21D-21E depict rats which have had prototype 3A (lyophilized and partially deacetylated p-GlcNAc membrane) abdominally implanted, as described, below, in Section 18.1. FIG. 21D shows a rat at day 0 of the implantation; FIG. 21E shows a rat at day 14 post-implantation.

FIG. 22. Dose-dependent vasoconstriction of isolated aortic rings by p-GlcNac, either with an intact endothelial layer, panel A, or after removal of the endothelial layer, panel B. The number of contraction measurements that were averaged to provide the values reported at each concentration of p-GlcNac tested, either with or without an intact endothelial layer, is indicated within the figure, above each p-GlcNAc concentration tested.

FIG. 23 A-E. Arterial vasoconstriction by p-GlcNac. FIG. 23 (A) depicts a cross-section of a porcine artery obtained 60 minutes after application of a gauze dressing to one side of the artery. FIG. 23 (B) depicts a cross-section of a porcine artery obtained 15 minutes after application of a p-GlcNac membrane to one side of the artery. FIG. 23 (C) depicts a cross-section of a porcine artery obtained 60 minutes after application of a p-GlcNac membrane to one side of the artery. FIG. 23 (D) depicts a cross-section of a porcine artery obtained 15 minutes after application of a fibrin-coated collagen dressing to one side of the artery. FIG. 23 (E) depicts a cross-section of a porcine artery obtained 60 minutes after application of a fibrin-coated collagen dressing to one side of the artery.

FIG. 24 Arterial vasoconstriction by p-GlcNac. FIG. 24 depicts the thickness of a porcine arterial wall that either was (1), or was not (2), in direct contact with the material tested, for 15 or 60 minutes, as indicated. The materials applied to one side of the artery were: (A) gauze dressing; (B) and (C) p-GlcNac membrane; (D) and (E) fibrin-coated collagen dressing.

DETAILED DESCRIPTION

The present invention relates to compositions and methods useful for effecting transient, localized modulation of vascular structure and/or function, by, e.g. (1) stimulation of endothelin-1 release, (2) vasoconstriction, and (3) reduction in blood flow out of a breached vessel, comprising topical administration of compositions and materials that comprise semi-crystalline poly-β-1→4-N-acetylglucosamine (p-GlcNac) polysaccharide polymers. Stimulation of endothelin-1 release, vasoconstriction, and reduction in blood flow out of a breached vessel in a target tissue may be achieved either by direct application of the materials of the present invention to the target tissue, or by application of those materials to the skin or other organ or tissue surface that is adjacent to or contiguous with the target tissue.

The present invention is therefore, also directed to compositions and methods that contribute to or directly effect cessation of bleeding. Administration of the materials of the invention, which comprise semi-crystalline poly-β-1→4-N-acetylglucosamine polymers, results in stimulation of endothelin-1 release, vasoconstriction, and decrease in blood flow out of a breached vessel. These physiological responses, individually and/or collectively, contribute to or directly effect cessation of bleeding, which may be a capillary, vein, or artery. While not wishing to be bound by a particular theory or mechanism, it is believed that such cessation occurs in a coagulation-independent manner. Moreover, achievement of cessation of bleeding using the compositions and methods of the present invention is also not dependent upon formation of a physical barrier or mechanical matrix that promotes clotting. That is, according to the present invention, the material need not be a barrier-forming material that provides a mechanical matrix that adheres to the site of application and seals the boundaries of the wound. In contrast, the compositions and methods of the present invention induce a transient, localized alteration of vascular structure and/or function, and it is that alteration, which is independent of clot formation, that, per se, contributes to or directly effects cessation of bleeding.

Furthermore, the preferred materials of the compositions and methods of the present invention comprise fully acetylated semi-crystalline poly-β-1→4-N-acetylglucosamine polymers, since, as demonstrated the Examples provided in Sections 16 and 17, as well as FIG. 22, infra, materials comprising 70%-deacetylated poly-β-1→4-N-acetylglucosamine polymers do not induce vasoconstriction and, therefore will not decrease the lumen of the vessel and, consequently, will not reduce blood flow out of a breached vessel.

This invention is based in part on Applicants\' discovery that topically-applied materials, which need not be barrier-forming materials, that comprise semi-crystalline poly-β-1→4-N-acetylglucosamine (p-GlcNac) polymers, induce vasoconstriction in isolated Sprague-Dawley rat aortic rings. In this blood-free system, fully acetylated poly-1-1→4-N-acetylglucosamine induced contraction of the isolated aortic rings in a concentration-dependent manner. As demonstrated infra, in the Example presented in Section 17, the degree of vasoconstriction obtained was substantially proportional to the concentration of p-GlcNac applied to the isolated aortic ring. In contrast, 70% deacetylated poly-β-1→4-N-acetylglucosamine, did not induce vasoconstriction of the isolated aortic rings, at any concentration tested.

This invention is also based in part on Applicants\' discovery that in vivo application of membrane membranes, which are formed from semi-crystalline poly-β-1→4-N-acetylglucosamine polymers, to experimental wounds in arteries, stimulated immediate vasoconstriction at the site of contact between the arterial tissue and the applied membrane. Histological analysis of treated tissue revealed that arterial constriction was greater on the side where the membrane was applied than on the opposite side of the artery. Furthermore, immunochemical analyses of these tissue samples also revealed the presence of a concentration gradient of endothelin-1 release, i.e., stimulation of endothelin-1 release was a localized physiological response. The extent of the stimulation of endothelin-1 release was greatest at the surface contacted by the semi-crystalline poly-β-1→4-N-acetylglucosamine polymer containing-membrane, and extended into adjacent tissue, although to an extent that decreased as the distance from the contact surface increased. A similar, localized stimulation of endothelin-1 release was observed in spleen tissue contacted with material comprising semi-crystalline poly-β-1→4-N-acetylglucosamine.

The methods of the present invention comprise topical administration of materials comprising a therapeutically effective form and a therapeutically effective amount of semi-crystalline poly-β-1→4-N-acetylglucosamine polymers, to a patient in order to achieve transient, localized: (1) enhancement of endothelin-1 release, (2) vasoconstriction, and/or (3) reduction of blood flow out of a breached vessel.

Presented below, is, first, a description of physical characteristics of the purified p-GlcNac starting material, and of its reformulations. Next, methods are described for the purification of the p-GlcNac starting material from microalgae, preferably diatom, starting sources. Third, reformulations of the p-GlcNac, and methods for the production of such reformulations are presented. Finally, uses are presented for the p-GlcNAc, p-GlcNAc derivatives and/or p-GlcNac reformulations of the starting material.

5.1. p-GlcNac

The p-GlcNac starting material can be made using techniques described herein, coupled with the teaching provided in U.S. Pat. Nos. 5,686,115, 5,624,679, 5,623,064, and 5,622,834, each of which is hereby incorporated by reference in its entirety. The p-GlcNac polymers used herein comprise about 50 to about 150,000 N-acetylglucosamine monosaccharides (FIG. 1). The purity of the p-GlcNac starting material is very high, as evidenced by chemical and physical criteria. Among these are chemical composition and non-polysaccharide contaminants. First, chemical composition data for the p-GlcNac produced using two different purification methods, both of which are described in Section 5.3, below, is shown in Table I below. As can be seen, the chemical composition of the p-GlcNac produced by both methods is, within the bounds of experimental error, the same as the formula compositions of p-GlcNac. Second, as is also shown in Table I, the p-GlcNac produced is free of detectable protein contaminants, is substantially free of other organic contaminants such as free amino acids, and is substantially free of inorganic contaminants such as ash and metal ions (the p-GlcNac starting material may deviate up to about 2% from the theoretical values of carbon, hydrogen, nitrogen and oxygen for pure p-GlcNac). Therefore, as used herein, the terms “substantially free of organic contaminants” and “substantially free of inorganic contaminants” refer to compositions of p-GlcNac having the profiles for carbon, hydrogen, nitrogen and oxygen which deviate no more than about 2% from the theoretical values, and preferably, the p-GlcNac starting material contain a profile as exemplified in the Experimental Data on p-GlcNac mats in Table I (allowing for the percent deviation). Further, the p-GlcNac starting material exhibits a low percentage of bound water.

TABLE I CHEMICAL ANALYSIS DATA (% by weight) Theoretical Values for Pure p-GlcNac: Carbon - 47.29 Hydrogen - 6.40 Nitrogen - 6.89 Oxygen - 39.41 Protein - 0.00 Experimental Data on p-GlcNac Mats: (Number of experimental batches for each membrane type being greater than 30 for each membrane type) MECHANICAL CHEMICAL/BIO- FORCE METHOD LOGICAL METHOD Normalized1 % Dev. Normalized1 % Dev. Carbon 47.21 ± 0.08 −0.17 47.31 ± 0.01 +0.04 Hydrogen  6.45 ± 0.08 +0.78  6.34 ± 0.08 −0.94 Nitrogen  6.97 ± 0.18 +0.87  6.94 ± 0.16 +0.73 Oxygen 39.55 ± 0.36 +0.36 39.41 ± 0.10 0.00 Average Values Average Values Protein 0.00 0.00 Ash 1.30 0.98 Moisture 2.0 1.2 1Raw analytical data have been normalized to account for ash and moisture content of the samples.

The pure p-GlcNac starting material exhibits a carbohydrate analysis profile substantially similar to that shown in FIG. 2. The primary monosaccharide of the pure p-GlcNac starting material is N-acetylglucosamine. Further, the pure p-GlcNac starting material does not contain the monosaccharide glucosamine.

The circular dichroism (CD) and sharp infra-red spectra (IR) of the p-GlcNac starting material are shown in FIG. 3A, and FIGS. 4A, 4D, and 4E, respectively, which present analyses of material produced using the methods described in Section 5.3, below. Such physical data corroborates that the p-GlcNac starting material is of high purity and semi-crystalline. The phrase “semi-crystalline” refers to the highly ordered nature of the material. One of skill in the art would readily appreciate that the sharp, well resolved peaks observed in the infra-red spectra of the p-GlcNAc polymers of the present invention reflect the highly ordered, crystalline nature of the material (i.e. “semi-crystalline”) examined. That artisan would also appreciate that broadened, poorly resolved peaks in such a IR spectra, as for example depicted in FIGS. 4B and 4C, would indicate loss or lack of a semi-crystalline nature. The methods used to obtain the CD and IR data are described, below, in the Working Example presented in Section 6.

NMR analysis of the pure p-GlcNac starting material exhibits a pattern substantially similar to that seen in FIGS. 5A, 14, 15 and 16. Such an NMR pattern indicates not only data which is consistent with the p-GlcNac starting material being a fully acetylated polymer, but also demonstrates the lack of contaminating organic matter within the p-GlcNac species. The electron micrographic structure of the p-GlcNac starting material, as produced using the methods described in Section 5.3, below and demonstrated in the Working Examples presented below in Section 8 and 9, is depicted in FIG. 6 through FIG. 9E.